Antibodies specific to human nectin 4

Abstract

The present invention provides a monoclonal antibody that recognizes human Nectin-4 with high affinity and specificity and inhibits its binding to the T cell immunoreceptor having Ig and ITIM domains (TIGIT). The present invention also provides pharmaceutical compositions comprising the antibody and methods for their use in cancer immunotherapy and diagnosis. [Selection diagram] None

Description

[Technical Field]

[0001] The present invention relates to the field of immunotherapy, and 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 further relates to therapeutic and diagnostic compositions comprising these antibodies, and to methods for treating and diagnosing diseases, particularly cancer, using these antibodies. [Background technology]

[0002] Immunotherapy is one of the most promising advances in cancer treatment over the past decade. Cancer immunotherapy is used to generate and enhance anti-tumor immune responses through treatments such as antigen-specific antibodies on tumor cells, fusion of antigen-presenting cells with tumor cells, or activation of anti-tumor T cells. The ability to mobilize immune cells (e.g., T cells) against tumor cells in a patient provides a treatment option to combat cancer types and metastases that were previously considered incurable.

[0003] The T cell-mediated immune response involves a series of steps regulated by a balance between costimulatory and coinhibitory signals that control the degree of the immune response. Inhibitory signals, known as immune checkpoints, are crucial for maintaining self-tolerance and limiting immune-mediated secondary tissue damage. These signals change as infection or immune induction resolves, 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, upregulation of programmed death ligand-1 (PD-L1) on the surface of cancer cells allows them to evade the host immune system by inhibiting T cells through binding to PD-1, otherwise these tumor cells could be attacked. Thus, immune checkpoints represent a significant barrier to the activation of functional cellular immunity against cancer. Therefore, antagonistic antibodies specific to inhibitory ligands in immune cells are considered viable anticancer agents, and they are used in cancer treatment (e.g., nivolumab and pembrolizumab). Another example of an immune checkpoint molecule is the "T cell immune receptor with Ig and ITIM domains" (TIGIT). TIGIT is a co-inhibitory molecule expressed in various 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 to TIGIT are disclosed, for example, in WO2016 / 028656 and WO2017 / 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 crucial role in angiogenesis and metastasis. Several patent applications, including U.S. Patent Application No. 2007 / 0041985, U.S. Patent Application No. 2009 / 0215175, and WO2017 / 149538, disclose molecules and antibodies that specifically bind to PVR, as well as their use against cancer.

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

[0007] In 2016 (Cancer Res. 2016;76:3003-13), Chailta-Eid et al. disclosed an anti-nectin 4 (enfortumab) antibody drug conjugate as a highly potent therapeutic agent in multiple preclinical cancer models. The antibody, conjugated with the microtubule inhibitor vedotin, binds to human, as well as rat and monkey nectin 4, inhibiting the proliferation of several cell lines and xenografts that express nectin 4.

[0008] Despite the successes achieved with cancer immunotherapy, there remains an unaddressed need for additional efforts and more effective and specific drug and drug combinations to enhance the immune system's ability to attack tumor cells. One such effort involves inhibiting nectin 4, which binds to TIGIT, with specific monoclonal antibodies. [Overview of the project]

[0009] The present invention provides 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 is partly based on the discovery that nectin 4, previously known as a receptor for measles virus and tumor antigens, is a ligand for the immunosuppressive molecule TIGIT, and is therefore first proposed as a target for inhibiting the suppressive effect of TIGIT in anti-cancer immunity. The present invention further provides, in some embodiments, chimeric antigen receptors (CARs) comprising a binding site with nectin 4.

[0010] The present invention provides highly effective monoclonal antibodies (mAbs) that are specific to human nectin 4, not only blocking the interaction between nectin 4 and the inhibitory receptor TIGIT, but also having a direct effect on target cells expressing this receptor. These antibodies have a binding constant to nectin 4 in the sub-nanomole range and, without being conjugated with any toxins or antitumor agents, reverse the immune system's TIGIT inhibition, directly promoting the elimination of tumor cells. Therefore, the antibodies of the present invention are useful, for example, in cancer immunotherapy for inhibiting the interaction between nectin 4 on target cells and TIGIT on immune cells. Furthermore, some intact antibodies described herein induce ADCC (antibody-dependent cell-mediated cytotoxicity) activity. Nectin 4 is specifically overexpressed in tumor cells. The high affinity of the antibodies of the present invention for nectin 4 and the ADCC activity induction accompanied by ADCC activity make them ideal candidates for immunotherapy.

[0011] This invention provides antibodies and fragments thereof that recognize the protein nectin 4, prevent its binding to the protein TIGIT, and inhibit its inhibitory activity in lymphocytes such as natural killer (NK) cells and T cells. The anti-nectin 4 antibodies disclosed herein can bind to nectin 4 present on target cells such as cancer cells. The antibodies and fragments of this invention are characterized by a unique set of complementarity-determining region (CDR) sequences, high affinity for human nectin 4, and high specificity, and are useful in cancer immunotherapy to combat tumor immune evasion, both as monotherapy and in combination with other anticancer agents. The antibodies are also useful in preventing viral infections, particularly measles infection.

[0012] The high-affinity anti-nectin 4 antibodies disclosed herein block the TIGIT-nectin 4 interaction and restore T cell and NK cell activity, as disclosed herein.

[0013] Some of the monoclonal antibodies of the present invention can also block the interaction between nectin 4 and nectin 1, demonstrating their ability to interfere with the invasiveness of tumors expressing nectin 4. Furthermore, anti-nectin 4 mAbs were able to induce NK cell activation in most target cells. Advantageously, the anti-nectin 4 mAbs according to the present invention have a direct effect on target cancer cells, inducing their death without the need for NK cells and / or toxins. It is further disclosed that the anti-nectin 4 antibodies of the present invention do not have a blocking effect on the signaling of costimulatory receptors such as DNAM1, and therefore are expected not to have adverse effects on other immune-inducing signals.

[0014] Interestingly, despite the high sequence similarity between human and rodent nectin 4 sequences, some of the antibodies of the present invention are highly specific to human nectin 4 and do not bind to rodent nectin 4.

[0015] Some of the anti-nectin 4 mAbs described herein were able to reduce tumor cell viability in an immune-independent manner by blocking nectin 4 on tumor cells. In some embodiments, the nectin 4 antibodies described herein inhibit tumor cell proliferation through immune-independent interference by binding to nectin 1 on tumor cells, without being conjugated with any toxic molecules.

[0016] In one embodiment, the present invention provides an isolated monoclonal antibody (mAb), or an antibody fragment thereof comprising at least an antigen-binding moiety, which specifically binds to human nectin 4 and inhibits its binding to TIGIT.

[0017] The present invention also provides an mAb or antibody fragment thereof that can inhibit the binding of human nectin 4 to human TIGIT for use in the treatment of cancer, together with T cell lymphocytes and / or natural killer (NK) cells.

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

[0019] According to some embodiments, the isolated antibody or antibody fragment comprises a set of six complementarity - determining region (CDR) sequences, 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 the sequence of said antibody or fragment, 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 the sequence of said antibody or fragment, and iii. three CDRs of the HC variable region comprising SEQ ID NO: 6 and 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 the sequence of said antibody or fragment, and is selected from the group consisting of.

[0020] According to some embodiments, the isolated antibody or antibody fragment comprises a set of six CDR sequences, iv. 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 the sequence of said antibody or fragment, v. 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 the sequence of said antibody or fragment, and vi. three CDRs of the HC variable region comprising SEQ ID NO: 37 and 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 the sequence of said antibody or fragment antibody, and is selected from the group consisting of.

[0021] There are several methods known in the art for determining the CDR sequence of a given antibody molecule, but there is no standard, definitive method. Determination of the CDR sequence from the heavy and light chain variable regions of an antibody can be carried out according to any method known in the art, but is not limited to methods known as KABAT, Chothia, and IMGT. A selected set of CDRs may include sequences identified by two or more methods, i.e., some CDR sequences are determined using KABAT and some using IMGT. According to some embodiments, the CDR sequence of the mAb variable region is determined using the IMGT method.

[0022] According to some embodiments, the isolated monoclonal antibody or fragment comprises the CDR sequence of the monoclonal antibody represented by hNec4.11 (or Nectin 4.11, or Clone 11), namely, three CDR sequences contained in the heavy chain variable region shown in SEQ ID NO: 39, and three CDR sequences contained in the light chain variable region shown in SEQ ID NO: 40.

[0023] According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR1 containing the sequence SYYIH (SEQ ID NO: 25). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR2 containing the sequence WIYPGNVNTKYNERFKG (SEQ ID NO: 26). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR3 containing the sequence SNPYVMDY (SEQ ID NO: 27).

[0024] According to a particular embodiment, the isolated monoclonal antibody or antibody fragment comprises (i) HC CDR1 containing the sequence SYYIH (SEQ ID NO: 25), (ii) HC CDR2 containing the sequence WIYPGNVNTKYNERFKG (SEQ ID NO: 26), and (iii) HC CDR3 containing the sequence SNPYVMDY (SEQ ID NO: 27).

[0025] According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR1 containing the sequence KASQSVNNDVA (SEQ ID NO: 28). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR2 containing the sequence YASNRFT (SEQ ID NO: 29). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR3 containing the sequence QQAYRSPYT (SEQ ID NO: 30).

[0026] According to a particular embodiment, the isolated monoclonal antibody or antibody fragment comprises (i) LC CDR1 containing the sequence KASQSVNNDVA (SEQ ID NO: 28), (ii) LC CDR2 containing the sequence YASNRFT (SEQ ID NO: 29), and (iii) HC CDR3 containing the sequence QQAYRSPYT (SEQ ID NO: 30).

[0027] According to some specific embodiments, the isolated monoclonal antibody or fragment includes a heavy chain CDR1 sequence containing the sequence SYYIH (SEQ ID NO: 25), a heavy chain CDR2 sequence containing the sequence WIYPGNVNTKYNERFKG (SEQ ID NO: 26), a heavy chain CDR3 sequence containing the sequence SNPYVMDY (SEQ ID NO: 27), a light chain CDR1 sequence containing the sequence KASQSVNNDVA (SEQ ID NO: 28), a light chain CDR2 sequence containing the sequence YASNRFT (SEQ ID NO: 29), and a light chain CDR3 sequence containing the sequence QQAYRSPYT (SEQ ID NO: 30), or analogues thereof containing 5% or less of amino acid substitutions, deletions, and / or insertions in the hypervariable region (HVR) sequence.

[0028] According to some specific embodiments, the isolated monoclonal antibody or fragment is i. Heavy chain CDR1 having the sequence shown in sequence number 25, ii. Heavy chain CDR2 having the sequence shown in Sequence ID No. 26, iii. Heavy chain CDR3 having the sequence shown in Sequence ID No. 27, iv. Light chain CDR1 having the sequence shown in sequence number 28, v. Light chain CDR2 having the sequence shown in sequence number 29, vi. Includes a set of six CDR sequences consisting of a light chain CDR3 having the sequence shown in sequence number 30.

[0029] According to some embodiments, the isolated monoclonal antibody or its fragment comprises a heavy chain variable region shown in SEQ ID NO: 39, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain variable region sequence.

[0030] According to some embodiments, the isolated monoclonal antibody or its fragment comprises a light chain variable region shown in SEQ ID NO: 40, or an analog thereof having at least 90% sequence identity with the light chain variable region sequence.

[0031] According to a particular embodiment, the isolated monoclonal antibody or its fragment 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.

[0032] The present invention also includes an antibody or antibody fragment that can bind with high affinity to an epitope in the human nectin 4 protein to which the monoclonal antibody hNec4.11 binds.

[0033] According to some embodiments, the isolated monoclonal antibody or fragment comprises the CDR sequence of the monoclonal antibody represented by hNec4.01 (or Nectin 4.01), namely, three CDR sequences contained in the heavy chain variable region shown in SEQ ID NO: 35 and three CDR sequences contained in the light chain variable region shown in SEQ ID NO: 36.

[0034] According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR1 containing the sequence AYNIH (SEQ ID NO: 9). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR2 containing the sequence YIYPNNGGSGYNQKFMN (SEQ ID NO: 10). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR3 containing the sequence FDYDEAWFIY (SEQ ID NO: 11).

[0035] According to a particular embodiment, the isolated monoclonal antibody or antibody fragment comprises (i) HC CDR1 containing the sequence AYNIH (SEQ ID NO: 9), (ii) HC CDR2 containing the sequence YIYPNNGGSGYNQKFMN (SEQ ID NO: 10), and (iii) HC CDR3 containing the sequence FDYDEAWFIY (SEQ ID NO: 11).

[0036] According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR1 containing the sequence SASSSVSYMH (SEQ ID NO: 12). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR2 containing the sequence DTSKLAS (SEQ ID NO: 13). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR3 containing the sequence FQGSGSPYT (SEQ ID NO: 14).

[0037] According to a particular embodiment, the isolated monoclonal antibody or antibody fragment comprises (i) LC CDR1 containing the sequence SASSSVSYMH (SEQ ID NO: 12), (ii) LC CDR2 containing the sequence DTSKLAS (SEQ ID NO: 13), and (iii) HC CDR3 containing the sequence FQGSGSPYT (SEQ ID NO: 14).

[0038] According to some specific embodiments, the isolated monoclonal antibody or fragment includes a heavy chain CDR1 sequence containing the sequence AYNIH (SEQ ID NO: 9), a heavy chain CDR2 sequence containing the sequence YIYPNNGGSGYNQKFMN (SEQ ID NO: 10), a heavy chain CDR3 sequence containing the sequence FDYDEAWFIY (SEQ ID NO: 11), a light chain CDR1 sequence containing the sequence SASSSVSYMH (SEQ ID NO: 12), a light chain CDR2 sequence containing the sequence DTSKLAS (SEQ ID NO: 13), and a light chain CDR3 sequence containing the sequence FQGSGSPYT (SEQ ID NO: 14), or analogues thereof containing 5% or less of amino acid substitutions, deletions, and / or insertions in the hypervariable region (HVR) sequence.

[0039] According to some specific embodiments, the isolated monoclonal antibody or fragment is i. Heavy chain CDR1 having the sequence shown in Sequence ID No. 9, ii. Heavy chain CDR2 having the sequence shown in Sequence ID No. 10, iii. Heavy chain CDR3 having the sequence shown in sequence number 11, iv. Light chain CDR1 having the sequence shown in sequence number 12, v. Light chain CDR2 having the sequence shown in sequence number 13, and vi. A set of six CDR sequences consisting of a light chain CDR3 having the sequence shown in sequence number 14.

[0040] According to some embodiments, the isolated monoclonal antibody or its fragment comprises a heavy chain variable region shown in SEQ ID NO: 35, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain variable region sequence.

[0041] According to some embodiments, the isolated monoclonal antibody or its fragment comprises a light chain variable region shown in SEQ ID NO: 36, or an analog thereof having at least 90% sequence identity with the light chain variable region sequence.

[0042] According to a particular embodiment, the isolated monoclonal antibody or its fragment 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.

[0043] The present invention also includes an antibody or antibody fragment that can bind with high affinity to an epitope in the human nectin 4 protein to which the monoclonal antibody hNec4.01 binds.

[0044] According to some embodiments, the isolated monoclonal antibody or fragment comprises a CDR sequence of the monoclonal antibody represented by hNec4.05 (or Nectin 4.05), namely, three CDR sequences contained in the heavy chain variable region shown in SEQ ID NO: 37 and three CDR sequences contained in the light chain variable region shown in SEQ ID NO: 38.

[0045] According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR1 containing the sequence TYYIH (SEQ ID NO: 15). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR2 containing the sequence WIYPGNVNTKNNEKFKV (SEQ ID NO: 16). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR3 containing the sequence SNPYVMDY (SEQ ID NO: 17).

[0046] According to a particular embodiment, the isolated monoclonal antibody or antibody fragment comprises (i) HC CDR1 containing the sequence TYYIH (SEQ ID NO: 15), (ii) HC CDR2 containing the sequence WIYPGNVNTKNNEKFKV (SEQ ID NO: 16), and (iii) HC CDR3 containing the sequence SNPYVMDY (SEQ ID NO: 17).

[0047] According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR1 containing the sequence KASQSVSNDVA (SEQ ID NO: 18). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR2 containing the sequence YASNRYT (SEQ ID NO: 19). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a light chain CDR3 containing the sequence QQDYSSPYT (SEQ ID NO: 20).

[0048] According to a particular embodiment, the isolated monoclonal antibody or antibody fragment comprises (i) LC CDR1 containing the sequence KASQSVSNDVA (SEQ ID NO: 18), (ii) LC CDR2 containing the sequence YASNRYT (SEQ ID NO: 19), and (iii) HC CDR3 containing the sequence QQDYSSPYT (SEQ ID NO: 20).

[0049] According to some specific embodiments, the isolated monoclonal antibody or fragment includes a heavy chain CDR1 sequence containing the sequence TYYIH (SEQ ID NO: 15), a heavy chain CDR2 sequence containing the sequence WIYPGNVNTKNNEKFKV (SEQ ID NO: 16), a heavy chain CDR3 sequence containing the sequence SNPYVMDY (SEQ ID NO: 17), a light chain CDR1 sequence containing the sequence KASQSVSNDVA (SEQ ID NO: 18), a light chain CDR2 sequence containing the sequence YASNRYT (SEQ ID NO: 19), and a light chain CDR3 sequence containing the sequence QQDYSSPYT (SEQ ID NO: 20), or analogues thereof containing 5% or less of amino acid substitutions, deletions, and / or insertions in the hypervariable region (HVR) sequence.

[0050] According to some specific embodiments, the isolated monoclonal antibody or fragment is i. Heavy chain CDR1 having the sequence shown in sequence number 15, ii. Heavy chain CDR2 having the sequence shown in Sequence ID No. 16, iii. Heavy chain CDR3 having the sequence shown in Sequence ID No. 17, iv. Light chain CDR1 having the sequence shown in sequence number 18, v. a light chain CDR2 having the sequence shown in SEQ ID NO: 19, and vi. a light chain CDR3 having the sequence shown in SEQ ID NO: 20, and comprises a set of six CDR sequences.

[0051] According to some embodiments, an isolated monoclonal antibody or a fragment thereof comprises a heavy chain variable region sequence shown in SEQ ID NO: 37, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain variable region sequence.

[0052] According to some embodiments, an isolated monoclonal antibody or a fragment thereof comprises a light chain variable region shown in SEQ ID NO: 38, or an analog thereof having at least 90% sequence identity with the light chain variable region sequence.

[0053] According to certain embodiments, an isolated monoclonal antibody or a 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 sequences.

[0054] The present invention also encompasses an antibody or antibody fragment that can bind with high affinity to an epitope within the human nectin-4 protein to which the monoclonal antibody hNec4.05 binds.

[0055] According to some embodiments, an isolated antibody or a fragment thereof recognizes human nectin-4 with an affinity of at least 10 -8 M. According to other embodiments, the antibody or antibody fragment binds to human nectin-4 with an affinity of 10 -8 M, 5×10 -9 M, 10 -9 M, 5×10 -10 M, 10 -10 M, 5×10 -11 M, or binds to human nectin-4 with an even higher affinity. According to some embodiments, the antibody or antibody fragment binds to human nectin-4 with an affinity of 10 -9 M to 10 -10It binds to human nectin with affinity for M. Each possibility represents a distinct embodiment of the present invention.

[0056] Isolated mAb analogs and derivatives, as well as the aforementioned fragments, are also within the scope of the present invention.

[0057] According to some embodiments, the antibody or antibody fragment analog has at least 90% sequence identity with the hypervariable region of the sequence of the reference antibody.

[0058] According to certain embodiments, an isolated antibody or an analog or derivative of its fragment 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 a distinct embodiment of the present invention.

[0059] According to some embodiments, the antibody or antibody fragment according to the present invention comprises a 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 to said sequence.

[0060] According to some embodiments, the antibody or antibody fragment comprises a light chain variable region shown in SEQ ID NO: 40, SEQ ID NO: 36, or SEQ ID NO: 38, or an analog having at least 95% sequence similarity to said sequence.

[0061] According to some embodiments, the antibody or antibody fragment comprises a heavy chain and a light chain, (i) the heavy chain comprising SEQ ID NO: 39 and the light chain comprising SEQ ID NO: 40, (ii) the heavy chain comprising SEQ ID NO: 35 and the light chain comprising SEQ ID NO: 36, or (iii) the heavy chain comprising SEQ ID NO: 37 and the light chain comprising SEQ ID NO: 38. Analogues of the antibody or fragment having at least 95% sequence similarity to the heavy chain or light chain are also included.

[0062] According to some embodiments, the analog has at least 96, 97, 98, or 99% sequence identity with the antibody light chain or heavy chain variable region described above. According to some embodiments, the analog includes not more than one amino acid substitution, deletion, or addition to one or more CDR sequences of the hypervariable region, i.e., any one of the CDR sequences shown in SEQ ID NOs. 25, 26, 27, 28, 29, 30, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. Each possibility represents a distinct embodiment of the present invention. According to some embodiments, the amino acid substitution is a conservative substitution.

[0063] According to some embodiments, the antibody or antibody fragment comprises a hypervariable region (HVR) having the light-chain and heavy-chain regions defined above, with 1, 2, 3, 4, or 5 amino acids substituted, deleted, and / or added. Each possibility represents a distinct embodiment of the present invention.

[0064] According to some embodiments, the antibody or antibody fragment comprises an HVR having the light chain and heavy chain regions defined above, with one amino acid substituted. According to some embodiments, the antibody or antibody fragment comprises a CDR defined above, with one amino acid substituted.

[0065] According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a CDR set. i. A set of six CDs, where 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). ii. A set of six CDs in which HC CDR1 is AYNIH (sequence number 9), HC CDR2 is YIYPNNGGSGYNQKFMN (sequence number 10), HC CDR3 is FDYDEAWFIY (sequence number 11), LC CDR1 is SASSSVSYMH (sequence number 12), LC CDR2 is DTSKLAS (sequence number 13), and LC CDR3 is FQGSGSPYT (sequence number 14), and iii. Select from a group consisting of six CDR sets, where the HC CDR1 sequence is TYYIH (sequence number 15), the HC CDR2 is WIYPGNVNTKNNEKFKV (sequence number 16), the HC CDR3 is SNPYVMDY (sequence number 17), the LC CDR1 is KASQSVSNDVA (sequence number 18), the LC CDR2 is YASNRYT (sequence number 19), and the LC CDR3 is QQDYSSPYT (sequence number 20).

[0066] The present invention also provides a monoclonal antibody comprising a heavy chain and a light chain and its conjugation fragment, wherein the chain comprises a set of heavy chain variable region sequences and a set of light chain variable region sequences, i. Sets including sequence numbers 39 and 40, ii. Sets including SEQ ID NOs. 35 and 36, and iii. Selected from the group consisting of the set containing sequence numbers 37 and 38.

[0067] According to some embodiments, an antibody or antibody fragment can inhibit human nectin 4 from binding to TIGIT expressed in T cells or NK cells.

[0068] According to certain embodiments, the mAb is selected from the group consisting of chimeric antibodies and antibody fragments comprising at least an antigen-binding portion of an antibody. According to certain embodiments, the antibody is a chimeric antibody. According to yet another embodiment, 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 region, single-chain variable region (scFV), single-chain antibody (scab), "bispecific antibody," and "linear antibody." Each possibility represents a distinct embodiment of the present invention.

[0069] Single-chain variable regions (scFVs) including heavy-chain and light-chain variable regions of antibodies described herein are also provided in accordance with the present invention. According to certain embodiments, there are hinge regions between the variable regions.

[0070] According to some embodiments, the scFV sequence is represented by SEQ ID NO: 32, SEQ ID NO: 34, or analogues thereof having at least 90% sequence similarity to said sequence.

[0071] According to some 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 distinct embodiment of the present invention.

[0072] According to some specific embodiments, a monoclonal antibody is a chimeric monoclonal antibody.

[0073] According to some embodiments, the chimeric antibody includes a constant region of human origin.

[0074] According to some embodiments, the human constant region of the chimeric antibody is selected from the group consisting of human IgG1, human IgG2, human IgG3, and human IgG4.

[0075] According to certain embodiments, the antibody is human IgG1. According to some embodiments, human IgG is provided that includes the variable region of the antibody described herein.

[0076] According to some embodiments, a conjugate comprising the antibody or fragment thereof described above is provided.

[0077] According to some embodiments, the conjugate includes a carrier protein.

[0078] A chimeric antigen receptor (CAR) comprising an extracellular component (binding domain) capable of binding to nectin 4 is provided according to another aspect of the present invention.

[0079] According to some embodiments, the CAR comprises an extracellular component containing one of the antibodies or fragments thereof provided herein.

[0080] According to some embodiments, the CAR comprises a nectin-4 binding site containing a CDR set, and the CDR set is i. A set of six CDs, where 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). ii. A set of six CDRs in which 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 SASSSVSYMH (SEQ ID NO: 12), LC CDR2 is DTSKLAS (SEQ ID NO: 13), and LC CDR3 is FQGSGSPYT (SEQ ID NO: 14), and iii. Select from a group consisting of six CDR sets, where the HC CDR1 sequence is TYYIH (sequence number 15), the HC CDR2 is WIYPGNVNTKNNEKFKV (sequence number 16), the HC CDR3 is SNPYVMDY (sequence number 17), the LC CDR1 is KASQSVSNDVA (sequence number 18), the LC CDR2 is YASNRYT (sequence number 19), and the LC CDR3 is QQDYSSPYT (sequence number 20).

[0081] According to some embodiments, the CAR comprises an antigen-binding domain including SEQ ID NO: 32 or 34, a transmembrane domain, and an intracellular T cell signaling domain.

[0082] According to one embodiment, the present invention provides an isolated nucleic acid molecule encoding a CAR comprising an antibody or antibody fragment comprising a nectin 4-binding domain comprising a CDR set, wherein the CDR set is i. A set of six CDs, where 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). ii. A set of six CDs in which HC CDR1 is AYNIH (sequence number 9), HC CDR2 is YIYPNNGGSGYNQKFMN (sequence number 10), HC CDR3 is FDYDEAWFIY (sequence number 11), LC CDR1 is SASSSVSYMH (sequence number 12), LC CDR2 is DTSKLAS (sequence number 13), and LC CDR3 is FQGSGSPYT (sequence number 14), and iii. Select from a group consisting of six CDR sets, where the HC CDR1 sequence is TYYIH (sequence number 15), the HC CDR2 is WIYPGNVNTKNNEKFKV (sequence number 16), the HC CDR3 is SNPYVMDY (sequence number 17), the LC CDR1 is KASQSVSNDVA (sequence number 18), the LC CDR2 is YASNRYT (sequence number 19), and the LC CDR3 is QQDYSSPYT (sequence number 20).

[0083] According to some embodiments, a vector is provided comprising a polynucleotide sequence represented by SEQ ID NO: 31 or SEQ ID NO: 33, or an analogue having at least 95% similarity to said sequence.

[0084] According to some embodiments, T cells engineered to express the CARs described herein are provided.

[0085] According to additional embodiments, NK cells engineered to express the CAR described herein are provided.

[0086] Polynucleotide sequences encoding monoclonal antibodies having high affinity and specificity for human nectin 4, as well as vectors and host cells having these polynucleotide sequences, are provided according to another aspect of the present invention.

[0087] According to some embodiments, polynucleotide sequences encoding the amino acid sequences of the heavy chain variable region and the light chain variable region are provided.

[0088] According to some embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain that can bind to an epitope in human nectin 4 protein that binds to (i) a monoclonal antibody having the heavy chain variable region of SEQ ID NO: 39 and the light chain variable region of SEQ ID NO: 40 (identified herein as hNec4.11), (ii) a monoclonal antibody having the heavy chain variable region of SEQ ID NO: 35 and the light chain variable region of SEQ ID NO: 36 (identified herein as hNec4.01), or (iii) a monoclonal antibody having the heavy chain variable region of SEQ ID NO: 37 and the light chain variable region of SEQ ID NO: 38 (identified herein as hNec4.05).

[0089] According to some embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain containing a sequence selected from the group consisting of SEQ ID NOs: 39 and 40, SEQ ID NOs: 35 and 36, or SEQ ID NOs: 37 and 38. Each possibility represents a distinct embodiment of the present invention.

[0090] Furthermore, according to some embodiments, the polynucleotide sequence according to the present invention is i. A set of six CDs, where 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). ii. A set of six CDs in which HC CDR1 is AYNIH (sequence number 9), HC CDR2 is YIYPNNGGSGYNQKFMN (sequence number 10), HC CDR3 is FDYDEAWFIY (sequence number 11), LC CDR1 is SASSSVSYMH (sequence number 12), LC CDR2 is DTSKLAS (sequence number 13), and LC CDR3 is FQGSGSPYT (sequence number 14), and iii. Encoding an antibody or antibody fragment or chain containing a set of six CDRs, where the HC CDR1 sequence is TYYIH (SEQ ID NO: 15), the HC CDR2 is WIYPGNVNTKNNEKFKV (SEQ ID NO: 16), the HC CDR3 is SNPYVMDY (SEQ ID NO: 17), the LC CDR1 is KASQSVSNDVA (SEQ ID NO: 18), the LC CDR2 is YASNRYT (SEQ ID NO: 19), and the LC CDR3 is QQDYSSPYT (SEQ ID NO: 20).

[0091] Each possibility represents a distinct embodiment of the present invention.

[0092] According to some embodiments, the polynucleotide sequence defined above encodes a molecule selected from the group consisting of an antibody, an antibody fragment including at least an antigen-binding portion, and an antibody conjugate containing the antibody or antibody fragment. Each possibility represents a distinct embodiment of the present invention.

[0093] According to some embodiments, the polynucleotide sequence encoding the variable region of the monoclonal antibody heavy chain includes the sequence shown in SEQ ID NO: 21 or a variant thereof having at least 90% sequence identity.

[0094] According to some embodiments, the polynucleotide sequence encoding the variable region of the monoclonal antibody heavy chain includes the sequence shown in SEQ ID NO: 1 or a variant thereof having at least 90% sequence identity.

[0095] According to some embodiments, the polynucleotide sequence encoding the variable region of the monoclonal antibody heavy chain includes the sequence shown in SEQ ID NO: 5, or a variant thereof having at least 90% sequence identity.

[0096] According to some embodiments, the polynucleotide sequence encoding the variable region of the monoclonal antibody light chain includes the sequence shown in SEQ ID NO: 23 or a variant thereof having at least 90% sequence identity.

[0097] According to some embodiments, the polynucleotide sequence encoding the variable region of the monoclonal antibody light chain includes the sequence shown in SEQ ID NO: 3 or a variant thereof having at least 90% sequence identity.

[0098] According to some embodiments, the polynucleotide sequence encoding the variable region of the monoclonal antibody light chain includes the sequence shown in SEQ ID NO: 7, or a variant thereof having at least 90% sequence identity.

[0099] According to some embodiments, the polynucleotide sequence encoding the variable region of the monoclonal antibody heavy chain includes the sequence shown in SEQ ID NO: 45 or a variant thereof having at least 90% sequence identity.

[0100] According to some embodiments, the polynucleotide sequence encoding the variable region of the monoclonal antibody heavy chain includes the sequence shown in SEQ ID NO: 41 or a variant thereof having at least 90% sequence identity.

[0101] According to some embodiments, the polynucleotide sequence encoding the variable region of the monoclonal antibody heavy chain includes the sequence shown in SEQ ID NO: 43, or a variant thereof having at least 90% sequence identity.

[0102] According to some embodiments, the polynucleotide sequence encoding the variable region of the monoclonal antibody light chain includes the sequence shown in SEQ ID NO: 46 or a variant thereof having at least 90% sequence identity.

[0103] According to some embodiments, the polynucleotide sequence encoding the variable region of the monoclonal antibody light chain includes the sequence shown in SEQ ID NO: 42 or a variant thereof having at least 90% sequence identity.

[0104] According to some embodiments, the polynucleotide sequence encoding the variable region of the monoclonal antibody light chain includes the sequence shown in SEQ ID NO: 44, or a variant thereof having at least 90% sequence identity.

[0105] According to some embodiments, the present invention provides a polypeptide comprising at least one sequence encoded by at least one polynucleotide sequence disclosed above.

[0106] In further embodiments, 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 some embodiments, the nucleic acid construct is a plasmid.

[0107] According to some embodiments, the plasmid comprises at least one polynucleotide sequence represented by a sequence selected from the group consisting of SEQ ID NOs: 45, 46, 41, 42, 43, and 44. Each possibility represents a distinct embodiment of the present invention.

[0108] In yet another aspect, the present invention provides cells capable of producing antibodies or antibody fragments comprising a specific CDR sequence and / or specific heavy and light chain variable regions as defined above.

[0109] According to some embodiments, cells are provided that include at least one polynucleotide sequence disclosed above.

[0110] According to some embodiments, cells are i. A set of six CDs, where 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). ii. A set of six CDs in which HC CDR1 is AYNIH (sequence number 9), HC CDR2 is YIYPNNGGSGYNQKFMN (sequence number 10), HC CDR3 is FDYDEAWFIY (sequence number 11), LC CDR1 is SASSSVSYMH (sequence number 12), LC CDR2 is DTSKLAS (sequence number 13), and LC CDR3 is FQGSGSPYT (sequence number 14), and iii. A monoclonal antibody can be produced containing a set of six CDRs, where the HC CDR1 sequence is TYYIH (SEQ ID NO: 15), the HC CDR2 sequence is WIYPGNVNTKNNEKFKV (SEQ ID NO: 16), the HC CDR3 sequence is SNPYVMDY (SEQ ID NO: 17), the LC CDR1 sequence is KASQSVSNDVA (SEQ ID NO: 18), the LC CDR2 sequence is YASNRYT (SEQ ID NO: 19), and the LC CDR3 sequence is QQDYSSPYT (SEQ ID NO: 20).

[0111] Each possibility represents a distinct embodiment of the present invention.

[0112] According to some embodiments, cells produce monoclonal antibodies in hybridoma cells.

[0113] The antibody or fragment thereof according to the present invention may be conjugated to a cytotoxic moiety, a radioactive moiety, or a identifiable moiety.

[0114] In 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 nectin 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 can inhibit the binding of human nectin 4 to human TIGIT.

[0115] According to some embodiments, the pharmaceutical composition comprises a nectin-4 specific mAb, and the mAb is not conjugated with any toxin or antitumor agent.

[0116] According to some embodiments, the pharmaceutical composition comprises a monoclonal antibody or a fragment thereof that can bind to an epitope in the human nectin 4 protein that binds to a monoclonal antibody selected from the group consisting of hNec4.11, hNec4.01, and hNec4.05 having the variable region and CDR sequence disclosed above.

[0117] According to some embodiments, the pharmaceutical composition i. A set of six CDs, where HC CDR1 is sequence number 25, HC CDR2 is sequence number 26, HC CDR3 is sequence number 27, LC CDR1 is sequence number 28, LC CDR2 is sequence number 29, and LC CDR3 is sequence number 30. ii. A set of six CDRs in which HC CDR1 is sequence number 9, HC CDR2 is sequence number 10, HC CDR3 is sequence number 11, LC CDR1 is sequence number 12, LC CDR2 is sequence number 13, and LC CDR3 is sequence number 14, or iii. A monoclonal antibody comprising at least one set of six CDRs, wherein the HC CDR1 sequence is sequence number 15, the HC CDR2 sequence is sequence number 16, the HC CDR3 sequence is sequence number 17, the LC CDR1 sequence is sequence number 18, the LC CDR2 sequence is sequence number 19, and the LC CDR3 sequence is sequence number 20.

[0118] Each possibility represents a distinct embodiment of the present invention.

[0119] According to some embodiments, the pharmaceutical composition comprises a monoclonal antibody or a fragment thereof containing 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.

[0120] According to some embodiments, the pharmaceutical composition comprises a monoclonal antibody or a 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.

[0121] According to a particular 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.

[0122] According to a particular 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.

[0123] According to a particular 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.

[0124] Furthermore, a pharmaceutical composition comprising at least one antibody, antibody fragment, or antibody conjugate according to the present invention is provided for use in restoring NK cytotoxicity by inhibiting the binding of nectin 4 to TIGIT expressed on NK cells.

[0125] According to some embodiments, an antibody, antibody fragment, or antibody conjugate can inhibit the binding of human nectin 4 to TIGIT expressed on T cells.

[0126] According to some embodiments, the pharmaceutical compositions according to the present invention are intended for use in cancer immunotherapy or for enhancing immune responses.

[0127] According to some embodiments, the pharmaceutical composition further comprises human lymphocytes expressing TIGIT.

[0128] According to some embodiments, human lymphocytes are killer cells selected from the group consisting of T cells, NK cells, and natural killer T cells (NKT cells).

[0129] According to some embodiments, killer cells are either self-derived or allogeneic.

[0130] According to some embodiments, the pharmaceutical composition comprises autologous or allogeneic NK cells expressing TIGIT.

[0131] The cancer treatable by the compositions according to the present invention may be any cancer that expresses nectin 4. According to some embodiments, the cancer overexpresses nectin 4. According to some embodiments of the present invention, the cancer is metastatic cancer. According to some embodiments, the pharmaceutical compositions according to the present invention are for use in inhibiting the formation or distribution of metastases or reducing the total number of metastases in a subject.

[0132] According to some embodiments of the present invention, cancer is selected from the group consisting of melanoma, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, colon cancer, cervical cancer, kidney cancer, lung cancer, thyroid cancer, prostate cancer, brain cancer, renal cancer, throat cancer, laryngeal cancer, bladder cancer, liver cancer, fibrosarcoma, endometrial cell carcinoma, glioblastoma, sarcoma, myeloid cancer, leukemia, and lymphoma. Each possibility represents a distinct embodiment of the present invention.

[0133] According to some embodiments, the cancer is a solid tumor. According to some specific embodiments, the solid tumor is selected from the group consisting of breast cancer, lung cancer, bladder cancer, pancreatic cancer, and ovarian cancer.

[0134] According to other embodiments, the cancer is a blood cancer. According to some embodiments, when the pharmaceutical composition is used for the treatment of cancer, it is accompanied by human lymphocytes.

[0135] According to some embodiments, human lymphocytes are killer cells selected from the group consisting of T cells, NK cells, and NKT cells.

[0136] According to some embodiments, killer cells are either self-derived or allogeneic.

[0137] According to some embodiments, killer cells are NK cells.

[0138] According to some embodiments, the pharmaceutical compositions of the present invention are intended for use in preventing or treating viral infections.

[0139] According to some embodiments, the pharmaceutical composition is intended for use in preventing infection by the measles virus.

[0140] In yet another aspect, the present invention provides a method for inhibiting the binding of human nectin 4 to TIGIT by using a monoclonal antibody or antibody fragment as defined above.

[0141] In yet 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 nectin 4, wherein the antibody or fragment thereof is not conjugated with any toxin or antitumor agent.

[0142] In an additional aspect, the present invention provides a method for enhancing an immune response in a subject requiring such enhancement, comprising administering to the subject a therapeutically effective dose of the monoclonal antibody, antibody fragment, or antibody conjugate defined above.

[0143] In yet another aspect, the present invention provides a method for treating cancer, comprising administering to a subject in need of such treatment a pharmaceutical composition comprising a therapeutically effective amount of at least one antibody or antibody fragment capable of recognizing human nectin 4 with high affinity and specificity and inhibiting its binding to its ligand TIGIT.

[0144] According to some embodiments of the present invention, the therapeutically effective dose results in a reduction in tumor size or the number of metastases in the subject.

[0145] According to some embodiments, the method involves administering a pharmaceutical composition comprising an mAb that is not conjugated with any toxin or antitumor agent.

[0146] According to some embodiments, a method for treating cancer includes administering or performing at least one additional anti-cancer therapy. According to certain embodiments, the additional anti-cancer therapy is surgery, chemotherapy, radiation therapy, or immunotherapy.

[0147] According to some embodiments, a method for treating cancer involves administering a monoclonal antibody that recognizes human nectin 4 with high affinity and specificity, and an additional anticancer agent. According to some embodiments, the additional anticancer agent is selected from the group consisting of immunomodulators, activated lymphocytes, kinase inhibitors, and chemotherapeutic agents.

[0148] According to other embodiments, the additional immunomodulator is an antibody, antibody fragment, or antibody conjugate that binds to an antigen other than human nectin 4.

[0149] According to some embodiments, the additional immunomodulator is an antibody against an immune checkpoint molecule. According to some embodiments, the additional immunomodulator is an antibody against an immune checkpoint molecule selected from the group consisting of human programmed cell death protein 1 (PD-1), PD-L1, and PD-L2, oncoemulsional antigen-associated cell adhesion molecule 1 (CEACAM1), lymphocyte activator gene 3 (LAG3), CD137, OX40 (also known as CD134), killer cell immunoglobulin-like receptor (KIR), TIGIT, PVR, CTLA-4, NKG2A, GITR, and other checkpoint molecules or combinations thereof. Each possibility represents a distinct embodiment of the present invention.

[0150] According to several 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, mechloretamine, melphalan, thiotepa, dacarbazine, bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, plicamycin, etoposide, teniposide, and any combination thereof. Each possibility represents a distinct embodiment of the present invention.

[0151] According to some embodiments, the anticancer agent is an epidermal growth factor receptor (EGFR) inhibitor. According to some embodiments, the EGFR inhibitor is selected from the group consisting of cetuximab (Erbitux®), panitumumab (Vectibix®), and nesitumumab (Portrazza®). According to some embodiments, the EGFR inhibitor is cetuximab (Erbitux®).

[0152] According to some embodiments of the present invention, the subject is a human subject.

[0153] According to some embodiments of the present invention, the use further includes the use of agents that downmodulate the activity or expression of immune co-inhibitory receptors.

[0154] According to some embodiments of the present invention, the immune cells are T cells.

[0155] According to several embodiments of the present invention, the immunocoinhibitory receptor is selected from the group consisting of PD-1, TIGIT, PVR, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, CD96, BY55, LAIR1, SIGLEC10, and 2B4. Each possibility represents a distinct embodiment of the present invention.

[0156] According to one embodiment, the present invention provides a method for modulating the function and / or activity of the immune system, which includes modulating the binding of nectin 4 to TIGIT using an antibody according to the present invention.

[0157] According to some embodiments, a method for treating cancer includes preventing or reducing the formation, growth, or spread of metastases in a subject.

[0158] According to some embodiments, a method for treating cancer comprises administering to a subject in need of such treatment a pharmaceutical composition comprising an mAb or an antibody fragment thereof that can inhibit the binding of human nectin 4 to human TIGIT, and further administering human lymphocytes of the subject.

[0159] According to some embodiments, human lymphocytes are killer cells selected from the group consisting of T cells, NK cells, and NKT cells.

[0160] According to some embodiments, killer cells are either self-derived or allogeneic.

[0161] According to some embodiments, killer cells are NK cells.

[0162] The present invention also provides a method for preventing or treating a viral infection, comprising administering to a subject at least one mAb specific to human nectin 4, or a fragment thereof comprising at least one antigen-binding domain, wherein the mAb or fragment can inhibit the binding of nectin 4 to TIGIT.

[0163] According to several embodiments, a method is provided for preventing infection by the measles virus, comprising administering a human nectin 4-specific mAb, or a fragment thereof containing at least an antigen-binding domain, the mAb or fragment thereof being able to inhibit the binding of the measles virus to human nectin 4 expressed in epithelial cells. According to several embodiments, the cells are epithelial cells. According to one aspect, the present invention provides a method for diagnosing or prognosing cancer or infectious disease in a subject, the method comprising determining the expression level of nectin 4 in a biological sample of the subject using at least one antibody as described herein.

[0164] In yet another aspect, the present invention provides a method for treating cancer, comprising administering a therapeutically effective amount of cells containing the CAR molecule described herein to a subject in need thereof.

[0165] The present invention further comprises, according to another embodiment, a method for determining or quantifying nectin 4 expression, 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 is i. A set of six CDs, where HC CDR1 is sequence number 25, HC CDR2 is sequence number 26, HC CDR3 is sequence number 27, LC CDR1 is sequence number 28, LC CDR2 is sequence number 29, and LC CDR3 is sequence number 30. ii. A set of six CDRs in which HC CDR1 is sequence number 9, HC CDR2 is sequence number 10, HC CDR3 is sequence number 11, LC CDR1 is sequence number 12, LC CDR2 is sequence number 13, and LC CDR3 is sequence number 14, or iii. A set of six CDRs, where the HC CDR1 sequence is sequence number 15, the HC CDR2 sequence is sequence number 16, the HCCDR3 sequence is sequence number 17, the LC CDR1 sequence is sequence number 18, the LC CDR2 sequence is sequence number 19, and the LC CDR3 sequence number is 20.

[0166] Methods for determination and quantification may be carried out in vitro or ex vivo according to several embodiments, or may be used to diagnose conditions associated with nectin 4 expression. The antibodies according to the present invention may also be used to constitute screening methods. For example, enzyme-linked immunosorbent assays (ELISA) or radioimmunoassays (RIA), as well as methods such as IHC or FACS, can be constructed to measure levels of secreted or cell-associated polypeptides using the antibodies and methods known in the art.

[0167] According to some embodiments, a method for detecting or quantifying the presence of nectin 4 expressed in cells or secreted into a biological medium is: i. A step of incubation the sample with an antibody specific to human nectin 4 or an antibody fragment containing at least an antigen-binding moiety. ii. The step includes detecting the bound nectin 4 using a detectable probe.

[0168] According to some embodiments, this method further, iii. The step of comparing the amount of (ii) with a standard curve obtained from a reference sample containing a known amount of nectin 4, and iv. This includes the step of calculating the amount of nectin-4 in the sample from a standard curve.

[0169] According to some specific embodiments, the sample is a body fluid.

[0170] According to some embodiments, the method is carried out in vitro or ex vivo.

[0171] A kit for measuring the expression or presence of nectin 4 in a biological sample is also provided, comprising at least one antibody or antibody fragment according to the present invention. According to some embodiments, the kit includes i. A set of six CDs, where HC CDR1 is sequence number 25, HC CDR2 is sequence number 26, HC CDR3 is sequence number 27, LC CDR1 is sequence number 28, LC CDR2 is sequence number 29, and LC CDR3 is sequence number 30. ii. A set of six CDRs in which HC CDR1 is sequence number 9, HC CDR2 is sequence number 10, HC CDR3 is sequence number 11, LC CDR1 is sequence number 12, LC CDR2 is sequence number 13, and LC CDR3 is sequence number 14, or iii. A set of six CDRs, where the HC CDR1 sequence is sequence number 15, the HC CDR2 sequence is sequence number 16, the HCCDR3 sequence is sequence number 17, the LC CDR1 sequence is sequence number 18, the LC CDR2 sequence is sequence number 19, and the LC CDR3 sequence number is 20.

[0172] According to one embodiment, the present invention provides a kit for detecting cancer, the diagnostic kit comprising an antibody or an antibody fragment thereof disclosed herein.

[0173] According to some embodiments, the present invention provides a method for diagnosing, assessing the severity of, or staging an immune-related or proliferative disorder, comprising using an antibody or fragment or conjugate thereof according to the present invention to determine the expression, concentration, or activity of nectin 4 in a sample derived from a subject, and comparing the expression or activity of nectin 4 to a reference level of nectin 4 expression, concentration, or activity. The reference level can be obtained from a normal subject, from samples taken from the same subject at different stages of the disease, or from clinical data of a large population of subjects.

[0174] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given below. However, it should be understood that the detailed description and specific examples, while illustrating preferred embodiments of the present invention, are given only as examples, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawing]

[0175] [Figure 1] Schematic diagram of receptors involved in TIGIT signaling in immune cells (NK cells) and tumor cells. Anti-nectin 4Ab is shown. [Figure 2] The antibody clones hNec4.05 and hNec4.01 exhibit the best blocking ability of the TIGIT-Nectin 4 interaction. The graph shows the mean fluorescence intensity (MFI) values ​​of FACS staining of RAJI Burkitt lymphoma cells transfected with Nectin 4. Cells were incubated with 0.2 μg of different clones (as shown), then incubated with 3 μg of TIGIT-Ig, and subsequently stained with anti-human secondary antibody. [Figure 3] Quantification of the binding affinity of antibody clones to nectin 4. Binding of fluorophore-labeled nectin 4-Ig to antibody clones hNec4.05 and hNec4.01 was observed by microscale thermophoresis. Measurements were repeated using at least three independent protein preparations, and the mean of the results is shown (±SEM). [Figure 4]Figures 4A-4B. Antibody clones hNec4.05 and hNec4.01 block Nectin 4 and increase NK cytotoxicity. [35S]methionine-labeled, (4A) Nectin 4-transfected RAJI Burkitt lymphoma cells and (4B) LNCap prostate cancer cells (spontaneously expressing Nectin 4) were incubated with 1 μg / well of mouse IgG1 or either the anti-Nectin 4 antibody hNec4.01 or hNec4.05 as a control antibody. After 1 hour, the cells were supplemented with NK cells and incubated for 5 hours. Mean specific death (±sd) of NK:cancer cells at various effector:target (E:T) ratios is plotted. * indicates a significant effect (p<0.05) of the hNec4.01 and hNec4.05 clones compared to the control antibody. The figure shows a representative experiment from one of three trials performed. The same effect was determined using MCF-7, MDA-MB-453, SK-BR-3, and T47D cells, all of which are breast cancer cell lines. [Figure 5] Figures 5A-5C. The antibody clone does not bind to mouse nectin 4. (A) Western blot of RAJI cells transfected with mouse nectin 4 (shown as mNectin 4) and detected by a commercially available anti-mouse nectin 4 mAb (clone 356704, which does not function by flow cytometry). Expression was compared to RAJI cells expressing an empty vector (shown as empty). hGAPDH staining was used as a loading control. (B-C) FACS staining (black line histogram) of RAJI cells transfected with mouse nectin 4. Cells were stained with 0.2 μg of (B) clone hNec4.01 or (C) clone hNec4.05. [Figure 6]Figures 6A-6C. Blockade of the Nectin 4-Nectin 1 interaction. (A-B) FACS staining of RAJI cells transfected with human Nectin 4. Cells were pre-incubated with 1 μg of (A) clone hNec4.01 or (B) clone hNec4.05, and then incubated with 3 μg of Nectin 1-Ig (black line histogram). Unblocked staining is shown by gray lines. Histograms filled in gray are background control staining with secondary antibody only. (C) Mean fluorescence intensity (MFI) values ​​of FACS staining for A and B. [Figure 7] Figures 7A-7C. In vivo effects of anti-nectin 4 mAb. (A) 5 × 10⁶ Raji cells transfected with either an empty vector (EV) or overexpression (OE) nectin 4 were subcutaneously transplanted into SCID beige mice alone (left panel, no NK) or with 1 × 10⁶ NK cells (right panel, NK). (B) 5 × 10⁶ Raji cells overexpressing nectin 4 were subcutaneously transplanted into SCID beige mice with 1 × 10⁶ NK cells. Mice were treated twice weekly by intraperitoneal infusion with either 75 ug of control Ab or anti-nectin 4 clone hNec4.05 mAb. (C) 5 × 10⁶ MDA-MB-453 cells were subcutaneously transplanted into SCID beige mice alone or with 7 × 10⁵ NK cells. Mice were then treated as in (B). Tumors were collected and weighed 21 days (A), 27 days (B), or 23 days (C) after tumor injection. N=7 in all mouse experimental groups. (*)p<0.05. [Figure 8]Figures 8A-8C. Antibody clones hNec4.05 and hNec4.11 that bind to cell surface nectin-4. (A) Antibodies binding to nectin-4 expressed in human cell line MDA-MD-453 were evaluated by FACS analysis. For each clone, the EC50 value calculated after titration of Ab binding (range 20-0.01 nM) and the maximum binding signal are shown. Notably, similar results were shown for chimeric Ab types in which mouse IgG1Fc was substituted with human Fc. (B) Antibodies that bind to CHO cells transfected with cynomolgus monkey (Cyno)-nectin-4 and analyzed similarly to A. (C) Antibodies that bind to CHO cells transfected with mouse-nectin-4 and analyzed similarly to A. ND indicates not detected. [Figure 9] Figures 9A–9D. Antibody clones hNec4.05 and hNec4.11 block the binding of nectin-4 to its ligands, TIGIT and nectin-1. Nectin-4 ligand binding was evaluated by FACS analysis. CHO cells transfected with human nectin-4 were incubated with either human TIGIT-Ig (A and C) or human nectin-1-Ig (B and D) at 20 ug / ml each, or with or without the anti-nectin-4 clone hNec4.05 (A and B) or clone hNec4.11 (C and D) at 8 ug / ml each. Potent binding inhibition by anti-nectin-4 antibodies is shown in all examples. Remaining signals may be due to ligands binding to other receptors expressed by CHO cells, such as PVR, which are unaffected by anti-nectin-4 antibodies. [Figure 10]Figures 10A-10B. Human IgG1 chimeric Ab clones hNec4.05 and hNec4.11 enhance NK cell activation in the presence of tumor cells. Human NK cells (effector, E) were incubated with target cells (T) HT1376 (A) and MDA-MD-453 (B) at an E:T ratio of 2:1. Incubation was performed in the presence of 12 ug / ml chimeric clones hNec4.05 and hNec4.11 or control hIgG1. After 2 hours, NK cells were assayed for degranulation and activation status by FACS analysis of CD107a expression. Degranulation of NK cells in the presence of control hIgG1 was set to 1, and the induction factor was calculated accordingly. Shown are the mean of 2-3 repeats and their normalized SD. [Figure 11] Figures 11A-11D. CAR-T cells driven by hNec4.11 result in specific T cell activation in the presence of tumor cells expressing nectin-4. (A) Schematic diagram of the CART construct. (B) Jurcut cells were transduced with lenti particles encoding the construct and GFP. Transduction efficacy exceeded 99% as determined by FACS analysis of GFP expression. (C) Parental Jurcut cells or Jurcut cells expressing the CART construct (Jurcut pHAGE2.4.11) were incubated with target cells HT1376 or MDA-MD-453 (MDA-453) for 48 hours, after which the culture medium was collected and tested for IL-2 concentration as a way to assess Jurcut activation. IL-2 secretion was significantly induced by CART expression (*=0.003, **=0.00014, two-sided Student's test). Two representative experiments performed are shown. (D) PBMCs derived from healthy donors were transduced using the CART construct. CART PBMCs were incubated with HT1376 cells over the range E:T (shown on the X axis). Areas marked with asterisks indicate significant target cell death (***p<7×10⁻⁵). [Modes for carrying out the invention]

[0176] This invention provides an effective monoclonal antibody that is specific to human nectin 4. The invention also provides the manufacture and use of mAbs as therapeutic agents. In particular, the mAbs of this invention can be used to restore and enhance the antitumor killing activity of immune cells and as diagnostic reagents.

[0177] While previous publications have shown the use of anti-nectin 4 mAbs to target drugs to tumor cells that overexpress nectin 4, this invention discloses for the first time a monoclonal antibody that directly enhances the immune system against tumor cells by inhibiting the binding of nectin 4 to the suppressor receptor TIGIT of immune cells such as NK cells.

[0178] The antibody of the present invention overcomes the shortcomings of TIGIT-specific antibodies currently being tested for cancer treatment. Reportedly, anti-TIGIT antibodies can distort the entire immune system toward activation by blocking all immune cells that express the TIGIT receptor, potentially causing autoimmune effects, whereas the anti-nectin 4 antibody of the present invention targets only nectin 4-expressing cells, which are known to be overexpressed in tumors.

[0179] Furthermore, some of the antibodies of the present invention may also induce immune-independent death of tumor cells through their ability to potentially block the interaction of nectin 4 with nectin 1.

[0180] As used herein, the term “antigen” refers to a molecule or part of a molecule that can induce antibody formation and can be specifically bound by an antibody. An antigen may have one or more epitopes. Specific binding as referred to above means that the antigen reacts with the corresponding antibody in a highly selective manner, but not with a number of other antibodies that may be induced by other antigens. An antigen in some embodiments of the present invention is the nectin 4 protein.

[0181] The term “nectin 4” or “nectin cell adhesion molecule 4” refers, as used herein, to a single-pass type I membrane protein with 510 amino acids and a molecular weight of 55454 Da, also known as PVRL4, LNIR, PRR4, and EDSS1. The nectin 4 protein contains two immunoglobulin-like (Ig-like) C2 domains and one Ig-like V domain. It is involved in cell adhesion through trans-alloaffinity and trans-heteroaffinity interactions. The soluble form is produced by proteolytic cleavage on the cell surface by the metalloproteinase ADAM17 / TACE, while the secreted form is found in both mammary tumor cell lines and mammary tumor patients. Exemplary nectin 4 according to the present invention is represented by the symbols or registration 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 in SwissPort, UniPort, and GenBank.

[0182] The antibody or fragment thereof according to the present invention binds to an epitope of nectin 4. Specifically, the antibody binds to an epitope within the extracellular domain (extracellular portion) of the nectin 4 protein sequence.

[0183] As used herein, the terms “antigen determinant” or “epitope” refer to a region of an antigen molecule that specifically reacts with a particular antibody. Peptide sequences derived from epitopes can be used alone or conjugated to a carrier moiety to immunize animals using methods known in the art to produce additional polyclonal or monoclonal antibodies. Isolated peptides derived from epitopes can be used in diagnostic methods for detecting antibodies.

[0184] 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, for example, using the dissociation constant Kd. It should be noted that a lower Kd reflects higher affinity.

[0185] Antibodies, or immunoglobulins, consist of two heavy chains and two light chains linked together by disulfide bonds, with each light chain linked to its respective heavy chain in a "Y" shape by disulfide bonds. Proteolytic digestion of antibodies yields Fv (variable fragment) and Fc (crystalline fragment) domains. The antigen-binding domain, Fab, contains a region where the polypeptide sequence changes. The term F(ab')2 represents the two Fab' arms linked together by disulfide bonds. The central axis of the antibody is called the Fc fragment. Each heavy chain has a variable domain (V) at one end. H ) has several constant domains (C H ) follows. Each light chain has a variable domain (V) at one end. L ), and other ends have a steady domain (C L The light chain variable domain is aligned with the heavy chain variable domain, and the light chain constant domain is aligned with the heavy chain's first constant domain (CH1). Each pair of variable domains in the light and heavy chains forms the antigen-binding site. The light and heavy chain domains have the same overall structure, and each domain contains four framework regions whose sequences are relatively conserved and are linked by three hypervariable domains known as complementarity-determining regions (CDR1-3). These domains contribute to the specificity and affinity of the antigen-binding site.

[0186] The identification or determination of CDRs from a given heavy or light chain variable sequence is usually performed using one of the few methods known in the art. For example, such determinations are made according to Kabat (Wu T.T and Kabat E.A., J Exp Med, 1970;132:211-50) and IMGT (Lefranc MP, et al., Dev Comp Immunol, 2003, 27:55-77).

[0187] When the term "CDR containing a sequence" or similar terms is used, it includes the option that the CDR contains a specified sequence, and the option that the CDR consists of a specified sequence.

[0188] Antigen specificity in antibodies is based on the hypervariable region (HVR), which is the unique CDR sequence in both the light and heavy chains that together form the antigen-binding site.

[0189] The heavy chain isotype (gamma, alpha, delta, epsilon, or mu) determines the immunoglobulin class (IgG, IgA, IgD, IgE, or IgM, respectively). The light chain is one of two isotypes (kappa, κ, or lambda, λ). Both isotopes are found in all antibody classes.

[0190] The term "antibody" is used in its 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 nectin 4.

[0191] The antibodies or a plurality of antibodies according to the present invention include intact antibodies such as polyclonal antibodies or monoclonal antibodies (mAbs), as well as their proteolytic fragments such as Fab or F(ab')2 fragments. Single-chain antibodies are also included within the scope of the present invention.

[0192] Antibody fragment An "antibody fragment" contains only a portion of an intact antibody, and generally includes the antigen-binding site of the intact antibody, thus retaining its ability to bind to an antigen. Examples of antibody fragments included by this definition include: (i) Fab fragments having VL, CL, VH, and CH1 domains; (ii) Fab' fragments having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iii) Fd fragments having VH and CH1 domains; (iv) Fd' fragments having VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) Fv fragments having VL and VH domains in a single arm of the antibody; (vi) dAb fragments consisting of a VH domain (Ward et al., Nature 1989, 341, 544-546); (vii) F(ab')2 fragments, which are bivalent fragments containing an isolated CDR region and (viii) two Fab' fragments linked by disulfide crosslinks at a hinge region; and (ix) single-chain antibody molecules (e.g., single-chain Fv, scFv) (Bird et al., Science (x) "Bispecific antibodies" having two antigen-binding sites and containing a heavy chain variable domain (VH) conjugated to a light chain variable domain (VL) on the same polypeptide chain (e.g., EP404, 097, WO93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 6444-6448), (xi) "Linear antibodies" containing a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding domains together with a complementary light chain polypeptide (Zapata et al. Protein This includes Eng., 1995, 8, 1057-1062, and U.S. Patent No. 5,641,870.

[0193] Various techniques have been developed for the generation of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., 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 generated directly by recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically conjugated to produce F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell cultures. Other techniques for the generation of 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).

[0194] A single-chain antibody is a single-chain complex polypeptide, i.e., linked V, that has antigen-binding ability and contains an amino acid sequence homologous or similar to the variable regions of immunoglobulin light and heavy chains. H -V L Alternatively, it can be a single-chain Fv (scFv). Techniques for producing single-chain antibodies (U.S. Patent No. 4,946,778) can be adapted to produce single-chain antibodies against nectin 4.

[0195] As used herein, the term “monoclonal antibody” (mAb) refers to an antibody obtained from a nearly homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for the possibility of spontaneous variation that may exist in small amounts. Monoclonal antibodies are highly specific and directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant of the antigen. The modifying phrase “monoclonal” should not be interpreted as requiring the production of the antibody by any particular method. mAbs can be obtained by methods known to those skilled in the art. For example, monoclonal antibodies used in accordance with the present invention can be produced by the hybridoma method first described by Kohler et al., Nature 1975, 256, 495, or by the recombinant DNA method (e.g., U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, Clackson et al., Nature 1991, 352, 624-628 or Marks et al., J. Mol. Biol., 1991, 222:581-597.

[0196] The rapid identification and cloning of functional variable heavy chain (VH) and variable light chain (VL) genes, the design and development of recombinant monovalent antigen-binding molecules derived from monoclonal antibodies, and the design and cloning of synthetic DNA sequences optimized for expression in recombinant bacteria are described in Fields et at. 2013, 8(6):1125-48.

[0197] The mAbs of the present invention may be of any immunoglobulin class, including IgG, IgM, IgE, IgA, and IgD. Hybridomas producing mAbs can be cultured in vitro or in vivo. High-titer mAbs can be obtained by in vivo production, in which cells from individual hybridomas are intraperitoneally injected into untreated, prepared Balb / c mice to produce ascites containing high concentrations of the desired mAb. The mAbs can be purified from such ascites or from the culture supernatant using methods well known to those skilled in the art.

[0198] The present invention also includes an anti-idiotype antibody that is specifically immunoreactive with respect to the hypervariable region of the antibody.

[0199] The present invention provides a monoclonal antibody or antibody fragment comprising an antigen-binding domain (ABD) containing three CDRs of a light chain and three CDRs of a heavy chain, wherein the ABD has at least 90% sequence identity or similarity to an ABD of a monoclonal mouse antibody comprising (i) a heavy variable chain containing the amino acid sequence of SEQ ID NO: 39 and a light variable chain containing the amino acid sequence of SEQ ID NO: 40 (identified herein as hNec4.11), (i) a heavy variable chain containing the amino acid sequence of SEQ ID NO: 35 and a light variable chain containing the amino acid sequence of SEQ ID NO: 36 (identified herein as hNec4.01), or (ii) a heavy variable chain containing the amino acid sequence of SEQ ID NO: 37 and a light variable chain containing the amino acid sequence of SEQ ID NO: 38 (identified herein as hNec4.05). Such antibodies may have an ABD domain with at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or similarity to the corresponding ABD of the antibody hNec4.11, hNec4.01, or hNec4.05, or with 100% sequence identity.

[0200] Sequence identity is the amount of amino acids or nucleotides that exactly match between two different sequences. Sequence similarity allows us to determine that conserved amino acid substitutions are identical amino acids.

[0201] The present invention also provides conserved amino acid variants of antibody molecules according to the present invention. Variants according to the present invention may also be generated as conserving the entire molecular structure of the encoded protein. Given the properties of the individual amino acids constituting the disclosed protein product, several reasonable substitutions will be recognized by those skilled in the art. Amino acid substitutions, i.e., “conservative substitutions,” may be made, for example, based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues they contain. As used herein, the term “antibody analog” refers to an antibody derived from another antibody by one or more conserved amino acid substitutions.

[0202] As used herein, the term “antibody variant” refers to any molecule containing the antibody of the present invention. For example, a fusion protein in which an antibody or its antigen-binding fragment is bound to another chemical entity is considered an antibody variant.

[0203] Antibody sequence analogues and variants are also within the scope of this application. These include, but are not limited to, conserved and non-conservative substitutions, insertions, and deletions of amino acids in the sequence. Such modifications and the resulting antibody analogues or variants are within the scope of the invention insofar as they confer, or even improve, the binding of the antibody to human nectin 4.

[0204] Conservative amino acid substitutions known to those skilled in the art are within the scope of this invention. Conservative amino acid substitutions include replacing one amino acid with another of the same type of functional group or side chain, e.g., aliphatic, aromatic, positively charged, or negatively charged. These substitutions can enhance oral bioavailability, permeability, and targeting to specific cell populations, immunogenicity, etc. Those skilled in the art will recognize that individual substitutions, deletions, or additions to a peptide, polypeptide, or protein sequence, which change, add, or delete a single amino acid or a small proportion of amino acids in the encoded sequence, are "conservatively modified variants" and that the change results in the substitution of an amino acid with a chemically similar amino acid. A list of conservative substitutions resulting in functionally similar amino acids is well known in the art. For example, one list known in the art includes the following six groups, each containing amino acids that are conserved substitutions of each other: 1) Alanine (A), Serine (S), Threonine (T), 2) Aspartic acid (D), glutamic acid (E), 3) Asparagine (N), glutamine (Q), 4) Arginine (R), Lysine (K), 5) Isoleucine (I), leucine (L), methionine (M), valine (V), and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0205] It should be emphasized that the mutant chain sequence is determined by sequencing methods using specific primers. Different sequencing methods used on the same sequence may result in slightly different sequences, particularly at the sequence ends, due to technical issues and different primers. Therefore, different variants of the anti-nectin 4 variable chain sequence are identified in accordance with this application.

[0206] As used herein, the terms “molecule having an antigen-binding moiety of an antibody” and “antigen-binding fragment” are intended to include not only intact immunoglobulin molecules produced by any animal cell line or microorganism in any isotype, but also their antigen-binding reactive fractions, but not limited to, for example, Fab fragments, Fab' fragments, F(ab')2 fragments, their variable portions of the heavy and / or light chains, Fab mini-antibodies (see, for example, WO93 / 15210, U.S. Patent Application No. 08 / 256,790, WO96 / 13583, U.S. Patent Application No. 08 / 817,788, WO96 / 37621, U.S. Patent Application No. 08 / 999,554), and single-chain antibodies incorporating such reactive fractions, as well as any other type of molecule into which an antibody-reactive fraction is physically inserted. Such molecules may be provided by any known technique, but not limited to, enzymatic cleavage, peptide synthesis, or recombination techniques.

[0207] Monoclonal antibodies as used herein include, in particular, “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, while the remaining portion of the chain is identical or homologous to a corresponding sequence in a fragment of an antibody derived from another species or belonging to another antibody class or subclass, and insofar as they exhibit the desired biological activity (U.S. Patent No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Furthermore, complementarity-determining region (CDR) grafting can be performed to modify certain properties of the antibody molecule, including affinity or specificity. Non-limiting examples of CDR grafts are disclosed in U.S. Patent No. 5,225,539.

[0208] Chimeric antibodies are molecules in which different parts originate from different animal species, such as those possessing a variable region derived from a mouse mAb and a constant region from a human immunoglobulin. Antibodies that have a variable region framework residue mostly derived from a human antibody (called an acceptor antibody) and a CDR mostly derived from a mouse antibody (called a donor antibody) are also called humanized antibodies. Chimeric antibodies are primarily used to reduce immunogenicity in application and increase yield in production. For example, mouse mAbs have a high yield from hybridomas but are highly immunogenic in humans, hence the use of human / mouse chimeric mAbs. Chimeric antibodies and methods for producing them are known in the art (e.g., PCT patent applications WO86 / 01533, WO97 / 02671, WO90 / 07861, WO92 / 22653, and U.S. Patents 5,693,762, 5,693,761, 5,585,089, 5,530,101, and 5,225,539).

[0209] According to some specific embodiments, a monoclonal antibody is a chimeric monoclonal antibody.

[0210] According to some embodiments, the chimeric antibody includes a constant region of human origin.

[0211] According to some embodiments, the human constant region of the chimeric antibody is selected from the group consisting of human IgG1, human IgG2, human IgG3, and human IgG4.

[0212] According to certain embodiments, a chimeric monoclonal antibody that recognizes human nectin 4 is provided. i. A set of six CD-Rs, where 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). ii. A set of six CD-Rs, where HC CDR1 is (sequence number 9), HC CDR2 is (sequence number 10), HC CDR3 is (sequence number 11), LC CDR1 is (sequence number 12), LC CDR2 is (sequence number 13), and LC CDR3 is (sequence number 14). iii. A set of six CD-Rs, where HC CDR1 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).

[0213] According to one embodiment, the present invention provides a CAR comprising an antibody fragment that specifically binds to nectin 4.

[0214] According to some embodiments, the CAR comprises i) a specific binding agent capable of specifically binding to nectin 4, ii) a spacer or hinge domain, iii) a transmembrane domain (TM) for immobilizing the CAR within the T cell membrane, and iv) an intracellular domain for transmitting signals within the T cell.

[0215] According to some embodiments, the CAR includes a set of CD-Rs. i. A set of six CDs, where 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). ii. A set of six CDs in which HC CDR1 is AYNIH (sequence number 9), HC CDR2 is YIYPNNGGSGYNQKFMN (sequence number 10), HC CDR3 is FDYDEAWFIY (sequence number 11), LC CDR1 is SASSSVSYMH (sequence number 12), LC CDR2 is DTSKLAS (sequence number 13), and LC CDR3 is FQGSGSPYT (sequence number 14), and iii. Select from a group consisting of six CDR sets, where the HC CDR1 sequence is TYYIH (sequence number 15), the HC CDR2 is WIYPGNVNTKNNEKFKV (sequence number 16), the HC CDR3 is SNPYVMDY (sequence number 17), the LC CDR1 is KASQSVSNDVA (sequence number 18), the LC CDR2 is YASNRYT (sequence number 19), and the LC CDR3 is QQDYSSPYT (sequence number 20).

[0216] According to one embodiment, the present invention provides an isolated nucleic acid molecule encoding a CAR comprising an antibody or antibody fragment comprising a nectin 4-binding domain comprising a CDR set, wherein the CDR set is i. A set of six CDs, where 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). ii. A set of six CDs in which HC CDR1 is AYNIH (sequence number 9), HC CDR2 is YIYPNNGGSGYNQKFMN (sequence number 10), HC CDR3 is FDYDEAWFIY (sequence number 11), LC CDR1 is SASSSVSYMH (sequence number 12), LC CDR2 is DTSKLAS (sequence number 13), and LC CDR3 is FQGSGSPYT (sequence number 14), and iii. Select from a group consisting of six CDR sets, where the HC CDR1 sequence is TYYIH (sequence number 15), the HC CDR2 is WIYPGNVNTKNNEKFKV (sequence number 16), the HC CDR3 is SNPYVMDY (sequence number 17), the LC CDR1 is KASQSVSNDVA (sequence number 18), the LC CDR2 is YASNRYT (sequence number 19), and the LC CDR3 is QQDYSSPYT (sequence number 20).

[0217] According to some embodiments, the vector comprises a 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.

[0218] According to some embodiments, T cells engineered to express the CARs described herein are provided.

[0219] With respect to the transmembrane domain, in various embodiments, the CAR can be designed to include a transmembrane domain that binds to the extracellular domain of the CAR. The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acids associated with the extracellular region of the transmembrane-inducing protein (e.g., one, two, three, four, five, or more amino acids of the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., one, two, three, four, five, or more amino acids of the intracellular region). According to some embodiments, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interaction with other members of the receptor complex.

[0220] According to some embodiments, the transmembrane domain is the transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T cell receptor, CD27, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. Each possibility represents a distinct embodiment of the present invention.

[0221] According to some embodiments, an antibody or antibody fragment containing a nectin 4-binding domain is linked by a transmembrane domain and a hinge region. According to some embodiments, the hinge is derived from a human protein. According to some embodiments, the hinge is a human Ig (immunoglobulin) hinge. According to certain embodiments, the hinge is an IgG4 hinge or a CD8a hinge.

[0222] The cytoplasmic domain or region of a CAR includes an intracellular signaling domain. The intracellular signaling domain is generally involved in the activation of at least one of the normal effector functions of the immune cell into which the CAR is introduced. The term "effector function" refers to a specific function of the cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity including cytokine secretion. Therefore, the term "intracellular signaling domain" refers to the portion of a protein that transmits effector function signals and directs the cell to perform a specific function. The term "intracellular signaling domain" means any cleaved portion of an intracellular signaling domain that is sufficient to transmit effector function signals. Examples of intracellular signaling domains for use in the CARs of the present invention include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act in coordination to initiate signaling after antigen receptor engagement, as well as any derivatives or variants of these sequences and any recombinant sequences having the same functional capabilities.

[0223] In some embodiments, signals arising solely via the TCR are insufficient for complete T cell activation, 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: those that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling domain), and those that act antigen-independently to produce secondary or co-stimulatory signals (secondary cytoplasmic domains, e.g., co-stimulatory domains).

[0224] According to some embodiments, the intracellular signaling domain is designed to include two or more co-stimulatory signaling domains. According to some embodiments, the two or more co-stimulatory signaling domains are separated by a linker molecule. According to some embodiments, the linker molecule is a glycine residue. According to some embodiments, the linker is an alanine residue.

[0225] According to some embodiments, the CAR comprises a co-stimulatory domain obtained from a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Each possibility represents a distinct embodiment of the present invention.

[0226] The present invention also relates to nucleic acid molecules encoding CARs, along with vectors and host cells. The present invention also relates to (heterogeneous) T cells comprising the CARs of the present invention, along with a pharmaceutical composition containing such CARs, along with a suitable carrier or excipient. The present invention also relates to autologous T cell therapy (and corresponding medical applications) incorporating the T cells, compositions, and CARs of the present invention.

[0227] According to some embodiments, the CAR-expressing cells described herein may further contain a second CAR, for example, the second CAR containing different antigen-binding domains for the same target (nectin 4) or different targets. According to some embodiments, if the CAR-expressing cells contain two or more different CARs, the antigen-binding domains of the different CARs may be such that the antigen-binding domains do not interact with each other.

[0228] According to some embodiments, a population of cells is provided in which at least one cell expresses a CAR having the anti-nectin 4 domain described herein, and a second cell expresses another substance, for example, a substance that enhances the activity of the CAR-expressing cell.

[0229] The present invention also relates to (heterogeneous) NK cells comprising the CAR of the present invention together with a pharmaceutical composition containing such CAR, along with a suitable carrier or excipient. The present invention also relates to autologous NK cell therapy (and corresponding medical applications) incorporating the NK cells, composition, and CAR of the present invention.

[0230] As used herein, “chimeric antigen receptor” or “CAR” refers to an artificially constructed hybrid polypeptide comprising an antigen-binding domain (e.g., the antigen-binding portion of an antibody (e.g., scFV)), a transmembrane domain, and a T cell or NK cell signaling and / or T cell or NK cell activation domain. CARs have the ability to redirect the specificity and reactivity of T cells or NK cells to selected targets by MHC-unrestricted means, leveraging the antigen-binding properties of monoclonal antibodies. MHC-unrestricted antigen recognition gives T cells or NK cells expressing CARs the ability to recognize antigens independently of antigen processing, thus evading the primary mechanism of tumor escape. Most commonly, the extracellular binding domain of a CAR consists of a single-chain variable fragment (scFv) derived from the fusion of variable heavy and light regions of a mouse or humanized monoclonal antibody.

[0231] Pharmacology In pharmaceutical and drug formulations, the activator is preferably used with one or more pharmaceutically acceptable carriers and optionally with any other therapeutic component. The carriers must be pharmaceutically acceptable in that they are compatible with the other components of the formulation and are not excessively harmful to the recipient. The activator is provided in an amount effective to achieve the desired pharmacological effect and in an appropriate amount to achieve the desired exposure.

[0232] Typically, the antibodies of the present invention, as well as their fragments and conjugates, which include an antigen-binding moiety of the antibody or another polypeptide containing a peptide mimetic, are suspended in sterile saline for therapeutic use. Alternatively, the pharmaceutical composition may be formulated to control the release of the active ingredient (the molecule containing the antigen-binding moiety of the antibody) or to prolong its presence in the patient's system. Numerous suitable drug delivery systems are known, including, for example, implantable drug release systems, hydrogels, hydroxymethylcellulose, microcapsules, liposomes, microemulsions, and microspheres. Controlled-release preparations can be prepared through the use of polymers to complexe or adsorb the molecules according to the present invention. For example, biocompatible polymers include poly(ethylene-co-vinyl acetate) matrices and polyanhydride copolymer matrices of stearate dimer and sevalic acid. The release rate of the molecules according to the present invention, i.e., antibodies or antibody fragments, from such matrices depends on the molecular weight of the molecules, the amount of molecules in the matrix, and the size of the dispersed particles.

[0233] The pharmaceutical compositions of the present invention may be administered by any suitable means, such as orally, topically, intranasally, subcutaneously, intramuscularly, intravenously, intraarterially, intraarterially, intralesionally, intratumorally, or parenterally. Intravenous (iv) administration is typically used to deliver antibodies.

[0234] It will be apparent to those skilled in the art that the therapeutically effective dose of the molecule according to the present invention depends, in particular, on the administration schedule, the unit dose of the molecule administered, whether the molecule is administered in combination with other therapeutic agents, the patient's immune status and health, the therapeutic activity of the molecule administered, its persistence in blood circulation, and the judgment of the treating physician.

[0235] As used herein, the term “therapeutic dose” refers to the amount of a drug that is effective in treating a disease or disorder in a mammal. In the case of cancer, a therapeutic dose of a drug may reduce the number of cancer cells, reduce tumor size, inhibit (i.e., delay, preferably halt) cancer cell invasion into peripheral organs, inhibit (i.e., delay, preferably halt) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more of the symptoms associated with the disorder. To the extent that the drug can inhibit growth and / or kill existing cancer cells, it may be cell proliferation inhibitory and / or cytotoxic. For cancer treatment, in vivo efficacy can be determined, for example, by evaluating survival time, time to disease progression (TTP), response rate (RR), duration of response, and / or quality of life.

[0236] Cancers that can be modified by the treatment according to the present invention include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric or stomach 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 or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, various types of head and neck cancers, and B-cell lymphoma (low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL)). NHL includes moderate / follicular NHL, moderate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small unsevered cell NHL, giant tumor lesion NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenström macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorders (PTLD), as well as nevus disorders, edema (such as those associated with brain tumors), and abnormal angiogenesis associated with Meggs syndrome. Preferably, the cancer is selected from the group consisting of breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkin lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid tumor, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma. Metastatic cancer is a malignant cancer condition that can be modified for the treatment of the present invention.

[0237] According to other embodiments, the pharmaceutical compositions according to the present invention are for use in the treatment of cancers characterized by overexpression of nectin 4. The types of cancer associated with overexpression of nectin 4 can be identified using known databases such as The Cancer Genome Atlas (TCGA). According to certain embodiments, cancers treatable by compositions according to the present invention include adrenocortical carcinoma (ACC), chromophobe renal cell carcinoma (KICH), hepatocellular carcinoma of the liver (LIHC), adenocarcinoma of the colon and rectum (COAD, READ), ductal adenocarcinoma of the pancreas (PAAD), pheochromocytoma and paraganglioma (PCPG), papillary renal carcinoma (KIRP), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), prostate adenocarcinoma (PRAD), endometrial carcinoma of the uterine body (UCEC), and cervical cancer. The group consists of cancer (CESC), cutaneous melanoma (SKCM), mesothelioma (MESO), urothelial bladder carcinoma (BLCA), clear cell kidney carcinoma (KIRC), pulmonary squamous cell carcinoma (LUSC), uterine carcinosarcoma (UCS), sarcoma (SARC), ovarian serous cystadenocarcinoma (OV), papillary thyroid carcinoma (THCA), glioblastoma pleomorphoni (GBM), breast cancer (BRCA), low-grade glioma (LGG), and diffuse large B-cell lymphoma (DLBC). Each possibility represents a distinct embodiment of the present invention.

[0238] The molecule of the present invention, as the active ingredient, is dissolved, dispersed, or mixed with an excipient that is pharmaceutically acceptable and compatible with the active ingredient, as is well known. Suitable excipients include, for example, water, physiological saline, phosphate-buffered saline (PBS), dextrose, glycerol, ethanol, and combinations thereof. Other suitable carriers are well known to those skilled in the art. Furthermore, the composition may optionally contain small amounts of auxiliary substances such as wetting agents or emulsifiers and pH buffers.

[0239] The pharmaceutical composition according to the present invention may be administered together with an antineoplastic composition.

[0240] As used herein, the term “treatment” refers to both therapeutic treatment and preventive or deterrent measures. Those requiring treatment include those who already have a disability, as well as those for which the disability should be prevented.

[0241] The terms “cancer” and “malignant” generally refer to or describe a physiological condition in mammals characterized by uncontrolled cell proliferation. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include melanoma, and cancers of the lung, thyroid, breast, colon, prostate, liver, bladder, kidney, cervix, pancreas, leukemia, lymphoma, myeloid, ovarian, uterine, sarcoma, bile duct, or endometrium.

[0242] According to some embodiments, a method for treating cancer involves administering the pharmaceutical composition as part of a treatment regimen that includes the administration of at least one additional anticancer agent.

[0243] According to some embodiments, the anticancer agent is selected from the group consisting of antimetabolites, mitotic inhibitors, taxanes, topoisomerase inhibitors, topoisomerase II inhibitors, asparaginases, alkylating agents, antitumor antibiotics, and combinations thereof. Each possibility represents a distinct embodiment of the present invention.

[0244] According to some embodiments, the antimetabolite is selected from the group consisting of cytarabine, glutarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, and hydroxyurea. According to some embodiments, the mitotic inhibitor is selected from the group consisting of vincristine, vinblastine, and vinorelbine. According to some embodiments, the topoisomerase inhibitor is selected from the group consisting of topotecan and irenotecan. According to some embodiments, the alkylating agent is selected from the group consisting of busulfan, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosamide, mechloretamine, melphalan, thiotepa, dacarbazine, and procarbazine. According to some embodiments, the antitumor antibiotic is selected from the group consisting of bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, and plicamycin. According to some embodiments, topoisomerase II is selected from the group consisting of etoposide and teniposide. Each possibility represents a distinct embodiment of the present invention.

[0245] According to some specific embodiments, additional anticancer agents are selected from the group consisting of bevacizumab, carboplatin, cyclophosphamide, doxorubicin hydrochloride, gemcitabine hydrochloride, topotecan hydrochloride, thiotepa, and combinations thereof. Each possibility represents a distinct embodiment of the present invention.

[0246] The monoclonal antibody according to the present invention may be used as part of a combination therapy with at least one anticancer agent. According to some embodiments, the additional anticancer agent may be an immunomodulator, activated lymphocytes, a kinase inhibitor, or a chemotherapeutic agent.

[0247] According to some embodiments, the anticancer agent is an immunomodulator, such as an antibody against an immune checkpoint molecule, whether it is an agonist or an antagonist.

[0248] Blocking immune checkpoints through immunotherapy is proving to be an exciting new frontier in cancer treatment. Immune checkpoint pathways consist of a set of co-stimulatory and inhibitory molecules that work together to maintain self-tolerance and protect tissues from immune system damage under physiological conditions. Tumors evade the immune system by utilizing specific checkpoint pathways. Therefore, inhibiting such pathways is emerging as a promising anti-cancer treatment strategy.

[0249] The anticytotoxic T lymphocyte 4 (CTLA-4) antibody ipilimumab (approved in 2011) was the first immunotherapy agent to demonstrate benefit in treating cancer patients. The antibody interferes with inhibitory signaling during antigen presentation to T cells. The anti-programmed cell death 1 (PD-1) antibody pembrolizumab (approved in 2014) blocks negative immunomodulatory signaling of the PD-1 receptor expressed by T cells. An additional anti-PD-1 agent was submitted for regulatory approval in 2014 for the treatment of non-small cell lung cancer (NSCLC). Active research is now exploring many other immune checkpoints, among others, CEACAM1, NKG2A, B7-H3, B7-H4, VISTA, CD112R, lymphocyte activator gene 3 (LAG3), CD137, OX40 (also known as CD134), and killer cell immunoglobulin-like receptors (KIRs).

[0250] According to some specific embodiments, immunomodulators are selected from the group consisting of 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 a distinct embodiment of the present invention. According to some specific embodiments, additional immunomodulators are 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.

[0251] According to other embodiments, additional anticancer agents are chemotherapeutic agents. The chemotherapeutic agents that can be administered together with or separately from the antibody according to the present invention may include, but are not limited to, any of the agents known in the art that exhibit anticancer activity, such as mitoxantrone, topoisomerase inhibitors, spindle toxins such as vinblastine, vincristine, vinorelbine (Taxol), paclitaxel, docetaxel; alkylating agents such as mechloretamine, chlorambucil, cyclophosphamide, melphalan, ifosfamide; methotrexate, 6-mercaptopurine, and 5-fluorouracil. This includes cytarabine, gemcitabine, podophyllotoxins such as etoposide, irinotecan, topotecan, and dacarbazine; antibiotics such as doxorubicin (adriamycin), bleomycin, and mitomycin; nitrosoureas such as carmustine (BCNU), lomustine, epirubicin, idarubicin, and daunorubicin; inorganic ions such as cisplatin and carboplatin; interferons, asparaginase, and hormonal agents such as tamoxifen, leuprolide, flutamide, and megestrol acetate.

[0252] According to some embodiments, the chemotherapeutic agent is selected from alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxin, antibiotics, L-asparaginase, topoisomerase inhibitors, interferons, platinum coordination complexes, anthracendione-substituted ureas, methylhydrazine derivatives, corticosteroids, corticosteroids, progestins, estrogens, antiestrogens, androgens, and gonadotropin-releasing hormone analogs. According to another embodiment, the chemotherapeutic agent is selected from the group consisting of 5-fluorouracil (5-FU), leucovorin (LV), irenotecan, oxaliplatin, capecitabine, paclitaxel, and doxetaxel. One or more chemotherapeutic agents may be used.

[0253] In some embodiments, the pharmaceutical composition according to the invention is for use in the treatment of cancer or for use in enhancing the immune response.

[0254] The term "enhancing the immune response" refers to increasing the responsiveness of the immune system and inducing or prolonging its memory. The pharmaceutical composition according to the invention can be used to stimulate the immune system during vaccination. Thus, in one embodiment, the pharmaceutical composition can be used to improve vaccination.

[0255] In certain embodiments, the cancer is selected from cancers of the lung, thyroid, breast, colon, melanoma, prostate, liver, bladder, kidney, cervix, pancreas, leukemia, lymphoma, myeloid, ovary, uterus, sarcoma, bile duct, and endometrial cells. Each possibility represents a separate embodiment of the invention.

[0256] According to some embodiments, a pharmaceutical composition comprising at least one antibody or a fragment thereof according to the invention, and a pharmaceutical composition comprising an additional immunomodulator or kinase inhibitor are used for the treatment of cancer by separate administrations.

[0257] In yet another aspect, the invention provides a method of treating cancer in a subject that needs it, comprising administering to the subject a therapeutically effective amount of a monoclonal antibody or antibody fragment according to the invention.

[0258] In an additional aspect, the invention provides a method for treating a disease associated with nectin 4 overexpression.

[0259] In one aspect, the invention provides a method of treating cancer in a subject that needs it, comprising administering to the subject a therapeutically effective amount of a plurality of T cells comprising a CAR molecule described herein.

[0260] As used herein, the term “effective dose” refers to a sufficient amount of monoclonal antibody of an antibody fragment that, when administered to a subject, has the intended therapeutic effect. The effective dose required to achieve the final therapeutic outcome may depend on many factors, including, for example, the specific type of tumor and the severity of the patient’s condition, as well as whether the combination therapy is further administered concurrently with radiation. In the context of the present invention, the effective dose of the activator should be sufficient to influence a beneficial therapeutic response in the subject over a period of time, and may include, but are not limited to, inhibition of tumor growth, reduction of tumor growth rate, prevention of tumor and metastatic growth, and improvement of survival.

[0261] The toxicity and therapeutic efficacy of the compositions described herein can be determined by standard pharmaceutical procedures in cell culture or experimental animals, for example, by determining the IC50 (concentration that yields 50% inhibition) and maximum tolerated dose for the compound in question. Data obtained from these cell culture assays and animal studies can be used to formulate a range of doses for use in humans. Dosages may vary depending, among other relevant factors, the dosage form used, the administration plan selected, the composition of the drug used for treatment, and the route of administration utilized. The appropriate formulation, route of administration, and dosage may be selected by the individual physician taking into account the patient's condition. Depending on the severity and responsiveness of the condition being treated, administration may also be a single dose of a sustained-release composition, and the treatment process may continue for several days to several weeks, or until a cure is achieved or a reduction in the condition is attained. The amount of composition administered will, of course, depend on the subject being treated, the severity of the illness, the means of administration, the judgment of the prescribing physician, and all other relevant factors.

[0262] The terms “administering” or “dosing” a substance, compound, or drug to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or drug can be administered enterally or parenterally. Enteral administration refers to administration via the gastrointestinal tract, including orally, sublingually, or rectally. Parenteral administration includes administration by vein, intradermal, intramuscular, intraperitoneal, subcutaneous, ocular, sublingual, nasal, inhalation, intraspinal, intracerebral, and transdermal (e.g., by absorption through the skin duct). Compounds or drugs can also be appropriately introduced by rechargeable or biodegradable polymer devices or other devices, such as patches and pumps, or by formulations, resulting in sustained release, sustained release, or controlled release of the compound or drug. Administration can also be carried out, for example, once, multiple times, and / or over one or more extended periods. In some embodiments, administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who administers medication to a patient himself or has another person administer the medication, and / or a physician who provides a prescription for medication to a patient, is administering medication to a patient.

[0263] Antibodies are generally administered in doses ranging from approximately 0.1 to 20 mg / kg of patient body weight, typically from approximately 0.5 to 10 mg / kg, and often from approximately 1 to 5 mg / kg. In this regard, it is preferable to use antibodies with a circulating half-life of at least 12 hours, preferably at least 4 days, and more preferably up to 21 days. Chimeric antibodies are expected to have a circulating half-life of 14 to 21 days. In some cases, administering a large loading dose followed by intermittent (e.g., weekly) maintenance doses over the course of treatment may be effective. Antibodies can also be delivered by sustained-release delivery systems, pumps, and other known delivery systems for continuous infusion.

[0264] The term "approximately" means that an acceptable error range, such as 5% or 10%, should be assumed for a particular value.

[0265] diagnosis The present invention further discloses a method for diagnosing and prognosing cancer.

[0266] According to one embodiment, the present invention provides a diagnostic and / or prognostic diagnostic method for cancer or infection in a subject, the method comprising the step of determining the expression level of nectin 4 in a biological sample of the subject using at least one antibody as described herein.

[0267] The term “biological sample” encompasses a variety of sample types obtained from organisms that may be used in diagnostic or monitoring assays. This term includes blood and other fluid samples of biological origin, solid tissue samples such as biopsy specimens, or tissue cultures or cells and their offspring derived therefrom. Furthermore, this term may include circulating tumors or other cells. Specifically, this term includes clinical samples, and further includes biological fluids such as cells in cell culture, cell supernatants, cell lysates, serum, plasma, urine, amniotic fluid, aqueous humor and vitreous humor for eye samples, as well as tissue samples. This term also includes samples that have been manipulated in any way after achieving treatment with reagents, solubilization, or enrichment of specific components.

[0268] The expression level of nectin 4 can be determined by a labeled anti-nectin 4 antibody as described herein. Expression determination can be performed, for example, by ELISA. The method of the present invention may further include a step of comparing the expression level with a control level.

[0269] The following embodiments are provided to further illustrate some embodiments of the present invention. They should not be construed as limiting the scope of the invention. [Examples]

[0270] Experimental Procedure Herein, for reference, the following embodiments are carried out, and together with the above description, the present invention will be explained in a non-limiting manner.

[0271] In general, the nomenclature used herein and the experimental procedures utilized in the present invention include molecular, biochemical, microbiological, immunological, and recombinant DNA techniques. Such techniques are well known in the art. Other general references that refer to well known procedures are provided throughout this document for the convenience of the reader.

[0272] method cell line 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 mouse nectin-4. For all cells except RAJI cells, the cells were grown in DMEM at 37°C, humidity >95%, and 5% CO2, and cultured in RPMI (medium and serum from Sigma-Aldrich) supplemented with 10% heat-inactivated FCS.

[0273] Flow cytometry Flow cytometry was performed using an anti-mouse nectin 4 mAb (clone 356704). Cells were incubated on ice for 30 minutes with 0.2 μg of mAb per 100,000 cells. Detection was performed on ice for 30 minutes with a secondary goat ab conjugated with AlexaFluor647 (Jackson ImmunoResearch).

[0274] In several cases, anti-nectin-4Ab was used at various concentrations and incubated with target cells on ice for 30 minutes. Detection was performed on ice for 30 minutes using a secondary antibody against mouse or human Fc conjugated with AlexaFluor647 (Jackson ImmunoResearch).

[0275] For staining with human TIGIT-Ig or human Nectin-1-Ig, cells were incubated with 3 μg of TIGIT-Ig per 100,000 cells on ice for 1 hour. Detection was performed on ice for 30 minutes using secondary AlexaFluor647 anti-human (Jackson ImmunoResearch). For blockade experiments, cells were pre-incubated with 1 μg of the indicated antibody and then stained with TIGIT-Ig or human Nectin-1-Ig. In some cases, cells were incubated with 8 ug / ml of the indicated antibody and human TIGIT-Ig or human Nectin-1-Ig (20 ug / ml). Ligand binding detection was performed using secondary AlexaFluor647 anti-human antibody (Jackson ImmunoResearch).

[0276] The analysis was performed using a FACS-Calibur (BD Biosciences) or Cytoflex BecmanCoulter flow cytometer and FCSExpress software.

[0277] Killing assay To evaluate the cytotoxic activity of NK cells against target cells, S 35 The release assay was performed as described (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 the addition of PHA and IL-2. Target cells were treated with radioactive methionine [S 35 The cells were incubated overnight in methionine-free medium with added [antibody]. Next, the cells were washed and incubated on ice with 1 μg of antibody per well (5000 cells). Then, the cells were incubated with NK cells for 5 hours. 35 The emission was measured using a β-counter TopCount (Packard). The result is expressed as (CPM(sample)-CPM(spontaneous emission)) / (CPM(total emission)-CPM(spontaneous emission))×100, where CPM represents the count per minute.

[0278] ADCC assay NK cells were isolated from healthy donors using the EasySep Human NK Cell Isolation Kit (19055 STEMCELL TECHNOLOGIES) and expanded with the addition of PHA and IL-2. Target cells were added to 96U plates at 2.5×10 4 cells per well and co-cultured with activated NK cells at an E:T ratio of 2:1. Incubation was performed in the presence of 12 μg / ml chimeric clones hNec4.05hIgG1 and hNec4.11hIgG1 or control hIgG1. After 2 hours, NK cells were analyzed by FACS for their CD107a (Biolegend catalog number 328619) degranulation marker expression.

[0279] Generation and functional assay of CAR-T The single chain of hNec4.11Ab was cloned in-frame into the CD8 stalk region, followed by the CD28TM domain, the 41BB intracellular domain, and the intracellular domain of the CD3Zeta chain. A schematic diagram of the CAR-T construct is shown in Figure 11A. The construct was introduced into hEf1a containing the promoter lentiviral vector (pHAGE2), followed by the introduction of the IRESGFP cassette to monitor the effectiveness of transduction. Jurkat cells were transduced with lentiviral particles encoding the construct. The transduction effectiveness exceeded 99% (Figure 11B).

[0280] Parent Jurkat cells or Jurkat cells expressing the CAR-T construct (Jurkat pHAGE2.4.11) (5×10 4 cells per well) 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 the IL-2 level was measured using Peprotech's IL-2 ELISA kit (catalog number 900-T12) according to the manufacturer's protocol.

[0281] PBMCs derived from healthy donors were pre-activated 72 hours prior to the use of ImmunoCult® human CD3 / CD28 T cell activator according to the manufacturer's protocol. Cells were transduced using a pHAGE 2.4.11 lentiform according to Kochenderfer JN et al. (J Immunotther. 2009 doi:10.1097 / CJI.0b013e3181ac6138), and expression was verified by GFP levels. CART PBMCs were transduced from HT1376 cells (2.5 × 10⁶). 4 The cells (individual cells / well) were incubated with cells from various E:T ranges. After 48 hours, effector cells were removed, target cells were washed three times, and viability was measured using the CellTiter-Glo® luminescent cell viability assay according to the manufacturer's protocol.

[0282] In vivo mouse tumor model All experiments were conducted using 6-8 week old SCID beige female mice. All mice were housed in a specific pathogen-free facility at the Hebrew University Faculty of Medicine (Ein Kerem, Jerusalem) under SPF conditions, with normal light-dark cycles, and at 22+ / -2°C, in accordance with the guidelines of the Ethics Committee. Each mouse group consisted of 7 females (n=7). Xenografts were prepared by subcutaneous injection of the indicated cells into the left flank region. Intraperitoneal injections of anti-nectin 4 clone .05 antibody and control antibodies (anti-mouse CD3, InVivoMAb clone 17A2) were administered twice weekly. Mice were monitored daily. On the day of the end of the assay (see legend in the figure), all mice were sacrificed and individual tumor weights were recorded. No differences were observed in their general health status at baseline among the different mouse groups.

[0283] Example 1. Production and selection of anti-nectin-4mAb The immunogen (Nec4-Fc) expression technique, based on mammalian HEK293T cells, is a method of particular choice for glycoproteins, providing the highest quality, stability, solubility, and yield. The Nec4-Fc protein, a fusion protein of the external domain of nectin-4 and the human IgG1Fc domain, was synthesized and purified by recombination as follows.

[0284] The coding sequence of human nectin 4 was cloned as a fusion with the Fc fragment of human IgG1 to generate a recombinant Fc-fusion protein. The extracellular domain (extracellular) portion of the human nectin 4 molecule, extending from residues 32 to 349, was used. The C-terminal serine residue at position 349 of the nectin 4 amino acid sequence was fused to the heavy chain hinge of the deglycosylated Fc (N297A) of human IgG1, followed by the CH2 and CH3 constant regions. The open reading frame (ORF) of the recombinant protein was codon-optimized for high-level expression in mammalian cells. The optimized DNA sequence was constructed using the GeneArt synthesis service (Invitrogen) by adding adjacent DNA sequences corresponding to EcoRI and NotI restriction sites to the 5' and 3' ends of the DNA fragment, respectively. The expression vector was constructed by double digesting the optimized DNA fragment with EcoRI and NotI, followed by ligation with pIRESpuro3 (Clontech Laboratories, Inc.). The resulting construct was transfected into HEK-293T cells using FuGENE6 transfection reagent (Roche Diagnostics). After 48 hours, the transfected cells underwent antibiotic selection with 5 g / mL puromycin (Sigma-Aldrich). The stable pool was analyzed for protein secretion by SDS / PAGE. The supernatant was collected and purified using a Poros20 protein G column at a High-Pressure Perfusion Chromatography Station, BioCAD (PerSeptive Biosystems). The resulting fusion protein immunogen is denoted as Nectin 4-Fc.

[0285] For immunization, BALB / c mice were infused with 50 μg of immunogen in complete Freund's adjuvant (CFA), followed 14 days after the initial immunization by infusion of 50 μg of immunogen in incomplete Freund's adjuvant (IFA). Serum was then analyzed by ELISA for anti-Nec-4-Fc antibody titer. Mice with the highest titer were further immunized with 50 μg of immunogen in PBS. Three days later, the spleens of the immunized mice were harvested, and after erythrocyte lysis, the splenic cells were fused with the SP2 / 0 cell line. Potential hybridoma cells were seeded in 20% RPMI1640 medium containing hypoxanthine, aminopterin, and thymidine (HAT) for 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, 30 wells in which the supernatants were positive for binding to Nec4-Fc coated on ELISA plates were retested for their positivity, and in parallel, cross-reactivity tests were performed on unrelated Fc fusion proteins. This resulted in 10 hybridoma cell lines that secreted antibodies specifically recognizing the nectin-4 extradomain. All of these candidates that showed specific signals in ELISA were tested for their ability to recognize native human nectin-4 protein in transfectant cell lines.

[0286] RAJI Burkitt lymphoma cells were transfected with human nectin 4 and analyzed by FACS to determine the binding of the five hybridoma clones shown. Cells were incubated with different clones and then incubated with human TIGIT-Ig and stained with secondary antibodies. The mean fluorescence intensity (MFI) values ​​for FACS staining are shown in Figure 2. As shown, monoclonal antibodies produced by clones hNec4.01 (clone 1) and hNec4.05 (clone 5) showed the best blocking ability of the TIGIT-Nectin 4 interaction.

[0287] Similar assays using JEG3 and LNCap cell lines that spontaneously express nectin 4 have also been performed, yielding similar results.

[0288] Five antibodies showed positive staining. Of these five, four were further selected for their IgG isotype (not IgM isotype). Finally, the remaining four candidates all showed strong binding ability to native human nectin-4 molecules expressed on the surface of living cells and were repeatedly tested with several unrelated fusion proteins to select those with zero cross-reactivity between nectin-4 and other ligands for immune cell receptors. However, only two of these were shown to be blocking antibodies against nectin-4. Thus, two stable clonal cell lines, hNec4.01 and hNec4.05, were generated, both of which are of isotype kappaIgG1. Next, large-scale ab production was carried out, and both monoclonal antibodies were purified in small amounts (a few milligrams) from serum-free medium using a GE AKTA PrimePlus liquid chromatography system and HiTrap Protein G columns.

[0289] Similarly, additional clones were identified, and clone 11 (hNec4.11) exhibited the best binding and blocking ability, so it was screened in parallel with hNec4.05 as described below.

[0290] Based on the above attempts, even in the case of immunogens very similar to the target protein nectin-4 (in that they are dimers, glycosylated by mammalian cellular mechanisms, and produced under non-denaturing conditions), the chances of obtaining hybridoma cell lines that secrete blocking anti-nectin-4 antibodies are less than 2% (per mille).

[0291] Example 2. Affinity of anti-nectin-4 mAb with human nectin-4 The affinity of antibodies hNec4.01 and hNec4.05 to fluorophore-labeled human nectin 4-Ig molecules was further determined using a microscale thermophoresis assay (Wienken et al. 2010, Nat. Commun. 1:100). Measurements were repeated with at least three independent protein preparations. Very high binding affinity was observed for both antibodies, as shown in Figure 3. The calculated Kd was 272 ± 154 pM for clone hNec4.05 and 107 ± 115 pM for clone hNec4.01.

[0292] Example 3. Sequence determination of anti-nectin-4mAb Two hybridoma clones, No. 0.1 and No. 0.5, which showed the best inhibition of nectin 4-TIGIT binding, were subjected to nucleotide and amino acid sequencing.

[0293] method Total RNA was isolated from hybridoma cells according to the technical manual for TRIzol® reagent (Ambion, catalog number: 15596-026). Next, the total RNA was reverse transcribed into cDNA using isotype-specific antisense primers or universal primers, according to the technical manual for the PrimeScript® 1st Strand cDNA synthesis kit (Takara, catalog number: 6110A). H and V L The antibody fragment was amplified according to the standard operating procedure (SOP) for rapid amplification of cDNA ends (RACE) in GenScript. The amplified antibody fragment was cloned separately into a standard cloning vector. Colony PCR was performed to screen for clones with the correct-sized insert. Five or more colonies with the correct-sized insert were sequenced for each fragment. The sequences of the different clones were aligned to obtain the consensus sequence of these clones.

[0294] The following tools were used for sequence analysis of the immunoglobulin variable region. i.NCBI nucleotide BLAST, ii. IMGT / V Quest program, and iii. NCB1gBLAST.

[0295] The resulting sequence is as follows: Clone hNec4.01: [ka] [ka] [ka] [ka] [ka] [ka] Clone hNec4.05 [ka] [ka] [ka] [ka] [ka] [ka] Clone hNec4.11 [ka] [ka] [ka] [ka] [ka] [ka]

[0296] Example 4. Blocking the nectin-4-TIGIT interaction with an anti-nectin 4 mAb enhanced the lethality of human cell lines by NK cells. Antibody clones hNec4.05 and hNec4.01 were tested for blocking the binding of Nectin 4 to TIGIT and inhibiting NK cytotoxicity. [ 35 [S]methionine-labeled RAJI Burkitt lymphoma cells and LNCaP prostate cancer cells (which intrinsically express nectin 4) were incubated with 1 μg / well of either mouse IgG1 or the mouse anti-human nectin 4 mAb hNec4.01 or hNec4.05 as control antibodies. After 1 hour, the cells were supplemented with NK cells and incubated for 5 hours. Mean specific death (±sd) of NK:cancer cells at various effector:target (E:T) ratios is plotted in Figure 4A (RAJI cells) and Figure 4B (LNCaP cells). * indicates a significant effect (p<0.05) of the hNec4.01 and hNec4.05 clones compared to the control antibody. Each figure shows a representative experiment from one of three trials performed. The same effect was determined when using the MCF-7-breast cancer cell line.

[0297] Example 5. Verify the binding specificity of the anti-nectin 4 antibody. Testing using FACS staining revealed that mAbs hNec4.01 and hNec4.05 are specific to human nectin 4 and do not bind to mouse proteins, as shown in Figures 5A–5C. RAJI cells were transfected with mouse nectin 4 (black line histogram). First, RAJI cells transfected with mouse nectin 4 lysate were used in a Western blot assay with a commercially available anti-mouse nectin 4 mAb (clone 356704) to verify mouse nectin 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 mouse Nectin 4.

[0298] Example 6. Anti-nectin 4 antibodies can block the nectin 4-nectin 1 interaction. The ability of anti-nectin 4 to block the nectin 4-nectin 1 interaction was also determined. FACS staining of RAJI cells transfected with nectin 4 is shown in Figures 6A-6C. Cells were pre-incubated with 1 μg of (Figure 6A) clone hNec4.01 or (Figure 6B) clone hNec4.05, and then incubated with 3 μg of nectin 1-Ig (black line). Staining without blockade is shown by the gray line. The gray-filled histogram is the background control staining of the secondary antibody only. It can be concluded that the mAb can block the nectin 4-nectin 1 interaction, which is suspected to increase the invasiveness of tumors expressing nectin 4.

[0299] Example 7. In vivo model The efficacy of anti-nectin 4 mAbs was determined in vivo in animal models. Cell lines expressing nectin 4 intrinsically (MDA-MB-453) or recombinantly (Raji nectin 4OE) were subcutaneously injected into mice (5 × 10 cells per mouse). 6 (1 x 10⁶ cells). SCID beige mice lacking NK cells, B cells, and T cells were used. To test the contribution of NK cells to tumor cell proliferation in these models, human NK cells were added in 1 x 10⁶ cells. 6It was injected individually into several treatment groups along with tumor cells.

[0300] The anti-nectin-4 mAb clone hNec4.05, or the control Ab (anti-mouse CD3, InVivoMAb clone 17A2), was tested in vivo for their effects on tumor growth, either directly or in conjunction with NK cells. mAbs were intraperitoneally injected at 75 μg per mouse twice weekly. Tumor weight was measured at the end of the study. As shown in Figure 7(A), overexpression (OE) of nectin-4 in Raji cells did not affect their growth compared to the growth of their parent cells transfected with an empty vector (EV). Nevertheless, in the presence of human NK cells, OE of nectin-4 increased tumor growth, suggesting a negative effect of nectin-4 on the NK-mediated tumor suppressor activity of NK cells. As shown in Figure 7B, the addition of anti-nectin-4 Ab blocked NK cell suppression, resulting in reduced tumor growth compared to control Ab-treated animals. Finally, as shown in Figure 7C, these effects were also observed when using a cell line that intrinsically expresses nectin 4 (MDA-MB-453). Ab had no effect in the absence of NK cells, but Ab significantly enhanced the antitumor effect of human NK cells co-injected with tumor cells. In summary, these experiments demonstrate that anti-nectin 4 antibodies such as hNec4.05mAb can enhance NK cytotoxicity and inhibit tumor growth in vivo.

[0301] Example 8. Binding of anti-nectin-4 clones to human, monkey, and mouse nectin-4 expressed in cells. The binding of mouse anti-human nectin-4 clones hNec4.05 and hNec4.11 to nectin-4 expressed in the human cell line MDA-MD-453 was evaluated by FACS analysis. Figure 8A shows the EC50 values ​​calculated after titer measurement of Ab binding (ranging from 20 to 0.01 nM) and the maximum binding signal for each clone. Similar values ​​were reached when chimeric mouse Ab types, in which the mouse IgG1Fc chain was replaced with a human IgG1Fc chain, were tested. Figure 8B shows antibody binding with CHO cells transfected with cynomolgus monkey (Cyno)-nectin-4, and Figure 8C shows antibody binding with CHO cells transfected with mouse-nectin-4. These data show EC50 values ​​against the human target in the sub-nanomolar range, which corresponds to the Kd value scale shown in Figure 3 for clones hNec4.01 and hNec4.05. Furthermore, these results demonstrate the cross-reactivity of these two clones to the monkey (cynomolgus monkey) nectin-4 target, binding to it with EC50 values ​​similar to those calculated for the human target. This could be important for Ab's preclinical trials. Finally, clone hNec4.11 was shown to cross-react with the mouse nectin-4 target, but not with clone hNec4.05. However, in this case, the calculated EC50 was approximately 10 times higher than that calculated for the human target.

[0302] Example 9. Blocking of nectin-4 ligand by anti-nectin-4 clones. The antibody clones hNec4.05 and hNec4.11 block the binding of nectin-4 to its ligands, TIGIT and Nectin-1. Nectin-4 ligand binding was evaluated by FACS analysis. CHO cells transfected with human nectin-4 were incubated with either human TIGIT-Ig (Figure 9A and C) or human nectin-1-Ig (Figure 9B and D) at 20 ug / ml, or with either anti-nectin-4 clone hNec4.05 (9A and 9B) or clone hNec4.11 (9C and 9D) at 8 ug / ml, or without. Potent inhibition of binding by anti-nectin-4 anti-ab was shown in all cases. Remaining signals may be due to ligand binding to other receptors expressed by CHO cells, such as PVR, which are unaffected by the anti-nectin-4 antibody. These results suggest that the nectin-4 clones hNec4.05 and hNec4.11 may affect targeted cancer cells by blocking nectin-4-mediated signaling on the cell surface. Furthermore, these antibodies may also affect effector cells expressing nectin-4 ligands such as the inhibitory ligand TIGIT.

[0303] Example 10. Human IgG1 chimeric Ab clones hNec4.05 and hNec4.1 enhance NK cell activation in the presence of tumor cells. Figure 11 shows the relative expression levels of the degranulation marker CD107a in NK cells. Human NK cells (effector, E) were incubated with target cells (T), HT1376 (Figure 10A) and MDA-MD-453 (Figure 10B), in an E:T ratio of 2:1. Incubation was performed in the presence of 12 ug / ml of chimeric clones hNec4.05hIgG1, hNec4.11hIgG1, or control hIgG1. After 2 hours, NK cells were assayed for their degranulation and activation status by FACS analysis of CD107a expression. Degranulation of NK cells in the presence of control hIgG1 was set to 1, and the induction factor was calculated accordingly. Shown are the mean of 2-3 repeats and their normalized standard deviations. These results suggest the anticancer effect of these antibodies by enhancing antitumor NK cell activity. A slight advantage of clone hNec4.11-hIgG1 over clone hNec4.05hIgG1 was shown in ADCC induction, which is consistent with the higher maximum binding of clone hNec4.11, as shown in Figure 8A.

[0304] Example 11. CAR-T cells driven by hNec4.11 result in the activation of specific T cells in the presence of tumor cells expressing nectin-4. A schematic diagram of the CAR-T construct is shown in Figure 11A. Transduction efficacy, as determined by GFP expression, exceeded 99% (Figure 11B). Jarcut cells expressing parental Jarcut cells or CAR-T constructs containing hNec4.11-based single-chain variable regions (scFV) (Jarcut pHAGE2.4.11) were incubated with target cells HT1376 and MDA-MD-453 (MDA-453) (Figure 11C). IL-2 secretion by Jarcut cells was significantly induced by CAR-T expression. Next, PBMCs were transduced using pHAGE2.4.11 lenti particles (Figure 11D). CAR-T PBMCs were incubated with HT1376 cells across the E:T range. After 48 hours, effector cells were removed, and target cell viability was evaluated using the CellTiter-Glo® bioluminescence viability assay. Target cell death was significant. In summary, these results suggest potential for further development of CAR-T therapies based on the nectin-4 antibody described herein. scFv array of CAR constructor Clone 11-Nucleic Acid (SEQ ID NO: 31) ATGGGATGGAGCTGTATCCATCCTCTTCTTGGGTAGCAACAGCTACAGGTTGCATTCACAGGTCCAGCTGCAGCAGTCTGGACCTGAACTGGTGAAGCCTGAGACTTCAGTGAAGATTTCCTGCAAGGCTTCTGGCTACACCTTCACAAGTTACTATATACACTGGGTGAAACAGAGGCCTGGACAGGGACTTGAGTGGATTGGCTGGATTTATCCTGGAAATGTTAATACTAAGTATAATGAGAGGTTTAAGGGCAAGGCCACTGCAGACAAATCCTCCAACACAGCCCAC ATGCAGCTCACCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAGATCGAACCCCTATGTTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGGTGGAGGTGGCTCCGGAGGAGGTGGTTCTGGAGGAGGTGGTTCTGATATCGTGAGCCCAGACTCCCAAATTCCTGCTTGTATCAGCAGGAGACAGAGTCACCATAACCTGCAAGGCCAGTCAGAGGTGAATAATGATGTGGCTGGTTACAACAGAAGCCAGGGCTGTCTCCTGAACTGCTT ATGTATTATGCATCCAATCGCTTCACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCTCTGTGCAGGCTGAAGACCTGGCAATTTATTTCTGTCAGCAGGCTTATAGGTCTCCGTACACGTTCGGAGGGGGACCAAGCTGGAAATTCAA クローン11-amino acids MGWSCIILFLVATATGVHSQVQLQQSGPELVKPETSVKISCKASGYTFTSYYIHWVKQRPGQGLEWIGWIYPGNVNTKYNERFKGKATLTADKSSNTAHMQLTSLTSEDSAVYFCARSNPYVMDYWGQG TSVTVSSGGGGSGGGGSGGGGSDIVMTQTPKFLLVSAGDRVTITCKASQSVNNDVAWYQQKPGLSPELLMYYASNRFTGVPDRFTGSGYGTDFTFTISSVQAEDLAIYFCQQAYRSPYTFGGGTKLEIQ Clone 5-Nucleic Acid (SEQ ID NO: 33) ATGGGATGGAGCTGTATCCATCCTCTTCTTGGGTAGCAACAGCTACAGGTGTGCATTCACAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAGGATATCCTGCAAGGCCTCTGGCTACACCTTCACAACCTACTATATACACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTG GAAATGTTAATACTAAGAACAATGAGAAGTTCAAGGTCCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACAGCAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTTATTCTGTGCAAGATCGAACCCCTATGTTATGGGACTACTGGGGTCAGGGAACCTCAGTCACCGTCTCCTCAGGTGGAGGTGCTCCGGAGGAGGTGGT TCTGGAGGAGGTGGTTCTAGTATTGTGATCGACCAGACTCCCAAATTCCTGCTTGTATCAGCAGGAGACAGGGTTACCATACCTGCAAGGCCAGTCAGAGTGTGAGTAATGATGTAGCTTGGTACCAACAGAAGCCAGGGCAGTCTCCTAAACTGCTGATATACTATG CATCCAATCGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCAGCGCTGTGCAGGCTGAAGACCTGGCAGTTTATTTCTGTCAGCAGGATTATAGCTCTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAA クラーン5-amino acid MGWSCIILFLVATATGVHSQVQLQQSGPELVKPGASVRISCKASGYTFTTYYIHWVKQRPGQGLEWIGWIYPGNVNTKNNEKFKVKATLTADKSSSTAYMQLSSLTSEDSAVYFCARSNPYVMDYWGQG TSVTVSSGGGGSGGGGSGGGGSSIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISAVQAEDLAVYFCQQDYSSPYTFGGGTKLEIK

[0305] The foregoing description of specific embodiments fully reveals the general nature of the invention; others can readily modify and / or adapt them to various uses of such specific embodiments without excessive experimentation and without departing from the general concept by applying current knowledge, and such adaptations and modifications should be understood, and are intended to be understood, within the meaning and scope of the equivalents of the disclosed embodiments. It should be understood that any expressions or terms used herein are for illustrative purposes only and not for limitation.

Claims

1. An isolated monoclonal antibody that binds to nectin 4, or an antibody fragment thereof containing at least an antigen-binding moiety, which can inhibit the binding of nectin 4 to a T cell immune receptor (TIGIT) having Ig and ITIM domains, The isolated antibody or antibody fragment comprises a set of six CDRs, and the set comprises (i) A set of six CDRs in which heavy chain (HC) CDR1 is SYYIH (SEQ ID NO: 25), HC CDR2 is WIYPGNVNTKYNERFKG (SEQ ID NO: 26), HC CDR3 is SNPYVMDY (SEQ ID NO: 27), light chain (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); and (ii) A set of six CDRs, where 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); Selected from the group consisting of, Isolated monoclonal antibody or its antibody fragment.

2. An isolated monoclonal antibody or antibody fragment according to claim 1, comprising a heavy chain and a light chain, The heavy chain includes sequence number 39, and the light chain includes sequence number 40, or The heavy chain includes sequence number 37, and the light chain includes sequence number 38. Isolated monoclonal antibody or antibody fragment.

3. An isolated monoclonal fragment according to claim 1 or 2, wherein the isolated monoclonal fragment is a single-chain Fv (scFv).

4. The isolated monoclonal fragment according to claim 3, comprising a sequence selected from the group consisting of SEQ ID NOs: 32 and SEQ ID NOs: 34, or a variant thereof having at least 90% sequence similarity to the aforementioned sequence.

5. The aforementioned monoclonal antibodies are human nectin 4 and 10 －９ M-10 －１０ It binds due to the affinity of M, or The antibody or its fragment inhibits the interaction between nectin 4 and nectin 1 in tumor cells. An isolated monoclonal antibody or antibody fragment according to any one of claims 1 to 4.

6. A nucleic acid comprising a polynucleotide sequence encoding an HC sequence and an LC sequence of a monoclonal antibody or antibody fragment according to any one of claims 1 to 5.

7. A plasmid or cell comprising at least one polynucleotide sequence according to claim 6.

8. A pharmaceutical composition comprising, as an active ingredient, an isolated antibody or a fragment thereof comprising at least one antigen-binding portion as described in any one of claims 1 to 5, and comprising a pharmaceutically acceptable excipient, diluent, salt, or carrier.

9. The pharmaceutical composition according to claim 8, wherein the monoclonal antibody is not conjugated with a cytotoxic moiety.

10. The pharmaceutical composition according to claim 9, for use in regulating the immune system by inhibiting the binding of nectin 4 to TIGIT, or for use in the treatment of cancer, or for use in the prevention or treatment of viral infection in a subject.

11. The pharmaceutical composition according to claim 10, wherein the use further comprises the use of an additional immunomodulator, activated lymphocytes, kinase inhibitor, chemotherapeutic agent, or any other anticancer agent.

12. A chimeric antigen receptor (CAR) comprising at least one antibody according to any one of claims 1 to 5, or an antibody fragment having at least an antigen-binding portion.

13. The CAR according to claim 12, comprising a sequence selected from the group consisting of SEQ ID NOs. 32 and 34, a transmembrane domain, and an intracellular T cell or NK cell signaling domain. or A population of T cells or NK cells manipulated to express the CAR described in claim 12.

14. The CAR according to claim 12 for use in the treatment of cancer.

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