TIGIT and CD112R blocking

Abstract

TIGIT and CD112R blocking are provided. [Solution] This specification provides TIGIT-binding proteins, CD112R-binding proteins, and combinations thereof. Also provided are compositions comprising TIGIT-binding proteins and CD112R-binding proteins, optionally further comprising PD-1-binding proteins. Related conjugates, fusion proteins, nucleic acids, vectors, host cells, and kits are further provided. Furthermore, pharmaceutical compositions comprising TIGIT-binding proteins, CD112R-binding proteins, or combinations thereof, optionally further comprising PD-1 antigen-binding proteins, or conjugates, fusion proteins, nucleic acids, vectors, or host cells, and pharmaceutically acceptable carriers, diluents, or excipients, as well as methods for treating subjects requiring such compositions, are provided.

Description

[Technical Field]

[0001] Cross-reference of related applications The interests under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 052,011, filed on 15 July 2020, and U.S. Provisional Patent Application No. 63 / 212,315, filed on 18 June 2021, are asserted herein by reference, and these disclosures are incorporated herein by reference in their entirety.

[0002] Incorporation by referencing electronically submitted materials The computer-readable nucleotide / amino acid sequence listing submitted concurrently with this specification and identified below is incorporated in its entirety by reference: a 5.22 MB ASCII (Text) file named "A-2443-WO-PCT_Seqlisting.txt" created on 28 June 2021. [Background technology]

[0003] The PD-1 / PD-L1 axis is involved in the suppression of the T-cell immune response in cancer. Antagonists of this pathway have been clinically validated in many solid tumor indications. Nivolumab and pembrolizumab are two such inhibitors that target the PD-1 pathway and are approved by the U.S. Food and Drug Administration (FDA) for the treatment of metastatic melanoma, respectively. Recently, researchers have tested the checkpoint inhibitor paradigm in the case of other tumor types. Although some progress has been made, checkpoint inhibitor therapy remains overshadowed by other cancer treatment options.

[0004] Studies of checkpoint inhibitors in combination with other drugs are ongoing or have recently been completed. For example, the combination of nivolumab and ipilimumab, CTLA-4 receptor blocking antibodies, was tested in a phase III clinical trial in patients with unresectable stage III or IV melanoma. In this study, the proportion of patients achieving complete response was highest among those receiving the nivolumab and ipilimumab combination, exceeding the outcomes shown in patients receiving either drug alone. However, the response to immunotherapy blocking CTLA-4 and PD-1 checkpoint receptors is not universal, and many mechanisms by which tumors evade response have been identified. As an approach to enhance overall efficacy and limit tumor resistance, combination therapies targeting multiple pathways represent the rationale for the next steps.

[0005] There is a need for safe and effective combination therapies that target multiple checkpoint inhibitory pathways. [Overview of the project] [Means for solving the problem]

[0006] This specification presents data demonstrating the induction of TIGIT and CD112R on activated human T cells and TILs (tumor-infiltrating leukocytes) in primary human tumor tissue, as well as data supporting the high co-expression levels of TIGIT and CD112R (CD155 and CD112) ligands on tumor cells. The data provided herein support that primary human T cell activity is enhanced by blocking a single interaction between TIGIT or CD112R and its ligand, while primary human T cell activity is significantly enhanced by simultaneously blocking the binding of both receptors (TIGIT and CD112R) to their respective ligands. The data further support that, in addition to blocking the interaction between TIGIT and CD112R, blocking a third interaction involving PD-1 and its ligand significantly increases overall primary human T cell activity. The increase in activity achieved by blocking all three molecules (PD-1, TIGIT, and CD112R) exceeds the increase in activity achieved by single blocking (TIGIT only or CD112R only) and double blocking (both TIGIT and CD112R, both TIGIT and PD-1, or both CD112R and PD-1).

[0007] Accordingly, this disclosure provides TIGIT antigen-binding proteins (e.g., antibodies and their antigen-binding fragments), CD112R antigen-binding proteins (e.g., antibodies and their antigen-binding fragments), and combinations thereof. Furthermore, compositions comprising TIGIT antigen-binding proteins, CD112R antigen-binding proteins, and PD-1 antigen-binding proteins are provided by this disclosure. In certain embodiments, the composition comprises a TIGIT antibody or its TIGIT-binding fragment, and / or a CD112R antibody or its CD112R-binding fragment, and / or a PD-1 antibody or its PD-1-binding fragment. In preferred embodiments, the composition comprises a TIGIT antibody and a CD112R antibody. Related conjugates, fusion proteins, nucleic acids, vectors, host cells, and kits are provided herein.

[0008] This disclosure also provides pharmaceutical compositions comprising a TIGIT antigen-binding protein, a CD112R antigen-binding protein, or a combination thereof, and optionally further comprising a PD-1 antigen-binding protein, or a conjugate, fusion protein, nucleic acid, vector or host cell, and a pharmaceutically acceptable carrier, diluent or excipient. In preferred embodiments, the pharmaceutical composition comprises a TIGIT antibody and a CD112R antibody in a 1:1 ratio.

[0009] A method for producing antigen-binding proteins is provided. Also provided is a method for treating a subject in need of treatment, which includes administering the pharmaceutical composition of this disclosure to the subject. In embodiments of the present invention, for example, the following items are provided. (Item 1) (a) Heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence as described in Table A1, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity, (b) HC CDR2 amino acid sequence as described in Table A1, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity, (c) HC CDR3 amino acid sequence as described in Table A1, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity, (d) Light chain (LC) CDR1 amino acid sequence as described in Table A1, or 1 to 4 CD112R antigen-binding protein comprising (e) the LC CDR2 amino acid sequence described in Table A1, or a variant sequence described in Table A1, which differs by only one to four amino acids, or which has at least or about 90% sequence identity, and (f) the LC CDR3 amino acid sequence described in Table A1, or a variant sequence described in Table A1, which differs by only one to four amino acids, or which has at least or about 90% sequence identity. (Item 2) The CD112R antigen-binding protein described in Item 1, comprising six CDR amino acid sequences listed in a single row in Table A1, or six CDR amino acid sequences selected from the group consisting of (a) SEQ ID NOs. 13-18, (b) SEQ ID NOs. 23-28, (c) SEQ ID NOs. 33-38, (d) SEQ ID NOs. 43-48, (e) SEQ ID NOs. 53-58, (f) SEQ ID NOs. 63-68, (g) SEQ ID NOs. 73-78, (h) SEQ ID NOs. 83-88, (i) SEQ ID NOs. 93-98, (j) SEQ ID NOs. 103-108, (k) SEQ ID NOs. 233-238, (l) SEQ ID NOs. 1973-1978, (m) SEQ ID NOs. 1983-1988, (n) SEQ ID NOs. 1993-1998, and (o) SEQ ID NOs. 2003-2008. (Item 3) (a) The HC variable region amino acid sequence described in Table B1, or a variant sequence thereof that differs from the HC variable region amino acid sequence in Table B1 by only 1 to 15 amino acids, or has at least or about 90% or about 95% sequence identity; (b) The LC variable region amino acid sequence described in Table B1, or a variant sequence thereof that differs from the LC variable region amino acid sequence in Table B1 by only 1 to 15 amino acids, or has at least or about 90% or about 95% sequence identity; or (c) A CD112R antigen-binding protein as described in item 1 or 2, including a combination of (a) and (b). (Item 4) The CD112R antigen-binding protein described in item 3, comprising a pair of HC variable region and LC variable region amino acid sequences listed in a single row in Table B1, or comprising a pair of amino acid sequences selected from the group consisting of (a) SEQ ID NOs: 11-12, (b) SEQ ID NOs: 21-22, (c) SEQ ID NOs: 31-32, (d) SEQ ID NOs: 41-42, (e) SEQ ID NOs: 51-52, (f) SEQ ID NOs: 61-62, (g) SEQ ID NOs: 71-72, (h) SEQ ID NOs: 81-82, (i) SEQ ID NOs: 91-92, (j) SEQ ID NOs: 101-102, (k) SEQ ID NOs: 231-232, (l) SEQ ID NOs: 1971-1972, (m) SEQ ID NOs: 1981-1982, (n) SEQ ID NOs: 1991-1992, and (o) SEQ ID NOs: 2001-2002. (Item 5) (a) The full-length (FL)HC amino acid sequence described in Table B1, or a variant sequence thereof that differs from the FL HC amino acid sequence in Table B1 by only 1 to 50 amino acids, or has at least or about 90% or about 95% sequence identity; (b) The FL LC amino acid sequence described in Table B1, or a variant sequence thereof that differs from the FL LC amino acid sequence in Table B1 by only 1 to 50 amino acids, or has at least or about 90% or about 95% sequence identity; or (c) A CD112R antigen-binding protein described in any one of items 1 to 4, including a combination of (a) and (b). (Item 6) CD112R antigen-binding proteins as described in item 5, comprising a pair of full-length (FL) HC and FL LC amino acid sequences listed in a single row in Table B, or comprising a pair of amino acid sequences selected from the group consisting of (a) SEQ ID NOs: 9-10, (b) SEQ ID NOs: 19-20, (c) SEQ ID NOs: 29-30, (d) SEQ ID NOs: 39-40, (e) SEQ ID NOs: 49-50, (f) SEQ ID NOs: 59-60, (g) SEQ ID NOs: 69-70, (h) SEQ ID NOs: 79-80, (i) SEQ ID NOs: 89-90, (j) SEQ ID NOs: 99-100, (k) SEQ ID NOs: 229-230, (l) SEQ ID NOs: 1969-1970, (m) SEQ ID NOs: 1979-1980, (n) SEQ ID NOs: 1989-1990, and (o) SEQ ID NOs: 1999-2000. (Item 7) An antibody, specifically the CD112R antigen-binding protein described in any one of items 1-6. (Item 8) a. The heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence of SEQ ID NO: 33, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (b) The HC CDR2 amino acid sequence of SEQ ID NO: 34, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (c) The HC CDR3 amino acid sequence of SEQ ID NO: 35, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (d) The light chain (LC) CDR1 amino acid sequence of SEQ ID NO: 36, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (e) The LC CDR2 amino acid sequence of SEQ ID NO: 37, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (f) The LC CDR1 amino acid sequence of SEQ ID NO: 38 The CDR3 amino acid sequence, or a variant sequence thereof that differs by only 1 to 4 amino acids, or has at least or approximately 90% sequence identity; or (b) the heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence of SEQ ID NO: 63, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (b) the HC CDR2 amino acid sequence of SEQ ID NO: 64, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (c) the HC CDR3 amino acid sequence of SEQ ID NO: 65, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (d) the light chain (LC) CDR1 amino acid sequence of SEQ ID NO: 66, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (e) the LC CDR2 amino acid sequence of SEQ ID NO: 67, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (f) the LC CDR1 amino acid sequence of SEQ ID NO: 68 The CDR3 amino acid sequence, or a variant sequence thereof that differs by only 1 to 4 amino acids, or has at least or approximately 90% sequence identity; or (c) The heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence of SEQ ID NO: 83, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (b) The HC CDR2 amino acid sequence of SEQ ID NO: 84, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (c) The HC CDR3 amino acid sequence of SEQ ID NO: 85, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (d) The light chain (LC) CDR1 amino acid sequence of SEQ ID NO: 86, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (e) The LC CDR2 amino acid sequence of SEQ ID NO: 87, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (f) The LC CDR1 amino acid sequence of SEQ ID NO: 88 The CDR3 amino acid sequence, or a variant sequence thereof that differs by only 1 to 4 amino acids, or has at least or approximately 90% sequence identity; or (d)(a) the HC variable region amino acid sequence of SEQ ID NO: 31, or a variant sequence thereof that differs from the HC variable region amino acid sequence of SEQ ID NO: 31 by only 1 to 15 amino acids, or has at least or about 90% or about 95% sequence identity; (b) the LC variable region amino acid sequence of SEQ ID NO: 32, or a variant sequence thereof that differs from the LC variable region amino acid sequence of SEQ ID NO: 32 by only 1 to 15 amino acids, or has at least or about 90% or about 95% sequence identity; or (c) a combination of (a) and (b); or (e)(a) The HC variable region amino acid sequence of SEQ ID NO: 61, or the sequence of the HC variable region amino acid sequence of SEQ ID NO: 61 differs from only 1 to 15 amino acids, or at least (b) the LC variable region amino acid sequence of SEQ ID NO: 62, or a variant sequence that differs from the LC variable region amino acid sequence of SEQ ID NO: 62 by only 1 to 15 amino acids, or that has at least 90% or approximately 95% sequence identity; or (c) a combination of (a) and (b); or (f)(a) the HC variable region amino acid sequence of SEQ ID NO: 81, or a variant sequence thereof that differs from the HC variable region amino acid sequence of SEQ ID NO: 81 by only 1 to 15 amino acids, or has at least or about 90% or about 95% sequence identity; (b) the LC variable region amino acid sequence of SEQ ID NO: 82, or a variant sequence thereof that differs from the LC variable region amino acid sequence of SEQ ID NO: 82 by only 1 to 15 amino acids, or has at least or about 90% or about 95% sequence identity; or (c) a combination of (a) and (b); or (g)(a) the full-length (FL)HC amino acid sequence of SEQ ID NO: 29, or a variant sequence thereof that differs from the FL HC amino acid sequence of SEQ ID NO: 29 by only 1 to 50 amino acids, or has at least or about 90% or about 95% sequence identity; (b) the FL LC amino acid sequence described in SEQ ID NO: 30, or a variant sequence thereof that differs from the FL LC amino acid sequence of SEQ ID NO: 30 by only 1 to 50 amino acids, or has at least or about 90% or about 95% sequence identity; or (c) a combination of (a) and (b); or (h)(a) the full-length (FL)HC amino acid sequence of SEQ ID NO: 59, or a variant sequence thereof that differs from the FL HC amino acid sequence of SEQ ID NO: 59 by only 1 to 50 amino acids, or has at least or about 90% or about 95% sequence identity; (b) the FL LC amino acid sequence described in SEQ ID NO: 60, or a variant sequence thereof that differs from the FL LC amino acid sequence of SEQ ID NO: 60 by only 1 to 50 amino acids, or has at least or about 90% or about 95% sequence identity; or (c) a combination of (a) and (b); or (i)(a) the full-length (FL)HC amino acid sequence of SEQ ID NO: 79, or a variant sequence thereof that differs from the FL HC amino acid sequence of SEQ ID NO: 79 by only 1 to 50 amino acids, or has at least or about 90% or about 95% sequence identity; (b) the FL LC amino acid sequence described in SEQ ID NO: 80, or a variant sequence thereof that differs from the FL LC amino acid sequence of SEQ ID NO: 80 by only 1 to 50 amino acids, or has at least or about 90% or about 95% sequence identity; or (c) a combination of (a) and (b). CD112R antigen-binding proteins, including those listed in item 7. (Item 9) The CD112R antigen-binding protein described in any one of items 1 to 6, which is an antigen-binding fragment of an antibody. (Item 10) An antibody protein product, optionally an scFv, which is a CD112R antigen-binding protein as described in any one of items 1 to 6. (Item 11) A nucleic acid encoding the CD112R antigen-binding protein described in any one of items 1 to 10. (Item 12) A nucleic acid encoding the light chain, heavy chain, or both the light and heavy chain of an antibody as described in item 7 or 8. (Item 13) (a) The HC variable region amino acid sequence described in Table B1, or a variant sequence having only 1 to 15 amino acids different from the HC variable region amino acid sequence in Table B1, or having at least 90% or about 95% sequence identity; (b) The LC variable region amino acid sequence described in Table B1, or 1 to 15 amino acids different from the LC variable region amino acid sequence in Table B1 A variant sequence of the same sequence that differs by only one amino acid, or has at least or about 90% or about 95% sequence identity; or a nucleic acid as described in item 11 or 12, comprising both (c)(a) and (b). (Item 14) A vector containing one or more nucleic acids as described in any one of items 11-13. (Item 15) A host cell containing one or more nucleic acids as described in any one of items 11-13, or one or more vectors as described in item 14. (Item 16) The host cell is the host cell described in item 15, which produces the CD112R antigen-binding protein described in any one of items 1 to 10. (Item 17) (a) Heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence as described in Table A2, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (b) HC CDR2 amino acid sequence as described in Table A2, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (c) HC CDR3 amino acid sequence as described in Table A2, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (d) Light chain (LC) CDR1 amino acid sequence as described in Table A2, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (e) LC CDR2 amino acid sequence as described in Table A2, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (f) LC as described in Table A2 A TIGIT antigen-binding protein comprising the CDR3 amino acid sequence, or a variant sequence thereof that differs by only 1 to 4 amino acids, or has at least or approximately 90% sequence identity. (Item 18) (a) LC CDR1 amino acid sequence containing Gln27 or its conserved amino acid substitution, Ser28 or its conserved amino acid substitution, or any combination thereof; LC CDR2 amino acid sequence containing Glu1 or its conserved amino acid substitution; and LC CDR3 amino acid sequence containing Ser91 or its conserved amino acid substitution, Ser92 or its conserved amino acid substitution, Ser93 or its conserved amino acid substitution, Leu94 or its conserved amino acid substitution, or any combination thereof; HC containing Val32 or its conserved amino acid substitution, Tyr33 or its conserved amino acid substitution, or any combination thereof CDR1 amino acid sequence; HC CDR2 amino acid sequence containing Tyr52 or its conserved amino acid substitution, Tyr54 or its conserved amino acid substitution, Tyr55 or its conserved amino acid substitution, Ser56 or its conserved amino acid substitution, Gly57 or its conserved amino acid substitution, Gly58 or its conserved amino acid substitution, Thr59 or its conserved amino acid substitution, Tyr60 or its conserved amino acid substitution, Pro63 or its conserved amino acid substitution, Arg66 or its conserved amino acid substitution, or any combination thereof; and HC CDR3 amino acid sequence containing Ile102 or its conserved amino acid substitution, Ala104 or its conserved amino acid substitution, Gly107 or its conserved amino acid substitution, Tyr108 or its conserved amino acid substitution, Phe109 or its conserved amino acid substitution, Tyr110 or its conserved amino acid substitution, Tyr111 or its conserved amino acid substitution, or any combination thereof (where the position numbers are relative positions in the LC variable region amino acid sequence of the TIGIT antigen-binding protein). (b) Gln27 or its conserved amino acid substitution, Ser28 or its conserved amino acid substitution, Val29 or its conserved amino acid substitution, Ser30 or its conserved amino acid substitution, Ser31 or its conserved amino acid substitution, Thr32 or its conserved amino acid substitution, Tyr33 or its conserved amino acid substitution, or any combination thereof LC CDR1 amino acid sequence containing combinations; LC CDR2 amino acid sequence containing Glu1 or its conserved amino acid substitution, Ile2 or its conserved amino acid substitution, Ser68 or its conserved amino acid substitution, Gly69 or its conserved amino acid substitution, or any combination thereof; LC CDR3 amino acid sequence containing Tyr92 or its conserved amino acid substitution, Asp93 or its conserved amino acid substitution, Val94 or its conserved amino acid substitution, Ser95 or its conserved amino acid substitution, Pro96 or its conserved amino acid substitution, Trp97 or its conserved amino acid substitution, or any combination thereof; HC containing Gly32 or its conserved amino acid substitution, Tyr35 or its conserved amino acid substitution, or any combination thereof CDR1 amino acid sequence; HC CDR2 amino acid sequence containing Tyr52 or its conserved amino acid substitution, Tyr54 or its conserved amino acid substitution, Tyr55 or its conserved amino acid substitution, Ser56 or its conserved amino acid substitution, Ser58 or its conserved amino acid substitution, Thr59 or its conserved amino acid substitution, Phe60 or its conserved amino acid substitution, Pro63 or its conserved amino acid substitution, Lys66 or its conserved amino acid substitution, or any combination thereof; HC CDR3 amino acid sequence containing Arg102 or its conserved amino acid substitution, Asn104 or its conserved amino acid substitution, Trp105 or its conserved amino acid substitution, Asn106 or its conserved amino acid substitution, Tyr107 or its conserved amino acid substitution, or any combination thereof (where the position numbers are relative positions in the LC variable region amino acid sequence of the TIGIT antigen-binding protein). (c) LC CDR1 amino acid sequences including Arg30 or its conserved amino acid substitution, Arg31 or its conserved amino acid substitution, Tyr32 or its conserved amino acid substitution, or any combination thereof; LC sequences including Ser91 or its conserved amino acid substitution, Tyr92 or its conserved amino acid substitution, Ser93 or its conserved amino acid substitution, Thr94 or its conserved amino acid substitution, or any combination thereof CDR3 amino acid sequence (where the position number is the relative position of the LC variable region amino acid sequence of the TIGIT antigen-binding protein); HC CDR1 amino acid sequence containing Thr30 or its conserved amino acid substitution, Gly31 or its conserved amino acid substitution, Tyr32 or its conserved amino acid substitution, Tyr33 or its conserved amino acid substitution, or any combination thereof; HC containing Trp47 or its conserved amino acid substitution, Trp50 or its conserved amino acid substitution, Ser52 or its conserved amino acid substitution, Thr54 or its conserved amino acid substitution, Ser55 or its conserved amino acid substitution, Ala57 or its conserved amino acid substitution, Thr58 or its conserved amino acid substitution, Gly59 or its conserved amino acid substitution, Tyr60 or its conserved amino acid substitution, Gln65 or its conserved amino acid substitution, or any combination thereof CDR2 amino acid sequence; HC CDR3 amino acid sequence containing Asn101 or its conserved amino acid substitution, Ser102 or its conserved amino acid substitution, Val103 or its conserved amino acid substitution, Leu104 or its conserved amino acid substitution, Tyr105 or its conserved amino acid substitution, Tyr106 or its conserved amino acid substitution, Tyr107 or its conserved amino acid substitution, or any combination thereof (where the position numbers are relative positions in the HC variable region amino acid sequence of the TIGIT antigen-binding protein). (d) LC CDR1 amino acid sequences including Gln27 or its conserved amino acid substitution, Leu30 or its conserved amino acid substitution, Ser32 or its conserved amino acid substitution, or any combination thereof; LC sequences including Ser96 or its conserved amino acid substitution, Ile97 or its conserved amino acid substitution, Gln98 or its conserved amino acid substitution, Leu99 or its conserved amino acid substitution, or any combination thereof CDR3 amino acid sequence; HC containing Asp33 or its conserved amino acid substitution; CDR1 amino acid sequence; Tyr52 or its conserved amino acid substitution, Tyr54 or its conserved amino acid substitution, Tyr55 or its conserved amino acid substitution, Ser56 or The TIGIT antigen-binding protein described in item 17, comprising an HC CDR2 amino acid sequence including its conserved amino acid substitution, Gly57 or its conserved amino acid substitution, Gly58 or its conserved amino acid substitution, Thr59 or its conserved amino acid substitution, Tyr60 or its conserved amino acid substitution, Pro63 or its conserved amino acid substitution, Lys66 or its conserved amino acid substitution, or any combination thereof; and an HC CDR3 amino acid sequence including Ile102 or its conserved amino acid substitution, Ala104 or its conserved amino acid substitution, Gly107 or its conserved amino acid substitution, Tyr108 or its conserved amino acid substitution, Phe109 or its conserved amino acid substitution, Tyr110 or its conserved amino acid substitution, Phe111 or its conserved amino acid substitution, or any combination thereof (where the position numbers are relative positions in the HC variable region amino acid sequence of the TIGIT antigen-binding protein). (Item 19) The TIGIT antigen-binding protein described in item 17 or 18, comprising six CDR amino acid sequences listed in a single row in Table A2, or six CDR amino acid sequences selected from the group consisting of (a) SEQ ID NOs. 113-118, (b) SEQ ID NOs. 123-128, (c) SEQ ID NOs. 133-138, (d) SEQ ID NOs. 143-148, (e) SEQ ID NOs. 153-158, (f) SEQ ID NOs. 163-168, (g) SEQ ID NOs. 173-178, (h) SEQ ID NOs. 183-188, (i) SEQ ID NOs. 193-198, (j) SEQ ID NOs. 203-208, (k) SEQ ID NOs. 213-218, (l) SEQ ID NOs. 223-228, and (m) SEQ ID NOs. 2013-2018. (Item 20) (a) The HC variable region amino acid sequence described in Table B2, or a variant sequence thereof that differs from the HC variable region amino acid sequence in Table B2 by only 1 to 15 amino acids, or has at least or about 90% or about 95% sequence identity; (b) The LC variable region amino acid sequence described in Table B2, or a variant sequence thereof that differs from the LC variable region amino acid sequence in Table B2 by only 1 to 15 amino acids, or has at least or about 90% or about 95% sequence identity; or (c) A TIGIT antigen-binding protein described in any one of items 17 to 19, including a combination of (a) and (b). (Item 21) The TIGIT antigen-binding protein described in item 20, comprising a pair of HC variable region and LC variable region amino acid sequences listed in a single row in Table B2, or comprising a pair of amino acid sequences selected from the group consisting of (a) SEQ ID NOs: 111-112, (b) SEQ ID NOs: 121-122, (c) SEQ ID NOs: 131-132, (d) SEQ ID NOs: 141-142, (e) SEQ ID NOs: 151-152, (f) SEQ ID NOs: 161-162, (g) SEQ ID NOs: 171-172, (h) SEQ ID NOs: 181-182, (i) SEQ ID NOs: 191-192, (j) SEQ ID NOs: 201-202, (k) SEQ ID NOs: 211-212, (l) SEQ ID NOs: 221-222, and (m) SEQ ID NOs: 2011-2012. (Item 22) (a) The full-length (FL)HC amino acid sequence described in Table B2, or a variant sequence thereof that differs from the FL HC amino acid sequence in Table B2 by only 1 to 50 amino acids, or has at least or about 90% or about 95% sequence identity; (b) The FL LC amino acid sequence described in Table B2, or a variant sequence thereof that differs from the FL LC amino acid sequence in Table B2 by only 1 to 50 amino acids, or has at least or about 90% or about 95% sequence identity; or (c) A TIGIT antigen-binding protein described in any one of items 17 to 21, including a combination of (a) and (b). (Item 23) Table B2 contains a pair of full-length (FL) HC and FL LC amino acid sequences listed in a single row, or (a) SEQ ID NOs: 109-110, (b) SEQ ID NOs: 119-120, (c) SEQ ID NOs: 129-130, (d) SEQ ID NOs: 139-140, (e) SEQ ID NOs: 149-150, (f) SEQ ID NOs: 159-160, (g) SEQ ID NOs: 169-170, (h) SEQ ID NOs: 1 A TIGIT antigen-binding protein as described in item 22, comprising a pair of amino acid sequences selected from the group consisting of (i) SEQ ID NOs: 79-180, (i) SEQ ID NOs: 189-190, (j) SEQ ID NOs: 199-200, (k) SEQ ID NOs: 209-210, (l) SEQ ID NOs: 219-220, and (m) SEQ ID NOs: 2009-2010. (Item 24) An antibody, specifically a TIGIT antigen-binding protein as described in any one of items 17-23. (Item 25) (a) The heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence of SEQ ID NO: 203, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (b) The HC CDR2 amino acid sequence of SEQ ID NO: 204, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (c) The HC CDR3 amino acid sequence of SEQ ID NO: 205, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (d) The light chain (LC) CDR1 amino acid sequence of SEQ ID NO: 206, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (e) The LC CDR2 amino acid sequence of SEQ ID NO: 207, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (f) The LC CDR1 amino acid sequence of SEQ ID NO: 208 The CDR3 amino acid sequence, or a variant sequence thereof that differs by only 1 to 4 amino acids, or has at least or approximately 90% sequence identity; or (b) the heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence of SEQ ID NO: 223, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (b) the HC CDR2 amino acid sequence of SEQ ID NO: 224, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (c) the HC CDR3 amino acid sequence of SEQ ID NO: 225, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (d) the light chain (LC) CDR1 amino acid sequence of SEQ ID NO: 226, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (e) the LC CDR2 amino acid sequence of SEQ ID NO: 227, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (f) the LC CDR1 amino acid sequence of SEQ ID NO: 228 The CDR3 amino acid sequence, or a variant sequence thereof that differs by only 1 to 4 amino acids, or has at least or approximately 90% sequence identity; or (c)(a) the HC variable region amino acid sequence of SEQ ID NO: 201, or a variant sequence thereof that differs from the HC variable region amino acid sequence of SEQ ID NO: 201 by only 1 to 15 amino acids, or has at least or about 90% or about 95% sequence identity; (b) the LC variable region amino acid sequence of SEQ ID NO: 202, or a variant sequence thereof that differs from the LC variable region amino acid sequence of SEQ ID NO: 202 by only 1 to 15 amino acids, or has at least or about 90% or about 95% sequence identity; or a combination of (c)(a) and (b); or (d)(a) the HC variable region amino acid sequence of SEQ ID NO: 221, or a variant sequence thereof that differs from the HC variable region amino acid sequence of SEQ ID NO: 221 by only 1 to 15 amino acids, or has at least or about 90% or about 95% sequence identity; (b) the LC variable region amino acid sequence of SEQ ID NO: 222, or a variant sequence thereof that differs from the LC variable region amino acid sequence of SEQ ID NO: 222 by only 1 to 15 amino acids, or has at least or about 90% or about 95% sequence identity; or (c) a combination of (a) and (b); or (e)(a) The full-length (FL)HC amino acid sequence of SEQ ID NO: 199, or a variant sequence thereof that differs from the FL HC amino acid sequence of SEQ ID NO: 199 by only 1 to 50 amino acids, or has at least or about 90% or about 95% sequence identity; (b ) The FL LC amino acid sequence described in SEQ ID NO: 200, or a variant sequence thereof that differs from the FL LC amino acid sequence of SEQ ID NO: 200 by only 1 to 50 amino acids, or has at least or about 90% or about 95% sequence identity; or (c) a combination of (a) and (b); or (f)(a) The full-length (FL)HC amino acid sequence of SEQ ID NO: 219, or a variant sequence thereof that differs from the FL HC amino acid sequence of SEQ ID NO: 219 by only 1 to 50 amino acids, or has at least or about 90% or about 95% sequence identity; (b) The FL LC amino acid sequence described in SEQ ID NO: 220, or a variant sequence thereof that differs from the FL LC amino acid sequence of SEQ ID NO: 220 by only 1 to 50 amino acids, or has at least or about 90% or about 95% sequence identity; or (c) A combination of (a) and (b). The TIGIT antigen-binding protein described in item 24, including the TIGIT antigen-binding protein. (Item 26) An antigen-binding fragment of an antibody, a TIGIT antigen-binding protein as described in any one of items 17-23. (Item 27) An antibody protein product, optionally an scFv, which is a TIGIT antigen-binding protein as described in any one of items 17-23. (Item 28) A nucleic acid encoding a TIGIT antigen-binding protein as described in any one of items 17-27. (Item 29) A nucleic acid encoding the light chain, heavy chain, or both the light and heavy chain of an antibody as described in item 24 or 25. (Item 30) The nucleotide sequence is (a) the HC variable region amino acid sequence described in Table B2, or a variant sequence thereof that differs from the HC variable region amino acid sequence in Table B2 by only 1 to 15 amino acids, or has at least or about 90% or about 95% sequence identity; (b) the LC variable region amino acid sequence described in Table B2, or a variant sequence thereof that differs from the LC variable region amino acid sequence in Table B2 by only 1 to 15 amino acids, or has at least or about 90% or about 95% sequence identity; or (c) the nucleic acid described in item 28 or 29 that encodes both (a) and (b). (Item 31) A vector containing one or more nucleic acids as described in any one of items 28-30. (Item 32) A host cell containing one or more nucleic acids as described in any one of items 28-30, or one or more vectors as described in item 31. (Item 33) The host cell is a TIGIT antigen-binding protein as described in any one of items 17 to 27. Host cells that produce nitrite, as described in item 31. (Item 34) A composition comprising the CD112R antigen-binding protein described in any one of items 1 to 10, and the TIGIT antigen-binding protein described in any one of items 17 to 27. (Item 35) (A) The CD112R antigen-binding protein is 1E1, 1E1.016, 24F1, 29E10, 24F1.001, 29E10_CONS.020, 29E10_CONS.021, 29E10_CONS.022, 29E10_CONS.025, 11E4, 31B3, 27G12, 28F9, 28H7, or 36C8, or, at any discretion, 24F1, 29E10_CONS.020, or 29E10_CONS.022, and (B) The TIGIT antigen-binding protein is listed in Table A2 or Table B2. The compositions described in item 34, which are any one of 55G7.041.008, 58A7.003.008.075, 4G10, 11A3, 28B8, 39D2, 43B7, 55G7, 66H9, 43B7.002.015, 58A7.003.08, 66H9.009, or 58A7, or any combination of 43B7.002.015 or 66H9.009, or a combination of (A) and (B). (Item 36) The compositions are (A) 24F1 and 43B7.002.015, (B) 24F1 and 66H9.009, (C) 29E10_CONS.020 and 43B7.002.015, (D) 29E10_CONS.020 and 66H9.009, (E) 29E10_CONS.022 and 43B7.002.015, (F) 29E10_CONS.022 and 66H9.009, (G) The compositions described in item 34 or 35, comprising 43B7.002.015 and 1E1.016, (H)43B7.002.015 and 24F1, (I)43B7.002.015 and 29E10, (J)66H9.009 and 1E1.016, (K)66H9.009 and 29E10, (L)43B7 and 29E10, (M)43B7 and 24F1, or (N)43B7 and 11E4. (Item 37) The composition according to any one of items 34 to 36, wherein the CD112R antigen-binding protein and the TIGIT antigen-binding protein are present in the composition in a ratio of approximately 1:1. (Item 38) A composition according to any one of items 34 to 37, further comprising a PD-1 antigen-binding protein. (Item 39) A kit comprising an antigen-binding protein as described in any one of items 1-10 and 17-27, a nucleic acid as described in any one of items 11-13 and 28-30, a vector as described in item 14 or 31, a host cell as described in any one of items 15, 16, 32 and 33, a composition as described in any one of items 34-38, or a combination thereof, and a container. (Item 40) The kit described in item 39 further contains PD-1 antigen-binding protein. (Item 41) A pharmaceutical composition comprising an antigen-binding protein as described in any one of items 1-10 and 17-27, a nucleic acid as described in any one of items 11-13 and 28-30, a vector as described in item 14 or 31, a host cell as described in any one of items 15, 16, 32 and 33, a composition as described in any one of items 33-36, or a combination thereof, and a pharmaceutically acceptable carrier, excipient or diluent. (Item 42) The pharmaceutical composition according to item 41, further comprising a PD-1 antigen-binding protein. (Item 43) To express the aforementioned CD112R antigen-binding protein, either item 15 or 16 A method for producing a CD112R antigen-binding protein, comprising the steps of culturing the host cells described in paragraph 1, and recovering the expressed CD112R antigen-binding protein. (Item 44) A method for producing a TIGIT antigen-binding protein, comprising the steps of: culturing host cells described in either item 32 or 33 to express the TIGIT antigen-binding protein; and recovering the expressed TIGIT antigen-binding protein. (Item 45) A method for treating a subject in need thereof, comprising administering to the subject in need of the pharmaceutical composition described in item 41 or 42 in an amount effective for treating the subject. (Item 46) The subject has a solid tumor, and the pharmaceutical composition is for treating the solid tumor of the subject. The method according to item 45, administered to the subject in an effective amount. (Item 47) A method for treating a subject in need thereof, comprising administering to the subject in need thereof a first pharmaceutical composition comprising a CD112R antigen-binding protein and a TIGIT antigen-binding protein, and a second pharmaceutical composition comprising a PD-1 inhibitor. (Item 48) The method according to item 47, wherein the subject has a solid tumor, and the first pharmaceutical composition and the second pharmaceutical composition are administered to the subject in an amount effective to treat the solid tumor in the subject. [Brief explanation of the drawing]

[0010] [Figure 1A-1] Figure 1A is a series of plots showing the co-expression profiles of cells for the indicated tumor indicators. The top row shows the co-expression of TIGIT family members with each other and with PD-1. The bottom row shows the co-expression of TIGIT family member ligands with each other and with PD-1. [Figure 1A-2] Figure 1A is a series of plots showing the co-expression profiles of cells for the indicated tumor indicators. The top row shows the co-expression of TIGIT family members with each other and with PD-1. The bottom row shows the co-expression of TIGIT family member ligands with each other and with PD-1. [Figure 1A-3] Figure 1A is a series of plots showing the co-expression profiles of cells for the indicated tumor indicators. The top row shows the co-expression of TIGIT family members with each other and with PD-1. The bottom row shows the co-expression of TIGIT family member ligands with each other and with PD-1. [Figure 1A-4] Figure 1A is a series of plots showing the co-expression profiles of cells for the indicated tumor indicators. The top row shows the co-expression of TIGIT family members with each other and with PD-1. The bottom row shows the co-expression of TIGIT family member ligands with each other and with PD-1. [Figure 1B] Figure 1B shows a series of plots indicating the expression of TIGIT, CD112R, CD226, or PD-1 (data based on single-cell RNA sequence data). [Figure 1C] Figure 1C shows a series of FACS plots illustrating the co-expression of TIGIT, CD112R, and PD-1. [Figure 1D] Figure 1D is a table listing the percentages of CD4 T cells, CD8 T cells, or natural killer (NK) cells that are positive for CD112R, TIGIT, or PD-1 expression in tumor-infiltrating T / NK cells. [Figure 1E] Figure 1E shows a series of plots illustrating the expression of Epcam, CD45, CD112, CD155, CD11c, and CD11b in tumors versus PBMCs. CD112 and CD155 expression, or CD11c and CD11b expression, were evaluated in Epcam+, CD45+Epcam+, or CD45+Epcam- populations. [Figure 2AB] Figure 2A shows the Jurkat reporter gene assay (RGA). The assay system uses modified CHO cells that stably express CD112 and CD3 engagers, as well as purified human pan T cells pre-activated with a CD3 / CD28 antibody. It is expected that when CD112R expressed on the surface of T cells binds to CD112 expressed on the surface of CHO cells, IL-2 release will be suppressed. Figure 2B is a graph showing the increase in CD112R expression in activated T cells (right) compared to unactivated T cells (left). The CD112R staining profile is shown against the isotype control. [Figure 2CD]Figure 2C is a graph plotted as a function of the concentration of tool antibodies (PL-52575, PL-52576, and PL-52577) or human or mouse IgG-corresponding control antibodies (HuIgG isotype and MuIgG isotype, respectively), showing the binding of antibodies or ligands to human CD112R-expressing CHO cells (expressed by GeoMean magnification). Binding of CD112 ligand is also shown. Figure 2D is a graph plotted as a function of the concentration of tool antibodies (PL-52575, PL-52576, and PL-52577) or human or mouse IgG-corresponding control antibodies (HuIgG isotype and MuIgG isotype, respectively), showing the calculated inhibition percentage of binding between ligands expressed on CHO cells and human CD112R. [Figure 2E] Figure 2E is a graph of IL-2 concentration (pg / mL) plotted as a function of the concentration of tool antibodies (PL-52575, PL-52576, and PL-52577) or human or mouse IgG-corresponding control antibodies (HuIgG isotype and MuIgG isotype, respectively) during interaction with CHO cells transfected with an empty vector (Vector) or a vector encoding CD112 (CD112). The EC50 of each tool antibody is shown in the table below the X axis. [Figure 2F] Figure 2F is a graph of IL-2 concentration (pg / mL) plotted as a function of the concentration of CD226 antibody or isotype-corresponding control antibody in an assay co-culturing CHO cells transfected with an empty vector (Vector-CHO) or a vector encoding CD112 (CD112-CHO) with T cells. As an example, T cells without antibody are shown. [Figure 3] Figure 3 is a schematic diagram of the screening cascade used to discover anti-CD112R antagonist antibodies. [Figure 4A]Figure 4A is a schematic diagram of the Jurkat reporter gene assay (RGA). CHO cells expressing the CD3 engager and CD112 are co-cultured with Jurkat T cells expressing the NFAT-luciferase construct and CD112R in the presence of an antibody or control. [Figure 4BC] Figure 4B is a graph of the induction ratio of luciferase activity plotted for harvested samples 6–9. Figure 4C is a graph of binding activity to primary cyno T cells plotted for a 350-hit panel and positive conjugate. [Figure 5] Figure 5A is a table listing the characteristics of harvested crops 1-3, and Figure 5B is a table listing the characteristics of harvested crops 6-9. [Figure 6] Figure 6 is a graph of relative binding activity for CD112R antibodies with an arbitrary cutoff threshold of 100 pM Kd. [Figure 7A] Figure 7A is a graph showing the cladding results illustrating the sequence diversity of antibodies. [Figure 7B] Figure 7B is a table listing exemplary EC50 values ​​for the antibodies shown, as well as germline and HC CDR3 sequence information. [Figure 8] Figure 8 is a graph of the binding inhibition rate % achieved by the indicated antibodies or tool antibodies (PL-52575, PL-52577). Unrelated mouse and human antibodies are used as controls. The table below the X axis lists the EC50 value and maximum inhibition rate % for each tool antibody. [Figure 9]Figure 9A is a graph of the signals between different stages of the competitive assay for three scenarios: A2, B2, and F2, where A2 is the case where two different antibodies are used to determine whether the second antibody competes with the first antibody for ligand binding. B2 is the case where the same antibody is used throughout the assay. F2 is the case where an unrelated control antibody is used. Figure 9B is a table listing the antibodies (Bin A) that compete with each other for ligand binding as determined by the competitive assay. Cultured human T cells and cynoPBMCs were incubated with various concentrations of antibody, starting at 3 μg / mL (assay using human T cells) or 5 μg / mL (assay using cynoPBMCs). The antibody was titrated at 1 / 3 for the minimum concentration of 0.001 μg / mL (in the assay using human T cells) and 0.002 μg / mL (in the assay using cynomolgus monkey PBMCs). GeoMeans were obtained using FCS Express, and the isotype control, titration curve, and EC50 value were determined using Screener. [Figure 10] Figures 10A and 10B are graphs of the magnification ratio relative to the isotype control, plotted as a function of the concentration of the antibody shown. The results of assays using cynoPBMCs and human T cells are shown in Figures 10A and 10B, respectively. The graphs in Figures 10A and 10B plot the magnification ratio relative to the isotype control signal, plotted as a function of the log concentration of the antibody shown. [Figure 11] Figure 11A is a graph of NFAT luciferase activity plotted as a function of the concentration of the antibody shown. Figure 11B is a table listing the EC50s of the antibodies determined by Jurkat RGA. [Figure 12AB] Figure 12A is a graph of the % inhibition of TIGIT binding to CD155-Fc, plotted as a function of TIGIT antibody concentration. Figure 12B is a graph of the % inhibition of binding to CD112-Fc, as a function of antibody concentration. [Figure 12CDE]Figure 12C is an explanatory diagram of a cell assay for testing the activity of the tool TIGIT antibody. Figure 12D is a graph of the binding of CD226-Fc to different cells in Figure 12C, without the antibody. Figure 12E is a graph of the binding of CD226-Fc in the presence of the tool antibody or control antibody at the indicated concentrations. [Figure 12FG] Figure 12F is a graph of the concentration of IFNγ produced by T cells in the presence of the indicated concentrations of the tool antibody or control antibody. Figure 12G is a graph of the concentration of NFAT luciferase activity induced in the presence of the indicated concentrations of the tool antibody or control antibody. [Figure 12HI] Figure 12H is a graph of binding in the presence of the indicated concentrations of the tool antibody or control antibody. 1F4 is a tool antibody such as 10A7 and MBSA43. Figure 12I is a series of plots showing the binding activity of the indicated antibodies against pre-activated primary cyno T cells compared to isotype controls. [Figure 13] Figure 13 is a schematic diagram of the screening assay used to discover anti-TIGIT antibodies. [Figure 14A] Figure 14A is a schematic diagram of the Jurkat reporter gene assay (RGA). CHO cells expressing CD3 engager and CD155 are co-cultured with Jurkat T cells expressing NFAT-luciferase construct and TIGIT in the presence of an antibody or control. [Figure 14B] Figure 14B is a graph of NFAT-luciferase activity induced in the presence of the indicated TIGIT antibody, tool antibody (MBSA43), or control antibody (human IgG4 isotype control, human IgG2 isotype control, mouse IgG1 isotype control). [Figure 14C] Figure 14C is a graph of IFNγ produced by T cells in the presence of the indicated TIGIT antibody, tool antibody (MBSA43), or control antibody (human IgG isotype control, mouse IgG1 isotype control). [Figure 15]Figure 15 is a graph of luciferase activity induced by Jurkat T cells transfected with the IL-2-luciferase report construct and TIGIT in the presence of the indicated TIGIT antibody (AB1 or AB2) or tool antibody (MBSA43). One set of cells was manipulated to knock out CD226 expression (CD226 KO). [Figure 16] Figure 16A is a schematic diagram of an IFNγ release assay using T cells expressing TIGIT, CD226, and CD112R, and CHO cells expressing CD155, CD112, and scFV anti-CD3. Figure 16B is a graph of IFNγ released in the presence of the antibody combinations shown. HuIgG1 and mIgG1 are isotype-corresponding control antibodies. The tool CD112R antibody includes PL-52577. [Figure 17AB] Figures 17A-17C each show graphs of IFNγ released in the presence of two or three antibodies or a single antibody in the indicated combinations. 3× = combination of anti-PD-1, anti-TIGIT, and anti-CD112R antibodies. [Figure 17CD] Figures 17A-17C each show graphs of IFNγ release in the presence of the indicated combinations of two or three antibodies or a single antibody. 3× = combination of anti-PD-1, anti-TIGIT, and anti-CD112R antibodies. Figure 17D is a graph of predicted versus measured results of the IFNγ release assay for the indicated antibody combination or single antibody. [Figure 17EFG] Figure 17E is a graph of IFNγ released in the presence of two or three antibodies or a single antibody in a human tumor tissue-derived cell assay. It shows a comparison of IFNγ concentrations in the supernatant collected on day 3 and day 6. Figure 17F is a graph of the percentage of cells positive for the expression of the indicated molecule in CD4 T cells in PBMCs or tumor cells. Figure 17G is a graph of the percentage of cells positive for the expression of the indicated molecule in CD8 T cells in PBMCs or tumor cells. [Figure 17HI]Figure 17H ​​is a graph of the percentage of cells positive for PD-1 expression on day 6 of tumor tissue-derived cell culture, where cells were treated with anti-TIGIT antibody, anti-CD112R antibody, or isotype control antibody. Figure 17I is a graph of the percentage of cells positive for TIGIT expression on day 6 of tumor tissue-derived cell culture, where cells were treated with anti-PD-1 antibody, anti-CD112R antibody, or isotype control antibody. [Figure 18-1] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-2] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-3] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-4] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-5] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-6] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-7] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-8] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-9] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-10] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-11] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-12] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-13] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-14] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-15] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-16] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-17] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-18] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-19] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-20] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-21] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-22] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18-23] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 18] Figure 18 is a compilation of Tables 2-5 and 12-19 referenced herein. [Figure 19] Figures 19A and 19B are graphs showing the amount of IFN-γ (pg / mL) produced by cytotoxic T lymphocytes when stimulated with formulations containing anti-CD112R mAb (24F1), anti-TIGIT mAb (43B7.002.015), and anti-PD-1 mAb in various ratios, respectively. The total antibody concentrations of the formulations in Figures 19A and 19B are 1.5 nM and 30 nM, respectively. [Figure 20]Figures 20A–20C are graphs of TIGIT, CD112R, and ligand CD155, CD112, and PD-L1 expression in ex vivo primary human tumor tissue and corresponding blood samples, as determined by FACS analysis. Figure 20A graphs the proportion of TIGIT+ and CD112R+ cells on CD8+ T cells and CD3-CD56+ NK cells among CD45+ immune cells derived from tumor tissue, while Figure 20B shows the proportion of TIGIT+ and CD112R+ cells expressed by tumor-infiltrating lymphocytes (TILs) or combined T / NK cells in blood. Figure 20C shows the proportion of CD155-, CD112-, and PD-L1-positive cells in EpcamHI tumor cells, based on ligand expression analysis of a subset of samples. Connecting lines indicate values ​​from individual donors. These data summarize results from tumor tissues for four indications (PANC, CRC, GIST, and TNBC). [Figure 21] Figure 21 is a graph of mean serum mAb concentrations plotted as a function of time after IV administration to male cynomolgus monkeys. The circular lines plot the mean serum concentrations of anti-TIGIT mAb in group 1 animals, the square lines plot the mean serum concentrations of anti-CD112R mAb in group 1 animals, the upward-pointing triangles plot the mean serum concentrations of anti-TIGIT mAb in group 2 animals, and the downward-pointing triangles plot the mean serum concentrations of anti-CD112R mAb in group 3 animals. [Figure 22AB]Figures 22A-22D show that CD112R+TIGIT blockade additively enhances human primary NK cell activity against tumor cells. Figure 22A is a graph of target-positive NK cells % whose target is CD226, TIGIT, CD112R, PD-1, or CD96, showing that purified human NK cells express CD226, TIGIT, and CD112R at high levels and PD-1 and CD96 at low levels. Figure 22B is a graph of ligand-positive cells % expressing CD155, CD112, or PD-L1, showing that the target tumor cells (SKBR3 tumor cell line) used in the assay express CD155 and CD112 at high levels and PD-L1 at low levels. Figure 22C is a graph showing the degree of tumor cell death (compared to isotype control antibodies) by purified human NK cells stimulated with anti-PD-1 antibody, anti-TIGIT antibody, anti-CD112R antibody, or a combination of anti-TIGIT antibody and anti-CD112R antibody, or a combination of anti-PD-1 antibody, anti-TIGIT antibody, and anti-CD112R antibody (3×). Figure 22D is a graph showing the degree of IFNγ production (compared to isotype control antibodies) stimulated by the indicated antibody mixture containing anti-PD-1 antibody, anti-TIGIT antibody, anti-CD112R antibody, or a combination of anti-TIGIT antibody and anti-CD112R antibody, or a combination of anti-PD-1 antibody, anti-TIGIT antibody, and anti-CD112R antibody (3×). Figures 22C and 22D show that individual blockade of TIGIT or CD112R enhanced NK cell activity compared to isotype (HuIgG1) or PD-1 antibody-treated cells, but blockade of both TIGIT and CD112R additively enhanced both tumor cell death and IFNg production at 16 hours. NK cell activity is shown as a fold change relative to the isotype control. Each mAb was added at 10 μg / mL. Single-cell suspensions prepared from dissociated ex vivo tumor tissue were cultured in the presence of the indicated antibody mixtures, including anti-PD-1 antibody, anti-TIGIT antibody, anti-CD112R antibody, or a combination of anti-TIGIT antibody and anti-CD112R antibody, or a combination of anti-PD-1 antibody, anti-TIGIT antibody, and anti-CD112R antibody (10 μg / mL each). T / NK cell activity was measured by IFNg levels in the supernatant on day 3. [Figure 22CD] Same as above. [Figure 23] Figure 23 is a graph of the increase (compared to isotype control antibodies) in the primary TIL response in dissociated ex vivo tumor tissue, showing that a mixture containing all three antibodies targeting CD112R+TIGIT+PD-1 in a 1:1:1 ratio stimulates the highest TIL response. Mean values ​​and error bars showing SEM from five different tissues are shown in this graph. [Figure 24AB] Figures 24A–24C show graphs of tumor growth responses over time in several different mouse tumor models. Figure 24A shows tumor growth in the CT26 syngeneic tumor model, with tumor volume plotted as a function of days after tumor transplantation in wild-type (WT) mice, TIGIT×CD112R KO (double KO) mice, CD112R KO mice, or TIGIT KO mice treated with isotype control or anti-PD1 antibody. The numbered groups correspond to (1) WT mice treated with isotype control antibody, (2) TIGIT×CD112R KO mice treated with isotype control antibody, (3) WT mice treated with anti-PD-1 antibody, (4) CD112R KO mice treated with anti-PD-1 antibody, (5) TIGIT KO mice treated with anti-PD-1 antibody, and (6) TIGIT×CD112R KO mice treated with anti-PD-1 antibody. Figure 24B is a graph of tumor volume in the B16F10 syngeneic tumor model, where the group numbers correspond to (1) WT mice treated with isotype control antibody, (2) WT mice treated with anti-PD-1 antibody, (3) TIGIT×CD112R KO(dKO) mice treated with isotype control antibody, and (4) TIGIT×CD112R KO(dKO) mice treated with anti-PD-1 antibody. Figure 24C is a graph of tumor volume measured over time in xenograft models treated with (1) isotype control antibody, (2) anti-PD-1 antibody, (3) combination formulations containing anti-TIGIT mAb and anti-CD112R mAb, and (4) combination formulations containing anti-TIGIT mAb, anti-CD112R mAb, and anti-PD-1 mAb. [Figure 24C] Same as above. [Figure 25AB] Figures 25A to 25D are graphs plotted as a function of time (weeks) of high molecular weight (HMW) species % after storage at -30°C (Figure 25A), 4°C (Figure 25B), 25°C (Figure 25C), and 40°C (Figure 25D), as measured by SEC, for (1) anti-CD112R mAb (CD112R), (2) anti-TIGIT mAb (TIGIT-10), (3) another anti-TIGIT mAb (TIGIT-12), (4) both anti-CD112R mAb and TIGIT-10 mAb, and (5) formulations containing both anti-CD112R mAb and TIGIT-12. [Figure 25CD] Figures 25A to 25D are graphs plotted as a function of time (weeks) of high molecular weight (HMW) species % after storage at -30°C (Figure 25A), 4°C (Figure 25B), 25°C (Figure 25C), and 40°C (Figure 25D), as measured by SEC, for (1) anti-CD112R mAb (CD112R), (2) anti-TIGIT mAb (TIGIT-10), (3) another anti-TIGIT mAb (TIGIT-12), (4) both anti-CD112R mAb and TIGIT-10 mAb, and (5) formulations containing both anti-CD112R mAb and TIGIT-12. [Figure 25EF] This is Figure 25EF. [Figure 25GH] This is Figure 25GH. [Figure 26] Figure 26 is a table listing the LMW+HMW peak % for formulations containing 70 mg / mL or 140 mg / mL of anti-CD112R mAb, TIGIT-10, or TIGIT-12, or formulations containing both anti-CD112R and TIGIT-10 in a 1:1 ratio, or both anti-CD112R and TIGIT-12 in a 1:1 ratio. [Figure 27] Figure 27 is a graph of viscosity (cP) for formulations containing 70 mg / mL or 140 mg / mL of anti-CD112R mAb, TIGIT-10, or TIGIT-12, or formulations containing both anti-CD112R and TIGIT-10 in a 1:1 ratio, or both anti-CD112R and TIGIT-12 in a 1:1 ratio. [Modes for carrying out the invention]

[0011] TIGIT and CD112R (also known as PVRIG) belong to a family of receptors that contain an immunoglobulin (Ig) domain in their extracellular region. These receptors also interact with ligands that contain an Ig domain. While there is evidence that each receptor can interact with multiple ligands within the family, TIGIT-CD155 and CD112R-CD112 represent primary receptor-ligand pairings based on affinity measurements. Another family member, CD226, can interact with both CD112 and CD155, both with weaker affinities than TIGIT-CD155 and CD112R-CD112. CD226 binding specifically enhances T / NK cell activity in the context of the T / NK cell response to tumor cells. This "co-stimulatory" signal is thought to be inhibited when TIGIT and CD112R are co-expressed at high levels, for the following reasons: 1) Both TIGIT and CD112R bind to their ligands with higher affinity than CD226, effectively restricting ligand access; and 2) the intracellular domains of TIGIT and CD112R contain ITIM or ITIM-like domains that are thought to generate inhibitory signals, but the nature of such signals is not broadly characterized in T cells.

[0012] Transcripts of CD155 and CD112, the primary ligands for TIGIT and CD112R, are present in a wide range of tissues and cell types. This contrasts with PD-L1, whose expression is more localized and preferentially expressed in antigen-presenting cells and tumor cells. These ligands have been shown to be induced by different types of stimuli; PD-L1 is upregulated by exposure to IFNγ, while CD155 and CD112 are not regulated by exposure to this cytokine, but are instead upregulated in response to DNA damage, viral infection, and reactive oxygen species (ROS). Thus, ligand-induced responses further distinguish between pathways involving TIGIT and CD112R and those involving PD-1.

[0013] The present disclosure provides antigen-binding proteins that bind to TIGIT, such as antibodies and antigen-binding fragments thereof, such as TIGIT-binding proteins (also referred to herein as TIGIT antigen-binding proteins). In preferred embodiments, the TIGIT antigen-binding protein is an antibody that specifically binds to TIGIT (e.g., a TIGIT antibody, an α-TIGIT antibody).

[0014] <00​​​​​​​​​​​​​​​​​​​​​​​​This is micromolar, nanomolar, picomolar, or femtomole. In exemplary embodiments, the K of the TIGIT antigen-binding protein or CD112R antigen-binding protein provided herein. D It is about 10 -4 ~10 -6 M, or 10 -7 ~10 -9 M, or 10 -10 ~10 -12 M, or 10 -13 ~10 -15 It is within the range.Optionally, the K of the TIGIT antigen-binding protein or CD112R antigen-binding protein provided herein. D It is about 10 -12 ~10 -8 M, 10 (optional selection) -11 ~10 -10 It is within the range of M.

[0016] In various embodiments, an antigen-binding protein, such as an antibody, binds to human TIGIT. The amino acid sequence of human TIGIT is provided herein as SEQ ID NO: 1. In particular, amino acids 1-21 of SEQ ID NO: 1 represent a signal peptide, and amino acids 22-244 of SEQ ID NO: 1 represent the mature human TIGIT amino acid sequence. In exemplary embodiments, the antigen-binding protein has a K content of about 50 nM or less (e.g., about 40 nM or less, about 30 nM or less, about 20 nM or less, or about 10 nM or less). D It binds to human TIGIT. In an exemplary embodiment, the antigen-binding protein has a K content of approximately 5 nM or less, approximately 4 nM or less, approximately 3 nM or less, approximately 2 nM or less, or approximately 1 nM or less. D It binds to human TIGIT. In various embodiments, the K of the antigen-binding protein to human TIGIT D The K of the antigen-binding protein for human TIGIT is less than 1 nM, for example, less than 0.75 nM, less than 0.5 nM, or less than 0.25 nM. Optionally, the K of the antigen-binding protein for human TIGIT is selected. D The K of the antigen-binding protein for human TIGIT is approximately 0.001 nM or greater, or approximately 0.01 nM or greater and less than 0.5 nM. In various embodiments, the K of the antigen-binding protein for human TIGIT is DThese ranges from approximately 0.01 nM to approximately 0.5 nM, approximately 0.02 nM to approximately 0.5 nM, approximately 0.03 nM to approximately 0.5 nM, approximately 0.04 nM to approximately 0.5 nM, approximately 0.05 nM to approximately 0.5 nM, approximately 0.06 nM to approximately 0.5 nM, approximately 0.07 nM to approximately 0.5 nM, approximately 0.08 nM to approximately 0.5 nM, approximately 0.09 nM to approximately 0.5 nM, approximately 0.1 nM to approximately 0.5 nM, approximately 0.2 nM to approximately 0.5 nM, approximately 0.3 nM to approximately 0.5 nM, approximately 0.4 nM to approximately 0.5 nM, The chromosome levels are approximately 0.01 nM to 0.4 nM, 0.01 nM to 0.3 nM, 0.01 nM to 0.2 nM, 0.01 nM to 0.1 nM, 0.01 nM to 0.09 nM, 0.01 nM to 0.08 nM, 0.01 nM to 0.07 nM, 0.01 nM to 0.06 nM, 0.01 nM to 0.05 nM, 0.01 nM to 0.04 nM, 0.01 nM to 0.03 nM, or 0.01 nM to 0.02 nM. In various embodiments, the antigen-binding protein also binds to cynomolgus monkey (cyno)TIGIT. The amino acid sequence of cynoTIGIT is provided herein as Sequence ID No. 2024. In particular, amino acids 1-21 of SEQ ID NO: 2024 represent the signal peptide, and amino acids 22-245 of SEQ ID NO: 2024 represent the mature cynoTIGIT protein. In exemplary embodiments, the antigen-binding protein has a K content of approximately 1 nM to approximately 25 nM, for example, approximately 5 nM to approximately 20 nM, or approximately 5 nM to approximately 15 nM. D It binds to TIGIT in cynomolgus monkeys. In an exemplary embodiment, the antigen-binding protein has a K content of approximately 8 nM to approximately 14 nM. D It binds to cynoTIGIT. In various embodiments, the antigen-binding protein binds with high affinity to both human TIGIT and cynoTIGIT. By choice, the K of the antigen-binding protein to human TIGIT is selected. D The K of the antigen-binding protein for cynoTIGIT is approximately 0.01 nM and less than 0.5 nM. D The K of the antigen-binding protein for human TIGIT is approximately 8 nM to 14 nM. In an example, the K of the antigen-binding protein for human TIGIT is approximately 8 nM to 14 nM. D This is the K of the antigen-binding protein for cynoTIGIT. DThese values ​​are approximately 100 times, 50 times, 25 times, 10 times, 5 times, or 2 times or less. In various embodiments, the EC50 value of TIGIT antigen-binding protein against human T cells expressing human TIGIT is approximately 100 times, 50 times, 25 times, 10 times, 5 times, or 2 times or less the EC50 value of TIGIT antigen-binding protein against cynoPBMCs expressing cynoTIGIT.

[0017] In various examples, antigen-binding proteins, such as antibodies, bind to human CD112R. The amino acid sequence of human CD112R is provided herein as Sequence ID No. 3. In particular, amino acid 53 of Sequence ID No. 3 represents the first amino acid of the extracellular domain. In exemplary embodiments, the antigen-binding protein binds to both human CD112R and cynoCD112R with high affinity. In exemplary embodiments, the antigen-binding protein has a K content of about 50 nM or less (e.g., about 40 nM or less, about 30 nM or less, about 20 nM or less, or about 10 nM or less). D It binds to human CD112R. In an exemplary embodiment, the antigen-binding protein has a K content of approximately 5 nM or less, approximately 4 nM or less, approximately 3 nM or less, approximately 2 nM or less, or approximately 1 nM or less. D It binds to human CD112R. In various embodiments, the antigen-binding protein K to human CD112R D The K of the antigen-binding protein for human CD112R is less than 1 nM, for example, less than 0.75 nM, less than 0.5 nM, or less than 0.25 nM. Optionally, the K of the antigen-binding protein for human CD112R is selected. D It is approximately 0.001 nM or greater, or approximately 0.01 nM or greater and less than 3 nM. In various embodiments, the K of the antigen-binding protein to human CD112R DThese ranges are approximately 0.01 nM to 5 nm, approximately 0.05 nM to 5 nm, approximately 0.10 nM to 5 nm, approximately 0.5 nM to 5 nm, approximately 1 nM to 5 nm, approximately 2 nM to 5 nm, approximately 3 nM to 5 nm, approximately 4 nM to 5 nm, approximately 0.01 nM to 4 nm, approximately 0.01 nM to 3 nm, approximately 0.01 nM to 2 nm, approximately 0.01 nM to 1 nm, approximately 0.01 nM to 0.5 nm, approximately 0.01 nM to 0.1 nm, or approximately 0.01 nM to 0.05 nm. In various embodiments, the antigen-binding protein binds to cynoCD112R. The amino acid sequence of cynoCD112R is provided herein as Sequence ID No. 2022, of which amino acid 53 is the first amino acid of the extracellular domain. In an exemplary embodiment, the antigen-binding protein has a K content of approximately 1 nM to approximately 25 nM, for example, approximately 5 nM to approximately 20 nM, or approximately 5 nM to approximately 15 nM. D It binds to cynomolgus monkey (cyno) CD112R. In an exemplary embodiment, the antigen-binding protein has a K content of approximately 0.05 nM to approximately 0.15 nM. D It binds to cynoCD112R. By choice, the K of the antigen-binding protein for human CD112R is selected. D The K of the antigen-binding protein for cynoCD112R is approximately 0.01 nM and less than 5 nM. D The magnitude is approximately 0.05 nM to approximately 0.15 nM.

[0018] In the example, the K of the antigen-binding protein for human CD112R D This is the K of the antigen-binding protein for cynoCD112R. D The EC50 values ​​of the CD112R antigen-binding protein against human T cells expressing human CD112R are approximately 100 times, 50 times, 25 times, 10 times, 5 times, or 2 times or less than the EC50 values ​​of the CD112R antigen-binding protein against cynoPBMCs expressing cynoCD112R.

[0019] In exemplary embodiments, an antigen-binding protein, such as an antibody, exhibits a binding affinity for its target (TIGIT or CD112R), and this affinity is elevated compared to the binding affinity of the innate interaction between TIGIT and CD155, TIGIT and CD112, or CD112R and CD112. The increase in binding affinity may be at least or about 5%, at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, or at least or about 95% compared to the binding affinity of human TIGIT to its ligand (CD155) or human CD112R to its ligand (CD112). In exemplary embodiments, the antigen-binding protein has approximately 2 times, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100 times, 105 times, 110 times, 115 times, 120 times, 125 times, 130 times, 135 times, 140 times, 145 times, 1 It shows an increase in binding affinity to its target (TIGIT or CD112R) of 50 times, 175 times, 200 times, 225 times, 250 times, 275 times, 300 times, 325 times, 350 times, 375 times, 400 times, 425 times, 450 times, 475 times, 500 times, 525 times, 550 times, 575 times, 600 times, 625 times, 650 times, 675 times, 700 times, 725 times, 750 times, 775 times, 800 times, 825 times, 850 times, 875 times, 900 times, 925 times, 950 times, 975 times, and 1000 times or more.

[0020] Competitive assay In various embodiments, antigen-binding proteins, such as antibodies, inhibit the binding interaction between human TIGIT and a reference antibody. This reference antibody is known to bind to TIGIT but is not the antigen-binding protein of this disclosure. In various examples, the TIGIT-binding protein of this disclosure competes with the reference antibody for binding to human TIGIT, thereby reducing the amount of human TIGIT that binds to the reference antibody, as determined by an in vitro competitive binding assay. In various embodiments, the antigen-binding protein of this disclosure inhibits the binding interaction between human TIGIT and a reference antibody, and this inhibition is IC. 50 Characterized by: In various embodiments, antigen-binding proteins exhibit an IC50 of less than approximately 250 nM in relation to the inhibition of the binding interaction between human TIGIT and the reference antibody. 50 This indicates that, in various aspects, antigen-binding proteins have ICs of less than approximately 200 nM, less than approximately 150 nM, less than approximately 100 nM, less than approximately 90 nm, less than approximately 80 nm, less than approximately 70 nm, less than approximately 60 nm, less than approximately 50 nm, less than approximately 40 nm, less than approximately 30 nm, less than approximately 20 nm, or less than approximately 10 nm. 50 This indicates that, in various aspects, the antigen-binding protein has an IC of less than approximately 9 nM, less than approximately 8 nM, less than approximately 7 nM, less than approximately 6 nM, less than approximately 5 nM, less than approximately 4 nM, less than approximately 3 nM, less than approximately 2 nM, less than approximately 1 nM, less than approximately 0.5 nM, or less than approximately 0.1 nM. 50This illustrates the following. In various examples, the antigen-binding protein of this disclosure competes with a reference antibody for binding to human TIGIT, thereby reducing the amount of human TIGIT bound to the reference antibody, as determined by a FACS-based assay. In this assay, the fluorescence of a fluorophore-conjugated secondary antibody bound to the Fc of the reference antibody is measured in the absence or presence of a specific amount of the antigen-binding protein of this disclosure. In various embodiments, the FACS-based assay is performed using a reference antibody, a fluorophore-conjugated secondary antibody, and cells expressing TIGIT. In various embodiments, cells are genetically engineered to overexpress TIGIT. In some embodiments, the cells are HEK293T cells transduced with a viral vector to express TIGIT. In other embodiments, the cells endogenously express TIGIT. Before performing the FACS-based assay, in some embodiments, the endogenously expressing TIGIT cells are pre-determined as low-TIGIT-expressing cells or high-TIGIT-expressing cells.

[0021] In exemplary embodiments, antigen-binding proteins, such as antibodies, inhibit the binding interaction between human TIGIT and its intrinsic ligands, such as CD155 and CD112. In various examples, antigen-binding proteins, such as antibodies, inhibit the binding interaction between human TIGIT and CD155, as determined by a FACS-based receptor-ligand competitive binding assay, such as the assay described in Example 5 of this specification. In various embodiments, more than 80% (e.g., more than 85%, more than 90%) of the binding interaction between human TIGIT and CD155 is inhibited in the presence of the antigen-binding proteins of this disclosure, such as antibodies. Optionally, more than 95% (e.g., more than 96%, more than 97%, more than 98%, more than 99%, or approximately 100%) of the binding interaction between human TIGIT and CD155, as determined by a FACS-based receptor-ligand competitive binding assay, such as the assay described in Example 5 of this specification, is inhibited in the presence of the antigen-binding proteins of this disclosure, such as antibodies.

[0022] In various embodiments, antigen-binding proteins, such as antibodies, inhibit the binding interaction between human CD112R and a reference antibody. This reference antibody is known to bind to CD112R but is not the antigen-binding protein of this disclosure. In various examples, the CD112R-binding protein of this disclosure competes with the reference antibody for binding to human CD112R, thereby reducing the amount of human CD112R that binds to the reference antibody, as determined by an in vitro competitive binding assay. In various embodiments, the antigen-binding protein of this disclosure inhibits the binding interaction between human CD112R and the reference antibody, and this inhibition is IC 50 Characterized by: In various embodiments, antigen-binding proteins exhibit an IC50 of less than 250 nM in relation to the inhibition of the binding interaction between human CD112R and the reference antibody. 50 This indicates that, in various aspects, antigen-binding proteins have ICs of less than approximately 200 nM, less than approximately 150 nM, less than approximately 100 nM, less than approximately 90 nm, less than approximately 80 nm, less than approximately 70 nm, less than approximately 60 nm, less than approximately 50 nm, less than approximately 40 nm, less than approximately 30 nm, less than approximately 20 nm, or less than approximately 10 nm. 50 This indicates that, in various aspects, the antigen-binding protein has an IC of less than approximately 9 nM, less than approximately 8 nM, less than approximately 7 nM, less than approximately 6 nM, less than approximately 5 nM, less than approximately 4 nM, less than approximately 3 nM, less than approximately 2 nM, less than approximately 1 nM, less than approximately 0.5 nM, or less than approximately 0.1 nM. 50 This shows that, by arbitrary selection, antigen-binding proteins have IC50s of approximately 0.05 nM to 0.5 nM (e.g., approximately 0.06 nM, 0.07 nM, 0.08 nM, 0.09 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM). 50This illustrates the following. In various examples, the antigen-binding protein of this disclosure competes with a reference antibody for binding to human CD112R, thereby reducing the amount of human CD112R bound to the reference antibody, as determined by a FACS-based assay. In this assay, the fluorescence of a fluorophore-conjugated secondary antibody bound to the Fc of the reference antibody is measured in the absence or presence of a specific amount of the antigen-binding protein of this disclosure. In various embodiments, the FACS-based assay is performed using a reference antibody, a fluorophore-conjugated secondary antibody, and cells expressing CD112R. In various embodiments, cells are genetically engineered to overexpress CD112R. In some embodiments, the cells are HEK293T cells transduced with a viral vector to express CD112R. In other embodiments, the cells endogenously express CD112R. Before performing the FACS-based assay, in some embodiments, the endogenously expressing CD112R cells are pre-determined as low-CD112R-expressing cells or high-CD112R-expressing cells.

[0023] In exemplary embodiments, antigen-binding proteins, such as antibodies, inhibit the binding interaction between human CD112R and its innate ligand, such as CD112. In various examples, antigen-binding proteins, such as antibodies, inhibit the binding interaction between human CD112R and CD112 as determined by a FACS-based receptor-ligand competitive binding assay, such as the assay described in Example 3 of this specification. In various embodiments, more than 90% of the binding interaction between human CD112R and CD112 is inhibited in the presence of the antigen-binding protein of this disclosure, such as antibodies. Optionally, more than 95% (e.g., more than 96%, more than 97%, more than 98%, more than 99%, or approximately 100%) of the binding interaction between human CD112R and CD112 as determined by a FACS-based receptor-ligand competitive binding assay, such as the assay described in Example 3 of this specification, is inhibited in the presence of the antigen-binding protein of this disclosure, such as antibodies.

[0024] Other binding assays, such as competitive binding assays or competitive assays that test the ability of an antibody to compete with another antigen-binding protein for binding to an antigen or its epitope, are known in the art. For example, see Trikha et al., Int J Cancer 110:326-335 (2004); Tam et al., Circulation 98(11):1085-1091 (1998); U.S. Patent Application Publication No. 2014 / 0178905; Chand et al., Biologicals 46:168-171 (2017); Liu et al., Anal Biochem 525:89-91 (2017); Goolia et al., J Vet Diagn Invest 29(2):250-253 (2017); Hunter and Cochran, Methods Enzymol 250:21-44 (2016); Cox et al., Immunoassay Methods, Immunoassay Methods. 2012 May 1 [Updated 2019 Jul 8]. In: Sittampalam GS, Grossman A, Brimacombe K, et al., editors. Assay Guidance Manual [Internet]. Bethesda(MD):Eli Lilly & Company and the National Center for Advancing Translational Sciences;2004-. Source: https: / / www.ncbi.nlm.nih.gov / books / NBK92434 / ; Clarke, William, “Immunoassays for Therapeutic Drug Monitoring and Clinical Toxicology”, Handbook of Analytical Separations, Volume 5, pages 95-112 (2004), and Goolia et al., J Vet Diagn Invest 29(2):250-253 (2017). Other methods for comparing two antibodies are also known in the art, including, for example, surface plasmon resonance (SPR). SPR can be used to determine the binding constants of one antibody and a second antibody, and the two binding constants can be compared.

[0025] Inhibitory and antagonistic effects In various embodiments, antigen-binding proteins, such as antibodies, bind to their target or antigen and inhibit the binding interaction between this target or antigen and its native ligand or binding partner. In exemplary embodiments, the TIGIT-binding protein of this disclosure binds to TIGIT, thereby inhibiting the binding interaction between TIGIT and CD155. In exemplary embodiments, the TIGIT-binding protein of this disclosure may, or may not, inhibit the binding interaction between TIGIT and another ligand (e.g., CD112). In exemplary embodiments, the CD112R-binding protein of this disclosure binds to CD112R, thereby inhibiting the binding interaction between CD112R and CD112. In exemplary embodiments, the CD112R-binding protein of this disclosure may, or may not, inhibit the binding interaction between CD112R and other ligands (e.g., CD96, CD226, TIGIT). In various embodiments, antigen-binding proteins, such as antibodies, are antagonists that inhibit the biological activity of a target or antigen. In various embodiments, CD112R-binding proteins bind to CD112R and inhibit signaling pathways activated when CD112 binds to CD112R. In various embodiments, TIGIT-binding proteins bind to TIGIT and inhibit signaling pathways activated when CD155 binds to TIGIT. Further signaling pathways may be inhibited when TIGIT-binding proteins bind to TIGIT, or when CD112R-binding proteins bind to CD112R.

[0026] The reduction or inhibition provided by antigen-binding proteins, such as antibodies, may not be 100% or complete inhibition, suppression, or reduction. Rather, there are various degrees of reduction or inhibition that a person skilled in the art would recognize as having a potential benefit or therapeutic effect. In this regard, antigen-binding proteins may inhibit the TIGIT and / or CD112R proteins to any amount or level. In exemplary embodiments, the reduction or inhibition provided by antigen-binding proteins is at least or about 10% reduction or inhibition (e.g., at least or about 20% reduction or inhibition, at least or about 30% reduction or inhibition, at least or about 40% reduction or inhibition, at least or about 50% reduction or inhibition, at least or about 60% reduction or inhibition, at least or about 70% reduction or inhibition, at least or about 80% reduction or inhibition, at least or about 90% reduction or inhibition, at least or about 95% reduction or inhibition, at least or about 98% reduction or inhibition).

[0027] In exemplary examples, the antigen-binding proteins of this disclosure inhibit the binding of CD112 to CD112R or the binding of TIGIT to CD155. In exemplary embodiments, inhibition may be characterized with respect to the maximum inhibitory concentration (IC50), which is a measure of the effectiveness of the antigen-binding protein in inhibiting a specific biological or biochemical function.

[0028] Methods suitable for measuring the inhibitory or antagonistic activity of the antigen-binding proteins of this disclosure are known in the art. In exemplary cases, the antagonistic or inhibitory activity of an antigen-binding protein is such that the co-binding of CD112R to CD112 and / or TIGIT to CD155, and the activation of the T cell receptor (TCR) on T cells, produce inhibitory signals that inactivate or block the TCR-mediated response, and thus block the interaction between CD112R and CD112 and / or between TIGIT and CD155 during TCR ligation, resulting in phosphorylation of TCR subunits, recruitment of Zap70 to TCR, LAT and / or Assuming that this leads to TCR-mediated activity including one or more of the following: SLP-76 phosphorylation, calcium mobilization or release from the endoplasmic reticulum (ER), PLC-γ activation, diacylglycerol (DAG) and inositol triphosphate (IP3) production, protein kinase C activation, MARPK / Erk signaling, NF-κB activation, NFAT activation, IL-2 promoter activation, IL-2 production, IFN-gamma production, and T cell proliferation, it can be assayed by measuring the level of TCR activation. See, for example, Smith-Garvin et al., Annu Rev Immunol 27:591-619 (2009). Thus, in various examples, the inhibitory or antagonistic activity of the antigen-binding proteins of this disclosure can be assayed by measuring, for example, IL-2 production, IFN-gamma production, and / or NF-κB and / or NFAT activation. In various forms, a luciferase reporter gene assay using Jurkat T cells is employed, in which luciferase activity is measured in the presence of TIGIT-binding protein and / or CD112R-binding protein upon TCR activation. In the presence of TIGIT-binding protein and / or CD112R-binding protein, luciferase activity is expected to be higher than that observed in the absence of TIGIT-binding protein and / or CD112R-binding protein. RGA is described in the examples herein (see, for example, Examples 3 and 5). In various embodiments, the antagonist activity of TIGIT-binding proteins and / or CD112R-binding proteins may be measured by receptor-ligand binding assays or Jurkat RGA. In various embodiments, the antagonist or inhibitory activity of antigen-binding proteins of this disclosure may be measured by Jurkat RGA, and the activity is measured by EC 50 It is expressed as follows: In various examples, the EC of CD112R antigen-binding protein or TIGIT antigen-binding protein 50 These ranges from approximately 0.01nM to 10nM, 0.01nM to 9nM, 0.01nM to 8nM, 0.01nM to 7nM, 0.01nM to 6nM, 0.01nM to 5nM, 0.01nM to 4nM, 0.01nM to 3nM, 0.01nM to 2nM, 0.01nM to 1nM, 0.01nM to 0.5nM, and 0.01nM to 0.1n M, approximately 0.01 nM to 0.05 nM, approximately 0.05 nM to 10 nM, approximately 0.1 nM to 10 nM, approximately 0.5 nM to 10 nM, approximately 1 nM to 10 nM, approximately 2 nM to 10 nM, approximately 3 nM to 10 nM, approximately 4 nM to 10 nM, approximately 5 nM to 10 nM, approximately 6 nM to 10 nM, approximately 7 nM to 10 nM, approximately 8 nM to 10 nM, or approximately 9 nM to 10 nM. By arbitrary selection, the CD112R antigen-binding protein in Jurkat RGA is as described above and / or as described in Figure 7B or Tables 2-5. 50 This indicates that, by choice, the TIGIT antigen-binding protein in Jurkat RGA is as described above and / or as described in Tables 13-15. 50 This indicates.

[0029] CD112R antigen-binding protein IC 50 In exemplary embodiments, the IC of the CD112R antigen-binding protein is less than approximately 10 nM, and optionally less than 5 nM. In exemplary embodiments, the IC of the CD112R antigen-binding protein is less than approximately 10 nM. 50 The IC of the CD112R antigen-binding protein is less than 2 nM or less than 1 nM. In an exemplary embodiment, the IC of the CD112R antigen-binding protein is less than 2 nM. 50The concentration is approximately 0.5 nM to 2 nM. In various cases, the IC of the CD112R antigen-binding protein 50 These ranges from approximately 0.01nM to 10nM, 0.01nM to 9nM, 0.01nM to 8nM, 0.01nM to 7nM, 0.01nM to 6nM, 0.01nM to 5nM, 0.01nM to 4nM, 0.01nM to 3nM, 0.01nM to 2nM, 0.01nM to 1nM, 0.01nM to 0.5nM, and 0.01nM to 0.1n The IC values ​​are approximately M, 0.01 nM to 0.05 nM, 0.05 nM to 10 nM, 0.1 nM to 10 nM, 0.5 nM to 10 nM, 1 nM to 10 nM, 2 nM to 10 nM, 3 nM to 10 nM, 4 nM to 10 nM, 5 nM to 10 nM, 6 nM to 10 nM, 7 nM to 10 nM, 8 nM to 10 nM, or 9 nM to 10 nM. In various embodiments, the IC of the CD112R antigen-binding protein 50 This is a measure of the effectiveness of the CD112R antigen-binding protein in inhibiting the binding interaction between CD112R and CD112, as determined by receptor-ligand binding assays. See, for example, Example 3.

[0030] IC of TIGIT antigen-binding protein 50 In exemplary embodiments, the IC of the TIGIT antigen-binding protein is less than approximately 10 nM, and optionally less than 5 nM. In exemplary embodiments, the IC of the TIGIT antigen-binding protein is less than approximately 10 nM. 50 The IC of the TIGIT antigen-binding protein is less than 2 nM or less than 1 nM. In an exemplary embodiment, the IC of the TIGIT antigen-binding protein is less than 2 nM. 50 The concentration is approximately 0.5 nM to 2 nM. In various cases, the IC of TIGIT antigen-binding protein 50These ranges from approximately 0.01nM to 10nM, 0.01nM to 9nM, 0.01nM to 8nM, 0.01nM to 7nM, 0.01nM to 6nM, 0.01nM to 5nM, 0.01nM to 4nM, 0.01nM to 3nM, 0.01nM to 2nM, 0.01nM to 1nM, 0.01nM to 0.5nM, and 0.01nM to 0.1n The IC values ​​are approximately M, 0.01 nM to 0.05 nM, 0.05 nM to 10 nM, 0.1 nM to 10 nM, 0.5 nM to 10 nM, 1 nM to 10 nM, 2 nM to 10 nM, 3 nM to 10 nM, 4 nM to 10 nM, 5 nM to 10 nM, 6 nM to 10 nM, 7 nM to 10 nM, 8 nM to 10 nM, or 9 nM to 10 nM. In various embodiments, the IC of TIGIT antigen-binding protein 50 This is a measure of the effectiveness of the TIGIT antigen-binding protein in inhibiting the binding interaction between TIGIT and CD155 or CD112, as determined by receptor-ligand binding assays. See, for example, Example 5.

[0031] Type of antigen-binding protein The antigen-binding protein of this disclosure can take any form of many of the antigen-binding proteins known in the art. In exemplary embodiments, the antigen-binding protein is an antibody or immunoglobulin, or an antigen-binding antibody fragment thereof, or an antibody protein product.

[0032] Collectively, antibodies form a family of plasma proteins known as immunoglobulins, which contain an immunoglobulin domain. (Janeway et al., Immunobiology: The Immune System in Health and Disease, 4) thed., Elsevier Science Ltd. / Garland Publishing, 1999. As used herein, the term “antibody” refers to a protein having a conventional immunoglobulin form, comprising heavy and light chains, and including a variable region and a constant region. For example, an antibody may be an IgG, which has a “Y-shaped” structure with two pairs of identical polypeptide chains, each pair having one “light” chain (typically with a molecular weight of about 25 kDa) and one “heavy” chain (typically with a molecular weight of about 50–70 kDa). Antibodies have a variable region and a constant region. In the IgG form, the variable region is generally about 100–110 or more amino acids, includes three complementarity-determining regions (CDRs), and is primarily involved in antigen recognition, substantially different from other antibodies that bind to different antigens. The constant region allows the antibody to recruit cells and molecules of the immune system. The variable region is made up of the N-terminal regions of each light and heavy chain, while the constant region is made up of the C-terminal portions of the heavy and light chains, respectively. (Janeway et al., “Structure of the Antibody Molecule and the Immunoglobulin Genes”, Immunobiology: The Immune System in Health and Disease, 4 th ed.Elsevier Science Ltd. / Garland Publishing, (1999)).

[0033] The general structure and properties of antibody CDRs are described in the art. In short, within the antibody backbone, CDRs are embedded within the framework of the variable regions of the heavy and light chains, where they constitute regions that play a significant role in antigen binding and recognition. The variable region typically contains at least three heavy chain or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service (NIH, Bethesda, Md.; Chothia and Lesk, 1987, J.Mol.Biol.196:901-917; see also Chothia et al., 1989, Nature 342:877-883), these fall within the framework areas (referred to as framework areas 1-4, FR1, FR2, FR3, and FR4, by Kabat et al., 1991; see also Chothia and Lesk, 1987, op. cit.).

[0034] Antibodies may include any constant region known in the art. Human light chains are classified into kappa and lambda light chains. Heavy chains are classified into mu, delta, gamma, alpha, or epsilon, defining antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but is not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but is not limited to, IgM1 and IgM2. Embodiments of this disclosure include all such classes or isotypes of antibodies. Light chain constant regions may be, for example, kappa or lambda light chain constant regions, e.g., human kappa or lambda light chain constant regions. Heavy chain constant regions may be, for example, alpha, delta, epsilon, gamma, or mu heavy chain constant regions, e.g., human alpha, delta, epsilon, gamma, or mu heavy chain constant regions. Therefore, in the exemplary embodiment, the antibody is an isotype IgA, IgD, IgE, IgG, or IgM antibody containing one of IgG1, IgG2, IgG3, or IgG4.

[0035] Antibodies can be monoclonal or polyclonal antibodies. In some embodiments, antibodies contain sequences substantially similar to naturally occurring antibodies produced by mammals, such as mice, rabbits, goats, horses, chickens, hamsters, and humans. In this respect, antibodies can be considered mammalian antibodies, such as mouse antibodies, rabbit antibodies, goat antibodies, horse antibodies, chicken antibodies, hamster antibodies, and human antibodies. In certain embodiments, antibodies are human antibodies. In certain embodiments, antibodies are chimeric antibodies or humanized antibodies. The term "chimeric antibody" refers to an antibody that contains domains derived from two or more different antibodies. A chimeric antibody may, for example, contain a constant domain from one species and a variable domain from another species, or more generally, a stretch of amino acid sequences from at least two species. A chimeric antibody may also contain domains from two or more different antibodies within the same species. When used in relation to antibodies, the term "humanization" refers to an antibody of non-human origin that has at least a CDR region and has been modified to have a structure and immune function more similar to a true human antibody than the original source antibody. For example, humanization may involve transplanting a CDR from a non-human antibody, such as a mouse antibody, to a human antibody. Humanization may also involve selective amino acid substitutions to make the non-human sequence more similar to a human sequence.

[0036] Antibodies can be cleaved into fragments by enzymes such as papain and pepsin. Papain cleaves the antibody to produce two Fab fragments and one Fc fragment. Pepsin cleaves the antibody to produce an F(ab')2 fragment and a pFc' fragment. In exemplary embodiments of this disclosure, the antigen-binding protein of this disclosure comprises an antigen-binding antibody fragment. As used herein, the term “antigen-binding antibody fragment” refers to the portion of the antibody molecule that can bind to an antigen, and is also known as “antigen-binding fragment” or “antigen-binding moiety.” In exemplary examples, the antigen-binding antibody fragment is a Fab fragment or an F(ab')2 fragment.

[0037] Antibody structures are used to create a wide range of alternative forms, which have monomeric (n=1), dimeric (n=2), trimeric (n=3), tetrameric (n=4), and potentially higher titer (n) ranges, spanning a molecular weight range of at least approximately 12–150 kDa. Such alternative forms are referred to herein as “antibody protein products.” Antibody protein products include those based on the complete antibody structure and those mimicking antibody fragments that retain complete antigen-binding ability, such as scFv, Fab, and VHH / VH (discussed below). The smallest antigen-binding antibody fragment that retains a complete antigen-binding site is the Fv fragment, which consists entirely of a variable (V) region. A soluble and flexible amino acid peptide linker is used to link the V region to an scFv (single-chain variable fragment) fragment to stabilize the molecule, or to add a constant (C) domain to the V region to generate a Fab fragment [fragment, antigen-binding]. Both scFv and Fab fragments can be readily produced in host cells, such as prokaryotic host cells. Other antibody protein products include dimeric and multimeric antibody types such as diabodies, triabodies, and tetrabodies, or minibodies (mini-Abs), which include different types consisting of disulfide bond-stabilized scFv (ds-scFv), single-chain Fab (scFab), and scFv linked to an oligomerized domain. The smallest fragment is VHH / VH of a camelid heavy chain Ab and a single-domain Ab (sdAb). The most frequently used building block for creating novel antibody types is a single-chain variable (V)-domain antibody fragment (scFv) containing V domains (VH domain and VL domain) derived from the heavy and light chains linked by a peptide linker of approximately 15 amino acid residues. Peptibodies or peptide-Fc fusions are yet another antibody protein product. The structure of a peptide body consists of a biologically active peptide grafted onto an Fc domain. Peptibodies are well described in the art. For example, see Shimamoto et al., mAbs 4(5):586-591 (2012).

[0038] Other antibody protein products include single-chain antibodies (SCAs), diabodies, triabodies, tetrabodies, and bispecific or trispecific antibodies. Bispecific antibodies can be classified into five main classes: BsIgG, IgG adducts, BsAb fragments, bispecific fusion proteins, and BsAb conjugates. See, for example, Spiess et al., Molecular Immunology 67(2) Part A:97-106 (2015).

[0039] In exemplary embodiments, the antigen-binding proteins of the Disclosure include one of these antibody protein products. In exemplary embodiments, the antigen-binding proteins of the Disclosure include one of scFv, Fab, VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibodies, multimeric antibodies (e.g., diabody, triabody, tetrabody), miniAb, peptibody VHH / VH of a heavy chain antibody of a camelid, sdAb, diabody; triabody; tetrabody; bispecific or triplicate antibodies, BsIgG, appended IgG, BsAb fragment, bispecific fusion proteins, and BsAb conjugates.

[0040] In exemplary examples, the antigen-binding proteins of this disclosure include antibody protein products in monomeric, polymeric, oligomeric, or polymeric forms. In certain embodiments, where the antibody comprises two or more distinct antigen-binding region fragments, the antibody is considered bispecific, triplicate, or multiplespecific, or bivalent, trivalent, or polyvalent, depending on the number of distinct epitopes recognized and bound to by the antibody. In exemplary embodiments, the antigen-binding protein of this disclosure is a bispecific antibody (bsAb) comprising two scFvs, one of which binds to TIGIT and the other to CD112R. In various embodiments, the scFv that binds to CD112R comprises a light chain variable region and a heavy chain variable region of 29E10, 24F1, or 11E4. In various embodiments, the scFv that binds to TIGIT comprises a light chain variable region and a heavy chain variable region of 43B7.002.015, 66H9.009, or 58A7.002.008. In an exemplary example, each scFv is optionally ligated to an IgG heavy chain and / or light chain.

[0041] Structure of antigen-binding proteins In exemplary embodiments, the CD112R antigen-binding protein (e.g., an antibody or its antigen-binding fragment) is: (a) the heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence described in Table A1, or a variant sequence thereof that differs by only 1 to 4 amino acids (e.g., 1, 2, 3, or 4 amino acids) or has at least or about 90% sequence identity; (b) the HC CDR2 amino acid sequence described in Table A1, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or about 90% sequence identity; (c) the HC CDR3 amino acid sequence described in Table A1, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or about 90% sequence identity; (d) the light chain (LC) CDR1 amino acid sequence described in Table A1, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or about 90% sequence identity; (e) the LC described in Table A1 (f) A CDR2 amino acid sequence, or a variant thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (g) An LC CDR3 amino acid sequence as described in Table A1, or a variant thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; or (g) Any combination of 2, 3, 4, 5, or 6 of (a) to (f).

[0042] [Table 1]

[0043] In exemplary embodiments, the CD112R antigen-binding protein (e.g., an antibody, or an antigen-binding fragment thereof) comprises at least one or two of the LC CDR1 amino acid sequence, LC CDR2 amino acid sequence, and LC CDR3 amino acid sequence described in Table A1, and the HC CDR amino acid sequence described in Table A1. In exemplary embodiments, the CD112R antigen-binding protein comprises the HC CDR1 amino acid sequence, HC CDR2 amino acid sequence, and HC CDR3 amino acid sequence described in Table A1. The CD112R antigen-binding protein includes the CDR3 amino acid sequence and at least one or two of the LC CDR amino acid sequences listed in Table A1. In some embodiments, the CD112R antibody-binding protein includes all three such CDRs. In exemplary embodiments, the CD112R antigen-binding protein includes all three, four, five, or six of the amino acid sequences shown by the single-row sequence numbers in Table A1. In exemplary embodiments, the CD112R antigen-binding protein includes each of the LC CDR amino acid sequences shown by the single-row sequence numbers in Table A1, and at least one or two of the HC CDR amino acid sequences shown by the same single-row or different single-row sequence numbers in Table A1. In exemplary embodiments, the CD112R antigen-binding protein includes each of the HC CDR amino acid sequences shown by the single-row sequence numbers in Table A1, and at least one or two of the LC CDR amino acid sequences shown by the same single-row or different single-row sequence numbers in Table A1. In exemplary embodiments, the CD112R antigen-binding protein comprises six CDR amino acid sequences listed in a single row in Table A1, or six CDR amino acid sequences selected from the group consisting of (a) SEQ ID NOs: 13-18, (b) SEQ ID NOs: 23-28, (c) SEQ ID NOs: 33-38, (d) SEQ ID NOs: 43-48, (e) SEQ ID NOs: 53-58, (f) SEQ ID NOs: 63-68, (g) SEQ ID NOs: 73-78, (h) SEQ ID NOs: 83-88, (i) SEQ ID NOs: 93-98, (j) SEQ ID NOs: 103-108, (k) SEQ ID NOs: 233-238, (l) SEQ ID NOs: 1973-1978, (m) SEQ ID NOs: 1983-1988, (n) SEQ ID NOs: 1993-1998, and (o) SEQ ID NOs: 2003-2008. In exemplary embodiments, the CD112R antigen-binding protein includes the six CDR amino acid sequences described above, and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 2019 or SEQ ID NO: 2020.

[0044] In the exemplary examples, the amino acid sequences in Table A1 are separated by at least one (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) intervening amino acids, such as framework residues. In the exemplary examples, there are approximately 10 to 20 amino acids between the sequences of LC CDR1 and LC CDR2, and approximately 25 to 40 amino acids between the sequences of LC CDR2 and LC CDR3. In the exemplary examples, there are approximately 14 to 16 amino acids between the sequences of LC CDR1 and LC CDR2, and approximately 30 to 35 amino acids between the sequences of LC CDR2 and LC CDR3. In the exemplary examples, there are approximately 10 to 20 amino acids between the sequences of HC CDR1 and HC CDR2, and approximately 25 to 40 amino acids between the sequences of HLC CDR2 and HC CDR3. In the exemplary example, there are approximately 14 to 16 amino acids between the sequences of HC CDR1 and HC CDR2, and approximately 30 to 35 amino acids between the sequences of HC CDR2 and HC CDR3. In the exemplary embodiment, the intervening amino acids include a framework region.

[0045] In exemplary embodiments, the TIGIT antigen-binding protein (e.g., an antibody or its antigen-binding fragment) is: (a) the heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence described in Table A2, or a variant sequence thereof that differs by only 1 to 4 amino acids (e.g., 1, 2, 3, or 4 amino acids) or has at least or about 90% sequence identity; (b) the HC CDR2 amino acid sequence described in Table A2, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or about 90% sequence identity; (c) the HC CDR3 amino acid sequence described in Table A2, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or about 90% sequence identity; (d) the light chain (LC) CDR1 amino acid sequence described in Table A2, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or about 90% sequence identity; (e) the LC described in Table A2 (f) A CDR2 amino acid sequence, or a variant thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (g) An LC CDR3 amino acid sequence as described in Table A2, or a variant thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; or (g) Any combination of 2, 3, 4, 5, or 6 of (a) to (f).

[0046] [Table 2]

[0047] In exemplary embodiments, the TIGIT antigen-binding protein (e.g., an antibody, or its antigen-binding fragment) comprises at least one or two of the LC CDR1, LC CDR2, and LC CDR3 amino acid sequences listed in Table A2, and the HC CDR amino acid sequences listed in Table A2. In exemplary embodiments, the TIGIT antigen-binding protein comprises at least one or two of the HC CDR1, HC CDR2, and HC CDR3 amino acid sequences listed in Table A2, and the LC CDR amino acid sequences listed in Table A2. In some embodiments, the TIGIT antibody-binding protein comprises all three such CDRs. In exemplary embodiments, the TIGIT antigen-binding protein comprises all three, four, five, or six of the amino acid sequences shown by the single row sequence numbers in Table A2. In exemplary embodiments, the TIGIT antigen-binding protein comprises each of the LC CDR amino acid sequences shown by the single row sequence numbers in Table A2, and at least one or two of the HC CDR amino acid sequences shown by the same single row or another single row sequence numbers in Table A2. In exemplary embodiments, the TIGIT antigen-binding protein comprises six CDR amino acid sequences listed in a single row in Table A2, or six CDR amino acid sequences selected from (a) SEQ ID NOs. 113-118, (b) SEQ ID NOs. 123-128, (c) SEQ ID NOs. 133-138, (d) SEQ ID NOs. 143-148, (e) SEQ ID NOs. 153-158, (f) SEQ ID NOs. 163-168, (g) SEQ ID NOs. 173-178, (h) SEQ ID NOs. 183-188, (i) SEQ ID NOs. 193-198, (j) SEQ ID NOs. 203-208, (k) SEQ ID NOs. 213-218, (l) SEQ ID NOs. 223-228, and (m) SEQ ID NOs. 2013-2018. In exemplary embodiments, the TIGIT antigen-binding protein comprises the above six CDR amino acid sequences and a heavy chain constant region comprising the amino acid sequence of SEQ ID NOs. 2019 or SEQ ID NOs. 2020.

[0048] In an exemplary example, the amino acid sequence in Table A2 is separated by at least one (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) intervening amino acids, such as framework residues. In the exemplary example, LC CDR1 and LC There are approximately 10 to 20 amino acids between the CDR2 sequences and approximately 25 to 40 amino acids between the LC CDR2 and LC CDR3 sequences. In an exemplary example, there are approximately 14 to 16 amino acids between the LC CDR1 and LC CDR2 sequences and approximately 30 to 35 amino acids between the LC CDR2 and LC CDR3 sequences. In an exemplary example, there are approximately 10 to 20 amino acids between the HC CDR1 and HC CDR2 sequences and approximately 25 to 40 amino acids between the HLC CDR2 and HC CDR3 sequences. In an exemplary example, there are approximately 14 to 16 amino acids between the HC CDR1 and HC CDR2 sequences and approximately 30 to 35 amino acids between the HC CDR2 and HC CDR3 sequences. In an exemplary embodiment, the intervening amino acids include a framework region.

[0049] In exemplary embodiments, the CD112R antigen-binding protein (e.g., an antibody or its antigen-binding fragment) includes a pair of amino acid sequences of the HC variable region and the LC variable region listed in a single row in Table B1, or includes one of the following pairs of amino acid sequences: (a) SEQ ID NOs. 11-12, (b) SEQ ID NOs. 21-22, (c) SEQ ID NOs. 31-32, (d) SEQ ID NOs. 41-42, (e) SEQ ID NOs. 51-52, (f) SEQ ID NOs. 61-62, (g) SEQ ID NOs. 71-72, (h) SEQ ID NOs. 81-82, (i) SEQ ID NOs. 91-92, (j) SEQ ID NOs. 101-102, (k) SEQ ID NOs. 231-232, (l) SEQ ID NOs. 1971-1972, (m) SEQ ID NOs. 1981-1982, (n) SEQ ID NOs. 1991-1992, or (o) SEQ ID NOs. 2001-2002. In an exemplary embodiment, the CD112R antigen-binding protein comprises a pair of amino acid sequences of the HC variable region and the LC variable region described above, and a heavy chain constant region containing the amino acid sequence of SEQ ID NO: 2019 or SEQ ID NO: 2020. In exemplary embodiments, the CD112R antigen-binding protein includes a pair of full-length (FL)HC and FL LC amino acid sequences listed in a single row in Table B1, or one of the following pairs of amino acid sequences: (a) SEQ ID NOs. 9-10, (b) SEQ ID NOs. 19-20, (c) SEQ ID NOs. 29-30, (d) SEQ ID NOs. 39-40, (e) SEQ ID NOs. 49-50, (f) SEQ ID NOs. 59-60, (g) SEQ ID NOs. 69-70, (h) SEQ ID NOs. 79-80, (i) SEQ ID NOs. 89-90, (j) SEQ ID NOs. 99-100, (k) SEQ ID NOs. 229-230, (l) SEQ ID NOs. 1969-1970, (m) SEQ ID NOs. 1979-1980, (n) SEQ ID NOs. 1989-1990, or (o) SEQ ID NOs. 1999-2000.

[0050] [Table 3]

[0051] In exemplary embodiments, the TIGIT antigen-binding protein (e.g., an antibody or its antigen-binding fragment) includes a pair of amino acid sequences of the HC variable region and the LC variable region listed in a single row in Table B2, or includes one of the following pairs of amino acid sequences: (a) SEQ ID NOs. 111-112, (b) SEQ ID NOs. 121-122, (c) SEQ ID NOs. 131-132, (d) SEQ ID NOs. 141-142, (e) SEQ ID NOs. 151-152, (f) SEQ ID NOs. 161-162, (g) SEQ ID NOs. 171-172, (h) SEQ ID NOs. 181-182, (i) SEQ ID NOs. 191-192, (j) SEQ ID NOs. 201-202, (k) SEQ ID NOs. 211-212, (l) SEQ ID NOs. 221-222, or (m) SEQ ID NOs. 2011-2012. In an exemplary embodiment, the TIGIT antigen-binding protein comprises a pair of amino acid sequences of the HC variable region and the LC variable region described above, and a heavy chain constant region containing the amino acid sequence of SEQ ID NO: 2019 or SEQ ID NO: 2020. In exemplary embodiments, the TIGIT antigen-binding protein comprises a pair of full-length (FL)HC and FL LC amino acid sequences listed in a single row in Table B2, or one of the following pairs of amino acid sequences: (a) SEQ ID NOs. 109-110, (b) SEQ ID NOs. 119-120, (c) SEQ ID NOs. 129-130, (d) SEQ ID NOs. 139-140, (e) SEQ ID NOs. 149-150, (f) SEQ ID NOs. 159-160, (g) SEQ ID NOs. 169-170, (h) SEQ ID NOs. 179-180, (i) SEQ ID NOs. 189-190, (j) SEQ ID NOs. 199-200, (k) SEQ ID NOs. 209-210, (l) SEQ ID NOs. 219-220, or (m) SEQ ID NOs. 2009-2010.

[0052] [Table 4]

[0053] In exemplary embodiments, the CD112R antigen-binding protein or the TIGIT antigen-binding protein contains an amino acid sequence similar to the above-described amino acid sequence, but the antigen-binding protein substantially retains its biological function, for example, the ability to bind to its target or antigen, such as human TIGIT or human CD112R, or the ability to reduce, block, inhibit, suppress or interfere with signal transduction resulting from the interaction between TIGIT and its binding partner CD155, or the interaction between CD112R and its binding partner CD112.

[0054] In exemplary embodiments, the CD112R antigen-binding protein (e.g., an antibody or its antigen-binding fragment) contains an amino acid sequence that differs by only one, two, three, four, five, six, or more amino acids from a parent amino acid sequence having the amino acid sequence described in Table A1 or Table B1. In exemplary embodiments, the TIGIT antigen-binding protein contains an amino acid sequence that differs by only one, two, three, four, five, six, or more amino acids from a parent amino acid sequence having the amino acid sequence described in Table A2 or Table B2. In exemplary embodiments, the antigen-binding protein (e.g., CD112R antigen-binding protein or TIGIT antigen-binding protein) contains a variant sequence of the parent sequence, which differs by only one or two amino acids from the parent sequence. In exemplary embodiments, the antigen-binding protein contains one or more amino acid substitutions located outside the CDR, for example, these one or more amino acid substitutions located within the framework region of the heavy or light chain. In exemplary embodiments, the antigen-binding protein contains one or more amino acid substitutions, but the antigen-binding protein retains the amino acid sequence of the six CDRs. In exemplary embodiments, the antigen-binding protein comprises an amino acid sequence having only one, two, three, four, five, six, or more conserved amino acid substitutions compared to the parent sequence. As used herein, the term “conserved amino acid substitution” refers to the substitution of one amino acid with another amino acid having similar properties (e.g., size, charge, hydrophobicity, hydrophilicity, and / or aromaticity), and includes substitutions in one of the following five groups: I. Aliphatic, nonpolar or slightly polar small residues: Ala, Ser, Thr, Pro, Gly; II. Negatively charged polar residues, and their amides and esters: Asp, Asn, Glu, Gln, cysteic acid, and homocysteic acid; III. Polarly polar, positively charged residues: His, Arg, Lys; ornithine (Orn) IV. Large, nonpolar aliphatic residues: Met, Leu, Ile, Val, Cys, norleucine (Nle), homocysteine V. Large aromatic residues: Phe, Tyr, Trp, acetylphenylalanine.

[0055] In exemplary embodiments, an antigen-binding protein (e.g., an antibody or its antigen-binding fragment) comprises an amino acid sequence containing at least one amino acid substitution relative to the parent amino acid sequence, wherein the amino acid substitution is a non-conservative amino acid substitution. As used herein, the term “non-conservative amino acid substitution” is defined herein as the substitution of one amino acid with another amino acid having different properties (e.g., size, charge, hydrophobicity, hydrophilicity, and / or aromaticity), and includes substitutions other than those in the five groups described above.

[0056] In exemplary embodiments, an antigen-binding protein (e.g., an antibody or its antigen-binding fragment) comprises an amino acid sequence containing at least one amino acid substitution relative to the parent amino acid sequence, wherein the substituted amino acid is a naturally occurring amino acid. “Naturally occurring amino acid,” “standard amino acid,” or “canonical amino acid” means one of the 20 eukaryotic alpha amino acids (Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gln, Cys, Gly, Pro, Arg, His, Lys, Asp, Glu) directly encoded by codons in the universal genetic code. In exemplary embodiments, an antigen-binding protein comprises an amino acid sequence containing at least one amino acid substitution relative to the parent amino acid sequence, wherein the substituted amino acid is a non-standard amino acid or an amino acid that is not incorporated into the protein during translation. Non-standard amino acids include, but are not limited to, selenocysteine, pyrrolicin, ornithine, norleucine, β-amino acids (e.g., β-alanine, β-aminoisobutyric acid, β-phenylalanine, β-homophenylalanine, β-glutamic acid, β-glutamine, β-homotryptophan, β-leucine, β-lysine), homo-amino acids (e.g., homophenylalanine, homoserine, homoarginine, monocysteine, homocysteine), N-methylamino acids (e.g., L-abulin, N-methylalanine, N-methylisoleucine, N-methylleucine), 2-aminocaprylic acid, 7-aminocephalosporanic acid, 4-aminocinnamic acid, alpha-aminocyclohexanepropionic acid, amino-(4-hydroxyphenyl)acetic acid, 4-aminonicotinic acid, and 3-aminophenylacetic acid.

[0057] In exemplary embodiments, the antigen-binding protein includes an amino acid sequence having approximately 30% or more, approximately 50% or more, or approximately 70% or more sequence identity with respect to the parent amino acid sequence. In exemplary embodiments, the antigen-binding protein includes an amino acid sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or more than 90% sequence identity with respect to the parent amino acid sequence. In exemplary embodiments, the antigen-binding protein includes an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, or more than 90% sequence identity along the full length of the parent amino acid sequence. In exemplary embodiments, the antigen-binding protein includes an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity along the full length of the parent amino acid sequence.

[0058] In various embodiments, the CD112R antigen-binding protein (e.g., an antibody or its antigen-binding fragment) comprises a variant sequence of the HC variable region amino acid sequence listed in Table B1, or a variant sequence of the LC variable region amino acid sequence listed in Table B1, wherein the variant sequence differs from the Table B1 sequence by only 1 to 12 amino acids (e.g., 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2) or has at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity). In various embodiments, the CD112R antigen-binding protein comprises a variant sequence of the FL HC amino acid sequence listed in Table B1, or a variant sequence of the FL LC amino acid sequence listed in Table B1, wherein the variant sequence differs from the Table B1 sequence by only 1 to 46 amino acids, or has at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity). In exemplary embodiments, the CD112R antigen-binding protein includes a pair of amino acid sequences of the HC variable region and the LC variable region listed in a single row in Table C1, or includes one of the following pairs of amino acid sequences: (a) the first 100 amino acids of SEQ ID NOs. 241 and 242, respectively; (b) the first 100 amino acids of SEQ ID NOs. 247 and 248, respectively; (c) the first 100 amino acids of SEQ ID NOs. 249 and 250, respectively; (d) the first 100 amino acids of SEQ ID NOs. 251 and 252, respectively; or (e) the first 100 amino acids of SEQ ID NOs. 263 and 264, respectively.In exemplary embodiments, the CD112R antigen-binding protein contains the first 105, first 106, first 107, first 108, first 109, first 110, first 111, first 112, first 113, first 114, or first 115 amino acids of SEQ ID NO: 242, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, or SEQ ID NO: 264, and / or contains the first 115, first 116, first 117, first 118, first 119, first 120, first 121, first 122, first 123, first 124, first 125, first 126, or first 127 amino acids of SEQ ID NO: 241, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, or SEQ ID NO: 263. vhCDR2, including SEQ ID NO: 251 or SEQ ID NO: 263, in exemplary embodiments, the CD112R antigen-binding protein includes a pair of full-length (FL)HC and FLLC amino acid sequences listed in a single row in Table C1, or one of the following pairs of amino acid sequences: (a) SEQ ID NOs: 241-242; (b) SEQ ID NOs: 247-248; (c) SEQ ID NOs: 249-250; (d) SEQ ID NOs: 251-252; or (e) SEQ ID NOs: 263-264. In various embodiments, the CD112R antigen-binding protein is a modified version of Table C1. *The CD112R antigen-binding protein includes FL LC amino acid sequences having odd-numbered sequence numbers listed in the column ", and FL HC amino acid sequences having a sequence number one greater than that of the FL LC sequence number. In various embodiments, the CD112R antigen-binding protein includes at least a portion of any one sequence number in Table C1, the portion of which includes the first 100 amino acids of the amino acid sequence, the first 105 amino acids of the amino acid sequence, the first 110 amino acids of the amino acid sequence, the first 115 amino acids of the amino acid sequence, or the first 120 amino acids of the amino acid sequence. In various examples, the CD112R antigen-binding protein includes at least 2, 3, 4, 5, or 6 CDRs of the amino acid sequences in Table C1. The CDRs of a given antibody HC or LC can be determined by any one or more methods known in the art. For example, Kabat et al., Sequences of Proteins of Immunological Interest, USDept.of Health and Human Services, NIH(1991); Chothia et al., J Mol Biol 196:901-917(1987); Al-Lazikani et al., J Mol Biol 273:927-948(1997); Abhinandan et al., Mol Immunol 45:3832-3839 (2008); Lefranc et al., The Immunologist 7:132-136 (1999); Lefranc et al., Dev Comp Immunol 27:55-77 (2003); and Honegger et al., J Mol Biol 309:657-670 (2001).

[0059] [Table 5]

[0060] In various embodiments, the TIGIT antigen-binding protein (e.g., an antibody or its antigen-binding fragment) comprises a variant sequence of the HC variable region amino acid sequence listed in Table B2, or a variant sequence of the LC variable region amino acid sequence listed in Table B2, wherein these variant sequences differ from the sequences in Table B2 by only 1 to 12 amino acids, or have at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity). In various embodiments, the TIGIT antigen-binding protein comprises a variant sequence of the FL HC amino acid sequence listed in Table B2, or a variant sequence of the FL LC amino acid sequence listed in Table B2, wherein these variant sequences differ from the sequences in Table B2 by only 1 to 46 amino acids, or have at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity). In exemplary embodiments, the TIGIT antigen-binding protein includes a pair of amino acid sequences of the HC variable region and the LC variable region listed in a single row in Table C2, or includes one of the following pairs of amino acid sequences: (a) the first 100 amino acids of SEQ ID NOs. 239 and 240, respectively; (b) the first 100 amino acids of SEQ ID NOs. 243 and 244, respectively; (c) the first 100 amino acids of SEQ ID NOs. 245 and 246, respectively; (d) the first 100 amino acids of SEQ ID NOs. 253 and 254, respectively; (e) the first 100 amino acids of SEQ ID NOs. 255 and 256, respectively; (f) the first 100 amino acids of SEQ ID NOs. 257 and 258, respectively; (g) the first 100 amino acids of SEQ ID NOs. 259 and 260, respectively; or (h) the first 100 amino acids of SEQ ID NOs. 261 and 262, respectively.In exemplary embodiments, the TIGIT antigen-binding protein contains the first 105, first 106, first 107, first 108, first 109, first 110, first 111, first 112, first 113, first 114, or first 115 amino acids of SEQ ID NO: 239, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, or SEQ ID NO: 261, and / or contains the first 115, first 116, first 117, first 118, first 119, first 120, first 121, first 122, first 123, first 124, first 125, first 126, or first 127 amino acids of SEQ ID NO: 239, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, or SEQ ID NO: 261. In exemplary embodiments, the TIGIT antigen-binding protein comprises a pair of full-length (FL)HC and FL LC amino acid sequences listed in a single row in Table C2, or one of the following pairs of amino acid sequences: (a) SEQ ID NOs. 239-240; (b) SEQ ID NOs. 243-244; (c) SEQ ID NOs. 245-246; (d) SEQ ID NOs. 253-254; (e) SEQ ID NOs. 255-256; (f) SEQ ID NOs. 257-258; (g) SEQ ID NOs. 259-260; or (h) SEQ ID NOs. 261-262. In various embodiments, the TIGIT antigen-binding protein is a modified version of the sequence in Table C2. * The TIGIT antigen-binding protein includes FL LC amino acid sequences having odd-numbered sequence numbers listed in the column ", and FL HC amino acid sequences having a sequence number one greater than that of the FL LC sequence number. In various embodiments, the TIGIT antigen-binding protein includes at least a portion of any one sequence number in Table C2, the portion of which includes the first 100 amino acids of the amino acid sequence, the first 105 amino acids of the amino acid sequence, the first 110 amino acids of the amino acid sequence, the first 115 amino acids of the amino acid sequence, or the first 120 amino acids of the amino acid sequence. In various examples, the TIGIT antigen-binding protein includes at least 2, 3, 4, 5, or 6 CDRs of the amino acid sequences in Table C2. The CDRs of a given antibody HC or LC can be determined by any one or more methods known in the art.

[0061] [Table 6]

[0062] In various embodiments, the CD112R antigen-binding protein (e.g., an antibody or its antigen-binding fragment) comprises an antibody, an antigen-binding fragment of an antibody (e.g., Fab), or an antibody protein product, e.g., scFv. In various embodiments, the CD112R antigen-binding protein is bivalent, containing two antigen-binding sites. In various embodiments, the TIGIT antigen-binding protein comprises an antibody, an antigen-binding fragment of an antibody (e.g., Fab), or an antibody protein product, e.g., scFv. In various embodiments, the TIGIT antigen-binding protein is bivalent, containing two antigen-binding sites.

[0063] In exemplary embodiments, a TIGIT antigen-binding protein, such as an anti-TIGIT antibody, contains an LC variable region amino acid sequence (e.g., at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity)) that is highly similar to the first 105-115 amino acids of SEQ ID NO: 192 or SEQ ID NO: 222 or SEQ ID NO: 240, and this LC variable region amino acid sequence has glutamic acid (Glu) at position 1. 1) or its conserved amino acid substitution, glutamine (Gln27) or its conserved amino acid substitution at position 27, serine (Ser28) or its conserved amino acid substitution at position 28, serine (Ser91) or its conserved amino acid substitution at position 91, serine (Ser92) or its conserved amino acid substitution at position 92, serine (Ser93) or its conserved amino acid substitution at position 93, leucine (Leu94) or its conserved amino acid substitution at position 94, or any combination thereof. In exemplary embodiments, a TIGIT antigen-binding protein, for example, an anti-TIGIT antibody, includes an LC CDR1 amino acid sequence containing Gln27 or its conserved amino acid substitution, Ser28 or its conserved amino acid substitution, or any combination thereof. In exemplary embodiments, a TIGIT antigen-binding protein, for example, an anti-TIGIT antibody, includes an LC CDR2 amino acid sequence containing Glu1 or its conserved amino acid substitution. In exemplary embodiments, a TIGIT antigen-binding protein, such as an anti-TIGIT antibody, includes an LC CDR3 amino acid sequence containing Ser91 or its conserved amino acid substitution, Ser92 or its conserved amino acid substitution, Ser93 or its conserved amino acid substitution, Leu94 or its conserved amino acid substitution, or any combination thereof. In exemplary embodiments, a TIGIT antigen-binding protein, such as an anti-TIGIT antibody, includes an LC variable region amino acid sequence containing Gln27 or its conserved amino acid substitution, which forms a hydrogen bond with an amino acid of TIGIT. In various embodiments, when a TIGIT antigen-binding protein, such as a TIGIT antibody, binds to TIGIT, the amino acid residues named above are located approximately 3 to 4 angstroms from the amino acids of TIGIT.

[0064] In exemplary embodiments, a TIGIT antigen-binding protein, such as an anti-TIGIT antibody, is highly similar to the first 115-127 amino acids of SEQ ID NO: 191, SEQ ID NO: 221, or SEQ ID NO: 239 (e.g., at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity)), with valine (Val32) or its conserved amino acid substitution at position 32, 3 Tyrosine (Tyr33) or its conserved amino acid substitution at position 3, tyrosine (Tyr52) or its conserved amino acid substitution at position 52, tyrosine (Tyr54) or its conserved amino acid substitution at position 54, tyrosine (Tyr55) or its conserved amino acid substitution at position 55, serine (Ser56) or its conserved amino acid substitution at position 56, glycine (Gly57) or its conserved amino acid substitution at position 57, glycine (Gly58) or The conserved amino acid substitutions include threonine (Thr59) or its conserved amino acid substitution at position 59, tyrosine (Tyr60) or its conserved amino acid substitution at position 60, proline (Pro63) or its conserved amino acid substitution at position 63, arginine (Arg66) or its conserved amino acid substitution at position 66, isoleucine (Ile102) or its conserved amino acid substitution at position 102, and alanine (Ala104) or its conserved amino acid substitution at position 104. The HC variable region amino acid sequence includes substitutions, glycine (Gly107) or its conserved amino acid substitution at position 107, tyrosine (Tyr108) or its conserved amino acid substitution at position 108, phenylalanine (Phe109) or its conserved amino acid substitution at position 109, tyrosine (Tyr110) or its conserved amino acid substitution at position 110, tyrosine (Tyr111) or its conserved amino acid substitution at position 111, or any combination thereof. In an exemplary embodiment, a TIGIT antigen-binding protein, such as an anti-TIGIT antibody, includes an HC CDR1 amino acid sequence including Val32 or its conserved amino acid substitution, Tyr33 or its conserved amino acid substitution, or any combination thereof.In exemplary embodiments, the TIGIT antigen-binding protein, for example, an anti-TIGIT antibody, comprises an HC CDR2 amino acid sequence including Tyr52 or its conserved amino acid substitution, Tyr54 or its conserved amino acid substitution, Tyr55 or its conserved amino acid substitution, Ser56 or its conserved amino acid substitution, Gly57 or its conserved amino acid substitution, Gly58 or its conserved amino acid substitution, Thr59 or its conserved amino acid substitution, Tyr60 or its conserved amino acid substitution, Pro63 or its conserved amino acid substitution, Arg66 or its conserved amino acid substitution, or any combination thereof. In exemplary embodiments, a TIGIT antigen-binding protein, such as an anti-TIGIT antibody, comprises an HC CDR3 amino acid sequence including Ile102 or its conserved amino acid substitution, Ala104 or its conserved amino acid substitution, Gly107 or its conserved amino acid substitution, Tyr108 or its conserved amino acid substitution, Phe109 or its conserved amino acid substitution, Tyr110 or its conserved amino acid substitution, Tyr111 or its conserved amino acid substitution, or any combination thereof. In various embodiments, each of Tyr52, Ser56, Thr59, Phe109, Tyr55, Tyr60, or their conserved amino acid substitutions, forms a hydrogen bond with an amino acid of TIGIT. In various embodiments, when a TIGIT antigen-binding protein, such as a TIGIT antibody, binds to TIGIT, each of the amino acid residues named above is located approximately 3 to 4 angstroms from an amino acid of TIGIT. In exemplary embodiments, a TIGIT antigen-binding protein, such as an anti-TIGIT antibody, includes an HC variable region amino acid sequence containing Arg66 or its conserved amino acid substitution, which forms a salt bridge with an amino acid of TIGIT. In various embodiments, Tyr52 and Thr59 form hydrogen bonds with the same amino acid of TIGIT. In various examples, Ser56 forms hydrogen bonds with two different amino acids of TIGIT.

[0065] In exemplary embodiments, a TIGIT antigen-binding protein, such as an anti-TIGIT antibody, is highly similar to the first 105-115 amino acids of SEQ ID NO: 122, 212, or 2012, or SEQ ID NO: 246 (e.g., at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity)), with glutamic acid (Glu1) or its conserved amino acid substitution at position 1, isoleucine (Ile2) or its conserved amino acid substitution at position 2, glutamine (Gln27) or its conserved amino acid substitution at position 27, serine (Ser28) or its conserved amino acid substitution at position 28, valine (Val29) or its conserved amino acid substitution at position 29, serine (Ser30) or its conserved amino acid substitution at position 31 The LC variable region amino acid sequence includes serine (Ser31) at position 32, threonine (Thr32) or its conserved amino acid substitution at position 32, tyrosine (Tyr33) or its conserved amino acid substitution at position 33, serine (Ser68) or its conserved amino acid substitution at position 68, glycine (Gly69) or its conserved amino acid substitution at position 69, tyrosine (Tyr92) or its conserved amino acid substitution at position 92, aspartic acid (Asp93) or its conserved amino acid substitution at position 93, valine (Val94) or its conserved amino acid substitution at position 94, serine (Ser95) or its conserved amino acid substitution at position 95, proline (Pro96) or its conserved amino acid substitution at position 96, tryptophan (Trp97) or its conserved amino acid substitution at position 97, or any combination thereof. In exemplary embodiments, the TIGIT antigen-binding protein, for example, an anti-TIGIT antibody, comprises an LC CDR1 amino acid sequence including Gln27 or its conserved amino acid substitution, Ser28 or its conserved amino acid substitution, Val29 or its conserved amino acid substitution, Ser30 or its conserved amino acid substitution, Ser31 or its conserved amino acid substitution, Thr32 or its conserved amino acid substitution, Tyr33 or its conserved amino acid substitution, or any combination thereof.In exemplary embodiments, the TIGIT antigen-binding protein, for example, an anti-TIGIT antibody, comprises an LC CDR2 amino acid sequence including Glu1 or its conserved amino acid substitution, Ile2 or its conserved amino acid substitution, Ser68 or its conserved amino acid substitution, Gly69 or its conserved amino acid substitution, or any combination thereof. In exemplary embodiments, a TIGIT antigen-binding protein, for example, an anti-TIGIT antibody, includes an LC CDR3 amino acid sequence containing Tyr92 or its conserved amino acid substitution, Asp93 or its conserved amino acid substitution, Val94 or its conserved amino acid substitution, Ser95 or its conserved amino acid substitution, Pro96 or its conserved amino acid substitution, Trp97 or its conserved amino acid substitution, or any combination thereof. In exemplary embodiments, a TIGIT antigen-binding protein, for example, an anti-TIGIT antibody, includes an LC variable region amino acid sequence containing Asp93 or its conserved amino acid substitution, Ser95 or its conserved amino acid substitution, Try33 or its conserved amino acid substitution (each of which forms a hydrogen bond with an amino acid of TIGIT). In exemplary embodiments, a TIGIT antigen-binding protein, for example, an anti-TIGIT antibody, includes an LC variable region amino acid sequence containing Asp93 or its conserved amino acid substitution that forms a salt bridge with an amino acid of TIGIT. In various embodiments, when a TIGIT antigen-binding protein, such as a TIGIT antibody, binds to TIGIT, the amino acid residues named above are located approximately 3 to 4 angstroms from the amino acids of TIGIT.

[0066] In exemplary embodiments, the TIGIT antigen-binding protein, for example, the anti-TIGIT antibody, is highly similar to the first 115-127 amino acids of SEQ ID NO: 121 or SEQ ID NO: 211 or SEQ ID NO: 245 (e.g., at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity)), with glycine (Gly32) or its conserved amino acid substitution at position 32, tyrosine (Tyr35) or its conserved amino acid substitution at position 35, tyrosine (Tyr52) or its conserved amino acid substitution at position 52, tyrosine (Tyr54) or its conserved amino acid substitution at position 54, tyrosine (Tyr55) or its conserved amino acid substitution at position 55, serine (Ser56) or its conserved amino acid substitution at position 56, and position 58 The HC variable region amino acid sequence includes serine (Ser58) or its conserved amino acid substitution at position 59, threonine (Thr59) or its conserved amino acid substitution at position 60, phenylalanine (Phe60) or its conserved amino acid substitution at position 63, proline (Pro63) or its conserved amino acid substitution at position 63, lysine (Lys66) or its conserved amino acid substitution at position 66, arginine (Arg102) or its conserved amino acid substitution at position 102, asparagine (Asn104) or its conserved amino acid substitution at position 104, tryptophan (Trp105) or its conserved amino acid substitution at position 105, asparagine (Asn106) or its conserved amino acid substitution at position 106, tyrosine (Tyr107) or its conserved amino acid substitution at position 107, or any combination thereof. In exemplary embodiments, the TIGIT antigen-binding protein, for example, an anti-TIGIT antibody, comprises an HC CDR1 amino acid sequence including Gly32 or its conserved amino acid substitution, Tyr35 or its conserved amino acid substitution, or any combination thereof.In exemplary embodiments, a TIGIT antigen-binding protein, for example, an anti-TIGIT antibody, includes an HC CDR2 amino acid sequence comprising Tyr52 or its conserved amino acid substitution, Tyr54 or its conserved amino acid substitution, Tyr55 or its conserved amino acid substitution, Ser56 or its conserved amino acid substitution, Ser58 or its conserved amino acid substitution, Thr59 or its conserved amino acid substitution, Phe60 or its conserved amino acid substitution, Pro63 or its conserved amino acid substitution, Lys66 or its conserved amino acid substitution, or any combination thereof. In exemplary embodiments, a TIGIT antigen-binding protein, for example, an anti-TIGIT antibody, includes an HC CDR3 amino acid sequence comprising Arg102 or its conserved amino acid substitution, Asn104 or its conserved amino acid substitution, Trp105 or its conserved amino acid substitution, Asn106 or its conserved amino acid substitution, Tyr107 or its conserved amino acid substitution, or any combination thereof. In various embodiments, each of Ser58, Asn106, Tyr107, Tyr35, Arg102, and Ser56, or any of these conserved amino acid substitutions, forms hydrogen bonds with the amino acids of TIGIT. In various embodiments, when a TIGIT antigen-binding protein, such as a TIGIT antibody, binds to TIGIT, each of the amino acid residues named above is located approximately 3 to 4 angstroms from the amino acids of TIGIT.

[0067] In exemplary embodiments, a TIGIT antigen-binding protein, such as an anti-TIGIT antibody, includes an LC variable region amino acid sequence that is highly similar to the first 105-115 amino acids of SEQ ID NO: 162 or SEQ ID NO: 262 (e.g., at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity)), and includes arginine (Arg30) or its conserved amino acid substitution at position 30, arginine (Arg31) or its conserved amino acid substitution at position 31, tyrosine (Tyr32) or its conserved amino acid substitution at position 32, serine (Ser91) or its conserved amino acid substitution at position 91, tyrosine (Tyr92) or its conserved amino acid substitution at position 92, serine (Ser93) or its conserved amino acid substitution at position 93, threonine (Thr94) or its conserved amino acid substitution at position 94, or any combination thereof. In exemplary embodiments, a TIGIT antigen-binding protein, for example, an anti-TIGIT antibody, includes an LC CDR1 amino acid sequence containing Arg30 or its conserved amino acid substitution, Arg31 or its conserved amino acid substitution, Tyr32 or its conserved amino acid substitution, or any combination thereof. In exemplary embodiments, a TIGIT antigen-binding protein, for example, an anti-TIGIT antibody, includes an LC CDR3 amino acid sequence containing Ser91 or its conserved amino acid substitution, Tyr92 or its conserved amino acid substitution, Ser93 or its conserved amino acid substitution, Thr94 or its conserved amino acid substitution, or any combination thereof. In exemplary embodiments, a TIGIT antigen-binding protein, for example, an anti-TIGIT antibody, includes an LC variable region amino acid sequence containing Tyr32, Tyr92, Thr94, Arg30, Arg31, or their conserved amino acid substitutions (each of which forms a hydrogen bond with an amino acid of TIGIT). In exemplary embodiments, a TIGIT antigen-binding protein, such as an anti-TIGIT antibody, includes an LC variable region amino acid sequence containing Arg30 or its conserved amino acid substitutions, which forms a salt bridge with the amino acids of TIGIT.In various embodiments, when a TIGIT antigen-binding protein, such as a TIGIT antibody, binds to TIGIT, the amino acid residues named above are located approximately 3 to 4 angstroms from the amino acids of TIGIT.

[0068] In exemplary embodiments, a TIGIT antigen-binding protein, such as an anti-TIGIT antibody, is highly similar to the first 115-127 amino acids of SEQ ID NO: 161 or SEQ ID NO: 261 (e.g., at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity)), with threonine (Thr30) or its conserved amino acid substitution at position 30, and glycine (Gly31) or its conserved amino acid substitution at position 31. Conservative amino acid substitution, tyrosine (Tyr32) or its conservative amino acid substitution at position 32, tyrosine (Tyr33) or its conservative amino acid substitution at position 33, tryptophan (Trp47) or its conservative amino acid substitution at position 47, tryptophan (Trp50) or a conservative amino acid at position 50, serine (Ser52) or its conservative amino acid substitution at position 52, threonine (Thr54) or its conservative amino acid substitution at position 54, serine (Ser55) or its conservative amino acid substitution at position 55 Conservative amino acid substitutions, alanine (Ala57) or its conservative amino acid substitution at position 57, threonine (Thr58) or its conservative amino acid substitution at position 58, glycine (Gly59) or its conservative amino acid substitution at position 59, tyrosine (Tyr60) or its conservative amino acid substitution at position 60, glutamine (Gln65) or its conservative amino acid substitution at position 65, asparagine (Asn101) or its conservative amino acid substitution at position 101, serine (Ser1) at position 102 02) The HC variable region amino acid sequence includes a valine (Val103) or its conserved amino acid substitution at position 103, a leucine (Leu104) or its conserved amino acid substitution at position 104, a tyrosine (Tyr105) or its conserved amino acid substitution at position 105, a tyrosine (Tyr106) or its conserved amino acid substitution at position 106, a tyrosine (Tyr107) or its conserved amino acid substitution at position 107, or any combination thereof.In exemplary embodiments, a TIGIT antigen-binding protein, for example, an anti-TIGIT antibody, includes an HC CDR1 amino acid sequence comprising Thr30 or its conserved amino acid substitution, Gly31 or its conserved amino acid substitution, Tyr32 or its conserved amino acid substitution, Tyr33 or its conserved amino acid substitution, or any combination thereof. In exemplary embodiments, a TIGIT antigen-binding protein, for example, an anti-TIGIT antibody, includes an HC CDR2 amino acid sequence comprising Trp47 or its conserved amino acid substitution, Trp50 or its conserved amino acid substitution, Ser52 or its conserved amino acid substitution, Thr54 or its conserved amino acid substitution, Ser55 or its conserved amino acid substitution, Ala57 or its conserved amino acid substitution, Thr58 or its conserved amino acid substitution, Gly59 or its conserved amino acid substitution, Tyr60 or its conserved amino acid substitution, Gln65 or its conserved amino acid substitution, or any combination thereof. In exemplary embodiments, a TIGIT antigen-binding protein, such as an anti-TIGIT antibody, comprises an HC CDR3 amino acid sequence including Asn101 or its conserved amino acid substitution, Ser102 or its conserved amino acid substitution, Val103 or its conserved amino acid substitution, Leu104 or its conserved amino acid substitution, Tyr105 or its conserved amino acid substitution, Tyr106 or its conserved amino acid substitution, Tyr107 or its conserved amino acid substitution, or any combination thereof. In various embodiments, each of Leu104, Tyr33, Asn101, Tyr107, Tyr60, Ser55, Ser52, or their conserved amino acid substitutions, forms a hydrogen bond with an amino acid of TIGIT. In various embodiments, when a TIGIT antigen-binding protein, such as a TIGIT antibody, binds to TIGIT, each of the above-named amino acid residues is located approximately 3 to 4 angstroms from an amino acid of TIGIT.

[0069] In exemplary embodiments, a TIGIT antigen-binding protein, such as an anti-TIGIT antibody, includes an LC variable region amino acid sequence that is highly similar to the first 105-115 amino acids of SEQ ID NO: 132 or SEQ ID NO: 254 (e.g., at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity)), and includes glutamic acid (Glu27) or its conserved amino acid substitution at position 27, leucine (Leu30) or its conserved amino acid substitution at position 30, serine (Ser32) or its conserved amino acid substitution at position 32, serine (Ser96) or its conserved amino acid substitution at position 96, isoleucine (Ile97) or its conserved amino acid substitution at position 97, glutamine (Gln98) or its conserved amino acid substitution at position 98, leucine (Leu99) or its conserved amino acid substitution at position 99, or any combination thereof. In exemplary embodiments, a TIGIT antigen-binding protein, for example, an anti-TIGIT antibody, includes an LC CDR1 amino acid sequence containing Gln27 or its conserved amino acid substitution, Leu30 or its conserved amino acid substitution, Ser32 or its conserved amino acid substitution, or any combination thereof. In exemplary embodiments, a TIGIT antigen-binding protein, for example, an anti-TIGIT antibody, includes an LC CDR3 amino acid sequence containing Ser96 or its conserved amino acid substitution, Ile97 or its conserved amino acid substitution, Gln98 or its conserved amino acid substitution, Leu99 or its conserved amino acid substitution, or any combination thereof. In exemplary embodiments, a TIGIT antigen-binding protein, for example, an anti-TIGIT antibody, includes an LC variable region amino acid sequence containing Ser32 or its conserved amino acid substitution, and / or Gln98 or its conserved amino acid substitution (each of which forms a hydrogen bond with an amino acid of TIGIT). In various embodiments, when a TIGIT antigen-binding protein, such as a TIGIT antibody, binds to TIGIT, the amino acid residues named above are located approximately 3 to 4 angstroms from the amino acids of TIGIT.

[0070] In exemplary embodiments, the TIGIT antigen-binding protein, e.g., anti-TIGIT antibody, is highly similar to the first 115-127 amino acids of SEQ ID NO: 131 or SEQ ID NO: 253 (e.g., at least or about 70% sequence identity (e.g., at least or about 80% sequence identity, at least or about 90% sequence identity, at least or about 95% sequence identity)), with aspartic acid (Asp33) or its conserved amino acid substitution at position 33, tyrosine (Tyr52) or its conserved amino acid substitution at position 52, tyrosine (Tyr54) or its conserved amino acid substitution at position 54, tyrosine (Tyr55) or its conserved amino acid substitution at position 55, serine (Ser56) or its conserved amino acid substitution at position 56, glycine (Gly57) or its conserved amino acid substitution at position 57, glycine (Gly58) or its conserved amino acid substitution at position 58, and treol The HC variable region amino acid sequence includes onine (Thr59) or its conserved amino acid substitution, tyrosine (Tyr60) or its conserved amino acid substitution at position 60, proline (Pro63) or its conserved amino acid substitution at position 63, lysine (Lys66) or its conserved amino acid substitution at position 66, isoleucine (Ile102) or its conserved amino acid substitution at position 102, alanine (Ala104) or its conserved amino acid substitution at position 104, glycine (Gly107) or its conserved amino acid substitution at position 107, tyrosine (Tyr108) or its conserved amino acid substitution at position 108, phenylalanine (Phe109) or its conserved amino acid substitution at position 109, tyrosine (Tyr110) or its conserved amino acid substitution at position 110, tyrosine (Tyr111) or its conserved amino acid substitution at position 111, or any combination thereof. In exemplary embodiments, the TIGIT antigen-binding protein, for example, an anti-TIGIT antibody, comprises an HC CDR1 amino acid sequence including Asp33 or its conserved amino acid substitution.In exemplary embodiments, the TIGIT antigen-binding protein, for example, an anti-TIGIT antibody, comprises an HC CDR2 amino acid sequence including Tyr52 or its conserved amino acid substitution, Tyr54 or its conserved amino acid substitution, Tyr55 or its conserved amino acid substitution, Ser56 or its conserved amino acid substitution, Gly57 or its conserved amino acid substitution, Gly58 or its conserved amino acid substitution, Thr59 or its conserved amino acid substitution, Tyr60 or its conserved amino acid substitution, Pro63 or its conserved amino acid substitution, Lys66 or its conserved amino acid substitution, or any combination thereof. In exemplary embodiments, a TIGIT antigen-binding protein, such as an anti-TIGIT antibody, comprises an HC CDR3 amino acid sequence including Ile102 or its conserved amino acid substitution, Ala104 or its conserved amino acid substitution, Gly107 or its conserved amino acid substitution, Tyr108 or its conserved amino acid substitution, Phe109 or its conserved amino acid substitution, Tyr110 or its conserved amino acid substitution, Phe111 or its conserved amino acid substitution, or any combination thereof. In various embodiments, each of Tyr52, Ser56, Thr59, Tyr60, Phe109, Tyr54, Tyr55, Lys66, or their conserved amino acid substitutions, forms a hydrogen bond with an amino acid of TIGIT. In various embodiments, when a TIGIT antigen-binding protein, such as a TIGIT antibody, binds to TIGIT, each of the above-named amino acid residues is located approximately 3 to 4 angstroms from an amino acid of TIGIT.

[0071] In exemplary embodiments, the antigen-binding protein comprises a heavy chain amino acid sequence containing a pair of charge-pair mutations, as described herein. In specific embodiments, the heavy chain amino acid sequence comprises charge-pair mutations selected from V1, V103, and V131 charge-pair mutations.

[0072] In further exemplary embodiments, the antigen-binding protein includes one or more amino acid modifications compared to its naturally occurring counterpart to improve half-life / stability or to make the antibody more suitable for expression / manufacturability. In exemplary examples, the antigen-binding protein is designed to block or reduce the interaction between Fc and the Fc receptor. In exemplary examples, the antigen-binding protein is a stable effector dysfunction (SEFL) antibody containing a constant region that lacks the ability to interact with the Fcγ receptor. SEFL antibodies are known in the art. See, for example, Liu et al., J Biol Chem 292:1876-1883 (2016); and Jacobsen et al., J. Biol. Chem. 292:1865-1875 (2017). In exemplary embodiments, the SEFL antibody contains one or more of the following mutations, numbered by the EU system: L242C, A287C, R292C, N297G, V302C, L306C, and / or K334C. In exemplary embodiments, the SEFL antibody contains N297G. In exemplary embodiments, the SEFL antibody contains A287C, N297G, and L306C. In other exemplary embodiments, the SEFL antibody contains R292C, N297G, and V302C (i.e., SEFL2-2). In various embodiments, the antigen-binding protein contains a heavy chain containing the amino acid sequence of SEQ ID NO: 2019, and optionally SEQ ID NO: 2020.

[0073] In various embodiments, the antigen-binding protein is an antibody containing an HC variable region encoded by the V gene segment of the VH1, VH3, or VH4 family of gene segments. In various embodiments, the antigen-binding protein is an antibody containing an HC variable region encoded by the D gene segment of the D1, D3, D5, D6, or D7 family of gene segments. In various embodiments, the antigen-binding protein is an antibody containing an HC variable region encoded by the J gene segment of the JH4 or JH6 family of gene segments. Optionally, the antigen-binding protein is an antibody containing an HC variable region encoded by the V gene segment of the VH3 family of gene segments, the D gene segment of the D1, D3, or D6 family of gene segments, and / or the J gene segment of the JH4 or JH6 family of gene segments. In various examples, the antigen-binding protein is an antibody containing an HC variable region encoded by the V gene segment of the VH3 family of gene segments, the D gene segment of the D1 family of gene segments, and the J gene segment of the JH6 family of gene segments. In various examples, the antigen-binding protein is an antibody containing an HC variable region encoded by the V gene segment of the VH3 family of gene segments, the D gene segment of the D3 family of gene segments, and the J gene segment of the JH6 family of gene segments. In various examples, the antigen-binding protein is an antibody containing an HC variable region encoded by the V gene segment of the VH3 family of gene segments, the D gene segment of the D6 family of gene segments, and the J gene segment of the JH6 family of gene segments. In various examples, the antigen-binding protein is an antibody containing an HC variable region encoded by the V gene segment of the VH1 family of gene segments, the D gene segment of the D5 family of gene segments, and the J gene segment of the JH6 family of gene segments.In various examples, antigen-binding proteins are antibodies that contain HC variable regions encoded by the V gene segment of the VH4 family of gene segments, the D gene segment of the D7 family of gene segments, and the J gene segment of the JH6 family of gene segments. In various examples, antigen-binding proteins are antibodies that contain HC variable regions encoded by the V gene segment of the VH3 family of gene segments, the D gene segment of the D1 family of gene segments, and the J gene segment of the JH4 family of gene segments.

[0074] polypeptide Polypeptides comprising, substantially comprising, or comprising one or more amino acid sequences from Table A1 or Table A2, Table B1 or Table B2, or Table C1 or Table C2 are provided herein. In various embodiments, the polypeptide comprises six of the CDRs from Table A1 or Table A2 having intervening amino acids. In various embodiments, the polypeptide comprises only one of the HC variable amino acid sequences or LC variable amino acid sequences from Table B1 or Table B2. In various embodiments, the polypeptide comprises both the HC variable amino acid sequence and the LC variable amino acid sequence from Table B1 or Table B2 fused as a single sequence, where optionally the HC variable amino acid sequence and the LC variable amino acid sequence are linked by a linker sequence. In various examples, the polypeptide comprises two copies of the HC variable region sequence and two copies of the LC variable region sequence, which are optionally linked by a linker sequence. In some embodiments, the polypeptide comprises the amino acid sequence scFv1, scFv2, or (scFv)2. In some embodiments, the polypeptide comprises amino acid sequences such as diabodies, triabodies, single-domain antibodies, single variable domains, tandem scFv, and tascFv. In various embodiments, the polypeptide comprises only one of the FL HC amino acid sequences or FL LC amino acid sequences from Table B1 or Table B2 or Table C1 or Table C2. In various embodiments, the polypeptide comprises both the FL HC amino acid sequence and the FL LC amino acid sequence from Table B1 or Table B2 or Table C1 or Table C2 fused as a single sequence, and optionally, the FL HC and FL LC are linked by a linker sequence. In exemplary embodiments, the polypeptide comprises a variant sequence of a parent sequence comprising an amino acid sequence from Table A1 or Table A2 or Table B1 or Table B2 or Table C1 or Table C2. In various embodiments, the polypeptide comprises an amino acid sequence that differs from the parent sequence by only one, two, three, four, five, six or more amino acids. In exemplary embodiments, the polypeptide comprises a variant sequence that differs from the parent sequence by only one or two amino acids.In exemplary embodiments, the polypeptide comprises a variant sequence having only one, two, three, four, five, six or more conserved amino acid substitutions compared to the above amino acid sequence.

[0075] In exemplary embodiments, the polypeptide comprises an amino acid sequence having about 30% or more, about 50% or more, or about 70% or more sequence identity with respect to the parent amino acid sequence. In exemplary embodiments, the polypeptide comprises an amino acid sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or more than 90% sequence identity with respect to the parent sequence. In exemplary embodiments, the polypeptide comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, or more than 90% sequence identity along the entire length of the parent sequence. In exemplary embodiments, the polypeptide comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity along the entire length of the parent sequence.

[0076] In alternative or additional embodiments of the present disclosure, polypeptides are further described herein by lipid addition (e.g., myristoylation, palmitoylation), glycosylation, amidation, carboxylation, phosphorylation, esterification, acylation, acetylation, cyclization, or conversion to an acid addition salt, and / or optionally by dimerization, polymerization, or conjugatement.

[0077] This specification provides peptide mimetic bodies designed to mimic the polypeptides of the present disclosure. The peptide mimetic bodies are substantially similar in structure to the polypeptides of the present disclosure, but have at least one structural difference. For example, a peptide mimetic body is a peptoid having one or more bonds in which one or more peptide bonds are substituted. In exemplary examples, a peptoid includes a side chain linked to the nitrogen of the peptide backbone, instead of the α-carbon as in the peptide. In some embodiments, the peptoids of the present disclosure lack amide hydrogens, which are involved in many of the secondary components in peptides and proteins. See, for example, Reyna et al., PNAS 89(20):9367-9371 (1992).

[0078] Peptide mimes and methods for producing them are known in the art. For example, see Advances in Amino Acid Mimetics and Peptidomimetics, Volumes 1 and 2, ed., Abell, A., JAI Press Inc., Greenwich, CT, 2006. In some embodiments, the peptide mime is a peptide mime of a D-peptide containing a D-isomer amino acid. In some embodiments, the peptide mime is a peptoid in which the side chain of the amino acid is bonded to the alpha nitrogen atom of the peptide backbone. Methods for producing peptoids are known in the art. For example, see Zuckermann et al., JACS 114(26):10646-10647 (1992) and Design, Synthesis, and Evaluation of Novel Peptoids, Fowler, Sarah, University of Wisconsin-Madison, 2008. In some embodiments, the peptide mime is a β-peptide containing a β-amino acid having an amino group bonded to the β-carbon rather than the α-carbon. Methods for producing β-peptides are known in the art. For example, see Seebach et al., Helvetica Chimica Acta 79(4):913-941 (1996).

[0079] Aptamer In some embodiments, the antigen-binding protein is an aptamer. Recent advances in the field of combinatorial science have identified short polymer sequences (e.g., oligonucleotides or peptide molecules) that have high affinity and specificity to a given target. For example, SELEX technology has been used to identify DNA and RNA aptamers with binding properties comparable to mammalian antibodies, antibodies or antibody fragments that bind to countless compounds have been generated and isolated in the field of immunology, and phage display has been used to discover novel peptide sequences with highly desirable binding properties. Based on the success of these molecular evolution techniques, it is certainly possible to construct molecules that bind to any target molecule. Loop structures are often involved in providing the desired binding attributes, as in the following examples: aptamers that often utilize hairpin loops constructed from short regions without complementary base pairing, naturally occurring antibodies that utilize combinatorial sequences of loop-shaped hypervariable regions, and novel phage display libraries that utilize cyclic peptides that have shown improved results compared to linear peptide phage display results. Therefore, there is sufficient evidence to suggest that high-affinity ligands can be generated and identified by combinatorial molecular evolution techniques. In this disclosure, molecular evolution techniques can be used to isolate TIGIT or CD112R-specific compounds that inhibit the binding interaction between TIGIT and CD155 or between CD112R and CD112. For details on aptamers, see Gold, L., Singer, B., He, YY, Brody. E., “Aptamers As Therapeutic And Diagnostic Agents,” J. Biotechnol. 74:5-13 (2000). Relevant techniques for generating aptamers can be found in U.S. Patent No. 6,699,843, which is incorporated in its entirety by reference.

[0080] Conjugate This disclosure also provides conjugates comprising one or more antigen-binding proteins of this disclosure linked to a heterologous moiety. As used herein, the term “heterologous moiety” is synonymous with the term “conjugate moiety” and refers to any molecule (chemical or biochemical, of natural origin or non-coding) that is different from the antigen-binding proteins described herein. Exemplary conjugate moieties that may be linked to any of the antigen-binding proteins described herein include, but are not limited to, heterologous peptides or polypeptides, targeting agents, radioisotopes, diagnostic labels such as fluorophores or enzyme labels, polymers including water-soluble polymers, or other therapeutic or diagnostic agents. In some embodiments, the conjugate comprises one or more antigen-binding proteins described herein and one or more peptides (different from the antigen-binding proteins described herein), polypeptides, nucleic acid molecules, another antibody or fragment thereof, polymers, quantum dots, small molecules, toxins, diagnostic agents, carbohydrates, and amino acids.

[0081] In exemplary embodiments, the conjugate of this disclosure comprises an antigen-binding protein as described herein and a heterologous portion which is a polypeptide (for example, a polypeptide different from any of the antigen-binding proteins described herein), wherein the conjugate is a fusion polypeptide or fusion protein, or a chimeric protein or chimeric polypeptide. Further descriptions of such conjugates are provided herein under “Fusion Protein.”

[0082] In some embodiments, the heterologous moiety is bound to the antigen-binding protein of the Disclosure by non-covalent or covalent bonds. In exemplary embodiments, the binding between the antigen-binding protein and the heterologous moiety is achieved by covalent chemical bonds, such as peptide bonds or disulfide bonds, or by physical forces, such as electrostatic, hydrogen, ionic, van der Waals, or hydrophobic or hydrophilic interactions. Various non-covalent bonding systems can be used, for example, including biotin-avidin, ligand / receptor, enzyme / substrate, nucleic acid / nucleic acid-binding protein, lipid / lipid-binding protein, cell adhesion molecule partner; or any binding partner or fragment thereof that have affinity for one another.

[0083] In exemplary embodiments, the antigen-binding protein is directly linked to the conjugate moiety via covalent bonds by reacting the target amino acid residues of the antigen-binding protein with an organic derivatizing agent capable of reacting with selected side chains or N-terminal or C-terminal residues of these target amino acids. Examples of reactive groups on the antigen-binding protein or conjugate moiety include aldehydes, aminos, esters, thiols, α-haloacetyls, maleimides, or hydrazino groups. Examples of derivatizing agents include maleimidobenzoylsulfosuccinimide (conjugation via cysteine ​​residues), N-hydroxysuccinimide (conjugation via lysine residues), glutaraldehyde, succinic anhydride, or other agents known in the art. Alternatively, the conjugate moiety can be indirectly linked to the antigen-binding protein via an intermediate carrier such as a polysaccharide or polypeptide carrier. An example of a polysaccharide carrier is aminodextran. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, copolymers thereof, and polymers of these amino acids with other substances that impart desirable solubility properties to the resulting carrier, such as serine.

[0084] Cysteinyl residues most commonly react with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to produce carboxymethyl or carboxyamidemethyl derivatives. Cysteinyl residues are also derivatized by reactions with bromotrifluoroacetone, alpha-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercrinebenzoic acid, 2-chloromercrine-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0085] The histidyl residue is derivatized by reaction with diethyl pyrocarbonate at pH 5.5–7.0 because this agent is relatively specific to the histidyl side chain. Para-bromophenacyl bromide is also useful, and this reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0.

[0086] The ricinyl and amino-terminal residues react with succinic acid or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the ricinyl residue. Other suitable reagents for derivatizing alpha-amino-containing residues include transaminase-catalyzed reactions with imide esters such as methyl picolineimide, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid; O-methylisourea, 2,4-pentanedione, and glyoxylate.

[0087] Arginine residues are modified by reaction with one or more conventional reagents, particularly phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Due to the high pKa of the guanidine functional group, derivatization of arginine residues requires the reaction to be carried out under alkaline conditions. Furthermore, these reagents can react with the lysine group and the arginine epsilon-amino group.

[0088] Specific modification of tyrosyl residues may be performed, particularly to introduce spectral labeling to tyrosyl residues through reactions with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidisole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively.

[0089] The carboxyl side group (aspartyl or glutamyl) is selectively modified by reaction with a carbodiimide (R'-N=C=N-R'), where R and R' are optionally different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, the aspartyl and glutamyl residues are converted to asparaginyl and glutamyl residues by reaction with an ammonium ion.

[0090] Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of ceryl or threonyl residues, methylation of alpha-amino groups of lysine side chains, arginine side chains, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983)), deamidation of asparagine or glutamine, acetylation of N-terminal amines, and / or amidation or esterification of C-terminal carboxylic acid groups.

[0091] Another type of covalent modification involves chemically or enzymatically coupling a glycoside to an antigen-binding protein. The sugar can be attached to (a) arginine and histidine, (b) a free carboxyl group, (c) a free sulfhydryl group such as that of cysteine, (d) a free hydroxyl group such as that of serine, threonine, or hydroxyproline, (e) an aromatic residue such as that of tyrosine or tryptophan, or (f) an amide group of glutamine. These methods are described in International Publication No. 87 / 05330, published on September 11, 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0092] In exemplary embodiments, heterologous portions are conjugated to the antigen-binding protein of the present disclosure via a linker. In some embodiments, the linker comprises a chain of atoms having a length of 1 to about 60, or 1 to 30 or more, 2 to 5, 2 to 10, 5 to 10, or 10 to 20 atoms. In some embodiments, all chain atoms are carbon atoms. In some embodiments, the chain atoms in the linker backbone are selected from the group consisting of C, O, N, and S. The chain atoms and linker may be selected based on their expected solubility (hydrophilicity) to provide a more soluble conjugate. In some embodiments, the linker provides a functional group that is cleaved by an enzyme or other catalyst or hydrolytic condition found in the target tissue or organ or cell. In some embodiments, the length of the linker is long enough to reduce the possibility of steric hindrance. If the linker is covalent or peptidyl and the conjugate is a polypeptide, the entire conjugate may be a fusion protein. Such a peptidyl linker may be of any length. Exemplary peptidyl linkers are approximately 1–50 amino acids, 5–50 amino acids, 3–5 amino acids, 5–10 amino acids, 5–15 amino acids, or 10–30 amino acids in length, and are flexible or rigid. In exemplary embodiments, the linker is a peptide containing approximately 2–20 amino acids. In exemplary embodiments, the linker is a peptide containing approximately 2–15 amino acids, approximately 2–10 amino acids, or approximately 2–5 amino acids. Suitable peptide linkers are known in the art. For example, Chen et al., Adv Drug Delivery. See Reviews 65(10):1357-1369(2013); Arai et al., Protein Eng Des Sel 14(8):529-532(2001); and Wriggers et al., Curr Trends in Peptide Science 80(6):736-746(2005). In various embodiments, the linker is a peptide containing the amino acid sequence GGGGS (SEQ ID NO: 2021).

[0093] Fusion protein In exemplary embodiments, the antigen-binding protein is conjugated to a polypeptide different from any of the antigen-binding proteins described herein, and the conjugate is a fusion polypeptide or fusion protein, or a chimeric protein or chimeric polypeptide. Accordingly, this disclosure provides a fusion polypeptide or fusion protein comprising the antigen-binding protein and a heterogeneous polypeptide or peptide of this disclosure. In exemplary embodiments, the fusion protein of this disclosure comprises an HC variable amino acid sequence fused to an LC variable amino acid sequence or an FL HC sequence fused to an FL LC sequence. In various embodiments, the fusion protein comprises a peptide linker between the HC variable amino acid sequence and the LC variable amino acid sequence, or between the FL HC sequence and the FL LC sequence.

[0094] nucleic acid This disclosure further provides nucleic acids comprising nucleotide sequences encoding antigen-binding proteins or polypeptides or fusion proteins of this disclosure. “Nucleic acid,” as used herein, includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or RNA or a modified form thereof, which may be single-stranded or double-stranded, may be synthesized or obtained from natural origin (e.g., isolated and / or purified), may contain natural, unnatural or modified nucleotides, and may contain natural, unnatural or modified nucleotide linkages, such as phosphoramidate links or phosphorothioate links, instead of phosphodiesters found between nucleotides in unmodified oligonucleotides. A nucleic acid may contain any nucleotide sequence encoding any of the antigen-binding proteins or polypeptides of this disclosure. In some embodiments, the nucleic acid may contain no insertions, deletions, inversions, and / or substitutions. In other embodiments, the nucleic acid may contain one or more insertions, deletions, inversions, and / or substitutions.

[0095] In some embodiments, the nucleic acids of this disclosure are recombinants. As used herein, the term “recombinant” means (i) a molecule constructed outside of a living cell by linking a natural or synthetic nucleic acid segment to a nucleic acid molecule that can replicate in a living cell, or (ii) a molecule resulting from replication of the one described in (i) above. For the purposes of this specification, replication may be in vitro replication or in vivo replication.

[0096] Nucleic acids in some embodiments are constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, for example, Sambrook et al. (cited above) and Ausubel et al. (cited above). For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to improve the biological stability of the molecule or the physical stability of the double helix formed during hybridization. Examples of modified nucleotides that can be used to produce nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylkeosin, inosine, N 6 -Isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-substituted adenine, 7-methylguanine, 5-methylammomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylkeosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6Examples include isopentenyl adenine, uracil-5-oxyacetic acid(v), weybutosine, pseudouracil, keosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of this disclosure can be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0097] In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an antigen-binding protein or polypeptide or fusion protein of the Disclosure. In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an amino acid sequence having the sequence numbers listed in Table A1 or Table A2 or Table B1 or Table B2 or Table C1 or Table C2. In various embodiments, the nucleotide sequence encodes an amino acid sequence comprising six of the CDRs of Table A1 or Table A2 having intervening amino acids. In various embodiments, the nucleotide sequence encodes only one of the HC-variable or LC-variable amino acid sequences of Table B1 or Table B2. In various embodiments, the nucleotide sequence encodes both the HC-variable amino acid sequence and the LC-variable amino acid sequence of Table B1 or Table B2 fused as a single sequence, where optionally, the HC-variable amino acid sequence and the LC-variable amino acid sequence are linked by a linker sequence. In various examples, the nucleotide sequence encodes a polypeptide as described herein. In various embodiments, the nucleotide sequence encodes only one of the FL-HC or FL-LC amino acid sequences of Table B1 or Table B2 or Table C1 or Table C2. In various embodiments, the nucleotide sequences are fused as a single sequence in the FL of Table B1 or Table B2 or Table C1 or Table C2. The nucleotide sequence includes both the HC amino acid sequence and the FL LC amino acid sequence, where, optionally, the FL HC and FL LC are linked by a linker sequence. In an exemplary embodiment, the nucleotide sequence encodes a variant sequence of a parent sequence containing the amino acid sequence of Table A1 or Table A2 or Table B1 or Table B2 or Table C1 or Table C2. In various embodiments, the nucleotide sequence encodes an amino acid sequence that differs from the parent sequence by only one, two, three, four, five, six or more amino acids. In an exemplary embodiment, the nucleotide sequence encodes a variant sequence that differs from the parent sequence by only one or two amino acids. In an exemplary embodiment, the nucleotide sequence encodes a variant sequence that has only one, two, three, four, five, six or more conserved amino acid substitutions compared to the above amino acid sequence. In an exemplary embodiment, the nucleic acid contains one nucleotide sequence of sequence numbers 2037 to 2092, as shown in Table D. In various embodiments, one nucleotide sequence of sequence numbers 2037-2092 includes a nucleotide sequence encoding a signal sequence. In various embodiments, the nucleic acid of the Disclosure includes one nucleotide sequence of sequence numbers 2037-2092 that does not include a nucleotide sequence encoding a signal sequence. In various embodiments, the nucleic acid includes one nucleotide sequence of sequence numbers 2037-2092 that does not include the first 60, 63, 66, or 69 of any one of sequence numbers 2037-2092.

[0098] [Table 7]

[0099] vector In some embodiments, the nucleic acids of the Disclosure are incorporated into a vector. In this regard, the Disclosure provides a vector comprising any of the nucleic acids of the Disclosure. In exemplary embodiments, the vector is a recombinant expression vector. For the purposes of this Spectrum, the term “recombinant expression vector” means a genetically modified oligonucleotide or polynucleotide construct that enables the expression of mRNA, protein, polypeptide, or peptide by a host cell, where the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector is brought into contact with a cell under conditions sufficient to have mRNA, protein, polypeptide, or peptide expressed in the cell. The vectors of the Disclosure as a whole do not exist in nature. However, parts of the vectors may exist in nature. The vectors of the Disclosure may contain any type of nucleotide (including, but not limited to, DNA and RNA, which may be single-stranded or double-stranded, may be partially obtained synthetically or from natural sources, and may contain natural, non-natural, or modified nucleotides. The vectors may contain nucleotide-nucleotide bonds that are naturally present or not naturally present, or both types of bonds. In some embodiments, modified nucleotides or internucleotide bonds that do not exist naturally do not interfere with the transcription or replication of the vector.

[0100] The vectors of this disclosure may be any suitable vector and may be used to transform or transfect any suitable host. Suitable vectors include those designed for growth and expansion, or for expression, or both, such as plasmids and viruses. Vectors can be selected from the group consisting of the pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJoIIa, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors such as λGTIO, λGTl1, λZapII (Stratagene), λEMBL4, and λNMl149 can also be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). In some embodiments, the vector is a viral vector, such as a retroviral vector.

[0101] The vectors of this disclosure can be prepared, for example, using standard recombinant DNA techniques described in Sambrook et al. (previously cited) and Ausubel et al. (previously cited)a. The circular or linear expression vector constructs can be prepared to include a functional replication system in prokaryotic or eukaryotic host cells. The replication system may be derived from, for example, CoIEl, 2μ plasmid, λ, SV40, bovine papilloma virus, etc.

[0102] In some embodiments, the vector is specific to the type of host into which the vector is introduced (e.g., bacteria, fungi, plants, or animals), and, if necessary, includes regulatory sequences such as start and stop codons for transcription and translation, taking into consideration whether the vector is DNA-based or RNA-based.

[0103] The vector may contain one or more marker genes that enable selection of the transformed or transfected host. Marker genes may include biocide resistance, such as resistance to antibiotics and heavy metals, and complementarity that provides protrophotrophy in a trophic host. Suitable marker genes for expression vectors in this disclosure include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0104] A vector may include a nucleotide sequence encoding a polypeptide (including its functional portion and functional variants), or a native or normative promoter operably ligated to a nucleotide sequence complementary to or hybridizing with a nucleotide sequence encoding an antigen-binding protein. The selection of promoters, e.g., strong, weak, inducible, tissue-specific, and development-specific promoters, is within the scope of the skill of those skilled in the art. Similarly, the combination of nucleotide sequences and promoters is also within the scope of the skill of those skilled in the art. Promoters may be non-viral promoters or viral promoters, e.g., cytomegalovirus (CMV) promoters, SV40 promoters, RSV promoters, and promoters found in the long-terminal repeats of mouse stem cell viruses.

[0105] In some embodiments, the vector encodes an antibody light chain, an antibody heavy chain, or both an antibody light chain and a heavy chain. A vector encoding an antibody light chain may be useful for producing the antibody of the present invention when expressed in cells further containing a vector encoding an antibody heavy chain. Similarly, a vector encoding an antibody heavy chain may be useful for producing the antibody of the present invention when expressed in cells further containing a vector encoding an antibody light chain. Thus, both light and heavy chains may be encoded on a single vector, although in certain embodiments the vector encodes a light chain but not a heavy chain. In other embodiments the vector encodes a heavy chain but not a light chain.

[0106] In various embodiments, the vectors of the present invention include a nucleotide sequence encoding an HC variable region or full-length HC, and a nucleotide sequence encoding an LC variable region or full-length LC. In alternative embodiments, the vectors of the present disclosure include a nucleotide sequence encoding an HC variable region or full-length HC as described in Table B1, C1, B2, or C2, or a nucleotide sequence encoding an LC variable region or full-length LC as described in Table B1, C1, B2, or C2.

[0107] host cell This specification provides host cells containing one or more nucleic acids or vectors of this disclosure. As used herein, the term “host cell” means any type of cell that may contain the vectors disclosed herein and may produce expression products encoded by nucleic acids (e.g., mRNA, proteins). In some embodiments, host cells are adherent cells or suspension cells, i.e., cells that grow in a suspension. In exemplary embodiments, host cells are cultured cells or primary cells, i.e., cells isolated directly from an organism. Host cells may be of any cell type, may originate from any type of tissue, and may be at any developmental stage.

[0108] In exemplary embodiments, the cell is a eukaryotic cell, and includes, but is not limited to, yeast cells, filamentous fungal cells, protist cells, algal cells, insect cells, or mammalian cells. Such host cells have been described in the art. For example, Frenzel, et al. See al., Front Immunol 4:217 (2013). In exemplary embodiments, eukaryotic cells are mammalian cells. In exemplary embodiments, mammalian cells are non-human mammalian cells. In some embodiments, the cells include Chinese hamster ovary (CHO) cells and their derivatives (e.g., CHO-K1, CHO pro-3, CS9), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2 / 0), cells engineered to lack dihydrofolate reductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or their derivatives (e.g., HEK293T, HEK293-EBNA), African green monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa), human osteosarcoma epithelial cells U2-OS, adenocarcinoma human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic carcinoma cells P19, and mouse embryonic fibroblast cells NIH These include 3T3, mouse fibroblast L929, mouse neuroblastoma N2a, human breast cancer MCF-7, retinoblastoma Y79, human retinoblastoma SO-Rb50, human liver cancer Hep G2, mouse B myeloma J558L, or neonatal hamster kidney (BHK) cells (Gaillet et al. 2007; Khan, Adv Pharm Bull 3(2):257-263(2013)). In certain embodiments, the host cell is CS9 (CHO cell line).

[0109] For the purpose of amplifying or replicating the vector, the host cell is, in some embodiments, a prokaryotic cell, such as a bacterial cell.

[0110] A population of cells comprising at least one type of host cell described herein is also provided by this disclosure. In some embodiments, the population of cells is a heterogeneous population comprising host cells containing the described vector, in addition to at least one other type of cell that does not contain any vector. Alternatively, in some embodiments, the population of cells is a substantially homogeneous population, which comprises (e.g., substantially consists of) host cells containing the vector. In some embodiments, the population is a clonal population of cells in which all cells in the population are clones of a single host cell containing the vector, such that all cells in the population contain the vector. In exemplary embodiments of this disclosure, the population of cells is a clonal population comprising host cells containing the vector described herein.

[0111] In various embodiments of this disclosure, the host cell comprises a first vector comprising a nucleotide sequence encoding an HC variable region or full-length HC as described in Table B1, C1, B2, or C2, or a second vector comprising a nucleotide sequence encoding an LC variable region or full-length LC as described in Table B1, C1, B2, or C2.

[0112] Pharmaceutical composition Compositions comprising antigen-binding proteins (e.g., TIGIT-binding protein, CD112R-binding protein), polypeptides, nucleic acids, vectors, host cells, conjugates, fusion proteins, or combinations thereof are provided herein. In some embodiments, compositions comprise antigen-binding proteins, polypeptides, conjugates, fusion proteins, nucleic acids, vectors, or host cells, or combinations thereof, in isolated and / or purified forms. In some embodiments, compositions comprise a single type (e.g., structure) of the antigen-binding proteins, polypeptides, conjugates, fusion proteins, nucleic acids, vectors, or host cells, or a combination of two or more different types (e.g., different structures) of the antigen-binding proteins, polypeptides, conjugates, fusion proteins, nucleic acids, vectors, or host cells.

[0113] In exemplary embodiments, a composition includes an agent that enhances the chemophysical characteristics of an antigen-binding protein, polypeptide, conjugate, fusion protein, nucleic acid, vector, or host cell, for example, by stabilization at a specific temperature (e.g., room temperature), increased shelf life, reduced degradation (e.g., oxidative protease-mediated degradation), or increased half-life of an antigen-binding protein. In some embodiments, a composition optionally includes, as a heterologous or conjugate portion, any of the agents disclosed herein in a mixture with the antigen-binding protein or polypeptide of this disclosure.

[0114] In exemplary embodiments of this disclosure, the composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, an antigen-binding protein, polypeptide, conjugate, fusion protein, nucleic acid, vector, or host cell (hereinafter referred to as the "active agent") disclosed herein is formulated together with a pharmaceutically acceptable carrier, diluent, or excipient into a pharmaceutical composition comprising the active agent. In this regard, this disclosure provides pharmaceutical compositions comprising an active agent intended for administration to a subject, for example, a mammal.

[0115] In some embodiments, the active agent is present in the pharmaceutical composition at a purity level suitable for administration to a patient. In some embodiments, the active agent has a purity level of at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, and has a pharmaceutically acceptable diluent, carrier, or excipient. In some embodiments, the composition contains a surfactant at a concentration of about 0.001 to about 30.0 mg / ml.

[0116] In exemplary embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” includes any standard pharmaceutical carrier, such as phosphate-buffered saline, water, emulsions such as oil / water or water / oil emulsions, and various types of wetting agents. The term also includes any agent approved by a U.S. federal regulatory authority or listed in the United States Pharmacopeia for use in animals, including humans.

[0117] This pharmaceutical composition may contain, for example, acidifying agents, additives, adsorbents, aerosol propellants, exhaust agents, alkalizing agents, anticaking agents, anticoagulants, antimicrobial preservatives, antioxidants, preservatives, bases, binders, buffers, chelating agents, coating agents, colorants, drying agents, surfactants, diluents, disinfectants, disintegrants, dispersants, dissolution accelerators, pigments, softeners, emulsifiers, emulsion stabilizers, fillers, film-forming agents, flavor enhancers, flavoring agents, flow accelerators, gelling agents, and other materials. It may contain any pharmaceutically acceptable ingredients, including granules, water-retaining agents, lubricants, mucosal adhesives, ointment bases, ointments, oily vehicles, organic bases, tablet bases, pigments, plasticizers, abrasives, preservatives, metal ion chelating agents, skin penetration agents, solubilizers, solvents, stabilizers, suppository bases, surfactants, surfactants, suspending agents, sweeteners, therapeutic agents, thickeners, isotonic agents, toxic agents, viscosity enhancers, water absorbents, water-miscible cosolvents, hard water softeners, or wetting agents. For example, see the Handbook of Pharmaceutical Excipients, Third Edition, AHKibbe (Pharmaceutical Press, London, UK, 2000). This document is incorporated by reference in its entirety. Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980). This document is referenced in its entirety.

[0118] In exemplary embodiments, the pharmaceutical composition comprises formulation materials that are non-toxic to the recipient at the dosage and concentration used. In specific embodiments, the pharmaceutical composition comprises an activator and one or more pharmaceutically acceptable salts, polyols, surfactants, osmotic balancers, isotonic agents, antioxidants, antibiotics, antifungals, fillers, lyophilization protectants, defoamers, chelating agents, preservatives, colorants, analgesics, or additional pharmaceutical agents. In exemplary embodiments, the pharmaceutical composition optionally comprises one or more excipients, for example, pharmaceutically acceptable salts, osmotic balancers (isotonic agents), antioxidants; antibiotics, antifungals, fillers, lyophilization protectants, defoamers; chelating agents, preservatives, colorants, and analgesics, in addition to one or more polyols and / or one or more surfactants.

[0119] In certain embodiments, the pharmaceutical composition may contain, for example, formulation materials for altering, maintaining, or preserving the composition's pH, volume molar osmotic pressure concentration, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeability. In such embodiments, suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antibacterial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulin); colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; and salt-forming counterions. Examples of preservatives include (sodium, etc.); preservatives (benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide, etc.); solvents (glycerin, propylene glycol, or polyethylene glycol, etc.); sugar alcohols (mannitol or sorbitol, etc.); suspending agents; surfactants or wetting agents (pluronic, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbatec, triton, tromethamine, lecithin, cholesterol, tyroxapol); stabilization enhancers; isotonic enhancers (alkali metal halides, etc., preferably sodium chloride or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants, but are not limited to these. See REMINGTON'S PHARMACEUTICAL SCIENCES, 18" Edition, (ARGenrmo, ed.), 1990, Mack Publishing Company.

[0120] The pharmaceutical composition can be formulated to achieve a physiologically compatible pH. In some embodiments, the pH of the pharmaceutical composition can be, for example, about 4 or about 5 to about 8.0, or about 4.5 to about 7.5, or about 5.0 to about 7.5.

[0121] Route of administration With respect to this disclosure, the active agent or a pharmaceutical composition containing the same may be administered to a subject by any preferred route of administration. For example, the active agent may be administered to a subject by parenteral, nasal, oral, pulmonary, topical, vaginal, or rectal administration. The following description of the route of administration is provided solely to illustrate exemplary embodiments and should not be construed as limiting the scope.

[0122] Suitable formulations for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the recipient's blood, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The term "parenteral" means administration via some other route, such as subcutaneous, intramuscular, intraspinal, or intravenous, rather than through the gastrointestinal tract. The active agents of this disclosure may be administered together with a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or liquid mixture, which may contain pharmaceutically acceptable surfactants such as water, physiological saline, aqueous dextrose and related sugar solutions, alcohols such as ethanol or hexadecyl alcohol, glycols such as propylene glycol or polyethylene glycol, dimethyl sulfoxide, glycerol, ketals such as 2,2-dimethyl-153-dioxolane-4-methanol, ethers, poly(ethylene glycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides with or without pharmaceutically acceptable surfactants such as soap or surfactants, pectin, carbomer, methylcellulose, hydroxypropyl methylcellulose, or carboxymethylcellulose, or emulsifiers and other pharmaceutical adjuvants.

[0123] Oils that can be used in parenteral formulations include petroleum, animal oils, vegetable oils, and synthetic oils. Specific examples of oils include peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum, and mineral oil. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0124] Suitable soaps for use in parenteral formulations include fatty acid alkali metal salts, ammonium salts, and triethanolamine salts, and suitable surfactants include (a) cationic surfactants, such as dimethyldialkylammonium halides and alkylpyridinium halides; (b) anionic surfactants, such as alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglycerides, sulfates and sulfosuccinates; (c) nonionic surfactants, such as fatty acid amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers; (d) amphoteric surfactants, such as alkyl-β-aminopropionates and 2-alkyl-imidazoline quaternary ammonium salts; and (e) mixtures thereof.

[0125] In some embodiments, the parenteral formulation contains about 0.5% to about 25% by weight of the active agent of the present disclosure in solution. Preservatives and buffers may be used. To minimize or eliminate irritation at the injection site, such compositions may contain one or more nonionic surfactants having a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such formulations is typically in the range of about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters such as sorbitan monooleate, and high molecular weight adducts of ethylene oxide having a hydrophobic base formed by the condensation of propylene oxide and propylene glycol. In some embodiments, the parenteral formulation is provided in sealed containers of unit dose or multiple doses, such as ampoules and vials, and can be stored in a freeze-dried state requiring only the addition of a sterile liquid additive for injection, such as water, immediately before use. In some embodiments, the immediate-compound injectable solutions and suspensions are prepared from the types of sterile powders, granules, and tablets previously described.

[0126] The injectable formulations shall be as described herein. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those skilled in the art (see, for example, Pharmaceuticals and Pharmacy Practice, JBLippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).

[0127] Dosage The active agents of this disclosure are considered useful in methods of inhibiting biological activity initiated by the binding of TIGIT to CD155 or CD112R to CD112, as described herein, and are therefore considered useful in methods of increasing immune responses, such as T cell-mediated immune responses, and in methods of treating or preventing one or more diseases, such as cancer. For the purposes of this disclosure, the amount or dose of the active agent administered should be sufficient to produce an effect, such as a therapeutic or prophylactic response, in a subject or animal over a reasonable period of time. For example, the dose of the active agent should be sufficient to treat cancer as described herein for a period of about 1 to 4 hours, 1 to 4 days, or 1 to 4 weeks or longer, for example, 5 to 20 weeks or longer, from the time of administration. In certain embodiments, the period may be longer. The dose is determined by the efficacy of the particular active agent and the condition of the animal (e.g., human) and the body weight of the animal being treated (e.g., human).

[0128] Assays for determining the dose to be administered are numerous and known in the art. For the purposes of this specification, it is possible to determine the starting dose to be administered to mammals using an assay that includes comparing the extent to which cancer is treated when a given volume of the active agent of this disclosure is administered to mammals, in groups of mammals each given different doses of the active agent. The extent to which cancer is treated when a particular dose is administered can be expressed, for example, by the cytotoxicity of the active agent or by the degree of tumor regression achieved with the active agent in a mouse xenograft model. Methods for measuring the cytotoxicity of antigen-binding proteins and methods for assaying tumor regression are known in the art. Briefly, to assay in vivo tumor regression, a tumor xenograft can be established by subcutaneously transplanting human tumor cells suspended in PBS (pH 7.4) into the right flank of a mouse. The tumor can be measured using a digital caliper. Tumor volume V (mm²) 3 The formula is V=(π / 6)LS 2This can be calculated using the formula (where L is the maximum surface diameter and S is the minimum surface diameter). For example, see Shi et al., mAbs 9(4):3740-3752 (2015).

[0129] The dosage of the active agents of this disclosure will also be determined by the presence, nature, and severity of adverse side effects that may occur with the administration of the particular active agent of this disclosure. Typically, the attending physician will determine the dosage of the active agent of this disclosure for each individual patient by considering various factors such as age, weight, overall health, diet, sex, the active agent of this disclosure being administered, the route of administration, and the severity of the condition being treated. For example (not intended to limit this disclosure), the dosage of the active agent of this disclosure may be approximately 0.0001 to 1 g / day per kg of body weight of the person being treated, approximately 0.0001 to 0.001 g / day per kg of body weight, or approximately 0.01 to 1 g / day per kg of body weight.

[0130] Controlled-release formulation In some embodiments, the active agents described herein may be modified into depot forms so that the manner in which the active agent is released into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Patent No. 4,450,150). The depot forms of the active agents of the disclosure may be, for example, an implantable composition comprising the active agent and a porous or non-porous material such as a polymer, wherein the active agent is encapsulated or diffused by or through the material and / or diffused by the decomposition of the non-porous material. The depot is then implanted at a desired location within the body of the subject, and the active agent is released from the implant at a predetermined rate.

[0131] A pharmaceutical composition containing an active agent in a particular embodiment may be modified to have any type of in vivo release profile. In some embodiments, the pharmaceutical composition may be an immediate-release, controlled-release, sustained-release, long-release, delayed-release, or two-stage-release formulation. Methods for formulating peptides for controlled-release are known in the art. See, for example, Qian et al., J Pharm 374:46-52 (2009) and International Publication Nos. 2008 / 130158, 2004 / 033036, 2000 / 032218, and 1999 / 040942.

[0132] The composition may further include, for example, micelles, liposomes, or other encapsulation forms, or it may be administered in a long-release form to provide long-term storage and / or delivery effects.

[0133] combination In some embodiments, the active agents described herein are administered alone, and in alternative embodiments, they are administered in combination with another therapeutic agent, for example, another active agent of the present disclosure of a different type (e.g., structure). Accordingly, the present disclosure provides combinations comprising a first antigen-binding protein targeting CD112R and a second antigen-binding protein targeting TIGIT (each of these being an antigen-binding protein according to the present disclosure). In various embodiments, the first antigen-binding protein is one of 1E1, 1E1.016, 24F1, 29E10, 24F1.001, 29E10_CONS.020, 29E10_CONS.021, 29E10_CONS.022, 29E10_CONS.025, 11E4, 31B3, 27G12, 28F9, 28H7, or 36C8, as listed in Table A1 or Table B1. In various embodiments, the second antigen-binding protein is one of 55G7.041.008, 58A7.003.008.075, 4G10, 11A3, 28B8, 39D2, 43B7, 55G7, 66H9, 43B7.002.015, 58A7.003.08, 66H9.009, or 58A7, as listed in Table A2 or Table B2. In various examples, the first antigen-binding protein is 24F1, 29E10_CONS.020, or 29E10_CONS.022. In exemplary embodiments, the second antigen-binding protein is 43B7.002.015, or 66H9.009. In one embodiment, the combination includes 24F1 and 43B7.002.015. In another embodiment, the combination includes 24F1 and 66H9.009. In one embodiment, the combination includes 29E10_CONS.020 and 43B7.002.015. In another embodiment, the combination includes 29E10_CONS.020 and 66H9.009. In one embodiment, the combination includes 29E10_CONS.022 and 43B7.002.015. In another embodiment, the combination includes 29E10_CONS.022 and 66H9.009. In yet another embodiment, the combination includes 43B7.002.015 and 1E1.016 or 24F1 or 29E10. In yet another embodiment, the combination includes 66H9.009 and 1E1.016 or 24F1 or 29E10.In exemplary embodiments, the combination includes 43B7 and 29E10 or 24F1 or 11E4.

[0134] This disclosure provides, in various examples, combinations as compositions, for example, pharmaceutical compositions. Accordingly, this disclosure provides compositions, for example, pharmaceutical compositions, comprising a first antigen-binding protein and a second antigen-binding protein. In various examples, the first antigen-binding protein is 24F1, 29E10_CONS.020, or 29E10_CONS.022. In exemplary embodiments, the second antigen-binding protein is 43B7.002.015, or 66H9.009. In one embodiment, the combination comprises 24F1 and 43B7.002.015. In another embodiment, the composition comprises 24F1 and 66H9.009. In one embodiment, the composition comprises 29E10_CONS.020 and 43B7.002.015. In another embodiment, the composition comprises 29E10_CONS.020 and 66H9.009. In one embodiment, the composition comprises 29E10_CONS.022 and 43B7.002.015. In another embodiment, the composition comprises 29E10_CONS.022 and 66H9.009. In yet another embodiment, the combination comprises 43B7.002.015 and 1E1.016 or 24F1 or 29E10. In yet another embodiment, the combination comprises 66H9.009 and 1E1.016 or 24F1 or 29E10. In an exemplary embodiment, the combination comprises 43B7 and 29E10 or 24F1 or 11E4. In the exemplary example, the first antigen-binding protein and the second antigen-binding protein are present in the composition in a ratio of approximately 1:1.

[0135] In some embodiments, the combination or composition further comprises an additional active agent, such as a third antigen-binding protein. Optionally, the third antigen-binding protein binds to negative regulators, immunosuppressives, or immune checkpoint proteins of the immune system, including, but not limited to, CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, CEACAM-1, TIGIT, LAG3, CD112, CD112R, CD96, TIM3, BTLA, or the costimulatory receptors ICOS, OX40, 41BB, CD27, GITR. In various examples, the additional active agent is a PD-1 binding protein, such as an anti-PD-1 antibody. Examples of anti-PD-1 antibodies include nivolumab (BMS-936558), pembrolizumab (MK3475), BMS 936558, BMS-936559, TSR-042 (Tesaro), ePDR001 (Novartis), and pidilizumab (CT-011). Optionally, the third antigen-binding protein is any PD-1 antigen-binding protein described in International Patent Application No. PCT / US2019 / 013205 (published as International Publication No. 2019 / 140196, the entire contents of which are incorporated herein by reference). In the exemplary example, the third antigen-binding protein includes the HC CDR1 amino acid sequence, HC CDR2 amino acid sequence, HC CDR3 amino acid sequence, LC CDR1 amino acid sequence, LC CDR2 amino acid sequence, and LC CDR3 amino acid sequence, respectively, of Sequence IDs 352-357 of International Publication No. 2019 / 140196. In the exemplary example, the third antigen-binding protein includes the HC CDR1 amino acid sequence, HC CDR2 amino acid sequence, and LC CDR3 amino acid sequence, respectively, of Sequence IDs 2027-2032. The third antigen-binding protein includes the CDR1 amino acid sequence, the HC CDR2 amino acid sequence, the HC CDR3 amino acid sequence, the LC CDR1 amino acid sequence, the LC CDR2 amino acid sequence, and the LC CDR3 amino acid sequence. In various embodiments, the third antigen-binding protein includes the HC variable region amino acid sequence and the LC variable region amino acid sequence of SEQ ID NO. 358 and SEQ ID NO. 359, respectively, in International Publication No. 2019 / 140196. In various embodiments, the third antigen-binding protein includes the HC variable region amino acid sequence and the LC variable region amino acid sequence of SEQ ID NO. 2033 and SEQ ID NO. 2034, respectively. In various examples, the third antigen-binding protein includes the FL HC amino acid sequence and the FL LC amino acid sequence of SEQ ID NO. 360 and SEQ ID NO. 361, respectively, in International Publication No. 2019 / 140196. In various examples, the third antigen-binding protein includes the FL HC amino acid sequence and the FL LC amino acid sequence of SEQ ID NO. 2035 and SEQ ID NO. 2036, respectively. In various examples, the third antigen-binding protein contains the FL HC amino acid sequence and the FL LC amino acid sequence of SEQ ID NO: 2093 and SEQ ID NO: 2094, respectively. Optionally, the first, second, and third antigen-binding proteins are present in the composition in a ratio of approximately 1:1:1.

[0136] In some embodiments, the other therapeutic agent is intended to treat or prevent cancer. In some embodiments, the other therapeutic agent is a chemotherapeutic agent. In some embodiments, the other therapeutic agent is an agent used in radiotherapy for the treatment of cancer. Accordingly, in some embodiments, the active agents described herein are administered in combination with one or more platinum-coordinate compounds, topoisomerase inhibitors, antibiotics, antimitotic alkaloids, and difluoronucleosides.

[0137] kit This disclosure further provides kits comprising antigen-binding proteins, polypeptides, conjugates, fusion proteins, nucleic acids, vectors, or host cells of the Disclosure, or combinations thereof. In exemplary embodiments, a kit contains, in a container, at least one antigen-binding protein, polypeptide, conjugate, fusion protein, nucleic acid, vector, or host cell of the Disclosure, or combinations thereof. In exemplary embodiments, at least one antigen-binding protein, polypeptide, conjugate, fusion protein, nucleic acid, vector, or host cell of the Disclosure is provided in the kit as a unit dose. For the purposes of this specification, “unit dose” refers to the amount dispersed in a suitable carrier. In exemplary embodiments, a unit dose is an amount sufficient to provide a desired effect to a subject, for example, the treatment of cancer. In exemplary embodiments, a kit contains several unit doses, for example, a unit dose for a week or a month's supply, which are optionally packaged individually or separated from other unit doses. In some embodiments, the components of the kit / unit dose are packaged together with instructions for administration to a patient. In some embodiments, the kit includes one or more devices for administration to a patient, such as a needle and a syringe. In some embodiments, at least one antigen-binding protein, polypeptide, conjugate, fusion protein, nucleic acid, vector, or host cell, or a combination thereof, is pre-packaged in a ready-to-use form, such as a syringe or infusion bag. In exemplary embodiments, the ready-to-use form is for single use. In exemplary embodiments, the kit includes multiple ready-to-use single-use forms of at least one antigen-binding protein, polypeptide, conjugate, fusion protein, nucleic acid, vector, or host cell of the disclosure. In some embodiments, the kit further includes other therapeutic or diagnostic agents or pharmaceutically acceptable carriers (e.g., solvents, buffers, diluents, etc.) containing any of those described herein.

[0138] In various embodiments, the kit comprises two or more antigen-binding proteins of this disclosure. In an exemplary example, the kit comprises a first antigen-binding protein that binds to CD112R and a second antigen-binding protein that binds to TIGIT. Optionally, the first antigen-binding protein is formulated together with the second antigen-binding protein. In some embodiments, the kit comprises a composition comprising the first antigen-binding protein and the second antigen-binding protein. In various embodiments, the first antigen-binding protein is packaged and / or formulated separately from the second antigen-binding agent. In various cases, the first antigen-binding protein is one of the following listed in Table A1 or Table B1: 1E1, 1E1.016, 24F1, 29E10, 24F1.001, 29E10_CONS.020, 29E10_CONS.021, 29E10_CONS.022, 29E10_CONS.025, 11E4, 31B3, 27G12, 28F9, 28H7, or 36C8. In various embodiments, the second antigen-binding protein is one of 55G7.041.008, 58A7.003.008.075, 4G10, 11A3, 28B8, 39D2, 43B7, 55G7, 66H9, 43B7.002.015, 58A7.003.08, 66H9.009, or 58A7, as listed in Table A2 or Table B2. In various examples, the first antigen-binding protein is 24F1, 29E10_CONS.020, or 29E10_CONS.022. In exemplary embodiments, the second antigen-binding protein is 43B7.002.015, or 66H9.009. In one embodiment, the kit includes 24F1 and 43B7.002.015. In another embodiment, the kit includes 24F1 and 66H9.009. In one embodiment, the kit includes 29E10_CONS.020 and 43B7.002.015. In another embodiment, the kit includes 29E10_CONS.020 and 66H9.009. In one embodiment, the kit includes 29E10_CONS.022 and 43B7.002.015. In another embodiment, the kit includes 29E10_CONS.022 and 66H9.009. In another embodiment, the kit includes 43B7.002.015 and 1E1.016 or 24F1 or 29E10.In yet another embodiment, the kit comprises 66H9.009 and 1E1.016 or 24F1 or 29E10. In an exemplary embodiment, the kit comprises 43B7 and 29E10 or 24F1 or 11E4. In the exemplary example, the first antigen-binding protein and the second antigen-binding protein are present in the composition in a ratio of about 1:1.

[0139] In various cases, the kit includes an additional active agent, such as a third antigen-binding protein. Optionally, the third antigen-binding protein may bind to negative regulators, immunosuppressives, or immune checkpoint proteins of the immune system, including, but not limited to, CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, CEACAM-1, TIGIT, LAG3, CD112, CD112R, CD96, TIM3, BTLA, or the costimulatory receptors ICOS, OX40, 41BB, CD27, GITR. In various cases, the additional active agent may be a PD-1 binding protein, such as an anti-PD-1 antibody. Examples of anti-PD-1 antibodies include nivolumab (BMS-936558), pembrolizumab (MK3475), BMS 936558, BMS-936559, TSR-042 (Tesaro), ePDR001 (Novartis), and pidilizumab (CT-011). Optionally, the third antigen-binding protein is any PD-1 antigen-binding protein described in international patent application PCT / US2019 / 013205 (published as international publication brochure 2019 / 140196, the entire contents of which are incorporated herein by reference). In exemplary examples, the third antigen-binding protein includes the HC CDR1 amino acid sequence, HC CDR2 amino acid sequence, HC CDR3 amino acid sequence, LC CDR1 amino acid sequence, LC CDR2 amino acid sequence, and LC CDR3 amino acid sequence, respectively, of Sequence IDs 352-357 of International Publication No. 2019 / 140196. In exemplary examples, the third antigen-binding protein includes the HC CDR1 amino acid sequence, HC CDR2 amino acid sequence, HC CDR3 amino acid sequence, LC CDR1 amino acid sequence, LC CDR2 amino acid sequence, and LC CDR3 amino acid sequence, respectively, of Sequence IDs 2027-2032. In various embodiments, the third antigen-binding protein includes the HC variable region amino acid sequence and the LC variable region amino acid sequence, respectively, of Sequence IDs 358 and 359 of International Publication No. 2019 / 140196.In various embodiments, the third antigen-binding protein includes the HC variable region amino acid sequence and the LC variable region amino acid sequence of SEQ ID NO: 2033 and SEQ ID NO: 2034, respectively. In various examples, the third antigen-binding protein includes the FL HC amino acid sequence and the FL LC amino acid sequence of SEQ ID NO: 360 and SEQ ID NO: 361 of International Publication No. 2019 / 140196, respectively. In various examples, the third antigen-binding protein includes the FL HC amino acid sequence and the FL LC amino acid sequence of SEQ ID NO: 2035 and SEQ ID NO: 2036, respectively. In various embodiments, each antigen-binding protein is packaged separately in a kit. Optionally, the kit includes a container, e.g., a vial, syringe, bag, etc., containing at least two of the first, second, and third antigen-binding proteins. Optionally, the kit includes all antigen-binding proteins as a mixture in the same container.

[0140] Treatment method Treatment methods are further provided by this disclosure. In exemplary embodiments, a method is a method for treating a subject in need thereof, comprising administering to the subject in need an amount effective for treating the subject a pharmaceutical composition of this disclosure.

[0141] The pharmaceutical compositions of this disclosure are useful for inhibiting TIGIT signaling and / or CD112R signaling and / or PD-1 signaling. While not bound by any particular theory, the TIGIT inhibitory activity and / or CD112R inhibitory activity and / or PD-1 inhibitory activity of the compositions provided herein enable such entities to enhance T cell activity and be useful in enhancing immune responses, particularly immune responses against tumors or cancer.

[0142] Therefore, provided herein are methods for enhancing T cell activity in a subject, enhancing T cell survival and effector function, limiting the loss of terminal differentiation and replicative ability, promoting T cell lifespan, and enhancing cytotoxicity against target (e.g., cancer) cells. In an exemplary embodiment, the method comprises administering to the subject an effective amount of a pharmaceutical composition of the present disclosure. In an exemplary aspect, the T cell activity or immune response is an activity directed against cancer cells or cancer tissue or tumor cells or tumors. In an exemplary aspect, the immune response is a humoral immune response. In an exemplary aspect, the immune response is a innate immune response. In an exemplary embodiment, the enhanced immune response is a T cell-mediated immune response.

[0143] As used herein, the term "enhancing" and derivatives thereof need not be a 100% or complete enhancement or increase. Rather, there are various degrees of enhancement that one of ordinary skill in the art would recognize as having a potential benefit or therapeutic effect. In this regard, the pharmaceutical compositions of the present disclosure can enhance T cell activity or enhance an immune response to any amount or level. In an exemplary embodiment, the enhancement provided by the methods of the present disclosure is an enhancement of at least or about 10% (e.g., at least or about 20% enhancement, at least or about 30% enhancement, at least or about 40% enhancement, at least or about 50% enhancement, at least or about 60% enhancement, at least or about 70% enhancement, at least or about 80% enhancement, at least or about 90% enhancement, at least or about 95% enhancement, at least or about 98% enhancement).

[0144] Methods for measuring T cell activity and immune responses are known in the art. T cell activity can be measured by cytotoxicity assays, such as those described in Fu et al., PLoS ONE 5(7):e11867 (2010). Other T cell activity assays are described in Bercovici et al., Clin Diagn Lab Immunol. 7(6):859-864 (2000). Methods for measuring immune responses are described, for example, in Macatangay et al., Clin Vaccine Immunol 17(9):1452-1459 (2010) and Clay et al., Clin Cancer Res. 7(5):1127-35 (2001).

[0145] Furthermore, this specification also provides a method for enhancing natural killer (NK) cell activity in a subject. In exemplary embodiments, the method comprises administering an effective amount of the pharmaceutical composition of this disclosure to the subject. In exemplary embodiments, NK cell activity is activity directed towards cancer cells or cancer tissue or tumor cells or tumors.

[0146] Furthermore, provided herein are methods of treating a subject having cancer and methods of treating a subject having a solid tumor. In an exemplary embodiment, the method comprises administering to the subject a pharmaceutical composition of the present disclosure in an amount effective to treat cancer or a solid tumor in the subject. The cancers treatable by the methods disclosed herein can be any cancer, e.g., any malignant tumor or neoplasm caused by abnormal, uncontrolled cell division that can spread to other parts of the body via the lymphatic system or bloodstream. The cancer can, in some embodiments, be acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal, anal canal or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical, gallbladder or pleural cancer, nasal, nasal cavity or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colorectal cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omentum and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, and bladder cancer, and is selected from the group consisting of. In certain embodiments, the cancer is selected from the group consisting of head and neck cancer, ovarian cancer, cervical cancer, bladder cancer and esophageal cancer, pancreatic cancer, gastrointestinal cancer, stomach cancer, breast cancer, endometrial cancer, colorectal cancer, hepatocellular carcinoma, glioblastoma, bladder cancer, lung cancer, e.g., non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma. In a particular embodiment, the tumor is non-small cell lung cancer (NSCLC), head and neck cancer, kidney cancer, triple negative breast cancer, and stomach cancer. In an exemplary embodiment, the subject has a tumor (e.g., a solid tumor, a hematological malignancy, or a lymphatic malignancy), and the pharmaceutical composition is administered to the subject in an amount effective to treat the subject's tumor. In other exemplary embodiments, the tumor is non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, kidney cancer, breast cancer, melanoma, ovarian cancer, liver cancer, pancreatic cancer, colorectal cancer, prostate cancer, stomach cancer, lymphoma or leukemia, and the pharmaceutical composition is administered to the subject in an amount effective to treat the subject's tumor.

[0147] Where used herein, the term “to treat” and related terms do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment that a person skilled in the art would recognize as having potential benefits or therapeutic effects. In this regard, the methods for treating cancer of this disclosure may provide any amount or level of treatment. Furthermore, the treatment provided by the methods of this disclosure may include treatment of one or more pathological conditions, symptoms, or signs of the cancer being treated. Also, the treatment provided by the methods of this disclosure may include slowing the progression of cancer. For example, the methods may treat cancer by enhancing T-cell activity or NK-cell activity or immune response against cancer, reducing tumor or cancer growth, reducing metastasis of tumor cells, or increasing cell death of tumor or cancer cells. In exemplary embodiments, the methods treat to delay the onset or recurrence of cancer by 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 30 days, 2 months, 4 months, 6 months, 1 year, 2 years, 4 years, or more. In an exemplary embodiment, the method is used to treat a subject in order to extend their survival time.

[0148] subject In some embodiments of this disclosure, the subject matter includes, but is not limited to, mammals of the order Rodentia, such as mice and hamsters; mammals of the order Logomorpha, such as rabbits; mammals of the order Carnivora, including felines (cats) and canines (dogs); mammals of the order Artiodactyla, including cats and pigs; or mammals of the order Persodactyla, including horses. In some embodiments, the mammals are mammals of the order Primates, Ceboids, or Simoids (monkeys), or Halophini (humans and apes). In some embodiments, the mammal is human.

[0149] Manufacturing method The antigen-binding proteins of this disclosure can be obtained by methods known in the art. A preferred method for de novo synthesis of polypeptides is, for example, Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford. This is described in University Press, Oxford, United Kingdom, 2005; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2000; and U.S. Patent No. 5,449,752. Further exemplary methods for producing the peptides of the present invention are described herein.

[0150] In some embodiments, the antigen-binding proteins described herein are commercially synthesized by companies such as Synpep (Dublin, CA), Peptide Technologies Corp (Gaithersburg, MD), Multiple Peptide Systems (San Diego, CA), Peptide 2.0 Inc (Chantilly, VA), and American Peptide Co. (Sunnyvale, CA). In this regard, antigen-binding proteins can be synthesized, recombinant, isolated, and / or purified.

[0151] Furthermore, in some embodiments, antigen-binding proteins are recombinantly produced using nucleic acids encoding the amino acid sequence of the peptide, employing standard recombination methods. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual. 3rd ed., Cold Spring Harbor. See Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994.

[0152] Methods for producing the antigen-binding proteins of this disclosure are further provided herein. In exemplary embodiments, the method comprises the steps of culturing host cells of this disclosure to express an antigen-binding protein, and recovering the expressed antigen-binding protein. The host cells may be any of the host cells described herein. In exemplary embodiments, the host cells are selected from the group consisting of CHO cells, NS0 cells, COS cells, VERO cells, and BHK cells. In exemplary embodiments, the step of culturing the host cells comprises culturing the host cells in a growth medium that supports the proliferation and expansion of the host cells. In exemplary embodiments, the growth medium increases cell density, culture viability, and productivity. In exemplary embodiments, the growth medium contains amino acids, vitamins, inorganic salts, glucose, and serum as sources of growth factors, hormones, and adhesion factors. In exemplary embodiments, the growth medium is a fully chemically defined medium consisting of amino acids, vitamins, trace elements, inorganic salts, lipids, and insulin or insulin-like growth factors. In addition to nutrients, the growth medium also helps maintain pH and osmotic pressure. Several growth media are commercially available and described in the art. For example, see Arora, “Cell Culture Media: A Review,” MATER METHODS 3:175 (2013).

[0153] In exemplary embodiments, the method for producing the antigen-binding protein of the present disclosure includes culturing host cells in a feed medium. In exemplary embodiments, the method includes culturing in a feed medium in a fed-batch mode. Methods for producing recombinant proteins are known in the art. See, for example, Li et al., “Cell culture processes for monoclonal antibody production” MAbs2(5):466-477(2010).

[0154] A method for producing antigen-binding proteins may include one or more steps for purifying proteins from cell cultures or their supernatants, and preferably recovering the purified proteins. In exemplary embodiments, the method includes one or more chromatographic steps, such as affinity chromatography (e.g., protein A affinity chromatography), ion exchange chromatography, and hydrophobic interaction chromatography. In exemplary embodiments, the method includes purifying proteins using a protein A affinity chromatography resin.

[0155] In exemplary embodiments, the method further includes a step for formulating purified protein and the like to obtain a formulation containing the purified protein. Such a step is described in *Formulation and Process Development Strategies for Manufacturing*, eds. Jameel and Hershenson, John Wiley & Sons, Inc. (Hoboken, NJ), 2010.

[0156] Exemplary Embodiments The following is a list of exemplary embodiments of the present disclosure: 1. (a) Heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence as described in Table A1, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (b) HC CDR2 amino acid sequence as described in Table A1, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (c) HC CDR3 amino acid sequence as described in Table A1, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (d) Light chain (LC) CDR1 amino acid sequence as described in Table A1, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (e) LC CDR2 amino acid sequence as described in Table A1, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (f) LC as described in Table A1 CD112R antigen-binding protein, optionally an antibody or its antigen-binding fragment, comprising the CDR3 amino acid sequence, or a variant thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity, or any two or more combinations of (g)(a) to (f). 2. A CD112R antigen-binding protein of Embodiment 1, comprising six CDR amino acid sequences listed in a single row in Table A1, or six CDR amino acid sequences selected from the group consisting of (a) SEQ ID NOs: 13-18, (b) SEQ ID NOs: 23-28, (c) SEQ ID NOs: 33-38, (d) SEQ ID NOs: 43-48, (e) SEQ ID NOs: 53-58, (f) SEQ ID NOs: 63-68, (g) SEQ ID NOs: 73-78, (h) SEQ ID NOs: 83-88, (i) SEQ ID NOs: 93-98, (j) SEQ ID NOs: 103-108, (k) SEQ ID NOs: 233-238, (l) SEQ ID NOs: 1973-1978, (m) SEQ ID NOs: 1983-1988, (n) SEQ ID NOs: 1993-1998, and (o) SEQ ID NOs: 2003-2008. 3. (a) The HC variable region amino acid sequence described in Table B1, or a variant sequence thereof that differs from the HC variable region amino acid sequence in Table B1 by only 1 to 15 amino acids, or has at least or about 90% or about 95% sequence identity; (b) The LC variable region amino acid sequence described in Table B1, or a variant sequence thereof that differs from the LC variable region amino acid sequence in Table B1 by only 1 to 15 amino acids, or has at least or about 90% or about 95% sequence identity; or (c) A CD112R antigen-binding protein of Embodiment 1 or 2, comprising a combination of (a) and (b). 4. A CD112R antigen-binding protein of Embodiment 3, comprising a pair of HC variable region and LC variable region amino acid sequences listed in a single row in Table B1, or a pair of amino acid sequences selected from the group consisting of (a) SEQ ID NOs: 11-12, (b) SEQ ID NOs: 21-22, (c) SEQ ID NOs: 31-32, (d) SEQ ID NOs: 41-42, (e) SEQ ID NOs: 51-52, (f) SEQ ID NOs: 61-62, (g) SEQ ID NOs: 71-72, (h) SEQ ID NOs: 81-82, (i) SEQ ID NOs: 91-92, (j) SEQ ID NOs: 101-102, (k) SEQ ID NOs: 231-232, (l) SEQ ID NOs: 1971-1972, (m) SEQ ID NOs: 1981-1982, (n) SEQ ID NOs: 1991-1992, and (o) SEQ ID NOs: 2001-2002. 5. (a) A full-length (FL)HC amino acid sequence as described in Table B1, or a variant sequence thereof that differs from the FL HC amino acid sequence in Table B1 by only 1 to 50 amino acids, or has at least or about 90% or about 95% sequence identity; (b) A FL LC amino acid sequence as described in Table B1, or a variant sequence thereof that differs from the FL LC amino acid sequence in Table B1 by only 1 to 50 amino acids, or has at least or about 90% or about 95% sequence identity; or (c) Any one of Embodiments 1 to 4, comprising a combination of (a) and (b). 6. A CD112R antigen-binding protein of Embodiment 5 comprising a pair of full-length (FL) HC and FL LC amino acid sequences listed in a single row in Table B, or a pair of amino acid sequences selected from the group consisting of (a) SEQ ID NOs: 9-10, (b) SEQ ID NOs: 19-20, (c) SEQ ID NOs: 29-30, (d) SEQ ID NOs: 39-40, (e) SEQ ID NOs: 49-50, (f) SEQ ID NOs: 59-60, (g) SEQ ID NOs: 69-70, (h) SEQ ID NOs: 79-80, (i) SEQ ID NOs: 89-90, (j) SEQ ID NOs: 99-100, (k) SEQ ID NOs: 229-230, (l) SEQ ID NOs: 1969-1970, (m) SEQ ID NOs: 1979-1980, (n) SEQ ID NOs: 1989-1990, and (o) SEQ ID NOs: 1999-2000. 7. An antibody, which is a CD112R antigen-binding protein, one of the embodiments 1 to 6. 8. (a) the heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence of SEQ ID NO: 33, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (b) the HC CDR2 amino acid sequence of SEQ ID NO: 34, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (c) the HC CDR3 amino acid sequence of SEQ ID NO: 35, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (d) the light chain (LC) CDR1 amino acid sequence of SEQ ID NO: 36, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (e) the LC CDR2 amino acid sequence of SEQ ID NO: 37, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (f) the LC CDR1 amino acid sequence of SEQ ID NO: 38 The CDR3 amino acid sequence, or a variant sequence thereof that differs by only 1 to 4 amino acids, or has at least or approximately 90% sequence identity; or (b) the heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence of SEQ ID NO: 63, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (b) the HC CDR2 amino acid sequence of SEQ ID NO: 64, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (c) the HC CDR3 amino acid sequence of SEQ ID NO: 65, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (d) the light chain (LC) CDR1 amino acid sequence of SEQ ID NO: 66, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (e) the LC CDR2 amino acid sequence of SEQ ID NO: 67, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (f) the LC CDR1 amino acid sequence of SEQ ID NO: 68 The CDR3 amino acid sequence, or a variant sequence thereof that differs by only 1 to 4 amino acids, or has at least or approximately 90% sequence identity; or (c) The heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence of SEQ ID NO: 83, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (b) The HC CDR2 amino acid sequence of SEQ ID NO: 84, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (c) The HC CDR3 amino acid sequence of SEQ ID NO: 85, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (d) The light chain (LC) CDR1 amino acid sequence of SEQ ID NO: 86, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (e) The LC CDR2 amino acid sequence of SEQ ID NO: 87, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (f) The LC CDR1 amino acid sequence of SEQ ID NO: 88 The CDR3 amino acid sequence, or a variant sequence thereof that differs by only 1 to 4 amino acids, or has at least or approximately 90% sequence identity; or (d)(a) the HC variable region amino acid sequence of SEQ ID NO: 31, or a variant sequence thereof that differs from the HC variable region amino acid sequence of SEQ ID NO: 31 by only 1 to 15 amino acids, or has at least or approximately 90% or approximately 95% sequence identity; (b) the LC variable region amino acid sequence of SEQ ID NO: 32, or a variant sequence thereof that differs from the LC variable region amino acid sequence of SEQ ID NO: 32 by only 1 to 15 amino acids, or has at least or approximately 90% or approximately 95% sequence identity; or (c) a combination of (a) and (b); or (e)(a) the HC variable region amino acid sequence of SEQ ID NO: 61, or a variant sequence thereof that differs from the HC variable region amino acid sequence of SEQ ID NO: 61 by only 1 to 15 amino acids, or has at least or approximately 90% or approximately 95% sequence identity; (b) the LC variable region amino acid sequence of SEQ ID NO: 62, or a variant sequence thereof that differs from the LC variable region amino acid sequence of SEQ ID NO: 62 by only 1 to 15 amino acids, or has at least or approximately 90% or approximately 95% sequence identity; or (c) a combination of (a) and (b); or (f)(a) the HC variable region amino acid sequence of SEQ ID NO: 81, or a variant sequence thereof that differs from the HC variable region amino acid sequence of SEQ ID NO: 81 by only 1 to 15 amino acids, or has at least or approximately 90% or approximately 95% sequence identity; (b) the LC variable region amino acid sequence of SEQ ID NO: 82, or a variant sequence thereof that differs from the LC variable region amino acid sequence of SEQ ID NO: 82 by only 1 to 15 amino acids, or has at least or approximately 90% or approximately 95% sequence identity; or (c) a combination of (a) and (b); or (g)(a) The full-length (FL)HC amino acid sequence of SEQ ID NO: 29, or the FL of SEQ ID NO: 29 (b) A variant sequence having only 1 to 50 amino acids different from the HC amino acid sequence, or having at least or approximately 90% or approximately 95% sequence identity; (c) The FL LC amino acid sequence described in SEQ ID NO: 30, or a variant sequence having only 1 to 50 amino acids different from the FL LC amino acid sequence of SEQ ID NO: 30, or having at least or approximately 90% or approximately 95% sequence identity; or (c) A combination of (a) and (b); or (h)(a) The full-length (FL)HC amino acid sequence of SEQ ID NO: 59, or the FL of SEQ ID NO: 59 (b) A variant sequence having only 1 to 50 amino acids different from the HC amino acid sequence, or having at least or approximately 90% or approximately 95% sequence identity; (c) The FL LC amino acid sequence described in SEQ ID NO: 60, or a variant sequence having only 1 to 50 amino acids different from the FL LC amino acid sequence of SEQ ID NO: 60, or having at least or approximately 90% or approximately 95% sequence identity; or (c) A combination of (a) and (b); or (i)(a) The full-length (FL)HC amino acid sequence of SEQ ID NO: 79, or the FL of SEQ ID NO: 79 (b) A variant sequence having only 1 to 50 amino acids different from the HC amino acid sequence, or having at least or approximately 90% or approximately 95% sequence identity; (c) The FL LC amino acid sequence described in SEQ ID NO: 80, or a variant sequence having only 1 to 50 amino acids different from the FL LC amino acid sequence of SEQ ID NO: 80, or having at least or approximately 90% or approximately 95% sequence identity; or (c) A combination of (a) and (b). The CD112R antigen-binding protein of Embodiment 7, including the CD112R antigen-binding protein of Embodiment 7. 9. A CD112R antigen-binding protein, which is an antigen-binding fragment of an antibody, one of the CD112R antigen-binding proteins from Embodiments 1 to 8. 10. An antibody protein product, optionally being an scFv, which is one of the CD112R antigen-binding proteins from Embodiments 1 to 9. 11. A polypeptide comprising the amino acid sequence of SEQ ID NO: of Table A1, B1, or C1, or a variant sequence thereof having at least or about 90% or about 95% sequence identity to the amino acid sequence of SEQ ID NO: of that table, or a combination thereof. 12. A conjugate comprising any one of the CD112R antibody-binding proteins or polypeptides of embodiments 1 to 11 and a heterologous moiety. 13. The conjugate of embodiment 12, comprising the amino acid sequence of an antigen-binding protein or polypeptide fused to another amino acid sequence. 14. A nucleic acid encoding any one of the CD112R antigen-binding proteins or polypeptides or conjugates of embodiments 1 to 13. 15. A nucleic acid encoding the light chain, heavy chain, or both the light and heavy chains of the antibody of embodiment 9 or 10. 16. (a) The HC variable region amino acid sequence set forth in Table B1, or a variant sequence thereof having only 1 to 15 amino acids different from the HC variable region amino acid sequence of Table B1, or having at least or about 90% or about 95% sequence identity thereto; (b) The LC variable region amino acid sequence set forth in Table B1, or a variant sequence thereof having only 1 to 15 amino acids different from the LC variable region amino acid sequence of Table B1, or having at least or about 90% or about 95% sequence identity thereto; or (c) The nucleic acid of embodiment 14 or 15, comprising both (a) and (b). 17. A vector comprising one or more nucleic acids of any one of embodiments 14 to 16. 18. A host cell comprising one or more nucleic acids of any one of embodiments 14 to 16, or one or more vectors of embodiment 17. 19. The host cell of embodiment 18, wherein the host cell produces the CD112R antigen-binding protein of any one of embodiments 1 to 10. 20. (a) Heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence as described in Table A2, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (b) HC CDR2 amino acid sequence as described in Table A2, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (c) HC CDR3 amino acid sequence as described in Table A2, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (d) Light chain (LC) CDR1 amino acid sequence as described in Table A2, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (e) LC CDR2 amino acid sequence as described in Table A2, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (f) LC as described in Table A2 A TIGIT antigen-binding protein, optionally an antibody or its antigen-binding fragment, comprising the CDR3 amino acid sequence, or a variant thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity, or any two or more combinations of (g)(a) to (f). twenty one. a. LC CDR1 amino acid sequence containing Gln27 or its conserved amino acid substitution, Ser28 or its conserved amino acid substitution, or any combination thereof; LC CDR2 amino acid sequence containing Glu1 or its conserved amino acid substitution; and LC CDR3 amino acid sequence containing Ser91 or its conserved amino acid substitution, Ser92 or its conserved amino acid substitution, Ser93 or its conserved amino acid substitution, Leu94 or its conserved amino acid substitution, or any combination thereof; HC containing Val32 or its conserved amino acid substitution, Tyr33 or its conserved amino acid substitution, or any combination thereof CDR1 amino acid sequence; HC CDR2 amino acid sequence containing Tyr52 or its conserved amino acid substitution, Tyr54 or its conserved amino acid substitution, Tyr55 or its conserved amino acid substitution, Ser56 or its conserved amino acid substitution, Gly57 or its conserved amino acid substitution, Gly58 or its conserved amino acid substitution, Thr59 or its conserved amino acid substitution, Tyr60 or its conserved amino acid substitution, Pro63 or its conserved amino acid substitution, Arg66 or its conserved amino acid substitution, or any combination thereof; and HC CDR3 amino acid sequence containing Ile102 or its conserved amino acid substitution, Ala104 or its conserved amino acid substitution, Gly107 or its conserved amino acid substitution, Tyr108 or its conserved amino acid substitution, Phe109 or its conserved amino acid substitution, Tyr110 or its conserved amino acid substitution, Tyr111 or its conserved amino acid substitution, or any combination thereof (where the position numbers are relative positions in the LC variable region amino acid sequence of the TIGIT antigen-binding protein). b. LC CDR1 amino acid sequence containing Gln27 or its conserved amino acid substitution, Ser28 or its conserved amino acid substitution, Val29 or its conserved amino acid substitution, Ser30 or its conserved amino acid substitution, Ser31 or its conserved amino acid substitution, Thr32 or its conserved amino acid substitution, Tyr33 or its conserved amino acid substitution, or any combination thereof; LC CDR2 amino acid sequence containing Glu1 or its conserved amino acid substitution, Ile2 or its conserved amino acid substitution, Ser68 or its conserved amino acid substitution, Gly69 or its conserved amino acid substitution, or any combination thereof; LC CDR1 amino acid sequence containing Tyr92 or its conserved amino acid substitution, Asp93 or its conserved amino acid substitution, Val94 or its conserved amino acid substitution, Ser95 or its conserved amino acid substitution, Pro96 or its conserved amino acid substitution, Trp97 or its conserved amino acid substitution, or any combination thereof CDR3 amino acid sequence; HC CDR1 amino acid sequence containing Gly32 or its conserved amino acid substitution, Tyr35 or its conserved amino acid substitution, or any combination thereof; HC CDR2 amino acid sequence containing Tyr52 or its conserved amino acid substitution, Tyr54 or its conserved amino acid substitution, Tyr55 or its conserved amino acid substitution, Ser56 or its conserved amino acid substitution, Ser58 or its conserved amino acid substitution, Thr59 or its conserved amino acid substitution, Phe60 or its conserved amino acid substitution, Pro63 or its conserved amino acid substitution, Lys66 or its conserved amino acid substitution, or any combination thereof; HC CDR3 amino acid sequence containing Arg102 or its conserved amino acid substitution, Asn104 or its conserved amino acid substitution, Trp105 or its conserved amino acid substitution, Asn106 or its conserved amino acid substitution, Tyr107 or its conserved amino acid substitution, or any combination thereof (where the position numbers are relative positions in the LC variable region amino acid sequence of the TIGIT antigen-binding protein). c. LC CDR1 amino acid sequence containing Arg30 or its conserved amino acid substitution, Arg31 or its conserved amino acid substitution, Tyr32 or its conserved amino acid substitution, or any combination thereof; LC CDR3 amino acid sequence containing Ser91 or its conserved amino acid substitution, Tyr92 or its conserved amino acid substitution, Ser93 or its conserved amino acid substitution, Thr94 or its conserved amino acid substitution, or any combination thereof (where the position number is the relative position of the LC variable region amino acid sequence of the TIGIT antigen-binding protein); HC containing Thr30 or its conserved amino acid substitution, Gly31 or its conserved amino acid substitution, Tyr32 or its conserved amino acid substitution, Tyr33 or its conserved amino acid substitution, or any combination thereof CDR1 amino acid sequence; HC CDR2 amino acid sequence containing Trp47 or its conserved amino acid substitution, Trp50 or its conserved amino acid substitution, Ser52 or its conserved amino acid substitution, Thr54 or its conserved amino acid substitution, Ser55 or its conserved amino acid substitution, Ala57 or its conserved amino acid substitution, Thr58 or its conserved amino acid substitution, Gly59 or its conserved amino acid substitution, Tyr60 or its conserved amino acid substitution, Gln65 or its conserved amino acid substitution, or any combination thereof; HC CDR3 amino acid sequence containing Asn101 or its conserved amino acid substitution, Ser102 or its conserved amino acid substitution, Val103 or its conserved amino acid substitution, Leu104 or its conserved amino acid substitution, Tyr105 or its conserved amino acid substitution, Tyr106 or its conserved amino acid substitution, Tyr107 or its conserved amino acid substitution, or any combination thereof (where the position numbers are relative positions in the HC variable region amino acid sequence of the TIGIT antigen-binding protein). d. LC CDR1 amino acid sequence containing Gln27 or its conserved amino acid substitution, Leu30 or its conserved amino acid substitution, Ser32 or its conserved amino acid substitution, or any combination thereof; LC CDR3 amino acid sequence containing Ser96 or its conserved amino acid substitution, Ile97 or its conserved amino acid substitution, Gln98 or its conserved amino acid substitution, Leu99 or its conserved amino acid substitution, or any combination thereof; HC CDR1 amino acid sequence containing Asp33 or its conserved amino acid substitution; HC containing Tyr52 or its conserved amino acid substitution, Tyr54 or its conserved amino acid substitution, Tyr55 or its conserved amino acid substitution, Ser56 or its conserved amino acid substitution, Gly57 or its conserved amino acid substitution, Gly58 or its conserved amino acid substitution, Thr59 or its conserved amino acid substitution, Tyr60 or its conserved amino acid substitution, Pro63 or its conserved amino acid substitution, Lys66 or its conserved amino acid substitution, or any combination thereof CDR2 amino acid sequence; HC CDR3 amino acid sequence containing Ile102 or its conserved amino acid substitution, Ala104 or its conserved amino acid substitution, Gly107 or its conserved amino acid substitution, Tyr108 or its conserved amino acid substitution, Phe109 or its conserved amino acid substitution, Tyr110 or its conserved amino acid substitution, Phe111 or its conserved amino acid substitution, or any combination thereof (where the position numbers are relative positions in the HC variable region amino acid sequence of the TIGIT antigen-binding protein). A TIGIT antigen-binding protein of embodiment 20, including the TIGIT antigen-binding protein of embodiment 20. 22. A TIGIT antigen-binding protein of Embodiment 20 or 21, comprising six CDR amino acid sequences listed in a single row in Table A2, or six CDR amino acid sequences selected from the group consisting of (a) SEQ ID NOs: 113-118, (b) SEQ ID NOs: 123-128, (c) SEQ ID NOs: 133-138, (d) SEQ ID NOs: 143-148, (e) SEQ ID NOs: 153-158, (f) SEQ ID NOs: 163-168, (g) SEQ ID NOs: 173-178, (h) SEQ ID NOs: 183-188, (i) SEQ ID NOs: 193-198, (j) SEQ ID NOs: 203-208, (k) SEQ ID NOs: 213-218, (l) SEQ ID NOs: 223-228, and (m) SEQ ID NOs: 2013-2018. 23. (a) The HC variable region amino acid sequence described in Table B2, or a variant sequence thereof that differs from the HC variable region amino acid sequence in Table B2 by only 1 to 15 amino acids, or has at least or about 90% or about 95% sequence identity; (b) The LC variable region amino acid sequence described in Table B2, or a variant sequence thereof that differs from the LC variable region amino acid sequence in Table B2 by only 1 to 15 amino acids, or has at least or about 90% or about 95% sequence identity; or (c) Any one of the TIGIT antigen-binding proteins of Embodiments 20 to 22, including a combination of (a) and (b). 24. A TIGIT antigen-binding protein of Embodiment 23 comprising a pair of HC variable region and LC variable region amino acid sequences listed in a single row in Table B2, or a pair of amino acid sequences selected from the group consisting of (a) SEQ ID NOs: 111-112, (b) SEQ ID NOs: 121-122, (c) SEQ ID NOs: 131-132, (d) SEQ ID NOs: 141-142, (e) SEQ ID NOs: 151-152, (f) SEQ ID NOs: 161-162, (g) SEQ ID NOs: 171-172, (h) SEQ ID NOs: 181-182, (i) SEQ ID NOs: 191-192, (j) SEQ ID NOs: 201-202, (k) SEQ ID NOs: 211-212, (l) SEQ ID NOs: 221-222, and (m) SEQ ID NOs: 2011-2012. 25. (a) The full-length (FL)HC amino acid sequence described in Table B2, or a variant sequence thereof that differs from the FL HC amino acid sequence in Table B2 by only 1 to 50 amino acids, or has at least or about 90% or about 95% sequence identity; (b) The FL LC amino acid sequence described in Table B2, or a variant sequence thereof that differs from the FL LC amino acid sequence in Table B2 by only 1 to 50 amino acids, or has at least or about 90% or about 95% sequence identity; or (c) Any one of the TIGIT antigen-binding proteins of Embodiments 20 to 24, including a combination of (a) and (b). 26. A TIGIT antigen-binding protein of Embodiment 25 comprising a pair of full-length (FL) HC and FL LC amino acid sequences listed in a single row in Table B2, or a pair of amino acid sequences selected from the group consisting of (a) SEQ ID NOs: 109-110, (b) SEQ ID NOs: 119-120, (c) SEQ ID NOs: 129-130, (d) SEQ ID NOs: 139-140, (e) SEQ ID NOs: 149-150, (f) SEQ ID NOs: 159-160, (g) SEQ ID NOs: 169-170, (h) SEQ ID NOs: 179-180, (i) SEQ ID NOs: 189-190, (j) SEQ ID NOs: 199-200, (k) SEQ ID NOs: 209-210, (l) SEQ ID NOs: 219-220, and (m) SEQ ID NOs: 2009-2010. 27. An antibody, which is a TIGIT antigen-binding protein from any one of embodiments 20 to 26. 28. (a) The heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence of SEQ ID NO: 203, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (b) The HC CDR2 amino acid sequence of SEQ ID NO: 204, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (c) The HC CDR3 amino acid sequence of SEQ ID NO: 205, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (d) The light chain (LC) CDR1 amino acid sequence of SEQ ID NO: 206, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (e) The LC CDR2 amino acid sequence of SEQ ID NO: 207, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (f) The LC CDR1 amino acid sequence of SEQ ID NO: 208 The CDR3 amino acid sequence, or a variant sequence thereof that differs by only 1 to 4 amino acids, or has at least or approximately 90% sequence identity; or (b) the heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence of SEQ ID NO: 223, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (b) the HC CDR2 amino acid sequence of SEQ ID NO: 224, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (c) the HC CDR3 amino acid sequence of SEQ ID NO: 225, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (d) the light chain (LC) CDR1 amino acid sequence of SEQ ID NO: 226, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (e) the LC CDR2 amino acid sequence of SEQ ID NO: 227, or a variant sequence thereof that differs by only 1 to 4 amino acids or has at least or approximately 90% sequence identity; (f) the LC CDR1 amino acid sequence of SEQ ID NO: 228 The CDR3 amino acid sequence, or a variant sequence thereof that differs by only 1 to 4 amino acids, or has at least or approximately 90% sequence identity; or (c)(a) the HC variable region amino acid sequence of SEQ ID NO: 201, or a variant sequence thereof that differs from the HC variable region amino acid sequence of SEQ ID NO: 201 by only 1 to 15 amino acids, or has at least or approximately 90% or approximately 95% sequence identity; (b) the LC variable region amino acid sequence of SEQ ID NO: 202, or a variant sequence thereof that differs from the LC variable region amino acid sequence of SEQ ID NO: 202 by only 1 to 15 amino acids, or has at least or approximately 90% or approximately 95% sequence identity; or a combination of (c)(a) and (b); or (d)(a) the HC variable region amino acid sequence of SEQ ID NO: 221, or a variant sequence thereof that differs from the HC variable region amino acid sequence of SEQ ID NO: 221 by only 1 to 15 amino acids, or has at least or approximately 90% or approximately 95% sequence identity; (b) the LC variable region amino acid sequence of SEQ ID NO: 222, or a variant sequence thereof that differs from the LC variable region amino acid sequence of SEQ ID NO: 222 by only 1 to 15 amino acids, or has at least or approximately 90% or approximately 95% sequence identity; or (c) a combination of (a) and (b); or (e)(a) the full-length (FL)HC amino acid sequence of SEQ ID NO: 199, or a variant sequence thereof that differs from the FL HC amino acid sequence of SEQ ID NO: 199 by only 1 to 50 amino acids, or has at least or approximately 90% or approximately 95% sequence identity; (b) the FL LC amino acid sequence described in SEQ ID NO: 200, or a variant sequence thereof that differs from the FL LC amino acid sequence of SEQ ID NO: 200 by only 1 to 50 amino acids, or has at least or approximately 90% or approximately 95% sequence identity; or (c) a combination of (a) and (b); or (f)(a) The full-length (FL)HC amino acid sequence of SEQ ID NO: 219, or a variant sequence thereof that differs from the FL HC amino acid sequence of SEQ ID NO: 219 by only 1 to 50 amino acids, or has at least or approximately 90% or approximately 95% sequence identity; (b) The FL LC amino acid sequence described in SEQ ID NO: 220, or a variant sequence thereof that differs from the FL LC amino acid sequence of SEQ ID NO: 220 by only 1 to 50 amino acids, or has at least or approximately 90% or approximately 95% sequence identity; or (c) A combination of (a) and (b). The TIGIT antigen-binding protein of Embodiment 27, including the TIGIT antigen-binding protein of Embodiment 27. 29. A TIGIT antigen-binding protein, which is an antigen-binding fragment of an antibody, one of any one of embodiments 20 to 26. 30. An antibody protein product, optionally being an scFv, which is one of the TIGIT antigen-binding proteins from Embodiments 20 to 26. 31. A polypeptide comprising the amino acid sequence of the sequence number in Table A2, B2, or C2, or a variant sequence having at least or about 90% or about 95% sequence identity with the amino acid sequence of the sequence number in that table, or a combination thereof. 32. A conjugate comprising a TIGIT antibody-binding protein or polypeptide and a heterogeneous moiety, as described in any one of embodiments 20 to 31. 33. The conjugate of Embodiment 32, comprising an amino acid sequence of an antigen-binding protein or polypeptide fused to another amino acid sequence. 34. A nucleic acid encoding any one of the TIGIT antigen-binding proteins, polypeptides, or conjugates described in Embodiments 20 to 33. 35. A nucleic acid encoding the light chain, heavy chain, or both the light chain and heavy chain of the antibody of Embodiment 27 or 28. 36. (a) The HC variable region amino acid sequence described in Table B2, or a variant sequence thereof that differs from the HC variable region amino acid sequence in Table B2 by only 1 to 15 amino acids, or has at least or about 90% or about 95% sequence identity; (b) The LC variable region amino acid sequence described in Table B2, or a variant sequence thereof that differs from the LC variable region amino acid sequence in Table B2 by only 1 to 15 amino acids, or has at least or about 90% or about 95% sequence identity; or (c) Nucleic acids of Embodiment 34 or 35 comprising both (a) and (b). 37. A vector comprising one or more nucleic acids from any one of embodiments 34 to 36. 38. A host cell containing one or more nucleic acids from any one of embodiments 34 to 36, or one or more vectors from embodiment 37. 39. The host cell of Embodiment 37, which produces any of the TIGIT antigen-binding proteins of Embodiments 20 to 30. 40. A composition comprising one CD112R antigen-binding protein from any of Embodiments 1 to 10 and one TIGIT antigen-binding protein from any of Embodiments 20 to 30. 41. (A) The CD112R antigen-binding protein is 1E1, 1E1.016, 24F1, 29E10, 24F1.001, 29E10_CONS.020, 29E10_CONS.021, 29E10_CONS.022, 29E10_CONS.025, 11E4, 31B3, 27G12, 28F9, 28H7, or 36C8, as listed in Table A1 or Table B1, and optionally 24F1, 29E10_CONS.020, or 29E10_CONS.022. (B) The composition of Embodiment 40, wherein the TIGIT antigen-binding protein is one of the following listed in Table A2 or Table B2: 55G7.041.008, 58A7.003.008.075, 4G10, 11A3, 28B8, 39D2, 43B7, 55G7, 66H9, 43B7.002.015, 58A7.003.08, 66H9.009, or 58A7, or optionally 43B7.002.015 or 66H9.009, or a combination of (A) and (B). 42. (A) 24F1 and 43B7.002.015, (B) 24F1 and 66H9.009, (C) 29E10_CONS.020 and 43B7.002.015, (D) 29E10_CONS.020 and 66H9.009, (E) 29E10_CONS.022 and 43B7.002.015, (F) 29E10_CONS.022 and 66H9.009, (G) 43 A composition of Embodiment 40 or 41 comprising B7.002.015 and 1E1.016, (H)43B7.002.015 and 24F1, (I)43B7.002.015 and 29E10, (J)66H9.009 and 1E1.016, (K)66H9.009 and 29E10, (L)43B7 and 29E10, (M)43B7 and 24F1, or (N)43B7 and 11E4. 43. A composition of any one of claims 40 to 42, wherein the CD112R antigen-binding protein and the TIGIT antigen-binding protein are present in the composition in a ratio of approximately 1:1. 44. Any one of embodiments 40 to 43, further comprising a third antigen-binding protein that targets PD-1. 45. The composition of Embodiment 44, wherein the third antigen-binding protein is any PD-1 antigen-binding protein described in International Patent Application No. PCT / US2019 / 013205 (which has been published as International Publication Brochure No. 2019 / 140196). 46. The composition of Embodiment 44 or 45, wherein the third antigen-binding protein comprises the HC variable region amino acid sequence of SEQ ID NO: 2033 and the HC variable region amino acid sequence of SEQ ID NO: 2034. 47. A kit comprising one antigen-binding protein from any of Embodiments 1 to 10, a polypeptide from Embodiment 11, a conjugate from Embodiment 12 or 13, one nucleic acid from any of Embodiments 14 to 16, a vector from Embodiment 17, a host cell from Embodiment 18 or 19, one antigen-binding protein from any of Embodiments 20 to 30, a polypeptide from Embodiment 31, a conjugate from Embodiment 32 or 33, a nucleic acid from Embodiments 34 to 36, a vector from Embodiment 37, a host cell from Embodiment 38 or 39, one composition from any of Embodiments 40 to 46, or a combination thereof, and a container. 48. A pharmaceutical composition comprising any one antigen-binding protein from Embodiments 1 to 10, a polypeptide from Embodiment 11, a conjugate from Embodiment 12 or 13, a nucleic acid from any one of Embodiments 14 to 16, a vector from Embodiment 17, a host cell from Embodiment 18 or 19, an antigen-binding protein from any one of Embodiments 20 to 30, a polypeptide from Embodiment 31, a conjugate from Embodiment 32 or 33, a nucleic acid from Embodiments 34 to 36, a vector from Embodiment 37, a host cell from Embodiment 38 or 39, any one composition from Embodiments 40 to 46, or a combination thereof, and a pharmaceutically acceptable carrier, diluent excipient, or diluent. 49. A method for producing a CD112R antigen-binding protein, comprising the steps of: culturing a host cell from either Embodiment 18 or 19 to express the CD112R antigen-binding protein; and recovering the expressed CD112R antigen-binding protein. 50. A method for producing a TIGIT antigen-binding protein, comprising the steps of: culturing a host cell from either Embodiment 38 or 39 to express the TIGIT antigen-binding protein; and recovering the expressed TIGIT antigen-binding protein. 51. A method for treating a subject in need thereof, comprising administering to the subject in need the pharmaceutical composition of Embodiment 48 in an amount effective for treating the subject. 52. The method of Embodiment 51, wherein the subject has a solid tumor, and the pharmaceutical composition is administered to the subject in an amount effective to treat the solid tumor of the subject. 53. A method for treating a subject in need thereof, comprising administering to the subject in need a first pharmaceutical composition comprising a CD112R antigen-binding protein and a TIGIT antigen-binding protein, and a second pharmaceutical composition comprising a PD-1 inhibitor. 54. The method of Embodiment 53, wherein the subject has a solid tumor, and the first pharmaceutical composition and the second pharmaceutical composition are administered in amounts effective to treat the solid tumor of the subject.

[0157] The following embodiments are provided solely for illustrative purposes and are not intended to limit the scope of the present invention. [Examples]

[0158] Example 1 This example describes the expression of TIGIT family receptors and ligands in cancer and normal T cells.

[0159] Correlation analyses were performed for multiple tumor indications using RNA-seq data from The Cancer Genome Atlas (TCGA) to evaluate the interaction of TIGIT family members and co-expression with PD-1. The same analyses were performed for TIGIT family members and PD-1 ligands. Tumor indications included invasive breast cancer (BRCA), clear cell renal carcinoma (KIRC), cervical squamous cell carcinoma (HNSC), and cutaneous melanoma (SKCM).

[0160] As shown in the upper panel of Figure 1A, most TIGIT family receptors showed positive correlations with each other in most oncological indications, suggesting that many of these receptors are co-expressed in the cancer or tumor environment. In contrast, ligands of TIGIT family members showed limited correlations (lower panel of Figure 1A), with the best correlation being between CD112 and CD155. The rest did not show good correlations with each other or with PD-L1.

[0161] Single-cell RNA-seq data from tumor-infiltrating lymphocytes (TILs) derived from human hepatocellular carcinoma were also analyzed, suggesting that TIGIT and PD-1 expression overlapped, while CD112R expression was more widespread (Figure 1B). Similar results were obtained from additional single-cell RNA-seq datasets from TILs derived from colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) (data not shown).

[0162] CD112R is a recently added receptor to the TIGIT family. It has been previously shown to be expressed on NK cells and activated T cells, and primarily on CD8 T cells. CD112R expression was confirmed to be induced on activated CD8 T cells, and a significant proportion of these cells were also found to co-express PD-1 and TIGIT, consistent with patterns suggested by scRNAseq data (Figure 1C).

[0163] To confirm the expression of CD112R, TIGIT, and PD-1, as well as their ligands CD112 and CD155, in primary human cells, the expression of these molecules was evaluated in tumor-infiltrating immune cells and tumor cells derived from human tumor tissue. Within a limited number of samples, there was considerable variability in the relative expression of receptors detectable by FACS (Figure 1D). Furthermore, significant differences in ligand expression were observed between Epcam+CD45- tumor cells and Epcam-CD45+ immune cells. As shown in Figure 1E, CD112 and CD155 were co-expressed at high levels in Epcam+CD45- tumor cells, but expression levels were low in intratumor immune cells, with very few cells expressing both ligands. CD45+Epcam- myeloid cells in PBMCs showed only very few cells, if any, that co-expressed these ligands.

[0164] These results show the expression patterns of CD112R, TIGIT, and PD-1.

[0165] Example 2 This example demonstrates that CD112R blockade enhances the T cell response.

[0166] To demonstrate the function of CD112R in T cells, we developed an in vitro assay system using engineered CHO cells that stably express CD112 and CD3 engagers. Purified human pan T cells were pre-activated with a CD3 / CD28 antibody and then allowed to stand. It was expected that when CD112R expressed on the surface of T cells binds to CD112 expressed on the surface of CHO cells, IL-2 release would be suppressed. A diagram of this assay is shown in Figure 2A. It was confirmed that T cells induced CD112R expression on their cell surface (Figure 2B). The ability of tool antibodies to bind to CD112R and block IL-2 release was tested using this assay system. The tool antibodies (PL-52575, PL-52576, and PL-52577) showed dose-dependent binding to cells expressing huCD112R (Figure 2C), and the relative affinity / binding activity of these antibodies correlated with their ability to block ligand binding (Figure 2D). The following table shows an overview of the EC50 and IC50 values ​​for these tool antibodies.

[0167] [Table 8]

[0168] Using this assay system, the tool antibody dose-dependently enhanced T cell activity in the presence of CD112-expressing CHO cells (blue circles, red squares, and green triangles in Figure 2E). The antibody's ability to induce activity depended on the presence of CD112 on the CHO cells; when CHO cells were mock-transfected with an empty vector and did not express CD112, T cell activity was not enhanced. These data suggest that the interaction between CD112 and CD112R inhibits the T cell response.

[0169] As previously shown, CD112 binds to CD226 to induce a co-stimulatory signal. Importantly, CD112-mediated co-stimulation of T cells in the absence of CD112R is entirely driven by CD226 (Figure 2F), further confirming that CD112R primarily inhibits CD226-dependent co-stimulatory signaling by binding to the same ligand as CD226.

[0170] These results suggest that CD112R blockade is a good strategy for enhancing the T cell response.

[0171] Example 3 This example demonstrates the generation of CD112R monoclonal antibody (mAb).

[0172] A fully human antibody against human CD112R was prepared as follows.

[0173] Generation of an anti-CD112R immune response Mouse strain Fully human antibodies against human CD112R were produced by immunizing XENOMOUSE® transgenic mice (U.S. Patent Nos. 6,114,598; 6,162,963; 6,833,268; 7,049,426; 7,064,244 (these documents are incorporated herein by reference in their entirety); Green et al., 1994, Nature Genetics 7:13-21; Mendez et al., 1997, Nature Genetics 15:146-156; Green and Jakobovitis, 1998, J.Ex.Med, 188:483-495; Kellerman and Green, Current Opinion in Biotechnology 13,593-597, 2002). Animals derived from XMG4-K, XMG4-KL, XMG2-K, and XMG2-KL XENOMOUSE® strains were used for immunization. Furthermore, custom XMG2 CD112R KO mouse strains were created (Horizon Discovery).

[0174] immunization A repertoire of antibodies was constructed by applying multiple immunization strategies to various XenoMouse strains, including the XenoMouse knockout strain XMG2 CD112R KO. Blood was collected from animals, and plasma was collected at various time points during immunization testing between 4 and 10 weeks to evaluate CD112R-specific titers.

[0175] CD112R-specific serum titers were monitored by live-cell FACS analysis using an Accuri flow cytometer. Briefly, HEK293 cells were either mock-transfected or transiently transfected with human CD112R or cynomolgus monkey CD112R. Serum from the immunized animals was diluted 100-fold and incubated on the transfected cells on ice for 1 hour. The cells were then washed to remove unbound antibodies, and a Cy5-labeled secondary anti-human IgG Fc-specific antibody was incubated on the cells for a further 15 minutes at 4°C. The cells were washed once to remove unbound secondary antibodies, and the fluorescence signal on the cells was quantified by FACS. Animals with the highest antigen-specific serum titers directed towards human and cynomolgus monkey CD112R were used for hybridoma construction (Kohler and Milstein, 1975). The animal strains from harvest 1 and harvest 3 were XMG2 / XMG4, while the strain from harvest 2 was XMG2kl.

[0176] Preparation of monoclonal antibodies Hybridoma fabrication Animals exhibiting suitable serum titers were identified, and lymphocytes were obtained from the spleen and / or influx region lymph nodes. Pooled lymphocytes (from each harvest) were isolated from lymphoid tissue by grinding in a suitable medium (e.g., Dulbecco's Modified Eagle Medium (DMEM); Invitrogen, Carlsbad, CA). B cells were selected and / or proliferated using standard methods and fused with suitable fusion partners using techniques known in the art. Subsequently, antibody-producing hybridomas were plated using FACS-based antigen-specific sorting or by standard polyclonal plating techniques.

[0177] Antigen-specific staining of hybridoma cells: Hybridoma cells were removed from the flask and washed in sterile FACS buffer (2% FBS PBS). The cells were then stained with soluble human CD112R protein and incubated at 4°C for 1 hour. The cells were washed again in FACS buffer and stained with a 1 mL detection cocktail containing 5 μg / mL of Alexa Fluor 488 conjugate F(ab')2 fragment goat anti-human IgG Fc (Jackson, Cat: 109-546-098) and Alexa Fluor 647 conjugate streptavidin (Jackson, Cat: 016-600-084). The cells were then incubated in the dark at 4°C for 30 minutes. The cells were washed again in FACS buffer, resuspended in culture medium, and then passed through a 40-micron cell strainer to remove aggregated cells. Antigen-specific cells (IgG+ and antigen-binding cells) were selected by gatening populations exhibiting both Alexa Fluor488 and Alexa Fluor 647 fluorescence using BD FACSAria 3.

[0178] The selected cells were cultured in hybridoma medium for several days. After confirming the successful enrichment of CD112R-specific cells, the hybridomas were sorted into single cells on 384-well microtiter plates using BD FACSAria 3. After two weeks of culture, the supernatant was collected from the microtiter plates and screened for CD112R binding.

[0179] Initial selection of CD112R-specific binding antibodies Figure 3 shows the sequence of screening assays used to identify and select antibodies against human CD112R.

[0180] Human CD112R chromosome assay Using transfected cells, antibody binding specificity was evaluated using flow cytometry against host human embryonic kidney (HEK) 293T cells as follows: Proteins were expressed in HEK 293T cells transfected according to manufacturer-defined protocols using human CD112R, mouse CD112R, rat CD112R, human CD96, human CD226, or control expression vectors, Gibco® Opti-MEM® medium (Gibco, catalog no. 31985088), and 293Fectin® reagent (Invitrogen, catalog no. 12347019). After 24 hours, the transfected cells were resuspended in FACS buffer (PBS + 2% fetal bovine serum) and added to 96-well plates. Hybridoma supernatant samples were added to a final concentration of 2.5 μg / mL (with the exception of 11E4, which was tested at a final 1:10 dilution), the cells were resuspended, and incubated at 4°C for 1 hour. The plates were washed twice with FACS buffer, the cells were pelleted by centrifugation, the supernatant was removed, and the cells were resuspended in FACS buffer to remove unbound antibodies. Next, Alexa Fluor 488-goat anti-human IgG (Fcγ fragment specific) secondary antibody (Jackson ImmunoResearch, catalog number 109-545-098), prepared to 5 μg / mL in FACS buffer, was added to each well, the cells were resuspended, and incubated at 4°C for 15 minutes. The plates were washed twice with FACS buffer, the cells were pelleted by centrifugation, the supernatant was removed, and the cells were resuspended in FACS buffer to remove unbound secondary antibodies. Next, the samples were resuspended in FACS buffer and read using a BD Accuri® flow cytometer with an Intellicyt HyperCyt autosampler. The binder counts for human CD112R in harvest 1, harvest 2, and harvest 3 were 216, 539, and 569, respectively. The huCD112R antibody was selective for human CD112R and did not cross-react with mouse or rat CD112R (data not shown).

[0181] Jurkat Human CD112R / NFAT-Luciferase Reporter Gene Assay (RGA) To screen for hybridomas or purified anti-CD112R antibodies that can enhance T cell activity by blocking the CD112-CD112R interaction, we developed an NFAT reporter assay in Jurkat cells (Figure 4A). Jurkat cells stably expressing human CD112R and NFAT-luciferase reporters (generated in-house using Promega's Jurkat NFAT-luciferase cell line, catalog number CS176401) were cultured in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum (Sigma), 2 mM L-glutamine (Sigma), 10 mM HEPES (Hyclone, GE Healthcare Life Sciences), 1 × MEM EAA (Sigma), 1 × sodium pyruvate (Sigma), 500 μg / mL genethicin (Invitrogen), and 0.5 μg / mL puromycin (Invitrogen). Jurkat NFAT-luciferase / CD112R clone C4 cells were co-cultured with Chinese hamster ovary (CHO)-K1 cells (developed in-house) that stably express human CD112 and human T cell engagers, thereby binding to and stimulating T cell receptors. Nutrient Mixture In a complete growth medium containing F12 HAM (Sigma), 10% fetal bovine serum, 10 mM HEPES, 500 μg / mL genethicin, 200 μg / mL hygromycin B (Invitrogen), and 100 μg / mL zeosin (Invitrogen), 1 × 10 4 CHO-K1-CD112+ cells were seeded overnight in a white half-area 96-well plate (Costar catalog number 3688) at 37°C / 5% CO2. After overnight incubation, growth medium was used to sample 5 × 10¹⁶ hybridoma supernatants in assay medium (RPMI1640 medium supplemented with 1% fetal bovine serum, 2 mM L-glutamine, and 10 mM HEPES) or in the presence of antibodies (including their respective controls). 4Each well was replaced with Jurkat NFATluc / CD112R clone C4 cells and incubated at 37°C / 5% CO2 for 18 hours. The reporter signal in each well was determined using the Bio-Glo luciferase assay system (Promega catalog number G7940) according to the manufacturer's recommendations. Luminescence was detected using an EnVision plate reader (Perkin Elmer). In single-point assays, human IgG in the depleted hybridoma culture supernatant samples was quantified, normalized to a fixed concentration, and tested at 2.0 μg / mL. A total of 216 antibodies resulted in more than 3-fold induction of the NFAT-luciferase signal and proceeded to further screening.

[0182] Primary cell binding assay The binding of hybridoma supernatant to CD112R expressed in primary human and cynomolgus monkey cells was examined by flow cytometry. For the human primary cell binding assay, purified human T cells (Biological Specialty Corp.) were thawed and measured in 2.5 × 10⁶ cells. 6 The cells were suspended at a concentration of cells / mL. T cells were stimulated at 37°C / 5%CO2 for 72 hours in plates pre-coated with 5 μg / mL anti-mouse IgG Fc (Pierce) using 5 μg / mL anti-human CD3 clone OKT3 (eBioscience) and 1 μg / mL anti-human CD28 (BD Pharmingen). After 72 hours, the cells were removed, washed, and 0.5 × 10⁶ cells were mixed with 10 ng / mL IL-2 (Pepro Tech). 6 The cells were suspended at a concentration of cells / mL. The cells were then incubated at 37°C and 5% CO2 for a further 5 days. For the cynomolgus monkey primary cell binding assay, cynomolgus monkey PBMCs (SNBLs) were thawed and 4 × 10⁶ cells were attached. 6 ~5×10 6The cells were suspended at a concentration of cells / mL. PBMCs were stimulated at 37°C / 5%CO2 for 72 hours with 1 μg / mL anti-human CD3 clone SP34 (BD Pharmingen) and 1 μg / mL anti-human CD28 (BD Pharmingen) in plates pre-coated with 5 μg / mL anti-mouse IgG Fc (Pierce). After 72 hours, the cells were removed, washed, and 0.5 × 10⁶ cells were mixed with 20 ng / mL IL-2 (Pepro Tech). 6 The cells were suspended at a concentration of cells / mL. The cells were then incubated at 37°C and 5% CO2 for a further 7 days.

[0183] A complete culture medium change, including the addition of fresh IL-2, was performed every 48–72 hours for both human and cyno cells. At each medium change, 0.5 × 10 cells were added. 6 The cells were suspended at a concentration of cells / mL. After final incubation, the cells were prepared for flow cytometry by incubation with standardized hybridoma supernatant, positive control antibody, and isotype control antibody at a final concentration of 10 μg / mL. Alexa Fluor647AffiniPure F(ab') 2 Fragmented goat anti-hi IgG(H+L) (Jackson ImmunoResearch) was used for secondary detection at 5 μg / mL, and 8.25 nM YoPro1 (Invitrogen) was used for live / dead cell staining. Subsequently, cells were electrophoresed using a BD FACSCanto II flow cytometer to detect anti-CD112R antibody binding.

[0184] In wave 1, over 1200 antibodies were confirmed to bind to the human CD112R receptor transiently expressed on 293T cells. Of these, 216 antibodies were found to bind to the endogenous human CD112R receptor expressed on Jurkat cells and also function as antagonists of CD112R activity. To identify antibodies that cross-react with cynomolgus monkey orthologs of CD112R, a panel of over 1200 recombinant human binders was tested for binding to recombinant cynoCD112R transiently expressed on HEK293T cells. 274 antibodies in the panel were found to bind to cynoCD112R, and only two of these (11E4 and 1E1) were found to bind to endogenous cynoCD112R expressed on primary cynoT cells.

[0185] Selection of the second wave and antibody A second wave of Xenomouse immunization was performed, including the XMG2kl strain and a custom-generated XMG2CD112R knockout (KO) mouse strain. Hybridoma cells were generated substantially as described above, and antibodies against human CD112R were identified and selected using the screening assay described in Figure 3. Representative data from harvests 6–9 from the second wave are shown in Figures 4B and 4C. The second wave yielded over 1300 human CD112R-specific antibodies, of which 350 resulted in more than 3-fold induction of NFAT-luciferase signaling, as determined by Jurkat RGA. In the wave 2 panel of recombinant human CD112R binders, only 336 antibodies bound to recombinant cynoCD112R transiently expressed on 293T cells, and only 27 antibodies bound to endogenous cynoCD112R expressed on primary cynoT cells.

[0186] A summary of the characteristics of antibody harvested from the first and second waves is provided in Figures 5A and 5B. Antibodies that showed antagonist function by Jurkat RGA determination and antigen-binding activity by human and cynoprimary cell binding assays were subsequently subjected to characterization screening, sequencing, and affinity determination.

[0187] High-throughput KinExA affinity ranking of anti-hCD112R antibodies using soluble hCD112R Selective monoclonal antibodies specific to human CD112R were ranked by affinity using the high-throughput (HT) KinExA method with a Kd cutoff of 100 pM. This method is based on the theory that when an antibody equilibrates with an antigen concentration at the Kd cutoff and an Ab concentration below the Kd cutoff concentration, the free Ab present in equilibrium is less than 50% if its Kd is 100 pM or less.

[0188] In short, the experiment involved equilibrating each antibody at 25 pM in PBS / 0.05% NaN3 / 0.01% BSA with or without 100 pM hCD112R at room temperature for 24 hours. At equilibrium, free antibodies present in the equilibrium mixture and in individual antibody tubes were measured in KinExA. Free antibodies were captured using PMMA beads coated with hCD112R and detected using a mixture of Mu anti-hIgG2, G3, G4+ anti-muIgG(H+L)Alexa647.

[0189] The KinExA signal generated by the antibody alone is considered 100% free, and the inhibitory free fraction (IFF)% is calculated from the signal measured in the presence of the antigen as follows:

number

[0190] Antibodies exhibiting the lowest IFF% have the highest affinity, and conversely, antibodies exhibiting the highest IFF% have the lowest affinity; they can be ranked by plotting their IFF% on a graph. Monoclonal antibodies giving an IFF of 50% or less should have passed a 100 pM Kd cutoff, which means they should each have a Kd of 100 pM or less.

[0191] The results are shown in Figure 6. Of the 10 antibodies analyzed, 8 had K levels below 100 pM. D This indicates that only two types have K levels exceeding 100 pM. D This was shown.

[0192] Molecular rescue and sequencing of CD112R antagonist antibodies The heavy and light chains of selected CD112R antibodies were sequenced. Briefly, RNA (total RNA or mRNA) was purified from wells containing CD112R agonist antibody-producing hybridoma cells using the Qiagen RNeasy mini or Invitrogen mRNA catcher plus kit. Using the purified RNA, cDNA synthesis was performed by reverse transcription, followed by amplification of the variable region (V) genes of the antibody heavy and light chains by polymerase chain reaction (RT-PCR). The fully human antibody gamma heavy chain was obtained using the Qiagen 1-step reverse transcriptase PCR kit (Qiagen). Using this method, the initial cDNA strand was generated from an RNA template, and then the variable region of the gamma heavy chain was amplified by multiplex PCR. A 5' gamma chain-specific primer was annealed to the signal sequence of the antibody heavy chain, while a 3' primer was annealed to the gamma constant domain region. The fully human kappa light chain was obtained using the Qiagen 1-step reverse transcriptase PCR kit (Qiagen). Using this method, the initial cDNA strand was generated from an RNA template, and then the variable region of the kappa light chain was amplified by multiplex PCR. A 5' kappa light chain-specific primer was annealed to the signal sequence of the antibody light chain, while the 3' primer was annealed to the region of the kappa constant domain. The complete human lambda light chain was obtained using the Qiagen 1-step reverse transcriptase PCR kit (Qiagen). Using this method, the initial cDNA strand was generated from an RNA template, and then the variable region of the lambda light chain was amplified by multiplex PCR. A 5' lambda light chain-specific primer was annealed to the signal sequence of the light chain, while the 3' primer was annealed to the region of the lambda constant domain.

[0193] The amplified cDNA was enzymatically purified using exonuclease I and alkaline phosphatase, and the purified PCR product was directly sequenced. The amino acid sequence was predicted by bioinformatics from the corresponding nucleic acid sequence. To confirm that none of the observed mutations were the result of PCR, two additional independent RT-PCR amplification and sequencing cycles were completed for each hybridoma sample. Next, the obtained amino acid sequences were analyzed to determine the germline sequence origin of the antibody and to identify differences from the germline sequence. The amino acid sequences corresponding to the complementarity-determining regions (CDRs) of the sequenced antibody were aligned, and these alignments were used to group clones by similarity. The sequences were also analyzed for "hot spots" (residues that were predicted by computer or empirically determined to adversely affect molecular expression, purification, thermal stability, colloidal stability, long-term storage stability, in vivo pharmacokinetics, and / or immunogenicity). The assay results are shown in Figure 7A. Figure 7B lists the antibodies analyzed for sequence diversity and shows the VH germline and HC CDR3 residues. The EC50(nM) determined by Jurkat RGA for each antibody is also listed. To proceed to the next round of screening, the antibody list in Figure 7B was narrowed down to the following antibodies based on sequence diversity analysis and hotspot analysis: 1E1, 11E4, 27G12, 29E10, 31B3, and 24F1.

[0194] Human CD112R receptor-ligand competition assay The ability of human CD112R-conjugated hybridoma supernatants to block human CD112L was tested using flow cytometry on beads, as follows: Biotinylated human CD112R-Fc was captured on streptavidin polystyrene beads (Spherotech, catalog no. SVP-60-5) in FACS buffer and incubated at room temperature for 30 minutes. The beads were washed twice with FACS buffer, pelletized by centrifugation, the supernatant was removed, and the beads were resuspended in FACS buffer to remove unbound proteins. Biotinylated human CD112R-coated beads were added to a 96-well plate. Hybridoma supernatant samples were added to a final concentration of 5 μg / mL, the beads were resuspended, and incubated at room temperature for 1 hour. Zenon® Alexa Fluor 647 (Molecular Probes, catalog no. Z25408)-labeled CD112-huFc ligand, labeled according to the manufacturer's protocol and prepared in FACS buffer, was added at a final concentration of 370 ng / mL and incubated in the dark at room temperature for 15 minutes. The beads were washed once with FACS buffer, pelletized by centrifugation, the supernatant was removed, and the beads were resuspended in FACS buffer to remove unbound ligand. The samples were then resuspended in FACS buffer and read using a BD Accuri® flow cytometer with an Intellicyt HyperCyt autosampler.

[0195] The results are shown in Figure 8. As shown in this figure, all six antibodies (1E1, 11E4, 27G12, 29E10, 31B3, and 24F1) showed significant inhibitory activity, preventing over 90% of CD112 ligands from binding to the CD112R receptor. The inhibitory activity of the six antibodies was equivalent to that of two reference anti-CD112R antibodies, PL-52575 and PL-52577, which had IC50 values ​​of 0.12 nM and 0.10 nM, respectively.

[0196] Competition-based binning for CD112R antibody lead panels Selected human CD112R-conjugated hybridoma supernatants were tested for competition-based binning using the Octet HTX platform. Antibodies were loaded onto an Anti-HuFc (kinetic) biosensor ForteBio 18-5064 at 2 μg / mL for 2 minutes in assay buffer (pH 7.4) containing 10 mM Tris, 0.1% Triton, 150 mM NaCl, 1 mM CaCl2, and 0.1 mg / mL BSA. The biosensor was then blocked with 50 μg / mL of unrelated HuIgG2 in assay buffer for 5 minutes. 1 μg / mL of CD112R was conjugated in as...

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[Claim 1] The invention described in the specification.

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