Humanized antibodies against connexin 2 and drug conjugates thereof

Abstract

The present invention provides humanized monoclonal antibodies that recognize human connexin 2 with high affinity and specificity and inhibit its binding to CD112R. These antibodies carry a cytotoxic load and do not interact with the Fc gamma receptor. The invention further provides pharmaceutical compositions comprising said antibodies and methods of their use in cancer immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of immunotherapy and relates to anti-connexin 2 antibodies and antibody conjugates and therapeutic and diagnostic compositions containing them for treating diseases, especially cancer. Background Art

[0002] Cancer immunotherapy is used to generate and enhance anti-tumor immune responses, such as by treatment with antibodies that are specific for 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 a patient provides a therapeutic modality to combat cancer types and metastases that were originally considered incurable.

[0003] The T cell-mediated immune response consists of multiple sequential steps regulated by the balance between co-stimulatory and co-inhibitory signals that control the magnitude of the immune response. Inhibitory signals known as immune checkpoints are essential for maintaining self-tolerance and limiting immune-mediated collateral tissue damage. These inhibitory signals influence the T cell response and reshape the immune response.

[0004] Connexin 2, also known as poliovirus receptor-associated protein 2, poliovirus receptor-like 2, CD112, or PRR-2, is a single-pass transmembrane glycoprotein with two Ig-like C2-type domains and one Ig-like V-type domain. Connexin is involved in mediating cell adhesion to extracellular matrix molecules, serving as one of the plasma membrane components of adherens junctions. It also serves as an entry receptor for certain mutant strains of herpes simplex virus and pseudorabies virus and is involved in the cell-to-cell spread of these viruses. Variations in this gene have been associated with differences in the severity of multiple sclerosis. Importantly, connexin 2 can also act as a regulator of T-cell signaling. It can be a co-stimulator or co-inhibitor of T-cell function, depending on the receptor on these target cells to which it binds: upon binding to CD226 (DNAM-1), it stimulates T-cell proliferation and cytokine production, including that of IL-2 and IFNγ, whereas upon interaction with PVRIG (CD112R) and / or TIGIT (T-cell immunoreceptor with immunoglobulin and ITIM domains), it inhibits T-cell proliferation and activation. These contradictory interactions are competitive.

[0005] Connexin 2 has been shown to be overexpressed in various tumors including breast cancer and ovarian cancer (Oshima et al., Molecular Cancer, 2013, 12: 60). The presence of connexin 2 on tumor cells leads to poor prognosis and reduced T cell activity (Stamm et al., Oncogene (2018) 37: 5269-5280).

[0006] U.S. Patent Application No. 2017 / 0037133 discloses inhibitors (e.g., antibodies) against CD112 (connexin 2, PVRL2), CD155 (PVR), galectin 9, TIM-3 and / or TIGIT for the treatment of blood-borne cancers, particularly acute myeloid leukemia (AML).

[0007] WO 2020 / 144697 discloses a monoclonal antibody (mAb) that recognizes human connexin 2 and blocks the interaction with TIGIT and CD112R, which is used to treat cancer.

[0008] 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 their therapeutic window by limiting the specific delivery of these drugs to cells expressing the target antigen, thereby reducing their systemic exposure and toxicity.

[0009] An example of a possible load is Auristatin, a drug that destroys microtubules. It is derived from the marine shellless mollusk Dolabella auricularia and is called dolastatins. Various derivatives of Auristatin, such as monomethyl Auristatin E (MMAE) and monomethyl Auristatin F (MMAF), have been synthesized. MMAE and MMAF were developed by Seattle Genetics and used as loads for ADCs. MMAF and MMAE each have advantages and disadvantages. MMAE has stronger membrane permeability and a lower IC50 than MMAF. However, compared with MMAE, MMAF is more hydrophilic, has a lower tendency to aggregate, and shows lower systemic toxicity (park et al., Molecules 2019, 24, 2754).

[0010] There is an unmet need to provide humanized antibodies recognizing human connexin 2 that are safe and effective and can be used alone or as a targeting tool for diagnostic and therapeutic use in diseases involving connexin 2 expression, such as ADCs in cancer. Summary of the invention

[0011] The present invention provides humanized antibodies that specifically bind to connexin 2. The humanized antibodies of the present invention selected from a larger number of antibody clones have improved properties compared to other anti-connexin 2 antibodies. According to some embodiments, the present invention further provides a conjugate comprising the antibody and a diagnostic or therapeutic agent. In some embodiments, the conjugate comprises a cytotoxic portion that is targeted by the humanized antibody to tumor cells that present connexin 2 receptors on the surface.

[0012] 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. Advantageously, the newly designed humanized variable regions described herein retain residues that are critical to maintaining antibody conformation and binding affinity, while having a lower incidence of potential T cell epitopes, thereby minimizing the risk of producing adverse immune responses to antibodies. Antibodies disclosed herein are designed based on factors including homology, T cell epitopes, key residues, and predicted structures. Unexpectedly, compared to parent chimeric antibodies, humanized antibodies disclosed herein show improved biological stability.

[0013] It has been found that the humanized antibodies disclosed herein are very suitable for use as targeted anti-cancer therapies using therapeutic toxins. It is now disclosed that the anti-connexin 2 monoclonal humanized antibodies described herein coupled to a cytotoxic moiety exhibit potent lethality against various tumor cell lines. Direct targeting of toxins using the antibodies described herein has the potential to increase the anti-tumor efficacy of these toxins, reduce their systemic toxicity and thus increase the survival rate of cancer patients.

[0014] Human Fc receptors (FcγRs) activate Fc effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Some ADCs of the present invention comprise one or more Fc mutations that significantly reduce their binding by FcγRs expressed on immune cells, thereby improving their safety and specificity. It is now disclosed that ADCs comprising anti-connexin 2 monoclonal humanized antibodies having specific Fc mutations and linked to toxins are highly effective and safe and are suitable for anti-cancer therapy.

[0015] The inventors of the present invention have shown for the first time that ADCs targeting connexin 2 are very useful for treating cancer, especially solid tumors.

[0016] According to the first aspect, the present invention provides a humanized antibody that specifically binds to human connexin 2 or a fragment thereof that at least comprises an antigen binding site, wherein the antibody or its fragment comprises a heavy chain (HC) and a light chain (LC), 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: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; 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: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.

[0017] 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 90% identical to SEQ ID NO: 11, and the light chain comprises a variable region whose amino acid sequence is at least about 90% 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 variable region comprises the sequence set forth in SEQ ID NO:11, and the light chain variable region comprises the sequence set forth in SEQ ID NO:12.

[0019] 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, and others can be determined using IMGT. According to some embodiments, the CDR sequences of the mAb variable regions are determined using the IMGT method.

[0020] According to some embodiments, the humanized antibody or fragment thereof comprises a set of 6 CDR sequences, wherein the heavy chain CDR1 comprises the sequence SYW, the heavy chain CDR2 comprises the sequence VYPGNSDS (SEQ ID NO: 8), the heavy chain CDR3 comprises the sequence LVGTFDY (SEQ ID NO: 3), the light chain CDR1 comprises the sequence QNVGIN (SEQ ID NO: 10), the light chain CDR2 comprises the sequence SAS, and the light chain CDR3 comprises the sequence QQYNTNPFT (SEQ ID NO: 6).

[0021] According to some embodiments, the humanized antibody or fragment thereof comprises a set of 6 CDR sequences, wherein the heavy chain CDR1 comprises the sequence SYWIH (SEQ ID NO: 1), the heavy chain CDR2 comprises the sequence AVYPGNSDSNYNQKF(KA / QG) (SEQ ID NO: 2), the heavy chain CDR3 comprises the sequence LVGTFDY (SEQ ID NO: 3), the light chain CDR1 comprises the sequence (K / R)ASQNVGINV(V / A) (SEQ ID NO: 4), the light chain CDR2 comprises the sequence SASYRYS (SEQ ID NO: 5), and the light chain CDR3 comprises the sequence QQYNTNPFT (SEQ ID NO: 6).

[0022] According to some embodiments, the heavy chain CDR2 comprises the sequence AVYPGNSDSNYNQKFKA (SEQ ID NO: 41) or AVYPGNSDSNYNQKFQG (SEQ ID NO: 42). According to certain embodiments, the light chain CDR1 comprises a sequence selected from KASQNVGINVV (SEQ ID NO: 43), KASQNVGINVA (SEQ ID NO: 44), RASQNVGINVV (SEQ ID NO: 45) and RASQNVGINVA (SEQ ID NO: 46).

[0023] According to some embodiments, the humanized antibody or fragment thereof comprises a set of 6 CDR sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, and 6. According to other exemplary embodiments, the humanized antibody or fragment thereof comprises a set of 6 CDR sequences set forth in SEQ ID NOs: 7, 8, 9, 10, (SAS) and 6. According to other embodiments, the humanized antibody or fragment thereof comprises a set of 6 CDR sequences set forth in SEQ ID NOs: 1, 41, 3, 43, 5, and 6.

[0024] According to some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising:

[0025] i. a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 1-3; and

[0026] ii. a set of four heavy chain framework (FR) sequences, wherein FR-H1 is selected from SEQ ID NOs: 21, 25 and 27; FR-H2 is SEQ ID NO: 22; FR-H3 is selected from SEQ ID NOs: 23, 26, 28 and 29; and FR-H4 is SEQ ID NO: 24.

[0027] According to some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a light chain variable region comprising:

[0028] i. a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 4-6; and

[0029] ii. a set of four light chain framework (FR) sequences, wherein FR-L1 is selected from SEQ ID NOs: 30, 34, 37 and 39; FR-L2 is selected from SEQ ID NOs: 31 and 35; FR-L3 is selected from SEQ ID NOs: 32, 36, 38 and 40; and FR-L4 is SEQ ID NO: 33.

[0030] According to some embodiments, 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:

[0031] i. a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 1-3; and

[0032] ii. A set of four heavy chain framework sequences, wherein FR-H1 is selected from SEQ ID NOs: 21, 25 and 27; FR-H2 is SEQ ID NO: 22; FR-H3 is selected from SEQ ID NOs: 23, 26, 28 and 29; FR-H4 is SEQ ID NO: 24;

[0033] And the light chain variable region comprises:

[0034] i. a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 4-6; and

[0035] ii. a set of four light chain framework sequences, wherein FR-L1 is selected from SEQ ID NOs: 30, 34, 37 and 39; FR-L2 is selected from SEQ ID NOs: 31 and 35; FR-L3 is selected from SEQ ID NOs: 32, 36, 38 and 40; and FR-L4 is SEQ ID NO: 33.

[0036] According to some embodiments, 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:

[0037] i. a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 7-9; and

[0038] ii. A set of four heavy chain framework sequences, wherein: FR-H1 is selected from SEQ ID NO: 21, 25 and 27; FR-H2 is SEQ ID NO: 22; FR-H3 is selected from SEQ ID NO: 23, 26, 28 and 29; FR-H4 is SEQ ID NO: 24;

[0039] And the light chain variable region comprises:

[0040] i. a set of three CDR sequences comprising the sequence set forth in SEQ ID NO: 10, sequence SAS and SEQ ID NO: 6; and

[0041] ii. a set of four light chain framework sequences, wherein: FR-L1 is selected from SEQ ID NOs: 30, 34, 37 and 39; FR-L2 is selected from SEQ ID NOs: 31 and 35; FR-L3 is selected from SEQ ID NOs: 32, 36, 38 and 40; and FR-L4 is SEQ ID NO: 33.

[0042] According to some embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises an amino acid sequence having at least about 95% identity to a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16; and the light chain variable region comprises an amino acid sequence having at least about 95% identity to a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In certain embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises an amino acid sequence having at least about 97% identity to a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16; and the light chain variable region comprises an amino acid sequence having at least about 97% identity to a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In certain embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

[0043] 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).

[0044] According to some embodiments, the humanized antibody or fragment thereof is an IgG monoclonal antibody. According to some embodiments, the humanized monoclonal antibody comprises a heavy chain constant region selected from IgG4, IgG1 and IgG2. In certain embodiments, the humanized antibody or fragment thereof is an IgG4 subclass. In certain embodiments, the humanized antibody or antigen-binding fragment thereof is an IgG1 subclass.

[0045] According to some embodiments, the humanized monoclonal antibody comprises a kappa light chain constant region.

[0046] According to some embodiments, the humanized monoclonal antibody is an IgG1, whose heavy chain comprises an amino acid sequence having at least about 90%, 95% or 98% identity to the sequence set forth in SEQ ID NO: 47. According to certain exemplary embodiments, the humanized monoclonal antibody is an IgG1, whose heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 47.

[0047] According to some embodiments, the kappa light chain of the humanized monoclonal antibody comprises an amino acid sequence that is at least about 90%, 95%, or 98% identical to the sequence set forth in SEQ ID NO: 49. According to certain exemplary embodiments, the kappa light chain of the humanized monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO: 49.

[0048] According to some embodiments, the humanized antibody has a mutated Fc domain that prevents FcγR-mediated internalization.

[0049] 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 found on immune cells. According to certain exemplary embodiments, the Fc domain is ineffective for binding to CD64, CD32a, CD32b, CD16a and / or CD16b.

[0050] According to some embodiments, the humanized antibody comprises an IgG1 Fc domain. According to certain embodiments, the IgG1 Fc domain is ineffective for binding to Fcγ receptors. According to other embodiments, the IgG1 Fc domain is ineffective for binding to one or more Fcγ receptors. According to certain exemplary embodiments, the IgG1 Fc domain is ineffective for binding to CD64, CD32a, CD32b, CD16a and / or CD16b.

[0051] According to some embodiments, the humanized antibody comprises a human IgG selected from the following: (i) human IgG1 with mutation L235S; (ii) human IgG1 with mutation L235S and E272K; (iii) human IgG1 with mutation G237I; (iv) human IgG1 with mutation G237I and E272I; (v) human IgG1 with mutation G237I and V264R; (vi) human IgG1 with mutation V215A, E269R and K322A; (vii) human IgG1 with mutation L234A, L235A and P329G; (viii) human IgG4 with mutation S228P, L235P and V264R; (ix) human IgG2 with mutation P238H; or (x) human IgG2 with mutation P238H and V264R. Each possibility represents an independent embodiment of the present invention.

[0052] According to some embodiments, the humanized antibody comprises a heavy chain sequence selected from the group consisting of SEQ ID NOs: 61 - 70. Each possibility represents a separate embodiment of the invention.

[0053] According to some embodiments, a conjugate comprising the above-mentioned humanized antibody or fragment thereof is provided.

[0054] According to some embodiments, the antibody or fragment thereof according to the invention is attached to a cytotoxic moiety, a radioactive moiety or an affinity or marker label.

[0055] The antibody-drug conjugate (ADC) according to the present invention comprises a humanized antibody as described herein and optionally a linker, and a toxin.

[0056] According to some embodiments, the humanized antibody or fragment thereof is coupled to a toxin (payload) directly or through a linker.

[0057] 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.

[0058] 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).

[0059] According to some embodiments, the toxin is saporin.

[0060] According to some embodiments, the toxin is a maytansine derivative. According to certain embodiments, the maytansine derivative is DM4 or DM1.

[0061] According to some embodiments, the toxin is a quinoline alkaloid. According to certain embodiments, the quinoline alkaloid is SN-38.

[0062] According to some embodiments, the toxin is a DNA topoisomerase I (TOP1) inhibitor. According to some embodiments, the DNA topoisomerase I (TOP1) inhibitor is Exatecan or an Exatecan derivative. According to certain embodiments, the DNA topoisomerase I (TOP1) inhibitor is DXd.

[0063] According to some embodiments, the toxin is directly attached to the antibody. According to other embodiments, the antibody and toxin are connected via a linker or a spacer. According to some embodiments, the toxin is directly or via a linker or a spacer attached to the humanized antibody. Each possibility represents an independent embodiment of the present invention.

[0064] 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).

[0065] 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). Each possibility represents an independent embodiment of the present invention.

[0066] According to some embodiments, polynucleotide sequences encoding the amino acid sequences of the above-mentioned heavy chain variable region and light chain variable region are provided.

[0067] According to some embodiments, the polynucleotide sequence encodes a humanized antibody heavy chain variable region, the polynucleotide comprising a sequence selected from SEQ ID NO: 51-55 or a variant thereof having at least 80% sequence identity. Each possibility represents an independent embodiment of the present invention.

[0068] According to some embodiments, the polynucleotide sequence encodes a humanized antibody light chain variable region, the polynucleotide comprising a sequence selected from SEQ ID NO: 56-60 or a variant thereof having at least 80% sequence identity. Each possibility represents an independent embodiment of the present invention.

[0069] According to some embodiments, the polynucleotide sequence encodes a humanized antibody heavy chain variable region, and the polynucleotide comprises a sequence selected from SEQ ID NOs: 51 to 55. According to some embodiments, the polynucleotide sequence encodes a humanized antibody light chain variable region, and the polynucleotide comprises a sequence selected from SEQ ID NOs: 56 to 60.

[0070] According to some embodiments, the polynucleotide sequence encodes a humanized antibody heavy chain variable region, and the polynucleotide comprises the sequence set forth in SEQ ID NO: 51. According to some embodiments, the polynucleotide sequence encodes a humanized antibody light chain variable region, and the polynucleotide comprises the sequence set forth in SEQ ID NO:56.

[0071] According to some embodiments, polynucleotide sequences encoding the amino acid sequences of the above-mentioned humanized antibody heavy chains and light chains are provided.

[0072] According to some embodiments, the polynucleotide sequence encoding the humanized antibody heavy chain comprises the sequence set forth in SEQ ID NO: 48 or a variant thereof having at least 80% sequence identity. According to some embodiments, the polynucleotide sequence encoding the humanized antibody light chain comprises the sequence set forth in SEQ ID NO: 50 or a variant thereof having at least 80% sequence identity.

[0073] According to some embodiments, the DNA sequence encoding the amino acid chain of the humanized antibody described herein comprises a leader sequence. According to certain exemplary embodiments, the DNA sequence encodes the leader peptide sequence set forth in SEQ ID NO:71.

[0074] 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.

[0075] Also described is a cell line comprising a nucleic acid encoding an antibody of the present invention. The cell line is used to express the humanized antibody or fragment thereof described herein. In certain embodiments, the cell line is a mammalian cell line such as a Chinese hamster ovary (CHO) cell line.

[0076] 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.

[0077] According to some embodiments, the pharmaceutical composition is for use in treating cancer.

[0078] 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.

[0079] 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.

[0080] 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 or conjugate thereof as described herein.

[0081] According to some embodiments, the cancer is characterized by overexpression of Connexin2.

[0082] In certain embodiments, the cancer comprises a solid tumor.

[0083] In certain embodiments, the cancer is selected from prostate cancer, colorectal 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.

[0084] According to some embodiments, the cancer is breast cancer. According to some embodiments, the cancer is colorectal adenocarcinoma or lung adenocarcinoma.

[0085] 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.

[0086] According to some embodiments, the subject is a human.

[0087] 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.

[0088] According to some embodiments, the method of treating cancer comprises administering a humanized antibody described herein and an additional anti-cancer agent. According to some embodiments, the additional anti-cancer agent is selected from an immunomodulator, an activated lymphocyte, a kinase inhibitor, and a chemotherapeutic agent.

[0089] According to other embodiments, the additional immunomodulator is an antibody, antibody fragment or antibody conjugate that binds to an antigen other than human connexin 2.

[0090] According to some embodiments, the additional immunomodulator is an antibody against an immune checkpoint molecule. According to some embodiments, the additional immunomodulator is an antibody against an immune checkpoint molecule, and the immune checkpoint molecule is selected from human programmed cell death protein 1 (PD-1), PD-L1 and PD-L2, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), lymphocyte activation gene 3 (LAG3), CD137, OX40 (also known as CD134), lethal cell immunoglobulin-like receptor (KIR), TIGIT, PVR, CTLA-4, NKG2A, GITR and any other checkpoint molecule or combination thereof. Each possibility represents an independent embodiment of the present invention. According to certain embodiments, the additional immunomodulator is an antibody against PD-1. According to some embodiments, the additional immunomodulator is an antibody against CTLA-4.

[0091] 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.

[0092] According to some embodiments, the method of treating cancer involves preventing or reducing the formation, growth or spread of metastases in a subject.

[0093] According to another aspect, the present invention provides an antibody-drug conjugate comprising a humanized antibody that specifically binds to human connexin 2 and a toxin, for use in treating cancer.

[0094] The cancer and toxins are described above.

[0095] According to some embodiments, the humanized antibody comprises a mutated Fc domain.

[0096] According to one aspect, the present invention also provides a method for diagnosing or prognosing cancer in a subject, the method comprising determining the expression level of connexin 2 in a biological sample of the subject using at least one humanized antibody, fragment or conjugate described herein.

[0097] According to another aspect, the present invention also provides a method for determining or quantifying the expression of connexin 2, the method comprising contacting a biological sample with the antibody or antibody fragment described herein, and measuring the level of complex formation.

[0098] According to some embodiments, the method for detecting or quantifying the expression of connexin 2 comprises the following steps:

[0099] i. incubating the sample with an antibody specific for connexin 2 or an antibody fragment thereof comprising at least an antigen binding portion;

[0100] ii. Detecting bound connexin 2 using a detectable probe.

[0101] According to some embodiments, the method further comprises the steps of:

[0102] iii. comparing the amount of (ii) to a standard curve obtained from a reference sample containing a known amount of connexin 2; and

[0103] iv. Calculate the amount of connexin 2 in the sample from the standard curve.

[0104] 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.

[0105] Also provided is a kit for measuring the expression of connexin 2 in a biological sample, comprising at least one antibody or antibody fragment described herein and a device for measuring the expression of connexin 2. In some embodiments, the kit further comprises instructional materials directing the use of the kit.

[0106] From the detailed description given below, further embodiments and the full scope of applicability of the present invention will become apparent. However, it should be understood that the detailed description and specific examples, although indicating preferred embodiments of the present invention, are given by way of illustration only, because for those skilled in the art, various changes and modifications within the spirit and scope of the present invention will become apparent from the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figures 1A-1C Plotted are the correlations between connexin 2 mRNA levels (high or low as indicated) and low-grade gliomas ( Figure 1A ), renal clear cell carcinoma ( Figure 1B) and lung adenocarcinoma ( Figure 1C ) patients’ survival. 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.

[0108] Figure 2 Schematic diagram of receptors expressed on immune cells and their corresponding affinities for connexin 2 (CD112) expressed on tumors or on antigen presenting cells (APCs). TIGIT is a co-inhibitory receptor on many immune cells (e.g., T cells and NK cells); DNAM-1 (also known as CD226) is an activating receptor on many immune cells (e.g., T cells), and CD112R (also known as PVRIG) and TIGIT are co-inhibitory receptors on lymphoid immune cells (e.g., T cells and NK cells); connexin 2 acts as an inhibitory ligand for immune cells primarily through its high affinity binding to CD112R (rectangle).

[0109] Figure 3 is a graph showing the percentage of tumors positive for Connexin 2 expression. Data obtained from proteatlas.com using HPA012759 mAb (anti-Connexin 2 antibody; ). In 17 / 20 indications, membrane expression of connexin 2 was observed at moderate to high levels.

[0110] Figures 4A-4C Describe the improved binding on cells of humanized variants of parent (chimeric) anti-connexin 2 mAb. The binding of cells expressing connexin 2 to serially diluted mAb humanized variants was analyzed by FACS. The EC50 value of each variant was calculated and recorded relative to the EC50 value of chimeric Ab (H0K0), which was set to 1. Figure 4A -Fold change in EC50 for binding to 293T cells expressing human connexin2. Figure 4B - Fold change in EC50 for binding to Vero cells derived from African green monkeys (AFG, Chlorocebus) and naturally expressing Connexin 2 (XP_007995342.1). Figure 4C - Results of blocking CD112R by different humanized mAb variants, assessed by FACS analysis of CD112R-Fc binding to CHO(K)1 cells overexpressing human connexin 2 in the presence of anti-connexin 2 humanized clones at concentrations ranging from 66-0.81 nM. Variants that maintained >70% blocking capacity at the lowest dose were considered superior.

[0111] Figures 5A-5CThe lethal effects of humanized anti-Connexin2 ADC variants are depicted. The ADC is based on saporin (ZAP) and A549 target cells (lung adenocarcinoma) were confirmed to express Connexin2. Figure 5A The lead clone H3K3 was shown to be more lethal than other humanized variants tested at two concentrations. Figure 5B Comparable levels of ADC activity between humanized H3K3 and H4K2 variants are depicted. Figure 5C - Comparable levels of ADC activity between chimeric mAb (H0K0) and humanized H3K3 variants at three concentrations. H3K3 was also tested when grafted onto IgG2-P238H, which has an FcgR null Fc, and had no effect on the potency of this variant. All lethal results were significant (p<0.001, two-way t-test) unless indicated with NS (not significant).

[0112] Figures 6A-6C It was confirmed that humanized H3K3-FcgR conjugated to various toxins null Anti-connexin2 ADC leads to effective killing of various solid tumor cell lines. The figure shows that H3K3-FcgR null Significant lethal activity of ADCs at different ADC concentrations and between different cancer targets after 72 hours of incubation. Fig. 6A The killing of RKO cells (colorectal adenocarcinoma) is depicted. Figure 6B Depicted is the killing of MDA-MB-231 cells (triple negative breast cancer). Figure 6C Depicted is the killing of A549 cells (lung adenocarcinoma). Unless indicated by NS (not significant), the killing of target cells was significant (p<0.001). Although all ADCs had toxic effects, the extent varied depending on the nature of the coupled toxin and the target cells. Nevertheless, in all target cells, ADCs containing MMAE or MMAF were superior to all other ADCs.

[0113] Figure 7 Confirmed that humanized H3K3-FcgR null Anti-connexin2 ADC is not internalized through interaction with high-affinity FcgR-CD64. CHO-K1 cells or CHO-K1 cells overexpressing human CD64 (hCD64) but not connexin2 were incubated with two versions of H3K3-FcgR null ADC (coupled to MMAE or DM4) or control irrelevant ADC with hIgG4 (S228P) or hIgG1 Fc (both coupled to DM4) was incubated at 12ug / ml for 72 hours. When hIgG4 or hIgG1 was used, an increase in specific killing of CD64 positive cells was observed, but when FcgR nullOf note, DM4 induced background nonspecific killing regardless of whether the target cells expressed connexin 2 or CD64.

[0114] Figure 8 Confirmed that humanized H3K3-FcgR null Anti-Connexin2 ADCs lead to efficient killing of hematological cell lines. H3K3-FcgR null Abs were incubated with HL-60 cells (AML model) at concentrations of 9-1 μg / ml for 72 hours.Except for DM1, all linker-load combinations resulted in >90% lethality at high doses and were significantly lethal at all doses tested (p<0.001).

[0115] Figures 9A-9B Humanized H3K3-FcgR is depicted null In vivo efficacy of anti-connexin2 ADC against the hematological tumor cell line HL-60 transplanted subcutaneously into female nude mice. Fig.9A The efficacy of treatments using different linker-payload (LP) combinations was compared. Two LP combinations had significant effects: DM4 resulted in significant tumor growth inhibition (TGI, p<0.05), while complete tumor regression was observed with MMAE. Fig. 9B Other treatments are not included.

[0116] Fig.10 Humanized H3K3-FcgR is depicted null In vivo efficacy of anti-connexin2 ADC (1107-MC-VC-MMAE) against the solid (colorectal adenocarcinoma) tumor cell line RKO. Empty vehicle was used as control.

[0117] Fig.11 Depicted is the humanized H3K3-FcgR linked to Dxd compared to Trodelvy (Sacituzumab-govitecan), an ADC drug approved for TNBC null In vitro killing of MDA-MB-231 (triple negative breast cancer or TNBC) cells by anti-connexin 2 mAb (NTX1107). Both ADCs used the same class of cytotoxic payloads (i.e., topoisomerase I (TOP1) inhibitors). MDA-MB-231 cells were incubated in the presence of these ADCs at the indicated concentrations. Killing was assessed after 120 hours. Both ADCs induced strong killing of target cells, with Trodelvy having a significant advantage at both concentrations (***p<0.0005).

[0118] Fig.12Depicted is the effect of humanized H3K3-FcgR compared to Trodelvy null In vivo efficacy of anti-connexin 2 ADC (NTX1107) against the MDA-MB-231 subcutaneous tumor model in female nude mice. NTX1107-Dxd was able to significantly inhibit tumor growth, leading to tumor stasis, while Trodelvy had no effect on tumor growth under the same treatment regimen.

[0119] Fig.13 Compared with H3K3-FcgR null -Dxd (NTX1107-Dxd) and H3K3-FcgR null Effect of in vivo treatment with MMAE (NTX1107-MMAE) on the MDA-MB-231 subcutaneous tumor model in female nude mice. Complete tumor regression was observed for HNTX1107-MMAE, with 7 / 7 mice having no measurable tumors at the end of the study. Tumor regression was observed for NTX1107-Dxd, with 4 / 7 mice having no measurable tumors at the end of the study. DETAILED DESCRIPTION

[0120] The present invention provides a humanized monoclonal antibody that recognizes connexin 2. Advantageously, the antibodies of the present invention are almost completely humanized, thereby avoiding the risk of adverse immune responses to the antibodies and thus may be safe for use in humans. The antibodies of the present invention are characterized by having unique CDR sequences and novel humanized framework sequences and designs.

[0121] In some embodiments, the present invention further provides antibody-drug conjugates or ADCs comprising the humanized antibodies described herein, which can be used to treat cancer.

[0122] 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 appreciate that the embodiments provided can be practiced without these details. Unless the context requires otherwise, throughout this specification and the subsequent claims, the word "comprising" and its variations should be interpreted in an open, inclusive sense, i.e., interpreted as "including but not limited to". References without a specific number used in this specification and the appended claims include multiple references unless the content clearly provides otherwise. It should also be noted that the term "or" is generally used in its meaning including "and / or", unless the content clearly provides otherwise. In addition, the titles provided herein are for convenience only and do not explain the scope or meaning of the claimed embodiments.

[0123] As used herein, the term "about" refers to an amount that varies by 10% or less from the stated amount.

[0124] As used herein, the term "connexin 2" or "connexin cell adhesion molecule 2" refers to a human plasma membrane glycoprotein also known as CD112 and PVRL2. The connexin 2 protein is a type I single-pass transmembrane glycoprotein with two Ig-like C2-type domains and one Ig-like V-type domain. This protein is one of the plasma membrane components of adhesion junctions. It also serves as a portal for certain mutant strains of herpes simplex virus and pseudorabies virus and is involved in the cell-to-cell spread of these viruses. Exemplary connexin 2 according to the present invention are described in the following SwissPort, UniPort and GenBank symbols or accession numbers: Gene ID: 5819, Q92692, I68093, NP_001036189.1, NP_002847.1 and #Q92692.

[0125] According to one aspect, the present invention provides a humanized antibody that specifically binds to connexin 2, or a fragment thereof that comprises at least an antigen binding site, wherein the antibody or its fragment 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: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; 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: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.

[0126] According to another aspect, the present invention provides a humanized antibody that specifically binds to connexin 2 or a fragment thereof that at least comprises an antigen binding site, wherein the antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises:

[0127] (i) a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 1-3; and

[0128] (ii) a set of four heavy chain framework sequences, wherein: FR-H1 (framework heavy chain No. 1) is selected from SEQ ID NOs: 21, 25 and 27; FR-H2 is SEQ ID NO: 22; FR-H3 is selected from SEQ ID NOs: 23, 26, 28 and 29; FR-H4 is SEQ ID NO: 24;

[0129] And the light chain variable region comprises:

[0130] (i) a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 4-6; and

[0131] (ii) a set of four light chain framework sequences, wherein FR-L1 (framework light chain No. 1) is selected from SEQ ID NOs: 30, 34, 37 and 39; FR-L2 is selected from SEQ ID NOs: 31 and 35; FR-L3 is selected from SEQ ID NOs: 32, 36, 38 and 40; and FR-L4 is SEQ ID NO: 33.

[0132] The framework is a non-CDR sequence of the variable chain region. FR-H1 is the sequence before CDR1 in the variable heavy chain, FR-H2 is the sequence between CDR1 and CDR2, FR-H3 is the sequence between CDR2 and CDR3, and FR-H4 is the sequence after CDR3. FR-L1 is the sequence before CDR1 in the variable light chain, FR-L2 is the sequence between CDR1 and CDR2, FR-L3 is the sequence between CDR2 and CDR3, and FR-L4 is the sequence after CDR3.

[0133] According to another aspect, the present invention provides a humanized antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises:

[0134] i. a set of three CDR sequences, the CDR comprising the sequences SYW, SEQ ID NO: 8 and SEQ ID NO: 3; and

[0135] ii. A set of four heavy chain framework sequences, wherein: FR-H1 is selected from SEQ ID NO: 21, 25 and 27; FR-H2 is SEQ ID NO: 22; FR-H3 is selected from SEQ ID NO: 23, 26, 28 and 29; FR-H4 is SEQ ID NO: 24;

[0136] And the light chain variable region comprises:

[0137] i. a set of three CDR sequences comprising the sequences set forth in SEQ ID NO: 10, SAS and SEQ ID NO: 6; and

[0138] ii. a set of four light chain framework sequences, wherein: FR-L1 is selected from SEQ ID NOs: 30, 34, 37 and 39; FR-L2 is selected from SEQ ID NOs: 31 and 35; FR-L3 is selected from SEQ ID NOs: 32, 36, 38 and 40; and FR-L4 is SEQ ID NO: 33.

[0139] According to some embodiments, the humanized antibody or fragment thereof comprises: a heavy chain variable region comprising a set of three CDR sequences, wherein the CDR comprises the sequence SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9; and a light chain variable region comprising a set of three CDR sequences, wherein the CDR comprises the sequence SEQ ID NO: 10, SAS and SEQ ID NO: 6.

[0140] The antibodies provided include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies and multireactive antibodies) and antibody fragments. The antibodies include antibody conjugates and molecules such as chimeric molecules comprising the antibodies. Therefore, the antibodies include but are not limited to the full length and fragments and parts thereof retaining binding specificity, such as any specific binding portion thereof, including those with any immunoglobulin class and / or isotype (e.g., 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 contained in the monoclonal antibody composition is the same. Polyclonal antibodies are preparations that include different antibodies of different sequences, which are generally directed to two or more different determinants (epitopes). The monoclonal antibodies may include human IgG1 constant regions. The monoclonal antibody may comprise a human IgG4 constant region.

[0141] The term "antibody" herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including complete 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 bi-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.

[0142] CDR identification or determination from a given heavy or light chain variable sequence is generally performed using one of several methods known in the art. For example, such determinations are 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).

[0143] When the term "CDR having a 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.

[0144] The antigen specificity of an antibody is based on the hypervariable regions (HVRs), the unique CDR sequences of both the light and heavy chains that together form the antigen binding site.

[0145] The antibodies provided include antibody fragments. "Antibody fragments" refer to molecules other than intact antibodies that contain a portion of an intact antibody that binds to an 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.

[0146] "Humanized" antibodies are antibodies in which all or substantially all CDR amino acid residues are derived from non-human CDRs, and all or substantially all framework region (FR) amino acid residues are derived from human FRs. Humanized antibodies may optionally include at least a portion of antibody constant regions derived from human antibodies. "Humanized forms" of non-human antibodies refer to humanized variants of non-human antibodies, 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 by corresponding residues from non-human antibodies (e.g., antibodies from which CDR residues are derived), for example, to restore or improve the specificity or affinity of the antibody.

[0147] The amino acid residues in the Fc domain may be replaced to be ineffective, which means that the Fc domain does not bind to the Fc receptor, or may bind with such low affinity and / or avidity that no Fc receptor signaling is caused by binding. The Fc domain may be ineffective for binding to the Fcγ receptor. Some example Fcγ receptors to which the Fc domain may be ineffective for binding may 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 may have one or more, two or more, three or more, or four or more amino acid substitutions that reduce the binding of the Fc domain to the Fc receptor.

[0148] According to some embodiments, the humanized antibody has a mutated Fc domain that prevents FcγR-mediated internalization.

[0149] 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 Fc gamma receptor.

[0150] As used herein, "Fc null" refers to a domain that has weak or no binding to one or more Fcγ receptors.

[0151] Antibody-drug conjugates

[0152] According to one aspect, the invention provides a conjugate comprising a humanized antibody as described herein fused to a toxin.

[0153] According to some embodiments, the conjugate comprises an antibody or fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence that is at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16; and wherein the light chain comprises a variable region having an amino acid sequence that is at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

[0154] 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.

[0155] 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).

[0156] According to some embodiments, the toxin is a saponin.

[0157] According to some embodiments, the toxin is a maytansine derivative. According to certain embodiments, the maytansine derivative is DM4 or DM1.

[0158] According to some embodiments, the toxin is a quinoline alkaloid. According to certain embodiments, the quinoline alkaloid is SN-38.

[0159] According to other embodiments, the toxin is selected from MMAE, MMAF, saponin, DM4, DM1, SN-38, calicheamicin, DXd, PBD, duocarmycin, sandromycin, α-amanitin, chaetocin, daunorubicin, 17-AAG, Agrochelin A, doxorubicin, methotrexate, colchicine, cordycepin, hygrolidin, herboxidiene, ferulic acid, curvulin, Englerin A, taltobulin, triptolide, scutellarin and nemorubicin. Each possibility represents an independent embodiment of the present invention.

[0160] According to some embodiments, the toxin is a DNA topoisomerase I (TOP1) inhibitor. According to certain embodiments, the DNA topoisomerase I (TOP1) inhibitor is DXd.

[0161] Toxin names known in the art are used herein. Non-limiting examples of suitable payloads include:

[0162] -DM1——N 2 ′-Deacetyl-N 2 ′-(3-Mercapto-1-oxopropyl)-maytansine - a microtubule inhibitor.

[0163] -DM4——N 2 ′-Deacetyl-N 2 ′-(4-Mercapto-4-methyl-1-oxopentyl)-Ravtansine - a tubulin inhibitor.

[0164] -SN-38 is a potent DNA topoisomerase I inhibitor, a member of the pyranoindolizine quinoline class, i.e., (4S)-pyrano[3',4':6,7]indolizine[1,2-b]quinoline-3,14-dione, bearing two additional ethyl substituents at positions 4 and 11, and two additional hydroxyl substituents at positions 4 and 9.

[0165] -DXd - an exotecan derivative, a potent DNA topoisomerase I inhibitor (e.g., Catalog No. HY-13631D).

[0166] -MMAF – Monomethyl auristatin F – a microtubule inhibitor with the molecular formula C39H65N5O8.

[0167] -MMEA – Monomethyl auristatin E – a microtubule inhibitor with the molecular formula C39H67N5O7.

[0168] 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.

[0169] According to some embodiments, the linker is cleavable. According to other embodiments, the linker is non-cleavable.

[0170] 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.

[0171] 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.

[0172] According to other embodiments, the pharmaceutical composition according to the present invention is used to treat cancer characterized by overexpression of connexin 2. Cancer types associated with connexin 2 overexpression 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 invention is selected from the group consisting of adrenocortical carcinoma (ACC), chromophobe renal cell carcinoma (KICH), hepatocellular carcinoma (LIHC), colon and rectal adenocarcinoma (COAD, 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 an independent embodiment of the present invention.

[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 the compositions and methods can be used to treat. According to some embodiments, the individual is a mammal. According to some embodiments, the mammal is a mouse, a rat, a rabbit, a dog, a cat, a horse, a cow, a sheep, a pig, a goat, a llama, an alpaca, or a yak. ​​According to some embodiments, the individual is a human.

[0174] The term "effective amount" as used herein refers to the amount of a therapeutic agent that causes a biological effect when administered to a mammal. Biological effects include, but are not limited to, inhibiting or blocking receptor-ligand interactions (e.g., PVR-TIGIT, PD-1-PD-L1 / PD-L-2), inhibiting signal transduction pathways, reducing tumor growth, reducing tumor metastasis, or prolonging the survival of animals carrying tumors. "Therapeutic amount" is the concentration of a drug calculated to exert a therapeutic effect. The therapeutic amount covers a dosage range that can induce a therapeutic response in an individual population. The mammal may be a human individual. The human individual may have or be suspected of having a tumor.

[0175] The term "combination" or "combination therapy" as used herein may refer to simultaneous administration of the products to be combined or sequential administration of the products to be combined. As described herein, when the combination involves sequential administration of products, the products may be administered in any time order.

[0176] As used herein, "checkpoint inhibitors" refer to drugs that inhibit biological molecules ("checkpoint molecules") produced by an organism, which negatively regulate the anti-tumor / cancer activity of T cells in the organism. Checkpoint molecules include, but are not limited to, PD-1, PD-L-1, PD-L-2, CTLA4, TIM-3, LAG-3, VISTA, SIGLEC7, PVR, TIGIT, IDO, KIR, A2AR, B7-H3, B7H4, CEACAM1, and CD112R.

[0177] 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, etc. and combinations thereof. Other suitable carriers are well known to those skilled in the art. In addition, if necessary, the composition may contain a small amount of auxiliary substances, such as wetting agents or emulsifiers, pH buffers.

[0178] As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in whom the disorder is to be prevented.

[0179] The terms "cancer" and "tumor" relate to a physiological condition in mammals characterized by unregulated cell growth. Cancer is a class of diseases in which a group of cells exhibit uncontrolled growth or unwanted growth. Cancer cells can also spread to other places, which can lead to the formation of metastases. For example, the spread of cancer cells in the body can occur through the lymph or blood. Uncontrolled growth, invasion, and metastasis formation are also known as the malignant properties of cancer. These malignant properties distinguish cancer from benign tumors that usually do not invade or metastasize.

[0180] 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.

[0181] According to some embodiments, the anticancer agent is selected from 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.

[0182] 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.

[0183] According to another aspect, the present invention provides a method for treating cancer in an individual suffering from cancer, the method comprising administering to the individual a therapeutically effective amount of the humanized antibody or its antigen-binding fragment or the pharmaceutical composition, and an inhibitor of PD-1, PD-L1, CTLA-4 or CD112R signaling. In certain embodiments, the cancer comprises a solid tumor. In certain embodiments, the cancer is selected from cancer, colon cancer, glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, cervical cancer or prostate cancer. In certain embodiments, the inhibitor of PD-1 signaling is an antibody or fragment thereof that binds to PD-1. In certain embodiments, the antibody or fragment thereof that binds to PD-1 is pembrolizumab, nivolumab, AMP-514, tislelizumab, spartalizumab, or a PD-1 binding fragment thereof. In certain embodiments, the inhibitor of PD-1 signaling is an antibody that specifically binds to PD-L-1 or PD-L-2. In certain embodiments, the antibody that specifically binds to PD-L1 or PD-L2 includes durvalumab, atezolizumab, avelumab, BMS-936559 or FAZ053, or a PD-L1 or PD-L2 binding fragment thereof. In certain embodiments, the inhibitor of PD-1 signaling includes an Fc fusion protein that binds to PD-1, PD-L1 or PD-L2. In certain embodiments, the Fc fusion protein includes AMP-224 or a PD-1 binding fragment thereof. In certain embodiments, the inhibitor of PD-1 signaling includes a small molecule inhibitor of PD-1, PD-L1 or PD-L2.In certain embodiments, the small molecule inhibitor of PD-1, PD-L1 or PD-L2 signaling includes one or more of the following: N-{2-[({2-methoxy-6-[(2-methyl[1,1'-biphenyl]-3-yl)methoxy]pyridin-3-yl}methyl)amino]ethyl}acetamide (BMS202); (2-((3-cyanobenzyl)oxy)-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylbenzyl)oxy)-5-methylbenzyl)-D -Serine hydrochloride; (2R,4R)-1-(5-chloro-2-((3-cyanobenzyl)oxy)-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylbenzyl)oxy)benzyl)-4-hydroxypyrrolidine-2-carboxylic acid; 3-(4,6-dichloro-1,3,5-triazin-2-yl)-1-phenylindole; 3-(4,6-dichloro-1,3,5-triazin-2-yl)-1-phenyl-1h-indole; L-α-glutamine, N2,N6-bis(L-serine acyl-L-asparaginyl-L-threonyl-L-seryl-L-α-glutamyl-L-seryl-L-phenylalanyl)-L-lysyl-L-phenylalanyl-L-arginyl-L-valyl-L-threonyl-L-glutaminyl-L-leucyl-L-alanyl-L-prolyl-L-lysyl-L-alanyl-L-glutaminyl-L-isoleucyl-L-lysyl; (2S)-1-[[2,6-dimethoxy-4-[(2-methyl[1,1' -biphenyl]-3-yl)methoxy]phenyl]methyl]-2-piperidinic acid; glycinamide, N-(2-mercaptoacetyl)-L-phenylalanyl-N-methyl-L-alanyl-L-asparaginyl-L-prolyl-L-histidyl-L-leucyl-N-methylglycyl-L-tryptophyl-L-seryl-L-tryptophyl-N-methyl-L-norleucyl-N-methyl-L-norleucyl-L-arginyl-L-cysteinyl-, cyclic (1→14)-thioether; or a derivative or analog thereof.

[0184] Also described herein are methods for preparing a composition for treating cancer in an individual suffering from cancer, the method comprising mixing the humanized antibody or its antigen-binding fragment 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 colon cancer, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, glioblastoma, cervical cancer, prostate cancer and lung cancer.

[0185] Also described herein is a method of producing the humanized antibody or antigen-binding fragment thereof, the method comprising incubating the cell line described herein in a cell culture medium under conditions sufficient to allow expression and secretion of the humanized antibody or antigen-binding fragment thereof.

[0186] According to some specific embodiments, the additional anticancer agent is selected from bevacizumab, carboplatin, cyclophosphamide, doxorubicin hydrochloride, gemcitabine hydrochloride, topotecan hydrochloride, thiotepa and combinations thereof. Each possibility represents an independent embodiment of the present invention.

[0187] Example

[0188] Reference is now made to the following examples, which together with the above descriptions illustrate the invention in a non limiting fashion.

[0189] 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.

[0190] Example 1. High expression of connexin 2 mRNA is associated with low survival rates in patients with various cancers

[0191] The correlation between connexin 2 mRNA expression level and survival rate was examined on the data from the TCGA website and analyzed using the oncolnc.org website (https: / / doi.org / 10.7717 / peerj-cs.67). Connexin 2 mRNA expression level was the basis for dividing patients into two subgroups: low-expressors and high-expressors, as shown in Figure 1 with arrows for low-grade gliomas ( Figure 1A ; p = 5.22E-5), renal clear cell carcinoma ( Figure 1B ; p = 0.00037) and lung adenocarcinoma ( Figure 1C ; p=0.0319) as indicated by the patient.

[0192] Example 2. Connexin 2 binds to and affects immune cells via specific receptors

[0193] Figure 2The schematic diagram demonstrates receptors expressed on immune cells and their corresponding affinity to connexin 2 expressed by tumors or antigen presenting cells (APCs). TIGIT is associated with co-inhibitory receptors on immune cells such as T cells and NK cells; DNAM-1 (also known as CD226) is associated with activation receptors on immune cells (such as T cells), and CD112R (also known as PVRIG) is associated with co-inhibitory receptors on lymphoid immune cells (such as T cells and NK cells); connexin 2 (CD112) acts as an inhibitory ligand of immune cells mainly through its binding to CD112R. According to the present invention, humanized anti-connexin 2 mAbs block the interaction of connexin 2 with its high affinity receptor CD112R, and can target cancer cells for specific toxin delivery and lethality.

[0194] Example 3. Connexin 2 is expressed on most solid tumors

[0195] The Proteinatlas.com database was searched for all aliases of Connexin 2 (Connexin 2, CD112, HVEB, PRR2, PVRL2, PVRR2). Under the Pathology heading, data using three different mAbs were found. The HPA0127569 mAb had the highest validation score (Enhanced) because it was validated by an orthogonal approach. Therefore, only expression data for this clone between different tumors were selected, as shown in Table 1. Figure 3 The figure shows the percentage of tumors positive for the expression of connexin 2. In 17 / 20 indications, membrane expression of connexin 2 was observed at medium to high levels.

[0196] Example 4. Improved performance of humanized anti-connexin 2 mAbs

[0197] Murine anti-human connexin 2 clone 2.11, disclosed in patent application publication number WO2020144697, was selected as the lead mAb for humanization. Based on structural analysis, a preliminary large set of sequence segments were identified for creating humanized variants. Using iTope TM These segments were selected and analyzed using the TCED of T cell epitopes associated with known antibody sequences using in silico analysis of peptide binding to human class II MHC alleles (Perry et al., 2008). TM (Bryson et al., 2010). Identified as a significant non-human germline binder to human MHC class II or to TCED TMThe sequence segments with significant hits were discarded. This produced a reduced set of segments, and the combination of these segments was further analyzed as described above to ensure that the junctions between the segments did not contain potential T cell epitopes. The selected sequence segments were assembled into complete V region sequences without significant T cell epitopes. Five heavy chain (VH1 to VH5) and five light chain (VK1 to VK5) sequences were then selected. The data in Table 1 show the iTope TM Readout of the algorithm. To reduce the possibility of generating anti-drug antibodies (ADA), the sequences of the mAbs were analyzed for potential binding to MHCII. The main risk was associated with high affinity binding peptides, which were separated from the moderate affinity binding peptides.

[0198] Table 1 Improved properties of humanized anti-connexin2 mAbs. Illustration of predicted MHCII epitopes (iTope scores) of parental (V0) and humanized heavy (VH1-5) and light (Vk1-5) variable chains used to generate humanized variants for lead drug selection.

[0199] Heavy chain

[0200]

[0201] Light chain

[0202]

[0203] The parental heavy chain (VH0) had 8 predicted high affinity motifs, and the parental light chain (Vk0) had 4 such motifs. After the humanization process, the number of predicted high affinity MHCII epitopes for the heavy chain was reduced to 3 (VH2, VH3) or even 1 (VH4, VH5), and the predicted high affinity MHCII motifs for the light chain were reduced to 2 (VK3-5).

[0204] Thus, the humanization process eliminated most of the predicted high affinity MHCII motifs that could elicit immunogenicity against the mAb.

[0205] The stability of the humanized variants was assessed by thermal ramp stability experiments, which is an established method for ranking the stability of proteins and formulations. The denaturation curve of a protein provides information about its thermal stability and represents a structural "fingerprint" for assessing 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.

[0206] 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 conformational changes. An increase in the Tm is a desirable feature in Ab lead selection because it predicts a more stable Ab.

[0207] The lead humanized antibody variants (in duplicate) of purification are diluted to the final test concentration of 0.5mg / ml in PBS, to which Sypro orange (160x storage solution) is added to the final concentration of 20x solution. Each sample mixture of 9 μL is loaded in duplicate into Uni micro-colorimetric cell. Sample is subjected to 15-95 ℃ hot ramp, ramp rate is 0.3 ℃ / minute, and excitation wavelength is 473nm. Total emission spectrum is collected from 250-720nm, and the inflection point (Tonset and Tm) of transition curve is calculated using the area under the curve between 510-680nm. As seen in Table 2, for all antibodies tested, Tm1 and Tonset of humanized variants are higher than chimeric antibodies (VH0 / Vκ0).

[0208] Table 2 Summary of thermal stability values ​​of parental / chimeric (VH0 / Vk0) antibodies and six purified lead humanized variants determined using the UNcle biostability platform. Improvements in stability above 5 degrees Celsius for both Tonset and TM1 (a measure of unfolding) are considered significant and are marked in bold.

[0209]

[0210]

[0211] Example 5. Improved binding of humanized anti-connexin 2 mAbs to connexin 2 and blocking of CD112R binding

[0212] Binding of humanized anti-connexin2 mAbs to human connexin2 expressed by 293T cells (protein id: Q92692) and Chlorocebus (African green monkey, AFG) Vero cells was evaluated and EC50 values ​​established. AFG expresses a connexin2 protein (XP_007995342.1) with 97% similarity to human connexin2. Figure 4A and 4B Fold changes in EC50 values ​​for connexin 2 expressed in human and AFG cells are depicted, respectively. Both cell lines were cultured at 0.5x10 5Cells were plated. Chimeric (H0KO) and humanized mAbs were added at concentrations ranging from 20-0.001 nM. For detection, anti-human APC antibody (Jackson Immunoresearch AB_2340526) was used at a dilution of 1:200. Cells were analyzed by FACS, and EC50 was calculated and compared to the EC50 of the chimeric Ab (fold improvement). This analysis showed improved binding of the humanized variant compared to the chimeric mAb and a similar degree of cross-reactivity with monkey connexin 2 ( Figure 4A Compared to 4B). To evaluate the blocking ability of the humanized Ab, human connexin 2 was overexpressed in Chinese hamster ovary (CHO) cells. Figure 4C Shows when 10 5 Data generated when 2 / well of CHO-h connexin were incubated with 30nM human CD112R-mIgG2a in the presence of a specified humanized Ab in a concentration range of 66-0.81nM. Anti-mIgG2a-647 (Jackson Immunoresearch Cat 115-607-186) diluted 1:200 was used to detect bound CD112R, followed by FACS analysis. It can be seen that all humanized variants maintained complete blocking ability.

[0213] Example 6. Anti-human connexin 2 humanized variant H3K3 can act as an ADC driver in an Fc-independent manner

[0214] To evaluate the ability of mAbs to act as ADCs, streptavidin-saporin (ZAP) IT-27-250 (ATS) was used. The mAbs indicated in Figure 5 were biotinylated at a 1:1 ratio using the biotinylation kit Ab207195 (Abeam). Figure 5A Depicted are the results of an initial screen that included the top humanized clones and A549 (lung adenocarcinoma) cells as targets. 3 Cells were plated and allowed to adhere for a period of 4-6 hours. ADC was added and the cells were incubated with ADC. After 72 hours, the assay system was harvested and used Tumor cell lethality was assessed using the 2.0 cell viability assay (Promega G9242) following standard protocols. Only clone H3K3 showed strong lethality. Next, clone H3K3 was directly compared to clone H4K2 ( Figure 5B Both clones showed robust lethality and H3K3 was chosen for future analysis due to its excellent developable properties. The chimeric parental clone and clone H3K3(IgG1) were tested for FcgR under the above conditions. nullVariant (IgG2-P238H) (at FcgR null In the equation, g represents γ), the result is Figure 5C Both chimeric and humanized H3K3 variants showed significant lethality (p<0.001) at all concentrations of these ADCs, and FcgR null The variants resulted in equivalent levels of target cell killing.

[0215] Example 7. Anti-human connexin 2H3K3-FcgR null ADCs cause robust lethality of solid tumor cell lines in vitro

[0216] As shown in Table 3, additional H3K3-FcgRs were generated by mutating key residues in the hinge region of human IgG1. null mAbs.

[0217] Table 3 Humanized IgG1 FcgR for H3K3-based ADCs null Summary of Fc substitutions, release mechanisms, and relevant drug-antibody ratios (DARs) for linker-payload combinations

[0218]

[0219] The presented linker-payload combinations were selected based on the desired release mechanism and were produced by Abzena LTD according to standard protocols. Briefly, mAbs were reduced and incubated with excess linker-payload to obtain a desired drug-antibody ratio (DAR) of 4 for all linker-payloads except SN-38, for which the target DAR was 8. The final product was then purified and the DAR established by LC / MS methods. Selected tumor cell lines representing various solid tumors were administered to evaluate the in vitro efficacy of various linker-payload combinations. The indicated target cells were plated at 2×10 per well. 3 Cells were plated and allowed to adhere for a period of 4-6 hours. ADC was added at a concentration of 4-0.16 μg / ml using a 5-fold dilution and the cells were incubated with ADC. After 72 hours, the assay was harvested and used Tumor cell lethality was assessed using the 2.0 cell viability assay (Promega G9242) according to standard protocols. Figures 6A-6C The robust killing of RKO cells (colorectal adenocarcinoma), MDA-MB-231 cells (triple negative breast cancer) and A549 cells (lung adenocarcinoma) are depicted in Figure 1. Killing of target cells was significant (p<0.001) by two-tailed student t-test unless noted with NS (not significant, p>0.05).

[0220] Example 8. Selected FcgRnull Mutations prevent ADCs from killing via FcgR

[0221] To assess the extent of non-target specific lethality of the humanized anti-connexin 2 ADC (which is due to the binding of the Ab Fc to its FcγR and may cause non-specific effects of the ADC), CHO cells overexpressing the high affinity Fc receptor hCD64 (CHO-hCD64) were generated. These cells do not express human connexin 2, so no target specific lethality is expected. Parental CHO cells and CHO-hCD64 were plated at 2x10 3 Cells were plated and allowed to adhere for 4-6 hours. Different Fc variant ADCs were added at a concentration of 12 μg / ml. After 72 hours of incubation, the assay system was harvested and used The lethality of tumor cells was assessed by 2.0 cell viability assay (Promega G9242).

[0222] like Figure 7 As shown in Figure 2, both IgG4-S228P and hIgG1 positive controls resulted in significantly higher levels of lethality in CHO-hCD64 cells compared to parental CHO cells, as expected. On the other hand, IgG1 G237I substitution resulted in similar low levels of lethality (non-specific lethality) for both parental and hCD64 overexpressing cells. These results confirm that FcgR null The variants are not bound and internalized by the high affinity FcgR CD64.

[0223] Example 9. Anti-human connexin 2 humanized H3K3-FcgR null ADC causes potent lethality of the hematological tumor cell line HL-60 in vitro

[0224] HL-60 cells (AML model) were cultured at 2×10 3 Cells were plated and ADC was added at a concentration of 9-1 μg / ml using 3-fold dilutions. Cells were incubated with ADC for 72 hours. The assay was then harvested and used as described above. 2.0 cell viability assay (Promega G9242) was used to assess tumor cell lethality. Figure 8 As can be seen in the Figure 1, except for DM1 payload (indicated in the figure), all other compounds resulted in significant killing of the target at all tested concentrations, and at the highest concentration (>60 nM) resulted in more than 90% lethality. Based on these results, SMCC-DM1 linker-load was excluded from future experiments.

[0225] Example 10. Anti-human connexin 2H3K3-FcgR null-MC-VC-PAB-MMAE ADC leads to tumor regression in an aggressive AML model

[0226] Female nude mice (n=25) were treated with 10x10 6 Once the tumor reaches 205 mm 3 The mice were randomly divided into five groups (n=5 per group) and injected intravenously with PBS (vehicle), H3K3-FcgR null -MMAE, H3K3-FcgR null -MMAF, H3K3-FcgR null -DM4 or H3K3-FcgR null -SN-38. All treatments were given at 5 mg / kg every 4 days for 4 doses. Fig.9A As seen in Figure 2, on day 23 after randomization, H3K3-FcgR null -MMAF and SN-38 variants do not attenuate tumor growth. H3K3-FcgR null -DM4 resulted in a significant tumor growth inhibition (TGI) of 49%, while H3K3-FcgR null -MMAE resulted in significant tumor regression. Fig. 9B H3K3-FcgR null -Average effect of MMAE.

[0227] Example 11. Anti-human connexin 2H3K3-FcgR null -MC-VC-PAB-MMAE ADC induces tumor regression in an aggressive colon adenocarcinoma model

[0228] Female nude mice (n=5 per group) were treated with 5×10 6 Once the tumor reaches 160 mm 3 The mice were randomly divided into two groups and injected intravenously with PBS (vehicle) or H3K3-FcgR in a blinded manner. null -MMAE (1107-MC-VC-MMAE) was treated with 5 mg / kg once every 4 days for 4 consecutive doses. Fig.10 As seen in Figure 2, on day 13 after randomization, H3K3-FcgR null -MMAE resulted in significant tumor regression compared to the vehicle-treated group, representing a TGI of >80%.

[0229] Example 12. Anti-human connexin 2H3K3-FcgR nullmAb has similar in vitro lethal activity compared to Trodelvy

[0230] For in vitro lethality assays ( Fig.11 ), MDA-MB-231 target cells were plated at 2×10 3 Cells were plated and allowed to adhere for 4-6 hours. Next, H3K3-FcgR linked to Dxd was added at concentrations of 12 μg / ml (dark grey bars) and 3 μg / ml (light grey bars). null Anti-connexin 2 mAb (NTX1107) and Trodelvy (Sacituzumab govitecan), an approved ADC for TNBC. Notably, both ADCs use the same class of cytotoxic payloads (i.e., topoisomerase I (TOP1) inhibitors). Cells were incubated with ADCs for 120 hours and then treated with CellTiter Tumor cell killing was assessed using the 2.0 cell viability assay (Promega G9242) following standard protocols. Both ADCs induced robust killing of MDA-MB-231 cells, with Trodelvy having a significant advantage at both concentrations (***p<0.0005).

[0231] Example 13. Anti-human connexin 2H3K3-FcgR coupled to a topoisomerase 1 inhibitor null But not Trodelvy inhibits the in vivo growth of aggressive MDA-MB-231 tumors

[0232] Female nude mice (n=7 per group) were treated with 5×10 6 Once the tumor reaches 230 mm 3 The mice were randomized into treatment groups and treated in a blinded manner by intravenous injection of PBS (vehicle), NTX1107-Dxd, or Trodelvy every 4 days at a dose of 5 mg / kg for 3 doses. Trodelvy treatment had no effect on tumor growth.

[0233] As in Fig.12 As seen in Figure 2, NTX1107-Dxd was able to significantly inhibit tumor growth (*p<0.02, **<0.005, ***p<0.0005). These findings were unexpected because Trodelvy had a higher in vitro lethality than NTX1107-Dxd ( Fig.11 ), and Trodelvy is an approved ADC for TNBC. These observations suggest that anti-connexin 2 antibody (NTX1107) is a uniquely effective ADC for the treatment of solid tumors.

[0234] Example 14. Anti-human connexin 2H3K3-FcgR null Regression of aggressive MDA-MB-231 tumors in vivo when coupled to tubulin or TOP1 targeting agents

[0235] Female nude mice (n=7 per group) were treated with 5×10 6 Once the tumor reaches 210 mm 3 The mice were randomly divided into treatment groups and injected intravenously with PBS (vehicle), H3K3-FcgR null Treatment with (NTX1107)-MMAE or NTX1107-Dxd was performed every 4 days for 5 consecutive doses. Fig.13 As can be seen in Figure 2, all animals treated with NTX1107-MMAE had no detectable tumors at the end of the study. For animals treated with NTX1107 Dxd, four were tumor-free, while the remaining three had an average tumor volume of 210 mm 3 , indicating tumor stasis. These findings suggest that the humanized antibody targeting connexin 2 described herein (NTX1107) can form a unique and effective ADC for the treatment of solid tumors when used with various classes of payloads.

[0236] sequence

[0237] Table 4: CDR sequences:

[0238]

[0239]

[0240] SEQ ID NO: 11 - Heavy chain variable region NT20-11_VH3

[0241] QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYWIHWVRQPPGKGLEWIGAVYPGNSDSNYNQKFKARVTITAVTSTSTAYMELSSLRSEDTAVYYCTKLVGTFDYWGQGTTVTVSS

[0242] SEQ ID NO: 12—Light chain variable region NT20-11_Vk3

[0243] DIQMTQSPSTLSASVGDRVSVTCKASQNVGINVVWYQQKPGQPPKTLIYSASYRYSGVPDRFSGSGSGTDFTLTISSLQAEDLAEYFCQQYNTNPFTFGQGTKLEIK

[0244] SEQ ID NO: 13 - Heavy chain variable region NT20 - 11_VH1

[0245] EVQLVQSGTELKKPGSSVKVSCKASGYIFTSYWIHWVRQPPGKGLEWIGAVYPGNSDSNYNQKFKARATITAVTSTSTAYMELSSLTSEDSAVYYCTKLVGTFDYWGQGTTVTVSS

[0246] SEQ ID NO: 14 - Heavy chain variable region NT20 - 11_VH2

[0247] EVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYWIHWVRQPPGKGLEWIGAVYPGNSDSNYNQKFKARATITAVTSTSTAYMELSSLRSEDTAVYYCTKLVGTFDYWGQGTTVTVSS

[0248] SEQ ID NO: 15 - Heavy chain variable region NT20 - 11_VH4

[0249] QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYWIHWVRQPPGKGLEWIGAVYPGNSDSNYNQKFQGRVTITAVTSTSTAYMELSSLRSEDTAVYYCTKLVGTFDYWGQGTTVTVSS

[0250] SEQ ID NO: 16 - Heavy chain variable region NT20 - 11_VH5

[0251] QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYWIHWVRQPPGKGLEWIGAVYPGNSDSNYNQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTKLVGTFDYWGQGTTVTVSS

[0252] SEQ ID NO: 17 - Light chain variable region NT20 - 11_Vk1

[0253] DIVMTQSPSFLSASVGDRVSVTCKASQNVGINVVWYQQRAGQPPKTLIYSASYRYSGVPDRFTGSGSGTDFTLTISSLQSEDLAEYFCQQYNTNPFTFGQGTKLEIK

[0254] SEQ ID NO: 18—Light chain variable region NT20-11_Vk2

[0255] DIVMTQSPSTLSASVGDRVSVTCKASQNVGINVVWYQQKPGQPPKTLIYSASYRYSGVPDRFTGSGSGTDFTLTISSLQAEDLAEYFCQQYNTNPFTFGQGTKLEIK

[0256] SEQ ID NO: 19—Light chain variable region NT20-11_Vk4

[0257] DIQMTQSPSTLSASVGDRVTITCRASQNVGINVVWYQQKPGQPPKTLIYSASYRYSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYNTNPFTFGQGTKLEIK

[0258] SEQ ID NO: 20—Light chain variable region NT20-11_Vk5

[0259] DIQMTQSPSTLSASVGDRVTITCRASQNVGINVAWYQQKPGQPPKTLIYSASYRYSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYNTNPFTFGQGTKLEIK

[0260] Table 5: Framework (non-CDR) sequences of humanized heavy chain variable regions

[0261]

[0262]

[0263] Table 6: Framework (non-CDR) sequences of humanized light chain variable regions

[0264]

[0265]

[0266] SEQ ID NO: 41-HC CDR2AVYPGNSDSNYNQKFKA

[0267] SEQ ID NO: 42 - HC CDR2 AVYPGNSDSNYNQKFQG

[0268] SEQ ID NO: 43 - LC CDR1 KASQNVGINVV

[0269] SEQ ID NO: 44 - LC CDR1 KASQNVGINVA

[0270] SEQ ID NO: 45 - LC CDR1 RASQNVGINVV

[0271] SEQ ID NO: 46 - LC CDR1 RASQNVGINVA

[0272] SEQ ID NO: 47 - Amino acid sequence of full - length hIgG1 heavy chain VH3

[0273] QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYWIHWVRQPPGKGLEWIGAVYPGNSDSNYNQKFKARVTITAVTSTSTAYMELSSLRSEDTAVYYCTKLVGTFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0274] SEQ ID NO: 48 - DNA sequence of full - length hIgG1 VH3

[0275] caggtgcagctggtgcagagcggcgcggaagtgaaaaaaccgggcagcagcgtgaaagtg

[0276] agctgcaaagcgagcggctatatttttaccagctattggattcattgggtgcgccagccg

[0277] ccgggcaaaggcctggaatggattggcgcggtgtatccgggcaacagcgatagcaactat

[0278] aaccagaaatttaaagcgcgcgtgaccattaccgcggtgaccagcaccagcaccgcgtat

[0279] atggaactgagcagcctgcgcagcgaagataccgcggtgtattattgcaccaaactggtg

[0280] ggcacctttgattattggggccagggcaccaccgtgaccgtgagcagcgcgagcaccaaa

[0281] ggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcggcg

[0282] ctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagcggc

[0283] gcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtatagc

[0284] ctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgcaac

[0285] gtgaaccataaaccgagcaacaccaaagtggataaacgcgtggaaccgaaaagctgcgat

[0286] aaaacccatacctgcccgccgtgcccggcgccggaactgctgggcggcccgagcgtgttt

[0287] ctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacctgc

[0288] gtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggatggc

[0289] gtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctatcgc

[0290] gtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaatgc

[0291] aaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaaggc

[0292] cagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgaagaaatgaccaaaaac

[0293] caggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaatgg

[0294] gaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagcgat

[0295] ggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggcaac

[0296] gtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagcctg

[0297] agcctgagcccgggcaaa

[0298] SEQ ID NO: 49 - Amino acid sequence of full - length κ light chain Vk3

[0299] DIQMTQSPSTLSASVGDRVSVTCKASQNVGINVVWYQQKPGQPPKTLIYSASYRYSGVPDRFSGSGSGTDFTLTISSLQAEDLAEYFCQQYNTNPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0300] SEQ ID NO: 50 - DNA sequence of full - length κVk3

[0301] gatattcagatgacccagagcccgagcaccctgagcgcgagcgtgggcgatcgcgtgagc

[0302] gtgacctgcaaagcgagccagaacgtgggcattaacgtggtgtggtatcagcagaaaccg

[0303] ggccagccgccgaaaaccctgatttatagcgcgagctatcgctatagcggcgtgccggat

[0304] cgctttagcggcagcggcagcggcaccgattttaccctgaccattagcagcctgcaggcg

[0305] gaagatctggcggaatatttttgccagcagtataacaccaacccgtttacctttggccag

[0306] ggcaccaaactggaaattaaacgcaccgtggcggcgccgagcgtgtttatttttccgccg

[0307] agcgatgaacagctgaaaagcggcaccgcgagcgtggtgtgcctgctgaacaacttttat

[0308] ccgcgcgaagcgaaagtgcagtggaaagtggataacgcgctgcagagcggcaacagccag

[0309] gaaagcgtgaccgaacaggatagcaaagatagcacctatagcctgagcagcaccctgacc

[0310] ctgagcaaagcggattatgaaaaacataaagtgtatgcgtgcgaagtgacccatcagggc

[0311] ctgagcagcccggtgaccaaaagctttaaccgcggcgaatgc

[0312] SEQ ID NO: 51—VH3 nucleic acid

[0313] SEQ ID NO: 52—VH1 nucleic acid

[0314] SEQ ID NO: 53—VH2 nucleic acid

[0315] SEQ ID NO: 54—VH4 nucleic acid

[0316] SEQ ID NO: 55—VH5 nucleic acid

[0317] SEQ ID NO: 56—Vk3 nucleic acid

[0318] SEQ ID NO: 57—Vk1 nucleic acid

[0319] SEQ ID NO:58—Vk2 nucleic acid

[0320] SEQ ID NO: 59—Vk4 nucleic acid

[0321] SEQ ID NO:60—Vk5 nucleic acid

[0322] SEQ ID NO:61—Human IgG1 (L235S) (including VH3)

[0323] SEQ ID NO:62—Human IgG1 (L235S / E272K) (including VH3)

[0324] SEQ ID NO:63—Human IgG1 (G237I) (including VH3)

[0325] SEQ ID NO:64—Human IgG1 (G237I / E272I) (including VH3)

[0326] SEQ ID NO:65—Human IgG1 (G237I / V264R) (including VH3)

[0327] SEQ ID NO:66—Human IgG1 (V215A / E269R / K322A) (including VH3)

[0328] SEQ ID NO:67—Human IgG1 (L234A / L235A / P329G) (including VH3)

[0329] SEQ ID NO:68—Human IgG4 (S228P / L235P / V264R) (including VH3)

[0330] SEQ ID NO:69—Human IgG2 (P238H) (including VH3)

[0331] SEQ ID NO:70—Human IgG2 (P238H / V264R) (including VH3)

[0332] SEQ ID NO: 71 - Leader peptide sequence

Claims

1. A humanized antibody that specifically binds to human connexin 2 or a fragment thereof that at least comprises an antigen binding site, wherein the antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises: (i) a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 1-3; and (ii) a set of four heavy chain (HC) framework (FR) sequences, wherein: FR-H1 is selected from SEQ ID NOs: 21, 25 and 27; FR-H2 is SEQ ID NO: 22; FR-H3 is selected from SEQ ID NOs: 23, 26, 28 and 29; FR-H4 is SEQ ID NO: 24; And the light chain variable region comprises: (i) a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 4-6; and (ii) a set of four light chain (LC) framework (FR) sequences, wherein: FR-L1 is selected from SEQ ID NOs: 30, 34, 37 and 39; FR-L2 is selected from SEQ ID NOs: 31 and 35; FR-L3 is selected from SEQ ID NOs: 32, 36, 38 and 40; and FR-L4 is SEQ ID NO:

33.

2. The humanized antibody of claim 1, wherein the heavy chain comprises a variable region sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16; and wherein the light chain comprises a variable region sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO:

20.

3. The humanized antibody according to any one of claims 1 or 2, wherein the humanized antibody or fragment thereof comprises a heavy chain and a light chain selected from the group consisting of: (i) the heavy chain comprises a variable region having the amino acid sequence of SEQ ID NO: 11, and the light chain comprises a variable region having the amino acid sequence of SEQ ID NO: 12; (ii) the heavy chain comprises a variable region having the amino acid sequence of SEQ ID NO: 15, and the light chain comprises a variable region having the amino acid sequence of SEQ ID NO: 18; (iii) the heavy chain comprises a variable region having the amino acid sequence of SEQ ID NO: 13, and the light chain comprises a variable region having the amino acid sequence of SEQ ID NO: 17; (iv) the heavy chain comprises a variable region having the amino acid sequence of SEQ ID NO: 14, and the light chain comprises a variable region having the amino acid sequence of SEQ ID NO: 18; (v) the heavy chain comprises a variable region having the amino acid sequence of SEQ ID NO: 15, and the light chain comprises a variable region having the amino acid sequence of SEQ ID NO: 19; and (vi) the heavy chain comprises a variable region having the amino acid sequence of SEQ ID NO: 16, and the light chain comprises a variable region having the amino acid sequence of SEQ ID NO:

20.

4. The humanized antibody of any of the preceding claims, wherein 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 SEQ ID NO: 11, and the light chain comprises a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO:

12.

5. The humanized antibody according to claim 4, wherein 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: 11, and the light chain comprises a variable region having the amino acid sequence of SEQ ID NO:

12.

6. The humanized antibody according to any one of the preceding claims, wherein the humanized monoclonal antibody has a heavy chain constant region selected from IgG1, IgG4 and IgG2.

7. The humanized antibody according to any one of the preceding claims, wherein the humanized antibody has a mutated Fc domain that prevents FcγR-mediated internalization. The humanized antibody according to claim 7 , wherein the Fc domain is ineffective for binding to Fcγ receptors.

9. A conjugate comprising the antibody or fragment thereof according to any one of the preceding claims.

10. The conjugate of claim 9, wherein the humanized antibody or fragment thereof is attached to a cytotoxic moiety, a radioactive moiety, an affinity moiety or a labeling moiety.

11. The conjugate of claim 9, wherein the humanized antibody or fragment thereof is attached to a toxin (payload).

12. The conjugate of claim 11, wherein the toxin is selected from the group consisting of microtubule inhibitors, DNA synthesis inhibitors, topoisomerase inhibitors and RNA polymerase inhibitors.

13. The conjugate according to claim 12, wherein the microtubule inhibitor is auristatin or a derivative thereof. The conjugate according to claim 13 , wherein the auristatin derivative is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

15. The conjugate according to claim 12, wherein the toxin is a maytansine derivative.

16. The conjugate according to claim 15, wherein the maytansine derivative is DM4 or DM1.

17. The conjugate according to claim 12, wherein the toxin is a DNA topoisomerase I (TOP1) inhibitor.

18. The conjugate according to claim 17, wherein the DNA topoisomerase I (TOP1) inhibitor is DXd.

19. The conjugate according to claim 17, wherein the DNA topoisomerase I (TOP1) inhibitor is SN-38.

20. The conjugate of claim 11, wherein the antibody and the toxin are connected via a linker.

21. The conjugate of claim 20, wherein the linker is cleavable.

22. A polynucleotide sequence encoding the amino acid sequence of the heavy chain variable region and the light chain variable region of the humanized antibody or fragment thereof according to any one of claims 1 to 8.

23. A pharmaceutical composition comprising the humanized antibody or antigen-binding fragment according to any one of claims 1-8 and / or the conjugate according to any one of claims 9-20, and a pharmaceutically acceptable excipient, carrier or diluent.

24. The pharmaceutical composition according to claim 23, for use in treating cancer.

25. A method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of at least one humanized antibody or a fragment thereof according to any one of claims 1 to 8 or a conjugate according to any one of claims 9 to 21.

26. The method of claim 25, wherein the cancer is a solid cancer.

27. The method of claim 25, wherein the cancer is selected from the group consisting of prostate cancer, colorectal 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.

28. The method of claim 25, wherein the cancer is colorectal adenocarcinoma or lung adenocarcinoma.

29. The method of claim 25, wherein the cancer is a hematological cancer.

30. The method of any one of claims 25-29, wherein the method further comprises administering or performing at least one additional anti-cancer therapy.

31. A method for diagnosing or prognosing cancer in a subject, the method comprising determining the expression level of connexin 2 in a biological sample of the subject using at least one humanized antibody or fragment according to any one of claims 1-8.

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