Antibody-drug conjugates containing STING agonists

Abstract

Methods for treating diseases, disorders, and / or conditions that benefit from modulation of STING (stimulator of interferon genes) are provided. The present disclosure provides scaffolds and antibody-drug conjugates (ADCs) comprising stimulator of interferon genes (STING). The present disclosure also provides uses of the ADCs in treatments, such as cancer treatments.

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 004,108, filed April 2, 2020, U.S. Provisional Application No. 63 / 040,755, filed June 18, 2020, and U.S. Provisional Application No. 63 / 111,820, filed November 10, 2020, the contents of each of which are incorporated by reference herein in their entirety.

[0002] INCORPORATION BY REFERENCE OF SEQUENCE LISTING The contents of the text file named "MRSN-033_001WO_SeqList.txt", created on March 31, 2021, and 49KB in size, are incorporated by reference herein in their entirety. [Background technology]

[0003] background Stimulator of interferon genes (STING) is an endoplasmic reticulum receptor that mediates innate immune detection of cytosolic pathogen-derived DNA and self-DNA. STING is a 378-amino acid protein containing three main structural domains: (i) an N-terminal transmembrane domain (aa1-154); (ii) a central globular domain (aa155-341); and (iii) a C-terminal tail (aa342-379). STING can form a symmetric dimer in a V-shaped conformation upon binding to its ligand, but does not completely cover the bound ligand. STING agonists can enter and bind to the pocket region of STING. However, in some severe disease states, the STING activation process is easily inhibited, resulting in inactivation of the STING pathway. Therefore, screening and designing potent STING agonists is crucial for cancer immunotherapy and other infectious disease treatments, including, but not limited to, obesity, liver injury, glucose-lipid metabolism, and viral infections. Specific targeting of immune pathways offers opportunities for cancer therapy, potentially offering greater specificity than cell population-based therapeutic approaches.

[0004] Antibody-drug conjugates (ADCs) consist of a drug, such as a small molecule, covalently attached to an antibody. The antibody is the targeting mechanism, tailored to a specific site of action. Once at this site, the ADC is designed to release the small molecule drug, allowing it to perform its intended function, as opposed to diffusing systemically throughout the subject's body. This targeted approach allows for treatment with drugs that would otherwise require toxicly high doses when administered systemically.

[0005] A key feature of the innate immune system is its ability to recognize and eliminate foreign substances. Identification of these pathogenic invaders is achieved through host recognition of evolutionarily conserved microbial structures known as pathogen-associated molecular patterns (PAMPs). Host recognition can occur through multiple pathways, including pattern recognition receptor (PRR) activation, which ultimately leads to downstream signaling events and ultimately initiates an immune response.

[0006] The antibody-drug conjugates of the present disclosure modulate STING activity and may therefore provide beneficial therapeutic effects in the treatment of diseases, disorders, and / or conditions that benefit from modulation of STING (stimulator of interferon genes), including, but not limited to, inflammation, allergic and autoimmune diseases, infectious diseases, cancer, precancerous syndromes, and as vaccine adjuvants. There remains a need for new immunotherapies for treating diseases, particularly cancer. Summary of the Invention

[0007] overview In some aspects, the present disclosure provides a method for manufacturing a semiconductor device comprising: Formula (I): PBRM-[A 1 -(L C ) 0または1 -D]d 15 (I) or a pharmaceutically acceptable salt or solvate thereof, wherein PBRM stands for protein-based recognition molecule, L C is a linker unit, when present, A 1 L C When there is a PBRM, C or L C is a bivalent linker moiety connecting the PBRM to D when absent, D is a STING agonist drug moiety; d 15 is an integer from about 1 to about 20, Conjugates, or pharmaceutically acceptable salts or solvates thereof, are provided.

[0008] In some aspects, the present disclosure provides a method for manufacturing a semiconductor device comprising: Useful for conjugating with a PBRM, the compound of formula (II): A 1’ -(L C ) 0または1 -D (II) or a pharmaceutically acceptable salt or solvate thereof, wherein: PBRM stands for protein-based recognition molecule, L C is a linker unit, when present, A 1’ is a monovalent linker moiety containing a functional group capable of forming a covalent bond with a functional group of the PBRM; D is a STING agonist drug moiety; d 15 is an integer from about 1 to about 20, A scaffold, or a pharmaceutically acceptable salt or solvate thereof, is provided.

[0009] In some aspects, the present disclosure provides pharmaceutical compositions comprising a conjugate described herein and one or more pharmaceutically acceptable carriers or excipients.

[0010] In some aspects, the present disclosure provides a method of activating or enhancing the activity of stimulator of interferon genes (STING) in a subject, comprising administering to the subject a conjugate described herein or a pharmaceutically acceptable salt thereof.

[0011] In some aspects, the present disclosure provides a method of preventing or treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a conjugate described herein, or a pharmaceutically acceptable salt thereof.

[0012] In some aspects, the present disclosure provides a conjugate described herein, or a pharmaceutically acceptable salt thereof, for activating or enhancing STING activity in a subject.

[0013] In some aspects, the present disclosure provides a conjugate described herein, or a pharmaceutically acceptable salt thereof, for preventing or treating a disease or disorder in a subject.

[0014] In some aspects, the present disclosure provides the use of a conjugate described herein, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for activating or enhancing STING activity in a subject.

[0015] In some aspects, the present disclosure provides the use of a conjugate described herein, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for preventing or treating a disease or disorder in a subject.

[0016] [The present invention 1001] Formula (I): PBRM-[A 1 -(L C ) 0または1 -D]d 15 (I) or a pharmaceutically acceptable salt or solvate thereof, wherein PBRM stands for protein-based recognition molecule, L C is a linker unit, when present, A 1 L C When there is a PBRM, C or L C is a bivalent linker moiety connecting the PBRM to D when absent, D is a STING agonist drug moiety; d 15 is an integer from about 1 to about 20, The antibody-drug conjugate (ADC), or a pharmaceutically acceptable salt or solvate thereof. [The present invention 1002] Useful for conjugating with a PBRM, the compound of formula (II): A 1’ -(L C ) 0または1 -D (II) or a pharmaceutically acceptable salt or solvate thereof, wherein: L C is a linker unit, when present, A 1’ is a monovalent linker moiety containing a functional group capable of forming a covalent bond with a functional group of the PBRM; D is a STING agonist drug moiety; The scaffold, or a pharmaceutically acceptable salt or solvate thereof. [The present invention 1003] Each L C But when it exists, independently, TIFF0007777065000001.tif16128, wherein: # is A 1 ## indicates that it is attached to D. M A is, when present, a peptide moiety containing at least two amino acids; T 1 is a hydrophilic group, when present; L D is M A When exists, D is M A or M A When does not exist, D is replaced by A 1 is a bivalent linker moiety connecting Any of the conjugates or scaffolds of the present invention. [The present invention 1004] Formula (I-B'): Any of the conjugates of the present invention which is a conjugate of TIFF0007777065000002.tif19128, or a pharmaceutically acceptable salt or solvate thereof. [The present invention 1005] Each A 1 But independently, TIFF0007777065000003.tif51167, wherein: R 7 -O-, -NR 8 , -(C1~C 10 Alkyl)-, -(C1-C 10 alkenyl)-, -(C1-C 10 Alkynyl)-, -(C3-C8 cycloalkyl)-, -aryl-, -O-(C1-C8 alkyl)-, -O-(C1-C 10 alkenyl)-, -O-(C1-C 10 alkynyl)-, -(C1-C 10 alkyl)-(C3-C8 cycloalkyl)-, -(C1-C 10 Alkyl)-aryl-, -(C2-C 10 alkenyl)-(C3-C8 cycloalkyl)-, -(C2-C 10 alkenyl)-aryl-, -(C2-C 10 alkynyl)-(C3-C8 cycloalkyl)-, -(C2-C 10 alkynyl)-aryl-, -(C3-C8 cycloalkyl)-(C1-C 10 Alkyl, aryl (C1-C 10 Alkyl)-, -(C3-C8 cycloalkyl)-(C2-C 10 Alkenyl, aryl (C2-C 10 alkenyl)-, -(C3-C8 cycloalkyl)-(C2-C 10 Alkynyl)-, -aryl-(C2-C 10 alkynyl)-, -(3- to 8-membered heterocycloalkyl)-, -(5- to 8-membered heteroaryl)-, -(C1-C 10 alkyl)-(3-8 membered heterocycloalkyl)-, -(C1-C 10 alkyl)-(5-8 membered heteroaryl)-, -(C2-C 10alkenyl)-(3-8 membered heterocycloalkyl)-, -(C2-C 10 alkenyl)-(5-8 membered heteroaryl)-, -(C2-C 10 alkynyl)-(3-8 membered heterocycloalkyl)-, -(C2-C 10 Alkynyl)-(5-8 membered heteroaryl)-, -(3-8 membered heterocycloalkyl)-(C1-C 10 alkyl)-, -(5-8 membered heteroaryl)-(C1-C 10 alkyl)-, -(3-8 membered heterocycloalkyl)-(C2-C 10 alkenyl)-, -(5-8 membered heteroaryl)-(C2-C 10 alkenyl)-, -(5-8 membered heteroaryl)-(C2-C 10 alkynyl)-, -(5-8 membered heteroaryl)-(C2-C 10 Alkynyl)-, -OC(O)-(CH2CH2O) r -(CH2)2-, -(CH2CH2O) r - or -(CH2CH2O) r -(CH2)2-, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkyl, or heteroaryl is optionally substituted; R 8 is H, hydroxy, or C 1~4 is alkyl, r is an integer from about 1 to about 12; * indicates that it is attached to the PBRM, ** L C When exists, L C or L C When is not present, it indicates that it is attached to D. Any of the conjugates or scaffolds of the present invention. [The present invention 1006] Each A 1’ But independently, TIFF0007777065000004.tif61148, wherein: r is an integer from about 4 to about 6; ** L C When exists, L C or L C When is not present, it indicates that it is attached to D. Any of the conjugates or scaffolds of the present invention. [The present invention 1007] Each L D But independently, TIFF0007777065000005.tif12128, wherein: L E When present, -NH-[(CH2CH2O) p -(CH2) 0~2 ] q -C(O)-, -NH-(C1-C6 alkyl)-OC(O)-, or -NH-[(CH2CH2O) p -(CH2) 0~2 ] q -C(O)-NH-(C1-C6 alkyl)-OC(O)-, p is an integer of about 1 to about 20, q is an integer of about 1 to about 10, and each W is independently a natural amino acid unit or an unnatural amino acid unit; w is an integer from about 0 to about 12; *** is M A When exists, M A or M A When does not exist, A 1 indicates that it is attached to **** indicates that it is attached to D, Any of the conjugates or scaffolds of the present invention. [The present invention 1008] Any of the preceding conjugates or scaffolds of the invention, wherein w is 0, 1, 2, 3, 4, or 5. [The present invention 1009] L EWhen present, -NH-(CH2CH2O)2-(CH2)2-C(O)-, -NH-CH2-CH(CH3)-OC(O)-, or -NH-[(CH2CH2O) 1~4 Any of the conjugates or scaffolds of the present invention, wherein the conjugate or scaffold is -(CH2)2-C(O)-NH-(CH2)2-OC(O)-. [The present invention 1010] Each L D But when it exists, independently, TIFF0007777065000006.tif134151TIFF0007777065000007.tif213153TIFF0007777065000008.tif93147, wherein *** is M A When exists, M A or M A When does not exist, A 1 indicates that it is attached to **** indicates that it is attached to D, Any of the conjugates or scaffolds of the present invention. [The present invention 1011] L D but, TIFF0007777065000009.tif102164, wherein: *** is M A When exists, M A or M A When does not exist, A 1 indicates that it is attached to **** indicates that it is attached to D, Any of the conjugates or scaffolds of the present invention. [The present invention 1012] L D but, TIFF0007777065000010.tif16128, wherein: *** is M AWhen exists, M A or M A When does not exist, A 1 indicates that it is attached to **** indicates that D is attached to any of the conjugates or scaffolds of the present invention. [The present invention 1013] M A but, TIFF0007777065000011.tif22128, wherein: * is A 1 indicates that it is attached to ** is T 1 indicates that it is attached to *** L D any of the conjugates or scaffolds of the invention as described above, shown attached to [The present invention 1014] T 1 But -OH or TIFF0007777065000012.tif6128, wherein: n1 is an integer from 0 to about 6; Each R 58 are independently -H or C 1~8 is alkyl, R 60 is a bond, C 1~6 Alkyl linker, or -CHR 59 - and R 59 -H, C 1~8 alkyl, cycloalkyl, or arylalkyl; R 61 is CH2OR 62 , COOR 62 , -(CH2) n2 COOR 62 or heterocycloalkyl substituted with one or more hydroxyl groups; R 62 is -H or C 1~8 is alkyl, n2 is an integer from 1 to about 5; Any of the conjugates or scaffolds of the present invention. [The present invention 1015] T 1 but, Any of the conjugates or scaffolds of the present invention, which are TIFF0007777065000013.tif25128. [The present invention 1016] T 1 but, TIFF0007777065000014.tif15128, n4 is an integer from 1 to about 25; Each R 63 are independently hydrogen or C 1~8 is alkyl, R 64 is a bond or C 1~8 is an alkyl linker, R 65 is H, C 1~8 Alkyl, -(CH2) n2 COOR 62 , or -(CH2) n2 COR 66 and R 62 is H or C 1~8 is alkyl, R 66 H, TIFF0007777065000015.tif48144, n2 is an integer from 1 to about 5; Any of the conjugates or scaffolds of the present invention. [The present invention 1017] T 1 but, TIFF0007777065000016.tif18128, wherein: R 67 is (1)-OH; TIFF0007777065000017.tif72146, n4 is an integer of about 2 to about 20, about 4 to about 16, about 6 to about 12, or about 8 to about 12; Any of the conjugates or scaffolds of the present invention. [The present invention 1018] Any of the preceding conjugates or scaffolds of the invention, wherein n4 is 6, 7, 8, 9, 10, 11, or 12. [The present invention 1019] d 13 is an integer of about 2 to about 8. [The present invention 1020] d 13 is 6 or 8. [The present invention 1021] Each D independently represents a group of formula (A): TIFF0007777065000018.tif69128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, wherein: Y1, Y2, Z1, and Z2 are each independently O, S, C, or N; X1, X2, W1, and W2 are each independently C or N; X3 and X4 are independently S or NR f and X5 is N or CR A2 and X6 is N or CR A1 and R 3 and R 5 are each independently -CON(R d )(R f ), -CH2N(R d )(R f ), -N(R d )(R f ), -N(R d )CO(R f ), -CH2N(R d )CO(R f ) or R 3 and R 5 One of the two is -CON(R d )(R f ), -CH2N(Rd )(R f ), -N(R d )(R f ), -N(R d )CO(R f ), or -CH2N(R d )CO(R f ) and R 3 and R 5 The other of these is H, -COOH, or -CO2(R C ) and R c is C 1~4 is alkyl, R A2 and R A1 are each independently H, halogen, hydroxy, amino, amino(C 1~4 alkyl)-, optionally substituted (C 1~6 alkyl), or optionally substituted (C 1~6 alkyl)oxy-, and the optionally substituted (C 1~6 alkyl) or optionally substituted (C 1~6 Alkyl)oxy-C 1~6 Alkyl, each independently hydroxyl, C 1~4 Alkoxyl, -N(R e )(R f ), -CO2(R f ), -CON(R e )(R f ), and —COOH, Each R d are independently H, hydroxy, or C 1~4 is alkyl, R e is H, (C 1~4 alkyl), -CO(C 1~4 alkyl), -OCO(C 1~4 alkyl), and -CO2(C 1~4 alkyl), Each R f are independently H, hydroxy, or (C 1~4 alkyl), R 14and R C2 are each independently absent or C 1~4 alkyl, C 1~4 Alkyl is halogen, -OR c , -NR c R d , -CO2R c , -CONR c R d , -SO2NR c R d , and -OCONR c R d and optionally substituted with a substituent selected from R 16 and R C1 are each independently absent, H, or C 1~4 is alkyl, R 15 , R 17 , R 18 , or R 19 are each independently absent, H, or C 1~4 alkyl, C 1~4 Alkyl is halogen, -OR c , -NR c R d , -CO2R c , -CONR c R d , -SO2NR c R d , and -OCONR c R d and optionally substituted with a substituent selected from (i)R A2 and R A1 At least one of the following is present and R A2 and R A1 At least one of the C When there exists L C directly or indirectly connected to or C When does not exist, the R A2 and / or R A1 A via at least one functional group of 1 or (ii) R C2 and RC1 At least one of the following is present and R C2 and R C1 At least one of the C When there exists L C directly or indirectly connected to or C When does not exist, the R C2 and / or R C1 A via at least one functional group of 1 connected to Any of the conjugates or scaffolds of the present invention. [The present invention 1022] each D independently represents the formula (Aa), (Ab), (Ac), (Ad), (Ae), (Af), (A-f1), (A-f2), (Af)3, (A-f4), (A-f5), (Ag), (A-g1), (A-g2), (A-g3), (A-g4), (A-g5), (Ah), (A-h1), (A-h2), or (Ai): TIFF0007777065000019.tif71140TIFF0007777065000020.tif219140TIFF0007777065000021.tif210140TIFF0007777065000022.tif209140TIFF0007777065000023.tif80140, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof. [The present invention 1023] Each D is independent, TIFF0007777065000024.tif95128TIFF0007777065000025.tif196131TIFF0007777065000026.tif2 16128TIFF0007777065000027.tif211128TIFF0007777065000028.tif211126TIFF000777706500002 9.tif209128TIFF0007777065000030.tif207128TIFF0007777065000031.tif214131TIFF0007777065000032.tif191128TIFF0007777065000033.tif190128TIFF0007777065000034.tif191128, wherein R 2 is absent, -O-, or -NR 4 - and R 4 is H or C 1~3 is alkyl, TIFF0007777065000035.tif5128 is L C When exists, L C or L C When does not exist, A 1 indicates that it is attached to Any of the conjugates or scaffolds of the present invention. [The present invention 1024] Each D is independent, TIFF0007777065000036.tif185135, wherein: R 2 is absent, -O-, or -NR 4 - and R 4 is H or C 1~4 is alkyl, TIFF0007777065000037.tif5128 is L C When exists, L C or L C When does not exist, A1 indicates that it is attached to Any of the conjugates or scaffolds of the present invention. [The present invention 1025] Any of the scaffolds of the present invention selected from the scaffolds listed in Table A1. [The present invention 1026] Any of the scaffolds of the present invention selected from the scaffolds listed in Table A2. [The present invention 1027] Any of the conjugates of the present invention selected from the conjugates set forth in Table B1. [The present invention 1028] Any of the conjugates of the present invention selected from the conjugates set out in Table B2. [The present invention 1029] below Any of the conjugates of the present invention, which is TIFF0007777065000038.tif81149TIFF0007777065000039.tif195143TIFF0007777065000040.tif160140. [The present invention 1030] below Any of the conjugates of the present invention, which is TIFF0007777065000041.tif124144. [The present invention 1031] below Any of the conjugates of the present invention, which is TIFF0007777065000042.tif78137TIFF0007777065000043.tif168151TIFF0007777065000044.tif159161. [The present invention 1032] A pharmaceutical composition comprising any of the conjugates of the present invention and one or more pharmaceutically acceptable carriers or excipients. [The present invention 1033] A pharmaceutical composition comprising any of the conjugates of the present invention, further comprising at least one immunomodulator or at least one immunostimulant. [The present invention 1034] A method for activating or enhancing the activity of stimulator of interferon genes (STING) in a subject, comprising the step of administering to the subject any of the conjugates of the present invention or pharmaceutically acceptable salts thereof. [This invention 1035] A method for preventing or treating a disease or disorder in a subject, comprising the step of administering to the subject a therapeutically effective amount of any of the conjugates of the present invention or a pharmaceutically acceptable salt thereof. [The present invention 1036] Any of the conjugates of the present invention or pharmaceutically acceptable salts thereof for activating or enhancing the activity of stimulator of interferon genes (STING) in a subject. [This invention 1037] Any of the conjugates of the present invention or pharmaceutically acceptable salts thereof for preventing or treating a disease or disorder in a subject. [The present invention 1038] Use of any of the conjugates of the present invention, or pharmaceutically acceptable salts thereof, in the manufacture of a medicament for activating or enhancing the activity of stimulator of interferon genes (STING) in a subject. [This invention 1039] Use of any of the conjugates of the present invention, or pharmaceutically acceptable salts thereof, in the manufacture of a medicament for preventing or treating a disease or disorder in a subject. [The present invention 1040] Any of the methods, conjugates for use, or uses of the present invention, wherein the disease or disorder is associated with agonism of STING. [This invention 1041] Any of the methods, conjugates for use, or uses of the present invention, wherein the disease or disorder is cancer. [The present invention 1042] Any of the methods, conjugates for use, or uses of the present invention, wherein the disease or disorder is bladder cancer, breast cancer, colorectal cancer, colon cancer, endometrial cancer, gastric cancer, head and neck squamous cell carcinoma, melanoma, lung cancer, ovarian cancer, esophageal cancer, biliary tract cancer, urothelial cancer, cervical cancer, papillary thyroid cancer, papillary renal cell carcinoma, bile duct cancer, salivary duct cancer, kidney cancer, or pancreatic cancer. [This invention 1043] Formula BB TIFF0007777065000045.tif70138 or a pharmaceutically acceptable salt thereof, the conjugate comprises an XMT-1519 antibody comprising a variable heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence FTFSSYSMN (SEQ ID NO:20); a variable heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO:21); a variable heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence GGHGYFDL (SEQ ID NO:22); and a variable light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence RASQSVSSSYLA (SEQ ID NO:27); a variable light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence GASSRAT (SEQ ID NO:28); and a variable light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence QQYHHSPLT (SEQ ID NO:29); 15 is about 8, The conjugate or a pharmaceutically acceptable salt thereof. [This invention 1044] A method for preventing or treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a conjugate of the present invention 1043 or a pharmaceutically acceptable salt thereof. [This invention 1045] The method of claim 1044, wherein the disease or disorder is cancer. [The present invention 1046] The method of claim 1045, wherein the cancer is breast cancer, gastric cancer, colorectal cancer, colon cancer, esophageal cancer, biliary tract cancer, endometrial cancer, urothelial cancer, or non-small cell lung cancer. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein, the singular also includes the plural unless clearly dictated by context. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. References cited herein are not admitted to be prior art to the present invention. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are merely illustrative and not intended to be limiting. If there is a conflict between a chemical structure and the name of a compound disclosed herein, the chemical structure will control.

[0017] Other features and advantages of the present disclosure will be apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0018] [Figure 1A] FIG. 1A plots the red object confluency as a function of time for conjugate 8b-1 and conjugate 8f (100 nM, 10 nM, and 1 nM, respectively) and conjugate 8c-1 and compound 1 (100 nM and 10 nM, respectively) (conjugate concentrations were based on payload). [Figure 1B] Figure 1B plots red object confluency as a function of time for conjugate 8l and compound 1 (100 nM, 10 nM, and 1 nM, respectively) and conjugate 8m (100 nM) (conjugate concentrations were based on payload). [Figure 2] FIG. 2 shows CD14- / CD3+ cells in PBMC, enriched monocytes, and CD16-depleted monocyte populations. [Figure 3]Figures 3A and 3B plot red object confluency as a function of time and show killing of STING wild-type (sgNT-2) and knockout (sg#3-2) SKBR3 NucRed cells by PBMCs for conjugate 8a-3, conjugate 8j, conjugate 8c-2, and compound 1 at 100 nM, 25 nM, 5 nM, and 1 nM (conjugate concentrations based on payload), respectively. [Figure 4] Figure 4A plots red object confluency as a function of dose response for conjugate 8a-3, conjugate 8-j, wild-type Fc-trastuzumab, and AAG Fc-mutant trastuzumab for STING wild-type SKBR3 cancer cell killing activity in SKBR3 cancer cell / PBMC coculture. Figure 4B plots red object confluency as a function of dose response for conjugate 8a-3, conjugate 8-j, wild-type Fc-trastuzumab, and AAG Fc-mutant trastuzumab for STING knockout SKBR3 cancer cell killing activity in SKBR3 cancer cell / PBMC coculture. [Figure 5A] Figure 5A plots the change in red object confluency of OVCAR3-NucRed cancer cells by PBMCs as a function of time for conjugate 8b-1 and compound 1, both at 20 nM and 4 nM (conjugate concentrations based on payload). [Figure 5B] Figure 5B plots the change in red object confluency of OVCAR3-NucRed cancer cells as a function of time by enriched monocytes targeting conjugate 8b-1 and compound 1, both at 20 nM and 4 nM (conjugate concentrations based on payload). [Figure 5C] Figure 5C plots the change in red object confluency of OVCAR3-NucRed cancer cells as a function of time by CD16-depleted monocytes targeting conjugate 8b-1 and compound 1, both at 20 nM and 4 nM (conjugate concentrations based on payload). [Figure 6]Figure 6 is a graph showing the anti-tumor efficacy of trastuzumab (3 / 0 mg / kg), diABZI STING agonist (0 / 5 mg / kg), conjugate 8c-1 (1 / 0.04 mg / kg or 3 / 0.12 mg / kg), conjugate 8a-1 (1 / 0.03 mg / kg or 3 / 0.09 mg / kg) (all doses indicated by antibody / payload) in a SKOV3 xenograft mouse model. [Figure 7-1] Figures 7A-7H show cytokine levels of murine CXCL-10 (IP-10; Figure 7A), IL-6 (Figure 7B), TNFα (Figure 7C), IFNγ (Figure 7D), CXCL1(KC) (Figure 7E), MIG (Figure 7F), MIP-1a (Figure 7G), and RANTES (Figure 7H) as a function of time after administration of diABZI STING agonist (0 / 5 mg / kg), conjugate 8c-1 (3 / 0.12 mg / kg), or conjugate 8a-1 (3 / 0.09 mg / kg) (all doses are written as antibody / payload) to the SKOV3 mouse model. Insets in each plot show cytokine levels induced by conjugate 8a-1 and conjugate 8c-1 relative to vehicle. [Figure 7-2] See description of Figure 7-1. [Figure 7-3] See description of Figure 7-1. [Figure 7-4] See description of Figure 7-1. [Figure 8A] Figures 8A-8C show mouse CXCL10 (Figure 8A), interferon-β (Figure 8B), and IL-6 (Figure 8C) mRNA levels in a SKOV3 xenograft mouse model 12 and 72 hours after administration of conjugate 8c-1 (3 / 0.12 mg / kg), conjugate 8a-1 (3 / 0.09 mg / kg), conjugate 8c-1 (3 / 0.12 mg / kg), or conjugate 8a-1 (3 / 0.09 mg / kg) (all doses are written as antibody / payload). [Figure 8B] See legend to Figure 8A. [Figure 8C]See legend to Figure 8A. [Figure 9] Figure 9 shows CD45 immunohistochemistry (IHC) staining with rabbit anti-CD45 monoclonal antibody at 12 and 72 hours for conjugate 8a-1 (3 / 0.09 mg / kg), conjugate 8c-1 (3 / 0.12 mg / kg), or vehicle. [Figure 10] FIG. 10 is a graph showing circulating plasma concentrations of total antibody and conjugated drug after administration of conjugate 8a-1 (3 / 0.1 mg / kg) (doses indicated by antibody / payload) to CB.17 SCID mice. [Figure 11] Figure 11 shows the effect of IFNλ1 (IL29) or IFNλ2 (IL28A) neutralizing antibodies (10 μg / mL, 2 μg / mL, 0.4 μg / mL, 0.08 μg / mL) on the killing activity of conjugate 8a-3 (1 nM or 0.1 nM, based on payload) in co-cultures of cancer cells and PBMCs. [Figure 12] Figure 12 is a graph showing the anti-tumor efficacy of diABZI STING agonist (0 / 5 mg / kg), conjugate 8c-1 (3 / 0.12 mg / kg), conjugate 8b-1 (3 / 0.09 mg / kg), or conjugate 8f (3 / 0.12 mg / kg) (all doses indicated by antibody / payload) in OVCAR3 xenografts in mice. [Figure 13] FIG. 13 is a graph showing the antitumor efficacy of conjugate 8d-2 (3 / 0.1 mg / kg) or conjugate 8e (3 / 0.1 mg / kg) (all doses shown as antibody / payload) in OVCAR3 xenografts in mice. [Figure 14] Figure 14 is a graph showing the antitumor efficacy of diABZI STING agonist (0 / 5 mg / kg), conjugate 8c-1 (1 / 0.04 mg / kg), or conjugate 8h (1 / 0.04 mg / kg) (all doses shown as antibody / payload) in triple-negative breast cancer xenografts in mice. [Figure 15A]FIG. 15A is a graph showing the antitumor efficacy of conjugate 8d-3 (1 / 0.04 mg / kg) or conjugate 8g (0.88 / 0.04 mg / kg) (all doses shown as antibody / payload) in a syngenic mouse model of colon cancer. [Figure 15B] Figure 15B shows Kaplan-Meier survival curves for conjugate 8d-3 (1 / 0.04 mg / kg) or conjugate 8g (0.88 / 0.04 mg / kg) (all doses are shown as antibody / payload) in a syngenic mouse model of colon cancer. [Figure 16-1] FIG. 16A is a graph showing the antitumor efficacy of conjugate 8g (0.9 / 0.04 mg / kg) (all doses shown as antibody / payload) in individual mice in a syngenic mouse model of colon cancer. [Figure 16-2] Figure 16B is a graph showing the antitumor efficacy of conjugate 8g (0.9 / 0.04 mg / kg) (all doses shown as antibody / payload) when rechallenged with syngenic mouse colon cancer cells. Figure 16C is a graph showing the antitumor efficacy of conjugate 8g (0.9 / 0.04 mg / kg) (all doses shown as antibody / payload) when rechallenged with syngenic mouse lung cancer cells. [Figure 17] Figure 17 is a graph showing the antitumor efficacy of diABZI STING agonist (0 / 5 mg / kg), conjugate 8d-3 (5.5 / 0.18 mg / kg), or conjugate 8i (3.2 / 0.18 mg / kg) (all doses shown as antibody / payload) in a syngenic mouse embryonic carcinoma model. [Figure 18] FIG. 18 is a graph showing the antitumor efficacy of conjugate 20a (3 / 0.09 mg / kg), conjugate 34a (3 / 0.11 mg / kg), conjugate 34 (3 / 0.11 mg / kg), or conjugate 20-1 (3 / 0.10 mg / kg) (all doses shown as antibody / payload) in SKOV3 xenografts in mice. [Figure 19] FIG. 19 is a graph showing the antitumor efficacy of conjugate 28 (0.3 / 0.01 mg / kg or 1 / 0.03 mg / kg), conjugate 29 (0.2 / 0.01 mg / kg or 0.8 / 0.02 mg / kg), conjugate 8-2 (0.3 / 0.01 mg / kg or 1 / 0.04 mg / kg), conjugate 25 (0.3 / 0.01 mg / kg or 1 / 0.04 mg / kg), or conjugate 45 (0.3 / 0.01 mg / kg or 1 / 0.04 mg / kg) (all doses shown as antibody / payload) in SKOV3 xenografts in mice. [Figure 20] Figure 20 shows the effect of diABZI STING agonist (1.5 mg / kg q3dx3 or 0.128 mg / kg qdx1), compound 30 (1.5 mg / kg q3dx3 or 0.128 mg / kg qdx1), conjugate 32b-2 (3.42 / 0.128 mg / kg qdx1), XMT-1519 (3.00 mg / kg qdx1), conjugate 32-5 (0.100 / 0.004, 0.300 / 0.013, 1.00 / 0.042, or 3.00 / 0.128 mg / kg qdx1), conjugate 32e (1.00 / 0.039 or 3.00 / 0.117 mg / kg qdx1) on SKOV3 xenografts in mice. 1 is a graph showing the antitumor efficacy of qdx1)) (all doses are shown as antibody / payload). [Figure 21] Figure 21A is a graph showing the antitumor efficacy of conjugate 8d-3 (1 / 0.04 mg / kg) or conjugate 8k (0.9 / 0.04 mg / kg) in a syngenic mouse model. Figure 21B shows the antitumor efficacy of conjugate 8d-3 (1 / 0.04 mg / kg) in individual mouse syngenic mouse models. Figure 21C shows the antitumor efficacy of conjugate 8k (0.9 / 0.04 mg / kg) in individual mouse syngenic mouse models. [Figure 22]Figure 22 is a graph showing the anti-tumor efficacy of conjugate 8c-2 (3.17 / 0.10 mg / kg), conjugate 8a-2 (2.7 / 0.10 mg / kg or 0.81 / 0.03 mg / kg), conjugate 8j (2.71 / 0.10 mg / kg or 0.81 / 0.03 mg / kg), or diABZI IV STING agonist (0 / 5 mg / kg) (all doses shown as antibody / payload) in SKOV3 xenografts in mice. [Figure 23] Figure 23 is a graph showing the antitumor efficacy of conjugate 32b-1 (3.39 / 0.10 mg / kg), conjugate 32a (0.93 / 0.03 or 3.12 / 0.10 mg / kg), conjugate 32c (2.12 / 0.10 mg / kg), conjugate 32d (2.20 / 0.10 mg / kg), or diABZI IV STING agonist (0 / 5 mg / kg) (all doses shown as antibody / payload) in OVCAR3 xenografts in mice. [Figure 24]Figure 24 shows the results of the combination of rituximab AF-HPA ADC (0.75 / 0.023 mg / kg) and conjugate 8c-2 (4.0 / 0.126 mg / kg); XMT-1535 AF-HPA ADC (0.75 / 0.024 mg / kg); conjugate 8b-2 (2.0 / 0.071 or 4.0 / 0.142 mg / kg); XMT-1535 AF-HPA ADC (0.75 / 0.024 mg / kg) and conjugate 8c-2 (4.0 / 0.126 mg / kg); rituximab AF-HPA ADC (0.75 / 0.023 mg / kg) and conjugate 8b-2 (4.0 / 0.142 mg / kg); rituximab AF-HPA 8A-8D are graphs showing the antitumor efficacy of the combination of XMT-1535 AF-HPA ADC (0.75 / 0.023 mg / kg) with conjugate 8b-2 (2.0 / 0.071 mg / kg); the combination of XMT-1535 AF-HPA ADC (0.75 / 0.024 mg / kg) with conjugate 8b-2 (4.0 / 0.142 mg / kg); the combination of XMT-1535 AF-HPA ADC (0.75 / 0.024 mg / kg) with conjugate 8b-2 (2.0 / 0.071 mg / kg); the combination of XMT-1535 AF-HPA ADC (0.75 / 0.024 mg / kg) with XMT-1535 (4.0 / 0 mg / kg); or XMT-1535 (4.75 / 0 mg / kg). [Figure 25] FIG. 25 is a graph showing the antitumor efficacy of conjugate 32b (0.85 / 0.03 mg / kg), conjugate 32-2 (0.90 / 0.03 mg / kg), conjugate 88 (0.87 / 0.03 mg / kg), conjugate 85 (2.87 / 0.10 mg / kg), conjugate 92 (2.36 / 0.10 mg / kg), conjugate 100 (2.23 / 0.10 mg / kg), conjugate 89 (0.99 / 0.030 mg / kg), conjugate 85a (2.59 / 0.10 mg / kg), conjugate 93 (2.85 / 0.10 mg / kg), or conjugate 101 (2.70 / 0.10 mg / kg) in a SKOV3 xenograft mouse model. [Figure 26]FIG. 26 is a graph showing the antitumor efficacy of vehicle, conjugate 28 (0.99 / 0.0325 mg / kg), or conjugate 62 (0.92 / 0.0325 mg / kg) in a SKOV3 xenograft mouse model. [Figure 27] FIG. 27 is a graph showing circulating plasma concentrations of conjugate drug following administration of conjugate 28 (3.0 / 0.10 mg / kg) or conjugate 62 (2.84 / 0.10 mg / kg) (doses indicated by antibody / payload) to CB.17 SCID mice. DETAILED DESCRIPTION OF THE INVENTION

[0019] Detailed Description The present disclosure provides novel antibody-drug conjugates, synthetic methods for making the conjugates or scaffolds, pharmaceutical compositions containing them, and various uses of the conjugates.

[0020] definition The chemical names given to the intermediate compounds and / or compounds of the present disclosure described herein may refer to any one of the tautomeric representations of such compounds (in some cases, such aliases are given to experimental compounds). Any reference to a compound named (an intermediate compound or compound of the present disclosure) or a compound whose structure is depicted (an intermediate compound or compound of the present disclosure) is to be understood as intended to encompass all tautomeric forms of such compounds, including zwitterionic forms and any mixtures thereof.

[0021] It should be understood that the terms "in some embodiments," "in some embodiments of the present disclosure," and "in some embodiments of compounds of the present disclosure" may be used interchangeably where appropriate.

[0022] The terms "about," "approximately," or "approximate," when used in connection with a numerical value, are meant to include a collection or range of numerical values. In some embodiments, "about X" includes a range of values ​​of ±25%, ±20%, ±15%, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of the numerical value X. In some embodiments, the term "about" refers to a range of values ​​5% above or below the specified value. In some embodiments, the term "about" refers to a range of values ​​2% above or below the specified value. In some embodiments, the term "about" refers to a range of values ​​1% above or below the specified value.

[0023] The recitation of a range of values, unless otherwise specified herein, is intended to serve merely as a shorthand method of referring to each different value within the range, one by one. Each different value is incorporated herein as if recited individually. As used herein, ranges include the two limits of the range unless otherwise specified. In some embodiments, the phrases "x being an integer between 1 and 6" and "x being an integer of 1 to 6" both mean "x is 1, 2, 3, 4, 5, or 6." That is, the terms "between X and Y" and "range from X to Y" include X and Y and any integer therebetween.

[0024] As used herein, the term "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. Antibody amino acid numbering is according to the Kabat EU Index (see Kabat, EA, et al., Sequences of Protein of Immunological Interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)).

[0025] The term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0026] As used herein, the term "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay. Conversely, the reference antibody blocks the binding of the "antibody that binds to the same epitope" as the reference antibody to its antigen by 50% or more in a competitive assay. An exemplary competitive assay is provided herein.

[0027] The term "class" of an antibody refers to the type of constant domain or constant region possessed by the antibody heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), e.g., IgGi, IgG2, IgG3, IgG4, IgAi, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0028] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, for example, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variations, e.g., containing natural mutations or arising during the generation of the monoclonal antibody preparation. Such variations are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (e.g., epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci. Such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0029] The term "epitope" refers to a specific site on an antigen molecule to which an antibody binds.

[0030] The term "protein-based recognition molecule" or "PBRM" refers to a molecule that recognizes and binds to cell surface markers or receptors, such as transmembrane proteins, surface-immobilized proteins, or proteoglycans. In some embodiments, the PBRM contains an engineered cysteine. Examples of PBRMs include, but are not limited to, antibodies, peptides, lipocalins, proteins, peptides, or peptidomimetics. In addition to targeting conjugates to specific cells, tissues, or locations, protein-based recognition molecules may also have certain therapeutic effects, such as antiproliferative (cytostatic and / or cytotoxic) activity against target cells or pathways. Protein-based recognition molecules contain or can be engineered to contain at least one chemically reactive group, such as -COOH, primary amine, secondary amine -NHR, -SH, or a chemically reactive amino acid moiety or side chain, such as tyrosine, histidine, cysteine, or lysine. In some embodiments, a PBRM may be a ligand (LG) or targeting moiety that specifically binds to or complexes with a cell surface molecule, such as a cell surface receptor or antigen, of a predetermined target cell population. After specific binding or complexation between the ligand and its receptor, the cell allows the uptake of the ligand or ligand-drug conjugate, which is then internalized by the cell. As used herein, a ligand that "specifically binds to or complexes with" or "targets" a cell surface molecule preferentially associates with the cell surface molecule through intermolecular forces. In some embodiments, the ligand can preferentially associate with the cell surface molecule with a Kd of less than about 50 nM, less than about 5 nM, or less than 500 pM. Techniques for measuring the binding affinity of a ligand to a cell surface molecule are well known. For example, one suitable technique is called surface plasmon resonance (SPR). In some embodiments, the ligand used for targeting has no detectable therapeutic effect when separated from the drug it delivers.In some embodiments, the ligand functions both as a targeting moiety and as a therapeutic agent or an immunomodulatory agent (e.g., to enhance the activity of an active drug or prodrug). As used herein, the term "PEG unit" refers to a group represented by the formula: TIFF0007777065000046.tif10128. In some embodiments, the PEG unit comprises multiple PEG subunits.

[0031] As used herein, the term "alkyl" refers to a saturated, straight- or branched-chain hydrocarbon group having the specified number of carbon atoms. 1~6 The term "alkyl" refers to a methyl moiety or a straight- or branched-chain alkyl moiety containing 2 to 6 carbon atoms. Exemplary alkyls include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, pentyl, and hexyl.

[0032] As used herein, the term "halo(alkyl)" refers to a saturated, straight or branched chain hydrocarbon group having the specified number (n) of carbon atoms and one or more (up to 2n+1) halogen atoms. 1~4 Examples of "(alkyl)" groups include, but are not limited to, -CF3 (trifluoromethyl), -CCl3 (trichloromethyl), 1,1-difluoroethyl, 2,2,2-trifluoroethyl, and hexafluoroisopropyl.

[0033] As used herein, the term "alkenyl" refers to a straight or branched chain hydrocarbon group having the specified number of carbon atoms and at least one and up to three carbon-carbon double bonds. Examples include ethenyl and propenyl.

[0034] The term "alkynyl," as used herein, refers to a straight- or branched-chain hydrocarbon group having the specified number of carbon atoms and at least one, and up to three, carbon-carbon triple bonds. Examples include ethynyl and propynyl.

[0035] The term "alkoxy-" or "(alkyl)oxy-" as used herein refers to an "alkyl-oxy-" group that includes an alkyl portion having the specified number of carbon atoms attached through an oxygen linking atom. 1~4 Alkoxy-" or "(C 1~4 Alkyl)oxy-" groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, s-butoxy, and t-butoxy.

[0036] As used herein, the term "halo(alkoxy)-" represents a saturated, straight or branched chain hydrocarbon group having the specified number (n) of carbon atoms and one or more (up to 2n+1) halogen atoms attached through an oxygen linking atom. Exemplary "halo(C 1~4 Alkoxy)-" groups include, but are not limited to, -OCHF2 (difluoromethoxy), -OCF3 (trifluoromethoxy), -OCH2CF3 (trifluoroethoxy), and -OCH(CF3)2 (hexafluoroisopropoxy).

[0037] As used herein, the term "amino" refers to a substituent containing at least one nitrogen atom. Specifically, -NH, -NH(C 1~4 alkyl), alkylamino, or (C 1~4 alkyl)amino- or (C 1~4 Alkyl)(C 1~4 Included within the term "amino" are alkyl)amino- or dialkylamino, amido-, carbamido-, urea, and sulfamido substituents.

[0038] As used herein, the term "carbocyclic group or moiety" refers to a cyclic group or moiety whose ring members are carbon atoms, which may be saturated, partially unsaturated (non-aromatic), or fully unsaturated (aromatic).

[0039] As used herein, the term "cycloalkyl" refers to a non-aromatic saturated hydrocarbon ring group containing a specified number of carbon atoms in the ring. For example, "C 3~6 The term "cycloalkyl" refers to a cyclic group having 3 to 6 ring carbon atoms. 3~6 "Cycloalkyl" groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0040] The term "aryl" as used herein refers to a group having aromaticity, including "conjugated" or polycyclic structures, having one or more aromatic rings and no heteroatoms in the ring structure. The term aryl includes both monovalent and divalent species. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl, and the like. In some embodiments, the aryl is phenyl.

[0041] As used herein, the term "heterocyclic group or moiety" refers to a cyclic group or moiety having atoms of at least two different elements as ring members, which cyclic group or moiety may be saturated, partially unsaturated (non-aromatic), or fully unsaturated (aromatic).

[0042] The term "heteroatom" as used herein refers to a nitrogen, sulfur, or oxygen atom, e.g., a nitrogen atom or an oxygen atom.

[0043] The term "heterocycloalkyl," as used herein, refers to a non-aromatic monocyclic or bicyclic group containing 3 to 10 ring atoms and containing one or more (typically one or two) heteroatom ring members independently selected from oxygen, sulfur, and nitrogen. The point of attachment of the heterocycloalkyl group can be any suitable carbon or nitrogen atom.

[0044] As used herein, the term "heteroaryl" refers to an aromatic monocyclic or bicyclic group containing 5 to 10 ring atoms, including 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein at least a portion of the group is aromatic. For example, the term encompasses bicyclic heterocyclic-aryl groups containing a phenyl ring fused to a heterocyclic moiety or a heteroaryl ring moiety fused to a carbocyclic moiety. Attachment of the heteroaryl group can be at any suitable carbon or nitrogen atom.

[0045] As used herein, the terms "halogen" and "halo" refer to a halogen radical, for example, a fluoro, chloro, bromo, or iodo substituent.

[0046] The term "oxo" as used herein refers to a double-bonded oxygen moiety, e.g., when attached directly to a carbon atom it forms a carbonyl moiety (C=O).

[0047] As used herein, the term "hydroxy" or "hydroxyl" is intended to mean the radical --OH.

[0048] As used herein, the term "cyano" refers to the nitrile group -C≡N.

[0049] As used herein, the term "optionally substituted" indicates that a group (e.g., an alkyl, cycloalkyl, alkoxy, heterocycloalkyl, aryl, or heteroaryl group) or ring or moiety can be unsubstituted, or that said group, ring, or moiety can be substituted with one or more substituents. When groups can be selected from a number of alternative groups, the selected groups can be the same or different. Suitable substituents may include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonato, sulfamoyl, sulfamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

[0050] As used herein, the term "independently" means that multiple substituents are selected from a number of possible substituents, which may be the same or different.

[0051] As used herein, the term "pharmaceutically acceptable" refers to compounds, conjugates, materials, compositions, and dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0052] The term "treating" or "treat," as used herein, refers to the management and care of a patient to combat a disease, condition, or disorder, and includes administering a compound of the present disclosure, or a pharmaceutically acceptable salt, polymorph, or solvate thereof, to alleviate the symptoms or complications of the disease, condition, or disorder, or to eliminate the disease, condition, or disorder. The term "treating" can also include the treatment of cells in vitro or in animal models.

[0053] As used herein, the terms "preventing," "prevent," or "protecting from" refer to reducing or eliminating the onset of symptoms or complications of such diseases, conditions, or disorders.

[0054] The term "subject" refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation, or experiment.

[0055] The term "therapeutically effective amount" refers to the amount of an active compound or pharmaceutical agent, including a conjugate of the present disclosure, that elicits the biological or medicinal response in a tissue system, animal, or human that a researcher, veterinarian, physician, or other clinician is seeking, including alleviation or partial alleviation of the symptoms of the disease, syndrome, condition, or disorder being treated.

[0056] A therapeutically "effective amount" is intended to mean an amount of a conjugate that, when administered to a patient in need of such treatment, is sufficient to effectively treat or prevent as defined herein. The amount of a given conjugate that corresponds to such an amount may be determined by the specific conjugate (e.g., efficacy (pIC)). so ), efficacy (EC 50The dosage will vary depending on factors such as the biological half-life of the particular conjugate, the disease state and its severity, the individual patient's personal information (e.g., age, size, and weight) requiring treatment, but can nevertheless be routinely determined by one of ordinary skill in the art. Similarly, the duration of treatment and the duration of administration of the conjugate (dosing and timing, e.g., before / with / after meals) will vary depending on the individual mammal's personal information (e.g., body weight), the particular conjugate and its properties (e.g., pharmacokinetic properties), the disease or disorder and its severity, and the particular compositions and methods being used, but can nevertheless be determined by one of ordinary skill in the art.

[0057] The term "composition" refers to a product containing the specified ingredients in therapeutically effective amounts, as well as any product resulting directly or indirectly from the combination of the specified ingredients in the specified amounts.

[0058] As used herein, the term "pharmaceutically acceptable excipient" generally refers to an excipient that is safe, non-toxic, and not biologically or otherwise undesirable, useful in preparing pharmaceutical compositions, and includes excipients that are acceptable for veterinary and human pharmaceutical use. As used in the specification and claims, "pharmaceutically acceptable excipient" includes both one and more than one such excipient.

[0059] As used herein, the term "STING agonist" refers to a compound or moiety that can interact with STING, e.g., by binding to STING, and / or by inducing downstream signaling (e.g., characterized by activation of molecules associated with STING function). This includes direct phosphorylation of STING, IRF3, and / or NF-kB, and may also include STAT6. In some embodiments, activation of the STING pathway increases the production of type 1 interferons (primarily IFN-a and IFN-b) and / or the expression of genes stimulated by interferon.

[0060] As used herein, the term "STING agonist drug moiety" refers to a moiety derived from a STING agonist that is capable of interacting with STING. In some embodiments, the STING agonist drug moiety is a moiety derived from a STING agonist that allows the moiety to be linked to the remainder of a conjugate of the present disclosure.

[0061] The conjugates of the present disclosure are useful in methods for treating or ameliorating viral infections, diseases, syndromes, conditions, or disorders affected by STING agonism. Such methods comprise, consist of, and / or consist essentially of administering a therapeutically effective amount of the conjugate of the present disclosure, or its enantiomer, diastereomer, solvate, or pharmaceutically acceptable salt, to a subject, including animals, mammals, and humans, in need of such treatment, amelioration, and / or prevention.

[0062] In some embodiments, the conjugates of the present disclosure, or enantiomers, diastereomers, solvates, or pharmaceutically acceptable salt forms thereof, are useful for treating or ameliorating diseases, syndromes, conditions, or disorders, such as melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, fibrosarcoma, and hepatitis B.

[0063] As used herein, the term "conjugate(s) of the disclosure" or "conjugate(s) of the present disclosure" refers to a conjugate as defined herein in any form, i.e., any tautomeric form, any isomeric form, any salt or non-salt form (e.g., a free acid or free base form, or a salt, particularly a pharmaceutically acceptable salt thereof), and any physical form thereof (e.g., a non-solid form (e.g., a liquid or semi-solid form), as well as a solid form (e.g., an amorphous or crystalline form, a particular polymorphic form, a solvate form, including a hydrate form (e.g., a monohydrate, a dihydrate, and a hemihydrate)), as well as mixtures of the various forms.

[0064] Thus, conjugates in any salt or non-salt form and in any physical form thereof, as well as mixtures of various forms, are included in the disclosure herein, with the understanding that conjugates of the present disclosure in any salt or non-salt form and in any physical form thereof may have different levels of activity, different bioavailability, and different handling characteristics for formulation purposes.

[0065] As used herein, the phrases "one or more of A, B, or C," "one or more of A, B, or C," "one or more of A, B, and C," "one or more of A, B, and C," "selected from the group consisting of A, B, and C," "selected from A, B, and C," etc. are used interchangeably and, unless otherwise specified, all refer to something selected from the group consisting of A, B, and / or C, i.e., one or more A, one or more B, one or more C, or any combination thereof.

[0066] Throughout this specification, when a composition is described as having, including, or comprising certain components, it is understood that it is also intended to consist essentially of or consist of the recited components. Similarly, when a method or process is described as having, including, or comprising certain process steps, it similarly means that the process consists essentially of or consists of the recited process steps. Furthermore, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0067] All percentages and ratios used herein are by weight unless otherwise specified. Other features and advantages of the present disclosure are apparent from the different examples. The examples shown illustrate different components and methodologies useful in implementing the present disclosure. The examples do not limit the claimed disclosure. Based on the present disclosure, one skilled in the art can identify and use other components and methodologies useful in implementing the present disclosure.

[0068] All publications and patent documents cited herein are incorporated by reference to the same extent as if each such publication or document was specifically and individually indicated to be incorporated by reference herein. Citation of publications and patent documents is not intended as an admission of the pertinent prior art, and does not constitute an admission as to the contents or date of the publications and patent documents. Now that the invention has been described through a written description, those skilled in the art will recognize that the invention can be practiced in various embodiments, and that the foregoing description and the following examples are intended to be illustrative, not limiting, of the scope of the claims that follow.

[0069] Conjugates and Scaffolds of the Present Disclosure In some aspects, the present disclosure provides a method for manufacturing a semiconductor device comprising: Formula (I): PBRM-[A 1 -(L C ) 0または1 -D]d 15 (I) or a pharmaceutically acceptable salt or solvate thereof, wherein PBRM stands for protein-based recognition molecule, L C is a linker unit, when present, A 1 L C When there is a PBRM, C or L C is a bivalent linker moiety connecting the PBRM to D when absent, D is a STING agonist drug moiety; d 15 is an integer from about 1 to about 20, Conjugates, or pharmaceutically acceptable salts or solvates thereof, are provided.

[0070] In some embodiments, the conjugate comprises: Formula (IA): PBRM-[A 1 -D]d 15(IA) or a pharmaceutically acceptable salt or solvate thereof.

[0071] In some embodiments, the conjugate comprises: Formula (IB): PBRM-[A 1 -L C -D]d 15 (IB) or a pharmaceutically acceptable salt or solvate thereof.

[0072] In some embodiments, the conjugate has formula (I-B'): TIFF0007777065000047.tif19128, or a pharmaceutically acceptable salt or solvate thereof.

[0073] In some aspects, the present disclosure provides compounds of formula (II): A 1’ -(L C ) 0または1 -D (II) or a pharmaceutically acceptable salt or solvate thereof, wherein: PBRM stands for protein-based recognition molecule, L C is a linker unit, when present, A 1’ is a monovalent linker moiety containing a functional group capable of forming a covalent bond with a functional group of the PBRM; D is a STING agonist drug moiety; A scaffold, or a pharmaceutically acceptable salt or solvate thereof, is provided.

[0074] In some embodiments, the scaffold has Formula (II-A): A 1’ -D (II-A) or a pharmaceutically acceptable salt or solvate thereof.

[0075] In some embodiments, the scaffold has Formula (II-B): A 1’ -L C -D (II-B) or a pharmaceutically acceptable salt or solvate thereof.

[0076] In some embodiments, the scaffold has the formula (II-B'): TIFF0007777065000048.tif17128, or a pharmaceutically acceptable salt or solvate thereof.

[0077] For a conjugate of any one of formulas (I), (IA), (IB), (I-B'), (II), (II-A), (II-B), or (II-B'), or a pharmaceutically acceptable salt or solvate thereof, the variables PBRM, L C , A 1 , T 1 , M A , L D , D, and d 15 may each be selected from the group described herein, where applicable, and the variables PBRM, L C , A 1 , T 1 , M A , L D , D, and d 15 Any group described herein for any of the variables PBRM, L, C , A 1 , T 1 , M A , L D , D, and d 15 It is understood that any of the groups described herein may be combined with one or more of the remaining groups described herein.

[0078] Variable d 15 In some embodiments, d15 is an integer of about 2 to about 14, about 2 to about 12, about 2 to about 10, about 2 to about 8, about 2 to about 6, about 2 to about 4, about 4 to about 10, about 4 to about 8, about 4 to about 6, about 6 to about 14, about 6 to about 12, about 6 to about 10, about 6 to about 8, about 8 to about 14, about 8 to about 12, or about 8 to about 10.

[0079] In some embodiments, d 15 is an integer of about 2 to about 8.

[0080] In some embodiments, d 15 is 2, 4, 6, or 8. In some embodiments, d 15 is 6 or 8.

[0081] In some embodiments, d 15 is 8. In some embodiments, d 15 is 6.

[0082] Variable A 1 and A 1’ In some embodiments, each A 1 is independently, L C When there is a PBRM, C or L C When absent, is a bivalent linker moiety connecting the PBRM to D.

[0083] In some embodiments, each A 1 is, independently, TIFF0007777065000049.tif49128, wherein: R 7 -O-, -NR 8 , -(C1~C 10 Alkyl)-, -(C1-C 10 alkenyl)-, -(C1-C 10 Alkynyl)-, -(C3-C8 cycloalkyl)-, -aryl-, -O-(C1-C8 alkyl)-, -O-(C1-C 10 alkenyl)-, -O-(C1-C 10alkynyl)-, -(C1-C 10 alkyl)-(C3-C8 cycloalkyl)-, -(C1-C 10 Alkyl)-aryl-, -(C2-C 10 alkenyl)-(C3-C8 cycloalkyl)-, -(C2-C 10 alkenyl)-aryl-, -(C2-C 10 alkynyl)-(C3-C8 cycloalkyl)-, -(C2-C 10 alkynyl)-aryl-, -(C3-C8 cycloalkyl)-(C1-C 10 Alky-, -aryl-(C1-C 10 Alkyl)-, -(C3-C8 cycloalkyl)-(C2-C 10 Alkenyl, aryl (C2-C 10 alkenyl)-, -(C3-C8 cycloalkyl)-(C2-C 10 Alkynyl)-, -aryl-(C2-C 10 alkynyl)-, -(3- to 8-membered heterocycloalkyl)-, -(5- to 8-membered heteroaryl)-, -(C1-C 10 alkyl)-(3-8 membered heterocycloalkyl)-, -(C1-C 10 alkyl)-(5-8 membered heteroaryl)-, -(C2-C 10 alkenyl)-(3-8 membered heterocycloalkyl)-, -(C2-C 10 alkenyl)-(5-8 membered heteroaryl)-, -(C2-C 10 alkynyl)-(3-8 membered heterocycloalkyl)-, -(C2-C 10 Alkynyl)-(5-8 membered heteroaryl)-, -(3-8 membered heterocycloalkyl)-(C1-C 10 alkyl)-, -(5-8 membered heteroaryl)-(C1-C 10 alkyl)-, -(3-8 membered heterocycloalkyl)-(C2-C 10 alkenyl)-, -(5-8 membered heteroaryl)-(C2-C 10 alkenyl)-, -(5-8 membered heteroaryl)-(C2-C 10 alkynyl)-, -(5-8 membered heteroaryl)-(C2-C 10Alkynyl)-, -OC(O)-(CH2CH2O) r -(CH2)2-, -(CH2CH2O) r - or -(CH2CH2O) r -(CH)-, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycloalkyl, or heteroaryl is optionally substituted; R 8 is H, hydroxy, or C 1~4 is alkyl, r is an integer from about 1 to about 12; * indicates that it is attached to the PBRM, ** L C When exists, L C indicates that it is attached to C When is absent, it indicates that it is attached to D.

[0084] In some embodiments, R 7 -O-, -NR 8 , -(C1~C 10 alkyl)-, -(C3-C8 cycloalkyl)-, -aryl-, -O-(C1-C8 alkyl)-, -(C1-C 10 Alkyl)-aryl-, -aryl-(C1-C 10 Alkyl)-, -(C1-C 10 alkyl)-(C3-C8 cycloalkyl)-, -(C3-C8 cycloalkyl)-(C1-C 10 alkyl)-, -(3- to 8-membered heterocycloalkyl)-, -(5- to 8-membered heteroaryl)-, -(C1-C 10 alkyl)-(3-8 membered heterocycloalkyl)-, -(C1-C 10 alkyl)-(5-8 membered heteroaryl)-, -(3-8 membered heterocycloalkyl)-(C1-C 10 alkyl)-, -(5-8 membered heteroaryl)-(C1-C 10 alkyl)-, -OC(O)-(CH2CH2O) r -(CH2)2-, -(CH2CH2O) r - or -(CH2CH2O)r -(CH2)2-.

[0085] In some embodiments, R 7 is -(C1~C 10 alkyl)-, -O-(C1-C8 alkyl)-, -(CH2CH2O) r -, -OC(O)-(CH2CH2O) r -(CH2)2- or -(CH2CH2O) r -(CH2)2-.

[0086] In some embodiments, R 7 is -O-, -NH, -N(CH3), -CH2-, -(CH2)2-, -(CH2)5-, -OC(O)-(CH2CH2O)6-(CH2)2-, -(CH2CH2O)-(CH2)2-, -(CH2CH2O)2-(CH2)2-, -(CH2CH2O)4-(CH2)2-, or -(CH2CH2O)6-(CH2)2-.

[0087] In some embodiments, each A 1 is, independently, TIFF0007777065000050.tif48128, where R 8 is H, hydroxy, or C 1~4 alkyl, and r is an integer from about 4 to about 6; * indicates that it is attached to the PBRM, ** L C When exists, L C indicates that it is attached to C When is absent, it indicates that it is attached to D.

[0088] Each A 1 Before being connected to the PBRM, 1’ is understood to correspond to

[0089] In some embodiments, each A 1’ is, independently, TIFF0007777065000051.tif50128, wherein: R 7 , R 8 and r is as described herein; ** L C When exists, L C indicates that it is attached to C When is absent, it indicates that it is attached to D.

[0090] In some embodiments, each A 1’ is, independently, TIFF0007777065000052.tif55142, wherein: r is an integer from about 4 to about 6; ** L C When exists, L C indicates that it is attached to C When is absent, it indicates that it is attached to D.

[0091] Variable L C In some embodiments, each L C When present, independently, TIFF0007777065000053.tif14128, wherein: # is A 1 ## indicates that it is attached to D. M A is, when present, a peptide moiety containing at least two amino acids; T 1 is a hydrophilic group, when present; L D is M A When exists, D is M A or M A When does not exist, D is replaced by A 1 is a bivalent linker moiety that connects

[0092] In some embodiments, each L D contains at least one cleavable bond such that when the bond is broken, D is released in an active form for its intended therapeutic effect.

[0093] In some embodiments, each L C When present, independently, The file is TIFF0007777065000054.tif6128.

[0094] In some embodiments, each L C When present, independently, The file is TIFF0007777065000055.tif12128.

[0095] Variable L D In some embodiments, each L D independently, M A When exists, D is M A or M A When does not exist, D is replaced by A 1 is a bivalent linker moiety that connects

[0096] In some embodiments, each L D contains at least one cleavable bond such that when the bond is broken, D is released in an active form for its intended therapeutic effect.

[0097] In some embodiments, L D In some embodiments, L D contains multiple cleavable sites or bonds.

[0098] Each L D Before being connected to D, it is independently a monovalent moiety L D is understood to correspond to '.

[0099] In some embodiments, L D' includes a functional group capable of forming a cleavable bond. Functional groups capable of forming a cleavable bond can include, for example, a sulfhydryl group for forming a disulfide bond, an aldehyde, ketone, or hydrazine group for forming a hydrazone bond, a hydroxylamine group for forming an oxime bond, a carboxy or amino group for forming a peptide bond, a carboxy or hydroxy group for forming an ester bond, and a sugar for forming a glycosidic bond.

[0100] In some embodiments, each L D In some embodiments, L comprises a disulfide bond cleavable by disulfide exchange, an acid labile bond cleavable at acidic pH, and / or a bond cleavable by a hydrolytic enzyme. D contains a carbamate bond (i.e., -OC(O)-NR-, where R is hydrogen or alkyl, etc.).

[0101] In some embodiments, L D The structure and sequence of the single cleavable bond in may be such that the single bond is cleaved by the action of an enzyme present at the target site. In some embodiments, the single cleavable bond may be cleavable by other mechanisms.

[0102] In some embodiments, L D The structure and arrangement of the multiple cleavable bonds in may be such that the multiple bonds are cleaved by the action of an enzyme present at the target site. In some embodiments, the multiple cleavable bonds may be cleavable by other mechanisms.

[0103] In some embodiments, the cleavable bond can be enzymatically cleaved by one or more enzymes, including tumor-associated proteases, to liberate the Drug unit or D, and the conjugate of the present disclosure, or an intermediate thereof, or scaffold, is protonated in vivo to provide the Drug unit or D upon release.

[0104] In some embodiments, each L D is, independently, The file is TIFF0007777065000056.tif10128.

[0105] L E When present, NH-[(CH2CH2O) p -(CH2) 0~2 ] q -C(O)-, -NH-(C1-C6 alkyl)-OC(O)-, or -NH-[(CH2CH2O) p -(CH2) 0~2 ] q -C(O)-NH-(C1-C6 alkyl)-OC(O)-, p is an integer of about 1 to about 20, and q is an integer of about 1 to about 10; each W is independently a natural amino acid unit or an unnatural amino acid unit; w is an integer from about 0 to about 12; *** is M A When exists, M A or M A When does not exist, A 1 indicates that it is attached to **** indicates that it is attached to D.

[0106] In some embodiments, each L D is, independently, TIFF0007777065000057.tif10128. In some embodiments, each L D is, independently, TIFF0007777065000058.tif11128. In some embodiments, each L D is, independently, The file is TIFF0007777065000059.tif10128.

[0107] In some embodiments, L Ecomprises at least one PEG unit.

[0108] In some embodiments, the PEG unit comprises at least 1 subunit, at least 2 subunits, at least 3 subunits, at least 4 subunits, at least 5 subunits, or at least 6 subunits. In some embodiments, the PEG unit comprises at least 4 subunits, at least 3 subunits, at least 2 subunits, or at least 1 subunit. In some embodiments, the PEG unit comprises at least 1 subunit. In some embodiments, the PEG unit comprises at least 2 subunits.

[0109] In some embodiments, p is an integer from about 1 to about 15, from about 1 to about 10, from about 1 to about 9, from about 1 to about 8, from about 1 to about 7, from about 1 to about 6, or from about 1 to about 5.

[0110] In some embodiments, p is an integer from about 1 to about 6. In some embodiments, p is an integer from about 1 to about 4. In some embodiments, p is an integer from about 1 to about 2.

[0111] In some embodiments, p is 2.

[0112] In some embodiments, q is an integer from about 1 to about 15, from about 1 to about 10, from about 1 to about 9, from about 1 to about 8, from about 1 to about 7, from about 1 to about 6, or from about 1 to about 5.

[0113] In some embodiments, q is 1, 2, 3, 4, or 5. In some embodiments, q is 2.

[0114] In some embodiments, L E When present, NH-(CH2CH2O) 1~4 In some embodiments, L is —(CH)—C(O)—. E When present, L is NH—(CHCHO)—(CH)—C(O)—. EWhen present, NH-(CH2CH2O)3-(CH2) 0~2 In some embodiments, L is —C(O)—. E When present, L is NH—(CHCHO)—(CH)—C(O)—. E When present, L is NH—(CHCHO)—(CH)—C(O)—. E When present, NH-CH2CH2O-(CH2) 0~2 In some embodiments, L is —C(O)—. E When present, L is NH—CHCHO—C(O)—. E When present, L is NH—(C1-C6 alkyl)—OC(O)—. E When present, L is NH—CH—CH(CH)—OC(O)—. E When present, NH-[(CH2CH2O) 1~4 In some embodiments, L is —(CH)—C(O)—NH—(C-C alkyl)-OC(O)—. E When present, is NH—CHCHO—(CH)—C(O)—NH—(CH)—OC(O)—.

[0115] In some embodiments, w is an integer from about 1 to about 12 (e.g., 1 to 6, or 1 to 4, or 1 to 3, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12).

[0116] In some embodiments, w is 0, 1, 2, 3, 4, or 5. In some embodiments, w is 1, 2, 3, 4, or 5.

[0117] In some embodiments, w is 1. In some embodiments, w is 2. In some embodiments, w is 3.

[0118] In some embodiments, each W is independently a natural or unnatural amino acid and / or a D or L isomer.

[0119] In some embodiments, each W is independently a natural or unnatural α, β, or γ amino acid.

[0120] In some embodiments, at least one W is a natural amino acid. In some embodiments, at least one W is a non-natural amino acid.

[0121] In some embodiments, W w does not contain any naturally occurring amino acids. w does not include unnatural amino acids.

[0122] In some embodiments, W w In some embodiments, W comprises a natural amino acid linked to an unnatural amino acid. w comprises a natural amino acid linked with a D-isomer of the natural amino acid.

[0123] In some embodiments, W w is a dipeptide, for example, -Val-Cit-, -Phe-Lys-, -Val-Ala-, or Glu-Ala-.

[0124] In some embodiments, W w is a monopeptide, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide unit.

[0125] In some embodiments, W w is a peptide (e.g., a peptide of 1-12 amino acids) that is directly conjugated to D. In some embodiments, the peptide is a single amino acid. In some embodiments, the peptide is a dipeptide. In some embodiments, the peptide is a tripeptide.

[0126] In some embodiments, W w is independently selected from alanine, beta-alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, cysteine, methionine, selenocysteine, ornithine, penicillamine, aminoalkanoic acids, aminoalkynic acids, aminoalkanedioic acids, aminobenzoic acids, amino-heterocyclo-alkanoic acids, heterocyclo-carboxylic acids, citrulline, statins, diaminoalkanoic acids, and derivatives thereof.

[0127] In some embodiments, W w wherein each amino acid is independently selected from alanine, beta-alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, citrulline, and derivatives thereof.

[0128] In some embodiments, W w Each amino acid in is independently selected from proteinogenic and non-proteinogenic amino acids.

[0129] In some embodiments, W w is independently selected from the D- or L-isomers of the following amino acids: alanine, beta-alanine, arginine, aspartic acid, asparagine, cysteine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, methionine, serine, tyrosine, threonine, tryptophan, proline, ornithine, penicillamine, aminoalkynol acids, aminoalkanedioic acids, heterocyclo-carboxylic acids, citrulline, statins, diaminoalkanoic acids, valine, citrulline, and derivatives thereof.

[0130] In some embodiments, W w Each amino acid in is independently cysteine, homocysteine, penicillamine, ornithine, lysine, serine, threonine, glycine, glutamine, alanine, aspartic acid, glutamic acid, selenocysteine, proline, glycine, isoleucine, leucine, methionine, valine, citrulline, or alanine.

[0131] In some embodiments, W w is independently selected from the L-isomers of the following amino acids: alanine, beta-alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, tryptophan, citrulline, and valine.

[0132] In some embodiments, W w is independently selected from the D-isomers of the following amino acids: alanine, beta-alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, tryptophan, citrulline, and valine.

[0133] In some embodiments, W w Each amino acid in is alanine, beta-alanine, glycine, glutamic acid, isoglutamic acid, isoaspartic acid, valine, citrulline, or aspartic acid.

[0134] In some embodiments, W w In some embodiments, W comprises β-alanine. In some embodiments, W comprises (β-alanine)-(alanine). In some embodiments, W w(β-alanine), optionally glutamic acid, isoglutamic acid, aspartic acid, isoaspartic acid, valine, (valine)-(alanine), (alanine)-(alanine), or (valine)-(citrulline).

[0135] In some embodiments, W w contains (glutamic acid)-(alanine).

[0136] In some embodiments, W w comprises glutamic acid, and optionally alanine, glycine, isoglutamic acid, aspartic acid, isoaspartic acid, valine, (valine)-(alanine), (alanine)-(alanine), or (valine)-(citrulline).

[0137] In some embodiments, W w In some embodiments, W comprises 2,3-diaminopropanoic acid. w In some embodiments, W comprises (R)-2,3-diaminopropane. w In some embodiments, W comprises glutamic acid. w comprises (glutamic acid)-(alanine). In some embodiments, W w contains (glutamic acid)-(glycine)-(alanine).

[0138] In some embodiments, W w includes L-glutamic acid, D-glutamic acid, (L-glutamic acid)-(L-alanine), (L-glutamic acid)-(D-alanine), (D-glutamic acid)-(L-alanine), (D-glutamic acid)-(D-alanine), (L-glutamic acid)-(glycine)-(L-alanine), D-glutamic acid)-(glycine)-(D-alanine), (L-glutamic acid)-(glycine)-(D-alanine), or (D-glutamic acid)-(glycine)-(L-alanine).

[0139] In some embodiments, W w contains one or more amino acids plus a carbamate bond.

[0140] In some embodiments, L D (For example, W w ) is selective for enzymatic cleavage (e.g., by a particular enzyme). In some embodiments, the particular enzyme is a tumor-associated protease.

[0141] In some embodiments, L D (For example, W w ) contains a bond whose cleavage is catalyzed by cathepsin B, cathepsin C, cathepsin D, or plasmin proteases.

[0142] In some embodiments, L D contains the glycosylation site.

[0143] In some embodiments, L D comprises a sugar moiety (Su) linked to a self-immolative group via an oxygen glycosidic bond.

[0144] In some embodiments, the "self-immolative group" is M A When present, it contains three spaced chemical moieties: a sugar moiety (via a glycosidic bond), a drug unit (direct or indirect), and an M A may be trifunctional chemical moieties capable of covalently linking (directly or indirectly) together, M A When does not exist, A 1 That's fine too.

[0145] In some embodiments, glycosidic bonds can be cleaved at the target site to initiate a self-immolative reaction sequence leading to release of the drug.

[0146] In some embodiments, each L D When present, independently, TIFF0007777065000060.tif138163TIFF0007777065000061.tif223163TIFF0007777065000062.tif32163, wherein*** is M A When exists, M A or M A When does not exist, A 1 indicates that it can be attached to **** indicates that it is attached to D.

[0147] In some embodiments, each L D When present, independently, TIFF0007777065000063.tif86163, wherein: *** is M A When exists, M A or M A When does not exist, A 1 indicates that it is attached to **** indicates that it is attached to D.

[0148] In some embodiments, each L D When present, independently, TIFF0007777065000064.tif15128, wherein: *** is M A indicates that it is attached to **** indicates that it is attached to D.

[0149] Variable M A In some embodiments, M A comprises a peptide portion of at least two amino acids.

[0150] In some embodiments, a single amino acid is referred to herein as "AA" and multiple amino acids are referred to as "AA's."

[0151] In some embodiments, M A -L D -A moiety that can form a covalent bond with the D unit, allowing for the attachment of multiple drugs.

[0152] In some embodiments, M A contains one AA unit, or has two or more (e.g., 2-10, 2-6, or 2, 3, 4, 5, or 6) AA units, each of which is independently a natural or unnatural amino acid, an amino alcohol, an amino aldehyde, a diamine, a polyamine, or a combination thereof.

[0153] In some embodiments, to have the required number of attachments, at least one of the AA units has a -L D -D units have functionalized side chains for attachment. In some embodiments, exemplary functionalized AA units (e.g., amino acids, amino alcohols, or amino aldehydes) include, for example, azide- or alkyne-functionalized AA units (e.g., amino acids, amino alcohols, or amino aldehydes modified to have an azide or alkyne group).

[0154] In some embodiments, M A In some embodiments, M comprises 2 to 12 AA units. A In some embodiments, M comprises 2 to 10 AA units. A In some embodiments, M contains 2 to 6 AA units. A contains 2, 3, 4, 5, or 6 AA units.

[0155] In some embodiments, M A has 2 AA units. In some embodiments, the peptide moiety has 3 AA units. In some embodiments, the peptide moiety has 4 AA units. In some embodiments, the peptide moiety has 5 AA units. In some embodiments, the peptide moiety has 6 AA units.

[0156] In some embodiments, M AAttachment within or to other components of the conjugate, its intermediate, or scaffold can be, for example, via amino, carboxy, or other functionality.

[0157] In some embodiments, M A Each amino acid in may independently be a D or L isomer of a thiol-containing amino acid. A Each amino acid in may independently be a D isomer of a thiol-containing amino acid. A Each amino acid in may independently be the L-isomer of a thiol-containing amino acid. In some embodiments, the thiol-containing amino acid may be, for example, cysteine, homocysteine, or penicillamine.

[0158] In some embodiments, M A Each amino acid in may independently be the L- or D-isomer of the following amino acids: alanine (including β-alanine), arginine, aspartic acid, asparagine, cysteine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, methionine, serine, tyrosine, threonine, tryptophan, proline, ornithine, penicillamine, aminoalkynol, aminoalkanedioic acid, heterocyclo-carboxylic acid, citrulline, statin, diaminoalkanoic acid, stereoisomers thereof, or derivatives thereof.

[0159] In some embodiments, M A Each amino acid in is independently cysteine, homocysteine, penicillamine, ornithine, lysine, serine, threonine, glycine, glutamine, alanine, aspartic acid, glutamic acid, selenocysteine, proline, glycine, isoleucine, leucine, methionine, valine, alanine, or a stereoisomer thereof.

[0160] In some embodiments, M AIn some embodiments, M comprises a monopeptide, dipeptide, tripeptide, tetrapeptide, or pentapeptide. A contains a pentapeptide.

[0161] In some embodiments, M A In some embodiments, M comprises at least about 5 amino acids (e.g., 5, 6, 7, 8, 9, or 10 amino acids). A contains a maximum of about 10 amino acids.

[0162] In some embodiments, M A Each amino acid in is independently glycine, serine, glutamic acid, lysine, aspartic acid, and cysteine.

[0163] In some embodiments, M A contains at least four glycines and at least one glutamic acid, e.g., (glycine)4 and glutamic acid, where the glutamic acid is located anywhere along the peptide chain, e.g., (glutamic acid)-(glycine)4; (glycine)-(glutamic acid)-(glycine)3; (glycine)2-(glutamic acid)-(glycine)2; (glycine)3-(glutamic acid)-(glycine); or (glycine)4-(glutamic acid).

[0164] In some embodiments, M A comprises (glycine)4-(glutamic acid). In some embodiments, the peptide moiety comprises (glutamic acid)-(glycine)4.

[0165] In some embodiments, M A contains at least four glycines and at least one serine, e.g., (glycine)4 and serine, where the serine is located anywhere along the peptide chain, e.g., (serine)-(glycine)4; (glycine)-(serine)-(glycine)3; (glycine)2-(serine)-(glycine)2; (glycine)3-(serine)-(glycine); or (glycine)4-(serine).

[0166] In some embodiments, M A comprises (glycine)4-(serine). In some embodiments, the peptide moiety comprises (serine)-(glycine)4.

[0167] In some embodiments, M A comprises (β-alanine)-(glycine)-(serine), where the serine is anywhere along the peptide chain, e.g., (β-alanine)-(serine)-(glycine)-; (β-alanine)-(glycine)-(serine)-(glycine)-; (β-alanine)-(glycine)-(serine)-(glycine); or (β-alanine)-(glycine)-(serine).

[0168] In some embodiments, M A comprises (glycine)4-(serine)-(glutamic acid), where the serine is located anywhere along the peptide chain, e.g., (serine)-(glycine)4-(glutamic acid); (glycine)-(serine)-(glycine)3-(glutamic acid); (glycine)2-(serine)-(glycine)2-(glutamic acid); (glycine)3-(serine)-(glycine)-(glutamic acid); or (glycine)4-(serine)-(glutamic acid). In some embodiments, the peptide moiety comprises (β-alanine)-(glycine)4-(serine)-(glutamic acid), where the serine is located anywhere along the peptide chain, e.g., (β-alanine)-(serine)-(glycine)4-(glutamic acid); (β-alanine)-(glycine)-(serine)-(glycine)3-(glutamic acid); (β-alanine)-(glycine)2-(serine)-(glycine)2-(glutamic acid); (β-alanine)-(glycine)3-(serine)-(glycine)-(glutamic acid); or (β-alanine)-(glycine)4-(serine)-(glutamic acid).

[0169] In some embodiments, M A comprises (glycine)4-(serine). In some embodiments, the peptide moiety comprises (serine)-(glycine)4.

[0170] In some embodiments, M A contains (β-alanine)-(glycine)4-(serine), where the serine can be anywhere along the peptide chain.

[0171] In some embodiments, M A contains (glycine)4-(serine)-(glutamic acid), where the serine can be anywhere along the peptide chain.

[0172] In some embodiments, M A contains (β-alanine)-(glycine)-4-(serine)-(glutamic acid), where the serine can be anywhere along the peptide chain.

[0173] In some embodiments, M A (glutamic acid)-(glycine) 1~4 Including M A A via one of the glutamic acids 1 Attached to M A T via glycine 1 Attached to M A is released via glutamate D Can be attached to.

[0174] In some embodiments, M A teeth, Contains TIFF0007777065000065.tif21128.

[0175] In some embodiments, M A contains (glutamic acid)-(glycine)4, and M A A is produced via glutamate 1 Attached to M A is linked to T via one of the glycines. 1 Attached to M A is released via glutamate D Can be attached to.

[0176] In some embodiments, M A teeth, Includes TIFF0007777065000066.tif22128.

[0177] In some embodiments, M A contains (glutamic acid)-(glycine), and M A A is produced via glutamate 1 Attached to M A T via glycine 1 Attached to M A is released via glutamate D Can be attached to.

[0178] In some embodiments, the peptide moiety is Contains TIFF0007777065000067.tif26128.

[0179] In some embodiments, M A (glycine) 1~4 - (glutamic acid), M A A via one of the glycines 1 Attached to M A T is activated via glutamate 1 Attached to M A is released via glutamate D Can be attached to.

[0180] In some embodiments, M A teeth, Includes TIFF0007777065000068.tif23128.

[0181] In some embodiments, M A contains (glycine)4-(glutamic acid), and M A A is produced via glutamate 1 Attached to M A T via glycine 1 Attached to M A is released via glutamate D Can be attached to.

[0182] In some embodiments, M A teeth, Includes TIFF0007777065000069.tif23128.

[0183] In some embodiments, M A contains (glycine)-(glutamic acid), and M A A is catalyzed by glycine 1 Attached to M A T is activated via glutamate 1 Attached to M A is released via glutamate D Can be attached to.

[0184] In some embodiments, M A teeth, Includes TIFF0007777065000070.tif21128.

[0185] In some embodiments, M A (glycine) 1~4 - (serine), M A A via one of the glycines 1 Attached to M A T via serine 1 Attached to M A L via serine D Can be attached to.

[0186] In some embodiments, M A teeth, Includes TIFF0007777065000071.tif13128.

[0187] In some embodiments, M A contains (glycine)-(serine), and M A A is catalyzed by glycine 1 Attached to M A T via serine 1 Attached to M A L via serine D Can be attached to.

[0188] In some embodiments, M A teeth, Includes TIFF0007777065000072.tif15128.

[0189] In some embodiments, M A contains (glycine)4-(serine), and M A A via one of the glycines 1 Attached to M A T via serine 1 Attached to M A L via serine D Can be attached to.

[0190] In some embodiments, M A teeth, Includes TIFF0007777065000073.tif15128.

[0191] In some embodiments, M A (Serine)-(Glycine) 1~4 Including M A A via serine 1 Attached to M A is linked to T via one of the glycines. 1 Attached to M A L via serine D Can be attached to.

[0192] In some embodiments, M A teeth, Includes TIFF0007777065000074.tif15128.

[0193] In some embodiments, M A contains (serine)-(glycine)4, and M A A via serine 1 Attached to M A is linked to T via one of the glycines. 1 Attached to M A L via serine D Can be attached to.

[0194] In some embodiments, M A teeth, Contains TIFF0007777065000075.tif15128.

[0195] In some embodiments, M A contains (serine)-(glycine), and M A A via serine 1 Attached to M A is linked to T via one of the glycines. 1 Attached to M A L via serine D Can be attached to.

[0196] In some embodiments, M A teeth, Includes TIFF0007777065000076.tif20128.

[0197] In some embodiments, M A is (β-alanine)-(glycine) 1~4 - (serine), M A A via β-alanine 1 Attached to M A T via serine 1 Attached to M A L via serine D Can be attached to.

[0198] In some embodiments, M A teeth, Includes TIFF0007777065000077.tif20128.

[0199] In some embodiments, M A contains (β-alanine)-(glycine)4-(serine), and M A A via β-alanine 1 Attached to M A T via serine 1 Attached to M A L via serine D Can be attached to.

[0200] In some embodiments, MA teeth, Contains TIFF0007777065000078.tif21128.

[0201] In some embodiments, the peptide moiety comprises (β-alanine)-(glycine)-(glutamic acid), and the peptide moiety comprises L 3 When there exists L 3 or L 3 In the absence of β-alanine, L M The peptide moiety is attached to T 1 When glutamate is present, T 1 and the peptide moiety is attached to L D When glutamic acid is present, L D Can be attached to.

[0202] In some embodiments, the peptide moiety is Contains TIFF0007777065000079.tif25128.

[0203] M A Regarding the above aspect, * is A 1 indicates that it is attached to ** is T 1 indicates that it is attached to *** L D is understood to indicate that the

[0204] Hydrophilic group (variable T 1 ) In some embodiments, the hydrophilic group included in the conjugate or scaffold of the present disclosure is a water-soluble and substantially non-antigenic polymer. Examples of hydrophilic groups include, but are not limited to, polyhydric alcohols, polyethers, polyanions, polycations, polyphosphates, polyamines, polysaccharides, polyhydroxy compounds, polylysine, and derivatives thereof. In some embodiments, one end of the hydrophilic group is connected to M by a non-cleavable linkage or via a cleavable linkage. A Linkers (e.g., MA The hydrophilic group may be functionalized to allow covalent attachment to an amino acid (e.g., an amino acid within the linker). In some embodiments, functionalization may be via, for example, an amine, thiol, NHS ester, maleimide, alkyne, azide, carbonyl, or other functional group. In some embodiments, the other end (or ends) of the hydrophilic group is free and untethered. In some embodiments, "untethered" means that the hydrophilic group is not attached to another moiety, such as a D or Drug unit, or other component of a conjugate or scaffold of the present disclosure. In some embodiments, the free and untethered end of the hydrophilic group may comprise a methoxy, carboxylic acid, alcohol, or other suitable functional group. In some embodiments, the methoxy, carboxylic acid, alcohol, or other suitable functional group serves as a cap for one or more ends of the hydrophilic group.

[0205] In some embodiments, a cleavable linkage refers to a linkage that is substantially insusceptible to cleavage while circulating in plasma, but is susceptible to cleavage in an intracellular or intratumoral environment. In some embodiments, a non-cleavable linkage is a linkage that is substantially insusceptible to cleavage in any biological environment. In some embodiments, chemical hydrolysis of a hydrazone, reduction of a disulfide, and enzymatic cleavage of a peptide or glycosidic bond are examples of cleavable linkages. In some embodiments, exemplary attachment of a hydrophilic group is via an amide linkage, an ether linkage, an ester linkage, a hydrazone linkage, an oxime linkage, a disulfide linkage, a peptide linkage, or a triazole linkage. In some embodiments, M A Linkers (e.g., M A The attachment of the hydrophilic group to the amino acid (which is in the linker) is via an amide linkage.

[0206] In some embodiments, a conjugate or scaffold of the present disclosure comprises multiple hydrophilic groups, which may be the same chemical moiety or different chemical moieties (e.g., hydrophilic groups of different molecular weights, number of subunits, or chemical structures). In some embodiments, multiple hydrophilic groups are attached to only one M at a single attachment site. A M at different sites may be attached to a linker A It may be attached to a linker.

[0207] In some embodiments, the addition of a hydrophilic group may have two potential effects on the pharmacokinetics of the resulting conjugate. In some embodiments, the desired effect is decreased clearance (and consequently increased exposure) due to reduced nonspecific interactions induced by exposed hydrophobic elements on the drug or drug-linker. In some embodiments, the undesired effect is a decreased volume and rate of distribution, which may result from an increased molecular weight of the conjugate. In some embodiments, increasing the molecular weight of the hydrophilic group may increase the hydrodynamic radius of the conjugate, resulting in a decreased diffusivity and thereby reducing the ability of the conjugate to enter tumors. Because of these two competing pharmacokinetic effects, it may be desirable to use a hydrophilic group that is large enough to decrease conjugate clearance and thus increase plasma exposure, but not large enough to significantly decrease its diffusivity, which may therefore reduce the conjugate's ability to reach its intended target cell population.

[0208] In some embodiments, the hydrophilic group includes, but is not limited to, a sugar alcohol (also known as a polyalcohol, polyhydric alcohol, alditol, or glycitol, e.g., inositol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, galactitol, mannitol, sorbitol, etc.), or a derivative thereof (e.g., an amino polyalcohol), a carbohydrate (e.g., a sugar), polyvinyl alcohol, a carbohydrate-based polymer (e.g., dextran), hydroxypropyl methacrylamide (HPMA), a polyalkylene oxide, and / or a copolymer thereof.

[0209] In some embodiments, T 1 includes moieties incorporating multiple hydroxyl ("-OH") groups, eg, monosaccharides, oligosaccharides, polysaccharides, etc.

[0210] In some embodiments, T 1 is R 58 is -H or C 1~8 Multiple -(CR 58 OH)-group.

[0211] In some embodiments, T 1 is -OH or TIFF0007777065000080.tif6128, wherein: n1 is an integer from 0 to about 6; Each R 58 are independently -H or C 1~8 is alkyl, R 60 is a bond, C 1~6 Alkyl linker, or -CHR 59 - and R 59 -H, C 1~8 alkyl, cycloalkyl, or arylalkyl; R 61 is CH2OR 62 , COOR 62 , -(CH2) n2COOR 62 or heterocycloalkyl substituted with one or more hydroxyl groups; R 62 is -H or C 1~8 is alkyl, n2 is an integer of 1 to about 5.

[0212] In some embodiments, T 1 is —OH. In some embodiments, T 1 teeth, The file is TIFF0007777065000081.tif5128.

[0213] In some embodiments, R 58 is -H and R 60 is a bond or C 1~6 is an alkyl linker, n1 is an integer from 1 to about 6, and R 61 is CH2OH or COOH.

[0214] In some embodiments, R 58 is -H and R 60 Ha-CHR 59 -, n1 is 0, and R 61 is a heterocycloalkyl substituted with one or more hydroxyls, e.g., a monosaccharide.

[0215] In some embodiments, T 1 comprises a glucosyl-amine, diamine, or triamine.

[0216] In some embodiments, T 1 is the following fragment or stereoisomer thereof: TIFF0007777065000082.tif177169, wherein R 59 -H, C 1~8 alkyl, cycloalkyl, or arylalkyl, where n1 is an integer from 1 to about 6, n2 is an integer from 1 to about 5, and n3 is an integer from about 1 to about 3.

[0217] It is understood that all stereochemical forms of the hydrophilic group are contemplated herein. For example, in the above formula, the hydrophilic group may be derived from ribose, xylose, glucose, mannose, galactose, or other sugars, retaining the stereochemical configuration of the pendant hydroxyl and alkyl groups present in these molecules.

[0218] It should be understood that various deoxy compounds are also contemplated in the above formula. Illustratively, where applicable, one or more of the following characteristics for the hydrophilic group are contemplated:

[0219] In some embodiments, n3 is 2 or 3.

[0220] In some embodiments, n1 is 1, 2, or 3.

[0221] In some embodiments, n2 is 1.

[0222] In some embodiments, R 59 is hydrogen.

[0223] In some embodiments, T 1 teeth, TIFF0007777065000083.tif16128. In some embodiments, T 1 teeth, TIFF0007777065000084.tif24128. In some embodiments, T 1 teeth, The file is TIFF0007777065000085.tif24128.

[0224] In some embodiments, T 1 teeth, TIFF0007777065000086.tif15128, wherein: n4 is an integer from 1 to about 25; Each R 63 are independently -H or C 1~8 is alkyl, R 64is a bond or C 1~8 is an alkyl linker, R 65 -H, C 1~8 Alkyl, -(CH2) n2 COOR 62 , or -(CH2) n2 COR 66 and R 62 is H or C 1~8 is alkyl, R 66 H, TIFF0007777065000087.tif48144, n2 is an integer of 1 to about 5.

[0225] In some embodiments, T 1 teeth, TIFF0007777065000088.tif15128, where R 67 is (1)-OH; TIFF0007777065000089.tif64149, and n4 is an integer of about 2 to about 20, about 4 to about 16, about 6 to about 12, or about 8 to about 12.

[0226] In some embodiments, T 1 teeth, The file is TIFF0007777065000090.tif12128.

[0227] In some embodiments, n4 is an integer from about 2 to about 20, from about 4 to about 16, from about 6 to about 12, or from about 8 to about 12.

[0228] In some embodiments, n4 is 6, 7, 8, 9, 10, 11, or 12.

[0229] In some embodiments, n4 is 8 or 12.

[0230] In some embodiments, T 1 teeth, TIFF0007777065000091.tif42128, and n4 is an integer of from about 2 to about 24, from about 4 to about 16, from about 6 to about 12, or from about 8 to about 12.

[0231] In some embodiments, n4 is 6, 7, 8, 9, 10, 11, or 12.

[0232] In some embodiments, n4 is 8 or 12. In some embodiments, n4 is 8.

[0233] In some embodiments, T 1 teeth, TIFF0007777065000092.tif26128, where n4 is 8.

[0234] In some embodiments, T 1 include polyethers, such as polyalkylene glycols (PAOs). PAOs include, but are not limited to, polymers of lower alkylene oxides, particularly polymers of ethylene oxide, such as propylene oxide, polypropylene glycol, polyethylene glycol (PEG), polyoxyethylenated polyols, copolymers thereof, and block copolymers thereof.

[0235] In some embodiments, the polyalkylene glycol is polyethylene glycol (PEG), including but not limited to polydisperse PEG, monodisperse PEG, and discrete PEG.Polydisperse PEG is a heterogeneous mixture of sizes and molecular weights, while monodisperse PEG is typically purified from a heterogeneous mixture and therefore exhibits a single chain length and molecular weight.In some embodiments, the PEG unit is discrete PEG, which represents one type of molecule with a defined and specified chain length.In some embodiments, the polyethylene glycol is mPEG.

[0236] In some embodiments, T 1comprises a PEG unit comprising one or more PEG chains. The PEG chains may be linked together, for example, in a linear, branched, or star configuration. In addition to comprising repeating PEG subunits, the PEG unit may also comprise non-PEG material (e.g., to facilitate coupling of multiple PEG chains to each other or to an amino acid). Non-PEG material refers to atoms in the PEG chain that are not part of the repeating -CH2CHO- subunits. In some embodiments, the PEG chain may comprise two monomeric PEG chains linked to each other via a non-PEG element. In some embodiments, the PEG unit may comprise two linear PEG chains attached to a central core that is attached to an amino acid (i.e., the PEG unit itself is branched).

[0237] PEG units are attached to M via reactive groups. A Linkers (e.g., M A The reactive group may be covalently attached to an amino acid within the linker. A reactive group is a group (e.g., a free amino group or a carboxyl group) to which an activated PEG molecule can be attached. In some embodiments, the N-terminal amino acid and lysine (K) have a free amino group, and the C-terminal amino acid residue has a free carboxyl group. Sulfhydryl groups (e.g., found on cysteine ​​residues) may also be used as reactive groups for attaching PEG.

[0238] In some embodiments, the PEG units are prepared by using methoxylated PEGs ("mPEG") with different reactive moieties, including, but not limited to, succinimidyl succinate (SS), succinimidyl carbonate (SC), mPEG-imidate, para-nitrophenyl carbonate (NPC), succinimidyl propionate (SPA), and cyanuric chloride. A Linkers (e.g., M AExamples of mPEG include mPEG-succinimidyl succinate (mPEG-SS), mPEG2-succinimidyl succinate (mPEG2-SS), mPEG-succinimidyl carbonate (mPEG-SC), mPEG2-succinimidyl carbonate (mPEG2-SC), mPEG-imidate, mPEG-para-nitrophenyl carbonate (mPEG-NPC), mPEG-imidate, mPEG2-para-nitrophenyl carbonate (mPEG2-NP C), mPEG-succinimidyl propionate (mPEG-SPA), mPEG2-succinimidyl propionate (mPEG2-SPA), mPEG-N-hydroxy-succinimide (mPEG-NHS), mPEG2-N-hydroxy-succinimide (mPEG2-NHS), mPEG-cyanuric chloride, mPEG2-cyanuric chloride, mPEG2-lysinol-NPC, and mPEG2-Lys-NHS. A wide variety of PEG species can be used, and virtually any suitable reactive PEG reagent can be used. In some embodiments, the reactive PEG reagent is a multifunctional linker or MPEG-NHS. A Linkers (e.g., M AThey form carbamate or amide bonds when attached to amino acids within the linker. Reactive PEG reagents include mPEG-N-hydroxy-succinimide (mPEG-NHS), bifunctional PEG propionaldehyde (mPEG-ALD), multi-arm PEGs, maleimide-containing PEGs (mPEG(MAL)2, mPEG2(MAL)), mPEG-NH2, mPEG-succinimidyl propionate (mPEG-SPA), mPEG succinimide butanoic acid (mPEG-SBA), mPEG-thioesters, mPEG-double esters, mPEG-BTC, mPEG-ButyrALD, mPEG-acetaldehyde diethyl acetal (mPEG-ACET), heterofunctional PEGs (e.g., NH2-PEG-COOH, Boc-PEG-NHS, Fmoc-PEG-NHS, NHS-PEG-vinyl sulfone (NHS-PEG-VS), or NH These include, but are not limited to, the SUNBRITE™ series of multi-armed PEGs, including glycerin-based PEGs activated by a chemical reaction selected by one of skill in the art, any of the SUNBRITE activated PEGs (including, but not limited to, carboxyl-PEG, p-NP-PEG, tresyl-PEG, aldehyde-PEG, acetal-PEG, amino-PEG, thiol-PEG, maleimide-PEG, hydroxyl-PEG-amine, amino-PEG-COOK hydroxyl-PEG-aldehyde, carboxylic acid anhydride-PEG, functionalized PEG-phospholipids, and other similar and / or suitable reactive PEGs).

[0239] In some embodiments, the PEG unit comprises at least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, at least 12 subunits, at least 13 subunits, at least 14 subunits, at least 15 subunits, at least 16 subunits, at least 17 subunits, at least 18 subunits, at least 19 subunits, at least 20 subunits, at least 21 subunits, at least 22 subunits, at least 23 subunits, or at least 24 subunits, In some such embodiments, the PEG unit comprises about 72 or fewer subunits.

[0240] In some embodiments, the PEG unit comprises at least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, at least 12 subunits, at least 13 subunits, at least 14 subunits, at least 15 subunits, at least 16 subunits, at least 17 subunits, at least 18 subunits, at least 19 subunits, or at least 20 subunits.

[0241] In some embodiments, the PEG unit comprises at least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, at least 12 subunits, at least 13 subunits, at least 14 subunits, at least 15 subunits, at least 16 subunits, at least 17 subunits, or at least 18 subunits.

[0242] In some embodiments, the PEG unit comprises at least 6 subunits, at least 7 subunits, at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, or at least 12 subunits.

[0243] In some embodiments, the PEG unit comprises at least 8 subunits, at least 9 subunits, at least 10 subunits, at least 11 subunits, or at least 12 subunits.

[0244] In some embodiments, the PEG unit comprises at least 6 subunits, at least 7 subunits, or at least 8 subunits.

[0245] In some embodiments, the linear PEG unit is TIFF0007777065000093.tif29147, wherein: TIFF0007777065000094.tif9128 is M A Linkers (e.g., M A indicates the site of attachment of the linker to the amino acid Y 71 is the PEG attachment unit, Y 72 is the PEG capping unit, Y 73 is a PEG coupling unit (i.e., a unit for coupling multiple PEG subunit chains together), d9 is an integer between 2 and 72, Each d 10 are independently an integer from 1 to 72, d 11 is an integer between 2 and 5.

[0246] In some embodiments, d9 is an integer from 2 to 24. In some embodiments, d9 is an integer from 4 to 24. In some embodiments, d9 is an integer from 6 to 24, 8 to 24, 10 to 24, or 12 to 24.

[0247] In some embodiments, the PEG unit has at least 6 PEG subunits. In some embodiments, the PEG unit has at least 8 PEG subunits. In some embodiments, the PEG unit has at least 10 PEG subunits. In some embodiments, the PEG unit has at least 12 PEG subunits.

[0248] In some embodiments, d9 is 8 or about 8, 12 or about 12, 24 or about 24.

[0249] In some embodiments, each Y 72 independently, C 1~10 Alkyl, -C 2~10 Alkyl-CO2H, -C 2~10 Alkyl-OH, -C 2~10 Alkyl-NH2, -C 2~10 Alkyl-NH(C 1~3 alkyl), or C 2~10 Alkyl-N(C 1~3 alkyl)2.

[0250] In some embodiments, Y 72 -C 1~10 Alkyl, -C 2~10 Alkyl-CO2H, -C 2~10 Alkyl-OH, or -C 2~10 It is alkyl-NH2.

[0251] In some embodiments, the PEG coupling unit is a non-PEG material that is part of a PEG unit and serves to connect two or more chains of repeating CH2CH2O- subunits. 73 -C 2~10 Alkyl-C(O)-NH-, -C 2~10 Alkyl-NH-C(O)-, -C 2~10 Alkyl-NH-, -C 2~10 Alkyl-C(O)-, -C 2~10 Alkyl-O- or -C 2~10 It is alkyl-S-.

[0252] In some embodiments, each Y 73 independently, C 1~10 Alkyl-C(O)-NH-, -C 1~10 Alkyl-NH-C(O)-, -C 2~10 Alkyl-NH-, -C 2~10 Alkyl-O-, -C 1~10 Alkyl-S- or -C 1~10 It is alkyl-NH-.

[0253] In some embodiments, the PEG attachment unit is part of a PEG unit, and the PEG unit is A Linkers (e.g., M A The PEG unit functions to link to an amino acid (an amino acid within a linker). In some embodiments, the amino acid has a functional group that forms a bond with a PEG unit. In some embodiments, functional groups for attaching a PEG unit to an amino acid include a sulfhydryl group for forming a disulfide or thioether bond, an aldehyde, ketone, or hydrazine group for forming a hydrazone bond, a hydroxylamine for forming an oxime bond, a carboxy or amino group for forming a peptide bond, a carboxy or hydroxy group for forming an ester bond, a sulfonic acid for forming a sulfonamide bond, an alcohol for forming a carbamate bond, and an amine for forming a sulfonamide, carbamate, or amide bond. In some embodiments, the PEG unit can be attached to an amino acid via, for example, a disulfide, thioester, hydrazine, oxime, peptide, ester, sulfonamide, carbamate, or amide bond. In some embodiments, the reaction for attaching the PEG unit can be a cycloaddition, addition, addition / elimination, or substitution reaction, or a combination thereof, if applicable.

[0254] Examples of linear PEG units include: TIFF0007777065000095.tif38148, wherein: TIFF0007777065000096.tif8128 is a multifunctional linker or M A In the linker (e.g., M A Each d9 independently represents an integer of 4 to 24, 6 to 24, 8 to 24, 10 to 24, 12 to 24, 14 to 24, or 16 to 24.

[0255] In some embodiments, d9 is about 8, about 12, or about 24. In some embodiments, d9 is about 8.

[0256] In some embodiments, the PEG unit is about 300 Da to about 5 kDa; about 300 Da to about 4 kDa; about 300 Da to about 3 kDa; about 300 Da to about 2 kDa; or about 300 Da to about 1 kDa. In some embodiments, the PEG unit has at least 6 subunits, or at least 8, 10, or 12 subunits. In some embodiments, the PEG unit has at least 6 subunits, or at least 8, 10, or 12 subunits, but not more than 24 subunits.

[0257] In some embodiments, suitable polyethylene glycols may have a free hydroxy group at each end of the polymer molecule, or one hydroxy group may be etherified with a lower alkyl, e.g., methyl, group. In some embodiments, suitable polyethylene glycols are derivatives of polyethylene glycols having an esterifiable carboxy group. In some embodiments, polyethylene glycols are commercially available under the trade name PEG, typically as a polymer mixture characterized by average molecular weight. In some embodiments, polyethylene glycols have an average molecular weight of about 300 to about 5,000. In some embodiments, polyethylene glycols have an average molecular weight of about 600 to about 1,000.

[0258] In some embodiments, examples of hydrophilic groups suitable for the conjugates, scaffolds, and methods disclosed herein can be found, for example, in US 8,367,065 column 13; US 8,524,696 column 6; WO 2015 / 057699 and WO 2014 / 062697, the contents of each of which are incorporated by reference herein in their entirety.

[0259] STING agonist drug moiety (variable D) In some embodiments, the STING agonist drug moiety (D) has the formula (A): TIFF0007777065000097.tif62128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, wherein: Y1, Y2, Z1, and Z2 are each independently O, S, C, or N; X1, X2, W1, and W2 are each independently C or N; X3 and X4 are independently S or NR f and X5 is N or CR A2 and X6 is N or CR A1 and R 3 and R 5 are each independently, CON(R d )(R f ), -CH2N(R d )(R f ), -N(R d )(R f ), -N(R d )CO(R f ), -CH2N(R d )CO(R f ) or R 3 and R 5 One of the two is -CON(R d )(R f ), -CH2N(R d )(R f ), -N(R d )(R f ), -N(R d )CO(Rf ) or -CH2N(R d )CO(R f ) and R 3 and R 5 The other is H, -COOH, or -CO2(R C ) and R c is C 1~4 is alkyl, R A2 and R A1 are each independently H, halogen, hydroxy, amino, amino (C 1~4 alkyl)-, optionally substituted (C 1~6 alkyl), or optionally substituted (C 1~6 alkyl)oxy-, and the optionally substituted (C 1~6 alkyl) or optionally substituted (C 1~6 Alkyl)oxy-C 1~6 Alkyl is independently hydroxyl, C 1~4 Alkoxyl, -N(R e )(R f ), -CO2(R f ), -CON(R e )(R f ), and —COOH, Each R d are independently H, hydroxy, or C 1~4 is alkyl, R e is H, (C 1~4 alkyl), -CO(C 1~4 alkyl), -OCO(C 1~4 alkyl), and -CO2(C 1~4 alkyl), Each R f are independently H, hydroxy, or (C 1~4 alkyl), R 14 and R C2 are not independently present or C 1~4 alkyl, C 1~4 Alkyl is halogen, -ORc , -NR c R d , -CO2R c , -CONR c R d , -SO2NR c R d , and -OCONR c R d and optionally substituted with a substituent selected from R 16 and R C1 are each independently absent, H, or C 1~4 is alkyl, R 15 , R 17 , R 18 , or R 19 are each independently absent, H, or C 1~4 alkyl, C 1~4 Alkyl is halogen, -OR c , -NR c R d , -CO2R c , -CONR c R d , -SO2NR c R d , and -OCONR c R d and optionally substituted with a substituent selected from (i)R A2 and R A1 There is at least one of R A2 and R A1 At least one of A2 and / or R A1 via at least one functional group of L D or (ii) R C2 and R C1 There is at least one of R C2 and R C1 At least one of C2 and / or R C1 via at least one functional group of L D is connected to.

[0260] In some embodiments, the STING agonist drug moiety has the formula (Aa): TIFF0007777065000098.tif75128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, wherein: Y1, Y2, Z1, Z2, X1, X2, W1, W2, X3, X4, R 3 , R 5 , R c , R d , R e , R f , R 14 , R C2 , R 16 , R C1 , R 15 , R 17 , R 18 , and R 19 is as defined in formula (A), X5 is CR A2 and R A2 is a halogen, hydroxyl, optionally substituted (C 1~6 alkyl), substituted (C 1~6 alkyl)oxy-, optionally substituted (C 1~6 alkyl)amino-, or optionally substituted (C 1~6 Alkyl)(C 1~4 alkyl)amino-, and the optionally substituted (C 1~6 alkyl) or substituted (C 1~6 Alkyl)oxy-C 1~6 Alkyl is independently hydroxyl, C 1~4 Alkoxyl, -N(R e )(R f ), -CO2(R f ), -CON(R e )(R f ), and —COOH, (i)R A2 is R A2 Through the functional group of L D or (ii) R C2 and R C1There is at least one of R C2 and R C1 At least one of C2 and / or R C1 via at least one functional group of L D is connected to.

[0261] In some embodiments, the STING agonist drug moiety has the formula (Ab): TIFF0007777065000099.tif75128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, wherein: Y1, Y2, Z1, Z2, X1, X2, W1, W2, X4, X5, X6, R 3 , R 5 , R c , R A2 , R A1 , R d , R e , R f , R 14 , R C2 , R 16 , R C1 , R 15 , R 17 , R 18 , and R 19 is as defined in formula (A), (i)R A2 and R A1 There is at least one of R A2 and R A1 At least one of A2 and / or R A1 via at least one functional group of L D or (ii) R C2 and R C1 There is at least one of R C2 and R C1 At least one of C2 and / or R C1 via at least one functional group of L D is connected to.

[0262] In some embodiments, the STING agonist drug moiety has the formula (Ac): TIFF0007777065000100.tif75128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, wherein: Y2, Z2, X2, W2, X3, X4, X5, X6, R 3 , R 5 , R c , R A2 , R A1 , R d , R e , R f , R 14 , R C2 , R 16 , R C1 , R 15 , R 17 , R 18 , and R 19 is as defined in formula (A), one of W1, X1, Y1, and Z1 is N and a further W1, X1, Y1, and Z1 is O, S, or C; (i)R A2 and R A1 There is at least one of R A2 and R A1 At least one of A2 and / or R A1 via at least one functional group of L D or (ii) R C2 and R C1 There is at least one of R C2 and R C1 At least one of C2 and / or R C1 via at least one functional group of L D is connected to.

[0263] In some embodiments, the STING agonist drug moiety has the formula (Ad): TIFF0007777065000101.tif75128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof; Y1, Y2, Z1, Z2, X1, X2, W1, W2, X3, R 3 , R 5 , R c , R d , R e , R f , R 14 , R A2 , R C2 , R 16 , R C1 , R 15 , R 17 , R 18 , and R 19 is as defined in formula (A), X5 is CR A2 and (i)R A2 is R A2 Through the functional group of L D or (ii) R C2 and R C1 There is at least one of R C2 and R C1 At least one of C2 and / or R C1 via at least one functional group of L D is connected to.

[0264] In some embodiments, the STING agonist drug moiety has the formula (Ae): TIFF0007777065000102.tif76128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, wherein: Y1, Y2, Z1, Z2, X1, X2, X3, W1, W2, R A1 , R3, R 5 , R c , R d , R e , R f , R 14 , R C2 , R 16 , R C1 , R 15 , R 17 , R 18 , and R 19 is as defined in formula (A), X6 is CRA1 and (i)R A1 is R A1 Through the functional group of L D or (ii) R C2 and R C1 There is at least one of R C2 and R C1 At least one of C2 and / or R C1 via at least one functional group of L D is connected to.

[0265] In some embodiments, the STING agonist drug moiety has the formula (Af): TIFF0007777065000103.tif71128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, wherein: X2, X3, X4, X5, X6, W2, Y2, Z2, R 3 , R 5 , R c , R A2 , R A1 , R d , R e , R f , R 16 , R 17 , R 18 , R 19 , R C2 , and R C1 is as defined in formula (A), (i)R A2 and R A1 There is at least one of R A2 and R A1 At least one of A2 and / or R A1 via at least one functional group of L D or (ii) R C2 and R C1 There is at least one of R C2 and R C1 At least one of C2 and / or R C1via at least one functional group of L D is connected to.

[0266] In some embodiments, the STING agonist drug moiety has formula (A-f1): TIFF0007777065000104.tif71128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, wherein: X2, X3, X4, W2, Y2, Z2, R 3 , R 5 , R c , R d , R e , R f , R 16 , R A2 , R 17 , R 18 , R 19 , R C2 , and R C1 is as defined in formula (A), X5 is CR A2 and (i)R A2 is R A2 Through the functional group of L D or (ii) R C2 and R C1 There is at least one of R C2 and R C1 At least one of C2 and / or R C1 via at least one functional group of L D is connected to.

[0267] In some embodiments, the STING agonist drug moiety has formula (A-f2): TIFF0007777065000105.tif71128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, wherein: X2, X4, X5, X6, W2, Y2, Z2, R 3 , R 5 , R c , R A2 , R A1 , Rd , R e , R C1 , R C2 , R 16 , R 17 , R 18 , R 19 , and R f is as defined in formula (A), (i)R A2 and R A1 There is at least one of R A2 and R A1 At least one of A2 and / or R A1 via at least one functional group of L D or (ii) R C2 and R C1 There is at least one of R C2 and R C1 At least one of C2 and / or R C1 via at least one functional group of L D is connected to.

[0268] In some embodiments, the STING agonist drug moiety has formula (A-f3): TIFF0007777065000106.tif71128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, wherein: X2, X4, W2, Y2, Z2, R 3 , R 5 , R c , R d , R e , R f , R 16 , R A2 , R C2 , R 16 , R 17 , R 18 , R 19 , and R C1 is as defined in formula (A), X5 is CR A2 and (i)R A2 is R A2 Through the functional group of LD or (ii) R C2 and R C1 There is at least one of R C2 and R C1 At least one of C2 and / or R C1 via at least one functional group of L D is connected to.

[0269] In some embodiments, the STING agonist drug moiety has formula (A-f4): TIFF0007777065000107.tif71128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, wherein: X2, X4, W2, Y2, Z2, R 3 , R 5 , R c , R d , R e , R f , R 16 , R A2 , R C2 , R 16 , R 17 , R 18 , R 19 , and R C1 is as defined in formula (A), X5 is CR A2 and (i)R A2 is R A2 Through the functional group of L D or (ii) R C2 and R C1 There is at least one of R C2 and R C1 At least one of C2 and / or R C1 via at least one functional group of L D is connected to.

[0270] In some embodiments, the STING agonist drug moiety has formula (A-f5): TIFF0007777065000108.tif71128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, wherein: X2, W2, Y2, Z2, R 3 , R 5 , R c , R d , R e , R f , R 16 , R A2 , R C2 , R 16 , R 17 , R 18 , R 19 , and R C1 is as defined in formula (A), X5 is CR A2 and (i)R A2 is R A2 Through the functional group of L D or (ii) R C2 and R C1 There is at least one of R C2 and R C1 At least one of C2 and / or R C1 via at least one functional group of L D is connected to.

[0271] In some embodiments, the STING agonist drug moiety has the formula (Ag): TIFF0007777065000109.tif71128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, wherein: X3, X4, X5, X6, R 3 , R 5 , R c , R A2 , R A1 , R C2 , R 17 , R 18 , R 19 , R d , R e , R f , R 16 , and RC1 is as defined in formula (A), Y2 and Z2 are each independently O, S, C, or N; X2 and W2 are each independently C or N; (i)R A2 and R A1 There is at least one of R A2 and R A1 At least one of A2 and / or R A1 via at least one functional group of L D or (ii) R C2 and R C1 There is at least one of R C2 and R C1 At least one of C2 and / or R C1 via at least one functional group of L D is connected to.

[0272] In some embodiments, the STING agonist drug moiety has formula (A-g1): TIFF0007777065000110.tif71128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, wherein: X2, W2, Y2, Z2, X3, X4, X5, R 3 , R 5 , R c , R d , R e , R f , R A2 , R C2 , R 17 , R 18 , R 19 , R 16 , and R C1 is as defined in formula (A), (i)R A2 is R A2 Through the functional group of L D or (ii) R C2 and R C1 There is at least one of R C2and R C1 At least one of C2 and / or R C1 via at least one functional group of L D is connected to.

[0273] In some embodiments, the STING agonist drug moiety has formula (A-g2): TIFF0007777065000111.tif71128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, wherein: X2, X4, X5, X6, W2, Y2, Z2, R 3 , R 5 , R c , R A2 , R A1 , R C2 , R 17 , R 18 , R 19 , R d , R e , R f , R 16 , and R C1 is as defined in formula (A), (i)R A2 and R A1 There is at least one of R A2 and R A1 At least one of A2 and / or R A1 via at least one functional group of L D or (ii) R C2 and R C1 There is at least one of R C2 and R C1 At least one of C2 and / or R C1 via at least one functional group of L D is connected to.

[0274] In some embodiments, the STING agonist drug moiety has formula (A-g3): TIFF0007777065000112.tif71128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, wherein: X2, X4, W2, Y2, Z2, R 3 , R 5 , R c , R A2 , R C2 , R 17 , R 18 , R 19 , R 16 , and R C1 is as defined in formula (A), X5 is CR A2 and (i)R A2 is R A2 Through the functional group of L D or (ii) R C2 and R C1 There is at least one of R C2 and R C1 At least one of C2 and / or R C1 via at least one functional group of L D connected to Optionally, R A2 is R A2 Through the functional group of L D is connected to.

[0275] In some embodiments, the STING agonist drug moiety has formula (A-g4): TIFF0007777065000113.tif71128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, wherein: X2, X4, W2, Y2, Z2, R 3 , R 5 , R c , R d , R e , R f , R A2 , R C2 , R 17 , R 18 , R 19 , R 16, and R C1 is as defined in formula (A), X5 is CR A2 and (i)R A2 is R A2 Through the functional group of L D or (ii) R C2 and R C1 There is at least one of R C2 and R C1 At least one of C2 and / or R C1 via at least one functional group of L D is connected to.

[0276] In some embodiments, the STING agonist drug moiety has the formula (A-g5): TIFF0007777065000114.tif71128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof; X2, W2, Y2, Z2, R 3 , R 5 , R c , R d , R e , R f , R A2 , R C2 , R 17 , R 18 , R 19 , R 16 , and R C1 is as defined in formula (A), X5 is CR A2 and (i)R A2 is R A2 Through the functional group of L D or (ii) R C2 and R C1 There is at least one of R C2 and R C1 At least one of C2 and / or R C1 via at least one functional group of L D is connected to.

[0277] In some embodiments, the STING agonist drug moiety has the formula (Ah): TIFF0007777065000115.tif78128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, wherein: X1, W1, Y1, Z1, X3, X4, X5, X6, R 3 , R 5 , R c , R A2 , R A1 , R d , R e , R f , R 14 , R 15 , R 18 , R 19 , R 16 , and R C1 is as defined in formula (A), (i)R A2 and R A1 There is at least one of R A2 and R A1 At least one of A2 and / or R A1 via at least one functional group of L D or (ii) R C2 and R C1 There is at least one of R C2 and R C1 At least one of C2 and / or R C1 via at least one functional group of L D is connected to.

[0278] In some embodiments, the STING agonist drug moiety has formula (A-h1): TIFF0007777065000116.tif78128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, wherein: X1, X3, W1, Y1, Z1, X5, X6, R 3 , R 5 , R c , RA2 , R A1 , R d , R e , R f , R 14 , R 15 , R 18 , R 19 , R 16 , and R C1 is as defined in formula (A), (i)R A2 and R A1 There is at least one of R A2 and R A1 At least one of A2 and / or R A1 via at least one functional group of L D or (ii) R C2 and R C1 There is at least one of R C2 and R C1 At least one of C2 and / or R C1 via at least one functional group of L D is connected to.

[0279] In some embodiments, the STING agonist drug moiety has formula (A-h2): TIFF0007777065000117.tif81128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof, wherein: X1, X3, W1, Y1, Z1, R 3 , R 5 , R c , R d , R e , R f , R A2 , R 14 , R 15 , R 18 , R 19 , R 16 , and R C1 is as defined in formula (A), X5 is CR A2 and (i)R A2 and RA1 There is at least one of R A2 and R A1 At least one of A2 and / or R A1 via at least one functional group of L D or (ii) R C2 and R C1 There is at least one of R C2 and R C1 At least one of C2 and / or R C1 via at least one functional group of L D is connected to.

[0280] In some embodiments, the STING agonist drug moiety (D) is a compound of formula (A), wherein said compound has formula (Ai): TIFF0007777065000118.tif57128, or a prodrug, solvate, pharmaceutically acceptable salt, or tautomer thereof; Y1, Y2, Z1, Z2, X1, X2, X3, X6, W1, W2, R A1 , R A2 , R c , R d , R e , R f , R 14 , R C2 , R 16 , R C1 , R 15 , R 17 , R 18 , and R 19 is as defined in formula (A), (i)R A2 and R A1 There is at least one of R A2 and R A1 At least one of A2 and / or R A1 via at least one functional group of L D or (ii) R C2 and R C1 There is at least one of R C2 and RC1 At least one of C2 and / or R C1 via at least one functional group of L D is connected to.

[0281] In some embodiments, each STING agonist drug moiety (D) independently comprises: TIFF0007777065000119.tif202131TIFF0007777065000120.tif215128TIFF0007777065000121.tif 218128TIFF0007777065000122.tif215126TIFF0007777065000123.tif218128TIFF00077770650001 24.tif214128TIFF0007777065000125.tif219131TIFF0007777065000126.tif208126TIFF0007777065000127.tif197126TIFF0007777065000128.tif200126TIFF0007777065000129.tif96128, wherein R 2 is absent, -O-, or -NR 4 - and R 4 is H or C 1~3 is alkyl, TIFF0007777065000130.tif4128 is L D Indicates that the device is attached to a

[0282] In some embodiments, each STING agonist drug moiety (D) independently comprises: TIFF0007777065000131.tif52128TIFF0007777065000132.tif216130TIFF0007777065000133.tif168130TIFF0007777065000134.tif103128, wherein R 2 is absent, -O-, or -NR 4 - and R 4is H or C 1~3 is alkyl, TIFF0007777065000135.tif4128 is L D Indicates that the device is attached to a

[0283] In some embodiments, each STING agonist drug moiety (D) independently comprises: TIFF0007777065000136.tif162128, wherein: R 2 is absent, -O-, or -NR 4 - and R 4 is H or C 1~3 is alkyl, TIFF0007777065000137.tif4128 is L D Indicates that the device is attached to a

[0284] In some embodiments, each STING agonist drug moiety (D) independently comprises: TIFF0007777065000138.tif153139, wherein: R 2 is absent, -O-, or -NR 4 - and R 4 is H or C 1~3 is alkyl, TIFF0007777065000139.tif4128 is L D Indicates that the device is attached to a

[0285] In some embodiments, each STING agonist drug moiety (D) independently comprises: TIFF0007777065000140.tif109139, wherein: R 2 is absent, -O-, or -NR 4 - and R 4 is H or C 1~3 is alkyl, TIFF0007777065000141.tif4128 is LD Indicates that the device is attached to a

[0286] Protein-based recognition molecules (PBRMs) In some embodiments, the protein-based recognition molecule directs the conjugate to a specific tissue, cell, or intracellular location. In some embodiments, the protein-based recognition molecule can direct the conjugate in culture, in a whole organism, or both. In each case, the protein-based recognition molecule may have a ligand present on the cell surface of the target cell to which the protein-based recognition molecule binds with effective specificity, affinity, and avidity. In some embodiments, the protein-based recognition molecule targets the conjugate to a tissue other than the liver. In some embodiments, the protein-based recognition molecule targets the conjugate to a specific tissue, such as the liver, kidney, lung, or pancreas. The protein-based recognition molecule can target the conjugate to a receptor expressed on a target cell, such as a cancer cell, a matrix tissue, or a protein associated with cancer, such as a tumor antigen. Alternatively, cells containing tumor vasculature may be targeted. The protein-based recognition molecule can direct the conjugate to a specific type of cell, such as specifically targeting hepatocytes in the liver as opposed to Kupffer cells. In some embodiments, the protein-based recognition molecule can direct the conjugate to cells of the reticuloendothelial system or lymphatic system, or to professional phagocytes such as macrophages or eosinophils, hi some embodiments, the conjugate itself can be an effective delivery system without the need for specific targeting.

[0287] In some embodiments, the protein-based recognition molecule can target the conjugate to a location within the cell, such as the nucleus, cytoplasm, or endosome, hi some embodiments, the protein-based recognition molecule can enhance cellular binding to a receptor or cytoplasmic transport to the nucleus and nuclear import or release from endosomes or other intracellular vesicles.

[0288] In some embodiments, the protein-based recognition molecule is an antibody, an antibody fragment, a protein, a peptide, or a peptidomimetic.

[0289] In some embodiments, the protein-based recognition molecule is an antibody. In some embodiments, the protein-based recognition molecule is an antibody fragment. In some embodiments, the protein-based recognition molecule is a protein. In some embodiments, the protein-based recognition molecule is a peptide. In some embodiments, the protein-based recognition molecule is a peptidomimetic.

[0290] In some embodiments, the antibody or antibody fragment is an antibody or antibody fragment in which one or more amino acids of the corresponding parent antibody or antibody fragment (e.g., the corresponding wild-type antibody or antibody fragment) have been substituted with a cysteine ​​(e.g., an engineered cysteine). In some embodiments, the parent antibody or antibody fragment may be wild-type or mutated.

[0291] In some embodiments, the antibody or antibody fragment may be a mutated antibody or antibody fragment. In some embodiments, a monoclonal antibody known in the art is engineered to form the antibody. In some embodiments, an antibody fragment known in the art (e.g., a Fab antibody fragment) is engineered to form the antibody fragment (e.g., a cysteine-engineered Fab antibody fragment). In some embodiments, a single mutation in the Fab results in one residue in the Fab, whereas a single mutation in the antibody results in two amino acids in the resulting antibody due to the dimeric nature of IgG antibodies.

[0292] In some embodiments, the antibody or antibody fragment retains the antigen-binding ability of its corresponding wild-type antibody or antibody fragment, hi some embodiments, the antibody or antibody fragment is capable of binding to one or more antigens of its corresponding wild-type antibody or antibody fragment.

[0293] In some embodiments, exemplary antibodies or antibodies derived from Fab, Fab2, scFv, or camelid antibody heavy chain fragments specific for cell surface markers include 5T4, AOC3, ALK, AXL, B7-H4, C242, C4.4a, CA-125, CCL11, CCR5, CD2, CD3, CD4, CD5, CD15, CA15-3, CD18, CD19, CA19-9, CDH6, CD20, CD22, CD23, CD25, CD28, CD30, CD31, CD33, CD37, CD38, CD40, CD41, CD44, CD44v6, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L ...P, CD62P, CD62P, CD62P, CD62P, CD62P, CD62P, CD62P, CD62P, CD62P, CD62P, CD62P, CD62P, CD62P, CD62P, CD62P, CD62P, CD62P, CD62P, CD62P, CD62P, CD6 D70, CD74, CD79-B, CD80, CD125, CD103, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM-5, clumping factor, Clec9A, CSFR1, CTLA-4, CXCR2, DEC205, EGFR (HER1), ErbB1, ErbB2, ErbB3, EpCAM, EPHA2, EPHB2, EPHB4, FAP, FGFR (i.e., FGFR1, FGFR2, FGFR3, FGFR4), FLT3, fibronectin-EDB, folate receptor, GD2, GD3, GPNMB, GCC (GUCY2C), HGF, HER2, HER3, and HMI.24, ICAM, ICOS-L, IGF-1 receptor, VEGFR1, EphA2, TRPV1, CFTR, gpNMB, CA9, Cripto, c-KIT, c-MET, ACE, APP, adrenergic receptor-β2, claudin 3, LIV1, LY6E, mesothelin, MUC1, MUC13, NaPi2b, NOTCH1, NOTCH2, NOTCH3, NOTCH4, RON, ROR1, PD-L1, PD-L2, PTK7, B7-H3, B7-B4, IL-2 receptor, IL-4 receptor, IL-13 receptor, TROP-2, frizzled-7, integrin (including α4, αvβ3, αvβ5, αvβ6, α1β4, α4β1, α4β7, α5β1, α6β4, and αIIbβ3 integrins), IFN-α, IFN-γ, IgE, IgE, IGF-1 receptor, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, ITGB2 (CD18), LFA-1 (CD11a), CD11b, L-selectin (CD62L), mucin, myostatin, NCA-90, NGF, PDGFRα, phosphatidylserine, prostate cancer cells, Pseudomonas aeruginosa aeruginosa), rabies, RANKL, respiratory syncytial virus, Rh factor, SLAMF7, sphingosine-1-phosphate, TAG-72, T cell receptor, tenascin-C, TGF-1, TGF-β2, TGF-β, TNF-α, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR2, vimentin, and the like.

[0294] In some embodiments, antibodies or antibodies derived from Fab, Fab2, scFv, or camelid antibody heavy chain fragments specific for cell surface markers include, but are not limited to, CA-125, C242, CD3, CD11b, CD19, CD22, CD25, CD30, CD31, CD33, CD37, CD40, CD44, CD51, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD103, CD138, CD141, CD326, CEA, Clec9A, CSFR1, CTLA-4, DEC205, EGFR (HER1), ErbB2, ErbB3, FAP, fibronectin-EDB, folate receptor, IGF-1 receptor, GD3, GPNMB, HGF, HER2, VEGF-A, VEG FR2, VEGFR1, EphA2, EpCAM, 5T4, PTK7, TAG-72, tenascin-C, TRPV1, CFTR, gpNMB, CA9, Cripto, ACE, APP, PDGFRα, phosphatidylserine, prostate cancer cells, adrenergic receptor-β2, claudin 3, mucin, MUC1, NaPi2b, B7H3, B7H4, C4.4a, CEACAM-5, MUC13, TROP-2, frizzled-7, mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, and integrins (including αvβ3, αvβ5, αvβ6, α1β4, α4β1, α5β1, α6β4 interdin), tenascin-C, TRAIL-R2, and vimentin.

[0295] In some embodiments, the antibody is selected from the group consisting of 5T4, CA-125, CEA, CDH6, CD3, CD11b, CD19, CD20, CD22, CD30, CD33, CD40, CD44, CD51, CD-103, CTLA-4, CEACAM5, Clec9A, CSFR1, DEC205, EpCAM, HER2, EGFR (HER1), FAP, fibronectin-EDB, folate receptor, GCC (GU), CY2C), HGF, integrin αvβ3, integrin α5β1, IGF-1 receptor, GD3, GPNMB, mucin, LIV1, LY6E, mesothelin, MUC1, MUC13, NaPi2b, PTK7, phosphatidylserine, prostate cancer cells, PDGFRα, TAG-72, tenascin-C, TRAIL-R2, VEGF-A, and VEGFR2 cell surface markers. In this embodiment, the antibody may be selected from the group consisting of abagovomab, adecatumumab, alacizumab, altumomab, anatumomab, arcitumomab, bavituximab, bevacizumab (AVASTIN®), bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, capromab, cetuximab, sitatuzumab, clivatuzumab, conatumumab, dacetuzumab, edrecolomab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, figitumumab, gemtuzumab, glembatumumab, ibritumomab, igovomab, intetumumab, inotuzumab, labetuzumab, lexatumumab, These include, but are not limited to, lintuzumab, lucatumumab, matuzumab, mitumomab, naptumomab-estafenatox, necitumumab, oportuzumab, oregovomab, panitumumab, pemtumomab, pertuzumab, pritumumab, rituximab (RITUXAN®), rilotumumab, lobatumumab, satumomab, sibrotuzumab, taplitumomab, tenatumomab, tenatumomab, ticilimumab, (tremelimumab), tigatuzumab, trastuzumab (HERCEPTIN®), tositumomab, tremelimumab, tucotuzumab-celmoleukin, volociximab, and zalutumumab.

[0296] In some embodiments, antibodies made against cell surface markers are, for HER2, pertuzumab or trastuzumab; for EGFR (HER1), the antibody is cetuximab or panitumumab; for CD20, the antibody is rituximab; for VEGF-A, the antibody is bevacizumab; for CD-22, the antibody is epratuzumab or veltuzumab; and for CEA, the antibody is labetuzumab.

[0297] Exemplary peptides or peptide mimics include integrin targeting peptides (RGD peptides), LHRH receptor targeting peptides, ErbB2 (HER2) receptor targeting peptides, prostate-specific membrane-bound antigen (PSMA) targeting peptides, lipoprotein receptor LRP1 targeting, ApoE protein-derived peptides, ApoA protein peptides, somatostatin receptor targeting peptides, chlorotoxin-derived peptides, and bombesin.

[0298] In some embodiments, the peptides or peptidomimetics are LHRH receptor targeting peptides and ErbB2 (HER2) receptor targeting peptides.

[0299] Exemplary proteins include insulin, transferrin, fibrinogen-γ fragment, thrombospondin, claudins, apolipoprotein E, affibody molecules such as ABY-025, ankyrin repeat proteins, ankyrin-like repeat proteins, and synthetic peptides.

[0300] In some embodiments, the protein-drug conjugate comprises a broad-spectrum cytotoxin in combination with a cell surface marker, e.g., pertuzumab or trastuzumab for HER2, e.g., cetuximab and panitumumab for EGFR, e.g., labetuzumab for CEA, e.g., rituximab for CD20, e.g., bevacizumab for VEGF-A, or epratuzumab or veltuzumab for CD-22.

[0301] In some embodiments, a protein-drug conjugate or protein conjugate used in the present disclosure includes a combination of two or more protein-based recognition molecules, for example, a combination of a bispecific antibody directed against EGF receptor (EGFR) on tumor cells and CD3 and CD28 on T cells; a combination of an antibody or an antibody derived from a Fab, Fab2, scFv, or camelid antibody heavy chain fragment and a peptide or peptidomimetic; a combination of an antibody or an antibody derived from a Fab, Fab2, scFv, or camelid antibody heavy chain fragment and a protein; or a combination of two bispecific antibodies, for example, a CD3-CD19+CD28-CD22 bispecific antibody.

[0302] In some embodiments, the protein-drug conjugate or protein conjugate used in the present disclosure is selected from the group consisting of, for example, trastuzumab, cetuximab, rituximab, bevacizumab, epratuzumab, veltuzumab, labetuzumab, B7-H4, B7-H3, CD11b, CD103, CA125, CDH6, CD33, CXCR2, CEACAM5, Clec9A, CSFR1, DEC205, E These include protein-based recognition molecules that are antibodies against antigens such as GFR, FAP, fibronectin-EDB, FGFR1, FGFR2, FGFR3, FGFR4, GCC (GUCY2C), HER2, LIV1, LY6E, NaPi2b, c-Met, mesothelin, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PD-L1, PTK7, c-Kit, MUC1, MUC13, and 5T4.

[0303] In some embodiments, the protein-drug conjugate or protein conjugate of the present disclosure comprises a protein-based recognition molecule that is CSRF1, CD11b, DEC205, clec9A, CD103, B7H4, mesothelin, PTK7, Ly6E, FAP, fibronectin-EDB, Her-2, or a NaPi2b antibody.

[0304] NaPi2b antibody In some embodiments, the NaPi2b antibody suitable for conjugation binds to the extracellular region of SLC34A2. In some embodiments, the present disclosure provides a NaPi2b-targeting monoclonal antibody that specifically recognizes NaPi2b, also known as sodium-dependent phosphate transporter protein 2B. In some embodiments, the NaPi2b antibody used in the conjugates disclosed herein can modulate, for example, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with, at least one biological activity of NaPi2b, and is useful for modulating, for example, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with. In some embodiments, the antibodies disclosed herein also include antibodies that bind to soluble NaPi2b. In some embodiments, the NaPi2b antibody specifically binds to an epitope on the extracellular domain (ECD) of human NaPi2b. These antibodies are collectively referred to herein as "NaPi2b" antibodies.

[0305] In some embodiments, the NaPi2b antibody-drug conjugates provided herein bind to a NaPi2b epitope with an equilibrium dissociation constant (K d or K D In some embodiments, the NaPi2b antibodies used in the antibody-drug conjugates disclosed herein have a K in the range of about ≦1 nM to about 1 pM. d Shows.

[0306] In some embodiments, the NaPi2b antibody-drug conjugates provided herein may comprise an antibody that acts to regulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with the functional activity of NaPi2b. In some embodiments, the functional activity of NaPi2b includes, for example, participating in transcellular inorganic phosphate (Pi) absorption, thereby contributing to the maintenance of phosphate homeostasis in the body. In some embodiments, the NaPi2b antibody completely or partially inhibits NaPi2b functional activity by partially or completely regulating, blocking, inhibiting, reducing, antagonizing, neutralizing, or otherwise interfering with transcellular inorganic phosphate absorption.

[0307] In some embodiments, a NaPi2b antibody is considered to completely modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with NaPi2b functional activity when the level of NaPi2b functional activity in the presence of the NaPi2b antibody is reduced by at least 95%, e.g., 96%, 97%, 98%, 99%, or 100%, compared to the level of NaPi2b functional activity in the absence of binding to a NaPi2b antibody described herein. In some embodiments, a NaPi2b antibody is considered to partially modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with NaPi2b functional activity when the level of NaPi2b activity in the presence of the NaPi2b antibody is reduced by less than 95%, e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, or 90%, compared to the level of NaPi2b activity in the absence of binding to a NaPi2b antibody described herein.

[0308] In some embodiments, exemplary antibodies disclosed herein include the XMT-1535 antibody, which exhibits specificity for human NaPi2b and has been shown to inhibit NaPi2b activity.

[0309] The NaPi2b human or humanized monoclonal antibody, XMT-1535, comprises a heavy chain (HC), a heavy chain variable region (VH), a light chain (LC), and a light chain variable region (VL), as shown in the amino acid sequences and corresponding nucleic acid sequences set forth in Table I below. In the amino acid sequences below, the variable heavy and variable light chain regions of each antibody are shaded. In the amino acid sequences set forth below, the complementarity determining regions (CDRs) of the heavy and light chains are underlined. The amino acids comprising the complementarity determining regions (CDRs) of the XMT-1535 antibody are disclosed in U.S. Patent No. 8,603,474.

[0310] Table I: NaPi2b human or humanized monoclonal antibody XMT-1535 sequence TIFF0007777065000142.tif76161

[0311] The antibodies disclosed herein specifically bind to an epitope on the extracellular domain (ECD) of human NaPi2b.

[0312] In some embodiments, one skilled in the art will recognize that, without undue experimentation, one can determine whether a monoclonal antibody has the same specificity as a monoclonal antibody disclosed herein (e.g., XMT-1535, 10H1.11.4B) by determining whether the former prevents the latter from binding to its natural binding partner or other molecules known to bind to NaPi2b. If the monoclonal antibody being tested competes with the monoclonal antibody disclosed herein, as indicated by reduced binding by the monoclonal antibody disclosed herein, then the two monoclonal antibodies bind to the same epitope or closely related epitopes.

[0313] An alternative method for determining whether a monoclonal antibody has the specificity of the monoclonal antibodies disclosed herein is to preincubate the monoclonal antibodies disclosed herein with soluble NaPi2b (which normally reacts with the monoclonal antibodies disclosed herein), and then add the monoclonal antibody being tested to determine whether it is inhibited in its ability to bind to NaPi2b. If the monoclonal antibody being tested is inhibited, then it likely has the same or a functionally equivalent epitope specificity as the monoclonal antibodies disclosed herein.

[0314] The monoclonal antibodies disclosed herein can also be screened, for example, by measuring NaPi2b-mediated activity to determine whether the test monoclonal antibody is capable of modulating, blocking, inhibiting, reducing, antagonizing, neutralizing, or otherwise interfering with NaPi2b activity.

[0315] In some embodiments, the antibodies disclosed herein comprise a heavy chain variable region having an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a sequence selected from SEQ ID NO:3, and a light chain variable region having an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a sequence selected from SEQ ID NO:4.

[0316] In some embodiments, the antibodies disclosed herein comprise a heavy chain amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:1 and a light chain amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:2.

[0317] In some embodiments, the antibodies disclosed herein comprise the heavy chain variable region amino acid sequence of SEQ ID NO:3 and the light chain variable region amino acid sequence of SEQ ID NO:4.

[0318] In some embodiments, the antibodies disclosed herein comprise the heavy chain amino acid sequence of SEQ ID NO:1 and the light chain amino acid sequence of SEQ ID NO:2.

[0319] In some embodiments, the antibodies disclosed herein comprise a CDRH1 amino acid sequence of SEQ ID NO:5, a CDRH2 amino acid sequence of SEQ ID NO:6, a CDRH3 amino acid sequence of SEQ ID NO:7, a CDRL1 amino acid sequence of SEQ ID NO:8, a CDRL2 amino acid sequence of SEQ ID NO:9, and a CDRL3 amino acid sequence of SEQ ID NO:10.

[0320] In some embodiments, an antibody disclosed herein comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:5; a CDRH2 comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:6; CDRH3 comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:7; CDRL1 comprising an amino acid sequence 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:8; CDRL2 comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:9; and A CDRL3 comprising an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of NO:10.

[0321] In some embodiments, the antibodies disclosed herein comprise one or more conservative amino acid substitutions in the variable domain sequence, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more conservative substitutions in the variable domain sequence. In some embodiments, these conservative amino acid substitutions are in the CDR regions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more conservative substitutions are made cumulatively across all CDRs, and in some particular embodiments, there may be 1, 2, 3, or up to 4 conservative amino acid substitutions in each CDR sequence, e.g., in SEQ ID NOs:5-10.

[0322] In some embodiments, one skilled in the art will recognize that, without undue experimentation, one can determine whether a monoclonal antibody has the same specificity as monoclonal antibody XMT-1535 by determining whether the former prevents the latter from binding to its natural binding partner or other molecules known to bind to NaPi2b. If the monoclonal antibody being tested competes with the monoclonal antibodies disclosed herein, as indicated by reduced binding by the monoclonal antibodies disclosed herein, then the two monoclonal antibodies bind to the same epitope or closely related epitopes.

[0323] In some embodiments, an alternative method for determining whether a monoclonal antibody has the specificity of the monoclonal antibodies disclosed herein is to preincubate the monoclonal antibodies disclosed herein with soluble NaPi2b (which normally reacts with the monoclonal antibodies disclosed herein), and then add the monoclonal antibody being tested to determine whether it is inhibited in its ability to bind to NaPi2b. In some embodiments, if the monoclonal antibody being tested is inhibited, it has the same or a functionally equivalent epitope specificity as the monoclonal antibodies disclosed herein.

[0324] Screening of the monoclonal antibodies disclosed herein can also be carried out, for example, by measuring NaPi2b-mediated activity and determining whether the test monoclonal antibody is capable of modulating, blocking, inhibiting, reducing, antagonizing, neutralizing, or otherwise interfering with NaPi2b activity.

[0325] In some embodiments, NaPi2b antibodies suitable for conjugation can be produced and purified by well-known techniques, e.g., WO2009 / 097128, WO2017 / 160754, and US16 / 136,706, each of which is incorporated by reference herein in its entirety.

[0326] HER2 antibody In some embodiments, HER2 antibodies suitable for conjugation bind to human HER2 in soluble or membrane-bound form (i.e., when expressed on the cell surface). In some embodiments, the present disclosure provides monoclonal antibodies that bind to HER2 and are humanized or fully human. In some embodiments, the present disclosure provides monoclonal antibodies that specifically bind to HER2. These antibodies are collectively referred to herein as "HER2" antibodies.

[0327] In some embodiments, a HER2 antibody suitable for conjugation binds to a HER2 epitope with an equilibrium dissociation constant (K d or K D In some embodiments, the present disclosure provides monoclonal antibodies that bind to HER2 and are humanized or fully human. For example, the HER2 antibodies provided herein have a K in the range of about ≦1 nM to about 1 pM. d Shows.

[0328] In some embodiments, the HER2 antibody disclosed herein acts to regulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with HER2 functional activity.In some embodiments, HER2 functional activity includes, for example, regulating PI3K-Akt pathway activity.In some embodiments, the HER2 antibody completely or partially inhibits HER2 functional activity by partially or completely regulating, blocking, inhibiting, reducing, antagonizing, neutralizing or otherwise interfering with PI3K-Akt pathway activity.PI3K-Akt pathway activity can be assessed using any art-recognized method for detecting PI3K-Akt pathway activity, including but not limited to, detecting the level of phosphorylated Akt in the presence and absence of the antibody or antigen-binding fragment disclosed herein.

[0329] In some embodiments, a HER2 antibody is considered to completely modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with HER2 functional activity when the level of HER2 functional activity in the presence of the HER2 antibody is reduced by at least 80%, e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, compared to the level of HER2 functional activity in the absence of binding with a HER2 antibody described herein. In some embodiments, a HER2 antibody is considered to partially modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with HER2 functional activity when the level of HER2 activity in the presence of the HER2 antibody is reduced by less than 95%, e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, or 90%, compared to the level of HER2 activity in the absence of binding to a HER2 antibody described herein.

[0330] In some embodiments, exemplary antibodies disclosed herein include the XMT-1519 antibody, which exhibits specificity for human HER2 and has been shown to inhibit HER2 functional activity in vitro.

[0331] The HER-2 monoclonal antibody XMT-1519 comprises a heavy chain (HC), a heavy chain variable region (VH), a light chain (LC), and a light chain variable region (VL), as shown in the amino acid sequences and corresponding nucleic acid sequences set forth in Table II below. The variable heavy and variable light chain regions of each antibody are shaded in the amino acid sequences below. The complementarity-determining regions (CDRs) of the heavy and light chains are underlined in the amino acid sequences set forth below.

[0332] Table II: HER2 human or humanized monoclonal antibody XMT-1519 sequence TIFF0007777065000143.tif80161

[0333] The antibodies and antigen-binding fragments thereof disclosed herein specifically bind to an epitope on the full-length human HER2 receptor, comprising the amino acid sequence of SEQ ID NO:16.

[0334] The antibodies and antigen-binding fragments thereof disclosed herein specifically bind to an epitope on the extracellular domain (ECD) of the human HER2 receptor, comprising the amino acid sequence of SEQ ID NO:31.

[0335] In some embodiments, the antibody of the present disclosure exhibits different HER2 binding properties from the antibody described in the art.In some embodiments, the antibody disclosed herein cross-blocks each other, but does not cross-block trastuzumab, pertuzumab, Fab37 or chA21 from binding to HER2, so binds to different HER2 epitopes.In addition, in contrast to known antibody, the antibody disclosed herein can be efficiently internalized into HER2-expressing cells without promoting cell proliferation.

[0336] In some embodiments, the antibodies disclosed herein are fully human monoclonal antibodies that bind to novel epitopes and / or have other favorable properties for therapeutic use. In some embodiments, exemplary properties include, but are not limited to, favorable binding properties to cancer cells that express high or low levels of human HER2, specific binding to recombinant human and cynomolgus monkey HER2, efficient internalization upon binding to HER2, high ability to kill cancer cells that express high or low levels of HER2 when administered as an antibody-drug conjugate (ADC), lack of significant agonistic effect on the growth of HER2-expressing cancer cells, and / or effective antibody-dependent cellular cytotoxicity (ADCC)-mediated killing of HER2-expressing cells, and any combination of the aforementioned properties.

[0337] In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments thereof that specifically bind to an epitope of the human HER2 receptor, including residues 452-531 of the extracellular domain of the human HER2 receptor, residues 474-553 of SEQ ID NO:16, or residues 452-531 of SEQ ID NO:31.

[0338] In some embodiments, the antibodies disclosed herein include antibodies or antigen-binding fragments thereof that bind to at least a portion of the N-terminus of domain IV of the human HER2 receptor but do not cross-compete with antibodies that bind to epitope 4D5 of the human HER2 receptor. In some embodiments, because trastuzumab is known to bind to epitope 4D5 of the human HER2 receptor, the antibodies or antigen-binding fragments thereof described herein do not cross-compete with trastuzumab for binding to the human HER2 receptor. As used herein, the term epitope 4D5 of the human HER2 receptor refers to amino acid residues 529-627 of the extracellular domain of the human HER2 receptor, residues 551-649 of SEQ ID NO:16, or residues 529-627 of SEQ ID NO:31. In some embodiments, the antibodies or antigen-binding fragments thereof also bind to at least one epitope on the cynomolgus monkey HER2 receptor.

[0339] In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments thereof that specifically bind to an epitope of the human HER2 receptor, including residues 452-500 of the extracellular domain of the human HER2 receptor, residues 474-522 of SEQ ID NO:16, or residues 452-500 of SEQ ID NO:31.

[0340] In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments thereof that specifically bind to an epitope of the human HER2 receptor comprising at least one amino acid residue selected from amino acid residues E521, L525, and R530 of the extracellular domain of the human HER2 receptor, e.g., residues 543, 547, and 552 of SEQ ID NO: 16 and residues 521, 525, and 530 of SEQ ID NO: 31. In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments thereof that specifically bind to an epitope of the human HER2 receptor comprising at least two amino acid residues selected from amino acid residues E521, L525, and R530 of the extracellular domain of the human HER2 receptor. In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments thereof that specifically bind to an epitope of the human HER2 receptor comprising at least amino acid residues E521, L525, and R530 of the extracellular domain of the human HER2 receptor. In some embodiments, any or all of these antibodies or antigen-binding fragments thereof also bind to at least one epitope on the cynomolgus monkey HER2 receptor.

[0341] In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments thereof that bind to at least a portion of domain III and at least a portion of the N-terminus of domain IV of the human HER2 receptor, but do not cross-compete with Fab37 monoclonal antibody or antibodies that bind to epitope 4D5 of the human HER2 receptor. In some embodiments, the antibodies or antigen-binding fragments thereof described herein do not cross-compete with Fab37 monoclonal antibody and / or trastuzumab for binding to the human HER2 receptor. In some embodiments, the antibodies or antigen-binding fragments thereof also bind to at least one epitope on the cynomolgus monkey HER2 receptor.

[0342] In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments thereof that specifically bind to an epitope of the human HER2 receptor that includes residues 520-531 of the extracellular domain of the human HER2 receptor, residues 542-553 of SEQ ID NO:16, or residues 520-531 of SEQ ID NO:31.

[0343] In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments thereof that specifically bind to an epitope of the human HER2 receptor that includes at least one amino acid residue selected from residues C453, H456, H473, N476, R495, G496, H497, and W499 of the extracellular domain of the human HER2 receptor, e.g., residues 475, 478, 495, 498, 517, 518, 519, and 521 of SEQ ID NO: 16, or residues 453, 456, 473, 476, 495, 496, 497, and 499 of SEQ ID NO: 31. In some embodiments, the antibodies disclosed herein include antibodies or antigen-binding fragments thereof that specifically bind to an epitope in the extracellular domain of the human HER2 receptor that includes at least two, at least three, at least four, at least five, or at least six amino acid residues selected from amino acid residues C453, H456, H473, N476, R495, G496, H497, and W499 in the extracellular domain of the human HER2 receptor. In some embodiments, the antibodies disclosed herein include antibodies or antigen-binding fragments thereof that specifically bind to an epitope in the extracellular domain of the human HER2 receptor that includes at least amino acid residues C453, H456, H473, N476, R495, G496, H497, and W499 in the extracellular domain of the human HER2 receptor. In some embodiments, any or all of these antibodies or antigen-binding fragments thereof also bind to at least one epitope on the cynomolgus monkey HER2 receptor.

[0344] In some embodiments, the antibodies disclosed herein also include antibodies or antigen-binding fragments thereof that specifically bind to an epitope of the human HER2 receptor that includes at least one amino acid residue selected from residues C453, H473, N476, R495, H497, and W499 of the extracellular domain of the human HER2 receptor, e.g., residues 475, 495, 498, 517, 519, and 521 of SEQ ID NO:16, or residues 453, 473, 476, 495, 497, and 499 of SEQ ID NO:31. In some embodiments, the antibodies disclosed herein include antibodies or antigen-binding fragments thereof that specifically bind to an epitope in the extracellular domain of the human HER2 receptor that includes at least two, at least three, at least four, at least five, or at least six amino acid residues selected from amino acid residues C453, H473, N476, R495, H497, and W499 in the extracellular domain of the human HER2 receptor. In some embodiments, the antibodies disclosed herein include antibodies or antigen-binding fragments thereof that specifically bind to an epitope in the extracellular domain of the human HER2 receptor that includes at least amino acid residues C453, H473, N476, R495, H497, and W499 in the extracellular domain of the human HER2 receptor. In some embodiments, any or all of these antibodies or antigen-binding fragments thereof also bind to at least one epitope on the cynomolgus monkey HER2 receptor.

[0345] In some embodiments, these antibodies exhibit specificity for human HER2 and have been shown to modulate, e.g., block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with, the PI3K-Akt pathway, which promotes cell survival by reducing phosphorylated AKT levels. In some embodiments, these antibodies are internalized from the cell surface of HER2-expressing cells at a rate that is the same as or substantially similar to the rate at which trastuzumab or a biosimilar thereof is internalized. In some embodiments, these antibodies and antigen-binding fragments have an internalization rate that internalizes about 50% of the total surface bound at time 0 by 4 hours.

[0346] In some embodiments, the antibodies disclosed herein comprise a heavy chain variable region having an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a sequence selected from SEQ ID NO:17, and a light chain variable region having an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a sequence selected from SEQ ID NO:24.

[0347] In some embodiments, the antibodies disclosed herein comprise a heavy chain amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:19 and a light chain amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:26.

[0348] In some embodiments, the antibodies disclosed herein comprise the heavy chain variable region amino acid sequence of SEQ ID NO:17 and the light chain variable region amino acid sequence of SEQ ID NO:24.

[0349] In some embodiments, the antibodies disclosed herein comprise a heavy chain amino acid sequence of SEQ ID NO:19 and a light chain amino acid sequence of SEQ ID NO:26.

[0350] In some embodiments, the antibodies disclosed herein comprise a CDRH1 amino acid sequence of SEQ ID NO:20, a CDRH2 amino acid sequence of SEQ ID NO:21, a CDRH3 amino acid sequence of SEQ ID NO:22, a CDRL1 amino acid sequence of SEQ ID NO:27, a CDRL2 amino acid sequence of SEQ ID NO:28, and a CDRL3 amino acid sequence of SEQ ID NO:29.

[0351] In some embodiments, the antibodies disclosed herein comprise one or more conservative amino acid substitutions in the variable domain sequence, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more conservative substitutions in the variable domain sequence. In some embodiments, these conservative amino acid substitutions are in the CDR regions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more conservative substitutions are made cumulatively across all CDRs. In certain embodiments, there may be 1, 2, 3, or up to 4 conservative amino acid substitutions in each CDR sequence, e.g., in SEQ ID NOs: 20-22 and 27-29.

[0352] Those skilled in the art will recognize that, without undue experimentation, it is possible to determine whether a monoclonal antibody has the same specificity as monoclonal antibody XMT-1519 by determining whether the former prevents the latter from binding to its natural binding partner or other molecules known to bind to HER2. In some embodiments, if the monoclonal antibody being tested competes with the monoclonal antibody disclosed herein, as indicated by reduced binding by the monoclonal antibody disclosed herein, then the two monoclonal antibodies bind to the same epitope or closely related epitopes.

[0353] In some embodiments, an alternative method for determining whether a monoclonal antibody has the specificity of the monoclonal antibodies disclosed herein is to preincubate the monoclonal antibodies disclosed herein with soluble HER2 (which normally reacts with the monoclonal antibodies disclosed herein), and then add the monoclonal antibody being tested to determine whether it is inhibited in its ability to bind to HER2. If the monoclonal antibody being tested is inhibited, it likely has the same or a functionally equivalent epitope specificity as the monoclonal antibodies disclosed herein.

[0354] In some embodiments, the screening of the monoclonal antibodies disclosed herein can also be carried out, for example, by measuring the PI3K-Akt pathway activity mediated by HER2, and determining whether the test monoclonal antibody can regulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with the PI3K-Akt pathway activity. In some embodiments, HER2 antibodies suitable for conjugation can be produced and purified by well-known techniques, for example, WO2015 / 195917 and PCT / US2018 / 019873. Each of WO2015 / 195917 and PCT / US2018 / 019873 is incorporated herein by reference in its entirety.

[0355] Conjugates In some embodiments, a conjugate of the present disclosure comprises one or more occurrences of D, wherein D is a STING agonist, and the one or more occurrences of D can be the same or different.

[0356] In some embodiments, one or more occurrences of PBRM are attached to a linker-drug moiety, and the one or more occurrences of PBRM can be the same or different. In some embodiments, one or more linker-drug moieties comprising one or more occurrences of D are connected to one PBRM (e.g., an antibody).

[0357] In some embodiments, the conjugates of the present disclosure are about 40 kDa or greater (e.g., about 60 kDa or greater; about 80 kDa or greater; about 100 kDa or greater; about 120 kDa or greater; about 140 kDa or greater; about 160 kDa or greater; about 180 kDa or greater; or about 200 kDa or greater, or about 40-200 kDa, about 40-180 kDa, about 40-140 kDa). a, about 60-200 kDa, about 60-180 kDa, about 60-140 kDa, about 80-200 kDa, about 80-180 kDa, about 80-140 kDa, about 100-200 kDa, about 100-180 kDa, or about 100-140 kDa) and include PBRMs having sulfhydryl (i.e., -SH or thiol) groups.

[0358] In some embodiments, the total number of sulfide bonds (or total number of attachment points) formed between the linker-drug moiety and the PBRM is 10 or less (eg, 8, 6, 4, or 2).

[0359] In some embodiments, for conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of about 40 kDa or more (e.g., about 60 kDa or more, about 80 kDa or more, about 100 kDa or more, about 120 kDa or more, about 140 kDa or more, about 160 kDa or more, or about 180 kDa or more; or about 40-200 kDa, about 40-180 kDa, about 40-140 kDa, about 60-200 kDa, about 60-180 kDa, about 60-140 kDa, about 80-200 kDa, about 80-180 kDa, about 80-140 kDa, about 100-200 kDa, about 100-180 kDa, or about 100-140 kDa).

[0360] In some embodiments, the PBRM has a molecular weight of about 40 kDa to about 200 kDa due to conjugation with one or more linker-drug moieties, hi some embodiments, the PBRM has a molecular weight of about 40 kDa to about 80 kDa due to conjugation with one or more linker-drug moieties.

[0361] In some embodiments, the PBRM has a molecular weight of 40 kDa to 200 kDa for conjugation with one or more linker-drug moieties, hi some embodiments, the PBRM has a molecular weight of 40 kDa to 80 kDa for conjugation with one or more linker-drug moieties.

[0362] In some embodiments, PBRMs within this molecular weight range include, but are not limited to, antibody fragments such as, for example, Fabs.

[0363] In some embodiments, due to conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of about 60 kDa to about 120 kDa.

[0364] In some embodiments, due to conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of 60 kDa to 120 kDa.

[0365] In some embodiments, PBRMs within this molecular weight range include, but are not limited to, camelids, Fab2s, scFvFc, and the like.

[0366] In some embodiments, due to conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of about 140 kDa to about 180 kDa.

[0367] In some embodiments, due to conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of 140 kDa to 180 kDa.

[0368] In some embodiments, PBRMs within this molecular weight range include, but are not limited to, full-length antibodies, eg, IgG, IgM.

[0369] In some embodiments, for example, the targeting ligands, linkers, and drug or prodrug fragments described herein can be assembled to construct the conjugates or scaffolds of the present disclosure, according to the techniques and methods disclosed. Therapeutic and targeting conjugates of the present disclosure and methods for producing them are described below as non-limiting examples.

[0370] In some embodiments, the total number of sulfide bonds (or total number of attachment points) formed between the linker-drug moiety and the PBRM is eight or less.

[0371] In some embodiments, the total number of sulfide bonds (or total number of attachment points) formed between the linker-drug moiety and the PBRM is 8. In some embodiments, the total number of sulfide bonds (or total number of attachment points) formed between the linker-drug moiety and the PBRM is 6. In some embodiments, the total number of sulfide bonds (or total number of attachment points) formed between the linker-drug moiety and the PBRM is 5. In some embodiments, the total number of sulfide bonds (or total number of attachment points) formed between the linker-drug moiety and the PBRM is 4. In some embodiments, the total number of sulfide bonds (or total number of attachment points) formed between the linker-drug moiety and the PBRM is 3. In some embodiments, the total number of sulfide bonds (or total number of attachment points) formed between the linker-drug moiety and the PBRM is 2.

[0372] In some embodiments, the ratio of linker-drug moiety to PBRM is about 1:1 to about 8:1. In some embodiments, the ratio of linker-drug moiety to PBRM is about 1:1 to about 6:1. In some embodiments, the ratio of linker-drug moiety to PBRM is about 1:1 to about 4:1. In some embodiments, the ratio of linker-drug moiety to PBRM is about 2:1 to about 2:1.

[0373] In some embodiments, the ratio of linker-drug moiety to PBRM is about 6:1 to about 8:1.

[0374] In some embodiments, the ratio of linker-drug moiety to PBRM is about 8:1.

[0375] In some embodiments, the ratio of linker-drug moiety to PBRM is about 6:1.

[0376] In some embodiments, the present disclosure also relates to a linker-drug moiety comprising at least two moieties, each of which is capable of conjugating to a thiol group present on the PBRM to form a protein-linker-drug conjugate.

[0377] In some embodiments, one or more thiol groups of the PBRM are generated by reducing a protein. The one or more thiol groups of the PBRM can then be reacted with one or more linker-drug moieties that are capable of conjugating the thiol groups from the PBRM to the linker-drug moiety. In some embodiments, at least two moieties connected to the PBRM are maleimide groups.

[0378] In some embodiments, the antibody may be activated for conjugation with a linker-drug moiety by treatment with a reducing agent, such as DTT (Cleland's reagent, dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine hydrochloride). In some embodiments, a full-length monoclonal antibody can be reduced with an excess of TCEP to reduce disulfide bonds (e.g., between cysteines present in the corresponding parent antibody) to obtain a reduced form of the antibody. The newly introduced and unpaired cysteine ​​may still be available for reaction with a linker-drug moiety to form the antibody conjugate of the present disclosure. In some embodiments, an excess of the linker-drug moiety is added to effect conjugation to form the antibody-drug conjugate, and the conjugation mixture is purified to remove excess linker-drug intermediates and other impurities.

[0379] In some embodiments, for linker-drug moiety conjugation, the PBRM has a molecular weight of 40 kDa or greater (e.g., 60 kDa or greater; 80 kDa or greater; or 100 kDa or greater; 120 kDa or greater; 140 kDa or greater; 160 kDa or greater; or 180 kDa or greater). In some embodiments, the ratio of PBRM to linker-drug moiety is about 1:1 to about 1:8; about 1:1 and about 1:6; about 1:1 to about 1:5; about 1:1 to about 1:4; about 1:1 to about 1:3; or about 1:1 to about 1:2.

[0380] PBRMs within this molecular weight range include, but are not limited to, full-length antibodies, such as IgG and IgM.

[0381] In some embodiments, for conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of 60 kDa to 120 kDa. In some embodiments, the ratio of PBRM to linker-drug moiety is between about 1:1 and about 1:8; about 1:1 to about 1:6; about 1:1 to about 1:5; about 1:1 to about 1:4; about 1:1 to about 1:3; or about 1:1 to about 1:2.

[0382] PBRMs within this molecular weight range include, but are not limited to, antibody fragments such as Fab2, scFcFv, and camelids.

[0383] In some embodiments, for conjugation with one or more linker-drug moieties, the PBRM has a molecular weight of 40 kDa to 80 kDa. In some embodiments, the ratio of PBRM to linker-drug moiety is about 1:1 and about 1:8; about 1:1 to about 1:6; about 1:1 to about 1:5; 1:1 to about 1:4; about 1:1 to about 1:3, or about 1:1 to about 1:2.

[0384] In some embodiments, PBRMs within this molecular weight range include, but are not limited to, for example, antibody fragments, eg, Fabs.

[0385] In some embodiments, the present disclosure features scaffolds useful for conjugating with either or both a protein-based recognition molecule (PBRM) and a STING agonist moiety (D).

[0386] In some embodiments, the drug-carrying scaffolds described herein (ie, without linkage to a PBRM) each typically have a polydispersity index (PDI) of 1.

[0387] The conjugates and scaffolds disclosed herein can be purified by large-scale diafiltration (i.e., removal of all starting materials). If necessary, further purification can be performed by size exclusion chromatography to remove any aggregated conjugates. Generally, when purified, the conjugates typically contain less than 5% (e.g., <2% w / w) aggregated conjugates as determined by SEC; less than 0.5% (e.g., <0.1% w / w) free (unconjugated) drug as determined by RP-HPLC; less than 1% drug-carrying peptide-containing scaffolds as determined by SEC; and less than 2% (e.g., <1% w / w) unconjugated PBRM as determined by HIC-HPLC.

[0388] In some embodiments, the scaffold is selected from the scaffolds listed in Table A1.

[0389] In some embodiments, the scaffold is selected from the scaffolds listed in Table A2.

[0390] In some embodiments, the conjugate is selected from the conjugates set forth in Table B1.

[0391] In some embodiments, the conjugate is selected from the conjugates set forth in Table B2.

[0392] [Table A1] TIFF0007777065000145.tif176161TIFF0007777065000146.tif199161TIFF0007777065000147.tif183161TI FF0007777065000148.tif197161TIFF0007777065000149.tif186161TIFF0007777065000150.tif175161In formula, R 14 , R 15 , R16 , R 17 , R 18 , R 19 , R C1 , R C2 , X3, X4, X6, X1, W1, Y1, Z1, X2, W2, Y2, Z2 are as defined herein.

[0393] [Table A2] TIFF0007777065000152.tif150170TIFF0007777065000153.tif165170TIFF0007777065000154.tif15717 0TIFF0007777065000155.tif206170TIFF0007777065000156.tif148170TIFF0007777065000157.tif19517 0TIFF0007777065000158.tif197170TIFF0007777065000159.tif199170TIFF0007777065000160.tif19817 0TIFF0007777065000161.tif186170TIFF0007777065000162.tif214170TIFF0007777065000163.tif17517 0TIFF0007777065000164.tif200170TIFF0007777065000165.tif177170TIFF0007777065000166.tif18317 0TIFF0007777065000167.tif190170TIFF0007777065000168.tif176170TIFF0007777065000169.tif19517 0TIFF0007777065000170.tif200170TIFF0007777065000171.tif204170TIFF0007777065000172.tif20417 0TIFF0007777065000173.tif204170TIFF0007777065000174.tif204170TIFF0007777065000175.tif74170

[0394]

Table B1

[0395] [Table B2] TIFF0007777065000208.tif217170TIFF0007777065000209.tif198170TIFF0007777065000210.tif202170TIFF0007777065000211.tif182170TIFF000 7777065000212.tif151170TIFF0007777065000213.tif185170TIFF0007777 065000214.tif205170TIFF0007777065000215.tif211170TIFF00077770650 00216.tif210170TIFF0007777065000217.tif180170TIFF0007777065000218.tif215170TIFF0007777065000219.tif178170TIFF0007777065000220.t if192170TIFF0007777065000221.tif211170TIFF0007777065000222.tif179170TIFF0007777065000223.tif182170TIFF0007777065000224.tif200170

[0396] In some embodiments, the conjugate comprises: TIFF0007777065000225.tif198156TIFF0007777065000226.tif157131, wherein R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , RC1 , R C2 , X3, X4, X6, X1, W1, Y1, Z1, X2, W2, Y2, Z2 are as defined herein.

[0397] In some embodiments, the conjugate comprises: TIFF0007777065000227.tif123144, wherein: d 15 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R C1 , R C2 , X3, X4, X6, X1, W1, Y1, Z1, X2, W2, Y2, Z2 are as defined herein.

[0398] In some embodiments, the conjugate comprises: TIFF0007777065000228.tif161138TIFF0007777065000229.tif201156TIFF0007777065000230.tif38167, In the formula, d 15 is as defined herein.

[0399] Pharmaceutical Compositions In some aspects, the present disclosure provides pharmaceutical compositions comprising a conjugate described herein and one or more pharmaceutically acceptable carriers or excipients.

[0400] Pharmaceutical compositions containing the conjugates of the present disclosure can be prepared in a generally known manner, for example, by conventional mixing, dissolving, granulating, dragee-making, pulverizing, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions can be formulated in a conventional manner using one or more pharmaceutically acceptable carriers containing excipients and / or auxiliaries that facilitate processing of the conjugates into pharmaceutically usable preparations. Of course, the appropriate formulation will depend on the selected route of administration.

[0401] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringeability exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0402] Sterile injectable solution can be prepared by incorporating the required amount of conjugate into suitable solvent with one or combination of the components listed above as needed, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating conjugate into a sterile vehicle that contains basic dispersion medium and other components required from the components listed above.For the sterile powder used to prepare sterile injectable solution, the preparation method is vacuum drying and freeze-drying, which produces a powder of active ingredient and any other desired components from the solution that has been previously sterilized by filtration.

[0403] Oral compositions generally contain an inert diluent or an edible pharmaceutically acceptable carrier. Oral compositions may be placed in gelatin capsules or compressed into tablets. For oral therapeutic administration, the conjugate may be incorporated with an excipient and used in the form of a tablet, troche, or capsule. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, in which the conjugate dissolved in the fluid carrier is orally applied, swished, and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, troches, etc. may contain any of the following ingredients: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose, a disintegrant such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a flow agent such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring, or compounds of a similar nature.

[0404] For administration by inhalation, the conjugates are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0405] Systemic administration can also be via transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, surfactants, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved using nasal sprays or suppositories. For transdermal administration, the conjugate is formulated into ointments, salves, gels, or creams generally known in the art.

[0406] The conjugate can be prepared using a pharmaceutically acceptable carrier that protects the conjugate from rapid elimination from the body, such as a sustained-release formulation including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are clear to those skilled in the art.

[0407] For ease of administration and uniformity of dosage, it may be particularly advantageous to formulate oral or parenteral compositions into unit dosage form.As used herein, unit dosage form refers to a physically separate unit suitable for administration to the subject to be treated individually.Each unit containing a predetermined amount of conjugate can be calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier.The specifications of the unit dosage form of the present disclosure are determined and directly depend on the unique characteristics of the conjugate and the specific therapeutic effect to be achieved.

[0408] In therapeutic applications, the dosage of pharmaceutical compositions used in accordance with the present disclosure will vary depending on the agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors influencing the selected dosage. Generally, the dose should be sufficient to slow, and preferably regress, the symptoms of the disease, and preferably sufficient to cause complete regression of the disease.

[0409] It will be appreciated that the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0410] How to use In some embodiments, the present disclosure provides a method of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a conjugate disclosed herein.

[0411] In some embodiments, the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a conjugate disclosed herein.

[0412] In some embodiments, the present disclosure provides a method of activating or enhancing the activity of STING in a subject, the method comprising administering to the subject a conjugate disclosed herein.

[0413] In some embodiments, the present disclosure relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a conjugate disclosed herein.

[0414] In some embodiments, the present disclosure provides a conjugate disclosed herein for use in treating or preventing a disease or disorder in a subject in need thereof.

[0415] In some embodiments, the present disclosure provides a conjugate disclosed herein for use in treating a disease or disorder in a subject in need thereof.

[0416] In some embodiments, the present disclosure provides a conjugate disclosed herein for treating a disease or disorder mediated by STING in a subject.

[0417] In some embodiments, the present disclosure provides a use of a conjugate disclosed herein for treating cancer in a subject in need thereof.

[0418] In some embodiments, the present disclosure provides for the use of a conjugate disclosed herein in the manufacture of a medicament for treating a disease or disorder in a subject in need thereof.

[0419] In some embodiments, the present disclosure provides for the use of a conjugate disclosed herein in the manufacture of a medicament for treating or preventing a disease or disorder in a subject in need thereof.

[0420] In some embodiments, the present disclosure provides for the use of a conjugate disclosed herein in the manufacture of a medicament for treating a disease or disorder mediated by STING in a subject.

[0421] In some embodiments, the present disclosure provides for the use of a conjugate disclosed herein in the manufacture of a medicament for treating cancer in a subject in need thereof.

[0422] In some embodiments, the present disclosure provides for the use of a conjugate disclosed herein to treat or prevent a disease or disorder in a subject in need thereof.

[0423] In some embodiments, the present disclosure provides a use of a conjugate disclosed herein for treating a disease or disorder in a subject in need thereof.

[0424] In some embodiments, the present disclosure provides a use of a conjugate disclosed herein for treating a disease or disorder mediated by STING in a subject.

[0425] In some embodiments, the present disclosure provides a use of a conjugate disclosed herein for treating cancer in a subject in need thereof.

[0426] In some embodiments, the conjugates disclosed herein are administered to a subject.

[0427] In some embodiments, the present disclosure provides a method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject a sufficient amount of at least one conjugate of the present disclosure, wherein the conjugate releases one or more therapeutic agents upon biodegradation.

[0428] In some embodiments, the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a sufficient amount of at least one conjugate of the present disclosure, wherein the conjugate releases one or more therapeutic agents upon biodegradation.

[0429] In some embodiments, the present disclosure, the conjugate is an antibody-STING agonist conjugate. In some embodiments, the disease or disorder is cancer.

[0430] In some embodiments, the present disclosure provides methods for treating or preventing STING-mediated diseases and disorders. Exemplary diseases / disorders include, but are not limited to, cancer, infectious diseases (e.g., HIV, HBV, HCV, HPV, and influenza), and vaccine adjuvants.

[0431] In some embodiments, the STING pathway can induce anti-tumor immunity by upregulating IFNβ and interferon (IFN)-activated genes (ISGs) in many cell types within tumors in response to agonistic cytosolic nucleic acids.

[0432] In some aspects, the present disclosure provides a conjugate disclosed herein for use as a vaccine adjuvant. Thus, an immunogenic composition or vaccine adjuvant comprising a conjugate disclosed herein is provided.

[0433] In some embodiments, a composition is provided comprising a conjugate disclosed herein and one or more immunostimulants.

[0434] In some embodiments, the present disclosure provides for the use of the conjugates disclosed herein in the manufacture of vaccines. In some embodiments, the present disclosure provides for the use of the conjugates disclosed herein to manufacture immunogenic or vaccine compositions comprising an antigen or antigenic composition for treating or preventing disease.

[0435] In some aspects, the present disclosure relates to a method of treating or preventing a disease, comprising administering to a human subject suffering from or susceptible to the disease an immunogenic or vaccine composition comprising an antigen or antigenic composition and a conjugate disclosed herein.

[0436] In some embodiments, the disease or disorder is inflammation, autoimmune disease, allergic disease, infectious disease, HIV infection, AIDS infection, HCV infection, influenza or human papillomavirus (HPV) infection. The range of diseases will be readily recognized by those skilled in the art. In some embodiments, these diseases are as described in PCT Application No. PCT / US2020 / 044538, the contents of which are incorporated herein by reference in their entirety.

[0437] As used herein, the terms "cancer," "neoplasm," and "tumor" are used interchangeably and refer to cells undergoing malignant transformation, whether singular or plural, that render them pathological to the host organism. Primary cancer cells can be easily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. The definition of cancer cells used herein includes not only primary cancer cells but also any cells derived from cancer cell ancestors. This includes metastasized cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. When referring to types of cancer that typically manifest as solid tumors, a "clinically detectable" tumor is one that can be detected based on tumor burden, e.g., by treatment, e.g., computed tomography (CT) scan, magnetic resonance imaging (MRI), X-ray, ultrasound, or palpation during physical examination, and / or by the expression of one or more cancer-specific antigens in samples available from the patient. The tumor may be a hematopoietic (or hematologic or hematological or blood-related) cancer, such as a cancer derived from blood cells or immune cells, which are sometimes referred to as "liquid tumors." Specific examples of clinical conditions based on hematopoietic tumors include leukemias, such as chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, and acute lymphocytic leukemia; plasma cell malignancies, such as multiple myeloma, MGUS, and Waldenstrom's macroglobulinemia; lymphomas, such as non-Hodgkin's lymphoma, Hodgkin's lymphoma, etc.

[0438] In some embodiments, the disease or disorder is a precancerous syndrome.

[0439] The conjugate of the present disclosure can be used to treat inflammation of any tissue and organ of the body, including musculoskeletal inflammation, vascular inflammation, neuroinflammation, digestive system inflammation, eye inflammation, reproductive system inflammation, and other inflammation.The range of diseases will be easily recognized by those skilled in the art.In some embodiments, these diseases are as described in PCT Application No. PCT / US2020 / 044538, the contents of which are incorporated herein by reference in their entirety.

[0440] Examples of cancer diseases and conditions in which the conjugates of the present disclosure may have potentially beneficial anti-tumor effects include cancer of the lung, bone, pancreas, skin, head, neck, uterus, ovary, stomach, colon, breast, esophagus, biliary tract, small intestine, bowel, endocrine system, thyroid, parathyroid, adrenal gland, urethra, prostate, penis, testis, ureter or urothelium, bladder, kidney or liver; rectal cancer; cancer of the anal region; cancer of the fallopian tube, endometrium, cervix, vagina, vulva, renal pelvis, renal cell; soft tissue sarcoma; myxoma; rhabdomyoma; fibroma; lipoma; teratoma; cholangiocarcinoma; hepatoblastoma; angiosarcoma; hemangioma; hepatoma; fibrosarcoma; chondrosarcoma; myeloma; chronic or acute leukemia; lymphocytic lymphoma; primary CNS lymphoma; neoplasms of the CNS; spinal axis axis tumor; squamous cell carcinoma; synovial sarcoma; malignant pleural mesothelioma; brain stem glioma; pituitary adenoma; bronchial adenoma; chondromatous hamartoma; mesothelioma; Hodgkin's disease or a combination of one or more of the foregoing cancers.

[0441] In some embodiments, the disease or disorder is a solid tumor. In one aspect, the tumor is selected from head and neck cancer, gastric cancer, melanoma, renal cell carcinoma (RCC), esophageal cancer, biliary tract cancer, non-small cell lung cancer (NSCLC), prostate cancer, colorectal cancer (CRC), colon cancer, ovarian cancer, endometrial cancer, urothelial cancer, cervical cancer, bladder cancer, papillary thyroid cancer, papillary renal cell carcinoma, bile duct cancer, salivary duct cancer, kidney cancer, cervical cancer, and pancreatic cancer. In some embodiments, the human has a liquid tumor, such as diffuse large B-cell lymphoma (DLBCL), multiple myeloma, chronic lymphoblastic leukemia (CLL), follicular lymphoma, acute myeloid leukemia, and chronic myeloid leukemia. In some embodiments, the disease or disorder is skin cancer (e.g., non-melanoma skin cancer, squamous cell carcinoma, basal cell carcinoma) or actinic keratosis. In addition to the field effect to eliminate superficial skin cancers, the conjugates of the present disclosure may prevent the development of subsequent skin cancers and premalignant actinic keratoses in treated subjects.

[0442] In some embodiments, the disease or disorder is bladder cancer, breast cancer, colorectal cancer, colon cancer, endometrial cancer, gastric cancer, esophageal cancer, biliary tract cancer, urothelial cancer, head and neck squamous cell carcinoma, melanoma, non-small cell lung cancer, ovarian cancer, or pancreatic cancer. In some embodiments, the disease or disorder is breast cancer, gastric cancer, colorectal cancer, colon cancer, esophageal cancer, biliary tract cancer, endometrial cancer, urothelial cancer, or non-small cell lung cancer.

[0443] In some embodiments, the breast cancer is a HER2 amplified / overexpressed breast cancer or a HER2 low breast cancer.

[0444] In some embodiments, the endometrial cancer is serous endometrial cancer.

[0445] The conjugates of the present disclosure may also be useful in treating one or more diseases afflicting mammals characterized by cell proliferation in areas of neovascularization and / or vascular permeability-related disorders, fibrotic disorders, and metabolic disorders. The range of diseases will be readily recognized by those skilled in the art. In some embodiments, these diseases are as described in PCT Application No. PCT / US2020 / 044538, the contents of which are incorporated herein by reference in their entirety.

[0446] In some embodiments, the disease or disorder is a neurodegenerative disease.Exemplary neurodegenerative diseases include, but are not limited to, multiple sclerosis, Huntington's disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS).The range of diseases can be easily recognized by those skilled in the art.In some embodiments, these diseases are as described in U.S. Provisional Patent Application Nos. 62 / 882,081, 62 / 944,643, and 62 / 982,935, the contents of which are incorporated herein by reference in their entirety.

[0447] In some embodiments, the disease or disorder is an infectious disease, which is any disease caused by or occurs simultaneously with infection by a pathogen derived from a bacterium from the DNA virus family or the RNA virus family.The range of diseases will be easily recognized by those skilled in the art.In some embodiments, these diseases are as described in PCT Application No. PCT / US2020 / 044538, the contents of which are incorporated herein by reference in their entirety.

[0448] The conjugates of the present disclosure may be used alone or in combination with other therapeutic agents. In the treatment of diseases and conditions in which modulation of STING is beneficial, the conjugates of the present disclosure may also be used as monotherapy as immune response modulators or in combination with other therapeutic agents. Thus, a combination therapy according to the present disclosure comprises the administration of a conjugate of the present disclosure, or a pharmaceutically acceptable salt thereof, and at least one other therapeutically active agent. In some embodiments, a combination therapy according to the present disclosure comprises the administration of at least one conjugate of the present disclosure, or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent. The conjugate of the present disclosure, its pharmaceutically acceptable salt, and the other therapeutic agent may be administered together in a single pharmaceutical composition or separately. When administered separately, they may be administered simultaneously or sequentially in any order. The amounts of the conjugate of the present disclosure, its pharmaceutically acceptable salt, and the other therapeutic agent, as well as the relative timing of administration, are selected to achieve the desired combined therapeutic effect. Thus, in a further aspect, there is provided a combination comprising a conjugate of the present disclosure, or a pharmaceutically acceptable salt thereof, together with one or more other therapeutic agents.

[0449] The conjugates of the present disclosure, and pharmaceutically acceptable salts thereof, may be used in combination with one or more other therapeutic agents that may be useful in the prevention or treatment of allergic, inflammatory, or autoimmune diseases, such as; antigen immunotherapy, antihistamines, steroids, NSAIDs, bronchodilators, methotrexate, leukotriene modulators, monoclonal antibody therapy, receptor therapy, or antigen non-specific immunotherapy.

[0450] The conjugates of the present disclosure and their pharmaceutically acceptable salts may be used in combination with at least one other therapeutic agent that may be useful in radiation therapy and / or surgery and / or the treatment of cancer and precancerous syndromes. Any anti-cancer agent, microtubule inhibitor, mitotic inhibitor, hormone, hormone analog signaling pathway inhibitor, protein tyrosine kinase, or anti-angiogenic therapeutic agent may be used in combination. The range of other therapeutic agents will be readily recognized by those skilled in the art. In some embodiments, the other therapeutic agent is as described in PCT Application No. PCT / US2020 / 044538, the contents of which are incorporated herein by reference in their entirety.

[0451] Agents used in immunotherapeutic regimens, therapeutic agents used in pro-apoptotic regimens, or cell cycle signaling inhibitors may also be useful in combination with the conjugates of the present disclosure.

[0452] In some embodiments, the combination of the present disclosure comprises a conjugate of the present disclosure or a salt thereof, particularly a pharmaceutically acceptable salt thereof, and at least one anti-neoplastic agent, microtubule inhibitor, anti-mitotic agent, hormone, hormone analog signaling pathway inhibitor, protein tyrosine kinase, or anti-angiogenic therapeutic agent, or a combination thereof.

[0453] Further examples of other therapeutic agents (e.g., anti-neoplastic agents) for use in combination with or for co-administration with a conjugate of the present disclosure, or a pharmaceutically acceptable salt thereof, are immunomodulators.

[0454] In some embodiments, the combinations of the present disclosure comprise a conjugate of the present disclosure or a salt thereof, particularly a pharmaceutically acceptable salt, and at least one immunomodulator or at least one immunostimulant.

[0455] As used herein, "immunomodulator" refers to any substance, including monoclonal antibodies, that affects the immune system. Immunomodulators can be used as anti-cancer agents to treat cancer. For example, immunomodulators include, but are not limited to, anti-CTLA-4 antibodies and anti-PD-1 antibodies. Other immunomodulators include, but are not limited to, ICOS antibodies, OX-40 antibodies, PD-L1 antibodies, LAG3 antibodies, TIM-3 antibodies, 41BB antibodies, and GITR antibodies.

[0456] Further examples of other therapeutic agents (anti-neoplastic agents) for use in combination with or for co-administration with the conjugates of the present disclosure are anti-PD-Ll agents (i.e., anti-PD-Ll antibodies) or PD-1 antagonists.

[0457] Thus, in some embodiments, a method for treating a human in need thereof is provided, comprising administering a conjugate of the present disclosure or a salt thereof and at least one immunomodulator. In some embodiments, the immunomodulator is selected from an ICOS agonist antibody, an OX-40 antibody, and a PD-1 antibody. In some embodiments, the human has cancer. Also provided herein is the use of a conjugate of the present disclosure or a salt thereof in combination with at least one immunomodulator to treat a human in need thereof.

[0458] As used herein, "immunostimulatory agent" refers to any agent that can stimulate the immune system.As used herein, immunostimulatory agent includes, but is not limited to, vaccine adjuvants, such as Toll-like receptor agonists, T cell checkpoint blockers, such as mAbs against PD-1 and CTL4, and T cell checkpoint agonists, such as agonist mAbs against OX-40 and ICOS.As used herein, "immunostimulatory agent" refers to any agent that can stimulate the immune system.As used herein, immunostimulatory agent includes, but is not limited to, vaccine adjuvants.

[0459] As used herein, the term "Toll-like receptor" (or "TLR") refers to a member or fragment of the Toll-like receptor family of proteins that sense microbial products and / or initiate adaptive immune responses. In some embodiments, TLRs activate dendritic cells (DCs). Toll-like receptors (TLRs) are a family of pattern recognition receptors originally identified as innate immune system sensors that recognize microbial pathogens. TLRs recognize unique structures on microorganisms often referred to as "PAMPs" (pathogen-associated molecular patterns). Ligand binding to TLRs triggers a cascade of intracellular signaling pathways that induce the production of factors involved in inflammation and immunity.

[0460] In some embodiments, the immunostimulant for use in combination with the conjugates of the present disclosure is a TLR4 agonist.

[0461] Thus, in some embodiments, a method for treating a human in need of treatment is provided, comprising administering a conjugate of the present disclosure or a salt thereof and at least one immunostimulant. In some embodiments, the immunostimulant is a TLR4 agonist. In some embodiments, the immunostimulant is an AGP. In some embodiments, the human has cancer. Also provided herein is the use of a conjugate of the present disclosure or a salt thereof in combination with at least one immunostimulant for treating a human in need of treatment.

[0462] In addition to the immunostimulants described above, the compositions of the present disclosure may further contain other therapeutic agents that have adjuvant properties and can stimulate the immune system to respond to cancer antigens present on inactivated tumor cells. Such adjuvants include, but are not limited to, lipids, liposomes, inactivated bacteria that induce innate immunity (e.g., inactivated or attenuated Listeria monocytogenes), and compositions that mediate innate immune activation via NOD-like receptors (NLRs), retinoic acid inducible gene-based (RIG)-I-like receptors (RLRs), and / or C-type lectin receptors (CLRs).

[0463] Due to the adjuvant properties of TLR agonists, they can be used in combination with other vaccines, adjuvants, and / or immunomodulators, and can be combined in various combinations.In some embodiments, the conjugate of the present disclosure binds to STING and induces STING-dependent TBKI activation.For therapeutic purposes, the inactivated tumor cells that express and secrete one or more cytokines that stimulate DC induction, recruitment, and / or maturation as described herein can be administered together with one or more TLR agonists.

[0464] An additional active ingredient (antineoplastic agent) for use in combination with or co-administered with the conjugates of the present disclosure is an IDO inhibitor.

[0465] In some embodiments, the conjugates of the present disclosure may be used in combination with at least one other therapeutic agent useful in the prevention or treatment of an infectious disease, a bacterial infection, a viral infection, a Kaposi's sarcoma-associated herpesvirus infection, a TB infection, a Chlamydia, a Plasmodium infection, a Staphylococcus infection, amyotrophic lateral sclerosis (ALS), multiple sclerosis, systemic lupus erythematosus and related lupus disorders, psoriasis, or Sjogren's syndrome.

[0466] The conjugates of the present disclosure can be administered by any suitable route of administration, including both systemic and local administration. Systemic administration includes oral administration, parenteral administration, transdermal administration, rectal administration, and administration by inhalation. Parenteral administration refers to a route of administration other than enteral, transdermal, or inhalation administration, and is typically administration by injection or infusion. Parenteral administration includes intravenous, intramuscular, and subcutaneous injection or infusion. Inhalation refers to administration to the patient's lungs, whether inhaled through the mouth or nasal passages. Topical administration includes application to the skin.

[0467] In addition to the above-mentioned administration routes suitable for oncology treatment, pharmaceutical compositions may be adapted for administration by intratumoral or peritumoral injection. Intratumoral or peritumoral injection of the conjugates of the present disclosure directly into or adjacent to a single solid tumor is expected to induce an immune response capable of attacking and destroying cancer cells throughout the body, significantly reducing and possibly permanently eliminating tumors from the affected subject. This activation of the immune system to kill tumors at distant sites is commonly known as the abscopal effect, and has been demonstrated in animals with multiple therapeutic modalities. A further advantage of local, intratumoral, or peritumoral administration is that comparable efficacy can be achieved at significantly lower doses, thereby minimizing or eliminating adverse events that may be observed with significantly higher systemic doses.

[0468] The conjugates of the present disclosure may be administered once or according to a dosing regimen, in which multiple doses are administered at various intervals over a predetermined period of time. For example, doses may be administered once, twice, three times, or four times daily. Doses may be administered until a desired therapeutic effect is achieved or indefinitely to maintain the desired therapeutic effect. Suitable dosing regimens for the conjugates of the present disclosure depend on the pharmacokinetic properties of the conjugate, such as absorption, distribution, and half-life, which can be ascertained by one of ordinary skill in the art. Furthermore, suitable dosing regimens for the conjugates of the present disclosure, including the duration over which such regimens are administered, depend on the disease or disorder being treated, the severity of the disease or disorder being treated, the age and physical condition of the patient being treated, the medical history of the patient being treated, any concurrent therapies, the desired therapeutic effect, and similar factors within the knowledge and expertise of one of ordinary skill in the art. It will further be understood by such skilled artisans that suitable dosing regimens may require adjustment over time to take into account an individual patient's response to the dosing regimen or as individual patients require changes. The total daily dose is 1 mg to 2000 mg, preferably 1 mg to 250 mg.

[0469] For use in therapy, the conjugates of the present disclosure are usually, but not necessarily, formulated into pharmaceutical compositions before being administered to a patient. Accordingly, the present disclosure also relates to pharmaceutical compositions comprising a conjugate of the present disclosure and at least one pharmaceutically acceptable excipient.

[0470] The pharmaceutical compositions of the present disclosure may be prepared and packaged in bulk or unit dosage form. For oral administration, for example, one or more tablets or capsules may be administered. A dose of the pharmaceutical composition contains at least a therapeutically effective amount of the conjugate of the present disclosure (i.e., the conjugate of the present disclosure or a salt thereof, particularly a pharmaceutically acceptable salt). When prepared in unit dosage form, the pharmaceutical composition may contain 1 mg to 1000 mg of the conjugate of the present disclosure.

[0471] As provided herein, a unit dosage form (pharmaceutical composition) containing 1 mg to 1000 mg of a conjugate of the present disclosure may be administered once, twice, three times, or four times per day, preferably once, twice, or three times per day, more preferably once or twice per day, to treat a disease or disorder mediated by STING.

[0472] The pharmaceutical composition of the present disclosure typically contains one kind of conjugate of the present disclosure.However, in certain embodiments, the pharmaceutical composition of the present disclosure contains multiple kinds of conjugates of the present disclosure.In addition, the pharmaceutical composition of the present disclosure may optionally further contain one or more kinds of additional therapeutic agents (e.g., pharmaceutically active conjugates).

[0473] As used herein, "pharmaceutically acceptable excipient" refers to a pharmaceutically acceptable material, composition, or vehicle involved in imparting form or consistency to a pharmaceutical composition. Each excipient must be compatible with the other components of the pharmaceutical composition when mixed to avoid interactions that would significantly reduce the efficacy of the conjugates of the present disclosure when administered to a patient and interactions that would result in a pharmaceutically unacceptable pharmaceutical composition. Furthermore, each excipient must, of course, be of sufficiently high purity to be pharmaceutically acceptable.

[0474] The conjugates of the present disclosure and pharmaceutically acceptable excipients are typically formulated into dosage forms suitable for administration to patients via desired administration routes.Conventional dosage forms include: (1) dosage forms suitable for oral administration, such as tablets, capsules, caplets, pills, lozenges, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and wafers; (2) dosage forms suitable for parenteral administration, such as sterile solutions, suspensions, and powders for reconstitution; (3) dosage forms suitable for transdermal administration, such as transdermal patches; (4) dosage forms suitable for rectal administration, such as suppositories; (5) dosage forms suitable for inhalation, such as aerosols and solutions; and (6) dosage forms suitable for topical administration, such as creams, ointments, lotions, solutions, pastes, sprays, foams, and gels.

[0475] Suitable pharmaceutically acceptable excipients will vary depending on the particular dosage form selected. Furthermore, suitable pharmaceutically acceptable excipients may be selected for a particular function they may serve in the composition. For example, certain pharmaceutically acceptable excipients may be selected for their ability to facilitate the production of a uniform dosage form. Certain pharmaceutically acceptable excipients may be selected for their ability to facilitate the production of a stable dosage form. Certain pharmaceutically acceptable excipients may be selected for their ability to facilitate the transport or transportation of the conjugates of the present disclosure upon administration from one organ or part of the body to another organ or part of the body in a patient. Certain pharmaceutically acceptable excipients may be selected for their ability to enhance patient compliance.

[0476] Suitable pharmaceutically acceptable excipients include the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, flow agents, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, flavor masking agents, colorants, anti-caking agents, humectants, chelating agents, plasticizers, thickeners, antioxidants, preservatives, stabilizers, surfactants, and buffers. Those skilled in the art will understand that a particular pharmaceutically acceptable excipient may serve multiple functions and may serve alternative functions depending on how much of the excipient is present in the formulation and what other ingredients are present in the formulation.

[0477] Those skilled in the art possess the knowledge and skill to select appropriate pharmaceutically acceptable excipients in appropriate amounts for use in the present disclosure. Furthermore, there are numerous resources available to those skilled in the art that describe pharmaceutically acceptable excipients and may be useful in selecting appropriate pharmaceutically acceptable excipients. Examples include Remington's Pharmaceutical Sciences (Mack Publishing Company), The Handbook of Pharmaceutical Additives (Gower Publishing Limited), and The Handbook of Pharmaceutical Excipients (the American Pharmaceutical Association and the Pharmaceutical Press).

[0478] In one aspect, the present disclosure relates to a solid oral dosage form, such as a tablet or capsule, comprising an effective amount of a conjugate of the present disclosure and a diluent or filler. The oral solid dosage form may further comprise a disintegrant or lubricant.

[0479] It is understood that the conjugates of the present disclosure may also be formulated with vaccines as adjuvants to modulate the activity of the vaccine. Such compositions may contain an antibody(ies) or antibody fragment or antigenic component, optionally together with one or more other components with adjuvant activity.

[0480] Certain compounds and / or conjugates of the present disclosure can be potent immunomodulators and therefore care must be taken when handling them. Having described the present disclosure, the following examples are offered by way of illustration and not by way of limitation.

[0481] In some embodiments, the conjugate has the formula BB TIFF0007777065000231.tif69138, wherein the conjugate comprises a variable heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence FTFSSYSMN (SEQ ID NO:20); variable heavy chain complementarity determining region 2 (CDRH2) comprising TIFF0007777065000232.tif4128; variable heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence GGHGYFDL (SEQ ID NO:22); and amino acid sequence TIFF0007777065000233.tif4128; variable light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence GASSRAT (SEQ ID NO:28); and variable light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence QQYHHSPLT (SEQ ID NO:29), 15 is about 8.

[0482] In some embodiments, the conjugate has the formula CC TIFF0007777065000234.tif79160, and the conjugate has the amino acid sequence CDRH1 containing TIFF0007777065000235.tif4128; amino acid sequence CDRH2 containing TIFF0007777065000236.tif4128; amino acid sequence CDRH3 containing TIFF0007777065000237.tif4128; amino acid sequence TIFF0007777065000238.tif4128; CDRL2 comprising the amino acid sequence YTSSLYS (SEQ ID NO:9); CDRL3 comprising the amino acid sequence QQYSKLPLT (SEQ ID NO:10); and XMT-1519 antibody comprising: 15 is about 8.

[0483] In some embodiments, the conjugate of formula BB or formula CC is useful for treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the conjugate of formula BB or formula CC. In some embodiments, the disease or disorder is cancer.

[0484] In some embodiments, for the conjugate of formula BB, the cancer is breast cancer, gastric cancer, colorectal cancer, esophageal cancer, biliary tract cancer, endometrial cancer, urothelial cancer, or non-small cell lung cancer. In some embodiments, the breast cancer is HER2-amplified / overexpressed breast cancer or HER2-low breast cancer. In some embodiments, the endometrial cancer is serous endometrial cancer.

[0485] In some embodiments, the conjugate of formula CC is useful for treating a NaPi2b-expressing tumor in a subject in need thereof. In some embodiments, the NaPi2b-expressing tumor is ovarian cancer, non-small cell lung cancer (NSCLC), papillary thyroid cancer, endometrial cancer, cholangiocarcinoma, papillary renal cell carcinoma, clear cell renal carcinoma, breast cancer, kidney cancer, cervical cancer, or salivary duct cancer.

[0486] In some embodiments, the subject has epithelial ovarian cancer, fallopian tube cancer, primary peritoneal cancer, platinum-resistant ovarian cancer, non-squamous NSCLC cancer, advanced, radioactive iodine-refractory, locally recurrent, or metastatic disease, papillary thyroid cancer, or epithelial endometrial cancer.

[0487] In some embodiments, the conjugate of Formula BB is useful for treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the conjugate of Formula BB in combination with one or more therapeutic agents. In some embodiments, the therapeutic agent is an immunomodulatory agent or immunostimulatory agent. In some embodiments, the immunomodulatory agent is an anti-CTLA-4 antibody, an anti-PD-1 antibody, an ICOS antibody, an OX-40 antibody, a PD-L1 antibody, a LAG3 antibody, a TIM-3 antibody, a 41BB antibody, or a GITR antibody.

[0488] In some embodiments, the conjugate of formula BB is useful for treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the conjugate of formula BB in combination with one or more HER2 antibodies that bind to a different HER2 epitope than the HER2 antibody XMT-1519. In some embodiments, the HER2 antibody that binds to HER2 different from the HER2 antibody XMT-1519 is trastuzumab, pertuzumab, Fab37, or chA21. [Example]

[0489] The following examples illustrate the present disclosure. These examples are not intended to limit the scope of the present disclosure, but rather to provide guidance to those skilled in the art for preparing and using the compounds, compositions, and methods of the present disclosure. Although specific embodiments of the present disclosure have been described, those skilled in the art will understand that various changes and modifications can be made without departing from the spirit and scope of the present disclosure.

[0490] It is understood that certain compounds of the present disclosure may be potent immunomodulators and therefore care must be taken in their handling.

[0491] The reactions described herein may be carried out with a variety of different substituents (e.g., R 1 , R 2 The present invention can be applied to produce compounds of the present disclosure having, for example, a hydroxyl group ...

[0492] Abbreviation The following abbreviations are used in the reaction schemes and synthetic examples that follow: This list is not intended to be a comprehensive list of abbreviations used in this application as additional standard abbreviations that are readily understood by those skilled in the art of organic synthesis may also be used in the synthetic schemes and examples. TIFF0007777065000239.tif139154

[0493] General information Unless otherwise specified, all reagents were purchased from the relevant suppliers.

[0494] The diABZI STING agonist was prepared as described by Ramanjulu et al. (Nature, 564(7736):439-443 (2018)).

[0495] XMT-1535 (anti-NaPi2b antibody) is disclosed in co-pending application US 15 / 457,574, filed March 13, 2017, the contents of which are incorporated herein by reference. XMT-1519 (anti-HER2 antibody) is disclosed in US 9,555,112, issued January 31, 2017, and US 9,738,720, issued August 22, 2017, the entire contents of which are incorporated herein by reference.

[0496] XMT-1535 AF-HPA ADC, DAR 5.9 and rituximab AF-HPA ADC, DAR 5.5 were prepared as described in co-pending applications US62 / 958,916, filed January 9, 2020, and US63 / 040,735, filed June 18, 2020, the entire contents of which are incorporated by reference herein.

[0497] HPLC purification was performed on a Phenomenex Gemini 5 μm C18 110 Å, 250×10 mm, semi-preparative column.

[0498] Where applicable, the drug content of the conjugates was determined spectrophotometrically, otherwise by RP-HPLC or LC / MS performed for quantitative determination of drug content.

[0499] The protein content of the antibody-drug conjugates was determined spectrophotometrically or by ELISA.

[0500] If necessary, the antibody-drug conjugate, drug-carrying scaffold, or antibody scaffold was purified (i.e., to remove any remaining unreacted drug, unconjugated antibody, enzyme, or starting material) by large-scale diafiltration, CHT chromatography, or HIC. If necessary, further purification was performed by SEC or HIC to remove aggregated antibody-drug conjugates. Generally, when purified, the antibody-drug conjugate contained <5% (w / w) (e.g., <2% (w / w)) aggregated antibody-drug conjugate as determined by SEC, <0.5% (w / w) (e.g., <0.1% (w / w)) free (unconjugated) drug as determined by RP-HPLC and / or LC-MS / MS, <1% (w / w) free drug conjugate as determined by SEC and / or RP-HPLC, and <10% (w / w) (e.g., <1% (w / w)) unconjugated antibody or antibody fragment as determined by HIC-HPLC and / or RP-HPLC. Reduced or partially reduced antibodies were prepared using procedures described in the literature. See, e.g., Francisco et al., Blood 102 (4):1458-1465 (2003). Total drug (conjugated and unconjugated) concentrations were determined by UV-Vis spectrophotometry or RP-HPLC.

[0501] To determine the concentration of free drug in biological samples, acidified samples were treated with acetonitrile. The free drug was extracted, and the acetonitrile supernatant was analyzed. To determine the concentration of conjugated STING agonist in nonclinical samples, samples were subjected to immunocapture using anti-human Fc antibody magnetic beads, followed by exhaustive basic hydrolysis. The acetonitrile supernatant containing the released drug was analyzed by LC-MS / MS. Total antibodies in nonclinical samples were measured using an MSD ECL immunoassay.

[0502] Free drug analysis was performed by RP-HPLC using a C-4 column and an acetonitrile gradient. Tandem mass spectrometry MRM peak areas were integrated and compared with auristatin F (AF) and auristatin F hydroxypropylamide (AF-HPA) standards. This method quantifies AF-HPA and AF in plasma and tissue homogenates linearly over the concentration range of 0.1 to 150 ng / mL. Total drug released after NaOH hydrolysis was measured under the same conditions. The dynamic range was 1 ng / mL to 5000 ng / mL. Total antibody standards ranged from 0.009 μg / mL to 20 μg / mL.

[0503] The drug:antibody ratio (DAR) was determined by measuring the absorbance of the conjugate. DAR values ​​were calculated using the appropriate molar extinction coefficients of the antibody and STING agonist payload.

[0504] Tumors were measured twice weekly using digital calipers and tumor volume was calculated using the formula: tumor volume (mm 3 )=(width 2 The tumor volume was calculated using the formula: (x length) / 2. Body weights were recorded daily for the first week and twice weekly thereafter. Animals were monitored for tumor volumes >1000 mm2. 3 Until it reaches ≥ 1500mm 3 The study continued until the body weight reached 0.05 or as indicated. The percent body weight change was calculated using the formula: Body Weight Change (%) = ((Body Weight) 試験X日目 -body weight 試験1日目 ) / body weight 試験1日目 ) * 100. Tumor volumes were reported as the mean ± standard error of the mean (SEM). Tumor growth inhibition (%TGI) was defined as the percent difference in mean tumor volume (MTV) between the treatment and control groups. To determine tumor growth inhibition (TGI), tumor size was measured throughout each efficacy study. Percent tumor regression was calculated using the formula: % regression = (1 - (mean tumor volume) 最終 ) / (mean tumor volume 1日目 )) *A partial response (PR) was defined as a tumor volume of 50% or less of the volume on day 1 for three consecutive measurements or a tumor volume of 13.5 mm on at least one of these three measurements. 3 Complete response (CR) was defined as a tumor volume equal to or greater than 13.5 mm for three consecutive measurements. 3 Tumor-free survivors (TFS) are classified as CR at the end of the study.

[0505] Example 1: Synthesis of XMT-1519 conjugate 8 TIFF0007777065000240.tif180160

[0506] Part A: To a mixture of compound 1 (prepared as described in WO2017175147A1, 0.5 g, 0.64 mmol), Boc-L-alanine (0.242 g, 1.28 mmol), DMAP (7.8 mg, 0.064 mmol), and DCC (0.264 g, 1.28 mmol) was added DMF (2 mL). The suspension was stirred overnight at room temperature, then the solution was concentrated and the residue was purified on silica gel (0-40% MeOH in DCM) to give compound 2 as a light yellow solid (0.52 g, 85% yield). 46 H 58 N 13 O 10 [M+H] + ESI-MS m / z calculated: 952.4; found 952.4.

[0507] Part B: To a suspension of compound 2 (0.52 g, 0.55 mmol) in dioxane (10 mL) was added 4N HCl (2 mL, 8.19 mmol). The reaction mixture was stirred at room temperature for 2 hours, and then the suspension was concentrated and used in the next step without further purification. Compound 3 was obtained as a white solid. 41 H 50 N 13 ESI-MS m / z calculated for O8 [M+H]: 852.4; found 852.3.

[0508] Part C: To a solution of compound 3 (0.586 g, 0.661 mmol) in DMF (5 mL) was added 2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatetradecanoic-14-oic acid (0.202 g, 0.727 mmol), followed by DIPEA (0.230 mL, 1.322 mmol). The reaction mixture was stirred at room temperature for 5 minutes, and then HATU (0.376 g, 0.992 mmol) and HOBt (0.153 g, 0.992 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. An additional aliquot of DIPEA (0.460 mL, 2.6 mmol) was added. After an additional hour, the reaction mixture was concentrated to an oil. The residue was purified on silica gel (0-40% MeOH in DCM) to give compound 4 (0.9 g, >95% yield) as a white solid. 53 H 71 N 14 O 13 [M+H] + ESI-MS m / z calculated: 1111.5; found 1111.5.

[0509] Part D: To a suspension of compound 4 (0.9 g, 0.810 mmol) in dioxane (10 mL) was added 4N HCl (3.04 mL, 12.15 mmol). The reaction mixture was stirred at room temperature for 1.5 hours. The suspension was concentrated to give compound 5 as a colorless solid (0.56 g, 66.0% yield). 48 H 63 N 14 O 11 [M+H] + ESI-MS calculated value: 1011.5; found value 1011.4.

[0510] Part E: To a solution of scaffold 6 (100 mg, 0.072 mmol, prepared as described in PCT / US2018 / 06719) and compound 5 (72.7 mg, 0.072 mmol) dissolved in DMF (2 mL) was added PyBOP (37.5 mg, 0.072 mmol) and DIPEA (0.075 mL, 0.431 mmol). The reaction mixture was stirred at room temperature for 2 h, then the solution was concentrated and the residue was purified by preparative HPLC (0-80% ACN in water) to give compound 7 (109 mg, 64% yield). 103 H 156 N 24 O 41 [M+2H] 2+ ESI-MS m / z calculated: 1192.5; found 1192.5.

[0511] Part F: To a solution of XMT-1519 (10 mg, 0.069 μmol) dissolved in 50 mM HEPES, 1 mM EDTA, pH 7 buffer, TCEP (0.059 mg, 0.207 μmol) was added, and the mixture was shaken at 37 °C for 90 minutes. Compound 7 (0.987 mg, 0.414 μmol dissolved in 200 μL DMA) was added to the reduced antibody. The resulting mixture was shaken at 37 °C for 60 minutes. The reaction was quenched with cysteine ​​(15 equivalents, 0.125 mg, 1.035 μmol dissolved in 125 μL of 50 mM HEPES, 1 mM EDTA, pH 7) and rotated at room temperature for 1 hour. The resulting conjugate 8 was purified by ultrafiltration or CHT chromatography. Details of antibody-drug conjugates 8-1 and 8-2 are shown below. Conjugates 8-1 and 8-2 were prepared as described, except that a higher TCEP:mAb ratio (4:1 compared to 3:1) and a higher compound 7:mAb ratio (8:1 compared to 6:1) were used for the synthesis of 8-2 compared to 8-1. TIFF0007777065000241.tif16128

[0512] Example 1a: Synthesis of trastuzumab conjugate 8a TIFF0007777065000242.tif74137

[0513] Conjugate 8a was prepared and characterized as described in Example 1, except that trastuzumab was used instead of XMT-1519. Details of antibody-drug conjugates 8a-1, 8a-2, and 8a-3 are provided below. TIFF0007777065000243.tif20128

[0514] Example 1b: Synthesis of XMT-1535 conjugate 8b, DAR 5.7 TIFF0007777065000244.tif74132

[0515] Conjugate 8b was prepared and characterized as described in Example 1, except that XMT-1535 was used instead of XMT-1519. Details of antibody-drug conjugates 8b-1, 8b-2, and 8b-3 are provided below. TIFF0007777065000245.tif20128

[0516] Example 1c: Synthesis of Palivizumab Conjugate 8c TIFF0007777065000246.tif71136

[0517] Conjugate 8c was prepared and characterized as described in Example 1, except that palivizumab was used instead of XMT-1519. Details of antibody-drug conjugates 8c-1 and 8c-2 are provided below. TIFF0007777065000247.tif16128

[0518] Example 1d: Synthesis of Palivizumab mIgG2a Conjugate 8d TIFF0007777065000248.tif74150

[0519] Conjugate 8d was prepared and characterized as described in Example 1, except that palivizumab mIgG2a was used instead of XMT-1519. Details of antibody-drug conjugates 8d-1, 8d-2, and 8d-3 are provided below. TIFF0007777065000249.tif20128

[0520] Example 1e: Synthesis of XMT-1535 hIgG1-mIgG2a conjugate 8e, DAR 7.0 TIFF0007777065000250.tif73158

[0521] Conjugate 8e was prepared and characterized as described in Example 1, except that XMT-1535 mIgG2a was used instead of XMT-1519. The STING agonist:XMT-1535 hIgG1-mIgG2a ratio of purified conjugate 8e was 7.0.

[0522] Example 1f: Synthesis of XMT-1535 AAG conjugate 8f, DAR 7.4 TIFF0007777065000251.tif73142

[0523] Conjugate 8f was prepared and characterized as described in Example 1, except that XMT-1535 AAG was used instead of XMT-1519. The STING agonist:XMT-1535 AAG ratio of purified conjugate 8f was 7.4.

[0524] Example 1g: Synthesis of Target D mIgG2a Conjugate 8g, DAR 7.4 TIFF0007777065000252.tif76141

[0525] Conjugate 8g was prepared and characterized as described in Example 1, except that target D mIgG2a was used instead of XMT-1519. The STING agonist:target D_mIgG2a ratio of purified conjugate 8g was 7.4.

[0526] Example 1h: Synthesis of Target C hIgG1a conjugate 8h, DAR 6.9 TIFF0007777065000253.tif74139

[0527] Conjugate 8h was prepared and characterized as described in Example 1, except target C hIgG1a was used instead of XMT-1519. The STING agonist:target C hIgG1a ratio of purified conjugate 8g was 6.9.

[0528] Example 1i: Synthesis of Target E mIgG2a Conjugate 8i, DAR 10.0 TIFF0007777065000254.tif75140

[0529] Conjugate 8i was prepared and characterized as described in Example 1, except that target E mIgG2a was used instead of XMT-1519. The STING agonist:target E mIgG2a ratio of purified conjugate 8g was 10.0.

[0530] Example 1j: Synthesis of trastuzumab AAG conjugate 8j, DAR 7.0 TIFF0007777065000255.tif76148

[0531] Conjugate 8j was prepared and characterized as described in Example 1, except that trastuzumab AAG was used instead of XMT-1519.

[0532] Details of conjugates 8j and 8j-1 are shown below. TIFF0007777065000256.tif15128

[0533] Example 1k: Synthesis of trastuzumab mIgG2a conjugate 8k, DAR 8.1 TIFF0007777065000257.tif75154

[0534] Conjugate 8k was prepared and characterized as described in Example 1, except that trastuzumab mIgG2a was used instead of XMT-1519. The STING agonist:trastuzumab mIgG2a ratio of purified conjugate 8k was 8.1.

[0535] Example 1l: Synthesis of XMT-1535 conjugate 8l, DAR 4.7 TIFF0007777065000258.tif86160

[0536] Conjugate 8l was prepared and characterized as described in Example 1, except that XMT-1535 was used instead of XMT-1519. 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)acetic acid was incorporated into the compound structure instead of (3-(2-(2-aminoethoxy)ethoxy)propanoyl)-L-alanine. The STING agonist:XMT-1535 ratio of purified conjugate 8l was 4.7.

[0537] Example 1m: Synthesis of Palivizumab Conjugate 8m, DAR 4.5 TIFF0007777065000259.tif84154

[0538] Conjugate 8m was prepared and characterized as described in Example 1, except that palivizumab was used instead of XMT-1519. 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)acetic acid was incorporated into the compound structure instead of (3-(2-(2-aminoethoxy)ethoxy)propanoyl)-L-alanine. The STING agonist:palivizumab ratio of purified conjugate 8m was 4.5.

[0539] Example 2: Synthesis of XMT-1519 conjugate 16, DAR 5.3 TIFF0007777065000260.tif93160

[0540] Part A: To a solution of compound 9 (0.100 g, 0.429 mmol) dissolved in THF (5 mL) was added HATU (0.196 g, 0.514 mmol) and HOBt (0.079 g, 0.514 mmol). The reaction mixture was stirred at 0 °C for 10 min, and then 2-(benzyloxy)ethan-1-amine (0.0648 mg, 0.429 mmol) and DIPEA (0.112 mL, 0.643 mmol) were added. The reaction mixture was stirred at room temperature overnight, the solution was concentrated, and the residue was purified on silica gel (0-10% MeOH in DCM) to give compound 10 (0.2 g, 100% yield). 19 H 30 N2O5Na[(M+Na] + ) ESI-MS m / z calculated: calculated 389.2; found 389.2.

[0541] Part B: A solution of compound 10 (150 mg, 0.409 mmol) dissolved in EtOH (10 mL) was degassed with N, followed by the addition of Pd—C (43.6 mg, 0.409 mmol). The mixture was then degassed with H. The reaction mixture was stirred overnight at room temperature under H (1 atm). The solid was filtered through a pad of Celite, and the filtrate was concentrated to give compound 11. The crude compound 11 was used in the next step without further purification. 12 H 24 N2O5Na[(M+Na] + ) ESI-MS m / z calculated: calculated 299.2; found 299.2.

[0542] Part C: A 100 mL flask containing the residue of compound 11 (128 mg, 0.463 mmol) and N,N-dimethylpyridin-4-amine (11.32 mg, 0.093 mmol) was flushed with argon, then triethylamine (129 μL, 0.926 mmol), acetonitrile (1.544 mL), and DMF (0.772 mL) were added. The reaction mixture was cooled to 0 °C and stirred for 5 min. After that, 4-nitrophenyl carbonochloridate (140 mg, 0.695 mmol) was added, and the resulting mixture was stirred at 20 °C for 2 h and then concentrated to an oil. The residue was purified on silica gel (0-100% ethyl acetate in hexanes) to give compound 12 (50 mg, 24% yield) as a white solid. 14 H 19 N3O7[(M-Boc+H] + ) ESI-MS m / z calculated: calculated 342.2; found 342.1.

[0543] Part D: To a solution of compound 12 (50 mg, 0.113 mmol) and compound 1 (36.9 mg, 0.047 mmol), DMAP (1.153 mg, 9.44 μmol) dissolved in DMF (500 μL) was added. The reaction mixture was heated at 80°C for 3 h, and then compound 12 (50 mg, 0.113 mmol) was added. The reaction mixture was stirred for an additional 3 h and then cooled to room temperature. The mixture was concentrated, and the residue was purified on silica gel (0-30% MeOH in DCM) to give compound 13 (20 mg, 39% yield) as a white solid. 51 H 67 N 14 O 13 [(M+H)] + ESI-MS m / z calculated for: calculated 1083.4; found 1083.5.

[0544] Part E: To a solution of compound 13 (20 mg, 0.047 mmol) dissolved in DCM (0.5 mL) was added TFA (0.1 mL). The reaction mixture was stirred at room temperature for 4 hours and then concentrated to give compound 14 (10 mg, 55% yield) as a solid. 46 H59 N 14 O 11 [M+H] + ESI-MS m / z calculated for: calculated 983.4; found 983.5.

[0545] Part F: To a solution of scaffold 6 (14.15 mg, 10.17 μmol) and compound 14 (10 mg, 10.17 μmol) dissolved in DMF (1 mL), HATU (4.64 mg, 0.012 mmol), HOAt (1.881 mg, 0.012 mmol), and DIPEA (0.018 mL, 0.102 mmol) were added. The reaction mixture was stirred at room temperature for 2 h, then concentrated, and the residue was purified by preparative RP HPLC (0-80% ACN in water) to give scaffold 15 (20 mg, 83% yield). 101 H 152 N 24 O 41 [(M+2H] 2+ ) ESI-MS m / z calculated: calculated 1178.7; found 1178.59.0.

[0546] Part G: A solution of XMT-1519 (10 mg, 0.069 μmol) was conjugated with scaffold 15 (0.823 mg, 0.347 μmol dissolved in 200 μL DMA) as described in Example 1. The purified conjugate 16 had a STING agonist:XMT-1519 ratio of 5.3.

[0547] Example 3: Synthesis of trastuzumab conjugate 20 TIFF0007777065000261.tif180160

[0548] Part A: A solution of Boc-ala-ala-OH (67 mg, 256 μmol), CDI (70 mg, 435 μmol), and DMF (2 mL) was stirred at room temperature for 23 h. Compound 1a (prepared as described in WO2017175147A1, 100 mg, 128 μmol) and DIPEA (67 μL, 384 μmol) were then added, and the reaction was stirred at room temperature for 23 h. The reaction mixture was concentrated, and the residue was chromatographed on silica gel (0-20% MeOH-DCM eluent). The product, compound 17, was isolated as a yellow foam (109 mg, 83% yield). 49 H 64 N 15 O 10 [M+H] + ESI-MS m / zi calculated: 1022.5; found: 1022.4.

[0549] Part B: A mixture of compound 17 (108 mg, 106 μmol) and 2M HCl-dioxane (6 mL) was stirred at room temperature for 2 hours. The reaction mixture was concentrated, and the residue was dried under high vacuum to give compound 18 as an off-white foam (103 mg, quant.). 44 H 56 N 15 ESI-MS m / z calculated for O8 [M+H]: 922.4; found 922.4.

[0550] Part C: A mixture of compound 18 (80 mg, 84 μmol), scaffold 6 (117 mg, 84 μmol), HOAt (12 mg, 84 μmol), DiPEA (59 μL, 336 μmol), and DMF (3 mL) was stirred at room temperature for 5 min. HATU (42 mg, 109 μmol) was then added, and the reaction mixture was stirred at room temperature for 20 h. The reaction mixture was then concentrated and chromatographed by reverse phase (10–100% ACN-water w / 0.1% HCOOH eluent). Scaffold 19 was isolated as a white fluffy solid (35 mg, 18% yield). 99 H 149 N 25 O 38 [M+2H] 2+ESI-MS m / z calculated for: calculated 1148.0; found 1148.4.

[0551] Part D: Trastuzumab (10 mg, 0.067 μmol) was conjugated with scaffold 19 (1.237 mg, 0.539 μmol dissolved in 200 μL DMA) as described in Example 1, followed by purification using CHT type II chromatography to yield conjugate 20. Details of antibody-drug conjugates 20-1 and 20-2 are provided below. TIFF0007777065000262.tif16128

[0552] Example 3a: Synthesis of Palivizumab Conjugate 20a, DAR 5.5 TIFF0007777065000263.tif71160

[0553] Conjugate 20a was prepared and characterized as described in Example 1, except that palivizumab was used instead of XMT-1519. The STING agonist:palivizumab ratio of purified conjugate 20a was 5.5.

[0554] Example 4: Synthesis of trastuzumab conjugate 25, DAR 6.6 TIFF0007777065000264.tif142157

[0555] Part A: To a mixture of compound 21 (prepared as described in US Pat. No. 6,982,935, 38 mg, 0.047 mmol) and tert-butyl (S)-(2-hydroxypropyl)carbamate (9.85 mg, 0.056 mmol) dissolved in DMF (2 mL) was added 3-((ethylamino)methylene)amino)-N,N-dimethylpropan-1-amine hydrochloride (13.48 mg, 0.070 mmol), HOBt (10.77 mg, 0.070 mmol), DIPEA (0.016 mL, 0.094 mmol), and DMAP (5.73 mg, 0.047 mmol). The suspension was then stirred at room temperature for 2 days. The mixture was concentrated to give a residue, which was then purified on silica gel (0-30% MeOH in DCM) to give compound 22 as a light yellow solid (20 mg, 44% yield). 47 H 58 N 11 O 12 [M+H] + ESI-MS m / z calculated for: calculated 968.4; found 968.3.

[0556] Part B: To a suspension of compound 22 (20 mg, 0.021 mmol) in dioxane (4 mL) was added 4N HCl (0.52 mL, 8.19 mmol). The reaction mixture was stirred at room temperature for 5 hours, concentrated, and used in the next step without purification. The product, compound 23 (17 mg, 95% yield), was a white solid. 42 H 50 N 11 O 10 [M+H] + ESI-MS m / z calculated: 868.3; found: 868.4.

[0557] Part C: To a solution of compound 23 (17 mg, 0.020 mmol) and scaffold 6 (32.1 mg, 0.023 mmol) dissolved in DMF (2 mL) was added PyBOP (15.3 mg, 0.03 mmol) and DIPEA (0.034 mL, 0.196 mmol). The reaction mixture was stirred at room temperature for 2 h and concentrated to give a residue, which was then purified by preparative RP HPLC (0-80% ACN in water) to give scaffold 24 (26 mg, 59% yield). 97 H 143 N 21 O 40 [M+2H] 2+ ESI-MS m / z calculated: 1120.98; found 1121.06.

[0558] Part D: XMT-1519 antibody (5 mg, 0.0347 μmol) was conjugated with scaffold 24 (0.622 mg, 0.278 μmol) as described in Example 1. The crude reaction mixture was purified by CHT column chromatography to give conjugate 25 (3.19 mg, 64% yield). The STING agonist:XMT-1519 ratio of purified conjugate 25 was 6.6.

[0559] Example 5: Synthesis of XMT-1519 conjugate 28, DAR 6.0 TIFF0007777065000265.tif130160

[0560] Part A: Compound 26 was prepared as described in Example 1, except that 26 (prepared as described in US Pat. No. 6,298,935) was used instead of compound 1. Compound 27 was obtained as a colorless solid (71.0 mg, 40% yield). 103 H 154 N 22 O 43 [M+2H] 2+ ESI-MS m / z calculated: 1193.52; found 1193.48.

[0561] Part B: Conjugate 28 was prepared as described in Example 1 to yield conjugate 28. The STING agonist:XMT-1519 ratio of purified conjugate 28 was 6.0.

[0562] Example 6: Synthesis of XMT-1519 conjugate 29, DAR 5.5 TIFF0007777065000266.tif61165

[0563] Conjugate 28 (6.5 mg) was formulated in PBS, pH 8, by three cycles of concentration and dilution using a 30 kDa MWCO ultrafiltration unit. The reformulated conjugate was then incubated at 37°C for 24 hours and then reformulated into trehalose buffer, pH 5.5. LCMS analysis of the heavy and light chains after antibody reduction confirmed ring-opening. Good resolution was observed between the various light chain species: unconjugated, conjugated with intact succinimide, and conjugated with ring-opened succinimide. While ring-opening was also observed in the corresponding heavy chain species, resolution between the various species was poor. Therefore, the extent of ring-opening was estimated by focusing on the light chain species. Using this approach, the percentage of ring-opened product in conjugate 29 compared to the intact succinimide was estimated to be 94%. The STING agonist:XMT-1519 ratio of conjugate 29 was 5.5.

[0564] Example 7: Synthesis of XMT-1519 conjugate 32-1, DAR 6.5 TIFF0007777065000267.tif130160

[0565] Part A: Scaffold 31 was prepared as described in Example 1, except that compound 30 (prepared as described in US Pat. No. 6,298,935) was used instead of compound 1. Scaffold 31 was obtained as a colorless solid (9 mg, 8% yield). 101 H 151 N 23 O42 [M+2H] 2+ ESI-MS m / z calculated: 1179.02; found 1179.21.

[0566] Part B: Conjugates 32-1, 32-2, 32-3, and 32-4 were prepared as described in Example 1 to yield the title conjugates. The STING agonist:XMT-1519 ratios for purified conjugates 32-1, 32-2, 32-3, 32-4, and 32-5 were as listed in the table below. The mAb concentration of conjugate 32-5 was >10 mg / mL and contained <1% unconjugated mAb and <1% high molecular weight species. TIFF0007777065000268.tif30128

[0567] Example 7a: Synthesis of XMT-1535 conjugate 32a, DAR 6.2 TIFF0007777065000269.tif68139

[0568] Conjugates 32a, 32a-1, 32a-2, 32a-3, and 32a-4 were prepared and characterized as described in Example 1, except that XMT-1535 was used instead of XMT-1519. The STING agonist:XMT-1535 ratios for purified conjugates 32a, 32a-1, 32a-2, 32a-3, and 32a-4 were as listed in the table below. TIFF0007777065000270.tif30128

[0569] Example 7b: Synthesis of Palivizumab Conjugate 32b, DAR 6.8 TIFF0007777065000271.tif68143

[0570] Conjugates 32b, 32b-1, and 32b-2 were prepared and characterized as described in Example 1, except that palivizumab was used instead of XMT-1519. The STING agonist:palivizumab ratios of purified conjugates 32b, 32b-1, and 32b-2 were as listed in the table below. TIFF0007777065000272.tif20128

[0571] Example 7c: Synthesis of Palivizumab mIgG2a Conjugate 32c, DAR 9.1 TIFF0007777065000273.tif68152

[0572] Conjugate 32c was prepared and characterized as described above in Example 1, except that palivizumab mIgG2a was used instead of XMT-1519. The STING agonist:palivizumab ratio of purified conjugate 32c was 9.1.

[0573] Example 7d: Synthesis of XMT-1535 mIgG2a conjugate 32d, DAR 8.8 TIFF0007777065000274.tif63139

[0574] Conjugate 32d was prepared and characterized as described above in Example 1, except that XMT-1535 mIgG2a was used instead of XMT-1519. The STING agonist:XMT-1535 mIgG2a ratio of purified conjugate 32d was 8.8.

[0575] Example 7e: Synthesis of XMT-1519 AAG conjugate 32e, DAR 7.4 TIFF0007777065000275.tif75143

[0576] Conjugate 32e was prepared and characterized as described in Example 1, except that XMT-1519 AAG was used instead of XMT-1519. The STING agonist:XMT-1519 AAG ratio of purified conjugate 32e was 7.4.

[0577] Example 7-1: Alternative synthesis of compound 31 TIFF0007777065000276.tif94159

[0578] Part A: Compound 30 (prepared as described in US Pat. No. 6,298,935) (500 mg, 0.663 mmol, 1 eq), Boc-PEG2-Ala-OH (0.693 g, 1.99 mmol, 3 eq), EDC-HCl (381 mg, 1.99 mmol, 3 eq), and DMAP (243 mg, 1.99 mmol) were dissolved in DMF (26.5 mL) and mixed in a vial under air. The reaction was complete in 3 h. The mixture was quenched with AcOH (0.76 mL, 10 eq), concentrated, and purified on silica gel (DCM:MeOH) to give compound 30a as a white solid. 51 H 66 N 13 O 14 [M+H] + ESI-MS m / z calculated: 1084.5; found 1084.4.

[0579] Part B: Compound 30a (0.663 mmol) was suspended in dioxane (10 mL) in a flask under air. HCl (4 M in dioxane, 6 mL) was added, and the mixture was stirred at room temperature for 1 h. The mixture was concentrated to give a white solid. The solid was dissolved in pure water and purified by reverse-phase chromatography (0-25% ACN in water) to give compound 30b (444 mg, 77%) as a white solid. 46 H 58 N 13 O 12 [M+H] + ESI-MS m / z calculated: 984.4; found 984.2.

[0580] Part C: To a solution of scaffold 6 (450 mg, 0.32 mmol, prepared as described in PCT / US2018 / 06719) and compound 30b (350 mg, 0.072 mmol) dissolved in DMF (6.5 mL) was added PyBOP (185 mg, 0.36 mmol) and triethylamine (0.23 mL, 1.62 mmol). The mixture was stirred at RT for 15 min, then quenched with AcOH (0.23 mL, 3.99 mmol) and purified by reverse-phase purification (0-40% ACN in water w / 0.1% acetic acid) to give compound 31 (408 mg, 55% yield). 101 H 151 N 23 O 42 [M+2H] 2+ ESI-MS m / z calculated: 1179.52; found 1179.27.

[0581] Example 8: Synthesis of trastuzumab conjugate 34, DAR 6.9 TIFF0007777065000277.tif77160

[0582] Part A: To a solution of compound 1a (prepared as described in WO2017175147A1, 0.030 g, 0.038 mmol) dissolved in DMF (1.5 mL) was added N-ethyl-N-isopropylpropan-2-amine (0.067 mL, 0.385 mmol). After stirring the solution at room temperature for 5 minutes, 2,5-dioxopyrrolidin-1-yl 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15,18-hexaoxaheneicosan-21-oate (0.027 g, 0.050 mmol) dissolved in DMF (0.5 mL) was added, and the reaction mixture was stirred at room temperature for 15 minutes. Then, after adding acetic acid (0.1 mL), the mixture was purified on a preparative HPLC column (C18, 21.2 mm x 100 mm) using 10-100% MeCN (0.1% HOAc) in HO (0.1% HOAc, 20 min gradient) to give scaffold 33 (0.006 g, 13.05% yield). 57 H 75N 14 O 15 [M+H] + ESI-MS m / z calculated for: calculated 1195.55; found 1195.47.

[0583] Part B: Trastuzumab (10 mg, 0.069 μmol) was conjugated with scaffold 33 (0.658 mg, 0.550 μmol dissolved in DMA) as described in Example 1. The crude conjugate 34 was purified by CHT type II chromatography. The STING agonist:trastuzumab ratio of the purified conjugate 34 was 6.9.

[0584] Example 8a: Synthesis of Palivizumab Conjugate 34a, DAR 7.0 TIFF0007777065000278.tif45152

[0585] Conjugate 34a was prepared and characterized as described in Example 8, except that palivizumab was used instead of trastuzumab. The STING agonist:palivizumab ratio of purified conjugate 34a was 7.0.

[0586] Example 9: Synthesis of XMT-1519 conjugate 45, DAR 6.5 TIFF0007777065000279.tif145160

[0587] Part A: To a mixture of compound 35 (400 mg, 1688 μmol) and t-butyl acetate (8 mL) was added 70% HClO (302 mg, 2.11 mmol). The resulting solution was then stirred at room temperature for 21 hours and then neutralized with saturated NaHCO solution. The aqueous phase was washed with EtOAc (2x), and the combined organic solvents were then washed with brine, dried (NaSO), filtered, and concentrated to give compound 36 as an opaque oil (530 mg, quant.). 16 H 24 NO4[M+H] +ESI-MS m / z calculated: 294.2; found 294.2.

[0588] Part B: A mixture of compound 36 (279 mg, 782 μmol), compound 37 (370 mg, 938 μmol), HOAt (128 mg, 938 μmol), DIPEA (409 μL, 2.35 mmol), and DMF (4 mL) was stirred at room temperature for 5 min. HATU (416 mg, 1095 μmol) was then added, and the reaction was stirred at room temperature for 2.5 h. The reaction mixture was then concentrated, and the residue was chromatographed on silica gel (20-100% EtOAc / hexane eluent). Compound 38 was isolated as a white fluffy solid (254 mg, 362 μmol, 46% yield). 39 H 48 N2O9[M+H] + ESI-MS m / z calculated: 702.3; found 702.4.

[0589] Part C: A mixture of compound 38 (254 mg, 362 μmol), MeOH (6 mL), and 10% Pd—C catalyst (50 mg) was stirred under H (1 atm) at room temperature for 2 hours. The reaction mixture was then filtered, and the filtrate was concentrated to give compound 39 as a clear oil (214 mg, 97% yield). 32 H 42 N3O9[M+H] + ESI-MS m / z calculated: 612.3; found 612.3.

[0590] Part D: A mixture of compound 39 (58 mg, 64 μmol), compound 40 (prepared as described in US Pat. No. 6,982,935, 47 mg, 77 μmol), HOAt (10 mg, 77 μmol), DiPEA (56 μL, 320 μL), and DMF (2 mL) was stirred at room temperature for 5 minutes. HATU (36 mg, 96 μmol) was then added, and the reaction mixture was stirred at room temperature for 16 hours, then concentrated to give compound 41, a yellow oil (90 mg, quant.). 70 H 83 N 14 O16 [M+H] + ESI-MS m / z calculated: 1375.6; found: 1375.4.

[0591] Part E: A solution of compound 41 (90 mg, 64 μmol) in 20% piperidine in DMF (2 mL) was stirred at room temperature for 1 h. The reaction mixture was then concentrated and chromatographed on silica gel (0-40% MeOH-DCM eluent) to give compound 42 as a yellow oil (30 mg, 41% yield). 55 H 73 N 14 O 14 ESI-MS m / z calculated for [M+H]: 1153.5; found 1153.4.

[0592] Part F: A solution of compound 42 (30 mg, 37 μmol), 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (8 mg, 44 μmol), TEA (7 μL, 74 μmol), and DMF (1 mL) was stirred at room temperature for 17 hours. The reaction mixture was then concentrated to give scaffold 43 as a yellow oil (40 mg, quant.). 61 H 76 N 15 O 17 [M+H] + ESI-MS m / z calculated: 1290.6; found: 1290.3.

[0593] Part G: A solution of scaffold 43 (30 mg, 26 μmol) dissolved in 10% TFA-DCM (2 mL) was stirred at room temperature for 1 h. The reaction mixture was then concentrated, and the residue was subjected to HPLC chromatography (10–100% ACN-water w / 0.1% HCOOH eluent). Scaffold 44 was isolated as an off-white fluffy solid (6 mg, 20% yield). 52 H 60 N 15 O 15 [M+H] +ESI-MS m / z calculated: 1134.4; found 1134.2.

[0594] Part H: Conjugate 45 was prepared as described in Example 1, except that 4 equivalents of TCEP were used. The STING agonist:XMT-1519 ratio of purified conjugate 45 was 6.5.

[0595] Example 10: Synthesis of XMT-1519 conjugate 50, DAR 8.2 TIFF0007777065000280.tif67160

[0596] Part A: A mixture of compound 26 (prepared as described in US Pat. No. 6,982,935, 50 mg, 64 μmol), Fmoc-D-glutamic acid-O-tBu (54 mg, 128 μmol), DCC (26 mg, 128 μmol), DMAP (1 mg, 6 μmol), and DMF (2 mL) was stirred at room temperature for 17 hours. The reaction mixture was concentrated and used in the next step without purification. Compound 46 was obtained as a yellow oil (135 mg). 62 H 68 N 11 O 14 [M+H] + ESI-MS m / z calculated: 1190.5; found 1190.3.

[0597] Part B: A mixture of compound 46 (135 mg, 64 μmol) dissolved in DMF (2.4 mL) and 33% TEA was stirred at room temperature for 4.5 hours. The reaction mixture was concentrated, and the residue was purified by reverse-phase HPLC (10-100% ACN-water w / 0.1% HCOOH eluent). Compound 47 was isolated as a yellow powder (27 mg, 44% yield). 47 H 58 N 11 O 12 [M+H] + ESI-MS m / z calculated: 968.4; found: 968.2.

[0598] Part C: A solution of compound 47 (25 mg, 26 μmol), 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (8 mg, 31 μmol), TEA (11 μL, 78 μmol), and DMF (1 mL) was stirred at room temperature for 1 hour. The reaction mixture was concentrated and used in the next step without purification. Scaffold 48 is a yellow oil (40 mg, quant.). 53 H 61 N 12 O 15 [M+H] + ESI-MS m / z calculated: 1105.4; found 1105.2.

[0599] Part D: A solution of scaffold 48 (40 mg, 24 μmol) in DCM (1 mL) and 15% TFA was stirred at room temperature for 2 h. The reaction mixture was then concentrated and subjected to HPLC chromatography (10–100% ACN-water with 0.1% HCOOH eluent). Scaffold 49 was isolated as a white fluffy solid (5 mg, 20% yield). 49 H 53 N 12 O 15 [M+H] + ESI-MS m / z calculated: 1049.4; found 1049.2.

[0600] Part E: XMT-1519 (10 mg, 0.069 μmol) was conjugated to scaffold 49 as described in Example 1. Conjugate 50 was purified by CHT type II chromatography. The STING agonist:XMT-1519 ratio of purified conjugate 50 was 8.2.

[0601] Example 11: Synthesis of XMT-1519 conjugate 52, DAR 7.7 TIFF0007777065000281.tif48160

[0602] Conjugate 52 was prepared from scaffold 51 as described in Example 10, except that Fmoc-L-Glu(O-tBu) was used instead of Fmoc-D-Glu(O-tBu). The STING agonist:XMT-1519 ratio of purified conjugate 52 was 7.7.

[0603] Example 12: Synthesis of XMT-1519 conjugate 58, DAR 6.5 TIFF0007777065000282.tif120160

[0604] Part A: To a mixture of compound 26 (prepared as described in US Pat. No. 6,982,935, 0.105 g, 0.134 mmol), Boc-L-alanine (50.8 mg, 0.268 mmol), DMAP (50.8 mg, 0.067 mmol), and DCC (0.111 g, 0.537 mmol) was added DMF (2 mL). The suspension was then stirred at room temperature for 2 days. The mixture was concentrated and purified on silica gel (0-40% MeOH in DCM) to give compound 53 as a light yellow solid (0.102 g, 80% yield). 46 H 56 N 11 O 12 [M+H] + ESI-MS m / z calculated: 954.4; found 954.4.

[0605] Part B: To a suspension of compound 53 (0.102 g, 0.107 mmol) dissolved in dioxane (10 mL) was added 4N HCl (0.508 mL, 2.031 mmol). The reaction mixture was stirred at room temperature for 3 hours. The suspension was then concentrated and used in the next step without purification. Compound 54 was obtained as a pale yellow solid. 41 H 48 N 11 O 10 [M+H] + ESI-MS m / z calculated: 854.3; found 854.3.

[0606] Part C: To a solution of compound 54 (0.015 g, 0.017 mmol) dissolved in DMF (1 mL) was added Boc-D-Glu(Otu)-OH (7.67 mg, 0.025 mmol), followed by DIPEA (0.026 mL, 0.152 mmol). The reaction mixture was stirred at room temperature for 5 min. PyBOP (13.15 mg, 0.025 mmol) was then added, and the mixture was stirred at room temperature for 1 h, concentrated, and the residue was purified on silica gel (0-30% MeOH in DCM) to give compound 55 (11 mg, 57.3% yield) as a white solid. 55 H 71 N 12 O 15 [M+H] + ESI-MS m / z calculated: 1139.5; found 1139.5.

[0607] Part D: To a suspension of compound 55 (11 mg, 0.00966 mmol) in dioxane (3 mL) was added 4N HCl (0.241 mL, 0.966 mmol). The reaction mixture was stirred at room temperature for 2 hours. The suspension was concentrated and used in the next step without purification. Compound 56 was a white solid. 46 H 55 N 12 O 13 [M+H] + ESI-MS m / z calculated: 983.4; found 983.4.

[0608] Part E: To a solution of compound 56 (9.5 mg, 0.00966 mmol) dissolved in DMF (3 mL) was added DIEA (8.44 μL, 0.048 mmol) and 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) acetate (3.17 mg, 0.013 mmol). The reaction mixture was stirred at room temperature for 1 h, neutralized to pH 6-7 with HOAc, and then purified by preparative RP HPLC (0-75% ACN in water) to give scaffold 57 (3.3 mg, 31% yield) as a white solid. 52 H 58 N 13 O 16[M+H] + ESI-MS m / z calculated: 1120.4; found 1120.4.

[0609] Part F: XMT-1519 (10 mg, 0.069 μmol) was conjugated with scaffold 57 (0.700 mg, 0.625 μmol dissolved in 200 μL DMA) as described in Example 1. Conjugate 58 was purified by CHT type II chromatography. The STING agonist:XMT-1519 ratio of purified conjugate 58 was 6.5.

[0610] Example 13: Synthesis of XMT-1519 conjugate 60 TIFF0007777065000283.tif41160

[0611] Conjugate 60 was prepared from 59 as described in Example 12, except that Boc-L-Glu(Otu)-OH was used instead of Boc-D-Glu-O-tBu. Details of antibody-drug conjugates 60-1 and 60-2 are provided below. TIFF0007777065000284.tif16128

[0612] Example 14: Synthesis of XMT-1519 conjugate 62, DAR 6.5 TIFF0007777065000285.tif41160

[0613] Conjugate 62 was prepared from scaffold 61 as described in Example 12, except that Boc-L-Glu(Otu)-OH was used instead of Boc-D-Glu-O-tBu and Boc-D-Ala was used instead of Boc-L-Ala. The purified conjugate 62 had a STING agonist:XMT-1519 ratio of 6.5.

[0614] Example 15: Synthesis of XMT-1519 conjugate 64, DAR 6.4 TIFF0007777065000286.tif41159

[0615] Conjugate 64 was prepared from scaffold 63 as described in Example 12, except that Boc-D-Ala was used instead of Boc-L-Ala. The STING agonist:XMT-1519 ratio of purified conjugate 64 was 6.4.

[0616] Example 16: Synthesis of XMT-1519 conjugate 66 TIFF0007777065000287.tif41160

[0617] Conjugate 66 was prepared from scaffold 65 as described in Example 12, except that Boc-2-amino-2-methylpropanoic acid was used instead of Boc-L-Ala. Details of antibody-drug conjugates 66-1 and 66-2 are shown below. TIFF0007777065000288.tif16128

[0618] Example 17: Synthesis of XMT-1519 conjugate 74, DAR 6.9 TIFF0007777065000289.tif84160

[0619] Part A: A mixture of compound 26 (75 mg, 0.096 mmol, prepared as described in US Pat. No. 6,982,935), N-Boc-(D)-Ala-OH (91 mg, 0.48 mmol), DCC (99 mg, 0.48 mmol), and DMAP (1.2 mg, 9.58 μmol) dissolved in DMF (3 mL) was stirred at room temperature for 1 hour and then concentrated under reduced pressure. Purification on silica gel (DCM:MeOH 60:40 v / v) afforded compound 67 (82 mg, 90% yield) as a pale yellow solid. 46 H 56 N 11 O 12 [M+H] + ESI-MS m / z calculated: 954.40, found: 954.43.

[0620] Part B: To a suspension of compound 67 (80 mg, 0.084 mmol) in dioxane (5 mL) was added HCl (4 M in dioxane, 0.42 mL, 1.68 mmol), and the mixture was stirred at room temperature for 4 hours. The mixture was concentrated under reduced pressure to give compound 68 (72 mg, 100% yield) as a pale yellow solid. 41 H 48 N 11 O 10 [M+H] + ESI-MS m / z calculated: 854.35, found: 854.38.

[0621] Part C: To a stirred solution of compound 68 (48 mg, 0.056 mmol), N-Boc-glycine (15 mg, 0.084 mmol), and PyBOP (44 mg, 0.084 mmol) dissolved in DMF (3 mL) was added DIPEA (0.088 mL, 0.51 mmol), and the mixture was stirred at room temperature for 2 h. The mixture was concentrated, and the residue was purified on silica gel (DCM:MeOH 60:40 v / v) to give compound 69 (53 mg, 93% yield) as a white solid. 48 H 59 N 12 O 13 [M+H] + ESI-MS m / z calculated: 1011.42, found: 1011.45.

[0622] Part D: To a suspension of compound 69 (50 mg, 0.049 mmol) in dioxane (5 mL) was added HCl (4 M in dioxane, 1 mL, 20% v / v), and the mixture was stirred at room temperature for 2 hours, then concentrated to give compound 70 (45 mg, 100% yield) as a white solid. 43 H 51 N 12 O 11 [M+H] + ESI-MS m / z calculated: 911.37, found: 911.39.

[0623] Part E: To a stirred solution of compound 70 (20 mg, 0.022 mmol), N-Boc-(D)-Glu(OtBu)-OH (10 mg, 0.033 mmol), and PyBOP (17 mg, 0.033 mmol) dissolved in DMF (2 mL) was added DIPEA (0.03 mL, 0.22 mmol), and the mixture was stirred at room temperature for 2 h. The mixture was concentrated, and the residue was purified on silica gel (DCM:MeOH 60:40 v / v) to give compound 71 (24 mg, 90% yield) as a white solid. 57 H 74 N 13 O 16 [M+H] + ESI-MS m / z calculated: 1196.53; found: 1196.55.

[0624] Part F: To a suspension of compound 71 (24 mg, 0.02 mmol) in DCM (5 mL) was added TFA (1 mL, 20% v / v), and the mixture was stirred at room temperature for 12 hours. The mixture was concentrated to give compound 72 (21 mg, 100% yield) as a pale yellow solid. 48 H 58 N 13 O 14 [M+H] + ESI-MS m / z calculated: 1040.41; found: 1040.23.

[0625] Part G: To a stirred solution of compound 72 (21 mg, 0.02 mmol), 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (7.6 mg, 0.03 mmol) dissolved in DMF (2 mL) was added DIPEA (0.035 mL, 0.20 mmol), and the mixture was stirred at room temperature for 1 h. The mixture was concentrated, and the residue was purified by RP HPLC to give scaffold 73 (4.5 mg, 19% yield) as a white solid. 54 H 61 N 14 O 17 [M+H] + ESI-MS m / z calculated: 1177.43; found: 1177.40.

[0626] Part H: Conjugate 74 was prepared from scaffold 73 as described in Example 12. The purified conjugate 74 had a STING agonist:XMT-1519 ratio of 6.9.

[0627] Example 18: Synthesis of XMT-1519 conjugate 76, DAR 7.5 TIFF0007777065000290.tif36160

[0628] Conjugate 76 was prepared from scaffold 75 as described in Example 17, except that N-Boc-(L)-Ala-OH was used instead of N-Boc-(D)-Ala-OH and N-Boc-(L)-Glu(OtBu)-OH was used instead of N-Boc-(D)-Glu(OtBu)-OH. The purified conjugate 76 had a STING agonist:XMT-1519 ratio of 7.5.

[0629] Example 19: Synthesis of XMT-1519 conjugate 78, DAR 7.4 TIFF0007777065000291.tif36160

[0630] Conjugate 78 was prepared from scaffold 70 as described in Example 17, except that N-Boc-(L)-Glu(OtBu)-OH was used instead of N-Boc-(D)-Glu(OtBu)-OH. The purified conjugate 78 had a STING agonist:XMT-1519 ratio of 7.4.

[0631] Example 20: Synthesis of XMT-1519 conjugate 80, DAR 7.5 TIFF0007777065000292.tif36160

[0632] Conjugate 80 was prepared from scaffold 79 as described in Example 17, except that N-Boc-(L)-Ala-OH was used instead of N-Boc-(D)-Ala-OH. The STING agonist:XMT-1519 ratio of purified conjugate 80 was 7.5.

[0633] Example 21: Synthesis of XMT-1519 conjugate 82, DAR 5.7 TIFF0007777065000293.tif41160

[0634] Conjugate 82 was prepared from scaffold 81 as described in Example 12, except that Boc-glycine was used instead of Boc-(L)-Ala-OH. The STING agonist:XMT-1519 ratio of purified conjugate 81 was 5.7.

[0635] Example 22: Synthesis of XMT-1519 conjugate 85, DAR 6.5 TIFF0007777065000294.tif130160

[0636] Part A: Scaffold 84 was prepared as described in Example 1, except that compound 83 (prepared as described in US Pat. No. 6,229,935) was used instead of compound 1. Scaffold 84 was obtained as a white fluffy solid (3.3 mg, 0.5% yield over 5 steps). 101 H 148 N 22 O 42 S[M+2H] 2+ ESI-MS m / z calculated: 1187.49; found 1187.78.

[0637] Part B: Conjugate 85 was prepared as described in Example 1 to yield the title conjugate. The purified conjugate 85 had a STING agonist:XMT-1519 ratio of 6.5.

[0638] Example 22a: Synthesis of Palivizumab Conjugate 85a, DAR 7.4 TIFF0007777065000295.tif66141

[0639] Conjugate 85a was prepared and characterized as described in Example 1, except that palivizumab was used instead of XMT-1519. The STING agonist:palivizumab ratio of purified conjugate 85a was 7.4.

[0640] Example 23: Synthesis of XMT-1519 conjugate 88, DAR 6.6 TIFF0007777065000296.tif81128

[0641] Part A: Scaffold 87 was prepared as described in Example 12, except that compound 86 (prepared as described in US Pat. No. 6,982,935) was used instead of compound 26. 50 H 55 N 14 O 15 [M+H] + ESI-MS m / z calculated: 1091.4; found 1091.2.

[0642] Part B: Conjugate 88 was prepared as described in Example 12, except that scaffold 87 was used instead of scaffold 57. The purified conjugate had a STING agonist:XMT-1519 ratio of 6.6.

[0643] Example 23a: Synthesis of Palivizumab Conjugate 89, DAR 5.9 TIFF0007777065000297.tif54128

[0644] Conjugate 89 was prepared and characterized as described in Example 12, except palivizumab was used instead of XMT-1519. The STING agonist:palivizumab ratio of purified conjugate 89 was 5.9.

[0645] Example 23b: Synthesis of CTL-48132_mIgG2a conjugate 89a, DAR 8.8 TIFF0007777065000298.tif47128

[0646] Conjugate 89a was prepared and characterized as described in Example 12, except that CTL-48132_mIgG2a was used instead of XMT-1519. The STING agonist:CTL-48132_mIgG2a ratio of purified conjugate 89a was 8.8.

[0647] Example 23c: Synthesis of MFP5_mIgG2a conjugate 89b, DAR 9.0 TIFF0007777065000299.tif47128

[0648] Conjugate 89b was prepared and characterized as described in Example 12, except that CTL-48132_mIgG2a was used instead of XMT-1519. The STING agonist:MFP5_mIgG2a ratio of purified conjugate 89b was 9.0.

[0649] Example 24: Synthesis of XMT-1519 conjugate 92, DAR 7.6 TIFF0007777065000300.tif79128

[0650] Part A: Scaffold 91 was prepared as described in Example 12, except that compound 90 (prepared as described in US Pat. No. 6,229,935) was used instead of compound 26. 52 H 58 N 13 O 15 S[M+H] + ESI-MS m / z calculated: 1136.4; found 1136.2.

[0651] Part B: Conjugate 88 was prepared as described in Example 12, except that scaffold 87 was used instead of scaffold 57. The purified conjugate had a STING agonist:XMT-1519 ratio of 7.6.

[0652] Example 24a: Synthesis of Palivizumab Conjugate 93, DAR 6.7 TIFF0007777065000301.tif51128

[0653] Conjugate 93 was prepared and characterized as described in Example 12, except palivizumab was used instead of XMT-1519. The STING agonist:palivizumab ratio of purified conjugate 93 was 6.7.

[0654] Example 25: Synthesis of XMT-1519 conjugate 100, DAR 7.8 TIFF0007777065000302.tif109170

[0655] Part A: To a stirred solution of compound 94 (45 mg, 0.054 mmol, prepared as described in US Pat. No. 6,298,935) dissolved in DMF (3 mL) was added (S)-1-(Boc-amino)propan-2-ol (19 mg, 0.11 mmol), EDC (17 mg, 0.109 mmol), and DMAP (3.3 mg, 0.027 mmol), and the mixture was stirred at room temperature for 12 h. The reaction was concentrated under reduced pressure, and the residue was purified on silica gel (DCM:MeOH 60:40 v / v) to give 95 (49 mg, 92% yield) as a white solid. ESI-MS: C 47 H 57 N 10 O 12 S(M+H): Calculated 985.38, Found: 985.21.

[0656] Part B: To a stirred suspension of compound 95 (49 mg, 0.05 mmol) in DCM (5 mL) was added TFA (1 mL, 20% v / v DCM) and the mixture was stirred at room temperature for 12 hours. The resulting mixture was concentrated to give compound 96 (44 mg, 100% yield) as a pale yellow solid. ESI-MS: C 42 H 49 N 10 O 10 S(M+H): calculated value 885.33, found value: 885.18.

[0657] Part C: To a stirred solution of compound 96 (50 mg, 0.05 mmol) in DMF (2 mL) was added Boc-Glu-OtBu (86 mg, 0.28 mmol), PyBOP (118 mg, 0.23 mmol), and DIPEA (0.12 mL, 0.68 mmol), and the mixture was stirred at room temperature for 30 min. The mixture was concentrated under reduced pressure, and the residue was purified on silica gel (DCM:MeOH 60:40 v / v) to give compound 97 (45 mg, 77% yield) as a white solid. ESI-MS: C 56 H 72 N 11 O 15 S(M+H): calculated 1170.49, found: 1170.29.

[0658] Part D: To a stirred suspension of compound 97 (45 mg, 0.038 mmol) in DCM (5 mL) was added TFA (1 mL, 20% v / v DCM) and the mixture was stirred at room temperature for 12 h. The mixture was concentrated under reduced pressure to give compound 98 (38 mg, 100% yield) as a pale yellow solid. ESI-MS:C 47 H 56 N 11 O 13 S(M+H): calculated value 1014.37, found value: 1014.20.

[0659] Part E: To a stirred solution of compound 98 (20 mg, 0.02 mmol) dissolved in DMF (2 mL), 2,5-dioxopyrrolidin-1-yl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetate (5 mg, 0.02 mmol) and DIPEA (0.034 mL, 0.20 mmol) were added, and the mixture was stirred at room temperature for 15 min. The reaction was quenched with acetic acid (0.034 mL, 1:1 v / v DIPEA) and directly purified by HPLC using a C18 stationary phase (water:ACN) to give scaffold 99 (4.7 mg, 20% yield) as a white solid. ESI-MS: C 53 H 59 N 12 O 16 S(M+H): calculated 1151.38, found: 1151.18.

[0660] Part F: Conjugate 100 was prepared as described in Example 12, except that scaffold 99 was used instead of scaffold 57. The purified conjugate had a STING agonist:XMT-1519 ratio of 7.8.

[0661] Example 25a: Synthesis of Palivizumab Conjugate 101, DAR 6.5 Conjugate 101 was prepared as described in Example 25, except that palivizumab was used instead of XMT-1519. The purified conjugate had a STING agonist:palivizumab ratio of 6.5.

[0662] Example 26A: Cancer cell-targeting wild-type or Fc-mutant STING-ADC activity in cancer cell / THP1 luciferase reporter cell co-cultures Generation of NaPi2b Fc-silenced antibody: A NaPi2b mAb (anti-NaPi2b-(AAG)) with an engineered Fc region to eliminate Fc effector function was engineered with three mutations in the heavy chain constant region: L234A, L235A, and P329G (AAG; Kabat-Eu numbering). It was generated using standard molecular biology procedures. The antibody was expressed and purified. Briefly, DNA encoding the heavy chain variable region of NaPi2b, the human IgG1 constant region with the L234A, L235A, and P329G mutations, the light chain variable region of anti-NaPi2b, and the human kappa light chain were cloned into a mammalian expression vector. The heavy and light chains of NaPi2b-(AAG) were coexpressed in HEK293 cells, and the antibody was purified from the cell supernatant by standard protein A affinity chromatography.

[0663] Induction of the STING pathway in immune cells: Induction of the STING pathway in immune cells by the NaPi2b-targeting STING ADC was evaluated using a cancer cell / THP1-IRF3-luciferase reporter cell coculture assay. OVCAR3 human ovarian cancer cells were seeded into 96-well CellBind surface tissue culture plates (15,000 cells / well) and allowed to attach for 6 hours in RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin. Various dilutions of the test articles conjugate 8b-1, conjugate 8f, conjugate 8d-1, and compound 1 (0.4 nM to 100 nM based on payload; 3-fold serial dilutions in growth medium) were added to each well, and the plate was incubated at 37°C for 20 minutes. Then, THP1-dual reporter cells (30,000 cells) were added to each well, and incubation continued for 20 hours at 37°C in a humidified atmosphere of 5% CO2. Cell culture supernatant (20 μl) from each incubation sample was added to resuspended QUANTI-Luc (50 μl), and the luminescence signal was immediately measured using a SpectraMax M5 plate reader (Molecular Devices). 50The EC values ​​were calculated from dose-response curves. Table 1A shows the EC values ​​in THP1-Dual cells co-cultured with OVCAR3 cancer cells. 50 Indicates the value.

[0664] [Table 1A]

[0665] As shown in Table 1A, conjugate 8b-1, which has wild-type Fc effector function, exhibits over 100-fold increased activity compared to the free agonist, compound 1, and approximately 1000-fold increased activity compared to conjugates 8f and 8d-1, confirming the role of Fc receptors in achieving activity. Results shown are EC values ​​of a representative experiment. 50 value.

[0666] Example 26B: Cancer cell-targeting wild-type or Fc-mutant STING-ADC activity in cancer cell / THP1 luciferase reporter cell co-cultures Generation of HER-2 Fc-silenced antibodies: An Fc region-engineered trastuzumab mAb (anti-Her2-(AAG)) that abolishes Fc effector function was designed with three mutations in the heavy chain constant region: L234A, L235A, and P329G (AAG; Kabat Eu numbering) and was generated as described in Example 26A.

[0667] Induction of the STING pathway in immune cells: Induction of the STING pathway in immune cells by HER2-targeted STING ADCs was assessed in a cancer cell / THP1-IRF3-luciferase reporter cell co-culture assay using SKBR3 human breast cancer cells and test articles conjugate 8a-2, conjugate 8j, conjugate 8c-2, and compound 1), as described in Example 26A. Table 1A shows the EC10 activity in THP1-Dual cells co-cultured with SKBR3 cancer cells. 50 Indicates the value.

[0668] [Table 1B]

[0669] As shown in Table 1B, conjugate 8a-2, which possesses wild-type Fc effector function, exhibits approximately 50-fold increased activity compared to the free agonist, compound 1, and approximately 1000-fold increased activity compared to conjugates 8j and 8c-2, confirming the role of Fc receptors in achieving activity. Results shown are EC values ​​of representative experiments. 50 value.

[0670] Example 27A: Cancer cell-targeting wild-type or Fc-mutated STING ADC activity in THP1 luciferase reporter cells cultured on tumor cell antigen-coated plates Human NaPi2b-derived peptide TIFF0007777065000306.tif4128 was coated onto the surface of each well of a 96-well plate by incubating with this peptide (1 μg / mL in PBS) overnight at 4 °C. The wells were then washed 1x with PBS-T and blocked by incubating with BSA (3% in PBS-T) at room temperature for 1 h. After washing with PBS-T (2x), PBS (1x), and growth medium (RPMI 1640, 10% FBS, 1% penicillin / streptomycin, 1x), various dilutions (0.4 nM to 100 nM based on payload; 3-fold serial dilutions in growth medium) of test articles (conjugate 8b-1, conjugate 8f, conjugate 8c-1, and compound 1) were added to each well, and the plate was incubated at 37 °C for 20 min. THP1-dual reporter cells (50,000 cells) were added to each well and incubated for 20 hours at 37°C in a humidified atmosphere of 5% CO2. Cell culture supernatant (20 μl) from each incubation sample was added to resuspended QUANTI-Luc (50 μl), and the luminescence signal was immediately measured using a SpectraMax M5 plate reader (Molecular Devices). EC 50Table 2A shows the EC values ​​obtained from dose-response curves in THP1-Dual cells cultured on NaPi2b recombinant peptide-coated plates. 50 Indicates the value.

[0671] [Table 2A]

[0672] As shown in Table 2, conjugate 8b-1 with wild-type Fc exhibits approximately 100-fold greater activity than compound 1. Conjugate 8f has no activity, and conjugate 8c-1 has approximately 1000-fold less activity than conjugate 8b-1. This confirms the role of Fc receptors in achieving activity. Results shown are EC values ​​of representative experiments. 50 value.

[0673] Example 27B: Cancer cell-targeting wild-type or Fc-mutant STING ADC activity in THP1 luciferase reporter cells cultured on tumor cell antigen-coated plates A peptide derived from human HER2 / ErbB2 protein (His-tagged, ECD, domain IV, 17.1 kDa) was coated onto the surface of each well of a 96-well plate by overnight incubation at 4°C with the peptide (1 μg / mL in PBS). The assay was performed as described in Example 27A, except that 3-fold serial dilutions (0.09 nM to 200 nM based on payload) of the test articles conjugate 8a-3, conjugate 8j, conjugate 8c-2, and compound 1 were used. Table 2B shows the EC1 activity in THP1-Dual cells cultured on Her2 recombinant protein-coated plates. 50 Indicates the value.

[0674] [Table 2B]

[0675] As shown in Table 2B, conjugate 8a-3 with wild-type Fc exhibits approximately 100-fold increased activity compared to compound 1. Conjugates 8j and 8c-2 lack activity, confirming the role of Fc receptors in achieving activity. Results shown are EC values ​​of representative experiments. 50 value.

[0676] Example 28A: Activity of tumor cell-targeting NaPi2b ADC in cancer cell / PBMC co-cultures OVCAR3 human ovarian cancer cells stably expressing the nuclear-restricted mKate fluorescent red protein were generated by transduction with IncuCyte® NucLight Red Lentivirus Reagent. Stably transduced cells (designated OVCAR3-NucRed cells) were selected for 2 days in puromycin-containing medium (2 μg / mL) and seeded into 96-well tissue culture plates (8,000 cells / well) and allowed to attach overnight in RPMI 1640 medium containing 10% FBS and 1% penicillin / streptomycin. The culture medium was replaced with fresh medium (50 μL). Test articles (3x concentrated, conjugate 8b-1 (100nM, 10nM, and 1nM), conjugate 8f (100nM, 10nM, and 1nM), conjugate 8c-1 (100nM and 10nM), and compound 1 (100nM and 10nM); conjugate concentrations were based on payload) were then added to each well containing medium (50μL), and the plate was incubated at 37°C for 20 minutes. Frozen human peripheral blood mononuclear cells (PBMCs) were thawed according to the supplier's instructions and added to each well (40,000 PBMCs dissolved in 50μL of medium), and the plate was placed in an IncuCyte© live cell imaging instrument in an incubator (37°C, 5% O2) and scanned every 4 hours for 2 days. The number of red objects (cancer cells) was quantified using IncuCyte© Zoom software. The red object confluency within each well was normalized to its own T=0 time point red object confluency.

[0677] Figure 1A plots red object confluency as a function of time and shows robust induction of cancer cell killing by PBMCs in response to conjugate 8b-1 at a 100-fold lower payload concentration compared to compound 1. Conjugate 8f also exhibited activity, but at a lower level compared to conjugate 8b. Conjugate 8c-1 exhibited significantly lower activity compared to conjugate 8b-1.

[0678] Example 28B: Activity of tumor cell-targeting NaPi2b ADC in cancer cell / PBMC co-cultures OVCAR3-NucRed cells were seeded into 96-well tissue culture plates (20,000 cells / well) and allowed to attach for 6 hours in RPMI 1640 medium containing 10% FBS and 1% penicillin / streptomycin. The culture medium was replaced with fresh medium (50 μL). Test articles (3x concentrated, conjugate 8l and compound 1 (100 nM, 10 nM, and 1 nM, respectively) and conjugate 8m (100 nM); conjugate concentrations were based on payload) were then added to each well containing medium (50 μL). The assay was performed as described in Example 28A, except that 30,000 PBMCs were used. The number of red objects (cancer cells) was quantified using IncuCyte ©Zoom software. The number of red objects in each well was normalized to its own T=0 time point number.

[0679] Figure 1B plots red object counts as a function of time and shows robust induction of cancer cell killing by PBMCs in response to conjugate 8l at a 100-fold lower payload concentration compared to compound 1. Conjugate 8m had no significant activity, and the increase in red object counts (cell proliferation) over time was similar to that of the untreated control. The inset shows that none of the test articles (100 nM) inhibited proliferation of OVCAR3-NucRed cells in monoculture.

[0680] Example 29: Flow cytometry analysis of CD14, Fcγ receptor, and CD3 expression in PBMCs and isolated monocyte subpopulations Frozen human PBMCs (1x10 8 The cells (50,000 cells) were thawed and aliquoted into three tubes. One aliquot was subjected to human monocyte enrichment [StemCell Technologies] ("CD16-depleted monocytes"), one aliquot was subjected to human monocyte enrichment without CD16 depletion [StemCell Technologies] ("enriched monocytes"), and one aliquot was not subjected to enrichment ("PBMCs"). For flow cytometry, cells (50,000 cells) from each group were transferred in quadruplicate to U-bottom 96-well plates, washed with PBS, and stained with live / dead fixable Aqua dead cell staining dye (Molecular Probes) followed by fluorophore-conjugated target-specific (in triplicate) or isotype control antibodies (Pacific Blue anti-human CD14, FITC anti-human CD3, APC / Cy7 CD16, PE anti-human CD32, and PE / Cy7 anti-human CD64). Cells were fixed, and surface expression of proteins of interest was determined by flow cytometry analysis on a MACSQuant flow cytometer. Data analysis was performed using FlowJo software. Table 3 shows the frequencies (% of single / viable cells) of CD14- / CD16+, CD14+ / CD16+, CD14- / CD32+, CD14+ / CD32+, CD14- / CD64+, CD14+ / CD64+, and CD14- / CD3+ cells in PBMC, enriched monocyte, and CD16-depleted monocyte populations.

[0681] [Table 3] * Gating on single / live cells

[0682] Table 3 shows efficient CD3+ cell depletion and monocyte enrichment, with low levels of CD16-positive cells in CD16-depleted monocytes after isolation. CD64 staining results show that all CD14-positive cells express CD64 (FcγRI) in PBMCs and enriched monocyte subpopulations.

[0683] FIG. 2 demonstrates efficient CD3+ cell depletion and monocyte expansion, as well as low levels of CD14-positive cells in CD16-depleted monocytes after isolation.

[0684] Example 30: Cancer cell-killing activity of Her2 ADCs targeting Fc-mutated tumor cells in in vitro co-cultures of PBMCs with STING wild-type or knockout SKBR3 cells Generation of STING knockout single cell clones expressing nuclear-restricted mKate fluorescent protein: SKBR3 cells were seeded in 24-well plates (50,000 cells / well) and transfected with a non-targeting sgRNA and three different sgRNAs targeting the human STING gene using Thermo Fisher's TrueGuide™ Synthetic gRNA, TrueCut™ Cas9 Protein v2, and Lipofectamine™ CRISPRMAX™ Transfection Reagent according to the manufacturer's protocol. The sgRNA sequences were: sgNT (non-targeting): TIFF0007777065000310.tif18161. Seven days after transfection, single cells were sorted and placed in 96-well plates containing 100 μL of DMEM containing 20% ​​FBS and 1% penicillin / streptomycin. Clones were allowed to form over 2–3 weeks (medium was refreshed 1–2 times per week). Multiple clones were trypsinized, expanded, and analyzed for STING expression by Western blot using rabbit monoclonal anti-STING (Cell Signaling Technologies) and anti-β-actin (Licor) antibodies. Clones lacking STING protein expression (confirmed by Western blot) were selected for stable expression of nuclear-restricted mKate fluorescent protein as described in Example 28.

[0685] Killing assay: NucRed-expressing STING wild-type (sgNT-2: non-targeting sgRNA, clone 2) and knockout (sg#3-2: sgRNA#3 clone 2) SKBR3 cells were seeded in 96-well plates (15,000 cells / well) in RPMI containing 10% FBS and 1% penicillin / streptomycin, and PBMC killing assays were performed using 100 nM, 25 nM, 5 nM, and 1 nM of conjugate 8a-3, conjugate 8j, conjugate 8c-2, and compound 1, respectively, as described in Example 29 (conjugate concentrations were based on payload).

[0686] Figures 3A and 3B plot red object confluency as a function of time, showing the killing of STING wild-type (sgNT-2) SKBR3 cells / PBMC cocultures and STING knockout (sg#3-2) SKBR3 cells / PBMC cocultures, respectively. Conjugate 8a-3 induced robust killing of STING wild-type and knockout SKBR3 cells / PBMC cocultures at all doses tested. Conjugate 8j had high activity at 100 nM, 25 nM, and 5 nM in STING wild-type (sgNT-2) SKBR3 cells / PBMC cocultures and low activity in STING knockout (sg#3-2) SKBR3 cells / PBMC cocultures. Compound 1 was only active at 100 nM in STING wild-type (sgNT-2) SKBR3 cells / PBMC cocultures and inactive at all doses in STING knockout (sg#3-2) SKBR3 cells / PBMC cocultures. Conjugate 8c-2 had no activity in either STING wild-type (sgNT-2) or STING knockout (sg#3-2) SKBR3 cells / PBMC cocultures.

[0687] Example 30A: Activity of tumor cell-targeting Her2 ADCs and Her2 antibodies in STING wild-type or STING knockout SKBR3 cancer cell / PBMC cocultures NucRed-expressing STING wild-type (sgNT-2: non-targeting sgRNA, clone 2) and knockout (sg#3-2: sgRNA#3 clone 2) SKBR3 cells were cocultured with PBMCs, and killing assays were performed using a range of doses of conjugates 8a-3 and 8-j (200 nM, 4x dilutions based on payload) as described in Example 30. Unconjugated wild-type Fc-trastuzumab and AAG Fc-mutant trastuzumab were dosed at antibody concentrations corresponding to the conjugate 8a-3 antibody concentration. As shown in Figures 4A and 4B, conjugate 8a-3 demonstrated robust killing of both STING wild-type and knockout SKBR3 cancer cells. In contrast, conjugate 8-j, an Fc-mutant Her2-targeting ADC, only exhibited killing activity in STING wild-type SKBR3 cocultures, which was nearly eliminated in STING knockout SKBR3 cocultures. Both the Fc wild-type and AAG mutant unconjugated trastuzumab antibody showed low activity in both STING wild-type and STING knockout cancer cell cocultures. These data demonstrate that the cancer cell-killing activity of Fc mutant cancer cell-targeting STING-ADCs in immune cell cocultures results from intrinsic STING activation in tumor cells.

[0688] Example 31: Activity of tumor cell-targeting NaPi2b ADC in cancer cell / human monocyte co-cultures in the absence of T cells OVCAR3-NucRed cells (prepared as described in Example 28) were seeded (15,000 cells / well) into 96-well CellBind surface tissue culture plates (Corning) and allowed to attach for 6 hours in RPMI1640 medium containing 10% FBS and 1% penicillin / streptomycin. The culture medium was replaced with fresh medium (50 μL). Test articles (conjugate 8b-1 and compound 1, 20 nM and 4 nM, respectively, with conjugate concentrations based on payload concentration) were then added to each well containing medium (50 μL). The plates were incubated at 37° C. for 20 minutes. PBMCs, enriched monocytes, and CD16-depleted monocytes were prepared as described in Example 29. Live PBMCs (30,000 cells / well), enriched monocytes (20,000 cells / well), and CD16-depleted monocytes (20,000 cells / well) were then added to wells containing 50 μL of culture medium. The plates were placed in an IncuCyte® live-cell imaging instrument in an incubator (37°C, 5% O2) and scanned every 4 hours for 2 days. The number of red objects (cancer cells) was quantified using IncuCyte® Zoom software. The red object confluency in each well was normalized to its own red object confluency at T=0. The red object confluency is plotted as a function of time in Figures 5A-5C. This demonstrates that CD3+ T cell-depleted monocyte populations have comparable cancer cell killing activity in response to tumor cell-targeting ADC activity. Conjugate 8b-1 at 4 nM and 20 nM payload concentrations induced robust killing of OVCAR3-NucRed cancer cells by PBMCs (Figure 5A), enriched monocytes (Figure 5B), and CD16-depleted monocytes (Figure 5C). Compound 1 at 20 nM induced cancer cell killing only in enriched and CD16-depleted monocyte cocultures.

[0689] Example 32: In vitro measurement of human CXCL10 and cellular binding of HER2-targeting antibody-drug conjugates to the HCC1954 human breast cancer cell line HCC1954 breast cancer cells were grown to approximately 80-95% confluency in RPMI 1640 medium supplemented with FBS (10%) and penicillin / streptomycin (1%). Cells were harvested and added to wells of a 96-well flat-bottom plate (40,000 cells / well) and incubated overnight at 37°C and 5% CO2. The cells were then treated with test HER2 antibody-drug conjugates (20 μL) at concentrations ranging from 1 pM to 10 μM as indicated in Table 4 and incubated for 24 hours at 37°C and 5% CO2. The plates were centrifuged (300 g, 5 min), and the supernatants were collected (100 mL) and subjected to ELISA analysis for human CXCL10 (human CXCL10 / IP-10). The developed plates were analyzed by OD analysis using a SpectraMax M5 plate reader. 450 The values ​​for each treatment were plotted and the EC 50 Values ​​were calculated using 4-parameter curve fitting with GraphPad Prism software.

[0690] To confirm cellular binding of HER2 antibody-drug conjugates to HCC1954 cells, HCC1954 cells were grown to approximately 80-95% confluency in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin / streptomycin. Cells were harvested and added to wells of a 96-well V-bottom plate (50,000 cells / well). Cells were pelleted (300 x g, 5 minutes) and resuspended in solutions of the test HER2 antibody-drug conjugates at concentrations ranging from 0.01 nM to 100 nM, as shown in Table 4, and incubated on ice for 3 hours. The cells were then washed 3x with ice-cold PBS, pelleted (300g, 5 min), and incubated with the detection antibody (goat anti-human IgG-Alexa-647 (H+L chain)) for 1 h at 4 °C. The cell suspension was pelleted (300g, 5 min), washed three times with ice-cold PBS, and fixed by resuspension in a solution of paraformaldehyde (2%). The resuspended cells were then subjected to flow cytometry analysis using a MACS Quantitative Flow Cytometry system. Single events (10,000) were collected for analysis. Population gating and mean fluorescence intensity (MFI) analysis were performed using FlowJo software.

[0691] Table 4 shows the mean EC20 of cell binding and CXCL10 induction in HCC1954. 50 This is a summary of the above.

[0692] [Table 4] ND=Not determined

[0693] As shown in Table 4, treatment of HCC1954 cells with Her2-targeting antibody-drug conjugates resulted in sub-nM to low nM EC for CXCL10 induction. 50 EC values ​​were obtained for the binding of Her2-targeting ADCs to HCC1954 cells. 50 Values, when determined, were in the low nM range.

[0694] Example 33: Activity of tumor cell-targeting HER2 antibody-drug conjugates in cancer cell / PBMC co-cultures Cancer cell killing activity: SKBR3 human breast cancer cells stably expressing the nuclear-restricted mKate fluorescent protein were generated as described in Example 28A and designated SKBR3 NucRed cells. 20,000 cells (per well) of SKBR3 NucRed cells were seeded into a 96-well tissue culture plate and allowed to attach for 6 hours in 50 μL of RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin. 50 μL of test articles (Conjugate 8a-2, Conjugate 8-j, Conjugate 8c-2, and Compound 1) at various dilutions (0.0.1 nM to 200 nM based on payload; 4-fold serial dilutions in growth medium) were added to each well, and the plate was incubated at 37°C for 20 minutes. PBMCs or primary human monocytes (isolated from PBMCs as described in Example 29) (50,000 cells) were then added to each well, and the assay was performed as described in Example 28. Table 5 shows the IC of test articles in cancer cell / PBMC and isolated primary human monocyte co-cultures (cancer cell killing activity). 50 The values ​​(killing activity) are shown.

[0695] [Table 5]

[0696] As shown in Table 5, conjugate 8a-2 exhibited approximately 300x and 150x greater potency than compound 1 in PBMC and monocyte cocultures, respectively. Conjugate 8-j was approximately 30- and 80-fold less potent than conjugate 8a-2 in PBMC and monocyte cocultures, respectively. Conjugate 8c-2 had only slight activity in both PBMC and monocyte cocultures.

[0697] CXCL10 induction: Table 6 shows the EC values ​​of test articles in cancer cell / PBMC and isolated primary human monocyte co-cultures for CXCL10 induction.50 Indicates the value.

[0698] [Table 6]

[0699] As shown in Table 6, conjugate 8a-2 exhibited approximately 400x and 700x greater CXCL10 induction potency compared to compound 1 in PBMC and monocyte cocultures, respectively. Conjugate 8-j was approximately 50-fold less potent than conjugate 8a-2 in both PBMC and monocyte cocultures. Conjugate 8c-2 had only slight activity in both PBMC and monocyte cocultures.

[0700] Type III IFN induction: Co-cultures of SKBR3 cells and PBMCs were prepared as described above to analyze type III interferon induction in the supernatants 24 hours after treatment using a human IL29 / IL28b ELISA kit. Table 7 shows the EC values ​​of test articles in cancer cell / PBMC co-cultures for IL29 / IL28b induction (IFNλ1 / λ3). 50 Indicates the value.

[0701] [Table 7]

[0702] As shown in Table 7, conjugate 8a-2 exhibited approximately 400x greater IL29 / IL28b induction potency in PBMC coculture than compound 1. Conjugate 8-j was approximately 6-fold less potent than conjugate 8a-2 in PBMC coculture. Conjugate 8c-2 had only slight activity in PBMC coculture.

[0703] The data in Tables 7-9 demonstrate that mutations in the Fc region of ADCs that inhibit FcγR interactions reduce, but do not eliminate, the cancer cell-killing activity of targeted ADCs, suggesting an Fc-independent contribution of the ADCs.

[0704] Example 34: Induction of the STING pathway in immune cells Induction of the STING pathway in immune cells by the Her2-targeting STING ADC was evaluated using a cancer cell / THP1-IRF3-luciferase reporter cell coculture assay. SKOV3 human ovarian adenocarcinoma cells were seeded into 96-well CellBind surface tissue culture plates (20,000 cells / well) and allowed to attach for 6 hours in McCoy's 5a medium containing 10% FBS and 1% penicillin / streptomycin. Various dilutions (0.01 nM to 300 nM based on payload; 4-fold serial dilutions in grow...

Claims

1. below or a pharmaceutically acceptable salt or stereoisomer thereof, wherein The HER2 antibody comprises a variable heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence FTFSSYSMN (SEQ ID NO:20); a variable heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO:21); a variable heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence GGHGYFDL (SEQ ID NO:22); a variable light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence RASQSVSSSYLA (SEQ ID NO:27); a variable light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence GASSRAT (SEQ ID NO:28); and a variable light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence QQYHHSPLT (SEQ ID NO:29), and 15 represents an integer in the range of 1 to 20, The antibody-drug conjugate (ADC), or a pharmaceutically acceptable salt or stereoisomer thereof.

2. below or a pharmaceutically acceptable salt or stereoisomer thereof, wherein The HER2 antibody comprises a variable heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence FTFSSYSMN (SEQ ID NO:20); a variable heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO:21); a variable heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence GGHGYFDL (SEQ ID NO:22); a variable light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence RASQSVSSSYLA (SEQ ID NO:27); a variable light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence GASSRAT (SEQ ID NO:28); and a variable light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence QQYHHSPLT (SEQ ID NO:29), and 15 represents an integer in the range of 1 to 20, The antibody-drug conjugate (ADC), or a pharmaceutically acceptable salt or stereoisomer thereof.

3. Formula (I): PBRM-[A 1 -(L C )-D] d15 (I) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: A 1 teeth, where: * indicates that it is attached to the PBRM, ** L C indicates that it is attached to L C teeth, where: # is A 1 ## indicates that it is attached to D. M A teeth, where: * is A 1 indicates that it is attached to ** is T 1 indicates that it is attached to *** is L D indicates that it is attached to L D is ***-NH-(CH 2 CH 2 O) 2 -(CH 2 ) 2 -C(O)-(alanine)-****, wherein *** is M A indicates that it is attached to **** indicates that it is attached to D, T 1 teeth where n 4 is 8, D is where: R 2 is -O-, L D indicates that it is attached to PBRM is a HER2 antibody comprising a variable heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence FTFSSYSMN (SEQ ID NO:20); a variable heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO:21); a variable heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence GGHGYFDL (SEQ ID NO:22); a variable light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence RASQSVSSSYLA (SEQ ID NO:27); a variable light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence GASSRAT (SEQ ID NO:28); and a variable light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence QQYHHSPLT (SEQ ID NO:29), and 15 represents an integer between 1 and 20, The antibody-drug conjugate, or a pharmaceutically acceptable salt or stereoisomer thereof.

4. L D but, where: *** is M A indicates that it is attached to **** indicates that it is attached to D, 4. The conjugate of claim 3, or a pharmaceutically acceptable salt or stereoisomer thereof.

5. 5. The conjugate of any one of claims 1 to 4, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the HER2 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

24.

6. 6. The conjugate of any one of claims 1 to 5, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the HER2 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

26.

7. d 15 7. The conjugate of any one of claims 1 to 6, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein represents an integer from 2 to 14.

8. d 15 8. The conjugate of any one of claims 1 to 7, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein represents an integer from 2 to 8.

9. d 15 The conjugate of any one of claims 1 to 8, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein is 2, 4, 6, or 8.

10. d 15 The conjugate of any one of claims 1 to 7, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein is an integer from 6 to 10.

11. d 15 11. The conjugate of claim 10, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein

12. d 15 11. The conjugate of claim 10, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

13. d 15 11. The conjugate of claim 10, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

14. 14. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 13, or a pharmaceutically acceptable salt or stereoisomer thereof, and one or more pharmaceutically acceptable carriers or excipients.

15. 15. The pharmaceutical composition of claim 14, wherein the composition is administered in combination with at least one immunomodulator or at least one immunostimulant.

16. 14. A pharmaceutical composition for activating or enhancing the activity of stimulator of interferon genes (STING) in a subject, comprising the conjugate of any one of claims 1 to 13, or a pharmaceutically acceptable salt or stereoisomer thereof.

17. 14. A pharmaceutical composition for preventing or treating a disease or disorder in a subject, comprising a therapeutically effective amount of the conjugate of any one of claims 1 to 13, or a pharmaceutically acceptable salt or stereoisomer thereof.

18. 18. The pharmaceutical composition of claim 17, wherein the disease or disorder is associated with agonism of STING.

19. 19. The pharmaceutical composition of claim 17 or claim 18, wherein the disease or disorder is cancer.

20. 20. The pharmaceutical composition of any one of claims 17-19, wherein the disease or disorder is bladder cancer, breast cancer, colorectal cancer, colon cancer, endometrial cancer, gastric cancer, head and neck squamous cell carcinoma, melanoma, lung cancer, ovarian cancer, esophageal cancer, biliary tract cancer, urothelial cancer, cervical cancer, papillary thyroid cancer, papillary renal cell carcinoma, bile duct cancer, salivary duct cancer, kidney cancer, or pancreatic cancer.

21. 21. The pharmaceutical composition of any one of claims 17 to 20, wherein the disease or disorder is breast cancer, colorectal cancer, gastric cancer, or lung cancer.

22. 16. Use of the conjugate of any one of claims 1 to 13, or a pharmaceutically acceptable salt or stereoisomer thereof, or the pharmaceutical composition of claim 14 or claim 15, in the manufacture of a medicament for activating or enhancing the activity of stimulator of interferon genes (STING) in a subject.

23. 16. Use of the conjugate of any one of claims 1 to 13, or a pharmaceutically acceptable salt or stereoisomer thereof, or the pharmaceutical composition of claim 14 or claim 15, in the manufacture of a medicament for preventing or treating a disease or disorder in a subject.

24. 24. The use of claim 23, wherein the disease or disorder is associated with agonism of STING.

25. 25. The use of claim 23 or claim 24, wherein the disease or disorder is cancer.

26. 26. The use of any one of claims 23 to 25, wherein the disease or disorder is bladder cancer, breast cancer, colorectal cancer, colon cancer, endometrial cancer, gastric cancer, head and neck squamous cell carcinoma, melanoma, lung cancer, ovarian cancer, esophageal cancer, biliary tract cancer, urothelial cancer, cervical cancer, papillary thyroid cancer, papillary renal cell carcinoma, bile duct cancer, salivary duct cancer, kidney cancer, or pancreatic cancer.

27. 27. The use of any one of claims 23 to 26, wherein the disease or disorder is breast cancer, colorectal cancer, gastric cancer, or lung cancer.

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