Constructs, compositions, and methods for treating extra domain b splice variant of fibronectin-expressing diseases and disorders

Abstract

The present disclosure provides methods of treatment comprising anti-EDB antibodies and antibody-drug conjugates. The present disclosure further provides methods of treating EDB+ FN-expressing disorders, such as cancer. In some aspects, the cancer is selected from the group consisting of ER+HR+Her2-breast cancer, triple negative breast cancer (TNBC), head and neck cancer, hepatocellular carcinoma, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, renal cancer, soft tissue sarcoma (sarcoma), and thyroid cancer.

Description

[0001] CONSTRUCTS, COMPOSITIONS, AND METHODS FOR TREATING EXTRA DOMAIN B SPLICE VARIANT OF FIBRONECTIN-EXP RES SING DISEASES AND DISORDERS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 571.754. filed March 29, 2024; and U.S. Provisional Patent Application No. 63 / 714,650, filed October 31, 2024, the disclosures of each of which are hereby incorporated herein by reference in their entireties.

[0004] REFERENCE TO SEQUENCE LISTING

[0005] The Sequence Listing concurrently submitted herewith as an XML-formatted text file named “384953-1023WOl_sequence_listing.xml” created on March 26, 2025, and having a size of 66,906 bytes, is herein incorporated by reference in its entirety pursuant to 37 C.F.R. 1 52(e)(5).

[0006] BACKGROUND

[0007] Fibronectin (FN) is a ubiquitously expressed high-molecular-weight ECM glycoprotein that exists in various isoforms. Fibronectin contains repeats of 3 types of domains: types I. II, and III. Three sites of alternative splicing within the type III repeats have been identified: type III homology extra domain A (EDA), type III homology extra domain B (EDB), and type III homology connecting segment. The extra domain B splice variant of fibronectin (EDB+FN) is synthesized, secreted, and deposited to the ECM by numerous cell types including endothelial cells of newly formed blood vessels, myofibroblasts, and tumor cells. It can be detected at the abluminal site of endothelial linings of newly formed blood vessels and between stromal structures. Although physiologic expression of EDB+FN is low in healthy adults, it can occur in chronic pathological conditions associated with new blood vessel formation, such as ocular-retinal diseases, severe atherosclerosis, and inflammatory rheumatoid disease. EDB+FN is abundant in tissues of almost all human solid cancers, irrespective of histotype, and its expression has been reported to be upregulated in several cancer indications compared to normal tissue such as non-small cell lung cancer (NSCLC), pancreatic ductal adenocarcinoma, and breast cancer.

[0008] An antibody that specifically binds to the EDB domain of fibronectin (FN). the LI 9 antibody, has been isolated by phage display technology. The L19 antibody is able to stain tumor blood vessels in a wide range of experimental tumor models and on sections of human tumors and other angiogenic disorders.

[0009] Although various therapies for EDB+FN-expressing disorders or diseases, such as LI 9 antibody based therapies, have been disclosed, there remains a significant clinical need for the development of further improved and optimized EDB+FN-targeting therapies. In particular, there remains a significant need for treatment options for those patients with EDB+FN-expressing disorders or diseases, such as cancers associated with EDB+FN expression and / or EDB+FN-expressing cancers.

[0010] BRIEF SUMMARY

[0011] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof. In some aspects, the method comprises administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, that binds to extra domain B of fibronectin; (b) a linker; and (c) a drug; wherein the EDB+FN-expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2- breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, sarcoma, and thyroid cancer.

[0012] In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting PR+ER+HER2-breast cancer, head and neck cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is HR+HER2- breast cancer. In some aspects, the EDB+FN-expressing disorder or disease is hepatocellular carcinoma. In some aspects, the EDB+FN-expressing disorder or disease is head and neck cancer. In some aspects, the EDB+FN-expressing disorder or disease is renal cancer. In some aspects, the EDB+FN-expressing disorder or disease is non-small cell lung cancer. In some aspects, the EDB+FN-expressing disorder or disease is sarcoma. In some aspects, the EDB+FN-expressing disorder or disease is thyroid cancer. In some aspects, the EDB+FN expressing disorder or disease is PR+ER+HER2-breast cancer. In some aspects, the EDB+FN expressing disorder or disease is a subtype of sarcoma selected from the group consisting of chordoma, leiomyosarcoma, liposarcoma, myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, and soft tissue sarcoma.

[0013] In some aspects, the antibody, or antigen binding fragment thereof, comprises at least one amino acid residue modification for site specific conjugation in a constant region of the antibody, or antigen binding fragment thereof. In some aspects, the antibody, or antigen binding fragment thereof, comprises at least one cysteine substitution or site-specific conjugation. In some aspects, the antibody, or antigen binding fragment thereof comprises: a heavy chain constant region comprising an engineered cysteine residue at position: 1 18 (114 according to the numbering of Kabat), 246, 249, 265, 267, 270, 276, 278, 283, 290, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 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. 25 428. 431, 432, 437, 438, 439. 443 or 444, or any combination thereof , according to the numbering of the EU index; or (b) a light chain constant region comprising: (i) an engineered cysteine residue at position 110, 111, 125, 149, 155, 158, 161, 183, 185. 188, 189, 191, 197, 205, 207, 208 or 210, or any 30 combination thereof, according to the numbering of Kabat; (ii) an engineered cysteine residue at a position corresponding to residue 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 engineered cysteine residue at a position corresponding to 5 residue 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). In some aspects, the antibody, or antigen binding fragment, comprises a VH region comprising the VH CDR1 region, VH CDR2 region, and VH CDR3 region of the VH amino acid sequence of SEQ ID NO:21, and a VL region comprising VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NO: 10. In some aspects, 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. In some aspects, 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. In some aspects, the antibody, or antigen binding fragment, comprises: 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 aspects, 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. In some aspects, antibody or antigen binding fragment thereof comprises: 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. In some aspects, the antibody, or antigen binding fragment thereof, comprises at least one of the following: a cysteine at position 290 of the heavy chain constant region according to the numbering of the EU index; and / or a cysteine at position 183 of the light chain constant region according to the numbering of the EU index. In some aspects, the antibody comprises at least one of the following: a heavy chain comprising SEQ ID NO:25; a light chain comprising SEQ ID NO:31.

[0014] In some aspects, the linker is a cleavable linker. In some aspects, the cleavable linker is selected from the group consisting of: vc having the structure diS having the structure diS-C2OCO having the structure AcLys-vc having the structure

[0015] In some aspects, the cleavable linker has the structure:

[0016] In some aspects, the drug is a cytotoxic agent. In some aspects, the cytotoxic agent is an auristatin. In some aspects, the auristatin is selected from the group consisting of: 0101 having the structure

[0017] 9411 having the structure

[0018] 4574 having the structure

[0019] In some aspects, the auristatin is auristatin 0101. In some aspects, the cytotoxic agent has the structure:

[0020] In some aspects, the cytotoxic agent, the linker, and the antibody, or antigen binding fragment thereof, are linked to generate the structure: wherein:

[0021] X-S represents the antibody, or antigen binding fragment thereof, wherein S is the sulfur atom of at least one cysteine residue in the antibody, or antigen binding fragment thereof.

[0022] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, that binds to extra domain B of fibronectin; (b) a cleavable linker; and (c) a cytotoxic drug.

[0023] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, that binds to extra domain B of fibronectin; (b) a cleavable linker; and (c) an auristatin.

[0024] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, that binds to extra domain B of fibronectin; (b) a vc linker; and (c) a 0101 drug.

[0025] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, comprising a VH region comprising the VH CDR1 region, VH CDR2 region, and VH CDR3 region of the VH amino acid sequence of SEQ ID NO:21. and a VL region comprising VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NO: 10; (b) a cleavable linker; and (c) a cytotoxic drug.

[0026] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, comprising a VH region comprising the VH CDR1 region, VH CDR2 region, and VH CDR3 region of the VH amino acid sequence of SEQ ID NO:21, and a VL region comprising VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NO: 10; (b) a cleavable linker; and (c) an auristatin.

[0027] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, comprising a VH region comprising the VH CDR1 region, VH CDR2 region, and VH CDR3 region of the VH amino acid sequence of SEQ ID NO:21, and a VL region comprising VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NO: 10; (b) a vc linker; and (c) a 0101 drug.

[0028] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and (c) a cytotoxic drug. In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and (c) an auristatin.

[0029] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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) a 0101 drug.

[0030] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate compnsing: (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 cleavable linker; and (c) a cytotoxic drug.

[0031] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and (c) an auristatin.

[0032] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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) a 0101 drug.

[0033] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and (c) a cytotoxic drug.

[0034] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and (c) an auristatin.

[0035] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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) a 0101 drug.

[0036] In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2 -breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting of PR+ER+HER2-breast cancer, head and neck cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is HR+HER2- breast cancer. In some aspects, the EDB+FN-expressing disorder or disease is hepatocellular (liver) cancer. In some aspects, the EDB+FN-expressing disorder or disease is head and neck cancer. In some aspects, the EDB+FN-expressing disorder or disease is renal cancer. In some aspects, the EDB+FN-expressing disorder or disease is sarcoma. In some aspects, the EDB+FN- expressing disorder or disease is thyroid cancer. In some aspects, the EDB+FN expressing disorder or disease is PR+ER+HER2-breast cancer. In some aspects, the EDB+FN expressing disorder or disease is a subtype of sarcoma selected from the group consisting of chordoma, leiomyosarcoma, liposarcoma, myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, and soft tissue sarcoma.

[0037] BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

[0039] FIG. 1 presents a schematic representation of a patient derived xenograft (PDX) study conducted for each PDX model in studies described herein. FIG. 2A-FIG. 2D present graphical representations of data related to PDX studies of PDX models for thyroid cancer. FIG. 2A presents a graphical representation of data related to CTG-1976. FIG. 2B presents a graphical representation of data related to THXF TH-OOI. FIG. 2C presents a graphical representation of data related to CTG-1921. FIG. 2D presents a graphical representation of data related to THXF TH-007.

[0040] FIG. 3 A-FIG. 31 present graphical representations of data related to PDX studies of PDX models for head and neck cancer. FIG. 3A presents a graphical representation of data related to HNXF-536. FIG. 3B presents a graphical representation of data related to HNXF- 1838. FIG. 3C presents a graphical representation of data related to CTG-2130. FIG. 3D presents a graphical representation of data related to CTG-1131. FIG. 3E presents a graphical representation of data related to HNXF-1842. FIG. 3F presents a graphical representation of data related to CTG-0776. FIG. 3G presents a graphical representation of data related to CTG-3130. FIG. 3H presents a graphical representation of data related to CTG-2719. FIG. 31 presents a graphical representation of data related to CTG-0719.

[0041] FIG. 4A-FIG. 4N present graphical representations of data related to PDX studies of PDX models for non-small cell lung cancer. FIG. 4A presents a graphical representation of data related to LXFA-677. FIG. 4B presents a graphical representation of data related to LXFA-2184. FIG. 4C presents a graphical representation of data related to LXFE-2276. FIG. 4D presents a graphical representation of data related to CTG-1992. FIG. 4E presents a graphical representation of data related to LXFL-2377. FIG. 4F presents a graphical representation of data related to LXFE-397. FIG. 4G presents a graphical representation of data related to CTG-2540. FIG. 4H presents a graphical representation of data related to LXFA-923. FIG. 41 presents a graphical representation of data related to CTG-3414. FIG. 4J presents a graphical representation of data related to CTG-1444. FIG. 4K presents a graphical representation of data related to CTG-2539. FIG. 4L presents a graphical representation of data related to CTG-3477. FIG. 4M presents a graphical representation of data related to CTG-0838. FIG. 4N presents a graphical representation of data related to LXFE-1422.

[0042] FIG. 5A-FIG. 5M present graphical representations of data related to PDX studies of PDX models for pancreatic cancer. FIG. 5A presents a graphical representation of data related to PAXF-736. FIG. 5B presents a graphical representation of data related to PAXF- 1881. FIG. 5C presents a graphical representation of data related to PAXF-2132. FIG. 5D presents a graphical representation of data related to CTG-2413. FIG. 5E presents a graphical representation of data related to PAXF-2196. FIG. 5F presents a graphical representation of data related to CTG-2118. FIG. 5G presents a graphical representation of data related to CTG-0288. FIG. 5H presents a graphical representation of data related to PAXF-2057. FIG. 51 presents a graphical representation of data related to PAXF-1657. FIG. 5J presents a graphical representation of data related to CTG-0283. FIG. 5K presents a graphical representation of data related to CTG-0306. Fig. 5L presents a graphical representation of data related to CTG-3348. FIG. 5M presents a graphical representation of data related to CTG-1375.

[0043] FIG. 6A-FIG. 6N present graphical representations of data related to PDX studies of PDX models for sarcoma. FIG. 6A presents a graphical representation of data related to SXFS-463. FIG. 6B presents a graphical representation of data related to SXFS-2406. FIG. 6C presents a graphical representation of data related to CTG-0243. FIG. 6D presents a graphical representation of data related to SXFS-174. FIG. 6E presents a graphical representation of data related to SXFS-2147. FIG. 6F presents a graphical representation of data related to SXFS-1301. FIG. 6G presents a graphical representation of data related to CTG-2426. FIG. 6H presents a graphical representation of data related to SXFS-1937. FIG. 61 presents a graphical representation of data related to SXFS-627. FIG. 6J presents a graphical representation of data related to CTG-1451. FIG. 6K presents a graphical representation of data related to CTG-2291. FIG. 6L presents a graphical representation of data related to CTG-1116. FIG. 6M presents a graphical representation of data related to CTG-1339. FIG. 6N presents a graphical representation of data related to CTG-2252.

[0044] FIG. 7A-FIG. 7M presents a graphical representation of data related to PDX studies of PDX models for breast cancer. FIG. 7A presents a graphical representation of data related to CTG-3283. FIG. 7B presents a graphical representation of data related to CTG-0437. FIG. 7C presents a graphical representation of data related to CTG-3090. FIG. 7D presents a graphical representation of data related to CTG-1520. FIG. 7E presents a graphical representation of data related to CTG-1167. FIG. 7F presents a graphical representation of data related to MAXFTN-BR5. FIG. 7G presents a graphical representation of data related to MAXFTN-401. FIG. 7H presents a graphical representation of data related to CTG-2055. FIG. 71 presents a graphical representation of data related to CTG-1883. FIG. 7J presents a graphical representation of data related to CTG-2849. FIG. 7K presents a graphical representation of data related to CTG-1941. FIG. 7L presents a graphical representation of data related to CTG-2308. FIG. 7M presents a graphical representation of data related to CTG-1059.

[0045] FIG. 8A-FIG. 8F present graphical representations of data related to PDX studies of PDX models for renal cancer. FIG. 8A presents a graphical representation of data related to RXF-1781. FIG. 8B presents a graphical representation of data related to RXF-393. FIG. 8C presents a graphical representation of data related to RXF-2178. FIG. 8D presents a graphical representation of data related to CTG-3003. FIG. 8E presents a graphical representation of data related to RXF-2357. FIG. 8F presents a graphical representation of data related to CTG-3461.

[0046] FIG. 9A-FIG. 9B present graphical representations of data related to PDX studies of PDX models for hepatocellular cancer. FIG. 9A presents a graphical representation of data related to LIXAH-575. FIG. 9B presents a graphical representation of data related to CTG- 0370.

[0047] FIG. 1 OA-FIG. 101 present graphical representations of data related to PDX studies of PDX models for ovarian cancer. FIG. 10A presents a graphical representation of data related to CTG-1086. FIG. 10B presents a graphical representation of data related to CTG-0703. FIG. 10C presents a graphical representation of data related to CTG-2561. FIG. 10D presents a graphical representation of data related to OVXF OV-109. FIG. 10E presents a graphical representation of data related to CTG-3718. FIG. 10F presents a graphical representation of data related to OVXF OV-003. FIG. 10G presents a graphical representation of data related to OVXF OV55. FIG. 10H presents a graphical representation of data related to CTG-0964. 101 presents a graphical representation of data related to CTG-0868.

[0048] FIG. 11A-FIG. 11 J present graphical representations of data related to long term tumor reduction in ten PDX studies. FIG. 11A presents a graphical representation of data related to CTG-3090, a PDX model for triple negative breast cancer. FIG. 1 IB presents a graphical representation of data related to LXFA-2184, a PDX model for non-small cell lung cancer. FIG. 11C presents a graphical representation of data related to CTG-1444, a PDX model for non-small cell lung cancer. FIG. 1 ID presents a graphical representation of data related to CTG-1992, a PDX model for non-small cell lung cancer. FIG. 1 IE presents a graphical representation of data related to CTG-2130, a PDX model for head and neck cancer. FIG. 1 IF presents a graphical representation of data related to CTG-0719, a PDX model for head and neck cancer. FIG. 11G presents a graphical representation of data related to CTG-3130, a PDX model for head and neck cancer. FIG. 11H presents a graphical representation of data related to PAXF-1881, a PDX model for pancreatic cancer. FIG. 1 II presents a graphical representation of data related to SXFS-463, a PDX model for sarcoma. FIG. 11 J presents a graphical representation of data related to SXFS-174. a PDX model for sarcoma.

[0049] FIG. 12A-FIG. 12B present graphical representations of data related to bystander activity of PYX-201 in vitro. FIG. 12A presents a graphical representation of data related to HT-29 cells mixed with Caki2 cells. FIG. 12B presents a graphical representation of data related to HT-29 cells mixed with WI-38 VA-13 2RA cells.

[0050] DETAILED DESCRIPTION

[0051] DEFINITIONS

[0052] Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and / or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

[0053] Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and nucleic acid hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry’ described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0054] Furthermore, the experiments described herein, unless otherwise indicated, use conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, e g., Ausubel, et al., ed.. Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (Fourth Edition) by MR Green and J. Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition). Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3. from 1 to 4, from 1 to 5. from 2 to 4, from 2 to 6, from 3 to 6 and so forth, as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0055] As used herein, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. By way of example, ‘"an element” means one element or more than one element. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B."

[0056] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%. more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0057] In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. patent law and can mean “includes.” “including,” and the like; “consisting essentially of’ or “consists essentially” likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

[0058] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by any degree of suppression, remission, or eradication of a disease state.

[0059] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0060] As used herein, to “alleviate” a disease means reducing the severity of one or more symptoms of the disease.

[0061] The terms “patient”, “subject”, and “individual” are used interchangeably and are intended to include living organisms that may be subjected to treatment for a given disease. e.g., mammals. A “subject”, “patient”, or “individual”, as used herein, can be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, and murine mammals, as well as simian and non- human primate mammals. Preferably, the subject is human.

[0062] As used herein, the terms “effective amount” and “therapeutically effective amount” are used interchangeably and refer to the amount required to reduce or improve at least one symptom or change in a clinical marker of a disease relative to an untreated patient. The effective amount of the treatment used for therapeutic treatment of the disease varies depending upon the manner of the specific disorder, condition or disease, extent of the disorder, condition or disease, and administration of the cells, as well as the age, body weight, and general health of the subject. The effective amount is capable of achieving a particular desired biological result and / or provides a therapeutic or prophylactic benefit.

[0063] As used herein, the term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides ( / .£., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0064] As used herein, the term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0065] As used herein, the term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0066] As used herein, the term “identity” refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g, if half (e.g.. five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.

[0067] As used herein, the terms "conservative variation" or "conservative substitution" generally refers to the replacement of an amino acid residue by another, biologically similar residue. Conservative variations or substitutions are not likely to change the shape of the peptide chain. Examples of conservative variations, or substitutions, include the replacement of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine.

[0068] As used herein, the term '‘isolated” means altered or removed from the natural state. For example, a nucleic acid or a polypeptide naturally present in a living animal is not “isolated,” but the same nucleic acid or polypeptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0069] An "isolated nucleic acid" refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, z.e., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids that have been substantially purified from other components which naturally accompany the nucleic acid, i.e.. RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokary ote or eukary ote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence. The term "recombinant polypeptide" as used herein is defined as a polypeptide produced by using recombinant DNA methods.

[0070] The term "recombinant DNA" as used herein is defined as DNA produced by joining pieces of DNA from different sources.

[0071] "Variant" as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide may differ in amino acid sequence by one or more substitutions, additions, or deletions in any combination. A variant of a nucleic acid or peptide may be a naturally occurring such as an allelic variant, or may be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.

[0072] By the term “modified'’ as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.

[0073] As used herein, the term “oligonucleotide” typically refers to short polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), this also includes an RNA sequence (i.e.. A, U. C, G) in which “U” replaces “T.” Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0074] “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, or infusion techniques.

[0075] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, “nucleic acid” and “polynucleotide’' as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides” and which comprise one or more “nucleotide sequence(s)”. The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences (i.e., “nucleotide sequences”) which are obtained by any means available in the art, including, without limitation, recombinant means, i.e.. the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0076] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0077] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be constmed to include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to. Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0078] As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences: efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA: sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. There are numerous expression control sequences, including promoters which are native, constitutive, inducible and / or tissue-specific, are known in the art that may be used in the compositions of the invention. “Operably linked” should be construed to include RNA expression and control sequences in addition to DNA expression and control sequences.

[0079] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0080] As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence, which is required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements, which are required for expression of the gene product. The promoter / regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

[0081] A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0082] An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.

[0083] As used herein, the term “pharmaceutical composition” refers to a mixture of at least one compound useful within the invention with other chemical components, such as carriers, stabilizers, diluents, adjuvants, dispersing agents, suspending agents, thickening agents, and / or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to: intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.

[0084] The language “pharmaceutically acceptable carrier” includes a pharmaceutically acceptable salt, pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subject such that it may perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each salt or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin: talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil. cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide: alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; diluent; granulating agent; lubricant; binder; disintegrating agent; wetting agent; emulsifier; coloring agent; release agent; coating agent; sweetening agent; flavoring agent; perfuming agent; preservative; antioxidant; plasticizer; gelling agent; thickener; hardener; setting agent; suspending agent; surfactant; humectant; carrier; stabilizer; and other non-toxic compatible substances employed in pharmaceutical formulations, or any combination thereof. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions.

[0085] An “analyte” as used herein means any biomolecule (e.g., polypeptide, nucleic acid, lipid, and the like), drug (e.g., therapeutic drugs, drugs-of-abuse, metabolites, and the like), small molecule (e.g.. natural moisturizing factors, nicotine, and the like, with the understanding that small molecules can also be drugs), warfare agent, environmental contaminant (e.g., pesticides, etc.), microbe (e.g., bacterium, virus, fungus, yeast, and the like) and the like that is present in or on the tissue and can be extracted from the tissue of interest (e.g., skin, a mucosal membrane, and the like) and detected, analyzed, and / or quantified.

[0086] By the term “specifically binds,” as used herein with respect to an antibody, or antigen binding fragment thereof, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. Conversely, an antibody that specifically binds to an antigen may only bind to an antigen from a single species. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross react, city does not itself alter the classification of an antibody as specific. Conversely, an antibody that specifically binds to an allelic form of then antigen may only bind to a single allelic form. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0087] The term “epitope” as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response, inducing B and / or T cell responses. An antigen can have one or more epitopes. Most antigens have many epitopes; i.e.. they are multivalent. In general, an epitope is roughly about 10 amino acids and / or sugars in size. In some instances, the epitope is about 4-18 amino acids, about 5-16 amino acids, about 6-14 amino acids, about 7-12, or about 8-10 amino acids. One skilled in the art understands that generally the overall three-dimensional structure, rather than the specific linear sequence of the molecule, is the main criterion of antigenic specificity and therefore distinguishes one epitope from another. Based on the present disclosure, a peptide used in the present invention can be an epitope.

[0088] As used herein, the term “host cell” includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in 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 this invention. Methods of Treatment and Anti-EDB Antibodies and Antibody-Drug Conjugates

[0089] PYX-201 (also known as PF-06878792 and EDB-ADC) is an antibody-drug conjugate (ADC) being developed for the treatment of advanced solid tumors that have relapsed, been nonresponsive, or have progressed with all available therapies. The monoclonal antibody (mAb) portion of PYX-201 targets the extra domain B splice variant of fibronectin (EDB+FN), an insoluble component of the extracellular matrix (ECM) found in many solid tumors with comparatively lower expression in healthy adult tissues. The PYX- 201 payload is an auristatin derivative, AurOlOl (also known as PF-06380101), a microtubule depolymerizing agent designed to have enhanced bystander cytotoxicity that can be cleaved from the antibody by proteolytic enzymes. AurOlOl results in cell cycle arrest and subsequent cell toxicity. In addition to inducing cytotoxicity of cells in the tumor microenvironment (TME), targeting EDB+FN with an AurOlOl-based ADC can potentiate immune cell infiltration into syngeneic mouse tumors. PYX-201 is thus a potent antimitotic and cytotoxic ADC targeting EDB+FN in the tumor microenvironment with the potential for antitumor activity via multiple mechanisms of action.

[0090] As such, the present disclosure relates to methods for treating EDB+FN-expressing disorders or diseases, such as non-cancers or cancers associated with EDB+FN expression and / or EDB+FN-expressing cancers.

[0091] Methods of Treatment

[0092] In one aspect, the present disclosure relates to a method of treating an EDB+FN- expressing disorder or disease in a subject of need thereof. In certain embodiments, the method comprises administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, that binds to extra domain B of fibronectin; (b) a linker; and (c) a drug.

[0093] In certain embodiments, the EDB+FN-expressing disorder or disease is selected from the group consisting of breast cancer, hormone receptor positive human epidermal growth factor negative (HR+HER2-) breast cancer, estrogen receptor positive progesterone receptor positive human epidermal growth factor receptor negative (PR+ER+HER2-) breast cancer, triple negative breast cancer (TNBC), colorectal cancer, hepatocellular (liver) cancer (HCC), head and neck cancer, renal cancer, lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2-breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, ovarian cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN- expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2 -breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, sarcoma, and thyroid cancer. In some aspects, the HR+HER2- breast cancer is positive for either or both estrogen receptor and progesterone receptor. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting PR+ER+HER2-breast cancer, head and neck cancer, ovarian cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting PR+ER+HER2-breast cancer, head and neck cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is HR+HER2- breast cancer. In some aspects, the EDB+FN-expressing disorder or disease is head and neck cancer. In some aspects, the EDB+FN-expressing disorder or disease is renal cancer. In some aspects, the EDB+FN-expressing disorder or disease is non-small cell lung cancer. In some aspects, the EDB+FN-expressing disorder or disease is ovarian cancer. In some aspects, the EDB+FN-expressing disorder or disease is pancreatic cancer. In some aspects, the EDB+FN- expressing disorder or disease is triple negative breast cancer. In some aspects, the EDB+FN-expressing disorder or disease is sarcoma. In some aspects, the EDB+FN- expressing disorder or disease is thyroid cancer. In some aspects, the EDB+FN expressing disorder or disease is PR+ER+HER2-breast cancer. In some aspects, the EDB+FN expressing disorder or disease is a subtype of sarcoma selected from the group consisting of chordoma, leiomyosarcoma, liposarcoma, myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, and soft tissue sarcoma. In some aspects, the EDB+FN expressing disorder or disease is a subtype of ovarian cancer selected from the group consisting of serous carcinoma, serous papillary carcinoma, adenocarcinoma, and endometrioid carcinoma.

[0094] In some aspects, the antibody, or antigen binding fragment thereof, that binds to extra domain B of fibronectin; the linker; and the drug are any of the antibodies or antigen binding fragments thereof, linkers, or drugs as discussed herein.

[0095] In one aspect, the present disclosure relates to a method of treating an EDB+FN- expressing disorder or disease in a subject of need thereof. In certain embodiments, the method comprises administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, that binds to extra domain B of fibronectin; (b) a linker; (c) a drug. In certain embodiments, the EDB+FN-expressing disorder or disease is selected from the group consisting of breast cancer. HR+HER2- breast cancer, PR+ER+HER2- breast cancer, triple negative breast cancer, colorectal cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, sarcoma, and thyroid cancer.

[0096] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, that binds to extra domain B of fibronectin; (b) a cleavable linker; and (c) a cytotoxic drug.

[0097] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, that binds to extra domain B of fibronectin; (b) a cleavable linker; and (c) an auristatin.

[0098] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, that binds to extra domain B of fibronectin; (b) a vc linker; and (c) a 0101 drug.

[0099] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, comprising a VH region comprising the VH CDR1 region, VH CDR2 region, and VH CDR3 region of the VH amino acid sequence of SEQ ID NO:21, and a VL region comprising VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NO: 10; (b) a cleavable linker; and (c) a cytotoxic drug.

[0100] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, comprising a VH region comprising the VH CDR1 region, VH CDR2 region, and VH CDR3 region of the VH amino acid sequence of SEQ ID NO:21, and a VL region comprising VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NO: 10; (b) a cleavable linker; and (c) an auristatin.

[0101] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, comprising a VH region comprising the VH CDR1 region, VH CDR2 region, and VH CDR3 region of the VH amino acid sequence of SEQ ID NO:21, and a VL region comprising VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NO: 10; (b) a vc linker; and (c) a 0101 drug.

[0102] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and (c) a cytotoxic drug.

[0103] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and (c) an auristatin.

[0104] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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) a 0101 drug.

[0105] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and (c) a cytotoxic drug.

[0106] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and (c) an auristatin.

[0107] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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) a 0101 drug.

[0108] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and (c) a cytotoxic drug.

[0109] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and (c) an auristatin.

[0110] In one aspect, the present disclosure generally relates to a method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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) a 0101 drug. In some aspects, the EDB+FN expressing disorder or disease is PR+ER+HER2 -breast cancer. In some aspects, the EDB+FN expressing disorder or disease is a subtype of sarcoma selected from the group consisting of chordoma, leiomyosarcoma, liposarcoma. myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, and soft tissue sarcoma. In some aspects, the EDB+FN expressing disorder or disease is a subtype of non-small cell lung cancer selected from the group consisting of adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. In some aspects, the EDB+FN expressing disorder or disease is a subtype of ovarian cancer selected from the group consisting of serous carcinoma, serous papillary carcinoma, adenocarcinoma, and endometrioid carcinoma. In some aspects, the EDB+FN-expressing disorder or disease is head and neck squamous cell carcinoma.

[0111] In some aspects, the antibody, or antigen binding fragment thereof, comprises at least one amino acid residue modification for site specific conjugation in a constant region of the antibody, or antigen binding fragment thereof. In some aspects, the antibody, or antigen binding fragment thereof, comprises at least one cysteine substitution or site-specific conjugation. In some aspects, the antibody, or antigen binding fragment thereof comprises: a heavy chain constant region comprising an engineered cysteine residue at position: 1 18 (114 according to the numbering of Kabat), 246, 249, 265, 267, 270, 276, 278, 283, 290, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 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, 25 428. 431, 432, 437, 438, 439. 443 or 444, or any combination thereof , according to the numbering of the EU index; or (b) a light chain constant region comprising: (i) an engineered cysteine residue at position 110, 111, 125, 149, 155, 158, 161, 183, 185, 188, 189, 191, 197, 205, 207, 208 or 210, or any 30 combination thereof, according to the numbering of Kabat; (ii) an engineered cysteine residue at a position corresponding to residue 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 engineered cysteine residue at a position corresponding to 5 residue 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).

[0112] In one aspect, the present disclosure relates to a method of treating an EDB+FN- expressing disorder or disease in a subject of need thereof. In certain embodiments, the method comprises administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, that binds to extra domain B of fibronectin; (b) a cleavable linker, including optionally val-cit, phe-lys, vc, AcLys-vc, diS, diS-C2OCO, me, me, MalPeg6C2, or a hydrazone linker; (c) a cytotoxic drug, including optionally, an anthracycline, an auristatin, a CPI / CBI dimer, CC-1065, a dolastatin, a duocarmycin, an enediyne, a geldanamycin, a maytansine, a puromycin, a taxane, a vinca alkaloid, SN-38, tubulysin, hemiasterlin, and stereoisomers, isosteres, analogs or derivatives thereof. In certain embodiments, the EDB+FN-expressing disorder or disease is selected from the group consisting of breast cancer, HR+HER2- breast cancer, PR+ER+HER2- breast cancer, triple negative breast cancer, colorectal cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, sarcoma, and thyroid cancer. In certain embodiments, the EDB+FN expressing disorder or disease is a subtype of sarcoma selected from the group consisting of chordoma, leiomyosarcoma, liposarcoma. myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, and soft tissue sarcoma. In certain embodiments, the EDB+FN expressing disorder or disease is a subtype of non-small cell lung cancer selected from the group consisting of adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. In certain embodiments, the EDB+FN expressing disorder or disease is a subtype of ovarian cancer selected from the group consisting of serous carcinoma, serous papillary carcinoma, adenocarcinoma, and endometrioid carcinoma. In certain embodiments, the EDB+FN-expressing disorder or disease is head and neck squamous cell carcinoma. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2-breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, ovarian cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN- expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2 -breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting PR+ER+HER2-breast cancer, head and neck cancer, ovarian cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN- expressing disorder or disease is selected from the group consisting PR+ER+HER2-breast cancer, head and neck cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN- expressing disorder or disease is HR+HER2- breast cancer. In some aspects, the EDB+FN- expressing disorder or disease is head and neck cancer. In some aspects, the EDB+FN- expressing disorder or disease is renal cancer. In some aspects, the EDB+FN-expressing disorder or disease is non-small cell lung cancer. In some aspects, the EDB+FN-expressing disorder or disease is ovarian cancer. In some aspects, the EDB+FN-expressing disorder or disease is pancreatic cancer. In some aspects, the EDB+FN-expressing disorder or disease is triple negative breast cancer. In some aspects, the EDB+FN-expressing disorder or disease is sarcoma. In some aspects, the EDB+FN-expressing disorder or disease is thyroid cancer. In some aspects, the EDB+FN expressing disorder or disease is PR+ER+HER2-breast cancer. In some aspects, the EDB+FN expressing disorder or disease is a subtype of sarcoma selected from the group consisting of chordoma, leiomyosarcoma, liposarcoma, myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, and soft tissue sarcoma. In some aspects, the EDB+FN expressing disorder or disease is a subtype of ovarian cancer selected from the group consisting of serous carcinoma, serous papillary carcinoma, adenocarcinoma, and endometrioid carcinoma. In one aspect, the present disclosure relates to a method of treating an EDB+FN- expressing disorder or disease in a subject of need thereof. In certain embodiments, the method comprises administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, comprising a heavy chain variable region (VH) comprising the VH CDR1 region, VH CDR2 region, and VH CDR3 region of the VH amino acid sequence of SEQ ID NO:21. and a light chain variable region (VL) comprising VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NO: 10; (b) a linker; (c) a drug. In certain embodiments, the EDB+FN-expressing disorder or disease is selected from the group consisting of breast cancer, HR+HER2- breast cancer, PR+ER+HER2- breast cancer, triple negative breast cancer, colorectal cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, sarcoma, and thyroid cancer. In certain embodiments, the EDB+FN expressing disorder or disease is a subtype of sarcoma selected from the group consisting of chordoma, leiomyosarcoma, liposarcoma, myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, and soft tissue sarcoma. In certain embodiments, the EDB+FN expressing disorder or disease is a subtype of non- small cell lung cancer selected from the group consisting of adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. In certain embodiments, the EDB+FN expressing disorder or disease is a subtype of ovarian cancer selected from the group consisting of serous carcinoma, serous papillary carcinoma, adenocarcinoma, and endometrioid carcinoma. In certain embodiments, the EDB+FN-expressing disorder or disease is head and neck squamous cell carcinoma. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2 -breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, ovarian cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2 -breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting PR+ER+HER2-breast cancer, head and neck cancer, ovarian cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting PR+ER+HER2 -breast cancer, head and neck cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is HR+HER2- breast cancer. In some aspects, the EDB+FN-expressing disorder or disease is head and neck cancer. In some aspects, the EDB+FN-expressing disorder or disease is renal cancer. In some aspects, the EDB+FN-expressing disorder or disease is non-small cell lung cancer. In some aspects, the EDB+FN-expressing disorder or disease is ovarian cancer. In some aspects, the EDB+FN-expressing disorder or disease is pancreatic cancer. In some aspects, the EDB+FN-expressing disorder or disease is triple negative breast cancer. In some aspects, the EDB+FN-expressing disorder or disease is sarcoma. In some aspects, the EDB+FN-expressing disorder or disease is thyroid cancer. In some aspects, the EDB+FN expressing disorder or disease is PR+ER+HER2-breast cancer. In some aspects, the EDB+FN expressing disorder or disease is a subtype of sarcoma selected from the group consisting of chordoma, leiomyosarcoma, liposarcoma, myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, and soft tissue sarcoma. In some aspects, the EDB+FN expressing disorder or disease is a subtype of ovarian cancer selected from the group consisting of serous carcinoma, serous papillary carcinoma, adenocarcinoma, and endometrioid carcinoma.

[0113] In one aspect, the present disclosure relates to a method of treating an EDB+FN- expressing disorder or disease in a subject of need thereof. In certain embodiments, the method comprises administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, comprising a VH region comprising the VH CDR1 region, VH CDR2 region, and VH CDR3 region of the VH amino acid sequence of SEQ ID NO:21, and a VL region comprising VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NO: 10; (b) a cleavable linker, including optionally, val-cit, phe-lys, vc. AcLys-vc. diS, diS-C2OCO, me, me, MalPeg6C2, or a hydrazone linker; (c) a cytotoxic drug, including optionally, an anthracy cline, an auristatin, a CPI / CBI dimer, CC-1065, a dolastatin, a duocarmycin, an enediyne, a geldanamycin, a maytansine, a puromycin, a taxane, a vinca alkaloid, SN-38, tubulysin, hemiasterlin, and stereoisomers, isosteres, analogs or derivatives thereof. In certain embodiments, the EDB+FN-expressing disorder or disease is selected from the group consisting of breast cancer, HR+HER2- breast cancer, PR+ER+HER2- breast cancer, triple negative breast cancer, colorectal cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, sarcoma, and thyroid cancer. In certain embodiments, the EDB+FN expressing disorder or disease is a subtype of sarcoma selected from the group consisting of chordoma, leiomyosarcoma, liposarcoma, myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, and soft tissue sarcoma. In certain embodiments, the EDB+FN expressing disorder or disease is a subtype of non-small cell lung cancer selected from the group consisting of adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. In certain embodiments, the EDB+FN expressing disorder or disease is a subtype of ovarian cancer selected from the group consisting of serous carcinoma, serous papillary carcinoma, adenocarcinoma, and endometrioid carcinoma. In certain embodiments, the EDB+FN-expressing disorder or disease is head and neck squamous cell carcinoma. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2-breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, ovarian cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN- expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2 -breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting PR+ER+HER2-breast cancer, head and neck cancer, ovarian cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN- expressing disorder or disease is selected from the group consisting PR+ER+HER2-breast cancer, head and neck cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN- expressing disorder or disease is HR+HER2- breast cancer. In some aspects, the EDB+FN- expressing disorder or disease is head and neck cancer. In some aspects, the EDB+FN- expressing disorder or disease is renal cancer. In some aspects, the EDB+FN-expressing disorder or disease is non-small cell lung cancer. In some aspects, the EDB+FN-expressing disorder or disease is ovarian cancer. In some aspects, the EDB+FN-expressing disorder or disease is pancreatic cancer. In some aspects, the EDB+FN-expressing disorder or disease is triple negative breast cancer. In some aspects, the EDB+FN-expressing disorder or disease is sarcoma. In some aspects, the EDB+FN-expressing disorder or disease is thyroid cancer. In some aspects, the EDB+FN expressing disorder or disease is PR+ER+HER2-breast cancer. In some aspects, the EDB+FN expressing disorder or disease is a subtype of sarcoma selected from the group consisting of chordoma, leiomyosarcoma, liposarcoma, myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, and soft tissue sarcoma. In some aspects, the EDB+FN expressing disorder or disease is a subtype of ovarian cancer selected from the group consisting of serous carcinoma, serous papillary carcinoma, adenocarcinoma, and endometrioid carcinoma.

[0114] In one aspect, the present disclosure relates to a method of treating an EDB+FN- expressing disorder or disease in a subject of need thereof. In certain embodiments, the method comprises administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, comprising a VH region comprising the VH CDR1 region, VH CDR2 region, and VH CDR3 region of the VH amino acid sequence of SEQ ID NO:21, including optionally, SEQ ID NOs: 3, 5 and 7, and a VL region comprising VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NO: 10, including optionally, SEQ ID NOs: 12, 13 and 14; (b) a vc linker; (c) a 0101 drug. In certain embodiments, the EDB+FN-expressing disorder or disease is selected from the group consisting of breast cancer, HR+HER2- breast cancer, PR+ER+HER2- breast cancer, triple negative breast cancer, colorectal cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, sarcoma, and thyroid cancer. In certain embodiments, the EDB+FN expressing disorder or disease is a subtype of sarcoma selected from the group consisting of chordoma, leiomyosarcoma, liposarcoma, myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, and soft tissue sarcoma. In certain embodiments, the EDB+FN expressing disorder or disease is a subtype of non-small cell lung cancer selected from the group consisting of adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. In certain embodiments, the EDB+FN expressing disorder or disease is a subtype of ovarian cancer selected from the group consisting of serous carcinoma, serous papillary carcinoma, adenocarcinoma, and endometrioid carcinoma. In certain embodiments, the EDB+FN- expressing disorder or disease is head and neck squamous cell carcinoma. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2-breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, ovarian cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2-breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN- expressing disorder or disease is selected from the group consisting PR+ER+HER2-breast cancer, head and neck cancer, ovarian cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting PR+ER+HER2-breast cancer, head and neck cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is HR+HER2- breast cancer. In some aspects, the EDB+FN-expressing disorder or disease is head and neck cancer. In some aspects, the EDB+FN-expressing disorder or disease is renal cancer. In some aspects, the EDB+FN-expressing disorder or disease is non-small cell lung cancer. In some aspects, the EDB+FN-expressing disorder or disease is ovarian cancer. In some aspects, the EDB+FN- expressing disorder or disease is pancreatic cancer. In some aspects, the EDB+FN- expressing disorder or disease is triple negative breast cancer. In some aspects, the EDB+FN- expressing disorder or disease is sarcoma. In some aspects, the EDB+FN-expressing disorder or disease is thyroid cancer. In some aspects, the EDB+FN expressing disorder or disease is PR+ER+HER2 -breast cancer. In some aspects, the EDB+FN expressing disorder or disease is a subtype of sarcoma selected from the group consisting of chordoma, leiomyosarcoma, liposarcoma, myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, and soft tissue sarcoma. In some aspects, the EDB+FN expressing disorder or disease is a subtype of ovarian cancer selected from the group consisting of serous carcinoma, serous papillary carcinoma, adenocarcinoma, and endometrioid carcinoma.

[0115] In one aspect, the present disclosure relates to a method of treating an EDB+FN- expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, comprising a VH region comprising SEQ ID NO: 21 and a VL region comprising SEQ ID NO: 10; (b) a vc linker; (c) a 0101 drug. In certain embodiments, the EDB+FN-expressing disorder or disease is selected from the group consisting of breast cancer, HR+HER2- breast cancer, PR+ER+HER2- breast cancer, triple negative breast cancer, colorectal cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, sarcoma, and thyroid cancer. In certain embodiments, the EDB+FN expressing disorder or disease is a subtype of sarcoma selected from the group consisting of chordoma, leiomyosarcoma, liposarcoma. myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, and soft tissue sarcoma. In certain embodiments, the EDB+FN expressing disorder or disease is a subtype of non- small cell lung cancer selected from the group consisting of adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. In certain embodiments, the EDB+FN expressing disorder or disease is a subtype of ovarian cancer selected from the group consisting of serous carcinoma, serous papillary carcinoma, adenocarcinoma, and endometrioid carcinoma. In certain embodiments, the EDB+FN-expressing disorder or disease is head and neck squamous cell carcinoma. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2 -breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, ovarian cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2 -breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting PR+ER+HER2-breast cancer, head and neck cancer, ovarian cancer. sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting PR+ER+HER2-breast cancer, head and neck cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is HR+HER2- breast cancer. In some aspects, the EDB+FN-expressing disorder or disease is head and neck cancer. In some aspects, the EDB+FN-expressing disorder or disease is renal cancer. In some aspects, the EDB+FN-expressing disorder or disease is non-small cell lung cancer. In some aspects, the EDB+FN-expressing disorder or disease is ovarian cancer. In some aspects, the EDB+FN-expressing disorder or disease is pancreatic cancer. In some aspects, the EDB+FN-expressing disorder or disease is triple negative breast cancer. In some aspects, the EDB+FN-expressing disorder or disease is sarcoma. In some aspects, the EDB+FN-expressing disorder or disease is thyroid cancer. In some aspects, the EDB+FN expressing disorder or disease is PR+ER+HER2-breast cancer. In some aspects, the EDB+FN expressing disorder or disease is a subtype of sarcoma selected from the group consisting of chordoma, leiomyosarcoma, liposarcoma, myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, and soft tissue sarcoma. In some aspects, the EDB+FN expressing disorder or disease is a subtype of ovarian cancer selected from the group consisting of serous carcinoma, serous papillary carcinoma, adenocarcinoma, and endometrioid carcinoma.

[0116] In one aspect, the present disclosure relates to a method of treating an EDB+FN- expressing disorder or disease in a subject of need thereof. In certain embodiments, the method comprises administering an effective amount of 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; (c) a 0101 drug. In certain embodiments, the EDB+FN-expressing disorder or disease is selected from the group consisting of breast cancer, HR+HER2- breast cancer, PR+ER+HER2- breast cancer, triple negative breast cancer, colorectal cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, sarcoma, and thyroid cancer. In certain embodiments, the EDB+FN expressing disorder or disease is a subtype of sarcoma selected from the group consisting of chordoma, leiomyosarcoma, liposarcoma. myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, and soft tissue sarcoma. In certain embodiments, the EDB+FN expressing disorder or disease is a subtype of non-small cell lung cancer selected from the group consisting of adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. In certain embodiments, the EDB+FN expressing disorder or disease is a subtype of ovarian cancer selected from the group consisting of serous carcinoma, serous papillary carcinoma, adenocarcinoma, and endometrioid carcinoma. In certain embodiments, the EDB+FN-expressing disorder or disease is head and neck squamous cell carcinoma. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2-breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, ovarian cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN- expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer. PR+ER+HER2 -breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting PR+ER+HER2 -breast cancer, head and neck cancer, ovarian cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN- expressing disorder or disease is selected from the group consisting PR+ER+HER2-breast cancer, head and neck cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN- expressing disorder or disease is HR+HER2- breast cancer. In some aspects, the EDB+FN- expressing disorder or disease is head and neck cancer. In some aspects, the EDB+FN- expressing disorder or disease is renal cancer. In some aspects, the EDB+FN-expressing disorder or disease is non-small cell lung cancer. In some aspects, the EDB+FN-expressing disorder or disease is ovarian cancer. In some aspects, the EDB+FN-expressing disorder or disease is pancreatic cancer. In some aspects, the EDB+FN-expressing disorder or disease is triple negative breast cancer. In some aspects, the EDB+FN-expressing disorder or disease is sarcoma. In some aspects, the EDB+FN-expressing disorder or disease is thyroid cancer. In some aspects, the EDB+FN expressing disorder or disease is PR+ER+FIER2-breast cancer. In some aspects, the EDB+FN expressing disorder or disease is a subtype of sarcoma selected from the group consisting of chordoma, leiomyosarcoma, liposarcoma, myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, and soft tissue sarcoma. In some aspects, the EDB+FN expressing disorder or disease is a subtype of ovarian cancer selected from the group consisting of serous carcinoma, serous papillary carcinoma, adenocarcinoma, and endometrioid carcinoma.

[0117] In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting of breast cancer, HR+HER2- breast cancer, PR+ER+HER2- breast cancer, triple negative breast cancer, hepatocellular carcinoma, head and neck cancer, renal cancer, non-small cell lung cancer, pancreatic cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2- breast cancer, triple negative breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, ovarian cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting of PR+ER+HER2- breast cancer, triple negative breast cancer, head and neck cancer, renal cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting of head and neck cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2- breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, ovarian cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2- breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting PR+ER+HER2-breast cancer, head and neck cancer, ovarian cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is selected from the group consisting PR+ER+HER2-breast cancer, head and neck cancer, sarcoma, and thyroid cancer. In some aspects, the EDB+FN-expressing disorder or disease is HR+HER2- breast cancer. In some aspects, the EDB+FN-expressing disorder or disease is head and neck cancer. In some aspects, the EDB+FN-expressing disorder or disease is renal cancer. In some aspects, the EDB+FN-expressing disorder or disease is non-small cell lung cancer. In some aspects, the EDB+FN-expressing disorder or disease is ovarian cancer. In some aspects, the EDB+FN-expressing disorder or disease is pancreatic cancer. In some aspects, the EDB+FN-expressing disorder or disease is triple negative breast cancer. In some aspects, the EDB+FN-expressing disorder or disease is sarcoma. In some aspects, the EDB+FN-expressing disorder or disease is thyroid cancer. In some aspects, the EDB+FN expressing disorder or disease is PR+ER+HER2-breast cancer. In some aspects, the EDB+FN expressing disorder or disease is a subtype of sarcoma selected from the group consisting of chordoma, leiomyosarcoma, liposarcoma, myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, and soft tissue sarcoma. In some aspects, the EDB+FN expressing disorder or disease is a subtype of ovarian cancer selected from the group consisting of serous carcinoma, serous papillary carcinoma, adenocarcinoma, and endometrioid carcinoma.

[0118] In certain embodiments, the EDB+FN-expressing disorder or disease is breast cancer. In certain embodiments, the EDB+FN-expressing disorder or disease is HR+HER2- breast cancer. In certain embodiments, the EDB+FN-expressing disorder or disease is PR+ER+HER2- breast cancer. In certain embodiments, the EDB+FN-expressing disorder or disease is triple negative breast cancer. In certain embodiments, the EDB+FN-expressing disorder or disease is colorectal cancer. In certain embodiments, the EDB+FN-expressing disorder or disease is hepatocellular (liver) cancer. In certain embodiments, the EDB+FN- expressing disorder or disease is head and neck cancer. In certain embodiments, the EDB+FN-expressing disorder or disease is renal cancer. In certain embodiments, the EDB+FN-expressing disorder or disease is lymphoma. In certain embodiments, the EDB+FN-expressing disorder or disease is non-small cell lung cancer. In certain embodiments, the EDB+FN-expressing disorder or disease is ovarian cancer. In certain embodiments, the EDB+FN-expressing disorder or disease is pancreatic cancer. In certain embodiments, the EDB+FN-expressing disorder or disease is sarcoma. In certain embodiments, the EDB+FN-expressing disorder or disease is thyroid cancer.

[0119] In certain embodiments, the EDB+FN expressing disorder or disease is the sarcoma subtype chordoma. In certain embodiments, the EDB+FN expressing disorder or disease is the sarcoma subtype leiomyosarcoma. In certain embodiments, the EDB+FN expressing disorder or disease is the sarcoma subtype liposarcoma. In certain embodiments, the EDB+FN expressing disorder or disease is the sarcoma subtype myxofibrosarcoma. In certain embodiments, the EDB+FN expressing disorder or disease is the sarcoma subtype osteosarcoma. In certain embodiments, the EDB+FN expressing disorder or disease is the sarcoma subtype rhabdomyosarcoma. In certain embodiments, the EDB+FN expressing disorder or disease is the sarcoma subtype soft tissue sarcoma. In certain embodiments, the EDB+FN expressing disorder or disease is the non-small cell lung cancer subtype adenocarcinoma. In certain embodiments, the EDB+FN expressing disorder or disease is the non-small cell lung cancer subtype squamous cell carcinoma. In certain embodiments, the EDB+FN expressing disorder or disease is the non-small cell lung cancer subtype large cell carcinoma. In certain embodiments, the EDB+FN expressing disorder or disease is the ovarian cancer subtype serous carcinoma. In certain embodiments, the EDB+FN expressing disorder or disease is the ovarian cancer subtype serous papillary carcinoma. In certain embodiments, the EDB+FN expressing disorder or disease is the ovarian cancer subtype adenocarcinoma. In certain embodiments, the EDB+FN expressing disorder or disease is the ovarian cancer subtype endometrioid carcinoma. In certain embodiments, the EDB+FN- expressing disorder or disease is the head and neck cancer subtype squamous cell carcinoma.

[0120] In some aspects, the disclosure provides a method of inhibiting tumor growth or progression in a subject who has an EDB-expressing disorder, such as non-cancers or cancers associated with EDB+FN expression and / or EDB-expressing cancers. In certain embodiments, the method comprises administering to the subject in need thereof an effective amount of an ADC as described herein and / or a composition (i.e., a pharmaceutical composition) having one or more EDB ADCs described herein. In other aspects of the disclosure, provided is a method of inhibiting metastasis of cancer cells associated with EDB+FN expression and / or EDB+FN-expressing cancers in a subject. In certain embodiments, the method comprises administering to the subject in need thereof an effective amount of a composition (i.e., a pharmaceutical composition) having one or more EDB ADCs described herein. In other aspects of the disclosure, provided is a method of inducing regression of a tumor associated with EDB+FN expression and / or EDB+FN-expressing cancers in a subject. In certain embodiments, the method comprises administering to the subject in need thereof an effective amount of a composition (i.e., a pharmaceutical composition) having one or more EDB ADCs described herein.

[0121] In some aspects, the EDB+FN expression may be detected in the extracellular matrix (ECM) adjacent to tumor cells. EDB+FN may be expressed by cells other than fibroblasts in the tumor microenvironment, including tumor cells. The secreted EDB+FN may be then deposited in the matrix adjacent to tumor cells, or on the plasma membrane of tumor cells. In other aspects, the disclosure provides a pharmaceutical composition comprising one or more EDB ADCs described herein for use in a method as described above. In other aspects, the disclosure provides the use of one or more EDB ADCs as described herein or a pharmaceutical composition comprising the EDB ADCs as described herein in the manufacture of a medicament for use in the methods described above.

[0122] Cancers associated with EDB+FN expression and / or EDB+FN-expressing cancers may generally include any cancer associated with tissue remolding. Further, cancers associated with EDB+FN expression and / or EDB+FN-expressing cancers may include, but are not limited to, solid tumors and blood cancers. In some aspects, solid tumors include, but are not limited to, thyroid cancer, sarcoma, breast cancer, TNBC, HR+HER2- breast cancer, ER+PR+HER2- 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, thymus cancer, brain cancer, adrenal cancer, eye cancer cervical cancer and lung cancer. In some aspects, blood cancers include, but are not limited to, leukemia, lymphoma and myeloma.

[0123] The EDB ADCs of the present disclosure are useful in treating EDB+FN-expressing disorders, such as cancers associated with EDB+FN expression and / or EDB+FN-expressing cancers. EDB ADCs of the disclosure may be used to treat cancers that express high levels of EDB+FN, moderate levels of EDB+FN or low levels of EDB+FN.

[0124] Thus, patients to be treated with EDB ADCs of the disclosure may 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 results in a patient population 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 the cells staining. In other cases, patients may be selected for certain cancer types, and subtypes, that have been demonstrated herein to respond to EDB ADCs.

[0125] Cancer growth or abnormal proliferation refers to any one of a number of indices that suggest change within cells to a more developed cancer form or disorder state. Inhibition of growth of cancer cells or cells of a non-neoplastic proliferative disorder may be assayed by methods known in the art, such as delayed tumor growth and inhibition of metastasis. Other indices for measuring inhibition of cancer growth include a decrease in cancer cell survival, a decrease in tumor volume or morphology (for example, as determined using computed tomographic (CT), sonography, or other imaging method), destruction of tumor vasculature, improved performance in delayed hypersensitivity skin test, an increase in the activity of cytolytic T-lymphocytes, and a decrease in levels of tumor-specific antigens.

[0126] Desired outcomes of the disclosed therapeutic methods are generally quantifiable measures as compared to a control or baseline measurement. As used herein, relative terms such as "improve," "increase," or "reduce" indicate values relative to a control or comparative molecule, such as a measurement in the same individual prior to initiation of treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of the treatment described herein. A representative control individual is an individual afflicted with the same form of cancer as the individual being treated, who is about the same age as the individual being treated (to ensure that the stages of the disorder in the treated individual and the control individual are comparable.

[0127] Changes or improvements in response to therapy can be generally observed or statistically significant. As used herein, the term "significance" or "significant" relates to a statistical analysis of the probability that there is a non-random association between two or more entities. To determine whether or not a relationship is "significant" or has "significance," statistical manipulations of the data can be "p-value." Those p-values that fall below' a user-defined cut-off point are regarded as significant. A p-value less than or equal to 0. 1, less than 0.05. less than 0.01, less than 0.005. or less than 0.001 may be regarded as significant. Dose and Administration

[0128] The present disclosure provides for EDB ADCs administered in an effective dosage. For prophylactic use, beneficial or desired results include eliminating or reducing the risk, lessening the severity, or delaying the outset of the disorder, including biochemical, histological and / or behavioral symptoms of the disorder, its complications and intermediate pathological phenotypes presenting during development of the disorder. For therapeutic use. beneficial or desired results include clinical results such as reducing incidence or amelioration of one or more symptoms of various EDB+FN-expressing disorders, such as cancer, decreasing the dose of other medications required to treat the disorder, enhancing the effect of another medication, and / or delaying the progression of the EDB+FN-expressing disorders of patients.

[0129] 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 may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.

[0130] For example, when administered to a cancer-bearing subject, an effective amount includes an amount sufficient to elicit anti-cancer activity, including cancer cell cytolysis, inhibition of cancer cell proliferation, induction of cancer cell apoptosis, reduction of cancer cell antigens, delayed tumor growth, and / or inhibition of metastasis. Tumor shrinkage is well accepted as a clinical surrogate marker for efficacy. Another well accepted marker for efficacy is progression-free survival.

[0131] The EDB ADCs of the present disclosure can be administered to an individual via any suitable route. It should be understood by persons skilled in the art that the examples described herein are not intended to be limiting but to be illustrative of the techniques available. Accordingly, in some aspects of the disclosure, the EDB ADC is administered to an individual in accord with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, intracranial, transdermal, subcutaneous, intra-articular, sublingually, intrasy novial, via insufflation, intrathecal, oral, inhalation or topical routes. Administration can be systemic, e.g., intravenous administration, 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 powder can be nebulized after reconstitution. Alternatively, the EDB ADC may be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.

[0132] In some aspects of the disclosure, the EDB ADCs are administered via site-specific or targeted local delivery techniques. Examples of site-specific or targeted local delivery techniques include various implantable depot sources of an EDC 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.

[0133] For the purpose of the present disclosure, the appropriate dosage of an EDB ADC may depend on the particular EDB ADC (or compositions thereof) employed, the type and severity of symptoms to be treated, whether the agent is administered for therapeutic purposes, previous therapy, 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 an EDB ADC until a dosage is reached that achieves the desired result and beyond. Dose and / or frequency can vary over course of treatment, but may stay constant as well. Empirical considerations, such as the half-life, generally will contribute to the determination of the dosage. For example, antibodies that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, may be used to prolong half-life of the antibody and to prevent the antibody being attacked by the host’s immune system. Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and / or suppression and / or amelioration and / or delay of symptoms, e.g., tumor growth inhibition or delay, etc. Alternatively, sustained continuous release formulations of EDB ADCs may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0134] For the purpose of the present disclosure, a typical daily dosage might range from about any of 3 pg / kg to 30 pg / kg to 300 pg / kg to 3 mg / kg, to 30 mg / kg, to 100 mg / kg or more, depending on the factors mentioned above. For example, dosage of about 1 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, and about 25 mg / kg may be used. For repeated administrations over several days or longer, depending on the disorder, the treatment is sustained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved, for example, to inhibit or delay tumor growth / progression or metastasis of cancer cells. Exemplary dosing regimens may include administering increasing doses (e.g.. initial dose of 1 mg / kg and gradual increase to one or more higher doses every week or longer time period). Other dosage regimens may also be useful, depending on the pattern of pharmacokinetic decay that the practitioner wishes to achieve. For example, in some aspects of the disclosure, dosing from one to four times a week is contemplated. In other aspects, dosing once a month or once every other month or every three months is contemplated, as well as weekly, bi-weekly and every three weeks. The progress of this therapy may be easily monitored by conventional techniques and assays. The dosing regimen (including the EDB ADC used) can vary' over time.

[0135] In some aspects of the disclosure, dosages for an EDB ADC may be determined empirically in individuals who have been given one or more administration(s) of an EDB ADC. Individuals may be given incremental dosages of an EDB ADC. To assess efficacy, an indicator of the disorder can be followed.

[0136] Administration of an EDB ADC in accordance with the method in the present disclosure can be continuous or intermittent, depending, for example, upon the recipient's physiological disorder, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of an EDB ADC may be essentially continuous over a preselected period of time or may be in a series of spaced doses.

[0137] In respect to comparing dosing levels for relevant animal studies to dosage levels in humans, the FDA provided guidance in their document (“FDA Dosing Guidance") entitled “Guidance for Industry - Estimating the Maximum Safe Starting Dose in Initial Clinical Trial for Therapeutics in Adult Healthy Volunteers'’ Center for Drug Evaluation and Research (CDER) 7.0.001 (2005). Human equivalent dose (HED) calculations are provided therein based upon body surface area with conversion factors and divisors provided in Table 1 of the FDA Dosing Guidance. The standard conversion factor set forth in the document to convert a mouse (murine) dose to a HED is to either divide the murine dose by 12.3 or multiple the murine dose by 0.03. For example, the conversion calculation of a murine dose of 3 mg / kg would be approximately 0.24 mg / kg HED. Further calculations are provided for exemplary species such as hamster, rat, ferret, guinea pig, rabbit, dose, various primates, and pigs.

[0138] Combination Therapies

[0139] In some aspects of the disclosure, the methods described herein further include a step of treating a subject with an additional form of therapy. In some aspects, the additional form of therapy is an additional anti-cancer therapy including, but not limited to, be used in chemotherapy, radiation, surgery', hormone therapy, and / or additional immunotherapy.

[0140] The disclosed EDB ADCs may be administered as an initial treatment, or for treatment of cancers that are unresponsive to conventional therapies. In addition, the EDB ADCs may combination with other therapies (e.g., surgical excision, radiation, additional anti-cancer drugs, etc.) to thereby elicit additive or potentiated therapeutic effects and / or reduce cytotoxicity of some anti-cancer agents. EDB ADCs of the disclosure may be coadministered or co-formulated with additional agents, or formulated for consecutive administration with additional agents in any order.

[0141] EDB ADCs of the disclosure may be used in combination with other therapeutic agents including, but not limited to, therapeutic antibodies, ADCs, immunomodulating agents, cytotoxic agents, and cytostatic agents. Representative agents useful for combination therapy also include any of the drugs described herein above as useful for preparation of an EDB ADC under the subheading "Drugs."

[0142] Therapeutic agents include, but are not limited to, the administration of a chemotherapeutic agent, a vaccine, a CAR-T cell-based therapy, radiotherapy, a cytokine therapy, a vaccine, a bispecific antibody, an ADC, an inhibitor of other immunosuppressive pathways, an inhibitors of angiogenesis, a T cell activator, an inhibitor of a metabolic pathway, an mTOR inhibitor, an inhibitor of an adenosine pathway, a tyrosine kinase inhibitor including but not limited to inlyta, ALK inhibitors and sunitinib, a BRAF inhibitor, an epigenetic modifier, an inhibitors or depletor of Treg cells and / or of myeloid-derived suppressor cells, a JAK inhibitor, a STAT inhibitor, a cyclin-dependent kinase inhibitor, a biotherapeutic agent (including but not limited to antibodies to VEGF, VEGFR, EGFR, Her2 / neu, other growth factor receptors. CD20, CD40, CD-40L, CTLA-4, OX-40, 4- IBB, and ICOS), an immunogenic agent (for example, attenuated cancerous cells, tumor antigens, antigen presenting cells such as dendritic cells pulsed with tumor derived antigen or nucleic acids, immune stimulating cytokines (for example, IL-2, IFNa2, GM- CSF), and cells transfected with genes encoding immune stimulating cytokines such as but not limited to GM-CSF).

[0143] Further representative antibodies, which may be used alone or as an ADC, include, but are not limited to, anti-5T4 antibodies (e.g., Al, A2, and A3), anti-CD19 antibodies, anti- CD20 antibodies (e.g., RITUXAN®, ZEVALIN®, BEXXAR®), anti-CD22 antibodies, antiantibodies (e.g., MYLOTARG®), anti CD33 antibody-drug conjugates. anti-Lewis Y antibodies (e.g., Hu3S193, Mthu3S193, AGmthu3S193), anti-HER-2 antibodies (e.g., HERCEPTIN® (trastuzumab), MDX-210, OMNITARG.RTM. (pertuzumab, rhuMAb 2C4)). 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-l / b), anti-CEA 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).

[0144] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide. triethylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC- 1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophy cin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin gammal 1 and calicheamicin phiM . see, e.g., Agnew, Chem. Inti. Ed. Engl., 33: 183-186 (1994); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin. detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2- pyrrolino- doxorubicin, and deoxydoxorubicin), pegylated liposomal doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin. potfiromycin, puromycin, quelamycin, rodorubicin. streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine. 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2, 2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel and doxetaxel; 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-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0145] Also included are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens 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 enzy me aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)- imidazoles, aminoglutethimide, megestrol acetate, exemestane. formestane, fadrozole. vorozole. letrozole. and anastrozole; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0146] In some aspects, EDB ADCs may be used in combination crizotinib, palbociclib, gemcitabine, cyclophosphamide, fluorouracil. FOLFOX, folinic acid, oxaliplatin, axitinib, sunitinib malate, tofacitinib, bevacizumab, rituximab, and traztuzumab. In some aspects, after treatment with EDB ADCs an increase in tumor infiltrating lymphocytes, an increase in CD8 / CD4 ratios, an increase in F4 / 80+ macrophages, and / or an increase in immunomodulatory proteins such as PDL1 and 4 IBB, or any combination thereof, may occur. Thus, the combination of an EDB ADC and an immune checkpoint inhibitor or IO agent, such as an anti-4 IBB agonist and / or anti-PDLl antagonist monoclonal antibody may be effective. (See Example 12). Further, EDB ADCs of the disclosure alone may have immunodulatory, and immune-oncology (IO) agent enabling mechanisms, that maybe increased with combination therapy.

[0147] In some aspects, an EDB ADC may be used in combination with one or more other therapeutic agents targeting an immune checkpoint modulator, including but not limited to, an agent (such as an antibody) targeting 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, CD1 12, LAIR1 , 2B4, BTLA, CD160, TIM1, TIM-3, TIM4, VISTA (PD-H1 ), 0X40, OX40L, GITR, GITRL , CD70, CD27 , 4- 1BB, 4-BBL, DR3, TL1A. CD40, CD40L, CD30. CD30L, LIGHT. HVEM, SLAM (SLAMF1 , CD 150). SLAMF2 (CD48). SLAMF3 (CD229), SLAMF4 (2B4. CD244). SLAMF5 (CD84), SLAMF6 (NTB-A), SLAMCF7 (CS1 ), SLAMF8 (BLAME), SLAMF9 (CD2F), CD28, CEACAM1 (CD66a ), CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM1 -3AS CEACAM3C2, CEACAM1 -15, PSG1 -1 1 , CEACAM1 -4C1 . CEACAM1 -4S. CEACAM1 - 4L, IDO, TDO, CCR2, CD39-CD73- adenosine pathway (A2AR), BTKs, TIKs, CXCR2, CCR4, CCR8, CCR5, VEGF pathway, CSF-1 , or an innate immune response modulator.

[0148] For combination therapies, an EDB ADC and / or one or more additional therapeutic agents are administered within any time frame suitable for performance of the intended therapy. Thus, the single agents may be administered substantially simultaneously (i.e., as a single formulation or within minutes or hours) or consecutively in any order. For example, single agent treatments may be administered within about 1 year of each other, such as within about 10. 8, 6, 4, or 2 months, or within 4. 3, 2 or 1 week(s), or within about 5, 4, 3, 2 or 1 day(s).

[0149] The disclosed combination therapies may elicit 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 may be an effect of at least about two-fold greater than the therapeutic effect elicited by a single agent, or the sum of the therapeutic effects elicited by the single agents of a given combination, or at least about five-fold greater, or at least about ten-fold greater, or at least about twenty-fold greater, or at least about fifty-fold greater, or at least about one hundred-fold greater. A synergistic therapeutic effect may also be observed as an increase in therapeutic effect of at least 10% compared to the therapeutic effect elicited by a single agent, or the sum of the therapeutic effects elicited by the single agents of a given combination, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or more. A synergistic effect is also an effect that permits reduced dosing of therapeutic agents when they are used in combination.

[0150] The EDB ADC may also be given in combination with any one or more of the additional therapeutic agents disclosed herein, such that the EDB ADC is given to resensitize a cancer which has become resistant to a first agent, wherein the EDB ADC reestablishes the therapeutic efficacy of the first agent.

[0151] EDB-ADC Structures

[0152] Various different EDB-ADC structures are presented in the table below.

[0153] Table 1.

[0154] Antibodies and Antibody-Drug Conjugates

[0155] ADCs comprise an antibody component conjugated to a drug, typically through the use of a linker. ADCs generated by conventional conjugation technology randomly link the drug to the antibody through lysine or cysteine residues that are endogenously on the antibody heavy and / or light chain. Accordingly, such ADCs are a heterogeneous mixture of species having different drug: antibody ratios (DAR). ADCs generated by site-specific conjugation technology link the drug to the antibody at particular engineered residues on the antibody heavy and / or light chain. As such, the site-specific conjugated ADCs are a homogeneous mixture of ADCs comprised of a species with a defined drug: antibody ratio (DAR). Thus, site-specific conjugated ADCs demonstrate uniform stoichiometry resulting in improved pharmacokinetics, biodistribution and safety profile.

[0156] ADCs of the present disclosure include anti-EDB antibodies conjugated to one or more drugs via a linker (i.e. forming linker-drug moieties). The present disclosure provides for ADCs having (a) an antibody, or antigen binding fragment thereof, that binds to EDB; (b) a linker and (c) a drug. The present disclosure further provides for ADCs of the formula Ab- (L-D), wherein (a) Ab is an antibody, or antigen-binding fragment thereof, that binds to EDB, and (b) L-D is a linker-drug moiety, wherein L is a linker, and D is a drug. In some aspects, the present disclosure provides for ADCs of the formula Ab-(L-D) / ?, wherein (a) Ab is an antibody, or antigen-binding fragment thereof, that binds to EDB, (b) L-D is a linker-drug moiety, wherein L is a linker, and D is a drug and (c) p is the number of linker-drug moieties attached to the antibody.

[0157] The number of linker-drug moieties attached to an antibody can be any number preferred for development of an ADC. In some aspects, the number of linker-drug moieties per antibody is 4. In other aspects, the number of linker-drug moieties per antibody is 3. In some aspects, the number of linker-drug moieties per antibody is 2. In some aspects, the number of linker-drug moieties per antibody is 1. In other aspects, the number of linker-drug moieties per antibody is greater than 4, such as 5, 6, 7, 8, 9, 10, 11, 12 or greater than 12 linker-drug moieties per antibody.

[0158] Further the present disclosure provides for ADCs, wherein the linker-drug moieties are attached to the antibody via conventional or site-specific conjugation technology. In some aspects, the anti -EDB antibodies, or antigen-binding fragments thereof, are conjugated or linked to a drug such as a cytotoxic, cytostatic, and / or therapeutic agent, as described further herein. For example, a cytotoxic agent can be linked or conjugated to an anti-EDB antibody as described herein for targeted local delivery of the cytotoxic agent. Also provided are methods of preparing and manufacturing such ADCs, and use of the same in clinical applications.

[0159] In contrast to other ADCs being developed to target internalizing cell surface expressed proteins, the ADCs of the present disclosure target EDB. a protein expressed in the extracellular matrix (ECM). Targeting a protein expressed in the ECM may provide benefits over targeting a protein expressed on the tumor cells. The ADC may directly access the target without having to penetrate through the stromal and ECM barriers common in many difficult- to-treat human cancers. Further, targeting EDB in the ECM with an ADC provides a specific mechanism to access many difficult to target cell types in the tumor microenvironment. This may result in the extracellular release of a cytotoxic payload or drug, resulting in the killing of a variety of cells, via mechanisms such as cell death / cell-cycle arrest of tumor cells and / or stromal cells by bystander mechanism. In addition, further mechanisms include, but are not limited to disregulated angiogenesis or cytotoxic vascular targeting / collapse, vascular normalization, immunomodulation and induction of cellular differentiation and / or impediment of the epithelial to mesenchymal transition.

[0160] Extra-domain B of Fibronectin

[0161] As used herein “EDB+FN” and “EDB” are used interchangeable and refer to fibronectin (FN) containing the extra-domain B (EDB). Further, “anti-EDB antibodies'’ and “anti-EDB+FN antibodies'’ are used interchangeable and refer to antibodies that bind to EDB. "‘Anti-EDB antibody-drug conjugates”, “EDB antibody-drug conjugates”, “anti-EDB ADCs”, “EDB ADCs” are used interchangeable and refer to ADCs comprising antibodies, or antigenbinding fragments thereof, that bind to EDB and are 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 embryogenesis, wound healing, blood coagulation, host defense, and metastasis. FN typically exists as a dimer formed by two nearly identical ~250kDa subunits covalently linked near their C-terminus by a pair of disulfide bonds. Each monomer consists of three types of repeating units: type I, type II and type III FN repeats. A single 75-kb gene encodes FN, however there are twenty protein variants observed in humans. Alternative splicing of the FN gene occurs in three regions resulting in the inclusion or exclusion of either one of the two type III repeats, called extra domain A (EDA) and extra domain B (EDB), and of a segment connecting tw o other Npe III repeats, called type III connecting segment (IIICS). EDB is a 91 amino acid sequence that is 100% identical in mice, rats, rabbits, dogs, cynomologus monkey and humans. A representative EDB+FN nucleotide sequence is provided under Accession No. NM_001306129.1 and corresponding amino acid sequence is provided under Accession No. NP_001293058.1. EDB and recombinant human 7-EDB-8-9 amino acid sequences are provided in Table 2. Recombinant human 7-EDB-8-9 comprises EDB flanked by domain 7 on the amino terminus and [domain 8| and domain 9 at the carboxy terminus of EDB.

[0162] Table 2: EDB AND 7-EDB-8-9 SEQUENCES

[0163] Anti-EDB Antibodies

[0164] Antibodies of the present disclosure specifically bind to EDB. For preparation of ADCs of the disclosure, an antibody, or antigen-binding fragment thereof, may be any antibody (including antibodies described herein), or antigen-binding fragment thereof, that specifically binds to EDB. The antibody, or antigen-binding fragment thereof, may be isolated, purified, or derivatized for use in preparation of an EDB ADC.

[0165] 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., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. The term can encompass any type of antibody, including but not limited to monoclonal antibodies, polyclonal antibodies, “antigenbinding fragments'’ (or portion), such as Fab, Fab’, F(ab’)2, Fd, Fv, Fc, etc., of intact antibodies that retain the ability to specifically bind to a given antigen (e.g. EDB), an isolated complementarity determining region (CDR), bispecific antibodies, heteroconjugate antibodies, mutants thereof, fusion proteins having an antibody, or antigen-binding fragment thereof, (e.g., a domain antibody), single chain (ScFv) and single domain antibodies (e.g., shark and camelid antibodies), maxibodies, minibodies, intrabodies, diabodies, tiiabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Holliger and Hudson, 2005, Nature Biotechnology 23(9): 1126-1136), humanized antibodies, chimeric antibodies and any other modified configuration of the immunoglobulin molecule that includes an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibodies may be murine, rat, human, or any other origin (including chimeric or humanized antibodies). In some aspects of the disclosure, the antibody, or antigen-binding fragment thereof, of the disclosed EDB ADCs is a chimeric, humanized, or a recombinant human antibody, or EDB- binding fragment thereof.

[0166] Native or naturally occurring antibodies and native immunoglobulins are typically heterotetrameric glycoproteins of about 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 a number of constant domains or regions (e.g. hinge. CHI. CH2 or CH3). referred to as “CH domains”. Each light chain has a variable domain (VL) and a constant domain, referred to as “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. The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as Fc receptor (FcR) binding, participation of the antibody in antibody-dependent cellular toxicity (ADCC), opsonization, initiation of complement dependent cytotoxicity, and mast cell degranulation. The constant regions of the EDB antibodies may be derived from constant regions of any one of IgA, IgD, IgE, IgG, IgM, any isotypes thereof (e.g., IgGl, IgG2, IgG3, or IgG4 isotypes of IgG), as well as subclasses and mutated versions thereof.

[0167] CHI domain includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain that extends, e.g. from about positions 118-215 according to the EU index of Kabat. The CHI domain is adjacent to the VH domain and amino terminal to the hinge region of an immunoglobulin heavy chain molecule, and does not form a part of the Fc region of an immunoglobulin heavy chain.

[0168] The hinge region includes the portion of a heavy chain molecule that joins the CHI 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. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains.

[0169] CH2 domain includes the portion of a heavy chain immunoglobulin molecule that extends, e.g. from about positions 231-340 according to the EU index of Kabat. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. In some aspects, the antibody (or fragment thereof) of the disclosure comprises a CH2 domain derived from an IgG molecule, such as IgGl, IgG2, IgG3, or IgG4. In some aspects, the IgG is a human IgG.

[0170] CH3 domain includes the portion of a heavy chain immunoglobulin molecule that extends approximately 110 residues from N-terminus of the CH2 domain, e.g. from about positions 341-447 according to the EU index of Kabat. The CH3 domain typically forms the C -terminal portion of the antibody. In some immunoglobulins, however, additional domains may extend from CH3 domain to form the C-terminal portion of the molecule (e.g. the CH4 domain in the p chain of IgM and the s chain of IgE). In some aspects, the antibody (or fragment thereof) of the disclosure comprises a CH3 domain derived from an IgG molecule, such as IgGl, IgG2, IgG3, or IgG4. In some aspects, the IgG is a human IgG.

[0171] CL domain includes the constant region domain of an immunoglobulin light chain that extends, e.g. from about positions 108-214 according to the EU index of Kabat. The CL domain is adjacent to the VL domain. In some aspects, the antibody (or fragment thereof) of the disclosure comprises a kappa light chain constant domain (CLK). In some aspects, the antibody (or fragment thereof) comprises a lambda light chain constant domain (CL ). CLK has known polymorphic loci CLK-V / A45and CLK-L / V83 (using Kabat numbering) thus allowing for polymorphisms Km(l): CLK-V45 / L83; Km(l,2): CLK-A45 / L83; and Km(3): CLK-A45 / V83. Polypeptides, antibodies and ADCs of the disclosure may have antibody components with any of these light chain constant regions.

[0172] The Fc region generally comprises a CH2 domain and a CH3 domain. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230 (according to the EU index of Kabat), to the carboxyl-terminus thereof. A 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). A “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 know n in the art, the variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies.

[0173] A CDR of a variable domain may be identified in accordance w ith the definitions of the Kabat (Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C), Chothia (Chothia et al., Nature 342:877-883, (1989)), the accumulation of both Kabat and Chothia. AbM definition (derived using Oxford Molecular's AbM antibody modeling software, now Accelrys®), contact definition (based on observed antigen contacts, set forth in MacCallum et al., J. Mol. Biol., 262:732-745, (1996)), and / or conformational definition (Makabe et al., Journal of Biological Chemistry, 283:1156- 1166, 2008) or any method of CDR determination well known in the art. As used herein, a CDR may refer to CDRs defined by any approach known in the art. including combinations of approaches. For the present disclosure, the CDRs set forth in Table 3 below w ere derived using Kabat and Chothia definitions. The anti-EDB antibodies, or antigen-binding fragment thereof, of the present disclosure include one or more CDR(s) (such as one, two, three, four, five, or all six CDRs).

[0174] An "isolated antibody", as used herein, refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds EDB is substantially free of antibodies that specifically bind antigens other than EDB). Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals. It is also understood that by reading this definition, for example, an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target.

[0175] In some aspects of the disclosure, an EDB ADC includes an antibody that competes for binding to human EDB with, and / or binds the same epitope as, an antibody, or antigenbinding fragment thereof, described herein.

[0176] The term “compete”, as used herein with regard to an antibody, means that a first antibody, or an antigen-binding fragment thereof, binds to an epitope in a manner sufficiently similar to the binding of a second antibody, or an antigen-binding fragment thereof, such that the result of binding of the first antibody with its cognate epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody, can, but need not be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope. However, where each antibody detectably inhibits the binding of the other antibody with its cognate epitope or ligand, whether to the same, greater, or lesser extent, the 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 disclosure. 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), the skilled artisan would appreciate, based upon the teachings provided herein, that such competing and / or cross-competing antibodies are encompassed and can be useful for the methods disclosed herein.

[0177] The 'Ll 9" antibody, herein also referenced as “EDB-L19” antibody, is a human antibody that binds EDB. The LI 9 antibody is disclosed and characterized in PCT International Publication Nos. WO 1997 / 045544, WO1999 / 058570 and W02001 / 062800, which are incorporated herein by reference in their entirety, and the L19-EDB sequences are provided herein in Table 3 (SEQ ID NOs. 1-16).

[0178] In some aspects of the disclosure, antibodies used to prepare EDB ADCs may be monoclonal antibodies. 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 highly specific, being directed against a single antigenic site. 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, and is not to be construed as requiring production of the antibody by any particular method.

[0179] In some aspects of the disclosure, antibodies used to prepare ADCs of the disclosure may be monovalent, i.e., having one antigen binding site per molecule (e g., IgG or Fab). In some instances, a monovalent antibody can have more than one antigen binding sites, but the binding sites are from different antigens. In some aspects of the disclosure, the antibody, or antigen-binding fragment thereof, of an ADC of the disclosure may include a “bivalent antibody”, i.e., having two antigen binding sites per molecule (e.g.. IgG). In some instances, the two binding sites have the same antigen specificities. Alternatively, bivalent antibodies may be bispecific. A “bispecific,” “dual-specific” or “bifunctional” antibody is a hybrid antibody having two different antigen binding sites. The two antigen binding sites of a bispecific antibody bind to two different epitopes, which may reside on the same or different protein targets.

[0180] Antibodies of the disclosure can be produced using techniques well know n in the art, e.g., recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies or other technologies readily known in the art (see, for example. Jayasena, S.D.. Clin. Chem.. 45: 1628-50 (1999) and Fellouse, F.A., et al, J. Mol. Biol., 373(4):924-40 (2007)). Additional guidance may be found in Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000); Ausubel et al, Short Protocols in Molecular Biolog}': 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, N.Y. (1998); and Coligan et al, Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003).

[0181] Nucleic acids encoding the heavy and light chains of the antibodies used to prepare the ADCs of the disclosure can be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest may be maintained in vector in a host cell and the host cell can then be expanded and frozen for future use. Production of recombinant monoclonal antibodies in cell culture can be carried out through cloning of antibody genes from B cells by means known in the art. See, e.g. Tiller et al. J. Immunol. Methods 329: 112- 124, 2008; U.S. Patent No. 7,314,622.

[0182] For all heavy chain constant region amino acid positions discussed in the present disclosure, numbering is according to the Eu index first described in Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1 ):78-85, describing the amino acid sequence of myeloma protein Eu. which is the first human IgGl 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 "EU index of Kabat" or “EU Index” refers to the residue numbering system based on the human IgGl Eu antibody of Edelman et al. as set forth in Kabat 1991. The numbering system used for the light chain constant region amino acid sequence is that set forth in Kabat 1991.

[0183] The EDB ADCs of the present disclosure may be conjugated to the drug / payload using conventional cysteine technology or site-specific conjugation technology. To accommodate site-specific conjugation via engineered cysteines, the constant domain may be modified to provide for a reactive cysteine residue engineered at one or more specific sites (sometimes referred to as “Cys” mutants). To accommodate site-specific conjugation via transglutaminase-based conjugation, an acyl donor glutamine-containing (“Q”) tag or an endogenous glutamine is made reactive by polypeptide engineering in the presence of transglutaminase and an amine.

[0184] The present disclosure provides for optimization of the L19-EDB antibody by generation of a non-immunogenic antibody. In some aspects, the L19-EDB human IgGl constant region comprising a Glm(a) allotype having aspartic acid (D) at position 356 and leucine (L) at position 358, may be substituted with a non-Glm(a) allotype having glutamic acid (E) at position 356 and methionine (M) at position 358 (according to the numbering of the EU index of Kabat).

[0185] Further, to reduce potential chemical liabilities and antigen binding a putative protein glycation site, anti-EDB antibodies of the present disclosure may have a heavy chain variable region comprising a mutation of the lysine (K) at position 94 (according to the numbering of the EU index of Kabat) to an arginine (R). e.g. (K94R).

[0186] For site-specific conjugation via engineered cysteines, the anti-EDB antibody heavy chain constant domain may comprise a reactive engineered cysteine residue at position 290 (K290C), according to the numbering of the EU index of Kabat. Additional cysteine substitutions may be introduced. In some aspects, the anti-EDB antibody light chain constant domain may comprise a reactive engineered cysteine residue at position 183 (KKI 83C), according to the numbering of Kabat. Additional cysteine substitutions may be introduced.

[0187] For site-specific conjugation via engineered glutamine residues, the anti-EDB antibody heavy chain constant domain may comprise an engineered FU6-glutamine- containing tag LLQG (SEQ ID NO: 40). Further, 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 may be substituted with an arginine (R). e.g. (K222R).

[0188] Amino acid modifications can be made by any method known in the art and many such methods are well known and routine for the skilled artisan, e.g. mutations, substitutions, deletions, and / or additions. For example, but not by way of limitation, amino acid substitutions, deletions and insertions may be accomplished using any well-known PCR- based technique. Amino acid substitutions may be made by site-directed mutagenesis (see, for example, Zoller and Smith, 1982, Nucl. Acids Res. 10:6487-6500; and Kunkel, 1985, PNAS 82:488).

[0189] In some aspects of the disclosure, the EDB ADCs include an antibody, or antigen binding fragment thereof, having a heavy chain and / or a light chain comprising an amino acid sequence that is at least 90%, 95%, 98%, or 99% identical to any of the heavy or light chains disclosed herein. Residues that have been altered can be in the variable region or in the constant region of the antibody. In some aspects, there are no more than 1, 2, 3, 4 or 5 residues that have been altered as compared to any of the heavy or light chains disclosed herein.

[0190] For use in preparation of EDB ADCs, antibodies described herein may 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, yet more preferably, at least 98% pure, and most preferably, at least 99% pure.

[0191] Tables 3 and 4 provide the amino acid (protein) sequences and associated nucleic acid (DNA) sequences of anti-EDB antibodies of the present disclosure. The CDRs are as defined by Kabat and Chothia. The shaded residues identify amino acid mutations, substitutions and / or insertions relating to antibody optimization and underlined residues identify amino acid mutations, substitutions and / or insertions relating to site-specific conjugation technology7.

[0192] Table 3: ANTI-EDB ANTIBODY SEQUENCES

[0193]

[0194] In some aspects of the disclosure, an EDB ADC includes an antibody, or antigen binding fragment thereof, that binds to extra domain B (EDB) of fibronectin (FN).

[0195] In some aspects of the disclosure, an antibody of the present disclosure, or antigen binding fragment thereof, has a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the VH CDR1 region, VH CDR2 region, and VH CDR3 region of the VH amino acid sequence of SEQ ID NO:21, and wherein the VL comprises the VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NO: 10. In some aspects of the disclosure, an antibody of the present disclosure, or antigen binding fragment thereof, has a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH has three CDRs comprising SEQ ID NOs: 3, 5 and 7. In some aspects of the disclosure, an antibody, or antigen binding fragment thereof, has a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VL has three CDRs comprising SEQ ID NOs: 12, 13 and 14. An 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.

[0196] In some aspects, an antibody of the present disclosure, or antigen binding fragment thereof, 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 NOs: 7. In some aspects, an antibody, or antigen binding fragment thereof, may have a light chain variable region (VL) 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 (according to Kabat and Chothia). In a further aspect, an 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 NOs: 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.

[0197] In some aspects of the disclosure, an antibody, or antigen-binding fragment thereof, may have a heavy chain variable region comprising the VH CDR1 region, VH CDR2 region, and VH CDR3 region of the VH amino acid sequence of SEQ ID NO:21 and a non-CDR region that is at least 90% identical to SEQ ID NO: 21 ; and a light chain variable region comprising the VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NO: 10 and a non-CDR region that is at least 90% identical to SEQ ID NO: 10.

[0198] In some aspects of the disclosure, an antibody, or antigen-binding fragment thereof, may heave a heavy chain variable region comprising SEQ ID NOs: 1 or 21 and / or a light chain variable region comprising SEQ ID NO: 10. An antibody, or antigen-binding fragment thereof, may comprise: a heavy chain variable region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and a light chain variable region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 10; a heavy chain variable region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 21 and a light chain variable region having an amino acid sequence that is 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.

[0199] In some aspects of the disclosure, an 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 NOs: 15 or 31.

[0200] An antibody of the present disclosure, or antigen-binding fragment thereof, may comprise: a heavy chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 8 and a light chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 15; a heavy chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 8 and a light chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 31; a heavy' chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 17 and a light chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 15; a heavy chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 17 and a light chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 31; a heavy chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 19 and a light chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 15; a heavy chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 19 and a light chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 31; a heavy chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 23 and a light chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 15; a heavy chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 23 and a light chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 31; a heavy chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 25 and a light chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 15; a heavy chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 25 and a light chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 31; a heavy chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 27 and a light chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 15; a heavy chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 27 and a light chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 31; or a heavy' chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 29 and a light chain having an amino acid sequence that is at least 90% identical to SEQ ID NO: 15.

[0201] An antibody of the present disclosure, or antigen-binding fragment thereof, 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.

[0202] Representative DNAs encoding anti-EDB antibody heavy chain and light chain variable regions comprise SEQ ID NOs: 2 and 22 and SEQ ID NO: 1 1. respectively. Representative DNAs encoding anti-EDB antibody heavy chains and light chains comprise SEQ ID NOs: 9, 18, 20, 24, 26, 28 and 30, and SEQ ID NOs: 16 and 32, respectively.

[0203] Table 4: SEQ ID NOS FOR VARIOUS ANTI-EDB ANTIBODIES

[0204] (CDRs in Kabat and (Chothia))

[0205] Drugs Drugs useful in preparation of the disclosed EDB ADCs include any substance having biological or detectable activity, for example, therapeutic agents, detectable labels, binding agents, etc., and prodrugs, which are metabolized to an active agent in vivo. A drug may also be a drug derivative, wherein a drug has been functionalized to enable conjugation with an antibody of the disclosure.

[0206] 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 immunomodulating agents. A cytotoxic effect refers to the depletion, elimination and / or the killing 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 immunomodulating agent refers to an agent that stimulates the immune response though the production of cytokines and / or antibodies and / or modulating T cell function thereby inhibiting or reducing the growth of a subset of cells (i.e., tumor cells) either directly or indirectly by allowing another agent to be more efficacious.

[0207] In some aspects the drug is a membrane permeable drug. In such aspects, the payload can elicit a bystander effect wherein cells that may not express EDB+FN or have EDB+FN bound to their surface, but surround the cell that is bound by the ADC are killed by the cell permeable payload. This occurs when the payload is released from the antibody (i.e., by cleaving of a cleavable linker) and crosses the cellular membrane and, upon diffusion, induces the killing of surrounding cells.

[0208] In accordance with the disclosed methods, the EDB ADCs may be produced or generated having (a) an antibody, or antigen binding fragment thereof, 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 a plurality of ADCs may be characterized by an average DAR. DAR and average DAR can be determined by various conventional means such as UV spectroscopy, mass spectroscopy, ELISA assay, radiometric methods, hydrophobic interaction chromatography (HIC), electrophoresis and HPLC.

[0209] In aspects of the disclosure, an EDB ADC may have a DAR of 1, a DAR of 2, a DAR of 3, a DAR of 4, a DAR of 5, a DAR of 6, a DAR of 7, a DAR of 8, a DAR of 9, a DAR of 10, a DAR of 11. a DAR of 12 or a DAR greater than 12. In aspects of the disclosure, an EDB ADC may have one 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 greater than 12 molecules.

[0210] In aspects of the disclosure, an EDB ADC may have 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 aspects the compositions, batches and / or formulations of EDB ADCs may have an average DAR of about 1, or an average DAR of about 2, 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. As used in the foregoing ranges of average DAR, the term “about” means + / - 0.5%.

[0211] A composition, batch, and / or formulation of EDB ADCs 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. Further, a composition, batch, and / or formulation of EDB ADCs 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.

[0212] In some aspects of the disclosure, a composition, batch, and / or formulation of EDB ADCs 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 some aspects, a composition, batch, and / or formulation of EDB ADCs 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 some aspects, a composition, batch, and / or formulation of EDB ADCs 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 some aspects, a composition, batch, and / or formulation of EDB ADCs 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.

[0213] In some aspects, a composition, batch, and / or formulation of EDB ADCs 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.

[0214] In other aspects, a composition, batch, and / or formulation of EDB ADCs 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.

[0215] In some aspects of the present disclosure, the methods for conventional conjugation via cysteine residues and purification conditions disclosed herein provide a composition, batch, and / or formulation of EDB ADCs with an optimized average DAR in the range of about 3 to 5, preferably about 4.

[0216] In some aspects of the present disclosure, the methods for site-specific conjugation via engineered cysteine residues and purification conditions disclosed herein provide a composition, batch, and / or formulation of EDB ADCs with an optimized average DAR in the range of about 3 to 5, preferably about 4.

[0217] In some aspects of the present disclosure, the methods for site-specific conjugation via transglutaminase-based conjugation and purification conditions disclosed herein provide a composition, batch, and / or formulation of EDB ADCs with an optimized average DAR in the range of about 1 to 3, preferably about 2.

[0218] In some aspects of the present disclosure, the therapeutic agent may be a cytotoxic agent. Examples of cytotoxic agents include, but are not limited to an anthracycline. an auristatin, CC-1065, a dolastatin, a duocarmycin, an enediyne, a geldanamycin, a maytansine, a puromycin, a taxane, a vinca alkaloid, SN-38, tubulysin, hemiasterlin, and stereoisomers, isosteres, analogs or derivatives thereof. Plant toxins, other bioactive proteins, enzymes (i.e.. ADEPT), radioisotopes, photosensitizers (i.e., for photodynamic therapy) may also be used. Further examples of a cytotoxic agent include, but are not limited to, an anthracycline, an auristatin (e.g., dolestatin, MMAD, MMAE, MMAF, PF-06380101, PF-06463377 and PF- 06456780), a spliceostatin, a CBI / CPI dimer (including “mixed” dimers comprising both CBI and CPI components, as described in U.S. Provisional patent application 61 / 932.118), a calicheamicin, a duocarmycin, an enediyne, a geldanamycin, a maytansine, a puromycin, a taxane, a vinca alkaloid, SN-38, a tubulysin, a hemiasterlin, a camptothecin, a combretastatin, a dolastatin, an indolino-benzodiazepine dimer, a pyrrolobenzodiazepine dimer and a pladienolide. and stereoisomers, isosteres, analogs, or derivatives thereof.

[0219] The anthracy clines are derived from bacteria Strepomyces and have been used to treat a wide range of cancers, such as leukemias, lymphomas, breast, uterine, ovarian, and lung cancers. Exemplary' anthracy clines include, but are not limited to, daunorubicin, doxorubicin (i.e., adriamycin), epirubicin, idarubicin, valrubicin. and mitoxantrone.

[0220] Dolastatins and their peptidic analogs and derivatives, 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-methyl valine- valine- dolaisoleuine-dolaproine-phenylalanine), MMAE (Monomethyl Auristatin E or N- methylvaline-valine-dolaisoleuine-dolaproine-norephedrine), and 5-benzoylvaleric acid-AE ester (AEVB).

[0221] In some aspects, the drug / payload is an auristatin. Auristatins inhibit cell proliferation by inhibiting the formation of microtubules during mitosis through inhibition of tubulin polymerization. PCT International Publication No. WO 2013 / 072813, which is incorporated herein by reference in its entirety’, discloses auristatins that are useful in the EDB ADCs of the present disclosure and provides methods of producing the auristatins. For example, payload 0101 having the structure: payload 1569 having the structure: payload 9411 having the structure: pay load 4574 having the structure: and payload Cemadotin having the structure:

[0222] Camptothecin is a cytotoxic quinoline alkaloid which inhibits the enzyme topoisomerase I. Examples of camptothecin and its derivatives include, but are not limited to, topotecan and irinotecan, and their metabolites, such as SN-38.

[0223] Combretastatins are natural phenols with vascular disruption properties in tumors. Exemplary combretastatins and their derivatives include, but are not limited to. combretastatin A-4 (CA-4) and ombrabulin.

[0224] 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, (+)-docarmycin A and (+)-duocarmycin SA, and (+)-CC-1065.

[0225] In some aspects, the drug / payload is a CPI or CBI dimer. CPI dimers induce interstrand DNA crosslinking and potent cytotoxicity. PCT International Publication No. WO2015 / 1 10935, which is incorporated herein by reference in its entirety, discloses CPI and CBI dimers that are useful in the EDB ADCs of the present disclosure and provides methods of producing the CPI and CBI dimers. For example, payload CPI-8314 dimer having the structure: payload CPI-0326 having the structure:

[0226] Enediynes are a class of anti-tumor bacterial products characterized by either nine- and ten-membered rings or the presence of a cyclic system of conjugated triple-double-triple bonds. Exemplary enediynes include, but are not limited to, calicheamicin, esperamicin, and dynemicin. Calicheamicin. also called the LL-E33288 complex, for example. 0- calicheamicin, y-calicheamicin or N-acetyl-y-calicheamicin (gamma-calicheamicin (yi)), is an enediyne antibiotic that was 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-strand DNA breaks and subsequently induces apoptosis in target cells (Zein et al. Science 27:240(4856): 1198-1201, 1988; Nicolaou et al. Chem. Biol. Sep;l(l):57-66, 1994; Prokop et al. Oncogene 22:9107-9120, 2003). The disulfide analog is N-acetyl- y -calicheamicin dimethyl hydrazide.

[0227] Hemiasterlin and its analogues (e.g.. HTI-286) bind to the tubulin, disrupt normal microtubule dynamics, and, at stoichiometric amounts, depolymerize microtubules.

[0228] Geldanamycins are benzoquinone ansamycin antibiotic that bind to Hsp90 (Heat Shock Protein 90) and have been used antitumor drugs. Exemplary geldanamycins include, but are not limited to, 17-AAG (17-N-Allylamino-17-Demethoxygeldanamycin) and 17- DMAG (17-Dimethylaminoethylamino-17-demethoxygeldanamycin).

[0229] Maytansines or their derivatives maytansinoids inhibit cell proliferation by inhibiting the microtubules formation during mitosis through inhibition of polymerization of tubulin. See Remillard et al., Science 189: 1002-1005, 1975. Exemplary maytansines and maytansinoids include, but are not limited to, mertansine (DM1) having the structure: and its derivatives as well as ansamitocin.

[0230] Pyrrolobenzodiazepine dimers (PBDs) and indolino-benzodiazepine dimers (IGNs) are anti-tumor agents that contain one or more immine functional groups, or their equivalents, that bind to duplex DNA. PBD and IGN molecules are based on the natural product anthramycin, and interact with DNA in a sequence-selective manner, with a preference for purine-guanine-p urine sequences. Exemplary PBDs and their analogs include, but are not limited to, SJG-136.

[0231] Spliceostatins and pladienolides are anti-tumor compounds which inhibit splicing and interacts with spliceosome, SF3b. Examples of spliceostatins include, but are not limited to, spliceostatin A, FR901464. Examples of pladienolides include, but are not limited to, Pladi enolide B, Pladi enolide D, and E7107.

[0232] Taxanes are diterpenes that act as anti -tubulin agents or mitotic inhibitors. Exemplary taxanes include, but are not limited to, paclitaxel (e.g., TAXOL®) and docetaxel (TAXOTERE®).

[0233] Vinca alkyloids are also anti-tubulin agents. Exemplary vinca alkyloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine.

[0234] In some aspects of the disclosure, the pay load is an immunomodulating agent. Examples of an immunomodulating agent include, but are not limited to, gancyclovier, etanercept, tacrolimus, sirolimus, voclosporin, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolgate mofetil, methotrextrate, glucocorticoid and its analogs, 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., interferons -a, -P and -y), the stem cell growth factor designated “SI factor,” ery thropoietin and thrombopoietin, or a combination thereof.

[0235] In some embodiments, the agent moiety is a toxin polypeptide (or a toxin protein). Examples of a toxin polypeptide include, but are not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca cimericana proteins (PAPI. PAPIL and PAP-S). Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, tricothecenes, inhibitor cysteine knot (ICK) peptides (e.g., ceratotoxins), and conotoxin (e.g., KIIIA or Smilla).

[0236] Immunomodulatory agents useful in the disclosure also include anti-hormones that block hormone action on tumors and immunosuppressive agents that suppress cytokine production, down-regulate self-antigen expression, or mask MHC antigens. Representative anti-hormones include anti-estrogens including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 1 17018, onapnstone, and toremifene; and anti -androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and anti-adrenal agents. Representative immunosuppressive agents include 2-amino-6-aryl-5 -substituted pyrimidines, azathioprine, cyclophosphamide, bromocryptine, danazol, dapsone, glutaraldehyde, anti-idiotypic antibodies for MHC antigens and MHC fragments, cyclosporin A, steroids such as glucocorticosteroids, cytokine or cytokine receptor antagonists (e.g., anti -interferon antibodies, anti-ILlO antibodies, anti-TNFa antibodies, anti-IL2 antibodies), streptokinase, TGFp, rapamycin, T-cell receptor, T-cell receptor fragments, and T cell receptor antibodies.

[0237] In some aspects of the disclosure, the drug is a therapeutic protein including, but is not limited to, a toxin, a hormone, an enzyme, and a grow th factor. Examples of a toxin protein (or polypeptide) include, but are not limited to, dipththeria (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 proteins, dianthin proteins, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin- A, pokeweed antiviral protein, gelonin, diphtherin toxin, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, mitogellin, restrictocin. phenomycin. enomycin, tricothecenes, inhibitor cystine knot (ICK) peptides (e g., ceratotoxins), and conotoxin (e.g., KIIIA or Smilla).

[0238] Examples of hormones include, but are not limited to, estrogens, androgens, progestins and corticosteroids.

[0239] In some aspects of the disclosure, the drug is an oligonucleotide, such as anti-sense oligonucleotides.

[0240] Additional drugs useful in the disclosure include anti-angiogenic agents that inhibit blood vessel formation, for example, famesyltransferase inhibitors, COX-2 inhibitors, VEGF inhibitors, bFGF inhibitors, steroid sulphatase inhibitors (e.g., 2-methoxyoestradiol bissulphamate (2-MeOE2bisMATE)), interleukin-24, thrombospondin, metallospondin proteins, class I interferons, interleukin 12, protamine, angiostatin, laminin, endostatin, and prolactin fragments.

[0241] Anti-proliferative agents and pro-apoptotic agents include activators of PPAR-gamma (e.g.. cyclopentenone prostaglandins (cyPGs)), retinoids, triterpinoids (e.g., cycloartane, lupane, ursane, oleanane, friedelane, dammarane, cucurbitacin, and limonoid triterpenoids), inhibitors of EGF receptor (e.g., HER4), rampamycin, CALCITRIOL® (1,25- dihydroxycholecalciferol (vitamin D)), aromatase inhibitors (FEMARA® (letrozone)), telomerase inhibitors, iron chelators (e.g., 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (Triapine)), apoptin (viral protein 3 - VP3 from chicken aneamia 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).

[0242] Representative chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziidines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechiorethamine, mechiorethamine oxide hydrochloride, melphalan. novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard: nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin. carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin. 6-diazo-5-oxo-L-norleucine. doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tuberci din, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5- fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-EU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenal such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea: lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2'-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C): cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology of Princeton, N.J.) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer of Antony, France); chiorambucil; 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; aininopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoic acid; esperamicins; and capecitabine.

[0243] Additional therapeutic agents that may be used in accordance with the present disclosure include photosensitizing agents, such as U.S. Publication No. 20020197262 and U.S. Patent No. 5,952,329, which are incorporated herein by reference in its entirety, for photodynamic therapy; magnetic particles for thermotherapy, such as U.S. Publication No. 20030032995, which is incorporated herein by reference in its entirety; binding agents, such as peptides, ligands, cell adhesion ligands, etc., and prodrugs such as phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate containing prodrugs, peptide containing prodrugs, |3-lactam-containing prodrugs, substituted phenoxyacetamide-containing prodrugs or substituted phenylacetamide-containing prodrugs, 5 -fluorocytosine and other 5- fluorouridine prodrugs that may be converted to the more active cytotoxic free drug.

[0244] For diagnostic methods using anti-EDB antibodies, a drug may include a detectable label used to detect the presence of EDB+FN-expressing ECM or cells in vitro or in vivo. Radioisotopes that are detectable in vivo, such as those labels that are detectable using scintigraphy, magnetic resonance imaging, or ultrasound, may be used in clinical diagnostic applications. Useful scintigraphic labels include positron emitters and y-emitters. Representative contrast agents for magnetic source imaging are 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 ultrasonic detection, gases or liquids may be entrapped 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.

[0245] Thus, in some aspects of the disclosure, the drug is an imaging agent (e g., a fluorophore or a PET (Positron Emission Tomography) label. SPECT (Single-Photon Emission Computed Tomorgraphy) label), or MRI (Magnetic Resonance Imaging) label.

[0246] The term “label” when used herein refers to a detectable compound or composition that is conjugated directly or indirectly to the antibody so as to generate a “labeled” antibody . The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable. Radionuclides that can serve as detectable labels include, for example, 1-131, 1-123, 1-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, and Pd-109. The label might also be a non-detectable entity such as a toxin.

[0247] Examples of fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluorescein amidite (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein, erythrosine, ALEXA FLUOR® (Molecular Probes, Inc.; fluorescent chemicals and biomolecule labeling kits) (e.g., ALEXA FLUOR® 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).

[0248] Therapeutic or diagnostic radioisotopes or other labels (e.g., PET or SPECT labels) can be incorporated in the agent for conjugation to the anti-EDB antibodies as described herein. The isotope may be directly bound to the antibody, for example, at a cysteine residue present in the antibody, or a chelator may be used to mediate the binding of the antibody and the radioisotope. Radioisotopes suitable for radiotherapy include but are not limited to a- emitters, P-emitters, and auger electrons. For diagnostic applications, useful radioisotopes include positron emitters and y-emitters. An anti-EDB antibody of the disclosure may further be iodinated, for example, on a tyrosine residue of the antibody, to facilitate detection or therapeutic effect of the antibody.

[0249] Examples of a radioisotope or other labels include, but are not limited to,3H,nC,13N, 14C,15N,150,35S,18F,32P,33P,47SC,51Cr,57Co,58Co,59Fe,62Cu,64Cu,67Cu,67Ga,68Ga,75Se,

[0250] 165Tm,166Dy,166H.167Tm,168Tm,169Yb.177Lu,186Re,188Re.189Re,197Pt,198Au.199Au,2O1T1, 203Hg,211At,212BI,212Pb,213BI,223Ra,224Ac, and225Ac.

[0251] Linkers

[0252] EDB ADCs of the present disclosure may be prepared using a linker to directly or indirectly link or conjugate a drug to an antibody. A linker is a bifunctional compound that links a drug and an antibody to form an ADC. Such ADCs allow the selective delivery of drugs via antibodies that bind to specific antigens or proteins. Suitable linkers include, for example, cleavable and non-cleavable linkers. A cleavable linker is typically susceptible to cleavage and release of drug by specific intracellular and extracellular conditions. Major mechanisms by which a conjugated drug may be cleaved from an antibody intracellularly include hydrolysis in the acidic pH of the lysosomes (hydrazones, acetals, and cis-aconitate- like amides), peptide cleavage by lysosomal enzymes (the cathepsins and other lysosomal enzymes), and reduction of disulfides. A conjugated drug may be cleaved from an antibody extracellulary by proteases in a tumor microenvironment (TME), such as cathepsins. As a result of these varying mechanisms for cleavage, mechanisms of linking the drug to the antibody also vary widely and any suitable linker can be used.

[0253] Suitable linkers may include any cleavable linker. In some aspects, the cleavable linkers are self-immolating cleavable linkers. In some aspects, 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 contain a dipeptide attached to additional immolation elements, such as N~2~-acetyl-L-lysyl-L-valyl- L-citruline-p-aminobenzyloxycarbonyl-N,N’-dimethylaminoethyl-CO- (AcLys-vc) linker, suitable for transglutaminase-based conjugation technology. In some aspects, suitable linkers include disulfide linkers, such as sulfanyl pyridine (diS) linker and 2-(pyridin-2- yldisulfanyljethyl carbamoyl (diS-C2OCO) linker. In some aspects, the linker may be a non- cleavable linker, such as maleimidocaproyl (me), maleimido-heptanoyl (me) and maleimido- Peg6C2 (MalPeg6C2). In other aspects, suitable linkers include linkers hydrolyzable at a specific pH or a pH range, such as a hydrazone linker.

[0254] The linker may be covalently bound to the antibody through a thioester linkage, for instance by reaction of a maleimide or haloacetamide. present on the linker with a native or engineered cysteine residue present on the antibody. In some aspects, the linker may be covalently bound to the antibody through amide linkages to lysine residues present on the antibody, for instance by reaction of an N-hydroxy-succinimide activated carboxylic acid present on the linker with a free amine of a lysine residue. In some aspects, the linker may be covalently bound to the antibody through amide linkages to the side chains of glutamine residues present or engineered into the antibody, for instance by enzymatic reaction catalyzed by a transglutaminase enzyme that creates a new amide linkage from a primary' amine present on the linker with a side chain amide of a glutamine residue.

[0255] In some aspects, linkers of the present disclosure include:

[0256] ■‘mc-vc-PABC” or “vc-PABC” or “vc” linker having the structure: diS linker having the structure: g the structure:

[0257] Methods of Preparing ADCs

[0258] Provided herein are methods for preparing EDB ADCs of the present disclosure. The present disclosure further provides for a process for producing or generating conventionally and site-specific conjugated EDB ADCs as disclosed herein and may include (a) linking the linker to the drug; (b) conjugating the linker-drug moiety to the antibody; and (c) purifying the antibody drug conjugate. See Examples 3 and 4 of WO 2018 / 073680.

[0259] In some aspects, EDB ADCs may be generated using conventional, non-specific conjugation of linker-payload moieties through one or more cysteine residues of an anti -EDB antibody, or an antigen binding fragment thereof.

[0260] In some aspects, EDB ADCs may be generated using site-specific conjugation of linker-payload moieties though one or more reactive cysteine residues engineered into an anti-EDB antibody constant domain. Methods of preparing antibodies for site-specific conjugation via engineered cysteine residues are described in PCT International Publication No. WO2013 / 093809, which is incorporated herein by reference in its entirety.

[0261] One or more amino acid residues of an anti-EDB antibody heavy chain may be substituted to another amino acid, such as a cysteine residue, for the purpose of conjugation to a drug or payload. In some aspects, the disclosure provides an anti-EDB antibody, or antigen binding fragment thereof, comprising an antibody heavy chain constant region comprising an engineered cysteine residue at position: 118 (114 according to Kabat), 246,

[0262] 249, 265, 267, 270, 276, 278, 283, 290, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320,

[0263] 327, 332. 333, 334, 336, 345, 347. 354, 355, 358, 360, 362, 370, 373. 375, 376, 378, 380,

[0264] 382, 386. 388, 390, 392. 393, 401. 404, 411, 413, 414, 416. 418, 419. 421, 428, 431. 432,

[0265] 437, 438, 439, 443 or 444, or any combination thereof , according to the numbering of the EU index of Kabat). 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. In some aspects, the disclosure 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 numbering of the EU index of Kabat.

[0266] One or more amino acid residues of an anti-EDB antibody light chain constant domain may be substituted to another amino acid, such as a cysteine residue, for the purpose of conjugation to a drug or payload. In some aspects, the disclosure provides an anti-EDB antibody, or antigen binding fragment thereof, comprising an antibody light chain constant region comprising (i) an engineered cysteine residue at position 110, 111, 125, 149, 155, 158, 161, 183. 185, 188, 189, 191, 197, 205, 207, 208 or 210, or any combination thereof, according to the numbering of Kabat); (ii) an engineered cysteine residue at a position corresponding to residue 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 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, 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.

[0267] In some aspects, the disclosure 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 1 11 or 210), according to the numbering of Kabat; or (ii) an engineered cysteine residue at a position corresponding to residue 4, 42, 81, 100, 103, or any combination thereof, of SEQ ID NO: 37 (preferably residue 4 or 103), when the constant domain is aligned with SEQ ID NO: 37.

[0268] In some aspects, the disclosure 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 the numbering of Kabat; 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 of SEQ ID NO: 38 (preferably residue 4, 5, 19, 43, or 49), when the constant domain is aligned with SEQ ID NO:38.

[0269] In some aspects, the disclosure 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 (K 183C), according to the numbering of Kabat; or (ii) an engineered cysteine residue at a position corresponding to residue 76 of SEQ ID NO: 37. when said constant domain is aligned with SEQ ID NO: 37.

[0270] SEQ ID NO: 37 (CK constant domain)

[0271] RTVAAPSVFI FPPSDEQLKS GTASWCLLN NFYPREAKVQ WKVDNALQSG

[0272] NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK S FNRGEC

[0273] SEQ ID NO 38 (CA constant domain)

[0274] GQPKANPTVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADGS PVK

[0275] AGVETTKPSK QSNNKYAASS YLSLTPEQWK SHRSYSCQVT HEGSTVEKTV APTECS

[0276] In some aspects, EDB ADCs may be generated using site-specific conjugation technology though one or more engineered acyl donor glutamine-containing tags or endogenous glutamine residues made reactive in an anti-EDB antibody constant region. Methods of preparing antibodies for site-specific conjugation via acyl donor glutamine- containing tags or glutamine residues are described in PCT International Publication No. WO2012 / 059882, which is incorporated herein by reference in its entirety.

[0277] In some aspects, the acyl donor glutamine-containing tag comprises at least one glutamine (Q) and may be attached to different position of the heavy and / or light chain (i.e., at the N-terminus, C-terminus or internally). In some aspects, the acyl donor glutamine- containing tag may 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, an acyl donor glutamine-containing tag replaces wild type amino acid positions in a heavy chain constant domain. In some aspects, an anti-EDB antibody may comprise an acyl glutamine-containing tag having the amino acid sequence LLQG (SEQ ID NO: 40) that replaces the amino acids at positions E294- N297 (according to the EU index of Kabat) of the heavy chain.

[0278] Optimal reaction conditions for the generation of ADCs may be empirically determined by a variation of reaction variables such as temperature, pH, linker-payload moiety input, and additive concentration. Conditions suitable for conjugation of other drugs may 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.

[0279] Following conjugation, the conjugates may be separated, purified from unconjugated reactants and / or aggregated forms of the conjugates, and characterized by conventional methods. This includes processes such as, but not limited to, mass spectrometry', size exclusion chromatography (SEC), ultrafiltration / diafiltration, ion exchange chromatography (IEC), chromatofocusing (CF), site-directed mutagenesis, fluorescence-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 includes, but is not limited to, Phenyl Sepharose 6 Fast Flow chromatographic medium, Butyl Sepharose 4 Fast Flow chromatographic medium, Octyl Sepharose 4 Fast Flow chromatographic medium, Toyopearl Ether-650M chromatographic medium, Macro-Prep methyl HIC medium or Macro-Prep t-Butyl HIC medium.

[0280] Table 1 provides EDB ADCs produced according to the conjugation and purification methods described herein and used to generate data provided in the Examples.

[0281] In some aspects of the disclosure, EDB ADCs of the present disclosure comprise (a) an antibody, or antigen binding fragment thereof, that binds to EDB; (b) a linker and (c) a drug.

[0282] In some aspects of the disclosure, EDB ADCs of the present disclosure comprise (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 aspects, the linker is vc, diS. diS-C2OCO or AcLys-vc.

[0283] In some aspects of the disclosure, EDB ADCs of the present disclosure comprise (a) an antibody, or antigen binding fragment thereof, that binds to EDB; (b) a linker and (c) a drug, wherein the drug is cytotoxic agent. In some aspects, the drug is an auristatin. In some aspects, the drug is a CPI or CBI dimer. In some aspects, the auristatin is 0101, 1569, 9411 or 4574. In some aspects, the CPI dimer is CPI-8314 or CPI-0326.

[0284] In some aspects of the disclosure, EDB ADCs of the present disclosure comprise (a) an antibody, or antigen binding fragment thereof comprising: a VH region comprising the VH CDR1 region, VH CDR2 region, and VH CDR3 region of the VH amino acid sequence of SEQ ID NO:21, and a VL region comprising VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NOTO; (b) a linker; and (c) a cytotoxic drug. In some aspects the linker is a cleavable linker. In some aspects, the cleavable linker is vc, diS, diS-C20C0 or AcLys-vc. In some aspects, the drug is an auristatin. In some aspects, the drug is a CPI or CBI dimer. In some aspects, the auristatin is 0101, 1569, 9411 or 4574. In some aspects, the CPI dimer is CPI-8314 or CPI-0326.

[0285] In some aspects of the disclosure, EDB ADCs of the present disclosure 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; 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 aspects, the linker is a cleavable or non-cleavable linker. In some aspects, the linker is vc, diS, diS-C20C0 or AcLys-vc. In some aspects, the drug is cytotoxic agent. In some aspects, the drug is an auristatin. In some aspects, the drug is a CPI or CBI dimer. In some aspects, the auristatin is 0101, 1569, 9411 or 4574. In some aspects, the CPI dimer is CPI-8314 or CPI-0326.

[0286] Formulations

[0287] The present disclosure further provides pharmaceutical compositions including any of the EDB ADCs disclosed herein and a pharmaceutically acceptable carrier. Further, the compositions may include more than one EDB ADC disclosed herein.

[0288] The composition used in the present disclosure may further include pharmaceutically acceptable carriers, excipients, or stabilizers (Remington: The Science and practice of Pharmacy 21st Ed., 2005, Lippincott Williams and Wilkins, Ed. K. E. Hoover), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations, and may include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, buh l or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; 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 dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). "‘Pharmaceutically acceptable salt” as used herein refers to pharmaceutically acceptable organic or inorganic salts of a molecule or macromolecule. Pharmaceutically acceptable excipients are further described herein.

[0289] Various formulations of the EDB ADCs may be used for administration, including but not limited to, formulations comprising a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients are known in the art, and are relatively inert substances that facilitate administration of a pharmacologically effective substance. For example, an excipient can give form or consistency, or act as a diluent. Suitable excipients include but are not limited to stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers. Excipients as well as formulations for parenteral and nonparenteral drug delivery are set forth in Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000.

[0290] In some aspects of the disclosure, these agents may be formulated for administration by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Accordingly, these agents can be combined with pharmaceutically acceptable vehicles such as saline, Ringer’s solution, dextrose solution, and the like. The particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular individual and that individual’s medical history.

[0291] Therapeutic formulations of EDB ADCs used in accordance with the present disclosure may be prepared for storage by mixing an antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzy l alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; 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 counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0292] 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. The compositions according to the present disclosure may be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

[0293] Suitable surface-active agents include, in particular, non-ionic agents, such as polyoxyethylenesorbitans (e.g. Tween™ 20, 40. 60. 80 or 85) and other sorbitans (e.g. Span™ 20, 40, 60, 80 or 85). Compositions with a surface-active agent will conveniently include between 0.05 and 5% surface-active agent, and can be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added, for example mannitol or other pharmaceutically acceptable vehicles, if necessary.

[0294] Suitable emulsions may be prepared using commercially available fat emulsions, such as INTRALIPID™, LIPOSYN™, INFONUTROL™, LIPOFUNDIN™ and LIPIPHYSAN™. The active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in an oil (e.g. soybean oil, safflower oil. cottonseed oil, sesame oil. com oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g. egg phospholipids, soybean phospholipids or soybean lecithin) and water. It will be appreciated that other ingredients may be added, for example glycerol or glucose, to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, for example, between 5 and 20%. The fat emulsion can include fat droplets between 0. 1 and 1.0 pm, particularly 0. 1 and 0.5 pm, and have a pH in the range of 5.5 to 8.0. The emulsion compositions can be those prepared by mixing an EDB ADC with INTRALIPID™ or the components thereof (soybean oil, egg phospholipids, glycerol and water).

[0295] The disclosure also provides kits for use in the instant methods. Kits of the disclosure include one or more containers including an EDB antibody or an EDB ADC as described herein and instructions for use in accordance w ith any of the methods of the disclosure described herein. Generally, these instructions include a description of administration of the EDB antibody or EDB ADC for the above described diagnostic or therapeutic treatments.

[0296] The instructions relating to the use of an EBD antibody or an EDB ADC as described herein generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0297] The kits of this disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. A kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an EDB antibody or EDB ADC. The container may further include a second pharmaceutically active agent.

[0298] Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit includes a container and a label or package insert(s) on or associated with the container.

[0299] Table 5: SEQUENCE TABLE

[0300] EXAMPLES

[0301] The instant specification further describes in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the instant specification should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0302] Example 1: Characteristics of the anti-EDB+FN Antibody Drug Conjugate

[0303] An anti-EDB+FN antibody-drug conjugate known as PYX-201 was utilized in the following examples. The PYX-201 used in the examples is composed of: an anti-EDB+FN monoclonal antibody mAb (fully human IgGl) derived from the LI 9 clone, with engineered cysteines site-specific conjugation at KK183C and K290C and a final mAb defined as an anti- EDB+FN-K(94)R-hulgGl-K290C- KK183C; an mcValCitPABC linker conjugated to the anti-EDB+FN antibody at each of the engineered cysteine sites; and an AuristatinOlOl payload conjugate to the linker; as further described in WO 2018 / 073680, which is incorporated herein it its entirety.

[0304] Example 2: Patient Derived Xenograft Study - Experimental Design

[0305] 135 PDX models were obtained to test the efficacy of PYX-201, an anti-EDB+FN antibody drug conjugate with site-specific conjugation, a cleavable linker and a cytotoxic payload, across a range of tumor types. The study included 10 solid tumor indications including HR+Her2- / PR+ER+HER2- breast cancer, triple negative breast cancer (TNBC), head and neck cancer, hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, renal cancer, sarcoma, and thyroid cancer, with various tumor subtypes, and each PDX model with distinct: EDB+FN expression, % of stroma content, and doubling-time.

[0306] PDX tumor tissue fragments from donor mice were unilaterally implanted subcutaneously in athymic recipient female mice (5-8 weeks old) and passage number was recorded. Mice were randomized into a control and a treated groups (n=3 mice per group) when tumor volume reached 50-300 mm3(mean tumor volume of 80-250 mm3).

[0307] As shown in FIG. 1, dosing started within 24 hours of randomization and mice received 4 doses of treatment every 4 days (Q4Dx4) via intravenous injection as following: control group was given a dose volume of lOmL / kg 0.9% sodium chloride for injection, USP (saline) based on last body weight measurement; and treated group was given 3mg / kg of PYX-201 (or PYX-201 DS or L19-mcValCitPABC_Aur-06380101 ) in a dose volume of 10 mL / kg 0.9% Sodium Chloride for injection. USP (saline) based on last body weight measurement. Per the FDA Dosing Guidance discuss hereinabove, this murine dose of 3 mg / kg would convert to approximately 0.24 mg / kg HED.

[0308] Tumor sizes and body weight were measured at least twice weekly using an electronic caliper and tumor volumes (TV) were calculated as following:

[0309] 1

[0310] Tumor Volume (TV) = — (Length x Width2)

[0311] Mice were monitored until tumor volume reached max tumor volume and / or if mice needed to be euthanized for humane reasons. Anti-tumor activity was determined by calculating tumor growth inhibition (TGI) index as disclosed herein.

[0312] As used herein, the term '‘tumor growth inhibition (TGI) index” refers to the value used to represent the degree in which an agent (e.g., a cytotoxic EDB+FN targeting ADC such as PYX-201 described herein) inhibits the grow th of a tumor when compared to an untreated control. TGI is calculated for a particular time point (e.g.. a specific number of days into a study or clinical trial) according to the following formula: where “Tx Day X” denotes the end date of the trial, which occurs when the end point criteria has been met. The end point criteria may be chosen for a number of reasons. In the case of murine PDX trials, as a non-limiting example. Tx Day X may be when the control group has reached a humane limit of total tumor volume, (e.g. approximately 1000mm3, 2000mm3, 3000mm3, etc.) and the control animals are humanely sacrificed. Mean volumes for all tumors in the treated and control groups are ty pically utilized. As a non-limiting example of calculating TGI where: Tx Day X is 28 days because the study has reached its end point criteria, the mean tumor volume in the treated group at day 28 is 100mm3, and the mean tumor volume in the control group at day 28 is 500mm3, the TGI index on day 28 is 100%[l - (100mm3 / 500mm3)] = 80%.

[0313] Each participant in a trial should begin with a similar initial tumor burden in terms of volume, or TGI should be adjusted based upon the average initial tumor burden as follows:

[0314] TGI = where “Tx Day 0” denotes the start date of the trial, and “Tx Day X” denotes the end date of the trial. As a non-limiting example of calculating TGI where the starting volume of the treated and control group are not equal: Tx Day 0 is Day 1 of a trial and Tx Day X is day 28 of a trial, the mean tumor volume in the treated group at day 1 is 200mm3and the mean tumor volume in the treated group at day 28 is 50mm3, the mean tumor volume in the control group at day 0 is 100mm3and the mean tumor volume in the control group at day 28 is 500mm3, the TGI index on day 28 is 100%[ 1 - ((50mm3 / 500mm3) (100mm3 / 200mm3))] = 95%.

[0315] Example 3: Overall Tumor Growth Inhibition by Cancer Type

[0316] Results for 135 PDX studies were aggregated across ten tumor types: HR+Her2- / PR+ER+HER2- breast, TNBC, head & neck, hepatocellular, NSCLC, ovarian, pancreatic, renal, sarcoma, and thyroid cancers. The total number of PDX studies conducted in each cancer indication is shown in Table 6, as well as the number of PDX models within each indication that averaged above 50% tumor grow th inhibition and the average tumor grow th inhibition of treatment versus control group across all PDX models within that cancer indication. Certain exemplary PDX studies are presented for specific cancer types and subtypes in Examples 4-13.

[0317] Table 6

[0318] Example 4: Thyroid Cancer PDX Studies

[0319] Four PDX conducted in thyroid cancer across three subtypes including squamous carcinoma, pleomorphic, and anaplastic squamous carcinoma are shown in Table 7. As shown in Table 7, percent inhibition of tumor growth between control and treatment ranged from almost complete disappearance of tumors at 99.42% to a robust response of 71.48%. FIG. 2A-FIG. 2D display a substantial tumor grow th inhibition of greater than 90% in three of the PDX models (CTG-1976 (FIG. 2A), THXF TH-001 (FIG. 2B), CTG-1921 (FIG. 2Q) treated with PYX-201.

[0320] Table 7

[0321] Example 5: Head and Neck Cancer PDX Studies

[0322] Nine PDX studies conducted in head and neck cancer using a single subtype, squamous cell carcinoma are shown in Table 8. As shown in Table 8, percent inhibition of tumor growth between control and treatment in the selected models range from almost complete disappearance of tumors at 96.89% to a robust response of 62.68%. FIG. 3A-FIG. 31 display a substantial tumor growth inhibition of greater than 90% in four of the PDX models (HNXF 536 (FIG. 3A), HNXF 1838 (FIG. 3B), CTG-2130 (FIG. 3C), and CTG-1131 (FIG. 3D)) treated with PYX-201.

[0323] Table 8

[0324] Example 6: Non-small Cell Lung Cancer

[0325] Fourteen PDX studies conducted in non-small cell lung cancer using three different subtypes, including adenocarcinoma, squamous cell carcinoma, and large cell carcinoma are shown in Table 9. As shown in Table 9, percent inhibition of tumor growth between control and treatment in the selected models range from almost complete disappearance of tumors at 99.81% to a robust response of 56. 18%. FIG. 4A-FIG. 4N display a substantial tumor growth inhibition of greater than 90% in seven of the PDX models (LXFA-677 (FIG. 4A), LXFA- 2184 (FIG. 4B), LXFE-2276 (FIG. 4C), CTG-1992 (FIG. 4D), LXFL-2377 (FIG. 4E), LXFE-397 (FIG. 4F), and CTG-2540 (FIG. 4G)) treated with PYX-201.

[0326] Table 9

[0327] Example 7: Pancreatic Cancer Thirteen PDX studies conducted in pancreatic cancer using at least three different subtypes, including adenocarcinoma, carcinoma, and invasive ductal carcinoma are shown in Table 10. As show n in Table 10, percent inhibition of tumor growth between control and treatment in the selected models range from complete disappearance at 100% to a robust response of 52.92%. FIG. 5A-FIG. 5M display a substantial tumor growth inhibition of greater than 90% in five of the PDX models (PAXF 736 (FIG. 5A). PAXF 1881 (FIG. 5B), PAXF 2132 (FIG. 5C), CTG-2413 (FIG. 5D), and PAXF 2196 (FIG. 5E)) treated with PYX- 201.

[0328] Table 10

[0329] Example 8: Sarcoma

[0330] Sarcoma are mesenchymally -derived cancers that are usually rich in extracellular matrix proteins. The high tumor heterogeneity exhibited by sarcomas complicate treatment options, which options are often ineffective. A study was conducted to characterize the prevalence and distribution of EDB+FN expression in sarcoma subtypes using an immunohistochemistry (IHC) assay for detection of a dynamic range of EDB+FN protein expression in formalin-fixed paraffin-embedded (FFPE) tumor tissue. An H-score approach was initially developed for a pathologist to visually separately score EDB+FN expression in three areas: tumor-induced stroma, tumor cell membrane, and tumor cell cytoplasm according to the following formula:

[0331] H = (1 x % area, level 1) + (2 x % area, level 2) + (3 x % area, level 3).

[0332] A digital pathology’ algorithm was subsequently developed for streamlined assessment of EDB+FN expression in nonclinical human tumor samples. Briefly, using digital pathology softw are, a tumor / stroma classifier was trained with multiple examples of tumor and stroma morphology’ across a range of indications and this classifier w as applied to categorize individual cells within each slide scan. Thresholds for level 1, level 2, and level 3 expression intensity were set using pathologist-scored samples (as described above) as a guide.

[0333] Combined areas of each cell category and each intensity threshold were used to compute EDB+FN digital H-score (dH-score).

[0334] Digital H scores for overall EDB+FN IHC staining: Scores including the median dH- score and range of dH-scores across samples from commercially sourced tumor tissue microarrays for different sarcoma subtypes can be seen in Table 11 A. A high median dH- score indicating strong EDB+FN expression in a sarcoma subtype may serve as an indicator that treatment with an anti-EDB cytotoxic ADC, including the anti-EDB site-specific cytotoxic ADC PYX-201, will result in tumor growth inhibition.

[0335] Table 11A

[0336] Fourteen PDX studies conducted in sarcoma using at least eight different subtypes, including rhabdomyosarcoma, soft tissue sarcoma, osteosarcoma, anaplastic squamous carcinoma, myxofibrosarcoma, liposarcoma, dedifferentiated liposarcoma and leiomyosarcoma are shown in Table 1 IB. As shown in Table 1 IB, percent inhibition of tumor growth between control and treatment in the selected models range from almost complete disappearance at 99.54% to a robust response of 61.19%. FIG. 6A-FIG. 6N display a substantial tumor growth inhibition of greater than 90% in seven of the PDX models (SXFS 463 (FIG. 6A). SXFS 2406 (FIG. 6B). CTG-0243 (FIG. 6C), SXFS 174 (FIG. 6D), CTG- 1 1 16 (FIG. 6L), CTG-1339 (FIG. 6M), SXFS 2147 (FIG. 6F)) treated with PYX-201. Table 11B

[0337] Example 9: Breast Cancer

[0338] Thirteen PDX studies conducted in breast cancer for both triple negative breast cancer (‘ TNBC”) and hormone receptor positive, human epidermal growth factor receptor negative (“HR+Her2-) breast cancer, more specifically estrogen receptor positive, progesterone receptor positive, human epidermal growth factor receptor negative ("ER+PR+Her2-") breast cancer, using five different subtypes, including adenocarcinoma, ductal carcinoma, invasive ductal carcinoma, metaplastic spindle cell carcinoma and intraductal carcinoma are shown in Table 12. As show n in Table 12, percent inhibition of tumor grow th between control and treatment in the selected models range from almost complete disappearance at 98.45% to a robust response of 51.69%. FIG. 7A-FIG. 7M display a substantial tumor growth inhibition of greater than 90% in four of the PDX models (CTG-0437 (FIG. 7B), CTG-3090 (FIG. 7C), CTG-1520 (FIG. 7D), CTG-1167 (FIG. 7E)) treated with PYX-201.

[0339] Table 12

[0340] Example 10: Renal Cancer

[0341] Six PDX studies conducted in renal cancer using three different subtypes, including squamous cell carcinoma, clear cell carcinoma and adenocarcinoma are shown in Table 13. As shown in Table 13, percent inhibition of tumor growth between control and treatment in the selected models range from almost complete disappearance at 98.70% to a robust response of 71.32%. FIG. 8A-FIG. 8F display a substantial tumor growth inhibition of greater than 90% in two of the PDX models (RXF 1781 (FIG. 8A), RXF 393 (FIG. 8B)) treated with PYX-201.

[0342] Table 13

[0343] Example 11: Hepatocellular (Liver) Cancer

[0344] Two PDX studies conducted in hepatocellular (liver) cancer using two different subtypes, including cribiform carcinoma and cholangiosarcoma are show' in Table 14. As show n in Table 14, percent inhibition of tumor growth between control and treatment in the selected models range from almost complete disappearance at 98.78% to a robust response of 68.79%. FIG. 9A-FIG. 9B display a substantial tumor growth inhibition of greater than 90% in one of the PDX models (LIXAH 575 (FIG. 9A)) treated with PYX-201.

[0345] Table 14

[0346] Example 12: Ovarian Cancer

[0347] Nine PDX studies conducted in ovarian cancer using at least five different subtypes, including serous papillary carcinoma, serous adenocarcinoma, clear cell adenocarcinoma, endometrioid carcinoma and adenocarcinoma are shown in Table 15. As shown in Table 15, percent inhibition of tumor growth between control and treatment in the selected models range from almost complete disappearance at 93.57% to a robust response of 50.99%. FIG. 10A-FIG. 101 display a substantial tumor growth inhibition of greater than 90% in one of the PDX models (CTG-1086 (FIG. 10A)) treated with PYX-201.

[0348] Table 15

[0349] Example 13: Durability of Response

[0350] The durability of the PYX-201 response was evaluated over the course of 90 to 100 days in select PDX models including breast cancer (TNBC: CTG-3090; FIG. 11 A), non-small cell lung cancer (NSCLC: LXFA-2184, CTG-1444, CTG-1992; FIGS. 1 IB - 1 ID), head and neck cancer (H&N: CTG-2130, CTG-0719, CTG-3130; FIGS. HE - 11G), pancreatic cancer (PDAC: PAXF 1881; FIG. 11H), and sarcoma (STS: SXFS 463, SXFS 174; FIGS. 111 - 11 J). The results of FIG. 11 A through FIG. 11 J demonstrate the durability of the PYX-201 response over an extended period long after PYX-201 administration has completed, indicating a durable response in these exemplary PDX models. Example 14: Bystander Activity of PYX-201 In-Vitro

[0351] HT-29 cells which were negative for EDB+FN were modified to express the Luciferase reporter and cultured as a monolayer or mixed with Caki2 (FIG. 12A) or WI-38 VA-13 2RA (FIG. 12B) (both were positive for EDB+FN) at a 1:3 ratio). Cells were treated with a non-targeted ADC or PYX-201 for 5 days at 8,000 ng / ml, and Luciferase activity was measured to evaluate the amount of HT-29 cells in each well.

[0352] Referring now to FIG. 12A-FIG. 12B. HT-29 cells were sensitive to PYX-201 only when co-cultured with EDB+FN positive cells. The non-targeted ADC did not induce cell killing compared to the untreated condition. These data demonstrated the bystander effect of PYX-201 in an in vitro setting.

[0353] Enumerated Embodiments

[0354] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0355] Embodiment 1 : A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, that binds to extra domain B of fibronectin; (b) a linker; and (c) a drug; wherein the EDB+FN-expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2-breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, sarcoma, and thyroid cancer.

[0356] Embodiment 2: The method of embodiment 1, wherein the EDB+FN-expressing disorder or disease is selected from the group consisting PR+ER+HER2-breast cancer, head and neck cancer, sarcoma, and thyroid cancer.

[0357] Embodiment 3: The method of embodiment 1, wherein the EDB+FN-expressing disorder or disease is HR+HER2- breast cancer.

[0358] Embodiment 4: The method of embodiment 1, wherein the EDB+FN-expressing disorder or disease is hepatocellular carcinoma.

[0359] Embodiment 5: The method of embodiment 1. wherein the EDB+FN-expressing disorder or disease is head and neck cancer.

[0360] Embodiment 6: The method of embodiment 1, wherein the EDB+FN-expressing disorder or disease is renal cancer.

[0361] Embodiment 7: The method of embodiment 1. wherein the EDB+FN-expressing disorder or disease is sarcoma. Embodiment 8: The method of embodiment 1, wherein the EDB+FN-expressing disorder or disease is thyroid cancer.

[0362] Embodiment 9: The method of embodiment 1, wherein the EDB+FN-expressing disorder or disease is PR+ER+HER2-breast cancer.

[0363] Embodiment 10: The method of embodiment 1, wherein the EDB+FN expressing disorder or disease is a subtype of sarcoma selected from the group consisting of chordoma, leiomyosarcoma, liposarcoma. myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, and soft tissue sarcoma.

[0364] Embodiment 11 : The method of embodiment 1 , wherein the EDB+FN expressing disorder or disease is chordoma.

[0365] Embodiment 12: The method of any one of the preceding embodiments, wherein the antibody, or antigen binding fragment thereof, comprises at least one amino acid residue modification for site specific conjugation in a constant region of the antibody, or antigen binding fragment thereof.

[0366] Embodiment 13: The method of any one of the preceding embodiments, wherein the antibody, or antigen binding fragment thereof, comprises at least one cysteine substitution or site-specific conjugation.

[0367] Embodiment 14: The method of any one of the preceding embodiments, wherein the antibody, or antigen binding fragment thereof comprises: a heavy chain constant region comprising an engineered cysteine residue at position: 118 (114 according to the numbering of Kabat), 246, 249, 265, 267, 270, 276, 278, 283, 290, 292, 293, 294, 300, 302, 303, 314, 315, 318, 320, 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, 25 428, 431. 432, 437, 438, 439, 443 or 444, or any combination thereof , according to the numbering of the EU index; or (b) a light chain constant region comprising: (i) an engineered cysteine residue at position 110, 111, 125, 149, 155, 158, 161, 183, 185, 188, 189, 191, 197, 205, 207, 208 or 210, or any 30 combination thereof, according to the numbering of Kabat; (ii) an engineered cysteine residue at a position corresponding to residue 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 engineered cysteine residue at a position corresponding to 5 residue 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).

[0368] Embodiment 15: The method of any one of the preceding embodiments, wherein the antibody, or antigen binding fragment, comprises a VH region comprising the VH CDR1 region, VH CDR2 region, and VH CDR3 region of the VH amino acid sequence of SEQ ID NO:21, and a VL region comprising VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NO: 10.

[0369] Embodiment 16: The method of any one of the preceding embodiments, 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.

[0370] Embodiment 17 : The method of any one of the preceding embodiments, 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.

[0371] Embodiment 18: The method of any one of the preceding embodiments, wherein the antibody, or antigen binding fragment, comprises: 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.

[0372] Embodiment 19: The method of any one of the preceding embodiments, 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.

[0373] Embodiment 20: The method of any one of the preceding embodiments, wherein the antibody or antigen binding fragment thereof comprises: 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.

[0374] Embodiment 21 : The method of any one of the preceding embodiments, wherein the antibody, or antigen binding fragment thereof, comprises at least one of the following: a cysteine at position 290 of the heavy chain constant region according to the numbering of the EU index; and / or a cysteine at position 183 of the light chain constant region according to the numbering of the EU index.

[0375] Embodiment 22: The method of any one of the preceding embodiments, wherein the antibody comprises at least one of the following: a heavy chain comprising SEQ ID NO:25; a light chain comprising SEQ ID NO:31.

[0376] Embodiment 23: The method of any one of the foregoing embodiments, wherein the linker is a cleavable linker.

[0377] Embodiment 24: The method of embodiment 23, wherein the cleavable linker is selected from the group consisting of: vc having the structure diS having the structure diS-C2OCO having the structure

[0378] AcLys-vc having the structure

[0379] Embodiment 25 : The method of embodiment 23 or embodiment 24, wherein the cleavable linker has the structure:

[0380]

[0381] Embodiment 26: The method of any one of the foregoing embodiments, wherein the drug is a cytotoxic agent.

[0382] Embodiment 27: The method of embodiment 26, wherein the cytotoxic agent is an auristatin.

[0383] Embodiment 28: The method of embodiment 27, wherein the auristatin is selected from the group consisting of: 0101 having the structure

[0384] 9411 having the structure

[0385] 4574 having the structure

[0386] Embodiment 29: The method of any one of embodiments 27-28, wherein the auristatin is auristatin 0101. Embodiment 30: The method of embodiment 26, wherein the cytotoxic agent has the structure:

[0387] Embodiment 31 : The method of embodiment 26, wherein the cytotoxic agent, the linker, and the antibody, or antigen binding fragment thereof, are linked to generate the structure: wherein X-S represents the antibody, or antigen binding fragment thereof, wherein S is the sulfur atom of at least one cysteine residue in the antibody, or antigen binding fragment thereof.

[0388] Embodiment 32: A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, that binds to extra domain B of fibronectin; (b) a cleavable linker; and (c) a cytotoxic drug.

[0389] Embodiment 33: A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, that binds to extra domain B of fibronectin; (b) a cleavable linker; and (c) an auristatin.

[0390] Embodiment 34: A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, that binds to extra domain B of fibronectin; (b) a vc linker; and (c) a 0101 drug.

[0391] Embodiment 35: A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, comprising a VH region comprising the VH CDR1 region, VH CDR2 region, and VH CDR3 region of the VH amino acid sequence of SEQ ID NO:21, and a VL region comprising VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NO: 10; (b) a cleavable linker; and (c) a cytotoxic drug.

[0392] Embodiment 36: A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, comprising a VH region comprising the VH CDR1 region, VH CDR2 region, and VH CDR3 region of the VH amino acid sequence of SEQ ID NO:21. and a VL region comprising VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NO: 10; (b) a cleavable linker; and (c) an auristatin.

[0393] Embodiment 37: A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising: (a) an antibody, or antigen binding fragment thereof, comprising a VH region comprising the VH CDR1 region, VH CDR2 region, and VH CDR3 region of the VH amino acid sequence of SEQ ID NO:21, and a VL region comprising VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NO: 10; (b) a vc linker; and (c) a 0101 drug.

[0394] Embodiment 38: A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and (c) a cytotoxic drug.

[0395] Embodiment 39: A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and (c) an auristatin.

[0396] Embodiment 40: A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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) a 0101 drug. Embodiment 41 : A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and (c) a cytotoxic drug.

[0397] Embodiment 42: A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and (c) an auristatin.

[0398] Embodiment 43: A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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) a 0101 drug.

[0399] Embodiment 44: A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and (c) a cytotoxic drug.

[0400] Embodiment 45: A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and (c) an auristatin. Embodiment 46: A method of treating an EDB+FN- expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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) a 0101 drug.

[0401] Embodiment 47: The method of any one of embodiments 32-46, wherein the EDB+FN-expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2-breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, sarcoma, and thyroid cancer.

[0402] Embodiment 48: The method of any one of embodiments 32-46, wherein the EDB+FN-expressing disorder or disease is selected from the group consisting of PR+ER+HER2-breast cancer, head and neck cancer, sarcoma, and thyroid cancer.

[0403] Embodiment 49: The method of any one of embodiments 32-46, wherein the EDB+FN-expressing disorder or disease is HR+HER2- breast cancer.

[0404] Embodiment 50: The method of any one of embodiments 32-46, wherein the EDB+FN-expressing disorder or disease is hepatocellular (liver) cancer.

[0405] Embodiment 51 : The method of any one of embodiments 32-46, wherein the EDB+FN-expressing disorder or disease is head and neck cancer.

[0406] Embodiment 52: The method of any one of embodiments 32-46, wherein the EDB+FN-expressing disorder or disease is renal cancer.

[0407] Embodiment 53: The method of any one of embodiments 32-46, wherein the EDB+FN-expressing disorder or disease is sarcoma.

[0408] Embodiment 54: The method of any one of embodiments 32-46, wherein the EDB+FN-expressing disorder or disease is thyroid cancer.

[0409] Embodiment 55: The method of any one of embodiments 32-46, wherein the EDB+FN expressing disorder or disease is PR+ER+HER2-breast cancer.

[0410] Embodiment 56: The method of any one of embodiments 32-46, wherein the EDB+FN expressing disorder or disease is a subtype of sarcoma selected from the group consisting of chordoma, leiomyosarcoma, liposarcoma, myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, and soft tissue sarcoma.

[0411] Embodiment 57: The method of any one of embodiments 32-46, wherein the EDB+FN expressing disorder or disease is chordoma.

[0412] The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for earn ing out the same purposes and / or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure.

[0413] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0414] In sum, while this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMSWhat is claimed is:

1. A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising:(a) an antibody, or antigen binding fragment thereof, that binds to extra domain B of fibronectin;(b) a linker; and(c) a drug; wherein the EDB+FN-expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2-breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, sarcoma, and thyroid cancer.

2. The method of claim 1, wherein the EDB+FN-expressing disorder or disease is selected from the group consisting PR+ER+HER2-breast cancer, head and neck cancer, sarcoma, and thyroid cancer.

3. The method of claim 1, wherein the EDB+FN-expressing disorder or disease is HR+HER2- breast cancer.

4. The method of claim 1, wherein the EDB+FN-expressing disorder or disease is hepatocellular carcinoma.

5. The method of claim 1, wherein the EDB+FN-expressing disorder or disease is head and neck cancer.

6. The method of claim 1, wherein the EDB+FN-expressing disorder or disease is renal cancer.

7. The method of claim 1, wherein the EDB+FN-expressing disorder or disease is sarcoma.

8. The method of claim 1, wherein the EDB+FN-expressing disorder or disease is thyroid cancer.

9. The method of claim 1, wherein the EDB+FN-expressing disorder or disease is PR+ER+HER2- breast cancer.

10. The method of claim 1, wherein the EDB+FN expressing disorder or disease is a subtype of sarcoma selected from the group consisting of chordoma, leiomyosarcoma, liposarcoma, myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, and soft tissue sarcoma.

11. The method of claim 1 , wherein the EDB+FN expressing disorder or disease is chordoma.

12. The method of any one of the preceding claims, wherein the antibody, or antigen binding fragment thereof, comprises at least one amino acid residue modification for site specific conjugation in a constant region of the antibody, or antigen binding fragment thereof.

13. The method of any one of the preceding claims, wherein the antibody, or antigen binding fragment thereof, comprises at least one cysteine substitution or site-specific conjugation.

14. The method of any one of the preceding claims, wherein the antibody, or antigen binding fragment thereof comprises: a heavy chain constant region comprising an engineered cysteine residue at position: 118 (114 according to the numbering of Kabat), 246, 249. 265, 267, 270, 276, 278. 283, 290, 292, 293, 294, 300, 302. 303, 314, 315. 318, 320. 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, 41 1, 413, 414, 416, 418, 419, 421, 25 428, 431, 432, 437, 438, 439, 443 or 444, or any combination thereof , according to the numbering of the EU index; or (b) a light chain constant region comprising: (i) an engineered cysteine residue at position 110. I l l, 125. 149, 155, 158, 161, 183, 185, 188, 189, 191, 197, 205, 207, 208 or 210, or any 30combination thereof, according to the numbering of Kabat; (ii) an engineered cysteine residue at a position corresponding to residue 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 engineered cysteine residue at a position corresponding to 5 residue 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).

15. The method of any one of the preceding claims, wherein the antibody, or antigen binding fragment, comprises a VH region comprising the VH CDR1 region, VH CDR2 region, and VH CDR3 region of the VH amino acid sequence of SEQ ID NO:21, and a VL region comprising VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NOTO.

16. The method of any one of the preceding claims, 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.

17. The method of any one of the preceding claims, 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.

18. The method of any one of the preceding claims, wherein the antibody, or antigen binding fragment, comprises: 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.

19. The method of any one of the preceding claims, 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.

20. The method of any one of the preceding claims, antibody or antigen binding fragment thereof comprises: 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 heavy7chain 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 SEQID NO: 15; or a heavy chain comprising SEQ ID NO: 29 and a light chain comprising SEQ ID NO: 31.

21. The method of any one of the preceding claims, wherein the antibody, or antigen binding fragment thereof, comprises at least one of the following: a cysteine at position 290 of the heavy chain constant region according to thenumbering of the EU index; and / or a cysteine at position 183 of the light chain constant region according to the numbering of the EU index.

22. The method of any one of the preceding claims, wherein the antibody comprises at least one of the following: a heavy chain comprising SEQ ID NO:25; a light chain comprising SEQ ID NO:31.

23. The method of any one of the preceding claims, wherein the linker is a cleavable linker.

24. The method of claim 23, wherein the cleavable linker is selected from the group consisting of: vc having the structurediS having the structurediS-C2OCO having the structureAcLys-vc having the structureThe method of claim 23, wherein the cleavable linker has the structure:

26. The method of any one of the preceding claims, wherein the drug is a cytotoxic agent.

27. The method of claim 26, wherein the cytotoxic agent is an auristatin.

28. The method of claim 27, wherein the auristatin is selected from the group consisting of:1569 having the structure4574 having the structure29. The method of claim 27 or claim 28, wherein the auristatin is auristatin 0101 .

30. The method of claim 26, wherein the cytotoxic agent has the structure:

31. The method of claim 26, wherein the cytotoxic agent, the linker, and the antibody, or antigen binding fragment thereof, are linked to generate the structure:wherein:X-S represents the antibody, or antigen binding fragment thereof, wherein S is the sulfur atom of at least one cysteine residue in the antibody, or antigen binding fragment thereof.

32. A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising:(a) an antibody, or antigen binding fragment thereof, that binds to extra domain B of fibronectin;(b) a cleavable linker; and(c) a cytotoxic drug.

33. A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising:(a) an antibody, or antigen binding fragment thereof, that binds to extra domain B of fibronectin;(b) a cleavable linker; and(c) an auristatin.

34. A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising:(a) an antibody, or antigen binding fragment thereof, that binds to extra domain B of fibronectin;(b) a vc linker; and(c) a 0101 drug.

35. A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising:(d) an antibody, or antigen binding fragment thereof, comprising a VH region comprising the VH CDR1 region, VH CDR2 region, and VH CDR3 region of the VH amino acid sequence of SEQ ID NO:21, and a VL region comprising VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NO: 10;(e) a cleavable linker; and(f) a cytotoxic drug.

36. A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising:(a) an antibody, or antigen binding fragment thereof, comprising a VH region comprising the VH CDR1 region, VH CDR2 region, and VH CDR3 region ofthe VH amino acid sequence of SEQ ID NO:21, and a VL region comprising VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NO: 10;(b) a cleavable linker; and(c) an auristatin.

37. A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of an antibody-drug conjugate comprising:(a) an antibody, or antigen binding fragment thereof, comprising a VH region comprising the VH CDR1 region, VH CDR2 region, and VH CDR3 region of the VH amino acid sequence of SEQ ID NO:21, and a VL region comprising VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NO: 10;(b) a vc linker; and(c) a 0101 drug.

38. A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and(c) a cytotoxic drug.

39. A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and(c) an auristatin.

40. A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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) a 0101 drug.

41. A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and(c) a cytotoxic drug.

42. A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and(c) an auristatin.

43. A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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) a 0101 drug.

44. A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and(c) a cytotoxic drug.

45. A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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 cleavable linker; and(c) an auristatin.

46. A method of treating an EDB+FN-expressing disorder or disease in a subject of need thereof, comprising administering an effective amount of 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) a 0101 drug.

47. The method of any one of claims 32-46, wherein the EDB+FN-expressing disorder or disease is selected from the group consisting of HR+HER2- breast cancer, PR+ER+HER2-breast cancer, hepatocellular (liver) cancer, head and neck cancer, renal cancer, sarcoma, and thyroid cancer.

48. The method of any one of claims 32-46, wherein the EDB+FN-expressing disorder or disease is selected from the group consisting of PR+ER+HER2-breast cancer, head and neck cancer, sarcoma, and thyroid cancer.

49. The method of any one of claims 32-46, wherein the EDB+FN-expressing disorder or disease is HR+HER2- breast cancer.

50. The method of any one of claims 32-46, wherein the EDB+FN-expressing disorder or disease is hepatocellular (liver) cancer.

51. The method of any one of claims 32-46, wherein the EDB+FN-expressing disorder or disease is head and neck cancer.

52. The method of any one of claims 32-46, wherein the EDB+FN-expressing disorder or disease is renal cancer.

53. The method of any one of claims 32-46, wherein the EDB+FN-expressing disorder or disease is sarcoma.

54. The method of any one of claims 32-46, wherein the EDB+FN-expressing disorder or disease is thyroid cancer.

55. The method of any one of claims 32-46, wherein the EDB+FN-expressing disorder or disease is PR+ER+HER2-breast cancer.

56. The method of any one of claims 32-46, wherein the EDB+FN expressing disorder or disease is a subtype of sarcoma selected from the group consisting of chordoma, leiomyosarcoma, liposarcoma. myxofibrosarcoma, osteosarcoma, rhabdomyosarcoma, and soft tissue sarcoma.

57. The method of any one of claims 32-46, wherein the EDB+FN-expressing disorder or disease is chordoma.

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