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

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 method comprises administration of an anti-EDB+FN ADC to a patient having cancer, such as, for example, non-small cell lung cancer (NSCLC), breast cancer, head and neck cancer, ovarian cancer, thyroid cancer, pancreatic cancer, soft tissue sarcoma, chordoma, hepatocellular carcinoma, kidney cancer, cervical cancer, and endometrial cancer.

Description

[0001] Attorney Docket No. 384953-1024W01(00350)

[0002] CONSTRUCTS, COMPOSITIONS, AND METHODS FOR TREATING DISEASES AND DISORDERS EXPRESSING EXTRA DOMAIN B SPLICE VARIANT OF FIBRONECTIN

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U. S. C. § 119(e) to U. S. Provisional Patent Application No. 63 / 722,504, filed November 19, 2024, the disclosure of which is hereby incorporated herein by reference in its entirety.

[0004] REFERENCE TO SEQUENCE LISTING

[0005] The Sequence Listing concurrently submitted herewith as an XML-formatted text file named '‘384953-1024WOl_sequence_listing.xml:’ created on November 19, 2025, and having a size of 67,088 bytes, is herein incorporated by reference in its entirety pursuant to 37 C. F. R. 1.52(e)(5).

[0006] BACKGROUND

[0007] Fibronectin 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. 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 LI 9 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] 1

[0010] 567343972 Attorney Docket No. 384953-1024W01(00350)

[0011] 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 wi th EDB+ FN-expressing disorders or diseases, such as cancers associated with EDB+ FN expression and / or EDB+ FN-expressing cancers.

[0012] BRIEF SUMMARY

[0013] In one aspect, the present disclosure generally relates to a method of treating a patient having a proliferative disorder, In certain embodiments, the method comprises administering to the patient an effective amount of an anti-extra domain B (EDB)+fibronectin (FN) antibody-drug conjugate (ADC) at least twice every week, at least weekly (QW), at least every 2 weeks (Q2W), at least every 3 weeks (Q3W), at least every 4 weeks (Q4W), at least every 5 weeks (Q5W), or at least every 6 weeks (Q6W), wherein the anti-EDB+FN ADC comprises an anti-EDB+FN antibody, or antigen binding fragment thereof, conjugated to a drug. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of 3.6 mg / kg to 5.4 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient every' 3 weeks (Q3W). In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of about 0.3 mg / kg to about 8 mg / kg, about 0.6 mg / kg to about 8 mg / kg, about 1.2 mg / kg to about 8 mg / kg. about 2.4 mg / kg to about 8 mg / kg, about 3.6 mg / kg to about 8 mg / kg, about 4.4 mg / kg to about 8 mg / kg, about 5.4 mg / kg to about 8 mg / kg, about 6.6 mg / kg to about 8 mg / kg, about 0.3 mg / kg to about 6.6 mg / kg, about 0.6 mg / kg to about 6.6 mg / kg, about 1.2 mg / kg to about 6.6 mg / kg, about 2.4 mg / kg to about 6.6 mg / kg. about 3.6 mg / kg to about 6.6 mg / kg, about 4.4 mg / kg to about 6.6 mg / kg. about 5.4 mg / kg to about 6.6 mg / kg, about 0.3 mg / kg to about 5.4 mg / kg, about 0.6 mg / kg to about 5.4 mg / kg, about 1.2 mg / kg to about 5.4 mg / kg, about 2.4 mg / kg to about 5.4 mg / kg, about 3.6 mg / kg to about 5.4 mg / kg, about 4.4 mg / kg to about 5.4 mg / kg, about 0.3 mg / kg to about 4.4 mg / kg, about 0.6 mg / kg to about 4.4 mg / kg, about 1.2 mg / kg to about 4.4 mg / kg. about 2.4 mg / kg to about 4.4 mg / kg, about 0.3 mg / kg to about 3.6 mg / kg, about 0.6 mg / kg to about 3.6 mg / kg, about 1.2 mg / kg to about 3.6 mg / kg, about 2.4 mg / kg to about 3.6 mg / kg, about 0.3 mg / kg to about 2.4 mg / kg, about 0.6 mg / kg to about 2.4 mg / kg, about 1.2 mg / kg to about 2.4 mg / kg, about 0.3 mg / kg to about 1.2 mg / kg, about 0.6 mg / kg to about 1.2 mg / kg, or about 0.3 mg / kg to about 0.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of 0.3 mg / kg to 8 mg / kg, 0.6 mg / kg to 6.6 mg / kg, 1.2 mg / kg to 5.4 mg / kg, 2.4 to 5.4 mg / kg, 3.6

[0014] 2

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[0016] mg / kg to 5.4 mg / kg, or 4.4 mg / kg to 5.4 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of 3.6 mg / kg to 8 mg / kg. 3.6 mg / kg to 6.6 mg / kg, 3.6 mg / kg to 5.4 mg / kg, 3.6 mg / kg to 4.4 mg / kg, 4.4 mg / kg to 8 mg / kg, 4.4 mg / kg to 6.6 mg / kg, 4.4 mg / kg to 5.4 mg / kg, 5.4 mg / kg to 8 mg / kg, 5.4 mg / kg to 6.6 mg / kg, 6.6 mg / kg to 8 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of about 0.3 mg / kg, about 0.6 mg / kg. about 1.2 mg / kg, about 2.4 mg / kg, about 3.6 mg / kg, about 4.4 mg / kg, about 5.4 mg / kg, about 6.6 mg / kg or about 8 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of 0.3 mg / kg, 0.6 mg / kg, 1.2 mg / kg, 2.4 mg / kg, 3.6 mg / kg, 4.4 mg / kg, 5.4 mg / kg, 6.6 mg / kg or 8 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of 3.6 mg / kg or greater. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of 3.6 mg / kg to 6.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of 3.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of 4.4 mg / kg or greater. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of 4.4 mg / kg to 6.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of 4.4 mg / kg to 5.4 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of 4.4 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of 5.4 mg / kg or greater. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of 5.4 mg / kg to 6.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of 5.4 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of 6.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of 8 mg / kg.

[0017] In some aspects, the proliferative disorder is cancer. In some aspects, the cancer is selected from a group consisting of non-small cell lung cancer (NSCLC). breast cancer, head and neck cancer, ovarian cancer, thyroid cancer, pancreatic cancer, soft tissue sarcoma, chordoma, hepatocellular carcinoma, kidney cancer, cervical cancer, and endometrial cancer. In some aspects, the cancer is selected from a group consisting of non-small cell lung cancer, triple negative breast cancer, hormone receptor positive breast cancer, head and neck cancer, ovarian cancer, pancreatic cancer, soft tissue sarcoma, and chordoma. In some aspects, the cancer is non-small cell lung cancer. In some aspects, the cancer is triple negative breast cancer. In some aspects, the cancer is hormone receptor positive breast cancer. In some aspects, the cancer is hormone receptor positive, human epidermal growth factor receptor 2 negative, breast cancer. In some aspects, the cancer is head and neck cancer. In some aspects, the cancer is head and neck squamous cell carcinoma. In some aspects, the cancer is ovarian 3

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[0019] cancer In some aspects, the cancer is pancreatic cancer. In some aspects, the cancer is pancreatic ductal adeno carcinoma. In some aspects, the cancer is soft tissue sarcoma. In some aspects, the cancer is chordoma.

[0020] In some aspects, the drug is a cytotoxic agent. In some aspects, the drug is a microtubule polymerization inhibitor. In some aspects, the drug is a dolastatin, auristatin, or derivative thereof. In some aspects, the drug is an auristatin. In some aspects, the drug is selected from the group consisting of: MMAD, MMAE, MMAF, auristatin 1569, auristatin 9411, auristatin 4574 and auristatin 0101. In some aspects, the drug is auristatin 0101. In some aspects, the drug antibody ratio (DAR) is 1, 2, 3, or 4. In some aspects, the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises: (a) a heavy chain comprising a heavy chain constant region (CH) and heavy chain variable region (VH), (b) a light chain comprising a light chain constant region (CL) and light chain variable region (VL), and (c) 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 anti-EDB+FN antibody, or antigen binding fragment thereof, comprises at least one cysteine substitution for site specific conjugation in a constant region of the antibody, or antigen binding fragment thereof. In some aspects, (a) the CH region comprises 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, 428, 431, 432, 437, 438, 439, 443 or 444, or any combination thereof, according to the numbering of the EU index; or (b) the CL region comprises: (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). In some aspects, (a) the CH region comprises an engineered cysteine residue at position 290 according to the numbering of the EU index; or (b) the CL region comprises an engineered cysteine residue at position 183 according to the numbering of Kabat. In some aspects, (a) the CH region comprises an engineered cysteine residue at position 290 according to the numbering of the 4

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[0022] EU index; and (b) the CL region comprises an engineered cysteine residue at position 183 according to the numbering of Kabat. In some aspects, the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises: (a) a heavy chain comprising a heavy chain variable region (VH), the 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 (b) a light chain comprising a light chain variable region (VL). the VL comprising 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, the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises: (a) a heavy chain comprising a heavy7chain variable region (VH), the VH comprising the VH amino acid sequence of SEQ ID NO: 21; or (b) a light chain comprising a light chain variable region (VL), the VL comprising the VL amino acid sequence of SEQ ID NO: 10. In some aspects, the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises: (a) a heavy chain comprising a heavy chain variable region (VH), the VH comprising the VH amino acid sequence of SEQ ID NO: 21; and (b) a light chain comprising a light chain variable region (VL), the VL comprising the VL amino acid sequence of SEQ ID NO: 10. In some aspects, the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 25; or (b) a light chain comprising the amino acid sequence of SEQ ID NO: 31. In some aspects, the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 25; and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 31.

[0023] In some aspects, the anti-EDB+FN ADC further comprises a linker, wherein the linker conjugates the anti-cancer drug to the antibody, or antigen binding fragment thereof. In some aspects, the linker is a cleavable linker. In some aspects, the linker is selected from the group consisting of val-cit, phe-lys, vc, AcLys-vc, diS, diS-C2OCO, me, me, MalPeg6C2, and a hydrazone linker. In some aspects, the linker is selected from the group consisting of vc, diS, diS-C2OCO, and AcLys-vc. In some aspects, the linker is vc. In some aspects, the ADC is PYX-201.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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.

[0026] FIG. 1 presents a schematic representation of the overall design of a clinical study 5

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[0028] described herein.

[0029] FIG. 2 presents a graphical representation of data related to the change in tumor volume from baseline at dose levels 2.4 to 4.4 mg / kg for various different indications.

[0030] FIG. 3 presents a graphical representation of data related to the change in tumor volume from baseline at dose levels 5.4 to 6.6 mg / kg for various different indications.

[0031] FIG. 4 presents a graphical representation of data related to a patient-by-patient breakdown by dose of the change in tumor volume from baseline for head and neck cancer, more specifically head and neck squamous cell carcinoma (HNSCC).

[0032] FIG. 5 presents a graphical representation of data related to a patient-by-patient breakdown by dose of the change in tumor volume from baseline for pancreatic cancer, more specifically pancreatic ductal adeno carcinoma (PDAC).

[0033] FIG. 6 presents a graphical representation of data related to a patient-by-patient breakdown by dose of the change in tumor volume from baseline for non-small cell lung cancer (NSCLC).

[0034] FIG. 7 presents a graphical representation of data related to a patient-by-patient breakdown by dose of the change in tumor volume from baseline for triple negative breast cancer (TNBC).

[0035] FIG. 8 presents a graphical representation of data related to a patient-by-patient breakdown by dose of the change in tumor volume from baseline for hormone receptor positive human epidermal growth factor receptor 2 negative (HR+Her2-) breast cancer.

[0036] FIG. 9 presents a graphical representation of data related to a patient-by-patient breakdown by dose of the change in tumor volume from baseline for ovarian cancer.

[0037] FIG. 10 presents a graphical representation of data related to a patient-by-patient breakdown by dose of the change in tumor volume from baseline for soft tissue sarcoma (STS) excluding chordoma.

[0038] FIG. 11 presents a graphical representation of data related to a patient-by-patient breakdow n by dose of the change in tumor volume from baseline for chordoma.

[0039] FIG. 12 presents a graphical representation of data related to a patient-by-patient breakdown by dose of the change in tumor volume from baseline for all soft tissue sarcomas including chordoma.

[0040] FIG. 13 presents a graphical representation of data related to a patient-by-patient breakdow n by dose of the change in tumor volume from baseline for additional cancer types including hepatocellular carcinoma (HCC) and thyroid cancer.

[0041] In FIG. 2-FIG. 13, it is noted that: (i) PD: At least a 20% increase in the sum of the 6

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[0043] diameters of target lesions, taking as reference the smallest sum on study recorded since the treatment started or the appearance of one or more new lesions. In addition to 20% increase, the sum must also demonstrate an absolute increase of at least 5 mm; (ii) SD: Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum LD since the treatment started; (iii) PR: At least a 30% decrease in the sum of the diameters of target lesions, taking as reference the Baseline sum diameters; and (iv) CR: Disappearance of all target lesions. Pathologic nodes must have a reduction in the short axis to <10 mm.

[0044] DETAILED DESCRIPTION DEFINITIONS

[0045] 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.

[0046] 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.

[0047] 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 w ell known to the skilled worker, and are explained fully in 7

[0048] 567343972 Attorney Docket No. 384953-1024W01(00350)

[0049] 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).

[0050] 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.

[0051] 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.”

[0052] “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.

[0053] 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.

[0054] 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.

[0055] To “treat” a disease as the term is used herein, means to reduce the frequency or 8

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[0057] severity' of at least one sign or symptom of a disease or disorder experienced by a subject.

[0058] As used herein, to "‘alleviate’' a disease means reducing the severity of one or more symptoms of the disease.

[0059] 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.

[0060] 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.

[0061] 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 (z.e., 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.

[0062] 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.

[0063] 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 9

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[0065] 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

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[0072] recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (z.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzy me digestion) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

[0073] The term “recombinant polypeptide” as used herein is defined as a polypeptide produced by using recombinant DNA methods.

[0074] The term “recombinant DNA” as used herein is defined as DNA produced by joining pieces of DNA from different sources.

[0075] “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.

[0076] 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.

[0077] 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).

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[0080] “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.). intramuscular (i.m.), or intrastemal injection, or infusion techniques.

[0081] The term “polynucleotide” as used herein is defined as a chain of nucleotides.

[0082] 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 (z.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.

[0083] 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.

[0084] 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 construed 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,

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[0087] adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0088] 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.

[0089] 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.

[0090] 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, w hich 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.

[0091] 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.

[0092] 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.

[0093] 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.

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[0096] 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.

[0097] 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;

[0098] 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.

[0099] 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

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[0102] 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.

[0103] 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 reactivity’ 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.

[0104] 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.

[0105] 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 15

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[0107] 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.

[0108] Methods of Treatment and Anti-EDB Antibodies and Antibody-Drug Conjugates PYX-201 (also known as PF-06878792 and EDB-ADC and EDB+FN 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 cy totoxicity' 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 infdtration 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.

[0109] The studies discussed in the present disclosure demonstrate the surprising and unexpected result of being able to administer doses of PYX-201 with its Auristatin 0101 payload, an optimized derivative of monomethyl auristatin E (MMAE), at levels of 3.6 mg / kg and above, including 3.6 mg / kg, 4.4 mg / kg, 5.4 mg / kg, and in some cases even 6.6 mg / kg and 8 mg / kg, to a patient without observing dose-hmiting toxicity. These results are surprising as previous studies have shown that toxicities of ADCs with auristatin payloads, including specifically MMAE payloads and a DAR of 4 generally have a maximum tolerated dose (MTD) of around 1.8 mg / kg irrespective of the target antigen. See, for instance, Rubahamya B, Dong S, Thurber GM. Clinical translation of antibody drug conjugate dosing in solid tumors from preclinical mouse data. Sci Adv. 2024 May 31;10(22):eadkl894. doi:

[0110] 10.1126 / sciadv.adkl894. Epub 2024 May 31. PMID: 38820153; PMCID: PMC11141632. For example, the recommended dose for PADCEV®, anectin-4 targeting ADC with a MMAE pay load (vedotin). is 1.25 mg / kg (see, PADCEV® Dosing and Administration Guide) and the recommended dose for Tivdak®, a tissue factor targeting ADC with an 16

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[0112] MMAE payload (vedotin), is 2 mg / kg (see, Tivdak® Prescribing Information).

[0113] Additionally, the working examples also demonstrate that the ADCs used in the methods herein, e.g., the EDB+FN ADCs described herein, have favorable safety and tolerability of higher dosing levels not found in the art. See, for instance, Example 3, which presents data demonstrating the favorable safety and tolerability of PYX-201 at higher doses as compared to other ADCs known in the field.

[0114] Furthermore, as compared to FDA approved ADCs with auristatin payloads, PYX-201 has a more favorable profile at similar dose levels to those found in the art of 1.2 mg / kg and 2.4 mg / kg, and even at higher dosage levels than those found in the art including 3.6 mg / kg, 4.4 mg / kg, 5.4 mg / kg and 6.6 mg / kg as compared to the FDA approved auristatin based ADCs at much lower dosing levels between 1 and 2 mg / kg. For example, patients treated with PYX-201 at 1.2 mg / kg experienced no TRAEs leading to treatment discontinuation, dose reduction or dose delay. At 2.4 mg / kg patients experienced no TRAEs leading to treatment discontinuation, and only 13% of patients experienced a TRAE leading to dose reduction or delay. Even at higher dose levels than found in the art, PYX-201 at 3.6 mg / kg saw no TRAEs leading to treatment discontinuation or dose delay, and only 9% of patients experience a TRAE leading to dose reduction; at 4.4 mg / kg 0% of patients experienced a TRAE leading to treatment continuation, dose reduction or dose delay; and at 5.4 mg / kg only 3% of patients experienced a TRAE leading to treatment discontinuation, and 28% and 18% experienced a TRAE leading to dose reduction or dose delay, respectively.

[0115] These safety results for PYX-201 compare favorably to treatment discontinuation, dose reduction and dose delay in approved auristatin based ADCs. For example PADCEV® at 1.25 mg / kg dosed weeks one through three with each fourth week off, per the PADCEV® safety profile in post-platinum, post-PD-(L)l inhibitor patients, 47% of patients treated experienced a serious adverse reaction, 61% of patients had their dose interrupted due to an adverse reaction, 34% of patients had their dose reduced due to an adverse reaction, 34% of patients had their dose reduced due to an adverse reaction. As another example, per the Tivdak® innovaTV 301 safety study results, for patients receiving 2 mg / kg Q3W, serious adverse reactions occurred in 33% of patients, fatal adverse reactions occurred in 1.6% of patients, adverse reactions leading to permanent discontinuation occurred in 15% of patients, and adverse reactions leading to dose reduction occurred in 30% of patients.

[0116] Furthermore, in a Phase 1 study of the same Auristatin 0101 payload conjugated via a VC linker to a trastuzumab antibody targeting HER2, adverse events (AE) leading to treatment discontinuation occurred in 47.3% of patients, more specifically in Part 1A with 17

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[0118] dosage levels from 0.15 mg / kg to 5 mg / kg, 25.5% of patients discontinued study drug due to AEs, in Part 2A with dosage levels of 3 mg / kg or 4 mg / kg, 56.5% discontinued study drug due to AEs, and dose levels at 5.0 mg / kg or higher were determined to be intolerable.. See, for instance, Meric-Bemstam F, Calvo E, Lee KS, Moreno V, Park YH, Rha SY, Chalasani P, Zhong W, Zhou L, Pirie-Shepherd S, Leung ACF, Curigliano G. Safety7and Tolerability of a Novel Anti-HER2 Antibody-Drug Conjugate (PF-06804103) in Patients with HER2-Expressing Solid Tumors: A Phase 1 Dose-Escalation Study. Mol Cancer Ther. 2023 Oct 2;22(10): 1191-1203. doi: 10.1158 / 1535-7163. MCT-23-0101. PMID: 37420274; PMCID: PMC 10543980. The increased tolerability7of PYX-201 enabling higher dosing further enables increased disease control rates (DCR) and overall response rates (ORR). Across a tumor types in the Phase 1 Clinical Study described further in Example 1 and Example 2, PYX-201 at dosing levels of 3.6 mg / kg or higher PYX-201 saw a DCR of 76% and ORR of 22%. In certain cancer types, such as head and neck cancer, for dosages of 3.6 mg / kg and above DCR reached 100% and ORR 33%. As further non-limiting examples, for non-small lung cancer patients treated with PYX-201 at 3.6 mg / kg or above DCR was 78% and ORR was 22%, for ovarian cancer DCR was 100% and ORR 50%, for soft-tissue sarcoma including chordoma DCR was 78% with ORR of 22%.

[0119] Furthermore, the results presented in the working examples demonstrate that the methods described herein can provide a therapeutic benefit to patients who had previously received a cancer treatment. In particular, the working examples presented herein demonstrate that the methods described herein can provide a therapeutic benefit to patients who had previously received a chemotherapy, an immunotherapy, a targeted therapy or another antibody-drug conjugate. Furthermore, the working examples presented herein demonstrate that the methods described herein can provide a therapeutic benefit to patients who had previously received a taxane-based cancer treatment. The working examples presented herein also demonstrate that the methods described herein can provide a therapeutic benefit to patients who had previously received a platinum-based therapy. The working examples presented herein further demonstrate that the methods described herein can provide a therapeutic benefit to patients who had previously received a PD1 or PDL1 inhibitor. The working examples presented herein additionally7demonstrate that the methods described herein can provide a therapeutic benefit to patients who had previously received a PARP or CDK4 / 6 inhibitor. The working examples presented herein also demonstrate that the methods described herein can provide a therapeutic benefit to patients who had previously received another antibody-drug conjugate such as those targeting Trop-2 and 5T4

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[0122] Oncotrophoblast Glycoprotein, with either a topoisomerase inhibiting payload or an auristatin payload. See, for instance. Example 4, which presents data demonstrating tumor regression with PYX-201 treatment across a range of cancer types following treatment with a variety of anti-cancer agents.

[0123] 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.

[0124] Methods of Treatment, Amelioration, and / or Prevention

[0125] In one aspect, the present disclosure relates to a method of treating, ameliorating, and / or preventing 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. 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. In certain embodiments, the EDB+ FN-expressing disorder or disease comprises and / or is cancer.

[0126] In one aspect, the present disclosure relates to method of treating a patient having a proliferative disorder, such as cancer, comprising administering to the patient an effective amount of an anti-extra domain B (EDB)+fibronectin (EDB+FN) antibody -drug conjugate (ADC) at least twice every week, at least weekly (QW), at least every 2 weeks (Q2W), at least every 3 weeks (Q3W), at least every 4 weeks (Q4W), at least every 5 weeks (Q5W), or at least every 6 weeks (Q6W), wherein the anti-EDB+FN ADC comprises an anti-EDB+FN antibody, or antigen binding fragment thereof, conjugated to a drug, such as an anti-cancer drug.

[0127] In some aspects, the anti-EDB+FN ADC is administered at least twice every week. In some aspects, the anti-EDB+FN ADC is administered at least weekly (QW). In some aspects, the anti-EDB+FN ADC is administered at least every 2 weeks (Q2W). In some aspects, the anti-EDB+FN ADC is administered at least every 3 weeks (Q3W). In some aspects, the anti-EDB+FN ADC is administered at least every 4 weeks (Q4W). In some aspects, the anti-EDB+FN ADC is administered at least every 5 weeks (Q5W). In some aspects, the anti-EDB+FN ADC is administered at least every 6 weeks (Q6W). In some aspects, the anti-EDB+FN ADC is administered every 3 weeks (Q3W).

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[0130] In some aspects, the anti-EDB+FN ADC is administered at a dose of about 0.3 mg / kg to about 8 mg / kg, about 0.6 mg / kg to about 8 mg / kg, about 1.2 mg / kg to about 8 mg / kg, about 2.4 to about 8 mg / kg, about 3.6 mg / kg to about 8 mg / kg, about 4.4 mg / kg to about 8 mg / kg, about 5.4 mg / kg to about 8 mg / kg, about 6.6 mg / kg to about 8 mg / kg, about 0.3 mg / kg to about 6.6 mg / kg, about 0.6 mg / kg to about 6.6 mg / kg, about 1.2 mg / kg to about 6.6 mg / kg, about 2.4 to about 6.6 mg / kg, about 3.6 mg / kg to about 6.6 mg / kg, about 4.4 mg / kg to about 6.6 mg / kg, about 5.4 mg / kg to about 6.6 mg / kg, about 0.3 mg / kg to about 5.4 mg / kg, about 0.6 mg / kg to about 5.4 mg / kg, about 1.2 mg / kg to about 5.4 mg / kg, about 2.4 to about 5.4 mg / kg, about 3.6 mg / kg to about 5.4 mg / kg, about 4.4 mg / kg to about 5.4 mg / kg, about 0.3 mg / kg to about 4.4 mg / kg, about 0.6 mg / kg to about 4.4 mg / kg, about 1.2 mg / kg to about 4.4 mg / kg, about 2.4 to about 4.4 mg / kg, about 0.3 mg / kg to about 3.6 mg / kg, about 0.6 mg / kg to about 3.6 mg / kg, about 1.2 mg / kg to about 3.6 mg / kg, about 2.4 to about 3.6 mg / kg, about 0.3 mg / kg to about 2.4 mg / kg, about 0.6 mg / kg to about 2.4 mg / kg, about 1.2 mg / kg to about 2.4 mg / kg, about 0.3 mg / kg to about 1.2 mg / kg, about 0.6 mg / kg to about 1.2 mg / kg, or about 0.3 mg / kg to about 0.6 mg / kg.

[0131] In some aspects, the anti-EDB+FN ADC is administered at a dose of about 0.3 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a dose of about 0.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a dose of about 1.2 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a dose of about 2.4 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a dose of about 3.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a dose of about 4.4 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a dose of about 5.4 mg / kg In some aspects, the anti-EDB+FN ADC is administered at a dose of about 6.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a dose of about 8 mg / kg.

[0132] In some aspects, the anti-EDB+FN ADC is administered at a dose of about 0.3 mg / kg to about 8 mg / kg, 0.6 mg / kg to 6.6 mg / kg, about 1.2 mg / kg to about 6.6 mg / kg, about 1.2 mg / kg to about 5.4 mg / kg, about 2.4 to about 6.6 mg / kg, about 2.4 to about 5.4 mg / kg, about 3.6 to about 6.6 mg / kg, about 3.6 mg / kg to about 5.4 mg / kg, about 4.4 mg / kg to about 6.6 mg / kg, about 4.4 mg / kg to about 5.4 mg / kg or about 5.4 mg / kg to 6.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a dose of 0.3 mg / kg to 8 mg / kg, 0.6 mg / kg to 6.6 mg / kg, 1.2 mg / kg to 6.6 mg / kg, 2.4 to 6.6 mg / kg, 3.6 mg / kg to 6.6 mg / kg, 4.4 mg / kg to 6.6 mg / kg, 5.4 to 6.6 mg / kg, 3.6 mg / kg to 5.4 mg / kg, or 4.4 mg / kg to 5.4 mg / kg.

[0133] In some aspects, the anti-EDB+FN ADC is administered at a dose of about 3.6 mg / kg to about 8 mg / kg, about 3.6 mg / kg to about 6.6 mg / kg, about 3.6 mg / kg to about 5.4 mg / kg,

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[0136] about 3.6 mg / kg to about 4.4 mg / kg, about 4.4 mg / kg to about 8 mg / kg, about 4.4 mg / kg to about 6.6 mg / kg. about 4.4 mg / kg to about 5.4 mg / kg. about 5.4 mg / kg to about 8 mg / kg, about 5.4 mg / kg to about 6.6 mg / kg, about 6.6 mg / kg to about 8 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a dose of 3.6 mg / kg to 8 mg / kg, 3.6 mg / kg to 6.6 mg / kg, 3.6 mg / kg to 5.4 mg / kg, 3.6 mg / kg to 4.4 mg / kg, 4.4 mg / kg to 8 mg / kg, 4.4 mg / kg to 6.6 mg / kg, 4.4 mg / kg to 5.4 mg / kg, 5.4 mg / kg to 8 mg / kg, 5.4 mg / kg to 6.6 mg / kg, 6.6 mg / kg to 8 mg / kg.

[0137] In some aspects, the anti-EDB+FN ADC is administered at a dose of about 0.3 mg / kg, about 0.6 mg / kg, about 1.2 mg / kg, about 2.4 mg / kg, about 3.6 mg / kg, about 4.4 mg / kg, about 5.4 mg / kg, about 6.6 mg / kg or about 8 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a dose of 0.3 mg / kg. 0.6 mg / kg. 1.2 mg / kg, 2.4 mg / kg, 3.6 mg / kg, 4.4 mg / kg, 5.4 mg / kg, 6.6 mg / kg or 8 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a dose of 3.6 mg / kg or greater. In some aspects, the anti-EDB+FN ADC is administered at a dose of 3.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a dose of 4.4 mg / kg or greater. In some aspects, the anti-EDB+FN ADC is administered at a dose of 4.4 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a dose of 5.4 mg / kg or greater. In some aspects, the anti-EDB+FN ADC is administered at a dose of 5.4 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a dose of 6.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a dose of 8 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of 3.6 mg / kg to 6.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of 3.6 mg / kg to 5.4 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of 4.4 mg / kg to 6.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of 4.4 mg / kg to 5.4 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a dose of 5.4 mg / kg to 6.6 mg / kg.

[0138] In some aspects, the proliferative disorder is cancer. In some aspects, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), breast cancer, head and neck cancer, ovarian cancer, thyroid cancer, pancreatic cancer, soft tissue sarcoma, chordoma, hepatocellular carcinoma, kidney cancer, cervical cancer, and endometrial cancer. In some aspects, the cancer is selected from the group consisting of non-small cell lung cancer, triple negative breast cancer, hormone receptor positive breast cancer, head and neck cancer, ovarian cancer, pancreatic cancer, soft tissue sarcoma, and chordoma. In some aspects, the cancer is non-small cell lung cancer. In some aspects, the cancer is triple negative breast cancer. In some aspects, the cancer is hormone receptor positive breast cancer. In some 21

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[0140] aspects, the cancer is hormone receptor positive, human epidermal grow th factor receptor 2 negative, breast cancer. In some aspects, the cancer is head and neck cancer. In some aspects, the cancer is head and neck squamous cell carcinoma. In some aspects, the cancer is ovarian cancer. In some aspects, the cancer is pancreatic cancer. In some aspects, the cancer is pancreatic ductal adeno carcinoma. In some aspects, the cancer is soft tissue sarcoma. In some aspects, the cancer is chordoma.

[0141] In some aspects, the anti-cancer drug is a cytotoxic agent, such as any of the cytotoxic agents described herein. In some aspects, the anti-cancer drug is a microtubule polymerization inhibitor. In some aspects, the anti-cancer drug is a dolastatin, auristatin, or derivative thereof. In some aspects, the anti-cancer drug is an auristatin. In some aspects, the anti-cancer drug is selected from the group consisting of: MMAE, MMAF. DuostatinS, Duostatin5.2, SHR152852, MMAD, LP2, PF-0638010I / AurOlOl, Auristatin F-HPA, Auristatin W analog, F55443, MMAU, ZD02044. In some aspects, the anti-cancer drug is selected from the group consisting of MMAD, MMAE, MMAF, auristatin 1569, auristatin 9411, auristatin 4574 and auristatin 0101. In some aspects, the anti-cancer drug is auristatin 0101. In some aspects, the drug antibody ratio (DAR) is 1, 2, 3, or 4.

[0142] In some aspects, the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises: a. a heavy chain comprising a heavy chain constant region (CH) and heavy chain variable region (VH); b. a light chain comprising a light chain constant region (CL) and light chain variable region (VL); and c. 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 anti-EDB+FN antibody, or antigen binding fragment thereof, comprises at least one cysteine substitution for site specific conjugation in a constant region of the antibody, or antigen binding fragment thereof. In some aspects, a. the CH region comprises 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, 428, 431, 432, 437, 438, 439, 443 or 444, or any combination thereof, according to the numbering of the EU index; or b. the CL region comprises: 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 22

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[0144] 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). In some aspects, a. the CH region comprises an engineered cysteine residue at position 290 according to the numbering of the EU index; or b. the CL region comprises an engineered cysteine residue at position 183 according to the numbering of Kabat. In some aspects, the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises: a. a heavy chain comprising a heavy chain variable region (VH), the 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 b. a light chain comprising a light chain variable region (VL), the VL comprising 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, the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises: a. a heavy chain comprising a heavy chain variable region (VH), the VH comprising the VH amino acid sequence of SEQ ID NO: 21; or b. a light chain comprising a light chain variable region (VL). the VL comprising the VL amino acid sequence of SEQ ID NO: 10. In some aspects, the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises: a. a heavy chain comprising a heavy chain variable region (VH), the VH comprising the VH amino acid sequence of SEQ ID NO: 21; and b. a light chain comprising a light chain variable region (VL), the VL comprising the VL amino acid sequence of SEQ ID NO: 10. In some aspects, the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises: a. a heavy chain comprising the amino acid sequence of SEQ ID NO: 25; or b. a light chain comprising the amino acid sequence of SEQ ID NO: 31. In some aspects, the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises: a. a heavy chain comprising the amino acid sequence of SEQ ID NO: 25; and b. a light chain comprising the amino acid sequence of SEQ ID NO: 31.

[0145] In some aspects, the anti-EDB+FN ADC further comprises a linker, wherein the linker conjugates the anti-cancer drug to the antibody, or antigen binding fragment thereof. In some aspects, the linker is a cleavable linker. In some aspects, the linker is selected from the group consisting of val-cit, phe-lys, vc, AcLys-vc, diS, diS-C2OCO, me, me, MalPeg6C2, and a hydrazone linker. In some aspects, the linker is selected from the group consisting of vc, diS, diS-C2OCO, and AcLys-vc. In some aspects, the linker is vc. In some aspects, the ADC is PYX-201.

[0146] 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 23

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[0148] 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+FN 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+FN 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+FN ADCs described herein.

[0149] 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+FN ADCs described herein for use in a method as described above. In other aspects, the disclosure provides the use of one or more EDB+FN ADCs as described herein or a pharmaceutical composition comprising the EDB+FN ADCs as described herein in the manufacture of a medicament for use in the methods described above.

[0150] Cancers associated with EDB+ FN expression and / or EDB+ FN-expressing cancers may generally include any cancer associated with tissue remodeling. 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, estrogen receptor positive, progesterone receptor positive, human epidermal growth factor receptor 2 negative (ER+PR+HER2-) breast cancer, pancreatic cancer, PDAC, glioblastoma, gallbladder cancer, kidney cancer, skin cancer, uterine cancer, mesothelioma, colorectal cancer, head and neck cancer, HNSCC, 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.

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[0153] The EDB+FN 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+FN 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.

[0154] Thus, patients to be treated with EDB+FN 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 tvpes. and subtypes, that have been demonstrated herein to respond to EDB+FN ADCs.

[0155] 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 grow th 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.

[0156] 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.

[0157] 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 25

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[0159] 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.

[0160] Administration / Dosage / Formulations

[0161] The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations, such as those comprising one or more of the anti-EDB+FN ADCs described herein, may be administered to the subject either prior to, during, or after the onset of a disease or disorder contemplated herein (in a non-limiting aspect, cancer, such as a cancer type described herein). Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0162] Administration of the compositions and / or compounds described herein, such as those comprising one or more anti-EDB+FN ADCs, to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat, prevent and / or ameliorate cancer in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may v ary according to factors such as the state of the disease or disorder in the patient; the age, sex. and weight of the patient; and the ability of the therapeutic compound to treat, prevent and / or ameliorate cancer in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A nonlimiting example of an effective dose range for a therapeutic compound described herein is from about 1 and 5,000 mg / kg of body weight / per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0163] Actual dosage levels of the active ingredients in the pharmaceutical compositions described herein may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0164] In particular, the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of

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[0167] excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex. weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.

[0168] A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds described herein employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0169] In some aspects, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the compound(s) described herein are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic compound.

[0170] In some aspects, the compositions described herein are formulated using one or more pharmaceutically acceptable excipients or carriers. In some aspects, the pharmaceutical compositions described herein comprise a therapeutically effective amount of a compound described herein and a pharmaceutically acceptable carrier.

[0171] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or poly alcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

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[0174] In some aspects, the compositions described herein are administered to the patient in dosages that range from one to five times per day or more. In some aspects, the compositions described herein are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, once every two weeks, once every three weeks, one every four weeks, once every five weeks, or once every six weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions described herein varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, administration of the compounds and compositions described herein should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physician taking all other factors about the patient into account.

[0175] The compound(s) described herein for administration may be in the range of from about 1 pg to about 10,000 mg, about 20 pg to about 9,500 mg, about 40 pg to about 9,000 mg, about 75 pg to about 8,500 mg, about 150 pg to about 7,500 mg, about 200 pg to about 7,000 mg, about 350 pg to about 6,000 mg, about 500 pg to about 5,000 mg, about 750 pg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg. about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.

[0176] In some aspects, the dose of a compound described herein is from about 1 mg and about 2,500 mg. In some aspects, a dose of a compound described herein used in compositions described herein is less than about 10.000 mg, or less than about 8.000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some aspects, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg. or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg. or less than about 10 mg, or less than about 5 mg, or less than about 2 mg. or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

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[0179] In some aspects, a composition as described herein is a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound described herein, alone or in combination with a second pharmaceutical agent: and instructions for using the compound to treat, ameliorate, and / or reduce one or more symptoms of viral infection in a patient.

[0180] Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.

[0181] Routes of administration of any of the compositions described herein include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds for use in the compositions described herein can be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0182] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions described herein are not limited to the particular formulations and compositions that are described herein.

[0183] Dosing

[0184] In some aspects, a compound comprises one or more of the anti-EDB+FN ADCs described herein. The therapeutically effective amount or dose of compounds described herein depends on the age, sex and weight of the patient, the current medical condition of the 29

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[0186] patient and the progression of a viral infection in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.

[0187] A suitable dose of a compound described herein can be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day. The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.

[0188] Another suitable dose of a compound described herein can be in the range of from about 0.01 mg / kg to about 100 mg / kg per day or about 0.1 pg / kg to about 100 pg / kg per day, wherein the amount of the compound administered depends on the weight of the patient. The dose of compounds described herein can be about 0.1 mg / kg to about 100 mg / kg, about 0.5 mg / kg to about 100 mg / kg, about 1 mg / kg to about 50 mg / kg, 0.5 mg / kg to about 25 mg / kg, about 0.3 mg / kg to about 8 mg / kg. about 0.6 mg / kg to about 8 mg / kg, about 1.2 mg / kg to about 8 mg / kg, about 2.4 to about 8 mg / kg, about 3.6 mg / kg to about 8 mg / kg, about 4.4 mg / kg to about 8 mg / kg, about 5.4 mg / kg to about 8 mg / kg, about 6.6 mg / kg to about 8 mg / kg, about 0.3 mg / kg to about 6.6 mg / kg, about 0.6 mg / kg to about 6.6 mg / kg, about 1.2 mg / kg to about 6.6 mg / kg, about 2.4 to about 6.6 mg / kg, about 3.6 mg / kg to about 6.6 mg / kg, about 4.4 mg / kg to about 6.6 mg / kg. about 5.4 mg / kg to about 6.6 mg / kg, about 0.3 mg / kg to about 5.4 mg / kg, about 0.6 mg / kg to about 5.4 mg / kg, about 1.2 mg / kg to about 5.4 mg / kg, about 2.4 to about 5.4 mg / kg, about 3.6 mg / kg to about 5.4 mg / kg, about 4.4 mg / kg to about 5.4 mg / kg, about 0.3 mg / kg to about 4.4 mg / kg, about 0.6 mg / kg to about 4.4 mg / kg, about 1.2 mg / kg to about 4.4 mg / kg, about 2.4 to about 4.4 mg / kg, about 0.3 mg / kg to about 3.6 mg / kg, about 0.6 mg / kg to about 3.6 mg / kg, about 1.2 mg / kg to about 3.6 mg / kg, about 2.4 to about 3.6 mg / kg, about 0.3 mg / kg to about 2.4 mg / kg, about 0.6 mg / kg to about 2.4 mg / kg, about 1.2 mg / kg to about 2.4 mg / kg, about 0.3 mg / kg to about 1.2 mg / kg, about 0.6 mg / kg to about 1.2 mg / kg, or about 0.3 mg / kg to about 0.6 mg / kg. In some aspects, the dose of a compound described herein can be greater than, less than, or at least about 0.01 mg / kg. 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 65 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 85 mg / kg, 90 mg / kg. 95 mg / kg, or about 100 mg / kg. In some aspects, the dose of a

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[0191] compound described herein can be greater than, less than, or at least about 0.3 mg / kg, 0.6 mg / kg, 1.2 mg / kg, 2.4 mg / kg, 3.6 mg / kg, 4.4 mg / kg, 5.4 mg / kg, 6.6 mg / kg, and 8 mg / kg.

[0192] It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, every 5 days, every week, every two weeks, every three weeks, every four weeks, every five weeks, or every’ six weeks. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.

[0193] In the case wherein the patient’s status does improve, upon the doctor’s discretion the administration of the compound(s) described herein is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (z.e., a ‘’drug holiday”). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days. 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The length of the drug holiday can also be measured in weeks, and vary between 1 and 52 weeks, including by way of example only, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 12 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, or 50 weeks. The dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0194] Once improvement of the patient’s conditions has occurred, a maintenance dose is administered if necessary’. Subsequently, the dosage or the frequency of administration, or both, is reduced to a level at which the improved disease is retained. In some aspects, patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and / or infection.

[0195] The compounds described herein can be formulated in unit dosage form. The term “unit dosage form” refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of multiple daily doses (e., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

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[0198] Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LDso and EDso. The data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity7. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.

[0199] Further Dosing and Administration

[0200] The present disclosure provides for EDB+FN 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.

[0201] 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.

[0202] 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.

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[0205] The EDB+FN 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+FN 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, intrasynovial, 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+FN ADC may be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.

[0206] In some aspects of the disclosure, the EDB+FN ADCs are administered via sitespecific or targeted local delivery techniques. Examples of site-specific or targeted local delivery techniques include various implantable depot sources of an EDB+FN ADC or local delivery7catheters, 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.

[0207] For the purpose of the present disclosure, the appropriate dosage of an EDB+FN ADC may depend on the particular EDB+FN ADC (or compositions thereof) employed, the type and severity7of 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+FN 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 33

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[0209] inhibition or delay, etc. Alternatively, sustained continuous release formulations of EDB+FN ADCs may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0210] 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. In some aspects, the typical daily dosage is any of the dosages described herein. For instance, in some aspects, the anti-EDB+FN ADC is administered at a daily dose of about 0.3 mg / kg to about 8 mg / kg, about 0.6 mg / kg to about 8 mg / kg, about 1.2 mg / kg to about 8 mg / kg, about 2.4 to about 8 mg / kg, about 3.6 mg / kg to about 8 mg / kg, about 4.4 mg / kg to about 8 mg / kg, about 5.4 mg / kg to about 8 mg / kg, about 6.6 mg / kg to about 8 mg / kg, about 0.3 mg / kg to about 6.6 mg / kg, about 0.6 mg / kg to about 6.6 mg / kg, about 1.2 mg / kg to about 6.6 mg / kg, about 2.4 to about 6.6 mg / kg, about 3.6 mg / kg to about 6.6 mg / kg, about 4.4 mg / kg to about 6.6 mg / kg, about 5.4 mg / kg to about 6.6 mg / kg. about 0.3 mg / kg to about 5.4 mg / kg, about 0.6 mg / kg to about 5.4 mg / kg, about 1.2 mg / kg to about 5.4 mg / kg, about 2.4 to about 5.4 mg / kg, about 3.6 mg / kg to about 5.4 mg / kg, about 4.4 mg / kg to about 5.4 mg / kg, about 0.3 mg / kg to about 4.4 mg / kg, about 0.6 mg / kg to about 4.4 mg / kg, about 1.2 mg / kg to about 4.4 mg / kg, about 2.4 to about 4.4 mg / kg, about 0.3 mg / kg to about 3.6 mg / kg, about 0.6 mg / kg to about 3.6 mg / kg. about 1.2 mg / kg to about 3.6 mg / kg. about 2.4 to about 3.6 mg / kg, about 0.3 mg / kg to about 2.4 mg / kg, about 0.6 mg / kg to about 2.4 mg / kg, about 1.2 mg / kg to about 2.4 mg / kg, about 0.3 mg / kg to about 1.2 mg / kg, about 0.6 mg / kg to about 1.2 mg / kg, or about 0.3 mg / kg to about 0.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a daily dose of about 0.3 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a daily dose of about 0.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a daily dose of about 1.2 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a daily dose of about 2.4 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a daily dose of about 3.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a daily dose of about 4.4 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a daily dose of about 5.4 mg / kg In some aspects, the anti-EDB+FN ADC is administered at a daily dose of about 6.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a daily dose of about 8 mg / kg.

[0211] In some aspects, the anti-EDB+FN ADC is administered at a daily dose of about 0.3 mg / kg to about 8 mg / kg, 0.6 mg / kg to 6.6 mg / kg, about 1.2 mg / kg to about 6.6 mg / kg, about 34

[0212] 567343972 Attorney Docket No. 384953-1024W01(00350)

[0213] 1.2 mg / kg to about 5.4 mg / kg, about 2.4 to about 6.6 mg / kg, about 2.4 to about 5.4 mg / kg, about 3.6 to about 6.6 mg / kg, about 3.6 mg / kg to about 5.4 mg / kg, about 4.4 mg / kg to about 6.6 mg / kg, about 4.4 mg / kg to about 5.4 mg / kg or about 5.4 mg / kg to 6.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a daily dose of 0.3 mg / kg to 8 mg / kg, 0.6 mg / kg to 6.6 mg / kg, 1.2 mg / kg to 6.6 mg / kg, 2.4 to 6.6 mg / kg, 3.6 mg / kg to 6.6 mg / kg, 4.4 mg / kg to 6.6 mg / kg, 5.4 to 6.6 mg / kg, 3.6 mg / kg to 5.4 mg / kg, or 4.4 mg / kg to 5.4 mg / kg.

[0214] In some aspects, the anti-EDB+FN ADC is administered at a daily dose of about 3.6 mg / kg to about 8 mg / kg, about 3.6 mg / kg to about 6.6 mg / kg, about 3.6 mg / kg to about 5.4 mg / kg, about 3.6 mg / kg to about 4.4 mg / kg, about 4.4 mg / kg to about 8 mg / kg, about 4.4 mg / kg to about 6.6 mg / kg, about 4.4 mg / kg to about 5.4 mg / kg, about 5.4 mg / kg to about 8 mg / kg, about 5.4 mg / kg to about 6.6 mg / kg. about 6.6 mg / kg to about 8 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a daily dose of 3.6 mg / kg to 8 mg / kg, 3.6 mg / kg to 6.6 mg / kg, 3.6 mg / kg to 5.4 mg / kg, 3.6 mg / kg to 4.4 mg / kg, 4.4 mg / kg to 8 mg / kg, 4.4 mg / kg to 6.6 mg / kg, 4.4 mg / kg to 5.4 mg / kg, 5.4 mg / kg to 8 mg / kg, 5.4 mg / kg to 6.6 mg / kg, 6.6 mg / kg to 8 mg / kg.

[0215] In some aspects, the anti-EDB+FN ADC is administered at a daily dose of about 0.3 mg / kg, about 0.6 mg / kg, about 1.2 mg / kg, about 2.4 mg / kg, about 3.6 mg / kg, about 4.4 mg / kg, about 5.4 mg / kg, about 6.6 mg / kg or about 8 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a daily dose of 0.3 mg / kg, 0.6 mg / kg, 1.2 mg / kg, 2.4 mg / kg. 3.6 mg / kg, 4.4 mg / kg, 5.4 mg / kg, 6.6 mg / kg or 8 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a daily dose of 3.6 mg / kg or greater. In some aspects, the anti-EDB+FN ADC is administered at a daily dose of 3.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a daily dose of 4.4 mg / kg or greater. In some aspects, the anti-EDB+FN ADC is administered at a daily dose of 4.4 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a daily dose of 5.4 mg / kg or greater. In some aspects, the anti-EDB+FN ADC is administered at a daily dose of 5.4 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a daily dose of 6.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered at a daily dose of 8 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a daily dose of 3.6 mg / kg to 6.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a daily dose of 3.6 mg / kg to 5.4 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a daily dose of 4.4 mg / kg to 6.6 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a daily dose of 4.4 mg / kg to 5.4 mg / kg. In some aspects, the anti-EDB+FN ADC is administered to the patient at a daily dose of 5.4 mg / kg to 6.6 mg / kg.

[0216] 35

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[0218] 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+FN ADC used) can vary over time.

[0219] In some aspects of the disclosure, dosages for an EDB+FN ADC may be determined empirically in individuals who have been given one or more administration(s) of an EDB+FN ADC. Individuals may be given incremental dosages of an EDB+FN ADC. To assess efficacy, an indicator of the disorder can be followed.

[0220] Administration of an EDB+FN 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+FN ADC may be essentially continuous over a preselected period of time or may be in a series of spaced doses.

[0221] Combination Therapies

[0222] 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.

[0223] The disclosed EDB+FN ADCs may be administered as an initial treatment, or for treatment of cancers that are unresponsive to conventional therapies. In addition, the EDB+FN 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+FN ADCs of the disclosure may be co-administered or co-formulated with additional agents, or formulated for consecutive 36

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[0225] administration with additional agents in any order.

[0226] EDB+FN ADCs of the disclosure may be used in combination with, or following the treatment 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+FN ADC under the subheading ‘“Drugs."

[0227] 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).

[0228] 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, A3, ASN004), anti-CD19 antibodies, anti-CD20 antibodies (e.g., RITUXAN®, ZEVALIN®, BEXXAR®), anti-CD22 antibodies, anti- antibodies (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 (pamtumumab)), 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

[0229] 37

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[0231] antibodies (e.g., GIVAREX®), anti-fibrin antibodies (e.g., MH-1), anti-Nectin-4 antibodies (e.g., enfortumab), and anti-TF (tissue factor) antibodies (e.g. tisotumab).

[0232] 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); br ostatin; callystatin; CC- 1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobim 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 38

[0233] 567343972 Attorney Docket No. 384953-1024W01(00350)

[0234] 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"- tri chlorotriethylamine; 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; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0235] 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 enzyme 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.

[0236] In some aspects, EDB+FN ADCs may be used in combination with, or following the treatment with, crizotinib, palbociclib, gemcitabine, cyclophosphamide, fluorouracil, FOLFOX, folinic acid, oxaliplatin, axitinib, sunitinib malate, tofacitinib, bevacizumab, rituximab, and traztuzumab.

[0237] In some aspects, after treatment with EDB+FN 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 41BB, or any combination thereof, may occur. Thus, the combination of an EDB+FN ADC and an immune checkpoint inhibitor or IO agent, such as an anti-41BB agonist and / or anti-PDLl antagonist monoclonal antibody may be effective. (See Example 12 of WO 2018 / 073680). Further, EDB+FN ADCs 39

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[0239] of the disclosure alone may have immunomodulatory, and immune-oncology (IO) agent enabling mechanisms, that maybe increased with combination therapy.

[0240] In some aspects, an EDB+FN ADC may be used in combination with, or following the treatment 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. In some aspects, an EDB+FN ADC may be used in combination with, or directly proceeding, one or more other ADCs including gemtuzumab ozogamicin (Mylotarg®), brentuximab v edotin (Adcetris®), trastuzumab emtansine (Kadcyla®), inotuzumab ozogamicin (Besponsa®), polatuzumab vedotin-piiq (Polivy®), enfortumab vedotin (Padcev®), sacituzumab govitecan (Trodelvy®), trastuzumab deruxtecan (Enhertu®), loncastuximab tesirine-lpyl (ZYNLONTA®), tisotumab vedotin-tftv (Tivdak®), and mirvetuximab soravtansine (ELAHERE®).

[0241] For combination therapies, an EDB+FN 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).

[0242] 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 40

[0243] 567343972 Attorney Docket No. 384953-1024W01(00350)

[0244] 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.

[0245] \

[0246] The EDB+FN ADC may also be gi oven in combination with any one or more of the additional therapeutic agents disclosed hereiSnS, j such that the EDB+FN ADC is given to

[0247] / \O;s

[0248] resensitize a cancer which has become resistant to a first agent, wherein the EDB+FN ADC f ) p O X

[0249] reestablishes the therapeutic efficacy of the first agent <. \...

[0250] A E.. A

[0251] EDB+FN ADC Structures cn

[0252] Various different EDB+FN ADC structures are presented in the table below.

[0253] Table 1.

[0254] ADC# ADC Structure Method EDB- ADC1 L19-vc- I H J 1 H O A 0101

[0255] EDB- Hi" —

[0256] ADC2 (KK183C- oHO VH\_ / B K290C)- vc-0101

[0257] EDB- ADC3 (K94R)- A vc-0101

[0258] EDB- (KK183C- ADC4 K94R- OHO IH\= / B K290C)- vc-0101

[0259] EDB- ADC5 L19-diS- D

[0260] DM1

[0261]

[0262] 41

[0263] 567343972 Attorney Docket No. 384953-1024W01(00350)

[0264] EDB- ADC6 L19-diS- D C2OCO- 1569

[0265] EDB- ADC7 L19-vc- A 9411

[0266] ADC8 EDB-diS- L 19-4574 D \ d

[0267] J EDB- U

[0268] (H16- /

[0269] A K222R)- \ J.

[0270] DC9 AcLys- C Y « rr

[0271] vc-CPI- 8314

[0272] HNv

[0273] Y° OAH, p

[0274] ^

[0275] EDB- tr>

[0276] ADC 10 1 \ / H0\

[0277] L 9-vc- (n A 1569 '■?v. dddd XT A-O

[0278] r

[0279]

[0280] Antibodies and Antibody-Drug Conjugates

[0281] 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.

[0282] 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)

[0283] 42

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[0285] 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)p, 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.

[0286] 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.

[0287] 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.

[0288] 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 variety7of 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

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[0291] impediment of the epithelial to mesenchymal transition.

[0292] Extra-domain B of Fibronectin

[0293] 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 antigen-binding 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.

[0294] 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.

[0295]

[0296] Table 2: EDB AND 7-EDB-8-9 SEQUENCES

[0297] SEQ ID Description Sequence

[0298] NO.

[0299] EDB EVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAAGEGIPIFE

[0300] 33 DFVDSSVGYYTVTGLEPGIDYDISVITLINGGESAPTTLTQQT Human FN- VVTOLSPPTNLHLEANPDTGVLAVSWERSTTPDITGYRITTTPTN 34 7-EDB-89- GOOGNSLEEVVHADOSSCTFDNLSPGLEYNVSVYTVKDDKESV

[0301]

[0302] HIS protein PISDTIIPEVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAA

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[0305] GEGIPIFEDFVDSSVGYYTVTGLEPGIDYDISVITLINGGESAPTTL TQQT|AVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVK NEEDY AELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLR GRQKT GLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSG RPRED1 YFPHSRNSITLTNLTPGTEYWSIVALNGREESPLLIGRSRSYWA I II II 111

[0306] Cynomolgu VVTPLSPPTNLHLETNPDTGVLTVSWERSTTPDITGYRITTTPTNG s FN-7- OOGYSLEEVVHADOSSCTFDNLSPGLEYNVSVYTVKDDKESVPI EDB-89- SDTIIPEVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAAGE HIS protein GIPIFEDFVDSSVGYYTVTGLEPGIDYDISVITLINGGESAPTTLTQ 35 QljAVPPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNE EDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGR

[0307] Q^GLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHMSGR PREDRVPPSRNSITLTNLTPGTEYWSIVALNGREESPLLIGRSRSiSYiA HHH

[0308] Rat FN-7- VVTPLSPPTNLHLEANPDTGVLTVSWERSTTPDITGYRITTTPTN EDB-89- GOOGTALEEVVHADOSSCTFENLNPGLEYNVSVYTVKDDKESA HIS protein PISDTVIPEVPOLTDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAA GEGIPIFEDFVDSSVGYYTVTGLEPGIDYDISVITLINGGESAPTTL

[0309] 36 TQQT|AVPPPTDLRFTNIGPDTMRVTWAPPPSIELTNLLVRYSPVK|

[0310] NEEDY AELSISPSDNAVVLTNLLPGTEYLVSVSSVYEQHESIPLRI GRQKT GLDSPTGFDSSDVTANSFTVHWVAPRAPITGYIIRHHAEHSA GRPRQi DRVPPSRNSITLTNLNPGTEYIVTIIAVNGREESPPLIGRSRSiA

[0311]

[0312] HHHHH

[0313] Anti-EDB Antibodies

[0314] 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+FN ADC.

[0315] 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

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[0318] 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, triabodies, 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+FN ADCs is a chimeric, humanized, or a recombinant human antibody, or EDB-binding fragment thereof.

[0319] 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.

[0320] 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.

[0321] 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.

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[0324] Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains.

[0325] 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.

[0326] 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.

[0327] 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.

[0328] 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).

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[0331] 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 know n 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.

[0332] A CDR of a variable domain may Be identified in accordance with 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).

[0333] 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.

[0334] In some aspects of the disclosure, an EDB+FN ADC includes an antibody that competes for binding to human EDB with, and / or binds the same epitope as, an antibody, or antigen-binding fragment thereof, described herein.

[0335] 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 delectably decreased in 48

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[0337] 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.

[0338] 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).

[0339] In some aspects of the disclosure, antibodies used to prepare EDB+FN 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.

[0340] 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 49

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[0342] 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.

[0343] Antibodies of the disclosure can be produced using techniques well known 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 Biology': A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory^ Press, Cold Spring Harbor, N. Y. (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003).

[0344] 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.

[0345] 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.

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[0348] The numbering system used for the light chain constant region amino acid sequence is that set forth in Kabat 1991.

[0349] The EDB+FN 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.

[0350] The present disclosure provides for optimization of the L19-EDB antibody by generation of anon-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 anon-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).

[0351] 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).

[0352] For site-specific conjugation via engineered cysteines, one or more amino acid residues of an anti-EDB antibody heavy chain may be substituted for a cysteine residue at position: 118 (114 according to 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, 428, 431, 432, 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.

[0353] For site-specific conjugation via engineered cysteines, one or more amino acid residues of an anti-EDB antibody light chain may be substituted for a cysteine residue (i) 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) at a position corresponding to residue 4, 42, 81, 100, 103, or any combination thereof, of SEQ ID NO: 37,

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[0356] when the constant domain is aligned with SEQ ID NO: 37 (kappa light chain); or (iii) 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.

[0357] 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 (KK183C), according to the numbering of Kabat. Additional cysteine substitutions may be introduced.

[0358] For site-specific conjugation via engineered glutamine residues, the anti-EDB antibody heavy chain constant domain may comprise an engineered H16-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).

[0359] 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).

[0360] In some aspects of the disclosure, the EDB+FN 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.

[0361] For use in preparation of EDB+FN 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,

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[0364] and most preferably, at least 99% pure.

[0365] 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 underlined bold 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 technology.

[0366] Table 3: ANTI-EDB ANTIBODY SEQUENCES SEQ ID Description Sequence

[0367] NO.

[0368] 1 EDB-L19 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAP Protein GKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSS

[0369] 2 EDB-L19 VH GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAG DNA CCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA TTCACCTTTAGCAGTTTTTCGATGAGCTGGGTCCGCCAGG CTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAGTG GTAGTTCGGGTACCACATACTACGCAGACTCCGTGAAGG GCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGC TGTATCTGCAAATGAACAGCCTGAGAGCCGAAGACACG GCCGTATATTACTGTGCGAAACCGTTTCCGTATTTTGACT ACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT

[0370] 3 EDB L19 VH SFSMS

[0371] CDR1

[0372] Kabat

[0373] 4 EDB-L19 VH GFTFSSF

[0374] CDR1

[0375] Chothia

[0376] 5 EDB-L19 VH SISGSSGTTYYADSVKG

[0377] CDR2

[0378] Kabat

[0379] 6 EDB-L19 VH SGSSGT

[0380] CDR2

[0381] Chothia

[0382] 7 EDB-L19 VH PFPYFDY

[0383] CDR3

[0384] Kabat / Chothia

[0385] 8 EDB-L19 HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAP Human IgGl GKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQ Protein MNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

[0386]

[0387] KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 53

[0388] 567343972 Attorney Docket No. 384953-1024W01(00350)

[0389] 9 EDB-L19 HC GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAG DNA CCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA TTCACCTTTAGCAGTTTTTCGATGAGCTGGGTCCGCCAGG CTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAGTG GTAGTTCGGGTACCACATACTACGCAGACTCCGTGAAGG GCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGC TGTATCTGCAAATGAACAGCCTGAGAGCCGAAGACACG GCCGTATATTACTGTGCGAAACCGTTTCCGTATTTTGACT ACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTGCGT CGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCT CCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCA GCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGA CCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCA AGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAA ACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTG GGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAA GTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGC CAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGT ACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT GCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCA GCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACA TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGC TCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGC AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAA

[0390] 10 EDB-L19 VL EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPG Protein QAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA VYYCQQTGRIPPTFGQGTKVEIK

[0391] 11 EDB-L19 VL GAAATTGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGT DNA CTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTC AGAGTGTTAGCAGCAGCTTTTTAGCCTGGTACCAGCAGA AACCTGGCCAGGCTCCCAGGCTCCTCATCTATTATGCAT CCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCA GTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGAC TGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGA CGGGTCGTATTCCGCCGACGTTCGGCCAAGGGACCAAGG TGGAAATCAAA

[0392] 12 EDB- L19 VL RASQSVSSSFLA

[0393] CDR1

[0394] Kabat / Chothia

[0395] 13 EDB-L19 VL YASSRAT

[0396] CDR2

[0397] Kabat / Chothia

[0398]

[0399] 14 EDB-L19 VL QQTGRIPPT

[0400] 54

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[0402] CDR3

[0403] Kabat / Chothia

[0404] 15 EDB-L19 LC EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPG Human Kappa QAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA Protein VYYCQQTGRIPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC

[0405] 16 EDB-L19 LC GAAATTGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGT DNA CTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTC AGAGTGTTAGCAGCAGCTTTTTAGCCTGGTACCAGCAGA AACCTGGCCAGGCTCCCAGGCTCCTCATCTATTATGCAT CCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCA GTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGAC TGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGA CGGGTCGTATTCCGCCGACGTTCGGCCAAGGGACCAAGG TGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCA TCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTG CCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGA GGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAAT CGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGC AAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTG AGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTG CGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAA GAGCTTCAACAGGGGAGAGTGT

[0406] 17 EDB-PFE HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAP Protein GKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0407] 18 EDB-PFE HC GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAG DNA CCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA TTCACCTTTAGCAGTTTTTCGATGAGCTGGGTCCGCCAGG CTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAGTG GTAGTTCGGGTACCACATACTACGCAGACTCCGTGAAGG GCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGC TGTATCTGCAAATGAACAGCCTGAGAGCCGAAGACACG GCCGTATATTACTGTGCGAAACCGTTTCCGTATTTTGACT ACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTGCGT CGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCT CCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCA GCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGA CCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCA AGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAA

[0408]

[0409] ACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTG

[0410] 55

[0411] 567343972 Attorney Docket No. 384953-1024W01(00350)

[0412] GGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAA GTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGC CAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGT ACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT GCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCA GCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACA TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGC TCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGC AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCCCCGGGT

[0413] 19 EDB-(K290C) HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAP Protein GKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTCPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0414] 20 EDB-(K290C) HC GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAG DNA CCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA TTCACCTTTAGCAGTTTTTCGATGAGCTGGGTCCGCCAGG CTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAGTG GTAGTTCGGGTACCACATACTACGCAGACTCCGTGAAGG GCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGC TGTATCTGCAAATGAACAGCCTGAGAGCCGAAGACACG GCCGTATATTACTGTGCGAAACCGTTTCCGTATTTTGACT ACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTGCGT CGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCT CCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCA GCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGA CCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCA AGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAA ACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTG GGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAA GTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGC CAAGACATGCCCGCGGGAGGAGCAGTACAACAGCACGT ACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA

[0415]

[0416] GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT 56

[0417] 567343972 Attorney Docket No. 384953-1024W01(00350)

[0418] GCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCA GCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACA TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGC TCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGC AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCCCCGGGT

[0419] 21 EDB-(K94R) VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAP Protein GKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCARPFPYFDYWGQGTLVTVSS

[0420] 22 EDB-(K94R) VH GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAG DNA CCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA TTCACCTTTAGCAGTTTTTCGATGAGCTGGGTCCGCCAGG CTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAGTG GTAGTTCGGGTACCACATACTACGCAGACTCCGTGAAGG GCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGC TGTATCTGCAAATGAACAGCCTGAGAGCCGAAGACACG GCCGTATATTACTGTGCGAGACCGTTTCCGTATTTTGACT ACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT

[0421] 23 EDB-(K94R) HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAP Protein GKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCARPFPYFDYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0422] 24 EDB-(K94R) HC GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAG DNA CCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA TTCACCTTTAGCAGTTTTTCGATGAGCTGGGTCCGCCAGG CTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAGTG GTAGTTCGGGTACCACATACTACGCAGACTCCGTGAAGG GCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGC TGTATCTGCAAATGAACAGCCTGAGAGCCGAAGACACG GCCGTATATTACTGTGCGAGACCGTTTCCGTATTTTGACT ACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTGCGT CGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCT CCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCA GCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGA CCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCA AGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAA ACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTG GGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAA GTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGC

[0423]

[0424] CAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGT

[0425] 57

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[0427] ACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT GCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCA GCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACA TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGC TCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGC AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCCCCGGGT

[0428] 25 EDB-(K94R- EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAP K290C) HC GKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQ Protein MNSLRAEDTAVYYCARPFPYFDYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTCPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0429] 26 EDB-(K94R- GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAG K290C) HC CCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA DNA TTCACCTTTAGCAGTTTTTCGATGAGCTGGGTCCGCCAGG CTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAGTG GTAGTTCGGGTACCACATACTACGCAGACTCCGTGAAGG GCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGC TGTATCTGCAAATGAACAGCCTGAGAGCCGAAGACACG GCCGTATATTACTGTGCGAGACCGTTTCCGTATTTTGACT ACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTGCGT CGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCT CCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCA GCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGA CCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCA AGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAA ACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTG GGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAA GTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGC CAAGACATGCCCGCGGGAGGAGCAGTACAACAGCACGT ACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT GCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCA GCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACA TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGC

[0430]

[0431] TCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGC 58

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[0433] AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCCCCCGGA

[0434] 27 EDB-(H16- EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAP K222R) HC GKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQ Protein MNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDRTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPRELXQGSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0435] 28 EDB-(H16- GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAG K222R) HC CCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA DNA TTCACCTTTAGCAGTTTTTCGATGAGCTGGGTCCGCCAGG CTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAGTG GTAGTTCGGGTACCACATACTACGCAGACTCCGTGAAGG GCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGC TGTATCTGCAAATGAACAGCCTGAGAGCCGAAGACACG GCCGTATATTACTGTGCGAAACCGTTTCCGTATTTTGACT ACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTGCGT CGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCT CCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCA GCGTAGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGA CCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCA AGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACCGC ACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTG GGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAA GTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGC CAAGACAAAGCCGCGGGAGCTGCTGCAGGGGAGCACGT ACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT GCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCA GCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACA TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGC TCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGC AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGT

[0436] 29 EDB-(K94R-H16- EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAP K222R) HC GKGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYLQ Protein MNSLRAEDTAVYYCARPFPYFDYWGQGTLVTVSSASTKGP

[0437] SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT

[0438]

[0439] SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP

[0440] 59

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[0442] SNTKVDKKVEPKSCDRTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREIAQGSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0443] 30 EDB-(K94R-H16- GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAG K222R) HC CCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGA DNA TTCACCTTTAGCAGTTTTTCGATGAGCTGGGTCCGCCAGG CTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAGTG GTAGTTCGGGTACCACATACTACGCAGACTCCGTGAAGG GCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGC TGTATCTGCAAATGAACAGCCTGAGAGCCGAAGACACG GCCGTATATTACTGTGCGAGACCGTTTCCGTATTTTGACT ACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGTGCGT CGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCT CCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCA GCGTAGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGA CCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCA AGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACCGC ACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTG GGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAA GTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGC CAAGACAAAGCCGCGGGAGCTGCTGCAGGGGAGCACGT ACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT GCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCA GCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACA TCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGC TCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGC AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG CATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCCCCCGGA

[0444] 31 EDB-(KK183C) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPG LC QAPRLLIYYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA

[0445] Protein VYYCQQTGRIPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSCADYEKHKVYACEVTHQGLSSPVT KSFNRGEC

[0446] 32 EDB-(KK183C) GAAATTGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGT LC CTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTC DNA AGAGTGTTAGCAGCAGCTTTTTAGCCTGGTACCAGCAGA AACCTGGCCAGGCTCCCAGGCTCCTCATCTATTATGCAT CCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCA GTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGAC

[0447]

[0448] TGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGA

[0449] 60

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[0451] CGGGTCGTATTCCGCCGACGTTCGGCCAAGGGACCAAGG TGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCA TCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTG CCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGA GGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAAT CGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGC AAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTG AGCTGCGCAGACTACGAGAAACACAAAGTCTACGCCTG CGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAA

[0452]

[0453] GAGCTTCAACAGGGGAGAGTGT

[0454] In some aspects of the disclosure, an EDB+FN ADC includes an antibody, or antigen binding fragment thereof, that binds to extra domain B (EDB) of fibronectin (FN).

[0455] 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.

[0456] 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.

[0457] 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:

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[0460] 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.

[0461] 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.

[0462] In some aspects of the disclosure, an antibody, or antigen-binding fragment thereof, may have 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.

[0463] 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.

[0464] 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 62

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[0466] 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.

[0467] 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 alight chain comprising SEQ ID NO: 15; a heavy chain comprising SEQ ID NO: 17 and alight 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.

[0468] Representative DNAs encoding anti-EDB antibody heavy chain and light chain 63

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[0470] variable regions comprise SEQ ID NOs: 2 and 22 and SEQ ID NO: 11, respectively.

[0471] 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.

[0472] Table 4: SEQ ID NOS FOR VARIOUS ANTI-EDB ANTIBODIES (CDRs in Kabat and (Chothia))

[0473] VH VH VH VH VL VL VL CDR1 CDR2 CDR3 HC VL CDR1 CDR2 CDR3 LC EDB-L19 1 3 (4) 5 (6) 7 8 10 12 13 14 15 EDB-PFE 1 3 (4) 5 (6) 7 17 10 12 13 14 15 EDB- (KK183C- 1 3 (4) 5 (6) 7 19 10 12 13 14 31 K290C)

[0474] EDB-(K94R) 21 3 (4) 5 (6) 7 23 10 12 13 14 15 EDB- (KK183C- 21 7 25 10 12 13 14 31 K94R- 3 (4) 5 (6)

[0475] K290C)

[0476] EDB-(H16- 1 3 (4) 5 (6) 7 27 10 12 13 14 15 K222R)

[0477] EDB-(K94R- 21 7 29 10 12 13 14 15

[0478]

[0479] H16-K222R) 3 (4) 5 (6)

[0480] Drugs

[0481] Drugs useful in preparation of the disclosed EDB+FN 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.

[0482] 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 grow th 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

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[0485] 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.

[0486] 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.

[0487] In accordance with the disclosed methods, the EDB+FN 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.

[0488] In aspects of the disclosure, an EDB+FN 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+FN 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.

[0489] In aspects of the disclosure, an EDB+FN 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+FN 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 65

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[0491] 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%.

[0492] A composition, batch, and / or formulation of EDB+FN 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+FN 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.

[0493] In some aspects of the disclosure, a composition, batch, and / or formulation of EDB+FN 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+FN 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+FN 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+FN 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.

[0494] In some aspects, a composition, batch, and / or formulation of EDB+FN 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.

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[0497] In other aspects, a composition, batch, and / or formulation of EDB+FN 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.

[0498] 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+FN ADCs with an optimized average DAR in the range of about 3 to 5, preferably about 4.

[0499] 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+FN ADCs with an optimized average DAR in the range of about 3 to 5, preferably about 4.

[0500] 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+FN ADCs with an optimized average DAR in the range of about 1 to 3, preferably about 2.

[0501] 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, apuromycin, ataxane, 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.

[0502] 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 anthracyclines include, but are not limited to, daunorubicin, doxorubicin 67

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[0504] (i.e., adriamycin), epirubicin, idarubicin, valrubicin, and mitoxantrone.

[0505] 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 orN-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine), MMAE (Monomethyl Auristatin E or N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine), and 5-benzoylvaleric acid-AE ester (AEVB).

[0506] 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+FN ADCs of the present disclosure and provides methods of producing the auristatins. For example, payload 0101 having the structure:

[0507]

[0508] payload 1569 having the structure:

[0509]

[0510] payload 9411 having the structure:

[0511]

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[0514] payload 4574 having the structure:

[0515]

[0516] and

[0517] payload Cemadotin having the structure:

[0518]

[0519] 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.

[0520] 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.

[0521] 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.

[0522] Exemplary dolastatins include, but are not limited to, (+)-docarmycin A and (+)-duocarmycin SA, and (+)-CC-1065.

[0523] 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.

[0524] WO2015 / 110935, which is incorporated herein by reference in its entirety, discloses CPI and CBI dimers that are useful in the EDB+FN ADCs of the present disclosure and provides methods of producing the CPI and CBI dimers. For example, payload CPI-8314 dimer having the structure:

[0525]

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[0528] payload CPI-0326 having the structure:

[0529]

[0530] 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):l 198-1201, 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.

[0531] Sep;l(l):57-66, 1994; Prokop et al. Oncogene 22:9107-9120. 2003). The disulfide analog is N-acetyl- y -calicheamicin dimethyl hydrazide.

[0532] Hemiasterlin and its analogues (e.g., HTI-286) bind to the tubulin, disrupt normal microtubule dynamics, and, at stoichiometric amounts, depolymerize microtubules.

[0533] 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).

[0534] 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:

[0535]

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[0538] and its derivatives as well as ansamitocin.

[0539] 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-purine sequences. Exemplary PBDs and their analogs include, but are not limited to, SJG-136.

[0540] 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.

[0541] 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®).

[0542] Vinca alkyloids are also anti-tubulin agents. Exemplary vinca alkyloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine.

[0543] In some aspects of the disclosure, the pay load is an immunomodulating agent.

[0544] 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 grow th 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, - and -y), the stem cell growth factor designated “SI factor,’’ erythropoietin and thrombopoietin, or a combination thereof.

[0545] 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 americana proteins (PAPI, PAPII, 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).

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[0548] 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 117018, onapnstone, and toremifene; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide. and goserelm; and anti-adrenal agents. Representative immunosuppressive agents include 2-amino-6-ary 1-5 -substituted pyrimidines, azathioprine, cyclophosphamide, bromocryptine, danazol, dapsone, glutaraldehyde, anti-idioty pic 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, TGF|3, rapamycin, T-cell receptor, T-cell receptor fragments, and T cell receptor antibodies.

[0549] 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 growth factor.

[0550] 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).

[0551] Examples of hormones include, but are not limited to, estrogens, androgens, progestins and corticosteroids.

[0552] In some aspects of the disclosure, the drug is an oligonucleotide, such as anti-sense oligonucleotides.

[0553] 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-methoxy oestradiol bissulphamate (2-MeOE2bisMATE)), interleukin-24, thrombospondin, metallospondin proteins, class I interferons, interleukin 12, protamine, angiostatin, laminin, endostatin, and prolactin fragments.

[0554] Anti-proliferative agents and pro-apoptotic agents include activators of PPAR-gamma 72

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[0556] (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 (FEM ARA " (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).

[0557] 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, 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, 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;

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[0560] 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; difluoromethylornithine (DMFO); retinoic acid; esperamicins; and capecitabine.

[0561] 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.

[0562] 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, 0-lactam-containing prodrugs, substituted phenoxy acetamide-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.

[0563] 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.

[0564] 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.

[0565] Thus, in some aspects of the disclosure, the drug is an imaging agent (e.g., a

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[0568] fluorophore or a PET (Positron Emission Tomography) label, SPECT (Single-Photon Emission Computed Tomorgraphy) label), or MRI (Magnetic Resonance Imaging) label.

[0569] 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.

[0570] 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).

[0571] 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.

[0572] 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,76Br,77Br,86Y,89Zr,90Y.94Tc,95Ru,97Ru. " Tc,103RU,105Rh,105Ru,107Hg,109Pd.mAg,inIn, 113jn121ye122ye123j 124j 125j 125-pe126j 13 Ij 131jn133j 142pf143pf153p^ 153gm161rpfo

[0573]

[0574] 165Tm,166Dy,166H,167Tm,168Tm,169Yb,177Lu,186Re,188Re,189Re,197Pt,198Au,199Au,2O1T1,203Hg,211At,212Bi,212Pb,213Bi,223Ra,224Ac, and225Ac.

[0575] Linkers

[0576] EDB+FN ADCs of the present disclosure may be prepared using a linker to directly 75

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[0578] 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.

[0579] 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-citrulhne-p-aminobenzyloxy carbonyl (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- (AcLy s-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-yldisulfanyl)ethyl 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.

[0580] 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 enzvme that creates a new amide linkage from a primary amine present on the linker with a side chain amide of a glutamine residue.

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[0583] In some aspects, linkers of the present disclosure include:

[0584] “mc-vc-PABC’?or “vc-PABC’?orL‘vc’?linker having the structure:

[0585] “AcLys-vc-PABC-DMAE-CO” or “AcLys-vc” linker having the structure:

[0586]

[0587] diS linker having the structure:

[0588] diS-C2OCO linker having the structure:

[0589]

[0590] Methods of Preparing ADCs

[0591] Provided herein are methods for preparing EDB+FN ADCs of the present disclosure. The present disclosure further provides for a process for producing or generating conventionally and site-specific conjugated EDB+FN 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.

[0592] In some aspects, EDB+FN 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.

[0593] In some aspects, EDB+FN ADCs may be generated using site-specific conjugation of

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[0596] 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. W02013 / 093809, which is incorporated herein by reference in its entirety.

[0597] 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, 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, 428, 431, 432, 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.

[0598] 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.

[0599] 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.

[0600] In some aspects, the disclosure provides an anti-EDB antibody or antigen binding 78

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[0602] fragment thereof comprising an antibody kappa light chain constant region comprising (i) an engineered cysteine residue at position 111, 149, 188. 207, 210, or any combination thereof (preferably 111 or 210), according to 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.

[0603] 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.

[0604] 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.

[0605] SEQ ID NO: 37 (CK constant domain)

[0606] RTVAAPSVFI FPPSDEQLKS GTASWCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC SEQ ID NO 38 (CX constant domain)

[0607] GQPKANPTVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADGSPVK AGVETTKPSK QSNNKYAASS YLSLTPEQWK SHRSYSCQVT HEGSTVEKTV APTECS

[0608] In some aspects, EDB+FN 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.

[0609] 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.,

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[0612] 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.

[0613] 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 of WO 2018 / 073680.

[0614] 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. Toy opearl Ether-650M chromatographic medium, Macro-Prep methyl HIC medium or Macro-Prep t-Butyl HIC medium.

[0615] Table 1 provides EDB+FN ADCs produced according to the conjugation and purification methods described herein and used to generate data provided in the Examples.

[0616] In some aspects of the disclosure, EDB+FN ADCs of the present disclosure comprise 80

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[0618] (a) an antibody, or antigen binding fragment thereof, that binds to EDB; (b) a linker and (c) a drug.

[0619] In some aspects of the disclosure, EDB+FN 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.

[0620] In some aspects of the disclosure, EDB+FN 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.

[0621] In some aspects of the disclosure, EDB+FN 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 ammo acid sequence of SEQ ID NO: 10; (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-C2OCO 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.

[0622] In some aspects of the disclosure, EDB+FN ADCs of the present disclosure comprise (a) an antibody, or antigen binding fragment thereof, comprising: a heavy chain comprising SEQ ID NO: 8 and alight 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 alight chain comprising SEQ ID NO: 31; aheavy 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; aheavy' chain comprising SEQ ID NO: 29 and alight chain 81

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[0624] 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-C2OCO 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.

[0625] Formulations

[0626] The present disclosure further provides pharmaceutical compositions including any of the EDB+FN ADCs disclosed herein and a pharmaceutically acceptable carrier. Further, the compositions may include more than one EDB+FN ADC disclosed herein.

[0627] 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, butyl 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.

[0628] Various formulations of the EDB+FN 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 82

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[0630] 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.

[0631] 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.

[0632] Therapeutic formulations of EDB+FN ADCs used in accordance with the present disclosure may be prepared for storage by mixing an antibody having the desired degree of purify 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 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 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).

[0633] Therapeutic EDB+FN 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 83

[0634] 567343972 Attorney Docket No. 384953-1024W01(00350)

[0635] administration by inhalation or insufflation.

[0636] 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.

[0637] 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.

[0638] 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+FN ADC with INTRALIPID™ or the components thereof (soybean oil, egg phospholipids, glycerol and water).

[0639] 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+FN ADC as described herein and instructions for use in accordance with any of the methods of the disclosure described herein. Generally, these instructions include a description of administration of the EDB antibody or EDB+FN ADC for the above described diagnostic or therapeutic treatments.

[0640] The instructions relating to the use of an EBD antibody or an EDB+FN 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.

[0641] The kits of this disclosure are in suitable packaging. Suitable packaging includes, but 84

[0642] 567343972 Attorney Docket No. 384953-1024W01(00350)

[0643] 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+FN ADC. The container may further include a second pharmaceutically active agent.

[0644] 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.

[0645] Table 5: SEQUENCE TABLE

[0646] SEQ ID SEQUENCE NO

[0647] 1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVS SISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKP FPYFDYWGQGTLVTVSS

[0648] 2 gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctg gattcacctttagcagtttttcgatgagctgggtccgccaggctccagggaaggggctggagtgggtctcatctattag tggtagttcgggtaccacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaaca cgctgtatctgcaaatgaacagcctgagagccgaagacacggccgtatattactgtgcgaaaccgtttccgtattttga ctactggggccagggaaccctggtcaccgtctcgagt

[0649] 3 SFSMS

[0650] 4 GFTFSSF

[0651] 5 SISGSSGTTYYADSVK

[0652] 6 SGSSGT

[0653] 7 PFPYFDY

[0654] 8 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVS SISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK PFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLM1SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK

[0655] 9 gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctct ggattcacctttagcagtttttcgatgagctgggtccgccaggctccagggaaggggctggagtgggtctcatctatt agtggtagttcgggtaccacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga

[0656]

[0657] acacgctgtatctgcaaatgaacagcctgagagccgaagacacggccgtatattactgtgcgaaaccgtttccgtat 85

[0658] 567343972 Attorney Docket No. 384953-1024W01(00350)

[0659] tttgactactggggccagggaaccctggtcaccgtctcgagtgcgtcgaccaagggcccatcggtcttccccctgg caccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccg gtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcagga ctctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatc acaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgt gcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctc ccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgt ggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtc agcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctc ccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccc catcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcg ccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacg gctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgt gatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa 10 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYY ASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQG TKVEIK

[0660] 11 gaaattgtgttaacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggcca gtcagagtgttagcagcagctttttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctattatg catccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatca gcagactggagcctgaagattttgcagtgtattactgtcagcagacgggtcgtattccgccgacgttcggccaagg gaccaaggtggaaatcaaa

[0661] 12 RASQSVSSSFLA

[0662] 13 YASSRAT

[0663] 14 QQTGRIPPT

[0664] 15 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYY ASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQG TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC

[0665] 16 gaaattgtgttaacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggcca gtcagagtgttagcagcagctttttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctattatg catccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatca gcagactggagcctgaagattttgcagtgtattactgtcagcagacgggtcgtattccgccgacgttcggccaagg gaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaat ctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataac gccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagca gcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctg agctcgcccgtcacaaagagcttcaacaggggagagtgt

[0666] 17 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVS SISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK PFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG

[0667]

[0668] 18 gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctct 86

[0669] 567343972 Attorney Docket No. 384953-1024W01(00350)

[0670] ggattcacctttagcagtttttcgatgagctgggtccgccaggctccagggaaggggctggagtgggtctcatctatt agtggtagttcgggtaccacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaagacacggccgtatattactgtgcgaaaccgtttccgtat tttgactactggggccagggaaccctggtcaccgtctcgagtgcgtcgaccaagggcccatcggtcttccccctgg caccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccg gtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcagga ctctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatc acaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgt gcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctc ccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgt ggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtc agcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctc ccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccc catcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcg ccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacg gctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtg atgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggt

[0671] 19 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVS SISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK PFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTCPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG

[0672] 20 gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctct ggattcacctttagcagtttttcgatgagctgggtccgccaggctccagggaaggggctggagtgggtctcatctatt agtggtagttcgggtaccacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaagacacggccgtatattactgtgcgaaaccgtttccgtat tttgactactggggccagggaaccctggtcaccgtctcgagtgcgtcgaccaagggcccatcggtcttccccctgg caccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccg gtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcagga ctctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatc acaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgt gcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctc ccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgt ggacggcgtggaggtgcataatgccaagacatgcccgcgggaggagcagtacaacagcacgtaccgtgtggtc agcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctc ccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccc catcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcg ccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacg gctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtg atgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggt

[0673] 21 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVS SISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR PFPYFDYWGQGTLVTVS S

[0674] 22 gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctct

[0675]

[0676] ggattcacctttagcagtttttcgatgagctgggtccgccaggctccagggaaggggctggagtgggtctcatctatt 87

[0677] 567343972 Attorney Docket No. 384953-1024W01(00350)

[0678] agtggtagttcgggtaccacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaagacacggccgtatattactgtgcgagaccgtttccgtat tttgactactggggccagggaaccctggtcaccgtctcgagt

[0679] 23 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVS SISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR PFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG

[0680] 24 gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctct ggattcacctttagcagtttttcgatgagctgggtccgccaggctccagggaaggggctggagtgggtctcatctatt agtggtagttcgggtaccacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaagacacggccgtatattactgtgcgagaccgtttccgtat tttgactactggggccagggaaccctggtcaccgtctcgagtgcgtcgaccaagggcccatcggtcttccccctgg caccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccg gtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcagga ctctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatc acaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgt gcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctc ccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgt ggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtc agcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctc ccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccc catcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcg ccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacg gctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtg atgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggt

[0681] 25 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVS SISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR PFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTCPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG

[0682] 26 gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctct ggattcacctttagcagtttttcgatgagctgggtccgccaggctccagggaaggggctggagtgggtctcatctatt agtggtagttcgggtaccacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaagacacggccgtatattactgtgcgagaccgtttccgtat tttgactactggggccagggaaccctggtcaccgtctcgagtgcgtcgaccaagggcccatcggtcttccccctgg caccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccg gtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcagga ctctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatc

[0683]

[0684] acaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgt 88

[0685] 567343972 Attorney Docket No. 384953-1024W01(00350)

[0686] gcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctc ccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgt ggacggcgtggaggtgcataatgccaagacatgcccgcgggaggagcagtacaacagcacgtaccgtgtggtc agcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctc ccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccc catcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcg ccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacg gctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtg atgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtcccccgga

[0687] 27 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVS SISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK PFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDRTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRELLQ GSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG

[0688] 28 gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctct ggattcacctttagcagtttttcgatgagctgggtccgccaggctccagggaaggggctggagtgggtctcatctatt agtggtagttcgggtaccacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga acacgctgtatctgcaaatgaacagcctgagagccgaagacacggccgtatattactgtgcgaaaccgtttccgtat tttgactactggggccagggaaccctggtcaccgtctcgagtgcgtcgaccaagggcccatcggtcttccccctgg caccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccg gtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcagga ctctactccctcagcagcgtagtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatc acaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgaccgcactcacacatgcccaccgt gcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctc ccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgt ggacggcgtggaggtgcataatgccaagacaaagccgcgggagctgctgcaggggagcacgtaccgtgtggtc agcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctc ccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccc catcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcg ccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacg gctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtg atgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggt

[0689] 29 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVS SISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR PFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDRTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRELLQ GSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG

[0690] 30 gaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctct ggattcacctttagcagtttttcgatgagctgggtccgccaggctccagggaaggggctggagtgggtctcatctatt

[0691]

[0692] agtggtagttcgggtaccacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaaga 89

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[0694] acacgctgtatctgcaaatgaacagcctgagagccgaagacacggccgtatattactgtgcgagaccgtttccgtat tttgactactggggccagggaaccctggtcaccgtctcgagtgcgtcgaccaagggcccatcggtcttccccctgg caccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccg gtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcagga ctctactccctcagcagcgtagtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatc acaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgaccgcactcacacatgcccaccgt gcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctc ccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgt ggacggcgtggaggtgcataatgccaagacaaagccgcgggagctgctgcaggggagcacgtaccgtgtggtc agcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctc ccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccc catcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcg ccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacg gctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtg atgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtcccccgga

[0695] 31 E1VLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYY ASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQG TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSCADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC

[0696] 32 gaaattgtgttaacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggcca gtcagagtgttagcagcagctttttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctattatg catccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatca gcagactggagcctgaagattttgcagtgtattactgtcagcagacgggtcgtattccgccgacgttcggccaagg gaccaaggtggaaatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatc tggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacg ccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcag caccctgacgctgagctgcgcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctga gctcgcccgtcacaaagagcttcaacaggggagagtgt

[0697] 33 EVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAAGEGIPIFEDFVDS SVGYYTVTGLEPGIDYDISVITLINGGESAPTTLTQQT

[0698] 34 VVTQLSPPTNLHLEANPDTGVLAVSWERSTTPDITGYRITTTPTNGQQGN SLEEVVHADQSSCTFDNLSPGLEYNVSVYTVKDDKESVPISDTIIPEVPQL TDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAAGEGIPIFEDFVDSSVGY YTVTGLEPGIDYDISVITLINGGESAPTTLTQQTAVPPPTDLRFTNIGPDTM RVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTE YVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRA TITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGR EESPLLIGRSRSHHHHHH

[0699] 35 VVTPLSPPTNLHLETNPDTGVLTVSWERSTTPDITGYRITTTPTNGQQGY SLEEVVHADQSSCTFDNLSPGLEYNVSVYTVKDDKESVPISDTIIPEVPQL TDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAAGEGIPIFEDFVDSSVGY YTVTGLEPGIDYDISVITLINGGESAPTTLTQQTAVPPPTDLRFTNIGPDTM RVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTE YVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRA TITGYRIRHHPEHMSGRPREDRVPPSRNSITLTNLTPGTEYVVSIVALNGR EESPLLIGRSRSHHHHHH

[0700] 36 VVTPLSPPTNLHLEANPDTGVLTVSWERSTTPDITGYRITTTPTNGQQGT

[0701] ALEEVVHADQSSCTFENLNPGLEYNVSVYTVKDDKESAPISDTVIPEVPQ

[0702]

[0703] LTDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAAGEGIPIFEDFVDSSVGY 90

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[0705] YTVTGLEPGIDYDISVITLINGGESAPTTLTQQTAVPPPTDLRFTNIGPDTM RVTWAPPPSIELTNLLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTE YLVSVSSVYEQHESIPLRGRQKTGLDSPTGFDSSDVTANSFTVHWVAPR APITGYIIRHHAEHSAGRPRQDRVPPSRNSITLTNLNPGTEYIVTIIAVNGR EESPPLIGRSRSHHHHHH

[0706] 37 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC

[0707] 38 GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPV KAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEK TVAPTECS

[0708] 39 LLQGG

[0709] 40 LLQG

[0710] 41 LSLSQG

[0711] 42 GGGLLQGG

[0712] 43 GLLQG

[0713] 44 GSPLAQSHGG

[0714] 45 GLLQGGG

[0715] 46 GLLQGG

[0716] 47 GLLQ

[0717] 48 LLQLLQGA

[0718] 49 LLQGA

[0719] 50 LLQYQGA

[0720] 51 LLQGSG

[0721] 52 LLQYQG

[0722] 53 LLQLLQG

[0723] 54 SLLQG

[0724] 55 LLQLQ

[0725] 56 LLQLLQ

[0726]

[0727] 57 LLQGR

[0728] EXAMPLES

[0729] 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.

[0730] Example 1: PYX-201 Phase 1 Clinical Study Overview

[0731] A. Study Overview

[0732] This example describes a Phase 1, first in human, open-label, multicenter, 2-part, non¬

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[0735] randomized dose-escalation (Part 1) followed by a dose-expansion (Part 2) study to evaluate the safety, tolerability, PK, PD, and antitumor activity of PYX-201, an EDB+FN targeting ADC with site specific conjugation of an enhanced auristatin payload (aurOlOl) via a cleavable maleimidocaproyl valine-citrulline p-aminocarbamate (vc) linker at engineered cysteines sites kK183 and K290. The overall study design is shown in FIG. 1.

[0736] In Part 1, patients received escalating doses of PYX-201 starting at 0.3 mg / kg, with initial dosing on day one and subsequent doses each 21 days (D1Q3W). The patients included those with advanced solid tumors who have relapsed, been non-responsive to, or have progressed on or after standard available therapies, with a focus on those tumor types known to have expression of the target antigen. Part 1 of the study was performed to evaluate the safety, tolerability. PK, PD, and antitumor activity of PYX-201.

[0737] In Part 2, patients in selected cohorts, including eligible participants with: inoperable recurrent / metastatic HNSCC (Cohort A), triple negative breast cancer (Cohort B), HR+ breast cancer (Cohort C), and various other advanced tumor types (Cohort D), will receive a predetermined dose of PYX-201. The predetermined dose will be a dose that was determined to be safe during Part 1 of the study. Enrollment to some cohorts or specific tumor types may be gated based on emerging data from Part 1 of the study. Additional tumor types and / or the evaluation of alternative dosing regimens may also be considered based on emerging data.

[0738] B. Patient Population

[0739] In Part 1: Dose Escalation, approximately 80 participants will be enrolled to the doseescalation cohorts to estimate the MTD or the recommended dose(s) / schedule of PYX-201 for Part 2. Additional participants may be enrolled by backfill enrollment.

[0740] In Part 2: Dose Expansion, participants will be enrolled according to Table 6.

[0741] Table 6 - PYX-201 Phase 1 Part 2 Cohorts

[0742] Cohort Number of Participants

[0743] Cohort A (HNSCC) 46

[0744] Cohort B (TNBC) 10-20

[0745] Cohort C (HR+ breast cancer 10-20

[0746]

[0747] Cohort D (Advanced cancers) 10-20

[0748] All participants enter study screening following direct informed consent. Participants are then screened based upon eligibility criteria. A screen failure is a consented participant who has been deemed ineligible on the basis of one or more eligibility criteria or who has 92

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[0750] withdrawn consent prior to treatment assignment. An enrolled participant is one who has been deemed eligible and has been assigned to a treatment cohort.

[0751] To be eligible for Part 1 and Part 2 of the study, participants must meet the following general inclusion criteria: male or non-pregnant, non-lactating female participants age >18 years, with study selected histologically or cytologically confirmed solid tumor.

[0752] For Part 1, participants must have developed disease progression through standard therapy, and for whom standard of care therapy according to the study investigator that prolongs survival is unavailable or unsuitable in the following study selected solid tumors: NSCLC, locally advanced / metastatic breast cancer, HNSCC, ovarian cancer, thyroid cancer, PDAC, soft tissue sarcoma, chordoma, hepatocellular carcinoma, kidney cancer, cervical cancer, and endometrial cancer.

[0753] For Part 2, participant must meet the inclusion criteria for one of the study selected cohorts A-D wherein Cohort A is HNSCC, Cohort B is triple negative breast cancer, Cohort C is HR+ breast cancer, and Cohort D is various other advanced tumor types including NSCLC, HR- HER2+ breast cancer, thyroid cancer, soft tissue sarcoma, chordoma, cervical cancer, endometrial cancer, or any other cancer supported by emerging data from Part 1 and approved by study sponsor.

[0754] Cancer Type Specific Requirements

[0755] (i) For NSCLC, participants must have a histologic diagnosis of advanced NSCLC not amenable to curative therapy and have disease progression on / after >1 prior systemic regimen for Part 1 and no more than 2 prior regimens for Part 2, which will have included a checkpoint inhibitor and a platinum-based chemotherapy. Participants with driver mutations / translocations such as EGFR, ROS. RAF, and anaplastic lymphoma kinase must have progressed on / after targeted therapy prior to entry.

[0756] (ii) For HR+ HER2- breast cancer, participants must have locally advanced, unresectable / metastatic HR+ and HER2- breast cancer and have disease progression on / after ±3 prior regimens for Part 1 and no more than 3 prior therapies for Part 2, including hormonal therapy and a CDK4 / 6 inhibitor.

[0757] (iii) For triple negative breast cancer, participants must have locally advanced / metastatic TNBC not amendable to surgical intervention and have experienced disease progression on / after >2 prior regimens for Part 1 and no more than 2 prior regimens for Part 2.

[0758] (iv) For HR- and HER2+ breast cancer, participants must have locally advanced,

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[0761] unresectable / metastatic HER2+ and HR- breast cancer and have experienced disease progression on / after >2 prior regimens, including fam-trastuzumab-deruxtecan-nxki for Part 1 and no more than 2 regimens for Part 2.

[0762] (v) For HNSCC, participants must have locally advanced / metastatic HNSCC and have disease progression on / after >1 prior regimen including platinum-based therapy and a checkpoint inhibitor (if eligible for a checkpoint inhibitor) for Part 1. Participants in Part 2 must have received 1 but no more than 2 prior systemic therapies.

[0763] (vi) For ovarian cancer (Part 1 only), participants must have locally advanced / metastatic ovarian cancer and have disease progression >1 prior regimens of therapy which will have consisted of platinum-based therapy.

[0764] (vii) For thyroid cancer, participants must have recurrent / relapsed, advanced thyroid cancer that has progressed after prior regimens or are otherwise ineligible for the standard of care therapy. Participants with papillary thyroid cancer must have received prior radioactive iodine if they were eligible to receive radioactive iodine.

[0765] (viii) For PDAC (Part 1 only), participants must have locally advanced / metastatic PDAC not amendable to surgical intervention and have disease progression after >1 prior regimen given for recurrent disease.

[0766] (ix) For soft tissue sarcoma including chordoma, participants must have locally advanced / metastatic STS, not amenable to curative therapy, and have received at least 1 prior line of systemic therapy.

[0767] (x) For hepatocellular carcinoma (Part 1 only), participants must have locally advanced / metastatic HCC and have disease progression on / after >2 prior regimens including a checkpoint inhibitor. Variant histologies are allowed provided that HCC is the predominant histology.

[0768] (xi) For kidney cancer (Part 1 only), participants must have locally advanced / metastatic kidney cancer and have disease progression on / after a tyrosine kinase inhibitor and a checkpoint inhibitor. Each therapy may have been administered alone or in combination.

[0769] (xii) For cervical cancer in Part 2, participants must have locally advanced / metastatic disease and have been treated with no more than 2 prior systemic regimens including therapy that contained ICI and a platinum-based chemotherapy regimen.

[0770] (xiii) For endometrial cancer in Part 2, participants must have locally advanced / metastatic disease and have been treated with no more than 2 prior systemic regimens.

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[0773] Additionally, participants in Parts 1 and 2 of the study must have a ECOG Performance Status of 0-1, have at least one measurable lesion by RECIST Version 1.1 criteria that has not been previously irradiated, a life expectancy greater than 3 month in the opinion of the investigator, adequate cardiac function and no significant cardiac issues, adequate hematological function, adequate hepatic function, adequate renal function, adequate coagulation profile, ability to understand and willingness to sign a written consent form, willingness and ability7to comply with study procedures and follow-ups, and willingness and ability7to consent to study biopsy requirements. Additionally, male participants who can father a child, and female participants of childbearing potential, must consent to study criteria regarding pregnancy, breastfeeding, and contraception. For Part 2 only, participants must consent to additional study nutrition and biopsy requirements.

[0774] Participants were excluded if they met any of the following key exclusion criteria: history of another malignancy except certain minor non-invasive cancers or fully resolved previous solid tumors without metastasis; untreated or symptomatic brain metastases at the time of signing the informed consent; significant cardiovascular disease within 6 months prior to start of PYX-201 treatment; evidence of active infection requirement treatment at start of PYX-201 treatment; known active HBV, HCV, HIV or AIDS; failure to recover to baseline severity or Grade <1 CTCAE Version 5.0 from acute non-hematologic toxicity; major surgery within 4 weeks prior to start of PYX-201 treatment; prior solid organ or bone marrow progenitor cell transplantation; prior high-dose chemotherapy requiring stem cell rescue; concurrent anticancer therapy; certain previous exposure to radiation therapy; live vaccine administered w ithin 28 days prior to first dose of PYX-201; previous administration of an EDB+FN targeting treatment; certain diagnoses of neuropathy; uncontrolled diabetes mellitus; certain chronic illness; administration of certain other drugs; certain comeal or visual acuity disease or impairments; certain lung diseases; epidermal necrolysis or related conditions; or a tumor that invades, encases, and / or abuts a major named blood vessel(s) such that tumor necrosis might lead to a major bleeding event.

[0775] C. Treatment Schedule

[0776] Part 1 - Dose Escalation with PYX-201

[0777] The objective of Part 1 w as to evaluate the safety, tolerability, PK, PD, and antitumor activity of PYX-201 in patients with advanced solid tumors who have relapsed, been non- 95

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[0779] responsive to, or have progressed on or after standard available therapies, with a focus on those tumor types known to have expression of the target antigen. Participants were dosed with a PYX-201 intravenous infusion on a D1Q3W schedule at escalating dose levels.

[0780] Dose Escalation began with one participant treated at the lowest planned dose level of PYX-201, 0.3 mg / kg. The first participant in the first cohort of Arm 1 (D1Q3W) was observed for 7 days before subsequent participants were enrolled. Once cohorts of size 3 were reached the following rule was applied to dose escalation.

[0781] If DLTs were observed:

[0782] (i) If the observed DLT rate at the current dose is <0.236, escalate the dose to the next higher dose level;

[0783] (ii) If at the current dose a DLT has been observed and the rate is <0.236 and if >50% of participants at the current dose have clinically significant drug-related Grade >2 toxicity(ies) a dose increment of up to 30% will be applied;

[0784] (iii) If the observed DLT rate at the current dose is >0.359, de-escalate to either the next lower dose level already evaluated or an intermediate level based on the available safety data from all relevant dose levels;

[0785] (iv) Otherwise, stay at the current dose.

[0786] In the absence of DLTs:

[0787] (i) If <50% of participants at the current dose have clinically significant drug-related Grade >2 toxicity(ies), dosing will escalate sequentially in up to 100% increments; (ii) If >50% of participants at the current dose have clinically significant drug-related Grade >2 toxicity(ies), dosing will escalate a dose increment of up to 50%.

[0788] Per the dose escalation rules herein, dose escalation in Part 1 proceeded according to Table 7 including the study of intermediate doses.

[0789] Table 7 - PYX-201 Phase 1 Part 1 Dose Escalation Levels

[0790] PYX-201 Dose

[0791] Cohort

[0792] (mg / kg)

[0793] 1 0.3

[0794] 2 0.6

[0795] 3 1.2

[0796] 4 2.4

[0797] 5 3.6

[0798] 6 5.4

[0799]

[0800] 7 8

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[0803] 8 6.6

[0804]

[0805] 9 4.4

[0806] Treatment with PYX-201 in Part 1 continued until radiographically documented evidence of disease progression, unacceptable or dose limiting toxicity (DLT), the start of new anticancer treatment, or any other criteria for withdrawal from the study or study drug occurred, or for a maximum of 2 years, whichever occurred first. Stable or responding participants who experienced DLTs were able to continue therapy at a lower dose once DLTs resolved. Participants who in the opinion of the Investigator derived clinical benefit were able to continue to receive treatment beyond radiographical disease progression.

[0807] In Part 1 the primary endpoints were overall safety and tolerability of PYX-201 as a single agent, which includes DLT rate, incidence of AEs characterized overall and by type, seriousness, relationship to study treatment, timing and severity', and change in clinical laboratory parameters, vital signs, and ECG parameters.

[0808] Part 2 - Dose Expansion with PYX-201

[0809] Part 2 of the study is a dose expansion portion in participants with advanced solid tumors, through indication-specific cohorts. Participants will be dosed with a PYX-201 intravenous infusion on a D1Q3W schedule at a dose of 5.4 mg / kg, which was determined to be safe in Part 1 of the study, although other doses including, but not limited to, 3.6 mg / kg, 4.4 mg / kg, 6.6 mg / kg, and 8 mg / kg could potentially be used as a dose in Part 2.

[0810] Treatment with PYX-201 in Part 2 will continue until clear radiographic disease progression per RECIST vl.l, clinical disease progression requiring immediate treatment with a new anticancer therapy (including salvage / palliative irradiation or surgery), unacceptable toxicity', study termination by Sponsor, and / or upon meeting any other criteria for w ithdraw al from the study or PYX-201, or for a maximum of 2 years, whichever occurs first.

[0811] During the study, participants will be evaluated for response 6 weeks after cycle 1 day 1 (C1D1), 12 weeks after C1D1. and then every 9 weeks until disease progression or start of new anticancer therapy, using a CT and / or MRI scan according to RECIST Version 1.1. If a participant discontinues PYX-201 for any reason, a disease assessment is required at the EOT Visit. In Part 2, disease assessment will be performed using a CT scan with contrast and / or MRI with and / or w ithout contrast (and may include PET imaging, if relevant) of the head, neck, chest, abdomen, and pelvis (and other relevant areas if they include target lesions

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[0814] and / or areas of known or suspected disease).

[0815] D. Overall Results - Part 1 All Patients

[0816] Patient responses across all tumor types were observed and classified as either progressive disease (PD), stable disease (SD), partial response (PD) or complete response (CR) according to the following:

[0817] (i) PD: At least a 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study recorded since the treatment started or the appearance of one or more new lesions. In addition to 20% increase, the sum must also demonstrate an absolute increase of at least 5 mm;

[0818] (ii) SD: Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum LD since the treatment started;

[0819] (iii) PR: At least a 30% decrease in the sum of the diameters of target lesions, taking as reference the Baseline sum diameters; and

[0820] (iv) CR: Disappearance of all target lesions. Pathologic nodes must have a reduction in the short axis to <10 mm.

[0821] The patient responses were then aggregated to determine the overall disease control rate (DCR) and overall response rate (ORR). DCR was calculated as the sum of the patients exhibiting SD, PR, or CR divided by the total number of patients, and ORR was calculated as the sum of the patient exhibiting PR or CR divided by the total number of patients.

[0822] In Phase 1 Part 1 of the PYX-201 clinical study, in a heavily pretreated population across a diverse set of hard-to-treat cancer types, treatment with PYX-201 resulted in a disease control rate (DCR) of 70% at a treatment dose of 3.6 mg / kg, 100% at 4.4 mg / kg, 78% at a treatment dose of 5.4 mg / kg, 75% at a treatment dose of 6.6 mg / kg, and 50% at a treatment dose of 8 mg / kg; and an overall response rate of 20% at a treatment dose of 3.6 mg / kg, 22% at a treatment dose of 5.4 mg / kg, 25% at a treatment dose of 6.6 mg / kg and 50% at a treatment dose of 8 mg / kg (Table 8). Overall, across all dose levels 3.6 - 8 mg / kg a 73% DCR was observed with 55% of patients experiencing stable disease, 20% achieving a partial response, and one patient achieving a complete response.

[0823] Table 8 - PYX-201 All Patients Best Overall Response By Dose Level

[0824] 98

[0825] 567343972 Attorney Docket No. 384953-1024W01(00350)

[0826] Dose n = PD SD PR

[0827] (mg / kg) CR DCR ORR 3.6 10 3 (30%) 5 (50%) 2 (20%) 0 (0%) 70% 20% 4.4 3 0 (0%) 3 (100%) 0 (0%) 0 (0%) 100% 0% 5.4 32 7 (22%) 18 (56%) 6 (19%) 1 (3%) 78% 22% 6.6 4 1 (25%) 2 (50%) 1 (25%) 0 (0%) 75% 25% 8 2 1 (50%) 0 (0%) 1 (50%) 0 (0%) 50% 50%

[0828] 51 12 (24%) 28 (55%) 9 (20%) 1 (2%) 76% 22%

[0829]

[0830] Table 9 shows the best overall response as a percentage change from baseline (%A) in tumor volume and best overall response (BOR) for each individual patient for specific indications at dose levels 2.4 to 4.4 mg / kg. Patients among this group experience up to a 50% reduction (-50%) in tumor volume with a substantial number of patients experiencing a clinically meaningful benefit as show in FIG. 2.

[0831] Table 9 - PYX-201 All Patients % Change from Baseline 2.4-4.4 mg / kg Indication Dose (mg / kg) %A BOR

[0832] NSCLC 2.4 20.7 PD

[0833] TNBC 2.4 8.4 PD

[0834] PDAC 2.4 8.3 SD

[0835] HR+ Breast 2.4 0.0 SD

[0836] NSCLC 2.4 -2.8 SD

[0837] PDAC 2.4 -2.8 PD

[0838] PDAC 2.4 -6.3 SD

[0839] STS 2.4 -24.7 SD

[0840] STS 3.6 29.6 PD

[0841] NSCLC 3.6 19.5 SD

[0842] TNBC 3.6 17.8 PD

[0843] PDAC 3.6 13.8 PD

[0844] PDAC 3.6 8.0 SD

[0845] H& N 3.6 5.2 PD

[0846] NSCLC 3.6 -3.6 SD

[0847] PDAC 3.6 -13.4 SD

[0848] Ovarian 3.6 -34.4 PR

[0849] H& N 3.6 -50.0 PR

[0850] PDAC 4.4 10.2 SD

[0851] Chordoma 4.4 0.0 SD

[0852]

[0853] STS 4.4 -17.1 SD

[0854] 99

[0855] 567343972 Attorney Docket No. 384953-1024W01(00350)

[0856] Table 10 shows the best overall response as a percentage change from baseline (%A) in tumor volume and best overall response (BOR) for each individual patient for specific indications at dose levels 5.4 to 6.6 mg / kg. Patients among this group experience up to a complete response with 100% reduction (-100%) in tumor volume with a substantial number of patients experiencing a clinically meaningful benefit as show in FIG. 3.

[0857] Table 10 - PYX-201 All Patients % Change from Baseline 5.4-6.6 mg / kg Indication Dose (mg / kg) %A BOR

[0858] PDAC 5.4 86.8 PD

[0859] PDAC 5.4 46.3 PD

[0860] TNBC 5.4 30.4 PD

[0861] PDAC 5.4 24.6 PD

[0862] H& N 5.4 19.3 SD

[0863] STS 5.4 19.2 SD

[0864] Chordoma 5.4 18.5 SD

[0865] NSCLC 5.4 16.7 PD

[0866] NSCLC 5.4 6.9 PD

[0867] PDAC 5.4 6.5 PD

[0868] Ovarian 5.4 5.6 SD

[0869] Ovarian 5.4 2.5 SD

[0870] Thyroid 5.4 0.8 SD

[0871] Thyroid 5.4 0.1 SD

[0872] NSCLC 5.4 0.0 SD

[0873] HCC 5.4 -0.7 SD

[0874] NSCLC 5.4 -1.2 SD

[0875] PDAC 5.4 -4.2 SD

[0876] Thyroid 5.4 -10.2 SD

[0877] H& N 5.4 -11.1 SD

[0878] Chordoma 5.4 -14.6 SD

[0879] H& N 5.4 -15.5 SD

[0880] TNBC 5.4 -21.6 SD

[0881] NSCLC 5.4 -25.6 SD

[0882] TNBC 5.4 -26.4 SD

[0883] PDAC 5.4 -31.5 PR

[0884] H& N 5.4 -34.9 PR

[0885] NSCLC 5.4 -42.0 PR

[0886] HR+ Breast 5.4 -54.9 PR

[0887] STS 5.4 -55.0 PR

[0888] Ovarian 5.4 -72.0 PR

[0889] H& N 5.4 -100.0 CR

[0890] STS 6.6 29.1 PD

[0891]

[0892] H& N 6.6 -7.8 SD

[0893] 100

[0894] 567343972 Attorney Docket No. 384953-1024W01(00350)

[0895] H& N 6.6 -25.9 SD

[0896]

[0897] NSCLC 6.6 -31.1 PR

[0898] Example 2: PYX-201 Phase 1 Clinical Study Results by Indication

[0899] Data from patients in Phase 1 Part 1 of the PYX-201 clinical study of Example 1 were then analyzed based upon type / indi cation of cancer within the following cancer types: head and neck cancer, pancreatic cancer, non-small cell lung cancer, triple negative breast cancer. HR+HER2- breast cancer, ovarian cancer, soft tissue sarcoma, and chordoma, with a particular focus on dose levels of 3.6 - 8 mg / kg. As in Example 1, patients were categorized as exhibiting PD, SD, PR, or CR, and an overall DCR and ORR was calculated for each dose level for each cancer type / mdication.

[0900] Head and Neck Cancer

[0901] In total, 9 head and neck patients were evaluated with all 9 receiving a dose between 3.6 and 8 mg / kg. As seen in Table 11, at dosage levels of 3.6 mg / kg or greater, 67% of head and neck cancer patients experienced stable disease, 22% experienced a partial response, and one patient (11%) achieved a complete response. Overall disease control rate at dosage levels of 3.6 mg / kg or above was 100% with an overall response rate of 33%. At 3.6 mg / kg, of two patients one demonstrated stable disease and the other a partial response for a DCR of 100% and an ORR of 50%. At 5.4 mg / kg 60% of patients experienced stable disease, 20% achieved a partial response, and 20% achieved a complete response, for a disease control rate of 100% and an overall response rate of 40%. At 6.6 mg / kg 100% of patients achieved stable disease for a DCR of 100%. In Table 12 and FIG. 4 a patient-by-patient breakdown is shown by dose with percentage change from baseline (%A) of up to 100% reduction in tumor volume and the best overall response (BOR) for each individual according to PD, SD, PR, or CR.

[0902] Table 11 - PYX-201 Head and Neck Best Overall Response Per Dose Dose n = 1 PD SD PR

[0903] (mg / kg) CR DCR ORR 3.6 2 1 (50%) 1 (50%) 100% 50% 4.4

[0904] 5.4 5 3 (60%) 1 (20%) 1 (20%) 100% 40% 6.6 2 2 (100%) 100% 0% 8

[0905]

[0906] Total 9 6 (67%) 2 (22%) 1 (H%) 100% 33%

[0907] 101

[0908] 567343972 Attorney Docket No. 384953-1024W01(00350)

[0909] Table 12 - PYX-201 Head and Neck Percent Change from Baseline By Patient Dose (mg / kg) %A BOR

[0910] 5.4 19.3 SD

[0911] 3.6 5.2 SD

[0912] 6.6 -7.8 SD

[0913] 5.4 -11.1 SD

[0914] 5.4 -15.5 SD

[0915] 6.6 -25.9 SD

[0916] 5.4 -34.9 PR

[0917] 3.6 -50 PR

[0918]

[0919] 5.4 -100 CR

[0920] Pancreatic Ductal Adeno Carcinoma (Pancreatic Cancer)

[0921] In total. 16 pancreatic cancer patients were evaluated with 11 receiving a dose between 3.6 and 8 mg / kg. As seen in Table 13, at dosage levels of 3.6 mg / kg or greater, DCR was 45% with 5 of 11 pancreatic cancer patients experiencing a stable disease or a partial response. Overall disease control rate at a dosage level of 3.6 mg / kg was 67%. One out of one patient at 4.4 mg / kg achieved stable disease for a DCR of 100%. At 5.4 mg / kg 17% of patients achieved stable disease and 17% achieved a partial response, for a DCR of 33% and an ORR of 17%. In Table 14 and FIG. 5 a patient-by-patient breakdown is shown by dose with percentage change from baseline (%A) of up to a 31.5% reduction in tumor volume and the best overall response (BOR) for each individual according to PD, SD, PR, or CR.

[0922] Table 13 - PYX-201 PDAC Best Overall Response Per Dose Dose

[0923] (mg / kg) n = PD SD PR CR DCR ORR 3.6 3 1 (33%) 2 (67%) 67% 0% 4.4 1 1 (100%) 100% 0% 5.4 6 4 (67%) 1 (17%) 1 (17%) 33% 17% 6.6

[0924] 8 1 1 (100%) 0% 0%

[0925]

[0926] Total 11 6 (55%) 4 (36%) 1 (9%) 45% 9%

[0927] Table 14 - PYX-201 PDAC Percent Change from Baseline By Patient Dose (mg / kg) %A BOR

[0928]

[0929] 5.4 86.8 PD

[0930] 102

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[0932] 8.0 55.8 PD

[0933] 5.4 46.3 PD

[0934] 1.2 31.6 PD

[0935] 5.4 24.6 PD

[0936] 0.6 20.0 SD

[0937] 3.6 13.8 PD

[0938] 4.4 10.2 SD

[0939] 2.4 8.3 SD

[0940] 3.6 8.0 SD

[0941] 5.4 6.5 PD

[0942] 2.4 -2.8 PD

[0943] 5.4 -4.2 SD

[0944] 2.4 -6.3 SD

[0945] 3.6 -13.4 SD

[0946]

[0947] 5.4 -31.5 PR

[0948] Non-Small Cell Lung Cancer

[0949] In total, 12 non-small cell lung cancer patients were evaluated with 9 receiving a dose between 3.6 and 8 mg / kg. As seen in Table 15, at dosage levels of 3.6 mg / kg or greater, 56% of NSCLC patients experienced stable disease and 22% experienced a partial response for a DCR of 78% and an ORR of 22%. Overall disease control rate at the dosage level of 3.6 mg / kg was 100%. At 5.4 mg / kg 50% of patients achieved stable disease and 17% achieved a partial response, for a disease control rate of 67% and an overall response rate of 17%. One patient was treated at 6.6 mg / kg and achieved a partial response for a DCR and ORR of 100%. In Table 16 and FIG. 6 a patient-by-patient breakdown is shown by dose with percentage change from baseline (%A) of up to 42% reduction in tumor volume and the best overall response (BOR) for each individual according to PD, SD, PR, or CR.

[0950] Table 15 - PYX-201 NSCLC Best Overall Response Per Dose Dose

[0951] (mg / kg) n = PD SD PR CR DCR ORR 3.6 2 2 (100%) 100% 0% 4.4

[0952] 5.4 6 2 (33%) 3 (50%) 1 (17%) 67% 17% 6.6 1 1 (100%) 100% 100% 8

[0953]

[0954] Total 9 2 (22%) 5 (56%) 2 (22%) 78% 22%

[0955] Table 16 - PYX- 201 NSCLC Percent Change from Baseline By Patient

[0956] 103

[0957] 567343972 Attorney Docket No. 384953-1024W01(00350)

[0958] Dose (mg / kg) %A BOR

[0959] 0.3 40.1 PD

[0960] 2.4 20.7 PD

[0961] 3.6 19.5 SD

[0962] 5.4 16.7 PD

[0963] 5.4 7.0 PD

[0964] 3.6 -3.6 SD

[0965] 5.4 0.0 SD

[0966] 5.4 -1.2 SD

[0967] 2.4 -2.8 PD

[0968] 5.4 -25.6 SD

[0969] 6.6 -31.1 PR

[0970]

[0971] 5.4 -42.0 PR

[0972] Triple Negative Breast Cancer

[0973] In total. 7 triple negative breast cancer patients were evaluated with 4 receiving a dose between 3.6 and 8 mg / kg. As seen in Table 17, at dosage levels of 3.6 mg / kg or greater, 50% of TNBC patients experienced stable disease for a DCR of 50%. At 5.4 mg / kg 67% of patients experienced stable disease for a DCR of 67%. In Table 18 and FIG. 7 a patient-by-patient breakdown is shown by dose with percentage change from baseline (%A) of up to 26.4% reduction in tumor volume and the best overall response (BOR) for each individual according to PD, SD, PR, or CR.

[0974] Table 17 - PYX-201 TNBC Best Overall Response Per Dose Dose

[0975] (mg / kg) n = PD SD PR CR DCR ORR 3.6 1 1 (100%) 0% 0% 4.4

[0976] 5.4 3 1 (50%) 2 (50%) 67% 0% 6.6

[0977] 8

[0978]

[0979] Total 4 2 (50%) 2 (50%) 50% 0%

[0980] Table 18 - PYX-201 TNBC Percent Change from Baseline By Patient Dose (mg / kg) %A BOR

[0981] 5.4 30.4 PD

[0982] 3.6 17.8 PD

[0983] 2.4 8.4 PD

[0984] 1.2 5.9 SD

[0985]

[0986] 0.3 3.7 SD

[0987] 104

[0988] 567343972 Attorney Docket No. 384953-1024W01(00350)

[0989] 5.4 -21.6 SD

[0990]

[0991] 5.4 -26.4 SD

[0992] HR Her2- Breast Cancer

[0993] In total, 3 HR+Her2- breast cancer patients were evaluated with one patient receiving a dose between 3.6 and 8 mg / kg. As seen in Table 19, the patient at dosage levels of 3.6 mg / kg or greater, in this case 5.4 mg / kg. achieved a partial response for a DCR of 100% and an ORR of 100%. In Table 20 and FIG. 8 a patient-by -patient breakdown is shown by dose with percentage change from baseline (%A) of up to 51.2% reduction in tumor volume and the best overall response (BOR) for each individual according to PD, SD, PR, or CR.

[0994] Table 19 - PYX-201 HR+ Breast Best Overall Response Per Dose Dose

[0995] (mg / kg) n = PD SD PR CR DCR ORR 3.6

[0996] 4.4

[0997] 5.4 1 1 (100%) 100% 100% 6.6

[0998] 8

[0999]

[1000] Total 1 1 (50%) 100% 100%

[1001] Table 20 - PYX-201 HR+ Breast Percent Change from Baseline By Patient Dose (mg / kg) %A BOR

[1002] 2.4 0.0 SD

[1003] 1.2 -50.0 PR

[1004]

[1005] 5.4 -51.2 PR

[1006] Ovarian Cancer

[1007] In total, 5 ovarian cancer patients were evaluated with 4 receiving a dose between 3.6 and 8 mg / kg. As seen in Table 21, at dosage levels of 3.6 mg / kg or greater, 50% of ovarian cancer patients experienced stable disease and 50% a partial response for a DCR of 100% and an ORR of 50%. Specifically, one of one patient at 3.6 mg / kg experienced a partial response for a DCR and ORR of 100%. At 5.4 mg / kg, 66% of patients exhibit stable disease and 33% of patients experienced a partial response, for a DCR of 100% and an ORR of 33%. In Table 22 and FIG. 9 a patient-by -patient breakdown is shown by dose with percentage change from baseline (%A) of up to 73% reduction in tumor volume and the best overall response (BOR) for each individual according to PD, SD, PR, or CR.

[1008] 105

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[1010] Table 21 - PYX-201 Ovarian Best Overall Response Per Dose Dose

[1011] n = PD SD PR

[1012] (mg / kg) CR DCR ORR 3.6 1 1 (100%) 100% 100% 4.4

[1013] 5.4 3 2 (67%) 1 (33%) 100% 33% 6.6

[1014] 8

[1015] 2 2

[1016]

[1017] Total 4 (50%) (50%) 100% 50%

[1018] Table 22 - PYX- 201 Ovarian Percent Change from Baseline By Patient Dose (mg / kg) %A BOR

[1019] 0.6 25.7 PD

[1020] 5.4 5.6 SD

[1021] 5.4 2.5 SD

[1022] 3.6 -34.4 PR

[1023]

[1024] 5.4 -73 PR

[1025] Soft Tissue Sarcoma (STS) Excluding Chordoma

[1026] In total, 7 soft tissue sarcoma patients (not including chordoma) were evaluated with 5 receiving a dose between 3.6 and 8 mg / kg. As seen in Table 23, at dosage levels of 3.6 mg / kg or greater, 40% of soft tissue sarcoma (excluding chordoma) patients experienced stable disease and 20% a partial response for a DCR of 60% and an ORR of 20%.

[1027] Specifically, one of one patient at 4.4 mg / kg achieved stable disease for a DCR of 100%. At 5.4 mg / kg, 50% of patients exhibit stable disease and 50% of patients experienced a partial response, for a DCR of 100% and an ORR of 50%. In Table 24 and FIG. 10 a patient-by- patient breakdown is shown by dose with percentage change from baseline (%A) of up to 55% reduction in tumor volume and the best overall response (BOR) for each individual according to PD, SD, PR, or CR, as well as the subtype of sarcoma for each patient.

[1028] Table 23 - PYX-201 STS Excluding Chordoma Best Overall Response Per Dose Dose

[1029] n = PD SD PR CR DCR ORR (mg / kg)

[1030] 3.6 1 1 (100%) 0% 0% 4.4 1 1 (100%) 100% 0% 5.4 2 1 (50%) 1 (50%) 100% 50% 6.6 1 1 (100%) 0% 0%

[1031]

[1032] 8

[1033] 106

[1034] 567343972 Attorney Docket No. 384953-1024W01(00350)

[1035]

[1036] Total 5 2 (40%) 2 (40%) 1 (20%) 60% 20%

[1037] Table 24 - PYX-201 STS Excluding Chordoma Percent Change from Baseline By Patient

[1038] Dose (mg / kg) %A BOR Subtype

[1039] 3.6 29.6 PD Leiomyosarcoma

[1040] 6.6 29.1 PD Synovial sarcoma

[1041] 5.4 19.2 SD Leiomyosarcoma

[1042] 0.3 -4.2 SD Spindle cell neoplasm

[1043] 4.4 -17.1 SD Leiomyosarcoma

[1044] Pulmonary Epithelioid

[1045] 2.4 -24.7 SD

[1046] Hemangioendothelioma

[1047]

[1048] 5.4 -55.0 PR Endometrial

[1049] Chordoma

[1050] In total, 4 chordoma patients were evaluated with all 4 receiving a dose between 3.6 and 8 mg / kg. As seen in Table 25, at dosage levels of 3.6 mg / kg or greater, 75% of chordoma patients experienced stable disease and 25% achieved a partial response for a DCR of 100% and an ORR of 25%. Specifically, one of one patient at 4.4 mg / kg achieved stable disease for a DCR of 100%. At 5.4 mg / kg, 100% of patients exhibit stable disease for a DCR of 100%. One out of one patient at 8 mg / kg experienced a partial response, for a DCR and ORR of 100%. In Table 26 and FIG. 11 a patient-by-patient breakdown is shown by dose with percentage change from baseline (%A) of up to 55.5% reduction in tumor volume and the best overall response (BOR) for each individual according to PD, SD, PR, or CR.

[1051] Table 25 - PYX-201 Chordoma Best Overall Response Per Dose Dose

[1052] n = PD SD PR CR DCR ORR (mg / kg)

[1053] 3.6

[1054] 4.4 1 1 (100%) 100% 0% 5.4 2 2 (100%) 100% 0% 6.6

[1055] 8 1 1 (100%) 100% 100%

[1056]

[1057] Total 4 3 (75%) 1 (25%) 100% 25%

[1058] Table 26 - PYX-201 Chordoma Percent Change from Baseline By Patient Dose (mg / kg) %A BOR

[1059] 5.4 18.5 SD

[1060]

[1061] 4.4 0.0 SD

[1062] 107

[1063] 567343972 Attorney Docket No. 384953-1024W01(00350)

[1064] 5.4 -14.6 SD

[1065]

[1066] 8.0 -55.5 PR

[1067] Soft Tissue Sarcoma (STS) including Chordoma

[1068] As seen in Table 27, if chordoma is included with the other subtypes of soft tissue sarcoma, at doses above 3.6 mg / kg 56% of patients experience SD and 22% experience a partial response for an overall DCR of 78% and ORR of 22%. More specifically, at 4.4 mg / kg 100% of patients experienced SD for a DCR of 100%, at 5.4 mg / kg 75% of patients experienced SD and 25% achieved a PR for a DCR of 100% and ORR of 25%, and at 8 mg / kg one of one patients experienced a PR for a DCR and ORR of 100%. In Table 28 and FIG. 12 a patient-by-patient breakdown is shown by dose with percentage change from baseline (%A) of up to 55.5% reduction in tumor volume, the best overall response (BOR) for each individual according to PD, SD, PR, or CR, and the subtype of soft tissue sarcoma for each patient.

[1069] Table 27 - PYX-201 STS including Chordoma Best Overall Response Per Dose Dose

[1070] n = PD SD PR CR DCR ORR (mg / kg)

[1071] 3.6 1 1 (100%) 0% 0% 4.4 2 2 (100%) 100% 0% 5.4 4 3 (75%) 1 (25%) 100% 25% 6.6 1 1 (100%) 0% 0% 8 1 1 (100%) 100% 100%

[1072]

[1073] Total 9 2 (22%) 5 (56%) 2 (22) 78% 22%

[1074] Table 28 - PYX-201 STS including Chordoma Percent Change from Baseline By Patient

[1075] Dose (mg / kg) %A BOR Subtype

[1076] 3.6 29.6 PD Leiomyosarcoma 6.6 29.1 PD Synovialsarcoma 5.4 19.2 SD Leiomyosarcoma 5.4 18.5 SD Chordoma

[1077] 4.4 0.0 SD Chordoma

[1078] 0.3 -4.2 SD Spindlecell neolplasm

[1079] 5.4 -14.6 SD Chordoma

[1080] 4.4 -17.1 SD Leiomyosarcoma 2.4 -24.7 SD Pulmonary Epithelioid Hemangioendothelioma

[1081] 5.4 -55.0 PR Endometrial

[1082]

[1083] 108

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[1085]

[1086] 8.0 -55.5 PR Chordoma

[1087] Additional Cancer Types

[1088] In addition to the eight indications above, three patients with thyroid cancer and one patient with hepatocellular carcinoma were evaluated, all of which received a dose of 5.6 mg / kg. As seen in Table 29, two of these patients experienced stable disease for a DCR of 50%. In Table 30 and FIG. 13 a patient-by-patient breakdown is show n by dose with percentage change from baseline (%A) of up to 10.2% reduction in tumor volume and the best overall response (BOR) for each individual according to PD. SD, PR, or CR

[1089] Table 29 - PYX-201 Other Indications Best Overall Response Per Dose Dose n = PD SD PR CR DCR ORR (mg / kg)

[1090] 3.6

[1091] 4.4

[1092] 5.4 4 2 (50%) 2 (50%) 50% 0% 6.6

[1093] 8

[1094] Total 4 0

[1095]

[1096] (0%) 3 (50%) 50% 0% Table 30 - PYX-201 Other Indications Percent Change from Baseline By Patient Indication Dose (mg / kg) %A BOR

[1097] Thyroid 5.4 0.1 PD

[1098] Thyroid 5.4 0.8 PD

[1099] HCC 5.4 -0.7 SD

[1100]

[1101] Thyroid 5.4 -10.2 SD

[1102] Example 3: PYX-201 Phase 1 Clinical Study Safety Profile

[1103] PYX-201 exhibited a favorable safety and tolerability profile with a low incidence of grade three or higher treatment related adverse effects (TRAEs). As seen in Table 31, overall grade 3 or 4 TRAEs were low with no grade 3+ TRAEs below 2.4 mg / kg, 25% incidence at 2.4 mg / kg, 9% incidence at 3.6 mg / kg, 33% incidence at 4.4 mg / kg, 36% incidence at 5.4 mg / kg, with an overall grade 3+ TRAE incidence of 29% across all dosage levels. Only one patient in the trial experience a TRAE leading to treatment discontinuation for an incidence rate of 1%. No treatment related deaths (Grade 5 TRAEs) were reported.

[1104] Table 31 - PYX-201 Treatment Related Adverse Effects (TRAEs)

[1105] 109

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[1107] 0.3 0.6 1.2 2.4 3.6 4.4 5.4 6.6 8.0

[1108] mg / kg mg / kg mg / kg mg / kg mg / kg mg / kg mg / kg mg / kg mg / kg TOTAL N 3 3 3 8 11 3 39 4 3 77

[1109] 1 1 3 6 9 3 36 4 3 66 All TRAEs (33%) (33%) (100%) (75%) (82%) (100%) (92%) (100%) (100%) (86%) Grade 3 / 4 2 1 1 14 1 22 TRAEs 0 0 0 (25%) (9%) (33%) (36%) 3 (75%) (33%) (29%) TRAEs leading

[1110] to treatment 1 1 discontinuation 0 0 0 0 0 0 (3%) 0 0 (1%) TRAEs leading

[1111] to dose 1 1 11 1 1 15 reduction 0 0 0 (13%) (9%) 0 (28%) (25%) (33%) (20%) TRAEs leading 1 7 3 1 12 to dose delay 0 0 0 (13%) 0 0 (18%) (75%) (33%) (16%) Treatment

[1112] related deaths

[1113] (Grade 5

[1114]

[1115] TRAE) 0 0 0 0 0 0 0 0 0 0

[1116] As seen in Table 32, compared to the percentage of grade 3+ TRAEs in the safety

[1117] profile of other approved antibody drug conjugates, as listed in their respective FDA

[1118] approved drug labels, the safety and tolerability of PYX-201 is favorable. For patients

[1119] 5 treated with PYX-201 at 5.4 mg / kg (N = 39), no (0%) of patients experienced an ocular or neuropathy grade 3+ TRAE, only 8% of patients experienced grade 3+ neutropenia or

[1120] cutaneous toxicity, and only 1% experienced grade 3+ pneumonitis. As seen in Table 32, PYX-201 possesses favorable tolerability even at the higher dosing level of 5.4 mg / kg as compared to FDA approved auristatin based ADCs such as Padcev® and Tivdak® which are 10 given at lower dosing levels 1.25 mg / kg and 2 mg / kg. respectively. Compared to 0% of

[1121] patients experiencing neuropathy at a PYX-201 dose of 5.4 mg / kg, 5% of Padcev® and 6% of Tivdak® patients experienced a grade 3 or higher TRAE for neuropathy. At a lower dose than PYX-201, Padcev® exhibited higher occurrences of grade 3+ neutropenia and cutaneous TRAEs (10% and 14%, respectively) as compared to PYX-201 (8% and 8%, respectively).

[1122] 15 Additionally, Tivdak® at its lower dosing level also resulted in an ocular TRAE grade 3 or higher in 3% of patients, whereas PYX-201 observed no ocular toxicity7in its study. Even as compared ADCs with other payloads other than auristatins, at similar dosing levels, such as Elahere (DM4 maytansine payload dosed at 6 mg / kg), Enhertu (topoisomerase inhibitor

[1123] payload dosed at 5.4 mg / kg) and Trodelvy (SN38 payload dosed at 10 mg / kg), PYX-201

[1124] 20 retains a favorable tolerability profile.

[1125] 110

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[1127] ACD Table 32 - PYX-201 Grade 3+ TRAEs Compared to Approved ADCs

[1128] Dose

[1129] (mg / kg)

[1130] Target

[1131] PYX-201 5.4 EDB+FN Auristatin 0101 0% 0% 8% 8% 1% Padcev 1.25 Nectin-4 Vedotin 0% 5% 10% 14% 0.80% Tivdak 2 JF Vedotin 3% 6% 3% <1% 0%

[1132] 6 DM4

[1133] Elahere Emtansine Folate 11% 3% 2% 0% 1% Enhertu 5.4 HER2 TOPO1 <0.1% <0.1% 17% <0.2% 1%

[1134]

[1135] Trodelvy 10 TROP2 SN38 0% 0% 49% <1% 2%

[1136] Example 4: PYX-201 Efficacy Following Previous Cancer Treatments

[1137] Participants in the Phase 1 Clinical Trial were in advanced stages of cancer with a median time from initial diagnosis of 39 months, and were heavily pretreated with a median of four prior cancer treatments. 71% of participants had previously received a taxane based

[1138] therapy, 69% had previously received a platinum based therapy, 43% had previously received an immunotherapy, and 18% had previously received an antibody-drug conjugate based treatment.

[1139] Importantly, as seen in Table 33, these heavily pretreated patients experienced tumor regression across a range of tumor types after being treated with other anti-cancer agents. More specifically, patients who had previously failed taxane-based therapy with head and neck cancer, ovarian cancer, NSCLC, HR+ breast cancer, TNBC, and sarcoma experienced tumor regression with PYX-201 treatment. Similarly, patients who had previously failed a platinum-based therapy across head and neck cancer, ovarian cancer, NSCLC, HR+ breast cancer, and TNBC experienced tumor regression. Further, patients in head and neck, NSCLC, and TNBC that were previously treated with a PD1 / PDL1 inhibitor experience tumor regression with PYX-201 treatment. Patients with HR+ breast cancer who previously were treated with CDK4 / 6 inhibitors experienced tumor regression with PYX-201 treatment. Patients with TNBC with prior Trop-2 ADC treatment with a topoisomerase inhibiting payload experience tumor regression with PYX-201 treatments, and patients with NSCLC previously treated with an ADC targeting 5T4 oncofetal antigen with an auristatin payload experience tumor regression with PYX-201 treatment.

[1140] Table 33 - PYX-201 Tumor Regression After Prior Treatments

[1141] 111

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[1143] Head HR+

[1144] Prior and Ovarian NSCLC HER2- TNBC Sarcoma Treatment Neck Breast

[1145] Taxanes X X X X X X Chemotherapeutics

[1146] Platinum X X X X X PD1 / PDL1

[1147] Immunotherapy X X X Inhibitors

[1148] PART X X X

[1149] Targeted Inhibitors

[1150] Therapies CDK4 / 6

[1151] X

[1152] Inhibitors

[1153] Trop-2 ADCs X Antibody-Drug

[1154] Conjugates 5T4 oncofetal X

[1155]

[1156] antigen ADCs

[1157] Enumerated Embodiments

[1158] In some aspects, the present invention is directed to the following non-limiting embodiments:

[1159] Embodiment 1: A method of treating a patient having a proliferative disorder, the method comprising administering to the patient an effective amount of an anti-extra domain B (EDB)+fibronectin (FN) antibody-drug conjugate (ADC) at least twice every week, at least weekly (QW), at least every' 2 weeks (Q2W), at least every' 3 weeks (Q3W), at least every' 4 weeks (Q4W), at least every 5 weeks (Q5W), or at least every' 6 weeks (Q6W), wherein the anti-EDB+FN ADC comprises an anti-EDB+FN antibody, or antigen binding fragment thereof, conjugated to a drug.

[1160] Embodiment 2: The method of embodiment 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 3.6 mg / kg to 5.4 mg / kg.

[1161] Embodiment 3: The method of embodiment 1. wherein the anti-EDB+FN ADC is administered to the patient at a dose of 3.6 mg / kg or greater.

[1162] Embodiment 4: The method of embodiment 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 4.4 mg / kg or greater.

[1163] Embodiment 5: The method embodiment 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 5.4 mg / kg.

[1164] Embodiment 6: The method of embodiment 1, wherein the anti-EDB+FN ADC is administered to the patient every' 3 weeks (Q3W).

[1165] Embodiment 7: The method of embodiment 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of about 0.3 mg / kg to about 8 mg / kg, about 0.6 mg / kg to about 8 mg / kg, about 1.2 mg / kg to about 8 mg / kg, about 2.4 mg / kg to about 8 mg / kg, about 112

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[1167] 3.6 mg / kg to about 8 mg / kg, about 4.4 mg / kg to about 8 mg / kg, about 5.4 mg / kg to about 8 mg / kg, about 6.6 mg / kg to about 8 mg / kg. about 0.3 mg / kg to about 6.6 mg / kg, about 0.6 mg / kg to about 6.6 mg / kg, about 1.2 mg / kg to about 6.6 mg / kg, about 2.4 mg / kg to about 6.6 mg / kg, about 3.6 mg / kg to about 6.6 mg / kg, about 4.4 mg / kg to about 6.6 mg / kg, about 5.4 mg / kg to about 6.6 mg / kg, about 0.3 mg / kg to about 5.4 mg / kg, about 0.6 mg / kg to about 5.4 mg / kg, about 1.2 mg / kg to about 5.4 mg / kg. about 2.4 mg / kg to about 5.4 mg / kg, about 3.6 mg / kg to about 5.4 mg / kg, about 4.4 mg / kg to about 5.4 mg / kg, about 0.3 mg / kg to about 4.4 mg / kg, about 0.6 mg / kg to about 4.4 mg / kg, about 1.2 mg / kg to about 4.4 mg / kg, about 2.4 mg / kg to about 4.4 mg / kg, about 0.3 mg / kg to about 3.6 mg / kg, about 0.6 mg / kg to about 3.6 mg / kg, about 1.2 mg / kg to about 3.6 mg / kg, about 2.4 mg / kg to about 3.6 mg / kg, about 0.3 mg / kg to about 2.4 mg / kg. about 0.6 mg / kg to about 2.4 mg / kg. about 1.2 mg / kg to about 2.4 mg / kg, about 0.3 mg / kg to about 1.2 mg / kg, about 0.6 mg / kg to about 1.2 mg / kg, or about 0.3 mg / kg to about 0.6 mg / kg.

[1168] Embodiment 8: The method of embodiment 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 0.3 mg / kg to 8 mg / kg. 0.6 mg / kg to 6.6 mg / kg, 1.2 mg / kg to 5.4 mg / kg, 2.4 to 5.4 mg / kg, 3.6 mg / kg to 5.4 mg / kg, or 4.4 mg / kg to 5.4 mg / kg.

[1169] Embodiment 9: The method of embodiment 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 3.6 mg / kg to 8 mg / kg, 3.6 mg / kg to 6.6 mg / kg, 3.6 mg / kg to 5.4 mg / kg, 3.6 mg / kg to 4.4 mg / kg, 4.4 mg / kg to 8 mg / kg, 4.4 mg / kg to 6.6 mg / kg.

[1170] 4.4 mg / kg to 5.4 mg / kg, 5.4 mg / kg to 8 mg / kg, 5.4 mg / kg to 6.6 mg / kg, 6.6 mg / kg to 8 mg / kg.

[1171] Embodiment 10: The method of embodiment 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of about 0.3 mg / kg, about 0.6 mg / kg, about 1.2 mg / kg, about 2.4 mg / kg. about 3.6 mg / kg. about 4.4 mg / kg, about 5.4 mg / kg, about 6.6 mg / kg or about 8 mg / kg.

[1172] Embodiment 11: The method of embodiment 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 0.3 mg / kg, 0.6 mg / kg, 1.2 mg / kg, 2.4 mg / kg, 3.6 mg / kg, 4.4 mg / kg, 5.4 mg / kg, 6.6 mg / kg or 8 mg / kg.

[1173] Embodiment 12: The method of embodiment 1. wherein the anti-EDB+FN ADC is administered to the patient at a dose of 3.6 mg / kg to 6.6 mg / kg.

[1174] Embodiment 13: The method of embodiment 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 3.6 mg / kg.

[1175] Embodiment 14: The method of embodiment 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 4.4 mg / kg to 6.6 mg / kg.

[1176] 113

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[1178] Embodiment 15: The method of embodiment 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 4.4 mg / kg to 5.4 mg / kg.

[1179] Embodiment 16: The method of embodiment 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 4.4 mg / kg.

[1180] Embodiment 17: The method of embodiment 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 5.4 mg / kg or greater.

[1181] Embodiment 18: The method of embodiment 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 5.4 mg / kg to 6.6 mg / kg.

[1182] Embodiment 19: The method of embodiment 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 6.6 mg / kg.

[1183] Embodiment 20: The method of embodiment 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 8 mg / kg.

[1184] Embodiment 21: The method of any one of embodiments 1-20, wherein the proliferative disorder is cancer.

[1185] Embodiment 22: The method of embodiment 21, wherein the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), breast cancer, head and neck cancer, ovarian cancer, thyroid cancer, pancreatic cancer, soft tissue sarcoma, chordoma, hepatocellular carcinoma, kidney cancer, cervical cancer, and endometrial cancer.

[1186] Embodiment 23: The method of embodiment 21, wherein the cancer is selected from the group consisting of non-small cell lung cancer, triple negative breast cancer, hormone receptor positive breast cancer, head and neck cancer, ovarian cancer, pancreatic cancer, soft tissue sarcoma, and chordoma.

[1187] Embodiment 24: The method embodiment 21, wherein the cancer is non-small cell lung cancer.

[1188] Embodiment 25: The method of embodiment 21, wherein the cancer is triple negative breast cancer.

[1189] Embodiment 26: The method of embodiment 21, wherein the cancer is hormone receptor positive breast cancer.

[1190] Embodiment 27: The method embodiment 21, wherein the cancer is hormone receptor positive, human epidermal grow th factor receptor 2 negative, breast cancer.

[1191] Embodiment 28: The method of embodiment 21, wherein the cancer is head and neck cancer.

[1192] Embodiment 29: The method of embodiment 21, wherein the cancer is head and neck squamous cell carcinoma.

[1193] 114

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[1195] Embodiment 30: The method of embodiment 21, wherein the cancer is ovarian cancer.

[1196] Embodiment 31: The method of embodiment 21, wherein the cancer is pancreatic cancer.

[1197] Embodiment 32: The method of embodiment 21, wherein the cancer is pancreatic ductal adeno carcinoma.

[1198] Embodiment 33: The method of embodiment 21, wherein the cancer is soft tissue sarcoma.

[1199] Embodiment 34: The method of embodiment 21, wherein the cancer is chordoma. Embodiment 35: The method of embodiments 1-34, wherein the drug is a cytotoxic agent.

[1200] Embodiment 36: The method of any one of embodiments 1-34, wherein the drug is a microtubule polymerization inhibitor.

[1201] Embodiment 37: The method of any one of embodiments 1-34, wherein the drug is a dolastatin, auristatin. or derivative thereof.

[1202] Embodiment 38: The method of any one of embodiments 1-34, wherein the drug is an auristatin.

[1203] Embodiment 39: The method of any one of embodiments 1-34, wherein the drug is selected from the group consisting of MMAD, MMAE, MMAF, auristatin 1569, auristatin 9411, auristatin 4574 and auristatin 0101.

[1204] Embodiment 40: The method of any one of embodiments I -34, wherein the drug is auristatin 0101.

[1205] Embodiment 41: The method of any one of embodiments 1-34, wherein the drug antibody ratio (DAR) is 1, 2. 3, or 4.

[1206] Embodiment 42: The method of any one of embodiments 1-41, wherein the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises: a. a heavy chain comprising a heavy chain constant region (CH) and heavy chain variable region (VH); b. a light chain comprising a light chain constant region (CL) and light chain variable region (VL); and c. at least one amino acid residue modification for site specific conjugation in a constant region of the antibody, or antigen binding fragment thereof.

[1207] Embodiment 43: The method of embodiment 42 wherein the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises at least one cysteine substitution for site specific conjugation in a constant region of the antibody, or antigen binding fragment thereof.

[1208] Embodiment 44: The method of embodiment 42, wherein: a. the CH region comprises 115

[1209] 567343972 Attorney Docket No. 384953-1024W01(00350)

[1210] 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, 428, 431, 432, 437, 438, 439, 443 or 444, or any combination thereof, according to the numbering of the EU index; or b. the CL region comprises: 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 111. 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).

[1211] Embodiment 45: The method of embodiment 42, wherein: a. the CH region comprises an engineered cysteine residue at position 290 according to the numbering of the EU index; or b. the CL region comprises an engineered cysteine residue at position 183 according to the numbering of Kabat.

[1212] Embodiment 46: The method of embodiment 42, wherein: a. the CH region comprises an engineered cysteine residue at position 290 according to the numbering of the EU index; and b. the CL region comprises an engineered cysteine residue at position 183 according to the numbering of Kabat.

[1213] Embodiment 47: The method of any one of embodiments 1-46, wherein the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises: a. a heavy chain comprising a heavy chain variable region (VH). the 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 b. a light chain comprising a light chain variable region (VL), the VL comprising the VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NO: 10.

[1214] Embodiment 48: The method of any one of embodiments 1-46, wherein the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises: a. a heavy chain comprising a heavy' chain variable region (VH), the VH comprising the VH amino acid sequence of SEQ ID NO: 21; or b. a light chain comprising a light chain variable region (VL), the VL comprising the VL amino acid sequence of SEQ ID NO: 10.

[1215] Embodiment 49: The method of any one of embodiments 1-46, wherein the anti- 116

[1216] 567343972 Attorney Docket No. 384953-1...

Claims

Attorney Docket No. 384953-1024W01(00350)CLAIMSWhat is claimed is:

1. A method of treating a patient having a proliferative disorder,the method comprising administering to the patient an effective amount of an anti-extra domain B (EDB)+fibronectin (FN) antibody-drug conjugate (ADC) at least twice every week, at least weekly (QW). at least even’ 2 weeks (Q2W), at least every 3 weeks (Q3W), at least every 4 weeks (Q4W), at least every 5 weeks (Q5W), or at least every 6 weeks (Q6W),wherein the anti-EDB+FN ADC comprises an anti-EDB+FN antibody, or antigen binding fragment thereof, conjugated to a drug.

2. The method of claim 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 3.6 mg / kg to 5.4 mg / kg.

3. The method of claim 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 3.6 mg / kg or greater.

4. The method of claim 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 4.4 mg / kg or greater.

5. The method of claim 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 5.4 mg / kg.

6. The method of claim 1, wherein the anti-EDB+FN ADC is administered to the patient every 3 weeks (Q3W).

7. The method of claim 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of about 0.3 mg / kg to about 8 mg / kg, about 0.6 mg / kg to about 8 mg / kg, about 1.2 mg / kg to about 8 mg / kg, about 2.4 mg / kg to about 8 mg / kg, about 3.6 mg / kg to about 8 mg / kg, about 4.4 mg / kg to about 8 mg / kg, about 5.4 mg / kg to about 8 mg / kg, about 6.6 mg / kg to about 8 mg / kg, about 0.3 mg / kg to about 6.6 mg / kg. about 0.6 mg / kg to about 6.6 mg / kg, about 1.2 mg / kg to about 6.6 mg / kg, about 2.4 mg / kg to about 6.6 mg / kg, about 3.6 mg / kg to about 6.6 mg / kg, about 4.4 mg / kg to about 6.6 mg / kg, about 5.4 mg / kg to about 6.6 mg / kg, about 0.3 mg / kg to about 5.4 mg / kg, about 0.6 mg / kg to about 5.4 mg / kg, about 1.2 mg / kg to about 5.4 mg / kg, about 2.4 mg / kg to about 5.4 mg / kg. about 3.6 mg / kg to about 5.4 mg / kg. about 4.4119567343972Attorney Docket No. 384953-1024W01(00350)mg / kg to about 5.4 mg / kg, about 0.3 mg / kg to about 4.4 mg / kg, about 0.6 mg / kg to about 4.4 mg / kg, about 1.2 mg / kg to about 4.4 mg / kg. about 2.4 mg / kg to about 4.4 mg / kg, about 0.3 mg / kg to about 3.6 mg / kg, about 0.6 mg / kg to about 3.6 mg / kg, about 1.2 mg / kg to about 3.6 mg / kg, about 2.4 mg / kg to about 3.6 mg / kg, about 0.3 mg / kg to about 2.4 mg / kg, about 0.6 mg / kg to about 2.4 mg / kg, about 1.2 mg / kg to about 2.4 mg / kg, about 0.3 mg / kg to about 1.2 mg / kg. about 0.6 mg / kg to about 1.2 mg / kg. or about 0.3 mg / kg to about 0.6 mg / kg.

8. The method of claim 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 0.3 mg / kg to 8 mg / kg, 0.6 mg / kg to 6.6 mg / kg, 1.2 mg / kg to 5.4 mg / kg, 2.4 to 5.4 mg / kg, 3.6 mg / kg to 5.4 mg / kg, or 4.4 mg / kg to 5.4 mg / kg.

9. The method of claim 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 3.6 mg / kg to 8 mg / kg, 3.6 mg / kg to 6.6 mg / kg, 3.6 mg / kg to 5.4 mg / kg, 3.6 mg / kg to 4.4 mg / kg, 4.4 mg / kg to 8 mg / kg, 4.4 mg / kg to 6.6 mg / kg, 4.4 mg / kg to 5.4 mg / kg, 5.4 mg / kg to 8 mg / kg, 5.4 mg / kg to 6.6 mg / kg, 6.6 mg / kg to 8 mg / kg.

10. The method of claim 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of about 0.3 mg / kg, about 0.6 mg / kg, about 1.2 mg / kg, about 2.4 mg / kg, about 3.6 mg / kg, about 4.4 mg / kg, about 5.4 mg / kg, about 6.6 mg / kg or about 8 mg / kg.

11. The method of claim 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 0.3 mg / kg, 0.6 mg / kg, 1.2 mg / kg, 2.4 mg / kg, 3.6 mg / kg, 4.4 mg / kg, 5.4 mg / kg, 6.6 mg / kg or 8 mg / kg.

12. The method of claim 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 3.6 mg / kg to 6.6 mg / kg.

13. The method of claim 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 3.6 mg / kg.

14. The method of claim 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 4.4 mg / kg to 6.6 mg / kg.

15. The method of claim 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 4.4 mg / kg to 5.4 mg / kg.120567343972Attorney Docket No. 384953-1024W01(00350)16. The method of claim 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 4.4 mg / kg.

17. The method of claim 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 5.4 mg / kg or greater.

18. The method of claim 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 5.4 mg / kg to 6.6 mg / kg.

19. The method of claim 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 6.6 mg / kg.

20. The method of claim 1, wherein the anti-EDB+FN ADC is administered to the patient at a dose of 8 mg / kg.

21. The method of any one of claims 1-20, wherein the proliferative disorder is cancer.

22. The method of claim 21, wherein the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), breast cancer, head and neck cancer, ovarian cancer, thyroid cancer, pancreatic cancer, soft tissue sarcoma, chordoma, hepatocellular carcinoma, kidney cancer, cervical cancer, and endometrial cancer.

23. The method of claim 21, wherein the cancer is selected from the group consisting of non-small cell lung cancer, triple negative breast cancer, hormone receptor positive breast cancer, head and neck cancer, ovarian cancer, pancreatic cancer, soft tissue sarcoma, and chordoma.

24. The method of claim 21, wherein the cancer is non-small cell lung cancer.

25. The method of claim 21, wherein the cancer is triple negative breast cancer.

26. The method of claim 21, wherein the cancer is hormone receptor positive breast cancer.

27. The method of claim 21, wherein the cancer is hormone receptor positive, human epidermal growth factor receptor 2 negative, breast cancer.

28. The method of claim 21, wherein the cancer is head and neck cancer.121567343972Attorney Docket No. 384953-1024W01(00350)29. The method of claim 21, wherein the cancer is head and neck squamous cell carcinoma.

30. The method of claim 21, wherein the cancer is ovarian cancer31. The method of claim 21, wherein the cancer is pancreatic cancer.

32. The method of claim 21, wherein the cancer is pancreatic ductal adeno carcinoma.

33. The method of claim 21, wherein the cancer is soft tissue sarcoma.

34. The method of claim 21, wherein the cancer is chordoma.

35. The method of claims 1-34, wherein the drug is a cytotoxic agent.

36. The method of any one of claims 1-34, wherein the drug is a microtubule polymerization inhibitor.

37. The method of any one of claims 1-34, wherein the drug is a dolastatin, auristatin, or derivative thereof.

38. The method of any one of claims 1-34, wherein the drug is an auristatin.

39. The method of any one of claims 1-34, wherein the drug is selected from the group consisting of MMAD, MMAE, MMAF, auristatin 1569, auristatin 9411, auristatin 4574 and auristatin 0101.

40. The method of any one of claims 1-34, wherein the drug is auristatin 0101.

41. The method of any one of claims 1-34, wherein the drug antibody ratio (DAR) is 1, 2, 3, or 4.

42. The method of any one of claims 1-41, wherein the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises:(a) a heavy chain comprising a heavy chain constant region (CH) and heavy chain variable region (VH),(b) a light chain comprising a light chain constant region (CL) and light chain variable region (VL), and(c) at least one amino acid residue modification for site specific conjugation in a constant region of the antibody, or antigen binding fragment thereof.122567343972Attorney Docket No. 384953-1024W01(00350)43. The method of claim 42 wherein the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises at least one cysteine substitution for site specific conjugation in a constant region of the antibody, or antigen binding fragment thereof.

44. The method of claim 42, wherein:(a) the CH region comprises 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, 428, 431, 432, 437, 438, 439, 443 or 444, or any combination thereof, according to the numbering of the EU index; or(b) the CL region comprises:(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).

45. The method of claim 42, wherein:(a) the CH region comprises an engineered cysteine residue at position 290 according to the numbering of the EU index; or(b) the CL region comprises an engineered cysteine residue at position 183 according to the numbering of Kabat.

46. The method of claim 42, wherein:(a) the CH region comprises an engineered cysteine residue at position 290 according to the numbering of the EU index; and123567343972Attorney Docket No. 384953-1024W01(00350)(b) the CL region comprises an engineered cysteine residue at position 183 according to the numbering of Kabat.

47. The method of any one of claims 1-46, wherein the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises:(a) a heavy chain comprising a heavy chain variable region (VH), the 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(b) a light chain comprising a light chain variable region (VL), the VL comprising the VL CDR1 region, VL CDR2 region, and VL CDR3 region of the VL amino acid sequence of SEQ ID NO: 10.

48. The method of any one of claims 1-46, wherein the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises:(a) a heavy chain comprising a heavy chain variable region (VH), the VH comprising the VH amino acid sequence of SEQ ID NO: 21; or (b) a light chain comprising a light chain variable region (VL), the VL comprising the VL amino acid sequence of SEQ ID NO: 10.

49. The method of any one of claims 1-46, wherein the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises:(a) a heavy chain comprising a heavy chain variable region (VH). the VH comprising the VH amino acid sequence of SEQ ID NO: 21; and (b) a light chain comprising a light chain variable region (VL), the VL comprising the VL amino acid sequence of SEQ ID NO: 10.

50. The method of any one of claims 1-46, wherein the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises:(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 25; or (b) a light chain comprising the amino acid sequence of SEQ ID NO:

31.

51. The method of any one of claims 1-46, wherein the anti-EDB+FN antibody, or antigen binding fragment thereof, comprises:(a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 25; and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 31.124567343972Attorney Docket No. 384953-1024W01(00350)52. The method of any one of claims 1- 1, wherein the anti-EDB+FN ADC further comprises a linker, wherein the linker conjugates the anti-cancer drug to the antibody, or antigen binding fragment thereof.

53. The method of claim 52, wherein the linker is a cleavable linker.

54. The method of claim 52, wherein the linker is selected from the group consisting of val-cit, phe-lys, vc. AcLys-vc, diS, diS-C2OCO, me, me, MalPeg6C2, and a hydrazone linker.

55. The method of any one of claims 52, wherein the linker is selected from the group consisting of vc, diS, diS-C2OCO, and AcLys-vc.

56. The method of any one of claims 52, wherein the linker is vc.

57. The method of any one of claims 1-56, wherein the ADC is PYX-201.125567343972

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