Antibodies specific to human connexin 4

Abstract

The present invention provides monoclonal antibodies that recognize human connexin 4 (Nectin4) with high affinity and specificity and inhibit its binding to T cell immune receptors (TIGIT) having Ig and ITIM domains. The invention also provides pharmaceutical compositions comprising the antibodies and methods of their use in cancer immunotherapy and diagnosis.

Description

Technical Field

[0001] The present invention belongs to the field of immunotherapy and relates to antibodies and fragments thereof that bind to the human protein Nectin 4, polynucleotide sequences encoding these antibodies, and cells that produce these antibodies. The present invention also relates to therapeutic and diagnostic compositions comprising these antibodies, and methods for treating and diagnosing diseases, particularly cancer, using these antibodies. Background Art

[0002] Immunotherapy is one of the most promising advances made in cancer treatment over the past decade. Cancer immunotherapy is used to generate and enhance anti-tumor immune responses, for example, by treatment with antibodies specific for antigens on tumor cells, treatment using fusions of antigen-presenting cells with tumor cells, or by activation of anti-tumor T cells. The ability to recruit immune cells (e.g., T cells) against tumor cells in patients provides a treatment modality against types of cancer and metastases that have been considered incurable so far.

[0003] The T cell-mediated immune response consists of multiple sequential steps that are regulated by the balance between co-stimulatory and co-inhibitory signals that control the intensity of the immune response. Inhibitory signals known as immune checkpoints are critical for maintaining self-tolerance and limiting immune-mediated collateral tissue damage. These signals change when infection or immune challenge is cleared, worsens, or persists, and these changes influence and reshape the immune response.

[0004] The expression of immune checkpoint proteins can be regulated by tumors. For example, programmed death ligand-1 (PD-L1) is upregulated on the surface of cancer cells, which inhibits T cells that might otherwise attack these tumor cells by binding to PD-1, allowing them to avoid the host immune system. Therefore, immune checkpoints represent an important barrier to activating functional cellular immunity against cancer. Therefore, antagonistic antibodies specifically targeting inhibitory ligands on immune cells are considered to be viable anticancer agents, and they are being used in cancer treatment (e.g., nivolumab and pembrolizumab). Another example of an immune checkpoint molecule is "T cell immune receptor with Ig and ITIM domains" (TIGIT). TIGIT is a co-inhibitory molecule expressed on a variety of different immune cells, including T cells and natural killer cells (NK cells). TIGIT binds to the poliovirus receptor (PVR, CD155) with high affinity. Monoclonal antibodies (mAbs) specific for TIGIT are disclosed in, for example, WO 2016 / 028656 and WO 2017 / 037707.

[0005] The poliovirus receptor (PVR) is a transmembrane glycoprotein involved in mediating cell adhesion to extracellular matrix molecules. PVR is a known tumor antigen and a target for therapeutic intervention. Blocking PVR on tumor cells is expected to reduce tumor cell viability. PVR also plays a key role in angiogenesis and tumor metastasis. Several patent applications, including U.S. Patent Application No. 20070041985, U.S. Patent Application No. 20090215175, and WO 2017149538, disclose molecules and antibodies that specifically bind to PVR and their use in combating cancer.

[0006] Connexin cell adhesion molecule 4 (Connexin 4), also known as poliovirus receptor-related protein 4 (PVRL4), is a type I transmembrane protein and a member of the connexin family of related immunoglobulin-like adhesion molecules. Connexin 4 is a tumor-associated marker for many tumors, including lung, breast, colon, and ovarian cancers.

[0007] Chailta-Eid et al., 2016 (Cancer Res. 2016; 76: 3003-13) disclosed that anti-connexin 4 antibody (Enfortumab)-drug conjugates are highly effective therapeutic agents in multiple preclinical cancer models. The antibody coupled to the tubulin inhibitor restatin binds to human as well as rat and monkey connexin 4 and inhibits the growth of several cell lines and xenografts expressing connexin 4.

[0008] Despite the success in cancer immunotherapy, there remains an unmet need for other methods of enhancing cells of the immune system to attack tumor cells and for more effective and specific agents and drug combinations. One such approach involves inhibiting the binding of connexin 4 to TIGIT by specific monoclonal antibodies. Summary of the invention

[0009] The present invention provides antibodies and fragments thereof that bind to the human protein connexin 4, polynucleotide sequences encoding these antibodies, and cells that produce these antibodies. The present invention is based in part on the discovery that connexin 4, previously known as a receptor for measles virus and a tumor antigen, is a ligand for the immunosuppressive molecule TIGIT, and thus it was first proposed as a target for inhibiting the inhibitory effect of TIGIT on anti-cancer immunity. In certain embodiments, the present invention also provides a chimeric antigen receptor (CAR) comprising a binding site for connexin 4.

[0010] The present invention provides highly effective monoclonal antibodies (mAbs) specific for human connexin 4, which not only block the interaction between connexin 4 and the inhibitory receptor TIGIT, but also have a direct effect on target cells expressing this receptor. These antibodies with binding constants to connexin 4 in the subnanomolar range reverse the TIGIT inhibition of the immune system without being coupled to any toxin or anti-tumor agent, and directly enhance the elimination of tumor cells. Therefore, the antibodies of the present invention can be used, for example, to inhibit the interaction between connexin 4 on target cells and TIGIT on immune cells in cancer immunotherapy. In addition, some complete antibodies described herein induce ADCC (antibody-dependent cell-mediated cytotoxicity) activity. Connexin 4 is specifically overexpressed on tumor cells. The ADCC activity induction of the antibodies of the present invention, together with their high affinity for connexin 4 and their ADCC activity, make them ideal candidates for immunotherapy.

[0011] The present invention provides antibodies and fragments thereof that recognize connexin 4, prevent it from binding to TIGIT protein and inhibit the inhibitory activity of lymphocytes such as natural killer (NK) cells and T-cells. The anti-connexin 4 antibodies disclosed herein are capable of binding to connexin 4 present on target cells such as cancer cells. The antibodies and fragments of the present invention are characterized by having a unique set of complementary determining region (CDR) sequences, high affinity and high specificity for human connexin 4, and can be used as an independent therapy and in combination with other anticancer agents for cancer immunotherapy to combat tumor immune escape. The antibodies can also be used to prevent viral infections, in particular to prevent measles infections.

[0012] It is now disclosed that the high affinity anti-Connexin4 antibodies disclosed herein block TIGIT-Connexin4 interaction and restore T and NK cell activity.

[0013] Certain monoclonal antibodies of the present invention are also able to block the interaction between connexin 4 and connexin 1, indicating their ability to interfere with the invasiveness of tumors expressing connexin 4. In addition, anti-connexin 4 mAbs are able to induce NK cell activation in most target cells. Advantageously, the anti-connexin 4 mAbs according to the present invention have a direct effect on target cancer cells, inducing their killing without the need for NK cells and / or toxins. It has also been disclosed that the anti-connexin 4 antibodies of the present invention have no blocking effect on the signaling of costimulatory receptors such as DNAM1, and therefore it is expected that they have no deleterious effects on other immune-inducing signals.

[0014] Interestingly, despite the high sequence similarity between human and rodent connexin4 sequences, certain antibodies of the invention are highly specific for human connexin4 and do not bind to rodent connexin4.

[0015] Certain anti-connexin 4 mAbs described herein can reduce tumor cell survival in an immune-independent manner by blocking connexin 4 on tumor cells. In certain embodiments, the connexin 4 antibodies described herein inhibit tumor cell proliferation by interfering with the binding of connexin 1 on tumor cells in an immune-independent manner without binding to any toxic molecules.

[0016] According to one aspect, the present invention provides an isolated monoclonal antibody (mAb) or an antibody fragment thereof, which comprises at least an antigen binding portion that specifically binds to human connexin 4 and inhibits its binding to TIGIT.

[0017] The present invention also provides a mAb or an antibody fragment thereof capable of inhibiting the binding of human connexin 4 to human TIGIT, which is used together with T-lymphocytes and / or natural killer (NK) cells for the treatment of cancer.

[0018] According to certain embodiments, the mAb is not conjugated to any toxin or anti-tumor agent.

[0019] According to certain embodiments, the isolated antibody or antibody fragment comprises a group of 6 complementary determining region (CDR) sequences selected from the following:

[0020] i. three CDRs of the heavy chain (HC) variable region comprising SEQ ID NO: 22 and three CDRs of the light chain (LC) variable region comprising SEQ ID NO: 24, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence;

[0021] ii. three CDRs of the HC variable region comprising SEQ ID NO: 2 and three CDRs of the LC variable region comprising SEQ ID NO: 4, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence; and

[0022] iii. The three CDRs of the HC variable region comprising SEQ ID NO: 6 and the three CDRs of the LC variable region comprising SEQ ID NO: 8, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence.

[0023] According to certain embodiments, the isolated antibody or antibody fragment comprises a group of 6 CDR sequences selected from the following:

[0024] i. three CDRs of the HC variable region comprising SEQ ID NO: 39 and three CDRs of the LC variable region comprising SEQ ID NO: 40, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence;

[0025] ii. three CDRs of the HC variable region comprising SEQ ID NO: 35 and three CDRs of the LC variable region comprising SEQ ID NO: 36, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence; and

[0026] iii. The three CDRs of the HC variable region comprising SEQ ID NO: 37 and the three CDRs of the LC variable region comprising SEQ ID NO: 38, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence.

[0027] There are several methods known in the art for determining the CDR sequences of a given antibody molecule, but there is no standard clear method. The determination of the CDR sequences from the antibody heavy chain and light chain variable regions can be made according to any method known in the art, including but not limited to the methods known as KABAT, Chothia and IMGT. The selected CDR group can include sequences identified by more than one method, i.e., for example, some CDR sequences can be determined using KABAT, and some sequences are determined using IMGT. According to some embodiments, the CDR sequences of the mAb variable regions are determined using the IMGT method.

[0028] According to certain embodiments, the isolated monoclonal antibody or fragment comprises the CDR sequences of the monoclonal antibody known as hNec4.11 (or connexin 4.11 or clone 11), namely, the three CDR sequences in the heavy chain variable region shown in SEQ ID NO: 39 and the three CDR sequences in the light chain variable region shown in SEQ ID NO: 40.

[0029] According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR1 comprising the sequence SYYIH (SEQ ID NO: 25). According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR2 comprising the sequence WIYPGNVNTKYNERFKG (SEQ ID NO: 26). According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR3 comprising the sequence SNPYVMDY (SEQ ID NO: 27).

[0030] According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) a HC CDR1 comprising the sequence SYYIH (SEQ ID NO: 25); (ii) a HC CDR2 comprising the sequence WIYPGNVNTKYNERFKG (SEQ ID NO: 26); and (iii) a HC CDR3 comprising the sequence SNPYVMDY (SEQ ID NO: 27).

[0031] According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR1 comprising the sequence KASQSVNNDVA (SEQ ID NO: 28). According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR2 comprising the sequence YASNRFT (SEQ ID NO: 29). According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR3 comprising the sequence QQAYRSPYT (SEQ ID NO: 30).

[0032] According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) a LC CDR1 comprising the sequence KASQSVNNDVA (SEQ ID NO: 28); (ii) a LC CDR2 comprising the sequence YASNRFT (SEQ ID NO: 29); and (iii) a HC CDR3 comprising the sequence QQAYRSPYT (SEQ ID NO: 30).

[0033] According to certain specific embodiments, the isolated monoclonal antibody or fragment comprises a heavy chain CDR1 sequence comprising the sequence SYYIH (SEQ ID NO: 25), a heavy chain CDR2 comprising the sequence WIYPGNVNTKYNERFKG (SEQ ID NO: 26), a heavy chain CDR3 comprising the sequence SNPYVMDY (SEQ ID NO: 27), a light chain CDR1 comprising the sequence KASQSVNNDVA (SEQ ID NO: 28), a light chain CDR2 comprising the sequence YASNRFT (SEQ ID NO: 29) and a light chain CDR3 comprising the sequence QQAYRSPYT (SEQ ID NO: 30), or an analog thereof comprising no more than 5% amino acid substitutions, deletions and / or insertions in the hypervariable region (HVR) sequence.

[0034] According to certain specific embodiments, the isolated monoclonal antibody or fragment comprises a set of 6 CDR sequences consisting of:

[0035] i. a heavy chain CDR1 having the sequence shown in SEQ ID NO: 25;

[0036] ii. a heavy chain CDR2 having the sequence shown in SEQ ID NO: 26;

[0037] iii. a heavy chain CDR3 having the sequence shown in SEQ ID NO: 27;

[0038] iv. a light chain CDR1 having the sequence shown in SEQ ID NO: 28;

[0039] v. a light chain CDR2 having the sequence shown in SEQ ID NO: 29; and

[0040] vi. A light chain CDR3 having the sequence shown in SEQ ID NO:30.

[0041] According to certain embodiments, the isolated monoclonal antibody or fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO: 39 or an analog or derivative thereof having at least 90% sequence identity to the heavy chain variable region sequence.

[0042] According to certain embodiments, the isolated monoclonal antibody or fragment thereof comprises a light chain variable region set forth in SEQ ID NO: 40 or an analog or derivative thereof having at least 90% sequence identity to the light chain variable region sequence.

[0043] According to a specific embodiment, the isolated monoclonal antibody or fragment thereof comprises a heavy chain variable region having the sequence shown in SEQ ID NO: 39 and a light chain variable region having the sequence shown in SEQ ID NO: 40, or an analog thereof having at least 90% sequence identity with the light chain and / or heavy chain sequence.

[0044] The present invention also encompasses antibodies or antibody fragments that are capable of binding with high affinity to the epitope within human connexin 4 to which the monoclonal antibody hNec4.11 binds.

[0045] According to certain embodiments, the isolated monoclonal antibody or fragment comprises the CDR sequences of the monoclonal antibody known as hNec4.01 (or connexin 4.01), namely the three CDR sequences contained in the heavy chain variable region shown in SEQ ID NO: 35 and the three CDR sequences contained in the light chain variable region shown in SEQ ID NO: 36.

[0046] According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR1 comprising the sequence AYNIH (SEQ ID NO: 9). According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR2 comprising the sequence YIYPNNGGSGYNQKFMN (SEQ ID NO: 10). According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR3 comprising the sequence FDYDEAWFIY (SEQ ID NO: 11).

[0047] According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) a HC CDR1 comprising the sequence AYNIH (SEQ ID NO: 9); (ii) a HC CDR2 comprising the sequence YIYPNNGGSGYNQKFMN (SEQ ID NO: 10); and (iii) a HC CDR3 comprising the sequence FDYDEAWFIY (SEQ ID NO: 11).

[0048] According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR1 comprising the sequence SASSSVSYMH (SEQ ID NO: 12). According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR2 comprising the sequence DTSKLAS (SEQ ID NO: 13). According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR3 comprising the sequence FQGSGSPYT (SEQ ID NO: 14).

[0049] According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) a LC CDR1 comprising the sequence SASSVSYMH (SEQ ID NO: 12); (ii) a LC CDR2 comprising the sequence DTSKLAS (SEQ ID NO: 13); and (iii) a HC CDR3 comprising the sequence FQGSGSPYT (SEQ ID NO: 14).

[0050] According to certain specific embodiments, the isolated monoclonal antibody or fragment comprises a heavy chain CDR1 sequence comprising the sequence AYNIH (SEQ ID NO: 9), a heavy chain CDR2 comprising the sequence YIYPNNGGSGYNQKFMN (SEQ ID NO: 10), a heavy chain CDR3 comprising the sequence FDYDEAWFIY (SEQ ID NO: 11), a light chain CDR1 comprising the sequence SASSVSYMH (SEQ ID NO: 12), a light chain CDR2 comprising the sequence DTSKLAS (SEQ ID NO: 13) and a light chain CDR3 comprising the sequence FQGSGSPYT (SEQ ID NO: 14), or an analog thereof comprising no more than 5% amino acid substitutions, deletions and / or insertions in the hypervariable region (HVR) sequence.

[0051] According to certain specific embodiments, the isolated monoclonal antibody or fragment comprises a set of 6 CDR sequences consisting of:

[0052] i. a heavy chain CDR1 having the sequence shown in SEQ ID NO: 9;

[0053] ii. a heavy chain CDR2 having the sequence shown in SEQ ID NO: 10;

[0054] iii. a heavy chain CDR3 having the sequence shown in SEQ ID NO: 11;

[0055] iv. a light chain CDR1 having the sequence shown in SEQ ID NO: 12;

[0056] v. a light chain CDR2 having the sequence shown in SEQ ID NO: 13; and

[0057] vi. A light chain CDR3 having the sequence shown in SEQ ID NO:14.

[0058] According to certain embodiments, the isolated monoclonal antibody or fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO: 35 or an analog or derivative thereof having at least 90% sequence identity to the heavy chain variable region sequence.

[0059] According to certain embodiments, the isolated monoclonal antibody or fragment thereof comprises a light chain variable region set forth in SEQ ID NO: 36 or an analog or derivative thereof having at least 90% sequence identity to the light chain variable region sequence.

[0060] According to a specific embodiment, the isolated monoclonal antibody or fragment thereof comprises a heavy chain variable region having the sequence shown in SEQ ID NO: 35 and a light chain variable region having the sequence shown in SEQ ID NO: 36, or an analog thereof having at least 90% sequence identity with the light chain and / or heavy chain sequence.

[0061] The present invention also encompasses antibodies or antibody fragments that are capable of binding with high affinity to the epitope within human connexin 4 to which the monoclonal antibody hNec4.01 binds.

[0062] According to certain embodiments, the isolated monoclonal antibody or fragment comprises the CDR sequences of the monoclonal antibody known as hNec4.05 (or connexin 4.05), namely, the three CDR sequences contained in the heavy chain variable region shown in SEQ ID NO: 37 and the three CDR sequences contained in the light chain variable region shown in SEQ ID NO: 38.

[0063] According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR1 comprising the sequence TYYIH (SEQ ID NO: 15). According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR2 comprising the sequence WIYPGNVNTKNNEKFKV (SEQ ID NO: 16). According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR3 comprising the sequence SNPYVMDY (SEQ ID NO: 17).

[0064] According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) a HC CDR1 comprising the sequence TYYIH (SEQ ID NO: 15); (ii) a HC CDR2 comprising the sequence WIYPGNVNTKNNEKFKV (SEQ ID NO: 16); and (iii) a HC CDR3 comprising the sequence SNPYVMDY (SEQ ID NO: 17).

[0065] According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR1 comprising the sequence KASQSVSNDVA (SEQ ID NO: 18). According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR2 comprising the sequence YASNRYT (SEQ ID NO: 19). According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR3 comprising the sequence QQDYSSPYT (SEQ ID NO: 20).

[0066] According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) a LC CDR1 comprising the sequence KASQSVSNDVA (SEQ ID NO: 18); (ii) a LC CDR2 comprising the sequence YASNRYT (SEQ ID NO: 19); and (iii) a HC CDR3 comprising the sequence QQDYSSPYT (SEQ ID NO: 20).

[0067] According to certain specific embodiments, the isolated monoclonal antibody or fragment comprises a heavy chain CDR1 sequence comprising the sequence TYYIH (SEQ ID NO: 15), a heavy chain CDR2 comprising the sequence WIYPGNVNTKNNEKFKV (SEQ ID NO: 16), a heavy chain CDR3 comprising the sequence NPYVMDY (SEQ ID NO: 17), a light chain CDR1 comprising the sequence KASQSVSNDVA (SEQ ID NO: 18), a light chain CDR2 comprising the sequence YASNRYT (SEQ ID NO: 19) and a light chain CDR3 comprising the sequence QQDYSSPYT (SEQ ID NO: 20), or an analog thereof comprising no more than 5% amino acid substitutions, deletions and / or insertions in the hypervariable region (HVR) sequence.

[0068] According to certain specific embodiments, the isolated monoclonal antibody or fragment comprises a set of 6 CDR sequences consisting of:

[0069] i. a heavy chain CDR1 having the sequence shown in SEQ ID NO: 15;

[0070] ii. a heavy chain CDR2 having the sequence shown in SEQ ID NO: 16;

[0071] iii. a heavy chain CDR3 having the sequence shown in SEQ ID NO: 17;

[0072] iv. a light chain CDR1 having the sequence shown in SEQ ID NO: 18;

[0073] v. a light chain CDR2 having the sequence shown in SEQ ID NO: 19; and

[0074] vi. A light chain CDR3 having the sequence shown in SEQ ID NO:20.

[0075] According to certain embodiments, the isolated monoclonal antibody or fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO: 37 or an analog or derivative thereof having at least 90% sequence identity to the heavy chain variable region sequence.

[0076] According to certain embodiments, the isolated monoclonal antibody or fragment thereof comprises a light chain variable region set forth in SEQ ID NO: 38 or an analog or derivative thereof having at least 90% sequence identity to the light chain variable region sequence.

[0077] According to a specific embodiment, the isolated monoclonal antibody or fragment thereof comprises a heavy chain variable region having the sequence shown in SEQ ID NO: 37 and a light chain variable region having the sequence shown in SEQ ID NO: 38, or an analog thereof having at least 90% sequence identity with the light chain and / or heavy chain sequence.

[0078] The present invention also encompasses antibodies or antibody fragments that are capable of binding with high affinity to the epitope within human connexin 4 to which the monoclonal antibody hNec4.05 binds.

[0079] According to certain embodiments, the isolated antibody or fragment thereof is present in an amount of at least 10 -8 According to other embodiments, the antibody or antibody fragment recognizes human connexin 4 with an affinity of 10 -8 M, 5x10 -9 M, 10 -9 M, 5x10 -10 M, 10 -10 M, 5x10 -11 M or even higher affinity to human connexin 4. According to certain embodiments, the antibody or antibody fragment is 10 -9 M to 10 -10 M binds with affinity to human connexin 4. Each possibility represents a separate embodiment of the present invention.

[0080] Analogs and derivatives of the above-described isolated mAbs and fragments are also within the scope of the present invention.

[0081] According to certain embodiments, the antibody or antibody fragment analog has at least 90% sequence identity with the hypervariable region of the reference antibody sequence.

[0082] According to certain embodiments, the isolated antibody or fragment thereof analog or derivative has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with the variable region of the reference antibody sequence. Each possibility represents an independent embodiment of the present invention.

[0083] According to certain embodiments, the antibody or antibody fragment according to the present invention comprises the heavy chain variable region shown in SEQ ID NO: 39, SEQ ID NO: 35 or SEQ ID NO: 37 or an analog having at least 95% sequence similarity thereto.

[0084] According to certain embodiments, the antibody or antibody fragment comprises a light chain variable region set forth in SEQ ID NO: 40, SEQ ID NO: 36 or SEQ ID NO: 38, or an analog having at least 95% sequence similarity thereto.

[0085] According to certain embodiments, the antibody or antibody fragment comprises a heavy chain and a light chain, wherein: (i) the heavy chain comprises SEQ ID NO: 39 and the light chain comprises SEQ ID NO: 40; (ii) the heavy chain comprises SEQ ID NO: 35 and the light chain comprises SEQ ID NO: 36; or (iii) the heavy chain comprises SEQ ID NO: 37 and the light chain comprises SEQ ID NO: 38. Also included are analogs of the antibodies or fragments having at least 95% sequence similarity to the heavy or light chain.

[0086] According to certain embodiments, the analog has at least 96, 97, 98 or 99% sequence identity with the above-mentioned antibody light chain or heavy chain variable region. According to certain embodiments, the analog comprises no more than one amino acid substitution, deletion or addition to one or more CDR sequences of the hypervariable region, i.e., any of the CDR sequences shown in SEQ ID NO: 25, 26, 27, 28, 29, 30, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20. Each possibility represents an independent embodiment of the present invention. According to certain embodiments, the amino acid substitution is a conservative substitution.

[0087] According to certain embodiments, the antibody or antibody fragment comprises a hypervariable region (HVR) having a light chain and a heavy chain region as defined above, wherein 1, 2, 3, 4 or 5 amino acids are substituted, deleted and / or added. Each possibility represents an independent embodiment of the present invention.

[0088] According to certain embodiments, the antibody or antibody fragment comprises an HVR having a light chain and a heavy chain region as defined above, wherein one amino acid is replaced. According to a specific embodiment, the antibody or antibody fragment comprises a CDR as defined above, wherein one amino acid is replaced.

[0089] According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises a CDR group selected from the following:

[0090] i. A set of 6 CDRs, wherein: HC CDR1 is SYYIH (SEQ ID NO: 25); HC CDR2 is WIYPGNVNTKYNERFKG (SEQ ID NO: 26); HC CDR3 is SNPYVMDY (SEQ ID NO: 27); LC CDR1 is KASQSVNNDVA (SEQ ID NO: 28); LC CDR2 is YASNRFT (SEQ ID NO: 29); and LC CDR3 is QQAYRSPYT (SEQ ID NO: 30);

[0091] ii. a group of 6 CDRs, wherein: HC CDR1 is AYNIH (SEQ ID NO: 9), HC CDR2 is YIYPNNGGSGYNQKFMN (SEQ ID NO: 10), HC CDR3 is FDYDEAWFIY (SEQ ID NO: 11), LC CDR1 is SASSVSYMH (SEQ ID NO: 12), LC CDR2 is DTSKLAS (SEQ ID NO: 13), and LC CDR3 is FQGSGSPYT (SEQ ID NO: 14); and

[0092] iii. A group of 6 CDRs, wherein: HC CDR1 is TYYIH (SEQ ID NO: 15), HC CDR2 is WIYPGNVNTKNNEKFKV (SEQ ID NO: 16), HC CDR3 is SNPYVMDY (SEQ ID NO: 17), LC CDR1 is KASQSVSNDVA (SEQ ID NO: 18), LC CDR2 is YASNRYT (SEQ ID NO: 19), and LC CDR3 is QQDYSSPYT (SEQ ID NO: 20).

[0093] The present invention also provides a monoclonal antibody and a binding fragment thereof, which comprises a heavy chain and a light chain, wherein the chain comprises a group of heavy chain variable region sequences and light chain variable region sequences, the group being selected from:

[0094] i. A group comprising SEQ ID NOs: 39 and 40;

[0095] ii. a group comprising SEQ ID NOs: 35 and 36; and

[0096] iii. a group comprising SEQ ID NOs: 37 and 38.

[0097] According to certain embodiments, the antibody or antibody fragment is capable of inhibiting the binding of human connexin 4 to TIGIT expressed on T cells or NK cells.

[0098] According to certain embodiments, the mAb is selected from a chimeric antibody and an antibody fragment comprising at least the antigen binding portion of an antibody. According to certain embodiments, the antibody is a chimeric antibody. According to other embodiments, the chimeric antibody comprises a human constant region. According to certain embodiments, the antibody fragment is selected from the group consisting of Fab, Fab', F(ab'), 2 , Fd, Fd', Fv, dAb, isolated CDR regions, single chain variable region (scFV), single chain antibody (scab), "diabody" and "linear antibody". Each possibility represents a separate embodiment of the present invention.

[0099] According to the present invention, there is also provided a single chain variable region (scFV) comprising the heavy chain and light chain variable regions of the antibodies described herein. According to certain embodiments, there is a hinge region between the variable regions.

[0100] According to certain embodiments, the scFV sequence is set forth in SEQ ID NO: 32, SEQ ID NO: 34, or an analog thereof having at least 90% sequence similarity thereto.

[0101] According to certain embodiments, the antibody comprises a constant region sequence selected from the group consisting of mouse IgG1, mouse IgG2a, mouse IgG2b, mouse IgG3, human IgG1, human IgG2, human IgG3 and human IgG4. Each possibility represents a separate embodiment of the invention.

[0102] According to certain specific embodiments, the monoclonal antibody is a chimeric monoclonal antibody.

[0103] According to certain embodiments, the chimeric antibody comprises a constant region derived from a human.

[0104] According to certain embodiments, the human constant region of the chimeric antibody is selected from human IgG1, human IgG2, human IgG3 and human IgG4.

[0105] According to certain embodiments, the antibody is a human IgG1. According to certain embodiments, there is provided a human IgG comprising the variable region of an antibody described herein.

[0106] According to certain embodiments, a conjugate comprising the antibody or fragment thereof as described above is provided.

[0107] According to certain embodiments, the conjugate comprises a carrier protein.

[0108] According to another aspect of the present invention, a chimeric antigen receptor (CAR) is provided, which comprises an extracellular portion (binding domain) capable of binding to connexin 4.

[0109] According to certain embodiments, the CAR comprises an extracellular portion comprising any of the provided antibodies or fragments thereof described herein.

[0110] According to certain embodiments, the CAR comprises a connexin 4 binding site comprising a CDR group selected from the following:

[0111] i. A set of 6 CDRs, wherein: HC CDR1 is SYYIH (SEQ ID NO: 25); HC CDR2 is WIYPGNVNTKYNERFKG (SEQ ID NO: 26); HC CDR3 is SNPYVMDY (SEQ ID NO: 27); LC CDR1 is KASQSVNNDVA (SEQ ID NO: 28); LC CDR2 is YASNRFT (SEQ ID NO: 29); and LC CDR3 is QQAYRSPYT (SEQ ID NO: 30);

[0112] ii. a group of 6 CDRs, wherein: HC CDR1 is AYNIH (SEQ ID NO: 9), HC CDR2 is YIYPNNGGSGYNQKFMN (SEQ ID NO: 10), HC CDR3 is FDYDEAWFIY (SEQ ID NO: 11), LC CDR1 is SASSVSYMH (SEQ ID NO: 12), LC CDR2 is DTSKLAS (SEQ ID NO: 13), and LC CDR3 is FQGSGSPYT (SEQ ID NO: 14); and

[0113] iii. A group of 6 CDRs, wherein: HC CDR1 is TYYIH (SEQ ID NO: 15), HC CDR2 is WIYPGNVNTKNNEKFKV (SEQ ID NO: 16), HC CDR3 is SNPYVMDY (SEQ ID NO: 17), LC CDR1 is KASQSVSNDVA (SEQ ID NO: 18), LC CDR2 is YASNRYT (SEQ ID NO: 19), and LC CDR3 is QQDYSSPYT (SEQ ID NO: 20).

[0114] According to certain embodiments, the CAR comprises an antigen binding domain comprising SEQ ID NO: 32 or 34, a transmembrane domain, and an intracellular T cell signaling domain.

[0115] According to one aspect, the present invention provides an isolated nucleic acid molecule encoding a CAR comprising an antibody or antibody fragment, wherein the antibody or antibody fragment comprises a connexin 4 binding domain, wherein the binding domain comprises a CDR group selected from the following:

[0116] i. A set of 6 CDRs, wherein: HC CDR1 is SYYIH (SEQ ID NO: 25); HC CDR2 is WIYPGNVNTKYNERFKG (SEQ ID NO: 26); HC CDR3 is SNPYVMDY (SEQ ID NO: 27); LC CDR1 is KASQSVNNDVA (SEQ ID NO: 28); LC CDR2 is YASNRFT (SEQ ID NO: 29); and LC CDR3 is QQAYRSPYT (SEQ ID NO: 30);

[0117] ii. a group of 6 CDRs, wherein: HC CDR1 is AYNIH (SEQ ID NO: 9), HC CDR2 is YIYPNNGGSGYNQKFMN (SEQ ID NO: 10), HC CDR3 is FDYDEAWFIY (SEQ ID NO: 11), LC CDR1 is SASSVSYMH (SEQ ID NO: 12), LC CDR2 is DTSKLAS (SEQ ID NO: 13), and LC CDR3 is FQGSGSPYT (SEQ ID NO: 14); and

[0118] iii. A group of 6 CDRs, wherein: HC CDR1 is TYYIH (SEQ ID NO: 15), HC CDR2 is WIYPGNVNTKNNEKFKV (SEQ ID NO: 16), HC CDR3 is SNPYVMDY (SEQ ID NO: 17), LC CDR1 is KASQSVSNDVA (SEQ ID NO: 18), LC CDR2 is YASNRYT (SEQ ID NO: 19), and LC CDR3 is QQDYSSPYT (SEQ ID NO: 20).

[0119] According to certain embodiments, a vector is provided, comprising the polynucleotide sequence shown in SEQ ID NO: 31 or SEQ ID NO: 33 or an analog having at least 95% similarity to the sequence.

[0120] According to certain embodiments, a T cell engineered to express a CAR described herein is provided.

[0121] According to another embodiment, a NK cell engineered to express the CAR described herein is provided.

[0122] According to another aspect of the present invention, provided are polynucleotide sequences encoding monoclonal antibodies having high affinity and specificity for human connexin 4, as well as vectors and host cells carrying these polynucleotide sequences.

[0123] According to certain embodiments, a polynucleotide sequence encoding the amino acid sequence of the heavy chain variable region and the light chain variable region is provided.

[0124] According to certain embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain capable of binding to an epitope within human connexin 4 that is bound by: (i) a monoclonal antibody having a heavy chain variable region of SEQ ID NO: 39 and a light chain variable region of SEQ ID NO: 40 (identified herein as hNec4.11); (ii) a monoclonal antibody having a heavy chain variable region of SEQ ID NO: 35 and a light chain variable region of SEQ ID NO: 36 (identified herein as hNec4.01); or (iii) a monoclonal antibody having a heavy chain variable region of SEQ ID NO: 37 and a light chain variable region of SEQ ID NO: 38 (identified herein as hNec4.05).

[0125] According to certain embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain comprising the sequence set forth in the sequence selected from the group consisting of: SEQ ID NO: 39 and SEQ ID NO: 40; SEQ ID NO: 35 and SEQ ID NO: 36; or SEQ ID NO: 37 and SEQ ID NO: 38. Each possibility represents a separate embodiment of the invention.

[0126] According to other embodiments, the polynucleotide sequence according to the present invention encodes an antibody or an antibody fragment or chain, which comprises:

[0127] i. A set of 6 CDRs, wherein: HC CDR1 is SYYIH (SEQ ID NO: 25); HC CDR2 is WIYPGNVNTKYNERFKG (SEQ ID NO: 26); HC CDR3 is SNPYVMDY (SEQ ID NO: 27); LC CDR1 is KASQSVNNDVA (SEQ ID NO: 28); LC CDR2 is YASNRFT (SEQ ID NO: 29); and LC CDR3 is QQAYRSPYT (SEQ ID NO: 30);

[0128] ii. a group of 6 CDRs, wherein: HC CDR1 is AYNIH (SEQ ID NO: 9), HC CDR2 is YIYPNNGGSGYNQKFMN (SEQ ID NO: 10), HC CDR3 is FDYDEAWFIY (SEQ ID NO: 11), LC CDR1 is SASSVSYMH (SEQ ID NO: 12), LC CDR2 is DTSKLAS (SEQ ID NO: 13), and LC CDR3 is FQGSGSPYT (SEQ ID NO: 14); and

[0129] iii. A group of 6 CDRs, wherein: HC CDR1 is TYYIH (SEQ ID NO: 15), HC CDR2 is WIYPGNVNTKNNEKFKV (SEQ ID NO: 16), HC CDR3 is SNPYVMDY (SEQ ID NO: 17), LC CDR1 is KASQSVSNDVA (SEQ ID NO: 18), LC CDR2 is YASNRYT (SEQ ID NO: 19), and LC CDR3 is QQDYSSPYT (SEQ ID NO: 20).

[0130] Each possibility represents a separate embodiment of the invention.

[0131] According to certain embodiments, the polynucleotide sequence defined above encodes a molecule selected from the group consisting of an antibody, an antibody fragment comprising at least an antigen binding portion, and an antibody conjugate comprising said antibody or antibody fragment. Each possibility represents an independent embodiment of the present invention.

[0132] According to certain embodiments, the polynucleotide sequence encoding the heavy chain variable region of the monoclonal antibody comprises the sequence shown in SEQ ID NO: 21 or a variant thereof having at least 90% sequence identity.

[0133] According to certain embodiments, the polynucleotide sequence encoding the heavy chain variable region of the monoclonal antibody comprises the sequence shown in SEQ ID NO: 1 or a variant thereof having at least 90% sequence identity.

[0134] According to certain embodiments, the polynucleotide sequence encoding the heavy chain variable region of the monoclonal antibody comprises the sequence shown in SEQ ID NO: 5 or a variant thereof having at least 90% sequence identity.

[0135] According to certain embodiments, the polynucleotide sequence encoding the monoclonal antibody light chain variable region comprises the sequence shown in SEQ ID NO: 23 or a variant thereof having at least 90% sequence identity.

[0136] According to certain embodiments, the polynucleotide sequence encoding the monoclonal antibody light chain variable region comprises the sequence shown in SEQ ID NO: 3 or a variant thereof having at least 90% sequence identity.

[0137] According to certain embodiments, the polynucleotide sequence encoding the monoclonal antibody light chain variable region comprises the sequence shown in SEQ ID NO: 7 or a variant thereof having at least 90% sequence identity.

[0138] According to certain embodiments, the polynucleotide sequence encoding the heavy chain variable region of the monoclonal antibody comprises the sequence shown in SEQ ID NO: 45 or a variant thereof having at least 90% sequence identity.

[0139] According to certain embodiments, the polynucleotide sequence encoding the heavy chain variable region of the monoclonal antibody comprises the sequence shown in SEQ ID NO: 41 or a variant thereof having at least 90% sequence identity.

[0140] According to certain embodiments, the polynucleotide sequence encoding the heavy chain variable region of the monoclonal antibody comprises the sequence shown in SEQ ID NO: 43 or a variant thereof having at least 90% sequence identity.

[0141] According to certain embodiments, the polynucleotide sequence encoding the monoclonal antibody light chain variable region comprises the sequence shown in SEQ ID NO: 46 or a variant thereof having at least 90% sequence identity.

[0142] According to certain embodiments, the polynucleotide sequence encoding the monoclonal antibody light chain variable region comprises the sequence shown in SEQ ID NO: 42 or a variant thereof having at least 90% sequence identity.

[0143] According to certain embodiments, the polynucleotide sequence encoding the monoclonal antibody light chain variable region comprises the sequence shown in SEQ ID NO: 44 or a variant thereof having at least 90% sequence identity.

[0144] According to certain embodiments, the present invention provides a polypeptide comprising at least one sequence encoded by at least one polynucleotide sequence disclosed above.

[0145] In another aspect, the present invention provides a nucleic acid construct comprising a nucleic acid molecule encoding at least one antibody chain or fragment thereof according to the present invention. According to certain embodiments, the nucleic acid construct is a plasmid.

[0146] According to certain embodiments, the plasmid comprises at least one polynucleotide sequence set forth in a sequence selected from the group consisting of: SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44. Each possibility represents a separate embodiment of the invention.

[0147] In another aspect, the present invention provides a cell capable of producing an antibody or antibody fragment comprising the specific CDR sequences and / or specific heavy and light chain variable regions as defined above.

[0148] According to certain embodiments, a cell is provided, comprising at least one polynucleotide sequence as defined above.

[0149] According to certain embodiments, the cell is capable of producing a monoclonal antibody comprising the following set of CDRs:

[0150] i. A set of 6 CDRs, wherein: HC CDR1 is SYYIH (SEQ ID NO: 25); HC CDR2 is WIYPGNVNTKYNERFKG (SEQ ID NO: 26); HC CDR3 is SNPYVMDY (SEQ ID NO: 27); LC CDR1 is KASQSVNNDVA (SEQ ID NO: 28); LC CDR2 is YASNRFT (SEQ ID NO: 29); and LC CDR3 is QQAYRSPYT (SEQ ID NO: 30);

[0151] ii. a group of 6 CDRs, wherein: HC CDR1 is AYNIH (SEQ ID NO: 9), HC CDR2 is YIYPNNGGSGYNQKFMN (SEQ ID NO: 10), HC CDR3 is FDYDEAWFIY (SEQ ID NO: 11), LC CDR1 is SASSVSYMH (SEQ ID NO: 12), LC CDR2 is DTSKLAS (SEQ ID NO: 13), and LC CDR3 is FQGSGSPYT (SEQ ID NO: 14); and

[0152] iii. A group of 6 CDRs, wherein: HC CDR1 is TYYIH (SEQ ID NO: 15), HC CDR2 is WIYPGNVNTKNNEKFKV (SEQ ID NO: 16), HC CDR3 is SNPYVMDY (SEQ ID NO: 17), LC CDR1 is KASQSVSNDVA (SEQ ID NO: 18), LC CDR2 is YASNRYT (SEQ ID NO: 19), and LC CDR3 is QQDYSSPYT (SEQ ID NO: 20).

[0153] Each possibility represents a separate embodiment of the invention.

[0154] According to certain embodiments, the monoclonal antibody-producing cell is a hybridoma cell.

[0155] The antibodies or fragments thereof according to the present invention may be attached to a cytotoxic moiety, a radioactive moiety or a recognizable moiety.

[0156] According to another aspect, the present invention provides a pharmaceutical composition comprising as an active ingredient at least one antibody, antibody fragment or conjugate thereof that recognizes human connexin 4 with high affinity and specificity, and optionally at least one pharmaceutically acceptable excipient, diluent, salt or carrier, wherein the at least one antibody or antibody fragment is capable of inhibiting the binding of human connexin 4 to human TIGIT.

[0157] According to certain embodiments, the pharmaceutical composition comprises a mAb specific for connexin 4, wherein the mAb is not conjugated to any toxin or anti-tumor agent.

[0158] According to certain embodiments, the pharmaceutical composition comprises a monoclonal antibody or a fragment thereof capable of binding to an epitope within human connexin 4 bound by a monoclonal antibody selected from the group consisting of hNec4.11, hNec4.01 and hNec4.05 having the variable region and CDR sequences disclosed above.

[0159] According to certain embodiments, the pharmaceutical composition comprises at least one monoclonal antibody comprising the following CDR set:

[0160] i. A set of 6 CDRs, wherein: HC CDR1 is SEQ ID NO: 25; HC CDR2 is SEQ ID NO: 26; HC CDR3 is SEQ ID NO: 27; LC CDR1 is SEQ ID NO: 28; LC CDR2 is SEQ ID NO: 29; and LC CDR3 is SEQ ID NO: 30;

[0161] ii. a set of 6 CDRs, wherein: HC CDR1 is SEQ ID NO: 9; HC CDR2 is SEQ ID NO: 10; HC CDR3 is SEQ ID NO: 11; LC CDR1 is SEQ ID NO: 12; LC CDR2 is SEQ ID NO: 13; and LC CDR3 is SEQ ID NO: 14; or

[0162] iii. A set of 6 CDRs, wherein: the HC CDR1 sequence is SEQ ID NO: 15; HC CDR2 is SEQ ID NO: 16; HC CDR3 is SEQ ID NO: 17; LC CDR1 is SEQ ID NO: 18; LC CDR2 is SEQ ID NO: 19; and LC CDR3 is SEQ ID NO: 20.

[0163] Each possibility represents a separate embodiment of the invention.

[0164] According to certain embodiments, the pharmaceutical composition comprises a monoclonal antibody or fragment thereof comprising a heavy chain variable region having a sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 35 and SEQ ID NO: 37. Each possibility represents a separate embodiment of the present invention.

[0165] According to certain embodiments, the pharmaceutical composition comprises a monoclonal antibody or fragment thereof comprising a light chain variable region having a sequence selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 36 and SEQ ID NO: 38. Each possibility represents a separate embodiment of the present invention.

[0166] According to a specific embodiment, the pharmaceutical composition comprises a monoclonal antibody or a fragment thereof comprising a heavy chain variable region having the sequence shown in SEQ ID NO:39 and a light chain variable region having the sequence shown in SEQ ID NO:40.

[0167] According to a specific embodiment, the pharmaceutical composition comprises a monoclonal antibody or a fragment thereof comprising a heavy chain variable region having the sequence shown in SEQ ID NO:35 and a light chain variable region having the sequence shown in SEQ ID NO:36.

[0168] According to a specific embodiment, the pharmaceutical composition comprises a monoclonal antibody or a fragment thereof comprising a heavy chain variable region having the sequence shown in SEQ ID NO:37 and a light chain variable region having the sequence shown in SEQ ID NO:38.

[0169] Also provided is a pharmaceutical composition comprising at least one antibody, antibody fragment or antibody conjugate according to the present invention, which is used to restore NK cell cytotoxicity by inhibiting the binding of connexin 4 to TIGIT expressed on NK cells.

[0170] According to certain embodiments, the antibody, antibody fragment or antibody conjugate is capable of inhibiting the binding of human connexin 4 to TIGIT expressed on T-cells.

[0171] According to certain embodiments, the pharmaceutical composition according to the present invention is used for cancer immunotherapy or enhancing immune response.

[0172] According to certain embodiments, the pharmaceutical composition further comprises human lymphocytes expressing TIGIT.

[0173] According to certain embodiments, the human lymphocytes are killer cells selected from T cells, NK cells and natural killer T (NKT) cells.

[0174] According to certain embodiments, the killer cells are autologous or allogeneic.

[0175] According to certain embodiments, the pharmaceutical composition comprises autologous or allogeneic NK cells expressing TIGIT.

[0176] The cancer that can be treated using the composition according to the present invention can be any cancer that expresses connexin 4. According to some embodiments, the cancer overexpresses connexin 4. According to some embodiments of the present invention, the cancer is a metastatic cancer. According to some embodiments, the pharmaceutical composition according to the present invention is used to inhibit the formation or distribution of metastases or reduce the total number of metastases in a subject.

[0177] According to some embodiments of the invention, the cancer is selected from melanoma, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, colon cancer, cervical cancer, kidney cancer, lung cancer, thyroid cancer, prostate cancer, brain cancer, kidney cancer, laryngeal cancer, pharyngeal cancer, bladder cancer, liver cancer, fibrosarcoma, endometrial cell cancer, glioblastoma, sarcoma, myeloid leukemia and lymphoma. Each possibility represents an independent embodiment of the invention.

[0178] According to certain embodiments, the cancer is a solid cancer. According to certain specific embodiments, the solid cancer is selected from breast cancer, lung cancer, bladder cancer, pancreatic cancer and ovarian cancer.

[0179] According to other embodiments, the cancer is a blood cancer. According to certain embodiments, the pharmaceutical composition, if used to treat cancer, is used with human lymphocytes.

[0180] According to certain embodiments, the human lymphocytes are killer cells selected from the group consisting of T cells, NK cells and NKT cells.

[0181] According to certain embodiments, the killer cells are autologous or allogeneic.

[0182] According to certain embodiments, the killer cells are NK cells.

[0183] According to certain embodiments, the pharmaceutical composition according to the present invention is used to prevent or treat viral infection.

[0184] According to certain embodiments, the pharmaceutical composition is used to prevent measles virus infection.

[0185] According to another aspect, the present invention provides a method for inhibiting the binding of human connexin 4 to TIGIT using the monoclonal antibody or antibody fragment defined above.

[0186] According to another aspect, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof an antibody or fragment thereof that binds to connexin 4, wherein the antibody or fragment thereof is not conjugated to any toxin or anti-tumor agent.

[0187] According to another aspect, the present invention provides a method of enhancing an immune response in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the monoclonal antibody, antibody fragment or antibody conjugate as defined above.

[0188] According to another aspect, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of at least one antibody or its antibody fragment that recognizes human connexin 4 with high affinity and specificity and is capable of inhibiting its binding to its ligand TIGIT.

[0189] According to some embodiments of the invention, the therapeutically effective amount results in a decrease in tumor size or number of metastases in the subject.

[0190] According to certain embodiments, the method comprises administering a pharmaceutical composition comprising the mAb that is not conjugated to any toxin or anti-tumor agent.

[0191] According to certain 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.

[0192] According to certain embodiments, the method of treating cancer comprises administering a monoclonal antibody that recognizes human connexin 4 with high affinity and specificity and an additional anticancer agent. According to certain embodiments, the additional anticancer agent is selected from immunomodulators, activated lymphocytes, kinase inhibitors and chemotherapeutic agents.

[0193] According to other embodiments, the additional immunomodulatory agent is an antibody, antibody fragment or antibody conjugate that binds to an antigen other than human connexin 4.

[0194] According to certain embodiments, the additional immunomodulator is an antibody against an immune checkpoint molecule. According to certain embodiments, the additional immunomodulator is an antibody against an immune checkpoint molecule selected from the following: 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), killer 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.

[0195] According to certain embodiments, the anticancer agent is selected from the group consisting of: Erbitux, cytarabine, fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, vincristine, vinblastine, vinorelbine, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosfamide, dichloromethyl diethylamine, 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.

[0196] According to certain embodiments, the anticancer agent is an epidermal growth factor receptor (EGFR) inhibitor. According to certain embodiments, the EGFR inhibitor is selected from cetuximab Panitumumab Nexituzumab According to certain embodiments, the EGFR inhibitor is cetuximab

[0197] According to some embodiments of the invention, the subject is a human subject.

[0198] According to some embodiments of the present invention, the method further comprises using an agent that downregulates the activity or expression of an immune co-inhibitory receptor.

[0199] According to some embodiments of the invention, the immune cell is a T cell.

[0200] According to certain embodiments of the present invention, the immune co-inhibitory receptor is selected from PD-1, TIGIT, PVR, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, CD96, BY55, LAIR1, SIGLEC10 and 2B4. Each possibility represents an independent embodiment of the present invention.

[0201] According to one aspect, the present invention provides a method for regulating immune system function and / or activity, the method comprising using the antibody according to the present invention to regulate the binding of connexin 4 to TIGIT.

[0202] According to certain embodiments, the method of treating cancer comprises preventing or reducing the formation, growth or spread of metastases in a subject.

[0203] According to certain embodiments, the method for treating cancer comprises administering to a subject in need thereof a pharmaceutical composition comprising a mAb or an antibody fragment thereof capable of inhibiting the binding of human connexin 4 to human TIGIT, and further administering human lymphocytes to the subject.

[0204] According to certain embodiments, the human lymphocytes are killer cells selected from the group consisting of T cells, NK cells and NKT cells.

[0205] According to certain embodiments, the killer cells are autologous or allogeneic.

[0206] According to certain embodiments, the killer cells are NK cells.

[0207] The present invention also provides a method for preventing or treating viral infection, which comprises administering to a subject at least one mAb specific for human connexin 4 or a fragment thereof comprising at least an antigen binding domain, wherein the mAb or its fragment is capable of inhibiting the binding of connexin 4 to TIGIT.

[0208] According to certain embodiments, a method for preventing measles virus infection is provided, the method comprising administering a mAb specific for human connexin 4 or a fragment thereof comprising at least an antigen binding domain, wherein the mAb or fragment thereof is capable of inhibiting the binding of measles virus to human connexin 4 expressed on epithelial cells. According to certain embodiments, the cell is an epithelial cell.

[0209] According to one aspect, the present invention provides a method for diagnosing or prognosing cancer or an infectious disease in a subject, the method comprising determining the expression level of connexin 4 in a biological sample of the subject using at least one antibody described herein.

[0210] 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 cells comprising the CAR molecule described herein.

[0211] According to another aspect, the present invention also includes a method for determining or quantifying the expression of connexin 4, the method comprising contacting a biological sample with an antibody or antibody fragment and measuring the level of complex formation, wherein the antibody or antibody fragment comprises the following CDR set:

[0212] i. A set of 6 CDRs, wherein: HC CDR1 is SEQ ID NO: 25; HC CDR2 is SEQ ID NO: 26; HC CDR3 is SEQ ID NO: 27; LC CDR1 is SEQ ID NO: 28; LC CDR2 is SEQ ID NO: 29; and LC CDR3 is SEQ ID NO: 30;

[0213] ii. a set of 6 CDRs, wherein: HC CDR1 is SEQ ID NO: 9; HC CDR2 is SEQ ID NO: 10; HC CDR3 is SEQ ID NO: 11; LC CDR1 is SEQ ID NO: 12; LC CDR2 is SEQ ID NO: 13; and LC CDR3 is SEQ ID NO: 14; or

[0214] iii. A set of 6 CDRs, wherein: the HC CDR1 sequence is SEQ ID NO: 15; HC CDR2 is SEQ ID NO: 16; HC CDR3 is SEQ ID NO: 17; LC CDR1 is SEQ ID NO: 18; LC CDR2 is SEQ ID NO: 19; and LC CDR3 is SEQ ID NO: 20.

[0215] According to certain embodiments, the determination and quantification methods can be performed in vitro or ex vivo, or can be used to diagnose conditions associated with the expression of connexin 4. The antibodies according to the present invention can also be used to set up screening methods. For example, enzyme-linked immunosorbent assays (ELISA) or radioimmunoassays (RIA) and methods such as IHC or FACS can be constructed to measure the level of secreted or cell-bound polypeptides using the antibodies and methods known in the art.

[0216] According to certain embodiments, the method for detecting or quantifying the presence of connexin 4 expressed on cells or secreted into a biological culture medium comprises the following steps:

[0217] i. incubating the sample with an antibody specific for human connexin 4 or an antibody fragment thereof comprising at least an antigen binding portion;

[0218] ii. Detecting bound connexin 4 using a detectable probe.

[0219] According to some embodiments, the method further comprises the steps of:

[0220] iii. comparing the amount of (ii) to a standard curve obtained from a reference sample containing a known amount of connexin 4; and

[0221] iv. calculating the amount of connexin 4 in the sample from the standard curve.

[0222] According to certain specific embodiments, the sample is a body fluid.

[0223] According to certain embodiments, the method is performed in vitro or ex vivo.

[0224] Also provided is a kit for measuring the expression or presence of connexin 4 in a biological sample, the kit comprising at least one antibody or antibody fragment according to the present invention. According to certain embodiments, the antibody or antibody fragment contained in the kit comprises the following CDR group:

[0225] i. A set of 6 CDRs, wherein: HC CDR1 is SEQ ID NO: 25; HC CDR2 is SEQ ID NO: 26; HC CDR3 is SEQ ID NO: 27; LC CDR1 is SEQ ID NO: 28; LC CDR2 is SEQ ID NO: 29; and LC CDR3 is SEQ ID NO: 30;

[0226] ii. a set of 6 CDRs, wherein: HC CDR1 is SEQ ID NO: 9; HC CDR2 is SEQ ID NO: 10; HC CDR3 is SEQ ID NO: 11; LC CDR1 is SEQ ID NO: 12; LC CDR2 is SEQ ID NO: 13; and LC CDR3 is SEQ ID NO: 14; or

[0227] iii. A set of 6 CDRs, wherein: the HC CDR1 sequence is SEQ ID NO: 15; HC CDR2 is SEQ ID NO: 16; HC CDR3 is SEQ ID NO: 17; LC CDR1 is SEQ ID NO: 18; LC CDR2 is SEQ ID NO: 19; and LC CDR3 is SEQ ID NO: 20.

[0228] According to one aspect, the present invention provides a kit for detecting cancer, the diagnostic kit comprising the antibody or antibody fragment thereof disclosed herein.

[0229] According to certain embodiments, the present invention provides a method for diagnosing, assessing the severity or staging an immune-related disease or a proliferative disease, the method comprising using the antibody or fragment or conjugate thereof according to the present invention to determine the expression, concentration or activity of connexin 4 in a sample from a subject, and comparing the expression or activity of connexin 4 with a reference amount of the expression, concentration or activity of connexin 4. The reference amount can be obtained from a sample obtained from a normal subject, from the same subject at a different stage of the disease, or determined from clinical data of a large group of subjects.

[0230] From the detailed description given below, other 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 the preferred embodiments of the present invention, are provided only as illustrations, 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

[0231] Figure 1 Schematic representation of the receptors involved in TIGIT signaling on immune (NK) and tumor cells. Anti-Connexin4 Ab is depicted.

[0232] Figure 2 Antibody clones hNec4.05 and hNec4.01 showed the best blocking ability of TIGIT-connexin 4 interaction. The figure depicts the mean fluorescence intensity (MFI) values ​​of FACS staining of RAJI Burkitt's lymphoma cells transfected with connexin 4. The cells were incubated with 0.2 μg of different clones (as indicated) and then incubated with 3 μg of TIGIT-Ig and then stained with anti-human secondary antibody.

[0233] Figure 3 Quantification of the binding affinity of antibody clones to connexin 4. Binding of fluorophore-labeled connexin 4-Ig to antibody clone hNec4.05 and clone hNec4.01 was visualized using microthermophoresis. The measurements were repeated with at least three independent protein preparations, and the mean (± SEM.) of the results is shown.

[0234] Figure 4A and 4B Antibody clones hNec4.05 and hNec4.01 blocked connexin 4 and enhanced NK cell cytotoxicity. 35S] Methionine-labeled (4A) RAJI Burkitt lymphoma cells transfected with connexin 4 and (4B) LNCap prostate cancer cells (naturally expressing connexin 4) were incubated with 1 μg / well of mouse IgG1 as a control antibody or anti-connexin 4 antibodies hNec4.01 or hNec4.05. After 1 h, NK cells were added to the cells and incubated for 5 hours. The average specific killing (± sd) at various effector cell: target cell (E:T) ratios of NK: cancer cells was plotted. * indicates a significant effect of hNec4.01 and hNec4.05 clones compared to the control antibody (p < 0.05. The figure shows a representative experiment from three experiments performed. The same effect was determined using MCF-7, MDA-MB-453, SK-BR-3 and T47D cells, all breast cancer cell lines.

[0235] Figures 5A-5C The antibody clones do not bind to murine connexin 4. (A) Western blot of RAJI cells transfected with murine connexin 4 (labeled as murine connexin 4) and probed with a commercial anti-murine connexin 4 mAb (clone 356704, which cannot be used for flow cytometry). Expression was compared to RAJI cells expressing empty vector (labeled as empty). hGAPDH staining was used as a loading control. (BC) FACS staining of RAJI cells transfected with murine connexin 4 (black line histogram). Cells were stained with 0.2 μg of (B) clone hNec4.01 or (C) clone hNec4.05.

[0236] Figures 6A-6C Blocking of connexin 4-connexin 1 interaction. (AB) FACS staining of RAJI cells transfected with human connexin 4. Cells were pre-incubated with 1 μg of (A) clone hNec4.01 or (B) clone hNec4.05 and then incubated with 3 μg connexin 1-Ig (black line bar graph). Staining without blocking is shown as gray line. The gray solid bar graph is the background control staining of the secondary antibody only. (C) Mean fluorescence intensity (MFI) values ​​of FACS staining of A and B.

[0237] Figures 7A-7C In vivo effects of anti-connexin4 mAb. (A) SCID-beige mice were treated with 5×10 6 Raji cells transfected with empty vector (EV) or overexpressing (OE) connexin4 were either isolated (left panel, without NK) or treated with 1×10 6 NK cells (right, NK) were implanted subcutaneously. (B) SCID-beige mice were treated with 5×10 6 Raji cells overexpressing connexin 4 were compared with 1×10 6NK cells were implanted subcutaneously. Mice were treated with 75 μg of control Ab or anti-connexin 4 clone hNec4.05 mAb twice a week by intraperitoneal injection. (C) SCID-beige mice were implanted with 5×10 6 MDA-MB-453 cells alone or with 7×10 5 NK cells were implanted subcutaneously. Mice were then treated as described in (B). Tumors were harvested and weighed on day 21 (A), 27 (B), or 23 (C) after tumor injection. N=7 for all mouse experimental groups. (*) p<0.05.

[0238] Figures 8A-8C Antibody clones hNec4.05 and hNec4.11 bind to cell surface connexin-4. (A) Antibodies that bind to connexin-4 expressed on human cell line MDA-MD-453 are evaluated by FACS analysis. EC50 values ​​and maximum binding signals calculated after titration (range 20-0.01 nM) of Ab binding for each clone are shown. It is noteworthy that similar results were observed when using a chimeric version of Ab in which murine IgG1 Fc was replaced by human Fc. (B) Antibodies bind to CHO cells transfected with cynomolgus monkey (Cyno)-connexin-4 and analyzed as described in A. (C) Antibodies bind to CHO cells transfected with murine connexin-4 and analyzed as described in A. ND-Not Detected.

[0239] Figures 9A-9D Antibody clones hNec4.05 and hNec4.11 block the binding of connexin-4 to its ligands TIGIT and connexin-1. The binding of connexin-4 ligands was evaluated by FACS analysis. CHO cells transfected with human connexin-4 were incubated with 20 μg / ml of human TIGIT-Ig (A&C) or human connexin-1-Ig (B&D) in the presence or absence of 8 μg / ml of anti-connexin-4 clones hNec4.05 (A&B) or clones hNec4.11 (C&D). In all cases, strong binding inhibition of anti-connexin-4 Ab was observed. The residual signal may be caused by ligand binding to other receptors expressed by CHO cells, such as PVR, which is not affected by the anti-connexin-4 Ab.

[0240] Figures 10A-10BHuman IgG1 chimeric Ab clones hNec4.05 and hNec4.11 enhance NK cell activation in the presence of tumor cells. Human NK cells (effector cells, E) were incubated with target cells (T) HT1376 (A) and MDA-MD-453 (B) at an E:T ratio of 2:1. The incubation was carried out in the presence of 12 μg / ml chimeric clones hNec4.05 and hNec4.11 or control hIgG1. After 2 hours, the degranulation and activation state of NK cells were determined by FACS analysis of CD107a expression. The degranulation of NK cells in the presence of control hIgG1 was set to 1, and the corresponding induction multiple was calculated. Shown is the mean value of 2-3 repeated experiments and their normalized SD.

[0241] Figures 11A-11D CAR-T driven by hNec4.11 causes specific T cell activation in the presence of tumor cells expressing connexin-4. (A) Schematic diagram of CART construct. (B) Jurkat cells are transduced with lentiviral particles encoding the construct and GFP. As judged by FACS analysis of GFP expression, the transduction efficiency is higher than 99%. (C) Parental Jurkat cells or Jurkat cells (Jurkat pHAGE2.4.11) expressing the CART construct are incubated with target cells HT1376 or MDA-MD-453 (MDA-453) for 48 hours, and then the culture medium is collected and the IL-2 concentration is tested as a way to evaluate Jurkat activation. IL-2 secretion is significantly induced by CART expression (* = 0.003, ** = 0.00014; two-tailed student test). Representative experiments in two experiments performed are shown. (D) PBMCs from healthy donors are transduced with the CART construct. The CART PBMCs were incubated with HT1376 cells at multiple E:T ratios (marked on the X axis). Killing of target cells was significant where marked with an asterisk (***p<7*10 -5 ). DETAILED DESCRIPTION

[0242] The present invention provides an effective monoclonal antibody specifically targeting human connexin 4. The present invention also provides the production of the mAb and the use as a therapeutic agent. Specifically, the mAb of the present invention can be used to restore and enhance the anti-tumor killing activity of immune cells and can be used as a diagnostic reagent.

[0243] Although existing publications show the use of anti-connexin 4 mAbs for targeting drugs to tumor cells overexpressing connexin 4, the present invention discloses for the first time a monoclonal antibody that directly enhances immune cells to fight tumor cells by inhibiting the binding of connexin 4 to the inhibitory receptor TIGIT of immune cells such as NK cells.

[0244] The antibodies of the present invention overcome the shortcomings of antibodies specific to TIGIT that are currently being tested for the treatment of cancer. It is said that anti-TIGIT antibodies can activate the entire immune system by blocking all immune cells expressing TIGIT receptors and may cause autoimmunity, while the anti-connexin 4 antibodies of the present invention only target cells expressing connexin 4, which is known to be overexpressed in tumors.

[0245] Furthermore, certain antibodies of the invention may also result in immune-independent killing of tumor cells, possibly through their ability to block the interaction of connexin4 with connexin1.

[0246] As used herein, the term "antigen" refers to a molecule or a portion of a molecule that is capable of inducing antibody formation and is specifically bound by an antibody. An antigen may have one or more epitopes. The specific binding mentioned above means that the antigen reacts with its corresponding antibody in a highly selective manner and does not react with a large number of other antibodies that may be elicited by other antigens. According to certain embodiments of the present invention, the antigen is connexin 4.

[0247] As used herein, the term "connexin 4" or "connexin cell adhesion molecule 4" refers to a single transmembrane type I membrane protein with 510 amino acids and a molecular weight of 55454Da, also known as PVRL4, LNIR, PRR4 and EDSS1. The connexin 4 contains two immunoglobulin-like (Ig-like) C2-type domains and one Ig-like V-type domain. It participates in cell adhesion through trans-homophilic and -heterophilic interactions. The soluble form is produced by proteolytic cleavage by the metalloproteinase ADAM17 / TACE at the cell surface, and the secreted form is present in both breast tumor cell lines and breast tumor patients. Exemplary connexin 4 according to the invention are shown in the following SwissPort, UniPort and GenBank symbols or accession numbers: Q96NY8-NECT4_HUMAN; Q96NY8; B4DQW3; Q96K15; Q96NY8-1; Q96NY8-2; ENSP00000356991; NP_112178.2; XP_005245565.1; XP_011508323.1; XP_011508324.1; or XP_011508325.1.

[0248] The antibody or fragment thereof according to the present invention binds to an epitope in connexin 4. Specifically, the antibody binds to an epitope within the ectodomain (extracellular part) of the connexin 4 protein sequence.

[0249] As used herein, the term "antigenic determinant" or "epitope" refers to a region of an antigen molecule that specifically reacts with a particular antibody. Using methods known in the art, peptide sequences derived from epitopes can be used alone or in combination with a carrier to immunize animals and generate additional polyclonal or monoclonal antibodies. Isolated peptides derived from epitopes can be used in diagnostic methods to detect antibodies.

[0250] It should be noted that affinity can be quantified using known methods such as surface plasmon resonance (SPR) (described in Scarano S, Mascini M, Turner AP, Minunni M., Surface plasmon resonance imaging for affinity-based biosensors, Biosens Bioelectron. 2010, 25:957-66) and can be calculated using, for example, the dissociation constant Kd, such that a lower Kd reflects a higher affinity.

[0251] Antibodies or immunoglobulins contain two heavy chains and two light chains linked together by disulfide bonds, with each light chain linked to a corresponding heavy chain by disulfide bonds in a "Y" shaped configuration. Proteolytic digestion of antibodies yields the Fv (variable fragment) and Fc (fragment crystallographic) domains. The antigen binding domain Fab includes the region where the polypeptide sequence varies. The term F(ab') 2 The Fab' arm is two Fab' arms connected by disulfide bonds. The central axis of the antibody is called the Fc fragment. Each heavy chain has a variable domain (V H ), followed by multiple constant domains (C H Each light chain has a variable domain at one end (V L ) and has a constant domain (C L ), the light chain variable domain is aligned with the variable domain of the heavy chain, and the light chain constant domain is aligned with the first constant domain (CH1) of the heavy chain. Each pair of variable domains of the light chain and the heavy chain forms an antigen binding site. The domains on the light chain and the heavy chain have the same overall structure, and each domain contains 4 relatively conservative framework regions, which are connected by 3 hypervariable domains called complementary determining regions (CDR 1-3). These domains contribute to the specificity and affinity of the antigen binding site.

[0252] The identification or determination of the CDR from a given heavy or light chain variable sequence is usually carried out using one of several methods known in the art. For example, this determination is carried out 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).

[0253] When the term "CDR having a certain sequence" or similar terms is used, it includes the option wherein the CDR comprises the specified sequence as well as the option wherein the CDR consists of the specified sequence.

[0254] 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.

[0255] The isotype of the heavy chain (gamma, alpha, delta, epsilon, or mu) determines the immunoglobulin class (IgG, IgA, IgD, IgE, or IgM, respectively). The light chain has two isotypes (kappa or lambda). Both isotypes are present in all antibody classes.

[0256] The term "antibody" is used in the broadest sense and includes monoclonal antibodies (including full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, and antibody fragments of sufficient length to exhibit the desired biological activity, i.e., binding to human connexin 4.

[0257] The one or more antibodies according to the present invention include complete antibodies such as polyclonal antibodies or monoclonal antibodies (mAb) and proteolytic fragments thereof such as Fab or F(ab'). 2 Single chain antibodies are also within the scope of the present invention.

[0258] Antibody fragments

[0259] "Antibody fragments" contain only a portion of an intact antibody, typically including the antigen binding site of the intact antibody, and thus retain the ability to bind antigen. Examples of antibody fragments encompassed by this definition include (i) Fab fragments, which have VL, CL, VH and CH1 domains; (ii) Fab' fragments, which are Fab fragments with one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iii) Fd fragments with VH and CH1 domains; (iv) Fd' fragments with VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) Fv fragments with the VL and VH domains of a single arm of an antibody; (vi) dAb fragments consisting of a VH domain (Ward et al., Nature 1989, 341, 544-546); (vii) isolated CDR regions; (viii) F(ab') 2 (ix) single-chain antibody molecules (e.g., single-chain Fv; scFv) (Bird et al., Science 1988, 242, 423-426; and Huston et al., Proc. Natl. Acad. Sci. (USA) 1988, 85, 5879-5883); (x) "diabodies" with two antigen-binding sites, which comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 404,097). 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 6444-6448); (xi) "linear antibodies" which comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al., Protein Eng., 1995, 8, 1057-1062; and U.S. Pat. No. 5,641,870).

[0260] Various different technologies have been developed for producing antibody fragments. Traditionally, these fragments are produced by proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, the antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab') 2Fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another method, F(ab') 2 Fragments can be isolated directly from recombinant host cell cultures. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv).

[0261] A single-chain antibody can be a single-chain composite polypeptide having antigen-binding ability and comprising an amino acid sequence homologous or similar to the variable regions of the light chain and heavy chain of an immunoglobulin, i.e., a linked V H -V L Or single-chain Fv (scFv).Techniques used to produce single-chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single-chain antibodies against connexin 4.

[0262] When used in this article, the term "monoclonal antibody" (mAb) refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the population are consistent except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and are directed against a single antigen. In addition, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" should not be interpreted as requiring the production of the antibody by any ad hoc method. mAb can be obtained by methods known to those skilled in the art. For example, the monoclonal antibodies used in accordance with the present invention can be manufactured by the hybridoma method first described by Kohler et al., Nature 1975, 256, 495, or can be manufactured by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature 1991, 352, 624-628 or Marks et al., J. Mol. Biol., 1991, 222: 581-597.

[0263] The design and development of recombinant monovalent antigen-binding molecules derived from monoclonal antibodies by rapid identification and cloning of functional variable heavy chain (VH) and variable light chain (VL) genes and the design and cloning of synthetic DNA sequences optimized for expression in recombinant bacteria are described in Fields et al., 2013, 8(6): 1125-48.

[0264] mAb of the present invention can be any immunoglobulin class including IgG, IgM, IgE, IgA and IgD. The hybridoma producing mAb can be cultivated in vitro or in vivo. The high titer of mAb can be obtained by in vivo production, wherein the cell intraperitoneal injection coming from individual hybridoma is injected into the original Balb / c mouse of first immunity, to produce ascites containing high concentration of required mAb. mAb can use methods well known to those skilled in the art from these ascites or from culture supernatant purification.

[0265] Anti-idiotypic antibodies specifically immunoreactive with the hypervariable regions of the antibodies of the invention are also contemplated.

[0266] The present invention provides a monoclonal antibody or antibody fragment comprising an antigen binding domain (ABD), wherein the antigen binding domain comprises three CDRs of a light chain and three CDRs of a heavy chain, wherein the ABD has at least 90% sequence identity or similarity with the ABD of a monoclonal mouse antibody comprising the following chains: (i) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 39 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 40 (identified herein as hNec4.11); (ii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 35 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 36 (identified herein as hNec4.01); or (iii) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 37 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 38 (identified herein as hNec4.05). These antibodies may have an ABD domain having at least 93%, at least 94%, at least 95%, at least 96, at least 97, at least 98, at least 99% sequence identity or similarity, or 100% sequence identity to the corresponding ABD of antibody hNec4.11, hNec4.01 or hNec4.05.

[0267] Sequence identity is the number of exact matches of amino acids or nucleotides between two different sequences. Sequence similarity allows conservative substitutions of amino acids to be identified as identical amino acids.

[0268] The present invention also provides conservative amino acid variants of antibody molecules according to the present invention. Variants according to the present invention can also be made to retain the overall molecular structure of the encoded protein. Given the properties of the individual amino acids constituting the disclosed protein product, professionals will recognize some reasonable replacements. Amino acid replacements, i.e., "conservative replacements," can be made, for example, on the basis of similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues involved. When used herein, the term "antibody analog" refers to an antibody derived from another antibody by one or more conservative amino acid replacements.

[0269] When used herein, the term "antibody variant" refers to any molecule comprising an antibody of the invention. For example, a fusion protein in which the antibody or antigen-binding fragment thereof is linked to another chemical moiety is considered an antibody variant.

[0270] Analogs and variants of the antibody sequences are also within the scope of the present invention. They include, but are not limited to, conservative and non-conservative substitutions, insertions and deletions of amino acids within the sequences. These modifications and resulting antibody analogs or variants are within the scope of the present invention as long as they provide or even improve the binding of the antibody to human connexin 4.

[0271] Conservative substitutions of amino acids known to those skilled in the art are within the scope of the present invention. Conservative amino acid substitutions include replacing one amino acid with another amino acid having the same type of functional group or side chain, such as aliphatic, aromatic, positively charged, negatively charged functional groups or side chains. These substitutions may enhance oral bioavailability, permeability, targeting to specific cell populations, immunogenicity, etc. Professional and technical personnel will recognize that changes, additions or deletions made to a peptide, polypeptide or protein sequence, each replacement, deletion or addition of a single amino acid or a small proportion of amino acids in the encoded sequence, where the changes result in amino acids being replaced by chemically similar amino acids, are "conservatively modified variants". Conservative substitution tables that provide functionally similar amino acids are well known in the art. For example, according to a table known in the art, the following 6 groups each contain amino acids that are conservatively substituted for each other:

[0272] 1) Alanine (A), serine (S), threonine (T);

[0273] 2) Aspartic acid (D), Glutamic acid (E);

[0274] 3) Asparagine (N), glutamine (Q);

[0275] 4) Arginine (R), Lysine (K);

[0276] 5) isoleucine (I), leucine (L), methionine (M), valine (V); and

[0277] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0278] It should be emphasized that the variant chain sequence is determined by sequencing using specific primers. Using different sequencing methods for the same sequence may produce slightly different sequences, especially at the end of the sequence, due to technical problems and different primers. Therefore, different variants of the anti-connexin 4 antibody variable chain sequence are described in detail with this application.

[0279] When used herein, the terms "molecule having an antigen-binding portion of an antibody" and "antigen-binding fragment" are intended to include not only intact immunoglobulin molecules of any isotype and produced by any animal cell line or microorganism, but also antigen-binding reactive portions thereof, including but not limited to Fab fragments, Fab' fragments, F(ab') fragments, 2 Fragments, variable portions of heavy and / or light chains thereof, Fab minibodies (see, e.g., WO 93 / 15210, U.S. Patent Application 08 / 256,790, WO 96 / 13583, U.S. Patent Application 08 / 817,788, WO 96 / 37621, U.S. Patent Application 08 / 999,554) and single chain antibodies incorporating such reactive portions, as well as any other type of molecule into which these antibody reactive portions have been physically inserted. These molecules can be provided by any known technique including, but not limited to, enzymatic cleavage, peptide synthesis or recombinant technology.

[0280] The monoclonal antibodies herein specifically include "chimeric" antibodies, in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of these "chimeric" antibodies, as long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). In addition, complementary determining regions (CDRs) can be grafted to change certain properties of the antibody molecule, including affinity or specificity. Non-limiting examples of CDR grafting are disclosed in U.S. Pat. No. 5,225,539.

[0281] Chimeric antibodies are molecules whose different parts are derived from different animal species, such as those with the variable region and human immunoglobulin constant region derived from murine mAb. Antibodies with variable region framework residues substantially derived from human antibodies (referred to as receptor antibodies) and antibodies substantially derived from the CDR of mouse antibodies (referred to as donor antibodies) are also referred to as humanized antibodies. Chimeric antibodies are mainly used to reduce immunogenicity in applications and to increase yield in production, such as in the case where murine mAb has a higher yield produced from hybridomas but higher immunogenicity in humans, using human / mouse chimeric mAb. Chimeric antibodies and their production methods are known in the art (e.g. PCT patent applications WO 86 / 01533, WO 97 / 02671, WO 90 / 07861, WO 92 / 22653 and U.S. Patents 5,693,762, 5,693,761, 5,585,089, 5,530,101 and 5,225,539).

[0282] According to certain specific embodiments, the monoclonal antibody is a chimeric monoclonal antibody.

[0283] According to certain embodiments, the chimeric antibody comprises a constant region derived from a human.

[0284] According to certain embodiments, the human constant region of the chimeric antibody is selected from human IgG1, human IgG2, human IgG3 and human IgG4.

[0285] According to a specific embodiment, there is provided a chimeric monoclonal antibody recognizing human connexin 4, comprising:

[0286] i. A set of 6 CDRs, wherein: HC CDR1 is (SEQ ID NO: 25); HC CDR2 is (SEQ ID NO: 26); HC CDR3 is (SEQ ID NO: 27); LC CDR1 is (SEQ ID NO: 28); LC CDR2 is (SEQ ID NO: 29); and LC CDR3 is (SEQ ID NO: 30);

[0287] ii. a set of 6 CDRs, wherein: HC CDR1 is (SEQ ID NO: 9); HC CDR2 is (SEQ ID NO: 10); HC CDR3 is (SEQ ID NO: 11); LC CDR1 is (SEQ ID NO: 12); LC CDR2 is (SEQ ID NO: 13); and LC CDR3 is (SEQ ID NO: 14); or

[0288] iii. A group of 6 CDRs, wherein: the HC CDR1 sequence is (SEQ ID NO: 15); HC CDR2 is (SEQ ID NO: 16); HC CDR3 is (SEQ ID NO: 17); LC CDR1 is (SEQ ID NO: 18); LC CDR2 is (SEQ ID NO: 19); and LC CDR3 is (SEQ ID NO: 20).

[0289] According to one aspect, the present invention provides a CAR comprising an antibody fragment that specifically binds to connexin 4.

[0290] According to certain embodiments, the CAR comprises: i) a specific binding agent that can specifically bind to connexin 4; ii) a spacer or hinge domain; iii) a transmembrane domain (TM) that anchors the CAR in the T cell membrane; iv) an intracellular domain that transmits signals within the T cell.

[0291] According to certain embodiments, the CAR comprises a CDR group selected from the following:

[0292] i. A set of 6 CDRs, wherein: HC CDR1 is SYYIH (SEQ ID NO: 25); HC CDR2 is WIYPGNVNTKYNERFKG (SEQ ID NO: 26); HC CDR3 is SNPYVMDY (SEQ ID NO: 27); LC CDR1 is KASQSVNNDVA (SEQ ID NO: 28); LC CDR2 is YASNRFT (SEQ ID NO: 29); and LC CDR3 is QQAYRSPYT (SEQ ID NO: 30);

[0293] ii. a set of 6 CDRs, wherein: HC CDR1 is AYNIH (SEQ ID NO: 9); HC CDR2 is YIYPNNGGSGYNQKFMN (SEQ ID NO: 10); HC CDR3 is FDYDEAWFIY (SEQ ID NO: 11); LC CDR1 is SASSVSYMH (SEQ ID NO: 12); LC CDR2 is DTSKLAS (SEQ ID NO: 13); and LC CDR3 is FQGSGSPYT (SEQ ID NO: 14); and

[0294] iii. A group of 6 CDRs, wherein: HC CDR1 sequence is TYYIH (SEQ ID NO: 15); HC CDR2 is WIYPGNVNTKNNEKFKV (SEQ ID NO: 16); HC CDR3 is SNPYVMDY (SEQ ID NO: 17); LC CDR1 is KASQSVSNDVA (SEQ ID NO: 18); LC CDR2 is YASNRYT (SEQ ID NO: 19); and LC CDR3 is QQDYSSPYT (SEQ ID NO: 20).

[0295] According to one aspect, the present invention provides an isolated nucleic acid molecule encoding a CAR, wherein the CAR comprises an antibody or antibody fragment comprising a connexin 4 binding domain, wherein the binding domain comprises a CDR group selected from the following:

[0296] i. A set of 6 CDRs, wherein: HC CDR1 is SYYIH (SEQ ID NO: 25); HC CDR2 is WIYPGNVNTKYNERFKG (SEQ ID NO: 26); HC CDR3 is SNPYVMDY (SEQ ID NO: 27); LC CDR1 is KASQSVNNDVA (SEQ ID NO: 28); LC CDR2 is YASNRFT (SEQ ID NO: 29); and LC CDR3 is QQAYRSPYT (SEQ ID NO: 30);

[0297] ii. a set of 6 CDRs, wherein: HC CDR1 is AYNIH (SEQ ID NO: 9); HC CDR2 is YIYPNNGGSGYNQKFMN (SEQ ID NO: 10); HC CDR3 is FDYDEAWFIY (SEQ ID NO: 11); LC CDR1 is SASSVSYMH (SEQ ID NO: 12); LC CDR2 is DTSKLAS (SEQ ID NO: 13); and LC CDR3 is FQGSGSPYT (SEQ ID NO: 14); and

[0298] iii. A group of 6 CDRs, wherein: HC CDR1 sequence is TYYIH (SEQ ID NO: 15); HC CDR2 is WIYPGNVNTKNNEKFKV (SEQ ID NO: 16); HC CDR3 is SNPYVMDY (SEQ ID NO: 17); LC CDR1 is KASQSVSNDVA (SEQ ID NO: 18); LC CDR2 is YASNRYT (SEQ ID NO: 19); and LC CDR3 is QQDYSSPYT (SEQ ID NO: 20).

[0299] According to certain embodiments, a vector is provided, comprising the polynucleotide sequence shown in SEQ ID NO: 31 or SEQ ID NO: 33. According to certain embodiments, the vector is a viral vector. According to certain embodiments, the viral vector is a lentiviral vector.

[0300] According to certain embodiments, a T cell engineered to express a CAR described herein is provided.

[0301] For the transmembrane domain, in various embodiments, CAR can be designed to include a transmembrane domain attached to the extracellular domain of the CAR. A transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, such as one or more amino acids associated with the extracellular region of the protein from which the transmembrane region is derived (e.g., 1, 2, 3, 4, 5 or more amino acids in the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane region is derived (e.g., 1, 2, 3, 4, 5 or more amino acids in the intracellular region). According to certain embodiments, the transmembrane domain may be selected or modified by amino acid replacement to avoid binding of these domains to the transmembrane domains of the same or different surface membrane proteins, such as to minimize interactions with other members of the receptor complex.

[0302] According to certain embodiments, the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of the α, β or ζ chains of the T-cell receptor, CD27, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. Each possibility represents an independent embodiment of the present invention.

[0303] According to certain embodiments, the antibody or antibody fragment comprising the connexin 4 binding domain is connected to the transmembrane domain via a hinge region. According to certain embodiments, the hinge is from a human protein. According to certain embodiments, the hinge is a human Ig (immunoglobulin) hinge. According to certain embodiments, the hinge is an IgG4 hinge or a CD8a hinge.

[0304] The cytoplasmic domain or region of the CAR includes an intracellular signaling domain. The intracellular signaling domain is generally responsible for the activation of at least one normal effector function of the immune cell into which the CAR has been introduced. The term "effector function" refers to a special function of a cell. For example, the effector function of a T cell can be a cell lytic activity or an auxiliary activity, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals and instructs the cell to perform a special function. The term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal. Examples of intracellular signaling domains used in the CAR of the present invention include cytoplasmic sequences of T cell receptors (TCRs) and auxiliary receptors that act synergistically to initiate signal transduction after antigen receptor engagement, as well as any derivatives or variants of these sequences and any recombinant sequences with the same functional capabilities.

[0305] In certain embodiments, the signal generated by the TCR alone is insufficient to fully activate the T cell, and secondary and / or co-stimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: signaling sequences that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and signaling sequences that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic domains, e.g., co-stimulatory domains).

[0306] According to certain embodiments, the intracellular signaling domain is designed to include two or more costimulatory signaling domains. According to certain embodiments, the two or more costimulatory signaling domains are separated by a linker molecule. According to certain embodiments, the linker molecule is a glycine residue. According to certain embodiments, the linker is an alanine residue.

[0307] According to certain embodiments, the CAR comprises a co-stimulatory domain obtained from a protein selected from OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) and 4-1BB (CD137). Each possibility represents an independent embodiment of the present invention.

[0308] The present invention also relates to nucleic acid molecules, vectors and host cells encoding the CAR. The present invention also relates to (heterologous) T cells comprising the CAR of the present invention, and pharmaceutical compositions comprising these CARs and suitable carriers or excipients. The present invention also relates to autologous T cell therapies (and corresponding medical uses) comprising T cells, compositions and CARs of the present invention.

[0309] According to certain embodiments, the cell expressing CAR described herein may further include a second CAR, for example, including a second CAR for different antigen binding domains for, for example, the same target (connexin 4) or different targets. According to certain embodiments, when the cell expressing CAR includes two or more different CARs, the antigen binding domains of the different CARs may cause the antigen binding domains to not interact with each other.

[0310] According to certain embodiments, a cell population is provided, wherein at least one cell in the population expresses a CAR having an anti-connexin 4 domain as described herein, and a second cell expresses another agent, such as an agent that enhances the activity of the cell expressing the CAR.

[0311] The present invention also relates to (heterogeneous) NK cells comprising the CAR of the present invention, and pharmaceutical compositions comprising these CARs and suitable carriers or excipients. The present invention also relates to autologous NK cell therapies (and corresponding medical uses) comprising NK cells, compositions and CAR of the present invention.

[0312] As used herein, "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide comprising an antigen binding domain (e.g., an antigen binding portion of an antibody (e.g., scFV)), a transmembrane domain, and T-cell or NK-cell signaling and / or T-cell or NK-cell activation domains. CAR has the ability to redirect the specificity and reactivity of T-cells or NK-cells to selected targets using the antigen binding properties of monoclonal antibodies in a non-MHC restricted manner. The non-MHC restricted antigen recognition provides CAR-expressing T-cells or NK-cells with the ability to recognize antigens independently of antigen processing, thereby bypassing the main mechanism of tumor escape. Most commonly, the extracellular binding domain of the CAR is composed of a single-chain variable fragment (scFv) produced by fusing the heavy and light chain variable regions of a murine or humanized monoclonal antibody.

[0313] Pharmacology

[0314] In pharmaceutical and drug dosage forms, the active agent is preferably used together with one or more pharmaceutically acceptable carriers and optionally any other therapeutic ingredients. The carrier must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the dosage form and not unduly deleterious to the recipient thereof. The active agent is provided in an amount effective to achieve the desired pharmacological effect as described above and in an amount suitable to achieve the desired exposure.

[0315] Typically, the antibody of the present invention and its fragment and conjugate comprising the antigen-binding portion of the antibody or comprising another polypeptide including a peptide mimetic are suspended in a sterile saline solution for therapeutic use. The pharmaceutical composition can be optionally formulated to control the release of the active ingredient (molecule comprising the antigen-binding portion of the antibody) or prolong its presence in the patient's system. A large number of suitable drug delivery systems are known, and include, for example, implantable drug release systems, hydrogels, hydroxymethylcellulose, microcapsules, liposomes, microemulsions, microspheres, etc. Controlled release formulations can be prepared by using polymers to compound or adsorb molecules according to the present invention. For example, biocompatible polymers include a matrix of poly(ethylene-co-vinyl acetate) and a matrix of a polyanhydride copolymer of stearic acid dimer and sebacic acid. The rate at which the molecule according to the present invention, i.e., the antibody or antibody fragment, is released from this matrix depends on the molecular weight of the molecule, the amount of the molecule in the matrix, and the size of the dispersed particles.

[0316] The pharmaceutical compositions of the invention can be administered by any suitable means, such as oral, topical, intranasal, subcutaneous, intramuscular, intravenous, intraarterial, intraarticular, intralesional, intratumoral or parenteral administration. Typically, intravenous (iv) administration is used to deliver the antibody.

[0317] It will be apparent to one of ordinary skill in the art that a therapeutically effective amount of a molecule according to the invention will depend, inter alia, on the dosing schedule, the unit dose of the molecule being administered, whether the molecule is administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic activity of the molecule being administered, its persistence in the blood circulation, and the judgment of the attending physician.

[0318] As used herein, the term "therapeutically effective amount" refers to the amount of a drug that effectively treats a disease or disorder in a mammal. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow down and preferably stop to a certain extent) cancer cell infiltration into surrounding organs, inhibit (i.e., slow down and preferably stop to a certain extent) tumor metastasis, inhibit tumor growth to a certain extent, and / or alleviate one or more symptoms associated with the disorder to a certain extent. In the case where the drug can prevent the growth of existing cancer cells and / or kill existing cancer cells, it may have cell inhibition and / or cytotoxicity. For cancer therapy, in vivo efficacy can be measured, for example, by assessing the length of survival time, time to disease progression (TTP), response rate (RR), response duration and / or quality of life.

[0319] Cancers suitable for treatment by the present invention include, but are not limited to, epithelial cancers, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of these cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, and various types of head and neck cancers, as well as B-cell lymphomas (including low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-lytic cell NHL, bulky disease) NHL, mantle cell lymphoma, AIDS-related lymphoma and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy cell leukemia, chronic myeloid leukemia and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with nevus, edema (e.g., edema associated with brain tumors) and Meigs syndrome. Preferably, the cancer is selected from breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkin lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid, head and neck cancer, melanoma, ovarian cancer, mesothelioma and multiple myeloma. Cancerous conditions suitable for treatment of the present invention include metastatic cancers.

[0320] According to other embodiments, the pharmaceutical composition according to the present invention is used to treat cancer characterized by overexpression of connexin 4. Cancer types associated with overexpression of connexin 4 can be identified using known databases such as The Cancer Genome Atlas (TCGA). According to certain embodiments, the cancer that can be treated using the composition according to the 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), multiforme glioblastoma (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.

[0321] The molecules of the present invention as active ingredients are dissolved, dispersed or mixed in known pharmaceutically acceptable excipients that are compatible with the active ingredient. Suitable excipients are, for example, water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, etc. and combinations thereof. Other suitable carriers are well known to those skilled in the art. In addition, if desired, the composition may contain small amounts of auxiliary substances such as wetting or emulsifying agents, pH buffers.

[0322] The pharmaceutical composition according to the present invention can be administered together with an anti-tumor composition.

[0323] As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic measures. Those in need of treatment include those already suffering from the disorder as well as those in which the disorder is to be prevented.

[0324] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, epithelial cancers, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of these cancers include melanoma, lung cancer, thyroid cancer, breast cancer, colon cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, cervical cancer, pancreatic cancer, leukemia, lymphoma, bone marrow cancer, ovarian cancer, uterine cancer, sarcoma, bile duct cancer, or endometrial cancer.

[0325] According to certain embodiments, the method of treating cancer comprises administering the pharmaceutical composition as part of a treatment regimen that includes administering at least one additional anti-cancer agent.

[0326] According to certain embodiments, the anticancer agent is selected from the group consisting of antimetabolites, taxanes, topoisomerase inhibitors, topoisomerase II inhibitors, asparaginases, alkylating agents, antitumor antibiotics and combinations thereof. Each possibility represents a separate embodiment of the present invention.

[0327] According to certain embodiments, the antimetabolite is selected from cytarabine, fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine and hydroxyurea. According to certain embodiments, the mitotic inhibitor is selected from vincristine, vinblastine and vinorelbine. According to certain embodiments, the topoisomerase inhibitor is selected from topotecan and irinotecan. According to certain embodiments, the alkylating agent is selected from busulfan, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosfamide, dichloromethyl diethylamine, melphalan, thiotepa, dacarbazine and procarbazine. According to certain embodiments, the antitumor antibiotic is selected from bleomycin, actinomycin D, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone and plicamycin. According to certain embodiments, the topoisomerase II inhibitor is selected from etoposide and teniposide. Each possibility represents an independent embodiment of the present invention.

[0328] According to certain 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.

[0329] The monoclonal antibodies according to the present invention may be used as part of a combination therapy with at least one anticancer agent. According to certain embodiments, the additional anticancer agent is an immunomodulator, activated lymphocytes, a kinase inhibitor or a chemotherapeutic agent.

[0330] According to certain embodiments, the anti-cancer agent is an immunomodulatory agent that acts as an agonist or antagonist, such as an antibody directed against an immune checkpoint molecule.

[0331] Checkpoint immunotherapy blockade has proven to be an exciting new venue for cancer treatment. Immune checkpoint pathways consist of a series of co-stimulatory and inhibitory molecules that work together to maintain self-tolerance and protect tissues from immune system damage under physiological conditions. Tumors exploit certain checkpoint pathways in order to evade the immune system. Therefore, inhibition of these pathways has emerged as a promising anti-cancer therapeutic strategy.

[0332] The anti-cytotoxic T lymphocyte 4 (CTLA-4) antibody ipilimumab (approved in 2011) was the first immunotherapeutic agent to show benefit in the treatment of cancer patients. The antibody interferes with inhibitory signals during antigen presentation to T cells. The anti-programmed cell death 1 (PD-1) antibody pembrolizumab (approved in 2014) blocks negative immune regulatory signaling by the PD-1 receptor expressed by T cells. Another anti-PD-1 agent was submitted for regulatory approval in 2014 for the treatment of non-small cell lung cancer (NSCLC). Active research is currently exploring many other immune checkpoints, including CEACAM1, NKG2A, B7-H3, B7-H4, VISTA, CD112R, lymphocyte activation gene 3 (LAG3), CD137, OX40 (also known as CD134), and killer cell immunoglobulin-like receptors (KIRs).

[0333] According to certain specific embodiments, the immunomodulator is selected from: antibodies that inhibit CTLA-4, anti-human programmed cell death protein 1 (PD-1), PD-L1 and PD-L2 antibodies, activated cytotoxic lymphocytes, lymphocyte activators, antibodies against CEACAM, antibodies against TIGIT and RAF / MEK pathway inhibitors. Each possibility represents an independent embodiment of the present invention. According to certain specific embodiments, the additional immunomodulator is selected from mAbs against PD-1, mAbs against PD-L1, mAbs against PD-L2, mAbs against CEACAM1, mAbs against CTLA-4, mAbs against TIGIT, PVR, interleukin 2 (IL-2) or lymphokine-activated killer (LAK) cells.

[0334] According to other embodiments, the additional anticancer agent is a chemotherapeutic agent. Chemotherapeutic agents that can be administered together or separately with the antibody according to the present invention may include any such agent known in the art that exhibits anticancer activity, including but not limited to: mitoxantrone, topoisomerase inhibitors, spindle toxins Vinca roseus: vinblastine, vincristine, vinorelbine (taxol), paclitaxel, docetaxel; alkylating agents: dichloromethyl diethylamine, chlorambucil, cyclophosphamide, melphalan, ifosfamide; methotrexate; 6- Mercaptopurine; 5-fluorouracil, cytarabine, gemcitabine; podophyllotoxins: etoposide, irinotecan, topotecan, dacarbazine; antibiotics: doxorubicin (adriamycin), bleomycin, mitomycin; nitrosoureas: carmustine (BCNU), lomustine, epirubicin, idarubicin, daunorubicin; inorganic ions: cisplatin, carboplatin; interferon, asparaginase; hormones: tamoxifen, leuprolide, flutamide and megestrol acetate.

[0335] According to certain embodiments, the chemotherapeutic agent is selected from alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxins, antibiotics, L-asparaginase, topoisomerase inhibitors, interferons, platinum coordination complexes, ureas substituted with anthracenediones, methylhydrazine derivatives, adrenocortical hormone inhibitors, adrenocortical steroids, progestins, estrogens, antiestrogens, androgens, antiandrogens and gonadotropin-releasing hormone analogs. According to another embodiment, the chemotherapeutic agent is selected from 5-fluorouracil (5-FU), leucovorin (LV), irinotecan, oxaliplatin, capecitabine, paclitaxel and docetaxel. One or more chemotherapeutic agents can be used.

[0336] In certain embodiments, the pharmaceutical composition according to the present invention is used to treat cancer or to enhance immune response.

[0337] The term "enhancing immune response" refers to increasing the responsiveness of the immune system and inducing or prolonging its memory. The pharmaceutical composition according to the present invention can be used to stimulate the immune system after vaccination. Therefore, in one embodiment, the pharmaceutical composition can be used to improve vaccination.

[0338] In certain embodiments, the cancer is selected from lung cancer, thyroid cancer, breast cancer, colon cancer, melanoma, prostate cancer, liver cancer, bladder cancer, kidney cancer, cervical cancer, pancreatic cancer, leukemia, lymphoma, bone marrow cancer, ovarian cancer, uterine cancer, sarcoma, bile duct cancer and endometrial cancer. Each possibility represents an independent embodiment of the present invention.

[0339] According to certain embodiments, a pharmaceutical composition comprising at least one antibody or fragment thereof according to the present invention and a pharmaceutical composition comprising an additional immunomodulator or kinase inhibitor are used for treating cancer by separate administration.

[0340] According to another aspect, the present invention provides a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the monoclonal antibody or antibody fragment according to the present invention.

[0341] According to another aspect, the present invention provides a method for treating a disease associated with overexpression of connexin 4.

[0342] According to one aspect, the present invention provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a plurality of T-cells comprising a CAR molecule described herein.

[0343] As used herein, the term "effective amount" refers to an amount of the monoclonal antibody or antibody fragment sufficient to have a target therapeutic effect when administered to a subject. The effective amount required to achieve the final result of the treatment may depend on a variety of factors, including, for example, the specific type of tumor and the severity of the patient's condition, and whether the combination is further co-administered with radiation. In the context of the present invention, the effective amount (dose) of the active agent should be sufficient to achieve a beneficial therapeutic response in the subject over time, including but not limited to inhibition of tumor growth, reduction in tumor growth rate, prevention of tumor and metastatic tumor growth, and improved survival.

[0344] The toxicity and therapeutic efficacy of the compositions described herein can be determined in cell culture or experimental animals by standard pharmaceutical procedures, for example, by determining the IC50 (concentration providing 50% inhibition) and the highest tolerated dose of the subject compound. The data obtained from these cell culture assays and animal studies can be used to formulate a variety of doses used in humans. The dosage may vary, in particular, with the dosage form used, the selected dosage regimen, the composition of the medicament used for treatment, the route of administration used, and other relevant factors. The precise dosage form, route of administration, and dosage can be selected by each physician according to the patient's condition. Depending on the severity and responsiveness of the condition to be treated, administration can also be a single administration of a sustained-release composition, and the course of treatment lasts for several days to several weeks or until a cure is achieved or the reduction of the disease state is achieved. The amount of the composition to be administered certainly depends on the object to be treated, the severity of the disease, the mode of administration, the judgment of the prescribing physician, and all other relevant factors.

[0345] The term "administering" a substance, compound or agent to a subject can be performed using one of various methods known to those skilled in the art. For example, a compound or agent can be administered enterally or parenterally. Enteral refers to administration through the gastrointestinal tract, including oral, sublingual or rectal administration. Parenteral administration includes intravenous, intradermal, intramuscular, intraperitoneal, subcutaneous, ocular, sublingual, intranasal, by inhalation, intraspinal, intracerebral and transdermal (absorbed by, for example, a percutaneous catheter). Compounds or agents can also be suitably introduced by refillable or biodegradable polymer devices or other devices such as patches and pumps, or by providing a dosage form that prolongs, slowly or controls the release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more extended time periods. In certain embodiments, the administration includes both direct administration (including self-administration) and indirect administration (including the action of prescribing a drug). For example, when used herein, a physician who instructs a patient to self-administer a drug or administers a drug by another person and / or a physician who provides a drug prescription to a patient is administering the drug to the patient.

[0346] The antibody is typically administered in a dosage range of about 0.1 to about 20 mg / kg patient body weight, typically about 0.5 to about 10 mg / kg, typically about 1 to about 5 mg / kg. In this regard, antibodies with a circulation half-life of at least 12 hours, preferably at least 4 days, more preferably up to 21 days are preferably used. Chimeric antibodies are expected to have a circulation half-life of up to 14-21 days. In some cases, it may be advantageous to administer a large loading dose followed by regular (e.g., weekly) maintenance doses during the treatment period. The antibody may also be delivered by sustained release delivery systems, pumps, and other known delivery systems for continuous infusion.

[0347] The term "about" means that an acceptable error range of, for example, up to 5% or 10% should be assumed for the particular value.

[0348] diagnosis

[0349] The invention also discloses methods for diagnosing and prognosing cancer.

[0350] According to one aspect, the present invention provides a method for diagnosis and / or prognosis of cancer or a sensory disease in a subject, the method comprising the step of determining the expression level of connexin 4 in a biological sample of the subject using at least one antibody described herein.

[0351] The term "biological sample" encompasses a variety of sample types obtained from an organism that can be used for diagnostic or monitoring assays. The term encompasses blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures, or cells and their progeny derived therefrom. In addition, the term encompasses circulating tumors or other cells. The term particularly encompasses clinical samples, and also includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, urine, amniotic fluid, biological fluids and tissue samples including aqueous humor and vitreous humor for ocular samples. The term also encompasses samples that have been treated in any way after acquisition, such as samples treated with reagents, solubilized, or enriched for certain components.

[0352] Determining the expression level of connexin 4 can be performed by using the labeled anti-connexin 4 antibodies described herein. Determining the expression can be performed, for example, by ELISA. The method of the present invention can also include the step of comparing the expression level with a control level.

[0353] The following examples are presented to more fully illustrate certain embodiments of the present invention. They should in no way be construed as limiting the scope of the present invention.

[0354] Example

[0355] Experimental Procedure

[0356] Reference will now be made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

[0357] 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 relating to well-known procedures are provided throughout this document.

[0358] method

[0359] Cell lines

[0360] The cell lines used were LNCap, JEG3, MCF-7, RAJI, MDA-MD-453, HT1376, and CHO cells. In some cases, CHO cells were stably transfected with Cynomolgus monkey (Cyno) or murine connexin-4. For all cells except RAJI cells, cells were maintained at 37°C, >95% humidity, and 5% CO. 2 The cells were grown in DMEM under normal conditions, while RAJI cells were cultured in RPMI supplemented with 10% heat-inactivated FCS (medium and serum were from Sigma-Aldrich).

[0361] Flow cytometry

[0362] Flow cytometry was performed using anti-murine connexin 4 mAb (clone 356704). Cells were incubated with 0.2 μg mAb per 100,000 cells for 30 minutes on ice. Detection was performed using a goat secondary antibody conjugated to AlexaFluor 647 (Jackson ImmunoResearch) for 30 minutes on ice.

[0363] In some cases, anti-connexin-4 Ab was used at various concentrations and incubated with target cells on ice for 30 minutes. Detection was performed using secondary antibodies against murine or human Fc conjugated to AlexaFluor 647 (Jackson ImmunoResearch) on ice for 30 minutes.

[0364] For staining of human TIGIT-Ig or human connexin 1-Ig, cells were incubated on ice for 1 hour with 3 μg of TIGIT-Ig per 100,000 cells. Detection was performed on ice for 30 min using Alexa Fluor 647 anti-human secondary antibody (Jackson ImmunoResearch). For blocking experiments, cells were pre-incubated with 1 μg of the indicated antibody before staining with TIGIT-Ig or human connexin 1-Ig. In some cases, cells were incubated with 8 μg / ml of the indicated Ab and human TIGIT-Ig or human connexin-1-Ig (20 μg / ml). Detection of ligand binding was performed using Alexa Fluor 647 anti-human secondary antibody (Jackson ImmunoResearch).

[0365] Analysis was performed using a FACS-Calibur (BD Biosciences) or Cytoflex Becman Coulter flow cytometer and FCS express software.

[0366] Killing assay

[0367] To evaluate the cytotoxic activity of NK cells against target cells, S 35 Release assay (Mandelboim et al., Exp. Med. 184(3):913-22). NK cells were isolated from healthy donors using the EasySep Human NK Isolation Kit (19055 STEMCELL TECHNOLOGIES) and grown with PHA and IL-2. Target cells were incubated in a 4% culture medium containing radioactive methionine [S 35 ] in methionine-free medium overnight. Next, the cells were washed and incubated on ice with 1 μg of antibody per 5000 cells per well. The cells were then incubated with NK cells for 5 hours. 35 The release was measured using a beta counter TopCount (Packard). The results were expressed as: (CPM(sample)-CPM(spontaneous release)) / (CPM(total release)-CPM(spontaneous release))×100, where CPM means counts per minute.

[0368] ADCC assay

[0369] NK cells were isolated from healthy donors using the EasySep Human NK Isolation Kit (19055 STEMCELL TECHNOLOGIES) and grown with PHA and IL-2. Target cells were plated at 2.5*10 4The density of cells was plated in 96U plates and co-incubated with activated NK cells at an E:T ratio of 2: 1. The incubation was carried out in the presence of 12 μg / ml chimeric clones hNec4.05hIgG1 and hNec4.11hIgG1 or control hIgG1. After 2 hours, the expression of the CD107a (Biolegend, catalog number 328619) degranulation marker of NK cells was analyzed by FACS.

[0370] CAR-T generation and functional assays

[0371] The single chain of hNec4.11 Ab was cloned in frame into the CD8 stem region, followed by the CD28TM domain, the 41BB intracellular domain, and the intracellular domain of the CD3ζ chain. A schematic diagram of the CAR-T construct is shown in Fig.11A The construct was introduced into a lentiviral vector (pHAGE2) containing the hEf1a promoter, and then an IRES GFP cassette was introduced to monitor transduction efficiency. Jurkat cells were transduced with lentiviral particles encoding the construct. The transduction efficiency was greater than 99% ( Fig. 11B ).

[0372] Parental Jurkat cells or Jurkat cells expressing the CAR-T construct (Jurkat pHAGE2.4.11) (5*10 4 ) were incubated with target cells HT1376 and MDA-MD-453 at an E:T ratio of 1: 1 for 48 hours. The supernatant was collected after centrifugation and IL-2 levels were measured using Peprotech's IL-2 ELISA kit (Catalog No. 900-T12) according to the manufacturer's protocol.

[0373] Using ImmunoCult TM Human CD3 / CD28 T cell activator, PBMC from healthy donors were preactivated for 72 hours according to the manufacturer's protocol. The cells were transduced with pHAGE2.4.11 lentivirus as described by Kochenderfer JN et al. (JImmunother.2009doi:10.1097 / CJI.0b013e3181ac6138), and expression was confirmed by GFP level. The CART PBMCs were mixed with HT1376 (2.5*10 4 The cells were incubated at various E:T ratios. After 48 hours, the effector cells were removed, the target cells were washed three times, and the cells were cultured using CellTiter- Luminescent Cell Viability Assay Viability was measured following the manufacturer's protocol.

[0374] In vivo mouse tumor model

[0375] All experiments were performed using 6-8 week old SCID light brown female mice. All mice were raised in a specific pathogen-free facility at the Hebrew University Medical School (Ein-Kerem, Jerusalem) and in accordance with the guidelines of the Ethics Committee under SPF conditions, normal light / dark cycles and 22+ / -2°C. Each mouse group contained 7 female mice (n=7). Xenografts were produced by subcutaneous injection of the indicated cells into the left flank area. Anti-connexin 4 antibody clone 05 and control (anti-murine CD3 antibody, InVivoMAb clone 17A2) antibody injections were administered intraperitoneally twice a week. The mice were monitored every day. On the day when the assay ended (see legend), all mice were killed and the weight of each tumor was recorded. At baseline, no differences were observed in overall health between the various different mouse groups.

[0376] Example 1. Generation and selection of anti-connexin-4 mAbs

[0377] The immunogen (Nec4-Fc) expression technology is based on mammalian HEK 293T cells and is a technology of choice that gives the best quality, stability, solubility and yield, especially in the case of glycoproteins. Nec4-Fc protein, a fusion protein of the extracellular domain of connexin-4 with the human IgG1 Fc domain, was produced and purified by recombinant methods as described below:

[0378] The coding sequence of human connexin 4 is cloned into the Fc fragment fusion with human IgG1 to produce recombinant Fc-fusion protein. The extracellular domain (extracellular) part of human connexin 4 molecule is used, which spans the 32nd to 349th residues. The C-terminal serine residue of connexin 4 amino acid sequence at the 349th position is fused to the heavy chain hinge of the deglycosylated Fc (N297A) of human IgG1, followed by CH2 and CH3 constant regions. The open reading frame (ORF) of the recombinant protein is codon optimized for high-level expression in mammalian cells. The optimized DNA sequence is produced by GeneArt synthesis service (Invitrogen), and the flanking DNA sequences corresponding to EcoRI and NotI restriction sites are added to the 5' and 3' ends of the DNA fragment, respectively. By double digesting the optimized DNA fragment with EcoRI and NotI, and then connecting it to pIRESpuro3 (Clontech Laboratories, Inc.), an expression vector is constructed. The resulting construct was transfected into HEK-293T cells using FuGENE 6 transfection reagent (Roche Diagnostics). After 48 h, 5 g / mL puromycin (Sigma-Aldrich) was used to select the transfected cells for antibiotics. Protein secretion of the stable amalgam was analyzed by SDS / PAGE. The supernatant was collected and purified on a Poros 20 protein G column in a high pressure perfusion chromatography station BioCAD (PerSeptive Biosystems). The resulting fusion protein immunogen was referred to as connexin 4-Fc.

[0379] For immunization, BALB / c mice were injected with 50 μg of immunogen in Freund's complete adjuvant (CFA), followed by 50 μg of immunogen in Freund's incomplete adjuvant (IFA) on the 14th day after the first immunization. Next, the anti-Nec-4-Fc antibody titer of the serum was analyzed by ELISA. The mice with the highest titer were boosted with 50 μg of immunogen in PBS. Three days later, the spleen of the immunized mice was taken out, and after lysing the red blood cells, the splenocytes were fused with the SP2 / 0 cell line. Potential hybridoma cells were inoculated in 20% RPMI 1640 culture medium containing hypoxanthine, aminopterin and thymidine (HAT) for the selection of stable hybridoma cell lines. The above procedure was repeated twice, and a total of 1034 wells were screened for anti-Nec4-Fc antibody secretion by ELISA. Next, the 30 wells that were positive for binding of the supernatant to the coated Nec4-Fc on the ELISA plate were retested for their positivity and cross-reactivity was tested in parallel on an unrelated Fc fusion protein. This yielded 10 hybridoma cell lines that secreted antibodies that specifically recognized the extracellular domain of connexin-4. All of these candidates that showed a specific signal in the ELISA were tested for their ability to recognize native human connexin-4 on the transfected cell line.

[0380] RAJI Burkitt lymphoma cells were transfected with human connexin 4 and analyzed by FACS to determine the binding of the indicated 5 hybridoma clones. Cells were incubated with the different clones and then incubated with human TIGIT-Ig and stained with secondary antibodies. The mean fluorescence intensity (MFI) values ​​of the FACS staining are depicted in Figure 2 As shown, monoclonal antibodies generated from clones hNec4.01 (clone 1) and hNec4.05 (clone 5) showed the best blocking ability of TIGIT-connexin4 interaction.

[0381] Similar assays were also performed using JEG3 and LNCap cell lines that naturally express connexin 4, and similar results were obtained.

[0382] 5 kinds of antibodies show positive staining.Among these 5 kinds of antibodies, 4 kinds are further selected to obtain their IgG isotype (rather than IgM isotype).Finally, the 4 remaining candidates all show strong binding ability to the native human connexin-4 molecules expressed on the surface of living cells, and repeated tests are carried out on several unrelated fusion proteins to select the antibody without cross-reactivity between connexin-4 and other parts of immune cell receptors.However, only 2 kinds of blocking antibodies for connexin-4 are shown among them.Therefore, 2 stable clonal cell lines hNec4.01 and hNec4.05 are produced, both of which are isotype κ IgG1.Next, large-scale Ab production is carried out, and two monoclonal antibodies are purified from serum-free medium with several milligrams using GE AKTA PrimePlus liquid chromatography system and HiTrap protein G column.

[0383] Similar to above, additional clones were identified, of which clone 11 (hNec4.11) had the best binding and blocking capabilities and was therefore screened in parallel with hNec4.05 as described below.

[0384] Based on the above attempts it can be concluded that even in case the immunogen is very similar to the target protein Connexin-4 (in terms of being a dimer, glycosylated by the mammalian cell machinery and produced under non-denaturing conditions), the chance of obtaining a hybridoma cell line secreting blocking anti-Connexin-4 antibodies is less than 2% (per ml).

[0385] Example 2. Affinity of anti-connexin-4 mAbs for human connexin 4

[0386] The affinity of antibodies hNec4.01 and hNec4.05 for fluorophore-labeled human connexin 4-Ig molecules was further determined using microthermophoresis (Wienken et al., 2010, Nat. Commun. 1:100). The measurements were repeated using at least three independent protein preparations. Figure 3 As shown in , very high binding affinities were observed for both antibodies. The calculated Kd was 272±154 pM for clone hNec4.05 and 107±115 pM for clone hNec4.01.

[0387] Example 3. Sequencing of anti-connexin-4 mAb

[0388] The two hybridoma clones No.0.1 and No.0.5 that were confirmed to have the best inhibition of connexin 4-TIGIT binding were sent for nucleotide and amino acid sequencing.

[0389] method

[0390] Follow Total RNA was isolated from the hybridoma cells using the PrimeScript TM Total RNA was reverse transcribed into cDNA using isoform-specific antisense primers or universal primers according to the technical manual of the First Strand cDNA Synthesis Kit (Takara, catalog number 6110A). V was amplified according to the standard operating procedure (SOP) of GenScript's Rapid Amplification of cDNA Ends (RACE). H and V L The antibody fragments of the amplification were cloned into standard cloning vectors. Colony PCR was performed to screen clones with inserts of the correct size. For each fragment, no less than 5 clones with inserts of the correct size were sequenced. The sequences of different clones were compared and the consensus sequences of these clones were provided.

[0391] The following tools for sequence analysis of immunoglobulin variable regions were used:

[0392] i. NCBI nucleotide BLAST;

[0393] ii.IMGT / V Quest Program; and

[0394] iii.NCBI IgBLAST.

[0395] The obtained sequence is:

[0396] Clone hNec4.01:

[0397] Heavy chain variable region DNA sequence, 414 base pairs (SEQ ID NO: 1):

[0398] Preamble sequence - FR1 -CDR1- FR2 -CDR2- FR3 -CDR3- FR4 ATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGCAGGCTTCCACTCT GAGGTCCAACTTCAGCAGTCAGGACCTGAACTGGTGAAACCTGGGGCCT CAGTGAAGATTGCCTGCAGGGCCTCTGGATACACATTCACT GCCTACAATATCCAC TGGGTGAGCCAGAGACATGGA AAGAGCCTTGAATGGATTGGA TATATCTATCCTAACAATGGTGGTTCTGGCTACAACCAGAAATTCATGAAC AAGGC CACATTGACTGTAGACCATTCCTCCAATACAGCCTACATGGAGCTCCGCAGCCTGACGTCTGAGGACTCTGCAGTCT ATTACTGTGCAATA TTTGATTACGACGAGGCCTGGTTTATTTAC TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0399] Heavy chain variable region polypeptide sequence, 138 amino acids (SEQ ID NO: 2):

[0400] Preamble sequence - FR1 -CDR1- FR2 -CDR2- FR3 -CDR3- FR4 MGWSWIFLFLLSGTAGFHS EVQLQQSGPE LVKPGASVKIACRASGYTFT AYNIH WVSQRHGKSLEWIG YIYPNNGGSGYNQKFMN KATLTVDHSSNTAYMELRSLT SEDSAVYYCAI FDYDEAWFIY WGQGTLVTVSA

[0401] Heavy chain variable region polypeptide sequence ( FR1 -CDR1- FR2 -CDR2- FR3 -CDR3- FR4 (SEQ ID NO: 35)

[0402] EVQLQQSGPELVKPGASVKIACRASGYTFTAYNIHWVSQRHGKSLEWIGYIYPNNGGSGYNQKFMNKATLTVDHSSNTAYMELRSLTSEDSAVYYCAIFDYDEAWFIYWGQGTLVTVSA

[0403] Light chain variable region DNA sequence, 384 base pairs (SEQ ID NO: 3):

[0404] Preamble sequence - FR1 -CDR1- FR2 -CDR2- FR3 -CDR3- FR4

[0405] ATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATGTCCAGAGGA GAA AATGTTCTCACCCAGTCTCCAGAAATCATGTCTGCATCTCCCGGGGAAGAGGTCACCATGACCTGT AGTGCCAGCTCAAGTGTTAGTTACATGCAC TGGTTCCAGCAGAAGTCAACTATCTCCCAAACTCTGGATTTAT GACACATCCAAACTGGCTTCT GGAGTCCCCGGTCGCTTCAGTGGCAGTGGGTCTGGCAAGTCTTACTCTCTCACGATCAGAAACATGGAG GCTGAAGATGTTGCCACCTATTACTGT TTTCAGGGGAGTGGGAGCCCGTACACG TTCGGAGGGGGGACCAAGCTGGA AATTAAA

[0406] Light chain variable region polypeptide sequence, 128 amino acids (SEQ ID NO: 4):

[0407] Preamble sequence - FR1 -CDR1- FR2 -CDR2- FR3 -CDR3- FR4 MDFQVQIFSFLLISASVIMSRG ENVLTQS PEIMSASPGEEVTMTC SASSSVSYMH WFQQKSTISPKLWIY DTSKLAS GVPGRFSGSGSGKSYSLTIRNMEAEDVAT YYC FQGSGSPYT FGGGTKLEIK

[0408] Light chain variable region polypeptide sequence ( FR1 -CDR1- FR2 -CDR2- FR3 -CDR3- FR4 (SEQ ID NO: 36)

[0409] ENVLTQSPEIMSASPGEEVTMTCSASSSVSYMHWFQQKSTISPKLWIYDTSKLASGVPGRFSGSGSGKSYSLTIRNMEAEDVATYYCFQGSGSPYTFGGGTKLEIK

[0410] Clone hNec4.05

[0411] Heavy chain variable region DNA sequence, 408 base pairs (SEQ ID NO: 5):

[0412] Preamble sequence - FR1 -CDR1- FR2 -CDR2- FR3 -CDR3- FR4 ATGGGATGGAGCCGGATCTTTCTCTTCCTCCTGTCAATAATTGCAGGTGTCCATTGC CAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTT <h2 style=";text-align:left;direction:ltr">CAGTGAGGATATCCTGCAAGGCCTCTGGCTACACCTTCACA ACCTACTATATACAC <h2 style=";text-align:left;direction:ltr"> TGGGTGAAGCAGAGGCCTGGA <h2 style=";text-align:left;direction:ltr"> CAGGGACTTGAGTGGATTGGA TGGATTTATCCTGGAAATGTTAATACTAAGAACAATGAGAAGTTCAAGGTC <h2 style=";text-align:left;direction:ltr"> AAGGC <h2 style=";text-align:left;direction:ltr"> CACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGGTCT <h2 style=";text-align:left;direction:ltr"> ATTTCTGTGCAAGA TCGAACCCCTATGTTATGGACTAC <h2 style=";text-align:left;direction:ltr"> TGGGGTCAGGGAACCTCAGTCACCGTCTCCTCA

[0413] Heavy chain variable region polypeptide sequence, 136 amino acids (SEQ ID NO: 6):

[0414] Preamble sequence - <h2 style=";text-align:left;direction:ltr"> FR1 -CDR1- <h2 style=";text-align:left;direction:ltr"> FR2 -CDR2- <h2 style=";text-align:left;direction:ltr"> FR3 -CDR3- <h2 style=";text-align:left;direction:ltr"> FR4 MGWSRIFLFLLSIIAGVHC <h2 style=";text-align:left;direction:ltr"> QVQLQQSGPE <h2 style=";text-align:left;direction:ltr"> LVKPGASVRISCKASGYTFT TYYIH <h2 style=";text-align:left;direction:ltr"> WVKQRPGQGLEWIG WIYPGNVNTKNNEKFKV <h2 style=";text-align:left;direction:ltr"> KATLTADKSSSTAYMQLSSL <h2 style=";text-align:left;direction:ltr"> TSEDSAVYFCAR SNPYVMDY <h2 style=";text-align:left;direction:ltr"> WGQGTSVTVSS Heavy chain variable region polypeptide sequence ( <h2 style=";text-align:left;direction:ltr"> FR1 -CDR1- <h2 style=";text-align:left;direction:ltr"> FR2 -CDR2- <h2 style=";text-align:left;direction:ltr"> FR3 -CDR3- <h2 style=";text-align:left;direction:ltr"> FR4(SEQ ID NO:37)

[0415] QVQLQQSGPELVKPGASVRISCKASGYTFTTYYIHWVKQRPGQGLEWIGWIYPGNVNTKNNEKFKVKATLTADKSSSTAYMQLSSLTSEDSAVYFCARSNPYVMDYWGQGTSVTVSS

[0416] Light chain variable region DNA sequence, 381 base pairs (SEQ ID NO: 7):

[0417] Preamble sequence - <h2 style=";text-align:left;direction:ltr"> FR1 -CDR1- <h2 style=";text-align:left;direction:ltr"> FR2 -CDR2- <h2 style=";text-align:left;direction:ltr"> FR3 -CDR3- <h2 style=";text-align:left;direction:ltr"> FR4

[0418] ATGAAGTCACAGACCCAGGTCTTCGTATTTCTACTGCTCTGTGTGTCTGGTGCTCATGGG <h2 style=";text-align:left;direction:ltr"> AGTATTGTG <h2 style=";text-align:left;direction:ltr"> ATGACCCAGAACTCCCAAATTCCTGCTTGTATCAGCAGGAACAGGGTTACCATAACCTGC AAGGCCAGTCAGAGTGTGAGTAATGATGTAGCT <h2 style=";text-align:left;direction:ltr"> TGGTACCAACAGAAGCCAGGCAGTCTCCTAAACTGCTGATATAC TATGCATCCAATCGCTACACT <h2 style=";text-align:left;direction:ltr"> GGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCGCTGTGCAGGCT <h2 style=";text-align:left;direction:ltr"> GAAGACCTGGCAGTTTATTTCTGT CAGCAGGATTATAGCTCTCCGTACACG <h2 style=";text-align:left;direction:ltr"> TTCGGAGGGGGGACCAAGCTGGAAAT <h2 style=";text-align:left;direction:ltr"> AAAA

[0419] Light chain variable region polypeptide sequence, 127 amino acids (SEQ ID NO: 8):

[0420] Preamble sequence - <h2 style=";text-align:left;direction:ltr"> FR1-CDR1- <h2 style=";text-align:left;direction:ltr"> FR2 -CDR2- <h2 style=";text-align:left;direction:ltr"> FR3 -CDR3- <h2 style=";text-align:left;direction:ltr"> FR4

[0421] MKSQTQVFVFLLLCVSGAHG <h2 style=";text-align:left;direction:ltr"> SIVMTQTPKFLLVSAGDRVTITC KASQSVSNDVA <h2 style=";text-align:left;direction:ltr"> WYQQKPGQSPKLLIY YASNRYT <h2 style=";text-align:left;direction:ltr"> GVPDRFTGSGYGTDFTFTISAVQAEDLAVYFC QQDYSSPYT <h2 style=";text-align:left;direction:ltr"> FGGGTKLEIK

[0422] Light chain variable region polypeptide sequence ( <h2 style=";text-align:left;direction:ltr"> FR1 -CDR1- <h2 style=";text-align:left;direction:ltr"> FR2 -CDR2- <h2 style=";text-align:left;direction:ltr"> FR3 -CDR3- <h2 style=";text-align:left;direction:ltr"> FR4(SEQ ID NO:38)

[0423] SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISAVQAEDLAVYFCQQDYSSPYTFGGGTKLEIK

[0424] Cloning hNec4.11

[0425] Heavy chain variable region DNA sequence, 408 base pairs (SEQ ID NO: 21):

[0426] Preamble sequence - <h2 style=";text-align:left;direction:ltr"> FR1 -CDR1- <h2 style=";text-align:left;direction:ltr"> FR2 -CDR2- <h2 style=";text-align:left;direction:ltr"> FR3 -CDR3- <h2 style=";text-align:left;direction:ltr"> FR4

[0427] ATGGGATGGAGCCGGATCTTTCTCTTCCTCCTGTCAATAATTGCAGGTGTCCATTGC <h2 style=";text-align:left;direction:ltr"> CAGGTCCAGCTG <h2 style=";text-align:left;direction:ltr"> CAGCAGTCTGGACCTGAACTGGTGAAGCCTGAGACTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACCTTCAC A AGTTACTATATACAC <h2 style=";text-align:left;direction:ltr"> TGGGTGAAACAGAGGCCTGGACAGGGACTTGAGTGGATTGGC TGGATTTATCCTGGAAATGTTAATACTAAGTATAATGAGAGGTTTAAGGGC <h2 style=";text-align:left;direction:ltr"> AAGGCCACTCTGACTGCAGACAAATCCCTCCAACACAGCCCACATG <h2 style=";text-align:left;direction:ltr"> CAGCTCACCAGCCTGACCCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAGA TCGAACCCCTATGTTATGGACTAC <h2 style=";text-align:left;direction:ltr"> TG <h2 style=";text-align:left;direction:ltr"> GGGTCAAGGAACCTCAGTCACCGTCTCCTCA

[0428] Heavy chain variable region polypeptide sequence, 136 amino acids (SEQ ID NO: 22):

[0429] Preamble sequence - <h2 style=";text-align:left;direction:ltr"> FR1-CDR1- <h2 style=";text-align:left;direction:ltr"> FR2 -CDR2- <h2 style=";text-align:left;direction:ltr"> FR3 -CDR3- <h2 style=";text-align:left;direction:ltr"> FR4

[0430] MGWSRIFLFLLSIIAGVHC QVQLQQSGPELVKPETSVKISCKASGYTFT YY WVKQRPGQGLEWIG WIYPGNVNTKYNERFKG KATLTADKSSNTAHMQLTSLTSEDSAVYFCAR SNPYVMDY WGQGTSVTVSS

[0431] Heavy chain variable region polypeptide sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (SEQ ID NO: 39)

[0432] QVQLQQSGPELVKPETSVKISCKASGYTFTSYYIHWVKQRPGQGLEWIGWIYPGNVNTKYNERFKGKAT LTADKSSNTAHMQLTSLTSEDSAVYFCARSNPYVMDYWGQGTSVTVSS

[0433] Light chain variable region DNA sequence, 381 base pairs (SEQ ID NO: 23):

[0434] Preamble sequence - FR1 -CDR1- FR2 -CDR2- FR3 -CDR3- FR4

[0435] ATGAAGTCACAGACCCAGGTCTTCGTATTTCTACTGCTCTGTGTGTCTGGTGCTCATGGG AGTATTGTG ATGACCCAGACTCCCAAATTCCTGCTTGTATCAGCAGGAGACAGAGTCACCATAACCTGCAAGGCCAGTCAGAGTGT GAATAATGATGTGGCTTGGTATCAACAGAAGCCAGGGCTGTCTCCTGAACTGCTTATGTAT TATGCATCCAATCGCTTCACT GGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCTCTGTGCAGGCT GAAGACCTGGCAATTTATTTCTGT CAGCAGGCTTATAGGTCTCCGTACACG TTCGGAGGGGGGACCAAGCTGGAAAT TCAA

[0436] Light chain variable region polypeptide sequence, 127 amino acids (SEQ ID NO: 24):

[0437] Preamble sequence - FR1 -CDR1- FR2 -CDR2- FR3 -CDR3- FR4 MKSQTQVFVFLLLCVSGAHG SIVMTQTPK FLLVSAGDRVTITC KASQSVNNDVA WYQQKPGLSPELLMY YASNRFT GVPDRFTGSGYGTDFTFTISSVQAEDLAIY FC QQAYRSPYT FGGGTKLEIQ

[0438] Light chain variable region polypeptide sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (SEQ ID NO: 40)

[0439] SIVMTQTPKFLLVSAGDRVTITCKASQSVNNDVAWYQQKPGLSPELLMYYASNRFTGVPDRFTGSGYGTDFTFTISSVQAEDLAIYFCQQAYRSPYTFGGGTKLEIQ

[0440] Example 4. Blockade of Connexin 4-TIGIT Interaction with Anti-Connexin 4 mAb Enhances NK Cell Killing of Human Cell Lines

[0441] Antibody clones hNec4.05 and hNec4.01 were tested for their ability to block connexin 4 binding to TIGIT and inhibit NK cell cytotoxicity. 35 S] Methionine-labeled RAJI Burkitt lymphoma cells transfected with connexin 4 and LNCap prostate cancer cells (naturally expressing connexin 4) were incubated with 1 μg / well of mouse IgG1 as a control antibody or mouse anti-human connexin 4 mAb hNec4.01 or hNec4.05. After 1 h, NK cells were added to the cells and incubated for 5 h. The average specific killing (± sd) at various effector cell: target cell (E:T) ratios of NK: cancer cells is plotted in Figure 4A (RAJI cells) and Figure 4B (LNCap cells). * indicates a significant effect of hNec4.01 and hNec4.05 clones compared to control antibody (p<0.05). Each figure shows one representative experiment out of three performed. The same effect was determined when using the MCF-7 breast cancer cell line.

[0442] Example 5. Confirmation of Binding Specificity of Anti-Connexin 4 Antibodies

[0443] As tested using FACS staining and in Figures 5A-5C As demonstrated in , mAbs hNec4.01 and hNec4.05 are specific for human connexin 4 and do not bind to the murine protein. RAJI cells were transfected with murine connexin 4 (black line bar graph). First, RAJI cell lysates transfected with murine connexin 4 were used for Western blot assays using a commercial anti-murine connexin 4 mAb (clone 356704) to confirm murine connexin 4 expression (A). Next, the cells were stained with 0.2 μg of (B) clone hNec4.01 or (C) clone hNec4.05. Both hNec4.01 and hNec4.05 mAbs did not show binding to murine connexin 4.

[0444] Example 6. Anti-Connexin 4 Antibodies Can Block Connexin 4-Connexin 1 Interaction

[0445] The ability of anti-connexin4 to block connexin4-connexin1 interaction was also determined. FACS staining of RAJI cells transfected with connexin4 showed Figures 6A-6C The cells were incubated with 1 μg of ( Fig. 6A ) clone hNec4.01 or ( Figure 6B ) clone hNec4.05 was pre-incubated and then incubated with 3 μg of connexin 1-Ig (black line). Staining without blocking is shown as gray line. The solid gray bar graph is the background control staining of the secondary antibody alone. The conclusion is that the mAb is able to block the connexin 4-connexin 1 interaction, which is suspected to increase the invasiveness of tumors expressing connexin 4.

[0446] Example 7. In vivo model

[0447] The in vivo efficacy of anti-Nectin4 mAbs was determined in an animal model. Cell lines expressing either natural (MDA-MB-453) or recombinant (Raji Nectin4 OE) Nectin4 were injected SC into mice (5 × 10 cells per mouse). 6 SCID beige mice lacking NK, B, and T cells were used. To investigate the contribution of NK cells to tumor cell growth in these models, human NK cells were inoculated at 1×10 6 The amount is co-injected with the tumor cells.

[0448] The in vivo effects of the anti-connexin 4 mAb clone hNec4.05 or a control Ab (anti-murine CD3 antibody, InVivoMAb-clone 17A2) on tumor growth directly or together with NK cells were tested. The mAb was injected IP twice a week at 75 μg per mouse. Tumor weights were measured at the end of the study. As can be seen in Figure 7(A), overexpression (OE) of connexin-4 on Raji cells did not affect their growth compared to the growth of parental cells (EV) transfected with an empty vector. However, in the presence of human NK cells, OE of connexin 4 increased tumor growth, indicating a negative effect of connexin 4 on the NK-mediated tumor suppressor activity of NK cells. As shown in Figure 7B As seen in Figure 2, the addition of anti-connexin4 Ab blocked NK cell inhibition, resulting in reduced tumor growth compared to control Ab treated animals. Figure 7CAs seen in the , these effects were also observed when using a cell line (MDA-MB-453) that naturally expresses connexin 4. In the absence of NK cells, the Ab had no effect, while the Ab significantly enhanced the anti-tumor effect of human NK cells co-injected with tumor cells. Taken together, these experiments show that anti-connexin 4 antibodies such as hNec4.05 mAb can enhance NK cell toxicity in vivo and lead to tumor growth inhibition.

[0449] Example 8. Binding of anti-connexin-4 clones to human, monkey and mouse connexin-4 expressed on cells

[0450] Binding of murine anti-human connexin-4 clones hNec4.05 and hNec4.11 to connexin-4 expressed on the human cell line MDA-MD-453 was assessed by FACS analysis. Fig. 8A Depicted are the EC50 values ​​and maximum binding signals calculated for each clone after titration of Ab binding (range 20-0.01 nM).Similar values ​​were achieved when testing chimeric versions of the murine Ab in which the murine IgG1 Fc chain was replaced with a human IgG1 Fc chain. Figure 8B Depicts the binding of antibodies to CHO cells transfected with cynomolgus monkey (Cyno) connexin-4, Figure 8C Binding of the antibodies to CHO cells transfected with murine connexin-4 is depicted. These data demonstrate that EC50 values ​​for the human target are in the subnanomolar range, which is Figure 3 In the Figure 2, the levels of Kd values ​​presented for clones hNec4.01 and hNec4.05 are shown. In addition, these results confirm the cross-reactivity of these two clones to the monkey (cynomolgus monkey) connexin-4 target, as they bind to it with EC50 values ​​close to the EC50 values ​​calculated for the human target. This may be important for preclinical studies of the Ab. Finally, clone hNec4.11 but not clone hNec4.05 was shown to also cross-react with the murine connexin-4 target. In addition, the EC50 calculated in this case is about 10 times higher than the EC50 calculated for the human target.

[0451] Example 9. Blocking of Connexin-4 Ligands by Anti-Connexin-4 Clones

[0452] Antibody clones hNec4.05 and hNec4.11 blocked the binding of connexin-4 to its ligands TIGIT and connexin-1. Binding of connexin-4 ligands was assessed by FACS analysis. CHO cells transfected with human connexin-4 were incubated with human TIGIT-Ig (9C and 9D) at 20 μg / ml in the presence or absence of anti-connexin-4 clones hNec4.05 (9A and 9B) or clones hNec4.11 (9C and 9D), both at 8 μg / ml. Fig.9A and C) or human connexin-1-Ig ( Fig. 9B and D) incubation. In all cases, strong binding inhibition of the anti-connexin-4 Ab was observed. The residual signal may be caused by the binding of the ligand to other receptors expressed by CHO cells, such as PVR, which is not affected by the anti-connexin-4 Ab. These results indicate that the connexin-4 clones hNec4.05 and hNec4.11 may affect the target cancer cells by blocking the signal transduction of the cell surface connexin-4. In addition, these Abs may also affect effector cells expressing connexin-4 ligands such as the inhibitory ligand TIGIT.

[0453] Example 10. Human IgG1 chimeric Ab clones hNec4.05 and hNec4.1 enhance NK cell activation in the presence of tumor cells.

[0454] The relative expression levels of the degranulation marker CD107a on NK cells are depicted in Figure 11. Human NK cells (effector cells, E) were compared with target cells (T) HT1376 ( Fig. 10A ) and MDA-MD-453( Fig. 10B ) were incubated at an E:T ratio of 2:1. The incubation was carried out in the presence of 12 μg / ml chimeric clones hNec4.05hIgG1, hNec4.11hIgG1 or control hIgG1. After 2 hours, the degranulation and activation state of NK cells were determined by FACS analysis of CD107a expression. The degranulation of NK cells in the presence of control hIgG1 was set to 1 and the corresponding induction fold was calculated. The mean values ​​of 2-3 repeated experiments and their normalized SD are shown. These results indicate the anti-cancer effect of these Abs by enhancing the activity of anti-tumor NK cells. It shows a slight advantage of clone hNec4.11-hIgG1 in ADCC induction compared with clone hNec4.05hIgG1, which is consistent with Fig. 8A This is consistent with the higher maximal binding of clone hNec4.11 shown in .

[0455] Example 11. CAR-T driven by hNec4.11 causes specific T cell activation in the presence of tumor cells expressing connexin-4.

[0456] Fig.11A A schematic diagram of the CAR-T construct is shown in FIG. The transduction efficiency, as determined by GFP expression, was greater than 99% ( Fig. 11B Parental Jurkat cells or Jurkat cells expressing CAR-T constructs with single-chain variable regions (scFV) based on hNec4.11 (Jurkat pHAGE2.4.11) were incubated with target cells HT1376 and MDA-MD-453 (MDA-453) ( Fig. 11C ). IL-2 secretion of Jurkat cells was significantly induced by CAR-T expression. Next, pHAGE2.4.11 lentiviral particles were used to transduce PBMCs ( Fig.11D ). CAR-T PBMCs were incubated with HT1376 cells at various E:T levels. Effector cells were removed 48 hours later and the cells were cultured using CellTiter- Luminescent cell viability assays assessed target cell viability. Killing of target cells was significant. Taken together, these observations point to the possibility of further developing CR-T therapies based on the connexin 4 Abs described herein.

[0457] ScFv sequences of CAR constructs

[0458] Clone 11 - nucleic acid (SEQ ID NO: 31)

[0459] ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTGCATTCACAGGTCCAGCTGCAGCAGTCTGGACCTGAACTGGTGAAGCCTGAGACTTCAGTGAAGATTTCCTGCAAGGCTTCTGGCTACACCTTCACAAGTTACTATATACACTGGGTGAAACAGAGGCCTGGACAGGGACTTGAGTGGATTGGCTGGATTTATCCTGGAAATGTTAATACTAAGTATAATGAGAGGTTTAAGGGCAAGGCCACTCTGACTGCAGACAAATCCTCCAACACAGCCCACATGCAGCTCACCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAGATCGAACCCCTATGTTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGGTGGAGGTGGCTCCGGAGGAGGTGGTTCTGGAGGAGGTGGTTCTGATATCGTGATGACCCAGACTCCCAAATTCCTGCTTGTATCAGCAGGAGACAGAGTCACCATAACCTGCAAGGCCAGTCAGAGTGTGAATAATGATGTGGCTTGGTATCAACAGAAGCCAGGGCTGTCTCCTGAACTGCTTATGTATTATGCATCCAATCGCTTCACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCTCTGTGCAGGCTGAAGACCTGGCAATTTATTTCTGTCAGCAGGCTTATAGGTCTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATTCAA

[0460] Clone 11 - amino acid (SEQ ID NO: 32)

[0461] MGWSCIILFLVATATGVHSQVQLQQSGPELVKPETSVKISCKASGYTFTSYYIHWVKQRPGQGLEWIGWIYPGNVNTKYNERFKGKATLTADKSSNTAHMQLTSLTSEDSAVYFCARSNPYVMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQTPKFLLVSAGDRVTITCKASQSVNNDVAWYQQKPGLSPELLMYYASNRFTGVPDRFTGSGYGTDFTFTISSVQAEDLAIYFCQQAYRSPYTFGGGTKLEIQ

[0462] Clone 5 - nucleic acid (SEQ ID NO: 33)

[0463] ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTGCATTCACAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAGGATATCCTGCAAGGCCTCTGGCTACACCTTCACAACCTACTATATACACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTGGAAATGTTAATACTAAGAACAATGAGAAGTTCAAGGTCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAGATCGAACCCCTATGTTATGGACTACTGGGGTCAGGGAACCTCAGTCACCGTCTCCTCAGGTGGAGGTGGCTCCGGAGGAGGTGGTTCTGGAGGAGGTGGTTCTAGTATTGTGATGACCCAGACTCCCAAATTCCTGCTTGTATCAGCAGGAGACAGGGTTACCATAACCTGCAAGGCCAGTCAGAGTGTGAGTAATGATGTAGCTTGGTACCAACAGAAGCCAGGGCAGTCTCCTAAACTGCTGATATACTATGCATCCAATCGCTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCGCTGTGCAGGCTGAAGACCTGGCAGTTTATTTCTGTCAGCAGGATTATAGCTCTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[0464] Clone 5-amino acid (SEQ ID NO: 34)

[0465] MGWSCIILFLVATATGVHSQVQLQQSGPELVKPGASVRISCKASGYTFTTYYIHWVKQRPGQGLEWIGWIYPGNVNTKNNEKFKVKATLTADKSSSTAYMQLSSLTSEDSAVYFCARSNPYVMDYWGQG TSVTVSSGGGGSGGGGSGGGGSSIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISAVQAEDLAVYFCQQDYSSPYTFGGGTKLEIK

[0466] The description of the foregoing specific embodiments is very sufficient to reveal the overall nature of the present invention, so that others can easily modify and / or adapt these specific embodiments to various different applications by applying current knowledge without excessive experimentation and without departing from the general concept, and therefore, these adaptations and modifications should and are intended to be included in the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the phrases or terms used herein are for the purpose of description rather than limitation.

Claims

1. An isolated monoclonal antibody or an antibody fragment thereof comprising at least an antigen binding portion thereof that binds to connexin 4, wherein the isolated monoclonal antibody or antibody fragment comprises a group of 6 complementary determining regions (CDRs) selected from the following: i. A group of 6 CDRs, wherein: Heavy chain (HC) CDR1 is TYYIH (SEQ ID NO: 15), HC CDR2 is WIYPGNVNTKNNEKFKV (SEQ ID NO: 16), HC CDR3 is SNPYVMDY (SEQ ID NO: 17), light chain (LC) CDR1 is KASQSVSNDVA (SEQ ID NO: 18), LC CDR2 is YASNRYT (SEQ ID NO: 19), and LC CDR3 is QQDYSSPYT (SEQ ID NO: 20); ii. A group of 6 CDRs, wherein: HC CDR1 is AYNIH (SEQ ID NO: 9), HC CDR2 is YIYPNNGGSGYNQKFMN (SEQ ID NO: 10), HC CDR3 is FDYDEAWFIY (SEQ ID NO: 11), LC CDR1 is SASSVSYMH (SEQ ID NO: 12), LC CDR2 is DTSKLAS (SEQ ID NO: 13), and LC CDR3 is FQGSGSPYT (SEQ ID NO: 14).

2. The isolated monoclonal antibody or antibody fragment according to claim 1, comprising a heavy chain variable region of SEQ ID NO: 37, SEQ ID NO: 35, or an analog having at least 95% sequence similarity to the heavy chain variable region sequence.

3. The isolated monoclonal antibody or antibody fragment of claim 1, comprising a light chain variable region sequence of SEQ ID NO: 38, SEQ ID NO: 36, or an analog having at least 95% sequence similarity to the light chain variable region sequence.

4. The isolated monoclonal antibody or fragment thereof according to claim 1, wherein the fragment is a single-chain Fv (scFv). 5 . A polynucleotide comprising a sequence encoding the HC variable region and the LC variable region of the monoclonal antibody or antibody fragment of claim 1 .

6. A plasmid comprising at least one polynucleotide according to claim 5.

7. A cell comprising the polynucleotide according to claim 5.

8. A pharmaceutical composition comprising at least one isolated antibody or fragment thereof according to claim 1 as an active ingredient, and a pharmaceutically acceptable excipient, diluent, salt or carrier.

9. Use of the pharmaceutical composition according to claim 8 in the preparation of a medicament for treating cancer, wherein the cancer is selected from lymphoma, prostate cancer, breast cancer and bladder cancer.

10. A chimeric antigen receptor (CAR) comprising at least one antibody or antibody fragment according to claim 1, wherein the CAR comprises a transmembrane domain and an intracellular T or NK cell signaling domain.

11. Use of the antibody or antibody fragment according to claim 1 in the preparation of a kit for detecting or quantifying the presence of connexin 4 expressed on cells or secreted into a biological medium, the use comprising contacting a biological sample with the antibody or antibody fragment according to claim 1.

12. A kit for diagnosing cancer in a subject, comprising at least one antibody or antibody fragment according to claim 1.

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