Combination therapy for cancer treatment

Abstract

A combination therapy is provided for treating cancers with KRAS G12C mutations, comprising a construct having a TIGIT-binding moiety and a PVRIG-binding moiety, and a KRAS G12C inhibitor. In some cases, the construct is a multispecific or bispecific antibody that recognizes PVRIG and TIGIT.

Description

[Technical Field]

[0001] This application claims the interests of PCT application number PCT / CN2023 / 106333, filed on 7 July 2023, which is incorporated herein by reference in its entirety.

[0002] This application relates to a combination therapy for treating cancer using a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and a KRAS G12C inhibitor. [Background technology]

[0003] The tumor immunomicroenvironment is a crucial component of a patient's response to immunotherapy. The same treatment can elicit a wide range of responses, from minimal or no clinical benefit to significant clinical response. One factor in the therapeutic response is T cell exhaustion. In the tumor microenvironment, persistent antigen stimulation can lead to T cell exhaustion (a state of T cell dysfunction), and co-inhibitory receptors such as PD-1, LAG-3, TIM3, and TIGIT may be overexpressed.

[0004] TIGIT (T cell immune receptor with Ig and ITIM domains), also known as Vstm3 and WUCAM, is a co-inhibitory receptor expressed on NK and CD8+ T cells, as well as CD4+ T cell subsets such as immunosuppressive regulatory T cells (Tregs) [1-4]. Four ligands for TIGIT are known: poliovirus receptor (PVR), PVRL2, PVRL3, and PVRL4, all of which are overexpressed in tumors and antigen-presenting cells, causing immunosuppression [3, 5-8]. These ligands also bind to the co-stimulatory molecule CD226 and the co-inhibitory molecules PVRIG and CD96 (the latter sometimes considered a co-stimulatory molecule). TIGIT antagonist antibodies prevent TIGIT from binding to its ligand, blocking its inhibitory signal and inducing a potent anti-tumor immune response by making CD226-mediated activation signals dominant [9-11]. TIGIT, like other fatigue markers such as PD-1, LAG3, and TIM3 [15, 16], has been identified as a fatigue marker with increased expression in cancer and inflammatory diseases [12-14]. Furthermore, TIGIT has been identified as an important inhibitory receptor for stem cell-like memory T cells, a new population of T cells that may be a preferred target for anti-PD-(L)1 efficacy [17, 18].

[0005] Poliovirus receptor-associated Ig domain-containing protein (PVRIG, also known as CD112R) is a co-inhibitory immune checkpoint protein. PVRIG plays an important role in the recovery of T cells from exhaustion and the increased activation of NK cells [19-22]. PVRIG belongs to the nectin and nectin-like family, which also includes TIGIT, DNAM-1 (CD226), and CD96. PVRIG is expressed in NK cells and T cells, and its expression increases further in T cells when activated [19, 20]. The interaction between PVRIG and its ligand PVRL2 (CD112), which is expressed in APCs and several tumor cells, suppresses the activation of T cells and NK cells [21, 22]. PVRL2 is also a ligand for CD226, and CD226 activates human T cells and human NK cells through interaction with its ligand [23-25]. Although PVRIG and TIGIT share many similarities, scientists have reported clear differences that indicate these pathways are not overlapping [20, 22].

[0006] KRAS is a key regulator of cytokines that shape the tumor microenvironment. KRAS mutations play a role in many cancers. For example, the KRAS G12C mutation accounts for approximately 44% of all KRAS mutations. G12C is a point mutation involving a glycine-to-cysteine ​​substitution at codon 12 of the KRAS protein, supporting an active GTP-binding conformation. This promotes constitutive activation of signaling pathways that lead to tumorigenesis.

[0007] All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are incorporated herein by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference. [Overview of the Initiative]

[0008] In one embodiment, the present application provides a method for treating cancer in an individual, comprising administering to the individual 1) a construct comprising an effective amount of a TIGIT binding moiety and a PVRIG binding moiety, and 2) an effective amount of a KRAS G12C inhibitor.

[0009] In some embodiments, the TIGIT binding portion comprises an antibody portion comprising a heavy chain variable region (VH) containing heavy chain CDR (HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) containing light chain CDR (LCDR)1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 1; HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 2; and HCDR3 comprises the amino acid sequence of SEQ ID NO: 3. The amino acid sequences include an acid sequence or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 3. LCDR1 includes the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 4. LCDR2 includes the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 5. LCDR3 includes the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 6. Optionally, the substitutions are conservative substitutions. In some embodiments, HCDR1 includes the amino acid sequence of SEQ ID NO: 1, HCDR2 includes the amino acid sequence of SEQ ID NO: 2, HCDR3 includes the amino acid sequence of SEQ ID NO: 3, LCDR1 includes the amino acid sequence of SEQ ID NO: 4, LCDR2 includes the amino acid sequence of SEQ ID NO: 5, and LCDR3 includes the amino acid sequence of SEQ ID NO: 6.

[0010] In some embodiments of any of the above methods, the PVRIG binding moiety comprises an antibody moiety comprising VH containing HCDR1, HCDR2, and HCDR3, and VL containing LCDR1, LCDR2, and LCDR3, where HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 7; HCDR2 comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 8; and HCDR3 comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence of SEQ ID NO: 9 The amino acid sequences include those having one, two, or three or fewer amino acid substitutions compared to each other. For example, LCDR1 includes the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 10; LCDR2 includes the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 11; and LCDR3 includes the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 12. Optionally, the substitutions are conservative substitutions. In some embodiments, HCDR1 includes the amino acid sequence of SEQ ID NO: 7, HCDR2 includes the amino acid sequence of SEQ ID NO: 8, HCDR3 includes the amino acid sequence of SEQ ID NO: 9, LCDR1 includes the amino acid sequence of SEQ ID NO: 10, LCDR2 includes the amino acid sequence of SEQ ID NO: 11, and LCDR3 includes the amino acid sequence of SEQ ID NO: 12.

[0011] In some embodiments of any of the above methods, the TIGIT coupling portion and the PVRIG coupling portion are either in Fab format or scFv format. In some embodiments, the TIGIT coupling portion is in Fab format and the PVRIG coupling portion is in scFv format, or the TIGIT coupling portion is in scFv format and the PVRIG coupling portion is in Fab format, or both the TIGIT coupling portion and the PVRIG coupling portion are in Fab format.

[0012] In some embodiments of any of the above methods, the VH portion of the TIGIT binding portion includes the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 13, and / or the VL portion of the TIGIT binding portion includes the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 14.

[0013] In some embodiments of any of the above methods, the VH of the PVRIG binding portion includes the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 15, and / or the VL of the PVRIG binding portion includes the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 16.

[0014] In some embodiments of any of the above methods, the construct further comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region, or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variant comprises one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend its half-life.

[0015] In some embodiments of any of the above methods, the structure includes (a) an Fc region operably connected to a TIGIT coupling portion and a PVRIG coupling portion, (b) a TIGIT coupling portion operably connected to a PVRIG coupling portion and a PVRIG coupling portion operably connected to an Fc region, or (c) a PVRIG coupling portion operably connected to a TIGIT coupling portion and a TIGIT coupling portion operably connected to an Fc region.

[0016] In some embodiments of any of the above methods, the structure includes one, two or more TIGIT coupling portions and one, two or more PVRIG coupling portions.

[0017] In some embodiments of any of the above methods, the construct includes identical or different TIGIT coupling portions and / or identical or different PVRIG coupling portions.

[0018] In some embodiments of any of the above methods, the construct comprises (i) first and second heavy chains including sequence number 17 and first and second light chains including sequence number 18, or (ii) first and second heavy chains including sequence number 19 and first and second light chains including sequence number 20.

[0019] In some embodiments of any of the above methods, the construct comprises two heavy chains and two light chains, wherein the TIGIT binding portion is in Fab format and the PVRIG binding portion is in scFv format, and from the N-terminus to the C-terminus, the first and second heavy chains each comprise a domain operably linked to the following format, VH1-CH1-Fc-scFv or scFv-VH1-CH1-Fc, and the first and second light chains each comprise a domain operably linked to the following format, VL1-CL, where VH1-CH1 and VL1-CL originate from the TIGIT binding portion and scFv originates from the PVRIG binding portion.

[0020] In some embodiments of any of the above methods, the construct comprises two heavy chains and two light chains, wherein the TIGIT binding portion is in scFv format and the PVRIG binding portion is in Fab format, and from the N-terminus to the C-terminus, the first and second heavy chains each comprise a domain operably linked to the following format, scFv-VH2-CH1-Fc or VH2-CH1-Fc-scFv, and the first and second light chains each comprise a domain operably linked to the following format, VL2-CL, where scFv originates from the TIGIT binding portion and VH2-CH1 and VL2-CL originate from the PVRIG binding portion.

[0021] In some embodiments of any of the above methods, the construct comprises two heavy chains and four light chains, wherein the TIGIT binding portion and the PVRIG binding portion are in Fab format, and from the N-terminus to the C-terminus, the first and second heavy chains each comprise a domain operably linked to the following format: VH1-CH1-Fc-VH2-CH1, VH2-CH1-Fc-VH1-CH1, VH1-CH1-VH2-CH1-Fc or VH2-CH1-VH1-CH1-Fc, the first and second light chains each comprise a domain operably linked to the following format: VL1-CL, and the third and fourth light chains each comprise a domain operably linked to the following format: VL2-CL, where VH1-CH1 and VL1-CL originate from the TIGIT binding portion and VH2-CH1 and VL2-CL originate from the PVRIG binding portion.

[0022] In some embodiments of any of the above methods, the construct comprises two heavy chains and four light chains, the TIGIT binding construct comprises two heavy chains and four light chains, the TIGIT binding portion and the PVRIG binding portion are in Fab format, and from the N-terminus to the C-terminus, the first and second heavy chains each comprise a domain operably linked to the following format: VH1-C1-Fc-VH2-CH1, VH2-CH1-Fc-VH1-C1, VH1-C1-VH2-CH1-Fc or VH2-CH1-VH1-C1-Fc, the first and second light chains each comprise a domain operably linked to the following format: VL1-C2, and the third and fourth light chains each comprise a domain operably linked to the following format: VL2-CL, where VH1-C1 and VL1-C2 originate from the TIGIT binding portion and VH2-CH1 and VL2-CL originate from the PVRIG binding portion.

[0023] In some embodiments of any of the above methods, the construct comprises two heavy chains and four light chains, wherein the TIGIT binding portion and the PVRIG binding portion are in Fab format, and from the N-terminus to the C-terminus, the first and second heavy chains each comprise a domain operably linked to the following format: VH1-CH1-Fc-VH2-C1, VH2-C1-Fc-VH1-CH1, VH1-CH1-VH2-C1-Fc or VH2-C1-VH1-CH1-Fc, the first and second light chains each comprise a domain operably linked to the following format: VL1-CL, and the third and fourth light chains each comprise a domain operably linked to the following format: VL2-C2, where VH1-CH1 and VL1-CL originate from the TIGIT binding portion and VH2-C1 and VL2-C2 originate from the PVRIG binding portion.

[0024] In some embodiments of some of the above methods, scFv includes a VH region operably linked to a VL region, where the VH region is at the N-terminus of the VL region, or the VL region is at the N-terminus of the VH region.

[0025] In some embodiments of any of the above methods, the KRAS G12C inhibitor is a small molecule. In some embodiments, the KRAS G12C inhibitor is selected from the group consisting of sotrasib, adagrasib, JAB-21822, GDC-6036, JDQ443, D-1553, GH35, GFH925, BPI-421286 and LY3537982, RMC-6291, HBI-2438, BI 1823911, MK-1084 and JNJ-74699157. In some embodiments, the KRAS G12C inhibitor is represented by formula (III) or a pharmaceutically acceptable salt thereof. [ka]

[0026] During the ceremony,

[0027] T1 is selected from O and N.

[0028] R1 is selected from C6-10 aryls and 5-10 membered heteroaryls, where the C6-10 aryls and 5-10 membered heteroaryls are optionally substituted with 1, 2, 3, 4, or 5 Ra units.

[0029] If T1 is O, then R2 does not exist.

[0030] When T1 is N, R2 is selected from H, C1~3 alkyl, -C(=O)-C1~3 alkyl, and -S(=O)2-C1~3 alkyl, where C1~3 alkyl, -C(=O)-C1~3 alkyl, and -S(=O)2-C1~3 alkyl are optionally substituted with one, two, or three Rb atoms.

[0031] R3 is a C1-3 alkyl group, where the C1-3 alkyl group is optionally substituted with one, two, or three Rc atoms.

[0032] R4 is selected from H and C1-3 alkyl groups, where C1-3 alkyl groups are optionally substituted with one, two, or three Rd groups.

[0033] R5, R6, and R7 are each independently selected from H, F, Cl, Br, I, and C1-3 alkyl groups, where the C1-3 alkyl groups are optionally substituted with one, two, or three F atoms.

[0034] R8 is selected from H and CH3.

[0035] Ra is independently selected from F, Cl, Br, I, OH, NH2, CN, C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl, where C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl are optionally substituted with one, two, or three F atoms.

[0036] Rb is independently selected from F, Cl, Br, I, OH, and NH2.

[0037] Each Rc is independently selected from a 4- to 8-membered heterocycloalkyl group, where the 4- to 8-membered heterocycloalkyl group is optionally substituted with one, two, or three R atoms.

[0038] Rd is independently selected from F, Cl, Br, I, OH, NH2, and CN.

[0039] R is independently selected from H, F, Cl, Br, OH, CN, C1-3 alkyl, C1-3 alkoxy, and -C1-3 alkyl-OC(=O)-C1-3 alkylamino.

[0040] However, if R1 is naphthyl, naphthyl can be optionally substituted with F, Cl, Br, OH, NH2, CF3, CH2CH3, and -C≡CH, and R5, R6, and R7 are each independently H.

[0041] In some embodiments of any of the above methods, the construct is administered intravenously or subcutaneously.

[0042] In some embodiments of any of the above methods, the KRAS G12C inhibitor is administered orally.

[0043] In some embodiments of any of the above methods, the cancer comprises one or more cancer cells expressing the KRAS G12C mutant protein.

[0044] In some embodiments of any of the above methods, cancers include colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, stomach cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma.

[0045] In some embodiments of any of the above methods, the cancer is an advanced, unresectable, and / or metastatic solid tumor.

[0046] In some embodiments of any of the above methods, the construct and the KRAS G12C inhibitor are administered simultaneously.

[0047] In some embodiments of any of the methods described above, the construct and the KRAS G12C inhibitor are administered in parallel.

[0048] In some embodiments of any of the above methods, the construct and the KRAS G12C inhibitor are administered sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct.

[0049] In some embodiments of any of the methods described above, the method further includes administering an effective dose of a third therapy to the individual. In some embodiments, the third therapy comprises another anticancer agent. In some embodiments, the anticancer agent is selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents.

[0050] In some embodiments of any of the methods described above, the individual is human.

[0051] In some embodiments of any of the methods described above, the method includes selecting an individual for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0052] In another embodiment, the present application provides a kit for treating cancer in an individual, comprising 1) a construct comprising a TIGIT-binding moiety and a PVRIG-binding moiety, and 2) a KRAS G12C inhibitor. In some embodiments, the construct binds to PVRIG and / or TIGIT. In some embodiments, the construct that binds to TIGIT is an anti-TIGIT antibody or an immunologically active fragment thereof. In some embodiments, the construct that binds to PVRIG is an anti-PVRIG antibody or an immunologically active fragment thereof. In some embodiments, the anti-TIGIT antibody or an immunologically active fragment thereof is (a) CDR-H1 comprising (1) SEQ ID NO: 1, (2) CDR-H2 comprising SEQ ID NO: 2, and (3) CDR-H3 comprising SEQ ID NO: 3. H (b) (1) CDR-L1 containing sequence number 4, (2) CDR-L2 containing sequence number 5, and (3) CDR-L3 containing sequence number 6 LThe CDR sequence is defined according to the Kabat numbering system. In some embodiments, the anti-PVRIG antibody or its immunologically active fragment is (a) (1) CDR-H1 containing SEQ ID NO: 7, (2) CDR-H2 containing SEQ ID NO: 8, and (3) CDR-H3 containing SEQ ID NO: 9. H (b) (1) CDR-L1 containing sequence number 10, (2) CDR-L2 containing sequence number 11, and (3) CDR-L3 containing sequence number 12 L The CDR sequence is defined according to the Kabat numbering system. In some embodiments, the V of the anti-TIGIT antibody or its immunologically active fragment is included. H The domain contains an amino acid sequence that has at least 85% identity with SEQ ID NO: 13, and is the V of an anti-TIGIT antibody or an immunologically active fragment thereof. L This includes an amino acid sequence having at least 85% identity with SEQ ID NO: 14. In some embodiments, the V of an anti-TIGIT antibody or an immunologically active fragment thereof is used. H The domain contains an amino acid sequence that has at least 85% identity with SEQ ID NO: 15, and is the V of an anti-TIGIT antibody or an immunologically active fragment thereof. LIt comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 16. In some embodiments, the construct comprises (i) first and second heavy chains comprising SEQ ID NO: 17 and first and second light chains comprising SEQ ID NO: 18, or (ii) first and second heavy chains comprising SEQ ID NO: 19 and first and second light chains comprising SEQ ID NO: 20. In some embodiments, the kit further comprises a KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is an antibody, peptide, protein, antisense oligonucleotide, or small molecule that inhibits the activity of the KRAS G12C mutant protein. In some embodiments, the KRAS G12C inhibitor is a small molecule inhibitor. In some embodiments, the small molecule is selected from the group consisting of compound 17, sotrasib, adaglasib, JAB-21822, GDC-6036, JDQ443, D-1553, GH35, GFH925, BPI-421286 and LY3537982, RMC-6291, HBI-2438, BI 1823911, MK-1084 and JNJ-74699157. In some embodiments, the small molecule is compound 17. In some embodiments, cancers comprising one or more cancer cells expressing the KRAS G12C mutant protein include lung cancer, colon adenocarcinoma, colorectal adenocarcinoma, pancreatic cancer, bile duct cancer, endometrial cancer, ovarian cancer, peritoneal cancer, bladder cancer, gastric cancer, thyroid cancer, melanoma, breast cancer, head and neck cancer, multiple myeloma, acute myeloid leukemia (AML), uterine cancer, gastroesophageal cancer, or rectal adenocarcinoma.

[0053] In another embodiment, this application provides the use of a construct comprising a TIGIT binding moiety and a PVRIG binding moiety for the manufacture of a pharmaceutical product for the treatment of cancer, wherein the treatment is in combination with a KRAS G12C inhibitor. In some embodiments, the cancer is selected from colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, the cancer is colon cancer.

[0054] It should be understood that one, some, or all of the characteristics of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other embodiments of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention will be further described by the following detailed description. [Brief explanation of the drawing]

[0055] [Figure 1A] Figure 1A shows a gating strategy for identifying various myeloid cell populations.

[0056] [Figure 1B] Figure 1B shows a gating strategy for identifying various T cell populations.

[0057] [Figure 2] Figure 2 shows strategies for quantifying CD226-positive cells in various cell type populations.

[0058] [Figure 3] Figure 3 shows a gating strategy for identifying various cell populations for subsequent immune checkpoint expression analysis.

[0059] [Figure 4] Figure 4 shows strategies for quantifying cells positive for various markers in whole T cell and CD8+ T cell populations, myeloid cells, and tumor cells.

[0060] [Figure 5] Figure 5 shows the percentage of CD45+ cells in a population of living cells.

[0061] [Figure 6] Figure 6 shows the percentage of myeloid cell populations and T cell populations in CD45+ cells.

[0062] [Figure 7] Figure 7 shows the percentage of the myeloid-derived suppressor cell (MDSC) subpopulation in CD45+ cells.

[0063] [Figure 8] Figure 8 shows the percentages of M1 and M2 macrophages.

[0064] [Figure 9] Figure 9 shows the percentage of CD8+ T cells and CD4+ T cells in CD45+ cells.

[0065] [Figure 10] Figure 10 shows the expression of CD226 in T cells and various T cell subpopulations. MFI indicates the average fluorescence intensity.

[0066] [Figure 11A] Figure 11A shows the median fluorescence intensity (MFI) of PVRIG signaling in T cells and CD8+ T cells before and after treatment with compound 17.

[0067] [Figure 11B] Figure 11B shows the PVRIG positivity rates in T cell and CD8+ T cell populations before and after treatment with compound 17.

[0068] [Figure 12A] Figure 12A shows the median fluorescence intensity (MFI) of the TIGIT signal in T cells and CD8+ T cells before and after treatment with compound 17.

[0069] [Figure 12B] Figure 12B shows the TIGIT positivity rates in T cell and CD8+ T cell populations before and after treatment with compound 17.

[0070] [Figure 13A] Figure 13A shows the median fluorescence intensity (MFI) of the CD112 signal in myeloid cells and tumor cells before and after treatment with compound 17.

[0071] [Figure 13B] Figure 13B shows the CD112 positivity rates in myeloid and tumor cell populations before and after treatment with compound 17.

[0072] [Figure 14A] Figure 14A shows the median fluorescence intensity (MFI) of the CD155 signal in myeloid cells and tumor cells before and after treatment with compound 17.

[0073] [Figure 14B] Figure 14B shows the CD155 positivity rates in myeloid and tumor cell populations before and after treatment with compound 17.

[0074] [Figure 15] Figure 15 shows the average percentage change in body weight per treatment group. N indicates the number of mice in the treatment group, PO indicates oral administration, QD indicates once daily, Q2W indicates treatment every two weeks, and IP+PO indicates that compound 17 was administered orally and antibody msG15 was administered intraperitoneally (IP).

[0075] [Figure 16] Figure 16 shows the average tumor volume change over time for each treatment group.

[0076] [Figure 17A]Figure 17A shows the individual tumor volume growth curves of mice administered the vehicle control.

[0077] [Figure 17B] Figure 17B shows the individual tumor volume growth curves of mice treated with compound 17 at a dose of 10 mg / kg.

[0078] [Figure 17C] Figure 17C shows the individual tumor volume growth curves of mice treated with compound 17 at a dose of 30 mg / kg.

[0079] [Figure 17D] Figure 17D shows the individual tumor volume growth curves of mice treated with the antibody msG15 at a dose of 13.3 mg / kg.

[0080] [Figure 17E] Figure 17E shows the individual tumor volume growth curves of mice treated with antibody msG15 (13.3 mg / kg) and compound 17 (10 mg / kg).

[0081] [Figure 17F] Figure 17F shows the individual tumor volume growth curves of mice treated with antibody msG15 (13.3 mg / kg) and compound 17 (30 mg / kg).

[0082] [Figure 18] Figure 18 shows the mean survival rate over time for each treatment group.

[0083] [Figure 19] Figure 19 shows the complete response rate (CR) for groups that received different treatments. [Modes for carrying out the invention]

[0084] This application provides a combination therapy for treating KRAS-mutated cancer (e.g., KRAS G12C cancer) comprising a construct containing a TIGIT ("T cell immune receptor having Ig and ITIM domains") binding moiety and a PVRIG ("containing a poliovirus receptor-associated immunoglobulin domain") binding moiety, and a KRAS inhibitor (e.g., a KRAS G12C inhibitor). In some embodiments, the construct contains a TIGIT / PVRIG multispecific antibody. As shown in the examples, this combination therapy provides advantageous effects in treating KRAS-mutated cancer while being safely tolerated in vivo. See, for example, Example 2.

[0085] definition Before describing embodiments in detail, it should be understood that this disclosure is not limited to any particular composition or biological system and is naturally subject to change. It should also be understood that the terminology used herein is intended solely to describe specific embodiments and is not intended to limit them.

[0086] As used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple objects unless the content explicitly indicates otherwise. Thus, for example, a reference to "molecule" may optionally include a combination of two or more such molecules.

[0087] As used herein, the term “about” refers to the normal range of error for each value, which is readily known to those skilled in the art. References to values ​​or parameters “about” herein include (and are described) embodiments relating to the value or parameter itself.

[0088] The aspects and embodiments of this disclosure are understood to include the terms "comprising," "consisting," and "consisting essentially of."

[0089] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues, or aggregates of polymers of multiple amino acid residues. This term applies to amino acid polymers, in which one or more amino acid residues are artificial chemical mimics of corresponding natural amino acids, as well as to natural and non-natural amino acid polymers. The term “amino acid” refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function similarly to natural amino acids. Natural amino acids are those encoded by the genetic code, as well as later modified amino acids, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as natural amino acids, i.e., hydrogen, a carboxyl group, an amino group, and an alpha carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as natural amino acids. The alpha carbon refers to the first carbon atom bonded to a functional group such as a carbonyl group. A beta carbon refers to a second carbon atom linked to an alpha carbon, and this system continues to name carbons alphabetically using Greek letters. Amino acid mimes refer to chemical compounds that have a different structure from the typical chemical structure of amino acids but function similarly to natural amino acids. The term "protein" typically refers to a large polypeptide. The term "peptide" typically refers to a short polypeptide. Polypeptide sequences are usually written so that the left end of the polypeptide sequence is the amino terminus (N terminus) and the right end is the carboxyl terminus (C terminus). As used herein, "construct" refers to a complex comprising one or more polypeptides that associate to perform a particular function. In certain embodiments, the polypeptides are immune-related. In some embodiments, the construct comprises at least two polypeptide chains.

[0090] The terms "operatably linked" or "operatably linked" refer to juxtaposing two or more biological sequences of interest with or without spacers or linkers to create a relationship that enables them to function as intended. When used in relation to polypeptides, it is intended to mean that the polypeptide sequence is linked in such a way that the linked product will have its intended biological function. For example, an antibody variable region may be operatably linked to a constant region to yield a stable product with antigen-binding activity. The term may also be used in relation to polynucleotides. For example, when a polynucleotide encoding a polypeptide is operatably linked to a control sequence (e.g., a promoter, enhancer, or silencer sequence), it is intended to mean that the polynucleotide sequence is linked in such a way that it enables the controlled expression of the polypeptide from the polynucleotide.

[0091] As used herein, the term “antibody” is used in its broadest sense and specifically includes intact antibodies (e.g., full-length antibodies), antibody fragments (but not limited to Fab, F(ab')2, scFv, scFv-Fc, single-domain antibodies, etc.), monoclonal antibodies, and polyclonal antibodies, insofar as they exhibit the desired biological activity (e.g., epitope binding).

[0092] A “blocking” antibody or “antagonist” antibody inhibits or reduces the biological activity of the antigen to which it binds. In some embodiments, the blocking antibody or antagonist antibody substantially inhibits (e.g., inhibits by at least 50%, 60%, 70%, 80%, 90%, or 95%) or completely inhibits the biological activity of the antigen. For example, the bispecific antibodies of this application block TIGIT and PVRIG-mediated signaling.

[0093] As used herein, the term “isolated” antibody may refer to an antibody that is substantially free of other cellular material. In one embodiment, the isolated antibody is substantially free of other proteins from the same species. In another embodiment, the isolated antibody is expressed by cells from different species and is substantially free of other proteins from different species. In some embodiments, the “isolated” antibody is identified, separated and / or recovered from components of its natural environment. The contaminants of its natural environment may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes that would interfere with the diagnostic or therapeutic use of the antibody. Antibodies can be made substantially free of naturally occurring components (or components associated with the cell expression system used to produce the antibody) by isolating them using protein purification techniques well known in the art. In some embodiments, the antibody may be purified (1) to more than 75% by weight, most preferably more than 80%, 90%, 95%, or 99% by weight, if obtained by the Lowry method, or (2) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. The isolated antibody contains the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment is absent. However, typically, the isolated antibody is prepared by at least one purification step.

[0094] As used herein, the term “epitope” means any antigenic determinant on an antigen to which an antibody paratope binds. Epitope determinants typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural properties and specific charge properties.

[0095] As used herein, the term “variable” refers to the fact that certain portions of the variable domain have significantly different sequences among antibodies, and that these are used for the binding and specificity of each particular antibody to its particular antigen. However, this variability is not uniformly distributed throughout the variable domain of an antibody. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in both the light chain and heavy chain variable domains. The more highly conserved portions of the variable domain are called frameworks (FRs). The natural heavy and light chain variable domains each primarily employ a β-sheet structure and contain four FR regions connected by three CDRs, which form loops that connect, and sometimes form part of, the β-sheet structure. The CDRs of each chain are held together in close proximity by the FR regions and, together with the CDR of the other chain, contribute to the formation of the antibody’s antigen-binding site. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, National Institute of Health, Bethesda, Maryland (1991). The constant domain does not directly participate in antibody binding to the antigen, but exhibits various effector functions, such as antibody involvement in antibody-dependent cytotoxicity. The variable region sequence of interest includes the humanized variable region sequence of multispecific antibodies, which is described in detail elsewhere in this specification.

[0096] The terms "hypervariable regions (HVRs)" or "complementarity-determining regions (CDRs)" may refer to subregions of the VH and VL domains characterized by increased sequence variability and / or the formation of defined loops. These include the three CDRs (H1, H2, H3) in the VH domain and the three CDRs (L1, L2, L3) in the VL domain. H3 is thought to be important for conferring fine binding specificity, while L3 and H3 exhibit the highest levels of variability. See the chapter by Johnson and Wu in Methods in Molecular Biology 248:1-25 (editor Lo, Human Press, Totowa, New Jersey, 2003).

[0097] Several CDR / HVR depictions are known. The Kabat complementarity-determining region (CDR) is based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (1991)). Alternatively, Chothia refers to the location of the structural loop (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). AbM HVR represents the compromise between Kabat HVR and the Chothia structural loop and is used by Oxford Molecular's AbM antibody modeling software. The "contact" HVR is based on an analysis of available complex crystal structures. The residues from each of these HVR / CDRs are shown below. "Framework" or "FR" residues are variable domain residues other than the HVR / CDR residues. Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B(Kabat numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35(Chothia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101

[0098] "Extended" HVRs, namely 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in VL, and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH (Kabat numbering), are also known.

[0099] "Kabat numbering" may refer to the numbering system used for the heavy chain variable domain or light chain variable domain in the antibody sequence collection by Kabat et al. mentioned above. The actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of FR or HVR in the variable domain. Kabat numbering of residues can be determined for a given antibody by alignment of the antibody sequence with the homology region of the "standard" Kabat numbering sequence. Typically, Kabat numbering is used when referring to residues in the variable domain (roughly light chain residues 1-107 and heavy chain residues 1-113), while the EU numbering system or index (e.g., the EU index in Kabat, numbered according to EU IgG1) is generally used when referring to residues in the heavy chain constant region.

[0100] As used herein, “monoclonal” antibody refers to a substantially homogeneous antibody, for example, an antibody obtained from a population of antibodies that are substantially identical but allow for some background mutations and / or modifications. “Monoclonal” exhibits substantially homogeneous characteristics of the antibody and does not require antibody production by a specific method. In some embodiments, monoclonal antibodies are selected by their HVR, VH and / or VL sequences and / or binding characteristics, for example, from a pool of clones (e.g., recombinant, hybridoma, or phage-derived). Monoclonal antibodies can be manipulated to include one or more mutations, for example, to affect the binding affinity or other properties of the antibody, to produce humanized or chimeric antibodies, to improve antibody production and / or homogeneity, and to manipulate multispecific antibodies, the resulting antibodies are still considered essentially monoclonal. A population of monoclonal antibodies can be distinguished from polyclonal antibodies because the individual monoclonal antibodies in that population recognize the same antigenic site. Various techniques are known for producing monoclonal antibodies, such as the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975), Hongo et al., Hybridoma, 14(3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd edition, 1988), and the chapter by Hammerling et al. in Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, New York, 1981)), recombinant DNA method (e.g., see U.S. Patent No. 4,816,567), and phage display technology (e.g., Clackson et al., Nature, 352:624-628 (1991), Marks et al.) al., J.Mol.Biol.222:581-597(1992), Sidhu et al.,J.Mol.Biol.338(2):299-310(2004), Lee et al.,J.Mol.Biol.See 340(5):1073-1093(2004), Fellouse, Proc Natl. Acad. Sci. USA 101(34):12467-12472(2004), and Lee et al., J. Immunol. Methods 284(1-2):119-132(2004), as well as techniques for producing human or human-like antibodies in animals having some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, for example, International Publication No. 1998 / 24893, International Publication No. 1996 / 34096, International Publication No. 1996 / 33735, International Publication No. 1991 / 10741, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551(1993), Jakobovits et al., Nature 362:255-258(1993), Bruggemann et al., Year in Immunol.7:33(1993), U.S. Patent No. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, Marks et al., Bio / Technology 10:779-783 (1992), Lonberg et al., Nature 368:856-859(1994), Morrison, Nature 368:812-813(1994), Fishwild et al., Nature See Biotechnol. 14:845-851 (1996), Neuberger, Nature Biotechnol. 14:826 (1996), and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0101] A “chimeric” antibody may refer to an antibody having a heavy and / or light chain portion from a particular isotype, class, or organism, and another portion from a different isotype, class, or organism. In some embodiments, the variable region may originate from one origin or organism, and the constant region may originate from another origin or organism.

[0102] The term "humanized antibody" may refer to an antibody that primarily contains human sequences and very few non-human (e.g., mouse or chicken) sequences. In some embodiments, a humanized antibody has one or more HVR sequences (having the desired binding specificity) derived from antibodies of a non-human (e.g., mouse or chicken) organism grafted onto a human recipient antibody framework (FR). In some embodiments, non-human residues are further grafted onto the human framework (which are not present in either the source antibody or the recipient antibody) to improve antibody properties, for example. Generally, a humanized antibody contains substantially all of at least one, typically two, variable domains, all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. A humanized antibody may also optionally contain at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of the immunoglobulin constant region (Fc) of a human immunoglobulin. See Jones et al., Nature 321:522-525 (1986), Riechmann et al., Nature 332:323-329 (1988), and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0103] "Human" antibodies may refer to antibodies having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, and / or antibodies produced using any of the techniques for producing human antibodies disclosed herein. Human antibodies are a variety of techniques known in the art, including phage display libraries, Hoogenboom and Winter, J.Mol.Biol., 227:381 (1991), Marks et al., J.Mol.Biol., 222:581 (1991), Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985), Boerner et al. These can be produced using methods such as the preparation of human monoclonal antibodies as described in al., J.Immunol., 147(1):86-95 (1991), or by administering an antigen to transgenic animals modified to produce antibodies in response to antigen stimulation (with the endogenous gene locus deactivated), such as immunized heterologous mice (see, for example, U.S. Patents 6,075,181 and 6,150,584 relating to XENOMOUSE® technology), or chickens possessing human immunoglobulin sequences (see, for example, International Publication Nos. 2012162422, 2011019844, and 2013059159).

[0104] Immunoglobulins have five main classes: IgA, IgD, IgE, IgG, and IgM. Some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0105] As used herein, the term “antibody fragment” and all its grammatical variations are defined, in certain cases, as part of an intact antibody that includes an antigen-binding site or variable region of the intact antibody, excluding the constant heavy chain domain of the Fc region of the intact antibody (i.e., CH2, CH3, and / or CH4, depending on the antibody isotype). Examples of antibody fragments include, but are not limited to, (1) single-chain Fv(scFv) molecules, (2) single-chain polypeptides containing only one light chain variable domain, or fragments thereof containing three CDRs of the light chain variable domain and not containing the associated heavy chain portion, and (3) single-chain polypeptides containing only one heavy chain variable region, or fragments thereof containing three CDRs of the heavy chain variable region and not containing the associated light chain portion, as well as multispecific or multivalent structures formed from antibody fragments. In an antibody fragment comprising one or more heavy chains, the heavy chains may contain any constant domain sequence found in the non-Fc region of an intact antibody (e.g., CH1 in an IgG isotype) and / or any hinge region sequence found in an intact antibody and / or a leucine zipper sequence fused to or located within the hinge region sequence or constant domain sequence of the heavy chain.

[0106] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, each having a single antigen-binding site, and the remaining "Fc" fragment, whose name reflects its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment with two antigen-binding sites that can still crosslink antigens. "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. In double-stranded Fv species, this region consists of a dimer in which one heavy-chain variable domain and one light-chain variable domain are closely and non-covalently bonded. In single-stranded Fv species (scFv), one heavy-chain variable domain and one light-chain variable domain can be covalently bonded by a flexible peptide linker so that the light and heavy chains can associate in a "dimer" structure similar to that of double-stranded Fv species. In this configuration, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. In summary, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only the three antigen-specific CDRs) has the ability to recognize and bind to the antigen, albeit with lower affinity than the entire binding site. See, for example, the Pluckthun chapter in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269–315 (1994).

[0107] The Fab fragment also contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by adding several residues to the carboxyl terminus of the heavy chain CH1 domain, which contains one or more cysteines derived from the antibody hinge region. Fab'-SH is the herein designation for Fab' having a free thiol group in the cysteine ​​residue of the constant domain. The F(ab')2 antibody fragments were originally prepared as pairs of Fab' fragments having a hinge cysteine ​​between them. Other chemical couplings of antibody fragments are also known.

[0108] As used herein, the term “monoclonal antibody” (mAb) refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies within the population are identical except for a very small number of potentially naturally occurring variations. Monoclonal antibodies are highly specific and directed to a single antigenic site. Each mAb is directed to a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized by hybridoma cultures that are not contaminated with other immunoglobulins. The modifier “monoclonal” indicates a characteristic of antibodies obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring antibody production by a specific method. For example, monoclonal antibodies used in accordance with the present invention can be produced in immortalized B cells or their hybridomas, or by recombinant DNA methods.

[0109] Monoclonal antibodies as used herein include hybrid antibodies and recombinant antibodies produced by splicing a variable (including hypervariable) domain of an antibody with a constant domain (e.g., a "humanized" antibody), or by splicing a light chain with a heavy chain, or by splicing a chain from one species with a chain from another species, or by fusing with a heterologous protein, regardless of the designation of the species of origin or immunoglobulin class or subclass, as well as antibody fragments (e.g., Fab, F(ab')2, and Fv) insofar as they exhibit the desired biological activity.

[0110] The monoclonal antibodies described herein specifically include chimeric antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies (insofar as they exhibit the desired biological activity).

[0111] As used herein, the term “treatment” refers to a clinical intervention designed to alter the natural course of an individual or cell being treated in the course of clinicopathology. Desired effects of treatment include a reduction in the rate of disease progression, improvement or mitigation of the disease state, and remission or improved prognosis. For example, an individual is successfully “treated” if, but not limited to, one or more symptoms associated with cancer are alleviated or eliminated, there is a reduction (or destruction) of cancerous cell proliferation, a reduction in symptoms caused by the disease, an improvement in the quality of life of the person with the disease, a reduction in the dosage of other drugs required to treat the disease, and / or an extension of the individual’s lifespan. In some embodiments, “treating” a disease such as cancer means delaying the progression of the disease, i.e., postponing, hindering, slowing, delaying, stabilizing, and / or postponing the progression of the disease (such as cancer). This delay can be of varying lengths depending on the disease being treated and / or the individual’s medical history. As will be apparent to those skilled in the art, a sufficient or significant delay may encompass prevention in that the individual does not develop the disease. For example, the development of late-stage cancer, such as metastasis, may be delayed.

[0112] An “effective dose” is the minimum amount necessary to achieve at least a measurable improvement or prevention of a particular disease (e.g., cancer). The effective dose as used herein may vary depending on factors such as the patient’s disease state, age, sex, and weight, and the ability of the therapeutic agent (or combination of therapeutic agents) to induce a desired response in the individual. An effective dose is also the amount in which the therapeutically beneficial effect outweighs any toxic or adverse effects of the treatment. With respect to therapeutic use, beneficial or desired outcomes include clinical results such as a reduction in one or more symptoms caused by the disease, an improvement in the quality of life of the person affected, a reduction in the dose of other drugs necessary for treating the disease, enhancement of the effect of another drug by targeting, etc., a delay in disease progression, and / or an extension of survival. In the case of cancer or tumors, an effective dose of a drug may be effective in reducing the number of cancer cells, shrinking tumor size, inhibiting (i.e., slowing to some extent or preferably stopping) cancer cell invasion into peripheral organs, inhibiting (i.e., slowing to some extent and preferably stopping) tumor metastasis, inhibiting tumor growth to some extent, and / or alleviating to some extent one or more symptoms associated with the disorder. An effective dose can be administered in one or more doses. In this disclosure, an effective dose of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve a therapeutic action, either directly or indirectly. As understood in a clinical context, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, “effective dose” may be considered in a situation in which one or more therapeutic agents are administered, and a single agent may be considered given in an effective dose if, in combination with one or more other agents, the desired outcome can or does occur.

[0113] As used herein, the term “subject” for therapeutic purposes refers to any animal classified as a mammal. This includes humans, livestock and domesticated animals, as well as animals kept in zoos, for sports, or as pets (such as dogs, horses, cats, and cows). Preferably, the mammal is a human.

[0114] The terms “subject” and “patient” are used interchangeably and include mammals, such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species. This term does not necessarily indicate that the subject has been diagnosed with a specific disease and typically refers to an individual under medical supervision.

[0115] As used herein in the context of preventing a condition, the terms “prevent,” “prevent,” or “prevention” generally mean preventing or delaying the onset of a disease, or preventing the manifestation of its clinical or subclinical symptoms in an object (whether human or animal), for example, preventing the disease from developing in an object that is predisposed to a condition or disease but has not yet been diagnosed as having it.

[0116] As used herein, the term “pharmaceutically acceptable” means that its vehicle, diluent, excipient and / or salt is chemically and / or physically compatible with the other components of the formulation and physiologically compatible with the recipient.

[0117] As used herein, the term “pharmaceutically acceptable carriers and / or excipients” means carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and activator, and are well known in the art (see, for example, Remington's Pharmaceutical Sciences, Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0118] As used herein, the term “adjuvant” refers to a nonspecific immunostimulant that, when delivered to an organism with an antigen or prior to the antigen, can enhance the immune response to an antigen or alter the type of immune response in the organism. Various adjuvants exist, including, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund’s adjuvants (e.g., complete Freund’s adjuvant and incomplete Freund’s adjuvant), Corynebacterium parvum, lipopolysaccharides, and cytokines. Freund’s adjuvants are currently the most commonly used adjuvants in animal studies. Aluminum hydroxide adjuvants are more commonly used in clinical trials.

[0119] The pharmaceutically acceptable salts disclosed herein can be prepared from parent compounds containing an acidic or basic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting a compound in free acidic or basic form with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture thereof.

[0120] The compounds disclosed herein may exist in specific geometric or stereoisomeric forms. This disclosure envisions all such compounds, including cis and trans isomers, (-)-enantiomers and (+)-enantiomers, (R)-enantiomers and (S)-enantiomers, diastereoisomers, (D)-isomers, (L)-isomers, and racemic and other mixtures, such as enantiomers or diastereoisomers concentrated in a mixture, all of which are encompassed within the scope of disclosure herein. Substituents such as alkyl groups may further have chiral carbon atoms. All of these isomers and mixtures thereof are encompassed within the scope of disclosure herein.

[0121] The compounds disclosed herein may contain unnatural proportions of atomic isotopes in one or more of the atoms constituting the compound. For example, compounds can be labeled with radioactive isotopes such as tritium (3H), iodine-125 (125I), or C-14 (14C). In another example, hydrogen can be replaced with deuterium to form deuterated drugs. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared to non-deuterated drugs, deuterated drugs have advantages such as reduced toxic side effects, increased drug stability, improved efficacy, and extended biological half-life. All variations in the isotopic composition of the compounds disclosed herein, regardless of radioactivity, are included within the scope of this disclosure.

[0122] The terms "optional" or "optional" mean that a subsequent event or state may occur but is not required, and that this term includes both instances in which the event or state occurs and instances in which it does not occur.

[0123] The term "substitution" means that one or more hydrogen atoms on a particular atom are substituted by substituents such as deuterium and hydrogen mutants, while the valence of that particular atom remains normal and the substituted compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are substituted. Oxo substitution is not possible at positions on an aromatic ring. The term "optionally substituted" means that an atom may or may not be substituted by a substituent, and unless otherwise specified, the type and number of substituents may be arbitrary as long as it is chemically achievable.

[0124] When an arbitrary variable (e.g., R) appears multiple times in the composition or structure of a compound, the definition of the variable is independent for each appearance. Therefore, for example, if a group is substituted with 0 to 2 R atoms, that group may be substituted with up to 2 R atoms of arbitrary choice, and the definition of R is independent for each appearance. Furthermore, combinations of substituents and / or their variants are only permitted if the combination results in a stable compound.

[0125] When the number of linking groups is 0, as in -(CRR)0-, it means that the linking groups are single bonds.

[0126] If one of the variables is a single bond, it means that the two groups linked by the single bond are directly connected. For example, if L in ALZ represents a single bond, then the structure of ALZ is actually AZ.

[0127] If a listed linking unit does not indicate its linking direction, the linking direction is arbitrary. For example, [ka] If the linking group L in is -MW-, then -MW- links ring A and ring B in the same direction as the reading order from left to right. [ka] It is possible to construct a ring A and ring B in the reverse order of reading from left to right. [ka] This can constitute a compound. Combinations of linking groups, substituents, and / or their variants are permitted only if such combinations result in a stable compound.

[0128] Unless otherwise specified, if a group has one or more connectable sites, any one or more sites of the group may be connected to other groups via chemical bonds. If the bond position of a chemical bond is variable and there are hydrogen atoms in a connectable site, when a connectable site with hydrogen atoms is connected to a chemical bond, the number of hydrogen atoms in this site decreases in proportion to the increase in the number of chemical bonds, and the group becomes a group with the corresponding valency. The chemical bond between that site and other groups is a linear solid bond. [ka] , Linear dashed line combination [ka] or wavy line [ka] It can be represented by this. For example, a straight solid bond in -OCH3 indicates that a group is bonded to another group via the oxygen atom within that group. [ka] The dashed linear bond indicates that a group is bonded to another group via two ends of the nitrogen atom within that group. [ka] The wavy lines indicate that the group is bonded to other groups via the 1- and 2-carbon atoms in the phenyl group. [ka] Any connectable site on the piperidinyl group has at least four connection modes. [ka] , [ka] , [ka] and [ka] This demonstrates that it is possible to connect to other groups via a single chemical bond, even if the H atom is attracted to the -N-. [ka] is still [ka] This includes the bonding mode in which, when just one chemical bond is joined, the number of H atoms at that site decreases by one, and that group becomes the corresponding monovalent piperidinyl group.

[0129] Unless otherwise specified, wedge-shaped solid bonds [ka] and wedge-shaped dashed line connections [ka] This indicates the absolute arrangement of the center of the solid, and the solid lines represent the connections. [ka] and straight dashed line connections [ka] The wavy line indicates the relative arrangement of the center of the solid. [ka] wedge-shaped solid bond [ka] Or a wedge-shaped dashed line connection. [ka] Indicate, or use a wavy line. [ka] This is a combination of straight lines and solid lines. [ka] and straight dashed line connections [ka] This indicates, for example, [ka] teeth [Chemistry] and [Chemistry] represent [Chemistry] is [Chemistry] and [Chemistry] represent.

[0130] Unless otherwise specified, the terms "one isomer is concentrated", "isomer concentration", "one enantiomer is concentrated", or "enantiomer concentration" mean that the content of one isomer or enantiomer is less than 100%, and the content of that isomer or enantiomer is 60% or more, or 70% or more, or 80% or more, or 90% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, or 99.5% or more, or 99.6% or more, or 99.7% or more, or 99.8% or more, or 99.9% or more.

[0131] Optically active (R)- and (S)-isomers, or D and L isomers, can be prepared using chiral synthesis, chiral reagents, or other prior art. To obtain a single enantiomer of a particular compound disclosed herein, the pure enantiomer of the desired type can be obtained by asymmetric synthesis or derivatization of a chiral auxiliary, followed by separation of the resulting diastereomer mixture and cleavage of the auxiliary groups. Alternatively, if the molecule contains a basic functional group (such as amino) or an acidic functional group (such as carboxyl), the compound can be reacted with a suitable optically active acid or base to form a salt of the diastereomer isomer, which is then subjected to diastereomer resolution by conventional methods in the art to obtain the pure enantiomer. Furthermore, enantiomers and diastereoisomers are generally isolated by chromatography using a chiral stationary phase and optionally combined with chemical derivation methods (e.g., carbamates produced from amines).

[0132] Unless otherwise specified, the term "C1-6 alkyl" is used to refer to a linear or branched saturated hydrocarbon group composed of 1 to 6 carbon atoms. C1-6 alkyls include C1-5, C1-4, C1-3, C1-2, C2-6, C2-4, C6, and C5 alkyls, among others. They can be monovalent (such as methyl), divalent (such as methylene), or polyvalent (such as methenyl). Examples of C1-6 alkyls include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (such as n-butyl, isobutyl, s-butyl, and t-butyl), pentyl (such as n-pentyl, isopentyl, and neopentyl), and hexyl.

[0133] Unless otherwise specified, the term "C1-3 alkyl" is used to represent a straight-chain or branched saturated hydrocarbon group composed of 1 to 3 carbon atoms. Examples of C1-3 alkyl include C1-2 alkyl, C2-3 alkyl, etc. It can be monovalent (such as methyl), divalent (such as methylene), or polyvalent (such as methenyl). Examples of C1-3 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (such as n-propyl and isopropyl).

[0134] Unless otherwise specified, the term "C1-3 alkoxy" means an alkyl group containing 1 to 3 carbon atoms and bonded to the rest of the molecule by an oxygen atom. Examples of C1-3 alkoxy groups include C1-2 alkoxy groups, C2-3 alkoxy groups, C3 alkoxy groups, and C2 alkoxy groups. Examples of C1-3 alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (such as n-propoxy and isopropyl). <>

[0135] Unless otherwise specified, the term "C1-3 alkylamino" means an alkyl group containing 1 to 3 carbon atoms and bonded to the rest of the molecule by an amino group. Examples of C1-3 alkylamino groups include C1-2, C3, C2 alkylamino groups. Examples of C1-3 alkylamino groups include, but are not limited to, -NHCH3, -N(CH3)2, -NHCH2CH3, -N(CH3)CH2CH3, -NHCH2CH2CH3, -NHCH2(CH3)2.

[0136] Unless otherwise specified, "C2-3 alkenyl" is used to represent a straight-chain or branched-chain hydrocarbon group composed of 2 to 3 carbon atoms containing at least one carbon-carbon double bond, and the carbon-carbon double bond can be located at any position in the group. C2-3 alkenyl includes C3 and C2 alkenyl. C2-3 alkenyl can be monovalent, divalent, or polyvalent. Examples of C2-3 alkenyl include, but are not limited to, vinyl, propenyl.

[0137] Unless otherwise specified, "C2-3 alkynyl" is used to represent a straight-chain or branched-chain hydrocarbon group consisting of 2-3 carbon atoms containing at least one carbon-carbon triple bond, where the carbon-carbon triple bond can be located at any position on the group. C2-3 alkynyl includes C3 and C2 alkynyls. Examples of C2-3 alkynyls include, but are not limited to, ethynyl and propynyl.

[0138] Unless otherwise specified, the terms “C6-10 aromatic ring” and “C6-10 aryl” may be used interchangeably in this disclosure. The terms “C6-10 aromatic ring” or “C6-10 aryl” mean a cyclic hydrocarbon group having a conjugated pi-electron system and composed of 6 to 10 carbon atoms. It may be a monocyclic, fused bicyclic, or fused tricyclic ring system, where each ring is aromatic. It may be monovalent, divalent, or polyvalent. Examples of C6-10 aryls include C6-9 aryls, C9 aryls, C10 aryls, and C6 aryls. Examples of C6-10 aryls include, but are not limited to, phenyl and naphthyl (1-naphthyl and 2-naphthyl, etc.).

[0139] Unless otherwise specified, the terms “5-10 membered heteroaromatic ring” and “5-10 membered heteroaryl” may be used interchangeably. The term “5-10 membered heteroaryl” refers to a cyclic group having a conjugated pi-electron system and composed of 5-10 ring atoms, where 1, 2, 3, or 4 ring atoms are heteroatoms independently selected from O, S, and N, and the remainder are carbon atoms. This may be a monocyclic, fused bicyclic, or fused tricyclic cyclic system, each ring being aromatic, the nitrogen atom optionally quaternized, and the nitrogen and sulfur heteroatoms optionally oxidized (i.e., NO and S(O)p, p is 1 or 2). 5-10 membered heteroaryls can be bonded to the rest of the molecule via heteroatoms or carbon atoms. Examples of 5-10 membered heteroaryl groups include 5-8 membered, 5-7 membered, 5-6 membered, 5-membered, and 6-membered heteroaryl groups. Examples of 5-10 member heteroaryls include pyrrolyl (N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, etc.), pyrazolyl (2-pyrrolyl and 3-pyrrolyl, etc.), imidazolyl (N-imidazolyl, 2-imidazolyl, 4-imidazolyl, and 5-imidazolyl, etc.), oxazolyl (2-oxazolyl, 4-oxazolyl, and 5-oxazolyl, etc.), triazolyl (1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl, and 4H-1,2,4-triazolyl, etc.), tetrazolyl, isoxazolyl (3-isoxazolyl, 4-isoxazolyl, and 5-isoxazolyl, etc.), thiazolyl (2-thiazolyl, 4- Examples include, but are not limited to, thiazolyl (and 5-thiazolyl, etc.), furyl (2-furyl and 3-furyl, etc.), thienyl (2-thienyl and 3-thienyl, etc.), pyridyl (2-pyridyl, 3-pyridyl and 4-pyridyl, etc.), pyrazinyl or pyrimidinyl (2-pyridinyl and 4-pyridinyl, etc.), benzothiazolyl (5-benzothiazolyl, etc.), prinyl, benzimidazolyl (2-benzimidazolyl, etc.), benzoxazolyl, indolyl (5-indolyl, etc.), isoquinolyl (1-isoquinolyl, 5-isoquinolyl, etc.), quinoxalinyl (2-quinoxalinyl, 5-quinoxalinyl, etc.), or quinolyl (3-quinolyl, 6-quinolyl, etc.).

[0140] Unless otherwise specified, the term "4- to 8-membered heterocycloalkyl" refers, either alone or in combination with other terms, to a saturated cyclic group composed of 4 to 8 ring atoms, where 1, 2, 3, or 4 ring atoms are heteroatoms independently selected from O, S, and N, the remainder being carbon atoms, the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms are optionally oxidized (i.e., NO and S(O)p, where p is 1 or 2). The rings include monocyclic and bicyclic ring systems, and the bicyclic ring systems include spiro rings, fused rings, and bridging rings. Furthermore, with respect to "4- to 8-membered heterocycloalkyl," heteroatoms may be present in positions where the heterocycloalkyl group is bonded to the remainder of the molecule. Examples of 4- to 8-membered heterocycloalkyls include 4- to 6-membered, 5- to 6-membered, 4-membered, 5-membered, and 6-membered heterocycloalkyls. Examples of 4- to 8-membered heterocycloalkyls include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl (tetrahydrothien-2-yl and tetrahydrothien-3-yl, etc.), tetrahydrofuranyl (tetrahydrofuran-2-yl, etc.), tetrahydropyranyl, piperidinyl (1-piperidinyl, 2-piperidinyl and 3-piperidinyl, etc.), piperazinyl (1-piperadinyl and 2-piperadinyl, etc.), morpholinyl (3-morpholinyl and 4-morpholinyl, etc.), dioxanyl, dithianyl, isoxazolidinyl, isothiazolidinyl, 1,2-oxazinyl, 1,2-thiadinyl, hexahydropyridazinyl, homopiperadinyl, homopiperidinyl, or dioxepanyl.

[0141] Unless otherwise specified, Cn~n+m or Cn~Cn+m includes any specific case of n~n+m carbon atoms, for example C1~12 includes C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11 and C12, and also includes any range of n~n+m, for example C1~12 includes C1~3, C1~6, C1~9, C3~6, C3~9, C3~12, C6~9, C6~12 and C9~1 This includes 2, etc. Similarly, n-membered to n+m-membered indicates that the number of atoms on the ring is n to n+m. For example, 3-12 membered rings include 3-membered rings, 4-membered rings, 5-membered rings, 6-membered rings, 7-membered rings, 8-membered rings, 9-membered rings, 10-membered rings, 11-membered rings, and 12-membered rings, and also include any range of n to n+m. For example, 3-12 membered rings include 3-6 membered rings, 3-9 membered rings, 5-6 membered rings, 5-7 membered rings, 6-7 membered rings, 6-8 membered rings, and 6-10 membered rings, etc.

[0142] The compounds disclosed herein can be prepared by various synthetic methods well known to those skilled in the art, including the embodiments listed below, embodiments formed by the embodiments listed below in combination with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Alternative embodiments include, but are not limited to, the examples disclosed herein.

[0143] The structures of the compounds disclosed herein can be determined by conventional methods well known to those skilled in the art. Where the disclosure relates to the absolute configuration of a compound, the absolute configuration can be determined by conventional techniques in the art, such as single-crystal X-ray diffraction (SXRD). In single-crystal X-ray diffraction (SXRD), diffraction intensity data of a cultured single crystal is collected using a Bruker D8 venture diffractometer equipped with a CuKα light source in φ / ω scan mode. After collecting this data, the crystal structure is further analyzed by a direct method (Shelxs97) to determine the absolute configuration.

[0144] The solvents used in this disclosure are commercially available.

[0145] Compounds are named according to general naming principles in the art, or by ChemDraw® software, and commercially available compounds are named according to their vendor's directory name.

[0146] The pharmaceutically acceptable salts disclosed herein can be prepared from parent compounds containing an acidic or basic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting a compound in free acidic or basic form with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture thereof.

[0147] All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are incorporated herein by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference.

[0148] Methods of treating cancer This specification provides a method for treating a cancer having a KRAS G12C mutation (i.e., a cancer containing one or more cancer cells expressing a KRAS G12C mutant protein) in a subject, the method comprising administering to the subject a construct comprising an effective amount of a TIGIT-binding moiety and a PVRIG-binding moiety (i.e., a TIGIT / PVRIG-binding construct) and an effective amount of a KRAS G12C inhibitor. In some embodiments, the TIGIT / PVRIG-binding construct is used in the manufacture of a pharmaceutical product for treating cancer, and the treatment is carried out in combination with a KRAS G12C inhibitor.

[0149] In some embodiments, a method is provided for treating cancer (e.g., cancer involving KRAS mutations) in an individual (e.g., a human patient), comprising administering to the individual 1) a construct comprising a TIGIT-binding moiety and a PVRIG-binding moiety, and 2) a KRAS G12C inhibitor. In some embodiments, the TIGIT-binding moiety and the PVRIG-binding moiety are either in Fab or scFv format. In some embodiments, the TIGIT-binding moiety is in Fab format and the PVRIG-binding moiety is in scFv format, or the TIGIT-binding moiety is in scFv format and the PVRIG-binding moiety is in Fab format, or both the TIGIT-binding moiety and the PVRIG-binding moiety are in Fab format. In some embodiments, the construct further comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region, or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variants include one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend their half-life. In some embodiments, the construct includes (a) an Fc region operably linked to a TIGIT binding moiety and a PVRIG binding moiety, (b) a TIGIT binding moiety operably linked to a PVRIG binding moiety and a PVRIG binding moiety operably linked to an Fc region, or (c) a PVRIG binding moiety operably linked to a TIGIT binding moiety and a TIGIT binding moiety operably linked to an Fc region. In some embodiments, one of the TIGIT binding moiety and the PVRIG binding moiety is a full-length antibody, and the other is an scFv or VHH operably linked to the full-length antibody (for example, by optionally linking to the N-terminus or C-terminus of one or both of the heavy or light chains of the full-length antibody via a linker).In some embodiments, the KRAS G12C inhibitor is optionally a small molecule selected from the group consisting of sotrasib, adaglasib, compound 17, JAB-21822, GDC-6036, JDQ443, D-1553, GH35, GFH925, BPI-421286 and LY3537982, RMC-6291, HBI-2438, BI 1823911, MK-1084 and JNJ-74699157, and any of the compounds disclosed in U.S. Patent Application Publication No. 20230151004 (e.g., 8A and 17). In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally. In some embodiments, cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, cancer is progressive, unresectable, and / or metastatic solid tumors. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to an individual an effective dose of a third therapy comprising another anticancer agent, optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents. In some embodiments, the method comprises selecting an individual for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0150] In some embodiments, a method is provided for treating cancer in an individual (e.g., cancer involving a KRAS mutation), comprising administering to the individual 1) a construct comprising a TIGIT-binding moiety and a PVRIG-binding moiety, and 2) a KRAS G12C inhibitor, wherein the TIGIT-binding moiety comprises a full-length antibody comprising two heavy chains and two light chains, the PVRIG-binding moiety comprises an scFv, and the anti-PVRIG scFv is ligated to the N-terminus or C-terminus of both heavy chains of the anti-TIGIT full-length antibody. In some embodiments, a method is provided for treating cancer in an individual (e.g., cancer involving a KRAS mutation), comprising administering to the individual 1) a construct comprising a TIGIT-binding moiety and a PVRIG-binding moiety, and 2) a KRAS G12C inhibitor, wherein the PVRIG-binding moiety comprises a full-length antibody comprising two heavy chains and two light chains, the TIGIT-binding moiety comprises an scFv, and the anti-TIGIT scFv is ligated to the N-terminus or C-terminus of both heavy chains of the anti-PVRIG full-length antibody. In some embodiments, the full-length antibody includes an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region, or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variant includes one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend its half-life. In some embodiments, the KRAS G12C inhibitor is optionally a small molecule selected from the group consisting of sotrasib, adaglasib, compound 17, JAB-21822, GDC-6036, JDQ443, D-1553, GH35, GFH925, BPI-421286 and LY3537982, RMC-6291, HBI-2438, BI 1823911, MK-1084 and JNJ-74699157, and any of the compounds disclosed in U.S. Patent Application Publication No. 20230151004 (e.g., 8A and 17). In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally.In some embodiments, cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, cancer is progressive, unresectable, and / or metastatic solid tumors. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to an individual an effective dose of a third therapy comprising another anticancer agent, optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents. In some embodiments, the method comprises selecting an individual for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0151] In some embodiments, a method is provided for treating cancer in an individual (e.g., cancer including KRAS mutations), comprising administering to the individual 1) a construct comprising a TIGIT-binding moiety and a PVRIG-binding moiety, and 2) a KRAS G12C inhibitor, wherein the TIGIT-binding moiety comprises an antibody moiety comprising a heavy chain variable region (VH) comprising heavy chain CDRs (HCDRs) 1, HCDRs 2 and HCDRs 3, and a light chain variable region (VL) comprising light chain CDRs (LCDRs) 1, LCDRs 2 and LCDRs 3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the TIGIT-binding moiety and the PVRIG-binding moiety are either in Fab or scFv format. In some embodiments, the TIGIT binding portion is in Fab format and the PVRIG binding portion is in scFv format, or the TIGIT binding portion is in scFv format and the PVRIG binding portion is in Fab format, or both the TIGIT binding portion and the PVRIG binding portion are in Fab format. In some embodiments, the construct further comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variant comprises one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend its half-life. In some embodiments, the structure includes (a) an Fc region operably connected to a TIGIT coupling portion and a PVRIG coupling portion, (b) a TIGIT coupling portion operably connected to a PVRIG coupling portion and a PVRIG coupling portion operably connected to an Fc region, or (c) a PVRIG coupling portion operably connected to a TIGIT coupling portion and a TIGIT coupling portion operably connected to an Fc region.In some embodiments, one of the TIGIT binding moieties and the PVRIG binding moiety is a full-length antibody, and the other is an scFv or VHH operably linked to the full-length antibody (for example, by optionally linking to the N-terminus or C-terminus of one or both of the heavy or light chains of the full-length antibody via a linker). In some embodiments, the KRAS G12C inhibitor is optionally a small molecule selected from the group consisting of sotrasib, adaglasib, compound 17, JAB-21822, GDC-6036, JDQ443, D-1553, GH35, GFH925, BPI-421286 and LY3537982, RMC-6291, HBI-2438, BI 1823911, MK-1084 and JNJ-74699157, and any of the compounds disclosed in U.S. Patent Application Publication No. 20230151004 (e.g., 8A and 17). In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally. In some embodiments, cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, cancer is progressive, unresectable, and / or metastatic solid tumors. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to an individual an effective amount of a third therapy comprising another anticancer agent, which is optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents.In some embodiments, the method includes selecting individuals for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0152] In some embodiments, a method is provided for treating cancer in an individual (e.g., cancer involving KRAS mutations), comprising administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the PVRIG binding moiety comprises an antibody moiety comprising a heavy chain variable region (VH) comprising heavy chain CDR (HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising light chain CDR (LCDR)1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, HCDR2 comprises the amino acid sequence of SEQ ID NO: 8, HCDR3 comprises the amino acid sequence of SEQ ID NO: 9, LCDR1 comprises the amino acid sequence of SEQ ID NO: 10, LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the TIGIT binding moiety and the PVRIG binding moiety are either in Fab or scFv format. In some embodiments, the TIGIT binding portion is in Fab format and the PVRIG binding portion is in scFv format, or the TIGIT binding portion is in scFv format and the PVRIG binding portion is in Fab format, or both the TIGIT binding portion and the PVRIG binding portion are in Fab format. In some embodiments, the construct further comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variant comprises one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend its half-life. In some embodiments, the structure includes (a) an Fc region operably connected to a TIGIT coupling portion and a PVRIG coupling portion, (b) a TIGIT coupling portion operably connected to a PVRIG coupling portion and a PVRIG coupling portion operably connected to an Fc region, or (c) a PVRIG coupling portion operably connected to a TIGIT coupling portion and a TIGIT coupling portion operably connected to an Fc region.In some embodiments, one of the TIGIT binding moieties and the PVRIG binding moiety is a full-length antibody, and the other is an scFv or VHH operably linked to the full-length antibody (for example, by optionally linking to the N-terminus or C-terminus of one or both of the heavy or light chains of the full-length antibody via a linker). In some embodiments, the KRAS G12C inhibitor is optionally a small molecule selected from the group consisting of sotrasib, adaglasib, compound 17, JAB-21822, GDC-6036, JDQ443, D-1553, GH35, GFH925, BPI-421286 and LY3537982, RMC-6291, HBI-2438, BI 1823911, MK-1084 and JNJ-74699157, and any of the compounds disclosed in U.S. Patent Application Publication No. 20230151004 (e.g., 8A and 17). In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally. In some embodiments, cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, cancer is progressive, unresectable, and / or metastatic solid tumors. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to an individual an effective amount of a third therapy comprising another anticancer agent, which is optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents.In some embodiments, the method includes selecting individuals for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0153] In some embodiments, a method for treating cancer in an individual (e.g., cancer including KRAS mutations) comprises administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the TIGIT binding moiety comprises an antibody moiety comprising a heavy chain variable region (VH) comprising heavy chain CDR (HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising light chain CDR (LCDR)1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6 A method is provided in which the PVRIG binding portion comprises an antibody portion comprising a heavy chain variable region (VH) containing heavy chain CDR (HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) containing light chain CDR (LCDR)1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, HCDR2 comprises the amino acid sequence of SEQ ID NO: 8, HCDR3 comprises the amino acid sequence of SEQ ID NO: 9, LCDR1 comprises the amino acid sequence of SEQ ID NO: 10, LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the TIGIT binding portion and the PVRIG binding portion are either in Fab or scFv format. In some embodiments, the TIGIT binding portion is in Fab format and the PVRIG binding portion is in scFv format, or the TIGIT binding portion is in scFv format and the PVRIG binding portion is in Fab format, or both the TIGIT binding portion and the PVRIG binding portion are in Fab format. In some embodiments, the construct further comprises an immunoglobulin constant region, such as an IgG constant region, such as a human IgG1, IgG4, IgG2, or IgG3 Fc region, or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof.In some embodiments, the variants include one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend their half-life. In some embodiments, the construct includes (a) an Fc region operably linked to a TIGIT binding moiety and a PVRIG binding moiety, (b) a TIGIT binding moiety operably linked to a PVRIG binding moiety and a PVRIG binding moiety operably linked to an Fc region, or (c) a PVRIG binding moiety operably linked to a TIGIT binding moiety and a TIGIT binding moiety operably linked to an Fc region. In some embodiments, one of the TIGIT binding moiety and the PVRIG binding moiety is a full-length antibody, and the other is an scFv or VHH operably linked to the full-length antibody (for example, by optionally linking to the N-terminus or C-terminus of one or both of the heavy or light chains of the full-length antibody via a linker). In some embodiments, the KRAS G12C inhibitor is optionally a small molecule selected from the group consisting of sotrasib, adaglasib, compound 17, JAB-21822, GDC-6036, JDQ443, D-1553, GH35, GFH925, BPI-421286 and LY3537982, RMC-6291, HBI-2438, BI 1823911, MK-1084 and JNJ-74699157, and any of the compounds disclosed in U.S. Patent Application Publication No. 20230151004 (e.g., 8A and 17). In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally. In some embodiments, cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, stomach cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, cancer is progressive, unresectable, and / or metastatic solid tumors.In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to the individual an effective amount of a third therapy comprising another anti-cancer agent, which optionally is selected from the group consisting of an immune checkpoint inhibitor, a cytotoxic agent, a cytostatic agent, an anti-angiogenic agent, a tumor reducing agent, a chemotherapeutic agent, an antibody-drug conjugate, radiotherapy and radiotherapy agents, a targeted anti-cancer agent, a BRM, a therapeutic antibody, a cancer vaccine, a cytokine, hormonal therapy, radiotherapy, and an anti-metastatic agent. In some embodiments, the method comprises selecting an individual as a treatment subject based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0154] In some embodiments, a method for treating cancer in an individual (e.g., cancer involving a KRAS mutation) comprising: 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety; and 2) KRAS A method is provided comprising administering a G12C inhibitor to an individual, wherein the TIGIT binding portion comprises an antibody portion including a heavy chain variable region (VH) and a light chain variable region (VL), the VH of the TIGIT binding portion comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence identical to at least 85%, 90%, or 95% of SEQ ID NO: 13, and / or the VL of the TIGIT binding portion comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence identical to at least 85%, 90%, or 95% of SEQ ID NO: 14, and / or the PVRIG binding portion comprises an antibody portion including a heavy chain variable region (VH) and a light chain variable region (VL), the VH of the PVRIG binding portion comprises the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence identical to at least 85%, 90%, or 95% of SEQ ID NO: 15, and / or the VL of the PVRIG binding portion comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence identical to at least 85%, 90%, or 95% of SEQ ID NO: 16. In some embodiments, the VH of the TIGIT binding moiety contains the amino acid sequence of SEQ ID NO: 13, and the VL of the TIGIT binding moiety contains the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH of the PVRIG binding moiety contains the amino acid sequence of SEQ ID NO: 15, and / or the VL of the PVRIG binding moiety contains the amino acid sequence of SEQ ID NO: 16. In some embodiments, the TIGIT binding moiety and the PVRIG binding moiety are either in Fab or scFv format. In some embodiments, the TIGIT binding moiety is in Fab format and the PVRIG binding moiety is in scFv format, or the TIGIT binding moiety is in scFv format and the PVRIG binding moiety is in Fab format, or both the TIGIT binding moiety and the PVRIG binding moiety are in Fab format. In some embodiments, the construct further includes an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region, or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof.In some embodiments, the variants include one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend their half-life. In some embodiments, the construct includes (a) an Fc region operably linked to a TIGIT binding moiety and a PVRIG binding moiety, (b) a TIGIT binding moiety operably linked to a PVRIG binding moiety and a PVRIG binding moiety operably linked to an Fc region, or (c) a PVRIG binding moiety operably linked to a TIGIT binding moiety and a TIGIT binding moiety operably linked to an Fc region. In some embodiments, one of the TIGIT binding moiety and the PVRIG binding moiety is a full-length antibody, and the other is an scFv or VHH operably linked to the full-length antibody (for example, by optionally linking to the N-terminus or C-terminus of one or both of the heavy or light chains of the full-length antibody via a linker). In some embodiments, the KRAS G12C inhibitor is optionally a small molecule selected from the group consisting of sotrasib, adaglasib, compound 17, JAB-21822, GDC-6036, JDQ443, D-1553, GH35, GFH925, BPI-421286 and LY3537982, RMC-6291, HBI-2438, BI 1823911, MK-1084 and JNJ-74699157, and any of the compounds disclosed in U.S. Patent Application Publication No. 20230151004 (e.g., 8A and 17). In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally. In some embodiments, cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, stomach cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, cancer is progressive, unresectable, and / or metastatic solid tumors.In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to the individual an effective dose of a third therapy comprising another anticancer agent, optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents. In some embodiments, the method comprises selecting an individual for treatment based on the presence of one or more cancer cells expressing a KRAS G12C mutant protein.

[0155] In some embodiments, a method for treating cancer in an individual (e.g., cancer including KRAS mutations) comprises administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the TIGIT binding moiety comprises a full-length antibody comprising a heavy chain variable region (VH) comprising heavy chain CDR (HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising light chain CDR (LCDR)1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6 A method is provided in which the PVRIG binding moiety comprises an scFv comprising a heavy chain variable region (VH) containing heavy chain CDR(HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) containing light chain CDR(LCDR)1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, HCDR2 comprises the amino acid sequence of SEQ ID NO: 8, HCDR3 comprises the amino acid sequence of SEQ ID NO: 9, LCDR1 comprises the amino acid sequence of SEQ ID NO: 10, LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 12, and the anti-PVRIG scFv is ligated to the N-terminus or C-terminus of the two heavy chains of an anti-TIGIT full-length antibody. In some embodiments, the full-length antibody comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variants include one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend their half-life.In some embodiments, the KRAS G12C inhibitor is optionally a small molecule selected from the group consisting of sotrasib, adaglasib, compound 17, JAB-21822, GDC-6036, JDQ443, D-1553, GH35, GFH925, BPI-421286 and LY3537982, RMC-6291, HBI-2438, BI 1823911, MK-1084 and JNJ-74699157, and any of the compounds disclosed in U.S. Patent Application Publication No. 20230151004 (e.g., 8A and 17). In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally. In some embodiments, cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, cancer is progressive, unresectable, and / or metastatic solid tumors. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to an individual an effective dose of a third therapy comprising another anticancer agent, optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents. In some embodiments, the method comprises selecting an individual for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0156] In some embodiments, a method for treating cancer in an individual (e.g., cancer including KRAS mutations) comprises administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the TIGIT binding moiety comprises an scFv comprising a heavy chain variable region (VH) comprising heavy chain CDR (HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising light chain CDR (LCDR)1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6 A method is provided in which a PVRIG binding moiety comprises a full-length antibody comprising a heavy-chain variable region (VH) containing heavy-chain CDR(HCDR)1, HCDR2, and HCDR3, and a light-chain variable region (VL) containing light-chain CDR(LCDR)1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, HCDR2 comprises the amino acid sequence of SEQ ID NO: 8, HCDR3 comprises the amino acid sequence of SEQ ID NO: 9, LCDR1 comprises the amino acid sequence of SEQ ID NO: 10, LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 12, and anti-TIGIT scFv is ligated to the N-terminus or C-terminus of the two heavy chains of the anti-PVRIG full-length antibody. In some embodiments, the full-length antibody comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variants include one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend their half-life.In some embodiments, the KRAS G12C inhibitor is optionally a small molecule selected from the group consisting of sotrasib, adaglasib, compound 17, JAB-21822, GDC-6036, JDQ443, D-1553, GH35, GFH925, BPI-421286 and LY3537982, RMC-6291, HBI-2438, BI 1823911, MK-1084 and JNJ-74699157, and any of the compounds disclosed in U.S. Patent Application Publication No. 20230151004 (e.g., 8A and 17). In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally. In some embodiments, cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, cancer is progressive, unresectable, and / or metastatic solid tumors. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to an individual an effective dose of a third therapy comprising another anticancer agent, optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents. In some embodiments, the method comprises selecting an individual for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0157] In some embodiments, a method is provided for treating cancer in an individual (e.g., cancer including a KRAS mutation), comprising administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the construct comprises first and second heavy chains comprising SEQ ID NO: 17 and first and second light chains comprising SEQ ID NO: 18. In some embodiments, a method is provided for treating cancer in an individual (e.g., a human patient), comprising administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the construct comprises first and second heavy chains comprising SEQ ID NO: 19 and first and second light chains comprising SEQ ID NO: 20.

[0158] In some embodiments, a method for treating cancer in an individual (e.g., cancer involving a KRAS mutation) comprises administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the KRAS G12C inhibitor is of formula (III) [ka] (In the formula, T1 is selected from O and N, and R1 is selected from C6-10 aryls and 5-10 membered heteroaryls, where C6-10 aryls and 5-10 membered heteroaryls are optionally substituted with 1, 2, 3, 4, or 5 Ra atoms. When T1 is O, R2 is absent, and when T1 is N, R2 is selected from H, C1-3 alkyl, -C(=O)-C1-3 alkyl, and -S(=O)2-C1-3 alkyl, where C1-3 alkyl, -C(=O)-C1-3 alkyl, and -S(=O)2-C1-3 alkyl are optionally substituted with 1, 2, 3, 4, or 5 Ra atoms. R3 is substituted with one or three Rb atoms, R3 is a C1-3 alkyl group, where the C1-3 alkyl group is optionally substituted with one, two, or three Rc atoms, R4 is selected from H and C1-3 alkyl groups, where the C1-3 alkyl group is optionally substituted with one, two, or three Rd atoms, R5, R6, and R7 are independently selected from H, F, Cl, Br, I, and C1-3 alkyl groups, where the C1-3 alkyl group is optionally substituted with one, two, or three F atoms, R8 is selected from H and CH3, and Ra is independently The Rb is selected from F, Cl, Br, I, OH, NH2, CN, C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl, where the C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl are optionally substituted with 1, 2, or 3 F atoms; Rb is independently selected from F, Cl, Br, I, OH, and NH2; and Rc is independently selected from 4-8 member heterocycloalkyl groups, where the 4-8 member heterocycloalkyl groups are optionally substituted with 1, 2, or 3 R atoms. The compounds are represented by (where Rd is independently selected from F, Cl, Br, I, OH, NH2, and CN, and R is independently selected from H, F, Cl, Br, OH, CN, C1-3 alkyl, C1-3 alkoxy, and -C1-3 alkyl-OC(=O)-C1-3 alkylamino, provided that if R1 is naphthyl, naphthyl is optionally substituted with F, Cl, Br, OH, NH2, CF3, CH2CH3, and -C≡CH, and R5, R6, and R7 are independently H) or their pharmaceutically acceptable salts.A method is provided. In some embodiments, the TIGIT binding moiety and the PVRIG binding moiety are either in Fab or scFv format. In some embodiments, the TIGIT binding moiety is in Fab format and the PVRIG binding moiety is in scFv format, or the TIGIT binding moiety is in scFv format and the PVRIG binding moiety is in Fab format, or both the TIGIT binding moiety and the PVRIG binding moiety are in Fab format. In some embodiments, the construct further comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variant comprises one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend its half-life. In some embodiments, the construct includes (a) an Fc region operably linked to a TIGIT binding moiety and a PVRIG binding moiety, (b) a TIGIT binding moiety operably linked to a PVRIG binding moiety and a PVRIG binding moiety operably linked to an Fc region, or (c) a PVRIG binding moiety operably linked to a TIGIT binding moiety and a TIGIT binding moiety operably linked to an Fc region. In some embodiments, one of the TIGIT binding moiety and the PVRIG binding moiety is a full-length antibody, and the other is an scFv or VHH operably linked to the full-length antibody (for example, by optionally linking to the N-terminus or C-terminus of one or both of the heavy or light chains of the full-length antibody via a linker). In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally. In some embodiments, cancers include colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, stomach cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma,The cancers are Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, the cancer is an advanced, unresectable, and / or metastatic solid tumor. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to the individual an effective dose of a third therapy comprising another anticancer agent, optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents. In some embodiments, the method comprises selecting an individual for treatment based on the presence of one or more cancer cells expressing a KRAS G12C mutant protein.

[0159] In some embodiments, a method is provided for treating cancer in an individual (e.g., cancer involving a KRAS mutation), comprising administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the TIGIT binding moiety comprises a full-length antibody comprising two heavy chains and two light chains, and the PVRIG binding moiety comprises an scFv, with the anti-PVRIG scFv being ligated to the N-terminus or C-terminus of both heavy chains of the anti-TIGIT full-length antibody. In some embodiments, a method for treating cancer in an individual (e.g., cancer involving a KRAS mutation) comprises administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the PVRIG binding moiety comprises a full-length antibody comprising two heavy chains and two light chains, the TIGIT binding moiety comprises an scFv, the anti-TIGIT scFv is ligated to the N-terminus or C-terminus of both heavy chains of the anti-PVRIG full-length antibody, and the KRAS G12C inhibitor is of formula (III) [ka] (In the formula, T1 is selected from O and N, and R1 is selected from C6-10 aryls and 5-10 membered heteroaryls, where C6-10 aryls and 5-10 membered heteroaryls are optionally substituted with 1, 2, 3, 4, or 5 Ra atoms. When T1 is O, R2 is absent, and when T1 is N, R2 is selected from H, C1-3 alkyl, -C(=O)-C1-3 alkyl, and -S(=O)2-C1-3 alkyl, where C1-3 alkyl, -C(=O)-C1-3 alkyl, and -S(=O)2-C1-3 alkyl are optionally substituted with 1, 2, 3, 4, or 5 Ra atoms. R3 is substituted with one or three Rb atoms, R3 is a C1-3 alkyl group, where the C1-3 alkyl group is optionally substituted with one, two, or three Rc atoms, R4 is selected from H and C1-3 alkyl groups, where the C1-3 alkyl group is optionally substituted with one, two, or three Rd atoms, R5, R6, and R7 are independently selected from H, F, Cl, Br, I, and C1-3 alkyl groups, where the C1-3 alkyl group is optionally substituted with one, two, or three F atoms, R8 is selected from H and CH3, and Ra is independently The Rb is selected from F, Cl, Br, I, OH, NH2, CN, C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl, where the C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl are optionally substituted with 1, 2, or 3 F atoms; Rb is independently selected from F, Cl, Br, I, OH, and NH2; and Rc is independently selected from 4-8 member heterocycloalkyl groups, where the 4-8 member heterocycloalkyl groups are optionally substituted with 1, 2, or 3 R atoms. The compounds are represented by (where Rd is independently selected from F, Cl, Br, I, OH, NH2, and CN, and R is independently selected from H, F, Cl, Br, OH, CN, C1-3 alkyl, C1-3 alkoxy, and -C1-3 alkyl-OC(=O)-C1-3 alkylamino, provided that if R1 is naphthyl, naphthyl is optionally substituted with F, Cl, Br, OH, NH2, CF3, CH2CH3, and -C≡CH, and R5, R6, and R7 are independently H) or their pharmaceutically acceptable salts.A method is provided. In some embodiments, the full-length antibody comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region, or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variant comprises one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend its half-life. In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally. In some embodiments, cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, cancer is progressive, unresectable, and / or metastatic solid tumors. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to an individual an effective dose of a third therapy comprising another anticancer agent, optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents. In some embodiments, the method comprises selecting an individual for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0160] In some embodiments, a method for treating cancer in an individual (e.g., cancer including KRAS mutations) comprises administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the TIGIT binding moiety comprises an antibody moiety comprising a heavy chain variable region (VH) comprising heavy chain CDR (HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising light chain CDR (LCDR)1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, LCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the KRAS G12C inhibitor is of formula (III) [ka] (In the formula, T1 is selected from O and N, R1 is selected from C6-10 aryls and 5-10 membered heteroaryls, where C6-10 aryls and 5-10 membered heteroaryls are optionally substituted with 1, 2, 3, 4, or 5 Ra atoms, when T1 is O, R2 is absent, and when T1 is N, R2 is selected from H, C1-3 alkyl, -C(=O)-C1-3 alkyl and -S(=O)2-C1-3 alkyl, where C1-3 alkyl, -C(=O)-C1-3 alkyl and -S(=O)2-C1-3 alkyl are optionally substituted with 1, 2, or 3 Rb atoms)

[0161] R3 is a C1-3 alkyl group, where the C1-3 alkyl group is optionally substituted with 1, 2, or 3 Rc atoms; R4 is selected from H and a C1-3 alkyl group, where the C1-3 alkyl group is optionally substituted with 1, 2, or 3 Rd atoms; and R5, R6, and R7 are independently selected from H, F, Cl, Br, I, and a C1-3 alkyl group, where the C1-3 alkyl group is optionally... R8 is substituted with one, two, or three F atoms, R8 is selected from H and CH3, and Ra is independently selected from F, Cl, Br, I, OH, NH2, CN, C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl, where C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl are optionally substituted with one, two, or three F atoms. A method is provided in which Rb is independently selected from F, Cl, Br, I, OH, and NH2, Rc is independently selected from 4- to 8-membered heterocycloalkyl groups, where R5, R6, and R7 are optionally substituted with 1, 2, or 3 Rs, Rd is independently selected from F, Cl, Br, I, OH, NH2, and CN, and R is independently selected from H, F, Cl, Br, OH, CN, C1-3 alkyl, C1-3 alkoxy, and -C1-3 alkyl-OC(=O)-C1-3 alkylamino groups, where R1 is naphthyl, where naphthyl is optionally substituted with F, Cl, Br, OH, NH2, CF3, CH2CH3, and -C≡CH, and R5, R6, and R7 are independently H) or a pharmaceutically acceptable salt thereof. In some embodiments, the TIGIT and PVRIG coupling portions are either in Fab or scFv format. In some embodiments, the TIGIT coupling portion is in Fab format and the PVRIG coupling portion is in scFv format, or the TIGIT coupling portion is in scFv format and the PVRIG coupling portion is in Fab format, or both the TIGIT and PVRIG coupling portions are in Fab format.In some embodiments, the construct further comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variant comprises one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend its half-life. In some embodiments, the construct comprises (a) an Fc region operably linked to a TIGIT binding moiety and a PVRIG binding moiety, (b) a TIGIT binding moiety operably linked to a PVRIG binding moiety and a PVRIG binding moiety operably linked to an Fc region, or (c) a PVRIG binding moiety operably linked to a TIGIT binding moiety and a TIGIT binding moiety operably linked to an Fc region. In some embodiments, one of the TIGIT-binding moieties and the PVRIG-binding moiety is a full-length antibody, and the other is an scFv or VHH operably linked to the full-length antibody (for example, by optionally linking to the N-terminus or C-terminus of one or both of the heavy or light chains of the full-length antibody via a linker). In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally. In some embodiments, cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, cancer is progressive, unresectable, and / or metastatic solid tumors. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct.In some embodiments, the method further comprises administering to an individual an effective dose of a third therapy comprising another anticancer agent, optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents. In some embodiments, the method comprises selecting an individual for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0162] In some embodiments, a method for treating cancer in an individual (e.g., cancer including KRAS mutations) comprises administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the PVRIG binding moiety comprises an antibody moiety comprising a heavy chain variable region (VH) comprising heavy chain CDR (HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising light chain CDR (LCDR)1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, HCDR2 comprises the amino acid sequence of SEQ ID NO: 8, HCDR3 comprises the amino acid sequence of SEQ ID NO: 9, LCDR1 comprises the amino acid sequence of SEQ ID NO: 10, LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 12, and the KRAS G12C inhibitor is of formula (III) [ka] (In the formula, T1 is selected from O and N, R1 is selected from C6-10 aryls and 5-10 membered heteroaryls, where C6-10 aryls and 5-10 membered heteroaryls are optionally substituted with 1, 2, 3, 4, or 5 Ra atoms, when T1 is O, R2 is absent, and when T1 is N, R2 is selected from H, C1-3 alkyl, -C(=O)-C1-3 alkyl and -S(=O)2-C1-3 alkyl, where C1-3 alkyl, -C(=O)-C1-3 alkyl and -S(=O)2-C1-3 alkyl are optionally substituted with 1, 2, or 3 Rb atoms)

[0163] R3 is a C1-3 alkyl group, where the C1-3 alkyl group is optionally substituted with 1, 2, or 3 Rc atoms; R4 is selected from H and a C1-3 alkyl group, where the C1-3 alkyl group is optionally substituted with 1, 2, or 3 Rd atoms; and R5, R6, and R7 are independently selected from H, F, Cl, Br, I, and a C1-3 alkyl group, where the C1-3 alkyl group is optionally... R8 is substituted with one, two, or three F atoms, R8 is selected from H and CH3, and Ra is independently selected from F, Cl, Br, I, OH, NH2, CN, C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl, where C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl are optionally substituted with one, two, or three F atoms. A method is provided in which Rb is independently selected from F, Cl, Br, I, OH, and NH2, Rc is independently selected from 4- to 8-membered heterocycloalkyl groups, where R5, R6, and R7 are optionally substituted with 1, 2, or 3 Rs, Rd is independently selected from F, Cl, Br, I, OH, NH2, and CN, and R is independently selected from H, F, Cl, Br, OH, CN, C1-3 alkyl, C1-3 alkoxy, and -C1-3 alkyl-OC(=O)-C1-3 alkylamino groups, where R1 is naphthyl, where naphthyl is optionally substituted with F, Cl, Br, OH, NH2, CF3, CH2CH3, and -C≡CH, and R5, R6, and R7 are independently H) or a pharmaceutically acceptable salt thereof. In some embodiments, the TIGIT and PVRIG coupling portions are either in Fab or scFv format. In some embodiments, the TIGIT coupling portion is in Fab format and the PVRIG coupling portion is in scFv format, or the TIGIT coupling portion is in scFv format and the PVRIG coupling portion is in Fab format, or both the TIGIT and PVRIG coupling portions are in Fab format.In some embodiments, the construct further comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variant comprises one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend its half-life. In some embodiments, the construct comprises (a) an Fc region operably linked to a TIGIT binding moiety and a PVRIG binding moiety, (b) a TIGIT binding moiety operably linked to a PVRIG binding moiety and a PVRIG binding moiety operably linked to an Fc region, or (c) a PVRIG binding moiety operably linked to a TIGIT binding moiety and a TIGIT binding moiety operably linked to an Fc region. In some embodiments, one of the TIGIT-binding moieties and the PVRIG-binding moiety is a full-length antibody, and the other is an scFv or VHH operably linked to the full-length antibody (for example, by optionally linking to the N-terminus or C-terminus of one or both of the heavy or light chains of the full-length antibody via a linker). In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally. In some embodiments, cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, cancer is progressive, unresectable, and / or metastatic solid tumors. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct.In some embodiments, the method further comprises administering to an individual an effective dose of a third therapy comprising another anticancer agent, optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents. In some embodiments, the method comprises selecting an individual for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0164] In some embodiments, a method for treating cancer in an individual (e.g., cancer including KRAS mutations) comprises administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the TIGIT binding moiety comprises an antibody moiety comprising a heavy chain variable region (VH) comprising heavy chain CDR (HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising light chain CDR (LCDR)1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6 The antibody moiety comprises an acid sequence, and the PVRIG binding moiety comprises a heavy chain variable region (VH) containing heavy chain CDR (HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) containing light chain CDR (LCDR)1, LCDR2, and LCDR3, wherein HCDR1 contains the amino acid sequence of SEQ ID NO: 7, HCDR2 contains the amino acid sequence of SEQ ID NO: 8, HCDR3 contains the amino acid sequence of SEQ ID NO: 9, LCDR1 contains the amino acid sequence of SEQ ID NO: 10, LCDR2 contains the amino acid sequence of SEQ ID NO: 11, and LCDR3 contains the amino acid sequence of SEQ ID NO: 12, and the KRAS G12C inhibitor is of formula (III) [ka] (In the formula, T1 is selected from O and N, and R1 is selected from C6-10 aryls and 5-10 membered heteroaryls, where C6-10 aryls and 5-10 membered heteroaryls are optionally substituted with 1, 2, 3, 4, or 5 Ra atoms. When T1 is O, R2 is absent, and when T1 is N, R2 is selected from H, C1-3 alkyl, -C(=O)-C1-3 alkyl, and -S(=O)2-C1-3 alkyl, where C1-3 alkyl, -C(=O)-C1-3 alkyl, and -S(=O)2-C1-3 alkyl are optionally substituted with 1, 2, 3, 4, or 5 Ra atoms. R3 is substituted with one or three Rb atoms, R3 is a C1-3 alkyl group, where the C1-3 alkyl group is optionally substituted with one, two, or three Rc atoms, R4 is selected from H and C1-3 alkyl groups, where the C1-3 alkyl group is optionally substituted with one, two, or three Rd atoms, R5, R6, and R7 are independently selected from H, F, Cl, Br, I, and C1-3 alkyl groups, where the C1-3 alkyl group is optionally substituted with one, two, or three F atoms, R8 is selected from H and CH3, and Ra is independently The Rb is selected from F, Cl, Br, I, OH, NH2, CN, C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl, where the C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl are optionally substituted with 1, 2, or 3 F atoms; Rb is independently selected from F, Cl, Br, I, OH, and NH2; and Rc is independently selected from 4-8 member heterocycloalkyl groups, where the 4-8 member heterocycloalkyl groups are optionally substituted with 1, 2, or 3 R atoms. The compounds are represented by (where Rd is independently selected from F, Cl, Br, I, OH, NH2, and CN, and R is independently selected from H, F, Cl, Br, OH, CN, C1-3 alkyl, C1-3 alkoxy, and -C1-3 alkyl-OC(=O)-C1-3 alkylamino, provided that if R1 is naphthyl, naphthyl is optionally substituted with F, Cl, Br, OH, NH2, CF3, CH2CH3, and -C≡CH, and R5, R6, and R7 are independently H) or their pharmaceutically acceptable salts.A method is provided. In some embodiments, the TIGIT binding moiety and the PVRIG binding moiety are either in Fab or scFv format. In some embodiments, the TIGIT binding moiety is in Fab format and the PVRIG binding moiety is in scFv format, or the TIGIT binding moiety is in scFv format and the PVRIG binding moiety is in Fab format, or both the TIGIT binding moiety and the PVRIG binding moiety are in Fab format. In some embodiments, the construct further comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variant comprises one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend its half-life. In some embodiments, the construct includes (a) an Fc region operably linked to a TIGIT binding moiety and a PVRIG binding moiety, (b) a TIGIT binding moiety operably linked to a PVRIG binding moiety and a PVRIG binding moiety operably linked to an Fc region, or (c) a PVRIG binding moiety operably linked to a TIGIT binding moiety and a TIGIT binding moiety operably linked to an Fc region. In some embodiments, one of the TIGIT binding moiety and the PVRIG binding moiety is a full-length antibody, and the other is an scFv or VHH operably linked to the full-length antibody (for example, by optionally linking to the N-terminus or C-terminus of one or both of the heavy or light chains of the full-length antibody via a linker). In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally. In some embodiments, cancers include colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, stomach cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma,The cancers are Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, the cancer is an advanced, unresectable, and / or metastatic solid tumor. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to the individual an effective dose of a third therapy comprising another anticancer agent, optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents. In some embodiments, the method comprises selecting an individual for treatment based on the presence of one or more cancer cells expressing a KRAS G12C mutant protein.

[0165] In some embodiments, a method for treating cancer in an individual (e.g., cancer involving a KRAS mutation) comprising: 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety; and 2) KRAS A method is provided comprising administering a G12C inhibitor to an individual, wherein the TIGIT binding portion comprises an antibody portion including a heavy chain variable region (VH) and a light chain variable region (VL), the VH of the TIGIT binding portion comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence identical to at least 85%, 90%, or 95% of SEQ ID NO: 13, and / or the VL of the TIGIT binding portion comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence identical to at least 85%, 90%, or 95% of SEQ ID NO: 14, and / or the PVRIG binding portion comprises an antibody portion including a heavy chain variable region (VH) and a light chain variable region (VL), the VH of the PVRIG binding portion comprises the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence identical to at least 85%, 90%, or 95% of SEQ ID NO: 15, and / or the VL of the PVRIG binding portion comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence identical to at least 85%, 90%, or 95% of SEQ ID NO: 16. In some embodiments, the VH of the TIGIT binding moiety comprises the amino acid sequence of SEQ ID NO: 13, and the VL of the TIGIT binding moiety comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH of the PVRIG binding moiety comprises the amino acid sequence of SEQ ID NO: 15, and / or the VL of the PVRIG binding moiety comprises the amino acid sequence of SEQ ID NO: 16, and the KRAS G12C inhibitor is of formula (III) [ka] (In the formula, T1 is selected from O and N, R1 is selected from C6-10 aryls and 5-10 membered heteroaryls, where C6-10 aryls and 5-10 membered heteroaryls are optionally substituted with 1, 2, 3, 4, or 5 Ra atoms, when T1 is O, R2 is absent, and when T1 is N, R2 is selected from H, C1-3 alkyl, -C(=O)-C1-3 alkyl and -S(=O)2-C1-3 alkyl, where C1-3 alkyl, -C(=O)-C1-3 alkyl and -S(=O)2-C1-3 alkyl are optionally substituted with 1, 2, or 3 Rb atoms)

[0166] R3 is a C1-3 alkyl group, where the C1-3 alkyl group is optionally substituted with 1, 2, or 3 Rc atoms; R4 is selected from H and a C1-3 alkyl group, where the C1-3 alkyl group is optionally substituted with 1, 2, or 3 Rd atoms; and R5, R6, and R7 are independently selected from H, F, Cl, Br, I, and a C1-3 alkyl group, where the C1-3 alkyl group is optionally... R8 is substituted with one, two, or three F atoms, R8 is selected from H and CH3, and Ra is independently selected from F, Cl, Br, I, OH, NH2, CN, C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl, where C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl are optionally substituted with one, two, or three F atoms. A method is provided in which Rb is independently selected from F, Cl, Br, I, OH, and NH2, Rc is independently selected from 4- to 8-membered heterocycloalkyl groups, where R5, R6, and R7 are optionally substituted with 1, 2, or 3 Rs, Rd is independently selected from F, Cl, Br, I, OH, NH2, and CN, and R is independently selected from H, F, Cl, Br, OH, CN, C1-3 alkyl, C1-3 alkoxy, and -C1-3 alkyl-OC(=O)-C1-3 alkylamino groups, where R1 is naphthyl, where naphthyl is optionally substituted with F, Cl, Br, OH, NH2, CF3, CH2CH3, and -C≡CH, and R5, R6, and R7 are independently H) or a pharmaceutically acceptable salt thereof. In some embodiments, the TIGIT and PVRIG coupling portions are either in Fab or scFv format. In some embodiments, the TIGIT coupling portion is in Fab format and the PVRIG coupling portion is in scFv format, or the TIGIT coupling portion is in scFv format and the PVRIG coupling portion is in Fab format, or both the TIGIT and PVRIG coupling portions are in Fab format.In some embodiments, the construct further comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variant comprises one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend its half-life. In some embodiments, the construct comprises (a) an Fc region operably linked to a TIGIT binding moiety and a PVRIG binding moiety, (b) a TIGIT binding moiety operably linked to a PVRIG binding moiety and a PVRIG binding moiety operably linked to an Fc region, or (c) a PVRIG binding moiety operably linked to a TIGIT binding moiety and a TIGIT binding moiety operably linked to an Fc region. In some embodiments, one of the TIGIT-binding moieties and the PVRIG-binding moiety is a full-length antibody, and the other is an scFv or VHH operably linked to the full-length antibody (for example, by optionally linking to the N-terminus or C-terminus of one or both of the heavy or light chains of the full-length antibody via a linker). In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally. In some embodiments, cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, cancer is progressive, unresectable, and / or metastatic solid tumors. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct.In some embodiments, the method further comprises administering to an individual an effective dose of a third therapy comprising another anticancer agent, optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents. In some embodiments, the method comprises selecting an individual for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0167] In some embodiments, a method for treating cancer in an individual (e.g., cancer including KRAS mutations) comprises administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the TIGIT binding moiety comprises a full-length antibody comprising a heavy chain variable region (VH) comprising heavy chain CDR (HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising light chain CDR (LCDR)1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6 The PVRIG binding moiety includes an scFv comprising a heavy chain variable region (VH) containing heavy chain CDR(HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) containing light chain CDR(LCDR)1, LCDR2, and LCDR3, wherein HCDR1 contains the amino acid sequence of SEQ ID NO: 7, HCDR2 contains the amino acid sequence of SEQ ID NO: 8, HCDR3 contains the amino acid sequence of SEQ ID NO: 9, LCDR1 contains the amino acid sequence of SEQ ID NO: 10, LCDR2 contains the amino acid sequence of SEQ ID NO: 11, and LCDR3 contains the amino acid sequence of SEQ ID NO: 12, and the anti-PVRIG scFv is ligated to the N-terminus or C-terminus of the two heavy chains of the anti-TIGIT full-length antibody, and the KRAS G12C inhibitor is of formula (III) [ka] (In the formula, T1 is selected from O and N, and R1 is selected from C6-10 aryls and 5-10 membered heteroaryls, where C6-10 aryls and 5-10 membered heteroaryls are optionally substituted with 1, 2, 3, 4, or 5 Ra atoms. When T1 is O, R2 is absent, and when T1 is N, R2 is selected from H, C1-3 alkyl, -C(=O)-C1-3 alkyl, and -S(=O)2-C1-3 alkyl, where C1-3 alkyl, -C(=O)-C1-3 alkyl, and -S(=O)2-C1-3 alkyl are optionally substituted with 1, 2, 3, 4, or 5 Ra atoms. R3 is substituted with one or three Rb atoms, R3 is a C1-3 alkyl group, where the C1-3 alkyl group is optionally substituted with one, two, or three Rc atoms, R4 is selected from H and C1-3 alkyl groups, where the C1-3 alkyl group is optionally substituted with one, two, or three Rd atoms, R5, R6, and R7 are independently selected from H, F, Cl, Br, I, and C1-3 alkyl groups, where the C1-3 alkyl group is optionally substituted with one, two, or three F atoms, R8 is selected from H and CH3, and Ra is independently The Rb is selected from F, Cl, Br, I, OH, NH2, CN, C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl, where the C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl are optionally substituted with 1, 2, or 3 F atoms; Rb is independently selected from F, Cl, Br, I, OH, and NH2; and Rc is independently selected from 4-8 member heterocycloalkyl groups, where the 4-8 member heterocycloalkyl groups are optionally substituted with 1, 2, or 3 R atoms. The compounds are represented by (where Rd is independently selected from F, Cl, Br, I, OH, NH2, and CN, and R is independently selected from H, F, Cl, Br, OH, CN, C1-3 alkyl, C1-3 alkoxy, and -C1-3 alkyl-OC(=O)-C1-3 alkylamino, provided that if R1 is naphthyl, naphthyl is optionally substituted with F, Cl, Br, OH, NH2, CF3, CH2CH3, and -C≡CH, and R5, R6, and R7 are independently H) or their pharmaceutically acceptable salts.A method is provided. In some embodiments, the full-length antibody comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region, or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variant comprises one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend its half-life. In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally. In some embodiments, cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, cancer is progressive, unresectable, and / or metastatic solid tumors. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to an individual an effective dose of a third therapy comprising another anticancer agent, optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents. In some embodiments, the method comprises selecting an individual for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0168] In some embodiments, a method for treating cancer in an individual (e.g., cancer including KRAS mutations) comprises administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the TIGIT binding moiety comprises an scFv comprising a heavy chain variable region (VH) comprising heavy chain CDR (HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising light chain CDR (LCDR)1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6 The antibody comprises a full-length antibody including a column, where the PVRIG binding moiety comprises a heavy chain variable region (VH) containing heavy chain CDR(HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) containing light chain CDR(LCDR)1, LCDR2, and LCDR3, wherein HCDR1 contains the amino acid sequence of SEQ ID NO: 7, HCDR2 contains the amino acid sequence of SEQ ID NO: 8, HCDR3 contains the amino acid sequence of SEQ ID NO: 9, LCDR1 contains the amino acid sequence of SEQ ID NO: 10, LCDR2 contains the amino acid sequence of SEQ ID NO: 11, and LCDR3 contains the amino acid sequence of SEQ ID NO: 12, and anti-TIGIT scFv is ligated to the N-terminus or C-terminus of the two heavy chains of the anti-PVRIG full-length antibody, and the KRAS G12C inhibitor is of formula (III) [ka] (In the formula, T1 is selected from O and N, R1 is selected from C6-10 aryls and 5-10 membered heteroaryls, where C6-10 aryls and 5-10 membered heteroaryls are optionally substituted with 1, 2, 3, 4, or 5 Ra atoms, when T1 is O, R2 is absent, and when T1 is N, R2 is selected from H, C1-3 alkyl, -C(=O)-C1-3 alkyl and -S(=O)2-C1-3 alkyl, where C1-3 alkyl, -C(=O)-C1-3 alkyl and -S(=O)2-C1-3 alkyl are optionally substituted with 1, 2, or 3 Rb atoms)

[0169] R3 is a C1-3 alkyl group, where the C1-3 alkyl group is optionally substituted with 1, 2, or 3 Rc atoms; R4 is selected from H and a C1-3 alkyl group, where the C1-3 alkyl group is optionally substituted with 1, 2, or 3 Rd atoms; and R5, R6, and R7 are independently selected from H, F, Cl, Br, I, and a C1-3 alkyl group, where the C1-3 alkyl group is optionally... R8 is substituted with one, two, or three F atoms, R8 is selected from H and CH3, and Ra is independently selected from F, Cl, Br, I, OH, NH2, CN, C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl, where C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl are optionally substituted with one, two, or three F atoms. A method is provided in which Rb is independently selected from F, Cl, Br, I, OH, and NH2, Rc is independently selected from 4- to 8-membered heterocycloalkyl groups, where R5, R6, and R7 are optionally substituted with 1, 2, or 3 Rs, Rd is independently selected from F, Cl, Br, I, OH, NH2, and CN, and R is independently selected from H, F, Cl, Br, OH, CN, C1-3 alkyl, C1-3 alkoxy, and -C1-3 alkyl-OC(=O)-C1-3 alkylamino groups, where R1 is naphthyl, where naphthyl is optionally substituted with F, Cl, Br, OH, NH2, CF3, CH2CH3, and -C≡CH, and R5, R6, and R7 are independently H) or a pharmaceutically acceptable salt thereof. In some embodiments, the full-length antibody comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region, or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variant comprises one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend its half-life. In some embodiments, the construct is administered intravenously or subcutaneously.In some embodiments, the KRAS G12C inhibitor is administered orally. In some embodiments, the cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, the cancer is progressive, unresectable, and / or metastatic solid tumor. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to the individual an effective dose of a third therapy comprising another anticancer agent, which is optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents. In some embodiments, the method comprises selecting an individual for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0170] In some embodiments, a method is provided for treating cancer in an individual (e.g., cancer including a KRAS mutation), comprising administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the construct comprises first and second heavy chains comprising SEQ ID NO: 17 and first and second light chains comprising SEQ ID NO: 18. In some embodiments, a method is provided for treating cancer in an individual (e.g., a human patient), comprising administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the construct comprises first and second heavy chains comprising SEQ ID NO: 19 and first and second light chains comprising SEQ ID NO: 20, and the KRAS G12C inhibitor is of formula (III) [ka] (In the formula, T1 is selected from O and N, R1 is selected from C6-10 aryls and 5-10 membered heteroaryls, where C6-10 aryls and 5-10 membered heteroaryls are optionally substituted with 1, 2, 3, 4, or 5 Ra atoms, when T1 is O, R2 is absent, and when T1 is N, R2 is selected from H, C1-3 alkyl, -C(=O)-C1-3 alkyl and -S(=O)2-C1-3 alkyl, where C1-3 alkyl, -C(=O)-C1-3 alkyl and -S(=O)2-C1-3 alkyl are optionally substituted with 1, 2, or 3 Rb atoms)

[0171] R3 is a C1-3 alkyl group, where the C1-3 alkyl group is optionally substituted with 1, 2, or 3 Rc atoms; R4 is selected from H and a C1-3 alkyl group, where the C1-3 alkyl group is optionally substituted with 1, 2, or 3 Rd atoms; and R5, R6, and R7 are independently selected from H, F, Cl, Br, I, and a C1-3 alkyl group, where the C1-3 alkyl group is optionally... R8 is substituted with one, two, or three F atoms, R8 is selected from H and CH3, and Ra is independently selected from F, Cl, Br, I, OH, NH2, CN, C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl, where C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl are optionally substituted with one, two, or three F atoms. A method is provided in which Rb is independently selected from F, Cl, Br, I, OH, and NH2, Rc is independently selected from 4- to 8-membered heterocycloalkyl groups, where R5, R6, and R7 are optionally substituted with 1, 2, or 3 Rs, Rd is independently selected from F, Cl, Br, I, OH, NH2, and CN, and R is independently selected from H, F, Cl, Br, OH, CN, C1-3 alkyl, C1-3 alkoxy, and -C1-3 alkyl-OC(=O)-C1-3 alkylamino groups, where R1 is naphthyl, where naphthyl is optionally substituted with F, Cl, Br, OH, NH2, CF3, CH2CH3, and -C≡CH, and R5, R6, and R7 are independently H) or a pharmaceutically acceptable salt thereof. In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally. In some embodiments, the cancers are colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma.In some embodiments, the cancer is an advanced, unresectable, and / or metastatic solid tumor. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to the individual an effective dose of a third therapy comprising another anticancer agent, optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents. In some embodiments, the method comprises selecting an individual for treatment based on the presence of one or more cancer cells expressing a KRAS G12C mutant protein.

[0172] In some embodiments, a method for treating cancer (e.g., cancer involving KRAS mutations) in an individual (e.g., a human patient) comprises administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the KRAS G12C inhibitor [ka] , [ka] , or [ka] A method is provided in which the compound is selected from or a pharmaceutically acceptable salt thereof. In some embodiments, the KRAS G12C inhibitor is compound 17 or a pharmaceutically acceptable salt thereof. In some embodiments, the TIGIT binding moiety and the PVRIG binding moiety are either in Fab or scFv format. In some embodiments, the TIGIT binding moiety is in Fab format and the PVRIG binding moiety is in scFv format, or the TIGIT binding moiety is in scFv format and the PVRIG binding moiety is in Fab format, or both the TIGIT binding moiety and the PVRIG binding moiety are in Fab format. In some embodiments, the construct further comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variant comprises one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend its half-life. In some embodiments, the construct includes (a) an Fc region operably linked to a TIGIT binding moiety and a PVRIG binding moiety, (b) a TIGIT binding moiety operably linked to a PVRIG binding moiety and a PVRIG binding moiety operably linked to an Fc region, or (c) a PVRIG binding moiety operably linked to a TIGIT binding moiety and a TIGIT binding moiety operably linked to an Fc region. In some embodiments, one of the TIGIT binding moiety and the PVRIG binding moiety is a full-length antibody, and the other is an scFv or VHH operably linked to the full-length antibody (for example, by optionally linking to the N-terminus or C-terminus of one or both of the heavy or light chains of the full-length antibody via a linker). In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally.In some embodiments, cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, cancer is progressive, unresectable, and / or metastatic solid tumors. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to an individual an effective dose of a third therapy comprising another anticancer agent, optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents. In some embodiments, the method comprises selecting an individual for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0173] In some embodiments, a method is provided for treating cancer in an individual (e.g., cancer involving a KRAS mutation), comprising administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the TIGIT binding moiety comprises a full-length antibody comprising two heavy chains and two light chains, the PVRIG binding moiety comprises an scFv, and the anti-PVRIG scFv is ligated to the N-terminus or C-terminus of both heavy chains of the anti-TIGIT full-length antibody, and the KRAS G12C inhibitor is provided. In some embodiments, a method is provided for treating cancer in an individual (e.g., cancer involving a KRAS mutation), comprising administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the PVRIG binding moiety comprises a full-length antibody comprising two heavy chains and two light chains, the TIGIT binding moiety comprises an scFv, and the anti-TIGIT scFv is ligated to the N-terminus or C-terminus of both heavy chains of the anti-PVRIG full-length antibody, and the KRAS G12C inhibitor is [ka] , [ka] , or [ka] A method is provided in which the construct is a compound selected from or a pharmaceutically acceptable salt thereof. In some embodiments, the KRAS G12C inhibitor is compound 17 or a pharmaceutically acceptable salt thereof. In some embodiments, the full-length antibody comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variant comprises one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend its half-life. In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally. In some embodiments, cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, cancer is progressive, unresectable, and / or metastatic solid tumors. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to an individual an effective amount of a third therapy comprising another anticancer agent, which is optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents.In some embodiments, the method includes selecting individuals for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0174] In some embodiments, a method for treating cancer in an individual (e.g., cancer including KRAS mutations) comprises administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the TIGIT binding moiety comprises an antibody moiety comprising a heavy chain variable region (VH) comprising heavy chain CDR (HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising light chain CDR (LCDR)1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, LCDR3 comprises the amino acid sequence of SEQ ID NO: 6, and the KRAS G12C inhibitor is [ka] , [ka] , or [ka] A method is provided in which the compound is selected from or a pharmaceutically acceptable salt thereof. In some embodiments, the KRAS G12C inhibitor is compound 17 or a pharmaceutically acceptable salt thereof. In some embodiments, the TIGIT binding moiety and the PVRIG binding moiety are either in Fab or scFv format. In some embodiments, the TIGIT binding moiety is in Fab format and the PVRIG binding moiety is in scFv format, or the TIGIT binding moiety is in scFv format and the PVRIG binding moiety is in Fab format, or both the TIGIT binding moiety and the PVRIG binding moiety are in Fab format. In some embodiments, the construct further comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variant comprises one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend its half-life. In some embodiments, the construct includes (a) an Fc region operably linked to a TIGIT binding moiety and a PVRIG binding moiety, (b) a TIGIT binding moiety operably linked to a PVRIG binding moiety and a PVRIG binding moiety operably linked to an Fc region, or (c) a PVRIG binding moiety operably linked to a TIGIT binding moiety and a TIGIT binding moiety operably linked to an Fc region. In some embodiments, one of the TIGIT binding moiety and the PVRIG binding moiety is a full-length antibody, and the other is an scFv or VHH operably linked to the full-length antibody (for example, by optionally linking to the N-terminus or C-terminus of one or both of the heavy or light chains of the full-length antibody via a linker). In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally.In some embodiments, cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, cancer is progressive, unresectable, and / or metastatic solid tumors. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to an individual an effective dose of a third therapy comprising another anticancer agent, optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents. In some embodiments, the method comprises selecting an individual for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0175] In some embodiments, a method for treating cancer in an individual (e.g., cancer including KRAS mutations) comprises administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the PVRIG binding moiety comprises an antibody moiety comprising a heavy chain variable region (VH) comprising heavy chain CDR (HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising light chain CDR (LCDR)1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, HCDR2 comprises the amino acid sequence of SEQ ID NO: 8, HCDR3 comprises the amino acid sequence of SEQ ID NO: 9, LCDR1 comprises the amino acid sequence of SEQ ID NO: 10, LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, LCDR3 comprises the amino acid sequence of SEQ ID NO: 12, and the KRAS G12C inhibitor is [ka] , [ka] , or [ka] A method is provided in which the compound is selected from or a pharmaceutically acceptable salt thereof. In some embodiments, the KRAS G12C inhibitor is compound 17 or a pharmaceutically acceptable salt thereof. In some embodiments, the TIGIT binding moiety and the PVRIG binding moiety are either in Fab or scFv format. In some embodiments, the TIGIT binding moiety is in Fab format and the PVRIG binding moiety is in scFv format, or the TIGIT binding moiety is in scFv format and the PVRIG binding moiety is in Fab format, or both the TIGIT binding moiety and the PVRIG binding moiety are in Fab format. In some embodiments, the construct further comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variant comprises one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend its half-life. In some embodiments, the construct includes (a) an Fc region operably linked to a TIGIT binding moiety and a PVRIG binding moiety, (b) a TIGIT binding moiety operably linked to a PVRIG binding moiety and a PVRIG binding moiety operably linked to an Fc region, or (c) a PVRIG binding moiety operably linked to a TIGIT binding moiety and a TIGIT binding moiety operably linked to an Fc region. In some embodiments, one of the TIGIT binding moiety and the PVRIG binding moiety is a full-length antibody, and the other is an scFv or VHH operably linked to the full-length antibody (for example, by optionally linking to the N-terminus or C-terminus of one or both of the heavy or light chains of the full-length antibody via a linker). In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally.In some embodiments, cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, cancer is progressive, unresectable, and / or metastatic solid tumors. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to an individual an effective dose of a third therapy comprising another anticancer agent, optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents. In some embodiments, the method comprises selecting an individual for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0176] In some embodiments, a method for treating cancer in an individual (e.g., cancer including KRAS mutations) comprises administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the TIGIT binding moiety comprises an antibody moiety comprising a heavy chain variable region (VH) comprising heavy chain CDR (HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising light chain CDR (LCDR)1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6 The antibody moiety comprises an acid sequence, and the PVRIG binding moiety comprises a heavy chain variable region (VH) containing heavy chain CDR (HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) containing light chain CDR (LCDR)1, LCDR2, and LCDR3, wherein HCDR1 contains the amino acid sequence of SEQ ID NO: 7, HCDR2 contains the amino acid sequence of SEQ ID NO: 8, HCDR3 contains the amino acid sequence of SEQ ID NO: 9, LCDR1 contains the amino acid sequence of SEQ ID NO: 10, LCDR2 contains the amino acid sequence of SEQ ID NO: 11, and LCDR3 contains the amino acid sequence of SEQ ID NO: 12, and the KRAS G12C inhibitor is [ka] , [ka] , or [ka] A method is provided in which the compound is selected from or a pharmaceutically acceptable salt thereof. In some embodiments, the KRAS G12C inhibitor is compound 17 or a pharmaceutically acceptable salt thereof. In some embodiments, the TIGIT binding moiety and the PVRIG binding moiety are either in Fab or scFv format. In some embodiments, the TIGIT binding moiety is in Fab format and the PVRIG binding moiety is in scFv format, or the TIGIT binding moiety is in scFv format and the PVRIG binding moiety is in Fab format, or both the TIGIT binding moiety and the PVRIG binding moiety are in Fab format. In some embodiments, the construct further comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variant comprises one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend its half-life. In some embodiments, the construct includes (a) an Fc region operably linked to a TIGIT binding moiety and a PVRIG binding moiety, (b) a TIGIT binding moiety operably linked to a PVRIG binding moiety and a PVRIG binding moiety operably linked to an Fc region, or (c) a PVRIG binding moiety operably linked to a TIGIT binding moiety and a TIGIT binding moiety operably linked to an Fc region. In some embodiments, one of the TIGIT binding moiety and the PVRIG binding moiety is a full-length antibody, and the other is an scFv or VHH operably linked to the full-length antibody (for example, by optionally linking to the N-terminus or C-terminus of one or both of the heavy or light chains of the full-length antibody via a linker). In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally.In some embodiments, cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, cancer is progressive, unresectable, and / or metastatic solid tumors. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to an individual an effective dose of a third therapy comprising another anticancer agent, optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents. In some embodiments, the method comprises selecting an individual for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0177] In some embodiments, a method for treating cancer in an individual (e.g., cancer involving a KRAS mutation) comprising: 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety; and 2) KRAS A method is provided comprising administering a G12C inhibitor to an individual, wherein the TIGIT binding portion comprises an antibody portion including a heavy chain variable region (VH) and a light chain variable region (VL), the VH of the TIGIT binding portion comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence identical to at least 85%, 90%, or 95% of SEQ ID NO: 13, and / or the VL of the TIGIT binding portion comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence identical to at least 85%, 90%, or 95% of SEQ ID NO: 14, and / or the PVRIG binding portion comprises an antibody portion including a heavy chain variable region (VH) and a light chain variable region (VL), the VH of the PVRIG binding portion comprises the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence identical to at least 85%, 90%, or 95% of SEQ ID NO: 15, and / or the VL of the PVRIG binding portion comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence identical to at least 85%, 90%, or 95% of SEQ ID NO: 16. In some embodiments, the VH portion of the TIGIT binding moiety contains the amino acid sequence of SEQ ID NO: 13, and the VL portion of the TIGIT binding moiety contains the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH portion of the PVRIG binding moiety contains the amino acid sequence of SEQ ID NO: 15, and / or the VL portion of the PVRIG binding moiety contains the amino acid sequence of SEQ ID NO: 16, and the KRAS G12C inhibitor is [ka] , [ka] , or [ka] A method is provided in which the compound is selected from or a pharmaceutically acceptable salt thereof. In some embodiments, the KRAS G12C inhibitor is compound 17 or a pharmaceutically acceptable salt thereof. In some embodiments, the TIGIT binding moiety and the PVRIG binding moiety are either in Fab or scFv format. In some embodiments, the TIGIT binding moiety is in Fab format and the PVRIG binding moiety is in scFv format, or the TIGIT binding moiety is in scFv format and the PVRIG binding moiety is in Fab format, or both the TIGIT binding moiety and the PVRIG binding moiety are in Fab format. In some embodiments, the construct further comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variant comprises one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend its half-life. In some embodiments, the construct includes (a) an Fc region operably linked to a TIGIT binding moiety and a PVRIG binding moiety, (b) a TIGIT binding moiety operably linked to a PVRIG binding moiety and a PVRIG binding moiety operably linked to an Fc region, or (c) a PVRIG binding moiety operably linked to a TIGIT binding moiety and a TIGIT binding moiety operably linked to an Fc region. In some embodiments, one of the TIGIT binding moiety and the PVRIG binding moiety is a full-length antibody, and the other is an scFv or VHH operably linked to the full-length antibody (for example, by optionally linking to the N-terminus or C-terminus of one or both of the heavy or light chains of the full-length antibody via a linker). In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally.In some embodiments, cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, cancer is progressive, unresectable, and / or metastatic solid tumors. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to an individual an effective dose of a third therapy comprising another anticancer agent, optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents. In some embodiments, the method comprises selecting an individual for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0178] In some embodiments, a method for treating cancer in an individual (e.g., cancer including KRAS mutations) comprises administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the TIGIT binding moiety comprises a full-length antibody comprising a heavy chain variable region (VH) comprising heavy chain CDR (HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising light chain CDR (LCDR)1, LCDR2, and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6 The sequence includes an scFv in which the PVRIG binding moiety includes a heavy chain variable region (VH) containing heavy chain CDR(HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) containing light chain CDR(LCDR)1, LCDR2, and LCDR3, wherein HCDR1 contains the amino acid sequence of SEQ ID NO: 7, HCDR2 contains the amino acid sequence of SEQ ID NO: 8, HCDR3 contains the amino acid sequence of SEQ ID NO: 9, LCDR1 contains the amino acid sequence of SEQ ID NO: 10, LCDR2 contains the amino acid sequence of SEQ ID NO: 11, and LCDR3 contains the amino acid sequence of SEQ ID NO: 12, and the anti-PVRIG scFv is ligated to the N-terminus or C-terminus of the two heavy chains of the anti-TIGIT full-length antibody, and the KRAS G12C inhibitor is [ka] , [ka] , or [ka] A method is provided in which the construct is a compound selected from or a pharmaceutically acceptable salt thereof. In some embodiments, the KRAS G12C inhibitor is compound 17 or a pharmaceutically acceptable salt thereof. In some embodiments, the full-length antibody comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variant comprises one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend its half-life. In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally. In some embodiments, cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, cancer is progressive, unresectable, and / or metastatic solid tumors. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to an individual an effective amount of a third therapy comprising another anticancer agent, which is optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents.In some embodiments, the method includes selecting individuals for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0179] In some embodiments, a method for treating cancer in an individual (e.g., cancer including KRAS mutations) comprises administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety and 2) a KRAS G12C inhibitor, wherein the TIGIT binding moiety comprises an scFv comprising a heavy chain variable region (VH) comprising heavy chain CDR (HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising light chain CDR (LCDR)1, LCDR2, and LCDR3, where HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6 The anti-TIGIT scFv is linked to the N-terminus or C-terminus of the two heavy chains of the anti-PVRIG full-length antibody, and the PVRIG binding moiety comprises a full-length antibody comprising a heavy chain variable region (VH) containing heavy chain CDR(HCDR)1, HCDR2, and HCDR3, and a light chain variable region (VL) containing light chain CDR(LCDR)1, LCDR2, and LCDR3, where HCDR1 contains the amino acid sequence of SEQ ID NO: 7, HCDR2 contains the amino acid sequence of SEQ ID NO: 8, HCDR3 contains the amino acid sequence of SEQ ID NO: 9, LCDR1 contains the amino acid sequence of SEQ ID NO: 10, LCDR2 contains the amino acid sequence of SEQ ID NO: 11, and LCDR3 contains the amino acid sequence of SEQ ID NO: 12. [ka] , [ka] , or [ka] A method is provided in which the construct is a compound selected from or a pharmaceutically acceptable salt thereof. In some embodiments, the KRAS G12C inhibitor is compound 17 or a pharmaceutically acceptable salt thereof. In some embodiments, the full-length antibody comprises an immunoglobulin constant region, e.g., an IgG constant region, e.g., a human IgG1, IgG4, IgG2, IgG3 Fc region or a variant thereof. In some embodiments, the human IgG Fc region is a human IgG1 Fc region or a variant thereof. In some embodiments, the variant comprises one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend its half-life. In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally. In some embodiments, cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, cancer is progressive, unresectable, and / or metastatic solid tumors. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to an individual an effective amount of a third therapy comprising another anticancer agent, which is optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents.In some embodiments, the method includes selecting individuals for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

[0180] In some embodiments, a method is provided for treating cancer in an individual (e.g., cancer including a KRAS mutation), comprising administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the construct comprises first and second heavy chains comprising SEQ ID NO: 17 and first and second light chains comprising SEQ ID NO: 18. In some embodiments, a method is provided for treating cancer in an individual (e.g., cancer including a KRAS mutation), comprising administering to the individual 1) a construct comprising a TIGIT binding moiety and a PVRIG binding moiety, and 2) a KRAS G12C inhibitor, wherein the construct comprises first and second heavy chains comprising SEQ ID NO: 19 and first and second light chains comprising SEQ ID NO: 20, and the KRAS G12C inhibitor is [ka] , [ka] , or [ka] A method is provided in which the KRAS G12C inhibitor is a compound selected from or a pharmaceutically acceptable salt thereof. In some embodiments, the KRAS G12C inhibitor is compound 17 or a pharmaceutically acceptable salt thereof. In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally. In some embodiments, the cancers are colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, the cancer is an advanced, unresectable, and / or metastatic solid tumor. In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously, in parallel, or sequentially. In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct. In some embodiments, the method further comprises administering to the individual an effective dose of a third therapy comprising another anticancer agent, optionally selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents. In some embodiments, the method comprises selecting an individual for treatment based on the presence of one or more cancer cells expressing a KRAS G12C mutant protein.

[0181] In some embodiments, the subject is human. The cancers described herein include all types of cancer having a KRAS G12C mutation. A cancer (or population of cancer cells) containing one or more cancer cells expressing the KRAS G12C mutant protein is hereafter referred to herein as a "KRAS G12C mutant cancer." In some embodiments, cancers (such as KRAS G12C mutant cancers) include colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma. In some embodiments, the cancer is a progressive, unresectable, and / or metastatic solid tumor.

[0182] In some embodiments, the construct is coupled to TIGIT and PVRIG. Exemplary constructs used with this method are described in further detail below.

[0183] In some embodiments, the KRAS G12C inhibitor is, for example, a polypeptide (such as an antibody), peptide, antisense oligonucleotide, or small molecule that inhibits the activity of the KRAS G12C mutant protein. In some embodiments, the KRAS G12C inhibitor is a small molecule. Examples of small molecule KRAS G12C inhibitors that can be used with the methods provided herein include, but are not limited to, compound 17, sotrasib, adaglasib, JAB-21822, GDC-6036, JDQ443, D-1553, GH35, GFH925, BPI-421286 and LY3537982, RMC-6291, HBI-2438, BI 1823911, MK-1084 and JNJ-74699157. In some embodiments, the KRAS G12C inhibitor is compound 17. Further details regarding these and other exemplary small molecule KRAS G12C inhibitors are provided below.

[0184] In some embodiments, the construct is administered intravenously or subcutaneously. In some embodiments, the KRAS G12C inhibitor is administered orally.

[0185] In some embodiments, the construct and the KRAS G12C inhibitor are administered simultaneously. In some embodiments, “simultaneous administration” means that the TIGIT / PVRIG conjugate construct and the KRAS G12C inhibitor are administered at time intervals of about 15 minutes or less, for example, about 10 minutes, 5 minutes, or 1 minute or less. In some embodiments, simultaneous administration of the TIGIT / PVRIG conjugate construct and the KRAS G12C inhibitor can be combined with an additional dose of the TIGIT / PVRIG conjugate construct and / or the KRAS G12C inhibitor. In some embodiments, the TIGIT / PVRIG conjugate construct and the KRAS G12C inhibitor are administered sequentially. In some embodiments, “sequential administration” means that the TIGIT / PVRIG conjugate construct and the KRAS G12C inhibitor are administered at time intervals exceeding about 15 minutes, for example, about 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, or more. For example, in some embodiments, the TIGIT / PVRIG conjugate is administered before the small molecule KRAS G12C inhibitor. In some embodiments, the small molecule KRAS G12C inhibitor is administered before the TIGIT / PVRIG conjugate. In some embodiments, the administration of the TIGIT / PVRIG conjugate and the KRAS G12C inhibitor is parallel, i.e., the administration periods of the TIGIT / PVRIG conjugate and the KRAS G12C inhibitor overlap. In some embodiments, the administration of the TIGIT / PVRIG conjugate and the KRAS G12C inhibitor is not parallel.

[0186] In some embodiments, the construct is administered before the KRAS G12C inhibitor. In some embodiments, the KRAS G12C inhibitor is administered prior to the construct.

[0187] In some embodiments, the methods disclosed herein further include administering an effective amount of a third therapy. In some embodiments, the third therapy is another anticancer agent. The terms “anticancer agent” or “antiproliferative agent” mean any agent that can be used to treat a cell proliferation disorder such as cancer, and include, but are not limited to, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy and anti-metastatic agents, and immunotherapy agents. It will be understood that such anticancer agents may include a conjugate in the selected embodiments described above, and may be conjugated with the antibody of this disclosure before administration. More specifically, in certain embodiments, as described above, a manipulated conjugate is provided by linking a selected anticancer agent to an unpaired cysteine ​​of a manipulated antibody. Thus, such a manipulated conjugate is explicitly intended to be within the scope of this disclosure. In other embodiments, the anticancer agents of this disclosure are administered in combination with a site-specific conjugate containing a different therapeutic agent, as described above.

[0188] In some embodiments, the KRAS G12C inhibitor (e.g., compound 17) is administered at a dose of 10 mg / kg. In some embodiments, the KRAS G12C inhibitor (e.g., compound 17) is administered at a dose of 30 mg / kg. In some embodiments, the KRAS G12C inhibitor (e.g., compound 17) is administered daily. In some embodiments, the KRAS G12C inhibitor (e.g., compound 17) is administered orally.

[0189] In some embodiments, the construct having a TIGIT-binding moiety and a PVRIG-binding moiety (e.g., a bispecific TIGIT / PVRIG antibody) is administered at a dose of approximately 13.3 mg / kg. In some embodiments, the construct having a TIGIT-binding moiety and a PVRIG-binding moiety (e.g., a bispecific TIGIT / PVRIG antibody) is administered twice a week. The construct having a TIGIT-binding moiety and a PVRIG-binding moiety (e.g., a bispecific TIGIT / PVRIG antibody) is administered intraperitoneally.

[0190] Structures including TIGIT and PVRIG joints The constructs provided herein include, but are not limited to, multispecific antibodies, their antigen-binding moieties, and fusion proteins comprising such multispecific antibodies or their antigen-binding moieties.

[0191] In some embodiments, the TIGIT / PVRIG binding constructs described herein include a first antigen-binding moiety ("PVRIG binding moiety") that specifically binds to PVRIG (e.g., human PVRIG) and a second antigen-binding moiety ("TIGIT binding moiety") that specifically binds to TIGIT (e.g., human TIGIT).

[0192] A.TIGIT TIGIT, also known as WUCAM, Vstm3, and VSIG9, is a member of the Ig superfamily. Its expression has been reported in several human cancers, including melanoma, NSCLC, and colorectal cancer. The TIGIT receptor consists of an Ig variable domain, a transmembrane domain, and an immunoreceptor inhibitory tyrosine motif. Activated T cells, which are both regulatory CD4+ and effector CD8+, as well as NK cells, express TIGIT on their cell surface and interact with the poliovirus receptor (PVR), also known as CD155, with maximum binding affinity, and with weaker affinity to the nectin-2 receptor, poliovirus receptor-associated 2 (PVRL2), or CD112. Similarly, the costimulatory receptor CD226 is among the ligands for TIGIT and has lower affinity for TIGIT binding compared to CD155. CD155 is an adhesion molecule preferentially expressed on dendritic cells and macrophages and acts as a recognition molecule for NK cells. The interaction between CD155 and its ligand, TIGIT, was studied in various malignancies, including melanoma and NSCLC. In lung adenocarcinoma, immunohistochemical (IHC) overexpression of TIGIT / CD155 emerged as an unfavorable prognostic factor. The CD155 / TIGIT interaction is responsible for negative regulation of innate and adaptive immune responses at different levels. In response to activation of the CD155 / TIGIT pathway, T cell receptor expression is reduced, leading to impaired NK cell and CD8 T cell effector function. Impaired T cell activation is also a result of reduced release of immunosuppressive cytokines such as interleukin-10 and decreased interleukin-12 production by dendritic cells, which is facilitated by TIGIT involvement. Inhibition of CD226 signaling by inhibiting homodimerization is one known mechanism of TIGIT inhibition in T cells. For information on TIGIT expression in humans, please refer to Rousseau et al., ESMO Open. 2023 Apr;8(2):101184, which describes a late event in the cancer immune cycle that occurs after chronic tumor antigen exposure.

[0193] TIGIT continues to attract interest in drug development, but the clinical findings of novel checkpoint-targeting antibodies are mixed. Anti-TIGIT monotherapy has yielded objective response rates (ORRs) ranging from 0% to 5% in several trials in advanced solid tumors.

[0194] TIGIT can compete for ligand binding with CD226, replacing CD155 binding with CD226, and thus impairing antitumor immunity as demonstrated in mouse and human. Furthermore, TIGIT signaling in regulatory T cells (Tregs) enhances their immunosuppressive function. In mouse and human, TIGIT is highly expressed by a subset of native Tregs, and its upregulation in Tregs is associated with hypomethylation and Foxp3 binding at the TIGIT locus. TIGIT+Tregs upregulate many Treg gene signature markers in tumors, including Foxp3, Helios, neuropilin-1, CTLA-4, PD-1, TIM-3, and LAG-3. Similar to PD-1 / PD-L1, TIGIT binding to its ligand can suppress T cell function. This pathway does not overlap with the PD-1 / PD-L1 axis but shows more than two similarities. Both PD-1 and TIGIT are increasingly upregulated in activated T lymphocytes to prevent excessive immune responses. Similar to PD-1 / PD-L1, binding of TIGIT to its ligand can suppress T cell function. This pathway does not overlap with the PD-1 / PD-L1 axis but shows more than two similarities. Both PD-1 and TIGIT are increasingly upregulated in activated T lymphocytes to prevent excessive immune responses. To date, accumulating data supports that inhibition of the TIGIT receptor releases the immune system against cancer cells, thereby counteracting the phenomenon of immune evasion. Preclinical evidence and early-stage clinical trials demonstrate the feasibility of novel drugs addressing combinations of ICIs such as TIGIT and PD-(L)1. See Rousseau et al., ESMO Open. 2023 Apr;8(2):101184.

[0195] Dual blockade of PD-1 and TIGIT is a promising combination immunotherapy for cancer. While each single blockade does not significantly inhibit CT26 tumor growth in mice, dual TIGIT and PD-1 / PD-L1 blockade synergistically enhance the proliferation and function of antitumor CD8+ T cells, resulting in protective memory T cells, complete tumor rejection, and long-term overall survival. These effects abolish upon CD8+ T cell depletion, supporting the crucial role of CD8+ T cell-mediated tumor reactivity. See Chauvin et al., J Immunother Cancer. 2020 Sep;8(2):e000957.

[0196] Recent reports indicate that three trials are currently investigating anti-TIGIT therapies: a) vivostrimab in a Phase III trial, b) etigirimab in a Phase I trial, and c) tiragolumab in a Phase III trial. All of these trials involve combinations of TIGIT inhibitors and PD-1 / PD-L1 inhibitors for the treatment of NSCLC. See Rousseau et al., ESMO Open. 2023 Apr;8(2):101184.

[0197] Regarding tiragolumab, in March 2022, a press release announced the interim results of the Phase III SKYSCRAPER-01 trial of tiragolumab plus atezolizumab as first-line treatment for metastatic NSCLC with high PD-L1 expression. This trial did not meet its co-primary endpoint of PFS. However, the trial is still ongoing as it was premature to evaluate the other primary endpoint, OS. The atezolizumab group in CITYSCAPE performed poorly, with a median OS of 14.5 months for the overall population and 12.8 months for the high-expression PD-L1 group, compared to 18 months for the overall population and 20 months for the high-expression PD-L1 group in IMpower110. Thus, the positive results in Phase II could be explained by this poorly performing control arm, leading to the failure of Phase III. See Rousseau et al., ESMO Open. 2023 Apr;8(2):101184.

[0198] B. PVRIG PVRIG, also known as CD112R, was discovered in 2016. As a recently discovered inhibitory receptor within this family, research on this receptor is significantly limited compared to studies on TIGIT, CD96, and CD226. PVRIG is expressed on T cells and NK cells, with its expression on T cells increasing with cell activation. Upregulation of PVRIG on CD8+ T cells leads to their exhaustion in lymphocytic choriomeningitis virus infection. CD8+ T cells from PVRIG-deficient mice exhibit stronger antigen-specific effector function during acute Listeria monocytogenes infection. Furthermore, PVRIG-deficient mice show a significant reduction in tumor growth due to enhanced CD8+ T cell function. Additionally, gene knockout of PVRIG or treatment with anti-PVRIG mAbs (both early and late treatment) in mice significantly inhibited NK cell exhaustion and delayed tumor growth in several mouse tumor models. See Li et al., J Hematol Oncol. 2021 Jun 26;14(1):100.

[0199] PVRIG may compete with TIGIT and CD226 for binding to CD112, and it is hypothesized that blocking PVRIG / CD112 in addition to blocking TIGIT / PVR may allow CD226 to engage its ligand without interference. PVRIG is expressed on TILs and intratumor NK cells. In vitro experiments have demonstrated that a combination of anti-PVRIG and anti-TIGIT mAbs enhances human TIL and NK cell effector function. A combination of anti-PVRIG and anti-PD-L1 reduces tumor growth in PVRIG- / - mice, as anti-PD-L1 treatment does, suggesting that the combination of anti-PVRIG and anti-TIGIT may be another strategy worth investigating. See Chiang et al., Journal for ImmunoTherapy of Cancer 2022;10:e004711.

[0200] C. TIGIT Binding Moiety The TIGIT binding moieties discussed herein include any moiety that can bind to TIGIT (e.g., human TIGIT). In some embodiments, the TIGIT binding moiety is an antibody moiety. In some embodiments, the TIGIT binding moiety is a small molecule. In some embodiments, the TIGIT binding moiety blocks the binding of TIGIT (e.g., human TIGIT) and CD155 (e.g., human CD155). In some embodiments, the TIGIT binding moiety blocks the binding of TIGIT (e.g., human TIGIT) and CD112 (e.g., human CD112). See Zhong et al., Journal for ImmunoTherapy of Cancer 2020;8:doi:10.1136 / jitc-2020-SITC2020.0184.

[0201] In some embodiments, the TIGIT binding moiety is a small molecule that specifically targets TIGIT. In some embodiments, the TIGIT binding moiety includes liotyrosine. See Zhou et al., Cell Commun Signal 18,142(2020).

[0202] In some embodiments, the TIGIT binding moiety is an antibody moiety. In some embodiments, the TIGIT binding moiety (or bispecific TIGIT / PVRIG antibody) binds to the TIGIT protein (e.g., human, monkey or mouse TIGIT protein) with a K -10 of 1×10 D M or less, a K -11 of 8×10 D M or less, a K -11 of 6×10 D M or less, a K -11 of 4×10 D M or less, a K -11 of 2×10 D M or less. In some embodiments, the TIGIT binding moiety (or bispecific TIGIT / PVRIG antibody) has a K -9 of 5×10 D M or less, a K of 4×10-9 K below M D , or 3 × 10 -9 K below M D It then binds to the TIGIT protein (for example, the TIGIT protein of humans, monkeys, or mice).

[0203] In some embodiments, the TIGIT binding moiety comprises six CDRs, VHs and / or VLs, or the complete sequence of an anti-TIGIT antibody known in the art. Exemplary anti-TIGIT antibodies include tiragolumab (RG6058), donbanalimab (AB154), vivostrimab (MK-7684), osiperlimab (BGB-A1217), BMS-986207, BMS-98620, EOS-448, MBSA43, ASP-8374, OMP-313M32, COM-902, etigirimab, IBI-939, AGEN-1307, PTZ-201 (ASP8374), CASC-674, NB-6253, PH-804, M6223, MTIG7192A, osiperlimab, and eos88448. See Rotte et al., Biomedicines. 2021 Sep;9(9):1277.

[0204] In some embodiments, the TIGIT coupling portion is published in U.S. Patent No. 11,225,523, U.S. Patent No. 9,499,596, International Publication No. 2016 / 191643, International Publication No. 2017 / 053748, International Publication No. 2016 / 191643, International Publication No. 2016 / 028656, International Publication No. 2017 / 030823, U.S. Patent Application Publication No. 2016 / 0176963, International Publication No. 2017 / 037707, International Publication No. 2017 / 059095, International Publication The invention includes the complete sequences of six CDRs, VHs and / or VLs, or anti-TIGIT antibodies, as disclosed in any of the following publications: 2016 / 106302, 2017281764, 2015 / 009856, 20170037133, 2017 / 048824, U.S. Patent No. 9,713,364, or 2016 / 028656 (all of which are incorporated herein in their entirety). In some embodiments, the TIGIT binding moiety includes the complete sequences of six CDRs, VHs and / or VLs, or 10A7, 1F4, 14A6, 28H5, 31C6, 15A6, 22G2, 11G11, or 10D7.

[0205] In some embodiments, the TIGIT binding region includes a heavy chain variable region (VH) and / or a light chain variable region (VL), where A) VH comprises (i) HCDR1, which includes the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having one, two, or three or fewer amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 1; (ii) HCDR2, which includes the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having one, two, or three or fewer amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 2; and (iii) HCDR2, which includes the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having one, two, or three or fewer amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 3. A) A light chain CDR (LCDR) comprising a heavy chain CDR (HCDR) containing HCDR3, and a light chain CDR (LCDR) comprising (i) an amino acid sequence LCDR1 containing the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having one, two, or three or fewer amino acids added, deleted, and / or substituted compared to SEQ ID NO: 4, (ii) an amino acid sequence LCDR2 containing the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having one, two, or three or fewer amino acids added, deleted, and / or substituted compared to SEQ ID NO: 5, and (iii) an amino acid sequence LCDR3 containing the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having one, two, or three or fewer amino acids added, deleted, and / or substituted compared to SEQ ID NO: 6.

[0206] In some embodiments, the TIGIT binding region includes a heavy chain variable region (VH) and a light chain variable region (VL), where (A) VH comprises a heavy chain CDR (HCDR) including (i) an HCDR1 containing the amino acid sequence of SEQ ID NO: 1, (ii) an HCDR2 containing the amino acid sequence of SEQ ID NO: 2, and (iii) an HCDR3 containing the amino acid sequence of SEQ ID NO: 3, and B) VL comprises a light chain CDR (LCDR) including (i) an LCDR1 containing the amino acid sequence of SEQ ID NO: 4, (ii) an LCDR2 containing the amino acid sequence of SEQ ID NO: 5, and (iii) an LCDR3 containing the amino acid sequence of SEQ ID NO: 6.

[0207] In some embodiments, the TIGIT binding region comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), where (A) VH is (i) the amino acid sequence of SEQ ID NO: 13, (ii) an amino acid sequence identical to SEQ ID NO: 13 by at least about 80%, 85%, 90%, or 95% (preferably at least 90%, more preferably at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%), or (iii) one or more amino acids (e.g., 1, 2, 3 or more, preferably 1, 2, or 3, more preferably 1 or 2) compared to SEQ ID NO: 13, with additions, deletions, and / or Or comprising a substituted amino acid sequence, and / or (B)VL comprises (i) the amino acid sequence of SEQ ID NO: 14, (ii) an amino acid sequence that is at least about 80%, 85%, at least 90%, or at least 95% (preferably at least 90%, more preferably at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%)) identical to SEQ ID NO: 14, or (iii) an amino acid sequence in which one or more amino acids (e.g., one, two, three or more, preferably one, two, or three, more preferably one or two) are added, deleted, and / or substituted compared to SEQ ID NO: 14.

[0208] In some embodiments, the TIGIT binding portion includes an scFv containing the sequence shown in SEQ ID NO: 23, or an amino acid sequence that is at least about 80%, 85%, 90%, or 95% (preferably at least 90%, more preferably at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%)) identical to SEQ ID NO: 23.

[0209] D.PVRIG binding part The PVRIG-binding moieties discussed herein include any moiety capable of binding to PVRIG (e.g., human PVRIG). In some embodiments, the PVRIG-binding moiety is an antibody moiety. In some embodiments, the PVRIG-binding moiety is a small molecule. In some embodiments, the PVRIG-binding moiety blocks the binding of PVRIG (e.g., human PVRIG) and PVRL2 (e.g., human PVRL2).

[0210] In some embodiments, the PVRIG binding moiety is a small molecule that specifically targets PVRIG.

[0211] In some embodiments, the PVRIG binding portion is the antibody portion. In some embodiments, the PVRIG binding portion (or multispecific TIGIT / PVRIG antibody) is 1 × 10 -10 K below M D , 8×10 -11 K below M D , 6×10 -11 K below M D , 4×10 -11 K below M D , or 2 × 10 -11 K below M D It binds to the PVRIG protein (e.g., human, monkey, or mouse PVRIG protein). In some embodiments, the PVRIG binding moiety (or multispecific TIGIT / PVRIG antibody) is measured by surface plasmon resonance at 5 × 10⁻¹⁴ -9 K below M D , 4×10 -9 K below M D , or 3 × 10 -9 K below M D It then binds to the PVRIG protein (e.g., human, monkey, or mouse PVRIG protein).

[0212] In some embodiments, an anti-PVRIG antibody (containing an antigen-binding fragment) binds to PVRIG and prevents its activation by PVLR2 (for example, by blocking the interaction between PVRIG and PVLR2).

[0213] In some embodiments, the PVRIG binding moiety comprises six CDRs, VHs and / or VLs, or the complete sequence of an anti-PVRIG antibody known in the art. An example of a PVRIG antibody is COM701.

[0214] In some embodiments, the PVRIG-binding moiety includes six CDRs, VHs and / or VLs, or complete sequences of anti-PVRIG antibodies disclosed in any of the following: U.S. Patent Publication No. 2021018974, U.S. Patent Publication No. 2021000952, U.S. Patent Publication No. 20190382477, U.S. Patent Publication No. 20230057899, International Publication No. 2022069940, and European Patent No. 3259597 (all of which are incorporated herein in their entirety).

[0215] In some embodiments, the PVRIG binding region includes a heavy chain variable region (VH) and / or a light chain variable region (VL), where A) VH comprises (i) HCDR1, which includes the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having one, two, or three or fewer additions, deletions, and / or substitutions compared to SEQ ID NO: 7; (ii) HCDR2, which includes the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having one, two, or three or fewer additions, deletions, and / or substitutions compared to SEQ ID NO: 8; and (iii) H, which includes the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having one, two, or three or fewer additions, deletions, and / or substitutions compared to SEQ ID NO: 9. A) A light chain CDR (LCDR) comprising a heavy chain CDR (HCDR) including CDR3, and a light chain CDR (LCDR) comprising (i) an amino acid sequence LCDR1 having the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having the addition, deletion, and / or substitution of 1, 2, or 3 or fewer amino acids compared to SEQ ID NO: 10, (ii) an amino acid sequence LCDR2 having the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having the addition, deletion, and / or substitution of 1, 2, or 3 or fewer amino acids compared to SEQ ID NO: 11, and (iii) an amino acid sequence LCDR3 having the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having the addition, deletion, and / or substitution of 1, 2, or 3 or fewer amino acids compared to SEQ ID NO: 12.

[0216] In some embodiments, the PVRIG binding region includes a heavy chain variable region (VH) and a light chain variable region (VL), where (A) VH comprises a heavy chain CDR (HCDR) including (i) an HCDR1 containing the amino acid sequence of SEQ ID NO: 7, (ii) an HCDR2 containing the amino acid sequence of SEQ ID NO: 8, and (iii) an HCDR3 containing the amino acid sequence of SEQ ID NO: 9, and B) VL comprises a light chain CDR (LCDR) including (i) an LCDR1 containing the amino acid sequence of SEQ ID NO: 10, (ii) an LCDR2 containing the amino acid sequence of SEQ ID NO: 11, and (iii) an LCDR3 containing the amino acid sequence of SEQ ID NO: 12.

[0217] In some embodiments, the PVRIG binding region comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), where (A) VH is (i) the amino acid sequence of SEQ ID NO: 15, (ii) an amino acid sequence identical to SEQ ID NO: 15 by at least about 80%, 85%, 90%, or 95% (preferably at least 90%, more preferably at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%), or (iii) one or more amino acids (e.g., one, two, three or more, preferably one, two, or three, more preferably one or two) compared to SEQ ID NO: 15, with addition, deletion, and / or Or comprising a substituted amino acid sequence, and / or (B)VL comprises (i) the amino acid sequence of SEQ ID NO: 16, (ii) an amino acid sequence that is at least about 80%, 85%, at least 90%, or at least 95% (preferably at least 90%, more preferably at least 95%) (e.g., 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 16, or (iii) an amino acid sequence in which one or more amino acids (e.g., one, two, three or more, preferably one, two, or three, more preferably one or two) are added, deleted, and / or substituted compared to SEQ ID NO: 16.

[0218] In some embodiments, the PVRIG binding portion includes an scFv containing the sequence shown in SEQ ID NO: 23, or an amino acid sequence that is at least about 80%, 85%, 90%, or 95% (preferably at least 90%, more preferably at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%)) identical to SEQ ID NO: 23.

[0219] The binding of antibodies to TIGIT and PVRIG can be evaluated using one or more well-established techniques in the art, such as ELISA or FACS, which measure the binding of antibodies to soluble TIGIT / PVRIG proteins or to TIGIT / PVRIG proteins expressed on the cell surface, respectively. For example, antibodies can be tested by flow cytometry assays in which the antibodies are reacted with cell lines expressing human TIGIT or human PVRIG, such as CHO cells transfected to express TIGIT or PVRIG on the cell surface, or TIGIT or PVRIG-positive cell lines, or TIGIT and PVRIG bipositive cell lines.

[0220] In some embodiments, the multispecific antibodies disclosed herein are characterized by specific functional features or properties. In some embodiments, the antibody has one or more of the following properties: (a) specifically binds to human, cynomolgus monkey, and mouse TIGIT; (b) specifically binds to human and cynomolgus monkey PVRIG; (c) can simultaneously bind to both TIGIT and PVRIG; (d) efficiently blocks the binding of PVRIG to PVRL2 and the binding of TIGIT to PVR; (e) has no cross-activity with paralog proteins of TIGIT and PVRIG; (f) shows better efficacy than control antibodies (WBP364-BMK1 and WBPT117-BMK1), or even combinations of control antibodies, in reporter gene assays, NK cell killing, and T cell activation assays; (g) has good antibody development suitability, such as thermal stability, solubility, hydrophobicity, and stress stability; (h) has significantly improved efficacy in cancer treatment when combined with an anti-PD-L1 antibody, as demonstrated in an in vivo mouse model; and / or (i) has an acceptable pharmacokinetic profile in monkeys.

[0221] In some embodiments, the control antibody is a monoclonal antibody, such as a monoclonal anti-TIGIT antibody. In some embodiments, the control antibody is WBP364-BMK1, as shown in Table 2. In some embodiments, the control antibody is a monoclonal anti-PVRIG antibody. In some embodiments, the control antibody is WBPT117-BMK1, as shown in Table 2. In some embodiments, the control antibody is the parent antibody from which a multispecific antibody is derived and constructed. In some embodiments, the control antibody is W3642 and / or WT1175.

[0222] In some embodiments, the multispecific constructs disclosed herein have a higher binding affinity to TIGIT compared to monospecific anti-TIGIT antibodies or other anti-TIGIT / PVRIG multispecific antibodies. In some embodiments, the multispecific constructs disclosed herein have a binding affinity to TIGIT that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than monospecific anti-TIGIT antibodies, as measured by SPR or FACS.

[0223] In some embodiments, the multispecific constructs disclosed herein have a higher or equivalent binding affinity to PVRIG compared to monospecific anti-PVRIG antibodies or other anti-TIGIT / PVRIG multispecific antibodies. In some embodiments, the multispecific constructs disclosed herein, as measured by FACS, have a binding affinity to PVRIG that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than monospecific anti-PVRIG antibodies or other anti-TIGIT / PVRIG multispecific antibodies.

[0224] The anti-TIGIT / PVRIG antibodies disclosed herein can inhibit the interactions between TIGIT and its ligand PVR (CD155), and between PVRIG and PVRL2 (CD112). By blocking these signaling pathways, functional responses by T cells to antigen stimulation (e.g., proliferation, cytokine production, target cell killing) can be restored from a dysfunctional state.

[0225] The ability of an antibody to inhibit the interaction between an antigen and its ligand may be evaluated in a binding assay by measuring, for example, whether the physical interaction between TIGIT and PVR is reduced. In some embodiments, the binding assay is a competitive binding assay. The assay may be carried out in various formats, including, but is not limited to, ELISA assays, flow cytometry, surface plasmon resonance (SPR) assays (e.g., Biacore®), or biolayer interferometry (e.g., ForteBio Octet®).

[0226] E. Antibody portion in the construct (e.g., anti-TIGIT or anti-PVRIG antibody portion) The antibody portion described herein (such as the anti-TIGIT antibody portion or the anti-PVRIG antibody portion) may have one or more of the following characteristics.

[0227] In some embodiments, the antibody portion comprises an Fc fragment. In some embodiments, the antibody portion comprises an scFv. In some embodiments, the antibody portion comprises an scFv fused to an Fc fragment. In some embodiments, the antibody portion comprises an scFv fused to an Fc fragment via a peptide linker. In some embodiments, the Fc fragment is a human IgG1 Fc fragment. In some embodiments, the Fc fragment comprises one or more mutations to increase clearance or decrease half-life.

[0228] In some embodiments, the Fc fragment includes a constant region of the immunoglobulin IgG heavy chain containing a hinge region (beginning at Cys226), an IgG CH2 domain, and a CH3 domain. As used herein, the terms “hinge region” or “hinge sequence” refer to the amino acid sequence located between the linker and the CH2 domain. In some embodiments, the fusion protein includes an Fc fragment containing a hinge region. In some embodiments, the Fc fragment of the fusion protein begins at a hinge region and extends to the C-terminus of the IgG heavy chain. In some embodiments, the fusion protein includes an Fc fragment that does not contain a hinge region.

[0229] In some embodiments, the antibody moiety comprises an Fc fragment selected from the group consisting of IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the Fc fragment is derived from human IgG. In some embodiments, the Fc fragment comprises the Fc region of human IgG1, IgG2, IgG3, IgG4, or combinations or hybrids of IgG. In some embodiments, the Fc fragment is an IgG1 Fc fragment. In some embodiments, the Fc fragment comprises the CH2 and CH3 domains of IgG1. In some embodiments, the Fc fragment is an IgG4 Fc fragment. In some embodiments, the Fc fragment comprises the CH2 and CH3 domains of IgG4. IgG4 Fc is known to exhibit lower effector activity than IgG1 Fc and may therefore be desirable for some applications. In some embodiments, the Fc fragment is derived from mouse immunoglobulin.

[0230] In some embodiments, the IgG CH2 domain begins at Ala231. In some embodiments, the CH3 domain begins at Gly341. It is understood that the C-terminal Lys residue of human IgG may be optionally absent. It is also understood that conservative amino acid substitutions of the Fc region that do not affect the desired structure and / or stability of Fc are intended within the scope of the invention.

[0231] Heterodimerization of non-identical polypeptides in the antibody portion of the Fc fragment can be facilitated by methods known in the art, including, but not limited to, heterodimerization by knob-into-hole technology. The structure and assembly method of knob-into-hole technology can be found, for example, in U.S. Patent No. 5,821,333, U.S. Patent No. 7,642,228, U.S. Patent No. 2011 / 0287009 and International Application PCT / US2012 / 059810, which are incorporated herein by reference in their entirety. This technology was developed by introducing a “knob” (or protrusion) by substituting a small amino acid residue with a large amino acid residue in the CH3 domain of one Fc and introducing a “hole” (or cavity) by substituting one or more large amino acid residues with small amino acid residues in the CH3 domain of the other Fc. In some embodiments, one chain of the Fc fragment in the fusion protein contains a knob and a second chain of the Fc fragment contains a hole.

[0232] Preferred residues for knob formation are generally native amino acid residues, preferably selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Most preferred are tryptophan and tyrosine. In one embodiment, the original residues for knob formation, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine, have a small side-chain volume. Exemplary amino acid substitutions in the CH3 domain for knob formation include, but are not limited to, T366W, T366Y, or F405W substitutions.

[0233] Preferred residues for hole formation are typically native amino acid residues, preferably selected from alanine (A), serine (S), threonine (T), and valine (V). In one embodiment, the original residue for hole formation, e.g., tyrosine, arginine, phenylalanine, or tryptophan, has a large side-chain volume. Exemplary amino acid substitutions in the CH3 domain for generating holes include, but are not limited to, the T366S, L368A, F405A, Y407A, Y407T, and Y407V substitutions. In certain embodiments, the knob includes the T366W substitution, and the hole includes the T366S / L368A / Y407V substitution. Other modifications to the Fc region known in the Art that promote heterodimerization are also contemplated and understood to be included in this application.

[0234] Other antibody moieties are intended to include any of the variants described herein (e.g., Fc variants, effector function variants, glycosylation variants, cysteine ​​manipulation variants) or combinations thereof.

[0235] a) Antibody affinity The binding specificity of the antibody moiety can be experimentally measured by methods known in the art. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA, EIA, BIACORE® tests, and peptide scanning.

[0236] In some embodiments, the KD of binding between the antibody moiety and the target antigen (e.g., TIGIT or PVRIG) is approximately 10 -7 M~about 10 -12 M, about 10 -7 M~about 10 -8 M, about 10 -8 M~about 10 -9 M, about 10 -9 M~about 10 -10 M, about 10 -10 M~about 10 -11 M, about 10 -11 M~about 10 -12 M, about 10 -7 M~about 10-12 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12 M, about 10 -7 M to about 10 -11 M, about 10 -8 M to about 10 -11 M, about 10 -9 M to about 10 -11 M, about 10 -7 M to about 10 -10 M, about 10 -8 M to about 10 -10 M, or about 10 -7 M to about 10 -9 M. In some embodiments, the K of the binding between the antibody portion and the target antigen (e.g., TIGIT or PVRIG) D is about 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 the M is smaller than any one of them. In some embodiments, the target antigen (e.g., TIGIT or PVRIG) is a human antigen.

[0237] In some embodiments, the K of the binding between the antibody portion and the target antigen (e.g., TIGIT or PVRIG) on is about 10 3 M -1 s -1 to about 10 8 M -1 s<​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​-1 s -1 , about 10 6 M -1 s -1 ~about 10 7 M -1 s -1 , or about 10 7 M -1 s -1 ~about 10 8 M -1 s -1 In some embodiments, the K of binding between the antibody moiety and the target antigen (e.g., TIGIT or PVRIG) is on It is about 10 3 M -1 s -1 ~about 10 5 M -1 s -1 , about 10 4 M -1 s -1 ~about 10 6 M -1 s -1 , about 10 5 M -1 s -1 ~about 10 7 M -1 s -1 , about 10 6 M -1 s -1 ~about 10 8 M -1 s -1 , about 10 4 M -1 s -1 ~about 10 7 M -1 s -1 , or about 10 5 M -1 s -1 ~about 10 8 M -1 s -1 In some embodiments, the K of binding between the antibody moiety and the target antigen (e.g., TIGIT or PVRIG) is on It is about 10 3 M -1 s -1 , 10 4 M -1 s -1 , 10 5 M -1 s-1 , 10 6 M -1 s -1 , 10 7 M -1 s -1 or 10 8 M -1 s -1 It is one or less of the following. In some embodiments, the target antigen (e.g., TIGIT or PVRIG) is a human antigen.

[0238] In some embodiments, the K2 binding between the antibody moiety and the target antigen (e.g., TIGIT or PVRIG) off It takes about 1 second -1 ~about 10 -6 s -1 Approximately 1 second -1 ~about 10 -2 s -1 , about 10 -2 s -1 ~about 10 -3 s -1 , about 10 -3 s -1 ~about 10 -4 s -1 , about 10 -4 s -1 ~about 10 -5 s -1 , about 10 -5 s -1 ~about 10 -6 s -1 Approximately 1 second -1 ~about 10 -5 s -1 , about 10 -2 s -1 ~about 10 -6 s -1 , about 10 -3 s -1 ~about 10 -6 s -1 , about 10 -4 s -1 ~about 10 -6 s -1 , about 10 -2 s -1 ~about 10 -5 s -1 , or about 10 -3 s -1 ~about 10 -5 s-1 In some embodiments, the K of binding between the antibody moiety and the target antigen (e.g., TIGIT or PVRIG) is off It takes at least about 1 second -1 , 10 -2 s -1 , 10 -3 s -1 , 10 -4 s -1 , 10 -5 s -1 or 10 -6 s -1 It is one of the following. In some embodiments, the target antigen (e.g., TIGIT or PVRIG) is a human antigen.

[0239] b) Chimeric antibody or humanized antibody In some embodiments, the antibody portion is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In some embodiments, the chimeric antibody includes a non-human variable region (e.g., a mouse-derived variable region) and a human constant region. In some embodiments, the chimeric antibody is a “class-switched” antibody in which the class or subclass is modified from that of the parent antibody. The chimeric antibody includes its antigen-binding fragment.

[0240] In some embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity against humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody contains one or more variable domains, where HVR, e.g., CDR (or a portion thereof), is derived from the non-human antibody and FR (or a portion thereof) is derived from the human antibody sequence. The humanized antibody may optionally also contain at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues derived from the non-human antibody (e.g., the antibody from which the HVR residues are derived) to restore or improve the specificity or affinity of the antibody, for example.

[0241] Humanized antibodies and their production methods are outlined, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further, for example, in Riechmann et al., Nature 332:323-329 (1988), Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989), U.S. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321, and No. 7,087,409, and Kashmiri et al., Methods This is described in 36:25-34 (2005) (SDR(a-CDR) grafting), Padlan, Mol.Immunol.28:489-498 (1991) ("Resurfacing" is described), Dall'Acqua et al., Methods 36:43-60 (2005) ("FR shuffle" is described), and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br.J.Cancer,83:252-260 (2000) ("Guide selection" approach to FR shuffle is described).

[0242] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (e.g., Sims et al., J.Immunol. 151:2296 (1993)), framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain variable regions or heavy chain variable regions (see, for example, Carter et al. Proc.Natl.Acad.Sci.USA, 89:4285 (1992) and Presta et al. J.Immunol., 151:2623 (1993)), human maturation (somatic mutation) framework regions or human germline framework regions (see, for example, Almagro and Fransson, Front.Biosci. 13:1619-1633 (2008)), and framework regions derived from screening of FR libraries (e.g., Baca et al. See also al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0243] c) Human antibodies In some embodiments, the antibody portion is a human antibody (known as a human domain antibody or human DAb). Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001), Lonberg, Curr. Opin. Immunol. 20:450-459 (2008), and Chen, Mol. Immunol. 47(4):912-21 (2010). Transgenic mice or rats capable of producing fully human single-domain antibodies (or DAbs) are known in the art. For example, see U.S. Patent Publication No. 20090307787, U.S. Patent No. 8,754,287, U.S. Patent Publication No. 20150289489, U.S. Patent Publication No. 20100122358, and International Publication No. 2004049794.

[0244] Human antibodies (e.g., human DAbs) can be prepared by administering immunogens to transgenic animals modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci, which either replace endogenous immunoglobulin loci, are located extrachromosomally, or are randomly incorporated into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE® technology, U.S. Patent No. 5,770,429 describing HuMab® technology, U.S. Patent No. 7,041,870 describing KM MOUSE® technology, and U.S. Patent Application Publication 2007 / 0061900 describing VelociMouse® technology. Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining them with different human constant regions.

[0245] Human antibodies (e.g., human DAbs) can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987) and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced via human B-cell hybridoma technology have also been described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include, for example, those described in U.S. Patent No. 7,189,826 (production of monoclonal human IgM antibody from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0246] Human antibodies (e.g., human DAbs) can also be produced by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0247] d) Antibodies derived from the library

[0248] The antibody portion can be isolated by screening a combinatorial library for antibodies having one or more desired activities. For example, various methods for preparing phage display libraries and screening such libraries for antibodies having desired binding properties are known in the art. Such methods are outlined, for example, in the chapter by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (edited by O'Brien et al., Human Press, Totowa, New Jersey, 2001), and further, for example, in the McCafferty et al., Nature 348:552-554, Clackson et al., Nature 352:624-628 (1991), Marks et al., J.Mol.Biol.222:581-597 (1992), in the chapters by Marks and Bradbury in Methods in Molecular Biology 248:161-175 (edited by Lo, Human Press, Totowa, New Jersey, 2003), and in Sidhu et al., J.Mol.Biol.338(2):299-310 (2004), Lee This is described in et al., J.Mol.Biol.340(5):1073-1093 (2004), Fellouse, Proc.Natl.Acad.Sci.USA 101(34):12467-12472 (2004), and Lee et al., J.Immunol.Methods 284(1-2):119-132 (2004). Methods for constructing single-domain antibody libraries are described; see, for example, U.S. Patent No. 7,371,849.

[0249] In certain phage display methods, the repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR), randomly recombined within a phage library, and then screened for antigen-binding phages, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). The phages typically present antibody fragments as either scFv or Fab fragments. Libraries from immunized sources provide high-affinity antibodies against immunogens and do not require the construction of hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12:725-734 (1993), naive repertoires can be cloned without immunization (e.g., from humans) to provide a single antibody source against a wide range of non-self and autoantigens. Finally, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992), naive libraries can also be synthetically constructed by cloning an unreorganized V gene segment from stem cells, encoding a highly variable CDR3 region using PCR primers containing random sequences, and achieving rearrangement in vitro. Examples of patent publications describing human antibody phage libraries include U.S. Patent No. 5,750,373, and U.S. Patent Application Publications 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0250] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments in this specification. e) Substitutions, insertions, deletions, and variants

[0251] In some embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVR (or CDR) and FR. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More substantial substitutions are shown in Table 1 under the heading "Exemplary Substitutions" and are described in further detail below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into antibodies of interest and screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0252] [Table 1]

[0253] Amino acids can be classified according to their general side-chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0254] Non-conservative substitution involves swapping one member of one class with another.

[0255] One type of substitution mutant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting mutants selected for further study have altered (e.g., improved) specific biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody, and / or substantially retain the specific biological properties of the parent antibody. Exemplary substitution mutants are affinity-mature antibodies, which can be readily generated using, for example, phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more HVR residues are mutated, the mutant antibody is presented on a phage, and it is screened for specific biological activity (e.g., binding affinity).

[0256] For example, to improve antibody affinity, modifications (e.g., substitutions) may be made to HVRs. Such modifications may be made at HVR "hot spots," i.e., codons that are frequently mutated during the somatic cell maturation process (e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues encoded by SDRs (a-CDRs), and the resulting mutant VH or VL is tested for binding affinity. Affinity maturation by secondary library construction and re-selection from secondary libraries is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., Human Press, Totowa, New Jersey (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by one of various methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-specific mutagenesis). A secondary library is then constructed. Next, this library is screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves an HVR-directed approach that randomizes several HVR residues (e.g., 4-6 residues at a time). HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are often targeted in particular.

[0257] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, provided that such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) can be made in an HVR. Such changes may occur outside of an HVR "hotspot" or CDR. (See previously provided variant V) H In some embodiments of the H sequence, each HVR is either unchanged or contains one, two, or three or fewer amino acid substitutions.

[0258] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, target residues or groups of residues (e.g., charged residues, e.g., Arg, Asp, His, Lys, and Glu) are identified and replaced with uncharged or negatively charged amino acids (e.g., alanine or polyalanine) to investigate whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex may be used to identify contact points between the antibody and antigen. Such contact residues and adjacent residues can be targeted or excluded as candidates for substitution. Mutants can be screened to determine whether they possess the desired properties.

[0259] Amino acid sequence insertions include amino-terminus and / or carboxyl-terminus fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of terminal insertion is an antibody with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusion of antibodies to the N-terminus or C-terminus with enzymes (e.g., for ADEPT) or polypeptides that increase the serum half-life of the antibody.

[0260] f) Glycosylated mutants In some embodiments, the antibody moiety is modified to increase or decrease the degree to which the construct is glycosylated. Addition or deletion of glycosylation sites to the antibody can be conveniently achieved by altering the amino acid sequence so that one or more glycosylation sites are created or removed.

[0261] If the antibody moiety includes an Fc region, the carbohydrate bound to it may change. Native antibodies produced by mammalian cells typically have a C region in the Fc region. HThe molecule contains a branched, bibranched oligosaccharide linked to Asn297 of two domains by an N-bond. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose linked to GlcNAc in the "stem" of the bibranched oligosaccharide structure. In some embodiments, modification of the oligosaccharide in the antibody moiety may be performed to produce antibody variants with specific improved properties.

[0262] In some embodiments, the antibody moiety has a carbohydrate structure lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such an antibody may be 1%–80%, 1%–65%, 5%–65%, or 20%–40%. The amount of fucose is determined by calculating the average amount of fucose in the glycans at Asn297 relative to the total amount of all sugar structures bound to Asn297 (e.g., complex, hybrid, and high-mannose structures) as measured by MALDI-TOF mass spectrometry, as described, for example, in International Publication No. 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 of the Fc region (EU numbering of Fc region residues), although Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to slight sequence variations in the antibody. Such fucosylated variants may have improved ADCC function. For example, see U.S. Patent Publication No. 2003 / 0157108 (Presta, L.) and U.S. Patent Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include U.S. Patent Publication No. 2003 / 0157108, International Publication No. 2000 / 61739, International Publication No. 2001 / 29246, U.S. Patent Publication No. 2003 / 0115614, U.S. Patent Publication No. 2002 / 0164328, U.S. Patent Publication No. 2004 / 0093621, and U.S. Patent Publication No. 2004 / 0132140. Examples include U.S. Patent Application Publication No. 2004 / 0110704, U.S. Patent Application Publication No. 2004 / 0110282, U.S. Patent Application Publication No. 2004 / 0109865, International Publication No. 2003 / 085119, International Publication No. 2003 / 084570, International Publication No. 2005 / 035586, International Publication No. 2005 / 035778, International Publication No. 2005 / 053742, International Publication No. 2002 / 031140, Okazaki et al. J.Mol.Biol.336:1239-1249 (2004), and Yamane-Ohnuki et al. Biotech.Bioeng.87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986), U.S. Patent Application Publication No. 2003 / 0157108 by Presta, L., and particularly Example 11 of International Publication No. 2004 / 056312 by Adams et al.), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, and knockout CHO cells (e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004), Kanda, Y. et al.). See also al., Biotechnol. Bioeng., 94(4):680-688 (2006), and International Publication No. 2003 / 085107.

[0263] In some embodiments, the antibody moiety has a bisected oligosaccharide, for example, a branched oligosaccharide bound to the Fc region of the antibody that is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in International Publication 2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), and U.S. Patent Application Publication 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide bound to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in International Publication 1997 / 30087 (Patel et al.), International Publication 1998 / 58964 (Raju, S.), and International Publication 1999 / 22764 (Raju, S.).

[0264] g) Fc region variant In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibody moiety to generate Fc region variants. The Fc region variant may include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0265] In some embodiments, Fc fragments possess some, but not all, effector functions, making them desirable candidates for applications where the half-life of the antibody portion in vivo is important, but specific effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxic assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the main cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of a target molecule are described in U.S. Patent No. 5,500,362 (e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (e.g., ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, California), and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wisconsin)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively or additionally, the ADCC activity of the target molecule can be evaluated in vivo, for example, in an animal model, such as the animal model disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in International Publication Nos. 2006 / 029879 and International Publication Nos. 2005 / 100402. To evaluate complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J.Immunol.Methods 202:163 (1996), Cragg, MS et al., Blood 101:1045-1052 (2003), and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life can also be measured using methods known in the art (e.g., Petkova, S B et al., Int'l.Immunol. 18(12):1759-1769 (2006)).

[0266] Antibodies with reduced effector function include antibodies in which one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 are substituted (U.S. Patent No. 6,737,056). Such Fc variants include the so-called "DANA" Fc variant (U.S. Patent No. 7,332,581), in which residues 265 and 297 are substituted with alanine, and other Fc variants having two or more substitutions among the amino acids at positions 265, 269, 270, 297, and 327.

[0267] Certain antibody variants with improved or reduced binding to FcR have been described (see, for example, U.S. Patent No. 6,737,056, International Publication No. 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0268] In some embodiments, the Fc fragment is an IgG1 Fc fragment. In some embodiments, the IgG1 Fc fragment contains the L234A mutation and / or the L235A mutation. In some embodiments, the Fc fragment is an IgG2 or IgG4 Fc fragment. In some embodiments, the Fc fragment is an IgG4 Fc fragment containing the S228P, F234A, and / or L235A mutation.

[0269] In some embodiments, the antibody moiety includes an Fc region having one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333 and / or 334 (residue EU numbering) of the Fc region.

[0270] In some embodiments, modifications are made to the Fc region, as described, for example, in U.S. Patent No. 6,194,551, International Publication No. 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000), resulting in altered (i.e., improved or decreased) C1q binding and / or complement-dependent cytotoxic activity (CDC).

[0271] In some embodiments, antibody partial variants include a variant Fc region containing one or more amino acid substitutions that alter the half-life and / or alter the binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J.Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)) are described in U.S. Patent Application Publication No. 2005 / 0014934 (Hinton et al.). These antibodies include an Fc region having one or more substitutions that alter the binding of the Fc region to FcRn. Such Fc variants include those having substitutions in one or more Fc region residues, for example, those having a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0272] See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent Nos. 5,648,260, 5,624,821, and International Publication No. 94 / 29351 on other examples of Fc region variants.

[0273] h) Cysteine-modified antibody variants In some embodiments, it may be desirable to produce a cysteine-modified antibody moiety, e.g., “thioMAb”, in which one or more residues of the antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues are located in an accessible site of the antibody. By substituting these residues with cysteine, the reactive thiol group is thereby positioned in an accessible site of the antibody and can be used to conjugate the antibody to other parts, e.g., a drug moiety or a linker-drug moiety, to produce an immunoconjugate, as further described herein. In some embodiments, one or more of the following residues, A118 (EU numbering) of the heavy chain and S400 (EU numbering) of the heavy chain Fc region, may be substituted with cysteine. The cysteine-modified antibody moiety may be produced, for example, as described in U.S. Patent No. 7,521,541.

[0274] i) Antibody derivative In some embodiments, the antibody moieties described herein may be further modified to include further non-proteinoid moieties that are known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. This polymer may have any molecular weight and may be branched or unbranched. The number of polymers that bind to an antibody can vary, and if two or more polymers bind, they may be the same or different molecules. In general, the number and / or types of polymers used in derivatization can be determined based on considerations such as the specific properties or functions of the antibody being improved, and whether the antibody derivative will be used for diagnostic purposes under defined conditions, but this is not limiting.

[0275] In some embodiments, the antibody moiety may be further modified to include one or more biologically active proteins, polypeptides, or fragments thereof. “Bioactive” or “biologically active,” when used interchangeably herein, means exhibiting biological activity in the body to perform a particular function. For example, it may mean combination with certain biomolecules such as proteins and DNA, and then promotion or inhibition of the activity of such biomolecules. In some embodiments, biologically active proteins or fragments thereof include proteins and polypeptides administered to patients as active drug substances for the prevention or treatment of a disease or condition, as well as proteins and polypeptides used for diagnostic purposes, such as enzymes used in diagnostic tests or in vitro assays, and proteins and polypeptides such as vaccines administered to patients to prevent disease.

[0276] Morphology of F. TIGIT / PVRIG multispecific construct and morphology of exemplary TIGIT / PVRIG multispecific antibodies The multispecific constructs described herein may take any form, as long as the construct maintains its function of binding to both TIGIT and PVRIG.

[0277] In some embodiments, the TIGIT antibody moiety comprises a full-length antibody containing two heavy chains and two light chains. In some embodiments, the anti-PVRIG antibody moiety is fused to one or two heavy chains of the full-length antibody. In some embodiments, the anti-PVRIG antibody moiety is fused to the N-terminus of one or two heavy chains of the full-length antibody. In some embodiments, the anti-PVRIG antibody moiety is fused to the C-terminus of one or two heavy chains of the full-length antibody. In some embodiments, the anti-PVRIG antibody moiety is fused to one or two light chains of the full-length antibody. In some embodiments, the anti-PVRIG antibody moiety is fused to the N-terminus of one or two light chains of the full-length antibody. In some embodiments, the anti-PVRIG antibody moiety is fused to the C-terminus of one or two light chains of the full-length antibody. In some embodiments, the anti-PVRIG antibody portion is an scFv antibody, such as an scFv antibody containing HCDR1 containing the amino acid sequence of SEQ ID NO: 7, HCDR2 containing the amino acid sequence of SEQ ID NO: 8, HCDR3 containing the amino acid sequence of SEQ ID NO: 9, LCDR1 containing the amino acid sequence of SEQ ID NO: 10, LCDR2 containing the amino acid sequence of SEQ ID NO: 11, and LCDR3 containing the amino acid sequence of SEQ ID NO: 12.

[0278] In some embodiments, the anti-PVRIG antibody moiety comprises a full-length antibody containing two heavy chains and two light chains. In some embodiments, the anti-TIGIT antibody moiety is fused to one or two heavy chains of the full-length antibody. In some embodiments, the anti-TIGIT antibody moiety is fused to the N-terminus of one or two heavy chains of the full-length antibody. In some embodiments, the anti-TIGIT antibody moiety is fused to the C-terminus of one or two heavy chains of the full-length antibody. In some embodiments, the anti-TIGIT antibody moiety is fused to one or two light chains of the full-length antibody. In some embodiments, the anti-TIGIT antibody moiety is fused to the N-terminus of one or two light chains of the full-length antibody. In some embodiments, the anti-TIGIT antibody moiety is fused to the C-terminus of one or two light chains of the full-length antibody. In some embodiments, the anti-TIGIT antibody portion is an scFv antibody, such as an scFv antibody containing HCDR1 containing the amino acid sequence of SEQ ID NO: 1, HCDR2 containing the amino acid sequence of SEQ ID NO: 2, HCDR3 containing the amino acid sequence of SEQ ID NO: 3, LCDR1 containing the amino acid sequence of SEQ ID NO: 4, LCDR2 containing the amino acid sequence of SEQ ID NO: 5, and LCDR3 containing the amino acid sequence of SEQ ID NO: 6.

[0279] In some embodiments, the TIGIT / PVRIG binding constructs disclosed herein include two or more antigen-binding moieties that specifically bind to PVRIG and / or two or more antigen-binding moieties that specifically bind to TIGIT. Typically, in the case of a multispecific antibody, the two or more antigen-binding moieties either have the same variable region (and therefore target the same antigen / epitope) or are completely identical in the variable region and the constant region (if any). For example, an antibody may include two identical PVRIG binding moieties and one TIGIT binding moiety, or one PVRIG binding moiety and two identical TIGIT binding moieties, or two identical PVRIG binding moieties and two identical TIGIT binding moieties. Furthermore, if two PVRIG binding moieties or two TIGIT binding moieties are present, these moieties can take the following formats. That is, (i) the TIGIT binding portion is Fab while the PVRIG binding portion is scFv, or (ii) the TIGIT binding portion is scFv while the PVRIG binding portion is Fab, or (iii) both the TIGIT binding portion and the PVRIG binding portion are Fab.

[0280] In some specific embodiments, the TIGIT / PVRIG conjugation construct includes a TIGIT conjugation portion in Fab format comprising a first heavy chain variable domain (VH1) (VH1-CH1) operably linked to the antibody heavy chain CH1 domain and a first light chain variable domain (VL1) (VL1-CL) operably linked to the antibody light chain constant (CL) domain, and the PVRIG conjugation portion is in scFv format comprising a second heavy chain variable domain (VH2) (VH2-VL2) operably linked to a second light chain variable domain (VL2). The PVRIG conjugation scFv may be located at the N-terminus or C-terminus of the heavy or light chain of the multispecificity construct, and within the scFv, VH2 may be at the N-terminus of VL2, or vice versa. In some embodiments, the domains are operably linked by a linker. In some embodiments, the linker is a polypeptide linker.

[0281] In some specific embodiments, the TIGIT binding portion is a Fab format comprising a first heavy chain variable domain (VH1) operably linked to the antibody heavy chain CH1 domain (VH1-CH1) and a first light chain variable domain (VL1) operably linked to the antibody light chain constant (CL) domain (VL1-CL), and the PVRIG binding portion is an scFv format comprising a second heavy chain variable domain (VH2) operably linked to a second light chain variable domain (VL2) (VH2-VL2). In some embodiments, the PVRIG-binding scFv is located at the C-terminus of the heavy chain of the multispecificity construct, and within the scFv, VH2 may be at the N-terminus of VL2, or vice versa. In some embodiments, the domains are operably linked by a linker. In some embodiments, the linker is a polypeptide linker.

[0282] In some embodiments, PVRIG scFv includes (and in some embodiments consists of) the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least about 85%, 90%, or 95% sequence identity (preferably at least 90%, more preferably at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%)). In some embodiments, PVRIG scFv includes the amino acid sequence of SEQ ID NO: 24. In some embodiments, PVRIG scFv is the amino acid sequence of SEQ ID NO: 24.

[0283] In some embodiments, the PVRIG-bound scFv is located at the C-terminus of the heavy chain of the multispecific construct, and within the scFv, VH2 is located at the N-terminus of VL2 (VH2-VL2). In some embodiments, the multispecific construct includes (and in some embodiments consists of) the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 85%, 90%, or 95% sequence identity (preferably at least 90%, more preferably at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%)). In some embodiments, the construct includes the amino acid sequence of SEQ ID NO: 17. In some embodiments, the construct is the amino acid sequence of SEQ ID NO: 17.

[0284] In some embodiments, the VL1-CL1 of the TIGIT binding moiety includes (and in some embodiments consists of) the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least about 85%, 90%, or 95% sequence identity (preferably at least 90%, more preferably at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%)). In some embodiments, the construct includes the amino acid sequence of SEQ ID NO: 18. In some embodiments, the construct is the amino acid sequence of SEQ ID NO: 18.

[0285] In some specific embodiments, the TIGIT binding portion is an scFv format comprising a first VH operably linked to a first VL (VH1-VL1), and the PVRIG binding portion is a Fab format comprising a second VH operably linked to an antibody heavy chain CH1 domain (VH2-CH1) and a second VL operably linked to an antibody light chain constant (CL) domain (VL2-CL). The TIGIT-bound scFv may be located at the N-terminus or C-terminus of the heavy or light chain of the multispecificity construct, and within the scFv, VH1 may be at the N-terminus of VL1, or vice versa. In some embodiments, the domains are operably linked by a linker. In some embodiments, the linker is a polypeptide linker.

[0286] In some specific embodiments, the TIGIT binding portion is an scFv format including a first VH operably linked to a first VL (VH1-VL1), and the PVRIG binding portion is a Fab format including a second VH operably linked to an antibody heavy chain CH1 domain (VH2-CH1) and a second VL operably linked to an antibody light chain constant (CL) domain (VL2-CL). In some embodiments, the TIGIT-binding scFv is located at the N-terminus of the heavy chain of the multispecificity construct, and within the scFv, VH1 may be at the N-terminus of VL1, or vice versa. In some specific embodiments, the TIGIT binding portion is an scFv format including a first VH operably linked to a first VL (VH1-VL1), and the PVRIG binding portion is a Fab format including a second VH operably linked to an antibody heavy chain CH1 domain (VH2-CH1) and a second VL operably linked to an antibody light chain constant (CL) domain (VL2-CL).

[0287] In some specific embodiments, the TIGIT binding portion is an scFv format comprising a first VH operably linked to a first VL (VH1-VL1), and the PVRIG binding portion is a Fab format comprising a second VH operably linked to an antibody heavy chain CH1 domain (VH2-CH1) and a second VL operably linked to an antibody light chain constant (CL) domain (VL2-CL). In some embodiments, the TIGIT binding scFv is located at the N-terminus of the heavy chain of the multispecificity construct. In some embodiments, VH1 may be at the N-terminus of VL1 within the TIGIT binding scFv.

[0288] In some embodiments, the TIGIT-bound scFv is located at the C-terminus of the heavy chain of the multispecific construct, and within the scFv, VH2 is the N-terminus of VL2 (VH2-VL2). In some embodiments, the TIGIT-bound scFv includes (and in some embodiments consists of) the amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least about 85%, 90%, or 95% sequence identity (preferably at least 90%, more preferably at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%)). In some embodiments, the TIGIT-bound scFv contains the amino acid sequence of SEQ ID NO: 23. In some embodiments, the TIGIT-bound scFv is the amino acid sequence of SEQ ID NO: 23.

[0289] In some specific embodiments, the TIGIT binding portion is an scFv format including a first VH operably linked to a first VL (VH1-VL1), and the PVRIG binding portion is a Fab format including a second VH operably linked to an antibody heavy chain CH1 domain (VH2-CH1) and a second VL operably linked to an antibody light chain constant (CL) domain (VL2-CL). In some embodiments, the TIGIT binding portion is an scFv format comprising a first VH operably linked to a first VL (VH1-VL1), and the PVRIG binding portion is a Fab format comprising a second VH operably linked to an antibody heavy chain CH1 domain (VH2-CH1), comprising (and in some embodiments comprising) the amino acid sequence of SEQ ID NO: 19, or a variant thereof having at least about 85%, 90%, or 95% sequence identity (preferably at least 90%, more preferably at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%)). In some embodiments, the construct comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the construct is the amino acid sequence of SEQ ID NO: 19.

[0290] In some embodiments, a second VL operably linked to the antibody light chain constant (CL) domain (VL2-CL) includes (and in some embodiments consists of) the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least about 85%, 90%, or 95% sequence identity (preferably at least 90%, more preferably at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%)). In some embodiments, the VL2-CL complex includes the amino acid sequence of SEQ ID NO: 20. In some embodiments, VL2-CL includes the amino acid sequence of SEQ ID NO: 20.

[0291] The percentage of identity between two amino acid sequences can be determined using the E. Meyers and W. Miller algorithm (Comput.Appl.Biosci.,4:11-17(1988)) incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, gap length penalty 12, and gap penalty 4. Furthermore, the percentage of identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J.Mol.Biol.48:444-453(1970)) incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com), using either the Blossum62 matrix or the PAM250 matrix, gap weights 16, 14, 12, 10, 8, 6, or 4, and length weights 1, 2, 3, 4, 5, or 6.

[0292] Additionally or alternatively, the protein sequences of this disclosure can be used as "query sequences" to perform searches against public databases, for example, to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J.MoI. Biol. 215:403-10. The BLAST protein search can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the antibody molecules of this disclosure. To obtain gapped alignments for comparative purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When using the BLAST program and the Gapped BLAST program, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0293] As an example, the construct disclosed herein may comprise two heavy chains and two light chains, wherein the TIGIT binding portion is in Fab format and the PVRIG binding portion is in scFv format, wherein, from the N-terminus to the C-terminus, (a) the first and second heavy chains each comprise an operably linked domain such as VH1-CH1-hinge-Fc-scFv and the first and second light chains each comprise an operably linked domain such as VL1-CL, or (b) the first and second heavy chains each comprise an operably linked domain such as scFv-VH1-CH1-hinge-Fc and the first and second light chains each comprise an operably linked domain such as VL1-CL, wherein VH1-CH1 and VL1-CL originate from the TIGIT binding portion and scFv (from the N-terminus to the C-terminus, VH2-VL2 or VL2-VH2) originates from the PVRIG binding portion.

[0294] As another example, the construct disclosed herein may comprise two heavy chains and two light chains, where the TIGIT binding portion is in scFv format and the PVRIG binding portion is in Fab format, where (a) the first and second heavy chains each comprise an operably linked domain such as VH2-CH1-hinge-Fc-scFv and the first and second light chains each comprise an operably linked domain such as VL2-CL, from the N-terminus to the C-terminus, or (b) the first and second heavy chains each comprise an operably linked domain such as scFv-VH2-CH1-hinge-Fc and the first and second light chains each comprise an operably linked domain such as VL2-CL, where scFv (VH1-VL1 or VL1-VH1, from the N-terminus to the C-terminus) is derived from the TIGIT binding portion and VH2-CH1 and VL2-CL are derived from the PVRIG binding portion.

[0295] In some specific embodiments, the TIGIT binding portion is a Fab format comprising a first VH (VH1-CH1) operably linked to the antibody heavy chain CH1 domain and a first VL (VL1-CL) operably linked to the antibody light chain constant (CL) domain, and the PVRIG binding portion is also a Fab format comprising a second heavy chain variable domain (VH2-CH1) operably linked to the antibody heavy chain CH1 domain and a second VL (VL2-CL) operably linked to the antibody light chain constant (CL) domain. The TIGIT binding portion may be located at the N-terminus of the Fc region and the PVRIG binding portion at the C-terminus of the Fc region, or the TIGIT binding portion may be located at the C-terminus of the Fc region and the PVRIG binding portion at the N-terminus of the Fc region. In other embodiments, the Fc region is located between the TIGIT binding portion and the PVRIG binding portion. In some other embodiments, both the TIGIT binding portion and the PVRIG binding portion are located at the N-terminus of the Fc region. For dimerization stability, the TIGIT-binding Fab and the PVRIG-binding Fab can be distinguished by substituting the CH1 and CL domains of the TIGIT-binding or PVRIG-binding domain with a pair of TCR constant regions. Therefore, in some embodiments, the CH1 domain is substituted with a first TCR constant region (C1, i.e., the wild-type or manipulated TCR beta-chain constant region), and the CL domain is substituted with a second TCR constant region (C2, i.e., the wild-type or manipulated TCR alpha-chain constant region). Alternatively, they may be swapped to form the TCR beta-chain constant region in the light chain and the TCR alpha-chain constant region in the heavy chain.

[0296] The T cell receptor (TCR) is a heterodimeric T cell surface protein belonging to the immunoglobulin superfamily, similar to a semi-antibody having a single heavy chain and a single light chain. Natural TCRs have an extracellular portion, a transmembrane portion, and an intracellular portion. The extracellular domain of the TCR has a membrane-proximal constant region and a membrane-distal variable region. The introduction of the TCR constant region to replace commonly used CH1 and CL domains has been shown to increase the stability and expression levels of the resulting antibody format. A detailed description of the usefulness of the TCR constant region in assembling two parental antibodies into a multispecific molecule with desired titer and functionality is disclosed in International Publication No. 2019 / 057122, which is incorporated herein by reference in its entirety. Substitution with the TCR constant region results in a chimeric Fab with a unique light-heavy chain interface orthogonal to that of a normal antibody Fab. The assembly of chimeric Fabs of different formats and normal Fabs can create a variety of multispecific molecules with different structures and titers.

[0297] A pair of TCR constant regions in a chimeric Fab includes a TCR alpha constant region in the light chain (wild-type or preferably manipulated) and a TCR beta constant region in the heavy chain (wild-type or preferably manipulated). In some embodiments, the chimeric Fab includes a first TCR constant region and a second TCR constant region linked via a non-natural interchain disulfide bond. The TCR constant regions may be manipulated to introduce a non-natural disulfide bond at the light-heavy chain interface. In some other embodiments, the chimeric Fab includes a pair of wild-type TCR constant regions, e.g., wild-type human TCR beta and alpha constant regions.

[0298] The sequences of the constant regions of the wild-type human TCR beta and alpha chains can be found at NCBI accession numbers A0A5B9 (www.uniprot.org / uniprot / A0A5B9) and P01848 (www.uniprot.org / uniprot / P01848). The pair of TCR constant regions used to construct multispecific antibodies in this specification are derived from wild-type TCR constant regions and have one or more substitutions, additions, or deletions of one or more amino acids. The multispecific antibody may also include the manipulated TCR beta chain constant region and the manipulated TCR alpha chain constant region (referred to herein as C1 and C2). As illustrated in this application, the multispecific antibody comprises a manipulated TCR beta chain constant region (C1) having the sequence shown in SEQ ID NO: 31 (LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGR) and SEQ ID NO: 32

[0299] The modified TCR alpha chain constant region (C2) having the sequence shown in (PDIQNPDCAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPECTFFPS) may also include. Multiple TCR constant region variants for constructing multispecific antibody formats are disclosed in PCT / CN2021 / 072601, the entire contents of which are incorporated herein by reference.

[0300] As an example, the constructs disclosed herein comprise two heavy chains and four light chains, wherein the TIGIT binding portion is in Fab format and the PVRIG binding portion is in Fab format, wherein, from the N-terminus to the C-terminus, the first and second heavy chains each comprise operably linked domains such as VH1-C1-VH2-CH1-hinge-Fc or VH2-CH1-VH1-C1-hinge-Fc (where VH1-C1 is derived from the TIGIT binding portion and VH2-CH1 is derived from the PVRIG binding portion), two light chains comprise operably linked domains such as VL1-C2 (where VL1-C2 is derived from the TIGIT binding portion), and two other light chains comprise operably linked domains such as VL2-CL (where VL2-CL is derived from the PVRIG binding portion). In some embodiments, the domains are operably linked b...

Claims

1. A method for treating cancer in an individual, comprising administering to the individual: 1) a construct comprising an effective amount of a TIGIT binding moiety and a PVRIG binding moiety; and 2) an effective amount of a KRAS G12C inhibitor.

2. The method according to claim 1, wherein the construct comprises a multispecific antibody including a TIGIT antibody portion and a PVRIG antibody portion, and optionally the construct comprises a bispecific antibody.

3. The method according to claim 1 or claim 2, wherein the TIGIT bonding portion blocks the bonding between TIGIT and CD155, and the PVRIG bonding portion blocks the bonding between PVRIG and PVRL2.

4. The method according to any one of claims 1 to 3, wherein the TIGIT is human TIGIT.

5. The method according to any one of claims 1 to 4, wherein the PVRIG is a human PVRIG.

6. The method according to any one of claims 1 to 5, wherein the TIGIT coupling portion and the PVRIG coupling portion are either Fab format or scFv format.

7. The TIGIT merging portion is in Fab format and the PVRIG merging portion is in scFv format, or The TIGIT merging portion is in scFv format and the PVRIG merging portion is in Fab format, or The method according to claim 6, wherein both the TIGIT junction and the PVRIG junction are in Fab format.

8. The method according to any one of claims 1 to 7, wherein the construct further comprises an immunoglobulin constant region, for example, an IgG constant region, for example, a human IgG1, IgG4, IgG2, IgG3 Fc region or a variant thereof.

9. The method according to claim 8, wherein the human IgG Fc region is a human IgG1 Fc region or a variant thereof.

10. The method according to claim 8 or 9, wherein the mutant comprises one or more substitutions for regulating receptor binding or effector function, promoting dimerization, preventing glycosylation, and / or extending its half-life.

11. The aforementioned structure, (a) The TIGIT coupling portion operably connected to the Fc region, and the Fc region operably connected to the PVRIG coupling portion, or (b) The TIGIT coupling portion operably connected to the PVRIG coupling portion, and the PVRIG coupling portion operably connected to the Fc region, (c) The method according to any one of claims 1 to 10, comprising a PVRIG coupling portion operably connected to the TIGIT coupling portion, and a TIGIT coupling portion operably connected to the Fc region.

12. The method according to any one of claims 1 to 11, wherein the structure comprises one, two or more TIGIT bonding portions and one, two or more PVRIG bonding portions.

13. The method according to any one of claims 1 to 12, wherein the structure includes identical or different TIGIT coupling portions and / or identical or different PVRIG coupling portions.

14. The method according to any one of claims 1 to 13, wherein the construct comprises a full-length TIGIT antibody comprising two heavy chains and two light chains, and an anti-PVRIG scFv, wherein the anti-PVRIG scFv is fused to the N-terminus or C-terminus of the two heavy chains of the full-length TIGIT antibody, and optionally the anti-PVRIG scFv is fused to the N-terminus or C-terminus of the two heavy chains of the full-length TIGIT antibody via a linker, and optionally the linker is a peptide linker, and optionally the linker is a GS linker.

15. The method according to any one of claims 1 to 13, wherein the construct comprises a full-length PVRIG antibody comprising two heavy chains and two light chains, and an anti-TIGIT scFv, wherein the anti-TIGIT scFv is fused to the N-terminus or C-terminus of the two heavy chains of the full-length PVRIG antibody, optionally, the anti-TIGIT scFv is fused to the N-terminus or C-terminus of the two heavy chains of the full-length PVRIG antibody via a linker, optionally, the linker is a peptide linker, and optionally, the linker is a GS linker.

16. The TIGIT binding portion comprises an antibody portion comprising a heavy chain variable region (VH) containing heavy chain CDR (HCDR) 1, HCDR 2, and HCDR 3, and a light chain variable region (VL) containing light chain CDR (LCDR) 1, LCDR 2, and LCDR 3. The method according to any one of claims 1 to 15, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 1; HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 2; HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 3; LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 4; LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 5; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 6, and optionally the substitutions are conservative substitutions.

17. The method according to claim 16, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO:

6.

18. The PVRIG binding portion comprises an antibody portion comprising VH containing HCDR1, HCDR2, and HCDR3, and VL containing LCDR1, LCDR2, and LCDR3. The method according to any one of claims 1 to 17, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 7; HCDR2 comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 8; HCDR3 comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 9; LCDR1 comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 10; LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 11; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 12, and optionally the substitutions are conservative substitutions.

19. The method according to claim 18, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, HCDR2 comprises the amino acid sequence of SEQ ID NO: 8, HCDR3 comprises the amino acid sequence of SEQ ID NO: 9, LCDR1 comprises the amino acid sequence of SEQ ID NO: 10, LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, and LCDR3 comprises the amino acid sequence of SEQ ID NO:

12.

20. The VH of the TIGIT binding portion includes the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 13, and / or The method according to any one of claims 1 to 19, wherein the VL of the TIGIT binding portion includes the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO:

14.

21. The VH of the PVRIG binding portion includes the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 15, and / or The method according to any one of claims 1 to 20, wherein the VL of the PVRIG binding portion includes the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO:

16.

22. The aforementioned structure, (i) First and second heavy chains including SEQ ID NO: 17, and first and second light chains including SEQ ID NO: 18, or (ii) The method according to any one of claims 1 to 21, comprising first and second heavy chains including sequence number 19, and first and second light chains including sequence number 20.

23. The method according to any one of claims 1 to 22, wherein the KRAS G12C inhibitor is a small molecule.

24. The method according to claim 23, wherein the KRAS G12C inhibitor is selected from the group consisting of sotrasib, adagrasib, JAB-21822, GDC-6036, JDQ443, D-1553, GH35, GFH925, BPI-421286 and LY3537982, RMC-6291, HBI-2438, BI 1823911, MK-1084 and JNJ-74699157.

25. The aforementioned KRAS G12C inhibitor is of formula (III) 【Chemistry 1】 (In the formula, T1 is selected from O and N. R1 is selected from C6-10 aryls and 5-10 membered heteroaryls, where C6-10 aryls and 5-10 membered heteroaryls are optionally substituted with 1, 2, 3, 4, or 5 Ra atoms. If T1 is O, then R2 does not exist. When T1 is N, R2 is selected from H, C1-3 alkyl, -C(=O)-C1-3 alkyl, and -S(=O)2-C1-3 alkyl, where C1-3 alkyl, -C(=O)-C1-3 alkyl, and -S(=O)2-C1-3 alkyl are optionally substituted with one, two, or three Rb atoms. R3 is a C1-3 alkyl group, where the C1-3 alkyl group is optionally substituted with one, two, or three Rc atoms. R4 is selected from H and C1-3 alkyl groups, where C1-3 alkyl groups are optionally substituted with one, two, or three Rd groups. R5, R6, and R7 are each independently selected from H, F, Cl, Br, I, and C1-3 alkyl groups, where C1-3 alkyl groups are optionally substituted with one, two, or three F atoms. R8 is selected from H and CH3. Ra is independently selected from F, Cl, Br, I, OH, NH2, CN, C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl, where C1-3 alkyl, C1-3 alkoxy, C2-3 alkynyl, and C2-3 alkenyl are optionally substituted with one, two, or three F atoms. Rb is independently selected from F, Cl, Br, I, OH, and NH2. Each Rc is independently selected from 4- to 8-membered heterocycloalkyl groups, where the 4- to 8-membered heterocycloalkyl group is optionally substituted with one, two, or three R atoms. Rd is independently selected from F, Cl, Br, I, OH, NH2, and CN. R is independently selected from H, F, Cl, Br, OH, CN, C1-3 alkyl, C1-3 alkoxy, and -C1-3 alkyl-O-C(=O)-C1-3 alkylamino. However, if R1 is naphthyl, naphthyl can be optionally substituted with F, Cl, Br, OH, NH2, CF3, CH2CH3, and -C≡CH, and R5, R6, and R7 are each independently H. It is represented by or a pharmaceutically acceptable salt thereof. The method according to claim 23, wherein optionally the compound is compound 17.

26. The method according to any one of claims 1 to 25, wherein the construct is a) administered intravenously or subcutaneously and / or b) administered once every two weeks.

27. The method according to any one of claims 1 to 26, wherein the KRAS G12C inhibitor is a) administered orally and / or b) administered daily.

28. The method according to any one of claims 1 to 27, wherein the cancer comprises one or more cancer cells expressing the KRAS G12C mutant protein.

29. The method according to any one of claims 1 to 28, wherein the cancer is colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, stomach cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell carcinoma of the kidney, head and neck cancer, germ cell carcinoma, bone cancer, thyroid cancer, skin cancer, neoplasm of the central nervous system, mesothelioma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma, myeloma, and sarcoma.

30. The method according to any one of claims 1 to 29, wherein the cancer is an advanced, unresectable and / or metastatic solid tumor.

31. The method according to any one of claims 1 to 30, wherein the construct and the KRAS G12C inhibitor are administered simultaneously.

32. The method according to any one of claims 1 to 30, wherein the construct and the KRAS G12C inhibitor are administered simultaneously.

33. The method according to any one of claims 1 to 30, wherein the construct and the KRAS G12C inhibitor are administered sequentially.

34. The method according to any one of claims 1 to 33, further comprising administering an effective amount of the third therapy to the individual.

35. The method according to claim 34, wherein the third therapy comprises another anticancer agent.

36. The method according to claim 35, wherein the anticancer agent is selected from the group consisting of immune checkpoint inhibitors, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, tumor debulking agents, chemotherapeutic agents, antibody-drug conjugates, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic agents.

37. The method according to any one of claims 1 to 36, wherein the individual is a human.

38. The method according to any one of claims 1 to 37, comprising selecting an individual as a target for treatment based on the presence of one or more cancer cells expressing the KRAS G12C mutant protein.

39. A kit for treating cancer in an individual, A kit comprising 1) a construct containing a TIGIT binding site and a PVRIG binding site, and 2) a KRAS G12C inhibitor.

40. The kit according to claim 39, wherein the construct is bound to human PVRIG and / or human TIGIT.

41. The kit according to claim 39 or 40, wherein the construct that binds to TIGIT is an anti-TIGIT antibody or an immunologically active fragment thereof.

42. The kit according to any one of claims 39 to 41, wherein the construct that binds to PVRIG is an anti-PVRIG antibody or an immunologically active fragment thereof.

43. The anti-TIGIT antibody or its immunoactive fragment, (a) (1) CDR-H1 containing SEQ ID NO: 1, (2) CDR-H2 containing SEQ ID NO: 2, and (3) CDR-H3 containing SEQ ID NO: 3 Ｈ and, (b) (1) CDR-L1 containing SEQ ID NO: 4, (2) CDR-L2 containing SEQ ID NO: 5, and (3) CDR-L3 containing SEQ ID NO: 6 Ｌ The kit according to any one of claims 39 to 42, comprising, wherein the sequence of the CDR is defined according to the Kabat numbering system.

44. The anti-PVRIG antibody or its immunoactive fragment is 1. (a) (1) CDR-H1 containing SEQ ID NO: 7, (2) CDR-H2 containing SEQ ID NO: 8, and (3) V containing CDR-H3 containing SEQ ID NO: 9 Ｈ and, 2. (b) (1) CDR-L1 containing SEQ ID NO: 10, (2) CDR-L2 containing SEQ ID NO: 11, and (3) CDR-L3 containing SEQ ID NO: 12 Ｌ The kit according to any one of claims 39 to 43, wherein the sequence of the CDR is defined according to the Kabat numbering system.

45. The V of the anti-TIGIT antibody or its immunologically active fragment Ｈ The domain contains an amino acid sequence having at least 85% identity with SEQ ID NO: 13, and the V of the anti-TIGIT antibody or its immunologically active fragment. Ｌ The kit according to any one of claims 39 to 44, wherein the amino acid sequence comprises an amino acid sequence having at least 85% identity with SEQ ID NO:

14.

46. The V of the anti-TIGIT antibody or its immunologically active fragment Ｈ The domain contains an amino acid sequence having at least 85% identity with SEQ ID NO: 15, and the V of the anti-TIGIT antibody or its immunologically active fragment. Ｌ The kit according to any one of claims 39 to 44, wherein the amino acid sequence comprises an amino acid sequence having at least 85% identity with SEQ ID NO:

16.

47. The aforementioned structure, (i) First and second heavy chains including SEQ ID NO: 17, and first and second light chains including SEQ ID NO: 18, or (ii) The kit according to any one of claims 39 to 46, comprising a first and second heavy chain containing SEQ ID NO: 19, and a first and second light chain containing SEQ ID NO:

20.

48. The kit according to any one of claims 39 to 47, wherein the KRAS G12C inhibitor is an antibody, peptide, protein, antisense oligonucleotide, or small molecule that inhibits the activity of the KRAS G12C mutant protein.

49. The kit according to claim 48, wherein the KRAS G12C inhibitor is a small molecule inhibitor.

50. The kit according to claim 49, wherein the small molecule is selected from the group consisting of compound 17, sotrasib, adaglasib, JAB-21822, GDC-6036, JDQ443, D-1553, GH35, GFH925, BPI-421286 and LY3537982, RMC-6291, HBI-2438, BI 1823911, MK-1084 and JNJ-74699157.

51. The kit according to claim 49, wherein the small molecule is compound 17.

52. The kit according to any one of claims 39 to 51, wherein the cancer comprises one or more cancer cells expressing the KRAS G12C mutant protein.

53. Use of a construct comprising a TIGIT binding moiety and a PVRIG binding moiety for the manufacture of a drug for the treatment of cancer, wherein the treatment is in combination with a KRAS G12C inhibitor.

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