RU2826228C2 - Antibody-sting agonist conjugates and use thereof in immunotherapy - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to antibody-drug conjugates containing a cyclic dinucleotide of formula IIk, which is a STING agonist, conjugated with an antibody, methods for preparing them and using them.

EFFECT: antibody-sting agonist conjugates and their use in immunotherapy are disclosed.

39 cl, 5 dwg, 4 tbl, 7 ex

Description

1. List of sequences

This application contains a Sequence Listing, which was filed electronically in ASCII format and is incorporated herein by reference in its entirety. Said ASCII file, created on July 17, 2020, is named 400160_005US_SL_ST25.txt and is 44502 bytes in size.

2. Field of technology

The present invention relates, inter alia, to the use of STING agonists in antibody-drug conjugates (ADCs) for immunotherapy; compositions comprising ADCs, methods for producing ADCs, and methods for using ADCs to treat cancer.

3. Prerequisites for the creation of an invention

The cytoplasmic DNA-sensing pathway plays an important role in the initiation and maintenance of the immune response against malignant tumors, in addition to its primary function of protecting the host from infection by DNA-containing microbes. Cytoplasmic DNA from damaged tumor cells triggers the activation of an enzyme called cyclic AMP-GMP synthase (cGAS), which synthesizes 2'3'-cyclic AMP-GMP (cGAMP). As an endogenous ligand, cGAMP binds and activates the ER adaptor protein stimulator of interferon genes (STING), leading to the release of interferons and inflammatory cytokines, the production and maturation of antigen-presenting cells, and ultimately the antitumor immune response mediated by T cells and natural killer (NK) cells. A number of STING agonists that are similar to cGAMP but have improved therapeutic properties are in development for use in cancer immunotherapy. Although these drugs show promise for the treatment of a number of solid tumors, they have obvious limitations. These compounds require intratumoral administration because they can induce a systemic cytokine response when injected at other sites. Even with intratumoral injection, an undesirable cytokine response may still occur due to rapid leakage to peripheral tissues. The short contact of these compounds with immune cells in the tumor environment also reduces their efficacy. Therefore, there is an urgent need for new therapeutic agents that can specifically deliver a STING agonist to the tumor environment and have an extended residence time.

4. Brief description of the invention

The present invention describes certain cyclic dinucleotides (CDNs) that can be conjugated to antibodies or antigen-binding fragments that target specific antigens in the microenvironment of diseased cells or tissues. For example, antibodies or antigen-binding fragments thereof can bind to cancer antigens present on cells in the tumor microenvironment, such as tumor cells or immune cells. The CDNs of the present invention exert an agonistic effect on STING, thereby stimulating the immune system. When the CDNs of the present invention are conjugated to antibodies that target antigens in a diseased cell or tissue, they provide sufficient contact time with the CDN in the microenvironment of the diseased cells or tissues, while reducing the detrimental side effects associated with large leakage of CDNs into peripheral tissues.

In one aspect, the present invention features antibody drug conjugates (ADCs) having the structure of formula I:

(Formula I) Ab-[-L-(D) _m ] _n

Where:

“D” represents a CDN (e.g., a CDN described in this application, such as having formula II);

“Ab” is an antibody or its binding fragment that binds to a target antigen;

“L” in each case independently represents a linker linking one or more D to Ab;

“m” is the number of Ds linked to a given linker; and

“n” is the number of linkers associated with Ab.

In some embodiments, the present invention features ADCs having the structure of Formula Ia:

(Formula Ia) Ab-[-LD] _n

Where:

“D” represents a CDN (e.g., a CDN described in this application, such as having formula II);

“Ab” is an antibody or its binding fragment that binds to a target antigen;

“L” in each case independently represents a linker that connects D to Ab; and

“n” is the number of D linked to Ab via a linker (L).

In another aspect, the present invention provides specific CDNs (D) that can be administered alone or as part of an ADC of formula I. These CDNs may have a structure of formula II:

Formula II

Where

R ¹ is C _1-6 alkyl, such as C _2-6 alkyl or C _2-3 alkyl, substituted with a hydroxyl, thiol, amino, C _1-6 alkylamino or -PEG-OH group;

R ³ and R ⁴ independently represent a hydrogen atom, a halogen, C _1-6 alkyl, C _2-6 alkenyl or C _2-6 alkynyl, wherein C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl may independently be optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy, C _1-6 hydroxyalkoxy, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino, C _1-6 alkylamino, di(C _1-6 alkyl)amino, oxo and azido;

R ² , R ⁵ and R ⁶ independently represent a hydrogen atom, a halogen, a hydroxyl, an azido group, an amino group, a C _1-6 alkylamino group, a di(C _1-6 alkyl)amino group, a C _1-6 alkyl, a C _1-6 alkoxy group, a C _2-6 alkenyl, a C _3-6 alkenyl-O-, a C _2-6 alkynyl or a C _3-6 alkynyl-O-, wherein the C _1-6 alkyl, C _1-6 alkoxy group, a C _2-6 alkenyl, a C _3-6 alkenyl-O-, a C _2-6 alkynyl and a C _3-6 alkynyl-O- independently may be optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy groups, C _1-6 hydroxyalkoxy groups, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino groups, C _1-6 alkylamino groups, di(C _1-6 alkyl)amino groups, oxo groups and azido groups; or R ⁶ and R ⁵ together represent =CH ₂ ; or R ⁶ and R ⁴ together form a bridge through a ring containing V ² selected from ethylene, -O-CH ₂ - and -NH-CH ₂ -;

V ¹ and V ² independently represent O, S or CH ₂ ;

BG ¹ starting from a carbon atom in the ring containing V ¹ and BG ² starting from a carbon atom in the ring containing V ² are independently -OP(O)R ^p -O-, -OP(S)R ^p -O-, -OP(O)R ^p -S-, -OP(S)R ^p -S-, -SP(O)R ^p -O-, -SP(S)R ^p -O-, -SP(O)R ^p -S-, -SP(S)R ^p -S- or -NH-SO ₂ -NH-; where

R ^p at each occurrence independently represents hydroxyl, thiol, C _1-6 alkyl, C _1-6 alkoxy, C _3-6 alkenyl-O-, C _3-6 alkynyl-O-, -PEG-OH, borano (-BH ₃ ⁻ ) or -NR'R", wherein the C _1-6 alkyl, C _1-6 alkoxy, C _3-6 alkenyl-O- and C _3-6 alkynyl-O- may independently be optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy, C _1-6 hydroxyalkoxy, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino group, C _1-6 alkylamino group, di(C _1-6 alkyl)amino group, oxo group and azido group; and

R' and R" independently represent a hydrogen atom or a C _1-6 alkyl optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy, C _1-6 hydroxyalkoxy, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino, C _1-6 alkylamino, di(C _1-6 alkyl)amino, oxo and azido; or R' and R", together attached to one nitrogen atom, form a C _3-5 heterocyclic ring;

R ^a1 , R ^b1 , R ^a2 and R ^b2 independently represent a hydrogen atom or C _1-3 alkyl; and

B ¹ and B ² are independently selected from:

And ,

Where

V ³ is O or S, in particular O;

Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ and Z ⁶ in each case independently represent CR ^z or N;

Z ^a is O (except when Z ⁵ is N) or NR'; where

R ^z in each case independently represents a hydrogen atom, a halogen, an azido group, an amino group, a C _1-6 alkylamino group, a di(C _1-6 alkyl)amino group, a C _1-6 alkyl, a C _1-6 alkoxy group, a C _2-6 alkenyl, a C _3-6 alkenyl-O-, a C _2-6 alkynyl, a C _3-6 alkynyl-O-, -NO ₂ , -CN, -C(O)C _1-6 alkyl, -CO ₂ H, -CO ₂ C _1-6 alkyl, -S(O)C _1-6 alkyl, -S(O) ₂ C _1-6 alkyl, -C(O)NR', -C(O)NR'R”, -SO ₂ NR'R”, -OC(O)C _1-6 alkyl, -NR'C(O)C _1-6 alkyl, -N(R')C(O)NR'R”, -N(R')SO ₂ NR'R”, -N(R)SO ₂ C _1-6 alkyl or -OC(O)NR'R”, where

C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyl, C _3-6 alkenyl-O-, C _2-6 alkynyl and C _3-6 alkynyl-O- are each independently optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy, C _1-6 hydroxyalkoxy, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino, C _1-6 alkylamino, di(C _1-6 alkyl)amino, oxo and azido; and

R' and R" in each case independently represent a hydrogen atom or a C _1-6 alkyl optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy, C _1-6 hydroxyalkoxy, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino, C _1-6 alkylamino, di(C _1-6 alkyl)amino, oxo and azido; or R' and R", attached to the same nitrogen atom, together form a C _3-5 heterocyclic ring;

or a pharmaceutically acceptable salt thereof.

The present invention describes methods for producing an ADC of formula I by conjugating a CDN of formula II to an antibody via a linker. The CDN of formula II can be conjugated to the antibody via a cleavable or non-cleavable linker. In particular embodiments, the CDN is released in tumor cells, cancer immune cells, or in the tumor microenvironment by cleavage of the linker.

In the ADC of formula I, wherein the CDN (D) has formula II, the CDN may be covalently bonded to the linker (L) via a hydroxyl, thiol, amino, C _1-6 alkylamino, or -PEG-OH group at the R ¹ position of the CDN of formula II.

In one embodiment of the present invention, the CDN of formula II has formula IIe:

Formula IIe

where R ¹ , R ⁵ and R ⁶ ; R ^P ; and B ¹ and B ² have the meanings specified above for formula II;

or a pharmaceutically acceptable salt thereof.

In one embodiment of the present invention, the CDN of formula II has formula IIk:

Formula IIk

Where

W, X, Y and Z independently represent CH or N; and

R ^P in each case independently has the meaning specified above for formula II;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides a composition comprising CDN and a base, wherein the CDN is a compound of formula II, such as formula IIe or IIk depicted above, or formula IIn or IIo depicted below. In some of these embodiments, the composition consists of CDN and a base. In some embodiments, the base is an amine base, such as pyridine. In some of these embodiments, the composition is anhydrous.

In some embodiments, the present invention provides a composition comprising a CDN and a linker or cross-linking agent, or a linker and a cross-linking agent together, wherein the CDN is a compound of formula II, such as formula IIe or IIk depicted above, or formula IIn or IIo depicted below, and wherein the cross-linking agent provides coupling of the CDN to the linker, such as by generating an activated ester on the linker. In some embodiments, the composition further comprises an aprotic polar solvent. In some embodiments, the composition is anhydrous.

In some embodiments, the present invention features a compound that is a cyclic dinucleotide (CDN) linked to a linker (L) of the formula L-CDN. In some embodiments, the CDN is linked to the linker L via a thioether, amide, ester, carbamate, carbonate, urea, disulfide, or ether group, especially via an amide, carbamate, or disulfide group. In some embodiments, the CDN has a formula II, such as formula IIe or IIk depicted above, or formula IIm, IIn, or IIo depicted below, and the CDN is linked to L via a thiol group, an amino group, or a C _1-6 alkylamino group in R ¹ of formula II, and L comprises a site capable of binding to a complementary site on an antibody.

In one embodiment, the ADC of Formula I has a structure corresponding to Formula III:

Formula III

where the variables W, X, Y, Z, R ^P and n have the meanings specified above for formulas I and II.

In one embodiment, the ADC of Formula I has a structure corresponding to Formula IV:

Formula IV

where the variables W, X, Y, Z, R ^P and n have the meanings specified above for formulas I and II.

In one embodiment, the ADC of Formula I or III is derived from a CDN(D) having the following structure (CDN-A):

or a pharmaceutically acceptable salt thereof. It should be understood that the term "derived from" means that the amine (-NH ₂ ) functional group at the R ¹ position of CDN is covalently bonded to the corresponding position in the linker. For example, in some embodiments, the amine group is covalently bonded to a carbonyl moiety in the linker, thereby forming an amide or carbamate bond.

In another embodiment, the ADC of formula I or IV is derived from CDN(D) having the following structure (CDN-B):

or a pharmaceutically acceptable salt thereof. It should be understood that the term "derived from" means that the thiol (-SH) functional group at the R ¹ position of CDN is covalently bonded to the corresponding position in the linker. For example, in some embodiments, the thiol group of CDN is covalently bonded to a thiol group in the linker, thereby forming a disulfide bond.

In another embodiment, the ADC of formula I or III is derived from CDN(D) having the following structure:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the ADC of formula I or III is derived from CDN(D) having the following structure:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the ADC of formula I or III is derived from CDN(D) having the following structure:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the antibody or antigen-binding fragment of an ADC of Formula I, III, or IV targets a specific antigen that is expressed on tumor cells or immune cells in the tumor microenvironment. In particular embodiments, the antibody or antigen-binding fragment of an ADC of Formula I, III, or IV targets the PD-L1 receptor. In other particular embodiments, the antibody or antigen-binding fragment thereof specifically binds to a tumor antigen relevant to cancer that is a growth factor receptor (GFR). In some embodiments, the tumor antigen relevant to cancer is a GFR from the EGFR/ErbB/HER family.

In one aspect, the present invention describes methods for eliciting an immune response in a subject (e.g., a human) by administering a therapeutically effective amount of an ADC of Formula I, III, or IV. For example, an ADC of Formula I, III, or IV can be used to induce interferon-β (IFNβ) production in a human.

An ADC of Formula I, III, or IV may be administered in combination with one or more additional therapeutic agents. The additional therapeutic agent(s) may be administered prior to, concurrently with, or subsequent to the administration of the additional therapeutic agent(s). In a particular embodiment, an ADC of Formula I, III, or IV may be administered in combination with an immune checkpoint inhibitor. For example, an ADC of Formula I, III, or IV may be administered with an inhibitor of PD-1, PD-L1, or CTLA-4, or a combination thereof.

In another embodiment, the ADC of Formula I, III, or IV may be administered with free CDN that is not conjugated to the antibody or antigen-binding fragment of Formula I. In such cases, the free CDN may be the same as or different from the CDN conjugated to the antibody in the ADC of Formula I, III, or IV.

5. Brief description of drawings

Fig. 1A-1D show the structural characteristics and activity of ADCI. Fig. 1A shows the chemical structure of ADCI. Fig. 1B and 1C show the IFN-stimulating activity potential in a luciferase reporter gene assay using mouse RAWLucia ISG cells and human THP1-Lucia ISG cells, respectively. Fig. 1D shows tumor development in C57BL6 mice bearing B16-F10 tumors under the indicated treatments. The anti-PDL1 antibody used as a reference was Ab-A1.

Fig. 2A-2C show the structural characteristics and activity of ADCII. Fig. 2A depicts the chemical structure of ADCII. Fig. 2B and 2C show the IFN-stimulating activity potential in a luciferase reporter gene assay using mouse RAWLucia ISG cells and human THP1-Lucia ISG cells, respectively.

Fig. 3A-3B show the structural characteristics and activity of ADCIII. Fig. 3A shows the chemical structure of ADCIII. Fig. 3B shows the IFN-stimulating activity potential in a luciferase reporter gene assay using mouse RAWLucia ISG cells. The reference anti-EGFR antibody is Ab-B1.

Fig. 4A-4F show the structural characteristics and activity of ADCIV. Fig. 4A shows the chemical structure of ADCIV. Fig. 4B shows the IFN-stimulating activity potential in a luciferase reporter gene assay using mouse RAWLucia ISG cells (fold change compared to cells stimulated with PBS). Fig. 4C and 4D show tumor development and survival, respectively, in EGFR-expressing C57BL6 mice bearing B16F10 tumors under the indicated treatments. Fig. 4E and 4F show tumor development and survival, respectively, in EGFR-expressing C57BL6 mice bearing B16F10 tumors under the indicated treatments. The reference anti-EGFR antibody was Ab-B1, and the reference anti-PDL1 antibody was Ab-A3.

Fig. 5A-5B show the structural characteristics and activity of ADCV. Fig. 5A shows the chemical structure of ADCV. Fig. 5B shows the IFN-stimulating activity potential in a luciferase reporter gene assay using mouse RAWLucia ISG cells. The anti-PDL1 antibody used as a reference was Ab-A3.

Fig. 6A-6F show the structural characteristics and activity of ADCVI. Fig. 6A shows the chemical structure of ADCVI. Fig. 6B shows the IFN-stimulating activity potential in a luciferase reporter gene assay using mouse RAWLucia ISG cells. Fig. 6C and 6D show tumor development and survival, respectively, in EGFR-expressing C57BL6 mice bearing B16F10 tumors under the indicated treatments. The reference anti-PDL1 antibody was Ab-A2. Fig. 6E and 6F show tumor development and survival, respectively, in EGFR-expressing C57BL6 mice bearing B16F10 tumors under the indicated treatments.

Figures 7A-7F show the structural characteristics and activity of ADCVII. Figure 7A depicts the chemical structure of ADCVII. Figure 7B shows the IFN-stimulating activity potential in a luciferase reporter gene assay using mouse RAWLucia ISG cells (fold change compared to cells stimulated with PBS). Figures 7C and 7D show tumor development and survival, respectively, in EGFR-expressing C57BL6 mice bearing B16F10 tumors under the indicated treatments. I.p. (intraperitoneal) injections were performed on days 7, 11, and 15. Figures 7E and 7F show tumor development and survival, respectively, in EGFR-expressing C57BL6 mice bearing B16F10 tumors under the indicated treatments. injections were given on days 7, 11, and 15. The anti-EGFR antibody used as a reference was Ab-B2, and the anti-PDL1 antibody used as a reference was Ab-A3.

Fig. 8A-8D show the structural characteristics and activity of ADCVIII. Fig. 8A shows the chemical structure of ADCVIII. Fig. 8B shows the IFN-stimulating activity potential in a luciferase reporter gene assay using murine RAWLucia ISG cells. Fig. 8C and 8D show tumor development and survival, respectively, in EGFR-expressing C57BL6 mice bearing B16F10 tumors under the indicated treatments. I.p. injections were performed on days 7, 11, and 15. The anti-PDL1 antibody used as a reference was Ab-A3.

Fig. 9A-9D show the structural characteristics and activity of ADCIX. Fig. 9A depicts the chemical structure of ADCIX. Fig. 9B shows the IFN-stimulating activity potential in a luciferase reporter gene assay using murine RAWLucia ISG cells. Fig. 9C and 9D show tumor development and survival, respectively, in EGFR-expressing C57BL6 mice bearing B16F10 tumors under the indicated treatments. I.p. injections were performed on days 6, 9, and 13. The anti-PDL1 antibody used as a reference was Ab-A3.

Figs. 10A-10E show the structural characteristics and activity of ADCX. Fig. 10A shows the chemical structure of ADCX. Fig. 10B shows the IFN-stimulating activity potential in a luciferase reporter gene assay using mouse RAWLucia ISG cells. Figs. 10C and 10D show tumor development and survival, respectively, in EGFR-expressing C57BL6 mice bearing B16F10 tumors with the indicated treatments. I.p. injections were given on days 6, 10, and 13. The reference anti-HER2 antibody was Ab-C1 (trastuzumab), and the reference anti-PDL1 antibody was Ab-A3. Fig. 10E shows tumor development in EGFR-expressing C57BL6 mice bearing B16F10 tumors with the indicated treatments. I.p. and intratumoral (i.t.) injections were performed on days 7 and 11.

6. Detailed description of the invention

The present invention describes antibody-drug conjugates (ADCs), each comprising an antibody, one or more cyclic dinucleotides (CDNs), and one or more linkers that connect the one or more CDNs to the antibody. The ADCs of the present invention are capable of exerting an agonistic effect and/or binding to STING and eliciting an immune response.

6.1 Antibody-Drug Conjugates (ADCs)

In some embodiments, the ADCs of the present invention have a structure of Formula I:

(Formula I) Ab-[-L-(D) _m ] _n

Where:

“D” represents a CDN (e.g., a CDN described in this application, such as having formula II);

“Ab” is an antibody or its binding fragment that binds to a target antigen;

“L” in each case independently represents a linker linking one or more D to Ab;

“m” is the number of Ds linked to a given linker; and

“n” is the number of linkers associated with Ab.

In some embodiments of Formula I, m is an integer selected from the range of 1 to 10, including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, m is in the range of 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10, such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, or 1 to 6. In other embodiments, Formula I describes an ADC as a mixture of multiple ADCs in which m is in a certain range of values, such as m is in the range of 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10, such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, or 1 to 6. In some embodiments, Formula I describes an ADC as a mixture of several ADCs, such as? that greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the ADC in the mixture have an m value of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, Formula I describes an ADC as a mixture of multiple ADCs such that greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the ADC in the mixture have an m value in the range of 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10, such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, or from 1 to 6.

In other embodiments, Formula I describes an ADC as a mixture of several ADCs, and m is replaced by “m _cp ”, which is an average value of m for the mixture, i.e., the average number of CDNs linked to a given linker (L) in the mixture, which can be calculated by dividing the total number of CDNs linked to the antibody by the total number of CDN-containing linkers (L) in the mixture. In such embodiments, m _cp is an integer or a non-integer number in the range of 1 to 10, such as in the range of 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, or 1 to 9, such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, or 1 to 6.

In some embodiments of Formula I, n is an integer selected from the range of 1 to 20, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, n is in the range of 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, or 1 to 20, such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, or 1 to 8. In other embodiments, Formula I describes an ADC as a mixture of multiple ADCs in which n is in a certain range of values, such as n is in the range of 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, or 1 to 20, such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10. In some embodiments, Formula I describes an ADC as a mixture of multiple ADCs such that greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the ADCs in the mixture have an n value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, Formula I describes an ADC as a mixture of multiple ADCs such that? that more than 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the ADCs in this mixture have an n value ranging from 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, from 1 to 12, from 1 to 13, from 1 to 14, from 1 to 15, from 1 to 16, from 1 to 17, from 1 to 18, from 1 to 19, or from 1 to 20, for example from 1 to 2, from 1 to 3, from 1 to 4, from 1 to 5, from 1 to 6, from 1 to 7, from 1 to 8, 1 to 9, or 1 to 10.

In other embodiments, Formula I describes an ADC as a mixture of several ADCs, and n is replaced by “n _cp ,” which is the average value of n for the mixture, i.e., the average number of linkers (L) linked to a given antibody (Ab) in the mixture, which can be calculated by dividing the total number of linkers (L) linked to the antibody by the total number of linker-containing antibodies (Ab) in the mixture. In such embodiments, n _cp is an integer or non-integer in the range from 1 to 20, for example in the range from 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 1 4, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, or 1 to 20, for example 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10.

In some embodiments, m is 1, in which case the ADC has a 1:1 ratio of linker to CDN and can be represented by formula Ia:

(Formula Ia) Ab-[-LD] _n

Where:

“D” represents a CDN (e.g., a CDN described in this application, such as having formula II);

“Ab” is an antibody or its binding fragment that binds to a target antigen;

“L” in each instance independently represents a linker linking one or more D to Ab; and

“n” is the number of D linked to Ab via a linker (L).

In some embodiments of Formula Ia, n is an integer selected from the range of 1 to 20, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, n is in the range of 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, or 1 to 20, such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, or 1 to 8. In other embodiments, Formula Ia describes an ADC as a mixture of multiple ADCs in which n is in a certain range of values, such as n is in the range of 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, or 1 to 20, such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, or 1 to 8. In some embodiments, Formula Ia describes an ADC as a mixture of multiple ADCs such that greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the ADCs in the mixture have an n value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, Formula Ia describes an ADC as a mixture of multiple ADCs such that greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% ADCs in this mixture have n values ranging from 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, or 1 to 20, such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, or 1 to 8.

In other embodiments, Formula Ia describes an ADC as a mixture of several ADCs, and n is replaced by “n _cp ,” which is the average value of n for the mixture, i.e., the average number of linkers (L) linked to a given antibody (Ab) in the mixture, which can be calculated by dividing the total number of linkers (L) linked to the antibody by the total number of linker-containing antibodies (Ab) in the mixture. In such embodiments, n _cp is an integer or non-integer in the range from 1 to 20, for example in the range from 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 1 4, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, or 1 to 20, for example 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10.

The ADCs of the present invention are “modular” in nature, i.e., each of them comprises modular components Ab, L, and D. Various specific non-limiting embodiments and examples of these modular components are described in the present invention. The modules of all specific embodiments described in the present invention can be combined with each other as if each individual combination was expressly described separately.

One of skill in the art will also appreciate that the various ADCs described herein may be in the form of salts and, in certain particular embodiments, in the form of pharmaceutically acceptable salts.

6.2. Cyclic dinucleotides (CDN)

The CDNs used in the present invention comprise two nucleosides linked by two bridging groups. In some embodiments, the CDNs are 2'3'-CDNs, i.e., each nucleoside comprises a cyclic 5-carbon sugar (pentose), wherein the first nucleoside is linked at the 2' position of its sugar to the 5' position of the sugar of a second nucleoside, such as via a bridging group, to form a 2'-5' linkage, and the second nucleoside is linked at the 3' position of its sugar to the 5' position of the sugar of the first nucleoside, such as via a bridging group, to form a 3'-5' linkage. Examples of suitable 2'3'-CDNs include 2'3'-cGAMP and analogs or derivatives thereof, including pharmaceutically acceptable salts. The CDNs of formula II below are 2'3'-CDNs.

In other embodiments, the CDNs are 3'3'-CDNs, wherein the first nucleotide is linked to the second nucleotide, such as via a bridging group, in a 3'-5' linkage similar to that described above, and the second nucleotide is linked to the first nucleotide, such as via a bridging group, also in a 3'-5' linkage similar to that described above. Examples of suitable 3'3'-CDNs include 3'3'-cGAMP and analogs or derivatives thereof, including pharmaceutically acceptable salts.

In other embodiments, the CDNs are 2'2'-CDNs, wherein the first nucleotide is linked to the second nucleotide, such as via a bridging group, in a 2'-5' linkage similar to that described above, and the second nucleotide is linked to the first nucleotide, such as via a bridging group, also in a 2'-5' linkage similar to that described above. Examples of suitable 2'2'-CDNs include 2'2'-cGAMP and analogs or derivatives thereof, including pharmaceutically acceptable salts.

In other embodiments, the CDNs are 3'2'-CDNs, wherein the first nucleotide is linked to the second nucleotide, such as via a bridging group, in a 3'-5' linkage, similar to that described above, and the second nucleotide is linked to the first nucleotide, such as via a bridging group, in a 2'-5' linkage, similar to that described above. Examples of suitable 3'2'-CDNs include 3'2'-cGAMP and analogs or derivatives thereof, including pharmaceutically acceptable salts.

6.2.1. Nucleosides

In some cases, each nucleoside in a CDN contains a nucleobase, which independently of the others may be a pyrimidine base or a purine base. For example, each nucleobase may be a canonical nucleobase (such as adenine, guanine, thymine, uracil, or cytosine) or a non-canonical, modified, non-natural nucleobase (such as xanthine, hypoxanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine, 1-methylcytosine, 2,6-diaminopurine, 6,8-diaminopurine, 2-aminoimidazo[1,2a][1,3,5]triazin-4(1H)-one, 6-amino-5-nitropyridin-2-one, iso-guanine, isocytosine, 5-(2,4-diaminopyrimidine), 4-thiouracil, pseudouracil, etc.). Suitable nucleobases include those described below for variables B ¹ and B ² .

In some cases, the CDN comprises two nucleosides that comprise two pyrimidine bases or two purine bases, or one pyrimidine base and one purine base. In some embodiments, the two nucleosides comprise two purine bases, such as an adenine and a guanine, two adenines or two guanines, in particular adenine and guanine or two adenines. It should be understood that the indication of a particular nucleobase or other chemical structure (e.g., "adenine") indicates that it encompasses both the canonical nucleobase and its modified versions.

In some embodiments, the CDN comprises two nucleosides, each of which comprises a cyclic 5-carbon sugar (pentose), such as D- or L-ribose or deoxyribose, D- or L-arabinose, D- or L-lyxose, D- or L-xylose, or modified forms thereof. In some cases, one of the pentoses in the CDN (e.g., ribose) is connected to a linker (L) at the 3' position of the sugar, such as through a C _1-6 alkyl group (e.g., a C _2-6 alkyl group) at the 3' position, and the C _1-6 alkyl group is optionally substituted with a hydroxyl, thiol, amino, C _1-6 alkylamino group, or -PEG-OH group; a hydroxyl, thiol, amino, or C _1-6 alkylamino group; a thiol, amino, or C _1-6 alkylamino group; or a thiol or amino group. In other embodiments, one of the pentoses in CDN (e.g., ribose) is connected to the linker (L) at the 2' position of the sugar, such as through a C _1-6 alkyl group (e.g., C _2-6 alkyl group) at the 2' position, and the C _1-6 alkyl group is optionally substituted with a hydroxyl, thiol, amino, C _1-6 alkylamino, or -PEG-OH group; a hydroxyl, thiol, amino, or C _1-6 alkylamino group; a thiol, amino, or C _1-6 alkylamino group; or a thiol or amino group. In some embodiments, the linker (L) is attached to the CDN by replacing a proton in a hydroxyl, thiol, amino, C _1-6 alkylamino, or -PEG-OH group.

The following descriptions of the group at the 3' position of the pentose also apply to the group at the 2' position of the pentose.

In some cases, the group at the 3' position of the pentose (e.g., ribose) is a _C1-6 alkyl group, such as a _C2-6 alkyl group substituted with hydroxyl, such as -ethylene-OH, -propylene-OH, -butylene-OH, or -pentylene-OH. In some embodiments, the linker (L) is attached by replacing a proton in a hydroxyl group in one of the above groups.

In some cases, the group at the 3' position of the pentose (e.g., ribose) is a _C1-6 alkyl group, such as a _C2-6 alkyl group substituted with a thiol group, such as -ethylene-SH, -propylene-SH, -butylene-SH, or -pentylene-SH. In some embodiments, the linker (L) is attached by replacing a proton in the thiol group with one of the above groups.

In some cases, the group at the 3' position of the pentose (e.g., ribose) is a _C1-6 alkyl group, such as a _C2-6 alkyl group substituted with an amino group, such as -ethylene- _NH2 , -propylene- _NH2 , -butylene- _NH2 , or -pentylene- _NH2 . In some embodiments, the linker (L) is attached by replacing a proton in the amino group with one of the above groups.

In some embodiments, the group at the 3' position of the pentose (e.g., ribose) is a _C1-6 alkyl group, such as a _C2-6 alkyl group substituted with a _C1-6 alkylamino group, such as -ethylene-N( _C1-6 alkyl)H, -propylene-N( _C1-6 alkyl)H, -butylene-N( _C1-6 alkyl)H, or -pentylene-N( _C1-6 alkyl)H. In some embodiments, the linker (L) is attached by replacing a proton on the _C1-6 alkylamino group with one of the above groups.

In some embodiments, the group at the 3' position of the pentose (e.g., ribose) is a _C1-6 alkyl group, such as a _C2-6 alkyl group substituted with a -PEG-OH group, such as -ethylene-PEG-OH, -propylene-PEG-OH, -butylene-PEG-OH, or -pentylene-PEG-OH. In some embodiments, the linker (L) is attached by replacing a proton at the terminal hydroxyl of the -PEG-OH group. It should be understood that “PEG” means a polymer of polyethylene glycol, polyethylene oxide or polyoxyethylene, contains repeating units -(O-CH ₂ -CH ₂ )x- and has an average molecular weight of from 200 to 10,000 g/mol, such as from 200, 400, 800, 1000, 2000 or 4000 to 5000, 6000 8000 or 10,000 g/mol, including ranges from 400 to 8000 g/mol, from 400 to 2000 g/mol, from 5000 to 10,000 g/mol, from 1000 to 4000 g/mol, from 1000 to 6000 g/mol or from 2000 to 6000 g/mol, including values less than 4000, 5000 or 6000 g/mol.

In some cases, the group at the 3' position of the pentose (e.g., ribose) is a _C2-3 alkyl group substituted with a hydroxyl group, a thiol group, an amino group, a _C1-6 alkylamino group, or a -PEG-OH group, such as -ethylene-OH, -propylene-OH, -ethylene-SH, -propylene-SH, -ethylene- _NH2 , -propylene- _NH2 , -ethylene-N( _C1-6 alkyl)H, -propylene-N( _C1-6 alkyl)H, -ethylene-PEG-OH, or -propylene-PEG-OH. In some embodiments, the linker (L) is attached by replacing a proton in a hydroxyl, thiol, amino group, or a _C1-6 alkylamino group or a -PEG-OH group with one of the above groups.

In some cases, the group at the 3'-position of the pentose (e.g., ribose) is a _C2 alkyl group (ethyl group) substituted with a hydroxyl group, a thiol group, an amino group, a _C1-6 alkylamino group, or a -PEG-OH group, such as -ethylene-OH, -ethylene-SH, -ethylene- _NH2 , -ethylene-N( _C1-6 alkyl)H, or -ethylene-PEG-OH. In some embodiments, the linker (L) is attached by replacing a proton in a hydroxyl, thiol, amino group, or a _C1-6 alkylamino group or a -PEG-OH group with one of the above groups.

In some cases, the group at the 3'-position of the pentose (e.g., ribose) is a _C3 alkyl group (propyl group) substituted with a hydroxyl group, thiol, amino group, _C1-6 alkylamino group, or -PEG-OH group, such as -propylene-OH, -propylene-SH, -propylene- _NH2 , -propylene-N( _C1-6 alkyl)H, or -propylene-PEG-OH. In some embodiments, the linker (L) is attached by replacing a proton in a hydroxyl, thiol, amino group, _C1-6 alkylamino group, or -PEG-OH group with one of the above groups.

In some embodiments, one of the pentoses in the CDN (e.g., ribose) is connected to the linker (L) at the 3' position of the sugar via a substituted methyl group at the 3' position, wherein the methyl group is substituted with, for example, a hydroxyl, thiol, amino, _C1-6 alkylamino, or -PEG-OH group; a hydroxyl, thiol, amino, or _C1-6 alkylamino group; a thiol, amino, or _C1-6 alkylamino group; a thiol or amino group; or a thiol group. In some embodiments, the linker (L) is attached to the CDN by replacing a proton in the hydroxyl, thiol, amino, _C1-6 alkylamino, or -PEG-OH group.

In some embodiments, the CDN comprises two nucleosides, each comprising a ribose, wherein one of the riboses is attached to a linker (L) through a _C2-6 alkyl group (such as a _C2 alkyl, _C3 alkyl, or _C2-3 alkyl group) at the 3' position of the ribose ring, wherein the _C2-6 alkyl group is optionally substituted with a hydroxyl, thiol, amino, _C1-6 alkylamino, or -PEG-OH group; a hydroxyl, thiol, amino, or _C1-6 alkylamino group; a thiol, amino, or _C1-6 alkylamino group; or a thiol or amino group; where the linker (L) is attached to the CDN by replacing a proton in a hydroxyl, thiol, amino, C _1-6 alkylamino, or -PEG-OH group.

In some embodiments, the CDN comprises two nucleosides, each comprising a ribose, wherein one of the riboses is attached to a linker (L) via a substituted ethyl group at the 3' position of the ribose ring, wherein the ethyl group is substituted with, for example, a hydroxyl, thiol, amino group, a C _1-6 alkylamino group, or a -PEG-OH group; a hydroxyl, thiol, amino group, or a C _1-6 alkylamino group; a thiol, amino group, or a C _1-6 alkylamino group; or a thiol or amino group; wherein the linker (L) is attached to the CDN by replacing a proton in the hydroxyl, thiol, amino group, a C _1-6 alkylamino group, or a -PEG-OH group.

In some embodiments, the CDN comprises two nucleosides, each comprising a ribose, wherein one of the riboses is connected to a linker (L) via a substituted ethyl group at the 3' position of the ribose ring selected from _{-CH2CH2 -} OH, _{-CH2CH2 -} SH and _-CH2CH2 - _NH2 , in particular from _{-CH2CH2 -} SH and _{-CH2CH2 - NH2} ; wherein the linker (L) is attached to the CDN by replacing _a proton in a hydroxyl, thiol or amino group.

6.2.2. Bridge groups

As a consequence of their cyclic structure, CDNs include two bridging groups that connect the nucleosides described above to form the CDN macrocycle. In some embodiments, the bridging groups independently include 2 to 5 atoms in the bridge between the two nucleoside sugars, such as 3 atoms. For example, -O-P(=O)(OH)-O- can be a bridging group, where it is understood that the sugars are connected at the terminal oxygen atoms and the bridge contains three atoms. The bridging groups can independently include only heteroatoms in the bridge, heteroatoms and carbon atoms in the bridge, or only carbon atoms in the bridge.

In some cases, the bridging groups are divalent phosphate or thiophosphate groups or modified versions thereof, such as -OP(=O)(OH)-O- or -OP(=O)(SH)-O-. For example, two bridging groups can be independently selected from -OP(O)R ^P -O-, -OP(S)R ^P -O-, -OP(O)R ^P -S-, -OP(S)R ^P -S-, -SP(O)R ^P -O-, -SP(S)R ^P -O-, -SP(O)R ^P -S- or -SP(S)R ^P -S-, wherein R ^P has the meaning given below. In some embodiments, R ^P in the bridging groups described above is independently selected from hydroxyl or thiol at each occurrence.

It should be understood that when both bridging groups are phosphate groups or modified versions thereof, each bridging group in combination with the nucleoside described above is a nucleotide, and both sets of bridging groups and nucleosides yield a cyclic dinucleotide. However, the CDNs of the present invention are not necessarily limited to bridging groups that are phosphate groups.

6.2.3. Private options of the CDN

The present invention describes CDNs (D), which can be administered as such or as part of an ADC of formula I. In some cases, the CDN has a structure corresponding to formula II, shown below. It should be understood that reference to formula I also includes reference to subformula Ia. Likewise, reference to formula II also includes reference to its subformulas, such as Formula IIa, IIb, etc. Formula II has the structure:

Formula II

Where

R ¹ is C _1-6 alkyl, such as C _2-6 alkyl or C _2-3 alkyl, substituted with a hydroxyl, thiol, amino, C _1-6 alkylamino or -PEG-OH group;

R ³ and R ⁴ independently represent a hydrogen atom, a halogen, C _1-6 alkyl, C _2-6 alkenyl or C _2-6 alkynyl, wherein C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl may independently be optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy, C _1-6 hydroxyalkoxy, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino, C _1-6 alkylamino, di(C _1-6 alkyl)amino, oxo and azido;

R ² , R ⁵ and R ⁶ independently represent a hydrogen atom, a halogen, a hydroxyl, an azido group, an amino group, a C _1-6 alkylamino group, a di(C _1-6 alkyl)amino group, a C _1-6 alkyl, a C _1-6 alkoxy group, a C _2-6 alkenyl, a C _3-6 alkenyl-O-, a C _2-6 alkynyl or a C _3-6 alkynyl-O-, wherein the C _1-6 alkyl, C _1-6 alkoxy group, a C _2-6 alkenyl, a C _3-6 alkenyl-O-, a C _2-6 alkynyl and a C _3-6 alkynyl-O- independently may be optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy groups, C _1-6 hydroxyalkoxy groups, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino groups, C _1-6 alkylamino groups, di(C _1-6 alkyl)amino groups, oxo groups and azido groups; or R ⁶ and R ⁵ together represent =CH ₂ ; or R ⁶ and R ⁴ together form a bridge through a ring containing V ² selected from ethylene, -O-CH ₂ - and -NH-CH ₂ -;

V ¹ and V ² independently represent O, S or CH ₂ ;

BG ¹ starting from a carbon atom in the ring containing V ¹ and BG ² starting from a carbon atom in the ring containing V ² are independently -OP(O)R ^p -O-, -OP(S)R ^p -O-, -OP(O)R ^p -S-, -OP(S)R ^p -S-, -SP(O)R ^p -O-, -SP(S)R ^p -O-, -SP(O)R ^p -S-, -SP(S)R ^p -S-, -NH-P(O)R ^p -O-, -OP(O)R ^p -NH-, -NH-P(S)R ^p -O-, -OP(S)R ^p -NH- or -NH-SO ₂ -NH-; where

R ^p at each occurrence independently represents hydroxyl, thiol, C _1-6 alkyl, C _1-6 alkoxy, C _3-6 alkenyl-O-, C _3-6 alkynyl-O-, -PEG-OH, borano (-BH ₃ ⁻ ), or -NR'R", wherein the C _1-6 alkyl, C _1-6 alkoxy, C _3-6 alkenyl-O- and C _3-6 alkynyl-O- may independently be optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy, C _1-6 hydroxyalkoxy, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino group, C _1-6 alkylamino group, di(C _1-6 alkyl)amino group, oxo group and azido group; and

R' and R" independently represent a hydrogen atom or a C _1-6 alkyl optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy, C _1-6 hydroxyalkoxy, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino, C _1-6 alkylamino, di(C _1-6 alkyl)amino, oxo and azido; or R' and R", attached to the same nitrogen atom, together form a C _3-5 heterocyclic ring;

R ^a1 , R ^b1 , R ^a2 and R ^b2 independently represent a hydrogen atom or C _1-3 alkyl; and

B ¹ and B ² are independently selected from:

And , Where

V ³ is O or S, in particular O;

Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ and Z ⁶ in each case independently represent CR ^z or N;

Z ^a is O (except when Z ⁵ is N) or NR'; where

R ^z in each case independently represents a hydrogen atom, a halogen, an azido group, an amino group, a C _1-6 alkylamino group, a di(C _1-6 alkyl)amino group, a C _1-6 alkyl, a C _1-6 alkoxy group, a C _2-6 alkenyl, a C _3-6 alkenyl-O-, a C _2-6 alkynyl, a C _3-6 alkynyl-O-, -NO ₂ , -CN, -C(O)C _1-6 alkyl, -CO ₂ H, -CO ₂ C _1-6 alkyl, -S(O)C _1-6 alkyl, -S(O) ₂ C _1-6 alkyl, -C(O)NR', -C(O)NR'R”, -SO ₂ NR'R”, -OC(O)C _1-6 alkyl, -NR'C(O)C _1-6 alkyl, -N(R')C(O)NR'R”, -N(R')SO ₂ NR'R”, -N(R)SO ₂ C _1-6 alkyl or -OC(O)NR'R”, where

C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyl, C _3-6 alkenyl-O-, C _2-6 alkynyl and C _3-6 alkynyl-O- are each independently optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy, C _1-6 hydroxyalkoxy, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino, C _1-6 alkylamino, di(C _1-6 alkyl)amino, oxo and azido; and

R' and R" in each case independently represent a hydrogen atom or a C _1-6 alkyl optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy, C _1-6 hydroxyalkoxy, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino, C _1-6 alkylamino, di(C _1-6 alkyl)amino, oxo and azido; or R' and R", attached to the same nitrogen atom, together form a C _3-5 heterocyclic ring;

or a pharmaceutically acceptable salt thereof.

In some cases, when a CDN (D) of Formula II is covalently linked to a linker (L) in an ADC of Formula I or Ia, the CDN is covalently linked to the linker through a hydroxyl, thiol, amino group, C _1-6 alkylamino group, or -PEG-OH group at the R ¹ position.

In some embodiments, R ¹ is C _2-6 alkyl substituted with a hydroxyl, thiol, amino, C _1-6 alkylamino, or -PEG-OH group. In some embodiments, R ¹ is C _2-6 alkyl substituted with a hydroxyl, thiol, amino, or C _1-6 alkylamino group. In some embodiments, R ¹ is C _2-6 alkyl substituted with a thiol, amino, or C _1-6 alkylamino group. In some embodiments, R ¹ is C _2-6 alkyl substituted with a thiol or amino group. In some embodiments, R ¹ is C _2-6 alkyl substituted with a thiol group. In some embodiments, R ¹ is C _2-6 alkyl substituted with an amino group. In any of these embodiments, R ¹ can be C _2-4 alkyl, such as C _2-3 alkyl substituted with hydroxy, thiol, amino, or C _1-6 alkylamino, such as ethyl substituted with hydroxy, thiol, amino, or C _1-6 alkylamino.

In some embodiments, R ¹ is C _2-4 alkyl substituted with a hydroxyl group. In some such embodiments, R ¹ is C ₂ alkyl substituted with a hydroxyl group. In other such embodiments, R ¹ is C ₃ alkyl substituted with a hydroxyl group. In other such embodiments, R ¹ is C ₄ alkyl substituted with a hydroxyl group.

In some embodiments, R ¹ is C _2-4 alkyl substituted with an amino group (-NH ₂ ). In some such embodiments, R ¹ is C ₂ alkyl substituted with an amino group. In other such embodiments, R ¹ is C ₃ alkyl substituted with an amino group. In other such embodiments, R ¹ is C ₄ alkyl substituted with an amino group.

In some embodiments, R ¹ is C _2-4 alkyl substituted with a thiol (-SH) group. In some such embodiments, R ¹ is C ₂ alkyl substituted with a thiol group. In other such embodiments, R ¹ is C ₃ alkyl substituted with a thiol group. In other such embodiments, R ¹ is C ₄ alkyl substituted with a thiol group.

In some embodiments, R ¹ is C _2-4 alkyl substituted with a C _1-6 alkylamino group. In some such embodiments, R ¹ is C ₂ alkyl substituted with a methylamino group. In other such embodiments, R ¹ is C ₃ alkyl substituted with a methylamino group. In other such embodiments, R ¹ is C ₃ alkyl substituted with a methylamino group.

In some embodiments, R ³ and R ⁴ are independently hydrogen, halogen, C _1-6 alkyl, C _2-6 alkenyl, or C _2-6 alkynyl, wherein the C _1-6 alkyl, C _2-6 alkenyl, and C _2-6 alkynyl are unsubstituted. In some embodiments, R ³ and R ⁴ are independently hydrogen, C _1-6 alkyl, or C _2-6 alkynyl, wherein the C _1-6 alkyl and C _2-6 alkynyl are unsubstituted.

In some embodiments, one of R ³ and R ⁴ is hydrogen and the other is halogen, C _1-6 alkyl, C _2-6 alkenyl, or C _2-6 alkynyl, wherein the C _1-6 alkyl, C _2-6 alkenyl, and C _2-6 alkynyl may independently be optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy, C _1-6 hydroxyalkoxy, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino, C _1-6 alkylamino, di(C _1-6 alkyl)amino, oxo, and azido groups. In some embodiments, one of R ³ and R ⁴ is a hydrogen atom and the other is a halogen, C _1-6 alkyl, C _2-6 alkenyl, or C _2-6 alkynyl, wherein the C _1-6 alkyl, C _2-6 alkenyl, and C _2-6 alkynyl are unsubstituted. In some embodiments, one of R ³ and R ⁴ is a hydrogen atom and the other is C _1-6 alkyl or C _2-6 alkynyl, wherein the C _1-6 alkyl and C _2-6 alkynyl are unsubstituted. In some embodiments, R ³ and R ⁴ are both a hydrogen atom.

In some embodiments, R ² is hydrogen, halogen, hydroxyl, azido, amino, C _1-6 alkyl, or C _1-6 alkoxy, wherein the C _1-6 alkyl and C _1-6 alkoxy groups may independently be optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy, C _1-6 hydroxyalkoxy, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino, C _1-6 alkylamino, di(C _1-6 alkyl)amino, oxo, and azido groups. In some embodiments, R ² is a hydrogen atom, a halogen, hydroxyl, azido, amino, C _1-6 alkyl, or C _1-6 alkoxy, wherein the C _1-6 alkyl and C _1-6 alkoxy are unsubstituted. In some embodiments, R ² is a hydrogen atom or a halogen, such as fluorine. In some embodiments, R ² is a hydrogen atom.

In some embodiments, R ⁵ and R ⁶ are independently hydrogen, halogen, hydroxyl, azido, amino, C _1-6 alkyl, or C _1-6 alkoxy, wherein the C _1-6 alkyl and C _1-6 alkoxy groups may independently be optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy, C _1-6 hydroxyalkoxy, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino, C _1-6 alkylamino, di(C _1-6 alkyl)amino, oxo, and azido groups. In some embodiments, R ⁵ and R ⁶ are independently hydrogen, halogen, hydroxyl, azido, amino, C _1-6 alkyl, or C _1-6 alkoxy, wherein the C _1-6 alkyl and C _1-6 alkoxy are unsubstituted. In some embodiments, R ⁵ and R ⁶ are independently hydrogen, halogen, or hydroxyl. In some embodiments, R ⁵ and R ⁶ together are =CH ₂ .

In some embodiments, R ⁵ is hydrogen and R ⁶ is hydrogen, halogen, hydroxyl, azido, amino, C _1-6 alkylamino, di(C _1-6 alkyl)amino, C 1-6 alkyl, C _{1-6 alkoxy, C 2-6 alkenyl} , C _3-6 alkenyl-O-, C _2-6 alkynyl, or C _3-6 alkynyl-O-, wherein the C _1-6 alkyl, C 1-6 alkoxy, C _2-6 alkenyl _{, C 3-6 alkenyl-O-, C 2-6 alkynyl, and C 3-6 alkynyl - O-} may independently be optionally substituted _with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy groups, C _1-6 hydroxyalkoxy groups, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino groups, C _1-6 alkylamino groups, di(C _1-6 alkyl)amino groups, oxo groups and azido groups. In some embodiments, R ⁵ is hydrogen and R ⁶ is hydrogen, halogen, hydroxyl, azido, amino, _1-6 alkyl, or C _1-6 alkoxy, wherein the C _1-6 alkyl and C _1-6 alkoxy groups may independently be optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy, C _1-6 hydroxyalkoxy, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino, _1-6 alkylamino, di(C _1-6 alkyl)amino, oxo and azido groups. In some embodiments, R ⁵ is a hydrogen atom and R ⁶ is a hydrogen atom, a halogen, hydroxyl, azido, amino, C _1-6 alkyl, or C _1-6 alkoxy, wherein the C _1-6 alkyl and C _1-6 alkoxy are unsubstituted. In some embodiments, R ⁵ is a hydrogen atom and R ⁶ is a hydrogen atom, a halogen (such as fluorine or chlorine), hydroxyl, or an unsubstituted C _1-6 alkoxy group (such as methoxy). In some embodiments, R ⁵ is a hydrogen atom and R ⁶ is a halogen, such as fluorine or chlorine. In some cases, R ⁵ is a hydrogen atom and R ⁶ is hydroxyl. In other embodiments, R ⁵ is a hydrogen atom and R ⁶ is an unsubstituted C _1-6 alkoxy group, such as a methoxy group.

In some embodiments, R ⁶ is hydrogen and R ⁵ is hydrogen, halogen, hydroxyl, azido, amino, C _1-6 alkylamino, di(C _1-6 alkyl)amino, C 1-6 alkyl, C _{1-6 alkoxy, C 2-6 alkenyl} , C _3-6 alkenyl-O-, C _2-6 alkynyl, or C _3-6 alkynyl-O-, wherein the C _1-6 alkyl, C 1-6 alkoxy, C _2-6 alkenyl _{, C 3-6 alkenyl-O-, C 2-6 alkynyl, and C 3-6 alkynyl - O-} may independently be optionally substituted _with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy groups, C _1-6 hydroxyalkoxy groups, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino groups, C _1-6 alkylamino groups, di(C _1-6 alkyl)amino groups, oxo groups and azido groups. In some embodiments, R ⁶ is hydrogen and R ⁵ is hydrogen, halogen, hydroxyl, azido, amino, C _1-6 alkyl, or C _1-6 alkoxy, wherein the C _1-6 alkyl and C _1-6 alkoxy groups may independently be optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy, C _1-6 hydroxyalkoxy, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino, C _1-6 alkylamino, di(C _1-6 alkyl)amino, oxo groups and azido groups. In some embodiments, R ⁶ is a hydrogen atom and R ⁵ is a hydrogen atom, a halogen, hydroxyl, azido, amino, C _1-6 alkyl, or C _1-6 alkoxy, wherein the C _1-6 alkyl and C _1-6 alkoxy are unsubstituted. In some embodiments, R ⁶ is a hydrogen atom and R ⁵ is a hydrogen atom, a halogen (such as fluorine or chlorine), hydroxyl, or an unsubstituted C _1-6 alkoxy group (such as methoxy). In some embodiments, R ⁶ is a hydrogen atom and R ⁵ is a halogen, such as fluorine or chlorine. In some cases, R ⁶ is a hydrogen atom and R ⁵ is hydroxyl. In other embodiments, R ⁵ is a hydrogen atom and R ⁶ is an unsubstituted C _1-6 alkoxy group, such as a methoxy group.

In some embodiments, one of R ⁵ and R ⁶ is a hydrogen atom and the other is a halogen, such as fluorine. In some embodiments, R ⁵ is a hydrogen atom and R ⁶ is a halogen, such as fluorine. In some embodiments, R ⁶ is a hydrogen atom and R ⁵ is a halogen, such as fluorine.

In some cases, R ⁵ and R ⁶ are both a hydrogen atom or a halogen, for example, R ⁵ and R ⁶ are both a hydrogen atom, or R ⁵ and R ⁶ are both a halogen, such as fluorine.

In some embodiments, R ⁵ is a hydrogen atom. In other embodiments, R ⁵ is hydroxyl. In other embodiments, R ⁵ is halogen. For example, R ⁵ can be fluorine, bromine, or chlorine.

In some embodiments, R ⁶ is a hydrogen atom. In other embodiments, R ⁶ is hydroxyl. In other embodiments, R ⁶ is methoxy. In other embodiments, R ⁶ is halogen. For example, R ⁶ can be fluorine, bromine, or chlorine.

In some embodiments, V ¹ and V ² are independently O or S. In some embodiments, V ¹ and V ² are independently O or CH ₂ . In some embodiments, V ¹ and V ² are both O. In some embodiments, V ¹ and V ² are both S. In some embodiments, at least one of V ¹ and V ² is O. In some embodiments, at least one of V ¹ and V ² is S. In some embodiments, at least one of V ¹ and V ² is CH ₂ .

In some embodiments, BG ¹ and BG ² are independently -OP(O)R ^P -O- or -OP(S)R ^P -O-. In some embodiments, at least one of BG ¹ and BG ² is -OP(O)R ^P -O-. In some embodiments, at least one of BG ¹ and BG ² is -OP(S)R ^P -O-. In some embodiments, BG ¹ is -OP(O)R ^P -O- and BG ² is -OP(S)R ^P -O-. In some embodiments, BG ¹ is -OP(O)R ^P -O- and BG ² is -OP(S)R ^P -O-. In some embodiments, BG ¹ and BG ² are both -OP(O)R ^P -O-.

In some embodiments, R ^p is independently hydroxyl, thiol, C _1-6 alkyl, borano (-BH ₃ ⁻ ), or -NR'R". In some embodiments, R ^P is independently hydroxyl or thiol at each occurrence. In some embodiments, R ^P is hydroxyl. In some embodiments, R ^P is thiol. In some embodiments, one R ^P is hydroxyl and the other is thiol.

In some embodiments, BG ¹ and BG ² are both -OP(O) ^RP -O-, and ^Rp is independently hydroxyl, thiol, _C1-6 alkyl, borano ( _-BH3- ⁾ , or -NR'R". In some embodiments, BG ¹ and BG ² are both -OP(O) ^RP -O-, and ^Rp is independently hydroxyl or thiol. In some embodiments, BG ¹ and BG ² are both -OP(O) ^RP -O-, and ^Rp is hydroxyl. In other embodiments, BG ¹ and BG ² are both -OP(O) ^RP -O-, and ^Rp is thiol. In some embodiments, BG ¹ is -OP(O)R ^P -O-, where R ^P is hydroxyl, and BG ² is -OP(O)R ^P -O-, where R ^P is a thiol. In other embodiments, BG ¹ is -OP(O)R ^P -O-, where R ^P is thiol, and BG ² is -OP(O)R ^P -O-, where R ^P is hydroxyl.

In some cases, at least one of BG ¹ and BG ² is -NH-P(O)R ^P -O-, -OP(O)R ^P -NH-, -NH-P(S)R ^P -O-, or -OP(S)R ^P -NH-. In some cases, at least one of BG ¹ and BG ² is -NH-P(O)R ^P -O- or -OP(O)R ^P -NH-. In some embodiments, BG ¹ and BG ² are both -NH-P(O)R ^P -O- or -OP(O)R ^P -NH-. In some cases, at least one of BG ¹ and BG ² is -NH-P(O)R ^P -O-, -OP(O)R ^P -NH-, -NH-P(S)R ^P -O-, or -OP(S)R ^P -NH-; and R ^P in each case is independently hydroxyl or thiol. In some cases, at least one of BG ¹ and BG ² is -NH-P(O) ^RP -O- or -OP(O) ^RP -NH-; and ^Rp is independently hydroxyl or a thiol at each occurrence. In some embodiments, BG ¹ and BG ² are both -NH-P(O) ^RP -O- or -OP(O) ^RP -NH-; and ^Rp is independently hydroxyl or a thiol at each occurrence. In some embodiments, BG ¹ and BG ² are both -NH-P(O) ^RP -O- or -OP(O) ^RP -NH-; and ^Rp is hydroxyl. In other embodiments, BG ¹ and BG ² are both -NH-P(O) ^RP -O- or -OP(O) ^RP -NH-; and ^Rp is thiol.

In some embodiments, when R ^P is -NR'R", R' and R" are independently a hydrogen atom or an unsubstituted C _1-6 alkyl, or R' and R" together attached to a single nitrogen atom form a C _3-5 heterocyclic ring, such as morpholine, pyrrolidine, or piperazine.

In some embodiments, B ¹ and B ² are the same nucleobase. In other embodiments, B ¹ and B ² are different nucleobases. In some embodiments, B ¹ and B ² are both a purine nucleobase. In some embodiments, B ¹ and B ² are both a pyrimidine nucleobase. In some embodiments, B ¹ is a purine nucleobase and B ² is a pyrimidine nucleobase. In some embodiments, B ¹ is a pyrimidine nucleobase and B ² is a purine nucleobase.

In some cases, B ¹ and B ² are independently selected from adenine, guanine, thymine, uracil, and cytosine, as well as modified variants thereof. In some embodiments, B ¹ and B ² are both adenine or a modified variant thereof. In some embodiments, B ¹ and B ² are both guanine or a modified variant thereof. In some embodiments, B ¹ is guanine or a modified variant thereof, and B ² is adenine or a modified variant thereof. In some embodiments, B ¹ is adenine or a modified variant thereof, and B ² is guanine or a modified variant thereof.

In some embodiments, B ¹ and B ² are independently selected from:

And , Where

Z ¹ , Z ² , Z ³ , Z ⁴ , Z ⁵ and Z ⁶ in each case independently represent CR ^z or N;

Z ^a is O (except when Z ⁵ is N) or NR'; where

R ^z in each case independently represents a hydrogen atom, a halogen, an azido group, an amino group, a C _1-6 alkylamino group, a di(C _1-6 alkyl)amino group, a C _1-6 alkyl, a C _1-6 alkoxy group, -C(O)C _1-6 alkyl, -CO ₂ H, -CO ₂ C _1-6 alkyl, -C(O)NR', -C(O)NR'R”, -OC(O)C _1-6 alkyl, -NR'C(O)C _1-6 alkyl, -N(R')C(O)NR'R” or -OC(O)NR'R”, where

C _1-6 alkyl and C _1-6 alkoxy are each independently optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy, C _1-6 hydroxyalkoxy, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino, C _1-6 alkylamino, di(C _1-6 alkyl)amino, oxo and azido; and

R' and R" in each case independently represent a hydrogen atom or a C _1-6 alkyl optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy group, C _1-6 hydroxyalkoxy group, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino group, C _1-6 alkylamino group, di(C _1-6 alkyl)amino group, oxo group and azido group; or R' and R", attached to the same nitrogen atom, together form a C _3-5 heterocyclic ring.

In some embodiments, for B ¹ and B ² , one or both occurrences of Z ¹ are CR ^z (such as CH or CNH ₂ ). In some embodiments, one or both occurrences of Z ⁵ are CR ^z (such as CH or CNH ₂ ). In some embodiments, one or both occurrences of Z ¹ are CR ^z (such as CH or CNH ₂ ) and one or both occurrences of Z ⁵ are CR ^z (such as CH or CNH ₂ ). In some embodiments, both occurrences of Z ¹ are CR ^z (such as CH or CNH ₂ ) and both occurrences of Z ⁵ are CR ^z (such as CH or CNH ₂ ).

In some embodiments of ^B1 and ^{B2, Z3} is ^CRz (such as CH or _CNH2 ). In some embodiments, ^Z3 is ^CRz (such as CH or _CNH2 ) and one or both occurrences of ^Z1 are ^CRz (such as CH or _CNH2 ). In some embodiments, ^Z3 is ^CRz (such as CH or _CNH2 ) and one or both occurrences of ^Z5 are ^CRz (such as CH or _CNH2 ). In some embodiments, ^Z3 is ^CRz (such as CH or _CNH2 ), both occurrences of ^Z1 are ^CRz (such as CH or _CNH2 ), and both occurrences of ^Z5 are ^CRz (such as CH or _CNH2 ).

In some cases for B ¹ and B ² , Z ^a is NR', such as NH or NC _1-6 alkyl. In other embodiments, Z ^a is O.

In some embodiments, one or both Z ² are N. In some embodiments, Z ⁴ is N. In some embodiments, one or both Z ² are N and Z ⁴ is N. In some embodiments, both Z ² are N and Z ⁴ is N.

In some embodiments, one or both Z ⁶ are N. In some embodiments, Z ⁴ is N. In some embodiments, one or both Z ⁶ are N and Z ⁴ is N. In some embodiments, both Z ⁶ are N and Z ⁴ is N. In some embodiments, one or both Z ⁶ are N and one or both Z ² are N. In some embodiments, both Z ⁶ are N and both Z ² are N.

In some embodiments, B ¹ and B ² are independently selected from:

And , Where

Z ¹ , Z ³ and Z ⁵ in each case independently represent CR ^z or N;

Z ^a is O (except when Z ⁵ is N) or NR'; where

R ^z in each case independently represents a hydrogen atom, a halogen, an azido group, an amino group, a C _1-6 alkylamino group, a di(C _1-6 alkyl)amino group, a C _1-6 alkyl, a C _1-6 alkoxy group, -C(O)C _1-6 alkyl, -CO ₂ H, -CO ₂ C _1-6 alkyl, -C(O)NR', -C(O)NR'R”, -OC(O)C _1-6 alkyl, -NR'C(O)C _1-6 alkyl, -N(R')C(O)NR'R” or -OC(O)NR'R”, where

C _1-6 alkyl and C _1-6 alkoxy are each independently optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy, C _1-6 hydroxyalkoxy, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino, C _1-6 alkylamino, di(C _1-6 alkyl)amino, oxo and azido; and

R' and R" in each case independently represent a hydrogen atom or a C _1-6 alkyl optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy group, C _1-6 hydroxyalkoxy group, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino group, C _1-6 alkylamino group, di(C _1-6 alkyl)amino group, oxo group and azido group; or R' and R" attached to the same nitrogen atom, together form a C _3-5 heterocyclic ring.

In some embodiments, B ¹ and B ² are independently selected from:

And , Where

Z ^a represents NR'; where

R ^z in each case independently represents a hydrogen atom, a halogen, an azido group, an amino group, a C _1-6 alkylamino group, a di(C _1-6 alkyl)amino group, a C _1-6 alkyl, a C _1-6 alkoxy group, -C(O)C _1-6 alkyl, -CO ₂ H, -CO ₂ C _1-6 alkyl, -C(O)NR', -C(O)NR'R”, -OC(O)C _1-6 alkyl, -NR'C(O)C _1-6 alkyl, -N(R')C(O)NR'R” or -OC(O)NR'R”, where

C _1-6 alkyl and C _1-6 alkoxy are each independently optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy, C _1-6 hydroxyalkoxy, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino, C _1-6 alkylamino, di(C _1-6 alkyl)amino, oxo and azido; and

R' and R" in each case independently represent a hydrogen atom or a C _1-6 alkyl optionally substituted with one or more groups selected from halogen, thiol, hydroxyl, carboxyl, C _1-6 alkoxy group, C _1-6 hydroxyalkoxy group, -OC(O)C _1-6 alkyl, -N(H)C(O)C _1-6 alkyl, -N(C _1-3 alkyl)C(O)C _1-6 alkyl, amino group, C _1-6 alkylamino group, di(C _1-6 alkyl)amino group, oxo group and azido group; or R' and R", attached to the same nitrogen atom, together form a C _3-5 heterocyclic ring.

In some embodiments, B ¹ and B ² are independently selected from:

And .

In some embodiments, R ^a1 , R ^b1 , R ^a2 and R ^b2 are all hydrogen. In some embodiments, at least one of R ^a1 , R ^b1 , R ^a2 and R ^b2 is C _1-3 alkyl, such as methyl. In some cases, R ^a1 and R ^a2 are C _1-3 alkyl, such as methyl. In some embodiments, one of R ^a1 , R ^b1 , R ^a2 and R ^b2 is C _1-3 alkyl, such as methyl.

In some cases, the CDN has a structure corresponding to formula IIa:

Formula IIa

where R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ ; V ¹ and V ² ; BG ¹ and BG ² ; and B ¹ and B ² have the meanings specified above for formula II;

or a pharmaceutically acceptable salt thereof.

In some embodiments of formula IIa, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a hydroxyl group, a thiol group, or an amino group. In some embodiments of formula IIa, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a thiol group or an amino group.

In some cases, CDN has a structure corresponding to formula IIb:

Formula IIb

where R ¹ , R ⁵ and R ⁶ ; V ¹ and V ² ; BG ¹ and BG ² ; and B ¹ and B ² have the meanings specified above for formula II;

or a pharmaceutically acceptable salt thereof.

In some embodiments of formula IIb, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a hydroxyl group, a thiol group, or an amino group. In some embodiments of formula IIb, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a thiol group or an amino group.

In some cases, CDN has a structure corresponding to formula IIc:

Formula IIc

where R ¹ , R ⁵ and R ⁶ ; BG ¹ and BG ² ; and B ¹ and B ² have the meanings specified above for formula II;

or a pharmaceutically acceptable salt thereof.

In some embodiments of formula IIc, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a hydroxyl group, a thiol group, or an amino group. In some embodiments of formula IIc, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a thiol group or an amino group.

In some cases, CDN has a structure corresponding to formula IId:

Formula IId

where R ¹ , R ⁵ , R ⁶ and R ^P ; V ¹ and V ² ; and B ¹ and B ² have the meanings specified above for formula II;

or a pharmaceutically acceptable salt thereof.

In some embodiments of formula IId, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a hydroxyl group, a thiol group, or an amino group. In some embodiments of formula IId, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a thiol group or an amino group.

In some cases, the CDN has a structure corresponding to formula IIe:

Formula IIe

where R ¹ , R ⁵ , R ⁶ and R ^P ; and B ¹ and B ² have the meanings specified above for formula II;

or a pharmaceutically acceptable salt thereof.

In some embodiments of formula IIe, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a hydroxyl group, a thiol group, or an amino group. In some embodiments of formula IIe, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a thiol group or an amino group.

In some cases, the CDN has a structure that corresponds to the formula IIf:

Formula IIf

Where

Y and Z independently represent CH or N; and

R ¹ , R ⁵ , R ⁶ , R ^P and B ¹ have the meanings specified above for formula II;

or a pharmaceutically acceptable salt thereof.

In some embodiments of formula IIf, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a hydroxyl group, a thiol group, or an amino group. In some embodiments of formula IIf, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a thiol group or an amino group.

In some cases, CDN has a structure corresponding to formula IIg:

Formula IIg

Where

W and X independently represent CH or N; and

R ¹ , R ⁵ , R ⁶ , R ^P and B ² have the meanings specified above for formula II;

or a pharmaceutically acceptable salt thereof.

In some embodiments of formula IIg, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a hydroxyl group, a thiol group, or an amino group. In some embodiments of formula IIg, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a thiol group or an amino group.

In some cases, CDN has a structure corresponding to formula IIh:

Formula IIh

Where

W, X, Y and Z independently represent CH or N; and

R ¹ , R ⁵ , R ⁶ and R ^P have the meanings specified above for formula II;

or a pharmaceutically acceptable salt thereof.

In some embodiments of formula IIh, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a hydroxyl group, a thiol group, or an amino group. In some embodiments of formula IIh, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a thiol group or an amino group.

In some cases, the CDN has a structure corresponding to formula IIi:

Formula IIi

Where

W, X, Y and Z independently represent CH or N; and

R ¹ , R ⁵ , R ⁶ and R ^P have the meanings specified above for formula II;

or a pharmaceutically acceptable salt thereof.

In some embodiments of formula IIi, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a hydroxyl group, a thiol group, or an amino group. In some embodiments of formula IIi, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a thiol group or an amino group.

In some cases, the CDN has a structure corresponding to formula IIj:

Formula IIj

Where

W, X, Y and Z independently represent CH or N; and

R ¹ , R ⁵ , R ⁶ and R ^P have the meanings specified above for formula II;

or a pharmaceutically acceptable salt thereof.

In some embodiments of formula IIj, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a hydroxyl group, a thiol group, or an amino group. In some embodiments of formula IIj, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a thiol group or an amino group.

In some cases, the CDN has a structure corresponding to formula IIk:

Formula IIk

Where

W, X, Y and Z independently represent CH or N; and

R ¹ and R ^P have the meanings specified above for formula II;

or a pharmaceutically acceptable salt thereof.

In some embodiments of formula IIk, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a hydroxyl group, a thiol group, or an amino group. In some embodiments of formula IIk, R ¹ is C _2-4 alkyl, such as ethyl, substituted with a thiol group or an amino group.

In some cases, the CDN has a structure corresponding to formula IIm:

Formula IIm

Where

R ¹ is C _2-4 alkyl, such as ethyl, substituted with a thiol group or an amino group;

X and Z independently represent CH or N;

R ^P in each case independently represents hydroxyl or thiol;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CDN has a structure corresponding to formula IIn:

Formula IIn

Where

X and Z independently represent CH or N; and

R ^P in each case independently represents hydroxyl or thiol;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the CDN has a structure corresponding to formula IIo:

Formula IIo

Where

X and Z independently represent CH or N; and

R ^P in each case independently represents hydroxyl or thiol;

or a pharmaceutically acceptable salt thereof.

In some embodiments, in formulas IId-k and IIm-o, both occurrences of R ^P are hydroxyl. In some embodiments, in formulas IId-k and IIm-o, both occurrences of R ^P are thiol. In some embodiments, in formulas IId-k and IIm-o, the occurrence of R ^P corresponding to BG ¹ (upper left R ^P ) is hydroxyl, and the occurrence of R ^P corresponding to BG2 (lower right R ^P ) is thiol. In some embodiments, in formulas IId-k and IIm-o, the occurrence of R ^P corresponding to BG ¹ is thiol, and the occurrence of R ^P corresponding to BG2 is hydroxyl.

In one embodiment, the CDN has the following structure (CDN-A):

or a pharmaceutically acceptable salt thereof.

In one embodiment, the CDN has the following structure:

or a pharmaceutically acceptable salt thereof.

In one embodiment, the CDN has the following structure:

or a pharmaceutically acceptable salt thereof.

In one embodiment, the CDN has the following structure:

or a pharmaceutically acceptable salt thereof.

In one embodiment, the CDN has the following structure (CDN-B):

or a pharmaceutically acceptable salt thereof.

In one embodiment, the CDN has the following structure:

or a pharmaceutically acceptable salt thereof.

In one embodiment, the CDN has the following structure:

or a pharmaceutically acceptable salt thereof.

In one embodiment, the CDN has the following structure:

or a pharmaceutically acceptable salt thereof.

The present invention describes methods for producing an ADC of formula I by conjugating a CDN of formula II (e.g., a CDN-A or a CDN-B) to an antibody via a linker. The CDN of formula II can be conjugated to the antibody via a cleavable or non-cleavable linker. In particular embodiments, the CDN is released into the tumor cell, cancer immune cell, or tumor microenvironment upon cleavage of the linker.

In an ADC of Formula I wherein the CDN (D) is of Formula II (e.g., CDN-A or CDN-B), the CDN may be covalently bonded to the linker (L) via a hydroxyl, thiol, amino, C _1-6 alkylamino, or -PEG-OH group at R ¹ in the CDN of Formula II. It should be understood that the CDN of Formula II may also be unbonded to the linker (L) or the ADC and may be administered by the routes described herein, alone or in combination with an ADC of the present invention, in combination with other active agents (such as immuno-oncology agents such as immune checkpoint inhibitors, including anti-PD1, anti-PD-L1, and anti-CTLA-4 antibodies), or in combination with both an ADC of the present invention and other active agents (such as immuno-oncology agents).

CDNs of Formula II (e.g., CDN-A or CDN-B) are capable of exerting an agonistic effect on STING when used alone or as a component of an ADC of Formula I. In particular embodiments, CDNs can be conjugated to an antibody or antigen-binding moiety via a linker. As described herein, CDNs can be covalently linked to a linker by a chemical reaction between a hydroxyl, amino group, thiol, _C1-6 alkylamino group, or -PEG-OH group in ^R1 of a CDN of Formula II and the corresponding group in the linker. In other words, in some embodiments, the CDN is coupled to a linker (L) of one of the following linkages at R ¹ of the CDN: R ¹ -OL, R ¹ -NH-L, R ¹ -SL, R ¹ -N(C _1-6 alkyl)-L, or R ¹ -PEG-OL, wherein R ¹ is the remainder of the R ¹ moiety except for a hydroxyl, amino group, thiol, C _1-6 alkylamino group, or -PEG-OH group in R ¹ . Particular embodiments of antibodies, antigen-binding fragments, and linkers are described below.

6.3 Antibodies and Antigen-binding fragments

As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to a particular target antigen. Antibodies may be of human or non-human origin. Antibodies may be conjugated to the CDNs described in Section 6.2 via a linker. Antibodies may be polyclonal, monoclonal, genetically modified, and/or otherwise modified. Antibodies comprising the ADCs of the present invention are suitable for administration to humans, for example, as humanized antibodies or fully human antibodies.

Antibodies have heavy and light chains containing hypervariable regions known as complementarity determining regions (CDRs) that mediate binding of the antibody to a target antigen. Antibodies typically have a heavy chain containing a variable region (VH) having three CDRs, namely VH CDR#1, VH CDR#2 and VH CDR#3, and a light chain containing a variable region (VL) having three CDRs, namely VL CDR#1, VL CDR#2 and VL CDR#3. Particular embodiments of the ADCs of the present invention include, but are not limited to, those that contain antibodies and/or antigen-binding fragments comprising these exemplary CDRs and/or VH and/or VL sequences.

Antibodies comprising the ADCs of the present invention may be in the form of full-length antibodies, which may be or be derived from any antibody isotype, including, for example, IgA, IgD, IgE, IgG, IgM, or IgY. In some embodiments, the antibody comprising the ADC is IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the antibodies comprise all or a portion of an antibody constant region.

The antibodies included in the ADCs of the present invention may be bispecific antibodies, dual variable domain antibodies, multichain or single chain antibodies, single domain antibodies, camel antibodies, scFv-Fc antibodies, surrogate antibodies (including surrogate light chain constructs), etc.

The ADCs of the present invention may comprise full-length (intact) antibody molecules as well as antigen-binding fragments. As used herein, the term "fragment" means a portion of an intact antibody that contains fewer amino acid residues than the intact antibody. As used herein, the term "antigen-binding fragment" means a polypeptide fragment of an antibody that binds to an antigen or competes with the intact antibody for binding to an antigen. Suitable examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, scFv, dAb, Fd, or an isolated complementarity determining region (CDR) having sufficient structure for binding. As known to those skilled in the art, fragments can be produced by genetic engineering techniques, or by chemical or enzymatic manipulation of an intact antibody or antibody chain, or by recombinant techniques.

The antibodies or antigen-binding fragments thereof are not limited to any particular method of their generation or production, and can be produced by well-known techniques such as hybridoma techniques, recombinant techniques, phage display technologies, transgenic animals, or combinations thereof.

6.4 Target antigens and antibodies

The antibodies or antigen-binding fragments thereof included in the ADCs of the present invention specifically bind to one or more antigens associated with tumor or immune cells related to cancer.

In some embodiments, the antigen associated with cancer tumor or immune cells is a T cell co-inhibitory molecule. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a tumor-associated antigen selected from PD-L1, PD-L2, CD47, CD80, CD86, HVEM, UL144, CD155, CD112, CD113, galectin-1, galectin-3, galectin-9, CD48, LIGHT, BTLA, and CD160. In some embodiments, the tumor-associated antigen is a molecule that binds to a T cell molecule selected from BTLA, Tim-3, PD-1, CTLA-4, TIGIT, CD244, and CD223.

In some embodiments, the antibody is an anti-PD-L1 antibody such as atezolizumab, durvalumab, avelumab, or an antigen-binding fragment thereof, or an antibody or antigen-binding fragment thereof comprising an equivalent amino acid sequence thereof.

In some embodiments, the antibody is an anti-CD47 antibody such as Hu5F9-G4, IBI188, CC-90002, ZL1201, TTI-621, AO-176, SGN-CD47M antibody, ALX148 antigen-binding domain, or an antigen-binding fragment thereof, or an antibody or antigen-binding fragment thereof comprising an equivalent amino acid sequence thereof.

In other embodiments, the antibody or antigen-binding fragment thereof specifically binds to a cancer tumor antigen that is a growth factor receptor (GFR). In some embodiments, the cancer tumor antigen is a GFR of the EGFR/ErbB/HER family. In some embodiments, the cancer tumor antigen is selected from EGFR/HER1 (ErbB1), HER2/c-Neu (ErbB2), Her3 (ErbB3), and Her4 (ErbB4) receptor. In some embodiments, the cancer tumor antigen is a GFR of the IGFR family. In some embodiments, the cancer tumor antigen is an IGF1R or an IGF2R receptor. In some embodiments, the cancer tumor antigen is a GFR of the TGF-βR (TβR) family. In some embodiments, the cancer tumor antigen is a TβR I or TβR II receptor. In some embodiments, the cancer tumor antigen is a GFR of the VEGFR family. In some embodiments, the cancer tumor antigen is a VEGFR1, VEGFR2, or VEGFR3 receptor. In some embodiments, the cancer tumor antigen is a GFR of the PDGFR family. In some embodiments, the cancer tumor antigen is a PDGFR-α or PDGFR-β receptor. In some embodiments, the cancer tumor antigen is a GFR of the FGFR family. In some embodiments, the cancer tumor antigen is a FGFR1, FGFR2, FGFR3, or FGFR4 receptor.

In some embodiments, the antibody is an anti-EGFR/HER1 (ErbB1) antibody, such as cetuximab, panitumumab, necitumumab, or an antigen-binding fragment thereof, or an antibody or antigen-binding fragment thereof comprising an equivalent amino acid sequence thereof. In some embodiments, the antibody is an anti-HER2 (ErbB2) antibody, such as trastuzumab, pertuzumab, or an antigen-binding fragment thereof, or an antibody or antigen-binding fragment thereof comprising an equivalent amino acid sequence thereof. In some embodiments, the antibody is an anti-VEGFR2 antibody, such as ramucirumab or an antigen-binding fragment thereof, or an antibody or antigen-binding fragment thereof comprising an equivalent amino acid sequence thereof. In some embodiments, the antibody is an anti-PDGFR-α antibody, such as olaratumab or an antigen-binding fragment thereof, or an antibody or antigen-binding fragment thereof comprising an equivalent amino acid sequence thereof.

In other embodiments, the antibody or antigen-binding fragment thereof specifically binds to an antigen associated with lymphoma. In some embodiments, the antigen associated with lymphoma is CD20, CD30, CD19/CD3, CD22, or CD33.

In some embodiments, the antibody is an anti-CD20 antibody such as rituximab, ibritumomab, ofatumumab, obinutuzumab, or an antigen-binding fragment thereof, or an antibody or antigen-binding fragment thereof comprising an equivalent amino acid sequence thereof. In some embodiments, the antibody is an anti-CD30 antibody such as brentuximab or an antigen-binding fragment thereof, or an antibody or antigen-binding fragment thereof comprising an equivalent amino acid sequence thereof. In some embodiments, the antibody is an anti-CD19/CD3 antibody such as blinatumomab or an antigen-binding fragment thereof, or an antibody or antigen-binding fragment thereof comprising an equivalent amino acid sequence thereof. In some embodiments, the antibody is an anti-CD22 antibody such as inotuzumab or an antigen-binding fragment thereof, or an antibody or antigen-binding fragment thereof comprising an equivalent amino acid sequence thereof. In some embodiments, the antibody is an anti-CD33 antibody, such as gemtuzumab or an antigen-binding fragment thereof, or an antibody or antigen-binding fragment thereof comprising an equivalent amino acid sequence thereto.

In other embodiments, the antibody or antigen-binding fragment thereof specifically binds to an antigen associated with myeloma. In some embodiments, the antigen associated with lymphoma is SLAMF7 or CD38.

In some embodiments, the antibody is an anti-SLAMF7 antibody, such as elotuzumab or an antigen-binding fragment thereof, or an antibody or an antigen-binding fragment thereof comprising an equivalent amino acid sequence thereof. In some embodiments, the antibody is an anti-CD38 antibody, such as daratumumab or an antigen-binding fragment thereof, or an antibody or an antigen-binding fragment thereof comprising an equivalent amino acid sequence thereof.

In other embodiments, the antibody or antigen-binding fragment thereof specifically binds to a blastoma-associated antigen. In some embodiments, the blastoma-associated antigen is GD2.

In some embodiments, the antibody is an anti-GD2 antibody, such as dinutuximab or an antigen-binding fragment thereof, or an antibody or antigen-binding fragment thereof comprising an equivalent amino acid sequence.

In other embodiments, the antibody or antigen-binding fragment thereof specifically binds to a RANK ligand.

In some embodiments, the antibody is an anti-RANK ligand antibody such as denosumab or an antigen-binding fragment thereof, or an antibody or antigen-binding fragment thereof comprising an equivalent amino acid sequence thereto.

In some embodiments, the antibody is an antibody that binds to an antigen that is predominantly expressed or overexpressed on cancer cells, such as PD-L1 and EGFR.

In some embodiments, the antibody is an antibody that binds to an antigen produced by a microbe that infects human cells.

When used in this text, “α” and “anti” are used interchangeably, for example when describing an “anti-PD-L1” antibody or an “α-PD-L1” antibody.

When used in this text, hyphenated protein names are used interchangeably with their unhyphenated names (e.g., “PD-L1” and “PDL1” are used interchangeably).

When used in this text, amino acid residue numbering in immunoglobulins follows the Eu numbering system unless otherwise indicated.

6.5. Linkers

In the ADC of the present invention, the CDN is linked to the antibody or antigen-binding fragment via a polyvalent linker. Linkers link the CDN to the antibody or antigen-binding fragment by forming a covalent linkage to the CDN at one position in the linker, and a covalent linkage to the antibody or antigen-binding fragment at another position in the linker. Linkers may be monovalent with respect to the CDN (e.g., in Formula Ia), such that they covalently link one CDN to one site in the antibody or fragment thereof. Linkers may also be polyvalent with respect to the CDN (e.g., in Formula I when m>1), such that they covalently link more than one CDN to one site in the antibody or fragment thereof. As used herein, the term "linker" includes unconjugated, partially conjugated (i.e., with CDN or Ab only), and fully conjugated forms of the linker (i.e., with both CDN and Ab). In particular embodiments, moieties comprising functional groups in the antibody and linker that form a covalent bond between the antibody and linker are specifically illustrated as RX and RY, respectively.

The linkers that link the CDN to the antibody or fragment thereof may be long, short, flexible, rigid, hydrophilic or hydrophobic in nature, or may include segments with different characteristics. A wide variety of linkers are known in the art that can be used to link drugs to antibodies or fragments thereof in an ADC. These linkers, as well as other linkers, can be used to link the CDN to the antibody or antigen-binding fragment of the ADC of the present invention.

In some embodiments, the linkers comprise 2-100, 2-75, 2-50, 2-25, 2-10, 5-100, 5-75, 5-50, 5-25, 5-10, 10-100, 10-75, 10-50, or 10-25 atoms in the chain that connects the CDN to the antibody or antigen-binding fragment (including any atoms at the ends of the linker that may come from the CDN or the antibody or antigen-binding fragment). Similarly, linkers used in CDN-conjugated linkers (as described below) can also contain 2-100, 2-75, 2-50, 2-25, 2-10, 5-100, 5-75, 5-50, 5-25, 5-10, 10-100, 10-75, 10-50, or 10-25 in the chain that connects the CDN to a site in the linker capable of conjugating to a complementary site in the antibody or antigen-binding fragment.

The linker may be chemically stable in the extracellular environment and blood plasma, or may include linkers that are purposefully unstable and may release CDN into the extracellular space or tumor microenvironment.

In some embodiments, the linkers include ligaments that are specifically designed to release the CDN upon internalization of the ADC into the cell. In some particular embodiments, the linkers include ligaments that are specifically designed to be cleaved and/or otherwise specifically or non-specifically destroyed in cells.

The number of CDNs linked to an antibody or antigen-binding fragment thereof in an ADC may vary (the so-called “drug/antibody ratio” or “DAR”) and is limited by the number of available attachment sites in the antibody or antigen-binding fragment thereof and the number of CDNs linked to a single linker. In ADCs that include more than one CDN, each CDN may be the same or different. Provided that the CDN does not exhibit an unacceptable degree of aggregation under the conditions of use and/or storage employed, ADCs with a DAR of 10 or even higher are encompassed by the present invention. In some embodiments, the ADCs of the present invention may have a DAR in the range of 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, or 1 to 4. In some embodiments, the ADCs of the present invention may have a DAR in the range of 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, or 2 to 4. In some particular embodiments, the ADCs may have a DAR value of 1, 2, 3, or 4. In other particular embodiments, the ADCs may have a DAR value of 5, 6, 7, or 8. In some particular embodiments, the ADCs may have a DAR value of 1.

By way of example and without limitation, certain cleavable and non-cleavable linkers that may be included in the ADCs of the present invention are described below.

6.5.1 Cleavable Linkers

In some embodiments, the linker is capable of being cleaved in vivo by chemical or enzymatic processes, releasing the CDN. In some cases, the CDN is cleaved from the linker, releasing the same CDN that was present prior to conjugation to the linker. In other embodiments, the released CDN is a CDN that is a modification of the CDN originally attached to the linker, wherein the modified CDN contains residual functionality from the linker, but retains potency or even exhibits increased potency compared to the original CDN. In general, cleavable linkers contain one or more chemical bonds that are chemically or enzymatically cleavable, while the remainder of the linker is non-cleavable.

In some embodiments, the cleavable linker comprises a chemically labile group. Chemically labile groups have different properties in plasma and cytoplasmic compartments, for example, their properties in the acidic environment of endosomes and lysosomes differ from the properties in conditions of high thiol (e.g., glutathione) concentration in the cytosol. In some embodiments, the plasma stability of a linker comprising a chemically labile group can be increased or decreased by varying the steric hindrance around the chemically labile group using substituents.

In some embodiments, the chemically labile group in the cleavable linker is an acid labile group. The acid labile group remains unchanged when circulating in the blood at neutral pH and undergoes hydrolysis under acidic conditions to release CDN, such as in the acidic microenvironment of a tumor or upon neutralization in endosomal (pH 5.0-6.5) and lysosomal (pH 4.5-5.0) cellular compartments. This pH-dependent release mechanism of the cleavable linker can be optimized by chemical modification, such as with different substituents to adjust the release of CDN at a particular pH. In some embodiments, the cleavable linker comprises an acid labile group such as a hydrazone, hydrazine, cis-acotinyl, acetal, orthoester, or imine group. In some embodiments, the acid labile group is cleaved in the tumor microenvironment, in the endosomes of the tumor or immune cell, in the lysosomes of the tumor or immune cell, in an acidic intracellular compartment of the tumor or immune cell, or combinations thereof. In some embodiments, the acid labile group is not cleaved in the tumor microenvironment, in the endosomes of the tumor or immune cell, in the lysosomes of the tumor or immune cell, and/or in an acidic intracellular compartment of the tumor or immune cell. The cleavability of the acid labile group can be determined by the pH sensitivity of the fully conjugated linker in the ADC. Acid labile linkers can contain additional cleavage sites, such as additional acid labile cleavage sites and/or enzymatically labile cleavage sites.

In some embodiments, the cleavable linker comprises a disulfide group. The disulfide groups are intended to release the drug upon internalization into the cell, where the cytosol has a more reducing environment compared to the extracellular environment. Cleavage of disulfide bonds typically requires the presence of a cytosolic thiol cofactor, such as (reduced) glutathione (GSH), so that disulfide-containing linkers are sufficiently stable in the circulatory system to selectively release the drug into the cytosol. The intracellular enzyme protein disulfide isomerase or similar enzymes capable of cleaving disulfide bonds may also be involved in disulfide cleavage. Tumor cells may induce a hypoxic state due to impaired blood flow, resulting in increased reducing enzyme activity and even higher glutathione concentrations.

ADCs with examples of disulfide-containing linkers include the following formulas:

(V) (VI) (VII)

Where

D represents the CDN (e.g. CDN-A or CDN-B);

S in S-D is taken from CDN, NH in Ab-NH is taken from Ab, S in Ab-S is taken from Ab;

Ab is an antibody or its binding fragment;

“n” represents the number of D linked to Ab via the linker; and

R in each case independently represents a hydrogen atom or C _1-3 alkyl.

In some embodiments, the linker is the structure

, where NH in *-NH can be taken from Ab, and represents the point of attachment of a linker directly or indirectly to the CDN (e.g., at a hydroxyl, amino group, thiol, etc. in R ¹ of the CDN, e.g., CDN-A or CDN-B). In some embodiments, represents the point of direct attachment of the linker to the thiol in the R ¹ group of CDN, where S, bound to , is part of the thiol from the R ¹ group.

Another type of cleavable linker in the ADC of the present invention is an enzymatically cleavable linker. Such linkers are typically peptides or include peptide portions and may be more stable in plasma and the extracellular space than chemically labile linkers. Peptide linkages are generally stable in plasma due to the higher pH than in lysosomes and the presence of endogenous inhibitors of lysosomal proteolytic enzymes. Release of CDN from the ADC may occur upon exposure to lysosomal proteases, such as cathepsin and plasmin, which may be present at elevated levels in some tumor cells. In some embodiments, the cleavable peptide is cleaved by a lysosomal enzyme. In some embodiments, the cleavable peptide is cleaved by a cathepsin (e.g., cathepsin B) or plasmin.

As will be appreciated by one skilled in the art, proteolytic cleavage of a peptide linker directly attached to a CDN can yield an amino acid adduct of the CDN upon hydrolysis of the amide bond. Thus, the ADCs of the present invention also include an enzymatically cleavable linker, which is a self-cleaving linker that spatially separates the CDN from the cleavage site. Use of a self-cleaving linker allows for the cleavage of a fully active, chemically unmodified CDN of formula II upon hydrolysis of the amide bond.

One encompassing self-cleaving linker of the present invention is a bifunctional para-aminobenzyl alcohol group that is linked at one end via a benzyl hydroxyl group to the amine group of a carbamate-functionalized CDN and at the other end is linked via an amino group to form an amide bond to a peptide (PABC group). Upon protease cleavage of the peptide, the resulting CDN is activated, resulting in a 1,6-elimination reaction that releases unmodified CDN, carbon dioxide, and linker residues. In some embodiments, the cleavable linker comprises a PABC group. Further encompassing self-cleaving linkers are the heterocyclic variants of PABC described in the literature (see, e.g., U.S. Pat. No. 7,989,434, which is incorporated herein by reference).

In some embodiments, the enzymatically cleavable linker is a non-peptide linker. In some embodiments, the non-peptide linker is a peptidomimetic. In some embodiments, the non-peptide linker is cleaved by tumor-specific proteases. In some embodiments, the non-peptide linker is cleaved by tumor-specific proteases that are enriched in tumors and/or the tumor microenvironment. In some embodiments, the non-peptide linker is cleaved by cathepsin B. In some embodiments, the non-peptide linker is cyclobutane-1,1-dicarboxamide.

In some embodiments, the enzymatically cleavable linker is a β-glucuronic acid-based linker. Cleavage of the β-glucuronide glycosidic bond can be accomplished by the lysosomal enzyme β-glucuronidase, which is found in lysosomes and is overexpressed in some types of tumors, while it has low activity outside of cells.

Cleavable linkers may include non-cleavable portions or segments, and/or cleavable portions or regions may be included in a non-cleavable linker to render it cleavable. By way of example only, polyethylene glycol (PEG) and related polymers may contain cleavable groups in their backbone. For example, a polyethylene glycol or polymer linker may contain one or more cleavable groups such as a disulfide, hydrazine, hydrazone, dipeptide, or cyclobutane-1,1-dicarboxamide.

In some embodiments, the linker comprises an enzymatically cleavable peptide moiety, such as a linker having formula VIIIa, VIIIb, VIIIc, VIIId, VIIIe, VIIIf, or VIIIg:

(VIIIa) (VIIIb) (VIIIc) (VIIId) (VIIIe) (VIIIf) (VIIIg)

Where:

"peptide" is a peptide or peptidomimetic chain (illustrated in the N→C direction and with the carboxyl and amino "termini" not shown) that is cleaved by a lysosomal enzyme;

T is a chain (e.g., a polymer chain) containing one or more ethylene glycol moieties or an alkylene chain, or combinations thereof;

R ^a is selected from a hydrogen atom, C _1-6 alkyl, sulfonate and methylsulfonate;

R ^b is selected from the hydrogen atom, , , , , , , , And ;

n is an integer in the range 2 to 10, such as 3 to 6, in particular 5;

p is an integer between 0 and 5;

q is an integer in the range 0 to 5, in particular 3;

w is 0 or 1, and -S- in *-S- can be from Ab;

x is 0 or 1, and NH in *-NH can be from Ab;

y is 0 or 1; and NH in *-NH can be from Ab;

z is 0 or 1;

represents the point of attachment of the linker directly or indirectly to the CDN (e.g., at the hydroxyl, amino group, thiol, etc. at R ¹ in CDN, e.g., CDN-A or CDN-B); and

* represents the point of attachment to the rest of the linker either directly or indirectly to the antibody;

or its salt.

In some embodiments, the cleavable peptide fragment or “peptide” in Formulas VIIIa-d is the following structure:

, where the terminal -NH- can be from Ab if x, y and w are 0.

In some embodiments, the cleavable peptide fragment or “peptide” in formula VIIIe is the following structure:

, where w is equal to 1.

In some embodiments, the cleavable peptide fragment or “peptide” in formulas VIIIa-g comprises 2-20 amino acid residues, such as 2-15, 2-10, 2-7, or 2-5 residues, including a tetrapeptide, tripeptide, or dipeptide. In particular embodiments, the cleavable peptide fragment or “peptide” is a dipeptide, such as a dipeptide selected from: Ala-Ala, Ala-(D)Asp, Ala-Cit, Ala-Lys, Ala-Val, Asn-Cit, Asp-Cit, Asn-Lys, Asn-(D)Lys, Asp-Val, Cit-Ala, Cit-Asn, Cit-Asp, Cit-Cit, Cit-Lys, Cit-Ser, Cit-Val, Glu-Val, PhenylGly-(D)Lys, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, Lys-Cit, Me3Lys-Pro, Met-Lys, Met-(D)Lys, Phe-Arg, Phe-Cit, Phe-Lys, Pro-(D)Lys, Ser-Cit, Trp-Cit, Val-Ala, Val-(D)Asp, NorVal-(D)Asp, Val-Cit, Val-Glu, Val-Lys, and salts thereof. In some embodiments, the dipeptide is Val-Cit. In some embodiments, the cleavable peptide fragment or “peptide” is a tripeptide, such as Glu-Val-Cit. In some embodiments, the cleavable peptide fragment or “peptide” is a tetrapeptide, such as Gly-Phe-Leu-Gly or Ala-Leu-Ala-Leu.

In some embodiments, the linker has formula VIIIa and has the following structure:

, where “peptide” is Glu-Val-Cit.

In some embodiments, the linker has formula VIIIc and has the following structure:

, where “peptide” is Val-Cit.

In some embodiments, the cleavable peptide fragment or “peptide” in Formula VIIId is a dipeptide, such as a dipeptide selected from: Ala-Ala, Ala-(D)Asp, Ala-Cit, Ala-Lys, Ala-Val, Asn-Cit, Asp-Cit, Asn-Lys, Asn-(D)Lys, Asp-Val, Cit-Ala, Cit-Asn, Cit-Asp, Cit-Cit, Cit-Lys, Cit-Ser, Cit-Val, Glu-Val, PhenylGly-(D)Lys, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, Lys-Cit, Me3Lys-Pro, Met-Lys, Met-(D)Lys, Phe-Arg, Phe-Cit, Phe-Lys, Pro-(D)Lys, Ser-Cit, Trp-Cit, Val-Ala, Val-(D)Asp, NorVal-(D)Asp, Val-Cit, Val-Glu, Val-Lys, and salts thereof. In some embodiments, the dipeptide of formula VIIId is Val-Cit. In some embodiments, the cleavable peptide fragment or “peptide” of formula VIIId is a tripeptide, such as Glu-Val-Cit. In some embodiments, the cleavable peptide fragment or “peptide” of formula VIIId is a tetrapeptide, such as Gly-Phe-Leu-Gly or Ala-Leu-Ala-Leu.

In some embodiments, the linker has formula VIIId and has the following structure:

, where “peptide” is Val-Cit.

In some embodiments, the linker has formula VIIIe and has the following structure:

, where “peptide” is .

6.5.2. Groups used to attach linkers to antibodies

Various attachment groups can be used to attach linker-CDN synthons to antibodies to produce ADCs. Attachment groups in a linker-CDN synthon are typically electrophilic in nature. In some embodiments, the attachment group is selected from a maleimide group; an activated disulfide such as DSDM, SPDB, or sulfo-SPDB; an active ester such as an NHS ester or HOBt ester; a haloformate, an acyl halide, and an alkyl or benzyl halide such as a haloacetamide. In some embodiments, the resulting linkage between the linker (L) and the antibody (Ab) is a thioether, amide, ester, carbamate, carbonate, urea group, disulfide, or ether.

For the ADCs of the present invention, “self-stabilizing” maleimides and “bridged disulfides” are also encompassed. An example of a “self-stabilizing” maleimide group is described in US 2013/0309256, incorporated herein by reference. Examples of “bridged disulfides” are described in Badescu et al., 2014, Bioconjugate Chem. 25:1124-1136, and WO 2013/085925, each of which is incorporated herein by reference.

6.5.3. ADCs with cathepsin-cleavable linkers

As described above, in some embodiments of the present invention, the CDN (e.g., CDN-A or CDN-B) and the antibodies comprising the ADC of the present invention are linked via a cathepsin-cleavable linker. In one such embodiment, the ADC has a structure corresponding to formula III:

Formula III

where the variables W, X, Y, Z, R ^P and n have the meanings given above for formulas I and II. In the schematic view shown above, is a covalent linkage of a cathepsin-cleavable linker with an antibody or its antigen-binding fragment (Ab).

In one embodiment, the pyrrolidine-2,5-dione group in the linker of formula III is attached at its 3-position to the antibody (Ab) via a thiol group. For example, the pyrrolidine-2,5-dione can be covalently attached at its 3-position to the antibody via a cysteine residue in the antibody. The resulting ADC has a structure corresponding to formula IIIa:

Formula IIIa

where the variables W, X, Y, Z, R ^P and n have the meanings specified above for formulas I and II.

In one embodiment, the ADC has the following structure:

.

6.5.4. ADCs with glutathione-cleavable linkers

As described above, in some embodiments of the present invention, the CDN (e.g., CDN-A or CDN-B) and antibodies comprising the ADC of the present invention are linked via a glutathione-cleavable linker. In one such embodiment, the ADC has a structure corresponding to formula IV:

Formula IV

where the variables W, X, Y, Z, R ^P and n have the meanings given above in formulas I and II. In the schematic view shown above, is a covalent bond of a glutathione-cleavable linker with an antibody or its antigen-binding fragment (Ab).

In one embodiment, the carbonyl group of the linker in formula IV can be covalently linked to the antibody via a lysine residue or another amino acid in the antibody having a side chain with an amino group, such that an amide bond is formed with the amino group at bond connected to the carbonyl group. The resulting ADC has a structure corresponding to formula IVa:

Formula IVa

where the variables W, X, Y, Z, R ^P and n have the values specified above in formulas I and II.

In one embodiment, the ADC has the following structure:

.

6.6. Methods for producing antibody-drug conjugates

In general, ADC of formula I can be prepared according to the following scheme:

Ab-R ^X + R ^Y -L-(D) _m → (Formula I) Ab-[-L-(D) _m ] _n

where Ab, L, D, m and n have the meanings given above for formula I, and RX and RY are complementary groups capable of forming covalent bonds with each other.

Accordingly, ADC of formula Ia can be obtained according to the following scheme:

Ab-R ^X + R ^Y -LD → (Formula Ia) Ab-[-LD] _n

where Ab, L, D and n have the meanings given above for formula I, and RX and RY are complementary groups capable of forming covalent bonds with each other as described above.

The nature of the RX and RY groups depends on the chemistry used to link the RY-L-(D)m or RY-L-D synthon to the antibody. Synthons are typically linked to the side chains of amino acid residues in the antibody, including, for example, a free thiol group of an accessible cysteine residue or a primary amino group of an accessible lysine residue. In some embodiments, RX is a group in the side chain of an amino acid in the antibody, such as an amino group or a thiol group. In some embodiments, RX is an amino group in the side chain of an amino acid, such as lysine, 5-hydroxylysine, ornithine, or a statin, in particular lysine. In some embodiments, RX is a thiol group in the side chain of an amino acid, such as cysteine or homocysteine, in particular cysteine. In such linkages, free thiol groups can be generated by first completely or partially reducing the antibody to break the interchain disulfide bridges between cysteine residues. A number of RY functional groups and corresponding chemistries can be used to form linkages with thiol groups, and include, but are not limited to, maleimides and halogenated acetyls.

The ADCs of the present invention also encompass engineered antibodies with mutations at one or more codons that lead to the disruption of one or more disulfide bridges, and include, as a non-limiting example, a mutation of one cysteine residue in an interchain disulfide bridge with a serine residue that results in a free thiol group from the unpaired cysteine. The ADCs of the present invention also encompass engineered antibodies with mutations at one or more codons that lead to the insertion of a residue containing a thiol group for conjugation to a linker, and include, as a non-limiting example, a mutation of one or more cysteine residues or the inclusion of additional cysteine residues in the amino acid sequence of the antibody or antigen-binding fragment thereof.

In some embodiments, RX is a thiol, such as from a cysteine residue in an antibody, and RY is a group selected from haloacetyl, maleimide, aziridine, acryloyl, vinyl sulfone, pyridyl disulfide, TNB thiol, and an alkylating or arylating agent. In some embodiments, RX is a thiol group, such as from a cysteine residue in an antibody, and RY is a maleimide group. In some embodiments, RX is a primary amino group of a lysine residue. A number of RY functional groups and corresponding chemistries can be used to form linkages to lysine, and include, but are not limited to, NHS esters and isothiocyanates.

In some embodiments, RX is an amine, such as from a lysine residue in an antibody, and RY is a group capable of alkylating or acylating the amine. In some embodiments, RX is an amine, such as from a lysine residue in an antibody, and RY is a group selected from an isothiocyanate, an isocyanate, an acyl azide, an NHS ester, a sulfonyl chloride, an aldehyde, a glyoxal, an epoxide, an oxirane, a carbonate, an aryl halide, an imido ester, a carbodiimide, an anhydride, and a fluorophenyl ether. In some embodiments, RX is an amine, such as from a lysine residue in an antibody, and RY is an NHS ester.

For the ADC of the present invention, the chemistry of the conjugation process is not limited by the available groups in the side chain. The antibody can be designed to contain amino acid residues for conjugation, and these include, as non-limiting examples, the conversion of groups such as amino groups in the side chains to other suitable groups, such as thiol groups, by coupling an appropriate small molecule to the amine. For example, a side chain containing a primary amino group of an amino acid can be converted to a thiol-containing side chain, such as -NH-C _1-6 alkyl-SH, including -NH-CH ₂ -CH ₂ -SH, where -NH- comes from a primary amino group.

As will be appreciated by one skilled in the art, the number of CDNs linked to an antibody molecule can vary, resulting in a heterogeneous ADC preparation in which some antibodies have one CDN attached, some two, some three, etc. (and some have none). The degree of heterogeneity depends, among other factors, on the chemistry of the process used to link the CDNs. For example, when an IgG1 antibody is reduced, providing thiol groups for attachment, heterogeneous mixtures of antibodies containing 0, 1, 2, 3, 4, 5, 6, 7, or 8 linked CDNs per molecule are often formed. Additionally, by adjusting the molar ratio of RX and RY, ADCs can be prepared containing 0, 1, 2, 3, 4, 5, 6, 7, or 8 linked CDNs per molecule. Therefore, it should be understood that, depending on the context, the drug/antibody ratios (DARs) stated may represent average values for a set of ADCs. For example, “DAR3” means a heterogeneous ADC preparation in which the average drug/antibody ratio is 3, such as a mixture of ADCs having equal amounts of DAR2 and DAR4.

Purity can be assessed by various methods known in the art. As a specific example, an ADC preparation can be analyzed by HPLC, hydrophobic exchange, ion exchange, gel permeation or other types of chromatography, and the degree of purity is estimated by analyzing the areas of the corresponding peaks.

In some embodiments, the present invention provides a method for producing an ADC comprising (a) conjugating one or more CDNs of the present invention (i.e., a CDN of Formula II) to a linker (e.g., as described herein as L) to produce a linker linked to the one or more CDNs; and (b) conjugating the linker linked to the one or more CDNs to an antibody or antigen-binding fragment thereof (e.g., as described herein as Ab) to produce the ADC.

In some embodiments, the present invention relates to a method for producing an ADC comprising (b) conjugating a linker linked to one or more CDNs (i.e., a CDN of formula II and, for example, L described herein) to an antibody or antigen-binding fragment thereof (e.g., Ab described herein) to produce the ADC.

In some embodiments, the present invention relates to a method for producing linkers linked to one or more CDNs, comprising (a) conjugating one or more CDNs of the present invention (i.e., a CDN of formula II) to a linker (e.g., as described herein L) to produce linkers linked to one or more CDNs.

In other embodiments, the present invention provides a method of producing an ADC comprising (a) conjugating one or more linkers (e.g., those described herein as L) to an antibody or antigen-binding fragment thereof (e.g., those described herein as Ab) to produce an antibody linked to a linker; and (b) conjugating one or more CDNs of the present invention (i.e., CDNs of formula II) to the antibody linked to a linker to produce an ADC.

In some embodiments, the present invention provides a method of producing an ADC comprising (b) conjugating one or more CDNs of the present invention (i.e., a CDN of Formula II) to an antibody or antigen-binding fragment thereof linked to a linker (e.g., one or more Ls described herein, and, e.g., an Ab described herein) to produce the ADC.

In some embodiments, the present invention relates to a method of producing an antibody linked to a linker, comprising (a) conjugating one or more linkers (e.g., those described herein as L) to an antibody or antigen-binding fragment thereof (e.g., those described herein as Ab) to produce the antibody linked to the linker.

In some embodiments, the present invention provides a method of making an ADC comprising (a) conjugating a CDN of the present invention (i.e., a CDN of Formula II) to a linker (e.g., L described herein) to form a CDN-linked linker; and (b) conjugating multiple CDN-linked linkers to an antibody or antigen-binding fragment thereof (e.g., Ab described herein) to form an ADC.

In other embodiments, the present invention relates to a method of producing an ADC comprising (a) conjugating multiple linkers (e.g., L as described herein) to an antibody or antigen-binding fragment thereof (e.g., Ab as described herein) to produce a linker-linked antibody; and (b) conjugating one or more CDNs of the present invention (i.e., CDNs of formula II) to the linker-linked antibody to produce an ADC.

6.6.1. CDN compositions for working with CDN before conjugation

As described above, the CDNs of the present invention (i.e., the CDNs of formula II, such as CDN-A or CDN-B) can be conjugated to a linker (e.g., L described herein) to produce linkers linked to one or more CDNs, or they can be conjugated to an antibody linked to a linker (e.g., one or more L described herein and, e.g., Ab described herein) to produce an ADC. Therefore, described herein are CDN compositions that facilitate purification, drying, or handling of the CDNs used in these conjugation steps.

In this context, the present invention describes a CDN composition comprising a CDN of formula II (including subformulas of formula II such as Formula IIa, IIb, etc.) and a base such as an amine base. In some embodiments, the amine base is liquid at room temperature and pressure, such as pyridine, piperidine, pyrrolidine, morpholine, lutidine (e.g., 2,6-lutidine), triethylamine (TEA), or diisopropylethylamine (DIPEA), in particular pyridine. In some embodiments, the CDN composition comprising a CDN and a base is an anhydrous composition comprising less than 100, 50, 25, or 10 ppm of water. The CDN composition comprising a CDN and a base can be dried by evaporation under high vacuum prior to use in the conjugation step.

In some embodiments, a CDN composition comprises a CDN of Formula II, such as Formula IIk, IIm, IIn, or IIo, and an amine base, such as pyridine. In some embodiments, a CDN composition comprises a CDN of Formula II having an amino group at the R ¹ position, and an amine base, such as pyridine. In some embodiments, a CDN composition comprises a CDN of Formula IIn or IIo (and optionally having an amino group at the R ¹ position), and an amine base, such as pyridine. In some embodiments, a CDN composition comprises a CDN-A as described herein, or a pharmaceutically acceptable salt thereof, and an amine base, such as pyridine, and is optionally anhydrous. In some embodiments, a CDN composition comprises a CDN-B as described herein, or a pharmaceutically acceptable salt thereof, and an amine base, such as pyridine, and is optionally anhydrous.

The present invention also provides a CDN composition, which is an aqueous solution comprising a CDN of Formula II (including subformulas of Formula II such as Formula IIa, IIb, etc.) and a buffer allowing to adjust the pH to a range of 5 to 10, including a range of 5 to 8, such as pH 5, 5.5, 5.8, 6, 6.2, 6.5, 7, 7.5, or 8, including any pH ranges within these pH values as boundaries, such as a range of 5 to 7. In some embodiments, the buffer allows to adjust the pH to 6 +/- 0.2. In some embodiments, the buffer is a phosphate buffer. In some embodiments, the CDN composition is an aqueous solution comprising a CDN of Formula II having a thiol group at the R ¹ position, such as Formula IIo, and a buffer allowing to adjust the pH to 6 +/- 0.2, such as a phosphate buffer. In particular embodiments, the CDN composition is an aqueous solution comprising CDN of formula IIo and a buffer allowing the pH to be adjusted to 6 +/- 0.2, such as a phosphate buffer. In one embodiment, the CDN composition is an aqueous solution comprising CDN-B or a pharmaceutically acceptable salt thereof and a buffer allowing the pH to be adjusted to 6 +/- 0.2, in particular a phosphate buffer.

6.6.2. CDN compositions for conjugation of CDN with a linker

The preparation of the above-described linker coupled to a CDN (i.e., comprising a CDN of formula II, such as CDN-A or CDN-B, coupled to a linker, such as described in this application L), may involve the preparation of one or more CDN compositions for conjugating the CDN to the linker.

In this context, the present invention describes a CDN composition comprising a CDN of formula II (including subformulas of formula II such as Formula IIa, IIb, etc.) and a linker (e.g., as described herein by L) or a crosslinking agent, or both a linker and a crosslinking agent, wherein the crosslinking agent enables coupling of the CDN to the linker. In some embodiments, the crosslinking agent is capable of activating the linker for coupling with the CDN, such as by forming an activated ester on the linker, such that the CDN is then capable of reacting with the activated ester on the linker to conjugate the CDN to the linker. Examples of suitable crosslinking agents include hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and azabenzotriazole tetramethyluronium hexafluorophosphate (HATU). In some embodiments, the CDN composition further comprises an aprotic polar solvent such as dimethylformamide (DMF), dimethylacetamide (DMA), acetonitrile or tetrahydrofuran (THF), particularly DMF. In some embodiments, the CDN composition comprising CDN and a linker or a crosslinking agent (or both) is an anhydrous composition comprising less than 100, 50, 25 or 10 ppm of water.

In some embodiments, the CDN composition comprises a CDN of formula II, such as formula IIk, IIm, IIn, or IIo (and comprising an amino or thiol group at the R ¹ position), and a linker (e.g., as described herein by L) or a crosslinking agent, or a linker and a crosslinking agent together. In some embodiments, the CDN composition comprises a CDN of formula II having an amino or thiol group at the R ¹ position, and a linker or a crosslinking agent, or a linker and a crosslinking agent together. In some embodiments, the CDN composition comprises a CDN of formula IIn or IIo (and optionally comprising an amino or thiol group at the R ¹ position), and a linker or a crosslinking agent, or a linker and a crosslinking agent together. In some embodiments, the CDN composition comprises CDN-A or CDN-B, or a pharmaceutically acceptable salt thereof, as described herein, and a linker or cross-linking agent, or a linker and cross-linking agent together, and optionally an aprotic polar solvent as described above. In some embodiments, the CDN composition comprises CDN-A or CDN-B, or a pharmaceutically acceptable salt thereof, a linker, and an aprotic polar solvent. In some embodiments, the CDN composition comprises CDN-A or CDN-B, or a pharmaceutically acceptable salt thereof, a cross-linking agent, and an aprotic polar solvent.

6.6.3. Linkers associated with CDN

As described above, linkers linked to CDNs can be useful intermediates in the preparation of the ADCs of the present invention. In some embodiments, a linker linked to a CDN comprises a CDN of Formula II (e.g., CDN-A or CDN-B) conjugated to a linker, such as L described herein. For example, a linker linked to a CDN can have the formula L-CDN, wherein L comprises a site capable of binding to a complementary site in an antibody or antigen-binding moiety, wherein the CDN has Formula II (including subformulas such as Formulas IIk, IIm, IIn, and IIo), and wherein the CDN is covalently bonded to the linker at a hydroxyl, thiol, amino, _C1-6 alkylamino, or -PEG-OH group at the ^R1 position of Formula II.

In some embodiments, the CDN and the linker in the CDN-linked linker are connected via a thioether, amide, ester, carbamate, carbonate, urea, disulfide group or ether group, especially via an amide, carbamate or disulfide group. For example, in some embodiments, the CDN and the linker in the CDN-linked linker are connected via an amide group. In such embodiments, the linker associated with the CDN may have the formula LC(O)NH-CDN or LC(O)N(C _1-6 alkyl)-CDN, wherein the CDN has the formula II (including subformulas such as formulas IIk, IIm, and IIn), wherein R ¹ in formula II is C _1-6 alkyl, such as C _2-6 alkyl or C _2-3 alkyl, substituted with an amino group or a C _1-6 alkylamino group, any of which forms the amine portion of the amide group in the formula. In one such embodiment, the linker associated with CDN has the formula LC(O)NH-CDN, wherein CDN has the formula II (including subformulas such as formulas IIk, IIm and IIn), wherein R ¹ in formula II is C _2-3 alkyl, such as ethyl, substituted with an amino group that forms the amine portion of the amide group in the formula.

In some embodiments, the linker associated with the CDN has a structure corresponding to formula IXa:

Formula IXa

where R ^L represents the remainder of the linker L, and the variables W, X, Y, Z and R ^P have the meanings specified above for formulas I and II.

In other embodiments, the CDN and the linker in the CDN-linked linker are connected via a carbamate group. In such embodiments, the linker associated with the CDN may have the formula L-OC(O)NH-CDN or L-OC(O)N(C _1-6 alkyl)-CDN, wherein the CDN has the formula II (including subformulas such as formulae IIk, IIm, and IIn), wherein R ¹ in formula II is C _1-6 alkyl, such as C _2-6 alkyl or C _2-3 alkyl substituted with an amino group or a C _1-6 alkylamino group, any of which forms the amine portion of the carbamate group in the formula. In one such embodiment, the linker associated with CDN has the formula L-OC(O)NH-CDN, wherein CDN has the formula II (including subformulas such as formulas IIk, IIm, and IIn), wherein R ¹ in formula II is C _2-3 alkyl, such as ethyl, substituted with an amino group that forms the amine portion of the carbamate group in the formula.

In some embodiments, the linker associated with the CDN has a structure corresponding to formula IXb:

Formula IXb

where R ^L represents the remainder of the linker L, and the variables W, X, Y, Z and R ^P have the meanings specified above for formulas I and II.

In some embodiments, the linker associated with the CDN has the following structure:

where the variables W, X, Y, Z and R ^P have the meanings specified above for formulas I and II.

In other embodiments, the CDN and the linker in the linker bound to the CDN are connected through a urea group. In such embodiments, the linker bound to the CDN can have the formula L-NHC(O)NH-CDN or L-NHC(O)N(C _1-6 alkyl)-CDN, wherein the CDN has the formula II (including subformulas such as formulae IIk, IIm, and IIn), wherein R ¹ in formula II is C _1-6 alkyl, such as C _2-6 alkyl or C _2-3 alkyl substituted with an amino group or a C _1-6 alkylamino group, any of which forms the right-hand amine portion of the urea group in the formula. In one such embodiment, the linker associated with CDN has the formula LNHC(O)NH-CDN, wherein CDN has the formula II (including subformulas such as formulas IIk, IIm, and IIn), wherein R ¹ in formula II is C _2-3 alkyl, such as ethyl, substituted with an amino group that forms the right-hand amine portion of the urea group in the formula.

In some embodiments, the linker associated with the CDN has a structure corresponding to formula IXc:

Formula IXc

where R ^L represents the remainder of the linker L, and the variables W, X, Y, Z and R ^P have the meanings specified above for formulas I and II.

In other embodiments, the CDN and the linker in the CDN-linked linker are connected via an ester group. In such embodiments, the linker associated with the CDN may have the formula LC(O)O-CDN, wherein the CDN has the formula II (including subformulas such as formulas IIk, IIm, and IIn), wherein R ¹ in formula II is C _1-6 alkyl, such as C _2-6 alkyl or C _2-3 alkyl, substituted with hydroxyl or a -PEG-OH group, any of which forms the alcohol portion of the ester group in the formula. In one such embodiment, the linker associated with CDN has the formula LC(O)O-CDN, wherein CDN has the formula II (including subformulas such as formulas IIk, IIm, and IIn), wherein R ¹ in formula II is C _2-3 alkyl, such as ethyl, substituted with a hydroxyl group that forms the alcohol portion of the ester group in the formula.

In some embodiments, the linker associated with the CDN has a structure corresponding to formula IXd:

Formula IXd

where R ^L represents the remainder of the linker L, and the variables W, X, Y, Z and R ^P have the meanings specified above for formulas I and II.

In other embodiments, the CDN and the linker in the CDN-linked linker are connected via a carbonate group. In such embodiments, the CDN-linked linker may have the formula L-OC(O)O-CDN, wherein the CDN has the formula II (including subformulas such as formulae IIk, IIm, and IIn), wherein R ¹ in formula II is C _1-6 alkyl, such as C _2-6 alkyl or C _2-3 alkyl, substituted with hydroxyl or a -PEG-OH group, any of which forms the right alcohol portion of the carbonate group in the formula. In one such embodiment, the linker associated with CDN has the formula L-OC(O)O-CDN, wherein CDN has the formula II (including subformulas such as formulas IIk, IIm, and IIn), wherein R ¹ in formula II is C _2-3 alkyl, such as ethyl, substituted with a hydroxyl group, which forms the right alcohol portion of the carbonate group in the formula.

In some embodiments, the linker associated with the CDN has a structure corresponding to formula IXe:

Formula IXe

where R ^L represents the remainder of the linker L, and the variables W, X, Y, Z and R ^P have the meanings specified above for formulas I and II.

In other embodiments, the CDN and the linker in the CDN-linked linker are linked through a disulfide group. In such embodiments, the CDN-linked linker may have the formula LSS-CDN, wherein the CDN has the formula II (including subformulas such as formulas IIk, IIm, and IIo), wherein R ¹ in formula II is C _1-6 alkyl, such as C _2-6 alkyl or C _2-3 alkyl, substituted with a thiol group that forms the right-hand side of the disulfide group in the formula. In one such embodiment, the linker associated with CDN has the formula LS-S-CDN, wherein CDN has the formula II (including subformulas such as formulas IIk, IIm and IIo), wherein R ¹ in formula II is C _2-3 alkyl, such as ethyl, substituted with a thiol group that forms the right-hand side of the disulfide group in the formula.

In some embodiments, the linker associated with the CDN has a structure corresponding to formula IXf:

Formula IXf

where R ^L represents the remainder of the linker L, and the variables W, X, Y, Z and R ^P have the meanings specified above for formulas I and II.

In some embodiments, the linker associated with the CDN has the following structure:

where the variables W, X, Y, Z and R ^P have the meanings specified above for formulas I and II.

6.7. Pharmaceutical compositions and medicinal products

The ADC and/or CDN of the present invention may be in the form of a pharmaceutical composition comprising the ADC or CDN and one or more carriers, excipients and/or diluents. The compositions may be prepared for pharmaceutical use in humans and may include a pharmaceutically acceptable carrier such as an aqueous, optionally buffered, solution, suspension or dispersion. The compositions may also be lyophilized solid compositions intended for reconstitution prior to use.

The pharmaceutical compositions of the present invention may be in any suitable form and may be administered to a patient by various routes, such as intravenously, intratumorally, subcutaneously, intramuscularly, orally, intranasally, intrathecally, transdermally, topically, or locally. The route of administration in each case depends on the particular antibody and/or ADC, the subject, the nature and severity of the disease, and the physical condition of the subject. In some embodiments, the pharmaceutical composition is administered intravenously, intratumorally, subcutaneously, or intramuscularly in the form of a liquid preparation.

In some embodiments, the ADC and/or CDN of the present invention, including pharmaceutical compositions thereof, are administered systemically, such as subcutaneously, intraperitoneally, intramuscularly or intravenously, in particular intravenously. In other embodiments, the ADC and/or CDN of the present invention, including pharmaceutical compositions thereof, are administered locally to the tumor site, such as within the tumor or in the tumor microenvironment.

The present invention also describes CDNs of formula II (including subformulas of formula II such as formula IIa, IIb, etc.) for use in the manufacture of medicaments for therapy, such as agents for promoting an immune response and/or for the treatment of cancer in a subject, including one or more cancers described below, and in combination with one or more additional therapeutic agents as described below.

6.8. Treatment methods

In some embodiments, the ADCs of the present invention (i.e., the ADCs of Formula I) or the CDNs of the present invention (i.e., the CDNs of Formula II, such as CDN-A or CDN-B) can be used in therapy. In some embodiments, the present invention relates to pharmaceutical compositions of the present invention comprising the ADCs of the present invention (i.e., the ADCs of Formula I) or the CDNs of the present invention (i.e., the CDNs of Formula II) for use in therapy.

The ADC and/or CDN of the present invention can be used alone or in combination with each other and/or with other therapeutic agents. As shown in the examples described below, the ADC and CDN are capable of promoting an immune response when administered to a subject. For example, the ADC and CDN of the present invention, alone or in combination, are capable of inducing interferon-β (IFNβ) in humans. The ability of the ADC and CDN of the present invention to promote an immune response is attributed, in part, to their ability to exert an agonistic effect on STING. The ADC is capable of delivering the CDN of the present invention (e.g., a compound of formula II) to target tumor or cancer immune cells, or to the tumor microenvironment, triggering activation of STING and the corresponding immune response. Conjugation of CDN to an antibody or its antigen-binding fragment that binds to an antigen in tumor or cancer immune cells is aimed at delivering CDN and prolonging and enhancing the immune response.

The ADCs of the present invention are capable of promoting an immune response that is stronger than that of unconjugated CDN or an antibody comprising an ADC. It has been unexpectedly found that the ADCs of the present invention are capable of promoting an immune response that is stronger than the additive immune response of unconjugated CDN and an antibody comprising an ADC. In other words, a synergistic effect can be achieved by conjugating the CDN of the present invention to a specific immunotherapeutic antibody. In other embodiments, as described herein, the CDN of the present invention can also be administered other than as part of an ADC.

Accordingly, in one aspect, the present invention provides methods of inducing or promoting an immune response in a subject comprising administering an effective amount of an ADC of the present invention (i.e., an ADC of Formula I). In another aspect, the present invention provides methods of inducing or promoting an immune response in a subject comprising administering an effective amount of a CDN of the present invention (i.e., a CDN of Formula II). And in another aspect, the present invention provides methods of inducing or promoting an immune response in a subject comprising administering an effective amount of an ADC of the present invention in combination with a CDN of the present invention. In some embodiments, the ADC and/or CDN are administered to mammals in need thereof. In particular embodiments, the ADC and/or CDN are administered to humans in need thereof.

In particular embodiments, the ADC or CDN of the present invention is used to treat cancer. For example, the ADC or CDN of the present invention can be used to treat cancer of the lung, bone, pancreas, skin, head, neck, uterus, ovary, stomach, intestine, breast, esophagus, small intestine, colon, endocrine system, thyroid, parathyroid, adrenal, ureter, prostate, penis, testicle, urethra, bladder, kidney, or liver. Other cancers that can be treated with the ADC or CDN of the present invention include colorectal cancer; anal cancer; carcinoma of the fallopian tubes, endometrium, cervix, vagina, vulva, renal pelvis, and renal cell; soft tissue sarcoma; myxoma; rhabdomyoma; fibroma; lipoma; teratoma; cholangiocarcinoma; hepatoblastoma; angiosarcoma; hematoma; hepatoma; fibrosarcoma; chondrosarcoma; myeloma; chronic or acute leukemia; lymphocytic lymphoma; primary CNS lymphoma; CNS neoplasms; spinal axis tumors; squamous cell carcinoma; synovial sarcoma; malignant pleural mesothelioma; brainstem glioma; pituitary adenoma; bronchial adenoma; chondromatous hamartoma; mesothelioma; Hodgkin's disease; or a combination of one or more of the above.

Accordingly, in one aspect, the present invention provides methods of treating cancer in a subject comprising administering a pharmaceutical composition comprising a pharmaceutically acceptable amount of an ADC of the present invention (i.e., an ADC of Formula I). In another aspect, the present invention provides methods of treating cancer in a subject comprising administering a pharmaceutical composition comprising a pharmaceutically acceptable amount of a CDN of the present invention (i.e., a CDN of Formula II). In some embodiments, the pharmaceutical composition is administered to mammals in need thereof. In particular embodiments, the pharmaceutical composition is administered to humans in need thereof.

In another aspect, the present invention provides methods of treating cancer in a subject by administering a pharmaceutical composition comprising a pharmaceutically acceptable amount of an ADC of the present invention (i.e., an ADC of Formula I) with at least one additional anti-cancer agent to the subject (e.g., a human). The ADC and one or more additional anti-cancer agents may be administered together or separately, and when administered separately, the administration may be simultaneous or sequential, in any order and by any suitable route, as separate or combined pharmaceutical compositions. In some embodiments, the additional anti-cancer agent enhances expression of a target antigen of the antibody or antigen-binding fragment thereof in the ADC of Formula I. The amount of the ADC and other pharmaceutically active anti-cancer agent(s) and the relative timing of administration are selected in accordance with the objective of achieving the desired combination therapeutic effect.

In some cases, the present invention provides methods for treating cancer in a subject by administering a pharmaceutical composition comprising a pharmaceutically acceptable amount of a CDN of the present invention (i.e., a CDN of Formula II) with at least one additional anti-cancer agent to the subject (e.g., a human). The CDN and one or more additional anti-cancer agents may be administered together or separately, and when administered separately, the administration may be simultaneous or sequential, in any order and by any suitable route, as separate or combined pharmaceutical compositions. The amount of the CDN and the other pharmaceutically active anti-cancer agent(s) and the relative timing of administration are selected in accordance with the objective of achieving the desired combined therapeutic effect.

The combination of the ADC or CDN of the present invention and one or more anticancer agents may be administered together in a single pharmaceutical composition. Alternatively, the ADC or CDN and one or more anticancer agents may be in separate formulations. When in separate formulations, they may be in any suitable compositions formulated according to principles known to those skilled in the art.

Accordingly, the ADC or CDN of the present invention can be used with other therapeutic methods for treating cancer, such as antitumor therapy, combination therapy with immune checkpoint inhibitors, other chemotherapeutic agents, hormonal agents, antibodies, as well as surgical methods and/or radiation therapy methods, in particular radiation therapy.

In one embodiment, an ADC of the present invention (i.e., an ADC of Formula I) or a CDN of the present invention (i.e., a CDN of Formula II), or an ADC and CDN together, are used in combination with an immune checkpoint inhibitor to treat cancer. Immune checkpoint inhibitors, such as humanized antibodies to PD-1, PD-L1, and CTLA4, have recently shown great success in the treatment of several types of metastatic cancer, including melanoma, non-small cell lung cancer, renal cell carcinoma, and bladder cancer (Sharma and Allison, 2015, Science 348, 56). However, to date, only a small percentage of cancer patients have benefited from immune checkpoint inhibitor therapy, in part because insufficient numbers of anti-tumor immune cells, such as CD8 T cells, are generated and/or infiltrated into tumors. As shown in the examples described herein, the combination of an ADC and/or CDN of the present invention and an immune checkpoint inhibitor is capable of working synergistically to treat cancers that are resistant to immune checkpoint inhibitor monotherapy.

In one embodiment, the present invention provides methods of treating cancer in a subject by administering a pharmaceutical composition comprising a pharmaceutically acceptable amount of an ADC of the present invention (i.e., an ADC of Formula I) or a CDN of the present invention (i.e., a CDN of Formula II), or both an ADC and a CDN of the present invention, in combination with a PD-L1 inhibitor. Examples of PD-L1 inhibitors that can be used in combination with an ADC of the present invention include, but are not limited to, atezolizumab (Tecentriq®), avelumab (Bavencio®), durvalumab (Imfinzi®), BMS-936559, and CK-301. In some embodiments, an ADC and/or a CDN of the present invention is not administered in combination with a PD-L1 inhibitor, including those listed above.

In one embodiment, the present invention provides methods of treating cancer in a subject by administering a pharmaceutical composition comprising a pharmaceutically acceptable amount of an ADC of the present invention (i.e., an ADC of Formula I) or a CDN of the present invention (i.e., a CDN of Formula II), or both an ADC and a CDN of the present invention, in combination with a PD-1 inhibitor. Examples of PD-L1 inhibitors that can be used in combination with an ADC of the present invention include, but are not limited to, pembrolizumab (Keytruda®), nivolumab (Opdivo®), cemiplimab (Libtayo®), AMP-224, AMP-514, and PDR001. In some embodiments, an ADC and/or a CDN of the present invention is not administered in combination with a PD-L1 inhibitor, including those listed above.

In one embodiment, the present invention provides methods of treating cancer in a subject by administering a pharmaceutical composition comprising a pharmaceutically acceptable amount of an ADC of the present invention (i.e., an ADC of Formula I) or a CDN of the present invention (i.e., a CDN of Formula II), or both an ADC and a CDN of the present invention, in combination with a CTLA-4 inhibitor. Examples of CTLA-4 inhibitors that can be used in combination with an ADC of the present invention include, but are not limited to, ipilmumab (Yervoy®) and tremelimumab. In some embodiments, an ADC and/or a CDN of the present invention is not administered in combination with a CTLA-4 inhibitor, including those listed above.

In another embodiment, the present invention features methods of treating cancer in a subject by administering a pharmaceutical composition comprising a pharmaceutically acceptable amount of an ADC of the present invention (i.e., an ADC of Formula I) or a CDN of the present invention (i.e., a CDN of Formula II), or an ADC and CDN of the present invention together, with one or more tubulin polymerization inhibitors such as diterpenoids and vinca alkaloids; platinum coordination complexes; alkylating agents such as nitrogen mustards, oxazaphosphorinanes, alkyl sulfonates, nitrosoureas, and triazeparizenes; antibiotics such as anthracyclines, actinomycins, and bleomycins; topoisomerase II inhibitors such as epipodophyllotoxins; antimetabolites such as purine and pyrimidine analogs and antifolate compounds; topoisomerase I inhibitors such as campothecins; hormones and hormone analogues; signal transduction pathway inhibitors; angiogenesis inhibitors; immunotherapeutic agents; proapoptotic agents; and/or cell cycle signaling inhibitors.

An ADC of Formula I can be used, for example, to treat cancer or to induce or promote an immune response, in combination with a STING agonist that is not conjugated to an antibody or antigen-binding fragment. In some embodiments, the STING agonist that is not conjugated to an antibody or antigen-binding fragment is a CDN, such as one of those described herein, i.e., 2'3'-CDN. In other embodiments, the STING agonist is 3'3'-CDN, 2'2'-CDN, or 3'2'-CDN. In some embodiments, the STING agonist is a benzophenone analog. In other embodiments, the STING agonist is a dimeric aminobenzimidazole. Examples of STING agonists unconjugated to an antibody or antigen-binding fragment that can be used in combination with the ADC of the present invention include IMSA101, ADU-S100 (MIW815), BMS-986301, CRD5500, CMA (10-carboxymethyl-9-acridanone), diABZI STING agonist-1 (e.g., CAS No.: 2138299-34-8), DMXAA (ASA404/vadimesan), E7766, GSK-532, GSK-3745417, MK-1454, MK-2118, SB-11285, SRCB-0074, TAK-676, and TTI-10001. The STING agonist can be administered prior to, concurrently with, or following administration of the ADC of Formula I.

An ADC of Formula I can be used, for example, to treat cancer or to induce or promote an immune response, in combination with a "free" CDN that is not conjugated to an antibody or antigen-binding fragment of Formula I. The free CDN can be administered prior to, simultaneously with, or subsequent to the administration of the ADC of Formula I. In such cases, the free CDN can be the same as or different from the CDN conjugated to the antibody in the ADC of Formula I. The free CDN can be a cGAMP, such as 2'3'-cGAMP or an analog or derivative thereof, or a pharmaceutically acceptable salt thereof. In other embodiments, the free CDN is 3'3'-cGAMP, 2'2'-cGAMP, 3'2'-cGAMP or an analog or derivative thereof, or a pharmaceutically acceptable salt thereof.

Accordingly, the present invention provides methods for treating cancer in a subject by administering a pharmaceutical composition comprising a pharmaceutically acceptable amount of an ADC of the present invention (i.e., an ADC of Formula I) with at least one CDN that is not conjugated to an antibody ("free CDN"). The ADC of Formula I and the free CDN may be administered together or separately, and when administered separately, the administration may be simultaneous or sequential, in any order, in any suitable manner, separately or in combination.

In one embodiment, the free CDN that is administered in combination with the ADC of Formula I is the compound depicted below or a pharmaceutically acceptable salt thereof:

.

In another embodiment, the free CDN that is administered in combination with the ADC of Formula I is the compound depicted below or a pharmaceutically acceptable salt thereof:

.

In another embodiment, the free CDN that is administered in combination with the ADC of Formula I is the compound depicted below or a pharmaceutically acceptable salt thereof:

.

In another embodiment, the free CDN that is administered in combination with the ADC of Formula I is the compound depicted below or a pharmaceutically acceptable salt thereof:

.

In another embodiment, the free CDN that is administered in combination with the ADC of Formula I is the compound depicted below or a pharmaceutically acceptable salt thereof:

.

In another embodiment, the free CDN that is administered in combination with the ADC of Formula I is the compound depicted below or a pharmaceutically acceptable salt thereof:

.

In another embodiment, the free CDN that is administered in combination with the ADC of Formula I is the compound depicted below or a pharmaceutically acceptable salt thereof:

.

In another embodiment, the free CDN that is administered in combination with the ADC of Formula I is the compound depicted below or a pharmaceutically acceptable salt thereof:

.

In another embodiment, the free CDN that is administered in combination with the ADC of Formula I is the compound depicted below or a pharmaceutically acceptable salt thereof:

.

In another embodiment, the free CDN that is administered in combination with the ADC of Formula I is the compound depicted below or a pharmaceutically acceptable salt thereof:

.

In another embodiment, the free CDN that is administered in combination with the ADC of Formula I is the compound depicted below or a pharmaceutically acceptable salt thereof:

.

In another embodiment, the free CDN that is administered in combination with the ADC of Formula I is the compound depicted below or a pharmaceutically acceptable salt thereof:

.

In another embodiment, the free CDN that is administered in combination with the ADC of Formula I is the compound depicted below or a pharmaceutically acceptable salt thereof:

.

In another embodiment, the free CDN that is administered in combination with the ADC of Formula I is the compound depicted below or a pharmaceutically acceptable salt thereof:

.

In another embodiment, the free CDN that is administered in combination with the ADC of Formula I is the compound depicted below or a pharmaceutically acceptable salt thereof:

.

In another embodiment, the free CDN that is administered in combination with the ADC of Formula I is the compound depicted below or a pharmaceutically acceptable salt thereof:

.

In another embodiment, the free CDN that is administered in combination with the ADC of Formula I is the compound depicted below or a pharmaceutically acceptable salt thereof:

.

In another embodiment, the free CDN that is administered in combination with the ADC of Formula I is the compound depicted below or a pharmaceutically acceptable salt thereof:

.

In another embodiment, the free CDN that is administered in combination with the ADC of Formula I is the compound depicted below or a pharmaceutically acceptable salt thereof:

.

7. Examples

The following Examples, which highlight certain distinctive properties and characteristics of illustrative embodiments of CDNs, ADCs, and methods of using these ADCs to treat patients, are provided for illustrative purposes only and are not limiting.

Abbreviations

¹ H-NMR Proton nuclear magnetic resonance spectroscopy ¹⁹ F-NMR Nuclear Magnetic Resonance Spectroscopy on ¹⁹ F Nuclei ³¹ P-NMR Nuclear magnetic resonance spectroscopy on ³¹ P nuclei G Guanine A Adenine A ^Bz 6N-benzoyladenine Gib 2N-isobutyryl Bz Benzoyl DHUK Dichloroacetic acid DXM Dichloromethane DMOCP 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide DMT 4,4'-dimethoxytrityl DMTCl 4,4'-dimethoxytrityl chloride PBS Phosphate buffered saline Py. Pyridine TBS tert-butyldimethylsilyl TrCl Tris(hydroxymethyl)aminomethane hydrochloride IBX 2-iodoxybenzoic acid LAH Lithium aluminum hydride DMF Dimethylformamide NMM N-Methylmorpholine Et ₃ N Triethylamine

Example 1. Obtaining CDN-A

The synthesis of CDN (“CDN-A”) of the present invention is depicted below in Schemes A1 and A2. The synthesis and characterization of CDN and its synthetic intermediates are described below.

Synthesis of intermediate 19 from 1

Scheme A1. Synthesis of intermediate 19

Scheme A1. Synthesis of intermediate 19 (continued)

Step 1: Synthesis of (3aR,5R,6S,6aR)-5-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-6-ol

A solution of ( _2R ,4S,5R)-5-(hydroxymethyl)tetrahydrofuran-2,3,4-triol (100 g, 0.67 mol) in acetone (2.6 L) containing _H2SO4 (conc., 184.00 g, 1.88 mol, 100 mL, 2.8 equiv) was stirred at 20 °C for 1 h. A solution of _Na2CO3 ( ₁₃₀ g, 1.23 mol, 1.8 equiv) in _H2O (600 mL) was carefully added at 0 ^o C. The resulting mixture was stirred for another 2.5 h, then a second portion _{of Na2CO3} (70 g, 0.66 mol) was added. After 0.5 h, the precipitate was isolated by filtration and washed with acetone (0.5 L × 3). The filtrate was evaporated and purified by column chromatography (SiO ₂ , DCM:MeOH = 10:1 to 5:1) to give (3aR,5R,6R)-5-(hydroxymethyl)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-ol (53.5 g, 0.28 mol, 85% yield) as a yellow oil. (MS: [M+Na]+ 213.0).

Step 2: Synthesis of 3aR,5R,6S,6aR)-2,2-dimethyl-5-((trityloxy)methyl)tetrahydrofuro[2,3-d][1,3]dioxol-6-ol

To a solution of (3aR,5R,6R)-5-(hydroxymethyl)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-ol (125 g, 0.66 mol) in pyridine (600 mL) was added TrCl (219.9 g, 0.79 mol, 1.2 equiv). After 16 h at 60 °C, the mixture was cooled and evaporated. The residue was partitioned between CH ₂ Cl ₂ (400 mL) and aq. NaHCO ₃ (sat., 800 mL). The aqueous phase was extracted with dichloromethane (600 mL × 2). The combined organic layers were dried over _Na2SO4 , filtered, evaporated and purified by column chromatography ( _{SiO2 ,} petroleum ether/ethyl acetate = 10:1 to 5:1) to give (3aR,5R,6R)-2,2-dimethyl-5-(trityloxymethyl)-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-ol (250 g, 0.58 mol, 88% yield) as a white solid. (MS: [M+Na]+ 455.0).

Step 3: Synthesis of (3aR,5R,6aS)-2,2-dimethyl-5-((trityloxy)methyl) dihydrofuro[2,3-d][1,3]dioxol-6(3aH)-one

To a solution of (3aR,5R,6R)-2,2-dimethyl-5-(trityloxymethyl)-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-ol (250 g, 0.58 mol) in _CH3CN (1.5 L) was added IBX (323 g, 1.2 mol, 2.00 equiv). The resulting mixture was stirred at 90°C for 6 h. After cooling, the mixture was filtered. The filtrate was evaporated to give (3aR,5R,6aS)-2,2-dimethyl-5-(trityloxymethyl)-3a,6a-dihydrofuro[2,3-d][1,3]dioxol-6-one (240 g, 0.56 mol, 96.5% yield) as a light yellow oil. (MS: [M+Na]+ 453.0).

Step 4: Synthesis of (E)-methyl 2-((3aR,5S,6aR)-2,2-dimethyl-5-((trityloxy)methyl)furo[2,3-d][1,3]dioxol-6(3aH,5H,6aH)-ylidene)acetate

Methyl 2-dimethoxyphosphoryl acetate (66 g, 0.36 mol, 60% in oil, 1.3 equiv) was added dropwise to a mixture of NaH (14.5 g, 0.36 mol, 60% in oil, 1.3 equiv) and THF (1.00 L) at 0°C over 15 min. The resulting mixture was stirred at the same temperature for 45 min, then a solution of (3aR,5R,6aS)-2,2-dimethyl-5-(trityloxymethyl)-3a,6a-dihydrofuro[2,3-d][1,3]dioxol-6-one (120 g, 0.28 mol, 1 equiv) in THF (500 mL) was added dropwise. After 15 h at 25 °C, the reaction was quenched by adding NH ₄ Cl (sat., 50 mL) at 0 °C. The resulting mixture was evaporated and partitioned between brine (500 mL) and CH ₂ Cl ₂ (500 mL x 3). The combined organic layers were dried over Na ₂ SO ₄ , filtered, evaporated and purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 15:1 to 5:1) to give methyl (2E)-2-[(3aR,5S,6aR)-2,2-dimethyl-5-(trityloxymethyl)-3a,6a-dihydrofuro[2,3-d][1,3]dioxol-6-ylidene]acetate (65 g, 0.53 mol, 96% yield) as a light yellow oil. (MS: [M+Na]+ 509.0).

Step 5: Synthesis of methyl 2-((3aR,5S,6R,6aR)-2,2-dimethyl-5-((trityloxy)methyl)tetrahydrofuro[2,3-d][1,3]dioxol-6-yl)acetate

To a solution of methyl (2E)-2-[(3aR,5S,6aR)-2,2-dimethyl-5-(trityloxymethyl)-3a,6a-dihydrofuro[2,3-d][1,3]dioxol-6-ylidene]acetate (260 g, 0.53 mol) in EtOAc (700 mL) was added Pd/C (10% on carbon, 10 g) under nitrogen atmosphere. The suspension was degassed and purged with hydrogen three times. The resulting mixture was stirred under hydrogen atmosphere (20 psi) at 25°C for 16 h. The catalyst was isolated by filtration. The filtrate was evaporated and purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 15:1 to 10:1) to give methyl 2-[(3aR,5S,6S)-2,2-dimethyl-5-(trityloxymethyl)-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-yl]acetate (210 g, 0.43 mol, 80.4% yield) as a white solid. (MS: [M+Na]+ 511.1).

Step 6: Synthesis of 2-((3aR,5S,6R,6aR)-2,2-dimethyl-5-((trityloxy)methyl)tetrahydrofuro[2,3-d][1,3]dioxol-6-yl)ethanol

To a mixture of LiAlH ₄ (15.5 g, 0.41 mol, 2 equiv.) and THF (500 mL) was slowly added a solution of methyl 2-[(3aR,5S,6S)-2,2-dimethyl-5-(trityloxymethyl)-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-yl]acetate (100 g, 0.20 mol) in THF (20 mL) at 0 °C. After stirring for 2.5 h at 25 °C, the reaction was quenched by adding water (15 mL) and NaOH (aq., 15%, 15 mL) at 0 °C. The resulting crude product was dried over _{Na2SO4 ,} filtered, evaporated and purified by column chromatography ( _SiO2 , petroleum ether/ethyl acetate = 5:1 to 2:1) to give 2-[(3aR,5S,6S)-2,2-dimethyl-5-(trityloxymethyl)-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-yl]ethanol (80 g, 0.35 mol, 85% yield) as a light yellow oil.

¹ H-NMR (400 MHz, CDCl3) δ= 7.43 - 7.35 (m, 6H), 7.25 - 7.18 (m, 6H), 7.18 - 7.11 (m, 3H), 5.82 (d, J = 3.8 Hz, 1H), 4.62 (t, J = 4.2 Hz, 1H), 3.86 (td, J = 3.5, 10.2 Hz, 1H), 3.61 - 3.47 (m, 2H), 3.37 (dd, J = 2.8, 10.7 Hz, 1H), 3.02 (dd, J = 4.1, 10.7 Hz, 1H), 2.13 (tt, J = 4.8, 9.9 Hz, 1H), 1.73 - 1.62 (m, 2H), 1.42 (s, 3H), 1.40 - 1.31 (m, 1H), 1.26 (s, 3H). MS: [M+Na]+ 483.2.

Step 7: Synthesis of (3aR,5S,6R,6aR)-6-(2-(benzyloxy)ethyl)-2,2-dimethyl-5-((trityloxy)methyl)tetrahydrofuro[2,3-d][1,3]dioxole

To a mixture of NaH (27.1 g, 0.68 mol, 60% in oil, 4.00 equiv.) and THF (500 mL) was added dropwise a solution of 2-[(3aR,5S,6S)-2,2-dimethyl-5-(trityloxymethyl)-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-6-yl]ethanol (78 g, 0.17 mol) in THF (200 mL) at -20 °C over 5 min. After the addition, the mixture was stirred at 25 °C for 2 h. BnBr (60.3 mL, 0.51 mol, 3.00 equiv.) was added dropwise. The resulting mixture was stirred at 80 °C for 14 h. After cooling to 0 °C, the reaction was quenched by addition of aq. NH ₄ Cl (sat., 20 mL), diluted with water (400 mL) and extracted with dichloromethane (400 mL x 3). The combined organic layers were dried over Na ₂ SO ₄ , filtered, evaporated and purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 15:1 to 5:1) to give (3aR,5S,6S)-6-(2-benzyloxyethyl)-2,2-dimethyl-5-(trityloxymethyl)-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxole (90 g, 0.16 mol, 97% yield) as a white solid. (MS: [M+Na]+ = 573.1).

Step 8: Synthesis of (3aR,5S,6R,6aR)-6-(2-(benzyloxy)ethyl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)methanol

To a solution of (3aR,5S,6S)-6-(2-benzyloxyethyl)-2,2-dimethyl-5-(trityloxymethyl)-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxole (90 g, 0.16 mol) in _CH2Cl2 (300 _mL ) was added _CHCl2COOH (30 mL, 0.16 mol, 1.00 equiv). After 3 h at 25 °C, the reaction mixture was neutralized with aqueous _NaHCO3 (sat., 500 mL) to pH~7.0 at 0 °C. The resulting crude product was extracted with dichloromethane (100 mL x 3). The combined organic layers were dried over MgSO ₄ , filtered, evaporated and purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 5:1 to 2:1) to give [(3aR,5S,6S)-6-(2-benzyloxyethyl)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]methanol (44 g, 0.14 mol, 87% yield) as a yellow oil.

Step 9: Synthesis of (3aR,5S,6R,6aR)-6-(2-(benzyloxy)ethyl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)methyl benzoate

To a solution of [(3aR,5S,6S)-6-(2-benzyloxyethyl)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]methanol (62 g, _0.2 mol) in _CH2Cl2 (200 mL) were added BzCl (35 mL, 0.3 mol, 1.50 equiv) and _Et3N (55.7 mL, 0.4 mol, 2 equiv). After 1 h at 25°C, the reaction mixture was evaporated and purified by column chromatography ( _SiO2 , petroleum ether/ethyl acetate = 15:1 to 10:1) to give [(3aR,5S,6S)-6-(2-benzyloxyethyl)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]methyl benzoate (80 g, 0.19 mol, 97% yield) as a light yellow oil. (MS: [M+Na]+ 435.1).

Step 10: Synthesis of ((2S,3S,4R)-3-(2-(benzyloxy)ethyl)-4,5-dihydroxytetrahydrofuran-2-yl)methyl benzoate

To a mixture of [(3aR,5S,6S)-6-(2-benzyloxyethyl)-2,2-dimethyl-3a,5,6,6a-tetrahydrofuro[2,3-d][1,3]dioxol-5-yl]methyl benzoate (20 g, 49 mmol) and _H2O (6 mL) was added HOAc (28 mL, 10 equiv). The resulting mixture was stirred at 100 °C for 5 h. After cooling, the reaction mixture was neutralized with aqueous _NaHCO3 (sat., 2 L) and extracted with dichloromethane (400 mL x 3). The combined organic layers were evaporated to give [(2S,3R,5R)-3-(2-benzyloxyethyl)-4,5-dihydroxy-tetrahydrofuran-2-yl]methyl benzoate (17.5 g, 47 mmol, 96% yield) as a light yellow oil, which was used in the next step without purification (MS: [M+ Na]+ 395.1).

Step 11: Synthesis of (3R,4R,5S)-5-((benzoyloxy)methyl)-4-(2-(benzyloxy)ethyl) tetrahydrofuran-2,3-diyl acetate

Ac ₂ O (0.75 mol, 70.4 mL, 4.0 equiv) was added to a solution of [(2S,3R,5R)-3-(2-benzyloxyethyl)-4,5-dihydroxy-tetrahydrofuran-2-yl]methyl benzoate (70 g, 0.19 mol) in pyridine (300 mL). The resulting mixture was stirred at 60 °C for 4 h. After cooling to 25 °C, the reaction mixture was neutralized with aqueous NaHCO ₃ (sat.) to pH~7 and extracted with dichloromethane (300 mL x 3). The organic layers were evaporated and purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 10:1 to 5:1) to give [(2S,3S,5S)-4,5-diacetoxy-3-(2-benzyloxyethyl)tetrahydrofuran-2-yl]methyl benzoate (80 g, 93% yield) as a white solid. (MS: [M+ Na]+ 479.1).

Step 12: Synthesis of ((2S,3R,4R,5R)-4-acetoxy-3-(2-(benzyloxy)ethyl)-5-(2-isobutyramido-6-oxo-1H-purine-9(6H)- yl)tetrahydrofuran-2-yl)methyl benzoate

To a solution of 2-methyl-N-(6-oxo-1,9-dihydropurin-2-yl)propanamide (18.9 g, 85.4 mmol, 1.30 equiv.) in _CH3CN (300 mL) was added BSA (84.5 mL, 341.7 mmol, 5.2 equiv.) at 20°C. After stirring at 65°C for 0.5 h, the mixture was cooled and evaporated. The residue was dissolved in MeCN (600 mL), then a solution of [(2S,3S,5S)-4,5-diacetoxy-3-(2-benzyloxyethyl)tetrahydrofuran-2-yl]methyl benzoate (30 g, 65.7 mmol) in MeCN (150 mL) and TMSOTf (17.8 mL, 98.6 mmol, 1.5 equiv) was added at -15 °C. The resulting mixture was stirred at 65 °C for 15 h. After cooling, the mixture was evaporated and purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 5:1 to 1:1) to give ((2S,3R,4R,5R)-4-acetoxy-3-(2-(benzyloxy)ethyl)-5-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-2-yl)methyl benzoate (30 g, 48.6 mmol, 74% yield) as a white solid. (MS: [M+1]+ 618.1).

1H-NMR (400 MHz, chloroform-d) δ = 12.00 (s, 1H), 9.11 (s, 1H), 7.92 - 7.84 (m, 2H), 7.82 - 7, 76 (m, 1H), 7.58 (t, J = 7.1 Hz, 1H), 7.46 - 7.37 (m, 2H), 7.27 - 7.16 (m, 5H), 5 .90 - 5.85 (m, 1H), 5.74 (d, J = 5.3 Hz, 1H), 4.78 - 4.61 (m, 2H), 4.55 - 4.38 (m , 3H), 3.55 (t, J = 5.8 Hz, 2H), 3.23 - 3.14 (m, 1H), 2.47 (spt, J = 6.9 Hz, 1H), 2.22 - 2.10 (m, 3H), 1.83 (q, J = 6.1 Hz , 2H), 1.17 (dd, J = 6.9, 8.9 Hz, 6H).

Step 13: Synthesis of ((2S,3R,4R,5R)-4-acetoxy-3-(2-hydroxyethyl)-5-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran -2-yl)methyl benzoate

To a solution of [(2S,3S,5R)-4-acetoxy-3-(2-benzyloxyethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methyl benzoate (25 g, 40.5 mmol) in EtOH (500 mL) were added Pd/C (38 g, 10% on carbon) and HOAc (25.00 mL, 437.1 mmol, 11 equiv) under N ₂ . The resulting suspension was purged with hydrogen 3 times and stirred under hydrogen atmosphere (40 psi) for 48 h at 50 °C. After cooling, the reaction mixture was filtered. The filtrate was evaporated and purified by column chromatography ( _SiO2 , petroleum ether/ethyl acetate = 5:1 to 2:1) to give [(2S,3S,5R)-4-acetoxy-3-(2-hydroxyethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methyl benzoate (20 g, 37.9 mmol, 94% yield) as a white solid. (MS: [M+1]+ 528.3).

Step 14: Synthesis of [(2S,3R,5R)-4-acetoxy-3-(2-iodoethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methyl benzoate

To a solution of [(2S,3R,5R)-4-acetoxy-3-(2-hydroxyethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methyl benzoate (3 g, 5.69 mmol, 1 equiv.) in THF (90 mL) were added imidazole (1.16 g, 17.06 mmol, 3 equiv.) and triphenylphosphine (4.47 g, 17.06 mmol, 3 equiv.) in one portion, then a solution of I ₂ (2.60 g, 10.24 mmol, 1.8 equiv.) in THF (10 mL) was slowly added. The reaction mixture was stirred at 25 °C for 16 h, quenched with saturated aqueous Na ₂ SO ₃ (8 mL) and evaporated to give a residue. The residue was dissolved in ethyl acetate (80 mL) and washed with water (80 mL). The aqueous layer was extracted with ethyl acetate (150 mL x 3). The combined organic layers were dried over anhydrous Na ₂ SO ₄ and evaporated under reduced pressure. The residue was purified by column chromatography ( _SiO2 , petroleum ether/ethyl acetate = 1:1 to 1:3) to give [(2S,3R,5R)-4-acetoxy-3-(2-iodoethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methyl benzoate (2.3 g, 64% yield) as a yellow solid. (MS: [M+1]+ 638.2).

Step 15: Synthesis of [(2S,3R,4R,5R)-4-acetoxy-3-(2-azidoethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl] tetrahydrofuran-2-yl]methyl benzoate

To a solution of [(2S,3R,4R,5R)-4-acetoxy-3-(2-iodoethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methyl benzoate (3.8 g, 5.96 mmol, 1 equiv.) in THF (40 mL) were added NaN ₃ (2.52 g, 38.75 mmol, 6.5 equiv.) and H ₂ O (10 mL). The resulting mixture was stirred at 50 °C for 2 h. The reaction mixture was quenched with saturated aqueous Na ₂ CO ₃ (50 mL) and extracted with ethyl acetate (100 mL x 3). The combined organic layers were dried over _{anhydrous Na2SO4} and evaporated under reduced pressure to give [(2S,3R,4R,5R)-4-acetoxy-3-(2-azidoethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methyl benzoate (2 g, 61% yield) as a yellow solid. (MS: [M+1]+ 553.1).

Step 16: Synthesis of N-[9-[(2R,3R,4S,5S)-4-(2-azidoethyl)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methylpropanamide

To a solution of [(2S,3R,5R)-4-acetoxy-3-(2-azidoethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methyl benzoate (2.55 g, 4.62 mmol, 1 equiv.) in EtOH (220 mL) was added aq. NaOH (2M, 23 mL, 10 equiv.) at 0 °C. The resulting mixture was stirred at room temperature for 0.5 h. HCOOH was added to the reaction mixture to adjust pH = 7~8 at 0 °C, and the mixture was evaporated under reduced pressure to give a residue. The residue was purified by prep-HPLC (0.1% TFA in MeCN and water, 0%~70%) to give N-[9-[(2R,3R,4S,5S)-4-(2-azidoethyl)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methylpropanamide (1.6 g, 3.94 mmol, 85% yield) as a white solid. (MS: [M+1]+407.1).

Step 17: Synthesis of N-[9-[(2R,3R,4S,5S)-4-(2-azidoethyl)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-hydroxy-tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide

To a solution of N-[9-[(2R,3R,4S,5S)-4-(2-azidoethyl)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methylpropanamide (1.6 g, 3.94 mmol) in pyridine (15 mL) was added DMTCl (1.60 g, 4.72 mmol, 1.2 equiv). The resulting mixture was stirred at 25 °C for 3 h. The reaction mixture was quenched by adding MeOH (10 mL) at 25 °C. The resulting mixture was evaporated under reduced pressure to yield a residue as a yellow oil. The residue was purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 1/1 to EtOH/ethyl acetate = 1:250) to give N-[9-[(2R,3R,4S,5S)-4-(2-azidoethyl)-5-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]-3-hydroxy-tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide (950 mg, 1.34 mmol, 34% yield) as a yellow solid. (MS: [M+1]+709.4).

Step 18: Synthesis of [(2R,3R,4R,5S)-4-(2-azidoethyl)-5-(hydroxymethyl)-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxyphosphinic acid

To a solution of N-[9-[(2R,3R,4S,5S)-4-(2-azidoethyl)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-hydroxytetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methylpropanamide (700 mg, 988 μmol) in pyridine (6 mL) was added diphenyl phosphite (809.5 mg, 3.46 mmol, 664 μL, 3.5 equiv). After 1 h at 25 °C, DCM (5 mL) and Et ₃ N (3 mL) were added, and the mixture was stirred at 25 °C for another 1.5 h. The resulting mixture was evaporated, and the residue was partitioned between DCM (20 mL) and aqueous solution. NaHCO ₃ (5%, 20 mL). The organic phase was evaporated to give a crude intermediate, which was redissolved in a mixture of H ₂ O (3 mL) and 2,2-dichloroacetic acid (382 mg, 2.96 mmol, 243 µL, 3 eq.) in DCM (10 mL). The resulting mixture was stirred at 25 °C for 0.5 h. The reaction was quenched by adding Et ₃ N (3.0 mL), then the mixture was evaporated to give a residue. The residue was purified on a reverse phase column (0.1% TEA in MeCN and water, 0%~70%) to give [(2R,3R,4R,5S)-4-(2-azidoethyl)-5-(hydroxymethyl)-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxyphosphinic acid (400 mg, 82% yield, 95% purity) as a white solid. (MS: [M+1]+ 471.0).

Synthesis of CDNA-A from intermediate 19

Diagram A2. Synthesis of CDN-A

Step 1: Synthesis of [(2R,3R,4R,5R)-2-[[[(2R,3R,4R,5S)-4-(2-azidoethyl)-5-(hydroxymethyl)-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxy-(2-cyanoethoxy)phosphoryl]oxymethyl]-5-(6-benzamidopurin-9-yl)-4-[tert-butyl(dimethyl)silyl]oxy-tetrahydrofuran-3-yl]oxyphosphinic acid

To a solution of [(2R,3R,4R,5R)-5-(6-benzamidopurin-9-yl)-4-[tert-butyl(dimethyl)silyl]oxy-2-(hydroxymethyl)tetrahydrofuran-3-yl]oxyphosphinic acid (400 mg, 727.8 μmol) in _CH3CN (3 mL) was added a solution of tetrazole (0.45 M in MeCN, 6.47 mL, 4 equiv). The resulting mixture was stirred at 25°C for 5 min. N-[9-[(2R,3R,4R,5S)-4-(2-azidoethyl)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[2-cyanoethoxy-(diisopropylamino)phosphanyl]oxytetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide (595 mg, 655 μmol, 0.9 equiv) was added. After 0.5 h at 25 °C, 2-hydroperoxy-2-methyl-propane (197 mg, 2.18 mmol, 209 μL, 3 equiv) was added and the mixture was stirred at 25 °C for 0.5 h. Then 2,2-dichloroacetic acid (938 mg, 7.28 mmol, 598 µL, 10 equiv) in DCM (10 mL) was added. The resulting mixture was stirred at 25 °C for 25 min and quenched with saturated aqueous Na ₂ SO ₃ (2.0 mL), then pyridine (2.0 mL) was added for neutralization. The resulting mixture was evaporated to give a residue. The residue was purified by reverse phase column (0.1% TEA in MeCN and water, 0%~70%) to give [(2R,3R,4R,5R)-2-[[[(2R,3R,4R,5S)-4-(2-azidoethyl)-5-(hydroxylmethyl)-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxy-(2-cyanoethoxy)phosphoryl]oxymethyl]-5-(6-benzamidopurin-9-yl)-4-[tert-butyl(dimethyl)silyl]oxy-tetrahydrofuran-3-yl]oxyphosphinic acid (600 mg, 62% yield, 80% purity) as a white solid. (MS: [M+1]+ 1071.5).

Step 2: Synthesis of N-[9-[(27S,28R,29R,30R,31R,32R,33R,34R)-29-(2-azidoethyl)-32-[tert-butyl(dimethyl)silyl]oxy-67,68-dihydroxy-33-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]-67,68-dioxo-59,60,61,62,63,64-hexaoxa-67,68-diphosphatetricyclooctadecan-34-yl]purin-6-yl]benzamide

To a solution of [(2R,3R,4R,5S)-4-(2-azidoethyl)-5-[[[(2R,3R,4R,5R)-5-(6-benzamidopurin-9-yl)-4-[tert-butyl(dimethyl)silyl]oxy-2-(hydroxymethyl)tetrahydrofuran-3-yl]oxy-(2-cyanoethoxy)phosphoryl]oxymethyl]-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxyphosphinic acid (400 mg, 373.48 μmol) in pyridine (9 mL) was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (345 mg, 1.87 mmol, 5 equiv.). After 15 min at 25 °C, I ₂ (379 mg, 1.49 mmol, 4 equiv.) and H ₂ O (13.5 mg, 747.0 μmol, 13.5 μL, 2 equiv.) were added and the mixture was stirred at 25 °C for 0.5 h. The reaction was quenched with saturated aqueous NaHCO ₃ solution (2.0 mL) and saturated aqueous Na ₂ SO ₃ solution (2.0 mL). After evaporation, the residue was dissolved in CH ₃ CN (10 mL) and 2-methylpropan-2-amine (10 mL) was added. The resulting mixture was stirred at 25 °C for 1 h. The reaction mixture was evaporated to give a residue. The residue was purified on a reverse phase column (0.1% TEA in MeCN and water, 0%~70%) to give N-[9-[(27S,28R,29R,30R,31R,32R,33R, 34R)-29-(2-azidoethyl)-32-[tert-butyl(dimethyl)silyl]oxy-67,68-dihydroxy-33-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]-67,68-dioxo-59,60,61,62,63,64-hexaoxa-67,68-diphosphate tricyclooctadecan-34-yl]purin-6-yl]benzamide (350 mg, 310 μmol, 83% yield, 90% purity) as a white solid. (MS: [M+1]+ 1016.4).

Step 3: Synthesis of 2-amino-9-[(19S,20R,21R,22R,23R,24R,25R,26R)-26-(6-aminopurin-9-yl)-21-(2-azidoethyl)-24-[tert-butyl(dimethyl)silyl]oxy-54,55-dihydroxy-54,55-dioxo-46,47,48,49,50,51-hexaoxa-54,55-diphosphatetricyclooctadecan-25-yl]-1H-purin-6-one

N-[9-[(27S,28R,29R,30R,31R,32R,33R,34R)-29-(2-azidoethyl)-32-[tert-butyl(dimethyl)silyl]oxy-67,68-dihydroxy-33-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]-67,68-dioxo-59,60,61,62,63,64-hexaoxa-67,68-diphosphate tricyclooctadecan-34-yl]purin-6-yl]benzamide (300 mg, 295 μmol) was dissolved in _MeNH2 /EtOH (5M, 2.95 mL), and the mixture was stirred at 25 °C for 2 h. The resulting mixture was evaporated to give a residue. The residue was purified by reverse phase column (0.1% TEA in MeCN and water, 0%~35%) to give 2-amino-9-[(19S,20R,21R,22R,23R,24R,25R,26R)-26-(6-aminopurin-9-yl)-21-(2-azidoethyl)-24-[tert-butyl(dimethyl)silyl]oxy-54,55-dihydroxy-54,55-dioxo-46,47,48,49,50,51-hexaoxa-54,55-diphosphatetricyclooctadecan-25-yl]-1H-purin-6-one (160 mg, 52% yield, 80% purity) as a white solid. (MS: [M+1]+ 842.3).

Step 4: Synthesis of 2-amino-9-[(19S,20R,21R,22R,23R,24R,25R,26R)-21-(2-aminoethyl)-26-(6-aminopurin-9-yl)-24-[tert-butyl(dimethyl)silyl]oxy-52,53-dihydroxy-52,53-dioxo-44,45,46,47,48,49-hexaoxa-52,53-diphosphatetricyclooctadecan-25-yl]-1H-purin-6-one

To a solution of 2-amino-9-[(19S,20R,21R,22R,23R,24R,25R,26R)-26-(6-aminopurin-9-yl)-21-(2-azidoethyl)-24-[tert-butyl(dimethyl)silyl]oxy-54,55-dihydroxy-54,55-dioxo-46,47,48,49,50,51-hexaoxa-54,55-diphosphatetricyclooctadecan-25-yl]-1H-purin-6-one (100 mg, 118.8 μmol) in MeOH (8 mL) was added Pd/C (30 mg, 10% purity) in one portion, and the mixture was stirred at 25 °C under hydrogen atmosphere (10 psi) for 5 h. The resulting mixture was filtered and the filtrate was evaporated to give a residue. The residue was purified on a reverse phase column (0.1% TEA in MeCN and water, 0%~30%) to give 2-amino-9-[(19S,20R,21R,22R,23R,24R,25R,26R)-21-(2-aminoethyl)-26-(6-aminopurin-9-yl)-24-[tert-butyl(dimethyl)silyl]oxy-52,53-dihydroxy-52,53-dioxo-44,45,46,47,48,49-hexaoxa-52,53-diphosphatetricyclooctadecan-25-yl]-1H-purin-6-one (70 mg, 77.2 μmol, 65% yield, 90% purity) as a white solid. (MS: [M+1]+ 816.5).

Step 5: Synthesis of 2-amino-9-[(14S,15R,16R,17R,18S,19R,20R,21R)-16-(2-aminoethyl)-20-(6-aminopurin-9-yl)-17 ,45,46-trihydroxy-45,46-dioxo-39,40,41,42,43,44-hexaoxa-45,46-diphosphatetricyclooctadecan-21-yl]-1H-purin-6-one (CDN-A)

To a solution of 2-amino-9-[(19S,20R,21R,22R,23R,24R,25R,26R)-21-(2-aminoethyl)-26-(6-aminopurin-9-yl)-24-[tert-butyl(dimethyl)silyl]oxy-52,53-dihydroxy-52,53-dioxo-44,45,46,47,48,49-hexaoxa-52,53-diphosphatetricyclooctadecan-25-yl]-1H-purin-6-one (35 mg, 42.9 μmol) in MeOH (3 mL) was added NH ₄ F (127 mg, 3.43 mmol, 80 equiv) in one portion, and the mixture was stirred at 70 °C for 0.25 h. After cooling to room temperature, the mixture was evaporated to give a residue. The residue was purified by reverse phase column (0.1% HCOOH in MeCN and water, 0%~30%) to give 2-amino-9-[(14S,15R,16R,17R,18S,19R,20R,21R)-16-(2-aminoethyl)-20-(6-aminopurin-9-yl)-17,45,46-trihydroxy-45,46-dioxo-39,40,41,42,43,44-hexaoxa-45,46-diphosphatetricyclooctadecan-21-yl]-1H-purin-6-one (CDNA, 8.6 mg, 11.9 μmol, 28% yield, 97% purity) as a white solid. (MS: [M+1]+ 702.0).

1H NMR (D2O+buffer, 400 MHz): δ (ppm) 8.19 (s, 1H), 8.18 (s, 1H), 7.76 (s, 1H), 6.08 (s , 1H), 5.83-5.81 (m, 1H), 5.71-5.67 (m, 1H), 5.03-5.01 (m, 1H), 4.42-4.37 (m, 2H), 4.30-4.28 (m, 1H), 4.18-4.16 (m, 1H), 4.07-4.01 (m, 2H), 3.13-3 .11 (m, 2H), 2.68-2.66 (m, 1H), 2.33-2.30 (m, 1H), 1.85-1.83 (m, 1H). 31P NMR (D2O+buffer): δ (ppm) -1.15, -2.47.

Example 2. Obtaining CDN-B

Below, Schemes B1 and B2 depict the synthesis of CDN (“CDN-B”) of the present invention. The synthesis and characterization of this CDN and the synthetic intermediates used are described below.

Synthesis of intermediate 29 from intermediate 14

Scheme B1: Synthesis of intermediate 29

Step 1: Synthesis of [(2S,3R,4R,5R)-4-acetoxy-3-(2-chloroethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl] tetrahydrofuran-2-yl]methyl benzoate

To a solution of [(2S,3R,4R,5R)-4-acetoxy-3-(2-hydroxyethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methyl benzoate (19 g, 36.0 mmol) in DMF (100 mL) were added PPh ₃ (23.6 g, 90.0 mmol, 2.5 equiv) and CCl ₄ (17.3 mL, 180.1 mmol, 5 equiv). After stirring for 16 h at 25 °C, the reaction was quenched by adding saturated aqueous NaHCO ₃ solution (150 mL) and extracted with ethyl acetate (80 mL × 2). The combined organic layers were evaporated and purified by column chromatography (SiO ₂ , petroleum ether/ethyl acetate = 10:1 to 1:1) to give [(2S,3R,4R,5R)-4-acetoxy-3-(2-chloroethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methyl benzoate (13 g, 23.8 mmol, 66% yield) as a light yellow solid. (MS: [M+1]+ 546.2).

Step 2: Synthesis of [(2S,3R,4R,5R)-4-acetoxy-3-(2-acetylsulfanylethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl] tetrahydrofuran-2-yl]methyl benzoate

To a solution of [(2S,3R,4R,5R)-4-acetoxy-3-(2-chloroethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methyl benzoate (15 g, 27.5 mmol) in DMF (100 mL) was added AcSK (7.84 g, 68.7 mmol, 2.5 equiv). The reaction mixture was stirred at 50 °C for 16 h. After cooling, the reaction mixture was diluted with dichloromethane (200 mL) and washed with aqueous NaHCO ₃ solution (sat., 200 mL). The organic layer was evaporated to give [(2S,3R,4R,5R)-4-acetoxy-3-(2-acetylsulfanylethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methyl benzoate (16 g) as a light yellow oil, which was used in the next step without purification. (MS: [M+1]+ 586.3).

Step 3: Synthesis of N-[9-[(2R,3R,4S,5S)-3-hydroxy-5-(hydroxymethyl)-4-(2-sulfanylethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide

To a solution of [(2S,3R,4R,5R)-4-acetoxy-3-(2-acetylsulfanylethyl)-5-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methyl benzoate (16 g, 27.3 mmol) in EtOH (160 mL) was added NaOH (2 M, 68.3 mL, 5 equiv) at 0 °C. The reaction mixture was stirred at 0 °C for 0.5 h. The pH was adjusted to 7 with HOAc. The resulting mixture was evaporated in vacuo to remove most of the solvent. The formed brown precipitate was separated and treated with DCM/MTBE (1/100, v/v, 200 mL). After filtration, the filtrate was evaporated to give N-[9-[(2R,3R,4S,5S)-3-hydroxy-5-(hydroxymethyl)-4-(2-sulfanylethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide (11 g, crude, ~10% disulfide) as a brown solid, which was used in the next step without further purification. (MS: [M+1]+ 398.1).

Step 4: Synthesis of N-[9-[(2R,3R,4S,5S)-3-hydroxy-5-[[(3-methoxyphenyl)-(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-[2-[(3-methoxyphenyl)-(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide

To a solution of N-[9-[(2R,3R,4S,5S)-3-hydroxy-5-(hydroxymethyl)-4-(2-sulfanylethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methylpropanamide (11 g, 27.7 mmol) in pyridine (110 mL) was added DMTCl (28.1 g, 83.0 mmol, 3 equiv). After 16 h at 25 °C, the reaction was quenched with aqueous NaHCO ₃ (sat., 200 mL) and extracted with ethyl acetate (200 mL x 2). The organic phase was dried over _Na2SO4 , filtered and purified by silica gel chromatography using PE:EE (EA:EtOH = 3:1) = 10:1 to ~ 2:1 as eluent to give N-[9-[( _2R ,3R,4S,5S)-3-hydroxy-5-[[(3-methoxyphenyl)-(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-[2-[(3-methoxyphenyl)-(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide (14 g, 47.0% yield, 93% purity) as a light yellow solid. (MS: [M+1]+ 1002.5).

Step 5: Synthesis of [(2R,3R,4R,5S)-5-(hydroxymethyl)-4-[2-[(3-methoxyphenyl)-(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxyphosphinic acid

To a solution of N-[9-[(2R,3R,4S,5S)-3-hydroxy-5-[[(3-methoxyphenyl)-(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-[2-[(3-methoxyphenyl)-(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methylpropanamide (12 g, 8.4 mmol) in pyridine (120 mL) was added phenoxyphosphonoyloxybenzene (7.51 mL, 29.3 mmol, 3.5 equiv) at 25 °C. After 1 h, Et ₃ N/H ₂ O (100 mL, 1:1) was added. After 0.5 h, the mixture was extracted with ethyl acetate (200 mL x 2). The organic phase was evaporated and then dissolved in aqueous AcOH (80%, 120 mL). The resulting mixture was stirred at 25 °C for 1 h. The reaction mixture was neutralized by adding saturated aqueous Na ₂ CO ₃ solution at 0 °C until pH ~ 7. The resulting mixture was directly purified by reversed-phase column (CH ₃ CN/H ₂ O, 0 ~ 60%) to afford [(2R,3R,4R,5S)-5-(hydroxymethyl)-4-[2-[(3-methoxyphenyl)-(4-methoxyphenyl)-phenylmethyl]sulfanylethyl]-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxyphosphinic acid (4.2 g, 5.5 mmol, 65% yield) as a white solid. (MS: [M+1]+ 764.4).

Synthesis of CDN-B from intermediate 29

Scheme B2. Synthesis of CDN-B

Step 1: Synthesis of [(2R,3R,4R,5S)-5-[[[(2R,3R,4R,5R)-5-(6-benzamidopurin-9-yl)-4-[tert-butyl(dimethyl)silyl]oxy-2-(hydroxymethyl)tetrahydrofuran-3-yl]oxy-(2-cyanoethoxy)phosphoryl]oxymethyl]-4-[2-[bis(4-methoxyphenyl)-phenylmethyl]sulfanylethyl]-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxyphosphinic acid

To a solution of [(2R,5S)-4-[2-[bis(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]-5-(hydroxymethyl)-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxyphosphinic acid (0.75 g, 0.98 mmol) in 1H-tetrazole (0.45 M in MeCN, 22.50 mL, 10 equiv) was added N-[9-[(2R,3R,4R,5R)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[tert-butyl(dimethyl)silyl]oxy-4-[2-cyanoethoxy-(diisopropylamino)phosphanyl]oxy-tetrahydrofuran-2-yl]purin-6-yl]benzamide (1.1 g, 1.1 mmol, 1.1 equiv). After 1 h, TBHP (0.43 mL, 65% in decane, 3 equiv) was added. The reaction mixture was stirred at 25 °C for 0.5 h. The reaction was quenched by adding aqueous sodium bisulfite solution (33%, 4 mL) at 0 °C and extracted with ethyl acetate (100 mL x 2). The organic phase was evaporated and dissolved in aqueous AcOH (80%, 20 ml). After 1 h, the reaction mixture was neutralized with aqueous _Na2CO3 (sat _. ) at 0°C. The resulting mixture was purified by reverse phase column ( _CH3CN / _H2O , neutral conditions, 0~60%) to give [(2R,3R,4R,5S)-5-[[[(2R,3R,4R,5R)-5-(6-benzamidopurin-9-yl)-4-[tert-butyl(dimethyl)silyl]oxy-2-(hydroxymethyl)tetrahydrofuran-3-yl]oxy-(2-cyanoethoxy)phosphoryl]oxymethyl]-4-[2-[bis(4-methoxyphenyl)-phenylmethyl]sulfanylethyl]-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxyphosphinic acid (0.79 g, 48% yield, 80% purity) as a white solid. (MS: [M+1]+ 1364.0).

Step 2: Synthesis of N-[9-[(47S,48R,49R,50R,51R,52R,53R,54R)-49-[2-[bis(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]-52-[tert-butyl(dimethyl)silyl]oxy-87,88-dihydroxy-53-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]-87,88-dioxo-77,78,79,80,81,82-hexaoxa-87,88-diphosphatetricyclooctadecan-54-yl]purin-6-yl]benzamide

To a solution of [(2R,3R,4R,5S)-5-[[[(2R,3R,4R,5R)-5-(6-benzamidopurin-9-yl)-4-[tert-butyl (dimethyl)silyl]oxy-2-(hydroxymethyl)tetrahydrofuran-3-yl]oxy-(2-cyanoethoxy) phosphoryl]oxymethyl]-4-[2-[bis(4-methoxyphenyl)-phenylmethyl]sulfanylethyl]-2-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl]oxyphosphinic acid (0.79 g, 0.58 mmol) in pyridine (16 mL) was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (0.7 g, 3.8 mmol, 6.5 equiv). After 0.5 h, CCl ₄ (3.2 g, 20.6 mmol, 1.98 mL, 35.5 equiv), H ₂ O (0.16 mL, 8.79 mmol) and NMM (0.79 mL) were added. The reaction mixture was stirred at 25 °C for 0.5 h, then poured into aqueous NaHSO ₃ (sat., 10 mL). After 5 min, aqueous NaHCO ₃ (sat., 20 mL) was slowly added. The resulting mixture was extracted with ethyl acetate (30 mL x 2). The organic phase was evaporated and dissolved in CH ₃ CN (8 mL) and tBuNH ₂ (8 mL). After 0.5 h, the mixture was evaporated and purified on a reversed-phase column (CH ₃ CN/H ₂ O, neutral conditions, 0~40%) to give N-[9-[(47S,48R,49R,50R,51R,52R,53R,54R)-49-[2-[bis(4-methoxyphenyl)-phenylmethyl]sulfanylethyl]-52-[tert-butyl(dimethyl)silyl]oxy-87,88-dihydroxy-53-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]-87,88-dioxo-77,78,79,80,81,82-hexaoxa-87,88-diphosphate tricyclooctadecan-54-yl]purin-6-yl]benzamide (0.5 g, 60% yield, 90.8% purity) as a white solid. (MS: [M+1]+ 1309.8).

Step 3: Synthesis of 2-amino-9-[(39S,40R,41R,42R,43R,44R,45R,46R)-46-(6-aminopurin-9-yl)-41-[2-[bis(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]-44-[tert-butyl(dimethyl)silyl]oxy-74,75-dihydroxy-74,75-dioxo-64,65,66,67,68,69-hexaoxa-74,75-diphosphatetricyclooctadecan-45-yl]-1H-purin-6-one

A mixture of N-[9-[(47S,48R,49R,50R,51R,52R,53R,54R)-49-[2-[bis(4-methoxyphenyl)-phenyl-methyl]sulfanylethyl]-52-[tert-butyl(dimethyl)silyl]oxy-87,88-dihydroxy-53-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]-87,88-dioxo-77,78,79,80,81,82-hexaoxa-87,88-diphosphate tricyclooctadecan-54-yl]purin-6-yl]benzamide (500 mg, 0.38 mmol) and an alcoholic solution of methylamine (10 ml, 30%) was stirred at 25°C for 4 hours. The reaction mixture was evaporated in vacuo. The residue was purified by reverse phase column ( _CH3CN / _H2O , neutral conditions, 0~30%) to give 2-amino-9-[(39S,40R,41R,42R,43R,44R, 45R,46R)-46-(6-aminopurin-9-yl)-41-[2-[bis(4-methoxyphenyl)-phenylmethyl]sulfanylethyl]-44-[tert-butyl(dimethyl)silyl]oxy-74,75-dihydroxy-74,75-dioxo-64,65,66,67,68,69-hexaoxa-74,75-diphosphatetricyclooctadecan-45-yl]-1H-purin-6-one (220 mg, 48% yield, 95% purity) as a white solid.

Step 4: Synthesis of 2-amino-9-[(19S,20R,21R,22R,23R,24R,25R,26R)-26-(6-aminopurin-9-yl)-24-[tert-butyl(dimethyl) silyl]oxy-51,52-dihydroxy-51,52-dioxo-21-(2-sulfanylethyl)-43,44,45,46,47,48-hexaoxa-51,52-diphosphatetricyclooctadecan-25-yl]-1H -purine-6-ona

To a solution of 2-amino-9-[(39S,40R,41R,42R,43R,44R,45R,46R)-46-(6-aminopurin-9-yl)-41-[2-[bis(4-methoxyphenyl)-phenylmethyl]sulfanylethyl]-44-[tert-butyl(dimethyl)silyl]oxy-74,75-dihydroxy-74,75-dioxo-64,65,66,67,68,69-hexaoxa-74,75-diphosphatetricyclooctadecan-45-yl]-1H-purin-6-one (190 mg, 0.17 mmol) in DCM (4 mL) was added 2,2-dichloroacetic acid (0.8 mL, 9.74 mmol, 58 equiv.). The resulting mixture was stirred at 25°C for 1 h and neutralized with water/ _Et3N (1:1, v/v, 3 mL) at 0°C. The resulting mixture was evaporated and purified on a reversed-phase column (CH ₃ CN/H ₂ O, containing 0.05% TEA, from 0% to 40%) to give 2-amino-9-[(19S,20R,21R,22R, 23R,24R,25R,26R)-26-(6-aminopurin-9-yl)-24-[tert-butyl(dimethyl)silyl]oxy-51,52-dihydroxy-51,52-dioxo-21-(2-sulfanylethyl)-43,44,45,46,47,48-hexaoxa-51,52-diphosphate tricyclooctadecan-25-yl]-1H-purin-6-one (36 mg, 25% yield, 98% purity, TEA salt) as white solid. (MS: [M+1]+ 833.3).

Step 5: Synthesis of 2-amino-9-[(14S,15R,16R,17R,18S,19R,20R,21R)-20-(6-aminopurin-9-yl)-17,44,45-trihydroxy-44 ,45-dioxo-16-(2-sulfanylethyl)-38,39,40,41, 42,43-hexaoxa-44,45-diphosphatetricyclooctadecan-21-yl]-1H-purin-6-one (CDN-B)

To a solution of 2-amino-9-[(19S,20R,21R,22R,23R,24R,25R,26R)-26-(6-aminopurin-9-yl)-24-[tert-butyl(dimethyl)silyl]oxy-51,52-dihydroxy-51,52-dioxo-21-(2-sulfanylethyl)-43,44,45,46,47,48-hexaoxa-51,52-diphosphatetricyclooctadecan-25-yl]-1H-purin-6-one (36 mg, 0.043 mmol) in MeOH (4 mL) was added NH ₄ F (0.16 g, 4.32 mmol, 100 equiv.). The resulting mixture was stirred at 70°C for 1 h, evaporated and purified on a reversed-phase column (CH ₃ CN/H ₂ O, containing 0.05% formic acid, 0% to 30%) to give 2-amino-9-[(14S,15R,16R,17R,18S,19R,20R,21R)-20-(6-aminopurin-9-yl)-17,44,45-trihydroxy-44,45-dioxo-16-(2-sulfanylethyl)-38,39,40,41,42,43-hexaoxa-44,45-diphosphate tricyclooctadecan-21-yl]-1H-purin-6-one (CDNB, 7 mg, 22.5% yield, 99.8% purity) as a white solid.

1H NMR (400 MHz, D2O) δ = 8.25 (s, 1H), 8.19 (s, 1H), 7.77 (s, 1H), 6.10 (s, 1H), 5.80 ( d, J = 8.3 Hz, 1H), 5.67 (kv, J = 8.2 Hz, 1H), 5.05 - 4.98 (m, 1H), 4.43 (d, J = 9 .0 Hz, 1H), 4.36 (d, J = 12.1 Hz, 1H), 4.31 (b.s, 1H), 4.18 (d, J = 11.7 Hz, 1H), 4.08 - 3.97 (m, 2H), 3.11 (kv, J = 7.2 Hz, 1H), 2.79 - 2.66 (m, 2H), 2.62 - 2.51 (m, 1H), 2.28 - 2.13 (m, 1H), 1.84 - 1.73 (m, 1H ), 1.19 (t, J = 7.3 Hz, 1H). 31P NMR: -0.951, -2.201. MS: [M+1]+ 718.9.

Example 3. Obtaining target-binding antibodies

Anti-PD-L1 antibodies

Ab-A1 (mu-anti-PDL1): Expression vectors encoding the mouse anti-human PDL1 antibody AbA1 (mu-anti-PDL1) having the heavy chain of SEQ ID NO:3 and the light chain of SEQ ID NO:5 were prepared by cloning cDNAs encoding the heavy chain variable region (VH) sequence of SEQ ID NO:1 and the light chain variable region (VL) sequence of SEQ ID NO:2 into separate pFUSEssCHIgmG2a (mouse IgG2a heavy chain constant region) and pFUSE2ss-CLIg-mk (mouse kappa light chain constant region) expression vectors, respectively. The VH and VL sequences in Ab-A1 are based on atezolizumab.

Ab-A2 (mu-anti-PDL1-cys): Expression vectors encoding the murine anti-human PDL1 antibody AbA2 (mu-anti-PDL1-cys) having the heavy chain of SEQ ID NO:4 and the light chain of SEQ ID NO:5 were prepared using a procedure similar to that used for Ab-A1. The VH and VL sequences in Ab-A2 are based on atezolizumab. The heavy chain constant region of Ab-A2 has two mutations relative to wild-type mouse IgG2a, where the first is a substitution of leucine to phenylalanine at position 234 in the CH2 domain (i.e., L234F, numbering according to the wild-type mouse IgG2a sequence corresponding to the human IgG1 sequence, according to Eu numbering), and the second is a substitution of serine to cysteine at position 239 in the CH2 domain to create an additional cysteine residue for conjugation (i.e., S239C, according to the same numbering system).

The heavy chain sequences of the anti-human PD-L1 antibodies Ab-A1 and AbA2 are shown below as SEQ ID NOs:3 and 4, respectively. The heavy chain variable region is underlined in each sequence, is the same for both, and corresponds to SEQ ID NO:1. The L234F and S239C mutations of the CH2 domain in SEQ ID NO:4 for Ab-A2 are highlighted in bold and in brackets {}.

Heavy chain in Ab-A1 (SEQ ID NO:3)

SKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

Heavy chain in Ab-A2 (SEQ ID NO:4)

The sequence of the light chains in the anti-human PD-L1 antibody Ab-A1 and Ab-A2 is shown below as SEQ ID NO:5. The variable region of the light chain is underlined in the sequence and corresponds to SEQ ID NO:2.

Light chain in Ab-A1 and Ab-A2 (SEQ ID NO:5)

Ab-A3 (rt-anti-PDL1): Rat anti-mouse PD-L1 antibody Ab-A3, containing the constant region of rat IgG2b heavy chain, was purchased from the commercial supplier BioXcell (BE0101).

Anti-EGFR antibodies

Ab-B1 (mu-anti-EGFR): Mouse anti-human EGFR antibody Ab-B1, containing the constant region of the mouse IgG2a heavy chain, was purchased from the commercial supplier BioXcell (BE0279).

Ab-B2 (mu-anti-EGFR-cys): Expression vectors encoding the mouse anti-human EGFR antibody AbB2 having the heavy chain of SEQ ID NO:8 and the light chain of SEQ ID NO:9 were generated by cloning cDNAs encoding the heavy chain variable region sequence of SEQ ID NO:6 and the light chain variable region (VL) sequence of SEQ ID NO:7 into separate pFUSEssCHIgmG2a (mouse IgG2a heavy chain constant region) and pFUSE2ss-CLIg-mk (mouse kappa light chain constant region) expression vectors, respectively. The VH and VL sequences of Ab-B2 are based on cetuximab. The CH2 domain of the heavy chain of Ab-B2 contains the L234F and S239C mutations relative to wild-type mouse IgG2a, as described above for Ab-A2.

The heavy chain sequence of the anti-human EGFR antibody Ab-B2 is shown below as SEQ ID NO:8. The variable region of the heavy chain is underlined and corresponds to SEQ ID NO:6. The mutations L234F and S239C in the CH2 domain are highlighted in bold and in brackets {}.

Heavy chain in Ab-B2 (SEQ ID NO:8)

The light chain sequence of Ab-B2 is shown below as SEQ ID NO:9. The variable region is underlined and corresponds to SEQ ID NO:7.

Light chain in Ab-B2 (SEQ ID NO:9)

Ab-B3 (hu-anti-EGFR): Expression vectors encoding the mouse-human chimeric anti-human EGFR antibody AbB3, having the heavy chain of SEQ ID NO:12 and the light chain of SEQ ID NO:13, were generated by cloning cDNAs encoding the heavy chain variable region sequence of SEQ ID NO:10 plus the human IgG1 heavy chain constant region and the light chain variable region sequence of SEQ ID NO:11 plus the human Ig-kappa light chain constant region into separate pcDNA3.4 expression vectors. The full-length heavy and light chain sequences in Ab-B2 are cetuximab-based.

The heavy chain sequence of the anti-human EGFR antibody Ab-B3 is shown below as SEQ ID NO:12. The variable region of the heavy chain is underlined and corresponds to SEQ ID NO:10.

Heavy chain in Ab-B3 (SEQ ID NO:12)

The light chain sequence of Ab-B3 is shown below as SEQ ID NO:13. The variable region is underlined and corresponds to SEQ ID NO:11.

Light chain in Ab-B3 (SEQ ID NO:13)

Ab-B4 (hu-anti-EGFR-A/C,V/C): An expression vector encoding human anti-human EGFR antibody AbB4 having a heavy chain of SEQ ID NO:16 and a light chain of SEQ ID NO:17 was generated by cloning cDNAs encoding the heavy chain variable region sequence of SEQ ID NO:14 plus the heavy chain constant region of human IgG1 and the light chain variable region sequence of SEQ ID NO:15 plus the light chain constant region of modified human Ig-kappa into separate pcDNA3.4 expression vectors. The VH and VL sequences of Ab-B4 are based on cetuximab, with VH having an alanine to cysteine mutation at position 109 adjacent to the CH1 domain (i.e., A109C). The light chain in Ab-B4 has a valine to cysteine mutation at position 205 (i.e. V205C).

The heavy chain sequence of the anti-human EGFR antibody Ab-B4 is shown below as SEQ ID NO:16. The variable region of the heavy chain is underlined and corresponds to SEQ ID NO:14. The A109C mutation is highlighted in bold and in brackets {}.

Heavy chain in Ab-B4 (SEQ ID NO:16)

The light chain sequence of Ab-B4 is shown below as SEQ ID NO:17. The variable region is underlined and corresponds to SEQ ID NO:15. The V205C mutation is highlighted in bold and in brackets {}.

Light chain in Ab-B4 (SEQ ID NO:17)

Anti-HER2 antibodies

Ab-C1 (anti-HER2): Humanized anti-human HER2 antibody Ab-C1 was purchased from a commercial supplier as trastuzumab (Herceptin®).

The heavy chain sequence of the anti-human HER2 antibody Ab-C1 is shown below as SEQ ID NO:20. The variable region of the heavy chain is underlined and corresponds to SEQ ID NO:18.

Heavy chain in Ab-C1 (SEQ ID NO:20)

The light chain sequence of Ab-C1 is shown below as SEQ ID NO:21. The variable region is underlined and corresponds to SEQ ID NO:19.

Light chain in Ab-C1 (SEQ ID NO:21)

Anti-CD47 antibodies

Ab-D1 (rt-anti-CD47): Rat anti-mouse CD47 antibody Ab-D1, which has the constant region of the rat IgG2b heavy chain, was purchased from the commercial supplier BioXcell (BE0270).

Expression and purification

Plasmids encoding the heavy chain and light chain of target-binding antibodies were transfected into CHO cells for antibody expression using ExpiFectamine™ CHO Transfection Kit (ThermoFisher Scientific, Cat No: A29129) according to the manufacturer's protocol. The total amount of plasmids for transfection was 0.5 μg/ml CHO cells, and the ratio of plasmids for heavy chain to light chain was 2:3. Six days after transfection, CHO cells were separated by centrifugation, filtered from the medium, then loaded onto protein A beads (HiTrap Protein A HP, GE, Cat No: 17-0403-01) and eluted with 0.1 M glycine solution (pH 3.0). Eluted antibody fractions were pooled and evaporated to 1 ml, then buffer exchanged with PBS by size separation (ENrich Sec650, Bio-Rad, Cat No: 780-1650) before storage at -80°C.

Example 4. Obtaining CDN-A ADC

Synthesis of CDNA-A-linker

CDNA (27 mg, 0.0385 mmol) was co-evaporated with pyridine (3 x 3 mL) and dried in vacuo before use. 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (54 mg, 0.0732 mmol, 2.0 equiv), HOBt (5.2 mg, 0.0385 mmol, 1.0 equiv), and DMF (2.5 mL) were added with stirring. DIPEA (67 µL, 0.385 mmol, 10 equiv) was added. The resulting mixture was stirred at room temperature under N ₂ for 42 h. The reaction mixture was diluted with a solvent mixture of ethyl acetate (25 mL), tert-butyl methyl ether (75 mL), and acetic acid (30 μL, 0.521 mmol). The solid precipitate was collected by centrifugation and washed with tert-butyl methyl ether (3 × 75 mL) to give 44 mg of crude product. 20 mg of the resulting crude product was further purified on a reversed-phase column (CH ₃ CN/H ₂ O, 0.1% formic acid, 0% to 30%) to give the desired product (7 mg, 31% yield, 97% purity). (MS: [M-1]- 1298.2).

Conjugation of antibodies to CDNA

Reduction of disulfides in the antibody: Target-binding antibodies were first reduced with tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP) (2.3 molar equivalents of TCEP) at room temperature for 1.5 hours, and with 40 molar equivalents of TCEP at 37°C for 2 hours. Excess TCEP was removed using a desalting column (HiTrap Desalting column, GE, Cat. No: 29048684).

Reoxidation of hinge cysteines: The hinge cysteines in the reduced antibody were reoxidized with 200 molar equivalents of dehydroascorbic acid (DHA, 0.5 M in DMSO) at 37°C for 4 h. Reoxidation was performed by SDS-PAGE under non-reducing conditions. Excess DHA was removed using a 30 kDa molecular weight cutoff centrifugal filter (Amicon Ultra-15, Merck Millipore, Cat No: UFC903024).

Conjugation with CDNA: (Type I for reduced antibody) The reaction mixture with reduced antibody was cooled at 4°C for 20 min. CDNA-Linker (4.8 molar equivalents relative to antibody) in histidine buffer (20 mM, pH 7.4) was added and incubated at 4°C for 80 min. (Type II for reoxidized antibody) The reoxidized antibody was mixed with 10 molar equivalents of CDNA-Linker and incubated at room temperature for 4 h. After conjugation, the reaction was quenched with N-acetylcysteine and excess CDNA-Linker and N-acetylcysteine were removed on a 5 ml desalting column (GE) under FPLC control in phosphate-buffered saline (PBS). The resulting ADCs were evaporated and stored at 4°C.

Measuring DAR

To determine the DAR in ADCs containing CDN-A as the CDN, the OD260/OD280 ratio was determined using a UV spectrometer and compared with a standard curve constructed from mixtures of CDN-A and antibody with known proportions. As shown in the table below, the DAR value in the example ADCs ranged from 1 to 6.3. The DAR value in ADCs using Ab-A2 antibody (mu-anti-PDL1-cys) containing an additional cysteine after substitution was higher than that using the wild-type Ab-A1 antibody (mu-anti-PDL1).

Table 1: Examples of CDN-A ADC

ADC Ab Note on Ab CDN DAR ADC-IV Ab-B1 mu-anti-EGFR (BioXcell BE0279) CDN-A 1 ADC-V Ab-A3 rt-anti-PDL1 (BioXcell BE0101) CDN-A 1 ADC-VI Ab-A2 mu-anti-PDL1-cys CDN-A 2~4 ADC-VII Ab-B2 mu-anti-EGFR-cys CDN-A 2 ADC-VIII Ab-B3 hu-anti-EGFR CDN-A 6.3 ADC-IX Ab-B4 hu-anti-EGFR-A/C,V/C CDN-A 3 ADC-X Ab-C1 hu-anti-HER2 (trastuzumab) CDN-A 4.8 ADC-XI Ab-D1 rt-anti-CD47 (BioXCell BE0270) CDN-A 2.5

Example 5. Obtaining CDN-B ADC

Synthesis of 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfaneyl)pentanoate (linker I)

EDC (HCl salt, 61 mg, 0.32 mmol) was added to a solution of 4-(pyridin-2-yldisulfaneyl)pentanoic acid (24 mg, 0.1 mmol) and NHS (14 mg, 0.12 mmol) in DMA. The resulting solution was stirred at room temperature overnight. After filtration, the filtrate was evaporated and purified by column (MeOH/DCM = 0% to 10%) to give 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfaneyl)pentanoate as a white solid (8 mg, 23.5%). (MS: [M+1]+ 341.1).

Conjugation of antibodies to CDN-B

0.2 ml of 3 mM 2-amino-9-[(14S,15R,16R,17R,18S,19R,20R,21R)-20-(6-aminopurin-9-yl)-17,44,45-trihydroxy-44,45-dioxo-16-(2-sulfanylethyl)-38,39,40,41,42,43-hexaoxa-44,45diphosphatetricyclooctadecan-21-yl]-1H-purin-6-one solution in phosphate buffer pH 6.0 was mixed with 0.2 ml of 2 mM 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfaneyl)pentanoate in DMA. After incubation at room temperature overnight, 3.6 mg of target-binding antibody in 2 ml of PBS was added and incubated at room temperature for another 2 h. The resulting mixture was evaporated, and the resulting conjugate was purified on a 5-ml desalting column under FPLC control equilibrated with phosphate-buffered saline (PBS). The resulting ADCs were evaporated and stored at 4°C.

Measuring DAR

To release CDN-B from ADC, DTT was added to ADC to a final concentration of 10 mM, incubated at 95°C for 5 min, and passed through a filter with a nominal molecular weight cutoff of 30 kDa. The filtrate was diluted and added to THP1-Lucia ISG cells whose membrane permeabilization had been artificially increased by the addition of 0.5 nM perfringolysin (PFO). After 16 h, luciferase activity was measured, the concentration of active compounds released from the ADC was calculated, and DAR values were calculated by comparison with standard curves. As shown in the table below, the DAR value for the example ADCs ranged from 0.33 to 1.66.

Table 2: Examples of CDN-B ADC

ADC Ab Note on Ab CDN DAR ADC-I Ab-A1 mu-anti-PDL1 CDN-B 1.66 ADC-II Ab-A3 rt-anti-PDL1 (BioXcell BE0101) CDN-B 0.36 ADC-III Ab-B1 mu-anti-EGFR (BioXcell BE0279) CDN-B 0.33

Example 6. Cellular activity of ADCs with CDNA-A and CDNA-B

Interferon-stimulating activity

Two reporter cell lines, murine RAWLucia ISG macrophages and human THP1-Lucia ISG monocytes, were used to assess ADC activity. Both cell lines contain interferon-stimulated response elements (ISREs) linked to the ISG54 minimal promoter. ADC at different concentrations (0.3, 1.0, and/or 3 μM) were added to reporter cells and incubated for 16 h. Luciferase activity, corresponding to induced interferon expression, was compared with serial dilutions of a standard compound (cGAMP).

ADC-I - ADC-XI showed effective interferon-stimulating activity of varying potency as shown in Table 3. The reported “cGAMP equivalent” value refers to the concentration of cGAMP (μM) required to achieve the same level of response as that provided by 1 μM ADC.

Table 3: Strength of interferon-stimulating activity in reporter cells

ADC mouse RAW-Lucia ISG
(cGAMP equivalent) human THP1-Lucia ISG
(cGAMP equivalent) ADC-I 89 28 ADC-II 3.4 4.2 ADC-III ~7 0.58 ADC-IV ~10 weakly ADC-V ~10 weakly ADC-VI >100 weakly ADC-VII 3.1 not defined ADC-VIII 33 not defined ADC-IX 31 not defined ADC-X 10 not defined ADC-XI 3 not defined

All the ADCs showed high interferon-stimulating activity in the mouse RAWLucia ISG reporter cell assay (Table 3). ADC-I and ADC-VI had the highest potency (Figs. 1B and 6B, respectively), followed by ADC-VIII, ADC-IX (Figs. 8B and 9B, respectively), then ADC-IV, ADC-V and ADC-X (Figs. 4B, 5B and 10, respectively), and then ADC-III (Fig. 3B), ADC-II (Fig. IIB) and ADC-XI. As shown in Figs. 3B, 4B, 5B and 6B, the addition of the ADCs of the present invention showed synergism in interferon-stimulating activity compared to the addition of antibody alone or CDN alone, or the combined value of antibody and agonist.

CDNA-A antibody conjugates also demonstrated interferon-stimulating activity in a test using human THP1-Lucia ISG monocytes as reporter cells (Table 3). Of those tested, ADC-I (Fig. 1C) showed the highest potency, followed by ADC-II (Fig. 1C) and ADC-III.

The activity of ADC-VIII and ADC-IX was further tested in the THP1-lucia ISG cell line, which stably expresses human EGFR. Both ADCs showed potent interferon-stimulating activity in these cells (Table 4). The reported “cGAMP equivalent” value indicates the concentration of cGAMP (nM) required to achieve the same level of response as 1 nM of ADC. EC ₅₀ is the concentration of half-maximal effect.

Table 4: Activity of ADC-VIII and ADC-IX on THP1-EGFR-lucia ISG cells

ADC cGAMP equivalent EC50 ADC-VIII 977 11.7 nM ADC-IX 912 8.2 nM

Example 7. Antitumor efficacy of ADCs with CDN-A and CDN-B

The antitumor efficacy of selected ADCs was tested in the syngeneic mouse tumor models of B16F10 metastatic melanoma, human EGFR-transfected B16F10 (B16F10-EGFR), and human HER2-transfected Lewis lung carcinoma (LLC1-HER2). Briefly, ¹⁰⁶ logarithmic-phase tumor cells in 100 μl PBS were injected subcutaneously into the right flank of C57BL6 mice. After 4–6 days, when the tumor volume was 50–100 mm ³ , mice were reassorted into groups based on their tumor size and injected intraperitoneally with 50–200 μg ADC or unconjugated antibody, unconjugated CDN, or PBS placebo control (see Figure legends for details).

Tumor volume and survival of mice were monitored. ADC-IV, ADC-VI, ADC-VII, ADC-VIII, and ADC-IX showed high antitumor efficacy in both volume reduction and survival, and both are potential candidates for use in the treatment of human tumors. ADCX was shown to slow tumor growth. ADC-I also showed tumor growth slowdown, but toxicity was observed.

Tumor volume

In B16F10 tumor-bearing mice, administration of ADCI (anti-PDL1-CDNB) slowed tumor progression compared with placebo, anti-PDL1 antibody alone, CDNB alone, and the combination of anti-PDL1 antibody and unconjugated CDNB (Fig. 1C).

In mice bearing B16F10-EGFR tumors, administration of ADCIV (anti-EGFR-CDN-A) slowed tumor growth and reduced tumor volume compared to placebo or anti-PDL1 antibody (Figs. 4C and 4E). Comparative administration of anti-EGFR antibody plus unconjugated CDN did not arrest tumor growth (Fig. 4C), suggesting improved efficacy when using ADCs to target CDN. When ADCIV was combined with anti-PDL1 antibody, complete inhibition of tumor growth was observed (Fig. 4E), suggesting improved efficacy when combining CDN ADCs with immune checkpoint inhibitors.

In mice bearing B16F10-EGFR tumors, intraperitoneal administration of ADC-VI (anti-PDL1-CDNA) at a dose of 200 μg (Fig. 6C) or intratumoral administration of 10 μg or 50 μg (Fig. 6D) prevented tumor growth. Within two weeks after ADCVI administration, B16F10-EGFR tumors showed complete remission, while administration of anti-PDL1 antibody alone or placebo did not stop tumor growth.

In B16F10-EGFR tumor-bearing mice, administration of ADC-VII (anti-EGFR-CDN-A) resulted in a significant reduction in tumor growth compared to placebo or anti-PDL1 antibody (Figs. 7C and 7E). Comparative administration of anti-EGFR antibody plus unconjugated CDN did not arrest tumor growth (Fig. 7C), suggesting improved efficacy when using ADCs to target CDN. Enhanced tumor suppression was observed when ADC-VII was combined with anti-PDL1 antibody, whereas anti-PDL1 antibody alone or placebo did not arrest tumor growth (Fig. 7E), suggesting improved efficacy when combining CDN ADCs with immune checkpoint inhibitors.

In mice bearing B16F10-EGFR tumors, administration of ADCVIII (anti-EGFR CDNA-A) resulted in a significant reduction in tumor growth (Fig. 8C). This effect was enhanced when ADC-VIII was combined with anti-PDL1 antibody, whereas administration of anti-PDL1 antibody alone or placebo did not stop tumor growth, suggesting improved efficacy when CDNA ADCs are combined with immune checkpoint inhibitors.

In mice bearing B16F10-EGFR tumors, administration of ADC-IX (anti-EGFR-CDNA) resulted in decreased tumor growth, and a similar effect was observed when ADC-IX was combined with anti-PDL1 antibody (Fig. 9C). Administration of anti-PDL1 antibody alone or placebo did not stop tumor growth.

In LLC1-HER2 tumor-bearing mice, administration of ADC-X (anti-HER2-CDN-A) reduced tumor growth in two separate mouse assays (Figs. 10C and 10E), whereas administration of anti-PDL1 antibody alone or placebo did not arrest tumor growth. Comparative administration of anti-HER2 antibody plus unconjugated CDN did not arrest tumor growth, suggesting improved efficacy when using ADCs to target CDN. Fig. 10E further shows that intratumoral administration of 30 μg ADC-X resulted in complete tumor remission.

Survival

The survival of mice bearing B16F10-EGFR or LLC1-HER2 tumors was monitored. Treatment with ADCIV (anti-EGFR-CDNA) increased the survival time of B16F10-EGFR tumor-bearing mice (3/5 at Day 37, Fig. 4D; 0/5 at Day 39, Fig. 4F) compared with anti-PDL1 antibody alone (0/5 at Day 26, Fig. 4D; 0/5 at Day 29, Fig. 4F) and placebo (0/5 at Day 23, Fig. 4D; 0/5 at Day 20, Fig. 4F). Survival following ADC-IV administration was superior to that of the comparative anti-EGFR antibody plus unconjugated CDN (0/5 at Day 34, Fig. 4D), suggesting improved efficacy when using ADCs to target CDN. Combination administration of ADC-IV with anti-PDL1 antibody increased survival to 80% of mice (4/5 at Day 42), suggesting improved efficacy when combining CDN ADCs with immune checkpoint inhibitors.

Administration of ADC-VI (anti-PDL1-CDNA) intraperitoneally resulted in complete survival of B16F10-EGFR tumor-bearing mice (5/5 at Day 43) compared with administration of anti-PDL1 antibody alone (0/5 at Day 29) or placebo (0/5 at Day 27) (Fig. 6D). Similarly, intratumor administration of ADC-VI resulted in complete survival of tumor-bearing mice (Fig. 6F).

Administration of ADCVII (anti-EGFR-CDNA-A) increased survival of B16F10-EGFR tumor-bearing mice at the endpoint of the experiment (2/5 at Day 36, Figs. 7D and 7F) compared with administration of anti-PDL1 antibody alone (0/5 at Day 27, Fig. 7F) or placebo (0/5 at Day 23, Figs. 7D and 7F). Survival after administration of ADC-VII was superior to that of the comparative administration of anti-EGFR antibody plus unconjugated CDNA (0/5 at Day 22, Fig. 7D), suggesting improved efficacy when using ADC to target CDNA. Combination administration of ADC-VII with anti-PDL1 antibody increased survival by 60% of mice (3/5 at Day 36, Fig. 7F) compared to ADC-VII alone, suggesting improved efficacy when combining CDN ADCs with immune checkpoint inhibitors.

Administration of ADCVIII (anti-EGFR-CDNA) increased survival of B16F10-EGFR tumor-bearing mice (1/5 at Day 35) compared with anti-PDL1 antibody alone (0/5 at Day 27) or placebo (0/5 at Day 25) (Fig. 8D). Combination administration of ADC-VIII and anti-PD-L1 antibody increased survival to 80% of mice (4/5 at Day 35), suggesting improved efficacy when combining CDNA ADC with immune checkpoint inhibitors.

Administration of ADCIX (anti-EGFR-CDNA) increased survival of B16F10-EGFR tumor-bearing mice (3/5 at Day 30) compared with anti-PDL1 antibody alone (0/5 at Day 23) or placebo (0/5 at Day 19) (Fig. 9D). Combination administration of ADC-IX and anti-PD-L1 antibody increased survival to 60% of mice (3/5 at Day 30), suggesting improved efficacy when combining CDNA ADC with immune checkpoint inhibitors.

Administration of ADCX (anti-HER2-CDNA-A) increased the survival time of LLC1-HER2 tumor-bearing mice (0/5 at Day 41) compared to placebo (0/5 at Day 32) (Fig. 10D). Survival time after administration of ADC-X was superior to that of the comparative administration of anti-HER2 antibody plus unconjugated CDNA (0/5 at Day 32), indicating improved efficacy when using ADCs to target CDNA.

--->

SEQUENCE LIST

<110> ImmuneSensor Therapeutics, Inc.

The Board of Regents of the University of Texas System

<120> ANTIBODY-STING AGONIST CONJUGATES AND THEIR APPLICATIONS

IN IMMUNOTHERAPY

<130> 400160-005WO

<150> US 63/019,212

<151> 2020-05-01

<150> US 62/876,590

<151> 2019-07-19

<160> 21

<170> PatentIn version 3.5

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Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

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Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

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Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

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Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

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Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Ala Asp Ala Ala

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Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly

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Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

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Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

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Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

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Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

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<212> PRT

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Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

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Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

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Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

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Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Ala Pro Ser Val Tyr

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Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu

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Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

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Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser

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Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys

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Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Phe Leu Gly Gly Pro

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Cys Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser

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Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp

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Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr

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Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val

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Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu

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Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg

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Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val

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Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr

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Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr

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Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu

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Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys

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Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

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Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

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Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

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Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala

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Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly

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Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

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Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

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Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

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Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

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Phe Asn Arg Asn Glu Cys

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<210> 10

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<212> PRT

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Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

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Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

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Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

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Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

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Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

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Thr Leu Val Thr Val Ser Ala

115

<210> 11

<211> 107

<212> PRT

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Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

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Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

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Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

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Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

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Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

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<210> 12

<211> 449

<212> PRT

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Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

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Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

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Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

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Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

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Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

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Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe

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Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

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Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

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Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

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Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

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Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 13

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

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Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

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Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

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Phe Asn Arg Gly Glu Cys

210

<210> 14

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

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Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Cys

115

<210> 15

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 15

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 16

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 16

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Cys Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 17

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 17

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Cys Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 18

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 18

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 19

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 19

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 20

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 20

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 21

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 21

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<---

Claims (106)

1. An antibody-drug conjugate (ADC) having a structure of formula Ia: (Formula Ia) Ab-[-LD] _n Where: “Ab” is an antibody or its binding fragment that binds to an antigen associated with tumor or cancer-related immune cells; “L” in each case independently represents a linker linking one or more D to Ab; “D” represents the drug; “n” is the number of D linked to Ab via a linker (L), wherein the ADC is distinguished in that the said medicinal product is a cyclic dinucleotide (CDN) having a structure of formula IIk: Where W and Y represent CH; X and Z represent N; R ¹ is C _2-4 alkyl substituted by a thiol group, an amino group, or a C _1-6 alkylamino group; R ^P in each case independently represents hydroxyl or thiol; or a pharmaceutically acceptable salt thereof; where CDN (D) is covalently bonded to a linker (L) via a thiol group, amino group, or C _1-6 alkylamino group at the R ¹ position of CDN. 2. ADC according to claim 1, wherein R ¹ is C _2-4 alkyl substituted with an amino group. 3. ADC according to claim 1, wherein R ¹ is C _2-4 alkyl substituted with a thiol group. 4. An ADC according to claim 1, wherein the ADC is obtained from a structure of formula IIn: Formula IIn or a pharmaceutically acceptable salt thereof. 5. An ADC according to claim 1, wherein the ADC is derived from a structure of formula IIo: Formula IIo or a pharmaceutically acceptable salt thereof. 6. The ADC of claim 1, wherein the ADC has a structure corresponding to formula III: Formula III. 7. The ADC of claim 1, wherein the ADC has a structure corresponding to formula IV: Formula IV. 8. ADC according to claim 1, wherein the ADC is obtained from a CDN having the following structure: or a pharmaceutically acceptable salt thereof. 9. ADC according to claim 1, wherein the ADC is obtained from a CDN having the following structure: or a pharmaceutically acceptable salt thereof. 10. An ADC according to any one of claims 1-9, wherein L comprises one or more cleavable linkers. 11. An ADC according to any one of claims 1 to 10, wherein L is linked to Ab via a maleimide group, an activated disulfide, an active ester, a haloformate, an acid halide, an alkyl halide, or a benzyl halide. 12. An ADC according to any one of claims 1-11, having a covalent linkage formed between L and Ab via a thiol group or a primary amino group on Ab. 13. The ADC of claim 1, wherein the ADC has the following structure: 14. The ADC of claim 1, wherein the ADC has the following structure: . 15. An ADC according to any one of claims 1-14, wherein the Ab is an antibody that is bound to PD-L1, CD47, an antigen preferentially expressed or overexpressed in cancer cells, or an antigen obtained from a microbe that infects human cells. 16. An ADC according to any one of claims 1-14, wherein the Ab is an antibody that is bound to an antigen that is a growth factor receptor (GFR). 17. The ADC of claim 16, wherein the GFR is EGFR. 18. The ADC of claim 16, wherein the GFR is ErbB. 19. The ADC of claim 16, wherein the GFR is ErbB2 (HER2). 20. The ADC of claim 16, wherein the GFR is a receptor from the HER family. 21. A pharmaceutical composition for stimulating an immune response, comprising a pharmaceutically acceptable amount of an ADC according to any one of claims 1-20. 22. A method for stimulating an immune response in a human patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition of claim 21. 23. A method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable amount of an ADC according to any one of claims 1-20, wherein the cancer is selected from lung cancer, breast cancer, bowel cancer and kidney cancer. 24. A compound which is a compound of formula IIk: Formula IIk Where W and Y represent CH; X and Z represent N; R ¹ is ethyl substituted by an amino group or a C _1-6 alkylamino group; R ^P in each case independently represents hydroxyl or thiol; or a pharmaceutically acceptable salt thereof. 25. The compound according to claim 24, wherein R ¹ is ethyl substituted with an amino group. 26. A compound according to any one of claims 24-25, wherein in at least one instance R ^P is hydroxyl. 27. A compound according to any one of claims 24-25, wherein in at least one instance R ^P is a thiol. 28. The compound of claim 24, wherein the compound is or a pharmaceutically acceptable salt thereof. 29. A composition for conjugating a cyclic dinucleotide (CDN) to a linker, comprising: a pharmaceutically acceptable amount of CDN; and a linker or a cross-linking agent, or both a linker and a cross-linking agent; wherein CDN is a compound according to any one of claims 24-28, and the crosslinking agent ensures crosslinking of CDN with the linker. 30. The composition according to item 29, where the CDN has the following structure: or a pharmaceutically acceptable salt thereof. 31. A compound of formula L-CDNH, where L is a linker that contains a site capable of binding to a complementary site in an antibody or antigen-binding fragment; CDN is a cyclic dinucleotide with the structure of formula IIk: Formula IIk Where W and Y represent CH; X and Z represent N; R ¹ is C _1-6 alkyl substituted by a thiol group, an amino group, or a C _1-6 alkylamino group; R ^P in each case independently represents hydroxyl or thiol; or a pharmaceutically acceptable salt thereof; and CDN is linked to L via the thiol group, amino group, or C _1-6 alkylamino group in R ¹ . 32. The compound according to claim 31, wherein CDN is bonded to L via a thioether, amide, ester, carbamate, carbonate, urea, disulfide group or ether group. 33. The compound according to claim 32, wherein the compound has a structure corresponding to formula IXb: Formula IXb where R ^L represents the remainder of the linker L. 34. The compound according to claim 32, wherein the compound has a structure corresponding to formula IXf: Formula IXf where R ^L represents the remainder of the linker L. 35. A compound according to any one of claims 31 to 34, wherein R ¹ is ethyl substituted with an amino group. 36. A compound according to any one of claims 31 to 35, wherein in at least one instance R ^P is hydroxyl. 37. A compound according to any one of claims 31 to 35, wherein in at least one instance R ^P is a thiol. 38. The compound according to claim 31, wherein the CDN has the following structure: or a pharmaceutically acceptable salt thereof. 39. The compound according to claim 31, wherein the CDN has the following structure: or a pharmaceutically acceptable salt thereof.