Anti-EDB antibodies and antibody-drug conjugates

Abstract

To provide development of further improved and optimized EDB+FN targeting therapy for patients with EDB+FN expressing disorders or diseases, such as antibody-drug conjugates.SOLUTION: The invention provides an antibody-drug conjugate comprising: (a) an antibody or antigen binding fragment thereof that binds to extra domain B (EDB) of fibronectin (FN); (b) a linker; and (c) a drug. In some aspects, the antibody-drug conjugate comprises an antibody or antigen binding fragment thereof that may comprise a heavy chain variable region comprising three CDRs comprising SEQ ID NOs: 3, 5 and 7, and a light chain variable region comprising three CDRs comprising SEQ ID NOs: 12, 13 and 14.SELECTED DRAWING: Figure 5-1

Description

[Technical Field]

[0001] The present invention relates to anti-EDB antibodies and EDB antibody-drug conjugates (ADCs). The present invention further relates to methods of using such antibodies and ADCs for the treatment of EDB+FN-expressing disorders, such as cancer. [Background technology]

[0002] Fibronectin is a high-molecular-weight adhesive glycoprotein that exists in a soluble form in plasma and other body fluids and in an insoluble form in the extracellular matrix (ECM). The extra domain B splice variant of fibronectin 1 (EDB+FN or EDB) is a non-internalized ECM protein. EDB is a 91-amino acid type III homology domain that is inserted into the fibronectin molecule by an alternative splicing mechanism at the level of the primary transcript whenever tissue remodeling occurs. EDB+FN has been shown to preferentially accumulate in the stroma surrounding new blood vessels in tumors and other pathologies but is largely absent in normal adult vasculature. Zardi et al., Embo J. 6(8):2337-42 (1987). EDB+FN is expressed in many high-grade tumors and, depending on the tumor type, exhibits either a predominantly vascular or diffuse stromal pattern of expression. Carnemolla et al., J. Cell Biol. 108(3):1139–48 (1989).

[0003] The L19 antibody, which specifically binds to the EDB domain of fibronectin (FN), has been isolated using phage display technology. Carnemolla et al., Int. J. Cancer 68(3):397-405 (1996); Neri et al., Nat. Biotechnol. 15(12):1271-5 (1997); Pini et al., J. Biol. Chem. 273(34):21769-76 (1998). The L19 antibody can stain tumor blood vessels in a wide range of experimental tumor models and on sections of human tumors and other angiogenic disorders. Carnemolla et al., J. Cell Biol. 108(3):1139-48 (1989); Kaczmarek et al., Int. J. Cancer 59(1):11-6 (1994); Berndt et al., Histochem. Cell Biol. 109(3):249-55 (1998).

[0004] Various targeting strategies have been investigated using different formats of the L19 antibody in the treatment of cancer, such as the scFv(L19) monoclonal antibody fragment, Birchler et al., Nat Biotechnol. 17:984-8 (1999), a fusion protein containing interleukin-12 (IL-12) and tumor necrosis factor (TNF-alpha) fused to scFv(L19), Halin C. et al., Cancer Res. 63(12):3202-10 (2003), and the L19 small immune protein (SIP) alone and conjugated to a photosensitizer, Fabbrini M. et al., Int J Cancer 118(7):1805-13 (2006). Summary of the Invention [Problem to be solved by the invention]

[0005] Although various L19 antibody-based therapies have been disclosed, there remains a significant clinical need for the development of further improved and optimized EDB+FN-targeted therapies, e.g., antibody-drug conjugates, for patients with EDB+FN-expressing disorders or diseases, such as cancers associated with EDB+FN expression and / or EDB+FN-expressing cancers. [Means for solving the problem]

[0006] The present invention provides antibody-drug conjugates comprising (a) an antibody or antigen-binding fragment thereof that binds to fibronectin (FN) extra domain B (EDB), (b) a linker, and (c) a drug. In some embodiments, the antibody-drug conjugate comprises an antibody or antigen-binding fragment that may comprise a heavy chain variable region comprising three CDRs comprising SEQ ID NOs: 3, 5, and 7, and a light chain variable region comprising three CDRs comprising SEQ ID NOs: 12, 13, and 14. In some embodiments, the antibody-drug conjugate comprises an antibody or antigen-binding fragment that may comprise a heavy chain variable region comprising SEQ ID NO: 1 or 21, and a light chain variable region comprising SEQ ID NO: 10.

[0007] The present invention also provides antibody-drug conjugates comprising an antibody or antigen-binding fragment, which may comprise a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO: 10; or a heavy chain variable region comprising SEQ ID NO: 21 and a light chain variable region comprising SEQ ID NO: 10. In some embodiments, the antibody-drug conjugate comprises an antibody or antigen-binding fragment comprising a heavy chain comprising SEQ ID NO: 8, 17, 19, 23, 25, 27, or 29 and a light chain comprising SEQ ID NO: 15 or 31.

[0008] The present invention also provides antibody-drug conjugates comprising an antibody or antigen-binding fragment comprising a heavy chain comprising SEQ ID NO:8 and a light chain comprising SEQ ID NO:15; a heavy chain comprising SEQ ID NO:8 and a light chain comprising SEQ ID NO:31; a heavy chain comprising SEQ ID NO:17 and a light chain comprising SEQ ID NO:15; a heavy chain comprising SEQ ID NO:17 and a light chain comprising SEQ ID NO:31; a heavy chain comprising SEQ ID NO:19 and a light chain comprising SEQ ID NO:15; a heavy chain comprising SEQ ID NO:19 and a light chain comprising SEQ ID NO:31; a heavy chain comprising SEQ ID NO:23 and a light chain comprising SEQ ID NO:15; a heavy chain comprising SEQ ID NO:23 and a light chain comprising SEQ ID NO:31; a heavy chain comprising SEQ ID NO:25 and a light chain comprising SEQ ID NO:15; a heavy chain comprising SEQ ID NO:25 and a light chain comprising SEQ ID NO:31; a heavy chain comprising SEQ ID NO:27 and a light chain comprising SEQ ID NO:15; a heavy chain comprising SEQ ID NO:27 and a light chain comprising SEQ ID NO:31; a heavy chain comprising SEQ ID NO:29 and a light chain comprising SEQ ID NO:15; or a heavy chain comprising SEQ ID NO:29 and a light chain comprising SEQ ID NO:31.

[0009] The present invention also provides antibody-drug conjugates comprising antibodies or antigen-binding fragments having heavy and / or light chain constant regions comprising an engineered cysteine ​​residue for site-specific conjugation. In some embodiments, the antibody-drug conjugate has a heavy chain constant region comprising an engineered cysteine ​​residue at position 290 (K290C) according to the EU index of Kabat numbering. In some embodiments, the antibody-drug conjugate has a light chain constant region comprising an engineered cysteine ​​residue at position 183 (κK183C) according to the Kabat numbering. In some embodiments, the antibody-drug conjugate has a heavy chain constant region comprising an engineered cysteine ​​residue at position 290 (K290C) according to the EU index of Kabat numbering, and a light chain constant region comprising an engineered cysteine ​​residue at position 183 (κK183C) according to the Kabat numbering.

[0010] The present invention further provides antibody-drug conjugates having antibodies or antigen-binding fragments comprising a heavy chain constant region comprising an engineered glutamine-containing tag inserted into the antibody or replacing one or more endogenous amino acids in the antibody. In some embodiments, the antibody-drug conjugate has an engineered glutamine-containing tag inserted into the antibody at positions E294 to N297. In some embodiments, the antibody-drug conjugate has a glutamine-containing tag comprising the amino acid sequence LLQG (SEQ ID NO: 40). In some embodiments, the antibody-drug conjugate has a heavy chain constant region further comprising an arginine (R) substituting a lysine (K) at position 222 (K222R) according to the EU index of Kabat numbering.

[0011] The present invention also provides an antibody-drug conjugate having an antibody or antigen-binding fragment comprising a heavy chain variable region comprising an arginine (R) substituting lysine (K) at position 94 (K94R) according to Kabat numbering.

[0012] The present invention further provides antibody-drug conjugates having a linker that is a cleavable linker. In some embodiments, the cleavable linker is selected from the group consisting of vc, diS, diS-COCO, and AcLys-vc.

[0013] The present invention further provides antibody-drug conjugates having a drug that is a cytotoxic agent. In some embodiments, the cytotoxic agent is an auristatin. In some embodiments, the auristatin is selected from the group consisting of 0101, 1569, 9411, and 4574. In some embodiments, the cytotoxic agent is a CPI dimer. In some embodiments, the CPI dimer is CPI-8314 or CPI-0326.

[0014] The present invention also provides an antibody-drug conjugate comprising (a) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising three CDRs comprising SEQ ID NOs: 3, 5, and 7 and a light chain variable region comprising three CDRs comprising SEQ ID NOs: 12, 13, and 14; (b) a vc linker; and (c) an O101 drug.

[0015] The present invention also provides an antibody-drug conjugate comprising: (a) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO: 21 and a light chain variable region comprising SEQ ID NO: 10; (b) a vc linker; and (c) an O101 drug.

[0016] The present invention also provides an antibody-drug conjugate comprising: (a) an antibody or antigen-binding fragment thereof comprising a heavy chain comprising SEQ ID NO: 25 and a light chain comprising SEQ ID NO: 31; (b) a vc linker; and (c) an O101 drug.

[0017] The present invention further provides a pharmaceutical composition comprising an antibody-drug conjugate of the present invention and a pharmaceutically acceptable carrier. The present invention also provides a composition comprising a plurality of antibody-drug conjugates of the present invention and, optionally, a pharmaceutical carrier, wherein the composition has an average DAR ranging from 3 to 5. The present invention also provides a composition comprising a plurality of antibody-drug conjugates according to any one of claims 1 to 25 and, optionally, a pharmaceutical carrier, wherein the composition has an average DAR ranging from 1 to 3.

[0018] The present invention provides nucleic acids encoding the heavy or light chain of an antibody of the present invention. In some embodiments, the nucleic acid may comprise SEQ ID NO: 9, 18, 20, 24, 26, 28, or 30 encoding the heavy chain, or SEQ ID NO: 16 or 32 encoding the light chain. The present invention further provides vectors comprising any of the nucleic acids of the present invention. The present invention also provides host cells comprising any of the nucleic acids of the present invention.

[0019] The present invention provides a process for producing an antibody-drug conjugate of the present invention, comprising the steps of: (a) linking a linker to a drug; (b) conjugating the linker and drug to the antibody; and (c) purifying the antibody-drug conjugate. In some aspects, the conjugating is site-specific on one or more engineered cysteine ​​and / or glutamine residues on the antibody.

[0020] The present invention also provides a method for treating an EDB+FN-expressing disorder or disease, the method comprising administering an effective amount of a composition comprising an antibody-drug conjugate of the present invention to a subject in need thereof. In some embodiments, the EDB+FN-expressing disorder or disease is cancer. In some embodiments, the cancer is a solid tumor or a blood cancer. In some embodiments, the solid tumor is thyroid cancer, sarcoma, breast cancer, pancreatic cancer, glioblastoma, gallbladder cancer, kidney cancer, skin cancer, uterine cancer, mesothelioma, colorectal cancer, head and neck cancer, ovarian cancer, bladder cancer, testicular cancer, prostate cancer, liver cancer, endocrine cancer, thymic cancer, brain tumor, adrenal cancer, eye cancer, cervical cancer, and lung cancer. In some embodiments, the blood cancer is leukemia, lymphoma, or myeloma.

[0021] The present invention further provides use of the antibody-drug conjugate of the present invention in the manufacture of a medicament for treating an EDB+FN-expressing disorder or disease in a subject. In some embodiments, the EDB+FN-expressing disorder or disease is cancer. In some embodiments, the cancer is a solid tumor or a blood cancer. In some embodiments, the solid tumor is thyroid cancer, sarcoma, breast cancer, pancreatic cancer, glioblastoma, gallbladder cancer, kidney cancer, skin cancer, uterine cancer, mesothelioma, colorectal cancer, head and neck cancer, ovarian cancer, bladder cancer, testicular cancer, prostate cancer, liver cancer, endocrine cancer, thymic cancer, brain tumor, adrenal cancer, eye cancer, cervical cancer, and lung cancer. In some embodiments, the blood cancer is leukemia, lymphoma, or myeloma. In particular embodiments, for example, the following items are provided: (Item 1) An antibody-drug conjugate comprising: (a) an antibody or antigen-binding fragment thereof that binds to the extra domain B of fibronectin; (b) a linker; and (c) a drug. (Item 2) 2. The antibody-drug conjugate of item 1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising three CDRs comprising SEQ ID NOs: 3, 5, and 7, and a light chain variable region comprising three CDRs comprising SEQ ID NOs: 12, 13, and 14. (Item 3) 3. The antibody-drug conjugate of any one of items 1 to 2, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising SEQ ID NO: 1 or 21 and a light chain variable region comprising SEQ ID NO: 10. (Item 4) the antibody or antigen-binding fragment: a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO: 10; or A heavy chain variable region comprising SEQ ID NO: 21 and a light chain variable region comprising SEQ ID NO: 10 4. The antibody-drug conjugate of any one of items 1 to 3, comprising: (Item 5) 5. The antibody-drug conjugate of any one of items 1 to 4, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising SEQ ID NO: 8, 17, 19, 23, 25, 27 or 29 and a light chain comprising SEQ ID NO: 15 or 31. (Item 6) the antibody or antigen-binding fragment: a heavy chain comprising SEQ ID NO:8 and a light chain comprising SEQ ID NO:15; a heavy chain comprising SEQ ID NO:8 and a light chain comprising SEQ ID NO:31; a heavy chain comprising SEQ ID NO: 17 and a light chain comprising SEQ ID NO: 15; a heavy chain comprising SEQ ID NO: 17 and a light chain comprising SEQ ID NO: 31; a heavy chain comprising SEQ ID NO: 19 and a light chain comprising SEQ ID NO: 15; a heavy chain comprising SEQ ID NO: 19 and a light chain comprising SEQ ID NO: 31; a heavy chain comprising SEQ ID NO: 23 and a light chain comprising SEQ ID NO: 15; a heavy chain comprising SEQ ID NO: 23 and a light chain comprising SEQ ID NO: 31; a heavy chain comprising SEQ ID NO: 25 and a light chain comprising SEQ ID NO: 15; a heavy chain comprising SEQ ID NO: 25 and a light chain comprising SEQ ID NO: 31; a heavy chain comprising SEQ ID NO: 27 and a light chain comprising SEQ ID NO: 15; a heavy chain comprising SEQ ID NO: 27 and a light chain comprising SEQ ID NO: 31; a heavy chain comprising SEQ ID NO:29 and a light chain comprising SEQ ID NO:15; or A heavy chain comprising SEQ ID NO: 29 and a light chain comprising SEQ ID NO: 31 6. The antibody-drug conjugate of any one of items 1 to 5, comprising: (Item 7) 2. The antibody-drug conjugate of item 1, wherein the antibody or antigen-binding fragment comprises a heavy chain constant region and / or a light chain constant region comprising an engineered cysteine ​​residue for site-specific conjugation. (Item 8) 8. The antibody-drug conjugate of item 7, wherein the heavy chain constant region comprises an engineered cysteine ​​residue at position 290 (K290C) according to the EU index of Kabat numbering. (Item 9) 8. The antibody-drug conjugate of item 7, wherein the light chain constant region comprises an engineered cysteine ​​residue at position 183 according to Kabat numbering (κK183C). (Item 10) 2. The antibody-drug conjugate of item 1, wherein the heavy chain constant region comprises an engineered cysteine ​​residue at position 290 according to EU index of Kabat numbering (K290C), and the light chain constant region comprises an engineered cysteine ​​residue at position 183 according to Kabat numbering (κK183C). (Item 11) 2. The antibody-drug conjugate of item 1, wherein the antibody or antigen-binding fragment comprises a heavy chain constant region comprising an engineered glutamine-containing tag inserted into or replacing one or more endogenous amino acids in the antibody. (Item 12) 12. The antibody-drug conjugate of item 11, wherein the engineered glutamine-containing tag is inserted into the antibody at positions E294 to N297. (Item 13) 13. The antibody-drug conjugate of item 12, wherein the glutamine-containing tag comprises the amino acid sequence LLQG (SEQ ID NO: 40). (Item 14) 12. The antibody-drug conjugate of item 11, wherein the heavy chain constant region further comprises an arginine (R) substituting a lysine (K) at position 222 (K222R) according to the EU index of Kabat numbering. (Item 15) 2. The antibody-drug conjugate of item 1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an arginine (R) substituting a lysine (K) at position 94 (K94R) according to the Kabat numbering system. (Item 16) 16. The antibody-drug conjugate of any one of items 1 to 15, wherein the linker is a cleavable linker. (Item 17) 17. The antibody-drug conjugate of item 16, wherein the cleavable linker is selected from the group consisting of vc, diS, diS-COCO, and AcLys-vc. (Item 18) 18. The antibody-drug conjugate of any one of items 1 to 17, wherein the drug is a cytotoxic agent. (Item 19) 19. The antibody-drug conjugate of item 18, wherein the cytotoxic agent is an auristatin. (Item 20) 20. The antibody-drug conjugate of item 19, wherein the auristatin is selected from the group consisting of 0101, 1569, 9411 and 4574. (Item 21) 19. The antibody-drug conjugate of any one of items 1 to 18, wherein the cytotoxic agent is a CPI dimer. (Item 22) 22. The antibody-drug conjugate of item 21, wherein the CPI dimer is CPI-8314 or CPI-0326. (Item 23) An antibody or antigen-binding fragment thereof comprising: (a) a heavy chain variable region comprising three CDRs comprising SEQ ID NOs: 3, 5, and 7, and a light chain variable region comprising three CDRs comprising SEQ ID NOs: 12, 13, and 14; (b) a vc linker; and (c) an O101 drug. (Item 24) (a) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO: 21 and a light chain variable region comprising SEQ ID NO: 10; (b) a vc linker; and (c) an antibody-drug conjugate comprising an O101 drug. (Item 25) (a) an antibody or antigen-binding fragment thereof comprising a heavy chain comprising SEQ ID NO: 25 and a light chain comprising SEQ ID NO: 31; (b) a vc linker; and (c) an antibody-drug conjugate comprising an O101 drug. (Item 26) 26. A pharmaceutical composition comprising the antibody-drug conjugate of any one of items 1 to 25 and a pharmaceutically acceptable carrier. (Item 27) 26. A composition comprising a plurality of antibody-drug conjugates according to any one of items 1 to 25 and optionally a pharmaceutical carrier, wherein the composition has an average DAR ranging from 3 to 5. (Item 28) 26. A composition comprising a plurality of antibody-drug conjugates according to any one of items 1 to 25 and optionally a pharmaceutical carrier, wherein the composition has an average DAR ranging from 1 to 3. (Item 29) 26. A nucleic acid encoding the heavy or light chain of the antibody of any one of items 1 to 25. (Item 30) A nucleic acid of any of SEQ ID NOs: 9, 18, 20, 24, 26, 28 or 30 encoding a heavy chain, or any of SEQ ID NOs: 16 or 32 encoding a light chain. (Item 31) A vector comprising the nucleic acid of any one of items 29 to 30. (Item 32) A host cell comprising the nucleic acid of any one of items 29 to 30. (Item 33) 26. A process for producing the antibody-drug conjugate of any one of items 1 to 25, comprising: (a) linking the linker to the drug; (b) conjugating the linker and drug to the antibody; and (c) purifying the antibody-drug conjugate A process involving: (Item 34) 34. The process of claim 33, wherein the conjugating step is site-specific on one or more engineered cysteine ​​and / or glutamine residues on the antibody. (Item 35) 26. A method for treating an EDB+FN-expressing disorder or disease, comprising administering to a subject in need thereof an effective amount of a composition comprising the antibody-drug conjugate of any one of items 1 to 25. (Item 36) 36. The method of item 35, wherein the EDB+FN-expressing disorder or disease is cancer. (Item 37) 37. The method of claim 36, wherein the cancer is a solid tumor or a blood cancer. (Item 38) 38. The method of item 37, wherein the solid tumor is thyroid cancer, sarcoma, breast cancer, pancreatic cancer, glioblastoma, gallbladder cancer, kidney cancer, skin cancer, uterine cancer, mesothelioma, colorectal cancer, head and neck cancer, ovarian cancer, bladder cancer, testicular cancer, prostate cancer, liver cancer, endocrine cancer, thymic cancer, brain tumor, adrenal cancer, eye cancer, cervical cancer, or lung cancer. (Item 39) 37. The method of claim 36, wherein the blood cancer is leukemia, lymphoma, or myeloma. (Item 40) 26. Use of the antibody-drug conjugate of any one of items 1 to 25 in the manufacture of a medicament for the treatment of an EDB+FN-expressing disorder or disease in a subject. (Item 41) 41. The use of item 40, wherein the EDB+FN-expressing disorder or disease is cancer. (Item 42) 42. The use of item 41, wherein the cancer is a solid tumor or a blood cancer. (Item 43) 43. The use according to item 42, wherein the solid tumor is thyroid cancer, sarcoma, breast cancer, pancreatic cancer, glioblastoma, gallbladder cancer, kidney cancer, skin cancer, uterine cancer, mesothelioma, colorectal cancer, head and neck cancer, ovarian cancer, bladder cancer, testicular cancer, prostate cancer, liver cancer, endocrine cancer, thymic cancer, brain tumor, adrenal cancer, eye cancer, cervical cancer and lung cancer. (Item 44) 44. The use of item 43, wherein the blood cancer is leukemia, lymphoma or myeloma. [Brief explanation of the drawings]

[0022] [Figure 1] 1A and 1B show the binding characteristics of [A] EDB-L19, EDB-PFE, and EDB-(K94R) antibodies; and [B] EDB-(K94R) and EDB-(κK183C-K94R-290C) antibodies. [Figure 2] Figure 2 shows EDB+FN expression using RNA-Seq analysis in human patient-derived xenograft (PDX) cancer models. [Figure 3] 3A and 3B show ELISA binding curves for [A] EDB-L19 antibody and EDB-L19-vc-0101 ADC and EDB-(κK183C-K94R-290C) antibody and EDB-(κK183C-K94R-290C)-vc-0101 ADC; and [B] EDB-(K94R) antibody and EDB-(K94R)-vc-0101 ADC and EDB-(κK183C-K290C) antibody and EDB-(κK183C-K290C)-vc0101 ADC. [Figure 4] FIG. 4 shows EDB+FN expression by Western blot in WI38-VA13 and HT-29 cells. [Figure 5-1] Figure 5A [Figure 5-2] Figures 5B and 5C [Figure 5-3] Figure 5D [Figure 5-4]Figures 5E, 5F: Figures 5A-5F show tumor growth inhibition curves for each individual tumor-bearing mouse, showing the site-specific results for [A] 0.3, 0.75, 1.5, and 3 mg / kg EDB-L19-vc-0101; [B] 3 mg / kg EDB-L19-vc-0101 and 10 mg / kg disulfide-linked EDB-L19-diS-DM1; [C] 1 and 3 mg / kg EDB-L19-vc-0101 and 5 mg / kg disulfide-linked EDB-L19-diS-COCO-1569; and [D] 0.3, 1 and 3 mg / kg and 1.5 mg / kg doses, respectively. [E] Site-specifically conjugated EDB-(κK183C-K94R-K290C)-vc-0101 at doses of 0.3, 1, and 3 mg / kg; and [F] EDB-(κK183C-K94R-K290C)-vc-0101 dosed at 3 mg / kg are shown to be effective in PDX-NSX-11122, a patient-derived xenograft (PDX) model of human cancer, NSCLC, with high EDB+FN expression. [Figure 6-1] Figure 6A [Figure 6-2] Figures 6B and 6C [Figure 6-3] Figure 6D [Figure 6-4]Figures 6E, 6F: Figures 6A-6F show: [A] EDB-L19-vc-0101 at 0.3, 0.75, 1.5, and 3 mg / kg; [B] EDB-L19-vc-0101 and EDB-L19-vc-1569 at 0.3, 1, and 3 mg / kg; [C] EDB-L19-vc-0101 and EDB-(H16-K222R)-AcLys-vc-CPI-8314 at 0.5, 1.5, and 3 mg / kg and 0.1, 0.3, and 1 mg / kg, respectively; [D] Site-specifically conjugated EDB- Figure 1 shows the antitumor efficacy of (κK183C+K290C)-vc-0101 and conventionally conjugated EDB-L19-vc-0101; [E] 1 and 3 mg / kg EDB-L19-vc-0101 and EDB-(K94R)-vc-0101; and [F] 1 and 3 mg / kg EDB-(κK183C+K290C)-vc-0101 and EDB-(κK183C-K94R-K290C)-vc-0101 in H-1975, a moderate-to-high EDB+FN-expressing NSCLC cell line xenograft (CLX) model of human cancer. [Figure 7] FIG. 7 shows the antitumor efficacy of 3 mg / kg EDB-L19-vc-0101 and EDB-L19-vc-9411 in HT29, a moderately EDB+FN-expressing colon CLX model of human cancer. [Figure 8] Figures 8A and 8B show the antitumor efficacy of 0.3, 1, and 3 mg / kg EDB-L19-vc-0101 in [A] PDX-PAX-13565, a moderate-to-high EDB+FN-expressing pancreatic PDX; and [B] PDX-PAX-12534, a low-to-moderate EDB+FN-expressing pancreatic PDX. [Figure 9] FIG. 9 shows the antitumor efficacy of 1 and 3 mg / kg EDB-L19-vc-0101 in Ramos, a moderately EDB+FN-expressing lymphoma CLX model of human cancer. [Figure 10]Figures 10A and 10B show the antitumor efficacy in EMT-6, a mouse syngeneic breast cancer model, of [A] 4.5 mg / kg EDB-(κK183C-K94R-K290C)-vc-0101; and [B] the EDB-(κK183C-K94R-K290C)-vc-0101 group dosed at 4.5 mg / kg as tumor growth inhibition curves for each individual tumor-bearing mouse. [Figure 11] FIG. 11 shows absolute neutrophil counts for 5 mg / kg conventionally conjugated EDB-L19-vc-0101 compared to 6 mg / kg site-specifically conjugated EDB-(κK183C-K94R-K290C)-vc-0101 (ADC4). DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention provides antibodies and antibody-drug conjugates (ADCs) that bind to the extra domain B (EDB) of fibronectin (FN), interchangeably referred to as "EDB+FN" or "EDB." The present invention also provides processes for preparing ADCs using anti-EDB antibodies, linkers, and drugs (payloads). The present invention further provides ADCs produced using conventional and / or site-specific conjugation technologies. The antibodies and ADCs of the present invention are useful in the preparation and manufacture of compositions, such as medicaments, that can be used in the diagnosis, prevention, and / or treatment of hyperproliferative disorders characterized by or associated with EDB+FN expression, e.g., cancer. ... Nucleic acids encoding anti-EDB antibodies for use in generating ADCs are also provided.

[0024] ADCs typically comprise an antibody component conjugated to a drug via the use of a linker. ADCs produced by conventional conjugation technology randomly link drugs to antibodies via endogenously present lysine or cysteine ​​residues on the antibody heavy and / or light chains. Thus, such ADCs are heterogeneous mixtures of species with different drug:antibody ratios (DARs). ADCs produced by site-specific conjugation technology link drugs to antibodies at specific engineered residues on the antibody heavy and / or light chains. Thus, site-specifically conjugated ADCs are homogeneous mixtures of ADCs composed of species with defined drug:antibody ratios (DARs). Thus, site-specifically conjugated ADCs demonstrate uniform stoichiometry, resulting in improved pharmacokinetics, biodistribution, and safety profiles.

[0025] The ADCs of the invention comprise an anti-EDB antibody conjugated to one or more drugs via a linker (i.e., forming a linker-drug moiety). The invention provides ADCs having (a) an antibody or antigen-binding fragment thereof that binds to EDB; (b) a linker; and (c) a drug. The invention further provides ADCs of the formula Ab-(LD), where (a) Ab is an antibody or antigen-binding fragment thereof that binds to EDB, (b) LD is a linker-drug moiety, L is a linker, and D is a drug. In another aspect, the invention provides ADCs of the formula Ab-(LD)p, where (a) Ab is an antibody or antigen-binding fragment thereof that binds to EDB, (b) LD is a linker-drug moiety, L is a linker, D is a drug, and (c) p is the number of linker-drug moieties attached to the antibody.

[0026] The number of linker-drug moieties attached to an antibody can be any number preferred for ADC development. In some embodiments, the number of linker-drug moieties per antibody is 4. In other embodiments, the number of linker-drug moieties per antibody is 3. In another embodiment, the number of linker-drug moieties per antibody is 2. In another embodiment, the number of linker-drug moieties per antibody is 1. In other embodiments, the number of linker-drug moieties per antibody is greater than 4, e.g., 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 linker-drug moieties per antibody.

[0027] Furthermore, the present invention provides ADCs in which the linker-drug moiety is attached to the antibody via conventional or site-specific conjugation technology. In some embodiments, the anti-EDB antibody or its antigen-binding fragment is conjugated or linked to a drug, such as a cytotoxic agent, a cytostatic agent, and / or a therapeutic agent, as further described herein. For example, a cytotoxic agent can be linked or linked to the anti-EDB antibody described herein for targeted, localized delivery of the cytotoxic agent. Methods for preparing and manufacturing such ADCs, as well as their use in clinical applications, are also provided.

[0028] In contrast to other ADCs developed to target internalized cell surface-expressed proteins, the ADCs of the present invention target EDB, a protein expressed in the extracellular matrix (ECM). Targeting proteins expressed in the ECM may offer advantages over targeting proteins expressed on tumor cells. ADCs can directly access their targets without the need to penetrate the stromal and ECM barriers common in many difficult-to-treat human cancers. Furthermore, targeting EDB in the ECM with ADCs provides a specific mechanism for accessing many difficult-to-target cell types in the tumor microenvironment. This can result in the extracellular release of cytotoxic payloads or drugs, resulting in the killing of various cells through mechanisms such as cell death / cell cycle arrest of tumor cells and / or stromal cells by bystander mechanisms. Furthermore, additional mechanisms include, but are not limited to, dysregulated angiogenesis or cytotoxic vascular targeting / collapse, vascular normalization, immune modulation, and induction of cell differentiation and / or disruption of epithelial-mesenchymal transition.

[0029] The examples provided herein demonstrate improved characteristics obtained during anti-EDB antibody and EDB ADC production, such as allotype optimization to reduce immunogenicity, removal of COOH-terminal lysine to increase product homogeneity, and introduction of mutations to mitigate potential glycosylation tendency and reduce heterogeneity (see Examples 1 and 2). Additionally, as shown in the examples, EDB ADCs produced using a variety of conventional and site-specific conjugation technologies (i.e., cysteine, lysine, and / or acyl donor glutamine-containing ("Q") tags) and a variety of linker-drug moieties. The ADCs have demonstrated robust in vitro and in vivo efficacy (see Examples 6-8). The examples provided herein also show that EDB ADCs generated using site-specific conjugation via engineered cysteine ​​residues demonstrated improved characteristics compared to EDB ADCs generated using conventional conjugation via cysteine ​​residues, such as improved pharmacokinetic (PK) profiles (i.e., increased exposure and conjugation stability resulting in fewer off-target toxic effects), favorable thermal stability, and profiled nonclinical safety (i.e., reduced myelosuppression) (see Examples 9, 10, and 11, respectively). Furthermore, the improved characteristics of EDB ADCs generated using site-specific conjugation technology may enable higher dosages in human treatment, thus providing increased efficacy. In some embodiments, these EDB ADCs may comprise a substitution of a lysine (K) at position 290 (according to EU index of Kabat) in the human IgG1 heavy chain constant region with a reactive cysteine ​​(C) (K290C), and / or a substitution of a lysine (K) at position 183 (according to Kabat) in the human kappa light chain constant region with a reactive cysteine ​​(C) (κK183C) to allow for site-specific conjugation.

[0030] Fibronectin extra domain B As used herein, "EDB+FN" and "EDB" are used interchangeably to refer to fibronectin (FN) containing extra domain B (EDB). Furthermore, "anti-EDB antibody" and "anti-EDB+FN antibody" are used interchangeably to refer to an antibody that binds to EDB. "Anti-EDB antibody-drug conjugate," "EDB antibody-drug conjugate," "anti-EDB ADC," and "EDB ADC" are used interchangeably to refer to an ADC that binds to EDB and contains an antibody or its antigen-binding fragment conjugated or linked to a drug. FN is a high-molecular-weight glycoprotein present in the extracellular matrix (ECM) and is involved in cell adhesion and migration processes, including embryonic development, wound healing, blood coagulation, host defense, and metastasis. FN typically exists as a dimer formed by two nearly identical, approximately 250 kDa, subunits covalently linked near their C-termini by a pair of disulfide bonds. Each monomer consists of three types of repeating units: type I, type II, and type III FN repeats. Although a single 75-kb gene encodes FN, 20 protein variants are observed in humans. Alternative splicing of the FN gene occurs in three regions, resulting in the inclusion or exclusion of one of two type III repeats, termed extra domain A (EDA) and extra domain B (EDB), as well as the segment connecting two other type III repeats, termed the type III connecting segment (IIICS). EDB is a 91-amino acid sequence that is 100% identical in mouse, rat, rabbit, dog, cynomolgus monkey, and human. A representative EDB+FN nucleotide sequence is provided under accession number NM_001306129.1, and the corresponding amino acid sequence is provided under accession number NP_001293058.1. The amino acid sequences of EDB and recombinant human 7-EDB-8-9 are provided in Table 1. Recombinant human 7-EDB-8-9 contains EDB flanked at the amino terminus by domain 7 (underlined) and at the carboxy terminus by domain 8 (boxed) and domain 9 (italicized).

[0031] [Table 1]

[0032] Anti-EDB antibody The antibody of the present invention specifically binds to EDB. For the preparation of the ADC of the present invention, the antibody or its antigen-binding fragment can be any antibody (including the antibodies described herein) or its antigen-binding fragment that specifically binds to EDB. This antibody or its antigen-binding fragment can be isolated, purified, or derivatized for use in the preparation of EDB ADC.

[0033] As used herein, "antibody" or "Ab" refers to an immunoglobulin molecule capable of recognizing and binding to a specific target or antigen, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. The term includes monoclonal antibodies, polyclonal antibodies, "antigen-binding fragments" (or portions), such as Fab, Fab', F(ab')2, Fd, Fv, Fc, etc., of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., EDB), isolated complementarity-determining regions (CDRs), bispecific antibodies, heteroconjugate antibodies, variants thereof, fusion proteins comprising an antibody or antigen-binding fragment thereof (e.g., domain antibodies), single-chain (ScFv) and single-domain antibodies (e.g., shark and camelid antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (e.g., Hollige The antibodies may encompass any type of antibody, including, but not limited to, antibodies such as, but not limited to, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. These antibodies may be murine, rat, human, or of any other origin (including chimeric or humanized antibodies). In some embodiments of the present invention, the antibodies or antigen-binding fragments of the disclosed EDB ADCs are chimeric, humanized, or recombinant human antibodies, or EDB-binding fragments thereof.

[0034] Native or naturally occurring antibodies and native immunoglobulins are typically heterotetrameric glycoproteins of approximately 150,000 daltons composed of two identical light chains (LC) and two identical heavy chains (HC). Each heavy chain has a variable domain (VH) followed by several constant domains or regions (e.g., hinge, CH1, CH2, or CH3) called "CH domains." Each light chain has a variable domain (VL) and a constant domain called a "CL domain." The term "constant region" or "constant domain" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination. These constant domains are not directly involved in binding the antibody to an antigen but exhibit various effector functions, such as Fc receptor (FcR) binding, antibody participation in antibody-dependent cellular cytotoxicity (ADCC), opsonization, initiation of complement-dependent cytotoxicity, and mast cell degranulation. The constant region of the EDB antibody may be derived from the constant region of any one of IgA, IgD, IgE, IgG, IgM, any isotype thereof (e.g., IgG of IgG1, IgG2, IgG3, or IgG4 isotype), and subclasses and mutated versions thereof.

[0035] The CH1 domain comprises, for example, the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain, extending from about positions 118 to 215 according to the EU index of Kabat. The CH1 domain is adjacent to the VH domain of an immunoglobulin heavy chain molecule, amino-terminal to the hinge region, and does not form part of the Fc region of the immunoglobulin heavy chain.

[0036] The hinge region comprises the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains.

[0037] The CH2 domain comprises, for example, the portion of a heavy chain immunoglobulin molecule extending from about positions 231 to 340 according to the EU index of Kabat. The CH2 domain is unique in that it is not tightly paired with another domain. Rather, two N-linked branched carbohydrate chains are located between the two CH2 domains in an intact native IgG molecule. In some embodiments, the antibody (or fragment thereof) of the present invention comprises a CH2 domain derived from an IgG molecule, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the IgG is a human IgG.

[0038] The CH3 domain comprises the portion of a heavy chain immunoglobulin molecule extending approximately 110 residues from the C-terminus of the CH2 domain, e.g., from about positions 341 to 447 according to the EU index of Kabat. The CH3 domain typically forms the C-terminal portion of the antibody. However, in some immunoglobulins, additional domains may extend from the CH3 domain to form the C-terminal portion of the molecule (e.g., the CH4 domain in the μ chain of IgM and the ε chain of IgE). In some embodiments, the antibody (or fragment thereof) of the present invention comprises a CH3 domain derived from an IgG molecule, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the IgG is a human IgG.

[0039] The CL domain comprises, for example, the constant region domain of an immunoglobulin light chain extending from about position 108 to 214 according to the EU index of Kabat. The CL domain is adjacent to the VL domain. In some embodiments, the antibodies (or fragments thereof) of the present invention comprise a kappa light chain constant domain (CLκ). In some embodiments, the antibodies (or fragments thereof) comprise a lambda light chain constant domain (CLλ). CLκ has known polymorphic loci CLκ-V / A45 and CLκ-L / V83 (using Kabat numbering), thus allowing for the polymorphisms Km(1):CLκ-V45 / L83; Km(1,2):CLκ-A45 / L83; and Km(3):CLκ-A45 / V83. The polypeptides, antibodies, and ADCs of the present invention may have antibody components having any of these light chain constant regions.

[0040] The Fc region generally comprises a CH2 domain and a CH3 domain. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually defined to stretch from the amino acid residue at position Cys226, or from position Pro230 (according to the EU index of Kabat), to the carboxyl terminus. The Fc region may be a native sequence Fc region or a variant Fc region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0041] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As is known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held in close proximity by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody.

[0042] The CDRs of the variable domains may be defined according to the Kabat definition (Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington DC), Chothia (Chothia et al., Nature 342:877-883, (1989)), a combination of both Kabat and Chothia, the AbM definition (derived using Oxford Molecular's AbM antibody modeling software (now Accelrys®)), the contact definition (based on observed antigen contacts as set forth in MacCallum et al., J. Mol. Biol., 262:732-745, (1996)) and / or the conformational definition (Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008), or any method of CDR determination known in the art. As used herein, CDRs may refer to CDRs defined by any approach known in the art, including a combination of approaches. For the present invention, the CDRs shown in Table 2 below were derived using the definitions of Kabat and Chothia. The anti-EDB antibodies or antigen-binding fragments thereof of the present invention comprise one or more CDRs (e.g., one, two, three, four, five, or all six CDRs).

[0043] Antibodies, ADCs, or polypeptides that "specifically bind" or "preferentially bind" (used interchangeably herein) to a target or antigen (e.g., an EDB protein) are terms well understood in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance more frequently, more rapidly, with a longer duration, and / or with a higher affinity than with alternative cells or substances. An antibody "specifically binds" or "preferentially binds" to a target or antigen if it binds with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to an EDB epitope is one that binds this epitope with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other EDB epitopes or non-EDB epitopes.

[0044] The term "binding affinity" or "K D " as used herein is intended to refer to the equilibrium dissociation constant of a particular antigen-antibody interaction. D is the rate of association, i.e., the "on-rate" or "k a "off-rate" or "k d Also called is the ratio of the dissociation rates observed. D is k d / k a and is expressed as molar concentration (M). D The smaller the K, the stronger the binding affinity. D has a K of 1 nM D The K for the antibody shows weaker binding affinity compared to the D The K value can be determined using methods well established in the art. DOne method for determining is by using surface plasmon resonance, typically using a biosensor system such as a BIAcore® system. Other standard assays for assessing the binding ability of a ligand, such as an antibody, to a target are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometry analysis.

[0045] An "isolated antibody," as used herein, refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds EDB is substantially free of antibodies that specifically bind antigens other than EDB). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals. It is also understood by reading this definition that, for example, an antibody (or portion or epitope) that specifically or preferentially binds a first target may or may not specifically or preferentially bind a second target.

[0046] In some embodiments of the invention, the EDB ADC comprises an antibody that competes with an antibody or antigen-binding fragment thereof described herein for binding to human EDB and / or an antibody that binds the same epitope as an antibody or antigen-binding fragment thereof described herein.

[0047] As used herein, the term "compete" with respect to antibodies means that a first antibody or antigen-binding fragment thereof binds to an epitope in a manner sufficiently similar to that of a second antibody or antigen-binding fragment thereof such that the result of binding of the first antibody to its cognate epitope is detectably reduced in the presence of the second antibody, compared to binding of the first antibody in the absence of the second antibody. The option may, but need not, be true that binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody. That is, a first antibody can inhibit binding of a second antibody to its epitope without the second antibody inhibiting binding of the first antibody to its respective epitope. However, if each antibody detectably inhibits binding of the other antibody to its cognate epitope or ligand, whether to the same extent, a greater extent, or a lesser extent, these antibodies are said to "cross-compete" with each other for binding of their respective epitope(s). Both competing and cross-competing antibodies are encompassed by the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), one of skill in the art will understand, based on the teachings provided herein, that such competing and / or cross-competing antibodies are encompassed and may be useful in the methods disclosed herein.

[0048] The "L19" antibody, also referred to herein as the "EDB-L19" antibody, is a human antibody that binds EDB. The L19 antibody is disclosed and characterized in PCT International Patent Application Publication Nos. WO1997 / 045544, WO1999 / 058570, and WO2001 / 062800, which are incorporated by reference in their entireties, and the L19-EDB sequence is provided herein in Table 2 (SEQ ID NOS: 1-16).

[0049] In some embodiments of the present invention, the antibody used to prepare the EDB ADC may be a monoclonal antibody. The term "monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are directed against a single antigenic site and are highly specific. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, but should not be construed as requiring production of the antibody by any particular method.

[0050] In some embodiments of the invention, antibodies used to prepare the ADCs of the invention may be monovalent, i.e., have one antigen-binding site per molecule (e.g., IgG or Fab). In some cases, a monovalent antibody may have more than one antigen-binding site, but these binding sites are from different antibodies. In some embodiments of the invention, the antibodies or antigen-binding fragments thereof of the ADCs of the invention may comprise "bivalent antibodies," i.e., have two antigen-binding sites per molecule (e.g., IgG). In some cases, these two binding sites have the same antigen specificity. Alternatively, a bivalent antibody may be bispecific. A "bispecific," "dual-specific," or "bifunctional" antibody is one that binds two different antigens. A bispecific antibody is a hybrid antibody having two antigen-binding sites, each of which binds to two different epitopes that may be present on the same or different protein targets.

[0051] The term "chimeric antibody" is intended to refer to an antibody in which some or all of the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., an antibody in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0052] As used herein, "humanized" or "CDR-grafted" antibodies refer to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from one or more CDRs of the recipient are replaced by residues from one or more CDRs of a non-human species (donor antibody), e.g., mouse, rat, or rabbit, having the desired specificity, affinity, and capacity.

[0053] Antibodies of the present invention can be produced using techniques well known in the art, such as recombinant technology, phage display technology, synthetic technology, or a combination of such technologies or other technologies readily known in the art (see, e.g., Jayasena, SD, Clin. Chem., 45:1628-50 (1999) and Fellouse, FA et al., J. MoI. Biol., 373(4):924-40 (2007)). Further guidance can be found in Sambrook J. and Russell D. Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003).

[0054] The nucleic acids encoding the heavy and light chains of the antibody used to prepare the ADC of the present invention can be cloned into a vector for expression or expansion.The sequence encoding the antibody of interest can be maintained in a vector in a host cell, and then the host cell can be expanded and frozen for further use.The production of recombinant monoclonal antibodies in cell culture can be carried out by cloning antibody genes from B cells by means known in the art.See, for example, Tiller et al., J.Immunol.Methods 329:112-124, 2008; U.S. Patent No. 7,314,622.

[0055] As used herein, the term " vector " refers to the construct that can deliver one or more genes or sequences of interest, and preferably express them in host cell.Examples of vector include but are not limited to virus vector, naked DNA or RNA expression vector, plasmid, cosmid or phage vector, DNA or RNA expression vector that is associated with cationic condensing agent, DNA or RNA expression vector that is encapsulated in liposome, and certain eukaryotic cells, such as production cells.

[0056] As used herein, the term "host cell" includes an individual cell or cell culture that can be or has been a recipient of a vector(s) for incorporation of a polynucleotide insert. A host cell includes the progeny of a single host cell, which progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of the invention.

[0057] As known in the art, "polynucleotide," "nucleic acid / nucleotide," and "oligonucleotide" are used interchangeably herein and include polymeric forms of any length of nucleotides, either deoxyribonucleotides or ribonucleotides, their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. Polynucleotides can be naturally occurring, synthetic, recombinant, or any combination thereof.

[0058] For all heavy chain constant region amino acid positions discussed herein, numbering follows the Eu index first described in Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85, which describes the amino acid sequence of myeloma protein Eu, the first human IgG1 to be sequenced. The Eu index of Edelman et al. is also set forth in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed., United States Public Health Service, National Institutes of Health, Bethesda. Thus, "the EU index as set forth in Kabat" or "Kabat's EU index" refers to the residue numbering system based on the human IgG1 Eu antibody of Edelman et al., as set forth in Kabat 1991.

[0059] The numbering system used for the light chain constant region amino acid sequence is that shown in Kabat 1991.

[0060] The EDB ADCs of the present invention can be conjugated to drugs / payloads using conventional cysteine ​​technology or site-specific conjugation technology. To accommodate site-specific conjugation via engineered cysteines, the constant domains can be modified to provide engineered reactive cysteine ​​residues at one or more specific sites (also referred to as "Cys" mutants). To accommodate site-specific conjugation via transglutaminase-based conjugation, acyl donor glutamine-containing ("Q") tags or endogenous glutamines are made reactive by polypeptide engineering in the presence of transglutaminase and amines.

[0061] The present invention provides for the optimization of the L19-EDB antibody by generating a non-immunogenic antibody. In some embodiments, the L19-EDB human IgG1 constant region, which comprises the G1m(a) allotype having aspartic acid (D) at position 356 and leucine (L) at position 358, can be substituted with a non-G1m(a) allotype having glutamic acid (E) at position 356 and methionine (M) at position 358 (according to the EU index numbering of Kabat).

[0062] Furthermore, to reduce potential chemical propensity and antigen binding putative protein glycosylation sites, the anti-EDB antibodies of the invention may have a heavy chain variable region containing a lysine (K) to arginine (R) mutation at position 94 (according to the EU index numbering of Kabat), e.g., (K94R).

[0063] For site-specific conjugation via an engineered cysteine, the anti-EDB antibody heavy chain constant domain may contain a reactive engineered cysteine ​​residue at position 290 (K290C) according to the EU index numbering of Kabat. Additional cysteine ​​substitutions may be introduced. In another embodiment, the anti-EDB antibody light chain constant domain may contain a reactive engineered cysteine ​​residue at position 183 (κK183C) according to the Kabat numbering. Additional cysteine ​​substitutions may be introduced.

[0064] For site-specific conjugation via an engineered glutamine residue, the anti-EDB antibody heavy chain constant domain can contain an engineered H16-glutamine-containing tag LLQG (SEQ ID NO: 40). Furthermore, to optimize this site-specific conjugation, the lysine (K) amino acid at position 222 (according to the EU index of Kabat) on the heavy chain can be substituted with arginine (R), for example (K222R).

[0065] Amino acid modification can be carried out by any method known in the art, and many such methods, such as mutation, substitution, deletion and / or addition, are well known and routine for those skilled in the art.For example, but not limited to, amino acid substitution, deletion and insertion can be achieved using any well-known PCR-based technique.Amino acid substitution can be carried out by site-directed mutagenesis (see, for example, Zoller and Smith, 1982, Nucl. Acids Res. 10:6487-6500; and Kunkel, 1985, PNAS 82:488).

[0066] In some embodiments of the present invention, the EDB ADC comprises an antibody or antigen-binding fragment thereof having a heavy and / or light chain comprising an amino acid sequence at least 90%, 95%, 98%, or 99% identical to any of the heavy or light chains disclosed herein. The altered residues may be in the variable or constant regions of the antibody. In some embodiments, no more than 1, 2, 3, 4, or 5 residues are altered compared to any of the heavy or light chains disclosed herein.

[0067] The term "percent identical," with respect to amino acid sequences, refers to the number of residues in two sequences that are the same when aligned for maximum correspondence. There are several different algorithms known in the art that can be used to measure amino acid percent identity (i.e., Basic Local Alignment Tool or BLAST®). Unless otherwise specified, default parameters for a particular program or algorithm are used.

[0068] For use in preparing EDB ADCs, the antibodies described herein can be substantially pure, i.e., at least 50% pure (i.e., free from contaminants), more preferably at least 90% pure, more preferably at least 95% pure, even more preferably at least 98% pure, and most preferably at least 99% pure.

[0069] Tables 2 and 3 provide the amino acid (protein) and related nucleic acid (DNA) sequences of the anti-EDB antibodies of the invention. CDRs are as defined by Kabat and Chothia. Shaded residues identify amino acid mutations, substitutions, and / or insertions relevant to antibody optimization, and underlined residues identify amino acid mutations, substitutions, and / or insertions relevant to site-specific conjugation technology.

[0070] [Table 2-1]

[0071] [Table 2-2]

[0072] [Table 2-3]

[0073] [Table 2-4]

[0074] [Table 2-5]

[0075] [Table 2-6]

[0076] [Table 2-7]

[0077] [Table 2-8]

[0078] [Table 2-9]

[0079] In some embodiments of the invention, the EDB ADC comprises an antibody or antigen-binding fragment thereof that binds to extra domain B (EDB) of fibronectin (FN).

[0080] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof has a heavy chain variable region (VH) and a light chain variable region (VL), where the VH has three CDRs comprising SEQ ID NOs: 3, 5, and 7. In some embodiments of the present invention, the antibody or antigen-binding fragment thereof has a heavy chain variable region (VH) and a light chain variable region (VL), where the VL has three CDRs comprising SEQ ID NOs: 12, 13, and 14. The antibody or antigen-binding fragment thereof may have a VH having three CDRs comprising SEQ ID NOs: 3, 5, and 7; and a VL having three CDRs comprising SEQ ID NOs: 12, 13, and 14.

[0081] In another embodiment, the antibody or antigen-binding fragment thereof of the invention may have a heavy chain variable region (VH) comprising a VH CDR1 of SEQ ID NO: 3, a VH CDR2 of SEQ ID NO: 5, and a VH CDR3 of SEQ ID NO: 7 (according to Kabat), or a VH CDR1 of SEQ ID NO: 4, a VH CDR2 of SEQ ID NO: 6, and a VH CDR3 of SEQ ID NO: 7 (according to Chothia), or a VH CDR1 of SEQ ID NO: 3 or 4, a VH CDR2 of SEQ ID NO: 5 or 6, and a VH CDR3 of SEQ ID NO: 7. In another embodiment, the antibody or antigen-binding fragment thereof may have a heavy chain variable region (VH) comprising a VL CDR1 of SEQ ID NO: 12, a VL CDR2 of SEQ ID NO: 13, and a VL CDR3 of SEQ ID NO: 14. and a light chain variable region (VL) comprising CDR3 (according to Kabat and Chothia). In a further embodiment, the antibody or antigen-binding fragment thereof may have a VH CDR1 of SEQ ID NO: 3 or 4, a VH CDR2 of SEQ ID NO: 5 or 6, and a VH CDR3 of SEQ ID NO: 7, and a VL CDR1 of SEQ ID NO: 12, a VL CDR2 of SEQ ID NO: 13, and a VL CDR3 of SEQ ID NO: 14.

[0082] In some embodiments of the invention, the antibody or antigen-binding fragment thereof may have a heavy chain variable region comprising SEQ ID NO: 1 or 21 and / or a light chain variable region comprising SEQ ID NO: 10. The antibody or antigen-binding fragment thereof may comprise a heavy chain variable region having an amino acid sequence at least 90% identical to SEQ ID NO: 1 and a light chain variable region having an amino acid sequence at least 90% identical to SEQ ID NO: 10; a heavy chain variable region having an amino acid sequence at least 90% identical to SEQ ID NO: 21 and a light chain variable region having an amino acid sequence at least 90% identical to SEQ ID NO: 10; a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO: 10; or a heavy chain variable region comprising SEQ ID NO: 21 and a light chain variable region comprising SEQ ID NO: 10.

[0083] In another embodiment of the invention, the antibody or antigen-binding fragment thereof may have a heavy chain comprising any one of SEQ ID NOs: 8, 17, 19, 23, 25, 27 and 29 and / or a light chain comprising SEQ ID NO: 15 or 31.

[0084] The antibody or antigen-binding fragment thereof of the present invention may comprise a heavy chain having an amino acid sequence at least 90% identical to SEQ ID NO:8 and a light chain having an amino acid sequence at least 90% identical to SEQ ID NO:15; a heavy chain having an amino acid sequence at least 90% identical to SEQ ID NO:8 and a light chain having an amino acid sequence at least 90% identical to SEQ ID NO:31; a heavy chain having an amino acid sequence at least 90% identical to SEQ ID NO:17 and a light chain having an amino acid sequence at least 90% identical to SEQ ID NO:15; a heavy chain having an amino acid sequence at least 90% identical to SEQ ID NO:17 and a light chain having an amino acid sequence at least 90% identical to SEQ ID NO:31; a heavy chain having an amino acid sequence at least 90% identical to SEQ ID NO:19 and a light chain having an amino acid sequence at least 90% identical to SEQ ID NO:15; a heavy chain having an amino acid sequence at least 90% identical to SEQ ID NO:19 and a light chain having an amino acid sequence at least 90% identical to SEQ ID NO:31; a heavy chain having an amino acid sequence at least 90% identical to SEQ ID NO: 27 and a light chain having an amino acid sequence at least 90% identical to SEQ ID NO: 15; a heavy chain having an amino acid sequence at least 90% identical to SEQ ID NO: 27 and a light chain having an amino acid sequence at least 90% identical to SEQ ID NO: 15; a heavy chain having an amino acid sequence at least 90% identical to SEQ ID NO: 23 and a light chain having an amino acid sequence at least 90% identical to SEQ ID NO: 31; a heavy chain having an amino acid sequence at least 90% identical to SEQ ID NO: 25 and a light chain having an amino acid sequence at least 90% identical to SEQ ID NO: 15; a heavy chain having an amino acid sequence at least 90% identical to SEQ ID NO: 27 and a light chain having an amino acid sequence at least 90% identical to SEQ ID NO: 31; or a heavy chain having an amino acid sequence at least 90% identical to SEQ ID NO: 29 and a light chain having an amino acid sequence at least 90% identical to SEQ ID NO: 15.

[0085] An antibody or antigen-binding fragment thereof of the present invention may comprise a heavy chain comprising SEQ ID NO:8 and a light chain comprising SEQ ID NO:15; a heavy chain comprising SEQ ID NO:8 and a light chain comprising SEQ ID NO:31; a heavy chain comprising SEQ ID NO:17 and a light chain comprising SEQ ID NO:15; a heavy chain comprising SEQ ID NO:17 and a light chain comprising SEQ ID NO:31; a heavy chain comprising SEQ ID NO:19 and a light chain comprising SEQ ID NO:15; a heavy chain comprising SEQ ID NO:19 and a light chain comprising SEQ ID NO:31; a heavy chain comprising SEQ ID NO:23 and a light chain comprising SEQ ID NO:15; a heavy chain comprising SEQ ID NO:23 and a light chain comprising SEQ ID NO:31; a heavy chain comprising SEQ ID NO:25 and a light chain comprising SEQ ID NO:15; a heavy chain comprising SEQ ID NO:25 and a light chain comprising SEQ ID NO:31; a heavy chain comprising SEQ ID NO:27 and a light chain comprising SEQ ID NO:15; a heavy chain comprising SEQ ID NO:27 and a light chain comprising SEQ ID NO:31; a heavy chain comprising SEQ ID NO:29 and a light chain comprising SEQ ID NO:15; or a heavy chain comprising SEQ ID NO:29 and a light chain comprising SEQ ID NO:31.

[0086] Representative DNAs encoding the anti-EDB antibody heavy and light chain variable regions include SEQ ID NOs: 2 and 22, and SEQ ID NO: 11. Representative DNAs encoding the anti-EDB antibody heavy and light chain variable regions include SEQ ID NOs: 9, 18, 20, 24, 26, 28, and 30, and SEQ ID NOs: 16 and 32, respectively.

[0087] [Table 3]

[0088] drugs Drugs useful in preparing the disclosed EDB ADCs include any substance with biological or detectable activity, such as therapeutic agents, detectable labels, binding agents, etc., and prodrugs that are metabolized to active agents in vivo. The drug may also be a drug derivative, where the drug is functionalized to allow for conjugation to an antibody of the invention.

[0089] A therapeutic agent is an agent that exerts a cytotoxic, cytostatic, and / or immunomodulatory effect on cancer cells or activated immune cells. Examples of therapeutic agents include cytotoxic agents, chemotherapeutic agents, cytostatic agents, and immunomodulatory agents. A cytotoxic effect refers to the depletion, elimination, and / or death of a target cell(s). A cytotoxic agent refers to an agent that has a cytotoxic and / or cytostatic effect on a cell. A cytostatic effect refers to the inhibition of cell proliferation. A cytostatic agent refers to an agent that has a cytostatic effect on a cell, thereby inhibiting the growth and / or expansion of a specific subset of cells. A chemotherapeutic agent refers to an agent that is a chemical compound useful in the treatment of cancer. An immunomodulatory agent refers to an agent that stimulates an immune response through the production of cytokines and / or antibodies and / or alteration of T-cell function, either directly or indirectly by making another agent more effective, thereby inhibiting or reducing the growth of a subset of cells (i.e., tumor cells).

[0090] In some embodiments, the drug is a membrane-permeable drug. In such embodiments, the payload can induce a bystander effect, in which cells surrounding cells bound by the ADC are killed by the cell-permeable payload, even though they may not express EDB+FN or have EDB+FN bound to their surface. This occurs when the payload is released from the antibody (i.e., by cleavage of the cleavable linker), crosses the cell membrane, and induces the death of surrounding cells upon diffusion.

[0091] According to the disclosed method, an EDB ADC can be produced or generated, which has (a) an antibody or its antigen-binding fragment that binds to EDB; (b) a linker; and (c) a drug. The drug-to-antibody ratio (DAR) or drug loading indicates the number of drug molecules conjugated per antibody. Compositions, batches, and / or formulations of multiple ADCs can be characterized by an average DAR. The DAR and average DAR can be determined by various conventional means, such as UV spectroscopy, mass spectrometry, ELISA assay, radiometric method, hydrophobic interaction chromatography (HIC), electrophoresis, and HPLC.

[0092] In embodiments of the invention, an EDB ADC can have a DAR of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or a DAR greater than 12. In embodiments of the invention, an EDB ADC can have 1 drug molecule, or 2 drug molecules, or 3 drug molecules, or 4 drug molecules, or 5 drug molecules, or 6 drug molecules, or 7 drug molecules, or 8 drug molecules, or 9 drug molecules, or 10 drug molecules, or 11 drug molecules, or 12 drug molecules, or more than 12 molecules.

[0093] In embodiments of the invention, the EDB ADC may have an average DAR in the range of about 2 to about 4, or an average DAR in the range of about 3 to about 5, or an average DAR in the range of about 4 to about 6, or an average DAR in the range of about 5 to about 7, or an average DAR in the range of about 6 to about 8, or an average DAR in the range of about 7 to about 9, or an average DAR in the range of about 8 to about 10, or an average DAR in the range of about 9 to about 11, or an average DAR in the range of about 10 to about 12, etc. In some embodiments, the EDB ADC compositions, batches and / or formulations may have an average DAR of about 1, or an average DAR of about 2, or an average DAR of about 3, or an average DAR of about 4, or an average DAR of about 5, or an average DAR of about 6, or an average DAR of about 7, or an average DAR of about 8, or an average DAR of about 9, or an average DAR of about 10, or an average DAR of about 11, or an average DAR of about 12, or an average DAR greater than 12. When used in the above ranges of average DAR, the term "about" means + / - 0.5%.

[0094] The EDB ADC compositions, batches, and / or formulations may be characterized by a preferred range of average DAR, e.g., an average DAR in the range of about 3 to about 5, an average DAR in the range of about 3 to about 4, or an average DAR in the range of about 4 to about 5. Additionally, the EDB ADC compositions, batches, and / or formulations may be characterized by a preferred range of average DAR, e.g., an average DAR in the range of 3 to 5, an average DAR in the range of 3 to 4, or an average DAR in the range of 4 to 5.

[0095] In some embodiments of the invention, EDB ADC compositions, batches, and / or formulations may be characterized by an average DAR of about 1.0, or an average DAR of 1.0, or an average DAR of 1.1, or an average DAR of 1.2, or an average DAR of 1.3, or an average DAR of 1.4, or an average DAR of 1.5, or an average DAR of 1.6, or an average DAR of 1.7, or an average DAR of 1.8, or an average DAR of 1.9. In other embodiments, EDB ADC compositions, batches, and / or formulations may be characterized by an average DAR of about 2.0, or an average DAR of 2.0, or an average DAR of 2.1, or an average DAR of 2.2, or an average DAR of 2.3, or an average DAR of 2.4, or an average DAR of 2.5, or an average DAR of 2.6, or an average DAR of 2.7, or an average DAR of 2.8, or an average DAR of 2.9. In another embodiment, EDB ADC compositions, batches, and / or formulations may be characterized by an average DAR of about 3.0, or an average DAR of 3.0, or an average DAR of 3.1, or an average DAR of 3.2, or an average DAR of 3.3, or an average DAR of 3.4, or an average DAR of 3.5, or an average DAR of 3.6, or an average DAR of 3.7, or an average DAR of 3.8, or an average DAR of 3.9. In another embodiment, EDB ADC compositions, batches, and / or formulations may be characterized by an average DAR of about 4.0, or an average DAR of 4.0, or an average DAR of 4.1, or an average DAR of 4.2, or an average DAR of 4.3, or an average DAR of 4.4, or an average DAR of 4.5, or an average DAR of 4.6, or an average DAR of 4.7, or an average DAR of 4.8, or an average DAR of 4.9, or an average DAR of 5.0.

[0096] In another embodiment, the EDB ADC compositions, batches and / or formulations may be characterized by an average DAR of 12 or less, an average DAR of 11 or less, an average DAR of 10 or less, an average DAR of 9 or less, an average DAR of 8 or less, an average DAR of 7 or less, an average DAR of 6 or less, an average DAR of 5 or less, an average DAR of 4 or less, an average DAR of 3 or less, an average DAR of 2 or less, or an average DAR of 1 or less.

[0097] In other embodiments, EDB ADC compositions, batches and / or formulations may be characterized by an average DAR of 11.5 or less, an average DAR of 10.5 or less, an average DAR of 9.5 or less, an average DAR of 8.5 or less, an average DAR of 7.5 or less, an average DAR of 6.5 or less, an average DAR of 5.5 or less, an average DAR of 4.5 or less, an average DAR of 3.5 or less, an average DAR of 2.5 or less, an average DAR of 1.5 or less.

[0098] In some embodiments of the invention, the methods and purification conditions for conventional conjugation via cysteine ​​residues disclosed herein provide compositions, batches and / or formulations of EDB ADCs with an optimized average DAR in the range of about 3-5, preferably about 4.

[0099] In some embodiments of the present invention, the methods and purification conditions for site-specific conjugation via engineered cysteine ​​residues disclosed herein provide compositions, batches and / or formulations of EDB ADCs with an optimized average DAR in the range of about 3-5, preferably about 4.

[0100] In some embodiments of the invention, the methods and purification conditions for site-specific conjugation via transglutaminase-based conjugation disclosed herein provide compositions, batches and / or formulations of EDB ADCs with an optimized average DAR in the range of about 1 to 3, preferably about 2.

[0101] Examples of cytotoxic agents include anthracyclines, auristatins, CC-1065, dolastatins, duocarmycins, enediynes, geldanamycin, maytansine, puromycin, taxanes, vinca alkaloids, SN-38, tubulysins. , hemiasterin, and their stereoisomers, isosteres, analogs, or derivatives. Plant toxins, other bioactive proteins, enzymes (i.e., ADEPT), radioisotopes, photosensitizers (i.e., for photodynamic therapy) may also be used.

[0102] Anthracyclines are derived from the bacterium Streptomyces and have been used to treat a wide range of cancers, including leukemia, lymphoma, breast, uterine, ovarian, and lung cancer. Exemplary anthracyclines include, but are not limited to, daunorubicin, doxorubicin (i.e., adriamycin), epirubicin, idarubicin, valrubicin, and mitoxantrone.

[0103] Dolastatins and their peptidic analogs and derivatives, the auristatins, are highly potent antimitotic agents that have been shown to have anticancer and antifungal activity. See, e.g., U.S. Patent No. 5,663,149 and Pettit et al., Antimicrob. Agents Chemother. 42:2961-2965, (1998). Exemplary dolastatins and auristatins include, but are not limited to, dolastatin 10, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), MMAD (monomethyl auristatin D or monomethyl dolastatin 10), MMAF (monomethyl auristatin F or N-methylvaline-valine-dolaisoleucine-dolaproline-phenylalanine), MMAE (monomethyl auristatin E or N-methylvaline-valine-dolaisoleucine-dolaproline-norephedrine), 5-benzoylvaleric acid-AE ester (AEVB), and other novel substances.

[0104] In some embodiments, the drug / payload is an auristatin. Auristatins inhibit cell proliferation by inhibiting the formation of microtubules during mitosis through the inhibition of tubulin polymerization. PCT International Patent Application Publication No. WO2013 / 072813, the entire contents of which are incorporated herein by reference, discloses auristatins useful in the EDB ADCs of the present invention and provides methods for producing auristatins. For example, the structure:

[0105] [ka] Payload 0101, with structure:

[0106] [ka] Payload 1569, with structure:

[0107] [ka] Payload 9411, having the structure:

[0108] [ka] Payload 4574, with structure:

[0109] [ka] Payload DM1 and structure:

[0110] [ka] The payload cemadotin.

[0111] Duocarmycin and CC-1065 are CPI-based monomers that act as DNA alkylating agents with cytotoxic potency. See Boger and Johnson, PNAS 92:3642-3649, 1995. Exemplary dolastatins include, but are not limited to, (+)-duocarmycin A and (+)-duocarmycin SA, and (+)-CC-1065.

[0112] In some embodiments, the drug / payload is CPI or CBI dimer.CPI dimer induces interstrand DNA cross-linking and potent cytotoxicity.PCT International Patent Application Publication No. WO2015 / 110935, the entirety of which is incorporated herein by reference, discloses the CPI and CBI dimer useful in the EDB ADC of the present invention, and provides a method for producing CPI and CBI dimer.For example, the structure:

[0113] [ka] The payload CPI-8314 dimer and structure:

[0114] [ka] The payload is CPI-0326.

[0115] Enediynes are a class of antitumor bacterial products characterized by either 9- and 10-membered rings or the presence of conjugated triple-double-triple bond ring systems. Exemplary enediynes include, but are not limited to, calicheamicin, esperamicin, and dynemicin. Calicheamicin, also referred to as LL-E33288 complex, e.g., β-calicheamicin, γ-calicheamicin, or N-acetyl-γ-calicheamicin (gamma-calicheamicin (γ1)), is an enediyne antibiotic originally isolated as a natural product from the soil organism Micromonospora echinospora ssp. calichensis (Zein et al., Science 27;240(4856):1198-1201, 1988); it generates double-stranded DNA breaks in target cells and subsequently induces apoptosis (Zein et al., Science 27;240(4856):1198-1201, 1988; Nicolaou et al., Chem. Biol. September;1(1):57-66, 1994; Prokop et al., Oncogene 22:9107-9120, 2003). The disulfide analog is N-acetyl-γ-calicheamicin dimethylhydrazide.

[0116] Geldanamycin is a benzoquinone ansamycin antibiotic that binds to Hsp90 (heat shock protein 90) and has been used as an antitumor drug. Exemplary geldanamycins include, but are not limited to, 17-AAG (17-N-allylamino-17-demethoxygeldanamycin) and 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin).

[0117] Maytansine or its derivatives, maytansinoids, inhibit cell proliferation by inhibiting microtubule formation during mitosis through the inhibition of tubulin polymerization. See Remillard et al., Science 189:1002-1005, 1975. Exemplary maytansines and maytansinoids include, but are not limited to, mertansine (DM1) and its derivatives, and ansamitocin. It will not be done.

[0118] Taxanes are diterpenes that act as antitubulin or antimitotic agents. Exemplary taxanes include, but are not limited to, paclitaxel (e.g., TAXOL®) and docetaxel (TAXOTERE®).

[0119] Vinca alkaloids are also antitubulin agents. Exemplary vinca alkaloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine.

[0120] In some aspects of the invention, the agent is an immunomodulatory agent. Examples of immunomodulatory agents include, but are not limited to, ganciclovir, etanercept, tacrolimus, sirolimus, voclosporin, cyclosporin, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, glucocorticoids and analogs thereof, cytokines, xanthines, stem cell growth factors, lymphotoxins, tumor necrosis factor (TNF), hematopoietic factors, interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony-stimulating factors (e.g., granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF)), interferons (e.g., interferon-α, -β, and -γ), stem cell growth factors referred to as "S1 factors," erythropoietin, and thrombopoietin, or combinations thereof.

[0121] The immunomodulatory agent useful in the present invention also includes antihormonal drugs that block hormone action on tumors, and immunosuppressants that suppress cytokine production, downregulate self-antigen expression, or mask MHC antigens.Representative antihormonal drugs include, for example, antiestrogens, including tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and antiadrenal agents. Representative immunosuppressants include 2-amino-6-aryl-5-substituted pyrimidines, azathioprine, cyclophosphamide, bromocriptine, danazol, dapsone, glutaraldehyde, anti-idiotypic antibodies against MHC antigens and MHC fragments, cyclosporin A, steroids such as glucocorticosteroids, cytokine or cytokine receptor antagonists (e.g., anti-interferon antibodies, anti-IL10 antibodies, anti-TNFα antibodies, anti-IL2 antibodies), streptokinase, TGFβ, rapamycin, T cell receptors, T cell receptor fragments, and T cell receptor antibodies.

[0122] In some aspects of the invention, the drug is a therapeutic protein, including but not limited to toxins, hormones, enzymes and growth factors.

[0123] Examples of toxic proteins (or polypeptides) include diphtheria (e.g., diphtheria A chain), Pseudomonas exotoxin and endotoxin, ricin (e.g., ricin A chain), abrin (e.g., abrin A chain), modeccin (e.g., modeccin A chain), alpha-sarcin, Aleurites fordii protein, dianthin protein, ribonuclease (RNase), DNase I, Staphylococcus aureus enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, pokeweed proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitors, curcin, crotin, soapwort inhibitors, mitogellin, restrictocin, phenomycin, enomycin, trichothecin, inhibitor cystine knot (ICK) peptides (e.g., ceratotoxin) and conotoxins (e.g., KIIIA or SmIIIa).

[0124] Examples of hormones include, but are not limited to, estrogens, androgens, progestins, and corticosteroids.

[0125] In some aspects of the invention, the drug is an oligonucleotide, for example, an antisense oligonucleotide.

[0126] Additional drugs useful in the present invention include antiangiogenic agents that inhibit blood vessel formation, such as farnesyltransferase inhibitors, COX-2 inhibitors, VEGF inhibitors, bFGF inhibitors, steroid sulfatase inhibitors (e.g., 2-methoxyestradiol bis-sulfamate (2-MeOE2bisMATE)), interleukin-24, thrombospondin, metallospondin proteins, class I interferons, interleukin-12, protamine, angiostatin, laminin, endostatin, and prolactin fragments.

[0127] Antiproliferative and proapoptotic agents include activators of PPAR-gamma (e.g., cyclopentenone prostaglandins (cyPGs)), retinoids, triterpenoids (e.g., cycloartane, lupane, ursane, oleanane, friedelane, dammarane, cucurbitacins, and limonoid triterpenoids), inhibitors of EGF receptors (e.g., HER4), rapamycin, CALCITRIOL® (1,25-dihydroxycholecalciferol (vitamin D)), aromatase inhibitors (FEMARA® (letrozole)), tetanus, erythromycin ... These include thrombinase inhibitors, iron chelators (e.g., 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (Triapine)), apoptin (viral protein 3-VP3 from chicken anemia virus), inhibitors of Bcl-2 and Bcl-X(L), TNF-alpha, FAS ligand, TNF-related apoptosis-inducing ligand (TRAIL / Apo2L), activators of TNF-alpha / FAS ligand / TNF-related apoptosis-inducing ligand (TRAIL / Apo2L) signaling, and inhibitors of PI3K-Akt survival pathway signaling (e.g., UCN-01 and geldanamycin).

[0128] Representative chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines, and altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine. Methylmelamine; nitrogen mustards, e.g., chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, e.g., carmustine, clo lozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin , 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tuberculin antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, Cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-EU; androgens, e.g., calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal drugs, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestrabucil; bisantrene ( Bisantrene; Edatrexate; Defofamine; Demecolcine; Diaziquone; Eflornithine; Elliptinium acetate; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Podophyllinic acid; 2-Ethyl thiazolehydrazide; procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2'-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ)) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; and capecitabine.

[0129] Additional therapeutic agents that may be used in accordance with the present invention include photosensitizing agents for photodynamic therapy, e.g., U.S. Patent Application Publication No. 20020197262 and U.S. Patent No. 5,952,329, which are incorporated herein by reference in their entireties; magnetic particles for hyperthermia, e.g., U.S. Patent Application Publication No. 20030032995, which are incorporated herein by reference in their entireties; binding agents, e.g., peptides, ligands, cell adhesion ligands, and the like, and prodrugs that can be converted to more active cytotoxic free drugs, e.g., phosphate-, thiophosphate-, sulfate-, peptide-, β-lactam-, substituted phenoxyacetamide-, or phenylacetamide-containing prodrugs, 5-fluorocytosine, and other 5-fluorouridine prodrugs.

[0130] For diagnostic methods using anti-EDB antibodies, the agent may include a detectable label used to detect the presence of EDB+FN-expressing ECM or cells in vitro or in vivo. Radioisotopes detectable in vivo, such as labels detectable using scintigraphy, magnetic resonance imaging, or ultrasound, may be used in clinical diagnostic applications. Useful scintigraphy labels include positron emitters and gamma-emitters. Representative contrast agents for magnetic field imaging include paramagnetic or superparamagnetic ions (e.g., iron, copper, manganese, chromium, erbium, europium, dysprosium, holmium, and gadolinium), iron oxide particles, and water-soluble contrast agents. For ultrasound detection, gases or liquids can be trapped in porous inorganic particles that are released as microbubble contrast agents. For in vitro detection, useful detectable labels include fluorophores, detectable epitopes or binding agents, and radioactive labels.

[0131] Thus, in some embodiments of the invention, the agent is an imaging agent (e.g., a fluorophore or a PET (positron emission tomography) label, a SPECT (single photon emission computed tomography) label, or an MRI (magnetic resonance imaging) label).

[0132] The term "label," as used herein, refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody to produce a "labeled" antibody. The label may be detectable itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable. Radionuclides that can function as detectable labels include, for example, I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, and Pd-109. The label may also be a non-detectable entity, such as a toxin.

[0133] Examples of fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluoresceinamidite (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein, erythrosine, Alexa Fluor® (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, or 750), carboxytetramethylrhodamine (TAMRA) (e.g., 5,-TAMRA), tetramethylrhodamine (TMR), and sulforhodamine (SR) (e.g., SR101).

[0134] Therapeutic or diagnostic radioisotopes or other labels (e.g., PET or SPECT labels) can also be incorporated into the anti-EDB antibodies described herein for conjugation. The isotopes can be directly attached to the antibody, for example, at a cysteine ​​residue present in the antibody, or a chelator can be used to mediate the binding of the antibody to the radioisotope. Radioisotopes suitable for radiotherapy include, but are not limited to, α-emitters, β-emitters, and Auger electrons. For diagnostic applications, useful radioisotopes include positron emitters and γ-emitters. The anti-EDB antibodies of the present invention can be further iodinated, for example, at tyrosine residues of the antibody, to facilitate detection or therapeutic effect of the antibody.

[0135] Examples of radioisotopes or other labels include: 3 H, 11 C. 13 N, 14 C. 15 N, 15 O. 35 S, 18 F, 32 P, 33 P, 47 Sc, 51 Cr, 57 Co, 58 Co, 59 Fe, 62 Cu, 64Cu, 67 Cu, 67 Ga, 68 Ga, 75 Se, 76 Br, 77 Br, 86 Y, 89 Zr, 90 Y, 94 Tc, 95 Ru, 97 Ru, 99 Tc, 103 Ru, 105 Rh, 105 Ru, 107 Hg, 109 Pd, 111 Ag, 111 In, 113 In, 121 Te, 122 Te, 123 I, 124 I, 125 I, 125 Te, 126 I, 131 I, 131 In, 133 I, 142 Pr, 143 Pr, 153 Pb, 153 Sm, 161 Tb, 165 Tm, 166 Dy, 166 H, 167 Tm, 168 Tm, 169 Yb, 177 Lu, 186 Re, 188 Re, 189 Re, 197 Pt, 198 Au, 199 Au, 201 Tl, 203 Hg, 211 At, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 224 Ac and 225 Includes but is not limited to Ac.

[0136] Linker The EDB ADCs of the present invention can be prepared using a linker to directly or indirectly link or conjugate a drug to an antibody. The linker is a bifunctional compound that connects the drug and the antibody to form an ADC. Such an ADC enables selective delivery of a drug via an antibody that binds to a specific antigen or protein. Suitable linkers include, for example, cleavable and non-cleavable linkers. Cleavable linkers are typically susceptible to drug cleavage and release under specific intracellular and extracellular conditions. The main mechanisms by which a conjugated drug can be cleaved from an antibody intracellularly include hydrolysis (hydrazones, acetals, and cis-aconitate-like amides) at the acidic pH of lysosomes, peptide cleavage by lysosomal enzymes (cathepsins and other lysosomal enzymes), and disulfide reduction. Conjugated drugs can be cleaved extracellularly from antibodies by proteases such as cathepsins in the tumor microenvironment (TME). As a result of these different mechanisms for cleavage, the mechanism for linking a drug to an antibody also varies widely, and any appropriate linker can be used.

[0137] Suitable linkers may include any cleavable linker. In some embodiments, suitable linkers include a valine-citrulline (val-cit) linker, a phenylalanine-lysine (phe-lys) linker, or a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (vc) linker, or a dipeptide linked to an additional immolation element suitable for transglutaminase-based conjugation technology, such as an N-2-acetyl-L-lysyl-L-valyl-L-citrulline-p-aminobenzyloxycarbonyl-N,N'-dimethylaminoethyl-CO-(AcLys-vc) linker. In other embodiments, suitable linkers include disulfide linkers, such as a sulfanylpyridine (diS) linker and a 2-(pyridin-2-yldisulfanyl)ethylcarbamoyl (diS-COCO) linker. In another embodiment, the linker can be a non-cleavable linker, such as maleimidocaproyl (mc), maleimido-heptanoyl (me), and maleimido-Peg6C2 (MalPeg6C2). In other embodiments, suitable linkers include linkers that are hydrolyzable at a specific pH or pH range, such as hydrazone linkers.

[0138] The linker can be covalently attached to the antibody via a thioester linkage, for example, by reaction of a maleimide or haloacetamide present on the linker with a native or engineered cysteine ​​residue present on the antibody. In another embodiment, the linker can be covalently attached to the antibody via an amide linkage to a lysine residue present on the antibody, for example, by reaction of an N-hydroxy-succinimide-activated carboxylic acid present on the linker with the free amine of the lysine residue. In another embodiment, the linker can be covalently attached to the antibody via an amide linkage to the side chain of a glutamine residue present in or engineered into the antibody, for example, by an enzymatic reaction catalyzed by a transglutaminase enzyme, which creates a new amide linkage from the primary amine present on the linker to the side chain amide of the glutamine residue.

[0139] In some aspects, the linkers of the present invention have the structure:

[0140] [ka] a "mc-vc-PABC" or "vc-PABC" or "vc" linker having the structure:

[0141] [ka] "AcLys-vc-PABC-DMAE-CO" or "AcLys-vc" linker having the structure:

[0142] [ka] diS linker and structure:

[0143] [ka] The diS-COCO linker has the formula:

[0144] Method for preparing EDB ADC Methods for preparing the EDB ADCs of the present invention are provided herein. The present invention also provides a process for producing or generating the conventionally and site-specifically conjugated EDB ADCs disclosed herein, which may include the steps of (a) linking a linker to a drug; (b) conjugating a linker-drug moiety to an antibody; and (c) purifying the antibody-drug conjugate. See Examples 3 and 4.

[0145] In some embodiments, EDB ADCs can be generated using conventional non-specific conjugation of a linker-payload moiety via one or more cysteine ​​residues of an anti-EDB antibody or antigen-binding fragment thereof.

[0146] In another embodiment, EDB ADCs can be generated using site-specific conjugation of linker-payload moieties via one or more reactive cysteine ​​residues engineered into the anti-EDB antibody constant domain. Methods for preparing antibodies for site-specific conjugation via engineered cysteine ​​residues are described in PCT International Patent Application Publication No. WO2013 / 093809, which is incorporated herein by reference in its entirety.

[0147] One or more amino acid residues of an anti-EDB antibody heavy chain can be substituted with another amino acid, such as a cysteine ​​residue, for the purpose of conjugation to a drug or payload. In one aspect, the present invention provides an anti-EDB antibody or antigen-binding fragment thereof comprising an antibody heavy chain constant region comprising engineered cysteine ​​residues at the following positions: 118 (114 according to Kabat), 246, 249, 265, 267, 270, 276, 278, 283, 290, 292, 293, 294, 300, 302, 303, 314, 315, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 38 20, 327, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 375, 376, 378, 380, 382, ​​386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443 or 444, or any combination thereof. In particular, positions 118 (114 according to Kabat), 290, 334, 347, 373, 375, 380, 388, 392, 421, 443, or any combination thereof may be used. Additional cysteine ​​substitutions may be introduced.

[0148] In another aspect, the invention provides an anti-EDB antibody or antigen-binding fragment thereof comprising a heavy chain constant domain comprising an engineered cysteine ​​residue at position 290 (K290C) according to the EU index of Kabat numbering.

[0149] One or more amino acid residues in the anti-EDB antibody light chain constant domain may be substituted with another amino acid, such as a cysteine ​​residue, for the purpose of conjugation to a drug or payload. In one aspect, the present invention provides an anti-EDB antibody light chain constant domain comprising: (i) an engineered cysteine ​​residue at positions 110, 111, 125, 149, 155, 158, 161, 183, 185, 188, 189, 191, 197, 205, 207, 208, or 210, or any combination thereof, according to Kabat numbering; (ii) an engineered cysteine ​​residue at residues 4, 42, 81, 100, 103, or any combination thereof, of SEQ ID NO: 37, when the constant domain is aligned with SEQ ID NO: 37 (kappa light chain). or (iii) an antibody light chain constant region comprising engineered cysteine ​​residues at positions corresponding to residues 4, 5, 19, 43, 49, 52, 55, 78, 81, 82, 84, 90, 96, 97, 98, 99, 101, or any combination thereof, of SEQ ID NO: 38 when the constant domain is aligned with SEQ ID NO: 38 (lambda light chain). Additional cysteine ​​substitutions may be introduced.

[0150] In another aspect, the present invention provides an anti-EDB antibody or antigen-binding fragment thereof comprising an antibody kappa light chain constant region comprising: (i) an engineered cysteine ​​residue at position 111, 149, 188, 207, 210, or any combination thereof (preferably 111 or 210), according to Kabat numbering; or (ii) an engineered cysteine ​​residue at a position corresponding to residue 4, 42, 81, 100, 103, or any combination thereof (preferably residue 4 or 103) of SEQ ID NO:37, when the constant domain is aligned with SEQ ID NO:37.

[0151] In another aspect, the present invention provides an anti-EDB antibody or antigen-binding fragment thereof comprising an antibody lambda light chain constant region comprising: (i) an engineered cysteine ​​residue at position 110, 111, 125, 149, 155, 158, 161, 185, 188, 189, 191, 197, 205, 206, 207, 208, 210, or any combination thereof (preferably 110, 111, 125, 149, or 155), according to Kabat numbering; or (ii) an engineered cysteine ​​residue at a position corresponding to residue 4, 5, 19, 43, 49, 52, 55, 78, 81, 82, 84, 90, 96, 97, 98, 99, 101, or any combination thereof (preferably residue 4, 5, 19, 43, or 49) of SEQ ID NO:38, when the constant domain is aligned with SEQ ID NO:38.

[0152] In another aspect, the invention provides an anti-EDB antibody or antigen-binding fragment thereof comprising a light chain constant domain comprising: (i) an engineered cysteine ​​residue at position 183 according to Kabat numbering (κK183C); or (ii) an engineered cysteine ​​residue at a position corresponding to residue 76 of SEQ ID NO:37 when the constant domain is aligned with SEQ ID NO:37.

[0153] SEQ ID NO: 37 (Cκ constant domain) RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLS K ADYE KHKVYACEVT HQGLSSPVTK SFNRGEC SEQ ID NO: 38 (Cλ constant domain) GQPKANPTVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADGSPVK AGVETTKPSK QSNNKYAASS YLSLTPEQWK SHRSYSCQVT HEGSTVEKTV APTECS

[0154] In another embodiment, EDB ADCs can be generated using site-specific conjugation technology via one or more engineered acyl donor glutamine-containing tags or reactive endogenous glutamine residues in the anti-EDB antibody constant region. Methods for preparing antibodies for site-specific conjugation via acyl donor glutamine-containing tags or glutamine residues are described in PCT International Patent Application Publication No. WO 2012 / 059882, which is incorporated herein by reference in its entirety.

[0155] In some embodiments, the acyl donor glutamine-containing tag comprises at least one glutamine (Q) and can be attached at different positions (ie, N-terminal, C-terminal, or internal) on the heavy and / or light chain. In another aspect, the acyl donor glutamine-containing tag can comprise an amino acid sequence selected from LLQGG (SEQ ID NO:39), LLQG (SEQ ID NO:40), LSLSQG (SEQ ID NO:41), GGGLLQGG (SEQ ID NO:42), GLLQG (SEQ ID NO:43), LLQ, GSPLAQSHGG (SEQ ID NO:44), GLLQGGG (SEQ ID NO:45), GLLQGG (SEQ ID NO:46), GLLQ (SEQ ID NO:47), LLQLLQGA (SEQ ID NO:48), LLQGA (SEQ ID NO:49), LLQYQGA (SEQ ID NO:50), LLQGSG (SEQ ID NO:51), LLQYQG (SEQ ID NO:52), LLQLLQG (SEQ ID NO:53), SLLQG (SEQ ID NO:54), LLQLQ (SEQ ID NO:55), LLQLLQ (SEQ ID NO:56), and LLQGR (SEQ ID NO:57). In some aspects, the acyl donor glutamine-containing tag replaces a wild-type amino acid position in the heavy chain constant domain. In some embodiments, the anti-EDB antibody may comprise an acylglutamine-containing tag having the amino acid sequence LLQG (SEQ ID NO: 40), replacing amino acids at positions E294 to N297 (according to EU index of Kabat) of the heavy chain.

[0156] The optimal reaction conditions for producing ADCs can be empirically determined by varying reaction variables such as temperature, pH, linker-payload moiety input amount, and additive concentration. Suitable conditions for the conjugation of other drugs can be determined by those skilled in the art without undue experimentation. Representative methods for conjugating and characterizing EDB ADCs are described in Examples 3 and 4.

[0157] After conjugation, the conjugates can be separated from unconjugated reactants and / or aggregated forms of the conjugate, purified, and characterized by conventional methods, including, but not limited to, mass spectrometry, size exclusion chromatography (SEC), ultrafiltration / diafiltration, ion exchange chromatography (IEC), chromatofocusing (CF), site-directed mutagenesis, fluorescent labeling, X-ray crystallography, high-performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), Sephacryl S-200 chromatography, or hydrophobic interaction chromatography (HIC). Suitable HIC media include, but are not limited to, Phenyl Sepharose 6 Fast Flow chromatography media, Butyl Sepharose 4 Fast Flow chromatography media, Octyl Sepharose 4 Fast Flow chromatography media, Toyopearl Ether-650M chromatography media, Macro-Prep methyl HIC media, or Macro-Prep t-Butyl HIC media.

[0158] Table 13 provides the EDB ADCs produced according to the conjugation and purification methods described herein and used to generate the data provided in the Examples.

[0159] In some embodiments of the invention, the EDB ADCs of the invention comprise (a) an antibody or antigen-binding fragment thereof that binds to EDB; (b) a linker; and (c) a drug.

[0160] In another embodiment of the invention, the EDB ADC of the invention comprises (a) an antibody or antigen-binding fragment thereof that binds to EDB; (b) a linker; and (c) a drug, wherein the linker is a cleavable or non-cleavable linker. In some embodiments, the linker is vc, diS, diS-COCO, or AcLys-vc.

[0161] In another embodiment of the present invention, the EDB ADC of the present invention comprises (a) an antibody or antigen-binding fragment thereof that binds to EDB; (b) a linker; and (c) a drug, wherein the drug is a cytotoxic agent. In some embodiments, the drug is an auristatin. In some embodiments, the drug is a CPI or CBI dimer. In some embodiments, the auristatin is 0101, 1569, 9411, or 4574. In some embodiments, the CPI dimer is CPI-8314 or CPI-0326.

[0162] In some embodiments of the invention, the EDB ADCs of the invention comprise (a) an antibody or antigen-binding fragment thereof comprising a heavy chain comprising SEQ ID NO:8 and a light chain comprising SEQ ID NO:15; a heavy chain comprising SEQ ID NO:8 and a light chain comprising SEQ ID NO:31; a heavy chain comprising SEQ ID NO:17 and a light chain comprising SEQ ID NO:15; a heavy chain comprising SEQ ID NO:17 and a light chain comprising SEQ ID NO:31; a heavy chain comprising SEQ ID NO:19 and a light chain comprising SEQ ID NO:15; a heavy chain comprising SEQ ID NO:19 and a light chain comprising SEQ ID NO:31; a heavy chain comprising SEQ ID NO:23 and a light chain comprising SEQ ID NO:15; a heavy chain comprising SEQ ID NO:23 and a light chain comprising SEQ ID NO:31; a heavy chain comprising SEQ ID NO:25 and a light chain comprising SEQ ID NO:15; a heavy chain comprising SEQ ID NO:25 and a light chain comprising SEQ ID NO:31; a heavy chain comprising SEQ ID NO:27 and a light chain comprising SEQ ID NO:15; a heavy chain comprising SEQ ID NO:27 and a light chain comprising SEQ ID NO:31; a heavy chain comprising SEQ ID NO:29 and a light chain comprising SEQ ID NO:15; or a heavy chain comprising SEQ ID NO:29 and a light chain comprising SEQ ID NO:31; (b) a linker, and (c) a drug. In some embodiments, the linker is a cleavable or non-cleavable linker. In some embodiments, the linker is vc, diS, diS-COCO, or AcLys-vc. In some embodiments, the drug is a cytotoxic agent. In some embodiments, the drug is an auristatin. In some embodiments, the drug is a CPI or CBI dimer. In some embodiments, the auristatin is 0101, 1569, 9411, or 4574. In some embodiments, the CPI dimer is CPI-8314 or CPI-0326.

[0163] Using EDB ADC The anti-EDB antibodies and EDB ADCs of the invention are useful in a variety of applications, including, but not limited to, therapeutic and diagnostic treatment methods.

[0164] The present invention provides methods for treating an EDB+FN-expressing disorder or disease in a subject, such as a non-cancer or cancer associated with EDB+FN expression and / or an EDB+FN-expressing cancer. The present invention also provides an EDB ADC or pharmaceutical composition described herein for use in a method for treating an EDB+FN-expressing disorder in a subject, such as a non-cancer or cancer associated with EDB+FN expression and / or an EDB+FN-expressing cancer. The present invention further provides use of an EDB ADC or pharmaceutical composition described herein in the manufacture of a medicament for treating an EDB+FN-expressing disorder in a subject, such as a non-cancer or cancer associated with EDB+FN expression and / or an EDB+FN-expressing cancer.

[0165] In some aspects, the present invention provides methods for inhibiting tumor growth or progression in a subject with an EDB-expressing disorder, e.g., a non-cancer or cancer associated with EDB+FN expression and / or an EDB-expressing cancer, comprising administering to a subject in need thereof an effective amount of a composition (i.e., pharmaceutical composition) comprising one or more EDB ADCs described herein. In other aspects of the present invention, methods for inhibiting metastasis of cancer cells associated with EDB+FN expression and / or an EDB+FN-expressing cancer in a subject are provided, comprising administering to a subject in need thereof an effective amount of a composition (i.e., pharmaceutical composition) comprising one or more EDB ADCs described herein. In other aspects of the present invention, methods for inducing regression of tumors associated with EDB+FN expression and / or an EDB+FN-expressing cancer in a subject are provided, comprising administering to a subject in need thereof an effective amount of a composition (i.e., pharmaceutical composition) comprising one or more EDB ADCs described herein.

[0166] In some aspects, this EDB+FN expression can be detected in the extracellular matrix (ECM) adjacent to tumor cells. EDB+FN can be expressed by cells other than fibroblasts in the tumor microenvironment, including tumor cells. The secreted EDB+FN can then be deposited in the matrix adjacent to tumor cells or on the plasma membrane of tumor cells. In other aspects, the present invention provides pharmaceutical compositions comprising one or more EDB ADCs described herein for use in the above methods. In other aspects, the present invention provides the use of one or more EDB ADCs described herein, or pharmaceutical compositions comprising EDB ADCs described herein, in the manufacture of a medicament for use in the above methods.

[0167] Cancers associated with EDB+FN expression and / or EDB+FN-expressing cancers can generally include any cancer associated with tissue remodeling. Furthermore, cancers associated with EDB+FN expression and / or EDB+FN-expressing cancers can include, but are not limited to, solid tumors and blood cancers. In some embodiments, solid tumors include, but are not limited to, thyroid cancer, sarcoma, breast cancer, pancreatic cancer, glioblastoma, gallbladder cancer, kidney cancer, skin cancer, uterine cancer, mesothelioma, colorectal cancer, head and neck cancer, ovarian cancer, bladder cancer, testicular cancer, prostate cancer, liver cancer, endocrine cancer, thymic cancer, brain tumor, adrenal cancer, eye cancer, cervical cancer and lung cancer. In another embodiment, blood cancers include, but are not limited to, leukemia, lymphoma and myeloma.

[0168] The EDB ADCs of the invention are useful in treating EDB+FN-expressing disorders, such as cancers associated with EDB+FN expression and / or EDB+FN-expressing cancers. The EDB ADCs of the invention can be used to treat cancers that express high levels of EDB+FN, moderate levels of EDB+FN, or low levels of EDB+FN.

[0169] Thus, patients for treatment with the EDB ADCs of the invention can be selected based on biomarker expression, including, but not limited to, mRNA (qPCR) of bulk tumor samples, and elevated expression of EDB+FN protein, which occurs in patient populations selected for enriched target expression rather than tumor origin or histology. Target expression can be measured as a function of the number of cells staining combined with the intensity of cell staining.

[0170] Cancer growth or abnormal proliferation refers to any one of several indicators that suggest intracellular changes to more advanced cancerous forms or disorder states.The inhibition of the growth of cancer cells or cells of non-neoplastic proliferative disorders can be measured by methods known in the art, for example, by inhibiting delayed tumor growth and metastasis.Other indicators for measuring the inhibition of cancer growth include the reduction in cancer cell survival, the reduction in tumor volume or morphology (for example, by using computed tomography (CT), ultrasound examination or other imaging methods), the destruction of tumor vasculature, the improved performance in delayed-type hypersensitivity skin test, the increase in the activity of cytotoxic T lymphocytes, and the reduction in the level of tumor-specific antigens.

[0171] The desired outcome of the disclosed treatment method is generally a quantifiable measure, compared with control or baseline measurement.As used herein, relative terms, such as " improve ", " increase " or " reduce " refer to the value compared with the measurement value of control or comparison molecule, for example, the measurement value of the same individual before the treatment described herein is started, or the measurement value of control individual (or multiple control individuals) in the absence of treatment described herein.Representative control individual is the individual who suffers from the same form of cancer as the individual who is treated, and is about the same age as the individual who is treated (to ensure that the stage of the disease in the individual who is treated and the control individual is comparable).

[0172] The change or improvement in response to treatment is generally statistically significant.As used herein, the term "significance" or "significant" refers to the statistical analysis of the probability that a non-random relationship exists between two or more entities.The statistical operation of data to determine whether a relationship is "significant" or has "significance" can be "p-value".A p-value below a user-defined cutoff point is considered significant.A p-value of 0.1 or less, less than 0.05, less than 0.01, less than 0.005, or less than 0.001 can be considered significant.

[0173] In vivo detection and diagnosis In another aspect, provided is a method for detecting, diagnosing and / or monitoring EDB+FN expressing disorders, such as cancers associated with EDB+FN expression and / or EDB+FN expressing cancers.For example, the anti-EDB antibody described herein can be labeled with a detectable moiety, such as imaging agent and enzyme-substrate label.The antibody described herein can also be used in in vivo diagnostic assays, for example, in vivo imaging (for example, PET or SPECT), or staining reagents.

[0174] After administering to a subject an EDB ADC in which the drug is a detectable label, and after allowing sufficient time for binding, the biodistribution of antibody-bound EDB+FN protein can be visualized. The disclosed diagnostic method can be used in combination with a treatment method. Furthermore, the EDB ADC of the present invention can be administered for the dual purposes of detection and treatment.

[0175] Representative non-invasive detection methods include scintigraphy (e.g., SPECT (single photon emission computed tomography), PET (positron emission tomography), gamma camera imaging, and linear scanning), magnetic resonance imaging (e.g., conventional magnetic resonance imaging, magnetization transfer imaging (MTI), proton magnetic resonance spectroscopy (MRS), diffusion weighted imaging (DWI), and functional MR imaging (fMRI)), and ultrasound.

[0176] formulation The present invention further provides pharmaceutical compositions comprising any of the EDB ADCs disclosed herein and a pharmaceutically acceptable carrier. Moreover, these compositions may contain more than one EDB ADC disclosed herein.

[0177] The compositions used in the present invention may further comprise pharmaceutically acceptable carriers, excipients, or stabilizers (Remington: The Science and Practice of Pharmacy, 21st Edition, 2005, Lippincott Williams and Wilkins, ed. K.E. Hoover) in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at dosages and concentrations, and include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine antioxidants; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, e.g., methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as Examples of suitable excipients include serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). As used herein, "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a molecule or polymer. Pharmaceutically acceptable excipients are further described herein.

[0178] Various formulations of EDB ADC can be used for administration, including but not limited to formulations containing pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients are known in the art and are relatively inert substances that facilitate the administration of pharmacologically effective substances. For example, excipients can provide form or consistency, or act as diluents. Suitable excipients include, but are not limited to, stabilizers, wetting agents and emulsifiers, salts for adjusting osmolality, encapsulating agents, buffers, and skin penetration enhancers. Excipients and formulations for parenteral and nonparenteral drug delivery are listed in Remington, The Science and Practice of Pharmacy, 20th Edition, Mack Publishing, 2000.

[0179] In some aspects of the invention, these agents may be formulated for administration by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Thus, these agents may be combined with a pharmaceutically acceptable vehicle, such as saline, Ringer's solution, dextrose solution, etc. The particular dosing regimen, i.e., dosage, timing, and repetition, will depend on the particular individual and their medical history.

[0180] Therapeutic formulations of EDB ADC used in accordance with the invention can be prepared for storage by mixing antibody having the desired degree of purity, in the form of a lyophilized formulation or aqueous solution, with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington, The Science and Practice of Pharmacy, 21st ed., Mack Publishing, 2005). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers, e.g., phosphates, citrates, and other organic acids; salts, e.g., sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, e.g., methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; tacrolimus derivatives; and the like. The surfactant may include proteins such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0181] Therapeutic EDB ADC compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. Compositions in accordance with the invention may be in unit dosage form, such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral, or rectal administration, or for administration by inhalation or insufflation.

[0182] Suitable surfactants include, in particular, nonionic agents such as polyoxyethylene sorbitan (e.g., Tween™ 20, 40, 60, 80, or 85) and other sorbitan (e.g., Span™ 20, 40, 60, 80, or 85). Compositions that include a surfactant conveniently contain between 0.05% and 5% surfactant, and may be between 0.1% and 2.5%. It is understood that other ingredients, such as mannitol or other pharmaceutically acceptable vehicles, may be added as needed.

[0183] Suitable emulsions can be prepared using commercially available fat emulsions, such as INTRALIPIDS™, LIPOSYN™, INFONUTROL™, LIPOFUNDIN™ and LIPIPHYSAN™. The active ingredient can be dissolved in a premixed emulsion composition, or alternatively, can be dissolved in an emulsion formed by mixing oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and phospholipid (e.g., egg phospholipid, soybean phospholipid or soybean lecithin) with water. It is understood that other ingredients, such as glycerol or glucose, can be added to adjust the osmotic pressure of the emulsion. Suitable emulsions typically contain up to 20% oil, for example, between 5% and 20% oil. The fat emulsion may contain lipid droplets between 0.1 μm and 1.0 μm, particularly between 0.1 μm and 0.5 μm, and may have a pH ranging from 5.5 to 8.0. The emulsion composition may be a composition prepared by mixing EDB ADC with INTRALIPIDS™ or its components (soybean oil, egg phospholipids, glycerol, and water).

[0184] The present invention also provides kits for use in the methods of the present invention. The kits of the present invention include one or more containers containing an EDB antibody or EDB ADC described herein and instructions for use in accordance with any of the methods of the present invention described herein. Typically, these instructions include instructions for administering the EDB antibody or EDB ADC for the diagnostic or therapeutic treatment.

[0185] Instructions for use of the EBD antibodies or EDB ADCs described herein generally include information regarding dosage, dosing schedule, and route of administration for the intended treatment. These containers may be unit dose, bulk packaging (e.g., multi-dose packaging), or sub-unit doses. Instructions provided in kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0186] The kit of the present invention is in a suitable packaging material. Suitable packaging materials include, but are not limited to, vials, bottles, jars, flexible packaging materials (e.g., sealed Mylar or plastic bags), etc. Packaging for use in combination with certain devices, such as inhalers, nasal administration devices (e.g., atomizers), or injection devices, such as minipumps, is also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an EDB antibody or EDB ADC. The container may further contain a second pharmaceutically active agent.

[0187] Kits may optionally provide additional components, such as buffers and interpretive information. Typically, kits include a container and a label or package insert(s) on or associated with the container.

[0188] Dosage and Administration The present invention provides EDB ADCs administered in an effective dosage. The phrases "effective dosage" or "effective amount," as used herein, refer to the amount of an ADC, drug, payload, compound, or pharmaceutical composition required to achieve any one or more beneficial or desired therapeutic results. For prophylactic use, beneficial or desired results include eliminating or reducing the risk of, reducing the severity of, or delaying the onset of a disorder, including the biochemical, histological, and / or behavioral symptoms of the disorder, its complications, and intermediate pathological phenotypes present during the development of the disorder. For therapeutic use, beneficial or desired results include clinical outcomes, such as reducing the incidence or ameliorating one or more symptoms of various EDB+FN-expressing disorders, such as cancer, reducing the dose of other medications required to treat the disorder, enhancing the effectiveness of another medication, and / or delaying the progression of an EDB+FN-expressing disorder in a patient.

[0189] An effective dosage can be administered in one or more administrations. An effective dosage of an ADC, drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective dosage" can be considered in terms of administering one or more therapeutic agents, and a single agent can be considered to be given in an effective amount if a desired result can be or is achieved in conjunction with one or more other agents.

[0190] For example, when administered to a cancer-bearing subject, an effective amount includes an amount sufficient to induce anti-cancer activity, including cancer cell cytolysis, inhibition of cancer cell proliferation, induction of cancer cell apoptosis, reduction of cancer cell antigen, delay of tumor growth and / or inhibition of metastasis.Tumor shrinkage is well-accepted as a clinical surrogate marker of efficacy.Another well-accepted marker of efficacy is progression-free survival.

[0191] The EDB ADC of the present invention can be administered to an individual via any suitable route. Those skilled in the art should understand that the examples described herein are intended to be illustrative of available techniques, not limiting. Thus, in some embodiments of the present invention, the EDB ADC is administered to an individual according to known methods, for example, intravenously, e.g., as a bolus, or by continuous infusion over a period of time, intramuscularly, intraperitoneally, intracerebrospinal, intracranially, transdermally, subcutaneously, intraarticularly, sublingually, intrasynovially, by insufflation, intrathecally, orally, by inhalation, or topically. Administration can be systemic, e.g., intravenous, or localized. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be directly nebulized, and lyophilized powders can be nebulized after reconstitution. Alternatively, the EDB ADC can be aerosolized using a fluorocarbon formulation and a metered-dose inhaler, or inhaled as a lyophilized and milled powder.

[0192] In some embodiments of the invention, the EDB ADC is administered via a site-specific or targeted local delivery technique. Examples of site-specific or targeted local delivery techniques include various implantable depot sources of the EDB ADC or local delivery catheters, such as infusion catheters, indwelling catheters or needle catheters, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site-specific carriers, direct injection, or direct application.

[0193] For purposes of the present invention, the appropriate dosage of an EDB ADC may depend on the particular EDB ADC (or composition thereof) used, the type and severity of the condition being treated, whether the agent is administered for therapeutic purposes, previous treatments, the patient's clinical history and response to the agent, the patient's clearance rate for the administered agent, and the discretion of the attending physician. The clinician may administer the EDB ADC until a dosage is reached that achieves and exceeds the desired result. The dose and / or frequency may vary over the course of treatment, but may also remain constant. Empirical considerations such as half-life will generally contribute to determining the dosage. For example, antibodies compatible with the human immune system, such as humanized or fully human antibodies, may be used to extend the half-life of the antibody and prevent the antibody from being attacked by the host's immune system. The frequency of administration may be determined and adjusted over the course of treatment and is generally, but not necessarily, based on the treatment and / or suppression and / or amelioration and / or delay of symptoms, such as inhibition or delay of tumor growth. Alternatively, continuous sustained-release formulations of EDB ADC may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0194] For purposes of the present invention, a typical daily dosage can range anywhere from about 3 μg / kg, up to 30 μg / kg, up to 300 μg / kg, up to 3 mg / kg, up to 30 mg / kg, up to 100 mg / kg, or more, depending on the factors mentioned above. For example, dosages of about 1 mg / kg, about 2.5 mg / kg, about 5 mg / kg, about 10 mg / kg, and about 25 mg / kg can be used. For repeated administration over several days or longer, depending on the disorder, treatment is sustained until a desired suppression of symptoms occurs or until a therapeutic level sufficient to, for example, inhibit or delay tumor growth / cancer cell progression or metastasis is achieved. An exemplary dosing regimen can include administering escalating doses (e.g., an initial dose of 1 mg / kg and gradual increases to one or more higher doses weekly or over a longer period). Other dosing regimens may also be useful, depending on the pharmacokinetic decay pattern the practitioner desires to achieve. For example, in some embodiments of the present invention, administration is contemplated once to four times a week. In other embodiments, administration is contemplated once a month, once every two months, or once every three months, as well as weekly, every other week, and every three weeks. The progress of this treatment can be easily monitored by conventional techniques and assays. The dosing regimen (including the EDB ADC used) can vary over time.

[0195] In some embodiments of the present invention, the dosage of the EDB ADC can be empirically determined in individuals who have received one or more doses of the EDB ADC. Individuals can be given incremental doses of the EDB ADC. To assess efficacy, the indicators of impairment can be as follows:

[0196] Administration of an EDB ADC according to the methods of the invention can be continuous or intermittent, depending, for example, on the physiological disorder of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those of skill in the art. Administration of the EDB ADC can be essentially continuous over a preselected period of time, or can be in a series of spaced doses.

[0197] Combination treatment In some aspects of the present invention, the methods described herein further comprise treating the subject with an additional form of therapy, which in some aspects is an additional anti-cancer therapy, including but not limited to those used in chemotherapy, radiation, surgery, hormonal therapy, and / or additional immunotherapy.

[0198] The disclosed EDB ADCs can be administered as initial treatments or for the treatment of cancers that are unresponsive to conventional therapies. Furthermore, EDB ADCs can be used in combination with other treatments (e.g., surgical resection, radiation, additional anticancer drugs, etc.) to thereby induce additive or enhanced therapeutic effects and / or reduce the cytotoxicity of some anticancer drugs. The EDB ADCs of the present invention can be co-administered or co-formulated with additional agents, or can be formulated for sequential administration with additional agents in any order.

[0199] The EDB ADCs of the invention can be used in combination with other therapeutic agents, including, but not limited to, therapeutic antibodies, ADCs, immunomodulatory agents, cytotoxic agents, and cytostatic agents. Exemplary agents useful in combination therapy also include any of the drugs described herein above under the subheading "Drugs" as useful in preparing EDB ADCs.

[0200] Therapeutic agents include, but are not limited to, chemotherapeutic agents, vaccines, CAR-T cell-based therapy, radiation therapy, cytokine therapy, vaccines, bispecific antibodies, ADCs, inhibitors of other immunosuppressive pathways, inhibitors of angiogenesis, T cell activators, inhibitors of metabolic pathways, mTOR inhibitors, inhibitors of the adenosine pathway, inlytas, ALK inhibitors, and sunitinib, tyrosine kinase inhibitors, BRAF inhibitors, epigenetic modifiers, inhibitors or depletors of Treg cells and / or myeloid-derived suppressor cells, JAK inhibitors, STAT inhibitors, cyclin-dependent kinase inhibitors, biotherapeutics (VEGF), These include, but are not limited to, administration of immunogenic agents (e.g., attenuated cancerous cells, tumor antigens, antigen-presenting cells, e.g., dendritic cells pulsed with tumor-derived antigens or nucleic acids, immunostimulatory cytokines (e.g., IL-2, IFNa2, GM-CSF), and cells transfected with a gene encoding an immunostimulatory cytokine, e.g., but not limited to, GM-CSF).

[0201] Additional representative antibodies that can be used alone or as ADCs include anti-5T4 antibodies (e.g., A1, A2, and A3), anti-CD19 antibodies, anti-CD20 antibodies (e.g., RITUXAN®, ZEVALIN®, BEXXAR®), anti-CD22 antibodies, anti-CD33 antibodies (e.g., MYLOTARG®), anti-CD33 antibody-drug conjugates, anti-Lewis antibodies, and the like. Y antibodies (e.g., Hu3S193, Mthu3S193, AGmthu3S193), anti-HER-2 antibodies (e.g., HERCEPTIN® (trastuzumab), MDX-210, OMNITARG® (pertuzumab, rhuMAb2C4)), anti-CD52 antibodies (e.g., CAMPATH®), anti-EGFR antibodies (e.g., ERBITUX® (cetuximab), ABX-EGF (panitumumab)), anti-VEGF antibodies (e.g., AVASTIN® (bevacizumab)), anti-DNA / histone complex antibodies (e.g., ch-TNT-1 / b), anti-C These include, but are not limited to, EA antibodies (e.g., CEA-Cide, YMB-1003) hLM609, anti-CD47 antibodies (e.g., 6H9), anti-VEGFR2 (or kinase insert domain-containing receptor, KDR) antibodies (e.g., IMC-1C11), anti-Ep-CAM antibodies (e.g., ING-1), anti-FAP antibodies (e.g., sibrotuzumab), anti-DR4 antibodies (e.g., TRAIL-R), anti-progesterone receptor antibodies (e.g., 2C5), anti-CA19.9 antibodies (e.g., GIVAREX®), and anti-fibrin antibodies (e.g., MH-1).

[0202] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, metuledopa, and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin, and biceresin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including the synthetic analogs KW-2189 and CBI-TMI); eluterobin Pancratistatin; Sarcodictyin; Spongistatin; Nitrogen mustards, e.g., chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembine, phenesterine, prednimustine, trofosfamide, uracil mustard; Nitroso ureas, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, e.g., enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1 and calicheamicin phiM, see e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); dynemicins, including dynemicin A; bisphosphonates, e.g., clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino ... sorbicin and deoxydoxorubicin), pegylated liposomal doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, e.g., mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folic acid analogs, e.g., purine analogues, for example, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues, for example, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, for example, calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal drugs, for example, aminoglutethimide, mitotane, Trilostane; folic acid supplements, e.g., folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziconazole; eflornithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, e.g., maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; fenamet;Pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziconazole; 2,2',2"-trichlorotriethylamine; trichothecines (especially T-2 toxin, verrucarin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabino amide ("Ara-C"); cyclophosphamide; thiotepa; taxoids such as paclitaxel and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0203] Also included are antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY1 17018, onapristone, and toremifene (Fareston); aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, formestane, fadrozole, vorozole, letrozole, and anastrozole; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0204] In some embodiments, the EDB ADC may be used in combination with crizotinib, palbociclib, gemcitabine, cyclophosphamide, fluorouracil, FOLFOX, folinic acid, oxaliplatin, axitinib, sunitinib malate, tofacitinib, bevacizumab, rituximab, and trastuzumab.

[0205] In one embodiment, treatment with an EDB ADC can result in an increase in tumor-infiltrating lymphocytes, an increase in the CD8 / CD4 ratio, an increase in F4 / 80+ macrophages, and / or an increase in immunomodulatory proteins such as PDL1 and 41BB, or any combination thereof. Thus, the combination of an EDB ADC with an immune checkpoint inhibitor or IO agent, such as an anti-41BB agonist and / or an anti-PDL1 antagonist monoclonal antibody, can be effective (see Example 12). Furthermore, the EDB ADC of the present invention alone can have immunomodulatory and immuno-oncology (IO) agent-enabling mechanisms that can be augmented using combined therapy.

[0206] In some embodiments, the EDB ADC is selected from the group consisting of PD-1, PD-L1, CTLA-4, LAG-3, B7-H3, B7-H4, B7-DC(PD-L2), B7-H5, B7-H6, B7-H8, B7-H2, B7-1, B7-2, ICOS, ICOS-L, TIGIT, CD2, CD47, CD80, CD86, CD48, CD58, CD226, CD155, CD112, LAIR1, 2B4, BTLA, CD160, TIM1, TIM-3, TIM4, VISTA(PD-H1), OX40, OX40L, GITR, GITRL, CD70, CD27, 4-1BB, 4-BBL, DR 3, TL1A, CD40, CD40L, CD30, CD30L, LIGHT, HVEM, SLAM (SLAMF1, CD150), SLAMF2 (CD48), SLAMF3 (CD229), SLAMF4 (2B4, CD244), SLAMF5 (CD84), SLAMF6 (N TB-A), SLAMCF7(CS1), SLAMF8(BLAME), SLAMF9(CD2F), CD28, CEACAM1(CD66a), CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM1-3AS They may be used in combination with one or more other therapeutic agents that target immune checkpoint modulators, including, but not limited to, agents (e.g., antibodies) that target CEACAM3C2, CEACAM1-15, PSG1-11, CEACAM1-4C1, CEACAM1-4S, CEACAM1-4L, IDO, TDO, CCR2, the CD39-CD73-adenosine pathway (A2AR), BTK, TIK, CXCR2, CCR4, CCR8, CCR5, the VEGF pathway, CSF-1, or innate immune response modulators.

[0207] For combination therapy, the EDB ADC and / or one or more additional therapeutic agents are administered within any time frame appropriate for the intended therapeutic outcome. Thus, single agents can be administered substantially simultaneously (i.e., as a single formulation, or within minutes or hours) or sequentially in any order. For example, single agent treatments can be administered within about 1 year, e.g., within about 10, 8, 6, 4, or 2 months, or within 4, 3, 2, or 1 week, or within about 5, 4, 3, 2, or 1 day of each other.

[0208] The disclosed combination treatments can induce a synergistic therapeutic effect, i.e., an effect greater than the sum of their individual effects or therapeutic outcomes. For example, a synergistic therapeutic effect can be at least about 2-fold greater, at least about 5-fold greater, at least about 10-fold greater, at least about 20-fold greater, at least about 50-fold greater, or at least about 100-fold greater than the therapeutic effect induced by a single agent or the sum of the therapeutic effects induced by the single agents of a given combination. A synergistic therapeutic effect can also be observed as an increase in therapeutic effect of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% or more compared to the therapeutic effect induced by a single agent or the sum of the therapeutic effects induced by the single agents of a given combination. A synergistic effect can also allow for a reduction in the dosage of therapeutic agents when used in combination. [Example]

[0209] The following examples of specific modes for carrying out the present invention are offered for illustrative purposes only and are not intended to limit the scope of the invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

[0210] Example 1 Generation of anti-EDB antibodies and preparation for conjugation Generation of anti-EDB antibodies cDNAs encoding various fully human antibodies that bind to EDB were constructed using standard molecular biology methodologies and derived from the L19 human monoclonal antibody (hereinafter "anti-EDB-L19" or "EDB-L19" antibody) that specifically binds to EDB. The EDB-L19 antibody contains a human IgG1 constant region with the G1m(a) allotype, with aspartic acid (D) at position 356 and leucine (L) at position 358 (according to the EU index of Kabat), and a human kappa light chain constant region. The EDB-L19 antibody heavy and light chain variable regions are set forth in SEQ ID NOs: 1 and 10, respectively, and the heavy and light chains are set forth in SEQ ID NOs: 8 and 15, respectively.

[0211] To generate a non-immunogenic antibody, a non-Glm(a) allotype with glutamic acid (E) at position 356 and methionine (M) at position 358 (according to the EU index of Kabat) was introduced into the EDB-L19 heavy chain. To generate the heavy chain, the nucleotide sequence encoding the EDB-L19 heavy chain variable region was cloned into Glm(a) z The EDB-L19 antibody IgG1 constant region was fused to human IgG1 constant region cDNAs with non-(a), non-(x) allotypes. In some embodiments, these antibodies were further modified by eliminating the C-terminal lysine (K) of the EDB-L19 antibody IgG1 constant region to generate EDB-PFE HC (SEQ ID NO: 17), thereby reducing antibody charge variants and increasing antibody homogeneity. The EDB-PFE antibody heavy and light chains are set forth in SEQ ID NOs: 17 and 15, respectively.

[0212] Imaging capillary electrophoresis (iCE) was performed using an iCE3 equipped with a Prince Autosampler to determine the percent charge variants for the antibody preparations, as shown in Table 4. The EDB-L19 antibody had a significant increase in basic species and a decrease in the main antibody peak as a result of incomplete C-terminal lysine processing during cell culture, compared to the EDB-PFE antibody.

[0213] [Table 4]

[0214] Antibodies for site-specific conjugation via engineered cysteine ​​residues Methods for preparing anti-EDB antibodies for site-specific conjugation to various linker-payloads via reactive engineered cysteine ​​residues were generally carried out as described in PCT International Patent Application Publication No. WO2013 / 093809, the entire contents of which are incorporated herein by reference. One or more residues on the heavy chain, e.g., position K290 (according to the EU index of Kabat), or one or more residues on the light chain, e.g., position K183 (according to Kabat), were altered to cysteine ​​(C) residues by site-directed mutagenesis.

[0215] In some embodiments, position K290 (according to EU index of Kabat) in the human IgG1 heavy chain constant region of the EDB-PFE antibody was substituted with a reactive cysteine ​​(C) to allow site-specific conjugation to generate EDB-(K290C)HC (SEQ ID NO: 19). In other embodiments, residue K183 (according to Kabat) in the human kappa light chain constant region was substituted with a reactive cysteine ​​(C) to allow site-specific conjugation to generate EDB-(κK183C)LC (SEQ ID NO: 31).

[0216] Antibodies for site-specific conjugation via engineered glutamine residues Anti-EDB antibodies with human IgG1 subtypes engineered with reactive glutamine residues, such as glutamine-containing ("Q") tags, at various amino acid positions were expressed for conjugation to various linker-payloads. Methods for preparing anti-EDB antibodies for site-specific conjugation via reactive glutamine residues were generally carried out as described in PCT International Patent Application Publication No. WO2012 / 059882, the entire contents of which are incorporated herein by reference.

[0217] In some embodiments, an H16-glutamine tag, LLQG (SEQ ID NO: 40), was engineered into the human IgG1-Fc region of the EDB-PFE antibody to enable DAR 2 transglutaminase-mediated site-specific conjugation. For example, in the EDB-PFE antibody heavy chain, amino acids at positions E294 to N297 (according to EU index of Kabat) were replaced with the H16-glutamine-containing tag, LLQG (SEQ ID NO: 40). In other embodiments, these antibodies were further modified to increase the specificity of conjugation to the engineered H16-glutamine-containing tag. The lysine (K) amino acid at position 222 (according to EU index of Kabat) on the heavy chain was substituted with arginine (R) to generate EDB-(H16-K222R)HC (SEQ ID NO: 27). The K222R substitution provided an increase in homogeneous ADC, improved intermolecular cross-linking between antibody and linker-payload, and / or a significant decrease in interchain cross-linking with the H16-glutamine-containing tag on the C-terminus of the antibody light chain.

[0218] Potential Chemical Trends Potential chemical trends, particularly within the CDRs, can affect molecular heterogeneity and result in antigens that bind putative protein glycosylation sites. Protein glycosylation is a non-enzymatic glycosylation that can occur in recombinant antibodies during cell culture, and glycosylated proteins can undergo further reactions to generate poorly characterized heterogeneous products collectively referred to as advanced glycation end products. To mitigate potential glycosylation trends, position K94 (Kabat numbering) adjacent to CDR3 in the EDB-L19 heavy chain variable region was mutated to arginine (R) to generate EDB-(K94R)VH (SEQ ID NO: 21), which was then fused to a human IgG1 constant region to generate EDB-(K94R)HC (SEQ ID NO: 23). A K94R glycosylation mutation was also introduced into the EDB-(K290C) and EDB-(H16-K222R) heavy chains engineered for site-specific conjugation to generate EDB-(K94R-K290C)HC (SEQ ID NO: 25) and EDB-(K94R-H16-K222R)HC (SEQ ID NO: 29), respectively.

[0219] Example 2 Characterization of EDB antibody variant binding properties Binding affinity analysis Surface plasmon resonance (SPR) was used to characterize the binding kinetics of anti-EDB antibody variants to recombinant human, cynomolgus monkey, and rat 7-EDB-89 (SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively), confirming that the binding properties of the anti-EDB antibody with the K94R glycosylation mutation were fully retained. Binding is detected by surface SPR of laser light refracted from the surface. Analysis of the signal kinetics (on-rate (k) and off-rate (kd)) allows for the differentiation between nonspecific and specific interactions.

[0220] Anti-human IgG antibodies (GE Healthcare) were covalently amine-coupled to all four flow cells of a CM5 carboxymethylated dextran-coated sensor chip to a density of approximately 10,000 resonance units (RU) according to the manufacturer's protocol. Each anti-EDB antibody variant was then captured to a level of approximately 60-90 RU. The running and sample buffer used was HBS-EP+ buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, and 0.05% v / v surfactant P20 pH 7.4). A three-fold serial dilution series of 7-EDB-89, ranging from 600 nM to 11.1 nM, was injected over the surface at a flow rate of 50 μL / min for 60 s of association and 120 s of dissociation. The surface was then regenerated with a 30-second pulse of 3 M MgCl, a 30-second pulse of ionic regeneration buffer (0.46 M KSCN, 1.83 M MgCl, 0.92 M urea, and 1.83 M guanidine-HCl pH 7.4), and then equilibrated with a 30-second pulse of HBS-EP+ running buffer. All SPR assays were performed at 25°C using a BIAcore® T200 instrument (GE Healthcare) at a data collection rate of 1 Hz. The resulting sensorgrams were double-referenced using both a control surface and a buffer injection (Myszka, DG, J. Mol. Recognit., 12:279-284, 1999). Rate constants were calculated using the BIAcore® T200 evaluation software v2.0 and the equation K D =k d / k a The K was determined by fitting the data to a 1:1 Langmuir model using the formula: Each experiment was performed in duplicate and the average K D As shown in Table 5, the EDB-L19 and EDB-(K94R) antibodies show comparable binding to human 7-EDB-89. t = half-life, R = maximum response, RU = resonance units.

[0221] [Table 5]

[0222] Furthermore, the binding affinities of the EDB-L19 and EDB-(κK183C-K94R-K290C) antibodies to human, cynomolgus monkey, and rat 7-EDB-89 were determined. As shown in Table 6, the binding affinities of the EDB-L19 and EDB-(κK183C-K94R-K290C) antibodies were similar. As shown in Table 7, the binding affinities of the EDB-(κK183C-K94R-K290C) antibody to human, cynomolgus monkey, and rat 7-EDB-89 were comparable, confirming that cross-species reactivity was retained after engineering the EDB-L19 antibody to allow site-specific conjugation and removal of putative glycosylation sites.

[0223] [Table 6]

[0224] [Table 7]

[0225] Competitive binding by ELISA The binding properties of the EDB-(K94R) and EDB-(κK183C-K94R-K290C) antibodies were further evaluated using a competitive ELISA with biotinylated EDB-L19 to confirm that binding to EDB was fully maintained. Human 7-EDB-89 (SEQ ID NO: 34) was immobilized (100 ng / well) on a 96-well ELISA plate, and 20 ng / mL biotinylated EDB-L19 antibody was added to compete with varying concentrations of modified anti-EDB antibody samples. Binding was detected using an anti-streptavidin-HRP antibody (Southern Biotech, Birmingham, AL).

[0226] As shown in Figure 1A and Table 8, the EDB-L19 and EDB-(K94R) antibodies had similar half-maximal inhibitory concentration values. Figure 1B and Table 9 show that the EDB-(K94R) and EDB-(κK183C-K94R-K290C) antibodies also had similar half-maximal inhibitory concentration values. This indicates that the EDB-(K94R) and EDB-(κK183C-K94R-K290C) modified antibodies retained EDB-binding properties and that the (K94R) modification of the heavy chain and / or the introduction of a reactive engineered cysteine ​​for site-specific conjugation did not alter binding to EDB.

[0227] [Table 8]

[0228] [Table 9]

[0229] Bond strength analysis The affinity for the EDB-L19 antibody to bind EDB was determined to be approximately 230 nM, a low binding interaction. Therefore, SPR was used to investigate whether binding strength affected differential target levels in the tumor microenvironment. Varying densities of human 7-EDB-89 (SEQ ID NO: 34) were covalently amine-coupled onto individual flow cells of a CM5 carboxymethylated dextran-coated sensor chip. Running and sample buffers were as described above for binding affinity analysis. A 3-fold serial dilution series of EDB-L19 antibody, ranging from 6 nM to 0.074 nM, was injected at a flow rate of 50 μL / min over a 110-second association period and a 900-second dissociation period. The surface was then regenerated with two 30-second pulses of ionic regeneration buffer (0.46 M KSCN, 1.83 M MgCl, 0.92 M urea, and 1.83 M guanidine-HCl pH 7.4) and then equilibrated with a 30-second pulse of HBS-EP+ running buffer. Each experiment was performed in duplicate, and the average k, kd, and K were measured. DIt was decided that:

[0230] As shown in Table 10, the results showed that as the level of immobilized human 7-EDB-89 increased, the off-rate (kd) slowed and the affinity subsequently increased. D The values ​​were proportional to the immobilization level of human 7-EDB-89, confirming that the EDB-L19 antibody binds EDB with a large avidity component.

[0231] [Table 10]

[0232] Multiple reactivity of anti-EDB antibodies Multiple reactivity is associated with rapid clearance in vivo (Hotzel et al., mAbs 4(6):753-760, 2012) and undesirable protein-protein interactions (Xu et al., Protein Eng Des Sel 26(10):663-670 (2013)). DNA and insulin direct binding ELISAs have been shown to correlate with known pharmacokinetics (PK) of clinically validated antibodies. Serial dilutions of antibodies starting at 10 µg / mL in quadruplicate were evaluated in low stringency assays for binding to either DNA or insulin coated directly onto ELISA plates.

[0233] As shown in Table 11, both the EDB-(K94R) and EDB-(κK183C-K94R-K290C) antibodies had very low multireactivity scores that were comparable to or better than the negative control, which had optimal PK properties. Furthermore, the multireactivity scores were significantly lower than the positive control antibody, which had poor PK and resulted in rapid clearance.

[0234] [Table 11]

[0235] FcRn chromatography FcRn chromatography was utilized to investigate the potential charge-mediated impact of introducing reactive engineered cysteines into the wild-type IgG1 constant region on FcRn-dependent pharmacokinetics. Evaluation of antibodies using FcRn column methodology demonstrated that elution time positively correlated with human and non-human primate clearance (Schoch A. et al., PNAS, 2015, Vol. 112). The FcRn affinity column was prepared according to Schlothauer et al., MAbs 5(4):576-586, 2013. Next, 50 μg of EDB-(κK183C-K94R-K290C) antibody or EDB-(κK183C-K94R-K290C)-vc-0101 ADC was injected, followed by centrifugation in 20 mM MES, 150 mM NaCl, pH 5.5, and 20 mM Tris, 150 mM NaCl. NaCl, pH 8.8, was used as the eluent and eluted with a linear pH gradient (30 CV) from pH 5.5 to 8.8 within 60 min.

[0236] As shown in Table 12, the FcRn column relative elution times of the EDB-(κK183C-K94R-K290C) antibody and EDB-(κK183C-K94R-K290C)-vc-0101 ADC were consistent with acceptable PK parameters. These data demonstrate that incorporation of the reactive engineered cysteine ​​residue K290C in the IgG1 constant region does not affect FcRn binding.

[0237] [Table 12]

[0238] Example 3 Bioconjugation of EDB ADC The anti-EDB antibodies of the present invention were conjugated to a drug / payload via a linker to produce EDB ADCs. The conjugation method used was either conventional conjugation (i.e., via a random cysteine ​​residue) or site-specific conjugation (i.e., via an engineered cysteine ​​residue or an engineered glutamine residue). Table 13 shows the conjugation methods used for various EDB ADCs.

[0239] Method A: Conventional conjugation via cysteine ​​residues Anti-EDB antibody at 27 mg / ml in PBS, pH 7.2, was reduced with 2.3-2.6x (m / m) TCEP for 2 hours at 37°C and then conjugated. The molar ratio was generally 2.5x but was optimized depending on the amount of antibody conjugate to achieve an optimal final average DAR of approximately 4.0. The partially reduced antibody was conjugated with 6-7x (m / m) linker-payload in PBS with 10% DMA for 1 hour at 25°C. Excess linker-payload was quenched with L-cysteine ​​for 15 minutes at 25°C. The crude ADC was dialyzed overnight in PBS at 4-6°C.

[0240] The crude ADC was purified by size-exclusion chromatography (SEC) on Superdex 200 in PBS, and the collected monomer peak was stored at 4-6°C or dialyzed in 20 mM histidine, 8.5% sucrose, pH 5.8; sterile filtered, and frozen at -70°C. The negative control huNeg-8.8 antibody was conjugated by the same method.

[0241] Method B: Site-specific conjugation via engineered cysteine ​​residues Two grams of anti-EDB antibody produced with reactive engineered cysteine ​​residues, 27.2 mg / ml in PBS, pH 7.2, was reduced with 15x (m / m) TCEP for 7 hours at 37°C and desalted with Sephadex® G-25 in PBS to remove excess TCEP. Interchain cysteines were oxidized with 30x DHA (m / m) overnight at 4-6°C. DHA was removed by desalting with Sephadex® G-25 in PBS. For ADCs with a higher degree of glutathione capping instead of the preferred cysteine ​​capping of site-specific cysteines, 100x TCEP (m / m) was used for reduction.

[0242] The reduced and oxidized antibody was conjugated with 9x (m / m) linker-payload in PBS with 10% DMA for 2 hours at 25°C. Excess linker-payload was quenched with 9x (m / m) L-cysteine ​​for 15 minutes at 25°C. The crude ADC was dialyzed overnight in PBS at 4-6°C.

[0243] The crude ADC was purified by SEC on Superdex 200 in PBS, and the collected monomer peak was dialyzed in 20 mM histidine, 8.5% sucrose, pH 5.8; sterile filtered, and frozen at −70° C. The negative control huNeg-8.8 antibody was conjugated by the same method.

[0244] Method C: Site-specific conjugation via engineered glutamine residues Anti-EDB antibodies produced with reactive engineered glutamine residues were dialyzed in reaction buffer: 100 mM phosphate, 200 mM NaCl, pH 7.0. 20 mg / ml of antibody was conjugated to linker-payload (10x m / m) using 1 unit of commercially available purified transglutaminase (TG) per mg of antibody in 100 mM potassium phosphate, 200 mM NaCl, 10% DMSO for 15 hours at room temperature. The crude ADC was centrifuged, and the supernatant was purified by SEC.

[0245] The crude ADC was purified by SEC on Superdex 200 in PBS, and the collected monomer peak was dialyzed in 20 mM histidine, 8.5% sucrose, pH 5.8; sterile filtered, and frozen at −70° C. The negative control huNeg-8.8 antibody was conjugated by the same method.

[0246] Method D: Conventional conjugation via cysteine ​​residues using a disulfide linker Anti-EDB antibody at 27 mg / ml in PBS, pH 7.2, was partially reduced with 5x (m / m) TCEP for 2 hours at 37°C and desalted using Sephadex® G-25 SEC.

[0247] The partially reduced antibody was diluted in 67 mM DMSO with 0.7 mM DTPA and 7% DMA. Conjugation was performed with 12–15x (m / m) reduced linker-payload in HEPES, pH 7.0, for 15 min at 25°C. Excess linker-payload was quenched with 20x NEM (m / m) for 15 min at 25°C.

[0248] The crude ADC was purified by SEC on Superdex 200 in PBS with 50 mM DHA and 50 mM DTPA, and the collected monomer peak was stored at 4-6° C. A negative control was conjugated by the same method.

[0249] [Table 13-1]

[0250] [Table 13-2]

[0251] Example 4 EDB ADC characterization The EDB ADCs of the present invention were characterized using a combination of size exclusion chromatography (SEC), LC-MS, and hydrophobic interaction chromatography (HIC). The average drug:antibody ratio (DAR) was determined by mass spectrometry (MS). Table 14 provides analytical characteristics of various EDB ADCs.

[0252] LC-MS: Column = Waters BEH300-C4, 2.1 x 100 mm (P / N = 186004496); Instrument = Acquity UPLC with SQD2 mass spectrometer detector; Flow rate = 0.7 mL / min; Temperature = 80 °C; Buffer A = water + 0.1% formic acid; Buffer B = acetonitrile + 0.1% formic acid. The gradient was from 3% B to 95% B over 2 min, held at 95% B for 0.75 min, and then re-equilibrated to 3% B. Samples were reduced with TCEP or DTT immediately prior to injection. The eluate was monitored by LCMS (400-2000 daltons), and protein peaks were deconvoluted using MaxEnt1. DAR is reported as weight average loading, as previously described.

[0253] SEC: Column: Superdex200 (5 / 150 GL); Mobile phase: phosphate buffer solution containing 2% acetonitrile, pH 7.4; Flow rate = 0.25 mL / min; Temperature = ambient; Instrument: Agilent 1100 HPLC.

[0254] HIC: Column: TSKGel Butyl NPR, 4.6 mm x 3.5 cm (P / N=S0557-835); Buffer A = 1.5 M ammonium sulfate containing 10 mM phosphate, pH 7; Buffer B = 10 mM phosphate, pH 7 + 20% isopropyl alcohol; Flow rate = 0.8 mL / min; Temperature = ambient; Gradient = 0% B to 100% B over 12 min, hold at 100% B for 2 min, then re-equilibrate to 100% A; Instrument: Agilent 1100 HPLC.

[0255] [Table 14]

[0256] Example 5 EDB+FN expression To conduct a broad survey of cancer indications for EDB ADC-based therapy, EDB+FN expression was analyzed at the protein and mRNA levels in human tumors and PDX models.

[0257] RNA-Seq analysis of EDB+FN expression RNA-Seq data from 10,660 individual tumor samples collected as part of The Cancer Genome Atlas (TCGA) project (National Cancer Institute, HI, Bethesda, MD) spanning 31 tumor types were analyzed. Isoform-level expression data were obtained from OmicSoft software (Cary, NC). EDB+FN expression was calculated as the sum of the expression levels of EDB-bearing fibronectin (FN1) isoforms. Expression levels were calculated as fragments per kilobase of transcript per million reads. The number of transcripts per million reads (FPKM) was measured for each tumor type. Summary statistics for EDB+FN expression levels are shown in Table 15. Generally, a gene is considered expressed if the FPKM is about 1 or greater.

[0258] Table 15 shows RNA-Seq analysis of EDB+FN in human tumors. EDB+FN expression is demonstrated in a wide range of human tumor indications, including, but not limited to, thyroid carcinoma, sarcoma, breast cancer, pancreatic adenocarcinoma, glioblastoma, cholangiocarcinoma, lung adenocarcinoma, renal carcinoma, melanoma, uterine carcinosarcoma, mesothelioma, lung squamous cell carcinoma, rectal and colon adenocarcinoma, liver hepatocellular carcinoma, colon carcinoma, ovarian serous cystadenocarcinoma, and bladder carcinoma.

[0259] [Table 15]

[0260] Gene expression quantification was performed using the RSEM program on RNA-Seq data from 160 Pfizer internal patient-derived xenograft (PDX) models derived from breast, ovarian, head and neck, colorectal, melanoma, pancreatic, non-small cell lung cancer (NSCLC), and small cell lung cancer. See Li et al., BMC Bioinformatics, 12:323, 2011. EDB+FN expression was calculated as the sum of the expression levels of EDB-bearing fibronectin (FN1) isoforms. As shown in Figure 2, EDB+FN was expressed at varying levels across all tumor types analyzed (all samples had levels >1). Data are presented as fragments per kilobase of transcript per million reads (FPKM).

[0261] Immunohistochemistry (IHC) detection of EDB+FN expression EDB+FN protein expression in human cancers was verified by IHC using the EDB-L19 antibody on frozen sections. Freshly frozen 8-micron tissue sections embedded in Tissue-Tek OCT Compound (Sakura Finetek) were fixed in a 3:1 mixture of acetone and 100% ethanol for 4 minutes and then immersed in 10% neutral-buffered formalin for 20 seconds. Slides were rinsed in TBS. Endogenous peroxidase activity was inactivated with Peroxidazed 1 (Biocare Medical) for 10 minutes. Nonspecific protein interactions were blocked with Background Punisher (Biocare Medical) for 10 minutes. The EDB-L19 antibody or the isotype-negative control huNeg-8.8 antibody was precomplexed with rabbit anti-human IgG (Jackson ImmunoResearch) at final concentrations of 3 μg / ml and 0.5 μg / ml, respectively, for 1 hour at room temperature. The precomplexed mixture was incubated with excess whole human IgG (Jackson ImmunoResearch) for 15 minutes at room temperature and then added to slides for 1 hour. Sections were washed in TBS and incubated with SignalStain Boost Rabbit HRP (CellSignaling Technologies) for 30 minutes. The color signal was developed with DAB+ (Dako) for 5 minutes and subsequently quenched with distilled H2O. Slides were briefly counterstained with CAT hematoxylin (Biocare Medical), washed in water, dehydrated in graded alcohols, cleared in xylene, and coverslipped with Permount Mounting Medium (Fisher Chemicals). Expression analysis was performed and confirmed.

[0262] As shown in Table 16, EDB+FN protein was expressed at moderate to high levels across all human cancer indications profiled, including head and neck cancer (data not shown), pancreatic cancer, non-small cell lung cancer (NSCLC), ovarian cancer, and breast cancer. Expression in all tumors was predominantly stromal (including fibroblastic and vasculature-associated), although some staining of tumor cells was also observed.

[0263] [Table 16]

[0264] Example 6 In vitro binding of EDB ADC To evaluate the relative binding of anti-EDB antibodies and EDB ADCs to EDB, MaxiSorp 96-well plates were coated with 0.5 or 1 μg / ml of human 7-EDB-89 (SEQ ID NO: 34) in PBS and incubated overnight at 4°C with gentle shaking. The plates were then emptied, washed with 200 μl of PBS, and blocked with 100 μl of blocking buffer (ThermoScientific) at room temperature for 3 hours. The blocking buffer was removed, and the wells were washed with PBS and incubated with 100 μl of serially diluted (4-fold) anti-EDB antibodies or EDB ADCs in ELISA assay buffer (EAB; 0.5% BSA / 0.02% Tween-20 / PBS). The first column of the plate was left empty, and the last column of the plate was filled with EAB as a blank control. The plate was incubated at room temperature for 3 hours. The reagent was removed, and the plate was washed with 200 μl of 0.03% Tween-20 in PBS (PBST). Anti-human IgG-Fc-HRP (Thermo / Pierce), diluted 1:5000 in EAB, was added to the wells in 100 μl of solution and incubated at room temperature for 15 minutes. The plate was washed with 200 μl of PBST, and then 100 μl of BioFX TMB (Fisher) was added and developed at room temperature for 4 minutes. The reaction was stopped with 100 μl of 0.2 N sulfuric acid, and the absorbance at 450 nm was read on a Victor plate reader (Perkin Elmer, Waltham, MA).

[0265] Table 17 provides the relative binding of anti-EDB antibodies and EDB ADCs to the human 7-EDB-89 protein fragment bound to a 96-well plate in an ELISA format. All antibodies and ADCs targeting EDB bound to the target protein with similar affinities ranging from 19 pM to 58 pM. In contrast, non-EDB-targeting antibodies and ADCs had high EC of >10,000 pM. 50 Representative ELISA binding curves are shown in Figures 3A and 3B.

[0266] [Table 17]

[0267] Example 7 In vitro cytotoxicity of EDB ADCs cell culture WI38-VA13 are SV40-transformed human lung fibroblasts obtained from ATCC and maintained in MEM Eagle's medium (Cell-Gro) supplemented with 10% FBS, 1% MEM non-essential amino acids, 1% sodium pyruvate, 100 units / ml penicillin-streptomycin, and 2 mM GlutaMax. HT29 are derived from a human colorectal carcinoma (ATCC) and maintained in DMEM medium supplemented with 10% FBS and 1% glutamine.

[0268] Detection of EDB+FN transcripts For gene expression and transcript analysis of EDB+FN, adherent, growing WI38-VA13 and HT29 cells were dissociated from cell culture flasks using TrypLE Express (Gibco). Total RNA was purified from the collected cell pellet using the RNeasy Mini Kit (Qiagen). Residual DNA was removed during RNA purification with an RNase-Free DNase Set (Qiagen). The High Capacity RNA-to-cDNA Kit (Applied Biosystems) was used for reverse transcription of total RNA to cDNA. cDNA was analyzed by quantitative real-time PCR using TaqMan Universal Master Mix II with UNG (Applied Biosystems). EDB+FN signals were detected with TaqMan primer Hs01565271_m1 and normalized to the average of both ACTB (TaqMan primer Hs99999903_m1) and GAPDH (TaqMan primer Hs99999905_m1) signals. All primers were from ThermoFisher Scientific. Data from a representative experiment are shown.

[0269] Detection of EDB+FN protein by Western blotting For detection of EDB+FN by Western blotting, adherent, growing WI38-VA13 and HT29 cells were harvested by cell detachment. Cell lysates were prepared in cell lysis buffer (Cell Signaling Technology) containing protease and phosphatase inhibitors. Tumor lysates were lysed in RIPA lysis buffer or 2x cell lysis buffer (Cell Signaling Technology) containing protease and phosphatase inhibitors. Cell Signaling Technology). Protein lysates were analyzed by SDS-PAGE followed by Western blotting. Proteins were transferred to nitrocellulose membranes, blocked with 5% milk / TBS, and then incubated with EDB-L19 and anti-GAPDH antibodies (Cell Signaling Technology) overnight at 4°C. After washing, the anti-EDB blots were incubated with ECL HRP-linked anti-human IgG secondary antibody (GE Healthcare) for 1 hour at room temperature. After washing, the EDB + FN signal was developed with Pierce ECL 2 Western blotting substrate (Thermo Scientific) and detected with X-ray film. The anti-GAPDH blots were incubated with Alexa Fluor 680-conjugated anti-rabbit IgG secondary antibody (Invitrogen) in blocking buffer for 1 hour at room temperature. After washing, the GAPDH signal was detected with a LI-COR Odyssey Imaging System. Densitometric analysis of EDB+FN Western blots was performed using a Bio-Rad GS-800 Calibrated Imaging Densitometer and quantified using Quantity One version 4.6.9 software. Data from a representative experiment are shown.

[0270] Figure 4 shows EDB+FN expression by Western blot in WI38-VA13 and HT29 cells. EDB+FN is expressed in the WI38-VA13 cell line, while the HT29 colon cancer cell line is negative when grown in vitro.

[0271] Detection of EDB+FN protein by flow cytometry The expression of EDB+FN on the cell surface of WI38-VA13 or HT29 cells was measured by flow cytometry using the EDB-L19 antibody. Cells were dissociated with non-enzymatic cell dissociation buffer (Gibco) and incubated with cold flow buffer (FB, 3% BSA / PBS + Ca + Mg) on ​​ice for blocking. Cells were then incubated with primary antibodies in FB on ice. After incubation, cells were washed with cold PBS-Ca-Mg and then incubated with viability stain (Biosciences) according to the manufacturer's protocol to distinguish live and dead cells. Signals were analyzed on a BD Fortessa flow cytometer, and data were analyzed using BD FACS DIVA software. Data from a representative experiment are shown.

[0272] Table 18 summarizes the results from Western blot, qRT-PCR, and flow cytometry. The data demonstrate that WI38-VA13 is EDB+FN positive and HT29 is EDB+FN negative.

[0273] [Table 18]

[0274] In vitro cytotoxicity assay Proliferating WI38-VA13 or HT29 cells were harvested from culture flasks using non-enzymatic cell dissociation buffer and cultured overnight in 96-well plates (Corning) at 1000 cells / well in a humidified chamber (37°C, 5% CO2). The following day, cells were treated with EDB ADCs or isotype control non-EDB-binding ADCs by adding 50 μl of 3x stock in duplicate at 10 concentrations. In some experiments, cells were plated at 1500 cells / well and treated on the same day. Cells were then incubated with EDB ADCs or isotype control non-EDB-binding ADCs for 4 days. On the day of harvest, 50 μl of Cell Titer Glo (Promega) was added to the cells and incubated at room temperature for 0.5 hours. Luminescence was measured using a Victor plate reader (Perkin The IC was measured using a 100% RT-PCR kit (Elmer, Waltham, MA). Relative cell viability was determined as a percentage of untreated control wells. 50 Values ​​were calculated using XLfit v4.2 (IDBS) using a four parameter logistic model #203.

[0275] Table 19 shows the IC of EDB ADC treatment in cytotoxicity assays performed against WI38-VA13 (an EDB+FN-positive tumor cell line) and HT29 colon cancer cells (an EDB+FN-negative tumor cell line). 50 (ng / ml of antibody) are shown. EDB ADC induced cell death in EDB+FN-expressing cell lines. IC 50 Values ​​ranged from approximately 184 ng / ml to 216 ng / ml and were similar for all EDB ADCs with vc-0101 linker-payload (EDB-L19-vc-0101, EDB-(κK183C-K290C)-vc-0101, EDB-(K94R)-vc-0101, EDB-(κK183C-K94R-K290C)-vc-0101). The ADC is quite weak, and the IC 50 The IC values ​​were approximately 70-200 times higher than those of the EDB-vc-0101 ADC. All vc-0101 ADCs had IC values ​​46-83 times higher in the EDB+FN-negative tumor cell line HT29.50 Thus, the EDB ADC relied on EDB+FN expression for its in vitro cytotoxicity.

[0276] Other auristatin-based EDB ADCs with "vc" protease-cleavable linkers, EDB-L19-vc-9411 and EDB-L19-vc-1569, also demonstrated potent cytotoxicity in WA38-VA13 cells, with approximately 50- to 180-fold higher selectivity compared to the corresponding negative control ADCs and approximately 25- to 140-fold higher selectivity compared to non-expressing cell lines. The EDB-L19-diS-DM1 ADC had similar potency to the vc-0101 ADC but was significantly less selective compared to the negative control ADC (approximately 3-fold) and HT29 cells (approximately 0.9-fold).

[0277] [Table 19]

[0278] As shown in Table 20, the unconjugated payload was highly potent in both cell lines, regardless of EDB+FN expression, indicating that these cells were sensitive to the cytotoxic agents used as ADC payloads.

[0279] [Table 20]

[0280] Example 8 In vivo efficacy of EDB ADC EDB ADCs were evaluated in cell line xenografts (CLXs), patient-derived xenografts (PDXs), and syngeneic tumor models. EDB+FN expression was detected using immunohistochemistry (IHC) assays as previously described herein.

[0281] To generate the CLX model, 8 × 10 6 ~10×10 6H-1975, HT29, or Ramos tumor lines of cells were implanted subcutaneously into female athymic nude mice. Ramos and H-1975 cells for inoculation were suspended in 50% and 100% Matrigel (BD Biosciences), respectively. For the Ramos model, animals received total body irradiation (4 Gy) prior to cell inoculation to promote tumor establishment. Average tumor volumes were approximately 160–320 mm. 3 Once tumors reached 100 mg / kg / day, animals were randomized into treatment groups with 8-10 mice in each group. ADC or vehicle (PBS) was administered intravenously on day 0, and animals were then dosed once every 4 days for 4-8 doses. Tumors were measured once or twice weekly, and tumor volumes were recorded as volume (mm 3 ) = (width × width × length) / 2. Animal weights were monitored for 4 to 9 weeks, and no weight loss was observed in any of the treatment groups.

[0282] To generate PDX models, tumors were harvested from donor animals, and approximately 3 × 3 mm tumor fragments were implanted subcutaneously into the flanks of female athymic nude mice (for the PDX-NSX-11122 model) or NOD SCID mice (for the PDX-PAX-13565 and PDX-PAX-12534 models) using a 10-gauge trocar. The mean tumor volume was approximately 160–260 mm. 3 Once tumor growth reached 100%, mice were randomized into treatment groups with 7–10 mice in each group. The ADC or vehicle (PBS) dosing regimen and route of administration, as well as tumor measurement procedures, were the same as those described above for the CLX model. Animal weights were monitored for 5–14 weeks, and no weight loss was observed in any of the treatment groups. Tumor growth inhibition is plotted as the mean tumor size ± standard error of the mean.

[0283] Expression of EDB+FN As shown in Table 21, expression of EDB+FN in the H-1975, HT29, and Ramos CLX models, the PDX-NSX-11122, PDX-PAX-13565, and PDX-PAX-12534 PDX models, and the EMT-6 syngeneic tumor model was measured by EDB-L19 antibody binding and subsequent detection in an IHC assay. The CLX HT-29 was a moderately expressing CLX, but was negative when tested in vitro due to most of the protein expression in CLX coming from the tumor stroma.

[0284] [Table 21]

[0285] PDX-NSX-11122 NSCLC PDX The efficacy of various ADCs was evaluated in PDX-NSX-11122, a human NSCLC PDX model expressing high levels of EDB+FN. Figure 5A shows the antitumor activity of EDB-L19-vc-0101 at 0.3, 0.75, 1.5, and 3 mg / kg. The data demonstrate that EDB-L19-vc-0101 exhibited tumor regression in a dose-dependent manner at 3 mg / kg and 1.5 mg / kg.

[0286] The antitumor efficacy of vc-linked ADCs was compared with disulfide-linked ADCs. Figures 5B and 5C show the antitumor activity of 3 mg / kg EDB-L19-vc-0101 compared with 10 mg / kg disulfide-linked EDB-L19-diS-DM1, and the antitumor activity of 1 and 3 mg / kg EDB-L19-vc-0101 compared with 5 mg / kg disulfide-linked EDB-L19-diS-COCO-1569, respectively. As shown in Figures 5B and 5C, EDB-L19-vc-0101 demonstrated greater efficacy than the isotype-negative control ADC and the ADCs generated using disulfide linkers, EDB-L19-diS-DM1 and EDB-L19-dis-COCO-1569. Furthermore, tumor-bearing animals treated with EDB-L19-vc-0101 had delayed tumor growth at 1 mg / kg and complete regression at 3 mg / kg. This data demonstrates that EDB-L19-vc-0101 (ADC1) inhibits the growth of PDX-NSX-11122 NSCLC xenografts in a dose-dependent manner.

[0287] The activity of site-specifically and conventionally conjugated ADCs was evaluated. Figure 5D shows the antitumor efficacy of site-specifically conjugated EDB-(κK183C+K290C)-vc-0101 at doses of 0.3, 1, and 3 mg / kg compared to conventionally conjugated EDB-L19-vc-0101 at a dose of 1.5 mg / kg. Dose-level efficacy was comparable, with EDB-(κK183C+K290C)-vc-0101 causing tumor regression in a dose-dependent manner.

[0288] The activity of vc-0101 EDB ADCs with various mutations was evaluated. Figure 5E shows the antitumor efficacy of site-specifically conjugated EDB-(κK183C-K94R-K290C)-vc-0101 at doses of 0.3, 1, and 3 mg / kg. EDB-(κK183C-K94R-K290C)-vc-0101 induced tumor regression at 1 and 3 mg / kg. Figure 5F shows tumor growth inhibition curves for 10 individual tumor-bearing mice in the EDB-(κK183C-K94R-K290C)-vc-0101 group dosed at 3 mg / kg in Figure 5E. Tumor regression in the 3 mg / kg group was complete and durable in 8 of 10 mice (80%) at the end of the study (day 95).

[0289] H-1975 NSCLC CLX The efficacy of various vc-linked auristatin and CPI ADCs was evaluated in H-1975, a moderate-to-high EDB+FN-expressing NSCLC CLX model of human cancer. Figure 6A shows EDB-L19-vc-0101 evaluated for antitumor activity at 0.3, 0.75, 1.5, and 3 mg / kg. The data demonstrate that EDB-L19-vc-0101 exhibited tumor regression in a dose-dependent manner at 3 mg / kg and as low as 1.5 mg / kg. Figure 6B shows EDB-L19-vc-0101 and EDB-L19-vc-1569 evaluated for antitumor activity at 0.3, 1, and 3 mg / kg. The data demonstrate that EDB-L19-vc-0101 and EDB-L19-vc-1569 exhibited tumor regression in a dose-dependent manner.

[0290] The antitumor activity of vc-linked auristatin ADCs was compared with that of CPI ADCs. As shown in Figure 6C, EDB-L19-vc-0101 and EDB-(H16-K222R)-AcLys-vc-CPI-8314 were evaluated at 0.5, 1.5, and 3 mg / kg and 0.1, 0.3, and 1 mg / kg, respectively. Both EDB-L19-vc-0101 and EDB-(H16-K222R)-AcLys-vc-CPI-8314 demonstrated tumor regression at the highest dose evaluated.

[0291] The activity of site-specifically and conventionally conjugated EDB ADCs was evaluated. Figure 6D shows the antitumor efficacy of site-specifically conjugated EDB-(κK183C+K290C)-vc-0101 compared to conventionally conjugated EDB-L19-vc-0101 at doses of 0.5, 1.5, and 3 mg / kg. Dose-level efficacy was comparable, with EDB-(κK183C+K290C)-vc-0101 producing tumor regression in a dose-dependent manner.

[0292] The activity of vc-0101 EDB ADCs with various mutations was evaluated. Figure 6E shows the antitumor efficacy of EDB-L19-vc-0101 and EDB-(K94R)-vc-0101 at 1 and 3 mg / kg. Figure 6F shows the antitumor efficacy of site-specific EDB-(κK183C+K290C)-vc-0101 and EDB-(κK183C-K94R-K290C)-vc-0101 at 1 and 3 mg / kg. The four ADCs demonstrated similar efficacy in the H-1975 model regardless of whether they contained the κK183C-K290C and / or K94R mutations. Furthermore, all tested ADCs produced robust antitumor efficacy, including tumor regression, at 3 mg / kg. These data demonstrate that the introduction of the κ K183C-K290C and / or K94R mutations did not negatively impact the efficacy of the ADC.

[0293] HT29 colon CLX The efficacy of various vc-linked auristatin ADCs was evaluated in HT29, a moderately EDB+FN-expressing colon CLX model of human cancer. As shown in Figure 7, EDB-L19-vc-0101 and EDB-L19-vc-9411 were tested for antitumor activity at 3 mg / kg. Both EDB-L19-vc-0101 and EDB-L19-vc-9411 demonstrated tumor regression over time at the 3 mg / kg dose.

[0294] PDX-PAX-13565 and PDX-PAX-12534 pancreatic PDXs The antitumor efficacy of EDB-L19-vc-0101 was evaluated in human pancreatic PDX models. As shown in Figure 8A, EDB-L19-vc-0101 was evaluated at 0.3, 1, and 3 mg / kg in PDX-PAX-13565, a moderate-to-high EDB+FN-expressing pancreatic PDX. As shown in Figure 8B, EDB-L19-vc-0101 was evaluated at 0.3, 1, and 3 mg / kg in PDX-PAX-12534, a low-to-moderate EDB+FN-expressing pancreatic PDX. EDB-L19-vc-0101 demonstrated tumor regression in a dose-dependent manner in both pancreatic PDX models evaluated.

[0295] Ramos lymphoma CLX The antitumor efficacy of EDB-L19-vc-0101 was evaluated in Ramos, a moderately EDB+FN-expressing lymphoma CLX model. EDB-L19-vc-0101 was evaluated for antitumor activity at 1 and 3 mg / kg. As shown in Figure 9, EDB-L19-vc-0101 showed tumor regression in a dose-dependent manner at the 3 mg / kg dose.

[0296] EMT-6 breast cancer syngeneic model The antitumor efficacy of EDB-(κK183C-K94R-K290C)-vc-0101 was evaluated in EMT-6, a mouse syngeneic breast cancer model in an immunocompetent background. As shown in Figure 10A, EDB-(κK183C-K94R-K290C)-vc-0101 demonstrated tumor growth inhibition at 4.5 mg / kg. Tumor growth inhibition was plotted as the mean tumor size ± standard error of the mean in 11 tumor-bearing animals. Figure 10B shows tumor growth inhibition curves for 11 individual tumor-bearing mice in the EDB-(κK183C-K94R-K290C)-vc-0101 group dosed at 4.5 mg / kg. Tumor regression in the 4.5 mg / kg group was complete and durable in 9 of 11 mice (82%) at the end of the study (day 34).

[0297] ovaries The activity of EDB-(κK183C-K94R-K290C)-vc-0101 was tested in EDB+FN-expressing ovarian and breast cancer human PDX models. Activity was observed at dose levels of 3 mg / kg and 10 mg / kg (data not shown).

[0298] Example 9 Pharmacokinetics (PK) Exposure of conventionally conjugated EDB-L19-vc-0101 and site-specifically conjugated EDB-(κK183C-K94R-K290C)-vc-0101 conjugated antibody-drug conjugates was determined in cynomolgus monkeys after intravenous (IV) bolus administration of either 5 or 6 mg / kg, respectively. Concentrations of total antibody (total Ab; a measure of both conjugated and unconjugated mAb), ADC (mAb conjugated to at least one drug molecule) were measured using a ligand binding assay (LBA), and the concentration of released payload 0101 was measured using mass spectrometry. Quantification of total Ab and ADC concentrations was achieved by ligand binding assay (LBA) using a Gyrolab® workstation with fluorescence detection. The biotinylated capture protein used was sheep anti-hIgG, and the detection antibody was Alexa Fluor 647 goat anti-hIgG for total antibodies or Alexa Fluor 647 anti-0101 mAb for ADCs (data processed by the Watson v7.4 LIMS system). In vivo samples were prepared for unconjugated payload analysis using protein precipitation and infused onto an AB Sciex API5500 (QTRAP) mass spectrometer using positive Turbo IonSpray electrospray ionization (ESI) and multiple reaction monitoring (MRM) mode. The transitions 743.6 → 188.0 and 751.6 → 188.0 were used for the analyte and deuterated internal standard, respectively. Data acquisition and processing were performed using Analyst software version 1.5.2 (Applied Biosystems / MDS Sciex, Canada).

[0299] The pharmacokinetics of total Ab, ADC, and released payload from cynomolgus monkeys dosed with EDB-L19-vc-0101 ADC (5 mg / kg) and EDB-(κK183C-K94R-K290C)-vc-0101 ADC (6 mg / kg) are shown in Table 22. The exposure of the site-specifically conjugated EDB-(κK183C-K94R-K290C)-vc-0101 ADC showed both increased exposure (approximately 2.3× increase as measured by dose-normalized AUC) and increased conjugation stability when compared to the conventional conjugate. Conjugation stability was compared to the conventional EDB-L19-vc-0101 ADC, with a higher ADC / Ab ratio (84% vs. 75%) and lower released payload exposure (dose-normalized AUC; 0.0058 vs. 0.0082 μg) for the site-specifically conjugated EDB-(κK183C-K94R-K290C)-vc-0101 ADC, respectively. * NA = not applicable.

[0300] [Table 22]

[0301] Example 10 Thermal stability evaluation of EDB ADC Differential scanning calorimetry (DCS) was used to determine the thermal stability of anti-EDB antibody variants and the corresponding conventionally and site-specifically conjugated EDB ADCs. Samples formulated in PBS-CMF pH 7.2 were dispensed into the sample tray of a MicroCal VP-Capillary DSC with Autosampler (GE Healthcare Bio-Sciences, Piscataway, NJ), equilibrated at 10°C for 5 minutes, and then scanned to 110°C at a rate of 100°C per hour. A 16-second filtering period was selected. Raw data were baseline corrected and protein concentration normalized. Data were fitted to an MN2-State model with the appropriate number of transitions using Origin Software 7.0 (OriginLab Corporation, Northampton, MA).

[0302] As shown in Table 23, various anti-EDB antibodies and EDB ADCs were evaluated using both site-specific and conventional conjugation technologies and showed favorable thermal stability as determined by a first melting transition (Tm1) >65° C. These results demonstrate that the EDB-(κK183C-K94R-K290C) antibody and the κK183C-K94R-K290C-vc-0101 ADC, which incorporate engineered cysteine ​​residues, were thermally stable.

[0303] [Table 23]

[0304] Example 11 toxicity research The nonclinical safety profiles of conventionally conjugated EDB-L19-vc-0101 and site-specifically conjugated EDB-(κK183C-K94R-K290C)-vc-0101 were characterized in exploratory repeated-dose (Q3W x 3) studies in Wistar-Han rats and cynomolgus monkeys. Rats and cynomolgus monkeys were considered pharmacologically relevant nonclinical species for toxicity evaluation because of 100% protein sequence homology with human EDB and similar binding affinities of antibodies EDB-L19 and EDB-(κK183C-K94R-K290C) to rats, humans, and monkeys by Biacore assay, as demonstrated in Example 2.

[0305] EDB-L19-vc-0101 was evaluated in Wistar Han rats and cynomolgus monkeys at doses up to 10 and 5 mg / kg, respectively, and EDB-(κK183C-K94R-K290C)-vc-0101 was evaluated in cynomolgus monkeys at doses up to 12 mg / kg. Rats or monkeys were intravenously dosed once every three weeks (on days 1, 22, and 43) and euthanized on day 46 (three days after the third dose). Animals were evaluated for clinical signs, changes in body weight, food intake, clinical pathology parameters, organ weights, and macroscopic and microscopic observations. No mortality or significant changes in the clinical condition of the animals were observed in these studies.

[0306] In rats and monkeys, there were no signs of target-dependent toxicity in EDB+FN-expressing tissues / organs. In both species, the primary toxicity was reversible myelosuppression with associated hematological changes. In monkeys, as shown in Table 24 and Figure 11, significant transient neutropenia was seen with conventionally conjugated EDB-L19-vc-0101 at 5 mg / kg / dose, whereas only minimal effect on neutrophil counts was seen with site-specifically conjugated EDB-(κK183C-K94R-K290C)-vc-0101 at 6 mg / kg / dose. Points indicate the mean, and error bars indicate ±1 standard deviation (SD) from the mean.

[0307] The data demonstrate significant reduction of myelosuppression with site-specific conjugation. The toxicity profiles of EDB-L19-vc-0101 and EDB-(κK183C-K94R-K290C)-vc-0101 are consistent with the target-independent effects of these conjugates, with the highest non-severely toxic dose (HNSTD) for EDB-L19-vc-0101 and EDB-(κK183C-K94R-K290C)-vc-0101 being ≥ 5 mg / kg / dose and ≥ 5 mg / kg / dose, respectively. and ≥ 12 mg / kg / dose.

[0308] [Table 24]

[0309] Example 12 Combined with IO When cancer cells die, they release antigens that are taken up and presented by dendritic cells (DCs). Due to mutations in these tumor cells, some of these antigens contain cancer neoepitopes that have the potential to be presented to T cells by mature DCs, thereby activating them and inducing anti-tumor targeting. However, negative regulatory mechanisms are upregulated in cancer patients. For example, signaling through checkpoints, such as the PD-1 / PD-L1 pathway, can limit neoepitope recognition and T cell activation.

[0310] Payloads conjugated to antibodies in ADC formats can engage dendritic cell maturation pathways, resulting in increased tumor antigen cross-presentation, enabling T cell priming and increased tumor T cell targeting. The EDB ADCs of the present invention, including various payloads, such as payload -0101, have been utilized to improve immune recognition of tumor neoantigens by creating an immunogenic tumor environment. When EDB ADCs and immuno-oncology agents are administered in combination, these environments become responsive to the immuno-oncology agents, blocking negative regulatory pathways.

[0311] Data from an efficacy study of EMT6 syngeneic tumors treated with EDB-L19-vc-0101 suggest that an effector response to Payload-0101 was induced. Increased infiltration of CD3+ T cells was observed in EDB-L19-vc-0101-treated tumors compared to vehicle controls. Furthermore, increased expression of PDL1 in treated tumors was observed, suggesting IFNγ release due to increased effector T cell responses.

[0312] Combining EDB ADCs with agents that target immune regulatory pathways, such as anti-PDL1 antagonistic or anti-41BB agonist antibodies, is likely to improve antitumor efficacy and provide more durable responses.

[0313] Example 13 Biomarkers / Mechanism of Action The NSCLC PDX models PDX-NSX-11122 were developed in nude mice as previously described. EDB-(K94R)-vc-0101, EDB-(κK183C-K94R-K290C)-vc-0101, and Neg-vc-0101 The ADC was administered at 3 mg / kg via tail vein injection (4 animals per time point per group). 96 hours after the single administration, the animals were anesthetized and perfused with saline. After saline perfusion, the tumors were removed and prepared for antibody and ADC measurement via ligand binding assay (LBA) or for immunohistochemistry (IHC).

[0314] Ligand Binding Assay (LBA) For the LBA assay, 5x buffer was added to tumor samples. Tissue extraction reagent (Invitrogen) contained 1% protease inhibitors (Sigma) (v / w) at a final dilution of 6x (µg / mL homogenate → µg / g tissue). Stainless steel beads were added, and the tissue was homogenized using a Mini-Beadbeater-96 (BioSpec). The homogenate (approximately 100-300 µL, depending on sample size) was transferred to an appropriate vial (Marsh tube) and centrifuged at 14,000 rpm for 10 min (4°C). The centrifuged homogenate was diluted with Super Block™ (MRD) for analysis according to the assay protocol.

[0315] Ligand binding assays were used to determine mean total antibody and ADC plasma concentrations (μg / mL) and tumor concentrations (μg / g) following single-dose administration of the EDB ADC, as shown in Table 25. The data demonstrate that EDB-(K94R)-vc-0101 and EDB-(κK183C-K94R-K290C)-vc-0101 were detected at tumor sites at increased levels, as measured by total antibody and ADC, compared to Neg-vc-0101. Furthermore, decreased plasma-to-tumor ratios for both ADC and total antibody were observed for EDB-(K94R)-vc-0101 and EDB-(κK183C-K94R-K290C)-vc-0101 compared to Neg-vc-0101, indicating increased efficiency of tumor-specific targeting of the EDB-targeted ADC.

[0316] [Table 25]

[0317] Immunohistochemistry (IHC) For immunohistochemical detection of ADC distribution and downstream biomarkers of response, samples were fixed in 10% neutral buffered formalin for 48 hours. After fixation, samples were embedded in paraffin and sectioned at 5 μM. Cut paraffin sections were deparaffinized in xylene substitute and rehydrated through graded alcohols up to distilled water. Antigens were retrieved in a pressure cooker (Electron Microscopy Sciences) in 10 mM citrate buffer pH 6.0 (Invitrogen) for phospho-histone H3 and cleaved caspase 3 detection, or in Borg Decloaker buffer pH 9.5 (Biocare Medical) for anti-human IgG and anti-O101 detection, and cooled to room temperature. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 minutes. Nonspecific protein interactions were blocked with protein block (DAKO) for 20 minutes. Tissue sections were incubated with primary antibodies at room temperature for 1 hour. The primary antibodies were as follows: 0.3 μg / mL anti-human pan-IgG antibody (Epitomics); 10 μg / mL anti-0101 Ab; 0.13 μg / mL anti-phospho-histone H3 (pHH3, Cell Signaling Technologies); and 1.3 μg / mL anti-cleaved caspase 3 (Cell Signaling Technologies). To avoid mouse-to-mouse detection, the anti-0101 isotype antibody was labeled with AlexaFluor 488 using an AlexaFluor 488 protein labeling kit (Life Technologies). Unlabeled primary antibodies were detected with Signalstain Boost reagent (Cell Signaling Technologies) for 30 minutes at room temperature. AlexaFluor 488-labeled primary antibodies were detected with 1 μg / mL rabbit anti-AlexaFluor 488 (Life Technologies) for 45 minutes at room temperature, followed by incubation with Signalstain Boost reagent (Cell Signaling Technologies) for 30 minutes at room temperature. Color was developed using DAB+ (3',3'-diaminobenzidine; Dako) for 5 minutes.Sections were briefly counterstained in hematoxylin, washed in water, dehydrated in graded alcohols, cleared in xylene substitutes, and coverslipped with Permount Mounting Medium.

[0318] Ninety-six hours after a single dose, both conventional EDB-(K94R)-vc-0101 and site-specifically conjugated EDB-(κK183C-K94R-K290C)-vc-0101 were similarly detected by anti-human IgG IHC in the PDX-NSX-11122 PDX model. An increase in pHH3-positive cells, a marker of mitotic arrest, was observed in tumors treated with both EDB-(K94R)-vc-0101 and EDB-(κK183C-K94R-K290C)-vc-0101 compared with tumors treated with the negative control ADC (Neg-vc-0101). The majority of cells bearing the pHH3 mitotic arrest marker were neoplastic, suggesting a bystander effect. Cleaved caspase 3 staining showed increased apoptosis in tumors treated with EDB-(K94R)-vc-0101 (and EDB-(κK183C-K94R-K290C)-vc-0101) compared with tumors treated with the negative control ADC (Neg-vc-0101).

Claims

[Claim 1] An object, method or system as described in the specification.

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