Antibody specific to human nectin-2

Abstract

The present disclosure provides a monoclonal antibody that recognizes human nectin-2 (nectin-2, poliovirus receptor-related protein 2, poliovirus receptor-like 2, CD112, or PRR-2, a single-pass transmembrane glycoprotein containing two Ig-like C2-type domains and an Ig-like V-type domain) with high affinity and specificity and inhibits its binding to TIGIT and / or CD112R. This antibody recognizes the nectin-2 protein (CD112) and prevents its binding to T cell immunoreceptor (TIGIT) and CD112R (PVRIG), which contain Ig and ITIM domains, and inhibits its suppressive activity on lymphocytes such as natural killer (NK) cells and T cells. The present disclosure also provides pharmaceuticals and methods for use in cancer immunotherapy and diagnosis. Finally, the present disclosure further provides a chimeric antigen receptor (CAR) comprising an scFv antibody that binds to nectin-2.

Description

[Technical field]

[0001] The present invention is in the field of immunotherapy and relates to antibodies and fragments thereof that bind to the human protein Nectin-2 (CD112), polynucleotide sequences encoding these antibodies and fragments, and cells producing them. The present invention further relates to therapeutic and diagnostic compositions comprising these antibodies and fragments, and methods of using them to treat and diagnose diseases, particularly cancer. [Background technology]

[0002] Cancer immunotherapy is utilized to generate and enhance anti-tumor immune responses, for example, by treatment with antibodies specific to antigens on tumor cells, by fusion of antigen-presenting cells with tumor cells, or by specific activation of anti-tumor T cells. The ability to recruit immune cells (e.g., T cells) against tumor cells in patients provides a treatment to combat types of cancer and metastasis that were previously considered incurable.

[0003] T cell-mediated immune responses involve multiple sequential steps regulated by the balance between costimulatory and co-inhibitory signals that control the extent of the immune response. Inhibitory signals, called immune checkpoints, are important for maintaining self-tolerance and limiting immune-mediated secondary tissue damage. These inhibitory signals change as an infection or immune challenge resolves, worsens, or persists, and these changes affect T cell responses to reshape the immune response.

[0004] The expression of immune checkpoint proteins is altered by tumors. For example, upregulation of programmed cell death ligand 1 (PD-L1) on the surface of cancer cells allows them to bind to the checkpoint molecule PD-1 expressed on T cells. This results in the inhibition of T cells that would otherwise attack tumor cells and allow them to evade the host immune system. Thus, immune checkpoints represent a significant barrier to the activation of functional cellular immunity against cancer. Thus, antagonistic antibodies specific for inhibitory ligands on T cells (e.g., PD-1) are examples of targeted agents against immune checkpoints that have been used in cancer therapy (e.g., nivolumab and pembrolizumab). Another example of an immune checkpoint molecule is the T cell immunoreceptor with Ig and ITIM domains (TIGIT). TIGIT is a co-inhibitory molecule expressed on various immune cells, including T cells and natural killer cells (NK cells). TIGIT binds with high affinity to the poliovirus receptor (PVR) and to nectin-2.

[0005] Nectin-2, also known as poliovirus receptor-related protein 2, poliovirus receptor-like 2, CD112, or PRR-2, is a single-pass transmembrane glycoprotein with two Ig-like C2-type domains and an Ig-like V-type domain. Nectin-2 is involved in mediating cell adhesion to extracellular matrix molecules and functions as one of the plasma membrane components of adherens junctions. Nectin-2 also functions as an entry receptor for certain mutant strains of herpes simplex virus and pseudorabies virus and is involved in the cell-to-cell spread of these viruses. Mutations in this gene have been associated with differences in the severity of multiple sclerosis. Importantly, nectin-2 can also function as a regulator of T cell signaling. Depending on the receptor it binds, nectin-2 can be either a co-stimulator or co-inhibitor of T cell function. Upon binding to CD226 (DNAM-1), it stimulates T cell proliferation and cytokine production, including IL-2 and IFNγ, and upon interaction with PVRIG (CD112R) and / or TIGIT, it inhibits T cell proliferation. These two opposing interactions are competitive.

[0006] Nectin-2 has been shown to be overexpressed in a variety of tumors, including breast and ovarian cancer (Oshima et al. Molecular Cancer 2013). Its presence in tumor cells is associated with poor prognosis and reduced T cell activity (Stamm et al. Oncogene 2018).

[0007] US Patent Application No. 2017 / 0037133 discloses inhibitors against CD112 (Nectin-2, PVRL2), CD155 (PVR), Galectin-9, TIM-3 and / or TIGIT for use in the treatment of blood-borne cancers, particularly acute myeloid leukemia (AML). The inhibitors may be antibody constructs.

[0008] There is an unmet need to provide additional, more effective, specific, safe and / or stable agents that can enhance the cells of the immune system to attack tumor or virus-infected cells, either alone or in combination with other agents. Monoclonal antibodies that inhibit the binding of Nectin-2 to CD112R and TIGIT may be such agents. Summary of the Invention

[0009] The present invention provides antibodies and fragments thereof that recognize the Nectin-2 protein (CD112), prevent its binding to T cell immunoreceptor with Ig and ITIM domains (TIGIT) and CD112R (PVRIG), and inhibit suppressive activity on lymphocytes such as natural killer (NK) cells and T cells. The anti-Nectin-2 antibodies disclosed herein can bind to Nectin-2 present on cancer cells. These antibodies and fragments thereof are characterized by having a unique set of CDR sequences, high affinity and high specificity for human Nectin-2, and are useful in cancer immunotherapy to combat tumor immune evasion, both as monotherapy and in combination with other anti-cancer agents. The antibodies are also useful in the treatment of viral infections and can be used in the diagnosis of cancer. The present invention further includes CAR-T cells and methods of their use for adoptive therapy.

[0010] It is disclosed herein that the high affinity anti-nectin-2 antibodies described herein block TIGIT- and / or CD112R-nectin-2 interaction, and subsequently restore the activity of T cells and NK cells. The antibodies of the present invention are highly specific to human nectin-2. These properties make the monoclonal antibodies of the present invention valuable candidates for use in cancer immunotherapy, allowing the administration of lower doses with fewer side effects.

[0011] Advantageously, anti-nectin-2 mAbs according to the present invention were found to induce T cell proliferation in a manner similar to that induced by anti-PD-1 and anti-CTLA-4 mAbs. Combinations of some anti-nectin-2 mAbs with clinically approved therapeutic anti-PD-1 and anti-CTLA4 mAbs resulted in a significant increase in activity over the activity levels induced by either of the individual mAbs. Surprisingly, some of the combinations of anti-nectin-2 mAbs and anti-PD-1 described herein demonstrated a synergistic effect in killing tumor cells. The induction effect was shown in human peripheral blood mononuclear cells (PBMCs) and primarily in T cells. In addition, nectin-2 mAbs were able to induce the activation of NK cells in the presence of target cancer cells. Furthermore, it has been disclosed that some anti-nectin-2 antibodies did not have a blocking effect on the co-stimulatory signaling of DNAM-1 and therefore did not have a detrimental effect on immune induction signals. In addition, the antibodies described herein were found to be highly specific for human or cynomolgus nectin-2 protein.

[0012] The anti-Nectin-2 mAbs disclosed herein may have a functional heavy chain (Fc) that may further induce an anti-cancer immune response.

[0013] Some of the anti-Nectin-2 mAbs described herein may be able to reduce tumor cell viability in an immune-independent manner.

[0014] According to one aspect, the present invention provides an antibody, or an antibody fragment thereof comprising at least an antigen-binding portion, that specifically binds to human Nectin-2 and inhibits its binding to at least one of the receptors TIGIT and CD112R, the antibody having an affinity of at least 10 for human Nectin-2. -9 It has an affinity for M.

[0015] The present invention also provides an antibody or antibody fragment thereof capable of inhibiting the binding of human Nectin-2 to human TIGIT or to CD112R, for use in the treatment of cancer, together with T cell lymphocytes and / or natural killer (NK) cells, the antibody having a specific binding activity of at least 10 to human Nectin-2. -9 It has an affinity for M.

[0016] According to some embodiments, the antibody specifically binds to human Nectin-2 and inhibits its binding to TIGIT and CD112R.

[0017] According to some embodiments, the antibody or antibody fragment comprises: i. the three complementarity determining regions (CDRs) of the heavy chain (HC) variable region comprising SEQ ID NO:7 and the three CDRs of the light chain (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 ii. A set of six CDR sequences selected from the group consisting of the three CDRs of a heavy chain variable region comprising SEQ ID NO: 17 and the three CDRs of a light chain variable region comprising SEQ ID NO: 18, or an analog or derivative thereof having at least 90% sequence identity with the sequence of said antibody or fragment.

[0018] There are several methods known in the art for determining the CDR sequences of a given antibody molecule, but there is no standard, defined method. The determination of CDR sequences from the heavy and light chain variable regions of an antibody can be performed according to any method known in the art, including but not limited to the methods known as KABAT, Chothia, and IMGT. The set of selected CDRs can include sequences that are identified by more than one method, i.e., for example, some CDR sequences are determined using KABAT and some use IMGT. According to some embodiments, the CDR sequences of mAb variable regions are determined using the IMGT method.

[0019] According to some embodiments, the antibody or fragment comprises the CDR sequences of the monoclonal antibody designated clone 7, i.e., the three CDR sequences contained in the heavy chain variable region as set forth in SEQ ID NO: 7 and the three CDR sequences contained in the light chain variable region as set forth in SEQ ID NO: 8, or the monoclonal antibody described in clone 11, i.e., the three CDR sequences contained in the heavy chain variable region as set forth in SEQ ID NO: 17 and the three CDR sequences contained in the light chain variable region as set forth in SEQ ID NO: 18.

[0020] According to some embodiments, the antibody or antibody fragment comprises a heavy chain CDR1 comprising the sequence RFTMS (SEQ ID NO: 1). According to some embodiments, the antibody or antibody fragment comprises a heavy chain CDR2 comprising the sequence TISSGGSYTYYPDSVKG (SEQ ID NO: 2). According to some embodiments, the antibody or antibody fragment comprises a heavy chain CDR3 comprising the sequence DRDFYGPYYAMDY (SEQ ID NO: 3).

[0021] According to certain embodiments, the antibody or antibody fragment comprises (i) an HC CDR1 comprising the sequence RFTMS (SEQ ID NO:1), (ii) an HC CDR2 comprising the sequence TISSGGSYTYYPDSVKG (SEQ ID NO:2), and (iii) an HC CDR3 comprising the sequence DRDFYGPYYAMDY (SEQ ID NO:3).

[0022] According to some embodiments, the antibody or antibody fragment comprises a light chain CDR1 comprising the sequence KSSQSLLNSGNQKNYLA (SEQ ID NO: 4). According to some embodiments, the antibody or antibody fragment comprises a light chain CDR2 comprising the sequence FASTRES (SEQ ID NO: 5). According to some embodiments, the antibody or antibody fragment comprises a light chain CDR3 comprising the sequence QQHYTTPLT (SEQ ID NO: 6).

[0023] According to certain embodiments, the antibody or antibody fragment comprises: (i) an LC CDR1 comprising the sequence KSSQSLLNSGNQKNYLA (SEQ ID NO: 4), (ii) an LC CDR2 comprising the sequence FASTRES (SEQ ID NO: 5), and (iii) an HC CDR3 comprising the sequence QQHYTTPLT (SEQ ID NO: 6).

[0024] According to some particular embodiments, the antibody or fragment comprises a heavy chain CDR1 comprising the sequence RFTMS (SEQ ID NO:1), a heavy chain CDR2 comprising the sequence TISSGGSYTYYPDSVKG (SEQ ID NO:2), a heavy chain CDR3 comprising the sequence DRDFYGPYYAMDY (SEQ ID NO:3), a light chain CDR1 comprising the sequence KSSQSLLNSGNQKNYLA (SEQ ID NO:4), a light chain CDR2 comprising the sequence FASTRES (SEQ ID NO:5), and a light chain CDR3 comprising the sequence QQHYTTPLT (SEQ ID NO:6), or analogs thereof comprising 5% or less amino acid substitutions, deletions, and / or insertions in the hypervariable region (HVR) sequences.

[0025] According to some particular embodiments, the antibody or fragment comprises: i. a heavy chain CDR1 having the sequence shown in SEQ ID NO:1; ii. a heavy chain CDR2 having the sequence shown in SEQ ID NO:2; iii. A heavy chain CDR3 having the sequence set forth in SEQ ID NO:3; iv. A light chain CDR1 having the sequence shown in SEQ ID NO:4; v. a light chain CDR2 having the sequence set forth in SEQ ID NO:5, and vi. A light chain CDR3 having the sequence shown in SEQ ID NO:6.

[0026] According to some embodiments, the antibody or fragment thereof comprises a heavy chain variable region as set forth in SEQ ID NO:7, or an analog or derivative thereof having at least 90% sequence identity to the heavy chain variable region sequence.

[0027] According to some embodiments, the antibody or fragment thereof comprises a light chain variable region as set forth in SEQ ID NO:8, or an analog thereof having at least 90% sequence identity to the light chain variable region sequence.

[0028] According to certain embodiments, the antibody or fragment thereof comprises a heavy chain variable region having the sequence set forth in SEQ ID NO:7, and a light chain variable region having the sequence set forth in SEQ ID NO:8, or an analog thereof having at least 90% sequence identity with the light and / or heavy chain sequences.

[0029] According to some embodiments, the antibody or fragment comprises the CDR sequences of the monoclonal antibody shown in clone 11, i.e., the three CDR sequences contained in the heavy chain variable region shown in SEQ ID NO: 17 and the three CDR sequences contained in the light chain variable region shown in SEQ ID NO: 18.

[0030] According to some embodiments, the antibody or antibody fragment comprises a heavy chain CDR1 comprising the sequence SYWIH (SEQ ID NO: 11). According to some embodiments, the antibody or antibody fragment comprises a heavy chain CDR2 comprising the sequence AVYPGNSDSNYNQKFKA (SEQ ID NO: 12). According to some embodiments, the antibody or antibody fragment comprises a heavy chain CDR3 comprising the sequence LVGTFDY (SEQ ID NO: 13).

[0031] According to certain embodiments, the antibody or antibody fragment comprises (i) an HC CDR1 comprising the sequence SYWIH (SEQ ID NO: 11), (ii) an HC CDR2 comprising the sequence AVYPGNSDSNYNQKFKA (SEQ ID NO: 12), and (iii) an HC CDR3 comprising the sequence LVGTFDY (SEQ ID NO: 13).

[0032] According to some embodiments, the antibody or antibody fragment comprises a light chain CDR1 comprising the sequence KASQNVGINVV (SEQ ID NO: 14). According to some embodiments, the antibody or antibody fragment comprises a light chain CDR2 comprising the sequence SASYRYS (SEQ ID NO: 15). According to some embodiments, the antibody or antibody fragment comprises a light chain CDR3 comprising the sequence QQYNTNPFT (SEQ ID NO: 16).

[0033] According to certain embodiments, the antibody or antibody fragment comprises (i) an LC CDR1 comprising the sequence KASQNVGINVV (SEQ ID NO: 14), (ii) an LC CDR2 comprising the sequence SASYRYS (SEQ ID NO: 15), and (iii) an HC CDR3 comprising the sequence QQYNTNPFT (SEQ ID NO: 16).

[0034] According to some particular embodiments, the antibody or fragment comprises a heavy chain CDR1 comprising the sequence SYWIH (SEQ ID NO: 11), a heavy chain CDR2 comprising the sequence AVYPGNSDSNYNQKFKA (SEQ ID NO: 12), a heavy chain CDR3 comprising the sequence LVGTFDY (SEQ ID NO: 13), a light chain CDR1 comprising the sequence KASQNVGINVV (SEQ ID NO: 14), a light chain CDR2 comprising the sequence SASYRYS (SEQ ID NO: 15), and a light chain CDR3 comprising the sequence QQYNTNPFT (SEQ ID NO: 16), or analogs thereof comprising no more than 5% amino acid substitutions, deletions, and / or insertions in the hypervariable region (HVR) sequences.

[0035] According to some particular embodiments, the antibody or fragment comprises: i. a heavy chain CDR1 having the sequence shown in SEQ ID NO:11; ii. a heavy chain CDR2 having the sequence set forth in SEQ ID NO: 12; iii. A heavy chain CDR3 having the sequence set forth in SEQ ID NO: 13; iv. A light chain CDR1 having the sequence set forth in SEQ ID NO: 14; v. a light chain CDR2 having the sequence set forth in SEQ ID NO:15, and vi. A light chain CDR3 having the sequence shown in SEQ ID NO:16.

[0036] According to some embodiments, the antibody or fragment thereof comprises a heavy chain variable region as set forth in SEQ ID NO: 17, or an analog or derivative thereof having at least 90% sequence identity to the heavy chain variable region sequence.

[0037] According to some embodiments, the antibody or fragment thereof comprises a light chain variable region set forth in SEQ ID NO: 18, or an analog thereof having at least 90% sequence identity to the light chain variable region sequence.

[0038] According to certain embodiments, the antibody or fragment thereof comprises a heavy chain variable region having the sequence set forth in SEQ ID NO: 17, and a light chain variable region having the sequence set forth in SEQ ID NO: 18, or an analog thereof having at least 90% sequence identity with the light and / or heavy chain sequences.

[0039] According to some embodiments, the antibody is an isolated monoclonal antibody.

[0040] According to some embodiments, the antibody or fragment thereof is at least 5×10 -9 According to another embodiment, the antibody or antibody fragment recognizes human Nectin-2 with an affinity of 5×10 -9 M, 10 -9 M, 5×10 -10 M, 10 -10 M, 5×10 -11 According to some embodiments, the antibody or antibody fragment binds to human Nectin-2 with a binding affinity of 10 -9 M~10 -11 According to some embodiments, the antibody or antibody fragment binds to human Nectin-2 with an affinity of 10 -9 M~10 -10 According to some embodiments, the antibody or antibody fragment binds to human Nectin-2 with an affinity of 10 -10 M~10 -11binds to human Nectin-2 with an affinity of M. Each possibility represents a separate embodiment of the present invention.

[0041] Analogs and derivatives of the above isolated antibodies and fragments are also within the scope of the present invention.

[0042] According to some embodiments, the antibody or antibody fragment analog has at least 90% sequence identity with the hypervariable regions of the sequence of the reference antibody.

[0043] According to certain embodiments, the isolated antibody or fragment thereof analog or derivative has at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with the variable region of the reference antibody sequence. Each possibility represents a separate embodiment of the present invention.

[0044] According to some embodiments, the antibody or antibody fragment according to the invention comprises a heavy chain variable region as shown in SEQ ID NO:7 or SEQ ID NO:17, or an analog having at least 95% sequence similarity to said sequences.

[0045] According to some embodiments, the antibody or antibody fragment according to the invention comprises a light chain variable region as shown in SEQ ID NO:8 or SEQ ID NO:18, or an analog having at least 95% sequence similarity thereto.

[0046] According to some embodiments, the antibody or antibody fragment comprises a heavy chain and a light chain, where (i) the heavy chain comprises SEQ ID NO: 7 and the light chain comprises SEQ ID NO: 8, or (ii) the heavy chain comprises SEQ ID NO: 17 and the light chain comprises SEQ ID NO: 18. Also included are analogs of the antibody or fragment having at least 95% sequence similarity to the heavy or light chain.

[0047] According to some embodiments, the analog has at least 96, 97, 98, or 99% sequence similarity with the antibody light or heavy chain variable region described above. According to some embodiments, the analog contains only one amino acid substitution, deletion, or addition in one or more CDR sequences of the hypervariable regions, i.e., any one of the CDR sequences shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 11, 12, 13, 14, 15, and 16. Each possibility represents a separate embodiment of the invention. According to some embodiments, the amino acid substitution is a conservative substitution.

[0048] According to some embodiments, the antibody or antibody fragment comprises a hypervariable region (HVR) having the light and heavy chain regions defined above, in which 1, 2, 3, 4, or 5 amino acids have been substituted, deleted, and / or added. Each possibility represents a separate embodiment of the invention.

[0049] According to some embodiments, the antibody or antibody fragment comprises HVRs having the light and heavy chain regions defined above, with one amino acid substitution. According to certain embodiments, the antibody or antibody fragment comprises CDRs defined above, with one amino acid substitution.

[0050] According to some embodiments, the antibody or antibody fragment comprises: i. a set of six CDRs, where HC CDR1 is RFTMS (SEQ ID NO: 1), HC CDR2 is TISSGGSYTYYPDSVKG (SEQ ID NO: 2), HC CDR3 is DRDFYGPYYAMDY (SEQ ID NO: 3), LC CDR1 is KSSQSLLNSGNQKNYLA (SEQ ID NO: 4), LC CDR2 is FASTRES (SEQ ID NO: 5), and LC CDR3 is QQHYTTPLT (SEQ ID NO: 6), and ii. The set of six CDRs, wherein the HC CDR1 sequence is SYWIH (SEQ ID NO:11), the HC CDR2 is AVYPGNSDSNYNQKFKA (SEQ ID NO:12), the HC CDR3 is LVGTFDY (SEQ ID NO:13), the LC CDR1 is KASQNVGINVV (SEQ ID NO:14), the LC CDR2 is SASYRYS (SEQ ID NO:15), and the LC CDR3 is QQYNTNPFT (SEQ ID NO:16).

[0051] The present invention also provides an antibody or binding fragment thereof comprising a heavy chain and a light chain, the chain comprising a set of heavy chain variable region sequences and light chain variable region sequences, the set comprising i. SEQ ID NOs: 7 and 8, and ii. SEQ ID NO: 17 and 18.

[0052] According to some embodiments, the antibody or antibody fragment is capable of inhibiting the binding of human Nectin-2 to TIGIT or CD112R expressed on T cells or NK cells.

[0053] According to some embodiments, the antibody or antibody fragment is capable of inhibiting the binding of human Nectin-2 to TIGIT or CD112R expressed on T cells or NK cells.

[0054] According to certain embodiments, the antibody is selected from the group consisting of chimeric antibodies and antibody fragments comprising at least an antigen-binding portion of the antibody. According to certain embodiments, the antibody is a chimeric antibody. According to yet other embodiments, the chimeric antibody comprises a human constant region. According to certain embodiments, the antibody fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fd', Fv, dAb, isolated CDR regions, single chain variable region (scFV), single chain antibodies (scab), "diabodies" and "linear antibodies". Each possibility represents a separate embodiment of the invention.

[0055] According to some embodiments, the antibody or antibody fragment comprises a constant region 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 representing a separate embodiment of the present invention.

[0056] According to some particular embodiments, the monoclonal antibody is a chimeric monoclonal antibody.

[0057] According to some embodiments, the chimeric antibody comprises a constant region of human origin.

[0058] 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.

[0059] According to some embodiments, the human constant region of the chimeric antibody is selected from the group consisting of human IgG1 and human IgG2.

[0060] According to some embodiments, there is provided a conjugate comprising the antibody or fragment thereof described above.

[0061] The antibodies or fragments thereof according to the invention may be conjugated to a cytotoxic, radioactive, or identifiable moiety.

[0062] Polynucleotide sequences encoding antibodies with high affinity and specificity for human Nectin-2, as well as vectors and host cells harboring these polynucleotide sequences, are provided according to another aspect of the present invention.

[0063] According to some embodiments, polynucleotide sequences are provided that encode the amino acid sequences of the heavy and light chain variable regions described above.

[0064] According to some embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain capable of binding to an epitope within human Nectin-2 protein bound by (i) an antibody having a heavy chain variable region of SEQ ID NO: 7 and a light chain variable region of SEQ ID NO: 8 (identified herein as Clone 7), or (ii) an antibody having a heavy chain variable region of SEQ ID NO: 17 and a light chain variable region of SEQ ID NO: 18 (identified herein as Clone 11).

[0065] According to some embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain comprising a sequence set forth in a sequence selected from the group consisting of: (i) SEQ ID NO: 7 and SEQ ID NO: 8, and (ii) SEQ ID NO: 17 and SEQ ID NO: 18. Each possibility represents a separate embodiment of the present invention.

[0066] In some embodiments, the polynucleotide sequence according to the invention comprises: i. a set of six CDRs, where HC CDR1 is RFTMS (SEQ ID NO:1), HC CDR2 is TISSGGSYTYYPDSVKG (SEQ ID NO:2), HC CDR3 is DRDFYGPYYAMDY (SEQ ID NO:3), LC CDR1 is KSSQSLLNSGNQKNYLA (SEQ ID NO:4), LC CDR2 is FASTRES (SEQ ID NO:5), and LC CDR3 is QQHYTTPLT (SEQ ID NO:6), or ii. Encode an antibody or antibody fragment or chain that comprises a set of six CDRs, where the HC CDR1 sequence is SYWIH (SEQ ID NO:11), the HC CDR2 is AVYPGNSDSNYNQKFKA (SEQ ID NO:12), the HC CDR3 is LVGTFDY (SEQ ID NO:13), the LC CDR1 is KASQNVGINVV (SEQ ID NO:14), the LC CDR2 is SASYRYS (SEQ ID NO:15), and the LC CDR3 is QQYNTNPFT (SEQ ID NO:16). Each possibility represents a separate embodiment of the invention.

[0067] According to some embodiments, the polynucleotide sequence defined above encodes a molecule selected from the group consisting of an antibody, an antibody fragment comprising at least an antigen-binding portion, and an antibody conjugate comprising said antibody or antibody fragment, each possibility representing a separate embodiment of the present invention.

[0068] According to some embodiments, the polynucleotide sequence encodes a monoclonal antibody heavy chain variable region comprising the sequence shown in SEQ ID NO:7, or a variant thereof having at least 90% sequence identity.

[0069] According to some embodiments, the polynucleotide sequence encodes a monoclonal antibody heavy chain variable region comprising the sequence shown in SEQ ID NO: 17, or a variant thereof having at least 90% sequence identity.

[0070] According to some embodiments, the polynucleotide sequence encodes a monoclonal antibody light chain variable region comprising the sequence shown in SEQ ID NO:8, or a variant thereof having at least 90% sequence identity.

[0071] According to some embodiments, the polynucleotide sequence encodes a monoclonal antibody light chain variable region comprising the sequence set forth in SEQ ID NO: 18, or a variant thereof having at least 90% sequence identity.

[0072] The present invention, according to some embodiments, provides a polypeptide comprising at least one sequence encoded by at least one polynucleotide sequence disclosed above.

[0073] In a further aspect, the present invention provides a nucleic acid construct comprising a nucleic acid molecule encoding at least one antibody chain or fragment thereof according to the invention. According to some embodiments, the nucleic acid construct is a plasmid.

[0074] According to some embodiments, the plasmid comprises at least one polynucleotide sequence set forth in a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 19, and SEQ ID NO: 20. Each possibility represents a separate embodiment of the present invention.

[0075] In yet another aspect, the present invention provides cells capable of producing antibodies or antibody fragments comprising particular CDR sequences and / or particular heavy and light chain variable regions as defined above.

[0076] According to some embodiments, a cell is provided, comprising at least one polynucleotide sequence disclosed above.

[0077] According to some embodiments, the cells produce monoclonal antibodies in hybridoma cells.

[0078] According to another aspect, the present invention provides a pharmaceutical composition comprising as an active ingredient at least one antibody, antibody fragment, or conjugate thereof that recognizes human Nectin-2 with high affinity and specificity, and optionally at least one pharma- ceutically acceptable excipient, diluent, salt, or carrier, wherein the at least one antibody or antibody fragment is capable of inhibiting the binding of human Nectin-2 to human TIGIT and / or CD112R.

[0079] According to some embodiments, the pharmaceutical composition comprises a monoclonal antibody or fragment thereof capable of binding to an epitope in human Nectin-2 protein that binds to a monoclonal antibody selected from the group consisting of clone 7 and clone 11 having the variable region and CDR sequences disclosed above.

[0080] According to some embodiments, the pharmaceutical composition comprises: i. a set of six CDRs, where HC CDR1 is SEQ ID NO:1, HC CDR2 is SEQ ID NO:2, HC CDR3 is SEQ ID NO:3, LC CDR1 is SEQ ID NO:4, LC CDR2 is SEQ ID NO:5, and LC CDR3 is SEQ ID NO:6, or ii. At least one monoclonal antibody comprising a set of six CDRs, where HC CDR1 is (SEQ ID NO:11), HC CDR2 is (SEQ ID NO:12), HC CDR3 is (SEQ ID NO:13), LC CDR1 is (SEQ ID NO:14), LC CDR2 is (SEQ ID NO:15), and LC CDR3 is (SEQ ID NO:16).

[0081] Each possibility represents a separate embodiment of the present invention.

[0082] According to some embodiments, the pharmaceutical composition comprises an antibody or fragment thereof comprising a heavy chain variable region having a sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 17. Each possibility represents a separate embodiment of the present invention.

[0083] According to some embodiments, the pharmaceutical composition comprises an antibody or fragment thereof comprising a light chain variable region having a sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 18. Each possibility represents a separate embodiment of the present invention.

[0084] According to certain embodiments, the pharmaceutical composition comprises an antibody or fragment thereof comprising a heavy chain variable region having the sequence set forth in SEQ ID NO:7 and a light chain variable region having the sequence set forth in SEQ ID NO:8.

[0085] According to certain embodiments, the pharmaceutical composition comprises an antibody or fragment thereof comprising a heavy chain variable region having the sequence set forth in SEQ ID NO:17 and a light chain variable region having the sequence set forth in SEQ ID NO:18.

[0086] Single chain variable region (scFv) molecules of the antibody of the present invention are also provided. The scFv molecule comprises the antigen-binding site of the antibody expressed in one polypeptide chain. According to some embodiments, the present invention provides scFv molecules comprising the heavy chain variable region and the light chain variable region of an anti-Nectin-2 antibody. According to certain embodiments, the scFv comprises a hinge region between the two variable regions.

[0087] According to some embodiments, the scFv sequence is set forth in SEQ ID NO:22, SEQ ID NO:24, or an analog thereof having at least 85% sequence similarity thereto. According to some embodiments, the scFv analog has at least 90% sequence identity to a sequence selected from SEQ ID NO:22 and SEQ ID NO:24.

[0088] According to another aspect of the present invention, there is provided a chimeric antigen receptor (CAR) comprising an extracellular portion (binding domain) capable of binding to Nectin-2.

[0089] According to some embodiments, the CAR comprises an extracellular portion comprising any of the provided antibodies or fragments thereof described herein.

[0090] According to some embodiments, the CAR comprises: i. the three complementarity determining regions (CDRs) of the heavy chain (HC) variable region comprising SEQ ID NO:7 and the three CDRs of the light chain (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 ii. A Nectin-2 binding site comprising a set of six CDR sequences selected from the group consisting of the three CDRs of a heavy chain variable region comprising SEQ ID NO: 17 and the three CDRs of a light chain variable region comprising SEQ ID NO: 18, or an analog or derivative thereof having at least 90% sequence identity with the sequence of said antibody or fragment.

[0091] According to some embodiments, the CAR comprises: i. a set of six CDRs, where HC CDR1 is RFTMS (SEQ ID NO: 1), HC CDR2 is TISSGGSYTYYPDSVKG (SEQ ID NO: 2), HC CDR3 is DRDFYGPYYAMDY (SEQ ID NO: 3), LC CDR1 is KSSQSLLNSGNQKNYLA (SEQ ID NO: 4), LC CDR2 is FASTRES (SEQ ID NO: 5), and LC CDR3 is QQHYTTPLT (SEQ ID NO: 6), and ii. The set of six CDRs, wherein HC CDR1 sequence is SYWIH (SEQ ID NO: 11), HC CDR2 is AVYPGNSDSNYNQKFKA (SEQ ID NO: 12), HC CDR3 is LVGTFDY (SEQ ID NO: 13), LC CDR1 is KASQNVGINVV (SEQ ID NO: 14), LC CDR2 is SASYRYS (SEQ ID NO: 15), and LC CDR3 is QQYNTNPFT (SEQ ID NO: 16).

[0092] According to some embodiments, the CAR comprises an antigen binding domain comprising SEQ ID NO: 22 or 24, a transmembrane domain, and an intracellular T cell signaling domain.

[0093] According to some embodiments, lymphocytes engineered to express a CAR described herein are provided.

[0094] According to some embodiments, T cells engineered to express a CAR described herein are provided and are designated CAR-T. According to certain embodiments, NK cells engineered to express a CAR described herein are provided and are designated CAR-NK.

[0095] According to some embodiments, populations of lymphocytes engineered to express a CAR as described herein are provided. According to certain embodiments, populations of T cells or NK cells engineered to express a CAR as described herein are provided.

[0096] According to some embodiments, the CAR comprises a single chain variable region (scFV) comprising the heavy and light chain variable regions of an antibody described herein.

[0097] Also provided according to the invention are single chain variable domains (scFVs) comprising the heavy and light chain variable domains of the antibodies described herein. According to certain embodiments, there is a hinge region between the variable domains.

[0098] According to some embodiments, the scFV sequence is set forth in SEQ ID NO: 22, SEQ ID NO: 24, or an analog thereof having at least 85% sequence similarity thereto.

[0099] The present invention further provides, in some embodiments, a polynucleotide encoding a CAR comprising the sequence set forth in SEQ ID NO:21 or SEQ ID NO:23.

[0100] According to some embodiments, the CAR comprises at least one protein domain selected from the group consisting of an scFv sequence, a CD8 stalk domain, a CD28 TM domain, a 41BB domain, and a CD3ζ (CD3Z, Zetta) domain. According to some embodiments, the CAR comprises an scFv domain. According to some embodiments, the CAR comprises a CD8 stalk domain. According to some embodiments, the CAR comprises a CD28 TM domain. According to some embodiments, the CAR comprises a CD3Z domain. According to some embodiments, the CAR comprises a 41BB domain. According to certain embodiments, the CAR comprises a CD8 stalk domain, a CD28 TM domain, a 41BB domain, and a CD3Z domain.

[0101] According to some embodiments, the CAR comprises an scFv sequence comprising the Nectin-2 binding site of an antibody disclosed herein and at least one domain selected from the group consisting of a CD8 stalk domain, a CD28 TM domain, a 41BB domain, and a CD3Z domain. According to certain embodiments, the CAR comprises an scFv sequence comprising the Nectin-2 binding site of an antibody disclosed herein, a CD8 stalk domain, a CD28 TM domain, a 41BB domain, and a CD3Z domain.

[0102] According to certain embodiments, engineered T cells are provided that express an scFv sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24, or an analog thereof having at least 85% sequence similarity to any of said sequences, a CD8 stalk domain, a CD28 TM domain, a 41BB domain, and a CD3Z domain.

[0103] According to some embodiments, a population of T cells is provided that comprises T cells expressing an scFv sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24, or an analog thereof having at least 85% sequence similarity to any of said sequences, a CD8 stalk domain, a CD28 TM domain, a 41BB domain, and a CD3Z domain.

[0104] According to one aspect, the invention provides a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of at least one lymphocyte comprising a CAR as described herein.

[0105] Also provided is a pharmaceutical composition comprising at least one antibody, antibody fragment, or antibody conjugate according to the invention for use in restoring NK cytotoxicity by inhibiting binding of Nectin-2 to TIGIT and / or CD112R expressed on NK cells.

[0106] According to other embodiments, the antibody, antibody fragment, or antibody conjugate is capable of inhibiting the binding of human Nectin-2 to TIGIT and / or CD112R expressed on T cells.

[0107] According to some embodiments, the pharmaceutical composition according to the invention is for use in cancer immunotherapy or in enhancing an immune response.

[0108] According to some embodiments, the pharmaceutical composition further comprises human lymphocytes expressing TIGIT and / or CD112R.

[0109] According to some embodiments, the human lymphocytes are killer cells selected from the group consisting of T cells, NK cells, and natural killer T (NKT cells).

[0110] According to some embodiments, the killer cells are autologous or allogeneic.

[0111] According to some embodiments, the pharmaceutical composition comprises autologous or allogeneic NK cells expressing TIGIT and / or CD112R.

[0112] The cancer treatable by the composition according to the present invention may be any cancer that expresses Nectin-2. According to some embodiments, the cancer overexpresses Nectin-2. According to some embodiments of the present invention, the cancer is a metastatic cancer. According to some embodiments, the pharmaceutical composition according to the present invention is for use in inhibiting the formation or distribution of metastases or reducing the total number of metastases in a subject.

[0113] According to some embodiments of the invention, the cancer is selected from the group consisting of melanoma, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, colon cancer, cervical cancer, renal 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, leukemia, and lymphoma. Each possibility represents a separate embodiment of the present invention.

[0114] According to some embodiments, the cancer is a solid cancer. According to some particular embodiments, the solid cancer is selected from the group consisting of breast cancer, lung cancer, bladder cancer, pancreatic cancer, and ovarian cancer.

[0115] According to some embodiments, the solid tumor is treated with CAR-T or CAR-NK. According to certain embodiments, the solid tumor is treated with CAR-T. According to additional embodiments, the hematological cancer is treated with CAR-NK or CAR-T cells. According to certain embodiments, the hematological cancer is treated with CAR-NK cells.

[0116] According to some embodiments, the cancer is low-grade glioma. According to some embodiments, the cancer is kidney clear cell carcinoma (KIRC). According to some embodiments, the cancer is lung adenocarcinoma.

[0117] According to certain embodiments, the cancer is selected from the group consisting of melanoma, breast cancer, colorectal cancer, renal cancer, lung cancer, prostate cancer, and brain cancer, with each possibility representing a separate embodiment of the present invention.

[0118] According to other embodiments, the cancer is a hematological cancer. According to some embodiments, the pharmaceutical composition, when used to treat cancer, is associated with human lymphocytes.

[0119] According to some embodiments, the human lymphocytes are killer cells selected from the group consisting of T cells, NK cells, and NKT cells. Each possibility represents a separate embodiment of the present invention.

[0120] According to some embodiments, the killer cells are autologous or allogeneic.

[0121] According to some embodiments, the pharmaceutical composition according to the invention is for use in preventing or treating a viral infection.

[0122] According to yet another aspect, the present invention provides a method for inhibiting the binding of human Nectin-2 to TIGIT or CD112R by using a monoclonal antibody or antibody fragment as defined herein.

[0123] According to an additional aspect, the present invention provides a method for enhancing an immune response in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody, antibody fragment, or antibody conjugate described herein.

[0124] According to yet another aspect, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of at least one antibody, antibody fragment or conjugate thereof capable of recognizing human Nectin-2 with high affinity and specificity and inhibiting its binding to its ligands.

[0125] According to some embodiments of the invention, the therapeutically effective amount results in a reduction in tumor size or number of metastases in the subject.

[0126] According to some embodiments, the method of treating cancer comprises administering or performing at least one additional anti-cancer therapy. According to certain embodiments, the additional anti-cancer therapy is surgery, chemotherapy, radiation therapy, or immunotherapy.

[0127] According to some embodiments, the method of treating cancer comprises administering an antibody that recognizes human Nectin-2 with high affinity and specificity and an additional anti-cancer agent. According to some embodiments, the additional anti-cancer agent is selected from the group consisting of an immunomodulatory agent, an activated lymphocyte cell, a kinase inhibitor, and a chemotherapeutic agent.

[0128] According to other embodiments, the additional immunomodulatory agent is an antibody, antibody fragment, or antibody conjugate that binds to an antigen other than human Nectin-2.

[0129] According to some embodiments, the additional immunomodulatory agent is an antibody against an immune checkpoint molecule. According to some embodiments, the additional immunomodulatory agent 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, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), lymphocyte activation gene 3 (LAG3), CD137, OX40 (also called CD134), killer cell immunoglobulin-like receptor (KIR), TIGIT, PVR, CTLA-4, NKG2A, GITR, and other checkpoint molecules or combinations thereof. Each possibility represents a separate embodiment of the invention. According to certain embodiments, the additional immunomodulatory agent is an antibody against PD-1. According to some embodiments, the additional immunomodulatory agent is an antibody against CTLA-4.

[0130] According to some embodiments, the anticancer drug 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, mechlorethamine, melphalan, thiotepa, dacarbazine, bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, plicamycin, etoposide, teniposide, and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0131] According to some embodiments, the anti-cancer 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 necitumumab (Portrazza®). According to some embodiments, the EGFR inhibitor is cetuximab (Erbitux®).

[0132] According to some embodiments of the invention, the subject is a human subject.

[0133] According to some embodiments of the invention, the use further comprises the use of an agent that downregulates the activity or expression of an immune co-inhibitory receptor.

[0134] According to some embodiments of the invention, the immune cell is a T cell.

[0135] According to some embodiments of the invention, the immune co-inhibitory receptor is selected from the group consisting of PD-1, TIGIT, PVR, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, CD96, BY55 (CD 160), LAIR1, SIGLEC10, and 2B4, with each possibility representing a separate embodiment of the present invention.

[0136] According to one aspect, the present invention provides a method for regulating the function and / or activity of the immune system, comprising modulating the binding of Nectin-2 to TIGIT and / or CD112R using an antibody according to the present invention.

[0137] According to some embodiments, the methods of treating cancer include preventing or reducing the formation, growth, or spread of metastases in a subject.

[0138] According to some embodiments, a method for treating cancer comprises administering to a subject in need thereof a pharmaceutical composition comprising an antibody or antibody fragment thereof capable of inhibiting the binding of human Nectin-2 to human TIGIT and / or CD112R, and further administering human lymphocytes from the subject.

[0139] According to some embodiments, the human lymphocytes are killer cells selected from the group consisting of T cells, NK cells, and NKT cells.

[0140] According to some embodiments, the killer cells are autologous or allogeneic.

[0141] The present invention also provides a method for preventing or treating a viral infection, comprising administering to a subject at least one antibody specific to human Nectin-2, or a fragment thereof comprising at least the antigen-binding domain, wherein the mAb or fragment thereof is capable of inhibiting the binding of Nectin-2 to TIGIT or CD112R.

[0142] According to one aspect, the present invention provides a method for diagnosing or prognosing cancer in a subject, the method comprising determining the expression level of Nectin-2 in a biological sample of the subject using at least one antibody described herein.

[0143] The present invention further comprises, according to another aspect, a method for determining or quantifying Nectin-2 in a sample, the method comprising contacting a biological sample with an antibody or antibody fragment and measuring the level of complex formation, the antibody or antibody fragment being i. a set of six CDRs, where HC CDR1 is SEQ ID NO:1, HC CDR2 is SEQ ID NO:2, HC CDR3 is SEQ ID NO:3, LC CDR1 is SEQ ID NO:4, LC CDR2 is SEQ ID NO:5, and LC CDR3 is SEQ ID NO:6, or ii. A set of six CDRs, where HC CDR1 is (SEQ ID NO:11), HC CDR2 is (SEQ ID NO:12), HC CDR3 is (SEQ ID NO:13), LC CDR1 is (SEQ ID NO:14), LC CDR2 is (SEQ ID NO:15), and LC CDR3 is (SEQ ID NO:16).

[0144] The method of determining and quantifying can be carried out in vitro or ex vivo according to some embodiments, or can be used to diagnose conditions associated with the expression of Nectin-2. The antibody according to the present invention can also be used to construct a screening method. For example, methods such as enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), as well as IHC or FACS, can be constructed to measure the level of secreted or cell-associated polypeptides using the antibody and methods known in the art.

[0145] According to some embodiments, a method for detecting or quantifying the presence of Nectin-2 expressed in a cell or secreted into a biological medium comprises: i. incubating the sample with an antibody specific for human Nectin-2 or an antibody fragment thereof comprising at least the antigen-binding portion; ii. detecting the bound Nectin-2 using a detectable probe.

[0146] According to some embodiments, the method further comprises: iii. comparing the amount of (ii) with a standard curve obtained from a reference sample containing known amounts of Nectin-2; iv. calculating the amount of Nectin-2 in the sample from the standard curve.

[0147] According to some particular embodiments, the sample is a bodily fluid.

[0148] According to some embodiments, the method is performed in vitro or ex vivo.

[0149] A kit for measuring the expression or presence of Nectin-2 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 comprises: i. a set of six CDRs, where HC CDR1 is SEQ ID NO:1, HC CDR2 is SEQ ID NO:2, HC CDR3 is SEQ ID NO:3, LC CDR1 is SEQ ID NO:4, LC CDR2 is SEQ ID NO:5, and LC CDR3 is SEQ ID NO:6, or ii. An antibody or antibody fragment comprising a set of six CDRs, where HC CDR1 is (SEQ ID NO:11), HC CDR2 is (SEQ ID NO:12), HC CDR3 is (SEQ ID NO:13), LC CDR1 is (SEQ ID NO:14), LC CDR2 is (SEQ ID NO:15), and LC CDR3 is (SEQ ID NO:16).

[0150] According to one aspect, the present invention provides a kit for detecting cancer, the diagnostic kit comprising an antibody or antibody fragment thereof disclosed herein.

[0151] According to some embodiments, the present invention provides a method for diagnosing, early detecting, assessing the severity or staging of an immune-related or proliferative disease, comprising determining the expression, concentration or activity of Nectin-2 in a sample derived from a subject using an antibody or a fragment or conjugate thereof according to the present invention, and comparing the expression or activity of Nectin-2 with a reference amount of expression, concentration or activity of Nectin-2, which can be obtained from a normal subject, from samples taken from the same subject at different stages of the disease, or determined from clinical data of a large population of subjects.

[0152] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since 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 description of the drawings]

[0153] [Figure 1A]Correlation between Nectin-2 expression levels (high or low as indicated) and survival probability for patients with low-grade glioma (A), renal clear cell carcinoma (B) and lung adenocarcinoma (C). Datasets were obtained from the TCGA site and analyzed using the oncolnc.org site (https: / / doi.org / 10.7717 / peerj-cs.67). [Figure 1B] Correlation between Nectin-2 expression levels (high or low as indicated) and survival probability for patients with low-grade glioma (A), renal clear cell carcinoma (B) and lung adenocarcinoma (C). Datasets were obtained from the TCGA site and analyzed using the oncolnc.org site (https: / / doi.org / 10.7717 / peerj-cs.67). [Figure 1C] Correlation between Nectin-2 expression levels (high or low as indicated) and survival probability for patients with low-grade glioma (A), renal clear cell carcinoma (B) and lung adenocarcinoma (C). Datasets were obtained from the TCGA site and analyzed using the oncolnc.org site (https: / / doi.org / 10.7717 / peerj-cs.67). [Diagram 2] Schematic diagram of receptors expressed on immune cells and their respective affinities for Nectin-2 expressed on tumors or antigen-presenting cells (APCs). TIGIT is a co-inhibitory receptor for many immune cells (e.g., T cells or NK cells), DNAM-1 (also called CD226) is an activating receptor for many immune cells (e.g., T cells), CD112R (also called PVRIG) is a co-inhibitory receptor for lymphoid immune cells (e.g., T cells and NK cells), and Nectin-2 (CD112) is an inhibitory ligand for immune cells primarily via binding to CD112R, based on the affinities displayed. [Figure 3A] Analysis of the binding and blocking properties of the generated anti-nectin-2 mAbs. Figure 3A shows the binding of anti-nectin-2 clones to MDA-231 cells that endogenously express nectin-2 or 8866-h nectin-2 cells that overexpress nectin-2. [Figure 3B]Analysis of the binding and blocking properties of the generated anti-nectin-2 mAbs. Figure 3B shows the results of FACS analysis of CD112R-Fc binding to 8866-h nectin-2 cells. Among the generated antibodies, three clones (#9, 11, and 13) partially blocked these interactions, and one clone (#7) completely blocked them. [Figure 3C] Analysis of the binding and blocking properties of the generated anti-nectin-2 mAbs. Figure 3C shows the results of FACS analysis of DNAM-1-Fc binding to 8866-h nectin-2 cells. Except for clone 15, none of the other clones (#7-13) blocked the binding of the activating receptor DNAM-1 to nectin-2. [Figure 3D] Analysis of the binding and blocking properties of the generated anti-nectin-2 mAbs. Figure 3D shows the results of FACS analysis of TIGIT-Fc binding to CHOK1-hNectin2 cells in the presence of anti-nectin-2 clones 7 and 11. Both clones block >66% of TIGIT-Fc binding. [Figure 4A] Figure 4 shows that blockade of Nectin-2 with anti-Nectin-2 mAb (shown on the X-axis) enhances NK cell activation. NK activation was measured by induction of surface expression of CD107a and is expressed as fold change relative to control IgG (Y-axis). Results are shown for human cancer cell lines A549 (lung adenocarcinoma) (Figure 4A) and MDA-MB-231 (breast adenocarcinoma) (Figure 4B). The most significant effects were observed for clones #3, 7, and 11. *=P<0.04, **p<0.02, ***p<0.002 by two-tailed Student's t-test. Representative data for one of five donors are shown. Human IgG1 chimeric variants of clones 7 and 11 further increased degranulation (Figure 4C), resulting in >200% degranulation compared to isotype control. ***p<0.002. Representative data for one of two donors are shown. [Figure 4B]Figure 4 shows that blockade of Nectin-2 with anti-Nectin-2 mAb (shown on the X-axis) enhances NK cell activation. NK activation was measured by induction of surface expression of CD107a and is expressed as fold change relative to control IgG (Y-axis). Results are shown for human cancer cell lines A549 (lung adenocarcinoma) (Figure 4A) and MDA-MB-231 (breast adenocarcinoma) (Figure 4B). The most significant effects were observed for clones #3, 7, and 11. *=P<0.04, **p<0.02, ***p<0.002 by two-tailed Student's t-test. Representative data for one of five donors are shown. Human IgG1 chimeric variants of clones 7 and 11 further increased degranulation (Figure 4C), resulting in >200% degranulation compared to isotype control. ***p<0.002. Representative data for one of two donors are shown. [Figure 4C] Figure 4 shows that blockade of Nectin-2 with anti-Nectin-2 mAb (shown on the X-axis) enhances NK cell activation. NK activation was measured by induction of surface expression of CD107a and is expressed as fold change relative to control IgG (Y-axis). Results are shown for human cancer cell lines A549 (lung adenocarcinoma) (Figure 4A) and MDA-MB-231 (breast adenocarcinoma) (Figure 4B). The most significant effects were observed for clones #3, 7, and 11. *=P<0.04, **p<0.02, ***p<0.002 by two-tailed Student's t-test. Representative data for one of five donors are shown. Human IgG1 chimeric variants of clones 7 and 11 further increased degranulation (Figure 4C), resulting in >200% degranulation compared to isotype control. ***p<0.002. Representative data for one of two donors are shown. [Figure 5A]We demonstrate that the binding of mAbs, clones 7 and 11 to human and monkey nectin-2 is similar. Figure 5A shows overlay binding curves of both mAbs added at concentrations ranging from 13.3 nM to 0.02 nM in a series of 3-fold dilutions to CHO cells expressing human or cynomolgus monkey (Cyno) (macaca fascicularis) nectin-2 (protein id: XP_005589607.1). Results of FACS analysis of this assay are expressed as relative binding intensity compared to maximum binding, which was set at 100%. For detection, goat anti-mouse-647 Ab was used at a 1:250 dilution. A summary of the data analysis of this assay is shown as well, further demonstrating that both mAbs bind to human and cynomolgus monkey (Cyno) (macaca fascicularis) nectin-2 (protein id: XP_005589607.1) with high and similar affinity. Binding of anti-nectin-2 mAb was examined using Chlorocebus (African green monkey) nectin-2 (XP_007995342.1 expressed by Vero cells). [Figure 5B] We demonstrate that the binding of mAbs, clones 7 and 11, to human and monkey nectin-2 is similar. Figure 5B shows the binding of anti-nectin-2 mAbs to endogenous human nectin-2 (expressed in 293T cells) and endogenous African green monkey nectin-2 (expressed in Vero cells) tested by FACS analysis described in Figure 5A (Ab range: 20 - 0.0003 nM). This analysis reveals similar Ab binding to both human and monkey nectin-2 targets, with high affinity, for both anti-nectin-2 clones, which is also evident in the summary table. [Figure 6A] Figure 6 shows the effect of anti-hNectin-2 antibodies on T cell proliferation. Human PBMCs were CFSE-labeled and incubated with target cells MDA-MB-231 (6A) or A549 (6B) in the presence of PHA-L and the indicated antibodies. Results are presented as fold increase in proliferation compared to control. Shown are results from one PBMC donor representative of seven tested. [Figure 6B]Figure 6 shows the effect of anti-hNectin-2 antibodies on T cell proliferation. Human PBMCs were CFSE-labeled and incubated with target cells MDA-MB-231 (6A) or A549 (6B) in the presence of PHA-L and the indicated antibodies. Results are presented as fold increase in proliferation compared to control. Shown are results from one PBMC donor representative of seven tested. [Figure 7A] Figure 7 shows the effect of anti-hNectin-2 antibodies alone or in combination with known checkpoint blockade on CD8+ T cell proliferation. Human PBMCs were CFSE-labeled and incubated with target cells RKO (7A) or A549 (Figure 7B) in the presence of PHA-L and the indicated antibodies. Results are shown as fold increase in proliferation compared to control. All combinations tested significantly increased CD8+ T cell proliferation over individual treatments. Results from one PBMC donor out of two donors tested are shown. [Figure 7B] Figure 7 shows the effect of anti-hNectin-2 antibodies alone or in combination with known checkpoint blockade on CD8+ T cell proliferation. Human PBMCs were CFSE-labeled and incubated with target cells RKO (7A) or A549 (Figure 7B) in the presence of PHA-L and the indicated antibodies. Results are shown as fold increase in proliferation compared to control. All combinations tested significantly increased CD8+ T cell proliferation over individual treatments. Results from one PBMC donor out of two donors tested are shown. [Figure 8A] Figure 1 shows the effect of anti-hNectin-2 antibody on IFNγ secretion. Human PBMCs were incubated with target cells as described in Figure 7. After 96 hours, plates were centrifuged and supernatants were collected. IFNγ quantification was performed using Biolegend's Human IFN-γ ELISA MAX™ Deluxe according to the manufacturer's protocol. Results are shown for one PBMC donor out of five donors tested. [Figure 8B]Figure 1 shows the effect of anti-hNectin-2 antibody on IFNγ secretion. Human PBMCs were incubated with target cells as described in Figure 7. After 96 hours, plates were centrifuged and supernatants were collected. IFNγ quantification was performed using Biolegend's Human IFN-γ ELISA MAX™ Deluxe according to the manufacturer's protocol. Results are shown for one PBMC donor out of five donors tested. [Figure 9A] Figure 1 shows the effect of anti-hNectin-2 antibody alone or in combination with known checkpoint blockade on tumor cell killing by hPBMC. Assays were performed as described in Figure 7. After 96-120 hours, immune cells were removed, tumor cells were washed extensively, and CellTiter-Glo® was used to establish viability of adherent tumor cells according to the manufacturer's protocol. All results were within the linear range of the kit. Results are presented as fold increase in tumor cell killing compared to control. All combinations tested significantly increased tumor cell killing compared to individual treatments. Results from one PBMC donor out of two donors tested are shown. [Figure 9B] Figure 1 shows the effect of anti-hNectin-2 antibody alone or in combination with known checkpoint blockade on tumor cell killing by hPBMC. Assays were performed as described in Figure 7. After 96-120 hours, immune cells were removed, tumor cells were washed extensively, and CellTiter-Glo® was used to establish viability of adherent tumor cells according to the manufacturer's protocol. All results were within the linear range of the kit. Results are presented as fold increase in tumor cell killing compared to control. All combinations tested significantly increased tumor cell killing compared to individual treatments. Results from one PBMC donor out of two donors tested are shown. [Figure 10]Figure 1 shows the effect of Nectin-2 mAb on tumor development in vivo. Scid female mice (n=33) were injected subcutaneously with 5x106 MDA-MB-231 cells in Matrigel. When tumors reached 80-120 mm3, mice were randomly divided into three groups and treated twice weekly in a blinded manner by intravenous (iv) injection of either PBS (light grey diamonds), hIgG1 control Ab (grey squares), or clone-7-human IgG1 (2.7.1) (filled circles), both at 3 mg / kg. *p<0.04, **p<0.02, ***p<0.008. [Figure 11A] Figure 11 shows the effect of Nectin-2 mAb with hIgG2 Fc, either alone or in combination with PD-1, on tumor cell killing and PBMC proliferation. A549 cells were co-incubated with PBMC for 96 hours at an E:T ratio of 7:1 in the presence of 4ug / ml PHA-L with either no Ab, clone-11-human IgG2 (2.11.2), Keytruda™ (both at 3.5ug / ml), or their combination (each at 3.5ug / ml). Tumor cell killing (Figure 11A) and PBMC (T cell) proliferation (Figure 11B) are shown. *p<0.01, **p<0.002, ***p<0.0008. [Figure 11B] Figure 11 shows the effect of Nectin-2 mAb with hIgG2 Fc, either alone or in combination with PD-1, on tumor cell killing and PBMC proliferation. A549 cells were co-incubated with PBMC for 96 hours at an E:T ratio of 7:1 in the presence of 4ug / ml PHA-L with either no Ab, clone-11-human IgG2 (2.11.2), Keytruda™ (both at 3.5ug / ml), or their combination (each at 3.5ug / ml). Tumor cell killing (Figure 11A) and PBMC (T cell) proliferation (Figure 11B) are shown. *p<0.01, **p<0.002, ***p<0.0008. [Figure 12A]Figure 12 shows the effect of CAR-T expressing scFv derived from clone 7 and clone 11 antibodies (CAR-T2.07 and CAR-T2.11, respectively) on specific T cell activation in the presence of tumor cells expressing Nectin-2. PBMCs from healthy donors were transduced with CAR-T constructs. A general schematic of these constructs is shown in Figure 12A, where scFv represents the single chain of Nectin-2 mAb described herein. Nectin-2 CAR-T PBMCs were incubated with U937 or BT-474 target cells at various E:T ratios. Target cell killing (Figures 12B and 12D) as well as IFNγ secretion by activated PBMCs (Figures 12C and 12E, p<0.03) are shown. Figures 12B-E show a representative experiment out of three performed for each cell line (CAR-T2.07 grey bars, CAR-T2.11 black bars). [Figure 12B] Figure 12 shows the effect of CAR-T expressing scFv derived from clone 7 and clone 11 antibodies (CAR-T2.07 and CAR-T2.11, respectively) on specific T cell activation in the presence of tumor cells expressing Nectin-2. PBMCs from healthy donors were transduced with CAR-T constructs. A general schematic of these constructs is shown in Figure 12A, where scFv represents the single chain of Nectin-2 mAb described herein. Nectin-2 CAR-T PBMCs were incubated with U937 or BT-474 target cells at various E:T ratios. Target cell killing (Figures 12B and 12D) as well as IFNγ secretion by activated PBMCs (Figures 12C and 12E, p<0.03) are shown. Figures 12B-E show a representative experiment out of three performed for each cell line (CAR-T2.07 grey bars, CAR-T2.11 black bars). [Figure 12C]Figure 12 shows the effect of CAR-T expressing scFv derived from clone 7 and clone 11 antibodies (CAR-T2.07 and CAR-T2.11, respectively) on specific T cell activation in the presence of tumor cells expressing Nectin-2. PBMCs from healthy donors were transduced with CAR-T constructs. A general schematic of these constructs is shown in Figure 12A, where scFv represents the single chain of Nectin-2 mAb described herein. Nectin-2 CAR-T PBMCs were incubated with U937 or BT-474 target cells at various E:T ratios. Target cell killing (Figures 12B and 12D) as well as IFNγ secretion by activated PBMCs (Figures 12C and 12E, p<0.03) are shown. Figures 12B-E show a representative experiment out of three performed for each cell line (CAR-T2.07 grey bars, CAR-T2.11 black bars). [Figure 12D] Figure 12 shows the effect of CAR-T expressing scFv derived from clone 7 and clone 11 antibodies (CAR-T2.07 and CAR-T2.11, respectively) on specific T cell activation in the presence of tumor cells expressing Nectin-2. PBMCs from healthy donors were transduced with CAR-T constructs. A general schematic of these constructs is shown in Figure 12A, where scFv represents the single chain of Nectin-2 mAb described herein. Nectin-2 CAR-T PBMCs were incubated with U937 or BT-474 target cells at various E:T ratios. Target cell killing (Figures 12B and 12D) as well as IFNγ secretion by activated PBMCs (Figures 12C and 12E, p<0.03) are shown. Figures 12B-E show a representative experiment out of three performed for each cell line (CAR-T2.07 grey bars, CAR-T2.11 black bars). [Figure 12E]Figure 12 shows the effect of CAR-T expressing scFv derived from clone 7 and clone 11 antibodies (CAR-T2.07 and CAR-T2.11, respectively) on specific T cell activation in the presence of tumor cells expressing Nectin-2. PBMCs from healthy donors were transduced with CAR-T constructs. A general schematic of these constructs is shown in Figure 12A, where scFv represents the single chain of Nectin-2 mAb described herein. Nectin-2 CAR-T PBMCs were incubated with U937 or BT-474 target cells at various E:T ratios. Target cell killing (Figures 12B and 12D) as well as IFNγ secretion by activated PBMCs (Figures 12C and 12E, p<0.03) are shown. Figures 12B-E show a representative experiment out of three performed for each cell line (CAR-T2.07 grey bars, CAR-T2.11 black bars). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0154] The present invention provides an effective antibody specific to human Nectin-2. The present invention also provides the production and use of the antibody as a therapeutic agent. In particular, the mAb of the present invention can be used to enhance antitumor killing activity and as a diagnostic reagent. In some embodiments, the present invention provides an antibody specific to Nectin-2 for efficient restoration of immune activity against cancer cells that overexpress Nectin-2. In other embodiments, the antibody described herein is for use in treating viral infection. The antibody prevents herpes virus from entering cells by blocking Nectin-2.

[0155] The term "antigen" as used herein refers to a molecule or a part of a molecule that can induce antibody formation and can be specifically bound by an antibody. An antigen can 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 does not react with many other antibodies that may be induced by other antigens. The antigen according to some embodiments of the present invention is Nectin-2 protein.

[0156] The term "nectin-2" or "nectin cell adhesion molecule 2" as used herein refers to a human plasma membrane glycoprotein also known as CD112 and PVRL2. Nectin-2 protein is a single-pass transmembrane glycoprotein with two Ig-like C2-type domains and an Ig-like V-type domain. This protein is one of the plasma membrane components of the adherens junction. Nectin-2 also functions as an invader for certain mutant strains of herpes simplex virus and pseudorabies virus and is involved in the cell-to-cell spread of these viruses. Exemplary nectin-2 according to the present invention are described in SwissPort, UniPort and GenBank symbols or accession numbers: Gene ID: 5819, Q92692, I68093, NP_001036189.1, NP_002847.1, and #Q92692.

[0157] The antibody or fragment thereof according to the present invention binds to an epitope of Nectin-2. Specifically, the antibody binds to an epitope within the ectodomain (extracellular portion) of Nectin-2 protein.

[0158] The term "antigenic determinant" or "epitope" as used herein refers to a region of an antigen molecule that specifically reacts with a particular antibody. Peptide sequences derived from epitopes can be used alone or in conjunction with a carrier moiety to immunize animals to generate additional polyclonal or monoclonal antibodies, applying methods known in the art. Isolated peptides derived from epitopes can be used in diagnostic methods to detect antibodies.

[0159] 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, noting that a lower Kd reflects a higher affinity.

[0160] Antibodies, or immunoglobulins, contain two heavy chains and two light chains linked together by disulfide bonds, with each light chain attached to its respective heavy chain by a disulfide bond in a "Y" configuration. Proteolytic digestion of antibodies produces Fv (Fragment Variable) and Fc (Fragment Crystallizable) domains. The antigen-binding domain, Fab, contains the region of variable polypeptide sequence. The term F(ab')2 refers to two Fab' arms linked together by disulfide bonds. Each heavy chain contains a variable domain (V) at one end and a variable domain (V) at the other end. H ) and has several constant domains (C H Each light chain has at one end a variable domain (V L ), and a constant domain (C L ), where the light chain variable domain is aligned with the variable domain of the heavy chain and the light chain constant domain is aligned with the first constant domain (CH1) of the heavy chain. The variable domains of each pair of light and heavy chains form 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 connected by three hypervariable domains known as complementarity determining regions (CDR1-3). These domains contribute to the specificity and affinity of the antigen-binding site.

[0161] Identification or determination of CDRs from a given heavy or light chain variable sequence is usually carried out using one of a few methods known in the art.For example, such determination is carried out according to Kabat (Wu TT and Kabat EA, J Exp Med, 1970; 132: 211-50) and IMGT (Lefranc MP, et al., Dev Comp Immunol, 2003, 27: 55-77).

[0162] When the term "CDR having a sequence" or similar terminology is used, it includes the option that the CDR comprises the sequence specified, and also the option that the CDR consists of the sequence specified.

[0163] The antigen specificity of an antibody is based on the unique CDR sequences of both the light and heavy chains which together form the hypervariable region (HVR), i.e., the antigen-binding site.

[0164] The heavy chain isotype (gamma, alpha, delta, epsilon, or mu) determines the immunoglobulin class (IgG, IgA, IgD, IgE, or IgM, respectively). Light chains are one of two isotypes (kappa, κ or lambda, λ). Both isotopes are found in all antibody classes.

[0165] The term "antibody" is used in the broadest sense and includes monoclonal antibodies (including full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, and antibody fragments of sufficient length to exhibit the desired biological activity, i.e., binding to human Nectin-2.

[0166] The antibody or antibodies according to the invention include intact antibodies, such as polyclonal or monoclonal antibodies (mAb), as well as proteolytic fragments thereof, such as Fab or F(ab')2 fragments. Single chain antibodies are also included within the scope of the invention.

[0167] Antibody fragments An "antibody fragment" comprises only a portion of an intact antibody, generally including the antigen-binding site of the intact antibody, and thus retains the ability to bind antigen. Examples of antibody fragments encompassed by this definition include (i) a Fab fragment having the VL, CL, VH, and CH1 domains; (ii) a Fab' fragment, which is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iii) an Fd fragment having the VH and CH1 domains; (iv) an Fd' fragment having the VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) an Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) a dAb fragment (Ward et al., Nature 1989, 341, 544-546), which consists of a VH domain; (vii) isolated CDR regions; (viii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab' fragments linked by a disulfide bridge at the hinge region; (ix) a single chain antibody molecule (e.g., single chain Fv, scFv) (Bird et al., Science 1988, 242, 423-426, and Huston et al., Proc. Natl. Acad. Sci. (USA) 1988, 85, 5879-5883), (x) "bispecific antibodies" which have two antigen-binding sites and comprise a heavy chain variable domain (VH) combined with a light chain variable domain (VL) in the same polypeptide chain (e.g., EP 404,097, WO 93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 6444-6448), (xi) "linear antibodies" which comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which form a pair of antigen-binding regions together with a complementary light chain polypeptide (Zapata et al. Protein Int. Gazette, 1999, 10, 11161, and WO 93 / 11161). Eng., 1995, 8, 1057-1062, and U.S. Pat. No. 5,641,870.

[0168] Various techniques have been developed for the production 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 produced 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 coupled 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 culture. Other techniques for the production 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).

[0169] Single-chain antibodies are single-chain composite polypeptides that have antigen-binding capacity and contain amino acid sequences that are homologous or similar to the variable regions of immunoglobulin light and heavy chains, i.e. linked V H -V L or a single chain Fv (scFv). Techniques for producing single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies against Nectin-2.

[0170] The term "monoclonal antibody" (mAb) as used herein refers to an antibody obtained from a population of nearly homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations, which usually contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" should not be construed as requiring 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 can be produced by recombinant DNA methods (e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 1991, 352, 624-628 or Marks et al., J. Mol. Biol., 1991, 222:581-597.

[0171] The design and development of recombinant monovalent antigen-binding molecules derived from monoclonal antibodies through the rapid identification and cloning of functional variable heavy (VH) and variable light (VL) chain genes, as well as the design and cloning of synthetic DNA sequences optimized for expression in recombinant bacteria, is described in Fields et al. 2013, 8(6):1125-48.

[0172] The mAbs of the present invention can 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, where cells from individual hybridomas are injected intraperitoneally into naive primed Balb / c mice to generate ascites fluid containing high concentrations of the desired mAb. mAbs can be purified from such ascites fluid or from culture supernatants using methods well known to those skilled in the art.

[0173] Also included are anti-idiotypic antibodies that are specifically immunoreactive with the hypervariable regions of the antibodies of the invention.

[0174] The present invention provides a monoclonal antibody or antibody fragment comprising an antigen binding domain (ABD) comprising three CDRs of a light chain and three CDRs of a heavy chain, the ABD having at least 90% sequence identity or similarity to the ABD of a monoclonal murine antibody comprising (i) a heavy variable chain comprising the amino acid sequence of SEQ ID NO:7 and a light variable chain comprising the amino acid sequence of SEQ ID NO:8 (identified herein as clone 7), or (ii) a heavy variable chain comprising the amino acid sequence of SEQ ID NO:17 and a light variable chain comprising the amino acid sequence of SEQ ID NO:18 (identified herein as clone 11). Such an antibody may have an ABD domain having at least 93%, at least 94%, at least 95%, at least 96, at least 97, at least 98, at least 99% sequence identity or similarity, or 100% sequence identity to the corresponding ABD of antibody clone 7, or clone 11.

[0175] Sequence identity is the amount of amino acids or nucleotides that are exactly the same between two different sequences.Sequence similarity allows conservative substitution of amino acids to be determined as the same amino acid.The polynucleotide sequences described herein can be codon-optimized for expression in a particular cell, such as a human cell.Codon optimization does not change the encoded amino acid sequence of the antibody chain, but may increase expression in a cell, for example.

[0176] The present invention also provides conservative amino acid variants of the antibody molecules according to the present invention. Variants according to the present invention can also be generated that preserve the overall molecular structure of the encoded protein. Given the properties of the individual amino acids that make up the disclosed protein products, some reasonable substitutions will be recognized by those skilled in the art. Amino acid substitutions, i.e. "conservative substitutions", can be made, for example, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues involved. The term "antibody analog" as used herein refers to an antibody derived from another antibody by one or more conservative amino acid substitutions.

[0177] The term "antibody variant" as used herein refers to any molecule comprising an antibody of the invention. For example, a fusion protein in which an antibody or antigen-binding fragment thereof is linked to another chemical entity is considered an antibody variant.

[0178] Analogs and variants of the antibody sequence are also within the scope of this application. These include, but are not limited to, conservative and non-conservative substitutions, insertions, and deletions of amino acids within the sequence. Such modifications and the resulting antibody analogs or variants are within the scope of the present invention as long as they confer or even improve the binding of the antibody to human Nectin-2.

[0179] Conservative substitutions of amino acids known to those skilled in the art are within the scope of the present invention. Conservative amino acid substitutions include replacing one amino acid with another that has the same type of functional group or side chain, e.g., aliphatic, aromatic, positive charge, negative charge. These substitutions may enhance oral bioavailability, penetration, and targeting to specific cell populations, immunogenicity, and the like. Those skilled in the art will recognize that individual substitutions, deletions, or additions to peptide, polypeptide, or protein sequences that change, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants," where the changes result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution lists that result in functionally similar amino acids are well known in the art. For example, according to one list known in the art, the following six groups each contain amino acids that are conservative substitutions for 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).

[0180] It should be emphasized that the mutant strand sequence is determined by the sequencing method using a specific primer. Different sequencing methods used on the same sequence may result in slightly different sequences, especially at the ends of the sequence, due to technical issues and different primers.

[0181] The terms "molecule having an antigen-binding portion of an antibody" and "antigen-binding fragment" as used herein are intended to include intact immunoglobulin molecules of any isotype and produced by any animal cell line or microorganism, as well as antigen-binding reactive fractions thereof, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, variable portions of the heavy and / or light chains thereof, Fab mini-antibodies (see, e.g., WO93 / 15210, U.S. Patent Application Nos. 08 / 256,790, WO96 / 13583, U.S. Patent Application Nos. 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, including, but not limited to, enzymatic cleavage, peptide synthesis, or recombinant techniques.

[0182] Antibodies herein include in particular "chimeric" antibodies, in which a portion of the heavy and / or light chain is identical or homologous to the 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(s) is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as in fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). In addition, complementarity determining region (CDR) grafting may be performed to alter certain properties of antibody molecules, including affinity or specificity. A non-limiting example of CDR grafting is disclosed in U.S. Pat. No. 5,225,539.

[0183] Chimeric antibodies are molecules in which different parts are derived from different animal species, such as those with variable regions derived from murine mAbs and human immunoglobulin constant regions. Antibodies with variable region framework residues mostly derived from human antibodies (called acceptor antibodies) and CDRs mostly derived from mouse antibodies (called donor antibodies) are also called humanized antibodies. Chimeric antibodies are primarily used to reduce immunogenicity in applications and increase yields in production, for example, murine mAbs have high yields from hybridomas but are highly immunogenic in humans, so human / murine chimeric mAbs are used. Chimeric antibodies and methods for their production are known in the art (e.g., PCT Patent Applications WO86 / 01533, WO97 / 02671, WO90 / 07861, WO92 / 22653, and U.S. Patent Nos. 5,693,762, 5,693,761, 5,585,089, 5,530,101, and 5,225,539).

[0184] According to some embodiments, the antibody is a monoclonal antibody.

[0185] According to some particular embodiments, the monoclonal antibody is a chimeric monoclonal antibody.

[0186] According to some embodiments, the chimeric antibody comprises a constant region of human origin.

[0187] 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.

[0188] According to some embodiments, the human constant region of the chimeric antibody is selected from the group consisting of human IgG1 and human IgG2.

[0189] According to certain embodiments, a chimeric monoclonal antibody recognizing human Nectin-2 is provided, which comprises: i. a set of six CDRs, where HC CDR1 is SEQ ID NO:1, HC CDR2 is SEQ ID NO:2, HC CDR3 is SEQ ID NO:3, LC CDR1 is SEQ ID NO:4, LC CDR2 is SEQ ID NO:5, and LC CDR3 is SEQ ID NO:6, or ii. An antibody or antibody fragment comprising a set of six CDRs, where HC CDR1 is (SEQ ID NO:11), HC CDR2 is (SEQ ID NO:12), HC CDR3 is (SEQ ID NO:13), LC CDR1 is (SEQ ID NO:14), LC CDR2 is (SEQ ID NO:15), and LC CDR3 is (SEQ ID NO:16).

[0190] Pharmacology In pharmaceutical and drug formulations, the active agent is preferably utilized together with one or more pharma- ceutically acceptable carrier(s) and, optionally, any other therapeutic ingredients. The carrier(s) must be pharma- ceutically acceptable in the sense that it is compatible with the other ingredients of the formulation and is not unduly deleterious to its recipient. The active agent is provided in an amount effective to achieve the desired pharmacological effect, as described above, and in an amount appropriate to achieve the desired exposure.

[0191] Typically, the antibodies and their fragments and conjugates of the present invention, including the antigen-binding portion of the antibody or another polypeptide containing a peptidomimetic, are suspended in sterile saline for therapeutic use. Alternatively, the pharmaceutical composition can be formulated to control the release of the active ingredient (the molecule including the antigen-binding portion of the antibody) or to prolong its presence in the patient's system. Many suitable drug delivery systems are known, including, for example, implantable drug release systems, hydrogels, hydroxymethylcellulose, microcapsules, liposomes, microemulsions, microspheres, and the like. Controlled release preparations can be prepared through the use of polymers to complex or adsorb the molecules according to the present invention. For example, biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of polyanhydride copolymers of stearic acid dimer and sebacic 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 molecule, the amount of the molecule in the matrix, and the size of the particles dispersed.

[0192] The pharmaceutical compositions of the invention may be administered by any suitable means, such as orally, topically, intranasally, subcutaneously, intramuscularly, intravenously, intraarterially, intraarticularly, intralesionally, intratumorally, or parenterally. Typically, intravenous (iv) administration is used to deliver antibodies.

[0193] It will be apparent to those skilled in the art that a therapeutically effective amount of a molecule according to the invention will depend on, among other things, the administration schedule, the unit dose of the molecule administered, whether the molecule is administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic activity of the administered molecule, its persistence in the circulation, and the judgment of the treating physician.

[0194] As used herein, the term "therapeutically effective amount" refers to an amount of an agent that is effective to treat a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of an agent may reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow to some extent, preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent, preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with the disorder. To the extent that an agent may prevent growth and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. For cancer treatment, efficacy in vivo may be determined, for example, by assessing survival time, time to disease progression (TTP), response rate (RR), duration of response, and / or quality of life.

[0195] Cancers that are amenable to treatment according to the present invention include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia 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, renal or kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, various types of head and neck cancer, and B-cell lymphoma (low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) These include NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small noncleaved cell NHL, bulky mass disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel growth associated with nematoses, edema (such as that associated with brain tumors), and Meigs' syndrome. Preferably, the cancer is selected from the group consisting of breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkin's 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.Cancerous conditions modifiable for treatment according to the invention include metastatic cancer.

[0196] According to another embodiment, the pharmaceutical composition according to the invention is for use in the treatment of cancers characterized by overexpression of Nectin-2. The types of cancers associated with overexpression of Nectin-2 can be identified using known databases, such as The Cancer Genome Atlas (TCGA). According to certain embodiments, the cancers treatable by the composition according to the invention are adrenocortical carcinoma (ACC), chromophobe renal carcinoma (KICH), liver hepatocellular carcinoma (LIHC), colon and rectal adenocarcinoma (COAD, READ), pancreatic ductal adenocarcinoma (PAAD), pheochromocytoma and paraganglioma (PCPG), papillary renal carcinoma (KIRP), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), prostate adenocarcinoma (PRAD), uterine endometrial carcinoma (UCEC), uterine endometrial carcinoma (U ... Cervical cancer (CESC), cutaneous melanoma (SKCM), mesothelioma (MESO), urothelial bladder cancer (BLCA), clear cell renal carcinoma (KIRC), lung squamous cell carcinoma (LUSC), uterine carcinosarcoma (UCS), sarcoma (SARC), ovarian serous cystadenocarcinoma (OV), papillary thyroid carcinoma (THCA), glioblastoma multiforme (GBM), breast cancer (BRCA), low grade glioma (LGG), and diffuse large B-cell lymphoma (DLBC). Each possibility represents a separate embodiment of the present invention.

[0197] As an active ingredient, the molecules of the present invention are dissolved, dispersed or mixed in a pharma-ceutically acceptable excipient that is compatible with the active ingredient, as is well known.Suitable excipients are, for example, water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, etc., and combinations thereof.Other suitable carriers are well known to those skilled in the art.In addition, if necessary, the composition can contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, etc.

[0198] A pharmaceutical composition according to the invention may be administered in conjunction with an anti-neoplastic composition.

[0199] As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.

[0200] The term "cancer" generally refers to or describes a physiological condition in mammals characterized by uncontrolled cell proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include melanoma, lung, thyroid, breast, colon, prostate, liver, bladder, kidney, cervical, pancreatic, leukemia, lymphoma, myeloid, ovarian, uterine, sarcoma, bile duct, or endometrial cancer.

[0201] According to some embodiments, the method of treating cancer comprises administering the pharmaceutical composition as part of a treatment regimen that includes administration of at least one additional anti-cancer agent.

[0202] According to some embodiments, the anti-cancer agent is selected from the group consisting of antimetabolites, antimitotics, taxanes, topoisomerase inhibitors, topoisomerase II inhibitors, asparaginases, alkylating agents, antitumor antibiotics, and combinations thereof, each possibility representing a separate embodiment of the present invention.

[0203] According to some embodiments, the antimetabolite is selected from the group consisting of cytarabine, fludarabine, 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, ifosfamide, mechlorethamine, 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, the topoisomerase II is selected from the group consisting of etoposide and teniposide. Each possibility represents a separate embodiment of the present invention.

[0204] According to some specific embodiments, the additional anticancer agent is selected from the group consisting of bevacizumab, carboplatin, cyclophosphamide, doxorubicin hydrochloride, gemcitabine hydrochloride, topotecan hydrochloride, thiotepa, and combinations thereof, each possibility representing a separate embodiment of the present invention.

[0205] The monoclonal antibodies according to the invention may be used as part of a combination therapy with at least one anti-cancer agent. According to some embodiments, the additional anti-cancer agent is an immunomodulatory agent, an activated lymphocyte cell, a kinase inhibitor, or a chemotherapeutic agent.

[0206] According to some embodiments, the anti-cancer agent is an immunomodulatory agent, such as an antibody against an immune checkpoint molecule, whether an agonist or antagonist.

[0207] Checkpoint immunotherapeutic blockade is proving to be an exciting new arena for cancer treatment. Immune checkpoint pathways consist of a set of costimulatory and inhibitory molecules that work in concert to maintain self-tolerance and protect tissues from damage by the immune system under physiological conditions. Tumors exploit specific checkpoint pathways to evade the immune system. Thus, inhibition of such pathways has emerged as a promising anticancer therapeutic strategy.

[0208] The anti-cytotoxic T-lymphocyte 4 (CTLA-4) antibody ipilimumab (approved in 2011) was the first immunotherapy agent to show benefit in treating cancer patients. The antibody interferes with inhibitory signals during antigen presentation to T cells. The anti-programmed cell death 1 (PD-1) antibody pembrolizumab (approved in 2014) blocks the negative immunoregulatory signaling of the PD-1 receptor expressed by T cells. Additional anti-PD-1 agents were submitted for regulatory approval in 2014 for the treatment of non-small cell lung cancer (NSCLC). Currently, active research is exploring many other immune checkpoints, notably CEACAM1, NKG2A, B7-H3, B7-H4, VISTA, CD112R, lymphocyte activation gene 3 (LAG3), CD137, OX40 (also known as CD134), and killer cell immunoglobulin-like receptors (KIRs).

[0209] According to some particular embodiments, the immunomodulatory agent is selected from the group consisting of an antibody that inhibits CTLA-4, an anti-human programmed cell death protein 1 (PD-1), PD-L1, and PD-L2 antibody, an activated cytotoxic lymphocyte cell, a lymphocyte activator, an antibody against CEACAM, an antibody against TIGIT, and a RAF / MEK pathway inhibitor. Each possibility represents a separate embodiment of the invention. According to some particular embodiments, the additional immunomodulatory agent is selected from a mAb against PD-1, a mAb against PD-L1, a mAb against PD-L2, a mAb against CEACAM1, a mAb against CTLA-4, a mAb against TIGIT, PVR, interleukin 2 (IL-2), or a lymphokine-activated killer (LAK) cell.

[0210] According to another embodiment, the additional anti-cancer agent is a chemotherapeutic agent. Chemotherapeutic agents that can be administered together with the antibody according to the invention or separately can include any agent known in the art that exhibits anti-cancer activity, including but not limited to mitoxantrone, topoisomerase inhibitors, spindle poisons such as vincas vinblastine, vincristine, vinorelbine (taxol), paclitaxel, docetaxel; alkylating agents such as mechlorethamine, chlorambucil, cyclophosphamide, melphalan, ifosfamide; methotrexate, 6-mercaptopurine, 5-fluorouracil. , cytarabine, gemcitabine, the podophyllotoxins etoposide, irinotecan, topotecan, dacarbazine; the antibiotics doxorubicin (adriamycin), bleomycin, mitomycin; the nitrosoureas carmustine (BCNU), lomustine, epirubicin, idarubicin, daunorubicin, the inorganic ions cisplatin, carboplatin; interferons, asparaginase, the hormones tamoxifen, leuprolide, flutamide, and megestrol acetate.

[0211] 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, epipodophyllotoxins, antibiotics, L-asparaginase, topoisomerase inhibitors, interferons, platinum coordination complexes, anthracenedione-substituted ureas, methylhydrazine derivatives, adrenal cortical suppressants, corticosteroids, progestins, estrogens, antiestrogens, androgens, antiandrogens, 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), irinotecan, oxaliplatin, capecitabine, paclitaxel, and docetaxel. One or more chemotherapeutic agents can be used.

[0212] In some embodiments, a pharmaceutical composition according to the invention is for use in the treatment of cancer or for use in enhancing an immune response.

[0213] 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 present invention can be used to stimulate the immune system during vaccination. Thus, in one embodiment, the pharmaceutical composition can be used to improve vaccination.

[0214] In certain embodiments, the cancer is selected from lung, thyroid, breast, colon, melanoma, prostate, liver, bladder, kidney, cervical, pancreatic, leukemia, lymphoma, myeloid, ovarian, uterine, sarcoma, bile duct, and endometrial cell cancer, with each possibility representing a separate embodiment of the present invention.

[0215] According to some embodiments, a pharmaceutical composition comprising at least one antibody or 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 administration.

[0216] According to yet another aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a monoclonal antibody or antibody fragment according to the present invention.

[0217] The term "effective amount" as used herein refers to a sufficient amount of the monoclonal antibody of the antibody fragment that has the intended therapeutic effect when administered to a subject. The effective amount required to achieve the end result of treatment may depend on many factors, including, for example, the specific type of tumor and the severity of the patient's condition, and whether the combination is also co-administered with radiation. The effective amount (dosage) of the active agent in the context of the present invention should be sufficient to affect a beneficial therapeutic response in the subject over a period of time, including, but not limited to, inhibiting tumor growth, reducing the rate of tumor growth, preventing the growth of tumors and metastases, and improving survival rates.

[0218] Toxicity and therapeutic efficacy of the compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the IC50 (concentration resulting in 50% inhibition) and maximum tolerated dose for the compound of interest. Data obtained from these cell culture assays, and animal studies can be used to formulate a range of dosages for use in humans. Dosages can vary depending upon, among other relevant factors, the dosage form used, the dosing regimen selected, the composition of the agent used for treatment, and the route of administration utilized. The exact formulation, route of administration, and dosage can be selected by the individual physician in view of the patient's condition. Depending on the severity and responsiveness of the condition being treated, administration can also be a single dose of a sustained release composition, with the course of treatment lasting from several days to several weeks, or until a cure is effected or a diminution of the disease state is achieved. The amount of composition administered will, of course, depend on the subject being treated, the severity of the affliction, the means of administration, the judgment of the prescribing physician, and all other relevant factors.

[0219] The term "administering" or "administration of" a substance, compound, or agent to a subject can be performed using one of a variety of methods known to those skilled in the art. For example, the compound or agent can be administered enterally or parenterally. Enteral refers to administration via the gastrointestinal tract, including orally, sublingually, or rectally. Parenteral administration includes administration intravenously, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, intranasally, by inhalation, intraspinal, intracerebral, and transdermally (e.g., by absorption through the skin tract). The compound or agent can also be suitably introduced by rechargeable or biodegradable polymeric or other devices, such as patches and pumps, or formulations, which provide sustained, sustained, or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or for one or more extended periods of time. 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 instructs a patient to self-administer a drug or have another person administer the drug, and / or provides the patient with a prescription for a drug, administers the drug to a patient.

[0220] Antibodies are generally administered in the range of about 0.1 to about 20 mg / kg patient weight, generally about 0.5 to about 10 mg / kg, and often about 1 to about 5 mg / kg. In this regard, it is preferred to use antibodies that have 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 up to 14 to 21 days. In some instances, it may be effective to administer a large loading dose followed by intermittent (e.g., weekly) maintenance doses over the course of the treatment. Antibodies can also be delivered by sustained release delivery systems, pumps, and other known delivery systems for continuous infusion.

[0221] The antibodies of the present invention can be used in CAR-based adoptive immunotherapy, which utilizes engineered lymphocytes containing CARs to treat cancer. The CAR-T system is described herein as a non-limiting example.

[0222] T cell therapy utilizes chimeric antigen receptors (CARs) for the treatment of cancer or tumors (i.e., CAR-T cell therapy). CAR-T cell therapy is a cellular immunotherapy that involves administering genetically engineered T cells to cancer patients that act on tumor cells to cause apoptosis of the tumor cells. The genetically engineered T cells are prepared by expressing CARs on T cells using gene transfer technology, which have the variable regions (VL and VH) of an antibody bound to an intracellular domain, such as a fragment of the CD3 ζ chain sequence. CAR is a collective term for chimeric proteins in which the light and heavy chains of the variable regions of a monoclonal antibody specific to a tumor antigen bind to each other and then bind to a T cell receptor (TCR) at the C-terminus.

[0223] According to some embodiments, the CAR comprises at least one protein domain selected from the group consisting of a CD8 stalk domain, a CD28 TM domain, a 41BB domain, and a CD3ζ domain. According to some embodiments, the CAR comprises a CD8 stalk domain. According to some embodiments, the CAR comprises a CD28 TM domain. According to some embodiments, the CAR comprises a CD3ζ signaling domain. According to some embodiments, the CAR comprises a 41BB domain. According to certain embodiments, the CAR comprises a CD8 stalk domain, a CD28 TM domain, a 41BB domain, and a CD3ζ domain.

[0224] According to some embodiments, the CAR comprises a costimulatory domain derived from 4-1BB (or 41BB or CD137), ICOS, OX40, CD27, KIR2DS2, MYD88-CD40, or CD28. In some embodiments, the CAR comprises the signaling domains of CD3zeta, 41BB, and CD28.

[0225] According to some embodiments, the CAR comprises a transmembrane domain (TM) selected from CD28 TM, DAP12 TM, CD8 TM, CD3ζ TM, DAP10 TM, and ICOS TM.

[0226] According to some embodiments, the CAR comprises a hinge region sequence. According to some embodiments, the hinge region sequence is derived from a CD8, CD28, or IgG4 hinge.

[0227] According to some embodiments, a chimeric antigen receptor (CAR) is provided comprising a heavy chain variable region (VH) and a light chain variable region (VL) according to the present invention. According to certain embodiments, a genetically modified lymphocyte is provided having a CAR expressed on its surface. According to some particular embodiments, a genetically modified T cell (CAR-T cell) is provided having a CAR expressed on its surface.

[0228] According to some embodiments, the CAR comprises: i. the three complementarity determining regions (CDRs) of the heavy chain (HC) variable region comprising SEQ ID NO:7 and the three CDRs of the light chain (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 ii. A Nectin-2 binding site comprising a set of six CDR sequences selected from the group consisting of the three CDRs of a heavy chain variable region comprising SEQ ID NO: 17 and the three CDRs of a light chain variable region comprising SEQ ID NO: 18, or an analog or derivative thereof having at least 90% sequence identity with the sequence of said antibody or fragment.

[0229] According to some embodiments, an analog or derivative has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the antibody or fragment sequence.

[0230] According to some embodiments, the CAR comprises: i. the three complementarity determining regions (CDRs) of the heavy chain (HC) variable region comprising SEQ ID NO:7 and the three CDRs of the light chain (LC) variable region comprising SEQ ID NO:8; and ii. A Nectin-2 binding site comprising a set of six CDR sequences selected from the group consisting of: three CDRs of a heavy chain variable region comprising SEQ ID NO: 17 and three CDRs of a light chain variable region comprising SEQ ID NO: 18.

[0231] According to some embodiments, the CAR comprises: i. a set of six CDRs, where HC CDR1 is RFTMS (SEQ ID NO: 1), HC CDR2 is TISSGGSYTYYPDSVKG (SEQ ID NO: 2), HC CDR3 is DRDFYGPYYAMDY (SEQ ID NO: 3), LC CDR1 is KSSQSLLNSGNQKNYLA (SEQ ID NO: 4), LC CDR2 is FASTRES (SEQ ID NO: 5), and LC CDR3 is QQHYTTPLT (SEQ ID NO: 6), and ii. The set of six CDRs, wherein HC CDR1 sequence is SYWIH (SEQ ID NO: 11), HC CDR2 is AVYPGNSDSNYNQKFKA (SEQ ID NO: 12), HC CDR3 is LVGTFDY (SEQ ID NO: 13), LC CDR1 is KASQNVGINVV (SEQ ID NO: 14), LC CDR2 is SASYRYS (SEQ ID NO: 15), and LC CDR3 is QQYNTNPFT (SEQ ID NO: 16).

[0232] According to some embodiments, the CAR comprises an antigen binding domain comprising SEQ ID NO: 20 or 22, or an analog having at least 85% identity to SEQ ID NO: 20 or 22, a transmembrane domain, and an intracellular T cell signaling domain.

[0233] According to certain aspects, the present invention provides a cell comprising a CAR as described herein. According to some embodiments, the cell expresses or is capable of expressing the CAR of the present invention. According to some embodiments, the cell is a lymphocyte. According to some embodiments, the cell is selected from a T cell and a natural killer (NK) cell.

[0234] According to some embodiments, the cell, such as a T cell, comprises a nucleic acid molecule encoding a CAR of the invention. According to other embodiments, the cell, such as a T cell, comprises a nucleic acid construct comprising a nucleic acid molecule encoding a CAR of the invention. According to further embodiments, the invention provides a vector comprising a nucleic acid construct or molecule encoding a CAR of the invention. According to such embodiments, the T cell expresses or is capable of expressing a CAR of the invention.

[0235] According to some embodiments, lymphocytes are provided that are engineered to express a CAR as described herein. According to some embodiments, T cells are provided that are engineered to express a CAR as described herein.

[0236] According to additional embodiments, there is provided a NK cell engineered to express a CAR described herein.

[0237] The CAR of the present invention comprises a transmembrane domain (TM domain), a costimulatory domain, and an activation domain. According to some embodiments, the TM domain is a TM domain of a receptor selected from CD4, CD3zeta, CD28 and CD8, or an analog thereof having at least 85% amino acid identity to the original sequence, and / or the costimulatory domain is selected from a costimulatory domain of a protein selected from CD28, 4-1BB, OX40, iCOS, CD27, CD80, and CD70, an analog thereof having at least 85% amino acid identity to the original sequence, and any combination thereof, and / or the activation domain is selected from FcRgamma and CD3-zeta activation domains. According to some embodiments, the CAR comprises a primary peptide.

[0238] According to some embodiments, the invention provides a cell composition comprising a plurality of cells, e.g., CAR-presenting cells, of the invention.

[0239] The term "about" means that an acceptable range of error for a particular value, for example, up to 5% or 10%, should be assumed.

[0240] diagnosis The present invention further discloses methods for diagnosing and prognosing cancer.

[0241] According to one aspect, the present invention provides a method for the diagnosis and / or prognosis of cancer or an infectious disease in a subject, the method comprising determining the expression level of Nectin-2 in a biological sample of the subject using at least one antibody described herein.

[0242] The term "biological sample" encompasses various sample types obtained from an organism that may be used in diagnostic or monitoring assays. The term encompasses blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens, or tissue cultures or cells derived therefrom and their progeny. In addition, the term may encompass circulating tumor or other cells. The term specifically encompasses clinical samples, and further includes biological fluids including cells in cell culture, cell supernatants, cell lysates, serum, plasma, urine, amniotic fluid, aqueous humor and vitreous fluid for ocular samples, and tissue samples. The term also encompasses samples that have been manipulated in any way after treatment with reagents, solubilization, or enrichment for specific components, etc.

[0243] The determination of the expression level of Nectin-2 can be carried out by a labeled anti-Nectin-2 antibody described herein. The determination of expression can be carried out, for example, by ELISA.

[0244] The method of the invention may further comprise the step of comparing the expression level to a control level.

[0245] The following examples are presented to further illustrate some embodiments of the invention, but should not be construed as limiting the scope of the invention in any way. EXAMPLES

[0246] Reference is now made to the following examples which, together with the above descriptions, illustrate the invention in a non-limiting manner.

[0247] Generally, the nomenclature used herein and the laboratory 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 referring to well-known procedures are provided throughout this document for the convenience of the reader.

[0248] Example 1. High expression of Nectin-2 mRNA correlates with poor survival in patients with various cancers. The correlation between Nectin-2 mRNA expression and survival probability was examined with data from the TCGA site and analyzed using the oncolnc.org site (https: / / doi.org / 10.7717 / peerj-cs.67). This correlation is indicated by the arrows in Figure 1 for patients with low-grade glioma (Figure 1A; p=5.22E-5), renal clear cell carcinoma of the kidney (Figure 1B; p=0.00037), and lung adenocarcinoma (Figure 1C; p=0.0319).

[0249] Example 2. Nectin-2 binds to and affects immune cells via specific receptors. Schematic diagram of receptors expressed on immune cells and their respective affinities for Nectin-2 expressed on tumors or antigen-presenting cells (APCs) (Figure 2). TIGIT is associated with a co-inhibitory receptor on immune cells such as T cells or NK cells, DNAM-1 (also called CD226) is associated with an activating receptor on immune cells (e.g., T cells), CD112R (also called PVRIG) is associated with a co-inhibitory receptor on lymphoid immune cells (e.g., T cells and NK cells), and Nectin-2 (CD112) is an inhibitory ligand for immune cells, mainly via binding to CD112R. According to the present invention, anti-Nectin-2 mAb can block the interaction of Nectin-2 with its ligands CD112R and / or TIGIT, increasing the activation of immune cells.

[0250] Example 3. Analysis of the binding and blocking properties of anti-nectin-2 mAb. Nectin-2 clones did not bind to parental 8866 cells (EBV-positive Burkitt's lymphoma) that do not express nectin-2. Figure 3A shows binding of anti-nectin-2 clones to MDA-MB-231 (breast adenocarcinoma) cells that endogenously express nectin-2 (black bars) or to 8866-hnectin-2 cells that overexpress nectin-2 (gray bars). All of the mAbs were used at 30 ul / well from hybridoma supernatants. For detection, goat anti-mouse-647 Ab was used at 1:250 dilution. Figure 3B shows the results of FACS analysis of CD112R-Fc (extracellular domain of CD112R fused to the human Fc region of IgG1) binding to 8866-hnectin-2 cells. Of the antibodies generated, three clones (#9, 11, and 13) partially blocked these interactions and one clone (#7) completely blocked them. Figure 3C shows the results of FACS analysis of DNAM-1-Fc binding to 8866-hNectin2 cells. Except for clone 15, none of the other clones (#7-13) blocked the binding of the activating receptor DNAM-1 to Nectin-2. Figure 3D shows the results of FACS analysis of TIGIT-Fc binding to CHOK1-hNectin2 cells in the presence of anti-Nectin-2 clones 7 and 11. Both clones block >66% of TIGIT-Fc binding. All of the Fc proteins were used at 20ug / ml and co-incubated with 30ul / well of the indicated mAb supernatants. For detection, anti-human-APC Ab was used at 1:200 dilution (Jackson immunoresearch AB_2340526). These results suggest that some of the clones can prevent the binding of inhibitory receptors without any interference with the binding of activating receptors.

[0251] Example 4. Blockade of nectin-2 with anti-nectin-2 mAb promotes NK cell activation. NK cells from healthy donors were incubated for 2 hours at 37°C in the presence of different mAbs and target cell lines at an E:T ratio of 2:1. NK cell activation was measured by induction of surface expression of CD107a and is displayed as fold change relative to control IgG (Y-axis). All mAbs were used at 5ug / ml. Results for human cancer cell lines A549 (Figure 4A and 4C, lung adenocarcinoma) and MDA-MB-231 (Figure 4B, breast adenocarcinoma) are shown in Figure 4. The most significant effects were observed for clones #3, 7, and 11. *=P<0.04, **p<0.02, ***p<0.002 by two-tailed Student's t-test. Representative data for one of five donors are shown. Human IgG1 chimeric variants of clones 7 and 11 further increased degranulation (Figure 4C), resulting in >200% degranulation compared to isotype controls. ***p<0.002. Representative data from one of two donors is shown. The data suggest that blockade of Nectin-2 with specific clones increases NK cell activity against multiple targets. Furthermore, the presence of an effector Fc further increases NK activity, suggesting another possible mode of action for the mAb.

[0252] Example 5. Nectin-2 is expressed in various cancer cells. Expression of Nectin-2 and PVR in various human tumor cell lines was analyzed by FACS. Analysis was performed on melanoma cells, breast cancer cells, colorectal cancer (CRC) cells, kidney cells (HEK), lung cancer cells, prostate cancer cells, and brain tumor cells (GBM), all of which express PVR and Nectin-2. A commercially available anti-Nectin-2 (clone Tx31) and an in-house anti-PVR mAb were used. All mAbs were used at 2ug / ml. For detection, goat anti-mouse-647 was used at a 1:250 dilution. Nectin-2 was found to be highly expressed in various cancer cells.

[0253] Example 6. Similar binding of anti-nectin-2 mAb to human and cynomolgus nectin-2. Anti-nectin-2 mAbs (clones 7 and 11) were examined for binding to human nectin-2 (protein id: Q92692) and cynomolgus monkey (Cyno) (macaca fascicularis, protein id: XP_005589607.1) nectin-2. Protein blasting between cynomolgus and human nectin-2 revealed that the extracellular domain of the mature protein has 14 amino acid differences between the species. Figure 5A shows the overlay binding curves of both mAbs added in a series of 3-fold dilutions ranging from 13.3 nM to 0.02 nM to CHO cells expressing either human or Cyno nectin-2. The results of the FACS analysis of this assay are expressed as relative binding intensity compared to maximum binding, which was set at 100%. For detection, goat anti-mouse-647 Ab (Jackson immunoresearch AB_2338910) was used at a 1:250 dilution. A summary of the data analysis of this assay is also shown, further demonstrating that both mAbs bind to human and Cyno Nectin-2 with high and similar affinity.

[0254] Anti-nectin-2 mAb binding was also examined using Vero cells derived from Chlorocebus (African green monkey). This species expresses the nectin-2 protein (XP_007995342.1) with 97% similarity to human nectin-2. Figure 5B shows the binding of anti-nectin-2 mAb to endogenous human nectin-2 (expressed in 293T cells) and endogenous African green monkey-nectin-2 (expressed in Vero cells) tested by FACS analysis described in Figure 5A (Ab range: 20-0.0003 nM). This analysis revealed similar Ab binding to both human and monkey nectin-2 targets, with high affinity, for both anti-nectin-2 clones, which is also evident in the summary table.

[0255] Example 7. Anti-hNectin-2 mAb affects T cell proliferation. Human PBMCs were CFSE (C34554, ThermoFischer) labeled and incubated with target cells MDA-MB-231 (Figure 6A) or A549 (Figure 6B) in the presence of 0.2ug / ml PHA-L and 2ug / ml of the indicated antibodies. After incubation, immune cells were collected and stained with anti-human CD8. Cell proliferation of CD8+ T cells was assessed by CFSE signal intensity. CFSE levels of mIgG-treated cells were set to 1. Results are presented as fold increase in proliferation compared to this control. Experiments were performed in quadruplicate; all p values ​​were less than 0.02 by two-tailed Student's t-test. Shown are the results of one PBMC donor, representative of seven tested. Data show that blockade of Nectin-2 by the indicated clones significantly inhibited CD8 T cells in the presence of tumor cells from various origins. + These results suggest that it increases T cell proliferation.

[0256] Example 8. Anti-hNectin-2 mAb, alone or in combination with known checkpoint blockade, affects the proliferation of CD8+ T cells. To examine the effect of mAbs on T cell proliferation, human PBMCs were CFSE-labeled and incubated with targeting RKO (human colon cancer cells; Figure 7A) or A549 (Figure 7B) cells in the presence of 0.2ug / ml PHA-L and 2ug / ml of the indicated mAbs. For combination treatments, each mAb was added at 2ug / ml. After incubation, immune cells were collected and stained with anti-human CD8. Total population and CD8 proliferating cells were analyzed, and CFSE levels in mIgG-treated cells were set to 1. Experiments were performed in quadruplicate; all p values ​​were less than 0.02 by two-tailed Student's t-test. Results are presented as fold increase in proliferation compared to control. Results for one PBMC donor out of two donors tested are shown. All combinations tested significantly increased CD8+ T cell proliferation over individual treatments.

[0257] Example 9. Anti-hNectin-2 antibody affects the secretion of IFNγ. Human PBMCs were CFSE-labeled and incubated with target cells RKO (Figure 8A) or A549 (Figure 8B) in the presence of 0.2ug / ml PHA-L and 2ug / ml of the indicated antibodies. For combination treatments, each mAb was added at 2ug / ml. After 96 hours, plates were centrifuged and supernatants were collected. IFNγ quantification was performed using Biolegend's Human IFN-γ ELISA MAX™ Deluxe according to the manufacturer's protocol. Results are shown for one PBMC donor out of five donors tested. All of the treatments resulted in a significant increase in IFNγ secretion (p<0.001 two-tailed Student's t-test).

[0258] Example 10. Anti-hNectin-2 antibodies alone or in combination with known checkpoint blockade affect tumor cell killing by hPBMCs. The assay was performed as described in Example 8. After 96-120 hours, immune cells were removed, tumor cells were washed thoroughly, and CellTiter-Glo® was used to establish the viability of adherent tumor cells according to the manufacturer's protocol. All results were within the linear range of the kit. Tumor cell killing in wells treated with mIgG was set to 1. All individual treatments significantly increased tumor cell killing (p<0.01 two-tailed t-test) (Figure 9A, RKO; Figure 9B, A549). Results from one PBMC donor out of two donors tested are shown. Most combinations tested significantly increased tumor cell killing compared to individual treatments.

[0259] Example 11. Nectin-2 mAb significantly inhibits tumor development in vivo. Scid female mice (n = 33) were treated with 5 × 10 6 MDA-MB-231 cells were injected subcutaneously. 3Once the tumor cell count reached 100 mg / kg, mice were randomly divided into three groups and treated twice weekly in a blinded manner by intravenous (iv) injection of either PBS (light grey diamonds; Figure 10), hIgG1 control Ab (grey squares), or clone-7-human IgG1 (2.7.1) (black circles) (both at 3 mg / kg). As shown in Figure 10, significant tumor growth inhibition (TGI) was observed for clone 2.7.1 starting at day 7 post-treatment, reaching 54% at the end of the study. *p<0.04, **p<0.02, ***p<0.008.

[0260] Example 12. Chimeric nectin-2 mAb containing human IgG2 Fc synergizes with anti-PD-1 mAb to result in tumor cell killing and increased PBMC proliferation. A549 cells were co-incubated with PBMCs for 96 hours at an E:T ratio of 7:1 in the presence of 4ug / ml PHA-L with either no Ab or clone-11-hIgG2 (2.11.2), Keytruda (both at 3.5ug / ml), or their combination (each at 3.5ug / ml). The 2.11.2 treatment group saw a significant increase in tumor cell killing (Figure 11A) and PBMC (T cell) proliferation (Figure 11B), which was further increased when combined with the anti-PD-1 Ab Keytruda. *p<0.01, **p<0.002, ***p<0.0008.

[0261] Example 13. CAR-T cells expressing scFvs derived from clones 7 and 11 are specifically activated in the presence of tumor cells expressing Nectin-2. PBMCs from healthy donors were transduced with different CAR-T constructs comprising scFv molecules according to the invention and at least one regulatory, transmembrane and / or stimulatory domain. In the schematic diagram shown in Figure 12A, the CAR-T comprises an scFv and four domains; CD8 stalk, CD28 TM, 4-1BB and CD3ζ. Nectin-2 CAR-T2.07 (binding site derived from clone 7) or CAR-T 2.11 (binding site derived from clone 11) PBMCs were incubated with U937 or BT-474 target cells at various E:T ratios. Target cell killing (Figures 12B and 12D) was significant at most of the E:T ratios tested (p<0.005 except where indicated by NS) as well as IFNγ secretion by activated PBMCs (Figures 12C and 12E, p<0.03). Figures 12B-E show a representative experiment out of three performed for each cell line (CAR-T2.07 grey bars, CAR-T2.11 black bars).

[0262] Example 14. Antibody Sequences Table 1 details some of the antibody sequences of the present invention. [Table 1]

[0263] The foregoing description of the specific embodiments fully reveals the general nature of the present invention, so that others, by applying current knowledge, can easily modify and / or adapt to various uses of such specific embodiments without undue experimentation and without departing from the general concept, and therefore such adaptations and modifications should be understood within the meaning and range of equivalents of the embodiments of the present disclosure, and are so intended. It should be understood that the expressions or terms used herein are for the purpose of description and not for the purpose of limitation.

Claims

1. An antibody that binds to Nectin-2, or an antibody fragment thereof comprising at least an antigen-binding portion, The antibody or antibody fragment i. a set of six CDRs, wherein the amino acid sequence of HC CDR1 is RFTMS (SEQ ID NO: 1), the amino acid sequence of HC CDR2 is TISSGGSYTYYPDSVKG (SEQ ID NO: 2), the amino acid sequence of HC CDR3 is DRDFYGPYYAMDY (SEQ ID NO: 3), the amino acid sequence of LC CDR1 is KSSQSLLNSGNQKNYLA (SEQ ID NO: 4), the amino acid sequence of LC CDR2 is FASTRES (SEQ ID NO: 5), and the amino acid sequence of LC CDR3 is QQHYTTPLT (SEQ ID NO: 6); and ii. a set of six CDRs, wherein the amino acid sequence of HC CDR1 is SYWIH (SEQ ID NO:11), the amino acid sequence of HC CDR2 is AVYPGNSDSNYNQKFKA (SEQ ID NO:12), the amino acid sequence of HC CDR3 is LVGTFDY (SEQ ID NO:13), the amino acid sequence of LC CDR1 is KASQNVGINVV (SEQ ID NO:14), the amino acid sequence of LC CDR2 is SASYRYS (SEQ ID NO:15), and the amino acid sequence of LC CDR3 is QQYNTNPFT (SEQ ID NO:16); comprising a set of CDRs selected from the group consisting of: An antibody or antibody fragment.

2. a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:7 and the light chain comprises the amino acid sequence set forth in SEQ ID NO:8; or a heavy chain and a light chain, said heavy chain comprising the amino acid sequence set forth in SEQ ID NO:17 and said light chain comprising the amino acid sequence set forth in SEQ ID NO:18; The antibody or antibody fragment of claim 1.

3. The antibody fragment according to any one of claims 1 to 2, which is a single chain Fv (scFv).

4. A polynucleotide encoding the sequences of the heavy and light chain regions of the antibody or antibody fragment of any one of claims 1 to 3.

5. A cell comprising the polynucleotide of claim 4.

6. The antibody or antibody fragment of any one of claims 1 to 3 attached to a cytotoxic, radioactive, or identifiable moiety.

7. A pharmaceutical composition comprising at least one antibody or fragment thereof according to any one of claims 1 to 3 and 6 as an active ingredient, and a pharma- ceutically acceptable excipient, diluent, salt or carrier.

8. The pharmaceutical composition of claim 7, for use in treating cancer in a subject, or for use in regulating the immune system by inhibiting binding of Nectin-2 to TIGIT or CD112R, or for use in preventing or treating a viral infection in a subject.

9. 10. The pharmaceutical composition of claim 8 for use in treating cancer in a subject.

10. 10. The pharmaceutical composition of claim 9, wherein the use further comprises an additional anti-cancer therapy selected from surgery, chemotherapy, radiation therapy, and immunotherapy.

11. 10. The pharmaceutical composition of claim 9, wherein said use comprises administering an additional immunomodulatory agent, activated lymphocyte cells, kinase inhibitor, chemotherapeutic agent, or any other anti-cancer agent.

12. A chimeric antigen receptor (CAR) comprising an extracellular portion capable of binding to Nectin-2, said extracellular portion comprising at least one antibody or antibody fragment according to any one of claims 1 to 3.

13. A population of T cells or NK cells engineered to express the CAR of claim 12 comprising a CD8 stalk domain, a CD28 TM domain, a 41BB domain, and a CD3ζ domain.

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