TW202530697A - Biomarkers for eribulin-based antibody-drug conjugates and methods of use - Google Patents

Abstract

Biomarkers and methods of using them in treating folate receptor alpha-expressing cancers, such as platinum resistant ovarian cancer, are disclosed herein.

Description

Biomarkers and methods of use of eribulin-based antibody-drug conjugates

The present disclosure relates to biomarkers and methods for treating folate receptor alpha (FRA) phenotypical cancers using antibody-drug conjugates (ADCs) that bind to folate receptor alpha and provide anti-tubulin drug activity.

Cancer is a leading cause of morbidity and mortality worldwide, accounting for approximately 10 million cancer-related deaths in 2020. The most common causes of cancer death are lung cancer (1.8 million deaths); colon and rectal cancer (916,000); liver cancer (830,000 deaths); stomach cancer (769,000 deaths); and breast cancer (685,000 deaths). In addition, approximately 207,000 women died from ovarian cancer in 2020. The number of new cancer cases is expected to increase by approximately 70% over the next two decades, to approximately 22 million new cancer cases annually (World Cancer Report 2014).

Microtubules are dynamic, filamentous cytoskeletal proteins involved in a variety of cellular functions, including intracellular migration and transport, cell signaling, and maintenance of cell shape. Microtubules also play a key role in mitophagic cell division by forming the mitotic spindles required for chromosome segregation into two daughter cells. The biological functions of microtubules in all cells are largely regulated by their polymerization dynamics, which occurs through the reversible, non-covalent addition of α-tubulin dimers and β-tubulin dimers to the microtubule ends. This dynamic behavior and the resulting control of microtubule length are crucial for the proper function of the mitotic spindles. Even subtle changes in microtubule dynamics can activate the spindle checkpoint, arresting cell cycle progression during mitosis and subsequently leading to cell death (Mukhtar et al. (2014) Mol. Cancer Ther. 13:275-84). Because cancer cells divide rapidly, they are often more sensitive than normal cells to compounds that bind to tubulin and disrupt its normal function. For this reason, tubulin inhibitors and other agents targeting microtubules have become a promising class of cancer therapeutics (Dumontet and Jordan (2010) Nat. Rev. Drug Discov. 9:790-803).

Folate receptor alpha (FRA) is a glycophosphatidylinositol (GPI)-linked membrane protein that binds folate. Although the role of FRA in normal and cancerous tissue biology is not fully understood, it is highly overexpressed in a high proportion of epithelial ovarian cancers (O'Shannessy et al. (2013) Int. J. Gynecol. Pathol. 32(3):258-68) and in a proportion of non-small cell lung cancers (Christoph et al. (2014) Clin. Lung Cancer 15(5):320-30). FRA also has limited expression in normal tissues. These properties make FRA an attractive target for cancer immunotherapy.

Compounds with biological activity against FRA-expressing tumor cells, such as ADCs, for example, anti-FRA ADCs, such as MORAb-202 (also known as farletuzumab ecteribulin (FZEC)), have previously been shown to be effective in treating folate receptor alpha (FRA)-expressing cancers. MORAb-202 has also been shown to be effective in treating FRA-expressing cancers when administered based on body surface area, as disclosed in PCT/US2023/018214, which is incorporated herein by reference in its entirety. However, there remains a need for more refined methods and dosing regimens for treating subjects with FRA-phenotypical cancers with anti-FRA ADCs (e.g., MORAb-202), for example, to reduce the side effects of treatment and/or ensure that treatment is better targeted to those who are responsive to ADC therapy.

In various embodiments, the present disclosure provides a method for treating a cancer expressing a folate receptor alpha (FRA) phenotype, the method comprising (a) measuring the level of one or more biomarkers of tumor response in a baseline biological sample from a subject, and (b) administering to a subject in need thereof an amount of an antibody-drug conjugate having Formula (I): Ab-(L-D)p (I) wherein (i) Ab is an internalizing anti-folate receptor alpha antibody or an internalizing antigen-binding fragment thereof comprising: three heavy chain complementary determining regions (HCDRs) comprising 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 complementary determining regions (LCDRs) comprising SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2), and SEQ ID NO: 6 (LCDR3), as defined by the Kabat numbering system; or three heavy chain complementation determining regions (HCDRs) comprising 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 complementation determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQ ID NO: 12 (LCDR3), as defined by the IMGT numbering system; (ii)       D is eribulin; (iii)      L is a cleavable linker comprising Mal-(PEG)2-Val-Cit-pAB; and (iv)      p is an integer from 1 to 8, (c) measuring the level of one or more biomarkers of tumor response in a sample taken from the subject to determine a change from the level in a baseline sample, and (d) administering a further dose of the antibody-drug conjugate if a change in level is detected.

In some embodiments, the present disclosure provides a method for reducing the risk of interstitial lung disease (ILD) in a subject being treated for a cancer with a FRA phenotype, the method comprising: (a) measuring the level of one or more biomarkers of tumor response in a baseline biological sample from the subject, (b) administering to the subject in need thereof an amount of an antibody-drug conjugate having formula (I): Ab-(L-D)p     (I) wherein (i) Ab is an internalizing anti-folate receptor alpha antibody or an internalizing antigen-binding fragment thereof comprising: three heavy chain complementary determining regions (HCDRs) comprising 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 complementary determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2), and SEQ ID NO: 6 (LCDR3), as defined by the Kabat numbering system; or three heavy chain complementation determining regions (HCDRs) comprising 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 complementation determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQ ID NO: 12 (LCDR3), as defined by the IMGT numbering system; (ii)       D is eribulin; (iii)      L is a cleavable linker comprising Mal-(PEG)2-Val-Cit-pAB; and (iv)    p is an integer from 1 to 8, (c) measuring the level of one or more biomarkers of tumor response in a sample taken from the subject to determine a change from the level in a baseline sample, and (d) administering a further dose of the antibody-drug conjugate if a change in level is detected.

In some embodiments, the level of surfactant protein D (SP-D) in a sample taken from a subject after administration is increased relative to the level of SP-D in the baseline sample. In some embodiments, the level of SP-D is increased by at least 100% relative to the level of SP-D in the baseline sample. In some embodiments, the level of kallikrein-related peptidase 7 (KLK-7) in a sample taken from a subject after administration is decreased relative to the level of KLK-7 in the baseline sample. In some embodiments, the level of KLK-7 is decreased by at least 30% relative to the level of KLK-7 in the baseline sample. In some embodiments, the level of monocytic protein-1 (MCP-1) in a sample taken from a subject after administration is decreased relative to the level of MCP-1 in the baseline sample. In some embodiments, the level of MCP-1 is decreased by at least 15% relative to the level of MCP-1 in the baseline sample. In some embodiments, the level of angiopoietin 2 (ANG-2) in a sample taken from a subject after administration is reduced relative to the level of ANG-2 in the baseline sample. In some embodiments, the level of ANG-2 is reduced by at least 30% relative to the level of ANG-2 in the baseline sample. In some embodiments, the level of kallikrein-related peptidase 5 (KLK-5) in a sample taken from a subject after administration is reduced relative to the level of KLK-5 in the baseline sample. In some embodiments, the level of KLK-5 is reduced by at least 40% relative to the level of KLK-5 in the baseline sample. In some embodiments, the level of matrix metalloproteinase 12 (MMP12) in a sample taken from a subject after administration is reduced relative to the level of MMP12 in the baseline sample. In some embodiments, the level of MMP12 is reduced by at least 40% relative to the level of MMP12 in the baseline sample. In some embodiments, the level of ferritin (FRTN) in a sample taken from a subject after administration is reduced relative to the level of FRTN in a baseline sample. In some embodiments, the level of FRTN is reduced by at least 40% relative to the level of FRTN in a baseline sample. In some embodiments, the level of insulin-like growth factor binding protein 2 (IGFBP-2) in a sample taken from a subject after administration is reduced relative to the level of IGFBP-2 in a baseline sample. In some embodiments, the level of IGFBP-2 is reduced by at least 30% relative to the level of IGFBP-2 in a baseline sample.

In some embodiments, the present disclosure provides a method for treating a cancer with a folate receptor alpha (FRA) phenotype, the method comprising: (a) measuring the level of one or more biomarkers of tumor response in a baseline biological sample from a subject, (b) administering to a subject in need thereof an amount of an antibody-drug conjugate having formula (I): Ab-(L-D)p     (I) wherein (i) Ab is an internalizing anti-folate receptor alpha antibody or an internalizing antigen-binding fragment thereof comprising: three heavy chain complementary determining regions (HCDRs) comprising 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 complementary determining regions (LCDRs) comprising SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2), and SEQ ID NO: 6 (LCDR3), as defined by the Kabat numbering system; or three heavy chain complementation determining regions (HCDRs) comprising 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 complementation determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQ ID NO: 12 (LCDR3), as defined by the IMGT numbering system; (ii)       D is eribulin; (iii)      L is a cleavable linker comprising Mal-(PEG)2-Val-Cit-pAB; and (iv)      p is an integer from 1 to 8, (c) Measuring the level of one or more tumor-responsive biomarkers in a sample taken from the subject to determine a change from the level in a baseline sample, and (d) administering a further dose of the antibody-drug conjugate if no change in level is detected, wherein the biomarker is one or more of IGFBP-1, FLT3L, and VEGF-D. In some embodiments, the tumor-responsive biomarker is insulin-like growth factor binding protein 1 (IGFBP-1). In some embodiments, the tumor-responsive biomarker is fms-related receptor tyrosine kinase 3 ligand (FLT3LG). In some embodiments, the tumor-responsive biomarker is vascular endothelial growth factor D (VEGF-D).

In some embodiments, the present disclosure provides a method for treating a cancer with a folate receptor alpha (FRA) phenotype, the method comprising: (a) measuring the level of one or more biomarkers of tumor response in a baseline biological sample from a subject, (b) administering to a subject in need thereof an amount of an antibody-drug conjugate having formula (I): Ab-(L-D)p     (I) wherein (i) Ab is an internalizing anti-folate receptor alpha antibody or an internalizing antigen-binding fragment thereof comprising: three heavy chain complementary determining regions (HCDRs) comprising 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 complementary determining regions (LCDRs) comprising SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2), and SEQ ID NO: 6 (LCDR3), as defined by the Kabat numbering system; or three heavy chain complementation determining regions (HCDRs) comprising 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 complementation determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQ ID NO: 12 (LCDR3), as defined by the IMGT numbering system; (ii)       D is eribulin; (iii)      L is a cleavable linker comprising Mal-(PEG)2-Val-Cit-pAB; and (iv)      p is an integer from 1 to 8, (c) Measuring the level of one or more tumor response biomarkers in a sample taken from the subject to determine a change relative to the level in a baseline sample, and (d) determining the efficacy of the antibody-drug conjugate based on the change in the level of the one or more tumor response biomarkers.

In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and a light chain comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody-drug conjugate is MORAb-202. In some embodiments, p is 3 to 5.

In some embodiments, the dose is 8 mg to 44 mg/m2 of the subject's BSA. In some embodiments, the dose is 0.3 mg/kg to 1.2 mg/kg of the subject's body weight. In some embodiments, the antibody-drug conjugate is administered once every three weeks. In some embodiments, the antibody-drug conjugate is administered once every two weeks. In some embodiments, the antibody-drug conjugate is administered once weekly. In some embodiments, the method further comprises administering a corticosteroid. In some embodiments, the antibody-drug conjugate is administered intravenously.

In some embodiments, the FRA-expressing cancer is selected from the group consisting of gastric cancer, ovarian cancer, serous ovarian cancer, serous high-grade ovarian cancer, clear cell ovarian cancer, platinum-resistant ovarian cancer, lung cancer, non-small cell lung cancer, metastatic non-small cell lung cancer, lung carcinoid, colorectal cancer, breast cancer, triple-negative breast cancer, hormone receptor (HR)-positive and HER2-low breast cancer, endometrial cancer, serous endometrial cancer, peritoneal cancer, primary peritoneal cancer, fallopian tube cancer, pancreatic cancer, kidney cancer, renal cell cancer, cervical cancer, esophageal cancer, osteosarcoma, ependymoma, ependymoblastoma, neurotubercocyte germ cell tumor, nephroblastoma, acute myeloid leukemia, and head and neck cancer. In some embodiments, the FRA phenotype cancer is ovarian cancer. In some embodiments, the ovarian cancer is platinum-resistant ovarian cancer (PROC). In some embodiments, the FRA phenotype cancer is a refractory cancer. In some embodiments, the refractory cancer is refractory to targeted therapy. In some embodiments, the targeted therapy is directed against any of the following genes or variants thereof: EGFR, ALK, BRAF, RET, MET, NTRK, and ROS1. In some embodiments, the refractory cancer is refractory to both a platinum-based therapy and an immunotherapy-based therapy, wherein the therapies are administered concurrently or sequentially. In some embodiments, the platinum-based therapy is platinum doublet chemotherapy, and the immunotherapy-based therapy is a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, the subject does not have one or more of the following at the start of treatment: interstitial lung disease (ILD) and/or pneumonitis, history of ILD and/or pneumonitis, clinically significant specific lung disease, pleural effusion, pericardial effusion, previous lung resection, history of chest radiation within the past 2 years, autoimmune disorder with lung involvement, connective tissue disorder with lung involvement, or inflammatory disorder with lung involvement. In some embodiments, the subject does not have one or more of the following: a history of more than three prior therapies for the FRA phenotype cancer, a high neutrophil-to-lymphocyte ratio, or a serum albumin level less than 3 g/dL at the start of treatment.

The disclosed methods may be more readily understood by reference to the following detailed description in conjunction with the accompanying drawings, which form a part of this disclosure. It should be understood that the present disclosure is not limited to the specific methods disclosed and/or shown herein, and that the terminology used herein is for the purpose of disclosing specific embodiments by way of example only and is not intended to be limiting of the claimed methods.

Throughout this document, methods involving the use of compositions, such as anti-FRA ADCs, such as MORAb-202, are described. When the disclosure describes or claims features or embodiments in connection with methods of using the compositions, such features or embodiments apply equally to the compositions.

When a range of values is expressed, it includes embodiments using any specific value within the range. In addition, references to values stated as ranges include every value within the range. All ranges are inclusive and combinable. When a value is expressed as an approximation by using the word "about" in front, it should be understood that the specific value forms another embodiment. Unless the context clearly indicates otherwise, a reference to a specific numerical value includes at least that specific value. The use of "or" means "and/or" unless otherwise indicated in the specific context. All references cited herein are incorporated by reference for any purpose. In the event of a conflict between a reference and the present specification, the present specification shall prevail.

It will be appreciated that certain features of the disclosed methods that, for clarity, are disclosed herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, different features of the disclosed methods that, for brevity, are disclosed in the context of a single embodiment, may also be provided separately or in any subcombination.

Throughout this specification and the claims, various terms related to the described aspects are used. Unless otherwise indicated, such terms will be given their ordinary meanings in the art. Other specifically defined terms are to be interpreted in a manner consistent with the definitions provided herein.

As used herein, the singular forms "a," "an," and "the" include plural forms unless the context clearly dictates otherwise.

As will be apparent to those skilled in the art from the teachings contained herein, the terms "about" or "approximately" in the context of numerical values and ranges refer to values or ranges that are approximately or close to the recited value or range so that an embodiment can be performed as intended, such as having a desired amount of nucleic acid or polypeptide in a reaction mixture. This is due, at least in part, to the varying properties of nucleic acid composition, age, race, sex, anatomical and physiological variations, and the imprecision of biological systems. Thus, these terms encompass values that exceed those resulting from systematic error. It should be understood that the definition of "about" applies throughout this disclosure, unless otherwise specified in certain contexts, for example, when describing the average number of drug moieties for each individual antibody moiety or within a mixture of ADCs.

The term "agent" is used herein to refer to a compound, a mixture of compounds, a biomacromolecule, an extract prepared from biological material, or a combination thereof. The terms "therapeutic agent," "drug," or "drug moiety" refer to an agent that modulates a biological process and/or possesses biological activity.

The term "antibody" is used in the broadest sense to refer to immunoglobulin molecules that recognize and specifically bind to a target, such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or combination thereof, via at least one antigenic recognition site within the immunoglobulin molecule's variable region. The heavy chain of an antibody comprises a heavy chain variable domain ( _VH ) and a heavy chain constant region ( _CH ). The light chain comprises a light chain variable domain ( _VL ) and a light chain constant domain ( _CL ). For the purposes of this application, the mature heavy and light chain variable domains each comprise three complementary determining regions (CDR1, CDR2, and CDR3) within four framework regions (FR1, FR2, FR3, and FR4) arranged from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. An "antibody" can be naturally occurring or artificially produced, such as a monoclonal antibody produced by conventional fusion tumor technology. The term "antibody" includes full-length monoclonal and full-length polyclonal antibodies, as well as antibody fragments such as Fab, Fab', F(ab')2, Fv, and single-chain antibodies. The antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses thereof (e.g., isotype IgG1, IgG2, IgG3, IgG4). The term further encompasses human antibodies, chimeric antibodies, humanized antibodies, and any modified immunoglobulin molecules containing an antigen recognition site, so long as they exhibit the desired biological activity.

As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. In some cases, the variable regions of both the heavy and light chains correspond to those of an antibody 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 responses in the latter species.

As used herein, the term "human antibody" refers to an antibody produced by humans or an antibody having the amino acid sequence of an antibody produced by humans.

As used herein, the term "humanized antibody" refers to an antibody form that contains sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulins. Generally speaking, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are framework regions of human immunoglobulin sequences. Optionally, a humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. Humanized antibodies can be further modified by substitution of residues in the Fv framework region and/or within the replaced non-human residues to improve and optimize antibody specificity, affinity, and/or activity.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific for a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include multiple antibodies that are specific for or against different epitopes. The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous antibody population and should not be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies used in accordance with the present disclosure can be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or can be produced by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques such as those described in Clackson et al. (1991) Nature 352:624-8 and Marks et al. (1991) J. Mol. Biol. 222:581-97.

The monoclonal antibodies disclosed herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of one or more chains is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they specifically bind to the target antigen and/or exhibit the desired biological activity.

The terms "antibody-drug conjugate,""antibodyconjugate,""conjugate,""immunoconjugate," and "ADC" are used interchangeably to refer to a compound (e.g., eribulin) or a derivative thereof linked to an antibody (e.g., an anti-FRA antibody) and are defined by the following general formula: Ab-(LD) _p (Formula I), where Ab = the antibody portion (i.e., antibody or antigen-binding fragment), L = the linker portion, D = the drug portion, and p = the number of drug portions per antibody portion.

As used herein, the term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., FRA). Preferably, the antigen-binding fragment also retains the ability to be internalized into cells expressing the antigen. In some embodiments, the antigen-binding fragment also retains immune effector activity. Fragments of full-length antibodies have been shown to perform the antigen-binding function of the full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" or "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of a _VL domain, a _VH domain, a _CL domain, and a _CH1 domain; (ii) a F(ab') ₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of a _VH domain and a _CH1 binding domain; (iv) an Fv fragment consisting of the _VL and _VH domains of a single arm of an antibody; (v) a dAb fragment, which comprises a single variable domain, such as a _VH domain (see, e.g., Ward et al. (1989) Nature 341:544-6; and Winter et al., WO 90/05144); and (vi) Separate complementation-determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, _VL and _VH, are encoded by separate genes, they can be joined using recombinant methods via a synthetic linker that enables them to be made as a single protein chain, where the _VL and _VH regions pair to form a monovalent molecule known as a 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 included within the term "antigen-binding fragment" or "antigen-binding portion" of an antibody and are known in the art as an exemplary 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 can be incorporated into fusion proteins. Other forms of single-chain antibodies, such as diabodies, are also contemplated. Diabodies are bivalent, bispecific antibodies in which the _VH and _VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain. This forces the domains to pair with complementary domains from another chain, creating 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 obtained using conventional techniques known to those skilled in the art, and the binding fragments are screened for utility (e.g., binding affinity, internalization) in the same manner as intact antibodies. Antigen-binding fragments can be prepared by cleavage of the intact protein, for example by protease or chemical cleavage.

As used herein, "tumor-responsive biomarkers" refer to the following measurable substances from an organism that are indicators of biological processes relevant to tumor biology: SP-D, ANG-2, IGFBP-1, IgFBP-2, TNF-α, IL-10, ICAM-1, TGFA, VEGF, IL-6, IP-10, KLK-5, KLK-7, MCP-1, MMP12, Eosin-1, IL17C, EGF, FLT3LG, VEGF-D, HGF, OSM, CCL13, MIP-1β, CCL8, HER-2, FRTN, IL-18, and TIMP1. Biomarkers of tumor response may also include additional biomarkers in combination with those listed above, for example from a broader biomarker panel, such as a rules-based medicine (RBM) panel or the ^OLINK® Target-48 cytokine panel.

As used herein, a "biological sample" refers to biological material collected from a living organism. Exemplary biological samples include blood, blood components, tumor biopsies, urine, tissue, cell collections, and saliva.

As used herein, "body surface area" or "BSA" refers to the total surface area of a subject being treated using the methods disclosed herein. A subject's body surface area can be calculated to determine the optimal dose of a compound (e.g., an antibody-drug conjugate, such as an anti-FRA ADC) to administer to the subject. Generally, a subject's body surface area value can be calculated based on the subject's weight.

The term "cancer" refers to a physiological condition in mammals in which a cell population is characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, ovarian cancer (e.g., platinum-resistant ovarian cancer), breast cancer (e.g., triple-negative breast cancer), non-small cell lung cancer, endometrial cancer, peritoneal cancer, and fallopian tube cancer. Triple-negative breast cancer refers to breast cancer that is negative for the estrogen receptor (ER), progesterone receptor (PR), and Her2/neu genes. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, liver cancer, bladder cancer, hepatoma, osteosarcoma, melanoma, colon cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatoepithelial cancer, peritoneal cancer, fallopian tube cancer, and various types of head and neck cancer.

The terms "cancer cell" and "tumor cell" refer to individual cells or entire cell populations originating from a tumor, including non-tumorigenic cells and cancer stem cells. As used herein, the term "tumor cell" is modified by the term "non-tumorigenic" to distinguish tumor cells from cancer stem cells when referring only to those tumor cells that lack the ability to regenerate and differentiate.

The term "chemotherapeutic agent" or "anticancer agent" is used herein to refer to all compounds that are effective in treating cancer, regardless of their mechanism of action. Inhibition of metastasis or angiogenesis is often a property of chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, such as nitrogen mustards, ethyleneimine compounds, and alkyl sulfonates; anti-metabolites, such as folic acid, purine, or pyrimidine antagonists; anti-mitotic agents, such as anti-tubulin agents, such as eribulin or eribulin mesylate (Halaven™) or its derivatives, vinca alkaloids, and auristatins; cytotoxic antibiotics; compounds that damage or interfere with DNA expression or replication, such as DNA minor groove binders; and growth factor receptor antagonists. Additionally, chemotherapeutic agents include antibodies, biomolecules, and small molecules. Chemotherapeutic agents can be cytotoxic or cytostatic. The term "cytostatic" refers to an agent that inhibits or suppresses cell growth and/or cell proliferation.

The term "co-administration" or administration of one or more therapeutic agents "in combination" includes simultaneous administration as well as consecutive administration in any order.

As used herein, "corticosteroid" refers to any compound belonging to a class of steroid hormones normally produced in the adrenal cortex of vertebrates. The term, as used herein, further includes synthetic analogs of these hormones, such as pharmaceutical compositions that mimic the actions of naturally occurring corticosteroids. Corticosteroids are an exemplary embodiment of corticosteroids. Prednisone is also an exemplary embodiment of a corticosteroid. Methylprednisolone is another exemplary embodiment of a corticosteroid.

The term "cytotoxic agent" refers to a substance that causes cell death primarily by interfering with cellular activity and/or function. Examples of cytotoxic agents include, but are not limited to, antimitotic agents such as eribulin, auristatins (e.g., monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF)), maytansinoids (e.g., maytansine), urocandin, duostatin, nostoc, vinca alkaloids (e.g., vincristine, catharanthus alkaloids), taxanes, paclitaxel, and colchicine; anthracyclines (e.g., daunorubicin, adelamicin, dihydroxyanthraquinone); racindione); cytotoxic antibiotics (e.g., mitomycins, actinomycins, duomycins (e.g., CC-1065), auromycin/duomycin, calicheamicin, endomycin, phenomycin); alkylating agents (e.g., cisplatin); intercalating agents (e.g., ethidium bromide); topoisomerase inhibitors (e.g., etoposide, tenoposide); radioisotopes, such as At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² or Bi ²¹³ , P ³² and radioisotopes of tantalum (e.g. Lu ¹⁷⁷ ); and toxins of bacterial, fungal, plant or animal origin (e.g. ricin (e.g. ricin A chain), diphtheria toxin, Pseudomonas exotoxin A (e.g. PE40), endotoxins, mitogens, combrestatins, restrictin, gelonin, α-sarcin, abrin (e.g. abrin A chain), modisin (e.g. modisin A chain), curicin, crotonin, saponin ( Sapaonaria officinalis ) inhibitor), glucocorticoids).

As used herein, the term "dose" refers to the amount of a drug or medication taken at a specific time. A dose can provide an effective amount of an ADC. The drug or medication can be an ADC.

As disclosed herein, an "effective amount" of an ADC is an amount sufficient to carry out a specified purpose (e.g., to produce a therapeutic effect upon administration, such as a reduction in tumor growth rate or tumor size, a reduction in cancer symptoms, or some other indicator of therapeutic efficacy). An effective amount can be determined in a conventional manner relevant to the stated purpose. The term "therapeutically effective amount" refers to an amount of an ADC that is effective in treating a disease or disorder in a subject. In the context of cancer, a therapeutically effective amount of an ADC can reduce the number of cancer cells, reduce tumor size, inhibit (e.g., slow or stop) tumor metastasis, inhibit (e.g., slow or stop) tumor growth, and/or alleviate one or more symptoms. Treatment and therapeutically effective amounts include but do not require complete treatment. For example, the term includes but does not require complete cessation of tumor growth.

The term "epitope" refers to the portion of an antigen that is recognized and specifically bound by an antibody. When the antigen is a polypeptide, the epitope can be formed by consecutive amino acids or non-consecutive amino acids that are adjacent by tertiary folding of the polypeptide. The epitope to which the antibody binds can be identified using any epitope mapping technique known in the art, including X-ray crystallography for epitope identification (by direct visualization of the antigen-antibody complex), monitoring antibody binding to fragments or mutant variants of the antigen, or monitoring solvent accessibility of different portions of the antibody and antigen. Exemplary strategies for mapping antibody epitopes include, but are not limited to, array-based oligopeptide scanning, restricted proteolysis, site-directed mutagenesis, high-throughput mutagenesis 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).

As used herein, the term "eribulin" refers to a synthetic analog of spongin B, a macrocyclic compound originally isolated from the marine sponge Halichondria okadais . The term "eribulin drug moiety" refers to the ADC component that has the eribulin structure and is linked to the ADC linker via its C-35 amine. Eribulin is a microtubule dynamics inhibitor that is believed to bind to tubulin and induce cell cycle arrest in the G2/M phase by inhibiting mitotic spindle assembly. The term "eribulin mesylate" refers to the mesylate salt of eribulin, marketed under the trade name Halaven™.

As used herein, the term "folate receptor alpha" or "FRA" refers to any naturally occurring form of human FRA. The term encompasses full-length FRA (e.g., NCBI Reference Sequence: NP_000793; SEQ ID NO: 37), as well as any form of human FRA produced by cellular processing. The term also encompasses naturally occurring variants of FRA, including but not limited to splice variants, allelic variants, and isoforms. FRA can be isolated from humans or produced recombinantly or synthetically.

The terms "anti-FRA antibody" or "antibody that specifically binds to FRA" refer to any form of antibody or fragment thereof that specifically binds to FRA and encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, as long as such biologically functional antibody fragments specifically bind to FRA. Preferably, the anti-FRA antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment. MORAb-003 is an exemplary internalizing anti-human FRA antibody that can be used in the present disclosure, for example, as part of an anti-FRA ADC. As used herein, the terms "specific," "specifically binds," and "specifically binds" refer to the selective binding of an antibody to a target antigen epitope. The binding specificity of an antibody can be tested by comparing binding to an appropriate antigen with binding to an unrelated antigen or antigen mixture under a given set of conditions. An antibody is considered specific if it binds to the appropriate antigen with an affinity that is at least 2, 5, 7, and preferably 10 times greater than that of the unrelated antigen or antigen mixture. In one embodiment, a specific antibody is an anti-FRA antibody that binds only to the FRA antigen and does not bind (or exhibits minimal binding) to other antigens.

As used herein with respect to an antibody or antigen-binding fragment, "internalization" means that the antibody or antigen-binding fragment, after binding to a cell, is able to cross the lipid bilayer membrane of the cell and enter an internal compartment (i.e., "internalization"), preferably into the degradation compartment of the cell. For example, an internalizing anti-FRA antibody is one that is able to be taken up into the cell after binding to FRA on the cell membrane.

The term "interstitial lung disease" refers to any of a group of lung diseases characterized by inflammation that can lead to fibrosis of the lung. Interstitial lung disease (ILD) is known to be a potential adverse effect associated with immunotherapy treatment. An "adverse event" or "AE" is any untoward medical occurrence in a subject administered a compound that does not necessarily imply a causal relationship to the administered compound or that indicates that the compound cannot be used for treatment, but may limit the use of the compound. Methods for identifying and diagnosing interstitial lung disease are well known in the art, for example, the use of pulse oximetry to assess oxygen saturation and the use of chest computed tomography (CT) scans to assess ILD-related lung damage.

"Linker" or "linker moiety" refers to any chemical moiety that is capable of covalently linking a compound, typically a drug moiety such as a chemotherapeutic agent, to another moiety, such as an antibody moiety. The linker may be susceptible to or substantially resistant to acid-induced cleavage, peptidase-induced cleavage, light-based cleavage, esterase-induced cleavage, and/or disulfide bond cleavage under conditions that preserve the activity of the compound or antibody.

The term " p " or "antibody:drug ratio" or "drug to antibody ratio" or "DAR" refers to the number of drug moieties per antibody moiety, i.e., drug loading, or the number of -LD moieties per antibody or antigen-binding fragment (Ab) in an ADC having Formula I. In a composition comprising multiple copies of an ADC having Formula I, " p " refers to the average number of -LD moieties per antibody or antigen-binding fragment, also referred to as average drug loading.

"Pharmaceutically acceptable" means approved or approvable by a U.S. federal or state regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and more specifically in humans.

A "pharmaceutical composition" is a preparation that is in a form that permits administration of the active ingredient(s) and subsequently provides for the desired biological activity of one or more active ingredients and/or achieves a therapeutic effect, and does not contain additional components that are unacceptably toxic to a subject upon administration of the formulation. Pharmaceutical compositions may be sterile.

"Drug formulators" include substances such as excipients, carriers, pH adjusters and buffers, tonicity adjusters, wetting agents, preservatives, etc.

As used herein, "protein" means 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 by a peptide bond. Proteins can be composed of naturally occurring amino acids and peptide bonds, such as when the protein is made recombinantly using an expression system and a host cell. Alternatively, proteins can include synthetic amino acids (e.g., homophenylalanine, citrulline, ornithine, and norleucine) or peptide mimetic structures (i.e., "peptide or protein analogs," such as peptoids).

For amino acid sequences, sequence identity and/or similarity may be determined using standard techniques known in the art or by inspection, including but not limited to the following: the local sequence identity algorithm of Smith and Waterman, (1981) Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, the similarity search method of 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 Suite, Genetics Computer Group, 575 Preferably, percent identity is calculated using the best-fit sequence program described in Science Drive (Madison, WI), Devereux et al. (1984) Nucl. Acid Res. 12:387-95 (preferably using the default settings). Preferably, percent identity is calculated using FastDB based on the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and ligation 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).

An example of an applicable algorithm is PILEUP. PILEUP generates a multiple sequence alignment from a set of related sequences using progressive pairwise alignment. It also produces a tree diagram showing the cluster relationships used to generate the alignment. PILEUP uses a simplified form of the progressive alignment method of Feng and Doolittle (1987) J. Mol. Evol. 35:351-60; this method is similar to the method described by Higgins and Sharp (1989) CABIOS 5:151-3. Applicable PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

Another example of a suitable algorithm is the BLAST algorithm described in Altschul et al. (1990) J. Mol. Biol. 215:403-10; Altschul et al. (1997) Nucleic Acids Res. 25:3389-402; and Karin et al. (1993) Proc. Natl. Acad. Sci. USA 90:5873-87. A particularly suitable BLAST program is the WU-BLAST-2 program obtained from Altschul et al. (1996) Methods in Enzymology 266:460-80. WU-BLAST-2 uses several search parameters, most of which are set to default values. The adjustable parameters were set to the following values: overlap interval = 1, overlap fraction = 0.125, word threshold (T) = 11. The HSP S and HSP S2 parameters are dynamic values and are determined by the program itself based on the composition of the particular sequence and the composition of the particular database against which the target sequence is searched; however, these values can be adjusted to increase sensitivity.

An additional applicable algorithm is the Gapped BLAST algorithm described by Altschul et al. (1993) Nucl. Acids Res. 25:3389-402. Gapped BLAST uses BLOSUM-62 instead of scoring; the threshold parameter T is set to 9; a two-hit method is used to trigger ungapped stretches, adding a gap of length k at a cost of 10 + k; Xu is set to 16, and Xg is set to 40 during the database search phase and to 67 during the algorithm output phase. Gapped alignments are triggered by a score corresponding to approximately 22 bits.

Typically, the proteins and variants thereof disclosed herein (including variants of FRAs and variants of antibody variable domains (including individual variant CDRs)) have an amino acid homology, similarity or identity of at least 80% with the sequences described herein, and more typically, preferably, have an increased homology or identity of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, nearly 100%, or 100%.

Similarly, the "percent nucleic acid sequence identity (%)" for nucleic acid sequences of antibodies and other proteins identified herein is defined as the percentage of nucleotide residues in the candidate sequence that are identical to the nucleotide residues in the coding sequence of the antigen-binding protein. The specific method utilizes the BLASTN module of WU-BLAST-2 set to default parameters, with the overlap interval and overlap fraction set to 1 and 0.125, respectively.

The terms "subject," "patient," and "participant" are used interchangeably herein to refer to any animal, such as any mammal, including but not limited to humans, non-human primates, rodents, etc. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

The terms "tumor" and "neoplasm" refer to any tissue mass, benign or malignant, caused by excessive cell growth or proliferation, including precancerous lesions.

As used herein, "treat" or "therapeutic" and grammatically related terms refer to any improvement in any outcome of a disease, such as prolonged survival, halted or delayed tumor growth, reduced tumor size, lower morbidity, and/or reduction in side effects as a byproduct of alternative treatment modalities. As is readily understood in the art, complete eradication is preferred but not required for therapeutic action. As used herein, "treatment" or "treat" refers to administering, for example, a disclosed ADC to a subject, such as a patient. Treatment can be curing, healing, alleviating, alleviating, altering, remedying, ameliorating, alleviating, improving, or affecting a condition, a symptom of a condition, or a predisposition to a condition (e.g., cancer). The terms "treatment" and "therapy" are used interchangeably in this article.

In various embodiments, a method for reducing the risk of interstitial lung disease (ILD) in a subject treated with an anti-FRA ADC is provided herein. As used herein, " reducing risk " refers to a change (such as, a change in the subject quantity of ILD) relative to the ILD incidence rate for comparison treatment. For example, compared with a similar subject administered with an anti-FRA ADC in equal doses based on body weight within a given time period, the anti-FRA ADC treatment disclosed herein using a dosing regimen based on BSA can reduce the risk of ILD in some subjects.

As used herein, the term "upper quartile of weight" refers to weight values that are within the top 25% of all weight values for a given group of subjects (e.g., a group of adults in the general population or a group of subjects with FRA phenotype cancers). As used herein, the term includes values that represent the boundaries of the top 25%. For example, the upper quartile of weight for a given group of subjects can be determined by first arranging the weight values of the subjects in ascending order, then dividing the set of values into four equal parts (also called quartiles), and finally determining the values between the third and fourth quartiles. A subject in the upper quartile has a weight value that is at least equal to, if not greater than, the value between the third and fourth quartiles. In some embodiments, the value between the third and fourth quartiles for a given group of subjects is approximately or about 80 kilograms. Antibody-Drug Conjugates

The methods disclosed herein involve the use of compounds having anticancer activity. In particular, such compounds include an antibody portion (including antigen-binding fragments thereof) conjugated (i.e., covalently linked via a linker) to a drug moiety, wherein the drug moiety, for example, when not conjugated to the antibody moiety, exhibits cytotoxic or cytostatic activity. In various embodiments, the drug moiety exhibits reduced or no cytotoxicity when conjugated to the conjugate, but regains cytotoxicity after cleavage from the linker and the antibody moiety.

In some embodiments, the ADC comprises a peptide cleavable linker that links eribulin to an anti-FRA ADC. In some embodiments, the linker comprises a val-cit moiety. In some embodiments, the linker comprises a PEG spacer. In some embodiments, the linker comprises a Mal-(PEG) ₂ -Val-Cit-pAB linker that links eribulin to an anti-FRA antibody (e.g., an anti-FRA antibody such as MORAb-003). In some embodiments, the anti-FRA ADC is MORAb-202. "MORAb-202" refers to an anti-FRA ADC, wherein the anti-FRA antibody or antigen-binding fragment comprises the heavy chain amino acid sequence of SEQ ID NO: 15 and the light chain amino acid sequence of SEQ ID NO: 16, wherein the linker portion comprises Mal-(PEG) ₂ -Val-Cit-pAB, and wherein the linker is linked to eribulin via the C-35 amine. In some embodiments, the structure of MORAb-202 (including the sequence of the anti-FRA antibody portion of the ADC) is disclosed in PCT Application No. PCT/US2017/020529 (published as WO 2017/151979), which is incorporated herein by reference in its entirety.

In some embodiments, anti-FRA ADCs (e.g., MORAb-202) exhibit particularly advantageous properties within various classes, such as: (i) the ability to retain one or more therapeutic properties exhibited by separate antibody and drug moieties; (ii) the ability to maintain the specific binding properties of the antibody moiety; (iii) the ability to optimize drug loading and drug-to-antibody ratios; (iv) the ability to allow for delivery (e.g., intracellular delivery) of the drug moiety via stable attachment to the antibody moiety; (v) the ability to maintain the stability of the ADC as an intact conjugate until transport or delivery to the target site; (vi) minimizing aggregation of the ADC before or after administration; (vii) The ability to allow for the therapeutic effect of the drug moiety (e.g., cytotoxic effect) following cleavage in the cellular environment; (viii) similar or superior in vivo anticancer therapeutic efficacy to that of the separate antibody and drug moiety; (ix) minimization of off-target killing by the drug moiety; and/or (x) desirable pharmacokinetic and pharmacodynamic properties, formability, and toxicological/immunological profiles.

The disclosed ADC compounds can selectively deliver effective amounts of cytotoxic or cytostatic agents to FRA-expressing cancer cells or FRA-expressing tumor tissues. The disclosed ADCs have been found to exhibit potent cytotoxic and/or cell growth inhibitory activity against FRA-expressing cells. Exemplary FRA-expressing cancers include, but are not limited to, ovarian cancer (e.g., serous ovarian cancer, clear cell ovarian cancer, or platinum-resistant ovarian cancer), lung cancer (e.g., non-small cell lung cancer, e.g., metastatic non-small cell lung cancer), breast cancer (e.g., triple-negative breast cancer), and endometrial cancer.

An exemplary ADC has Formula I: Ab-(LD) _p (I) wherein Ab = internalizing anti-folate receptor alpha antibody or an internalizing antigen-binding fragment thereof, L = cleavable linker moiety, D = drug moiety, and p = number of drug moieties per antibody moiety.

In various embodiments, the antibody portion (Ab) of an ADC having Formula I includes within its scope any antibody or antigen-binding fragment that specifically binds to FRA on cancer cells. The antibody or antigen-binding fragment may bind to FRA with a dissociation constant ( _KD ) of ≤ 1 mM, ≤ 100 nM, or ≤ 10 nM, or any amount therebetween, as measured by, for example, BIAcore® analysis. In certain embodiments, the _KD is between 1 pM and 500 pM. In some embodiments, the _KD is between 500 pM and 1 µM, 1 µM and 100 nM, or 100 mM and 10 nM.

In some embodiments, the antibody portion is a four-chain antibody (also called an immunoglobulin) comprising two heavy chains and two light chains. In some embodiments, the antibody portion is a two-chain half antibody (one light chain and one heavy chain) or an antigen-binding fragment of an immunoglobulin.

In some embodiments, the antibody portion is an internalizing antibody or an internalizing antigen-binding fragment thereof. In some embodiments, the internalizing antibody binds to FRA expressed on the cell surface and enters the cell upon binding. In some embodiments, the FRA-targeting antibody portion is MORAb-003. In some embodiments, the drug portion of the ADC is released from the antibody portion of the ADC after ADC entry and is present in cells expressing the target cancer antigen (i.e., after the ADC has been internalized).

The amino acid and nucleic acid sequences of exemplary antibodies that can be used in the ADCs disclosed herein are listed in Tables 1-9. [ Table 1 ] Antibodies mAbs Class / Isotype target MORAb-003 Humanization Human folate receptor alpha [ Table 2 ] Amino acid sequences of mAb variable regions mAbs IgG chain SEQ ID NO Amino acid sequence 1 MORAb-003 Heavy Chain 13 EVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLEWVAMISSGGSYTYYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVYFCARHGDDPAWFAYWGQGTPVTVSS 2 MORAb-003 Light chain 14 DIQLTQSPSSSLSASVGDRVTITCSVSSSISSNNLHWYQQKPGKAPKPWIYGTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSYPYMYTFGQGTKVEIK [ Table 3 ] Nucleic acid sequences encoding mAb variable domains mAbs IgG chain SEQ ID NO Nucleic acid sequence 1 MORAb-003 Heavy Chain 29 GAGGTCCAACTGGTGGAGAGCGGTGGAGGTGTTGTGCAACCTGGCCGGTCCCTGCGCCTGTCCTGCTCCGCATCTGGCTTCACCTTCAGCGGCTATGGGTTGTCTTGGGTGAGACAGGCACCTGGAAAAGGTCTTGAGTGGGTTGCAATGATTAGTAGTGGTGGTAGTTATACCTACT ATGCAGACAGTGTGAAGGGTAGATTTGCAATATCGCGAGACAACGCCAAGAACACATTGTTCCTGCAAATGGACAGCCTGAGACCCGAAGACACCGGGGTCTATTTTTGTGCAAGACATGGGGACGATCCCGCCTGGTTCGCTTATTGGGGCCAAGGGACCCGGTCACCGTCTCCTCA 2 MORAb-003 Light chain 30 GACATCCAGCTGACCCAGAGCCCAAGCAGCCTGAGCGCCAGCGTGGGTGACAGAGTGACCATCACCTGTAGTGTCAGCTCAAGTATAAGTTCCAACAACTTGCACTGGTACCAGCAGAAGCCAGGTAAGGCTCCAAAGCCATGGATCTACGGCACATCCAACCTG GCTTCTGGTGTGCCAAGCAGATTCAGCGGTAGCGGTAGCGGTACCGACTACACCTTCACCATCAGCAGCCTCCAGCCAGAGGACATCGCCACCTACTACTGCCAACAGTGGAGTAGTTACCCGTACATGTACACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA [ Table 4 ] Amino acid sequences of mAb Kabat CDRs mAbs IgG chain SEQ ID NO Amino acid sequence 1 MORAb-003 HC CDR1 1 GYGLS 2 MORAb-003 HC CDR2 2 MISSGGSYTYYADSVKG 3 MORAb-003 HC CDR3 3 HGDDPAWFAY 4 MORAb-003 LC CDR1 4 SVSSSISSNNLH 5 MORAb-003 LC CDR2 5 GTSNLAS 6 MORAb-003 LC CDR3 6 QQWSSYPYMYT [ Table 5 ] Nucleic acid sequences encoding mAb Kabat CDRs mAbs IgG chain SEQ ID NO Nucleic acid sequence 1 MORAb-003 HC CDR1 17 GGCTATGGGTTGTCT 2 MORAb-003 HC CDR2 18 ATGATTAGTAGTGGTGGTAGTTATACCTACTATGCAGACAGTGTGAAGGGT 3 MORAb-003 HC CDR3 19 CATGGGGACGATCCCGCCTGGTTTCGCTTAT 4 MORAb-003 LC CDR1 20 AGTGTCAGCTCAAGTATAAGTTCCAACAACTTGCAC 5 MORAb-003 LC CDR2 twenty one GGCACATCCAACCTGGCTTCT 6 MORAb-003 LC CDR3 twenty two CAACAGTGGAGTAGTTACCCGTACATGTACACG [ Table 6 ] Amino acid sequences of mAb IMGT CDRs mAbs IgG chain SEQ ID NO Amino acid sequence 1 MORAb-003 HC CDR1 7 GFTFSGYG 2 MORAb-003 HC CDR2 8 ISSGGSYT 3 MORAb-003 HC CDR3 9 ARHGDDPAWFAY 4 MORAb-003 LC CDR1 10 SSISSNN 5 MORAb-003 LC CDR2 11 GTS 6 MORAb-003 LC CDR3 12 QQWSSYPYMYT [ Table 7 ] Nucleic acid sequences encoding mAb IMGT CDRs mAbs IgG chain SEQ ID NO Nucleic acid sequence 1 MORAb-003 HC CDR1 twenty three GGCTTCACCTTCAGCGGCTATGGG 2 MORAb-003 HC CDR2 twenty four ATTAGTAGTGGTGGTAGTTATACC 3 MORAb-003 HC CDR3 25 GCAAGACATGGGGGACGATCCCGCCTGGTTCGCTTAT 4 MORAb-003 LC CDR1 26 TCAAGTATAAGTTCCAACAAC 5 MORAb-003 LC CDR2 27 GGCACATCC 6 MORAb-003 LC CDR3 28 CAACAGTGGAGTAGTTACCCGTACATGTACACG [ Table 8 ] Amino acid sequence of full-length mAb Ig chain mAbs IgG chain SEQ ID NO Amino acid sequence 1 MORAb-003 Heavy Chain 15 EVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLEWVAMISSGGSYTYYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVYFCARHGDDPAWFAYWGQG TPVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 2 MORAb-003 Light chain 16 DIQLTQSPSSSLSASVGDRVTITCSVSSSISSNNLHWYQQKPGKAPKPWIYGTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSYPYMYTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [ Table 9 ] Nucleic acid ^sequences encoding full-length mAb Ig chains mAbs IgG chain SEQ ID NO Nucleic acid sequence 1 MORAb-003 Heavy Chain 31 GAGGTCCAACTGGTGGAGAGCGGTGGAGGTGTTGTGCAACCTGGCCGGTCCCTGCGCCTGTCCTGCTCCGCATCTGGCTTCACCTTCAGCGGCTATGGGTTGTCTTGGGTGAGACAGGCACCTGGAAAAGGTCTTGAGTGGGTTGCAATGATTAGTAGTGGTGGTAGT TATAACCTACTATGCAGACAGTGTGAAGGGTAGATTTGCAATATCGCGAGACAACGCCAAGAACACATTGTTCCTGCAAATGGACAGCCTGAGACCCGAAGACACCGGGGTCTATTTTTGTGCAAGACATGGGGACGATCCCGCCTGGTTCGCTTATTGGGGCCAAGGGA CCCCGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGT GCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACT CACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCA TCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAA GACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCTTATATTCAAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGTCCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAAATGA 2 MORAb-003 Light chain 32 GACATCCAGCTGACCCAGAGCCCAAGCAGCCTGAGCGCCAGCGTGGGTGACAGAGTGACCATCACCTGTAGTGTCAGCTCAAGTATAAGTTCCAACAACTTGCACTGGTACCAGCAGAAGCCAGGTAAGGCTCCAAAGCCATGGATCTACGGCACATCCAACC TGGCTTCTGGTGTGCCAAGCAGATTCAGCGGTAGCGGTAGCGGTACCGACTACACCTTCACCATCAGCAGCCTCCAGCCAGAGGACATCGCCACCTACTACTGCCACAGTGGAGTAGTTACCCGTACATGTACACGTTCGGCCAAGGGACCAAGGTGGAAATC AAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAA ⁺ The listed nucleic acid sequences do not include the leader sequence.

In various embodiments, the ADCs disclosed herein may comprise a set of MORAb-003 heavy and light chain variable domains listed in the table above, or a set of six MORAb-003 CDR sequences (Kabat and/or IMGT) from the heavy and light chain sequences listed in the table above. In some embodiments, the ADC further comprises human heavy and light chain constant domains or fragments thereof. For example, the ADC may comprise a human IgG heavy chain constant domain (e.g., IgG1) and a human kappa or lambda light chain constant domain. In various embodiments, the antibody portion of the disclosed ADC comprises a human immunoglobulin G subtype 1 (IgG1) heavy chain constant domain and a human Ig kappa light chain constant domain. In some embodiments, the anti-FRA antibody portion of the ADC comprises the complete heavy and light chain sequences listed in the table above.

In various embodiments, the anti-FRA antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: a heavy chain CDR1 (HCDR1) comprising SEQ ID NO: 1, a heavy chain CDR2 (HCDR2) comprising SEQ ID NO: 2, a heavy chain CDR3 (HCDR3) comprising SEQ ID NO: 3; a light chain CDR1 (LCDR1) comprising SEQ ID NO: 4, a light chain CDR2 (LCDR2) comprising SEQ ID NO: 5, and a light chain CDR3 (LCDR3) comprising SEQ ID NO: 6, as defined by the Kabat numbering system (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991))).

In some embodiments, the anti-FRA antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs and three light chain CDRs: a heavy chain CDR1 comprising SEQ ID NO:7, a heavy chain CDR2 comprising SEQ ID NO:8, a heavy chain CDR3 comprising SEQ ID NO:9; a light chain CDR1 comprising SEQ ID NO:10, a light chain CDR2 comprising SEQ ID NO:11, and a light chain CDR3 comprising SEQ ID NO:12, as defined by the IMGT numbering system (International ImMunoGeneTics Information System (IMGT®)).

In various embodiments, the anti-FRA antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-FRA antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO: 13 and the light chain variable region amino acid sequence of SEQ ID NO: 14, or sequences having at least 95% identity thereto. In some embodiments, the anti-FRA 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: 13 and 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: 14.

In various embodiments, the anti-FRA antibody comprises a human IgG1 heavy chain constant domain and a human Ig kappa light chain constant domain.

In various embodiments, the anti-FRA antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 15, or a sequence having at least 95% identity to SEQ ID NO: 15, and a light chain amino acid sequence of SEQ ID NO: 16, or a sequence having at least 95% identity to SEQ ID NO: 16. In specific embodiments, the antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 15 and a light chain amino acid sequence of SEQ ID NO: 16, or a sequence having at least 95% identity to such sequences. In some embodiments, the anti-FRA 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: 15 and/or a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 16. In some embodiments, the anti-FRA antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 15 and a light chain amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-FRA antibody comprises a heavy chain encoded by a nucleotide sequence of SEQ ID NO: 35 (with nucleotides encoding a leader sequence) or SEQ ID NO: 31 (without a leader sequence); and a light chain encoded by a nucleotide sequence of SEQ ID NO: 36 (with nucleotides encoding a leader sequence) or SEQ ID NO: 32 (without a leader sequence). In some embodiments, the heavy chain amino acid sequence lacks a C-terminal lysine. In various embodiments, the anti-FRA antibody has the amino acid sequence of an antibody produced by a cell line deposited with the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Va. 20110-2209, USA) under the terms of the Budapest Treaty on April 24, 2006, and designated PTA-7552, or such sequence lacking the heavy chain C-terminal lysine. In various embodiments, the anti-FRA antibody is MORAb-003 (USAN name: farletuzumab) (Ebel et al. (2007) Cancer Immunity 7:6), or an antigen-binding fragment thereof.

In various embodiments, amino acid substitutions are made with single residues. Insertions are typically on the order of about 1 to about 20 amino acid residues, although considerably larger insertions may be tolerated as long as the anti-FRA biological function is retained. Deletions are typically in the range of about 1 to about 20 amino acid residues, but in some cases deletions may be much larger. Substitutions, deletions, insertions, or any combination thereof may be used to obtain the final derivative or variant. Typically, such changes are made to a small number of amino acids, thereby minimizing changes in the molecule, particularly the immunogenicity and specificity of the antigen binding protein. However, in certain circumstances, more changes may be tolerated. Conservative substitutions are generally made according to the diagram depicted below in Table 10. [ Table 10 ] Original Residue Exemplary Substitutions Ala Ser Arg Lys Asn Gln、His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn、Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile、Leu

In some embodiments, the FRA-targeting antibody moiety is MORAb-003. In some embodiments, FRA-targeting antibody moieties such as MORAb-003 provide particularly improved drug:antibody ratios, tumor targeting, bystander killing, therapeutic efficacy, and reduced off-target killing. Improved therapeutic efficacy can be measured in vitro or in vivo and can include a reduced tumor growth rate and/or reduced tumor size. Linker

In various embodiments, the linker in an anti-FRA ADC (e.g., an ADC having Formula I) is extracellularly stable in a manner sufficient to be therapeutically effective. In some embodiments, the linker is stable outside of a cell such that the ADC remains intact when exposed to extracellular conditions (e.g., prior to transport or delivery to FRA-expressing cells). The term "intact," as used in the context of an ADC, means that the antibody moiety remains attached to the drug moiety. As used herein, "stable" in the context of a linker or an ADC comprising a linker means that when the ADC is exposed to extracellular conditions, no more than 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than about 3%, or no more than about 1% (or any percentage therebetween) of the linker in a sample of the ADC is cleaved (or, in the case of an otherwise incomplete ADC overall), when the ADC is exposed to extracellular conditions.

Whether the linker is stable outside of the cell can be determined, for example, by including an anti-FRA ADC in plasma for a predetermined period of time (e.g., 2, 4, 6, 8, 16, or 24 hours) and then quantifying the amount of free drug moiety present in the plasma. Stability can allow the ADC time to localize to target cancer cells and prevent premature drug release, which could reduce the therapeutic index of the ADC by indiscriminately damaging both normal and cancerous tissues. In some embodiments, the linker is stable outside of the target cell and releases the drug moiety from the ADC once inside the cell, allowing the drug moiety to bind to its target (e.g., to microtubules). Thus, the effective linker will: (i) maintain the specific binding properties of the antibody moiety; (ii) allow for the delivery (e.g., intracellular delivery) of the drug moiety via stable attachment to the antibody moiety; (iii) maintain stability and integrity until the ADC has been transported or delivered to the target site; and (iv) allow for therapeutic effects, e.g., cytotoxic effects, of the drug moiety following cleavage.

Linkers can be either "cleavable" or "non-cleavable" (Ducry and Stump, Bioconjugate Chem. (2010) 21:5-13). Cleavable linkers are designed to release the drug upon exposure to certain environmental cues (e.g., upon internalization into target cells), whereas non-cleavable linkers typically rely on degradation of the antibody moiety itself.

In some embodiments, the linker is a cleavable linker. A cleavable linker refers to any linker comprising a cleavable moiety. As used herein, the term "cleavable moiety" refers to any chemical bond that can be cleaved. Suitable cleavable chemical bonds are well known in the art and include, but are not limited to, acid-labile bonds, protease/peptidase-labile bonds, photolabile bonds, disulfide bonds, and esterase-labile bonds. Linkers comprising a cleavable moiety can allow the drug moiety to be released from the ADC via cleavage at a specific site in the linker. In various embodiments, the anti-FRA antibody cleaves the linked toxin to activate or increase the activity of the toxin.

In some embodiments, the linker is cleavable by a cleavage agent (e.g., an enzyme) present in the intracellular environment (e.g., within a lysosome, endosome, or caveolae). The linker can be, for example, a peptide linker that is cleaved by an intracellular peptidase or protease (including, but not limited to, a lysosomal or endosomal protease). In some embodiments, the linker is a cleavable peptide linker. As used herein, a cleavable peptide linker refers to any linker that comprises a cleavable peptide portion. The term "cleavable peptide portion" refers to any chemically linked amino acids (natural or synthetic amino acid derivatives) that can be cleaved by an agent present in the intracellular environment. For example, the linker can comprise a valine-citrulline (Val-Cit) sequence that can be cleaved by a peptidase such as a cathepsin, e.g., cathepsin B.

In some embodiments, the linker is an enzymatically cleavable linker and the cleavable peptide portion of the linker is cleavable by an enzyme. In some embodiments, the cleavable peptide portion is cleavable by cathepsin B. An exemplary dipeptide cleavable by cathepsin B is validine-citrulline (Val-Cit) (Dubowchik et al. (2002) Bioconjugate Chem. 13:855-69).

In some embodiments, the linker or the cleavable peptide portion of the linker comprises an amino acid unit. In some embodiments, the amino acid unit renders the linker cleavable by a protease, thereby promoting release of the drug moiety from the ADC upon 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). Exemplary amino acid units include, but are not limited to, dipeptides. Exemplary dipeptides include, but are not limited to, valeric acid-citrulline (Val-Cit). In some embodiments, the amino acid unit in the linker comprises Val-Cit. The amino acid units may comprise naturally occurring amino acid residues and/or secondary amino acids and/or non-naturally occurring amino acid analogs (such as citrulline).

In some embodiments, the linker in the anti-FRA ADCs disclosed herein comprises at least one spacer unit that connects the antibody portion to the drug portion. In some embodiments, the spacer unit connects a cleavage site (e.g., a cleavable peptide portion) in the linker to the antibody portion. In some embodiments, the linker comprises one or more polyethylene glycol (PEG) moieties, e.g., 1, 2, 3, 4, 5, or 6 PEG moieties. In some embodiments, the linker comprises 2 PEG moieties.

In some embodiments, the Spacer unit in the Linker comprises one or more PEG moieties. In some embodiments, the Spacer unit comprises -(PEG) _m -, and m is 2. In some embodiments, the Spacer unit comprises (PEG) ₂ .

In some embodiments, the Spacer unit indirectly links the Antibody moiety to the Drug moiety. In some embodiments, the Spacer unit indirectly links the Antibody moiety to the Drug moiety via a cleavable peptide moiety and a Linker moiety to link the Spacer unit to the Antibody moiety, e.g., a cis-imide moiety.

In various embodiments, the Spacer unit is linked to the anti-FRA antibody portion (i.e., anti-FRA antibody or antigen-binding fragment thereof) via a cis-butylenediimide moiety (Mal).

A spacer unit that is linked to an antibody or antigen-binding fragment via Mal is referred to herein as a "Mal-Spacer unit." As used herein, the term "cis-butylenediimide moiety" refers to a compound that contains a cis-butylenediimide group and is reactive with a sulfhydryl group, such as a sulfhydryl group of a cysteine residue on an antibody moiety. In some embodiments, a Mal-Spacer unit is reactive with a cysteine residue on an antibody or antigen-binding fragment. In some embodiments, a Mal-Spacer unit is linked to an antibody or antigen-binding fragment via a cysteine residue. In some embodiments, a Mal-Spacer unit comprises a (PEG) ₂ moiety.

In certain embodiments, the linker comprises a Mal-spacer unit and a cleavable peptide portion. In certain embodiments, the cleavable peptide portion comprises an amino acid unit. In certain embodiments, the amino acid unit comprises Val-Cit. In certain embodiments, the linker comprises Mal-(PEG) ₂ and Val-Cit.

In some embodiments, a Mal-Spacer unit links the anti-FRA antibody portion (i.e., an anti-FRA antibody or antigen-binding fragment thereof) to a cleavable moiety in a linker. In some embodiments, a Mal-Spacer unit links the antibody or antigen-binding fragment to a cleavable peptide portion. In some embodiments, the cleavable peptide portion comprises an amino acid unit. In some embodiments, the linker comprises a Mal-Spacer unit-amino acid unit. In some embodiments, the Mal-Spacer unit comprises a PEG moiety. In some embodiments, the amino acid unit comprises a Val-Cit.

In some embodiments, the linker comprises the following structure: Mal-SpacerUnit-Val-Cit. In some embodiments, the linker comprises the following structure: Mal-(PEG) ₂ -Val-Cit. In some embodiments, the linker comprises the following structure: Mal-(PEG) ₂ -Val-Cit-pAB.

In some embodiments, another spacer unit is used to link the cleavable moiety in the linker to the drug moiety, such as eribulin. In some embodiments, eribulin is linked to the cleavable moiety in the linker via a self-ablative spacer unit. In certain embodiments, eribulin is linked to the cleavable moiety in the linker via a self-ablative spacer unit, the cleavable moiety comprising Val-Cit, and another spacer unit comprising (PEG) ₂ links the cleavable moiety to the anti-FRA antibody moiety. In certain embodiments, eribulin is linked to the anti-FRA antibody via a MaI spacer unit in the linker, which is linked to the MaI spacer unit and the pAB self-ablative spacer unit.

A spacer unit can be "self-immolative" or "non-self-immolative." A "non-self-immolative" spacer unit is one in which a portion or all of the spacer unit remains bound to the drug moiety upon linker cleavage. Examples of non-self-immolative spacer units include, but are not limited to, glycine spacer units and glycine-glycine spacer units. Non-self-immolative spacer units may eventually degrade over time, but will not readily and completely release the linked natural drug under cellular conditions. A "self-immolative" spacer unit allows for the release of the natural drug moiety under intracellular conditions. A "natural drug" is a natural drug that does not retain portions of the spacer unit or other chemical modifications after spacer unit cleavage/degradation.

Self-ablative chemicals are known in the art and can be readily selected for the disclosed ADCs. In various embodiments, the spacer unit that connects the cleavable moiety in the linker to the drug moiety (e.g., eribulin) is self-ablative and undergoes self-ablation simultaneously with, before, or shortly after cleavage of the cleavable moiety under intracellular conditions.

In certain embodiments, the self-immolative spacer unit in the linker comprises a p-aminobenzyl unit. In some embodiments, p-aminobenzyl alcohol (pABOH) is linked 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-immolative spacer unit is or comprises a p-aminobenzyloxycarbonyl (pAB). Without being bound by theory, it is believed that self-immolation of pAB involves a spontaneous 1,6-elimination reaction (Jain et al. (2015) Pharm Res 32:3526-40).

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

In various embodiments, a self-immolative spacer unit links the cleavable moiety in the linker to the C-35 amine on eribulin. In some embodiments, the self-immolative spacer unit is pAB. In some embodiments, pAB links the cleavable moiety in the linker to the C-35 amine on eribulin. In some embodiments, pAB undergoes self-ablation after cleavage of the cleavable moiety, and eribulin is released from the ADC in its native, active form. In some embodiments, an anti-FRA antibody (e.g., MORAb-003) is linked to the C-35 amine of eribulin via a linker comprising Mal-(PEG) ₂ -Val-Cit-pAB.

In some embodiments, pAB undergoes self-ablation after cleavage of the cleavable peptide moiety in the linker. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the linker comprises an amino acid unit-pAB. In some embodiments, the amino acid unit is Val-Cit. In some embodiments, the linker comprises Val-Cit-pAB (VCP). In various other aspects, the antibody portion of the ADC is conjugated to the drug moiety via a linker, wherein the linker comprises a Mal-spacer unit, a cleavable amino acid unit, and pAB. In some embodiments, the spacer unit comprises a PEG moiety. In some embodiments, the linker comprises Mal-(PEG) ₂ -Val-Cit-pAB.

In some embodiments, the antibody moiety is conjugated to the drug moiety via a linker comprising a cis-imide moiety (Mal), a polyethylene glycol (PEG) moiety, valeric acid-citrulline (Val-Cit or "vc"), and pAB. In these embodiments, the cis-imide moiety covalently links the linker drug moiety to the antibody moiety, and pAB serves as a self-immolative spacer unit. Such linkers may be referred to as "m-vc-pAB" linkers, "Mal-VCP" linkers, "Mal-(PEG) ₂ -VCP" linkers, or "Mal-(PEG) ₂ -Val-Cit-pAB" linkers. In some embodiments, the drug moiety is eribulin. The structure of Mal-(PEG) ₂ -Val-Cit-pAB-eribulin is provided below. pAB was linked to the C-35 amine on eribulin using the Mal-(PEG) ₂ -Val-Cit-pAB linker.

It has been discovered that ADCs containing Mal-(PEG) ₂ -Val-Cit-pAB-eribulin exhibit a specific combination of desirable properties, particularly when paired with an anti-FRA antibody such as MORAb-003 or an antigen-binding fragment thereof. These functional properties are also exemplified in the examples provided in PCT application no. PCT/US2017/020529 (published as WO 2017/151979), which is incorporated herein by reference in its entirety.

In some embodiments, the ADC comprises Mal-(PEG) ₂ -Val-Cit-pAB-eribulin and an antibody portion comprising an internalizing anti-FRA antibody or an antigen-binding fragment thereof that retains the ability to target and internalize in tumor cells. In some embodiments, the ADC comprises Mal-(PEG) ₂ -Val-Cit-pAB-eribulin and an internalizing anti-FRA antibody or an internalizing antigen-binding fragment thereof that targets FRA-phenotypical tumor cells. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment thereof that targets FRA-expressing tumor cells comprises three heavy chain complementary determining regions (HCDRs) comprising 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 complementary determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2), and SEQ ID NO: 6 (LCDR3), as defined by the Kabat numbering system; or three heavy chain complementary determining regions (HCDRs) comprising 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 complementary determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 8 (HCDR1), SEQ ID NO: 9 (HCDR2), and SEQ ID NO: 10 (HCDR3). NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQ ID NO: 12 (LCDR3), as defined by the IMGT numbering system. In some embodiments, an internalizing antibody or internalizing antigen-binding fragment thereof that targets FRA-expressing tumor cells comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, an internalizing antibody or internalizing antigen-binding fragment thereof that targets FRA-expressing tumor cells comprises a human IgG1 heavy chain constant domain and an Ig kappa light chain constant domain.

In some embodiments, the ADC has Formula I: Ab-(LD) _p (I) wherein: (i) Ab is an internalizing anti-folate receptor alpha (FRA) antibody or an internalizing antigen-binding fragment thereof comprising: three heavy chain complementary determining regions (HCDRs) comprising 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 complementary determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2), and SEQ ID NO: 6 (LCDR3), as defined by the Kabat numbering system; or three heavy chain complementary determining regions (HCDRs) comprising SEQ ID NO: 7 (HCDR1), SEQ ID NO: 8 (HCDR2), and SEQ ID NO: 9 (HCDR3); and three light chain complementation determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQ ID NO: 12 (LCDR3), as defined by the IMGT numbering system; (ii) D is eribulin; (iii) L is a cleavable linker comprising Mal-(PEG) ₂ -Val-Cit-pAB; and (iv) p is an integer from 1 to 20.

In some embodiments, the internalizing antibody or internalizing antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the internalizing antibody is MORAb-003. In some embodiments, p is 1 to 8, or 1 to 6. In some embodiments, p is 2 to 8, or 2 to 5. In some embodiments, p is 3 to 4. In some embodiments, p is 4. Drug Moiety

In various embodiments, the drug moiety (D) of the ADCs disclosed herein (e.g., an ADC having Formula I) is an anti-tubulin agent, such as eribulin.

In some embodiments, the drug moiety is eribulin and the linker of the ADC is attached via the C-35 amine on eribulin.

In various embodiments, the native form of eribulin for attachment to the linker and antibody moieties is as follows:

In certain embodiments, ADCs are prepared by reacting an intermediate that serves as a precursor to a linker with eribulin under appropriate conditions. In certain embodiments, a reactive group is used on eribulin and/or the intermediate or linker. The reaction product between eribulin and the intermediate is then reacted with an anti-FRA antibody or antigen-binding fragment under appropriate conditions. Alternatively, the linker or intermediate can be reacted first with the antibody or derivatized antibody and then with eribulin.

A number of different reactions can be used to covalently link eribulin and/or the linker to the antibody moiety. This is typically achieved by reacting one or more amino acid residues on the antibody molecule (e.g., the hydroxyl group of cysteine). For example, nonspecific covalent attachment can be performed using a carbodiimide reaction to link a carboxyl (or amine) group on the compound to an amine (or carboxyl) group on the antibody moiety. Alternatively, bifunctional reagents such as dialdehydes or imidates can be used to link amine groups on the compound to amine groups on the antibody moiety. A Schiff base reaction can also be used to link a drug to a binding agent. This method involves the oxidation of a drug containing a diol or hydroxyl group with a periodate salt, thereby forming an aldehyde, which is then reacted with the binding agent. Attachment occurs via Schiff base formation with an amine group of the binding agent. Isothiocyanates can also be used as coupling agents for covalently linking the drug to the binding agent. Other techniques are known to those skilled in the art and are within the scope of this disclosure. Drug Loading

Drug loading is represented by p and is also referred to herein as the drug-to-antibody ratio (DAR). Drug loading can range from 1 to 20 drug moieties per antibody moiety. In some embodiments, p is an integer from 1 to 20. 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 3 to 4. In other embodiments, p is 1, 2, 3, 4, 5, or 6, preferably 3 or 4.

Drug loading can be limited by the number of attachment sites on the antibody moiety. In some embodiments, the linker portion (L) of the ADC is linked to the antibody moiety via a chemically reactive group on one or more amino acid residues on the antibody moiety. For example, the linker can be linked to the antibody moiety via a free amine, imine, hydroxyl, thiol, or carboxyl group (e.g., linked to the N-terminus or C-terminus, to an epsilon amino group of one or more lysine residues, to a free carboxylic acid group of one or more glutamine or aspartic acid residues, or to a sulfhydryl group of one or more cysteine residues). The site of attachment to the linker may be a natural residue in the amino acid sequence of the antibody portion, or it may be introduced into the antibody portion, for example, by DNA recombinant techniques (e.g., by introducing a cysteine residue into the amino acid sequence) or by protein biochemistry (e.g., by reduction, pH adjustment, or hydrolysis).

In some embodiments, the number of drug moieties that can be conjugated to the anti-FRA antibody portion is limited by the number of free cysteine residues. For example, in the case of a cysteine thiol group, the anti-FRA antibody may have only one or a few cysteine thiol groups, or may have only one or a few sufficiently reactive thiol groups through which the linker can be attached. Typically, antibodies do not contain many free reactive cysteine thiol groups that can be linked to the drug moiety. In fact, most cysteine thiol residues in antibodies exist as disulfide bonds. Excessive attachment of the linker-toxin to the antibody can destabilize the antibody by reducing cysteine residues available for disulfide bond formation. Therefore, the optimal drug:antibody ratio should increase the potency of the ADC (by increasing the number of drug moieties attached to each antibody) without destabilizing the antibody moiety. In some embodiments, the optimal ratio may be about 3-4.

In some embodiments, a linker linked to the antibody portion via a Mal moiety provides a ratio of about 3-4. In some embodiments, a linker comprising a short spacer unit (e.g., a short PEG spacer unit such as (PEG) ₂ ) provides a ratio of about 3-4. In some embodiments, a linker comprising a peptide cleavable moiety provides a ratio of about 3-4. In some embodiments, an ADC comprising Mal-(PEG) ₂ -Val-Cit-pAB-eribulin linked to an anti-FRA antibody such as MORAb-003 has a ratio of about 3-4.

In some embodiments, the antibody portion, such as MORAb-003, is exposed to reducing conditions prior to conjugation to generate 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 total reducing conditions to generate reactive cysteine thiol groups. In certain embodiments, the antibody can be subjected to denaturing conditions to reveal reactive nucleophilic groups on amino acid residues such as lysine or cysteine.

In a reaction mixture comprising multiple copies of an antibody moiety and a linker moiety, when more than one nucleophilic group reacts with a drug-linker intermediate or linker moiety reagent, followed by a drug moiety reagent, the resulting product can be a mixture of ADC compounds containing one or more drug moieties linked to each copy of the antibody moiety in the mixture. In some embodiments, the drug loading in the ADC mixture resulting from the conjugation reaction ranges from 1 to 20 linked drug moieties per antibody moiety. The average number of drug moieties per antibody moiety (i.e., average drug loading or average p ) can be calculated by any conventional method known in the art, such as mass spectrometry (e.g., reverse-phase LC-MS) and/or high-performance liquid chromatography (e.g., HIC-HPLC). In some embodiments, the average number of drug moieties per antibody moiety is determined by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC). In some embodiments, the average number of drug moieties per antibody moiety is determined by reverse phase liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the average number of drug moieties per antibody moiety is about 3 to about 4; about 3.1 to about 3.9; about 3.2 to about 3.8; about 3.2 to about 3.7; about 3.2 to about 3.6; about 3.3 to about 3.8; or about 3.3 to about 3.7. In some embodiments, the average number of drug moieties per antibody moiety is about 3.2 to about 3.8. In some embodiments, the average number of drug moieties per antibody moiety is about 3.8. In some embodiments, the average number of drug moieties per antibody moiety is 3 to 4; 3.1 to 3.9; 3.2 to 3.8; 3.2 to 3.7; 3.2 to 3.6; 3.3 to 3.8; or 3.3 to 3.7. In some embodiments, the average number of drug moieties per antibody moiety is 3.2 to 3.8. In some embodiments, the average number of drug moieties per antibody moiety is 3.8.

In some embodiments, the average number of drug moieties per antibody moiety is from about 3.5 to about 4.5; from about 3.6 to about 4.4; from about 3.7 to about 4.3; from about 3.7 to about 4.2; or from about 3.8 to about 4.2. In some embodiments, the average number of drug moieties per antibody moiety is from about 3.6 to about 4.4. In some embodiments, the average number of drug moieties per antibody moiety is about 4.0. In some embodiments, the average number of drug moieties per antibody moiety is from 3.5 to 4.5; from 3.6 to 4.4; from 3.7 to 4.3; from 3.7 to 4.2; or from 3.8 to 4.2. In some embodiments, the average number of drug moieties per antibody moiety is from 3.6 to 4.4. In some embodiments, the average number of drug moieties per antibody moiety is 4.0.

In various embodiments, the term "about" used with respect to the average number of drug moieties per individual antibody moiety or ADC mixture refers to +/- 10%. It should be understood that the definition of "about" applies to all descriptions of the average number of drug moieties per individual antibody moiety or ADC mixture in this disclosure.

Individual ADC complexes or "species" with specific DAR ratios can be identified in a mixture by mass spectrometry and separated by UPLC or HPLC (e.g., hydrophobic interaction chromatography (HIC-HPLC)). In certain embodiments, homogeneous or near-homogeneous ADCs with a single loading value can be separated from the complex mixture, for example, by electrophoresis or chromatography.

In some embodiments, the drug load and/or average drug load in the ADC (e.g., in MORAb-202) is about 4. In some embodiments, a drug load and/or average drug load of about 4 provides beneficial properties. See, e.g., PCT/US2017/020529 (published as WO 2017/151979), which is incorporated herein by reference in its entirety.

In some embodiments, the ADC has Formula I: Ab-(LD) _p (I) wherein: (i) Ab is an internalizing anti-folate receptor alpha antibody or an antigen-binding fragment thereof comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14; (ii) D is eribulin; (iii) L is a cleavable linker comprising Mal-(PEG) ₂ -Val-Cit-pAB; and (iv) p is an integer from 1 to 8.

In some embodiments, the ADC has Formula I: Ab-(LD) _p (I) wherein: (i) Ab is an internalizing anti-folate receptor alpha antibody or an antigen-binding fragment thereof comprising the heavy chain amino acid sequence of SEQ ID NO: 15 and the light chain amino acid sequence of SEQ ID NO: 16; (ii) D is eribulin; (iii) L is a cleavable linker comprising Mal-(PEG) ₂ -Val-Cit-pAB; and (iv) p is an integer of about 4. Therapeutic Uses

In various embodiments, disclosed herein are methods of using the disclosed anti-FRA ADCs (e.g., an ADC comprising the six CDR amino acid sequences of MORAb-003, e.g., MORAb-202, linked to a linker comprising Mal-(PEG) ₂ -Val-Cit-pAB) to treat a disorder (e.g., a tumor disorder, e.g., a FRA-phenotypical cancer, e.g., ovarian cancer (e.g., platinum-resistant ovarian cancer)) in a subject. The ADC can be administered alone or in combination (e.g., simultaneously or sequentially) with a second therapeutic agent (e.g., a corticosteroid, e.g., depoxetine, prednisone, or methylprednisolone), and can be administered in any pharmaceutically acceptable formulation. In particular, the methods disclosed herein provide for the use of the disclosed anti-FRA ADCs for treating a subject with a cancer exhibiting a FRA phenotype, wherein the ADC dosage is based on the subject's body surface area (BSA). In some embodiments, treatment using BSA dosing can reduce the risk of interstitial lung disease (ILD) in a subject requiring treatment with an anti-FRA ADC. In some embodiments, the methods disclosed herein provide for the use of the disclosed anti-FRA ADCs for treating a subject who has previously received at least one systemic anticancer therapy (e.g., a cytotoxic or targeted anticancer agent). In some embodiments, the methods disclosed herein provide for the use of the disclosed anti-FRA ADCs for treating a subject with a metastatic cancer, such as metastatic non-small cell lung cancer. In some embodiments, a subject with metastatic non-small cell lung cancer treated according to the disclosed methods has no genomic alterations. In some embodiments, a subject with a metastatic FRA phenotype cancer (e.g., metastatic non-small cell lung cancer) has at least one genomic alteration (e.g., at least one unknown or known genomic alteration). Examples of known genomic alterations include, but are not limited to, genomic alterations in any of the following genes: EGFR , ALK , PI3K , AKT , mTOR , RET , MET , BRAF , NTRK , ROS1 , and any gene involved in the RAS-MAPK pathway. In some embodiments, a subject with a metastatic FRA phenotype cancer has at least one known genomic alteration in at least one of any of the above genes. As used herein, "genomic alteration" refers to any change in the genome, including but not limited to somatic mutations, copy number variations, and gene fusions. In some embodiments, the methods disclosed herein provide for the use of the disclosed anti-FRA ADCs for treating subjects with refractory cancer. As used herein, "refractory cancer" is a cancer that has not responded to at least one prior therapy, i.e., is refractory. In some embodiments, the subject with refractory cancer is refractory to targeted therapy, for example, a therapy specifically targeting any of the following genes or variants thereof: epidermal growth factor receptor ( EGFR ), anaplastic lymphoma kinase ( ALK ), v-raf mouse sarcoma viral oncogene homolog 1 ( BRAF ), ret proto-oncogene ( RET ), MET proto-oncogene, receptor tyrosine kinase ( MET ), neurotrophic receptor tyrosine kinase ( NTRK ), and receptor tyrosine kinase ( ROS1 ). As used herein, "targeted therapy" refers to a cancer treatment that targets certain genes and/or proteins involved in cancer cell growth and/or survival. As used herein, the term "variant" is used to refer to any naturally occurring variant of a gene, including but not limited to splice variants, allelic variants, isoforms, and homologs (e.g., homologs or heterologs). In contrast to genes that contain genomic alterations, variants of a gene are not associated or correlated with a disease or disorder, such as cancer. In some embodiments, any of the above-mentioned genes (or variants thereof) that are specifically targeted by a targeted therapy may contain at least one genomic alteration. For example, a targeted therapy may be specific for a mutation in NTRK1 , while another targeted therapy may be specific for a mutation in NTRK2 .

In some embodiments, the subject with refractory cancer is refractory to platinum-based therapy (e.g., platinum doublet chemotherapy) and/or immunotherapy-based therapy (e.g., immune checkpoint inhibitors, e.g., PD-1 inhibitors or PD-L1 inhibitors). In some embodiments, the subject with refractory cancer is refractory to treatment with platinum doublet chemotherapy and immune checkpoint inhibitors (e.g., PD-1 inhibitors or PD-L1 inhibitors), wherein the platinum doublet chemotherapy and immune checkpoint inhibitors are administered simultaneously or sequentially. In various embodiments, the subject with refractory cancer is refractory to no more than three prior systemic therapies, e.g., no more than two prior systemic therapies. In some embodiments, the subject with refractory cancer has not responded to no more than 1 prior chemotherapy.

In various embodiments, ADC treatment efficacy can be assessed by measuring one or more biomarkers. In some embodiments, treatment is adjusted accordingly based on the detected levels or changes in the levels of the biomarkers. As explained in more detail below, in some embodiments, one or more biomarkers are measured before and after administration of the ADC. In some embodiments, any change in the levels of one or more biomarkers indicates treatment efficacy. In some embodiments, treatment is adjusted (e.g., changing the ADC dose or dosing regimen, continuing ADC treatment, or discontinuing treatment) after assessing changes in biomarkers before and after ADC administration. In some embodiments, toxicity or other indicators of ADC treatment efficacy can be assessed in conjunction with measurement of one or more biomarkers disclosed herein. Efficacy indicators include, but are not limited to, objective response rate (ORR). ORR can be measured after a given period of time after treatment, for example, at or after 24 weeks after the start of treatment. ORR can be determined based on tumor assessment according to RECIST, such as RECIST 1.1. As used herein, "RECIST" refers to the Response Evaluation Criteria in Solid Tumors (RECIST), a set of standardized guidelines used to measure the response of cancer patients to treatment (Therass et al. (2000) J Natl Cancer Inst . 92:205-16). As used herein, "RECIST 1.1" refers to RECIST version 1.1, in which the guidelines were revised and updated relative to earlier versions of RECIST (Eisenhauer et al. (2009) Eur J Cancer . 45:228-47).

In some embodiments, the methods disclosed herein for treating FRA phenotype cancers comprise administering to a subject in need thereof a therapeutically effective amount of an ADC disclosed herein having Formula (I), e.g., MORAb-202, wherein the ADC is administered to the subject in an amount based on the body surface area (BSA) of the subject.

In some embodiments, the methods disclosed herein for reducing the risk of ILD in a subject being treated for a cancer with an FRA phenotype comprise administering to the subject an ADC having Formula (I) as disclosed herein, such as MORAb-202, wherein the ADC is administered to the subject in an amount based on the subject's BSA.

In some embodiments, an ADC disclosed herein (e.g., MORAb-202) is administered at a dose of 0.3-1.2 mg/kg. In some embodiments, the ADC is administered at a dose of 0.3 mg/kg. In some embodiments, the ADC is administered at a dose of 0.45 mg/kg. In some embodiments, the ADC is administered at a dose of 0.68 mg/kg. In some embodiments, the ADC is administered at a dose of 0.9 mg/kg. In some embodiments, the ADC is administered at a dose of 1.2 mg/kg.

In some embodiments, an ADC disclosed herein (e.g., MORAb-202) is administered at a dose of 8 mg to 50 mg per square meter (m ² ) of subject BSA. In some embodiments, the ADC is administered at a dose of 8 mg to 44 mg per square meter (m ² ) of subject BSA. In some embodiments, the ADC is administered at a dose of 11 mg to 44 mg per square meter (m ² ) of subject BSA. In some embodiments, the ADC is administered at a dose of 8 mg to 10 mg per square meter (m ² ) of subject BSA. In some embodiments, the dose is 5-75 mg, 10-60 mg, 15-45 mg, or 20-40 mg, or 25-35 mg per square meter (m ² ) of subject BSA. In some embodiments, the ADC is administered at a dose of 33 mg/ ^m2 of the subject's BSA. In some embodiments, the ADC is administered at a dose of 25 mg/ ^m2 of the subject's BSA. In some embodiments, the ADC is administered at a dose of 17 mg/ ^m2 of the subject's BSA. In some embodiments, the ADC is administered at a dose of 15 mg/ ^m2 of the subject's BSA. In some embodiments, the ADC is administered at a dose of 12 mg/ ^m2 of the subject's BSA. In some embodiments, the ADC is administered at a dose of 10 mg/ ^m2 of the subject's BSA. In some embodiments, the ADC is administered at a dose of 8 mg/ ^m2 of the subject's BSA. Any of these dosages may be administered once a week, once every two weeks, or once every three weeks.

BSA can be calculated using any acceptable method known in the art. Formulas for calculating a subject's body surface area (BSA) include, for example, the Dubois and Dubois formula (or any variation thereof) (Dubois D, Dubois EF. (1916) Arch Intern Med. 1916;17:863-871), the Mosteller formula (or any variation thereof) (Mosteller RD. (1987) N Engl J Med. 22;317(17):1098), or the Haycock formula (Haycock GB et al. (1978) J Pediatr. Jul;93(1):62-6). Exemplary formulas for calculating BSA are provided below: (II) BSA (m ² ) = 0.20247 × height (m) ^0.725 × body weight (kg) ^0.425 (DuBois and DuBois); (III) BSA (m ² ) = 0.007184 × height (cm) ^0.725 × body weight (kg) ^0.425 (Dubois and Dubois variant); (IV) BSA (m ² ) = ([height (cm) × body weight (kg)]/3600) ^1/2 (Mosteller); or (V) BSA (m ² ) = 0.024265 × height (cm) ^0.3964 × BW (kg) ^0.5378 (Haycock).

In some embodiments, the actual dose of ADC to be administered to a subject can be calculated as follows: (VI) Predetermined dose (mg/m ² ) × Body surface area (BSA) (m ² ) = Actual dose (mg) As used herein, "predetermined dose" refers to a dose selected from the range of doses provided above (e.g., 8 mg to 50 mg) to be administered to a subject. For example, an ADC can be administered to a subject having an exemplary BSA of 1.8 square meters (m ² ) at a predetermined dose of 33 mg/m ² , as calculated using the assumed values of a height of 160 cm and a BW of 80 kg using Formula III above. In this particular example, according to Formula VI provided above, the amount of ADC administered to the subject, or the actual dose, is 60.5 mg.

In some embodiments, the treatment dose can be recalculated on the first day of each treatment cycle using the subject's height measured at the time of intake and the subject's weight measured on or before the first day of each treatment cycle (e.g., two days prior to the first day of each treatment cycle).

In some embodiments, the ADC is administered weekly, biweekly, triweekly, monthly, or any time period therebetween. In some embodiments, the ADC is administered every three weeks. In some embodiments, the ADC may be administered in a 21-day cycle. A treatment cycle of once every three weeks or a 21-day cycle may also be referred to as "Q3W." In some embodiments, the ADC is administered every two weeks. In some embodiments, the ADC may be administered in a 14-day cycle. A treatment cycle of once every two weeks or a 14-day cycle may also be referred to as "Q2W." In some embodiments, the ADC is administered once weekly. In some embodiments, the ADC may be administered in a 7-day cycle. A treatment cycle of once every week or a 7-day cycle may also be referred to as "QW."

In some embodiments, the ADC is administered once every three weeks at a dose of 0.3-1.2 mg/kg of body weight. In some embodiments, the ADC is administered once every three weeks at a dose of 0.3-1.2 mg/kg. In some embodiments, the ADC is administered once every three weeks at a dose of 0.3 mg/kg. In some embodiments, the ADC is administered once every three weeks at a dose of 0.45 mg/kg. In some embodiments, the ADC is administered once every three weeks at a dose of 0.68 mg/kg. In some embodiments, the ADC is administered once every three weeks at a dose of 0.9 mg/kg. In some embodiments, the ADC is administered once every three weeks at a dose of 1.2 mg/kg.

In some embodiments, the ADC is administered once every three weeks at a BSA-dependent dose of 8 mg/m ² to 50 mg/m ^2. In some embodiments, the ADC is administered once every two weeks at a BSA-dependent dose of 8 mg/m ² to 50 mg/m ^2. In some embodiments, the ADC is administered once weekly at a BSA-dependent dose of 8 mg/m ² to 50 mg/m ^2. In some embodiments, the ADC is administered once every three weeks at a BSA-dependent dose of 33 mg/m ^2. In some embodiments, the ADC is administered once every two weeks at a BSA-dependent dose of 33 mg/m ^2. In some embodiments, the ADC is administered once weekly at a BSA-dependent dose of 33 mg/m ² . In some embodiments, the ADC is administered once every three weeks at a BSA-dependent dose of 17 mg/m ^2. In some embodiments, the ADC is administered once every two weeks at a BSA-dependent dose of 17 mg/m ^2. In some embodiments, the ADC is administered once weekly at a BSA-dependent dose of 17 mg/m ^2. In some embodiments, the ADC is administered once every three weeks at a BSA-dependent dose of 15 mg/m ^2. In some embodiments, the ADC is administered once every two weeks at a BSA-dependent dose of 15 mg/m ^2. In some embodiments, the ADC is administered once weekly at a BSA-dependent dose of 15 mg/m ² . In some embodiments, the ADC is administered once every three weeks at a BSA-dependent dose of 8 mg/m ² to 10 mg/m ^2. In some embodiments, the ADC is administered once every two weeks at a BSA-dependent dose of 8 mg/m ² to 10 mg/m ^2. In some embodiments, the ADC is administered once weekly at a BSA-dependent dose of 8 mg/m ² to 10 mg/m ^2. In some embodiments, the ADC is administered once every three weeks at a BSA-dependent dose of 10 mg/m ^2. In some embodiments, the ADC is administered once every two weeks at a BSA-dependent dose of 10 mg/m ^2. In some embodiments, the ADC is administered once weekly at a BSA-dependent dose of 10 mg/m ² . In some embodiments, the ADC is administered once every three weeks at a BSA-dependent dose of 8 mg/m ^2. In some embodiments, the ADC is administered once every two weeks at a BSA-dependent dose of 8 mg/m ^2. In some embodiments, the ADC is administered once weekly at a BSA-dependent dose of 8 mg/m ^2. In some embodiments, the ADC is administered at a BSA-dependent dose of 12 mg/m ² on days 1 and 8 of each three-week cycle.

In some embodiments, the subject treated with the anti-FRA ADC disclosed herein has a weight value in the upper quartile of weight. In some embodiments, the subject treated with the anti-FRA ADC disclosed herein has a weight of at least 80 kg.

In some embodiments, the risk of ILD in subjects treated with an anti-FRA ADC disclosed herein is reduced by at least 5%, at least 10%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% after administration of the ADC based on BSA, compared to treatment in which the ADC is administered based on body weight (BW). In some embodiments, the control treatment is administered at a dose of 0.5 to 2 mg/kg of subject body weight (BW), e.g., at a dose of 0.9 mg to 1.2 mg/kg of BW. For example, in some embodiments, the control treatment can be administered at a dose of 0.9 mg/kg of subject BW, which is equivalent to a dose of 33 mg/m2 of subject BSA. In some embodiments, for an exemplary subject with a BW of 80 kg, administration of the ADC at a BW-based dose of 0.9 mg/kg would result in an actual dose of 72 mg; in contrast, administration of the ADC to the same subject at a BSA-based dose of 33 mg/m ² would result in an actual dose of 59.4 mg.

In some embodiments, the methods disclosed herein may further comprise administering one or more additional therapeutic agents, such as one or more additional anti-cancer agents. In some embodiments, the additional agent comprises a corticosteroid.

In some embodiments, corticosteroids can be administered prophylactically. As used herein, "prophylactic administration" refers to administering a treatment, such as a corticosteroid, to a subject before the subject has or develops ILD symptoms. In some embodiments, the corticosteroid and ADC are administered simultaneously or sequentially. In some embodiments, the corticosteroid is administered before or after the ADC is administered. In some embodiments, the corticosteroid is dipretinol. In some embodiments, the corticosteroid is prednisone. In some embodiments, the corticosteroid is methylprednisolone. In some embodiments, the corticosteroid (e.g., dipretinol, prednisone, or methylprednisolone) can be administered orally or intravenously. In some embodiments, when a corticosteroid (e.g., dipretinol or prednisone) is administered orally, it can be administered in an amount of 1-10 mg, e.g., 0.5-2 mg, e.g., 2-5 mg, e.g., 0.5 mg, 1 mg, 2 mg, or 4 mg. In some embodiments, dipretinol is administered in an amount determined to be therapeutically effective, e.g., 4 mg of dipretinol. In some embodiments, dipretinol is administered at least once a day, e.g., twice a day. In some embodiments, dipretinol is administered for several days (e.g., 1, 2, 3, 4, 5, or more days) prior to or at the start of treatment with the ADC. In some embodiments, dipretinol is administered for at least three days at the start of treatment with the ADC. In some embodiments, dipretinol is administered orally. In some embodiments, prednisone is administered in an amount determined to be therapeutically effective, for example, 0.5 mg, 1 mg, or 2 mg of prednisone. In some embodiments, prednisone is administered as 0.5 mg of prednisone. In some embodiments, prednisone is administered as 1 mg of prednisone. In some embodiments, prednisone is administered as 2 mg of prednisone. In some embodiments, prednisone is administered at least once daily. In some embodiments, prednisone is administered for several days (e.g., 10, 11, 12, 13, 14, or more days) prior to or at the start of treatment with the ADC. In some embodiments, prednisone is administered for at least 14 days prior to the start of treatment with the ADC. In some embodiments, prednisone is administered orally. In some embodiments, when a corticosteroid (e.g., methylprednisolone) is administered intravenously, it may be administered in an amount of 300-1200 mg, such as 400-1100 mg, such as 500-1000 mg, such as 500 mg, 750 mg, or 1000 mg. In some embodiments, methylprednisolone is administered in an amount of 30-130 mg, such as 40-125 mg. In some embodiments, methylprednisolone is administered at 1 mg/kg of subject body weight. In some embodiments, methylprednisolone is administered at 2 mg/kg of subject body weight. In some embodiments, methylprednisolone is administered intravenously. In some embodiments, methylprednisolone is administered orally. In some embodiments, when methylprednisolone is administered orally, it can be administered in an amount of 5-100 mg, such as 5-90 mg, such as 5-80 mg, such as 10-80 mg, such as 10-70 mg, such as 10-60 mg. In some embodiments, methylprednisolone is administered orally at 0.5-1.5 mg/kg of the subject's body weight. In some embodiments, methylprednisolone is administered orally at 0.5 mg/kg of the subject's body weight. In some embodiments, methylprednisolone is administered orally at 1 mg/kg of the subject's body weight. In some embodiments, methylprednisolone is administered orally at 1.5 mg/kg of the subject's body weight. In some embodiments, methylprednisolone is administered orally at least once daily. In some embodiments, methylprednisolone is administered for several days (e.g., 1, 2, 3, 4, 5, or more days) prior to or at the start of treatment with the ADC. In some embodiments, methylprednisolone is administered for at least three days prior to the start of treatment with the ADC.

The methods disclosed herein can be applied to human subjects in need of treatment, e.g., subjects suffering from cancer, such as a cancer with an FRA phenotype. In some embodiments, the methods disclosed herein can be applied to non-human mammals suffering from a cancer with an FRA phenotype for veterinary purposes or as animal models of human disease. In the latter case, such animal models can be used to evaluate the therapeutic efficacy of the disclosed methods (e.g., testing doses and administration schedules).

The ADCs disclosed herein can be administered to a subject via any suitable route of administration to produce a therapeutic effect. In some embodiments, the ADCs are administered intravenously to a subject.

In some embodiments, the disclosure features a method for treating a FRA-expressing cancer. The method can be used to treat any human or non-human mammalian subject suffering from a FRA-expressing cancer, e.g., those in whom disruption of tubulin provides therapeutic benefit. Methods for identifying subjects suffering from a FRA-expressing cancer are known in the art and can be used to identify subjects suitable for treatment with the disclosed ADCs. The FRA-expressing cancer can be a primary or metastatic FRA-expressing cancer, or a FRA-expressing cancer that is resistant to platinum-based therapy, e.g., a platinum-resistant cancer. Non-limiting examples of FRA-phenotypical cancers include gastric cancer, ovarian cancer (e.g., serous ovarian cancer, clear cell ovarian cancer, or platinum-resistant ovarian cancer), lung cancer (e.g., non-small cell lung cancer, e.g., metastatic non-small cell lung cancer), lung carcinoid, colorectal cancer, breast cancer (e.g., triple-negative breast cancer or hormone receptor (HR)-positive and HER2-low breast cancer), endometrial cancer (e.g., serous endometrial cancer), peritoneal cancer (e.g., primary peritoneal cancer), fallopian tube cancer, pancreatic cancer, kidney cancer (e.g., renal cell carcinoma), cervical cancer, esophageal cancer, and osteosarcoma.

In some embodiments, the FRA-expressing cancer is gastric cancer, ovarian cancer, serous ovarian cancer, serous high-grade ovarian cancer, clear cell ovarian cancer, platinum-resistant ovarian cancer, lung cancer, non-small cell lung cancer, metastatic non-small cell lung cancer, lung carcinoid, colorectal cancer, breast cancer, triple-negative breast cancer, hormone receptor (HR)-positive and HER2-low breast cancer, endometrial cancer, serous endometrial cancer, peritoneal cancer, primary peritoneal cancer, fallopian tube cancer, pancreatic cancer, kidney cancer, renal cell cancer, cervical cancer, esophageal cancer, osteosarcoma, ependymoma, ependymoblastoma, ductal germ cell tumor, nephroblastoma, acute myeloid leukemia, or head and neck cancer.

In some embodiments, the FRA-expressing cancer is gastric cancer, ovarian cancer, serous ovarian cancer, serous high-grade ovarian cancer, clear cell ovarian cancer, platinum-resistant ovarian cancer, lung cancer, non-small cell lung cancer, metastatic non-small cell lung cancer, lung carcinoid, breast cancer, triple-negative breast cancer, hormone receptor (HR)-positive and HER2-low breast cancer, endometrial cancer, serous endometrial cancer, peritoneal cancer, primary peritoneal cancer, fallopian tube cancer, pancreatic cancer, cervical cancer, esophageal cancer, osteosarcoma, ependymoma, ependymoblastoma, neurotubercocyte germ cell tumor, nephroblastoma, acute myeloid leukemia, or head and neck cancer.

In some embodiments, the FRA-expressing cancer is gastric cancer, ovarian cancer, serous ovarian cancer, serous high-grade ovarian cancer, clear cell ovarian cancer, platinum-resistant ovarian cancer, lung cancer, non-small cell lung cancer, metastatic non-small cell lung cancer, lung carcinoid, colorectal cancer, breast cancer, triple-negative breast cancer, hormone receptor (HR)-positive and HER2-low breast cancer, endometrial cancer, serous endometrial cancer, peritoneal cancer, primary peritoneal cancer, fallopian tube cancer, pancreatic cancer, kidney cancer, renal cell carcinoma, cervical cancer, esophageal cancer, osteosarcoma, and head and neck cancer.

In some embodiments, the FRA phenotype cancer is ovarian cancer, e.g., platinum-resistant ovarian cancer. In some embodiments, the FRA phenotype cancer is breast cancer, e.g., triple-negative breast cancer (TNBC). In some embodiments, the FRA phenotype cancer is non-small cell lung cancer (NSCLC), e.g., metastatic NSCLC. In some embodiments, the FRA phenotype cancer is endometrial cancer.

In some embodiments, the disclosure features a method for reducing the risk of ILD in a subject with a FRA-expressing cancer, wherein the subject is in need of treatment. The method can be used in any human or non-human mammalian subject with a FRA-expressing cancer for the purpose of reducing the risk of ILD. The FRA-expressing cancer can be a primary or metastatic FRA-expressing cancer, or a FRA-expressing cancer that is resistant to platinum-based therapy, e.g., a platinum-resistant cancer. Non-limiting examples of FRA-expressing cancers include gastric cancer, ovarian cancer (e.g., serous ovarian cancer, clear cell ovarian cancer, or platinum-resistant ovarian cancer), lung cancer (e.g., non-small cell lung cancer, e.g., metastatic non-small cell lung cancer), lung carcinoid, colorectal cancer, breast cancer (e.g., triple-negative breast cancer or hormone receptor (HR)-positive and HER2-low breast cancer), endometrial cancer (e.g., serous endometrial cancer), peritoneal cancer (e.g., primary peritoneal cancer), fallopian tube cancer, pancreatic cancer, kidney cancer (e.g., renal cell carcinoma), cervical cancer, esophageal cancer, and osteosarcoma. In some embodiments, the FRA-expressing cancer is ovarian cancer, e.g., platinum-resistant ovarian cancer. In some embodiments, the FRA-expressing cancer is breast cancer, e.g., triple-negative breast cancer (TNBC). In some embodiments, the FRA-expressing cancer is non-small cell lung cancer (NSCLC), such as metastatic NSCLC. In some embodiments, the FRA-expressing cancer is endometrial cancer.

In various embodiments, the methods disclosed herein, for example, using BSA-based dosing of an anti-FRA ADC such as MORAb- ²⁰² , are used to treat ovarian cancer, platinum-resistant ovarian cancer, breast cancer, triple-negative breast cancer, non-small cell lung cancer, or endometrial cancer, for example, at a BSA-dependent dose of 8 to 50 mg/m . In some embodiments, the methods disclosed herein are used to treat primary peritoneal cancer or fallopian tube cancer. In some embodiments, a BSA-dependent dose of 33 mg/m ² administered every three weeks (Q3W) is used to treat platinum-resistant ovarian cancer (PROC). In some embodiments, a BSA-dependent dose of 25 mg/m ² administered Q3W is used to treat PROC. In some embodiments, a BSA-dependent dose of 17 mg/m ² administered Q3W is used to treat PROC. In some embodiments, a BSA-dependent dose of 15 mg/m ² administered every two weeks (Q2W) is used to treat PROC. In some embodiments, a BSA-dependent dose of 8 mg/m ² to 10 mg/m ² administered once weekly (QW) is used to treat PROC. In some embodiments, PROC is serous ovarian cancer. In some embodiments, PROC is high-grade serous ovarian cancer. In some embodiments, a BSA-based dose of 33 mg/m ² Q3W is used to treat primary peritoneal cancer. In some embodiments, a BSA-based dose of 25 mg/m ² Q3W is used to treat primary peritoneal cancer. In some embodiments, a BSA-based dose of 17 mg/m ² Q3W is used to treat primary peritoneal cancer. In some embodiments, a BSA-based dose of 15 mg/m ² Q2W is used to treat primary peritoneal cancer. In some embodiments, a BSA-based dose of 8 mg/m ² to 10 mg/m ² QW is used to treat primary peritoneal cancer. In some embodiments, a BSA-based dose of 33 mg/m ² Q3W is used to treat fallopian tube cancer. In some embodiments, a BSA-based dose of 25 mg/m ² Q3W is used to treat fallopian tube cancer. In some embodiments, a BSA-based dose of 17 mg/m ² Q3W is used to treat fallopian tube cancer. In some embodiments, a BSA-based dose of 15 mg/m ² Q2W is used to treat fallopian tube cancer. In some embodiments, a BSA-based dose of 8 mg/m ² to 10 mg/m ² QW is used to treat fallopian tube cancer. In some embodiments, the subject treated according to the methods disclosed herein has cancer that recurred within six months of receiving platinum-based therapy.

In some embodiments, subjects who experience a treatment-related adverse event (TRAE) can subsequently have their dose level of MORAb-202 reduced. In some embodiments, subjects who are administered a BSA-based dose of 33 mg/ ^m2 and experience a TRAE can subsequently be administered a reduced dose of 25 mg/ ^m2 . In some embodiments, subjects who are administered a BSA-based dose of 25 mg/ ^m2 and experience a TRAE can subsequently be administered a reduced dose of 17 mg/ ^m2 . In some embodiments, subjects who are administered a BSA-based dose of 25 mg/ ^m2 and experience a TRAE can subsequently be administered a reduced dose of 15 mg/ ^m2 . In some embodiments, subjects who are administered a BSA-based dose of 25 mg/ ^m2 and experience a TRAE can subsequently be administered a reduced dose of 8 mg/ ^m2 to 10 mg/ ^m2 . In some embodiments, subjects who are administered a BSA-based dose of 15 mg/ ^m2 and experience a TRAE can subsequently be administered a reduced dose of 8 mg/ ^m2 to 10 mg/ ^m2 . In some embodiments, TRAEs are assessed and assigned a specific grade level according to NCI CTCAE v5. As used herein, NCI CTCAE v5 refers to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 5, a descriptive terminology used for adverse event reporting that provides a grading (severity) scale for each adverse event term. In some embodiments, a TRAE is a transfusion-related reaction of Grade 1 or higher (e.g., Grade 2, 3, or 4). In some embodiments, a TRAE is interstitial lung disease (ILD) or pneumonitis of Grade 1 or higher (e.g., Grade 2, 3, or 4). In some embodiments, a TRAE is a decrease in neutrophil count of Grade 3 or 4 or equal to or less than 1000 cells/μl in a subject's blood sample. In some embodiments, a TRAE is febrile neutropenia of Grade 3 or higher. In some embodiments, a TRAE is a decrease in platelet count of Grade 2 or higher or equal to or less than 75,000 cells/μl in a subject's blood sample. In some embodiments, a TRAE is any symptomatic or asymptomatic laboratory finding of Grade 3 or higher. In some embodiments, a TRAE is any non-hematologic toxicity of Grade 3 or higher.

In some embodiments, the methods disclosed herein, for example, using BSA-based dosing of an anti-FRA ADC such as MORAb-202, for example, at a BSA-dependent dose of 8 to 50 mg/m ² , reduce the risk of ILD.

Prior to initiating treatment, subjects may be evaluated using certain clinical criteria to identify those who may be at high risk for severe respiratory complications. If a subject is determined to be at high risk for severe respiratory complications, the subject may be excluded from treatment according to the methods disclosed herein. In some embodiments, subjects treated according to the aforementioned methods are evaluated using pulmonary function testing (PFT) prior to treatment. In some embodiments, subjects who are evaluated using PFTs and subsequently receive treatment do not have one or more of the following results: an FEV1/FVC ratio of less than 0.7, an FEV1 (forced expiratory volume in 1 second) of less than 80%, an FVC (forced vital capacity) of less than 80%, or a DLCO (diffusing capacity of the lungs for carbon monoxide) of less than 80%. In some embodiments, the subject treated according to the aforementioned method does not have one or more of the following at the start of treatment: interstitial lung disease (ILD) and/or pneumonia, a history of ILD and/or pneumonia, a clinically significant pulmonary idiopathic disease, pleural effusion, pericardial effusion, previous lung resection, history of chest radiation within the past 2 years, an autoimmune disorder with lung involvement, a connective tissue disorder with lung involvement, or an inflammatory disorder with lung involvement. Exemplary clinically significant pulmonary idiopathic diseases include, but are not limited to, any underlying pulmonary disorder (e.g., pulmonary embolism), asthma, chronic obstructive pulmonary disease (COPD), restrictive lung disease, or any other pulmonary idiopathic inflammatory disease or condition.

In some embodiments, the subject treated according to the aforementioned methods does not have one or more of the following: a history of more than three prior therapies for the FRA phenotype cancer, a high neutrophil-to-lymphocyte ratio, or a serum albumin level less than 3 g/dL at the start of treatment. As used herein, "high neutrophil-to-lymphocyte ratio" refers to a neutrophil-to-lymphocyte ratio, or "neutrophil-to-lymphocyte ratio" (NLR), in a blood sample from the subject that is higher than the mean NLR of a comparison population (e.g., a group of adults in the general population or a group of subjects with FRA phenotype cancer). In some embodiments, the high NLR can be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9. The NLR can be calculated using any method known in the art. For example, the NLR can be calculated by dividing the number of neutrophils by the number of lymphocytes. The number of neutrophils and lymphocytes can be measured in a peripheral blood sample from a subject. The NLR can be calculated using absolute neutrophil and/or lymphocyte counts or using relative percentages of neutrophils and/or lymphocytes. Biomarkers of Tumor Response

In various embodiments, one or more biomarkers of tumor response are measured in a subject in conjunction with the disclosed treatment methods (e.g., those using MORAb-202). In some embodiments, one or more biomarkers of tumor response are measured before and after one or more doses of MORAb-202 are administered to a subject. As used herein, "baseline" refers to a measurement at a time point before a specified intervention has occurred, for example, the measured level of a biomarker of tumor response in a biological sample taken from a subject before administration of an ADC (e.g., MORAb-202). In some embodiments, the baseline level of a biomarker is compared to the level of the biomarker after administration of the ADC to assess treatment efficacy and/or guide subsequent treatment decisions.

Biomarkers are measurable substances in an organism that are indicators of biological processes. These measurable substances can include nucleic acids, proteins, or other aspects of cellular activity. Tumor response biomarkers are measurable substances that are indicators of tumor biology. In some embodiments, tumor response biomarkers are indicators of angiogenesis, interleukins, inflammation, and/or other aspects of cancer biology and growth. A physician or other medical practitioner can assess the effects of a drug or treatment (e.g., MORAb-202) on a subject with a tumor by evaluating one or more biomarkers of tumor response before and after administration of the drug or treatment. Treatment decisions and treatment changes, such as continuing or discontinuing treatment, can also be made after evaluating one or more biomarkers of tumor response before and after administration of a drug or treatment (e.g., MORAb-202). Treatment changes can be made based on a specific change observed in a biomarker of tumor response, such as a specific percentage change in one or more biomarkers of tumor response after administration of a drug or treatment relative to a baseline level of one or more biomarkers of tumor response.

In various embodiments, a dose of a drug or treatment (e.g., MORAb-202) is administered to a tumor subject, and biological samples (e.g., blood samples) are collected before and after administration. In some embodiments, efficacy of the treatment is determined by measuring changes in one or more biomarkers of tumor response in samples collected before and after administration of the drug or treatment (or, in some cases, lack of change indicates efficacy of the treatment, while changes indicate lack of response to the treatment). A dose of a drug or treatment refers to the amount of the drug or treatment administered at a specific time. One or more doses can be administered between the baseline biomarker sampling and subsequent sampling. In some embodiments, after assessing the levels of one or more biomarkers of tumor response before and after administration of a drug or treatment, a physician can adjust the dose or frequency of administration of the drug or treatment (e.g., MORAb-202). In some embodiments, a change (or lack of change) in the levels of a biomarker associated with tumor response results in the administration of a larger dose of the drug or treatment. In some embodiments, a change (or lack of change) in the levels of a biomarker associated with tumor response results in the administration of a smaller dose of the drug or treatment. In some embodiments, a change (or lack of change) in the levels of a biomarker associated with tumor response results in the administration of a less frequent dose of the drug or treatment. In some embodiments, a change (or lack of change) in the levels of a biomarker associated with tumor response results in more frequent administration of a drug or treatment. In some embodiments, if a change (or lack of change) in the levels of one or more biomarkers of tumor response is detected, treatment is continued. In some embodiments, if a change (or lack of change) in the levels of one or more biomarkers of tumor response is detected, treatment is discontinued.

In some embodiments, baseline levels of one or more biomarkers of tumor response are measured in a biological sample taken from a subject prior to initiating treatment with MORAb-202. In some embodiments, further levels of one or more biomarkers of tumor response are measured in a biological sample taken from a subject following treatment with, for example, MORAb-202. In some embodiments, additional samples are collected, for example, after administration of additional doses of MORAb-202.

In some embodiments, the biological sample obtained from the subject is blood, a blood component, a tumor biopsy, urine, tissue, a cell collection, or saliva. Methods for obtaining such samples are known in the art and can be used herein. In some embodiments, the biological sample obtained from the subject is processed prior to measuring tumor-responsive biomarkers in the biological sample. In some embodiments, the levels of one or more tumor-responsive biomarkers in the sample are measured using methods known in the art, such as a Luminex-based platform, an OLINK-based multiplexing platform, or a SIMOA-based assay platform.

In some embodiments, after obtaining a baseline measurement of one or more biomarkers of tumor response, a drug or treatment, such as a treatment disclosed herein, is administered to a subject. In some embodiments, after administering the drug or treatment to a subject, additional measurements of the biomarkers of tumor response are measured in a biological sample taken from the subject. In some embodiments, the additional measurements of the one or more biomarkers of tumor response after administration of the drug or treatment are compared to the baseline measurement of the one or more biomarkers of tumor response. This comparison can be used to determine the impact of the drug or treatment on the biology of the subject's tumor. In some embodiments, more than one additional measurement of one or more tumor-responsive biomarkers is taken to monitor changes in biomarker levels during treatment. In some embodiments, additional measurements of tumor-responsive biomarkers are obtained from the subject periodically. In some embodiments, additional measurements of tumor-responsive biomarkers are obtained from the subject until a specific change in the level of the tumor-responsive biomarker is achieved. In some embodiments, treatment is discontinued once a specific level has been achieved.

In various embodiments, the ADCs and treatment methods described above can be monitored and/or guided by changes in one or more biomarkers measured in samples taken from a patient (e.g., before and after administration of an ADC disclosed herein (e.g., MORAb-202)).

In some embodiments, the ADC is administered until a change in the level of a biomarker is observed in a sample relative to the level of the biomarker in a sample taken from the subject before administration. Without being bound by theory, changes in biomarkers may reflect processes related to cancer or the tumor microenvironment. In some embodiments, the biomarkers are related to angiogenesis, interleukins, inflammation, and/or cancer.

In some embodiments, the level of a biomarker in a sample increases relative to the level of the biomarker in a sample taken from the subject prior to administration (baseline level). In some embodiments, if the measured level of the biomarker increases relative to the baseline level, treatment with the ADC is continued. In some embodiments, the level of a biomarker in a sample decreases relative to the baseline level. In some embodiments, if the measured level of the biomarker decreases relative to the baseline level, treatment with the ADC is continued.

A subject who receives a dose of an ADC (e.g., MORAb-202) may be a responder or a non-responder to the ADC treatment. Tumor response can be assessed using guidelines known in the art. In some embodiments, tumor response is assessed using the Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 guidelines. In some embodiments, the guideline-assessed tumor response is used to determine the subject's overall response rate or the subject's best overall response rate. In some embodiments, the subject's best overall response rate is complete response, partial response, stable disease, or progressive disease. In some embodiments, response can be supplemented or replaced by measuring one or more biomarkers discussed herein.

In some embodiments, a subject is a responder because, after receiving a dose of the ADC, the subject exhibits a complete response or partial response to the ADC treatment. If the subject is a responder to the ADC treatment, in some embodiments, the subject continues to receive the ADC treatment. In some embodiments, a subject is a non-responder because, after a dose of the ADC, the subject exhibits a stable disease state or a progressive disease state. If the subject is a non-responder to the ADC treatment, in some embodiments, the subject discontinues the ADC treatment. In some embodiments, the non-responder receives a different dose of the ADC, such as MORAb-202. In some embodiments, the non-responder subject is switched to a different tumor treatment.

In some embodiments, the biomarker is surfactant protein D (SP-D, NCBI Gene ID: 6441, SEQ ID NO: 39). In some embodiments, following administration of an ADC (e.g., MORAb-202), SP-D levels increase relative to baseline levels in subjects who have responded to treatment. In some embodiments, SP-D levels increase by at least 80%-120%, e.g., at least 100%, relative to baseline levels in subjects who have responded to treatment. Without being bound by theory, an increase in SP-D following administration of an ADC may indicate an improvement in the tumor microenvironment, such as reduced immunosuppression and decreased cancer growth and metastasis.

In some embodiments, the biomarker is a form of a kallikrein-related peptidase isoform. In some embodiments, the biomarker is kallikrein-related peptidase 5 (KLK-5, NCBI Gene ID: 25818, SEQ ID NO: 40). In some embodiments, following administration of the ADC, KLK-5 levels are reduced relative to baseline levels in subjects who respond to treatment. In some embodiments, KLK-5 levels are reduced by at least 20%-60%, such as at least 40%, relative to baseline levels in subjects who respond to treatment. In some embodiments, the biomarker is kallikrein-related peptidase 7 (KLK-7, NCBI Gene ID: 5650, SEQ ID NO: 41). In some embodiments, after administration of the ADC, KLK-7 levels are reduced relative to baseline levels in subjects who have responded to treatment. In some embodiments, KLK-7 levels are reduced by at least 10%-40%, for example, at least 30%, relative to baseline levels in subjects who have responded to treatment. Without being bound by theory, a decrease in KLK-5 and/or KLK-7 following administration of the ADC may indicate an improvement in the tumor microenvironment, such as reduced aberrant angiogenesis, reduced immunosuppression, reduced remodeling of the tumorigenic stroma and extracellular matrix, and reduced cancer growth and metastasis.

In some embodiments, the biomarker is a C-C motif interleukin ligand isoform. In some embodiments, the biomarker is C-C motif interleukin ligand 2 (MCP-1, NCBI Gene ID: 6347, SEQ ID NO: 42). In some embodiments, following administration of the ADC, MCP-1 levels are reduced relative to baseline levels in subjects who have responded to treatment. In some embodiments, MCP-1 levels are reduced by at least 5%-25%, for example, at least 15%, relative to baseline levels in subjects who have responded to treatment. Without being bound by theory, a reduction in MCP-1 following administration of the ADC may indicate an improvement in the tumor microenvironment, such as reduced aberrant angiogenesis, improved epithelial-mesenchymal transition, reduced immunosuppression, and reduced cancer growth and metastasis. In some embodiments, the biomarker is the C-C motif interleukin ligand 4 (MIP-1β, NCBI Gene ID: 6351, SEQ ID NO: 43). In some embodiments, after administration of the ADC, MIP-1β levels remain unchanged relative to baseline levels in subjects who respond to treatment, while MIP-1β levels increase relative to baseline levels in non-responders after administration of the ADC. In some embodiments, the biomarker is the C-C motif interleukin ligand 8 (CCL8, NCBI Gene ID: 6355, SEQ ID NO: 44). In some embodiments, after administration of the ADC, CCL8 levels remain unchanged relative to baseline levels in subjects who respond to treatment, while CCL8 levels increase relative to baseline levels in non-responders after administration of the ADC. In some embodiments, the biomarker is the C-C motif interleukin ligand 10 (IP-10, NCBI Gene ID: 3627, SEQ ID NO: 45). In some embodiments, after administration of the ADC, IP-10 levels remain unchanged relative to baseline levels in subjects who respond to treatment, while IP-10 levels increase relative to baseline levels in non-responders after administration of the ADC. In some embodiments, the biomarker is the C-C motif interleukin ligand 11 (eosin-1, NCBI Gene ID: 6356, SEQ ID NO: 46). In some embodiments, after administration of the ADC, eosin-1 levels decrease relative to baseline levels in subjects who respond to treatment. In some embodiments, the biomarker is the C-C motif chemokine ligand 13 (CCL13, NCBI Gene ID: 6357, SEQ ID NO: 47). In some embodiments, after administration of the ADC, CCL13 levels remain unchanged relative to baseline levels in subjects who respond to treatment, while after administration of the ADC, CCL13 levels increase relative to baseline levels in non-responders.

In some embodiments, the biomarker is angiopoietin 2 (ANG-2, NCBI Gene ID: 285, SEQ ID NO: 48). In some embodiments, following administration of the ADC, ANG-2 levels are reduced relative to baseline levels in subjects who have responded to treatment. In some embodiments, ANG-2 levels are reduced by at least 20%-40%, such as at least 30%, relative to baseline levels in subjects who have responded to treatment. Without being bound by theory, a reduction in ANG-2 following administration of the ADC may indicate an improvement in the tumor microenvironment, such as reduced aberrant angiogenesis, reduced hypoxia, and reduced cancer growth and metastasis.

In some embodiments, the biomarker is stromal metallopeptidase 12 (MMP12, NCBI Gene ID: 4321, SEQ ID NO: 49). In some embodiments, following administration of the ADC, MMP12 levels are reduced relative to baseline levels in subjects who have responded to treatment. In some embodiments, MMP12 levels are reduced by at least 20%-60%, for example, at least 40%, relative to baseline levels in subjects who have responded to treatment. Without being bound by theory, a reduction in MMP12 following administration of the ADC may indicate an improvement in the tumor microenvironment, such as reduced tumorigenic stroma and extracellular matrix remodeling, and reduced cancer growth and metastasis.

In some embodiments, the biomarker is ferritin (FRTN, NCBI Gene ID: 2395, SEQ ID NO: 50). In some embodiments, following administration of the ADC, FRTN levels are reduced relative to baseline levels in subjects who have responded to treatment. In some embodiments, FRTN levels are reduced by at least 20%-60%, for example, at least 40%, relative to baseline levels in subjects who have responded to treatment. Without being bound by theory, a reduction in FRTN following administration of the ADC may indicate an improvement in the tumor microenvironment, such as reduced abnormal angiogenesis, reduced immunosuppression, and reduced cancer growth and metastasis.

In some embodiments, the biomarker is an insulin-like growth factor binding protein isoform. In some embodiments, the biomarker is insulin-like growth factor binding protein 1 (IGFBP-1, NCBI Gene ID: 3484, SEQ ID NO: 51). In some embodiments, after administration of the ADC, IGFBP-1 levels remain unchanged relative to baseline levels in subjects who respond to treatment, while after administration of the ADC, IGFBP-1 levels increase relative to baseline levels in non-responders. In some embodiments, the biomarker is insulin-like growth factor binding protein 2 (IGFBP-2, NCBI Gene ID: 3485, SEQ ID NO: 52). In some embodiments, after administration of the ADC, IGFBP-2 levels decrease relative to baseline levels in subjects who respond to treatment. In some embodiments, the level of IGFBP-2 is reduced by at least 20%-40%, for example, at least 30%, relative to baseline levels in subjects who respond to treatment. Without being bound by theory, a decrease in IGFBP-2 following administration of an ADC may indicate an improvement in the tumor microenvironment, such as reduced abnormal angiogenesis, improved epithelial-mesenchymal transition, reduced immunosuppression, and reduced cancer growth and metastasis.

In some embodiments, the biomarker is fms-related receptor tyrosine kinase 3 ligand (FLT3LG, NCBI Gene ID: 2323, SEQ ID NO: 53). In some embodiments, after administration of the ADC, FLT3LG levels remain unchanged relative to baseline levels in subjects who respond to treatment, while after administration of the ADC, FLT3LG levels increase relative to baseline levels in non-responders.

In some embodiments, the biomarker is a vascular endothelial growth factor isoform. In some embodiments, the biomarker is vascular endothelial growth factor (VEGF, NCBI Gene ID: 7422, SEQ ID NO: 54). In some embodiments, after administration of the ADC, VEGF levels remain unchanged relative to baseline levels in subjects who respond to treatment, while VEGF levels increase relative to baseline levels in non-responders after administration of the ADC. In some embodiments, the biomarker is vascular endothelial growth factor D (VEGF-D, NCBI Gene ID: 2277, SEQ ID NO: 55). In some embodiments, after administration of the ADC, VEGF-D levels remain unchanged relative to baseline levels in subjects who respond to treatment, while VEGF-D levels increase relative to baseline levels in non-responders after administration of the ADC.

In some embodiments, the biomarker is an interleukin isoform. In some embodiments, the biomarker is interleukin 17C (IL17C, NCBI Gene ID: 27189, SEQ ID NO: 56). In some embodiments, after administration of the ADC, IL17C levels are reduced relative to baseline levels in subjects who have responded to treatment. In some embodiments, the biomarker is interleukin 10 (IL-10, NCBI Gene ID: 3586, SEQ ID NO: 57). In some embodiments, after administration of the ADC, IL-10 levels are reduced relative to baseline levels in subjects who have responded to treatment. In some embodiments, the biomarker is interleukin 6 (IL-6, NCBI Gene ID: 3569, SEQ ID NO: 58). In some embodiments, after administration of the ADC, IL-6 levels remain unchanged relative to baseline levels in subjects who respond to treatment, while IL-6 levels increase relative to baseline levels in non-responders after administration of the ADC. In some embodiments, the biomarker is interleukin 18 (IL-18, NCBI Gene ID: 3606, SEQ ID NO: 59). In some embodiments, after administration of the ADC, IL-18 levels remain unchanged relative to baseline levels in subjects who respond to treatment, while IL-18 levels increase relative to baseline levels in non-responders after administration of the ADC.

In some embodiments, the biomarker is epidermal growth factor (EGF, NCBI Gene ID: 1950, SEQ ID NO: 60). In some embodiments, after administration of the ADC, EGF levels remain unchanged relative to baseline levels in subjects who respond to treatment, while after administration of the ADC, EGF levels increase relative to baseline levels in non-responders.

In some embodiments, the biomarker is a tumor necrosis factor isoform. In some embodiments, the biomarker is tumor necrosis factor alpha (TNF-α, NCBI Gene ID: 7124, SEQ ID NO: 61). In some embodiments, following administration of the ADC, TNF-α levels remain unchanged relative to baseline levels in subjects who respond to treatment, whereas following administration of the ADC, TNF-α levels increase relative to baseline levels in non-responders.

In some embodiments, the biomarker is intercellular adhesion molecule 1 (ICAM-1, NCBI Gene ID: 3383, SEQ ID NO: 62). In some embodiments, after administration of the ADC, ICAM-1 levels remain unchanged relative to baseline levels in subjects who respond to treatment, while after administration of the ADC, ICAM-1 levels increase relative to baseline levels in non-responders.

In some embodiments, the biomarker is transforming growth factor alpha (TGFA, NCBI Gene ID: 7039, SEQ ID NO: 63). In some embodiments, after administration of the ADC, TGFA levels remain unchanged relative to baseline levels in subjects who respond to treatment, while after administration of the ADC, TGFA levels increase relative to baseline levels in non-responders.

In some embodiments, the biomarker is hepatocyte growth factor (HGF, NCBI Gene ID: 3082, SEQ ID NO: 64). In some embodiments, after administration of the ADC, HGF levels remain unchanged relative to baseline levels in subjects who respond to treatment, while after administration of the ADC, HGF levels increase relative to baseline levels in non-responders.

In some embodiments, the biomarker is oncostatin M (OSM, NCBI Gene ID: 5008, SEQ ID NO: 65). In some embodiments, after administration of the ADC, OSM levels remain unchanged relative to baseline levels in subjects who respond to treatment, while after administration of the ADC, OSM levels increase relative to baseline levels in non-responders.

In some embodiments, the biomarker is erb-b2 receptor tyrosine kinase 2 (HER-2, NCBI Gene ID: 2064, SEQ ID NO: 66). In some embodiments, after administration of the ADC, HER-2 levels remain unchanged relative to baseline levels in subjects who respond to treatment, while after administration of the ADC, HER-2 levels increase relative to baseline levels in non-responders.

In some embodiments, the biomarker is TIMP metallopeptidase inhibitor 1 (TIMP1, NCBI Gene ID: 7076, SEQ ID NO: 67). In some embodiments, after administration of the ADC, TIMP1 levels remain unchanged relative to baseline levels in subjects who respond to treatment, while after administration of the ADC, TIMP1 levels increase relative to baseline levels in non-responders.

In some embodiments, changes (or lack of changes) in combinations of two or more of the above-described biomarkers are used to assess the impact of a drug or treatment, or to help physicians adjust a subject's treatment. In some embodiments, following administration of an ADC (e.g., MORAb-202), levels of two or more biomarkers associated with tumor-promoting function are reduced relative to baseline levels in subjects who responded to treatment. In some embodiments, levels of two or more (e.g., all) of ANG-2, KLK-5, KLK-7, MCP-1, FRTN, and MMP12, or any combination thereof, are reduced relative to baseline levels in subjects who responded to treatment. Without theoretical constraints, reductions in a combination of biomarkers associated with pro-tumorigenic functions may indicate therapeutic improvements in the tumor microenvironment, such as reduced aberrant angiogenesis, reduced hypoxia, improved epithelial-mesenchymal transition, reduced immunosuppression, reduced tumorigenic stroma and extracellular matrix remodeling, and reduced cancer growth and metastasis.

In some embodiments, an increase in SP-D levels relative to baseline following administration of the ADC, combined with a decrease in IGFBP-2 and/or ANG-2 levels relative to baseline following administration of the ADC, indicates that the subject is responding to treatment. Without theoretical constraints, changes in this combination of biomarkers may indicate that the ADC (e.g., MORAb-202) is exerting a pharmacodynamic effect consistent with an anti-tumor response.

In some embodiments, a decrease in IGFBP-2 and/or ANG-2 levels relative to baseline following administration of the ADC indicates that the subject is responding to treatment. Without theoretical constraints, changes in this combination of biomarkers may indicate that the ADC (e.g., MORAb-202) is exerting pharmacodynamic effects consistent with an anti-tumor response.

In some embodiments, an increase in SP-D levels relative to baseline following administration of an ADC, combined with a decrease in KLK-7, MCP-1, MMP12, and/or IGFBP-2 levels, indicates that the subject is responding to treatment. Without theoretical constraints, changes in this combination of biomarkers are likely not confounded by drug exposure.

In some embodiments, a decrease in KLK-7, MCP-1, MMP12, and/or IGFBP-2 levels indicates that the subject is responding to treatment. Without theoretical constraints, changes in this combination of biomarkers are likely not confounded by drug exposure.

In some embodiments, following administration of an ADC, an increase in the levels of IGFBP-1, FLT3LB, MIP-1β, CCL8, CCL13, IP-10, VEGF, IL-6, IL-18, EGF, TNFα, ICAM-1, TGFA, HGF, OSM, HER-2, TIMP1, and/or VEGF-D relative to baseline levels indicates that the subject has not responded to treatment. Without being bound by theory, changes in this combination of biomarkers may indicate that the ADC (e.g., MORAb-202) inhibits tumor-promoting changes and/or may indicate a drug resistance pathway. Pharmaceutical Compositions and Formulations

The ADC used to implement the aforementioned methods can be formulated into a pharmaceutical composition suitable for administration to a subject, such as a human subject. In some embodiments, the pharmaceutical composition comprises the ADC and a pharmaceutically acceptable carrier suitable for the desired delivery method. Suitable carriers include any material that, when combined with the ADC disclosed herein, allows the ADC to retain its anti-tumor function and generally does not react with the subject's immune system. Pharmaceutically acceptable carriers can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, methanesulfonates, and the like, and combinations thereof. In some embodiments, the formulation includes one or more isotonic agents, such as sugars, polyols such as mannitol, sorbitol, or sodium chloride. Pharmaceutically acceptable carriers may contain very small amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the ADC.

The pharmaceutical compositions disclosed herein can be in a variety of forms. These include, for example, liquid, semisolid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic application.

The pharmaceutical composition can be dissolved and administered via any route capable of delivering the composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumoral, intradermal, intraorgan, orthotopic, and the like. The pharmaceutical composition can be lyophilized and stored as a sterile powder, preferably under vacuum, and then reconstituted in bacteriostatic water (containing, for example, benzyl alcohol as a preservative) or sterile water prior to injection. Administration can be systemic or local. The pharmaceutical composition can comprise an ADC or a pharmaceutically acceptable salt thereof (e.g., a mesylate). The pharmaceutical composition can further comprise a corticosteroid, such as decapeptidol, prednisone, or methylprednisolone. Alternatively, in some embodiments, the corticosteroid can be provided in a separate package.

In various embodiments, kits for the therapeutic applications disclosed herein are within the scope of the present disclosure. Such kits may include an ADC disclosed herein and a carrier, packaging, or container. The carrier, packaging, or container may be divided to accommodate one or more containers, such as vials, test tubes, etc., each of which contains one of the individual elements to be used in the methods disclosed herein and/or a label or insert containing instructions for use, such as the uses disclosed herein. The carrier, packaging, or container may also include a compartment for a corticosteroid.

The kit may further include one or more other containers associated therewith containing materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes; carriers, packages, container, vial, and/or sleeve labels listing the contents and/or instructions for use; and product inserts with instructions for use.

A label may be present on or in the container to indicate that the composition is for a particular therapy or for non-therapeutic use, such as prognostic, preventative, diagnostic, or laboratory use. The label may also indicate directions for in vivo or in vitro use, such as the uses disclosed herein. Directions and/or other information may also be included on one or more inserts or one or more labels included in or on the kit. The label may be on or associated with the container. The label may be on the container when the letters, numbers, or other characters forming the label are molded or etched into the container itself. When the label is present in a receptacle or carrier that also holds the container, the label may be associated with the container, for example, in the form of a package insert. The label may indicate that the composition is for use in diagnosing or treating a condition, such as cancer, as disclosed herein.

Those skilled in the art will readily appreciate that other suitable modifications and adaptations of the methods of the present invention disclosed herein are readily apparent and may be made using suitable equivalents without departing from the scope of the invention or the embodiments disclosed herein. Now that the present invention has been disclosed in detail, it will be more clearly understood by reference to the following examples, which are included for illustrative purposes only and are not intended to be limiting.

Example 1 : Biomarker Analysis of FZEC (MORAb-202) on Cancer Response in Platinum-Resistant Ovarian Cancer Patients. FZEC ( fatuzumab eribulin , also known as MORAb-202) is an antibody-drug conjugate composed of a humanized anti-folate receptor α (FRα) monoclonal antibody, fatuzumab, and the potent microtubule dynamics inhibitor eribulin, linked to a cathepsin B-cleavable linker. FZEC is cytotoxic to FRα-positive cancer cells (by direct binding) and adjacent tumor cells (by a bystander effect); it also exerts a non-cytotoxic bystander effect within the tumor microenvironment (TME). The effects of FZEC or eribulin on the tumor microenvironment (TME) (e.g., effects on reversal of tumor epithelial-mesenchymal transition, vascular normalization, and stromal differentiation) have been previously reported to correlate with antitumor activity in preclinical tumor cell or patient-derived xenograft models. Furthermore, the effects of eribulin on reversal of epithelial-mesenchymal transition, improved vascular perfusion, and enhanced immune responses in the TME have been reported in cancer patients. A first-in-human dose-escalation study (Study MORAb-202-J081-101) is underway in Japanese patients with selected solid tumors to determine the safety and preliminary efficacy of FZEC. Several FZEC doses have demonstrated antitumor activity in patients with platinum-resistant ovarian cancer (PROC) and FRα expression levels. We conducted an exploratory serum biomarker study to assess the association of FZEC-induced pharmacological effects with clinical responses in PROC patients and to investigate the clinical mechanism of action of FZEC.

Methods: PROC patients from the dose-escalation and dose-expansion phases of Study 101 were treated with FZEC (at doses of 0.3, 0.45, 0.68, 0.9, or 1.2 mg/kg; all doses administered every 3 weeks) and included in the biomarker analysis (Figure 1). In Study 101, weight-based dosing was used at the aforementioned doses. Subsequent studies led to body surface area-based dosing at 25 mg/ ^m2 in the continuation trial.

Biomarker profiling analyzed serum protein factors related to angiogenesis, interleukins, inflammation, and cancer biology. The study included 94 biomarkers analyzed by the Rules-Based Medicine (RBM) panel using Luminex-based or SIMOA-based assays, and 45 biomarkers analyzed using the ^OLINK® Target-48 interleukin panel.

An assay-specific lower limit of quantification (LLOQ) was established for each test. Biomarkers with ≥ 70% of data points above the LLOQ were included in the statistical analysis, while biomarkers with > 30% of data points below the LLOQ were removed. Data points below the LLOQ for any biomarker were replaced by half the LLOQ value for the corresponding biomarker (i.e., if the output for a data point was "< 54," a value of 27 was used for statistical analysis).

For analysis by confirmed best overall response (BOR) status, patients were dichotomized into responders (complete response [CR]/partial response [PR]) and nonresponders (stable disease [SD]/progressive disease [PD]).

At each postbaseline visit, the percentage change from baseline was analyzed within each BOR group using a 1-sample Wilcoxon signed-rank test, and the two groups were compared at each visit using a 2-sample Wilcoxon signed-rank test, with a one-sided alternative—one less, one greater. Alternatively, a logistic regression was performed, adjusting the AUC for FZEC exposure; one-sided P values were calculated using a Wald test for the logistic regression of the percentage change from baseline.

Fisher's method was used to generate a combined P value (also called a meta P value) to summarize the statistical differences between the visits between the two response groups. To determine the direction of the difference between responders and nonresponders, a Wilcoxon test was used with the alternatives being larger or smaller rather than two-sided. If the consensus across all tests was "larger," the change in the nonresponders was greater than that in the responders.

A cohort of combined FZEC doses (0.3, 0.45, 0.68, 0.9, and 1.2 mg/kg) was evaluated with and without adjustment for area under the curve (AUC). The 0.9 mg/kg cohort was evaluated without adjustment for AUC.

Results Patients For data generated by the RBM group, 58 PROC patients from the dose escalation and dose expansion phases of Study 101 were included in the biomarker analysis (Table 11). Patients were assigned: n = 1 in the FZEC 0.3 mg/kg group, n = 3 in the FZEC 0.45 mg/kg group, n = 2 in the FZEC 0.68 mg/kg group, n = 28 in the FZEC 0.9 mg/kg group, and n = 24 in the FZEC 1.2 mg/kg group.

For the data generated by the OLINK group, 35 PROC patients from the dose escalation and dose expansion phases of Study 101, FZEC 0.9 mg/kg (n = 22) and FZEC 1.2 mg/kg (n = 13), were included in the biomarker analysis (Table 11) . [ Table 11 ] Patients included in the biomarker analysis Study time point, patient n BOR status, patient n Group/Queue C1D1 C2D1 C3D1 C4D1 C5D1 C6D1 Responders (i.e., CR and PR) Nonresponders (ie, SD and PD) RBM/Combination 58 58 43 41 twenty four twenty one twenty three 35 OLINK/Combination 35 35 25 25 11 9 12 twenty three RBM/0.9 mg/kg 28 28 16 16 9 9 9 19 OLINK/0.9 mg/kg twenty two twenty two 12 12 5 5 5 17 BOR, best overall response; C#D#, cycle #days#; CR, complete response; PD, progressive disease; PR, partial response; RBM, rule-based medicine; SD, stable disease.

Of the 139 biomarkers measured, 98 had ≥ 70% of evaluable data points associated with biomarker changes associated with best overall response ( BOR ) and were therefore included in the statistical analysis. These 139 biomarkers included marker names that overlapped between the 2 analysis platforms.

We explored biomarkers with significant percentage changes from baseline between responders and nonresponders (1-sample Wilcoxon signed-rank P < 0.05). These changes were assessed at visits C2D1-C6D1 (i.e., second to sixth doses of FZEC).

For 29 biomarkers in the combined dose cohort (from the unadjusted AUC analysis) and 14 biomarkers in the FZEC 0.9 mg/kg cohort, percentage changes from baseline to at least 1 postdose visit were observed in either BOR group ( P < 0.05) and differences between responders and nonresponders (meta P < 0.05) were observed (Table 12).

In addition to the overall difference in changes between responders and nonresponders (meta P < 0.05) (Table 12), decreases in the biomarkers ANG-2, KLK-5, KLK-7, MCP-1, FRTN, MMP12, and IGFBP-2 and increases in SP-D in responders were sustained (P < 0.05 at ≥ 3 visits) in the combination cohort (Figure 2) or the FZEC 0.9 mg/kg cohort (Figure 3), suggesting that these changes may be mediated by FZEC treatment and associated with cancer response.

Following FZEC treatment, reductions in biomarkers with pro-tumor functions (ANG-2, KLK-5, KLK-7, MCP-1, FRTN, and MMP12) were observed in responders (Figures 2 and 3) and may contribute to the anti-tumor effects of FZEC/eribulin (Figure 4). The reduction in FRTN in responders suggests a link between iron metabolism and FZEC-mediated cancer responses. Iron metabolism is involved in shaping the immune landscape in cancer-associated inflammation. Following FZEC treatment, increases in SP-D (a marker involved in immune surveillance of cancer and inducing apoptosis in multiple patient-derived cancer cell lines and primary cancer cells) (Figures 2 and 3) suggest that this marker may contribute to the anti-tumor effects of FZEC/eribulin (Figure 4). [ Table 12 ] Biomarkers with changes associated with BOR Biomarkers with meta P < 0.05 between BOR groups Number of visits with significant percentage change from baseline ( P < 0.05 ) Combination dose queue FZEC 0.9 mg/kg dose queue Responders Non-responders Responders Non-responders SP-D 5↑ 4↑ 4↑ 1↑ ANG-2 5↓ 1↓ 1↓ — IGFBP-2 3↓ — 3↓ — IGFBP-1 — 3↑ — 2↑ TNF-α (OLINK) 1 1↑ — 1↑ ICAM-1 — 2↑ — 1↑ TGFA (OLINK) — 2↑ — 2↑ VEGF (OLINK) — 2↑ — 1↑ IL-6 (OLINK) — 2↑ — 1↑ IP-10 (OLINK) — 2↑ — 1↑ HGF (OLINK) — 2↑ — 2↑ KLK-5 5↓ 1↓ — — KLK-7 5↓ — — — MCP-1 5↓ — — — MMP12 (OLINK) 4↓ 1↓ — — MCP-1 (OLINK) 3↓ 1↑ — Eosin-1 (OLINK) 2↓ — — — IL17C (OLINK) 2↓ — — — IP-10 1↑ 3↑ — — EGF (OLINK) 1↓ — — — IL-10 (OLINK) 1↓ — — — FLT3LG (OLINK) — 3↑ — — VEGF-D — 3↑ — — VEGF — 2↑ — — OSM (OLINK) — 2↑ — — CCL13 (OLINK) — 1↑ — — MIP-1 β (OLINK) — 1↑ — — CCL8 (OLINK) — 1↑ — — HER-2 — 1↑ — — FRTN — — 4↓ — IL-18 (OLINK) TIMP1 — — — 1↑ TIMP1 — — — 1↑ Markers in shaded rows show overlap between changes in the combined dose cohort and the FZEC 0.9 mg/kg dose cohort. In the combined dose analysis, bold markers show changes starting on C2D1 ( P < 0.05). Dashes indicate P > 0.05 for all visits or meta- P > 0.05 for the corresponding marker. If a marker overlaps with a marker from the RBM group, the marker name from the OLINK group is labeled accordingly. Arrows indicate the direction of change. P values are not FDR-adjusted. BOR, best overall response; C#D#, cycle #days#; FDR, false discovery rate; FZEC, facilitation; RBM, rule-based medicine.

Eleven markers, including SP-D, IGFBP-2, and ANG-2, overlapped between the FZEC 0.9 mg/kg and combination dose cohorts, as demonstrated by changes from baseline in responders and/or nonresponders ( P < 0.05) and differences in percentage change between responders and nonresponders (meta- P < 0.05) (Table 12). These results support the view that FZEC exerts pharmacodynamics consistent with antitumor responses at the 0.9 mg/kg dose. Although the 0.9 mg/kg dose was initially selected for clinical use, it was no longer used in the ongoing study. In the future, additional data will be generated in continuation trials (using different dose levels) to confirm the findings from the 0.9 mg/kg dose.

Table 13 lists the markers for which more visits showed a change in responders ( P < 0.05) than a difference between nonresponders and the BOR group ( meta- P < 0.05) in at least one of the three analyses. The number of visits for each marker within the subcohort of responders or nonresponders that showed a significant change from baseline ( P < 0.05) is listed.

In the combined cohort analysis, some biomarkers (e.g., SP-D, KLK-7, MCP-1, MMP12, IGFBP-2) showed changes relative to baseline independent of AUC adjustment (Table 13), suggesting that the association of these biomarkers with tumor response may not be confounded by the association of changes in biomarker expression with drug exposure. This will be further explored at the lower doses currently being studied in clinical trials.

Although ANG-2 and KLK-5 levels appeared to differ between responders and nonresponders after treatment with FZEC in the combined dose cohort, these differences were not significant after adjusting for FZEC AUC (Table 13). While the hypotheses generated by this study require validation in further clinical studies, this may be because the changes in biomarker expression and associations with responders are highly correlated with drug exposure. The observed associations of biomarkers with cancer response may also be confounded by their association with drug exposure.

Biomarkers such as IGFBP-1, FLT3LG, and VEGF-D showed increases in nonresponders ( P < 0.05) (Table 12). This may indicate that FZEC inhibits tumor-promoting changes (Figure 4), or these changes may indicate resistance pathways. [ Table 13 ] Comparison of biomarkers associated with tumor responders biomarkers Number of visits for which the marker had a meta P < 0.05 response combination queue FZEC AUC - adjusted combined queue FZEC 0.9 mg/kg queue SP-D 5↑ 5↑ 4↑ KLK-7 5↓ 5↓ — MCP-1 5↓ 5↓ — ANG-2 5↓ — 1↓ KLK-5 5↓ — — MMP12 (OLINK) 4↓ 4↓ — FRTN — 2↓ 4↓ MCP-1 (OLINK) 3↓ 3↓ — IGFBP-2 3↓ 3↓ 3↓ TARC — — Eosin-1 (OLINK) 2↓ 2↓ — IL17C (OLINK) 2↓ 2↓ — EGF (OLINK) 1↓ 1↓ — IL-10 (OLINK) 1↓ 1↓ — MIG — — Markers showing a greater number of visits with a change in (1) responders compared with nonresponders ( P < 0.05) and (2) a difference between the BOR groups ( meta P < 0.05) in at least 1 of 3 analyses are listed. For each marker, the number of visits in which the responder cohort changed significantly from baseline ( P < 0.05) is listed. Dashes indicate P > 0.05 for all visits or meta P > 0.05 for the corresponding marker. AUC, area under the curve; BOR, best overall response; FZEC, facillinam-etibuzumab.

Summary and Conclusions The sustained changes in responder-associated biomarkers suggest that FZEC may modulate functions associated with these markers (e.g., vascular remodeling, immune regulation, mesenchymal/extracellular matrix remodeling, iron metabolism, and reverse epithelial-mesenchymal transition), which may contribute to the antitumor effects of FZEC and its cytotoxic effects on cancer cells from PROC patients (Figure 4).

In the limited number of patients analyzed, preliminary pharmacodynamic effects were consistent with the antitumor effects previously observed with FZEC 0.9 mg/kg. Selected sequences: SEQ ID NO: 1 (MORAb-003 HC CDR1; Kabat): GYGLS SEQ ID NO: 2 (MORAb-003 HC CDR2; Kabat): MISSGGSYTYYADSVKG SEQ ID NO: 3 (MORAb-003 HC CDR3; Kabat): HGDDPAWFAY SEQ ID NO: 4 (MORAb-003 LC CDR1; Kabat): SVSSSISSNNLH SEQ ID NO: 5 (MORAb-003 LC CDR2: Kabat): GTSNLAS SEQ ID NO: 6 (MORAb-003 LC CDR3; Kabat): QQWSSYPYMYT SEQ ID NO: 7 (MORAb-003 HC CDR1; IMGT): GFTFSGYG SEQ ID NO: 8 (MORAb-003 HC CDR2; IMGT): ISSGGSYT SEQ ID NO: 9 (MORAb-003 HC CDR3; IMGT): ARHGDDPAWFAY SEQ ID NO: 10 (MORAb-003 LC CDR1; IMGT): SSISSNN SEQ ID NO: 11 (MORAb-003 LC CDR2; IMGT): GTS SEQ ID NO: 12 (MORAb-003 LC CDR3; IMGT): QQWSSYPYMYT SEQ ID NO: 13 (MORAb-003 heavy chain variable domain (V _H )): 1 EVQLVESGGG VVQPGRSLRL SCSASGFTFS GYGLS WVRQA PGKGLEWVA M 51 ISSGGSYTYY ADSVKG RFAI SRDNAKNTLF LQMDSLRPED TGVYFCAR HG 101 DDPAWFAY WG QGTPVTVSSA SEQ ID NO: 14 (MORAb-003 light chain variable domain (V _L )): QQWSSYPYMYT 1 DIQLTQSPSS LSASVGDRVT ITCSVSSSIS SNNLHWYQQK PGKAPKPWIY 51 GTSNLASGVP SRFSGSGSGT DYTFTISSLQ PEDIATYYCQ QWSSYPYMYT 101 FGQGTKVEIK SEQ ID NO: 15 (MORAb-003 heavy chain (HC)) 1 EVQLVESGGG VVQPGRSLRL SCSASGFTFS GYGLS WVRQA PGKGLEWVA M 51 ISSGGSYTYY ADSVKG RFAI SRDNAKNTLF LQMDSLRPED TGVYFCAR HG 101 DDPAWFAY WG QGTPVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD 151 YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY 201 ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPELLGGP SVFLFPPKPK 251 DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS 301 TYRVVSVLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV 351 YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 401 DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK SEQ ID NO: 16 (MORAb-003 light chain (LC)) 1 DIQLTQSPSS LSASVGDRVT ITCSVSSSIS SNNLHWYQQK PGKAPKPWIY 51 GTSNLASGVP SRFSGSGSGT DYTFTISSLQ PEDIATYYCQ QWSSYPYMYT 101 FGQGTKVEIK RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ 151 WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT 201 HQGLSSPVTK SFNRGEC SEQ ID NO: 35 (MORAb-003 HC nt) 1 ATGGGATGGA GCTGTATCAT CCTCTTCTTG GTAGCAACAG CTACAGGTGT 51 CCACTCCGAG GTCCAACTGG TGGAGAGCGG TGGAGGTGTT GTGCAACCTG 101 GCCGGTCCCT GCGCCTGTCC TGCTCCGCAT CTGGCTTCAC CTTCAGCGGC 151 TATGGGTTGT CTTGGGTGAG ACAGGCACCT GGAAAAGGTC TTGAGTGGGT 201 TGCAATGATT AGTAGTGGTG GTAGTTATAC CTACTATGCA GACAGTGTGA 251 AGGGTAGATT TGCAATATCG CGAGACAACG CCAAGAACAC ATTGTTCCTG 301 CAAATGGACA GCCTGAGACC CGAAGACACC GGGGTCTATT TTTGTGCAAG 351 ACATGGGGAC GATCCCGCCT GGTTCGCTTA TTGGGGCCAA GGGACCCCGG 401 TCACCGTCTC CTCAGCCTCC ACCAAGGGCC CATCGGTCTT CCCCCTGGCA 451 CCCTCCTCCA AGAGCACCTC TGGGGGCACA GCGGCCCTGG GCTGCCTGGT 501 CAAGGACTAC TTCCCCGAAC CGGTGACGGT GTCGTGGAAC TCAGGCGCCC 551 TGACCAGCGG CGTGCACACC TTCCCGCCTG TCCTACAGTC CTCAGGACTC 601 TACTCCCTCA GCAGCGTGGT GACCGTGCCC TCCAGCAGCT TGGGCACCCA 651 GACCTACATC TGCAACGTGA ATCACAAGCC CAGCAACACC AAGGTGGACA 701 AGAAAGTTGA GCCCAAATCT TGTGACAAAA CTCACACATG CCCACCGTGC 751 CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA 801 ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG 851 TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG 901 GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA 951 CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT 1001 GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA 1051 GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC 1101 ACAGGTGTAC ACCCTGCCCC CATCCCGGGA TGAGCTGACC AAGAACCAGG 1151 TCAGCCTGAC CTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG 1201 GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC 1251 CGTGCTGGAC TCCGACGGCT CCTTCTTCTT ATATTCAAAG CTCACCGTGG 1301 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT 1351 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCCGG 1401 GAAATGA SEQ ID NO: 36 (MORAb-003 LC nt) 1 ATGGGATGGA GCTGTATCAT CCTCTTCTTG GTAGCAACAG CTACAGGTGT 51 CCACTCCGAC ATCCAGCTGA CCCAGAGCCC AAGCAGCCTG AGCGCCAGCG 101 TGGGTGACAG AGTGACCATC ACCTGTAGTG TCAGCTCAAG TATAAGTTCC 151 AACAACTTGC ACTGGTACCA GCAGAAGCCA GGTAAGGCTC CAAAGCCATG 201 GATCTACGGC ACATCCAACC TGGCTTCTGG TGTGCCAAGC AGATTCAGCG 251 GTAGCGGTAG CGGTACCGAC TACACCTTCA CCATCAGCAG CCTCCAGCCA 301 GAGGACATCG CCACCTACTA CTGCCAACAG TGGAGTAGTT ACCCGTACAT 351 GTACACGTTC GGCCAAGGGA CCAAGGTGGA AATCAAACGA ACTGTGGCTG 401 CACCATCTGT CTTCATCTTC CCGCCATCTG ATGAGCAGTT GAAATCTGGA 451 ACTGCCTCTG TTGTGTGCCT GCTGAATAAC TTCTATCCCA GAGAGGCCAA 501 AGTACAGTGG AAGGTGGATA ACGCCCTCCA ATCGGGTAAC TCCCAGGAGA 551 GTGTCACAGA GCAGGACAGC AAGGACAGCA CCTACAGCCT CAGCAGCACC 601 CTGACGCTGA GCAAAGCAGA CTACGAGAAA CACAAGTCT ACGCCTGCGA 651 AGTCACCCAT CAGGGCCTGA GCTCGCCCGT CACAAAGGC TTCAACAGGG 701 GAGAGTGTTA A SEQ ID NO: 37 (human FRA) 1 maqrmttqll lllvwvavvg eaqtriawar tellnvcmna khhkekpgpe dklheqcrpw 61 rknaccstnt sqeahkdvsy lyrfnwnhcg emapackrhf iqdtclyecs pnlgpwiqqv 121 dqswrkervl nvplckedce qwwedcrtsy tcksnwhkgw nwtsgfnkca vgaacqpfhf 181 yfptptvlcn eiwthsykvs nysrgsgrci qmwfdpaqgn pneevarfya aamsgagpwa 241 awpfllslal mllwlls SEQ ID NO: 38 (human FRA nucleotide) 1 cattccttgg tgccactgac cacagctctt tcttcaggga cagacatggc tcagcggatg 61 acaacacagc tgctgctcct tctagtgtgg gtggctgtag taggggaggc tcagacaagg 121 attgcatggg ccaggactga gcttctcaat gtctgcatga acgccaagca ccacaaggaa 181 aagccaggcc ccgaggacaa gttgcatgag cagtgtcgac cctggaggaa gaatgcctgc 241 tgttctacca acaccagcca ggaagcccat aaggatgttt cctacctata tagattcaac 301 tggaaccact gtggagagat ggcacctgcc tgcaaacggc atttcatcca ggacacctgc 361 ctctacgagt gctcccccaa cttggggccc tggatccagc aggtggatca gagctggcgc 421 aaagagcggg tactgaacgt gcccctgtgc aaagaggact gtgagcaatg gtgggaagat 481 tgtcgcacct cctacacctg caagagcaac tggcacaagg gctggaactg gacttcaggg 541 tttaacaagt gcgcagtggg agctgcctgc caacctttcc atttctactt ccccacaccc 601 actgttctgt gcaatgaaat ctggactcac tcctacaagg tcagcaacta cagccgaggg 661 agtggccgct gcatccagat gtggttcgac ccagcccagg gcaaccccaa tgaggaggtg 721 gcgaggttct atgctgcagc catgagtggg gctgggccct gggcagcctg gcctttcctg 781 cttagcctgg ccctaatgct gctgtggctg ctcagctgac ctccttttac cttctgatac 841 ctggaaatcc ctgccctgtt cagccccaca gctcccaact atttggttcc tgctccatgg 901 tcgggcctct gacagccact ttgaataaac cagacaccgc acatgtgtct tgagaattat 961 ttggaaaaaa aaaaaaaaaa aa [Table 14]. Biomarker sequences biomarkers SEQ ID sequence SP-D 39 MLLFLLSALVLLTQPLGYLEAEMKTYSHRTMPSACTLVMCSSVESGLPGRDGRDGREGPRGEKGDPGLPGAAGQAGMPGQAGPVGPKGDNGSVGEPGPKGDTGPSGPPGPPGVPGPAGREGPLGKQGNIGPQGKPGPKGEAGPKGEVGAPGMQGSAGARGLAGPKGERGVPGERGVPGNTGAAGSAG AMGPQGSPGARGPPGLKGDKGIPGDKGAKGESGLPDVASLRQQVEALQGQVQHLQAAFSQYKKVELFPNGQSVGEKIFKTAGFVKPFTEAQLLCTQAGGQLASPRSAAENAALQQLVVAKNEAAFLSMTDSKTEGKFTYPTGESLVYSNWAPGEPNDDGGSEDCVEIFTNGKWNDRACGEKRLVVCEF KLK-5 40 MATARPPWMWVLCALITALLLGVTEHVLANNDVSCDHPSNTVPSGSNQDLGAGAGEDARSDDSSSRIINGSDCDMHTQPWQAALLLRPNQLYCGAVLVHPQWLLTAAHCRKKVFRVRLGHYSLSPVYESGQQMFQGVKSIPHPGYS HPGHSNDLMLIKLNRRIRPTKDVRPINVSSHCPSAGTKCLVSGWGTTKSPQVHFPKVLQCLNISVLSQKRCEDAYPRQIDDTMFCAGDKAGRDSCQGDSGGPVVCNGSLQGLVSWGDYPCARPNRPGVYTNLCKFTKWIQETIQANS KLK-7 41 MARSLLPLQILLLSLALETAGEEAQGDKIIDGAPCARGSHPWQVALLSGNQLHCGGVLVNERWVLTAAHCKMNEYTVHLGSDTLGDRRAQRIKASKSFRHPGYSTQTHVNDLMLVKLNSQARLSS MVKKVRLPSRCEPPGTTCTVSGWGTTTSPDVTFPSDLMCVDVKLISPQDCTKVYKDLLENSMLCAGIPDSKKNACNGDSGGPLVCRGTLQGLVSWGTFPCGQPNDPGVYTQVCKFTKWINDTMKKHR MCP-1 42 MKVSAALLCLLLIAATFIPQGLAQPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT MIP-1 beta 43 MKLCVTVLSLLMLVAAFCSPALSAPMGSDPPTACCFSYTARKLPRNFVVDYYETSSLCSQPAVVFQTKRSKQVCADPSESWVQEYVYDLELN CCL8 44 MKVSAALLCLLLMAATFSPQGLAQPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRGKEVCADPKERWVRDSMKHLDQIFQNLKP IP-10 45 MNQTAILICCLIFLTLSGIQGVPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSP Eosin-1 46 MKVSAALLWLLLIAAAFSPQGLAGPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKDICADPKKKWVQDSMKYLDQKSPTPKP CCL13 47 MKVSAVLLCLLLMTAAFNPQGLAQPDALNVPSTCCFTFSSKKISLQRLKSYVITTSRCPQKAVIFRTKLGKEICADPKEKWVQNYMKHLGRKAHTLKT ANG-2 48 MWQIVFFTLSCDLVLAAAYNNFRKSMDSIGKKQYQVQHGSCSYTFLLPEMDNCRSSSSPYVSNAVQRDAPLEYDDSVQRLQVLENIMENNTQWLMKLENYIQDNMKKEMVEIQQNAVQNQTAVM IEIGTNLLNQTAEQTRKLTDVEAQVLNQTTRLELQLLEHSLSTNKLEKQILDQTSEINKLQDKNSFLEKKVLAMEDKHIIQLQSIKEEKDQLQVLVSKQNSIIEELEKKIVTATVNNSVLQKQQ HDLMETVNNLLTMMSTSNSAKDPTVAKEEQISFRDCAEVFKSGHTTNGIYTLTFPNSTEEIKAYCDMEAGGGGWTIIQRREDGSVDFQRTWKEYKVGFGNPSGEYWLGNEFVSQLTNQQRYVLK IHLKDWEGNEAYSLYEHFYLSSEELNYRIHLKGLTGTAGKISSISQPGNDFSTKDGDNDKCICKCSQMLTGGWWFDACGPSNLNGMYYPQRQNTNKFNGIKWYYWKGSGYSLKATTMMIRPADF MMP12 49 MKFLLILLLQATASGALPLNSSTSLEKNNVLFGERYLEKFYGLEINKLPVTKMKYSGNLMKEKIQEMQHFLGLKVTGQLDTSTLEMMHAPRCGVPDVHHFREMPGGPVWRKHYITYR INNYTPDMNREDVDYAIRKAFQVWSNVTPLKFSKINTGMADILVVFARGAHGDFHAFDGKGGILAHAFGPGSGIGGDAHFDEDEFWTTHSGGTNLFLTAVHEIGHSLGLGHSSDPKAV MFPTYKYVDINTFRLSADDIRGIQSLYGDPKENQRLPNPDNSEEPALCDPNLSFDAVTTVGNKIFFFKDRFFWLKVSERPKTSVNLISSLWPTLPSGIEAAYEIEARNQVFLFKDDKY WLISNLRPEPNYPKSIHSFGFPNFVKKIDAAVFNPRFYRTYFFVDNQYWRYDERRQMMDPGYPKLITKNFQGIGPKIDAVFYSKNKYYYFFQGSNQFEYDFLLQRITKTLKSNSWFGC FRTN 50 MWTLGRRAVAGLLASPSPAQAQTLTRVPRPAELAPLCGRRGLRTDIDATCTPRRASSNQRGLNQIWNVKKQSVYLMNLRKSGTLGHPGSLDETTYERLAEETLDS LAEFFEDLADKPYTFEDYDVSFGSGVLTVKLGGDLGTYVINKQTPNKQIWLSSSPSSGPKRYDWTGKNWVYSHDGVSLHELLAAELTKALKTKLDLSSLAYSGKDA IGFBP-1 51 MSEVPVARVWLVLLLLTVQVGVTAGAPWQCAPCSAEKLALCPPVSASCSEVTRSAGCGCCPMCALPLGAACGVATARCARGLSCRALPGEQQPLHALTRGQGACVQESDASAPHAAEAGSPESPESTEI TEEELLDNFHLMAPSEEDHSILWDAISTYDGSKALHVTNIKKWKEPCRIELYRVVESLAKAQETSGEEISKFYLPNCNKNGFYHSRQCETSMDGEAGLCWCVYPWNGKRIPGSPEIRGDPNCQIYFNVQN IGFBP-2 52 MLPRVGCPALPLPPPPLLPLLLLGASGGGGGARAEVLFRCPPCTPERLAACGPPPVAPPAAVAAVAGGARMPCAELVREPGCGCCSVCARLEGEACGVYTPRCGQGLRCYPHPGSELPLQALVMGEGTCEKRRDAEYGASPEQVADNGDDHSEGGLVENH VDSTMNMLGGGGSAGRKPLKSGMKELAVFREKVTEQHRQMGKGGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLHIPNCDKHGLYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEARGVHTQRMQ FLT3LG 53 MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPP SCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQPPLLLLLLLPVGLLLLAAAWCLHWQRTRRRTPRPGEQVPPVPSPQDLLLVEH VEGF 54 MTDRQTDTAPSPSYHLLPGRRRTVDAAASRGQGPEPAPGGGVEGVGARGVALKLFVQLLGCSRFGGAVVRAGEAEPSGAARSASSGREEPQPEEGEEEEEKEEERGPQWRLGARKPGSWTGEAAVCADSAPAARAPQALARASGRGGRVARRGAEESGPPHSPSRRGSASRAGPGRASETMNFLLSWVHWSLALLLY LHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQH IGEMSFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR VEGF-D 55 MYREWVVVNVFMMLYVQLVQGSSNEHGPVKRSSQSTLERSEQQIRAASSLEELLRITHSEDWKLWRCRLRLKSFTSMDSRSASHRSTRFAATFYDIETLKVIDEEWQRTQCSPRETCVEVASELGKSTNTFFKPPCVNVFRCGGCCNEESLICMNTTSSYISKQLFEIISVPLTSVPE LVPVKVANHTGCKCLPTAPRHPYSIIRRSIQIPEEDRCSHSKKLCPIDMLWDSNKCKCVLQEENPLAGTEDHSHLQEPALCGPHMMFDEDRCECVCKTPCPKDLIQHPKNCSCFECKESLETCCQKHKLFHPDTCSCEDRCPFHTRPCASGKTACAKHCRFPKEKRAAQGPHSRKNP IL17C 56 MTLLPGLLFLTWLHTCLAHHDPSLRGHPHSHGTPHCYSAEELPLGQAPPHLLARGAKWGQALPVALVSSLEAASHRGRHERPSATTQCPVLRPEEVLEADTHQRSISPWRYRVDTDEDRYPQKLAFAECLCRGCIDARTGRETAALNSVRLLQSLLVLRRRPCSRDGSGLPTPGAFAFHTEFIHVPVGCTCVLPRSV IL-10 57 MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN IL-6 58 MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGF NEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRALRQM IL-18 59 MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENLESDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED EGF 60 MLLTLIILLPVVSKFSFVSLSAPQHWSCPEGTLAGNGNSTCVGPAPFLIFSHGNSIFRIDTEGTNYEQLVVDAGVSVIMDFHYNEKRIYWVDLERQLLQRVFLNGSRQERVCNIEKNVSGMAINWINEEVIWSNQQEGIITVTDMKGNNS HILLSALKYPANVAVDPVERFIFWSSEVAGSLYRADLDGVGVKALLETSEKITAVSLDVLDKRLFWIQYNREGSNSLICSCDYDGGSVHISKHPTQHNLFAMSLFGDRIFYSTWKMKTIWIANKHTGKDMVRINLHSSFVPLGELKVVHPL AQPKAEDDTWEPEQKLCKLRKGNCSSTVCGQDLQSHLCMCAEGYALSRDRKYCEDVNECAFWNHGCTLGCKNTPGSYYCTCPVGFVLLPDGKRCHQLVSCPRNVSECSHDCVLTSEGPLCFCPEGSVLERDGKTCSGCSSPDNGGCSQLCV PLSPVSWECDCFPGYDLQLDEKSCAASGPQPFLLFANSQDIRHMHFDGTDYGTLLSQQMGMVYALDHDPVENKIYFAHTALKWIERANMDGSQRERLIEEGVDVPEGLAVDWIGRRFYWTDRGKSLIGRSDLNGKRSKIITKENISQPRGI AVHPMAKRLFWTDTGINPRIESSSLQGLGRLVIASSDLIWPSGITIDFLTDKLYWCDAKQSVIEMANLDGSKRRRLTQNDVGHPFAVAVFEDYVWFSDWAMPSVMRVNKRTGKDRVRLQGSMLKPSSLVVVHPLAKPGADPCLYQNGGCEH ICKKRLGTAWCSCREGFMKASDGKTCLALDGHQLLAGGEVDLKNQVTPLDILSKTRVSEDNITESQHMLVAEIMVSDQDDCAPVGCSMYARCISEGEDATCQCLKGFAGDGKLCSDIDECEMGVPVCPPASSKCINTEGGYVCRCSEGYQG DGIHCLDIDECQLGEHSCGENASCTNTEGGYTCMCAGRLSEPGLICPDSTPPPHLREDDHHYSVRNSDSECPLSHDGYCLHDGVCMYIELDKYACNCVVGYIGERCQYRDLKWWELRHAGHGQQQKVIVVAVCVVVLVMLLLLSLWGAHY YRTQKLLSKNPKNPYEESSRDVRSRRPADTEDGMSSCPQPWFVVIKEHQDLKNGGQPVAGEDGQAADGSMQPTSWRQEPQLCGMGTEQGCWIPVSSDKGSCPQVMERSFHMPSYGTQTLEGGVEKPHSLLSANPLWQQRALDPPHQMELTQ TNF-α 61 MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANG VELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL ICAM-1 62 MAPSSPRPALPALLVLLGALFPGPGNAQTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGIETPLPKKELLLPGNNRKVYELSNVQEDSQPMCYSNCPDGQSTAKTFLTVYWTPERVELAPLPSWQPVGKNLTL RCQVEGGAPRANLTVVLLRGEKELKREPAVGEPAEVTTTVLVRRDHHGANFSCRTELDLRPQGLELFENTSAPYQLQTFVLPATPPQLVSPRVLEVDTQGTVVCSLDGLFPVSEAQVHLALGDQRLNPTVTYG NDSFSAKASVSVTAEDEGTQRLTCAVILGNQSQETLQTVTIYSFPAPNVILTKPEVSEGTEVTVKCEAHPRAKVTLNGVPAQPLGPRAQLLLKATPEDNGRSFSCSATLEVAGQLIHKNQTRELRVLYGPRLD ERDCPGNWTWPENSQQTPMCQAWGNPLPELKCLKDGTFPLPIGESVTVTRDLEGTYLCRARSTQGEVTRKVTVNVLSPRYEIVIITVVAAAVIMGTAGLSTYLYNRQRKIKKYRLQQAQKGTPMKPNTQATPP TGFA 63 MVPSAGQLALFALGIVLAACQALENSTSPLSADPPVAAAVVSHFNDCPDSHTQFCFHGTCRFLVQEDKPACVCHSGYVGARCEHADLLAVVAASQKKQAITALVVVSIVALAVLIITCVLIHCCQVRKHCEWCRALICRHEKPSALLKGRTACCHSETVV HGF 64 MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGKDLQENYCRNPRG EEGGPWCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTDVPLETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGSESPW CFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNMEDLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNLDHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDYEA WLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLPNYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAGAEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKIILTYKVPQS OSM 65 MGVLLTQRTLLSLVLALLFPSMASMAAIGSSCKEYRVLLGQLQKQTDLMQDTSRLLDPYIRIQGLDVPKLREHCRERPGAFPSEETLRGLGRRGFLQTLNATLGCVLHRLADLEQRLPKAQDLERS GLNIEDLEKLQMARPNILGLRNNIYCMAQLLDNSDTAEPTKAGRGASQPPTPTPASDAFQRKLEGCRFLHGYHRFMHSVGRVFSKWGESPNRSRRHSPHQALRKGVRRTRPSRKGKRLMTRGQLPR HER-2 66 MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQ RNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTL VCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHH NTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCP INCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSR LLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERL PQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGL QSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV TIMP1 67 MAPFEPLASGILLLLWLIAPSRACTCVPPHPQTAFCNSDLVIRAKFVGTPEVNQTTLYQRYEIKMTKMYKGFQALGDAADIRFVYTPAMESVCGYFHRSHNRSEEFLIAGKLQDGLLHITTCSFVAPWNSLSLAQRRGFTKTYTVGCEECTVFPCLSIPCKLQSGTHCLWTDQLLQGSEKGFQSRHLACLPREPGLCTWQSLRSQIA

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Figure 1 shows the biomarker study design. C#: dosing period after the first dose, e.g., every three weeks; D#: days after the most recent dose; FZEC: fazolizumab ezetimibe; IV: intravenous; MoA: mechanism of action; Q3W: every three weeks.

Figure 2 shows changes in the levels of certain biomarkers in responders (blue) and non-responders (purple) in the MORAb-202 combination cohort. C#: # of cycles; D#: # of days.

Figure 3 shows changes in the levels of certain biomarkers in responders (blue) and nonresponders (purple) in the MORAb-202 0.9 mg/kg cohort. C#: cycle #; D#: day #.

Figure 4 shows the cytotoxicity and potential non-cytotoxic bystander effects of MORAb-202 administration. ECM: extracellular matrix; EMT: epithelial-mesenchymal transition; FRα: folate receptor α; FZEC: facillin-epibulin; MET: mesenchymal-epithelial transition.

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TW202530697A_113138705_SEQL.xml

Claims (43)

A method for treating a folate receptor alpha (FRA) phenotypical cancer, the method comprising: (a) measuring the level of one or more biomarkers of tumor response in a baseline biological sample from a subject, (b) administering to a subject in need thereof an amount of an antibody-drug conjugate having formula (I): Ab-(LD)p (I) wherein (i) Ab is an internalizing anti-folate receptor alpha antibody or an internalizing antigen-binding fragment thereof comprising: three heavy chain complementation determining regions (HCDRs) comprising 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 complementation determining regions (LCDRs) comprising SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2), and SEQ ID NO: 6 (LCDR3), as defined by the Kabat numbering system; or three heavy chain complementation determining regions (HCDRs) comprising 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 complementation determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQ ID NO: 12 (LCDR3), as defined by the IMGT numbering system; (ii) D is eribulin; (iii) L is a cleavable linker comprising Mal-(PEG) ₂ -Val-Cit-pAB; and (iv) p is an integer from 1 to 8, (c) measuring the level of one or more biomarkers of tumor response in a sample taken from the subject to determine a change relative to the level in the baseline sample, and (d) administering a further dose of the antibody-drug conjugate if a change in the level is detected. A method for reducing the risk of interstitial lung disease (ILD) in a subject being treated for a cancer with a FRA phenotype, the method comprising: (a) measuring the level of one or more biomarkers of tumor response in a baseline biological sample from the subject, (b) administering to the subject in need thereof an amount of an antibody-drug conjugate having formula (I): Ab-(LD)p (I) wherein (i) Ab is an internalizing anti-folate receptor alpha antibody or an internalizing antigen-binding fragment thereof comprising: three heavy chain complementary determining regions (HCDRs) comprising 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 complementary determining regions (LCDRs) comprising SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2), and SEQ ID NO: 6 (LCDR3), as defined by the Kabat numbering system; or three heavy chain complementation determining regions (HCDRs) comprising 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 complementation determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQ ID NO: 12 (LCDR3), as defined by the IMGT numbering system; (ii) D is eribulin; (iii) L is a cleavable linker comprising Mal-(PEG) ₂ -Val-Cit-pAB; and (iv) p is an integer from 1 to 8, (c) measuring the level of one or more biomarkers of tumor response in a sample taken from the subject to determine a change relative to the level in the baseline sample, and (d) administering a further dose of the antibody-drug conjugate if a change in the level is detected. The method of claim 1 or claim 2, wherein the level of surfactant protein D (SP-D) in a sample taken from the subject after administration is increased relative to the level of SP-D in the baseline sample. The method of claim 3, wherein the level of SP-D is increased by at least 100% relative to the level of SP-D in the baseline sample. The method of claim 1 or claim 2, wherein the level of kallikrein-related peptidase 7 (KLK-7) in a sample taken from the subject after administration is reduced relative to the level of KLK-7 in the baseline sample. The method of claim 5, wherein the level of KLK-7 is reduced by at least 30% relative to the level of KLK-7 in the baseline sample. The method of claim 1 or claim 2, wherein the level of monocyte cytochrome protein-1 (MCP-1) in a sample obtained from the subject after administration is reduced relative to the level of MCP-1 in the baseline sample. The method of claim 7, wherein the level of MCP-1 is reduced by at least 15% relative to the level of MCP-1 in the baseline sample. The method of claim 1 or claim 2, wherein the level of angiopoietin 2 (ANG-2) in a sample taken from the subject after administration is reduced relative to the level of ANG-2 in the baseline sample. The method of claim 9, wherein the level of ANG-2 is reduced by at least 30% relative to the level of ANG-2 in the baseline sample. The method of claim 1 or claim 2, wherein the level of kallikrein-related peptidase 5 (KLK-5) in a sample taken from the subject after administration is reduced relative to the level of KLK-5 in the baseline sample. The method of claim 11, wherein the level of KLK-5 is reduced by at least 40% relative to the level of KLK-5 in the baseline sample. The method of claim 1 or claim 2, wherein the level of matrix metalloproteinase 12 (MMP12) in a sample taken from the subject after administration is reduced relative to the level of MMP12 in the baseline sample. The method of claim 13, wherein the level of MMP12 is reduced by at least 40% relative to the level of MMP12 in the baseline sample. The method of claim 1 or claim 2, wherein the level of ferritin (FRTN) in a sample taken from the subject after administration is reduced relative to the level of FRTN in the baseline sample. The method of claim 15, wherein the level of FRTN is reduced by at least 40% relative to the level of FRTN in the baseline sample. The method of claim 1 or claim 2, wherein the level of insulin-like growth factor binding protein 2 (IGFBP-2) in a sample taken from the subject after administration is reduced relative to the level of IGFBP-2 in the baseline sample. The method of claim 17, wherein the level of IGFBP-2 is reduced by at least 30% relative to the level of IGFBP-2 in the baseline sample. A method for treating a folate receptor alpha (FRA) phenotypical cancer, the method comprising: (a) measuring the level of one or more biomarkers of tumor response in a baseline biological sample from a subject, (b) administering to a subject in need thereof an amount of an antibody-drug conjugate having formula (I): Ab-(LD)p (I) wherein (i) Ab is an internalizing anti-folate receptor alpha antibody or an internalizing antigen-binding fragment thereof comprising: three heavy chain complementation determining regions (HCDRs) comprising 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 complementation determining regions (LCDRs) comprising SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2), and SEQ ID NO: 6 (LCDR3), as defined by the Kabat numbering system; or three heavy chain complementation determining regions (HCDRs) comprising 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 complementation determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQ ID NO: 12 (LCDR3), as defined by the IMGT numbering system; (ii) D is eribulin; (iii) L is a cleavable linker comprising Mal-(PEG) ₂ -Val-Cit-pAB; and (iv) p is an integer from 1 to 8, (c) measuring the level of one or more biomarkers of tumor response in a sample taken from the subject to determine a change relative to the level in the baseline sample, and (d) administering a further dose of the antibody-drug conjugate if no change in the level is detected, wherein the biomarker is one or more of IGFBP-1, FLT3L, and VEGF-D. The method of claim 19, wherein the tumor response biomarker is insulin-like growth factor binding protein 1 (IGFBP-1). The method of claim 19, wherein the tumor response biomarker is fms-related receptor tyrosine kinase 3 ligand (FLT3LG). The method of claim 19, wherein the tumor response biomarker is vascular endothelial growth factor D (VEGF-D). A method for treating a folate receptor alpha (FRA) phenotypical cancer, the method comprising: (a) measuring the level of one or more biomarkers of tumor response in a baseline biological sample from a subject, (b) administering to a subject in need thereof an amount of an antibody-drug conjugate having formula (I): Ab-(LD)p (I) wherein (i) Ab is an internalizing anti-folate receptor alpha antibody or an internalizing antigen-binding fragment thereof comprising: three heavy chain complementation determining regions (HCDRs) comprising 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 complementation determining regions (LCDRs) comprising SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2), and SEQ ID NO: 6 (LCDR3), as defined by the Kabat numbering system; or three heavy chain complementation determining regions (HCDRs) comprising 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 complementation determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQ ID NO: 12 (LCDR3), as defined by the IMGT numbering system; (ii) D is eribulin; (iii) L is a cleavable linker comprising Mal-(PEG) ₂ -Val-Cit-pAB; and (iv) p is an integer from 1 to 8, (c) measuring the level of one or more tumor-responsive biomarkers in a sample taken from the subject to determine the change relative to the level in the baseline sample, and (d) determining the efficacy of the antibody-drug conjugate based on the change in the level of the one or more tumor-responsive biomarkers, wherein the tumor-responsive biomarkers are one or more of those of claims 1-22. The method of any one of claims 1 to 23, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and a light chain comprising the amino acid sequence of SEQ ID NO: 16. The method of any one of claims 1 to 24, wherein the antibody-drug conjugate is MORAb-202. The method of any one of claims 1 to 25, wherein p is 3 to 5. The method of any one of claims 1 to 26, wherein the dose is 8 mg to 44 mg/m ² of the subject's BSA. The method of any one of claims 1 to 26, wherein the dose is 0.3 mg/kg to 1.2 mg/kg of the subject's body weight. The method of any one of claims 1 to 28, wherein the antibody-drug conjugate is administered once every three weeks. The method of any one of claims 1 to 28, wherein the antibody-drug conjugate is administered once every two weeks. The method of any one of claims 1 to 28, wherein the antibody-drug conjugate is administered once a week. The method of any one of claims 1 to 31, further comprising administering a corticosteroid. The method of any one of claims 1 to 32, wherein the antibody-drug conjugate is administered intravenously. The method of any one of claims 1 to 33, wherein the FRA-expressing cancer is selected from the group consisting of gastric cancer, ovarian cancer, serous ovarian cancer, serous high-grade ovarian cancer, clear cell ovarian cancer, platinum-resistant ovarian cancer, lung cancer, non-small cell lung cancer, metastatic non-small cell lung cancer, lung carcinoid, colorectal cancer, breast cancer, triple-negative breast cancer, hormone receptor ( HR)-positive and low HER2 breast cancer, endometrial cancer, serous endometrial cancer, peritoneal cancer, primary peritoneal cancer, fallopian tube cancer, pancreatic cancer, kidney cancer, renal cell cancer, cervical cancer, esophageal cancer, osteosarcoma, choroidal plexus carcinoma, ependymoma, ependymoblastoma, medulloblastoma, nephroblastoma, acute myeloid leukemia, and head and neck cancer. The method of claim 34, wherein the FRA-expressing cancer is ovarian cancer. The method of claim 35, wherein the ovarian cancer is platinum-resistant ovarian cancer (PROC). The method of any one of claims 34 to 36, wherein the FRA phenotype cancer is a refractory cancer. The method of claim 37, wherein the refractory cancer is refractory to targeted therapy. The method of claim 38, wherein the targeted therapy is targeted therapy against any one of the following genes or variants thereof: EGFR , ALK , BRAF , RET , MET , NTRK , and ROS1 . The method of claim 37, wherein the refractory cancer is refractory to a platinum-based therapy and an immunotherapy-based therapy, wherein the therapies are administered simultaneously or sequentially. The method of claim 40, wherein the platinum-based therapy is platinum doublet chemotherapy, and wherein the immunotherapy-based therapy is a PD-1 inhibitor or a PD-L1 inhibitor. The method of any one of claims 1 to 41, wherein the subject does not have one or more of the following at the start of treatment: interstitial lung disease (ILD) and/or pneumonitis, a history of ILD and/or pneumonitis, a clinically significant specific lung disease, a pleural effusion, a pericardial effusion, a previous lung resection, a history of chest radiation within the past 2 years, an autoimmune disorder with lung involvement, a connective tissue disorder with lung involvement, or an inflammatory disorder with lung involvement. The method of any one of claims 1 to 41, wherein the subject does not have one or more of the following: a history of more than three prior therapies for the FRA-phenotype cancer, a high neutrophil-to-lymphocyte ratio, or a serum albumin level less than 3 g/dL at the start of treatment.