Humanized antibodies against connexin 4 and drug conjugates thereof

Abstract

The present invention provides humanized antibodies directed against Nectin-4 and antibody-drug conjugates of said humanized antibodies (ADCs) and their use in the treatment of diseases, in particular cancer.

Description

Technical Field

[0001] The present invention belongs to the field of immunotherapy and relates to humanized anti-nectin-4 antibodies and antibody conjugates and therapeutic and diagnostic compositions containing the same for treating diseases, in particular cancer. Background Art

[0002] Cancer immunotherapy is used to generate and enhance anti-tumor immune responses, for example by treating with antibodies that specifically target antigens on tumor cells, or by specifically activating anti-tumor T cells. The ability to recruit immune cells (e.g., T cells) against tumor cells in patients offers a therapeutic modality for combating cancer types and metastases that were previously considered incurable.

[0003] The cell adhesion molecule cohesin 4, also known as poliovirus receptor-related protein 4 (PVRL4), is a type I transmembrane protein and a member of the cohesin family of related immunoglobulin-like adhesion molecules. Cohesin 4 is a tumor-associated marker for many tumors, including bladder cancer, breast cancer, lung cancer, and other malignancies.

[0004] Chailta-Eid et al., 2016 (Cancer Res. 2016;76:3003–13) disclosed an anti-fibronectin 4 (ennosumab) antibody-drug conjugate as a highly effective therapeutic agent in multiple preclinical cancer models. The antibody, conjugated to the microtubule inhibitor vedotin, binds to human, rat, and monkey fibronectin 4 and inhibits the growth of several fibronectin 4-expressing cell lines and xenografts.

[0005] International patent application publication number WO 2019 / 215728 discloses a monoclonal antibody that recognizes human adhesion protein 4 with high affinity and specificity and inhibits its binding to the T cell immunoreceptor (TIGIT) containing Ig and ITIM domains.

[0006] Antibody-drug conjugates (ADCs) are promising tools for direct killing of tumor cells and subsequent activation of bystander immune cells. These therapeutic entities consist of mAbs linked to cytotoxic drugs (payloads) and are designed in principle to broaden the therapeutic window of these drugs by limiting their specific delivery to cells expressing the target antigen, thereby reducing their systemic exposure and toxicity.

[0007] Auristatin is a drug that disrupts microtubules. It is derived from the marine shellless mollusk Dolabella auricularia and is known as dolastatin. Various derivatives of auristatin have been synthesized, such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). MMAE and MMAF were developed by Seattle Genetics and used as payloads for ADCs. MMAF and MMAE each have advantages and disadvantages. MMAE has stronger membrane permeability and a lower IC50 value than MMAF. However, compared to MMAE, MMAF is more hydrophilic, has a lower tendency to aggregate, and exhibits lower systemic toxicity (park et al., Molecules 2019, 24, 2754).

[0008] International patent application publication number WO 2018 / 158398 discloses antibodies specific for cohesin 4, such as 14A5.2, and their use in treating cancer.

[0009] International Patent Application Publication No. WO 2017 / 042210 discloses an anti-fibronectin 4 antibody conjugated to MMAE that is more effective than enroku-vedotin (PADCEV). The antibody used in this application is of murine origin, making it less suitable for clinical use.

[0010] There is an unmet need to provide improved, safe and effective humanized antibodies that recognize human cohesin 4 and ADCs containing the same that can be used to treat cancers expressing cohesin 4. Summary of the Invention

[0011] The present invention provides humanized antibodies, or antigen-binding portions thereof, that specifically bind to fibronectin 4. The humanized antibodies of the present invention, selected from a larger pool of antibody clones, have improved properties compared to other anti-fibronectin 4 antibodies. According to some embodiments, the present invention further provides conjugates comprising the antibodies and a therapeutic or diagnostic agent. In some embodiments, the conjugates comprise a cytotoxic moiety and can be used to treat cancers in which tumor cells present fibronectin 4 receptors on their surfaces.

[0012] Notably, the present invention demonstrates that the NTX1105 ADC is more potent than the most advanced ADCs published to date carrying an anti-fibronectin 4 antibody (PADCEV or enroku-based ADCs). It is now shown that NTX1105-based ADCs have significant anti-tumor activity in treatment regimens where PADCEV or other enroku-based ADCs are inactive.

[0013] By combining specific sets of complementary determining region (CDR) sequences and human framework (FW) sequences, and introducing specific mutations in these sequences to produce antibodies with modified variable regions and improved properties, a large number of humanized antibodies have been produced. The antibodies disclosed herein are designed based on factors including homology, T cell epitopes, key residues and predicted structures. Advantageously, the newly designed humanized variable regions described herein retain residues that are crucial for maintaining antibody conformation and binding affinity, while having a lower incidence of potential T cell epitopes, thereby minimizing the risk of producing an adverse immune response to the antibody.

[0014] The humanized antibodies disclosed herein have excellent productivity characteristics and improved scalability (e.g., higher resistance to aggregation as measured by improved Tagg). The humanized antibodies disclosed herein have been found to be very suitable for use as targeted therapies using therapeutic toxins. It is now disclosed that the anti-adhesion protein 4 monoclonal humanized antibodies described herein coupled to a cytotoxic portion exhibit potent killing of various tumor cell lines. Direct targeting of toxins using the antibodies described herein has the potential to increase the anti-tumor activity of these toxins and improve the survival rate of cancer patients. Some ADCs of the present invention contain sequence modifications to their Fc regions, which significantly reduce their binding to normal cells carrying FcγRs, thereby improving their safety.

[0015] According to one aspect, the present invention provides a humanized antibody that specifically binds to human adhesion protein 4 or a fragment thereof that comprises at least an antigen binding site, wherein the antibody or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region whose amino acid sequence is at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and wherein the light chain comprises a variable region whose amino acid sequence is at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

[0016] According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO: 2. According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO: 3. According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO: 4. According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO: 5. According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO: 6.

[0017] According to some embodiments, the humanized antibody or fragment thereof comprises a light chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO: 8. According to some embodiments, the humanized antibody or fragment thereof comprises a light chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO: 9. According to some embodiments, the humanized antibody or fragment thereof comprises a light chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO: 10. According to some embodiments, the humanized antibody or fragment thereof comprises a light chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO: 11. According to some embodiments, the humanized antibody or fragment thereof comprises a light chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO: 12.

[0018] According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region whose amino acid sequence is at least about 95% identical to SEQ ID NO: 13, and the light chain comprises a variable region whose amino acid sequence is at least about 95% identical to SEQ ID NO: 14.

[0019] According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain comprising a variable region comprising the amino acids set forth in SEQ ID NO: 13. According to some embodiments, the humanized antibody or fragment thereof comprises a light chain comprising a variable region comprising the amino acids set forth in SEQ ID NO: 14. According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 13, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 14.

[0020] Several methods are known in the art for determining the CDR sequences of a given antibody molecule, but there is no standard, clear method. Determination of CDR sequences from antibody heavy and light chain variable regions can be performed according to any method known in the art, including but not limited to the methods known as KABAT, Chothia, and IMGT. A set of selected CDRs may include sequences identified by more than one method, i.e., for example, some CDR sequences may be determined using KABAT and others using IMGT. According to some embodiments, the CDR sequences of the mAb variable regions are determined using the IMGT method.

[0021] According to some embodiments, the humanized antibody or fragment thereof comprises a set of six CDR sequences, wherein the heavy chain CDR1 comprises the sequence SYY (SEQ ID NO: 26), the heavy chain CDR2 comprises the sequence IYPGNVNT (SEQ ID NO: 22), the heavy chain CDR3 comprises the sequence SNPYVMDY (SEQ ID NO: 17), the light chain CDR1 comprises the sequence QSVNND (SEQ ID NO: 24), the light chain CDR2 comprises the amino acid sequence YAS (SEQ ID NO: 25), and the light chain CDR3 comprises the sequence QQAYRSPYT (SEQ ID NO: 20).

[0022] According to some embodiments, the humanized antibody or fragment thereof comprises a set of six CDR sequences, wherein the heavy chain CDR1 comprises the sequence SYYIH (SEQ ID NO: 15), the heavy chain CDR2 comprises the sequence WIYPGNVNTKYNERF(K / Q)G (SEQ ID NO: 16), the heavy chain CDR3 comprises the sequence SNPYVMDY (SEQ ID NO: 17), the light chain CDR1 comprises the sequence (K / R)ASQSVNNDVA (SEQ ID NO: 18), the light chain CDR2 comprises the sequence YASNRFT (SEQ ID NO: 19), and the light chain CDR3 comprises the sequence QQAYRSPYT (SEQ ID NO: 20).

[0023] According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain CDR2 comprising the sequence WIYPGNVNTKYNERFKG (SEQ ID NO: 27). According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain CDR2 comprising the sequence WIYPGNVNTKYNERFQG ​​(SEQ ID NO: 28).

[0024] According to some embodiments, the humanized antibody or fragment thereof comprises a light chain CDR1 comprising the sequence KASQSVNNDVA (SEQ ID NO: 29). According to some embodiments, the humanized antibody or fragment thereof comprises a light chain CDR1 comprising the sequence RASQSVNNDVA (SEQ ID NO: 30).

[0025] According to some embodiments, the humanized antibody or fragment thereof comprises a set of six CDR sequences, wherein the heavy chain CDR1 comprises the sequence GYTFTSYY (SEQ ID NO: 21), the heavy chain CDR2 comprises the sequence IYPGNVNT (SEQ ID NO: 22), the heavy chain CDR3 comprises the sequence ARSNPYVMDY (SEQ ID NO: 23), the light chain CDR1 comprises the sequence QSVNND (SEQ ID NO: 24), the light chain CDR2 comprises the amino acid sequence YAS (SEQ ID NO: 25), and the light chain CDR3 comprises the sequence QQAYRSPYT (SEQ ID NO: 20).

[0026] According to some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a set of four heavy chain (HC) framework (FR) sequences: (A) FR-H1 selected from the group consisting of SEQ ID NOs: 31, 32, and 33; (B) FR-H2 selected from the group consisting of SEQ ID NOs: 34, 35, and 36; (C) FR-H3 selected from the group consisting of SEQ ID NOs: 37, 38, 39, and 40; (D) FR-H4 is SEQ ID NO: 41; and the light chain variable region comprises a set of four light chain (LC) framework (FR) sequences: (A) FR-L1 selected from the group consisting of SEQ ID NOs: 42, 43, and 44; (B) FR-L2 selected from the group consisting of SEQ ID NOs: 45 and 46; (C) FR-L3 selected from the group consisting of SEQ ID NOs: 47, 48, and 49; and (D) FR-L4 is SEQ ID NO: 50. Each possibility or combination of frameworks represents an independent embodiment.

[0027] According to some embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises an amino acid sequence that is at least about 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, and 6; and the light chain variable region of the humanized monoclonal antibody comprises an amino acid sequence that is at least about 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, and 12. Each possibility or combination of heavy and light chains represents a separate embodiment of the invention.

[0028] According to some embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, and 6; and the light chain variable region of the humanized monoclonal antibody comprises a sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, and 12. Each combination of heavy and light chain variable regions represents a separate embodiment of the present invention. According to certain exemplary embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises SEQ ID NO: 5, and the light chain variable region of the humanized monoclonal antibody comprises SEQ ID NO: 12 (represented herein as NTX1105 (H4 / k5)). According to other exemplary embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises SEQ ID NO: 6, and the light chain variable region of the humanized monoclonal antibody comprises SEQ ID NO: 12.

[0029] According to some embodiments, the humanized antibody or fragment thereof is a monoclonal antibody, Fab, F(ab)2, a single domain antibody, or a single chain variable fragment (scFv).

[0030] According to some embodiments, the humanized antibody or fragment thereof is an IgG monoclonal antibody. According to some embodiments, the humanized antibody has a heavy chain constant region selected from IgG1, IgG4, and IgG2. In certain embodiments, the humanized antibody or fragment thereof is of the IgG4 subclass. In certain embodiments, the humanized antibody or antigen-binding fragment thereof is of the IgG1 subclass. According to some embodiments, the antibody has a kappa light chain constant region.

[0031] According to some embodiments, the humanized antibody has a mutated Fc domain that prevents FcγR-mediated internalization.

[0032] According to some embodiments, the humanized antibody comprises an Fc null domain. According to certain embodiments, the Fc domain is ineffective for binding to Fc gamma receptors present on immune cells. According to certain exemplary embodiments, the Fc domain is ineffective for binding to CD64, CD32a, CD32b, CD16a and / or CD16b.

[0033] According to some embodiments, the humanized antibody comprises an Fc null domain having a LALAPG mutant.

[0034] According to some embodiments, the humanized antibody comprises the heavy chain sequence shown in SEQ ID NO: 51 and the light chain sequence shown in SEQ ID NO: 52 (with a LALAPG mutant (FcgR null )'s NTX1105(H4 / k5)).

[0035] According to some embodiments, a conjugate comprising the above-mentioned humanized antibody or fragment thereof is provided.

[0036] The antibodies or fragments thereof according to the present invention may be attached to a cytotoxic moiety, a radioactive moiety or a labeling tag.

[0037] According to some embodiments, the humanized antibody or fragment thereof is conjugated to a toxin (payload).

[0038] According to some embodiments, the toxin is selected from the group consisting of a microtubule inhibitor, a DNA synthesis inhibitor, a topoisomerase inhibitor, and an RNA polymerase inhibitor.

[0039] According to certain embodiments, the toxin is a microtubule disrupting drug. According to certain exemplary embodiments, the toxin is auristatin or a derivative thereof. According to certain embodiments, the auristatin derivative is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

[0040] According to some embodiments, the toxin is a saponin.

[0041] According to some embodiments, the toxin is a maytansine derivative. According to certain embodiments, the maytansine derivative is DM4 or DM1.

[0042] According to some embodiments, the toxin is a quinoline alkaloid. According to certain embodiments, the quinoline alkaloid is SN-38.

[0043] According to some embodiments, the toxin is directly linked to the antibody. According to other embodiments, the antibody and toxin are linked via a linker. According to some embodiments, the toxin is covalently linked to the humanized antibody directly or via a linker.

[0044] According to some embodiments, the linker is cleavable. According to other embodiments, the linker is non-cleavable. According to some embodiments, the linker is an enzyme-cleavable linker. According to certain embodiments, the linker is a pH-sensitive linker. According to some embodiments, the linker is a reducible linker (sulfo-SPDB).

[0045] According to some embodiments, the linker is selected from maleimidocaproyl (MC), maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-VC-PAB), maleimidomethylcyclohexane-1-carboxylate (SMCC), N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) and Lys-PAB-CO (lysine-p-aminobenzyl-C=O).

[0046] According to some embodiments, the drug to antibody ratio (DAR) is 4 to 8. According to certain embodiments, the DAR is 4 (DAR-4). According to certain embodiments, the DAR is 8 (DAR-8).

[0047] According to some embodiments, polynucleotide sequences encoding the amino acid sequences of the heavy chain variable region and the light chain variable region as described above are provided.

[0048] In another aspect, the present invention provides a nucleic acid construct comprising a nucleic acid molecule encoding at least one humanized antibody chain or fragment thereof as described herein. According to some embodiments, the nucleic acid construct is a plasmid.

[0049] According to some embodiments, a plasmid for expressing a humanized antibody or fragment thereof described herein is provided, the plasmid comprising a nucleic acid molecule encoding the antibody.

[0050] According to another aspect, the present invention provides a pharmaceutical composition comprising the humanized antibody or antigen-binding fragment described herein, or a conjugate comprising the antibody, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0051] According to some embodiments, the pharmaceutical composition is for use in treating cancer.

[0052] Any mode of administration can be used to deliver the compositions of the present invention to a subject in need thereof, including parenteral and enteral modes of administration.

[0053] According to some embodiments, the pharmaceutical composition is formulated for injection or infusion. According to some embodiments, the pharmaceutical composition is formulated for intravenous administration. In certain embodiments, the pharmaceutical composition is formulated for intratumoral administration.

[0054] According to another aspect, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of at least one humanized antibody, fragment thereof, or conjugate thereof as described herein.

[0055] According to some embodiments, the cancer comprises a solid tumor.

[0056] According to certain embodiments, the cancer is selected from the group consisting of prostate cancer, colorectal cancer, bladder cancer, liver cancer, ovarian cancer, endometrial cancer, gastric cancer, thyroid cancer, carcinoid tumors, head and neck cancer, breast cancer, pancreatic cancer, testicular cancer, urothelial cancer, cervical cancer, melanoma, lymphoma and lung cancer. Each possibility represents a separate embodiment of the present invention.

[0057] According to certain embodiments, the cancer is selected from pancreatic ductal adenocarcinoma, renal clear cell carcinoma, and cutaneous melanoma.

[0058] According to other embodiments, the cancer is a hematological cancer. According to some embodiments, the hematological cancer is selected from: leukemias, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL) and chronic lymphocytic leukemia (CLL); lymphomas, including Hodgkin's disease and non-Hodgkin's lymphoma; and multiple myeloma.

[0059] According to some embodiments, the subject is a human.

[0060] According to some embodiments, the method of treating cancer comprises administering or performing at least one additional anti-cancer therapy. According to certain embodiments, the additional anti-cancer therapy is surgery, chemotherapy, radiotherapy, or immunotherapy.

[0061] According to some embodiments, the method of treating cancer comprises administering a humanized antibody or conjugate described herein and an additional anti-cancer agent. According to some embodiments, the additional anti-cancer agent is selected from the group consisting of an immunomodulator, activated lymphocytes, a kinase inhibitor, and a chemotherapeutic agent.

[0062] According to some embodiments, the anticancer agent is selected from Erbitux, cytarabine, fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, vincristine, vinblastine, vinorelbine, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosfamide, nitrogen mustard, melphalan, thiotepa, dacarbazine, bleomycin, actinomycin D, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, plicamycin, etoposide, teniposide and any combination thereof. Each possibility represents an independent embodiment of the present invention.

[0063] According to some embodiments, the method of treating cancer involves preventing or reducing the formation, growth or spread of metastases in a subject.

[0064] According to another aspect, the present invention provides an antibody-drug conjugate (ADC) comprising a humanized anti-fibronectin 4 antibody or an antigen-binding portion thereof conjugated to a toxin (payload), wherein the antibody or antigen-binding portion thereof comprises a set of six CDR sequences, wherein the heavy chain CDR1 comprises the sequence SYY (SEQ ID NO: 26), the heavy chain CDR2 comprises the sequence IYPGNVNT (SEQ ID NO: 22), the heavy chain CDR3 comprises the sequence SNPYVMDY (SEQ ID NO: 17), the light chain CDR1 comprises the sequence QSVNND (SEQ ID NO: 24), the light chain CDR2 comprises the amino acid sequence YAS (SEQ ID NO: 25), and the light chain CDR3 comprises the sequence QQAYRSPYT (SEQ ID NO: 20).

[0065] According to some embodiments, the antibody-drug conjugate (ADC) comprises a humanized anti-fibronectin 4 antibody, or an antigen-binding portion thereof, comprising a set of six CDR sequences, wherein the heavy chain CDR1 comprises the sequence SYYIH (SEQ ID NO: 15), the heavy chain CDR2 comprises the sequence WIYPGNVNTKYNERF(K / Q)G (SEQ ID NO: 16), the heavy chain CDR3 comprises the sequence SNPYVMDY (SEQ ID NO: 17), the light chain CDR1 comprises the sequence (K / R)ASQSVNNDVA (SEQ ID NO: 18), the light chain CDR2 comprises the sequence YASNRFT (SEQ ID NO: 19), and the light chain CDR3 comprises the sequence QQAYRSPYT (SEQ ID NO: 20).

[0066] According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain CDR2 comprising the sequence WIYPGNVNTKYNERFKG (SEQ ID NO: 27). According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain CDR2 comprising the sequence WIYPGNVNTKYNERFQG ​​(SEQ ID NO: 28).

[0067] According to some embodiments, the humanized antibody or fragment thereof comprises a light chain CDR1 comprising the sequence KASQSVNNDVA (SEQ ID NO: 29). According to some embodiments, the humanized antibody or fragment thereof comprises a light chain CDR1 comprising the sequence RASQSVNNDVA (SEQ ID NO: 30).

[0068] According to some embodiments, the antibody or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and wherein the light chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

[0069] According to some embodiments, the heavy chain variable region of the humanized antibody comprises the amino acid sequence shown in SEQ ID NO. 13, and the light chain variable region of the humanized antibody comprises the amino acid sequence shown in SEQ ID NO: 14.

[0070] According to some embodiments, the heavy chain variable region of the humanized antibody comprises an amino acid sequence that is at least about 97% identical to a sequence selected from SEQ ID NOs: 2, 3, 4, 5, and 6; and the light chain variable region of the humanized monoclonal antibody comprises an amino acid sequence that is at least about 97% identical to a sequence selected from SEQ ID NOs: 8, 9, 10, 11, and 12.

[0071] According to some embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises the sequence shown in SEQ ID NO: 5, and the light chain variable region of the humanized monoclonal antibody comprises the sequence shown in SEQ ID NO: 12. According to other embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises the sequence shown in SEQ ID NO: 6, and the light chain variable region of the humanized monoclonal antibody comprises the sequence shown in SEQ ID NO: 12.

[0072] According to some embodiments, the antibody-drug conjugate comprises a toxin as described above.

[0073] Any chemical or biological entity that can kill tumor cells in vivo or inhibit their growth can be used as a toxin with the ADC of the present invention. According to some embodiments, the toxin is selected from microtubule inhibitors, DNA synthesis inhibitors, topoisomerase inhibitors and RNA polymerase inhibitors.

[0074] According to certain embodiments, the toxin is a microtubule disrupting drug. According to certain exemplary embodiments, the toxin is auristatin or a derivative thereof. According to certain embodiments, the auristatin derivative is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

[0075] According to some embodiments, the toxin is a saponin.

[0076] According to some embodiments, the toxin is a maytansine derivative. According to certain embodiments, the maytansine derivative is DM4 or DM1.

[0077] According to some embodiments, the toxin is a quinoline alkaloid. According to certain embodiments, the quinoline alkaloid is SN-38.

[0078] According to some embodiments, the toxin is selected from DM4, MMAE and SN-38. According to certain embodiments, the toxin is DM4 or MMAE.

[0079] According to some embodiments, the toxin is a topoisomerase I inhibitor. According to some embodiments, the toxin is a derivative of camptothecin. According to certain embodiments, the toxin is exitecan.

[0080] According to some embodiments, the toxin is directly linked to the antibody. According to other embodiments, the antibody and toxin are linked via a linker. According to some embodiments, the toxin is covalently linked to the humanized antibody directly or via a linker.

[0081] According to some embodiments, the linker is cleavable. According to other embodiments, the linker is non-cleavable. According to some embodiments, the cleavable linker is selected from an enzyme cleavable linker, a pH sensitive linker, and a reducible linker. According to some embodiments, the linker is an enzyme cleavable linker. According to certain embodiments, the linker is a pH sensitive linker. According to some embodiments, the linker is a reducible linker.

[0082] According to some embodiments, the linker is selected from maleimidocaproyl (MC), maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-VC-PAB), maleimidomethylcyclohexane-1-carboxylate (SMCC), N-succinimidyl-4-(2-pyridyldithio)butyrate (sulfo-SPDB), valine-alanine linker and Lys-PAB-CO (lysine-p-aminobenzyl-C=O).

[0083] According to some embodiments, the conjugate comprises the toxin MMAE and the linker MC-VC-PAB (denoted herein as NTX1105-MMAE). According to some embodiments, the conjugate comprises the toxin MMAF and the linker MC (denoted herein as NTX1105-MMAF). According to some embodiments, the conjugate comprises the toxin DM1 and the linker SMCC (denoted herein as NTX1105-DM1). According to some embodiments, the conjugate comprises the toxin DM4 and the linker SPDB (denoted herein as NTX1105-DM4). According to some embodiments, the conjugate comprises the toxin SN38 and the linker Lys-PAB-CO (denoted herein as NTX1105-SN38).

[0084] According to some embodiments, the ADC comprises an anti-fibronectin 4 antibody that competes with an antibody described herein for specific binding to a fibronectin 4 molecule. According to certain embodiments, the ADC comprises an anti-fibronectin 4 antibody that competes with the following antibody for specific binding to a fibronectin 4 molecule: the antibody comprises heavy and light chain variable regions, wherein the heavy chain CDR1 comprises the sequence SYYIH (SEQ ID NO: 15), the heavy chain CDR2 comprises the sequence WIYPGNVNTKYNERF(K / Q)G (SEQ ID NO: 16), the heavy chain CDR3 comprises the sequence SNPYVMDY (SEQ ID NO: 17), the light chain CDR1 comprises the sequence (K / R)ASQSVNNDVA (SEQ ID NO: 18), the light chain CDR2 comprises the sequence YASNRFT (SEQ ID NO: 19), and the light chain CDR3 comprises the sequence QQAYRSPYT (SEQ ID NO: 20).

[0085] According to another aspect, the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate described herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0086] Any mode of administration can be used to deliver the compositions of the present invention to a subject in need thereof, including parenteral and enteral modes of administration.

[0087] According to some embodiments, the pharmaceutical composition is formulated for injection or infusion. According to some embodiments, the pharmaceutical composition is formulated for intravenous (IV) administration. In certain embodiments, the pharmaceutical composition is formulated for intratumoral (IT) administration.

[0088] According to some embodiments, the conjugate or pharmaceutical composition is for use in treating cancer in an individual.

[0089] The cancer is as described above. In certain embodiments, the cancer comprises a solid tumor. In certain embodiments, the cancer is selected from liver cancer, lung cancer, colon cancer, glioblastoma, adrenal cancer, uterine cancer, testicular cancer, head and neck cancer, pancreatic cancer, and breast cancer. Each possibility represents a separate embodiment of the present invention.

[0090] According to some embodiments of the invention, the use further comprises use in combination with an additional ADC.

[0091] According to another aspect, the present invention provides a method for treating cancer in an individual in need of treatment, the method comprising administering to the individual a therapeutically effective amount of a conjugate or pharmaceutical composition described herein. In certain embodiments, the cancer is a solid tumor. According to other embodiments, the cancer is a non-solid tumor. In certain embodiments, the cancer is selected from glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, colon cancer, cervical cancer, prostate cancer and lung cancer. In certain embodiments, the method for treating cancer comprises preventing or reducing the formation, growth or spread of metastasis in the subject.

[0092] According to other embodiments, the cancer is a hematological cancer. According to some embodiments, the hematological cancer is selected from: leukemias, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL) and chronic lymphocytic leukemia (CLL); lymphomas, including Hodgkin's disease and non-Hodgkin's lymphoma; and multiple myeloma.

[0093] According to some embodiments, the individual is a human.

[0094] According to some embodiments, the method of treating cancer comprises administering or performing at least one additional anti-cancer therapy. According to certain embodiments, the additional anti-cancer therapy is surgery, chemotherapy, radiotherapy, or immunotherapy.

[0095] According to some embodiments, the method of treating cancer comprises administering a conjugate described herein and an additional anti-cancer agent. According to some embodiments, the additional anti-cancer agent is selected from the group consisting of an immunomodulator, activated lymphocytes, a kinase inhibitor, and a chemotherapeutic agent.

[0096] Also described herein are methods of preparing a composition for treating cancer in an individual having cancer, the method comprising admixing an ADC described herein with a pharmaceutically acceptable excipient, carrier, or diluent.

[0097] According to another aspect, the present invention provides a method of delivering at least one humanized antibody, fragment or conjugate thereof as described herein to a cell, the method comprising contacting the cell with the humanized antibody, fragment or conjugate thereof.

[0098] According to some embodiments, the method comprises administering the humanized antibody, fragment thereof, or conjugate to cells of a subject. According to certain embodiments, the subject is a human subject.

[0099] According to another aspect, the present invention provides a method of delivering at least one humanized antibody, fragment thereof, or conjugate thereof as described herein to a cell of a subject, the method comprising administering to the subject the at least one humanized antibody, fragment thereof, or conjugate. According to some embodiments, the cell is a tumor cell.

[0100] According to one aspect, the present invention further provides a method for diagnosing or prognosing cancer in a subject, the method comprising determining the expression level of cohesin 4 in a biological sample of the subject using at least one antibody conjugate described herein.

[0101] According to some embodiments, the method comprises detecting the bound antibody or antibody fragment and determining the expression level of cohesin-4 in the sample. According to certain embodiments, the method comprises comparing the expression level to a control. According to some embodiments, the control is a predefined value. According to certain embodiments, the control is a corresponding non-cancerous tissue. According to some embodiments, the comparison indicates or indicates whether the subject has cancer.

[0102] According to some embodiments, the method is used to diagnose cancer subtypes. According to some embodiments, the method is used to determine a patient's eligibility for anti-cancer therapy. According to certain embodiments, the anti-cancer therapy is an antibody-drug conjugate. According to some embodiments, the method is used to determine a patient's eligibility for therapy using an ADC described herein.

[0103] According to another aspect, the present invention further provides a method for diagnosing, determining or quantifying the expression of cohesin-4, the method comprising contacting a biological sample with the antibody conjugate described herein and measuring the level of complex formation.

[0104] According to some embodiments, the method for detecting or quantifying the expression of cohesin 4 comprises the following steps:

[0105] i. incubating the sample with the antibody conjugate described herein;

[0106] ii. Detecting bound fibronectin 4 using the conjugate.

[0107] According to some embodiments, the method further comprises the steps of:

[0108] iii. comparing the amount of step (ii) with a standard curve obtained from a reference sample containing a known amount of adhesion protein 4; and

[0109] iv. Calculate the amount of fibronectin 4 in the sample from the standard curve.

[0110] According to some specific embodiments, the sample is a body fluid or a solid tissue.In some embodiments, the method is performed in vitro or ex vivo.

[0111] Also provided is a kit for measuring the expression of cohesin 4 in a biological sample, comprising at least one conjugate described herein and means for measuring the expression of cohesin 4. In some embodiments, the kit further comprises instructional materials directing the use of the kit.

[0112] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] Figures 1A-1C Shown is the relationship between cohesin-4 expression mRNA levels (high or low as indicated) and pancreatic ductal adenocarcinoma ( Figure 1A ), renal clear cell carcinoma ( Figure 1B ) and skin melanoma ( Figure 1C ) patients' survival probability. The dataset was obtained from the TCGA website and analyzed using the oncolnc.org website (https: / / doi.org / 10.7717 / peerj-cs.67). N represents the number of patients included in the analysis.

[0114] Figure 2 A graph showing the percentage of tumors showing positive expression of cohesin 4 is shown. Data were obtained from proteinatlas.com using HPA010775 mAb (anti-cohesin 4 antibody, Moderate to high levels of membrane expression of cohesin-4 were observed in 13 of 20 indications.

[0115] Figure 3Depicted is the dose-dependent killing of the MDA-MB-468 cell line (breast adenocarcinoma cells) in vitro using various linker-payload combinations of NTX1105 (VH0 / Vκ0).

[0116] Figure 4 Depicted is the in vivo efficacy of NTX1105 (VH0 / Vκ0) ADC against the triple-negative breast cancer (TNBC) tumor cell line MDA-MB-468 implanted subcutaneously (sc) into NOD-SCID female mice. The effects of treatment with all linker-payload (LP) combinations of NTX1105 are shown.

[0117] Figure 5 Improved characteristics of some humanized variants were demonstrated.The productivity of the parental / chimeric (VH0 / Vκ0) antibody and six purified lead humanized variants was assessed.

[0118] Figures 6A-6B The robust in vivo activity of the humanized variants was demonstrated. The in vivo efficacy of the lead humanized NTX1105 (VH4 / Vκ5 and VH5 / Vκ5) ADCs conjugated to MC-VC-PAB-MMAE with a DAR of approximately 4 (~4) was evaluated against the TNBC tumor cell line MDA-MB-468. The results are shown as tumor volume ( Figure 6A ) and fold change of tumor volume over time ( Figure 6B ).

[0119] Figures 7A-7B The differential in vitro activity of NTX1105 (H4κ5) ADC compared to enroku-vedotin (PADCEV) is shown. The two ADCs were tested for binding on cells ( Figure 7A ) and in vitro killing activity ( Figure 7B ).

[0120] Figures 8A-8B The results show that NTX1105 (H4κ5) coupled with MC-VC-PAB-MMAE at a DAR of about 4 (~4) has better in vivo anti-tumor activity than enroku-vedotin (PADCEV). To compare the ADC with PADCEV, the activity of both ADCs was evaluated in vivo using H322 (NSCLC model). The ADC was administered at 1 mg / kg for 6 doses ( Figure 8A ) or 3 mg / kg for 3 doses ( Figure 8B ) administration.

[0121] Figure 9 Humanized NTX1105 (H4κ5-FcgR null) ADC against H322M (lung cancer model) sc tumor model in female nude mice.

[0122] Figure 10 Depicts humanized NTX1105 (H4κ5-FcgR null ) anti-adhesin 4 ADC compared with enroku-DAR-4 and ixetine in female nude mice against large (>450mm 3 ) In vivo advantages of the H322M tumor model. DETAILED DESCRIPTION

[0123] The present invention provides humanized anti-fibronectin 4 antibodies and antibody-drug conjugates (ADCs) containing them that can be used to treat cancer. Advantageously, the ADCs described herein comprise nearly fully humanized antibodies, thereby avoiding the risk of adverse immune responses to the antibodies and thus potentially being safe for use in humans. The ADCs described herein have been found to be highly effective and superior to other known anti-fibronectin 4 ADCs.

[0124] Certain specific details are set forth in the following description in order to provide a comprehensive understanding of the various embodiments. However, those skilled in the art will understand that the embodiments provided can be practiced without these details. Unless the context requires otherwise, throughout this specification and the claims that follow, the word "comprising" and its variations should be interpreted in an open, inclusive sense, that is, to mean "including but not limited to." References without a specific number used in this specification and the appended claims include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally used in its sense including "and / or" unless the content clearly dictates otherwise. In addition, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments. The term "about" as used herein refers to an amount that differs by 10% or less from the stated amount.

[0125] As used herein, the term "nectin-4" or "nectin cell adhesion molecule 4" refers to a single-pass transmembrane type I membrane protein of 510 amino acids and a molecular weight of 55,454 Da, also known as PVRL4, LNIR, PRR4, and EDSS1. The nectin-4 protein contains two immunoglobulin-like (Ig-like) C2-type domains and one Ig-like V-type domain. It participates in cell adhesion through trans homophilic and heterophilic interactions. The soluble form is produced by proteolytic cleavage at the cell surface by the metalloprotease ADAM17 / TACE, and the secreted form is found in breast tumor cell lines and breast tumor patients. Exemplary cohesin 4 according to the invention are set forth in the following SwissPort, UniPort and GenBank symbols or accession numbers: Q96NY8-NECT4_HUMAN, Q96NY8, B4DQW3, Q96K15, Q96NY8-1, Q96NY8-2, ENSP00000356991, NP_112178.2, XP_005245565.1, XP_011508323.1, XP_011508324.1, or XP_011508325.1.

[0126] According to one aspect, the present invention provides a humanized antibody that specifically binds to human adhesion protein 4 or a fragment thereof that comprises at least an antigen binding site, wherein the antibody or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region whose amino acid sequence is at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and wherein the light chain comprises a variable region whose amino acid sequence is at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

[0127] According to another aspect, the present invention provides an antibody-drug conjugate (ADC) comprising a humanized anti-fibronectin 4 antibody or an antigen-binding portion thereof conjugated to a toxin (payload), wherein the antibody or antigen-binding portion thereof comprises a set of six CDR sequences, wherein the heavy chain CDR1 comprises the sequence SYY (SEQ ID NO: 26), the heavy chain CDR2 comprises the sequence IYPGNVNT (SEQ ID NO: 22), the heavy chain CDR3 comprises the sequence SNPYVMDY (SEQ ID NO: 17), the light chain CDR1 comprises the sequence QSVNND (SEQ ID NO: 24), the light chain CDR2 comprises the amino acid sequence YAS (SEQ ID NO: 25), and the light chain CDR3 comprises the sequence QQAYRSPYT (SEQ ID NO: 20).

[0128] According to another aspect, the invention comprises an antibody-drug conjugate (ADC) comprising a humanized anti-fibronectin 4 antibody, or an antigen-binding portion thereof, conjugated to a toxin selected from the group consisting of MMAE, SN-38, DM1, DM4, and MMAFA, the humanized antibody, or antigen-binding portion thereof, comprising a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and wherein the light chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

[0129] According to some embodiments, the humanized antibody or antigen-binding fragment thereof comprises an amino acid sequence QVQL(Q / V)QSG(P / S)ELKKP(E / G)ASVK(I / V)SCKASGYTFTSYY IHWV(K / R)QAPGQGLEW(I / M)GWIYPGNVNTKYNERF(K / Q)GR(A / V)T(L / I)TA DKST(N / S)TA(H / Y)MELSSL(T / R)SED(S / T)AVY(F / Y)CARSNPYVMDYWGQGT SVTVSS (SEQ ID NO: 13), and a light chain variable region comprising the amino acid sequence of (S / D)I(V / Q)MTQSPS(F / T)L(L / S)AS(A / V)GDRVTITC(K / R)ASQSVNNDVAWYQQKPG(L / K)AP(E / K)LLMYYASNRFTGVPDRF(T / S)GSGYGTDFTLTISSLQAED(L / V)A(I / V)YFCQQAYRSPYTFGQGTKLEIK (SEQ ID NO: 14).

[0130] According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence at least about 95% identical to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and wherein the light chain comprises a variable region having an amino acid sequence at least about 95% identical to a sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. SEQ ID NOs: 2-6, 8-12 are humanized variants of NTX1105 selected on the basis of improved developability and low immunogenicity.

[0131] In general, depending on the context, "NTX1105" includes chimeric antibodies and / or humanized variants thereof. The parent chimeric antibody is disclosed in WO 2019 / 215728 (clone 11). The parent chimeric antibody is designated herein as NTX1105 (VH0 / Vk0).

[0132] The humanized variant of NTX1105 includes five heavy chains (VH1 to VH5) and five light chains (Vκ1 to Vκ5). According to some embodiments, the humanized variant comprises the sequence formula shown in SEQ ID NO: 13 (heavy chain) and SEQ ID NO: 14 (light chain).

[0133] According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having the amino acid sequence of SEQ ID NO: 5, and the light chain comprises a variable region having the amino acid sequence of SEQ ID NO: 12 (denoted herein as NTX1105 (VH5 / Vk5).

[0134] According to some embodiments, the humanized antibody comprises a combination of a heavy chain variable region and a light chain variable region, wherein the combination is selected from the group consisting of: SEQ ID NO: 2 and SEQ ID NO: 8, SEQ ID NO: 2 and SEQ ID NO: 9, SEQ ID NO: 2 and SEQ ID NO: 10, SEQ ID NO: 2 and SEQ ID NO: 11, SEQ ID NO: 2 and SEQ ID NO: 12, SEQ ID NO: 3 and SEQ ID NO: 8, SEQ ID NO: 3 and SEQ ID NO: 9, SEQ ID NO: 3 and SEQ ID NO: 10, SEQ ID NO: 3 and SEQ ID NO: 11, SEQ ID NO: 3 and SEQ ID NO: 12, SEQ ID NO: 4 and SEQ ID NO: 8, SEQ ID NO: 4 and SEQ ID NO: 9, SEQ ID NO: 4 and SEQ ID NO: 10, SEQ ID NO: 4 and SEQ ID NO: 11, SEQ ID NO: 4 and SEQ ID NO: 12, SEQ ID NO: 5 and SEQ ID NO: 8, SEQ ID NO: 5 and SEQ ID NO: 9, SEQ ID NO: 5 and SEQ ID NO: 10. NO: 10, SEQ ID NO: 5 and SEQ ID NO: 11, SEQ ID NO: 5 and SEQ ID NO: 12, SEQ ID NO: 6 and SEQ ID NO: 8, SEQ ID NO: 6 and SEQ ID NO: 9, SEQ ID NO: 6 and SEQ ID NO: 10, SEQ ID NO: 6 and SEQ ID NO: 11, and SEQ ID NO: 6 and SEQ ID NO: 12. Each possibility or combination of heavy and light chains represents a separate embodiment of the present invention.

[0135] According to some embodiments, the humanized antibody comprises a combination of a heavy chain variable region and a light chain variable region, wherein the combination is selected from:

[0136] i. the heavy chain variable region sequence shown in SEQ ID NO: 5 and the light chain variable region sequence shown in SEQ ID NO: 12; and

[0137] ii. The heavy chain variable region sequence shown in SEQ ID NO: 6 and the light chain variable region sequence shown in SEQ ID NO: 12.

[0138] The conjugate of the present invention comprises humanized antibodies described herein. The antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies and multireactive antibodies) and antibody fragments. Therefore, antibodies include but are not limited to full length and fragments and parts thereof that retain binding specificity, such as any specific binding portion thereof, including those with any immunoglobulin class and / or isotype (such as IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and IgM); and biologically relevant (antigen binding) fragments or their specific binding portions, including but not limited to Fab, F(ab')2, Fv and scFv (single chain or related entities). Monoclonal antibodies are generally one of substantially homogeneous antibody compositions; therefore, except for possible naturally occurring mutations that may be present in small amounts, any single antibody included in the monoclonal antibody composition is identical. The antibody may include a human IgG1 constant region. The antibody may include a human IgG4 constant region. The antibody may include a human κ light chain.

[0139] The term "antibody" as used herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including whole antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments (including single-chain variable fragments (sFv or scFv)) and single-domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified immunoglobulin forms, such as intracellular antibodies, peptibodies, fully human antibodies, humanized antibodies and heteroconjugate antibodies, multispecific (e.g., bispecific) antibodies, diabodies, triabodies and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise indicated, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD. The antibody may comprise a human IgG1 constant region. The antibody may comprise a human IgG4 constant region. The antibody may comprise a human kappa light chain.

[0140] Several methods are known in the art for determining the CDR sequences of a given antibody molecule, but there is no standard, clear method. Determining the CDR sequences from the antibody heavy and light chain variable regions can be performed according to any method known in the art, including but not limited to the methods known as KABAT, Chothia, and IMGT. A set of selected CDRs may include sequences identified by more than one method, i.e., for example, some CDR sequences can be determined using KABAT, while others can be determined using IMGT. According to some embodiments, the CDR sequences of the mAb variable regions are determined using the IMGT method. For example, CDR determination is performed according to Kabat (Wu TT and Kabat EA, J Exp Med, 1970; 132: 211–50) and IMGT (Lefranc MP et al., Dev Comp Immunol, 2003, 27: 55-77).

[0141] When the term "CDR having a certain sequence" or similar terms is used, it includes the option wherein the CDR comprises the specified sequence, as well as the option wherein the CDR consists of the specified sequence.

[0142] The antibodies provided include antibody fragments. An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv or sFv); and multispecific antibodies formed from antibody fragments. In a specific embodiment, the antibody is a single-chain antibody fragment comprising a variable heavy chain region and / or a variable light chain region, such as an scFv.

[0143] "Humanized" antibodies are antibodies in which all or substantially all CDR amino acid residues are derived from non-human CDR, and all or substantially all framework region (FR) amino acid residues are derived from people's FR. Humanized antibodies optionally may include at least a portion of an antibody constant region derived from a human antibody. "Humanized form" of a non-human antibody refers to a humanized variant of a non-human antibody, typically to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. According to some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0144] The amino acid residues in the Fc domain can be replaced to be ineffective, which means that the Fc domain does not bind to the Fc receptor, or can bind with such low affinity and / or avidity that no Fc receptor signaling is induced by binding. The Fc domain can be ineffective for binding to Fcγ receptors. Some example Fcγ receptors to which the Fc domain can be ineffective for binding can be, but are not limited to, FcγRI (CD64), FcγRIIA (CD32a), FcγRIIB (CD32b), FcγRIIIA (CD16a), FcγRIIIA (CD16a) F158 variant, FcγRIIIA (CD16a) V158 variant, or FcγRIIIB (CD16b). The Fc domain can have one or more, two or more, three or more, or four or more amino acid replacements that reduce the binding of the Fc domain to the Fc receptor.

[0145] According to some embodiments, the humanized antibody has a mutated Fc domain that prevents FcγR-mediated internalization.

[0146] According to some embodiments, the humanized antibody comprises an Fc null domain. According to certain embodiments, the Fc domain is null for binding to an Fcγ receptor.

[0147] As used herein, "Fc null" refers to a domain that has weak or no binding to one or more Fcγ receptors.

[0148] According to some embodiments, the humanized antibody comprises an Fc null domain. According to certain embodiments, the Fc domain is ineffective for binding to Fc gamma receptors present on immune cells. According to certain exemplary embodiments, the Fc domain is ineffective for binding to CD64, CD32a, CD32b, CD16a and / or CD16b.

[0149] According to some embodiments, the humanized antibody comprises an Fc null domain having a LALAPG mutant.

[0150] According to some embodiments, the humanized antibody comprises the heavy chain sequence shown in SEQ ID NO: 51 and the light chain sequence shown in SEQ ID NO: 52 (with a LALAPG mutant (FcgR null )'s NTX1105(H4 / k5)).

[0151] The present invention provides a conjugate comprising the humanized antibody disclosed herein and a toxin.

[0152] According to some embodiments, the toxin is selected from the group consisting of microtubule inhibitors, DNA synthesis inhibitors, topoisomerase inhibitors and RNA polymerase inhibitors. Each possibility represents a separate embodiment of the present invention.

[0153] According to certain embodiments, the toxin is a microtubule disrupting drug. According to certain exemplary embodiments, the toxin is auristatin or a derivative thereof. According to certain embodiments, the auristatin derivative is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). According to some embodiments, the toxin is exatecan.

[0154] According to some embodiments, the toxin is a saponin.

[0155] According to some embodiments, the toxin is a maytansine derivative. According to certain embodiments, the maytansine derivative is DM4 or DM1.

[0156] According to some embodiments, the toxin is a quinoline alkaloid. According to certain embodiments, the quinoline alkaloid is SN-38.

[0157] According to other embodiments, the toxin is selected from MMAE, MMAF, saponin, DM4, DM1, SN-38, calicheamicin, DXd, PBD, duocarmycin, sandromycin, α-amanitin, chaetocin, CYT997, daunorubicin, 17-AAG, agrochelin A, doxorubicin, methotrexate, colchicine, cordycepin, epothilone B, hygrolidin, herboxidiene, ferulic acid, curvulin, paclitaxel, Englerin A, taltobulin, triptolide, scutellarin and nemorubicin. Each possibility represents an independent embodiment of the present invention.

[0158] According to some embodiments, the toxin is SN-38. According to some embodiments, the toxin is DM1. According to some embodiments, the toxin is DM4. According to some embodiments, the toxin is MMAE. According to some embodiments, the toxin is MMAF. According to some embodiments, the toxin is exotecan.

[0159] According to some embodiments, the antibody is directly linked to the toxin. According to other embodiments, the antibody and toxin are linked via a linker. According to some embodiments, the humanized antibody described herein is covalently linked to the toxin.

[0160] According to some embodiments, the linker is cleavable. According to other embodiments, the linker is non-cleavable.

[0161] According to some embodiments, the linker is cleaved in response to a change in pH or redox potential. According to some embodiments, the linker is cleaved upon contact with a lysosomal enzyme.

[0162] According to some embodiments, the linker comprises a group selected from 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl (4-iodo-acetyl) aminobenzoate (SLAB), 6-maleimidocaproyl-valeric acid ester ... The present invention also provides a novel molecule comprising a moiety comprising amino-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB), Val-Cit-PABC, N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), N-succinimidyl 3-(pyridin-2-yldithio)-propionate (SPDP), Phe-Lys(Fmoc)-PAB, Aloc-D-Ala-Phe-Lys(Aloc)-PAB-PNP, Boc-Phe-(Alloc)Lys-PAB-PNP, and perfluorophenyl 3-(pyridin-2-yldisulfanyl)propionate. Each possibility represents a separate embodiment of the present invention.

[0163] According to some embodiments, the antibody is conjugated to two or more toxin molecules.

[0164] According to another aspect, the present invention provides a pharmaceutical composition comprising the conjugate described herein and a pharmaceutically acceptable excipient, carrier or diluent.

[0165] According to other embodiments, the pharmaceutical composition according to the present invention is used for the treatment of cancer.

[0166] Cancers that can be treated by the present invention include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer, as well as B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, The cancer is selected from the group consisting of: intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, large massive 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 post-transplant lymphoproliferative disorder (PTLD), and abnormal vascular proliferation associated with molar hamartomatosis, edema (e.g., associated with brain tumors) and Meigs syndrome. Preferably, the cancer is selected from the group consisting of breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkin lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid, head and neck cancer, melanoma, ovarian cancer, mesothelioma and multiple myeloma. Cancerous conditions suitable for treatment according to the present invention include metastatic cancers.

[0167] According to other embodiments, the pharmaceutical composition according to the present invention is used to treat cancer characterized by overexpression of cohesin 4. Cancer types associated with overexpression of cohesin 4 can be identified using known databases such as The Cancer Genome Atlas (TCGA). According to certain embodiments, the cancer that can be treated using the composition according to the present invention is selected from the group consisting of adrenocortical carcinoma (ACC), chromophobe renal cell carcinoma (KICH), hepatocellular carcinoma (LIHC), colon and rectal adenocarcinoma (COAD and READ), pancreatic ductal adenocarcinoma (PAAD), pheochromocytoma and paraganglioma (PCPG), papillary renal carcinoma (KIRP), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), prostate adenocarcinoma (PRAD), uterine corpus endometrial carcinoma (UCEC), cervical cancer (CESC), cutaneous melanoma (SKCM), mesothelioma (MESO), urothelial bladder cancer (BLCA), clear cell renal carcinoma (KIRC), lung squamous cell carcinoma (LUSC), uterine carcinosarcoma (UCS), sarcoma (SARC), ovarian serous cystadenocarcinoma (OV), papillary thyroid carcinoma (THCA), glioblastoma multiforme (GBM), breast cancer (BRCA), low-grade glioma (LGG) and diffuse large B-cell lymphoma (DLBC). Each possibility represents a separate embodiment of the present invention.

[0168] The molecules of the present invention as active ingredients are dissolved, dispersed or mixed in an excipient, which is well known to be pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, phosphate buffered saline (PBS), glucose, glycerol, ethanol, and the like, and combinations thereof. Other suitable carriers are well known to those skilled in the art. In addition, if desired, the composition may contain small amounts of auxiliary substances, such as wetting agents or emulsifiers, pH buffers.

[0169] The pharmaceutical composition according to the present invention can be administered together with an anti-tumor composition.

[0170] As used herein, the term "treating" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already suffering from the disorder as well as those in whom the disorder is to be prevented. The term "treating" also refers to alleviating or preventing the symptoms associated with the disease and / or lessening the severity of the disease.

[0171] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include melanoma, lung cancer, thyroid cancer, breast cancer, colon cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, cervical cancer, pancreatic cancer, leukemia, lymphoma, myeloid cell carcinoma, ovarian cancer, uterine cancer, sarcoma, biliary tract cancer, or endometrial cancer.

[0172] According to some embodiments, the method of treating cancer comprises administering the pharmaceutical composition as part of a treatment regimen comprising administration of at least one additional anti-cancer agent.

[0173] As used herein, the terms "individual," "patient," or "subject" refer to an individual diagnosed with, suspected of having, or at risk for developing at least one disease that can be treated using the compositions and methods. According to some embodiments, the individual is a mammal. According to some embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. ​​According to some embodiments, the individual is a human.

[0174] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that, when administered to a mammal, elicits a biological effect. Biological effects include, but are not limited to, reduced tumor growth, reduced tumor metastasis, or prolonged survival of a tumor-bearing animal. A "therapeutic amount" is a concentration of a drug calculated to exert a therapeutic effect. A therapeutic amount encompasses a range of doses capable of inducing a therapeutic response in a population of individuals. The mammal may be a human individual. The human individual may have or be suspected of having a tumor.

[0175] The molecules of the present invention as active ingredients are dissolved, dispersed or mixed in an excipient, which is well known to be pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, phosphate buffered saline (PBS), glucose, glycerol, ethanol, and the like, and combinations thereof. Other suitable carriers are well known to those skilled in the art. In addition, if desired, the composition may contain small amounts of auxiliary substances, such as wetting agents or emulsifiers, pH buffers.

[0176] According to some embodiments, the method of treating cancer comprises administering the pharmaceutical composition as part of a treatment regimen comprising administration of at least one additional anti-cancer agent.

[0177] According to some embodiments, the anticancer agent is selected from the group consisting of antimetabolites, mitotic inhibitors, taxanes, topoisomerase inhibitors, topoisomerase II inhibitors, asparaginase, alkylating agents, antitumor antibiotics, and combinations thereof. Each possibility represents a separate embodiment of the present invention.

[0178] According to some embodiments, the antimetabolite is selected from cytarabine, fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine and hydroxyurea. According to some embodiments, the mitotic inhibitor is selected from vincristine, vinblastine and vinorelbine. According to some embodiments, the topoisomerase inhibitor is selected from topotecan and irinotecan. According to some embodiments, the alkylating agent is selected from busulfan, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosfamide, nitrogen mustard, melphalan, thiotepa, dacarbazine and procarbazine. According to some embodiments, the antitumor antibiotic is selected from bleomycin, actinomycin D, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone and plicamycin. According to some embodiments, the topoisomerase II inhibitor is selected from etoposide and teniposide. Each possibility represents an independent embodiment of the present invention.

[0179] Also described herein is a method for preparing a composition for treating cancer in an individual suffering from cancer, the method comprising mixing a humanized antibody or ADC described herein with a pharmaceutically acceptable excipient, carrier, or diluent. In certain embodiments, the cancer comprises a solid tumor. In certain embodiments, the cancer is selected from glioblastoma, colon cancer, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, cervical cancer, prostate cancer, and lung cancer.

[0180] Example

[0181] Reference is now made to the following examples, which together with the above descriptions illustrate the invention in a non limiting fashion.

[0182] Generally, the nomenclature used herein and the laboratory procedures used in the present invention include molecular, biochemical, microbiological, immunological and recombinant DNA techniques. Such techniques are well known in the art. For the convenience of the reader, other general references citing well-known procedures are provided throughout this document.

[0183] Example 1. High expression of cohesin-4 mRNA is associated with low survival probability in patients with various cancers

[0184] The correlation between cohesin-4 mRNA expression and survival probability was examined in data from the TCGA website, analyzed using the oncolnc.org website (https: / / doi.org / 10.7717 / peerj-cs.67), and presented in Figures 1A-1CMiddle. Cohesin-4 mRNA expression levels served as the basis for stratifying patients into two subgroups: low and high expressers. As shown in the box, the survival advantage of patients with low expression confirms the deleterious role of cohesin-4 and the potential to improve survival by targeting this protein.

[0185] Example 2. Cohesin 4 is expressed at the protein level in most solid tumors

[0186] The Proteinatlas.com database was searched for all aliases of NECTIN 4 (NECTIN4, PVRL4). Under the Pathology heading, data using a single mAb were found (HPA010775). Expression levels between different tumors are depicted in Figure 2 The graph shows the percentage of tumors positive for cohesin 4. In all indications, at least one patient had surface expression of cohesin 4, and in 13 of 20 indications, membrane expression of cohesin 4 was observed at moderate to high levels.

[0187] Example 3. Various cytotoxic agents can be efficiently conjugated to mAbs targeting fibronectin 4 to generate fibronectin 4 ADCs

[0188] Various linker-payload combinations and drug-antibody ratios (DARs) for conjugating the anti-nectin 4 antibody Clone-11 (VH0 / Vκ0)-hIgG1 (also described in WO 2019 / 215728, belonging to the inventors of the present invention) to generate the NTX1105 (VH0 / Vκ0) nectin 4 ADC are described (Table 1). The cytotoxic agents described in Table 1 include tubulin and TOPO1 targeting agents, and the DARs were selected based on approved ADCs using the mentioned payloads.

[0189] surface 1. Various linker-payload combinations and drug-antibody ratios (DARs) used to conjugate Clone-11 (VH0 / Vκ0)-hIgG1 to generate the NTX1105 (VH0 / Vκ0) nectin 4 ADC

[0190] connector Payload Release mechanism Average DAR (LC-MS) MC-VC-PAB MMAE Proteolytic cleavage 4.2 MC MMAF degradation 3.8 SMCC DM1 degradation 3.6 SPDB DM4 Redox 3.5 Lys-PAB-CO SN38 pH 7.5

[0191] Example 4. Various cytotoxic agents can induce tumor cell killing in vitro when coupled to mAbs targeting fibronectin 4

[0192] Next, the dose-dependent killing activity of various linker-payload combinations of NTX1105 (VH0 / Vκ0) was examined. The in vitro assay for the TNBC model used the MDA-MB-468 cell line. Target cells were plated at 2*10 per well. 3The cells were plated at a density of 100 cells / mL and allowed to adhere for 4-6 hours. ADC was added at a concentration of 30-1.9 nM using a 2-fold dilution and the cells were incubated with ADC for an additional 72 hours. The assay was harvested after 72 hours and used Tumor cell killing was assessed using the 2.0 cell survival assay system (Promega G9242) according to standard protocols. The killing data showed strong potency, with EC-50 not reached for most payloads (except DM1 / 4, which reached EC-50 at ∼3.8 nM), indicating that the ADC had high potency regardless of the linker-payload combination selected. Figure 3 ).

[0193] Example 5. Various cytotoxic agents can induce tumor cell killing in vivo when coupled to mAbs targeting cohesin-4

[0194] The in vivo efficacy of NTX1105 (VH0 / Vκ0) anti-nectin 4 ADC was evaluated against the TNBC tumor cell line MDA-MB-468 (5M cells / mouse) implanted sc into 6-week-old NOD-SCID female mice. 3 Treatment was initiated at an average volume of 500 mg / kg and animals received 3 doses (Q4D) of 5 mg / kg NTX1105 with the indicated linker-payload. Figure 4 The effects of treatment with all linker-payload (LP) combinations of NTX1105 were compared. Three LP combinations showed significant effects: SN38 and DM4 resulted in significant tumor growth inhibition (TGI, p<0.05), while complete tumor regression was observed with MMAE. The fact that three of the five LPs tested resulted in significant anti-tumor activity demonstrates the unique utility of NTX1105 as an Ab for ADCs. Based on the results of these experiments, further development of a humanized anti-nectin 4 ADC based on the NTX1105 mAb was pursued.

[0195] Example 6. The preferred humanized cohesin-4 mAb retains human cohesin-4 binding and cross-reactivity with cynomolgus monkey cohesin-4

[0196] To evaluate the binding of all variants to human or cynomolgus monkey cohesin 4 antigen and select the lead humanized IgG with the closest affinity to the chimeric (VH0 / Vκ0) antibody, single-cycle kinetics were performed on supernatants from transfected cell cultures. Kinetic experiments were performed at 25°C on a Biacore T200 running Biacore T200 control software V2.0.1 and evaluation software V3.0 (Cytiva, Marlborough, USA). Single-cycle kinetic data were obtained using recombinant human (Acro Biosystems, Newark, USA) or cynomolgus monkey cohesin 4 (Acro Biosystems, Newark, USA) as the analyte, with a flow rate of 40 μl / min to minimize any potential mass transfer effects. Four two-fold dilutions of the antigen in running buffer (ranging from 1.25 nM to 10 nM) were used, with no regeneration between each concentration. For each time in the antigen injection that four concentrations increase gradually, monitor and combine stage 150 seconds, and measure single dissociation stage 250 seconds (in order to improve degree of fit, for human adhesion protein 4, dissociation is shortened to 175 seconds).Use single injection 3M MgCl2 to carry out regeneration on sensor chip surface.Based on the relative KD from the Biacore single cycle kinetic analysis of supernatant, six kinds of humanized variants (VH4 / Vκ1, VH4 / Vκ3, VH4 / Vκ4, VH4 / Vκ5, VH5 / Vκ4 and VH5 / Vκ5) are selected as the lead IgG of expanded production and purification.All selected variants are within 2.5 times of parent (VH0 / Vκ0) clones, show that described humanized variants have similar activity characteristics (table 2).

[0197] Table 2. Affinity values ​​of the top-ranked NTX1105 clones after humanization relative to the parental mAb (VH0 / Vκ0)

[0198]

[0199]

[0200] Example 7. Improved characteristics of humanized anti-adhesin 4 mAb (reduced immunogenicity)

[0201] As achieved through the application of Abzena's proprietary computer technology iTope TM The designed VH and Vκ sequences were analyzed for potential T cell epitopes as determined by (Perry et al., 2008). TMThe software predicts favorable interactions between the amino acid side chains of the peptide and specific binding pockets (particularly pocket positions p1, p4, p6, p7 and p9) within the open binding groove of 34 human class II MHC alleles. These alleles represent the most common HLA-DR alleles found worldwide, without weighting the most common alleles in any particular ethnic group. 20 of the alleles contain an "open" p1 configuration and 14 contain a "closed" configuration in which the glycine at position 83 is replaced by valine. The positions of the key binding residues are achieved by computer simulation generation of 9mer peptides that overlap 8 amino acids across the protein sequence tested. However, the results should be evaluated taking into account the fact that all prediction methods for class II MHC binding inherently overestimate the number of T cell epitopes because they do not allow for other important processes in the antigen presentation process, such as protein / peptide processing, recognition by T cell receptors or T cell tolerance to the peptide. iTope TM The analysis was performed using overlapping 9mer peptides (each peptide overlapped the last peptide by 8 residues). Each 9mer was scored based on its potential "fit" and interaction with each of the 34 MHC class II allotypes, resulting in a peptide score ranging from 0 to 1. The peptides were then analyzed by iTope as described below. TM Defining Class II MHC epitopes:

[0202] (1) Promiscuous high-affinity MHC class II binding peptides bound >50% of the alleles, with the majority (17 of 34 alleles) having binding scores >0.6.

[0203] (2) Promiscuous intermediate-affinity MHC class II binding peptides bound >50% of alleles with binding scores >0.55 (but not the majority >0.6).

[0204] In the case of the presence of large aromatic amino acids (i.e., F, W, Y) in the p1 anchor position, these criteria are altered, with the open p1 pocket of 20 of the 34 alleles allowing binding of large aromatic residues. When this occurs, a promiscuous peptide is defined as binding to 10 or more alleles in a subset of the 20 alleles. A number of promiscuous germline high and medium affinity class II MHC binding ligands were identified in the parent antibody and the designed variants, however, due to T cell tolerance, these epitopes were unlikely to have immunogenic potential and were therefore excluded from any further analysis. Overall, the humanization process produced significantly improved mAbs with lower immunogenicity scores, thereby reducing the associated risks. The variants with the lowest immunogenicity after this step included heavy chain variants 3-5 and light chain variant 5 (Table 3). Table 3Improved features of humanized anti-TNFR4 mAbs. Humanized variants showed reduced immunogenicity compared to the parent, as measured by predicted MHC II epitopes (fewer predicted epitopes correlated with lower immunogenicity) (iTope score). VH0 and humanized heavy (VH1-5) and light (Vκ1-5) chains were used to generate humanized variants for lead drug selection. Combinations of heavy and light chains capable of generating <6 high-affinity epitopes are shown in bold.

[0205] Heavy chain:

[0206]

[0207]

[0208] Light chain:

[0209] Predicted affinity for MHCII Vk0 Vk1 Vk2 Vk3 Vk4 Vk5 middle 9 4 3 3 3 4 high 9 7 7 6 5 4

[0210] Example 8. Improved Characteristics of Humanized Anti-Nephrin 4 mAbs (Improved Developability)

[0211] Thermal ramp stability experiments (Tm and Tagg) are well-established methods for ranking the stability of proteins and formulations. A protein's denaturation curve provides information about its thermal stability and represents a structural "fingerprint" for evaluating structural and formulation buffer modifications. A widely used measure of a protein's thermal structural stability is the temperature at which it unfolds from its native state to its denatured state.

[0212] For many proteins, this unfolding process occurs within a narrow temperature range, and the midpoint of this transition is called the "melting temperature" or "Tm". To determine the melting temperature of a protein, UNcle measures the fluorescence of Sypro Orange (which binds to exposed hydrophobic regions of the protein) as the protein undergoes a conformational change. Purified lead antibodies (in duplicate) were diluted in PBS to a final test concentration of 0.5 mg / ml, to which Sypro Orange (160x stock solution) was added to a final concentration of 20x solution. 9 μL of each sample mixture was loaded into a Uni microcuvette in duplicate. The samples underwent a thermal ramp from 25-95°C at a ramp rate of 0.3°C / min with an excitation wavelength of 473 nm. The full emission spectrum was collected from 250-720 nm, and the inflection point (Tonset and Tm) of the transition curve was calculated using the area under the curve between 510-680 nm. Monitoring static light scattering (SLS) at 473 nm allows detection of protein aggregation and calculation of Tagg (onset of aggregation) from the resulting SLS curve. TMData analysis was performed using the software version 4.0. No significant changes in TM or Tonset were observed, and significant improvements in stability (>5°C over VH0 / Vκ0) were observed for both humanized variants (VH4 / Vκ5 and VH5 / Vκ5) (Table 4).

[0213] Table 4. Characterization of some humanized variants. Depicted is a summary of the thermal stability values ​​of the parental / chimeric (VH0 / Vκ0) antibody and six purified lead humanized variants determined using the UNcle biostability platform. An increase in stability Tagg (a measure of aggregation) of more than 5 degrees Celsius was considered significant and is marked in bold.

[0214]

[0215] Example 9. Improved characteristics of humanized anti-adhesin 4 mAb (improved productivity)

[0216] The production of mAbs is a complex process that is affected by the amino acid composition and structure of the molecule. Transient production is a good indicator of the "productivity" that a mAb has. Combinations of chimeric (VH0 / Vκ0) and humanized heavy and light chain DNA constructs were transiently transfected into HEK293 EBNA adherent cells (LGC Standards, Teddington, UK) in 6-well plates using the PEI transfection method and incubated for 6 days after transfection. Samples were harvested and antibody concentrations were measured on an Octet QK 384 using a protein A biosensor (Molecular Devices, Wokingham, Berkshire, UK) using an IgG1 antibody as a standard. The humanization process generally improves the productivity of all mAbs (calculated by dividing the titer (mg / ml) of the relevant humanized variant by the titer of the parent / chimeric (VH0 / Vκ0)), as shown in Figure 2. Figure 5 As shown in , the productivity of the lead clones (VH4 / Vκ5 and VH5 / Vκ5) was improved >10-fold.

[0217] Example 10. Potent in vivo anti-tumor activity of the lead humanized mAb

[0218] The in vivo efficacy of the lead humanized NTX1105 (VH4 / Vκ5 and VH5 / Vκ5) anti-adhesin 4 ADC was evaluated against the TNBC tumor cell line MDA-MB-468 (5M cells / mouse) sc-implanted into 6-week-old NOD-SCID female mice. Based on previous results, the MMAE payload was selected. Animals were dosed with 5 mg / kg of the lead humanized ADC 4 times (Q4D). Figure 6AAs shown in the results, both lead ADCs resulted in tumor regression (reduction of more than 66% from the treatment start volume). Unexpectedly, clone H4κ5 was significantly superior to clone H5κ5 in maintaining the response over time. 40 days after the last dose, treatment with clone H5κ5 resulted in tumor volumes similar to those at the start of treatment (p = 0.8) (stagnation). In contrast, even 40 days after the last treatment, clone H4κ5 resulted in tumor volumes significantly lower than the treatment start date (p = 0.038) ( Figure 6B This result was unexpected and suggests that the humanized anti-fibronectin 4 clone H4κ5 is superior to other anti-fibronectin 4 humanized mAbs.

[0219] Example 11. Differential Binding and Cell Killing Between NTX1105 and Ennosumab-Vedotin in Vitro

[0220] Enrotumumab-vedotin (PADCEV) is the only approved ADC targeting cohesin 4. Despite its excellent clinical efficacy, PADCEV is also associated with serious adverse events, some of which can be attributed to on-target / off-tumor binding, as PADCEV is active even at the very low densities of cohesin 4 found in some normal tissues. Here, we hypothesized that some of these adverse events could be prevented by an ADC with significantly lower on-target binding / activity.

[0221] Binding was assessed on MDA-MB-468 cells. Briefly, 5 × 10 cells per well were injected with PADCEV or NTX1105 conjugated to MMAE at the same DAR as PADCEV. 4 ADC was added at a concentration of 3 μg / ml using a 3-fold dilution. 647 (AffiniPure Fab fragment goat anti-human IgG (H+L)) (cat109-607-003 Jackson ImmunoResearch) was used for detection. Both ADCs showed the same maximum binding, which is consistent with the literature. The EC-50 value of PADCEV was approximately 10 pM (black arrow), which was approximately 20 times stronger than that of NTX1105 (dashed gray arrow). Figure 7A To evaluate the killing ability of the two ADCs in vitro, MDA-MB-468 cells were plated at 2*10 3 Cells were plated at a density of 100 cells / mL and allowed to adhere for 4-6 hours. ADC was added at a concentration of 12-0.01 μg / ml using a 4-fold dilution and the cells were incubated with ADC for a further 72 hours. After 72 hours, the assay system was harvested and used Tumor cell killing was assessed using the 2.0 cell survival assay system (Promega G9242). The killing data showed that PADCEV was approximately 16-fold more potent than NTX1105 in vitro ( Figure 7B Overall, these results suggest that NTX1105 has a superior safety profile compared to PADCEV, the most advanced ADC targeting cohesin-4.

[0222] Example 12. NTX1105 lead humanized clone exhibits higher in vivo anti-tumor activity than enroku-vedotin (PADCEV)

[0223] Next, the in vivo activity of the lead humanized clone of NTX1105 (VH4 / Vκ5) was compared with that of enroku-vedotin (PADCEV). Enroku-vedotin is the leading anti-adhesin 4 ADC that has been approved for the treatment of patients with urothelial carcinoma. The lead ADC (humanized NTX1105 (VH4 / Vκ5), anti-adhesin 4 ADC) was compared with PADCEV using an in vivo efficacy assay against the NSCLC tumor cell line H322. Tumor cells (5M cells / mouse) were sc implanted into 6-week-old female nude mice. When tumors reached 140 mm 3 Treatment was initiated at 4 hr. Animals received six doses (Q4D) of PBS (vehicle) or 1 mg / kg PADCEV or NTX1105-MC-VC-PAB-MMAE DAR4 (same conjugation method and DAR as PADCEV), or three doses (Q4D) of 3 mg / kg PADCEV or NTX1105-MC-VC-PAB-MMAE DAR4 (same conjugation method and DAR as PADCEV). Figure 8A As shown in the Figure 2, at lower doses, PADCEV had no effect on tumor growth at any time point, and at the end of the study, the mean TV in the PADCEV-treated group was slightly higher than that in the PBS-treated group (924 vs. 693 mm). 3 ns, p=0.48), while NTX1105 was able to inhibit tumor growth (220 vs. 693 mm 3 , p = 0.04). In addition, NTX1105 mean TV did not increase significantly from the start date of treatment (p = 0.28 compared to the start date of treatment). At a dose of 3 mg / kg, both NTX1105 and PADCEV resulted in tumor regression (by day 20, mean TV decreased by 46% and 42% compared to the start date of treatment) ( Figure 8B). However, starting on day 34 after the first dose, NTX1105ADC was significantly better than PADCEV in preventing tumor growth throughout the study. Notably, at day 48 after the start of the study, the PADCEV-treated group was no longer significantly better than PBS in terms of tumor volume. The NTX1105 ADC-treated group resulted in a complete stagnation of tumor growth, and even at day 48, tumor volumes were lower than at the start of the study (110 vs. 138 mm on day 48). 3 These results were unexpected, particularly given the significantly stronger in vitro activity of PADCEV. Furthermore, this is the first time that an ADC targeting cohesin-4 (NTX1105) has been shown to exhibit significant anti-tumor activity in a model in which no biological activity was observed with PADCEV.

[0224] Example 13. Humanized NTX1105 (H4K5-FcgR null ADC can reverse H322 tumors in vivo

[0225] Use 5x10 6 H322 cells were injected SC into female nude mice (n=5 per group). Once the tumor reached 140 mm 3 Mice were treated with PBS, NTX1105-MMAE-DAR4 (3 mg / kg), or NTX1105-exitecan-DAR-8 (5 mg / kg) by iv injection every 4 days in a blinded manner for 2 doses. Figure 9 As shown in [ 2 ], both ADCs were able to regress established tumors to the same extent, a surprising result considering previous attempts using payloads targeting TOPO1 ( Figure 4 This demonstrates the potential utility of the NTX1105 ADC using MMAE and TOPO1 payloads.

[0226] Example 14. Humanized NTX1105 (H4K5-FcgR) with a payload targeting TOPO1 null )ADC outperforms enroku with the same payload and DAR

[0227] Use 5x10 6 H322 cells were injected SC into female nude mice (n=5 per group). Once the tumor reached 480 mm 3 The mice were treated in a blinded manner with two doses of 10 mg / kg PBS (vehicle), NTX1105-based or enroku ADC (both DAR-4 and ixetine) injected iv, 5 days apart. Figure 10As shown in Figure 2, only NTX1105 ADC was able to prevent tumor growth after two doses of 10 mg / kg, while enroku-ixitecan showed no effect compared with PBS. This suggests that NTX1105 ADC has potential advantages over other ADCs based on anti-TNF-α antibodies.

[0228] sequence

[0229] Table 5. Chimeric antibodies and humanized variable regions

[0230]

[0231]

[0232]

[0233] SEQ ID NO: 13 – Heavy chain – common sequence encompassing all humanized variants

[0234] QVQL(Q / V)QSG(P / S)ELKKP(E / G)ASVK(I / V)SCKASGYTFTSYYIHWV(K / R)QAPGQGLEW(I / M)GWIYPGNVNTKYNE RF(K / Q)GR(A / V)T(L / I)TADKST(N / S)TA(H / Y)MELSSL(T / R)SED(S / T)AVY(F / Y)CARSNPYVMDYWGQGTSVTVSS

[0235] SEQ ID NO: 14 – Light chain – common sequence encompassing all humanized variants

[0236] (S / D)I(V / Q)MTQSPS(F / T)L(L / S)AS(A / V)GDRVTITC(K / R)ASQSVNNDVAWYQQKPG(L / K)AP(E / K)LLMYYASNRFTGVPDRF(T / S)GSGYGTDFTLTISSLQAED(L / V)A(I / V)YFCQQAYRSPYTFGQGTKLEIK

[0237] Table 6. CDR sequences

[0238]

[0239]

[0240] Table 7: Framework (non-CDR) sequences of humanized heavy chain variable regions

[0241]

[0242]

[0243] Table 8. Framework (non-CDR) sequences of humanized light chain variable regions

[0244]

[0245]

[0246] NTX1105 (H4 / k5) with the LALAPG mutant (FcgR null ) - Heavy chain - SEQ ID NO: 51:

[0247] QVQLVQSGSELKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGWIYPGNVNTKYNERFKGRVTITADKSTNTAYMELSSLRSEDTAVYYCARSNPYVMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0248] NTX1105 (H4 / k5) - Light chain - SEQ ID NO: 52

[0249] DIQMTQSPSTLSASVGDRVTITCRASQSVNNDVAWYQQKPGKAPKLLMYYASNRFTGVPDRFSGSGYGTDFTLTISSLQAEDVAVYFCQQAYRSPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. A humanized antibody that specifically binds to human nectin-4 or a fragment thereof that at least comprises an antigen-binding site, wherein the antibody or the fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence with at least about 90% identity to a sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and wherein the light chain comprises a variable region having an amino acid sequence with at least about 90% identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO:

12.

2. The humanized antibody according to claim 1, wherein the antibody or the fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence with at least about 95% identity to a sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and wherein the light chain comprises a variable region having an amino acid sequence with at least about 90% identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO:

12.

3. The humanized antibody according to any one of claims 1 or 2, wherein the humanized antibody or the fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 13, and the light chain variable region comprises the sequence shown in SEQ ID NO:

14.

4. The humanized antibody according to any one of the preceding claims, wherein the humanized antibody or the fragment thereof comprises a set of six CDR sequences, wherein the heavy chain CDR1 comprises the sequence SYY (SEQ ID NO: 26), the heavy chain CDR2 comprises the sequence IYPGNVNT (SEQ ID NO: 22), the heavy chain CDR3 comprises the sequence SNPYVMDY (SEQ ID NO: 17), the light chain CDR1 comprises the sequence QSVNND (SEQ ID NO: 24), the light chain CDR2 comprises the amino acid sequence YAS (SEQ ID NO: 25), and the light chain CDR3 comprises the sequence QQAYRSPYT (SEQ ID NO: 20).

5. A humanized antibody according to any one of the preceding claims, wherein the humanized antibody or fragment thereof comprises a set of six CDR sequences, wherein the heavy chain CDR1 comprises the sequence SYYIH (SEQ ID NO: 15), the heavy chain CDR2 comprises the sequence WIYPGNVNTKYNERF(K / Q)G (SEQ ID NO: 16), the heavy chain CDR3 comprises the sequence SNPYVMDY (SEQ ID NO: 17), the light chain CDR1 comprises the sequence (K / R)ASQSVNNDVA (SEQ ID NO: 18), the light chain CDR2 comprises the sequence YASNRFT (SEQ ID NO: 19), and the light chain CDR3 comprises the sequence QQAYRSPYT (SEQ ID NO: 20).

6. A humanized antibody according to any one of the preceding claims, wherein the humanized antibody or fragment thereof comprises a heavy chain CDR2 containing the sequence WIYPGNVNTKYNERFKG (SEQ ID NO: 27) or WIYPGNVNTKYNERFQG (SEQ ID NO: 28).

7. A humanized antibody according to any one of claims 1 to 5, wherein the humanized antibody or fragment thereof comprises a light chain CDR1 containing the sequence KASQSVNNDVA (SEQ ID NO: 29) or RASQSVNNDVA (SEQ ID NO: 30).

8. A humanized antibody according to any one of the preceding claims, wherein the humanized antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a set of four heavy chain (HC) framework (FR) sequences: (A) FR-H1 is selected from SEQ ID NO: 31, 32 and 33; (B) FR-H2 is selected from SEQ ID NO: 34, 35 and 36; (C) FR-H3 is selected from SEQ ID NO: 37, 38, 39 and 40; (D) FR-H4 is SEQ ID NO: 41; and the light chain variable region comprising a set of four light chain (LC) framework (FR) sequences: (A) FR-L1 is selected from SEQ ID NO: 42, 43 and 44; (B) FR-L2 is selected from SEQ ID NO: 45 and 46; (C) FR-L3 is selected from SEQ ID NO: 47, 48 and 49; (D) FR-L4 is SEQ ID NO:

50.

9. A humanized antibody according to any one of the preceding claims, wherein the heavy chain variable region of the humanized monoclonal antibody comprises a sequence selected from SEQ ID NO: 2, 3, 4, 5 and 6; and the light chain variable region of the humanized monoclonal antibody comprises a sequence selected from SEQ ID NO: 8, 9, 10, 11 and 12.

10. The humanized antibody according to any one of the preceding claims, wherein the heavy chain variable region of the humanized monoclonal antibody comprises SEQ ID NO: 5, and the light chain variable region of the humanized monoclonal antibody comprises SEQ ID NO:

12.

11. A conjugate comprising the humanized antibody according to any one of the preceding claims.

12. The conjugate according to claim 11, which comprises a cytotoxic moiety, a radioactive moiety or a labeling tag.

13. A pharmaceutical composition comprising the humanized antibody or antigen-binding fragment according to any one of claims 1 to 10, or a conjugate comprising said antibody, and a pharmaceutically acceptable excipient, carrier or diluent.

14. The pharmaceutical composition according to claim 13, which is for treating cancer.

15. An antibody-drug conjugate (ADC) comprising a humanized anti-adhesion protein 4 antibody or an antigen-binding portion thereof conjugated to a toxin, said antibody or antigen-binding portion thereof comprising a set of six CDR sequences, wherein heavy chain CDR1 comprises the sequence SYY (SEQ ID NO: 26), heavy chain CDR2 comprises the sequence IYPGNVNT (SEQ ID NO: 22), heavy chain CDR3 comprises the sequence SNPYVMDY (SEQ ID NO: 17), light chain CDR1 comprises the sequence QSVNND (SEQ ID NO: 24), light chain CDR2 comprises the amino acid sequence YAS (SEQ ID NO: 25), and light chain CDR3 comprises the sequence QQAYRSPYT (SEQ ID NO: 20).

16. The antibody-drug conjugate according to claim 15, wherein said antibody or antigen-binding portion thereof comprises a set of six CDR sequences, wherein heavy chain CDR1 comprises the sequence SYYIH (SEQ ID NO: 15), heavy chain CDR2 comprises the sequence WIYPGNVNTKYNERF(K / Q)G (SEQ ID NO: 16), heavy chain CDR3 comprises the sequence SNPYVMDY (SEQ ID NO: 17), light chain CDR1 comprises the sequence (K / R)ASQSVNNDVA (SEQ ID NO: 18), light chain CDR2 comprises the sequence YASNRFT (SEQ IDNO: 19), and light chain CDR3 comprises the sequence QQAYRSPYT (SEQ ID NO: 20).

17. The antibody-drug conjugate according to any one of claims 15 or 16, wherein the antibody or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence with at least about 90% identity to a sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and wherein the light chain comprises a variable region having an amino acid sequence with at least about 90% identity to a sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO:

12.

18. The antibody-drug conjugate according to any one of claims 15 to 17, wherein the heavy chain variable region of the humanized antibody comprises the amino acid sequence shown in SEQ ID NO: 13, and the light chain variable region of the humanized antibody comprises the amino acid sequence shown in SEQ ID NO:

14.

19. The antibody-drug conjugate according to any one of claims 15 to 17, wherein the heavy chain variable region of the humanized antibody comprises the amino acid sequence shown in SEQ ID NO: 5, and the light chain variable region of the humanized antibody comprises the amino acid sequence shown in SEQ ID NO:

12.

20. The antibody-drug conjugate according to any one of claims 15 to 19, wherein the toxin is selected from microtubule inhibitors, DNA synthesis inhibitors, topoisomerase inhibitors, and RNA polymerase inhibitors.

21. The antibody-drug conjugate according to claim 20, wherein the toxin is selected from monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), saponins, DM4, DM1, irinotecan, and SN-38.

22. The antibody-drug conjugate according to any one of claims 15 to 21, wherein the antibody and the toxin are linked by a linker.

23. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 15 - 22 and a pharmaceutically acceptable excipient, carrier, or diluent.

24. The pharmaceutical composition according to claim 23, which is used for treating cancer in an individual.

25. The pharmaceutical composition for the use according to claim 24, wherein the cancer is selected from bladder cancer, liver cancer, lung cancer, colon cancer, glioblastoma, adrenal cancer, uterine cancer, testicular cancer, head and neck cancer, pancreatic cancer, and breast cancer.

26. A method for treating cancer in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of the pharmaceutical composition according to any one of claims 13 or 23.

27. The method according to claim 26, wherein the method for treating cancer comprises administering or performing at least one additional anti-cancer therapy.

28. The method according to claim 27, wherein the additional anti-cancer therapy is surgery, chemotherapy, radiotherapy, or immunotherapy.

29. The method according to claim 24, which comprises administering an additional anti-cancer agent selected from an immunomodulator, an activated lymphocyte, a kinase inhibitor, and a chemotherapeutic agent.

30. The humanized antibody or fragment thereof according to any one of claims 1 to 4 or the antibody-drug conjugate according to claim 11, wherein the humanized antibody comprises a set of six CDR sequences, wherein the heavy chain CDR1 comprises the sequence GYTFTSYY (SEQ ID NO: 21), the heavy chain CDR2 comprises the sequence IYPGNVNT (SEQ ID NO: 22), the heavy chain CDR3 comprises the sequence ARSNPYVMDY (SEQ ID NO: 23), the light chain CDR1 comprises the sequence QSVNND (SEQ ID NO: 24), the light chain CDR2 comprises the amino acid sequence YAS (SEQ ID NO: 25), and the light chain CDR3 comprises the sequence QQAYRSPYT (SEQ ID NO: 20).

31. The humanized antibody according to any one of claims 1 to 10, which comprises the heavy chain sequence set forth in SEQ ID NO: 51 and the light chain sequence set forth in SEQ ID NO: 52.

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