Compositions and methods for binding src

Abstract

The present disclosure relates to antibodies to Src and therapeutic compositions targeting Src. For example, the present disclosure relates to antibody-drug conjugates, antibody-radionuclide conjugates, and bispecific T cell engagers. The disclosure also provides small molecule conjugates useful for targeting Src. Also provided are methods of producing such compositions, nucleic acids encoding same, host cells genetically modified with the nucleic acids, as well as methods for treating cancer.

Description

Mintz Ref. No.: 048536-797001WO COMPOSITIONS AND METHODS FOR BINDING SRC CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to U.S. Provisional PatentApplication No.63 / 666,521, filed on July 1, 2024, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under P41 CA196276, CA191018,and GM097316 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD

[0003] The present disclosure relates to therapeutic compositions that bind Src. For example,the present disclosure relates to antibodies and antigen fragments thereof that bind Src. The disclosure also provides methods useful for producing such compositions, nucleic acids encoding same, host cells genetically modified with the nucleic acids, as well as methods for treatment of cancer. BACKGROUND

[0004] Src family kinases are a family of cytoplasmic tyrosine kinases. Src family kinaseshave a unique structure that includes an N-terminal region with a 14-carbon myristoyl group, two Src homology domains (SH2 and SH3), a catalytic tyrosine-protein kinase domain (SH1), and a short C-terminal tail. The SH1 domain has catalytic function, while the SH2 and SH3 domains have non-catalytic regulatory property, but all three domains are important for signal transduction. Src tyrosine kinase enzymes are involved in the signaling pathways that control a diverse spectrum of biological activities that include gene transcription, immune response, cell adhesion, cell cycle progression, cell differentiation, apoptosis, movement, transformation, proliferation, and other essential cellular functions.

[0005] Src is clinically relevant as it plays an important role in oncogenesis and is a knowncancer biomarker. Although Src expression and its specific activity are frequently elevated in a number of human cancers, it is rarely mutated. Nonetheless, Src is a key player in the maintenance of the neoplastic phenotype and promotes cell migration and invasion, which 1Mintz Ref. No.: 048536-797001WO have been linked to tumor progression and metastases. This has led to the development of several Src inhibitors as potential chemotherapeutic reagents. Unfortunately, generating highly selective inhibitors to block protein kinase function in biological studies remains a challenge and new strategies are required. Cancer biomarkers such as Src also require a high level of stringency for clinical translation. Most biomarkers require either a dramatic degree of protein-level upregulation or a completely new epitope (generated either from germline mutation or a unique post-translational modification) to be therapeutically leveraged.

[0006] Thus, there is a need in the art for novel Src ligands as therapeutics in cancer.SUMMARY

[0007] The present disclosure relates generally to antibodies against Src as well as conjugatecompounds and bispecific T cell engagers comprising these antibodies for potential use in cancer therapeutics.

[0008] The present disclosure provides an anti-Src antibody or antigen binding fragmentthereof comprising (a) a heavy chain variable region comprising a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3; and (b) a light chain variable region comprising a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 6.

[0009] The present disclosure also provides an anti-Src antibody or antigen binding fragmentthereof comprising (a) a heavy chain variable region comprising a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10.

[0010] The present disclosure also provides an anti-Src antibody or antigen binding fragmentthereof comprising (a) a heavy chain variable region comprising a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 136, and a heavy chain CDR3 domain 2Mintz Ref. No.: 048536-797001WO comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10.

[0011] The present disclosure also provides an anti-Src antibody or antigen binding fragmentthereof comprising (a) a heavy chain variable region comprising a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 137, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10.

[0012] The present disclosure also provides an anti-Src antibody or antigen binding fragmentthereof comprising (a) a heavy chain variable region comprising a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 136, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 137, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10.

[0013] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12.

[0014] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises a heavy chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 34 and a light chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0015] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14. 3Mintz Ref. No.: 048536-797001WO

[0016] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises a heavy chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 23 and a light chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 21.

[0017] In some embodiments, the anti-Src antibody or the antigen-binding fragment thereofis a monoclonal antibody, synthetic antibody, murine antibody, chimeric antibody, humanized antibody, or human antibody.

[0018] In some embodiments, the antibody or fragment thereof comprises full-lengthantibodies, Fab, F(ab′)2, Fd, Fv, scFv, domain antibodies, dual-specific antibodies, bibodies, minibodies, tribodies, bispecific antibodies, trispecific antibodies, multispecific antibodies, diabodies, triabodies, tetrabodies, intrabodies, nanobodies, small modular immunopharmaceuticals (SMTP), binding-domain immunoglobulin fusion proteins, camelized antibodies or VHH containing antibodies.

[0019] In some embodiments, the antibody comprises IgD antibodies, IgE antibodies, IgMantibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies.

[0020] Also provided herein is an antibody-drug conjugate comprising a cytotoxic ortherapeutic compound that is conjugated to an anti-Src antibody or antigen binding fragment thereof.

[0021] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises: (a) a heavy chain variable region comprising a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3; and (b) a light chain variable region comprising a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 6. In one embodiment, the anti-Src antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12.

[0022] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises a heavy chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 34 and a light chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 35. 4Mintz Ref. No.: 048536-797001WO

[0023] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises (a) a heavy chain variable region comprising a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10.

[0024] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises (a) a heavy chain variable region comprising a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 136, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10.

[0025] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises (a) a heavy chain variable region comprising a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 137, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10.

[0026] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises (a) a heavy chain variable region comprising a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 136, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 137, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10. 5Mintz Ref. No.: 048536-797001WO

[0027] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14.

[0028] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises a heavy chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 34 and a light chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0029] In some embodiments, the cytotoxic compound is selected from a tubulin inhibitor ora topoisomerase inhibitor.

[0030] In one embodiment, the cytotoxic compound is a tubulin inhibitor. In oneembodiment, the tubulin inhibitor is monomethyl auristatin E (MMAE).

[0031] In one embodiment, the cytotoxic compound is a topoisomerase inhibitor. In oneembodiment, the topoisomerase inhibitor is selected from SN38 and Dxd.

[0032] Also provided herein in is an antibody-radionuclide conjugate comprising aradionuclide that is conjugated to an anti-Src antibody or antigen binding fragment thereof.

[0033] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises (a) a heavy chain variable region comprising a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3; and (b) a light chain variable region comprising a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 6. In one embodiment, the anti-Src antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12.

[0034] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises a heavy chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 34 and a light chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0035] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises (a) a heavy chain variable region comprising a heavy chain CDR1 domain 6Mintz Ref. No.: 048536-797001WO comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10.

[0036] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises (a) a heavy chain variable region comprising a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 136, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10.

[0037] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises (a) a heavy chain variable region comprising a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 137, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10.

[0038] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14.

[0039] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises a heavy chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 34 and a light chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0040] In some embodiments, the radionuclide is a therapeutic radionuclide.

[0041] In some embodiments, the radionuclide is a diagnostic radionuclide.7Mintz Ref. No.: 048536-797001WO

[0042] Also provided herein is a bispecific T cell engager comprising (a) an anti-Srcantibody or antigen binding fragment thereof and (b) a T cell antigen-binding moiety.

[0043] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises (a) a heavy chain variable region comprising a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3; and (b) a light chain variable region comprising a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 6. In one embodiment, the anti-Src antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12. In one embodiment, the anti-Src antibody or antigen binding fragment thereof comprises a heavy chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 34 and a light chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0044] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises (a) a heavy chain variable region comprising a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10.

[0045] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises (a) a heavy chain variable region comprising a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 136, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10. 8Mintz Ref. No.: 048536-797001WO

[0046] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises (a) a heavy chain variable region comprising a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 137, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10.

[0047] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises (a) a heavy chain variable region comprising a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 136, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 137, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10.

[0048] In one embodiment, the anti-Src antibody or antigen binding fragment thereofcomprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14. In one embodiment, the anti-Src antibody or antigen binding fragment thereof comprises a heavy chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 34 and a light chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0049] In some embodiments, the T cell antigen-binding moiety binds to CD3.

[0050] Also provided herein is a conjugate compound comprising (a) a Src ligand and (b) acytotoxic or therapeutic compound.

[0051] In some embodiments, the conjugate compound is cell impermeable.

[0052] In some embodiments, the Src ligand is a small molecule. In some embodiments, thesmall molecule is dasatinib, sarcatinib, a DFG-out selective derivative, or a covalent derivative of those molecules. In some embodiments, the small molecule is azido-dasatinib.

[0053] In some embodiments, the cytotoxic compound is selected from a tubulin inhibitor ora topoisomerase inhibitor. 9Mintz Ref. No.: 048536-797001WO

[0054] In some embodiments, the cytotoxic compound is a tubulin inhibitor. In someembodiments, the tubulin inhibitor is monomethyl auristatin E (MMAE).

[0055] In some embodiments, the cytotoxic compound is a topoisomerase inhibitor. In someembodiments, the topoisomerase inhibitor is selected from SN38 and Dxd.

[0056] Also provided herein is a conjugate compound comprising (a) a Src ligand and (b) aradionuclide.

[0057] In some embodiments, the conjugate compound is cell impermeable.

[0058] In some embodiments, the Src ligand is a small molecule. In some embodiments, thesmall molecule is dasatinib, sarcatinib, a DFG-out selective derivative, or a covalent derivative of those molecules. In some embodiments, the small molecule is azido-dasatinib.

[0059] In some embodiments, the radionuclide is a therapeutic radionuclide.

[0060] In some embodiments, the radionuclide is a diagnostic radionuclide.

[0061] Also provided herein is a bifunctional molecule comprising (a) a Src ligand and (b) aT cell antigen-binding moiety.

[0062] In some embodiments, bifunctional molecule is cell impermeable.

[0063] In some embodiments, the Src ligand is a small molecule. In some embodiments,thesmall molecule is dasatinib, sarcatinib, a DFG-out selective derivative, or a covalent derivative of those molecules. In some embodiments, the small molecule is azido-dasatinib.

[0064] In some embodiments, the T cell antigen-binding moiety binds to CD3.

[0065] In some embodiments, the T cell antigen binding moiety is an anti-CD3 engagingFab. In some embodiments, the anti-CD3 Fab is a cyclooctyne-conjugated anti-CD3 engaging Fab.

[0066] Also provided herein is a compound represented by of Formula (I):(I), or a pharmaceutidefined herein.

[0067] The present disclosure also provides a pharmaceutical composition comprising theanti-Src antibody or antigen binding fragment thereof, antibody-drug conjugate, antibody- radionuclide conjugate, bispecific T cell engager, conjugate compound, or bifunctional molecule of any preceding claim.

[0068] The present disclosure also provides a nucleic acid encoding the anti-Src antibody orantigen binding fragment thereof according to the present disclosure 10Mintz Ref. No.: 048536-797001WO

[0069] The present disclosure also provides a vector comprising the nucleic acid according tothe present disclosure.

[0070] The present disclosure also provides a host cell comprising the nucleic acid accordingto the present disclosure.

[0071] The present disclosure also provides a method of treating cancer in a subjectcomprising administering, to the subject, the anti-Src antibody or antigen binding fragment thereof, antibody-drug conjugate, antibody-radionuclide conjugate, bispecific T cell engager, conjugate compound, or bifunctional molecule of the present disclosure.

[0072] In some embodiments, the cancer is selected from breast cancer, lung cancer,fibrosarcoma, melanoma, prostate cancer, colon cancer, pancreatic cancer.

[0073] The present disclosure also provides a method of inhibiting or reducing cancer cellgrowth comprising contacting the cancer cell with the anti-Src antibody or antigen binding fragment thereof, antibody-drug conjugate, antibody-radionuclide conjugate, bispecific T cell engager, conjugate compound, or bifunctional molecule of the present disclosure.

[0074] The present disclosure also provides a method of inhibiting or reducing cancer cellgrowth comprising contacting the cancer cell with a biological molecule that specifically binds a kinase selected from the group consisting of Src, Hck, and Lyn, wherein at least a portion of the kinase is located on the cell surface.

[0075] The present disclosure also provides a method of inhibiting or reducing cancer cellgrowth comprising contacting the cancer cell with a conjugate comprising a cytotoxic, therapeutic, or diagnostic compound that is conjugated a biological molecule, wherein the biological molecule specifically binds a kinase of the Src kinase family selected from the group consisting of Src, Hck, and Lyn, wherein at least a portion of the kinase is located on the cell surface.

[0076] The present disclosure also provides a method of inhibiting or reducing cancer cellgrowth comprising contacting the cancer cell with a bispecific T cell engager comprising a biological molecule and a T cell antigen-binding moiety, wherein the biological molecule specifically binds a kinase of the Src kinase family selected from the group consisting of Src, Hck, and Lyn, wherein at least a portion of the kinase is located on the cell surface.

[0077] The present disclosure also provides a method of inhibiting or reducing cancer cellgrowth comprising contacting the cancer cell with a bifunctional molecule comprising a biological molecule and a T cell antigen-binding moiety, wherein the biological molecule 11Mintz Ref. No.: 048536-797001WO specifically binds a kinase of the Src kinase family selected from the group consisting of Src, Hck, and Lyn, wherein at least a portion of the kinase is located on the cell surface.

[0078] The present disclosure also provides a biological molecule that specifically binds akinase of the Src kinase family, wherein the kinase is selected from the group consisting of Src, Hck, and Lyn, wherein at least a portion of the kinase is located on the cell surface.

[0079] The present disclosure also provides a conjugate comprising a cytotoxic, therapeutic,or diagnostic compound that is conjugated to the biological molecule of the present disclosure.

[0080] The present disclosure also provides a bispecific T cell engager comprising thebiological molecule of the present disclosure and a T cell antigen binding moiety.

[0081] The present disclosure also provides a bifunctional molecule comprising thebiological molecule of the present disclosure and a T cell antigen binding moiety.

[0082] The present disclosure also provides a pharmaceutical composition comprising thebiological molecule, conjugate, bispecific T cell engager, or bifunctional molecule of the present disclosure.

[0083] The present disclosure also provides a nucleic acid encoding the biological moleculeaccording to the present disclosure.

[0084] The present disclosure also provides a vector comprising the nucleic acid according tothe present disclosure.

[0085] The present disclosure also provides a host cell comprising the nucleic acid accordingto the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] The features of the present disclosure are set forth with particularity in the appendedclaims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0087] FIGs. 1A-1I show Src is secreted, presented, and active on the surface of a variety ofcancer cell types. FIG.1A shows HCC1569 breast cancer cells treated with or without ATP (1 mM) and with or without Src inhibitor 1 (1 µM) and stained with an anti-phosphotyrosine [J. Am. Chem. Soc.2018, 140, 48, 16615–16624] antibody conjugated AlexaFluor647 for 12Mintz Ref. No.: 048536-797001WO analysis by flow cytometry. HCC1569 cells were treated with Src inhibitor 1 (1 µM) or an equal volume of DMSO in the presence of ATP. Statistics were determined by one-wayANOVA corrected for multiple comparisons; p value is displayed above relevant comparison.FIGs.1B-1C show HCC1569 cells treated with or without tumor-relevant concentrations of ATP and with or without Src inhibitor 1. A pan anti-phosphotyrosine antibody coupled to a Dexter energy transfer iridium catalyst was then bound to the cells in the presence of a diazirine biotin followed by excitation with blue light to generate reactive carbenes and label phosphoproteins in situ. Cells were then lysed, and the biotinylated phosphoproteins were enriched on neutravidin, digested with trypsin, and tryptic peptides were analyzed by LCMS. FIG.1B is an UpSet plot representing unique proteins with at least two unique peptides identified in at least two of four replicates. FIG.1C shows protein abundance based on peptides areas were calculated by label-free quantitation. Log2 fold change of vehicle treated versus Src inhibitor 1 treated cells is shown. Protein with at least two unique peptides is displayed. FIGs.1D-1E show HCC1569 cells cultured in the presence of wild-type or loss of function recombinant Src kinase domain and ATP (FIG.1D), or likewise in the presence of ADAM17 inhibitor TAPI-2 (FIG.1E). Growth kinetics were monitored by imaging microscopy on an Incucyte SX5. FIG.1F shows cells from various tissues and pathologies stained with a commercial flow cytometry validated anti-Src antibody and analyzed by flow cytometry. FIG.1G shows normalized protein amounts of whole cell lysates separated by SDS-PAGE and transferred to nitrocellulose for probing with anti-Src antibody by Western blot with detection using a near-infrared dye conjugated antibody. Normalized total Src fluorescence by western blot plotted against the MFI of eSrc detected by flow cytometry. FIG.1H shows HCC1569 cells treated with human N-myristoyl transferase 1 / 2 inhibitor IMP-1088 (100 nM) in culture for 72 hours before analysis of eSrc expression by flow cytometry. FIG.1I shows PaTu8902 cells transfected with plasmid DNA encoding either Src-FLAG or Src(G2A)-FLAG. Live cells were analyzed by flow cytometry for Src and FLAG surface expression 48 hours post transfection.

[0088] FIGs. 2A-2B show representative histograms for FIG. 1A. FIG. 2A shows HCC1569breast cancer cells treated with or without ATP (1 mM) and stained with an anti- phosphotyrosine [J. Am. Chem. Soc.2018, 140, 48, 16615–16624] antibody conjugated AlexaFluor647, an isotype and fluorophore-matched control antibody, or vehicle for analysis by flow cytometry. FIG.2B shows HCC1569 breast cancer cells treated with or without ATP (1 mM) and with or without Src inhibitor 1 (1 μM) and stained with an anti-phosphotyrosine 13Mintz Ref. No.: 048536-797001WO [J. Am. Chem. Soc.2018, 140, 48, 16615–16624] antibody conjugated AlexaFluor647 for analysis by flow cytometry.

[0089] FIG. 3 shows a schematic for photoaffinity-based proximity labeling of extracellularSrc substrates. HCC1569 cells are exposed to extracellular ATP in the presence or DMSO or Src inhibitor 1. A high-affinity phosphotyrosine antibody (clone 4G10-G6) [J. Am. Chem. Soc.2018, 140, 48, 16615–16624] coupled to an iridium-based photocatalyst for Dexter energy transfer is then bound to surfaces. Biotin-diazirine is introduced to the live cells, which are then irradiated with blue (460 nm) light, exciting the Ir photocatalyst, which in turn generates carbene radicals from the biotin-diazirine in a short radius (<5 nm), as has been previously described [Science 2020, 367 (6482), 1091-1097]. The short half-life of the carbene radical results in short-distance labeling, favoring the phosphoprotein in complex with the Ab-photocatalyst. Biotinylated proteins are enriched, digested, and analyzed by liquid-chromatography mass spectrometry.

[0090] FIG. 4 shows quantitation of enriched proteins from photoaffinity labeling withphotocatalyst on 4G10-G6 or an isotype-control. JURKAT lysates were spiked with a fragment of antigen conjugated to an iridium-based photocatalyst (either anti- phosphotyrosine clone 4G10-G6 [J. Am. Chem. Soc.2018, 140, 48, 16615–16624] or an irrelevant control) and biotin-diazirine. Photocatalysts were activated with blue (460 nm) light. Lysates were desalted, and biotinylated proteins were enriched on NeutrAvidin resin, followed by on-resin digest, from which tryptic peptides were collected, desalted, separated by liquid chromatography, and analyzed by mass spectrometry. Peptides were matched to proteins using PEAKS [Mol. Cell. Proteomics 2012, 11(4)], and proteins containing 2 or more confident and unique peptides were compared by label-free quantitation. Phosphotyrosine-containing proteins identified previously by immunoprecipitation using clone 4G10-G6 [J. Am. Chem. Soc.2018, 140, 48, 16615–16624] are highlighted in red. Data was derived from three biological replicates per condition.

[0091] FIG. 5 shows quantitation of enriched proteins from photoaffinity labeling withphotocatalyst on 4G10-G6 or purified from whole-cell lysate. For photoaffinity-labeled samples, JURKAT lysates were spiked with a fragment of antigen binding conjugated to an iridium-based photocatalyst (anti-phosphotyrosine clone 4G10-G6 [J. Am. Chem. Soc.2018, 140, 48, 16615–16624] and biotin-diazirine. Photocatalysts were activated with blue (460 nm) light. Lysates were desalted, and biotinylated proteins were enriched on NeutrAvidin resin, followed by on-resin digest, from which tryptic peptides were collected, desalted, 14Mintz Ref. No.: 048536-797001WO separated by liquid chromatography, and analyzed by mass spectrometry. For whole- cell lysates, lysates were alkylated, digested, and analyzed by LC-MS. Peptides were matched to proteins using PEAKS, and proteins containing 2 or more confident and unique peptides were compared by label-free quantitation. Phosphotyrosine-containing proteins identified previously by immunoprecipitation using clone 4G10-G6 [J. Am. Chem. Soc.2018, 140, 48, 16615–16624] are highlighted in red. Data was derived from three biological replicates per condition.

[0092] FIG. 6 shows photoaffinity labeling yields direct enrichment of phosphotyrosine-containing peptides. For photoaffinity-labeled samples, JURKAT lysates were spiked with a fragment of antigen binding conjugated to an iridium-based photocatalyst (anti- phosphotyrosine clone 4G10-G6 [J. Am. Chem. Soc.2018, 140, 48, 16615–16624] and biotin-diazirine. Photocatalysts were activated with blue (460 nm) light. Lysates were desalted, and biotinylated proteins were enriched on NeutrAvidin resin, followed by on-resin digest, from which tryptic peptides were collected, desalted, separated by liquid chromatography, and analyzed by mass spectrometry. For whole-cell lysates, lysates were alkylated, digested, and analyzed by LC-MS. Peptides were matched to proteins using PEAKS [Mol. Cell. Proteomics 2012, 11(4)], and unique peptides identified in all samples were compared by label-free quantitation. Significantly enriched and confident phosphotyrosine containing peptides (PTM confidence <1% FDR) are highlighted in red. Data was derived from three biological replicates per condition.

[0093] FIGs. 7A-7B show photoaffinity-labeling identified known and putative Srcsubstrates using inhibitor and substrate-limited conditions. Live HCC1569 cells were treated with or without ATP and with or without Src inhibitor 1. The cells were washed and labeled with a fragment of antigen conjugated to an iridium-based photocatalyst (anti- phosphotyrosine clone 4G10-G6 [J. Am. Chem. Soc.2018, 140, 48, 16615–16624]). And Biotin-diazirine was added and photocatalysts were activated with blue (460 nm) light. Cells were washed, lysed, and biotinylated proteins were enriched on NeutrAvidin resin, followed by on-resin digest, from which tryptic peptides were collected, desalted, separated by liquid chromatography, and analyzed by mass spectrometry. Peptides were matched to proteins using PEAKS [Mol. Cell. Proteomics 2012, 11(4)], and proteins containing 2 or more confident and unique peptides were compared by label-free quantitation. Select proteins are highlighted in red. FIG.7A shows cells treated with ATP (VEH) compared to cells treated without ATP (NoTx). FIG.7B shows cells treated with ATP and Src inhibitor 1 (iSRC) 15Mintz Ref. No.: 048536-797001WO compared to cells treated without ATP (NoTx). Data was derived from four biological replicates per condition.

[0094] FIG. 8 shows in vitro validation of putative Src substrates by Western blot.Recombinant proteins (1 μM) were treated with recombinant Src kinase domain [Nucleic Acids Res 2021, 49(8) 480-489] (WT) or a loss-of-function (LOF) analogue (corresponding to D359A of the full-length Src) in the presence of 1 mM ATP in a kinase reaction buffer. Reactions were separated by SDS-PAGE, transferred to PVDF membranes, stained for total protein with REVERT Total Protein Stain, washed, blocked, probed with anti- phosphotyrosine clone 4G10-G6, and visualized with anti-hFc-CW800 and NeutrAvidin-680 using a LiCOR CLx.

[0095] FIGs. 9A-9C show visualization of Src-dependent tyrosine phosphorylation sites onErbb2, CD44, and ADAM17. Sites of Src-dependent tyrosine phosphorylation identified from LC-MS of recombinant phosphoproteins were visualized in Chimera. All identified tyrosines were solvent exposed. Confidence of phosphorylation identification is displayed next to the site ID in the caption: * corresponds to p < 0.05; ** corresponds to p < 0.01; **** corresponds to p < 0.001.

[0096] FIG. 10 shows Src stimulates ADAM17 activity in vitro. Recombinant ADAM17 wasimmobilized on a MaxiSorp plate, which was then blocked with BSA. Immobilized ADAM17 was treated with or without Src, washed, and then a fluorogenic TACE substrate was added. Fluorescence was measured using a fluorescence module on a Tecan M200Pro plate reader. Relevant comparison was measured using a Student’s t test, with the p-value displayed above the comparison. Samples were measured in technical triplicate.

[0097] FIG. 11 shows western blot analysis of HCC1569 secretions. Whole cell lysate(WCL) or conditioned media (CM) from HCC1569 cells cultured for 24 hours in serum-free media were separated by SDS-PAGE, transferred to PVDF membranes, blocked, probedprobed with anti- tubulin and anti-Src antibodies, and visualized with anti-rabbit IgG-CW800and anti-mouse IgG-IR680 using a LiCOR CLx.

[0098] FIGs. 12A-12C show SRC siRNA knockdown reduces eSrc abundance and total Srcexpression. HCC1569 cells were lipofected with an siRNA either complementing the coding region of SRC (iSRC), the gene encoding Src, or a non-complementary control (NC-1). Cells were analyzed 72 hours after transfection. In FIGs.12A-12B, cells were analyzed for eSrc abundance by flow cytometry. FIG.12A shows quantification data across three individual replicates. Error bars represent standard deviation. Statistics calculated by Student’s t-test. 16Mintz Ref. No.: 048536-797001WO Adjusted p-value is displayed above the comparison; * corresponds to p < 0.05. FIG.12Bshows representative cytograms for a single replicate of FIG. 12A. FIG. 12C shows cellswere lysed and lysates were probed by Western blot with either an anti-tubulin (left) or anti- Src (right) antibody.

[0099] FIG. 13 shows SRC transcript level is not correlated with eSrc expression. eSrc meanfluorescence intensity values were obtained from Figure 1F. SRC transcript quantitation values were obtained from the Human Protein Atlas [Nat. Biotech.201028, 1248-1250; proteinatlas.org]

[0100] FIGs. 14A-14B shows tyrosine ectokinase activity correlates with eSrc expression inisogenic MCF10A derivative cell lines. MCF10A cells transduced with empty vector (EV), an oncogenic KRAS mutation (KRAS), or an oncogenic HER2 mutation (HER2) previously to establish isogenic cell lines [Proc. Natl. Acad. Sci.2020, 117(14), 7764-7775]. FIG.14A shows MCF10A isogenic cell lines were stained with anti-Src and eSrc abundance was measured by flow cytometry. FIG.14B shows MCF10A isogenic cell lines with varying abundances of eSrc were incubated with or without ATP (1 mM) and with or without Src inhibitor 1 (1 µM). Cells were then stained with an anti-phosphotyrosine antibody to quantify extracellular surface phosphotyrosine abundance by flow cytometry.

[0101] FIG. 15 shows representative Western blots show variable levels total Src across celllines from whole cell lysate. Cells were lysed in denaturing lysis buffer with protease inhibitors and normalized protein quantities were separated by SDS-PAGE. Gels were transferred to PVDF membranes, probed with anti-Src (Invitrogen, #701396)) and visualized with goat anti-rabbit CW800 conjugate on a LiCOR CLx.

[0102] FIG. 16 shows Src total protein levels are highly upregulated across a variety ofcancers. Data are immunohistochemistry scoring of clinical tissue slices deposited to the Human Protein Atlas. [Nat. Biotech.201028, 1248-1250; proteinatlas.org]

[0103] FIGs. 17A-17B show inhibition of myristoyl transferases reduces eSrc abundance butdoes not affect total Src expression. FIG.17A shows representative histograms for data presented in Figure 1H. FIG.17B shows whole cell lysates of HCC1569 cells treated with DMSO or IMP-1088 were separated by SDS-PAGE, transferred to PVDF membranes, and probed for Src (left) or tubulin (right).

[0104] FIGs. 18A-18D show overexpression of SRC in PaTu8902 cells increases eSrcabundance in a myristoylation-dependent manner. PaTu8902 cells were transfected with plasmid DNA encoding Src-FLAG or Src(G2A)-FLAG or a mock transfection. FIGs.18A- 17Mintz Ref. No.: 048536-797001WO 18C show live cells were analyzed by flow cytometry for Src (FIG.18A) and FLAG (FIGs. 18B-18C) surface expression 48 hours post transfection. FIG.18D shows whole cell lysates of the transfectants were separated by SDS-PAGE, transferred to PVDF membranes, and probed for FLAG (left) or GAPDH (right).

[0105] FIGs. 19A-19B show surface abundance of lysosomal proteins correlates with eSrcabundance. FIG.19A shows previous work from our lab [Proc. Natl. Acad. Sci.2020, 117(14), 7764-7775] identified lysosomal markers LAMP1 and LAMP2 as highly enriched surface proteins in empty-vector (EV) versus KRAS(G12V) transduced isogenic MCF10A- derived cell lines. FIG.19B shows MCF10A-EV or MCF10A-KRAS(G12V) cell lines were analyzed for surface abundance of eSrc by flow cytometry.

[0106] FIGs. 20A-20B show surface abundance of Src and lysosomal markers is upregulatedin hypoxic environments. Previous datasets[refs] of cell surface proteomics (FIG.20A) and whole cell proteomics (FIG.20B) demonstrate that hypoxia, a unique characteristic of the tumor microenvironment, increases surface abundance of Src and lysosomal markers consistent with increased lysosomal exocytosis [Methods. Mol. Bio 2017, 1594, 205-211], while total Src levels are decreased.

[0107] FIGs. 21A-21H show Src is associated with autophagosomes and secreted byautolysosomal exocytosis. FIG.21A shows HCC1569 cells were treated with pharmacological inhibitors in culture for 72 hours before analysis of eSrc expression by flow cytometry. FIGs.21B-21C show HCC1569 were cultured in the presence or absence of N- myristoyltransferase inhibitor IMP-1088 (100 nM) for 72 hours prior to induction of autophagy by starvation and blockade of lysosome / autophagosome fusion with Bafilomycin A1 (10 nM) for 2 hours. Cells were harvested, lysed with a Dounce homogenizer, and intact autophagosomes were immuno-purified using anti-LC3B bound to Protein A / G magnetic beads. Autophagosomes were dissolved with 1% NP-40 in TBS and the contents were analyzed by Western blot. FIGs.21D-21E show HCC1569 were cultured in the presence or absence of N-myristoyltransferase inhibitor IMP-1088 (100 nM) tissue culture treated chambered glass slides in the presence of chloroquine (50 µM) to swell autophagosomes for imaging. Cells were fixed and stained with anti-Src and anti-LC3A / B, followed by DAPI, and imaged by confocal microscopy. Each data point represents a single cell. Colocalization analysis was performed in ImageJ using threshold colocalization to afford Pearson’s correlation coefficient. FIG.21F shows PaTu8902 cells were treated with inducers of autophagy or lysosomal exocytosis for 48 hours prior to analysis by flow cytometry. Statistics 18Mintz Ref. No.: 048536-797001WO were determined by a mixed-model analysis relative to the relevant DMSO control for each replicate, prior to normalization. FIG.21G shows leukemia and lymphoma cell lines were probed with commercial flow cytometry antibodies and analyzed by flow cytometry. FIG. 21H shows Raji B cells were cultured in the presence of Bafilomycin A1 (10 nM), IMP-1088 (100 nM), or an equal concentration of DMSO for 48 hours prior to analysis by flow cytometry.

[0108] FIGs. 22A-22B show representative histograms for data presented in FIG. 21A.

[0109] FIGs. 23A-23B show inhibitors of Src translocation to the cell surface do not altertotal Src levels. HCC1569 cells were treated with Vacuolin-1 (FIG.23A), Bafilomycin A1 (FIG.23A), or ATG-7-IN2 (FIG.23B) in culture for 72 hours. Cells were lysed, separated by SDS-PAGE, transferred to PVDF membranes, and probed with anti-Src and anti-tubulin antibodies.

[0110] FIG. 24 shows a proposed mechanism of Src translocation. Canonically, the geneencoding Src, SRC, is transcribed, translated, the protein is modified by an N- myristoyltransferase, and the lipid moiety inserts into the inner leaflet of the plasma membrane. The plasma membrane has been proposed to be a lipid reservoir for forming phagophore membranes, [Nature 2010, 12, 747-757] and therefore, without being bound by theory, that Src can remain associated with nucleating phagophore membranes. Upon autophagosome formation, Src could then remain localized both on the inner and outer leaflets of the autophagosome membranes. Without being bound by theory, lysosomal fusion can occur, yielding autolysosomes, which can be exocytosed in instances of high metabolic flux, resulting in cell surface presentation of Src.

[0111] FIG. 25 shows full Western blots of Figure 2B.

[0112] FIGs. 26A-26B show Dasatinib, but not trametinib, increases total Src protein levelsin PaTu8902 cells.

[0113] FIGs. 27A-27C show autophagic flux and eSrc abundance are not correlated inisogenic MCF10A derived cell lines. FIGs.27A-27B show autophagic flux for MCF10A-EV or MCF10A-KRAS(G12V) cell lines was measured using an Autophagy Green™ assay kit (AAT Bioquest #23002) and quantitated by flow cytometry. FIG.27C shows MCF10A-EV or MCF10A-KRAS(G12V) cell lines were analyzed for surface abundance of eSrc by flow cytometry.

[0114] FIG. 28 shows a scheme for phage panning for binders of Src. A library of phageexpressing a synthetic antibody fragment of antigen binding (Fab) on pIII has been 19Mintz Ref. No.: 048536-797001WO previously described by our lab. Phage was bound to immobilized Src kinase domain, washed, and eluted by catch-and-release with TEV protease, cleaving a TEV protease cutsite between the phage pIII protein and the Fab. Phage were analyzed by single-point ELISA for antigen binding, and hits were sequenced and analyzed by biolayer interferometry.

[0115] FIGs. 29A-29G show a synthetic anti-Src antibody shows high tumor specificity invivo. FIG.29A shows an Ab1 was assay for binding to HCC1569 cells using Ab1 Fab with a FLAG tag on the C-terminus of the light chain and detected with a fluorescent anti-FLAG antibody. HCC1569 cells transfected with a negative control (NC-1) or SRC targeting siRNA. FIG.29B shows binding kinetics for the fragment of antigen binding for Ab1 were determined by biolayer interferometry on conformationally-locked mutants of Src truncation bearing the SH2-SH3-kinase domains. FIG.29C shows Ab1 expressed as a human IgG1a isotype was cross-linked with DSSO to recombinant Src. Proteins were digested with trypsin and crosslinks were identified by mass spectrometry. FIG.29D shows crosslinks were fed into AlphaLink2.0 to predict a complex structure of the Ab1 Fv with Src. FIGs.29E-29G show Ab1 was conjugated to DFO and was then loaded with radioactive89Zr. Mice bearing HT-1080 tumors were injected with 300 µCi of radioligand-antibody conjugate and with or without 100-fold excess unlabeled Ab1. FIG.29E shows Representative images of HT-1080 xenograft bearing mice with (right) or without (left) blocking Ab124 hours after89Zr-Ab1 injection. FIG.29F shows time course of tumor uptake by region-of-interest (ROI) analysis of HT-1080 xenografts over seven days. Data are average of four mice. FIG.29G shows the biodistribution data of89Zr from HT-1080 xenograft bearing mice 24 hours after administration of89Zr-Ab1 with or without 100-fold excess Ab1. Data are averaged values from four animals. Statistics were calculated by multiple t-tests corrected for multiple comparisons. Statistically insignificant (FDR >5%) comparisons are omitted for clarity and * corresponds to FDR <5%.

[0116] FIGs. 30A-30B show Ab1 is specific for Src and cross reacts with mouse Src. FIG.30A shows multipoint biolayer interferometry curves of recombinant Fab for hSrc. Kinetics were computed in Octet Analysis Studio 12.0, and estimated a binding constant of 6 nM for human Src. FIG.30B shows single-point biolayer interferometry curves for Ab1 IgG binding to hSrc and mSrc. Kinetics were calculated in Prism 12. R2>0.98 for each curve.

[0117] FIGs. 31A-31F show Ab1 binds to eSrc+ cells. Cells were stained with recombinantAb1 Fab with a C-terminal FLAG tag fusion from the light chain and binding was measured by using an anti-FLAG antibody. Cells were analyzed on a CytoFLEX flow cytometry. FIG. 20Mintz Ref. No.: 048536-797001WO 31A shows average mean fluorescence intensity (MFI) fold change over isotype Fab with anti-FLAG for C04 and / or C04 / Ab1 Fab. FIG.31B shows C04 and / or C04 / Ab1 binding versus commercial anti-Src antibody binding. FIGs.31C-31F show representative histograms for MiaPaca2 (FIG.31C), Patu8902 (FIG.31D), HL-60 (FIG.31E), and HCC1569 (FIG.31F) cell lines are shown.

[0118] FIG. 32 shows a representative histogram of Figure 30A. iSRC denotes an siRNAencoding a complimentary sequence for SRC. NC1 denotes a negative control siRNA.

[0119] FIGs. 33A-33G show eSrc is a translational target for antibody-based therapies. InFIGs.33A-33E, a bispecific antibody consisting of Ab1 Fab on one arm and an anti-CD3 scFv derived from clone SP34 (BiTE) was assayed for efficacy at engaging T cells from PBMCs against target cells. Statistics were determined by two-way ANOVA corrected for multiple comparisons. In FIGs.33A-33D, BiTE was added to PBMCs in co-culture with target cells in a 10:1 ratio. Target cells were pre-labeled with a live cell dye. Cells were cultured for 72 hours in the presence of a membrane impermeable DNA-binding dye, Cytotox Red. Images were acquired every three hours on an Incucyte SX5. Specific lysis is defined as lysis of the target cell in the presence of BiTE minus the mean of the cells treated with PBMCs only. All data are representative of at least two independent experiments with different PBMC donors. FIG.33A shows efficacy across cell lines with varying eSrc expression. FIG.33B shows efficacy of BiTE or a loss of Src binding point mutant corresponding to Ab1 heavy chain Y31D on HCC1569 cells. FIG.33C shows HCC1569 cells were pretreated with N-myristoylation inhibitor IMP-1088 (100 nM) for 48 hours followed by drug wash out prior to co-culture with PBMC and BiTE addition. FIG.33D shows PaTu8902 cells were pretreated lysosomal exocytosis induced trametinib (50 nM) for 48 hours followed by drug wash out prior to co-culture with PBMC and BiTE addition. FIG. 33E shows HCC1569 tumors were implanted in NSG mice and grown to a size of 60-80 mm3at which time PBMCs were engrafted by intravenous injection, followed by BiTE administration (0.5 mg / kg, i.v.) on the following day and 7 days later, or saline. BiTE in vivo efficacy in humanized NSG mice. Five mice per arm were used. FIG.33F shows cells were treated with varying doses of Ab1 or isotype MMAE conjugates for 72 hours at 37 °C and 5% CO2. Cell death was monitored using CytotoxRed and fluorescence images were acquired using an Incucyte SX5. Efficacy of Ab1-MMAE conjugate across multiple cell lines at 50 nM is shown. Specific cytotoxicity is determined by normalizing measured cytotoxicity to doxorubicin (100%) and vehicle (0%). Statistics were determined by one-way ANOVA 21Mintz Ref. No.: 048536-797001WO corrected for multiple comparisons to KRAS wild-type diseased-matched cells. FIG.33G shows an Ab1-225Ac conjugate was injected into HT-1080 xenograft bearing NSG mice at 0.8 μCi on days 0 and 7. N = 8 mice for PBS arm and 16 mice for treatment arm. Representative of two independent experiments.

[0120] FIGs. 34A-34D show small molecule heterobifunctionals can be leveraged to targeteSrc-hi tumors. FIG.34A shows a Dasatinib-OKT3 Fab conjugate was added to PBMCs in co-culture with target cells in a 10:1 ratio. Target cells were pre-labeled with a live cell dye, Cytolight Rapid Green. Cells were cultured for 72 hours in the presence of a membrane impermeable DNA-binding dye, Sytox Deep Red. Images were acquired every two hours on an Incucyte SX5. Specific lysis is defined as lysis of the target cell in the presence of BiTE minus the mean of the cells treated with PBMCs only. Statistics were determined by Two- Way ANOVA corrected for multiple comparisons. FIG.34B shows efficacy of Dasatinib- MMAE conjugate across multiple cell lines at 100 nM. Specific cytotoxicity is determined by normalizing measured cytotoxicity to doxorubicin (100%) and vehicle (0%). FIGs.35C-35D show Dastatinib-DOTA conjugates were chelated to Cu64 and injected into HT-1080 xenograft bearing mice.

[0121] FIG. 35 shows SMiTE was added at the specified concentration to PBMCs in co-culture with target cells in a 10:1 ratio. Target cells were pre-labeled with a live cell dye. Cells were cultured for 72 hours in the presence of a membrane impermeable DNA-binding dye, Sytox DeepRed. Images were acquired every three hours on an Incucyte SX5. Specific lysis is defined as lysis of the target cell in the presence of BiTE minus the mean of the cells treated with PBMCs only. All data are representative of at least two independent experiments with different PBMC donors.

[0122] FIG. 36 shows SMiTE was added to PBMCs in co-culture with target cells in a 10:1ratio. Cells were cultured for 48 hours. The supernatant was aspirated and adherent cells were washed. Cell viability was then measured by ATP-Glo Cell Viability assay (Biotium). Specific lysis is defined as lysis of the target cell in the presence of SMiTE minus the mean of the cells treated with PBMCs only. All data are representative of at least two independent experiments with different PBMC donors.

[0123] FIG. 37 shows SMiTE was added to PBMCs in co-culture with target cells in a 10:1ratio. Cells were cultured for 48 hours. The supernatant was aspirated and adherent cells were washed. Cell viability was then measured by ATP-Glo Cell Viability assay. Specific lysis is defined as lysis of the target cell in the presence of SMiTE minus the mean of the cells 22Mintz Ref. No.: 048536-797001WO treated with PBMCs only. All data are representative of at least two independent experiments with different PBMC donors.

[0124] FIG. 38 shows HCC1569 cells were incubated on ice with either OKT3 Fv swapK149M bearing a C-terminal FLAG tag, or a conjugate with dasatinib via an oxaziridine linker. Binding of OKT3 or conjugate to the cell surface was measured by staining cells with an anti-FLAG antibody conjugated to BV421. Cells were analyzed by flow cytometry.

[0125] FIG. 39 shows SMiTE was added to PBMCs in co-culture with target cells in a 10:1ratio. Target cells were pre-labeled with a live cell dye. Cells were cultured for 72 hours in the presence of a membrane impermeable DNA-binding dye, Sytox DeepRed. Images were acquired every three hours on an Incucyte SX5. Specific lysis is defined as lysis of the target cell in the presence of SMiTE minus the mean of the cells treated with PBMCs only. All data are representative of at least two independent experiments with different PBMC donors. Dose responses are shown.

[0126] FIG. 40 shows SMiTE was added to PBMCs in co-culture with target cells in a 10:1ratio. Target cells were pre-labeled with a live cell dye. Cells were cultured for 72 hours in the presence of a membrane impermeable DNA-binding dye, Sytox DeepRed. Images were acquired every three hours on an Incucyte SX5. Specific lysis is defined as lysis of the target cell in the presence of SMiTE minus the mean of the cells treated with PBMCs only. All data are representative of at least two independent experiments with different PBMC donors. Single point responses at 600 pM are shown.

[0127] FIG. 41 shows SMiTE was added to PBMCs in co-culture with target cells in a 10:1ratio. Target cells were pre-labeled with a live cell dye. Cells were cultured for 72 hours in the presence of a membrane impermeable DNA-binding dye, Sytox DeepRed. Images were acquired every three hours on an Incucyte SX5. Specific lysis is defined as lysis of the target cell in the presence of SMiTE minus the mean of the cells treated with PBMCs only. All data are representative of at least two independent experiments with different PBMC donors. Dose response and representative images at 600 pM for LNCAP-C4 are shown.

[0128] FIG. 42 shows SMiTE was added to PBMCs in co-culture with target cells in a 10:1ratio. Target cells were pre-labeled with a live cell dye. Cells were cultured for 48 hours. Cells were labeled on ice with a fixable live / dead dye and immunoprofiling antibodies for flow cytometric analysis. Specific lysis is defined as lysis of the target cell in the presence of SMiTE minus the mean of the cells treated with PBMCs only. 23Mintz Ref. No.: 048536-797001WO

[0129] FIG. 43 shows a schematic representation of various formats of bispecific antibodiescontemplated in the disclosure, including scDb and tandem Fab-Fc.

[0130] FIG. 44 shows bispecific antibody Ab11 binding to eSrc. eSrc was immobilized on aBLI tip with a 7-point 5-fold dilution curve, starting at 5000 nM and diluted down to 0.32 nM. All data was background subtracted with a eSrc coated tip introduced to a blank,

[0131] FIG. 45 shows bispecific antibody Ab11 binding to CD3ed (CD3 epsilon and CD3delta). CD3ed (Acro) was immobilized on a BLI tip with a 7-point 5-fold dilution curve, starting at 5000 nM and diluted down to 0.32 nM. All data was background subtracted with a CD3ed coated tip introduced to a blank.

[0132] FIG. 46 shows two Src TCEs (Ab11 and Ab14) targeting primary murine T cells.TCR- T cells are shown in the left panel, and TCR+ T cells are shown in the right panel.

[0133] FIG. 47 shows two Src TCEs (Ab11 and Ab14) binding to murine cancer cell linesMC38, B16-F10 and LLC.

[0134] FIG. 48 shows four Src TCEs (Ab11, Ab12, Ab13, and Ab14) binding to murinecancer cell lines MC38, B16-F10 and LLC.

[0135] FIG. 49 shows targeted cell lysis by the Src-targeting TCEs (Ab11, Ab12, Ab13, andAb14) using RPMI-8226 Fluc assays. Ab11 showed 91% max lysis, Ab12 showed 95% max lysis, Ab13 showed 75% max lysis, and Ab14 showed >99% max lysis.

[0136] FIG. 50 shows cytotoxicity assays of targeted cell lysis in a tumor cell model usingSrc-targeting TCEs Ab11 and Ab29.

[0137] FIG. 51 shows the binding kinetics of exemplary dasatinib-based radioligand targetedchimeric heterobifunctional small molecules (RadTACs) to Src[256-536].

[0138] FIG. 52 shows the binding kinetics of an exemplary dasatinib-based RadTAC(Compound 3) to other Src family kinases, a Lyn kinase domain and a Hck kinase domain.

[0139] FIG. 53 shows the binding kinetics of an exemplary ponatinib-based (Compound 6),bosutinib-based (Compound 7) and a DGY-06-116-based (Compound 8) RadTAC to Src.

[0140] FIG. 54 shows exemplary data of RadTACs chelated to 64Cu uptake in tumors (left)and tumor-to-kidney ratios (right).

[0141] FIG. 55 shows images of positron emission tomography scans demonstrating theuptake of exemplary RadTACs chelated to64Cu in mice 24 hours post injection.

[0142] FIG. 56 demonstrates the tumor retention and tumor-to-kidney ratio of an exemplaryRadTAC (Compound 3) chelated to177Lu. 24Mintz Ref. No.: 048536-797001WO

[0143] FIG. 57 demonstrates the tumor growth inhibition (TGI) of an exemplary RadTACchelated to177Lu. The RadTAC was administered intravenously to mic bearing syngeneic EMT-6 subcutaneous tumors in either one 0.8 mCi dose on Day 0 or two 0.8 mCi doses on Day 0 and Day 3. DETAILED DESCRIPTION OF THE DISCLOSURE

[0144] Various aspects of the invention relate to antibodies against Src as well as conjugatecompounds and bispecific T cell engagers comprising these antibodies for potential use in cancer therapeutics. Also provided herein are anti-Src small molecule conjugates. These compositions have been developed around the surprising discovery that Src, the master regulatory kinase, is noncanonically translocated to the cell surface via autophago-lysosomal exocytosis and anchored to the external leaflet of the plasma membrane. As described herein, using cell lines with varying levels of eSrc, it has been found that extracellular / ecto Src (eSrc) is regulated both by autolysosomal flux and total Src protein level. It is demonstrated that eSrc is cancer-associated both in vitro, using immortalized cell lines and healthy primary cells, and in vivo, which can be due to Src total protein upregulation in cancer and dysregulated metabolic flux. Disclosed herein is a demonstration of the translational potential of eSrc as a diagnostic and therapeutic cancer antigen using a recombinant anti-Src antibody formulated as a bispecific T-cell engager, a drug conjugate, and a radioligand conjugate. Finally, classes of small molecule directed therapies, including small molecule inhibitor- based T cell engagers (SMITEs), cytotoxin targeted chimeric heterobifunctional small molecules (CytoTACs), radioligand targeted chimeric heterobifunctional small molecules (RadTACs) have been developed, and it is shown that eSrc can be targeted by these unconventional means.

[0145] In some embodiments, “Src” and “eSrc” are used interchangeably.DEFINITIONS

[0146] The singular form “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B.” 25Mintz Ref. No.: 048536-797001WO

[0147] The terms “administration” and “administering”, as used interchangeably herein, referto the delivery of a composition or formulation by an administration route including, but not limited to, intravenous, intra-arterial, intracerebral, intrathecal, intramuscular, intraperitoneal, subcutaneous, intramuscular, and combinations thereof. The term includes, but is not limited to, administration by a medical professional and self-administration.

[0148] The terms “host cell” and “recombinant cell” are used interchangeably herein. It isunderstood that such terms, as well as “cell culture”, “cell line”, refer not only to the particular subject cell or cell line but also to the progeny or potential progeny of such a cell or cell line, without regard to the number of transfers. It should be understood that not all progeny are exactly identical to the parental cell. This is because certain modifications may occur in succeeding generations due to either mutation (e.g., deliberate or inadvertent mutations) or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein, so long as the progeny retain the same functionality as that of the original cell or cell line.

[0149] The term “percent identity,” as used herein in the context of two or more nucleic acidsor proteins, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. See, e.g., the NCBI web site at ncbi.nlm.nih.gov / BLAST. This definition also refers to, or may be applied to, the complement of a test sequence. This definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. Sequence identity typically is calculated over a region that is at least about 20 amino acids or nucleotides in length, or over a region that is 10-100 amino acids or nucleotides in length, or over the entire length of a given sequence. Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res (1984) 12:387), BLASTP, BLASTN, FASTA (Atschul et al., J Mol Biol (1990) 215:403). Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of 26Mintz Ref. No.: 048536-797001WO Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis.53705), with the default parameters thereof

[0150] The term “treatment” used in reference to a disease or condition means that at least anamelioration of the symptoms associated with the condition afflicting an individual is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., a symptom, associated with the condition being treated. Treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or eliminated entirely such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition. Thus, treatment includes: (i) prevention (i.e., reducing the risk of development of clinical symptoms, including causing the clinical symptoms not to develop, e.g., preventing disease progression), and (ii) inhibition (i.e., arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease).

[0151] As used herein, and unless otherwise specified, a “therapeutically effective amount”of an agent is an amount sufficient to provide a therapeutic benefit in the treatment or management of the cancer, or to delay or minimize one or more symptoms associated with the cancer. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of the cancer. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the cancer, or enhances the therapeutic efficacy of another therapeutic agent. An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). The exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols.1-3, 2010); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (2016); Pickar, Dosage Calculations (2012); and Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Gennaro, Ed., Lippincott, Williams & Wilkins). 27Mintz Ref. No.: 048536-797001WO

[0152] As used herein, a “subject” or an “individual” includes animals, such as human (e.g.,human individuals) and non-human animals. In some embodiments, a “subject” or “individual” can be a patient under the care of a physician. Thus, the subject can be a human patient or an individual who has, is at risk of having, or is suspected of having a disease of interest (e.g., cancer) and / or one or more symptoms of the disease. The subject can also be an individual who is diagnosed with a risk of the condition of interest at the time of diagnosis or later. The term “non-human animals” includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, and non- mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, and the like.

[0153] Where a range of values is provided, it is understood that each intervening value, tothe tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0154] All ranges disclosed herein also encompass any and all possible sub-ranges andcombinations of sub-ranges thereof. Any listed range can be recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, and so forth. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, and so forth. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0155] It is appreciated that certain features of the disclosure, which are, for clarity, describedin the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are 28Mintz Ref. No.: 048536-797001WO specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub- combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub­combination was individually and explicitly disclosed herein.

[0156] Although features of the disclosures may be described in the context of a singleembodiment, the features may also be provided separately or in any suitable combination. Conversely, although the disclosures may be described herein in the context of separate embodiments for clarity, the disclosures may also be implemented in a single embodiment. Any published patent applications and any other published references, documents, manuscripts, and scientific literature cited herein are incorporated herein by reference for any purpose. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. COMPOSITIONS

[0157] The present disclosure provides, among others, antibodies specific to Src. In somenon-limiting exemplary embodiments, the present disclosure demonstrates the development of new Src-targeted compounds, including bispecific T-cell engagers, drug conjugates, radioligand conjugates, and small molecule directed therapies, including small molecule inhibitor-based T cell engagers (SMITEs), cytotoxin targeted chimeric heterobifunctional small molecules (CytoTACs), and radioligand targeted chimeric heterobifunctional small molecules (RadTACs).

[0158] The disclosure also provides, among others, nucleic acids that encode the antibodiesand therapeutics described herein, cells comprising nucleic acids encoding antibodies and therapeutics described herein, and pharmaceutical compositions comprising the antibodies and therapeutics described herein. The disclosure also provides methods of treatment using antibodies and therapeutics described herein, nucleic acids encoding antibodies and therapeutics described herein or pharmaceutical compositions comprising the antibodies and therapeutics described herein. The disclosure also provides compositions and methods useful for producing such agents, nucleic acids encoding same, host cells genetically modified with the nucleic acids, as well as methods for the treatment of cancers. 29Mintz Ref. No.: 048536-797001WO

[0159] In the following detailed description, reference is made to the accompanyingdrawings, which form a part hereof. In the drawings, similar symbols generally identify similar components, unless context dictates otherwise. The illustrative alternatives described in the detailed description, drawings, and claims are not meant to be limiting. Other alternatives may be used, and other changes may be made without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this application. ANTI-SRC ANTIBODIES

[0160] Described herein is an antibody that specifically binds Src expressed on the surface ofcells, for example, tumor cells. In one embodiment, the antibody is composed of two variable chains, one heavy and one light. On each variable chain, there are three CDRs that allow the antibody to bind to Src. On both variable chains, there are a total of six different CDRs.

[0161] In some embodiments, the invention provides a monoclonal anti-Src antibodycomprising two sets of six different complementarity-determining regions (CDRs), two sets of two different variable regions, two full heavy chains, two full light chains, and a human heavy chain constant region.

[0162] An antibody as used herein has its common meaning in the field and refers to animmunoglobulin molecule that recognizes and specifically binds to an epitope of a target (e.g., Src) through at least one antigen binding domain within the variable region of the immunoglobulin molecule. The target can be a peptide, e.g., an Src peptide. An antibody of this disclosure encompasses full length antibodies (including full length polyclonal antibodies and full length monoclonal antibodies), antigen-binding fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two full length antibodies, chimeric antibodies, humanized antibodies, human antibodies, synthetic antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity. An antibody can be of any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, 30Mintz Ref. No.: 048536-797001WO epsilon, gamma, and mu, respectively. In some embodiments, the antibody of the present disclosure is an IgG antibody. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations.

[0163] The antibody of the present disclosure can include one or more variable regions. Avariable region of an antibody refers to the variable region of the antibody light chain (VL) or the variable region of the antibody heavy chain (VH), either alone or in combination. The variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen- binding site of antibodies. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al (1997) J. Molec. Biol.273:927-948; Chothia & Lesk, 1987, J. Mol. Biol.196:901-917; Chothia et al., 1989, Nature 342:878-883; Oxford Molecular's AbM antibody modelling software and North numbering convention (North et al., A New Clustering of Antibody CDR Loop Conformations, Journal of Molecular Biology, 406:228-256 (2011))). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.

[0164] The Kabat numbering system is generally used when referring to a residue in thevariable region (e.g., Kabat et al., Sequences of Immunological Interest.5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The amino acid position numbering as in Kabat, refers to the numbering system used for heavy chain variable regions or light chain variable regions of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). The Kabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol.196:901-917 (1987)). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modelling software.

[0165] In some embodiments, the anti-Src antibodies provided herein are full lengthantibodies. A full-length antibody can include a four-polypeptide unit consisting of two 31Mintz Ref. No.: 048536-797001WO identical heavy chains and two identical light chains, as described in greater detail below, held together by disulfide bonds. The light chains are generally shorter, with lower molecular weights than the heavy chains. Each polypeptide chain has a constant region and a variable region. The variable region is specific to each particular antibody. The light chain variable region is referred to as VL and the light chain constant region as CL. Similarly, the heavy chain variable region is referred to as VH and the heavy chain constant regions as CH, with CH1, CH2, and CH3 each denoting a different portion of the constant region of the heavy chain. In some embodiments, carbohydrates can be normally attached to the CH2 domains of the heavy chains. Further, a full-length antibody can also contain a fragment crystallizable (Fc) region. The Fc region contains only constant regions from the heavy chains (CH2 and CH3). In contrast, the fragment antigen-binding region (Fab) can include both a constant domain and the variable domains of both the heavy and light chains (VH, VL, CH1 and CL). A fragment variable region (Fv) contains only the two variable domains.

[0166] As described above, the antibody of the present disclosure can include one or moreconstant regions. A “constant region” of an antibody is a well-known term in the art and refers to the part of the antibody that is relatively constant in amino acid sequence between different molecules. Typically, the heavy chain constant region is composed of three distinct regions, termed CH1, CH2, and CH3, numbered in the direction from the amino terminal (N- terminal) end to the carboxy terminal (C-terminal) end. A typical light chain only has one constant region, termed CL. The constant region of an antibody determines its particular effector function. One of skill in the art will readily understand the terminology and structural features of constant regions of antibodies.

[0167] An antibody provided herein can be a monoclonal antibody. A monoclonal antibodycan refer to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term “monoclonal antibody” encompasses both full length and full-length monoclonal antibodies as well as antigen-binding fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, “monoclonal antibody” refers to such antibodies made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals. 32Mintz Ref. No.: 048536-797001WO

[0168] The antibodies encompassed by the present disclosure can be human, non-human,humanized, murine, chimeric, synthetic, or resurfaced. In some embodiments, the antibody of the present disclosure can be a humanized antibody. As used herein, a humanized antibody refers to an antibody derived from a monoclonal antibody raised initially in a non-human animal, such as a rodent or rabbit. Certain amino acid residues in this monoclonal antibody, typically from non-antigen recognizing portions of the antibody, are modified to be homologous to corresponding residues in a human antibody of corresponding isotype. Humanization can be performed, for example, using various methods by substituting at least a portion of a rodent or rabbit variable region for the corresponding regions of a human antibody (see, e.g., United States Patent No.5,585,089, and No.5,693,762; Jones et al, 1986, Nature 321:522-525; Riechmann et al, 1988, Nature 332:323-27; and Verhoeyen et al, 1988, Science 239: 1534-1536).

[0169] Further, anti-Src antibodies of the present disclosure also include antigen-bindingfragments that specifically bind Src. An antigen-binding fragment as used herein refers to a portion of a full-length antibody. For instance, in some embodiments, an antigen-binding fragment of an antibody as used herein refers to the antigenic determining variable regions of a full-length antibody. Examples of antigen-binding fragments include, but are not limited to a Fab, Fab', F(ab')2, Fd, single chain Fv or scFv, disulfide linked Fv, domain antibodies, dual- specific antibodies, bibodies, minibodies, tribodies, bispecific antibodies, trispecific antibodies, multispecific antibodies, diabodies, triabodies, tetrabodies, intrabodies, nanobodies, small modular immunopharmaceuticals (SMTP), binding-domain immunoglobulin fusion proteins, camelized antibodies or VHH containing antibodies, DVD- Ig, Fcab, mAb2, (scFv)2, or scFv-Fc. In one embodiment, the antigen binding fragment is an scFv. In one embodiment, the antigen binding fragment is an scFv-Fc.

[0170] The antibodies and antigen binding fragments of the disclosure bind specifically toSrc. As used herein, “specifically binds” can mean that, under designated conditions, the antibody or antigen binding fragment thereof binds preferentially to a particular target protein, peptide or polysaccharide (i.e., Src) and does not bind in a significant amount to other proteins or polysaccharides present in the sample or subject. Specific binding can be determined by methods known in the art.

[0171] In one embodiment, the anti-Src antibodies and antigen binding fragments thereof donot exhibit an effect on Src activity upon binding. In one embodiment, the anti-Src antibodies and antigen binding fragments thereof do exhibit an effect on Src activity upon binding. In 33Mintz Ref. No.: 048536-797001WO one embodiment, the anti-Src antibodies and antigen binding fragments thereof inhibit the activity of the kinase domain in vitro. As used herein, an effect on Src activity can mean that one or more components of the src kinase signaling cascade are affected such that the function of the cell is altered. The components of the kinase signaling cascade include any protein that is directly or indirectly involved in the kinase signaling pathway, including second messengers and upstream and downstream targets.

[0172] In another embodiment, the anti-Src antibodies and antigen binding fragments thereofof the present disclosure are blocking antagonist antibodies, which inhibits or reduces biological activity of Src. In some embodiments, blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of Src. The biological activity of Src, can be reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100% comparing to its natural biological activity.

[0173] The term “epitope” or “antigenic determinant” are used interchangeably herein andrefer to that portion of an antigen capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding are typically lost upon protein denaturing.

[0174] In some embodiments, the anti-Src antibody or antigen binding fragments thereofbind to an epitope within Src having the amino acid sequence of SEQ ID NO: 38 below MGSNKSKPKDASQRRRSLEPAENVHGAGGGAFPASQTPSKPASADGHRGPSAAFAPAAAEPK LFGGFNSSDTVTSPQRAGPLAGGVTTFVALYDYESRTETDLSFKKGERLQIVNNTEGDWWLA HSLSTGQTGYIPSNYVAPSDSIQAEEWYFGKITRRESERLLLNAENPRGTFLVRESETTKGA YCLSVSDFDNAKGLNVKHYKIRKLDSGGFYITSRTQFNSLQQLVAYYSKHADGLCHRLTTVC PTSKPQTQGLAKDAWEIPRESLRLEVKLGQGCFGEVWMGTWNGTTRVAIKTLKPGTMSPEAF LQEAQVMKKLRHEKLVQLYAVVSEEPIYIVTEYMSKGSLLDFLKGETGKYLRLPQLVDMAAQ IASGMAYVERMNYVHRDLRAANILVGENLVCKVADFGLARLIEDNEYTARQGAKFPIKWTAP EAALYGRFTIKSDVWSFGILLTELTTKGRVPYPGMVNREVLDQVERGYRMPCPPECPESLHD LMCQCWRKEPEERPTFEYLQAFLEDYFTSTEPQYQPGENL

[0175] In some embodiments, the anti-Src antibody and antigen binding fragments thereofbinds to the kinase domain of Src. 34Mintz Ref. No.: 048536-797001WO

[0176] In one embodiment, the anti-Src antibodies and antigen binding fragments thereofbind to an epitope within amino acid residues R463-E492 of SEQ ID NO: 38. In one embodiment, the anti-Src antibodies and antigen binding fragments thereof bind to an epitope within amino acid residues V470-R480 and N417-Y439 of SEQ ID NO: 38.

[0177] In some embodiments, the antibody and antigen binding fragments thereof of thepresent disclosure binds to the open confirmation of Src.

[0178] In some embodiments, the anti-Src antibody or antigen binding fragment thereofspecifically binds to a monomeric form of Src.

[0179] In some the anti-Src antibody or antigen binding fragment thereof specifically bindsto human and mouse Src.

[0180] The anti-Src antibody or antigen-binding fragment particularly has specificity to Srcantigen and appropriate affinity to be used as an antibody therapeutic agent / diagnostic agent. In one embodiment, the affinity to Src is about 9 nM. In one embodiment, the affinity to Src is about 6 nM. The term “affinity”, as used herein, can refer the strength of the binding of an antibody or polypeptide to an epitope. Methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol.92:589-601 (1983), which references are entirely incorporated herein by reference. One standard method for determining the affinity of mAbs is the use of surface plasmon resonance (SPR) screening (such as by analysis with a BIAcore™ SPR analytical device).

[0181] In one embodiment, the antibody and antigen binding fragments thereof comprises sixCDRs comprising the following sequences according to Kabat numbering: CDR-H1: YYYIH (SEQ ID NO: 1) CDR-H2: SISSYSGYTSYADSVKG (SEQ ID NO: 2) CDR-H3: ASGYGAFFGFNY (SEQ ID NO: 3) CDR-L1: RASQSVGSALA (SEQ ID NO: 4) CDR-L2: SASSLYS (SEQ ID NO: 5) CDR-L3: QQYYDYGLIT (SEQ ID NO: 6).In some embodiments, the anti-Src antibody and antigen binding fragments of the present disclosure comprises CDRs about 80%, about 85%, about 90%, about 95%, or about 99% identical to a sequence selected from SEQ ID NOS: 1-6.

[0182] In one embodiment, the antibody and antigen binding fragments thereof comprises sixCDRs comprising the following sequences according to Kabat numbering: CDR-H1: SSSIH (SEQ ID NO: 7) CDR-H2: SISPYYGYTSYADSVKG (SEQ ID NO: 8) CDR-H3: 35Mintz Ref. No.: 048536-797001WO GPYHYYAIDY (SEQ ID NO: 9) CDR-L1: RASQSVGSALA (SEQ ID NO: 4) CDR-L2: SASSLYS (SEQ ID NO: 5) CDR-L3: QQSGYSPIT (SEQ ID NO: 10).

[0183] In one embodiment, the antibody and antigen binding fragments thereof comprises sixCDRs comprising the following sequences according to Kabat numbering: CDR-H1: SSSIH (SEQ ID NO: 7) CDR-H2: SISPDYGYTSYADSVKG (SEQ ID NO: 136) CDR-H3: GPYHYYAIDY (SEQ ID NO: 9) CDR-L1: RASQSVGSALA (SEQ ID NO: 4) CDR-L2: SASSLYS (SEQ ID NO: 5) CDR-L3: QQSGYSPIT (SEQ ID NO: 10).

[0184] In one embodiment, the antibody and antigen binding fragments thereof comprises sixCDRs comprising the following sequences according to Kabat numbering: CDR-H1: SSSIH (SEQ ID NO: 7) CDR-H2: SISPYYGYTSYADSVKG (SEQ ID NO: 8) CDR-H3: GPYHYYAIDY (SEQ ID NO: 9) CDR-L1: RASQDIGRALA (SEQ ID NO: 137) CDR-L2: SASSLYS (SEQ ID NO: 5) CDR-L3: QQSGYSPIT (SEQ ID NO: 10).

[0185] In one embodiment, the antibody and antigen binding fragments thereof comprises sixCDRs comprising the following sequences according to Kabat numbering: CDR-H1: SSSIH (SEQ ID NO: 7) CDR-H2: SISPDYGYTSYADSVKG (SEQ ID NO: 136) CDR-H3: GPYHYYAIDY (SEQ ID NO: 9) CDR-L1: RASQDIGRALA (SEQ ID NO: 137) CDR-L2: SASSLYS (SEQ ID NO: 5) CDR-L3: QQSGYSPIT (SEQ ID NO: 10).In some embodiments, the anti-Src antibody and antigen binding fragments of the present disclosure comprises CDRs about 80%, about 85%, about 90%, about 95%, or about 99% identical to a sequence selected from SEQ ID NOS: 4, 5, 7-10, and 136-137.

[0186] In some embodiments, the antibody and antigen binding fragments of this disclosurecomprises a CDR-H1 having the amino acid sequence shown as SEQ ID NO: 1, a CDR-H2 having the amino acid sequence shown as SEQ ID NO: 2; a CDR-H3 having the amino acid sequence shown as SEQ ID NO: 3, a CDR-L1 having the amino acid sequence shown as SEQ ID NO: 4, a CDR-L2 having the amino acid sequence shown as SEQ ID NO: 5; and a CDR- L3 having the amino acid sequence shown as SEQ ID NO: 6.

[0187] In some embodiments, the antibody and antigen binding fragments of this disclosurecomprises a CDR-H1 having the amino acid sequence shown as SEQ ID NO: 7, a CDR-H2 having the amino acid sequence shown as SEQ ID NO: 8; a CDR-H3 having the amino acid sequence shown as SEQ ID NO: 9, a CDR-L1 having the amino acid sequence shown as SEQ ID NO: 4, a CDR-L2 having the amino acid sequence shown as SEQ ID NO: 5; and a CDR- L3 having the amino acid sequence shown as SEQ ID NO: 10. 36Mintz Ref. No.: 048536-797001WO

[0188] In some embodiments, the antibody and antigen binding fragments of this disclosurecomprises a CDR-H1 having the amino acid sequence shown as SEQ ID NO: 7, a CDR-H2 having the amino acid sequence shown as SEQ ID NO: 136; a CDR-H3 having the amino acid sequence shown as SEQ ID NO: 9, a CDR-L1 having the amino acid sequence shown as SEQ ID NO: 4, a CDR-L2 having the amino acid sequence shown as SEQ ID NO: 5; and a CDR-L3 having the amino acid sequence shown as SEQ ID NO: 10.

[0189] In some embodiments, the antibody and antigen binding fragments of this disclosurecomprises a CDR-H1 having the amino acid sequence shown as SEQ ID NO: 7, a CDR-H2 having the amino acid sequence shown as SEQ ID NO: 8; a CDR-H3 having the amino acid sequence shown as SEQ ID NO: 9, a CDR-L1 having the amino acid sequence shown as SEQ ID NO: 137, a CDR-L2 having the amino acid sequence shown as SEQ ID NO: 5; and a CDR-L3 having the amino acid sequence shown as SEQ ID NO: 10.

[0190] In some embodiments, the antibody and antigen binding fragments of this disclosurecomprises a CDR-H1 having the amino acid sequence shown as SEQ ID NO: 7, a CDR-H2 having the amino acid sequence shown as SEQ ID NO: 136; a CDR-H3 having the amino acid sequence shown as SEQ ID NO: 9, a CDR-L1 having the amino acid sequence shown as SEQ ID NO: 137, a CDR-L2 having the amino acid sequence shown as SEQ ID NO: 5; and a CDR-L3 having the amino acid sequence shown as SEQ ID NO: 10.

[0191] In some embodiments, the antibody and antigen binding fragments of this disclosurecomprises a heavy chain variable region comprising the amino acid sequence shown as SEQ ID NO: 11: EVQLVESGGGLVQPGGSLRLSCAASGFNISYYYIHWVRQAPGKGLEWVASISSYSGYTSYAD SVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARASGYGAFFGFNYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVK (SEQ ID NO: 11); and a light chain variable region comprising the amino acid sequence shown as SEQ ID NO: 12: DIQMTQSPSSLSASVGDRVTITCRASQSVGSALAWYQQKPGKAPKLLIYSASSLYSGVPSRF SGSRSGTDFTLTISSLQPEDFATYYCQQYYDYGLITFGQGTKVEIK (SEQ ID NO: 12).

[0192] In some embodiments, the antibody and antigen binding fragments of the disclosurecomprises a heavy chain variable region comprising the nucleotide sequence shown as SEQ ID NO: 15: GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTC CTGTGCAGCTTCTGGCTTCAACATCTCTTATTATTATATCCACTGGGTGCGTCAGGCCCCGG 37Mintz Ref. No.: 048536-797001WO GTAAGGGCCTGGAATGGGTTGCATCTATTTCTTCTTATTCTGGCTATACTTCTTATGCCGAT AGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAAT GAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCGCTTCTGGTTACGGTG CTTTCTTCGGTTTTAACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCCTCCACC AAGGGTCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGC CCTGGGCTGCCTGGTCAAG

[0193] In some embodiments, the antibody and antigen binding fragments of the disclosurecomprises a light chain variable region comprising the nucleotide sequence shown as SEQ ID NO: 16: GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCAT CACCTGCCGTGCCAGTCAGTCTGTGGGATCCGCTCTCGCTTGGTATCAACAGAAACCAGGAA AAGCTCCGAAGCTTCTGATTTACTCGGCTAGCAGCCTCTACTCTGGAGTCCCTTCTCGCTTC TCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTT CGCAACTTATTACTGTCAGCAATACTACGACTACGGTCTGATCACGTTCGGACAGGGTACCA AGGTGGAGATCAAA

[0194] In some embodiments, the antibody and antigen binding fragments of this disclosurecomprises a heavy chain variable region comprising the amino acid sequence shown as SEQ ID NO: 13: EVQLVESGGGLVQPGGSLRLSCAASGFNLSSSSIHWVRQAPGKGLEWVASISPYYGYTSYAD SVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGPYHYYAIDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVK (SEQ ID NO: 13); and a light chain variable region comprising the amino acid sequence shown as SEQ ID NO: 14: DIQMTQSPSSLSASVGDRVTITCRASQSVGSALAWYQQKPGKAPKLLIYSASSLYSGVPSRF SGSRSGTDFTLTISSLQPEDFATYYCQQSGYSPITFGQGTKVEIK (SEQ ID NO: 14).

[0195] In some embodiments, the antibody and antigen binding fragments of the disclosurecomprises a heavy chain variable region comprising the nucleotide sequence shown as SEQ ID NO: 17: GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTC CTGTGCAGCTTCTGGCTTCAACCTCTCTTCTTCTTCTATCCACTGGGTGCGTCAGGCCCCGG GTAAGGGCCTGGAATGGGTTGCATCTATTTCTCCTTATTATGGCTATACTTCTTATGCCGAT AGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAAT GAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCGGTCCGTACCATTACT ACGCTATTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCCTCCACCAAGGGT 38Mintz Ref. No.: 048536-797001WO CCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGG CTGCCTGGTCAAG

[0196] In some embodiments, the antibody and antigen binding fragments of the disclosurecomprises a light chain variable region comprising the nucleotide sequence shown as SEQ ID NO: 18: GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCAT CACCTGCCGTGCCAGTCAGTCTGTGGGATCCGCTCTCGCTTGGTATCAACAGAAACCAGGAA AAGCTCCGAAGCTTCTGATTTACTCGGCTAGCAGCCTCTACTCTGGAGTCCCTTCTCGCTTC TCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTT CGCAACTTATTACTGTCAGCAATCTGGTTACTCTCCGATCACGTTCGGACAGGGTACCAAGG TGGAGATCAAA

[0197] In some embodiments, the antibody and antigen binding fragments of this disclosurecomprises a heavy chain variable region comprising the amino acid sequence shown as SEQ ID NO: 40: EVQLVESGGGLVQPGGSLRLSCAASGFNISYYYIHWVRQAPGKGLEWVASISSYSGYTSYAD SVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARASGYGAFFGFNYWGQGTLVTVSS (SEQ ID NO: 40); and a light chain variable region comprising the amino acid sequence shown as SEQ ID NO: 41: DIQMTQSPSSLSASVGDRVTITCRASQSVGSALAWYQQKPGKAPKLLIYSASSLYSGVPSRF SGSRSGTDFTLTISSLQPEDFATYYCQQYYDYGLITFGQGTKVEIK (SEQ ID NO: 41).

[0198] In some embodiments, the antibody and antigen binding fragments of this disclosurecomprises a heavy chain variable region comprising the amino acid sequence shown as SEQ ID NO: 42: EVQLVESGGGLVQPGGSLRLSCAASGFNLSSSSIHWVRQAPGKGLEWVASISPYYGYTSYAD SVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGPYHYYAIDYWGQGTLVTVSS (SEQ ID NO: 42); and a light chain variable region comprising the amino acid sequence shown as SEQ ID NO: 43: DIQMTQSPSSLSASVGDRVTITCRASQSVGSALAWYQQKPGKAPKLLIYSASSLYSGVPSRF SGSRSGTDFTLTISSLQPEDFATYYCQQSGYSPITFGQGTKVEIK (SEQ ID NO: 43).

[0199] In some embodiments, the antibody and antigen binding fragments of this disclosurecomprises a heavy chain variable region comprising the amino acid sequence shown as SEQ ID NO: 44: 39Mintz Ref. No.: 048536-797001WO EVQLVESGGGLVQPGGSLRLSCAASGFQLSSSSIHWVRQAPGKGLEWVASISPDYGYTSYAD SVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGPYHYYAIDYWGQGTLVTVSS (SEQ ID NO: 44); and a light chain variable region comprising the amino acid sequence shown as SEQ ID NO: 45: DIQMTQSPSSLSASVGDRVTITCRASQSVGSALAWYQQKPGKAPKLLIYSASSLYSGVPSRF SGSRSGTDFTLTISSLQPEDFATYYCQQSGYSPITFGQGTKVEIK (SEQ ID NO: 45).

[0200] In some embodiments, the antibody and antigen binding fragments of this disclosurecomprises a heavy chain variable region comprising the amino acid sequence shown as SEQ ID NO: 46: EVQLVESGGGLVQPGGSLRLSCAASGFNLSSSSIHWVRQAPGKGLEWVASISPYYGYTSYAD SVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGPYHYYAIDYWGQGTLVTVSS (SEQ ID NO: 46); and a light chain variable region comprising the amino acid sequence shown as SEQ ID NO: 47: DIQMTQSPSSLSASVGDRVTITCRASQDIGRALAWYQQKPGKAPKLLIYSASSLYSGVPSRF SGSRSGTDFTLTISSLQPEDFATYYCQQSGYSPITFGQGTKVEIK (SEQ ID NO: 47).

[0201] In some embodiments, the antibody and antigen binding fragments of thisdisclosure comprises a heavy chain variable region comprising the amino acid sequence shown as SEQ ID NO: 48: EVQLVESGGGLVQPGGSLRLSCAASGFQLSSSSIHWVRQAPGKGLEWVASISPDYGYTSYAD SVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGPYHYYAIDYWGQGTLVTVSS (SEQ ID NO: 48); and a light chain variable region comprising the amino acid sequence shown as SEQ ID NO: 49: DIQMTQSPSSLSASVGDRVTITCRASQDIGRALAWYQQKPGKAPKLLIYSASSLYSGVPSRF SGSRSGTDFTLTISSLQPEDFATYYCQQSGYSPITFGQGTKVEIK (SEQ ID NO: 49).

[0202] In some embodiments, the anti-Src antibody and antigen binding fragments thereof ofthe present disclosure comprise a VH sequence selected from SEQ ID NOS: 11 and 13 and a VL sequence selected from SEQ ID NOS: 12 and 14.

[0203] In some embodiments, the anti-Src antibody and antigen binding fragments thereof ofthe present disclosure comprises a VH sequence that is about 80%, about 85%, about 90%, about 95%, or about 99% identical to a sequence selected from SEQ ID NOS: 11, 13, 40, 42, 44, 46, and 48 and a VL sequence that is about 80%, about 85%, about 90%, about 95%, or about 99% identical to a sequence selected from SEQ ID NOS: 12 and 14. In some embodiments, the anti-Src antibody of the present disclosure comprise a VH sequence 40Mintz Ref. No.: 048536-797001WO selected from SEQ ID NOS: 11, 13, 40, 42, 44, 46, and 48 and a VL sequence selected from SEQ ID NOS: 12, 14, 41, 43, 45, 47, and 49. In some embodiments, the anti-Src antibody and antigen binding fragments of the present disclosure comprise a CL domain comprising the amino acid sequence as shown in SEQ ID NO: 35: RTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0204] In some embodiments, the anti-Src antibody and antigen binding fragments of thepresent disclosure comprise a CL domain encoded by the nucleotide sequence as shown in SEQ ID NO: 37: CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATTCACAGTTGAAATCTGG AACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGA AGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAG GACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAAAAACATAA AGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACA GGGGAGAGTGT

[0205] In some embodiments, the anti-Src antibody and antigen binding fragments of thepresent disclosure comprise a CL domain comprising the amino acid sequence as shown in SEQ ID NO: 69: ASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0206] In some embodiments, the anti-Src antibody of the present disclosure comprises a CHdomain comprising the amino acid sequence as shown in SEQ ID NO: 34: TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT

[0207] In some embodiments, the anti-Src antibody of the present disclosure comprises a CHdomain encoded by the nucleotide sequence as shown in SEQ ID NO: 36: ACCAAGGGTCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGC GGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAG GCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCC CTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGT GAATCACAAGCCCAGCAACACCAAGGTCGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAA CTCACACA 41Mintz Ref. No.: 048536-797001WO

[0208] In some embodiments, the anti-Src antibody of the present disclosure comprises a CHdomain comprising the amino acid sequence as shown in SEQ ID NO: 79: SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK

[0209] In some embodiments, the anti-Src antibody of the present disclosure comprise a CHdomain comprising the amino acid sequence as shown in SEQ ID NO: 80: SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQV SLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK

[0210] In some embodiments, the anti-Src antibody of the present disclosure comprises a CHdomain comprising the amino acid sequence as shown in SEQ ID NO: 34 and a CL domain comprising the amino acid sequence as shown in SEQ ID NO: 35.

[0211] Antibodies provided herein can include antibodies comprising chemicalmodifications, for example, antibodies which have been chemically modified, e.g., by covalent attachment of any type of molecule to the antibody. For example, but not by way of limitation, an anti-Src antibody can be glycosylated, acetylated, pegylated, phosphorylated, or amidated, can be derivitized via protective / blocking groups, or can further comprise a cellular ligand and or other protein or peptide (e.g., a heterologous protein or peptide), etc. For example, an antibody provided herein can be chemically modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Further, an anti-Src antibody described herein can contain one or more non-classical amino acids.

[0212] In one embodiment, an anti-Src antibody provided herein is a naked antibody which isnot linked, fused or conjugated (e.g., artificially linked, fused or conjugated) to another molecule, peptide or polypeptide (for example, a heterologous polypeptide). In another 42Mintz Ref. No.: 048536-797001WO embodiment, an anti-Src antibody provided herein is not an antibody-drug conjugate. In a particular embodiment, an anti-Src antibody provided herein is not a fusion protein. In particular embodiments, an anti-Src antibody described herein does not comprise any non- classical amino acids. Antibody Production

[0213] Anti-Src antibodies can be generated, for example, using various procedures knownwithin the art that may be used for the production of monoclonal antibodies directed against Src, or against derivatives, fragments, analogs homologs or orthologs thereof. (See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated herein by reference). Fully human antibodies are antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed “human antibodies” or “fully human antibodies” herein. Human monoclonal antibodies can be also prepared by using the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies may be utilized and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96).

[0214] Antibodies are purified by well-known techniques, such as affinity chromatographyusing protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen that is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol.14, No.8 (Apr.17, 2000), pp.25-28).

[0215] Monoclonal antibodies that modulate, block, inhibit, reduce, antagonize, neutralize orotherwise interfere with Src activity are generated, e.g., by immunizing an animal with membrane bound and / or soluble Src, such as, for example, murine, rat or human Src or an immunogenic fragment, derivative or variant thereof. Alternatively, the animal is immunized with cells transfected with a vector containing a nucleic acid molecule encoding Src such that 43Mintz Ref. No.: 048536-797001WO Src is expressed and associated with the surface of the transfected cells. Alternatively, the antibodies are obtained by screening a library that contains antibody or antigen binding domain sequences for binding to Src. This library is prepared, e.g., in bacteriophage as protein or peptide fusions to a bacteriophage coat protein that is expressed on the surface of assembled phage particles and the encoding DNA sequences contained within the phage particles (i.e., “phage displayed library”). Hybridomas resulting from myeloma / B cell fusions are then screened for reactivity to Src. Additionally, the antibodies by selected from, and optionally optimized in, yeast antibody display libraries and yeast library presentation systems as described in, e.g.: Blaise L, Wehnert A, Steukers M P, van den Beucken T, Hoogenboom H R, Hufton S E. Construction and diversification of yeast cell surface displayed libraries by yeast mating: application to the affinity maturation of Fab antibody fragments. Gene.2004 Nov.24; 342(2):211-8; Boder E T, Wittrup K D. Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol.1997 June; 15(6):553-7; Kuroda K, Ueda M. Cell surface engineering of yeast for applications in white biotechnology. Biotechnol Lett.2011 January; 33(1):1-9. doi: 10.1007 / s10529-010-0403-9. Review; Lauer T M, Agrawal N J, Chennamsetty N, Egodage K, Helk B, Trout B L. Developability index: a rapid in silico tool for the screening of antibody aggregation propensity. J Pharm Sci.2012 January; 101(1):102-15; Orcutt K. D. and Wittrup K. D. (2010), 207-233 doi: 10.1007 / 978-3-642-01144-3_15; Rakestraw J A, Aird D, Aha P M, Baynes B M, Lipovsek D. Secretion-and-capture cell-surface display for selection of target- binding proteins. Protein Eng Des Sel.2011 June; 24(6):525-30; U.S. Pat. Nos.8,258,082; 6,300,064; 6,696,248; 6,165,718; 6,500,644; 6,291,158; 6,291,159; 6,096,551; 6,368,805; 6,500,644. Exemplary yeast library presentation systems are described in, e.g., WO2008118476; WO2009 / 036379; WO2010105256; and WO2012009568. In certain embodiments, such yeast antibody display libraries or yeast library presentation systems are designed to mimic or reflect the diversity characteristic of the human preimmune antibody repertoire. In certain embodiments such yeast antibody display library diversity or yeast library presentation system diversity is generated in silico. In certain embodiments such yeast antibody display libraries or yeast library presentation systems comprise Saccharomyces yeast cells, such as Saccharomyces Cerevisiae cells. In certain embodiments such yeast antibody display libraries or yeast library presentation systems comprise Pichia cells.

[0216] Monoclonal antibodies are prepared, for example, using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a 44Mintz Ref. No.: 048536-797001WO mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

[0217] The immunizing agent will typically include the protein antigen, a fragment thereof ora fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.59- 103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.

[0218] Immortalized cell lines can be used that fuse efficiently and support stable high levelexpression of antibody by the selected antibody-producing cells. Additional immortalized cell lines include, without limitation, murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of monoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.51-63)).

[0219] The culture medium in which the hybridoma cells are cultured can then be assayed forthe presence of monoclonal antibodies directed against the antigen. In one embodiment, the binding specificity of monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (MA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Moreover, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies having a high degree of specificity and a high binding affinity for the target antigen. 45Mintz Ref. No.: 048536-797001WO

[0220] After the desired hybridoma cells are identified, the clones can be subcloned bylimiting dilution procedures and grown by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.59-103). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0221] The monoclonal antibodies secreted by the subclones can be isolated or purified fromthe culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0222] Monoclonal antibodies can also be made by recombinant DNA methods, such asthose described in U.S. Pat. No.4,816,567. DNA encoding the monoclonal antibodies disclosed herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells disclosed herein serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (see U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody disclosed herein, or can be substituted for the variable domains of one antigen-combining site of an antibody disclosed herein to create a chimeric bivalent antibody.

[0223] Monoclonal antibodies disclosed herein include fully human antibodies or humanizedantibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.

[0224] In other, alternative methods, an Src antibody is developed, for example, using phage-display methods using antibodies containing only human sequences. Such approaches are described in, e.g., in WO92 / 01047 and U.S. Pat. No.6,521,404, which are hereby 46Mintz Ref. No.: 048536-797001WO incorporated by reference. In this approach, a combinatorial library of phage carrying random pairs of light and heavy chains are screened using natural or recombinant source of Src.

[0225] The antibody described herein is isolated or purified. Generally, an isolated antibodyis one that is substantially free of other antibodies with different antigenic specificities than the isolated antibody. For example, in a particular embodiment, a preparation of an antibody described herein is substantially free of cellular material and / or chemical precursors. The language “substantially free of cellular material” includes preparations of an antibody in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes preparations of antibody having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous protein (also referred to herein as a “contaminating protein”) and / or variants of an antibody, for example, different post- translational modified forms of an antibody or other different versions of an antibody (e.g., antibody fragments). When the antibody is recombinantly produced, it is also generally substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When the antibody is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly, such preparations of the antibody have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In a specific embodiment, antibodies described herein are isolated or purified. ANTIBODY CONJUGATES

[0226] Also provided herein are anti-Src antibodies, or antigen-binding fragments thereof,conjugated or recombinantly fused to a diagnostic, detectable or therapeutic agent or any other molecule. The conjugated or recombinantly fused antibodies can be useful, e.g., for monitoring or prognosing the onset, development, progression and / or severity of an Src- associated disorder or disease, for example, as part of a clinical testing procedure, such as determining the efficacy of a particular therapy. The conjugated or recombinantly fused antibodies can be useful, e.g., for protecting against, treating or managing an Src-associated disorder, or for protecting against, treating or managing effects of an Src-associated disorder. 47Mintz Ref. No.: 048536-797001WO

[0227] In one aspect, the present disclosure relates to an antibody drug conjugate thatincludes a cytotoxic or therapeutic compound that is conjugated to an anti-Src antibody or antigen binding fragment thereof. The present disclosure also relates to antibody-radionuclide conjugates that include a radionuclide that is conjugated to an anti-Src antibody or antigen binding fragment thereof.

[0228] Anti-Src antibodies and antigen binding fragments thereof that can be used in theantibody conjugates are described above.

[0229] Whether conjugated to a cytotoxic / therapeutic compound or a radionuclide, the anti-Src antibody conjugates can include a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3; a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 6.

[0230] In some embodiments, the anti-Src antibody conjugates can include a heavy chainCDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10.

[0231] In some embodiments, the anti-Src antibody conjugates can include a heavy chainCDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 136, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10.

[0232] In some embodiments, the anti-Src antibody conjugates can include a heavy chainCDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; a light chain CDR1 domain 48Mintz Ref. No.: 048536-797001WO comprising the amino acid sequence of SEQ ID NO: 137, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10.

[0233] In some embodiments, the anti-Src antibody conjugates can include a heavy chainCDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 136, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 137, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 10.

[0234] In some embodiments, the anti-Src antibody conjugates include a variable heavychain sequence comprising the amino acid sequence set forth in SEQ ID NO: 11 and a variable light chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 12.

[0235] In some embodiments, the anti-Src antibody conjugates include a variable heavychain sequence comprising the amino acid sequence set forth in SEQ ID NO: 13 and a variable light chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 14.

[0236] In some embodiments, the anti-Src antibody conjugates include a variable heavychain sequence comprising the amino acid sequence set forth in SEQ ID NO: 40 and a variable light chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 41.

[0237] In some embodiments, the anti-Src antibody conjugates include a variable heavychain sequence comprising the amino acid sequence set forth in SEQ ID NO: 42 and a variable light chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 43.

[0238] In some embodiments, the anti-Src antibody conjugates include a variable heavychain sequence comprising the amino acid sequence set forth in SEQ ID NO: 44 and a variable light chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 45.

[0239] In some embodiments, the anti-Src antibody conjugates include a variable heavychain sequence comprising the amino acid sequence set forth in SEQ ID NO: 46 and a 49Mintz Ref. No.: 048536-797001WO variable light chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 47.

[0240] In some embodiments, the anti-Src antibody conjugates include a variable heavychain sequence comprising the amino acid sequence set forth in SEQ ID NO: 48 and a variable light chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 49.

[0241] In some embodiments, the anti-Src antibody conjugates of the present disclosurecomprises a VH sequence that is about 80%, about 85%, about 90%, about 95%, or about 99% identical to a sequence selected from SEQ ID NOS: 11, 13, 40, 42, 44, 46, and 48 and a VL sequence that is about 80%, about 85%, about 90%, about 95%, or about 99% identical to a sequence selected from SEQ ID NOS: 12, 14, 41, 43, 45, 47, and 49. In some embodiments, the anti-Src antibody of the present disclosure comprise a VH sequence selected from SEQ ID NOS: 11, 13, 40, 42, 44, 46, and 48 and a VL sequence selected from SEQ ID NOS: 12, 14, 41, 43, 45, 47, and 49. Antibody Drug Conjugate (ADC)

[0242] An antibody drug conjugate or ADC can refer to a binding protein, such as anantibody or antigen binding fragment thereof, chemically linked to one or more chemical drug(s) that may optionally be therapeutic or cytotoxic agents. In one embodiment, an ADC includes an antibody, a cytotoxic or therapeutic drug, and a linker that enables attachment or conjugation of the drug to the antibody. An ADC typically has anywhere from 1 to 8 drugs conjugated to the antibody, including drug loaded species of 2, 4, 6, or 8. Non- limiting examples of drugs that may be included in the ADCs are mitotic inhibitors, antitumor antibiotics, immunomodulating agents, vectors for gene therapy, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormones, antihormone agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, tyrosine kinase inhibitors, and radiosensitizers.

[0243] In some embodiments, the drug to be conjugated to the anti-Src antibody or antibodyfragment may be selected from the group consisting of an anthracycline, a camptothecin, a tubulin inhibitor, a maytansinoid, a calicheamycin, an auristatin, a nitrogen mustard, an ethylenimine derivative, an alkyl sulfonate, a nitrosourea, a triazene, a folic acid analog, a taxane, a COX-2 inhibitor, a pyrimidine analog, a purine analog, an antibiotic, an enzyme inhibitor, an epipodophyllotoxin, a platinum coordination complex, a vinca alkaloid, a 50Mintz Ref. No.: 048536-797001WO substituted urea, a methyl hydrazine derivative, an adrenocortical suppressant, a hormone antagonist, an antimetabolite, an alkylating agent, an antimitotic, an anti-angiogenic agent, a tyrosine kinase inhibitor, an mTOR inhibitor, a heat shock protein (HSP90) inhibitor, a proteosome inhibitor, an HDAC inhibitor, a pro-apoptotic agent, and a combination thereof.

[0244] Specific drugs of use may be selected from the group consisting of 5-fluorouracil,afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatinum, COX-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin, daunorubicin, DM1, DM3, DM4, doxorubicin, 2- pyrrolinodoxorubicine (2-PDox), a pro-drug form of 2-PDox (pro-2-PDox), cyano- morpholino doxorubicin, doxorubicin glucuronide, endostatin, epirubicin glucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib, entinostat, estrogen receptor binding agents, etoposide (VP16), etoposide glucuronide, etoposide phosphate, exemestane, fingolimod, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl-protein transferase inhibitors, flavopiridol, fostamatinib, ganetespib, GDC-0834, GS- 1101, gefitinib, gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, monomethylauristatin F (MMAF), monomethylauristatin D (MMAD), monomethylauristatin E (MMAE), navelbine, neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine, SN-38, sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide, transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine, vincristine, vinca alkaloids and ZD1839.

[0245] In some embodiments, the cytotoxic compound is a tubulin inhibitor. In one or moreembodiments, the drug is a tubulin inhibitor selected from the group consisting of dolastatin (dolastatin), auristatin (auristatin) and maytansine (maytansine). In one or more embodiments, the drug is an auristatin (auristatin) selected from the group consisting of monomethyl auristatin E (monomethyl auristatin E; MMAE), monomethyl auristatin F (monomethyl auristatin F; MMAF), and auristatin F (auristatin F; AF). In one embodiment, the tubulin inhibitor is MMAE. 51Mintz Ref. No.: 048536-797001WO

[0246] In some embodiments, the cytotoxic compound is a topoisomerase inhibitor. In one ormore embodiments, the drug is a topoisomerase inhibitor selected from the group consisting of irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9- nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, camptothecin derivative SN-38, exatecan derivative Dxd, 22-hydroxycamptothecin, topotecan, lurtoltecan, belotecan, irinotecan, silicon-based homocamptothecin (homosilatecan), 6, 8-dibromo-2-methyl-3- [2- (D-xylopyranosylamino) phenyl ] -4 (3H) - quinazolinone, 2-cyano-3- (3, 4-dihydroxyphenyl) N- (phenylmethyl) - (2E) -2-acrylamide, 2-cyano-3- (3, 4-dihydroxyphenyl) -N- (3-hydroxyphenylpropyl) - (E) -2-acrylamide, 12- β - D-glucopyranosyl-12, 13-dihydro-2, 10-hydroxy-2- [2- (D-xylopyranoside) phenyl ] -4 (3H) - quinazolinone, 2-cyano-3- (3, 4-dihydroxyphenyl) N- (phenylmethyl) - (2E) -2-acrylamide, 12- β -D-glucopyranosyl-12, 13-dihydro-2, 10-hydroxy-2- [2- (D-xylopyranoside ] methyl- [2- (4-hydroxypyrrolo-methyl) -5-amino ] 4- (3H) -quinazolinone, 5-hydroxy-4-methyl- [ 2- hydroxy ] ethyl ] amino ] carbonyl ] amino acid hydrochloride N- [2- (dimethylamino) ethyl ] -4-acridine carboxamide. In one embodiment, the topoisomerase inhibitor is SN38. In one embodiment, the topoisomerase inhibitor is Dxd. Antibody-radionuclide Conjugate

[0247] An antibody radionuclide conjugate can refer to a binding protein, such as an antibodyor antigen binding fragment thereof, chemically linked to one or more radionuclides.

[0248] In some embodiments, the radionuclide is one that can be used in medical imaging. Insome embodiments, the detectable label is a label that can be used for radiography, magnetic resonance imaging, nuclear medicine, ultrasound elastography, photoacoustic imaging, tomography, echocardiography, functional near-infrared spectroscopy, magnetic particle imaging. In embodiments, the detectable label is a label that can be used for tomography. In some embodiments, the detectable label is a label that can be used for positron emission tomography.

[0249] In some embodiments, the radionuclide is one that can be used in therapeutics.

[0250] In some embodiments, the radionuclide is one that can be used in diagnostics (e.g.,PET imaging).

[0251] Radioactive isotopes useful in the conjugates described herein include, but are notlimited to—111In,177Lu,212Bi,213Bi,211At,62Cu,67Cu,90Y,125I,131I,32P,33P,47Sc,111Ag,67Ga,142Pr,153Sm,161Tb,166Dy,166Ho,186Re,188Re,189Re,212Pb,223Ra,225Ac,59Fe,75Se,77As,89Sr,99Mo,105Rh,109Pd,143Pr,149Pm,169Er,194Ir,198Au,199Au,227Th and211Pb. 52Mintz Ref. No.: 048536-797001WO Additional potential radioisotopes of use include11C,13N,15O,75Br,198Au,224Ac,126I,133I,77Br,113mIn,95Ru,97Ru,103Ru,105Ru,107Hg,203Hg,121mTe,122mTe,125mTe,165Tm,167Tm,168Tm,197Pt,109Pd,105Rh,142Pr,143Pr,161Tb,166Ho,199Au,57Co,58Co,51Cr, 59Fe,75Se,201Tl,225Ac,76Br,169Yb,89Zr,64Cu,211At,227Th,225Ac,223Ra,213Bi, or212Bi,123I,124I,125I, or131I,18F,64Cu,68Ga,78Br,82Rb,86Y,89Zr,90Y,22Na,26Al,40K,83Sr, or124I and the like.

[0252] In embodiments, the detectable label is an iodine radioisotope. In embodiments, theradioisotope is123I,124I,125I, or131I. In embodiments, the radioisotope is123I. In embodiments, the radioisotope is 124I. In embodiments, the radioisotope is125I. In embodiments, the radioisotope is131I.

[0253] In embodiments, the radioisotope is a positron-emitting radioisotope. In embodiments,the positron-emitting radioisotope is11C,13N,15O,18F,64Cu,68Ga,78Br,82Rb,86Y,89Zr,90Y,22Na,26Al,40K,83Sr, or124I. In embodiments, the positron-emitting radioisotope is11C. In embodiments, the positron-emitting radioisotope is13N. In embodiments, the positron- emitting radioisotope is15O. In embodiments, the positron-emitting radioisotope is18F. In embodiments, the positron-emitting radioisotope is64Cu. In embodiments, the positron- emitting radioisotope is168Ga. In embodiments, the positron-emitting radioisotope is78Br. In embodiments, the positron-emitting radioisotope is82Rb. In embodiments, the positron- emitting radioisotope is86Y. In embodiments, the positron-emitting radioisotope is89Zr. In embodiments, the positron-emitting radioisotope is90Y. In embodiments, the positron- emitting radioisotope is22Na. In embodiments, the positron-emitting radioisotope is26Al. In embodiments, the positron-emitting radioisotope is40K. In embodiments, the positron- emitting radioisotope is 83Sr. In embodiments, the positron-emitting radioisotope is124I.

[0254] In embodiments, the radioisotope is an alpha-emitting radioisotope. In embodiments,the alpha-emitting radioisotope is211At,227Th,225Ac,223Ra,213Bi, or212Bi. In embodiments, the alpha-emitting radioisotope is211At. In embodiments, the alpha-emitting radioisotope is227Th. In embodiments, the alpha-emitting radioisotope is225Ac. In embodiments, the alpha- emitting radioisotope is223Ra. In embodiments, the alpha-emitting radioisotope is213Bi. In embodiments, the alpha-emitting radioisotope is212Bi. Conjugation Methods

[0255] As described above, antibodies or fragments thereof may be conjugated to one ormore therapeutic or diagnostic agents. The therapeutic agents do not need to be the same but can be different, e.g., a drug and a radioisotope. For example,131I can be incorporated into a tyrosine of an antibody or fusion protein and a drug attached to an epsilon amino group of a 53Mintz Ref. No.: 048536-797001WO lysine residue. Therapeutic and diagnostic agents also can be attached, for example to reduced SH groups and / or to carbohydrate side chains. Many methods for making covalent or non-covalent conjugates of therapeutic or diagnostic agents with antibodies or fusion proteins are known in the art and any such known method may be utilized. Radionuclides and other metals may be delivered, for example, using chelating groups attached to an antibody or conjugate. Macrocyclic chelates such as NOTA, DOTA, and TETA are of use with a variety of metals and radiometals, most particularly with radionuclides of gallium, yttrium, and copper, respectively. Such metal-chelate complexes can be made very stable by tailoring the ring size to the metal of interest. Other ring-type chelates, such as macrocyclic polyethers for complexing223Ra, may be used.

[0256] Conjugates of the antibody and cytotoxic agent can also be made using a variety ofbifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolylene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody (See WO94 / 11026).

[0257] Those of ordinary skill in the art will recognize that a large variety of possiblemoieties can be coupled to the resultant antibodies disclosed herein. (See, for example, “Conjugate Vaccines”, Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[0258] Coupling may be accomplished by any chemical reaction that will bind the twomolecules so long as the antibody and the other moiety retain their respective activities. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation. In one embodiment, the binding is covalent binding. Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of 54Mintz Ref. No.: 048536-797001WO the present disclosure, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom, Jour. Immun.133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987).

[0259] Preferred linkers are described in the literature. (See, for example, Ramakrishnan, S.et al., Cancer Res.44:201-208 (1984) describing use of MBS (M-maleimidobenzoyl-N- hydroxysuccinimide ester). See also, U.S. Pat. No.5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker. Particularly preferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2- pridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2- pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat. #21651G); (iv) Sulfo-LC- SPDP (sulfosuccinimidyl 6 [3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat. #2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC.

[0260] The linkers described above contain components that have different attributes, thusleading to conjugates with differing physico-chemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester containing linkers are less soluble than sulfo-NHS esters. Further, the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available. Sulfo-NHS, in particular, can enhance the stability of carbodimide couplings. Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone. BISPECIFIC T CELL ENGAGERS (BITES / TCES)

[0261] The present disclosure provides, inter alia, multi-specific (e.g., bi-, tri-, quad-specific)proteins, that are engineered to contain one or more T cell binding moieties that mediate 55Mintz Ref. No.: 048536-797001WO binding to and / or activation of a T cell. In one embodiment, the present disclosure relates to bispecific T cell engagers (BITES / TCEs) that include an anti-Src antibody or antigen binding fragment thereof and a T cell antigen binding moiety.

[0262] Anti-Src antibodies and antigen binding fragments thereof that can be used in thebispecific T cell enagager are described above.

[0263] T cell binding moieties can be selected from an antigen binding domain or ligand thatbinds to (e.g., and in some embodiments activates) one or more of CD3, TCRα, TCRβ, TCRγ, TCRζ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226. In other embodiments, the T cell binding moiety is selected from an antigen binding domain or ligand that binds to and does not activate one or more of CD3, TCRα, TCRβ, TCRγ, TCRζ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4- 1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226. In some embodiments, the T cell binding moiety binds to CD3.In some embodiments, T cell binding moieties bind to CD3. In some embodiments, the CD3 binding moiety comprises a GBR1302 antibody or an antigen binding fragment thereof. In some embodiments, a CD3 binding moiety comprises a heavy chain variable region comprising the amino acid sequence of: EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYY ADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVS S (SEQ ID NO: 50) and a light chain variable region comprising the amino acid sequence of: QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPA RFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGCGTKVEIK (SEQ ID NO: 51).

[0264] In some embodiments, the CD3 binding moiety comprises an SP34 antibody or anantigen binding fragment thereof. In some embodiments, a CD3 binding moiety comprises a heavy chain variable region comprising the amino acid sequence of: EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKCLEWVARIRSKYNNYATYY ADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVS S (SEQ ID NO: 52) and a light chain variable region comprising the amino acid sequence of: QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPA RFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGCGTKVEIK (SEQ ID NO: 53).

[0265] In some embodiments, the CD3 binding moiety comprises an Adimab-26906 antibodyor an antigen binding fragment thereof. In some embodiments, a CD3-binding moiety comprises a heavy chain variable region comprising the amino acid sequence of: QVQLVQSGAEVKKPGASVKVSCKASGFNIKDYYMHWVRQAPGQCLEWMGWIDLENANTIYDA 56Mintz Ref. No.: 048536-797001WO KFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDAYGRYFYDVWGQGTLVTVSS (SEQ ID NO: 54) and a light chain variable region comprising the amino acid sequence of: DIVMTQSPDSLAVSLGERATINCKSSQSLLNARTGKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYSRRTFGCGTKVEIK (SEQ ID NO: 55).

[0266] In some embodiments, the CD3 binding moiety comprises an Adimab-67447 antibodyor an antigen binding fragment thereof. In some embodiments, T cell binding moieties bind to CD3. In some embodiments, a CD3-binding moiety comprises a heavy chain variable region comprising the amino acid sequence of: QVQLVQSGAEVKKPGASVKVSCKASGFDIKDYYMHWVRQAPGQCLEWMGWIELENDDTIYDA KFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDAYGRYFYDVWGQGTLVTVSS (SEQ ID NO: 56) and a light chain variable region comprising the amino acid sequence of: DIVMTQSPDSLAVSLGERATINCKSSQSLLNARTGKNYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYSRRTFGCGTKVEIK (SEQ ID NO: 57).

[0267] In some embodiments, the CD3 binding moiety comprises a Glofitamab antibody oran antigen binding fragment thereof. In some embodiments, T cell binding moieties bind to CD3. In some embodiments, a CD3-binding moiety comprises a heavy chain variable region comprising the amino acid sequence of: EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYY ADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVS S (SEQ ID NO: 58) and a light chain variable region comprising the amino acid sequence of: QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPA RFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL (SEQ ID NO: 59).

[0268] Some T cell binding moieties that bind to CD3 are known in the art and include, forexample, SP34 scFV and OKT3 scFv.

[0269] In one embodiment, the T cell binding moiety includes an SP34 scFv. In oneembodiment, the SP34 scFV comprises the amino acid sequence of: EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYY ADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVS SGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQK PGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGG TKLTVL (SEQ ID NO: 19)

[0270] In one embodiment, the SP34 scFV comprises a sequence that is about 80%, about85%, about 90%, about 95%, or about 99% identical to SEQ ID NO: 19. 57Mintz Ref. No.: 048536-797001WO

[0271] In one embodiment, the T cell binding moiety includes an OKT3 scFv. In oneembodiment, the OKT3 scFV comprises the amino acid sequence of: QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQ KFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYSLDYWGQGTPVTVSSGGGGS GGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSK LASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR (SEQ ID NO: 20)

[0272] In one embodiment, the OKT3 scFV comprises a sequence that is about 80%, about85%, about 90%, about 95%, or about 99% identical to SEQ ID NO: 20.

[0273] Bispecific T cell engagers of the present disclosure can comprise different structuresincluding, but not limited to, bispecific immunoglobulin G, IgG appended with an additional antigen-binding moiety, and bispecific antibody fragments.

[0274] In one embodiment, the bispecific T cell engager as described herein is a bispecificimmunoglobulin G (BsIgG). BsIgG is a format that is monovalent for each antigen. Exemplary BsIgG formats include, but are not limited to, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-holes common LC, knobs-in-holes assembly, charge pair, Fab-arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, dk-body, and orthogonal Fab. See Spiess et al. Mol. Immunol.67(2015):95-106. In some embodiments, BsIgG comprises heavy chains that are engineered for heterodimerization. For example, heavy chains can be engineered for heterodimerization using a “knobs-into-holes” strategy, a SEED platform, a common heavy chain (e.g., in κλ-bodies), and use of heterodimeric Fc regions. See Spiess et al. Mol. Immunol.67(2015):95-106. Strategies that have been used to avoid heavy chain pairing of homodimers in BsIgG include knobs-in-holes, duobody, azymetric, charge pair, HA-TF, SEEDbody, and differential protein A affinity. BsIgG can be produced by separate expression of the component antibodies in different host cells and subsequent purification / assembly into a BsIgG. BsIgG can also be produced by expression of the component antibodies in a single host cell. BsIgG can be purified using affinity chromatography, e.g., using protein A and sequential pH elution.

[0275] In one embodiment, the bispecific T cell engager as described herein is an IgGappended with an additional antigen-binding moiety. For example, monospecific IgG can be engineered to have bispecificity by appending an additional antigen-binding unit onto the monospecific IgG, e.g., at the N- or C-terminus of either the heavy or light chain. Exemplary additional antigen-binding units include single domain antibodies (e.g., variable heavy chain 58Mintz Ref. No.: 048536-797001WO or variable light chain), engineered protein scaffolds, and paired antibody variable domains (e.g., single chain variable fragments or variable fragments). Examples of appended IgG formats include dual variable domain IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)- scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG- scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (four-in-one). See Spiess et al. Mol. Immunol.67(2015):95-106.

[0276] In one embodiment, the bispecific T cell engager as described herein is a bispecificantibody fragment. Bispecific antibody fragments (BsAb) are a format of bispecific antibody molecules that lack some or all of the antibody constant domains. For example, some BsAb lack an Fc region. In embodiments, bispecific antibody fragments include heavy and light chain regions that are connected by a peptide linker that permits efficient expression of the BsAb in a single host cell. Exemplary bispecific antibody fragments include but are not limited to nanobody, nanobody-HAS, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv- CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab′)2, F(ab′)2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, Diabody-Fc, tandem scFv-Fc, and intrabody. For example, the BiTE format comprises tandem scFvs, where the component scFvs bind to CD3 on T cells and eSrc cancer cells.

[0277] Exemplary bispecific T cell engagers also include, without limitation, a bivalent SrcFab -scFv (anti-CD3) fusion, tetravalent IgG with scFv (anti-CD3) fused to C-terminus of light chain of Src, knob-in-hole type bivalent IgG with scFv (anti-CD3) on one half-Fc and Src Fab on the other half Fc, knob-in-hole type bivalent IgG with CrossMab VH / VL swap (anti-CD3) on one half Fc and Src Fab on the other half Fc, and a trivalent IgG with Src Fab - CrossMab VH / VL swap (anti-CD3) on one half Fc and Src Fab on the other half Fc. In one embodiment, the bispecific T cell engager comprises a bivalent Src Fab -scFv (anti-CD3) fusion. In one embodiment, the bispecific T cell engager comprises tetravalent IgG with scFv (anti-CD3) fused to C-terminus of light chain of Src. In one embodiment, the bispecific T cell engager comprises knob-in-hole type bivalent IgG with scFv (anti-CD3) on one half-Fc and Src Fab on the other half Fc. In one embodiment, the bispecific T cell engager comprises a trivalent IgG with Src Fab -CrossMab VH / VL swap (anti-CD3) on one half Fc and Src Fab on the other half Fc.

[0278] The antibody molecules can be produced by recombinant expression, e.g., of at leastone or more component, in a host system. Exemplary host systems include eukaryotic cells 59Mintz Ref. No.: 048536-797001WO (e.g., mammalian cells, e.g., CHO cells, or insect cells, e.g., SF9 or S2 cells) and prokaryotic cells (e.g., E. coli). Bispecific antibody molecules can be produced by separate expression of the components in different host cells and subsequent purification / assembly. Alternatively, the antibody molecules can be produced by expression of the components in a single host cell. Purification of bispecific antibody molecules can be performed by various methods such as affinity chromatography, e.g., using protein A and sequential pH elution. In other embodiments, affinity tags can be used for purification, e.g., histidine-containing tag, myc tag, or streptavidin tag.

[0279] Various methods of producing multispecific antibodies have been disclosed to addressthe problem of incorrect heavy chain pairing. Exemplary methods are described below. Exemplary multispecific antibody formats and methods of making said multispecific antibodies are also disclosed in e.g., Speiss et al. Molecular Immunology 67 (2015) 95-106; and Klein et al mAbs 4:6, 653-663; November / December 2012; the entire contents of each of which are incorporated by reference herein.

[0280] Heterodimerized bispecific antibodies are based on the natural IgG structure, whereinthe two binding arms recognize different antigens. IgG derived formats that enable defined monovalent (and simultaneous) antigen binding are generated by forced heavy chain heterodimerization, combined with technologies that minimize light chain mispairing (e.g., common light chain). Forced heavy chain heterodimerization can be obtained using, e.g., knob-in-hole OR strand exchange engineered domains (SEED). ANTIBODY FORMATS Knob-In-Hole

[0281] Knob-in-Hole as described in U.S. Pat. Nos. 5,731,116, 7,476,724 and Ridgway, J. etal. (1996) Prot. Engineering 9(7): 617-621, broadly involves: (1) mutating the CH3 domain of one or both antibodies to promote heterodimerization; and (2) combining the mutated antibodies under conditions that promote heterodimerization. “Knobs” or “protuberances” are typically created by replacing a small amino acid in a parental antibody with a larger amino acid (e.g., T366Y or T366W); “Holes” or “cavities” are created by replacing a larger residue in a parental antibody with a smaller amino acid (e.g., Y407T, T366S, L368A and / or Y407V).

[0282] For bispecific antibodies including an Fc domain, introduction of specific mutationsinto the constant region of the heavy chains to promote the correct heterodimerization of the 60Mintz Ref. No.: 048536-797001WO Fc portion can be utilized. Several such techniques are reviewed in Klein et al. (mAbs (2012) 4:6, 1-11), the contents of which are incorporated herein by reference in their entirety. These techniques include the “knobs-into-holes” (KiH) approach which involves the introduction of a bulky residue into one of the CH3 domains of one of the antibody heavy chains. This bulky residue fits into a complementary “hole” in the other CH3 domain of the paired heavy chain so as to promote correct pairing of heavy chains (see e.g., U.S. Pat. No.7,642,228).

[0283] Exemplary KiH mutations include S354C, T366W in the “knob” heavy chain andY349C, T366S, L368A, Y407V in the “hole” heavy chain.

[0284] Heterodimeric Fc platforms that support the design of bispecific and asymmetricfusion proteins by devising strand-exchange engineered domain (SEED) C(H)3 heterodimers are known. These derivatives of human IgG and IgA C(H)3 domains create complementary human SEED C(H)3 heterodimers that are composed of alternating segments of human IgA and IgG C(H)3 sequences. The resulting pair of SEED C(H)3 domains preferentially associates to form heterodimers when expressed in mammalian cells. SEEDbody (Sb) fusion proteins consist of [IgG1 hinge]-C(H)2-[SEED C(H)3], that may be genetically linked to one or more fusion partners (see e.g., Davis J H et al. SEEDbodies: fusion proteins based on strand exchange engineered domain (SEED) CH3 heterodimers in an Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies. Protein Eng Des Sel 2010; 23:195-202; PMID:20299542 and U.S. Pat. No.8,871,912. The contents of each of which are incorporated by reference herein).

[0285] Exemplary Fc sequences are listed in Table 1 below:Table 1. Sequence name Fc sequence SEQ ID NO.6Mintz Ref. No.: 048536-797001WO KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GKuo o y “Duobody” technology to produce bispecific antibodies with correct heavy chain pairing are known. The DuoBody technology involves three basic steps to generate stable bispecific human IgGl antibodies in a post-production exchange reaction. In a first step, two IgG1s, each containing single matched mutations in the third constant (CH3) domain, are produced separately using standard mammalian recombinant cell lines. Subsequently, these IgG1 antibodies are purified according to standard processes for recovery and purification. After production and purification (post-production), the two antibodies are recombined under tailored laboratory conditions resulting in a bispecific antibody product with a very high yield (typically >95%) (see e.g., Labrijn et al, PNAS 2013; 110(13):5145-5150 and Labrijn et al. Nature Protocols 2014; 9(10):2450-63, the contents of each of which are incorporated by reference herein). Electrostatic Interactions

[0286] Methods of making multispecific antibodies using CH3 amino acid changes withcharged amino acids such that homodimer formation is electrostatically unfavorable are disclosed. EP1870459 and WO 2009089004 describe other strategies for favoring heterodimer formation upon co-expression of different antibody domains in a host cell. In these methods, one or more residues that make up the heavy chain constant domain 3 (CH3), 62Mintz Ref. No.: 048536-797001WO CH3-CH3 interfaces in both CH3 domains are replaced with a charged amino acid such that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favorable. Additional methods of making multispecific molecules using electrostatic interactions are described in the following references, the contents of each of which is incorporated by reference herein, include US20100015133, U.S. Pat. No. 8,592,562B2, U.S. Pat. No.9,200,060B2, US20140154254A1, and U.S. Pat. No. 9,358,286A1. Common Light Chain

[0287] Light chain mispairing needs to be avoided to generate homogenous preparations ofbispecific IgGs. One way to achieve this is through the use of the common light chain principle, i.e., combining two binders that share one light chain but still have separate specificities. An exemplary method of enhancing the formation of a desired bispecific antibody from a mixture of monomers is by providing a common variable light chain to interact with each of the heteromeric variable heavy chain regions of the bispecific antibody. Compositions and methods of producing bispecific antibodies with a common light chain as disclosed in, e.g., U.S. Pat. No.7,183,076B2, US20110177073A1, EP2847231A1, WO2016079081A1, and EP3055329A1, the contents of each of which is incorporated by reference herein. CrossMab

[0288] Another option to reduce light chain mispairing is the CrossMab technology whichavoids non-specific L chain mispairing by exchanging CHI and CL domains in the Fab of one half of the bispecific antibody. Such crossover variants retain binding specificity and affinity but make the two arms so different that L chain mispairing is prevented. The CrossMab technology (as reviewed in Klein et al. Supra) involves domain swapping between heavy and light chains so as to promote the formation of the correct pairings. Briefly, to construct a bispecific IgG-like CrossMab antibody that could bind to two antigens by using two distinct light chain-heavy chain pairs, a two-step modification process is applied. First, a dimerization interface is engineered into the C-terminus of each heavy chain using a heterodimerization approach, e.g., Knob-into-hole (KiH) technology, to ensure that only a heterodimer of two distinct heavy chains from one antibody (e.g., Antibody A) and a second antibody (e.g., Antibody B) is efficiently formed. Next, the constant heavy 1 (CH1) and constant light (CL) domains of one antibody are exchanged (Antibody A), keeping the variable heavy (VH) and variable light (VL) domains consistent. The exchange of the CH1 63Mintz Ref. No.: 048536-797001WO and CL domains ensured that the modified antibody (Antibody A) light chain would only efficiently dimerize with the modified antibody (antibody A) heavy chain, while the unmodified antibody (Antibody B) light chain would only efficiently dimerize with the unmodified antibody (Antibody B) heavy chain; and thus only the desired bispecific CrossMab would be efficiently formed (see e.g., Cain, C. SciBX 4(28); doi:10.1038 / scibx.2011.783, the contents of which are incorporated by reference herein). Common Heavy Chain

[0289] An exemplary method of enhancing the formation of a desired bispecific antibodyfrom a mixture of monomers is by providing a common variable heavy chain to interact with each of the heteromeric variable light chain regions of the bispecific antibody. Compositions and methods of producing bispecific antibodies with a common heavy chain are disclosed in, e.g., US20120184716, US20130317200, and US20160264685A1, the contents of each of which is incorporated by reference herein. Amino Acid Modifications

[0290] Alternative compositions and methods of producing multispecific antibodies withcorrect light chain pairing include various amino acid modifications. For example, Zymeworks describes heterodimers with one or more amino acid modifications in the CH1 and / or CL domains, one or more amino acid modifications in the VH and / or VL domains, or a combination thereof, which are part of the interface between the light chain and heavy chain and create preferential pairing between each heavy chain and a desired light chain such that when the two heavy chains and two light chains of the heterodimer pair are co-expressed in a cell, the heavy chain of the first heterodimer preferentially pairs with one of the light chains rather than the other (see e.g., WO2015181805). Other exemplary methods are described in WO2016026943 (Argen-X), US20150211001, US20140072581A1, US20160039947A1, and US20150368352. Lambda / Kappa Formats

[0291] Multispecific molecules (e.g., multispecific antibody molecules) that include thelambda light chain polypeptide and a kappa light chain polypeptides, can be used to allow for heterodimerization. Methods for generating bispecific antibody molecules comprising the lambda light chain polypeptide and a kappa light chain polypeptides are disclosed in U.S. Ser. No.62 / 399,319 filed on Sep.23, 2016, incorporated herein by reference in its entirety. 64Mintz Ref. No.: 048536-797001WO Exemplary BiTE Constructs

[0292] In some embodiments, the bispecific T cell engager of the present disclosure comprises an anti-Src Fab and an anti-CD3 scFv. An exemplary Fab-scFv BiTe can comprise the following amino acid sequences: Table 2. Chain Amino Acid Sequence SEQ ID NO

[0293] In some embodiments, the bispecific T cell engager of the present disclosure comprises anti-Src IgG and an anti-Cd3 scFv. In some embodiments, the BiTe is an IgG-scFv 65Mintz Ref. No.: 048536-797001WO tetrameric BiTe. An exemplary IgG-scFv BiTe can comprise the following amino acid sequences: Table 3 Chain Amino Acid Sequence SEQ ID NO

[0294] In some embodiments, the BiTe is a heterobifunctional IgG dimeric BiTe. An exemplary IgG-scFv BiTe can comprise the following amino acid sequences: 66Mintz Ref. No.: 048536-797001WO Table 4 Chain Amino Acid Sequence SEQ ID NO67Mintz Ref. No.: 048536-797001WO DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLL68Mintz Ref. No.: 048536-797001WO

[0295] In some embodiments, the BiTe is an IgG-scFv trimeric BiTe. An exemplary IgG- scFv BiTe can comprise the following amino acid sequences: Table 5 Chain Amino Acid Sequence SEQ ID NOMintz Ref. No.: 048536-797001WO GTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEIVVTQSPATL SVSPGERATLSCRSSTGAVTESNYANWVQEKPGQAFRGLIGGANKRAPGExemplary Src and CD3 formats

[0296] In some embodiments, alternative Src and / or CD3 binder formats are used.Exemplary formats are listed below, where “–” indicates linkage between the modules. Table 6. CD3 Binder FormatsMintz Ref. No.: 048536-797001WO CD3 VH - SEQ ID NO: 62- eSrc VL - SEQ ID scDb-Fc NO: 63- eSrc VH - SEQ ID NO: 62- CD3 VL - SEQ ID NO: 65 –SEQ ID NOs in the above table are listed below: Table 7 SEQ ID Se uence S E P V Q71Mintz Ref. No.: 048536-797001WO SEQ ID NO.62 GGGSGGGG E A Q S V K P S L E R L L Q S E P V S E P V Q S E P V

[0297] In some embodiments, anti-Src antibodies in IgG1 format or bispecific format (Src xCD3) are used. Exemplary antibody sequences are listed below. 72Mintz Ref. No.: 048536-797001WO Table 8 Antibody chain 1 sequence chain 2 sequence chain 3 sequence chain 4 sequence nameMintz Ref. No.: 048536-797001WO VNHKPSNTKVDKKVEP KVDNALQSGNSQ GSLRLSCAASGF KSCDKTHTCPPCPAPEA ESVTEQDSKDST TFNTYAMNWVR AGGPSVFLFPPKPKDTL YSLSSTLTLSKAD QAPGKCLEWVA74Mintz Ref. No.: 048536-797001WO YNSTYRVVSVLT VLHQDWLNGKE YKCKVSNKALPA75Mintz Ref. No.: 048536-797001WO ab7 SEQ ID NO: 90SEQ ID NO: 28 SEQ ID NO: 91 SEQ ID NO: 32 EVQLVESGGGLVQPGGS DIQMTQSPSSLSA EIVVTQSPATLSV EVQLVESGGGLV A E Y RF L V NS G A L N Q T D V SF76Mintz Ref. No.: 048536-797001WO ab8 SEQ ID NO: 92SEQ ID NO: 28 SEQ ID NO: 93 SEQ ID NO: 32 EVQLVESGGGLVQPGGS DIQMTQSPSSLSA EIVVTQSPATLSV EVQLVESGGGLV A E Y RF L V NS G A L N Q T D V SF77Mintz Ref. No.: 048536-797001WO ab9 SEQ ID NO: 94SEQ ID NO: 88 SEQ ID NO: 95 SEQ ID NO: 32 EVQLVESGGGLVQPGGS DIQMTQSPSSLSA EIVVTQSPATLSV EVQLVESGGGLV A E Y F L V S G A N Q T D SF78Mintz Ref. No.: 048536-797001WO RVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIE KTISKAKGQPREPQVYT A A I R T Y Q A L N Q T D SF79Mintz Ref. No.: 048536-797001WO ab12 SEQ ID NO: 101SEQ ID NO: 98 SEQ ID NO: 102 SEQ ID NO: 103 QAVVTQEPSLTVSPGGT EVQLLESGGGLV QVQLQESGPGLV DIQMTQSPSSLSA A Q Y RF TI Y G A K L N D K C T80Mintz Ref. No.: 048536-797001WO ab13 SEQ ID NO: 104SEQ ID NO: 98 SEQ ID NO: 105 SEQ ID NO: 106 QAVVTQEPSLTVSPGGT EVQLLESGGGLV QVQLVESGGGLV DIQMTQSPSSLSA A Q Y R T Y G A K L N D K C T81Mintz Ref. No.: 048536-797001WO ab14 SEQ ID NO: 107SEQ ID NO: 108 SEQ ID NO: 109 SEQ ID NO: 98 QVQLVQSGAEVKKPGS DIVMTQTPLSLPV QVQLVQSGAEV EVQLLESGGGLV A W A T G V E L Q N D K C T82Mintz Ref. No.: 048536-797001WO LGGPSVFLFPPKPKDTL YSLSSTLTLSKAD MISRTPEVTCVVVDVSH YEKHKVYACEV EDPEVKFNWYVDGVEV THQGLSSPVTKSF83Mintz Ref. No.: 048536-797001WO MISRTPEVTCVVVDVSH YEKHKVYACEV EDPEVKFNWYVDGVEV THQGLSSPVTKSF HNAKTKPREEQYNSTY NRGECMintz Ref. No.: 048536-797001WO EDPEVKFNWYVDGVEV EVTHQGLSSPVT HNAKTKPREEQYNSTY KSFNRGEC RVVSVLTVLHQDWLNG85Mintz Ref. No.: 048536-797001WO ab21 SEQ ID NO: 119SEQ ID NO: 65 SEQ ID NO: 98 SEQ ID NO: 100 EVQLVESGGGLVQPGGS DKTHTCPPCPAP EVQLLESGGGLV DIQMTQSPSSLSA A I R T Y Q A L N Q T D SF86Mintz Ref. No.: 048536-797001WO WYSNLWVFGGGTKLTV HTFPAVLQSSGL YSLSSTLTLSKAD LEPKSSDKTHTCPPCPAP YSLSSVVTVPSSS YEKHKVYACEV EAAGGPSVFLFPPKPKD LGTQTYICNVNH THQGLSSPVTKSF87Mintz Ref. No.: 048536-797001WO VEWESNGQPENN YKTTPPVLDSDG SFFLVSKLTVDKSMintz Ref. No.: 048536-797001WO GGTKLTVL89Mintz Ref. No.: 048536-797001WO DEAEYYCALWY SNLWVFGGGTKL TVLMintz Ref. No.: 048536-797001WO GGTKLTVL A A I R T Y Q A L N Q T D SF91Mintz Ref. No.: 048536-797001WO ab28 SEQ ID NO: 131SEQ ID NO: 98: SEQ ID NO: 132 SEQ ID NO: 133 QAVVTQEPSLTVSPGGT EVQLLESGGGLV EVQLVESGGGLV DIQMTQSPSSLSA A I SR T Y F V R L Q N D K C T92Mintz Ref. No.: 048536-797001WO ab29 SEQ ID NO: 134SEQ ID NO: 98: SEQ ID NO: 135 SEQ ID NO: 100 QAVVTQEPSLTVSPGGT EVQLLESGGGLV EVQLVESGGGLV DIQMTQSPSSLSA A I SR T Y Q A L N Q T D V SFSRC LIGAND CONJUGATES

[0298] The present invention also provides Src ligand-drug conjugates for targeted cancertherapy. The ligand targets cancer cells expressing Src and allows for delivery of the drug to the desired location. The conjugates provided here have three components: a targeting ligand, a therapeutic agent (drug), and a linker that connects the ligand to the drug.

[0299] In some embodiments, the Src ligand-drug conjugate is cell impermeable.

[0300] A number of known Src ligands can be used in the small molecule conjugatesdescribed herein. In some embodiments, the Src ligand is a Src kinase inhibitor. Src kinase inhibitors can include, without limitation, AZD0530 (saracatinib), Bosulif (vosutinib), 93Mintz Ref. No.: 048536-797001WO ENMD981693, KD020, KX01, Sprycel (dasatinib), Yervoy (ipilimumab), AP23464, AP23485, AP23588, AZD0424, c-Src kinase inhibitor KISSEI, CU201, KX2361, SKS927, SRN004, SUNK706, TG100435, TG100948, AP23451, dasatinib HETERO (dasatinib), dasatinib VALEANT (dasatinib), Fontrax (dasatinib), Src kinase inhibitors KINEX, VX680 (tozasertib lactate), XL228, SUNK706, VAL201, PUR1800, TOP1288, NXP900, VAL301, ASN006, CR13626, MLR-1023, and bafetinib. In some embodiments, the Src kinase inhibitor is dasatinib. In another embodiment, Src kinase inhibitors can be identified through methods known in the art (e.g., Sen, B. and Johnson, FM Regulation of Src Family Kinases in Human Cancers.2011. J, incorporated herein by reference). Signal Transduction.2011: 14 pages).

[0301] Src binding peptides are also contemplated for use as Src ligands in the conjugatesdescribed herein. Examples include the linear peptide (pY)EEI where (pY) is phosphotyrosine (Cell 1993, 72, 779-790), the cyclic peptides DNQYAARQ(dF)P and DNQYAFFQ(dF)P where (dF) is D-phenylalanine and peptides are cyclized by N terminus to C terminus amidation (Archives of Biochemistry and Biophysics 1998, 355(1), 124-130), and [RW]5 cyclized by N terminus to C terminus amidation (Bioorganic &Medicinal Chemistry Letters 2013, 23(11), 3230-3234).

[0302] In some embodiments, the drug to be conjugated to the Src ligand is a cytotoxiccompound. In some embodiments, the cytotoxic compound may be selected from the group consisting of an anthracycline, a camptothecin, a tubulin inhibitor, a maytansinoid, a calicheamycin, an auristatin, a nitrogen mustard, an ethylenimine derivative, an alkyl sulfonate, a nitrosourea, a triazene, a folic acid analog, a taxane, a COX-2 inhibitor, a pyrimidine analog, a purine analog, an antibiotic, an enzyme inhibitor, an epipodophyllotoxin, a platinum coordination complex, a vinca alkaloid, a substituted urea, a methyl hydrazine derivative, an adrenocortical suppressant, a hormone antagonist, an antimetabolite, an alkylating agent, an antimitotic, an anti-angiogenic agent, a tyrosine kinase inhibitor, an mTOR inhibitor, a heat shock protein (HSP90) inhibitor, a proteosome inhibitor, an HDAC inhibitor, a pro-apoptotic agent, and a combination thereof.

[0303] In some embodiments, the cytotoxic compound is cell impermeable.

[0304] Specific drugs of use may be selected from the group consisting of 5-fluorouracil,afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, 94Mintz Ref. No.: 048536-797001WO cisplatinum, COX-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin, daunorubicin, DM1, DM3, DM4, doxorubicin, 2- pyrrolinodoxorubicine (2-PDox), a pro-drug form of 2-PDox (pro-2-PDox), cyano- morpholino doxorubicin, doxorubicin glucuronide, endostatin, epirubicin glucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib, entinostat, estrogen receptor binding agents, etoposide (VP16), etoposide glucuronide, etoposide phosphate, exemestane, fingolimod, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl-protein transferase inhibitors, flavopiridol, fostamatinib, ganetespib, GDC-0834, GS- 1101, gefitinib, gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, monomethylauristatin F (MMAF), monomethylauristatin D (MMAD), monomethylauristatin E (MMAE), navelbine, neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine, SN-38, sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide, transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine, vincristine, vinca alkaloids and ZD1839.

[0305] In some embodiments, the cytotoxic compound is a tubulin inhibitor. In one or moreembodiments, the drug is a tubulin inhibitor selected from the group consisting of dolastatin (dolastatin), auristatin (auristatin) and maytansine (maytansine). In one or more embodiments, the drug is an auristatin (auristatin) selected from the group consisting of monomethyl auristatin E (monomethyl auristatin E; MMAE), monomethyl auristatin F (monomethyl auristatin F; MMAF), and auristatin F (auristatin F; AF). In one embodiment, the tubulin inhibitor is MMAE. In one embodiment, the MMAE is cycolooctyne-MMAE.

[0306] In some embodiments, the cytotoxic compound is a topoisomerase inhibitor. In one ormore embodiments, the drug is a topoisomerase inhibitor selected from the group consisting of irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9- nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, camptothecin derivative SN-38, exatecan derivative Dxd, 22-hydroxycamptothecin, topotecan, lurtoltecan, belotecan, irinotecan, silicon-based homocamptothecin (homosilatecan), 6, 8-dibromo-2-methyl-3- [2- (D-xylopyranosylamino) phenyl ] -4 (3H) - quinazolinone, 2-cyano-3- (3, 4-dihydroxyphenyl) N- (phenylmethyl) - (2E) -2-acrylamide, 95Mintz Ref. No.: 048536-797001WO 2-cyano-3- (3, 4-dihydroxyphenyl) -N- (3-hydroxyphenylpropyl) - (E) -2-acrylamide, 12- β - D-glucopyranosyl-12, 13-dihydro-2, 10-hydroxy-2- [2- (D-xylopyranoside) phenyl ] -4 (3H) - quinazolinone, 2-cyano-3- (3, 4-dihydroxyphenyl) N- (phenylmethyl) - (2E) -2-acrylamide, 12- β -D-glucopyranosyl-12, 13-dihydro-2, 10-hydroxy-2- [2- (D-xylopyranoside ] methyl- [2- (4-hydroxypyrrolo-methyl) -5-amino ] 4- (3H) -quinazolinone, 5-hydroxy-4-methyl- [ 2- hydroxy ] ethyl ] amino ] carbonyl ] amino acid hydrochloride N- [2- (dimethylamino) ethyl ] -4-acridine carboxamide. In one embodiment, the topoisomerase inhibitor is SN38. In one embodiment, the topoisomerase inhibitor is Dxd.

[0307] Radioactive isotopes can also be used for conjugation to Src ligands as describedherein. For example, radionuclides useful for treating diseased tissue include, but are not limited to—111In,177Lu,212Bi,213Bi,211At,62Cu,67Cu,90Y,125I,131I,32P,33P,47Sc,111Ag,67Ga,142Pr,153Sm,161Tb,166Dy,166Ho,186Re,188Re,189Re,212Pb,223Ra,225Ac,59Fe,75Se,77As,89Sr,99Mo,105Rh,109Pd,143Pr,149Pm,169Er,194Ir,198Au,199Au,227Th and211Pb. Additional potential radioisotopes of use include11C,13N,15O,75Br,198Au,224Ac,126I,133I,77Br,113mIn,95Ru,97Ru,103Ru,105Ru,107Hg,203Hg,121mTe,122mTe,125mTe,165Tm,167Tm,168Tm,197Pt,109Pd,105Rh,142Pr,143Pr,161Tb,166Ho,199Au,57Co,58Co,51Cr,59Fe,75Se,201Tl,225Ac,76Br,169Yb, and the like.

[0308] In embodiments, the radionuclide is an idoine radioisotope. In embodiments, theradioisotope is123I,124I, 125I, or131I. In embodiments, the radioisotope is123I. In embodiments, the radioisotope is124I. In embodiments, the radioisotope is125I. In embodiments, the radioisotope is131I.

[0309] In embodiments, the radioisotope is a positron-emitting radioisotope. In embodiments,the positron-emitting radioisotope is11C,13N,15O,18F,64Cu,68Ga,78Br,82Rb,86Y,89Zr,90Y,22Na,26Al,40K,83Sr, or124I. In embodiments, the positron-emitting radioisotope is11C. In embodiments, the positron-emitting radioisotope is13N. In embodiments, the positron- emitting radioisotope is15O. In embodiments, the positron-emitting radioisotope is18F. In embodiments, the positron-emitting radioisotope is64Cu. In embodiments, the positron- emitting radioisotope is168Ga. In embodiments, the positron-emitting radioisotope is78Br. In embodiments, the positron-emitting radioisotope is82Rb. In embodiments, the positron- emitting radioisotope is86Y. In embodiments, the positron-emitting radioisotope is89Zr. In embodiments, the positron-emitting radioisotope is90Y. In embodiments, the positron- emitting radioisotope is22Na. In embodiments, the positron-emitting radioisotope is26Al. In 96Mintz Ref. No.: 048536-797001WO embodiments, the positron-emitting radioisotope is40K. In embodiments, the positron- emitting radioisotope is83Sr. In embodiments, the positron-emitting radioisotope is124I.

[0310] In embodiments, the radioisotope is an alpha-emitting radioisotope. In embodiments,the alpha-emitting radioisotope is211At,227Th,225Ac,223Ra,213Bi, or212Bi. In embodiments, the alpha-emitting radioisotope is211At. In embodiments, the alpha-emitting radioisotope is227Th. In embodiments, the alpha-emitting radioisotope is225Ac. In embodiments, the alpha- emitting radioisotope is223Ra. In embodiments, the alpha-emitting radioisotope is213Bi. In embodiments, the alpha-emitting radioisotope is212Bi.

[0311] The targeting ligand is linked to the drug through any suitable linker. In general, thelinker has the following structure: x-y, where x and y can both react with groups on the ligand and drug to link the structures together. These groups on the ligand and drug include groups such as halogen atoms, COOH, NH2, OH and SH. Some examples of linkers include succinic ester, amino acid, peptide, diacid, bisamine, bis-alcohol, other anhydrides, CN or an alkyne group used for the click reaction, epoxy, hydrazine, azide, aldehyde, ketone, sulfonic acid, phosphoric acid, phosphoamidite, guanidine, short (C1-C6) alkyl, aromatic group, ester, amide, urea, thiourea, imidazole and its derivatives, thioester, acrylate, thiol ether, dithioate, selenide and phenyl selenide, diene, diketone, pyrimidine, purine and other heterocycle ring structure, crown ether (for chelating with metal), phenoldiazene (photochromic probe), nitrobenzene and its derivatives (photo quencher or as photocaged probe), iodo or bromo (for radioactivity labeling and heavy atom phasing), monosaccharide and oligosaccharide (e.g., cyclodextrin), azirine and benzophenone (for photo crosslinking), bipyridine (metal chelating), biphenol and aminophenol (redox electron or radical electron traps), other indole derivatives (as electrochemical crosslinker) and any radioactive atom or chelator for those atoms (for MRI or PET imaging applications). It is known in the art how to prepare suitable linkers with suitable groups and react linkers with groups to be linked, as well as to functionalize both the linkers and groups to be linked to cause the desired linkage to occur.

[0312] In some embodiments, the linker is azide. In some embodiments, the Src ligand isdasatinib functionalized with an azide. SMALL MOLECULE T CELL ENGAGERS

[0313] The present disclosure also relates to small molecule T cell engagers which arebifunctional molecules that include a Src ligand and a T cell antigen-binding moiety. 97Mintz Ref. No.: 048536-797001WO

[0314] A variety of Src ligands can be contemplated for use in the present disclosureincluding those described above. For example, in some embodiments, the Src ligand is a Src kinase inhibitor. Src kinase inhibitors can include, without limitation, AZD0530 (saracatinib), Bosulif (vosutinib), ENMD981693, KD020, KX01, Sprycel (dasatinib), Yervoy (ipilimumab), AP23464, AP23485, AP23588, AZD0424, c-Src kinase inhibitor KISSEI, CU201, KX2361, SKS927, SRN004, SUNK706, TG100435, TG100948, AP23451, dasatinib HETERO (dasatinib), dasatinib VALEANT (dasatinib), Fontrax (dasatinib), Src kinase inhibitors KINEX, VX680 (tozasertib lactate), XL228, SUNK706, VAL201, PUR1800, TOP1288, NXP900, VAL301, ASN006, CR13626, MLR-1023, and bafetinib. In some embodiments, the Src kinase inhibitor is dasatinib.

[0315] In some embodiments, the Src ligand is a Src binding peptide.

[0316] In some embodiments, the small molecule T cell engager is cell impermeable.

[0317] T cell antigen-binding moieties are also described above and include, withoutlimitation, antigen binding domains or ligands that bind to (e.g., and in some embodiments activates) one or more of CD3, TCRα, TCRβ, TCRγ, TCRζ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226. In other embodiments, the T cell antigen binding moiety is selected from an antigen binding domain or ligand that binds to and does not activate one or more of CD3, TCRα, TCRβ, TCRγ, TCRζ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226. In some embodiments, the T cell antigen binding moiety binds to CD3.

[0318] T cell binding moieties that bind to CD3 are known in the art and include, forexample, SP34 scFV and OKT3 scFv.

[0319] In one embodiment, the T cell binding moiety includes an SP34 scFv. In oneembodiment, the SP34 scFV comprises the amino acid sequence of: EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYY ADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVS SGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQK PGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGG TKLTVL (SEQ ID NO: 19)

[0320] In one embodiment, the SP34 scFV comprises a sequence that is about 80%, about85%, about 90%, about 95%, or about 99% identical to SEQ ID NO: 19. 98Mintz Ref. No.: 048536-797001WO

[0321] In one embodiment, the T cell binding moiety includes an OKT3 scFv. In oneembodiment, the OKT3 scFV comprises the amino acid sequence of: QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQ KFKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYSLDYWGQGTPVTVSSGGGGS GGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSK LASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR (SEQ ID NO: 20)

[0322] In one embodiment, the OKT3 scFV comprises a sequence that is about 80%, about85%, about 90%, about 95%, or about 99% identical to SEQ ID NO: 20.

[0323] In some embodiments, the T cell antigen binding moiety is cell impermeable. In oneembodiment, the T cell antigen binding moiety is cyclooctyne-conjugated anti-CD3. RADIOLIGAND TARGETED CHIMERIC HETEROBIFUNCTIONAL SMALL MOLECULES (RADTACS)

[0324] The present disclosure also relates to radioligand targeted chimeric heterobifunctionalsmall molecules (RadTACs) that include a Src ligand and a radionuclide.

[0325] A variety of Src ligands can be contemplated for use in the present disclosureincluding those described above. For example, in some embodiments, the Src ligand is a Src kinase inhibitor. Src kinase inhibitors can include, without limitation, AZD0530 (saracatinib), Bosulif (vosutinib), ENMD981693, KD020, KX01, Sprycel (dasatinib), Yervoy (ipilimumab), AP23464, AP23485, AP23588, AZD0424, c-Src kinase inhibitor KISSEI, CU201, KX2361, SKS927, SRN004, SUNK706, TG100435, TG100948, AP23451, dasatinib HETERO (dasatinib), dasatinib VALEANT (dasatinib), Fontrax (dasatinib), Src kinase inhibitors KINEX, VX680 (tozasertib lactate), XL228, ponatinib, bosutinib, DGY-06-116, SUNK706, VAL201, PUR1800, TOP1288, NXP900, VAL301, ASN006, CR13626, MLR- 1023, and bafetinib. In some embodiments, the Src kinase inhibitor is dasatinib. In some embodiments, the Src kinase inhibitor is ponatinib. In some embodiments, the Src kinase inhibitor is bosutinib. In some embodiments, the Src kinase inhibitor is ponatinib. In some embodiments, the Src kinase inhibitor is DGY-06-116.

[0326] In one aspect, provided herein, is a compound represented by Formula (I):(I),Mintz Ref. No.: 048536-797001WO or a pharmaceutically acceptable salt thereof, wherein: ,and A is a chelating agent capable of chelating a radionuclide.

[0327] As generally defined above, R1 is selected from the group consisting of,Mintz Ref. No.: 048536-797001WO ivethereof. In some embodiments In some embodiments,iss

[0328] As generally defined above, the linker is a part of the compound of Formula (I):(I)Mintz Ref. No.: 048536-797001WO

[0329] In some embodiments, the linker is present. In some embodiments, the linker is notpresent.

[0330] In some embodiments, the linker comprises C1-12 alkyl, polyethylene glycol (PEG), oran amino acid or an analog thereof. In some embodiments, the linker comprises C1-12alkyl.

[0331] In some embodiments, the linker comprises polyethylene glycol (PEG). The PEG canbe constructed from (PEG)n, wherein n is an integer between 1 to 20. In some embodiments, the linker comprises (PEG)3, (PEG)6, or (PEG)12. In some embodiments, the linker comprises (PEG)3. In some embodiments, the linker comprises (PEG)6. In some embodiments, the linker comprises (PEG)12.

[0332] In some embodiments, the linker comprises (PEG)3, (PEG)6, (PEG)12, C1-12 alkyl, oran amino acid. In some embodiments, the linker comprises an amino acid or an analog thereof. In some embodiments, the linker comprises an amino acid.

[0333] In some embodiments, the linker comprises glycine (Gly), lysine (Lys), aspartic acid(Asp), or serine (Ser). In some embodiments, the linker comprises Gly. In some embodiments, the linker comprises Lys. In some embodiments, the linker comprises Asp. In some embodiments, the linker comprises Ser. In some embodiments, the linker comprises Asp-Gly-Gly-Ser-Gly. HSA-Binder

[0334] As generally defined above, the HSA-binder is a part of the compound of Formula (I):(I)

[0335] Human sd, accounting for60% of total plasma protein content. HSA-binders bind to human serum albumin and can modulate the pharmacokinetics, distribution, and efficacy of pharmaceutical compounds. Non-limiting examples of HSA-binders include warfarin, ibuprofen, diazepam, thyroxine, indomethacin, tamoxifen, cisplatin, fatty acids, and 4-(p-iodophenyl)butyric acid (IBA).

[0336] In some embodiments, the HSA-binder is present. In some embodiments, the HSA-binder is not present. 102Mintz Ref. No.: 048536-797001WO

[0337] In some embodiments, the HSA-binder comprises I meembodiments, the HSA-binder comprise . Chelating Agent

[0338] The chelating agent may be acyclic or macrocyclic. Non-limiting examples ofchelating agents include 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); DOTA derivative: DO3A; diethylenetriamine-N,N,N',N'',N''-pentaacetic acid (DTPA); DTPA derivatives: 2-(p-SCN-Bz)-6-methyl-DTPA, CHX-A''-DTPA, and the cyclic anhydride of DTPA (CA-DTPA); 1,4,7-triazacyclononane-1,4-7-triacetic acid (NOTA); NOTA derivatives (e.g., BCNOTA, p-NCS-Bz-NOTA, BCNOT); 6-hydrazinonicotinamide (HYNIC); ethylenediamine tetraacetic acid (EDTA); N,N′-ethylene-di-L-cysteine; N,N′-bis(2,2- dimethyl-2-mercaptoethyl)ethylenediamine-N,N′-diacetic acid (6SS); 1-(4- carboxymethoxybenzyl)-N-N'-bis[(2-mercapto-2,2-dimethyl)ethyl]-1,2-ethylenediamine- N,N'-diacetic acid (B6SS); Deferoxamine (DFO); 1,1,1-tris(aminomethyl)ethane (TAME); tris(aminomethyl)ethane-N,N,N’,N’,N’’,N’’-hexaacetic acid (TAME Hex); O-hydroxybenzyl iminodiacetic acid; 1,4,7-triazacyclononane (TACN); 1,4,7,10-tretraazacyclododecane (cyclen); 1,4,7-triazacyclononane-1-succinic acid-4,7-diacetic acid (NODASA); 1-(1- carboxy-3-carboxypropyl)-4,7-bis-(carboxymethyl)-1,4,7-triazacyclononane (NODAGA); 1,4,7-tris(2-mercaptoethyl)-1,4,7-triazacylclonane (triazacyclononane−TM); 1,4,7- triazacyclononane-N,N′,N′′-tris(methylenephosphonic)acid (NOTP); 1, 4, 8, 11- tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (TETA); 1,4,7,10,13- pentaazacyclopentadecane-N,N′,N″,N''',N″″-pentaacetic acid (PEPA), 1,4,7,10,13,16- hexaazacyclohexadecane-N,N',N'',N''',N'''',N'''''-hexaacetic acid (HEHA); 1,4,7,10- tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (TCMC); and derivatives or analogs thereof.

[0339] In some embodiments, the chelating agents are polyaminocarboxylate agents, such asethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), 1,4,7,10-tetra-azacylcododecane-N,N′,N″,N‴-tetraacetic acid (DOTA), or derivatives thereof. They can coordinate with metals such as Fe, In, Ga, Zr, Y, Bi, Pb, or Ac. 103Mintz Ref. No.: 048536-797001WO A heating agents are macrocyclic agents: 1,4,7-Triazacyclononane-N,N,N -triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane- N,N′,N″,N‴-tetraacetic acid (TETA), 1,4,7,10,13-pentaazacyclopentadecane- N,N',N",N"',N""-pentaacetic acid (PEPA), 1,4,7,10,13,16- hexaazacyclohexadecane- N,N',N",N"',N"",N""'-hexaacetic acid (HEHA), or derivatives thereof.

[0341] Non-limiting examples of DTPA and derivatives thereof are:104Mintz Ref. No.: 048536-797001WO

[0342] Non-limiting examples of DOTA and derivatives thereof are:Mintz Ref. No.: 048536-797001WO lys-DOTA DOTAGA

[0343] In some embodiments, the conjugates of the present disclosure comprise DOTA,DOTAGA, or any derivative / analog thereof as a chelating agent. Any chelating agent disclosed in Eisenwiener et al., Bioorg Med Chem Lett., vol.10(18):2133 (2000), the contents of which are incorporated herein by reference in their entirety, may be used as a chelating agent, such as 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid, α-(2-carboxyethyl) (DOTAGA) or 1,4,7,10-Tetraazacyclododecane-1,4,7-triacetic acid, 10-(1,2-dicarboxyethyl) (DOTASA).DOTAGA n=2

[0344] Other non-limiting examples of chelating agents are:Mintz Ref. No.: 048536-797001WO NCS CO2H

[0345] As generally defined above, A is a chelating agent capable of chelating aradionuclide. In some embodiments, A comprises a chelating agent selected from DOTA, DOTAGA, NOTA, and TETA. In some embodiments, A comprises DOTA or DOTAGA. In some embodiments, A comprises DOTA. Radionuclides

[0346] In some embodiments, the compound is conjugated to a radionuclide.

[0347] In some embodiments, the radionuclide is one that can be used in therapeutics.

[0348] In some embodiments, the radionuclide is one that can be used in diagnostics (e.g.,PET imaging).

[0349] Radioactive isotopes useful in the conjugates described herein include, but are notlimited to—111In,177Lu,212Bi,213Bi,211At,62Cu,67Cu,90Y,125I,131I,32P,33P,47Sc,111Ag, 107Mintz Ref. No.: 048536-797001WO67Ga,142Pr,153Sm,161Tb,166Dy,166Ho,186Re,188Re,189Re,212Pb,223Ra,225Ac,59Fe,75Se,77As,89Sr,99Mo,105Rh,109Pd,143Pr,149Pm,169Er,194Ir,198Au,199Au,227Th and211Pb. Additional potential radioisotopes of use include11C,13N,15O,75Br,198Au,224Ac,126I,133I,77Br,113mIn,95Ru,97Ru,103Ru,105Ru,107Hg,203Hg,121mTe,122mTe,125mTe,165Tm,167Tm,168Tm,197Pt,109Pd,105Rh,142Pr,143Pr,161Tb,166Ho,199Au,57Co,58Co,51Cr, 59Fe,75Se,201Tl,225Ac,76Br,169Yb,89Zr,64Cu,211At,227Th,225Ac,223Ra,213Bi, or212Bi,123I,124I,125I, or131I,18F,64Cu,68Ga,78Br,82Rb,86Y,89Zr,90Y,22Na,26Al,40K,83Sr, or124I and the like.

[0350] In some embodiments, the radionuclide is 64Cu or 177Lu. In some embodiments, theradionuclide is64Cu. In some embodiments, the radionuclide is177Lu. Exemplary Compounds

[0351] In certain embodiments, the compound is a compound in Table 1 or apharmaceutically acceptable salt thereof. In certain embodiments, the compound is a compound in Table 9. Table 9. Compound Structure108Mintz Ref. No.: 048536-797001WOMintz Ref. No.: 048536-797001WO8 110Mintz Ref. No.: 048536-797001WO111Mintz Ref. No.: 048536-797001WO112Mintz Ref. No.: 048536-797001WONUCLEIC ACIDS

[0352] As discussed above, one aspect of the disclosure relates to recombinant nucleic acidsincluding a nucleic acid sequence that encodes an antibody of the disclosure. In some embodiments, the recombinant nucleic acids of the disclosure can be configured as expression cassettes or vectors containing these nucleic acid molecules operably linked to heterologous nucleic acid sequences such as, for example, regulatory sequences which allow in vivo expression of the antibody in a host cell.

[0353] Nucleic acid molecules of the present disclosure can be of any length, including forexample, between about 1 Kb and about 50 Kb, e.g., between about 1.2 Kb and about 10 Kb, between about 2 Kb and about 15 Kb, between about 5 Kb and about 20 Kb, between about 10 Kb and about 20 Kb, between about 5 Kb and about 40 Kb, between about 5 Kb and about 30 Kb, between about 5 Kb and about 20 Kb, or between about 10 Kb and about 50 Kb, for example between about 15 Kb to 30 Kb, between about 20 Kb and about 50 Kb, between about 20 Kb and about 40 Kb, about 5 Kb and about 25 Kb, or about 30 Kb and about 50 Kb.

[0354] Accordingly, in some embodiments, provided herein is a nucleic acid moleculeincluding a nucleotide sequence encoding an antibody of the disclosure. In certain embodiment, the nucleic acid molecule provided herein includes a nucleotide sequence encoding any of the polypeptide sequences disclosed herein. In some embodiments, the nucleotide sequence is incorporated into an expression cassette or an expression vector. It will be understood by the skilled artisan that an expression cassette generally includes a construct of genetic material that contains coding sequences of the antibody or antigen- binding fragment thereof and enough regulatory information to direct proper transcription 113Mintz Ref. No.: 048536-797001WO and / or translation of the coding sequences in a recipient cell, in vivo and / or ex vivo. Generally, the expression cassette can be inserted into a vector for targeting to a desired host cell and / or into an individual. As such, in some embodiments, an expression cassette of the disclosure includes a coding sequence for an antibody of the disclosure or an antigen-binding fragment thereof, which is operably linked to expression control elements, such as a promoter, and optionally, any or a combination of other nucleic acid sequences that affect the transcription or translation of the coding sequence.

[0355] An expression cassette can be inserted into a plasmid, cosmid, virus, autonomouslyreplicating polynucleotide molecule, phage, as a linear or circular, single-stranded or double- stranded, DNA or RNA polynucleotide molecule, derived from any source, capable of genomic integration or autonomous replication, including a nucleic acid molecule where one or more nucleic acid sequences has been linked in a functionally operative manner, e.g., operably linked.

[0356] In some embodiments, the nucleic acid molecule of the disclosure is incorporated intoan expression vector. It will be understood by one skilled in the art that the term “vector” generally refers to a recombinant polynucleotide construct designed for transfer between host cells, and that can be used for the purpose of transformation, e.g., the introduction of heterologous DNA into a host cell. As such, in some embodiments, the vector can be a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted so as to bring about the replication of the inserted segment. In some embodiments, the expression vector can be an integrating vector.

[0357] In some embodiments, the expression vector can be a viral vector. As will beappreciated by one of skill in the art, the term “viral vector” is widely used to refer either to a nucleic acid molecule (e.g., a transfer plasmid) that includes virus-derived nucleic acid elements that typically facilitate transfer of the nucleic acid molecule or integration into the genome of a cell or to a viral particle that mediates nucleic acid transfer. Viral particles will typically include various viral components and sometimes also host cell components in addition to nucleic acid(s). The term viral vector can refer either to a virus or viral particle capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are primarily derived from a virus. The term “retroviral vector” refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. The term “lentiviral vector” refers to a viral vector or plasmid 114Mintz Ref. No.: 048536-797001WO containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus, which is a genus of retrovirus.

[0358] The nucleic acid sequences encoding the antibodies and antigen-binding fragments asdisclosed herein can be optimized for expression in the host cell of interest. For example, the G-C content of the sequence can be adjusted to average levels for a given cellular host, as calculated by reference to known genes expressed in the host cell. Methods for codon usage optimization are known in the art. Codon usages within the coding sequence of the antibodies and antigen-binding fragment disclosed herein can be optimized to enhance expression in the host cell, such that about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons within the coding sequence have been optimized for expression in a particular host cell.

[0359] Also provided herein are vectors, plasmids, or viruses containing one or more of thenucleic acid molecules encoding any antibody or an antigen-binding fragment thereof as disclosed herein. The nucleic acid molecules can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transformed / transduced with the vector. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available, or readily prepared by a skilled artisan. See for example, Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferré, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in 115Mintz Ref. No.: 048536-797001WO Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference).

[0360] DNA vectors can be introduced into cells, e.g., eukaryotic cells via conventionaltransformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (2012, supra) and other standard molecular biology laboratory manuals, such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, nucleoporation, hydrodynamic shock, and infection.

[0361] Viral vectors that can be used in the disclosure include, for example, retrovirusvectors, adenovirus vectors, and adeno-associated virus vectors, lentivirus vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).

[0362] For example, an antibody or an antigen-binding fragment thereof as disclosed hereincan be produced in a eukaryotic host, such as a mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, VA). In selecting an expression system, it matters only that the components are compatible with one another. Artisans or ordinary skill are able to make such a determination. Furthermore, if guidance is required in selecting an expression system, skilled artisans can consult P. Jones, “Vectors: Cloning Applications”, John Wiley and Sons, New York, N.Y., 2009).

[0363] The nucleic acid molecules provided can contain naturally occurring sequences, orsequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide, e.g., antibody. These nucleic acid molecules can consist of RNA or DNA (for example, genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis), or combinations or modifications of the nucleotides within these types of nucleic acids. In addition, the nucleic acid molecules can be double-stranded or single-stranded (e.g., either a sense or an antisense strand).

[0364] The nucleic acid molecules are not limited to sequences that encode polypeptides(e.g., antibodies); some or all of the non-coding sequences that lie upstream or downstream from a coding sequence (e.g., the coding sequence of an antibody) can also be included. Those of ordinary skill in the art of molecular biology are familiar with routine procedures 116Mintz Ref. No.: 048536-797001WO for isolating nucleic acid molecules. In the event the nucleic acid molecule is a ribonucleic acid (RNA), molecules can be produced, for example, by in vitro transcription. RECOMBINANT CELL AND CELL CULTURES

[0365] The nucleic acid of the present disclosure can be introduced into a host cell, such as,for example, a Chinese hamster ovary (CHO) cell, to produce an engineered o recombinant cell containing the nucleic acid molecule. Introduction of the nucleic acid molecules (e.g., DNA or RNA, including mRNA) or vectors of the disclosure into cells can be achieved by methods known to those skilled in the art such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro-injection, nanoparticle-mediated nucleic acid delivery. For example, methods for introduction of heterologous nucleic acid molecules into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the nucleic acid molecule(s) in liposomes, lipid nanoparticle technology, biolistic injection and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors such as lentivirus or adeno-associated virus. As discussed in greater detail below, in some embodiments, an antibody or antigen-binding fragment thereof of the present disclosure can be introduced to a subject in nucleic acid form (e.g, DNA or RNA, including mRNA), such that the subject's own cells produce the antibody. The present disclosure further provides modifications to nucleotide sequences encoding the anti-Src antibodies described herein that result in increased antibody expression, increased antibody stability, increased nucleic acid (e.g., mRNA) stability, or improved affinity or specificity of the antibodies for the Src protein.

[0366] Accordingly, in some embodiments, the nucleic acid molecules can be delivered byviral or non-viral delivery vehicles known in the art. For example, the nucleic acid molecule can be stably integrated in the host genome, or can be episomally replicating, or present in the recombinant host cell as a mini-circle expression vector for transient expression. Accordingly, in some embodiments, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid molecule is stably integrated into the genome of the recombinant cell. Stable integration can 117Mintz Ref. No.: 048536-797001WO be achieved using classical random genomic recombination techniques or with more precise techniques such as guide RNA-directed CRISPR / Cas genome editing, or DNA-guided endonuclease genome editing with NgAgo (Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases). In some embodiments, the nucleic acid molecule is present in the recombinant host cell as a mini- circle expression vector for transient expression.

[0367] The nucleic acid molecules can be encapsulated in a viral capsid or a lipidnanoparticle or can be delivered by viral or non-viral delivery means and methods known in the art, such as electroporation. For example, introduction of nucleic acids into cells can be achieved by viral transduction. In a non-limiting example, adeno-associated virus (AAV) is engineered to deliver nucleic acids to target cells via viral transduction. Several AAV serotypes have been described, and all of the known serotypes can infect cells from multiple diverse tissue types. AAV is capable of transducing a wide range of species and tissues in vivo with no evidence of toxicity, and it generates relatively mild innate and adaptive immune responses.

[0368] Lentiviral-derived vector systems are also useful for nucleic acid delivery and genetherapy via viral transduction. Lentiviral vectors offer several attractive properties as gene- delivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the capability of infecting both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy-target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) a potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production.

[0369] In some embodiments, host cells can be genetically engineered (e.g., transduced ortransformed or transfected) with, for example, a vector construct of the present application that can be, for example, a viral vector or a vector for homologous recombination that includes nucleic acid sequences homologous to a portion of the genome of the host cell, or can be an expression vector for the expression of the polypeptides of interest. The antibodies of the present invention may be prepared and purified using known methods. For example, cDNA sequences encoding a HC may be cloned and engineered into an expression vector, using known methods. The engineered immunoglobulin expression vector may then be stably transfected into engineered cells. 118Mintz Ref. No.: 048536-797001WO

[0370] In some embodiments, the engineered cell is a eukaryotic cell. In some embodiments,the engineered cell is an animal cell. In some embodiments, the animal cell is a vertebrate animal cell or an invertebrate animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the animal cell is a human cell. In some embodiments, the animal cell is a non-human animal cell. In some embodiments, the engineered cell is a non-human primate cell. In some embodiments, the engineered cell is selected from the group consisting of a baby hamster kidney (BHK) cell, a Chinese hamster ovary cell (CHO cell), an African green monkey kidney cell (Vero cell), a human A549 cell, a human cervix cell, a human CHME5 cell, a human PER.C6 cell, a NS0 murine myeloma cell, a human epidermoid larynx cell, a human fibroblast cell, a human HEK-293 cell, a human HeLa cell, a human HepG2 cell, a human HUH-7 cell, a human MRC-5 cell, a human muscle cell, a mouse 3T3 cell, a mouse connective tissue cell, a mouse muscle cell, and a rabbit kidney cell. In some embodiments, the engineered cell is a Pichia pastoris cell or a Saccharomyces cerevisiae cell, all of which are also suitable for production of the antibodies that are described in the present invention.

[0371] In another aspect, provided herein are cell cultures including at least one recombinantcell as disclosed herein, and a culture medium. Generally, the culture medium can be any suitable culture medium for culturing the cells described herein. Techniques for transforming a wide variety of the above-mentioned host cells and species are known in the art and described in the technical and scientific literature. Accordingly, cell cultures including at least one recombinant cell as disclosed herein are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.

[0372] In some embodiments, the present disclosure provides methods for producing anantibody or antigen-binding fragment thereof as described herein. The method can include culturing the engineered or recombinant cell described herein under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment thereof.

[0373] Also provided, in another aspect, are animals including a recombinant nucleic acid ora vector as disclosed herein. In some embodiments, the disclosure provides a transgenic animal that is a non-human animal. In some embodiments, the transgenic animal produces an antibody or antigen-binding fragment as disclosed herein.

[0374] The transgenic non-human host animals of the disclosure are prepared using standardmethods known in the art for introducing exogenous nucleic acid into the genome of a non- 119Mintz Ref. No.: 048536-797001WO human animal. In some embodiments, the non-human animals of the disclosure are mice. Other animal species suitable for the compositions and methods of the disclosure include animals that are (i) suitable for transgenesis and (ii) capable of rearranging immunoglobulin gene segments to produce an antibody response. Examples of such species include but are not limited to rats, rabbits, chickens, goats, pigs, sheep and cows. Approaches and methods for preparing transgenic non-human animals are known in the art. Exemplary methods include pronuclear microinjection, DNA microinjection, lentiviral vector mediated DNA transfer into early embryos and sperm-mediated transgenesis, adenovirus mediated introduction of DNA into animal sperm (e.g., in pig), retroviral vectors (e.g., avian species), somatic cell nuclear transfer (e.g., in goats). The state of the art in the preparation of transgenic domestic farm animals is reviewed in Niemann, H. et al. (2005) Rev. Sci. Tech.24:285-298.

[0375] In some embodiments, the animal is a vertebrate animal or an invertebrate animal. Insome embodiments, the animal is a mammalian subject. In some embodiments, the mammalian animal is a non-human animal. In some embodiments, the transgenic animals of the disclosure can be made using classical random genomic recombination techniques or with more precise techniques such as guide RNA-directed CRISPR / Cas genome editing, or DNA- guided endonuclease genome editing with NgAgo (Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases). In some embodiments, the transgenic animals of the disclosure can be made using transgenic microinjection technology and do not require the use of homologous recombination technology and thus are considered to be easier to prepare and select than approaches using homologous recombination.

[0376] In another aspect, provided herein are methods for producing an antibody or antigen-binding fragment thereof, wherein the methods include growing (i) a transgenic animal as disclosed herein, or (ii) a recombinant cell as disclosed herein under conditions such that the antibody or antigen-binding fragment is produced.

[0377] In some embodiments, the methods for producing an antibody or antigen-bindingfragment thereof as described herein further include isolating the produced antibody or antigen-binding fragment from (i) the transgenic animal or (ii) recombinant cell and / or the medium in which the recombinant cell is cultured. In some embodiments, the mammalian animal is a non-human primate. Accordingly, the antibodies or antigen-binding fragments produced by the methods disclosed herein are also within the scope of the disclosure. 120Mintz Ref. No.: 048536-797001WO PHARMACEUTICAL COMPOSITIONS

[0378] The anti-Src antibodies, nucleic acids, of the disclosure can be incorporated intocompositions, including pharmaceutical compositions.

[0379] In another aspect, the anti-Src antibodies, bispecific T-cell engagers, drug conjugates,radioligand conjugates, small molecule directed therapies, nucleic acids, of the disclosure can be incorporated into compositions suitable for various downstream applications, for example, pharmaceutical compositions. Exemplary compositions of the disclosure include pharmaceutical compositions which generally include one or more of the antibodies, nucleic acids, and a pharmaceutically acceptable excipient, e.g., carrier. In some embodiments, the composition is a sterile composition. In some embodiments, the composition is formulated as a vaccine. In some embodiments, the composition further includes an adjuvant.

[0380] The pharmaceutical compositions provided herein can be in any form that allows forthe composition to be administered to an individual. In some specific embodiments, the pharmaceutical compositions are suitable for human administration. The scope of the present disclosure includes desiccated, e.g., freeze-dried, compositions comprising anti-Src antigen- binding polypeptides, e.g., antibody or antigen-binding fragment thereof, or a pharmaceutical composition thereof that includes a pharmaceutically acceptable carrier but substantially lacks water. As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeiae for use in animals, and more particularly in humans. The carrier can be a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, including injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin. In some embodiments, the pharmaceutical composition is sterilely formulated for administration into an individual or an animal (some non-limiting examples include a human, or a mammal). In some embodiments, the individual is a human.

[0381] In some embodiments, the pharmaceutical compositions of the present disclosure areformulated to be suitable for the intended route of administration to an individual. For 121Mintz Ref. No.: 048536-797001WO example, the pharmaceutical composition can be formulated to be suitable for parenteral, intraperitoneal, colorectal, intraperitoneal, and intratumoral administration. In some embodiments, the pharmaceutical composition can be formulated for oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal or intra-arterial administration. One of ordinary skilled in the art will appreciate that the formulation should suit the mode of administration.

[0382] For example, pharmaceutical compositions suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In some embodiments, the composition should be sterile and should be fluid to the extent that easy syringability exists. It can be stabilized under the conditions of manufacture and storage, and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, e.g., sodium dodecyl sulfate. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be generally to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and / or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0383] Sterile injectable solutions can be prepared by incorporating the active compound inthe required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. 122Mintz Ref. No.: 048536-797001WO METHODS

[0384] The present disclosure provides, among others, a method of treating cancer in asubject. The method includes administering to a subject in need thereof, a therapeutically effective amount of the anti-Src antibodies, bispecific T-cell engagers, drug conjugates, radioligand conjugates, and small molecule directed therapies, the nucleic acid, the vector, the engineered cell, or the pharmaceutical composition provided herein.

[0385] Some non-limiting cancers that can be treated by the various compositions describedherein include, without being limited to, breast cancer, B cell lymphoma, pancreatic cancer, Hodgkin’s lymphoma, ovarian cancer, prostate cancer, mesothelioma, lung cancer, non- Hodgkin’s B-cell (B-NHL), melanoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, neuroblastoma, glioma, glioblastoma, bladder cancer, and colorectal cancer. In some embodiments, the cancer is selected from the group consisting of breast cancer, lung cancer, fibrosarcoma, melanoma, prostate cancer, colon cancer, and pancreatic cancer. In one embodiment, the cancer is breast cancer. In one embodiment, the cancer is lung cancer. In one embodiment, the cancer is fibrosarcoma. In one embodiment, the cancer is melanoma. In one embodiment, the cancer is prostate cancer. In one embodiment, the cancer is colon cancer. In one embodiment, the cancer is pancreatic cancer. ADMINISTRATION

[0386] Administration of any one or more of the therapeutic compositions described herein,e.g., anti-Src antibodies, bispecific T-cell engagers, drug conjugates, radioligand conjugates, small molecule directed therapies, nucleic acids, recombinant cells, and pharmaceutical compositions, can be used to treat individuals having a condition described herein. In some embodiments, the anti-Src antibodies, bispecific T-cell engagers, drug conjugates, radioligand conjugates, small molecule directed therapies, nucleic acids, recombinant cells, and pharmaceutical compositions are incorporated into therapeutic compositions for use in methods down-regulating or inactivating T cells, such as CAR-T cells.

[0387] The therapeutic compositions of the disclosure are typically administered in solutionor suspension formulation by injection or infusion. In an embodiment, a therapeutic composition as disclosed herein is administered by injection directly into a tumor mass. In another embodiment, a therapeutic composition as disclosed herein is administered by systemic infusion. 123Mintz Ref. No.: 048536-797001WO

[0388] The effective dose of the therapeutic composition can be determined by a skilledperson in the field, e.g., a physician. The effective dose of any therapeutic composition may depend on the binding affinity for Src, and the degree of expression of Src. The range of effective concentrations, however, can be determined by one of ordinary skill in the art, using the disclosure and the experimental protocols provided herein. Similarly, using the effective concentration one can determine the effective dose or range of dosages required for administration.

[0389] Depending on the disease or disorder to be treated, the severity and extent of thedisease, the subject’s health, and the co-administration of other therapies, repeated doses may be administered. Alternatively, a continuous administration may be required. It is expected, however, that the therapeutic composition will remain in proximity to the cell. Administration of recombinant cells to an individual

[0390] In some embodiments, the methods involve administering the recombinant cells to anindividual who is in need of such method. This administering step can be accomplished using any method of implantation known in the art. For example, the recombinant cells can be injected directly into the individual’s bloodstream by intravenous infusion or otherwise administered to the individual.

[0391] The terms “administering”, “introducing”, and “transplanting” are usedinterchangeably herein to refer to methods of delivering recombinant cells expressing the bispecific binding agents provided herein to an individual. In some embodiments, the methods comprise administering recombinant cells to an individual by a method or route of administration that results in at least partial localization of the introduced cells at a desired site such that a desired effect(s) is / are produced. The recombinant cells or their differentiated progeny can be administered by any appropriate route that results in delivery to a desired location in the individual where at least a portion of the administered cells or components of the cells remain viable. The period of viability of the cells after administration to an individual can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years, or even long-term engraftment for the lifetime of the individual.

[0392] When provided prophylactically, in some embodiments, the recombinant cellsdescribed herein are administered to an individual in advance of any symptom of a disease or condition to be treated. Accordingly, in some embodiments the prophylactic administration of a recombinant stem cell population serves to prevent the occurrence of symptoms of the disease or condition. 124Mintz Ref. No.: 048536-797001WO

[0393] When provided therapeutically in some embodiments, recombinant stem cells areprovided at (or after) the onset of a symptom or indication of a disease or condition, e.g., upon the onset of disease or condition.

[0394] For use in the various embodiments described herein, an effective amount ofrecombinant cells as disclosed herein, can be at least 102cells, at least 5 × 102cells, at least 103cells, at least 5 × 103cells, at least 104cells, at least 5 × 104cells, at least 105cells, at least 2 × 105cells, at least 3 × 105cells, at least 4 × 105cells, at least 5 × 105cells, at least 6 × 105cells, at least 7 × 105cells, at least 8 × 105cells, at least 9 × 105cells, at least 1 × 106cells, at least 2 × 106cells, at least 3 × 106cells, at least 4 × 106cells, at least 5 × 106cells, at least 6 × 106cells, at least 7 × 106cells, at least 8 × 106cells, at least 9 × 106cells, or multiples thereof. The recombinant cells can be derived from one or more donors or can be obtained from an autologous source (i.e., the human subject being treated). In some embodiments, the recombinant cells are expanded in culture prior to administration to an individual in need thereof.

[0395] In some embodiments, the delivery of a composition comprising recombinant cells(i.e., a composition comprising a plurality of recombinant cells expressing a binding agent provided herein) into an individual by a method or route results in at least partial localization of the cell composition at a desired site. A cell composition can be administered by any appropriate route that results in effective treatment in the individual, e.g., administration results in delivery to a desired location in the individual where at least a portion of the composition delivered, e.g., at least 1 × 104cells, is delivered to the desired site for a period of time. Modes of administration include injection, infusion, instillation, and the like. Injection modes include, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In some embodiments, the route is intravenous. For the delivery of cells, administration by injection or infusion can be made.

[0396] In some embodiments, the recombinant cells are administered systemically, in otherwords a population of recombinant cells are administered other than directly into a target site, tissue, or organ, such that it enters, instead, the individual’s circulatory system and, thus, is subject to metabolism and other like processes. 125Mintz Ref. No.: 048536-797001WO

[0397] The efficacy of a treatment with a composition for the treatment of a disease orcondition can be determined by the skilled clinician. However, one skilled in the art will appreciate that a treatment is considered effective treatment if any one or all of the signs or symptoms or markers of disease are improved or ameliorated. Efficacy can also be measured by failure of an individual to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease in an individual or an animal (some non- limiting examples include a human, or a mammal) and includes: (1) inhibiting disease progression, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.

[0398] As discussed above, a therapeutically effective amount includes an amount of atherapeutic composition that is sufficient to promote a particular effect when administered to an individual, such as one who has, is suspected of having, or is at risk for a disease. In some embodiments, an effective amount includes an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.

[0399] In some embodiments, the individual is a mammal. In some embodiments, themammal is human. INHIBITION OF CANCER CELL GROWTH

[0400] In some embodiments, compositions of the disclosure are used to inhibit or reducecancer cell growth.

[0401] In some embodiments, Src is located on the cancer cell surface, and a Src-targetingcomposition of the disclosure is contacted with the cancer cell to inhibit or reduce its growth.

[0402] In some embodiments, Hck is located on the cancer cell surface, and a Hck-targetingcomposition of the disclosure is contacted with the cancer cell to inhibit or reduce its growth.

[0403] In some embodiments, Lyn is located on the cancer cell surface, and a Lyn-targetingcomposition of the disclosure is contacted with the cancer cell to inhibit or reduce its growth. 126Mintz Ref. No.: 048536-797001WO

[0404] Some non-limiting cancers which growth can be inhibited or reduced by the variouscompositions described herein include, without being limited to, breast cancer, B cell lymphoma, pancreatic cancer, Hodgkin’s lymphoma, ovarian cancer, prostate cancer, mesothelioma, lung cancer, non-Hodgkin’s B-cell (B-NHL), melanoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, neuroblastoma, glioma, glioblastoma, bladder cancer, and colorectal cancer. In some embodiments, the cancer is selected from the group consisting of breast cancer, lung cancer, fibrosarcoma, melanoma, prostate cancer, colon cancer, and pancreatic cancer. In one embodiment, the cancer is breast cancer. In one embodiment, the cancer is lung cancer. In one embodiment, the cancer is fibrosarcoma. In one embodiment, the cancer is melanoma. In one embodiment, the cancer is prostate cancer. In one embodiment, the cancer is colon cancer. In one embodiment, the cancer is pancreatic cancer.

[0405] In some embodiments, inhibiting or reducing cancer cell growth refers to amelioratingat least one symptom of the disease or disorder. In some embodiments, inhibiting or reducing cancer cell growth refers to impeding growth of the cancer, causing the cancer to shrink by weight or volume, extending the expected survival time of the patient, inhibiting tumor growth, reducing tumor mass, reducing size or number of metastatic lesions, inhibiting the development of new metastatic lesions, prolonging survival, prolonging progression- free survival, prolonging time to progression, and / or enhancing quality of life. SYSTEMS

[0406] Also provided herein are systems and kits including binding agents as disclosedherein, recombinant nucleic acids, recombinant cells, or pharmaceutical compositions provided and described herein as well as written instructions for making and using the same. For example, provided herein, in some embodiments, are systems and / or kits that include one or more of: a bispecific binding agent as described herein, a recombinant nucleic acid as described herein, a recombinant cell as described herein, or a pharmaceutical composition as described herein. In some embodiments, the systems and / or kits of the disclosure further include one or more syringes (including pre-filled syringes) and / or catheters used to administer one any of the provided binding agents, recombinant nucleic acids, recombinant cells, or pharmaceutical compositions to an individual. In some embodiments, a kit can have one or more additional therapeutic agents that can be administered simultaneously or sequentially with the other kit components for a desired purpose, e.g., for modulating an 127Mintz Ref. No.: 048536-797001WO activity of a cell, inhibiting a target cancer cell, or treating a disease in an individual in need thereof.

[0407] Any of the above-described systems and kits can further include one or moreadditional reagents, where such additional reagents can be selected from: dilution buffers; reconstitution solutions, wash buffers, control reagents, control expression vectors, negative control polypeptides, positive control polypeptides, reagents for in vitro production of the binding agents.

[0408] In some embodiments, a system or kit can further include instructions for using thecomponents of the kit to practice the methods. The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, and the like. The instructions can be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging), and the like. The instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, flash drive, and the like. In some instances, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet), can be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.

[0409] Additional embodiments are disclosed in further detail in the following examples,which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims. EXAMPLES

[0410] The practice of the present invention will employ, unless otherwise indicated,conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in 128Mintz Ref. No.: 048536-797001WO Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferré, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference.

[0411] Additional embodiments are disclosed in further detail in the following examples,which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.EXAMPLE 1: MATERIALS AND METHODS

[0412] Cell lines and cell culture. Cells were cultured according to recommended guidelinesfrom ATCC.

[0413] Cloning and plasmid preparation. Synthetic genes were ordered from IDT or TwistBiosciences. For mammalian expression, plasmids were cloned into a pCDNA3.4 vector. For bacterial expression, plasmids were cloned either into an in-house Fab expression vector [Hornbsy, et al.2015] or a 2B-T-10 vector (derived from Addgene #79700). Point mutations were introduced by PCR-based site directed mutagenesis using NEB Q5 SDM kits.

[0414] Protein Expression (Bacterial). Fragments of antigen binding (Fabs) were expressedC43(DE3) Pro+Tum+ E. coli (in house) via autoinduction. Bacteria were harvested 18-24 hours after inoculation by centrifugation (4000 rcf, 20 min), lysed with B-PER Complete Protein Extraction Reagent (Thermo Scientific, 89821), and purified on Protein A Resin (Thermo) according to the manufacturer’s instructions. Purified proteins were buffer exchanged by serial dilution in 10 kDa MWCO spin columns. Purified proteins were stored at -80 °C in 10% glycerol in PBS. 129Mintz Ref. No.: 048536-797001WO

[0415] Recombinant Src fusion proteins were expressed as N-terminal TwinStrep conjugatesin BL21(DE3) E. coli (NEB, C2527H) via autoinduction. Bacteria were harvested 18-24 hours after inoculation by centrifugation (4000 rcf, 20 min), lysed with B-PER Complete Protein Extraction Reagent (Thermo Scientific, 89821), and purified on StrepTactin-XT resin (IBA Lifesciences, 2-5010-002) according to the manufacturer’s instructions. Excess biotin was removed from the purified protein by serial buffer exchange in 10 kDa MWCO spin columns. Purified proteins were stored at -80 °C in 10% glycerol in PBS.

[0416] Protein Expression (Mammalian). Antibodies were expressed in ExpiCHO cellsusing the ExpiCHO expression system kit (Thermo Scientific, A29133) according to the manufacturer’s instructions. Supernatants were collected by centrifugation (4000 rcf, 20 min). Antibodies were purified on HiTrap Protein A cartridges (GE Lifesciences, 17-0402- 01) using a peristaltic pump according to the manufacturer’s instruction. Acid elution was quenched with an equimolar 1 M Tris base, which was removed from the purified protein by serial buffer exchange in 10 kDa MWCO spin columns (Millipore Sigma, UFC801096). Purified proteins were stored at -80 °C in 10% glycerol in PBS.

[0417] Flow cytometry. For flow cytometry experiments, adherent cells were lifted fromadherent monolayer culture with Versene (Fisher Scientific, 15-040-066) at 37 °C, harvested by centrifugation (500 rcf), resuspended in 1% BSA in PBS (500 rcf, 5 min). Cells were blocked with Fc Block (BD Biosciences) for 10 minutes at room temperature and then incubated on ice for 30 minutes with antibodies at the specified concentration in 1% BSA in PBS. Cells were washed two times with cold 1% BSA in PBS (500 rcf, 5 minutes), once with PBS (500 rcf, 5 minutes), and incubated for 15 minutes in PBS with Propidium Iodide Ready Flow (Thermo Scientific, R37169) live / dead cell stain. Cells were analyzed on a Beckman Coulter CytoFLEX flow cytometer. Data were analyzed on FloRead.io.

[0418] Bioconjugation of Fabs with iridium photocatalyst. Single point mutant R66MFabs, either anti-phopshotyrosine clone 4G10-G6 or a different Fab in the same scaffold that only binds denatured proteins, were conjugated to an oxaziridine azide, as previously described. In brief, Fab (50 μM) was incubated with oxaziridine azide (15 equivalents) in PBS for 30 minutes at room temperature. Excess azide was removed by buffer exchange on two 7 kDa Zeba spin columns (Thermo) pre-equilibrated with PBS. Iridium photocatalyst DBCO conjugate was then coupled to azide-bearing Fab by incubation with Fab (50 μM) and Ir-DBCO (5 equivalents) in PBS for 60 minutes at room temperature. Excess photocatalyst 130Mintz Ref. No.: 048536-797001WO was removed by buffer exchange on two 7 kDa Zeba spin columns (Thermo) pre-equilibrated with PBS.

[0419] Photoaffinity labeling of phosphoproteins. Photoaffinity labeling reactions werecarried out in PBS with Ir-Fab complex (250 nM) and biotin-diazirine (250 μM) by irradiating with blue light (450 nm) for 10 minutes. After removal of excess biotin, detailed below, protein concentration was quantitated by RapidGold BCA (Thermo Scientific, A53225). Normalized quantities of lysate, typically 400 μg in 200 μL, were immobilized on NeutrAvidin high-capacity agarose (Thermo) prewashed with RIPA at 4 °C for 16 hours. Resin was washed on a vacuum manifold in 2 mL columns, being careful to not let the resin dry, with three successive washes of 1 mL each of RIPA, PBS with 1 M NaCl, and 50 mM ammonium bicarbonate with 2 M urea. Resins were then alkylated and digested on-bead using Preomics iST 96x kits (Preomics, P.O.00027) according to the manufacturer’s protocol and eluted peptides were dried in vacuo. Peptides were resuspended in running solvent and quantitated using a Pierce Colorimetric Peptide Quantitation Assay (Thermo Fisher Scientific, 23275).

[0420] For generation of JURKAT whole cell lysates, cells were treated with sodiumpervanadate (1 mM) for 15 minutes prior to lysis in 1% NP-40 supplemented with HALT Protease + Phosphatase inhibitor cocktail (Thermo) and Benzonase nuclease. Lysates were clarified by centrifugation (21,000 rcf, 10 minutes, 4 °C) and desalted on PD-10 spin columns (Thermo). After photoaffinity labeling, lysates were desalted on PD-10 columns and then denatured by the addition of 10X RIPA lysis buffer.

[0421] For generation of live cell samples, HCC1569 cells were harvested with Versene andwashed with serum-free DMEM. Cells were then resuspended in a kinase-reaction buffer and treated with or without ATP (1 mM) and Src inhibitor 1 (10 μM). Kinase reactions were quenched by washing with ice-cold PBS. After photoaffinity labeling, cells were then washed three times with PBS and lysed in RIPA supplemented with HALT protease and phosphatase inhibitor (Thermo Fisher Scientific, 78440) and benzonase nuclease (Sigma Aldrich, E1014- 25KU) for 30 minutes at 4 °C. Lysates were clarified by centrifugation (21000 rcf, 15 minutes).

[0422] Liquid chromatography mass spectrometry. Desalted peptides (200 ng) wereloaded onto a timsTOF Pro equipped with a CaptiveSpray source and a nanoElute line (Bruker). The peptides were separated on a ReproSil C181.5 μM 100 Å 250 mm column (PepSep, PSC-25-150-15-UHP-nc) using a stepwise linear gradient method with H2O in 131Mintz Ref. No.: 048536-797001WO 0.1% Formic acid and acetonitrile with 0.1% formic acid (solvent B): 5-30% solvent B for 90 minutes at 0.5 μl / minute, 30-35% solvent B for 10 minutes at 0.6 μl / min, 35-95% solvent B for 4 minutes at 0.5 μl / minute, 95% hold for 4 minutes at 0.5 μl / minute). Acquired data was collected in a data-dependent acquisition mode with ion mobility activated in PASEF mode. MS and MS / MS spectra were collected with m / z ranging from 100 to 1700 in positive mode.

[0423] Mass spectrometry data analysis. All acquired data was searched using PEAKSonline Xpro 1.6 (Bioinformatics Solutions Inc.; Ontario, Canada).70 Spectral searches were performed using a custom FASTA-formatted dataset containing either a human or murine version of the Swissprot-reviewed species proteome file with gene ontology localized the plasma membrane (downloaded from Uniprot knowledge database, #entries). A precursor mass error tolerance was set to 20 ppm and a fragment mass error tolerance was set at 0.03 ppm. Peptides, ranging from 6 to 45 amino acids in length, were searched in semi-specific tryptic digest mode with a maximum of two missed cleavages. Carbidomethylation (+57.02 Da) on cysteines was set as a static modification and both methionine oxidation (+15.99 Da), N-terminal acetylation (+42.02 Da), and STY phosphorylation (+79.97 Da) were set as a variable modification. Lastly, peptides were filtered based on a false discovery rate (FDR) of 1%.

[0424] Phosphosite identification on purified phosphoproteins. CD44-Fc-AviTag N-terminal domain protein, Erbb2-Fc-AviTag ectodomain, or ADAM17-Fc-AviTag ectodomain (1 mg / mL) was incubated in the presence of recombinant Src kinase domain (10 nM), or catalytically inactive mutant (corresponding to D359A in the full-length sequence), in PBS with 10 mM MgCl2 and 1 mM ATP (Sigma Aldrich, A1852-1VL) for 30 minutes at 37 °C. Proteins were immobilized on High Capacity Neutravidin Agarose (Thermo Fisher Scientific, 29204) by incubating reactions on agarose for 1 hour at room temperature. Resin was washed on a vacuum manifold in 2 mL columns, being careful to not let the resin dry, with three successive washes of 1 mL each of RIPA, PBS with 1 M NaCl, and 50 mM ammonium bicarbonate with 2 M urea. Resins were then alkylated and digested on-bead using Preomics iST 96x kits (Preomics, P.O.00027) according to the manufacturer’s protocol and eluted peptides were dried in vacuo. Peptides were resuspended in running solvent and quantitated using a Pierce Colorimetric Peptide Quantitation Assay (Thermo Fisher Scientific, 23275). Peptides were separated and analyzed as described above. The spectra were searched against a FASTA-formatted dataset containing the sequences of the purified proteins used. 132Mintz Ref. No.: 048536-797001WO

[0425] ADAM17 activity assay. Recombinant ADAM17-Fc-AviTag (1 μM) was treatedwith recombinant Src kinase domain (50 nM) in PBS with 10 mM MgCl2 and 1 mM ATP (Sigma Aldrich, A1852-1VL) for 30 minutes at 37 °C. ADAM17 was then immobilized on NeutrAvidin-coated Maxisorp plates. Plates were then blocked with 1% BSA in HEPES (20 mM, pH 7.4). Commercial fluorogenic TACE substrate (Sigma) (200 μM) in 50 mM pH 8 Tris with 25 mM NaCl was added to wells and the plates were incubated for 15 minutes at 37 °C. Fluorescence (320 nm excitation / 420 nm emission) was measured using a Tecan M200Pro plate reader.

[0426] Recombinant Src-induced proliferation assay. HCC1569 cells were harvested withTrypsin / EDTA and seeded in 96 well plates at 5e3 cells per well in media supplemented with eATP (1 mM). To each well was added either recombinant Src kinase domain (10 nM) or catalytically inactive mutant (corresponding to D359A in the full-length sequence), and either ADAM17 / MMP inhibitor TAPI-2 (25 μM) or an equal volume of DMSO. Images of plates were acquired every two hours on an Incucyte SX5. Confluence was calculated using Incucyte analysis software using adherent cell-by-cell tracking.

[0427] siRNA knockdown of SRC. HCC1569 cells were seeded the day before transfectionat 350,000 cells per well in complete media in 6 well plates, such that the cells were 50-70% confluent the following day. The day of transfection, SRC silencer siRNA (Thermo) or a negative control siRNA (Thermo) were precomplexed in lipofectamine RNAiMAX (Thermo) according to the manufacturer’s instructions. Transfection complexes were then added to wells dropwise. After 72 hours, cells were harvested with Versene and analyzed by flow cytometry and Western blot.

[0428] Western blotting. For Western blotting experiments, cells were harvested, washedthree times with PBS and lysed in RIPA supplemented with HALT protease and phosphatase inhibitor (Thermo Fisher Scientific, 78440) and benzonase nuclease (Sigma Aldrich, E1014- 25KU) for 30 minutes at 4 °C. Lysates were clarified by centrifugation (21000 rcf, 15 minutes). Protein concentration in lysates quantitated by RapidGold BCA (Thermo Scientific, A53225). Lysate concentration was normalized and lysates were diluted in 4X SDS (BioRad) loading buffer supplemented with beta-mercaptoethanol (Sigma) according to the manufacturer’s instructions. Samples were heated at 95 °C for 5 minutes and 10 μg was loaded per well onto a 4-12% iBolt bisacrylamide gel (Invitrogen). Lysates were separated at 200 V for 30 minutes in MES buffer (BioRad) and transferred to PVDF membranes (Thermo) on an iBolt 2 transfer cassettes. When total protein loading was quantitated, REVERT Total 133Mintz Ref. No.: 048536-797001WO Protein Stain (LiCOR) was used at this stage, according to the manufacturer’s instructions, and removed after imaging on a LiCOR CLx, also according to the manufacturer’s instructions. Membranes were blocked with 5% BSA in TBS, probed with primary antibodies overnight at 4 °C in 2.5% BSA in TBS, washed thrice with TBS-T for 5 minutes each wash, probed with secondary antibodies for 1 hour at room temperature in 2.5% BSA in TBS, washed again thrice with TBS-T for 5 minutes each wash, and visualized on a LiCOR CLx. Images were analyzed in Image Studio Lite.

[0429] Assays of pharmacological modulators of eSrc expression. HCC1569 cells wereseeded at 350,000 cells per well in complete media in 6 well plates. Media was then supplemented with pharmacological modulator at the specified concentration or an equal volume of DMSO. When cells were treated with multiple compounds in the same plate, pharmacological reagents were diluted into equal volumes of DMSO. After 72 hours, cells were harvested with Versene and analyzed by flow cytometry and Western blot.

[0430] Autophagosome immunoisolation. To promote autophagosome production,HCC1569 cells were starved in HBSS (Gibco) for 2 hours at 37 °C in a 5% CO2 incubator, per previously established protocols. Cells were lifted by trypsin and harvested by centrifugation, before being washed twice with ice-cold PBS and resuspended in an ice-cold isotonic buffer (20 mM HEPES pH 7, 250 mM sucrose, 1 mM EDTA). The cell pellet was then lysed by 25 strokes of a Dounce homogenizer on ice. Lysate was centrifuged to pellet membranes and nuclear capsules (3000 rcf, 10 minutes, 4 °C). Autophagosomes were immunoisolated from 500 μg of homogenate using anti-LC3A / B antibodies (Thermo) on Protein A / G magnetic beads overnight at 4 °C. Autophagosomes were then eluted with 1% NP-40 in TBS. Eluates were analyzed by Western blot.

[0431] Confocal microscopy. Approximately 10,000 HCC1569 cells were seeded in Ibidi 8-well microchamber glass slides (Ibidi, 80826). In some experiments, IMP-1088 (100 nM) or DMSO (equal volume) was added to each well. After 24 hours, chloroquine (50 μM) was added to swell autophagosomes for ease of visualization by microscopy. After another 24 hours, cells were washed three times with cold 1% BSA in PBS and fixed in 10% neutral formalin for 20 minutes at room temperature in the dark. The cells were washed three times with PBS and then permeabilized and blocked with 0.1 mg / mL rabbit IgG (Jackson) and 0.1% Triton-X100 in PBS for 60 minutes at room temperature. Cells were then co-stained with primary antibodies in 1% BSA in PBS overnight at 4 °C. Cells were then washed three times with 1% BSA in PBS for 5 minutes each, stained with 1 μg / mL DAPI (Cell Signaling 134Mintz Ref. No.: 048536-797001WO Technologies, 4083S) in PBS for 5 min, and then washed three times with PBS before being immersed in Ibidi mounting medium (Ibidi, 50001). Cells were imaged on a Nikon Ti2 inverted fluorescence microscope with a Nikon C2 laser scanning confocal with 4 laser lines in the Center for Advanced Light Microscopy at the University of California, San Francisco. Images were analyzed in FIJI and colocalization analysis was performed using Coloc 2.

[0432] Phage display. Antibody fragment of antigen binding phage display was conductedaccording to previously published protocols [Miller et al 2012; Hornsby, et al 2015; Glasgow, et al.2022]. In brief, selections were performed using in-house phage library UCSF using an N-terminal His-TEV tagged Src[253-536] construct immobilized on HisPur Capture magnetic beads. Selections were performed in PBS supplemented with 0.2% bovine serum albumin (w / v), 0.05% Tween-20 (v / v), and 10 mM imidazole to reduce association of histidine residues to beads. Prior to each selection, phage that non-specifically bound to beads were depleted by incubation with HisPur Capture magnetic beads. Selections were then performed with decreasing amounts over four rounds (1 μM, 100 nM, 50 nM, 20 nM) of immobilized Src kinase domain (100 μL of antigen solution immobilized on 20 μL of pre- washed beads). Phage were eluted by a “catch and release” strategy, in which antigen-bound phage were eluted by incubation with TEV protease (2 μg / mL). Selections were performed on a Kingfisher 96x2 using a custom protocol.

[0433] After the fourth round, infected bacteria were plated, and single colonies weresequenced. Phage titers were performed as previously described. Single clones were grown in 96 deep well plates to confluence in the presence of M13K07 helper phage. Phage supernatants were then used to screen clones by antigen ELISA using Src kinase domain immobilized on MaxiSorp ELISA plates. Clones that bound specifically to Src versus immobilized BSA were expressed as recombinant Fabs and analyzed by biolayer interferometry and on-cell binding by flow cytometry.

[0434] Biolayer interferometry (BLI). BLI was performed on an Octet RED384 (Sartorius,formerly ForteBio). Assays were performed in PBS with 0.05% Tween and 0.2% high- quality BSA. Biotinylated anti-TwinStrep or anti-HisTag antibodies were loaded onto streptavidin sensors at a concentration of 50 nM for 180 seconds followed by blocking in the same buffer with 10 μM biotin. Either TwinStrep or His-tagged Src variant was loaded for 180 seconds at 200 nM. A reference lane of load-only was always included to monitor for dissociation of Src from the biosensor over the course of the assay. Fabs or IgGs were associated for 300 seconds and dissociated for 300 seconds. Dissociation constants were 135Mintz Ref. No.: 048536-797001WO determined from multipoint fits in Octet Analysis Studio 12.0. Single-point dissociation constants were calculated either in Octet Analysis Studio 12.0 or Prism 9.2.

[0435] Cross-linking mass spectrometry. Experiments were performed according toliterature protocols [Di Ianni, et al. J Proteome Res.2024 Mar 1;23(3):1049-1061].

[0436] Antibody-antigen complex prediction analysis. Experiments were performedaccording to literature protocols [Di Ianni, et al. J Proteome Res.2024 Mar 1;23(3):1049- 1061].

[0437] Antibody conjugations with radioligand chelators. Antibody was first labeled witha chelator, either DFO (in the case of 89Zr experiments) or macropa (in the case of 225Ac experiments) using isothiocyanate-derivates of the chelator. The reaction was incubated in 0.1 M sodium bicarbonate buffer (pH 9.0) and purified by buffer exchanged using a G25 column into 0.2 M sodium acetate (pH 7.0). Kinetic constants were confirmed by be unaltered by BLI after labeling.

[0438] 89Zr and 225Ac radiolabeling. A solution of 89Zr-oxalic acid (5 mCi; 40 μl) wasneutralized with 2 M Na2CO3 (18 μl). After 3 minutes, 0.30 ml of 0.5 M HEPES (pH 7.1– 7.3) and 1.5 mg of DFO-Ab1 (pH = 7) were added into the reaction vial. After incubation for 60 minutes at 37oC, the reaction progress was monitored by instant thin layer chromatography (ITLC) using a 20 mM citric acid (pH 4.9–5.1) mobile phase. The product was purified with a PD10 column. The decay corrected radiochemical yield was consistently > 95%.

[0439] A solution of 225Ac-nitrate (120 uCi) dissolved in 0.2 M HCl was added to anEppendorf vial, and the accurate activity was measured by using a dose calibrator. To this were added 2 M tetramethyl ammonium acetate buffer (25 uL), L-ascorbic acid (150 g / L; 10 uL), and the Ab1-macropa (120 ug). The pH of the reaction was determined by pH paper; pH of a typical reaction was 5.8. The mixture was incubated in 37.0oC for 1.5h. After this, the reaction was then quenched with 2 M DTPA (10 uL) and purified using an G25 desalting column that had been equilibrated previously with 1% human serum albumin in 0.5 M HEPES. The product was eluted in approximately 2 mL of 1% human serum albumin and analyzed by ITLC to determine the radiochemical purity. The decay corrected radiochemical yield was consistently > 95%.

[0440] Small animal PET / CT. All animal studies were conducted in compliance withInstitutional Animal Care and Use Committee at UCSF. For tumor imaging or treatment studies, three to five-week-old intact male athymic nu / nu mice were purchased from Charles 136Mintz Ref. No.: 048536-797001WO River and used for experiments after a brief period of acclimation. Mice were inoculated subcutaneously with cancer cells (~1.5×106) in the flank. The cells were injected in a 1:1 mixture (v / v) of media (DMEM / F12) and Matrigel (Corning). Xenografts were generally palpable within 14–18 days after injection.

[0441] Tumor-bearing mice received between ~200 μCi of 89Zr-Ab1 in 100 μL salinesolution volume intravenously using a custom mouse tail vein catheter with a 28-gauge needle and a 100–150 mm long polyethylene microtubing. After a dedicated period of uptake time, mice were anesthetized with isoflurane and imaged on a small animal PET / CT scanner (Inveon, Siemens Healthcare, Malvern, PA). Animals were scanned for 40 minutes for PET, and the CT acquisition was performed for 10 minutes.

[0442] The co-registration between PET and CT images was obtained using the rigidtransformation matrix generated prior to the imaging data acquisition since the geometry between PET and CT remained constant for each of PET / CT scans using the combined PET / CT scanner. The photon attenuation was corrected for PET reconstruction using the co- registered CT-based attenuation map to ensure the quantitative accuracy of the reconstructed PET data. Decay corrected images were analyzed using AMIDE software.

[0443] Biodistribution studies. At a dedicated time after radiotracer injection, animals wereeuthanized by cervical dislocation. Blood was harvested via cardiac puncture. Tissues were removed, weighed and counted on a Hidex automatic gamma counter for accumulation activity. The mass of the injected radiotracer was measured and used to determine the total number of CPM by comparison with a standard of known activity. The data were background- and decay-corrected and expressed as the percentage of the injected dose / weight of the biospecimen in grams (%ID / g).

[0444] In vitro T cell engager (TCE) assays. For in vitro TCE assays, target cells wereharvested from culture the day prior to the experiment. In some experiments, cells were pretreated with drugs for 48 hours prior to harvesting. Cells were labeled with CellTrace™ CFSE (Thermo) according to the manufacturer’s instructions and then seeded at 5e3 cells per well in a 96 well in complete media supplemented with Sytox DeepRed (Thermo) live / dead stain. The following day, 5e4 PBMCs in complete media were added to each well, followed by a 10X stock solution of TCE (or control) in PBS. Plates were imaged on an Incucyte SX5 and analyzed with the on-board analysis software.

[0445] In vivo TCE efficacy studies. HCC1569 human breast cancer cells (1e6) weresubcutaneously implanted in matrigel into the flank of NSG mice. Tumors were allowed to 137Mintz Ref. No.: 048536-797001WO grow until an average size of 80 mm3 (about two weeks). The day prior to administration (day -1), human PBMCs (1e7) pooled from multiple donors were intravenously injected into the mice to engraft a humanized immune system. TCE (or control) molecules were administered intravenously at the annotated concentration in sterile PBS on Day 0 and Day 7. Tumor volume and body weight were monitored for two weeks. Engraftment of PBMCs at that timepoint was confirmed by flow cytometry using DURAClone IM Phenotyping Kits (Beckman Coulter).

[0446] Antibody-drug conjugate (ADC) preparation. Antibodies were labeled withcytotoxic compounds using the corresponding toxin N-hydroxysuccinimidyl ester conjugate. In brief, antibody (1 mg / mL) was incubated with NHS-toxin (20 equivalents) for 1 hour at 37 °C in PBS (pH 7.4). Unconjugated toxin was removed by serial passages through two ZebaSpin 7 kDa MWCO desalting columns (Thermo) pre-equilibrated with PBS.

[0447] ADC in vitro efficacy studies. For in vitro ADC assays, target cells were harvestedfrom culture the day prior to the experiment. Cells were seeded at 5e3 cells per well in a 96 well in complete media supplemented with Sytox DeepRed (Thermo) live / dead stain. The following day, a 10X stock solution of ADC (or control) in PBS was added to each well. In some experiments, cells were co-treated with macropinocytosis inhibitor ethylisopropylamiloride hydrochloride. Plates were imaged on an Incucyte SX5 and analyzed with the on-board analysis software.

[0448] ADC in vivo efficacy studies. KP4 human pancreatic cancer cells (1e6) weresubcutaneously implanted in matrigel into the flank of NSG mice. Tumors were allowed to grow until an average size of 100 mm3 (about two weeks). ADC (or control) molecules were administered intravenously at the annotated concentration in sterile PBS on Day 0 and Day 7. Tumor volume and body weight were monitored for two weeks.

[0449] Radioligand therapy studies. Mice bearing unilateral subcutaneous HT-1080 tumorsreceived225Ac-Ab1 (~0.8 μCi / mouse) or vehicle (saline) at the indicated dose via tail vein. Animals were weighed at the time of injection, and once weekly until the completion of the study. Tumor volume measurements were calculated with calipers. For treatment studies, the primary endpoints were death due to tumor volume >3000 mm3 or ≥ 20% loss in mouse body weight.

[0450] Synthesis of dasatinib azide. Azido dasatinib with synthesized according topreviously published protocols [Li, et al. Bioconjugate Chem.2016, 27, 10, 2575–2579]. 138Mintz Ref. No.: 048536-797001WO

[0451] Synthesis of heterobifunctional small molecules. For heterobifunctional smallmolecules (i.e., CytoTACs and RadTACs), azido-dasatinib (10 mM) was incubated in a 1:1:1 mixture of water / dimethylsulfoxide / acetonitrile with an equimolar amount of a commercially available cyclooctyne-bearing toxin or chelator. Reactions were incubated at 37 °C for 1 hour. Heterobifunctional products were purified by reverse phage chromatography on an Agilent 1260 HPLC system with a Poroshell EC-C18 (Agilent) column.

[0452] Cytotoxin-targeted chimera (CytoTAC) in vitro efficacy assays. For in vitroCytoTAC assays, target cells were harvested from culture the day prior to the experiment. Cells were seeded at 5e3 cells per well in a 96 well in complete media supplemented with Sytox DeepRed (Thermo) live / dead stain. The following day, a 10X stock solution of CytoTAC (or control) in PBS was added to each well. Plates were imaged on an Incucyte SX5 and analyzed with the on-board analysis software.

[0453] Radioligand-targeted chimera (RadTAC) in vivo imaging studies. Dasatinib-DOTA was incubated with 64Cu under standard conditions (PBS, 55 °C) to generate the radiolabeled conjugates. The radioconjugate was purified by HPLC.

[0454] All animal studies were conducted in compliance with Institutional Animal Care andUse Committee at UCSF. For tumor imaging or treatment studies, three to five-week-old intact male athymic nu / nu mice were purchased from Charles River and used for experiments after a brief period of acclimation. Mice were inoculated subcutaneously with cancer cells (~1.5×106) in the flank. The cells were injected in a 1:1 mixture (v / v) of media (DMEM / F12) and Matrigel (Corning). Xenografts were generally palpable within 14–18 days after injection.

[0455] Tumor-bearing mice received between ~200 μCi of Dasatinib-DOTA(64Cu) in 100 μLsaline solution volume intravenously using a custom mouse tail vein catheter with a 28-gauge needle and a 100–150 mm long polyethylene microtubing. After a dedicated period of uptake time, mice were anesthetized with isoflurane and imaged on a small animal PET / CT scanner (Inveon, Siemens Healthcare, Malvern, PA). Animals were scanned for 40 minutes for PET, and the CT acquisition was performed for 10 minutes.

[0456] The co-registration between PET and CT images was obtained using the rigidtransformation matrix generated prior to the imaging data acquisition since the geometry between PET and CT remained constant for each of PET / CT scans using the combined PET / CT scanner. The photon attenuation was corrected for PET reconstruction using the co- 139Mintz Ref. No.: 048536-797001WO registered CT-based attenuation map to ensure the quantitative accuracy of the reconstructed PET data. Decay corrected images were analyzed using AMIDE software.

[0457] RadTAC in vivo efficacy studies. Dasatinib-DOTA is incubated with 225Ac understandard conditions (PBS, 55 °C) to generate the radiolabeled conjugates. The radioconjugate is purified by HPLC.

[0458] Mice bearing unilateral subcutaneous HT-1080 tumors receive Dasatinib-DOTA(225Ac) (~0.8 μCi / mouse) or vehicle (saline) at the indicated dose via tail vein. Animals are weighed at the time of injection, and once weekly until the completion of the study. Tumor volume measurements are calculated with calipers. For treatment studies, the primary endpoints are death due to tumor volume >3000 mm3 or ≥ 20% loss in mouse body weight.

[0459] Small molecule inhibitor targeted T cell engager (SMITE) bioconjugation. Fornon-specific lysine labeling, recombinant Fab’s (either an Fv-swap of OKT3 onto the 4D5 scaffold or an irrelevant Fab in the 4D5 framework) were first conjugated with an N- hydroxysuccinimidyl ester of bi[6.1.0]cyclononyne (BCN) (10 equivalents) in PBS (pH 7.4) at 37 °C for 1 hour. Unconjugated BCN was removed by serial passages through two ZebaSpin 7 kDa MWCO desalting columns (Thermo) pre-equilibrated with PBS. Then, azido dasatinib (5 equivalents) was incubated with the BCN-labeled Fab at 37 °C for 1 hour. Unconjugated dasatinib was removed by serial passages through two ZebaSpin 7 kDa MWCO desalting columns (Thermo) pre-equilibrated with PBS. Conjugation efficiency was confirmed by intact mass spectrometry.

[0460] For site-specific oxaziridine labeling, recombinant OKT3 (Fv swap onto the 4D5framework) or single methionine point mutants corresponding to light chain G67M, light chain K149M, or heavy chain T211M, were first conjugated to either alkynyl or azido oxaziridine, as previously described [Elledge, et al. Proc Natl Acad Sci, 2020, 117 (11) 5733- 5740]. In brief, Fab (50 μM) was incubated with oxaziridine (15 equivalents) at room temperature in PBS for 30 min. Excess oxaziridine was removed by serial passages through two ZebaSpin 7 kDa MWCO desalting columns (Thermo) pre-equilibrated with PBS. Then, corresponding complementary alkyne or azide (e.g., azido dasatinib) was added (5 equivalents), followed by precomplexed 2:1 THTPA / CuSO4 (0.8 equivalents), and finally sodium ascorbate (1.6 equivalents), all in PBS. Click reactions were incubated at 37 °C for 1 hour. Unconjugated targeting molecule was removed by serial passages through two 140Mintz Ref. No.: 048536-797001WO ZebaSpin 7 kDa MWCO desalting columns (Thermo) pre-equilibrated with PBS. Conjugation efficiency was confirmed by intact mass spectrometry.

[0461] SMITE efficacy by microscopy. For in vitro SMITE assays by microscopy, targetcells were harvested from culture the day prior to the experiment. Cells were labeled with CellTrace™ CFSE (Thermo) according to the manufacturer’s instructions and then seeded at 5e3 cells per well in a 96 well in complete media supplemented with Sytox DeepRed (Thermo) live / dead stain. The following day, 5e4 PBMCs in complete media were added to each well, followed by a 10X stock solution of SMITE (or control) in PBS. Plates were imaged on an Incucyte SX5 and analyzed with the on-board analysis software.

[0462] SMITE efficacy by ATP release assay. For in vitro SMITE assays by ATP release,target cells were harvested from culture the day prior to the experiment. In some experiments, cells were pretreated with drugs for 48 hours prior to harvesting. Cells were seeded at 1e4 cells per well in a white 96 well tissue culture treated plate in complete media. The following day, 1e5 PBMCs in complete media were added to each well, followed by a 10X stock solution of SMITE (or control) in PBS. After 48 hours, the media and PBMCs were aspirated and cells were washed thrice with PBS, before measuring ATP release of remaining live cells using ATP-Glo™ Bioluminometric Cell Viability assay (Biotium) per the manufacturer’s instructions. Plates were read on a Tecan M200Pro plate reader.

[0463] SMITE in vivo efficacy assays. Nomo1 human acute myeloid leukemia cells (1e6)stably expressing luciferase are intravenously injected into NSG mice. Five days after implantation, human PBMCs (1e7) pooled from multiple donors are intravenously injected into the mice to engraft a humanized immune system. SMITE (or control) molecules are administered intravenously at the annotated concentration in sterile PBS on Day 0 and Day 7. Tumor burden is measured by bioluminescence imaging, and body weight is monitored. Engraftment of PBMCs at that timepoint is confirmed by flow cytometry using DURAClone IM Phenotyping Kits (Beckman Coulter).EXAMPLE 2: SRC REMODELS THE CELL SURFACE PHOSPHOPROTEOME

[0464] Ectokinases have the unique potential in cancer to be chronically active due to theabundance of extracellular adenosine triphosphate. We initially sought to identify the resident tyrosine ectokinase(s) on HCC1569 breast cancer cells prior to profiling the cell surface tyrosine phosphoproteome, hypothesizing that phosphotyrosines could be leveraged as biomarkers using antibody-based technologies we have previously developed. We analyzed the cell surface proteome of HCC1569 cells using photoaffinity enrichment of surface 141Mintz Ref. No.: 048536-797001WO proteins followed by mass spectrometry. We identified known tyrosine ectokinases CK2 and Src in surface proteomics enrichments from these cells. When we incubated HCC1569 cells with tumor-relevant concentrations of extracellular ATP, we observed substantial tyrosine phosphorylation that was abrogated by co-incubation with a small molecule inhibitor of Src, Src inhibitor 1 (FIG.1A, FIGs.2A-2B). Therefore, and without being bound by theory, we hypothesized that Src, which has been described to be secreted (termed extracellular Src or eSrc) but has not been localized or mechanistically explained, can be responsible for cell surface tyrosine phosphorylation on HCC1569 cells.

[0465] We sought to define the scope of eSrc cell surface substrates, as this subset of proteinshas been neglected in prior studies of eSrc as an ectokinase. To selectively label and enrich extracellular phosphotyrosine-containing proteins, we devised a photoaffinity labeling (PAL) strategy. We leveraged a high affinity anti-phosphotyrosine antibody we have previously developed in conjugated to a short radius (< 5 nm) Dexter energy transfer (DET) photocatalyst. We proposed that this probe would enable selective and unbiased labeling on cell surfaces of extracellular phosphoproteins in complex with the antiphosphotyrosine antibody by employing a short-lived radical species, a biotin carbene derived from biotin diazirine by DET (FIG.1B, FIG.3). We validated this strategy by labeling lysates of pervanadate-treated JURKAT cells, a model system we have previously used to develop phosphotyrosine enrichment strategies. We observed that PAL with anti-phosphotyrosine Fabs enriched known tyrosine phosphorylated proteins from JURKAT lysates, while isotype Fab treated cell lysates (FIG.4) or whole-cell lysates (FIG.5) did not. Additionally, we observed significant enrichment of 6 of 7 phosphotyrosine peptides observed, adding confidence that our strategy enriches phosphotyrosine containing proteins (FIG.6).

[0466] We proceeded to use this reagent to analyze eSrc substrates on HCC1569 cells. Togenerate our surface phosphoproteome, we treated cells with or without ATP to visualize total endogenous ectokinase activity and with or without Src inhibitor 1 to assay the specific role of eSrc (FIGs.1B-1C). We observed 631 proteins shared among the three datasets and 53 proteins unique to the ATP only sample set, including the known Src substrates SRC8 and LYN and the cancer-associated metalloprotease ADAM17. In the label free quantitation analyses of shared proteins, we observed numerous proteins upregulated in the vehicle- treated dataset compared to Src inhibitor 1. Notably, we observed known Src substrates including cortactin, as well as known cancer antigens, including Erbb2, PTK7, PTPRF, and MUC18 / CD146. Concordant with the hypothesis that eSrc is the dominant tyrosine 142Mintz Ref. No.: 048536-797001WO ectokinase, little significant difference was observed between the Src inhibitor 1 treated samples and those without ATP supplemented (FIGs.7A-7B). To validate putative eSrc substrates ADAM17, Erbb2, CD44, PTK7, and MUC18 in vitro, we first treated recombinant protein with active Src kinase domain or a loss-of-function mutant, Src(D359A), and visualized phosphotyrosines by Western blot (FIG.8). We also analyzed active or D359A Src-treated ADAM17, CD44, and Erbb2 by mass spectrometry and observed 1, 3, and 2 phosphotyrosine residues, respectively (FIG.9). These results further validate the efficacy and utility of our PAL method for unbiased ecto-pY identification.

[0467] Previous literature has linked the activity of ADAM17 with Src activity in a fashionindependent of the ADAM17 cytoplasmic tail, suggesting that extracellular, rather than intracellular, Src may be responsible for the observed change. We hypothesized that, because we observed ADAM17 phosphorylation in the protease domain, Src-mediated phosphorylation could modulate protease activity and thus proliferation, a role that has been previously suggested for extracellular Src. Therefore, we treated ADAM17 with Src in vitro and found increased peptidase function (FIG.10). Furthermore, we found that treatment of HCC1569 cells with recombinant Src kinase domain but not the D359A loss-of-function analogue, resulted in increased proliferation, which was abrogated by the ADAM17 inhibitor TAPI-2 (FIGs.1D-1E).EXAMPLE 3: SRC IS NONCANONICALLY LOCALIZED TO THE CELL SURFACE ONIMMORTALIZED CELL LINES

[0468] Canonically, Src is associated with the inner leaflet of the plasma membrane by an N-terminal myristoyl lipid. Recent work proposed that Src may be associated with extracellular vesicles after observing its presence in media secretions of cultured cells. To establish the localization of extracellular Src (eSrc), we probed the cell surface (FIG.1F) and secretions (FIG.11) of HCC1569 cells with a commercial anti-Src antibody. While Src was undetectable in secretions, flow cytometry of live cells showed strong surface expression thatwas sensitive to siRNA knockdown of SRC ̧the gene encoding Src (FIG. 12).

[0469] To determine the generalizability of eSrc cell surface expression, we analyzedexpression across a variety of cancer-derived cell lines, immortalized models of healthy tissue, and healthy patient derived peripheral blood mononuclear cells (PBMCs) (FIG.1F). We found that eSrc was expressed by most, but not all, immortalized cell lines we tested and was not detectable on healthy donor PBMCs. eSrc expression was not correlated with 143Mintz Ref. No.: 048536-797001WO transcript levels of SRC, indicating a more complex mechanism of protein trafficking and homeostasis (FIG.13). Additionally, in isogenic cell lines, eSrc expression correlated with cell surface tyrosine phosphorylation activity, and that activity was inhibited by Src inhibitor 1 indicating that the secreted form of Src is catalytically active (FIG.14). Collectively, these results demonstrate that eSrc is expressed and active on the surface of a variety of cancer cell lines to varying degrees. We probed the total Src protein levels by Western blot from select cell lines (and PBMCs) and found that, eSrc expression correlated (R2 = 0.80) with total Src protein expression (FIG.1F, FIG.15). This result is notable as Src total protein expression is highly upregulated in a wide variety of cancers (FIG.16), implying that eSrc may be a broadly applicable tumor associated antigen.

[0470] Src is an N-myristoylated protein, and thus we hypothesized that the N-myristoyl lipidon the N-terminus of Src is necessary for eSrc to be anchored to the cell membrane. To test this hypothesis, we cultured eSrc+++ HCC1569 cells in the presence of a non-toxic inhibitor of the two human N-myristoyl transferases (IMP-1088), and observed that IMP-1088 potently reduced eSrc, congruent with our hypothesis (FIG.1G). IMP-1088 did not alter total Src protein levels (FIG.17). To confirm that these observations were due specifically to N- myristoylation of Src and not changes from global blockade of N-myristoylation, we also transiently transfected eSrc+ PaTu8902 cells with either C-terminally FLAG-tagged Src or Src(G2A), which cannot be myristoylated. We observed both FLAG and increased Src- staining of wild-type transfected cells, but not Src(G2A), compared to mock transfected cells (FIG.1H, FIG.18). Collectively, these data indicate that Src is anchored to the cell surface by its N-myristoyl lipid. EXAMPLE 4: ESRC IS NONCANONICALLY TRANSLOCATED TO THE CELL SURFACE BY AUTOPHAGOLYSOSOMALEXOCYTOSIS

[0471] In prior work we observed that the lysosomal membrane proteins LAMP1 andLAMP2 were two of the most significantly enriched surface proteins in empty-vector compared to KRAS- or HER2-transformed MCF10A cells (FIGs.19A-19B). We thus hypothesized that lysosomal exocytosis was upregulated in these cells, leading to membrane- bound lysosomal proteins being displayed on the surface, and further posited that Src may be such a protein. Consistent with this hypothesis, previous surface proteomics studies from our lab identified upregulation of lysosomal membrane proteins and Src when cultured in hypoxic environments (FIGs.20A-20B), a known stimulator of lysosomal exocytosis. We 144Mintz Ref. No.: 048536-797001WO tested our hypothesis by culturing HCC1569 cells in the presence of lysosomal exocytosis inhibitors Vacuolin-1 or Bafilomycin A1. We observed that either compound reduced eSrc abundance, whereas inhibitors of exosome biogenesis, endosomal exocytosis, or blebbing did not (FIG.21A; FIG.22-FIG.23).

[0472] Both Vacuolin-1 and Bafilomycin A1 also inhibit lysosome-autophagosome fusion,therefore we hypothesized that Src may enter the lysosome via autophagosomes. Autophagosomes nucleate inside the cytosol from curved membranes called phagophores to encapsulate cytoplasmic contents, and traffic to the lysosome for degradation. We thus hypothesized that if Src is anchored to phagophore membranes during nucleation, it would be anchored to the inner (and outer) leaflet of the autophagosome and then autolysosome. Thus, upon lysosomal exocytosis, Src would be exposed on the outer leaflet of the plasma membrane (FIG.24). We validated this hypothesis using an inhibitor of autophagosome formation, ATG7-IN-2, that potently abolished eSrc expression in HCC1569 cells without affecting total Src protein levels (FIG.21A; FIG.23).

[0473] To confirm that Src associates with autophagosomes, we used an antibody against theautophagosomal marker LC3B to isolate intact autophagosomes from HCC1569 homogenates and analyzed the contents by Western blot (FIGs.21B-21C, FIG.25). We detected robust Src signal in isolated autophagosomes, which was partially abrogated when cells were treated with IMP-1088 prior to vesicle isolation (p = 0.001). This confirms that Src is associated with autophagosomes, and that the N-myristoyl group can mediate this association.

[0474] Ultimately, we sought to co-localize Src with autophagosomes by microscopy. Usingconfocal microscopy, we analyzed cellular distribution of both Src and LC3A / B in chloroquine treated cells (FIGs.21D-21E). Chloroquine blocks autophagosome / lysosome fusion, which facilitates imaging by causing autophagosome swelling, and limits colocalization of lysosomal Src and LC3 after autophagosome / lysosome fusion. Notably, with this technique, we cannot discern inner leaflet from outer leaflet localization of Src on either the plasma membrane or autophagosome. However, we hypothesized that a small, transient subpopulation of Src should associate with autophagosomes en route to the cell surface and that this co-localization would be sensitive to IMP-1088. As expected, Src appeared across multiple cellular compartments, while LC3 was solely observed in swollen puncta. Colocalization analysis between LC3 and Src in chloroquine treated cells yielded a Pearson’s coefficient of R = 0.25, indicating a moderate degree of colocalization. 145Mintz Ref. No.: 048536-797001WO Furthermore, inhibition of N-myristoylation with IMP-1088 resulted in a significantly lower degree of colocalization (R = 0.15, p = 0.0059, Cohen’s d = 0.66). These results demonstrate that there is a modest colocalization of Src with autophagosomes that is significantly dependent on N-myristoylation.

[0475] Based on these data, we hypothesized that we could also pharmacologicallyupregulate eSrc expression by inducing autophagy, lysosomal exocytosis, or total Src expression. To facilitate gain-of-signal studies, we used the eSrc+ PaTu8902 cells and cultured them in the presence of rapamycin, to induce autophagy, or subtoxic concentrations of trametinib, which induces lysosomal exocytosis, or dasatinib, which increases total Src expression (FIG.21F, FIGs.26A-26B). We observed that trametinib and dasatinib, but not rapamycin, increased the expression of eSrc. We hypothesized that this was because lysosomal exocytosis is the direct means of translocation, whereas Src could be degraded in the lysosome as a normal part of autophagy. Consistent with these data, we observed that in isogenic MCF10A derivatives, eSrc levels correlated with lysosomal exocytosis, as evidenced by LAMP1 expression being higher in empty-vector, compared to KRAS, transformants, as opposed to autophagic flux, which was upregulated in KRAS-transformed cells (FIGs.27A- 27C). This is also consistent with our observations of cells cultured in a hypoxic environment, which is also known to upregulated lysosomal exocytosis.

[0476] These data collectively suggest that to be translocated, Src is first encapsulated byautophagosomes, which are then trafficked and fused to lysosomes, and ultimately secreted by lysosomal exocytosis.EXAMPLE 5: A SYNTHETIC ANTIBODY ENABLES DETECTION OF TUMOR-ASSOCIATED ESRC INVIVO.

[0477] The combination of protein-level upregulation and increased lysosomal exocytosis inmany cancers give eSrc the potential to be highly abundant and tumor-specific. To assess this, we used our Fab-phage libraries to find an antibody (FIG.28), which we termed Ab1, that was a single-digit nanomolar binder of human and murine Src, biophysically stable, and bound eSrc-expressing, but not SRC siRNA knocked-down, HCC1569 cells (FIGs.29A- 29B, FIGs.30-32). We did not observe a significant effect on Src activity upon Ab1 binding but did find that Ab1 preferred the open / active conformer of Src using conformationally- locked mutants (FIG.29C). Using an absolute fluorescence quantitation assay, we quantified binding events of Ab1 and a commercial anti-Src antibody, which binds the N-terminal 146Mintz Ref. No.: 048536-797001WO domain and thus should be insensitive to Src conformational changes, to the surface of HCC1569 to approximate eSrc copy number. We found that both assays calculated approximately 25,000 copies of eSrc on the surface of HCC1569 cells, indicating that most eSrc is in the open conformation. Notably, proteomics-based measurements estimate that in healthy murine tissue, Src is expressed on the order of 50-500 copies per cell, whereas cancerous cells contain up to 500,000 copies. Therefore, our estimates indicate that eSrc can exceed total Src in healthy cells by up to two orders of magnitude.

[0478] We hypothesized that the conformational preference of Ab1 could be explained bydifferences in the conformation of the open versus the closed conformers of Src. We generated a model of Src in complex with Ab1 using cross-linking mass spectrometry (FIG. 29C) to inform an AlphaLink2 model, which was then refined by ensemble docking in Rosetta (FIG.29D). We validated that the correct epitope had been predicted by demonstrating that mutagenesis of key contact residues on Src to alanine abrogated binding by biolayer interferometry. We observed that comparison of our predicted complex with published crystal structures showed a higher similarity of the conformation Ab1-binding epitope to the open conformer (PDB: 1y57) compared to a closed conformer (PBD: 2src), explaining the observed specificity of Ab1.

[0479] Using Ab1, we sought to determine whether eSrc is a tumor-associated target in vivo.We conjugated Ab1 to a heavy metal chelator, DFO, and then conjugated a PET-active89Zr to the DFO. Ab1-Zr89was injected into HT-1080 xenograft tumor bearing mice, which should bind eSrc on the tumor as well as any endogenous murine eSrc. We observed high tumor localization of the PET tracer, followed by expected signal from the89Zr probe as circulating antibody was metabolized. Tumor localization was observed to be both intense and sustained, with detectable89Zr signal past 168 hours and high tumor localization compared to other organs and tissues at 24 hours post injection (FIGs.29E-29G). Furthermore, coadministration of 100-fold excess unconjugated Ab1 potently abrogated tumor-specific uptake of89Zr-Ab1 but did not change89Zr signal in any other tissues (FIGs. 29E-29G). We observed similar results using subcutaneous EMT-6 tumor engraftments in syngeneic Balb / c mice. These data demonstrate that eSrc is expressed on tumors and has minimal, if any, endogenous expression on healthy cells in vivo.EXAMPLE 6: ESRC IS A TARGET FOR ANTIBODY-BASED THERAPIES147Mintz Ref. No.: 048536-797001WO

[0480] After demonstrating that eSrc is tumor-associated in vivo, we hypothesized that Ab1could be used for antibody-based cancer therapies. To investigate this possibility, we engineered Ab1 in multiple therapeutic formats to evaluate both the accessibility and efficacy of eSrc as a target.

[0481] We first sought to evaluate eSrc as a target for bispecific T cell engagers (BiTEs). In aBiTE assay, isolated T cells or PBMCs, which are predominantly T cells, are co-cultured with target tumor cells in the presence of BiTE, a bispecific antibody bearing a CD3 binding arm that engages T cells and a target cell binding arm. This ligation event elicits the cytotoxic activity of the T cell, killing the target cell. We observed BiTE-dependent cytotoxicity (specific lysis) across multiple cell lines using PBMCs from multiple healthy donors in a manner that correlated with eSrc abundance, i.e the highest degree of specific lysis was observed for eSrc+++ HCC1569 cells and no specific lysis was observed for eSrc- HL-60 cells (FIG.33A). We observed potent efficacy using multiple BiTE formats (knob-in-hole bispecific antibody, Fab-scFv fusion, IgG-scFv fusion), and found that monovalent controls (i.e., an Fc bearing only a single anti-Src or anti-CD3 arm or a BiTE bearing a Src non- binding point mutant) were ineffective at mediating specific lysis against HCC1569 cells (FIG.33B). We hypothesized that modulation of eSrc levels with pharmacological reagents could (de)sensitize cells to treatment with our Src-targeting BiTEs. Consistent with that hypothesis, we found that eSrc+++ HCC1569 cells could be rescued by pretreatment with IMP-1088, reducing eSrc expression (FIG.33C), and that eSrc+ PaTu8902 cells could be sensitized to BiTE-mediated cytotoxicity by pretreatment with trametinib, which induces eSrc expression (FIG.33D). We tested the efficacy of our BiTEs in a humanized mouse model, in which human PBMCs (pooled from multiple donors) were engrafted into HCC1569 xenograft-bearing NSG mice one day prior to BiTE administration (FIG.33E). We observed that an anti-Src BiTE was sufficient to induce tumor regression and T cell expansion in vivo.

[0482] We also investigated the potential of Ab1 as an antibody-drug conjugate (ADC). Wewere particularly interested in whether an Ab1-ADC would be cytotoxic, as there is no evidence that eSrc internalizes through a specific pathway. To assay this, we conjugated Ab1 to monomethyl auristatin E (MMAE), a tubulin inhibitor and clinical ADC payload. Upon treatment of cancer cell lines with an Ab1-MMAE conjugate, we observed dose-dependent cytotoxicity in KRAS-transformed cells but not disease-matched KRAS wild-type cells (FIG. 33F). We hypothesized that internalization could be possible through macropinocytosis, a pathway known to be upregulated in KRAS-transformed cell lines. To assay this, we treated 148Mintz Ref. No.: 048536-797001WO KRAS-transformed KP4 cells with the macropinocytosis inhibitor ethylisopropylamiloride hydrochloride and found that this abrogated the cytotoxic effects of Ab1-MMAE). We further tested the efficacy Ab1-MMAE against KP4 xenograft tumors implanted in NSG mice in vivo, however, did not see substantial efficacy, likely due to the comparatively low receptor count of eSrc and limited internalization compared to clinical ADC targets.

[0483] We hypothesized that a conjugate to a more potent agent that does not requireinternalization may show more efficacy in vivo than the Ab1-based ADC. Therefore, we turned to the growing field of radioligand therapy (RLT), in which radioactive nuclei (e.g.,225Ac) are coupled to the antibody via a chelator (e.g., macropa). Decaying nuclei then emit potently toxic radiation (e.g., an alpha particle in the case of225Ac) inside the tumor. We tested the efficacy of an Ab1-225Ac conjugate against eSrc+++ HT-1080 tumor cells in a xenograft mouse model (FIG.33G). At 0.8 μCi of Ab1-225Ac (~30 μCi / kg), we observed either tumor regression (2 / 16) or substantial reduction in tumor growth rate (14 / 16) compared to a saline control. Furthermore, we did not observe any acute toxicity upon initial dosing, however, general radiation toxicity was observed upon subsequent doses consistent with the general radiation tolerance of Balb / c mice.

[0484] Collectively these data demonstrate that eSrc is a therapeutically relevant target forantibody-based modalities in vitro and in vivo. We found that potent therapeutic mechanisms, specifically T cell engagers and radioactive nuclei, elicited tumor regression in vivo with a single dose at clinically relevant concentrations and were safe to administer even though Ab1 is cross-reactive for murine (e)Src.EXAMPLE 7: ESRC CAN BE THERAPEUTICALLY TARGETED USING HETEROBIFUNCTIONALSMALL MOLECULES AND SMALL MOLECULE CONJUGATES

[0485] Based on the promising results of the antibody-based eSrc targeting therapies, wehypothesized that, using the abundance of small molecule Src ligands, we could design cell- impermeable heterobifunctional molecules, functionally replacing the antibody with a small molecule Src ligand, which would provide a different pharmacokinetic profile, degree of tumor penetrance, and ease of synthesis compared to a biologic. We chose to use dasatinib, an FDA-approved small molecule Src family kinase inhibitor with picomolar affinity for Src, and functionalized the molecule with an azide, as previously described. We then used this functionalized ligand to make analogues of the antibody based therapies – small molecule inhibitor based T cell engagers (SMITEs) by analogy to BiTEs, cytotoxin targeted chimeras 149Mintz Ref. No.: 048536-797001WO (CytoTACs) by analogy to ADCs, and radioligand targeted chimeras (RadTACs) by analogy to RLT.

[0486] To create the SMITE, we coupled our azido-dastainib to a cyclooctyne conjugatedanti-CD3 engaging Fab to create a bifunctional molecule structurally an ADC but functionally a BiTE. We found that our SMITE induced dose-dependent specific lysis of eSrc+++ but not eSrc+ / - cell lines, and that an isotype-dasatinib conjugate did not (FIG. 34A).

[0487] For CytoTACs, we conjugated azido-dasatinib to cyclooctyne-MMAF (a cellimpermeable analogue of MMAE) to create a heterobifunctional small molecule that we hypothesized would be functionally like our Ab1-based ADCs. Indeed, again we observed dose-dependent toxicity in KRAS-transformed cell lines but not in KRAS-wild-type disease- matched controls (FIG.34B). These data suggest that CytoTAC behaves like ADC, remaining extracellular and only internalizing by macropinocytosis. We also demonstrated that alternative payloads, such as the Topoisomerase I inhibitors SN38 or DXd, were cytotoxic in KRAS-transformed cells, but not KRAS wild-type cells.

[0488] RadTACs were synthesized similarly to CytoTACs, coupling azido-dasatinib to acyclooctyne-conjugated metal chelator (DOTA). Using 64Cu isotopes for PET imaging, we observed dramatic tumor uptake of the RadTAC, that peaked near 4 hours post injection and with a half-life past 24 hours (FIGs.34C-34D). Notably, a cell-permeant18F-dasatinib PET probe is in clinical trials. We observed distinct staining patterns from that probe, indicating that the RadTAC is cell impermeant and is binding eSrc in vivo.EXAMPLE 8: SMALL MOLECULE T CELL ENGAGERS

[0489] T cell engagers (TCEs) have revolutionized the immunotherapy landscape. However,issues around bispecific antibody production and antibody format have provided several issues. Meanwhile, antibody drug conjugates (ADCs) have found strong purchase in the clinic and the bioconjugation of IgGs and recombinant fragments of antigen binding (Fabs) is comparably well established. Recent work has described use of the small molecule DUPA as a way to target an anti-CD3 Fab DUPA conjugate [Kim, et al. Proc. Natl. Acad. Sci.2013, 110 (44) 17796-17801; Lee, et al. Sci. Adv.2021, 7(33)], resulting in a hybrid architecture leveraging the potency of TCEs in the architecture of an ADC. However, there are a paucity of small molecule ligands to surface proteins. 150Mintz Ref. No.: 048536-797001WO

[0490] The discovery of Src as an inside-out protein greatly broadens the scope of potentialsmall molecule ligands to surface proteins. This provides unprecedented structural access to the cell surface. Therefore, it was proposed to develop small molecule conjugates to anti-CD3 resulting in small molecule directed T cell engagers (SMiTEs) that recruit T cells via the anti- CD3 arm to cancer cells via a small molecule targeting arm, prototypically the Src inhibitor dasatinib.

[0491] We first generated the precedented anti-CD3 clone OKT3 as an Fv-swapped into the4D5 (i.e., trastuzumab) scaffold, resulting in a hybrid anti-CD3 Fab that retains binding affinity to CD3 but is in on a constant domain that we have studied extensively for bioconjugation [Elledge, et al. Proc. Natl. Acad. Sci, 2020117 (11) 5733-5740]. Initially, we first conjugated dasatinib to OKT3 Fv-swap or an irrelevant non-binding Fab in the same scaffold by non-specific lysine (LysX) labeling. OKT3 was first labeled by NHS-ester chemistry using a commercially available bi[6.1.0]cyclononyne derivative, and then later coupled to a previously published azide-functionalized derivative of dasatinib [Li, et al. Bioconj Chem 2016 Oct 19;27(10):2575-2579] by strain-promoted azide alkyne cycloaddition. When these molecules were added to co-cultures of eSrc+ HCC1569 cells with human peripherial blood mononuclear cells (PBMCs) at an effector to target (E:T) ratio of 10:1, we observed dose-dependent killing with OKT3-LysX-dasatinib conjugate but not a CD3-nonbinding isotype-LysX-dasatinib control (Slide 1). Furthermore, testing the OKT3- LysX-Dasatinib on eSrc+ HT-1080 or eSrc- PaTu8902 showed that this SMiTE only induced specific lysis in eSrc+ cell lines (FIG.35).

[0492] Encouraged by preliminary results, we proceeded to generate site-specific conjugatesusing site-specific methionine labeling, as we have previously described for site-specific bioconjugation of Fabs in this scaffold via oxaziridne labeling [Elledge, et al. Proc. Natl. Acad. Sci, 2020117 (11) 5733-5740; Lin et al. Science 2017, 355(6325) 597-602]. We hypothesized that steric accessibility and geometry could impact SMiTE efficacy, as is well precedent in the TCE literature [Santich et al., Sci. Transl. Med.202012]. Therefore, we chose the three most stable Met-labeling sites in domains other than the VH based on our previous study of this scaffold, corresponding to: CH1 T211M, VL G67M, and CL K149M. Each of these sites readily conjugated to oxaziridine-alkyne and then azido-dasatinib via copper-catalyzed azide-alkyne cycloaddition. We then tested the activity of these SMiTEs against multiple cell lines expressing variable levels eSrc (FIGs.36-FIG.37). We observed consistent dose-dependent killing with the OKT3-K149M-dasatinib conjugate across multiple 151Mintz Ref. No.: 048536-797001WO cell lines (FIG.36), and that only OKT3-K149M-dasatinib consistently outperformed an unconjugated OKT3 control at 600 pM (FIG.37). We further confirmed that OKT3-K149M- dasatinib bound target cells via dasatinib by incubating either FLAG-tagged parental OKT3 or OKT3-K149M-dasatinib with eSrc+ HCC1569 cells on ice and visualizing bound Fab with a fluorescent anti-FLAG antibody by flow cytometry (FIG.38).

[0493] To demonstrate that the killing is mediated by the small molecule, we generated asecond conjugate of OKT3-K149M to the PSMA ligand DUPA. We then added one of these two SMiTEs to cocultures of PBMCs with eSrc+ PSMA- HT-1080, eSrc- PSMA+ LNCAP- C4, or eSrc- PSMA- DU-145 (FIGs 39-41). We observed dose-dependent (FIG.39) and subnanomolar (FIG.40) killing only when the cognate surface antigen was expressed (i.e., eSrc+ cells for OKT3-K149M-dasatinib or PSMA+ cells for OKT3-K149M-DUPA). These results indicate that T cell recruitment to target cells is driven by the small molecule ligand and that the specificity of the ligand dictates specificity.

[0494] Based on the observation that the small molecule dictates the target binding, weproposed to leverage the known polypharmacology around dasatinib. Dasatinib is known to potently bind Src family kinases (SFKs) beyond Src. We have previously observed that leukemia and lymphoma cell lines do not express appreciable levels of eSrc, presumably as they express low total Src as they express other SFKs. Therefore, we tested efficacy of the OKT3-K149M-dasatinib SMiTE on eSrc- but known eHck+ HL-60 and Raji cell lines (FIG. 42). We observed potent specific lysis of both of these cell lines, even though an anti-Src bispecific antibody TCE is ineffective against the lines. Therefore, the SMiTE offers a much broader range of target efficacy than a traditional antibody-based TCE. EXAMPLE 9: ANTI-SRC ANTIBODIES DEMONSTRATE SPECIFIC TARGETING OF ESRC

[0495] Anti-Src antibodies in IgG1 format and bispecific format (Src x CD3) were produced.Bio-Layer Interferometry (BLI) assays were performed to evaluate the binding of antibo...

Claims

Mintz Ref. No.: 048536-797001WO WHAT IS CLAIMED IS:

1. An anti-Src antibody or antigen binding fragment thereof comprising:(a) a heavy chain variable region comprising a heavy chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3; and (b) a light chain variable region comprising a light chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:

6.

2. An anti-Src antibody or antigen binding fragment thereof comprising:(a) a heavy chain variable region comprising a heavy chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:

10.

3. An anti-Src antibody or antigen binding fragment thereof comprising:(a) a heavy chain variable region comprising a heavy chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 136, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:

10.

4. An anti-Src antibody or antigen binding fragment thereof comprising:197Mintz Ref. No.: 048536-797001WO (a) a heavy chain variable region comprising a heavy chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 137, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:

10.

5. An anti-Src antibody or antigen binding fragment thereof comprising:(a) a heavy chain variable region comprising a heavy chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 136, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 137, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:

10.

6. The anti-Src antibody according to claim 1, wherein the anti-Src antibody or antigenbinding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

12.

7. The anti-Src antibody according to claim 1, wherein the anti-Src antibody or antigenbinding fragment thereof comprises a heavy chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 34 and a light chain sequence comprising the amino acid sequence set forth in SEQ ID NO:

35.

8. The anti-Src antibody according to claim 2, wherein the anti-Src antibody or antigenbinding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

14. 198Mintz Ref. No.: 048536-797001WO 9. The anti-Src antibody according to claim 1, wherein the anti-Src antibody or antigenbinding fragment thereof comprises a light chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 23 and a heavy chain sequence comprising the amino acid sequence set forth in SEQ ID NO:

21.

10. The anti-Src antibody according to claim 1, wherein the anti-Src antibody or antigenbinding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 40 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

41.

11. The anti-Src antibody according to claim 2, wherein the anti-Src antibody or antigenbinding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 42 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

43.

12. The anti-Src antibody according to claim 3, wherein the anti-Src antibody or antigenbinding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 44 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

45.

13. The anti-Src antibody according to claim 4, wherein the anti-Src antibody or antigenbinding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 46 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

47.

14. The anti-Src antibody according to claim 5, wherein the anti-Src antibody or antigenbinding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 48 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

49.

15. The anti-Src antibody or antigen binding fragment thereof of any one of claims 1-14,wherein the anti-Src antibody or antigen binding fragment thereof comprises the chain 1 or chain 2 sequence of Ab3, Ab10, Ab15, Ab16, Ab17, Ab18, or Ab19. 199Mintz Ref. No.: 048536-797001WO 16. The anti-Src antibody or antigen binding fragment thereof of any one of claims 1-15,comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 97-100.

17. The anti-Src antibody of any preceding claim, wherein the anti-Src antibody or theantigen binding fragment thereof is a monoclonal antibody, synthetic antibody, murine antibody, chimeric antibody, humanized antibody, or human antibody.

18. The anti-Src antibody of any preceding claim, wherein the antibody or fragmentthereof comprises full-length antibodies, Fab, F(ab')2, Fd, Fv, scFv, domain antibodies, dual- specific antibodies, bibodies, minibodies, tribodies, bispecific antibodies, trispecific antibodies, multispecific antibodies, diabodies, triabodies, tetrabodies, intrabodies, nanobodies, small modular immunopharmaceuticals (SMTP), binding-domain immunoglobulin fusion proteins, camelized antibodies or VHH containing antibodies.

19. The anti-Src antibody of any preceding claim, wherein the antibody comprises IgDantibodies, IgE antibodies, IgM antibodies, IgG 1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies.

20. An antibody-drug conjugate comprising a cytotoxic or therapeutic compound that isconjugated to an anti-Src antibody or antigen binding fragment thereof.

21. The antibody-drug conjugate according to claim 20, wherein the anti-Src antibody orantigen binding fragment thereof comprises: (a) a heavy chain variable region comprising a heavy chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3; and (b) a light chain variable region comprising a light chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:

6.

22. The antibody-drug conjugate according to claim 21, wherein the anti-Src antibody orantigen binding fragment thereof comprises a heavy chain variable region comprising the 200Mintz Ref. No.: 048536-797001WO amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

12.

23. The antibody-drug conjugate according to any one of claims 20-22, wherein the anti-Src antibody or antigen binding fragment thereof comprises a heavy chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 34 and a light chain sequence comprising the amino acid sequence set forth in SEQ ID NO:

35.

24. The antibody-drug conjugate according to claim 21, wherein the anti-Src antibody orantigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 40 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

41.

25. The antibody-drug conjugate according to claim 20, wherein the anti-Src antibody orantigen binding fragment thereof comprises: (a) a heavy chain variable region comprising a heavy chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:

10.

26. The antibody-drug conjugate according to claim 25, wherein the anti-Src antibody orantigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

14.

27. The antibody-drug conjugate according to claim 25, wherein the anti-Src antibody orantigen binding fragment thereof comprises a heavy chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 34 and a light chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 35 28. The antibody-drug conjugate according to claim 25, wherein the anti-Src antibody orantigen binding fragment thereof comprises a heavy chain variable region comprising the 201Mintz Ref. No.: 048536-797001WO amino acid sequence set forth in SEQ ID NO: 42 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

43.

29. The antibody-drug conjugate according to claim 20, wherein the anti-Src antibody orantigen binding fragment thereof comprises: (a) a heavy chain variable region comprising a heavy chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 136, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:

10.

30. The antibody-drug conjugate according to claim 29, wherein the anti-Src antibody orantigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 44 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

45.

31. The antibody-drug conjugate according to claim 20, wherein the anti-Src antibody orantigen binding fragment thereof comprises: (a) a heavy chain variable region comprising a heavy chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 137, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:

10.

32. The antibody-drug conjugate according to claim 31, wherein the anti-Src antibody orantigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 46 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

47. 202Mintz Ref. No.: 048536-797001WO 33. The antibody-drug conjugate according to claim 20, wherein the anti-Src antibody orantigen binding fragment thereof comprises: (a) a heavy chain variable region comprising a heavy chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 136, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 137, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:

10.

34. The antibody-drug conjugate according to claim 33, wherein the anti-Src antibody orantigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 48 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

49.

35. The antibody-drug conjugate of any one of claims 20-34, wherein the anti-Srcantibody or antigen binding fragment thereof comprises the chain 1 or chain 2 sequence of Ab3, Ab10, Ab15, Ab16, Ab17, Ab18, or Ab19.

36. The antibody-drug conjugate of any one of claims 20-35, comprising one or moreamino acid sequences selected from the group consisting of SEQ ID NOs: 97-100.

37. The antibody-drug conjugate according to any one of claims 20-35, wherein thecytotoxic compound is selected from a tubulin inhibitor or a topoisomerase inhibitor.

38. The antibody-drug conjugate according to claim 37, wherein the cytotoxic compoundis a tubulin inhibitor.

39. The antibody-drug conjugate according to claim 38, wherein the tubulin inhibitor ismonomethyl auristatin E (MMAE).

40. The antibody-drug conjugate according to claim 37, wherein the cytotoxic compoundis a topoisomerase inhibitor. 203Mintz Ref. No.: 048536-797001WO 41. The antibody-drug conjugate according to claim 40, wherein the topoisomeraseinhibitor is selected from SN38 and Dxd.

42. An antibody-radionuclide conjugate comprising a radionuclide that is conjugated toan anti-Src antibody or antigen binding fragment thereof.

43. The antibody-radionuclide conjugate according to claim 42, wherein the anti-Srcantibody or antigen binding fragment thereof comprises: (a) a heavy chain variable region comprising a heavy chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3; and (b) a light chain variable region comprising a light chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:

6.

44. The antibody-radionuclide conjugate according to claim 43, wherein the anti-Srcantibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

12.

45. The antibody-radionuclide conjugate according to claim 43, wherein the anti-Srcantibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 40 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

41.

46. The antibody-radionuclide conjugate according to any one of claims 43-44, whereinthe anti-Src antibody or antigen binding fragment thereof comprises a heavy chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 34 and a light chain sequence comprising the amino acid sequence set forth in SEQ ID NO:

35.

47. The antibody-radionuclide conjugate according to claim 42, wherein the anti-Srcantibody or antigen binding fragment thereof comprises: (a) a heavy chain variable region comprising a heavy chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the 204Mintz Ref. No.: 048536-797001WO amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:

10.

48. The antibody-radionuclide conjugate according to claim 47, wherein the anti-Srcantibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

14.

49. The antibody-radionuclide conjugate according to claim 47, wherein the anti-Srcantibody or antigen binding fragment thereof comprises a heavy chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 34 and a light chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 35 50. The antibody-radionuclide conjugate according to claim 47, wherein the anti-Srcantibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 42 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

43.

51. The antibody-radionuclide conjugate according to claim 42, wherein the anti-Srcantibody or antigen binding fragment thereof comprises: (a) a heavy chain variable region comprising a heavy chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 136, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:

10.

52. The antibody-radionuclide conjugate according to claim 51, wherein the anti-Srcantibody or antigen binding fragment thereof comprises a heavy chain variable region 205Mintz Ref. No.: 048536-797001WO comprising the amino acid sequence set forth in SEQ ID NO: 44 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

45.

53. The antibody-radionuclide conjugate according to claim 42, wherein the anti-Srcantibody or antigen binding fragment thereof comprises: (a) a heavy chain variable region comprising a heavy chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 137, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:

10.

54. The antibody-radionuclide conjugate according to claim 53, wherein the anti-Srcantibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 46 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

47.

55. The antibody-radionuclide conjugate according to claim 42, wherein the anti-Srcantibody or antigen binding fragment thereof comprises: (a) a heavy chain variable region comprising a heavy chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 136, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 137, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:

10.

56. The antibody-radionuclide conjugate according to claim 55, wherein the anti-Srcantibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 48 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

49. 206Mintz Ref. No.: 048536-797001WO 57. The antibody-radionuclide conjugate according to any one of claims 42-56 whereinthe anti-Src antibody or antigen binding fragment thereof comprises the chain 1 or chain 2 sequence of Ab3, Ab10, Ab15, Ab16, Ab17, Ab18, or Ab19..

58. The antibody-radionuclide conjugate according to any one of claims 42-57,comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 97-100.

59. The antibody-radionuclide conjugate according to any one of claims 42-58, whereinthe radionuclide is a therapeutic radionuclide.

60. The antibody-radionuclide conjugate according to any one of claims 42-58, whereinthe radionuclide is a diagnostic radionuclide.

61. A bispecific T cell engager comprising(a) an anti-Src antibody or antigen binding fragment thereof and (b) a T cell antigen-binding moiety.

62. The bispecific T cell engager according to claim 61, wherein the anti-Src antibody orantigen binding fragment thereof comprises: (a) a heavy chain variable region comprising a heavy chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3; and (b) a light chain variable region comprising a light chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:

6.

63. The bispecific T cell engager according to claim 62, wherein the anti-Src antibody orantigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

12. 207Mintz Ref. No.: 048536-797001WO 64. The bispecific T cell engager according to any one of claims 62-63, wherein the anti-Src antibody or antigen binding fragment thereof comprises a heavy chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 34 and a light chain sequence comprising the amino acid sequence set forth in SEQ ID NO:

35.

65. The bispecific T cell engager according to claim 62, wherein the anti-Src antibody orantigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 40 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

41.

66. The bispecific T cell engager according to claim 61, wherein the anti-Src antibody orantigen binding fragment thereof comprises: (a) a heavy chain variable region comprising a heavy chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:

10.

67. The bispecific T cell engager according to claim 66, wherein the anti-Src antibody orantigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

14.

68. The bispecific T cell engager according to any one of claims 66-67, wherein the anti-Src antibody or antigen binding fragment thereof comprises a heavy chain sequence comprising the amino acid sequence set forth in SEQ ID NO: 34 and a light chain sequence comprising the amino acid sequence set forth in SEQ ID NO:

35.

69. The bispecific T cell engager according to claim 66, wherein the anti-Src antibody orantigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 42 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

43. 208Mintz Ref. No.: 048536-797001WO 70. The bispecific T cell engager according to claim 61, wherein the anti-Src antibody orantigen binding fragment thereof comprises: (a) a heavy chain variable region comprising a heavy chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 136, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:

10.

71. The bispecific T cell engager according to claim 70, wherein the anti-Src antibody orantigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 44 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

45.

72. The bispecific T cell engager according to claim 61, wherein the anti-Src antibody orantigen binding fragment thereof comprises: (a) a heavy chain variable region comprising a heavy chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 137, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:

10.

73. The bispecific T cell engager according to claim 72, wherein the anti-Src antibody orantigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 46 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

47.

74. The bispecific T cell engager according to claim 61, wherein the anti-Src antibody orantigen binding fragment thereof comprises: 209Mintz Ref. No.: 048536-797001WO (a) a heavy chain variable region comprising a heavy chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 136, and a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region comprising a light chain CDRl domain comprising the amino acid sequence of SEQ ID NO: 137, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:

10.

75. The bispecific T cell engager according to claim 74, wherein the anti-Src antibody orantigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 48 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

49.

76. The bispecific T cell engager according to any one of claims 61-75, wherein the anti-Src antibody or antigen binding fragment thereof comprises the chain 1 or chain 2 sequence of Ab3, Ab10, Ab15, Ab16, Ab17, Ab18, or Ab19.

77. The bispecific T cell engager according to any one of claims 61-76, comprising one ormore amino acid sequences selected from the group consisting of SEQ ID NOs: 97-100.

78. The bispecific T cell engager according to any one of claims 61-77, wherein the T cellantigen-binding moiety binds to CD3.

79. A conjugate compound comprising:(a) a Src ligand and (b) a cytotoxic or therapeutic compound.

80. The conjugate compound according to claim 79, wherein the conjugate compound iscell impermeable.

81. The conjugate compound according to claim 79, wherein the Src ligand is a smallmolecule. 210Mintz Ref. No.: 048536-797001WO 82. The conjugate compound according to claim 81, wherein the small molecule isdasatinib, sarcatinib, a DFG-out selective derivative, or a covalent derivative of those molecules.

83. The conjugate compound according to claim 81, wherein the small molecule isazidodasatinib.

84. The conjugate compound according to any one of claims 79-83, wherein the cytotoxiccompound is selected from a tubulin inhibitor or a topoisomerase inhibitor.

85. The conjugate compound according to claim 84, wherein the cytotoxic compound is atubulin inhibitor.

86. The conjugate compound according to claim 85, wherein the tubulin inhibitor ismonomethyl auristatin E (MMAE).

87. The conjugate compound according to claim 84, wherein the cytotoxic compound is atopoisomerase inhibitor.

88. The antibody-drug conjugate according to claim 87, wherein the topoisomeraseinhibitor is selected from SN38 and Dxd.

89. A conjugate compound comprising:(a) a Src ligand and (b) a radionuclide.

90. The conjugate compound according to claim 89, wherein the conjugate compound iscell impermeable.

91. The conjugate compound according to claim 89, wherein the Src ligand is a smallmolecule.

92. The conjugate compound according to claim 91, wherein the small molecule isdasatinib, sarcatinib, a DFG-out selective derivative, or a covalent derivative of those molecules. 211Mintz Ref. No.: 048536-797001WO 93. The conjugate compound according to claim 91, wherein the small molecule isazidodasatinib.

94. The conjugate compound according to any one of claims 89-93, wherein theradionuclide is a therapeutic radionuclide.

95. The conjugate compound according to any one of claims 89-93, wherein theradionuclide is a diagnostic radionuclide.

96. A compound represented by of Formula (I):(I), or a pharmacewherein: ,[ ] denotes that the group is optional; 212Mintz Ref. No.: 048536-797001WO and A is a chelating agent capable of chelating a radionuclide.

97. The compound of claim 96, wherei .

98. The compound of claim 96, wherein R1 .

99. The compound of claim 96, wherei .

100. The compound of claim 96, wherei .

101. The compound of any one of claims 96-100, wherein the linker comprises C1-12alkyl, polyethylene glycol (PEG), or an amino acid or an analog thereof.

102. The compound of any one of claims 96-101, wherein the linker comprises(PEG)3, (PEG)6, (PEG)12, C1-12 alkyl, or an amino acid. 213Mintz Ref. No.: 048536-797001WO103. The compound of any one of claims 96-102, wherein the linker comprisespolyethylene glycol (PEG).

104. The compound of any one of claims 96-102, wherein the linker comprisesglycine (Gly), lysine (Lys), aspartic acid (Asp), or serine (Ser).

105. The compound of any one of claims 96-102, wherein the linker comprisesAsp-Gly-Gly-Ser-Gly.

106. The compound of any one of claims 96-105, wherein the HSA-binder.

107. The compound of any one of claims 96-106, wherein the HSA-binder.1007, wherein A comprises DOTA orDOTAGA.

109. The compound of any one of claims 96-108, wherein A comprises DOTA.

110. The compound of claim 96, wherein the compound is selected from the groupconsisting of 214Mintz Ref. No.: 048536-797001WO , ,215Mintz Ref. No.: 048536-797001WO ,216Mintz Ref. No.: 048536-797001WO , , ,Mintz Ref. No.: 048536-797001WO , ,Mintz Ref. No.: 048536-797001WO nd .. , e compound isconjugated to a radionuclide.

112. The compound of claim 111, wherein the radionuclide is 64Cu or 177Lu.

113. A bifunctional molecule comprising(a) a Src ligand and (b) a T cell antigen-binding moiety.

114. The bifunctional molecule according to claim 113, wherein the conjugatecompound is cell impermeable.

115. The bifunctional molecule according to claim 113, wherein the Src ligand is asmall molecule. 219Mintz Ref. No.: 048536-797001WO 116. The bifunctional molecule according to claim 115, wherein the small moleculeis dasatinib, sarcatinib, a DFG-out selective derivative, or a covalent derivative of those molecules.

117. The bifunctional molecule according to claim 115, wherein the small moleculeis azidodasatinib.

118. The bifunctional molecule according to any one of claims 113-117, whereinthe T cell antigen binding moiety binds to CD3.

119. The bifunctional molecule according to claim 118, wherein the T cell antigenbinding moiety is an anti-CD3 engaging Fab.

120. The bifunctional molecule according to claim 119, wherein the anti-CD3 Fabis a cyclooctyne-conjugated anti-CD3 engaging Fab.

121. A pharmaceutical composition comprising the anti-Src antibody or antigenbinding fragment thereof, antibody-drug conjugate, antibody-radionuclide conjugate, bispecific T cell engager, conjugate compound, or bifunctional molecule of any preceding claim.

122. A nucleic acid encoding the anti-Src antibody or antigen binding fragmentthereof according to any one of claims 1-19.

123. A vector comprising the nucleic acid according to claim 122.

124. A host cell comprising the nucleic acid according to claim 122.

125. A method of treating cancer in a subject comprising:administering, to the subject, the anti-Src antibody or antigen binding fragment thereof, antibody-drug conjugate, antibody-radionuclide conjugate, bispecific T cell engager, conjugate compound, or bifunctional molecule of any preceding claim. 220Mintz Ref. No.: 048536-797001WO 126. The method according to claim 125, wherein the cancer is selected from breastcancer, lung cancer, fibrosarcoma, melanoma, prostate cancer, colon cancer, pancreatic cancer.

127. A method of inhibiting or reducing cancer cell growth comprising contactingthe cancer cell with the anti-Src antibody or antigen binding fragment thereof, antibody-drug conjugate, antibody-radionuclide conjugate, bispecific T cell engager, conjugate compound, or bifunctional molecule of any preceding claim.

128. A method of inhibiting or reducing cancer cell growth comprising contactingthe cancer cell with a biological molecule that specifically binds a kinase selected from the group consisting of Src, Hck, and Lyn, wherein at least a portion of the kinase is located on the cell surface.

129. The method of claim 128, wherein the biological molecule is a smallmolecule.

130. The method of claim 128, wherein the biological molecule is an antibody orantigen binding fragment thereof.

131. The method of claim 130, wherein the antibody or the antigen bindingfragment thereof is a monoclonal antibody, synthetic antibody, murine antibody, chimeric antibody, humanized antibody, or human antibody.

132. The method of any one of claims 130-131, wherein the antibody or fragmentthereof comprises full-length antibodies, Fab, F(ab')2, Fd, Fv, scFv, domain antibodies, dual- specific antibodies, bibodies, minibodies, tribodies, bispecific antibodies, trispecific antibodies, multispecific antibodies, diabodies, triabodies, tetrabodies, intrabodies, nanobodies, small modular immunopharmaceuticals (SMTP), binding-domain immunoglobulin fusion proteins, camelized antibodies or VHH containing antibodies.

133. The method of any one of claims 130-132, wherein the antibody comprisesIgD antibodies, IgE antibodies, IgM antibodies, IgG 1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies. 221Mintz Ref. No.: 048536-797001WO 134. A method of inhibiting or reducing cancer cell growth comprising contactingthe cancer cell with a conjugate comprising a cytotoxic, therapeutic, or diagnostic compound that is conjugated a biological molecule, wherein the biological molecule specifically binds a kinase of the Src kinase family selected from the group consisting of Src, Hck, and Lyn, wherein at least a portion of the kinase is located on the cell surface.

135. The method of claim 134, wherein the cytotoxic compound is a tubulininhibitor or a topoisomerase inhibitor.

136. The method of claim 135, wherein the cytotoxic compound is a tubulininhibitor.

137. The method of claim 136, wherein the tubulin inhibitor is monomethylauristatin E (MMAE).

138. The method of claim 135, wherein the cytotoxic compound is a topoisomeraseinhibitor.

139. The method of claim 138, wherein the topoisomerase inhibitor is selectedfrom SN38 and Dxd.

140. The method of claim 134, wherein the therapeutic compound is a therapeuticradionuclide.

141. The method of claim 134, wherein the diagnostic compound is a diagnosticradionuclide.

142. The method of any one of claims 134-141, wherein the biological molecule isa small molecule.

143. The method of any one of claims 134-142, wherein the conjugate is cellimpermeable.

144. The method of any one of claims 142-143, wherein the small molecule isselected from the group consisting of AZD0530 (saracatinib), Bosulif (vosutinib), ENMD981693, KD020, KX0l, dasatinib, Yervoy (ipilimumab), AP23464, AP23485, AP23588, AZD0424, KISSEI, CU201, KX2361, SKS927, SRN004, SUNK706, TG100435, 222Mintz Ref. No.: 048536-797001WO TG100948, AP23451, KINEX, VX680 (tozasertib lactate), XL228, VAL201, PUR1800, TOP1288, NXP900, VAL301, ASN006, CR13626, MLR-1023, and bafetinib.

145. The method of claim 144, wherein the small molecule is dasatinib, saracatinib,a DFG-out selective derivative, or a covalent derivative of those molecules.

146. The method of claim 145, wherein the small molecule is azidodasatinib.

147. A method of inhibiting or reducing cancer cell growth comprising contactingthe cancer cell with a bispecific T cell engager comprising a biological molecule and a T cell antigen-binding moiety, wherein the biological molecule specifically binds a kinase of the Src kinase family selected from the group consisting of Src, Hck, and Lyn, wherein at least a portion of the kinase is located on the cell surface.

148. The method of claim 147, wherein the T cell antigen-binding moiety binds toCD3.

149. A method of inhibiting or reducing cancer cell growth comprising contactingthe cancer cell with a bifunctional molecule comprising a biological molecule and a T cell antigen-binding moiety, wherein the biological molecule specifically binds a kinase of the Src kinase family selected from the group consisting of Src, Hck, and Lyn, wherein at least a portion of the kinase is located on the cell surface.

150. The method of claim 149, wherein the bifunctional molecule is cellimpermeable.

151. The method of any one of claims 149-150, wherein the biological molecule isa small molecule.

152. The method of claim 151, wherein the small molecule is dasatinib, saracatinib,a DFG-out selective derivative, or a covalent derivative of those molecules.

153. The method of claim 152, wherein the small molecule is azidodasatinib.223Mintz Ref. No.: 048536-797001WO 154. The method of any one of claims 149-153, wherein the T cell antigen bindingmoiety binds to CD3.

155. The method of claim 154, wherein the T cell antigen binding moiety is an anti-CD3 engaging Fab.

156. The method of claim 155, wherein the anti-CD3 Fab is a cyclooctyne-conjugated anti-CD3 engaging Fab.

157. A biological molecule that specifically binds a kinase of the Src kinase family,wherein the kinase is selected from the group consisting of Src, Hck, and Lyn, wherein at least a portion of the kinase is located on the cell surface.

158. The biological molecule of claim 157, wherein the biological molecule is asmall molecule.

159. The biological molecule of claim 157, wherein the biological molecule is anantibody or antigen binding fragment thereof.

160. The biological molecule of claim 159, wherein the antibody or the antigenbinding fragment thereof is a monoclonal antibody, synthetic antibody, murine antibody, chimeric antibody, humanized antibody, or human antibody.

161. The biological molecule of any one of claims 159-160, wherein the antibodyor fragment thereof comprises full-length antibodies, Fab, F(ab')2, Fd, Fv, scFv, domain antibodies, dual-specific antibodies, bibodies, minibodies, tribodies, bispecific antibodies, trispecific antibodies, multispecific antibodies, diabodies, triabodies, tetrabodies, intrabodies, nanobodies, small modular immunopharmaceuticals (SMTP), binding-domain immunoglobulin fusion proteins, camelized antibodies or VHH containing antibodies.

162. The biological molecule of any one of claims 159-161, wherein the antibodycomprises IgD antibodies, IgE antibodies, IgM antibodies, IgG 1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies. 224Mintz Ref. No.: 048536-797001WO 163. A conjugate comprising a cytotoxic, therapeutic, or diagnostic compound thatis conjugated to the biological molecule any one of claims 157-162.

164. The conjugate of claim 163, wherein the cytotoxic compound is a tubulininhibitor or a topoisomerase inhibitor.

165. The conjugate of claim 164, wherein the cytotoxic compound is a tubulininhibitor.

166. The conjugate of claim 165, wherein the tubulin inhibitor is monomethylauristatin E (MMAE).

167. The conjugate of claim 164, wherein the cytotoxic compound is atopoisomerase inhibitor.

168. The conjugate of claim 167, wherein the topoisomerase inhibitor is selectedfrom SN38 and Dxd.

169. The conjugate of claim 163, wherein the therapeutic compound is atherapeutic radionuclide.

170. The conjugate of claim 163, wherein the diagnostic compound is a diagnosticradionuclide.

171. The conjugate of any one of claims 163-170, wherein the biological moleculeis a small molecule.

172. The conjugate of any one of claims 163-171, wherein the conjugate is cellimpermeable.

173. The conjugate of any one of claims 171-172, wherein the small molecule isselected from the group consisting of AZD0530 (saracatinib), Bosulif (vosutinib), 225Mintz Ref. No.: 048536-797001WO ENMD981693, KD020, KX0l, dasatinib, Yervoy (ipilimumab), AP23464, AP23485, AP23588, AZD0424, KISSEI, CU201, KX2361, SKS927, SRN004, SUNK706, TG100435, TG100948, AP23451, KINEX, VX680 (tozasertib lactate), XL228, VAL201, PUR1800, TOP1288, NXP900, VAL301, ASN006, CR13626, MLR-1023, and bafetinib.

174. The conjugate of claim 173, wherein the small molecule is dasatinib,saracatinib, a DFG-out selective derivative, or a covalent derivative of those molecules.

175. The conjugate according to claim 174, wherein the small molecule isazidodasatinib.

176. A bispecific T cell engager comprising(a) the biological molecule any one of claims 157-162, and (b) a T cell antigen-binding moiety.

177. The bispecific T cell engager of claim 176, wherein the T cell antigen-bindingmoiety binds to CD3.

178. A bifunctional molecule comprising(a) the biological molecule any one of claims 157-162, and (b) a T cell antigen-binding moiety.

179. The bifunctional molecule of claim 178, wherein the bifunctional molecule iscell impermeable.

180. The bifunctional molecule of any one of claims 178-179, wherein thebiological molecule is a small molecule.

181. The bifunctional molecule of claim 180, wherein the small molecule isdasatinib, saracatinib, a DFG-out selective derivative, or a covalent derivative of those molecules. 226Mintz Ref. No.: 048536-797001WO 182. The bifunctional molecule of claim 181, wherein the small molecule isazidodasatinib.

183. The bifunctional molecule of any one of claims 178-182, wherein the T cellantigen binding moiety binds to CD3.

184. The bifunctional molecule of claim 183, wherein the T cell antigen bindingmoiety is an anti-CD3 engaging Fab.

185. The bifunctional molecule of claim 184, wherein the anti-CD3 Fab is acyclooctyne-conjugated anti-CD3 engaging Fab.

186. A pharmaceutical composition comprising the biological molecule, conjugate,bispecific T cell engager, or bifunctional molecule of any one of claims 157-185.

187. A nucleic acid encoding the biological molecule of any one of claims 157-162.

188. A vector comprising the nucleic acid of claim 187.

189. A host cell comprising the nucleic acid of claim 188.

190. A method of inhibiting or reducing cancer cell growth comprising contactingthe cancer cell with the biological molecule, conjugate, compound, bispecific T cell engager, or bifunctional molecule of any one of claims 96-112 and 157-185.

191. A method of treating cancer in a subject comprising: administering, to thesubject, the biological molecule, conjugate, conjugate, bispecific T cell engager, or bifunctional molecule of any one of claims 96-112 and 157-185.

192. The method of claim 191, wherein the cancer is selected from breast cancer,lung cancer, fibrosarcoma, melanoma, prostate cancer, colon cancer, pancreatic cancer. 227

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