RU2799438C2 - Antibody and drug conjugates as a splicing modulator and methods of their use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to new conjugates of antibodies with drugs, and can be used in medicine. The invention discloses conjugates of antibodies capable of binding to tumor antigens with fragment-drugs that are splicing modulators, wherein the conjugated fragments are interconnected via a linker.

EFFECT: invention may be used in the treatment of neoplastic disorders, in particular cancer.

52 cl, 10 ex, 27 tbl, 26 dwg

Description

[01] The present invention claims the benefit of priority based on US Provisional Application No. 62/679,672, filed June 1, 2018; U.S. Provisional Application No. 62/679631 filed June 1, 2018 and U.S. Provisional Application No. 62/779324 filed December 13, 2018. All of the above applications are incorporated herein by reference in their entirety.

[02] The present invention relates to antibody drug conjugates (ADC) containing a splicing modulator and an antibody or antigen-binding fragment that binds a human tumor antigenic target. The present invention further relates to methods and compositions useful in the treatment or diagnosis of cancers that express a target antigen and/or are treatable by disrupting RNA splicing, as well as methods for preparing such compositions.

[03] Most protein-coding genes in the human genome consist of several exons (coding regions) that are separated by introns (non-coding regions). Gene expression leads to the formation of a single messenger RNA precursor (pre-mRNA). The intron sequences are subsequently removed from the pre-mRNA through a process called splicing, which results in mature messenger RNA (mRNA). By including different combinations of exons, alternative splicing causes the formation of mRNAs encoding different protein isoforms.

[04] RNA splicing is catalyzed by the spliceosome, a dynamic multiprotein RNA complex consisting of five small nuclear RNAs (snRNA U1, U2, U4, U5 and U6) and associated proteins. The spliceosome assembles onto pre-mRNA to provide a dynamic cascade of multiple RNA-protein interactions that catalyze intron excision and exon ligation (Matera and Wang (2014) Nat Rev Mol Cell Biol. 15(2):108-21). Accumulating evidence has linked human diseases to RNA splicing dysregulation that affects multiple genes (Scotti and Swanson (2016) Nat Rev Genet. 17(1):19-32).

[05] The spliceosome is an important target in oncobiology. Several studies have now documented significant changes in the splicing profile of cancer cells, as well as in the splicing factors themselves (Agrawal et al. (2018) Curr Opin Genet Dev. 48:67-74). Alternative splicing can result in differential exon inclusion/exclusion, intron retention, or the use of cryptic splicing sites (Seiler et al. (2018) Cell Rep. 23(1):282-296). Collectively, these events underlie functional changes that may contribute to tumorigenesis or therapy resistance (Siegfried and Karni (2018) Curr Opin Genet Dev. 48:16-21).

[06] Certain natural products can bind the SF3b spliceosome complex. These small molecules modulate splicing by promoting intron retention and/or exon skipping (Teng et al. (2017) Nat Commun. 8:15522). A significant proportion of the resulting transcripts contain premature stop codons that trigger nonsense-mediated mRNA degradation (NMD). In addition, since canonical splicing is disrupted, the number of canonical transcripts is greatly reduced, which can adversely affect cell function and viability. In this regard, splicing modulators are becoming a promising class of drugs for the treatment of cancer (Puthenveetil et al (2016) Bioconjugate Chem. 27:1880-8).

[07] Human epidermal growth factor proto-oncogenic receptor-2 ( HER2 ) encodes a transmembrane tyrosine kinase receptor that is part of the human epidermal growth factor receptor (EGFR) family (King et al. (1985) Science 229:974-6). HER2 overexpression mediates constitutive activation of growth factor signaling pathways, such as the PI3K-AKT-mTOR pathway, and thus acts as an oncogenic factor in several types of cancer, including approximately 20% of invasive breast carcinomas (Slamon et al. (1989) Science 244 :707-12; Gajria and Chandarlapaty (2011) Expert Rev Anticancer Ther. 11:263-75). Given that HER2 amplification mediates a transformed phenotype, and since HER2 expression is restricted largely to malignant cells, HER2 is a promising antigen for targeting certain cancers and/or delivering novel cancer therapies (Parakh et al. (2017) Cancer Treat Rev. 59:1-21). Additional antigens for targeted delivery of cancer therapies include, but are not limited to, CD138 (also referred to as syndecan-1) and ephrin type-A receptor 2 (EPHA2).

[08] CD138 is a cell surface heparan sulfate proteoglycan that is essential for maintaining cell morphology and its interaction with the surrounding microenvironment (Akl et al. (2015) Oncotarget 6(30):28693-715; Szatmári et al. (2015) Dis Markers 2015:796052). In general, loss of CD138 expression in carcinoma cells reduces cell adhesion to the extracellular matrix and increases cell motility and invasion (Teng et al. (2012) Matrix Biol. 31:3-16). Upregulation of CD138 expression in the stroma also alters fibronectin production and extracellular matrix organization (Yang et al. (2011) Am J Pathol. 178:325-35). Additionally, increased expression of CD138 in stromal fibroblasts has been associated with angiogenesis and cancer progression (Maeda et al. (2006) Oncogene 25:1408-12). CD138 expression is upregulated during B cell development and its presence is a hallmark of plasma cells (Ribatti (2017) Immunol Lett. 188:64-7). Expression of CD138 is conserved in multiple myeloma, a malignant neoplasm of plasma cells. Therefore, CD138 is an attractive antigen for the targeted treatment of several cancers and other hematological malignancies (Sherbenou et al. (2015) Blood Rev. 29(2):81-91; Wijdenes et al. (1996) Br J Haematol. 94 (2):318-23).

[09] EPHA2 is a transmembrane glycoprotein that is widely overexpressed in several cancer-derived cell lines and advanced cancers (Wykosky and Debinski (2008) Mol Cancer Ref. 6(12):1795-1806). For example, EPHA2 is highly overexpressed in approximately 61% of GBM patient tumors (Wykosky et al. (2008) Clin Cancer Res. 14:199-208), 76% of ovarian cancers (Thaker et al. (2004) Clin Cancer Res. 10 :5145-50) and 85% of prostate adenocarcinomas (Zeng et al. (2003) Am J Pathol. 163:2271-6). The EPHA2 protein is highly overexpressed in terms of the percentage of patient tumors and the percentage of cells within the tumor, and is a plasma membrane localized receptor that can be internalized when bound to a ligand (Walker-Daniels et al. (2002) Mol Cancer Res. 1:79-87). Moreover, EPHA2 expression is associated with poor prognosis, increased metastasis, and decreased survival. Thus, due to its expression pattern, localization, and functional importance in determining outcome in patients with cancer, EPHA2 represents another attractive antigen for targeted delivery of novel cancer therapies.

[10] In various embodiments, implementation of the present invention, among other things, presents new compounds with biological activity against neoplastic cells. The compounds may slow, inhibit and/or reverse tumor growth in mammals and may be useful in the treatment of human patients with cancer. In various embodiments, the present invention provides novel antibody-drug conjugates using novel compounds or other functional splicing inhibitor molecules.

[11] In various embodiments, the present invention more specifically relates to antibody drug conjugate (ADC) compounds that are capable of binding to and killing neoplastic cells. In various embodiments, the ADC compounds disclosed herein contain a linker that attaches the splicing modulator to a full-length antibody or antigen-binding fragment. In various embodiments, the ADC compounds are also capable of internalizing into the target cell after binding.

[12] In various embodiments, ADC compounds may be represented by formula (I):

Ab-(LD) _p (I),

where Ab is an antibody or an antigen-binding fragment thereof that specifically targets a neoplastic cell or other tumor-associated target;

D is a splicing modulator;

L is a linker that covalently attaches Ab to D; And

p is an integer from 1 to 15.

[13] In various embodiments, ADC compounds may be represented by formula (I):

Ab-(LD) _p (I),

where Ab is an antibody or antigen-binding fragment that specifically affects the neoplastic cell;

D is a splicing modulator of formula (II):

(II),

(II)

or a pharmaceutically acceptable salt thereof, where:

R ¹ is absent or selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups, -OC(=O)-(C ₁ -C ₆ alkyl) and -CD ₃ groups;

R ³ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups and -OC(=O)-(C ₁ -C ₆ alkyl) groups; And

each of R ⁴ , R ⁵ and R ⁸ is independently selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups and C ₁ -C ₆ alkyl groups;

each of R ⁶ and R ⁷ is independently selected from hydrogen, -OR ¹⁷ , -OC(=O)-R ¹⁷ , -OC(=O)-NR ¹⁵ R ¹⁶ , C ₁ -C ₆ alkyl groups and -NR ¹⁵ R ¹⁶ ;

each of R ¹⁵ and R ¹⁶ is independently selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C(=O)-OR ¹⁷ ;

R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups; And

,

,

,

и

;Z is selected from

,

,

,

And

;

where each of R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ¹⁵ , R ¹⁶ , and R ¹⁷ is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups,

-O-(C ₁ -C ₆ alkyl), -NR ¹⁵ R ¹⁶ , C ₃ -C ₈ cycloalkyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkylalkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups groups,

where at least one of R ⁶ and R ⁷ is hydrogen;

and where L is a linker that covalently attaches Ab to D; And

p is an integer from 1 to 15.

[14] In some embodiments, the antibody or antigen-binding fragment is capable of internalization into the target cell. In some embodiments, the linker is covalently attached to a splicing modulator of formula (II) ("L-D") and L-D has the structure of formula (II-A):

(II-A),

(II-A),

or a pharmaceutically acceptable salt thereof,

,

,

,

и

; иwhere Z' is selected from

,

,

,

And

; And

where all other variables are as defined for formula (II).

[15] In various other embodiments, ADC compounds may be represented by formula (I):

Ab-(LD) _p (I),

where Ab is an antibody or antigen-binding fragment that specifically affects the neoplastic cell;

D is a splicing modulator of formula (IV):

(IV),

(IV)

or a pharmaceutically acceptable salt thereof, where:

R ¹ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups, -OC(=O)-(C ₁ -C ₆ alkyl) and -CD ₃ groups;

R ³ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups and -OC(=O)-(C ₁ -C ₆ alkyl) groups; And

each of R ⁴ , R ⁵ and R ⁸ is independently selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups and C ₁ -C ₆ alkyl groups;

each of R ⁶ and R ⁷ is independently selected from hydrogen, -OR ¹⁷ , -OC(=O)-R ¹⁷ , -OC(=O)-NR ¹⁵ R ¹⁶ , C ₁ -C ₆ alkyl groups and -NR ¹⁵ R ¹⁶ ;

each of R ¹⁵ and R ¹⁶ is independently selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C(=O)-OR ¹⁷ ; And

R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups;

where each of R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ¹⁵ , R ¹⁶ , and R ¹⁷ is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups,

-O-(C ₁ -C ₆ alkyl), -NR ¹⁵ R ¹⁶ , C ₃ -C ₈ cycloalkyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkylalkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups groups,

where at least one of R ⁶ and R ⁷ is hydrogen;

and where L is a linker that covalently attaches Ab to D; And

p is an integer from 1 to 15.

[16] In some embodiments, the antibody or antigen-binding fragment is capable of internalization into the target cell. In some embodiments, the linker is covalently attached to a splicing modulator ("L-D") and L-D has the structure of formula (IV-A):

(IV-A),

(IV-A),

or a pharmaceutically acceptable salt thereof.

[17] In various other embodiments, ADC compounds may be represented by formula (I):

Ab-(LD) _p (I),

where Ab is an antibody or antigen-binding fragment that specifically affects the neoplastic cell;

D is a splicing modulator of formula (VI):

(VI),

(VI)

or a pharmaceutically acceptable salt thereof, where:

each of R ¹ and R ⁹ is independently selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups, -OC(=O)-(C ₁ -C ₆ alkyl) and -CD _{3 groups} ;

R ³ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups and -OC(=O)-(C ₁ -C ₆ alkyl) groups;

each of R ⁴ , R ⁵ and R ⁸ is independently selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups and C ₁ -C ₆ alkyl groups;

each of R ⁶ and R ⁷ is independently selected from hydrogen, -OR ¹⁷ , -OC(=O)-R ¹⁷ , -OC(=O)-NR ¹⁵ R ¹⁶ , C ₁ -C ₆ alkyl groups, -NR ¹⁵ R ¹⁶ and linker;

R ¹⁰ is selected from hydrogen, C ₁ -C ₆ alkyl groups, -C(=O)-(C ₁ -C ₆ alkyl) and -CD ₃ groups;

each of R ¹⁵ and R ¹⁶ is independently selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and

-C(=O)-OR ¹⁷ ;

R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups; And

a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

where each of R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹⁵ , R ¹⁶ , and R ¹⁷ is independently substituted with 0-3 groups independently selected from atoms halogen, hydroxyl groups, C ₁ -C ₆ alkyl groups, groups -O- (C ₁ -C ₆ alkyl), -NR ¹⁵ R ¹⁶ , C ₃ -C ₈ cycloalkyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ - C ₆ alkylalkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups;

where at least one of R ⁶ and R ⁷ is hydrogen; And

where both R ¹ and R ⁹ cannot be absent;

and where L is a linker that covalently attaches Ab to D; And

p is an integer from 1 to 15.

[18] In some embodiments, the antibody or antigen-binding fragment is capable of internalization into the target cell. In some embodiments, the linker is covalently attached to a splicing modulator ("L-D") and the L-D has the structure of formula (VI-A):

(VI-A),

(VI-A),

or a pharmaceutically acceptable salt thereof.

[19] In various other embodiments, ADC compounds may be represented by formula (I):

Ab-(LD) _p (I),

where Ab is an antibody or antigen-binding fragment that specifically affects the neoplastic cell;

D is a splicing modulator of formula (VIII):

(VIII),

(VIII)

or a pharmaceutically acceptable salt thereof, where:

R ¹ is absent or selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups, -OC(=O)-(C ₁ -C ₆ alkyl) and -CD ₃ groups;

R ³ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups and -OC(=O)-(C ₁ -C ₆ alkyl) groups;

R ⁴ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₆ alkyl), -OC(=O)-(C ₁ -C ₆ alkyl) groups, and C ₁ -C ₆ alkyl groups; And

R ¹⁰ is selected from 3-10 membered carbocycles and 3-10 membered heterocycles, each of which is substituted with 0-3 R ^a , where each R ^a is independently selected from halogen atoms, C ₁ -C ₆ alkyl groups, -O-( C ₁ -C ₆ )alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylhydroxy groups, -S(=O) _w -(4-7-membered heterocycles), 4-7-membered carbocycles and 4-7 -membered heterocycles;

each of R ¹⁵ and R ¹⁶ is independently selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C(=O)-OR ¹⁷ ; And

R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups;

where each of R ¹ , R ³ , R ⁴ , R ¹⁰ , R ¹⁵ , R ¹⁶ and R ¹⁷ is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, groups -O -(C ₁ -C ₆ alkyl), -NR ¹⁵ R ¹⁶ , C ₃ -C ₈ cycloalkyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkylalkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups; And

where each R ^a is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, -NR ¹⁵ R ¹⁶ , C ₁ -C ₆ alkyl groups, -(C=O)-(C ₁ -C ₆ alkyl) groups ,

-(C=O)-(C ₁ -C ₆ alkyl)-(C ₃ -C ₁₀ heterocyclyl groups), -S(=O) _w -(C ₃ -C ₈ heterocyclyl) groups and C ₁ -C ₆ groups alkylcarboxylic acids each substituted with 0, 1 or 2 groups independently selected from halogen atoms, hydroxyl groups, -NR ¹⁵ R ¹⁶ and C ₁ -C ₃ alkyl groups; And

w is 0, 1 or 2;

and where L is a linker that covalently attaches Ab to D; And

p is an integer from 1 to 15.

[20] In some embodiments, the antibody or antigen-binding fragment is capable of internalization into the target cell. In some embodiments, the linker is covalently attached to a splicing modulator ("L-D") and the L-D has the structure of formula (VIII-A):

(VIII-A),

(VIII-A),

or a pharmaceutically acceptable salt thereof.

[21] In some embodiments, the splicing modulator includes an SF3b complex modulator.

In some embodiments, the splicing modulator comprises a pladienolide or a pladienolide derivative. In some embodiments, the splicing modulator provides for pladienolide D or a pladienolide D derivative. In some embodiments, pladienolide D or a derivative provides for D2, D1, D4, D8, D10, D11 (E7107), D20, D21, D22, D12, or D25. In some embodiments, the pladienolide D or derivative provides for D2. In some embodiments, the pladienolide D or derivative provides for D1. In some embodiments, the pladienolide D or derivative provides for D4. In some embodiments, the pladienolide D or derivative provides for D12.

[22] In some embodiments, pladienolide D or a derivative provides for a zwitterionic pladienolide D or derivative thereof. In some embodiments, the zwitterionic pladienolide D or derivative provides D22 or D25.

[23] In some other embodiments, the splicing modulator provides for pladienolide B or a pladienolide B derivative. In some embodiments, pladienolide B or a derivative provides for D9, D18, D19, or D13.

In some embodiments, the splicing modulator comprises an aryl pladienolide. In some embodiments, the implementation of the aryl pladienolide provides D15, D14, D16, D17, D26 or D33. In some embodiments, the implementation of the aryl pladienolide provides D15. In some embodiments, the aryl pladienolide is a zwitterionic aryl pladienolide. In some embodiments, the zwitterionic aryl pladienolide provides for D33.

[24] In some embodiments, the splicing modulator provides D1:

(D1).

(D1).

[25] In some embodiments, the splicing modulator provides D2:

(D2).

(D2).

[26] In some embodiments, the splicing modulator provides D3:

(D3).

(D3).

[27] In some embodiments, the splicing modulator provides D4:

(D4).

(D4).

[28] In some embodiments, the splicing modulator provides D4':

(D4').

(D4').

[29] In some embodiments, the splicing modulator provides D5:

(D5).

(D5).

[30] In some embodiments, the splicing modulator provides D6:

(D6).

(D6).

[31] In some embodiments, the splicing modulator provides D7:

(D7).

(D7).

[32] In some embodiments, the splicing modulator provides D8:

(D8).

(D8).

[33] In some embodiments, the splicing modulator provides D9:

(D9).

(D9).

[34] In some embodiments, the splicing modulator provides D10:

(D10).

(D10).

[35] In some embodiments, the splicing modulator provides D11:

(D11).

(D11).

[36] In some embodiments, the splicing modulator provides D12:

(D12).

(D12).

[37] In some embodiments, the splicing modulator provides D13:

(D13).

(D13).

[38] In some embodiments, the splicing modulator provides D14:

(D14).

(D14).

[39] In some embodiments, the splicing modulator provides D15:

(D15).

(D15).

[40] In some embodiments, the splicing modulator provides D16:

(D16).

(D16).

[41] In some embodiments, the splicing modulator provides D17:

(D17).

(D17).

[42] In some embodiments, the splicing modulator provides D18:

(D18).

(D18).

[43] In some embodiments, the splicing modulator provides D19:

(D19).

(D19).

[44] In some embodiments, the splicing modulator provides D20:

(D20).

(D20).

[45] In some embodiments, the splicing modulator provides D21:

(D21).

(D21).

[46] In some embodiments, the splicing modulator provides D22:

(D22).

(D22).

[47] In some embodiments, the splicing modulator provides D23:

(D23).

(D23).

[48] In some embodiments, the splicing modulator provides D24:

(D24).

(D24).

[49] In some embodiments, the splicing modulator provides D25:

(D25).

(D25).

[50] In some embodiments, the splicing modulator provides D26:

(D26).

(D26).

[51] In some embodiments, the splicing modulator provides D27:

(D27).

(D27).

[52] In some embodiments, the splicing modulator provides D28:

(D28).

(D28).

[53] In some embodiments, the splicing modulator provides D29:

(D29).

(D29).

[54] In some embodiments, the splicing modulator provides D30:

(D30).

(D30).

[55] In some embodiments, the splicing modulator provides D31:

(D31).

(D31).

[56] In some embodiments, the splicing modulator provides D32:

(D32).

(D32).

[57] In some embodiments, the splicing modulator provides D33:

(D33).

(D33).

[58] In some embodiments, the splicing modulator provides D34:

(D34).

(D34).

[59] In some embodiments, the splicing modulator provides D35:

(D35).

(D35).

[60] In some embodiments, the splicing modulator provides one of the drug fragments listed in Table 7. In some embodiments, the splicing modulator provides D1, D2, D3, D4, D4', D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, D15, D16, D17, D18, D19, D20, D21, D22, D23, D24, D25, D26, D27, D28, D29, D30, D31, D32, D33, D34, and/or D35.

[61] In some embodiments, a splicing modulator is disclosed, as well as its use as a therapeutic drug or as part of an ADC. In some embodiments, the splicing modulator provides D4, D4', D12, D15, D8, D9, D10, D13, D18, D19, D20, D21, D22, D25, or D33.

[62] In some embodiments, the splicing modulator provides for D4 and the linker provides for MC-Val-Cit-pABC. In some embodiments, the splicing modulator provides D4 and the linker provides an MC-β-glucuronide. In some embodiments, the splicing modulator provides D12 and the linker provides MC-Val-Cit-pABC. In some embodiments, the splicing modulator provides D12 and the linker provides an MC-β-glucuronide. In some embodiments, the splicing modulator provides D15 and the linker provides MC-Val-Ala-pAB.

[63] In various embodiments, the linker used in the ADC disclosed herein is stable outside the cell, such that the ADC remains intact when in extracellular conditions, but is capable of being cleaved after internalization into a cell, for example, a tumor or cancer cell. cell. In some embodiments, the splicing modulator is cleaved from the antibody or antigen binding fragment when the ADC enters a cell that expresses the antigen that is targeted by the antibody or antigen binding fragment of the ADC. In some embodiments, the linker is a cleavable linker.

[64] In some embodiments, the implementation of the linker provides a cleavable peptide fragment. In some embodiments, the cleavable peptide fragment is cleaved by the action of an enzyme. In some embodiments, the cleavable peptide fragment or linker provides an amino acid unit. In some embodiments, the amino acid unit comprises valine-citrulline ("Val-Cit" or "VC"). In some other embodiments, the amino acid unit comprises valine-alanine ("Val-Ala" or "VA"). In some other embodiments, the amino acid unit comprises glutamic acid-valine-citrulline ("Glu-Val-Cit" or "EVC"). In some other embodiments, the amino acid unit comprises alanine-alanine-asparagine ("Ala-Ala-Asn" or "AAN").

[65] In some embodiments, the implementation of the linker provides a cleavable glucuronide fragment. In some embodiments, the cleavable glucuronide moiety is cleaved by the action of an enzyme. In some embodiments, the cleavable glucuronide moiety is cleaved by a glucuronidase. In some embodiments, the cleavable glucuronide moiety is cleaved by β-glucuronidase.

[66] In some embodiments, the implementation of the linker provides at least one spacer link. In some embodiments, the spacer or linker comprises a polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety is -(PEG) _m - and m is an integer from 1 to 10. In some embodiments, m is 2. In some other embodiments, the spacer unit or linker is an alkyl moiety. In some embodiments, the alkyl moiety is -(CH ₂ ) _n - and n is an integer from 1 to 10. In some embodiments, n is 2. In some embodiments, n is 5. In some embodiments, n is 6.

[67] In some embodiments, the spacer unit is attached to the antibody or antigen-binding fragment via a maleimide (Mal) fragment ("Mal spacer unit"). In some embodiments, the Mal spacer unit reacts with a cysteine residue on an antibody or antigen binding fragment. In some embodiments, the implementation of the Mal-spacer unit is connected to the antibody or antigennegative fragment through a cysteine residue on the antibody or antigennegative fragment.

[68] In some embodiments, the linker provides a Mal spacer unit and a cleavable peptide fragment. In some embodiments, the cleavable peptide fragment provides an amino acid unit. In some embodiments, the cleavable peptide fragment or amino acid unit is provided by Val-Cit. In some embodiments, the cleavable peptide fragment or amino acid unit is provided by Val-Ala. In some embodiments, the cleavable peptide fragment or amino acid unit is provided by Glu-Val-Cit. In some embodiments, the cleavable peptide fragment or amino acid unit is provided by Ala-Ala-Asn. In some embodiments, the Mal spacer unit provides an alkyl moiety. In some embodiments, the Mal spacer unit provides a PEG fragment. In some embodiments, the implementation of the Mal-spacer link provides maleimidocaproil (MC).

In some embodiments, a Mal spacer unit attaches an antibody or antigen-binding fragment to a cleavable fragment in a linker. In some embodiments, the cleavable fragment in the linker provides a cleavable peptide fragment. In some embodiments, the cleavable peptide fragment provides an amino acid unit. In some embodiments, the cleavable peptide fragment or amino acid unit is Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. In some embodiments, the implementation of the linker provides MC-Val-Cit. In some embodiments, the implementation of the linker provides MC-Val-Ala. In some embodiments, the implementation of the linker provides MC-Glu-Val-Cit. In some embodiments, the implementation of the linker provides MC-Ala-Ala-Asn. In some embodiments, the Mal spacer unit provides an alkyl moiety. In some embodiments, the Mal spacer unit provides a PEG fragment. In some embodiments, the implementation of the Mal-spacer link provides maleimidocaproil (MC).

[69] In some embodiments, the cleavable moiety in the linker is directly coupled to the splicing modulator, or the spacer unit attaches the cleavable moiety in the linker to the splicing modulator. In some embodiments, cleavage of the conjugate releases the splicing modulator from the antibody or antigen-binding fragment and the linker. In some embodiments, the spacer linking the cleavable fragment in the linker to the splicing modulator is self-cleaving.

[70] In some embodiments, the spacer linking the cleavable moiety in the linker to the splicing modulator is p-aminobenzyloxycarbonyl (pABC). In some embodiments, pABC attaches a cleavable fragment in a linker to a splicing modulator. In some embodiments, the cleavable fragment in the linker provides a cleavable peptide fragment. In some embodiments, the cleavable peptide fragment provides an amino acid unit. In some embodiments, the cleavable peptide fragment or amino acid unit is Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. In some embodiments, the implementation of the linker provides Val-Cit-pABC. In some other embodiments, the implementation of the linker provides Val-Ala-pABC. In some embodiments, the implementation of the linker provides Glu-Val-Cit-pABC. In some embodiments, the implementation of the linker provides Ala-Ala-Asn-pABC.

[71] In some embodiments, the spacer linking the cleavable fragment in the linker to the splicing modulator comprises p-aminobenzyl (pAB). In some embodiments, the pAB attaches a cleavable fragment in a linker to a splicing modulator. In some embodiments, the cleavable fragment in the linker provides a cleavable peptide fragment. In some embodiments, the cleavable peptide fragment provides an amino acid unit. In some embodiments, the cleavable peptide fragment or amino acid unit is Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. In some embodiments, the implementation of the linker provides Val-Cit-pAB. In some other embodiments, the implementation of the linker provides Val-Ala-pAB. In some other embodiments, the implementation of the linker provides Glu-Val-Cit-pAB. In some other embodiments, the implementation of the linker provides Ala-Ala-Asn-pAB.

[72] In various embodiments, the linker is a non-cleavable linker. In some embodiments, the splicing modulator is released from the ADC by disruption of the antibody or antigen-binding fragment. In some embodiments, the linker remains covalently linked to at least one amino acid of the antibody and drug after internalization into and degradation in the target cell.

[73] In some embodiments, the linker is a non-cleavable linker containing at least one spacer unit. In some embodiments, the spacer or linker comprises a polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety is -(PEG) _m - and m is an integer from 1 to 10. In some embodiments, m is 2. In some other embodiments, the spacer unit or linker is an alkyl moiety. In some embodiments, the implementation of the alkyl fragment provides -(CH ₂ ) _n - or -(CH ₂ ) _n -O-(CH ₂ ) _n and n is an integer from 1 to 10. In some embodiments, n is 2. In some embodiments , n is 5. In some embodiments, n is 6.

[74] In some embodiments, a spacer unit in a non-cleavable linker is attached to an antibody or antigen-binding fragment via a maleimide (Mal) fragment ("Mal spacer unit"). In some embodiments, the Mal spacer unit reacts with a cysteine residue on an antibody or antigen binding fragment. In some embodiments, the implementation of the Mal-spacer unit is connected to the antibody or antigennegative fragment through a cysteine residue on the antibody or antigennegative fragment. In some embodiments, the Mal spacer unit provides an alkyl moiety. In some embodiments, the Mal spacer unit provides a PEG fragment. In some embodiments, the implementation of the linker or Mal-spacer link provide maleimidocaproyl (MC). In some embodiments, the linker or Mal spacer unit comprises maleimidocaproyl (MC) and at least one additional spacer unit. In some embodiments, the implementation of the linker or Mal-spacer link provide MC-(PEG) ₂ . In some embodiments, the implementation of the linker or Mal-spacer unit provide MC-(PEG) ₂ and at least one additional spacer unit. In some embodiments, the implementation of the linker or Mal-spacer link provide Mal-Hex. In some embodiments, the implementation of the linker or Mal-spacer unit provide Mal-Hex and at least one additional spacer unit. In some embodiments, the implementation of the linker or Mal-spacer link provide Mal-Et. In some embodiments, the implementation of the linker or Mal-spacer unit provide Mal-Et and at least one additional spacer unit. In some embodiments, the implementation of the linker or Mal-spacer link provide Mal-Et-O-Et. In some embodiments, the implementation of the linker or Mal-spacer unit provide Mal-Et-O-Et and at least one additional spacer unit. In some embodiments, the implementation of the Mal-spacer unit attaches the antibody or antigennegative fragment to the splicing modulator.

[75] In various embodiments, ADC compounds may be represented by formula (I):

Ab-(LD) _p (I),

where Ab is an antibody or antigen-binding fragment thereof that specifically targets a neoplastic cell or other tumor-associated target, such as a cancer antigen (eg, any of the antibody or binding domain sequences disclosed herein); D is any small molecule suitable for the treatment of cancer (eg a splicing modulator, eg any of the splicing modulators disclosed herein); L is a linker that covalently attaches Ab to D (eg, any of the linkers disclosed herein); and p is an integer from 1 to 15.

[76] In some embodiments, the Ab is selected from any of the antibody sequences or binding domain disclosed herein. In some embodiments, Ab is an antibody or binding domain sequence that targets HER2 and/or a neoplastic cell expressing HER2. In some embodiments, the Ab is an antibody or binding domain sequence that targets CD138 and/or a CD138-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence that targets EPHA2 and/or a neoplastic cell expressing EPHA2. In some embodiments, the Ab is an antibody or binding domain sequence that targets the MSLN and/or the MSLN-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence that targets FOLH1 and/or a neoplastic cell expressing FOLH1. In some embodiments, Ab is an antibody or binding domain sequence that targets CDH6 and/or a CDH6-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence that targets CEACAM5 and/or a neoplastic cell expressing CEACAM5. In some embodiments, the Ab is an antibody or binding domain sequence that targets CFC1B and/or a CFC1B-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence that targets ENPP3 and/or a neoplastic cell expressing ENPP3. In some embodiments, the Ab is an antibody or binding domain sequence that targets FOLR1 and/or a FOLR1-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence that targets HAVCR1 and/or a neoplastic cell expressing HAVCR1. In some embodiments, the Ab is an antibody or binding domain sequence that targets a KIT and/or a KIT-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence that targets MET and/or a MET-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence that targets MUC16 and/or a neoplastic cell expressing MUC16. In some embodiments, Ab is an antibody or binding domain sequence that targets SLC39A6 and/or a neoplastic cell expressing SLC39A6. In some embodiments, Ab is an antibody or binding domain sequence that targets SLC44A4 and/or a neoplastic cell expressing SLC44A4. In some embodiments, Ab is an antibody or binding domain sequence that targets STEAP1 and/or a neoplastic cell expressing STEAP1. In some embodiments, the Ab is an antibody or binding domain sequence that targets another cancer antigen.

[77] In some embodiments, D is a splicing modulator. In some embodiments, D is selected from any of the splicing modulators disclosed herein. In some embodiments, D is a splicing modulator selected from D2, D1, D4, D8, D10, D11 (E7107), D20, D21, D22, D12, D25, D9, D18, D19, D13, D15, D14, D16 , D17, D26, and D33 or any derivative thereof. In some embodiments, D is a splicing modulator selected from D4, D12, D15, D8, D9, D10, D13, D18, D19, D20, D21, D22, D25, and D33, or any derivative thereof. In some embodiments, D is a splicing modulator comprising D2 or any derivative thereof. In some embodiments, D is a splicing modulator comprising D1 or any derivative thereof.

[78] In some embodiments, L is selected from any of the linkers disclosed herein or any combination of linker components disclosed herein. In some embodiments, L is a linker providing MC-Val-Cit-pABC, Mal-(PEG) ₂ -CO, MC-Val-Ala-pAB, MC-Val-Ala-pABC, MC-Val-Cit-pAB , Mal-Hex, Mal-Et or Mal-Et-O-Et. In some embodiments, the implementation of the linker may also provide one or more additional spacer units. In some embodiments, L is an ADL1, ADL2, ADL5, ADL6, ADL7, ADL10, ADL12, ADL13, ADL14, ADL15, ADL21, ADL22, or ADL23 linker. In some embodiments, L is an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker. In some embodiments, L is an ADL12, ADL14, or ADL15 linker. In some embodiments, the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker may also include one or more additional spacer units. In some embodiments, L is an ADL1 linker and may optionally include one or more additional spacer units. In some embodiments, L is an ADL2 linker and may optionally include one or more additional spacer units. In some embodiments, L is an ADL5 linker and may optionally include one or more additional spacer units. In some embodiments, L is an ADL6 linker and may optionally include one or more additional spacer units. In some embodiments, L is an ADL7 linker and may optionally include one or more additional spacer units. In some embodiments, L is an ADL12 linker and may optionally include one or more additional spacer units. In some embodiments, L is an ADL14 linker and may optionally include one or more additional spacer units. In some embodiments, L is an ADL15 linker and may optionally include one or more additional spacer units. In various embodiments of the ADC described herein, p is from 1 to 10. In various embodiments, p is from 2 to 8. In various embodiments, p is from 4 to 8. In some embodiments, p is 4. In some embodiments, p is 8.

[79] In some embodiments, L-D of formula (I) are ADL1-D1. In some embodiments, L-D of formula (I) are ADL6-D1. In some embodiments, L-D of formula (I) is ADL5-D2. In some embodiments, L-D of formula (I) are ADL1-D18. In some embodiments, L-D of formula (I) are ADL5-D19. In some embodiments, L-D of formula (I) are ADL14-D1. In some embodiments, L-D of formula (I) are ADL12-D1. In some embodiments, L-D of formula (I) is ADL15-D1. In some embodiments, L-D of formula (I) are ADL12-D20. In some embodiments, L-D of formula (I) are ADL10-D1. In some embodiments, L-D of formula (I) is ADL12-D2. In some embodiments, L-D of formula (I) are ADL15-D2. In some embodiments, L-D of formula (I) are ADL12-D21. In some embodiments, L-D of formula (I) are ADL6-D9. In some embodiments, L-D of formula (I) are ADL1-D4. In some embodiments, L-D of formula (I) are ADL1-D3. In some embodiments, L-D of formula (I) are ADL1-D12. In some embodiments, L-D of formula (I) are ADL1-D7. In some embodiments, L-D of formula (I) are ADL1-D6. In some embodiments, L-D of formula (I) are ADL1-D5. In some embodiments, L-D of formula (I) are ADL22-D4. In some embodiments, L-D of formula (I) are ADL5-D10. In some embodiments, L-D of formula (I) are ADL5-D11. In some embodiments, L-D of formula (I) are ADL1-D13. In some embodiments, L-D of formula (I) are ADL1-D8. In some embodiments, L-D of formula (I) are ADL1-D22. In some embodiments, L-D of formula (I) are ADL5-D25. In some embodiments, L-D of formula (I) are ADL12-D22. In some embodiments, L-D of formula (I) are ADL5-D15. In some embodiments, L-D of formula (I) are ADL1-D14. In some embodiments, L-D of formula (I) are ADL5-D26. In some embodiments, L-D of formula (I) are ADL1-D16. In some embodiments, L-D of formula (I) are ADL5-D17. In some embodiments, L-D of formula (I) are ADL1-D33. In some embodiments, L-D of formula (I) are ADL1-D28. In some embodiments, L-D of formula (I) are ADL1-D31. In some embodiments, L-D of formula (I) are ADL1-D29. In some embodiments, L-D of formula (I) are ADL1-D35. In some embodiments, L-D of formula (I) are ADL5-D32. In some embodiments, L-D of formula (I) are ADL5-D27. In some embodiments, L-D of formula (I) are ADL12-D35. In some embodiments, L-D of formula (I) are ADL12-D28. In some embodiments, L-D of formula (I) are ADL1-D23. In some embodiments, L-D of formula (I) are ADL1-D24.

[80] In some embodiments, an ADC pool is provided in which random conjugation occurs and the average p in the pool is from about 2 to about 8. In some embodiments, an ADC pool is provided in which random conjugation occurs and the average p in pool is from about 4 to about 8. In some embodiments, an ADC pool is represented in which random conjugation occurs and the average p in the pool is approximately 4. In some embodiments, an ADC pool is represented in which random conjugation occurs and the average p in the pool is about 8. This document provides compositions (for example, pharmaceutical compositions) containing multiple copies of any of the ADCs described, where the average drug load (average p ) of the ADC in the composition is from about 3.5 to about 5, 5 (eg, about 4) or from about 7 to about 9 (eg, about 8).

[81] In some embodiments, an antibody or antigen binding fragment (Ab) from an ADC targets a neoplastic cell derived from a hematologic malignancy or solid tumor. In some embodiments, the antibody or antigen-binding fragment targets a neoplastic cell derived from a hematologic malignancy. In some embodiments, the hematologic malignancy is selected from B-cell malignancy, leukemia (eg, acute myeloid leukemia), lymphoma, and myeloma (eg, multiple myeloma). In some embodiments, the hematologic malignancy is selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the antibody or antigen-binding fragment targets a neoplastic cell derived from a solid tumor. In some embodiments, the solid tumor is selected from breast cancer (e.g., HER2 positive breast cancer), stomach cancer (e.g., gastric adenocarcinoma), prostate cancer, ovarian cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer ( eg, endometrial serous carcinoma), salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer, and esophageal cancer. In some embodiments, the solid tumor is selected from HER2 positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

[82] In various embodiments, the antibody or antigen-binding fragment (Ab) from ADC is an anti-HER2 antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment binds to HER2 and targets HER2-expressing neoplastic cells (ie, the ADC targets HER2-expressing neoplastic cells). In some embodiments, the antibody or antigen-binding fragment from the ADC is an internalizing anti-HER2 antibody or internalizing antigen-binding fragment thereof.

[83] In some embodiments, the anti-HER2 antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) containing the amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2) and SEQ ID NO:6 (LCDR3). In some embodiments, the anti-HER2 antibody or antigen binding fragment is an internalizing antibody or an internalizing antigen binding fragment. In some embodiments, the anti-HER2 antibody or antigen-binding fragment comprises human framework sequences. In some embodiments, the anti-HER2 antibody or antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the anti-HER2 antibody or antigen-binding fragment comprises a human IgG heavy chain constant region. In some embodiments, the anti-HER2 antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the anti-HER2 antibody or antigen-binding fragment comprises a human Ig kappa or lambda light chain constant region. In some embodiments, an anti-HER2 antibody or antigen binding site competes for binding and/or binds to the same epitope as an antibody comprising a heavy chain variable domain of SEQ ID NO:19 and a light chain variable domain of SEQ ID NO:20.

[84] In various embodiments, the antibody or antigen-binding fragment (Ab) from ADC is an anti-CD138 antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment binds to CD138 and targets CD138-expressing neoplastic cells (ie, the ADC targets CD138-expressing neoplastic cells). In some embodiments, the antibody or antigen-binding fragment from the ADC is an internalized anti-CD138 antibody or internalized antigen-binding fragment thereof.

[85] In some embodiments, the anti-CD138 antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) containing the amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2) and SEQ ID NO:12 (LCDR3). In some embodiments, the anti-CD138 antibody or antigen binding fragment is an internalizing antibody or an internalizing antigen binding fragment. In some embodiments, the anti-CD138 antibody or antigen-binding fragment comprises human framework sequences. In some embodiments, the anti-CD138 antibody or antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:22. In some embodiments, the anti-CD138 antibody or antigen-binding fragment comprises a mouse IgG2a heavy chain constant region. In some embodiments, the anti-CD138 antibody or antigen binding fragment comprises a mouse Ig kappa light chain constant region. In some embodiments, the anti-CD138 antibody or antigen binding fragment comprises a human IgG heavy chain constant region. In some embodiments, the anti-CD138 antibody or antigen-binding fragment comprises a human IgG2a heavy chain constant region. In some embodiments, the anti-CD138 antibody or antigen binding fragment comprises a human Ig kappa or lambda light chain constant region. In some embodiments, an anti-CD138 antibody or antigen binding site competes for binding and/or binds to the same epitope as an antibody comprising a heavy chain variable domain of SEQ ID NO:21 and a light chain variable domain of SEQ ID NO:22.

[86] In various embodiments, the antibody or antigen-binding fragment (Ab) from ADC is an anti-EPHA2 antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment binds to EPHA2 and targets EPHA2-expressing neoplastic cells (i.e., the ADC targets EPHA2-expressing neoplastic cells). In some embodiments, the antibody or antigen-binding fragment from the ADC is an internalized anti-EPHA2 antibody or internalized antigen-binding fragment thereof.

[87] In some embodiments, the anti-EPHA2 antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) containing the amino acid sequences of SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2) and SEQ ID NO:18 (LCDR3). In some embodiments, the anti-EPHA2 antibody or antigen binding fragment is an internalizing antibody or an internalizing antigen binding fragment. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment comprises human framework sequences. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:24. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment comprises a human IgG heavy chain constant region. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment comprises a human Ig kappa or lambda light chain constant region. In some embodiments, an anti-EPHA2 antibody or antigen binding site competes for binding and/or binds to the same epitope as an antibody comprising a heavy chain variable domain of SEQ ID NO:23 and a light chain variable domain of SEQ ID NO:24.

[88] In various embodiments, this document also provides compounds containing a linker-drug, defined by the General formula: L-D, where L=linker fragment, and D=drug fragment (for example, a drug fragment that is a modulator splicing). In various embodiments, the linker-drug (L-D) compounds disclosed herein can be attached to an antibody or antigen-binding fragment and/or are suitable for use in the ADCs disclosed herein, e.g., in the ADCs of formula (I ).

[89] In various embodiments, the linker-drug compounds (L-D) disclosed herein provide a linker-drug structure according to formula (III). In various embodiments, the present invention provides a linker-drug compound (L-D) of formula (III):

(III),

(III)

or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is absent or selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups, -OC(=O)-(C ₁ -C ₆ alkyl) and -CD ₃ groups;

R ² is absent or is a linker;

R ³ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups and -OC(=O)-(C ₁ -C ₆ alkyl) groups; And

each of R ⁴ , R ⁵ and R ⁸ is independently selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups and C ₁ -C ₆ alkyl groups;

each of R ⁶ and R ⁷ is independently selected from hydrogen, -OR ¹⁷ , -OC(=O)-R ¹⁷ , -OC(=O)-NR ¹⁵ R ¹⁶ , C ₁ -C ₆ alkyl groups, -NR ¹⁵ R ¹⁶ and linker;

each of R ¹⁵ and R ¹⁶ is independently selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and

-C(=O)-OR ¹⁷ ;

R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups; And

,

,

,

и

;Z'' selected from

,

,

,

And

;

where each of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ¹⁵ , R ¹⁶ , and R ¹⁷ is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, groups -O-(C ₁ -C ₆ alkyl), -NR ¹⁵ R ¹⁶ , C ₃ -C ₈ cycloalkyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkylalkoxy groups , benzyl groups and C ₃ -C ₈ heterocyclyl groups;

where at least one of R ⁶ and R ⁷ is hydrogen; And

where, if R ² is a linker, then neither R ⁶ nor R ⁷ is a linker, and if R ⁶ or R ⁷ is a linker, then R ² is absent.

[90] In various other embodiments, the linker-drug compounds (L-D) disclosed herein provide a linker-drug structure according to formula (V). In various embodiments, the present invention provides a linker-drug compound (L-D) of formula (V):

(V),

(V)

or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is absent or selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups, -OC(=O)-(C ₁ -C ₆ alkyl) and -CD ₃ groups;

R ² is absent or is a linker;

R ³ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups and -OC(=O)-(C ₁ -C ₆ alkyl) groups; And

each of R ⁴ , R ⁵ and R ⁸ is independently selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups and C ₁ -C ₆ alkyl groups;

each of R ⁶ and R ⁷ is independently selected from hydrogen, -OR ¹⁷ , -OC(=O)-R ¹⁷ , -OC(=O)-NR ¹⁵ R ¹⁶ , C ₁ -C ₆ alkyl groups, -NR ¹⁵ R ¹⁶ and linker;

each of R ¹⁵ and R ¹⁶ is independently selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C(=O)-OR ¹⁷ ; And

R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups;

where each of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ¹⁵ , R ¹⁶ , and R ¹⁷ is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, groups -O-(C ₁ -C ₆ alkyl), -NR ¹⁵ R ¹⁶ , C ₃ -C ₈ cycloalkyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkylalkoxy groups , benzyl groups and C ₃ -C ₈ heterocyclyl groups;

where at least one of R ⁶ and R ⁷ is hydrogen; And

where, if R ² is a linker, then neither R ⁶ nor R ⁷ is a linker, and if R ⁶ or R ⁷ is a linker, then R ² is absent.

[91] In various other embodiments, the linker-drug compounds (L-D) disclosed herein provide a linker-drug structure according to formula (VII). In various embodiments, the present invention provides a linker-drug compound (L-D) of formula (VII):

(VII),

(VII)

or a pharmaceutically acceptable salt thereof, wherein:

each of R ¹ and R ⁹ is independently absent or selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups, -OC(=O)-(C ₁ -C ₆ alkyl) and -CD ₃ groups;

R ² is absent or is a linker;

R ³ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups and -OC(=O)-(C ₁ -C ₆ alkyl) groups;

each of R ⁴ , R ⁵ and R ⁸ is independently selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups and C ₁ -C ₆ alkyl groups;

each of R ⁶ and R ⁷ is independently selected from hydrogen, -OR ¹⁷ , -OC(=O)-R ¹⁷ , -OC(=O)-NR ¹⁵ R ¹⁶ , C ₁ -C ₆ alkyl groups, -NR ¹⁵ R ¹⁶ and linker;

R ¹⁰ is selected from hydrogen, C ₁ -C ₆ alkyl groups, -C(=O)-(C ₁ -C ₆ alkyl) and -CD ₃ groups;

each of R ¹⁵ and R ¹⁶ is independently selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and

-C(=O)-OR ¹⁷ ;

R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups; And

a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

where each of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹⁵ , R ¹⁶ , and R ¹⁷ is independently substituted with 0-3 groups, independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, -O-(C ₁ -C ₆ alkyl), -NR ¹⁵ R ¹⁶ groups, C ₃ -C ₈ cycloalkyl groups, C ₁ -C ₆ alkyl hydroxy groups, C ₁ -C ₆ alkylalkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups;

where at least one of R ⁶ and R ⁷ is hydrogen;

where, if R ² is a linker, then neither R ⁶ nor R ⁷ is a linker, and if R ⁶ or R ⁷ is a linker, then R ² is absent; And

where both R ¹ and R ⁹ cannot be absent.

[92] In various other embodiments, the linker-drug compounds (L-D) disclosed herein provide a linker-drug structure according to formula (IX). In various embodiments, the present invention provides a linker-drug compound (L-D) of formula (IX):

(IX),

(IX)

or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is absent or selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups, -OC(=O)-(C ₁ -C ₆ alkyl) and -CD ₃ groups;

R ² is a linker;

R ³ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups and -OC(=O)-(C ₁ -C ₆ alkyl) groups;

R ⁴ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₆ alkyl), -OC(=O)-(C ₁ -C ₆ alkyl) groups and C ₁ -C ₆ alkyl groups;

R ¹⁰ is selected from 3-10 membered carbocycles and 3-10 membered heterocycles, each of which is substituted with 0-3 R ^a , where each R ^a is independently selected from halogen atoms, C ₁ -C ₆ alkyl groups, -O-( C ₁ -C ₆ )alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylhydroxy groups, -S(=O) _w -(4-7-membered heterocycles), 4-7-membered carbocycles and 4-7 -membered heterocycles;

each of R ¹⁵ and R ¹⁶ is independently selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C(=O)-OR ¹⁷ ; And

R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups;

where each of R ¹ , R ² , R ³ , R ⁴ , R ¹⁰ , R ¹⁵ , R ¹⁶ and R ¹⁷ is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, -O-(C ₁ -C ₆ alkyl), -NR ¹⁵ R ¹⁶ , C ₃ -C ₈ cycloalkyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkylalkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups groups; And

where each R ^a is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, -NR ¹⁵ R ¹⁶ , C ₁ -C ₆ alkyl groups, -(C=O)-(C ₁ -C ₆ alkyl) groups , groups -(C=O)-(C ₁ -C ₆ alkyl)-(C ₃ -C ₁₀ heterocyclyl) and groups C ₁ -C ₆ alkylcarboxylic acid, each of which is substituted with 0, 1 or 2 groups independently selected from halogen atoms, hydroxyl groups, -NR ¹⁵ R ¹⁶ and C ₁ -C ₃ alkyl groups; And

w is 0, 1, or 2.

[93] Also, in various embodiments, this document provides ways of therapeutic use of the described ADC compounds and compositions based on them, for example, in the treatment of neoplastic disorders, such as cancer. In certain aspects, the present invention provides methods for treating a neoplastic disorder, such as cancer, that expresses an antigen that is targeted by an antibody or antigen-binding fragment from an ADC, such as HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1 , HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1.

[94] In certain aspects, the present invention provides methods for treating a subject who has or is suspected of having a neoplastic disorder by administering to the subject a therapeutically effective amount of any of the described ADCs or compositions thereof and/or administering an appropriate treatment regimen. In some embodiments, the neoplastic disorder is a hematologic malignancy or a solid tumor. In some embodiments, the neoplastic disorder is a hematologic malignancy. In some embodiments, the hematologic malignancy is selected from B-cell malignancy, leukemia, lymphoma, and myeloma. In some embodiments, the hematologic malignancy is selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the neoplastic disorder is a solid tumor. In some embodiments, the solid tumor is selected from breast cancer (e.g., HER2 positive breast cancer), stomach cancer (e.g., gastric adenocarcinoma), prostate cancer, ovarian cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer ( eg, endometrial serous carcinoma), salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer, and esophageal cancer. In some embodiments, the solid tumor is selected from HER2 positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

[95] In some embodiments, treatment with the antibody-drug conjugate or composition induces non-specific killing of neoplastic cells that do not express the target antigen but are adjacent to neoplastic cells that express the target antigen. In some embodiments, the subject has one or more neoplastic cells that express the target antigen.

[96] In some embodiments, the target antigen is HER2. In some embodiments, one or more neoplastic cells are derived from breast cancer, ovarian cancer, gastric cancer, lung cancer (e.g. lung adenocarcinoma), uterine cancer (e.g. uterine endometrial serous carcinoma), osteosarcoma or salivary duct carcinomas expressing HER2. In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-HER2 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or insufficiently responsive to treatment with a splicing modulator when administered alone.

[97] In some embodiments, the target antigen is CD138. In some embodiments, one or more neoplastic cells are derived from multiple myeloma expressing CD138. In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-CD138 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or insufficiently responsive to treatment with a splicing modulator when administered alone.

[98] In some embodiments, the target antigen is EPHA2. In some embodiments, one or more neoplastic cells are derived from breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer expressing EPHA2. In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-EPHA2 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or insufficiently responsive to treatment with a splicing modulator when administered alone.

[99] In some embodiments, the target antigen is MSLN. In some embodiments, one or more neoplastic cells are derived from ovarian cancer, cervical cancer, pancreatic cancer, or lung cancer (eg, lung adenocarcinoma) expressing MSLN. In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-MSLN antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or insufficiently responsive to treatment with a splicing modulator when administered alone.

[100] In some embodiments, the target antigen is FOLH1. In some embodiments, one or more neoplastic cells are derived from FOLH1-expressing prostate cancer. In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-FOLH1 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or insufficiently responsive to treatment with a splicing modulator when administered alone.

[101] In some embodiments, the target antigen is CDH6. In some embodiments, one or more neoplastic cells are derived from kidney cancer expressing CDH6. In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-CDH6 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or insufficiently responsive to treatment with a splicing modulator when administered alone.

[102] In some embodiments, the target antigen is CEACAM5. In some embodiments, one or more neoplastic cells are derived from colorectal cancer expressing CEACAM5. In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-CEACAM5 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or insufficiently responsive to treatment with a splicing modulator when administered alone.

[103] In some embodiments, the target antigen is CFC1B. In some embodiments, one or more neoplastic cells are derived from pancreatic cancer expressing CFC1B. In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-CFC1B antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or insufficiently responsive to treatment with a splicing modulator when administered alone.

[104] In some embodiments, the target antigen is ENPP3. In some embodiments, one or more neoplastic cells are derived from kidney cancer expressing ENPP3. In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-ENPP3 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or insufficiently responsive to treatment with a splicing modulator when administered alone.

[105] In some embodiments, the target antigen is FOLR1. In some embodiments, one or more neoplastic cells are derived from FOLR1-expressing ovarian cancer. In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-FOLR1 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or insufficiently responsive to treatment with a splicing modulator when administered alone.

[106] In some embodiments, the target antigen is HAVCR1. In some embodiments, one or more neoplastic cells are derived from kidney or esophageal cancer expressing HAVCR1. In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-HAVCR1 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or insufficiently responsive to treatment with a splicing modulator when administered alone.

[107] In some embodiments, the target antigen is a KIT. In some embodiments, one or more neoplastic cells are derived from kidney cancer expressing KIT. In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-KIT antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or insufficiently responsive to treatment with a splicing modulator when administered alone.

[108] In some embodiments, the target antigen is MET. In some embodiments, one or more neoplastic cells are derived from kidney or esophageal cancer expressing MET. In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-MET antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or insufficiently responsive to treatment with a splicing modulator when administered alone.

[109] In some embodiments, the target antigen is MUC16. In some embodiments, one or more neoplastic cells are derived from ovarian cancer, cervical cancer, or breast cancer expressing MUC16. In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-MUC16 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or insufficiently responsive to treatment with a splicing modulator when administered alone.

[110] In some embodiments, the target antigen is SLC39A6. In some embodiments, one or more neoplastic cells are derived from breast cancer or prostate cancer expressing SLC39A6. In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-SLC39A6 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or insufficiently responsive to treatment with a splicing modulator when administered alone.

[111] In some embodiments, the target antigen is SLC44A4. In some embodiments, one or more neoplastic cells are derived from prostate cancer expressing SLC44A4. In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-SLC44A4 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or insufficiently responsive to treatment with a splicing modulator when administered alone.

[112] In some embodiments, the target antigen is STEAP1. In some embodiments, one or more neoplastic cells are derived from prostate cancer expressing STEAP1. In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-STEAP1 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or insufficiently responsive to treatment with a splicing modulator when administered alone.

[113] In certain other aspects of the present invention, methods are provided for reducing or inhibiting tumor growth in a subject who has or is suspected of having a neoplastic disorder by administering to the subject a therapeutically effective amount of any of the described ADCs or compositions thereof and/or administering an appropriate treatment regimens.

[114] In some embodiments, treatment with the antibody drug conjugate or composition induces non-specific killing of neoplastic tumor cells that do not express the target antigen but are adjacent to neoplastic cells that express the target antigen. In some embodiments, the tumor contains one or more neoplastic cells that express the target antigen.

[115] In some embodiments, the target antigen is HER2. In some embodiments, one or more neoplastic cells are derived from breast cancer, ovarian cancer, gastric cancer, lung cancer (e.g. lung adenocarcinoma), uterine cancer (e.g. uterine endometrial serous carcinoma), osteosarcoma or salivary duct carcinomas expressing HER2. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-HER2 antibody when administered alone and/or (b) a splicing modulator when administered alone.

[116] In some embodiments, the target antigen is CD138. In some embodiments, one or more neoplastic cells are derived from multiple myeloma expressing CD138. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-CD138 antibody when administered alone and/or (b) a splicing modulator when administered alone.

[117] In some embodiments, the target antigen is EPHA2. In some embodiments, one or more neoplastic cells are derived from breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer expressing EPHA2. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-EPHA2 antibody when administered alone and/or (b) a splicing modulator when administered alone.

[118] In some embodiments, the target antigen is MSLN. In some embodiments, one or more neoplastic cells are derived from ovarian cancer, cervical cancer, pancreatic cancer, or lung cancer (eg, lung adenocarcinoma) expressing MSLN. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-MSLN antibody when administered alone and/or (b) a splicing modulator when administered alone.

[119] In some embodiments, the target antigen is FOLH1. In some embodiments, one or more neoplastic cells are derived from FOLH1-expressing prostate cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-FOLH1 antibody when administered alone and/or (b) a splicing modulator when administered alone.

[120] In some embodiments, the target antigen is CDH6. In some embodiments, one or more neoplastic cells are derived from kidney cancer expressing CDH6. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-CDH6 antibody when administered alone and/or (b) a splicing modulator when administered alone.

[121] In some embodiments, the target antigen is CEACAM5. In some embodiments, one or more neoplastic cells are derived from colorectal cancer expressing CEACAM5. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-CEACAM5 antibody when administered alone and/or (b) a splicing modulator when administered alone.

[122] In some embodiments, the target antigen is CFC1B. In some embodiments, one or more neoplastic cells are derived from pancreatic cancer expressing CFC1B. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-CFC1B antibody when administered alone and/or (b) a splicing modulator when administered alone.

[123] In some embodiments, the target antigen is ENPP3. In some embodiments, one or more neoplastic cells are derived from kidney cancer expressing ENPP3. In some embodiments, the tumor is resistant or refractory to treatment with (a) anti-ENPP3 antibody a when administered alone and/or (b) a splicing modulator when administered alone.

[124] In some embodiments, the target antigen is FOLR1. In some embodiments, one or more neoplastic cells are derived from FOLR1-expressing ovarian cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-FOLR1 antibody when administered alone and/or (b) a splicing modulator when administered alone.

[125] In some embodiments, the target antigen is HAVCR1. In some embodiments, one or more neoplastic cells are derived from kidney or esophageal cancer expressing HAVCR1. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-HAVCR1 antibody when administered alone and/or (b) a splicing modulator when administered alone.

[126] In some embodiments, the target antigen is a KIT. In some embodiments, one or more neoplastic cells are derived from kidney cancer expressing KIT. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-KIT antibody when administered alone and/or (b) a splicing modulator when administered alone.

[127] In some embodiments, the target antigen is MET. In some embodiments, one or more neoplastic cells are derived from kidney or esophageal cancer expressing MET. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-MET antibody when administered alone and/or (b) a splicing modulator when administered alone.

[128] In some embodiments, the target antigen is MUC16. In some embodiments, one or more neoplastic cells are derived from ovarian cancer, cervical cancer, or breast cancer expressing MUC16. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-MUC16 antibody when administered alone and/or (b) a splicing modulator when administered alone.

[129] In some embodiments, the target antigen is SLC39A6. In some embodiments, one or more neoplastic cells are derived from breast cancer or prostate cancer expressing SLC39A6. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-SLC39A6 antibody when administered alone and/or (b) a splicing modulator when administered alone.

[130] In some embodiments, the target antigen is SLC44A4. In some embodiments, one or more neoplastic cells are derived from prostate cancer expressing SLC44A4. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-SLC44A4 antibody when administered alone and/or (b) a splicing modulator when administered alone.

[131] In some embodiments, the target antigen is STEAP1. In some embodiments, one or more neoplastic cells are derived from prostate cancer expressing STEAP1. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-STEAP1 antibody when administered alone and/or (b) a splicing modulator when administered alone.

[132] In still other aspects of the present invention, methods are provided for determining whether a subject who has or is suspected of having a neoplastic disorder will be responsive to treatment with any of the described ADCs or compositions based thereon by obtaining a biological sample from the subject. and bringing the biological sample into contact with the ADC or composition. In some embodiments, the biological sample is a tumor sample. In some embodiments, the tumor sample is a tumor biopsy or a blood sample. In some embodiments, the blood sample is selected from blood, a blood fraction, or a cell derived from blood or a blood fraction. In some embodiments, the subject has one or more neoplastic cells that express the target antigen. In some embodiments, the target antigen is HER2. In some embodiments, one or more neoplastic cells are derived from breast cancer, ovarian cancer, gastric cancer, lung cancer (e.g. lung adenocarcinoma), uterine cancer (e.g. uterine endometrial serous carcinoma), osteosarcoma or salivary duct carcinomas expressing HER2. In some embodiments, the target antigen is CD138. In some embodiments, one or more neoplastic cells are derived from multiple myeloma expressing CD138. In some embodiments, the target antigen is EPHA2. In some embodiments, one or more neoplastic cells are derived from breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer expressing EPHA2. In some embodiments, the target antigen is MSLN. In some embodiments, one or more neoplastic cells are derived from ovarian cancer, cervical cancer, pancreatic cancer, or lung cancer (eg, lung adenocarcinoma) expressing MSLN. In some embodiments, the target antigen is FOLH1. In some embodiments, one or more neoplastic cells are derived from FOLH1-expressing prostate cancer. In some embodiments, the target antigen is CDH6. In some embodiments, one or more neoplastic cells are derived from kidney cancer expressing CDH6. In some embodiments, the target antigen is CEACAM5. In some embodiments, one or more neoplastic cells are derived from colorectal cancer expressing CEACAM5. In some embodiments, the target antigen is CFC1B. In some embodiments, one or more neoplastic cells are derived from pancreatic cancer expressing CFC1B. In some embodiments, the target antigen is ENPP3. In some embodiments, one or more neoplastic cells are derived from kidney cancer expressing ENPP3. In some embodiments, the target antigen is FOLR1. In some embodiments, one or more neoplastic cells are derived from FOLR1-expressing ovarian cancer. In some embodiments, the target antigen is HAVCR1. In some embodiments, one or more neoplastic cells are derived from kidney or esophageal cancer expressing HAVCR1. In some embodiments, the target antigen is a KIT. In some embodiments, one or more neoplastic cells are derived from kidney cancer expressing KIT. In some embodiments, the target antigen is MET. In some embodiments, one or more neoplastic cells are derived from kidney or esophageal cancer expressing MET. In some embodiments, the target antigen is MUC16. In some embodiments, one or more neoplastic cells are derived from ovarian cancer, cervical cancer, or breast cancer expressing MUC16. In some embodiments, the target antigen is SLC39A6. In some embodiments, one or more neoplastic cells are derived from breast cancer or prostate cancer expressing SLC39A6. In some embodiments, the target antigen is SLC44A4. In some embodiments, one or more neoplastic cells are derived from prostate cancer expressing SLC44A4. In some embodiments, the target antigen is STEAP1. In some embodiments, one or more neoplastic cells are derived from prostate cancer expressing STEAP1.

[133] In various embodiments, this document further provides pharmaceutical compositions containing an ADC and a pharmaceutically acceptable diluent, carrier, and/or excipient. Also disclosed are methods for preparing the described ADC compounds and compositions based on them.

BRIEF DESCRIPTION OF GRAPHICS

[134] FIG. 1 shows the dose-response effect of exemplary payload compounds in a competitive binding assay. Nuclear extracts from 293F cells overexpressing wt-flag SF3B1 were immunoprecipitated using an anti-SF3B1 antibody and a scintillation proximity assay (SPA) bead cocktail. Binding reaction mixtures contained a mixture of antibody-bead and increasing concentrations of the compound, after which a competitive assay was performed using a ³ H-labeled pladienolide B probe (PB). The y-axis represents the percent change (% response) in specific binding compared to control DMSO (0%). Data are presented as mean ± standard deviation (SD).

[135] FIG. 2 shows the modulation of splicing by exemplary payload compounds in an in vitro splicing assay. Nuclear extracts from HeLa S3 cells were incubated with pre-mRNA Ad2.2 and increasing concentrations of the compound, followed by quantification of splicing modulation by RT-PCR. The Ad2.2 sequence was derived from the adenoviral Ad2 pre-mRNA as a substrate with modifications near the sequence branch point. The y-axis represents the percentage change (% response) in splicing compared to the DMSO control (0%). Data are presented as mean ± SD.

[136] FIG. 3 shows the dose-dependent effect of exemplary payload compounds on cell viability on HER2-amplifying breast cancer cells (HCC1954). Cells were incubated with compound for 144 hours (6 days) and viability was read using the CellTiter-Glo® reagent. Data are presented as mean ± SD.

[137] FIG. 4 shows the results of the cell binding assay. Binding of exemplary HER2-ADCs to JIMT1 cells was assessed by flow cytometry. Average fluorescence intensity values were obtained to determine the binding of the conjugates, followed by staining with PE-labeled secondary antibody. Data are presented as mean ± SD.

[138] FIG. 5A shows the dose-dependent effect of exemplary HER2-ADCs on cell viability on HER2-amplifying breast cancer cells (HCC1954). In FIG. 5B shows the dose-dependent effect of exemplary HER2-ADCs on cell viability on HER2-amplified gastric cancer cells (N87). In FIG. 5C shows the dose-dependent effect of exemplary HER2-ADCs on cell viability on HER2-amplifying breast cancer cells (SKBR3). Cells were incubated with conjugates for 144 hours (6 days) and viability was read using the CellTiter-Glo® reagent. Data are presented as mean ± SD.

[139] FIG. 6 shows the dose-dependent effect of exemplary HER2-ADCs on cell viability on HER2-non-expressing breast cancer cells (MCF7). Cells were incubated with conjugates for 144 hours (6 days) and viability was read using the CellTiter-Glo® reagent. Data are presented as mean ± SD.

[140] FIG. 7 shows the results of a splicing analysis in the SLC25A19 gene on HER2 amplifying breast cancer cells (HCC1954). Cells were incubated with the conjugates for 24 hours and splicing in the SLC25A19 transcript was measured by real time qPCR using the Taqman specific primer probe set. The y-axis represents the percentage (%) response compared to the DMSO control (0%). Data are presented as mean ± SD.

[141] FIG. 8 shows the results of the non-specific killing analysis. H1568 cells overexpressing HER2 (target positive) or H1568 cells labeled with luciferase (target negative) seeded alone or incubated together in co-culture for 144 hours (6 days) were treated with exemplary HER2-ADC. The results of the analysis were read using the OneGlo® reagent. The y-axis represents the percentage (%) response compared to the PBS control (100%). Data are presented as mean ± SD.

[142] FIG. 9 shows tumor growth kinetics for each group of HCC1954-implanted CB17-SCID mice treated with intravenous administration of a single dose of exemplary HER2-ADC or an appropriate payload at the same dose (6-10 animals per group). Tumor volumes were measured twice a week after treatment. Data are presented as mean ± standard error of the mean (SEM).

[143] FIG. 10 shows the dose-dependent effect of exemplary CD138-ADCs on the viability of a multiple myeloma cell line expressing CD138. MOLP8 cells were incubated with the conjugates for 144 hours (6 days) and viability was read using the CellTiter-Glo® reagent. Data are presented as mean ± SD.

[144] FIG. 11 shows the dose-dependent effect of exemplary EPH2A-ADCs on the viability of a prostate cancer cell line expressing EPHA2. PC3 cells were incubated with the conjugates for 144 hours (6 days) and viability was read using the CellTiter-Glo® reagent. Data are presented as mean ± SD.

[145] FIG. 12A and FIG. 12B shows the results of an in vitro stability assay of exemplary anti-HER2 antibody ADCs, AB185-ADL1-D1. The y-axis represents the concentration of total antibody ( Fig. 12A ) and conjugated (intact) payload ( Fig. 12B ); the x-axis represents the incubation time at 37°C in hours.

[146] FIG. 13 shows plasma concentrations of exemplary anti-HER2 antibody ADCs, AB185-ADL1-D1, following single dose intravenous administration to N87 tumor CD17-SCID mice.

[147] FIG. 14 is a schematic diagram of an exemplary RNA sequencing and protein ligandome detection experiment.

[148] FIG. 15 is a schematic diagram of an exemplary T cell priming experiment.

[149] FIG. 16 shows the results of the FACS analysis. Monocytes were isolated from peripheral blood mononuclear cells (PBMC) and induced to differentiate into dendritic cells (DC) by culturing in a cytokine cocktail. FACS was performed to ascertain the differentiation of DC from monocytes and their maturation.

[150] FIG. 17A-D show the results of the ELISpot assay. In FIG. 17A shows ELISpot assay plates demonstrating activation of CD8+ T cell priming by neoantigen 1. Stimulation of CD8+ T cells was monitored by IFNγ secretion. In FIG. 17B shows the quantification of IFNγ spots (number of spots) in neoantigen 1 ELISpot assay plates ( FIG. 17A ). In FIG. 17C shows ELISpot assay plates demonstrating activation of CD8+ T cell priming by neoantigen 3. Stimulation of CD8+ T cells was monitored by IFNγ secretion. In FIG. 17D shows the quantification of IFNγ spots (fold change) in neoantigen 3 ELISpot assay plates ( FIG. 17C ).

[151] FIG. 18 shows a graph comparing exemplary anti-HER2 antibody-based splicing activity (IC50, qPCR) versus cell activity (GI50, CTG) on HCC1954 breast cancer cells. Values shown are sized according to cell lethal activity and hatched for response quality in alternative splicing.

[152] FIG. 19 shows a graph comparing exemplary anti-HER2 antibody-based ADCs for splicing (IC50, qPCR) versus cell activity (GI50, CTG) on N87 gastric cancer cells. Values shown are sized according to cell lethal activity and hatched for response quality in alternative splicing.

[153] FIG. 20 is a graph comparing the potency and potency of exemplary anti-HER2 antibody ADCs (on HCC1954 breast cancer cells) versus stability and permeability of the respective payloads. Values shown are sized according to payload stability and shaded by payload penetration.

[154] FIG. 21 shows tumor growth kinetics for each group of N87-implanted CB17-SCID mice treated with intravenous vehicle or 10 mg/kg trastuzumab, TDM1, or exemplary HER2-ADC compound Q7D for 2 cycles (N=8 per group) . Data are presented as mean ± standard error of the mean (SEM) (mm ³ ).

[155] FIG. 22 shows change in body weight for each group of N87-implanted CB17-SCID mice treated with intravenous vehicle medium or 10 mg/kg trastuzumab, TDM1, or exemplary HER2-ADC compound Q7D for 2 cycles (N=8 per group) . Data are presented as mean ± standard error of the mean (SEM) (%).

[156] FIG. 23 shows tumor growth kinetics (left) and change in body weight (right) for each group of N87-implanted CB17-SCID mice treated with intravenous vehicle or 10 mg/kg trastuzumab, TDM1, or exemplary HER2-ADC compound Q7D in for 2 cycles (N=8 per group). Data are presented as mean ± SEM (tumor volume, mm ³ ) or mean ± SEM (body weight, %).

[157] FIG. 24A - 24D show pharmacodynamic modulation of mRNA splice boundaries in N87-implanted CB17-SCID mice treated with intravenous vehicle or 10 mg/kg trastuzumab, TDM1, or exemplary HER2-ADC compound Q7D for 2 cycles. The presence of FBXW5 (mature mRNA transcript) was monitored by RT-qPCR and the results are shown in FIG. 24A and FIG. 24C . The presence of TAOK1 (new splice boundary transcript) was monitored by RT-qPCR and the results are shown in FIG. 24B and FIG. 24D . Animals (N=4 per group) were sampled either at the 48 hour time point ( FIG. 24A and FIG. 24B ) or at the indicated time points ( FIG. 24C and FIG. 24D ). Tumors were harvested for RNA isolation and RT-qPCR.

[158] FIG. 25 is a schematic diagram of an exemplary target detection analysis.

[159] FIG. 26 shows an exemplary bioconjugation scheme for ADC production using splicing modulators.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[160] The disclosed compositions and methods may be more readily understood with reference to the following detailed description, taken in conjunction with the accompanying figures, which form part of the present invention.

[161] Throughout this text, descriptions refer to compositions and methods of using the compositions. Where the present invention describes or claims a feature or embodiment associated with a composition, such feature or embodiment is equally applicable to methods of using the composition. Similarly, if the present invention describes or claims a feature or embodiment associated with a method of using a composition, such feature or embodiment is equally applicable to the composition.

[162] If a range of values is provided, it includes embodiments using any particular value within the range. In addition, reference to values stated in a range includes any and all values within that range. All ranges include their limit values and are combinable. If values are expressed as approximate values by using the preceding word "approximately", it will be understood that a particular value constitutes another embodiment. Reference to a specific numerical value includes at least that specific value, unless the context clearly dictates otherwise. The use of "or" will mean "and/or" unless the specific context of its use provides otherwise. All references cited in this document are incorporated by reference for any purpose. If a link and a description conflict, the description will take precedence.

[163] It should be understood that certain features of the disclosed compositions and methods, which for clarity are described herein in the context of separate embodiments, can also be presented in combination in one embodiment. Conversely, various features of the disclosed compositions and methods, which are described for brevity in the context of a single embodiment, may also be presented singly or in any subcombination.

Definitions

[164] Throughout the present application and the claims, various terms are used in connection with aspects of the present description. Unless otherwise indicated, such terms are given their usual meaning in the art. Other specifically defined terms should be interpreted in the sense that corresponds to the definitions presented in this document.

[165] As used herein, the singular forms include the plural forms, unless the context clearly requires otherwise.

[166] The terms "about" or "about" in the context of numerical values and ranges refer to values or ranges that are approximately equal to or close to the described values or ranges, so that the embodiment can be carried out as intended, as, for example, there is the desired amount of nucleic acids or polypeptides in the reaction mixture, as is obvious to a person skilled in the art based on the ideas contained in this document. In some embodiments, "about" means plus or minus 10% of the numerical amount.

[167] The terms "antibody drug conjugate,""antibody-basedconjugate,""conjugate,""immunoconjugate," and "ADC" are used interchangeably and refer to one or more therapeutic compounds (e.g., a splicing modulator) that are coupled to one or multiple antibodies or antigen-binding fragments and are defined by the general formula: Ab-(LD) _p (Formula I), where Ab=antibody or antigen-binding fragment, L=linker fragment, D=drug fragment (e.g., drug fragment representing is a splicing modulator), and p =number of drug fragments per antibody or antigen-binding fragment. An ADC containing a splicing modulator drug fragment may also be referred to herein more specifically as "antibody loaded with splicing modulator" or "SMLA." In ADCs containing a splice modulator drug moiety, " p " refers to the number of splice modulator compounds associated with the antibody or antigen-binding moiety. In some embodiments, the linker L may include a cleavable fragment between the antibody or antigen-binding fragment and the therapeutic compound. In some embodiments, the linker L may include a cleavable fragment that can be attached to either or both of the antibody or antigen-binding fragment and the therapeutic compound via spacer(s) unit(s). In some embodiments, if the spacer unit attaches a cleavable moiety to the therapeutic compound, it is a self-cleaving spacer unit. In other embodiments, linker L does not include a cleavable moiety and is a non-cleavable linker. In some embodiments, the linker L may include at least one spacer unit that can be directly attached to the antibody or antigen-binding fragment and to the therapeutic compound. Illustrative cleavable and non-cleavable linkers are described and exemplified herein.

[168] The term "antibody" is used in the broadest sense to refer to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or combinations of the above, through at least one antigen recognition site within variable region of an immunoglobulin molecule. The heavy chain of an antibody consists of a heavy chain variable domain (V _H ) and a heavy chain constant region ( _CH ). The light chain consists of a light chain variable domain (V _L ) and a light chain constant domain (C _L ). For the purposes of this application, each heavy chain and light chain mature variable domain contains three complementarity determining regions (CDR1, CDR2, and CDR3) within four framework regions (FR1, FR2, FR3, and FR4) extending from the N-terminus to the C- ends: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. An "antibody" may be naturally occurring or man-made, such as monoclonal antibodies produced using conventional hybridoma technology. The term "antibody" includes full length monoclonal antibodies and full length polyclonal antibodies, as well as antibody fragments such as Fab, Fab', F(ab') ₂ , Fv and single chain antibodies. An antibody may belong to any of the five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, or subclasses thereof (eg, IgG1, IgG2, IgG3, IgG4 isotypes). The term further encompasses human antibodies, chimeric antibodies, humanized antibodies, and any modified immunoglobulin molecule containing an antigen recognition site so long as it exhibits the desired biological activity (e.g., binds the target antigen, is internalized into a cell expressing the target antigen).

[169] As used herein, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in small quantities. Monoclonal antibodies are highly specific, directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a wide variety of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the characterization of the antibody as derived from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be obtained by the hybridoma method first described in Kohler et al. (1975) Nature 256:495, or can be obtained by recombinant DNA methods (see, for example, US patent No. 4816567). Monoclonal antibodies can also be isolated from antibody phage libraries using, for example, the techniques described in Clackson et al. (1991) Nature 352:624-8, and Marks et al. (1991) J Mol Biol. 222:581-97.

[170] The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain(s) are identical or homologous to the corresponding sequences in antibodies derived from a different species or belonging to a different class or subclass of antibodies, as well as fragments of such antibodies, provided that they specifically bind the target antigen and/ or exhibit the desired biological activity.

[171] As used herein, the term "human antibody" refers to an antibody produced in a human, or an antibody having the amino acid sequence of an antibody produced in a human.

[172] As used herein, the term "chimeric antibody" refers to antibodies in which the amino acid sequence of an immunoglobulin molecule is derived from two or more species. In some cases, the variable regions of both heavy and light chains correspond to the variable regions of antibodies derived from one species with the desired specificity, affinity, and activity, while the constant regions are homologous to antibodies derived from another species (e.g., human) to minimize immune response in the latter species.

[173] As used herein, the term "humanized antibody" refers to forms of antibodies that contain sequences from antibodies of a non-human species (eg, mouse) as well as human antibodies. Such antibodies are chimeric antibodies that contain a minimal sequence derived from a non-human immunoglobulin. In general, a humanized antibody will contain substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops correspond to human immunoglobulin loops, and all or substantially all of the framework (FR) regions are regions from the human immunoglobulin sequence. The humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region. The humanized antibody can be further modified by substitution of residues either within the Fv framework region and/or within the substituted non-human residues to improve and optimize the specificity, affinity and/or activity of the antibody.

[174] As used herein, the term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more fragments of an antibody or protein that retain the ability to specifically bind to an antigen (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KITP, MET, MUC16, SLC39A6, SLC44A4, STEAP1). Antigen-binding fragments may also retain the ability to internalize into a cell expressing the antigen. In some embodiments, antigen-binding fragments also retain immune effector activity. It has been shown that fragments of a full-length antibody can perform the antigen-binding function of a full-length antibody. Examples of binding fragments encompassed by the term "antigen binding fragment" or "antigen binding portion" of an antibody include: (i) Fab fragment, a monovalent fragment consisting of V domains_L, V_H, C_L and C_H1; (ii) F(ab')₂-fragment, a bivalent fragment containing two Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fd fragment consisting of V domains_H and C_H1; (iv) Fv fragment consisting of V domains_L and V_H one arm of the antibody; (v) a dAb fragment that contains a single variable domain, such as the V domain_H (See, for example, Ward et al. (1989) Nature 341:544-6; and International Publication No. WO 1990/005144); and (vi) a dedicated complementarity determining region (CDR). In addition, although the two domains of the Fv fragment, V_L and V_H, are encoded by separate genes, using recombinant methods, they can be connected using a synthetic linker, which allows them to be obtained as a single protein chain, in which the V_L and V_H pair to form a monovalent molecule (known as single chain Fv (scFv)). See, for example, Bird et al. (1988) Science 242:423-6; and Huston et al. (1988) Proc Natl Acad Sci. USA 85:5879-83. Such single chain antibodies are also intended to be encompassed by the term "antigen binding fragment" or "antigen binding portion" of an antibody, and are known in the art as an illustrative type of binding fragment that can be internalized into cells after binding (see, e.g., Zhu et al. (2010) 9:2131-41, He et al (2010) J Nucl Med 51:427-32 and Fitting et al (2015) MAbs 7:390-402). In certain embodiments, scFv molecules may be included in a fusion protein. Other forms of single chain antibodies, such as diabodies, are also covered. Diabodies are bivalent, bispecific antibodies in which the V domains_H and V_L are expressed on the same polypeptide chain, but using a linker that is too short to allow pairing between two domains on the same chain, thereby causing the domains to pair with complementary domains on the other chain and create two antigen-binding sites (see, e.g., Holliger et al (1993) Proc Natl Acad Sci USA 90:6444-8 and Poljak et al (1994) Structure 2:1121-3). Antigen binding fragments are prepared using conventional techniques known to those skilled in the art, and the binding fragments are screened for utility (eg, binding affinity, internalization) in the same manner as intact antibodies. Antigen-binding fragments can be obtained by cleavage of the intact protein, for example with a protease or chemical cleavage.

[175] “Internalizing,” as used herein when referring to an antibody or antigen-binding fragment, refers to an antibody or antigen-binding fragment that can be taken up through the lipid bilayer membrane of a cell into an internal compartment (i.e., “subject to internalization”) after binding with the cell, preferably in the degradation compartment of the cell. For example, an internalizing anti-HER2 antibody is an antibody that can be taken up into a cell after binding to HER2 on the cell membrane. In some embodiments, the antibody or antigen-binding fragment used in the ADCs disclosed herein targets a cell surface antigen (e.g., HER2) and is an internalizing antibody or an internalizing antigen-binding fragment (i.e., the ADC is transported across the cell membrane upon binding antigen). In some embodiments, the internalizing antibody or antigen-binding fragment binds a receptor on the cell surface. An internalizing antibody or an internalizing antigen-binding fragment that targets a receptor on the cell membrane can induce receptor-mediated endocytosis. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment is taken up into the cell via receptor-mediated endocytosis.

[176] "Non-internalizing", as used herein when referring to an antibody or antigen-binding fragment, refers to an antibody or antigen-binding fragment that is retained on the cell surface after binding to a cell. In some embodiments, the antibody or antigen-binding fragment used in the ADCs disclosed herein targets a cell surface antigen and is a non-internalizing antibody or non-internalizing antigen-binding fragment (i.e., the ADC remains on the cell surface and is not transported across the cell membrane after antigen binding). In some embodiments, the non-internalizing antibody or antigen-binding fragment binds a non-internalizing receptor or other cell surface antigen. Illustrative non-internalizing cell surface antigens include, without limitation, CA125 and CEA, and antibodies that bind to non-internalizing target antigens are also known in the art (see, for example, Bast et al. (1981) J Clin Invest. 68(5):1331 -7; Scholler and Urban (2007) Biomark Med. 1(4):513-23; and Boudousq et al. (2013) PLoS One 8(7):e69613).

[177] As used herein, the term "human epidermal growth factor receptor 2", "HER2" or "HER2/NEU" refers to any native form of human HER2. The term encompasses the full-length sequence of HER2 (eg, the reference sequence in UniProt: P04626; SEQ ID NO:31) as well as any form of human HER2 that may result from cellular processing. The term also encompasses functional variants or fragments of human HER2, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more of the biological functions of human HER2 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to the wild-type protein only). HER2 may be isolated from a human or may be produced recombinantly or by synthetic means.

[178] The term "anti-HER2 antibody" or "antibody that binds to HER2" refers to any form of antibody or fragment thereof that binds, e.g. specifically binds, to HER2, and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, eg bind specifically, to HER2. US Pat. No. 5,821,337, incorporated herein by reference, provides exemplary HER2 binding sequences, including exemplary anti-HER2 antibody sequences. In some embodiments, the anti-HER2 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. Trastuzumab (US Pat. No. 5,821,337; Molina et al. (2001) Cancer Res. 61(12):4744-9) is an exemplary anti-human HER2 antibody.

[179] As used herein, the term "syndecan-1", "SDC1", or "CD138" refers to any native form of human CD138. The term encompasses the full-length sequence of CD138 (eg, the reference sequence in UniProt: P18827; SEQ ID NO:32) as well as any form of human CD138 that may result from cellular processing. The term also encompasses functional variants or fragments of human CD138, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more of the biological functions of human CD138 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to the wild-type protein only). CD138 may be isolated from a human or may be produced recombinantly or by synthetic means.

[180] The term "anti-CD138 antibody" or "antibody that binds to CD138" refers to any form of antibody or fragment thereof that binds, e.g. specifically binds, to CD138, and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, eg bind specifically, to CD138. In some embodiments, the anti-CD138 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. B-B4 (Tassone et al. (2004) Blood 104:3688-96) is an exemplary anti-human CD138 antibody.

[181] As used herein, the term "ephrin type-A receptor 2" or "EPHA2" refers to any native form of human EPHA2. The term encompasses the full-length EPHA2 sequence (eg, the reference sequence in UniProt: P29317; SEQ ID NO:33), as well as any form of human EPHA2 that may result from cellular processing. The term also encompasses functional variants or fragments of human EPHA2, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more of the biological functions of human EPHA2 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to the wild-type protein only). EPHA2 may be isolated from a human or may be produced recombinantly or by synthetic means.

[182] The term "anti-EPHA2 antibody" or "antibody that binds to EPHA2" refers to any form of antibody or fragment thereof that binds, e.g. specifically binds, to EPHA2, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, eg bind specifically, to EPHA2. WO 2007/030642, incorporated herein by reference, provides exemplary EPHA2 binding sequences, including exemplary anti-EPHA2 antibody sequences. In some embodiments, the anti-EPHA2 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. 1C1 (WO 2007/030642; Jackson et al. (2008) Cancer Res. 68(22): 9367-74) is an exemplary anti-human EPHA2 antibody.

[183] As used herein, the term "mesothelin" or "MSLN" refers to any native form of human MSLN. The term encompasses the full-length MSLN sequence (eg, the reference sequence in UniProt: Q13421; SEQ ID NO:43) as well as any form of human MSLN that may be processed in a cell. The term also encompasses functional variants or fragments of human MSLNs, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more of the biological functions of a human MSLN (i.e., variants and fragments are covered unless the context indicates that the term used to refer to the wild-type protein only). The MSLN may be isolated from a human or may be produced recombinantly or by synthetic means.

[184] The term "anti-MSLN antibody" or "antibody that binds to MSLN" refers to any form of an antibody or fragment thereof that binds, e.g. specifically binds, to MSLNs, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, eg bind specifically, to MSLNs. WO 2011/074621, incorporated herein by reference, provides exemplary MSLN binding sequences, including exemplary anti-MSLN antibody sequences. In some embodiments, the anti-MSLN antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. 11-25, IC14-30, IC7-4, IC17-35, and 2-9 are exemplary anti-human MSLN antibodies.

[185] As used herein, the term "glutamate carboxypeptidase 2" or "FOLH1" refers to any native form of human FOLH1. The term encompasses the full length FOLH1 sequence (eg, the reference sequence in UniProt: Q04609; SEQ ID NO:44) as well as any form of human FOLH1 that may result from cellular processing. The term also encompasses functional variants or fragments of human FOLH1, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more of the biological functions of human FOLH1 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to the wild-type protein only). FOLH1 may be isolated from a human or may be produced recombinantly or by synthetic means.

[186] The term "anti-FOLH1 antibody" or "antibody that binds to FOLH1" refers to any form of an antibody or fragment thereof that binds, e.g., specifically binds, to FOLH1, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, eg bind specifically, to FOLH1. WO 2019/012260 and WO 2017/212250, incorporated herein by reference, provide exemplary FOLH1 binding sequences, including exemplary anti-FOLH1 antibody sequences. In some embodiments, the anti-FOLH1 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. J591 (deimmunized) is an exemplary anti-human FOLH1 antibody.

[187] As used herein, the term "cadherin-6" or "CDH6" refers to any native form of human CDH6. The term encompasses the full-length sequence of CDH6 (eg, the reference sequence in UniProt: P55285; SEQ ID NO:45) as well as any form of human CDH6 that may result from cellular processing. The term also encompasses functional variants or fragments of human CDH6, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more of the biological functions of human CDH6 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to the wild-type protein only). CDH6 may be isolated from a human or may be produced recombinantly or by synthetic means.

[188] The term "anti-CDH6 antibody" or "antibody that binds to CDH6" refers to any form of antibody or fragment thereof that binds, e.g. specifically binds, to CDH6, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, eg bind specifically, to CDH6. WO 2018/185618, incorporated herein by reference, provides exemplary CDH6 binding sequences, including exemplary anti-CDH6 antibody sequences. In some embodiments, the anti-CDH6 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[189] As used herein, the term "cell adhesion molecule 5 related to carcinoembryonic antigen" or "CEACAM5" refers to any native form of human CEACAM5. The term encompasses the full length sequence of CEACAM5 (eg, the reference sequence in UniProt: P06731; SEQ ID NO:46) as well as any form of human CEACAM5 that may be processed in a cell. The term also encompasses functional variants or fragments of human CEACAM5, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more of the biological functions of human CEACAM5 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to the wild-type protein only). CEACAM5 may be isolated from a human or may be produced recombinantly or by synthetic means.

[190] The term "anti-CEACAM5 antibody" or "antibody that binds to CEACAM5" refers to any form of antibody or fragment thereof that binds, e.g. specifically binds, to CEACAM5, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, eg bind specifically, to CEACAM5. US 2015/0125386, incorporated herein by reference, provides exemplary CEACAM5 binding sequences, including exemplary anti-CEACAM5 antibody sequences. In some embodiments, the anti-CEACAM5 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. hMN14 is an exemplary anti-human CEACAM5 antibody.

[191] As used herein, the term "cryptic protein family member 1B" or "CFC1B" refers to any native form of human CFC1B. The term encompasses the full-length sequence of CFC1B (eg, the reference sequence in UniProt: P0CG36; SEQ ID NO:47) as well as any form of human CFC1B that may result from cellular processing. The term also encompasses functional variants or fragments of human CFC1B, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more of the biological functions of human CFC1B (i.e., variants and fragments are covered unless the context indicates that the term used to refer to the wild-type protein only). CFC1B may be isolated from a human or may be produced recombinantly or by synthetic means.

[192] The term "anti-CFC1B antibody" or "antibody that binds to CFC1B" refers to any form of an antibody or fragment thereof that binds, e.g. specifically binds, to CFC1B, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, eg bind specifically, to CFC1B. WO 2002/088170, incorporated herein by reference, provides exemplary CFC1B binding sequences, including exemplary anti-CFC1B antibody sequences. In some embodiments, the anti-CFC1B antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[193] As used herein, the term "ectonucleotide pyrophosphatase/phosphodiesterase family member 3" or "ENPP3" refers to any native form of human ENPP3. The term encompasses the full length sequence of ENPP3 (eg, the reference sequence in UniProt: O14638; SEQ ID NO:48) as well as any form of human ENPP3 that may be processed in a cell. The term also encompasses functional variants or fragments of human ENPP3, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more of the biological functions of human ENPP3 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to the wild-type protein only). ENPP3 may be isolated from a human or may be produced recombinantly or by synthetic means.

[194] The term "anti-ENPP3 antibody" or "antibody that binds to ENPP3" refers to any form of an antibody or fragment thereof that binds, e.g., specifically binds, to ENPP3, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, eg bind specifically, to ENPP3. Donate et al. ((2016) Clin Cancer Res. 22(8):1989-99), incorporated herein by reference, provides exemplary ENPP3 binding sequences, including exemplary anti-ENPP3 antibody sequences. In some embodiments, the anti-ENPP3 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[195] As used herein, the term "folate receptor alpha" or "FOLR1" refers to any native form of human FOLR1. The term encompasses the full length sequence of FOLR1 (eg, the reference sequence in UniProt: P15328; SEQ ID NO:49) as well as any form of human FOLR1 that may be processed in the cell. The term also encompasses functional variants or fragments of human FOLR1, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more of the biological functions of human FOLR1 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to the wild-type protein only). FOLR1 may be isolated from a human or may be produced recombinantly or by synthetic means.

[196] The term "anti-FOLR1 antibody" or "antibody that binds to FOLR1" refers to any form of antibody or fragment thereof that binds, e.g. specifically binds, to FOLR1, and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided they bind, eg bind specifically, to FOLR1. In WO 2005/080431 and Coney et al. ((1991) Cancer Res. 51(22):6125-32), incorporated herein by reference, provides exemplary FOLR1 binding sequences, including exemplary anti-FOLR1 antibody sequences. In some embodiments, the anti-FOLR1 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. Farletuzumab and MOv19 are exemplary anti-human FOLR1 antibodies.

[197] As used herein, the term "hepatitis A virus cell receptor 1" or "HAVCR1" refers to any native form of human HAVCR1. The term encompasses the full length sequence of HAVCR1 (eg, the reference sequence in UniProt: Q96D42; SEQ ID NO:50) as well as any form of human HAVCR1 that may result from cellular processing. The term also encompasses functional variants or fragments of human HAVCR1, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more of the biological functions of human HAVCR1 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to the wild-type protein only). HAVCR1 may be isolated from a human or may be produced recombinantly or by synthetic means.

[198] The term "anti-HAVCR1 antibody" or "antibody that binds to HAVCR1" refers to any form of an antibody or fragment thereof that binds, e.g. specifically binds, to HAVCR1, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, eg bind specifically, to HAVCR1. Thomas et al. ((2016) Mol Cancer Ther. 15(12):2946-54), incorporated herein by reference, provides exemplary HAVCR1 binding sequences, including exemplary anti-HAVCR1 antibody sequences. In some embodiments, the anti-HAVCR1 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[199] As used herein, the term "mast/stem cell growth factor receptor Kit" or "KIT" refers to any native form of human KIT. The term encompasses the full-length sequence of KIT (eg, the reference sequence in UniProt: P10721; SEQ ID NO:51) as well as any form of human KIT that may result from cellular processing. The term also encompasses functional variants or fragments of a human KIT, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more of the biological functions of a human KIT (i.e., variants and fragments are covered unless the context indicates that the term used to refer to the wild-type protein only). The KIT may be isolated from a human or may be produced recombinantly or by synthetic means.

[200] The term "anti-KIT antibody" or "antibody that binds to KIT" refers to any form of antibody or fragment thereof that binds, e.g. specifically binds, to KIT, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, eg bind specifically, to the KIT. Shi et al. ((2016) Proc Natl Acad Sci USA 113(33):E4784-93) and Abrams et al. ((2018) Clin Cancer Res. 24(17):4297-308), incorporated herein by reference, provides exemplary KIT binding sequences, including exemplary anti-KIT antibody sequences. In some embodiments, the anti-KIT antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[201] As used herein, the term "hepatocyte growth factor receptor" or "MET" refers to any native form of human MET. The term encompasses the full-length sequence of MET (eg, the reference sequence in UniProt: P08581; SEQ ID NO:52) as well as any form of human MET that may be processed in a cell. The term also encompasses functional variants or fragments of human MET, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more of the biological functions of human MET (i.e., variants and fragments are covered unless the context indicates that the term used to refer to the wild-type protein only). MET may be isolated from a human or may be produced recombinantly or by synthetic means.

[202] The term "anti-MET antibody" or "antibody that binds to MET" refers to any form of an antibody or fragment thereof that binds, e.g., specifically binds, to MET, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, eg bind specifically, to MET. Yang et al. ((2019) Acta Pharmacol Sin.), incorporated herein by reference, provides exemplary MET binding sequences, including exemplary anti-MET antibody sequences. In some embodiments, the anti-MET antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[203] As used herein, the term "mucin-16" or "MUC16" refers to any native form of human MUC16. The term encompasses the full-length sequence of MUC16 (eg, the reference sequence in UniProt: Q8WXI7; SEQ ID NO:53) as well as any form of human MUC16 that may result from cellular processing. The term also encompasses functional variants or fragments of human MUC16, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more of the biological functions of human MUC16 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to the wild-type protein only). MUC16 may be isolated from a human or may be produced recombinantly or by synthetic means.

[204] The term "anti-MUC16 antibody" or "antibody that binds to MUC16" refers to any form of antibody or fragment thereof that binds, e.g. specifically binds, to MUC16, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, eg bind specifically, to MUC16. Liu et al. ((2016) Ann Oncol. 27(11):2124-30), incorporated herein by reference, provides exemplary MUC16 binding sequences, including exemplary anti-MUC16 antibody sequences. In some embodiments, the anti-MUC16 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[205] As used herein, the term "zinc transporter ZIP6" or "SLC39A6" refers to any native form of human SLC39A6. The term encompasses the full length sequence of SLC39A6 (eg, the reference sequence in UniProt:Q13433; SEQ ID NO:54) as well as any form of human SLC39A6 that may be processed in the cell. The term also encompasses functional variants or fragments of human SLC39A6, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more of the biological functions of human SLC39A6 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to the wild-type protein only). SLC39A6 may be isolated from a human or may be produced recombinantly or by synthetic means.

[206] The term "anti-SLC39A6 antibody" or "antibody that binds to SLC39A6" refers to any form of antibody or fragment thereof that binds, e.g. specifically binds, to SLC39A6, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, eg bind specifically, to SLC39A6. Sussman et al. ((2014) Mol Cancer Ther. 13(12):2991-3000), incorporated herein by reference, provides exemplary SLC39A6 binding sequences, including exemplary anti-SLC39A6 antibody sequences. In some embodiments, the anti-SLC39A6 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[207] As used herein, the term "choline transporter-like protein 4" or "SLC44A4" refers to any native form of human SLC44A4. The term encompasses the full-length sequence of SLC44A4 (eg, the reference sequence in UniProt: Q53GD3; SEQ ID NO:55) as well as any form of human SLC44A4 that may result from cellular processing. The term also encompasses functional variants or fragments of human SLC44A4, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more of the biological functions of human SLC44A4 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to the wild-type protein only). SLC44A4 may be isolated from a human or may be produced recombinantly or by synthetic means.

[208] The term "anti-SLC44A4 antibody" or "antibody that binds to SLC44A4" refers to any form of antibody or fragment thereof that binds, e.g. specifically binds, to SLC44A4, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided they bind, eg bind specifically, to SLC44A4. Mattie et al. ((2016) Mol Cancer Ther. 15(11):2679-87), incorporated herein by reference, provides exemplary SLC44A4 binding sequences, including exemplary anti-SLC44A4 antibody sequences. In some embodiments, the anti-SLC44A4 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[209] As used herein, the term "metal reductase STEAP1" or "STEAP1" refers to any native form of human STEAP1. The term encompasses the full length sequence of STEAP1 (eg, the reference sequence in UniProt: Q9UHE8; SEQ ID NO:56) as well as any form of human STEAP1 that may result from cellular processing. The term also encompasses functional variants or fragments of human STEAP1, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more of the biological functions of human STEAP1 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to the wild-type protein only). STEAP1 may be isolated from a human or may be produced recombinantly or by synthetic means.

[210] The term "anti-STEAP1 antibody" or "antibody that binds to STEAP1" refers to any form of an antibody or fragment thereof that binds, e.g. specifically binds, to STEAP1, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, eg bind specifically, to STEAP1. WO 2008/052187, incorporated herein by reference, provides exemplary STEAP1 binding sequences, including exemplary anti-STEAP1 antibody sequences. In some embodiments, the anti-STEAP1 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[211] As used herein, the term "specific""specificallybinds" or "binds in a specific manner" refers to a binding reaction between an antibody or antigen-binding fragment (eg, an anti-HER2 antibody) and a target antigen (eg, HER2) in a heterogeneous population of proteins and other biological molecules. Antibodies can be tested for binding specificity by comparing binding to an appropriate antigen with binding to an irrelevant antigen or mixture of antigens under a defined set of conditions. If an antibody binds to the corresponding antigen with an affinity interrupting at least 2, 5, 7 and preferably 10 or more times the affinity of an irrelevant antigen or mixture of antigens, then it is considered to be specific. A "specific antibody" or "target-specific antibody" is an antibody that binds only to a target antigen (eg, HER2) but does not bind (or shows minimal binding) to other antigens. In certain embodiments, an antibody or antigen-binding fragment that specifically binds a target antigen (eg, HER2) has a K _D of less than 1×10 ^-6 M, less than 1×10 ^-7 M, less than 1×10 ^-8 M, less than 1×10 ^-9 M, less than 1×10 ^-10 M, less than 1×10 ^-11 M, less than 1×10 ^-12 M, or less than 1×10 ^-13 M. In some embodiments, the implementation of K _D is from 1 pM up to 500 pM. In some embodiments, the K _D is 500 pM to 1 μM, 1 μM to 100 nM, or 100 mM to 10 nM.

[212] The term "epitope" refers to the portion of an antigen that an antibody can recognize or specifically bind to. If the antigen is a polypeptide, epitopes can be formed from contiguous amino acids or non-contiguous amino acids brought into contact by the tertiary folding of the polypeptide. An antibody-bound epitope can be identified using any epitope mapping technique known in the art, including X-ray diffraction crystallography to identify epitopes by directly visualizing the antigen-antibody complex, as well as monitoring antibody binding to antigen fragments or mutants, or monitoring solvent access to different parts of the antibody and antigen. Exemplary strategies used to map antibody epitopes include, but are not limited to, array-based oligopeptide scanning, limited proteolysis, site-directed mutagenesis, high-throughput mutagenesis-based mapping, hydrogen-deuterium exchange, and mass spectrometry (see, e.g., Gershoni et al. (2007) 21:145-56 and Hager-Braun and Tomer (2005) Expert Rev Proteomics 2:745-56).

[213] Competitive binding and epitope-specific sorting can also be used to identify antibodies that co-bind identical or overlapping epitopes. Competitive binding can be assessed using a cross-blocking assay, such as the assay described in "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory, Harlow and Lane ( ^1st ed. 1988, ^2nd ed. 2014). In some embodiments, competitive binding is identified if a test antibody or binding protein reduces the binding of a reference antibody or binding protein to a target antigen such as HER2 (e.g., a binding protein containing CDRs and/or variable domains selected from those identified in Tables 2- 4), at least about 50% in a cross-blocking assay (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5% or more, or any percentage between them) and/or vice versa. In some embodiments, competitive binding may be due to common or similar (e.g., partially overlapping) epitopes, or due to steric mismatch in which antibodies or binding proteins bind to closely spaced epitopes (see, e.g., Tzartos, Methods in Molecular Biology (Morris, ed. (1998) vol.66, pp. 55-66)). In some embodiments, competitive binding can be used to sort groups of binding proteins that have similar epitopes. For example, binding proteins that compete for binding can be "sorted" into a group of binding proteins that have overlapping or closely spaced epitopes, while proteins that do not compete are placed into a separate group of binding proteins that do not have overlapping or closely spaced epitopes. epitopes.

[214] The term "k _on " or "k _a " refers to the association rate constant when an antibody associates with an antigen to form an antibody/antigen complex. Velocity can be determined using standard assays such as surface plasmon resonance, biolayer inferometry, or ELISA analysis.

[215] The term "k _off " or "k _d " refers to the dissociation rate constant when an antibody dissociates from an antibody/antigen complex. Velocity can be determined using standard assays such as surface plasmon resonance, biolayer inferometry or ELISA.

[216] The term "K _D " refers to the equilibrium dissociation constant for a particular antibody-antigen interaction. K _D is calculated as k _a /k _d . Velocity can be determined using standard assays such as surface plasmon resonance, biolayer inferometry, or ELISA analysis.

[217] The term " p " or "drug loading" or "drug:antibody ratio" or "drug to antibody ratio" or "DAR" refers to the number of drug fragments per antibody or antigen-binding fragment, ie, drug loading, or number of -LD fragments per antibody or antigen binding fragment (Ab) in an ADC of formula (I). In ADCs containing a splice modulator drug moiety, " p " refers to the number of splice modulator compounds associated with the antibody or antigen-binding moiety. For example, if two splicing modulator compounds are bound to an antibody or antigen-binding fragment (eg, two compounds each having a D1 structure), p =2. In compositions containing multiple copies of ADCs of formula (I), "average p " refers to the average number of -LD fragments per antibody or antigen-binding fragment, also referred to as "average drug load".

[218] "Linker" or "linker moiety" is used herein to refer to any chemical moiety that is capable of covalently linking a compound, typically a drug moiety, such as a splicing modulator drug moiety, to another moiety, such as as an antibody or antigen-binding fragment. Linkers may be susceptible or substantially resistant to acid-induced cleavage, peptidase-induced cleavage, light-based cleavage, esterase-induced cleavage, and/or disulfide bond cleavage under conditions under which the compound or antibody remains active.

[219] The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract derived from biological materials. The term "therapeutic agent" or "drug" refers to an agent that is capable of modulating a biological process and/or has biological activity. The splicing modulator compounds described herein are exemplary therapeutic agents.

[220] The term "chemotherapeutic agent" or "anticancer agent" is used herein to refer to agents that are effective in the treatment of cancer, regardless of the mechanism of action. Inhibition of metastasis or angiogenesis is often a feature of a chemotherapeutic agent. Chemotherapeutic agents include antibodies, biological molecules, and small molecules, and encompass the splicing modulator compounds described herein. The chemotherapeutic agent may be a cytotoxic or cytostatic agent. The term "cytostatic agent" refers to an agent that inhibits or inhibits cell growth and/or cell reproduction. The term "cytotoxic agent" refers to a substance that causes cell death primarily by interfering with the expression activity and/or functioning of the cell.

[221] As used herein, the terms "splicing modulator", "spliceosome modulator", or "splice modulator" refer to compounds that exhibit anti-cancer activity by interacting with components of the spliceosome. In some embodiments, a splicing modulator changes the rate or shape of splicing in a target cell. Splicing modulators that act as inhibitory agents, for example, are capable of reducing uncontrolled cell proliferation. In some embodiments, splicing modulators may exert their effect by binding to the SF3b spliceosome complex. Such modulators may be natural compounds or synthetic compounds. Non-limiting examples of splicing modulators and categories of such modulators include pladienolide (e.g., pladienolide D or pladienolide B), pladienolide derivatives (e.g., pladienolide D or pladienolide B derivatives), herboxiene, herboxiene derivatives, splicestatin, splicestatin derivatives, sudemycin, or sudemycin derivatives. As used herein, the terms "derivative" and "analogue" when referring to a splicing modulator, etc., mean any such compound that retains substantially the same, similar, or improved biological function or activity as the parent compound, but has an altered chemical or biological structure. In some embodiments, the splicing modulator is a pladienolide or a pladienolide derivative.

[222] As used herein, "pladienolide derivative" refers to a compound that is structurally related to a member of the family of natural products known as pladienolides and that retains one or more of the biological functions of the parent compound. Pladienolides were first identified in bacteriaStreptomyces platensis(Mizui et al. (2004) J Antibiot. 57:188-96) as potential cytotoxic agents and they result in cell cycle arrest at the G1 and G2/M phases of the cell cycle (e.g. Bonnal et al. (2012) Nat Rev drugdis 11:847-59). Seven pladienolides occur naturally, pladienolide A-G (Mizui et al. (2004) J Antibiot. 57:188-96; Sakai et al. (2004) J Antibiotics 57:180-7). US Pat. Nos. 7,884,128 and 7,816,401 describe illustrative methods for the synthesis of pladienolide B and D, and are each incorporated herein by reference in the case of such methods. The synthesis of pladienolide B and D can also be carried out using the exemplary methods described in Kanada et al. ((2007) Angew Chem Int. Ed. 46:4350-5). In Kanada et al. and International Application Publication No. WO 2003/099813 describe illustrative methods for the synthesis of E7107 (D11) (compound 45 of WO 2003/099813) from pladienolide D (11107D of WO 2003/099813). The corresponding US Pat. No. 7,550,503 to Kotake et al. Each of these references is included in this document for the described methods of synthesis.

[223] As used herein, "splicing modulator drug moiety" refers to an ADC component or composition that provides the structure of a splicing modulator compound, such as the splicing modulator component (D) in an ADC of formula (I) or in a composition containing -L-D.

[224] As used herein, "spliceosome" refers to a ribonucleoprotein complex that removes introns from one or more RNA segments, such as pre-mRNA segments.

[225] The term "homolog" refers to a molecule that exhibits homology to another molecule, for example, has sequences of chemical residues that are the same or similar at the appropriate positions.

[226] As used herein, the term "inhibit" or "inhibition" means a reduction in a measurable amount and may include, but is not necessarily required, complete prevention or inhibition.

[227] The term "target negative", "target antigen negative", or "antigen negative" refers to the absence of target antigen expression in a cell or tissue. The term "target positive", "target antigen positive", or "antigen positive" refers to the presence of expression of the target antigen. For example, a cell or cell line that does not express the target antigen may be are described as target-negative, while a cell or cell line that expresses the target antigen can be described as target-positive.

[228] The term "non-specific killing" or "non-specific effect" refers to the killing of target-negative cells in the presence of target-positive cells, where killing of target-negative cells is not observed in the absence of target-positive cells. Cell-to-cell contact, or at least proximity, between target-positive and target-negative cells allows non-specific killing. This type of killing is distinguishable from "non-target killing", which refers to the indiscriminate killing of target-negative cells. "Off-target killing" can be observed in the absence of target-positive cells.

[229] The terms "neoplastic disorder" and "cancer" are used interchangeably herein to refer to the presence of cells having characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and/or certain morphological features. Often, cancer cells may be in the form of a tumor or mass, but such cells may exist singly in a subject or may circulate in the bloodstream as independent cells such as leukemic or lymphoma cells. The terms "neoplastic disorder" and "cancer" include all types of cancer and cancer metastases, including hematological malignancies, solid tumors, sarcomas, carcinomas, and other cancers that are solid tumors and non-solid tumors. Hematologic malignancies may include B-cell malignancies, blood cancers (leukemias), plasma cell cancers (myelomas, eg, multiple myeloma), or cancers of the lymph nodes (lymphomas). Illustrative B cell malignancies include chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, and diffuse large B cell lymphoma. Leukemias may include acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), acute monocytic leukemia (AMoL), etc. Lymphomas may include Hodgkin's lymphoma and non-Hodgkin's lymphoma. Other hematologic malignancies may include myelodysplastic syndrome (MDS). Solid tumors may include carcinomas such as adenocarcinoma, eg, breast cancer, pancreatic cancer, prostate cancer, colon or colorectal cancer, lung cancer, gastric cancer, cervical cancer, endometrial cancer, ovarian cancer, cholangiocarcinoma, glioma , melanoma, etc.

[230] The terms "tumor" and "neoplasm" refer to any mass of tissue that results from excess growth or proliferation of cells, whether benign or malignant, including precancerous lesions.

[231] The terms "tumor cell" and "neoplastic cell" are used interchangeably and refer to individual cells or to a general population of cells derived from a tumor or neoplasm, including both non-carcinogenic cells and cancer stem cells. As used herein, the term "tumor cell" will be modified by the term "non-carcinogenic" when it refers solely to those tumor cells that lack the ability to renew and differentiate to distinguish these tumor cells from cancer stem cells.

[232] The terms "subject" and "patient" are used interchangeably herein to refer to any animal, such as any mammal, including, without limitation, humans, non-human primates, rodents, and the like. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human. In some embodiments, the subject is a mouse. In some embodiments, the subject is a human.

[233] The term "co-administration" or administration "in combination with" one or more therapeutic agents includes simultaneous administration and sequential administration in any order.

[234] "Pharmaceutical composition" refers to a preparation that is in a form that allows administration and subsequently provides the intended biological activity of the active(s) ingredient(s) and/or the achievement of a therapeutic effect and which does not contain additional components that are unacceptably toxic to the subject to whom the formulation will be administered. The pharmaceutical composition may be sterile.

[235] A "pharmaceutical excipient" includes a material such as an adjuvant, a carrier, pH adjusters and buffers, tonicity adjusters, wetting agents, a preservative, and the like.

[236] "Pharmaceutical acceptable" means approved, or as may be approved, by a federal or state government regulatory agency, or listed in the United States Pharmacopeia or other generally accepted pharmacopoeia for use in animals and, more specifically, in humans.

[237] A "pharmaceutically acceptable salt" is a salt that retains the desired biological activity of the parent compound and does not cause undesirable toxicological effects. Examples of such salts are: (a) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like; and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, polygalacturonic acid, and the like; and (b) salts formed from elemental anions such as chlorine, bromine and iodine. Cm. For example, Haynes et al. "Commentary: Occurrence of Pharmaceutically Acceptable Anions and Cations in the Cambridge Structural Database," J Pharmaceutical Sciences, vol. 94, no. 10 (2005), and Berge et al. "Pharmaceutical Salts," J Pharmaceutical Sciences, vol. 66, no. 1 (1977), which

incorporated into this document by reference.

[238] As used herein, the term "effective amount" refers to an amount of a compound, ADC, or composition described herein (e.g., a splicing modulator or ADC) that is sufficient to accomplish a specific purpose, e.g., to effect a therapeutic effect after administration, such as a decrease in tumor growth rate or tumor volume, a decrease in a cancer symptom, or some other indication of the effectiveness of the treatment. An effective amount can be determined in a conventional manner with respect to the intended purpose. The term "therapeutically effective amount" refers to an amount of a compound, ADC, or composition described herein that is effective to detectably kill, reduce, and/or inhibit the growth or spread of tumor cells, the size or number of tumors, and/or other indication of level, stage , progression and/or severity of the cancer. A therapeutically effective amount may vary depending on the intended use (in vitro or in vivo) or the subject and disease state being treated, for example, the weight and age of the subject, the severity of the disease state, the route of administration, etc., which can be easily determined by a person skilled in the art. in this field of technology. The term is also applicable to a dose that will induce a specific response in target cells, such as inhibition of cell growth. The specific dosage may vary, for example, depending on the particular pharmaceutical composition, the subject and their age and current health or health risks, the regimen to be followed, the severity of the disease, whether it is administered in combination with by other means, the time of administration, the tissue into which it is administered, and the physical delivery system in which it is carried. In the case of cancer, a therapeutically effective amount of ADC can reduce the number of cancer cells, reduce tumor size, inhibit (eg, slow or stop) tumor metastasis, suppress (eg, slow or stop) tumor growth, and/or relieve one or more symptoms.

[239] A "prophylactically effective amount" refers to an amount, at dosages and over periods of time, effective in achieving the desired prophylactic result. Generally, since a prophylactic dose is administered to subjects prior to the onset of the disease or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[240] As used herein, "treat" or "therapeutic" and grammatically related terms refer to any improvement in any outcome of a disease, such as prolongation of survival, less clinical manifestations, and/or reduction in side effects that result from an alternative therapeutic mode of action. As is readily understood from the prior art, complete elimination of the disease is covered by, but not required for, curative action. As used herein, "treatment" or "treat" refers to the administration of the described ADC or composition to a subject, such as a patient. The treatment may be a cure, healing, amelioration, amelioration, alteration, elimination, alleviation, temporary amelioration, amelioration, or treatment of a disorder, the symptoms of a disorder, or a predisposition to a disorder, such as cancer. In some embodiments, in addition to treating a subject who has a condition, the composition disclosed herein can also be given prophylactically to prevent or reduce the likelihood of developing the condition.

[241] In some embodiments, a labeled ADC is used. Suitable "labels" include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like.

[242] As used herein, "protein" refers to at least two covalently linked amino acids. The term encompasses polypeptides, oligopeptides and peptides. In some embodiments, two or more covalently linked amino acids are linked via a peptide bond. The protein can be composed of naturally occurring amino acids and peptide bonds, for example, when the protein is produced in a recombinant manner using expression systems and host cells. Alternatively, the protein may include synthetic amino acids (eg, homophenylalanine, citrulline, ornithine, and norleucine) or peptidomimetic structures, ie, "peptide or protein analogs," such as peptoids. Peptoids are an illustrative class of peptidomimetics whose side chains are attached to the nitrogen atom of the peptide backbone, rather than to the α-carbons (as they are in amino acids), and have different hydrogen bonding and conformational characteristics compared to peptides (see e.g. , Simon et al (1992) Proc Natl Acad Sci USA 89:9367). In this regard, peptoids may be resistant to proteolysis or other physiological or storage conditions, and effective in penetrating cell membranes. Such synthetic amino acids can be included, in particular, when the antibody is synthesized in vitro using conventional methods widely known in the art. In addition, any combination of peptidomimetic, synthetic and naturally occurring residues/structures can be used. "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The "R group" or "side chain" of an amino acid may be in one of the (L) or (S) configurations. In a specific embodiment, the amino acids are in the (L)- or (S)-configuration.

[243] "Recombinant protein" is a protein obtained using recombinant techniques using any of the techniques and methods known in the art, ie, through the expression of a recombinant nucleic acid. Methods and techniques for obtaining recombinant proteins are widely known in the art.

[244] An "isolated" protein is not accompanied by at least some of the material with which it is normally associated in the natural state, such as at least about 5% or at least about 50% by weight of the total protein in said sample. . It is known that the isolated protein may comprise from 5% to 99.9% by weight of the total protein content, depending on the circumstances. For example, a protein can be produced at a significantly higher concentration through the use of an inducible promoter or a high expression promoter, whereby the protein is produced at higher concentration levels. The definition includes the production of an antibody in a wide variety of host organisms and/or cells that are known in the art.

[245] In the case of amino acid sequences, sequence identity and/or similarity can be determined using standard techniques known in the art, including, without limitation, the local sequence identity algorithm from Smith and Waterman (1981) Adv Appl Math. 2:482, an alignment algorithm for determining sequence identity from Needleman and Wunsch (1970) J Mol Biol. 48:443, similarity search method from Pearson and Lipman (1988) Proc Nat Acad Sci. USA 85:2444, computerized implementations of such algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA), the Best Fit sequence analysis program described by Devereux et al. (1984) Nucl Acid Res. 12:387-95, preferably using default settings, or by visual inspection. Preferably, percent identity is calculated by FastDB based on the following parameters: mismatch penalty - 1; gap penalty - 1; gap lengthening penalty of 0.33 and connection penalty of 30 ("Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149 (1988), Alan R. Liss, Inc. ).

[246] An example of an applicable algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive pairwise alignments. It can also build a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method from Feng & Doolittle (1987) J Mol Evol. 35:351-60; wherein the method is similar to that described in Higgins and Sharp (1989) CABIOS 5:151-3. Applicable parameters in PILEUP include a default gap insertion penalty of 3.00, a default gap extension penalty of 0.10, and estimated end gaps.

[247] Another example of a useful algorithm is the BLAST algorithm described in Altschul et al. (1990) J Mol Biol. 215:403-10; Altschul et al. (1997) Nucl Acid Res. 25:3389-402; and Karin et al. (1993) Proc Natl Acad Sci. USA 90:5873-87. A particularly useful BLAST program is the WU-BLAST-2 program, which was obtained from Altschul et al. (1996) Methods in Enzymology 266:460-80. WU-BLAST-2 applies several search parameters, most of which are set as default. The adjustable parameters are set to the following values: overlap length=l, overlap fraction=0.125, threshold word length (T)=II. The parameters HSP S and HSP S2 are dynamic values and are set by the program independently depending on the composition of a particular sequence and the composition of a particular database, against which the sequence of interest is searched; however, the values can be adjusted to increase sensitivity.

[248] An additional useful algorithm is gapped BLAST reported by Altschul et al. (1997) Nucl Acid Res. 25:3389-402. Gapped BLAST uses the BLOSUM-62 substitution matrix; threshold parameter T set to 9; "two matches" method for triggering extensions without gaps, the penalty for extending gaps by k is 10+k; Xu is set to 16 and Xg is set to 40 for the database search stage and 67 for the output stage of the algorithms. Gapped alignments start at a rate corresponding to approximately 22 bits.

[249] Generally, amino acid homology, similarity, or identity between the proteins disclosed herein and their variants, including target antigen variants (such as HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1 , KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1) and antibody variable domain variants (including individual CDR variants) is at least 80% with the sequences depicted herein, e.g., homology or identity is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, almost 100% or 100%.

[250] Similarly, "percent (%) nucleic acid sequence identity" with respect to the nucleic acid sequence of antibodies and other proteins identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical to nucleotide residues in the coding sequence of an antigen binding protein. The specific method uses the BLASTN module from WU-BLAST-2 set to default parameters with the overlap length and overlap ratio set to 1 and 0.125, respectively.

[251] Since the site or region of introduction of the amino acid sequence variation is predetermined, the mutation itself does not need to be predetermined. For example, to optimize the properties of a mutation at a given site, random mutagenesis can be performed at a target codon or region and expressed CDR variants of antigen binding proteins screened for the optimal combination of desired activity. Techniques for producing mutations that are substitutions at predetermined sites in DNA with a known sequence are widely known, such as mutagenesis with the MI3 primer and PCR mutagenesis.

[252] As used herein, "alkyl" or "alkyl group" means a straight, branched, or cyclic hydrocarbon that is fully saturated. In certain embodiments, the implementation of the alkyl groups may contain 1-8 carbon atoms ("C ₁ -C ₈ alkyl"). In certain embodiments, the implementation of the alkyl groups may contain 1-6 carbon atoms ("C ₁ -C ₆ alkyl"). In certain embodiments, the implementation of the alkyl groups contain 1-3 carbon atoms. In still other embodiments, the implementation of the alkyl groups contain 2-3 carbon atoms, and in other embodiments, the implementation of the alkyl groups contain 1-2 carbon atoms.

[253] As used herein, "alkylalkoxy" means an alkyl group substituted with an alkoxy group. As used herein, the term "alkoxy" refers to an alkyl group, as previously defined, attached to the main carbon chain via an oxygen atom ("alkoxy").

[254] As used herein, "alkylamino" means an alkyl group substituted with an amino group. As used herein, "amino" refers to -NH ₂ , -NH(alkyl) or -N(alkyl)(alkyl).

[255] As used herein, "alkylhydroxy" means an alkyl group substituted with an amino group. As used herein, "hydroxy" or "hydroxyl" refers to -OH.

[256] "Alkylene" refers to a divalent radical of an alkyl group. For example, -CH ₂ -, -CH ₂ CH ₂ -, -CH ₂ CH ₂ CH ₂ -, -CH ₂ CH ₂ CH ₂ CH ₂ -, -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ - and -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ - refers to methylene, ethylene, n-propylene, n-butylene, n-pentylene and n-hexylene, respectively.

[257] As used herein, "carbocycle" includes both aromatic (eg, aryl) and non-aromatic (eg, cycloalkyl) groups. In certain embodiments, the implementation of carbocyclic groups contain 3-10 carbon atoms ("3-10-membered carbocycle"). In certain embodiments, the implementation of carbocyclic groups contain 3-8 carbon atoms ("3-8-membered carbocycle"). In certain embodiments, the implementation of carbocyclic groups contain 3-6 carbon atoms ("3-6-membered carbocycle"). In certain embodiments, the implementation of carbocyclic groups contain 3-5 carbon atoms ("3-5-membered carbocycle").

[258] "Halogen" refers to any halogen radical, such as -F, -Cl, -Br, or -I.

[259] Used in this document, the terms "heterocycle", "heterocyclyl" and "heterocyclic" mean a monocyclic heterocycle, bicyclic heterocycle or tricyclic heterocycle containing at least one heteroatom in the ring.

[260] A monocyclic heterocycle is a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least one heteroatom independently selected from O, N, and S. In some embodiments, the heterocycle is A 3- or 4-membered ring containing one heteroatom selected from O, N, and S. In some embodiments, the heterocycle is a 5-membered ring containing no double bonds or containing one double bond and one, two, or three heteroatoms, selected from O, N, and S. In some embodiments, the heterocycle is a 6-, 7-, or 8-membered ring containing no double bonds, containing one or two double bonds, and one, two, or three heteroatoms selected from O, N and S. Illustrative examples of a monocyclic heterocycle include, without limitation, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, dihydropyranyl (including 3,4-dihydro-2H-pyran-6-yl), 1 ,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl , pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl (including tetrahydro-2H-pyran-4-yl), tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolidinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholinesulfone), thiopyranil and trithianyl.

[261] The bicyclic heterocycles of the present invention may include a monocyclic heterocycle fused to an aryl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle, having a total of 5 -12 atoms in the ring. Examples of bicyclic heterocycles include, without limitation, 3,4-dihydro-2H-pyranyl, 1,3-benzodioxolyl, 1,3-benzodithiolyl, 2,3-dihydro-1,4-benzodioxinyl, 2,3-dihydro-1-benzofuranyl , 2,3-dihydro-1-benzothienyl, 2,3-dihydro-1H-indolyl and 1,2,3,4-tetrahydroquinolinyl.

[262] The terms "heterocycle", "heterocyclyl", and "heterocyclic" encompass heteroaryls. "Heteroaryl" refers to a cyclic moiety containing one or more closed rings with one or more heteroatoms (oxygen, nitrogen, or sulfur) in at least one of the rings, where at least one of the rings is aromatic, and where the ring or rings are independently may be condensed and/or bridged. Examples include, without limitation, phenyl, thiophenyl, triazolyl, pyridinyl, pyrimidinyl, pyridazinyl, and pyrazinyl.

[263] As described herein, the compounds of the present invention may contain "optionally substituted" moieties. In general, the term "substituted", whether preceded by the term "optional" or not, means that one or more hydrogen atoms in the designated moiety have been replaced by a suitable substituent. Unless otherwise indicated, an "optionally substituted group" may have a suitable substituent at each substitutable position of the group, and if more than one position in any given structure can be substituted by more than one substituent selected from the specified group, then at each position the substituent may be either the same or different. The substituent combinations contemplated in the present invention are preferably those that result in stable or chemically feasible compounds.

[264] One of ordinary skill in the art will understand that "substitution" or "substituted" or "absent" includes the implicit condition that such substitution or lack thereof is in accordance with the allowable valency of the atom being replaced and the substituent, that the substitution or its absence results in a stable compound, e.g. which does not undergo spontaneous transformation, such as by rearrangement, cyclization, elimination, etc. For the purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any permitted substituents organic compounds described in this document, which satisfy the valencies of heteroatoms.

[265] "Stable" refers to compounds that are not substantially altered chemically and/or physically when subjected to conditions that enable them to be made, found, and, in certain embodiments, recovered, purified, and used to achieve one or more of the purposes disclosed. in this document. In some embodiments, a stable compound or a chemically feasible compound is a compound that is essentially unchanged during storage at 40° C. or less in the absence of moisture or other reactive conditions for at least a week. In some embodiments, the compounds disclosed herein are stable.

[266] Enantiomers set forth herein may include "enantiomerically pure" isomers that contain essentially one enantiomer, for example, exactly 90%, 92%, 95%, 98%, or 99% or more, or exactly 100% of one enantiomer for a particular asymmetric center or centers. "Asymmetric center" or "chiral center" refers to a tetrahedral carbon atom that contains four different substituents.

[267] The compounds described herein may also contain unnatural proportions of atomic isotopes on one or more of the atoms that make up these compounds. For example, compounds can be radioactively labeled with radioactive isotopes such as, for example, deuterium ( ² H), tritium ( ³ H), carbon-13 ( ¹³ C) or carbon-14 ( ¹⁴ C). It is implied that all isotopic variants of the compounds disclosed in this document, whether they are radioactive or not, are covered within the scope of the present invention. Furthermore, all tautomeric forms of the compounds described herein are intended to be within the scope of the claimed invention.

Antibody drug conjugates

[268] The antibody drug conjugate (ADC) compounds of the present invention include compounds with anticancer activity. In particular, ADC compounds include an antibody or antigen-binding fragment (including its antigen-binding fragment) conjugated (i.e., covalently attached via a linker) to a drug moiety (e.g., a splicing modulator), where the drug moiety, when not conjugated with an antibody or antigen-binding fragment, characterized by a cytotoxic or cytostatic effect. In various embodiments, the drug moiety, when not conjugated to an antibody or antigen-binding moiety, is capable of binding and/or interacting with the SF3b spliceosome complex. In various embodiments, the drug moiety, when not conjugated to an antibody or antigen-binding moiety, is capable of modulating RNA splicing in vitro and/or in vivo. In various embodiments, by targeting RNA splicing, the drug fragments and ADCs disclosed herein are active antiproliferative agents. In various embodiments, the drug fragments and ADCs disclosed herein can target both actively dividing and quiescent cells.

[269] In various embodiments, the present invention is based at least in part on the discovery that certain bioactive splicing modulators can provide improved properties when used in an ADC. While a splicing modulator may exhibit desirably enhanced features (e.g., robust binding of the SF3b spliceosome complex, strong modulation of RNA splicing) when used on its own, in various embodiments, the splicing modulator may exhibit fewer of the same desirably enhanced features when conjugated with an antibody or antigen-binding fragment. Thus, the development and production of an ADC for use as a human therapeutic agent, for example, as an agent for oncotherapy, may require more than identifying an antibody capable of binding to the desired target or targets and attaching to the drug used on its own. yourself for cancer treatment. The binding of an antibody to a drug can have significant effects on the activity of one or both of the antibody and the drug, with effects varying depending on the chosen type of linker and/or drug. Therefore, in some embodiments, the components of the ADC are selected to (i) retain one or more of the therapeutic properties exhibited by the antibody and drug fragments separately, (ii) maintain the specific binding properties of the antibody or antigen-binding fragment; (iii) optimize drug loading and drug to antibody ratios; (iv) provide delivery, such as intracellular delivery, of the drug fragment by stably attaching to the antibody or antigen-binding fragment; (v) keep the ADC stable as an intact conjugate until transport or delivery to the target site; (vi) minimize ADC aggregation before or after administration; (vii) provide a therapeutic effect, such as a cytotoxic effect, of the drug moiety upon cleavage or other release mechanism in the cellular environment; (viii) provide an in vivo anti-cancer treatment efficacy comparable to or superior to that of the antibody and drug fragments alone; (ix) minimize off-target killing by the drug moiety and/or (x) ensure that desired pharmacokinetic and pharmacodynamic properties, formulation capabilities and toxicological/immunological profiles are exhibited. Each of these properties may need to be identified to an improved ADC for therapeutic use (Ab et al. (2015) Mol Cancer Ther. 14:1605-13).

[270] In various embodiments, the ADCs disclosed herein exhibit surprisingly beneficial properties in some or each of the categories listed above. For example, in some embodiments, the ADC constructs disclosed herein exhibit unexpectedly favorable drug loading, aggregation, and/or stability profiles and/or retain antibody binding function, drug activity, and/or improved non-specific killing while reducing off-target killing. compared to ADCs containing an alternative linker and/or drug fragment (eg, an alternative splicing modulator). In some embodiments, the ADC constructs disclosed herein demonstrate superior stability, potency, efficacy, or other effect (measured in vivo or in vitro) compared to ADCs using an alternative linker and/or drug moiety (e.g., alternative splicing modulator). In some embodiments, the ADC constructs disclosed herein exhibit in vivo treatment efficacy when administered as a single dose. In some embodiments, the ADC constructs disclosed herein are surprisingly stable compared to ADCs using an alternative linker and/or drug moiety (eg, an alternative splicing modulator).

[271] The ADC compounds of the present invention can selectively deliver an effective dose of a cytotoxic or cytostatic agent to cancer cells or tumor tissue. The disclosed ADCs have been found to have potent cytotoxic and/or cytostatic activity against cells expressing the respective target antigen (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, STEAP1). In some embodiments, the implementation of the cytotoxic and/or cytostatic activity of ADC depends on the expression of the target antigen in the cell. In some embodiments, the disclosed ADCs are particularly effective in killing cancer cells expressing the target antigen while minimizing off-target killing. In some embodiments, the disclosed ADCs do not exhibit a cytotoxic and/or cytostatic effect on cancer cells that do not express the target antigen.

[272] Exemplary HER2-expressing cancers include, but are not limited to, breast cancer, gastric cancer, bladder cancer, urothelial cell carcinoma, esophageal cancer, lung cancer (e.g. lung adenocarcinoma), uterine cancer (e.g. uterine endometrial serous carcinoma) , salivary duct carcinoma, cervical cancer, endometrial cancer, and ovarian cancer (English et al. (2013) Mol Diagn Ther. 17:85-99).

[273] Exemplary CD138-expressing cancers include, but are not limited to, hilar cancer (eg, lung cancer, mesothelioma), skin cancer (eg, basal cell carcinoma, squamous cell carcinoma), head and neck cancer (eg, larynx, hypopharynx, nasopharynx) , breast cancer, urogenital cancer (eg, cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, urothelial cancer), hematologic malignancies (eg, myeloma such as multiple myeloma, Hodgkin's lymphoma), and cancer thyroid gland (Szatmári et al. (2015) Dis Markers 2015:796052).

[274] Illustrative cancers expressing EPHA2 include breast cancer, brain cancer, ovarian cancer, bladder cancer, pancreatic cancer, esophageal cancer, lung cancer, prostate cancer, melanoma, esophageal cancer, and gastric cancer (Tandon et al (2011) Expert Opin Ther Targets 15(1):31-51).

[275] In some embodiments, cleavage of the ADC releases the splicing modulator from the antibody or antigen-binding fragment and the linker. In some embodiments, a linker and/or splicing modulator is designed to facilitate non-specific killing (killing adjacent cells). In some embodiments, a linker and/or splicing modulator is designed to facilitate non-specific killing by cleavage after internalization into a cell and diffusion of the linker-drug moiety and/or drug moiety itself into neighboring cells. In some embodiments, the implementation of the linker promotes internalization into the cell. In some embodiments, a linker is designed to minimize extracellular degradation and thereby reduce toxicity to non-target tissue (e.g., non-cancerous tissue), while still retaining ADC binding to the target tissue and non-specific killing of cancer tissue that does not express the antigen, at which is targeted by an antibody or antigen-binding fragment of the ADC, but which surrounds the cancer target tissue expressing the antigen. In some embodiments, a drug moiety or a drug moiety catabolite derived from ADC cleavage is designed to facilitate uptake by target cells or neighboring cells (i.e., capable of entering the cell). Such drug and catabolite moieties may be referred to herein as "non-specifically active", while drug or catabolite moieties with reduced cellular penetration may be referred to as "non-specifically inactive".

[276] In some embodiments, the disclosed ADCs also exhibit non-specific killing activity but low off-target cytotoxicity. Without being limited by theory, non-specific killing activity in ADC may be particularly useful if its entry into a solid tumor is limited and/or expression of the target antigen among tumor cells is heterogeneous. In some embodiments, an ADC containing a cleavable linker is particularly effective in non-specific killing and/or exhibits increased non-specific killing activity compared to a comparable treatment with an ADC containing a non-cleavable linker. In some embodiments, the ADCs disclosed herein exhibit increased solubility and penetration into target cells compared to the drug moieties themselves. In some embodiments, the ADCs disclosed herein exhibit increased cytotoxicity compared to that of the drug moiety itself. In some embodiments, the ADCs disclosed herein use drug moieties that exhibit lower cytotoxicity when evaluated as a single drug, while surprisingly superior to ADCs containing other drug moieties, which are characterized by higher cytotoxicity when evaluated as a single drug. In some embodiments, cleavage and release of the splicing modulator improves the cytotoxicity of the ADC relative to comparable treatment with an ADC containing a non-cleavable linker. In other embodiments, cleavage and release of the splicing modulator is not required for the ADC to have the desired biological activity. In some embodiments, an ADC containing a non-cleavable linker with an extended spacer (e.g., ADL12) provides the same or similar cytotoxicity relative to a comparable treatment with an ADC containing a cleavable linker (e.g., ADL1, ADL5) and unexpectedly superior cytotoxicity relative to a comparable treatment. with an ADC containing a shorter non-cleavable linker. In some embodiments, an ADC containing a non-cleavable linker with an extended spacer without a carbonyl group (e.g., ADL12) provides the same or similar cytotoxicity relative to comparable treatment with an ADC containing a cleavable linker (e.g., ADL1, ADL5) and unexpectedly superior cytotoxicity relatively comparable ADC treatment containing a non-cleavable linker with a spacer of the same or similar length with a carbonyl group (eg ADL10). In some embodiments, the removal of the carbonyl group from a non-cleavable MC linker (e.g., ADL12) may result in an increase in cytotoxicity greater than 50-fold, greater than 75-fold, greater than 100-fold, greater than 150-fold, or greater than 200-fold relative to a comparative ADC treatment containing unmodified non-cleavable MC linker (eg ADL10). In some embodiments, removing the carbonyl group from a non-cleavable MC linker (e.g., ADL12) and increasing the length of the spacer (e.g., adding at least one spacer unit) can result in an increase in cytotoxicity of over 50-fold, over 75-fold, over 100-fold, more than 150 times or more than 200 times relative to comparative treatment with an ADC containing an unmodified, non-cleavable MC linker (eg, ADL10).

[277] Provided herein are ADC compounds comprising an antibody or antigen-binding fragment (Ab) thereof that targets a tumor cell, a drug fragment that is a splicing modulator (D), and a linker fragment (L) that covalently attaches Ab to D. In certain aspects, an antibody or antigen-binding fragment is capable of binding to a tumor-associated antigen (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4 , STEAP1) with high specificity and high affinity. In certain embodiments, the antibody or antigen-binding fragment is internalized into the target cell after binding, for example, into a degradation compartment in the cell. In various embodiments, ADCs that are internalized after binding to a target cell, undergo degradation, and release a splicing modulator drug moiety can be used to kill cancer cells. The splicing modulator drug moiety may be released from the antibody and/or the ADC linker moiety by enzymatic action, hydrolysis, oxidation, or any other mechanism.

[278] An exemplary ADC has formula (I):

Ab-(LD) _p (I),

where Ab=antibody or antigen-binding fragment, L=linker fragment, D=splicing modulator drug fragment, and p =number of splicing modulator drug fragments per antibody or antigen-binding fragment.

[279] In some preferred embodiments, the drug-targeting fragment for use in the described ADCs and compositions is an antibody or antigen-binding fragment. Other exemplary drug-targeting moieties for use in the described ADCs and compositions are also provided and exemplified herein. In some embodiments, the drug-targeting fragment may be any of a variety of cell-binding agents and non-antibody backbones. In some embodiments, the drug-targeting moiety is a cell-binding agent. As used herein, the term "cell-binding agent" refers to any agent that is capable of binding to an animal (e.g., human) cell and delivering a drug moiety (e.g., the splicing modulator drug moiety disclosed in this document). The term encompasses exemplary antibodies and antigen-binding fragments disclosed herein (eg, monoclonal antibodies and fragments thereof such as Fab and scFV). The term further encompasses exemplary cell-binding agents such as DARPin, duotel, bicyclic peptides, nanobodies, centirins, MSH (melanocyte-stimulating hormone), receptor-Fc fusion molecules, T-cell receptor structures, steroid hormones such as androgens and estrogens, growth factors, colony stimulating factors such as EGF, and other non-antibody backbones. In various embodiments, non-antibody backbones can be broadly divided into two structural classes, namely domain size compounds (about 6-20 kDa) and limited peptides (about 2-4 kDa). Exemplary backbones having a domain size include, but are not limited to, affitels, affilins, anticalins, atrimers, DARPins, FN3 backbones (e.g., adnectins and centirins), finomers, Kunitz domains, pronectins, O-bodies, and receptor-Fc fusion proteins, wherein exemplary limited peptides include avimers, bicyclic peptides, and Cys knots. In some embodiments, the drug-targeting moiety used in the described ADCs and compositions is selected from affitele, affilin, anticalin, atrimer, DARPin, FN3 backbone such as adnectin or centirin, finomer, Kunitz domain, pronectin, O-body, avimer , a bicyclic peptide, and a Cys node. In some embodiments, the drug-targeting moiety used in the described ADCs and compositions is a receptor-Fc fusion protein, eg, a chimeric HER2-Fc fusion protein. For a review of non-antibody backbones, see, for example, Vazquez-Lombardi et al. (2015) Drug Dis Today 20(10):1271-83.

Antibodies

[280] An antibody or antigen-binding fragment (Ab) of formula (I) includes within its scope any antibody or antigen-binding fragment that specifically binds to a target antigen on a cancer cell. An antibody or antigen-binding fragment can bind to a target antigen with a dissociation constant (K _D ) of ≦1 mM, ≦100 nM, or ≦10 nM, or any value in between, as measured, for example, by a ^BIAcore® assay. In certain embodiments, the implementation of K _D is from 1 pM to 500 pM. In some embodiments, the K _D is 500 pM to 1 μM, 1 μM to 100 nM, or 100 mM to 10 nM.

[281] In some embodiments, the antibody or antigen-binding fragment is a four-chain antibody (also referred to as an immunoglobulin or full-length or intact antibody) containing two heavy chains and two light chains. In some embodiments, the antibody or antigen-binding fragment is a double-chain semi-antibody (one light chain and one heavy chain) or an immunoglobulin antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is an antigen-binding fragment of an immunoglobulin that retains the ability to bind a cancer target antigen and/or confer immunoglobulin function.

[282] In some embodiments, the antibody or antigennegative fragment is an antibody or antigennegative fragment thereof. In some embodiments, the antibody or antigen-binding fragment is an internalizing antibody or an internalizing antigen-binding fragment thereof. In some embodiments, an internalizing antibody, or an internalizing antigen-binding fragment thereof, binds to a cancer target antigen expressed on a cell surface and enters the cell after binding. In some embodiments, the splice modulator drug fragment in the ADC is released from the antibody or antigen-binding fragment in the ADC after the ADC enters and resides in a cell expressing the cancer target antigen (i.e., after The ADC has been internalized), for example, by cleavage, by destruction of the antibody or antigen-binding fragment, or by any other suitable release mechanism.

[283] Amino acid sequences of exemplary antibodies of the present invention are shown in Tables 2-4.

Table 1. Antibodies

mAb Type Target Trastuzumab (AB185) humanized HER2/NEU B-B4 (AB205) murine CD138 (syndecan-1) 1C1 (AB206) humanized EPHA2

Table 2. Amino acid sequences of mAb variable regions

Table 3. Amino acid sequences of CDR mAb

mAb IgG chain SEQID NO Amino acid sequence Trastuzumab
(AB185) HCDR1 1 GFNIKDTYIH Trastuzumab
(AB185) HCDR2 2 RIYPTNGYTRYADSVKG Trastuzumab
(AB185) HDDR3 3 WGGDGFYAMDV Trastuzumab
(AB185) LCDR1 4 RASQDVNTAVAW Trastuzumab
(AB185) LCDR2 5 SASFLES Trastuzumab
(AB185) LCDR3 6 QQHYTTPPPT B-B4
(AB205) HCDR1 7 NYWIE B-B4
(AB205) HCDR2 8 ILPGTGRTIYNEKFKGKA B-B4
(AB205) HDDR3 9 RDYYGNFYYAMDY B-B4
(AB205) LCDR1 10 ASQGINNYLN B-B4
(AB205) LCDR2 eleven TSTLQS B-B4
(AB205) LCDR3 12 QQYSKLPRT 1C1
(AB206) HCDR1 13 HYMMA 1C1
(AB206) HCDR2 14 RIGPSGGPTHYADSVKG 1C1
(AB206) HDDR3 15 YDSGYDYVAVAGPAE-YFQH 1C1
(AB206) LCDR1 16 RASWSISTWLA 1C1
(AB206) LCDR2 17 KASNLHT 1C1
(AB206) LCDR3 18 QQYNSYS-RT

Table 4. Amino acid sequences of the Ig chains of the full-length mAb

mAb IgG chain Class SEQID NO Amino acid sequence Trastuzumab
(AB185) heavy chain IgG1 25 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Trastuzumab
(AB185) light chain kappa chain 26 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC B-B4
(AB205) heavy chain IgG2a 27 QVQLQQSGSELMMPGASVKISCKATGYTFSNYWIQRPGHGLEWIGEILPGTGRTIYNEKFKGKATFTADISSNTVQMQLSSLTSEDSAVYYCARRDYYGNFYYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSET VTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNT QPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG B-B4
(AB205) light chain kappa chain 28 DIQMTQSTSSLSASLGDRVTISCSASQGINNYLNWYQQKPDGTVELLIYYTSTLQSGVPSRFSGSGSGTDYSLTISNLEPEDIGTYYCQQYSKLPRTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSY TCEATHKTSTSPIVKSFNRNEC 1C1
(AB206) heavy chain IgG1 29 EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYMMAWVRQAPGKGLEWVSRIGPSGGPTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGYDSGYDYVAVAGPAEYFQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 1C1
(AB206) light chain kappa chain thirty DIQMTQSPSSLSASVGDRVTITCRASQSISTWLAWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTEFSLTISGLQPDDFATYYCQQYNSYSRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC

Table 5 Exemplary target antigen amino acid sequences

Antigen SEQID NO Amino acid sequence HER2/NEU 31 MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMC KGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYISA WPDSLPDLSVFQNLQVIRGRILHAYSLTLQGLGISWLGLRSLRELGSGLALIHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEG ACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQ DLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQ QGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTF KGTPTAENPEYLGLDVPV CD138 32 MRRAALWLWLCALALSLQPALPQIVATNLPPEDQDGSGDDSDNFSGSGAGALQDITLSQQTPSTWKDTQLLTAIPTSPEPTGLEATAASTSTLPAGEGPKEGEAVVLPEVEPGLTAREQEATPRPRETTQLPTTHLASTTTATTAQEPATSHPHRDMQPGHHETSTPAGPSQADLHTPHTEDGGPSATERAAEDGASSQLPAAEGSGEQDFTFETSG ENTAVVAVEPDRRNQSPVDQGATGASQGLLDRKEVLGGVIAGGLVGLIFAVCLVGFMLYRMKKKDEGSYSLEEPKQANGGAYQKPTKQEEFYA EPHA2 33 MELQAARACFALLWGCALAAAAAAAQGKEVVLLDFAAAGGELGWLTHPYGKGWDLMQNIMNDMPIYMYSVCNVMSGDQDNWLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQKRLFTKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRVY YKKCPELLQGLAHFPETIAGSDAPSLATVAGTCVDHAVVPPGGEEPRMHCAVDGEWLVPIGQCLCQAGYEKVEDACQACSPGFFKFEASESPCLECPEHTLPSPEGATSCECEEGFFRAPQDPASMPCTRPPSAPHYLTAVGMGAKVELRWTPPQDSGGREDIVYSVTCEQCWPESGECGPCEASVRYSEPPPHGLTRTSVTVSDLEPHMNYTFTVEAR NGVSGLVTSRSFRTASVSINQTEPPKVRLEGRSTTSLSVSWSIPPPQQSRVWKYEVTYRKKGDSNSYNVRRTEGFSVTLDDLAPDTTYLVQVQALTQEGQGAGSKVHEFQTLSPEGSGNLAVIGGVAVGVVLLLVLAGVFIHRRRKNQRARQSPEDVYFSKSEQLKPLKTYVDPHTYEDPNQAVLKFTTEIHPS CVTRQKVIGAGEFGEVYKGMLKTSSGKKEVPVAIKTLKAGYTEKQRVDFLGEAGIMGQFSHHNIIRLEGVISKYKPMMIITEYMENGALDKFLREKDGEFSVLQLVGMLRGIAAGMKYLANMNYVHRDLAARNILVNSNLVCKVSDFGLSRVLEDDPEATYTTSGGKIPIRWTAPEAISYRKFTSASDVWSFGIVMWEVMTYGERPYW ELSNHEVMKAINDGFRLPTPMDCPSAIYQLMMQCWQQERARRPKFADIVSILDKLIRAPDSLKTLADFDPRVSIRLPSTSGSEGVPFRTVSEWLESIKMQQYTEHFMAAGYTAIEKVVQMTNDDIKRIGVRLPGHQKRIAYSLLGLKDQVNTVGIPI MSLN 43 MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLD QDQQEAARAALQGGGPPYGPPSTWSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQAP RRPLPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGTPCLL GPGPVLTVLALLLASTLA FOLH1 44 MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKI VIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPTGNFSTQKVGMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQ SGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDY AVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA CDH6 45 MRTYRYFLLLFWVGQPYPTLSTPLSKRTSGFPAKKRALELSGNSKNELNRSKRSWMWNQFFLLEEYTGSDYQYVGKLHSDQDRGDGSLKYILSGDGAGDLFIINENTGDIQATKRLDREEKPVYILRAQAINRRTGRPVEPESEFIIKIHDINDNEPIFTKEVYTATVPEMSDVGTFVVQVTATDADDPTYGNSAKVV YSILQGQPYFSVESETGIIKTALLNMDRENREQYQVVIQAKDMGGQMGGLSGTTTVNITLTDVNDNPPRFPQSTYQFKTPESSPPGTPIGRIKASDADVGENAEIEYSITDGEGLDMFDVITDQETQEGIITVKKLLDFEKKKVYTLKVEASNPYVEPRFLYLGPFKDSATVRIVVEDVDEPPVFSKLAYILQIREDAQINTTI GSVTAQDPDAARNPVKYSVDRHTDMDRIFNIDSGNGSIFTSKLLDRETLLWHNITVIATEINNPKQSSRVPLYIKVLDVNDNAPEFAEFYETFVCEKAKADQLIQTLHAVDKDDPYSGHQFSFSLAPEAASGSNFTIQDNKDNTAGILTRKNGYNRHEMSTYLLPVVISDNDYPVQSSTGTVTVRVCACDHHGNMQSCHAEALIHPTGL STGALVAILLCIVILLVTVVLFAALRRQRKKEPLIISKEDIRDNIVSYNDEGGGEEDTQAFDIGTLRNPEAIEDNKLRRDIVPEALFLPRRTPTARDNTDVRDFINQRLKENDTDPTAPPYDSLATYAYEGTGSVADSLSSLESVTTDADQDYDYLSDWGPRFKKLADMYGGVDSDKDS CEACAM5 46 MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLS NGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNKLSVDHSDPV ILNVLYGPDDTPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCH SASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAGATVGIMIGVLVGVALI CFC1B 47 MTWRHHVRLLFTVSLALQIINLGNSYQREKHNGGREEVTKVATQKHRQSPLNWTSSHFGEVTGSAEGWGPEEPLPYSWAFGEGASARPRCCRNGGTCVLGSFCVCPAHFTGRYCEHDQRRSECGALEHGAWTLRACHLCRCIFGALHCLPLQTPDRCDPKDFLASHAHGPSAGGAPSLLLLPCALLHRLLRPDAPAHPR SLVPSVLQRERRPCGRPGGLGHRL ENPP3 48 MESTLTLATEQPVKKNTLKKYKIACIVLLALLVIMSLGLGLGLGLRKLEKQGSCRKKCFDASFRGLENCRCDVACKDRGDCCWDFEDTCVESTRIWMCNKFRCGETRLEASLCSCSDDCLQRKDCCADYKSVCQGETSWLEENCDTAQQSQCPEGFDLPPVILFSMDGFRAEYLYTWDTLMPNINKLKTCGIHSKYMRAMYPTKTFPNHYTIVTGLY PESHGIIDNNMYDVNLNKNFSLSSKEQNNPAWWHGQPMWLTAMYQGLKAATYFWPGSEVAINGSFPSIYMPYNGSVPFEERISTLLKWLDLPKAERPRFYTMYFEEPDSSGHAGGPVSARVIKALQVVDHAFGMLMEGLKQRNLHNCVNIILLADHGMDQTYCNKMEYMTDYFPRINFFYMYEGPAPRIRAHNIPHDFFSFNSEE IVRNLSCRKPDQHFKPYLTPDLPKRLHYAKNVRIDKVHLFVDQQWLAVRSKSNTNCGGGNHGYNNEFRSMEAIFLAHGPSFKEKTEVEPFENIEVYNLMCDLLRIQPAPNNGTHGSLNHLLKVPFYEPSHAEEVSKFSVCGFANPLPTESLDCFCPHLQNSTQLEQVNQMLNLTQEEITATVKVNLPFGRPRVLQK NVDHCLLYHREYVSGFGKAMRMPMWSSYTVPQLGDTSPLPPTVPDCLRADVRVPPSESQKCSFYLADKNITHGFLYPPASNRTSDSQYDALITSNLVPMYEEFRKMWDYFHSVLLIKHATERNGVNVVSGPIFDYNYDGHFDAPDEITKHLANTDVPIPTHYFVVLTSCKNKSHTPENCPGWLDVLPFIIPHRPTNVESCPEGK PEALWVEERFTAHIARVRDVELLTGLDFYQDKVQPVSEILQLKTYLPTFETTI FOLR1 49 MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTP TVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWAAWPFLLSLALMLLWLLS HAVCR1 50 MHPQVVILSLILHLADSVAGSVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTCQNGIVWTNGTHVTYRKDTRYKLLGDLSRRDVSLTIENTAVSDSGVYCCRVEHRGWFNDMKITVSLEIVPPKVTTTPIVTTVPTVTTVRTSTTVPTTTTVPMTTVPTTTVPTTMSIPTTTTVLTTMTVSTTTSVPTTTSIPTTTSVPVTTTVSTFVPPM PLPRQNHEPVATSPSSPQPAETHPTTLQGAIRREPTSSPLYSYTTDGNDTVTESSDGLWNNNQTQLFLEHSLLTANTTKGIYAGVCISVLVLLALLGVIIAKKYFFKKEVQQLSVSFSSLQIKALQNAVEKEVQAEDNIYIENSLYATD KIT 51 MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRLCLHCSVDQEGKSVELSEKFILK VRPAFKAVPVVSKASYLLREGEEFTVTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVY VNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPRNRLSFGKTLGAFGKVVE ATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKN DSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHDDV MET 52 MKAPAVLAPGILVLLFTLVQRSNGECKEALAKSEMNVNMKYQLPNFTAETPIQNVILHEHHIFLGATNYIYVLNEEDLQKVAEYKTGPVLEHPDCFPCQDCSSKANLSGGVWKDNINMALVVDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDCVVSALGAKVLSSVK DRFINFFVGNTINSSYFPDHPLHSISVRRLKETKDGFMFLTDQSYIDVLPEFRDSYPIKYVHAFESNFIYFLTVQRETLDAQTFHTRIIRFCSINSGLHSYMEMPLECILTEKRKKRSTKKEVFNILQAAYVSKPGAQLARQIGASLNDDILFGVFAQSKPDSAEPMDRSAMCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFNRTL LRNSSGCEARRDEYRTEFTTALQRVDLFMGQFSEVLLTSISTFIKGDLTIANLGTSEGRFMQVVVSRSGPSTPHVNFLLDSHPVSPEVIVEHTLNQNGYTLVITGKKITKIPLNGLGCRHFQSCSQCLSAPPFVQCGWCHDKCVRSEECLSGTWTQQICLPAIYKVFPNSAPLEGGTRLTICGWDFGFRRNNKFDLKKTRV LLGNESCTLTLSESTMNTLKCTVGPAMNKHFNMSIIISNGHGTTQYSTFSYVDPVITSISPKYGPMAGGTLLTLTGNYLNSGNSRHISIGGKTCTLKSVSNSILECYTPAQTISTEFAVKLKIDLANRETSIFSYREDPIVYEIHPTKSFISGGSTITGVGKNLNSVSVPRMVINVHEAGRNNFTVACQHRSNSEIIICCTTPSLQQLN LQLPLKTKAFFMLDGILSKYFDLIYVHNPVFKPFEKPVMISMGNENVLEIKGNDIDPEAVKGEVLKVGNKSCENIHLHSEAVLCTVPNDLLKLNSELNIEWKQAISSTVLGKVIVQPDQNFTGLIAGVVSISTALLLLLFLWLKKRKQIKDLGSELVRYDARVHTPHLDRLVSARSVSPTTEMVSNESVDYRATFPEDQ FPNSSQNGSCRQVQYPLTDMSPILTSGDSDISSPLLQNTVHIDLLSALNPELVQAVQHVVIGPSSLIVHFNEVIGRGHFGCVYHGTLLDNDGKKIHCAVKSLNRITDIGEVSQFLTEGIIMKDFSHPNVLSLLGICLRSEGSPLVVLPYMKHGDLRNFIRNETHNPTVKDLIGFGLQVAKGMKYLASKKFVHRDLAARNCMLDEK FTVKVADFGLARDMYDKEYYSVHNKTGAKLPVKWMALESLQTQKFTTKSDVWSFGVLLWELMTRGAPPYPDVNTFDITVYLLQGRRLLQPEYCPDPLYEVMLKCWHPKAEMRPSFSELVSRISAIFSTFIGEHYVHVNATYVNVKCVAPYPSLLSSEDNADDEVDTRPASFWETS MUC16 53 MLKPSGLPGSSSPTRSLMTGSRSTKATPEMDSGLTGATLSPKTSTGAIVVTEHTLPFTSPDKTLASPTSSVVGRTTQSLGVMSSALPESTSRGMTHSEQRTSPSLSPQVNGTPSRNYPATSMVSGLSSPRTRTSSTEGNFTKEASTYTLTVETTSGPVTEKYTVPTETSTTEGDSTETPWDTRYIPVKITSPMKTFADSTASKENAPVSMTPAETTVTDSHTPGR TNPSFGTLYSSFLDLSPKGTPNSRGETSLELILSTTGYPFSSPEPGSAGHSRISTSAPLSSSASVLDNKISETSIFSGQSLTSPLSPGVPEARASTMPNSAIPFSMTLSNAETSAERVRSTISSLGTPSISTKQTAETILTFHAFAETMDIPSTHIAKTLASEWLGSPGTLGGTSTSALTTTSPSTTLVSEETNTHHSTSGKETEGTLNTSMTPLETSAPGEESEMTA TLVPTLGFTTLDSKIRSPSQVSSSHPTRELRTTGSTSGRQSSSTAAHGSSDILRATTSSTSKASSWTSESTAQQQFSEPQHTQWVETSPSMKTERPPASTSVAAPITTSVPSVVSGFTTLKTSSTKGIWLEETSADTLIGESTAGPTTHQFAVPTGISMTGGSSTRGSQGTTHLLTRATASSETSADLTLATNGVPVSVSPAVSKTAAGSSPPGGTKPSYTMVSS VIPETSSLQSSAFREGTSLGLTPLNTRHPFSSPEPDSAGHTKISTSIPLLSSASVLEDKVSATSTFSHHKATSSITTGTPEISTKTKPSSAVLSSMTLSNAATSPERVRNATSPLTHPSPSGEETAGSVLTLSTSAETTDSPNIHPTGTLTSESSESPSTLSLPSVSGVKTTFSSSTPSTHLFTSGEETEETSNPSVSQPETSVSRVRTTLASTSVPTPVFPTMDT WPTRSAQFSSSHLVSELRATSSTSVTNSTGSALPKISHLTGTATMSQTNRDTFNDSAAPQSTTWPETSPRFKTGLPSATTTVSTSATSLSATVMVSKFTSPATSSMEATSIREPSTTILTTETTTNGPGSMAVASTNIPIGKGYITEGRLDTSHLPIGTTASSETSMDFTMAKESVSMSVSPSQSMDAAGSSTPGRTSQFVDTFSDDVYHLTSREITIPRDGTSSAL TPQMTATHPPSPDPGSARSTWLGILSSSPSSPTPKVTMSSTFSTQRVTTSMIMDTVETSRWNMPNLPSTTSLTPSNIPTSGAIGKSTLVPLDTPSPATSLEASEGGLPTLSTYPESTNTPSIHLGAHASSESPSTIKLTMASVVKPGSYTPLTFPSIETHIHVSTARMAYSSGSSPEMTAPGETNTGSTWDPTTYITTTDPKDTSSAQVSTPHSVRTLRTTE NHPKTESATPAAYSGSPKISSSPNLTSPATKAWTITDTTEHSTQLHYTKLAEKSSGFETQSAPGPVSVVIPTSPTIGSSTLELTSDVPGEPLVLAPSEQTTITLPMATWLSTSLTEEMASTDLDISSSPMSTFAIFPPMSTPSHELSKSEADTSAIRNTDSTTLDQHLGIRSLGRTGDLTTVPITPLTTTWTSVIEHSTQAQDTLSATMSPTHVTQSLK DQTSIPASASSPSHLTEVYPELGTQGRSSSEATTFWKPSTDTLSREIETGPTNIQSTPPMDNTTTGSSSSGVTLGIAHLPIGTSSPAETSTNMALERSSTATVSMAGTMGLLVTSAPGRSISQSLGRVSSVLSESTTEGVTDSSKGSSPRLNTQGNTALSSSLEPSYAEGSQMSTSIPLTSSPTTPDVEFIGGSTFWTKEVTTVMTSDISKSSARTESSSATLMSTAL GSTENTGKEKLRTASMDLPSPTPSMEVTPWISLTLSNAPNTTDSLDLSHGVHTSSAGTLATDRSLNTGVTRASRLENGSDTSSKSLSMGNSTHTSMTYTEKSEVSSSIHPRPETSAPGAETTLTSTPGNRAISLTLPFSSIPVEEVISTGITSGPDINSAPMTHSPITPPTIVWTSTGTIEQSTQPLHAVSSEKVSVQTQSTPYVNSVAVSASPTHENSSGSSTS SPYSSASLESLDSTISRRNAITSWLWDLTTSLPTTTWPSTSLSEALSSGHSGVSNPSSTTTEFPLFSAASTSAAKQRNPETETHGPQNTAASTLNTDASSVTGLSETPVGASISSEVPLPMAITSRSDVSGLTSESTANPSLGTASSAGTKLTRTISLPTSESLVSFRMNKDPWTVSIPLGSHPTTNTETSIPVNSAGPPGLSTVASDVIDTPSDGAESIPTVSFSPSPDTEVTT ISHFPEKTTHSFRTISSLTHELTSRVTPIPGDWMSSAMSTKPTGASPSITLGERRTITSAAPTTSPIVLTASFTETSTVSLDNETTVKTSDILDARKTNELPSDSSSSSDLINTSIASSTMDVTKTASISPTSISGMTASSSPSLFSSDRPQVPTSTTETNTATSPSVSSNTYSLDGGSNVGGTPSTLPPFTITHPVETSSALLAWSRPVRTFSTMVSTDTASGENPTSSNSV VTSVPAPGTWTSVGSTTDLPAMGFLKTSPAGEAHSLLASTIEPATAFTPHLSAAVVTGSATSEASSLLTTSESKAIHSSPQTPTTPTSGANWETSATPESLLVVTETSDTTLTSKILVTDTILFSTVSTPPSKFPSTGTLSGASFPTLLPDTPAIPLTATEPTSSLATSFDSTPLVTIASDSLGTVPETLTMSETSNGDALVLKTVSNPDRSIPGITIQGVTESPLHPS STSPSKIVAPRNTTYEGSITVALSTLPAGTTGSLVFSQSSENSETTALVDSSAGLERASVMPLTTGSQGMASSGGIRSGSTHSTGTKTFSSLPLTMNPGEVTAMSEITTNRLTATQSTAPKGIPVKPTSAESGLLTPVSASSSPSKAFASLTTAPPTWGIPQSTLTFEFSEVPSLDTKSASLPTPGQSLNTIPDSDASTASSSLSKSPEKNPRARMMTSTKAISASSFQSTGFTE TPEGSASPSMAGHEPRVPTSGTGDPRYASESMSYPDPSKASSAMTSTSLASKLTTLFSTGQAARSGSSSSPISLSTEKETSFLSPTASTSRKTSLFLGPSMARQPNILVHLQTSALTLSPTSTLNMSQEEPPELTSSQTIAEEEGTTAETQTLTFTPSETPTSLLPVSSPTEPTARRKSSPETWASSISVPAKTSLVETTDGTLVTTIKMSSQAAQGNSTWPAPAEET GSSPAGTSPGSPEMSTTLKIMSSKEPSISPEIRSTVRNSPWKTPETTVPMETTVEPVTLQSTALGSGSTSISHLPTGTTSPTKSPTENMLATERVSLSPSPPEAWTNLYSGTPGGTRQSLATMSSVSLESPTARSITGTGQQSSPELVSKTTGMEFSMWHGSTGGTTGDTHVSLSTSSNILEDPVTSPNSVSSLTDKSKHKTETWVSTTAIPSTVLNNKIMAAE QQTSRSVDEAYSSTSSWSDQTSGSDITLGASPDVTNTLYITSTAQTTSLVSLPSGDQGITSLTNPSGGKTSSASSVTSPSIGLETLRANVSAVKSDIAPTAGHLSQTSSPAEVSILDVTTAPTPGISTTITTMGTNSISTTTPNPEVGMSTMDSTPATERRTTSTEHPSTWSSTAASDSWTVTDMTSNLKVARSPGTISTMHTTSFLASSTELDSMSTPHGRITVIG TSLVTPSSDASAVKTETSTSERTLSPSDTTASTPISTFSRVQRMSISVPDILSTSWTPSSTEAEDVPVSMVSTDHASTKTDPNTPLSTFLFDSLSTLDWDTGRSLSSATATTSAPQGATTPQELTLETMISPATSQLPFSIGHITSAVTPAAMARSSGVTFSRPDPTSKKAEQTSTQLPTTTSAHPGQVPRSAATTLDVIPHTAKTPDATFQRQGQTALTTEARAT SDSWNEKEKSTPSAPWITEMMNSVSEDTIKEVTSSSSVLRTLNTLDINLESGTTSSPSWKSSPYERIAPSESTTDKEAIHPSTNTVETTGWVTSSEHASHSTIPAHSASSKLTSPVVTTSTREQAIVSMSTTTWPESTRARTEPNSFLTIELRDVSPYMDTSSTTQTSIISSPGSTAITKGPRTEITSSKRISSSFLAQSMRSSDSPSEAITRLSNFPAMTESGGMIL AMQTSPPGATSLSAPTLDTSATASWTGTPLATTQRFTYSEKTTLFSKGPEDTSQPSPPSVEETSSSSLVPIHATTSPSNILLTSQGHSPSSTPPVTSVFLSETSGLGKTTDMSRISLEPGTSLPPNLSSTAGEALSTYEASRDTKAIHHSADTAVTNMEATSSEYSPIPGHTKPSKATSPLVTSHIMGDITSSTSVFGSSETTEIETVSSVNQGLQERSTSQVA SSATETSTVITHVSSGDATTHVTKTQATFSSGTSISSPHQFITSTNTFTDVSTNPSTSLIMTESSGVTITTQTGPTGAATQGPYLLDTSTMPYLTETPLAVTPDFMQSEKTTLISKGPKDVSWTSPPSVAETSYPSSLTPFLVTTIPPATSTLQGQHTSSPVSATSVLTSGLVKTTDMLNTSMEPVTNSPQNLNNPSNEILATLAATTDIETIHPS INKAVTNMGTASSAHVLHSTLPVSSEPSTATSPMVPASSMGDALASISIPGSETTDIEGEPTSSLTAGRKENSTLQEMNSTTESNIILSNVSVGAITEATKMEVPSFDATFIPTPAQSTKFPDIFSVASSRLNSPPMTISTHMTTTQTGSSGATSKIPLALDTSTLETSAGTPSVVTEGFAHSKITTAMNNDVKDVSQTNPPFQDEASSPSSQAPVLVTTLPSSVA FTPQWHSTSSPVSMSSVLTSSLVKTAGKVDTSLETVTSSPQSMSNTLDDISVTSAATTDIETTHPSINTVVTNVGTTGSAFESHSTVSAYPEPSKVTSPNVTTSTMEDTTISRSIPKSSKTTRTETETTSSLTPKLRETSISQEITSSTETSTVPYKELTGATTEVSRTDVTSSSSTSFPGPDQSTVSLDISTETNTRLSTSPIMTESAEITITTQTGPHGATS QDTFTMDPSNTTPQAGIHSAMTHGFSQLDVTTLMSRIPQDVSWTSPPSVDKTSSPSSFLSSPAMTTPSLISSTLPEDKLSSPMTSLLTSGLVKITDILRTRLEPVTSSLPNFSSTSDKILATSKDSKDTKEIFPSINTEETNVKANNSGHESHSPALADSETPKATTQMVITTTVGDPAPSTSMPVHGSSETTNIKREPTYFLTPRLRETSTSQESSFPTDTSFLLS KVPTGTITEVSSTGVNSSSKISTPDHDKSTVPPDTFTGEIPRVFTSSIKTKSAEMTITTQASPPESASHSTLPLDTSTTLSQGGTHSTVTQGFPYSEVTTLMGMGPGNVSWMTTPPVEETSSVSSLMSSPAMTSPSPVSSTSPQSIPSSPLPVTALPTSVLVTTTDVLGTTSPESVTSSPPNLSSITHERPATYKDTAHTEAAMHHSTNTAVTNVGTSGSGH KSQSSVLADSETSKATPLMSTTTSTLGDTSVSTSTPNISQTNQIQTEPTASLSPRLRESSTSEKTSSTTETNTAFSYVPTGAITQASRTEISSSRTSISDLDRPTIAPDISTGMITRLFTSPIMTKSAEMTVTTQTTTPGATSQGILPWDTSTTLFQGGTHSTVSQGFPHSEITTLRSRTPGDVSWMTTPPVEETSSGFSLMSPSMTSPSPVSSTSPESIPSSPLPVTALL TSVLVTTTNVLGTTSPEPVTSSPPNLSSPTQERLTTYKDTAHTEAMHASMHTNTAVANVGTSISGHESQSSVPADSHTSKATSPMGITFAMGDTSVSTSTPAFFETRIQTESTSSLIPGLRDTRTSEEINTVTETSTVLSEVPTTTTTEVSRTEVITSSRTTISGPDHSKMSPYISTETITRLSTPFVTGSTEMAITNQTGPIGTISQATLTLDTSSTASWEG THSPVTQRFPHSEETTTMSRSTKGVSWQSPPSVEETSSPSSPVPLPAITSHSSLYSAVSGSSPTSALPVTSLLTSGRRKTIDMLDTHSELVTSSLPSASSFSGEILTSEASTNTETIHFSENTAETNMGTTNSMHKLHSSVSIHSQPSGHTPPKVTGSMMEDAIVSTSTPGSPETKNVDRDSTSPLTPELKEDSTALVMNSTTESNTVVSVSLDAATERAEVTY YDPTFMPASAQSTKSPDISPEASSSHSNSPPLTISTHKTIATQTGPSGVTSLGQLTLDTSTIATSAGTPSARTQDFVDSETTSVMNNDLNDVLKTSPFSAEEANSLSSQAPLLVTTSPSPVTSTLQEHSTSSLVSVTSVPTPTLAKITDMDTNLEPVTRSPQNLRNTLATSEATTDTHTMHPSINTAVANVGTTSSPNEFYFTVSPDSDPYKATSAVVI TSTSGDSIVSTSMPRSSAMKKIESETTFSLIFRLRETSTSQKIGSSSDTSTVFDKAFTAATTEVSRTELTSSSRTSIQGTEKPTMSPDTSTRSVTMLSTFAGLTKSEERTIATQTGPHRATSQGTLTWDTSITTSQAGTHSAMTHGFSQLDLSTLTSRVPEYISGTSPPSVEKTSSSSSLSLPAITSPSPSPVPTTLPESRPSSPVHLTSLPTSGLVKTTDMLAS VASLPPNLGSTSHKIPTTSEDIKDTEKMYPSTNIAVTNVGTTTSEKESYSSVPAYSEPPKVTSPMVTSFNIRDTIVSTSMPGSSEITRIEMESTFSLAHGLKGTSTSQDPIVSTEKSAVLHKLTTGATETSRTEVASSRRTSIPGPDHSTESPDISTEVIPSLPISLGITESSNMTIITRTGPPLGSTSQGTFTLDTPTTSSRAGTHSMATQEFPHSEMTTVMNK DPEILSWTIPPSIEKTSFSSSLMPSPAMTSPPVSSTLPKTIHTTPSPMTSLLTPSLVMTTDTLGTSPEPTTSSPPNLSTSHEILTTDEDTTAIEAMHPSTSTAATNVETTSSGHGSQSSVLADSEKTKATAPMDTTSTMGHTTVSTSMSVSSETTKIKRESTYSLTPGLRETSISQNASFSTDTSIVLSEVPTGTTAEVSRTEVTSSGRTSIPGPSQSTVLPEISTRTMTRLFASP TMTESAEMTIPTQTGPSGSTSQDTLTLDTSTTKSQAKTHSTLTQRFPHSEMTTLMSRGPGDMSWQSSPSLENPSSLPSLLSLPATTSPPPISSTLPVTISSSPLPVTSLLTSSPVTTTDMLHTSPELVTSSPPKLSHTSDERLTTGKDTTNTEAVHPSTNTAASNVEIPSSGHESPSSALADSETSKATSPMFITSTQEDTTVAISTPHFLETSRIQKESISSLSPKLRETG SSVETSSAIETSAVLSEVSIGATTEISRTEVTSSSRTSISGSAESTMLPEISTTRKIIKFPTSPILAESSEMTIKTQTSPPGSTSESTFTLDTSTTPSLVITHSTMTQRLPHSEITTLVSRGAGDVPRPSSLPVEETSPPSSQLSLSAMISPSPVSSTLPASSHSSSASVTSLLTPGQVKTTEVLDASAEPETSSPPSLSTSVEILATSEVTTDTEKIHPFSNTAVTKVGTSSS GHESPSSVLPDSETTKATSAMGTISIMGDTSVSTLTPALSNTRKIQSEPASSLTTRLRETSTSEETSLATEANTVLSKVSTGATTEVSRTEAISFSRTSMSGPEQSTMSQDISIGTIPRISASSVLTESAKMTITTQTGPSESTLNLNTATTPSWVETHSIVIQGFPHPEMTTSMGRGPGGVSWPSPPFVKETSPPSSPLSLPAVTSPHPVSTTFLAHIPPSPLP VTSLLTSGPATTTDILGTSTEPGTSSSSSLSTTSHERLTTYKDTAHTEAVHPSTNTGGTNVATTSSGYKSQSSVLADSSPMCTTSTMGDTSVLTSTPAFLETRRIQTELASSLTPGLRESSGSEGTSSGTKMSTVLSKVPTGATTEISKEDVTSIPGPAQSTISPDISTRTVSWFSTSPVMTESAEITMNTHTSPLGATTQGTSTLDTSSTTSLTMTHSTISQGFSH SQMSTLMRRGPEDVSWMSPPLLEKTRPSFSLMSSPATTSPSPVSSTLPESISSSPLPVTSLLTSGLAKTTDMLHKSSEPVTNSPANLSSTSVEILATSEVTTDTEKTHPSSNRTVTDVGTSSSGHESTSFVLADSQTSKVTSPMVITSTMEDTSVSTSTPGFFETSRIQTEPTSSLTLGLRKTSSSEGTSLATEMSTVLSGVPTGATAEVSRTEVTSSSRTSISGFAQ LTVSPETSTETITRLPTSSIMTESAEMMIKTQTDPPGSTPESTHTVDISTTPNWVETHSTVTQRFSHSEMTTLVSRSPGDMLWPSQSSVEETSSASSLLSLPATTSPSPVSSTLVEDFPSASLPVTSLLNPGLVITTDRMGISREPGTSSTSNLSSTSHERLTTLEDTVDTEDMQPSTHTAVTNVRTSISGHESQSSVLSDSETPKATSPMGTTYTMGETSISTSDFF ETSRIQIEPTSSLTSGLRETSSSERISSATEGSTVLSEVPSGATTEVSRTEVISSRGTSMSGPDQFTISPDISTEAITRLSTSPIMTESAESAITIETGSPGATSEGTLTLDTSTTTFWSGTHSTASPGFSHSEMTTLMSRTPGDVPWPSLPSVEEASSVSSSLSSPAMTSTSFFSTLPESISSSPHPVTALLTLGPVKTTDMLRTSSEPETSSPPNLSSTSAEILATSEVTKDREKI HPSSNTPVVNVGTVIYKHLSPSSVLADLVTTKPTSPMATTSTLGNTSVSTSTPAFPETMMTQPTSSLTSGLREISTSQETSSATERSASLSGMPTGATTKVSRTEALSLGRTSTPGPAQSTISPEISTETITRISTPLTTTGSAEMTITPKTGHSGASSQGTFTLDTSSRASWPGTHSAATHRSPHSGMTTPMSRGPEDVSWPSRPSVEKTSPPSSLVSLSAVTSP SPLYSTPSESSHSSPLRVTSLFTPVMMKTTDMLDTSLEPVTTSPPSMNITSDESLATSKATMETEAIQLSENTAVTQMGTISARQEFYSSYPGLPEPSKVTSPVVTSSTIKDIVSTTIPASSEITRIEMESTSTLTPTPRETTSTSQEIHSATKPSTVPYKALTSATIEDSMTQVMSSSRGPSPDQSTMSQDISTEVITRLSTSPIKTESTEMTITTQTGSPGATSRG TLTLDTSTTFMSGTHSTASQGFSHSQMTALMSRTPGDVPWLSHPSVEEASSASFSLSSPVMTSSSPVSSTLPDSIHSSSLPVTSLLTSGLVKTTELLGTSSEPETSSPPNLSSTSAEILAITEVTTDTEKLEMTNVVTSGYTHESPSSVLADSVTTKATSSMGITYPTGDTNVLTSTPAFSDTSRIQTKSKLSLTPGLMETSISEETSSATEKSTVLSSVPTGATTEVS RTEAISSSRTSIPGPAQSTMSSDTSMETITRISTPLTRKESTDMAITPKTGPSGATSQGTFTLDSSSTASWPGTHSATTQRFPQSVVTTPMSRGPEDVSWPSPLSVEKNSPPSSLVSSSSVTSPSPLYSTPSGSSHSSPVPVTSLFTSIMMKATDMLDASLEPETTSAPNMNITSDESLAASKATTETEAIHVFENTAASHVETTSATEELYSSSPGFSEPTKVISPVV TSSSIRDNMVSTTMPGSSGITRIEIESMSSLTPGLRETRTSQDITSSTETSTVLYKMPSGATPEVSRTEVMPSSRTSIPGPAQSTMSLDISDEVVTRLSTSPIMTESAEITITTQTGYSLATSQVTLPLGTSMTFLSGTHSTMSQGLSHSEMTNLMSRGPESLSWTSPRFVETTRSSSSLTSLPLTTSLSPVSSTLLDSSPSSPLPVTSLILPGLVKTTEVLDTSSEPK TSSSPNLSSTSVEIPATSEIMTDTEKIHPSSNTAVAKVRTSSSVHESHSSVLADSETTITIPSMGITSAVDDTTVFTSNPAFSETRRIPTEPTFSLTPGFRETSTSEETTSITETSAVLYGVPTSATTEVSMTEIMSSNRIHIPDSDQSTMSPDIITEVITRLSSSSMMSESTQMTITTQKSSPGATAQSTLTLATTTAPLARTHSTVPPRFLHSEMTTLMSRSPENPSWKSS LFVEKTSSSSLLSLPVTTSPSVSSTLPQSIPSSSFSVTSLLTPGMVKTTDTSTEPGTSLSPNLSGTSVEILAASEVTTDTEKIHPSSSMAVTNVGTTSSGHELYSSVSIHSEPSKATYPVGTPSSMAETSISTSMPANFETTGFEAEPFSHLTSRKTNMSLDTSSVTGFPTNTPSSPGTHLLQSSKTDFTSSAKTSSPDWPPASQYTEIPVDIITPFNAS PSITESTGITSFPESRFTMSVTESTHHLSTDLLPSAETISTGTVMPSLSEAMTSFATTGVPRAISGSGSPFSRTESGPGDATLSTIAESLPSSTPVPFSSSTFTTTDSSTIPALHEITSSSATPYRVDTSLGTESSTTEGRLVMVSTLDTSSQPGRTSSSPILDTRMTESVELGTVTSAYQVPSLSTRLTRTDGIMEHITKIPNEAAHRGTIRPVKGPQTSTSPASPKGLHT GGTKRMETTTTALKTTTTALKTTSRATLTTSVYTPTLGTLTPLNASMQMASTIPTEMMITTPYVFPDVPETTSSLATSLGAETSTALPRTTPSVFNRESETTASLVSRSGAERSPVIQTLDVSSSEPDTTASWVIHPAETIPTVSKTTPNFFHSELDTVSSTATSHGADVSSAIPTNISPSELDALTPLVTISGTDTSTTFPTLTKSPHETETRTTWLTHPAETS STIPRTIPNFSHHESDATPSIATSPGAETSSAIPIMTVSPGAEDLVTSQVTSSGTDRNMTIPTLTLSPGEPKTIASLVTHPEAQTSSAIPTSTISPAVSRLVTSMVTSLAAKTSTTNRALTNSPGEPATTVSLVTHPAQTSPTVPWTTSIFFHSKSDTTPSMTTSHGAESSSAVPTPTVSTEVPGVVTPLVTSSRAVISTTIPILTLSPGEPETTPSMATSHGEEASSAIPTPTVSPG VPGVVTSLVTSSRAVTSTTIPILTFSLGEPETTPSMATSHGTEAGSAVPTVLPEVPGMVTSLVASSRAVTSTTLPTLTLSPGEPETTPSMATSHGAEASSTVPTVSPEVPGVVTSLVTSSSGVNSTSIPTLILSPGELETTPSMATSHGAEASSAVPTPTVSPGVSGVVTPLVTSSRAVTSTTIPILTLSSSEPETTPSMATSHGVEASSAVLTVSPEVPGMVTSLVSSRAVTSTTI PTLTISSDEPETTTSLVTHSEAKMISAIPTLAVSPTVQGLVTSLVTSSGSETSAFSNLTVASSQPETIDSWVAHPGTEASSVVPTLTVSTGEPFTNISLVTHPAESSSTLPRTTSRFSHSELDTMPSTVTSPEAESSSAISTTISPGIPGVLTSLVTSSGRDISATFPTVPPESPHESEATASWVTHPAVTSTTVPRTTPNYSHSEPDTTPSIATSPGAEATSDFPTITVSPDVPDMV TSQVTSSGTDTSITIPTLTLSSGEPETTTSFITYSETHTSSAIPTLPVSPGASKMLTSLVISSGTDSTTTFPTLTETPYEPETTAIQLIHPAETNTMVPRTTPKFSHSKSDTTLPVAITSPGPEASSAVSTTTISPDMSDLVTSLVPSSGTDTSTTFPTLSETPYEPETTATWLTHPAETSTTVSGTIPNFSHRGSDTAPSMVTSPGVDTRSGVPTTTIPPSIPGVVTS QVTSSATDTSTAIPTLTPSPGEPETTASSATHPGTQTGFTVPIRTVPSSEPDTMASWVTHPPQTSTPVSRTTSSFSHSSPDATPVMATSPRTEASSAVLTTISPGAPEMVTSQITSSGAATSTTVPTLTHSPGMPETTALLSTHPRTETSKTFPASTVFPQVSETTASLTIRPGAETSTALPTQTTSSLFTLLVTGTSRVDLSPTASPGVSAKTAPLSTHPGTETSTMIPTSTLSL GLLETTGLLATSSSAETSTSTLTLTVSPAVSGLSSASITTDKPQTVTSWNTETSPSVTSVGPPEFSRTVTGTTMTLIPSEMPTPPKTSHGEGVSPTTILRTTMVEATNLATTGSSPTVAKTTTTFNTLAGSLFTPLTTPGMSTLASESVTSRTSYNHRSWISTTSSYNRRYWTPATSTPVTSTFSPGISTSSIPSSTAATVPFMVPFTLNFTITNLQYEEDMRHPG SRKFNATERELQGLLKPLFRNSSLEYLYSGCRLASLRPEKDSSATAVDAICTHRPDPEDLGLDRERLYWELSNLTNGIQELGPYTLDRNSLYVNGFTHRSSMPTTSTPGTSTVDVGTSGTPSSSPSPTTAGPLLMPFTLNFTITNLQYEEDMRRTGSRKFNTMESVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKDGAATGVDAICTHRL DPKSPGLNREQLYWELSKLTNDIEELGPYTLDRNSLYVNGFTHQSSVSTTSTPGTSTVDLRTSGTPSSLSSPTIMAAGPLLVPFTLNFTITNLQYGEDMGHPGSRKFNTTERVLQGLLGPIFKNTSVGPLYSGCRLTSLRSEKDGAATGVDAICIHHLDPKSPGLNRERLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHRTSVPTSSTPGTSTVDL GTSGTPFSLPSPATAGPLLVLFTLNFTITNLKYEEDMHRPGSRKFNTTERVLQTLLGPMFKNTSVGLLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHWIPVPTSSTPGTSTVDLGSGTPSSLPSPTTAGPLLVPFTLNFTITNLKYEEDMHCPGSRKFNTTERVLQSLLGPMF KNTSVGPLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHQTSAPNTSTPGTSTVDLGTSGTPSSLPSPTSAGPLLVPFTLNFTITNLQYEEDMHHPGSRKFNTTERVLQGLLGPMFKNTSVGLLYSGCRLTLLRPEKNGAATGMDAICSHRLDPKSPGLNREQLYWEL SQLTHGIKELGPYTLDRNSLYVNGFTHRSSVAPTSTPGTSTVDLGTSGTPSSLPSPTTAVPLLVPFTLNFTITNLQYGEDMRHPGSRKFNTTERVLQGLLGPLFKNSSVGPLYSGCRLISLRSEKDGAATGVDAICTHHLNPQSPGLDREQLYWQLSQMTNGIKELGPYTLDRNSLYVNGFTHRSSGLTTSTPWTSTVDLGTSGTPSPSPTT TGPLLVPFTLNFTITNLQYEENMGHPGSRKFNITESVLQGLLKPLFKSTSVGPLYSGCRLTLLRPEKDGVATRVDAICTHRPDPKIPGLDRQQLYWELSQLTHSITELGPYTLDRDSLYVNGFTQRSSVPTTSTPGTFTVQPETSETPSSLPGPTATGPVLLPFTLNFTITNLQYEEDMRRPGSRKFNTTERVLQGLLMPLFKNT SVSSLYSGCRLTLLRPEKDGAATRVDAVCTHRPDPKSPGLDRERLYWKLSQLTHGITELGPYTLDRHSLYVNGFTHQSSMTTTRTPDTSTMHLATSRTPASLSGPMTASPLLVLFTINFTITNLRYEENMHHPGSRKFNTTERVLQGLLRPVFKNTSVGPLYSGCRLTLLRPKKDGAATKVDAICTYRPDPKSPGLDREQLYWELSQLTHS ITELGPYTLDRDSLYVNGFTQRSSVPTTSIPGTPTVDLGTSGTPVSKPGPSAASPLLVLFTLNFTITNLRYEENMQHPGSRKFNTTERVLQGLLRRSLFKSTSVGPLYSGCRLTLLRPEKDGTATGVDAICTHHPDPKSPRLDREQLYWELSQLTHNITELGPYALDNDSLFVNGFTHRSSVSTTSTPGTPTVYLGASKTPASIFGPSAASHLLI LFTLNFTITNLRYEENMWPGSRKFNTTERVLQGLLRPLFKNTSVGPLYSGCRLTLLRPEKDGEATGVDAICTHRPDPTGPGLDREQLYLELSQLTHSITELGPYTLDRDSLYVNGFTHRSSVPTTSTGVVSEEPFTLNFTINNLRYMADMGQPGSLKFNITDNVMQHLLSPLFQRSSLGARYTGCRVIALRSVKNGAETRVDLLCTYLQPL SGPGLPIKQVHELSQQTHGITRLGPYSLDKDSLYLNGYNEPGPDEPPTTPKPATTFLPPLSEATTAMGYHLKTLTLNFTISNLQYSPDMGKGSATFNSTEGVLQHLLRPLFQKSSMGPFYLGCQLISLRPEKDGAATGVDTTCTYHPDPVGPGLDIQQQLYWELSQLTHGVTQLGFYVLDRDLSLFINGYAPQNLSIRGEYQ INFHIVNWNLSNPDPTSSEYITLLRDIQDKVTTLYKGSQLHDTFRFCLVTNLTMDSVLVTVKALFSSNLDPSLVEQVFLDKTLNASFHWLGSTYQLVDIHVTEMESSVYQPTSSSSTQHFYLNFTITNLPYSQDKAQPGTTNYQRNKRNIEDALNQLFRNSSIKSYFSDCQVSTFRSVPNRHHTGVDSLCNFFSPLARRV DRVAIYEEFLRMTRNGTQLQNFTLDRSSVLVDGYSPNRNEPLTGNSDLPFWAVILIGLAGLLGVITCLICGVLVTTRRRKKEGEYNVQQCPGYYQSHLDLEDLQ SLC39A6 54 MARKLSVILILTFALSVTNPLHELKAAAFPQTTEKISPNWESGINVDLAISTRQYHLQQLFYGENNSLSVEGFRKLLQNIGIDKIKRIHIHHDHDHHSDHEHHSDHERHSDHEHHSEHEHHSDHDHHSHHNHAASGKNKRKALCPDHDSDSSGKDPRNSQGKGAHRPEHASGRRNVKDSVSASEVTSTVYNTVSEGTHFLETIETPR PGKLFPKDVSSSTPPSVTSKSRVSRLAGRKTNESVSEPRKGFMYSRNTNENPQECFNASKLLTSHGMGIQVPLNATEFNYLCPAIINQIDARSCLIHTSEKKAEIPPKTYSLQIAWVGGFIAISIISFLSLLGVILVPLMNRVFFKFLLSFLVALAVGTLSGDAFLHLLPHASHHHSHSHEEPAMEMKRGPLFSHLSSQNIEESAYFDSTWKGLTAL GGLYFMFLVEHVLTLIKQFKDKKKKNQKKPENDDDVEIKKQLSKYESQLSTNEEKVDTDDRTEGYLRADSQEPSHFDSQQPAVLEEEEVMIAHAHPQEVYNEYVPRGCKNKCHSHFHDTLGQSDDLIHHHHDYHHILHHHHHQNHHPHSHSQRYSREELKDAGVATLAWMVIMGDGLHNFSDAIGAAF TEGLSSGLSTSVAVFCHELPHELGDFAVLLKAGMTVKQAVLYNALSAMLAYLGMATGIFIGHYAENVSMWIFALTAGLFMYVALVDMVPEMLHNDASDHGCSRWGYFFLQNAGMLLGFGIMLLISIFEHKIVFRINF SLC44A4 55 MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFILGYIVVGIVAWLYGDPRQVLYPRNSTGAYCGMGENKDKPYLLYFNIFSCILSSNIISVAENGLQCPTPQVCVSSCPEDPWTVGKNEFSQTVGEVFYTKNRNFCLPGVPWNMTVITSLQQELCPSFLLPSAPALGRCFPWTNVTPPALPGITNDT TIQQGISGLIDSLNARDISVKIFEDFAQSWYWILVALGVALVLSLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQLGFTTNLSAYQSVQETWLAALIVLAVLEAILLLMLIFLRQRIRIAIALLKEASKAVGQMMSTMFYPLVTFVLLLICIAYWAMTALYLATSGQPQYVLWASNISSPGCEK VPINTSCNPTAHLVNSSCPGLMCVFQGYSSKGLIQRSVFNLQIYGVLGLFWTLNWVLALGQCVLAGAFASFYWAFHKPQDIPTTFPLISAFIRTLRYHTGSLAFGALILTLVQIARVILEYIDHKLRGVQNPVARCIMCCFKCCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAFMLLMRNIVRVVVLDK VTDLLLFFGKLLVVGGVGVLSFFFFSGRIPGLGKDFKSPHLNYYWLPIMTSILGAYVIASGFFSVFGMCVDTLFLCFLEDLERNNGSLDRPYYMSKSLLKILGKKNEAPPDNKKRKK STEAP1 56 MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHQTAHADEFDCPSELQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLN WAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPIVVLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEICSQL

[284] In various embodiments, the ADC disclosed herein may comprise any set of heavy and light chain variable domains listed in the tables above, or a set of six CDR sequences from a set of heavy and light chains, for example, introduced by grafting six CDR into a selected human donor antibody framework. In various embodiments, the ADC disclosed herein may contain amino acid sequences that are homologous to the sequences listed in the tables above, provided that the ADC retains the ability to bind to its target cancer antigen (e.g., a K _D of less than 1× 10 ^-8 M) and retains one or more of the functional properties of the ADCs disclosed herein (eg, the ability to internalize, modulate RNA splicing, suppress cell growth, etc.).

[285] In some embodiments, the ADC further comprises human heavy and light chain constant domains or fragments thereof. For example, an ADC may comprise a human IgG heavy chain constant domain (such as IgG1) and a human kappa or lambda light chain constant domain. In various embodiments, the antibody or antigen-binding fragment of the disclosed ADCs comprise a human immunoglobulin G subtype 1 (IgG1) heavy chain constant domain with a human Ig kappa light chain constant domain.

[286] In various other embodiments, the cancer target antigen for ADC is human epidermal growth factor receptor 2 (HER2).

[287] In various embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: Heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:1, Heavy chain CDR2 (HCDR2), consisting of SEQ ID NO:2, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; Light chain CDR1 (LCDR1) consisting of SEQ ID NO:4, Light chain CDR2 (LCDR2) consisting of SEQ ID NO:5, and Light chain CDR3 (LCDR3) consisting of SEQ ID NO:6, as determined by the system numbering according to Kabat.

[288] In various embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO:19 and the light chain variable region amino acid sequence of SEQ ID NO:20, or sequences that are at least 95% identical to the disclosed sequences. . In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:19 , and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:20.

[289] In various embodiments, the anti-HER2 antibody or antigen-binding fragment thereof is an internalizing antibody or an internalizing antigen-binding fragment. In various embodiments, the anti-HER2 antibody comprises a human IgG1 heavy chain constant domain and a human Ig kappa light chain constant domain.

[290] In various embodiments, the anti-HER2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO:19 or a sequence that is at least 95% identical to SEQ ID NO:19 and a light chain amino acid sequence of SEQ ID NO:20 or a sequence that is at least 95% identical to SEQ ID NO:20. In specific embodiments, the anti-HER2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:19 and the light chain amino acid sequence of SEQ ID NO:20, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-HER2 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:19 and a light chain amino acid sequence , which is at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:20. In various embodiments, the anti-HER2 antibody is trastuzumab or an antigen-binding fragment thereof.

[291] In various embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs of trastuzumab, or wherein the CDRs comprise no more than one, two, three, four, five, or six amino acid additions, deletions, or substitutions in HCDR1 (SEQ ID NO:1), HCDR2 (SEQ ID NO:2), HCDR3 (SEQ ID NO:3); LCDR1 (SEQ ID NO:4), LCDR2 (SEQ ID NO:5) and LCDR3 (SEQ ID NO:6).

[292] In various other embodiments, the cancer target antigen for ADC is human syndecan-1 (CD138).

[293] In various embodiments, the anti-CD138 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:7, a heavy chain CDR2 (HCDR2), consisting of SEQ ID NO:8, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:9; Light chain CDR1 (LCDR1) consisting of SEQ ID NO:10, Light chain CDR2 (LCDR2) consisting of SEQ ID NO:11, and Light chain CDR3 (LCDR3) consisting of SEQ ID NO:12 as determined by the system numbering according to Kabat.

[294] In various embodiments, the anti-CD138 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:22. In some embodiments, the anti-CD138 antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO:21 and the light chain variable region amino acid sequence of SEQ ID NO:22, or sequences that are at least 95% identical to the disclosed sequences. . In some embodiments, the anti-CD138 antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:21 , and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:22.

[295] In various embodiments, the anti-CD138 antibody or antigen-binding fragment thereof is an internalizing antibody or an internalizing antigen-binding fragment. In various embodiments, the anti-CD138 antibody comprises a mouse IgG2a heavy chain constant domain and a mouse Ig kappa light chain constant domain. In various embodiments, the anti-CD138 antibody comprises a human IgG2a heavy chain constant domain and a human Ig kappa light chain constant domain.

[296] In various embodiments, the anti-CD138 antibody comprises a heavy chain amino acid sequence of SEQ ID NO:21 or a sequence that is at least 95% identical to SEQ ID NO:21 and a light chain amino acid sequence of SEQ ID NO:22 or a sequence that is at least 95% identical to SEQ ID NO:22. In specific embodiments, the anti-CD138 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:21 and the light chain amino acid sequence of SEQ ID NO:22, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, an anti-CD138 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:21 and a light chain amino acid sequence , which is at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:22. In various embodiments, the anti-CD138 antibody is B-B4 or an antigen-binding fragment thereof.

[297] In various embodiments, the anti-CD138 antibody, or antigen-binding fragment thereof, comprises three heavy chain CDRs and three B-B4 light chain CDRs, or wherein the CDRs comprise no more than one, two, three, four, five, or six amino acid additions, deletions, or substitutions in HCDR1 (SEQ ID NO:7), HCDR2 (SEQ ID NO:8), HCDR3 (SEQ ID NO:9); LCDR1 (SEQ ID NO:10), LCDR2 (SEQ ID NO:11) and LCDR3 (SEQ ID NO:12).

[298] In various other embodiments, the cancer target antigen for ADC is human aphrine type-A receptor 2 (EPHA2).

[299] In various embodiments, the anti-EPHA2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: Heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:13, Heavy chain CDR2 (HCDR2), consisting of SEQ ID NO:14, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:15; Light chain CDR1 (LCDR1) consisting of SEQ ID NO:16, Light chain CDR2 (LCDR2) consisting of SEQ ID NO:17, and Light chain CDR3 (LCDR3) consisting of SEQ ID NO:18, as determined by the system numbering according to Kabat.

[300] In various embodiments, the anti-EPHA2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:24. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO:23 and the light chain variable region amino acid sequence of SEQ ID NO:24, or sequences that are at least 95% identical to the disclosed sequences. . In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:23 , and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:24.

[301] In various embodiments, the anti-EPHA2 antibody or antigen-binding fragment thereof is an internalizing antibody or an internalizing antigen-binding fragment. In various embodiments, the anti-EPHA2 antibody comprises a human IgG1 heavy chain constant domain and a human Ig kappa light chain constant domain.

[302] In various embodiments, the anti-EPHA2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO:23 or a sequence that is at least 95% identical to SEQ ID NO:23 and a light chain amino acid sequence of SEQ ID NO:24 or a sequence that is at least 95% identical to SEQ ID NO:24. In specific embodiments, the anti-EPHA2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:23 and the light chain amino acid sequence of SEQ ID NO:24, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, an anti-EPHA2 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:23 and a light chain amino acid sequence , which is at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:24. In some embodiments, the implementation of the antibody to EPHA2 contains a heavy chain encoded by the nucleotide sequence under SEQ ID NO:23; and a light chain encoded by the nucleotide sequence of SEQ ID NO:24. In various embodiments, the anti-EPHA2 antibody is IC1 or an antigen-binding fragment thereof.

[303] In various embodiments, the anti-EPHA2 antibody, or antigen-binding fragment thereof, comprises three heavy chain CDRs and three 1C1 light chain CDRs, or wherein the CDRs comprise no more than one, two, three, four, five, or six amino acid additions, deletions, or substitutions in HCDR1 (SEQ ID NO:13), HCDR2 (SEQ ID NO:14), HCDR3 (SEQ ID NO:15); LCDR1 (SEQ ID NO:16), LCDR2 (SEQ ID NO:17) and LCDR3 (SEQ ID NO:18).

[304] In various embodiments, amino acid substitutions are single residue substitutions. Insertions will typically be on the order of about 1 to about 20 amino acid residues, although significantly longer insertions can be tolerated, provided the biological function (eg, binding to the target antigen) is maintained. Deletions typically range from about 1 to about 20 amino acid residues, although in some cases the deletions can be much longer. Substitutions, deletions, insertions, or any combination thereof may be used to achieve the final derivative or variant. Usually such changes are made on a small number of amino acids in order to minimize the change in the molecule, in particular the immunogenicity and specificity of the antigen binding protein. However, in some circumstances larger changes may be allowed. Conservative substitutions are usually made according to the following scheme, depicted as Table 6.

Table 6

[305] Significant changes in function or immunological identity are performed by selecting substitutions that are less conservative than those shown in Table 6. For example, substitutions can be made that more significantly affect: the structure of the polypeptide backbone in the area of change, for example, alpha- spiral or beta-pleated structure; the charge or hydrophobicity of the molecule at the target site, or the volume of the side chain. The substitutions that can generally result in the greatest changes in the properties of the polypeptide are those in which (a) a hydrophilic residue, such as seryl or threonyl, is replaced by (or replaced by) a hydrophobic residue, such as leucyl, isoleucyl, phenylalanyl, valyl, or allyl; (b) the cysteine or proline is substituted (or replaced) by any other residue; (c) a residue with an electropositive side chain, such as lysyl, arginyl, or histidyl, is substituted (or replaced) with an electronegative residue, such as glutamyl or aspartyl; or (d) a bulky side chain residue, eg phenylalanine, is substituted (or replaced) with a non-side chain residue, eg glycine.

[306] In various embodiments in which variant antibody sequences are used in the ADC, the variants will generally exhibit the same qualitative biological activity and will elicit the same immune response, although variants may also be selected to modify the characteristics of the antigen-binding proteins if desired. Alternatively, the variant can be designed such that the biological activity of the antigen binding protein is altered. For example, glycosylation sites may be altered or removed.

[307] Various antibodies can be used with the ADCs used herein to target cancer cells. As shown below, the linker payloads in the ADCs disclosed herein are surprisingly effective with antibodies targeting various tumor antigens. Suitable antigens expressed on tumor cells but not healthy cells, or expressed at a higher level on tumor cells than on healthy cells, are known in the art, as are antibodies directed against them. Such antibodies can be used with the linkers and splicing modulator payloads disclosed herein. In some embodiments, the antibody or antigen-binding fragment that targets HER2, and the antibody or antigen-binding fragment that targets HER2 is trastuzumab. In some embodiments, the antibody or antigen-binding fragment targets CD138, and the antibody or antigen-binding fragment that targets CD138 is B-B4. In some embodiments, the antibody or antigen-binding fragment that targets EPHA2, and the antibody or antigen-binding fragment that targets EPHA2 is 1C1. In some embodiments, in addition to the disclosed linkers and splicing modulator payloads being surprisingly effective with several antibodies targeting different tumors, antibodies targeting HER2 such as trastuzumab, antibodies targeting CD138, such as B-B4, and EPHA2-targeting antibodies such as 1C1 provided particularly improved drug:antibody ratio, aggregation rate, stability (i.e., in vitro and in vivo stability), tumor targeting (t e. cytotoxicity, potency) and/or treatment efficacy. Improved treatment efficacy may be measured in vitro or in vivo and may include a reduction in tumor growth rate and/or a reduction in tumor volume.

[308] In certain embodiments, alternative antibodies to the same targets or antibodies to different target antigens are used and provide at least some of the beneficial functional properties described above (e.g., improved stability, improved tumor targeting, improved treatment efficacy). etc.). In some embodiments, some or all of these beneficial functionalities are observed when the disclosed linkers and splicing modulator payloads are conjugated to an alternative antibody or antigen-binding fragment that targets HER2-, CD138-, or EPHA2. In some embodiments, some or all of these beneficial functional properties are observed when the disclosed linkers and splicing modulator payloads are conjugated to an antibody or antigen-binding fragment that targets HER2. In some embodiments, the antibody or antigen-binding fragment targets HER2. In some embodiments, the antibody or antigen-binding fragment that targets HER2 is trastuzumab. In some embodiments, some or all of these beneficial functionalities are observed when the disclosed linkers and splicing modulator payloads are conjugated to an antibody or antigen-binding fragment that targets CD138. In some embodiments, the antibody or antigen-binding fragment targets CD138. In some embodiments, the antibody or antigen-binding fragment that targets CD138 is B-B4. In some embodiments, some or all of these beneficial functionalities are observed when the disclosed linkers and splicing modulator payloads are conjugated to an antibody or antigen-binding fragment that targets EPHA2. In some embodiments, the antibody or antigen-binding fragment targets EPHA2. In some embodiments, the antibody or antigen-binding fragment that targets EPHA2 is 1C1.

Linkers

[309] In various embodiments, the implementation of the linker in the ADC is stable outside the cell sufficiently to be therapeutically effective. In some embodiments, the implementation of the linker is stable outside the cell, so that the ADC remains intact when it is in extracellular conditions (for example, prior to transport or delivery to the cell). The term "intact" as used in the context of ADC means that the antibody or antigen-binding fragment remains attached to the drug fragment (eg, splicing modulator). As used herein, "stable" in the context of a linker or an ADC containing a linker means not more than 20%, not more than about 15%, not more than about 10%, not more than about 5%, not more than about 3%, or not more than approximately 1% of the linkers (or any percentage in between) in an ADC sample are cleaved (or otherwise not intact in the case of total ADC) when the ADC is in extracellular conditions. In some embodiments, linkers and/or ADCs disclosed herein are surprisingly stable compared to alternative linkers and/or ADCs with alternative linkers and/or splicing modulator payloads. In some embodiments, the ADCs disclosed herein may remain intact for more than about 48 hours, more than 60 hours, more than about 72 hours, more than about 84 hours, or more than about 96 hours.

[310] Whether the linker is stable outside the cell can be determined, for example, by placing ADC in blood plasma for a predetermined period of time (for example, 2, 4, 6, 8, 16, 24, 48, or 72 hours) and then quantifying the amount free drug fragment present in blood plasma. Stability can provide time for ADC to find tumor cells for targeted action and prevent premature release of the drug moiety, which can reduce the therapeutic index of ADC when indiscriminately damaging both normal and tumor tissues. In some embodiments, the linker is stable outside the target cell and releases the drug moiety from the ADC upon entering the cell so that the drug can bind to its target (eg, the SF3b spliceosome complex). Thus, an effective linker will: (i) maintain the specific binding properties of the antibody or antigen-binding fragment; (ii) provide delivery, such as intracellular delivery, of the drug fragment by stably attaching to the antibody or antigen-binding fragment; (iii) remain stable and intact until the ADC has been transported or delivered to its target site; and (iv) provide a therapeutic effect, such as a cytotoxic effect, of the drug moiety after cleavage or an alternative release mechanism.

[311] Linkers can affect the physicochemical properties of the ADC. Because many cytotoxic agents are hydrophobic in nature, binding them to an antibody with an additional hydrophobic moiety can result in aggregation. ADC aggregates are insoluble and often limit the achievable drug load on an antibody, which can adversely affect ADC activity. Protein aggregates of biological substances, in general, have also been associated with increased immunogenicity. As shown below, the linkers disclosed herein lead to ADCs with low levels of aggregation and desired levels of drug loading.

[312] The linker may be "cleavable" or "non-cleavable" (Ducry and Stump (2010) Bioconjugate Chem. 21:5-13). Cleavable linkers are designed to release a drug moiety (eg, splicing modulator) upon exposure to certain environmental factors, such as internalization into a target cell, while non-cleavable linkers are typically dependent on degradation of the antibody or antigen-binding fragment itself.

[313] In some embodiments, the linker is a non-cleavable linker. In some embodiments, the splicing modulator drug fragment in the ADC is released upon degradation of the antibody or antigen-binding fragment. Non-cleavable linkers generally remain covalently associated with at least one amino acid of the antibody and drug after internalization into the target cell and are degraded within it. Numerous exemplary non-cleavable linkers are described herein and others are known in the art. Exemplary non-cleavable linkers may include a thioether, cyclohexyl, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1 carboxylate (SMCC) or N-hydroxysuccinimide (NHS), one or more polyethylene glycol (PEG) moieties, e.g. 1, 2, 3, 4, 5 or 6 PEG moieties, or one or more alkyl moieties.

[314] In some embodiments, the linker is a cleavable linker. A cleavable linker refers to any linker that contains a cleavable moiety. Used in this document, the term "cleavable fragment" refers to any chemical bond that can be split. Suitable cleavable chemical bonds are well known in the art and include, without limitation, acid labile bonds, protease/peptidase labile bonds, photolabile bonds, disulfide bonds, and esterase labile bonds. Linkers containing a cleavable moiety can allow the splicing modulator drug moiety to be released from the ADC by cleavage at a specific site in the linker.

[315] In some embodiments, the linker is cleaved under intracellular conditions such that cleavage of the linker sufficiently releases the splicing modulator drug fragment from the antibody or antigen-binding fragment in the intracellular environment to activate the drug and/or make the drug therapeutically efficient. In some embodiments, the splice modulator drug fragment is not cleaved from the antibody or antigen-binding fragment until the ADC enters a cell that expresses an antigen specific for the antibody or antigen-binding fragment in the ADC and the drug fragment , which is a splicing modulator, is cleaved from the antibody or antigen-binding fragment after entering the cell. In some embodiments, the linker provides a cleavable fragment that is positioned such that no portion of the linker or antibody or antigen-binding fragment remains associated with the splicing modulator drug fragment after cleavage. Exemplary cleavable linkers include acid labile linkers, protease/peptidase sensitive linkers, photolabile linkers, dimethyl, disulfide or sulfonamide containing linkers.

[316] In some embodiments, the implementation of the linker is a pH-sensitive linker, it is sensitive to hydrolysis at certain pH values. Typically, the pH-sensitive linker is cleaved under acidic conditions. This cleavage strategy typically takes advantage of the lower pH in the endosomal (pH ~5-6) and lysosomal (pH ~4.8) intracellular compartments compared to the cytosol (pH ~7.4) to drive hydrolysis of the acid-labile group in the linker, such as hydrazone (Jain et al. (2015) Pharm Res 32:3526-40). In some embodiments, the linker is an acid labile and/or hydrolysable linker. For example, an acid-labile linker that is lysosome hydrolysable and contains an acid-labile group (eg, hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic acid amide, orthoester, acetal, ketal, etc.) can be used. Cm., For example, U.S. Patent Nos. 5,122,368; 5824805; 5622929; Dubowchik and Walker (1999) Pharm Therapeutics 83:67-123; Neville et al. (1989) Biol Chem. 264:14653-61. Such linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable below pH 5.5 or 5.0, the approximate pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (such as, for example, a thioether attached to a therapeutic agent via an acylhydrazone bond) (see, for example, US Pat. No. 5,622,929).

[317] In some embodiments, the linker is cleaved under reducing conditions. In some embodiments, the linker is cleaved in the presence of a reducing agent such as glutathione or dithiothreitol. In some embodiments, the linker is a cleavable disulfide linker or a cleavable sulfonamide linker.

[318] In some embodiments, the linker is a cleavable disulfide linker. A variety of disulfide linkers are known in the art, including, for example, linkers that can be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N- succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)toluene), SPDB and SMPT. Cm., For example, Thorpe et al. (1987) Cancer Res. 47:5924-31; Wawrzynczak et al., in Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987). see also US Pat. No. 4,880,935. Disulfide linkers are typically used to exploit the abundance of intracellular thiols that can facilitate cleavage of their disulfide bonds. Intracellular concentrations of the most abundant intracellular thiol, reduced glutathione, are typically in the range of 1-10 nM, which is approximately 1000 times greater than the blood concentration of the most abundant low molecular weight thiol (i.e., cysteine) at approximately 5 μM (Goldmacher et al., in Cancer Drug Discovery and Development: Antibody-Drug Conjugates and Immunotoxins (G. L. Phillips ed., Springer, 2013)). Intracellular enzymes of the protein disulfide isomerase family can also contribute to the intracellular cleavage of the disulfide linker. As used herein, a cleavable disulfide linker refers to any linker that contains a cleavable disulfide moiety. The term "cleavable disulfide moiety" refers to a disulfide bond that can be cleaved and/or reduced, for example by the action of a thiol or an enzyme.

[319] In some embodiments, the linker is a cleavable sulfonamide linker. As used herein, a cleavable sulfonamide linker refers to any linker that contains a cleavable sulfonamide moiety. The term "cleavable sulfonamide moiety" refers to a sulfonamide group, ie, a sulfonyl group attached to an amino group, where the sulfur-nitrogen bond may be cleavable.

[320] In some embodiments, the linker may be a dendritic type linker for covalently attaching more than one drug moiety to an antibody or antigen-binding moiety via a branching, multifunctional linker moiety. Cm., For example, Sun et al. (2002) Bioorg Med Chem Lett. 12:2213-5; Sun et al. (2003) Bioorg Med Chem. 11:1761-8. Dendritic linkers can increase drug to antibody molar ratio, ie, drug loading, which is associated with ADC activity. Thus, if an antibody or antigen-binding fragment has only one reactive cysteine thiol group, for example, several splicing modulator drug fragments can be attached via a dendritic linker. In some embodiments, a linker moiety or a linker-moiety-drug can be attached to an antibody or antigen-binding moiety using disulfide reduction chemistry or lysine-limited technology. Cm., For example, International Application Publication Nos. WO 2013/173391 and WO 2013/173393.

[321] In some embodiments, the implementation of the linker is cleaved by the action of a cleaving agent, for example, an enzyme that is present in the intracellular environment (for example, within the lysosome, or endosome, or caveola). The linker may be, for example, a peptide linker that is cleaved by an intracellular peptidase or protease enzyme, including without limitation a lysosomal or endosomal protease.

[322] In some embodiments, the linker is a cleavable peptide linker. As used herein, a cleavable peptide linker refers to any linker that contains a cleavable peptide fragment. The term "cleavable peptide fragment" refers to any chemical bond that binds amino acids (natural amino acids or synthetic derivatives of amino acids) that can be cleaved by the action of an agent that is present in the intracellular environment. For example, the linker may contain a valine-alanine (Val-Ala) or a valine-citrulline (Val-Cit) sequence that is cleaved by a peptidase such as a cathepsin, such as cathepsin B. In some embodiments, the linker may contain a glutamic acid- valine-citrulline (Glu-Val-Cit). In some embodiments, the linker is an enzyme-cleavable linker and the cleavable peptide fragment in the linker is enzyme-cleavable. In some embodiments, the cleavable peptide fragment is cleaved by a lysosomal enzyme, such as cathepsin. In some embodiments, the linker is a cathepsin-cleavable linker. In some embodiments, the cleavable peptide fragment in the linker is cleaved by a lysosomal cysteine cathepsin, such as cathepsin B, C, F, H, K, L, O, S, V, X, or W. In some embodiments, the cleavable peptide fragment is cleaved cathepsin B. An exemplary dipeptide that can be cleaved by cathepsin B is valine-citrulline (Val-Cit) (Dubowchik et al. (2002) Bioconjugate Chem. 13:855-69).

[323] In some embodiments, the linker or cleavable peptide fragment in the linker provides an amino acid unit. In some embodiments, the amino acid unit allows the linker to be cleaved by a protease, thereby facilitating the release of the splicing modulator drug moiety from the ADC following exposure to one or more intracellular proteases, such as one or more lysosomal enzymes (Doronina et al. (2003) Nat Biotechnol 21:778-84 Dubowchik and Walker (1999) Pharm Therapeutics 83:67-123. Illustrative amino acid units include, without limitation, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Illustrative dipeptides include, without limitation, valine-alanine (Val-Ala), valine-citrulline (Val-Cit), alanine-asparagine (Ala-Asn), alanine-phenylalanine (Ala-Phe), phenylalanine-lysine (Phe-Lys) , Alanine-Lysine (Ala-Lys), Alanine-Valine (Ala-Val), Valine-Lysine (Val-Lys), Lysine-Lysine (Lys-Lys), Phenylalanine-Citrulline (Phe-Cit), Leucine-Citrulline ( Leu-Cit), isoleucine-citrulline (Ile-Cit), tryptophan-citrulline (Trp-Cit), and phenylalanine-alanine (Phe-Ala). Illustrative tripeptides include, without limitation, alanine-alanine-asparagine (Ala-Ala-Asn), glycine-valine-citrulline (Gly-Val-Cit), glycine-glycine-glycine (Gly-Gly-Gly), phenylalanine-phenylalanine-lysine ( Phe-Phe-Lys), glutamic acid-valine-citrulline (Glu-Val-Cit) (see e.g. Anami et al. (2018) Nat Comm. linkers providing Glu-Val-Cit) and glycine-phenylalanine-lysine (Gly-Phe-Lys). Other illustrative amino acid units include, without limitation, Gly-Phe-Gly-Gly (SEQ ID NO:34), Gly-Phe-Leu-Gly (SEQ ID NO:35), Ala-Leu-Ala-Leu (SEQ ID NO:36 ), Phe-N ⁹ -tosyl-Arg, and Phe-N ⁹ -nitro-Arg, as described in, for example, US Pat. No. 6,214,345. In some embodiments, the amino acid unit in the linker provides for Val-Ala. In some embodiments, the implementation of the amino acid unit in the linker provides Val-Cit. In some embodiments, the implementation of the amino acid unit in the linker provides Glu-Val-Cit. The amino acid unit may contain naturally occurring amino acid residues and/or minor amino acids and/or non-naturally occurring amino acid analogues such as citrulline. Amino acid units can be designed and optimized for enzymatic cleavage by a particular enzyme, such as a tumor associated protease, a lysosomal protease such as cathepsin B, C, D or S, or a plasmin protease.

[324] In some embodiments, the implementation of the linker is a cleavable β-glucuronide linker. As used herein, a cleavable β-glucuronide linker refers to any linker that contains a cleavable β-glucuronide moiety. An exemplary cleavable β-glucuronide linker contains the structure:

[325] The term "cleavable β-glucuronide moiety" refers to a glycosidic bond that can be cleaved by an agent having β-glucuronidase activity. In some embodiments, the linker contains a glycosidic bond that can be cleaved by β-glucuronidase. β-glucuronidase is a UDP-glucuronosyltransferase that catalyzes the hydrolysis of the glycosidic bond of β-configured glucuronides.

[326] In some embodiments, the implementation of the ADC disclosed in this document contains a cleavable β-glucuronide fragment in the linker, which is cleaved by the action of the enzyme. In some embodiments, the cleavable β-glucuronide moiety in the linker is cleaved by a lysosomal enzyme, such as β-glucuronidase. In some embodiments, the linker is a β-glucuronidase cleavable linker. In some embodiments, the cleavable β-glucuronide moiety in the linker allows the linker to be cleaved by β-glucuronidase after internalization of the ADC, thereby facilitating release of the drug moiety from the ADC into the cellular environment.

[327] In some embodiments, a linker in any of the ADCs disclosed herein may comprise at least one spacer linking an antibody or antigen-binding fragment to a drug moiety (e.g., a splicing modulator drug moiety) . In some embodiments, the spacer link between the antibody or antigen-binding fragment and the cleavage fragment, if present, connects the cleavage site (eg, cleavable peptide fragment) in the linker to the antibody or antigen-binding fragment. In some embodiments, a spacer link between the drug moiety and the cleavable moiety, if present, connects a cleavage site (eg, a cleavable peptide moiety) in the linker to the drug moiety. In some embodiments, no cleavage site is present and a spacer unit is used to bind the antibody or antigen-binding fragment to the drug fragment.

[328] In some embodiments, the implementation of the linker and/or the spacer unit in the linker are substantially hydrophilic. The hydrophilic linker can be used to reduce the extent to which a drug can be cleared from resistant cancer cells via the multidrug resistance protein (MDR) or functionally similar transporters. In some embodiments, the implementation of the hydrophilic linker may include one or more polyethylene glycol (PEG) fragments, such as 1, 2, 3, 4, 5, or 6 PEG fragments. In some embodiments, the implementation of the linker provides 2 PEG-fragment.

[329] In some embodiments, the implementation of the spacer unit in the linker provides one or more PEG fragments. In some embodiments, the spacer unit provides one or more -(PEG) _m - and m is an integer from 1 to 10 (i.e., m can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). In some embodiments, m is in the range from 1 to 10; from 2 to 8; from 2 to 6; from 2 to 5; 2 to 4, or 2 to 3. In some embodiments, m is 2. In some embodiments, the spacer unit comprises (PEG) ₂ , (PEG) ₃ , (PEG) ₄ , (PEG) ₅ , (PEG) ₆ , (PEG) ₇ , (PEG) ₈ , (PEG) ₉ or (PEG) ₁₀ . In some embodiments, the implementation of the spacer link provides (PEG) ₂ .

[330] In some embodiments, the implementation of the spacer unit in the linker provides an alkyl fragment. In some embodiments, the spacer unit provides one or more -(CH ₂ ) _n - and n is an integer from 1 to 10 (i.e., n can be 1, 2, 3, 4, 5, 6, 7, 8 , 9, or 10). In some embodiments , n ranges from 1 to 10; from 2 to 8; from 2 to 6; from 2 to 5; 2 to 4 or 2 to 3. In some embodiments, n is 2. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, the spacer unit provides (CH ₂ ) ₂ , (CH ₂ ) ₃ , (CH ₂ ) ₄ , (CH ₂ ) ₅ , (CH ₂ ) ₆ , (CH ₂ ) ₇ , (CH ₂ ) ₈ , (CH ₂ ) ₉ or (CH ₂ ) ₁₀ . In some embodiments, the implementation of the spacer link provides (CH ₂ ) ₂ ("Et"). In some embodiments, the implementation of the spacer link provides (CH ₂ ) ₆ ("Hex"). In some embodiments, the implementation of the spacer link provides (CH ₂ ) ₂ -O-(CH ₂ ) ₂ ("Et-O-Et").

[331] A spacer unit can be used, for example, to bind an antibody or antigen-binding fragment to a drug fragment, either directly or indirectly. In some embodiments, the spacer unit binds the antibody or antigen-binding fragment to the splicing modulator drug moiety directly. In some embodiments, the antibody or antigen-binding fragment and the splicing modulator drug fragment are linked via a spacer containing one or more PEG fragments (eg, (PEG) ₂ ) or one or more alkyl fragments (eg, (CH ₂ ) ₂ , (CH ₂ ) ₆ or (CH ₂ ) ₂ -O-(CH ₂ ) ₂ ). In some embodiments, the spacer unit indirectly binds the antibody or antigen-binding fragment to the splicing modulator drug moiety. In some embodiments, a spacer unit binds an antibody or antigen-binding fragment to a splicing modulator drug moiety indirectly via a cleavable fragment (e.g., a cleavable peptide or a cleavable β-glucuronide) and/or an attachment moiety to connect the spacer unit to the antibody or an antigen-binding moiety, such as a maleimide moiety.

[332] In various embodiments, the implementation of the spacer link is attached to the antibody or antigennegative fragment (ie, antibody or antigennegative fragment) through the maleimide (Mal) fragment.

[333] A spacer unit that attaches to an antibody or antigen-binding fragment via Mal is referred to herein as a "Mal spacer unit". As used herein, the term "Mal" or "maleimide moiety" means a compound that contains a maleimide group and that is reactive with a sulfhydryl group, for example, a sulfhydryl group of a cysteine residue on an antibody or antigen-binding fragment. Other functional groups that are reactive with sulfhydryl groups (thiols) include, without limitation, iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate. In some embodiments, the Mal spacer unit reacts with a cysteine residue on an antibody or antigen binding fragment. In some embodiments, the implementation of the Mal-spacer link is connected to the antibody or antigennegative fragment through a cysteine residue. In some embodiments, the Mal spacer unit provides a PEG fragment. In some embodiments, the implementation of the Mal-spacer unit provides an alkyl fragment.

[334] In certain embodiments, the linker provides a Mal spacer unit and a cleavable peptide fragment. In some embodiments, the cleavable peptide fragment provides an amino acid unit. In some embodiments, the amino acid unit provides for Val-Cit. In some embodiments, the implementation of the amino acid link provides Val-Ala. In some embodiments, the implementation of the amino acid link provides Glu-Val-Cit. In some embodiments, the implementation of the linker provides a Mal-spacer link and Val-Cit. In some embodiments, the implementation of the linker provides a Mal-spacer link and Val-Ala. In some embodiments, the linker provides a Mal spacer unit and Val-Cit, where the Mal spacer unit provides maleimidocaproyl (MC). In some embodiments, the linker provides a Mal spacer unit and Val-Ala, where the Mal spacer unit provides maleimidocaproyl (MC). In some embodiments, the linker provides a Mal spacer unit and a cleavable β-glucuronide moiety.

[335] In some embodiments, the implementation of the linker provides the structure: Mal-spacer unit. In some embodiments, the implementation of the Mal-spacer link provides maleimidocaproil (MC). In some embodiments, the implementation of the linker provides the structure: MC. In some embodiments, the implementation of the linker provides the structure: Mal-(CH ₂ ) ₂ ("Mal-Et"). In some embodiments, the implementation of the linker provides the structure: Mal-(CH ₂ ) ₆ ("Mal-Hex"). In some embodiments, the implementation of the linker provides the structure: Mal-(CH ₂ ) ₂ -O-(CH ₂ ) ₂ ("Mal-Et-O-Et"). In some embodiments, the implementation of the linker provides the structure: Mal-(PEG) ₂ . In some embodiments, the implementation of the linker provides the structure: Mal-(PEG) ₂ -CO.

[336] In various embodiments, the Mal spacer unit attaches an antibody or antigen-binding fragment to a cleavable peptide fragment. In some embodiments, the implementation of the linker provides a Mal-spacer link-peptide. In some embodiments, the implementation of the linker provides the structure: Mal-spacer link-Val-Cit. In some embodiments, the implementation of the Mal-spacer link provides maleimidocaproil (MC). In some embodiments, the implementation of the linker provides the structure: MC-Val-Cit.

[337] In some embodiments, the implementation of the linker provides the structure: Mal-spacer link-Val-Ala. In some embodiments, the implementation of the Mal-spacer link provides maleimidocaproil (MC). In some embodiments, the implementation of the linker provides the structure: MC-Val-Ala.

[338] In various embodiments, the Mal-spacer unit attaches an antibody or antigen-binding fragment to a cleavable β-glucuronide fragment. In some embodiments, the implementation of the linker provides Mal-spacer link-β-glucuronide. In some embodiments, the implementation of the linker provides MC-β-glucuronide.

[339] In various embodiments, the cleavable fragment in the linker is attached directly to the drug fragment, which is a splicing modulator. In other embodiments, a spacer unit is used to attach a cleavable moiety in a linker to a drug moiety that is a splicing modulator. In various embodiments, the splicing modulator is attached to the cleavable fragment in the linker with a spacer unit.

[340] The spacer unit may be "self-cleaving" or "non-self-cleaving." A "non-self-cleaving" spacer unit is a spacer unit in which part or all of the spacer unit remains associated with the splicing modulator drug moiety after cleavage of the linker. Examples of non-self-cleaving spacer units include, without limitation, a glycine spacer unit and a glycine-glycine spacer unit. Non-self-cleaving spacer units can sometimes break down over time, but do not readily release the bound native drug fragment entirely under cellular conditions. The "self-cleaving" spacer unit allows the release of the native drug fragment under intracellular conditions. A "native drug" or "native fragment-drug" is one in which no part of the spacer unit or other chemical modification remains after the spacer unit is cleaved/destroyed.

[341] Self-cleaving chemical structures are known in the art and can be easily selected for the disclosed ADCs. In various embodiments, the spacer linking the cleavable moiety in the linker to the splicing modulator drug moiety is self-cleaving and undergoes self-cleavage simultaneously or shortly before/shortly after cleavage of the cleavable moiety under intracellular conditions. In some embodiments, the splicing modulator is attached to the cleavable fragment in the linker with a self-cleaving spacer unit. In certain embodiments, the splicing modulator is attached to the cleavable fragment in the linker with a self-cleaving spacer unit, wherein the cleavage fragment provides Val-Cit and maleimidocaproyl (MC) connects the cleavable fragment to an antibody or antigen-binding fragment. In certain embodiments, the splicing modulator is attached to the cleavable fragment in the linker with a self-cleaving spacer unit, wherein the cleavage fragment provides Val-Ala and maleimidocaproyl (MC) connects the cleavable fragment to an antibody or antigen-binding fragment. In certain embodiments, the splicing modulator is attached to the cleavable fragment in the linker with a self-cleaving spacer unit, wherein the cleavage fragment provides Glu-Val-Cit and maleimidocaproyl (MC) connects the cleavable fragment to an antibody or antigen-binding fragment. In certain embodiments, the splicing modulator is coupled to the antibody or antigen-binding fragment via a Mal spacer (eg, MC) in a linker coupled to a Val-Cit cleavable fragment and a pABC or pAB self-cleaving spacer. In certain other embodiments, the splicing modulator is coupled to the antibody or antigen-binding fragment via a Mal spacer (eg, MC) in a linker coupled to a Val-Ala cleavable fragment and a pABC or pAB self-cleaving spacer. In certain other embodiments, the splicing modulator is connected to the antibody or antigen-binding fragment via a Mal spacer (eg, MC) in a linker connected to the Glu-Val-Cit cleavable fragment and the pABC or pAB self-cleaving spacer.

[342] In certain embodiments, the self-cleaving spacer unit in the linker provides a p-aminobenzyl unit. In some embodiments, p-aminobenzyl alcohol (pABOH) is attached to an amino acid unit or other cleavable moiety in the linker via an amide bond, and a carbamate, methyl carbamate, or carbonate is formed between pABOH and the drug moiety (Hamann et al. (2005) Expert Opin Ther Patents 15:1087-103). In some embodiments, the self-cleaving spacer unit is or provides for p-aminobenzyloxycarbonyl (pABC). Without being limited by theory, there is reason to believe that spontaneous 1,6-elimination reactions are involved in the self-degradation of pABC (Jain et al. (2015) Pharm Res. 32:3526-40).

[343] In various embodiments, the structure of p-aminobenzyloxycarbonylp (pABC) used in the disclosed ADCs is shown below:

[344] In various embodiments, a self-cleaving spacer unit attaches a cleavable moiety in a linker to a splicing modulator. In some embodiments, the self-cleaving spacer unit is pABC. In some embodiments, pABC attaches a cleavable fragment in a linker to a splicing modulator. In some embodiments, the pABC self-degrades upon cleavage of the cleavable moiety and the splicing modulator is released from the ADC in its native active form.

[345] In some embodiments, an anti-HER2 antibody or antigen-binding fragment is coupled to a splicing modulator via a linker providing MC-Val-Cit-pABC. In other embodiments, an anti-HER2 antibody or antigen binding fragment is coupled to a splicing modulator via a linker providing MC-Val-Ala-pABC.

[346] In some embodiments, an anti-CD138 antibody or antigen-binding fragment is coupled to a splicing modulator via a linker providing MC-Val-Cit-pABC. In other embodiments, an anti-CD138 antibody or antigen binding fragment is coupled to a splicing modulator via a linker providing MC-Val-Ala-pABC.

[347] In some embodiments, an anti-EPHA2 antibody or antigen-binding fragment is coupled to a splicing modulator via a linker providing MC-Val-Cit-pABC. In other embodiments, an anti-EPHA2 antibody or antigen binding fragment is coupled to a splicing modulator via a linker providing MC-Val-Ala-pABC.

[348] In some embodiments, pABC self-degrades after cleavage of the cleavable peptide fragment in the linker. In some embodiments, the cleavable peptide fragment provides an amino acid unit. In some embodiments, the implementation of the linker provides an amino acid link-pABC. In some embodiments, the amino acid unit is Val-Cit. In some embodiments, the implementation of the linker provides Val-Cit-pABC. In some embodiments, the amino acid unit is Val-Ala. In some embodiments, the implementation of the linker provides Val-Ala-pABC. In some embodiments, the amino acid unit is Glu-Val-Cit. In some embodiments, the implementation of the linker provides Glu-Val-Cit-pABC. In some embodiments, the amino acid unit is Ala-Ala-Asn. In some embodiments, the implementation of the linker provides Ala-Ala-Asn-pABC.

[349] In some embodiments, pABC self-degrades after cleavage of a cleavable β-glucuronide moiety in the linker. In some embodiments, the implementation of the linker provides β-glucuronide-pABC.

[350] In certain embodiments, the self-cleaving spacer unit in the linker provides a p-aminobenzyl unit. In some embodiments, the self-cleaving spacer unit in the linker provides p-aminobenzyl (pAB). In some embodiments, spontaneous 1,6-elimination reactions are involved in the self-degradation of pAB.

[351] In various embodiments, the structure of p-aminobenzyl (pAB) used in the disclosed ADCs is shown below:

[352] In various embodiments, the self-cleaving spacer unit attaches a cleavable moiety in a linker to a splicing modulator. In some embodiments, the self-cleaving spacer unit is pAB. In some embodiments, the pAB attaches a cleavable fragment in a linker to a splicing modulator. In some embodiments, the pAB undergoes self-degradation upon cleavage of the cleavable moiety and the splicing modulator is released from the ADC in its native active form.

[353] In some embodiments, an anti-HER2 antibody or antigen-binding fragment is coupled to a splicing modulator via a linker providing MC-Val-Cit-pAB. In other embodiments, an anti-HER2 antibody or antigen binding fragment is coupled to a splicing modulator via a linker providing MC-Val-Ala-pAB.

[354] In some embodiments, an anti-CD138 antibody or antigen binding fragment is coupled to a splicing modulator via a linker providing MC-Val-Cit-pAB. In other embodiments, an anti-CD138 antibody or antigen-binding fragment is coupled to a splicing modulator via a linker providing MC-Val-Ala-pAB.

[355] In some embodiments, an anti-EPHA2 antibody or antigen-binding fragment is coupled to a splicing modulator via a linker providing MC-Val-Cit-pAB. In other embodiments, an anti-EPHA2 antibody or antigen binding fragment is coupled to a splicing modulator via a linker providing MC-Val-Ala-pAB.

[356] In some embodiments, the implementation of the pAB undergoes self-degradation after cleavage of the cleavable peptide fragment in the linker. In some embodiments, the cleavable peptide fragment provides an amino acid unit. In some embodiments, the implementation of the linker provides an amino acid link-pAB. In some embodiments, the amino acid unit is Val-Cit. In some embodiments, the implementation of the linker provides Val-Cit-pAB. In some embodiments, the amino acid unit is Val-Ala. In some embodiments, the implementation of the linker provides Val-Ala-pAB. In some embodiments, the amino acid unit is Glu-Val-Cit. In some embodiments, the implementation of the linker provides Glu-Val-Cit-pAB. In some embodiments, the amino acid unit is Ala-Ala-Asn. In some embodiments, the implementation of the linker provides Ala-Ala-Asn-pAB.

[357] In some embodiments, pAB undergoes self-degradation after cleavage of a cleavable β-glucuronide moiety in the linker. In some embodiments, the implementation of the linker provides β-glucuronide-pAB.

[358] In some other embodiments, the splicing modulator is attached to the cleavable fragment in the linker with a non-self-cleaving spacer unit. In certain embodiments, the splicing modulator is attached to the cleavable fragment in the linker with a non-self-cleaving spacer unit, wherein the cleavage fragment provides Val-Cit and maleimidocaproyl (MC) connects the cleavable fragment to an antibody or antigen-binding fragment. In certain embodiments, the splicing modulator is attached to the cleavable fragment in the linker with a non-self-cleaving spacer unit, wherein the cleavage fragment provides Val-Ala and maleimidocaproyl (MC) connects the cleavable fragment to an antibody or antigen-binding fragment.

[359] In various aspects, the antibody or antigen binding fragment of the ADC is conjugated to the splicing modulator drug moiety via a linker, where the linker provides a Mal spacer unit (eg, MC), a cleavable amino acid unit, and pABC. In some embodiments, the spacer unit provides an alkyl moiety. In some embodiments, the implementation of the Mal-spacer link provides maleimidocaproil (MC). In some embodiments, the linker provides a Mal-spacer unit-amino acid unit-pABC. In some embodiments, the implementation of the linker provides MC-amino acid link-pABC. In some embodiments, the implementation of the linker provides MC-Val-Cit-pABC. In some embodiments, the implementation of the linker provides MC-Val-Ala-pABC. In some embodiments, the implementation of the linker provides MC-Glu-Val-Cit-pABC. In some embodiments, the implementation of the linker provides MC-Ala-Ala-Asn-pABC.

[360] In various other aspects, the antibody or antigen binding fragment of the ADC is conjugated to the splicing modulator drug moiety via a linker, where the linker provides a Mal spacer unit (e.g., MC), a cleavable amino acid unit, and pAB. In some embodiments, the spacer unit provides an alkyl moiety. In some embodiments, the implementation of the Mal-spacer link provides maleimidocaproil (MC). In some embodiments, the linker provides a Mal-spacer unit-amino acid unit-pAB. In some embodiments, the implementation of the linker provides MC-amino acid link-pAB. In some embodiments, the implementation of the linker provides MC-Val-Cit-pAB. In some embodiments, the implementation of the linker provides MC-Val-Ala-pAB. In some embodiments, the implementation of the linker provides MC-Glu-Val-Cit-pAB. In some embodiments, the implementation of the linker provides MC-Ala-Ala-Asn-pAB.

[361] In various other aspects, the antibody or antigen binding fragment of the ADC is conjugated to the splicing modulator drug moiety via a linker, where the linker provides a Mal spacer unit (eg, MC), a cleavable β-glucuronide, and pABC. In some embodiments, the implementation of the linker provides Mal-spacer link-β-glucuronide-pABC. In some embodiments, the implementation of the linker provides MC-β-glucuronide-pABC.

[362] In still other aspects, the antibody or antigen binding fragment of the ADC is conjugated to the splicing modulator drug moiety via a linker, wherein the linker provides a Mal spacer unit (e.g., MC), a cleavable β-glucuronide, and pAB. In some embodiments, the implementation of the linker provides Mal-spacer link-β-glucuronide-pAB. In some embodiments, the implementation of the linker provides MC-β-glucuronide-pAB.

[363] In various embodiments, the ADC compound has the formula (I):

Ab-(LD) _p (I),

where Ab is an antibody or antigen-binding fragment that specifically affects the neoplastic cell;

D is a splicing modulator;

L is a linker that covalently attaches Ab to D; And

p is an integer from 1 to 15.

[364] In some embodiments, an antibody or antigen-binding fragment (Ab) from an ADC is conjugated to a splicing modulator drug moiety via a linker, where the linker is any linker disclosed herein or incorporated by reference herein, or contains one or more components of any of the linkers disclosed in this document or included in it by reference.

[365] In some embodiments, the linker provides a cleavable fragment that is positioned such that no portion of the linker or antibody or antigen-binding fragment remains associated with the splicing modulator after cleavage. In some embodiments, the cleavable fragment is a cleavable peptide fragment, for example an amino acid unit such as Val-Cit or Val-Ala. In some embodiments, the amino acid unit or linker is provided by Val-Cit. In some embodiments, the amino acid unit or linker is Val-Ala. In some embodiments, the implementation of the amino acid unit or linker provide Glu-Val-Cit.

[366] In some embodiments, the implementation of the linker provides at least one spacer link connecting the antibody or antigennegative fragment with a cleavable fragment. In some embodiments, the linker provides at least one spacer link connecting the antibody or antigen-binding fragment to the drug fragment. In some embodiments, the implementation of the spacer unit or linker provide at least one alkyl fragment.

[367] In some embodiments, the implementation of the spacer unit in the linker is attached to the antibody or antigennegative fragment through the Mal-fragment ("Mal-spacer unit"). In some embodiments, the implementation of the Mal-spacer unit provides at least one alkyl fragment. In some embodiments, the implementation of the linker provides maleimidocaproil (MC). In some embodiments, the implementation of the linker provides Mal-(CH ₂ ) ₂ ("Mal-Et"). In some embodiments, the implementation of the linker provides Mal-(CH ₂ ) ₆ ("Mal-Hex"). In some embodiments, the implementation of the linker provides Mal-(CH ₂ ) ₂ -O-(CH ₂ ) ₂ ("Mal-Et-O-Et"). In some embodiments, the implementation of the linker provides Mal-(PEG) ₂ -CO. In some embodiments, a Mal spacer unit attaches an antibody or antigen-binding fragment to a drug fragment.

[368] In some embodiments, the implementation of the Mal-spacer unit or linker provide Mal-(PEG)₂, Mal-(PEG)₃, Mal-(PEG)₄, Mal-(PEG)₅, Mal-(PEG)₆, Mal-(PEG)₇ or Mal-(PEG)₈. In some embodiments, the implementation of the Mal-spacer unit or linker provide Mal-(PEG)₂. In some embodiments, the implementation of the Mal-spacer unit or linker provide Mal-(PEG)₂-CO Mal-(PEG)₃-CO Mal-(PEG)₄-CO, Mal-(PEG)₅-CO, Mal-(PEG)₆-CO, Mal-(PEG)₇-CO or Mal-(PEG)_8-CO. In some embodiments, the implementation of the Mal-spacer unit or linker provide Mal-(PEG)₂-CO. In some embodiments, the implementation of the Mal-spacer unit or linker provide Mal-(PEG)₂-CO and at least one additional spacer unit. In some embodiments, Mal-(PEG)₂-CO attaches the antibody or antigen-binding fragment to the drug fragment. In some embodiments, the implementation of the linker provides Mal-(PEG)₂-CO or consists of it. An example of a "Mal-(PEG)" linker₂-CO" is also referred to herein as "ADL2" or "ADL2" linker.

[369] In some embodiments, the implementation of the Mal-spacer unit or linker provide MC. In some embodiments, the implementation of the Mal-spacer unit or linker provide MC and at least one additional spacer unit. In some embodiments, the MC attaches an antibody or antigen-binding fragment to a drug fragment. In some embodiments, the implementation of the linker provides MC or consists of it. An exemplary "MC" linker is also referred to herein as "ADL10" or "ADL10" linker.

[370] In some embodiments, the implementation of the Mal-spacer unit or linker provide Mal-(CH ₂ ) ₆ ("Mal-Hex"). In some embodiments, the implementation of the Mal-spacer unit or linker provide Mal-Hex and at least one additional spacer unit. In some embodiments, Mal-Hex attaches an antibody or antigen-binding fragment to a drug fragment. In some embodiments, the implementation of the linker provides Mal-Hex. An example of a "Mal-Hex" linker is also referred to herein as "ADL12" or an "ADL12" linker.

[371] In some embodiments, the implementation of the Mal-spacer unit or linker provide Mal-(CH ₂ ) ₂ ("Mal-Et"). In some embodiments, the Mal-spacer unit or linker includes Mal-Et and at least one additional spacer unit. In some embodiments, Mal-Et attaches an antibody or antigen-binding fragment to a drug fragment. In some embodiments, the implementation of the linker provides Mal-Et. An example of a "Mal-Et" linker is also referred to herein as "ADL14" or an "ADL14" linker.

[372] In some embodiments, the implementation of the Mal-spacer unit or linker provide Mal-(CH ₂ ) ₂ -O-(CH ₂ ) ₂ ("Mal-Et-O-Et"). In some embodiments, the Mal-spacer unit or linker includes Mal-Et-O-Et and at least one additional spacer unit. In some embodiments, Mal-Et-O-Et attaches an antibody or antigen-binding fragment to a drug fragment. In some embodiments, the implementation of the linker provides Mal-Et-O-Et. An example of a "Mal-Et-O-Et" linker is also referred to herein as "ADL15" or "ADL15" linker.

[373] In some other embodiments, the Mal spacer unit attaches an antibody or antigen-binding fragment to a cleavable fragment in a linker. In some embodiments, the cleavable fragment in the linker is a cleavable peptide fragment, such as an amino acid unit. In some embodiments, the cleavable peptide fragment is Val-Cit or Val-Ala. In some embodiments, the implementation of the Mal-spacer unit or linker provide MC. In some embodiments, the implementation of the linker provides MC-Val-Cit. In some embodiments, the implementation of the linker provides MC-Val-Ala. In some embodiments, the implementation of the linker provides MC-Glu-Val-Cit. In some embodiments, the implementation of the linker provides MC-Ala-Ala-Asn.

[374] In some embodiments, the implementation of the spacer unit attaches a cleavable fragment in the linker to the splicing modulator. In some embodiments, the spacer that attaches the cleavable fragment to the splicing modulator is self-cleaving.

[375] In some embodiments, the implementation of the spacer link provides pABC. In some embodiments, the pABC attaches the cleavage fragment to the splicing modulator. In some embodiments, the cleavable fragment is a cleavable peptide fragment, such as an amino acid unit. In some embodiments, the implementation of the linker provides an amino acid link-pABC.

[376] In some embodiments, the implementation of the linker provides Val-Cit-pABC. In some embodiments, the implementation of the linker provides Val-Cit-pABC and MC-Mal-spacer link connecting the linker to the antibody or antigennegative fragment. In some embodiments, the implementation of the linker provides MC-Val-Cit-pABC. In some embodiments, the linker provides MC-Val-Cit-pABC and at least one additional spacer unit. An exemplary MC-Val-Cit-pABC linker is also referred to herein as "ADL1" or "ADL1" linker.

[377] In some embodiments, the implementation of the linker provides Val-Ala-pABC. In some embodiments, the implementation of the linker provides Val-Ala-pABC and MC-Mal-spacer element connecting the linker to the antibody or antigennegative fragment. In some embodiments, the implementation of the linker provides MC-Val-Ala-pABC. In some embodiments, the linker provides MC-Val-Ala-pABC and at least one additional spacer unit. An exemplary MC-Val-Ala-pABC linker is also referred to herein as "ADL6" or "ADL6" linker.

[378] In some embodiments, the implementation of the linker provides Glu-Val-Cit-pABC. In some embodiments, the implementation of the linker provides Glu-Val-Cit-pABC and MC Mal-spacer link connecting the linker to the antibody or antigennegative fragment. In some embodiments, the implementation of the linker provides MC-Glu-Val-Cit-pABC. In some embodiments, the linker provides MC-Glu-Val-Cit-pABC and at least one additional spacer unit. An exemplary MC-Glu-Val-Cit-pABC linker is also referred to herein as "ADL23" or "ADL23" linker.

[379] In some embodiments, the implementation of the linker provides Ala-Ala-Asn-pABC. In some embodiments, the implementation of the linker provides Ala-Ala-Asn-pABC and MC-Mal-spacer link connecting the linker to the antibody or antigennegative fragment. In some embodiments, the implementation of the linker provides MC-Ala-Ala-Asn-pABC. In some embodiments, the linker provides MC-Ala-Ala-Asn-pABC and at least one additional spacer unit. An exemplary MC-Ala-Ala-Asn-pABC linker is also referred to herein as "ADL21" or "ADL21" linker.

[380] In some other embodiments, the implementation of the spacer link provides pAB. In some embodiments, the pAB attaches the cleavage fragment to the splicing modulator. In some embodiments, the cleavable fragment is a cleavable peptide fragment, such as an amino acid unit. In some embodiments, the implementation of the linker provides an amino acid link-pAB.

[381] In some embodiments, the implementation of the linker provides Val-Ala-pAB. In some embodiments, the implementation of the linker provides Val-Ala-pAB and MC-Mal-spacer element connecting the linker to the antibody or antigennegative fragment. In some embodiments, the implementation of the linker provides MC-Val-Ala-pAB. In some embodiments, the implementation of the linker provides MC-Val-Ala-pAB and at least one additional spacer unit. An exemplary MC-Val-Ala-pAB linker is also referred to herein as "ADL5" or "ADL5" linker.

[382] In some embodiments, the implementation of the linker provides Val-Cit-pAB. In some embodiments, the implementation of the linker provides Val-Cit-pAB and MC-Mal-spacer element connecting the linker to the antibody or antigennegative fragment. In some embodiments, the implementation of the linker provides MC-Val-Cit-pAB. In some embodiments, the implementation of the linker provides MC-Val-Cit-pAB and at least one additional spacer unit. An exemplary MC-Val-Cit-pAB linker is also referred to herein as "ADL7" or "ADL7" linker.

[383] In some embodiments, the implementation of the linker provides β-glucuronide-pABC. In some embodiments, the implementation of the linker provides β-glucuronide-pABC and MC-Mal-spacer link connecting the linker to the antibody or antigennegative fragment. In some embodiments, the implementation of the linker provides MC-β-glucuronide-pABC. In some embodiments, the linker provides MC-β-glucuronide-pABC and at least one additional spacer unit. Example MC-β-glucuronide-pABC is also referred to herein as "ADL13" or "ADL13" linker.

[384] In some embodiments, the implementation of the linker provides β-glucuronide-pAB. In some embodiments, the implementation of the linker provides β-glucuronide-pAB and MC-Mal-spacer link connecting the linker to the antibody or antigennegative fragment. In some embodiments, the implementation of the linker provides MC-β-glucuronide-pAB.

[385] In some embodiments, the antibody or antigen binding fragment is conjugated to the splicing modulator drug moiety via an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker. In various embodiments, ADCs containing an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker and a splicing modulator drug moiety disclosed herein have been found to exhibit properties required for therapeutic ADC. In various embodiments, these properties include, but are not limited to, effective drug loading levels, low levels of aggregation, stability under storage or circulating conditions in the body (e.g., stability in blood serum), retention of affinity for cells expressing the target comparable to that of the unconjugated antibody, strong cytotoxicity against cells expressing the target, low levels of non-targeted cell killing, high levels of non-specific killing, and/or effective in vivo anti-cancer activity, all of these compared to ADCs using other payload linkers. For example, in various embodiments, ADCs comprising an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker and a splicing modulator drug moiety disclosed herein exhibit increased ability to inhibit growth and/or proliferation in cells expressing the target as compared to ADCs using other payload linkers (eg, an ADL10 linker and a splicing modulator drug fragment). In various embodiments, ADCs comprising an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker and a splicing modulator drug moiety disclosed herein exhibit surprisingly improved stability in in vivo (eg, plasma stability) compared to an ADC based on another splicing modulator (eg, Tylanstatin A ADC, for example, as reported in Puthenveetil et al. Bioconjugate Chem. (2016) 27:1880-8).

[386] In some embodiments, appropriate or superior functionality provided by a specific combination of an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker and a drug moiety disclosed herein represents a splicing modulator can be observed in the case of a payload linker conjugated to, for example, an anti-HER2 antibody such as trastuzumab; an anti-CD138 antibody such as B-B4; or an anti-EPHA2 antibody such as 1C1.

[387] In some embodiments, the ADC comprises an ADL1 splicing modulator and an antibody or antigen-binding fragment comprising an antibody or antigen-binding fragment thereof that retains the ability to target and internalize into a neoplastic cell. In some embodiments, the ADC comprises an ADL2 splicing modulator and an antibody or antigen-binding fragment comprising an antibody or antigen-binding fragment thereof that retains the ability to target and internalize to a neoplastic cell. In some embodiments, the ADC comprises an ADL5 splicing modulator and an antibody or antigen-binding fragment comprising an antibody or antigen-binding fragment thereof that retains the ability to target and internalize to a neoplastic cell. In some embodiments, the ADC comprises an ADL6 splicing modulator and an antibody or antigen-binding fragment comprising an antibody or antigen-binding fragment thereof that retains the ability to target and internalize to a neoplastic cell. In some embodiments, the ADC comprises an ADL7 splicing modulator and an antibody or antigen-binding fragment comprising an antibody or antigen-binding fragment thereof that retains the ability to target and internalize to a neoplastic cell. In some embodiments, the ADC comprises an ADL12 splicing modulator and an antibody or antigen-binding fragment comprising an antibody or antigen-binding fragment thereof that retains the ability to target and internalize into a neoplastic cell. In some embodiments, the ADC comprises an ADL13 splicing modulator and an antibody or antigen-binding fragment comprising an antibody or antigen-binding fragment thereof that retains the ability to target and internalize to a neoplastic cell. In some embodiments, the ADC comprises an ADL14 splicing modulator and an antibody or antigen-binding fragment comprising an antibody or antigen-binding fragment thereof that retains the ability to target and internalize to a neoplastic cell. In some embodiments, the ADC comprises an ADL15 splicing modulator and an antibody or antigen-binding fragment comprising an antibody or antigen-binding fragment thereof that retains the ability to target and internalize into a neoplastic cell.

[388] In some embodiments, the ADC comprises an ADL1 splicing modulator and an antibody or antigen-binding fragment thereof that target a neoplastic cell expressing HER2. In some embodiments, the ADC comprises an ADL2 splicing modulator and an antibody or antigen-binding fragment thereof that target a neoplastic cell expressing HER2. In some embodiments, the ADC comprises an ADL5 splicing modulator and an antibody or antigen-binding fragment thereof that target a neoplastic cell expressing HER2. In some embodiments, the ADC comprises an ADL6 splicing modulator and an antibody or antigen-binding fragment thereof that target a neoplastic cell expressing HER2. In some embodiments, the ADC comprises an ADL7 splicing modulator and an antibody or antigen-binding fragment thereof that target a neoplastic cell expressing HER2. In some embodiments, the ADC comprises an ADL12 splicing modulator and an antibody or antigen-binding fragment thereof that target a neoplastic cell expressing HER2. In some embodiments, the ADC comprises an ADL13 splicing modulator and an antibody or antigen-binding fragment thereof that target a neoplastic cell expressing HER2. In some embodiments, the ADC comprises an ADL14 splicing modulator and an antibody or antigen-binding fragment thereof that target a neoplastic cell expressing HER2. In some embodiments, the ADC comprises an ADL15 splicing modulator and an antibody or antigen-binding fragment thereof that target a neoplastic cell expressing HER2.

[389] In some embodiments, the antibody or antigen-binding fragment thereof that specifically targets a neoplastic cell expressing HER2 is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment thereof that targets a neoplastic cell expressing HER2 contains three heavy chain complementarity determining regions (HCDRs) containing the amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 ( HCDR2) and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions (LCDRs) containing the amino acid sequences under SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2) and SEQ ID NO:6 (LCDR3).

[390] In some embodiments, the implementation of the ADC has the formula (I):

Ab-(LD) _p (I),

Where:

(i) Ab is an anti-HER2 antibody, or an antigen-binding fragment thereof, comprising heavy chain complementarity determining regions (HCDRs) containing the amino acid sequences under SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO: 3 (HCDR3); and light chain complementarity determining regions (LCDRs) comprising the amino acid sequences under SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2) and SEQ ID NO:6 (LCDR3);

(ii) D is a splicing modulator;

(iii) L is a linker providing ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15; And

(iv) p is an integer from 1 to 15.

[391] In some embodiments, the antibody or antigen-binding fragment that specifically targets a neoplastic cell expressing HER2 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:19. NO:20. In some embodiments, the antibody or antigen-binding fragment thereof that targets a neoplastic cell expressing HER2 comprises a human IgG1 heavy chain constant domain and a human Ig kappa light chain constant domain. In some embodiments, the antibody is trastuzumab. In some embodiments, p is an integer from 1 to 10, 2 to 8, or 4 to 8. In some embodiments, p is 4. In some embodiments, p is 8.

[392] In some embodiments, the ADC comprises an ADL1 splicing modulator and an antibody or antigen-binding fragment thereof that target a CD138-expressing neoplastic cell. In some embodiments, the ADC comprises an ADL2 splicing modulator and an antibody or antigen-binding fragment thereof that target a CD138-expressing neoplastic cell. In some embodiments, the ADC comprises an ADL5 splicing modulator and an antibody or antigen-binding fragment thereof that target a CD138-expressing neoplastic cell. In some embodiments, the ADC comprises an ADL6 splicing modulator and an antibody or antigen-binding fragment thereof that target a CD138-expressing neoplastic cell. In some embodiments, the ADC comprises an ADL7 splicing modulator and an antibody or antigen-binding fragment thereof that target a CD138-expressing neoplastic cell. In some embodiments, the ADC comprises an ADL12 splicing modulator and an antibody or antigen-binding fragment thereof that target a CD138-expressing neoplastic cell. In some embodiments, the ADC comprises an ADL13 splicing modulator and an antibody or antigen-binding fragment thereof that target a CD138-expressing neoplastic cell. In some embodiments, the ADC comprises an ADL14 splicing modulator and an antibody or antigen-binding fragment thereof that target a CD138-expressing neoplastic cell. In some embodiments, the ADC comprises an ADL15 splicing modulator and an antibody or antigen-binding fragment thereof that target a CD138-expressing neoplastic cell.

[393] In some embodiments, the antibody or antigen-binding fragment thereof that specifically targets a CD138-expressing neoplastic cell is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment thereof that targets a CD138-expressing neoplastic cell contains three heavy chain complementarity determining regions (HCDRs) containing the amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 ( HCDR2) and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions (LCDRs) containing the amino acid sequences under SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2) and SEQ ID NO:12 (LCDR3).

[394] In some embodiments, the implementation of the ADC has the formula (I):

Ab-(LD) _p (I),

Where:

(i) Ab is an anti-CD138 antibody or antigen-binding fragment thereof containing heavy chain complementarity determining regions (HCDRs) containing the amino acid sequences under SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2) and SEQ ID NO: 9 (HCDR3); and complementarity determining regions of the light chain (LCDR) containing the amino acid sequences under SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2) and SEQ ID NO:12 (LCDR3);

(ii) D is a splicing modulator;

(iii) L is a linker providing ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15; And

(iv) p is an integer from 1 to 15.

[395] In some embodiments, the antibody or antigen binding fragment that specifically targets a neoplastic cell expressing CD138 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: NO:22. In some embodiments, the antibody or antigen-binding fragment thereof that targets a neoplastic cell expressing CD138 comprises a mouse IgG2a heavy chain constant domain and a mouse Ig kappa light chain constant domain. In some embodiments, the antibody or antigen-binding fragment thereof that targets a neoplastic cell expressing CD138 comprises a human IgG2a heavy chain constant domain and a human Ig kappa light chain constant domain. In some embodiments, the antibody is B-B4. In some embodiments, p is an integer from 1 to 10, 2 to 8, or 4 to 8. In some embodiments, p is 4. In some embodiments, p is 8.

[396] In some embodiments, the ADC comprises an ADL1 splicing modulator and an antibody or antigen-binding fragment thereof that target a neoplastic cell expressing EPHA2. In some embodiments, the ADC comprises an ADL2 splicing modulator and an antibody or antigen-binding fragment thereof that target a neoplastic cell expressing EPHA2. In some embodiments, the ADC comprises an ADL5 splicing modulator and an antibody or antigen-binding fragment thereof that target a neoplastic cell expressing EPHA2. In some embodiments, the ADC comprises an ADL6 splicing modulator and an antibody or antigen-binding fragment thereof that target a neoplastic cell expressing EPHA2. In some embodiments, the ADC comprises an ADL7 splicing modulator and an antibody or antigen-binding fragment thereof that target a neoplastic cell expressing EPHA2. In some embodiments, the ADC comprises an ADL12 splicing modulator and an antibody or antigen-binding fragment thereof that target a neoplastic cell expressing EPHA2. In some embodiments, the ADC comprises an ADL13 splicing modulator and an antibody or antigen-binding fragment thereof that target a neoplastic cell expressing EPHA2. In some embodiments, the ADC comprises an ADL14 splicing modulator and an antibody or antigen-binding fragment thereof that target a neoplastic cell expressing EPHA2. In some embodiments, the ADC comprises an ADL15 splicing modulator and an antibody or antigen-binding fragment thereof that target a neoplastic cell expressing EPHA2.

[397] In some embodiments, the antibody or antigen-binding fragment thereof that specifically targets the neoplastic cell expressing EPHA2 is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment thereof that targets a neoplastic cell expressing EPHA2 comprises three heavy chain complementarity determining regions (HCDRs) containing the amino acid sequences of SEQ ID NO:13 (HCDR1), SEQ ID NO:14 ( HCDR2) and SEQ ID NO:15 (HCDR3); and three light chain complementarity determining regions (LCDRs) containing the amino acid sequences under SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2) and SEQ ID NO:18 (LCDR3).

[398] In some embodiments, the implementation of the ADC has the formula (I):

Ab-(LD) _p (I),

Where:

(i) Ab is an anti-EPHA2 antibody, or an antigen-binding fragment thereof, comprising heavy chain complementarity determining regions (HCDRs) containing the amino acid sequences under SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO: 15 (HCDR3); and light chain complementarity determining regions (LCDRs) comprising the amino acid sequences under SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2) and SEQ ID NO:18 (LCDR3);

(ii) D is a splicing modulator;

(iii) L is a linker providing ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15; And

(iv) p is an integer from 1 to 15.

[399] In some embodiments, an antibody or antigen-binding fragment that specifically targets a neoplastic cell expressing EPHA2 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: NO:24. In some embodiments, the antibody or antigen-binding fragment thereof that targets a neoplastic cell expressing EPHA2 comprises a human IgG1 heavy chain constant domain and a human Ig kappa light chain constant domain. In some embodiments, the antibody is 1C1. In some embodiments, p is an integer from 1 to 10, 2 to 8, or 4 to 8. In some embodiments, p is 4. In some embodiments, p is 8.

Fragments-drugs

[400] The drug moiety (D) of the ADCs described herein can be any chemotherapeutic agent. Applicable classes of chemotherapeutic agents include, for example, RNA splicing modulators. In some preferred embodiments, the drug fragment is a splicing modulator. Exemplary splicing modulator compounds are described and exemplified herein.

[401] In various embodiments, the drug moiety is a splicing modulator compound of formula (II):

(II),

(II)

or a pharmaceutically acceptable salt thereof, where:

R ¹ is absent or selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups, -OC(=O)-(C ₁ -C ₆ alkyl) and -CD ₃ groups;

R ³ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups and -OC(=O)-(C ₁ -C ₆ alkyl) groups; And

each of R ⁴ , R ⁵ and R ⁸ is independently selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups and C ₁ -C ₆ alkyl groups;

each of R ⁶ and R ⁷ is independently selected from hydrogen, -OR ¹⁷ , -OC(=O)-R ¹⁷ , -OC(=O)-NR ¹⁵ R ¹⁶ , C ₁ -C ₆ alkyl groups and -NR ¹⁵ R ¹⁶ ;

each of R ¹⁵ and R ¹⁶ is independently selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C(=O)-OR ¹⁷ ;

R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups; And

,

,

,

и

;Z is selected from

,

,

,

And

;

where each of R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ¹⁵ , R ¹⁶ , and R ¹⁷ is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, -O-(C ₁ -C ₆ alkyl), -NR ¹⁵ R ¹⁶ , C ₃ -C ₈ cycloalkyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkylalkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups,

where at least one of R ⁶ and R ⁷ is hydrogen.

[402] In some embodiments, R ¹ is selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₁ -C ₄ alkylcarboxylic acid groups, and C ₃ -C ₈ cycloalkyl groups. In some embodiments, R ¹ is hydrogen. In some embodiments, R ¹ is a C ₁ -C ₄ alkyl group. In some embodiments, R ¹ is methyl. In some embodiments, R ¹ is ethyl. In some embodiments, R ¹ is a C ₁ -C ₄ alkyl carboxylic acid group. In some embodiments, R ¹ is -CH ₂ CH ₂ CH ₂ CO ₂ H. In some embodiments, R ¹ is a C ₃ -C ₈ cycloalkyl group. In some embodiments, R ¹ is cycloheptyl.

[403] In some embodiments, R ³ is selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₁ -C ₄ alkyl alkoxy groups, C ₁ -C ₄ alkyl carboxylic acid groups, and C ₁ -C ₄ alkyl hydroxy groups. In some embodiments, R ³ is selected from hydrogen and C ₁ -C ₄ alkyl carboxylic acid groups. In some embodiments, R ³ is hydrogen. In some embodiments, R ³ is a C ₁ -C ₄ alkyl carboxylic acid group. In some embodiments, R ³ is -CH ₂ CH ₂ CO ₂ H.

[404] In some embodiments, R ⁴ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl) groups, -OC(=O)-(C ₁ -C ₄ alkyl) groups, and C ₁ -C ₄ alkyl groups. In some embodiments, R ⁴ is hydrogen. In some embodiments, R ⁴ is hydroxyl. In some embodiments, R ⁴ is a -O-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁴ is -OCH ₃ . In some embodiments, R ⁴ is -OCH ₂ CH ₃ . In some embodiments, R ⁴ is a -OC(=O)-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁴ is -OC(=O)-CH ₃ . In some embodiments, R ⁴ is -OC(=O)-CH ₂ CH ₃ . In some embodiments, R ⁴ is a C ₁ -C ₄ alkyl group. In some embodiments, R ⁴ is methyl. In some embodiments, R ⁴ is ethyl.

[405] In some embodiments, R ⁵ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl) groups, and C ₁ -C ₄ alkyl groups. In some embodiments, R ⁵ is hydrogen. In some embodiments, R ⁵ is hydroxyl. In some embodiments, R ⁵ is a -O-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁵ is a C ₁ -C ₄ alkyl group.

[406] In some embodiments, R ⁶ is hydrogen. In some embodiments, R ⁷ is hydrogen. In some embodiments, R ⁶ is hydrogen and R ⁷ is —OR ¹⁷ . In some embodiments, R ⁶ is hydrogen and R ⁷ is -OR ¹⁷ , where R ¹⁷ is selected from hydrogen and C ¹ -C ⁴ alkyl groups. In some embodiments, R ⁶ is hydrogen and R ⁷ is —OR ¹⁷ , where R ¹⁷ is hydrogen. In some embodiments, R ⁶ is -OR ¹⁷ and R ⁷ is hydrogen. In some embodiments, R ⁶ is -OR ¹⁷ and R ⁷ is hydrogen, where R ¹⁷ is selected from hydrogen and C ¹ -C ⁴ alkyl groups. In some embodiments, R ⁶ is -OR ¹⁷ and R ⁷ is hydrogen, where R ¹⁷ is hydrogen. In some embodiments, R ⁶ is hydrogen and R ⁷ is —NR ¹⁵ R ¹⁶ . In some embodiments, R ⁶ is hydrogen and R ⁷ is -NR ¹⁵ R ¹⁶ where R ¹⁵ is H and R ¹⁶ is selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C (=O)-OR ¹⁷ . In some embodiments, R ⁶ is hydrogen and R ⁷ is -NR ¹⁵ R ¹⁶ where R ¹⁵ is H and R ¹⁶ is selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C (=O)-OR ¹⁷ , and where R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups and C ₃ -C ₈ heterocyclyl groups. In some embodiments, R ⁶ is -OR ¹⁷ . In some embodiments, R ⁶ is -OC(=O)-R ¹⁷ . In some embodiments, R ⁶ is C ₁ -C ₆ alkyl. In some embodiments, R ⁶ is C ₁ -C ₄ alkyl. In some embodiments, R ⁶ is C ₁ alkyl. In some embodiments, R ⁶ is -NR ¹⁵ R ¹⁶ . In some embodiments, R ⁷ is -OR ¹⁷ . In some embodiments, R ⁷ is -OC(=O)-R ¹⁷ . In some embodiments, R ⁷ is C ₁ -C ₆ alkyl. In some embodiments, R ⁷ is C ₁ -C ₄ alkyl. In some embodiments, R ⁷ is C ₁ alkyl. In some embodiments, R ⁷ is -NR ¹⁵ R ¹⁶ .

[407] In some embodiments, R ⁸ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl) and (C ₁ -C ₄ alkyl) groups. In some embodiments, R ⁸ is hydrogen. In some embodiments, R ⁸ is a hydroxyl group. In some embodiments, R ⁸ is a -O-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁸ is a -O-(C ₁ alkyl) group.

[408] In some embodiments, R ¹⁵ is hydrogen. In some embodiments, R ¹⁵ is R ¹⁷ . In some embodiments, R ¹⁵ is -C(=O)-R ¹⁷ . In some embodiments, R ¹⁵ is -C(=O)-OR ¹⁷ .

[409] In some embodiments, R ¹⁶ is hydrogen. In some embodiments, R ¹⁶ is R ¹⁷ . In some embodiments, R ¹⁶ is -C(=O)-R ¹⁷ . In some embodiments, R ¹⁶ is -C(=O)-OR ¹⁷ .

[410] In some embodiments, R ¹⁷ is selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₃ -C ₆ cycloalkyl groups, and C ₃ -C ₈ heterocyclyl groups. In some embodiments, R ¹⁷ is hydrogen. In some embodiments, R ¹⁷ is a C ₁ -C ₄ alkyl group. In some embodiments, R ¹⁷ is a C ₁ alkyl group. In some embodiments, R ¹⁷ is a C ₃ -C ₆ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₃ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₄ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₅ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₆ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₃ -C ₈ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₃ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₄ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₅ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₆ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₇ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₈ heterocyclyl group.

. В некоторых вариантах осуществления Z представляет собой

. В некоторых вариантах осуществления Z представляет собой

. В некоторых вариантах осуществления Z представляет собой

. В некоторых вариантах осуществления Z представляет собой

.[411] In some embodiments, Z is

. In some embodiments, Z is

. In some embodiments, Z is

. In some embodiments, Z is

. In some embodiments, Z is

.

[412] In some embodiments, a splicing modulator compound of formula (II) is attached to a linker L, for example, in an ADC of formula (I) as shown in formula (II-A):

(II-A),

(II-A),

,

,

,

и

; иwhere Z' is selected from

,

,

,

And

; And

where all other variables are as defined for formula (II).

[413] In various other embodiments, the drug moiety is a splicing modulator compound of formula (IV):

(IV),

(IV)

or a pharmaceutically acceptable salt thereof, where:

R ¹ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups, -OC(=O)-(C ₁ -C ₆ alkyl) and -CD ₃ groups;

R ³ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups and -OC(=O)-(C ₁ -C ₆ alkyl) groups; And

each of R ⁴ , R ⁵ and R ⁸ is independently selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups and C ₁ -C ₆ alkyl groups;

each of R ⁶ and R ⁷ is independently selected from hydrogen, -OR ¹⁷ , -OC(=O)-R ¹⁷ , -OC(=O)-NR ¹⁵ R ¹⁶ , C ₁ -C ₆ alkyl groups and -NR ¹⁵ R ¹⁶ ;

each of R ¹⁵ and R ¹⁶ is independently selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and

-C(=O)-OR ¹⁷ ; And

R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups;

where each of R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ¹⁵ , R ¹⁶ , and R ¹⁷ is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, -O-(C ₁ -C ₆ alkyl), -NR ¹⁵ R ¹⁶ , C ₃ -C ₈ cycloalkyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkylalkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups,

where at least one of R ⁶ and R ⁷ is hydrogen.

[414] In some embodiments, R ¹ is selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₁ -C ₄ alkylcarboxylic acid groups, and C ₃ -C ₈ cycloalkyl groups. In some embodiments, R ¹ is hydrogen. In some embodiments, R ¹ is a C ₁ -C ₄ alkyl group. In some embodiments, R ¹ is methyl. In some embodiments, R ¹ is ethyl. In some embodiments, R ¹ is a C ₁ -C ₄ alkyl carboxylic acid group. In some embodiments, R ¹ is -CH ₂ CH ₂ CH ₂ CO ₂ H. In some embodiments, R ¹ is a C ₃ -C ₈ cycloalkyl group. In some embodiments, R ¹ is cycloheptyl.

[415] In some embodiments, R ³ is selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₁ -C ₄ alkyl alkoxy groups, C ₁ -C ₄ alkyl carboxylic acid groups, and C ₁ -C ₄ alkyl hydroxy groups. In some embodiments, R ³ is selected from hydrogen and C ₁ -C ₄ alkyl carboxylic acid groups. In some embodiments, R ³ is hydrogen. In some embodiments, R ³ is a C ₁ -C ₄ alkyl carboxylic acid group. In some embodiments, R ³ is -CH ₂ CH ₂ CO ₂ H.

[416] In some embodiments, R ⁴ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl) groups, -OC(=O)-(C ₁ -C ₄ alkyl) groups, and C ₁ -C ₄ alkyl groups. In some embodiments, R ⁴ is hydrogen. In some embodiments, R ⁴ is hydroxyl. In some embodiments, R ⁴ is a -O-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁴ is -OCH ₃ . In some embodiments, R ⁴ is -OCH ₂ CH ₃ . In some embodiments, R ⁴ is a -OC(=O)-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁴ is -OC(=O)-CH ₃ . In some embodiments, R ⁴ is -OC(=O)-CH ₂ CH ₃ . In some embodiments, R ⁴ is a C ₁ -C ₄ alkyl group. In some embodiments, R ⁴ is methyl. In some embodiments, R ⁴ is ethyl.

[417] In some embodiments, R ⁵ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl) groups, and C ₁ -C ₄ alkyl groups. In some embodiments, R ⁵ is hydrogen. In some embodiments, R ⁵ is hydroxyl. In some embodiments, R ⁵ is a -O-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁵ is a C ₁ -C ₄ alkyl group.

[418] In some embodiments, R ⁶ is hydrogen. In some embodiments, R ⁷ is hydrogen. In some embodiments, R ⁶ is hydrogen and R ⁷ is —OR ¹⁷ . In some embodiments, R ⁶ is hydrogen and R ⁷ is -OR ¹⁷ , where R ¹⁷ is selected from hydrogen and C ¹ -C ⁴ alkyl groups. In some embodiments, R ⁶ is hydrogen and R ⁷ is —OR ¹⁷ , where R ¹⁷ is hydrogen. In some embodiments, R ⁶ is -OR ¹⁷ and R ⁷ is hydrogen. In some embodiments, R ⁶ is -OR ¹⁷ and R ⁷ is hydrogen, where R ¹⁷ is selected from hydrogen and C ¹ -C ⁴ alkyl groups. In some embodiments, R ⁶ is -OR ¹⁷ and R ⁷ is hydrogen, where R ¹⁷ is hydrogen. In some embodiments, R ⁶ is hydrogen and R ⁷ is —NR ¹⁵ R ¹⁶ . In some embodiments, R ⁶ is hydrogen and R ⁷ is -NR ¹⁵ R ¹⁶ where R ¹⁵ is H and R ¹⁶ is selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C (=O)-OR ¹⁷ . In some embodiments, R ⁶ is hydrogen and R ⁷ is -NR ¹⁵ R ¹⁶ where R ¹⁵ is H and R ¹⁶ is selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C (=O)-OR ¹⁷ , and where R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups and C ₃ -C ₈ heterocyclyl groups. In some embodiments, R ⁶ is -OR ¹⁷ . In some embodiments, R ⁶ is -OC(=O)-R ¹⁷ . In some embodiments, R ⁶ is C ₁ -C ₆ alkyl. In some embodiments, R ⁶ is C ₁ -C ₄ alkyl. In some embodiments, R ⁶ is C ₁ alkyl. In some embodiments, R ⁶ is -NR ¹⁵ R ¹⁶ .

[419] In some embodiments, R ⁷ is -OR ¹⁷ . In some embodiments, R ⁷ is -OC(=O)-R ¹⁷ . In some embodiments, R ⁷ is C ₁ -C ₆ alkyl. In some embodiments, R ⁷ is C ₁ -C ₄ alkyl. In some embodiments, R ⁷ is C ₁ alkyl. In some embodiments, R ⁷ is -NR ¹⁵ R ¹⁶ .

[420] In some embodiments, R ⁸ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl) and (C ₁ -C ₄ alkyl) groups. In some embodiments, R ⁸ is hydrogen. In some embodiments, R ⁸ is a hydroxyl group. In some embodiments, R ⁸ is a -O-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁸ is a -O-(C ₁ alkyl) group.

[421] In some embodiments, R ¹⁵ is hydrogen. In some embodiments, R ¹⁵ is R ¹⁷ . In some embodiments, R ¹⁵ is -C(=O)-R ¹⁷ . In some embodiments, R ¹⁵ is -C(=O)-OR ¹⁷ .

[422] In some embodiments, R ¹⁶ is hydrogen. In some embodiments, R ¹⁶ is R ¹⁷ . In some embodiments, R ¹⁶ is -C(=O)-R ¹⁷ . In some embodiments, R ¹⁶ is -C(=O)-OR ¹⁷ .

[423] In some embodiments, R ¹⁷ is selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₃ -C ₆ cycloalkyl groups, and C ₃ -C ₈ heterocyclyl groups. In some embodiments, R ¹⁷ is hydrogen. In some embodiments, R ¹⁷ is a C ₁ -C ₄ alkyl group. In some embodiments, R ¹⁷ is a C ₁ alkyl group. In some embodiments, R ¹⁷ is a C ₃ -C ₆ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₃ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₄ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₅ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₆ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₃ -C ₈ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₃ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₄ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₅ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₆ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₇ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₈ heterocyclyl group.

[424] In some embodiments, a splicing modulator compound of formula (IV) is attached to a linker L, for example, in an ADC of formula (I) as shown in formula (IV-A):

(IV-A).

(IV-A).

[425] In various other embodiments, the drug moiety is a splicing modulator compound of formula (VI):

(VI),

(VI)

or a pharmaceutically acceptable salt thereof, where:

each of R ¹ and R ⁹ is independently selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups, -OC(=O)-(C ₁ -C ₆ alkyl) and -CD _{3 groups} ;

R ³ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups and -OC(=O)-(C ₁ -C ₆ alkyl) groups;

each of R ⁴ , R ⁵ and R ⁸ is independently selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups and C ₁ -C ₆ alkyl groups;

each of R ⁶ and R ⁷ is independently selected from hydrogen, -OR ¹⁷ , -OC(=O)-R ¹⁷ , -OC(=O)-NR ¹⁵ R ¹⁶ , C ₁ -C ₆ alkyl groups, -NR ¹⁵ R ¹⁶ and linker;

R ¹⁰ is selected from hydrogen, C ₁ -C ₆ alkyl groups, -C(=O)-(C ₁ -C ₆ alkyl) and -CD ₃ groups;

each of R ¹⁵ and R ¹⁶ is independently selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C(=O)-OR ¹⁷ ;

R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups; And

a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

where each of R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹⁵ , R ¹⁶ , and R ¹⁷ is independently substituted with 0-3 groups independently selected from atoms halogen, hydroxyl groups, C ₁ -C ₆ alkyl groups, groups -O- (C ₁ -C ₆ alkyl), -NR ¹⁵ R ¹⁶ , C ₃ -C ₈ cycloalkyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ - C ₆ alkylalkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups;

where at least one of R ⁶ and R ⁷ is hydrogen; And

where both R ¹ and R ⁹ cannot be absent.

[426] In some embodiments, R ¹ is selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₁ -C ₄ alkylcarboxylic acid groups, and C ₃ -C ₈ cycloalkyl groups. In some embodiments, R ¹ is hydrogen. In some embodiments, R ¹ is a C ₁ -C ₄ alkyl group. In some embodiments, R ¹ is methyl. In some embodiments, R ¹ is ethyl. In some embodiments, R ¹ is a C ₁ -C ₄ alkyl carboxylic acid group. In some embodiments, R ¹ is -CH ₂ CH ₂ CH ₂ CO ₂ H. In some embodiments, R ¹ is a C ₃ -C ₈ cycloalkyl group. In some embodiments, R ¹ is cycloheptyl.

[427] In some embodiments, R ³ is selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₁ -C ₄ alkyl alkoxy groups, C ₁ -C ₄ alkyl carboxylic acid groups, and C ₁ -C ₄ alkyl hydroxy groups. In some embodiments, R ³ is selected from hydrogen and C ₁ -C ₄ alkyl carboxylic acid groups. In some embodiments, R ³ is hydrogen. In some embodiments, R ³ is a C ₁ -C ₄ alkyl carboxylic acid group. In some embodiments, R ³ is -CH ₂ CH ₂ CO ₂ H.

[428] In some embodiments, R ⁴ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl) groups, -OC(=O)-(C ₁ -C ₄ alkyl) groups, and C ₁ -C ₄ alkyl groups. In some embodiments, R ⁴ is hydrogen. In some embodiments, R ⁴ is hydroxyl. In some embodiments, R ⁴ is a -O-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁴ is -OCH ₃ . In some embodiments, R ⁴ is -OCH ₂ CH ₃ . In some embodiments, R ⁴ is a -OC(=O)-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁴ is -OC(=O)-CH ₃ . In some embodiments, R ⁴ is -OC(=O)-CH ₂ CH ₃ . In some embodiments, R ⁴ is a C ₁ -C ₄ alkyl group. In some embodiments, R ⁴ is methyl. In some embodiments, R ⁴ is ethyl.

[429] In some embodiments, R ⁵ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl) groups, and C ₁ -C ₄ alkyl groups. In some embodiments, R ⁵ is hydrogen. In some embodiments, R ⁵ is hydroxyl. In some embodiments, R ⁵ is a -O-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁵ is a C ₁ -C ₄ alkyl group.

[430] In some embodiments, R ⁹ is absent or selected from hydrogen, C ₁ -C ₄ alkyl groups, -(C=O)-(C ₁ -C ₄ alkyl) and -CD ₃ groups. In some embodiments, R ⁹ is absent. In some embodiments, R ⁹ is hydrogen. In some embodiments, R ⁹ is a C ₁ -C ₄ alkyl group. In some embodiments, the C ₁ -C ₄ alkyl group is methyl. In some embodiments, the C ₁ -C ₄ alkyl group is ethyl. In some embodiments, R ⁹ is -(C=O)-(C ₁ -C ₄ alkyl). In some embodiments, the -(C=O)-(C ₁ -C ₄ alkyl) group is -(C=O)-methyl. In some embodiments, R ⁹ is -CD ₃ .

[431] In some embodiments, R ¹⁰ is selected from hydrogen, C ₁ -C ₄ alkyl groups, -(C=O)-(C ₁ -C ₄ alkyl), and -CD _{3 groups} . In some embodiments, R ¹⁰ is hydrogen. In some embodiments, R ¹⁰ is a C ₁ -C ₄ alkyl group. In some embodiments, the C ₁ -C ₄ alkyl group is methyl. In some embodiments, the C ₁ -C ₄ alkyl group is ethyl. In some embodiments, R ¹⁰ is -(C=O)-(C ₁ -C ₄ alkyl). In some embodiments, the -(C=O)-(C ₁ -C ₄ alkyl) group is -(C=O)-methyl. In some embodiments, R ¹⁰ is -CD ₃ .

[432] In some embodiments, R ⁶ is hydrogen. In some embodiments, R ⁷ is hydrogen. In some embodiments, R ⁶ is hydrogen and R ⁷ is —OR ¹⁷ . In some embodiments, R ⁶ is hydrogen and R ⁷ is -OR ¹⁷ , where R ¹⁷ is selected from hydrogen and C ¹ -C ⁴ alkyl groups. In some embodiments, R ⁶ is hydrogen and R ⁷ is —OR ¹⁷ , where R ¹⁷ is hydrogen. In some embodiments, R ⁶ is -OR ¹⁷ and R ⁷ is hydrogen. In some embodiments, R ⁶ is -OR ¹⁷ and R ⁷ is hydrogen, where R ¹⁷ is selected from hydrogen and C ¹ -C ⁴ alkyl groups. In some embodiments, R ⁶ is -OR ¹⁷ and R ⁷ is hydrogen, where R ¹⁷ is hydrogen. In some embodiments, R ⁶ is hydrogen and R ⁷ is —NR ¹⁵ R ¹⁶ . In some embodiments, R ⁶ is hydrogen and R ⁷ is -NR ¹⁵ R ¹⁶ where R ¹⁵ is H and R ¹⁶ is selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C (=O)-OR ¹⁷ . In some embodiments, R ⁶ is hydrogen and R ⁷ is -NR ¹⁵ R ¹⁶ where R ¹⁵ is H and R ¹⁶ is selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C (=O)-OR ¹⁷ , and where R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups and C ₃ -C ₈ heterocyclyl groups. In some embodiments, R ⁶ is -OR ¹⁷ . In some embodiments, R ⁶ is -OC(=O)-R ¹⁷ . In some embodiments, R ⁶ is C ₁ -C ₆ alkyl. In some embodiments, R ⁶ is C ₁ -C ₄ alkyl. In some embodiments, R ⁶ is C ₁ alkyl. In some embodiments, R ⁶ is -NR ¹⁵ R ¹⁶ .

[433] In some embodiments, R ⁷ is -OR ¹⁷ . In some embodiments, R ⁷ is -OC(=O)-R ¹⁷ . In some embodiments, R ⁷ is C ₁ -C ₆ alkyl. In some embodiments, R ⁷ is C ₁ -C ₄ alkyl. In some embodiments, R ⁷ is C ₁ alkyl. In some embodiments, R ⁷ is -NR ¹⁵ R ¹⁶ .

[434] In some embodiments, R ⁸ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl) and (C ₁ -C ₄ alkyl) groups. In some embodiments, R ⁸ is hydrogen. In some embodiments, R ⁸ is a hydroxyl group. In some embodiments, R ⁸ is a -O-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁸ is a -O-(C ₁ alkyl) group.

[435] In some embodiments, R ¹⁵ is hydrogen. In some embodiments, R ¹⁵ is R ¹⁷ . In some embodiments, R ¹⁵ is -C(=O)-R ¹⁷ . In some embodiments, R ¹⁵ is -C(=O)-OR ¹⁷ .

[436] In some embodiments, R ¹⁶ is hydrogen. In some embodiments, R ¹⁶ is R ¹⁷ . In some embodiments, R ¹⁶ is -C(=O)-R ¹⁷ . In some embodiments, R ¹⁶ is -C(=O)-OR ¹⁷ .

[437] In some embodiments, R ¹⁷ is selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₃ -C ₆ cycloalkyl groups, and C ₃ -C ₈ heterocyclyl groups. In some embodiments, R ¹⁷ is hydrogen. In some embodiments, R ¹⁷ is a C ₁ -C ₄ alkyl group. In some embodiments, R ¹⁷ is a C ₁ alkyl group. In some embodiments, R ¹⁷ is a C ₃ -C ₆ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₃ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₄ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₅ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₆ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₃ -C ₈ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₃ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₄ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₅ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₆ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₇ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₈ heterocyclyl group.

[438] In some embodiments, a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, a is 1, 2, 3, 4, 5, or 6. In some embodiments, a is 1, 2, 3, 4, 5, or 6. In some in some embodiments, a is 1, 2, 3, 4, or 5. In some embodiments, a is 1, 2, 3, or 4. In some embodiments, a is 1, 2, or 3. In some embodiments, a is 1 or 2. In some embodiments, a is 1. In some embodiments, a is 2. In some embodiments, a is 3. In some embodiments, a is 4. In some embodiments, a is 5. In some embodiments, a is 6. In some embodiments, a is 6. in some embodiments, a is 7. In some embodiments, a is 8. In some embodiments, a is 9. In some embodiments, a is 10.

[439] In some embodiments, a splicing modulator compound of formula (VI) is attached to a linker L, for example, in an ADC of formula (I) as shown in formula (VI-A):

(VI-A).

(VI-A).

[440] In various other embodiments, the drug fragment is a splicing modulator compound of formula (VIII):

(VIII),

(VIII)

or a pharmaceutically acceptable salt thereof, where:

R ¹ is absent or selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups, -OC(=O)-(C ₁ -C ₆ alkyl) and -CD ₃ groups;

R ³ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups and -OC(=O)-(C ₁ -C ₆ alkyl) groups;

R ⁴ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₆ alkyl), -OC(=O)-(C ₁ -C ₆ alkyl) groups, and C ₁ -C ₆ alkyl groups; And

R ¹⁰ is selected from 3-10 membered carbocycles and 3-10 membered heterocycles, each of which is substituted with 0-3 R ^a , where each R ^a is independently selected from halogen atoms, C ₁ -C ₆ alkyl groups, -O-( C ₁ -C ₆ )alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylhydroxy groups, -S(=O) _w -(4-7-membered heterocycles), 4-7-membered carbocycles and 4-7 -membered heterocycles;

each of R ¹⁵ and R ¹⁶ is independently selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C(=O)-OR ¹⁷ ; And

R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups;

where each of R ¹ , R ³ , R ⁴ , R ¹⁰ , R ¹⁵ , R ¹⁶ and R ¹⁷ is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, groups -O -(C ₁ -C ₆ alkyl), -NR ¹⁵ R ¹⁶ , C ₃ -C ₈ cycloalkyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkylalkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups; And

where each R ^a is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, -NR ¹⁵ R ¹⁶ , C ₁ -C ₆ alkyl groups, -(C=O)-(C ₁ -C ₆ alkyl) groups , -(C=O)-(C ₁ -C ₆ alkyl)-(C ₃ -C ₁₀ heterocyclyl groups) and C ₁ -C ₆ alkylcarboxylic acid groups each substituted with 0, 1 or 2 groups independently selected from halogen atoms, hydroxyl groups, -NR ¹⁵ R ¹⁶ and C ₁ -C ₃ alkyl groups; And

w is 0, 1, or 2.

[441] In some embodiments, R ¹ is absent or selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₁ -C ₄ alkylcarboxylic acid groups, and C ₃ -C ₈ cycloalkyl groups. In some embodiments, R ¹ is hydrogen. In some embodiments, R ¹ is a C ₁ -C ₄ alkyl group. In some embodiments, R ¹ is methyl. In some embodiments, R ¹ is ethyl. In some embodiments, R ¹ is a C ₁ -C ₄ alkyl carboxylic acid group. In some embodiments, R ¹ is

-CH ₂ CH ₂ CH ₂ CO ₂ H. In some embodiments, R ¹ is a C ₃ -C ₈ cycloalkyl group. In some embodiments, R ¹ is cycloheptyl.

[442] In some embodiments, R ³ is selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₁ -C ₄ alkyl alkoxy groups, C ₁ -C ₄ alkyl carboxylic acid groups, and C ₁ -C ₄ alkyl hydroxy groups. In some embodiments, R ³ is selected from hydrogen and C ₁ -C ₄ alkyl carboxylic acid groups. In some embodiments, R ³ is hydrogen. In some embodiments, R ³ is a C ₁ -C ₄ alkyl carboxylic acid group. In some embodiments, R ³ is -CH ₂ CH ₂ CO ₂ H.

[443] In some embodiments, R ⁴ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl) groups, -OC(=O)-(C ₁ -C ₄ alkyl) groups, and C ₁ -C ₄ alkyl groups. In some embodiments, R ⁴ is hydrogen. In some embodiments, R ⁴ is hydroxyl. In some embodiments, R ⁴ is a -O-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁴ is -OCH ₃ . In some embodiments, R ⁴ is -OCH ₂ CH ₃ . In some embodiments, R ⁴ is a -OC(=O)-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁴ is -OC(=O)-CH ₃ . In some embodiments, R ⁴ is -OC(=O)-CH ₂ CH ₃ . In some embodiments, R ⁴ is a C ₁ -C ₄ alkyl group. In some embodiments, R ⁴ is methyl. In some embodiments, R ⁴ is ethyl.

[444] In some embodiments, R ¹⁰ is selected from 6-9 membered carbocycles and 6-9 membered heterocycles, each of which is substituted with 0-2 R ^a , where each R ^a is independently substituted with 0-3 groups independently selected from atoms halogen, hydroxyl groups, C ₁ -C ₆ alkyl groups, -(C=O)-(C ₁ -C ₆ alkyl) groups, -(C=O)-(C ₁ -C ₆ alkyl)-(3- 10-membered heterocycle) and C ₁ -C ₆ alkylcarboxylic acid groups.

.[445] In some embodiments, the carbocycle is phenyl substituted with 0-2 R ^a , where each R ^a is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, -( C=O)-(C ₁ -C ₆ alkyl), -(C=O)-(C ₁ -C ₆ alkyl)-(3-10 membered heterocycle) groups and C ₁ -C ₆ alkylcarboxylic acid groups. In some embodiments, phenyl is substituted with 2 R ^a , where each R ^a is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, -(C=O)-(C ₁ - C ₆ alkyl), -(C=O)-(C ₁ -C ₆ alkyl)-(3-10 membered heterocycle) groups, and C ₁ -C ₆ alkylcarboxylic acid groups. In some embodiments, phenyl is

.

.[446] In some embodiments, the heterocycle is a 9-membered heterocycle substituted with 0-2 R ^a , where each R ^a is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, groups -(C=O)-(C ₁ -C ₆ alkyl), groups -(C=O)-(C ₁ -C ₆ alkyl)-(3-10-membered heterocycle) and groups C ₁ -C ₆ alkylcarboxylic acids. In some embodiments, the 9-membered heterocycle is

.

,

,

и

.[447] In some embodiments, R ^a is selected from halogen atoms, 3-10 membered carbocycles, and 3-10 membered heterocycles, where each R ^a is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, -(C=O)-(C ₁ -C ₆ alkyl) groups, -(C=O)-(C ₁ -C ₆ alkyl)-(3-10 membered heterocycle) groups and C ₁ -C ₆ alkylcarboxylic acid. In some embodiments, R ^a is selected from halogen atoms,

,

,

And

.

[448] In some embodiments, R ¹⁵ is hydrogen. In some embodiments, R ¹⁵ is R ¹⁷ . In some embodiments, R ¹⁵ is -C(=O)-R ¹⁷ . In some embodiments, R ¹⁵ is -C(=O)-OR ¹⁷ .

[449] In some embodiments, R ¹⁶ is hydrogen. In some embodiments, R ¹⁶ is R ¹⁷ . In some embodiments, R ¹⁶ is -C(=O)-R ¹⁷ . In some embodiments, R ¹⁶ is -C(=O)-OR ¹⁷ .

[450] In some embodiments, R ¹⁷ is selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₃ -C ₆ cycloalkyl groups, and C ₃ -C ₈ heterocyclyl groups. In some embodiments, R ¹⁷ is hydrogen. In some embodiments, R ¹⁷ is a C ₁ -C ₄ alkyl group. In some embodiments, R ¹⁷ is a C ₁ alkyl group. In some embodiments, R ¹⁷ is a C ₃ -C ₆ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₃ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₄ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₅ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₆ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₃ -C ₈ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₃ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₄ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₅ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₆ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₇ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₈ heterocyclyl group.

[451] In some embodiments, a splicing modulator compound of formula (VIII) is attached to a linker L, for example, in an ADC of formula (I) as shown in formula (VIII-A):

(VIII-A).

(VIII-A).

[452] In various embodiments, the drug fragment is a splicing modulator selected from D2 and D1.

[453] In various embodiments, the drug moiety is D2. In various embodiments, the structure of the D2 drug moiety used in the disclosed ADCs is shown below:

D2.

[454] In various embodiments, a linker in an ADC (eg, an ADC of formula (I)) described herein is covalently attached to the D2 drug moiety via an amine on the piperazine group. In various embodiments, the drug moiety is a D2 derivative. In various embodiments, the D2 derivative retains at least one D2 biological function or activity (eg, SF3b complex binding, in vitro splicing activity, cytotoxicity) but has an altered chemical structure.

[455] In various embodiments, the drug moiety is D1 or a pharmaceutically acceptable salt thereof. In various embodiments, the structure of the D1 drug fragment used in the disclosed ADCs is shown below:

D1.

[456] In various embodiments, a linker in an ADC (eg, an ADC of formula (I)) described herein is covalently attached to the D1 drug moiety via an amine on the piperazine group. In various embodiments, the drug moiety is a D1 derivative. In various embodiments, the D1 derivative retains at least one biological function or activity of D1 (eg, SF3b complex binding, in vitro splicing activity, cytotoxicity) but has an altered chemical structure.

[457] In some embodiments, the splicing modulator provides D1:

(D1).

(D1).

[458] In some embodiments, the splicing modulator provides D2:

(D2).

(D2).

[459] In some embodiments, the splicing modulator provides D3:

(D3).

(D3).

[460] In some embodiments, the splicing modulator provides D4:

(D4).

(D4).

[461] In some embodiments, the splicing modulator provides D4':

(D4').

(D4').

[462] In some embodiments, the splicing modulator provides D5:

(D5).

(D5).

[463] In some embodiments, the splicing modulator provides D6:

(D6).

(D6).

[464] In some embodiments, the splicing modulator provides D7:

(D7).

(D7).

[465] In some embodiments, the splicing modulator provides D8:

(D8).

(D8).

[466] In some embodiments, the splicing modulator provides D9:

(D9).

(D9).

[467] In some embodiments, the splicing modulator provides D10:

(D10).

(D10).

[468] In some embodiments, the splicing modulator provides D11:

(D11).

(D11).

[469] In some embodiments, the splicing modulator provides D12:

(D12).

(D12).

[470] In some embodiments, the splicing modulator provides D13:

(D13).

(D13).

[471] In some embodiments, the splicing modulator provides D14:

(D14).

(D14).

[472] In some embodiments, the splicing modulator provides D15:

(D15).

(D15).

[473] In some embodiments, the splicing modulator provides D16:

(D16).

(D16).

[474] In some embodiments, the splicing modulator provides D17:

(D17).

(D17).

[475] In some embodiments, the splicing modulator provides D18:

(D18).

(D18).

[476] In some embodiments, the splicing modulator provides D19:

(D19).

(D19).

[477] In some embodiments, the splicing modulator provides D20:

(D20).

(D20).

[478] In some embodiments, the splicing modulator provides D21:

(D21).

(D21).

[479] In some embodiments, the splicing modulator provides D22:

(D22).

(D22).

[480] In some embodiments, the splicing modulator provides D23:

(D23).

(D23).

[481] In some embodiments, the splicing modulator provides D24:

(D24).

(D24).

[482] In some embodiments, the splicing modulator provides D25:

(D25).

(D25).

[483] In some embodiments, the splicing modulator provides D26:

(D26).

(D26).

[484] In some embodiments, the splicing modulator provides D27:

(D27).

(D27).

[485] In some embodiments, the splicing modulator provides D28:

(D28).

(D28).

[486] In some embodiments, the splicing modulator provides D29:

(D29).

(D29).

[487] In some embodiments, the splicing modulator provides D30:

(D30).

(D30).

[488] In some embodiments, the splicing modulator provides D31:

(D31).

(D31).

[489] In some embodiments, the splicing modulator provides D32:

(D32).

(D32).

[490] In some embodiments, the splicing modulator provides D33:

(D33).

(D33).

[491] In some embodiments, the splicing modulator provides D34:

(D34).

(D34).

[492] In some embodiments, the splicing modulator provides D35:

(D35).

(D35).

[493] An exemplary ADC has formula (I):

Ab-(LD) _p (I),

Where

Ab is an antibody or antigen-binding fragment that targets a neoplastic cell;

D is D2;

L is a linker that covalently attaches Ab to D; And

p is an integer from 1 to 15.

[494] In some embodiments, the antibody or antigen binding fragment targets a cell expressing HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, and /or STEAP1.

[495] In some embodiments, the antibody or antigen-binding fragment targets a cell that expresses HER2. In some embodiments, the antibody or antigen-binding fragment is an anti-HER2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) containing the amino acid sequences under SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2) and SEQ ID NO:6 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

[496] In some other embodiments, the antibody or antigen-binding fragment targets a cell that expresses CD138. In some embodiments, the antibody or antigen-binding fragment is an anti-CD138 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) containing the amino acid sequences under SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2) and SEQ ID NO:12 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:22. In some embodiments, the antibody or antigen-binding fragment comprises a mouse IgG2a heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a mouse Ig kappa light chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human IgG2a heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

[497] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing EPHA2. In some embodiments, the antibody or antigen-binding fragment is an anti-EPHA2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) containing the amino acid sequences under SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2) and SEQ ID NO:18 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:24. In some embodiments, the antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

[498] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing MSLN. In some embodiments, the antibody or antigen-binding fragment is an anti-MSLN antibody or antigen-binding fragment.

[499] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing FOLH1. In some embodiments, the antibody or antigen-binding fragment is an anti-FOLH1 antibody or antigen-binding fragment.

[500] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing CDH6. In some embodiments, the antibody or antigennegative fragment is an anti-CDH6 antibody or antigennegative fragment.

[501] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing CEACAM5. In some embodiments, the antibody or antigen-binding fragment is an anti-CEACAM5 antibody or antigen-binding fragment.

[502] In some other embodiments, the implementation of the antibody or antigennegative fragment purposefully affect a cell expressing CFC1B. In some embodiments, the antibody or antigennegative fragment is an anti-CFC1B antibody or antigennegative fragment.

[503] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing ENPP3. In some embodiments, the antibody or antigen-binding fragment is an anti-ENPP3 antibody or antigen-binding fragment.

[504] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing FOLR1. In some embodiments, the antibody or antigen-binding fragment is an anti-FOLR1 antibody or antigen-binding fragment.

[505] In some other embodiments, the implementation of the antibody or antigennegative fragment purposefully affect a cell expressing HAVCR1. In some embodiments, the antibody or antigen-binding fragment is an anti-HAVCR1 antibody or antigen-binding fragment.

[506] In some other embodiments, the implementation of the antibody or antigennegative fragment purposefully affect the cell expressing KIT. In some embodiments, the antibody or antigen-binding fragment is an anti-KIT antibody or antigen-binding fragment.

[507] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing MET. In some embodiments, the antibody or antigen-binding fragment is an anti-MET antibody or antigen-binding fragment.

[508] In some other embodiments, the antibody or antigen-binding fragment targets a cell that expresses MUC16. In some embodiments, the antibody or antigennegative fragment is an anti-MUC16 antibody or antigennegative fragment.

[509] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing SLC39A6. In some embodiments, the antibody or antigen-binding fragment is an anti-SLC39A6 antibody or antigen-binding fragment.

[510] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing SLC44A4. In some embodiments, the antibody or antigen-binding fragment is an anti-SLC44A4 antibody or antigen-binding fragment.

[511] In some other embodiments, the implementation of the antibody or antigennegative fragment purposefully affect the cell expressing STEAP1. In some embodiments, the antibody or antigen-binding fragment is an anti-STEAP1 antibody or antigen-binding fragment.

[512] In some embodiments, L is selected from any of the linkers disclosed herein or any combination of linker components disclosed herein. In some embodiments, L is a linker providing MC-Val-Cit-pABC, Mal-(PEG) ₂ -CO, MC-Val-Ala-pAB, MC-Val-Ala-pABC, MC-Val-Cit-pAB , Mal-Hex, Mal-Et or Mal-Et-O-Et. In some embodiments, the implementation of the linker may also provide one or more additional spacer units. In some embodiments, L is an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker. In some embodiments, L is an ADL12, ADL14, or ADL15 linker. In some embodiments, the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker may also include one or more additional spacer units.

[513] Another exemplary ADC has formula (I):

Ab-(LD) _p (I),

Where

Ab is an antibody or antigen-binding fragment that targets a neoplastic cell;

D is D1;

L is a linker that covalently attaches Ab to D; And

p is an integer from 1 to 15.

[514] In some embodiments, the antibody or antigen binding fragment targets a cell expressing HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, and /or STEAP1.

[515] In some embodiments, the antibody or antigen-binding fragment is targeted to a cell that expresses HER2. In some embodiments, the antibody or antigen-binding fragment is an anti-HER2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) containing the amino acid sequences under SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2) and SEQ ID NO:6 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

[516] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing CD138. In some embodiments, the antibody or antigen-binding fragment is an anti-CD138 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) containing the amino acid sequences under SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2) and SEQ ID NO:12 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:22. In some embodiments, the antibody or antigen-binding fragment comprises a mouse IgG2a heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a mouse Ig kappa light chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human IgG2a heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

[517] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing EPHA2. In some embodiments, the antibody or antigen-binding fragment is an anti-EPHA2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) containing the amino acid sequences under SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2) and SEQ ID NO:18 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:24. In some embodiments, the antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

[518] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing MSLN. In some embodiments, the antibody or antigen-binding fragment is an anti-MSLN antibody or antigen-binding fragment.

[519] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing FOLH1. In some embodiments, the antibody or antigen-binding fragment is an anti-FOLH1 antibody or antigen-binding fragment.

[520] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing CDH6. In some embodiments, the antibody or antigennegative fragment is an anti-CDH6 antibody or antigennegative fragment.

[521] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing CEACAM5. In some embodiments, the antibody or antigen-binding fragment is an anti-CEACAM5 antibody or antigen-binding fragment.

[522] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing CFC1B. In some embodiments, the antibody or antigennegative fragment is an anti-CFC1B antibody or antigennegative fragment.

[523] In some other embodiments, the antibody or antigen-binding fragment targets a cell that expresses ENPP3. In some embodiments, the antibody or antigen-binding fragment is an anti-ENPP3 antibody or antigen-binding fragment.

[524] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing FOLR1. In some embodiments, the antibody or antigen-binding fragment is an anti-FOLR1 antibody or antigen-binding fragment.

[525] In some other embodiments, the implementation of the antibody or antigennegative fragment purposefully affect a cell expressing HAVCR1. In some embodiments, the antibody or antigen-binding fragment is an anti-HAVCR1 antibody or antigen-binding fragment.

[526] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing the KIT. In some embodiments, the antibody or antigen-binding fragment is an anti-KIT antibody or antigen-binding fragment.

[527] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing MET. In some embodiments, the antibody or antigen-binding fragment is an anti-MET antibody or antigen-binding fragment.

[528] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing MUC16. In some embodiments, the antibody or antigennegative fragment is an anti-MUC16 antibody or antigennegative fragment.

[529] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing SLC39A6. In some embodiments, the antibody or antigen-binding fragment is an anti-SLC39A6 antibody or antigen-binding fragment.

[530] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing SLC44A4. In some embodiments, the antibody or antigen-binding fragment is an anti-SLC44A4 antibody or antigen-binding fragment.

[531] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing STEAP1. In some embodiments, the antibody or antigen-binding fragment is an anti-STEAP1 antibody or antigen-binding fragment.

[532] Another exemplary ADC has formula (I):

Ab-(LD) _p (I),

Where

Ab is an antibody or antigen-binding fragment that targets a neoplastic cell;

D is D4;

L is a linker that covalently attaches Ab to D; And

p is an integer from 1 to 15.

[533] In some embodiments, the antibody or antigen binding fragment targets a cell expressing HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, and /or STEAP1.

[534] In some embodiments, the antibody or antigen-binding fragment is targeted to a cell that expresses HER2. In some embodiments, the antibody or antigen-binding fragment is an anti-HER2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) containing the amino acid sequences under SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2) and SEQ ID NO:6 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

[535] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing CD138. In some embodiments, the antibody or antigen-binding fragment is an anti-CD138 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) containing the amino acid sequences under SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2) and SEQ ID NO:12 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:22. In some embodiments, the antibody or antigen-binding fragment comprises a mouse IgG2a heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a mouse Ig kappa light chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human IgG2a heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

[536] In some other embodiments, the implementation of the antibody or antigennegative fragment purposefully affect a cell expressing EPHA2. In some embodiments, the antibody or antigen-binding fragment is an anti-EPHA2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) containing the amino acid sequences under SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2) and SEQ ID NO:18 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:24. In some embodiments, the antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

[537] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing MSLN. In some embodiments, the antibody or antigen-binding fragment is an anti-MSLN antibody or antigen-binding fragment.

[538] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing FOLH1. In some embodiments, the antibody or antigen-binding fragment is an anti-FOLH1 antibody or antigen-binding fragment.

[539] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing CDH6. In some embodiments, the antibody or antigennegative fragment is an anti-CDH6 antibody or antigennegative fragment.

[540] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing CEACAM5. In some embodiments, the antibody or antigen-binding fragment is an anti-CEACAM5 antibody or antigen-binding fragment.

[541] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing CFC1B. In some embodiments, the antibody or antigennegative fragment is an anti-CFC1B antibody or antigennegative fragment.

[542] In some other embodiments, the antibody or antigen-binding fragment targets a cell that expresses ENPP3. In some embodiments, the antibody or antigen-binding fragment is an anti-ENPP3 antibody or antigen-binding fragment.

[543] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing FOLR1. In some embodiments, the antibody or antigen-binding fragment is an anti-FOLR1 antibody or antigen-binding fragment.

[544] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing HAVCR1. In some embodiments, the antibody or antigen-binding fragment is an anti-HAVCR1 antibody or antigen-binding fragment.

[545] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing the KIT. In some embodiments, the antibody or antigen-binding fragment is an anti-KIT antibody or antigen-binding fragment.

[546] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing MET. In some embodiments, the antibody or antigen-binding fragment is an anti-MET antibody or antigen-binding fragment.

[547] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing MUC16. In some embodiments, the antibody or antigennegative fragment is an anti-MUC16 antibody or antigennegative fragment.

[548] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing SLC39A6. In some embodiments, the antibody or antigen-binding fragment is an anti-SLC39A6 antibody or antigen-binding fragment.

[549] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing SLC44A4. In some embodiments, the antibody or antigen-binding fragment is an anti-SLC44A4 antibody or antigen-binding fragment.

[550] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing STEAP1. In some embodiments, the antibody or antigen-binding fragment is an anti-STEAP1 antibody or antigen-binding fragment.

[551] Another exemplary ADC has formula (I):

Ab-(LD) _p (I),

Where

Ab is an antibody or antigen-binding fragment that targets a neoplastic cell;

D is D12;

L is a linker that covalently attaches Ab to D; And

p is an integer from 1 to 15.

[552] In some embodiments, the antibody or antigen binding fragment targets a cell expressing HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, and /or STEAP1.

[553] In some embodiments, the antibody or antigen-binding fragment targets a cell that expresses HER2. In some embodiments, the antibody or antigen-binding fragment is an anti-HER2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) containing the amino acid sequences under SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2) and SEQ ID NO:6 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

[554] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing CD138. In some embodiments, the antibody or antigen-binding fragment is an anti-CD138 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) containing the amino acid sequences under SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2) and SEQ ID NO:12 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:22. In some embodiments, the antibody or antigen-binding fragment comprises a mouse IgG2a heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a mouse Ig kappa light chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human IgG2a heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

[555] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing EPHA2. In some embodiments, the antibody or antigen-binding fragment is an anti-EPHA2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) containing the amino acid sequences under SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2) and SEQ ID NO:18 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:24. In some embodiments, the antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

[556] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing MSLN. In some embodiments, the antibody or antigen-binding fragment is an anti-MSLN antibody or antigen-binding fragment.

[557] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing FOLH1. In some embodiments, the antibody or antigen-binding fragment is an anti-FOLH1 antibody or antigen-binding fragment.

[558] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing CDH6. In some embodiments, the antibody or antigennegative fragment is an anti-CDH6 antibody or antigennegative fragment.

[559] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing CEACAM5. In some embodiments, the antibody or antigen-binding fragment is an anti-CEACAM5 antibody or antigen-binding fragment.

[560] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing CFC1B. In some embodiments, the antibody or antigennegative fragment is an anti-CFC1B antibody or antigennegative fragment.

[561] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing ENPP3. In some embodiments, the antibody or antigen-binding fragment is an anti-ENPP3 antibody or antigen-binding fragment.

[562] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing FOLR1. In some embodiments, the antibody or antigen-binding fragment is an anti-FOLR1 antibody or antigen-binding fragment.

[563] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing HAVCR1. In some embodiments, the antibody or antigen-binding fragment is an anti-HAVCR1 antibody or antigen-binding fragment.

[564] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing the KIT. In some embodiments, the antibody or antigen-binding fragment is an anti-KIT antibody or antigen-binding fragment.

[565] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing MET. In some embodiments, the antibody or antigen-binding fragment is an anti-MET antibody or antigen-binding fragment.

[566] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing MUC16. In some embodiments, the antibody or antigennegative fragment is an anti-MUC16 antibody or antigennegative fragment.

[567] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing SLC39A6. In some embodiments, the antibody or antigen-binding fragment is an anti-SLC39A6 antibody or antigen-binding fragment.

[568] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing SLC44A4. In some embodiments, the antibody or antigen-binding fragment is an anti-SLC44A4 antibody or antigen-binding fragment.

[569] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing STEAP1. In some embodiments, the antibody or antigen-binding fragment is an anti-STEAP1 antibody or antigen-binding fragment.

[570] Another exemplary ADC has formula (I):

Ab-(LD) _p (I),

Where

Ab is an antibody or antigen-binding fragment that targets a neoplastic cell;

D is D15;

L is a linker that covalently attaches Ab to D; And

p is an integer from 1 to 15.

[571] In some embodiments, the antibody or antigen binding fragment targets a cell expressing HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, and /or STEAP1.

[572] In some embodiments, the antibody or antigen-binding fragment targets a cell that expresses HER2. In some embodiments, the antibody or antigen-binding fragment is an anti-HER2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) containing the amino acid sequences under SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2) and SEQ ID NO:6 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

[573] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing CD138. In some embodiments, the antibody or antigen-binding fragment is an anti-CD138 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) containing the amino acid sequences under SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2) and SEQ ID NO:12 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:22. In some embodiments, the antibody or antigen-binding fragment comprises a mouse IgG2a heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a mouse Ig kappa light chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human IgG2a heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

[574] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing EPHA2. In some embodiments, the antibody or antigen-binding fragment is an anti-EPHA2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) containing the amino acid sequences under SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2) and SEQ ID NO:18 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:24. In some embodiments, the antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

[575] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing MSLN. In some embodiments, the antibody or antigennegative fragment is an anti-MSLN antibody or antigennegative fragment.

[576] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing FOLH1. In some embodiments, the antibody or antigen-binding fragment is an anti-FOLH1 antibody or antigen-binding fragment.

[577] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing CDH6. In some embodiments, the antibody or antigennegative fragment is an anti-CDH6 antibody or antigennegative fragment.

[578] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing CEACAM5. In some embodiments, the antibody or antigen-binding fragment is an anti-CEACAM5 antibody or antigen-binding fragment.

[579] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing CFC1B. In some embodiments, the antibody or antigennegative fragment is an anti-CFC1B antibody or antigennegative fragment.

[580] In some other embodiments, the implementation of the antibody or antigennegative fragment purposefully affect a cell expressing ENPP3. In some embodiments, the antibody or antigen-binding fragment is an anti-ENPP3 antibody or antigen-binding fragment.

[581] In some other embodiments, the implementation of the antibody or antigennegative fragment purposefully affect a cell expressing FOLR1. In some embodiments, the antibody or antigen-binding fragment is an anti-FOLR1 antibody or antigen-binding fragment.

[582] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing HAVCR1. In some embodiments, the antibody or antigen-binding fragment is an anti-HAVCR1 antibody or antigen-binding fragment.

[583] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing the KIT. In some embodiments, the antibody or antigen-binding fragment is an anti-KIT antibody or antigen-binding fragment.

[584] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing MET. In some embodiments, the antibody or antigen-binding fragment is an anti-MET antibody or antigen-binding fragment.

[585] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing MUC16. In some embodiments, the antibody or antigennegative fragment is an anti-MUC16 antibody or antigennegative fragment.

[586] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing SLC39A6. In some embodiments, the antibody or antigen-binding fragment is an anti-SLC39A6 antibody or antigen-binding fragment.

[587] In some other embodiments, the antibody or antigen-binding fragment targets a cell expressing SLC44A4. In some embodiments, the antibody or antigen-binding fragment is an anti-SLC44A4 antibody or antigen-binding fragment.

[588] In some other embodiments, the antibody or antigen-binding fragment is targeted to a cell expressing STEAP1. In some embodiments, the antibody or antigen-binding fragment is an anti-STEAP1 antibody or antigen-binding fragment.

[589] In some embodiments, L is selected from any of the linkers disclosed herein, or any combination of linker components disclosed herein. In some embodiments, L is a cleavable linker. In some embodiments, L is a non-cleavable linker. In some embodiments, L is a linker providing MC-Val-Cit-pABC, MC-Val-Ala-pABC, MC-Val-Ala-pAB, MC-Glu-Val-Cit-pABC, Ala-Ala-Asn- pABC or β-glucuronide. In some embodiments, L is a linker providing Mal-Hex, Mal-Et, or Mal-Et-O-Et. In some embodiments, L is a linker providing MC-Val-Cit-pABC, Mal-(PEG) ₂ -CO, MC-Val-Ala-pAB, MC-Val-Ala-pABC, MC-Val-Cit-pAB , Mal-Hex, Mal-Et or Mal-Et-O-Et. In some embodiments, the implementation of the linker may also provide one or more additional spacer units. In some embodiments, L is an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker. In some embodiments, L is an ADL12, ADL14, or ADL15 linker. In some embodiments, the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker may also include one or more additional spacer units.

[590] In some embodiments , p is from 1 to 10. In some embodiments, p is from 2 to 8. In some embodiments, p is from 4 to 8. In some embodiments, p is 4. In some embodiments , p is 8.

[591] Another exemplary ADC has formula (I):

Ab-(L-D) _p (I)

where Ab is an anti-HER2 antibody or antigen binding fragment containing three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) containing the amino acid sequences under SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2) and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2) and SEQ ID NO:6 (LCDR3);

D is D2, D1, D4, D12 or D15;

L is a linker that covalently attaches Ab to D; And

p is an integer from 1 to 15.

[592] In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:20.

[593] In some embodiments, L is selected from any of the linkers disclosed herein, or any combination of linker components disclosed herein. In some embodiments, L is a linker providing MC-Val-Cit-pABC, Mal-(PEG) ₂ -CO, MC-Val-Ala-pAB, MC-Val-Ala-pABC, MC-Val-Cit-pAB , Mal-Hex, Mal-Et or Mal-Et-O-Et. In some embodiments, the implementation of the linker may also provide one or more additional spacer units. In some embodiments, L is an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker. In some embodiments, L is an ADL12, ADL14, or ADL15 linker. In some embodiments, the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker may also include one or more additional spacer units.

[594] Another exemplary ADC has formula (I):

Ab-(LD) _p (I),

where Ab is an anti-CD138 antibody or antigen binding fragment containing three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) containing the amino acid sequences under SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2) and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2) and SEQ ID NO:12 (LCDR3);

D is D2, D1, D4, D12 or D15;

L is a linker that covalently attaches Ab to D; And

p is an integer from 1 to 15.

[595] In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:22.

[596] In some embodiments, L is selected from any of the linkers disclosed herein or any combination of linker components disclosed herein. In some embodiments, L is a linker providing MC-Val-Cit-pABC, Mal-(PEG) ₂ -CO, MC-Val-Ala-pAB, MC-Val-Ala-pABC, MC-Val-Cit-pAB , Mal-Hex, Mal-Et or Mal-Et-O-Et. In some embodiments, the implementation of the linker may also provide one or more additional spacer units. In some embodiments, L is an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker. In some embodiments, L is an ADL12, ADL14, or ADL15 linker. In some embodiments, the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker may also include one or more additional spacer units.

[597] Another exemplary ADC has formula (I):

Ab-(LD) _p (I),

where Ab is an EPHA2 antibody or antigen binding fragment containing three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) containing the amino acid sequences under SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2) and SEQ ID NO:15 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2) and SEQ ID NO:18 (LCDR3);

D is D2, D1, D4, D12 or D15;

L is a linker that covalently attaches Ab to D; And

p is an integer from 1 to 15.

[598] In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:24.

[599] In some embodiments, L is selected from any of the linkers disclosed herein, or any combination of linker components disclosed herein. In some embodiments, L is a linker providing MC-Val-Cit-pABC, Mal-(PEG) ₂ -CO, MC-Val-Ala-pAB, MC-Val-Ala-pABC, MC-Val-Cit-pAB , Mal-Hex, Mal-Et or Mal-Et-O-Et. In some embodiments, the implementation of the linker may also provide one or more additional spacer units. In some embodiments, L is an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker. In some embodiments, L is an ADL12, ADL14, or ADL15 linker. In some embodiments, the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker may also include one or more additional spacer units.

[600] In various embodiments, ADCs containing a D2 or D1 drug fragment may include a cleavable or non-cleavable linker.

[601] In various embodiments, the linker is a cleavable linker. In various embodiments, the cleavable linker provides for MC-Val-Cit-pABC. In various embodiments, the cleavable linker provides for MC-Val-Ala-pABC. In various embodiments, the cleavable linker provides for MC-Val-Ala-pAB. In various embodiments, the cleavable linker comprises MC-Glu-Val-Cit-pABC. In various embodiments, the cleavable linker comprises MC-Ala-Ala-Asn-pABC.

[602] In various other embodiments, the linker is a non-cleavable linker. In various embodiments, the non-cleavable linker provides the MC alone or in combination with at least one additional spacer unit. In various embodiments, the non-cleavable linker provides Mal-Hex alone or in combination with at least one additional spacer unit. In various embodiments, the non-cleavable linker provides Mal-Et alone or in combination with at least one additional spacer unit. In various embodiments, the non-cleavable linker provides Mal-Et-O-Et alone or in combination with at least one additional spacer unit.

[603] In various embodiments, in ADCs containing a D2 or D1 drug fragment, p is from 1 to 10. In various embodiments, p is from 2 to 8. In various embodiments, p is from 4 to 8. In various embodiments , p is from 4 to 8. embodiments , p is 4. In various embodiments, p is 8.

[604] In various embodiments, an ADC disclosed herein comprising a D2 or D1 drug fragment exhibits an improved drug:antibody ratio, lower levels of aggregation, increased stability, increased targeted killing of cancer cells, reduced off-target killing of non-cancer cells, and /or increased cytotoxicity and/or activity compared to an ADC containing an alternative drug fragment (eg, an alternative drug fragment that is a splicing modulator). In some embodiments, an ADC disclosed herein comprising a D2 or D1 drug moiety provides appropriate or superior properties in one or more of the categories listed above, or across the spectrum of functional properties of a therapeutic ADC. In some embodiments, an ADC containing a D2 or D1 drug moiety exhibits unexpectedly effective activity and increased inhibition of cell growth and/or proliferation in cells that express an antigen targeted by the ADC compared to an ADC containing an alternative moiety - a drug (eg, an alternative drug fragment that is a splicing modulator). In some embodiments, activity can be measured by the concentration of a compound that results in a 50% reduction in cell proliferation (GI₅₀). In various embodiments, ADCs containing a D2 or D1 drug moiety exhibit surprisingly improved in vivo stability (e.g., plasma stability) compared to ADCs with an alternative drug moiety (e.g., an alternative drug moiety that is splicing modulator, e.g. Tilanstatin A). Cm., For example, ADC described in Puthenveetil et al. (Bioconjugate Chem. (2016) 27:1880-8) which shows the complete bioconversion of the payload in 72 hours (i.e., the acetate is completely hydrolysed).

[605] In certain embodiments, an intermediate that is a precursor to a linker moiety is reacted with a drug moiety (eg, a splicing modulator) under appropriate conditions. In certain embodiments, reactive groups are used on the drug and/or the intermediate or linker. The reaction product between the drug and the intermediate or derivatized drug (drug plus linker) is subsequently reacted with the antibody or antigen-binding fragment under appropriate conditions. Alternatively, the intermediate or linker can be reacted first with the antibody or antigen-binding fragment or derivatized antibody or antigen-binding fragment, and then reacted with the drug or derivatized drug.

[606] A number of different reactions are available for covalently attaching a drug moiety and/or a linker moiety to an antibody or antigen-binding moiety. This is often accomplished by reacting one or more amino acid residues of the antibody or antigen-binding moiety, including lysine amino groups, free carboxylic acid groups of glutamic acid and aspartic acid, cysteine sulfhydryl groups, and various aromatic amino acid moieties. For example, non-specific covalent attachment can be carried out using a carbodiimide reaction to link a carboxy (or amino) group on a drug moiety to an amino (or carboxy) group on an antibody or antigen-binding moiety. In addition, bifunctional agents such as dialdehydes or imidoesters can also be used to link the amino group on the drug moiety to the amino group on the antibody or antigen binding moiety. For attaching drugs (eg, splicing modulator) to binding agents, the reaction with the formation of Schiff bases is also suitable. This method involves the periodic oxidation of a drug that contains glycol or hydroxy groups, thereby forming an aldehyde, which then reacts with a binder. Attachment occurs through the formation of a Schiff base with amino groups of the binder. Isothiocyanates can also be used as coupling agents by covalently attaching drugs to binding agents. Other techniques are known to the person skilled in the art and are within the scope of the present invention. Examples of drug moieties that can be prepared and linked to an antibody or antigen binding moiety using various chemical structures known in the art include splicing modulators, such as the splicing modulators described and exemplified herein.

Linker Drug/Drug Compounds

[607] This document further discloses exemplary linker drug (L-D) compounds, as well as compositions containing multiple copies of such compounds. In various embodiments, linker-drug compounds disclosed herein may be defined by the general formula: L-D, where L=linker moiety, and D=drug moiety (e.g., splicing modulator drug moiety) . In certain embodiments, the disclosed L-D compounds are suitable for use in the ADCs described herein, for example, in the ADCs of formula (I).

[608] In various embodiments, linker-drug compounds (L-D) disclosed herein provide a linker-drug structure according to formula (III). In various embodiments, the present invention provides a linker-drug compound (L-D) of formula (III):

(III),

(III)

or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is absent or selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups, -OC(=O)-(C ₁ -C ₆ alkyl) and -CD ₃ groups;

R ² is absent or is a linker;

R ³ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups and -OC(=O)-(C ₁ -C ₆ alkyl) groups; And

each of R ⁴ , R ⁵ and R ⁸ is independently selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups and C ₁ -C ₆ alkyl groups;

each of R ⁶ and R ⁷ is independently selected from hydrogen, -OR ¹⁷ , -OC(=O)-R ¹⁷ , -OC(=O)-NR ¹⁵ R ¹⁶ , C ₁ -C ₆ alkyl groups, -NR ¹⁵ R ¹⁶ and linker;

each of R ¹⁵ and R ¹⁶ is independently selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C(=O)-OR ¹⁷ ;

R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups; And

,

,

,

и

;Z'' selected from

,

,

,

And

;

where each of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ¹⁵ , R ¹⁶ , and R ¹⁷ is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, groups -O-(C ₁ -C ₆ alkyl), -NR ¹⁵ R ¹⁶ , C ₃ -C ₈ cycloalkyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkylalkoxy groups , benzyl groups and C ₃ -C ₈ heterocyclyl groups;

where at least one of R ⁶ and R ⁷ is hydrogen; And

where, if R ² is a linker, then neither R ⁶ nor R ⁷ is a linker, and if R ⁶ or R ⁷ is a linker, then R ² is absent.

[609] In some embodiments, R ¹ is absent or selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₁ -C ₄ alkylcarboxylic acid groups, and C ₃ -C ₈ cycloalkyl groups. In some embodiments, R ¹ is absent. In some embodiments, R ¹ is hydrogen. In some embodiments, R ¹ is a C ₁ -C ₄ alkyl group. In some embodiments, R ¹ is methyl. In some embodiments, R ¹ is ethyl. In some embodiments, R ¹ is a C ₁ -C ₄ alkyl carboxylic acid group. In some embodiments, R ¹ is -CH ₂ CH ₂ CH ₂ CO ₂ H. In some embodiments, R ¹ is a C ₃ -C ₈ cycloalkyl group. In some embodiments, R ¹ is cycloheptyl.

[610] In some embodiments, R ³ is selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₁ -C ₄ alkyl alkoxy groups, C ₁ -C ₄ alkyl carboxylic acid groups, and C ₁ -C ₄ alkyl hydroxy groups. In some embodiments, R ³ is selected from hydrogen and C ₁ -C ₄ alkyl carboxylic acid groups. In some embodiments, R ³ is hydrogen. In some embodiments, R ³ is a C ₁ -C ₄ alkyl carboxylic acid group. In some embodiments, R ³ is -CH ₂ CH ₂ CO ₂ H.

[611] In some embodiments, R ⁴ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl) groups, -OC(=O)-(C ₁ -C ₄ alkyl) groups, and C ₁ -C ₄ alkyl groups. In some embodiments, R ⁴ is hydrogen. In some embodiments, R ⁴ is hydroxyl. In some embodiments, R ⁴ is a -O-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁴ is -OCH ₃ . In some embodiments, R ⁴ is -OCH ₂ CH ₃ . In some embodiments, R ⁴ is a -OC(=O)-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁴ is -OC(=O)-CH ₃ . In some embodiments, R ⁴ is -OC(=O)-CH ₂ CH ₃ . In some embodiments, R ⁴ is a C ₁ -C ₄ alkyl group. In some embodiments, R ⁴ is methyl. In some embodiments, R ⁴ is ethyl.

[612] In some embodiments, R ⁵ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl) groups, and C ₁ -C ₄ alkyl groups. In some embodiments, R ⁵ is hydrogen. In some embodiments, R ⁵ is hydroxyl. In some embodiments, R ⁵ is a -O-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁵ is a C ₁ -C ₄ alkyl group.

[613] In some embodiments, R ² is absent and R ⁶ is a linker. In some embodiments, R ² is absent and R ⁷ is a linker. In some embodiments, R ² is a linker. In some embodiments, R ⁶ is hydrogen. In some embodiments, R ⁷ is hydrogen.

[614] In some embodiments, R ⁶ is hydrogen and R ⁷ is —OR ¹⁷ . In some embodiments, R ⁶ is hydrogen and R ⁷ is -OR ¹⁷ , where R ¹⁷ is selected from hydrogen and C ¹ -C ⁴ alkyl groups. In some embodiments, R ⁶ is hydrogen and R ⁷ is —OR ¹⁷ , where R ¹⁷ is hydrogen. In some embodiments, R ⁶ is -OR ¹⁷ and R ⁷ is hydrogen. In some embodiments, R ⁶ is -OR ¹⁷ and R ⁷ is hydrogen, where R ¹⁷ is selected from hydrogen and C ¹ -C ⁴ alkyl groups. In some embodiments, R ⁶ is -OR ¹⁷ and R ⁷ is hydrogen, where R ¹⁷ is hydrogen. In some embodiments, R ⁶ is hydrogen and R ⁷ is —NR ¹⁵ R ¹⁶ . In some embodiments, R ⁶ is hydrogen and R ⁷ is -NR ¹⁵ R ¹⁶ where R ¹⁵ is H and R ¹⁶ is selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C (=O)-OR ¹⁷ . In some embodiments, R ⁶ is hydrogen and R ⁷ is -NR ¹⁵ R ¹⁶ where R ¹⁵ is H and R ¹⁶ is selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C (=O)-OR ¹⁷ , and where R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups and C ₃ -C ₈ heterocyclyl groups. In some embodiments, R ⁶ is -OR ¹⁷ . In some embodiments, R ⁶ is -OC(=O)-R ¹⁷ . In some embodiments, R ⁶ is C ₁ -C ₆ alkyl. In some embodiments, R ⁶ is C ₁ -C ₄ alkyl. In some embodiments, R ⁶ is C ₁ alkyl. In some embodiments, R ⁶ is -NR ¹⁵ R ¹⁶ .

[615] In some embodiments, R ⁷ is -OR ¹⁷ . In some embodiments, R ⁷ is -OC(=O)-R ¹⁷ . In some embodiments, R ⁷ is C ₁ -C ₆ alkyl. In some embodiments, R ⁷ is C ₁ -C ₄ alkyl. In some embodiments, R ⁷ is C ₁ alkyl. In some embodiments, R ⁷ is -NR ¹⁵ R ¹⁶ .

[616] In some embodiments, R ⁸ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl) and (C ₁ -C ₄ alkyl) groups. In some embodiments, R ⁸ is hydrogen. In some embodiments, R ⁸ is a hydroxyl group. In some embodiments, R ⁸ is a -O-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁸ is a -O-(C ₁ alkyl) group.

[617] In some embodiments, R ¹⁵ is hydrogen. In some embodiments, R ¹⁵ is R ¹⁷ . In some embodiments, R ¹⁵ is -C(=O)-R ¹⁷ . In some embodiments, R ¹⁵ is -C(=O)-OR ¹⁷ .

[618] In some embodiments, R ¹⁶ is hydrogen. In some embodiments, R ¹⁶ is R ¹⁷ . In some embodiments, R ¹⁶ is -C(=O)-R ¹⁷ . In some embodiments, R ¹⁶ is -C(=O)-OR ¹⁷ .

[619] In some embodiments, R ¹⁷ is selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₃ -C ₆ cycloalkyl groups, and C ₃ -C ₈ heterocyclyl groups. In some embodiments, R ¹⁷ is hydrogen. In some embodiments, R ¹⁷ is a C ₁ -C ₄ alkyl group. In some embodiments, R ¹⁷ is a C ₁ alkyl group. In some embodiments, R ¹⁷ is a C ₃ -C ₆ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₃ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₄ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₅ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₆ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₃ -C ₈ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₃ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₄ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₅ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₆ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₇ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₈ heterocyclyl group.

. В некоторых вариантах осуществления Z' представляет собой

. В некоторых вариантах осуществления Z' представляет собой

. В некоторых вариантах осуществления Z' представляет собой

. В некоторых вариантах осуществления Z' представляет собой

.[620] In some embodiments, Z' is

. In some embodiments, Z' is

. In some embodiments, Z' is

. In some embodiments, Z' is

. In some embodiments, Z' is

.

[621] In some embodiments, R ² is a linker. In some embodiments, the implementation of the linker provides at least one cleavable peptide fragment. In some embodiments, at least one cleavable peptide fragment is cleaved by the action of an enzyme. In some embodiments, the linker or cleavable peptide fragment comprises at least one amino acid unit. In some embodiments, at least one amino acid unit is selected from arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan and citrulline. In some embodiments, at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, the implementation of the linker provides citrulline and valine. In some embodiments, the implementation of the linker provides alanine and valine.

[622] In some embodiments, the implementation of the linker provides a fragment selected from sulfonamide, β-glucuronide, disulfide and carbonyl. In some embodiments, the implementation of the linker provides a sulfonamide. In some embodiments, the implementation of the linker provides β-glucuronide. In some embodiments, the implementation of the linker provides a disulfide. In some embodiments, the implementation of the linker provides a carbonyl.

[623] In some embodiments, the implementation of the linker provides a spacer link. In some embodiments, the spacer unit is selected from alkyl groups and polyethylene glycol (PEG) moieties. In some embodiments, the alkyl group is a C ₁ -C ₁₂ alkyl group. In some embodiments, the alkyl group is a C ₁ -C ₆ alkyl group. In some embodiments, the alkyl group is methylene. In some embodiments, the alkyl group is ethylene. In some embodiments, the alkyl group is n-propylene. In some embodiments, the alkyl group is n-butylene. In some embodiments, the alkyl group is n-pentylene. In some embodiments, the alkyl group is n-hexylene. In some embodiments, the PEG fragment provides -(PEG) _m -, where m is an integer from 1 to 10. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In in some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6.

[624] In some embodiments, the implementation of the linker provides a maleimide (Mal) fragment ("Mal-spacer link"). In some embodiments, the linker provides a self-cleaving spacer unit. In some embodiments, the self-cleaving spacer unit is selected from p-aminobenzyloxycarbonyl (pABC) and p-aminobenzyl (pAB).

[625] In some embodiments, the linker provides a Mal spacer unit, an alkyl group, at least one amino acid unit, and a self-cleaving spacer. In some embodiments, at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, at least one amino acid unit provides for alanine and valine. In some embodiments, at least one amino acid unit provides citrulline and valine. In some embodiments, the self-cleaving spacer is selected from pAB and pABC. In some embodiments, the self-cleaving spacer provides pAB. In some embodiments, the self-cleaving spacer provides pABC. In some embodiments, the alkyl group provides a C ₁ -C ₆ alkyl group.

[626] In some embodiments, the linker provides a Mal spacer unit, a PEG fragment, at least one amino acid unit, and a self-cleaving spacer. In some embodiments, at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, at least one amino acid unit provides for alanine and valine. In some embodiments, at least one amino acid unit provides citrulline and valine. In some embodiments, the self-cleaving spacer is selected from pAB and pABC. In some embodiments, the self-cleaving spacer provides pAB. In some embodiments, the self-cleaving spacer provides pABC. In some embodiments, the PEG fragment provides -(PEG) _m -, where m is an integer from 1 to 6.

[627] In various other embodiments, the linker-drug compounds (L-D) disclosed herein provide a linker-drug structure according to formula (V). In various embodiments, the present invention provides a linker-drug compound (L-D) of formula (V):

(V),

(V)

or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is absent or selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups, -OC(=O)-(C ₁ -C ₆ alkyl) and -CD ₃ groups;

R ² is absent or is a linker;

R ³ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups and -OC(=O)-(C ₁ -C ₆ alkyl) groups; And

each of R ⁴ , R ⁵ and R ⁸ is independently selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups and C ₁ -C ₆ alkyl groups;

each of R ⁶ and R ⁷ is independently selected from hydrogen, -OR ¹⁷ , -OC(=O)-R ¹⁷ , -OC(=O)-NR ¹⁵ R ¹⁶ , C ₁ -C ₆ alkyl groups, -NR ¹⁵ R ¹⁶ and linker;

each of R ¹⁵ and R ¹⁶ is independently selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C(=O)-OR ¹⁷ ; And

R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups;

where each of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ¹⁵ , R ¹⁶ and R ¹⁷ is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups , C ₁ -C ₆ alkyl groups, -O-(C ₁ -C ₆ alkyl), -NR ¹⁵ R ¹⁶ , C ₃ -C ₈ cycloalkyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkylalkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups;

where at least one of R ⁶ and R ⁷ is hydrogen; And

where, if R ² is a linker, then neither R ⁶ nor R ⁷ is a linker, and if R ⁶ or R ⁷ is a linker, then R ² is absent.

[628] In some embodiments, R ¹ is absent or selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₁ -C ₄ alkylcarboxylic acid groups, and C ₃ -C ₈ cycloalkyl groups. In some embodiments, R ¹ is absent. In some embodiments, R ¹ is hydrogen. In some embodiments, R ¹ is a C ₁ -C ₄ alkyl group. In some embodiments, R ¹ is methyl. In some embodiments, R ¹ is ethyl. In some embodiments, R ¹ is a C ₁ -C ₄ alkyl carboxylic acid group. In some embodiments, R ¹ is -CH ₂ CH ₂ CH ₂ CO ₂ H. In some embodiments, R ¹ is a C ₃ -C ₈ cycloalkyl group. In some embodiments, R ¹ is cycloheptyl.

[629] In some embodiments, R ³ is selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₁ -C ₄ alkyl alkoxy groups, C ₁ -C ₄ alkyl carboxylic acid groups, and C ₁ -C ₄ alkyl hydroxy groups. In some embodiments, R ³ is selected from hydrogen and C ₁ -C ₄ alkyl carboxylic acid groups. In some embodiments, R ³ is hydrogen. In some embodiments, R ³ is a C ₁ -C ₄ alkyl carboxylic acid group. In some embodiments, R ³ is -CH ₂ CH ₂ CO ₂ H.

[630] In some embodiments, R ⁴ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl), -OC(=O)-(C ₁ -C ₄ alkyl) groups, and C ₁ -C ₄ alkyl groups. In some embodiments, R ⁴ is hydrogen. In some embodiments, R ⁴ is hydroxyl. In some embodiments, R ⁴ is a -O-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁴ is -OCH ₃ . In some embodiments, R ⁴ is -OCH ₂ CH ₃ . In some embodiments, R ⁴ is a -OC(=O)-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁴ is -OC(=O)-CH ₃ . In some embodiments, R ⁴ is -OC(=O)-CH ₂ CH ₃ . In some embodiments, R ⁴ is a C ₁ -C ₄ alkyl group. In some embodiments, R ⁴ is methyl. In some embodiments, R ⁴ is ethyl.

[631] In some embodiments, R ⁵ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl) groups, and C ₁ -C ₄ alkyl groups. In some embodiments, R ⁵ is hydrogen. In some embodiments, R ⁵ is hydroxyl. In some embodiments, R ⁵ is -O-(C ₁ -C ₄ alkyl). In some embodiments, R ⁵ is a C ₁ -C ₄ alkyl group.

[632] In some embodiments, R ² is absent and R ⁶ is a linker. In some embodiments, R ² is absent and R ⁷ is a linker. In some embodiments, R ² is a linker. In some embodiments, R ⁶ is hydrogen. In some embodiments, R ⁷ is hydrogen.

[633] In some embodiments, R ⁶ is hydrogen and R ⁷ is —OR ¹⁷ . In some embodiments, R ⁶ is hydrogen and R ⁷ is -OR ¹⁷ , where R ¹⁷ is selected from hydrogen and C ¹ -C ⁴ alkyl groups. In some embodiments, R ⁶ is hydrogen and R ⁷ is —OR ¹⁷ , where R ¹⁷ is hydrogen. In some embodiments, R ⁶ is -OR ¹⁷ and R ⁷ is hydrogen. In some embodiments, R ⁶ is -OR ¹⁷ and R ⁷ is hydrogen, where R ¹⁷ is selected from hydrogen and C ¹ -C ⁴ alkyl groups. In some embodiments, R ⁶ is -OR ¹⁷ and R ⁷ is hydrogen, where R ¹⁷ is hydrogen. In some embodiments, R ⁶ is hydrogen and R ⁷ is —NR ¹⁵ R ¹⁶ . In some embodiments, R ⁶ is hydrogen and R ⁷ is -NR ¹⁵ R ¹⁶ where R ¹⁵ is H and R ¹⁶ is selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C (=O)-OR ¹⁷ . In some embodiments, R ⁶ is hydrogen and R ⁷ is -NR ¹⁵ R ¹⁶ where R ¹⁵ is H and R ¹⁶ is selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C (=O)-OR ¹⁷ , and where R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups and C ₃ -C ₈ heterocyclyl groups. In some embodiments, R ⁶ is -OR ¹⁷ . In some embodiments, R ⁶ is -OC(=O)-R ¹⁷ . In some embodiments, R ⁶ is C ₁ -C ₆ alkyl. In some embodiments, R ⁶ is C ₁ -C ₄ alkyl. In some embodiments, R ⁶ is C ₁ alkyl. In some embodiments, R ⁶ is -NR ¹⁵ R ¹⁶ .

[634] In some embodiments, R ⁷ is -OR ¹⁷ . In some embodiments, R ⁷ is -OC(=O)-R ¹⁷ . In some embodiments, R ⁷ is C ₁ -C ₆ alkyl. In some embodiments, R ⁷ is C ₁ -C ₄ alkyl. In some embodiments, R ⁷ is C ₁ alkyl. In some embodiments, R ⁷ is -NR ¹⁵ R ¹⁶ .

[635] In some embodiments, R ⁸ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl) and (C ₁ -C ₄ alkyl) groups. In some embodiments, R ⁸ is hydrogen. In some embodiments, R ⁸ is a hydroxyl group. In some embodiments, R ⁸ is a -O-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁸ is a -O-(C ₁ alkyl) group.

[636] In some embodiments, R ¹⁵ is hydrogen. In some embodiments, R ¹⁵ is R ¹⁷ . In some embodiments, R ¹⁵ is -C(=O)-R ¹⁷ . In some embodiments, R ¹⁵ is -C(=O)-OR ¹⁷ .

[637] In some embodiments, R ¹⁶ is hydrogen. In some embodiments, R ¹⁶ is R ¹⁷ . In some embodiments, R ¹⁶ is -C(=O)-R ¹⁷ . In some embodiments, R ¹⁶ is -C(=O)-OR ¹⁷ .

[638] In some embodiments, R ¹⁷ is selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₃ -C ₆ cycloalkyl groups, and C ₃ -C ₈ heterocyclyl groups. In some embodiments, R ¹⁷ is hydrogen. In some embodiments, R ¹⁷ is a C ₁ -C ₄ alkyl group. In some embodiments, R ¹⁷ is a C ₁ alkyl group. In some embodiments, R ¹⁷ is a C ₃ -C ₆ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₃ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₄ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₅ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₆ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₃ -C ₈ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₃ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₄ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₅ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₆ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₇ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₈ heterocyclyl group.

[639] In some embodiments, R ² is a linker. In some embodiments, the implementation of the linker provides at least one cleavable peptide fragment. In some embodiments, at least one cleavable peptide fragment is cleaved by the action of an enzyme. In some embodiments, the linker or cleavable peptide fragment comprises at least one amino acid unit. In some embodiments, at least one amino acid unit is selected from arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan and citrulline. In some embodiments, at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, the implementation of the linker provides citrulline and valine. In some embodiments, the implementation of the linker provides alanine and valine.

[640] In some embodiments, the implementation of the linker provides a fragment selected from sulfonamide, β-glucuronide, disulfide and carbonyl. In some embodiments, the implementation of the linker provides a sulfonamide. In some embodiments, the implementation of the linker provides β-glucuronide. In some embodiments, the implementation of the linker provides a disulfide. In some embodiments, the implementation of the linker provides a carbonyl.

[641] In some embodiments, the implementation of the linker provides a spacer link. In some embodiments, the spacer unit is selected from alkyl groups and polyethylene glycol (PEG) moieties. In some embodiments, the alkyl group is a C ₁ -C ₁₂ alkyl group. In some embodiments, the alkyl group is a C ₁ -C ₆ alkyl group. In some embodiments, the alkyl group is methylene. In some embodiments, the alkyl group is ethylene. In some embodiments, the alkyl group is n-propylene. In some embodiments, the alkyl group is n-butylene. In some embodiments, the alkyl group is n-pentylene. In some embodiments, the alkyl group is n-hexylene. In some embodiments, the PEG fragment provides -(PEG) _m -, where m is an integer from 1 to 10. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In in some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6.

[642] In some embodiments, the implementation of the linker provides a maleimide (Mal) fragment ("Mal-spacer link"). In some embodiments, the linker provides a self-cleaving spacer unit. In some embodiments, the self-cleaving spacer unit is selected from p-aminobenzyloxycarbonyl (pABC) and p-aminobenzyl (pAB).

[643] In some embodiments, the linker provides a Mal spacer unit, an alkyl group, at least one amino acid unit, and a self-cleaving spacer. In some embodiments, at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, at least one amino acid unit provides for alanine and valine. In some embodiments, at least one amino acid unit provides citrulline and valine. In some embodiments, the self-cleaving spacer is selected from pAB and pABC. In some embodiments, the self-cleaving spacer provides pAB. In some embodiments, the self-cleaving spacer provides pABC. In some embodiments, the alkyl group provides a C ₁ -C ₆ alkyl group.

[644] In some embodiments, the linker provides a Mal spacer unit, a PEG fragment, at least one amino acid unit, and a self-cleaving spacer. In some embodiments, at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, at least one amino acid unit provides for alanine and valine. In some embodiments, at least one amino acid unit provides citrulline and valine. In some embodiments, the self-cleaving spacer is selected from pAB and pABC. In some embodiments, the self-cleaving spacer provides pAB. In some embodiments, the self-cleaving spacer provides pABC. In some embodiments, the PEG fragment provides -(PEG) _m -, where m is an integer from 1 to 6.

[645] In various other embodiments, the linker-drug (L-D) compounds disclosed herein provide a linker-drug structure according to formula (VII). In various embodiments, the present invention provides a linker-drug compound (L-D) of formula (VII):

(VII),

(VII)

or a pharmaceutically acceptable salt thereof, wherein:

each of R ¹ and R ⁹ is independently absent or selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups, -OC(=O)-(C ₁ -C ₆ alkyl) and -CD ₃ groups;

R ² is absent or is a linker;

R ³ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups and -OC(=O)-(C ₁ -C ₆ alkyl) groups;

each of R ⁴ , R ⁵ and R ⁸ is independently selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups and C ₁ -C ₆ alkyl groups;

each of R ⁶ and R ⁷ is independently selected from hydrogen, -OR ¹⁷ , -OC(=O)-R ¹⁷ , -OC(=O)-NR ¹⁵ R ¹⁶ , C ₁ -C ₆ alkyl groups, -NR ¹⁵ R ¹⁶ and linker;

R ¹⁰ is selected from hydrogen, C ₁ -C ₆ alkyl groups, -C(=O)-(C ₁ -C ₆ alkyl) and -CD ₃ groups;

each of R ¹⁵ and R ¹⁶ is independently selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C(=O)-OR ¹⁷ ;

R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups; And

a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

where each of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹⁵ , R ¹⁶ , and R ¹⁷ is independently substituted with 0-3 groups, independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, -O-(C ₁ -C ₆ alkyl), -NR ¹⁵ R ¹⁶ groups, C ₃ -C ₈ cycloalkyl groups, C ₁ -C ₆ alkyl hydroxy groups, C ₁ -C ₆ alkylalkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups;

where at least one of R ⁶ and R ⁷ is hydrogen;

where, if R ² is a linker, then neither R ⁶ nor R ⁷ is a linker, and if R ⁶ or R ⁷ is a linker, then R ² is absent; And

where both R ¹ and R ⁹ cannot be absent.

[646] In some embodiments, R ¹ is absent or selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₁ -C ₄ alkylcarboxylic acid groups, and C ₃ -C ₈ cycloalkyl groups. In some embodiments, R ¹ is absent. In some embodiments, R ¹ is hydrogen. In some embodiments, R ¹ is a C ₁ -C ₄ alkyl group. In some embodiments, R ¹ is methyl. In some embodiments, R ¹ is ethyl. In some embodiments, R ¹ is a C ₁ -C ₄ alkyl carboxylic acid group. In some embodiments, R ¹ is -CH ₂ CH ₂ CH ₂ CO ₂ H. In some embodiments, R ¹ is a C ₃ -C ₈ cycloalkyl group. In some embodiments, R ¹ is cycloheptyl.

[647] In some embodiments, R ³ is selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₁ -C ₄ alkyl alkoxy groups, C ₁ -C ₄ alkyl carboxylic acid groups, and C ₁ -C ₄ alkyl hydroxy groups. In some embodiments, R ³ is selected from hydrogen and C ₁ -C ₄ alkyl carboxylic acid groups. In some embodiments, R ³ is hydrogen. In some embodiments, R ³ is a C ₁ -C ₄ alkyl carboxylic acid group. In some embodiments, R ³ is -CH ₂ CH ₂ CO ₂ H.

[648] In some embodiments, R ⁴ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl) groups, -OC(=O)-(C ₁ -C ₄ alkyl) groups, and C ₁ -C ₄ alkyl groups. In some embodiments, R ⁴ is hydrogen. In some embodiments, R ⁴ is hydroxyl. In some embodiments, R ⁴ is a -O-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁴ is -OCH ₃ . In some embodiments, R ⁴ is -OCH ₂ CH ₃ . In some embodiments, R ⁴ is a -OC(=O)-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁴ is -OC(=O)-CH ₃ . In some embodiments, R ⁴ is -OC(=O)-CH ₂ CH ₃ . In some embodiments, R ⁴ is a C ₁ -C ₄ alkyl group. In some embodiments, R ⁴ is methyl. In some embodiments, R ⁴ is ethyl.

[649] In some embodiments, R ⁵ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl) groups, and C ₁ -C ₄ alkyl groups. In some embodiments, R ⁵ is hydrogen. In some embodiments, R ⁵ is hydroxyl. In some embodiments, R ⁵ is a -O-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁵ is a C ₁ -C ₄ alkyl group.

[650] In some embodiments, R ⁹ is absent or selected from hydrogen, C ₁ -C ₄ alkyl groups, -(C=O)-(C ₁ -C ₄ alkyl) and -CD ₃ groups. In some embodiments, R ⁹ is absent. In some embodiments, R ⁹ is hydrogen. In some embodiments, R ⁹ is a C ₁ -C ₄ alkyl group. In some embodiments, the C ₁ -C ₄ alkyl group is methyl. In some embodiments, the C ₁ -C ₄ alkyl group is ethyl. In some embodiments, R ⁹ is -(C=O)-(C ₁ -C ₄ alkyl). In some embodiments, the -(C=O)-(C ₁ -C ₄ alkyl) group is -(C=O)-methyl. In some embodiments, R ⁹ is -CD ₃ .

[651] In some embodiments, R ¹⁰ is selected from hydrogen, C ₁ -C ₄ alkyl groups, -(C=O)-(C ₁ -C ₄ alkyl) and -CD _{3 groups} . In some embodiments, R ¹⁰ is hydrogen. In some embodiments, R ¹⁰ is a C ₁ -C ₄ alkyl group. In some embodiments, the C ₁ -C ₄ alkyl group is methyl. In some embodiments, the C ₁ -C ₄ alkyl group is ethyl. In some embodiments, R ¹⁰ is -(C=O)-(C ₁ -C ₄ alkyl). In some embodiments, the -(C=O)-(C ₁ -C ₄ alkyl) group is -(C=O)-methyl. In some embodiments, R ¹⁰ is -CD ₃ .

[652] In some embodiments, R ² is absent and R ⁶ is a linker. In some embodiments, R ² is absent and R ⁷ is a linker. In some embodiments, R ² is a linker. In some embodiments, R ⁶ is hydrogen. In some embodiments, R ⁷ is hydrogen. In some embodiments, R ⁶ is hydrogen and R ⁷ is —OR ¹⁷ . In some embodiments, R ⁶ is hydrogen and R ⁷ is -OR ¹⁷ , where R ¹⁷ is selected from hydrogen and C ¹ -C ⁴ alkyl groups. In some embodiments, R ⁶ is hydrogen and R ⁷ is —OR ¹⁷ , where R ¹⁷ is hydrogen. In some embodiments, R ⁶ is -OR ¹⁷ and R ⁷ is hydrogen. In some embodiments, R ⁶ is -OR ¹⁷ and R ⁷ is hydrogen, where R ¹⁷ is selected from hydrogen and C ¹ -C ⁴ alkyl groups. In some embodiments, R ⁶ is -OR ¹⁷ and R ⁷ is hydrogen, where R ¹⁷ is hydrogen. In some embodiments, R ⁶ is hydrogen and R ⁷ is —NR ¹⁵ R ¹⁶ . In some embodiments, R ⁶ is hydrogen and R ⁷ is -NR ¹⁵ R ¹⁶ where R ¹⁵ is H and R ¹⁶ is selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C (=O)-OR ¹⁷ . In some embodiments, R ⁶ is hydrogen and R ⁷ is -NR ¹⁵ R ¹⁶ where R ¹⁵ is H and R ¹⁶ is selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C (=O)-OR ¹⁷ , and where R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups and C ₃ -C ₈ heterocyclyl groups. In some embodiments, R ⁶ is -OR ¹⁷ . In some embodiments, R ⁶ is -OC(=O)-R ¹⁷ . In some embodiments, R ⁶ is C ₁ -C ₆ alkyl. In some embodiments, R ⁶ is C ₁ -C ₄ alkyl. In some embodiments, R ⁶ is C ₁ alkyl. In some embodiments, R ⁶ is -NR ¹⁵ R ¹⁶ .

[653] In some embodiments, R ⁷ is -OR ¹⁷ . In some embodiments, R ⁷ is -OC(=O)-R ¹⁷ . In some embodiments, R ⁷ is C ₁ -C ₆ alkyl. In some embodiments, R ⁷ is C ₁ -C ₄ alkyl. In some embodiments, R ⁷ is C ₁ alkyl. In some embodiments, R ⁷ is -NR ¹⁵ R ¹⁶ .

[654] In some embodiments, R ⁸ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl) and (C ₁ -C ₄ alkyl) groups. In some embodiments, R ⁸ is hydrogen. In some embodiments, R ⁸ is a hydroxyl group. In some embodiments, R ⁸ is a -O-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁸ is a -O-(C ₁ alkyl) group.

[655] In some embodiments, R ¹⁵ is hydrogen. In some embodiments, R ¹⁵ is R ¹⁷ . In some embodiments, R ¹⁵ is -C(=O)-R ¹⁷ . In some embodiments, R ¹⁵ is -C(=O)-OR ¹⁷ .

[656] In some embodiments, R ¹⁶ is hydrogen. In some embodiments, R ¹⁶ is R ¹⁷ . In some embodiments, R ¹⁶ is -C(=O)-R ¹⁷ . In some embodiments, R ¹⁶ is -C(=O)-OR ¹⁷ .

[657] In some embodiments, R ¹⁷ is selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₃ -C ₆ cycloalkyl groups, and C ₃ -C ₈ heterocyclyl groups. In some embodiments, R ¹⁷ is hydrogen. In some embodiments, R ¹⁷ is a C ₁ -C ₄ alkyl group. In some embodiments, R ¹⁷ is a C ₁ alkyl group. In some embodiments, R ¹⁷ is a C ₃ -C ₆ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₃ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₄ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₅ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₆ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₃ -C ₈ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₃ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₄ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₅ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₆ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₇ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₈ heterocyclyl group.

[658] In some embodiments, a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, a is 1, 2, 3, 4, 5, or 6. In some in some embodiments, a is 1, 2, 3, 4, or 5. In some embodiments, a is 1, 2, 3, or 4. In some embodiments, a is 1, 2, or 3. In some embodiments, a is 1 or 2. In some embodiments, a is 1. In some embodiments, a is 2. In some embodiments, a is 3. In some embodiments, a is 4. In some embodiments, a is 5. In some embodiments, a is 6. In some embodiments, a is 6. in some embodiments, a is 7. In some embodiments, a is 8. In some embodiments, a is 9. In some embodiments, a is 10.

[659] In some embodiments, R ² is a linker. In some embodiments, the implementation of the linker provides at least one cleavable peptide fragment. In some embodiments, at least one cleavable peptide fragment is cleaved by the action of an enzyme. In some embodiments, the linker or cleavable peptide fragment comprises at least one amino acid unit. In some embodiments, at least one amino acid unit is selected from arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan and citrulline. In some embodiments, at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, the implementation of the linker provides citrulline and valine. In some embodiments, the implementation of the linker provides alanine and valine.

[660] In some embodiments, the implementation of the linker provides a fragment selected from sulfonamide, β-glucuronide, disulfide and carbonyl. In some embodiments, the implementation of the linker provides a sulfonamide. In some embodiments, the implementation of the linker provides β-glucuronide. In some embodiments, the implementation of the linker provides a disulfide. In some embodiments, the implementation of the linker provides a carbonyl.

[661] In some embodiments, the implementation of the linker provides a spacer link. In some embodiments, the spacer unit is selected from alkyl groups and polyethylene glycol (PEG) moieties. In some embodiments, the alkyl group is a C ₁ -C ₁₂ alkyl group. In some embodiments, the alkyl group is a C ₁ -C ₆ alkyl group. In some embodiments, the alkyl group is methylene. In some embodiments, the alkyl group is ethylene. In some embodiments, the alkyl group is n-propylene. In some embodiments, the alkyl group is n-butylene. In some embodiments, the alkyl group is n-pentylene. In some embodiments, the alkyl group is n-hexylene. In some embodiments, the PEG fragment provides -(PEG) _m -, where m is an integer from 1 to 10. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In in some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6.

[662] In some embodiments, the implementation of the linker provides a maleimide (Mal) fragment ("Mal-spacer link"). In some embodiments, the linker provides a self-cleaving spacer unit. In some embodiments, the self-cleaving spacer unit is selected from p-aminobenzyloxycarbonyl (pABC) and p-aminobenzyl (pAB).

[663] In some embodiments, the linker provides a Mal spacer unit, an alkyl group, at least one amino acid unit, and a self-cleaving spacer. In some embodiments, at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, at least one amino acid unit provides for alanine and valine. In some embodiments, at least one amino acid unit provides citrulline and valine. In some embodiments, the self-cleaving spacer is selected from pAB and pABC. In some embodiments, the self-cleaving spacer provides pAB. In some embodiments, the self-cleaving spacer provides pABC. In some embodiments, the alkyl group provides a C ₁ -C ₆ alkyl group.

[664] In some embodiments, the linker provides a Mal spacer unit, a PEG fragment, at least one amino acid unit, and a self-cleaving spacer. In some embodiments, at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, at least one amino acid unit provides for alanine and valine. In some embodiments, at least one amino acid unit provides citrulline and valine. In some embodiments, the self-cleaving spacer is selected from pAB and pABC. In some embodiments, the self-cleaving spacer provides pAB. In some embodiments, the self-cleaving spacer provides pABC. In some embodiments, the PEG fragment provides -(PEG) _m -, where m is an integer from 1 to 6.

[665] In various other embodiments, the linker-drug compounds (L-D) disclosed herein provide a linker-drug structure according to formula (IX). In various embodiments, the present invention provides a linker-drug compound (L-D) of formula (IX):

(IX),

(IX)

or a pharmaceutically acceptable salt thereof, wherein:

R ¹ is absent or selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups, -OC(=O)-(C ₁ -C ₆ alkyl) and -CD ₃ groups;

R ² is a linker;

R ³ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups and -OC(=O)-(C ₁ -C ₆ alkyl) groups;

R ⁴ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₆ alkyl), -OC(=O)-(C ₁ -C ₆ alkyl) groups, and C ₁ -C ₆ alkyl groups;

R ¹⁰ is selected from 3-10-membered carbocycles and 3-10-membered heterocycles, each of which is substituted with 0-3 R ^a , where each R ^a is independently selected from halogen atoms, C ₁ -C ₆ alkyl groups, -O-( C ₁ -C ₆ )alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylhydroxy groups, -S(=O) _w -(4-7-membered heterocycles), 4-7-membered carbocycles and 4-7 -membered heterocycles;

each of R ¹⁵ and R ¹⁶ is independently selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C(=O)-OR ¹⁷ ; And

R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups;

where each of R ¹ , R ² , R ³ , R ⁴ , R ¹⁰ , R ¹⁵ , R ¹⁶ and R ¹⁷ is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, -O-(C ₁ -C ₆ alkyl), -NR ¹⁵ R ¹⁶ , C ₃ -C ₈ cycloalkyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkylalkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups groups; And

where each R ^a is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, -NR ¹⁵ R ¹⁶ , C ₁ -C ₆ alkyl groups, -(C=O)-(C ₁ -C ₆ alkyl) groups , -(C=O)-(C ₁ -C ₆ alkyl)-(C ₃ -C ₁₀ heterocyclyl groups) and C ₁ -C ₆ alkylcarboxylic acid groups each substituted with 0, 1 or 2 groups independently selected from halogen atoms, hydroxyl groups, -NR ¹⁵ R ¹⁶ and C ₁ -C ₃ alkyl groups; And

w is 0, 1, or 2.

[666] In some embodiments, R ¹ is absent or selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₁ -C ₄ alkylcarboxylic acid groups, and C ₃ -C ₈ cycloalkyl groups. In some embodiments, R ¹ is absent. In some embodiments, R ¹ is hydrogen. In some embodiments, R ¹ is a C ₁ -C ₄ alkyl group. In some embodiments, R ¹ is methyl. In some embodiments, R ¹ is ethyl. In some embodiments, R ¹ is a C ₁ -C ₄ alkyl carboxylic acid group. In some embodiments, R ¹ is -CH ₂ CH ₂ CH ₂ CO ₂ H. In some embodiments, R ¹ is a C ₃ -C ₈ cycloalkyl group. In some embodiments, R ¹ is cycloheptyl.

[667] In some embodiments, R ³ is selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₁ -C ₄ alkyl alkoxy groups, C ₁ -C ₄ alkyl carboxylic acid groups, and C ₁ -C ₄ alkyl hydroxy groups. In some embodiments, R ³ is selected from hydrogen and C ₁ -C ₄ alkyl carboxylic acid groups. In some embodiments, R ³ is hydrogen. In some embodiments, R ³ is a C ₁ -C ₄ alkyl carboxylic acid group. In some embodiments, R ³ is -CH ₂ CH ₂ CO ₂ H.

[668] In some embodiments, R ⁴ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl) groups, -OC(=O)-(C ₁ -C ₄ alkyl) groups, and C ₁ -C ₄ alkyl groups. In some embodiments, R ⁴ is hydrogen. In some embodiments, R ⁴ is hydroxyl. In some embodiments, R ⁴ is a -O-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁴ is -OCH ₃ . In some embodiments, R ⁴ is -OCH ₂ CH ₃ . In some embodiments, R ⁴ is a -OC(=O)-(C ₁ -C ₄ alkyl) group. In some embodiments, R ⁴ is -OC(=O)-CH ₃ . In some embodiments, R ⁴ is -OC(=O)-CH ₂ CH ₃ . In some embodiments, R ⁴ is a C ₁ -C ₄ alkyl group. In some embodiments, R ⁴ is methyl. In some embodiments, R ⁴ is ethyl.

[669] In some embodiments, R ¹⁰ is selected from 6-9 membered carbocycles and 6-9 membered heterocycles, each of which is substituted with 0-2 R ^a , where each R ^a is independently substituted with 0-3 groups independently selected from atoms halogen, hydroxyl groups, C ₁ -C ₆ alkyl groups, -(C=O)-(C ₁ -C ₆ alkyl) groups, -(C=O)-(C ₁ -C ₆ alkyl)-(3- 10-membered heterocycle) and C ₁ -C ₆ alkylcarboxylic acid groups.

.[670] In some embodiments, the carbocycle is phenyl substituted with 0-2 R ^a , where each R ^a is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, -( C=O)-(C ₁ -C ₆ alkyl), -(C=O)-(C ₁ -C ₆ alkyl)-(3-10 membered heterocycle), and C ₁ -C ₆ alkylcarboxylic acid groups. In some embodiments, phenyl is substituted with 2 R ^a , where each R ^a is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, -(C=O)-(C ₁ - C ₆ alkyl), -(C=O)-(C ₁ -C ₆ alkyl)-(3-10 membered heterocycle), and C ₁ -C ₆ alkylcarboxylic acid groups. In some embodiments, phenyl is

.

. [671] In some embodiments, the heterocycle is a 9-membered heterocycle substituted with 0-2 R ^a , where each R ^a is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, groups -(C=O)-(C ₁ -C ₆ alkyl), groups -(C=O)-(C ₁ -C ₆ alkyl)-(3-10-membered heterocycle) and groups C ₁ -C ₆ alkylcarboxylic acids. In some embodiments, the 9-membered heterocycle is

.

,

,

и

.[672] In some embodiments, R ^a is selected from halogen atoms, 3-10 membered carbocycles, and 3-10 membered heterocycles, where each R ^a is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, -(C=O)-(C ₁ -C ₆ alkyl) groups, -(C=O)-(C ₁ -C ₆ alkyl)-(3-10 membered heterocycle) groups and C ₁ -C ₆ alkylcarboxylic acid. In some embodiments, R ^a is selected from halogen atoms,

,

,

And

.

[673] In some embodiments, R ¹⁵ is hydrogen. In some embodiments, R ¹⁵ is R ¹⁷ . In some embodiments, R ¹⁵ is -C(=O)-R ¹⁷ . In some embodiments, R ¹⁵ is -C(=O)-OR ¹⁷ .

[674] In some embodiments, R ¹⁶ is hydrogen. In some embodiments, R ¹⁶ is R ¹⁷ . In some embodiments, R ¹⁶ is -C(=O)-R ¹⁷ . In some embodiments, R ¹⁶ is -C(=O)-OR ¹⁷ .

[675] In some embodiments, R ¹⁷ is selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₃ -C ₆ cycloalkyl groups, and C ₃ -C ₈ heterocyclyl groups. In some embodiments, R ¹⁷ is hydrogen. In some embodiments, R ¹⁷ is a C ₁ -C ₄ alkyl group. In some embodiments, R ¹⁷ is a C ₁ alkyl group. In some embodiments, R ¹⁷ is a C ₃ -C ₆ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₃ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₄ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₅ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₆ cycloalkyl group. In some embodiments, R ¹⁷ is a C ₃ -C ₈ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₃ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₄ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₅ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₆ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₇ heterocyclyl group. In some embodiments, R ¹⁷ is a C ₈ heterocyclyl group.

[676] In some embodiments, R ² is a linker. In some embodiments, the implementation of the linker provides at least one cleavable peptide fragment. In some embodiments, at least one cleavable peptide fragment is cleaved by the action of an enzyme. In some embodiments, the linker or cleavable peptide fragment comprises at least one amino acid unit. In some embodiments, at least one amino acid unit is selected from arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan and citrulline. In some embodiments, at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, the implementation of the linker provides citrulline and valine. In some embodiments, the implementation of the linker provides alanine and valine.

[677] In some embodiments, the implementation of the linker provides a fragment selected from sulfonamide, β-glucuronide, disulfide and carbonyl. In some embodiments, the implementation of the linker provides a sulfonamide. In some embodiments, the implementation of the linker provides β-glucuronide. In some embodiments, the implementation of the linker provides a disulfide. In some embodiments, the implementation of the linker provides a carbonyl.

[678] In some embodiments, the implementation of the linker provides a spacer link. In some embodiments, the spacer unit is selected from alkyl groups and polyethylene glycol (PEG) moieties. In some embodiments, the alkyl group is a C ₁ -C ₁₂ alkyl group. In some embodiments, the alkyl group is a C ₁ -C ₆ alkyl group. In some embodiments, the alkyl group is methylene. In some embodiments, the alkyl group is ethylene. In some embodiments, the alkyl group is n-propylene. In some embodiments, the alkyl group is n-butylene. In some embodiments, the alkyl group is n-pentylene. In some embodiments, the alkyl group is n-hexylene. In some embodiments, the PEG fragment provides -(PEG) _m -, where m is an integer from 1 to 10. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In in some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6.

[679] In some embodiments, the implementation of the linker provides a maleimide (Mal) fragment ("Mal-spacer link"). In some embodiments, the linker provides a self-cleaving spacer unit. In some embodiments, the self-cleaving spacer unit is selected from p-aminobenzyloxycarbonyl (pABC) and p-aminobenzyl (pAB).

[680] In some embodiments, the linker provides a Mal spacer unit, an alkyl group, at least one amino acid unit, and a self-cleaving spacer. In some embodiments, at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, at least one amino acid unit provides for alanine and valine. In some embodiments, at least one amino acid unit provides citrulline and valine. In some embodiments, the self-cleaving spacer is selected from pAB and pABC. In some embodiments, the self-cleaving spacer provides pAB. In some embodiments, the self-cleaving spacer provides pABC. In some embodiments, the alkyl group provides a C ₁ -C ₆ alkyl group.

[681] In some embodiments, the linker provides a Mal spacer unit, a PEG fragment, at least one amino acid unit, and a self-cleaving spacer. In some embodiments, at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, at least one amino acid unit provides for alanine and valine. In some embodiments, at least one amino acid unit provides citrulline and valine. In some embodiments, the self-cleaving spacer is selected from pAB and pABC. In some embodiments, the self-cleaving spacer provides pAB. In some embodiments, the self-cleaving spacer provides pABC. In some embodiments, the PEG fragment provides -(PEG) _m -, where m is an integer from 1 to 6.

[682] In some embodiments, the drug fragment is a splicing modulator selected from D1, D2, D3, D4, D4', D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, D15, D16, D17, D18, D19, D20, D21, D22, D23, D24, D25, D26, D27, D28, D29, D30, D31, D32, D33, D34, and D35.

[683] In various embodiments, the drug fragment is a splicing modulator selected from D2 and D1.

[684] In various embodiments, the drug moiety is D2. In various embodiments, the structure of the D2 drug moiety used in the disclosed linker drug compounds (L-D) is shown below:

D2.

[685] In various embodiments, the linker in the linker-drug (L-D) compounds described herein is covalently attached to the D2 drug moiety via an amine on the piperazine group. In various embodiments, the drug moiety is a D2 derivative. In various embodiments, the D2 derivative retains at least one D2 biological function or activity (eg, SF3b complex binding, in vitro splicing activity, cytotoxicity) but has an altered chemical structure.

[686] In various embodiments, the drug moiety is D1. In various embodiments, the structure of the D1 drug moiety used in the disclosed linker drug compounds (L-D) is shown below:

D1.

[687] In various embodiments, the linker in the linker-drug (L-D) compounds described herein is covalently attached to the D1 drug moiety via an amine on the piperazine group. In various embodiments, the drug moiety is a derivative of D1. In various embodiments, the D1 derivative retains at least one D1 biological function or activity (eg, SF3b complex binding, in vitro splicing activity, cytotoxicity) but has an altered chemical structure.

[688] Also disclosed herein are exemplary splicing modulator drug compounds for use alone or as drug fragments in the ADCs disclosed herein. In various embodiments, implementation of the present invention further provides compositions containing multiple copies of such compounds, for example, pharmaceutical compositions containing a compound based on the drug, which is a splicing modulator, and a pharmaceutically acceptable carrier.

[689] In various embodiments, the present invention provides a compound selected from a compound of the formula

(D4)

(D4)

and its pharmaceutically acceptable salts. In various embodiments, the present invention further provides a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[690] In various other embodiments, the present invention provides a compound selected from a compound of the formula

(D8)

(D8)

and its pharmaceutically acceptable salts. In various embodiments, the present invention further provides a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[691] In various other embodiments, the present invention provides a compound selected from a compound of the formula

(D9)

(D9)

and its pharmaceutically acceptable salts. In various embodiments, the present invention further provides a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[692] In various other embodiments, the present invention provides a compound selected from a compound of the formula

(D10)

(D10)

and its pharmaceutically acceptable salts. In various embodiments, the present invention further provides a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[693] In various other embodiments, the present invention provides a compound selected from a compound of the formula

(D13)

(D13)

and its pharmaceutically acceptable salts. In various embodiments, the present invention further provides a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[694] In various other embodiments, the present invention provides a compound selected from a compound of the formula

(D18)

(D18)

and its pharmaceutically acceptable salts. In various embodiments, the present invention further provides a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[695] In various other embodiments, the present invention provides a compound selected from a compound of the formula

(D19)

(D19)

and its pharmaceutically acceptable salts. In various embodiments, the present invention further provides a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[696] In various other embodiments, the present invention provides a compound selected from a compound of the formula

(D20)

(D20)

and its pharmaceutically acceptable salts. In various embodiments, the present invention further provides a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[697] In various other embodiments, the present invention provides a compound selected from a compound of the formula

(D21)

(D21)

and its pharmaceutically acceptable salts. In various embodiments, the present invention further provides a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[698] In various other embodiments, the present invention provides a compound selected from a compound of the formula

(D22)

(D22)

and its pharmaceutically acceptable salts. In various embodiments, the present invention further provides a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[699] In various other embodiments, the present invention provides a compound selected from a compound of the formula

(D25)

(D25)

and its pharmaceutically acceptable salts. In various embodiments, the present invention further provides a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[700] In various other embodiments, the present invention provides a compound selected from a compound of the formula

(D33)

(D33)

and its pharmaceutically acceptable salts. In various embodiments, the present invention further provides a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

Bioconjugation

[701] In some embodiments, a linker drug compound may be conjugated to an antibody or antigen-binding fragment in accordance with the illustrative scheme depicted in FIG. 26. Briefly, in some embodiments, an antibody or antigen-binding fragment can be treated with a reagent, such as a reducing agent, such as tris(2-carboxyethyl)phosphine, to activate the antibody or antigen-binding fragment by reducing one or more disulfide bonds. In some embodiments, the activated antibody or antigen-binding fragment is then processed using a linker-drug compound with a predetermined stoichiometry. Further, in some embodiments, the mixture is then subjected to purification by a specific technique, for example, using a size-exclusion resin or ultrafiltration, to obtain the desired antibody-drug conjugate.

[702] In some embodiments, provided herein is an antibody drug conjugate having the structure

(Ab-ADL1-D1),

(Ab-ADL1-D1),

where Ab is an antibody or antigen-binding fragment covalently linked to a maleimide group of a linker-drug compound (ADL1-D1) via a thiol sulfur atom on the antibody or antigen-binding fragment.

[703] In some embodiments, provided herein is an antibody drug conjugate having the structure

(Ab-ADL1-D4),

(Ab-ADL1-D4),

where Ab is an antibody or antigen-binding fragment covalently linked to the maleimide group of a linker-drug compound (ADL1-D4) via a thiol sulfur atom on the antibody or antigen-binding fragment.

[704] In some embodiments, provided herein is an antibody drug conjugate having the structure

(Ab-ADL1-D12),

(Ab-ADL1-D12),

where Ab is an antibody or antigen-binding fragment covalently linked to the maleimide group of a linker-drug compound (ADL1-D12) via a thiol sulfur atom on the antibody or antigen-binding fragment.

[705] In some embodiments, provided herein is an antibody drug conjugate having the structure

(Ab-ADL5-D2),

(Ab-ADL5-D2),

where Ab is an antibody or antigen-binding fragment covalently linked to the maleimide group of a linker-drug compound (ADL5-D2) via a thiol sulfur atom on the antibody or antigen-binding fragment.

[706] In some embodiments, provided herein is an antibody drug conjugate having the structure

(Ab-ADL5-D15),

(Ab-ADL5-D15),

where Ab is an antibody or antigen-binding fragment covalently linked to the maleimide group of a linker-drug compound (ADL5-D15) via a thiol sulfur atom on the antibody or antigen-binding fragment.

[707] In some embodiments, provided herein is an antibody drug conjugate having the structure

(Ab-ADL13-D4),

(Ab-ADL13-D4),

where Ab is an antibody or antigen-binding fragment covalently linked to the maleimide group of a linker-drug compound (ADL13-D4) via a thiol sulfur atom on the antibody or antigen-binding fragment.

[708] In some embodiments, the Ab is an anti-HER2 antibody or an antigen-binding fragment thereof. In some embodiments, the Ab binds to HER2 and targets neoplastic cells expressing HER2 (ie, the ADC targets neoplastic cells expressing HER2). In some embodiments, the Ab is an internalizing anti-HER2 antibody or an internalizing antigen-binding fragment thereof.

[709] In some embodiments, the anti-HER2 antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) containing the amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences under SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2) and SEQ ID NO:6 (LCDR3). In some embodiments, the anti-HER2 antibody or antigen binding fragment is an internalizing antibody or an internalizing antigen binding fragment. In some embodiments, the anti-HER2 antibody or antigen-binding fragment comprises human framework sequences. In some embodiments, the anti-HER2 antibody or antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the anti-HER2 antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the anti-HER2 antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

Drug loading

[710] Drug loading is expressed as p and is also referred to herein as the drug to antibody ratio (DAR). The drug load can range from 1 to 10 drug fragments per antibody or antigen binding fragment. In some embodiments, p is an integer from 1 to 10. In some embodiments, p is an integer from 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is an integer from 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In some embodiments, p is an integer from 1 to 8. In some embodiments, p is an integer from 2 to 4. In other embodiments , p is an integer from 4 to 8. In other embodiments, p is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 4 or 8.

[711] Drug loading may be limited by the number of attachment sites on an antibody or antigen-binding fragment. In some embodiments, the implementation of the linker fragment (L) ADC is attached to the antibody or antigennegative fragment through a reactive group on one or more amino acid residues on the antibody or antigennegative fragment. For example, a linker can be attached to an antibody or antigen-binding moiety through a free amino, imino, hydroxyl, thiol, or carboxyl group (e.g., to the N- or C-terminus, to the epsilon-amino group of one or more lysine residues, to the free carboxyl group of one or more glutamic acid or aspartic acid residues, or to the sulfhydryl group of one or more cysteine residues). The site to which the linker is attached may be a natural residue in the amino acid sequence of the antibody or antigen binding fragment, or it may be inserted into the antibody or antigen binding fragment, for example, by recombinant DNA technology (for example, by introducing a cysteine residue into the amino acid sequence) or by changing biochemical properties of the protein (for example, by reduction, change in pH, or hydrolysis).

[712] In some embodiments, the number of drug fragments that can be conjugated to an antibody or antigen-binding fragment is limited by the number of cysteine residues. For example, if the attachment is via a thiol group of a cysteine residue, the antibody may contain only one or more thiol groups of cysteine residues, or may contain one or more sufficiently reactive thiol groups through which a linker can be attached. Typically, antibodies do not contain many free and reactive thiol groups of cysteine residues that can be linked to the drug moiety. In fact, most of the thiol groups of cysteine residues in an antibody are involved in the formation of either interchain or intrachain disulfide bonds. Therefore, in some embodiments, cysteine conjugation may require at least partial recovery of the antibody. Excessive attachment of a toxin linker to an antibody can destabilize the antibody by reducing available cysteine residues to form disulfide bonds. Therefore, an optimal drug:antibody ratio would increase ADC activity (by increasing the number of drug fragments attached per antibody) without destabilizing the antibody or antigen-binding fragment. In some embodiments, the optimal ratio may be 2, 4, 6, or 8.

[713] In some embodiments, the antibody or antigen-binding fragment is subjected to reducing conditions prior to conjugation to form one or more free cysteine residues. In some embodiments, the antibody can be reduced with a reducing agent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) under partial or complete reduction conditions to form reactive cysteine thiol groups. Unpaired cysteines can be generated by partial reduction with limited molar equivalents of TCEP, which can reduce the intrachain disulfide bonds that connect the light chain and heavy chain (one pair per H-L pairing) and two heavy chains at the hinge region (two pairs per H-H pairing in the case of human IgG1), while intrachain disulfide bonds remain intact (Stefano et al. (2013) Methods Mol Biol. 1045:145-71). In one embodiment, the disulfide bonds in the antibody are electrochemically repaired, for example, by using a working electrode that applies a reducing and oxidizing electrical voltage. This approach may allow sequential combination of disulfide bond reduction with an analytical device (e.g., electrochemical detector, NMR spectrometer, or mass spectrometer), or chemical separation device (e.g., liquid chromatograph (e.g., HPLC), or electrophoresis device (see ., for example, US Patent Application Publication No. 20140069822)). In some embodiments, the antibody is subjected to denaturing conditions to detect reactive nucleophilic groups on amino acid residues such as cysteine.

[714] ADC drug loading can be controlled in a variety of ways, for example, by: (i) limiting the molar excess of the linker drug intermediate or linker reagent relative to the antibody; (ii) limitation of conjugation reaction time or temperature; (iii) partial or limiting reducing conditions for the modification of cysteine thiol groups; and/or (iv) constructing, by recombinant techniques, the amino acid sequence of the antibody such that the number and position of cysteine residues are modified to control the number and/or position of attachment sites for the drug linker.

[715] In some embodiments, free cysteine residues are introduced into the amino acid sequence of an antibody or antigen-binding fragment. For example, cysteine-modified antibodies can be made in which one or more amino acids in the parent antibody are replaced with the amino acid cysteine. Any form of antibody can be engineered in this way, that is, mutated. For example, a Fab fragment of a parent antibody can be engineered to produce a cysteine-modified Fab referred to as "ThioFab." Similarly, a parental monoclonal antibody can be engineered to produce "ThioMab." Mutation at one site results in one modified cysteine residue in ThioFab, while mutation at one site gives rise to two modified cysteine residues in ThioMab due to the dimeric nature of the IgG antibody. DNA encoding a variant amino acid sequence of the parent polypeptide can be obtained by a variety of methods known in the art (see, for example, the methods described in International Publication No. WO 2006/034488). Such methods include, without limitation, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of preformed DNA encoding a polypeptide. Recombinant antibody variants can also be constructed by restriction enzyme manipulation or by PCR with overlapping primers using synthetic oligonucleotides. ADCs of formula (I) include, without limitation, antibodies that have 1, 2, 3, or 4 modified amino acids cysteine (Lyon et al. (2012) Methods Enzymol. 502:123-38). In some embodiments, one or more free cysteine residues are already present in the antibody or antigen binding fragment without the use of genetic engineering, in which case the existing cysteine residues can be used to conjugate the antibody or antigen binding fragment to the drug fragment.

[716] In this case, more than one nucleophilic group reacts with an intermediate, which is a linker drug, or a reagent, which is a linker fragment, and then with a reagent, which is a drug fragment, in a reaction mixture containing multiple copies antibody or antigen-binding fragment and a linker fragment, then the resulting product may be a mixture of ADC compounds with a distribution of one or more drug fragments attached to each copy of the antibody or antigen-binding fragment in the mixture. In some embodiments, the drug load in the ADC mixture resulting from the conjugation reaction is in the range of 1 to 10 drug fragments attached to an antibody or antigen-binding fragment. The average number of drug fragments per antibody or antigen binding fragment (i.e., average drug load or average p ) can be calculated by any conventional method known in the art, for example, by mass spectrometry (for example, reverse phase LC -MS), and/or high performance liquid chromatography (eg HIC-HPLC). In some embodiments, the average number of drug fragments per antibody or antigen-binding fragment is determined by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC). In some embodiments, the average number of drug fragments per antibody or antigen-binding fragment is determined by reverse phase liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the average number of drug fragments per antibody or antigen binding fragment is from about 1.5 to about 3.5, from about 2.5 to about 4.5, from about 3.5 to about 5.5, from about 4.5 to about 6.5, about 5.5 to about 7.5, about 6.5 to about 8.5, or about 7.5 to about 9.5. In some embodiments, the average number of drug fragments per antibody or antigen binding fragment is from about 2 to about 4, from about 3 to about 5, from about 4 to about 6, from about 5 to about 7, from about 6 to about 8 , from about 7 to about 9, from about 2 to about 8, or from about 4 to about 8.

[717] In some embodiments, the average number of drug fragments per antibody or antigen binding fragment is about 2. In some embodiments, the average number of drug fragments per antibody or antigen binding fragment is about 1.5, about 1.6, about 1 .7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5. In some embodiments, the average number of drug fragments per antibody or antigen binding fragment is 2.

[718] In some embodiments, the average number of drug fragments per antibody or antigen binding fragment is about 4. In some embodiments, the average number of drug fragments per antibody or antigen binding fragment is about 3.5, about 3.6, about 3 .7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, or about 4.5. In some embodiments, the average number of drug fragments per antibody or antigen binding fragment is 4.

[719] In some embodiments, the average number of drug fragments per antibody or antigen binding fragment is about 8. In some embodiments, the average number of drug fragments per antibody or antigen binding fragment is about 7.5, about 7.6, about 7 .7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, or about 8.5. In some embodiments, the average number of drug fragments per antibody or antigen binding fragment is 8.

[720] In various embodiments, the expression "about," used in relation to the average number of drug fragments per antibody or antigen-binding fragment, means plus or minus 10%.

[721] Individual ADC compounds or "molecules" can be identified in a mixture by mass spectrometry and separated by UPLC or HPLC, such as hydrophobic interaction chromatography (HIC-HPLC). In some embodiments, a homogeneous or substantially homogeneous ADC product with a single loading value can be isolated from the conjugation mixture, for example, by electrophoresis or chromatography.

[722] In some embodiments, a higher drug load (eg, p > 8) may result in aggregation, insolubility, toxicity, and loss of cell penetration in certain antibody-drug conjugates. Higher drug loading can also adversely affect the pharmacokinetics (eg, clearance) of certain ADCs. In some embodiments, a lower drug load (eg, p < 2) may reduce the activity of certain ADCs on target-expressing cells and/or non-target cells. In some embodiments, the drug load for an ADC of the present invention ranges from about 2 to about 8; from about 2 to about 6; from about 2 to about 5; about 3 to about 5; about 2 to about 4, or about 4 to about 8.

[723] In some embodiments, a drug load and/or an average drug load of approximately 2 is provided, for example, by partial reduction of intrachain disulfides on the antibody or antigen-binding fragment, which provides beneficial properties. In some embodiments, a drug load and/or an average drug load of approximately 4 is provided, for example, by partial reduction of intrachain disulfides on the antibody or antigen-binding fragment, which provides beneficial properties. In some embodiments, a drug load and/or an average drug load of approximately 8 is provided, for example, by partial reduction of intrachain disulfides on the antibody or antigen-binding fragment, which provides beneficial properties. In some embodiments, a drug load and/or an average drug load of less than about 2 may result in unacceptably high levels of unconjugated antibody fragments that may compete with the ADC for binding to the target antigen and/or result in poor treatment efficacy. In some embodiments, a drug load and/or an average drug load greater than about 8 may result in an unacceptably high level of product heterogeneity and/or ADC aggregation. Drug loading and/or average drug loading greater than about 8 can also affect ADC stability due to the loss of one or more chemical bonds required to stabilize the antibody or antigen-binding fragment.

[724] Included in the present invention are methods for preparing the disclosed ADCs. Briefly, ADCs comprise an antibody or antigen-binding fragment as the antibody or antigen-binding fragment, a drug fragment (eg, a splicing modulator), and a linker that binds the drug fragment and the antibody or antigen-binding fragment. In some embodiments, ADCs can be made using a linker having reactive functional groups designed to covalently attach to the drug moiety and to the antibody or antigen-binding moiety. For example, in some embodiments, a cysteine thiol group in an antibody or antigen-binding fragment can form a bond with a reactive functional group of a linker or intermediate that is a linker drug (eg, with a maleimide moiety) to form an ADC. Obtaining ADC can be carried out by any technique known to the person skilled in the art.

[725] In some embodiments, an ADC is prepared by sequentially contacting an antibody or antigen-binding fragment with a linker and a drug moiety (e.g., a splicing modulator) such that the antibody or antigen-binding fragment is first covalently bound to the linker and then the preformed intermediate. the linker antibody compound reacted with the drug moiety. The antibody-linker intermediate may or may not be subjected to a purification step prior to being contacted with the drug moiety. In other embodiments, an ADC is produced by contacting an antibody or antigen-binding fragment with a linker-drug compound previously formed by reacting the linker with the drug fragment. The linker-drug preformed compound may or may not be subjected to a purification step prior to being contacted with an antibody or antigen-binding moiety. In other embodiments, the antibody or antigen-binding fragment is brought into contact with the linker and the drug moiety in a single reaction mixture, which allows for the simultaneous formation of covalent bonds between the antibody or antigen-binding fragment and the linker and between the linker and the drug moiety. This method of obtaining ADC may include a reaction in which the antibody or antigennegative fragment is brought into contact with the antibody or antigennegative fragment before adding the linker to the reaction mixture and vice versa. In some embodiments, an ADC is produced by reacting an antibody or antigen-binding fragment with a linker linked to a drug moiety, such as an ADL1 splicing modulator (e.g., ADL1-D1) or an ADL5 splicing modulator (e.g., ADL5-D2), when conditions that allow conjugation.

[726] ADC obtained in accordance with the methods described above, can be subjected to a purification step. The purification step may include any biochemical methods known in the art to purify proteins, or any combination of these methods. Such methods include, but are not limited to, tangential flow filtration (TFF), affinity chromatography, ion exchange chromatography, any charge transfer or isoelectric focusing chromatography, mixed mode chromatography such as CHT (ceramic hydroxyapatite), hydrophobic interaction chromatography, size exclusion chromatography, dialysis, filtration, selective precipitation, or any combination thereof.

Therapeutic Uses and Compositions

[727] Disclosed herein are methods of using ADCs and compositions in the treatment of a disorder in a subject, such as a neoplastic disorder. The ADC may be administered alone or in combination with a second therapeutic agent, and may be administered in any pharmaceutically acceptable formulation, dosage, and schedule. The efficacy of ADC treatment can be assessed for toxicity as well as efficacy rates and adjusted accordingly. Efficacy measures include, without limitation, cytostatic and/or cytotoxic effects observed in vitro or in vivo, tumor volume reduction, tumor growth inhibition, and/or long-term survival.

[728] Methods are known for determining whether an ADC has a cytostatic and/or cytotoxic effect on a cell. For example, the cytotoxic or cytostatic activity of ADC can be measured by: exposing mammalian cells expressing the target protein to ADC in a cell culture medium; culturing the cells for a period of time from about 6 hours to about 6 days and measuring cell viability. In vitro cell assays can also be used to measure viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) by ADC.

[729] To determine whether ADC has a cytostatic effect, thymidine incorporation assay can be used. For example, cancer cells expressing the target antigen at a density of 5000 cells/well in a 96-well plate can be cultured for a 72 hour period and exposed to 0.5 μCi ³ H-thymidine for the last 8 hours of the 72 hour period. ³ H-thymidine incorporation into culture cells is measured in the presence and absence of ADC.

[730] Necrosis or apoptosis (programmed cell death) can be measured to determine cytotoxicity. Necrosis is usually accompanied by increased permeability of the plasma membrane; swelling of the cell and rupture of the plasma membrane. Apoptosis can be quantified, for example, by measuring DNA fragmentation. Commercial photometric methods are available to quantify DNA fragmentation in vitro. Examples of such assays, including TUNEL (which detects the insertion of labeled nucleotides into fragmented DNA) and ELISA-based assays, are described in Biochemica (1999) No. 2, pp. 34-37 (Roche Molecular Biochemicals).

[731] Apoptosis can also be determined by measuring morphological changes in the cell. For example, as in the case of necrosis, disruption of the integrity of the plasma membrane can be determined by measuring the absorption of certain dyes (eg, a fluorescent dye, such as, for example, acridine orange or ethidium bromide). A method for measuring the number of apoptotic cells has been described by Duke and Cohen, Current Protocols in Immunology (Coligan et al., Eds. (1992) pp. 3.17.1-3.17.16). Cells can also be labeled with a DNA dye (eg, acridine orange, ethidium bromide, or propidium iodide) and the cells visually analyzed for chromatin condensation and edges along the inner nuclear membrane. In some embodiments, apoptosis can also be determined by screening for caspase activity. In some embodiments, a Caspase-Glo® assay can be used to measure caspase-3 and caspase-7 activity. In some embodiments, the assay provides a caspase-3/7 luminogenic substrate in a reagent optimized for caspase activity, luciferase activity, and cell lysis. In some embodiments, the addition of the Caspase-Glo® 3/7 reagent in an add-mix-measure format can result in cell lysis followed by cleavage of the substrate by caspases and generation of a luciferase "glow-type" luminescent signal. In some embodiments, luminescence may be proportional to the amount of caspase activity present and may be indicative of apoptosis. Other morphological changes that can be measured to determine apoptosis include, for example, cytoplasmic condensation, increased membrane blistering, and cell shrinkage. The determination of any of these effects on cancer cells indicates that ADC is useful in the treatment of cancer.

[732] Cell viability can be measured, for example, by determining cell uptake of a dye such as neutral red, trypan blue, crystal violet, or ALAMAR™ blue (see, for example, Page et al. (1993) Intl J Oncology 3:473 -6). In such an assay, cells are incubated in dye-containing media, the cells are washed, and the remaining dye, reflecting dye uptake by the cells, is measured spectrophotometrically. Cell viability can also be measured, for example, by quantifying ATP, a measure of metabolically active cells. In some embodiments, the in vitro activity and/or effect on cell viability of the resulting ADCs or splicing modulator compounds can be assessed using the CellTiter-Glo® Luminescent Cell Viability Assay described in the examples provided herein. In this assay, in certain embodiments, one reagent (CellTiter-Glo® reagent) is added directly to cells cultured in serum supplemented medium. The addition of the reagent leads to cell lysis and the generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in the culture. The protein-binding dye sulforodamine B (SRB) can also be used to measure cytotoxicity (Skehan et al. (1990) J Natl Cancer Inst. 82:1107-12).

[733] The disclosed ADCs can also be evaluated for killing activity. The activity of killing non-target cells can be determined, for example, using an analysis using two cell lines, one positive for the target antigen and the other negative for the target antigen. In certain embodiments, the assay design allows only target-negative cells to be detected. In certain embodiments, the cells are seeded under three conditions: (i) only target-negative cells (labeled or labelled); (ii) only target-positive cells; and (iii) co-culture of target-negative cells and target-positive cells. Cells are then treated with ADC followed by cytotoxicity monitoring. If the plate reaction assay is performed by reading using the CellTiter-Glo® reagent, viability of all cell populations can be monitored. When plates are read using the OneGlo® reagent, only labeled or labeled target-negative cells generate a signal. Killing target-negative cells when mixed with target-positive cells indicates killing non-target cells, while killing target-negative cells in the absence of target-positive cells indicates off-target killing.

[734] In certain aspects, the present invention provides a method of killing, suppressing or modulating growth or interfering with the metabolism of a cancer cell or tissue by disrupting RNA splicing. The method can be used in any subject in which disruption of RNA splicing provides a therapeutic effect. Subjects that may benefit from impaired RNA splicing include, without limitation, those who have or are at risk of developing a neoplastic disorder, such as a hematologic malignancy or a solid tumor. In certain embodiments, the hematologic malignancy is a B-cell malignancy, blood cancer (leukemia), plasma cell cancer (myeloma, eg, multiple myeloma), or cancer of the lymph nodes (lymphoma). In certain embodiments, the hematologic malignancy is selected from myelogenous leukemia or multiple myeloma. In some embodiments, the leukemia is acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), or acute monocytic leukemia (AMO). In some embodiments, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma. In certain embodiments, the hematologic malignancy is a myelodysplastic syndrome (MDS). In certain embodiments, the solid tumor is a carcinoma, such as breast cancer, pancreatic cancer, prostate cancer, colon or colorectal cancer, lung cancer, gastric cancer, cervical cancer, endometrial cancer, ovarian cancer, cholangiocarcinoma, glioma or melanoma. In certain embodiments, the solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer (e.g. lung adenocarcinoma), uterine cancer (e.g. uterine endometrial serous carcinoma), salivary duct carcinoma, melanoma, colon cancer colon, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer and esophageal cancer. In some embodiments, the lung cancer is an adenocarcinoma. In some embodiments, the implementation of the cancer of the uterus is a serous carcinoma of the endometrium of the uterus.

[735] In various embodiments, the disclosed ADCs can be administered to any cell or tissue that expresses HER2, such as a neoplastic cell or tissue that expresses HER2. An exemplary embodiment includes a method for suppressing HER2-mediated cell signaling or a method for killing a cell. The method can be used with any cell or tissue expressing HER2, such as a cancer cell or a metastatic site. Non-limiting examples of HER2-expressing cancers include breast cancer, gastric cancer, bladder cancer, urothelial cell carcinoma, esophageal cancer, lung cancer (eg lung adenocarcinoma), uterine cancer (eg uterine endometrial serous carcinoma), salivary duct carcinoma , cervical cancer, endometrial cancer and ovarian cancer (English et al. (2013) Mol Diagn Ther. 17:85-99). Non-limiting examples of cells expressing HER2 include HCC1954 and SKBR3, human breast ductal carcinoma, N87 cells, human gastric carcinoma cells, and cells containing a recombinant nucleic acid encoding HER2 or a portion thereof.

[736] In various embodiments, the disclosed ADCs can be administered to any cell or tissue that expresses CD138, such as a neoplastic cell or tissue that expresses CD138. An exemplary embodiment includes a method for suppressing CD138-mediated cell signaling or a method for killing a cell. The method can be used with any cell or tissue expressing CD138, such as a cancer cell or a metastatic site. Non-limiting examples of CD138-expressing cancers include hilar cancer (eg, lung cancer, mesothelioma), skin cancer (eg, basal cell carcinoma, squamous cell carcinoma), head and neck cancer (eg, larynx, hypopharynx, nasopharynx), breast cancer , urogenital cancer (eg, cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, urothelial cancer), hematologic malignancies (eg, myeloma such as multiple myeloma, Hodgkin's lymphoma), and thyroid cancer (Szatmári et al (2015) Dis Markers 2015:796052). Non-limiting examples of cells expressing CD138 include MOLP8, human multiple myeloma cells, and cells containing a recombinant nucleic acid encoding CD138 or a portion thereof.

[737] In various embodiments, the disclosed ADCs can be administered to any cell or tissue that expresses EPHA2, such as a neoplastic cell or tissue that expresses EPHA2. An exemplary embodiment includes a method for suppressing EPHA2 mediated cell signaling or a method for killing a cell. The method can be used with any cell or tissue expressing EPHA2, such as a cancer cell or a metastatic site. Non-limiting examples of EPHA2-expressing cancers include breast cancer, brain cancer, ovarian cancer, bladder cancer, pancreatic cancer, esophageal cancer, lung cancer, prostate cancer, melanoma, esophageal cancer, and gastric cancer (Tandon et al. (2011) Expert Opin Ther Targets 15(1):31-51 Non-limiting examples of cells expressing EPHA2 include PC3, human prostate cancer cells, and cells containing a recombinant nucleic acid encoding EPHA2 or a portion thereof.

[738] Exemplary methods include the steps of contacting a cell with the ADC described herein in an effective amount, ie, an amount sufficient to kill the cell. The method can be used in relation to cells in culture, for example in vitro, in vivo, ex vivo or in situ.For example, cells that express HER2 (eg, cells obtained from a tumor biopsy or metastatic lesion, cells from an established cancer cell line, or recombinant cells) can be cultured in vitro. in culture medium, the contacting step may be affected by the addition of ADC to the culture medium. This method will kill cells expressing HER2, including, in particular, tumor cells expressing HER2. Alternatively, the ADC may be administered to a subject via any suitable route of administration (eg, intravenous, subcutaneous, or direct contact with tumor tissue) to act in vivo. This approach can be used for antibodies targeting other cell surface antigens (eg, CD138, EPHA2).

[739] The in vivo effect of the described ADC-based therapeutic composition can be evaluated in a suitable animal model. For example, xenogenic cancer models can be used in which malignant tumor explants or passaged xenograft tissues are administered to immunocompromised animals such as nude mice or SCID mice (Klein et al. (1997) Nature Med. 3:402-8). Efficacy can be predicted using assays that measure suppression of tumorigenesis, tumor regression or metastasis, and the like.

[740] You can also use in vivo assays that evaluate the stimulation of tumor death through mechanisms such as apoptosis. In one embodiment, xenografts from tumor-bearing mice treated with a therapeutic composition can be assessed for the presence of apoptotic lesions and compared to untreated xenograft-bearing control mice. The extent to which apoptotic foci are found in the tumors of the treated mice is indicative of the therapeutic efficacy of the composition.

[741] In addition, this document provides methods for the treatment of neoplastic disorders, such as cancer. The ADCs described herein may be administered to a non-human mammal or human for the purpose of therapy. Therapeutic methods involve administering to a subject suffering from or suspected of having a neoplastic disorder a therapeutically effective amount of an ADC or a composition comprising a splicing modulator coupled to a targeting antibody that binds to an expressed antigen, is available to bind to, or is located on the surface of cancer cells. In some embodiments, treatment with the antibody drug conjugate or composition induces non-specific killing of neoplastic cells that do not express the target antigen but are adjacent to neoplastic cells that express the target antigen.

[742] An exemplary embodiment is a method for delivering a splicing modulator to a cell expressing HER2, comprising conjugating the splicing modulator to an antibody that immunospecifically binds to a HER2 epitope and exposes the cell to ADC. Exemplary tumor cells that express HER2 for which the ADCs of the present invention are provided include gastric carcinoma cells and ductal breast carcinoma cells.

[743] Another exemplary embodiment is a method for delivering a splicing modulator to a cell expressing CD138, comprising conjugating the splicing modulator to an antibody that immunospecifically binds to a CD138 epitope and exposes the cell to ADC. Exemplary tumor cells that express CD138, for which the ADCs of the present invention are indicated, include multiple myeloma cells.

[744] Another exemplary embodiment is a method for delivering a splicing modulator to a cell expressing EPHA2, comprising conjugating the splicing modulator to an antibody that immunospecifically binds to an EPHA2 epitope and exposes the cell to ADC. Exemplary tumor cells that express EPHA2, for which the ADCs of the present invention are indicated, include prostate cancer cells.

[745] Another exemplary embodiment is a method of reducing or inhibiting the growth of a tumor (e.g., HER2 expressing tumor, CD138 expressing tumor, EPHA2 expressing tumor) comprising administering a therapeutically effective amount of ADC or an ADC containing composition. In some embodiments, this treatment is sufficient to reduce or suppress tumor growth in a patient, reduce the number or size of metastatic lesions, reduce tumor burden, reduce primary tumor burden, reduce invasiveness, prolong survival time, and/or maintain or improve quality of life. In some embodiments, the tumor is resistant or refractory to treatment with an ADC antibody or antigen-binding fragment (e.g., anti-HER2 antibodies, anti-CD138 antibodies, anti-EPHA2 antibodies) when administered alone, and/or the tumor is resistant or refractory to treatment with a drug fragment. , which is a splicing modulator, with a separate introduction.

[746] In certain aspects, the present invention provides a method for reducing or suppressing the growth of a tumor expressing HER2. In certain embodiments, treatment with the antibody drug conjugate or composition induces non-specific killing of tumor cells that do not express HER2 but are adjacent to neoplastic tumor cells that express HER2. Exemplary cancers expressing HER2 include, but are not limited to, tumors derived from breast cancer, gastric cancer, bladder cancer, urothelial cell carcinoma, esophageal cancer, lung cancer (e.g. lung adenocarcinoma), uterine cancer (e.g. uterine endometrial serous carcinoma). ), salivary duct carcinomas, cervical cancer, endometrial cancer, and ovarian cancer expressing HER2. In some embodiments, the HER2-expressing tumor is a tumor derived from breast cancer, ovarian cancer, gastric cancer, lung cancer (eg, lung adenocarcinoma), uterine cancer (eg, uterine endometrial serous carcinoma), osteosarcoma, or salivary duct carcinoma expressing HER2. In some embodiments, the HER2-expressing tumor is lung adenocarcinoma or uterine endometrial serous carcinoma.

[747] In certain aspects, the present invention provides a method for reducing or suppressing the growth of a tumor expressing CD138. In certain embodiments, treatment with the antibody drug conjugate or composition induces non-specific killing of tumor cells that do not express CD138 but are adjacent to neoplastic tumor cells that express CD138. Illustrative tumor types expressing CD138 include, without limitation, tumors derived from hilar cancer (eg, lung cancer, mesothelioma), skin cancer (eg, basal cell carcinoma, squamous cell carcinoma), head and neck cancer (eg, larynx, hypopharynx, nasopharynx ), breast cancer, urogenital cancer (eg, cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, urothelial cancer) and thyroid cancer expressing CD138.

[748] In certain aspects, the present invention provides a method for reducing or suppressing the growth of a tumor expressing EPHA2. In certain embodiments, treatment with the antibody drug conjugate or composition induces non-specific killing of tumor cells that do not express EPHA2 but are adjacent to neoplastic tumor cells that express EPHA2. Exemplary tumor types expressing EPHA2 include, but are not limited to, tumors derived from breast cancer, brain cancer, ovarian cancer, bladder cancer, pancreatic cancer, esophageal cancer, lung cancer, prostate cancer, melanoma, esophageal cancer, and gastric cancer. expressing EPHA2. In certain embodiments, the EPHA2-expressing tumor is a tumor derived from breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer expressing EPHA2.

[749] Moreover, the antibodies of the present invention can be administered to a non-human mammal that expresses an antigen to which ADC is able to bind, for veterinary purposes or for use as an animal model of a human disease. With regard to the latter, such animal models may be useful for evaluating the therapeutic efficacy of the disclosed ADCs (eg, testing dosages and timing of administration).

[750] Additionally, this document presents options for therapeutic use of the disclosed ADC and compositions. In one exemplary embodiment, the use of an ADC in the treatment of a neoplastic disorder (eg, HER2 expressing cancer, CD138 expressing cancer, EPHA2 expressing cancer) is provided. In another exemplary embodiment, an ADC is provided for use in the treatment of a neoplastic disorder (eg, cancer expressing HER2, cancer expressing CD138, cancer expressing EPHA2). Methods for identifying subjects with a cancer that expresses a target antigen (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, STEAP1) are known. in the art and can be used to identify patients suitable for treatment with the disclosed ADC.

[751] In another exemplary embodiment, the use of an ADC in a method of making a medicament for the treatment of a neoplastic disorder (eg, HER2-expressing cancer, CD138-expressing cancer, EPHA2-expressing cancer) is provided.

[752] Therapeutic compositions used in the practice of the above methods can be formulated into pharmaceutical compositions containing a pharmaceutically acceptable carrier suitable for the desired method of delivery. In an exemplary embodiment, a pharmaceutical composition is provided comprising an ADC of the present invention and a pharmaceutically acceptable carrier. Suitable carriers include any material which, when combined with the therapeutic composition, retains the antitumor function of the therapeutic composition and does not generally interact with the patient's immune system. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of the following: water, physiological saline, phosphate-buffered physiological saline, dextrose, glycerol, ethanol, mesylate salt, and the like, as well as combinations thereof. In many cases, isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride are included in the composition. Pharmaceutically acceptable carriers may additionally contain small amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which increase the shelf life or effectiveness of the ADC.

[753] Therapeutic formulations can be solubilized and administered via any route that delivers the therapeutic composition to the site of the tumor. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumoral, intradermal, intraorgan, orthotopic, and the like. Protein-based therapeutics can be lyophilized and stored as sterile powders, e.g. in bacteriostatic water (containing, for example, the preservative benzyl alcohol) or in sterile water before injection. Therapeutic formulations may contain ADC or a pharmaceutically acceptable salt thereof, such as the mesylate salt.

[754] In some embodiments, the implementation of the ADC is administered to the patient daily, once every two months, or at any intermediate period of time. Dosages and administration protocols for the treatment of cancer using the aforementioned methods will vary depending on the method and target cancer and will generally depend on a number of other factors considered in the art.

[755] Various delivery systems are known that can be used to administer one or more ADCs of the present invention. Methods of administration of ADC include, without limitation, parenteral administration (eg, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes), epidural administration, intratumoral administration, and transmucosal administration (eg, intranasal and oral routes). In addition, pulmonary administration may be used, for example using an inhaler or nebulizer, as well as an aerosol formulation. See for example compositions and methods for pulmonary administration as described in US Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; and International Application Publication Nos. WO 1992/019244, WO 1997/032572, WO 1997/044013, WO 1998/031346 and WO 1999/066903. ADC can be administered by any convenient route, such as by infusion or bolus injection, or by absorption through epithelial or mucocutaneous membranes (eg oral mucosa, rectal mucosa and intestinal mucosa, etc.). The introduction can be systemic or local.

[756] Therapeutic compositions disclosed herein may be sterile and stable under the conditions of manufacture and storage. In some embodiments, one or more ADCs or one or more pharmaceutical compositions are supplied as a dry sterilized lyophilized powder or anhydrous concentrate in a hermetically sealed container, and they may be reconstituted (eg, with water or physiological saline) to a concentration suitable for administration to a subject. In some embodiments, one or more prophylactic or therapeutic agents or one or more pharmaceutical compositions are supplied as a dry sterile lyophilized powder in a sealed container in a dosage form of at least 5 mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg, or any amount in between. In some embodiments, the lyophilized ADCs or pharmaceutical compositions are stored at 2°C to 8°C in the original container. In some embodiments, one or more ADCs or one or more pharmaceutical compositions described herein are supplied in liquid form in a sealed container, such as a container with an indication of the amount and concentration of the agent. In some embodiments, the liquid form of the administered composition is provided in a sealed container containing at least 0.25 mg/mL, at least 0.5 mg/mL, at least 1 mg/mL, at least 2.5 mg /ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 50 mg/ml , at least 75 mg/ml or at least 100 mg/ml ADC. The liquid form can be stored between 2˚C and 8˚C in the original container.

[757] In some embodiments, the implementation of the disclosed ADC can be included in a pharmaceutical composition suitable for parenteral administration. The injectable solution may consist of either a liquid or lyophilized dosage form in a colorless or amber yellow vial, ampoule or pre-filled syringe or other known delivery or storage device.

[758] Described in this document, the composition can be in various forms. These include, for example, liquid, semi-solid and solid dosage forms such as liquid solutions (eg injection and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended route of administration and therapeutic application.

[759] In various embodiments, treatment comprises a single bolus or repeated administration of an ADC-based drug via an acceptable route of administration.

[760] Patients can be assessed for levels of the target antigen in a given sample (e.g., levels of cells expressing the target antigen) to help determine the most effective administration regimen, etc. An exemplary embodiment is a method for determining whether whether the patient will respond to treatment with the ADC of the present invention, which involves obtaining a biological sample from the patient and bringing the biological sample into contact with the ADC. Exemplary biological specimens include tissue or biological fluid such as inflammatory exudate, blood, serum, intestinal fluid, a stool specimen, or a tumor biopsy (e.g., a tumor biopsy obtained from a patient with cancer expressing a target antigen, e.g., cancer, expressing HER2, cancer expressing CD138, cancer expressing EPHA2, or at risk of developing it). In some embodiments, a sample (e.g., tissue and/or body fluid) may be obtained from a subject and a suitable immunological method may be used to detect and/or measure expression of a target antigen protein (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, STEAP1). Such scores are also used for monitoring purposes throughout therapy and are useful for assessing therapeutic success in conjunction with the evaluation of other parameters.

[761] In some embodiments, the effectiveness of the ADC can be assessed by contacting a tumor sample from a subject with the ADC and assessing the growth rate or volume of the tumor. In some embodiments, if the ADC has been determined to be effective, it may be administered to the subject.

[762] The above therapeutic approaches can be combined with any of a variety of additional modes of surgery, chemotherapy or radiation therapy. In some embodiments, the ADCs or compositions disclosed herein are formulated and/or co-administered with one or more additional therapeutic agents, such as one or more chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents such as nitrogen mustards, ethyleneimine compounds, and alkylsulfonates; antimetabolites, for example folic acid, purine or pyrimidine antagonists; antimitotic agents, eg tubulin inhibitors such as eribulin or eribulin mesylate (Halaven™), vinca alkaloids and auristatins; cytotoxic antibiotics; compounds that damage or interfere with the expression or replication of DNA, such as agents that bind to the minor groove of DNA; and growth factor receptor antagonists. In some embodiments, the chemotherapeutic agent may be a cytotoxic or cytostatic agent. Examples of cytotoxic agents include, without limitation, antimitotic agents such as eribulin or eribulin mesylate (Halaven™), auristatins (e.g., monomethylauristatin E (MMAE), monomethylauristatin F (MMAF)), maytansinoids (e.g., maytansine), dolastatins, duostatins, cryptophycins, vinca alkaloids (eg vincristine, vinblastine), taxanes, taxols and colchicines; anthracyclines (eg daunorubicin, doxorubicin, dihydroxyanthracindione); cytotoxic antibiotics (eg mitomycins, actinomycins, duocarmycins (eg CC-1065), auromycins, duomycins, calicheamicins, endomycins, phenomycins); alkylating agents (eg cisplatin); intercalating agents (eg ethidium bromide); topoisomerase inhibitors (eg etoposide, tenoposide); radioactive isotopes such as At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² or ²¹³ , P ³² and radioactive isotopes of lutetium (eg Lu ¹⁷⁷ ); and toxins of bacterial, fungal, plant or animal origin (eg, ricin (eg, ricin A chain), diphtheria toxin, Pseudomonas exotoxin A (eg, PE40), endotoxin, mitogellin, combrestatin, restrictocin, gelonin, alpha-sarcin, abrin (e.g. abrin A-chain), modeccin (e.g. modeccin A-chain), kuricin, crotin, inhibitor from Sapaonaria officinalis , glucocorticoid).

[763] This document also discloses options for using one or more of the disclosed ADCs in the manufacture of a medicament for the treatment of cancer, for example, in accordance with the methods described above. In some embodiments, the ADCs disclosed herein are used to treat cancer, for example, in accordance with the methods described above.

[764] In various embodiments, the implementation of the kits for research and therapeutic applications described in this document are within the scope of the present invention. Such kits may comprise a carrier, package, or container that is compartmentalized to hold one or more containers such as vials, test tubes, and the like, each container containing one of the individual items for use in the method disclosed herein. , along with a label or package insert containing instructions for use, such as the use described in this document. Kits may contain a container containing a drug fragment. The present invention also provides one or more ADCs or pharmaceutical compositions based on them, sealed in a container, such as an ampoule or sachet, with an indication of the amount of the agent.

[765] Kits may contain the container described above, and one or more other containers associated with it, which contain materials necessary in terms of commerce and from the user's point of view, including buffers, diluents, filters, needles, syringes; carrier, packaging, container, vial and/or tube, labeled with contents and/or instructions for use, as well as package inserts with instructions for use.

[766] A label may be present on or with the container to indicate that the composition is being used for a specific therapeutic use or use other than therapeutic, such as prognostic, prophylactic, diagnostic, or laboratory use. The label may also provide a prescription for in vivo or in vitro use, as described, for example, in this document. Prescriptions and other information may also be included in the package leaflet(s) or label(s) included with or affixed to the kit. The label may be on or associated with the container. The label may be on the container itself if the letters, numbers or other symbols forming the label are pressed or engraved on the container itself. The label may be associated with the container if present in a container or carrier which also holds the container, such as in the form of a leaflet. The label may indicate that the composition is used to diagnose or treat a condition, such as cancer, as described herein.

Neoantigens and methods of their use

[767] In various embodiments, this document also discloses methods of treating a patient by inducing the formation of antigens in tumor cells, which can be targeted by the patient's immune system, carrying out clearance. Without being limited by theory, in various embodiments, administration of a splicing modulator alone and/or as part of an ADC or composition may produce neoantigens that induce an immune response, induce an immune response to double-stranded RNA, for example, by re-expressing endogenous retroviruses present in the intron, and/or the production of neoantigens that induce immunogenic cell death.

[768] As used herein, the term "neoantigen" refers to any antigen not previously exposed to the immune system that results from one or more tumor-specific mutations and/or from exposure of a tumor to a drug (e.g., any one or more splicing modulators disclosed herein, individually and/or as part of an ADC or composition). Tumor-specific mutations can include missense mutations, frameshifts, translocations, and mRNA splicing variants, as well as mutations that affect post-translational processing such as phosphorylation and glycosylation. In various embodiments, these exemplary mutations may result from non-synonymous changes in the coding sequence and/or mutations that alter mRNA processing (eg, splicing). In various embodiments, all of these exemplary mutations can result in molecular changes that can be recognized by the respective T cell receptor. In various preferred embodiments, an exemplary neoantigen is a neoantigen induced by the delivery of a splicing modulator alone and/or as part of an ADC or composition. In various embodiments, delivery of a splicing modulator (e.g., any one or more of the splicing modulators disclosed herein) can induce a new mRNA splice variant that results in the translation of proteins containing one or more new peptide domains that the immune system has not previously been exposed to. was exposed. In various embodiments, the tumor-specific mutations may be mRNA splice variants resulting from the delivery or administration of a splicing modulator, ADC, or a composition comprising a splicing modulator, or ADC.

[769] Without being limited by theory, in various embodiments, delivery of splicing modulators, alone and as part of an ADC or composition, can induce a new mRNA splice variant (e.g., exon skipping, intron retention), resulting in a change in open reading frames and /or coding sequences of various genes. In various embodiments, these altered genes are translated into proteins containing one or more new peptide domains that are recognized as foreign by the immune system. In various embodiments, one or more new peptide domains do not exist in proteins or in any other part of the human proteome in the absence of splicing modulator treatment. In various embodiments, proteins containing one or more new peptide domains can be degraded by the proteasome to form new peptide fragments that act as substrates for immunopeptide presentation mechanisms, eg, via MHC presentation. In various embodiments, novel peptide fragments representing neoantigens can be presented in an MHC1-linked peptidome, for example, on tumor cells.

[770] In various embodiments, the delivery of splicing modulators, individually and as part of an ADC or composition, may result in one or more events specific to tumor cells (eg, arrest of cell growth). In various embodiments, tumor cell-specific event(s) can lead to (1) increased contact with phagocytes (Bracci et al. (2014) Cell Death Differ. 21(1):15-25); (2) moving new peptide fragments to the tumor-draining lymph node for contact with antigen-presenting cells; (3) processing in antigen-presenting cells of novel peptide fragments from the phagocytosed tumor cell and presentation of the fragments as neoantigens to populations of circulating naïve T cells; (4) interaction of novel peptide fragments with T cells expressing receptors that recognize the fragments as neoantigens; (5) maturation and activation of effector T cells that mediate responses (eg, CD4+ and/or CD8+ T cells); and/or (6) contacting T cells with additional tumor cells treated with a splicing modulator and presenting new peptide fragments representing neoantigens on their surface as part of complexes with MHC1. In various embodiments, tumor cell-specific event(s) may result either directly or indirectly in the recruitment of T cells with effector function and/or the destruction of neoantigen presenting tumor cells.

[771] In addition, without being limited by theory, in various embodiments, the delivery of splicing modulators individually and as part of an ADC or composition can cause re-expression of endogenous retroviruses present in the intron, leading to an immune response to double-stranded RNA.

[772] In addition, without being limited by theory, in various embodiments, the delivery of splicing modulators individually and as part of an ADC or composition can lead to immunogenic cell death caused by the release, induced by the splicing modulator, of neoantigens formed as a result of mutations. In various embodiments, delivery of splicing modulators alone and as part of an ADC or composition can induce an immune response to double-stranded RNA. In various embodiments, the implementation of the immune response to double-stranded RNA may result from the re-expression of endogenous retroviruses present in the intron. In various embodiments, the implementation of the immune response to double-stranded RNA can lead to the death of tumor cells. In various embodiments, delivery of splicing modulators alone and as part of an ADC or composition can induce immunogenic cell death. In various embodiments, immunogenic cell death may be caused by the release of mutated neoantigens and/or by the host's immune response to tumor cells.

[773] Accordingly, in various embodiments, methods of treatment are disclosed that provide for the induction of neoantigens by administering one or more splicing modulators and/or ADCs and/or compositions containing a splicing modulator or ADC, for example, any splicing modulator, ADC, or composition disclosed in this document. In various embodiments, the method comprises administering a splicing modulator, ADC, or composition at a reduced dosage than would be required in the absence of neoantigen induction. In some embodiments, the method involves administering one or more initial induction doses to produce neoantigens and induce an immune response (e.g., converting naive T cells to memory cells) followed by a reduction in dosage or frequency of administration (i.e., due to the combined effect of a splicing modulator, ADC or composition and immune targeting of neoantigens). In some embodiments, the treatment may involve administering a combination of a splicing modulator, an ADC, or a neoantigen-based immune response inducing composition, and at least one additional therapy (eg, a second cancer therapy). For example, in some embodiments, treatment may involve administering a combination of a splicing modulator, an ADC, or a neoantigen-based immune response inducing composition, and one or more immune response checkpoint inhibitors. In some embodiments, treatment may involve administering a combination of a splicing modulator, an ADC, or a neoantigen-based immune response inducing composition and one or more cytokines or cytokine analogs. In some embodiments, treatment may involve administering a combination of a splicing modulator, an ADC, or a neoantigen-based immune response inducing composition and one or more neoantigen vaccines. In some other embodiments, treatment may involve administering a combination of a splicing modulator, an ADC, or a neoantigen-based immune response inducing composition and one or more tumor-targeting engineered T cells (eg, CAR-T).

[774] In some embodiments, neoantigens can be used to monitor the effectiveness of treatment with a splicing modulator, ADC, or composition. For example, following administration of a splicing modulator, ADC, or composition, a patient sample (eg, tumor biopsy) may be obtained and screened for neoantigens or markers of an immune or inflammatory response. If a neoantigen and/or an immune response is detected, additional treatment may be given, eg at a reduced dosage.

[775] In various embodiments, methods of treatment are disclosed that provide for inducing an immune response to double-stranded RNA by administering one or more splicing modulators and/or ADCs and/or compositions containing a splicing modulator or ADC, e.g., any splicing modulator, ADC or compositions disclosed in this document.

[776] In various embodiments, methods of treatment are disclosed that provide for the induction of immunogenic cell death by administering one or more splicing modulators and/or ADCs and/or compositions containing a splicing modulator or ADC, for example, any splicing modulator, ADC, or composition. disclosed in this document.

[777] In various embodiments, the administration of a splicing modulator, an ADC, or a composition containing a splicing modulator can be combined with any known anti-cancer therapy. Examples of currently available immune activation strategies available for cancer treatment include, but are not limited to, treatment with immune checkpoint inhibitor (ICI) molecules, treatment with cytokines or cytokine analogs, vaccination with cancer vaccines, and T- tumor-targeting cells (e.g., proliferation of tumor-infiltrating lymphocytes or CAR-T). These technologies are predominantly aimed at enhancing or inducing an immune response to pre-existing tumor antigens (either mutations or aberrant expression of cell surface proteins). One or more of these strategies may involve one or more mutations that are capable of inducing the development of a T cell response to an antigen. For example, patient responses to checkpoint inhibition may correlate with mutation load with non-synonymous changes. In addition, cancer vaccine approaches that rely on pre-existing mutations and the antigenicity of those mutations can be used.

[778] Splicing modulators and/or ADCs containing such modulators can cause extensive transcriptome changes that occur across multiple lineages. Translation of these altered mRNAs can provide stable and reproducible altered proteins that produce MHC1-binding neopeptides with high affinity for various isotypes of HLA molecules. Without being limited by theory, due to the large number of changes in the transcriptome and proteome, treatment with splicing modulators and/or ADCs can increase the number of potentially reactive neoantigens for improved involvement in the adaptive immune response.

[779] As used herein, the terms "splicing modulator", "spliceosome modulator", or "splice modulator" refer to compounds that exhibit anticancer activity by interacting with components of the spliceosome. In some embodiments, a splicing modulator changes the rate or shape of splicing in a target cell. Splicing modulators that act as inhibitory agents, for example, are capable of reducing uncontrolled cell proliferation. In particular, in some embodiments, splicing modulators may exert their effect by inhibiting the SF3b spliceosome complex. In some embodiments, the splicing modulator is selected from any one or more of the splicing modulators disclosed herein. In some embodiments, the splicing modulator used is delivered to the cell and/or administered to the subject as a standalone agent. In some other embodiments, the splicing modulator used is delivered to a cell and/or administered to a subject as part of an ADC (eg, an ADC selected from any of the exemplary ADCs disclosed herein). In some other embodiments, the splicing modulator used is delivered to a cell and/or administered to a subject as part of a composition comprising multiple copies of the splicing modulator or multiple copies of an ADC carrying the splicing modulator. Such compositions are disclosed herein.

[780] In some embodiments, the splice modulator used, which is delivered to a cell and/or administered to a subject as part of an ADC (e.g., an ADC selected from any of the exemplary ADCs described herein), provides additional therapeutic benefits over the modulator used. splicing, which is delivered to the cell and/or administered to the subject as a separate agent. For example, in some embodiments, the splicing modulator used, which is delivered to a cell and/or administered to a subject as part of an ADC, provides targeted delivery of the splicing modulator to a neoplastic cell expressing the target antigen (i.e., the antigen is targeted by the ADC antibody fragment) . In some embodiments, such targeted delivery of a splicing modulator reduces off-target treatment and/or off-target cytotoxicity. In some embodiments, such targeted delivery promotes tumor selective presentation of the neoantigen on neoplastic cells, but not on healthy cells that do not express the target antigen. In some embodiments, such targeted delivery results in, for example, at least 75%, 80%, 85%, 90%, 95%, or 99% alternative splicing and the induction of new mRNAs and MHC-associated peptides re-presenting neoantigens in target neoplastic cells, but not in non-target cells. Thus, without being limited by theory, in some embodiments, after priming and/or expansion of effector T cells (e.g., using a neoantigen vaccine), the immune system may preferentially attack neoplastic cells presenting a neoantigen, but not healthy cells due to preferential expression of neoantigens. on tumor cells after treatment with the ADC described herein.

Immune response induction and treatment regimen :

[781] In various embodiments, the present invention provides a method of inducing the production of at least one neoantigen by contacting a neoplastic cell with an effective amount of a splicing modulator, an antibody drug conjugate (ADC) splicing modulator, or a composition comprising a splicing modulator or ADC. . In various embodiments, the present invention provides a method of inducing an immune response to double-stranded RNA by contacting a neoplastic cell with an effective amount of a splicing modulator, an antibody drug conjugate (ADC) splicing modulator, or a composition comprising a splicing modulator or ADC. In various embodiments, the present invention provides a method of inducing immunogenic cell death by contacting a neoplastic cell with an effective amount of a splicing modulator, an antibody drug conjugate (ADC) splicing modulator, or a composition comprising a splicing modulator or ADC.

[782] In some embodiments, the implementation of at least one neoantigen contains the amino acid sequence under any of SEQ ID NO: 37-65. In some embodiments, at least one neoantigen comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, at least one neoantigen comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, at least one neoantigen comprises the amino acid sequence of under any of SEQ ID NOs: 46-49.

[783] In some embodiments, the implementation of the neoplastic cell is in cell culture in vitro. In some embodiments, the neoplastic cell is obtained from a subject. In some embodiments, the implementation of the neoplastic cell is in the subject. In some embodiments, the neoplastic cell is derived from a hematologic malignancy or a solid tumor. In some embodiments, the hematologic malignancy is selected from B-cell malignancy, leukemia, lymphoma, and myeloma. In some embodiments, the hematologic malignancy is selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the solid tumor is selected from breast cancer (e.g., HER2 positive breast cancer), stomach cancer (e.g., gastric adenocarcinoma), prostate cancer, ovarian cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer ( eg, endometrial serous carcinoma), salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer, and esophageal cancer. In some embodiments, the solid tumor is selected from HER2 positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

[784] In some embodiments, the present invention further provides a method of inducing the production of at least one neoantigen and/or the development of a T cell response in a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a splicing modulator, ADC or a composition containing a splicing modulator or ADC. Also provided herein in various embodiments is a method of treating a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a splicing modulator, an ADC, or a composition comprising a splicing modulator or ADC, wherein administering the splicing modulator, an antibody conjugate, and drug or composition provides induction of the formation of at least one neoantigen and/or T-cell response.

[785] In various embodiments, the present invention provides a method of inducing an immune response to double-stranded RNA in a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a splicing modulator, ADC, or a composition comprising a splicing modulator or ADC. Also provided herein in various embodiments is a method of treating a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a splicing modulator, an ADC, or a composition comprising a splicing modulator or ADC, wherein administering the splicing modulator, an antibody conjugate, and drug or composition ensures the induction of an immune response to double-stranded RNA.

[786] In still other embodiments, the present invention provides a method of inducing immunogenic cell death in a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a splicing modulator, ADC, or a composition comprising a splicing modulator or ADC. Also provided herein in various embodiments is a method of treating a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a splicing modulator, an ADC, or a composition comprising a splicing modulator or ADC, wherein administering the splicing modulator, an antibody conjugate, and drug or composition provides the induction of immunogenic cell death.

[787] In some embodiments of the therapeutic methods described herein, at least one neoantigen contains the amino acid sequence of any of SEQ ID NOs: 37-65. In some embodiments, at least one neoantigen comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, at least one neoantigen comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, at least one neoantigen comprises the amino acid sequence of under any of SEQ ID NOs: 46-49.

[788] In some embodiments, the present invention further provides a method of treating a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a splicing modulator, ADC, or a composition comprising a splicing modulator or ADC, wherein administering the splicing modulator, conjugate antibodies and drugs or compositions in combination with one or more additional therapies involving a second agent, provides for the induction of immunogenic cell death.

[789] In some embodiments of the therapeutic methods described herein, the amount of splicing modulator, antibody drug conjugate, composition, or second agent administered is reduced due to induction of at least one neoantigen and/or T-cell response compared to standard dosage of the splicing modulator, antibody-drug conjugate, composition, or second agent. In some embodiments, the amount of splicing modulator, antibody drug conjugate, composition, or second agent administered is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75% , or 90% compared to the standard dosage of the splicing modulator, antibody-drug conjugate, composition, or second agent. In some embodiments, the splicing modulator, antibody drug conjugate, composition, or second agent is administered at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75 %, or 90% less frequently compared to the standard regimen for splicing modulator, antibody-drug conjugate, composition or second agent. In some embodiments, the amount administered and/or dosage administered of the splicing modulator, antibody drug conjugate, composition, or second agent results in lower systemic toxicity and/or improved tolerability.

[790] As used herein, the term "unit dosage" or "standard dosage regimen" refers to any conventional or conventional regimen for administering a therapeutic agent, such as one proposed by a manufacturer, approved by regulatory authorities, or otherwise tested in human subjects to satisfy the needs of the average patient. In some embodiments, the therapeutic agent is a splicing modulator, an antibody, or an antibody drug conjugate with anticancer activity.

[791] For example, a standard regimen for administering trastuzumab, the exemplary anti-HER2 antibody disclosed herein, may include intravenous administration of 8 mg/kg over 90 minutes. (week 1), followed by intravenous administration of 6 mg/kg over 30-90 minutes. every 3 weeks (from week 4 until end of cycle) (Herceptin® (trastuzumab), 2017 FDA Compliant Labeling Supplement).

[792] As another example, a standard regimen for administering ipilimumab, an exemplary checkpoint inhibitor anti-CTLA4 antibody, may include intravenous administration of 3 mg/kg over 90 minutes. every 3 weeks for 4 doses (Yervoy® (ipilimumab), 2018 FDA Compliant Labeling Supplement). Another standard regimen for administering ipilimumab may be intravenous administration of 10 mg/kg over 90 minutes. every 3 weeks for 4 doses, then 10 mg/kg every 12 weeks for up to 3 years (Yervoy® (ipilimumab), FDA Compliant Labeling Supplement, 2018).

[793] As another example, a standard schedule for administering nivolumab, an exemplary checkpoint inhibitor anti-PD1 antibody, may include intravenous administration of 3 mg/kg over 60 minutes. every 2 weeks (Opdivo® (nivolumab), FDA-compliant labeling, 2015).

[794] As another example, a standard schedule for administering atezolizumab, an exemplary anti-PDL1 checkpoint inhibitor antibody, may include intravenous administration of 1200 mg over 60 minutes. every 3 weeks (Tecentriq® (atezolizumab), FDA Compliant Labeling Supplement, 2018).

[795] As another example, a standard regimen for administering T-DM1, an exemplary anti-HER2 antibody drug conjugate, may include intravenous administration of 3.6 mg/kg over 90 minutes. every 3 weeks (Kadcyla® (T-DM1), FDA Compliant Labeling Supplement, 2016).

[796] In some embodiments, the methods described herein may further include administering at least one additional therapy (eg, checkpoint inhibitor, neoantigen vaccine, cytokine or cytokine analog, CAR-T, etc.). In some embodiments, the amount of splice modulator, antibody-drug conjugate, composition, and/or at least one additional therapy administered is reduced due to induction of at least one neoantigen and/or T cell response compared to the standard dosage of the splicing modulator, an antibody-drug conjugate, a composition, and/or at least one additional therapeutic agent. In some embodiments, the amount of splice modulator, antibody drug conjugate, composition, and/or at least one additional therapy administered is reduced due to the induction of an immune response to double-stranded RNA compared to a standard dosage of splice modulator, antibody drug conjugate, composition, and /or at least one additional therapy. In some embodiments, the amount of splice modulator, antibody drug conjugate, composition, and/or at least one additional therapy administered is reduced due to the induction of immunogenic cell death compared to a standard dosage of the splicing modulator, antibody drug conjugate, composition and/or at least one additional therapy. In some embodiments, the amount of splicing modulator, antibody drug conjugate, composition, and/or at least one additional therapy administered is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45 %, 50%, 75%, or 90% compared to the standard dosage of the splicing modulator, antibody-drug conjugate, composition, and/or at least one additional therapy. In some embodiments, the frequency of administration of the splicing modulator, antibody drug conjugate, composition, and/or at least one additional therapy is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45 %, 50%, 75%, or 90% compared to the standard schedule for administering the splicing modulator, antibody-drug conjugate, composition, and/or at least one additional therapy. In some embodiments, the amount administered and/or dosage administered of the splicing modulator, antibody drug conjugate, composition, and/or at least one additional therapy results in lower systemic toxicity and/or improved tolerability.

[797] In some embodiments, the splicing modulator, antibody drug conjugate, or composition is started prior to the administration of at least one additional therapy. In other embodiments, the splicing modulator, antibody-drug conjugate, or composition is administered following the administration of at least one additional therapy. In still other embodiments, the splicing modulator, antibody drug conjugate or composition is started concurrently with the administration of at least one additional therapy.

[798] In some embodiments, administration of the splicing modulator, antibody drug conjugate or composition is repeated at least once after the initial administration. In some embodiments, the amount of splicing modulator, antibody drug conjugate or composition used for reintroduction is reduced compared to the amount used for initial administration. In some embodiments, the amount of splicing modulator, antibody drug conjugate or composition used for reintroduction is reduced compared to the standard dosage of the splicing modulator, antibody drug conjugate or composition. In some embodiments, the amount of splicing modulator, antibody drug conjugate or composition used for reintroduction is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% compared to the standard dosage or starting dosage of the splicing modulator, antibody-drug conjugate or composition.

[799] In some embodiments, the administration of at least one additional therapy is repeated at least once after the initial administration. In some embodiments, the amount of at least one additional therapy used for repeated administration is reduced compared to the amount used for initial administration. In some embodiments, the amount of at least one additional therapy used for repeated administration is reduced compared to the standard dosage of at least one additional therapy. In some embodiments, the amount of at least one additional therapy used for repeated administration is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% compared to the standard dosage or initial dosage of at least one additional therapy.

[800] In some embodiments, the reintroduction of the splicing modulator, antibody-drug conjugate or composition is concurrent with the reintroduction of at least one additional therapy. In some embodiments, administration of the splicing modulator, antibody-drug conjugate or composition is performed sequentially or spaced apart in time with repeated administration of at least one additional therapy.

[801] In some embodiments, at least one additional therapy involves the administration of a checkpoint inhibitor, for example, any checkpoint inhibitor disclosed herein. In some embodiments, the subject is intolerant, unresponsive, or deficiently responsive to a checkpoint inhibitor when administered alone. In some embodiments, the checkpoint inhibitor targets PD1/PDL1, CTLA4, OX40, CD40, LAG3, TIM3, GITR, and/or KIR. In some embodiments, the checkpoint inhibitor targets CTLA4, OX40, CD40, and/or GITR. In some embodiments, the checkpoint inhibitor is an antibody having inhibitory or agonist activity on its target. In some embodiments, the checkpoint inhibitor is targeted by an inhibitory antibody or other similar inhibitory molecule. In other embodiments, the checkpoint inhibitor is targeted by an agonist antibody or other similar agonist molecule.

[802] In some other embodiments, at least one additional therapy involves the administration of a neoantigenic vaccine, for example, any neoantigenic vaccine disclosed herein. In some embodiments, the splicing modulator, ADC, or composition is administered prior to administration of the neoantigen vaccine. In some embodiments, the splicing modulator, ADC, or composition is administered following administration of the neoantigen vaccine. In some embodiments, the splicing modulator, ADC, or composition is administered concurrently with the administration of the neoantigen vaccine. In some embodiments, administration of the splicing modulator, ADC, or composition is repeated at least once after the initial administration. In some embodiments, the amount of splicing modulator, ADC, or composition used for reintroduction is reduced compared to the amount used for initial administration.

[803] In some embodiments, the neoantigenic vaccine comprises at least one neoantigenic peptide. In some embodiments, at least one neoantigenic peptide is in the range of about 10 to about 50 amino acids in length. In some embodiments, at least one neoantigenic peptide is in the range of about 10 to about 35 amino acids in length. In some embodiments, at least one neoantigenic peptide is in the range of about 15 to about 25 amino acids in length. In some embodiments, at least one neoantigenic peptide contains one or more neoantigenic sequences.

[804] In some embodiments, the implementation neoantigenic sequence contains the amino acid sequence under any of SEQ ID NO: 37-65. In some embodiments, the neoantigenic sequence comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the neoantigenic sequence comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the neoantigenic sequence comprises the amino acid sequence of any of SEQ ID NO: 46- 49.

[805] In some other embodiments, the neoantigenic sequence comprises the amino acid sequence of any of SEQ ID NOs: 66-93 or an antigenic portion of any of SEQ ID NOs: 66-93. In some embodiments, the neoantigenic sequence comprises the amino acid sequence of SEQ ID NO: 66 or the antigenic portion of SEQ ID NO: 66. In some embodiments, the neoantigenic sequence comprises the amino acid sequence of any of SEQ ID NOs: 74-77 or the antigenic portion of any of SEQ ID NO: 74-77. In some embodiments, the neoantigenic sequence and/or antigenic portion is in the range of about 10 to about 50 amino acids in length. In some embodiments, the neoantigenic sequence and/or antigenic portion is in the range of about 10 to about 35 amino acids in length. In some embodiments, the neoantigenic sequence and/or antigenic portion is in the range of about 15 to about 25 amino acids in length. In some embodiments, the neoantigenic sequence and/or antigenic portion is in the range of about 10 to about 20 amino acids in length. In some embodiments, the neoantigenic sequence and/or antigenic portion do not directly overlap with or consist of a canonical peptide sequence (eg, any of the illustrative canonical peptide sequences underlined in Table 21).

[806] As used herein, the term "antigenic portion" or "antigenic fragment" of a neoantigenic sequence refers to one or more fragments of a neoantigenic sequence that retain the ability to induce a T cell response (e.g., antigen-specific expansion and/or population maturation(- ii) effector T cells). In some embodiments, the antigenic portion may also retain the ability to be internalized, processed, and/or presented by antigen-presenting cells (eg, dendritic cells). In some embodiments, the antigenic portion also retains a T cell priming function. In some embodiments, the length of the antigenic portion of the neoantigenic sequence is in the range of about 10 to about 50 amino acids. In some embodiments, the length of the antigenic portion of the neoantigenic sequence is in the range of about 10 to about 35 amino acids. In some embodiments, the length of the antigenic portion of the neoantigenic sequence is in the range of about 15 to about 25 amino acids. In some embodiments, the length of the antigenic portion of the neoantigenic sequence is in the range of about 10 to about 20 amino acids. In some embodiments, the antigenic portion of the neoantigenic sequence (eg, the antigenic portion of any of SEQ ID NOs: 66-93) or the mRNA encoding it is formulated as a neoantigenic vaccine.

[807] An exemplary embodiment of the antigenic portion is the region(s) flanking amino acids 45-53 of SEQ ID NO: 66. Another exemplary embodiment of the antigenic portion is the region(s) flanking amino acids 82-90 of SEQ ID NO: 66. In some embodiments, the antigenic portion is capable of binding to at least one HLA allele expressed in the subject (eg, HLA-A*02:01). In some other embodiments, the antigenic moiety is capable of binding to at least one HLA allele expressed in at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from a neoplastic disorder. In some embodiments, the antigenic portion is capable of eliciting a T cell response against a tumor present in at least 1%, at least 5%, or at least 10% of a population of subjects suffering from a neoplastic disorder.

[808] In some embodiments, the antigenic portion does not directly overlap with or consist of the canonical peptide sequence. As used herein, the term "canonical peptide sequence" refers to any contiguous peptide sequence present in the human proteome in the absence of contact with a splicing modulator (e.g., in the absence of contact with a splicing modulator alone and/or as part of an ADC or composition), and/or to which the immune system has previously been exposed. In some embodiments, the canonical peptide sequence is derived from and/or encoded by the open reading frame of the canonical transcript. Illustrative canonical peptide sequences are underlined in Table 21.

[809] In some embodiments, if a splicing modulator is administered (e.g., alone and/or as part of an ADC or composition), the canonical peptide sequence can be derived from the 24 nucleotides located immediately at the 5' end of the frame prior to the aberrant splicing event induced splicing modulator, and/or may be encoded by them. Thus, in some embodiments, the implementation of the canonical peptide sequence contains or consists of 8 amino acids located directly from the N-terminus of the neoantigenic sequence, the formation of which is induced by the splicing modulator. In some embodiments, if the 5' exon sequence ends at the terminal nucleotide of the codon, the canonical peptide sequence ends at the end of the exon. In some other embodiments, if the 5' exon sequence ends in one or two of the three codon nucleotides, the canonical peptide sequence is derived from and/or encoded by the 24 nucleotides preceding the incomplete codon. In some embodiments, the 3' mRNA sequences in an aberrant splicing event may be translated in the same open reading frame generated by the 5' exon until a stop codon is reached, at which point translation may end. In some embodiments, if an aberrant splicing event (eg, exon skipping) results in the preservation of the open reading frame of the canonical transcript, the C-terminal sequence can be translated into an additional 24 nucleotides encoding the 8 C-terminal amino acids. In some embodiments, in this context, only a stretch along an aberrant exon junction may encode a neoantigenic sequence. In some embodiments, if the open reading frame is shifted (eg, while conserving an intron), the complete C-terminal sequence (encoded by the 3' mRNA) may encode a neoantigenic sequence.

[810] In some embodiments, the antigenic portion of a neoantigenic sequence is selected by comparing the neoantigenic sequence to a canonical peptide sequence and selecting a portion of the neoantigenic sequence that does not directly overlap, consist of, and/or align with the canonical peptide sequence. In some embodiments, the antigenic portion of a neoantigenic sequence can be screened for antigenicity and/or T cell priming function in the same manner as full length neoantigenic sequences (eg, the neoantigenic sequence from which the antigenic portion is derived). In some embodiments, an antigenic portion of a neoantigenic sequence is evaluated for antigenicity and/or T cell priming function using a T cell priming assay, such as the exemplary T cell priming experiments described herein.

[811] In some embodiments, the neoantigenic sequence is a subject-specific neoantigenic sequence. In some embodiments, the neoantigenic sequence is a personalized neoantigenic vaccine for a subject. In some embodiments, the neoantigen sequence used to create a personalized neoantigen vaccine for a subject is capable of binding to at least one HLA allele expressed in the subject. In some embodiments, the personalized neoantigen vaccine is selected by identifying neoantigens expressed in the subject's tumor, e.g., after administering a splicing modulator or ADC and selecting a vaccine containing the neoantigen sequence found in the patient's tumor, e.g., a vaccine containing the amino acid sequence of any of the SEQ IDs. NO: 37-65, which is found in the tumor.

[812] The term "personalized" when used to describe a neoantigen vaccine refers to a vaccine created by identifying one or more neoantigens produced in a patient, preferably the neoantigen identified in the patient after exposure to a splicing modulator, ADC, or composition, and then applying one or more of these neoantigens as the basis for a vaccine for the same patient. Accordingly, in some embodiments, a splicing modulator, ADC, or composition is administered to the patient and then screened for the presence of neoantigens produced as a result of the treatment. In some embodiments, the selected neoantigenic vaccine contains a neoantigenic peptide or mRNA as disclosed herein and whose presence in a patient is confirmed after exposure to a splicing modulator, ADC, or composition. In some embodiments, the splicing modulator, ADC or composition and/or peptide or mRNA vaccine can be administered to a patient once or repeatedly. Therefore, in some embodiments, one or more of these neoantigens is used to create a personalized vaccine that is administered to a patient. In some embodiments, one or more neoantigens used to create a personalized vaccine have binding affinity for one or more patient-specific HLA alleles. In some embodiments, the patient expresses one or more MHC1 alleles that bind to one or more neoantigens. Predicting whether a neoantigen will bind to a specific MHC1 allele can be done using any computational prediction method known in the art. Exemplary computational prediction methods are disclosed, for example, in Meydan et al. (2013) BMC Bioinformatics 14(Suppl. 2):S13, which is incorporated herein by reference for such methods.

[813] In some other embodiments, the neoantigenic sequence is a universal neoantigenic sequence. In some embodiments, the neoantigenic sequence is a generic neoantigenic vaccine.

[814] The term "universal" when used to describe a neoantigen vaccine refers to a vaccine containing a peptide or mRNA sequence that is based on the conventional(s) or known(s) neoantigen(s) detectable(- x) by sequencing neoantigens produced in multiple patients and/or in patient tissue samples, preferably after exposure to at least a splicing modulator, ADC, or composition. The peptide or mRNA sequence used in a vaccine does not have to be present in every patient, but is found in at least a few patients or in tissue samples from patients. In some embodiments, the splicing modulator, ADC or composition and/or peptide or mRNA vaccine can be administered to a patient once or repeatedly. Therefore, in some embodiments, the peptide or mRNA sequence is used to vaccinate additional patients. In some embodiments, a splicing modulator, ADC, or composition is administered to a patient, and then a peptide or mRNA vaccine based on a known neoantigen is administered to enhance the immune response to neoantigens produced by the splicing modulator, ADC, or composition. In some embodiments, the patient is given a generic peptide or mRNA vaccine and then given a single or repeated dose of a splicing modulator, ADC, or composition. In some embodiments, the neoantigenic sequence (or sequences) used to create a universal neoantigenic vaccine is selected based on the overall occurrence of the MHC1 allele in a given patient population (Maiers et al. (2007) Hum. Immunol. 68(9):779-88) .

[815] In some embodiments, a neoantigenic sequence (e.g., a universal neoantigen) is capable of binding to at least one HLA allele expressed in at least 10%, at least 15%, at least 20%, at least 25% , at least 30%, at least 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from a neoplastic disorder. In some embodiments, the neoantigenic sequence is capable of eliciting a T cell response to a tumor present in at least 1%, at least 5%, or at least 10% of a population of subjects suffering from a neoplastic disorder.

[816] In some embodiments, a neoantigenic sequence has been identified by sequencing at least one neoantigenic peptide or mRNA encoding it, the formation of which is induced in a subject by administration of an effective amount of a splicing modulator, antibody drug conjugate or composition. In some embodiments, at least one neoantigenic peptide comprises a neoantigenic sequence whose formation is induced by contacting a neoplastic cell with an effective amount of a splicing modulator, antibody drug conjugate or composition. In some embodiments, the neoplastic cell is in an in vitro cell culture. In some embodiments, the neoplastic cell is obtained from a subject. In some embodiments, the implementation of the neoplastic cell is in the subject.

[817] In some embodiments, the neoantigenic vaccine comprises at least one neoantigenic peptide and a pharmaceutically acceptable carrier (eg, any of the exemplary carriers described herein). In some embodiments, at least one neoantigenic peptide is associated with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is selected from a peptide, serum albumin, fisurella hemocyanin, immunoglobulin, thyroglobulin, ovalbumin, toxoid, or an attenuated derivative of toxoid, a cytokine, and a chemokine. In some embodiments, the neoantigenic peptide and the pharmaceutically acceptable carrier are covalently linked through a linker. In some embodiments, the neoantigenic peptide and the pharmaceutically acceptable carrier are expressed as a fusion protein. In some embodiments, the neoantigenic vaccine comprises at least one neoantigenic peptide and a pharmaceutically acceptable diluent. In some embodiments, the neoantigenic vaccine comprises at least one neoantigenic peptide and a pharmaceutically acceptable adjuvant.

[818] In some embodiments, the neoantigenic vaccine contains at least one neoantigenic mRNA. In some embodiments, at least one neoantigenic mRNA encodes one or more than one neoantigenic sequence. In some embodiments, the implementation neoantigenic sequence contains the amino acid sequence under any of SEQ ID NO: 37-65. In some embodiments, the neoantigenic sequence comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the neoantigenic sequence comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the neoantigenic sequence comprises the amino acid sequence of any of SEQ ID NO: 46- 49.

[819] In some other embodiments, the neoantigenic sequence comprises the amino acid sequence of any of SEQ ID NOs: 66-93 or an antigenic portion of any of SEQ ID NOs: 66-93. In some embodiments, the neoantigenic sequence comprises the amino acid sequence of SEQ ID NO: 66 or the antigenic portion of SEQ ID NO: 66. In some embodiments, the neoantigenic sequence comprises the amino acid sequence of any of SEQ ID NOs: 74-77 or the antigenic portion of any of SEQ ID NO: 74-77. In some embodiments, the neoantigenic sequence and/or antigenic portion is in the range of about 10 to about 50 amino acids in length. In some embodiments, the neoantigenic sequence and/or antigenic portion is in the range of about 10 to about 35 amino acids in length. In some embodiments, the neoantigenic sequence and/or antigenic portion is in the range of about 15 to about 25 amino acids in length. In some embodiments, the neoantigenic sequence and/or antigenic portion is in the range of about 10 to about 20 amino acids in length. In some embodiments, the neoantigenic sequence and/or antigenic portion do not directly overlap with or consist of a canonical peptide sequence (eg, any of the illustrative canonical peptide sequences underlined in Table 21).

[820] In some embodiments, the neoantigenic sequence is a subject-specific neoantigenic sequence. In some embodiments, the neoantigenic sequence is a personalized neoantigenic vaccine for a subject. In some embodiments, the neoantigenic sequence is capable of binding to at least one HLA allele expressed in the subject.

[821] In some other embodiments, the neoantigenic sequence is a universal neoantigenic sequence. In some embodiments, the neoantigenic sequence is a generic neoantigenic vaccine. In some embodiments, the neoantigenic sequence is capable of binding to at least one HLA allele expressed in at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least at least 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from a neoplastic disorder. In some embodiments, the neoantigenic sequence is capable of eliciting a T cell response to a tumor present in at least 1%, at least 5%, or at least 10% of a population of subjects suffering from a neoplastic disorder.

[822] In some embodiments, a neoantigenic sequence has been identified by sequencing the protein sequence of at least one neoantigen. In some embodiments, a neoantigenic sequence has been identified by sequencing at least one mRNA encoding a neoantigen that is induced in a subject by administration of an effective amount of a splicing modulator, antibody drug conjugate or composition. In some embodiments, at least one neoantigenic mRNA encodes a neoantigenic sequence whose formation is induced by contacting a neoplastic cell with an effective amount of a splicing modulator, antibody drug conjugate or composition. In some embodiments, the neoplastic cell is in an in vitro cell culture. In some embodiments, the neoplastic cell is obtained from a subject. In some embodiments, the implementation of the neoplastic cell is in the subject.

[823] In some embodiments, the neoantigenic vaccine comprises at least one neoantigenic mRNA and a pharmaceutically acceptable carrier (eg, any of the exemplary carriers described herein). In some embodiments, at least one neoantigenic mRNA is associated with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is selected from a peptide, serum albumin, fisurella hemocyanin, immunoglobulin, thyroglobulin, ovalbumin, toxoid, or an attenuated derivative of toxoid, a cytokine, and a chemokine. In some embodiments, the neoantigenic vaccine comprises at least one neoantigenic mRNA and a pharmaceutically acceptable diluent. In some embodiments, the neoantigenic vaccine comprises at least one neoantigenic mRNA and a pharmaceutically acceptable adjuvant. In some embodiments, the neoantigenic mRNA is encapsulated with an encapsulating agent. In some embodiments, the encapsulating agent is a liposome. In some embodiments, the implementation of the encapsulating agent is a nanoparticle.

[824] In some embodiments, at least one additional therapeutic agent involves the administration of a cytokine or cytokine analog, for example, any cytokine or cytokine analog disclosed herein. In some embodiments, the subject is characterized by intolerance, intolerance, or insufficient responsiveness to a cytokine or cytokine analog when administered alone. In some embodiments, the cytokine or cytokine analog provides an amplifying agent for T cells. In some embodiments, the cytokine or cytokine analog comprises IL-2, IL-10, IL-12, IL-15, IFNγ and/or TNFα. In some embodiments, the cytokine or cytokine analog comprises IL-2, IL-10, IL-12, and/or IL-15. In some embodiments, administration of a cytokine or cytokine analog enhances T cell priming following administration of a splicing modulator, antibody-drug conjugate or composition by inducing the production and presentation of neoantigens.

[825] In some embodiments, at least one additional therapy involves the administration of engineered T cells that target a tumor (i.e., CAR-T), for example, any CAR-T-based therapy disclosed herein .

[826] In some embodiments, the methods described herein may further include detecting one or more neoantigens and/or a T cell response in a subject after administration of a splicing modulator, antibody drug conjugate or composition, and optionally continuing to administer the splicing modulator, an antibody-drug conjugate or composition if one or more neoantigens and/or a T-cell response is detected. In some embodiments, detection of one or more neoantigens and/or a T cell response in a subject is indicative of efficacy of treatment with a splicing modulator, antibody drug conjugate or composition. In some embodiments, treatment with an additional therapy along with a splicing modulator, antibody drug conjugate or composition is continued if one or more neoantigens and/or a T cell response is detected. In some embodiments, treatment is continued at a reduced dosage and/or frequency if one or more neoantigens and/or a T cell response is detected.

[827] In some embodiments, the methods described herein may further comprise detecting double-stranded RNA immunity in a subject following administration of the splicing modulator, antibody drug conjugate or composition, and optionally continuing to administer the splicing modulator, antibody drug conjugate or composition. if an immune response to double-stranded RNA is detected. In some embodiments, detection of an immune response to double-stranded RNA in a subject is indicative of efficacy of treatment with the splicing modulator, antibody-drug conjugate or composition. In some embodiments, treatment with an additional therapy along with a splicing modulator, antibody-drug conjugate or composition is continued if an immune response to double-stranded RNA is detected. In some embodiments, treatment is continued at a reduced dosage and/or frequency if an immune response to double-stranded RNA is detected.

[828] In some embodiments, the methods described herein may further include detecting immunogenic cell death in a subject following administration of the splicing modulator, antibody drug conjugate or composition, and optionally continuing to administer the splicing modulator, antibody drug conjugate or composition, if immunogenic cell death is detected. In some embodiments, the detection of immunogenic cell death in a subject is indicative of the efficacy of treatment with the splicing modulator, antibody drug conjugate or composition. In some embodiments, treatment with an additional therapy along with a splicing modulator, antibody drug conjugate or composition is continued if immunogenic cell death is detected. In some embodiments, treatment is continued at a reduced dosage and/or frequency if immunogenic cell death is detected.

[829] In some embodiments, the implementation of the subject is characterized by a mutation load with non-synonymous changes of about 150 mutations or less. In some embodiments, the subject has a non-synonymous mutation burden of about 100 mutations or less. In some embodiments, the subject has a non-synonymous mutation burden of about 50 mutations or less. In some embodiments, the subject has or is suspected of having a neoplastic disorder, such as a hematologic malignancy or solid tumor. In some embodiments, the hematologic malignancy is selected from B-cell malignancy, leukemia, lymphoma, and myeloma. In some embodiments, the hematologic malignancy is selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer and cancer of the esophagus. In some embodiments, the solid tumor is selected from HER2 positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

[830] In various embodiments, the present invention further provides a method of treating a subject who has or is suspected of having a neoplastic disorder, comprising: (a) administering to the subject an effective amount of a splicing modulator, ADC, or a composition comprising a splicing modulator, or ADC, wherein administering a splicing modulator, antibody-drug conjugate or composition to induce the formation of at least one neoantigen and/or a T cell response; (b) detecting one or more neoantigens and/or a T cell response in the subject following administration of the splicing modulator, antibody drug conjugate or composition; and (c) continuing to administer the splicing modulator, antibody drug conjugate or composition if one or more neoantigens and/or a T cell response is detected. In some embodiments, detection of one or more neoantigens and/or a T cell response in a subject is indicative of efficacy of treatment with a splicing modulator, antibody drug conjugate or composition. In some embodiments, the implementation of one or more neoantigens contain the amino acid sequence under any of SEQ ID NO: 37-65. In some embodiments, one or more neoantigens comprise the amino acid sequence of SEQ ID NO: 37. In some embodiments, one or more neoantigens comprise the amino acid sequence of SEQ ID NO: 39. In some embodiments, one or more neoantigens comprise the amino acid sequence of any of SEQ ID NO: 46-49.

Combination of Splice Modulator/ADC and Inhibition of Immune Response Checkpoints

[831] In various embodiments, a patient having the cancer described herein may be treated with a combination of a splicing modulator, an ADC, or a checkpoint inhibitor composition and therapy. Immune response checkpoints are inhibitory pathways that slow or stop immune responses and prevent excessive tissue damage due to uncontrolled immune cell activity. As used herein, the term "checkpoint inhibitor" means any therapeutic agent, including any small molecular weight chemical compound, antibody, nucleic acid molecule or polypeptide, or any fragment thereof, that inhibits one or more inhibitory pathways, thereby providing a more functional immune system. activity.

[832] Treatment of patients with immune checkpoint inhibition has been shown to have good efficacy in certain clinical indications. The FDA recently approved the use of a checkpoint inhibitor in patients with tumors exhibiting high microsatellite instability, regardless of tissue origin. This registration was partly based on the observation that response rates are positively correlated with mutation load (Rizvi et al. (2015) Science 348(6230):124-8; Hellmann et al. (2018) Cancer Cell 33(5): 853-861). Estimates from the literature vary in absolute numbers and by lineage, but generally confirm that above a threshold of ~150-250 mutations, the likelihood of a response increases. Analysis of TCGA data demonstrates that a large percentage of developing adult tumors of different origins have a relatively small mutation load with non-synonymous changes (Vogelstein et al. (2013) Science 339:1549-58). Most lineages are characterized by mean non-synonymous mutation rates of ~30-80 per patient, well below the thresholds required to increase the likelihood of response to checkpoint inhibitors.

[833] For example, HER2 positive breast cancer has been shown to have an average of ~60 non-synonymous mutations present in a patient sample. However, the threshold for effective treatment with a checkpoint inhibitor, as mentioned above, is estimated to be in the range of ~150-250 non-synonymous mutations, i.e. patients above this threshold are more likely to experience complete remission, partial remission, and/or or stabilization of the disease, while patients with a value below this threshold are more likely to show progression of the disease. Therefore, strategies are required to increase the observed number of non-synonymous mutations and/or neoantigens present on tumor cells and may increase the overall likelihood of response to, for example, a checkpoint inhibitor therapy. Because cytokines (and their analogs) act through a similar mechanism of action, such strategies may also increase the overall likelihood of response to cytokine-based therapies.

[834] Currently, response rates for HER2-positive breast cancer are ~15-25% (CTI NCT02129556). In various embodiments disclosed herein, treatment with a splicing modulator, ADC, or composition in combination with a checkpoint inhibitor and/or cytokine therapy may improve this response rate. In various embodiments, treatment with a splicing modulator, an ADC, or a composition in combination with checkpoint inhibitor and/or cytokine therapy may be applicable to any tumor that occurs in adults, in particular those with a median non-synonymous mutation rate below the calculated threshold. values ~150 mutations. In some embodiments, exemplary cancers suitable for treatment with a splicing modulator, an ADC, or a composition of the present invention alone or in combination with an additional therapy (e.g., checkpoint inhibitor therapy, cytokine therapy) include without limitation, esophageal cancer, non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer, gastric cancer, endometrial cancer, pancreatic adenocarcinoma, ovarian cancer, prostate cancer, hepatocellular carcinoma, glioblastoma, breast cancer (e.g., HER2-positive breast cancer ), lung cancer (eg, non-small cell lung cancer), chronic lymphocytic leukemia, and acute myeloid leukemia. Other illustrative suitable types of cancer are defined, for example, in Vogelstein et al. (2013) Science 339:1549-58, which is incorporated herein by reference in its entirety.

[835] Since the use of many checkpoint inhibitor therapies is driven by persistent expression of tumor-associated antigens, regular booster treatments are required to be effective and to "re-stimulate" reactive T cell populations. The inducible nature of the splicing modulator or neoantigens generated by ADC provides therapeutic regimens that can be designed to enhance the immune response of neoantigen-reactive T cells while limiting T cell depletion often caused by chronic antigen stimulation. For example, in some embodiments, an initial dose of a splicing modulator, ADC, or composition is administered to a subject to initiate aberrant splicing and produce neoantigenic peptides. In some embodiments, after a period of time necessary for protein production and antigen presentation, the subject is then administered an initial dose of a checkpoint inhibitor to stimulate and/or enhance priming and expansion of effector T cells. In some embodiments, the waiting period between doses of the splicing modulator, ADC, or composition and the checkpoint inhibitor is about 2, about 3, about 4, about 5, about 6, or about 7 days. In some embodiments, the waiting period is from about 3 days to about 5 days. In some embodiments, the checkpoint inhibitor targets CTLA4, OX40, CD40, and/or GITR. In some embodiments, the combined therapeutic effect of the splicing modulator, ADC or composition and the checkpoint inhibitor may be additive or super-additive.

[836] In some embodiments, after a period allowing T cells to be primed and expanded, the subject is then administered a second or subsequent dose of a splicing modulator, ADC, or composition to initiate re-presentation of the neoantigenic peptides. In some embodiments, the waiting period between the initial dose of the checkpoint inhibitor and the second or subsequent dose of the splicing modulator, ADC, or composition is about 2, about 3, about 4, or about 5 weeks. In some embodiments, the waiting period is approximately 3 weeks. Following a second or subsequent dose of the splicing modulator, ADC, or composition, in some embodiments, the immune system may contact the neoantigen-presenting tumor cells and/or cause the tumor cells to be killed. In some embodiments, a second or subsequent dose of a checkpoint inhibitor is then administered to the subject to further expand the effector memory T cell population after T cell repriming and expansion has been achieved.

[837] In some embodiments, dosing of the splicing modulator, ADC, or composition following this illustrative initial treatment regimen may be pulsatile, i.e., the splicing modulator, ADC, or composition may be administered at long dose intervals (e.g., approximately every 4 weeks, approximately every 5 weeks, approximately every 6 weeks) to ensure antigen presentation, recruitment of T cells, and/or killing of tumor cells, and/or restoration of the population of memory T cells. At later time points, in some embodiments, treatment with a splicing modulator, ADC, or composition can be combined with one or more checkpoint inhibitors aimed at restoring effector functional capacity in depleted T cell populations. For example, in some embodiments, at later time points, treatment with a splicing modulator, ADC, or composition can be combined with one or more checkpoint inhibitors that target PD1/PDL1, LAG3, and/or TIM3. In some embodiments, the pulsatile nature of neoantigen presentation and priming may allow for less frequent and/or lower doses of the checkpoint inhibitor and/or splicing modulator, ADC, or composition to be administered. In some embodiments, the pulsatile nature of neoantigen presentation may provide one or more of the beneficial effects of treatment with a checkpoint inhibitor (e.g., an anti-CTLA4 antibody such as ipilimumab) compared to a checkpoint inhibitor administered without concomitant administration of a splicing modulator, ADC, or composition, e.g., by reducing the potential risk of adverse reactions often seen with the standard checkpoint inhibitor regimen.

[838] In certain embodiments, the checkpoint inhibitor is a cytotoxic T-lymphocyte-associated antigen (CTLA4) pathway inhibitor. CTLA4, also known as CD152, is a protein receptor that negatively regulates immune responses. CTLA4 is constitutively expressed on regulatory T cells, but its expression is upregulated in normal T cells only after activation. As used herein, the term "CTLA4 inhibitor" is intended to refer to any inhibitor of CTLA4 and/or the CTLA4 pathway. Exemplary CTLA4 inhibitors include, without limitation, anti-CTLA4 antibodies. Antibodies blocking CTLA4 for use in humans have been developed based on preclinical activity observed in mouse models of antitumor immunity. Exemplary anti-CTLA4 antibodies include, but are not limited to, ipilimumab (MDX-010) and tremelimumab (CP-675,206), both of which are fully human. Ipilimumab is an IgG1 with a plasma half-life of approximately 12-14 days; Tremelimumab is an IgG2 drug with a plasma half-life of approximately 22 days. Cm., for example, Phan et al. (2003) Proc Natl Acad Sci USA. 100:8372-7; Ribas et al. (2005) J Clin Oncol. 23:8968-77; Weber et al. (2008) J Clin Oncol. 26:5950-6. In some embodiments, the anti-CTLA4 antibody is ipilimumab.

[839] In certain embodiments, the checkpoint inhibitor is a programmed cell death protein 1 (PD1) pathway inhibitor. The programmed cell death protein 1 (PD1) pathway represents a master immune control switch that can be activated by tumor cells to overcome active immune surveillance of T cells. Ligands for PD1 (PDL1 and PDL2) are constitutively expressed or can be induced in various tumors. High levels of PDL1 (and to a lesser extent PDL2) expression on tumor cells have been found to correlate with poor prognosis and poor survival in various other types of solid tumors. In addition, it has been suggested that PD1 controls the proliferation of tumor-specific T cells in patients with malignant melanoma. These observations suggest that the PD1/PDL1 pathway plays a critical role in tumor evasion from recognition by the immune system and may be considered an attractive target for therapeutic intervention. As used herein, the term "PD1 inhibitor" is intended to refer to any inhibitor of PD1 and/or the PD1 pathway. Exemplary PD1 inhibitors include, without limitation, anti-PD1 and anti-PDL1 antibodies. In certain embodiments, the checkpoint inhibitor is an anti-PD1 antibody. Exemplary anti-PD1 antibodies include, but are not limited to, nivolumab and pemprolizumab (MK-3475). Nivolumab, for example, is a fully human antibody to the immunoglobulin G4 (IgG4) PD1 checkpoint inhibitor that disrupts the interaction of the PD1 receptor with its PDL1 and PDL2 ligands, thereby inhibiting the cellular immune response (Guo et al. (2017) ) J Cancer 8(3):410-6). In some embodiments, the anti-PD1 antibody is nivolumab. Pembrolizumab, for example, is a potent and highly selective humanized IgG4/kappa mAb designed to directly block the interaction between PD1 and its ligands, PDL1 and PDL2. Pembrolizumab significantly increases T-lymphocyte-mediated immune responses in cultured blood cells from healthy human donors, cancer patients, and primates. Pembrolizumab has also been reported to modulate levels of interleukin-2 (IL-2), tumor necrosis factor alpha (TNFα), interferon-gamma (IFNγ), and other cytokines. Exemplary anti-PDL1 antibodies include, without limitation, atezolizumab, avelumab, and durvalumab. Atezolizumab, for example, is a humanized IgG1 mAb that has been reported to block the PD1/PDL1 interaction by targeting expressed PDL1 on a variety of cancer cells. This blocking of the PD1/PDL1 pathway can stimulate immune defense mechanisms against tumors (Abdin et al. (2018) Cancers (Basel) 10(2):32). In some embodiments, the anti-PDL1 antibody is atezolizumab.

[840] In certain embodiments, the checkpoint inhibitor targets PD1/PDL1, CTLA4, OX40, CD40, LAG3, TIM3, GITR, and/or KIR. In certain embodiments, the checkpoint inhibitor targets CTLA4, OX40, CD40 and/or GITR. In certain embodiments, the checkpoint inhibitor is targeted as an inhibitory antibody or other similar inhibitory molecule (eg, an anti-CTLA4 or anti-PD1/PDL1 inhibitory antibody). In certain other embodiments, the checkpoint inhibitor is targeted as a target agonist; examples from this class include molecules that stimulate OX40, CD40 and/or GITR targets. In some embodiments, the checkpoint inhibitor that targets OX40, CD40, and/or GITR is an agonist antibody. Agonist antibodies directed against OX40 can play a dual role: inhibit suppression by regulatory T cells while enhancing the function of effector T cells. Anti-GITR agonists have also been shown to render effector T cells more resistant to inhibition induced by regulatory T cells (Karaki et al. (2016) Vaccines (Basel) 4(4):37). Similarly, anti-CD40 agonist antibodies exhibit T-cell dependent antitumor activity. Activation of CD40 on dendritic cells increases the cross-presentation of tumor antigens and hence the number of activated tumor-targeting effector T cells (Ellmark et al. (2015) Oncoimmunol. 4(7):e1011484).

[841] In certain embodiments, the checkpoint inhibitor targets CTLA4 (eg, is an anti-CTLA4 antibody). In certain embodiments, targeting CTLA4 facilitates priming and activation of naive T cells. In certain embodiments, the checkpoint inhibitor targets OX40 (eg, is an anti-OX40 antibody). In certain embodiments, targeting OX40 enhances the proliferation of effector T cells. In certain embodiments, the checkpoint inhibitor targets CD40 (eg, is an anti-CD40 antibody). In certain embodiments, targeting CD40 suppresses "tolerogenic" T cell priming and/or production of regulatory T cells. In certain embodiments, the checkpoint inhibitor targets GITR (eg, is an anti-GITR antibody). In certain embodiments, targeting GITR suppresses the activity of regulatory T cells. In certain embodiments, the efficacy of a combination therapy (e.g., effect on at least one symptom or risk/rate of disease progression) with a splicing modulator, ADC, or composition and an agent targeting CTLA4, OX40, CD40, and/or GITR is additive. In some embodiments, the effectiveness of combination therapy with a splicing modulator, ADC, or composition and an agent that targets CTLA4, OX40, CD40, and/or GITR is super-additive (ie, synergistic).

[842] Checkpoint inhibitor treatment strategies are based on the hypothesis that treatment facilitates and/or enhances T cell priming in responses to tumors that are weak or deficient in antigenicity (e.g., CTLA4), or that treatment restores and/or enhances T cells that respond to tumor antigens but are "depleted" due to persistent antigen presentation (eg, PD1, PDL1) (Chen and Mellman (2013) Immunity 39(1):1-10). Examples of suitable therapies and checkpoint inhibitors, eg anti-PD1, PDL1 or CTLA4 antibodies, are known in the art. Cm., For example, WO 2001/014424, WO 2013/173223, WO 2016/007235.

[843] Combining these primed T cell responses following checkpoint inhibitor therapy with treatment to induce neoantigens in tumor cells to which the primed immune system can respond may provide beneficial synergism. Since neoantigens produced by a splicing modulator or ADC have not yet been presented for T cell priming, a combination with a CTLA4 inhibitor may be particularly preferred. In some embodiments, the treatment comprises the administration of one or more splicing modulators, an ADC, or a composition to induce neoantigen production, and an initial administration of a CTLA4 inhibitor to promote CD8 T cell priming prior to, concurrent with, or subsequent to this administration. In some embodiments, the patient is given additional administrations of a CTLA4 inhibitor, eg, to further promote priming and/or activation of neoantigen-reactive CD8 populations. In some embodiments, additional administrations of a splicing modulator, ADC, or composition may be administered to a patient to increase neoantigen presentation by the tumor. Repeated administrations of the splicing modulator, ADC, or composition and checkpoint inhibitor therapy can be done simultaneously or at spaced apart time intervals. In some embodiments, the treatment further comprises co-treatment with a PD1/PDL1 inhibitor, for example, to restore the effector function of depleted T cells to target a neoantigen within the tumor microenvironment.

[844] As used herein, the terms "combination" or "combination therapy" refers to the administration of one or more splicing modulator, ADC, or composition together with an additional agent or therapy (e.g., checkpoint inhibitor, cytokine or cytokine analog, neoantigen vaccine, CAR-T) as part of a treatment regimen designed to provide a beneficial (i.e., additive or synergistic) effect from the combined action of one or more administered agents. In some embodiments, the combination may also include one or more additional agents, including, but not limited to, chemotherapeutic agents, angiogenesis inhibitors, and agents that reduce immune suppression (eg, a second checkpoint inhibitor). The beneficial effect of the combination includes, without limitation, the pharmacokinetic or pharmacodynamic synergy provided by the combination of therapeutic agents. The administration of such therapeutic agents in combination is typically carried out over a period of time (eg, minutes, hours, days, or weeks, depending on the combination chosen).

[845] As used herein, the terms administered "in combination" or "co-administration" means that two or more different treatments are delivered to a subject over a period of time during which the subject is suffering from a medical condition (e.g., a neoplastic disorder). For example, in some embodiments, two or more treatments are delivered after the subject has been diagnosed with a disease or disorder and before the disease or disorder has been treated or eliminated, or when the subject has been identified as at risk of developing it, but before after the subject developed symptoms of the disease. In some embodiments, one treatment is still being delivered when delivery of the second treatment is started so that there is overlap. In some embodiments, delivery of the first and second treatments is started at the same time. Such types of delivery are sometimes referred to herein as "simultaneous", "joint", or "accompanying" delivery. In other embodiments, delivery of one treatment ends before delivery of the second treatment begins. This type of delivery is sometimes referred to herein as "sequential" or "sequential" delivery.

[846] In some embodiments, two treatments (eg, splicing modulator, ADC, or composition and checkpoint inhibitor) are contained in the same composition. Such compositions may be administered in any suitable form and by any suitable route. In other embodiments, the two treatments (eg, splicing modulator, ADC, or composition, and checkpoint inhibitor) are administered in separate compositions, in any suitable form, and via any suitable route. For example, in some embodiments, a composition comprising a splicing modulator or ADC and a composition comprising a checkpoint inhibitor may be administered simultaneously or sequentially in any order at different time points; in any case, they should be administered close enough in time to provide the desired therapeutic or prophylactic effect.

[847] In embodiments involving either simultaneous or sequential delivery, treatment may be more effective due to combined administration. In some embodiments, the first treatment is more effective, e.g., an equivalent effect is observed with less of the first treatment (eg, at a lower dose) than would be observed if the first treatment was administered in the absence of the second treatment. In some embodiments, the first treatment is more effective such that the reduction in severity of a symptom or other parameter associated with the disease or disorder is greater than would be observed if the first treatment were delivered in the absence of the second treatment. In other embodiments, a similar situation is observed for the second treatment. In some embodiments, the beneficial effect of combination therapy (eg, effect on at least one symptom or risk/rate of disease progression) is additive. In some embodiments, the beneficial effect of the combination therapy is super-additive.

[848] In various embodiments, the present invention provides a method of treating a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a splicing modulator, ADC, or a composition comprising a splicing modulator, or ADC; and at least one additional therapy (ie, checkpoint inhibitor, cytokine or cytokine analog, neoantigen vaccine, CAR-T therapy). In some embodiments, administration of a splicing modulator, ADC, or composition provides for the induction of at least one neoantigen and/or a T cell response. In some embodiments, administration of a splicing modulator, ADC, or composition induces an immune response to double-stranded RNA. In some embodiments, administration of a splicing modulator, ADC, or composition provides for the induction of immunogenic cell death. In some embodiments, at least one additional therapy may include at least one, at least two, at least three, at least four, or at least five additional therapies. For example, in some embodiments, the splicing modulator, ADC, or composition may be administered in combination with checkpoint inhibitor therapies, ie, using two different checkpoint inhibitors. In some other embodiments, the splicing modulator, ADC, or composition may be administered in combination with a checkpoint inhibitor therapy and a neoantigen vaccine.

[849] In some combination therapy embodiments, the amount of splicing modulator, antibody drug conjugate or composition and/or at least one additional therapy administered is reduced by 10%, 15%, 20%, 25%, 30%, 35% , 40%, 45%, 50%, 75%, or 90% compared to the standard dosage of the splicing modulator, antibody drug conjugate or composition and/or at least one additional therapy. In some embodiments, the frequency of administration of the splicing modulator, antibody drug conjugate or composition and/or at least one additional therapy is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45 %, 50%, 75%, or 90% compared to the standard schedule for administering the splicing modulator, antibody-drug conjugate or composition and/or at least one additional therapy. In some embodiments, the amount administered and/or dosage administered of the splicing modulator, antibody drug conjugate or composition, and/or at least one additional therapy results in lower systemic toxicity and/or improved tolerability.

[850] In some embodiments, the splicing modulator, antibody drug conjugate, or composition is started prior to the administration of at least one additional therapy. In some embodiments, the splicing modulator, antibody-drug conjugate, or composition is administered following the administration of at least one additional therapy. In some embodiments, the splicing modulator, antibody-drug conjugate, or composition begins to be administered concurrently with the administration of at least one additional therapy.

[851] In some embodiments, administration of the splicing modulator, antibody drug conjugate or composition is repeated at least once after the initial administration. In some embodiments, the amount of splicing modulator, antibody drug conjugate or composition used for reintroduction is reduced compared to the amount used for initial administration. In some embodiments, the amount of splicing modulator, antibody drug conjugate or composition used for reintroduction is reduced compared to the standard dosage of the splicing modulator, antibody drug conjugate or composition. In some embodiments, the amount of splicing modulator, antibody-drug conjugate or composition used for reintroduction is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% compared to the standard dosage of the splicing modulator, antibody-drug conjugate or composition.

[852] In some embodiments, the administration of at least one additional therapy is repeated at least once after the initial administration. In some embodiments, the amount of at least one additional therapy used for reintroduction is reduced compared to the amount used for initial administration. In some embodiments, the amount of at least one additional therapy used for repeated administration is reduced compared to the standard dosage of at least one additional therapy. In some embodiments, the amount of at least one additional therapy used for repeated administration is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% compared to the standard dosage of at least one additional therapy.

[853] In some embodiments, the re-administration of the splicing modulator, antibody-drug conjugate or composition is concurrent with the re-administration of at least one additional therapy. In some embodiments, re-administration of the splicing modulator, antibody-drug conjugate or composition is performed sequentially or spaced apart in time with repeated administration of at least one additional therapy.

[854] In various embodiments, the present invention provides a method of treating a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a splicing modulator, ADC, or a composition comprising a splicing modulator, or ADC; and checkpoint inhibitor therapies. In some embodiments, the checkpoint inhibitor therapy comprises administering at least one checkpoint inhibitor. In some embodiments, the subject is intolerant, intolerant, or deficient in at least one checkpoint inhibitor when administered alone. In some embodiments, the subject may be considered to be resistant or deficient in responding to at least one checkpoint inhibitor, as determined using, for example, immune-dependent response criteria for immunotherapy (irRC) and/or immune-dependent criteria for assessing response to immunotherapy in solid tumors ( irRECIST). Cm., For example, Wolchok et al. (2009) Clin Cancer Res. 15(23):7412-20; Bohnsack et al. "Adaptation of the Immune-Related Response Criteria:irRECIST" (Abstract 4958) ESMO 2014. Illustrative criteria may include criteria used in the art to determine when tumors in cancer patients improve ("response") remain the same ("stabilization") or worsens ("progression") during treatment if the treatment being evaluated is an immune anticancer drug (eg, a checkpoint inhibitor). In some embodiments, a subject may be considered intolerant to at least one checkpoint inhibitor if the subject has one or more adverse (grade 2+) events identified for the corresponding checkpoint inhibitor (e.g., ipilimumab). For example, in some embodiments, a subject may be considered intolerant to an ipilimumab treatment if the subject has one or more adverse events selected from enterocolitis, hepatitis, dermatitis (including toxic epidermal necrolysis), neuropathy, and endocrinopathy (Yervoy ® (ipilimumab), 2018 FDA Compliant Labeling Supplement).

[855] In some embodiments, the checkpoint inhibitor targets PD1/PDL1, CTLA4, OX40, CD40, LAG3, TIM3, GITR, and/or KIR. In some embodiments, the checkpoint inhibitor targets CTLA4, OX40, CD40, and/or GITR. In some embodiments, the checkpoint inhibitor is targeted by an inhibitory antibody or other similar inhibitory molecule. In some other embodiments, the checkpoint inhibitor is targeted as an agonist antibody or other similar agonist molecule. In some embodiments, the checkpoint inhibitor comprises a cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) pathway inhibitor. In some embodiments, the CTLA4 inhibitor is an anti-CTLA4 antibody. In some embodiments, the anti-CTLA4 antibody is ipilimumab. In some embodiments, the checkpoint inhibitor comprises an inhibitor of programmed cell death protein 1 (PD1) pathway. In some embodiments, the PD1 inhibitor is an anti-PD1 antibody. In some embodiments, the anti-PD1 antibody is nivolumab. In some embodiments, the PD1 inhibitor is an anti-PDL1 antibody. In some embodiments, the anti-PDL1 antibody is atezolizumab. In some embodiments, the checkpoint inhibitor comprises a CTLA4 inhibitor and a PD1 inhibitor. In some embodiments, the checkpoint inhibitor targets OX40. In some embodiments, the checkpoint inhibitor targets CD40. In some embodiments, the checkpoint inhibitor targets the GITR. In some embodiments, the effectiveness of a combination therapy (e.g., effect on at least one symptom or risk/rate of disease progression) with a splicing modulator, ADC, or composition and a checkpoint inhibitor (e.g., an antibody or molecule that targets CTLA4, PD1/PDL1 , OX40, CD40 and/or GITR) is additive. In some embodiments, the efficacy of combination therapy with a splicing modulator, an ADC, or a composition and a checkpoint inhibitor agent (e.g., an antibody or molecule that targets CTLA4, PD1/PDL1, OX40, CD40, and/or GITR) is superadditive (i.e., e. synergistic).

[856] In various embodiments, the present invention provides a method of treating a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a splicing modulator, ADC, or a composition comprising a splicing modulator or ADC; and cytokine or cytokine analog therapy agents. In some embodiments, a cytokine or cytokine analog therapy means administering at least one cytokine or cytokine analog. In some embodiments, the subject is intolerant, intolerant, or deficient in at least one cytokine or cytokine analog when administered alone.

[857] In some embodiments, the cytokine or cytokine analog provides an amplifying agent for T cells. In some embodiments, the cytokine or cytokine analog comprises IL-2, IL-10, IL-12, IL-15, IFNγ and/or TNFα. In some embodiments, the cytokine or cytokine analog comprises IL-2, IL-10, IL-12, and/or IL-15. In some embodiments, administration of a cytokine or cytokine analog enhances T cell priming following administration of a splicing modulator, antibody-drug conjugate or composition by inducing the production and presentation of neoantigens.

[858] In some embodiments, the cytokine or cytokine analog provides for IL-2. In some embodiments, IL-2 enhances signals in effector cells to promote their proliferation (Rosenberg (2014) J Immunol. 192(12):5451-8). In some embodiments, the cytokine or cytokine analog comprises IL-10. In some embodiments, IL-10 enhances priming and activation of CD8+ T cells (Mumm et al. (2011) Cancer Cell 20(6):781-96). In some embodiments, the cytokine or cytokine analog comprises IL-12. In some embodiments, IL-12 couples innate and adaptive immune responses with increased antigen-specific priming and targeting (Tugues et al. (2015) Cell Death Differ. 22(2):237-46). In some embodiments, the cytokine or cytokine analog provides for IL-15. In some embodiments, IL-15 enhances priming and/or activation of effector T cells (CD8). In some embodiments, the cytokine or cytokine analog provides for IFNγ. In some embodiments, IFNγ supplements IFNγ secretion by effector T cells. In some embodiments, the cytokine or cytokine analog comprises TNFα. In some embodiments, TNFα complements TNFα secretion by effector T cells.

[859] In some embodiments, an initial dose of a splicing modulator, ADC, or composition is administered to a subject to initiate aberrant splicing and produce neoantigenic peptides. In some embodiments, after a period of time necessary for protein production and antigen presentation, the subject is then administered an initial dose of a cytokine or cytokine analog to stimulate and/or enhance priming and expansion of effector T cells. In some embodiments, the waiting period between doses of the splicing modulator, ADC, or composition and the cytokine or cytokine analog is about 2, about 3, about 4, about 5, about 6, or about 7 days. In some embodiments, the waiting period is from about 3 days to about 5 days. In some embodiments, the cytokine or cytokine analog is IL-2, IL-10, IL-12, IL-15, IFNγ and/or TNFα. In some embodiments, the combined therapeutic effect of the splicing modulator, ADC or composition and the cytokine or cytokine analog may be additive or super-additive.

[860] In some other embodiments, an initial dose of a cytokine or cytokine analog is administered to a subject to stimulate and/or enhance the priming and expansion of effector T cells. After a waiting period, in some embodiments, the subject is then administered an initial dose of a splicing modulator, ADC, or composition to initiate aberrant splicing and produce neoantigenic peptides. In some embodiments, the waiting period between doses of a cytokine and a cytokine analog and a splicing modulator, ADC, or composition is about 2, about 3, about 4, about 5, about 6, or about 7 days. In some embodiments, the waiting period is from about 3 days to about 5 days. In some embodiments, the cytokine or cytokine analog is IL-2, IL-10, IL-12, IL-15, IFNγ and/or TNFα. In some embodiments, the combined therapeutic effect of the cytokine or cytokine analog and the splicing modulator, ADC, or composition may be additive or super-additive.

[861] In some embodiments, after a period allowing T cells to be primed and expanded, the subject is then administered a second or subsequent dose of a splicing modulator, ADC, or composition to initiate re-presentation of the neoantigenic peptides. In some embodiments, the waiting period between the initial dose of the cytokine or cytokine analog and the second or subsequent dose of the splicing modulator, ADC, or composition is about 2, about 3, about 4, or about 5 weeks. In some embodiments, the waiting period is approximately 3 weeks. In some embodiments, subsequent doses of a cytokine or cytokine analog may be administered, for example, interspersed with subsequent doses of a splicing modulator, ADC, or composition. Following a second or subsequent dose of the splicing modulator, ADC, or composition, in some embodiments, the immune system may contact the neoantigen-presenting tumor cells and/or cause the tumor cells to be killed. In some embodiments, dosing of the splicing modulator, ADC, or composition following this illustrative initial treatment regimen may be pulsatile, i.e., the splicing modulator, ADC, or composition may be administered at long dose intervals (e.g., about every 4 weeks, about every 5 weeks, approximately every 6 weeks) to ensure antigen presentation, recruitment of T cells, and/or killing of tumor cells, and/or restoration of the memory T cell population.

[862] In some embodiments, the subject has a non-synonymous mutation burden of about 150 mutations or less. In some embodiments, the subject has a non-synonymous mutation burden of about 100 mutations or less. In some embodiments, the subject has a non-synonymous mutation burden of about 50 mutations or less. In some embodiments, the subject has or is suspected of having a neoplastic disorder, such as a hematologic malignancy or solid tumor. In some embodiments, the hematologic malignancy is selected from B-cell malignancy, leukemia, lymphoma, and myeloma. In some embodiments, the hematologic malignancy is selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer and cancer of the esophagus. In some embodiments, the solid tumor is selected from HER2 positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

Splicing modulator/ADC combination and neoantigen vaccine

[863] In various embodiments, a patient who has a cancer as described herein may be treated with a combination of a splicing modulator, ADC, or composition, and a neoantigen vaccine. Without being limited by theory, vaccines used alone or in combination with immune checkpoint inhibitor (ICI) molecules have shown promise in early phase clinical trials (Ott et al. (2017) Nature 547(7662):217- 21; Sahin et al. (2017) Nature 547(7662):222-6), but mutation sequencing is usually required in the patient's tumor (Ott et al. (2018) Bioorg Med Chem 26(10):2842-9). Thus, the effect of vaccines often depends on sufficient numbers of non-synonymous mutations that are antigenic. In general, there are few candidate antigens in tumors with very low mutation load, and in the case of rapidly growing tumors, there is limited time to identify and obtain patient-specific vaccines.

[864] Until now, attempts to develop vaccines that have a wide coverage of immunogenicity for a large percentage of patients have focused on proteins that either mutate frequently or are ectopically overexpressed or amplified, and/or that exist as "self" proteins in the body. . In addition, these proteins are often expressed in tissues that are not affected by the immune system (for example, neuronal markers expressed in types of neuroendocrine tumors), while other proteins may normally be expressed during embryogenesis (for example, cancerous embryonic antigens) . Thus, the usefulness of vaccines using such proteins as antigens is often limited to specific tumor lineages or tumor subgroups in which one or more antigens are present. The applicability of the vaccine will also need to be confirmed by sequencing the patient's tumor samples, which can be time consuming.

[865] Moreover, if such antigens exist as "self" proteins, the immune system is likely to be primed to recognize such proteins as "self" and thus not respond. Or, alternatively, if the immune system is capable of providing an effector response to such antigens, it may result in targeting side effects in tissues where the antigen may be expressed. In both of these cases, one of the key problems is that most antigenic peptides are derived from "passenger" genes (i.e., genes that mutate or amplified during oncogenesis, but do not play a critical role in the long-term survival or proliferation of the tumor itself along yourself). For this reason, these genes can be silenced without significant consequences for tumor progression, and thus may allow the tumor to "avoid" the immune response to these antigens. While not wishing to be bound by theory, this mechanism may play a role in tumor evolution, in which random mutations that are highly antigenic are often subject to "screening" by the tumor during the early stages of tumorigenesis (Dunn et al. (2004) Annu. Rev. Immunol. 22:329-60).

[866] In addition, some evidence also indicates that persistent antigen presentation and immune stimulation can lead to anergy and depletion of immune cells (Pardoll (2012) Nat. Rev. Cancer 12(4):252-64). Such phenotypes underlie the therapeutic rationale for currently used ICI-based therapies, as ICIs have been shown to either curb the immune cell depletion phenotype (α-PD1/PD-L1) or facilitate complementary immune cellular responses (α-CTLA4 ). Notably, in the case of α-CTLA4 based therapy, it has been reported that a subset of patients exhibited serious immune system-related adverse events that may be due to promoting T cell activation and disruption of immune tolerance mechanisms that limit autoreactive immune responses.

[867] Both of these approaches (i.e. triggering or enhancing de novo immune responses to neoantigens or re-activation when anergy or depletion of existing immune responses) are associated with permanent activation of the immune system. In this regard, these approaches are sensitive to anergy, editing and other tumor-mediated mechanisms aimed at suppressing the involvement of the immune system.

[868] In contrast, treatment with a splicing modulator, ADC, or composition disclosed herein can induce an immune response to novel neoantigen sequences. In some embodiments, the presentation of neoantigens provides the adaptive immune system with more diverse targets to contact and activate. In some embodiments, the ability of the splicing modulator, ADC, or composition to rapidly induce alternative splicing and neoantigen production may reduce the risk of immune system depletion due to chronic exposure to mutated neoantigens and/or limit the ability of tumor cells to adapt to avoid therapy. In some embodiments, administration of a splicing modulator, ADC, or composition in combination with a neoantigen vaccine enhances the immune response to neoantigens produced by the splicing modulator, ADC, or composition. In some embodiments, the splicing modulator, ADC, or composition is administered before, during, or after vaccination. In some embodiments, the splicing modulator, ADC, or composition and/or vaccine may be administered once or more than once during a course of treatment. In some embodiments, the vaccine is administered once and the splicing modulator, ADC, or composition is then administered multiple times during the course of treatment. In some embodiments, the vaccine is administered once and then one or more booster doses are administered during the course of treatment.

[869] As used herein, the term "neoantigenic vaccine" refers to a pooled sample of one or more immunogenic neoantigenic peptides or mRNAs, e.g., at least two, at least three, at least four, at least five, or more neoantigenic peptides. The term "vaccine" refers to an immune-promoting composition for the prevention and/or treatment of a disease (eg, a neoplastic disorder, eg, a hematologic malignancy or a solid tumor). Accordingly, vaccines are medicinal preparations that contain immunogenic agents and are intended for use in humans or animals to develop specific immune defense mechanisms and the formation of protective substances after vaccination. The neoantigenic vaccine may further include a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.

[870] As used herein, the term "immunogenic" refers to any agent or composition that can elicit an immune response, such as a T cell response. The immune response may be mediated by an antibody, or a cell, or both.

[871] In some embodiments, a splicing modulator, ADC, or composition is administered to a patient, and then a peptide or mRNA vaccine based on a known neoantigen is administered to enhance the immune response to neoantigens produced by the splicing modulator, ADC, or composition. In some embodiments, a splicing modulator, ADC, or composition is administered to the patient and then screened for the presence of neoantigens produced as a result of the treatment. Therefore, one or more of these neoantigens is used to create a personalized vaccine that is administered to a patient. In one of these embodiments, the splicing modulator, ADC or composition and/or peptide or mRNA vaccine can be administered to a patient once or repeatedly.

[872] In various embodiments, a suitable neoantigen for a vaccine can be identified by screening a panel of spliced and stable expression transcripts obtained from one or more patient tissue samples (eg, tumor biopsy). In some embodiments, variant protein sequences are identified in a screened sample based on translation along a junction in an aberrantly spliced mRNA while retaining portions of the protein sequence (no more than 12 amino acids) flanking the amino acid changes spanning the junction. In some embodiments, these peptide fragments spanning the splicing boundaries are examined for high binding affinity for MHC1 alleles, for example, using a tool such as NetMHC1 (Nielsen et al. (2003) Protein Sci 12(5):1007-17; Andreatta and Neilsen (2016) Bioinformatics 32(4):511-7). These results allow the filtering of neopeptides to neopeptides predicted to have a high binding affinity for the composition of HLA alleles unique to the patient, as well as to collect pools of neopeptides predicted to bind to a wide range of HLA alleles present at high frequency in different populations (Maiers et al. (2007) Hum Immunol 68(9):779-88). In various embodiments, the identified neopeptides are then formulated into a vaccine, e.g., by conjugation with a suitable carrier or adjuvant (Ott et al. (2017) Nature 547(7662):217-21), or for delivery as mRNA (Sahin et al. (2017) Nature 547(7662):222-6).

[873] In some embodiments, the choice of neoantigen is based on screening for an individual patient's tumor response to exposure to a splicing modulator, ADC, or composition, identifying one or more treatment-generated neoantigens for use in subsequent vaccination. In other embodiments, a neoantigen is selected, for example, based on screening a panel of samples obtained from different patients to identify common neoantigens produced by a splicing modulator, ADC, or composition, and then used as a universal vaccine for future patients.

[874] Without being limited by theory, in some embodiments, the use of a universal neoantigen vaccine will avoid the need for sequencing and analysis of the unique mutational status of each patient's tumor, since the selected neoantigens do not depend on mutation in the tumor, but rather mimic the neoantigen produced by the splicing modulator, ADC or compositions, and usually recognized as foreign by the body. In addition, in some embodiments, the use of a neoantigen vaccine may be particularly effective because the patient's tumor cells may be more likely to mutate without producing one or more neoantigens that depend on the mutation in the tumor, compared to cells that mimic the neoantigen produced under by the action of a splicing modulator, ADC, or composition. This may create the possibility of formulating a core vaccine that has a broad coverage of immunogenicity for a large percentage of patients, speeding up the start of the treatment regimen. Patients may be vaccinated according to the schedules set forth herein and may be further treated with a splicing modulator, ADC, or composition, for example, to induce expression of neoantigenic peptides, before the next completion of vaccination. In some embodiments, a splicing modulator, ADC, or composition may be administered to patients prior to or concurrent with vaccination. In some embodiments, patients are administered a splicing modulator, ADC, or composition that has been screened for one or more neoantigens found in the universal neoantigen panel and are vaccinated with a universal neoantigen vaccine containing at least one universal neoantigen identified in the subject. In some embodiments, patients may be administered a splicing modulator, ADC, or composition one or more times after vaccination. The splicing modulator or ADC or composition and/or vaccine may be administered once or more than once during a course of treatment.

[875] In some embodiments, the implementation of the vaccine may contain one or more than one neoantigenic peptide or mRNA. In various embodiments, the implementation of the vaccine may contain one or more than one long neoantigenic peptide. In various embodiments, such "long" neoantigenic peptides are efficiently internalized, processed, and cross-presented in specialized antigen-presenting cells, such as dendritic cells. Similarly, in other contexts, long vaccine peptides have been shown to induce cytotoxic T cells in humans (Melief and van der Burg (2008) Nat Rev Cancer 8(5):351-60). In various embodiments, the neoantigenic peptide is extended to include the neoantigenic peptide's own sequence in addition to the flanking amino acid sequences. In various embodiments, the extended peptide sequence facilitates uptake of the protein by antigen-presenting cells, such as dendritic cells. In various embodiments, the extended peptide sequence allows efficient antigen presentation and T cell priming in different HLA isotype models. In various embodiments, the longer neoantigenic peptide and/or the extended peptide sequence exhibit increased uptake by antigen presenting cells (e.g., dendritic cells), increased antigen presentation, and/or increased T cell priming compared to the shorter neoantigenic peptide and/or shorter peptide sequence. sequence (eg, with a peptide sequence that is less than about 10 or less than about 5 amino acids in length). In some embodiments, the length of the long neoantigenic peptide is in the range of about 5 to about 50 amino acids. In some embodiments, the length of the long neoantigenic peptide is in the range of about 10 to about 50 amino acids. In some embodiments, the length of the long neoantigenic peptide is in the range of about 10 to about 35 amino acids. In some embodiments, the length of the long neoantigenic peptide is in the range of about 15 to about 25 amino acids. Amino acid sequences of exemplary long neoantigenic peptides are shown in Table 21.

[876] As used herein, "neoantigenic peptide" or "mRNA vaccine" encompasses the use of a fragment of a neoantigenic peptide or mRNA encoding it, provided that the fragment retains immunogenic potential.

[877] In some embodiments, the neoantigenic vaccine comprises at least one neoantigenic peptide. In some embodiments, the neoantigen vaccine contains at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, or at least 20 neoantigenic peptides. In some embodiments, the neoantigenic peptide(s) is in the range of about 5 to about 50 amino acids in length. In some embodiments, the length of the neoantigenic(s) peptide(s) is in the range of about 10 to about 50 amino acids. In some embodiments, the length of the neoantigenic(s) peptide(s) is in the range of about 10 to about 35 amino acids. In some embodiments, the length of the neoantigenic(s) peptide(s) is in the range of about 15 to about 25 amino acids.

[878] In various embodiments, the present invention provides a method of treating a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a splicing modulator, ADC, or a composition comprising a splicing modulator, or ADC; and neoantigen vaccine. The neoantigen vaccine may be, for example, a peptide neoantigen vaccine or a neoantigen mRNA vaccine. In some embodiments, the splicing modulator, ADC, or composition is administered prior to administration of the neoantigen vaccine. In some embodiments, the splicing modulator, ADC, or composition is administered following administration of the neoantigen vaccine. In some embodiments, the splicing modulator, ADC, or composition is administered concurrently with the administration of the neoantigen vaccine. In some embodiments, administration of the splicing modulator, ADC, or composition is repeated at least once after the initial administration. In some embodiments, the amount of splicing modulator, ADC, or composition used for reintroduction is reduced compared to the amount used for initial administration.

[879] In various embodiments, the implementation of the present invention further provides a combination containing a splicing modulator, ADC or a composition containing a splicing modulator or ADC; and a neoantigen vaccine (eg, a generic neoantigen vaccine) for use in the treatment of a subject who has or is suspected of having a neoplastic disorder. In some embodiments, the neoantigen vaccine is a peptide neoantigen vaccine or a neoantigen mRNA vaccine. In some embodiments, the combination further comprises at least one additional therapy. In some embodiments, at least one additional therapy comprises at least one, at least two, at least three, at least four, or at least five additional therapies.

[880] In various embodiments, the present invention further provides a method of treating a subject who has or is suspected of having a neoplastic disorder by (a) administering to the subject an effective amount of a splicing modulator, ADC, or a composition comprising a splicing modulator, or ADC; (b) detecting one or more neoantigens in a subject after administration of a splicing modulator, ADC, or composition; (c) comparing one or more neoantigens to a panel of universal neoantigens; and (d) administering to the subject a universal neoantigen vaccine containing at least one universal neoantigen present in the subject. In some embodiments, the universal neoantigen vaccine is administered alone or in combination with at least one additional therapy. In some embodiments, at least one additional therapy comprises at least one, at least two, at least three, at least four, or at least five additional therapies.

[881] In some embodiments, at least one additional therapy involves the administration of a splicing modulator, ADC, or composition. In some embodiments, reintroduction of the splicing modulator, ADC, or composition is started after administration of the universal neoantigen vaccine. In some embodiments, the re-splicing modulator, ADC, or compositions are started after administration of the universal neoantigen vaccine. In some embodiments, reintroduction of the splicing modulator, ADC, or composition is initiated concurrently with the administration of the universal neoantigen vaccine. In some embodiments, the amount of splicing modulator, ADC, or composition used for reintroduction is reduced compared to the amount used for initial administration. In some embodiments, the amount of the splicing modulator, ADC, or composition used for initial and/or reintroduction is reduced from the standard dosage of the splicing modulator, ADC, or composition when used in the absence of vaccine treatment. In some embodiments, the amount of splicing modulator, ADC, or composition used for initial and/or reintroduction is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% compared to the standard dosage of the splicing modulator, ADC or composition.

[882] In some embodiments, at least one additional therapy involves the administration of a checkpoint inhibitor (eg, any of the exemplary checkpoint inhibitors described herein). In some embodiments, administration of the checkpoint inhibitor is started prior to administration of the universal neoantigen vaccine and/or reintroduction of the splicing modulator, ADC, or composition. In some embodiments, administration of the checkpoint inhibitor is initiated following administration of the universal neoantigen vaccine and/or the re-splicing modulator, ADC, or composition. In some embodiments, administration of the checkpoint inhibitor is started concurrently with administration of the universal neoantigen vaccine and/or reintroduction of the splicing modulator, ADC, or composition. In some embodiments, the checkpoint inhibitor administration is repeated at least once after the initial administration. In some embodiments, the amount of checkpoint inhibitor used for reintroduction is reduced compared to the amount used for initial administration. In some embodiments, the amount of checkpoint inhibitor used for reintroduction is reduced compared to the standard dosage of the checkpoint inhibitor. In some embodiments, the amount of checkpoint inhibitor used for reintroduction is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% according to compared to the standard dosage of checkpoint inhibitor. In some embodiments, the subject is intolerant, unresponsive, or deficiently responsive to a checkpoint inhibitor when administered alone.

[883] In various embodiments, this document also provides neoantigenic vaccines containing at least one neoantigenic peptide or at least one neoantigenic mRNA. In some embodiments, the neoantigenic vaccine comprises at least one neoantigenic peptide. In some other embodiments, the implementation of the neoantigenic vaccine contains at least one neoantigenic mRNA.

[884] Also provided herein in various embodiments are kits comprising a splicing modulator, ADC, or a composition comprising a splicing modulator, or ADC; and a neoantigen vaccine (eg, a universal neoantigen vaccine). In some embodiments, the neoantigen vaccine is a peptide neoantigen vaccine or a neoantigen mRNA vaccine. In some embodiments, the kit further comprises one or more additional components including, without limitation: instructions for use; other means, for example, one or more additional therapeutic agents; devices, containers, or other materials for receiving a splicing modulator, ADC, composition, and/or neoantigen vaccine for therapeutic administration; pharmaceutically acceptable carriers and devices, containers, or other materials for administering the splicing modulator, ADC, composition, and/or neoantigen vaccine to a patient. Instructions for use may include guidance for therapeutic applications, including intended dosages and/or modes of administration, for example, for a patient who has or is suspected of having a neoplastic disorder. In various embodiments, the kit further comprises instructions for a therapeutic use, such as the use of a splicing modulator, ADC, or composition, and a neoantigen vaccine to treat or prevent a neoplastic disorder in a patient. In various embodiments, the kit further comprises at least one additional therapeutic agent (eg, intended to be administered together with a splicing modulator, ADC, or composition and a neoantigen vaccine, eg, a checkpoint inhibitor). In various embodiments, the splicing modulator, ADC, composition, and/or neoantigenic vaccine are formulated into a pharmaceutical composition.

[885] In some embodiments of the methods and compositions disclosed herein, the neoantigenic vaccine comprises at least one neoantigenic peptide. In some embodiments, at least one neoantigenic peptide is in the range of about 10 to about 50 amino acids in length. In some embodiments, at least one neoantigenic peptide is in the range of about 10 to about 35 amino acids in length. In some embodiments, at least one neoantigenic peptide is in the range of about 15 to about 25 amino acids in length.

[886] In some embodiments, the implementation of at least one neoantigenic peptide contains one or more than one neoantigenic sequence. In some embodiments, the implementation neoantigenic sequence contains the amino acid sequence under any of SEQ ID NO: 37-65. In some embodiments, the neoantigenic sequence comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the neoantigenic sequence comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the neoantigenic sequence comprises the amino acid sequence of any of SEQ ID NO: 46- 49.

[887] In some other embodiments, the neoantigenic sequence comprises the amino acid sequence of any of SEQ ID NOs: 66-93 or an antigenic portion of any of SEQ ID NOs: 66-93. In some embodiments, the neoantigenic sequence comprises the amino acid sequence of SEQ ID NO: 66 or the antigenic portion of SEQ ID NO: 66. In some embodiments, the neoantigenic sequence comprises the amino acid sequence of any of SEQ ID NOs: 74-77 or the antigenic portion of any of SEQ ID NO: 74-77. In some embodiments, the neoantigenic sequence and/or antigenic portion is in the range of about 10 to about 50 amino acids in length. In some embodiments, the neoantigenic sequence and/or antigenic portion is in the range of about 10 to about 35 amino acids in length. In some embodiments, the neoantigenic sequence and/or antigenic portion is in the range of about 15 to about 25 amino acids in length. In some embodiments, the neoantigenic sequence and/or antigenic portion is in the range of about 10 to about 20 amino acids in length. In some embodiments, the neoantigenic sequence and/or antigenic portion do not directly overlap with or consist of a canonical peptide sequence (eg, any of the illustrative canonical peptide sequences underlined in Table 21).

[888] In some embodiments, the neoantigenic sequence is a subject-specific neoantigenic sequence. In some embodiments, the neoantigenic sequence is a personalized neoantigenic vaccine for a subject. In some embodiments, the neoantigenic sequence is capable of binding to at least one HLA allele expressed in the subject.

[889] In some other embodiments, the neoantigenic sequence is a universal neoantigenic sequence. In some embodiments, the neoantigenic sequence is a generic neoantigenic vaccine. In some embodiments, the neoantigenic sequence is capable of binding to at least one HLA allele expressed in at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least at least 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from a neoplastic disorder. In some embodiments, the neoantigenic sequence is capable of eliciting a T cell response to a tumor present in at least 1%, at least 5%, or at least 10% of a population of subjects suffering from a neoplastic disorder.

[890] In some embodiments, a neoantigenic sequence has been identified by sequencing at least one neoantigenic peptide induced in a subject by administration of an effective amount of a splicing modulator, antibody drug conjugate or composition. In some embodiments, at least one neoantigenic peptide comprises a neoantigenic sequence whose formation is induced by contacting a neoplastic cell with an effective amount of a splicing modulator, antibody drug conjugate or composition. In some embodiments, the neoplastic cell is in an in vitro cell culture. In some embodiments, the neoplastic cell is obtained from a subject. In some embodiments, the implementation of the neoplastic cell is in the subject.

[891] In some embodiments, the neoantigenic vaccine comprises at least one neoantigenic peptide or mRNA and a pharmaceutically acceptable carrier. In some embodiments, the neoantigenic peptide or mRNA can be attached to a suitable carrier to promote an immune response. Exemplary carriers for binding to immunogenic agents (e.g., a neoantigenic peptide or mRNA) include serum albumins, fisurella hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, or toxoid from other pathogenic bacteria such as diphtheria-causing bacteria, E. coli , bacteria that cause cholera, or H. pylori , or an attenuated toxin derivative. Other carriers for stimulating or enhancing an immune response include cytokines such as IL-1, α- and β-peptides IL-1, IL-2, γINF, IL-10, GM-CSF and chemokines such as M1P1α and β and RANTES . Immunogenic agents can also be attached to peptides that enhance tissue transport, as described, for example, in WO 97/17613 and WO 97/17614. In some embodiments, the pharmaceutically acceptable carrier is selected from a peptide, serum albumin, fisurella hemocyanin, immunoglobulin, thyroglobulin, ovalbumin, toxoid, or an attenuated derivative of toxoid, a cytokine, and a chemokine.

[892] In some embodiments, the neoantigenic peptide or mRNA may be fused to a pharmaceutically acceptable carrier. Immunogenic agents can be attached to carriers by chemical crosslinking. Techniques for attaching an immunogenic peptide to a carrier include disulfide bonding using N-succinimidyl-3-(2-pyridylthio)propionate (SPDP) and succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (if the peptide does not contain sulfhydryl group, this can be done by adding a cysteine residue). These reagents form a disulfide bond between themselves and the cysteine residues in the peptide on one protein and an amide bond through the epsilon-amino group on lysine or another free amino group on other amino acids. A number of such disulfide/amide bond forming agents are described in Jansen et al. ((1982) Immun Rev. 62:185). Other bifunctional coupling agents form a thioether bond instead of a disulfide bond. Many of these thioether bonding agents are commercially available and include the reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid and 2-iodoacetic acid, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid. Carboxyl groups can be activated by combining them with succinimide or sodium salt of 1-hydroxyl-2-nitro-4-sulfonic acid. In some embodiments, the neoantigenic peptide and the pharmaceutically acceptable carrier are covalently linked through a linker.

[893] Neoantigen and other such immunogenic peptides can also be expressed as fusion proteins with carriers. The immunogenic peptide may be attached at the amino-terminus, carboxy-terminus, or anywhere within the peptide (internal) to the carrier. In some embodiments, multiple repeats of the immunogenic peptide may be present in the fusion protein. In some embodiments, the neoantigenic peptide and the pharmaceutically acceptable carrier are expressed as a fusion protein.

[894] In some embodiments, the implementation of the neoantigenic vaccine contains at least one neoantigenic peptide or mRNA encoding it and a pharmaceutically acceptable diluent. In some embodiments, the neoantigenic vaccine comprises at least one neoantigenic peptide or mRNA encoding it and a pharmaceutically acceptable adjuvant (eg, the adjuvant described herein).

[895] In some embodiments of the methods and compositions disclosed herein, the neoantigenic vaccine contains at least one neoantigenic mRNA. In some embodiments, at least one neoantigenic mRNA encodes one or more than one neoantigenic sequence. In some embodiments, the implementation neoantigenic sequence contains the amino acid sequence under any of SEQ ID NO: 37-65. In some embodiments, the neoantigenic sequence comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the neoantigenic sequence comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the neoantigenic sequence comprises the amino acid sequence of any of SEQ ID NO: 46- 49.

[896] In some other embodiments, the neoantigenic sequence comprises the amino acid sequence of any of SEQ ID NOs: 66-93 or an antigenic portion of any of SEQ ID NOs: 66-93. In some embodiments, the neoantigenic sequence comprises the amino acid sequence of SEQ ID NO: 66 or the antigenic portion of SEQ ID NO: 66. In some embodiments, the neoantigenic sequence comprises the amino acid sequence of any of SEQ ID NOs: 74-77 or the antigenic portion of any of SEQ ID NO: 74-77. In some embodiments, the neoantigenic sequence and/or antigenic portion is in the range of about 10 to about 50 amino acids in length. In some embodiments, the neoantigenic sequence and/or antigenic portion is in the range of about 10 to about 35 amino acids in length. In some embodiments, the neoantigenic sequence and/or antigenic portion is in the range of about 15 to about 25 amino acids in length. In some embodiments, the neoantigenic sequence and/or antigenic portion is in the range of about 10 to about 20 amino acids in length. In some embodiments, the neoantigenic sequence and/or antigenic portion do not directly overlap with or consist of a canonical peptide sequence (eg, any of the illustrative canonical peptide sequences underlined in Table 21).

[897] In some embodiments, the neoantigenic sequence is a subject-specific neoantigenic sequence. In some embodiments, the neoantigenic sequence is a personalized neoantigenic vaccine for a subject. In some embodiments, the neoantigenic sequence is capable of binding to at least one HLA allele expressed in the subject.

[898] In some other embodiments, the neoantigenic sequence is a universal neoantigenic sequence. In some embodiments, the neoantigenic sequence is a generic neoantigenic vaccine. In some embodiments, the neoantigenic sequence is capable of binding to at least one HLA allele expressed in at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least at least 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from a neoplastic disorder. In some embodiments, the neoantigenic sequence is capable of eliciting a T cell response to a tumor present in at least 1%, at least 5%, or at least 10% of a population of subjects suffering from a neoplastic disorder.

[899] In some embodiments, a neoantigenic sequence has been identified by sequencing at least one mRNA induced in the subject by administration of an effective amount of a splicing modulator, antibody drug conjugate or composition. In some embodiments, at least one neoantigenic mRNA encodes a neoantigenic sequence whose formation is induced by contacting a neoplastic cell with an effective amount of a splicing modulator, antibody drug conjugate or composition. In some embodiments, the neoplastic cell is in an in vitro cell culture. In some embodiments, the neoplastic cell is obtained from a subject. In some embodiments, the implementation of the neoplastic cell is in the subject.

[900] In some embodiments, the neoantigenic vaccine comprises at least one neoantigenic mRNA and a pharmaceutically acceptable carrier. In some embodiments, at least one neoantigenic mRNA is associated with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is selected from a peptide, serum albumin, fisurella hemocyanin, immunoglobulin, thyroglobulin, ovalbumin, toxoid, or an attenuated derivative of toxoid, a cytokine, and a chemokine.

[901] In some embodiments, the neoantigenic vaccine comprises at least one neoantigenic mRNA and a pharmaceutically acceptable diluent. In some embodiments, the neoantigenic vaccine comprises at least one neoantigenic mRNA and a pharmaceutically acceptable adjuvant (eg, the adjuvant described herein).

[902] In some embodiments, the neoantigenic mRNA is encapsulated with an encapsulating agent. In some embodiments, the encapsulating agent protects the neoantigenic mRNA from degradation and improves vaccine delivery (McNamara et al. (2015) J Immunol Res. 2015:794528). In some embodiments, the encapsulating agent is a liposome. In some embodiments, the liposome is a cationic liposome, such as N-[1-(2,3-dioleoloxy)propyl]-N,N,N-trimethylammonium chloride 1 (DOTAP). In some embodiments, the implementation of the encapsulating agent is a nanoparticle. In some embodiments, the nanoparticle protects the neoantigenic mRNA from nuclease degradation and/or improves cell uptake and/or delivery efficiency. In some embodiments, the implementation of the nanoparticle can be designed in such a way that it is completely degradable. In some embodiments, the nanoparticle is a biodegradable core-shell nanoparticle comprising a pH-sensitive poly(b-aminoester) (PBAE) core surrounded by a phospholipid shell (Su et al. (2011) Mol Pharm. 8(3 ):774-87). In some embodiments, such nanoparticles are particularly effective in delivering mRNA in vivo and the development of an antitumor immune response.

[903] In some embodiments, the subject has a mutation load with non-synonymous changes of about 150 mutations or less. In some embodiments, the subject has a non-synonymous mutation burden of about 100 mutations or less. In some embodiments, the subject has a non-synonymous mutation burden of about 50 mutations or less. In some embodiments, the subject has or is suspected of having a neoplastic disorder, such as a hematologic malignancy or solid tumor. In some embodiments, the hematologic malignancy is selected from B-cell malignancy, leukemia, lymphoma, and myeloma. In some embodiments, the hematologic malignancy is selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer and cancer of the esophagus. In some embodiments, the solid tumor is selected from HER2 positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

[904] As used herein, the term "adjuvant" refers to a substance that is capable of increasing, enhancing, or modulating an immune response to a concomitant immunogenic agent, such as a neoantigenic peptide or mRNA. In certain embodiments, the neoantigen of the present invention may be administered in combination with adjuvants, ie substances that do not in themselves elicit adaptive immune responses, but enhance or modulate the response to a concomitant neoantigen. A variety of adjuvants can be used in combination with the disclosed neoantigens to promote the development of an immune response. In some embodiments, the adjuvant(s) are(are) selected to enhance the intrinsic response to the neoantigen without inducing conformational changes in the neoantigen that would affect the qualitative shape of the response. In some embodiments, the adjuvant(s) is/are selected to enhance priming and/or activation of effector T cells (eg, CD8).

[905] In certain embodiments, the implementation of the adjuvant is an aluminum salt (alum), such as aluminum hydroxide, aluminum phosphate and aluminum sulfate. Such adjuvants can be used with or without other specific immunostimulatory agents such as 3-O-deacylated monophosphoryl lipid A (MPL) or 3-DMP, polymeric or monomeric amino acids such as polyglutamic acid or polylysine. Such adjuvants may be used with other specific immunostimulatory agents such as muramyl peptides (e.g. N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetylnormuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), N-acetylglucosaminyl-N-acetylmuramyl -L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP)), or other components of the bacterial cell wall, or without them. Other adjuvants are oil-in-water emulsions and include (a) MF59 (WO 90/14837) containing 5% squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally containing varying amounts of MTP-PE ) formulated into submicron particles using a microfluidizer such as the Model 110Y Microfluidizer (Microfluidics), (b) SAF containing 10% squalene, 0.4% Tween 80, 5% Pluronic L121 block polymer and thr-MDP, or microfluidized into a submicron emulsion, or vortexed to form a larger particle size emulsion, and (c) Ribi™ Adjuvant System (RAS), (Ribi ImmunoChem) containing 2% squalene, 0.2% Tween 80, and one or more components of a bacterial a cell wall from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and a cell wall scaffold (CWS), such as MPL-FCWS (Detox™). In some embodiments, the implementation of the adjuvant is a saponin, such as Stimulon™ (QS21), or particles formed from it, such as ISCOM (immunostimulatory complexes) and ISCOMATRIX. Other adjuvants include complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA), cytokines such as interleukins (IL-1, IL-2 and IL-12), macrophage colony stimulating factor (M-CSF) and tumor necrosis factor (TNF). ).

[906] The adjuvant can be administered with the immunogenic agent (eg, neoantigenic peptide or mRNA) as a single composition, or it can be administered before, simultaneously with or after the administration of the immunogenic agent. In some embodiments, the immunogenic agent and adjuvant may be packaged and supplied in the same vial, or may be packaged in separate vials and mixed prior to use. In some embodiments, the immunogenic agent and adjuvant may be packaged with a label indicating the intended therapeutic use. In some embodiments, if the immunogenic agent and adjuvant are packaged separately, the package may include instructions for mixing prior to use. The choice of adjuvant and/or carrier depends on the stability of the immunogenic formulation containing the adjuvant, the route of administration, the dosing regimen, the effectiveness of the adjuvant in the animal species to be vaccinated and in humans, with a pharmaceutically acceptable adjuvant being an adjuvant that has been approved or may be approved. by the relevant regulatory authorities for human administration. For example, Freund's complete adjuvant is not suitable for human administration. However, alum, MPL, or incomplete Freund's adjuvant (Chang et al. (1998) Adv Drug Deliv Rev. 32:173-186) alone or optionally in combination with any of alum, QS21 and MPL, and all combinations thereof, are suitable for human administration.

[907] In various embodiments, the present invention further provides methods for screening and identifying at least one neoantigen. More specifically, in various embodiments, the present invention provides a method for identifying at least one neoantigen by (a) contacting a neoplastic cell with an effective amount of a splicing modulator, ADC, or a composition comprising a splicing modulator, or ADC; (b) detecting at least one alternatively spliced mRNA transcript after contacting the neoplastic cell with a splicing modulator, ADC, or composition; (c) predicting the translation of at least one alternatively spliced mRNA transcript into at least one peptide; and (d) comparing at least one peptide to a reference proteome, where at least one neoantigen is identified if at least one peptide does not match any peptides in the reference proteome. In various embodiments, the method further comprises bringing one or more additional neoplastic cells into contact to identify at least one universal neoantigen. In various embodiments, the method is repeated for one or more additional neoplastic cells or one or more samples (e.g., tissue biopsy) to confirm suitable neoantigens (e.g., for use in a neoantigen vaccine) and/or to identify one or more universal neoantigens.

[908] In various embodiments, the present invention provides a method for identifying at least one neoantigen by (a) contacting a neoplastic cell with an effective amount of a splicing modulator, ADC, or a composition comprising a splicing modulator, or ADC; (b) detecting at least one peptide containing a potential neoantigenic sequence after contacting a neoplastic cell with a splicing modulator, ADC, or composition; and (c) comparing at least one peptide to a reference proteome, where at least one neoantigen is identified if at least one peptide does not match any peptides in the reference proteome. In various embodiments, the method further comprises bringing one or more additional neoplastic cells into contact to identify at least one universal neoantigen. In various embodiments, the method is repeated for one or more additional neoplastic cells or one or more samples (e.g., tissue biopsy) to confirm suitable neoantigens (e.g., for use in a neoantigen vaccine) and/or to identify one or more universal neoantigens.

[909] In some embodiments of the neoantigen identification methods described herein, detection of at least one alternatively spliced mRNA transcript involves RNAseq. In some embodiments, predicting the translation of at least one alternatively spliced mRNA transcript involves quantifying the change in terms of percent spliced success rate (dPSI) value for at least one transcript. In some embodiments, translation prediction of at least one alternatively spliced mRNA transcript involves RiboSeq and/or ribosomal profiling.

[910] In some embodiments of the neoantigen identification methods described herein, the methods further comprise evaluating at least one peptide for predictive binding to major histocompatibility complex (MHC) molecules. In some embodiments, predictive binding to MHC molecules is determined by measuring predicted binding strength based on initial affinity for at least one peptide. In some embodiments, a predicted binding strength based on initial affinity of about 500 nM or greater indicates binding to MHC molecules. In some embodiments, predicted binding to MHC molecules is determined by identifying a distribution of predicted binding strength values for a series of random peptides and comparing the predicted binding strength of at least one peptide to that distribution. In some embodiments, the predicted binding strength in the top 2.0% of the distribution indicates weak binding to MHC molecules. In some embodiments, the predicted binding strength in the top 0.5% of the distribution indicates strong binding to MHC molecules.

[911] In some embodiments of the neoantigen identification methods described herein, the neoplastic cell is in an in vitro cell culture. In some embodiments, the neoplastic cell is obtained from a subject. In some embodiments, the implementation of the neoplastic cell is in the subject.

[912] In various embodiments, this document also provides methods for producing a neoantigenic vaccine by (a) identifying at least one neoantigen (e.g., at least one neoantigenic peptide or mRNA encoding it) using any of the exemplary identification methods disclosed in herein and (b) formulating at least one neoantigen together with a pharmaceutically acceptable carrier, diluent, or adjuvant (eg, any of the pharmaceutically acceptable carriers, diluents, or adjuvants described herein). In some embodiments, the implementation of at least one neoantigen used in the vaccine contains the amino acid sequence under any of SEQ ID NO: 37-65. In some embodiments, at least one neoantigen used in a vaccine contains the amino acid sequence of SEQ ID NO: 37. In some embodiments, at least one neoantigen used in a vaccine contains the amino acid sequence of SEQ ID NO: 39. In some embodiments, embodiments, the implementation of at least one neoantigen used in the vaccine contains the amino acid sequence under any of SEQ ID NO: 46-49.

[913] In some other embodiments, at least one neoantigen used in the vaccine comprises an amino acid sequence of any of SEQ ID NOs: 66-93 or an antigenic portion of any of SEQ ID NOs: 66-93. In some embodiments, at least one neoantigen used in a vaccine comprises the amino acid sequence of SEQ ID NO: 66 or an antigenic portion of SEQ ID NO: 66. In some embodiments, at least one neoantigen used in a vaccine comprises the amino acid sequence under any of SEQ ID NO: 74-77 or antigenic part under any of SEQ ID NO: 74-77. In some embodiments, at least one neoantigen and/or antigenic portion is in the range of about 10 to about 50 amino acids in length. In some embodiments, at least one neoantigen and/or antigenic portion is in the range of about 10 to about 35 amino acids in length. In some embodiments, at least one neoantigen and/or antigenic portion is in the range of about 15 to about 25 amino acids in length. In some embodiments, at least one neoantigen and/or antigenic portion is in the range of about 10 to about 20 amino acids in length. In some embodiments, at least one neoantigen and/or antigenic portion does not directly overlap with or consist of a canonical peptide sequence (eg, any of the illustrative canonical peptide sequences underlined in Table 21).

[914] In some embodiments, at least one neoantigen used in the vaccine is attached to a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is selected from a peptide, serum albumin, fisurella hemocyanin, immunoglobulin, thyroglobulin, ovalbumin, toxoid, or an attenuated derivative of toxoid, a cytokine, and a chemokine.

Splicing modulator/ADC and engineered T cells (CAR-T) combination:

[915] in various embodiments, a patient having a cancer described herein may be treated with a combination of a splicing modulator, ADC, or composition and one or more tumor-targeting engineered T cells (i.e., CAR-T). Thus, in some embodiments, the present invention provides a method of treating a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a splicing modulator, ADC, or a composition comprising a splicing modulator, or ADC; and engineered tumor-targeting T cells (i.e., CAR-T). In various embodiments, a chimeric T cell receptor can be constructed using antigen recognition sequences that react with an identified neoantigen.

[916] For example, in various embodiments, in order to target changes induced by a splicing modulator or ADC in the extracellular domains of cell surface proteins, a chimeric antigen-reactive T-cell receptor (CAR) can be constructed by first identifying antibodies that recognize the domain neoantigenic protein expressed on the cell surface. The antigen recognition sequences of such antibodies can then be fused to the T cell receptor domain for selective targeting and activation.

[917] In various other embodiments, the implementation uses the strategy of embedding the antigen-presenting mechanism of tumor cells together with a neoantigen formed under the action of a splicing modulator or ADC. In some embodiments, cells containing known and frequently presented HLA alleles (eg, HLA-A*02:01) can be treated with a splicing modulator, ADC, or composition and MHC1-associated neoantigens are identified by ligandomics. In some embodiments, these peptides can be used to prime and/or expand T cells from healthy donors expressing the same HLA allele. In some embodiments, such T cells can be isolated and the T cell receptor (TCR) α and β chains sequenced to identify cognate antigen recognition/variable regions. In some embodiments, a cognate CAR can then be constructed.

[918] In some embodiments, the CAR sequences are cloned into a population of patient-derived T cells and the cells are expanded using currently available protocols. In some embodiments, the engineered T cells are then transfused into the patient's bloodstream, followed by treatment with a splicing modulator, ADC, or composition. Following treatment with a splicing modulator, ADC, or composition, in some embodiments, antigen presentation by tumor cells may begin. In some embodiments, a population of engineered T cells can contact antigen-presenting tumor cells and destroy them.

[919] It will be apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present invention described herein are obvious and can be made using suitable equivalents without departing from the scope of the present invention or the embodiments disclosed herein. . After a detailed description of the present invention, the same will be more clear with reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.

EXAMPLES

EXAMPLE 1

[920] Methods for synthesizing payloads, linkers, and compounds based on a conjugated linker-payload (linker-drug, L-D) construct having the structure shown in Tables 7-9 are described. The linker-payload conjugate construct has been used in the preparation of antibody drug conjugates (ADCs). Exemplary ADCs are described in Examples 3-5.

1.1 Reagents and materials

[921] The starting materials used in the following synthesis methods are either commercially available, or they can be easily obtained using standard methods from known materials. The disclosed linker-payload conjugate construct can be generated using the reactions and techniques described herein. In the description of the synthesis methods described below, it should be understood that all suggested reaction conditions, including the choice of solvent, the atmosphere of the reaction mixture, the reaction temperature, the duration of the experiment, and the processing procedure, can be chosen as standard conditions for this reaction, unless otherwise indicated. . One skilled in the art of organic synthesis will appreciate that the functional groups present on the various moieties of the molecule must be compatible with the proposed reagents and reactions. Substituents incompatible with the reaction conditions are obvious to a person skilled in the art, and therefore alternative methods are indicated in this document.

[922] Preparative liquid chromatography-mass spectrometry (LC/MS) was performed using the Waters AutoPurification System and an XTerra MS C18 column (5 μm, 19 mm x 100 mm) under acidic mobile phase conditions. Nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz using an instrument from Varian (Agilent Technologies). Microwave heating was performed using a Biotage Emrys Liberator or Initiator microwave reactor. Column chromatography was performed using a Teledyne Isco Combiflash Rf200d. Solvent removal was carried out using either a rotary evaporator from Büchi or a centrifugal evaporator from Genevac.

[923] Terms/abbreviations . As used herein, the term "inert atmosphere" refers to the replacement of air in a reactor (eg, reaction vessel, flask, glass reactor) with an inert gas containing substantially no moisture, such as nitrogen or argon. The following abbreviations are used in this document: DCM=dichloromethane, DMF=dimethylformamide, HPLC=high performance liquid chromatography, KHMDS=potassium bis(trimethylsilyl)amide, LC/MS=liquid chromatography-mass spectrometry, MeOH=methanol, rt= room temperature, TBSCl=tert-butyldimethylsilyl chloride, THF=tetrahydrofuran, TLC=thin layer chromatography. Multiplicity is indicated using the following abbreviations: s=singlet, d=doublet, t=triplet, q=quartet, quint=quintet, sxt=sextet, m=multiplet, dd=doublet of doublets, ddd=doublet of doublets of doublets, dt=doublet of triplets , br s=wide singlet.

[924] LC/MS . Mobile phases=A (0.1% formic acid in H2O) and B (0.1% formic acid in acetonitrile). Gradient=B from 5% to 95% in 1.8 min. Column=Waters Acquity BEH C18 column (1.7 µm, 2.1×50 mm).

[925] References . US Pat. Nos. 7,884,128 and 7,816,401 describe illustrative methods for the synthesis of pladienolide B and D, and are each incorporated herein by reference in the case of such methods. The synthesis of pladienolide B and D can also be carried out using the exemplary methods described in Kanada et al. ((2007) Angew Chem Int. Ed. 46:4350-5). In Kanada et al. and International Application Publication No. WO 2003/099813 describe illustrative methods for the synthesis of E7107 (D11) (compound 45 of WO 2003/099813) from pladienolide D (11107D of WO 2003/099813). The corresponding US Pat. No. 7,550,503 to Kotake et al. Each of these references is included in this document for the described methods of synthesis.

Table 7. Structure of exemplary drug fragments (payloads)

Payload Structure/ID
(Payload Group)

D1
(Пладиенолид D)

D1
(Pladienolide D)

D2
(Pladienolide D)

D3
(Pladienolide D)

D4
(Pladienolide D)

D5
(Pladienolide D)

D6
(Pladienolide D)

D7
(Pladienolide D)

D4'
(Pladienolide D)

D8
(Pladienolide D)

D9
(Pladienolide B)

D10
(Pladienolide D)

D11 (E7107)
(Pladienolide D)

D12
(Pladienolide D)

D13
(Pladienolide B)

D14
(Arylpladienolide)

D15
(Arylpladienolide)

D16
(Arylpladienolide)

D17
(Arylpladienolide)

D18
(Pladienolide B)

D19
(Pladienolide B)

D20
(Pladienolide D)

D21
(Pladienolide D)

D22
(Zwitterionic pladienolide D)

D23
(Zwitterionic pladienolide D)

D24
(Zwitterionic pladienolide D)

D25
(Zwitterionic pladienolide D)

D26
(Arylpladienolide)

D27
(Arylpladienolide)

D28
(Arylpladienolide)

D29
(Arylpladienolide)

D30
(Arylpladienolide)

D31
(Arylpladienolide)

D32
(Arylpladienolide)

D33
(Zwitterionic arylpladienolide)

D34
(Zwitterionic arylpladienolide)

D35
(Zwitterionic arylpladienolide)

Table 8. Structure of exemplary linkers

Linker Structure/ID
(IUPAC name)

ADL1 - "MC-Val-Cit-pABC"
({4-[(2S)-5-(карбамоиламино)-2-[(2S)-2-[6-(2,5-диоксо-2,5-дигидро-1H-пиррол-1-ил)гексанамидо]-3-метилбутанамидо]пентанамидо]фенил}метилформиат)

ADL1 - "MC-Val-Cit-pABC"
({4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido] -3-methylbutanamido]pentanamido]phenyl}methyl formate)

ADL2 - "MC-(PEG) ₂ -CO"
(3-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}propanal)

ADL5 - "MC-Val-Ala-pAB"
(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-[(1S)-2-methyl-1-{[(1S)-1-[(4- methylphenyl)carbamoyl]ethyl]carbamoyl}propyl]hexanamide)

ADL6 - "MC-Val-Ala-pABC"
({4-[(2S)-2-[(2S)-2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido]-3-methylbutanamido]propanamido ]phenyl}methyl formate)

ADL7 - "MC-Val-Cit-pAB"
(N-[(1S)-1-{[(1S)-4-(carbamoylamino)-1-[(4-methylphenyl)carbamoyl]butyl]carbamoyl}-2-methylpropyl]-6-(2,5-dioxo -2,5-dihydro-1H-pyrrol-1-yl)hexanamide)

ADL10 - "MC"
(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanal)

ADL12 - "Mal Hex"
(1-hexyl-2,5-dihydro-1H-pyrrole-2,5-dione)

ADL13 - "MC-β-glucuronide"
((2S,3S,4S,5R,6S)-6-(2-{3-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido]propanamido}- 4-[(formyloxy)methyl]phenoxy)-3,4,5-trihydroxyoxane-2-carboxylic acid)

ADL14 - "Mal-Et"
(1-ethyl-2,5-dihydro-1H-pyrrole-2,5-dione)

ADL15 - "Mal-Et-O-Et"
(1-(2-ethoxyethyl)-2,5-dihydro-1H-pyrrole-2,5-dione)

ADL21 - "MC-Ala-Ala-Asp-pABC"

ADL22 - "Mc-PEG2-Val-Cit-pABC"

ADL23 - "MC-Glu-Val-Cit-pABC"

Table 9. Structures of exemplary connections based on conjugated linker-payload (L-D) constructs

ADL1-D1

ADL1-D1

ADL6-D1

ADL5-D2

ADL1-D18

ADL5-D19

ADL14-D1

ADL12-D1

ADL15-D1

ADL12-D20

ADL10-D1

ADL12-D2

ADL15-D2

ADL12-D21

ADL6-D9

ADL1-D4

ADL1-D3

ADL1-D12

ADL1-D7

ADL1-D6

ADL1-D5

ADL22-D4

ADL5-D10

ADL5-D11

ADL1-D13

ADL1-D8

ADL1-D22

ADL5-D25

ADL12-D22

ADL5-D15

ADL1-D14

ADL5-D26

ADL1-D16

ADL5-D17

ADL1-D33

ADL1-D28

ADL1-D31

ADL1-D29

ADL1-D35

ADL5-D32

ADL5-D27

ADL12-D35

ADL12-D28

ADL1-D23

ADL1-D24

ADL5-D30

ADL13-D4

1.2 Obtaining payloads based on pladienolide

1.2.1 Brief description. General procedure 1

Scheme 1

[926] Step 1 . (2S,3S,6S,7R,10R, E)-7,10-Dihydroxy-2-((R,2E,4E)-6-hydroxy-7-((2S,3S)-3-((2R, 3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl -acetate (1.7 g, 3.154 mmol), triethylamine (4.40 ml, 31.536 mmol), 1,2-dichloroethane (31.5 ml, 3.154 mmol) were combined and stirred at rt. 1 ml, 12.615 mmol) and stirred overnight. The brine solution was poured into the reaction mixture and stirred for 30 minutes. and the organic layer was separated. The aqueous layer was then back extracted with DCM (3X). The organic layers were combined, dried (anhydrous Na ₂ SO ₄ ), concentrated to dryness and chromatographed to give (2S,3S,6S,7R,10R, E)-7-hydroxy-3,7-dimethyl-2-(( R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2- yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-yl-acetate (1.274 g, 1.423 mmol , yield 45.1%).

[927] ¹ H-NMR (400 MHz, DMSO- d 6): δ ppm 0.51-0.64 (m, 18 H) 0.74-0.84 (m, 9 H) 0.90-0 .97 (m, 26 H) 1.04 (s, 3 H) 1.18 (s, 4 H) 1.37 (s, 3 H) 1.41-1.57 (m, 4 H) 1, 69 (s, 3 H) 1.84-1.93 (m, 1 H) 2.00-2.05 (m, 3 H) 2.24-2.35 (m, 1 H) 2.38- 2.45 (m, 1 H) 2.72-2.80 (m, 1 H) 3.28-3.30 (m, 1 H) 3.62-3.70 (m, 1 H) 3, 80-3.90 (m, 1 H) 4.56 (s, 1 H) 4.81-4.93 (m, 2 H) 5.41-5.52 (m, 1 H) 5.63- 5.73 (m, 1 H) 5.77-5.87 (m, 1 H) 6.01-6.12 (m, 1 H) 6.42 (dd, J=15.12, 11.11 Hz, 1H).

[928] Step 2 . To a solution of (2S,3S,6S,7R,10R, E)-7-hydroxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)- 7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl) -12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-yl-acetate (0.7 g, 0.782 mmol) in toluene (4.16 ml, 39.085 mmol) 1,8-naphthalenediamine was added , N, N,N',N'-tetramethyl- (1.173 g, 5.472 mmol) followed by the addition of methyl trifluoromethanesulfonate (0.354 ml, 3.127 mmol) at 0°C. Then ensured the heating of the reaction mixture to 50°C and heated it for 3 hours. The solvent was evaporated. Purification by column chromatography gave (2S,3S,6S,7R,10R,E)-7-methoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-(( triethylsilyl)oxy)-7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-diene -2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-yl-acetate (467 mg, 0.513 mmol, 65.7% yield) as a colorless oil.

[929] ¹ H-NMR (400 MHz, MeOH- d 4 ): δ ppm 0.62-0.70 (m, 18 H) 0.83-0.95 (m, 13 H) 0.99-1 .07 (m, 27 H) 1.22 (s, 3 H) 1.24-1.36 (m, 2 H), 1.28-1.28 (m, 1 H) 1.45 (s, 3 H) 1.47-1.65 (m, 8 H) 1.78 (d, J=0.88 Hz, 3 H) 1.91-2.00 (m, 1 H) 2.07 (s , 3 H) 2.37-2.45 (m, 1 H) 2.51-2.68, (m, 3 H) 2.88-2.92 (m, 1 H) 3.20-3, 24 (m, 1 H) 3.35 (s, 3 H) 3.74-3.82 (m, 1 H) 3.93-4.02 (m, 1 H) 4.94-4.99 ( m, 1 H) 5.08-5.14 (m, 1 H), 5.53-5.64 (m, 1 H) 5.70-5.79 (m, 1 H) 5.81-5 .88 (m, 1 H) 6.12-6.18 (m, 1 H) 6.47-6.58 (m, 1 H).

[930] Step 3 . (2S,3S,6S,7R,10R, E)-7-Hydroxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7- ((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12 -oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-yl-acetate (500 mg, 0.558 mmol) was dissolved in MeOH (6009 μl, 148.524 mmol) and potassium carbonate (232 mg, 1.675 mmol) was added . The reaction mixture was stirred at RT for 1 hour and the completion of the reaction was determined. The reaction mixture was diluted with water and extracted with EtOAc (3X). The combined organics were washed with brine, dried over sodium sulfate, filtered and concentrated. Purification by column chromatography gave (4R,7R,8S,11S,12S,E)-7,8-dihydroxy-7,11-dimethyl-12-((R,2E,4E)-6-methyl-6- ((triethylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4 -dien-2-yl)-4-((triethylsilyl)oxy)oxacyclododec-9-en-2-one (305 mg, 0.357 mmol, 64.0% yield).

[931] ¹ H-NMR (400 MHz, MeOH- d 4 ): δ ppm 0.61-0.72 (m, 18 H) 0.82-0.92 (m, 7 H) 0.92-1 .06 (m, 30 H) 1.19-1.40 (m, 7 H) 1.42-1.66 (m, 8 H) 1.51-1.52 (m, 1 H) 1.75 -1.81 (m, 3 H) 1.91-2.01 (m, 1 H) 2.34-2.45 (m, 1 H) 2.51-2.61 (m, 2 H) 2 .61-2.69 (m, 1H) 2.86-2.94 (m, 1H) 3.67-3.73 (m, 1H) 3.73-3.80 (m, 1H ) 3.89-3.96 (m, 1 H) 4.08-4.17 (m, 1 H) 4.93-4.99 (m, 1 H) 5.36-5.47 (m, 1 H) 5.67-5.78 (m, 1 H) 5.80-5.88 (m, 1 H) 6.10-6.19 (m, 1 H) 6.47-6.58 ( m, 1H).

[932] Step 4 . Combined (4R,7R,8S,11S,12S, E)-8-hydroxy-7-methoxy-7,11-dimethyl-12-((R,2E,4E)-6-methyl-6-((triethylsilyl) hydroxy)-7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2 -yl)-4-((triethylsilyl)oxy)oxacyclododec-9-en-2-one (0.386 g, 0.445 mmol), DCM (0.1 M), DMAP (1.0 eq.), Hunig's base (5 0 eq.), 4-nitrophenyl chloroformate (1.8 eq.) and stirred overnight. The reaction mixture was then extracted with 1N HCl. NaOH. The organic layer was dried (anhydrous Na ₂ SO ₄ ) and concentrated to dryness. The residue was mixed with DCM (0.1 M), Hunig's base (5.0 eq.), amine (2.0 eq.), which were combined and stirred for 1 hour. The reaction mixture was concentrated and chromatographed to give tri-TES-pladenolide carbamate.

[933] Step 5 . Combined tri-TES-pladienolide carbamate (1 eq.), DCM (0.04 M) and DIPEA (191 eq.) and cooled to -78°C. Hydrofluoride-pyridine (6.2 eq.) was added and the reaction mixture was allowed to warm to rt and stirred overnight. The reaction mixture was cooled in an ice bath, then sat. NaHCO ₃ , stirred and extracted with DCM. The organic layers were combined, dried over anhydrous Na ₂ SO ₄ , concentrated and chromatographed to give carbamate pladienolide.

1.2.1.1 D1

[934] General procedure 1 (specified in section 1.2.1) was used to synthesize D1.

[935] (2S,3S,6S,7R,10R, E)-7-Methoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy) -7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl )-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-ylpiperazin-1-carboxylate (240 mg, 0.245 mmol, 55.1% yield). LC/MS (ESI, mass/charge), 980.4 [M+H] ⁺ .

[936] ¹ H-NMR (400 MHz, CDCl ₃ - d ): δ ppm 0.56-0.66 (m, 18 H) 0.78-0.91 (m, 9 H) 0.96 (t , J=7.91 Hz, 27 H) 1.18-1.22 (m, 3 H) 1.23-1.28 (m, 1 H) 1.37-1.41 (m, 3 H) 1.41-1.63 (m, 7 H) 1.69-1.74 (m, 3 H) 1.89 (dd, J=13.87, 4.96 Hz, 1 H) 2.33- 2.62 (m, 4 H) 2.78-2.89 (m, 5 H), 3.35 (s, 3 H) 3.43-3.52 (m, 4 H) 3.73 (td , J=6.40, 3.51 Hz, 1 H) 3.86 (br dd, J=7.84, 3.95 Hz, 1 H) 4.94-5.13 (m, 2 H) 5 .58-5.76 (m, 3 H) 6.12, (br d, J=0.75 Hz, 1 H) 6.41 (dd, J=15.06, 11.04 Hz, 1 H) .

D1

[937] (2S,3S,6S,7R,10R, E)-10-Hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R ,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4- en-6-ylpiperazine-1-carboxylate.

[938] ¹ H-NMR (400 MHz, MeOH- d 4): δ ppm 0.83-1.01 (m, 10 H) 1.23 (s, 3 H) 1.25-1.32 (m , 1 H) 1.35 (s, 3 H) 1.40-1.61 (m, 6 H) 1.61-1.71 (m, 2 H) 1.79 (d, J=0.75 Hz, 3 H) 1.84-1.93 (m, 1 H) 2.45-2.63 (m, 3 H) 2.63-2.72 (m, 1 H) 2.77-2, 85 (m, 4 H) 2.86-2.94 (m, 1 H) 3.33-3.37 (m, 3 H) 3.40-3.57 (m, 5 H) 3.77- 3.89 (m, 1 H) 4.38-4.42 (m, 1 H) 5.01-5.12 (m, 2 H) 5.52-5.65 (m, 1 H) 5, 69-5.80 (m, 1 H) 5.84-5.92 (m, 1 H) 6.09-6.18 (m, 1 H) 6.49-6.60 (m, 1 H) .

1.2.1.2 D2

[939] General procedure 1 (specified in section 1.2.1) was used to synthesize D2.

[940] (2S,3S,6S,7R,10R, E)-7-Methoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy) -7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl )-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-yl-4-methylpiperazin-1-carboxylate (413 mg, 0.416 mmol, 90% yield). LC/MS (ESI, mass/charge), 994 [M+H] ⁺ .

[941] ¹ H-NMR (400 MHz, MeOH- d 4 ): δ ppm 0.61-0.71 (m, 18 H) 0.83-0.94 (m, 9 H) 0.98-1 .07 (m, 28 H) 1.23 (s, 3 H) 1.41-1.48 (m, 3 H) 1.48-1.65 (m, 7 H) 1.72-1.81 (m, 3 H) 1.93-1.99 (m, 1 H) 2.03 (s, 3 H) 2.32 (s, 3 H) 2.38-2.48 (m, 5 H) 2.52-2.67 (m, 3 H) 2.87-2.93 (m, 1 H) 3.47-3.61 (m, 4 H) 3.72-3.81 (m, 1 H) 3.96-4.04 (m, 1 H) 4.53-4.62 (m, 1 H) 4.93-5.07 (m, 2 H) 5.51-5.64 (m , 1 H) 5.69-5.79 (m, 1 H) 5.81-5.92 (m, 1 H) 6.09-6.21 (m, 1 H) 6.46-6.60 (m, 1H).

D2

[942] (2S,3S,6S,7R,10R, E)-10-Hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R ,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4- en-6-yl-4-methylpiperazine-1-carboxylate (37.8 mg, 0.058 mmol, 24.05% yield). LC/MS (ESI, mass/charge), 651.69 [M+H] ⁺ .

[943] ¹ H-NMR (400 MHz, DMSO- d 6): δ ppm 0.76-0.89 (m, 9 H) 1.10 (s, 3 H) 1.20-1.26 (m , 3 H) 1.30-1.42 (m, 4 H) 1.42-1.55 (m, 2 H) 1.66-1.75 (m, 3 H) 1.75-1.82 (m, 1 H) 2.13-2.21 (m, 3 H) 2.25 (br s, 4 H) 2.31-2.41 (m, 2 H) 2.54-2.60 ( m, 2 H) 2.72-2.82 (m, 1 H) 3.22 (s, 3 H) 3.36-3.40 (m, 3 H) 3.66-3.76 (m, 1 H) 4.36-4.46 (m, 1 H) 4.53-4.60 (m, 1 H) 4.78-4.85 (m, 1 H) 4.86-4.96 ( m, 2 H) 5.36-5.50 (m, 1 H) 5.60-5.74 (m, 1 H) 5.80-5.92 (m, 1 H) 6.02-6, 11 (m, 1 H) 6.34-6.46 (m, 1 H).

1.3 Obtaining the MC-Val-Cit-pABC-Payloads Linker Construction

1.3.1 Brief description. General procedure 1

Scheme 2

[944] Payload (1.0 eq.), Hunig base (3.0 eq.), DMF (0.1 M), and 4-((S)-2-((S)-2-(6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl)carbonate (1.2 eq) was combined and stirred at k. t. during the night. The reaction mixture was then concentrated and purified by column chromatography (MeOH in DCM) or reverse phase HPLC to give the product.

1.3.1.1 ADL1-D1

[945] Linker Payload (ADL1-D1) . General procedure 1 (specified in section 1.3.1) was used to synthesize 1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H -pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-((2S,3S,6S,7R,10R, E)-10-hydroxy-2-((R,2E ,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-diene -2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)piperazin-1,4-dicarboxylate (50 mg, 0.040 mmol, 42% yield). LC/MS (ESI mass/charge), 1258.5 [M+Na] ⁺ .

[946] ¹ H-NMR (400 MHz, MeOH- d 4 ): δ ppm 0.85-1.04 (m, 16 H) 1.18-1.26 (m, 3 H) 1.26-1 .38 (m, 6H) 1.40-1.71 (m, 14H) 1.80 (s, 3H) 1.85-1.98 (m, 2H) 2.02-2.15 (m, 1 H) 2.24-2.34 (m, 2 H) 2.44-2.64 (m, 3 H) 2.65-2.72 (m, 1 H) 2.87-2 .96 (m, 1H) 3.06 -3.27 (m, 2H) 3.37 (s, 6H) 3.43-3.61 (m, 12H) 3.79-3.90 (m, 1 H) 4.12-4.21 (m, 1 H) 4.48-4.55 (m, 1 H) 5.02-5.14 (m, 4 H) 5.55-5 .65 (m, 1 H), 5.69-5.81 (m, 1 H) 5.85-5.93 (m, 1 H) 6.12-6.19 (m, 1 H) 6, 49-6.60 (m, 1 H) 6.81 (s, 2 H) 7.29-7.38 (m, 2 H) 7.57-7.65 (m, 2 H).

1.3.1.2 ADL1-D18

[947] The D18 payload was generated using the procedures in section 1.2.1 using pladienolide B as starting material (SM) (R=H; Scheme 1).

[948] (2S,3S,6S,7R,10R, E)-10-Hydroxy-2-((S,2E,4E)-7-((2R,3R)-3-((2R,3S)- 3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6- ilpiperazine-1-carboxylate (205 mg, 0.330 mmol, 77% yield). LC/MS (ESI, mass/charge), 621.6 [M+H] ⁺ .

[949] ¹ H-NMR (400 MHz, MeOH- d 4 ): δ ppm 0.88-0.99 (m, 9 H) 1.10 (d, J =6.78 Hz, 3 H) 1, 24 (s, 4 H) 1.42-1.69 (m, 8 H) 1.77 (d, J =0.88 Hz, 3 H) 2.43-2.63 (m, 4 H) 2 .64-2.70 (m, 1 H) 2.71-2.82 (m, 5 H) 3.34 (br s, 3 H) 3.37 (s, 2 H) 3.42-3, 57 (m, 5 H) 3.79-3.89 (m, 1 H) 5.06 (s, 2 H), 5.54-5.63 (m, 1 H) 5.64-5.80 (m, 2 H) 6.07-6.16 (m, 1 H) 6.29-6.40 (m, 1 H).

[950] Linker Payload (ADL1-D18) . General procedure 1 (specified in section 1.3.1) was used to synthesize 1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H -pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-((2S,3S,6S,7R,10R, E)-10-hydroxy-2-((S,2E ,4E)-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl )-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)piperazin-1,4-dicarboxylate (72 mg, 0.059 mmol, 54.7% yield). LC/MS (ESI, mass/charge), 1220.08 [M+H] ⁺ .

[951] ¹ H-NMR (400 MHz, MeOH- d 4 ): δ ppm 0.87-1.02 (m, 15 H) 1.10 (d, J =6.78 Hz, 3 H) 1, 23 (s, 4 H) 1.29-1.38 (m, 2 H) 1.44-1.68 (m, 13 H), 1.54-1.55 (m, 1 H) 1.77 (d, J =0.75 Hz, 4 H) 1.87-1.95 (m, 1 H) 2.05-2.15 (m, 1 H) 2.24-2.32 (m, 2 H) 2.45-2.62 (m, 4 H) 2.65-2.70 (m, 1 H), 2.71-2.78 (m, 1 H) 3.13-3.18 ( m, 2 H) 3.46-3.58 (m, 11 H) 3.79-3.90 (m, 1 H) 4.13-4.21 (m, 1 H) 4.47-4, 56 (m, 1 H) 5.11 (s, 4 H) 5.53-5.81 (m, 3 H) 6.06-6.16 (m, 1 H) 6.29-6.40 ( m, 1 H) 6.81 (s, 2 H) 7.34 (d, J = 8.53 Hz, 2 H) 7.60 (d, J = 8.53 Hz, 2 H).

1.3.1.3 ADL1-D8 and ADL6-D8

[952] The D8 payload was synthesized according to the procedure below.

Scheme 3

[953] Step 1 . To a solution of tri-TES-pladienolide D (200 mg, 0.223 mmol) in dichloromethane (2 ml) at 0°C was added DMAP (409 mg, 3.35 mmol) and 4-nitrophenyl chloroformate (338 mg, 1.675 mmol). The reaction mixture was stirred at rt for 7 days, diluted with EtOAc and water, then the layers were separated. The aqueous layer was extracted with EtOAc (2X) and the combined organic extracts washed with brine. The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated in vacuo . Flash chromatography gave (2S,3S,6S,7R,10R, E)-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)- 7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl) -7-(((4-nitrophenoxy)carbonyl)oxy)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-yl-acetate (170 mg, 72% yield).

[954] ¹ H-NMR (400 MHz, CHCl ₃ - d ): δ ppm 0.54-0.67 (m, 18 H) 0.78-1.03 (m, 36 H) 1.19-1 .32 (m, 1H) 1.39 (s, 3H) 1.43-1.52 (m, 3H) 1.55-1.63 (m, 3H) 1.64 (s, 3 H) 1.74 (s, 3 H) 1.88 (dd, J = 13.80, 5.02 Hz, 1 H) 2.13 (s, 3 H) 2.23-2.37 (m, 1 H) 2.39-2.48 (m, 2 H) 2.51-2.63 (m, 2 H) 2.84 (s, 1 H) 3.69-3.77 (m, 1 H ) 3.82-4.00 (m, 1 H) 5.04 (d, J =10.79 Hz, 1 H) 5.24 (d, J =9.03 Hz, 1 H) 5.67- 5.84 (m, 3 H) 6.12 (d, J = 10.16 Hz, 1 H) 6.42 (dd, J = 15.06, 11.04 Hz, 1 H) 7.42 (d , J = 9.29 Hz, 2 H) 8.29 (d, J = 9.16 Hz, 2 H).

[955] Step 2 . In a solution of (2S,3S,6S,7R,10R, E)-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-(( 2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-7-( ((4-nitrophenoxy)carbonyl)oxy)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-yl-acetate (100 mg, 0.094 mmol) in DCM was added piperazine and DMAP. The resulting yellowish suspension was stirred for 6 hours. The reaction mixture was concentrated to give the crude product. Flash chromatography gave (2S,3S,6S,7R,10R, E)-6-acetoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl )oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien- 2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-7-ylpiperazin-1-carboxylate (95 mg, 100%). LC/MS (ESI, mass/charge), 1008.8 [M+H] ⁺ .

[956] ¹ H-NMR (400 MHz, CHCl ₃ - d ): δ ppm 0.42-0.70 (m, 22 H) 0.79-0.84 (m, 7 H) 0.86-0 .91 (m, 4 H) 0.92-1.03 (m, 30 H) 1.15-1.30 (m, 2 H) 1.37-1.42 (m, 3 H) 1.44 -1.52 (m, 3 H) 1.56-1.62 (m, 2 H) 1.62-1.68 (m, 1 H) 1.71-1.76 (m, 3 H) 1 .83-1.93 (m, 1H) 2.03-2.11 (m, 4H) 2.36-2.45 (m, 2H) 2.45-2.53 (m, 2H ) 2.54-2.64 (m, 1 H) 2.78-2.86 (m, 1 H) 2.86-3.07 (m, 4 H) 3.32-3.45 (m, 1 H) 3.45-3.64 (m, 3 H) 3.69-3.78 (m, 1 H) 3.79-3.94 (m, 1 H) 5.00 (d, J = 10.54 Hz, 1 H) 5.18 (s, 1 H) 5.54-5.79 (m, 3 H) 5.98-6.21 (m, 1 H) 6.33-6.57 (m, 1 H) 6.84-6.96 (m, 3 H) 8.02-8.35 (m, 2 H) 8.06-8.08 (m, 1 H).

[957] Step 3 . In a solution of (2S,3S,6S,7R,10R, E)-6-acetoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)- 7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl) -12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-7-ylpiperazin-1-carboxylate (95 mg, 0.094 mmol) in THF (3 ml) was added TBAF (0.424 ml, 1 M, 0.424 mmol ) and stirred at room temperature for 10 hours. The mixture was concentrated and diluted with EtOAc, washed with water and brine. The organic layer was separated and dried over Na ₂ SO ₄ , filtered and concentrated in vacuo . Purification by HPLC gave (2S,3S,6S,7R,10R, E)-6-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R )-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec -4-en-7-ylpiperazine-1-carboxylate (16 mg, 26%). LC/MS (ESI, mass/charge), 665.6 [M+H] ⁺ .

[958] ¹ H-NMR (400 MHz, CHCl ₃ - d ): δ ppm 0.90 (dd, J =6.84, 2.20 Hz, 6 H) 0.94 (t, J =7.40 Hz, 3H) 1.20-1.30 (m, 1H) 1.34 (s, 3H) 1.39-1.54 (m, 3H) 1.55 (s, 3H) 1 .59-1.73 (m, 3 H) 1.78 (d, J = 0.88 Hz, 3 H) 1.86 (dd, J = 13.99, 5.46 Hz, 1 H) 2, 05 (s, 3 H) 2.39-2.53 (m, 3 H) 2.55-2.65 (m, 1 H) 2.67 (dd, J = 8.03, 2.26 Hz, 1 H) 2.89 (s, 1 H) 3.22 (br s, 4 H) 3.50-3.57 (m, 1 H) 3.58-3.90 (m, 5 H) 5, 08 (d, J =10.67 Hz, 1 H) 5.18 (d, J = 9.03 Hz, 1 H) 5.58-5.78 (m, 2 H) 5.88 (d, J = 15.31 Hz, 1 H) 6.10-6.23 (m, 1 H) 6.53 (dd, J = 15.25, 10.98 Hz, 1 H).

[959] Step 4 .

[960] Linker Payload (ADL1-D8) . K 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5 -ureidopentanamido)benzyl-(4-nitrophenyl)carbonate (7.5 mg, 10.166 µmol) in DMF (315 µl, 4.066 mmol) Hunig's base (5.33 µl, 0.03 mmol) was added. The reaction mixture was cooled to 0°C and (2S,3S,6S,7R,10R, E)-6-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-(( 2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl- 12-oxooxacyclododec-4-en-7-yl-piperazine-1-carboxylate (7.50 mg, 0.011 mmol). The reaction mixture was stirred at room temperature until the starting material was consumed. The reaction mixture was concentrated in vacuo . Flash chromatography gave ADL1-D8 (7.2 mg, 5.70 µmol, 56.1% yield). LC/MS (ESI, mass/charge), 1263.8 [M+H] ⁺ . General procedure 1 (1.3.1) can also be used to obtain ADL1-110987.

[961] ¹ H-NMR (400 MHz, CHLOROFORM- d ): δ ppm 0.80-1.02 (m, 16 H) 1.20-1.35 (m, 6 H) 1.38-1, 48 (m, 2 H) 1.57-1.69 (m, 7 H) 1.74-1.81 (m, 4 H) 1.86-1.96 (m, 2 H) 2.00- 2.11 (m, 4 H) 2.31 (br d, J = 6.02 Hz, 2 H) 2.45-2.55 (m, 2 H) 2.59-2.74 (m, 2 H) 2.85-2.96 (m, 1H) 3.05-3.25 (m, 5H) 3.40-3.59 (m, 9H) 3.62-3.88 (m , 1 H) 4.15 (d, J = 7.53 Hz, 1 H) 5.05-5.22 (m, 5 H) 5.56-5.77 (m, 2 H) 5.84- 5.98 (m, 1 H) 6.11-6.22 (m, 1 H) 6.47-6.61 (m, 1 H) 7.31-7.40 (m, 2 H) 7, 56-7.65 (m, 2H).

[962] Linker Payload (ADL6-D8) . K 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido) benzyl (4-nitrophenyl) carbonate (5 mg, 7.673 µmol) in DMF (238 µl, 3.069 mmol) Hunig's base (4.02 µl, .023 mmol) was added. The reaction mixture was cooled to 0°C and (2S,3S,6S,7R,10R, E)-6-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-(( 2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl- 12-oxooxacyclododec-4-en-7-yl-piperazine-1-carboxylate. The reaction mixture was stirred at room temperature until the starting material was consumed. The reaction mixture was concentrated in vacuo . Purification by HPLC gave ADL6-D8 (1.2 mg, 1.019 µmol, 13.28% yield). LC/MS (ESI, mass/charge), 1199.9 [M+Na] ⁺ .

[963] ¹ H NMR (400 MHz, CHCl ₃ - d ): δ ppm 0.78-1.04 (m, 18 H) 1.15-1.52 (m, 58 H) 1.64-1, 72 (m, 3 H) 1.78 (d, J =6.53 Hz, 4 H) 2.04-2.19 (m, 3 H) 2.21-2.30 (m, 1 H) 2 .44-2.67 (m, 4 H) 2.76 (dd, J =6.09, 2.95 Hz, 1 H) 2.90-3.02 (m, 1 H) 3.42-3 .57 (m, 8 H) 3.61-3.70 (m, 1 H) 3.77 (br s, 1 H) 4.10-4.27 (m, 1 H) 4.58 (br d , J =6.65 Hz, 1 H) 5.01-5.26 (m, 4 H) 5.52-5.70 (m, 2 H) 5.88 (s, 1 H) 5.93- 6.02 (m, 1 H) 6.08-6.18 (m, 1 H) 6.47-6.58 (m, 1 H) 7.29-7.35 (m, 2 H) 7, 55 (d, J = 8.66 Hz, 1 H).

1.3.1.4 ADL1-D4

[964] Synthesized ADL1-D4 using the procedures below.

Scheme 4

[965] Step 1 . (2S,3S,6S,7R,10R, E)-3,7-Dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2R, 3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-6-((( 4-nitrophenoxy)carbonyl)oxy)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-7-yl-acetate.

[966] DMAP (32.7 mg, 0.268 mmol), triethylamine (0.75 ml , 5.36 mmol) and 4-nitrophenyl chloroformate (360 mg, 1.787 mmol). The reaction mixture was stirred at 40°C for 4 days and at 60°C for 2 hours. The reaction mixture was diluted with EtOAc and washed with water, then the layers were separated. The aqueous layer was extracted with EtOAc (2X). The combined organic extracts were washed successively with water and brine, dried over MgSO ₄ , filtered and concentrated in vacuo . Flash chromatography gave (2S,3S,6S,7R,10R, E)-7-acetoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl )oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien- 2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-ylpiperazin-1-carboxylate (150 mg, 79% yield).

[967] ¹ H-NMR (400 MHz, CHCl ₃ - d ): δ ppm 0.48-0.71 (m, 24 H) 0.78-0.85 (m, 7 H) 0.86-0 .93 (m, 5 H) 0.94-1.03 (m, 34 H) 1.18-1.22 (m, 2 H) 1.22-1.26 (m, 2 H) 1.35 -1.43 (m, 4 H) 1.43-1.52 (m, 4 H) 1.54 (s, 4 H) 1.56-1.65 (m, 3 H) 1.68-1 .72 (m, 3 H) 1.75 (br d, J = 0.75 Hz, 2 H) 1.84-1.95 (m, 1 H) 2.01-2.06 (m, 2 H ) 2.09 (s, 2 H) 2.11 (s, 2 H) 2.33-2.52 (m, 4 H) 2.57 (dd, J = 8.09, 2.07 Hz, 2 H) 2.80-2.90 (m, 1 H) 3.66-3.80 (m, 1 H) 3.82-3.93 (m, 2 H) 4.92-5.13 (m , 2 H) 5.63-5.68 (m, 1 H) 5.69-5.74 (m, 1 H) 5.75-5.83 (m, 2 H) 6.12 (br d, J = 10.67 Hz, 1 H) 6.41 (ddd, J = 15.15, 11.01, 5.08 Hz, 1 H) 7.50 (d, J = 9.41 Hz, 2 H) 8.35 (d, J =9.29 Hz, 2H).

[968]Stage 2. (2S,3S,6S,7R,10R, E)-7-Acetoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7- ((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12 -oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-ylpiperazin-1-carboxylate.

[969] To a solution of (2S,3S,6S,7R,10R, E)-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7 -((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)- 6-(((4-nitrophenoxy)carbonyl)oxy)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-7-yl-acetate in DCM (1 ml) was added piperazine (0.447 g, 5.195 mmol) and Hunig's base (0.9 ml, 5.195 mmol). The resulting yellowish suspension was stirred for 6 hours. The reaction mixture was concentrated and chromatographed on silica gel to give (2S,3S,6S,7R,10R, E)-7-acetoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6- ((triethylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4 -dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-ylpiperazin-1-carboxylate (1.0 g, 0.844 mmol, 81% yield). LC/MS (ESI, mass/charge), 1008.1 [M+H] ⁺ .

D4

[970] Step 3 . (2S,3S,6S,7R,10R, E)-7-Acetoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7- ((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12 -oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-ylpiperazine-1-carboxylate (1.09 g, 0.92 mmol), DCM (20.71 ml, 321.826 mmol) and DIPEA (19 .91 ml, 114.018 mmol) were combined and cooled to -78°C. Hydrofluoride-pyridine (0.518 g, 5.232 mmol) was added and the reaction mixture was allowed to warm to rt and stirred overnight. According to LC/MS, desilylation was expected. The reaction mixture was cooled in an ice bath. Saturated NaHCO ₃ solution was added and mixed and extracted with DCM. The organic layers were combined, dried over anhydrous Na ₂ SO ₄ and concentrated and chromatographed to give (2S,3S,6S,7R,10R, E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)- 6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl )-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazin-1-carboxylate (225 mg, 36.8%). LC/MS (ESI, mass/charge), 665.6 [M+H] ⁺ .

[971] ¹ H-NMR (400 MHz, CHCl ₃ - d ): δ ppm 0.87-0.92 (m, 6 H) 0.94 (t, J =7.40 Hz, 3 H) 1, 16-1.31 (m, 1 H) 1.35 (s, 3 H) 1.40-1.56 (m, 4 H) 1.59 (s, 3 H) 1.66 (br dd, J =14.68, 7.03 Hz, 3 H) 1.76-1.80 (m, 3 H) 1.87 (dd, J =14.12, 5.46 Hz, 1 H) 2.05 ( s, 3 H) 2.30-2.41 (m, 1 H) 2.50 (d, J =3.76 Hz, 2 H) 2.56-2.72 (m, 2 H) 2.90 (br d, J =2.01 Hz, 1 H) 3.19 (br t, J =5.14 Hz, 4 H) 3.50-3.59 (m, 1 H) 3.71 (br s , 4 H) 3.77-3.89 (m, 1 H) 5.01-5.13 (m, 2 H) 5.58-5.71 (m, 1 H) 5.71-5.81 (m, 1 H) 5.88 (d, J =15.31 Hz, 1 H) 6.15 (br d, J =10.79 Hz, 1 H) 6.53 (dd, J =15.18 , 10.92 Hz, 1 H).

AD1-D4

[972] K 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido )-5-ureidopentanamido)benzyl(4-nitrophenyl)carbonate (23 mg, 0.031 mmol) in DMF (966 μl) Hunig's base (16.33 μl, 0.094 mmol) was added in a round bottom flask. The reaction mixture was cooled to 0°C and D4 (22.80 mg, 0.034 mmol) was added and stirred at RT. The reaction mixture was concentrated in vacuo . Flash chromatography gave ADL1-D4 (30.5 mg, 0.024 mmol, 77% yield). LC/MS (ESI, mass/charge), 1263.8 [M+H] ⁺ .

[973] ¹ H-NMR (400 MHz, MeOH- d ₄ ): δ ppm 0.87-0.93 (m, 7 H) 0.93-1.01 (m, 8 H) 1.19-1 .34 (m, 4 H) 1.50 (s, 3 H) 1.57 (s, 5 H) 1.58-1.70 (m, 6 H) 1.70-1.77 (m, 1 H) 1.78 (s, 3 H) 1.83-1.95 (m, 2 H) 2.04 (s, 3 H) 2.05-2.13 (m, 1 H) 2.27 ( t, J = 7.40 Hz, 2 H) 2.32-2.42 (m, 1 H) 2.50 (d, J = 3.64 Hz, 2 H) 2.55-2.74 (m , 2 H) 2.90 (td, J = 5.83, 2.26 Hz, 1 H) 3.03-3.26 (m, 2 H) 3.35 (s, 13 H) 3.42- 3.61 (m, 11 H) 3.80 (br dd, J =9.85, 3.58 Hz, 1 H) 4.16 (d, J =7.40 Hz, 1 H) 4.50 ( dd, J = 8.91, 5.14 Hz, 1 H) 4.56 (s, 1 H) 5.05 (dd, J = 14.37, 10.10 Hz, 2 H) 5.09 (s , 2 H) 5.49 (s, 1 H) 5.59-5.69 (m, 1 H) 5.72-5.80 (m, 1 H) 5.87 (d, J =15.18 Hz, 1 H) 6.09-6.22 (m, 1 H) 6.44-6.60 (m, 1 H) 7.32 (d, J =8.66 Hz, 2 H) 7.58 (d, J = 8.53 Hz, 2 H).

1.3.1.5 ADL1-D9, ADL6-D9 and ADL1-D13

D9 and D13

[974] D9 and D13 were synthesized as a 3:1 mixture of isomers using the procedures outlined in Synthesis of D4 (Scheme 4) using tri-TES-pladienolide B.

[975] Payload (D9) : LC/MS (ESI Mass/Charge), 649.7 [M+H] ⁺ .

[976] ¹ H-NMR (400 MHz, CHCl ₃ - d ): δ ppm 0.87-1.01 (m, 10 H) 1.08 (d, J=6.78 Hz, 3 H) 1, 27-1.63 (m, 12 H) 1.66-1.74 (m, 1 H) 1.76 (s, 3 H) 1.97-2.10 (m, 3 H) 2.35- 2.57 (m, 5 H) 2.58-2.65 (m, 1 H) 2.65-2.71 (m, 1 H) 2.77 (td, J=5.93, 2.32 Hz, 1 H) 2.89-3.05 (m, 4 H) 3.07-3.34 (m, 8 H) 3.50-3.68 (m, 5 H) 3.72-3, 88 (m, 1 H) 4.88-5.09 (m, 1 H) 5.18 (d, J=10.67 Hz, 1 H) 5.50-5.84 (m, 3 H) 6 .01-6.13 (m, 1 H) 6.19-6.36 (m, 1 H).

[977] Payload (D13) : LC/MS (ESI Mass/Charge), 649.6 [M+H] ⁺ .

[978] ¹ H NMR (400 MHz, CHCl ₃ - d ) δ ppm 0.84-1.01 (m, 11 H) 1.08 (d, J =6.78 Hz, 3 H) 1.29- 1.64 (m, 10 H) 1.63-1.73 (m, 1 H) 1.76 (s, 3 H) 1.94-2.12 (m, 4 H) 2.37-2, 58 (m, 4 H) 2.59-2.65 (m, 1 H) 2.68 (dd, J =7.40, 2.26 Hz, 1 H) 2.77 (td, J =5, 93, 2.32 Hz, 1 H) 3.01-3.30 (m, 4 H) 3.49 (s, 1 H) 3.54-3.67 (m, 2 H) 3.69-3 .92 (m, 5 H) 4.13-4.78 (m, 11 H) 5.13-5.24 (m, 2 H) 5.48-5.61 (m, 1 H) 5.62 -5.74 (m, 2 H) 6.04-6.13 (m, 1 H) 6.18-6.32 (m, 1 H).

ADL1-D9.

[979] General procedure 1 (specified in section 1.3.1) was used to synthesize ADL1-D9.

[980] Linker payload (ADL1-D9) : (30.5 mg, 0.024 mmol, 77% yield). LC/MS (ESI, mass/charge), 1263.8 [M+H] ⁺ .

[981] ¹ H-NMR (400 MHz, MeOH- d ₄ ): δ ppm 0.87-0.93 (m, 7 H) 0.93-1.01 (m, 8 H) 1.19-1 .34 (m, 4 H) 1.50 (s, 3 H) 1.57 (s, 5 H) 1.58-1.70 (m, 6 H) 1.70-1.77 (m, 1 H) 1.78 (s, 3 H) 1.83-1.95 (m, 2 H) 2.04 (s, 3 H) 2.05-2.13 (m, 1 H) 2.27 ( t, J = 7.40 Hz, 2 H) 2.32-2.42 (m, 1 H) 2.50 (d, J = 3.64 Hz, 2 H) 2.55-2.74 (m , 2 H) 2.90 (td, J = 5.83, 2.26 Hz, 1 H) 3.03-3.26 (m, 2 H) 3.35 (s, 13 H) 3.42- 3.61 (m, 11 H) 3.80 (br dd, J =9.85, 3.58 Hz, 1 H) 4.16 (d, J =7.40 Hz, 1 H) 4.50 ( dd, J = 8.91, 5.14 Hz, 1 H) 4.56 (s, 1 H) 5.05 (dd, J = 14.37, 10.10 Hz, 2 H) 5.09 (s , 2 H) 5.49 (s, 1 H) 5.59-5.69 (m, 1 H) 5.72-5.80 (m, 1 H) 5.87 (d, J =15.18 Hz, 1 H) 6.09-6.22 (m, 1 H) 6.44-6.60 (m, 1 H) 7.32 (d, J =8.66 Hz, 2 H) 7.58 (d, J = 8.53 Hz, 2 H).

ADL6-D9

[982] General procedure 1 (specified in section 1.3.1) was used to synthesize ADL6-D9.

[983] Linker Payload (ADL6-D9) . To dilute 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido )benzyl (4-nitrophenyl) carbonate (16.5 mg, 0.025 mmol) in DMF (784 μl) Hunig's base (13.27 μl, 0.076 mmol) was added. The reaction mixture was cooled to 0°C and added D9. The reaction mixture was stirred at RT until completion of the reaction was determined by LC/MS. The reaction mixture was concentrated in vacuo . Flash chromatography of the residue on silica gel with DCM/MeOH afforded the title compound (16.7 mg, 57% yield). LC/MS (ESI mass/charge), 1184.6 [M+Na] ⁺ .

[984] ¹ H-NMR (400 MHz, MeOH- d ₄ ): δ ppm 0.80-1.02 (m, 16 H) 1.05-1.12 (m, 4 H) 1.13-1 .34 (m, 5 H) 1.38-1.51 (m, 7 H) 1.53-1.68 (m, 11 H) 1.71-1.80 (m, 4 H) 1.97 -2.16 (m, 4 H) 2.24-2.32 (m, 2 H) 2.32-2.42 (m, 1 H) 2.43-2.55 (m, 3 H) 2 .56-2.69 (m, 3 H) 2.73 (br d, J = 2.13 Hz, 1 H) 3.07-3.18 (m, 1 H) 3.42-3.61 ( m, 17 H) 3.75-3.91 (m, 1 H) 4.11-4.24 (m, 1 H) 4.43 (s, 2 H) 5.59-5.88 (m, 4 H) 6.06-6.17 (m, 1 H) 6.29-6.39 (m, 1 H) 7.24-7.39 (m, 2 H) 7.52-7.64 ( m, 2H).

ADL1-D13.

[985] Linker-Payload (AD1-D13) . K 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5 -ureidopentanamido)benzyl(4-nitrophenyl)carbonate (4.2 mg, 5.693 µmol) in DMF (176 µl, 2.277 mmol) Hunig's base (2.98 µl, 0.017 mmol) was added. The reaction mixture was cooled to 0°C and D13 (4.06 mg, 6.262 µmol) was added. The reaction mixture was stirred at RT until completion of the reaction was determined by LC/MS. The reaction mixture was concentrated in vacuo . Flash chromatography of the residue on silica gel with DCM/MeOH gave the title compound (4.8 mg, 68% yield). LC/MS (ESI mass/charge), 1248.0 [M+H] ⁺ .

[986] ¹ H-NMR (400 MHz, CHCl ₃ - d ): δ ppm 0.58-0.98 (m, 10 H) 0.99-1.06 (m, 2 H) 1.11-1 .31 (m, 6 H) 1.57-1.75 (m, 3 H) 2.11-2.26 (m, 1 H) 2.30-2.73 (m, 2 H) 3.28 -3.80 (m, 7 H) 4.14-4.33 (m, 1 H) 4.44-4.55 (m, 1 H) 4.63-4.81 (m, 1 H) 4 .92-5.06 (m, 1 H) 5.09 (s, 1 H) 5.49-5.74 (m, 1 H) 5.88-6.09 (m, 1 H) 6.14 -6.36 (m, 1 H) 6.99-7.08 (m, 1 H) 7.21-7.38 (m, 2 H) 7.53-7.67 (m, 1 H) 7 .95 (s, 1H).

1.3.1.6 ADL1-D14

[987] General procedure 1 (specified in section 1.3.1) was used to synthesize 1-((2S,3S,6R,7S,10R, E)-2-((E)-1-(1-(1-acetylpiperidine -4-yl)-4-fluoro-1H-benzo[d][1,2,3]triazol-6-yl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl- 12-oxooxacyclododec-4-en-6-yl)-4-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)piperazin-1,4-dicarboxylate (30 mg, 0.024 mmol, 39.2% yield). LC/MS (ESI, mass/charge), 1254.5 [M+H] ⁺ .

[988] ¹ H-NMR (400 MHz, DMSO- d 6): δ ppm 0.75-1.00 (m, 12 H), 1.06-1.26 (m, 4 H), 1.26 -1.77(m, 13H), 1.75-2.02(m, 7H), 2.08(s, 8H), 2.23-2.35(m, 2H), 2 .54-2.70 (m, 2 H), 2.78-3.10 (m, 4 H), 3.36 (br s, 13 H), 3.66-3.79 (m, 1 H ), 3.95-4.08 (m, 1 H), 4.11-4.25 (m, 1 H), 4.28-4.46 (m, 1 H), 4.46-4, 58 (m, 1 H), 4.58-4.66 (m, 1 H), 4.66-4.77 (m, 1 H), 5.01 (s, 3 H), 5.14- 5.27(m, 1H), 5.39(s, 4H), 5.71-5.79(m, 1H), 5.87-6.00(m, 1H), 6, 63-6.75(m, 1H), 6.99(s, 2H), 7.13-7.24(m, 1H), 7.24-7.34(m, 2H), 7.52-7.62(m, 2H), 7.65-7.74(m, 1H), 7.75-7.82(m, 1H), 7.98-8.13( m, 1H).

1.3.1.7 ADL1-D33

D33

[989] To a stirred solution of (2S,3S,6R,7S,10R, E)-2-((E)-1-(3-fluoro-5-(4-(2-methoxy-2-oxoethyl)piperazine- 1-yl)phenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazin-1-carboxylate (27 mg, 0.042 mmol) in THF/H ₂ O (3 ml/1 ml) at 0°C was added LiOH and provided a slow heating of the reaction mixture to 25°C. Synthesized (2S,3S,6R,7S,10R, E)-2-((E)-1-(3-fluoro-5-(4-(2-methoxy-2-oxoethyl)piperazin-1-yl)phenyl )prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazin-1-carboxylate using the procedures specified in International Patent Application No. PCT/ US2019/026992 (see, for example, procedures 17 and 19), which is incorporated herein by reference. After 6 hours the reaction mixture was neutralized with phosphate buffer (NaH ₂ PO ₄ , 1.0 M, 3 ml) and the phases were separated. The aqueous layer was extracted with ethyl acetate (3×2 ml) and the combined organic layers were dried with anhydrous sodium sulfate and concentrated in vacuo . Reverse phase column chromatography afforded the title compound (19.4 mg, 0.031 mmol, 73.4% yield). LC/MS (ESI, mass/charge), 631.4 [M+H] ⁺ .

[990] ¹ H-NMR (400 MHz, CHCl ₃ - d ): δ ppm 0.85-1.16 (m, 2 H) 1.23-1.52 (m, 1 H) 1.57-1 .77 (m, 1 H) 1.87 (s, 1 H) 1.92-2.13 (m, 1 H) 2.35-2.55 (m, 1 H) 2.50-2.74 (m, 1 H) 3.11 (br s, 1 H) 3.36-3.54 (m, 2 H) 3.64 (br d, J =10.42 Hz, 2 H) 3.77- 3.90 (m, 1 H) 4.76 (br s, 4 H) 5.07-5.19 (m, 1 H) 5.38-5.68 (m, 1 H) 6.40-6 .76 (m, 1 H) 8.02-8.66 (m, 1 H).

ADL1-D33.

[991] Linker Payload (ADL1-D33) . K 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5 -ureidopentanamido)benzyl(4-nitrophenyl)carbonate (17 mg, .023 mmol) in DMF (714 μl, 9.217 mmol) Hunig's base (12.07 μl, 0.069 mmol) was added. The reaction mixture was cooled to 0°C and 2-(4-(3-fluoro-5-((E)-2-((2S,3S,6R,7S,10R, E)-10-hydroxy-3.7 -dimethyl-12-oxo-6-((piperazin-1-carbonyl)oxy)oxacyclododec-4-en-2-yl)prop-1-en-1-yl)phenyl)piperazin-1-yl)acetic acid ( 16.71 mg, 0.026 mmol). The reaction mixture was stirred at RT until completion of the reaction was determined by LC/MS. The reaction mixture was concentrated in vacuo . Flash chromatography on silica gel and further purification by HPLC gave the title compound (13.5 mg, 10.98 µmol, 47.7% yield). LC/MS (ESI, mass/charge), 1229.5 [M+H] ⁺ .

[992] ¹ H-NMR (400 MHz, MeOH- d ₄ ): δ ppm 0.92-0.99 (m, 10 H) 1.01 (d, J =6.78 Hz, 3 H) 1, 24-1.46 (m, 9 H) 1.52-1.70 (m, 10 H) 1.70-1.82 (m, 1 H) 1.87 (d, J =1.00 Hz, 4 H) 1.89-2.00 (m, 2 H) 2.01-2.23 (m, 2 H) 2.27 (t, J =7.40 Hz, 2 H) 2.46 (dd , J =14.31, 5.27 Hz, 1 H) 2.57-2.71 (m, 2 H) 2.86 (d, J = 0.63 Hz, 1 H) 3.00 (s, 1 H) 3.06-3.27 (m, 4 H) 3.35 (s, 5 H) 3.41-3.53 (m, 19 H) 3.66 (s, 2 H) 3.76 -3.90 (m, 1 H) 4.07-4.21 (m, 1 H) 4.39-4.69 (m, 37 H) 5.09 (s, 3 H) 5.13 (d , J = 10.54 Hz, 1 H) 5.52 (dd, J = 14.56, 9.03 Hz, 2 H) 6.55 (br s, 2 H) 6.69 (d, J = 1 .38 Hz, 2 H) 7.33 (d, J = 8.66 Hz, 2 H) 7.59 (d, J = 8.53 Hz, 2 H) 7.88-8.04 (m, 1 H) 8.16-8.33 (m, 1 H).

1.3.2 ADL1-D4, ADL1-D5, ADL21-D4, ADL22-D4, ADL23-D4, ADL13-D4

1.3.2.1 General procedure for preparing D4 and D5

[993] The 4-step procedure below was used to synthesize the title compounds.

Stage 2

[994] DMAP (1.5 eq.), triethylamine (30 eq.) and 4-nitrophenyl chloroformate (10 eq.). The mixture was stirred at 40°C for 4 days and then for 2 hours at 60°C. The reaction mixture was diluted with EtOAc and washed with water, then the layers were separated and the aqueous layer was extracted with EtOAc (2x). The combined organic extracts were washed successively with water and brine, dried over magnesium sulfate, filtered and concentrated in vacuo. The resulting residue was purified by silica gel chromatography (EtOAc in hexanes) to give the intermediate compound as carbonate. Carbonate (1.0 eq.), DCM (0.2 M), triethylamine (3.0 eq.) and amine (2.0 eq.) were combined and stirred at RT for 1 hour. The resulting mixture was concentrated in vacuo and chromatographed (DCM/MeOH) to give the carbamate intermediate as a mixture of regioisomers.

Stage 3

[995] The mixture of regioisomeric carbamates obtained in step 2 (1.0 eq.) was dissolved in DCM (0.04 M). Hunig's base (124 eq.) was added and the resulting mixture was cooled to -78°C. HF-pyridine (30 eq) was added dropwise and the mixture was heated to rt and stirred overnight at rt. The mixture was then cooled to -78°C and saturated sodium bicarbonate was added dropwise to the reaction mixture and the mixture was heated to rt. The organic layers were separated and the aqueous layer was extracted with DCM (3X). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The resulting precipitate was purified by reverse phase HPLC to obtain each of the desired regioisomeric products.

D5

[996] The title compound was prepared using 2,5-diazabicyclo[2.2.1]heptane in step 2 via steps 2 and 3 of general procedure 1.3.2.1 .

Stage 2

[997] A mixture of (2S,3S,6S,7R,10R, E)-7-acetoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl) hydroxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2 -yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-yl-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate and (2S,3S,6S,7R ,10R, E)-6-acetoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2R,3R)-3 -((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl )oxy)oxacyclododec-4-en-7-yl-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (21.7 mg, 45.1%) as a colorless oil. LCMS (ESI, mass/charge), [M+H] ⁺ 1020.6

[998] ¹ H NMR (400 MHz, METHANOL-d) δ ppm 0.61-0.70 (m, 19 H) 0.82-0.95 (m, 10 H) 0.95-1.04 ( m, 29 H) 1.23 (br dd, J=7.15, 4.64 Hz, 1 H) 1.30 (s, 1 H) 1.42-1.64 (m, 12 H) 1, 78 (s, 4 H) 1.88-2.01 (m, 2 H) 2.06 (br d, J=10.92 Hz, 3 H) 2.14 (br s, 1 H) 2.43 (br d, J=4.64 Hz, 2 H) 2.49 (br s, 1 H) 2.63 (dd, J=8.16, 2.13 Hz, 2 H) 2.86-2, 91 (m, 1 H) 3.47-3.66 (m, 2 H) 3.76 (td, J=6.37, 3.33 Hz, 1 H) 3.95 (br d, J=4 .27 Hz, 1 H) 4.34 (br s, 1H) 6.15 (br d, J=10.92 Hz, 1 H) 6.52 (dd, J=15.12, 10.98 Hz, 1H)

Stage 3

D5

[999] Purification by HPLC gave (2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-(( 2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl- 12-oxooxacyclododec-4-en-6-yl-(1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (5 mg, 34.7%) as a white solid. LCMS (ESI, mass/charge), [M+H] ⁺ 677.6

[1000] 1H NMR (400 MHz, METHANOL- d 4) δ ppm 0.85-0.98 (m, 9 H) 1.25 (td, J =7.40, 4.14 Hz, 1 H) 1 .34 (s, 3 H) 1.42-1.69 (m, 9 H) 1.79 (s, 4 H) 1.82-1.96 (m, 2 H) 2.04 (d, J =9.29 Hz, 3 H) 2.33 (br d, J =10.04 Hz, 1 H) 2.47-2.54 (m, 2 H) 2.57-2.72 (m, 2 H) 2.87-2.92 (m, 1 H) 3.03 (br s.2 H) 3.33-3.43 (m, 2 H) 3.43-3.57 (m, 2 H ) 3.77-3.84 (m, 1 H) 3.86-3.94 (m, 1 H) 4.50 (br s, 1 H) 4.97-5.11 (m, 2 H) 5.60-5.68 (m, 1 H) 5.73-5.82 (m, 1 H) 5.88 (d, J =15.31 Hz, 1 H) 6.15 (br d, J =11.04 Hz, 1 H) 6.53 (dd, J =15.25, 10.98 Hz, 1 H) 8.54 (s, 1 H)

D4

[1001] The title compound was prepared using piperazine in step 2 via steps 2 and 3 of general procedure 1.3.2.1 .

Stage 2 .

[1002] Using step 2 of general procedure 1.3.2.1 using piperazine, (2S,3S,6S,7R,10R, E)-7-acetoxy-3,7-dimethyl-2-((R,2E,4E) -6-methyl-6-((triethylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2- yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-ylpiperazin-1-carboxylate (1.0 g, 0.844 mmol, yield 81%). LC/MS (ESI, mass/charge), 1008.1 [M+H] ⁺ .

Stage 3 . D4

[1003] Purification by HPLC gave (2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-(( 2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl- 12-oxooxacyclododec-4-en-6-ylpiperazine-1-carboxylate (225 mg, 36.8%). LC/MS (ESI, mass/charge), 665.6 [M+H] ⁺ .

[1004] ¹ H-NMR (400 MHz, CHCl ₃ - d ): δ ppm 0.87-0.92 (m, 6 H) 0.94 (t, J =7.40 Hz, 3 H) 1, 16-1.31 (m, 1 H) 1.35 (s, 3 H) 1.40-1.56 (m, 4 H) 1.59 (s, 3 H) 1.66 (br dd, J =14.68, 7.03 Hz, 3 H) 1.76-1.80 (m, 3 H) 1.87 (dd, J =14.12, 5.46 Hz, 1 H) 2.05 ( s, 3 H) 2.30-2.41 (m, 1 H) 2.50 (d, J =3.76 Hz, 2 H) 2.56-2.72 (m, 2 H) 2.90 (br d, J =2.01 Hz, 1 H) 3.19 (br t, J =5.14 Hz, 4 H) 3.50-3.59 (m, 1 H) 3.71 (br s , 4 H) 3.77-3.89 (m, 1 H) 5.01-5.13 (m, 2 H) 5.58-5.71 (m, 1 H) 5.71-5.81 (m, 1 H) 5.88 (d, J =15.31 Hz, 1 H) 6.15 (br d, J =10.79 Hz, 1 H) 6.53 (dd, J =15.18 , 10.92 Hz, 1 H).

ADL1-D4.

[1005] General procedure 1 (1.3.1) was used to obtain ADL1-D4. Flash chromatography gave ADL1-D4 (30.5 mg, 0.024 mmol, 77% yield).

[1006] LC/MS (ESI mass/charge), 1263.8 [M+H] ⁺ .

[1007] ¹ H-NMR (400 MHz, MeOH- d ₄ ): δ ppm 0.87-0.93 (m, 7 H) 0.93-1.01 (m, 8 H) 1.19-1 .34 (m, 4 H) 1.50 (s, 3 H) 1.57 (s, 5 H) 1.58-1.70 (m, 6 H) 1.70-1.77 (m, 1 H) 1.78 (s, 3 H) 1.83-1.95 (m, 2 H) 2.04 (s, 3 H) 2.05-2.13 (m, 1 H) 2.27 ( t, J = 7.40 Hz, 2 H) 2.32-2.42 (m, 1 H) 2.50 (d, J = 3.64 Hz, 2 H) 2.55-2.74 (m , 2 H) 2.90 (td, J = 5.83, 2.26 Hz, 1 H) 3.03-3.26 (m, 2 H) 3.35 (s, 13 H) 3.42- 3.61 (m, 11 H) 3.80 (br dd, J =9.85, 3.58 Hz, 1 H) 4.16 (d, J =7.40 Hz, 1 H) 4.50 ( dd, J = 8.91, 5.14 Hz, 1 H) 4.56 (s, 1 H) 5.05 (dd, J = 14.37, 10.10 Hz, 2 H) 5.09 (s , 2 H) 5.49 (s, 1 H) 5.59-5.69 (m, 1 H) 5.72-5.80 (m, 1 H) 5.87 (d, J =15.18 Hz, 1 H) 6.09-6.22 (m, 1 H) 6.44-6.60 (m, 1 H) 7.32 (d, J =8.66 Hz, 2 H) 7.58 (d, J = 8.53 Hz, 2 H).

ADL1-D5

[1008] General procedure 1 (1.3.1) was used to obtain ADL1-D5 using D5. Flash chromatography gave (14 mg, 51.6% yield). LCMS (ESI mass/charge), 1276.43 [M+H] ⁺

[1009] 1H NMR (400 MHz, METHANOL- d 4) δ ppm 0.86-1.00 (m, 16 H) 1.23-1.40 (m, 7 H) 1.42-1.69 ( m, 16 H) 1.73-1.80 (m, 4 H) 1.84-1.96 (m, 4H) 1.98-2.10 (m, 4 H) 2.27 (t, J =7.40 Hz, 2 H) 2.31-2.41 (m, 1 H) 2.50 (br d, J=3.39 Hz, 2 H) 2.56-2.64 (m, 1 H) 2.64-2.72 (m, 1 H) 2.80-2.93 (m, 1 H) 3.12 (br d, J=6.90 Hz, 1 H) 3.16-3 .26 (m, 1 H) 3.35-3.56 (m, 7 H) 3.76-3.86 (m, 1 H) 4.16 (d, J=7.40 Hz, 1 H) 4.48-4.58 (m, 3H) 4.93-5.16 (m, 4 H) 5.65 (br d, J=9.91 Hz, 1 H) 5.70-5.82 ( m, 1 H) 5.88 (d, J=15.18 Hz, 1 H) 6.14 (br d, J=11.04 Hz, 1 H) 6.50-6.58 (m, 1 H ), 6.79 (s, 2 H) 7.26-7.36 (m, 2 H) 7.56-7.63 (m, 2 H)

1.3.3 Synthesis of ADL22-D4

[1010] To a solution of (2S,3S,6S,7R,10R, E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R) -3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec- 4-en-6-ylpiperazin-1-carboxylate (12 mg, 0.018 mmol) in DMF (2 ml) was added 4-((2S,5S)-15-(2,5-dioxo-2,5-dihydro-1H -pyrrol-1-yl)-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-10,13-dioxa-3,6-diazapentadecanamido)benzyl(4-nitrophenyl)carbonate (16.98 mg , .022 mmol) and Hunig's base (7.00 mg, .054 mmol). The mixture was stirred for 1 hour at 20˚C. The mixture was concentrated in vacuo directly on silica gel and purified by column chromatography (MeOH/DCM 0-20%) to give 1-((2S,3S,6S,7R,10R, E)-7-acetoxy-10-hydroxy-2 -((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta -2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)-4-(4-((2S,5S)-15-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-10,13-dioxa-3,6-diazapentadecanamido)benzyl)piperazine -1,4-dicarboxylate (12 mg, 9.16 µmol, 50.8% yield) as a white solid. LCMS (ESI mass/charge), 1310.2 [M+H] ⁺

[1011] Chemical formula: C65H96N8O20. Molecular weight: 1309.52.

[1012] 1H NMR (400 MHz, DMSO- d 6) δ ppm 0.73-0.89 (m, 17 H) 1.03-1.13 (m, 1 H) 1.21-1.25 ( m, 4 H) 1.25-1.40 (m, 5 H) 1.42-1.47 (m, 4 H) 1.48, (br s, 3 H) 1.69 (s, 4 H ) 1.73-1.83 (m, 1 H) 1.99 (s, 2 H) 2.10-2.25 (m, 1 H) 2.36 (br s, 3 H) 2.40- 2.48 (m, 2 H) 2.54-2.66 (m, 2 H) 2.72-2.80 (m, 1 H) 2.88-3.08 (m, 2 H) 3 .34-3.44 (m, 9H) 3.44-3.48 (m, 3H) 3.48-3.52 (m, 2H) 3.52-3.59 (m, 3H ) 3.64-3.75 (m, 1 H) 4.24 (s, 1 H), 4.40 (br d, J=5.65 Hz, 2 H) 4.62 (d, J=5 .02 Hz, 1 H) 4.80-4.85 (m, 1 H) 4.90 (br d, J=9.03 Hz, 2 H) 5.01 (s, 2 H) 5.40 ( s, 2 H) 5.48-5.57 (m, 1 H), 5.66-5.76 (m, 1 H) 5.81-5.91 (m, 1 H) 5.94-6 .01 (m, 1 H) 6.02-6.11 (m, 1 H) 6.36-6.45 (m, 1 H) 7.02 (s, 2 H) 7.27-7.33 (m, 2 H) 7.58 (s, 2H) 7.81-7.89 (m, 1 H) 8.11 (br d, J=7.40 Hz, 1 H) 9.96-10, 02 (m, 1H)

1.3.4 General procedure for the synthesis of ADL21-D12, ADL21-D1, ADL21-D4

Stage 1

[1013] Payload (1.0 eq) and (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-(((S)-4-amino-1- ((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1,4-dioxobutan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropane -2-yl)carbamate (1.0 eq.) was dissolved in DMF (0.1 M) and Hunig's base (3.0 eq.) was added. The reaction mixture was stirred at RT for 30 minutes, after which the reaction mixture was concentrated in vacuo and the resulting residue was chromatographed (MeOH/DCM) to obtain the desired product.

Stage 2 and 3

[1014] The construct fmoc-Ala-Ala-asparagine-PABC-payload (1.0 eq.) was dissolved in N,N-dimethylformamide (0.05 M), then diethylamine (6.0 eq.) was added. The reaction mixture was stirred at RT for 1 hour and the mixture was evaporated to dryness. The crude product was diluted with N,N-dimethylformamide (0.05 M). 2,5-dioxopyrrolidin-1-yl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (1.2 eq.) and Hunig's base (2.5 eq. .) into the mixture and stirred at rt for 1 h. The mixture was then concentrated in vacuo and the resulting precipitate was purified by reverse phase HPLC to give the desired maleimidocaproyl-Ala-Ala-asparagine-PABC-payload linker design.

1.3.4.1 ADL21-D1

Stage 1

[1015] 1-(4-((5S,8S,11S)-11-(2-Amino-2-oxoethyl)-1-(9H-fluoren-9-yl)-5,8-dimethyl-3,6 ,9-trioxo-2-oxa-4,7,10-triazodecan-12-amido)benzyl)-4-((2S,3S,6S,7R,10R, E)-10-hydroxy-2-((R ,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4 -dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)piperazin-1,4-dicarboxylate (140.2 mg, 71.9%) LCMS ( ESI mass/charge) 1265.6 [M+H] ⁺

Chemical formula: C ₆₇ H ₈₉ N ₇ O ₁₇ .

Molecular weight: 1264.48.

Stages 2 and 3

[1016] 1-(4-((S)-4-Amino-2-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H -pyrrol-1-yl)hexanamido)propanamido)propanamido)-4-oxobutanamido)benzyl)-4-((2S,3S,6S,7R,10R, E)-10-hydroxy-2-((R,2E, 4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien- 2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)piperazin-1,4-dicarboxylate (49.1 mg, 35.8%). LCMS (ESI mass/charge) 1258.19 [M+Na] ⁺

[1017] ¹ H NMR (400 MHz, METHANOL- d 4) δ ppm 0.85-0.98 (m, 10 H) 1.22 (s, 4 H) 1.26-1.45 (m, 14 H) 1.45-1.69 (m, 12 H) 1.79 (s, 3 H) 1.90 (s, 2H) 2.20-2.27 (m, 2 H) 2.52 (br dd, J =10.10, 3.70 Hz, 3 H) 2.67 (br d, J =2.13 Hz, 1 H) 2.85 (t, J =6.15 Hz, 3 H) 3 .14 (t, J = 1.57 Hz, 1 H) 3.41-3.57, (m, 14 H) 3.78-3.88 (m, 1 H) 4.27 (dd, J = 12.36, 7.09 Hz, 2 H) 4.77 (s, 1 H) 4.97-5.13 (m, 6 H) 5.51-5.64 (m, 1 H) 5.72 (br d, J = 9.66 Hz, 1 H), 5.88 (d, J = 15.43 Hz, 1 H) 6.12-6.17 (m, 1 H) 6.47-6, 61 (m, 1 H) 6.79 (s, 2 H) 7.33 (m, J = 8.66 Hz, 2 H), 7.68 (m, J = 8.66 Hz, 2 H)

1.3.4.2 ADL21-D4

Stage 1

[1018] 1-((2S,3S,6S,7R,10R, E)-7-Acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R )-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec -4-en-6-yl)-4-(4-((5S,8S,11S)-11-(2-amino-2-oxoethyl)-1-(9H-fluoren-9-yl)-5, 8-dimethyl-3,6,9-trioxo-2-oxa-4,7,10-triazodecan-12-amido)benzyl)piperazine-1,4-dicarboxylate (125 mg, 85.6%). LCMS (ESI mass/charge) 1293.4 [M+H] ⁺

Stages 2 and 3

[1019] 1-((2S,3S,6S,7R,10R, E)-7-Acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R )-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec -4-en-6-yl)-4-(4-((S)-4-amino-2-((S)-2-((S)-2-(6-(2,5-dioxo- 2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)propanamido)-4-oxobutanamido)benzyl)piperazine-1,4-dicarboxylate (35 mg, 80%). LCMS (ESI mass/charge) 1285.0 [M+Na] ⁺

[1020] ¹ H NMR (400 MHz, DMSO-d6) δ ppm 0.71-0.87 (m, 9 H) 1.04-1.12 (m, 1 H) 1.14-1.26 ( m, 13 H) 1.27-1.40 (m, 3 H) 1.40-1.52 (m, 9 H) 1.52-1.62 (m, 1 H) 1.69 (s, 3 H) 1.74-1.83 (m, 1 H) 1.99 (d, J=4.27 Hz, 4 H) 2.03-2.14 (m, 2 H) 2.15-2 .26 (m, 1 H) 2.31-2.41 (m, 2 H) 2.54-2.65 (m, 4 H) 2.72-2.80 (m, 1 H) 3.17 (d, J=5.27 Hz, 3 H) 3.36 (br s, 9 H) 3.63-3.74 (m, 1 H) 3.99-4.12 (m, 2 H) 4 .14-4.22 (m, 1 H) 4.23-4.30 (m, 1 H) 4.40 (d, J=5.65 Hz, 1 H) 4.55-4.65 (m , 2 H) 4.82 (s, 1 H) 4.87-4.93 (m, 2 H) 5.01 (s, 2 H) 5.47-5.57 (m, 1 H) 5, 66-5.77 (m, 1 H) 6.01-6.09 (m, 1 H) 6.35-6.45 (m, 1 H) 6.89-6.96 (m, 1 H) 6.99 (s, 2 H) 7.30 (d, J=8.66 Hz, 2 H) 7.36-7.42 (m, 1 H) 7.62 (s, 2 H) 7.97 -8.21(m, 3H), 9.62-9.72(m, 1H).

1.3.5 ADL21-D12

[1021] To a solution of (2S,3S,6S,7R,10R, E)-7,10-dihydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3 -((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4- en-6-ylpiperazin-1-carboxylate (50 mg, 0.08 mmol) in DMF (1 ml) was added (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1- (((S)-4-amino-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1,4-dioxobutan-2-yl)amino)-1- oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate (73.9 mg, 0.096 mmol) followed by N-ethyl-N-isopropylpropan-2-amine (104 mg, 0.803 mmol). The reaction mixture was stirred for 1 hour. The reaction mixture was concentrated to dryness and purified by silica gel column chromatography. Fractions containing the desired compound were concentrated to give pure oil, which was used directly in the next step in an estimated yield of 100%. The resulting oil was then dissolved in DMF (2 ml) and diethylamine (58.7 mg, 0.803 mmol) was charged. The resulting mixture was stirred for 1 hour at 20˚C and concentrated in vacuo. The residue was redissolved in DMF (1 ml) and then 2,5-dioxopyrrolidin-1-yl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (32.2 mg, 0.104 mmol) and N-ethyl-N-isopropylpropan-2-amine (104 mg, 0.803 mmol). The resulting mixture was stirred for 1 hour. The reaction mixture was concentrated to dryness and purified by reverse phase HPLC to give 1-(4-((S)-4-amino-2-((S)-2-((S )-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)propanamido)-4-oxobutanamido)benzyl)-4-((2S,3S, 6S,7R,10R, E)-7,10-dihydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3- hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)piperazin-1, 4-dicarboxylate (7.5 mg, 6.14 µmol, 7.65% yield) as a white solid. LCMS (ESI mass/charge) 1222.28 [M+H] ⁺

1.3.6 ADL21-D8

[1022] To a solution of (2S,3S,6S,7R,10R, E)-6-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R) -3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec- 4-en-7-ylpiperazin-1-carboxylate (27.0 mg, .041 mmol) in DMF was added (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-( ((S)-4-amino-1-((4((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1,4-dioxobutan-2-yl)amino)-1-oxopropan- 2-yl)amino)-1-oxopropan-2-yl)carbamate (40.5 mg, .053 mmol) followed by the addition of Hunig's base (36.2 μl, 0.203 mmol). After stirring for 20 minutes at RT, the mixture was then concentrated in vacuo and the resulting residue was purified by column chromatography (0-20% MeOH/DCM) to give the desired intermediate. The residue was then combined with DMF (2 ml) and diethylamine (29.7 mg, 0.406 mmol) and stirred for 20 minutes at rt. The mixture was then concentrated in vacuo and the resulting precipitate was redissolved in DMF (2 ml) and treated with with 2,5-dioxopyrrolidin-1-yl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (16.28 mg, .053 mmol) and Hunig's base (36 .2 µl, 0.203 mmol). The resulting mixture was stirred for 20 minutes before being concentrated to dryness and the resulting residue was purified by column chromatography, eluting the intermediate with a 0-20% MeOH/DCM gradient. The resulting residue was then purified by reverse phase HPLC to give 1-((2S,3S,6S,7R,10R, E)-6-acetoxy-10-hydroxy-2-((R,2E,4E)-6 -hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl) -3,7-dimethyl-12-oxooxacyclododec-4-en-7-yl)-4-(4-((S)-4-amino-2-((S)-2-((S)-2- (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)propanamido)-4-oxobutanamido)benzyl)piperazine-1,4-dicarboxylate (3.5 mg, 2.77 µmol, 6.82% yield, isolated as a white solid. 1-((2S,3S,6S,7R,10R, E)-6-Acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3 -((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4- en-7-yl)-4-(4-((S)-4-amino-2-((S)-2-((S)-2-(6-(2,5-dioxo-2,5 -dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)propanamido)-4-oxobutanamido)benzyl)piperazine-1,4-dicarboxylate (3.5 mg, 6.8%). LCMS (ESI mass/charge) 1285.60 [M+Na] ⁺

[1023] ¹ H NMR: 0.68-0.90 (m, 11 H) 1.03-1.13 (m, 2 H) 1.15-1.26 (m, 14 H) 1.24- 1.38 (m, 4 H) 1.40-1.63 (m, 12 H), 1.69 (s, 3 H) 1.74-1.83 (m, 1 H) 2.03 (s , 3 H) 2.06-2.12 (m, 2 H) 2.24-2.41 (m, 3 H) 2.53-2.63 (m, 4 H), 3.79-3, 80 (m, 1H) 4.13-4.30 (m, 2H) 4.32-4.33 (m, 1H) 4.33-4.47 (m, 1H) 4.44-4 .48 (m, 1H) 4.52-4.73 (m, 2H) 4.74-4.97 (m, 2H) 5.03 (s, 3H) 5.47-5.61 (m, 1 H) 5.65-5.77 (m, 1 H) 5.79-5.92 (m, 1 H) 5.98-6.12 (m, 1 H) 6.30-6 .46 (m, 1H) 6.75-6.85 (m, 1H) 6.90-6.95 (m, 1H) 6.99 (s, 1H) 7.26-7.36 ( m, 2 H) 7.37-7.46 (m, 1 H) 7.56-7.73 (m, 2 H) 7.90-8.09 (m, 1 H) 8.07-8, 22 (m, 2 H) 8.46 (s, 1 H) 9.59-9.78 (m, 1 H)

1.3.7 Obtaining the construction linker DBCO-Val-Cit-pABC-payload, ADL25-D4

[1024] To a solution of 3-amino-1-(11,12-dihydrodibenzo[b,f]azocin-5(6H)-yl)propan-1-one (19.36 mg, 0.07 mmol) in DCM ( 5 ml) at 0˚C was added Hunig's base (10.26 mg, 0.079 mmol) followed by the addition of 4-nitrophenylcarbonyl chloride (14.12 mg, 0.07 mmol). The reaction was heated to 20˚C and stirred for 2 hours. The mixture was then concentrated to dryness and diluted with DMF. Hunig's base (10.26 mg, .079 mmol) and 1-((2S,3S,6S,7R,10R, E)-7-acetoxy-10-hydroxy-2-((R,2E, 4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien- 2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)-4-(4-((S)-2-((S)-2-amino-3-methylbutanamido)- 5-ureidopentanamido)benzyl)piperazine-1,4-dicarboxylate (50 mg, .047 mmol) and stirred for 1 hour. The mixture was then concentrated to dryness and purified by column chromatography followed by reverse phase purification to give 1- ((2S,3S,6S,7R,10R, E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-( (2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en- 6-yl)-4-(4-((9S,12S)-9-isopropyl-3,7,10-trioxo-12-(3-ureidopropyl)-2,6,8,11-tetraazatridecan-13-dibenzenecyclooctinamido )benzyl)piperazine-1,4-dicarboxylate (5.3 mg, 8.27%). LCMS (ESI mass/charge) 1372.7 [M] ⁺

1.3.8. Obtaining the construct linker maleimido-Glu-Val-Cit-pABC-payload

[1025] To a solution of (2S,3S,6S,7R,10R, E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R) -3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec- 4-en-6-ylpiperazin-1-carboxylate (20 mg, 0.03 mmol) in DMF (1 ml) was added (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino )-5-(((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo -5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)amino)-5-oxopentanoic acid (32.3 mg, 0.036 mmol) and Hunig's base (11.66 mg, 0.09 mmol) and stirred for 1 hour at 20˚C. Diethylamine (110 mg, 1.504 mmol) was then added and the mixture was stirred for an additional 30 minutes. at 20˚C. The mixture was then diluted with ethyl acetate and concentrated in vacuo. The resulting precipitate was diluted with DMF (1 ml), 2,5-dioxopyrrolidin-1-yl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (13.91 mg , 0.045 mmol) and Hunig's base (11.66 mg, 0.09 mmol) and the reaction mixture was stirred for 50 minutes. The reaction mixture was then diluted with ethyl acetate, concentrated to dryness and purified using reverse phase HPLC to give (S)-5-(((S)-1-(((S)-1-((4-(((4 -((((2S,3S,6S,7R,10R, E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)- 3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4 -en-6-yl)oxy)carbonyl)piperazine-1-carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2 -yl)amino)-4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-5-oxopentanoic acid (9.3 mg, 6.68 µmol, yield 22.20%) as a white solid; LCMS (ESI mass/charge) 1393.4 [M+H] ⁺

[1026] ¹ H NMR (400 MHz, DMSO- d 6) δ ppm 0.74-0.89 (m, 16 H) 1.03-1.66 (m, 25 H) 1.69 (s, 4 H) 1.74-1.94 (m, 2 H) 2.00 (s, 4 H) 2.09 (br d, J=4.39 Hz, 2 H) 2.21 (br d, J= 7.53 Hz, 3 H) 2.31-2.42 (m, 2 H) 2.53-2.84 (m, 3 H) 2.89-3.09 (m, 2 H) 3.34 -3.48 (m, 10 H) 3.65-3.74 (m, 1 H) 4.19, (dd, J=8.47, 6.59 Hz, 1 H) 4.26-4, 45 (m, 3 H) 4.57-4.64 (m, 1 H) 4.79-4.85 (m, 1 H) 4.90 (br d, J=9.03 Hz, 2 H) 5.01 (s, 2 H) 5.43 (br s, 2 H), 5.47-5.59 (m, 1 H) 5.67-5.80 (m, 1 H) 5.81- 5.93 (m, 1 H) 5.94-6.14 (m, 2 H) 6.35-6.47 (m, 1 H) 6.99 (s, 2 H) 7.30 (d, J=8.66Hz, 2H) 7.58, (d, J=8.53Hz, 2H) 7.63-7.71(m, 1H) 7.98-8.06(m, 1H) 8.15-8.27(m, 1H) 9.99-10.09(m, 1H)

1.3.10. ADL1-D3, ADL10-D3

[1027] The payload D3 was obtained using the procedure below.

D3

[1028] To a mixture of (2S,3S,6S,7R,10R, E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R) -3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec- 4-en-6-ylpiperazin-1-carboxylate (55 mg, 0.083 mmol) in DCM (3 ml) was added (9H-fluoren-9-yl)methyl-(2-oxoethyl)carbamate (46.5 mg, 0.165 mmol ) and sodium triacetoxyborohydride (52.6 mg, 0.248 mmol). The reaction mixture was stirred for 20 minutes at rt. The reaction mixture was then concentrated to dryness and purified by silica gel column chromatography (0-10% MeOH/DCM). The purified material was dissolved in DMF (3 ml). The mixture was then charged with diethylamine (121 mg, 1.655 mmol) and stirred at rt. The mixture was then concentrated in vacuo and purified by reverse phase HPLC purification to give (2S,3S,6S,7R,10R,E)- 7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxirane -2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl-4-(2-aminoethyl)piperazin-1- carboxylate (6 mg, 8.48 µmol, 10.25% yield) as a white solid.

[1029] LCMS (ESI Mass/Charge), 708.2 [M+H] ⁺

[1030] ¹ H NMR (400 MHz, DMSO- d 6) δ ppm 0.74-0.86 (m, 9 H) 1.04-1.15 (m, 1 H) 1.23 (s, 3 H) 1.26-1.40(m, 3H) 1.45(s, 4H) 1.47-1.51(m, 1H), 1.52-1.63(m, 1H ) 1.69 (s, 3 H) 1.73-1.82 (m, 1 H) 1.99 (s, 3 H) 2.13-2.42 (m, 9 H) 2.53-2 .65 (m, 4 H) 2.72-2.80 (m, 1 H), 3.35-3.41 (m, 3 H) 3.65-3.76 (m, 1 H) 4, 36-4.46 (m, 1 H) 4.57-4.66 (m, 1 H) 4.79-4.85 (m, 1 H) 4.87-4.95 (m, 2 H) 5.43-5.56 (m, 1 H) 5.64-5.77 (m, 1 H) 5.80-5.91 (m, 1 H) 6.01-6.12 (m, 1 H) 6.33-6.49 (m, 1 H).

1.3.10.1 ADL10-D3

[1031] To a solution of (2S,3S,6S,7R,10R, E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R) -3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec- 4-en-6-yl-4-(2-aminoethyl)piperazin-1-carboxylate (40 mg, 0.057 mmol) in DMF (2 ml) was added 2,5-dioxopyrrolidin-1-yl-6-(2.5 -dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (27.9 mg, 0.09 mmol) followed by Hunig's base (36.5 mg, 0.283 mmol) and stirred for 20 minutes. The resulting mixture was then concentrated on silica gel and purified by column chromatography (0-20% MeOH/DCM) to give (2S,3S,6S,7R,10R, E)-7-acetoxy-10-hydroxy-2-((R ,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4 -dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl-4-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole -1-yl)hexanamido)ethyl)piperazine-1-carboxylate (22 mg, 43.2% yield) as a yellowish solid. LCMS (ESI mass/charge) 902.1 [M+H] ⁺

[1032] ¹ H NMR (400 MHz, DMSO-d6) δ ppm 0.73-0.89 (m, 10 H) 0.99-1.29 (m, 10 H) 1.30-1.38 ( m, 2 H) 1.40-1.49 (m, 9 H) 1.52-1.64 (m, 1 H), 1.69 (s, 3 H) 1.74-1.83 (m , 1 H) 1.93-2.07 (m, 5 H) 2.34 (br d, J=16.94 Hz, 9 H) 2.59 (s, 3 H) 3.07-3.21 (m, 2 H) 3.36 (br d, J=14.05 Hz, 3 H) 3.66-3.78 (m, 1 H) 4.37-4.45 (m, 1 H) 4 .57-4.66 (m, 1 H) 4.79-4.86 (m, 1 H) 4.87-4.97 (m, 2 H) 5.46-5.57 (m, 1 H) ) 5.66-5.75 (m, 1 H) 5.81-5.91 (m, 1 H) 5.99-6.13 (m, 1 H) 6.31-6.47 (m, 1H) 7.00(s, 2H) 7.62-7.71(m, 1H)

1.3.10.2 ADL1-D3

[1033] To a solution of (2S,3S,6S,7R,10R, E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R) -3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec- 4-en-6-yl-4-(2-aminoethyl)piperazine-1-carboxylate (30mg, 0.042mmol) in DMF (2mL) was added (9H-fluoren-9-yl)methyl((S)-3 -methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino) -1-oxobutan-2-yl)carbamate (35.7 mg, 0.047 mmol) followed by the addition of Hunig's base (16.43 mg, 0.127 mmol) and the mixture was allowed to stir for 1 hour. The resulting mixture was concentrated directly on silica gel and purified using silica gel column chromatography to give (2S,3S,6S,7R,10R, E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R ,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12 -oxooxacyclododec-4-en-6-yl-4-(2-((((4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy) carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)ethyl)piperazine-1-carboxylate, which was used directly in the next step. To a solution of (2S,3S,6S,7R,10R, E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3- ((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-ene -6-yl-4-(2-((((4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3 -methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)ethyl)piperazine-1-carboxylate (30 mg, 0.022 mmol) in DMF (2 ml) was added diethylamine (82 mg, 1.123 mmol) and the mixture was allowed to stir in for 1 hour at 20°C. The resulting mixture was diluted with ethyl acetate and concentrated in vacuo. The resulting precipitate was then redissolved in DMF and 2,5-dioxopyrrolidin-1-yl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (8.31 mg, 0.027 mmol) and Hunig's base (8.71 mg, 0.067 mmol). The resulting mixture was stirred for 1 hour at 20˚C. The solution was then concentrated on silica gel and purified by column chromatography (0-20% MeOH/DCM) to give (2S,3S,6S,7R,10R, E)-7-acetoxy-10-hydroxy-2-((R, 2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4- dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl-4-(2-((((4-((S)-2-((S)-2- (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)ethyl)piperazin-1- carboxylate (25 mg, 0.019 mmol, 85% yield) as a white solid; LC/MS (ESI, mass/charge), 1306.2 [M] ⁺ .

[1034] ¹ H NMR (400 MHz, DMSO- d ₆ ) δ ppm 0.75-0.89 (m, 18 H) 1.05-1.12 (m, 1 H) 1.15-1.20 (m, 1 H) 1.21-1.26 (m, 4 H) 1.26-1.39 (m, 5 H) 1.40-1.40 (m.1 H) 1.42-1 .47 (m, 5 H) 1.57 (br s, 3 H) 1.69 (s, 4 H) 1.73-1.81 (m, 1 H) 1.92-1.98 (m, 1 H) 1.99 (d, J =4.39 Hz, 4 H) 2.14-2.26 (m, 1 H) 2.29-2.42 (m, 4H) 2.42-2, 47 (m, 1 H) 2.53-2.64 (m, 2 H) 2.73-2.78 (m, 1 H) 2.88-3.08 (m, 2 H) 3.35- 3.60 (m, 19 H) 3.65-3.75 (m, 1 H) 4.22 (dd, J = 8.47, 6.71 Hz, 1H) 4.32-4.45 (m , 2 H) 4.60-4.65 (m, 1 H) 4.79-4.85 (m, 1 H) 4.90 (br d, J =9.03 Hz, 2 H) 5.01 (s, 2H) 5.40 (s, 2H) 5.47-5.58 (m, 1H) 5.66-5.77 (m, 1H) 5.87 (s, 1H) 5 .97 (s, 1 H) 6.03-6.13 (m, 1 H) 6.35-6.45 (m, 1 H) 7.02 (s, 2 H) 7.30 (d, J = 8.53 Hz, 2 H) 7.59 (d, J = 8.53 Hz, 2 H) 7.81-7.88 (m, 1 H), 8.09-8.15 (m, 1 H) 9.90-10.09(m, 1H)

1.3.11 ADL1-D6 and ADL1-D7 were prepared using the general procedure described below.

[1035] To a solution of (2S,3S,6S,7R,10R, E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R) -3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec- 4-en-6-ylpiperazin-1-carboxylate (30 mg, 0.045 mmol) in DCM (0.1 M) was added (9H-fluoren-9-yl)methyl(2-oxoethyl)carbamate (5 eq.) and triacetoxyborohydride sodium (10 eq.) and stirred for 30 minutes. The mixture was then diluted with ethyl acetate and washed with water and brine. The organic layer was dried over anhydrous Na ₂ SO ₄ , concentrated and purified by column chromatography (0-10% MeOH/DCM gradient) to isolate the desired product. The resulting material was then diluted with DMF (0.1 M), loaded with diethylamine (20 eq.) and stirred for 20 minutes. The mixture was then concentrated to dryness and diluted again in DMF and charged with 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1 -yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl)carbonate (1.2 eq) followed by Hunig's base (3.0 eq). The mixture was stirred for 30 minutes, concentrated in vacuo and purified by purification using preparative HPLC to obtain the desired product.

1.3.11.2 ADL1-D7

[1036] General procedure 1 (1.3.11) gave (2S,3S,6S,7R,10R, E)-7,10-dihydroxy-2-((R,2E,4E)-6-hydroxy-7 -((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3.7 -dimethyl-12-oxooxacyclododec-4-en-6-yl-4-(2-(((4-((S)-2-((S)-2-(6-(2,5-dioxo-2 ,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)phenoxy)carbonyl)(methyl)amino)ethyl)piperazine-1-carboxylate (3.1 mg, yield 5, 03%). LC/MS (ESI, mass/charge), 1278.4 [M+H] ⁺ .

1.3.11.2 ADL1-D6

[1037] General procedure 1 (1.3.11) gave (2S,3S,6S,7R,10R, E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy -7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3 ,7-dimethyl-12-oxooxacyclododec-4-en-6-yl-4-(2-(((4-((S)-2-((S)-2-(6-(2,5-dioxo -2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)phenoxy)carbonyl)(methyl)amino)ethyl)piperazin-1-carboxylate (2 mg, yield 3, 36%) LC/MS (ESI, mass/charge), 1321.37 [M+H] ⁺ .

1.3.1.5 ADL1-D9, ADL6-D9 and ADL1-D13

D9 and D13

[1038] D9 and D13 were synthesized as a 3:1 mixture of isomers using the procedures outlined in Synthesis of D4 (Scheme 4) using tri-TES-pladienolide B.

[1039] Payload (D9) : LC/MS (ESI Mass/Charge), 649.7 [M+H] ⁺ .

[1040] ¹ H-NMR (400 MHz, CHCl ₃ - d ): δ ppm 0.87-1.01 (m, 10 H) 1.08 (d, J=6.78 Hz, 3 H) 1, 27-1.63 (m, 12 H) 1.66-1.74 (m, 1 H) 1.76 (s, 3 H) 1.97-2.10 (m, 3 H) 2.35- 2.57 (m, 5 H) 2.58-2.65 (m, 1 H) 2.65-2.71 (m, 1 H) 2.77 (td, J=5.93, 2.32 Hz, 1 H) 2.89-3.05 (m, 4 H) 3.07-3.34 (m, 8 H) 3.50-3.68 (m, 5 H) 3.72-3, 88 (m, 1 H) 4.88-5.09 (m, 1 H) 5.18 (d, J=10.67 Hz, 1 H) 5.50-5.84 (m, 3 H) 6 .01-6.13 (m, 1 H) 6.19-6.36 (m, 1 H).

[1041] Payload (D13) : LC/MS (ESI Mass/Charge), 649.6 [M+H] ⁺ .

[1042] ¹ H NMR (400 MHz, CHCl ₃ - d ) δ ppm 0.84-1.01 (m, 11 H) 1.08 (d, J =6.78 Hz, 3 H) 1.29- 1.64 (m, 10 H) 1.63-1.73 (m, 1 H) 1.76 (s, 3 H) 1.94-2.12 (m, 4 H) 2.37-2, 58 (m, 4 H) 2.59-2.65 (m, 1 H) 2.68 (dd, J =7.40, 2.26 Hz, 1 H) 2.77 (td, J =5, 93, 2.32 Hz, 1 H) 3.01-3.30 (m, 4 H) 3.49 (s, 1 H) 3.54-3.67 (m, 2 H) 3.69-3 .92 (m, 5 H) 4.13-4.78 (m, 11 H) 5.13-5.24 (m, 2 H) 5.48-5.61 (m, 1 H) 5.62 -5.74 (m, 2 H) 6.04-6.13 (m, 1 H) 6.18-6.32 (m, 1 H).

ADL1-D9

[1043] General procedure 1 (specified in section 1.3.1) was used to synthesize ADL1-D9.

[1044] Linker payload (ADL1-D9) : (30.5 mg, 0.024 mmol, 77% yield). LC/MS (ESI, mass/charge), 1263.8 [M+H] ⁺ .

[1045] ¹ H-NMR (400 MHz, MeOH- d ₄ ): δ ppm 0.87-0.93 (m, 7 H) 0.93-1.01 (m, 8 H) 1.19-1 .34 (m, 4 H) 1.50 (s, 3 H) 1.57 (s, 5 H) 1.58-1.70 (m, 6 H) 1.70-1.77 (m, 1 H) 1.78 (s, 3 H) 1.83-1.95 (m, 2 H) 2.04 (s, 3 H) 2.05-2.13 (m, 1 H) 2.27 ( t, J = 7.40 Hz, 2 H) 2.32-2.42 (m, 1 H) 2.50 (d, J = 3.64 Hz, 2 H) 2.55-2.74 (m , 2 H) 2.90 (td, J = 5.83, 2.26 Hz, 1 H) 3.03-3.26 (m, 2 H) 3.35 (s, 13 H) 3.42- 3.61 (m, 11 H) 3.80 (br dd, J =9.85, 3.58 Hz, 1 H) 4.16 (d, J =7.40 Hz, 1 H) 4.50 ( dd, J = 8.91, 5.14 Hz, 1 H) 4.56 (s, 1 H) 5.05 (dd, J = 14.37, 10.10 Hz, 2 H) 5.09 (s , 2 H) 5.49 (s, 1 H) 5.59-5.69 (m, 1 H) 5.72-5.80 (m, 1 H) 5.87 (d, J =15.18 Hz, 1 H) 6.09-6.22 (m, 1 H) 6.44-6.60 (m, 1 H) 7.32 (d, J =8.66 Hz, 2 H) 7.58 (d, J = 8.53 Hz, 2 H).

ADL6-D9

[1046] General procedure 1 (specified in section 1.3.1) was used to synthesize ADL6-D9.

[1047] Linker Payload (ADL6-D9) . To dilute 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido )benzyl (4-nitrophenyl) carbonate (16.5 mg, 0.025 mmol) in DMF (784 μl) Hunig's base (13.27 μl, 0.076 mmol) was added. The reaction mixture was cooled to 0°C and added D9. The reaction mixture was stirred at RT until completion of the reaction was determined by LC/MS. The reaction mixture was concentrated in vacuo . Flash chromatography of the residue on silica gel with DCM/MeOH afforded the title compound (16.7 mg, 57% yield). LC/MS (ESI mass/charge), 1184.6 [M+Na] ⁺ .

[1048] ¹ H-NMR (400 MHz, MeOH- d ₄ ): δ ppm 0.80-1.02 (m, 16 H) 1.05-1.12 (m, 4 H) 1.13-1 .34 (m, 5 H) 1.38-1.51 (m, 7 H) 1.53-1.68 (m, 11 H) 1.71-1.80 (m, 4 H) 1.97 -2.16 (m, 4 H) 2.24-2.32 (m, 2 H) 2.32-2.42 (m, 1 H) 2.43-2.55 (m, 3 H) 2 .56-2.69 (m, 3 H) 2.73 (br d, J = 2.13 Hz, 1 H) 3.07-3.18 (m, 1 H) 3.42-3.61 ( m, 17 H) 3.75-3.91 (m, 1 H) 4.11-4.24 (m, 1 H) 4.43 (s, 2 H) 5.59-5.88 (m, 4 H) 6.06-6.17 (m, 1 H) 6.29-6.39 (m, 1 H) 7.24-7.39 (m, 2 H) 7.52-7.64 ( m, 2H).

ADL1-D13.

[1049] Linker Payload (AD1-D13) . K 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5 -ureidopentanamido)benzyl(4-nitrophenyl)carbonate (4.2 mg, 5.693 µmol) in DMF (176 µl, 2.277 mmol) Hunig's base (2.98 µl, 0.017 mmol) was added. The reaction mixture was cooled to 0°C and D13 (4.06 mg, 6.262 µmol) was added. The reaction mixture was stirred at RT until completion of the reaction was determined by LC/MS. The reaction mixture was concentrated in vacuo . Flash chromatography of the residue on silica gel with DCM/MeOH gave the title compound (4.8 mg, 68% yield). LC/MS (ESI mass/charge), 1248.0 [M+H] ⁺ .

[1050] ¹ H-NMR (400 MHz, CHCl ₃ - d ): δ ppm 0.58-0.98 (m, 10 H) 0.99-1.06 (m, 2 H) 1.11-1 .31 (m, 6 H) 1.57-1.75 (m, 3 H) 2.11-2.26 (m, 1 H) 2.30-2.73 (m, 2 H) 3.28 -3.80 (m, 7 H) 4.14-4.33 (m, 1 H) 4.44-4.55 (m, 1 H) 4.63-4.81 (m, 1 H) 4 .92-5.06 (m, 1 H) 5.09 (s, 1 H) 5.49-5.74 (m, 1 H) 5.88-6.09 (m, 1 H) 6.14 -6.36 (m, 1 H) 6.99-7.08 (m, 1 H) 7.21-7.38 (m, 2 H) 7.53-7.67 (m, 1 H) 7 .95 (s, 1H).

1.3.1.6 ADL1-D14

[1051] General procedure 1 (specified in section 1.3.1) was used to synthesize 1-((2S,3S,6R,7S,10R, E)-2-((E)-1-(1-(1-acetylpiperidine -4-yl)-4-fluoro-1H-benzo[d][1,2,3]triazol-6-yl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl- 12-oxooxacyclododec-4-en-6-yl)-4-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)piperazin-1,4-dicarboxylate (30 mg, 0.024 mmol, 39.2% yield). LC/MS (ESI, mass/charge), 1254.5 [M+H] ⁺ .

[1052] ¹ H-NMR (400 MHz, DMSO- d ₆ ): δ ppm 0.75-1.00 (m, 12 H), 1.06-1.26 (m, 4 H), 1.26 -1.77(m, 13H), 1.75-2.02(m, 7H), 2.08(s, 8H), 2.23-2.35(m, 2H), 2 .54-2.70 (m, 2 H), 2.78-3.10 (m, 4 H), 3.36 (br s, 13 H), 3.66-3.79 (m, 1 H ), 3.95-4.08 (m, 1 H), 4.11-4.25 (m, 1 H), 4.28-4.46 (m, 1 H), 4.46-4, 58 (m, 1 H), 4.58-4.66 (m, 1 H), 4.66-4.77 (m, 1 H), 5.01 (s, 3 H), 5.14- 5.27(m, 1H), 5.39(s, 4H), 5.71-5.79(m, 1H), 5.87-6.00(m, 1H), 6, 63-6.75(m, 1H), 6.99(s, 2H), 7.13-7.24(m, 1H), 7.24-7.34(m, 2H), 7.52-7.62(m, 2H), 7.65-7.74(m, 1H), 7.75-7.82(m, 1H), 7.98-8.13( m, 1H).

1.3.1.7 ADL1-D33

D33

[1053] To a stirred solution of (2S,3S,6R,7S,10R, E)-2-((E)-1-(3-fluoro-5-(4-(2-methoxy-2-oxoethyl)piperazine- 1-yl)phenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazin-1-carboxylate (27 mg, 0.042 mmol) in THF/H ₂ O (3 ml/1 ml) at 0°C was added LiOH and provided a slow heating of the reaction mixture to 25°C. Synthesized (2S,3S,6R,7S,10R, E)-2-((E)-1-(3-fluoro-5-(4-(2-methoxy-2-oxoethyl)piperazin-1-yl)phenyl )prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazin-1-carboxylate using the procedures specified in International Patent Application No. PCT/ US2019/026992 (see, for example, procedures 17 and 19), which is incorporated herein by reference. After 6 hours the reaction mixture was neutralized with phosphate buffer (NaH ₂ PO ₄ , 1.0 M, 3 ml) and the phases were separated. The aqueous layer was extracted with ethyl acetate (3×2 ml) and the combined organic layers were dried with anhydrous sodium sulfate and concentrated in vacuo . Reverse phase column chromatography afforded the title compound (19.4 mg, 0.031 mmol, 73.4% yield). LC/MS (ESI, mass/charge), 631.4 [M+H] ⁺ .

[1054] ¹ H-NMR (400 MHz, CHCl ₃ - d ): δ ppm 0.85-1.16 (m, 2 H) 1.23-1.52 (m, 1 H) 1.57-1 .77 (m, 1 H) 1.87 (s, 1 H) 1.92-2.13 (m, 1 H) 2.35-2.55 (m, 1 H) 2.50-2.74 (m, 1 H) 3.11 (br s, 1 H) 3.36-3.54 (m, 2 H) 3.64 (br d, J =10.42 Hz, 2 H) 3.77- 3.90 (m, 1 H) 4.76 (br s, 4 H) 5.07-5.19 (m, 1 H) 5.38-5.68 (m, 1 H) 6.40-6 .76 (m, 1 H) 8.02-8.66 (m, 1 H).

ADL1-D33.

[1055] Linker Payload (ADL1-D33) . K 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5 -ureidopentanamido)benzyl(4-nitrophenyl)carbonate (17 mg, .023 mmol) in DMF (714 μl, 9.217 mmol) Hunig's base (12.07 μl, 0.069 mmol) was added. The reaction mixture was cooled to 0°C and 2-(4-(3-fluoro-5-((E)-2-((2S,3S,6R,7S,10R, E)-10-hydroxy-3.7 -dimethyl-12-oxo-6-((piperazin-1-carbonyl)oxy)oxacyclododec-4-en-2-yl)prop-1-en-1-yl)phenyl)piperazin-1-yl)acetic acid ( 16.71 mg, 0.026 mmol). The reaction mixture was stirred at RT until completion of the reaction was determined by LC/MS. The reaction mixture was then concentrated in vacuo . Flash chromatography on silica gel and further purification by HPLC gave the title compound (13.5 mg, 10.98 µmol, 47.7% yield). LC/MS (ESI, mass/charge), 1229.5 [M+H] ⁺ .

[1056] ¹ H-NMR (400 MHz, MeOH -d ₄ ): δ ppm 0.92-0.99 (m, 10 H) 1.01 (d, J =6.78 Hz, 3 H) 1, 24-1.46 (m, 9 H) 1.52-1.70 (m, 10 H) 1.70-1.82 (m, 1 H) 1.87 (d, J =1.00 Hz, 4 H) 1.89-2.00 (m, 2 H) 2.01-2.23 (m, 2 H) 2.27 (t, J =7.40 Hz, 2 H) 2.46 (dd , J =14.31, 5.27 Hz, 1 H) 2.57-2.71 (m, 2 H) 2.86 (d, J = 0.63 Hz, 1 H) 3.00 (s, 1 H) 3.06-3.27 (m, 4 H) 3.35 (s, 5 H) 3.41-3.53 (m, 19 H) 3.66 (s, 2 H) 3.76 -3.90 (m, 1 H) 4.07-4.21 (m, 1 H) 4.39-4.69 (m, 37 H) 5.09 (s, 3 H) 5.13 (d , J = 10.54 Hz, 1 H) 5.52 (dd, J = 14.56, 9.03 Hz, 2 H) 6.55 (br s, 2 H) 6.69 (d, J = 1 .38 Hz, 2 H) 7.33 (d, J = 8.66 Hz, 2 H) 7.59 (d, J = 8.53 Hz, 2 H) 7.88-8.04 (m, 1 H) 8.16-8.33 (m, 1 H).

ADL1-D28.

[1057] 4-((S)-2-((S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido) -5-ureidopentanamido)benzyl(4-nitrophenyl)carbonate (6.17 mg, 8.37 µmol) and (2S,3S,6R,7S,10R, E)-2-((E)-1-(3- fluoro-5-(4-((1-methyl-1H-imidazol-4-yl)sulfonyl)piperazin-1-yl)phenyl)prop-1-en-2-yl)-10-hydroxy-3,7- dimethyl 12-oxooxacyclododec-4-en-6-ylpiperazin-1-carboxylate (6 mg, 8.37 µmol) was dissolved in DMF (0.3 ml) and Hunig's base (4.39 µl, 0.025 mmol) was added. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated and purified by reverse phase HPLC to give 1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-((2S,3S,6R,7S,10R, E)-2-((E)-1-(3- fluoro-5-(4-((1-methyl-1H-pyrazol-4-yl)sulfonyl)piperazin-1-yl)phenyl)prop-1-en-2-yl)-10-hydroxy-3,7- dimethyl 12-oxooxacyclododec-4-en-6-yl)piperazin-1,4-dicarboxylate (1.5 mg, 13.6%). LC/MS (ESI, mass/charge), 1315.66 [M+H] ⁺ .

[1058] ¹ H NMR (400 MHz, METHANOL-d4) d ppm 0.85-0.93 (m, 12 H) 1.16-1.31 (m, 6 H) 1.43-1.57 ( m, 9 H) 1.62-1.70 (m, 1 H) 1.75 (s, 3 H) 1.77-1.90 (m, 2 H) 1.96-2.03 (m, 1 H) 2.13-2.20 (m, 2 H) 2.31-2.40 (m, 1 H) 2.46-2.55 (m, 2 H) 2.97-3.06 ( m, 2 H) 3.08-3.10 (m, 1 H) 3.15 (br d, J=6.27 Hz, 9 H) 3.33-3.43 (m, 10 H) 3, 71 (s, 4 H) 4.03-4.09 (m, 1 H) 4.37-4.44 (m, 1 H) 4.44-4.53 (m, 1 H) 4.96- 5.06 (m, 3 H) 5.33-5.51 (m, 2 H) 6.36-6.44 (m, 2 H) 6.45-6.53 (m, 2 H) 6, 69 (s, 2H) 7.19-7.25 (m, 2H) 7.47-7.52 (m, 2H) 7.63-7.70 (m, 2H)

ADL1-D31.

[1059] 4-((S)-2-((S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido) -5-ureidopentanamido)benzyl(4-nitrophenyl)carbonate (13.12 mg, 0.018 mmol) and (2S,3S,6R,7S,10R, E)-2-((E)-1-(5-chloropyridine- 3-yl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazin-1-carboxylate (9 mg, 0.018 mmol) was dissolved in DMF (179 µl, 2.312 mmol) and Hunig's base (9.32 µl, 0.053 mmol) was added. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated and purified by reverse phase HPLC to give 1-((2S,3S,6R,7S,10R, E)-2-((E)-1-(5-chloropyridin-3-yl)prop-1- en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)-4-(4-((S)-2-((S)-2-( 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)piperazine-1,4-dicarboxylate (6.9 mg, 34.09%). LC/MS (ESI, mass/charge), 1105.59 [M+H] ⁺ .

[1060] 1H NMR (400 MHz, METHANOL-d4) d ppm 0.88 (s, 12 H) 1.17-1.31 (m, 4 H) 1.43-1.57 (m, 8 H) 1.61-1.70 (m, 1 H) 1.80 (d, J=1.25 Hz, 5 H), 1.92-2.01 (m, 1 H) 2.14-2.20 (m, 2 H) 2.32-2.42 (m, 1 H) 2.48-2.59 (m, 2 H) 2.96-3.14 (m, 3 H) 3.38 (s , 10 H) 3.36-3.36 (m, 1 H) 3.38-3.40 (m, 3 H) 3.67-3.75 (m, 1 H) 4.03-4.07 (m, 1 H) 4.37-4.42 (m, 1 H) 4.97-5.01 (m, 2 H) 5.04-5.11 (m, 1 H) 5.35-5 .51 (m, 2H) 6.46-6.52, (m, 1H) 6.69 (s, 2H) 7.18-7.26 (m, 2H) 7.45-7, 54 (m, 2 H) 7.68-7.74 (m, 1 H) 8.29-8.34 (m, 2 H).

ADL1-D29.

[1061] 4-((S)-2-((S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido) -5-ureidopentanamido)benzyl(4-nitrophenyl)carbonate (8.62 mg, 0.012 mmol) and (2S,3S,6R,7S,10R, E)-2-((E)-1-(3-(dimethylamino )phenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazin-1-carboxylate (6 mg, 0.012 mmol) was dissolved in DMF ( 118 µl, 1.518 mmol) and Hunig's base (6.12 µl, 0.035 mmol). The reaction mixture was stirred at room temperature overnight. The solvent was evaporated and purified by reverse phase HPLC to give 1-((2S,3S,6R,7S,10R, E)-2-((E)-1-(3-(dimethylamino)phenyl)prop-1-ene -2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)-4-(4-((S)-2-((S)-2-(6 -(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)piperazine-1,4-dicarboxylate (4.0 mg, 30 .79%). LC/MS (ESI, mass/charge), 1112.59 [M+H] ⁺ .

[1062] ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ ppm 0.87 (s, 12 H) 1.17-1.23 (m, 3 H) 1.26-1.32 (m, 1 H) 1.44-1.57 (m, 8 H) 1.63-1.69 (m, 1 H) 1.74-1.81 (m, 4 H) 1.83-1.88 (m , 1 H) 1.93-2.03 (m, 1 H) 2.13-2.20 (m, 2 H) 2.34-2.42 (m, 1 H) 2.48-2.57 (m, 2H) 2.81 (s, 5H) 2.98-3.05 (m, 2H) 3.08-3.14 (m, 1H) 3.38 (br s, 9H) 3.65-3.75 (m, 1 H) 4.03-4.07 (m, 1 H) 4.35-4.43 (m, 1 H) 4.44-4.52 (m, 1 H) 4.96-5.01(m, 2H) 5.02-5.08(m, 1H) 5.34-5.51(m, 2H) 6.43-6.48(m , 1 H) 6.51-6.61 (m, 3 H) 6.69 (s, 2 H) 7.03-7.10 (m, 1 H) 7.20-7.26 (m, 2 H) 7.46-7.51 (m, 2H).

ADL1-D35.

[1063] 4-((S)-2-((S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido) -5-ureidopentanamido)benzyl-(4-nitrophenyl)carbonate (9.78 mg, 0.013 mmol) and (2S,3S,6R,7S,10R, E)-10-hydroxy-3,7-dimethyl-12-oxo -2-((E)-1-(3-(pyrrolidin-1-ylsulfonyl)phenyl)prop-1-en-2-yl)oxacyclododec-4-en-6-ylpiperazin-1-carboxylate (8 mg, 0.013 mmol) was dissolved in DMF (133 µl, 1.722 mmol) and Hunig's base (6.94 µl, 0.04 mmol) was added [two drops]. The resulting mixture was stirred at rt overnight, concentrated in vacuo and purified by reverse phase HPLC to give the product, 1-(4-((S)-2-((S)-2-(6-(2, 5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-((2S,3S,6R,7S,10R, E)- 10-hydroxy-3,7-dimethyl-12-oxo-2-((E)-1-(3-(pyrrolidin-1-ylsulfonyl)phenyl)prop-1-en-2-yl)oxacyclododec-4-ene -6-yl)piperazin-1,4-dicarboxylate (8.2 mg, 51.47%). LC/MS (ESI, mass/charge), 1203.53 [M+H] ⁺ .

[1064] 1H NMR (400 MHz, METHANOL-d4) d ppm 0.85-0.93 (m, 12 H) 1.17-1.34 (m, 4 H) 1.42-1.57 (m , 8 H) 1.61-1.69 (m, 5 H) 1.79 (s, 4 H) 1.83-1.90 (m, 1 H) 1.94-2.03 (m, 1 H) 2.12-2.20 (m, 2 H) 2.33-2.43 (m, 1 H) 2.48-2.59 (m, 2 H) 2.96-3.05 (m , 2 H) 3.06-3.17 (m, 6 H) 3.31 -3.41 (m, 10 H) 3.67-3.77 (m, 1 H) 4.02-4.10 (m, 1 H) 4.36-4.43 (m, 1 H) 4.96-5.02 (m, 2 H) 5.04-5.11 (m, 1 H) 5.35-5 .51 (m, 2H) 6.59 (s, 1H) 6.69 (s, 2H) 7.20-7.27 (m, 2H) 7.45-7.52 (m, 4H ) 7.58-7.65 (m, 2 H).

1.4 Obtaining the linker construct MC-Val-Ala-pABC-Payloads

1.4.1 Overview

Scheme 5

[1065] Step 1 . Macrocycle payload (1.0 eq.), DMF (0.1 M), (9H-fluoren-9-yl)methyl-((S)-3-methyl-1-(((S)-1 -((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)carbamate (1.5 eq. ; prepared using the procedure described in WO 2012/153193) and Hunig's base (3.0 eq.) were combined and stirred overnight. The reaction mixture was concentrated to dryness and chromatographed to give the fmoc-Val-Ala-pABC-payload construct.

[1066] Step 2 . The ammonium-fmoc-Val-Ala-pABC-payload construct (1.0 eq.) dissolved in DMF (0.1 M) and diethylamine (10 eq.) were combined and stirred for 30 minutes. The reaction mixture was concentrated to dryness under high vacuum. The crude product was dissolved in DMF (0.1 M) and Hunig's base (2 eq) was added, after which 2,5-dioxopyrrolidin-1-yl-6-(2,5-dioxo-2,5-dihydro-1H -pyrrol-1-yl)hexanoate (3 eq) and stirred overnight. The reaction mixture was then concentrated to dryness and chromatographed (0-30% MeOH in DCM) to give the desired MC-Val-Ala-pABC-payload construct.

1.4.1.1 ADL6-D1

[1067] Scheme 5, step 1 . According to the general procedure above (section 1.4.1), 1-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino )-3-methylbutanamido)propanamido)benzyl)-4-((2S,3S,6S,7R,10R, E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-( (2S,3S)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3 ,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)piperazin-1,4-dicarboxylate (32 mg, 0.027 mmol, 27.9% yield). LC/MS (ESI, mass/charge), [M+Na] ⁺ 1200.5.

[1068] Scheme 5, step 2 . According to the general procedure above (section 1.4.1), 1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H -pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl)-4-((2S,3S,6S,7R,10R, E)-10-hydroxy-2-((R,2E,4E) -6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2- yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)piperazin-1,4-dicarboxylate (24 mg, 0.021 mmol, 77% yield). LC/MS (ESI, mass/charge), 1149.2 [M+H] ⁺ .

[1069] ¹ H-NMR (400 MHz, MeOH- d 4 ): δ ppm 0.85-1.02 (m, 16 H), 1.20-1.23 (m, 3 H) 1.23- 1.32 (m, 3 H) 1.34 (s, 3 H) 1.41-1.46 (m, 4 H) 1.46-1.72 (m, 11 H) 1.78 (s, 3 H) 1.87 (dd, J=14.05, 5.40 Hz, 1 H) 2.08 (dq, J=13.77, 6.83 Hz, 1 H) 2.27 (t, J =7.40 Hz, 2 H) 2.43-2.63 (m, 3 H) 2.64-2.70 (m, 1 H) 2.90 (td, J=5.83, 2.26 Hz, 1 H) 3.32-3.33 (m, 4 H) 3.42-3.57 (m, 11 H) 3.82 (br dd, J=8.66, 3.89 Hz, 1 H) 4.15 (d, J=7.15 Hz, 1 H), 4.47 (q, J=7.15 Hz, 1 H) 4.99-5.12 (m, 4 H) 5, 57 (dd, J=15.18, 9.79 Hz, 1 H) 5.69-5.78 (m, 1 H) 5.87 (d, J=15.18 Hz, 1 H) 6.13 (d, J=10.92 Hz, 1 H), 6.53 (dd, J=15.31, 10.92 Hz, 1 H) 6.78 (s, 2 H) 7.32 (d, J =8.53 Hz, 2 H) 7.58 (d, J=8.53 Hz, 2 H).

1.4.1.2 ADL6-D16

[1070] Scheme 5, step 1 . According to the general procedure above (Section 1.4.1), 1-((2S,3S,6R,7S,10R, E)-2-((E)-1-(3-(4-(2-( 1H-pyrazol-1-yl)acetyl)piperazin-1-yl)-5-fluorophenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en- 6-yl)-4-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl )piperazine-1,4-dicarboxylate (0.060 g, 0.049 mmol, 60.8% yield). LC/MS (ESI mass/charge), 1223.68 [M+H].

[1071] Scheme 5, step 2 . According to the procedure above (Section 1.4.1), 1-((2S,3S,6R,7S,10R, E)-2-((E)-1-(3-(4-(2-(1H -pyrazol-1-yl)acetyl)piperazin-1-yl)-5-fluorophenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6 -yl)-4-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)- 3-methylbutanamido)propanamido)benzyl)piperazine-1,4-dicarboxylate (11.50 mg, 9.64 µmol, 12.05% yield). LC/MS (ESI, mass/charge), 1194.82 [M+H] ⁺ .

[1072] ¹ H-NMR (400 MHz, CHCl ₃ - d ): δ ppm 0.97 (br d, J=1.25 Hz, 12 H), 1.24-1.41 (m, 5 H) , 1.41-1.47(m, 3H), 1.52-1.69(m, 6H), 1.84-1.90(m, 3H), 1.92-2.01 (m, 1 H), 2.02-2.16 (m, 1 H), 2.23-2.30 (m, 2 H), 2.42-2.51 (m, 1 H), 2 .57-2.65(m, 2H), 3.19-3.28(m, 4H), 3.35(s, 4H), 3.40-3.54(m, 11H) , 3.69-3.77(m, 4H), 3.78-3.85(m, 1H), 4.12-4.18(m, 1H), 4.43-4.51 (m, 1 H), 5.07-5.16 (m, 3 H), 5.20 (s, 2 H), 5.42-5.62 (m, 2 H), 6.29-6 .38 (m, 1 H), 6.46-6.58 (m, 2 H), 6.59-6.68 (m, 2 H), 7.28-7.37 (m, 2 H) , 7.50-7.67 (m, 4H).

1.5 Obtaining the construct linker MC-Val-Ala-pAB-payloads

1.5.1 Overview

Scheme 6

[1073] Step 1 . (9H-Fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3- methyl 1-oxobutan-2-yl) carbamate (0.5 g, 0.97 mmol), 1,4-dioxane (9.70 ml, 0.97 mmol), triphenylphosphine (0.509 g, 1.939 mmol), N -bromosuccinimide (0.345 g, 1.939 mmol) and DMF (2.424 ml, 0.97 mmol) were combined and stirred for 3 hours at rt. The reaction mixture was concentrated and chromatographed to give (9H-fluoren-9-yl)methyl( (S)-1-(((S)-1-((4-(bromomethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (116 mg, 0.201 mmol, 20.68% yield). LC/MS (ESI, mass/charge), 580.14 [M+H].

[1074] Step 2 . Payload (1.0 eq.), (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(bromomethyl)phenyl)amino)-1-oxopropane -2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (0.069 g, 0.12 mmol), N,N-dimethylformamide (0.1 M) and Hunig's base (1.5 eq. .) were combined and stirred overnight. The reaction mixture was concentrated to dryness and chromatographed (0-10% MeOH in DCM) to give the quaternary ammonium-fmoc-Val-Ala-pAB-payload construct.

[1075] Step 3 . The quaternary ammonium-fmoc-Val-Ala-pAB-payload (1.0 eq.) construct dissolved in DMF (0.1 M) and diethylamine (10 eq.) were combined and stirred for 30 minutes. The reaction mixture was concentrated to dryness under high vacuum. The crude product was dissolved in DMF (0.1 M) and Hunig's base (2 eq) was added, after which 2,5-dioxopyrrolidin-1-yl-6-(2,5-dioxo-2,5-dihydro-1H -pyrrol-1-yl)hexanoate (3 eq) and stirred overnight. The reaction mixture was then concentrated to dryness and chromatographed (0-30% MeOH in DCM) to give the desired quaternary ammonium-payload linker design.

1.5.1.1 ADL5-D2

[1076] Scheme 6, step 2 . 1-(4-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)-4-( (((2S,3S,6S,7R,10R, E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2S,3S)-3-((2R, 3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-ene -6-yl)oxy)carbonyl)-1-methylpiperazin-1-ium bromide (30 mg, 0.024 mmol, 26.5% yield). LC/MS (ESI, mass/charge), 1149.77 [M+H] ⁺ .

[1077] Scheme 6, step 3 . Mono(1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3- methylbutanamido)propanamido)benzyl)-4-((((2S,3S,6S,7R,10R, E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2S ,3S)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7 -dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-ium-2-yl)monobromide (4.8 mg, 15.33% yield). LC/MS (ESI, mass/charge), 1120.99 [M+H] ⁺ .

[1078] ¹ H-NMR (400 MHz, MeOH- d 4 ): δ ppm 0.88 (s, 15 H), 1.09-1.26 (m, 9 H), 1.31-1.58 (m, 14 H), 1.42-1.44 (m, 1 H), 1.68 (s, 3 H), 1.73-1.81 (m, 1 H), 1.91-2 .03 (m, 1 H), 2.14-2.22 (m, 2 H), 2.35-2.52 (m, 3 H), 2.53-2.60 (m, 1 H) , 2.74-2.83(m, 1H), 2.97(s, 3H), 3.25(s, 1H), 3.28-3.46(m, 8H), 3 .52-3.66(m, 2H), 3.70-3.78(m, 1H), 3.97-4.11(m, 3H), 4.32-4.41(m , 1 H), 4.45-4.54 (m, 2 H), 4.91-5.01 (m, 2 H), 5.44-5.55 (m, 1 H), 5.57 -5.70(m, 1H), 5.72-5.82(m, 1H), 6.00-6.08(m, 1H), 6.43(dd, J=15.25 , 10.98 Hz, 1 H), 6.69 (s, 2 H), 7.36-7.47 (m, 2 H), 7.67-7.75 (m, 2 H).

1.5.1.2 ADL5-D19

[1079] The general procedure above (section 1.5.1) was used to synthesize ADL5-D19. The D19 payload was synthesized using the procedures outlined in section 1.2.1.

D19

[1080] (2S,3S,6S,7R,10R, E)-10-Hydroxy-2-((S,2E,4E)-7-((2R,3R)-3-((2R,3S)- 3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6- yl-4-methylpiperazine-1-carboxylate (114.2 mg, 0.180 mmol, 82% yield). LC/MS (ESI, mass/charge), 635.8 [M+H].

[1081] ¹ H-NMR (400 MHz, MeOH -d 4 ): δ ppm 0.88-1.00 (m, 9 H) 1.10 (d, J =6.65 Hz, 3 H) 1, 16-1.27 (m, 4 H) 1.40-1.70 (m, 8 H) 1.77 (d, J =0.88 Hz, 3 H) 2.28-2.36 (m, 3 H) 2.42 (br t, J = 5.08 Hz, 3 H) 2.47-2.61 (m, 4 H) 2.68 (dd, J = 8.22, 2.20 Hz, 1 H) 2.74 (td, J =5.99, 2.20 Hz, 1 H) 3.13 -3.17 (m, 1 H) 3.34-3.38 (m, 3 H) 3 .47-3.58 (m, 5H) 3.81-3.87 (m, 1H) 5.01-5.10 (m, 2H) 5.54-5.81 (m, 3H ) 6.06-6.15 (m, 1 H) 6.34 (dd, J =15.00, 10.98 Hz, 1 H).

[1082] 1-(4-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)- 4-((((2S,3S,6S,7R,10R, E)-10-hydroxy-2-((S,2E,4E)-7-((2R,3R)-3-((2R,3S )-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en- 6-yl)oxy)carbonyl)-1-methylpiperazin-1-y (25 mg, 45.6%). LC/MS (ESI, mass/charge), 1133.1 [M+] ⁺ .

[1083] ¹ H-NMR (400 MHz, MeOH -d 4): δ ppm 0.94 (br dd, J =18.45, 7.15 Hz, 18 H) 1.10 (d, J =6, 78 Hz, 3 H) 1.25 (s, 4 H) 1.28-1.35 (m, 1 H) 1.46 (br d, J =7.03 Hz, 9 H) 1.59-1 .70 (m, 2 H) 1.77 (s, 3 H) 2.05-2.12 (m, 1 H) 2.44-2.60 (m, 4 H) 2.63-2.72 (m, 1 H) 2.70-2.77 (m, 1 H) 3.06 (s, 3 H) 3.40-3.44 (m, 1 H) 3.50 (br d, J = 1.63 Hz, 4 H) 3.64-3.75 (m, 1 H) 3.80-3.88 (m, 1 H) 3.91-3.99 (m, 1 H) 4.04 -4.20 (m, 1 H) 4.21-4.28 (m, 1 H) 4.37-4.43 (m, 1 H) 4.59 (br s, 2 H) 4.93- 4.95 (m, 2H) 5.06-5.08 (m, 2H) 5.57-5.80 (m, 4H) 6.05-6.16 (m, 1H) 6, 27-6.41 (m, 1 H) 7.28-7.37 (m, 2 H) 7.38-7.45 (m, 2 H) 7.47-7.53 (m, 2 H) 7.65-7.72(m, 2H) 7.74-7.86(m, 4H).

ADL5-D19.

[1084] Linker Payload (ADL5-D19) . 1-(4-((S)-2-((S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido) propanamido)benzyl)-4-((((2S,3S,6S,7R,10R, E)-10-hydroxy-2-((S,2E,4E)-7-((2R,3R)-3- ((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec -4-en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-y (19.4 mg, 0.018 mmol, 80% yield). LC/MS (ESI, mass/charge), 1105.9 [M+H].

[1085] ¹ H NMR (400 MHz, MeOH -d 4 ): δ ppm 0.85-1.03 (m, 16 H) 1.10 (d, J =6.78 Hz, 3 H) 1.17 -1.27 (m, 4H) 1.29-1.37 (m, 3H) 1.44-1.70, (m, 15H) 1.74-1.80 (m, 3H) 2.04-2.14 (m, 1 H) 2.30 (t, J = 7.47 Hz, 2 H) 2.44-2.61 (m, 4 H) 2.64-2.69 ( m, 1 H) 2.72-2.78 (m, 1 H) 3.09 (s, 3 H) 3.40-3.59 (m, 9 H) 3.62-3.77 (m, 2 H) 3.82-3.89 (m, 1 H) 4.12-4.21 (m, 3 H) 4.40-4.55 (m, 1 H) 4.56-4.66 ( m, 3 H) 5.03-5.12 (m, 2 H) 5.55-5.81 (m, 3 H) 6.06-6.16 (m, 1 H) 6.26-6, 41 (m, 1 H) 6.81 (s, 2 H) 7.48-7.57 (m, 2 H) 7.78-7.86 (m, 2 H).

1.5.1.3 ADL5-D17

[1086] The general procedure above (section 1.5.1) was used to synthesize ADL5-D17. The D17 payload was synthesized using the procedures outlined in International Patent Application No. PCT/US2019/026992 (see, for example, procedure 2, procedure 3 and procedure 4 or 5), which is incorporated herein by reference.

[1087] Step 1 . 1-(4-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)-4-( (((2S,3S,6R,7S,10R, E)-2-((E)-1-(3-fluoro-5-morpholinophenyl)prop-1-en-2-yl)-10-hydroxy-3 ,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-y (12.9 mg, 69.8%). LC/MS (ESI, mass/charge), 1086.43 [M+H].

[1088] ¹ H-NMR (400 MHz, MeOH- d 4 ): δ ppm 0.96-1.07 (m, 12 H), 1.27-1.42 (m, 3 H), 1.46 (d, J=7.15 Hz, 3 H), 1.62-1.72 (m, 2 H), 1.88 (d, J=1.13 Hz, 3 H), 1.97-2 .06 (m, 1 H), 2.04-2.14 (m, 1 H), 2.44-2.53 (m, 1 H), 2.58-2.69 (m, 2 H) , 3.08 (s, 3 H), 3.13-3.19 (m, 4 H), 3.38-3.56 (m, 4 H), 3.61-3.75 (m, 2 H), 3.79-3.88 (m, 5 H), 3.90-3.98 (m, 1 H), 4.07-4.18 (m, 2 H), 4.20-4 .29 (m, 1 H), 4.35-4.45 (m, 2 H), 4.45-4.54 (m, 1 H), 4.61 (s, 2 H), 4.86 -4.92(m, 2H), 5.15(d, J=10.67Hz, 1H), 5.46-5.65(m, 2H), 6.46-6.67( m, 4 H), 7.28-7.37 (m, 2 H), 7.37-7.45 (m, 2 H), 7.47-7.53 (m, 2 H), 7, 66-7.71(m, 2H), 7.74-7.84(m, 4H).

[1089] Step 2 . 1-(4-((R)-2-((R)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido) propanamido)benzyl)-4-((((2S,3S,6R,7S,10R, E)-2-((E)-1-(3-fluoro-5-morpholinophenyl)prop-1-en-2- yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-ium bromide (7.6 mg, 60.5% yield) . LC/MS (ESI, mass/charge), 1057.62 [M+H].

[1090] ¹ H-NMR (400 MHz, MeOH -d 4 ): δ ppm 0.95-1.08 (m, 12 H), 1.24-1.42 (m, 5 H), 1.47 (d, J=7.15 Hz, 3 H), 1.52-1.71 (m, 6 H), 1.89 (d, J=1.13 Hz, 3 H), 1.95-2 .05 (m, 1 H), 2.05-2.15 (m, 1 H), 2.26-2.34 (m, 2 H), 2.46-2.53 (m, 1 H) , 2.58-2.69(m, 2H), 3.07-3.12(m, 3H), 3.14-3.19(m, 4H), 3.39-3.58 (m, 6 H), 3.61-3.76 (m, 2 H), 3.80-3.86 (m, 5 H), 4.09-4.18 (m, 3 H), 4 .43-4.51(m, 1H), 4.62(s, 2H), 4.87-4.93(m, 2H), 5.12-5.19(m, 1H) , 5.46-5.65(m, 2H), 6.47-6.66(m, 4H), 6.80(s, 2H), 7.53(d, J=8.66 Hz, 2 H), 7.82 (d, J=8.66 Hz, 2 H).

1.5.1.4 ADL5-D10

[1091] The general procedure above (section 1.5.1) was used to synthesize ADL5-D10. The D10 payload was synthesized using the procedures in section 1.2.1.

[1092] (2S,3S,6S,7R,10R, E)-7-Ethoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3 -((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4- en-6-yl-4-methylpiperazine-1-carboxylate (16.6 mg, 0.025 mmol, 38.7% yield). LC/MS (ESI, mass/charge), 665.7 [M+H] ⁺ .

[1093] ¹ H-NMR (400 MHz, METHANOL- d ): δ ppm 0.76-0.87 (m, 9 H), 1.03-1.09 (m, 3 H), 1.10- 1.13 (m, 3 H) 1.13-1.19 (m, 1 H) 1.20-1.27 (m, 3 H), 1.29-1.48 (m, 6 H) 1 .51-1.59 (m, 1 H) 1.65-1.71 (m, 3 H) 1.73-1.81 (m, 1 H) 2.20 (s, 3 H) , 2, 27-2.34 (m, 4 H) 2.34-2.44 (m, 2 H) 2.44-2.53 (m, 1 H) 2.53-2.59 (m, 1 H) 2.76-2.83 (m, 1 H) 3.23-3.26 (m, 1 H) 3.34-3.52 (m, 7 H) 3.67-3.75 (m, 1 H) 4.86-4.92 (m, 1 H) 4.92-4.99 (m.1 H), 5.41-5.51 (m, 1 H), 5.60-5.71 (m, 1 H), 5.72-5.82 (m, 1 H), 6.00-6.07 (m, 1 H), 6.37-6.48 (m, 1 H)

[1094] Step 1 . 1-(4-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)-4-( (((2S,3S,6S,7R,10R, E)-7-ethoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3- ((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-ene -6-yl)oxy)carbonyl)-1-methylpiperazin-1-ium (18.6 mg, 0.016 mmol, 87% yield) as a colorless film. LC/MS (ESI, mass/charge), 1163.01 [M] ⁺ .

[1095] ¹ H-NMR (400 MHz, MeOH- d 4 ): δ ppm 0.86-1.03 (m, 16 H), 1.20 (s, 3 H), 1.25 (s, 4 H), 1.36(s, 3H), 1.43-1.72(m, 10H), 1.81(s, 3H), 1.84-1.92(m, 1H) , 2.06-2.14 (m, 1 H), 2.46-2.63 (m, 3 H), 2.67-2.72 (m, 1 H), 2.88-2.94 (m, 1 H), 3.03-3.09 (m, 3 H), 3.38-3.45 (m, 3 H), 3.54 (s, 6 H), 3.63-3 .79 (m, 2H), 3.81-3.88 (m, 1H), 3.92-3.98 (m, 1H), 4.07-4.18 (m, 2H) , 4.21-4.28(m, 1H), 4.36-4.44(m, 2H), 4.47-4.53(m, 1H), 4.55-4.63 (m, 3 H), 5.02-5.11 (m, 3 H), 5.56-5.66 (m, 1 H), 5.75-5.84 (m, 1 H), 5 .86-5.92(m, 1H), 6.12-6.19(m, 1H), 6.49-6.61(m, 1H), 7.29-7.36(m , 2 H), 7.38-7.44 (m, 2 H), 7.47-7.53 (m, 2 H), 7.64-7.71 (m, 2 H), 7.80 (dd, J=15.06, 7.91 Hz, 4H).

[1096] Step 2 . 1-(4-((S)-2-((S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido) propanamido)benzyl)-4-((((2S,3S,6S,7R,10R, E)-7-ethoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-( (2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl -12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-ium (14.09 mg, 82% yield). LC/MS (ESI, mass/charge), 1057.62 [M+H] ⁺ .

[1097] ¹ H-NMR (400 MHz, MeOH- d 4 ): δ ppm 0.87-1.03 (m, 15 H), 1.20 (s, 3 H), 1.22-1.29 (m, 4H), 1.37(s, 6H), 1.45-1.72(m, 14H), 1.81(s, 3H), 1.84-1.95(m , 1 H), 2.05-2.16 (m, 1 H), 2.26-2.36 (m, 2 H), 2.47-2.62 (m, 3 H), 2.66 -2.74(m, 1H), 2.86-2.95(m, 1H), 3.11(s, 3H), 3.42-3.51(m, 4H), 3 .51-3.62(m, 5H), 3.64-3.78(m, 2H), 3.81-3.89(m, 1H), 4.09-4.20(m , 3 H), 4.43-4.52 (m, 1 H), 4.54-4.59 (m, 1 H), 4.55-4.67 (m, 2 H), 4.60 -4.68(m, 2H), 5.01-5.12(m, 2H), 5.54-5.65(m, 1H), 5.75-5.86(m, 1 H), 5.86-5.94(m, 1H), 6.10-6.21(m, 1H), 6.47-6.62(m, 1H), 6.81(s , 2 H), 7.49-7.60 (m, 2 H) 7.76-7.89 (m, 2 H).

1.5.1.5 ADL5-D15

[1098] The general procedure above (section 1.5.1) was used to synthesize ADL5-D15. The D15 payload was synthesized using the procedures outlined in International Patent Application No. PCT/US2019/026992, which is incorporated herein by reference .

[1099] Step 1 . 1-(4-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)-4-( (((2S,3S,6R,7S,10R, E)-2-((E)-1-(1-(1-acetylpiperidin-4-yl)-4-fluoro-1H-benzo[d][1 ,2,3]triazol-6-yl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1 -methylpiperazin-1-y (100 mg, 0.086 mmol, 57.3% yield). LC/MS (ESI mass/charge), 1167.76 [M+H].

[1100] Step 2 . 4-((((2S,3S,6R,7S,10R, E)-2-((E)-1-(1-(1-Acetylpiperidin-4-yl)-4-fluoro-1H-benzo[d ][1,2,3]triazol-6-yl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl )-1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3- methylbutanamido)propanamido)benzyl)-1-methylpiperazin-1-ium (32 mg, 0.028 mmol, 32.8% yield). LC/MS (ESI mass/charge), 1138.64 [M+H].

[1101] ¹ H-NMR (400 MHz, MeOH- d 4 ): δ ppm 0.99 (s, 10 H), 1.26-1.40 (m, 5 H), 1.42-1.49 (m, 3 H), 1.67 (br s, 6 H), 1.96 (d, J=1.00 Hz, 3 H), 2.00-2.13 (m, 2 H), 2 .16-2.42(m, 7H), 2.44-2.55(m, 1H), 2.59-2.76(m, 2H), 2.98-3.26(m , 4 H), 3.38-3.59 (m, 7 H), 3.60-3.76 (m, 2 H), 3.78-3.90 (m, 1 H), 4.13 (br d, J=7.15 Hz, 3 H), 4.41-4.52 (m, 1 H), 4.53-4.71 (m, 3 H), 4.89-4.97 (m, 1 H), 5.10-5.27 (m, 2 H), 5.48-5.67 (m, 2 H), 6.79 (s, 2 H) 7.07-7, 17(m, 1H), 7.48-7.61(m, 3H), 7.78-7.84(m, 2H).

1.5.1.5 ADL5-D32

[1102] The general procedure above (section 1.5.1) was used to synthesize ADL5-D32. The D15 payload was synthesized using the procedures outlined in International Patent Application No. PCT/US2019/026992, which is incorporated herein by reference .

[1103] Step 1 . 1-(4-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)-4-( (((2S,3S,6R,7S,10R, E)-2-((E)-1-(5-chloropyridin-3-yl)prop-1-en-2-yl)-10-hydroxy-3 ,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-ium (15 mg, 0.015 mmol, 69.6% yield) LC/MS (ESI, wt/ charge), 1018.4 [M] ⁺

[1104] 1H NMR (400 MHz, METHANOL-d4) d ppm 0.99 (s, 14 H) 1.29-1.41 (m, 3 H) 1.44-1.49 (m, 3 H) 1.61-1.71 (m, 2 H) 1.92 (d, J=1.25 Hz, 3 H), 1.95-2.13 (m, 2 H) 2.45-2.53 (m, 1H) 2.60-2.70 (m, 2H) 3.04-3.09 (m, 3H) 3.37 (s, 3H) 3.39-3.47 (m , 1 H) 3.47-3.54 (m, 2 H) 3.61-3.75, (m, 2 H) 3.78-3.87 (m, 1 H) 3.93-3, 99 (m, 1 H) 4.08-4.18 (m, 2 H) 4.22-4.29 (m, 1 H) 4.39-4.45 (m, 2 H) 4.45- 4.53 (m, 1 H) 4.56-4.63 (m.3 H) 5.17-5.23 (m, 1 H) 5.48-5.64 (m, 2 H) 6, 57-6.64 (m, 1 H) 7.28-7.36 (m.2 H) 7.37-7.45 (m, 2 H) 7.47-7.54 (m, 2 H) 7.64-7.70 (m, 2H) 7.75-7.84 (m, 5H) 8.41-8.48 (m, 2H)

[1105] Step 2. 4-((((2S,3S,6R,7S,10R, E)-2-((E)-1-(5-Chloropyridin-3-yl)prop-1-en-2 -yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-(4-((S)-2-((S)-2- (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl)-1-methylpiperazin-1-ium (6.2 mg, yield 42.5%). LC/MS (ESI, mass/charge), 990.34 [M+H].

[1106] 1H NMR (400 MHz, METHANOL-d4) d ppm 0.97-1.09 (m, 12 H) 1.24-1.41 (m, 5 H) 1.47 (d, J=7 .15 Hz, 3 H) 1.55-1.71 (m, 6 H) 1.92 (d, J=1.13 Hz, 3 H) 1.98-2.15 (m, 2 H) 2 .27-2.36 (m, 2H) 2.46-2.53 (m, 1H) 2.60-2.71 (m, 2H) 3.06-3.13 (m, 3H ) 3.35-3.38 (m, 1 H) 3.41-3.58 (m, 6 H) 3.62-3.76 (m, 2 H) 3.80-3.88 (m, 1 H) 4.08-4.19 (m, 3 H) 4.43-4.52 (m, 1 H) 4.57-4.66 (m, 2 H) 5.16-5.24 ( m, 1 H) 5.49-5.68, (m, 2 H) 6.62 (s, 1 H) 6.81 (s, 2 H) 7.49-7.57 (m, 2 H) 7.76-7.87(m, 3H) 8.39-8.49(m, 2H)

1.5.1.5 ADL5-D30

[1107] The general procedure above (section 1.5.1) was used to synthesize ADL5-D30. The D30 payload was synthesized using the procedures outlined in International Patent Application No. PCT/US2019/026992, which is incorporated herein by reference .

[1108] Step 1 . 1-(4-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)-4-( (((2S,3S,6R,7S,10R, E)-2-((E)-1-(3-(dimethylamino)phenyl)prop-1-en-2-yl)-10-hydroxy-3, 7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-ium (16 mg, 0.016 mmol, 51.4% yield). LC/MS (ESI, mass/charge), 1027.50 [M+H] ⁺

[1109] 1H NMR (400 MHz, METHANOL- d ₄ ) d ppm 0.95-1.07 (m, 13 H) 1.31-1.42 (m, 2 H) 1.44-1.50 ( m, 3H) 1.63-1.71 (m, 2H) 1.84-1.92 (m, 3H) 1.98-2.14 (m, 2H) 2.47-2.56 (m, 1H) 2.60-2.67 (m, 2H) 2.93 (s, 6H) 3.03-3.09 (m, 3H) 3.37 (s, 4H) 3.47-3.56 (m, 2 H) 3.63-3.75 (m, 2 H) 3.79-3.88 (m, 1 H) 3.92-3.98 (m, 1 H) 4.08-4.19 (m, 2 H) 4.21-4.27 (m, 1 H) 4.37-4.44 (m, 2 H) 4.45-4.54 (m , 1 H) 4.57-4.62 (m, 2H) 4.91-4.94 (m, 2 H) 5.15-5.20 (m, 1 H) 5.47-5.55 ( m, 1 H) 5.56-5.65 (m, 1 H) 6.58 (s, 1 H) 6.67 (d, J=1.51 Hz, 3 H) 7.15-7.24 (m, 1 H), 7.28-7.36 (m, 2 H) 7.38-7.45 (m, 2 H) 7.47-7.54 (m, 2 H) 7.64- 7.73(m, 2H) 7.75-7.85(m, 4H)

[1110] Step 2. 4-((((2S,3S,6R,7S,10R, E)-2-((E)-1-(5-Chloropyridin-3-yl)prop-1-en-2 -yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-(4-((S)-2-((S)-2- (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl)-1-methylpiperazin-1-ium (6.2 mg, yield 42.5%). LC/MS (ESI, mass/charge), 990.34 [M+H].

[1111] 1H NMR (400 MHz, METHANOL-d4) d ppm 0.88 (d, J=6.65 Hz, 12 H) 1.16-1.30 (m, 4 H) 1.32-1, 38 (m, 3H) 1.43-1.58 (m, 6H) 1.75-1.80, (m, 3H) 1.84-1.92 (m, 1H) 1.93 -2.02 (m, 1 H) 2.14-2.21 (m, 2 H) 2.35-2.42 (m, 1 H) 2.48-2.57 (m, 2 H) 2 .82 (s, 6 H) 2.94-3.02 (m, 3 H), 3.27-3.32 (m, 1 H) 3.35-3.45 (m, 4 H) 3, 50-3.64 (m, 2 H) 3.68-3.76 (m, 1 H) 3.97-4.07 (m, 3 H) 4.31-4.40 (m, 1 H) 4.47-4.53 (m, 2 H) 5.03-5.09 (m, 1 H) 5.35-5.44 (m, 1 H) 5.45-5.55 (m, 1 H) 6.42-6.50 (m, 1 H) 6.51-6.62 (m, 3 H) 6.69 (s, 2 H) 7.03-7.12 (m, 1 H) 7.38-7.44(m, 2H) 7.66-7.75(m, 2H) 8.42-8.47(m, 1H).

1.5.1.5 ADL5-D27

[1112] The general procedure above (section 1.5.1) was used to synthesize ADL5-D27. The D27 payload was synthesized using the procedures outlined in International Patent Application No. PCT/US2019/026992, which is incorporated herein by reference.

[1113] Step 1. 1-(4-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido) benzyl)-4-((((2S,3S,6R,7S,10R, E)-2-((E)-1-(3-fluoro-5-(4-((1-methyl-1H-pyrazole -4-yl)sulfonyl)piperazin-1-yl)phenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy) carbonyl)-1-methylpiperazin-1-y (25.2 mg, 0.020 mmol, 83% yield). LC/MS (ESI, mass/charge), 1230.54 [M+H] ⁺

Step 2. 1-(4-((S)-2-((S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3 -methylbutanamido)propanamido)benzyl)-4-((((2S,3S,6R,7S,10R, E)-2-((E)-1-(3-fluoro-5-(4-((1- methyl-1H-pyrazol-4-yl)sulfonyl)piperazin-1-yl)phenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6 -yl)oxy)carbonyl)-1-methylpiperazin-1-y (5.2 mg, 4.33 µmol, 21.13% yield). LC/MS (ESI, mass/charge), 1201.68 [M+H] ⁺

[1114] ¹ H NMR (400 MHz, METHANOL- d ₄ ) d ppm 0.87 (d, J=6.78 Hz, 12 H) 1.13-1.29 (m, 4 H) 1.31- 1.38 (m, 3 H) 1.43-1.59 (m, 6 H) 1.71-1.79, (m, 3 H) 1.83-2.02 (m, 2 H) 2 .14-2.21 (m, 2 H) 2.34-2.41 (m, 1 H) 2.56 (s, 8 H) 2.94-3.01 (m, 3 H) 3.12 -3.18 (m, 8 H) 3.28-3.45 (m, 5 H), 3.48-3.63 (m, 2 H) 3.70 (s, 4 H) 3.97- 4.07 (m, 3 H) 4.29-4.41 (m, 1 H) 4.49 (s, 2 H) 4.98-5.06 (m, 1 H) 5.34-5, 54 (m, 2H) 6.37-6.45 (m, 2H) 6.51 (s, 2H) 6.69 (s, 2H) 7.37-7.43 (m, 2H) 7.63-7.73(m, 4H)

1.5.1.6 ADL10-D1

[1115] (2S,3S,6S,7R,10R, E)-10-Hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2S,3S)-3-((2R ,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4- en-6-ylpiperazin-1-carboxylate (0.012 g, 0.019 mmol), 2,5-dioxopyrrolidin-1-yl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoate (0.012 g, 0.038 mmol) and DCM (0.188 ml, 0.019 mmol) were combined and stirred overnight. The mixture was directly loaded onto a silica gel column and chromatographed to give (2S,3S,6S,7R,10R, E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R, 3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7- dimethyl-12-oxooxacyclododec-4-en-6-yl-4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)piperazine-1-carboxylate (8, 4 mg, 10.12 µmol, 53.7% yield. LC/MS [M+Na] 853.5.

[1116] ¹ H-NMR (400 MHz, MeOH- d 4 ): δ ppm 0.89 (s, 9 H) 1.21-1.39 (m, 9 H) 1.41-1.56 (m , 5 H) 1.57-1.71 (m, 6 H) 1.69-1.71 (m, 1 H) 1.79, (d, J=0.88 Hz, 3 H) 1.82 -1.90 (m, 1 H) 2.34-2.43 (m, 1 H) 2.41-2.41 (m, 1 H) 2.46-2.63 (m, 3 H) 2 .65-2.68 (m, 2 H) 2.68 (s, 3 H) 2.86-2.92 (m, 1 H) 3.33 (s, 3 H) 3.51 (br d, J=7.03Hz, 6H) 3.54-3.60 (m, 5H) 3.77-3.88 (m, 1H) 5.06 (s, 2H) 5.53-5 .63 (m, 1 H) 5.70-5.80 (m, 1 H), 5.83-5.94 (m, 1 H) 6.08-6.19 (m, 1 H) 6, 47-6.59 (m, 1H) 6.80 (s, 2H).

1.6 Obtaining ADL12-D1, ADL14-D1 and ADL15-D1

1.6.1 Brief description. General procedure 1

Scheme 7

[1117] Stage 1 for ADL12-D1 . To a stirred solution of 1-(6-hydroxyhexyl)-1H-pyrrole-2,5-dione (0.200 g, 1.014 mmol) in DCM (20.28 ml, 1.014 mmol) was added PDC (3.81 g, 10.14 mmol ) and acetic acid (0.1 ml). The reaction mixture was stirred for 3 hours at rt. The reaction mixture was then filtered through a pad of silica gel and concentrated to dryness to give 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanal (40 mg, 0.205 mmol, 20.21% yield).

[1118] Stage 1 for ADL14-D1 and ADL15-D1 . To a stirred solution of maleimide alcohol (1.0 eq.) in DCM (0.05 M) was added pre-activated MS-4A powder, 2.6 mg per mg alcohol, N-methylmorpholine-N-oxide (1.2 eq. ) and TPAP (0.1 eq) in that order. The reaction mixture was stirred for 60 minutes. at rt. The reaction mixture was then filtered through a pad of silica gel and concentrated to dryness. The crude product was then transferred to the next stage (stage 2).

[1119] Step 2 . Mixed (2S,3S,6S,7R,10R, E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2S,3S)-3-((2R,3S )-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en- 6-ylpiperazin-1-carboxylate (1.0 eq.), 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetaldehyde (2.0-3.0 eq.) , DCM (0.1 M), sodium triacetoxyborohydride (3.0 eq) and stirred for 10 min. The reaction mixture was then directly loaded onto a silica gel column and chromatographed to obtain the desired linker-payload design.

1.6.1.1 ADL12-D1

[1120] Linker Payload (ADL12-D1) . The general procedure in section 1.6.1 was used to synthesize (2S,3S,6S,7R,10R, E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-(( 2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3, 7-dimethyl-12-oxooxacyclododec-4-en-6-yl-4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl)piperazin-1-carboxylate ( 41 mg, 0.050 mmol, 80% yield. LC/MS [M+] ⁺ 816.75.

[1121] ¹ H-NMR (400 MHz, MeOH- d 4 ): δ ppm 0.86-0.98 (m, 9 H) 1.20-1.23 (m, 3 H) 1.23-1 .40 (m, 8 H) 1.41-1.70 (m, 12 H) 1.79 (d, J=0.63 Hz, 3 H) 1.83-1.91 (m, 1 H) 2.33-2.40 (m, 2 H) 2.43 (br t, J=4.77 Hz, 4 H) 2.48-2.64 (m, 3 H) 2.65-2.69 (m, 1 H) 2.87-2.92 (m, 1 H) 3.32 (s, 3 H) 3.41-3.60 (m, 7 H) 3.79-3.87 (m , 1 H) 5.04 (dd, J=19.39, 10.23 Hz, 2 H) 5.57 (dd, J=15.18, 9.79 Hz, 1 H) 5.69-5, 79 (m, 1 H) 5.87 (d, J=15.31 Hz, 1 H) 6.11-6.17 (m, 1 H) 6.49-6.58 (m, 1 H) 6 .80 (s, 2H).

1.6.1.X ADL12-D28

[1122] Linker Payload (ADL12-D28). The general procedure given in section 1.6.1 was used to synthesize (2S,3S,6R,7S,10R, E)-2-((E)-1-(3-fluoro-5-(4-((1- methyl-1H-pyrazol-4-yl)sulfonyl)piperazin-1-yl)phenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6 -yl-4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl)piperazine-1-carboxylate (2.3 mg, 30.7%). LC/MS [M+] ⁺ 897.32.

[1123] ¹ H NMR (400 MHz, METHANOL- d ₄ ) δ ppm 0.85-0.93 (m, 6 H) 1.16-1.34 (m, 6 H) 1.37-1.58 (m, 6 H) 1.72-1.77 (m, 3 H) 1.80-1.88 (m, 1 H), 2.23-2.41 (m, 7 H) 2.47- 2.57 (m, 2 H) 3.08-3.19 (m, 9 H) 3.33-3.42 (m, 5 H) 3.70 (s, 4 H) 4.67-4, 72 (m, 1 H) 4.98-5.07 (m, 1 H) 5.33-5.50 (m, 2 H) 6.36-6.43 (m, 2 H) 6.44- 6.53(m, 2H) 6.70(s, 2H) 7.62-7.69(m, 2H)

1.6.1.X ADL12-D35

[1124] Linker Payload (ADL12-D35) . The general procedure in section 1.6.1 was used to synthesize (2S,3S,6R,7S,10R, E)-10-hydroxy-3,7-dimethyl-12-oxo-2-((E)-1- (3-(pyrrolidin-1-ylsulfonyl)phenyl)prop-1-en-2-yl)oxacyclododec-4-en-6-yl-4-(6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexyl)piperazine-1-carboxylate (4.0 mg, 5.11 µmol, 28.8% yield). LC/MS [M+H] ⁺ 783.5.

[1125] 1H NMR (400 MHz, METHANOL- d 4) d ppm 0.91 (dd, J =6.78, 2.51 Hz, 6 H) 1.17-1.33 (m, 6 H) 1 .38-1.59 (m, 6 H) 1.65 (s, 4 H) 1.80 (s, 3 H), 1.82-1.91 (m, 1 H) 2.23-2, 41 (m, 7 H) 2.49-2.61 (m, 2 H) 3.14 (s, 4 H) 3.38 (br d, J =7.15 Hz, 6 H) 3.67- 3.76 (m, 1 H) 4.68-4.73 (m, 1 H) 5.03-5.11 (m, 1 H) 5.34-5.52 (m, 2 H) 6, 55-6.62 (m, 1H) 6.70 (s, 2H) 7.46-7.54 (m, 2H) 7.58-7.65 (m, 2H)

1.6.1.X ADL12-D22(R)

[1126] Linker Payload (ADL12-D28). The general procedure given in Section 1.6.1 was used to synthesize 3-((R)-1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl)-4 -((((2S,3S,6S,7R,10R, E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-(( 2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4 -en-6-yl)oxy)carbonyl)piperazin-2-yl)propanoic acid (3.2 mg, 3.60 µmol, 15.39% yield). LC/MS [M+] ⁺ 888.5.

1.6.1.X ADL12-D22(S)

[1127] Linker Payload (ADL12-D28). The general procedure given in Section 1.6.1 was used to synthesize 3-((S)-1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl)-4 -((((2S,3S,6S,7R,10R, E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-(( 2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4 -en-6-yl)oxy)carbonyl)piperazin-2-yl)propanoic acid (3.6 mg, 3.60 μmol, 28.7% yield). LC/MS [M+] ⁺ 888.56.

1.6.1.2 ADL14-D1

[1128] Linker Payload (ADL14-D1) . The general procedure in section 1.6.1 was used to synthesize (2S,3S,6S,7R,10R, E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-(( 2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3, 7-dimethyl-12-oxooxacyclododec-4-en-6-yl-4-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)piperazin-1-carboxylate ( 8 mg, 10.53 mmol, 33.5% yield. LC/MS [M+H] 760.3.

[1129] ¹ H-NMR (400 MHz, MeOH- d 4 ): δ ppm 0.85-1.01 (m, 9 H), 1.17-1.33 (m, 5 H), 1.36 (s, 3H), 1.41-1.71(m, 7H), 1.80(s, 3H), 1.83-1.95(m, 1H), 2.48(br s, 10 H), 2.89-2.98 (m, 1 H), 3.40-3.57 (m, 5 H), 3.61-3.72 (m, 2 H), 3, 78-3.91(m, 1H), 4.98-5.14(m, 2H), 5.50-5.52(m, 1H), 5.52-5.65(m, 1 H), 5.69-5.82 (m, 1 H), 5.86-5.96 (m, 1 H), 6.09-6.21 (m, 1 H), 6.47- 6.60 (m, 1 H), 6.79-6.90 (m, 2 H).

1.6.1.3 ADL15-D1

[1130] Linker Payload (ADL15-D1) . The general procedure in section 1.6.1 was used to synthesize (2S,3S,6S,7R,10R, E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-(( 2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3, 7-dimethyl-12-oxooxacyclododec-4-en-6-yl-4-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)piperazine -1-carboxylate (7 mg, 8.71 µmol, 23.10% yield). LC/MS [M=+H] 804.1.

[1131] ¹ H-NMR (400 MHz, MeOH- d 4 ): δ ppm 0.85-1.02 (m, 9 H), 1.18-1.32 (m, 4 H), 1.36 (s, 3 H), 1.40-1.72 (m, 7 H), 1.78-1.83 (m, 3 H), 1.85-1.93 (m, 1 H), 2 .42-2.65(m, 9H), 2.67-2.73(m, 1H), 2.89-2.96(m, 1H), 3.37(s, 5H) , 3.41-3.58 (m, 5 H), 3.59-3.67 (m, 4 H), 3.68-3.75 (m, 2 H), 3.81-3.89 (m, 1 H), 4.94-5.15 (m, 2 H), 5.61 (br d, J=10.04 Hz, 1 H), 5.69-5.82 (m, 1 H), 5.89 (br d, J=15.18 Hz, 1 H), 6.16 (br d, J=10.92 Hz, 1 H), 6.56 (br s, 1 H), 6.77-6.91(m, 2H).

1.7 ADL12-D2

[1132] (2S,3S,6S,7R,10R, E)-10-Hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2S,3S)-3-((2R ,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4- en-6-yl-4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl)piperazin-1-carboxylate (26 mg, 0.032 mmol), acetonitrile (319 μl, 0.032 mmol), iodomethane (19.92 μl, 0.319 mmol) were combined and stirred overnight. The reaction mixture was concentrated to dryness and chromatographed (0-30% MeOH in DCM) to give 1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl)-4- ((((2S,3S,6S,7R,10R, E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R ,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4- en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-ium (14 mg, 0.017 mmol, 52.9% yield). LC/MS [M+] 831.6.

[1133] ¹ H-NMR (400 MHz, MeOH-d4): δ ppm 0.86-0.98 (m, 9 H) 1.26 (s, 4 H) 1.36 (s, 3 H) 1 .40-1.58 (m, 9 H) 1.60-1.71 (m, 4 H) 1.80 (d, J=0.88 Hz, 4 H) 1.85-1.91 (m , 1 H) 1.87-1.88 (m, 1 H) 2.48-2.63 (m, 3 H) 2.65-2.72 (m, 1 H) 2.88-2.94 (m, 1H) 3.18 (s, 3H) 3.34-3.41 (m, 3H) 3.53 (s, 10H) 3.73-3.90 (m, 3H) 3.90-4.06 (m, 2 H) 5.02-5.11 (m, 2 H) 5.56-5.66 (m, 1 H) 5.70-5.81 (m, 1 H) 5.84-5.94 (m, 1 H) 6.11-6.20 (m, 1 H) 6.50-6.60 (m, 1 H) 6.83 (s, 2 H) .

1.8 ADL15-D2

[1134] (2S,3S,6S,7R,10R, E)-10-Hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2S,3S)-3-((2R ,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4- en-6-yl-4-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)piperazine-1-carboxylate (6 mg, 7.463 µmol ), iodomethane (0.467 µl, 7.463 µmol) and acetonitrile (0.390 µl, 7.463 µmol) were combined and stirred at rt. The reaction mixture was concentrated to dryness to give (2S,3S,6S,7R,10R, E)-10 -hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl) -6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl-4-(2-(2-(2.5 -dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)-4-methyl-4l4-piperazine-1-carboxylate (6 mg, 7.33 µmol, 98% yield). LC/MS [M+] ⁺ 819.28.

[1135] ¹ H-NMR (400 MHz, MeOH -d 4): δ ppm 0.74-0.88 (m, 9 H) 1.18 (br d, J =4.27 Hz, 5 H) 1 .23-1.27 (m, 3 H) 1.28-1.49 (m, 5 H) 1.51-1.61 (m, 2 H) 1.66-1.71 (m, 3 H) ) 1.73-1.80 (m, 1 H) 2.36-2.53 (m, 3 H) 2.55-2.60 (m, 1 H) 2.78-2.83 (m, 1 H) 3.10-3.14 (m, 3 H) 3.23-3.25 (m, 3 H) 3.32-3.51 (m, 5 H) 3.52-3.67 ( m, 6 H) 3.67-3.87 (m, 7 H) 4.93-5.01 (m, 2 H) 5.44-5.55 (m, 1 H) 5.61-5, 70 (m, 1 H) 6.00-6.07 (m, 1 H) 6.38-6.48 (m, 1 H) 6.75 (s, 2 H).

1.9 ADL5-D25

Scheme 8

Stage 1 . D1 (40 mg, 0.063 mmol) was dissolved in N,N-dimethylformamide (730 µl, 9.422 mmol) and methyl bromobuterate (23.69 µl, 0.188 mmol) was added followed by

triethylamine (35.0 µl, 0.251 mmol). The solution was stirred at room temperature for 2 days. The solvent was evaporated. Purification by column chromatography gave (2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3 -((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12- oxooxacyclododec-4-en-6-yl-4-(4-methoxy-4-oxobutyl)piperazine-1-carboxylate (51 mg, 0.069 mmol, ~100% yield) as a colorless oil. LC/MS (ESI, mass/charge), 737.4 [M+H] ⁺ .

[1136] ¹ H-NMR (400 MHz, MeOH- d 4 ): δ ppm 0.76-0.86 (m, 9 H), 1.12 (s, 3 H), 1.14-1.19 (m, 1 H), 1.24 (s, 3 H), 1.34-1.50 (m, 5 H), 1.52-1.60 (m, 1 H), 1.69 (s , 6 H), 2.27 (s, 8 H), 2.37-2.49 (m, 3 H), 2.54-2.61 (m, 1 H), 2.77-2.83 (m, 1 H), 3.35-3.47 (m, 5 H), 3.56 (s, 3 H), 3.67-3.76 (m, 1 H), 4.46 (s , 3 H), 4.89-5.00 (m, 3 H), 5.42-5.52 (m, 1 H), 5.57-5.70 (m, 1 H), 5.73 -5.81 (m, 1 H), 5.98-6.08 (m, 1 H), 6.43 (dd, J =15.25, 10.98 Hz, 1 H).

[1137] Step 2 . Fmoc-Val-Ala-PAB-bromide (88 mg, 0.152 mmol) and (2S,3S,6S,7R,10R, E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy -7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7 -methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl-4-(4-methoxy-4-oxobutyl)piperazine-1-carboxylate (51 mg, 0.069 mmol) in N,N-dimethylformamide (670 µl, 8.651 mmol). The reaction mixture was stirred at room temperature overnight. The solvent was evaporated. Purification by column chromatography gave 1-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido )benzyl)-4-((((2S,3S,6S,7R,10R, E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R) -3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl- 12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-(4-methoxy-4-oxobutyl)piperazin-1-ium (42 mg, 0.034 mmol, 49.1% yield) as a waxy solid substances. LC/MS (ESI, mass/charge), 1235.4 [M] ⁺ .

[1138] Step 3 . 1-(4-((S)-2-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)-4-( (((2S,3S,6S,7R,10R, E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R, 3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-ene -6-yl)oxy)carbonyl)-1-(4-methoxy-4-oxobutyl)piperazin-1-ium (42 mg, 0.034 mmol) was dissolved in a ratio of 5:2:1 in THF (600 μl, 7.322 mmol) , MeOH (250 µl, 6.179 mmol), water (125 µl, 6.939 mmol) and lithium hydroxide (1.221 mg, 0.051 mmol) was added. The mixture was stirred at RT for 1 hour. The mixture was quenched with acetic acid (5.84 μl, 0.102 mmol) and the solvent was evaporated and purified by RP HPLC to give 1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido )benzyl)-1-(3-carboxypropyl)-4-((((2S,3S,6S,7R,10R, E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy- 7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7- methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)piperazin-1-ium as a waxy solid (18.4 mg, 0.018 mmol, 54.2% yield). LC/MS (ESI, mass/charge), 998.5 [M] ⁺ .

[1139] Step 4 . 1-(4-((S)-2-((S)-2-Amino-3-methylbutanamido)propanamido)benzyl)-1-(3-carboxypropyl)-4-((((2S,3S,6S, 7R,10R, E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2- yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl) piperazin-1-ium (18.4 mg, 0.018 mmol) was dissolved in N,N-dimethylformamide (356 μl, 4.603 mmol) and 2,5-dioxopyrrolidin-1-yl-6-(2,5-dioxo-2 ,5-dihydro-1H-pyrrol-1-yl)hexanoate (6.81 mg, 0.022 mmol) followed by the addition of Hunig's base (8.04 μl, 0.046 mmol). The reaction solution was stirred at room temperature for 30 minutes. The solvent was evaporated and the crude residue was purified by RP HPLC to give (1-(3-carboxypropyl)-1-(4-((S)-2-((R)-2-(6-(2,5-dioxo-2 ,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl)-4-((((2S,3S,6S,7R,10R, E)-10-hydroxy-2- ((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta- 2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)piperazin-1-ium (8.8 mg, 7, 38 µmol, 40.1% yield as white lyophilized solid LC/MS (ESI, mass/charge), 1191.5 [M] ⁺ .

[1140] ¹ H-NMR (400 MHz, DMSO- d 6): δ ppm 0.68-0.81 (m, 18 H), 0.98-1.07 (m, 4 H), 1.07 -1.14 (m, 3 H), 1.16 (br d, J =3.89 Hz, 5 H), 1.22-1.33 (m, 8 H), 1.36-1.47 (m, 7 H), 1.62 (s, 3 H) 1.66-1.73 (m, 1 H), 1.83-1.92 (m, 5 H), 1.98-2, 13 (m, 2 H), 2.26 (dt, J =3.54, 1.80 Hz, 1 H), 2.27-2.33 (m, 1 H), 2.47-2.51 (m, 2 H), 2.58-2.62 (m, 1 H), 2.66-2.72 (m, 1 H), 3.15 (s, 3 H) 3.45-3, 58 (m, 3 H), 3.60-3.68 (m, 2 H) 3.77-3.86 (m, 2 H), 4.06-4.13 (m, 1 H), 4 .27-4.37(m, 2H), 4.46-4.54(m, 3H), 4.72-4.78(m, 1H), 4.80-4.87(m , 2 H), 5.32-5.48 (m, 1 H), 5.53-5.67 (m, 1 H), 5.73-5.86 (m, 1 H), 5.93 -6.05(m, 1H), 6.16-6.26(m, 1H), 6.20(s, 1H), 6.23-6.41(m, 1H), 6 .27-6.38(m, 1H), 6.93(s, 2H), 6.98-7.06(m, 2H), 7.43-7.50(m, 2H) , 7.62-7.69(m, 2H), 7.74-7.80(m, 1H), 8.22-8.27(m, 1H), 8.44-8.49 (m, 4 H), 10.15-10.22 (m, 1 H).

1.10 ADL12-D20 and ADL12-D21

[1141] General procedure 1, specified in section 1.2.1, was used to synthesize D20.

[1142] (2S,3S,6S,7R,10R, E)-7-Methoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy) -7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl )-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-ylmethyl(2-(methylamino)ethyl)carbamate (43 mg, 0.044 mmol, 90% yield) as a colorless oil. LC/MS (ESI, mass/charge), 981.4 [M+H] ⁺ .

[1143] ¹ H-NMR (400 MHz, MeOH- d 4 ): δ ppm 0.62-0.73 (m, 18 H), 0.82-0.93 (m, 9 H), 0.95 -1.06 (m, 27 H), 1.24 (br s, 4 H), 1.45 (br s, 10 H), 1.78 (br s, 3 H), 1.92-2, 00 (m, 1 H), 2.46 (br s, 4 H), 2.51-2.67 (m, 3 H), 2.76-2.84 (m, 2 H), 2.86 -2.93(m, 1H), 2.93-3.02(m, 3H), 3.34-3.38(m, 3H), 3.43-3.49(m, 2 H), 3.72-3.82 (m, 1 H), 3.93-4.04 (m, 1 H), 4.94-5.07 (m, 2 H), 5.53-5 .64 (m, 1 H), 5.72-5.89 (m, 2 H), 6.07-6.21 (m, 1 H), 6.44-6.61 (m, 1 H) .

D20

[1144] (2S,3S,6S,7R,10R, E)-10-Hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R ,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4- en-6-ylmethyl(2-(methylamino)ethyl)carbamate (16.4 mg, 0.026 mmol, 58.6% yield) as a colorless oil. LC/MS (ESI, mass/charge), 639.7 [M+H] ⁺ .

[1145] ¹ H-NMR (400 MHz, MeOH -d 4): δ ppm 0.92 (br d, J = 6.53 Hz, 9 H), 1.25 (br s, 4 H), 1, 36 (s, 3 H), 1.41-1.74 (m, 6 H), 1.45-1.59 (m, 1 H), 1.64-1.72 (m, 1 H), 1.80 (s, 3 H), 1.85-1.94 (m, 1 H), 2.42 (s, 3 H), 2.50-2.63 (m, 3 H), 2, 65-2.73(m, 1H), 2.73-2.80(m, 2H), 2.88-3.01(m, 4H), 3.35-3.38(m, 3 H), 3.35-3.40 (m, 3 H), 3.41-3.48 (m, 2 H), 3.52-3.57 (m, 1 H), 3.78- 3.89(m, 1H), 5.00-5.13(m, 2H), 5.53-5.64(m, 1H), 5.71-5.82(m, 1H ), 5.84-5.94 (m, 1 H), 6.12-6.19 (m, 1 H), 6.49-6.61 (m, 1 H).

ADL12-D20.

[1146] The general procedure in section 1.6.1 was used to synthesize ALD12-D20 and obtained (13.0 mg, 0.016 mmol, 67.7%) as a colorless oil. LC/MS (ESI, mass/charge), 818.3 [M+H] ⁺ .

[1147] ¹ H-NMR (400 MHz, MeOH- d 4 ): δ ppm 0.91 (br s, 9 H), 1.21-1.31 (m, 4 H), 1.36 (br s , 7 H), 1.46-1.73 (m, 11 H), 1.80 (s, 3 H), 1.84-1.93 (m, 1 H), 2.50-2.72 (m, 7 H), 2.75-2.85 (m, 1 H), 2.87-3.09 (m, 7 H), 3.48-3.68 (m, 6 H) 3, 80-3.89(m, 1H), 5.02-5.13(m, 2H), 5.55-5.66(m, 1H), 5.71-5.82(m, 1 H), 5.85-5.94 (m, 1 H), 6.11-6.22 (m, 1 H), 6.49-6.61 (m, 1 H), 6.83 ( br d, J = 1.13 Hz, 2 H).

ADL12-D21.

[1148] ((2S,3S,6S,7R,10R, E)-10-Hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-(( 2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4 -en-6-yl-(2-((6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl)(methyl)amino)ethyl)(methyl)carbamate (7 mg, 8.557 µmol) was dissolved in N,N-dimethylformamide (99 µl, 1.284 mmol) and methyl iodide (2.68 µl, 0.043 mmol) was added. The solution was stirred at RT overnight. The solvent was evaporated. column chromatography gave 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-(((((2S,3S,6S,7R,10R, E)- 10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl )-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)(methyl)amino)ethyl) -N,N-dimethylhexane-1-aminium (7 mg, 8.40 µmol, 98% yield) LC/MS (ESI, mass/charge), 832.7 [M+H] ⁺ .

[1149] ¹ H-NMR (400 MHz, MeOH- d 4 ): δ ppm 0.75-0.87 (m, 9 H), 1.14 (s, 4 H), 1.25 (s, 3 H), 1.27-1.45(m, 9H), 1.48-1.61(m, 4H), 1.69(s, 4H), 1.72-1.79(m , 2 H), 2.35-2.53 (m, 3 H), 2.53-2.60 (m, 1 H), 2.75-2.83 (m, 1 H), 2.85 -2.95 (m, 3 H), 3.06 (br d, J =10.42 Hz, 6 H), 3.15-3.20 (m, 2 H), 3.25 (br d, J = 2.38 Hz, 3 H), 3.42 (br d, J = 6.90 Hz, 5 H), 3.53-3.69 (m, 2 H), 3.69-3.78 (m, 1 H), 4.88-5.01 (m, 2 H), 5.44-5.56 (m, 1 H), 5.59-5.72 (m, 1 H), 5 .73-5.83(m, 1H), 5.99-6.10(m, 1H), 6.40(s, 1H), 6.72(s, 2H).

1.11 ADL12-D22

[1150] General procedure 1, specified in section 1.2.1, was used to synthesize D22.

[1151] 3-(4-((((2S,3S,6S,7R,10R, E)-7-Methoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl- 6-((triethylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2 ,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-yl)oxy)carbonyl)piperazin-2-yl)propanoic acid (48 mg, 0.046 mmol , yield 34.4%) as a colorless oil. LC/MS (ESI, mass/charge), 1051.4 [M+H] ⁺ .

[1152] ¹ H-NMR (400 MHz, MeOH -d 4 ): δ ppm 0.62-0.70 (m, 18 H), 0.82-0.94 (m, 9 H), 1.01 (tt, J = 7.92, 3.18 Hz, 27 H), 1.22-1.28 (m, 4 H), 1.45 (s, 3 H), 1.47-1.66 ( m, 7 H), 1.76-1.87 (m, 5 H), 1.92-1.99 (m, 1 H), 2.36-2.47 (m, 3 H), 2, 50-2.68(m, 3H), 2.86-2.93(m, 1H), 2.98-3.08(m, 2H), 3.10-3.18(m, 1 H), 3.18-3.26 (m, 2 H), 3.27-3.31 (m, 1 H), 3.34-3.36 (m, 3 H), 3.36- 3.37(m, 1H) 3.72-3.81(m, 1H), 3.95-4.19(m, 3H), 4.93-5.00(m, 1H) , 5.01-5.07 (m, 1 H), 5.54-5.66 (m, 1 H) 5.72-5.81 (m, 1 H) 5.81-5.90 (m , 1 H), 6.12-6.19 (m, 1 H), 6.47-6.58 (m, 1 H).

D22

[1153] N-Ethyl-N-isopropylpropane-3-(4-((((2S,3S,6S,7R,10R, E)-10-hydroxy-2-((R,2E,4E)-6- hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)- 7-Methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)piperazin-2-yl)propanoic acid (23.5 mg, 0.031 mmol, 68.2% yield). LC/MS (ESI, mass/charge), 709.5 [M+H] ⁺ .

[1154] ¹ H-NMR (400 MHz, DMSO- d 6): δ ppm 0.71-0.77 (m, 9 H), 1.03 (s, 5 H), 1.14-1.20 (m, 6 H), 1.23-1.33 (m, 5 H), 1.36-1.48 (m, 5 H), 1.63 (s, 3 H), 1.67-1 .75 (m, 1 H), 2.13-2.24 (m, 4 H), 2.58-2.64 (m, 1 H), 2.68-2.81 (m, 2 H) , 3.20-3.20 (m, 3 H), 3.61-3.77 (m, 4 H), 4.28-4.39 (m, 1 H), 4.44-4.53 (m, 1 H), 4.79-4.88 (m, 2 H), 5.28-5.43 (m, 1 H), 5.52-5.66 (m, 1 H), 5 .73-5.85(m, 1H), 5.94-6.04(m, 1H), 6.27-6.41(m, 1H), 6.43-6.53(m , 2H), 8.19-8.29 (m, 1H).

ADL12-D22.

[1155] The general procedure outlined in section 1.6.1 was used to synthesize ADL12-D22 (3.6 mg, 4.05 µmol, 34.4% yield). LC/MS (ESI, mass/charge), 888.7 [M+H] ⁺ .

[1156] ¹ H-NMR (400 MHz, MeOH- d 4 ): δ ppm 0.77-0.89 (m, 9 H), 1.08-1.31 (m, 13 H), 1.35 -1.60(m, 12H), 1.69(s, 3H), 1.73-1.83(m, 1H), 1.85-1.97(m, 1H), 2 .15-2.27(m, 1H), 2.27-2.83(m, 11H), 2.84-2.98(m, 1H), 3.39(s, 7H) , 3.67-3.77(m, 1H), 4.89-5.01(m, 2H), 5.40-5.53(m, 1H), 5.59-5.69 (m, 1 H), 5.72-5.83 (m, 1 H), 5.99-6.11 (m, 1 H), 6.37-6.49 (m, 1 H), 6 .69 (s, 2H).

ADL1-D22.

[1157] General Procedure 1 in section 1.2.1 was used to synthesize ADL1-D22 (4.7 mg, 3.59 µmol, 38.6% yield) as a white lyophilized solid. LC/MS (ESI, mass/charge), 1307.6 [M+H] ⁺ .

[1158] ¹ H NMR (400 MHz, DMSO- d 6): δ ppm 0.68-0.79 (m, 16 H), 1.03 (s, 3 H), 1.16 (s, 5 H ), 1.23-1.44 (m, 13 H), 1.48-1.55 (m, 1 H), 1.62 (s, 4 H), 1.68-1.74 (m, 1 H), 1.86-1.93 (m, 1 H), 2.02 (s, 4 H), 2.24-2.37 (m, 3 H), 2.46-2.51 ( m, 2 H), 2.66-2.73 (m, 1 H), 2.79-2.99 (m, 4 H), 3.15 (s, 3 H), 3.21-3, 22 (m, 2 H), 3.30 (s, 4 H), 3.61-3.67 (m, 1 H), 3.73-3.88 (m, 3 H), 4.01- 4.08(m, 1H), 4.09-4.16(m, 1H), 4.29-4.38(m, 2H), 4.43-4.51(m, 1H ), 4.73-4.78 (m, 1 H), 4.79-4.88 (m, 2 H), 4.91-4.98 (m, 2 H), 5.29-5, 43 (m, 3 H), 5.54-5.67 (m, 1 H), 5.74-5.83 (m, 1 H), 5.95-6.07 (m, 1 H), 6.28-6.40(m, 1H), 6.54-6.62(m, 1H), 6.93(s, 2H), 7.17-7.26(m, 2H ), 7.44-7.57(m, 2H), 7.66-7.76(m, 1H), 7.95-8.03(m, 1H), 8.42-8, 49(m, 1H), 9.89-9.96(m, 1H).

ADL1-D23.

[1159] 4-((S)-2-((S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido) -5-ureidopentanamido)benzyl(4-nitrophenyl)carbonate (16.76 mg, .023 mmol) and 3-((R)-4-((((2S,3S,6S,7R,10R, E)-10 -hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl) -6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)piperazin-2-yl)propanoic acid (16.1 mg, 0.023 mmol) was dissolved in DMF (229 μl, 2.953 mmol) and Hunig's base (11.90 μl, 0.068 mmol) was added. The reaction mixture was stirred overnight at rt. The solvent was then evaporated and purified by reverse phase HPLC to give 3-((R)-1-(((4-((S)-2-((S) -2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)-4-(( ((2S,3S,6S,7R,10R, E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S )-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en- 6-yl)oxy)carbonyl)piperazin-2-yl)propanoic acid (3.9 mg, 2.98 µmol, 13.13% yield). LC/MS (ESI, mass/charge), 1308.6 [M+H] ⁺ .

ADL1-D24(S)

[1160] 3-((S)-4-((((2S,3S,6S,7R,10R, E)-10-Hydroxy-2-((R,2E,4E)-6-hydroxy-7- ((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy- 3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)piperazin-2-yl)propanoic acid (10mg, 0.014mmol), N,N-dimethylformamide (141µl, 0.014mmol) , Hunig's base (5 µl, 0.028 mmol), 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl )hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl)carbonate (20.8 mg, 0.028 mmol) was combined and stirred for 2 hours. Chromatographed (MeOH/CH ₂ Cl ₂ ) to give 3-((S)-1-(((4-((S)-2-((S)-2-(6-(2,5-dioxo-2 ,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)-4-((((2S,3S,6S,7R,10R, E) -10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2- yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)piperazin-2-yl) propanoic acid (5 mg, 3.82 µmol, 27.1% yield). LC/MS (ESI, mass/charge), 1308.4 [M+H] ⁺ .

EXAMPLE 2

[1162] Exemplary payloads of spliceosome modulators used in ADC generation were profiled. Payloads were evaluated for binding to the SF3b complex, in vitro splicing activity, and ability to inhibit cell growth.

2.1 SF3B1 Binding/Scintillation Proximity Assay (SPA)

[1163] A scintillation proximity assay was performed to measure the binding affinity of compounds ("payloads") to the SF3b complex. Immobilization of a portion of anti-SF3B1 antibody (MBL) onto anti-mouse Ig coated scintillation beads from PVT for SPA analysis (PerkinElmer) was performed as follows: for each 2.5 mg of nuclear extracts, 5 µg of anti-SF3B1 antibody and 1.5 mg of beads were mixed in 150 µl PBS. The antibody-bead mixture was incubated for 30 min. at k. t. and centrifuged at 18000g within 5 min. 150 μl of PBS was used to resuspend each 1.5 mg of the antibody-bead mixture. The granules were suspended and added to the resulting nuclear extracts. The suspension was incubated for 2 hours at 4°C with gentle stirring. The pellets were then collected by centrifugation at 18000g within 5 min. and washed twice with PBS+0.1% Triton X-100. After the final centrifugation step, each 1.5 mg of pellets were suspended using 150 μl of PBS. The SF3b complexes were tested for binding to a probe based on [³H]-labeled pladienolide ([³H]-PB) synthesized as previously described (Kotake et al. (2007) Nat Chem Biol. 3(9):570-5). 100 µl of binding reactions were prepared using 50 µl of bead suspension and by adding various concentrations of PB or PB-OH, and after 30 min. pre-incubation was added 2.5 nm [³H]-PB. The mixture was incubated for 30 min. and luminescence signals were read using a MicroBeta2 plate counter (PerkinElmer). Prism 7 (Graphpad) was used to fit the data to a non-linear regression curve.

[1164] Similar binding profiles were found across all payloads tested ( FIG. 1 ). In general, specific binding was in the lower nanomolar range, indicating that all of the tested payloads are agents with high binding activity to the SF3b complex and promising candidate compounds for use in ADC.

2.2 In vitro splicing (IVS)

[1165] An in vitro splicing assay was performed to evaluate load activity in a cell-free system. Payloads were incubated with nuclear extracts and a pre-mRNA-based construct as a substrate and minigenes.

[1166] Preparation of HeLa nuclear extract : pellets containing HeLa S3 cells were resuspended in hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl ₂ , 10 mM KCl, 0.2 mM PMSF and 0.5 mM DTT) and the suspension was adjusted to a total of 5 settled cell volumes (PCV). After centrifugation, the supernatant was discarded and the cells were adjusted to 3 PCV with hypotonic buffer and incubated on ice for 10 minutes. Cells were lysed using a Downs homogenizer and then centrifuged. The supernatant was discarded and the pellet was resuspended with ½ volume of precipitated nuclei (PNV) low salt buffer (20 mM HEPES, pH 7.9, 1.5 mM MgCl ₂ , 20 mM KCl, 0.2 mM EDTA, 25% glycerol, 0.2 mM PMSF, 0.5 mM DTT) then ½ PNV high salt buffer (same as low salt buffer except 1.4 M KCl was used). The cores were gently stirred for 30 minutes. before centrifugation. The supernatant (nuclear extract) was then dialyzed into storage buffer (20 mM HEPES, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 0.2 mM PMSF, 0.5 mM DTT). Protein concentration was determined using a NanoDrop 8000 UV-Vis spectrophotometer (ThermoFisher Scientific).

[1167]IVS: all sequences derived from Ad2 (Pellizzoni et al. (1998) Cell 95(5):615-24) were cloned into the vector pcDNA3.1(+) (Promega) using restriction sites at the 5' end for EcoRI and 3'-end for XbaI. Plasmids were linearized using XbaI and used as DNA templates in in vitro transcription reactions. Plasmid without intron FtzΔi (Luo and Reed (1999) 96(26):14937-42) was linearized using EcoRI. All RNAs were transcribed in vitro and then purified using the MEGAScript T7 (Invitrogen) and MegaClear (Invitrogen) kits, respectively. For splicing reactions using pre-mRNA Ad2 variants, a 1 μl reaction mixture was prepared using 8 μg of nuclear extracts derived from HeLa S3 cells, 2 ng pre-mRNA, 0.2 ng FTZ∆i, and varying concentrations of exemplary compounds or DMSO. After 15 min. pre-incubation at 30°C, 1 µl of buffer for splicing activation (0.5 mM ATP, 20 mM creatine phosphate, 1.6 mM MgCl₂) and the reactions were incubated for 90 min. at 30°C. The reactions were then quenched with 13 µl DMSO and 25 nl each was used for RT-qPCR. RT-qPCR reactions were prepared using a 1-step TaqMan RNA-to-C kit_T (Life Technologies), RNA from splashing reactions, primers-zond sets for MRNA AD2 (line: ActcttCCCCGCGTCGTCGTCCCAA; probe: CTGTGTGGCTCGCGTTG) and FTZ (Direct: T: T: T: T: T GGCATCAGATTGCAAAGAAGAC; reverse: ACGCCGGGTGATTATATAT; SUSK: CGAAACGCACCCCGTCAGACG). Prism 7 (Graphpad) was used to fit the spliced product data to a non-linear regression curve and normalized to a control sample (DMSO). Given that all tested payloads specifically bind to the SF3b complex and exhibit similar binding profiles (fig. 1), hypothesized that all should also modulate splicing to a comparable extent. All loadings significantly modulated pre-mRNA Ad2.2 splicing (fig. 2). In the presence of loading, a decrease in the amount of spliced product was observed.

2.3 Cell viability

[1168] The HCC1954 line (American Type Culture Collection (ATCC)) from breast ductal carcinoma cells was seeded at 2000 cells/well in flat-bottomed 96-well tissue culture plates (Corning) in a total volume of 90 μl of tissue culture medium with 10% fetal bovine serum (ThermoFisher Scientific). Cells were treated using a 3-fold serial dilution of the compound from 200 nM to 0.03 nM. Each concentration was tested in triplicate. At the time of treatment, the raw cell plate was evaluated using the CellTiter-Glo®2.0 Luminescent Cell Viability Assay as recommended by the manufacturer (Promega; No. G9241). CellTiter-Glo® 2.0 reagent was added to the medium, incubated and analyzed on an EnVision Multilabel Reader (PerkinElmer). Values are presented with time zero (T0). Viable cell count after 144 hours (T144) compound treatment was also determined using the CellTiter-Glo®2.0 Luminescent Cell Viability Assay. luminescence value at time zero (T0), growth control (C) in DMSO, and growth in the presence of test compound (T144) were used to calculate the percent growth for each compound concentration. Percent growth inhibition was calculated as: [(T144-T0)/(C-T0)] x 100 for concentrations where T144>/=T0, or [(T144-T0)/T0] x 100 for concentrations where T144<T0. Dose-response curves were plotted using Prism 7 (Graphpad) and fitted using non-linear regression analysis and logarithm (inhibitor) versus response - variable slope (four parameters).

[1169] A dose-dependent effect on cell viability was determined for all payloads in HCC1954 breast cancer cells with HER2 amplification. Most of the payloads tested exhibited GI ₅₀ values (i.e., the concentration of a compound that causes a 50% reduction in cell proliferation) in the single nanomolar range, with the exception of poorly penetrating payloads such as D25 ( FIG. 3 ). Illustrative penetration data is shown in Table 14.

EXAMPLE 3

[1170] The exemplary loads that were evaluated in Example 2 were conjugated to an exemplary anti-HER2 antibody (trastuzumab) via cysteine residues on the antibody. The production and evaluation of exemplary HER2-based ADCs are described below.

3.1 Antibody

[1171] The antibody trastuzumab ("AB185") (Molina et al. (2001) Cancer Res. 61(12):4744-9) was used to generate ADC based on an anti-HER2 antibody (also referred to herein as SMLA).

3.2 Bioconjugation

[1172] An antibody (trastuzumab) at 10 mg/mL in PBS buffer (pH 7.0) was mixed with 5 mM TCEP (2-4 molar equivalents) (ThermoFisher Scientific; No. 77720) to cleave interchain disulfide bonds. The reaction mixture was gently stirred at 22 ^° C. for 3 hours. Propylene glycol (15% v/v) was then added followed by the addition of 8 molar equivalents of payload linker (6 mM stock solution in DMSO) and the solution was thoroughly mixed. The reaction was placed on a turntable in an oven at 22 ^° C. After 2 hours of conjugation, the reaction mixture was purified to remove unconjugated payload using AKTA GE M150 (HiTrapTM 26/10 desalting columns; flow rate: 3 ml/min.) (GE Healthcare Bio-Sciences) in DPBS (pH 7.5). The resulting conjugate was concentrated by ultrafiltration through Amicon (30 kDa, Ultra-4) (EMD Millipore) and sterile filtered through a 0.22 μm PVDF disposable filter (EMD Millipore). The resulting clear solution was evaluated by UV-VIS to determine the antibody concentration ([mAb]; mol/l) and the concentration of the conjugated payload ([LD]; mol/l) according to the Beer-Lambert law (A= E * c * l) and according to the following equations:

A _{280 nm=} E ^mAb _{280 nm} * [mAb] * l+ E ^LD _{280 nm} * [LD] * l

A _{252 nm=} E ^mAb _{252 nm} * [mAb] * l+ E ^LD _{252 nm} * [LD] * l

E ^mAb _{280 nm} : trastuzumab=213.380 cm ^-1 M ^-1

E ^mAb _{252 nm} : trastuzumab=79.112 cm ^-1 M ^-1

E ^LD _{280 nm} ⁼ 800 cm^-1M^-1

E ^LD _{252 nm} ⁼ 31,000 cm^-1M^-1

Abbreviations: c - molar concentration; l - optical length (Nanodrop: 0.1 cm); E is the molar coefficient of light absorption; A - absorption.

3.3 Determination of biophysical characteristics

[1173] Drug to antibody ratio (DAR), percent aggregation, and percent unconjugated payload were determined for exemplary anti-HER2 antibody based ADCs by liquid chromatography-mass spectrometry (LC/MS), size exclusion chromatography (SEC), reverse phase high performance liquid chromatography (HPLC) respectively. In general, conjugates contain less than 2% free form drug and less than 10% aggregates.

3.3.1 LC/MS analysis - DAR

[1174] LC/MS analysis was performed using an Agilent 1290 UPLC system coupled to an Agilent G6224A Accurate Mass TOF time-of-flight mass spectrometer. The conjugate was deglycosylated with PNGase F (New England Biolabs; No. P0705L) for 4 hours at 37 ^° C, denatured with 8 M Gdn-HCl (Sigma; No. G9284), and finally resolved into light and heavy chain domains with using DTT (final concentration 5 mM) (Promega; no. V3151). The resulting sample was loaded onto an Agilent PLRP-S column (2.1×150 mm, 8 µm) and eluted with a gradient from 25% B to 50% B over 28 min. at room temperature (RT). Mobile phase A was water with 0.05% TFA, mobile phase B was acetonitrile with 0.04% TFA, with a flow rate of 1 ml/min. DAR was calculated from the post-deconvolution mass spectrum using weighted averages of the intensities of the unconjugated and conjugated drug peaks for the light chain (L0 or L1) and heavy chain (H0, H1, H2 and H3). The total DAR for the intact conjugate was calculated using the equation: (DAR _LC * 2) + (DAR _HC *2) = total DAR. DAR values for exemplary HER2 antibody based ADCs are shown in Tables 10-14.

3.3.2 SEC analysis - aggregation

[1175] Size exclusion chromatography was performed using a TOSON-G3000SWXL column (#008541) in 0.2 M potassium phosphate (pH 7) with 0.25 mM potassium chloride and 15% (v/v) IPA at a flow rate of 0.75 ml /min The area of the absorption peaks at 280 nm was determined for the high molecular weight and monomeric components of the conjugate by integrating the area under the curve. The percentage of monomers for exemplary HER2 antibody based ADCs is shown in Table 10.

3.3.3 HPLC Assay - Free Form Drug

[1176] The conjugate was precipitated using 10 volumes of acetonitrile on ice for 2 hours and precipitated by centrifugation. Supernatants containing residual unconjugated payload were then loaded onto an Agilent Poroshell 120 SB-C18 120A column (4.6×100 mm, 2.7 µm) and eluted with a gradient from 45% B to 70% B over 10 min. At RT. Mobile phase A was 100% water, mobile phase B was 100% acetonitrile, and the flow rate was 0.6 ml/min. with detection at 252 nm. The amount of residual free drug was quantified by UV detection compared to an external standard curve for the unconjugated payload linker. The percent free drug for exemplary anti-HER2 antibody ADCs is shown in Table 10.

3.4 Binding characteristics

3.4.1 Determination of binding characteristics to target-positive cells by FACS

[1177] Binding of unconjugated anti-HER2 antibody and ADC based on anti-HER2 antibody to target-positive cells was assessed by flow cytometry using indirect immunofluorescence. JIMT1 breast cancer cells (DSMZ) that endogenously express HER2 were seeded (5×10 ⁴ cells/well) in a 96-well v-bottom plate (Greiner Bio-One) and incubated for 2 hours at 4°C with test compounds diluted to various concentrations in assay medium (RPMI-1640 supplemented with 10% (w/v) fetal bovine serum albumin (Thermo Fisher Scientific)). Cells were then washed with PBS+2% FBS (FACS buffer) and stained with phycoerythrin-labeled (PE) goat anti-immunoglobulin G (IgG) antibody (Invitrogen) for 40 min. at 4°C in the dark. Cells were washed with cold FACS buffer and fixed with FluroFix buffer (Biolegend) for 30 min. at room temperature. Fixed cells were washed using FACS buffer. Fixed cells were analyzed for geometric mean PE fluorescence intensity using an LSRFortessa flow cytometer (BD Bioscience). Trastuzumab and T-DM1 (DM1 conjugated to trastuzumab) were included as controls.

[1178] All HER2 antibody-based ADCs showed stable binding to HER2 on JIMT1 cells. ADC binding was comparable to that of trastuzumab and T-DM1 ( Figure 4 ). This indicates that payload conjugation does not affect the antigen-binding affinity of the antibody.

3.5 In vitro analysis

3.5.1 Cell viability

[1179] Anti-HER2 ADCs were tested in several HER2-amplifying cell lines for their ability to inhibit cell growth. The cell lines used were HCC1954 (ATCC), 2000 cells/well), N87 (ATCC, 4000 cells/well), SKBR3 (ATCC, 3000 cells/well) and MCF7 (ATCC, 1500 cells/well). Cell viability analysis was performed as described in section 2.3.

[1180] Surprisingly, not all ADCs were active on HCC1954 cells, despite the fact that they have similar binding profiles ( FIG. 1 ) and payloads with similar biochemical properties. ADCs with certain linkers (eg, ADL10) and/or some payloads (eg, D14) were less able to inhibit cell growth, while ADCs with alternating linkers (ADL1, ADL5, ADL12) and/or payloads (eg, D1 , D25, D2, D4) were more active on HCC1954 cells ( Figure 5A and Tables 10, 11 and 14). Such trends were commonly observed among cell lines (HCC1954, N87, and SKBR3) and traits (HCC1954 and SKBR3 are breast cancer cell lines with high HER2 expression; N87 is a gastric cancer cell line with high expression of HER2) ( Fig. 5B-C and tables 11 and 12). Moreover, traditional non-cleavable linkers, such as ADL10, do not typically effectively deliver splicing modulator payloads. However, small modifications in the length of the same linkers make ADCs more active, for example when comparing AB185-ADL10-D1 and AB185-ADL12-D1 for a dose-dependent effect on cell viability ( FIG. 5A-C ).

[1181] To ensure that the anti-HER2 antibody-based ADC activity is antigen-dependent, HER2-negative MCF7 cells were treated. None of the tested ADCs were active at the same concentrations as sustainably targeted HER2-positive cells (fig. 6 and table 13). This indicates that the anti-HER2 antibody-based ADC activity is antigen-dependent.

Table 10 Characteristics of exemplary anti-HER2 antibody based ADCs

Таблица 10 (продолжение). Характеристики иллюстративных ADC на основе антитела к HER2

Table 10 (continued). Characteristics of exemplary anti-HER2 antibody-based ADCs

Table 10 (continued). Characteristics of exemplary anti-HER2 antibody-based ADCs

Table 10 (continued). Characteristics of exemplary anti-HER2 antibody-based ADCs

Table 10 (continued). Characteristics of exemplary anti-HER2 antibody-based ADCs

Table 11 Exemplary anti-HER2 ADCs - HCC1954 cells

Table 12. Exemplary anti-HER2 ADCs - N87 cells

Table 13 Exemplary Anti-HER2 ADCs - MCF7 Cells

3.5.2 Payload penetration of ADCs against Caco-2 cells

[1182] Caco-2 cells were cultured for 21 days in 24-well plates with Transwell inserts at 37°C, 95% humidity, 5% CO ₂ . Cell monolayer integrity was confirmed by TEER (transepithelial electrical resistance) and lucifer yellow stain. Payloads were applied in duplicate at 10 μm, separately on each side of the cell monolayer. Apical to basolateral (AB) and basolateral to apical (BA) penetration rates were determined by taking aliquots from both chambers immediately after treatment (t=0) and after incubation for 2 hours. The samples were protein precipitated with an organic solvent containing an internal standard and were analyzed by LC-MS/MS (SCIEX; API 5500). The ratio of payload peak area/internal standard versus time in both directions was used to obtain penetration values (cm/s). The efflux coefficient was calculated by dividing BA/AB. Control compounds for low and high penetration and efflux performed as expected. Penetration values are shown in Table 14.

3.5.3 Chemical stability of ADC with payload

[1183] Payloads were incubated in McIlvaine buffer (citrate-phosphate), pH 5.5 (Boston Bioproducts; No. BB-2466) at a final concentration of 20 μM (less than 0.5% DMSO from the stock solution). The payload solution and the internal standard were pipetted into 96-well plates, run a UPLC cycle (Waters Aquity H-class) and analyzed for the original chromatographic signal (t=0). The column was a UPLC HSS T3 1.8 µm, 2.1×50 mm column (#186003538). A gradient of mobile phase A from 95% to 10% was used over 1 min where A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile (flow rate 0.9 ml/min.). The remainder of the usable solution was incubated in a plate shaker at 37°C (Eppendorf ThermoMixer). Sample analyzes by UPLC were repeated at 24, 72 and 96 hours after incubation at 37°C. The ratio of peak area values for the payload and the internal standard was determined for three time points: for time point 0, day 1, and either day 3 or day 4. Time point 0 was defined as 100. The ratio of peak area values for later time points was compared with time 0. The percentage remaining was calculated as follows: (ratio of peak area values at day X/ratio of peak area values at time 0) * 100 = % remaining. A curved line was generated by Excel calculations comparing log % remaining and time point. Half-life values were calculated in Excel using the natural logarithm(2)/curve and are shown in Table 14.

Table 14 Characteristics of exemplary anti-HER2 antibody ADCs and associated payloads

Table 14 (continued). Characteristics of exemplary HER2 antibody-based ADCs and associated payloads

Table 14 (continued). Characteristics of exemplary HER2 antibody-based ADCs and associated payloads

3.5.4 PD analysis of splicing in a cell

[1184] To investigate in detail the mechanism of action of exemplary HER2-based ADCs, splicing of the mature SLC25A19 transcript was examined in HCC1954 cells treated with increasing concentrations of ADCs.

[1185] HCC1954 cells (ATCC) were seeded in RPMI+10% FBS without phenol red (ATCC) at 1×10 ⁵ cells per well in an amount of 90 μl per well. Cells were treated with the conjugates in a dose-dependent manner at a 3-fold dilution. After 24 hours or 6 hours, respectively, cells were lysed using 50 µl of CL buffer (IgePal CA-630, 5M NaCl, 1M Tris-HCl 1M pH 7.4 in water) containing 25 µl/ml RNasin® (Promega) and incubated for 45 min. at RT on an oscillating stirrer. The resulting mixture (0.5 μl) was used to assess splicing modulation in a reverse transcription PCR reaction using Taqman Fast Virus 1-Step MasterMix (Applied Biosystems) and the following Taqman primers as recommended by the manufacturer: SLC25A19 (Invitrogen, Hs00222265_m1); RPLPO (Invitrogen, Hs99999902_m1); 18S (Invitrogen, Hs99999901_s1).

[1186] The T-DM1 control (trastuzumab with a microtubule degrading agent as payload) did not have any effect on SLC25A19 splicing ( FIG. 7 ). In contrast, HER2 antibody-based ADCs, which exhibit strong growth-inhibitory activity in different cell lines ( Figure 5 ), also exhibit strong splicing modulation, indicative of a targeted mechanism of action ( Figure 7 ). Similarly, HER2 antibody-based ADCs that show low activity in the cell viability assay were less active in terms of splicing modulation.

3.5.5 Non-specific cell killing assay

[1187] The ability of exemplary anti-HER2 antibody conjugates to kill both target-positive cells and neighboring target-negative cells was measured in a non-specific cell killing assay. NCI-H1568 non-small cell lung cancer (ATCC) cells were engineered to express luciferase. cDNA was synthesized with GeneArt (ThermoFisher Scientific) and cloned into the pLVX-EF1a delivery vector (ThermoFisher Scientific). NCI-H1568 was separately engineered to express human HER2 (cloned into pLenti6.3/V5-DEST, ThermoFisher Scientific) using the TransIT® viral transfection system according to the manufacturer's instructions (Mirus Bio LLC; no. MIR6003). To evaluate the non-specific activity of the conjugates, 2000 target-positive (HER2-transduced) NCI-H1568 cells and 2000 target-negative (luciferase-labeled) NCI-H1568 cells were plated together in clear round-bottom 96-well plates (Corning) and incubated with conjugates added to 3-fold serial dilutions in triplicate, 30-0.005 nM. During processing, the plate with untreated cells was analyzed using the OneGlo® Luciferase Assay System (Promega) and the CellTiter-Glo®2.0 Luminescent Cell Viability Assay System (Promega) according to the manufacturer's recommendations for determining the moment time zero (T0). After 6 days in culture, the remaining cell population was analyzed by the OneGlo® Luciferase Assay System (Promega) to measure the fraction of surviving target negative cells. Overall viability was measured using the CellTiter-Glo®2.0 Luminescent Cell Viability Assay System (T144). Dose-response curves were constructed as described in section 2.3.

[1188] The design of the non-specific killing assay allows detection of only target-negative cells whose elimination is due to uptake of catabolite released by neighboring target-positive cells. As discussed above, cells were plated under three conditions: (1) target-negative cells only (luciferase labeled); (2) only target-positive cells; and (3) co-culture of target-negative and target-positive cells. If the reaction assay in the plates is performed by reading using the CellTiter-Glo® reagent, then viability of all cell populations can be determined to confirm antigen-dependent ADC activity. In contrast, if the OneGlo® reagent is used, only target-negative (luciferase labeled) cells will generate a signal.

[1189] Target-negative (luciferase-labeled) cells were insensitive to treatment with HER2 antibody-based ADC when cultured alone ( FIG. 8 ). However, when target-negative cells were cultured with target-positive cells, treatment with an anti-HER2 antibody ADC resulted in increased killing of target-negative cells. These data indicate that target-negative cells were more efficiently killed by anti-HER2 antibody-based ADCs when they were co-cultured with target-positive cells, referred to herein as non-specific killing. Non-specific killing is distinct from non-target killing, which is defined as the killing of self-target (antigen)-negative cells in the absence and regardless of co-culture with target-positive cells.

[1190] In this experiment, AB185-ADL5-D2 demonstrates an increased ability to kill populations of cancer cells that are heterogeneous in antigens, compared with AB185-ADL1-D1. Without being bound by theory, it is believed that this difference in potency may be due to the addition of one methyl group to D2, which provides a threefold increase in cell payload activity and penetration compared to D1 (see Table 14). ADC D2 treatment of a population of antigenically heterogeneous cancer cells is as effective as treatment with a control ADC based on AB185-MC-Val-Cit-MMAE.

3.6 In vivo analysis

[1191] Exemplary anti-HER2 antibody-based ADCs were evaluated in vivo and exhibited pronounced splicing modulation and cell growth inhibition.

3.6.1 Efficacy study using HCC1954 based xenograft

[1192] To investigate the efficacy of exemplary anti-HER2 antibody-based ADCs in the HCC1954 mouse xenograft cell (ATCC) xenograft model, breast ductal carcinoma cells (10×10 ⁶ cells/100 μl/1:1 RPMI:Matrigel by volume) were implanted subcutaneously into right side of female CB17-SCID mice. Mice were treated with a single IV bolus dose of the conjugates (formulated in DPBS, pH 7.4) or vehicle control. The amounts administered to the animals are shown in FIG. 9 . Twice weekly, animals were examined for tumor growth and body weight changes until they reached one of the following endpoints: (1) excessive tumor volume (calculated from the ellipsoid formula): (length x width ² )/2) or (2) occurrence any health disorder such as excessive body weight loss. All animal studies were performed in accordance with the Biomedical Guidelines for the Care and Use of Laboratory Animals H3.

[1193] Treatment with an anti-HER2 antibody ADC (AB185-ADL5-D2 or AB185-ADL1-D1) at a single dose of at least 2 mg/kg resulted in significant tumor regression ( FIG. 9 ). A dose-dependent effect of the conjugated payload was also observed, in which anti-HER2 antibody-based ADCs with higher DAR values (DAR ~ 4) showed increased efficacy and slowed tumor growth to a greater extent compared to the control T-DM1 antibody at the same dose. In addition, the tumor-targeting payload through the ADC was more efficient than the corresponding unconjugated payload.

EXAMPLE 4

[1194] To determine whether the properties of the exemplary HER2 antibody-based ADCs described in Example 3 can be extrapolated to other ADCs targeting alternative antigens and/or conditions, certain payloads were conjugated to an exemplary anti-CD138 antibody (B-B4 ) and exemplary anti-EPH2A antibody (1C1). CD138 is expressed on malignant plasma cells, such as in multiple myeloma, while EPH2A is more commonly expressed in a variety of malignancies. The production and evaluation of exemplary CD138 and EPH2A antibody based ADCs are described below.

4.1 Antibodies

[1195] Antibody B-B4 ("AB205") (Tassone et al. (2004) Blood 104:3688-96) and antibody 1C1 ("AB206") (Jackson et al. (2008) Cancer Res. 68(22) :9367-74) was used to generate an anti-CD138 ADC and an anti-EPH2A ADC, respectively.

4.2 Bioconjugation

[1196] Antibody (B-B4 or 1C1) at 10 mg/ml in PBS buffer (pH 7.0) was mixed with 5 mM TCEP (2-4 molar equivalents) (ThermoFisher Scientific; No. 77720) to cleave interchain disulfide bonds. The reaction mixture was gently stirred at 22 ^° C. for 3 hours. Propylene glycol (15% v/v) was then added followed by the addition of 8 molar equivalents of payload linker (6 mM stock solution in DMSO) and the solution was thoroughly mixed. The reaction was placed on a turntable in an oven at 22 ^° C. After 2 hours of conjugation, the reaction mixture was purified to remove unconjugated payload using AKTA GE M150 (HiTrapTM 26/10 desalting columns; flow rate: 3 ml/min.) (GE Healthcare Bio-Sciences) in DPBS (pH 7.5). The resulting conjugate was concentrated by ultrafiltration through Amicon (30 kDa, Ultra-4) (EMD Millipore) and sterile filtered through a 0.22 μm PVDF disposable filter (EMD Millipore). The resulting clear solution was evaluated by UV-VIS to determine the antibody concentration ([mAb]; mol/l) and the concentration of the conjugated payload ([LD]; mol/l) according to the Beer-Lambert law (A= E * c * l) and according to the following equations:

A _{280 nm=} E ^mAb _{280 nm} * [mAb] * l+ E ^LD _{280 nm} * [LD] * l

A _{252 nm=} E ^mAb _{252 nm} * [mAb] * l+ E ^LD _{252 nm} * [LD] * l

E ^mAb _{280 nm} : B-B4=224.320 cm ^-1 M ^-1

1C1=215.380 cm ^-1 M ^-1

E ^mAb _{252 nm} : B-B4=83.670 cm ^-1 M ^-1

1C1=80.337 cm ^-1 M ^-1 ;

E ^LD _{280 nm} ⁼ 800 cm^-1M^-1

E ^LD _{252 nm} ⁼ 31,000 cm^-1M^-1

Abbreviations: c - molar concentration; l - optical length (Nanodrop: 0.1 cm); E is the molar coefficient of light absorption; A - absorption.

4.3 Determination of biophysical characteristics

[1197] DAR, percent aggregation, and percent unconjugated payload were determined for exemplary anti-CD138 antibody and anti-EPHA2 antibody based ADCs by liquid chromatography-mass spectrometry (LC/MS), size exclusion chromatography (SEC), reverse phase high performance liquid chromatography (HPLC), respectively. All conjugates described herein contain less than 2% free form drug and contain less than 10% aggregates.

4.3.1 LC/MS analysis - DAR

[1198] LC/MS analysis was performed as described in section 3.3.1. DAR values for exemplary anti-CD138 and anti-EPHA2 ADCs are shown in Tables 15-18.

4.3.2 SEC analysis - aggregation

[1199] SEC analysis was performed as described in section 3.3.2. The percentage of monomers for exemplary anti-CD138 and anti-EPHA2 ADCs are shown in Tables 15-17.

4.3.3 HPLC Assay - Free Form Drug

[1200] HPLC analysis was performed as described in section 3.3.3. The percent free form drug for exemplary anti-CD138 and anti-EPHA2 ADCs is shown in Tables 15-17.

4.4 In vitro analysis

4.4.1 Cell viability

[1201] An anti-CD138 ADC was tested on a multiple myeloma cell line, MOLP8, expressing CD138. Similarly, the anti-EPHA2 ADC was tested on the PC3 prostate cancer cell line expressing EPHA2. Cell viability analysis was performed as described in section 2.3.

[1202] Conjugation of the payload with an anti-CD138 antibody ( FIG. 10 and Table 16) and an anti-EPHA2 antibody ( FIG. 11 and Table 18) resulted in patterns similar to those discussed in section 3.5. HER2. In particular, ADCs with certain linkers (eg ADL12) and/or payloads (eg D1, D25, D4) showed the highest activity against antigen expressing cells. Accordingly, alternative payloads (eg, D14) were less able to inhibit cell growth via antibody-mediated delivery despite being highly active small molecule compounds.

Table 15 Characteristics of exemplary anti-CD138 antibody based ADCs

Table 15 (continued). Characteristics of exemplary anti-CD138 antibody-based ADCs

[1203]

Table 16 Exemplary anti-CD138 ADCs - MOLP8 cells

Таблица 17. Характеристики иллюстративных ADC на основе антитела к EPHA2

Table 17 Characteristics of exemplary anti-EPHA2 antibody based ADCs

Table 17 (continued). Characteristics of exemplary anti-EPHA2 antibody based ADCs

Table 18 Exemplary anti-EPHA2 ADCs - PC3 cells

EXAMPLE 5

[1203] In vitro and/or in vivo stability of exemplary HER2 antibody-based ADCs was evaluated as described below.

5.1 Total antibody and conjugated payload from AB185-ADL1-D1 in plasma

[1204] After in vitro treatment (Section 5.1.1) or in vivo treatment (Section 5.1.2) using AB185-ADL1-D1, total antibody (TAb) and conjugated payload (CP) levels were quantified in plasma by the method immunoprecipitation followed by LC/MS/MS. Fitting was linear with 1/ ^x2 weights ranging from 0.5 to 100 μg/mL and from 0.5 to 500 ng/mL in mouse plasma for TAb and CP, respectively. Plasma samples were mixed with stable isotope labeled universal human monoclonal antibody ( ^SIuTM MAB, Sigma, St. Louis, MO) internal standard and 10 mM PBS, pH 7.4 and incubated at room temperature for one hour. . Dynabeads ^R MyOne ^TM Streptavidin T1 magnetic beads (ThermoFisher Scientific) were added to the mixture and incubated for an additional 30 min. at room temperature. A magnetic stand was used to bind the beads, which were washed successively with CHAPS buffer and then with PBS buffer. Analytes of interest were eluted from the beads using 30 mM HCl. Separate aliquots of the eluent were used to quantify TAb or CP. In the case of TAb was added Tris-HCl, pH 8.3, then trypsin and left to incubate overnight at 37°C. In the case of CP, 1M ammonium acetate was added, followed by the deuterated payload internal standard and cathepsin B, and left to incubate overnight at 25°C. Samples were analyzed by LC/MS/MS and quantitatively analyzed using the ratio of the peak areas of the analyte and the internal standard and compared with the corresponding calibration curve. To calculate the CP concentration, the TAb concentration was corrected for payload molecular weights, AB185-ADL1-D1 molecular weight, and DAR (Drug to Antibody Ratio).

5.1.1 In vitro stability of AB185-ADL1-D1 in mouse, rat and monkey plasma

[1205] AB185-ADL1-D1 (4.81 mg/mL) was added to pooled blood plasma (Bioreclamation) of mice, rats, and monkeys and PBS to achieve a final concentration of 50 μg/mL. Samples were incubated at 37°C, 95% humidity, 5% CO ₂ for 4 days in such a way as to stabilize the pH and minimize non-specific binding and evaporation. After 4, 24, 48, 72 and 96 hours, aliquots were removed from the oven and placed in storage at -70° C. until processed. Samples were thawed and processed for biological analysis using the methods described above (section 5.1) for total antibody and conjugated payload. Time 0 was defined as 100% for each matrix. The ratio of peak area values for later time points was compared to time point 0 separately for each matrix. The percentage remaining was calculated as follows: (ratio of peak area values at day X/ratio of peak area values at time 0) * 100 = % remaining. A curved line was generated by Excel calculations comparing log % remaining and time point. Half-life was calculated in Excel using the natural logarithm(2)/curve.

[1206] It was found that the total antibody from AB185-ADL1-D1 is stable in buffer and in all plasmas, showing only moderate precipitation over 4 days (fig. 12A). Conjugated (intact) D1 payload levels persisted for 4 days (fig. 12B).

5.1.2 Pharmacokinetic (PK) studies based on the Snapshot algorithm of mouse plasma after a single IV dose of AB185-ADL1-D1

[1207] Mice (CB17-SCID) were treated with a single IV dose of AB185-ADL1-D1 of 10 mg/kg or 20 mg/kg, followed by blood sampling at the time of sacrifice (cardiac puncture) in lithium- heparin (n=3 at time point per dosing group) at 24, 48 and 72 hours post-dose. The blood was centrifuged for 5 min. at 5000 rpm at 4°C to remove erythrocytes, and the blood plasma was stored at -80°C until further use. Samples were thawed and processed for biological analysis using the methods described above (section 5.1) for total antibody and conjugated payload.

[1208] After a single IV dose of AB185-ADL1-D1 greater than 400 ng/mL, the intact D1 payload remained conjugated to the AB185 antibody at 72 hours post-dose ( Figure 13 ). The AB185-ADL1-D1 ADC showed improved stability compared to an alternative ADC carrying a payload with a similar mechanism of action. Tilanstatin A ADC described in Puthenveetil et al. (Bioconjugate Chem. (2016) 27:1880-8) is comparatively less stable and shows complete bioconversion of the payload by 72 hours (i.e. the acetate is completely hydrolyzed).

5.2 Accelerated Stability Study of AB185-ADL14-D1 and AB185-ADL5-D25

[1209] Freshly prepared SMLAs (~1 mg each in Eppendorf tubes, 4~5 mg/mL) were centrifuged and measured by UV absorption (NanoDrop) at 280 nm to determine protein concentration. Samples were then incubated in a water bath at 37°C and samples were taken at four time points: 0 (freshly prepared), day 1, day 2 and day 4. Samples taken at different time points were stored at -80°C until the completion of the last collection samples. After thawing at 22°C, the samples were analyzed by SEC, hydrophobic interaction chromatography (HIC) and reverse phase MS(RP-HPLC) to quantify antibody aggregation/fragments and DAR. Percent aggregation, concentration and DAR values (HIC and RP-HPLC) for AB185-ADL14-D1 and AB185-ADL5-D25 at four time points are shown in Table 19.

Table 19 Accelerated Stability Study of Selected Anti-HER2 Antibody ADCs

EXAMPLE 6

[1210] To determine if cells treated with splicing modulator-based ADCs (i.e., the exemplary ADCs described herein) can produce neoantigens suitable for priming naive T cells for transition into effector cells (i.e., effector cells capable of acting on such neoantigens) conducted the following experiments.

6.1 RNA sequencing and protein ligandome experiment

6.1.1 Overview

[1211] Changes in the transcriptome were examined after processing using ADC. Two HER2 overexpressing cell lines, one engineered non-small cell lung cancer cell line (NCI-H1568) and another HER2 amplifying breast cancer cell line (HCC1954), were treated with ADC (AB185-ADL1-D1) at concentrations intended to initiate persistent aberrant mRNA splicing (4 nM and 1.3 nM, respectively). RNA sequencing experiments were performed at the time point 24 hours after treatment. These experiments demonstrated that many mRNA transcripts exhibit altered splicing patterns.

[1212] Next, to investigate whether the identified transcripts could be translated and presented as neoantigens on MHC1 and tumor cell complexes, a time point was chosen that would allow translation of the mRNA transcripts, but that would precede extensive cytotoxic effects (because they could impair selection). cellular material for analysis). Then, the cell lines NCI-H1568 and HCC1954 were treated with ADC at 3 nM for 48 hours and the MHC1-bound peptide was analyzed by LC-MS/MS. The peptidome data was filtered by initial peptide size to 8-14 amino acid fragments (defined as the size limit for binding to MHC1) and then filtered against known human proteins. The identified peptides were characterized as autoantigens regardless of the conditions under which they were found and in which cell line. All other peptide sequences were mapped to RNA sequencing data for 3 translation frames. Using strict criteria for evaluating peptide sequences (including the exclusion of: any peptide identified in any raw sample, any peptide that was not encoded in the canonical open reading frame, and any peptide that was not encoded by the exon junction spanning portion of the mRNA, where exon junctions were altered by processing with AB185-ADL1-D1) identified four neopeptides associated with MHC1. For a schematic of an exemplary RNA sequencing and protein ligandome experiment, see FIG. 14 .

6.1.2 Detailed description of methods

6.1.2.1 RNA sequencing of ADC-treated cells

[1213] NCI-H1568 cells (ATCC) overexpressing HCC1954 and HER2 were seeded at 1 x 10 ⁵ cells per individual well of a 6-well tissue culture plate (Corning). Cells were treated with PBS or AB185-ADL1-D1 at 1.3 nM and 4 nM, respectively, for 24 hours. Total RNA was extracted from cells using the RNAeasy Mini kit (Qiagen) and assessed qualitatively and quantitatively (RNA 6000 Nano LabChip kit on 2100 Bioanalyzer, both from Agilent). RNA sequencing libraries selected by polyadenylation were generated according to standard Illumina protocols. The cDNA libraries were qualitatively and quantitatively tested with the DNA-1000 kit on a 2100 Bioanalyzer (both from Agilent). The libraries were pooled and sequenced on a HiSeq 2000 (Illumina) to give 101 base paired end reads.

[1214] Raw reads obtained by sequencing were aligned with the human hg19 reference sequence by STAR 2.4.2a using two-pass alignment 35, and isoform quantification was performed using Kallisto 0.42.4 (Bray et al. (2016) Nat Biotechnol .34(5):525-7) against version 25 annotation in GENCODE mapped to GRCh37. The number of remaining introns was estimated by determining the 6 nucleotides (-3 nucleotides from the splicing site and +3 nucleotides after the splicing site) of the boundary regions of the exon-intron junction and counting the alignments that completely overlapped these regions. All raw data on the number of splicing boundaries, including exon-intron junctions, were pooled across all technical replicates for each cohort. The frequency of use of different splicing boundaries was assessed using a binomial z-test to identify differences in proportion between treated and untreated sample pools. Proportions were calculated based on all splice boundaries (and exon-intron junctions) that share a common splicing site, similar to the percentage spliced in (PSI) dimension. To maintain the normality assumption, only those total splice sites with a total number of splice boundaries (the sum of the raw count over all splice boundaries having that splice site) greater than or equal to 10 reads in the treated and untreated cohorts were considered in the analysis. Z-scores corresponding to a q-value ≤0.05 adjusted for false positive rate (FDR) for treated ("aberrant") splice boundaries and ≤0.20 for untreated ("canonical") splicing boundaries were considered significant.

[1215] Two or more splice boundaries that share a common splice site and have at least one splice boundary that is highly positively expressed in treated samples, or at least one splice boundary that is highly expressed in untreated samples, respectively, were considered aberrant or canonical splicing boundaries. Both aberrant and canonical splicing boundaries must have been present in an event for it to be considered distinctive splicing. In the case of intron conservation events, both exon-intron junctions (5' and 3' splicing sites), as well as the splicing boundary connecting them, should have been significant.

6.1.2.2 MHC1-related peptide of ADC-treated cells

[1216] NCI-H1568 cells (ATCC) overexpressing HCC1954 and HER2 were plated at 1×10 ⁸ cells per 15 cm ² tissue culture plates (Corning). Cells were treated with PBS or AB185-ADL1-D1 at 3 nM for 48 hours. Cells were harvested mechanically from the plates and processed according to Bassani-Sternberg et al. ((2015) Mol Cell Proteomics 14(3):658-73). Briefly, the procedure included solubilization of MHC-1 protein complexes from cell membranes, enrichment of the MHC-1 protein along with its associated peptides by immunoprecipitation, selective elution and purification of associated peptides, and identification and quantification of peptides by LC/MS/MS- analysis. For immunoprecipitation, the W6/32 monoclonal antibody isolated from the HB-95TM hybridoma cell line was used. Mass spectrometric analyzes were performed using a Q Exactive HF device. The raw data files obtained in this study were processed using the MaxQuant software package (version 1.5.7.13) for the identification and quantification of peptides and proteins using the human protein sequence database (version 22017). In addition to this database, predicted transcript variants based on RNAseq experiments with HCC1954 and NCI-H1568 cells treated with AB185-ADL1-D1 were included. MaxQuant performs a 6-frame translation comparison and integrates the resulting peptide sequences for matching with MS² spectra. The data were further manually filtered for peptide sequences that were characteristic of AB185-ADL1-D1-treated samples as well as peptides encoded by mRNA with splicing boundaries modulated by AB185-ADL1-D1 treatment ( Fig. 14 ). Four neopeptides were identified by stringent filtering of RNAseq data for the MHC-1 associated ligandome. These neopeptides were encoded by four genes. The protein sequences of four neopeptides, herein referred to as neoantigen 1, neoantigen 2, neoantigen 3 and neoantigen 4, were lengthened for subsequent experiments to initiate immune cell priming. The extended protein sequence simultaneously includes the neopeptide sequence itself in addition to the flanking amino acid sequences. The extended protein sequence better facilitates protein uptake by dendritic cells and promotes antigen presentation and T cell priming in multi-HLA isotype models.

6.2 T cell priming experiment

6.2.1 Overview

[1217] This experiment was an in vitro recovery interactions that occur in normal human secondary lymphatic organs (eg, tumor-draining lymph nodes). In this experiment, monocytes were isolated from peripheral blood mononuclear cells (PBMC) and induced to differentiate into dendritic cells (DC) by culturing in a cytokine cocktail. After differentiation to mature DCs, mature DCs were cultured with extended neopeptide sequences. During culture, mature DCs take up the peptides, process them into fragments, and present them on MHC1 for CD8 T cell priming. DC was then mixed with additional PBMCs from the same donor and incubated for approximately 2 weeks in a cytokine cocktail to stimulate CD8 T cell activation. After incubation, the cells were transferred to an ELISpot plate, which was recoated with the peptides used for priming, and restimulation of CD8 T cells was monitored for IFNγ secretion. The ELISpot plate was then processed and imaged, and IFNγ spots were counted to estimate the number of active CD8 T cells. For a schematic representation of an illustrative T cell priming experiment, seefig. 15.

6.2.2 Detailed description of methods

6.2.2.1 ELISpot assay of neoantigen-mediated T cell priming and activation

[1218] Peripheral blood mononuclear cell (PBMC) cell material was purchased from several vendors (StemExpress, HemaCare, Precision Medicine) and used to generate antigen-reactive CD8+ T cells according to Wölfl and Greenberg ((2014) Nat Prot. 9( 4):950-66; Fig. 15 ). Briefly, frozen PBMCs (5×10 ⁷ cells) were thawed and plated for 2-3 hours in T-75 plastic tissue culture flasks (Corning) in RPMI1640 supplemented with 5% human AB serum and 1% penicillin/streptomycin. Non-adherent cells were removed and discarded, and adherent cells were cultured for 5 days in RPMI1640/5% human serum supplemented with 20 ng/ml GM-CSF and 20 ng/ml IL-4. On day 6, additional TNF-α was added to the medium at 20 ng/ml. On day 7, mature DCs were harvested, counted, and loaded with neopeptides that were identified in experiments with the MHC1 peptidome ( Fig. 16 ). Cells were separated and loaded with each peptide at a final concentration of 10 μg/10 ⁶ cells/ml and incubated for 1 day. On day 8, DC was treated with mitomycin C at a concentration of 20 μg/ml for 30 minutes. and washed thoroughly. Simultaneously with DC 10 ⁸ treatment, the respective PBMCs were thawed and washed with RPMI1640. PBMC in T25 vials were then mixed with DC at a ratio of 10:1 in 10 ml RPMI1640/5% human AB serum supplemented with 30 ng/ml IL-21. After 3 days of co-culture (day 11), another 10 ml of fresh medium supplemented with IL-7 and IL-15 at a final concentration of 5 ng/ml was added. After an additional 3 days of co-culture (day 14), another 10 ml of fresh medium supplemented with IL-7 and IL-15 at a final concentration of 5 ng/ml was added and the co-culture was transferred to a T-75 flask. On day 17, another 20 ml of medium supplemented with IL-7 and IL-15 at a final concentration of 10 ng/ml was added and the cells were transferred to a T-175 flask. On day 20, cells were harvested and ELISpot assayed according to manufacturer's instructions (R&D Systems). Peptides were preliminarily added to the wells at a final concentration of 10 μg/ml per well, cells were added at a cell density of 2×10 ⁵ cells per well, seeded cells were duplicated and incubated overnight. IFNγ spots were developed and read on the AID ELISpot reader according to the manufacturer's instructions ( FIGS. 17A-D ).

[1219] Exemplary data from antigen priming experiments are shown in FIG. 17A-D . Batches of PBMC cells obtained from donors were subjected to DC differentiation and priming using neopeptides as shown in FIG. 15 . Analysis of ELISpot data shows that in PBMC from healthy donors, priming of naive T cells via DC-presentation of neopeptides can lead to antigen-specific growth and maturation of effector (CD8) T cell populations. These T cell populations exhibit antigen-specific reactivation as evidenced by IFNγ secretion only in the presence of the antigenic peptide, but not in the presence of peptides that do not initiate priming.

RT-qPCR

[1220] RNA was isolated from cell lines using RNeasy Mini with DNaseI (Qiagen) treatment and 1-2 μg of RNA was reverse transcribed using Superscript VILO reverse transcriptase (ThermoFisher Scientific) in 20 μl according to the manufacturer's instructions. RNA lysates were isolated and reverse transcribed using the TaqMan Gene Expression Cells-to-CT kit (ThermoFisher Scientific) according to the manufacturer's instructions. Quantitative PCR was performed using the TaqMan Gene Expression Master Mix reagent (ThermoFisher Scientific) with transcript probes targeting neoantigen splicing boundaries in GAPDH VIC-PL RNA duplex format (ThermoFisher Scientific) and quantitation was performed using the ΔΔCt method.

EXAMPLE 7

7.1 RNA sequencing and protein ligandome experiment

7.1.1 Methods

[1221] The RNA sequencing and protein ligandome experiment was performed as described in Example 6 (Section 6.1). See FIG. 14 .

7.1.2 Results

[1222] Twenty-nine neopeptides were identified by stringent filtering of RNAseq data by MHC1-associated ligandome (Table 20). Four neopeptides were selected and extended for further experiments to initiate immune cell priming. The four selected neopeptides are highlighted in bold in Table 20.

Table 20. Neopeptides

Neopeptide SEQID NO Connection (HG19) Gene Event type Observed in 1 SPTLPPRSL 37 chr12:49663470-49663610:+ TUBA1C Intron conservation H1568 2 HPSIKRGLSSL 38 chr12:42729776-42781257:+ PPHLN1 exon skipping H1568 3 LLLPHHVL 39 chr12:49663470-49663610:+ TUBA1C Intron conservation H1568 4 RTAPGVRPPF 40 chr14:35182767-35183743:- CFL2 Intron conservation H1568 5 RPQKSIQAL 41 chr10:28822963-28823162:+ WAC Intron conservation H1568 6 APAPPPLPA 42 chr17:80009840-80011149:+ GPS1 Intron conservation H1568 7 RPRPSFPVSL 43 chr7:55087058-55134942:+ EGFR Intron conservation H1568 8 RPKHGDGFSL 44 chr11:57472287-57472444:- MED19 Intron conservation H1568 9 GPAPGKTGL 45 chr7:75932393-75933118:+ HSBP1 Intron conservation H1568 10 EAARKGNSL 46 chr1:53480715-53504588:+ SCP2 exon skipping H1568 eleven RIKEKIEEL 47 chr9:72897499-72912881:+ SMC5 exon skipping H1568 12 EIKKRFRQF 48 chr1:28531860-28541450:- DNAJC8 exon skipping H1568 13 HESAAMAET 49 chr11:102272937-102323254:- TMEM123 exon skipping HCC1954 14 ALKLKQVGV 50 chr1:153610924-153617539:+ CHTOP exon skipping H1568 15 DLKKRHITF 51 chr13:41323417-41331008:- MRPS31 exon skipping H1568 16 DVKRNDIAM 52 chr1:41213277-41218822:+ NFYC exon skipping H1568 17 IPSDHILTPA 53 chr6:149718900-149720239:+ TAB2 exon skipping H1568 18 TVFSTSSLK 54 chr11:61197654-61213412:+ SDHAF2 exon skipping H1568 19 ITSCLLNF 55 chr5:137892555-137893090:- HSPA9 Intron conservation H1568 20 RASPVRGQL 56 chr7:75677544-75677893:+ MDH2 Intron conservation H1568 21 VVRKPVIAL 57 chr1:36923582-36929406:- MRPS15 exon skipping H1568 22 LLSEKKKIS 58 chr6:31750622-31750872:- VARS Intron conservation H1568 23 APASKPRPRL 59 chr19:3573798-3574380:+ HMG20B Intron conservation H1568 24 RYGQLSEKF 60 chr19:33076813-33078158:+ PDCD5 exon skipping HCC1954 25 VYISNVSKL 61 chr3:53920961-53925796:- SELK exon skipping HCC1954 26 LPTKETPSF 62 chr2:85133241-85133394:+ TMSB10 Alternative splicing site at 3' end HCC1954 27 GEAPPPPPA 63 chr17:80223672-80231181:- CSNK1D Intron conservation HCC1954 28 LEEISKQEI 64 chr17:27804724-27807385:+ TAOK1 exon skipping HCC1954 29 IYNHITVKI 65 chr4:2886393-2896308:+ ADD1 exon skipping HCC1954

[1223] The protein sequences of twenty-nine neopeptides can be extended. The extended protein sequence simultaneously includes the neopeptide sequence itself in addition to the flanking amino acid sequences. The extended protein sequence better facilitates protein uptake by dendritic cells and promotes antigen presentation and T cell priming in multi-HLA isotype models. The amino acid sequences of twenty-nine extended neopeptides are shown in Table 21.

Table 21. Amino acid sequences of extended neopeptides

Gene SEQID NO Amino acid sequence of extended neopeptide* TUBA1C 66 VDLEPTVI GELTSVTQVRSQGAGTGGLSWGGSAGHSPTLPPRSLSLLLLPHHVLQMKFALALTASSSTLSNSSQARKMLPITMPEGTTPLARRSLTSCWTEFASWLTSAPVFRASWFSTALVGELVLGSPRCSWNVSQLIMARSPSWSSPFTRRPRFPQL PPHLN1 67 APPPRSHPS IKRGLSSL CFL2 68 MVRRARWPGGRGEARKAPRTAPGVRPPF WAC 69 WVNCLFVSGRAAAGGGGGAVPPYLELAGPPFLLLTLIRIGLGRRSGRAGGRAGTQCGGERGPGFAAFRPLRPFRRLRVCAVCVRGSALGRSVGLPRGGAAGAPFSSSPAPHPRRVLCCLLFLFFSCHDRRGDSQPYQVPAEAGVEGLEGAGGGREGLLLERRPQKSIQALRCNTSETSTADPLKIPGLVPLALSSKV GPS1 70 MPLPVQVFNLQVTSRGRPGPPRPRAPRHWGRAEVEQGRGACARSRSGTLRAGPPRAARVGGCRAEGASPPWLRAAIGGRRAAPAPPPLPAAHGRGSRPPRR EGFR 71 QPAQPRTG APARRPRPRPSFPVSLRSAAPPTGTAGGTGRFVLRPGESGAGGGGDAWDTGLQARRGTAAGTSGAPNRSQLSSLTFPAQLRRIGVSGRKPGAGGRLGPGSRTCAPRCLPRARRGPGAHPRGGRCPPAETALFREAEEGTQKYSLPSDPAGQAAF MED19 72 FRLHTGPV SPVGGRRQMGRPKHGDGFSLQVCSFIMEQNG HSBP1 73 GVVEITG EPPCSCRGEEEASRAGRAGGVRLKRGSRGPGELNVGPAPGKTGLLIPLLRNWECGSLLRALSAL SCP2 74 KMGFPEA ARKGNSL SMC5 75 LEARIKEK IEELQQALI DNAJC8 76 EIKKRFRQ FKQAVYKQ TMEM123 77 AHESAAMA ETLQHVPS CHTOP 78 NRPSVQAA LKLKQVGV MRPS31 79 KTDDLKKR HITFTLGCGIC NFYC 80 MKLDEDVK RNDIAMAI TAB2 81 NSISQIPS DHILTPALFITFMTILDL SDHAF2 82 TVFSTSSL KLNQPQKYLKMKSWPC HSPA9 83 AEEDRRKK VITSCLLNFNLSKAQS MDH2 84 RSFSTSAQ VGQTRGGLQAEAPRPGPRASPVRGQL MRPS15 85 RGYVVRKP VIALSVKI VARS 86 VDMDFGTG GQGAGPVGRGKDWSCTLAVHLLSEKKKISFSQIDRAWGGSQGTVLDKWGPGVVSELHPSAKEVSVGRNSVESLMTWAS HMG20B 87 EKGSHEEE VRVPALSWGRPRAPAPASKPRPRLDLNCLWLRPQPIFLWKLRPRPVPAATPLTGPLPL PDCD5 88 RYGQLSEK FNRRKVMDS SELK 89 MVYISN VSKLCFSKM TMSB10 90 NTLPTKET PSFLLNPHTSWVPRPHREAPRLRVGVAAPLQRPLPALHSH CSNK1D 91 FGDIYLG EAPPPPPAARRPGPCGCQDQARSRKEVVAPAGSPRKSRHRRIVARTQRPLG TAOK1 92 GSASDLLE EISKQEISF ADD1 93 QLIYNHIT VKINLQGD

* Underlining indicates amino acids derived from the open reading frame of the canonical transcript (i.e., the canonical peptide sequence).

7.2 T cell priming experiment

7.2.1 Overview

[1224] See FIG. 15 .

7.2.2 Materials

[1225] Materials used for T cell priming:

RPMI 1640 medium (Thermo Fisher Scientific, cat. no. A10491-01)

Penicillin/streptomycin (100x, Thermo Fisher Scientific, cat. no. 15140122)

Human serum type AB (Sigma, # H3667-100ML)

Recombinant human IL-4 (Peprotech cat no. 200-04)

Recombinant human GM-CSF (Peprotech cat no. 300-03)

Recombinant human TNF-α (Peprotech cat no. 300-01A)

Human IL-7 (Peprotech cat no. 200-07)

Human IL-15 (Peprotech cat no. 200-15)

Human IL-21 (Peprotech cat no. 200-21)

Peptides (New England Peptide - 95% pure, dissolved in DMSO, 1000X stock solution at 10 µg/µl)

Human IFN-y ELISpot kit (R&D Systems, cat. no. EL285)

7.2.3 Methods

7.2.3.1 Generation and maturation of dendritic cells from human monocytes and loading with neopeptides

[1226] Frozen or fresh PBMCs were thawed and washed once with pre-warmed RPMI medium, and an aliquot of non-adherent cells was left for TCR sequencing at baseline. PBMC were resuspended in 10 ml of dendritic cell (DC) medium (RPMI 1640 supplemented with 5% human AB serum and 1% penicillin/streptomycin) at 5×10 ⁶ cells per ml, 50 million cells in total. Cells were transferred to a T-75 tissue culture plastic flask and incubated at 37° C., 5% CO ₂ for 2-3 hours. After gentle shaking, non-adherent cells were removed and discarded, and adherent (monocyte-enriched) cells were washed three times with prewarmed DC medium. 10 ml of DC medium supplemented with GM-CSF at a final concentration of 20 ng/ml and IL-4 at a final concentration of 20 ng/ml was added to the adherent cells. Attached cells were incubated at 37°C, 5% CO ₂ , 100% humidity for 5 days. On day 6, recombinant TNF-α was added to a final concentration of 20 ng/ml and cultured for an additional 48 hours. On day 7, DCs were collected and counted, then loaded with neopeptides (each at 10 μg/1×10 ⁶ cells/ml) or control without neopeptide. On day 8, cells were harvested as mature DCs. Mature DCs were treated with mitomycin C at a final concentration of 20 μg/ml for 30 min. at 37°C followed by intensive washing before co-incubation with T cells for priming.

7.2.3.2 Starting co-culture with autologous T cells

[1227] On day 0, frozen or fresh were thawed and washed once in prewarmed medium. PBMC were then resuspended and mixed with DC in T25 vials at a ratio of 10:1 (10×10⁶:1×10⁶ cells) in 10 ml RPMI1640/5% human AB serum supplemented with 30 ng/ml IL-21. After 3 days of co-culture (day 3), another 10 ml of fresh medium supplemented with IL-7 and IL-15 at a final concentration of 5 ng/ml was added. After an additional 3 days of co-culture (day 6), another 10 ml of fresh medium supplemented with IL-7 and IL-15 at a final concentration of 5 ng/ml was added and the co-culture was transferred to a T-75 flask. On day 9, another 20 ml of medium supplemented with IL-7 and IL-15 at a final concentration of 10 ng/ml was added and the cells were transferred to a T-175 flask. On days 11-12, cells were harvested, ELISpot assay was performed according to the manufacturer's instructions (R&D Systems) and TCR sequencing was performed (1×10⁶ cells). Peptides were preliminarily added to the wells at a final concentration of 10 μg/ml per well, cells were added at a cell density of 2 × 10⁵ cells per well, seeded cells were duplicated and incubated overnight. IFNγ spots were developed and read on an AID ELISpot reader according to the manufacturer's instructions.

7.2.4 Results

[1228] Illustrative data from antigen priming experiments are shown in Table 22.

Table 22 T cell priming data

Donor Answer in ELISpot Gene SEQID NO HLA
(predicted binding) 1 +++ DNAJC8 76 A*31:01/B*40:13 1 + TMEM123 77 A*02:01 2 +++ TMEM123 77 A*02:01 2 + SCP2 74 B*39:06 6 + DNAJC8 76 C*07:01/B*27 7 +++ DNAJC8 76 C*07:02/C*06:02 8 +++ DNAJC8 76 C*07:01 8 + SMC5 75 C*07:01/B*08:02 8 +++ SCP2 74 C*03:03

[1229] Batches of PBMC cells obtained from donors were subjected to DC differentiation and priming using neopeptides as shown in FIG. 15 . Analysis of ELISpot data shows that in PBMC from healthy donors, priming of naive T cells via DC-presentation of neopeptides can lead to antigen-specific growth and maturation of effector (CD8) T cell populations. These T cell populations exhibit antigen-specific reactivation as evidenced by IFNγ secretion only in the presence of the antigenic peptide, but not in the presence of peptides that do not initiate priming. The data also suggest that the response to neopeptides can not necessarily be predicted based on HLA.

EXAMPLE 8

8.1 Identification of neopeptides

8.1.1 Overview

[1230] To identify neopeptides resulting from modulated splicing, 8-11 amino acid long peptides that are believed to bind to MHC class I from the canonical and modulated splicing proteome were compared separately by tandem mass spectra using a standard approach. "target-trap". First, alternatively spliced mRNA transcripts were translated and compared to a reference proteome. Then, the spectra that did not correspond to the reference proteome were used to search for the corresponding new peptides. See FIG. 14 .

8.1.2 Methods

8.1.2.1 Building a database of potential neopeptides

[1231] To create a database of potential neopeptides, peptides spanning exon boundaries or containing introns not found in the reference proteome were identified (see Li et al. (2011) Mol. Cell Proteomics 10 (5): M110.006536). Peptides derived from the reference proteome were selected from manually analyzed protein sequences and loaded. In addition, proteins were filtered by splicing boundaries found in normal tissues. As an alternative approach, full-length proteins can be translated after splicing events, searched for peptides spanning exon splicing boundaries or containing introns, and then compared by BLAST to a reference proteome.

[1232] To avoid unnecessary statistical tests, only peptides that were predicted to bind to MHC were used. Protein sequences (both from the reference proteome and from translation of exon-skipping or intron-sparing transcripts) were evaluated for high affinity binding to MHC1 alleles using NetMHCPan and/or MHCnuggets software. For both the first and second software packages, hits with strong or weak binding were selected (i) from raw affinity binding strength prediction data (500 nM threshold for weak binding) or (ii) by selecting a large number of random existing peptides and determining the distribution predicted binding strength values for peptides. The estimated binding strength for any new peptides was then compared to this distribution. The strong and weak binding thresholds measured using the latter method (method (ii)) were predicted binding strengths for the highest 0.5% or highest 2.0% randomly selected existing peptides, respectively (see Nielsen and Andreatta (2016) Genome Med 8(1):33). Currently, prediction for MHC is limited to peptide sequences of 8-11 amino acids in length and is contained in the immune epitope database (IEDB).

8.1.2.2 Identification of splicing modulated transcripts using RNA sequencing (RNAseq) and ribosome profiling (RiboSeq)

[1233] A number of neopeptides are derived from larger peptide domains (eg, TUBA1C), while peptide fragments from the rest of the translated region may remain undetected. This may be due to increased degradation of undetected sites, binding affinity for MHC1, or technical problems associated with detection by mass spectrometry (MS). Typically, RNAseq analysis will overestimate the number of predicted neopeptides (because not all alternatively spliced transcripts will be translated), while the MHC1 ligandome will underestimate the number of predicted neopeptides.

[1234] To compile a list of neopeptides as a marker of translation into proteins, available variants of splicing boundaries formed as a result of splicing modulation with associated ribosomes were identified (Andreev et al. (2017) Nucleic Acids Res. 45 (2): 513-26). RiboSeq or ribosomal profiling revealed mRNA sequences (and variant domains) that had undergone active translation, as evidenced by the presence of transcript-bound ribosomes as well as specific exon junction domains.

EXAMPLE 9

9.1 In vitro analysis of exemplary anti-HER2 antibody based ADCs

9.1.1 Methods

[1235] HCC1954, NCI-N87 or MCF7 (ATCC) cells were seeded in RPMI without phenol red+10% FBS (ATCC) or EMEM+10% FBS+insulin (0.01 mg/mL) (for MCF7) in an amount 2.5 x 10 ³ cells per well at 90 µl per well. Cells (n=3 wells per condition) were treated with the conjugates in a dose-dependent manner at a 3-fold dilution. After 24 hours, cells were lysed using 50 µl of CL buffer (IgePal CA-630, 5M NaCl, 1M Tris-HCl 1M pH 7.4 in water) containing 25 µl/ml RNasin® (Promega) and incubated for 45 min. at RT on an oscillating stirrer. The resulting mixture (1 μl) was used to evaluate splicing modulation in reverse transcription PCR using Taqman Fast Virus 1-Step MasterMix (Applied Biosystems) and the following Taqman primers as recommended by the manufacturer: SLC25A19 (Invitrogen, Hs00222265_m1); FBXW5 (IDT, forward primer: CACACCAGATCGGCATCAA (SEQ ID NO:37), reverse primer: CGATGATGTGTCCGTGTATGT (SEQ ID NO:38), probe: ATCCTGCCACACCAGATGACCAC (SEQ ID NO:39), stain: FAM, quencher: ZEN/Iowa Black FQ ); TAOK1 (IDT, forward primer: CTGCTTCGGATTTACTAGAAGAGATA (SEQ ID NO:40), reverse primer: GCGTTCCCACAAAGGAATTG (SEQ ID NO:41), probe: TGCTGACTTTGGCTCTGCTTCCAT (SEQ ID NO:42), dye: FAM, quencher: ZEN/Iowa Black FQ ); RPLP0 (Invitrogen, Hs99999902_m1).

9.2 In vivo efficacy and pharmacodynamics (PD) of exemplary anti-HER2 antibody-based ADCs

9.2.1 Methods - in vivo efficacy in xenograft model based on NCI-N87 cells

[1236] CB17-SCID mice (Charles River Laboratories) were maintained in accordance with federal, state, city, and AAALAC animal research regulations in Cambridge, Massachusetts. The human gastric carcinoma cell line NCI-N87 was obtained from the American Type Culture Collection (ATCC No. CRL-5822). Passage 14 cells were used for tumor experiments. Cells were last tested for murine pathogens in September 2016 and for mycoplasmas in December 2018.

[1237] Single cell suspension of 5 million cells per 0.1 ml (50% ATCC-modified medium RPMI-1640, cat. no. A10491-01 (Thermo-Fisher Scientific) and 50% Corning Matrigel, cat. no. 356254 (VWR Scientific)) was injected subcutaneously into the outer area of the right flank using a syringe with a 25Ga needle. Mice were randomized on day 0 with a mean tumor volume of 180-183 mm ³ , n=8 per group for efficacy analysis and n=4 per group for pharmacodynamics (PD). On day 1, study drugs were administered (10 mg/kg) in a volume of 5 ml/kg IV every 7 days for 2 cycles to analyze efficacy; one treatment course was used for PD evaluation. Animals in the PD cohorts were sacrificed 24, 48, 72 and/or 96 hours after the single dose, depending on the study drug. Tumor volume and body weight were measured once or twice a week until completion (day 61) of the study. Tumor volume was calculated using the formula length x width ^2/2 . Mice that reached the endpoint (tumor size ≥ 20 mm ³ in any direction or tumor volume ≥ baseline body weight x 100) or had tumor ulceration or ocular infection were excluded from the study.

[1238] the Compounds were in a laminar box biological protection. The vehicle for the study drug was phosphate buffered saline, pH 7.4 (Cat. No. A10010-049 (Thermo-Fisher Scientific)). Groups are presented in table 23.

[1239] Antitumor activity was calculated as follows:

% T/C formula=treated/control*100

%TGI formula = (vehicle mean _{day X} - treated mean _{day X} )/vehicle mean _{day X} * 100, where day X was day 22.

9.2.2 Methods - in vivo PD (RT-qPCR)

[1240] RNA was isolated from tumor samples using a MagMAX-96 microarray kit and a MagMAX Express-96 Deep Well Magnetic Particle Processor (ThermoFisher Scientific). Tumor samples (weighing 50-100 mg) were collected in RNAlater buffer and stored at -80° C. until RNA isolation. Tissue homogenization was performed by adding 2-3 ceramic beads and 1 ml Tri-reagent to the tumor tube, followed by disintegration on an Omni Bead Ruptor 24 (Omni International). RNA isolation was performed according to the manufacturer's instructions.

[1241] RNA lysates were isolated and reverse transcribed using the TaqMan Fast Virus 1-Step Master Mixt reagent (ThermoFisher Scientific) according to the manufacturer's instructions.

[1242] Quantitative PCR with cDNA was performed using the TaqMan Gene Expression Master Mix reagent (ThermoFisher Scientific) with transcript probes targeting splicing boundaries in FBXW5 or TAOK1, in RPLP0 VIC-PL RNA duplex format (ThermoFisher Scientific), and quantified analysis was performed using the ΔΔCt method.

Table 23. Groups

Tested drug (efficacy) Test drug (PD) Carrier medium (PBS) Carrier medium (PBS) TDM1 TDM1 Trastuzumab (AB185) Trastuzumab (AB185) AB185-ADL5-D2 AB185-ADL5-D2 AB185-ADL1-D4 AB185-ADL1-D4 AB185-ADL5-D15 AB185-ADL5-D15 AB185-ADL14-D1 AB185-ADL14-D1

9.3 Results

In vitro analysis :

[1243] exemplary HER2 antibody-based ADCs were evaluated for antiproliferative activity in two HER2 positive cell lines of different origins (HCC1954 breast cancer cells and N87 gastric cancer cells) and a HER2 negative cell line (MCF7). The effectiveness of growth inhibition (GI50 activity) was measured, as well as the quality of the response (R _min compared to control). Alternative splicing in the housekeeping representative gene SLC25A19 was measured by qPCR after treatment of these cell lines with ADC. GI50 and splicing response quality were measured. The results of in vitro analysis are shown in table 25.

[1244] In general, the anti-HER2 antibody ADCs tested showed favorable efficacy against antigen positive cell lines and minimal viability and splicing response against antigen negative cell lines. ADCs differed in the quality of the response in killing cells and the completion of alternative splicing in SLC25A19. ADCs were classified into one of four categories with respect to their effect on each of the antigen-positive cell lines: 1) high response quality/high lethality and pronounced splicing response; 2) high response quality/high lethality and poor splicing response; 3) poor quality of response/low mortality and pronounced splicing response; or 4) low cell lethality and poor splicing response. Several ADCs have shown beneficial category 3 properties for the N87 gastric cancer cell line. Compound AB185-ADL13-D4 was consistently lethal and showed a high quality splicing response on both HCC1954 and N87 cells. Such persistent antigen-specific lethal activity is generally desirable for ADCs, and a water-soluble linker can provide even higher drug loading with favorable conjugate characteristics. Changing the linker to ADL23 (EVC tripeptide) unexpectedly changed the profile in both cell lines. The AB185-ADL1-D12 compound was also unique in that it is highly lethal to HCC1954 cells, but payload cell activity is consistently low for a number of solid and hematologic tumor cell lines. D12 is also the only low efflux payload in these groups. Thus, ADCs releasing this payload may be active against multidrug-resistant cancers that express the target antigen.

[1245] When comparing splicing activity (IC50 in qPCR) with activity against cells (GI50 in CTG) on HCC1954 breast cancer cells, the glucuronide linker and D12 payload showed favorable properties. The C6/C7 carbamate switch payload (D8) and dual base payload (D5) showed cell activity but did not show the most efficient alternative splicing. Changing the dipeptide to a tripeptide linker with a D4 or D1 payload reduces the efficiency of the alternative splicing, but the cell activity remains high. See fig. 18 (ranked by size according to cell lethal activity, hatched for quality of response as alternative splicing).

[1246] When comparing splicing activity (IC50 in qPCR) with activity against cells (GI50 in CTG) on N87 gastric cancer cells, the glucuronide linker (with D4) and D12 payload showed favorable properties, as well as on HCC1954 cells. Overall, all ADCs showed minimal splicing activity and activity against the HER2-negative MCF7 cell line. Despite the relatively low cell activity of the D12 payload, the activity of the anti-HER2 ADC with this payload was high and the payload showed a low efflux factor. These properties have the potential to improve the treatment of multidrug-resistant cancers. See fig. 19 (ranked by size according to cell lethal activity, hatched for quality of response as alternative splicing).

[1247] In vitro analysis was performed with additional ADC based on antibodies to HER2. Comparison of anti-HER2 antibody-based ADCs and corresponding payloads is shown in Table 26. When comparing the potency and lethality of anti-HER2 antibody-based ADCs with the stability and penetration of the respective payloads ( Figure 20 ), the AB185-ADL5-D15 compound showed a favorable combination of efficacy , lethal activity, chemical stability and catabolite permeability (eg for intended off-target destruction). As described below, AB185-ADL5-D15 was also active in vivo and showed strong antitumor activity in the NCI-N87 xenograft model.

In vivo efficiency :

[1248] The NCI-N87 gastric cancer cell line expressing human HER2 and a xenograft model based on it were obtained by inoculating NCI-N87 cells into SCID mice to evaluate the antitumor activity of exemplary anti-HER2 antibody-based ADCs. Trastuzumab and TDM1 were used as comparators.

[1249] The antitumor effects of intravenous trastuzumab, TDM1, and four anti-HER2 antibody-based ADCs in an NCI-N87 gastric carcinoma xenograft model are shown in FIG. 21 and FIG. 23 . The effect on body weight is shown in Fig. 22 and FIG. 23 . The test drug or carrier medium was administered intravenously (IV) Q7D for 2 cycles. Data are mean ± SEM (tumor volume, mm ³ ) or ± SEM (body weight,%) (N=8). * p < 0.0001 compared to vehicle group at day 22 using one-way ANOVA.

[1250] Tabular data on in vitro and in vivo efficacy on NCI-N87 are presented in table 24. Tabular data are percent (%) inhibition of tumor growth (TGI tumor volume) or % T/C (tumor volume) (N=8 ). Compound AB185-ADL5-D2 was surprisingly active in vivo despite low in vitro lethality and showed improved in vivo efficacy compared to, for example, AB185-ADL14-D1.

Table 24 Efficacy of exemplary anti-HER2 antibody ADC against NCI-N87 cell line

Day 22 (in vivo) NCI-N87 (in vitro) TGI (%) T/C (%) GI _{50 (nM)} LD _{50 (nM) Rmin} , % Carrier, I/O, Q7Dx2 - - - - - TDM1, 10 mg/kg, IV, Q7Dx2 71 29 0.1 40 -37 Trastuzumab 10 mg/kg IV Q7Dx2 4 96 >200 >200 98 AB185-ADL5-D2, 10 mg/kg, IV, Q7Dx2 73 27 0.2 >200 0 AB185-ADL1-D4, 10 mg/kg, IV, Q7Dx2 68 32 0.1 >200 -22 AB185-ADL5-D15, 10 mg/kg, IV, Q7Dx2 67 33 0.1 >200 -37 AB185-ADL14-D1, 10 mg/kg, IV, Q7Dx2 -7 107 0.1 >200 -40

[1251] Trastuzumab, a human anti-HER2 antibody that is the standard of care for HER2-positive breast cancer, did not demonstrate antitumor activity in this model. However, TDM1, a second-line drug containing trastuzumab with a covalently bound DM1 (cytotoxic agent), showed significantly better antitumor activity (TGI 71%) than trastuzumab alone (TGI 4%). Among the four ADCs tested, ABL185-ADL5-D2, AB185-ADL5-D15 and AB185-ADL1-D4 showed high antitumor activity with tumor growth inhibition (TGI) of 73%, 67% and 68%, respectively. Tumors in all responder groups resumed growth over time. A complete response was not observed in any group, with one of eight animals in each of the following groups showing a partial response: TDM1, ABL185-ADL5-D2 and AB185-ADL5-D15. All reagents and test preparations were well tolerated by mice, with minimal body weight loss. Overall, 3 out of 4 HER2 antibody-based ADCs tested demonstrated high antitumor activity compared to TDM1 in the NCI-N87 tumor model without significant body weight loss.

In vivo PD :

[1252] PD modulation of splice boundaries in mRNA by intravenous administration of trastuzumab, TDM1, and four anti-HER2 ADCs in an xenograft model based on NCI-N87 gastric carcinoma cells is shown in FIG. 24A - 24D . RT-qPCR monitored the presence of FBXW5 (mature mRNA transcript) and TAOK1 (new splice transcript). The test drug or carrier medium was administered intravenously (IV) at a dose of 10 mg/kg. Animals (N=4 per group) were collected either after 48 hours ( FIG. 24A and FIG. 24B ) or at the indicated time points ( FIG. 24C and FIG. 24D ). Tumors were harvested for RNA isolation and RT-qPCR. *p<0.05 compared to vehicle medium using one-way ANOVA ( FIG. 24A and FIG. 24B ) or two-way ANOVA ( FIG. 24C and FIG. 24D ).

[1253] Neither trastuzumab nor TDM1 showed significant changes when observed for FBXW5 or TAOK1 splicing. All AB185 and ADL5-D2/ADL1-D4/ADL5-D15 conjugates showed significant FBXW5 depletion, and AB185 and ADL5-D2/ADL1-D4 conjugates showed significant increases in TAOK1. ADL5-D15 showed an increase in TAOK1, but statistical significance was not reached. ADL14-D1 showed no statistically significant change in the copy number of any of the mRNA transcripts.

Table 25 Characteristics of exemplary anti-HER2 antibody ADCs and associated payloads

Table 25 (continued). Characteristics of exemplary HER2 antibody-based ADCs and associated payloads

Table 25 (continued). Characteristics of exemplary HER2 antibody-based ADCs and associated payloads

Table 26 Exemplary Anti-HER2 ADCs - HCC1954 Cells

EXAMPLE 10

10.1 Choice of tumor/target type based on bioinformatic analysis

[1254] To identify target antigens that are highly expressed in low mutation burden (TMB) tumors compared to normal tissue, a bioinformatic predictive analysis was performed. This example explains 1) how each filter was defined and 2) how the antigen/tumor type table was used to identify suitable target/tumor pairs. For a schematic diagram of an exemplary target detection assay, see FIG. 25 .

10.1.1 Overview

[1255] Target antigens and tumor types were identified using bioinformatic predictive analysis. Data from TCGA was processed to determine tumor mutation load (TMB), expression of target antigens for ADC delivery, and tumor infiltration by immune cells. Analyzes using published TMB cutoffs and infiltration to stratify predictive responders and non-responders to immune response checkpoint inhibitors (ICIs) were performed to identify tumor types and their corresponding antigens. Surface antigen expression was further filtered to evaluate for a population with probabilistic antigen expression that is at least 2-fold higher than in any normal tissue (estimated from the GTEx tissue-specific expression database). Tumor types (or subpopulations of tumor cells from TCGA) with an appropriate percentage of patients having one or more target antigens were further analyzed for mutation and immunity scores. The percentage of antigen-positive patients with a tumor mutation load below a cumulative rate of 10 mutations/million bases and a T-cell infiltration score greater than 0.5 was assessed. Tumor types for ~50% of patients meeting these criteria are listed in Table 27. Patients in these cohorts (but not limited to these cohorts) may respond to treatment with a splice modulator-based ADC (e.g., splice modulator-based ADC described herein). ).

10.1.2 Methods

[1256] Explanation of antigen and tumor type prediction table from Bulletin of Oncology :

Antigen : name of the gene encoding the antigen; only 59 genes that could be suitable targets for ADC from a literature review in the Journal of Oncology (see Moek et al. (2017) Annals of Oncology, 28: 3083-3091) were included in the table (the original complete analysis included a complete set of protein genes cell surface (surfasome) revealed by in silico analysis).

Cohort : name of the cohort according to TCGA

SUBTYPE : tumor subtype information according to TCGA

Total_cases : total number of cases

Cases.by.subtype : number of cases by.subtype

LTPM_median : median value of expression over log2(TPM+1)

LTPM_median_low TMB : median expression value by log2(TPM+1) for cases with low TMB (< 350)

LTMB_median : median value of log10(TMB)

LTMB_MAD : robust standard deviation measurement of MAD (absolute mean deviation) log10(TMB)

Infiltrate_median : median infiltration score according to T cell molecular signatures from PLoS One

Infiltrate_MAD : Robust measurement of the standard deviation of the MAD value (absolute deviation of the mean) of the infiltration score according to the T-cell molecular signatures from PLoS One

Correlation with purity : correlation of expression with tumor purity

GTEX_tissue : the name of the GTEx tissue with the highest median expression value

GTEX_LTPM_median : median value of antigen expression in tissue from GTEx with the highest median expression value

Surfasome.filter* : patient population after surfasome filtering: expression >2-fold higher than normal tissue from GTEx with highest median

Low TMB. Filter : Patient populations after low tumor mutation load (TMB) filter: < 350 per exome sequencer. (10 over 2 million bases)

Low TMB.Surfasome.Filter : patient populations after using both the surfasome filter and the tumor mutation load (tmb) filter

High.infilt.LowTMB.Surfasome.Filter : patient population after using three filters: surfasome expression filter, low tumor mutation load (tmb) filter, and high infiltration filter

Surface.Filter.Proc. proportion : percentage of the population after using the surfasome filter: expression > 2-fold higher than in normal tissue from GTEx with the highest median value

Low TMB.Filter.Proc. proportion : percentage of the population after using a low tumor mutation load (TMB) filter < 350

Low TMB.Surface.Filter.Proc. proportion : percentage of the population after using both the surfasome filter and the tumor mutation load (tmb) filter

High infiltrate.Low TMB.Surface.Filter.Proc. : percentage of population after using three filters: surfasome expression filter, low tumor mutation load (tmb) filter, and high infiltration filter

[1257] Probabilistic estimation of the patient population :

Collected Sources :

1. publication on the immune background in many types of cancer (Ready et al. (2019) Journal of Clinical Oncology, 37:992-1000)

2. RNA sequencing/exome sequencing dataset from TCGA obtained by Omicsoft

3. Publications on gene expression signatures in characterizing the immune microenvironment (Thorsson et al. (2018) Immunity, 48, 812-830)

4. Data from GTEx version 7

Tumor mutation load was calculated using Omicsoft software by recalling mutation data from TCGA obtained by exome sequencing. T cell signatures (CD3D, CD3E, CD2) from PLoS One were used to quantify immune cell infiltration. Z-score was calculated using the log2(TPM+1) value of RNA sequencing data from TCGA.

Surfasome.Filter : The probability that an individual patient has >2-fold overexpression (log2(TPM+1)) compared to normal GTEx tissue with the highest median value was calculated as follows:

1. identified normal tissue from GTEx, which is characterized by the highest median value for this gene among all normal tissues from GTEx, taking this indicator as T _h .

2. Obtained data from GTEx for this gene at 1000 patients in each tissue using the Minority Class Increment Method (SMOTE (Siemers et al. (2017) PLoS One, 12(7): e0179726)), while maintaining the distribution of tissues.

3. Count how many times (n times) this patient will have >2 times the tissue T _h data obtained from GTEx, with the probability that this patient will have >2 times the normal value from GTEx is n/1000

4. This probability was calculated for each patient

5. The sum of the individual probabilities is a probabilistic estimate of a population cohort with >2 excess over normal tissue from GTEx.

6. When using other filters at the same time, the sum of the individual probabilities will only apply to patients whose data has been passed through other filters.

10.1.3 Examples

[1258] Target antigens and tumor types determined using the exemplary bioinformatics predictive analysis described above are shown in Table 27.

Table 27 Exemplary Target Antigens and Tumor Types

Target antigen Type of tumor(s) MSLN ovarian cancer
Cervical cancer
Pancreas cancer
lung adenocarcinoma EPHA2 Esophageal carcinoma FOLH1 prostate cancer CDH6 kidney cancer CEACAM5 colorectal cancer CFC1B Pancreas cancer ENPP3 kidney cancer FOLR1 ovarian cancer HAVCR1 kidney cancer
Esophageal carcinoma KIT kidney cancer MET kidney cancer
Esophageal carcinoma MUC16 ovarian cancer
Cervical cancer
Mammary cancer SLC39A6 Mammary cancer
prostate cancer SLC44A4 prostate cancer STEAP1 prostate cancer

Claims (107)

1. A conjugate for the treatment of a neoplastic disorder, wherein the conjugate comprises formula (I) Ab-(LD) p (I) [formula (I)], where Ab is an antibody or antigen-binding fragment that specifically affects the neoplastic cell; D is a splicing modulator; L is a linker that covalently links Ab to D; And p is an integer from 1 to 15; characterized in that that L-D includes a compound of formula (III)

(III) or a pharmaceutically acceptable salt thereof, where: R ¹ is absent or selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups, -OC(=O)-(C ₁ -C ₆ alkyl) and -CD ₃ groups; R ² is an L linker containing at least one cleavable fragment; R ³ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₁ -C ₆ alkylalkoxy groups, C ₁ -C ₆ alkylamino groups, C ₁ -C ₆ alkylcarboxylic acid groups, C ₁ -C ₆ alkylhydroxy groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups, C ₃ -C ₈ heterocyclyl groups and -OC(=O)-(C ₁ -C ₆ alkyl) groups; each of R ⁴ , R ⁵ and R ⁸ is independently selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups and C ₁ -C ₆ alkyl groups; each of R ⁶ and R ⁷ is independently selected from hydrogen, -OR ¹⁷ , -OC(=O)-R ¹⁷ , -OC(=O)-NR ¹⁵ R ¹⁶ , C ₁ -C ₆ alkyl groups and -NR ¹⁵ R ¹⁶ ; each of R ¹⁵ and R ¹⁶ is independently selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C(=O)-OR ¹⁷ ; R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups; And Z'' selected from

,

,

,

And

, where each of R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ¹⁵ , R ¹⁶ and R ¹⁷ is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, -O-(C ₁ -C ₆ alkyl), -NR ¹⁵ R ¹⁶ , C ₃ -C ₈ cycloalkyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkylalkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups; where at least one of R ⁶ and R ⁷ is hydrogen; And if Z'' is

and R ³ , R ⁶ and R ⁷ are H, R ⁴ is methoxy and R ⁵ is hydroxyl, then R ¹ is neither absent nor methyl. 2. The conjugate according to claim 1, where: R ¹ is absent or selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₁ -C ₄ alkylcarboxylic acid groups, C ₁ -C ₄ alkylhydroxy groups and C ₃ -C ₈ cycloalkyl groups; R ² is an L linker containing at least one cleavable fragment; R ³ is selected from hydrogen, C ₁ -C ₄ alkyl groups, C ₁ -C ₄ alkylalkoxy groups, C ₁ -C ₄ alkylcarboxylic acid groups and C ₁ -C ₄ alkylhydroxy groups; R ⁴ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl), -OC(=O)-(C ₁ -C ₄ alkyl) groups, and C ₁ -C ₄ alkyl groups; R ⁵ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl) groups and C ₁ -C ₄ alkyl groups; R ⁶ is selected from hydrogen, -OR ¹⁷ , -OC(=O)-R ¹⁷ and C ¹ -C ⁴ alkyl groups; R ⁷ is selected from hydrogen, -OR ¹⁷ , -OC(=O)-R ¹⁷ , -OC(=O)-NR ¹⁵ R ¹⁶ , C ₁ -C ₆ alkyl groups and -NR ¹⁵ R ¹⁶ ; R ⁸ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl) groups and C ₁ -C ₄ alkyl groups; each of R ¹⁵ and R ¹⁶ is independently selected from hydrogen, R ¹⁷ , -C(=O)-R ¹⁷ and -C(=O)-OR ¹⁷ ; R ¹⁷ is selected from hydrogen, C ₁ -C ₆ alkyl groups, C ₃ -C ₈ cycloalkyl groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups; And Z'' selected from

,

,

,

And

; where each of R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ¹⁵ , R ¹⁶ and R ¹⁷ is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, -O-(C ₁ -C ₆ alkyl), -NR ¹⁵ R ¹⁶ , C ₃ -C ₈ cycloalkyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkylalkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups; where at least one of R ⁶ and R ⁷ is hydrogen; And if Z'' is

and R ³ , R ⁶ and R ⁷ are H, R ⁴ is methoxy and R ⁵ is hydroxyl, then R ¹ is neither absent nor methyl. 3. The conjugate according to claim 1, where: R ¹ is absent or selected from hydrogen, methyl and C ₁ -C ₄ alkylcarboxylic acid groups; R ² is an L linker containing at least one cleavable fragment; R ³ is selected from hydrogen and C ₁ -C ₄ alkylcarboxylic acid groups; R ⁴ is selected from hydrogen, hydroxyl groups, -O-(C ₁ -C ₄ alkyl), -OC(=O)-(C ₁ -C ₄ alkyl) groups, and C ₁ -C ₄ alkyl groups; R ⁵ is selected from hydrogen and hydroxyl groups; R ⁶ is hydrogen; R ⁷ is hydrogen; R ⁸ is selected from hydrogen and hydroxyl groups; And Z'' represents

; where each of R ¹ , R ³ , R ⁴ is independently substituted with 0-3 groups independently selected from halogen atoms, hydroxyl groups, C ₁ -C ₆ alkyl groups, groups -O-(C ₁ -C ₆ alkyl), C ₃ -C ₈ cycloalkyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkylalkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups; And where if R ³ , R ⁶ and R ⁷ are H, R ⁴ is methoxy and R ⁵ is hydroxy, then R ¹ is neither absent nor methyl. 4. The conjugate according to claim 1, where L-D is selected from a compound of formula

and its pharmaceutically acceptable salt. 5. The conjugate according to claim 1, where L-D is selected from a compound of formula

and its pharmaceutically acceptable salt. 6. The conjugate of claim 1, wherein at least one cleavable fragment in the L linker contains at least one cleavable peptide fragment. 7. The conjugate according to claim 6, where the cleavable peptide fragment contains valine-citrulline (Val-Cit), valine-alanine (Val-Ala), glutamine-valine-citrulline (Glu-Val-Cit) or alanine-alanine-asparagine ( Ala-Ala-Asn). 8. The conjugate of claim 7 wherein the linker contains a maleimide (Mal) moiety. 9. The conjugate of claim 8 wherein the Mal moiety comprises maleimidocaproyl (MC). 10. The conjugate of claim 9 wherein the linker contains MC-Val-Cit or MC-Val-Ala. 11. The conjugate according to claim 6, where the linker contains at least one spacer unit, and the spacer unit contains: (i) a polyethylene glycol (PEG) fragment, where the PEG fragment contains -(PEG) _m - and m is an integer from 1 to 10; or (ii) an alkyl moiety, where the alkyl moiety contains -(CH ₂ ) _n - and n is an integer from 1 to 10. 12. The conjugate of claim 11 wherein the spacer unit is attached to a maleimide (Mal) moiety ("Mal spacer unit"). 13. The conjugate of claim 6 wherein the cleavable moiety in linker L comprises valine (Val) attached to alanine (Ala), where Ala is covalently attached to the compound of formula (III) either directly or through an optional spacer unit. 14. The conjugate of claim 6 wherein the cleavable moiety in linker L comprises valine (Val) attached to citrulline (Cit) wherein Cit is covalently attached to the compound of formula (III) either directly or through an optional spacer unit. 15. The conjugate of claim 6 wherein the L linker is directly attached to the splicing modulator moiety or where the spacer unit attaches the L linker to the splicing modulator. 16. The conjugate of claim 15, wherein the spacer linking the cleavable fragment in the linker to the splicing modulator is self-cleaving. 17. The conjugate of claim 15, wherein the spacer linking the cleavable moiety in the linker to the splicing modulator contains p-aminobenzyloxycarbonyl (pABC) or p-aminobenzyl (pAB). 18. The conjugate of claim 17 wherein pABC or pAB attaches the cleavable fragment in the linker to the splicing modulator. 19. The conjugate of claim 18 wherein the cleavable peptide fragment comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. 20. The conjugate of claim 19 wherein the linker contains Val-Cit-pABC, Val-Ala-pABC, Val-Cit-pAB, or Val-Ala-pAB. 21. The conjugate of claim 20 wherein the linker contains MC-Val-Cit-pABC, MC-Val-Ala-pABC, MC-Val-Cit-pAB, or MC-Val-Ala-pAB. 22. The conjugate according to any one of paragraphs. 1-5 or a pharmaceutically acceptable salt thereof, wherein the linker L contains

. 23. The conjugate according to any one of claims 1-21, where p is from 1 to 10. 24. The conjugate according to any one of claims 1-21, where p is from 2 to 8. 25. The conjugate according to any one of claims 1-21, where p is from 4 to 8. 26. The conjugate of any one of claims 1 to 21, wherein the antibody or antigen-binding fragment specifically targets a neoplastic cell derived from a hematologic malignancy or solid tumor; wherein the hematologic malignancy is selected from B-cell malignancy, acute myeloid leukemia, and multiple myeloma; and wherein the solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer and cancer of the esophagus. 27. The conjugate according to any one of claims 1-21, where a) the antibody or antigennegative fragment thereof is targeted to a cell expressing HER2, where the antibody or antigennegative fragment is an anti-HER2 antibody or antigennegative fragment; and/or wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) containing the amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 2 (HCDR2), and SEQ ID NO: 3 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences of SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2) and SEQ ID NO: 6 (LCDR3); and/or where the antibody or antigennegative fragment contains a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 19, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 20; b) the antibody, or antigen-binding fragment thereof, targets a cell expressing CD138; where the antibody or antigennegative fragment is an antibody against CD138 or antigennegative fragment; and/or wherein the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) containing the amino acid sequences of SEQ ID NO: 7 (HCDR1), SEQ ID NO: 8 (HCDR2) and SEQ ID NO: 9 ( HDDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2) and SEQ ID NO: 12 (LCDR3); and/or where the antibody or antigennegative fragment contains a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 21, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 22; c) the antibody, or antigen-binding fragment thereof, targets a cell expressing EPHA2; where the antibody or antigennegative fragment is an antibody against EPHA2 or antigennegative fragment; and/or wherein the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) containing the amino acid sequences of SEQ ID NO: 13 (HCDR1), SEQ ID NO: 14 (HCDR2) and SEQ ID NO: 15 ( HDDR3); and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) containing the amino acid sequences of SEQ ID NO: 16 (LCDR1), SEQ ID NO: 17 (LCDR2) and SEQ ID NO: 18 (LCDR3); and/or where the antibody or antigennegative fragment contains a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 23, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 24; (d) the antibody or antigen-binding fragment targets a cell expressing CEACAM5; optionally wherein the antibody or antigen-binding fragment is an anti-CEACAM5 antibody or antigen-binding fragment thereof; or (e) the antibody, or antigen-binding fragment thereof, targets a cell expressing STEAP1; optionally wherein the antibody or antigen-binding fragment is an anti-STEAP1 antibody or antigen-binding fragment thereof. 28. The conjugate of claim 27 wherein the antibody or antigen binding fragment comprises a human IgG1 heavy chain constant region and/or wherein the antibody or antigen binding fragment comprises a human Ig kappa light chain constant region. 29. A pharmaceutical composition for the treatment of a neoplastic disorder, comprising an effective amount of a conjugate according to any one of claims 1 to 21 and a pharmaceutically acceptable carrier. 30. The pharmaceutical composition of claim 29 for use in treating a subject having a neoplastic disorder, wherein the neoplastic disorder is: (i) a hematologic malignancy selected from B-cell malignancy, acute myeloid leukemia, and multiple myeloma; or (ii) a solid tumor selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer and esophageal cancer. 31. A composition for use according to claim 30, wherein the average p value of the antibody drug conjugate in the composition is 3.5 to 5.5, or 7 to 9, or 4, or 8. 32. A method of treating a subject having a neoplastic disorder, comprising administering to the subject a therapeutically effective amount of the composition of claim 29. 33. The method of claim 32, wherein the neoplastic disorder is: (i) a hematologic malignancy selected from B-cell malignancy, acute myeloid leukemia, and multiple myeloma; or (ii) a solid tumor selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer and esophageal cancer. 34. The method of claim 33, wherein the average p value of the antibody drug conjugate in the composition is 3.5 to 5.5, or 7 to 9, or 4, or 8. 35. A method of reducing tumor growth in a subject suffering from a neoplastic disease, comprising administering to the subject a therapeutically effective amount of a composition according to claim 29. 36. The method of claim 35, wherein the neoplastic disorder is: (i) a hematologic malignancy selected from B-cell malignancy, acute myeloid leukemia, and multiple myeloma; or (ii) a solid tumor selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer and esophageal cancer. 37. The method of claim 36, wherein the average p value of the antibody drug conjugate in the composition is 3.5 to 5.5, or 7 to 9, or 4, or 8. 38. A method of inhibiting tumor growth in a subject having a neoplastic disorder, comprising administering to the subject a therapeutically effective amount of a composition according to claim 29. 39. The method of claim 38, wherein the neoplastic disorder is: (i) a hematologic malignancy selected from B-cell malignancy, acute myeloid leukemia, and multiple myeloma; or (ii) a solid tumor selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer and esophageal cancer. 40. The method of claim 39, wherein the average p value of the antibody drug conjugate in the composition is 3.5 to 5.5, or 7 to 9, or 4, or 8. 41. A pharmaceutical composition for the treatment of a neoplastic disorder, comprising an effective amount of the conjugate of claim 27 and a pharmaceutically acceptable carrier. 42. The pharmaceutical composition of claim 41 for use in treating a subject having a neoplastic disorder, wherein the neoplastic disorder is: (i) a hematologic malignancy selected from B-cell malignancy, acute myeloid leukemia, and multiple myeloma; or (ii) a solid tumor selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer and esophageal cancer. 43. A composition for use according to claim 42, wherein the average p value of the antibody drug conjugate in the composition is 3.5 to 5.5, or 7 to 9, or 4, or 8. 44. A method of treating a subject having a neoplastic disorder, comprising administering to the subject a therapeutically effective amount of the composition of claim 41. 45. The method of claim 44, wherein the neoplastic disorder is: (i) a hematologic malignancy selected from B-cell malignancy, acute myeloid leukemia, and multiple myeloma; or (ii) a solid tumor selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer and esophageal cancer. 46. The method of claim 45, wherein the average p value of the antibody drug conjugate in the composition is 3.5 to 5.5, or 7 to 9, or 4, or 8. 47. A method of reducing tumor growth in a subject having a neoplastic disorder, comprising administering to the subject a therapeutically effective amount of a composition according to claim 41. 48. The method of claim 47, wherein the neoplastic disorder is: (i) a hematologic malignancy selected from B-cell malignancy, acute myeloid leukemia, and multiple myeloma; or (ii) a solid tumor selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer and esophageal cancer. 49. The method of claim 48, wherein the average p value of the antibody drug conjugate in the composition is 3.5 to 5.5, or 7 to 9, or 4, or 8. 50. A method for inhibiting tumor growth in a subject having a neoplastic disorder, comprising administering to the subject a therapeutically effective amount of a composition according to claim 41. 51. The method of claim 50, wherein the neoplastic disorder is: (i) a hematologic malignancy selected from B-cell malignancy, acute myeloid leukemia, and multiple myeloma; or (ii) a solid tumor selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer and esophageal cancer. 52. The method of claim 51, wherein the average p value of the antibody drug conjugate in the composition is 3.5 to 5.5, or 7 to 9, or 4, or 8.

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