CDCP1-targeted therapies

Abstract

The present disclosure provides materials and methods for CUB domain-containing protein 1 (CDCP1) targeted therapy.

Description

Technical Field

[0001] The present disclosure relates to materials and methods for CUB domain-containing protein 1 (CDCP1) targeted therapy.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 758,442, filed on November 9, 2018, the entire contents of which are incorporated herein.

[0004] Parties to the Joint Research Statement

[0005] The presently claimed invention was made in part by or on behalf of the parties listed below under a joint research agreement that was in effect on or before the date the claimed invention was made, and the claimed invention was made in part as a result of activities performed within the scope of the joint research agreement. The parties to the joint research agreement are Beth Israel Deacones Medical Center and Pfizer Inc.

[0006] Description of the text file submitted electronically

[0007] This application contains a sequence listing, which is submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created around October 30, 2019, is named "BID-009PC_ST25.txt" and is approximately 126 KB in size. Background Art

[0008] Although many chemotherapeutic agents have been developed, they generally show unacceptable toxicity and or relative to non-cancer tissues, lack specificity to cancer cells. In order to avoid the nonspecific cytotoxic effects of chemotherapeutic agents, targeted antibody therapy has completely changed cancer treatment with several monoclonal antibodies showing clinical potential. Due to the lack of therapeutic activity of antibodies for tumor-specific antigens, they have been conjugated with cytotoxic agents, so that the effectiveness of chemotherapy and the targeted combination of antibodies. In principle, the systemic toxicity of traditional small molecule chemotherapeutic agents should be reduced by antibody binding to cytotoxic agents selectively delivered to specific tumor tissues. Due to successful antibody drug conjugates (ADC) method must be successfully bonded to target antigens so that toxic payload is delivered to target cells and not significantly bonded to non-target cells, it is crucial that ADC can deliver toxic payload to target cells, thus internalized, then once just release the payload inside the appropriate compartment in the cell. Summary of the Invention

[0009] The present disclosure particularly meets the aforementioned needs. CDCP1 is a target for therapeutic intervention in patients with various cancers, particularly cancers that are addicted to CDCP1 expression. Interestingly, the inventors of the present invention have found that CDCP1 is internalized by cells in a regulated manner, and therefore, this property can be used to develop anti-cancer therapies. In addition, the inventors have found that CDCP1 is affected by or affects various markers associated with cancer (including but not limited to LKB1, KRAS and AKT), thereby providing new treatment modalities using CDCP1 agents (including but not limited to antibodies that specifically bind to CDCP1 expressed on cells). Furthermore, the inventors have discovered interactions between CDCP1 and cancer markers including Src, PPP4R2 and PARG1, such interactions being activation state dependent and allowing specific cancer treatment.

[0010] In one aspect, the present disclosure provides a method for treating cancer in a patient in need thereof, the method comprising: (a) assessing the amount of mutant LKB1 and / or KRAS in a tumor sample; and (b) administering an agent that binds to CUB domain-containing protein 1 (CDCP1) to the cancer patient if the amount of mutant LKB1 and / or KRAS is higher than a reference sample.

[0011] In some embodiments, the tumor sample is a biopsy selected from the group consisting of a frozen tumor tissue specimen, cultured cells, circulating tumor cells, and a formalin-fixed paraffin-embedded tumor tissue specimen.

[0012] In some embodiments, the mutant KRAS is selected from G12C; G12A; G12D; G12R; G12S; G12V; G13C; and G13D mutants. In some embodiments, the assessment is performed by amplifying LKB1 and / or KRAS nucleic acid or a fragment suspected of containing a mutation from the tumor sample, and sequencing the amplified nucleic acid. In some embodiments, the assessment is performed by contacting an antibody or a form thereof against LKB1 and / or KRAS with the tumor sample, and quantifying the binding of the antibody or form thereof. In one aspect, the present disclosure provides a method for treating lung cancer in a patient in need thereof, the method comprising administering to the patient an agent that binds to CDCP1, wherein the lung cancer is characterized by AKT activation, and the agent that binds to CDCP1 is a CDCP1 activator.

[0013] In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC).In some embodiments, the method further comprises assessing AKT activation in the sample of the lung cancer.

[0014] In one aspect, the present disclosure provides a method of treating prostate cancer in a patient in need thereof, the method comprising administering to the patient an agent that binds to CDCP1, wherein the prostate cancer is characterized by AKT activation and the agent that binds to CDCP1 is a CDCP1 activator. In some embodiments, the method further comprises assessing AKT activation in a sample of the prostate cancer.

[0015] In some embodiments, the method further comprises administering an AKT inhibitor. In some embodiments, the patient is undergoing treatment with an AKT inhibitor. In some embodiments, the AKT inhibitor is selected from apremilast, ARQ 751, ARQ 092, AZD5363, BAY1125976, GSK2141795, GSK690693, etanercept, LY2780301, MK2206, and perifosine. In one aspect, the present disclosure provides a method for treating cancer in a patient in need thereof, the method comprising: (a) selecting an agent that binds to CDCP1 on a target cell and is internalized when it contacts CDCP1 on the target cell; and (b) administering the agent to the cancer patient.

[0016] In one aspect, the present disclosure provides a method for treating cancer in a patient in need thereof, the method comprising: (a) selecting an agent that binds to CDCP1 on a target cell and is internalized when it contacts CDCP1 on the target cell; and (b) administering the agent to the cancer patient, wherein the agent that binds to CDCP1 is an antibody that activates CDCP1 and is conjugated to a serine / threonine protein phosphatase 4 regulatory subunit 2 (PPP4R2) regulator.

[0017] In one aspect, the present disclosure provides a method for treating cancer in a patient in need thereof, the method comprising: (a) administering an agent that binds to CDCP1, wherein the agent that binds to CDCP1 is an antibody that does not activate CDCP1; and (b) administering an agent that regulates poly (ADP-ribose) glycohydrolase (PARG). In some embodiments, the agent that regulates PARG is a PARG inhibitor. In some embodiments, the PARG inhibitor is selected from olaparib, talazoparib, veliparib, rucaparib, iniparib, niraparib E7016, CEP9722, BGB-290, and 3-aminobenzamide.

[0018] In some embodiments, the agent that binds to CDCP1 is an antibody or an antigen-binding portion thereof that is specific for CDCP1.

[0019] In some embodiments, the antibody or antigen-binding portion thereof that is specific for CDCP1 is selected from one or more of the following: a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab′, Fab′-SH, F(ab′)2, Fv, a single-chain Fv, a diabody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, and a fusion protein comprising an antigen-binding portion of an antibody.

[0020] In some embodiments, the antibody or antigen binding portion thereof that is specific for CDCP1 is conjugated to a cytotoxic agent or a cytostatic agent. In some embodiments, the method further comprises administering the cytotoxic agent or cytostatic agent. In some embodiments, the administration is sequential or simultaneous.

[0021] In some embodiments, the cytotoxic agent is selected from the group consisting of taxol (paclitaxel), ricin, Pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenotoposide, colchicine, dihydroxyanthracenedione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, and mixtures thereof.

[0022] In some embodiments, the cytotoxic agent is an anti-tumor agent selected from the group consisting of methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine; alkylating agents such as mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea, cyclophosphamide, mechlorethamine, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum. (II) (DDP) cisplatin and carboplatin (Biltin); anthracyclines including daunorubicin, doxorubicin (adriamycin), detoxrubicin, carminomycin, idarubicin, epirubicin, mitoxantrone, and bisantrene; antibiotics including dactinomycin (actinomycin D), bleomycin, calicheamicin, mithramycin, and anthramycin (AMC); and antimitotics such as vinca alkaloids, vincristine, and vinblastine, and mixtures thereof. In some embodiments, the method further comprises administering the antitumor agent. In some embodiments, the administration is sequential or simultaneous. In some embodiments, the antitumor agent is a chemotherapeutic agent.

[0023] In some embodiments, the anti-tumor agent is a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is an agent targeting one of the following: TIM-3, BTLA, PD-1, CTLA-4, B7-H4, GITR, Galectin-9, HVEM, PD-L1, PD-L2, B7-H3, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, and TMIGD2. In some embodiments, the agent targeting PD-1 is an antibody or antigen-binding portion thereof that is specific for PD-1, optionally selected from nivolumab, pembrolizumab, and pidilizumab. In some embodiments, the agent targeting PD-L1 is an antibody or antigen-binding portion thereof that is specific for PD-L1, optionally selected from atezolizumab, avelumab, durvalumab, and BMS-936559. In some embodiments, the agent that targets CTLA-4 is an antibody or antigen-binding portion thereof specific for CTLA-4, optionally selected from ipilimumab and tremelimumab.

[0024] In some embodiments, the anti-tumor agent is a hypoxia-inducible factor-2 (HIF-2) inhibitor. In some embodiments, the HIF-2 inhibitor is selected from PT2385 and PT2977. In some embodiments, the anti-tumor agent is not a Src inhibitor, and the Src inhibitor is optionally selected from KX2-391, bosutinib, saracatinib, and dasatinib.

[0025] In some embodiments, the cancer is a tumor characterized by hypoxia.

[0026] In some embodiments, the cancer is selected from one or more of the following: basal cell carcinoma; biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatoma; intraepithelial neoplasia; kidney cancer or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; myeloma; neuroblastoma; oral cancer (lips, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urological cancer; vulvar cancer; lymphomas, including Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other cancers and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with keloids, edema (e.g., associated with brain tumors), and Meigs' syndrome.

[0027] In some embodiments, the cancer is head and neck cancer.

[0028] In one aspect, the present disclosure provides a method of determining whether a tumor will respond to treatment with an agent that binds to CDCP1, the method comprising determining the presence, absence, or amount of mutant LKB1 and / or KRAS protein or gene in a sample of the tumor, whereby an increase in the presence of mutant LKB1 and / or KRAS or the amount of mutant LKB1 and / or KRAS protein or gene relative to a reference sample indicates a likelihood of response to treatment with an agent that binds to CDCP1.

[0029] In some aspects, the present invention provides antibodies and antigen-binding fragments thereof that specifically bind to CDCP1, antibody drug conjugates comprising such antibodies, and uses and related methods thereof. Those skilled in the art will recognize or be able to ascertain many equivalents to the specific embodiments of the invention described herein using only routine experimentation. Such equivalents are intended to encompass the following embodiment (E).

[0030] E1. An isolated antibody or antigen-binding fragment thereof that specifically binds to CDCP1, the isolated antibody or antigen-binding fragment thereof comprising:

[0031] (i) a heavy chain variable region (VH), the heavy chain variable region comprising:

[0032] (a) VH complementarity determining region 1 (CDRH1), said VH complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 2,

[0033] (b) VH complementarity determining region 2 (CDRH2), said VH complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 3, and

[0034] (c) a VH complementarity determining region 3 (CDRH3), said VH complementarity determining region 3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 40 and SEQ ID NO: 45,

[0035] and (ii) a light chain variable region (VL), said light chain variable region comprising:

[0036] (a) VL complementarity determining region 1 (CDRL1), said VL complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 12,

[0037] (b) VL complementarity determining region 2 (CDRL2), said VL complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO: 13, and

[0038] (c) a VL complementarity determining region 3 (CDRL3), the VL complementarity determining region 3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 31.

[0039] E2. An isolated antibody or antigen-binding fragment thereof that specifically binds to CDCP1, the isolated antibody or antigen-binding fragment thereof comprising:

[0040] (i) a VH comprising:

[0041] (a) CDRH1 comprising the amino acid sequence of SEQ ID NO: 2,

[0042] (b) CDRH2 comprising the amino acid sequence of SEQ ID NO: 3; and

[0043] (c) CDRH3 comprising the amino acid sequence of SEQ ID NO: 27;

[0044] and (ii) a VL comprising:

[0045] (a) CDRL1 comprising the amino acid sequence of SEQ ID NO: 12,

[0046] (b) CDRL2 comprising the amino acid sequence of SEQ ID NO: 13; and

[0047] (c) CDRL3 comprising the amino acid sequence of SEQ ID NO: 31.

[0048] E3. An isolated antibody or antigen-binding fragment thereof as described in any of E1-E2, wherein the isolated antibody or antigen-binding fragment thereof comprises a VH, wherein the VH comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 26, SEQ ID NO: 39 or SEQ ID NO: 44.

[0049] E4. An isolated antibody or antigen-binding fragment thereof as described in any one of E1 to E3, wherein the isolated antibody or antigen-binding fragment thereof comprises a VL, wherein the VL comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 30, SEQ ID NO: 36 or SEQ ID NO: 11.

[0050] E5. The isolated antibody or antigen-binding fragment thereof of any one of E1 to E4, comprising a VH comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 26; and a VL comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 36.

[0051] E6. The isolated antibody or antigen-binding fragment thereof as described in any one of E1-E5, wherein the isolated antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 33; and a VL comprising the amino acid sequence of SEQ ID NO: 38.

[0052] E7. The isolated antibody or antigen-binding fragment thereof as described in any one of E1-E5, wherein the isolated antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 26; and a VL comprising the amino acid sequence of SEQ ID NO: 36.

[0053] E8. The isolated antibody or antigen-binding fragment thereof of any one of E1 to E5, comprising a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 30.

[0054] E9. The isolated antibody or antigen-binding fragment thereof as described in any one of E1-E4, wherein the isolated antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 39; and a VL comprising the amino acid sequence of SEQ ID NO: 36.

[0055] E10. The isolated antibody or antigen-binding fragment thereof as described in any one of E1 to E4, wherein the isolated antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 44; and a VL comprising the amino acid sequence of SEQ ID NO: 11.

[0056] E11. The isolated antibody or antigen-binding fragment thereof as described in any one of E1-E4, wherein the isolated antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 1; and a VL comprising the amino acid sequence of SEQ ID NO: 11.

[0057] E12. The antibody or antigen-binding fragment thereof as described in any one of E1 to E11, wherein the antibody or antigen-binding fragment thereof comprises an Fc domain.

[0058] E13. The antibody or antigen-binding fragment thereof as described in E12, wherein the Fc domain is the Fc domain of IgA, IgD, IgE, IgM or IgG.

[0059] E14. The antibody or antigen-binding fragment thereof as described in E13, wherein the Fc domain is an IgG Fc domain.

[0060] E15. The antibody or antigen-binding fragment thereof as described in E14, wherein the IgG is selected from the group consisting of: IgG1, IgG2, IgG3 or IgG4.

[0061] E16. The antibody or antigen-binding fragment thereof as described in E15, wherein the IgG is IgG1.

[0062] E17. An isolated antibody or antigen-binding fragment thereof as described in any one of E1 to E16, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10, 29, 41 or 46.

[0063] E18. An isolated antibody or antigen-binding fragment thereof as described in any one of E1 to E17, wherein the isolated antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 17, 32 or 37.

[0064] E19. The isolated antibody or antigen-binding fragment thereof of any one of E1 to E18, comprising a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID No: 29; and a light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 37.

[0065] E20. The isolated antibody or antigen-binding fragment thereof as described in any one of E1-E19, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29; and a light chain comprising the amino acid sequence of SEQ ID NO: 37.

[0066] E21. The isolated antibody or antigen-binding fragment thereof as described in any one of E1-E19, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29; and a light chain comprising the amino acid sequence of SEQ ID NO: 32.

[0067] E22. The isolated antibody or antigen-binding fragment thereof of any one of E1 to E18, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and a light chain comprising the amino acid sequence of SEQ ID NO: 37.

[0068] E23. The isolated antibody or antigen-binding fragment thereof as described in any one of E1-E18, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 46; and a light chain comprising the amino acid sequence of SEQ ID NO: 17.

[0069] E24. The isolated antibody or antigen-binding fragment thereof as described in any one of E1-E18, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 10; and a light chain comprising the amino acid sequence of SEQ ID NO: 17.

[0070] E25. An isolated antibody or antigen-binding fragment thereof that binds to an epitope on CDCP1, wherein the epitope comprises at least one amino acid residue selected from the group consisting of Thr124, Thr160, Ser162, Ala195, Leu196 and His197, according to the numbering of SEQ ID NO: 90.

[0071] E26. The isolated antibody or antigen-binding fragment thereof as described in E25, wherein the epitope further comprises at least one amino acid residue selected from the group consisting of: Lys45, Leu46, Gly47, Thr48, Pro49, Thr50, Ala53, Pro55, Glu92, Arg173 and Glu242, according to the numbering of SEQ ID NO: 90.

[0072] E27. The isolated antibody or antigen-binding fragment thereof as described in any one of E24-E25, wherein the epitope further comprises at least one amino acid residue selected from the group consisting of: Thr56, Tyr57, Thr66, Met67, Ile126, Val171, Arg173, according to the numbering of SEQ ID NO: 90.

[0073] E28. The isolated antibody or antigen-binding fragment thereof as described in any one of E25-E27, wherein the epitope further comprises a glycan linked to Asn122 according to the numbering of SEQ ID NO: 90.

[0074] E29. The isolated antibody or antigen-binding fragment thereof of any one of E25 to E28, wherein the antibody or antigen-binding fragment thereof comprises:

[0075] (i) a VH comprising:

[0076] (a) CDRH1 comprising the amino acid sequence of SEQ ID NO: 2

[0077] (b) CDRH2 comprising the amino acid sequence of SEQ ID NO: 3; and

[0078] (c) a CDRH3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 40, and SEQ ID NO: 45;

[0079] and (ii) a VL comprising:

[0080] (a) CDRL1 comprising the amino acid sequence of SEQ ID NO: 12,

[0081] (b) CDRL2 comprising the amino acid sequence of SEQ ID NO: 13; and

[0082] (c) CDRL3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 31.

[0083] E30. The isolated antibody or antigen-binding fragment thereof of any one of E25 to E29, wherein the antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 26; and a VL comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 36.

[0084] E31. The isolated antibody or antigen-binding fragment thereof as described in any one of E25-E30, wherein the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 26; and a VL comprising the amino acid sequence of SEQ ID NO: 36.

[0085] E32. The isolated antibody or antigen-binding fragment thereof of any one of E25 to E31, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID No: 29; and a light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 37.

[0086] E33. The isolated antibody or antigen-binding fragment thereof as described in any one of E25-E32, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29; and a light chain comprising the amino acid sequence of SEQ ID NO: 37.

[0087] E34. An isolated antibody or antigen-binding fragment thereof, which competes with the antibody or antigen-binding fragment thereof according to any one of E1 to E33 for binding to CDCP1.

[0088] E35. An isolated antibody or antigen-binding fragment thereof, which competes for binding to CDCP1 with an antibody or antigen-binding fragment thereof selected from the group consisting of CP13E10, CP13E10-183 / 290, CP13E10-H7C-K222R-N297A, CP13E10-54HC-89LC, CP13E10-54HC-89LC-183 / 290, CP13E10-54HC-89LC-H7C-K222R-N297A, CP13E10 10-54HC-89LCv1, CP13E10-54HC-89LCv1-183 / 290, CP13E10-54HC-89LCv1-H7C-K222R-N297A, CP13E10-54HCv13-89LCv1, CP13E10-54HCv13-89LCv1-183 / 290, CP13E10-54HCv13-89LCv1-H7C-K222R-N297A, CP13E10-291, Antibody 23, Antibody 24 and Antibody 76.

[0089] E36. An isolated antibody or antigen-binding fragment thereof that specifically binds to CDCP1, wherein the antibody or antigen-binding fragment thereof binds to substantially the same epitope as the antibody or antigen-binding fragment thereof of any one of E1-E35.

[0090] E37. An isolated antibody or antigen-binding fragment thereof that specifically binds to CDCP1, wherein the antibody or antigen-binding fragment thereof binds to substantially the same epitope as an antibody or antigen-binding fragment thereof selected from the group consisting of: CP13E10, CP13E10-183 / 290, CP13E10-H7C-K222R-N297A, CP13E10-54HC-89LC, CP13E10-54HC-89LC-183 / 290, CP13E10-54HC-89LC-H7C-K222R-N297A, CP13E10-54HC-89LCv1, CP13E10-54HC-89LCv1-183 / 290, CP13E10-54HC-89LCv1-H7C-K222R-N297A, CP13E10-54HCv13-89LCv1, CP13E10-54HCv13-89LCv1-183 / 290, CP13E10-54HCv13-89LCv1-H7C-K222R-N297A, CP13E10-291, Antibody 23, Antibody 24 and Antibody 76.

[0091] E38. The isolated antibody or antigen-binding fragment thereof as described in any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof binds to the antigen-binding protein with a binding affinity (K) of or less than about 350 nM, about 325 nM, about 323.10 nM, about 300 nM, about 286.44 nM, about 275 nM, about 250 nM, about 232.13 nM, about 225 nM, about 219.13 nM, about 200 nM, about 195.54 nM, about 175 nM, about 158 ​​nM, about 150 nM, about 125 nM or about 100 nM. D ) value combined with CDCP1.

[0092] E39. The isolated antibody or antigen-binding fragment thereof as described in any of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof has a K of or less than about 95 nM, about 90 nM, about 80 nM, about 79.89 nM, about 75 nM, about 70 nM, about 69.50 nM, about 65 nM, about 63.44 nM, about 60 nM, about 55 nM, about 52.88 nM, about 50 nM, about 45 nM, about 44.50 nM, about 41.99 nM, about 40 nM, about 35 nM, about 30 nM, about 25 nM, about 20 nM, about 10 nM, about 5 nM, or about 1 nM. D Values ​​bind CDCP1.

[0093] E40. The isolated antibody or antigen-binding fragment thereof as described in any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof has a K of or less than about 5 nM, about 4.5 nM, about 4 nM, about 3.5 nM, about 3.12 nM, about 3 nM, about 2.90 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 250 pM, about 200 pM, about 150 pM, about 100 pM, about 50 pM, about 40 pM, about 30 pM, about 25 pM, about 20 pM, about 15 pM, about 10 pM, about 5 pM, or about 1 pM. D Values ​​bind CDCP1.

[0094] E41. The isolated antibody or antigen-binding fragment thereof as described in any one of E38-E40, wherein the K D The values ​​are measured by surface plasmon resonance (SPR), optionally using a Biacore T200 instrument.

[0095] E42. The antibody or antigen-binding fragment thereof as described in any one of E38-E40, wherein the K DValues ​​are measured by biolayer interferometry (BLI), optionally using a ForteBio Octet instrument.

[0096] E43. The antibody or antigen-binding fragment thereof as described in any one of E38-E42, wherein the CDCP1 is human CDCP1, cynomolgus monkey CDCP1 or mouse CDCP1.

[0097] E44. The antibody or antigen-binding fragment thereof as described in any one of E38-E42, wherein the CDCP1 is human CDCP1, and the K D The value is about 40 nM, about 45 nM or about 50 nM.

[0098] E45. The antibody or antigen-binding fragment thereof as described in any one of E38-E42, wherein the CDCP1 is cynomolgus monkey CDCP1, and the K D The values ​​are about 62 nM, about 64 nM, about 66 nM, about 68 nM, or about 70 nM.

[0099] E46. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein said antibody or antigen-binding fragment thereof is internalized after binding to CDCP1 on mammalian cells.

[0100] E47. The antibody or antigen-binding fragment thereof as described in any of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof comprises an antibody heavy chain constant domain, which comprises an engineered cysteine ​​residue at position 290 according to the numbering of the Eu index of Kabat.

[0101] E48. The antibody or antigen-binding fragment thereof as described in E47, wherein the constant domain comprises an IgG, IgA, IgD, IgE or IgM heavy chain domain.

[0102] E49. The antibody or antigen-binding fragment thereof as described in E48, wherein the constant domain comprises an IgG1, IgG2, IgG3 or IgG4 heavy chain domain.

[0103] E50. The antibody or antigen-binding fragment thereof as described in E48, wherein the constant domain comprises an IgA1 or IgA2 heavy chain domain.

[0104] E51. The antibody or antigen-binding fragment thereof as described in any one of E47 to E50, wherein the constant domain is a human antibody constant domain.

[0105] E52. The antibody or antigen-binding fragment thereof as described in any one of E47 to E51, wherein the constant domain comprises an IgG1 heavy chain CH2 domain and an IgG1 heavy chain CH3 domain.

[0106] E53. The antibody or antigen-binding fragment thereof as described in any one of E47 to E52, wherein the antibody or antigen-binding fragment thereof further comprises an antibody light chain constant domain comprising an engineered cysteine ​​residue at position 183 according to Kabat numbering.

[0107] E54. The antibody or antigen-binding fragment thereof as described in E53, wherein the light chain constant domain comprises a kappa light chain constant domain (CLκ).

[0108] E55. The antibody or antigen-binding fragment thereof as described in E53, wherein the light chain constant domain comprises a lambda light chain constant domain (CLλ).

[0109] E56. An isolated antibody or antigen-binding fragment thereof as described in any one of E46 to E54, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 19, 33 or 42.

[0110] E57. An isolated antibody or antigen-binding fragment thereof as described in any one of E47 to E56, wherein the isolated antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 21, 34 or 38.

[0111] E58. The isolated antibody or antigen-binding fragment thereof of any one of E47 to E57, comprising a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID No: 33; and a light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 38.

[0112] E59. The isolated antibody or antigen-binding fragment thereof as described in any one of E47-E58, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 33; and a light chain comprising the amino acid sequence of SEQ ID NO: 38.

[0113] E60. The isolated antibody or antigen-binding fragment thereof as described in any one of E47 to E58, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 33; and a light chain comprising the amino acid sequence of SEQ ID NO: 34.

[0114] E61. The isolated antibody or antigen-binding fragment thereof as described in any one of E47 to E57, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 42; and a light chain comprising the amino acid sequence of SEQ ID NO: 38.

[0115] E62. The isolated antibody or antigen-binding fragment thereof as described in any one of E47 to E57, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19; and a light chain comprising the amino acid sequence of SEQ ID NO: 21.

[0116] E63. The antibody or antigen-binding fragment thereof as described in any one of E1-E46, wherein the antibody or antigen-binding fragment thereof comprises a tag sequence containing an acyl donor glutamine engineered at a specific site in the Fc region of the antibody.

[0117] E64. The antibody or antigen-binding fragment thereof as described in E63, wherein the glutamine-containing tag sequence comprises LLQG (SEQ ID NO: 91).

[0118] E65. The antibody or antigen-binding fragment thereof as described in any one of E63-E64, wherein the glutamine-containing tag is engineered after amino acid residue number 135 and before amino acid residue number 136 according to the numbering of the Eu index of Kabat.

[0119] E66. The antibody or antigen-binding fragment thereof as described in any one of E63 to E65, wherein the Fc domain of the antibody or antigen-binding fragment thereof comprises one or more amino acid substitutions selected from the group consisting of: N297A and K222R, numbered according to the Eu index of Kabat.

[0120] E67. An isolated antibody or antigen-binding fragment thereof as described in any one of E63 to E66, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25, 35 or 43.

[0121] E68. An isolated antibody or antigen-binding fragment thereof as described in any one of E63 to E67, wherein the isolated antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 17, 32 or 37.

[0122] E69. The isolated antibody or antigen-binding fragment thereof of any one of E63 to E68, comprising a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID No: 35; and a light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 37.

[0123] E70. The isolated antibody or antigen-binding fragment thereof as described in any one of E63 to E69, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35; and a light chain comprising the amino acid sequence of SEQ ID NO: 37.

[0124] E71. The isolated antibody or antigen-binding fragment thereof as described in any one of E63 to E69, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35; and a light chain comprising the amino acid sequence of SEQ ID NO: 32.

[0125] E72. The isolated antibody or antigen-binding fragment thereof as described in any one of E63 to E68, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 43; and a light chain comprising the amino acid sequence of SEQ ID NO: 37.

[0126] E73. The isolated antibody or antigen-binding fragment thereof as described in any one of E63 to E68, wherein the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 25; and a light chain comprising the amino acid sequence of SEQ ID NO: 17.

[0127] E74. An isolated nucleic acid molecule comprising one or more nucleic acid sequences encoding the antibody or antigen-binding fragment thereof according to any one of E1 to E73.

[0128] E75. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO: 75.

[0129] E76. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO: 76.

[0130] E77. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO: 77.

[0131] E78. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO: 78.

[0132] E79. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO: 79.

[0133] E80. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO: 80.

[0134] E81. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO: 81.

[0135] E82. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO: 82.

[0136] E83. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO: 83.

[0137] E84. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO: 84.

[0138] E85. A vector comprising the nucleic acid of any one of E74-E84.

[0139] E86. A host cell comprising at least one nucleic acid according to any one of E74-84.

[0140] E87. A host cell comprising the nucleic acid of SEQ ID NO: 85 and a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 86.

[0141] E88. The host cell of any one of E86-E87, wherein the cell is a mammalian cell.

[0142] E89. The host cell of E88, wherein the host cell is a CHO cell, a HEK-293 cell or a Sp2.0 cell.

[0143] E90. A method for preparing an antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell described in E86-89 under conditions where the host cell expresses the antibody or the antigen-binding fragment thereof.

[0144] E91. An antibody drug conjugate comprising the antibody or antigen-binding fragment thereof of any one of E1-E46, wherein the antibody is conjugated to a drug moiety.

[0145] E92. The antibody drug conjugate of E80, wherein the antibody is conjugated to the drug moiety via a linker.

[0146] E93. The antibody drug conjugate of any one of E91-E92, wherein the antibody is conjugated to the linker-drug moiety via one or more engineered cysteine ​​residues on the antibody.

[0147] E94. The antibody drug conjugate of E93, wherein the antibody or antigen-binding fragment thereof comprises the antibody or antigen-binding fragment thereof of any one of E47-62.

[0148] E95. The antibody drug conjugate of any one of E91 to E94, wherein the linker is selected from the group consisting of valine-citrulline (val-cit), 6-maleimidocaproyl (mc), methoxy-polyethylene glycol maleimide 6 (MalPeg6), p-aminobenzylcarbamate (PABC), dimethylaminoethanol (DMAE), maleimidopropionyl (MP), hydrolyzed Peg-maleimide, alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB). , 4-(2-pyridylthio) pentanoic acid N-succinimidyl ester (SPP), 4-(N-maleimidomethyl) cyclohexane-1 carboxylic acid N-succinimidyl ester (SMCC), (4-iodo-acetyl) aminobenzoic acid N-succinimidyl ester (SIAB), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-val-cit-PAB) and 6-maleimidocaproyl-valine-citrulline-p-aminobenzylcarbamate (mc-val-cit-PABC).

[0149] E96. The antibody drug conjugate of any one of E91-E92, wherein the antibody is conjugated to the linker using a tag sequence containing the acyl donor glutamine engineered on the antibody.

[0150] E97. The antibody drug conjugate of E96, wherein the tag sequence is LLQG (SEQ ID NO: 91).

[0151] E98. The antibody drug conjugate of E96-E97, wherein the antibody or antigen-binding fragment thereof comprises the antibody or antigen-binding fragment thereof of any one of E63-73.

[0152] E99. An antibody drug conjugate as described in any of E96-E98, wherein the linker is selected from the group consisting of: Ac-Lys-Gly (acetyl-lysine-glycine), aminocaproic acid, Ac-Lys-p-Ala (acetyl-lysine-p-alanine), amino-PEG2 (polyethylene glycol)-C2, amino-PEG3-C2, amino-PEG6-C2 (or aminoPEG6-propionyl), Ac-Lys-Val-Cit-PABC (acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl), amino-PEG6-C2-Val-Cit-PABC, aminohexanoyl-Val-Cit-PABC, [(3R,5R)-1-{3-[2-(2-aminoethoxy)ethoxy]propionyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, [(3S,5S)-1-{3-[2-(2-aminoethoxy)ethoxy]propionyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, putrescine, and Ac-Lys-putrescine.

[0153] E100. The antibody drug conjugate of any one of E91 to E99, wherein the drug moiety is a cytotoxic agent, an immunomodulatory agent, an imaging agent, a chemotherapeutic agent, or a therapeutic protein.

[0154] E101. The antibody drug conjugate of E100, wherein the cytotoxic agent is selected from the group consisting of anthracyclines, auristatins, CC-1065, dolastatin, duocarmycin, enediynes, geldanamycin, maytansine, puromycin, taxanes, vinca alkaloids, SN-38, tubulysin, hemicycline, and stereoisomers, isosteres, analogs or derivatives thereof.

[0155] E102. The antibody drug conjugate of E101, wherein the cytotoxic agent is an auristatin selected from the group consisting of:

[0156] MMAD (monomethyl auristatin D), 0101

[0158] 0131

[0160]

[0161] E103. The antibody drug conjugate of any one of E91-E102, wherein the linker is selected from the group consisting of valine-citrulline (val-cit), 6-maleimidocaproyl (mc), methoxy-polyethylene glycol maleimide 6 (MalPeg6), p-aminobenzylcarbamate (PABC), dimethylaminoethanol (DMAE), maleimidopropionyl (MP), hydrolyzed Peg-maleimide, alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), 4-(2- The invention relates to a novel medicament comprising N-succinimidyl pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1carboxylate (SMCC), N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-val-cit-PAB) and 6-maleimidocaproyl-valine-citrulline-p-aminobenzylcarbamate (mc-val-cit-PABC), and the drug moiety is auristatin.

[0162] E104. The antibody drug conjugate of E103, wherein the linker is mc-val-cit-PABC and the drug moiety is 0101 or 0131.

[0163] E105. An antibody drug conjugate comprising an antibody or an antigen-binding fragment thereof conjugated to a linker-drug moiety via one or more engineered cysteine ​​residues on the antibody, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 33; and a light chain comprising the amino acid sequence of SEQ ID NO: 38, and wherein the linker-drug moiety is mc-val-cit-PABC-0101.

[0164] E106. An antibody drug conjugate comprising an antibody or an antigen-binding fragment thereof conjugated to a linker-drug moiety via one or more engineered cysteine ​​residues on the antibody, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 33; and a light chain comprising the amino acid sequence of SEQ ID NO: 34, and wherein the linker-drug moiety is mc-val-cit-PABC-0101.

[0165] E107. An antibody drug conjugate as described in any of E91-E102, wherein the linker is selected from the group consisting of: Ac-Lys-Gly (acetyl-lysine-glycine), aminocaproic acid, Ac-Lys-p-Ala (acetyl-lysine-p-alanine), amino-PEG2 (polyethylene glycol)-C2, amino-PEG3-C2, amino-PEG6-C2 (or aminoPEG6-propionyl), Ac-Lys-Val-Cit-PABC (acetyl-lysine-valine-citrulline-p-aminobenzyloxy [(3R,5R)-1-{3-[2-(2-aminoethoxy)ethoxy]propionyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, [(3S,5S)-1-{3-[2-(2-aminoethoxy)ethoxy]propionyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, putrescine and Ac-Lys-putrescine, and the drug moiety is auristatin.

[0166] E108. The antibody drug conjugate of E107, wherein the linker is aminoPEG6-propionyl (ie, amino-PEG6-C2 or AMPeg6C2) and the drug moiety is 0101 or 0131.

[0167] E109. An antibody drug conjugate comprising an antibody or antigen-binding fragment thereof conjugated to a linker-drug moiety using an acyl donor glutamine-containing tag engineered at a specific site on the antibody, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35; and a light chain comprising the amino acid sequence of SEQ ID NO: 37, and wherein the linker-drug moiety is aminoPEG6-propionyl-0131 (AmPeg6C2-0131).

[0168] E110. An antibody drug conjugate comprising an antibody or antigen-binding fragment thereof conjugated to a linker-drug moiety using an acyl donor glutamine-containing tag engineered at a specific site on the antibody, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35; and a light chain comprising the amino acid sequence of SEQ ID NO: 32, and wherein the linker-drug moiety is aminoPEG6-propionyl-0131 (i.e., AmPeg6C2-0131).

[0169] E111. The antibody drug conjugate of any of E91-E110, wherein the antibody drug conjugate has a melting transition temperature greater than at least 60°C, at least 65°C, at least 70°C, at least 75°C, at least 80°C, at least 85°C, or at least 90°C.

[0170] E112. The antibody drug conjugate of E111, wherein the antibody drug conjugate has a first melting transition temperature greater than about 65°C.

[0171] E113. The antibody drug conjugate of any one of E91-E112, wherein the antibody drug conjugate has a K of about 50 nM, about 48 nM, about 46 nM, about 45 nM, about 44 nM, about 42 nM, or about 40 nM at pH 7.4. D Values ​​bind CDCP1.

[0172] E114. The antibody drug conjugate of any one of E91-E113, wherein the antibody drug conjugate has a K of about 70 nM, about 68 nM, about 66 nM, about 65 nM, about 64 nM, about 62 nM, or about 60 nM at pH 6.8. D Values ​​bind CDCP1.

[0173] E115. The antibody drug conjugate of any one of E91-E114, wherein the antibody drug conjugate has a half maximal inhibitory concentration (IC50) of no more than about 20,000 pM, about 15,000 pM, about 10,000 pM, about 9,500 pM, 8,000 pM, 7,000 pM, 6,000 pM, 5,000 pM, 4,000 pM, 3,000 pM, 2,000 pM, 1,000 pM, 900 pM, 800 pM, 700 pM, 650 pM, 600 pM, 500 pM, 400 pM, 300 pM, 250 pM, 200 pM or 100 pM. 50 )value.

[0174] E116. The antibody drug conjugate of any one of E91-E114, wherein the antibody drug conjugate has an IC50 value of no more than about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM, about 50 pM, about 40 pM, about 30 pM, about 20 pM, about 10 pM, about 9 pM, about 8 pM, about 7 pM, about 6 pM, about 5 pM, about 4 pM, about 3 pM, about 2 pM, or about 1 pM.

[0175] E117. The antibody drug conjugate of E115 or E116, wherein the IC50 value is determined using CDCP1 expressing cells.

[0176] E118. The antibody drug conjugate of any one of E91-E117, wherein the antibody drug conjugate reduces mean tumor volume by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to the mean tumor volume in an otherwise identical untreated tumor using a non-small cell lung cancer (NSCLC) patient-derived xenograft model.

[0177] E119. An antibody drug conjugate as described in any of E91-E118, wherein the antibody drug conjugate reduces the mean tumor volume in the treated tumor by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to the mean tumor volume in an otherwise identical untreated tumor in a head and neck cancer patient-derived xenograft model.

[0178] E120. A pharmaceutical composition comprising the antibody drug conjugate according to any one of E91-E119 and a pharmaceutically acceptable carrier.

[0179] E121. A method for treating cancer, autoimmune disease, inflammatory disease or infectious disease mediated by or associated with the expression of CDCP1 on cells, the method comprising administering to a subject in need thereof a therapeutically effective amount of the antibody drug conjugate of any one of E91-E119 or the composition of E120.

[0180] E122. The antibody drug conjugate of any one of E91-E119 or the composition of E120, for use in treating cancer, autoimmune disease, inflammatory disease or infectious disease mediated by or associated with the expression of CDCP1 on cells.

[0181] E123. Use of the antibody drug conjugate of any one of E89 to E112 or the composition of E113 for treating cancer, autoimmune disease, inflammatory disease or infectious disease mediated by or associated with the expression of CDCP1 on cells.

[0182] E124. Use of the antibody drug conjugate of any one of E89 to E112 or the composition of E113 in the manufacture of a medicament for treating cancer, autoimmune disease, inflammatory disease or infectious disease mediated by or associated with the expression of CDCP1 on cells.

[0183] E125. The cancer of any one of E121 to E124, wherein the cancer is selected from the group consisting of: basal cell carcinoma; biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatoma; intraepithelial neoplasia; renal or kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; myeloma; neuroblastoma; oral cancer (lips, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; ... ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; ovarian cancer; ovarian cancer; pancreatic cancer; prostate cancer; ovarian cancer; ovarian cancer; ovarian cancer; ovarian cancer; ovarian Uterine or endometrial cancer; urological cancers; vulvar cancer; lymphomas, including Hodgkin lymphoma and non-Hodgkin lymphoma, and B-cell lymphomas (including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other cancers and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with keloids, edema (e.g., edema associated with brain tumors), and Meigs' syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0184] Figure 1 is a box plot of bioinformatics analysis of expression microarray data from 790 cancer cell lines in the Sanger Cell Line Project, showing that CDCP1 expression is increased in cells expressing oncogenic KRAS and / or LKB1 compared to wild type.

[0185] Figure 2 Silencing CDCP1 was shown to shrink established H2009 NSCLC tumors.

[0186] Figure 3Graph showing the relative hydrophobicity of anti-CDCP1 antibodies based on elution time as detected by analytical hydrophobic interaction chromatography (HTC). Incorporation of three point mutations (Y(H100)H, W(H100C)H, Y(H100H)H) into CP13E10 CDRH3 (CP13E10-34 variant) significantly reduced hydrophobicity, as indicated by a decrease in elution time.

[0187] Figure 4 The elution times of the anti-CDCP1 variants as detected by analytical hydrophobic interaction chromatography (HIC) are shown. Incorporation of three point mutations (Y(H100)H, W(H100C)H, Y(H100H)H) into CP13E10 CDRH3 (CP13E10-34 variant) significantly reduced hydrophobicity as shown by the reduction in elution time.

[0188] Figure 5 is a line graph demonstrating that incorporation of V(H97)E into heavy chain CDR3 restores the CDCP1 binding properties of variant CP13E10-54 to those of the parent wild-type CP13E10 antibody.

[0189] Figure 6 Shown are the binding kinetics of anti-CDCP1 antibodies assayed against recombinant human, cynomolgus monkey and mouse CDCPl-ECD proteins.

[0190] Figure 7 Depicts a line graph demonstrating that the IGKV146 germline substitution incorporated into CP13E10-54HC-89LCv1 does not alter CDCP1 binding properties.

[0191] Figure 8 Shown is a line graph demonstrating that germline CP13E10-54HC-89LCv1 is identical to CP13E10-54HC-89LC in binding to CDCPl expressed on the surface of PC3 prostate cancer cells.

[0192] Figure 9 Depicted is a line graph demonstrating that CP13E10-54HCv13-89LCv1 comprising a G(H96)A mutation to remove the putative isomerization site in CDRH3 retains CDCP1 binding properties relative to CP13E10-54HC-89LCv1.

[0193] Figure 10 An overview of the complex formed by the extracellular domain (ECD) of CDCP1 and the Fab fragment of the antibody CP13E10-54HC-89LC is provided. The C-alpha trace at the top of the figure shows the position of CDCP1 and the termini are labeled. The ball-and-stick diagram indicates the positions of the heavy atoms in the amino acid residues of the antibody. The antibody is indicated by a black Fab heavy chain on the left and a light gray Fab light chain on the right. Labels indicate the positions of CDRH1 and CDRH3.

[0194] Figure 11 This is an antigen-centered close-up of the interface between the CDCP1 ECD and the Fab fragment of the antibody CP13E10-54HC-89LC. Figure 10 The light grey stick indicates the glycan attached to Asn122 of CDCP1. Figure 10 Same as indicated in the description.

[0195] Figure 12 Antigen-centered close-up showing the interface between CDCP1 ECD and the Fab fragment of antibody CP13E10-54HC-89LC. Figure 10 The light grey space-filling model indicates the glycan attached to CDCP1 Asn122. The ball and stick diagram indicates the heavy atoms of the amino acid residues in CDCP1. Without wishing to be bound by any particular theory, the labeled residue Phe (H100A) appears to play a key role in making Van Der Waals contacts with the six antigen residues (see Table 5).

[0196] Figure 13 Shows a reverse close-up of the interface between CDCP1 ECD and the Fab fragment of antibody CP13E10-54HC-89LC. Figure 12 , this view reflects a 180° rotation around an axis parallel to the vertical page axis. Figure 12 The positions of CDRL1, CDRL3, and CDRH2 are marked, as are the turns in framework 3 (FW3) of the heavy chain that make contact with the antigen. CDRL2 is located behind CDRL3 in this figure and is therefore not shown (for a different view showing CDRL2, see Figure 14 ).

[0197] Figure 14 Reverse close-up depicting the interface between the CDCP1 ECD and the VL of the antibody CP13E10-54HC-89LC. Figure 13 , this view reflects a 180° rotation around an axis parallel to the vertical page axis. Figure 12The positions of CDRL1, CDRL2, and CDRL3 are labeled. Amino acid residues 46-54 of CDCP1 (numbered with reference to SEQ ID NO: 90) surround Tyr (L32) of CDRL1.

[0198] Figure 15A A subset of interactions between the CDCP1 ECD and the Fab of the antibody CP13E10-54HC-89LC is shown. Sticks and C-alpha traces indicate CDCP1, and the ribbon and ball-and-stick diagram indicates the antibody. Key amino acid residues are labeled, with only the antibody labeling in brackets. Dashed lines indicate certain hydrogen bonds or salt bridges, and distance markers are in angstroms. Figure 15B A subset of interactions between CDCP1 ECD and the Fab of antibody CP13E10-54HC-89LC is shown. Figure 15A Alternative view of the same model shown in , indicating different distances. Amino acid residues 46-54 of CDCP1 (numbered with reference to SEQ ID NO: 90) surround Tyr (L32) of CDRL1.

[0199] Figure 16 A representative analysis of the time-dependent increase in the ratio of the median fluorescence intensity values ​​of the signals detected in the "membrane" and "internal" cellular compartments is shown. The slope of the regression line represents the internalization rate (Ke).

[0200] Figure 17 Shown is a bar graph showing that CDCP1-ADC (CP13E10-SS3-LP15) blocks tumor growth in NSCLC PDX models.

[0201] Figures 18A-18C Western blot images are shown, which show that in H1299 cells ( Figure 18A ), MDA-MB-231 cells ( Figure 18B ) and MCF10A cells ( Figure 18C ) CDCP1 activation by short-term (5 min) treatment of cells with antibodies CP13E10-291 and CPE10-54HC-89LC.

[0202] Figures 19A-19B Depicts Western blot images showing that breast cancer cells MDA-MB-231 ( Figure 19A ) and MDA-MB-468( Figure 19B ) of CDCP1 degradation.

[0203] Figure 20Shown are Western blot images showing CDCP1 degradation by prolonged treatment of H1299 cells with antibodies CP13E10-291 and CPE10-54HC-89LC, a time-dependent decrease in CDCP1 expression and tyrosine phosphorylation, and loss of Src activation.

[0204] Figures 21A-21B Depicts immunohistochemistry images and bar graphs showing that CDCP1 antibodies CP13E10-291 and CPE10-54HC-89LC reduce MDA-MB-231 cells ( Figure 21A ) and H1299 cells ( Figure 21B )migrate.

[0205] Figures 22A-22B Shown are images and bar graphs showing that CDCP1 antibodies CP13E10-291 and CPE10-54HC-89LC reduce MDA-MB-231 cells ( Figure 22A ) and H1299 cells ( Figure 22B ) invasion.

[0206] FIG. 23A to FIG. 23B Depicted are Western blot images showing that CDCP1-activating abs (CP13E10-291, CUB1, and antibody 23) reduced basal AKT activity in only some cells. Figure 23A showed a decrease in H1373 and H1299 cells, while Figure 23B No effect was shown in MCF10A, H1975 and HCT116 cells.

[0207] Figures 24A-24C Antibodies showing activation of CDCP1 also reduced basal AKT activity and AKT substrate phosphorylation in PC3 cells. Figure 24A Shown are activating Abs including 291, 76, 23, CUB1 and non-activating Ab 24 that does not affect P-AKT. Figure 24B and 24C The inhibition was short-lived, showing an 80% reduction within 20 minutes, and AKT phosphorylation returned to initial levels during prolonged antibody exposure, consistent with a decrease in CDCP1 protein expression.

[0208] Figure 25 Shown are experiments to identify new CDCP1 binding partners by using activated ("76") and ("24") anti-CDCP1 antibodies bound to intact PC3 cells.

[0209] Figure 26A and 26BShown is the dose-dependent binding of anti-CDCP1 antibodies to cells expressing CDCPl as determined by flow cytometry.

[0210] Figure 27 Shown are the binding kinetics of anti-CDCP1 CP13E10-54HC-89LCv1-183 / 290 antibody and CP13E10-54HC-89LCv1-183 / 290-vc0101 ADC assayed against recombinant human CDCP1-ECD protein.

[0211] Figure 28A It was demonstrated that incubation of PC3 cells with antibody CP13E10-54HC-89LCv1-183 / 290 or ADC CP13E10-54HC-89LCv1-183 / 290-vc0101 mediated cell killing (% dead cells) by co-incubated NK cells in a dose-dependent manner. Note that an isotype control antibody that cannot bind to PC3 cells did not induce NK cell killing. Figure 28B Prove that PC3 cells are incubated with antibody CP13E10-54HC-89LCv1-183 / 290 or ADC CP13E10-54HC-89LCv1-183 / 290-vc0101 in reporter gene Jurkat bioassay effector cells in a dose-dependent manner to mediate the induction of luciferase (measured in relative light units (RLU)). Luciferase induction shows that the antibody engaged by being bound to PC3 cells Fc γ RIII receptors on the reporter cells. Note that the isotype control antibody that cannot be bound to PC3 cells does not induce luciferase activity.

[0212] Figures 29A-29B Figure 2 shows tumor growth in a pancreatic cancer patient-derived xenograft (PDX) model treated with CDCP1 antibody drug conjugate (ADC). Pancreatic cancer PDX model PDX-PAX-24513 expresses high amounts of CDCP1 (H score as indicated). When the average tumor size reaches approximately 200 mm 3 Groups of mice were implanted with tumor cells and randomly assigned to treatment at 4 days (n=10 / treatment group). Each group received an intravenous (iv) injection of the indicated compound at the indicated concentration. A total of four iv injections were given at four-day intervals. Tumor growth was tracked as described over the indicated time course.

[0213] Figures 30A-30B Figure 2 shows tumor growth in pancreatic cancer PDX models treated with CDCP1 antibody drug conjugate (ADC). Pancreatic cancer PDX model PDX-PAX-24509 expresses high amounts of CDCP1 (H score as indicated). When the average tumor size reaches approximately 200 mm 3Cohorts were implanted with tumor cells at 4 days (n=10 / treatment group) and randomly assigned to treatment. Each group received an intravenous (iv) injection of the indicated compound at the indicated concentration. A total of four iv injections were given at four-day intervals. Tumor growth was assessed as described over the indicated time course.

[0214] Figure 31 Shown are the survival of the pancreatic cancer model PDX-PAX-24509 cohorts given 3 mg / kg (milligram / kilogram) of ADC or untreated negative controls administered phosphate buffered saline (PBS) without ADC from Figure 30. A statistically significant survival benefit was associated with CP13E10-54HC-89LC-183 / 290-vc0101 (mean ± SE, 47.1 ± 0.72 days) compared to untreated controls receiving PBS (mean ± SE, 16.9 ± 0.99 days; log rank, p < 0.0001) or 3 mg / kg of CP13E10-54HC-89LC-H7C-AmPEG6-0131 (mean ± SE, 20.8 ± 0.68 days; log rank, p < 0.0001).

[0215] Figure 32A Shown are tumor growth in the non-small cell lung cancer (NSCLC) model PDX-NSX-26101. All doses of CP13E10-54HC-89LC-H7C-AmPEG6-0131 resulted in progressive disease (PD). Figure 32B Figure 2 shows tumor growth in the NSCLC model PDX-NSX-26101. CP13E10-54HC-89LC-183 / 290-vc0101 also resulted in progressive disease (PD) when administered at doses of 0.3 and 1 mg / kg. However, administration of CP13E10-54HC-89LC-183 / 290-vc0101 at 3 mg / kg resulted in transient tumor regression, resulting in a partial response (PR) for at least two weeks after the final dose ( Figure 32B ).

[0216] Figure 33A Shown are tumor growth in the NSCLC model PDX-NSX-26113. CP13E10-54HC-89LC-H7C-AmPEG6-0131 doses of 0.3 and 1 mg / kg resulted in PD, while 3 mg / kg produced transient regressions, and PR was not observed until day 25 (13 days after the last dose). Figure 33BFigure 2 shows tumor growth in the NSCLC model PDX-NSX-26113. Both 0.3 and 1 mg / kg doses of CP13E10-54HC-89LC-183 / 290-vc0101 resulted in PD. Administration of CP13E10-54HC-89LC-183 / 290-vc0101 at 3 mg / kg resulted in transient tumor regression, resulting in a PR observed at least until day 42 (29 days after the last dose).

[0217] Figure 34A Figure 2 shows tumor growth in the NSCLC model PDX-NSX-15137. CP13E10-54HC-89LCv1-183 / 290-vc0101 produced a PR at doses of 1.5 mg / kg and 4.5 mg / kg. PRs were still observed at the 4.5 mg / kg dose by day 35, at which point tumors in the paclitaxel-treated group had increased in size beyond their starting volume. Figure 34B Tumor growth in the head and neck cancer model PDX-HNX-24715 is shown. Both 4.5 mg / kg of CP13E10-54HC-89LCv1-183 / 290-vc0101 and 3 mg / kg of CP13E10-54HC-89LC-183 / 290-vc0101 were superior to treatment with cisplatin.

[0218] Figure 35A Figure 2 shows tumor growth in the PDX tumor model PDX-NSX-26113 (H score 227). A tumor model was established and CP13E10-54HC-89LC-183 / 290-vc0101 was administered four times at doses of 0.3, 1, and 3 mg / kg, each at four-day intervals (q4dx4). A partial response was observed at 3 mg / kg on day 42. Thereafter, tumors began to grow continuously and were harvested on day 56 for re-implantation into a naive cohort of NOD / SCID mice. Figure 35B Shown implanted from Figure 35A The naive group of tumor cells collected from the 3 mg / kg group was given the 200 mm 3 Partial responses were observed in the 3 and 6 mg / kg groups, indicating that the tumors remained sensitive to the ADC and that Figure 35A The regrowth observed in was not a result of resistance to CP13E10-54HC-89LC-183 / 290-vc0101.

[0219] Figure 36Shown are the maximum mean changes in tumor size observed in pancreatic (PAX), head and neck (HNX), or non-small cell lung cancer (NSX) patient-derived xenograft (PDX) tumor models. These models were established in groups of mice (*n=10; for others n=4-5). When the mean tumor size reached 200 mm 3 Treatment with 3 mg / kg of CP13E10-54HC-89LC-183 / 290-vc0101 was initiated at 4 days post-inoculation. The percent change from the starting volume was determined as described in the text. An H-score, indicating CDCP1 expression levels, is given within each bar. Response evaluation criteria for solid tumors: complete response (1 / 14); partial response (10 / 14); progressive disease (3 / 14); and objective response rate (79%) (11 / 14).

[0220] Figure 37 The maximum mean change in tumor size observed in PAX, HNX, NSX, ovarian (OVX), breast (BRX), bladder (BLA), and small cell lung cancer (SCX) PDX tumor models is shown. These models were established in groups of mice (n=4-5). When the mean tumor size reached 200 mm 3 Treatment with 3 mg / kg of CP13E10-54HC-89LCv1-183 / 290-vc0101 was initiated at 4 days post-transplantation. The H score for CDCP1 expression is given within or above the individual bars, where indicated. Response Evaluation Criteria in Solid Tumors: complete response 8 / 40; partial response 17 / 40; stable disease 7 / 40; progressive disease 8 / 40; objective response rate 63% (25 / 40). DETAILED DESCRIPTION

[0221] The present invention is based, in part, on the unexpected discovery that CDCP1 exhibits interesting biological activities in the context of various cancers, which allows for specific therapeutic approaches and patient selection. Furthermore, the present invention provides novel antibodies and antibody drug conjugates based on said antibodies that specifically bind to CDCP1 and exhibit potential novel human therapeutic agents for diseases and disorders that demonstrate that they are mediated by or associated with CDCP1 expression on cells.

[0222] CDCP1 has the ability to internalize certain agents into cells, and this internalization provides utility in cancer treatment. Without wishing to be bound by theory, this internalization is carried out by, for example, the phosphorylation of CDCP1 by Src at tyrosine 734 of CDCP1. It has been previously proposed that this phosphorylation of CDCP1 is related to its cancer-promoting effect. Surprisingly, the present inventors have found that anti-tumor efficacy requires CDCP1 to be phosphorylated and therefore have internalization ability. In addition, although CDCP1 is widely expressed, targeting CDCP1 for internalization still provides a targeted anti-tumor effect. Therefore, the present invention particularly utilizes the discovery that the internalization of CDCP1 in certain cells, such as cells expressing Tyr734-phosphorylated CDCP1, allows drug delivery in a manner that mitigates off-target effects. Further, the present inventors have shown that certain CDCP1 targeting agents require long-term exposure to reduce CDCP1, and it is speculated (but not wishing to be bound by theory) that it is through internalization. Therefore, this pace of biological effect supports the scheme and combination therapy as described herein. Furthermore, the inventors have shown that in hypoxic environments, such as those of tumors (e.g., characterized by expression of HIF-2), internalization of CDCP1 is favored, thereby providing options for targeted oncological indications.

[0223] In various aspects, the present invention relates to a method of treating cancer in a patient in need thereof, comprising: (a) assessing CDCP1 (e.g., Tyr-734-phosphorylated CDCPI) expression on the surface of tumor cells in a tumor sample; and (b) administering an agent that binds to CDCP1 to the cancer patient.

[0224] Thus, in various aspects, the present invention relates to a method for treating cancer in a patient in need thereof, the method comprising: (a) assessing the amount of mutant LKB1 and / or KRAS in a tumor sample; and (b) administering an agent that binds to CDCP1 to the cancer patient if the amount of mutant LKB1 and / or KRAS is greater than that in a reference sample. In some embodiments, the tumor sample is a biopsy selected from the group consisting of a frozen tumor tissue specimen, cultured cells, circulating tumor cells, and a formalin-fixed, paraffin-embedded tumor tissue specimen. In some embodiments, the mutant KRAS is selected from the group consisting of G12C; G12A; G12D; G12R; G12S; G12V; G13C; and G13D mutants. In some embodiments, the assessment is performed by amplifying LKB1 and / or KRAS nucleic acid, or a fragment suspected of containing a mutation, from the tumor sample and sequencing the amplified nucleic acid. In some embodiments, the assessment is performed by contacting an antibody to LKB1 and / or KRAS, or a form thereof, with the tumor sample and quantifying binding of the antibody or form thereof.

[0225] In one aspect, the present disclosure provides a method of treating lung cancer in a patient in need thereof, the method comprising administering to the patient an agent that binds to CDCP1, wherein the lung cancer is characterized by AKT activation and the agent that binds to CDCP1 is a CDCP1 activator. In some embodiments, the lung cancer is NSCLC. In some embodiments, the method further comprises assessing AKT activation in a sample of the lung cancer.

[0226] In one aspect, the present disclosure provides a method for treating prostate cancer in a patient in need thereof, the method comprising administering to the patient an agent that binds to CDCP1, wherein the prostate cancer is characterized by AKT activation, and the agent that binds to CDCP1 is a CDCP1 activator. In some embodiments, the method further comprises assessing AKT activation in a sample of the prostate cancer. In some embodiments, the method further comprises administering an AKT inhibitor. In some embodiments, the patient is undergoing treatment with an AKT inhibitor. In some embodiments, the AKT inhibitor is selected from apremilast, ARQ 751, ARQ 092, AZD5363, BAY1125976, GSK2141795, GSK690693, etanercept, LY2780301, MK2206, and perifosine.

[0227] In some embodiments, the patient is not currently being treated with a Src inhibitor, optionally selected from KX2-391, bosutinib, saracatinib, and dasatinib. In some embodiments, the patient is not currently being treated with a Src inhibitor, optionally selected from KX2-391, bosutinib, saracatinib, and dasatinib.

[0228] In one aspect, the present disclosure provides a method for treating cancer in a patient in need thereof, the method comprising: (a) selecting an agent that binds to CDCP1 on a target cell and is internalized when it contacts CDCP1 on the target cell; and (b) administering the agent to the cancer patient, wherein the agent that binds to CDCP1 is an antibody that activates CDCP1 and is conjugated to a PPP4R2 modulator. In some embodiments, CDCP1-activating antibodies include but are not limited to CP13E10 and variants thereof, including CP13E10-54HC-89LCv1-183 / 290 and CP13E10-291, CUB1, antibody 23, and antibody 76.

[0229] In one aspect, the present disclosure provides a method for treating cancer in a patient in need thereof, the method comprising: (a) administering an agent that binds to CDCP1, wherein the agent that binds to CDCP1 is an antibody that does not activate CDCP1; and (b) administering an agent that modulates PARG. In some embodiments, the non-activating CDCP1 antibody includes but is not limited to antibody 24.

[0230] In one aspect, the present disclosure provides a method of determining whether a tumor will respond to treatment with an agent that binds to CDCP1, the method comprising determining the presence, absence, or amount of mutant LKB1 and / or KRAS protein or gene in a sample of the tumor, whereby an increase in the presence of mutant LKB1 and / or KRAS or the amount of mutant LKB1 and / or KRAS protein or gene relative to a reference sample indicates a likelihood of response to treatment with an agent that binds to CDCP1.

[0231] In some embodiments, the agent that binds to CDCP1 is an antibody or an antigen-binding portion thereof that is specific for CDCP1.

[0232] In some embodiments, the present disclosure provides a method for treating cancer in a patient, wherein an agent is selected for its ability to bind to CDCP1 on a target cell and to be internalized upon contact with CDCP1 on the target cell, and the agent is administered to the cancer patient. In some embodiments, internalization is mediated by phosphorylation of CDCP1 by Src, for example, at tyrosine 734 of CDCP1.

[0233] In various embodiments, the present invention relates to a method for treating cancer in a patient with a combination therapy of a checkpoint inhibitor and an agent selected for the ability to bind to CDCP1 (e.g., Tyr-734 phosphorylated CDCPI) on a target cell and internalized when it contacts the CDCP1 on the target cell. In various embodiments, the agent selected for the ability to bind to CDCP1 (e.g., Tyr-734 phosphorylated CDCPI) on a target cell and internalized when it contacts the CDCP1 on the target cell enhances the immune system's response to the checkpoint inhibitor. In various embodiments, the agent selected for the ability to bind to CDCP1 (e.g., Tyr-734 phosphorylated CDCPI) on a target cell and internalized when it contacts the CDCP1 on the target cell improves the patient's response to the checkpoint inhibitor (e.g., but not limited to, by improving the therapeutic effect of the checkpoint inhibitor, alleviating the side effects of the checkpoint inhibitor and / or converting a non-responder or a poor responder into a responder to the checkpoint inhibitor).

[0234] CUB domain-containing protein 1 (CDCP1)

[0235] CDCP1 has a large extracellular domain (665 amino acids in size) containing three CUB domains in the extracellular portion, which mediate protein-protein interactions and are thought to be involved in cell adhesion and interaction with the extracellular matrix. The CDCP1 gene has been found to be a gene strongly expressed in cancers such as lung cancer and head and neck cancer.

[0236] The transmembrane protein CDCP1 associates with Src and PKCδ, and when CDCP1 is activated, all three proteins display increased tyrosine phosphorylation. Src phosphorylates and binds to CDCP1, which then binds to the C2 domain, part of the regulatory domain of PKCδ. Tyr-734 has been identified as a site of phosphorylation by Src and Src family kinases, and therefore, P-Tyr-734 is a biomarker of CDCP1 activation. The full-length CDCP1 protein is 135 kDa, but in some cells, the extracellular domain is proteolytically cleaved to a transmembrane protein of approximately 75 kDa. CDCP1 antibodies or antibody-drug conjugates have been developed to knock down CDCP1 or otherwise target tumor cells expressing CDCP1. In some embodiments, the antibodies or ADCs must be internalized to be effective. CDCP1 has been identified as a therapeutic target for cancer, and new signaling pathways have been discovered that are affected by CDCP1 activation by targeted antibodies that bind to its extracellular domain.

[0237] In some aspects, the CDCP1 is human CDCP1. In some aspects, the CDCP1 is cynomolgus monkey (cyno) CDCP1. In some aspects, the CDCP1 is mouse CDCP1. In some aspects, the CDCP1 is primate CDCP1. Exemplary CDCP1 sequences are provided in Table 10.

[0238] CDCP1 Antibody

[0239] The term antibody is used in the broadest sense herein and specifically encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit the desired biological activity. Antibodies can be mouse antibodies, human antibodies, humanized antibodies, chimeric antibodies or antibodies derived from other species. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th edition, Garland Publishing, New York). Target antigens typically have many binding sites, also referred to as epitopes, recognized by CDRs on a variety of antibodies. Each antibody that specifically binds to different epitopes has a different structure. Therefore, an antigen can have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules containing an antigen binding site that immunospecifically binds to a target of interest or a portion thereof, such targets including but not limited to cancer cells or cells producing autoimmune antibodies associated with autoimmune diseases. Immunoglobulins disclosed herein can have immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, and IgA), classification (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Immunoglobulins can be derived from any species. However, in one aspect, immunoglobulins have human, mouse, or rabbit origins.

[0240] An "antigen-binding fragment" of an antibody refers to a fragment of a full-length antibody that retains the ability to specifically bind to an antigen (preferably with substantially the same binding affinity). Examples of antigen-binding fragments include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al., 1989 Nature 341: 544-546); and (vi) isolated complementarity-determining regions (CDRs), disulfide-linked Fv (dsFv), and anti-idiotypic (anti-Id) antibodies and intrabodies. In addition, although the two domains of the Fv fragment (VH and VL) are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that enables them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called single-chain Fv (scFv)); see, for example, Bird et al., Science 242: 423-426 (1988) and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883. Other forms of single-chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al., 1994, Structure 2:1121-1123).

[0241] An antibody "variable domain" refers to the variable region of an antibody light chain (VL) or the variable region of an antibody heavy chain (VH), alone or in combination. As known in the art, the variable regions of the heavy and light chains are each composed of four framework regions (FR) connected by three complementarity determining regions (CDRs) and contribute to the formation of the antigen binding site of an antibody.

[0242] "Complementarity determining regions" (CDRs) can be identified according to the Kabat, Chothia definitions, the cumulative, AbM, contact, North and / or conformational definitions of both Kabat and Chothia, or any CDR determination method well known in the art. See, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed. (hypervariable regions); Chothia et al., 1989, Nature 342:877-883 (structural loop structures). The identities of the amino acid residues in a particular antibody that comprise a CDR can be determined using methods well known in the art. The AbM definition of a CDR is a compromise between Kabat and Chothia, and is defined using Oxford Molecular's AbM antibody modeling software. The "contact" definition of CDRs is based on observed antigen contacts, as described in MacCallum et al., 1996, J. Mol. Biol., 262:732-745. The "conformational" definition of CDRs is based on residues that make an enthalpic contribution to antigen binding (see, e.g., Makabe et al., 2008, J. Biol. Chem., 283:1156-1166). North has identified canonical CDR conformations using a set of different preferred CDR definitions (North et al., 2011, J. Mol. Biol. 406:228-256). In another approach, referred to herein as the "conformational definition" of CDRs, positions of CDRs can be identified as residues that make an enthalpic contribution to antigen binding (Makabe et al., 2008, J Biol. Chem. 283:1156-1166). Other CDR boundary definitions may not strictly follow one of the above methods, but will still overlap with the Kabat CDRs, although they may be shortened or lengthened based on the prediction or experimental findings that a particular residue or group of residues, or even the entire CDR, does not significantly affect antigen binding. As used herein, a CDR may refer to a CDR defined by any method known in the art (including a combination of methods). The methods used herein can utilize CDRs defined according to any of these methods. For any given embodiment comprising more than one CDR, the CDRs (or other residues of an antibody) may be defined according to any of the Kabat, Chothia, North, extension, AbM, contact and / or conformational definitions.

[0243] The residues in the variable domain are numbered according to Kabat, which is a numbering system for heavy chain variable domains or light chain variable domains compiled for antibodies. See Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD. Using this numbering system, the actual linear amino acid sequence can contain less or additional amino acids, which correspond to the shortening of the FR or CDR of the variable domain or the insertion thereof. For example, the heavy chain variable domain can include a single amino acid insertion after residue 52 of H2 (according to residue 52a of Kabat) and an insertion residue after heavy chain FR residue 82 (e.g., according to residues 82a, 82b and 82c of Kabat). The Kabat numbering of residues can be determined for a given antibody by comparing the antibody sequence with a "standard" Kabat numbering sequence in a homology region. Various algorithms for specifying Kabat numbers are available. The algorithm implemented in Abysis version 2.3.3 release (www.abysis.org) is used herein to assign Kabat numbering to the variable regions CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3.

[0244] Specific amino acid residue positions in antibodies may also be numbered according to Kabat.

[0245] "Framework" (FR) residues are antibody variable domain residues other than CDR residues. The VH or VL domain framework comprises four framework subregions FR1, FR2, FR3, and FR4, with CDRs interspersed between them, in the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3--FR4.

[0246] "Epitope" refers to a region or area of ​​an antigen to which an antibody specifically binds, e.g., a region or area comprising residues that interact with the antibody. An epitope can be linear or conformational.

[0247] The term "paratope" is derived from the definition of "epitope" above by reversing the concept and refers to a region or area of ​​an antibody molecule that participates in the binding of an antigen, e.g., a region or area comprising residues that interact with the antigen. A paratope can be linear or conformational (e.g., discontinuous residues in a CDR).

[0248] A variety of experimental and computational epitope mapping methods can be used to define and characterize the epitope / paratope of a given antibody / antigen binding pair at varying levels of detail. Experimental methods include mutagenesis, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, hydrogen / deuterium exchange mass spectrometry (HX-MS), and various competitive binding methods.

[0249] At its most detailed level, the epitope / paratope of an interaction between an antibody (Ab) and an antigen (Ag) can be defined by defining the spatial coordinates of the atomic contacts present in the Ag-Ab interaction and information about their relative contributions to the binding thermodynamics. At one level, the epitope / paratope residues can be characterized by defining the spatial coordinates of the atomic contacts between the Ag and the Ab. In one aspect, the epitope / paratope residues can be defined by specific criteria, such as the distance between atoms in the Ab and Ag (e.g., equal to or less than about 100 nm from the heavy atoms of the cognate antibody and the heavy atoms of the antigen). distance). In another aspect, the epitope / paratope residues can be characterized as participating in hydrogen bond interactions with cognate antibodies / antigens or with water molecules that also hydrogen bond with cognate antibodies / antigens (water-mediated hydrogen bonding). In another aspect, the epitope / paratope residues can be characterized as forming salt bridges with residues of cognate antibodies / antigens. In another aspect, the epitope / paratope residues can be characterized as residues that have a non-zero change in buried surface area (BSA) due to interaction with cognate antibodies / antigens. At a less detailed level, the epitope / paratope can be characterized by function, for example, by competitive binding with other Abs. The epitope / paratope can also be defined more generally as comprising amino acid residues whose substitution by another amino acid will alter the characteristics of the interaction between the Ab and the Ag (e.g., alanine scanning).

[0250] An antibody that "preferentially binds" or "specifically binds" (used interchangeably herein) to an epitope is a term well known in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with a greater affinity than it reacts or associates with alternative cells or substances. An antibody "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to a CDCP1 epitope is an antibody that binds to this epitope with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other CDCP1 epitopes or non-CDCP1 epitopes. By reading this definition, it should also be understood that, for example, an antibody (or part or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Therefore, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Generally speaking, but not necessarily, reference to binding refers to preferential binding. "Specific binding" or "preferential binding" includes compounds that recognize and bind to a specific molecule, but do not substantially recognize or bind to other molecules in the sample, such as proteins, nucleic acids, antibodies, etc. For example, an antibody that recognizes and binds to its cognate antigen in a sample, but does not substantially recognize or bind to other molecules in the sample specifically binds to the cognate antigen. Therefore, under specified assay conditions, the specified binding portion (e.g., antibody or its antigen-binding portion thereof) preferentially binds to a specific target molecule and does not bind to other components present in the test sample in a large amount.

[0251] A variety of assays can be used to select antibodies or peptides that specifically bind to a target molecule. For example, solid phase ELISA immunoassays, immunoprecipitation, BIAcore TM (GE Healthcare, Piscataway, NJ), fluorescence-activated cell sorting (FACS), Octet TM (FortéBio, Inc., Menlo Park, CA) and Western blot analysis are among the many assays that can be used to identify antibodies that specifically react with an antigen or receptor or its ligand binding portion, or that specifically bind to a cognate ligand or binding partner. Typically, a specific or selective reaction will be at least two times the background signal or noise, and more typically more than 10 times the background, and even more specifically, when the equilibrium dissociation constant (K D) value is ≤1 μM, such as ≤100 nM, ≤10 nM, ≤100 pM, ≤10 pM, or ≤1 pM. An antibody is said to “specifically bind” an antigen when the antigen-binding protein (Ap) value is ≤1 μM, such as ≤100 nM, ≤10 nM, ≤100 pM, ≤10 pM, or ≤1 pM.

[0252] As used herein with respect to antibodies, the term "competition" refers to the fact that the binding of a first antibody, or its antigen-binding portion thereof, to an antigen reduces the subsequent binding of a second antibody, or its antigen-binding portion thereof, to the same antigen. Generally, binding to the first antibody results in steric hindrance, conformational change, or binding to a common epitope (or portion thereof) such that the binding of the second antibody to the same antigen is reduced. Standard competition assays can be used to determine whether two antibodies compete with each other. One suitable assay for antibody competition involves the use of Biacore technology, which can be measured using surface plasmon resonance (SPR) technology, typically using biosensor systems (e.g., The degree of interaction can be measured using a SPR system. For example, SPR can be used in an in vitro competitive binding inhibition assay to determine the ability of one antibody to inhibit the binding of a second antibody. Another assay for measuring antibody competition uses an ELISA-based method.

[0253] In addition, a high-throughput method for "binning" antibodies based on competition between antibodies is described in International Patent Application No. WO2003 / 48731. Competition exists if one antibody (or fragment) reduces the binding of another antibody (or fragment) to CDCP1. For example, a sequential binding competition assay can be used in which different antibodies are added sequentially. A first antibody can be added to achieve near-saturated binding. Then, a second antibody is added. If the binding of the second antibody to ROB02 is undetectable or significantly reduced (e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%) compared to a parallel assay in the absence of the first antibody (which value can be set to 100%), the two antibodies are considered to compete with each other.

[0254] "Antibody binding portion" comprises a portion of a full-length antibody, typically its antigen binding or variable region. Examples of antigen binding portions include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; fragments produced by Fab expression libraries, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity determining regions), and epitope binding fragments of any of the above items that immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In some embodiments, the antibody or its antigen binding portion is selected from a monoclonal antibody, a polyclonal antibody, an antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, a single-chain Fv, a diabody, a linear antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, and a fusion protein comprising the antigen binding portion of an antibody.

[0255] As used herein, the term monoclonal antibody refers to the antibody obtained from a substantially homogeneous antibody population, that is, except for the possible naturally occurring mutation that may exist in a small amount, the individual antibodies constituting the population are identical. Monoclonal antibodies have a high degree of specificity (for a single antigenic site). In addition, in contrast to the polyclonal antibody preparation comprising different antibodies for different determinants (epitopes), each monoclonal antibody is for a single determinant on the antigen. Except for its specificity, the advantage of monoclonal antibodies is that they can be synthesized but not contaminated by other antibodies. The modifier "monoclonal" indicates the characteristic of the antibody obtained from a substantially homogeneous antibody population and should not be construed as needing to produce the antibody by any ad hoc method. For example, the monoclonal antibody used according to the present invention can be prepared by the hybridoma method described by people such as Kohler, (1975) Nature 256:495, or can be prepared by a recombinant DNA method.

[0256] Fv is the smallest antibody fragment that contains a complete antigen recognition and antigen binding site. This region consists of a dimer of a heavy chain variable domain and a light chain variable domain in tight non-covalent association. It is the interaction of the three hypervariable regions of each variable domain that defines the antigen binding site on the surface of the VH-VL dimer. The six hypervariable regions together confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind to an antigen, although its affinity is lower than that of the complete binding site.

[0257] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a small number of residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab' in which one or more cysteine ​​residues of the constant domains have at least one free thiol group is referred to herein as Fab'SH. F(ab')2 antibody fragments were initially produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0258] The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0259] Single-chain Fv or scFv refers to a single-chain variable region antibody fragment that comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0260] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, comprising a variable heavy domain (VH) connected to a variable light domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and establish two antigen-binding sites.

[0261] Inhuman (for example rodent) antibody of humanized form is the chimeric antibody containing the minimum sequence that is derived from non-human immunoglobulin.In most cases, humanized antibody is the human immunoglobulin (receptor antibody) (acceptor antibody) wherein the residue from acceptor hypervariable region is replaced by the hypervariable region residue with required specificity, affinity and ability from non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate.In some cases, the framework region (FR) residue of human immunoglobulin is replaced by corresponding non-human residue.In addition, humanized antibody can comprise the residue that acceptor antibody or donor antibody do not exist.Carry out these modifications so that antibody performance is further improved.In general, humanized antibody will comprise at least one and usually two variable domains substantially all, wherein all or substantially all of hypervariable loops correspond to the hypervariable loops of non-human immunoglobulin, and all or substantially all of FRs are the FRs of human immunoglobulin sequences.

[0262] An isolated antibody is one that has been identified and separated and / or recovered from components of its natural environment. Contaminant components in its natural environment are substances that will interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. The antibody may be: (1) purified to greater than 95% by weight of the antibody as determined by the Lowry method, or greater than 99% by weight; (2) purified to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup protein sequencer; or (3) purified to homogeneity as determined by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. Isolated antibodies include antibodies in situ within recombinant cells, as at least one component of the antibody's natural environment will not be present. However, typically, isolated antibodies are prepared by at least one purification step.

[0263] In some aspects, the present invention provides antibodies and antigen-binding fragments thereof that specifically bind to CDCP1. The sequences of exemplary antibodies are shown in Table 10. As shown in the Examples (see, e.g., Examples 11 and 19), in some embodiments, the antibodies of the present invention are internalized after binding to CDCP1 on mammalian cells.

[0264] In some embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds CDCP1 comprises: (i) a VH comprising: (a) CDRH1 comprising the amino acid sequence of SEQ ID NO: 2, (b) CDRH2 comprising the amino acid sequence of SEQ ID NO: 3, and (c) CDRH3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 40, and SEQ ID NO: 45; and (ii) a VL comprising: (a) CDRL1 comprising the amino acid sequence of SEQ ID NO: 12, (b) CDRL2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) CDRL3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 31.

[0265] In some embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds CDCP1 comprises: (i) a VH comprising: (a) CDRH1 comprising the amino acid sequence of SEQ ID NO: 2, (b) CDRH2 comprising the amino acid sequence of SEQ ID NO: 3, and (c) CDRH3 comprising the amino acid sequence of SEQ ID NO: 27; and (ii) a VL comprising: (a) CDRL1 comprising the amino acid sequence of SEQ ID NO: 12, (b) CDRL2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) CDRL3 comprising the amino acid sequence of SEQ ID NO: 31.

[0266] In some embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds CDCP1 comprises: (i) a VH comprising: (a) CDRH1 comprising the amino acid sequence of SEQ ID NO: 2, (b) CDRH2 comprising the amino acid sequence of SEQ ID NO: 3, and (c) CDRH3 comprising the amino acid sequence of SEQ ID NO: 40; and (ii) a VL comprising: (a) CDRL1 comprising the amino acid sequence of SEQ ID NO: 12, (b) CDRL2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) CDRL3 comprising the amino acid sequence of SEQ ID NO: 31.

[0267] In some embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds CDCP1 comprises: (i) a VH comprising: (a) CDRH1 comprising the amino acid sequence of SEQ ID NO: 2, (b) CDRH2 comprising the amino acid sequence of SEQ ID NO: 3, and (c) CDRH3 comprising the amino acid sequence of SEQ ID NO: 45; and (ii) a VL comprising: (a) CDRL1 comprising the amino acid sequence of SEQ ID NO: 12, (b) CDRL2 comprising the amino acid sequence of SEQ ID NO: 13; and (c) CDRL3 comprising the amino acid sequence of SEQ ID NO: 14.

[0268] The antibody or antigen-binding fragment thereof may comprise a VH framework comprising a human germline VH framework sequence. The VH framework sequence may be derived from a human VH1 germline, a VH3 germline, a VH5 germline, or a VH4 germline. For example, a VH framework from the following germlines may be used: IGHV1-46, IGHV3-23, IGHV3-7, or IGHV1-69 (germline names are based on IMGT germline definitions). In some embodiments, the VH framework is entirely IGHV1-46*01 (DP-7) and does not include CDRH3.

[0269] Preferred human germline light chain frameworks are frameworks derived from Vκ or Vλ germlines. For example, VL frameworks from the following germlines can be used: IGKV3D-7, IGKV1-39, or IGKV3-20 (germline names are based on IMGT germline definitions). In some embodiments, the VL framework is IGKV3D-7*01 (DPK23). Alternatively or additionally, the framework sequence can be a human germline consensus framework sequence, such as a human Vλ1 consensus sequence, a VK1 consensus sequence, a VK2 consensus sequence, a VK3 consensus sequence, a VH3 germline consensus sequence, a VH1 germline consensus sequence, a VH5 germline consensus sequence, or a VH4 germline consensus sequence. The sequences of human germline frameworks can be obtained from various public databases (such as V-base, IMGT, NCBI, or Abysis).

[0270] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 26, SEQ ID NO: 39 or SEQ ID NO: 44.

[0271] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a VL comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 30, SEQ ID NO: 36 or SEQ ID NO: 11.

[0272] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 26; and a VL comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 36.

[0273] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 36.

[0274] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 30.

[0275] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 39; and a VL comprising the amino acid sequence of SEQ ID NO: 36.

[0276] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO:44; and a VL comprising the amino acid sequence of SEQ ID NO:11.

[0277] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 1; and a VL comprising the amino acid sequence of SEQ ID NO: 11.

[0278] Any combination of these VH and VL sequences is also encompassed by the present invention.

[0279] In certain embodiments, the antibodies described herein or their antigen-binding fragments include an Fc domain. The Fc domain may be derived from IgA (e.g., IgA1 or IgA2), IgG, IgE, or IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the Fc domain includes the wild-type sequence of the Fc domain. In some embodiments, the Fc domain includes one or more mutations that cause changes in biological activity. For example, mutations can be introduced into the Fc domain to increase the homogeneity during the recombinant protein production process. In some embodiments, the Fc domain is the Fc domain of human IgG1. In some embodiments, the lysine located in the C-terminal position of the Fc domain is missing to increase the homogeneity during the recombinant protein production process. In some embodiments, there is a lysine located in the C-terminal position of the Fc domain.

[0280] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10, 29, 41 or 46.

[0281] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 17, 32 or 37.

[0282] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 29; and a light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 37.

[0283] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29 and a light chain comprising the amino acid sequence of SEQ ID NO: 37 (referred to herein as antibody "CP13E10-54HC-89LCv1").

[0284] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29 and a light chain comprising the amino acid sequence of SEQ ID NO: 32 (referred to herein as antibody "CP13E10-54HC-89LC").

[0285] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 41 and a light chain comprising the amino acid sequence of SEQ ID NO: 37 (referred to herein as antibody "CP13E10-54HCv13-89LCv1").

[0286] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 46 and a light chain comprising the amino acid sequence of SEQ ID NO: 17 (referred to herein as antibody "CP13E10-291").

[0287] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain comprising the amino acid sequence of SEQ ID NO: 17 (referred to herein as antibody "CP13E10").

[0288] Crystal structure studies have shown that CDCP1 is crescent-shaped, and the CDCP1 antibody CP13E10-54HC-89LC is located on the inner side of the crescent. Six CDCP1 residues interact with the antibody near the center of the interface. Therefore, in some embodiments, the isolated antibody or antigen-binding fragment thereof binds to an epitope on CDCP1, wherein the epitope comprises at least one amino acid residue selected from the group consisting of Thr124, Thr160, Ser162, Ala195, Leu196, and His197, according to the numbering of SEQ ID NO: 90.

[0289] In some embodiments, the epitope further comprises at least one amino acid residue selected from the group consisting of Lys45, Leu46, Gly47, Thr48, Pro49, Thr50, Ala53, Pro55, Glu92, Arg173, and Glu242, according to the numbering of SEQ ID NO: 90. In some embodiments, the epitope further comprises at least one amino acid residue selected from the group consisting of Thr56, Tyr57, Thr66, Met67, Ile126, Val171, Arg173, according to the numbering of SEQ ID NO: 90.

[0290] In some embodiments, the epitope further comprises a glycan linked to Asn122 according to the numbering of SEQ ID NO:90.

[0291] The present invention also provides an antibody or antigen-binding fragment thereof that competes with any antibody or antigen-binding fragment thereof described herein, such as any antibody (or antigen-binding fragment thereof) provided herein for binding to CDCP1. For example, if binding of the antibody or antigen-binding portion thereof to CDCP1 blocks subsequent binding of CP13E10-54HC-89LCv1 to CDCP1, then the antibody or antigen-binding portion thereof competes with CP13E10-54HC-89LCv1 for binding to CDCP1.

[0292] The present invention also provides an antibody or antigen-binding fragment thereof that binds to the same CDCP1 epitope as any antibody or antigen-binding fragment thereof described herein, such as any antibody or antigen-binding fragment thereof provided herein. For example, antibody competition assays (and overlapping epitope analysis) can be evaluated by surface plasmon resonance (SPR) or biolayer interferometry (BLI), as described in detail herein.

[0293] The antibodies and antigen-binding fragments provided by the present invention include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chains (ScFv), mutants thereof, fusion proteins comprising antibody portions, domain antibodies (dAb), humanized antibodies, and any other modified configurations of immunoglobulin molecules comprising antigen recognition sites with desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies and antigen-binding fragments can be of mouse, rat, human, or any other origin (including chimeric or humanized antibodies). In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric, humanized, or human antibody. In certain embodiments, the antibody is a human antibody. In certain embodiments, the antibody is a humanized antibody.

[0294] The binding affinity of an antibody can be expressed as the equilibrium dissociation constant (K D ) value, which refers to the dissociation rate of a specific antigen-antibody interaction. D is the dissociation rate (also called the “dissociation rate (k off )") and the association rate or "association rate (k on )”. Therefore, K D Equal to k off / k on (dissociation / association), and expressed as molar concentration (M), and K D The smaller the K, the stronger the binding affinity. D Values ​​can be determined using methods well known in the art. Unless otherwise indicated, "binding affinity" refers to monovalent interactions (intrinsic activity; eg, binding of an antibody to an antigen through a monovalent interaction).

[0295] In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention have an affinity (K) of or less than about 350 nM, about 325 nM, about 323.10 nM, about 300 nM, about 286.44 nM, about 275 nM, about 250 nM, about 232.13 nM, about 225 nM, about 219.13 nM, about 200 nM, about 195.54 nM, about 175 nM, about 158 ​​nM, about 150 nM, about 125 nM, or about 100 nM. D )value.

[0296] In some embodiments, the antibody or antigen-binding fragment thereof has a K of or less than about 95 nM, about 90 nM, about 80 nM, about 79.89 nM, about 75 nM, about 70 nM, about 69.50 nM, about 65 nM, about 63.44 nM, about 60 nM, about 55 nM, about 52.88 nM, about 50 nM, about 45 nM, about 44.50 nM, about 41.99 nM, about 40 nM, about 35 nM, about 30 nM, about 25 nM, about 20 nM, about 10 nM, about 5 nM, or about 1 nM. D Values ​​bind CDCP1.

[0297] In some embodiments, the antibodies or antigen-binding fragments thereof have a K of or less than about 5 nM, about 4.5 nM, about 4 nM, about 3.5 nM, about 3.12 nM, about 3 nM, about 2.90 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 250 pM, about 200 pM, about 150 pM, about 100 pM, about 50 pM, about 40 pM, about 30 pM, about 25 pM, about 20 pM, about 15 pM, about 10 pM, about 5 pM, or about 1 pM. D Values ​​bind CDCP1.

[0298] K D The value of K can be determined directly by well-known methods and can be calculated even for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9: 340-362). For example, a double filter nitrocellulose filter binding assay can be used to establish K D , such as the assay disclosed by Wong and Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). Other standard assays for assessing the binding ability of a ligand, such as an antibody, to a target antigen are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometric analysis, as well as other assays exemplified elsewhere herein.

[0299] To measure binding affinity (K D An exemplary method for ) value is surface plasmon resonance (SPR), which is commonly used in biosensor systems such as system. SPR refers to the system that allows The system detects changes in protein concentration within the biosensor matrix to analyze optical phenomena of real-time biospecific interactions. BIAcore kinetic analysis involves analyzing the binding and dissociation of antigens from a chip to immobilized molecules (e.g., molecules containing an antigen-binding domain) on its surface, or the dissociation of antibodies or antigen-binding fragments from a chip from immobilized antigens.

[0300] In certain embodiments, using T100 or T200 instrument performs SPR measurement. For example, the standard determination conditions of surface plasmon resonance can be based on the antibody immobilization of about 100-500 response units (RU) of IgG on the SPR chip. The purified target protein is diluted to a final concentration within a certain range in buffer and injected at a necessary flow rate (e.g., 10-100 μl / min) to allow calculation of Ka. Dissociation is allowed to establish the dissociation rate, and then 3MMgCl2 (or 20mMNaOH) is used to regenerate the chip surface. The sensorgram is then analyzed using a kinetic evaluation software package. In an exemplary embodiment, the SPR determination is according to the conditions as described in the examples.

[0301] In certain embodiments, a solution-based kinetic exclusion assay (KinExA TM ) to measure binding affinity (K D ) value. In a specific embodiment, KinExA is used TM KinExA measurements were performed using a 3200 instrument (Sapidyne). TM ) is a universal immunoassay platform (essentially a fluorescence spectrophotometer) capable of measuring equilibrium dissociation constants as well as association and dissociation rate constants of antigen / antibody interactions. TM is performed after equilibrium has been achieved, so it is used to measure the K for high-affinity interactions. D KinExA is an advantageous technique for interactions where the off-rate of the interaction may be very slow. TM Methodology may generally be performed as described in Drake et al., (2004) Analytical Biochem. 328, 35-43.

[0302] For determining the K of antibodies D Another approach is to use biolayer interferometry (BLI), typically using a ForteBio Technology (e.g., Octet QKe system). In certain embodiments, BLI measurements are performed as follows: a sensor tip coated with a proprietary anti-human antibody (ForteBio) is immersed in a running buffer (e.g., 10 mM Hepes-buffered saline (HBS) containing 0.05% tween-20) for 120 seconds to stabilize the BLI signal. The antibody is then captured by immersing the sensor in a running buffer solution (the buffer may contain 1-10 ug / mL of antibody) for 300 seconds. The signal is then stabilized by immersing the sensor tip back in the running buffer for 120 seconds. The tip is then transferred to a solution containing the cognate antigen. Antibody-antigen binding is measured within 180 seconds before the sensor tip is transferred to the running buffer to monitor receptor dissociation within 180 seconds. In the case of CDCP1, a 7-point dose response of the antigen is typically measured (the range may be 1-2 nM in two-fold dilutions). In addition, the sensor tip without capture antibody is exposed to the antigen to monitor nonspecific binding of the receptor to the sensor tip. The second reference type also included a tip onto which the antibody was captured but then exposed to only running buffer without antigen. This allowed for double referencing to eliminate nonspecific binding as well as system noise and potential baseline drift due to antibody dissociation from the anti-human Fc sensor tip. The original was subjected to double reference subtraction and then fitted to a 1:1 Langmuir-type binding model to determine affinity and kinetic parameters.

[0303] In some embodiments, CDCP1 is human CDCP1, cynomolgus monkey CDCP1, or mouse CDCP1. Generally speaking, anti-CDCP1 antibodies should bind to CDCP1 with high affinity. It is desirable that anti-CDCP1 antibodies have a binding affinity (K) to human CDCP1 in the low nanomolar range (e.g., about 40 nM or lower). D In some embodiments, CDCP1 is human CDCP1, and K D The value is about 40 nM, about 45 nM or about 50 nM. In some embodiments, the CDCP1 is cynomolgus monkey CDCP1, and K D The values ​​are about 62 nM, about 64 nM, about 66 nM, about 68 nM, or about 70 nM.

[0304] Anti-drug conjugates

[0305] Antibody-drug conjugates or ADCs are an important class of highly effective biopharmaceutical drugs designed as targeted therapies for the treatment of cancer patients. Unlike chemotherapy, ADCs are intended to target and kill only cancer cells and excess healthy cells. ADCs are complex molecules consisting of antibodies connected to a biologically active cytotoxic (anti-cancer) payload or drug. As used herein, "antibody-drug conjugates" refer to an antibody or a portion of an antibody that is covalently linked to a cytotoxic or cytostatic drug / agent, wherein the drug / agent is also referred to herein as a "payload."

[0306] The term prodrug refers to a precursor or derivative form of a pharmaceutically active substance that has lower cytotoxicity to tumor cells than the parent drug and can be enzymatically activated or converted into a more active parent form. The prodrugs of the present disclosure include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine, and other 5-fluorouracil prodrugs that can be converted into more active non-cytotoxic drugs. Examples of cytotoxic drugs that can be derivatized into prodrug forms used in the present disclosure include, but are not limited to, those chemotherapeutic agents.

[0307] The antibody and the drug can be linked directly, or they can be linked via a moiety called a linker. A linker or linker refers to a chemical moiety comprising a covalent bond or chain of atoms that covalently links the antibody to the drug moiety. In various embodiments, the linker is designated L. Linkers include divalent groups such as alkyldiyl, aryldiyl, heteroaryldiyl, moieties such as: -(CR2)nO(CR2)n-, repeating units of alkoxy (e.g., polyethylenoxy, PEG, polymethyleneoxy) and repeating units of alkylamino (e.g., polyethyleneamino, Jeffamine TM ); and diacids and amides, including succinates, succinamides, diglycolates, malonates, and caproamides.

[0308] Anti-CDCP1 antibodies and conjugation sites

[0309] In some aspects, the present invention provides a conjugate (or immunoconjugate) of a CDCP1 antibody or antigen-binding fragment thereof as described herein, wherein the antibody or antigen-binding fragment is directly or indirectly conjugated via a linker to a drug (also referred to herein as a payload) for targeted immunotherapy (e.g., an antibody-drug conjugate, also referred to as an ADC). For example, a drug (e.g., a cytotoxic agent, which encompasses anti-tumor agents, etc.) can be linked or conjugated to a CDCP1 antibody or antigen-binding fragment thereof as described herein to deliver the drug moiety locally to cells expressing CDCP1 on the cell surface (e.g., tumors expressing CDCP1).

[0310] Methods for conjugating cytotoxic or other therapeutic agents to antibodies have been described in various publications. For example, antibodies can be chemically modified via lysine side chain amines or via cysteine ​​sulfhydryl groups activated by reduction of interchain disulfide bonds for the conjugation reaction to occur. See, for example, Tanaka et al., FEBS Letters 579:2092-2096, 2005 and Gentle et al., Bioconjugate Chem. 15:658-663, 2004. Reactive cysteine ​​residues engineered at specific sites in antibodies for specific drug conjugation with defined stoichiometry have also been described. See, for example, Junutula et al., Nature Biotechnology, 26:925-932, 2008. The use of tags containing the acyl donor glutamine or conjugation of reactive endogenous glutamine (i.e., the ability to form a covalent bond as an acyl donor) by polypeptide engineering in the presence of a transglutaminase and an amine (e.g., a cytotoxic agent comprising or linked to a reactive amine) is also described in International Applications WO 2012 / 059882 and WO 2015 / 015448, each of which is herein incorporated by reference in its entirety.

[0311] In some aspects, CDCP1 ADCs can be generated using site-specific conjugation of linker-payload moieties by engineering one or more reactive cysteine ​​residues into the constant domain of an anti-CDCP1 antibody (see, e.g., WO 2013 / 093809, US 2014 / 0127211, US 2017 / 0216452, and WO 2017 / 093844, each of which is incorporated herein by reference in its entirety). For conjugation to a drug or payload, one or more amino acid residues of the heavy chain of an anti-CDCP1 antibody can be substituted with another amino acid, such as a cysteine ​​residue. In one aspect, the present invention provides an anti-CDCP1 antibody or an antigen-binding fragment thereof, comprising an antibody heavy chain constant region comprising engineered cysteine ​​residues at the following positions according to the Eu index of Kabat: 118 (according to 114 of Kabat), 246, 249, 265, 267, 270, 276, 278, 283, 290, 292, 293, 294, 300, 302, 303, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360 8, 320, 327, 332, 333, 334, 336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 375, 376, 378, 380, 382, ​​386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416, 418, 419, 421, 428, 431, 432, 437, 438, 439, 443 or 444 or any combination thereof). In particular, position 118 (114 according to Kabat), 290, 334, 347, 373, 375, 380, 388, 392, 421, 443 or any combination thereof can be used. Additional cysteine ​​substitutions may be introduced.

[0312] In another aspect, the present invention provides an anti-CDCP1 antibody or an antigen-binding fragment thereof, comprising a heavy chain constant domain comprising an engineered cysteine ​​residue (K290C) at position 290, as numbered according to the Eu index of Kabat.

[0313] For conjugation to a drug or payload, one or more amino acid residues of the anti-CDCP1 antibody light chain constant domain may be substituted with another amino acid, such as a cysteine ​​residue (see, e.g., WO 2013 / 093809, US 2014 / 0127211, US 2017 / 0216452, and WO 2017 / 093844, each of which is incorporated herein by reference in its entirety). In one aspect, the present invention provides an anti-CDCP1 antibody or antigen-binding fragment thereof, comprising an antibody light chain constant region comprising an engineered cysteine ​​residue at positions 110, 111, 125, 149, 155, 158, 161, 183, 185, 188, 189, 191, 197, 205, 207, 208, or 210, or any combination thereof, according to Kabat numbering. Additional cysteine ​​substitutions may be introduced.

[0314] In another aspect, the present invention provides an anti-CDCP1 antibody or an antigen-binding fragment thereof, comprising a light chain constant domain comprising an engineered cysteine ​​residue at position 183 (κK183C) according to Kabat numbering.

[0315] In some respects, the invention provides an antibody-drug conjugate comprising an antibody or antigen-binding fragment, the antibody or antigen-binding fragment having a heavy chain and / or light chain constant region, the heavy chain and / or light chain constant region comprising an engineered cysteine ​​residue for site-specific conjugation. In some respects, the antibody-drug conjugate has a heavy chain constant region, the heavy chain constant region comprising the numbering according to the Eu index of Kabat, the engineered cysteine ​​residue (K290C) at position 290. In some respects, the antibody-drug conjugate has a light chain constant region, the light chain constant region comprising the numbering according to Kabat, the engineered cysteine ​​residue (κ K183C) at position 183. In some respects, the antibody-drug conjugate has a heavy chain constant region, the heavy chain constant region comprising the numbering according to the EU index of Kabat, the engineered cysteine ​​residue (K290C) at position 290; and a light chain constant region, the light chain constant region comprising the numbering according to Kabat, the engineered cysteine ​​residue (κ K183C) at position 183.

[0316] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 19, 33 or 42.

[0317] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 21, 34 or 38.

[0318] In some embodiments, the isolated antibody or antigen-binding fragment thereof as described in any one of E46-E56 comprises a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID No: 33; and a light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 38.

[0319] In some embodiments, the isolated antibody or antigen-binding fragment thereof of any one of E46-E57 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 33; and a light chain comprising the amino acid sequence of SEQ ID NO: 38.

[0320] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and a light chain comprising the amino acid sequence of SEQ ID NO: 34.

[0321] In some embodiments, the isolated antibody or antigen-binding fragment thereof of any one of E46-E56 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 42 and a light chain comprising the amino acid sequence of SEQ ID NO: 38.

[0322] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19; and a light chain comprising the amino acid sequence of SEQ ID NO: 21.

[0323] In another aspect, CDCP1 ADCs can be generated using site-specific conjugation technology by one or more engineered acyl donor glutamine-containing tags or reactive endogenous glutamine residues in the constant region of anti-CDCP1 antibodies. Methods for preparing antibodies for site-specific conjugation via acyl donor glutamine-containing tags or glutamine residues are described in PCT International Publication Nos. WO2012 / 059882 and WO2015 / 015448, each of which is incorporated herein by reference in its entirety.

[0324] In some aspects, the acyl donor glutamine-containing tag comprises at least one glutamine (Q) and can be attached to a specific position of the heavy and / or light chain (i.e., at the N-terminus, C-terminus, or internally). In another aspect, the acyl donor glutamine-containing tag can comprise an amino acid sequence selected from the group consisting of LLQG (SEQ ID NO: 91). In some aspects, the acyl donor glutamine-containing tag is inserted into a specific position of the heavy and / or light chain (i.e., at the N-terminus, C-terminus, or internally). In some aspects, an anti-CDCP1 antibody can comprise an acyl glutamine-containing tag having the amino acid sequence LLQG (SEQ ID NO: 91) inserted after position 135 and before position 136 of the heavy chain according to the Eu index of Kabat.

[0325] In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more substitutions selected from the group consisting of: N297A and K222R, numbered according to the Eu index of Kabat. In some embodiments, the antibody or antigen-binding fragment thereof comprises two substitutions, N297A and K222R, numbered according to the Eu index of Kabat.

[0326] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25, 35 or 43.

[0327] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 17, 32 or 37.

[0328] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 35; and a light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 37.

[0329] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35 and a light chain comprising the amino acid sequence of SEQ ID NO: 37.

[0330] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35 and a light chain comprising the amino acid sequence of SEQ ID NO: 32.

[0331] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:43; and a light chain comprising the amino acid sequence of SEQ ID NO:37.

[0332] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 25; and a light chain comprising the amino acid sequence of SEQ ID NO: 17.

[0333] Nucleic acids, vectors and host cells

[0334] The present invention also provides polynucleotides encoding any of the antibodies (including antibody fragments and modified antibodies described herein). The present invention also provides a method for preparing any of the polynucleotides described herein. The polynucleotides can be prepared and expressed by procedures known in the art.

[0335] Standard sequencing techniques can be used to determine the sequence of the desired antibody, defined antibody fragment, or antigen-binding fragment thereof, as well as the nucleic acid encoding such an antibody or fragment thereof. The nucleic acid sequence encoding the desired antibody, defined antibody fragment, or antigen-binding fragment thereof can be inserted into various vectors (e.g., cloning and expression vectors) for recombinant production and characterization. Nucleic acid encoding the heavy chain, defined antibody fragment, or antigen-binding fragment of the heavy chain and nucleic acid encoding the light chain, defined antibody fragment, or antigen-binding fragment of the light chain can be cloned into the same vector or different vectors.

[0336] In one aspect, the present invention provides a polynucleotide encoding the amino acid sequence of any one of the following CDCP1 antibodies and antigen-binding fragments thereof: CP13E10, CP13E10-183 / 290, CP13E10-H7C-K222R-N297A, CP13E10-54HC-89LC, CP13E10-54HC-89LC-183 / 290, CP13E10-54HC-89LC-H7C-K222R-N297A, CP13E10-54HC-89 LCv1, CP13E10-54HC-89LCv1-183 / 290, CP13E10-54HC-89LCv1-H7C-K222R-N297A, CP13E10-54HCv13-89LCv1, CP13E10-54HCv13-89LCv1-183 / 290, CP13E10-54HCv13-89LCv1-H7C-K222R-N297A, CP13E10-291, Antibody 23, Antibody 24, and Antibody 76. The polynucleotides encoding the above amino acid sequences encode an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, and more preferably identical, to the amino acid sequence of an antibody of the invention or an antigen-binding fragment thereof as disclosed herein.

[0337] The present invention provides polynucleotides encoding the amino acid sequences of antibodies or antigen-binding fragments thereof, wherein the antibodies or antigen-binding fragments thereof bind to substantially the same epitope as an antibody selected from the group consisting of: CP13E10, CP13E10-183 / 290, CP13E10-H7C-K222R-N297A, CP13E10-54HC-89LC, CP13E10-54HC-89LC-183 / 290, CP13E10-54HC-89LC-H7C-K222R-N297A, CP13E10 0-54HC-89LCv1, CP13E10-54HC-89LCv1-183 / 290, CP13E10-54HC-89LCv1-H7C-K222R-N297A, CP13E10-54HCv13-89LCv1, CP13E10-54HCv13-89LCv1-183 / 290, CP13E10-54HCv13-89LCv1-H7C-K222R-N297A, CP13E10-291, Antibody 23, Antibody 24 and Antibody 76.

[0338] The present invention provides a polynucleotide encoding the amino acid sequence of an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof competes for binding to CXCR5 with an antibody selected from the group consisting of: CP13E10, CP13E10-183 / 290, CP13E10-H7C-K222R-N297A, CP13E10-54HC-89LC, CP13E10-54HC-89LC-183 / 290, CP13E10-54HC-89LC-H7C-K222R-N297A, CP13E10 0-54HC-89LCv1, CP13E10-54HC-89LCv1-183 / 290, CP13E10-54HC-89LCv1-H7C-K222R-N297A, CP13E10-54HCv13-89LCv1, CP13E10-54HCv13-89LCv1-183 / 290, CP13E10-54HCv13-89LCv1-H7C-K222R-N297A, CP13E10-291, Antibody 23, Antibody 24 and Antibody 76.

[0339] The present invention provides polynucleotides encoding one or more proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-74.

[0340] In some embodiments, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO: 75. In some embodiments, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO: 76. In some embodiments, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO: 77. In some embodiments, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO: 78. In some embodiments, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO: 79. In some embodiments, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO: 80. In some embodiments, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO: 81. In some embodiments, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO: 82. In some embodiments, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO: 83. In some embodiments, the isolated nucleic acid comprises the nucleotide sequence of SEQ ID NO: 84.

[0341] The invention provides cells comprising one or more nucleic acid molecules as set forth in one or more of SEQ ID NOs: 75-84. The invention provides cells comprising one or more nucleic acid molecules as set forth in SEQ ID NOs: 85 and 86.

[0342] In another aspect, the present invention provides polynucleotides encoding anti-CDCP1 antibodies and variants thereof, wherein such variant polynucleotides share at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of the specific nucleic acid sequences disclosed herein. These amounts are not meant to be limiting, and increments between the recited percentages are specifically contemplated as part of this disclosure.

[0343] The present invention provides polypeptides encoded by the nucleic acid molecules described herein.

[0344] In one embodiment, the VH and VL domains, or antigen-binding fragments thereof, or full-length HC or LC, are encoded by separate polynucleotides. Alternatively, the VH and VL, or antigen-binding fragments thereof, or HC and LC, are all encoded by a single polynucleotide.

[0345] Polynucleotides complementary to any such sequences are also encompassed by the present disclosure. Polynucleotides can be single-stranded (coding or antisense) or double-stranded and can be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules and mRNA molecules that contain introns and correspond to DNA molecules in a one-to-one manner. Additional coding sequences or non-coding sequences can, but need not, be present in the polynucleotides of the present disclosure, and polynucleotides can, but need not, be connected to other molecules and / or carrier materials.

[0346] Polynucleotide can comprise native sequence (that is, endogenous sequence encoding antibody or its part) or can comprise variant of such sequence.Polynucleotide variant contains one or more substitutions, additions, deletions and / or insertions, so that relative to natural immune reactive molecules, the immunoreactivity of the encoded polypeptide does not reduce.The impact on the immunoreactivity of the encoded polypeptide can usually be assessed as described herein.In some embodiments, variant shows at least about 70% identity, in some embodiments at least about 80% identity, in some embodiments at least about 90% identity and in some embodiments at least about 95% identity with the polynucleotide sequence encoding natural antibody or its part.These amounts are not meant to be restrictive, and the increments between the enumerated percentages are particularly contemplated as a part of the present disclosure.

[0347] Two polynucleotide or polypeptide sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20 consecutive positions, typically 30 to about 75 or 40 to about 50 consecutive positions, over which a sequence can be compared to a reference sequence of the same number of consecutive positions after the two sequences have been optimally aligned.

[0348] Optimal alignment of sequences for comparison can be performed using Bioinformatics software suite ( Inc., Madison, WI) Program, using default parameters. This program includes several alignment schemes described in the following references: Dayhoff, MO, 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC, Vol. 5, Supplement 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes, pp. 626-645; Methods in Enzymology, Vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, 1989, CABIOS 5: 151-153; Myers, EW and Muller W., 1988, CABIOS 4:11-17; Robinson, ED, 1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4: 406-425; Sneath, PHA and Sokal, RR, 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJ and Lipman, DJ, 1983, Proc. Natl. Acad. Sci. USA 80:726-730.

[0349] In some embodiments, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may contain 20% or less, typically 5% to 15% or 10% to 12% additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the identical nucleic acid base or amino acid residue occurs in the two sequences to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to produce the percentage of sequence identity.

[0350] Variants may also or alternatively be substantially homologous to a native gene or a portion or complement thereof.Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a native antibody (or a complementary sequence).

[0351] Suitable "moderately stringent conditions" include a prewash in 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization overnight at 50°C-65°C, 5X SSC; and then washing twice each in 2X, 0.5X, and 0.2X SSC containing 0.1% SDS at 65°C for 20 minutes.

[0352] As used herein, "high stringency conditions" or "high stringency conditions" are those that: (1) use low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) use a denaturing agent, such as formamide, e.g., 50% (v / v) formamide in 50 mM sodium phosphate buffer, pH 6.5, with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 750 mM sodium chloride, 75 mM sodium citrate at 42°C during hybridization; or (3) use 50% formamide, 5X SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X Denhardt's solution, sonicated salmon sperm DNA (50 μg / mL), 0.1% SDS, and 10% dextran sulfate, with washes in 0.2X SSC (sodium chloride / sodium citrate) at 42° C. and 50% formamide at 55° C., followed by a high stringency wash consisting of 0.1X SSC with EDTA at 55° C. Those skilled in the art will recognize how to adjust temperature, ionic strength, etc. as needed to accommodate factors such as probe length.

[0353] Those of ordinary skill in the art will appreciate that, due to the degeneracy of the genetic code, there are many nucleotide sequences encoding polypeptides as described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any natural gene. However, the present disclosure specifically contemplates polynucleotides that vary due to differences in codon usage. In addition, alleles of genes comprising the polynucleotide sequences provided herein are within the scope of the present disclosure. Alleles are endogenous genes that change due to one or more mutations (such as deletions, additions, and / or substitutions) of nucleotides. The mRNA and protein obtained may, but need not, have a changed structure or function. Standard techniques (such as hybridization, amplification, and / or database sequence comparisons) can be used to identify alleles.

[0354] Polynucleotides of the present disclosure can be obtained using chemical synthesis, recombinant methods or PCR. The method of chemical polynucleotide synthesis is well known in the art and need not be described in detail herein. Those skilled in the art can use the sequence provided herein and a commercial DNA synthesizer to produce the desired DNA sequence.

[0355] In order to prepare polynucleotides using recombinant methods, the polynucleotides comprising the desired sequence can be inserted into a suitable vector, and then the vector can be introduced into a suitable host cell for replication and amplification, as further discussed herein. The polynucleotides can be inserted into the host cell by any means known in the art. Cells are transformed by introducing exogenous polynucleotides through direct uptake, endocytosis, transfection, F-mating or electroporation. Once introduced, the exogenous polynucleotides can be maintained in the cell as a non-integrating vector (such as a plasmid) or integrated into the host cell genome. The polynucleotides amplified in this way can be separated from the host cell by methods well known in the art. See, for example, Sambrook et al., 1989.

[0356] Alternatively, PCR allows for the replication of DNA sequences. PCR techniques are well known in the art and are described in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202, and in PCR: The Polymerase Chain Reaction, Mullis et al., eds., Birkauswer Press, Boston, 1994.

[0357] RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into an appropriate host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to those skilled in the art, for example, as described in Sambrook et al., 1989.

[0358] In some embodiments, the first vector comprises a polynucleotide encoding a heavy chain, and the second vector comprises a polynucleotide encoding a light chain. In some embodiments, the first vector and the second vector are transfected into a host cell in similar amounts (such as similar molar amounts or similar masses). In some embodiments, the first vector and the second vector are transfected into a host cell at a molar ratio or mass ratio of 5:1 to 1:5. In some embodiments, a mass ratio of 1:1 to 1:5 between a vector encoding a heavy chain and a vector encoding a light chain is used. In some embodiments, a mass ratio of 1:2 between a vector encoding a heavy chain and a vector encoding a light chain is used.

[0359] carrier

[0360] In some embodiments, a vector is selected that is optimized for expression of the polypeptide in CHO or CHO-derived cells or in NSO cells. Exemplary vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0361] Suitable cloning and expression vectors can include various components, such as promoters, enhancers and other transcriptional regulatory sequences. Carriers can also be constructed to allow antibody variable domains to be subsequently cloned into different carriers. Suitable cloning vectors can be constructed according to standard techniques, or can be selected from a large number of cloning vectors available in the art. Although the cloning vector selected can vary according to the host cell used in accordance with the expectation, useful cloning vectors will generally have the ability to self-replicate, can have a single target for specific restriction endonucleases, and / or can carry genes that can be used to select markers for cloning containing carriers. Suitable examples include plasmids and bacterial viruses, for example, pUC18, pUC19, Bluescript (for example, pBS SK+) and derivatives thereof, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors can be obtained from commercial suppliers such as BioRad, Stratagene and Invitrogen. Expression vectors are further provided. An expression vector is typically a replicable polynucleotide construct containing a polynucleotide according to the present disclosure. This implies that the expression vector can typically be replicated in a host cell as an episome or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors disclosed in PCT Publication No. WO 87 / 04462, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vectors. Vector components may typically include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcription control elements (such as promoters, enhancers, and terminators). In order to express (i.e., translate), one or more translation control elements, such as ribosome binding sites, translation initiation sites, and stop codons, are typically also required.

[0362] The vector containing the polynucleotide of interest can be introduced into the host cell by any of a variety of appropriate means, including electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microprojectile bombardment; lipofection; and infection (for example, in the case where the vector is an infectious agent such as vaccinia virus). The choice of the vector or polynucleotide to be introduced will generally depend on the characteristics of the host cell.

[0363] host cells

[0364] Suitable host cell recombinant preparation antibody or its Fab can be used.The nucleic acid encoding antibody or its Fab can be cloned into expression vector, and then expression vector is introduced into host cell, such as Escherichia coli cells, yeast cells, insect cells, monkey COS cells, Chinese hamster ovary (CHO) cells or myeloma cells (wherein said cell otherwise can not produce immunoglobulin), thereby obtaining the synthesis of antibody in recombinant host cells. In many cells well known in the art, preferred host cells include Chinese hamster ovary (CHO) cells, human embryonic kidney HEK-293 cells or Sp2.0 cells. Antibody fragment can be produced by proteolysis or other degradation of full-length antibodies, by recombinant method or by chemical synthesis. The polypeptide fragment of antibody (particularly about 50 amino acid whose shorter polypeptide at the most) can be easily prepared by chemical synthesis. The method for chemical synthesis of protein and peptide is known in the art and commercially available.

[0365] In various embodiments, the anti-CDCP1 heavy chain and / or anti-CDCP1 light chain can be expressed in prokaryotic cells such as bacterial cells; or in eukaryotic cells such as fungal cells (e.g., yeast), plant cells, insect cells, and mammalian cells. Such expression can be performed, for example, according to methods known in the art. Exemplary eukaryotic cells that can be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44.Lec13 CHO cells, and FUT8 CHO cells; cells (Crucell); and NSO cells. In some embodiments, the anti-CDCP1 heavy chain and / or anti-CDCP1 light chain can be expressed in yeast. See, for example, U.S. Publication No. US2006 / 0270045 A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to perform the desired post-translational modification of the anti-CDCP1 heavy chain and / or anti-CDCP1 light chain. For example, in some embodiments, CHO cells produce polypeptides that are more sialylated than the same polypeptides produced in 293 cells.

[0366] Introduction of one or more nucleic acids into the desired host cells can be accomplished by any method, including but not limited to calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected into the desired host cells according to any suitable method.

[0367] Anti-CXCR5 antibodies can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include CDCP1 ECD and ligands that bind to the constant region of the antibody. For example, protein A, protein G, protein A / G, or antibody affinity columns can be used to bind to the constant region and purify anti-CXCR5 antibodies. Hydrophobic interaction chromatography (e.g., butyl or phenyl columns) may also be suitable for purifying some polypeptides. Many methods for purifying polypeptides are known in the art.

[0368] In some embodiments, anti-CDCP1 antibodies are produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21: 695-713 (2003).

[0369] drug

[0370] Drugs that can be used to prepare the disclosed CDCP1 ADCs include any substance with biological or detectable activity, such as therapeutic agents, detectable labels, binding agents, and the like, as well as prodrugs that are metabolized in vivo to active agents. The drug can also be a drug derivative in which the drug has been functionalized to enable conjugation to the antibodies of the invention.

[0371] Therapeutic agents are agents that exert cytotoxic, cytostatic and / or immunomodulatory effects on cancer cells or activated immune cells. Examples of therapeutic agents include cytotoxic agents, chemotherapeutic agents, cytostatic agents and immunomodulators. Cytotoxicity refers to the depletion, elimination and / or killing of target cells. Cytotoxic agents refer to agents that have cytotoxic and / or cytostatic effects on cells. Cytostatic effects refer to the inhibition of cell proliferation. Cytostatic agents refer to agents that have cytostatic effects on cells, thereby inhibiting the growth and / or amplification of specific cell subpopulations. Chemotherapeutic agents refer to chemical compounds that can be used to treat cancer. Immunomodulators refer to agents that stimulate an immune response by directly or indirectly inhibiting or reducing the growth of cell subpopulations (i.e., tumor cells) through the production of cytokines and / or antibodies and / or regulating T cell function, thereby making another agent more effective.

[0372] According to the disclosed methods, a CDCP1 ADC having the following can be produced or generated: (a) an antibody or antigen-binding fragment thereof that binds to CDCP1; (b) a linker and (c) a drug. The drug-to-antibody ratio (DAR) or drug loading indicates the number of drug (D) molecules conjugated per antibody. The number of linker-drug moieties attached to the antibody can be any number preferred for developing an ADC. In some aspects, the number of linker-drug moieties per antibody is 4. In other aspects, the number of linker-drug moieties per antibody is 3. In another aspect, the number of linker-drug moieties per antibody is 2. In another aspect, the number of linker-drug moieties per antibody is 1. In other aspects, the number of linker-drug moieties per antibody is greater than 4, such as 5, 6, 7, 8, 9, 10, 11, 12 or greater than 12 linker-drug moieties per antibody. The DAR can be determined by various conventional means, such as UV spectroscopy, mass spectrometry, ELISA assays, radiometry, hydrophobic interaction chromatography (HIC), electrophoresis, and HPLC.

[0373] Examples of cytotoxic agents include, but are not limited to, anthracyclines, auristatins, CC-1065, dolastatin, duocarmycin, enediynes, geldanamycin, maytansine, puromycin, taxanes, vinca alkaloids, SN-38, tubulysin, hemicycline, and stereoisomers, isosteres, analogs, or derivatives thereof. Plant toxins, other biologically active proteins, enzymes (i.e., ADEPT), radioactive isotopes, and photosensitizers (i.e., for photodynamic therapy) may also be used.

[0374] Anthracyclines are derived from Streptomyces bacteria and have been used to treat a variety of cancers, such as leukemia, lymphoma, breast cancer, uterine cancer, ovarian cancer, and lung cancer. Exemplary anthracyclines include, but are not limited to, daunorubicin, doxorubicin (i.e., adriamycin), epirubicin, idarubicin, valrubicin, and mitoxantrone.

[0375] Aplysia and its peptide analogs and derivatives auristatins are highly potent antimitotic agents that have been shown to have anticancer and antifungal activity. See, for example, U.S. Pat. No. 5,663,149 and Pettit et al., Antimicrob. Agents Chemother. 42: 2961-2965, (1998). Exemplary Aplysias and Auristatins include, but are not limited to, Aplysia 10, Auristatin E, Auristatin EB (AEB), Auristatin EFP (AEFP), MMAD (monomethyl auristatin D or monomethyl Aplysia 10), MMAF (monomethyl auristatin F or N-methyl valine-valine-aplysia isoleucine-aplysia proline-phenylalanine), MMAE (monomethyl auristatin E or N-methyl valine-valine-aplysia isoleucine-aplysia proline-norephedrine), 5-benzoylvaleric acid-AE ester (AEVB).

[0376] In some aspects, the drug / payload is auristatin. Auristatin inhibits cell proliferation by inhibiting the formation of microtubules during mitosis by inhibiting tubulin polymerization. PCT International Publication No. WO 2013 / 072813 (which is incorporated herein by reference in its entirety) discloses auristatins that can be used in the CDCP1 ADC of the present invention and provides methods for producing auristatins. Non-limiting examples of auristatins include: Payload 0101 (designated #54 in WO 2013 / 072813) having the following structure:

[0377]

[0378] (2-methylalanyl- / V-[(34S,5S)-3-methoxy-1-{(2S)-2-[(12)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxohept-4-yl]- / V-methyl-L-valinamide), 3-[(34S,5S)-3-methoxy-1-{(2S)-2-[(12)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxohept-4-yl]- / V-methyl-L-valinamide) 377 (N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide) and

[0379] Payload 0131 (designated as #118 in WO 2013 / 072813) with the following structure

[0380]

[0381] (2-Methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxohept-4-yl]-N-methyl-L-valinamide).

[0382] Duocarmycin and CC-1065 are CPI-based monomers that can act as DNA alkylating agents with cytotoxic potency. See Boger and Johnson, PNAS 92:3642-3649, 1995. Exemplary Aplysias include, but are not limited to, (+)-duocarmycin A and (+)-duocarmycin SA, and (+)-CC-1065.

[0383] In some aspects, the drug / payload is a CPI or CBI dimer. CPI dimers induce interchain DNA crosslinks and potent cytotoxicity. PCT International Publication No. WO2015 / 110935 (incorporated herein by reference in its entirety) discloses CPI and CBI dimers that can be used to produce the CDCP1 ADC of the present invention, and provides methods for producing CPI and CBI dimers. Non-limiting examples of CPI dimers include: a payload CPI-8314 dimer having the following structure:

[0384]

[0385] Enediynes are a class of antitumor bacterial products characterized by ring systems with nine- and ten-membered rings or the presence of conjugated triple-double-triple bonds. Exemplary enediynes include, but are not limited to, calicheamicin, esperamicin, and dynemicin. Calcheamicin, also known as the LL-E33288 complex, such as β-calicheamicin, γ-calicheamicin, or N-acetyl-γ-calicheamicin (γ-calicheamicin (γ1)), is an enediyne antibiotic originally isolated as a natural product from the soil microorganism Micromonospora calichensis (Zein et al., Science 27; 240(4856): 1198-1201, 1988); it produces double-stranded DNA breaks and subsequently induces apoptosis in target cells (Zein et al., Science 27; 240(4856): 1198-1201, 1988; Nicolaou et al., Chem. Biol. Sep; 1(1): 57-66, 1994; Prokop et al., Oncogene 22: 9107-9120, 2003). The disulfide analog is N-acetyl-γ-calicheamicin dimethylhydrazide.

[0386] Geldanamycin is a benzoquinone ansamycin antibiotic that binds to Hsp90 (heat shock protein 90) and has been used as an anti-tumor drug. Exemplary geldanamycins include, but are not limited to, 17-AAG (17-N-allylamino-17-demethoxygeldanamycin) and 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin).

[0387] Maytansine or its derivatives, maytansinoids, inhibit cell proliferation by inhibiting the polymerization of tubulin, thereby inhibiting microtubule formation during mitosis. See Remillard et al., Science 189: 1002-1005, 1975. Exemplary maytansine and maytansinoids include, but are not limited to, Mertansine (DM1) and its derivatives, and ansamitocin.

[0388] Taxanes are diterpenes that act as anti-tubulin agents or mitotic inhibitors. Exemplary taxanes include, but are not limited to, paclitaxel (e.g. ) and docetaxel

[0389] Vinca alkaloids are also anti-tubulin agents. Exemplary vinca alkaloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine.

[0390] In some aspects of the invention, the agent is an immunomodulatory agent. Examples of immunomodulators include, but are not limited to, gancyclovier, etanercept, tacrolimus, sirolimus, voclosporin, cyclosporin, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, glucocorticoids and their analogs, cytokines, xanthines, stem cell growth factors, lymphotoxins, tumor necrosis factor (TNF), hematopoietic factors, interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony stimulating factors (e.g., granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF)), interferons (e.g., interferon-α, -β, and -γ), a stem cell growth factor designated "S1 factor," erythropoietin, and thrombopoietin, or a combination thereof.

[0391] Immunomodulators useful in the present invention also include anti-hormones that block the effects of hormones on tumors and immunosuppressants that suppress cytokine production, downregulate autoantigen expression, or mask MHC antigens. Representative anti-hormones include anti-estrogens, including, for example, tamoxifen, raloxifene, 4 (5) -imidazoles that inhibit aromatase, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapnstone, and toremifene; and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and anti-adrenal agents. Representative immunosuppressive agents include 2-amino-6-aryl-5-substituted pyrimidines, azathioprine, cyclophosphamide, bromocriptine, danazol, dapsone, glutaraldehyde, anti-idiotypic antibodies to MHC antigens and MHC fragments, cyclosporin A, steroids such as glucocorticoids, cytokines or cytokine receptor antagonists (e.g., anti-interferon antibodies, anti-IL10 antibodies, anti-TNFα antibodies, anti-IL2 antibodies), streptokinase, TGFβ, rapamycin, T-cell receptors, T-cell receptor fragments, and T-cell receptor antibodies.

[0392] In some aspects of the invention, the drug is a therapeutic protein, including but not limited to toxins, hormones, enzymes, and growth factors.

[0393] Examples of toxin proteins (or polypeptides) include, but are not limited to, diphtheria (e.g., diphtheria A chain), Pseudomonas exotoxin and endotoxin, ricin (e.g., ricin A chain), abrin (e.g., abrin A chain), modeccin (e.g., modeccin A chain), alpha-sarcin, Aleurites fordii protein, dianthin protein, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, pokeweed proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotonin, saponin inhibitor, mitogellin, restrictocin, phenomycin, enomycin, trichothecenes, inhibitor cystine knot (ICK) peptides (e.g., ceratotoxin), and conotoxins (e.g., KIIIA or SmIIIa).

[0394] Examples of hormones include, but are not limited to, estrogens, androgens, progestins, and corticosteroids.

[0395] In some aspects of the invention, the drug is an oligonucleotide, such as an antisense oligonucleotide.

[0396] Additional drugs that can be used in the present invention include anti-angiogenic agents that inhibit blood vessel formation, for example, farnesyl transferase inhibitors, COX-2 inhibitors, VEGF inhibitors, bFGF inhibitors, steroid sulfatase inhibitors (e.g., 2-methoxyestradiol bis-sulfamate (2-MeOE2bisMATE)), interleukin 24, thrombospondin, metallospondin, type I interferon, interleukin 12, protamine, angiostatin, laminin, endostatin, and prolactin fragments.

[0397] Antiproliferative and pro-apoptotic agents include activators of PPAR-γ (e.g., cyclopentenone prostaglandins (cyPG)), retinoids, triterpinoids (e.g., cyclobrane, lupeane, ursane, oleanane, suberane, dammarane, cucurbitacin and limonoid triterpenoids), inhibitors of EGF receptors (e.g., HER4), rapamycin, (1,25-dihydroxycholecalciferol (vitamin D)), aromatase inhibitors ( (Letrozone), telomerase inhibitors, iron chelators (e.g., 3-aminopyridine-2-pyrrolecarboxaldehyde thiosemicarbazone (Triapine)), apoptotic proteins (viral protein 3-VP3 from chicken anemia virus), inhibitors of Bcl-2 and Bcl-X(L), TNF-α, FAS ligand, TNF-related apoptosis-inducing ligand (TRAIL / Apo2L), activators of TNF-α / FAS ligand / TNF-related apoptosis-inducing ligand (TRAIL / Apo2L) signaling, and inhibitors of PI3K-Akt survival pathway signaling (e.g., UCN-01 and geldanamycin).

[0398] Representative chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziidines such as benzodopa, carboquinone, amitripaquin and euridopa; ethyleneimines and methylmelamines including hexamethylmelamine, triethylenemethylmelamine, triethylenephosphoramide, triethylenethiophosphoramide and trishydroxymethylmethylmelamine; nitrogen mustards such as chlorambucil, naphthyl mustard, clofosamide, estramustine, ifosfamide, nitrogen mustard, nitrogen mustard oxide hydrochloride, melphalan, nebiquinone, phenylephrine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorambucil, chloramphenicol, chloramphenicol, estramustine, ifosfamide, nitrogen mustard, nitrogen mustard oxide hydrochloride, melphalan, nebiquinone, phenylephrine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorambucil, chloramphenicol, chlorambucil, estramustine, estramustine, ifosfamide, estramustine ... , fotemustine, lomustine, nimustine, ranimustine; antibiotics, such as aclarubicin, dactinomycin, anthramycin, azaserine, bleomycin, dactinomycin, calicheamicin, carbapenem, carminomycin, chromomycin, dactinomycin, daunorubicin, detopicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, mexicomycin, mitomycin, mycophenolic acid, noramycin, olivemycin, peplomycin, porphyromycin, puromycin, triferon-doxorubicin, rhodorubicin, streptozocin, streptozotocin, tuberculin, ubenimex, jinsistatin, daunorubicin; antimetabolites, Such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as dimethylfolate, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiopurine, and thioguanine; pyrimidine analogs such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and 5-FU; androgens such as calutosterone, drostanolone propionate, cyclothiosteroid, melastane, and testolactone; adrenal inhibitors such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; aceglucuronolide; aldophosphamide glucoside; aminolevulinic acid; amsacrine; and betribuconazole Bisantrene; edatrexate; diflufamidate; demeclocycline; diazocine; efamicin; elixirs; etoglucagon; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguanidine; mitoxantrone; mopidarol; diamine nitrazepam; pentostatin; methambucil; pirarubicin; podophyllic acid; 2-ethylhydrazine; procarbazine; razoxane; sizolan; spirogermanamine; tricholomaric acid; triazoline; 2,2′,2′-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulanol; pipobroman; garcitocin; cytarabine (“Ara-C”); cyclophosphamide; thiotepa; taxanes, such as paclitaxel ( Bristol-Myers Squibb Oncology of Princeton, NJ) and docetaxel ( Rhone-Poulenc Rorer of Antony, France), chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; noranthorpe; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; and capecitabine.

[0399] Additional therapeutic agents that can be used in accordance with the present invention include photosensitizers for photodynamic therapy, such as U.S. Publication No. 20020197262 and U.S. Patent No. 5,952,329 (which are incorporated herein by reference in their entirety); magnetic particles for hyperthermia, such as U.S. Publication No. 20030032995 (which are incorporated herein by reference in their entirety); binding agents, such as peptides, ligands, cell adhesion ligands, and the like, and prodrugs, such as phosphate-containing prodrugs, phosphorothioate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, β-lactam-containing prodrugs, substituted phenoxyacetamide-containing prodrugs, or substituted phenylacetamide-containing prodrugs, 5-fluorocytosine, and other 5-fluorouracil prodrugs that can be converted into more active non-cytotoxic drugs.

[0400] For the diagnostic method using anti-CDCP1 antibodies, the drug may include a detectable label for detecting the presence of tumor cells expressing CDCP1 in vitro or in vivo. Radioisotopes detectable in vivo (such as those detectable using scintigraphy, magnetic resonance imaging or ultrasound) can be used in clinical diagnostic applications. Useful scintigraphy labels include positron emitters and gamma emitters. Representative contrast agents for magnetic source imaging are paramagnetic or superparamagnetic ions (e.g., iron, copper, manganese, chromium, erbium europium, dysprosium, holmium and gadolinium), iron oxide particles and water-soluble contrast agents. In order to perform ultrasonic detection, gas or liquid can be captured in porous inorganic particles released as microbubble contrast agents. For in vitro detection, useful detectable labels include fluorophores, detectable epitopes or binding agents and radioactive labels.

[0401] Thus, in some aspects of the invention, the drug is an imaging agent (eg, a fluorophore or a PET (positron emission tomography) marker, a SPECT (single photon emission computed tomography) marker) or an MRI (magnetic resonance imaging) marker.

[0402] The term "label" as used herein refers to a detectable compound or composition that is conjugated directly or indirectly to an antibody to produce a "labeled" antibody. The label can be detectable per se (e.g., a radioisotope label or a fluorescent label), or in the case of an enzyme label, can catalyze a chemical alteration of a detectable substrate compound or composition. Radionuclides that can serve as detectable labels include, for example, I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, and Pd-109. A label can also be an undetectable entity, such as a toxin.

[0403] Examples of fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluorescein phosphoramidite (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein, erythrosine, Alexa Fluor, (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, or 750), carboxytetramethylrhodamine (TAMRA) (e.g., 5-TAMRA), tetramethylrhodamine (TMR), and sulforhodamine (SR) (e.g., SR101).

[0404] Therapeutic or diagnostic radioisotopes or other labels (e.g., PET or SPECT labels) can be incorporated into agents for conjugation to anti-CDCP1 antibodies as described herein. The isotope can be directly bound to the antibody, for example, at a cysteine ​​residue present in the antibody, or a chelating agent can be used to mediate the binding of the antibody to the radioisotope. Radioisotopes suitable for radiotherapy include, but are not limited to, alpha emitters, beta emitters, and Auger electrons. For diagnostic applications, useful radioisotopes include positron emitters and gamma emitters. The anti-CDCP1 antibodies of the present invention can be further iodinated, for example, at tyrosine residues of the antibody to facilitate detection or therapeutic effects of the antibody.

[0405] Examples of radioisotopes or other labels include, but are not limited to, 3 H, 11 C. 13 N. 14 C. 15 N. 15 O. 35 S. 18 F. 32 p、 33 p、 47 Sc, 51 Cr, 57 Co、 58 Co、 59 Fe,62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Se, 76 Br, 77 ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​The CDCP1 ADC of the present invention can be prepared by using a linker to directly or indirectly connect or conjugate the drug to the antibody. A linker is a bifunctional compound that connects the drug and the antibody to form an ADC. Such ADCs allow selective delivery of the drug via antibodies that are bound to specific antigens or proteins. Suitable linkers include, for example, cleavable linkers and non-cleavable linkers. Cleavable linkers are generally easy to cleave and release the drug under specific intracellular and extracellular conditions. The main mechanisms by which conjugated drugs can be cleaved from antibodies within the cell include hydrolysis in the acidic pH of the lysosome (hydrazone, acetal, and cis-aconitic acid-like amide), peptide cleavage by lysosomal enzymes (cathepsins and other lysosomal enzymes), and reduction of disulfides. The conjugated drug can be cleaved from the antibody extracellularly by proteases (such as cathepsins) in the tumor microenvironment (TME). Due to these different cleavage mechanisms, the mechanism of connecting the drug to the antibody also varies widely, and any suitable linker can be used.

[0408] Suitable linkers can include any cleavable linker. In some aspects, suitable linkers include valine-citrulline (val-cit) linkers, phenylalanine-lysine (phe-lys) linkers, maleimidoacryloyl-valine-citrulline-p-aminobenzyloxycarbonyl linkers or 6-maleimidocaproyl-valine-citrulline-p-aminobenzylcarbamate (mc-val-cit-PABC or vc) linkers, or dipeptides containing a link to other additional sacrificial elements, such as -acetyl-L-lysyl-L-valyl-L-citrulline-p-aminobenzyloxycarbonyl-N, N'-dimethylaminoethyl-CO-linker, suitable for transglutaminase-based conjugation technology. On the other hand, suitable linkers include disulfide linkers, such as sulfanylpyridine (diS) linkers and 2-(pyridin-2-yldisulfanyl)ethylcarbamoyl (diS-COCO) linkers. On the other hand, the linker can be a non-cleavable linker, such as maleimidocaproyl (mc), maleimido-heptanoyl (me) and maleimido-Peg6C2 (MalPeg6C2). In other aspects, suitable linkers include linkers that are hydrolyzable at a specific pH or pH range, such as hydrazone linkers.

[0409] The linker can be covalently bound to the antibody via a thioester linkage, for example, by reaction of a maleimide or haloacetamide present on the linker with a natural or engineered cysteine ​​residue present on the antibody. In another aspect, the linker can be covalently bound to the antibody via an amide linkage to a lysine residue present on the antibody, for example, by reaction of an N-hydroxy-succinimide activated carboxylic acid present on the linker with the free amine of the lysine residue. In another aspect, the linker can be covalently bound to the antibody via an amide linkage to the side chain of a glutamine residue present or engineered into the antibody, for example, by an enzymatic reaction catalyzed by transglutaminase, which generates a new amide linkage from a primary amine present on the linker with the side chain amide of the glutamine residue.

[0410] In some aspects, the linker is selected from the group consisting of valine-citrulline (val-cit), 6-maleimidocaproyl (mc), methoxy-polyethylene glycol maleimide 6 (MalPeg6), p-aminobenzylcarbamate (PABC), dimethylaminoethanol (DMAE), maleimidopropionyl (MP), hydrolyzed Peg-maleimide, alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), 4-(2-pyridylthio)pentanoic acid The esters of the present invention include N-succinimidyl ester (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (SMCC), N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-val-cit-PAB), and 6-maleimidocaproyl-valine-citrulline-p-aminobenzylcarbamate (mc-val-cit-PABC or vc).

[0411] In some aspects, the linker is a linker selected from the group consisting of: Ac-Lys-Gly (acetyl-lysine-glycine), aminocaproic acid, Ac-Lys-p-Ala (acetyl-lysine-p-alanine), amino-PEG2 (polyethylene glycol)-C2, amino-PEG3-C2, amino-PEG6-C2 (or aminoPEG6-propionyl), Ac-Lys-Val-Cit-PABC (acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl) , amino-PEG6-C2-Val-Cit-PABC, aminocaproyl-Val-Cit-PABC, [(3R,5R)-1-{3-[2-(2-aminoethoxy)ethoxy]propionyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, [(3S,5S)-1-{3-[2-(2-aminoethoxy)ethoxy]propionyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, putrescine and Ac-Lys-putrescine.

[0412] In some embodiments, the linker is:

[0413] An "mc-val-cit-PABC" or "vc" linker having the following structure:

[0414]

[0415] In some embodiments, the linker is amino-PEG6-C2 (or aminoPEG6-propionyl).

[0416] In some embodiments, the antibody drug conjugate comprises an anti-CDCP1 antibody as described herein, wherein the antibody is conjugated to a linker-drug moiety via one or more engineered cysteine ​​residues on the antibody. The linker can be selected from any linker described herein. In some embodiments, the linker is selected from the group consisting of: valine-citrulline (val-cit), 6-maleimidocaproyl (me), methoxy-polyethylene glycol maleimide 6 (MalPeg6), p-aminobenzylcarbamate (PABC), dimethylaminoethanol (DMAE), maleimidopropionyl (MP), hydrolyzed Peg-maleimide, alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), 4-(2-pyridylthio) In some embodiments, the linker is mc-val-cit-PABC. In some embodiments, the drug moiety is auristatin. In some embodiments, the drug moiety is 0101 or 0131.

[0417] In some aspects, the present invention provides an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof conjugated to a linker-drug moiety via one or more engineered cysteine ​​residues on the antibody, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 33; and a light chain comprising the amino acid sequence of SEQ ID NO: 38, and wherein the linker-drug moiety is mc-val-cit-PABC-0101.

[0418] In some aspects, the present invention provides an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof conjugated to a linker-drug moiety via one or more engineered cysteine ​​residues on the antibody, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 33; and a light chain comprising the amino acid sequence of SEQ ID NO: 34, and wherein the linker-drug moiety is mc-val-cit-PABC-0101.

[0419] In some embodiments, the antibody drug conjugate comprises an anti-CDCP1 antibody as described herein, wherein the antibody is conjugated to a linker drug using a tag containing an acyl donor glutamine engineered at a specific site on the antibody. The linker can be selected from any linker described herein. In some embodiments, the linker is selected from the group consisting of: Ac-Lys-Gly (acetyl-lysine-glycine), aminocaproic acid, Ac-Lys-p-Ala (acetyl-lysine-p-alanine), amino-PEG2 (polyethylene glycol) -C2, amino-PEG3-C2, amino-PEG6-C2 (or aminoPEG6-propionyl), Ac-Lys-Val-Cit-PABC (acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl) , amino-PEG6-C2-Val-Cit-PABC, aminohexanoyl-Val-Cit-PABC, [(3R,5R)-1-{3-[2-(2-aminoethoxy)ethoxy]propionyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, [(3S,5S)-1-{3-[2-(2-aminoethoxy)ethoxy]propionyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, putrescine, and Ac-Lys-putrescine. In some embodiments, the linker is aminoPEG6-propionyl (i.e., aminoPEG6-C2 or AMPeg6C2). In some embodiments, the drug moiety is auristatin. In some embodiments, the drug moiety is 0101 or 0131.

[0420] In some aspects, the present invention provides an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof conjugated to a linker-drug moiety using an acyl donor glutamine-containing tag engineered at a specific site on the antibody, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35; and a light chain comprising the amino acid sequence of SEQ ID NO: 37, and wherein the linker-drug moiety is aminoPEG6-propionyl-0131 (i.e., AmPeg6C2-0131).

[0421] In some aspects, the present invention provides an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof conjugated to a linker-drug moiety via one or more engineered cysteine ​​residues on the antibody, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35; and a light chain comprising the amino acid sequence of SEQ ID NO: 32, and wherein the linker-drug moiety is aminoPEG6-propionyl-0131 (ie, AmPeg6C2-0131).

[0422] Optimal reaction conditions for producing ADCs can be determined empirically by varying reaction variables such as temperature, pH, linker-payload moiety input, and additive concentrations. Suitable conditions for conjugating other drugs can be determined by one skilled in the art without undue experimentation. Representative methods for conjugating and characterizing CDCP1 ADCs are described in Examples 17 and 18.

[0423] After conjugation, the conjugate can be isolated, purified from unconjugated reactants and / or aggregated forms of the conjugate, and characterized by conventional methods. This includes methods such as, but not limited to, mass spectrometry, size exclusion chromatography (SEC), ultrafiltration / diafiltration, ion exchange chromatography (IEC), chromatofocusing (CF), site-directed mutagenesis, fluorescent labeling, X-ray crystallography, high performance liquid chromatography (HPLC), fast protein liquid chromatography (FPLC), polyacrylamide dextran S-200 chromatography, or hydrophobic interaction chromatography (HIC). Suitable HIC media include, but are not limited to, phenyl sepharose 6 fast flow chromatography media, butyl sepharose 4 fast flow chromatography media, octyl sepharose 4 fast flow chromatography media, Toyopearl ether-650M chromatography media, Macro-Prep methyl HIC media, or Macro-Prep tert-butyl HIC media.

[0424] In some embodiments, the antibody drug conjugates as described herein have a melting transition temperature greater than at least 60° C., at least 65° C., at least 70° C., at least 75° C., at least 80° C., at least 85° C., or at least 90° C. In some embodiments, the antibody drug conjugates have a melting transition temperature greater than about 65° C.

[0425] In some embodiments, the antibody drug conjugates as described herein have a K of or less than about 50 nM, about 48 nM, about 46 nM, about 45 nM, about 44 nM, about 42 nM, or about 40 nM at pH 7.4. D In some embodiments, the antibody drug conjugate binds to CDCP1 at a K of about 70 nM, about 68 nM, about 66 nM, about 65 nM, about 64 nM, about 62 nM, or about 60 nM at pH 6.8.D Values ​​bind CDCP1.

[0426] In some embodiments, the antibody drug conjugates as described herein have a half maximal inhibitory concentration (IC50) of no more than about 20,000 pM, about 15,000 pM, about 10,000 pM, about 9,500 pM, 8,000 pM, 7,000 pM, 6,000 pM, 5,000 pM, 4,000 pM, 3,000 pM, 2,000 pM, 1,000 pM, 900 pM, 800 pM, 700 pM, 650 pM, 600 pM, 500 pM, 400 pM, 300 pM, 250 pM, 200 pM, or 100 pM. 50 In some embodiments, the antibody drug conjugates described herein have an IC of no more than about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM, about 50 pM, about 40 pM, about 30 pM, about 20 pM, about 10 pM, about 9 pM, about 8 pM, about 7 pM, about 6 pM, about 5 pM, about 4 pM, about 3 pM, about 2 pM, or about 1 pM. 50 In some embodiments, the IC50 value is determined in cells expressing CDCP1.

[0427] In some embodiments, the antibody drug conjugates described herein reduce mean tumor volume by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the mean tumor volume in untreated controls in NSCLC PDX models. In some embodiments, the antibody drug conjugates reduce mean tumor volume by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the mean tumor volume in untreated controls in patient-derived xenograft models of head and neck cancer.

[0428] Uses of CDCP1-specific antibodies and ADCs

[0429] The anti-CDCP1 antibodies and CDCP1 ADCs of the present invention can be used in various applications, including but not limited to therapeutic treatment methods and diagnostic treatment methods.

[0430] In some aspects, the present invention provides a method for treating a condition associated with CDCP1 expression in a subject. In some embodiments, the method for treating a condition associated with CDCP1 expression in a subject comprises administering to a subject in need thereof an effective amount of a composition (e.g., a pharmaceutical composition) comprising a CDCP1 antibody or CDCP1 antibody conjugate as described herein. Conditions associated with CDCP1 expression include, but are not limited to, abnormal CDCP1 expression, altered or abnormal CDCP1 expression, malignant cells expressing CDCP1, and proliferative disorders (e.g., cancer), autoimmune disorders, inflammatory diseases, or infectious diseases.

[0431] Therefore, in some aspects, the present invention provides a method for treating cancer, autoimmune disease, inflammatory disease or infectious disease in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of a composition comprising an anti-CDCP1 antibody or CDCP1 antibody conjugate as described herein (or a pharmaceutical composition comprising an anti-CDCP1 antibody or CDCP1 antibody drug conjugate).

[0432] The present invention provides methods for treating tumors, wherein the tumor cells express CDCP1 on their surface and wherein the surrounding non-tumor tissue expresses little or no detectable cell-surface CDCP1. The methods comprise administering an antibody or antigen-binding fragment thereof that specifically binds to CDCP1 and is internalized by the cells. Preferably, the antibody or antigen-binding fragment thereof is an antibody drug conjugate comprising a cytotoxic payload. Without wishing to be bound by any particular theory, CDCP1 is activated because it is phosphorylated at tyrosine 734 (P-734-tyr), and numbering is with reference to SEQ ID NO: 90. Even more preferably, and without wishing to be bound by any particular theory, the methods comprise treating tumors in a hypoxic environment such that CDCP1 is phosphorylated at Tyr734 at a higher level than CDCP1 in tumors not in a hypoxic environment. Such tumors particularly include lung cancer tumors and head and neck cancer tumors. Once armed with the teachings herein, one skilled in the art will appreciate that any tumor associated with a hypoxic environment and / or higher levels of P-tyr-734 compared to other identical tumors or tissues can be treated using the antibodies and / or ADCs of the present invention.

[0433] cancer

[0434] Cancer or tumor refers to uncontrolled cell growth and / or abnormal increase in cell survival and / or inhibition of apoptosis, which interferes with the normal function of body organs and systems. Includes benign and malignant cancers, polyps, hyperplasia and dormant tumors or micrometastasis. In addition, includes cells with abnormal proliferation that is not hindered by the immune system (for example, virally infected cells). Cancer can be primary cancer or metastatic cancer. Primary cancer can be a cancer cell area at the clinically detectable origin site, and can be a primary tumor. In contrast, metastatic cancer can be a disease that spreads from one organ or part to another non-adjacent organ or part. Metastatic cancer can be caused by cancer cells, and the cancer cells have the ability to penetrate and infiltrate the surrounding normal tissues in the local area, thereby forming new tumors, which can be local metastasis. Cancer cells can also be caused by cancer cells, and the cancer cells have the ability to penetrate lymphatic vessels and / or blood vessel walls, after which cancer cells can circulate through the bloodstream (thus becoming circulating tumor cells) to other parts and tissues in the body. Cancer can be caused by processes such as lymphatic or hematogenous spread. Cancer can also be caused by tumor cells that lodge in another location, re-penetrate a blood vessel or wall, continue to multiply, and eventually form another clinically detectable tumor. Cancer can be this new tumor, which can be a metastatic (or secondary) tumor.

[0435] Cancer can be caused by tumor cells that have metastasized, which can be secondary or metastatic tumors. The cells of the tumor can be similar to the cells in the original tumor. For example, if breast cancer or colon cancer metastasizes to the liver, the secondary tumor, although present in the liver, is composed of abnormal breast cancer or colon cells rather than abnormal liver cells. Therefore, the tumor in the liver can be metastatic breast cancer or metastatic colon cancer, rather than liver cancer. Cancer can originate from any tissue. Cancer can originate from melanoma, colon, breast or prostate; and therefore can be composed of cells that were originally skin, colon, breast or prostate tissue, respectively. Cancer can also be a hematological malignancy, which can be a leukemia or lymphoma. Cancer can invade tissues such as the liver, lungs, bladder or intestines.

[0436] In some embodiments, knockdown of CDCP1 results in upregulation of P38, extracellular signal-regulated kinase 1 (ERK1 and 2), Jun proto-oncogene (JUN isoforms 1, 2, and 3), AKT serine / threonine kinase 1 (AKT isoforms 1, 2, and 3), AMP-activated protein kinase (AMPK), signal transducer and activator of transcription (STAT2), STAT5 A / B, choline / ethanolamine kinase (CHK-2), and MET proto-oncogene, receptor tyrosine kinase (MET).

[0437] In one aspect, the present disclosure provides a method for treating cancer in a patient in need thereof, the method comprising: (a) assessing the amount of mutant LKB1 and / or KRAS in a tumor sample; and (b) administering an agent that binds to CDCP1 to the cancer patient if the amount of mutant LKB1 and / or KRAS is higher than a reference sample.

[0438] In one aspect, the present disclosure provides a method of determining whether a tumor will respond to treatment with an agent that binds to CDCP1, the method comprising determining the presence, absence, or amount of mutant LKB1 and / or KRAS protein or gene in a sample of the tumor, whereby an increase in the presence of mutant LKB1 and / or KRAS or the amount of mutant LKB1 and / or KRAS protein or gene relative to a reference sample indicates a likelihood of response to treatment with an agent that binds to CDCP1.

[0439] In one aspect, the present disclosure provides a method for treating cancer in a patient in need thereof, the method comprising: (a) selecting an agent that binds to CDCP1 on a target cell and is internalized when it contacts CDCP1 on the target cell; and (b) administering the agent to the cancer patient, wherein the agent that binds to CDCP1 is an antibody that activates CDCP1 and is conjugated to a PPP4R2 modulator.

[0440] In one aspect, the present disclosure provides a method for treating cancer in a patient in need thereof, comprising: (a) administering an agent that binds to CDCP1, wherein the agent that binds to CDCP1 is an antibody that does not activate CDCP1; and (b) administering an agent that modulates PARG.

[0441] Representative cancers and / or tumors of the present invention include, but are not limited to, basal cell carcinoma; biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatoma; intraepithelial neoplasia; renal or kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; myeloma; neuroblastoma; oral cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary tract cancer; vulvar cancer. cancers; lymphomas, including Hodgkin and non-Hodgkin lymphomas, and B-cell lymphomas (including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other cancers and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with keloids, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0442] In some embodiments, the cancer is lung cancer. In some embodiments, the cancer subtype is SCLC, NSCLC, or mesothelioma. In one aspect, the present disclosure provides a method of treating lung cancer in a patient in need thereof, the method comprising administering to the patient an agent that binds to CDCP1, wherein the lung cancer is characterized by AKT activation, and the agent that binds to CDCP1 is a CDCP1 activator.

[0443] In some embodiments, the cancer is prostate cancer.In one aspect, the present disclosure provides a method of treating prostate cancer in a patient in need thereof, the method comprising administering to the patient an agent that binds to CDCP1, wherein the prostate cancer is characterized by AKT activation and the agent that binds to CDCP1 is a CDCP1 activator.

[0444] In some embodiments, the cancer is head and neck cancer.

[0445] In some embodiments, the methods of the present disclosure provide a prognosis for a subject who is determined to have a proliferative disorder, such as cancer (e.g., NSCLC, prostate cancer, or head and neck cancer). The prognosis can be, for example, a poor prognosis or a good prognosis, measured by a shortened survival period or an extended survival period, respectively. In addition, overall survival (OS), disease-free survival (DFS), or recurrence-free survival (RFS) can be measured as survival. Cancer can be primary or recurrent and can have any type (as described above), staging (e.g., I, II, III, or IV phase or the equivalent of other staging systems) and / or histology. The patient can have any age, sex, behavioral status, and / or degree of remission and duration.

[0446] In some embodiments, knockdown of CDCP1 results in downregulation of HCK proto-oncogene, focal adhesion kinase (FAK), p70S6K, and phospholipase Cγ1 (PLCγ1).

[0447] In some embodiments, the cancer is not bladder cancer.

[0448] Combination therapy / conjugates

[0449] As described herein, in various embodiments, the present invention is directed to anti-tumor agents that can be part of the conjugates of the invention or used in the context of various combination therapies encompassed by the invention.

[0450] Combination therapy includes the administration of an antibody-drug conjugate and another therapeutic agent as part of a specific treatment regimen, optionally including a maintenance phase, intended to provide a beneficial effect from the synergistic effect of these therapeutic agents. The beneficial effect of the combination includes, but is not limited to, the pharmacokinetic or pharmacodynamic co-action produced by the combination of therapeutic agents. The combined administration of these therapeutic agents is typically carried out within a defined time period (depending on the selected combination, typically minutes, hours, days or weeks). Combination therapy is generally not intended to encompass the administration of two or more of these therapeutic agents as part of a separate monotherapy regimen that is incidental and arbitrarily produces a combination of the present invention.

[0451] Combination therapy includes administering these therapeutic agents in a sequential manner, i.e., wherein each therapeutic agent is administered at different times, and administering these therapeutic agents or at least two of the therapeutic agents in a substantially simultaneous manner. Sequential or substantially simultaneous administration of each therapeutic agent can be achieved by any appropriate route including, but not limited to, oral route, intravenous route, intramuscular route, subcutaneous route, and direct absorption by mucosal tissue. The therapeutic agent can be administered by the same route or by different routes. For example, a first therapeutic agent (e.g., a chemotherapeutic agent) can be administered orally, and a second agent (e.g., ADC) can be administered intravenously. In addition, the first therapeutic agent of the selected combination can be used by intravenous injection, while the other therapeutic agents of the combination can be administered orally. Alternatively, for example, both therapeutic agents can be administered by intravenous or subcutaneous injection.

[0452] In the present disclosure, unless otherwise stated, the term sequential means characterized by a regular order or sequence, for example, if a dosage regimen includes the administration of an ADC and a chemotherapeutic agent, a sequential dosage regimen may include administering the ADC before, simultaneously, substantially simultaneously, or after the administration of the chemotherapeutic agent, but the two agents will be administered in a regular order or sequence. Unless otherwise stated, the term alone refers to being separate from each other. Unless otherwise stated, the term simultaneously refers to occurring or proceeding simultaneously, that is, the compounds of the present invention are administered simultaneously. The term substantially simultaneously refers to the administration of compounds within a few minutes of each other (e.g., within 10 minutes of each other), and is intended to include combined administration and continuous administration, but if administration is continuous, it is only separated in time by a shorter time (e.g., the time required for a practitioner to administer the two compounds separately). As used herein, concurrent administration and substantially simultaneous administration are used interchangeably. Sequential administration refers to the administration of ADC and chemotherapeutic agents separated in time.

[0453] In some embodiments, the chemotherapeutic agent is selected from the group consisting of alkylating agents, such as thiotepa and CYTOXAN cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquinone, tebuconazole, and euridopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trishydroxymethylmelamine; polyacetylamines (e.g., bratacin and bratacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; CC-1065 (including its synthetic analogues adolesine, carzelesin, and biszelesin); nostoc (e.g., nostoc 1 and nostoc 8); and Aplysia caudatum. duocarcins (including synthetic analogs KW-2189 and CB1-TM1); arbutin; hyoscyamine; stolon; spongistatin; nitrogen mustards, such as chlorambucil, naphthiazolin, clofosamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nembicidin, phenylephrine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozolin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin gamma 11 and calicheamicin omega 11 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); daptomycins, including daptomycin A; bisphosphonates such as clodronate; esperamicins; and the neocarcin chromophores and related chromoprotein enediyne antibiotic chromophores), aclarubicin, actinomycin, anthramycin, azaserine, bleomycin, actinomycin C, carrubicin, carmomycin, carmomycin, chromomycin, dactinomycin, daunorubicin, detoximcin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN Doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, mexicomycin, mitomycins (such as mitomycin C), mycophenolic acid, noramycin, olivomycin, peplomycin, porfibrinocin, puromycin, triferric doxorubicin, rhodorubicin, streptozotocin, streptozotocin, tuberculin, ubenimex, zoloft, daunorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as dimethylformamide, methotrexate, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiopurine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as captestosterone, drostanolone propionate, cyclothiocarbamate, melastane, and testolactone; antiadrenergic drugs, such as aminoglutethimide, mitotane, and trilostan; folic acid supplements, such as folinic acid; and acetaminophen. esters; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; betribucil; bisantrene; edatrexate; colcemid; diazocone; eflornithine; elliptonium acetate; epothilones; etoglucagon; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansine alkaloids, such as maytansine and ansamitocin; mitoguanidine; mitoxantrone; mopidarol; diamine nitrazepam; pentostatin; methambucil; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizolan; spirogermanamine; tricholomanic acid; triazoline; 2,2′,2″-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verrucosporin A, baculosporin A, and serpentin); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; garcitocin; cytarabine (“Ara-C”); cyclophosphamide; thiotepa; taxanes, such as TAXOL paclitaxel, ABRAXANE cremophor-free albumin-engineered paclitaxel nanoparticle formulations (American Pharmaceutical Partners, Schaumberg, IL).) and TAXOTERE docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE; vinorelbine; Noantol; teniposide; edatrexate; daunorubicin; aminopterin; Xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including regimens combining irinotecan with 5-FU and leucovorin); the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretadine; folinic acid (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)), and VEGF-A that reduce cell proliferation, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0454] In some embodiments, the anti-tumor agent is a cytotoxic agent. In some embodiments, the cytotoxic agent is selected from methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine; alkylating agents such as nitrogen mustard, thiotepa chlorambucil;

[0015] Examples of the present invention include chlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea, cyclophosphamide, nitrogen mustard, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP), cisplatin, and carboplatin (Baldin); anthracyclines, including daunorubicin, doxorubicin (adriamycin), detorubicin, carminomycin, idarubicin, epirubicin, mitoxantrone, and bisantrene; antibiotics, including dactinomycin (actinomycin D), bleomycin, calicheamicin, mithramycin, and anthramycin (AMC); and antimitotic agents, such as vinca alkaloids, vincristine, and vinblastine, and mixtures thereof.

[0455] In some embodiments, the cytotoxic agent is selected from the group consisting of taxol (paclitaxel), ricin, Pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenotoposide, colchicine, dihydroxyanthracenedione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, and mixtures thereof.

[0456] In some embodiments, for example, in the treatment of various cancers, the compositions and methods of the present invention can be used in combination with checkpoint inhibitors. For example, the compositions and methods of the present invention can supplement checkpoint inhibitor-based cancer therapies, for example, by improving the patient's response to the checkpoint inhibitor (e.g., by converting non-responders to responders, and / or increasing the intensity of the therapeutic response, and / or reducing the dose or regimen required for the therapeutic response, and / or alleviating one or more side effects of checkpoint inhibitor-based cancer therapies).

[0457] In some embodiments, the checkpoint inhibitor is an agent that targets one of the following: TIM-3, BTLA, PD-1, CTLA-4, B7-H4, GITR, Galectin-9, HVEM, PD-L1, PD-L2, B7-H3, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, and TMIGD2.

[0458] In some embodiments, the immune checkpoint immunotherapeutic modulates PD-1. In some embodiments, the agent targeting PD-1 is an antibody or antigen-binding portion thereof specific for PD-1, optionally selected from nivolumab, pembrolizumab, and pidilizumab. In some embodiments, the antibody or antigen-binding portion thereof specific for PD-1 is nivolumab and can be administered at 240 mg every 2 weeks. In some embodiments, the antibody or antigen-binding portion thereof specific for PD-1 is pembrolizumab and can be administered at 200 mg every 3 weeks. In some embodiments, the antibody or antigen-binding portion thereof specific for PD-1 is pidilizumab and can be administered at 200 mg every 3 weeks.

[0459] In some embodiments, the immune checkpoint immunotherapy agent modulates PD-L1. In some embodiments, the agent that modulates PD-L1 is an antibody or antigen-binding portion thereof that is specific for PD-L1. In some embodiments, the antibody or antigen-binding portion thereof that is specific for PD-L1 is selected from atezolizumab, avelumab, durvalumab, and BMS-936559. In some embodiments, the antibody or antigen-binding portion thereof that is specific for PD-L1 is BMS-936559 and can be administered at 0.1 mg / kg every 2 weeks. In some embodiments, the antibody or antigen-binding portion thereof that is specific for PD-L1 is atezolizumab and can be administered at 1200 mg every 3 weeks. In some embodiments, the antibody or antigen-binding portion thereof that is specific for PD-L1 is avelumab and can be administered at 10 mg / kg every 2 weeks. In some embodiments, the antibody or antigen-binding portion thereof that is specific for PD-L1 is durvalumab and can be administered at 10 mg / kg every 2 weeks.

[0460] In some embodiments, the agent targeting CTLA-4 is an antibody or antigen-binding portion thereof specific for CTLA-4, optionally selected from ipilimumab and tremelimumab. In some embodiments, the antibody or antigen-binding portion thereof specific for CTLA-4 is tremelimumab and can be administered at 3 mg / kg, 6 mg / kg, or 10 mg / kg. In some embodiments, the antibody or antigen-binding portion thereof specific for CTLA-4 is ipilimumab and can be administered at 5 mg / mL every 12 weeks.

[0461] CDCP1 and hypoxia-inducible factor 2α (HIF-2α)

[0462] CDCP1 plays a key role in the survival of cells during metastasis and at distant metastatic sites, and exhibits a unique role under conditions of oxygen deprivation (hypoxia). Therefore, there is a biochemical pathway in which CDCP1 is involved in the activation of Src family members and the combination of SFK activation and phosphorylation and regulation of protein kinase Cδ (PKC-δ). Hypoxia triggers the increase of hypoxia-inducible factors HIF-1α and HIF-2α by blocking von Hippel Lindau (VHL)-dependent HIF-α degradation. HIF is a heterodimer of two basic helix-loop-helix / PAS proteins, HIF-α and aromatic hydrocarbon nuclear translocation protein (ARNT or HIF-β). HIF-α and ARNT subunits are ubiquitously expressed; however, the α-subunit is unstable under normal oxygen (5%-21% O2) conditions. Under hypoxic conditions (0.5%-5% O2), HIF-α subunits stabilize, dimerize with ARNT, translocate to the nucleus, and subsequently bind to hypoxia response elements (HREs) within target genes. Among HIF transcriptional targets are genes involved in glucose metabolism, angiogenesis, and metastasis, thus tightly linking HIF-mediated transcription to tumorigenesis. HIF-1α and HIF-2α are overexpressed in many primary and metastatic human cancers.

[0463] In some embodiments, the anti-tumor agent is a hypoxia-inducible factor-2 (HIF-2) inhibitor. In some embodiments, the HIF-2 inhibitor is selected from PT2385 and PT2977.

[0464] In some embodiments, the anti-tumor agent is not a Src inhibitor, optionally selected from KX2-391, bosutinib, saracatinib, and dasatinib. In some embodiments, the cancer is a tumor characterized by hypoxia.

[0465] Pharmaceutical composition

[0466] The present invention further provides a pharmaceutical composition comprising any anti-CDCP1 antibody, antigen-binding fragment thereof, or CDCP1 ADC disclosed herein and a pharmaceutically acceptable carrier. In addition, the composition may comprise more than one anti-CDCP1 antibody and / or more than one CDCP1 ADC disclosed herein.

[0467] The compositions of the present invention may also contain pharmaceutically acceptable carriers, excipients, or stabilizers (Remington: The Science and practice of Pharmacy 21st edition, 2005, Lippincott Williams and Wilkins, ed. KE Hoover) in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations described and may include: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or chelating agents such as TWEEN TM 、PLURONICS TM or polyethylene glycol (PEG) nonionic surfactants. As used herein, "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic salt or inorganic salt of a molecule or macromolecule.

[0468] Another embodiment of the present disclosure is a pharmaceutical composition or use of a pharmaceutical composition comprising an agent that binds to CDCP1 in a cancer patient if the amount of mutant LKB1 and / or KRAS is higher than a reference sample. In some embodiments, the pharmaceutical composition comprises an agent that binds to CDCP1 and is characterized by AKT activation or an agent that is a CDCP1 activator. In some embodiments, the pharmaceutical composition comprises an agent that activates CDCP1 and conjugates to PPP4R2. In some embodiments, the pharmaceutical composition comprises an agent that binds to CDCP1 on a target cell and is internalized when it contacts CDCP1 on the target cell. In the case of clinical applications, the pharmaceutical composition can be prepared in a form suitable for the intended application. Generally speaking, this will require preparing a composition that is substantially free of pyrogens and other impurities that may be harmful to humans or animals.

[0469] In some embodiments, a pharmaceutical composition comprises an agent of the present disclosure and a pharmaceutically acceptable carrier.An effective dose is an amount sufficient to affect a beneficial or desired clinical outcome.

[0470] Beneficial or desired clinical results may include, in particular, a reduction in tumor size and / or tumor growth and / or a reduction in cancer markers associated with the presence of cancer compared to that observed without the administration of a small molecule or peptide agent. Beneficial or desired clinical results may also include, in particular, an increase in the presence of markers associated with cancer alleviation compared to that observed without the administration of a small molecule or peptide agent. Beneficial or desired clinical results also include, in particular, an increase in the amount of genes comprising markers associated with cancer etiology compared to that observed without the administration of an inhibitor. Genes comprising markers associated with cancer etiology may include, for example, immune checkpoint genes such as TIM-3, BTLA, PD-1, CTLA-4, B7-H4, GITR, Galectin-9, HVEM, PD-L1, PD-L2, B7-H3, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, and TMIGD2.

[0471] Genes comprising markers associated with cancer etiology may include, for example, HIF-2 inhibitors.

[0472] Dosing and administration

[0473] It is generally desirable to use appropriate salts and buffers to stabilize the delivery vehicle and allow uptake by target cells. The aqueous composition of the present invention comprises an effective amount of a delivery vehicle comprising an agent of the present invention (e.g., liposomes or other complexes or expression vectors) dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase pharmaceutically acceptable or pharmacologically acceptable refers to a molecular entity and composition that does not produce side effects, allergies, or other adverse reactions when administered to an animal such as a human. As used herein, pharmaceutically acceptable carriers include solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents acceptable for use in a formulated drug, such as a drug suitable for administration to a human. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active ingredient of the present invention, it is contemplated that it will be used in therapeutic compositions. Supplementary active ingredients may also be incorporated into the composition as long as they do not inactivate the carrier or polynucleotide of the composition.

[0474] The active compositions of the present disclosure may include classical pharmaceutical formulations. Administration of these compositions according to the present invention may be via any conventional route, provided that the target tissue is accessible via said route. This includes oral, nasal, or buccal administration. Alternatively, administration may be by intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection, or by direct injection into the cancerous tissue. The agents disclosed herein may also be administered via a catheter system. Such compositions will normally be administered as pharmaceutically acceptable compositions as described herein.

[0475] After preparation, the solution can be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The preparation can be easily administered in a variety of dosage forms such as injectable solutions, drug release capsules, etc. With regard to parenteral administration in the form of an aqueous solution, for example, the solution is generally appropriately buffered and the liquid diluent is first made isotonic with, for example, sufficient normal saline or glucose. Such aqueous solutions can be used, for example, for intratumoral, intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Preferably, a sterile aqueous medium is used, which is known to those skilled in the art, particularly in light of the present disclosure. As an illustration, a single dose can be dissolved in 1 ml of isotonic NaCl solution, or added to 1000 ml of subcutaneous perfusion fluid, or injected into a proposed infusion site (see, for example, Remington's Pharmaceutical Sciences, 15th edition, pp. 1035-1038 and 1570-1580, the contents of which are hereby incorporated by reference). Depending on the disease of the subject being treated, a certain dosage variation will necessarily occur. The individual responsible for administration will in any case determine the appropriate dosage for the individual subject. Furthermore, for human administration, preparations must meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.

[0476] In some embodiments, the first and second doses can be administered in any order (eg, first then second, or second then first) or simultaneously.

[0477] In some embodiments, the present disclosure includes an agent described herein, and a second agent that is or comprises at least one other cancer biologic, therapeutic, chemotherapeutic, or drug.

[0478] In some embodiments, the agents of the present disclosure may be administered over any suitable period of time, such as a period of time from αβovτ1 day to about 12 months. In some embodiments, for example, the administration period may be from about 1 day to 90 days; preferably, from about 1 day to 60 days; from about 1 day to 30 days; from about 1 day to 20 days; from about 1 day to 10 days; from about 1 day to 7 days. In some embodiments, the administration period may be from about 1 week to 50 weeks; from about 1 week to 40 weeks; from about 1 week to 30 weeks; from about 1 week to 24 weeks; from about 1 week to 20 weeks; from about 1 week to 16 weeks; from about 1 week to 12 weeks; from about 1 week to 8 weeks; from about 1 week to 4 weeks; from about 1 week to 3 weeks; from about 1 week to 2 weeks; from about 2 weeks to 3 weeks; from about 2 weeks to 4 weeks; from about 2 weeks to 6 weeks; from about 2 weeks to 8 weeks; from about 3 weeks to 8 weeks; from about 3 weeks to 12 weeks; or from about 4 weeks to 20 weeks.

[0479] In some embodiments, an agent of the disclosure may be administered daily, every other day, weekly, every 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, or every 20 weeks, or monthly.

[0480] In some embodiments, the therapeutically effective amount of the compositions or agents of the present disclosure can be from about 0.01 mg / kg to about 10 mg / kg per day. In some embodiments, the dosage can be in the range of about 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 3 mg / kg, 5 mg / kg, 6 mg / kg, 7.5 mg / kg, or about 10 mg / kg. In some embodiments, the dosage will be in the range of about 0.1 mg / day to about 5 mg / kg; about 0.1 mg / day to about 10 mg / kg; about 0.1 mg / day to about 20 mg / kg; about 0.1 mg to about 30 mg / kg; or about 0.1 mg to about 40 mg / kg.

[0481] In some embodiments, the therapeutically effective amount of the composition or agent of the present disclosure may be from about 0.1 mg to about 50 mg / kg, or in single, divided or sequential doses (the doses may be based on the patient's body weight (kg), body surface area (m2), or 2 ) and age (years)).

[0482] In some embodiments, a therapeutically effective amount of a composition or agent of the present disclosure can be from about 1 mg / kg to about 1000 mg / kg, from about 5 mg / kg to about 950 mg / kg, from about 10 mg / kg to about 900 mg / kg, from about 15 mg / kg to about 850 mg / kg, from about 20 mg / kg to about 800 mg / kg, from about 25 mg / kg to about 750 mg / kg, from about 30 mg / kg to about 700 mg / kg, from about 35 mg / kg to about 650 mg / kg, from about 40 mg / kg to about 600 mg / kg. kg, about 45 mg / kg to about 550 mg / kg, about 50 mg / kg to about 500 mg / kg, about 55 mg / kg to about 450 mg / kg, about 60 mg / kg to about 400 mg / kg, about 65 mg / kg to about 350 mg / kg, about 70 mg / kg to about 300 mg / kg, about 75 mg / kg to about 250 mg / kg, about 80 mg / kg to about 200 mg / kg, about 85 mg / kg to about 150 mg / kg, and about 90 mg / kg to about 100 mg / kg.

[0483] In some embodiments, a therapeutically effective amount of a composition or agent of the present disclosure is 0.01 mg / kg to about 500 mg / kg, for example, about 0.1 mg / kg to about 200 mg / kg (such as about 100 mg / kg), or about 0.1 mg / kg to about 10 mg / kg (such as about 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 3 mg / kg, 5 mg / kg, 6 mg / kg, 7.5 mg / kg, or about 10 mg / kg).

[0484] Medicine kit of the present disclosure

[0485] The present invention also provides a kit that simplifies the administration of any agent described herein (e.g., an agent that binds to CDCP1 in a cancer patient if the amount of mutant LKB1 and / or KRAS is higher than a reference sample). In some embodiments, the agent binds to CDCP1 and is characterized by AKT activation, or the agent is a CDCP1 activator. In some embodiments, the agent activates CDCP1 and is conjugated to a PPP4R2 modulator. In some embodiments, the agent that binds to CDCP1 is conjugated to a PARG modulator.

[0486] Exemplary medicine boxes of the present invention include any compositions described herein in unit dosage form. In some embodiments, the unit dosage form is a container, such as a sterile pre-filled syringe, which contains any agent described herein and a pharmaceutically acceptable carrier, diluent, excipient or vehicle. The medicine box may also include a label or printed instructions for indicating the use of any agent described herein. The medicine box may also include an eyelid speculum, a local anesthetic and a detergent for application site. The medicine box may also include one or more other agents, such as biological products, therapeutic agents, chemotherapeutic agents or medicines as described herein. In some embodiments, the medicine box includes a container containing an effective amount of the composition of the present invention and an effective amount of another composition (such as those described herein).

[0487] Example

[0488] The present invention will be further described in detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise indicated. Therefore, the present invention should not be construed in any way as being limited to the following examples, but rather should be construed to encompass any and all variations that become apparent from the teachings provided herein.

[0489] Example 1: CDCP1 expression in human patient tumors

[0490] CDCP1 expression was assessed in human tumor microarrays by immunohistochemistry. Tumor tissue was collected and analyzed according to informed consent signed by the donors under procedures approved by the Institutional Review Board. Tumor cores (1.5 mm to 2.0 mm in diameter) were obtained from formalin-fixed, paraffin-embedded patient tumor samples, and microarrays were constructed in paraffin blocks using standard methods. Sections were cut at 5 μm thickness, mounted on slides, and processed for immunohistochemistry. Slides were pretreated with Epitope Retrieval Solution 2 (AR9640; Leica Biosystems, Buffalo Grove, IL) to expose antigenic sites, and CDCP1 was then labeled using a modified Bond Polymer Refine (DS9800; Leica Biosystems, Buffalo Grove, IL) detection protocol, which includes a peroxide blocker, a protein blocker to minimize nonspecific antibody binding, a post-primary polymer reagent, a DAB chromogen detection system, and a hematoxylin counterstain. CDCP1 protein was detected on the cell membrane using a rabbit anti-CDCP1 antibody (Cell Signaling Technologies, catalog number 4115) diluted 1:100 in Leica Bond primary antibody diluent (AR9352; Leica Biosystems, Buffalo Grove, IL).

[0491] The staining of the slides was assessed by a pathologist using an optical microscope to determine a semi-quantitative H score. The H score was calculated by multiplying the estimated percentage of tumor cells with membrane staining for each subjective staining intensity by the intensity value (0 = negative, 1 = low, 2 = medium, 3 = high) and then adding all the products together to obtain a total score. For example, a tumor in which 50% of the cells stained positive and all had medium intensity, while the other 50% of the cells were unstained, would have an H score of 100 ([50x0] + [0x1] + [50x2] + [0x3]) = 100). The higher the H score value, the greater the membrane staining intensity and / or distribution of the target protein in the sample. For ease of interpretation, for target protein expression, scores in the range of 201-300 were arbitrarily considered high, scores in the range of 101-200 were arbitrarily considered medium, and scores in the range of 1-100 were arbitrarily considered low.

[0492] Table 1 shows the results of many tumors evaluated. CDCP1 expression was noted in all tumor types measured. The H scores of many independent primary human tumors are given. Different patient samples are indicated by a given identifier (non-small cell lung cancer, abbreviated as NSCLC). The average score of tumors directly derived from patients is often lower than the average score of human tumors grown in immunocompromised mice (PDX tumors, see below). This finding has multiple possible explanations, including different tumor growth rates between mice and humans, species differences in the tumor microenvironment, and differences in sample handling and processing. Despite these differences, there is significant overlap between the H scores observed in mouse cancer models and human tumors for the cancer types measured.

[0493] Table 1.

[0494]

[0495]

[0496]

[0497] Example 2: CDCP1 expression in patient-derived xenograft (PDX) tumors

[0498] CDCP1 expression was investigated by immunohistochemistry in patient-derived xenograft (PDX) tumors isolated from mice. Tumors were processed using standard methods for formalin-fixed, paraffin-embedded tissue. Sections were cut at 5 μm thickness, mounted on slides, and processed for immunohistochemistry. Slides were pretreated with Epitope Retrieval Solution 2 (AR9640; Leica Biosystems, Buffalo Grove, IL) to expose antigenic sites, and CDCP1 was then labeled using a modified Bond Polymer Refine (DS9800; Leica Biosystems, Buffalo Grove, IL) detection protocol, including a peroxide blocker, a protein blocker to minimize nonspecific antibody binding, a postprimary polymer reagent, a DAB chromogen detection system, and a hematoxylin counterstain. CDCP1 protein was detected on the cell membrane using a rabbit anti-CDCP1 antibody (Cell Signaling Technologies, catalog number 4115) diluted 1:100 in Leica Bond primary antibody diluent (AR9352; Leica Biosystems, Buffalo Grove, IL).

[0499] As described in Example 1, the staining of the slides was evaluated by a pathologist using an optical microscope to determine the H score for each core in the microarray. Table 2 shows the results of many PDX tumors measured by the above method. H scores were given to tumors from many independent PDX tumors. Different rows are indicated by PDX identifiers (non-small cell lung cancer is abbreviated as NSCLC, and small cell lung cancer is abbreviated as SCLC). CDCP1 expression was noted in all tumor types measured. The highest average score was associated with the ovarian cancer model, while the highest absolute score was associated with ovarian cancer and non-small cell lung cancer (NSCLC). Overall, 31.6% of the models belonged to the high expression category, 61.4% belonged to the medium expression category, and 7% belonged to the low expression category. These data indicate a variety of tumors that may respond to drugs specifically targeting CDCP1.

[0500] Table 2

[0501]

[0502]

[0503]

[0504] Example 3: Expression of CDCP1 in human cancer cell lines and primary cells

[0505] CDCP1 protein expression was characterized by quantitative fluorescence flow cytometry on the surface of various human cancer and normal cell lines, including PC3 (prostate cancer), H1299 (non-small cell lung cancer), SCC-25 (head and neck cancer), H2009 (lung adenocarcinoma), PE / CA-PJ-49 (oral squamous cell carcinoma), and primary human aortic smooth muscle cells (HuAoSMC). To this end, the anti-CDCP1 antibody CP 13E10-54HC-89LCv1 was used as a detection reagent by directly conjugating it to the fluorescent dye Alexa 647 using the Alexa Fluor 647 Antibody Labeling Kit (ThermoFisher Scientific #A20186) according to the manufacturer's recommended protocol. Adherent cells were dissociated using non-enzymatic cell dissociation buffer (Life Technologies #13150-016), washed in FACS buffer (Hanks balanced salt solution, 2% FBS, 25 mM HEPES, 2 mM EDTA), and finally resuspended in a staining solution containing 5 μg / mL of Alexa 647-conjugated CP13E10-54HC-89LCv1 plus 7-AAD viability dye. For cells prepared as background controls, the staining buffer omitted Alexa 647-conjugated CP13E10-54HC-89LCv1 and contained only 7-AAD viability dye. The stained cells were washed and resuspended in FACS buffer and immediately analyzed using a BD FACSDiva TM The software was used for acquisition on a BD FACSAria instrument. Dead cells that stained positive for 7-AAD were selected, and the geometric mean fluorescence intensity (gMFI) of Alexa 647-positive cells in the APC channel minus the background was determined using FlowJo software.

[0506] CDCP1 levels are calculated from the gMFI of background subtraction by using Bangs Laboratories Quantum A647 MESF test kit (catalog number (Cat. No.) 647). In brief, blank beads and MESF (equivalent soluble fluorescent dye molecules) beads are diluted into PBS respectively, and run under the same date and identical fluorescence setting as the stained cells, to set up a calibration curve using the quantitative analysis template (QuickCal) provided with test kit. The gMFI value of background subtraction was identified on the calibration curve to determine the MESF unit of every cell line. MESF units are further divided by the F / P (fluorophore to protein) ratio of the CP13E10-54HC-89LCv1 antibody that Alexa647 is put together, to calculate the quantity (only surface-exposed molecules) of the CDCP1 molecules of each cell. The cell line measured by this method shows an expression range (table 3) from as low as 12814 molecules / cell (HuAoSMC) to as high as 233567 molecules / cell (PC3).

[0507] Table 3

[0508]

[0509] Example 4: Involvement of CDCP1 expression and phosphorylation in various cancers

[0510] The transmembrane protein CDCP1 associates with Src and PKCδ, and when CDCP1 is activated, all three proteins show an increase in tyrosine phosphorylation. Tyr-734 has been identified as a site phosphorylated by Src and Src family kinases. Bioinformatics analysis of CDCP1 expression indicates that CDCP1 is involved in cancer metastasis and reduces patient survival, including the following: CDCP1 expression levels in multiple tumor types (breast, colon, pancreas, bladder, kidney, ovary, lung) are higher than in corresponding normal tissues, high levels of CDCP1 expression can predict shorter patient survival in lung adenocarcinoma, and high levels of CDCP1 are associated with an increased 5-year recurrence rate in colorectal cancer; and CDCP1 tyrosine phosphorylation is higher in triple-negative breast cancer, which is known to have a poor prognosis in metastatic cases. In addition, different levels of CDCP1 expression were measured in patients with clear cell renal cell carcinoma, and it was determined that the survival rate (Kaplan-Meier plot) of patients with high CDCP1 expression levels was shorter than that of those with low CDCP1 levels. Previous studies have described that tyrosine phosphorylation of CDCP1 in lung cancer cells is greater than that found in normal tissues. Correlation analysis of protein tyrosine phosphorylation in human lung cancer tumor samples has previously been shown, and a strong positive correlation was found between tyrosine phosphorylation of CDCP1 and multiple SFKs. Similarly, human non-small cell lung cancer (NSCLC) cell lines and mice with activated KRAS and / or inactivated LKB1 tumor mutations exhibit high levels of phosphorylated CDCP1 and SFKs.

[0511] Since CDCP1 is a target gene of hypoxia-inducible factor 2α (HIF-2α), HIF-2α and CDCP1 expression may play a key role in promoting tumor metastasis in cells exposed to low oxygen levels. Hypoxia triggers the expression of HIF-2α and the activation of CDCP1 and Src, while stable knockdown of HIF-2α blocks the tyrosine phosphorylation of CDCP1 and Src by hypoxia. It has been shown that injection of A375 cancer cells overexpressing HIF-2α into mice results in the formation of larger tumors compared to control A375 cells, and tumors with high HIF-2α levels also contain enhanced CDCP1 protein expression. Notably, HIF-2α and CDCP1 expression show a strong correlation with epidermal growth factor receptor (EGFR) and Met hepatocyte growth factor receptor, which are known to be regulated by hypoxia and are HIF-2α target genes.

[0512] In the Sanger Project, CDCP1 expression was compared for 45 of the most common tumor suppressor and oncogenes in wild-type and mutant cells across a spectrum of 790 cancer cell lines. Two genes that showed significantly higher CDCP1 expression in mutants compared to wild-type cells were KRAS and LKB1 ( Figure 1 The strong correlation in KRAS suggests that CDCP1 overexpression may be triggered by oncogenic KRAS. Human clinical data show that KRAS+LKB1 mutant tumors have a much higher metastatic rate than tumors with only KRAS mutations, and patients with KRAS+LKB1 mutant tumors have the worst clinical outcomes.

[0513] Example 5: Validation of CDCP1 as a therapeutic target in cancer

[0514] The particular focus on documenting the role of CDCP1 in lung cancer provided an initial step toward developing anti-CDCP1 therapeutics. Therefore, as a first step toward validating CDCP1 as a therapeutic target for metastatic tumors and gaining information relevant to future precision medicine strategies, experiments were performed to determine the effects of silencing CDCP1 in mouse NSCLC cell xenograft tumors.

[0515] To obtain better clinical relevance, an orthotopic xenograft model of metastatic lung cancer was established. Tumors were first induced in mice by subcutaneous inoculation of A549 cells (with luciferase and shRNA). A small portion of the tumor was harvested and placed in the lungs of other mice, which were then treated with doxycycline. Lung tumors formed in mice with A549 cells containing control shRNA, but when CDCP1 was silenced in A549 cells containing CDCP1-specific shRNA, tumor growth was blocked.

[0516] In another experiment, H2009 NSCLC cells with luciferase and shRNA were subcutaneously inoculated into mice ( Figure 2 Until the tumor is formed (about 100cm 3 ) before treatment began, and then the mice were fed doxycycline. After 3-4 weeks, tumor size and body weight were significantly reduced in doxycycline-treated mice inoculated with cells harboring CDCP1-specific shRNA, but not in mice inoculated with cells harboring control shRNA. These studies demonstrate that silencing CDCP1 expression can reduce the size of established tumors. These in vivo experiments confirm that CDCP1 promotes the growth and survival of metastatic tumors and validate CDCP1 as a therapeutic target for reducing tumor growth and the size of established tumors.

[0517] Example 6: Selection of anti-CDCP1 antibodies

[0518] Anti-CDCP1 scFvs were selected from a phagemid-based human scFv naive antibody library and displayed on M13 phage. Selection was based on phage binding to biotinylated human and mouse CDCP1 extracellular domains (ECD; human, RefSeq NP_073753.3, amino acids 1-663; mouse, RefSeq NP_598735.2, amino acids 1-667) immobilized on streptavidin-coated magnetic beads. Human, mouse, and cynomolgus monkey CDCP1 ECDs (cynomolgus monkey; RefSeq XP_005546930.1, amino acids 30-663) were fused to a 6-histidine purification tag at their N-termini, transiently expressed in HEK293 mammalian cell culture, purified by Ni-NTA affinity chromatography, and chemically biotinylated with EZ-Link NHS-PEG4-Biotin (Thermo Pierce).

[0519] Carried out three rounds of phage display selection, then amplified the selected phage library, used people CDCP1 ECD or alternating people-mouse-people (HmH) CDCP1 ECD in all 3 rounds.After the third round, 2400 kinds of cultures of independent bacterium colony origin were grown, and people, mouse and cynomolgus monkey CDCP1 ECD in culture supernatant or periplasmic bacterial extract were screened by enzyme-linked immunosorbent assay (ELISA) for binding. There were 285 positive ELISA clones, 261 of which were bound to people CDCP1 ECD, 184 to mouse CDCP1 ECD, and 228 to cynomolgus monkey CDCP1 ECD. ELISA positive clones were further characterized by flow cytometry, which assessed the combination with human tumor cell line H1299, which expressed CDCP1 on its cell surface. For all binders identified in this assay, the scFv region of phagemid DNA was sequenced to determine unique clones. Sequencing identified 82 unique scFv sequences, 48 ​​of which bound to cynomolgus monkey, mouse, and human CDCP1 ECD. These clones were reformatted into human IgG1 and prioritized based on in vitro toxicity to human tumor cells (using an anti-human antibody toxin conjugate as a secondary reagent) and cell-based and biochemical binding assays. This process identified a candidate, designated CP13E10, which has been selected for further optimization through rational engineering as described herein.

[0520] The CP13E10 antibody heavy and light chain variable domains are set forth in SEQ ID NOs. 1 and 11, respectively, and the heavy and light chains are set forth in SEQ ID NOs. 10 and 17, respectively. Additional candidate antibodies identified through this evaluation process were Antibody 23, Antibody 24, and Antibody 76. The Antibody 23 heavy and light chain variable domains are set forth in SEQ ID NOs. 47 and 52, respectively, and the heavy and light chains are set forth in SEQ ID NOs. 51 and 58, respectively. The Antibody 24 heavy and light chain variable domains are set forth in SEQ ID NOs. 59 and 65, respectively, and the heavy and light chains are set forth in SEQ ID NOs. 64 and 70, respectively. The Antibody 76 heavy and light chain variable domains are set forth in SEQ ID NOs. 47 and 71, respectively, and the heavy and light chains are set forth in SEQ ID NOs. 51 and 74, respectively.

[0521] Example 7: Optimization of anti-CDCP1 antibodies

[0522] Rational design engineering was used to facilitate multi-parameter optimization of the anti-CDCP1 antibody CP13E10. The structure of the CP13E10 Fab in complex with CDCP1-ECD is shown in Figure 2. The resolution was analyzed and used to design rational phage display libraries to identify CP13E10 variants with improved biophysical properties while maintaining CDCP1 binding properties. By incorporating three point mutations (Y(H100)H, W(H100C)H, Y(H100H)H) into the CP13E10 CDRH3 (CP13E10-34 variant), the hydrophobicity of the CP13E10 antibody was reduced, thereby facilitating an efficient conjugation process for the production of antibody drug conjugates (ADCs). Proteins for characterization were produced by transiently transfecting DNA encoding the anti-CP13E10-34 antibody variant and the parent CP13E10 antibody into HEK-293 cells, and the resulting proteins were affinity purified by protein A and buffer exchanged into PBS-CMF pH 7.2 using a G-25 column. M to 0 MThe resulting CP13E10-34 antibody variants with Y(H100)H, W(H100C)H, and Y(H100H)H exhibited significantly reduced hydrophobicity as detected by analytical hydrophobic interaction chromatography (HIC) on a TSK gel butyl-NPR column with ammonium sulfate gradient elution, allowing for ranking of relative hydrophobicity based on elution time. The CP13E10 antibody eluted outside the human IgG pool elution envelope control at an elution time of 48.63 minutes, associated with high hydrophobicity, while the CP13E10 with three CDRH3 point mutations (Y(H100)H, W(H100C)H, and Y(H100H)H) eluted at 28.42 minutes, within the human IgG pool elution envelope control profile ( Figure 3-4 ).

[0523] Although the CP13E10-34 variants are significantly less hydrophobic than the parental CP13E10 antibody, incorporation of these CDRH3 mutations resulted in >7-fold lower binding to CDCP1 expressed on the surface of prostate cancer (PC3) cells (assessed by using a competition fluorescence-activated cell sorting (FACS) assay with a biotinylated CP13E10 antibody as the reporter antibody ( Figure 5 Specifically, the CDCP1 binding properties of the CP13E10 antibody variants were evaluated using a competition FACS assay (with biotinylated CP13E10 antibody as the reporter antibody) to determine whether the CP13E10 variants could effectively compete with the wild-type CP13E10 antibody for binding to the CDCP1 antigen expressed on PC3 cells. For this competition FACS assay, the parent anti-CDCP1 CP13E10 reporter antibody was biotinylated using EZ-link Sulfo-NHS-Biotin Sulfosuccinimidyl Biotin (Thermo / Pierce, catalog number 21217) at a molar coupling ratio of 20:1 according to the manufacturer's protocol. The proteins used in this assay were produced by transiently transfecting DNA encoding the anti-CDCP1 CP13E10 variants and the parent CP13E10 antibody into HEK-293 cells, and the resulting proteins were affinity purified using Protein A and buffer exchanged into PBS-CMF pH 7.2 using a G-25 column. For this competition FACS assay procedure, PC3 cells were detached from the flask using cell dissociation buffer (Gibco, cat. no. 13151-014), washed once with ice-cold FACS buffer (PBS-CMF pH 7.2 + 3% FBS + 0.1% sodium azide), and 2.5 x 10 510 cells were added to each well in a 96-well V-bottom plate (Corbing catalog number 3894). Biotinylated CP13E10 antibody diluted to 1.5 μg / mL in FACS buffer was mixed with anti-CDCP1 CP13E10 variants or parent (WT) CP13E10 antibodies at different concentrations as a positive control, and the samples were added to the plate containing the cells and incubated on ice for one hour. Next, the cells were washed twice with FACS buffer. Streptavidin-PE (Invitrogen / eBioscience catalog number 12-4317-87) diluted 1:200 was added and incubated at room temperature for 30 minutes. The cells were washed twice with FACS buffer and fixed on ice for 15 minutes by adding 100 μl of BD Cytofix (BD Biosciences catalog number 554655) to each well. The cells were washed twice with FACS buffer. Fluorescence intensity was measured using BD FACS CANTO II, and the results were in Figure 5 Incorporation of the CDRH3 point mutation V(H97)E into the CP13E10-34 variant having Y(H100)H, W(H100C)H, and Y(H100H)H restored the CDCP1 binding properties of this variant CP13E10-54 to those of the parent CP13E10 antibody ( Figure 5 These data demonstrate that incorporation of V(H97)E into the heavy chain CDR3 restores the CDCP1 binding properties of this variant CP13E10-54 to those of the parent wild-type CP13E10 antibody. That is, the CP13E10-54 variant competes equally with the biotinylated reporter anti-CDCP1 CP13E10 antibody for CDCP1 binding.

[0524] The CDRL3 mutations N(L93)Q and V(L94)E were then introduced into the CP13E10-54 variant to generate CP13E10-54HC-89LC, and the binding kinetics of the recombinant CDCP1 extracellular domain (ECD) protein were determined by surface plasmon resonance. Specifically, the binding kinetics of anti-CDCP1 antibodies against recombinant human, cynomolgus monkey, and mouse CDCP1-ECD were determined using surface plasmon resonance (SPR) and a Biacore T200 instrument (GE Healthcare). Using the manufacturer's recommendations, anti-human IgG antibodies were amine coupled to a CM5 carboxymethylated dextran sensor chip surface (GE Healthcare) to a density of approximately 10,000-13,000 response units (RU). Each anti-CDCP1 antibody was diluted to 0.5 μg / mL in 10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% P-20 (HBS-EP+) and captured for approximately 20-23 seconds at a flow rate of 10 μL / min. Four 3-fold dilutions of CDCP1-ECD ranging from 1800 nM to 66.7 nM were injected at a flow rate of 50 μL / min, associated for 54 seconds, and dissociated for 90 seconds. The sensor chip surface was regenerated with three 30-second pulses of 3 M MgCl2 at a flow rate of 50 μL / min. All injections used HBS-EP+ as running buffer and sample buffer and were performed at 25°C with a data acquisition rate of 1 Hz. The sensorgrams were double-referenced by using a control surface and buffer injection. The data were fitted to a 1:1 model and equation K using Biacore T200 Evaluation Software v3.0. D =k d / k a The rate constants were determined. The CP13E10-54HC-89LC antibody exhibited 4-fold and 3-fold higher affinity for human and cynomolgus monkey CDCP1-ECD, respectively, relative to the parent CP13E10 antibody, and >11-fold higher affinity for human CDCP1 than the CP13E10-34 variant ( Figure 6 Furthermore, the affinity of CP13E10-54HC-89LC for mouse CDCP1-ECD was slightly increased (approximately 1.6-fold) relative to the parental CP13E10 antibody and significantly increased (20-fold) compared to the CP13E10-34 variant ( Figure 6That is, in addition to the V(H97)E amino acid change that increases binding to CDCP1, incorporating N(L93)Q and V(L94)E into CP13E10-54CDRL3 to generate CP13E10-54HC-89LC further increased affinity for human, cynomolgus monkey, and mouse CDCP1. In addition, for CP13E10-54HC-89LC, the favorable hydrophobicity reduction observed by incorporating the Y(H100)H, W(H100C)H, Y(H100H)H mutations into the CP13E10-WT antibody was maintained. Figure 3-4 ).

[0525] The CP13E10-54HC-89LC antibody heavy and light chain variable domains are set forth in SEQ ID NOs. 26 and 30, respectively, and the heavy and light chains are set forth in SEQ ID NOs. 29 and 32, respectively. In another instance, the V(H97)E, H(H100C)Q, L(H100D)V, L(H100E)Y, D(H100F)N mutations were incorporated into the CP13E10-34 antibody to generate CP13E10-291, resulting in this variant exhibiting a significant >200-fold increase in affinity for human CDCPl and a 14-fold increase in affinity for mouse CDCPl relative to CP13E10-34, as determined by SPR ( Figure 6 ). In addition, Figure 6 As shown, the CP13E10-291 variant exhibited >70-fold increased binding to cynomolgus monkey CDCP1 relative to the CP13E10-WT antibody. The CP13E10-291 antibody heavy and light chain variable domains are set forth in SEQ ID NOs. 44 and 11, respectively, and the heavy and light chains are set forth in SEQ ID NOs. 46 and 17, respectively.

[0526] CP13E10-54HC-89LC antibody heavy chain variable region (V H ) is entirely IGHV1-46*01 (DP-7) germline, except for CDRH3, however the light chain variable region (V L ) contains three non-germline framework residues. To reduce the risk of immunogenicity, IGKV146 (DPK23) was selected for V L In addition, the IGKV146 (IGKV3D-7*01, DPK23) germline was used to replace the V by incorporating the E(L79)Q germline change. LComputer-predicted T cell epitopes in framework 3. Three substitutions (L(L4)M, R(L39)K, E(L79)Q) based on the IGKV146 germline were incorporated into CP13E10-54HC-89LC to generate the CP13E10-54HC-89LCv1 antibody. CP13E10-54HC-89LCv1 retained equivalent CDCP1 binding properties relative to CP13E10-54HC-89LC, as demonstrated by results obtained using a competition ELISA assay.

[0527] Specifically, the CDCP1 binding properties of the germlined antibody variant CP13E10-54HC-89LCv1 were evaluated using a competition ELISA (using a biotinylated CP13E10-54HC-89LC antibody as a reporter antibody) to determine whether the germlined variant could effectively compete with the CP13E10-54HC-89LC antibody for binding to the CDCP1 antigen. For this competition ELISA assay, the CP13E10-54HC-89LC reporter antibody was biotinylated using EZ-link Sulfo-NHS-Biotin Sulfosuccinimidyl Biotin (Thermo / Pierce, catalog number 21217) at a 20:1 molar coupling ratio according to the manufacturer's protocol. The protein used for this assay was produced by transiently transfecting DNA encoding the anti-CP13E10 variant into HEK-293 cells, and the resulting protein was affinity purified by protein A and buffer exchanged into PBS-CMF pH 7.2 using a G-25 column. For this competitive ELISA procedure, a 96-well plate (Costar catalog number 3590) was coated with human extracellular domain recombinant human CDCP1 protein (CDCP1-ECD). The CDCP1-ECD protein was diluted to 1 μg / ml in PBS-CMF pH 7.2, 100 μl was added to each well of the plate, and the plate was incubated overnight at 4°C. The contents of the plate were discarded and then blocked with PBS-CMF pH 7.2 + 0.02% casein for 3 hours at room temperature. 20 ng / mL of biotinylated CP13E10-54HC-89LC antibody in PBS + 0.5% BSA + 0.02% tween-20 was mixed with different concentrations of anti-CDCP1 CP13E10 variants or parent (WT) CP13E10 antibody as a positive control, and the samples were added to the CDCP1 coated and blocked plates and incubated for 2 hours at room temperature. The wells were washed four times with PBS-CMF pH 7.2 + 0.03% tween-20. Streptavidin-HRP (Cat. No. 7100-05, Southern Biotech, (Birmingham, Alabama) diluted 1:10,000 was added and incubated at room temperature for 30 minutes. The wells were washed four times with PBS-CMF pH 7.2 + 0.03% tween-20, and then TMB (BioFx) was added. The reaction was allowed to proceed for 5 to 10 minutes and then quenched with 0.18N H2SO4. The absorbance at 450 nm was measured and the results are shown in FIG. Figure 7 middle.

[0528] Three amino acid substitutions (L(L4)M, R(L39)K, E(L79)Q) based on the IGKV146 germline were incorporated into the CP13E10-54HC-89LC variable light chain framework region to generate CP13E10-54HC-89LCv1, and this variant fully retained human CDCP1 binding properties, as detected in a complete ELISA assay ( Figure 7 Furthermore, the binding kinetics of germlined CP13E10-54HC-89LCv1 against recombinant human, cynomolgus monkey, and mouse CDCP1-ECD were determined using a previously described surface plasmon resonance (SPR) method and were identical to those of the CP13E10-54HC-89LC variant, further confirming the V L Germline substitutions do not alter CDCP1 binding properties ( Figure 6 ).

[0529] The binding of CP13E10-54HC-89LCv1 to CDCP1 expressed on the surface of PC3 prostate cancer cells was assessed using fluorescence activated cell sorting (FACS). For this method, PC3 cells were detached from the flask using cell dissociation buffer (Gibco, catalog number 13151-014), washed once with ice-cold FACS buffer (PBS-CMF supplemented with 3% FBS and 0.1% w / v NaN3, ice-cold), and 2.5 x 10 5Each cell is added to each well in a 96-well V bottom plate (Corning catalog number 3894). Anti-CDCP1 antibody is serially diluted in FACS buffer, and 50 μL is added to each well containing PC3 cells and incubated on ice for 1 hour. Next, the 96-well plate is centrifuged at 1300 rpm for 4 minutes, the supernatant is discarded, and the cells are washed twice with 150 μL / well FACS buffer. The second detection antibody R-PE-conjugated goat anti-human IgGFc (Jackson Immuno Research Labs, catalog number 109-115-098) is diluted 1: 200 in FACS buffer, and 100 μL is added to each well and incubated on ice for 30 minutes. As described above, the cells are washed twice, 100 μL BD Cytofix (BD Biosciences#554655) is added to each well, and the cells are incubated on ice for 15 minutes. The cells were washed twice as described above and then resuspended in 100 uL FACS buffer and the fluorescence intensity was determined using a BD FACS CANTO II. These results demonstrate that using fluorescence activated cell sorting (FACS), the germlined CP13E10-54HC-89LCv1 that binds to CDCP1 expressed on the surface of PC3 prostate cancer cells is indistinguishable from CP13E10-54HC-89LC ( Figure 8 The heavy and light chain variable domains of the CP13E10-54HC-89LCv1 antibody are set forth in SEQ ID NOs. 26 and 36, respectively, and the heavy and light chains are set forth in SEQ ID NOs. 29 and 37, respectively.

[0530] The CP13E10-54HC-89LCv1 antibody contains a putative isomerization sequence propensity in CDRH3, specifically D(H95)-G(H96). Incorporation of the G(H96)A mutation into CP13E10-54HC-89LCv1 generated a variant CP13E10-54HCv13-89LCv1 that eliminated the potential isomerization sequence potential but retained CDCP1 binding properties in a competition ELISA relative to CP13E10-54HC-89LCv1 ( Figure 9 ).

[0531] Example 8: Engineering Antibodies to Achieve Site-Specific Conjugation of Linkers-Payloads via Cysteine ​​Conjugation

[0532] Methods for preparing anti-CDCP1 antibodies for site-specific conjugation to various linker-payloads via reactive cysteine ​​residues are generally performed as described in PCT International Publication No. WO2013 / 093809. One or more residues on the heavy chain (e.g., position 290 according to EU numbering according to Kabat) or the light chain (e.g., numbering 183 according to Kabat) are altered to cysteine ​​(C) residues by site-directed mutagenesis. In some aspects, position K290 (EU numbering according to Kabat, or numbering 307 using Kabat) in the human IgG1 heavy chain constant region of the anti-CDCP1 antibody variants is substituted with a reactive cysteine ​​(C) to achieve site-specific conjugation: CP13E10-183 / 290HC (SEQ ID NO. 19), CP13E10-54HC-89LC-183 / 290HC (SEQ ID NO. 33), and CP13E10-54HCv13-89LCv1-183 / 290HC (SEQ ID NO. 42). In other aspects, residue K183 in the human kappa light chain constant region was substituted with a reactive cysteine ​​(C) to enable site-specific conjugation: CP13E10-183 / 290LC (SEQ ID NO. 21), CP13E10-54HC-89LC-183 / 290LC (SEQ ID NO. 34), and CP13E10-54HC-89LCv1-183 / 290LC (SEQ ID NO. 38). Proteins for conjugation were produced by stably transfecting CHO-K1 SV 10E9 host cells with vectors encoding antibodies engineered with reactive cysteine. The resulting stable CHO pools were cultured, 11% DTNB (Ellman's reagent, 5,5'-dithio-bis-[2-nitrobenzoic acid]) was added on day 10, and the conditioned medium was harvested on day 12. The resulting conditioned medium was purified using a two-column process: Protein-A MabSelect SuRe LX platform followed by TMAE (50 mM HEPES 65 mM NaCl pH 7.0) to remove HMMS process-related impurities.

[0533] Conjugation via transglutaminase (TG)

[0534] Anti-CDCP1 antibodies with human IgG1 constant regions engineered with a transglutaminase ("Q") tag containing an acyl donor and glutamine were generated. The tag was inserted after threonine (T) position 135 and before serine (S) position 136 according to Kabat's Eu numbering for conjugation to various linker-payloads. Methods for preparing anti-CDCP1 antibodies for site-specific conjugation through glutamine residues were generally performed as described in PCT International Publication No. WO2012 / 059882. In some aspects, in addition to the substitution N297A (EU numbering according to Kabat) that allows efficient transglutaminase-mediated site-specific conjugation of endogenous glutamine (Q) at position 295 (EU numbering according to Kabat) to achieve DAR 4 site-specific conjugation of ADCs, an H7C-glutamine tag LLQG (SEQ ID NO: 91) was also engineered into the anti-CDCP1 antibody after position T135 and before position S136 within the human IgG1-CH1 region. The antibody was further altered to improve the specificity of transglutaminase-mediated conjugation to an engineered H7C-glutamine tag and endogenous glutamine (Q) at position 295 by replacing the lysine (K) amino acid at position 222 (EU numbering according to Kabat) on the heavy chain with arginine (R). These H7C-LLQG glutamine tag, N297A and K222R engineered anti-CDCP1 heavy chains CP13E10-H7C-K222R-N297A HC, CP13E10-54HC-89LC-H7C-K222R-N297A HC, and CP13E10-54HCv13-89LCv1-H7C-K222R-N297A HC are listed in SEQ ID NOs. 25, 35, and 43, respectively. The protein used for conjugation was produced by stably transfecting CHO-K1 SV 10E9 host cells with a vector encoding an antibody engineered with a glutamine-containing transglutaminase ("Q") tag and bearing N297A and K222R mutations. The resulting stable CHO pool was cultured and the conditioned medium was harvested on day 12. The resulting conditioned medium was purified using a two-column process: Protein-A MabSelect SuRe LX platform, followed by TMAE (50 mM HEPES 65 mM NaCl pH 7.0) to remove impurities related to the HMMS process.

[0535] Example 9: Crystallographic Identification of the CDCP1 Epitope of the Fab Fragment of the Anti-CDCP1 Antibody CP13E10-54HC-89LC

[0536] A complex of the human CDCP1 extracellular domain (ECD) and the antibody CP13E10-54HC-89LC Fab was formed using a 1:1.1 molar ratio, concentrated to 15.2 mg / mL, and crystallized at 18°C ​​using a hanging drop technique using a 1:1 well solution to protein solution ratio. Crystals were obtained using 20% ​​PEG6K, 200 mM magnesium chloride, 200 mM sodium chloride, 100 mM sodium acetate (pH 5.0) as a precipitant. For data collection, the crystals were cryoprotected in a reservoir solution with 20% ethylene glycol. The crystals were collected using a synchrotron radiation source at the APS in Argonne, IL. The crystals belong to the P 212121 space group, with unit cell parameters The data were processed and scaled using autoPROC. The structure was solved by molecular replacement using the structure of a proprietary antibody whose structure was not disclosed herein. The entire complex model was rebuilt and refined using the COOT and autoBuster programs. The final refined model had Rwork and Rfree values ​​of 20.4% and 21.6%, respectively.

[0537] Epitope and paratope analysis.

[0538] The asymmetric unit contains a single copy of the Fab / antigen complex. Figure 10-11 Antigen and antibody residues are shown Table 4-5 lists the corresponding epitope / paratope contacts involving the complementary determining regions (CDRs) of antibodies. In summary, Figure 10-14 The position of each CDR relative to the antigen is shown; all six CDRs make antigen contacts. In addition, framework residues H71, H73, H74, L49, and L67 also contact CDCP1, the first two having side chains that hydrogen bond to the side chain of CDCP1 Glu242.

[0539] Table 4: Contacts between CDCP1 ECD and the light chain complementarity determining regions (CDRs) of Fab CP13E10-54HC-89LC

[0540]

[0541]

[0542] Table 5: Contacts between CDCP1 ECD and the heavy chain complementarity determining regions (CDRs) of Fab CP13E10-54HC-89LC

[0543]

[0544] The structure reveals an N-linked glycan attached to Asn122 of the antigen (see Figure 12-13 Both antibody chains make contacts with the glycan. Figure 15A and 15B Detailed characterization of the paratope / epitope interaction is shown.

[0545] As in Figure 10 It is obvious that the antigen is crescent-shaped and the antibody is located inside the crescent. Figure 12 Phe (H100A) is shown to be located near the center of the interface, contacting 6 CDCP1 residues (Table 5), more than any other CDR residue. The Phe side chain fills a pocket whose center is approximately identified by the backbone of CDCP1 Leu196 and whose boundaries are approximately defined by the side chains of CDCP1 residues Thr124, Thr160, Ser162, Ala195, and His197. On either side of Phe are charge interactions (see Figure 15A and 15B ), such as between His(H100) / Glu92, Glu(H97) / Lys45, Asp(H100B) / Arg173, and Arg(H71) / Glu242. Another residue making multiple close contacts is Tyr(L32), which is surrounded by the Leu46-Pro55 region of CDCP1 on its phenolic moiety. Arg(L91) interacts with the backbone of the same region. The binding of the antibody covers approximately antigenic surface (including the glycan linked to Asn122).

[0546] Example 10: Internalization of CP13E10-54HC-89LC

[0547] Antibody internalization is a key feature for delivering ADC cytotoxic payloads to the interior of tumor cells. Therefore, CDCP1 internalization induced by the antibody CP13E10-54HC-89LC was evaluated using the human prostate cancer cell line PC3 expressing CDCP1.

[0548] For this purpose, Invitrogen TM SAIVI TM Alexa Fluor TM The antibody CP13E10-54HC-89LC was conjugated to Alexa Fluor 647 using the 647 Antibody / Protein 1mg Labeling Kit (ThermoFisher Scientific). TM The dye was conjugated and purified according to the manufacturer's instructions. Approximately 0.2-0.5 x 10 5 cells / cm2 PC3 cells at 400 nmol / L were exposed to 2 μg / mL of purified AlexaFluor 647 in growth medium (RPMI-1640 [Gibco], 10% heat-inactivated fetal bovine serum (Gibco)). TM Conjugated antibody CP13E10-54HC-89LC was added and incubated at 37°C for time intervals ranging from 5 to 120 minutes. At the end of the incubation period, the plate was cooled on ice, the medium was aspirated, and the plate was washed with excess Ca-free 2+ Mg 2+ The cells were washed with ice-cold phosphate-buffered saline (PBS [Gibco]). The cells were then detached using 0.25% trypsin-EDTA (Gibco), and the trypsin was neutralized with ice-cold growth medium. The detached cells were pelleted by centrifugation, rinsed once in ice-cold PBS, and the final pellet was collected and fixed in 4% paraformaldehyde (PFA) at 4°C for 20 minutes. Following this incubation, the PFA was removed by washing with PBS.

[0549] Using Amnis Image Fixed samples were analyzed using an X Mark II (EMD Millipore) imaging cytometer with a 40X objective lens at low speed / high sensitivity settings. Alexa Fluor 647 was detected in Ch11 using an excitation / emission setting of 642 nm laser. TM The fluorescence signal of The software processed the raw image data. Single cells were gated based on area M01 (bright field) versus aspect ratio M01 (bright field) dot plots. Cells in focus were then gated based on the gradient RMS bright field histogram. Digital masking of image segmentation of total compartments, membrane compartments, and internal compartments was used to determine the Alexa Fluor 647 expression in each of those compartments for each analyzed cell. TM The relative signal intensity of the cells was calculated and expressed as the median value of the cell population in a given sample. The quantitative data for each experiment were then exported to Excel, and the ratio between the values ​​of the internal compartment and the membrane compartment was calculated and plotted against time. The endocytic internalization rate constant, Ke, was determined using GraphPad Prism software based on a published procedure (Wiley et al., 1982, J. Biol. Chem. 257: 4222-4229) to obtain the slope of the best-fit linear regression line for the initial linear phase of internalization.

[0550] like Figure 16 As shown, in the PC3 cell line, the target internalization rate (Ke) induced by CP13E10-54HC-89LC was 0.013±0.001min -1A positive Ke indicates that the antibody is efficiently internalized from the cell membrane into the cell. Under these conditions, antibody concentration and relative cell density were not found to have any effect on the internalization rate.

[0551] Example 11: Evaluation of CDCP1 Antibodies on CDCP1 Activity and Cellular Processes

[0552] Since CDCP1 protein expression and tyrosine phosphorylation levels were observed to be high in NSCLC cell lines and lung tissue, this provided a method to test the efficacy of CDCP1 ADCs using lung tumor patient-derived autologous xenografts (PDXs). Lung tumors (<200 mm) of NSCLC cells were established in mice within a 21-day pretreatment period. 3 ), and these tumors had high expression of CDCP1 protein. After 21 days, four treatments were delivered at four-day intervals. Tumor masses in vehicle-treated mice progressed steadily, reaching their maximum size within 12 days ( Figure 17 ). In contrast, treatment with the CDCP1-ADC (CP13E10-SS3-LP15) rapidly suppressed tumor growth, with no signs of detectable disease at day 32 of treatment. It was not surprising that the negative ADC (linked to a negative control antibody) had some efficacy. The key finding was that the CDCP1-ADC successfully blocked lung tumor growth, which, along with other data, suggests that this antibody can be further developed as a lead candidate. Therefore, multiple CDCP1-ADCs were tested against a variety of PDX tumor models.

[0553] In various experimental approaches, CDCP1 antibodies were also tested for their ability to activate CDCP1 and its downstream targets. In several lung cancer (H1299) and breast cancer (MDA-MB-231) cell lines and normal mammary epithelial (MCF10A) cell lines, short-term treatment of cells with antibodies stimulated CDCP1 tyrosine phosphorylation and downstream phosphorylation of SFKs and PKCδ ( Figures 18A-18C In contrast, prolonged treatment with CDCP1 antibodies resulted in a decrease in CDCP1 expression and phosphorylation (possibly due to antibody-mediated internalization), as well as a decrease in downstream signaling, including a decrease in Src activation ( Figures 19A-19B and Figure 20 ).

[0554] Due to the known active role of CDCP1 in regulating these cellular processes, the effect of CDCP1 antibody treatment on cell migration and invasion was also tested. Surprisingly, treatment of H1299 and MDA-MB-231 cells with several different CDCP1 antibodies produced a decrease in cell migration in the Transwell assay ( Figures 21A-21B ), and a reduction in cell invasion in a three-dimensional tumor cell invasion assay ( Figures 22A-22B These findings demonstrate CDCP1 activity and its downstream cellular processes.

[0555] Due to the known active role of CDCP1 in regulating these cellular processes, the effects of CDCP1 antibody treatment on cell migration and invasion were also tested. Surprisingly, treatment of H1299 and MDA-MB-231 cells with several different CDCP1 antibodies produced a reduction in cell migration in a Transwell assay, as well as a reduction in cell invasion in a three-dimensional tumor cell invasion assay. Treatment of MDA-MB-231 cells with CDCP1 antibodies also reduced spheroid size and invasion. In contrast, treatment of MCF10A and DLD-1 cells with CDCP1 antibodies produced an increase in cell migration. The opposing effects of CDCP1 antibodies on processes associated with tumor formation and growth may be due to the contrasting effects of CDCP1 activating antibodies on AKT activity in different cells, as AKT plays an active role in the migration of most cells. CDCP1 activating antibodies reduced basal AKT phosphorylation (activity) in H1299 and H1373 cells, but had no effect on AKT in MCF10A, H1975, and HCT116 cells ( Figures 23A-23B The contrasting effects of the antibodies on AKT activity in H1299 and MCF10A cells are consistent with their corresponding roles in cell invasion.

[0556] CDCP1 antibodies also reduced basal AKT activity and AKT substrate phosphorylation in prostate cancer (PC3) cells (see Figures 24A-24C The inhibition was short-lived, with an 80% reduction within 20 minutes. During prolonged antibody exposure, AKT phosphorylation returned to its initial level, consistent with decreased CDCP1 protein expression. The reduction in AKT phosphorylation was blocked by Src inhibition, consistent with AKT inhibition being downstream of CDCP1 phosphorylation by Src. AKT activation downstream of G protein-coupled receptor activation (IGF1, P2Y2, LPA, muscarinic agents) was also blocked by CDCP1 activation.

[0557] Example 12: CDCP1 signaling and crosstalk with other proteins

[0558] To better understand the biological role of CDCP1 and the effects of CDCP1 antibody therapy, studies were performed to evaluate the involvement of CDCP1 on other downstream proteins and signaling pathways. Figure 25Various Western blot experiments are shown for immunoprecipitation studies using an activating antibody ("76") and a non-activating antibody ("24") in PC3 cells. Antibodies were added to intact cells (80 minutes, 4°C). Negative control antibodies and 24 (5 μg / ml); 76 (20 ng / ml). Preferential CDCP1 partner binding was observed for the following: 76 mAb (activating): SRC, PPP4R2 and 24 mAb (non-activating): PARG1. Non-preferential binding was observed for 76 and 24 for β-catenin, transferrin receptor, and importin-7.

[0559] Example 13: Binding of Antibodies CP13E10-54VH-89VL and CP13E10-54HC-89LCv1 to Human Cancer Cell Lines

[0560] As determined by flow cytometry, CP13E10-54HC-89LC and CP13E10-54HC-89LCv1 exhibited dose-dependent binding to cells expressing CDCP1. Binding was assessed on three human cancer cell lines, PC3 (prostate cancer), H1299 (non-small cell lung cancer), and H2009 (lung adenocarcinoma). To this end, adherent cells were first dissociated with cell dissociation buffer (Life Technologies #13150-016), pelleted by centrifugation, and resuspended in FACS buffer (PBS-CMF, 3% FBS, 0.1% weight / volume (w / v) sodium azide). The cells were then mixed with CP13E10-54HC-89LC or CP13E10-54HC-89LCv1 diluted in the same buffer to generate a 3-fold 12-point dilution series with a final antibody concentration range of 100 nM to 0.565 pM. Cells were maintained on ice for one hour with antibody, washed twice with ice-cold FACS buffer, and then stained with anti-human IgG Fc conjugated to R-PE (Jackson ImmunoRsearch Labs#109-115-098). After incubation on ice for 30 minutes in the dark, cells were washed twice, merged with eFluor 660 fixable viability dye (eBioscience#65-0864-18), and fixed (BD Cytofix, BD Biosciences#554655). BD FACSDiva software was used to obtain stained cells on LSR Fortessa instruments. FloWJo software was used to determine the gMFI of the background-subtracted viable cells, and Graphpad Prism software was used to map the ECs. 50 ( Figure 26A and 26BThe calculated values ​​for CP13E10-54HC-89LC were in the range of 0.73-1.69 nM. The EC values ​​for CP13E10-54HC-89LCv1 were determined only for PC3 cells. 50 It is 4.06 nM (Table 6).

[0561] Table 6

[0562]

[0563] Example 14: Preparation of anti-CDCP1 antibodies, CP13E10-54HC-89LC, and CP13E10-54HC-89LCv1 for site-specific conjugation of linker-cytotoxic drug payloads

[0564] Methods for preparing CP13E10-54HC-89LC and CP13E10-54HC-89LCv1 derivatives for site-specific conjugation via cysteine ​​residues are generally performed as described in PCT Publication WO2013 / 093809 (incorporated herein in its entirety) and as outlined in detail above. Methods for preparing CP13E10-54HC-89LC derivatives for site-specific conjugation via glutamine residues are generally performed as described in PCT Publication WO2012 / 059882 and / or WO2016 / 166629 (incorporated herein in their entirety) and as outlined in detail above.

[0565] In order to produce cysteine-modified antibodies CP13E10-54HC-89LC-183 / 290 or CP13E10-54HC-89LCv1-183 / 290 or glutamine-modified antibodies CP13E10-54HC-89LC-H7C-K222R-N297A, CHO cells were transfected with DNA constructs encoding the corresponding antibodies, and stable high-production pools were separated using standard procedures well known in the art. Antibodies were purified from transfected cell-conditioned medium using a two-column method. In brief, antibodies were affinity purified using protein-A (MabSelect SuReLX platform) and then purified using TMAE columns. In some cases, a final purification step using phenyl sepharose hydrophobic interaction chromatography (HIC) was employed.

[0566] The final purified product is analyzed by SoloVPE slope spectroscopy, SDS-PAGE and analytical SEC (YMC-PackDiol-200). Endotoxin is tested using Endosafe PTS RMPTS964 and Endosafe test paper PTS-20 from Charles River Laboratories. The quantity of CHO host cell protein and protein A impurity has been assessed by Cygnus ELISA (catalog number F550 and F610, respectively). Preparations typically exhibit endotoxin levels lower than 1EU / mg, host cell protein contamination lower than 100 ng / mg, protein A contamination lower than 10ng / mg and high molecular weight substances lower than 1%, and the target peak contains 99% total protein.

[0567] Example 15: Generation of Cytotoxic Payload Drug Compounds

[0568] Auristatin drug compounds 0101 and 0131 were prepared according to the methods described in PCT Publication No. WO 2013 / 072813, which is incorporated herein in its entirety. In the published application, auristatin compounds are denoted by the numbering system shown in Table 7.

[0569] Table 7

[0570] Auristatin drug compounds Name in WO2013 / 072813 0101 #54 0131 #118

[0571] According to PCT publication WO2013 / 072813, pharmaceutical compound 0101 was prepared according to the following procedure.

[0572]

[0573] General procedure as described in WO 2013 / 072813 (incorporated herein by reference):

[0574] General Procedure A: Removal of 9-fluorenylmethoxycarbonyl (FMOC) using diethylamine or piperidine. To a solution of the Fmoc-containing compound in dichloromethane or N,N-dimethylformamide (also known as DMF) is added an equal volume of diethylamine or piperidine. The progress of the reaction is monitored by LC-MS (or HPLC or TLC). The solvent is removed under vacuum, and in some cases, the residue is azeotroped 1 to 4 times with heptane. The residue is typically diluted with dichloromethane and a small amount of methanol, then reduced onto silica and purified by silica gel chromatography, eluting with methanol in dichloromethane (or other appropriate solvent mixtures) to give the desired material (or the crude material is used as is).

[0575] General Procedure D: Coupling with 0-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU). To a stirred solution of the amine (1.0 equiv) and the acid (1.0-2.0 equiv) in dichloromethane, N,N-dimethylformamide (also known as DMF), or a mixture of the two, is added HATU (1.0-2.0 equiv). This is followed by the addition of triethylamine (2.0-4.0 equiv) or diisopropylethylamine (2.0-4.0 equiv, also known as Hunin's base). The progress of the reaction is monitored by LC-MS (or HPLC or TLC); the reaction is typically complete within three hours. The solvent is removed under vacuum. The residue is purified by silica gel or reverse phase chromatography, or in some cases azeotroped three times with heptane, diluted with a small amount of ethyl acetate, then reduced onto silica or C18-bonded silica, and purified by silica gel or reverse phase chromatography.

[0576] Step 1. Synthesis of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxohept-4-yl]-N-methyl-L-valinamide (#53). The crude desired material was synthesized from #32 (2.05 g, 2.83 mmol, 1 eq), amine #19 (2.5 g, 3.4 mmol, 1.2 eq), HATU (1.29 g, 3.38 mmol, 1.2 eq), and triethylamine (1.57 mL, 11.3 mmol, 4 eq) in dichloromethane (20 mL, 0.1 M) and N,N-dimethylformamide (3 mL) according to General Procedure D and purified by silica gel chromatography (Gradient: 0% to 55% acetone in heptane) to yield #53 (2.42 g, 74%) as a solid. LC-MS: m / z 965.7 [M+H] + ],987.6[M+Na + ], retention time = 1.04 minutes; HPLC (Protocol A): m / z 965.4 [M+H + ], retention time = 11.344 min (purity > 97%); 1H NMR (400 MHz, DMSO-d6), it is assumed to be a mixture of rotamers, characteristic signals: δ 7.86-7.91 (m, 2H), [7.77 (d, J = 3.3 Hz) and 7.79 (d, J = 3.2 Hz), total 1H], 7.67-7.74 (m, 2H), [7.63 (d, J = 3.2 Hz) and 7.65 (d, J = 3.2 Hz), total 1H], 7.38-7.44 (m, 2H), 7.30-7.36 3H). Melting temperature: 4000 ℃ and 8000 ℃ (m, 2H), 7.11-7.30 (m, 5H), [5.39 (ddd, J = 11.4, 8.4, 4.1 Hz) and 5.52 (ddd, J = 11.7, 8.8, 4.2 Hz), 1H in total], [4.49 (dd, J = 8.6, 7.6 Hz) and 4.59 (dd, J = 8.6, 6.8 Hz), 1H in total], 3.13, 3.17, 3.18 and 3.24 (4 s, 6H in total), 2.90 and 3.00 (2 br s, 3H in total), 1.31 and 1.36 (2 br s, 6H in total), [1.05 (d, J = 6.7 Hz) and 1.09 (d, J = 6.7 Hz), 3H in total].

[0577] Step 2. Synthesis of 2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxohept-4-yl]-N-methyl-L-valinamide (referred to herein as #54 or 0101). The desired crude material was synthesized from #53 (701 mg, 0.726 mmol) in dichloromethane (10 mL, 0.07 M) according to General Procedure A, which was purified by silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane). The residue was diluted with ether and heptane and concentrated in vacuo to afford #54 (also referred to herein as 0101) (406 mg, 75%) as a white solid. LC-MS: m / z 743.6 [M+H + ], retention time = 0.70 min; HPLC (Protocol A): m / z 743.4 [M+H + ], retention time = 6.903 minutes, (purity > 97%); 1H NMR (400 MHz, DMSO-d6), presumably a mixture of rotamers, characteristic signals: δ [8.64 (br d, J = 8.5 Hz) and 8.86 (br d, J = 8.7 Hz), total 1H], [8.04 (br d, J = 9.3 Hz) and 8.08 (br d, J = 9.3 Hz), total 1H], [7.77 (d, J = 3.3 Hz) and 7.80 (d, J = 3.2 Hz), total 1H], [7.63 (d, J = 3.3 Hz) and 7.66 (d, J = 3.2 Hz), total 1H], 7.13-7.31 (m, 5H), [5.39 (ddd, J = 11, 8.5, 4 Hz) and 5.53 (ddd, J = 12, 9, 4 Hz), total 1H], [4.49 (dd, J = 9, 8 Hz) and 4.60 (dd, J = 9, 7 Hz), total 1H], 3.16, 3.20, 3.21 and 3.25 (4s, total 6H), 2.93 and 3.02 (2br s, 3H total), 1.21 (s, 3H), 1.13 and 1.13 (2s, 3H total), [1.05 (d, J = 6.7 Hz) and 1.10 (d, J = 6.7 Hz), 3H total], 0.73-0.80 (m, 3H).

[0578] In some embodiments, drug 0101 is linked to a linker, 6-maleimidocaproyl-valine-citrulline-p-aminobenzylcarbamate (also referred to herein as "mc-val-cit-PABC," "mcValCitPABC," or "vc"). The linker-cytotoxic drug payload (LP), mc-val-cit-PABC-0101 (also referred to herein as "vc0101"), is prepared according to the methods described in PCT Publication WO2013 / 072813, which is incorporated herein by reference in its entirety.

[0579] According to PCT publication WO2013 / 072813, pharmaceutical compound 0131 was prepared according to the following procedure.

[0580]

[0581] Step I. Synthesis of 1-(tert-butoxycarbonyl)-2-methyl-L-prolyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-methoxy-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]4-oxobutyl}-N-methyl-L-valinamide (#116). To a stirred solution of #114 (1.02 g, 1.61 mmol, 1.0 equiv) and 1-(tert-butoxycarbonyl)-2-methyl-L-proline (443 mg, 1.93 mmol, 1.2 equiv) in 12 mL of dichloromethane was added HATU (735 mg, 1.93 mmol, 1.2 equiv) followed by Hunin's base (1.12 mL, 6.45 mmol, 4.0 equiv). The reaction was allowed to stir at room temperature for 2 hours. The reaction was reduced, diluted with ethyl acetate, then washed with 0.5 N HCl and brine. The organics, dried over sodium sulfate, were then reduced to a smaller volume and then reduced onto silica. Silica chromatography (Gradient: 0% to 45% acetone in heptane) then produced #116 (1.02 g, 74%) as a white solid. LC-MS (Protocol Q): m / z 844.3 [M+H+], 867.2 [M+Na+], retention time = 2.15 min.

[0582]

[0146] Step 2A. Synthesis of 2-methyl-L-prolyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-benzyl-2-methoxy-2-oxoethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methoxypropyl]-4-oxobutyl}-N-methyl-L-valinamide, trifluoroacetic acid salt (#117). To a stirred solution of #116 (450 mg, 0.533 mmol, 1.0 equiv) in 7 mL of dichloromethane at 0°C was added TFA (3 mL, 40 mmol, 70 equiv). The reaction was stirred at 0°C for 5 minutes and then allowed to warm to room temperature while stirring for 20 minutes. The reaction was reduced, diluted with dichloromethane and a small amount of methanol, and then reduced onto silica. Silica chromatography (Gradient: 0% to 20% methanol in ethyl acetate) then produced #117 (396 mg, 89%) as a white solid. LC-MS (Protocol Q): m / z 744.5 [M+H+], 767.2 [M+Na+], retention time = 1.40 min; HPLC (Protocol A at 45°C): m / z 744.5 [M+H+], retention time = 7.149 min (purity > 91%). 1H NMR (400 MHz, DMSOd6), 88.73-9.14 (m), 8.66 (br d), 8.50(d), 8.22(d), 7.12-7.25(m), 4.67-4.74(m), 4.41-4.63(m), 3.93-4.00(m), 3.73(dd), 3.63(d), 3.46- 3.57(m), 3.38-3.45(m), 3.26-3.23(m), 3.22-3.25(m), 3.06-3.22(m), 2.99-3.05(m), 2.93-2.97(m), 2.80-2.8 9(m), 2.75-2.78(m), 2.64-2.67(m), 2.46-2.50(m), 2.27-2.43(m), 2.00-2.26(m), 1.85-1.99(m), 1.70-1.83( m), 1.52-1.69(m), 1.33-1.51(m), 1.18-1.31(m), 0.98-1.07(m), 0.93-0.97(m), 0.82-0.92(m), 0.71-0.78(m).

[0583]

[0266] Step 2B. 2-Methyl-prolyl-N-[(3R,4S5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxohept-4-yl]-N-methyl-L-valinamide, trifluoroacetate salt (#118). To a stirred solution of #116 (435 mg, 0.515 mmol) in 4 mL of THF under nitrogen was added LiOH (24.7 mg, 1.03 mmol, 2.0 equiv) dissolved in 2 mL of water. The reaction was stirred at room temperature until LC-MS indicated saponification of the methyl ester. The reaction was concentrated in vacuo and then placed under vacuum. The reaction was diluted with dichloromethane and placed under nitrogen. To this stirred mixture was added TFA (3 mL, 40.5 mmol, 80 equiv). The reaction was allowed to stir at room temperature for 30 h. The reaction was then reduced. The residue was purified by medium pressure reverse phase C18 chromatography (Gradient: 5% to 60% acetonitrile in water with 0.02% TFA in each phase) #118 (396 mg, 89%) as a white solid. LC-MS (Protocol Q): m / z 730.2 [M+H+], retention time = 1.18 min; HPLC (Protocol A at 45°C): m / z 730.5 [M+H+], retention time = 7.088 min (purity > 98%). 1H NMR(400MHz, DMSO-d6), 89.04-9.13(m), 8.75-8.87(m), 8.70(d), 8.38(d), 8.ll(d), 7.10-7.24(m), 4.66-4.74( m), 4.48-4.64(m), 4.37-4.47(m), 3.91-3.99(m), 3.77(m), 3.47-3.56(m), 3.33-3.47(m), 3.08-3.30(m), 2.93-3 .07(m), 2.75-2.86(m), 2.63-2.69(m), 2.45-2.50(m), 2.28-2.44(m), 2.03-2.27(m), 1.88-2.02(m), 1.68-1.86 (m), 1.55-1.67(m), 1.30-1.47(m), 1.17-1.29(m), 0.98-1.05(m), 0.93-0.97(m), 0.83-0.92(m), 0.71-0.79(m).

[0584] According to PCT Publication WO 2016 / 166629, which is incorporated herein in its entirety, the auristatin compound 0131 (chemical name 2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxohept-4-yl]-N-methyl-L-valinamide) coupled to an amine donor linker that allows conjugation to an acyl donor antibody is referred to herein as amino-PEG6-C2-0131 and has the following structure:

[0585]

[0586] Example 16: Bioconjugation of CP13E10-54HC-89LC-183 / 290, CP13E10-54HC-89LCv1-183 / 290, and CP13E10-54HC-89LC-H7C-K222R-N297A Antibodies

[0587] The antibodies of the present invention are conjugated to cytotoxic drug payloads via a linker to produce antibody-drug conjugates (ADCs). The conjugation method used is site-specific (ie, through a specific cysteine ​​residue or a specific glutamine residue).

[0588] The ADCs, CP13E10-54HC-89LC-183 / 290-vc0101 and CP13E10-54HC-89LCv1-183 / 290-vc0101, were produced by chemical conjugation using a cysteine ​​site-specific approach. The linker-cytotoxic drug payload (LP), mc-Val-Cit-PABC-0101 (also referred to herein as vc0101), was conjugated to the anti-CDCP1 antibody CP13E10-54HC-89LC-183 / 290 or CP13E10-54HC-89LCv1-183 / 290, prepared as described in Example 14, via its engineered cysteine ​​residue. As a first step, the 5-thio-2-nitrobenzoic acid (TNB)-capped antibody was reduced with a 20-fold molar excess of tris(3-sulfonatophenyl)phosphine (TSPP) at 37°C for 3 hours, followed by desalting to remove excess TSPP. The reduced antibody was incubated in a 2-fold molar excess of dehydroascorbic acid (DHA) at 25°C for 0.5 hours to reconstitute interchain disulfide bonds. LP was added to the reaction mixture at a LP / antibody molar ratio of 10 and reacted in the presence of 15% (vol / vol) dimethylacetamide (DMA) at 25°C for an additional 1.5 hours. Following incubation, a 20-fold molar excess of L-cysteine ​​was added to quench any unreacted LP.

[0589] The reaction mixture was then desalted to remove free LP and purified by hydrophobic interaction chromatography (HIC). The purified ADC was dialyzed into 20 mM histidine, 85 mg / mL sucrose (pH 5.8) formulation buffer and stored at -80°C. Protein concentration was determined by UV spectrophotometry. The purity of the ADC was further characterized by SEC; reverse phase (RP) UPLC and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI MS) were used to calculate drug loading profiles and drug-antibody ratios (DAR). The final ADC formulation typically had a monomer purity greater than 95% and less than 5% high molecular weight species and a DAR of approximately 4.

[0590] The ADC, CP13E10-54HC-89LC-H7C-K222R-N297A-amino-PEG6-C2-0131 (i.e., CP13E10-54HC-89LC-H7C-K222R-N297A-AmPEG6C2-0131), was produced by chemical conjugation using a glutamine site-specific transamidation method. In the transamidation reaction, glutamine on the antibody acts as an acyl donor, and an amine-containing compound on the linker-cytotoxic payload amino-PEG6-C2-0131 (also referred to herein as aminoPEG6-propionyl or AmPeg6C2-0131) (having a structure described in PCT Publication WO2016 / 166629 (incorporated herein in its entirety)) acts as an acyl acceptor (amine donor). Purified antibody CP13E10-54HC-89LC-H7C (acyl donor) was incubated with a 10-25 molar excess of acyl acceptor, AmPeg6C2-0131 (final concentration) ranging from 1 to 2 mM, in the presence of 0.75 units / mg antibody (final concentration) of Streptoverticillus mobara transglutaminase in 200 mM sodium chloride and Tris HCl buffer at pH 7.5-8.5. After incubation at 25° C. for 14-20 hours, the antibody drug conjugate was purified by butyl sepharose HIC-FPLC (GE Healthcare, Piscataway, NJ) using standard chromatography methods known to those skilled in the art.

[0591] The purified ADC was dialyzed into 20 mM histidine, 85 mg / mL sucrose (pH 5.8) formulation buffer and stored at -80°C. Protein concentration was determined by UV spectrophotometry. The purity of the ADC was further characterized by SEC; reverse phase (RP) UPLC and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI MS) were used to calculate drug loading profiles and drug-antibody ratios (DARs). The final ADC formulation typically had a monomer purity greater than 95% and less than 5% high molecular weight species, with a DAR of approximately 4.

[0592] Example 17: Characterization of CP13E10-54HC-89LCv1-183 / 290-vc0101 ADC

[0593] After conjugation (see Examples 16 and 17), the CP13E10-54HC-89LCv1-183 / 290-vc0101 ADC was formulated into 20 mM histidine, 85 mg / mL sucrose (pH 5.8), and at 3.27 mg / mL, there were no significant issues with solubility, viscosity, or aggregate formation. The purity of CP13E10-54HC-89LCv1-183 / 290-vc0101 was characterized by size exclusion chromatography (SEC); reversed-phase (RP) UPLC and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI MS) were used to calculate drug loading profiles and drug-antibody ratios (DAR). These results indicate that the overall yield of DAR 4.0 ADC was 51%, and free drug was below LOQ. To determine the integrity of the CP13E10-54HC-89LCv1-183 / 290-vc0101 ADC, percent purity was calculated using non-reducing and reducing capillary gel electrophoresis (cGE, Caliper LabChip GXII: PerkinElmer, Waltham, MA). The CP13E10-54HC-89LCv1-183 / 290-vc0101 ADC demonstrated excellent integrity, with the preparation being virtually free of HMMS or LMMS at 99.47% intact ADC using non-reducing cGE and 99.91% of the heavy and light chains under reducing conditions. Differential scanning calorimetry (DCS) was used to determine the thermal stability of the CP13E10-54HC-89LCv1-183 / 290-vc0101 ADC. For this analysis, ADC formulated into 20 mM histidine, 8.5% sucrose, 0.005% EDTA (pH 5.8) was dispensed into the sample tray of a MicroCal VP-capillary DSC with an autosampler (GE Healthcare Bio-Sciences, Piscataway, NJ), equilibrated at 10°C for 5 minutes, and then scanned at a rate of 100°C per hour up to 110°C. A filtration time of 16 seconds was selected. The raw data were baseline corrected and protein concentrations were normalized. Data were fitted to the MN2-State model using Origin software 7.0 (OriginLab Corporation, Northampton, MA) using the appropriate number of transitions. The CP13...

Claims

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to CDCP1, the isolated antibody or antigen-binding fragment thereof comprising: (i) a VH comprising: (a) CDRH1, wherein the CDRH1 consists of the amino acid sequence of SEQ ID NO: 2, (b) CDRH2 consisting of the amino acid sequence of SEQ ID NO: 3; and (c) CDRH3 consisting of the amino acid sequence of SEQ ID NO: 27; and (ii) a VL comprising: (a) CDRL1, wherein the CDRL1 consists of the amino acid sequence of SEQ ID NO: 12, (b) CDRL2 consisting of the amino acid sequence of SEQ ID NO: 13; and (c) CDRL3, wherein the CDRL3 consists of the amino acid sequence of SEQ ID NO:

31.

2. The isolated antibody or antigen-binding fragment thereof of claim 1, comprising a VH comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:26; and a VL comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:30 or SEQ ID NO:

36.

3. The isolated antibody or antigen-binding fragment thereof of claim 2, comprising a VH comprising the amino acid sequence of SEQ ID NO: 26; and a VL comprising the amino acid sequence of SEQ ID NO:

36.

4. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 3, comprising a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID No: 29; and a light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO:

37.

5. The isolated antibody or antigen-binding fragment thereof of claim 4, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 29; and a light chain comprising the amino acid sequence of SEQ ID NO:

37.

6. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 3, comprising a heavy chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID No: 33; and a light chain comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO:

38.

7. An isolated antibody or antigen-binding fragment thereof that specifically binds to CDCP1, the isolated antibody or antigen-binding fragment thereof comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 33; and a light chain comprising the amino acid sequence of SEQ ID NO:

38.

8. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-7.

9. The isolated nucleic acid of claim 8, wherein the nucleic acid comprises: a nucleic acid sequence of SEQ ID NO:79 and a nucleic acid sequence of SEQ ID NO:

83.

10. The isolated nucleic acid of claim 8, wherein the nucleic acid comprises: a nucleic acid sequence of SEQ ID NO: 85 and a nucleic acid sequence of SEQ ID NO:

86. A vector comprising the nucleic acid according to any one of claims 8 to 10. A host cell comprising the vector of claim 11 .

13. The host cell of claim 12, wherein the host cell is a mammalian cell selected from the group consisting of: CHO cells, COS cells, HEK-293 cells, NS0 cells, PER. cells or Sp2.0 cells.

14. A method for preparing an antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell according to any one of claims 12 to 13 under conditions where the host cell expresses the antibody or the antigen-binding fragment thereof.

15. The method of claim 14, further comprising isolating the antibody or antigen-binding fragment thereof.

16. An antibody drug conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody is conjugated to a drug moiety.

17. The antibody drug conjugate of claim 16, wherein the antibody is conjugated to the drug moiety via a linker.

18. The antibody drug conjugate of claim 17, wherein the linker is selected from the group consisting of valine-citrulline (val-cit), 6-maleimidocaproyl (mc), methoxy-polyethylene glycol maleimide 6 (MalPeg6), p-aminobenzylcarbamate (PABC), dimethylaminoethanol (DMAE), maleimidopropionyl (MP), hydrolyzed Peg-maleimide, alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), 4- (2-pyridylthio) pentanoic acid N-succinimidyl ester (SPP), 4-(N-maleimidomethyl) cyclohexane-1 carboxylic acid N-succinimidyl ester (SMCC), (4-iodo-acetyl) aminobenzoic acid N-succinimidyl ester (SIAB), 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-val-cit-PAB) and 6-maleimidocaproyl-valine-citrulline-p-aminobenzylcarbamate (mc-val-cit-PABC).

19. The antibody drug conjugate of any one of claims 16-18, wherein the drug moiety is a cytotoxic agent, an immunomodulatory agent, an imaging agent, a chemotherapeutic agent, or a therapeutic protein.

20. An antibody drug conjugate comprising an antibody that specifically binds to CDCP1, wherein the antibody comprises at least one engineered cysteine ​​and a heavy chain comprising the amino acid sequence of SEQ ID NO: 33 and a light chain comprising the amino acid sequence of SEQ ID NO: 38, wherein the antibody is conjugated to a linker-drug moiety mc-val-cit-PABC-0101 via the at least one engineered cysteine.

21. The antibody drug conjugate of claim 20, wherein the antibody comprises two engineered cysteines at position 290 on the antibody heavy chain constant domain according to the numbering of the Eu index of Kabat and at position 183 on the light chain constant domain according to the numbering of Kabat.

22. A pharmaceutical composition comprising the antibody of any one of claims 1-7 or the antibody-drug conjugate of any one of claims 16-21; and a pharmaceutically acceptable carrier.

23. Use of the antibody of any one of claims 1-7, the antibody drug conjugate of any one of claims 16-21, or the composition of claim 22 in the preparation of a medicament for treating cancer in a subject in need thereof, wherein the cancer is selected from one or more of the following: breast cancer; squamous cell carcinoma; lung cancer; lung adenocarcinoma; ovarian cancer; pancreatic cancer; and prostate cancer.

24. The use according to claim 23, wherein the cancer is small cell lung cancer.

25. The use of claim 23, wherein the cancer is non-small cell lung cancer.

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