Pharmaceutical composition containing PD-1 / PVRIG / TIGIT binding protein and medical application thereof

Abstract

The invention relates to a pharmaceutical composition containing a PD-1 / PVRIG / TIGIT binding protein and a medical application of the pharmaceutical composition. In particular, the present disclosure relates to pharmaceutical compositions comprising a PD-1 / PVRIG / TIGIT binding protein and a buffering agent. The pharmaceutical composition disclosed by the invention has good biological activity and stability.

Description

A pharmaceutical composition containing PD-1 / PVRIG / TIGIT binding protein and its medical use

[0001] This application claims priority to Chinese patent application No. 202310250609.3 filed on March 15, 2023. Technical Field

[0002] The present disclosure relates to the field of pharmaceutical preparations, and in particular to a pharmaceutical composition comprising a PD-1 / PVRIG / TIGIT binding protein, and its pharmaceutical uses. Background Art

[0003] Immunotherapy is a hot topic in the field of cancer treatment. Immune pathway therapeutic drugs represented by anti-PD-1 antibodies have demonstrated high effectiveness and safety in clinical practice.

[0004] PD-1 (Programmed Cell Death-1) belongs to the CD28 receptor family and is an immunosuppressive receptor (Riley et al. 2009, Immunol. Rev. 29:114-25). This family also includes CD28, CTLA-4, ICOS, PD-1, and BTLA. PD-1 is a type I transmembrane protein with a structure very similar to CTLA-4, but PD-1 lacks the MYPPPY sequence that binds to B7-1 and B7-2. PD-1 is primarily expressed on activated B cells, T cells, and myeloid cells (Chen et al. 2013, Nat. Rev. Immunol. 13:227-42).

[0005] PD-1 has two cell surface glycoprotein ligands, PD ligand 1 (PD-L1, also known as CD274, B7-H1) and PD ligand 2 (PD-L2, also known as B7-DC). Neither PD-L1 nor PD-L2 binds to other CD28 receptor family members. PD-L1 is widely expressed on lymphocytes (such as CD4 + T cells and CD8 + T cells, macrophages, etc.) as well as peripheral tissues, various tumor cells, and virus-infected cells. PD-L2 is primarily expressed on activated dendritic cells and macrophages (Dong et al. 1999, Nat. Med. 5:1365-9). Upon binding to its ligands PD-L1 or PD-L2, PD-1 downregulates T cell function, including reducing T cell activation, differentiation and proliferation, and cytokine secretion.

[0006] PVRIG (Poliovirus receptor-related Ig domain containing protein), also known as CD112R, belongs to the B7 / CD28 superfamily along with TIGIT (T cell immunoglobulin and ITIM domain), CD96 and CD226, and plays an important role in the immune system. When PVRIG's ligand PVRL2 (also known as CD112) binds to PVRIG, it activates the ITIM domain in the intracellular region of PVRIG, causing PVRIG to play an immunosuppressive role. PVRIG is mainly expressed in CD4 + T cells, CD8 + PVRIG and its ligand PVRL2 are highly expressed on the surfaces of T cells and NK cells. PVRIG and its ligand PVRL2 are highly expressed in a variety of solid tumors, including lung, breast, ovarian, renal, gastric, endometrial, and head and neck cancers. PVRIG expression in these cancers is highly correlated with TIGIT and PD-1. Similar to PD-1 and TIGIT, PVRIG-positive T cells are also Eomes-positive and Tbet-negative, suggesting that PVRIG is associated with T cell exhaustion. Therefore, PVRIG may represent a new immune checkpoint in addition to PD-1 and TIGIT, acting as a redundant checkpoint. In vitro cell experiments and mouse models have shown that knockout or inhibition of PVRIG in mice can effectively inhibit cancer growth and synergize with PD-1 and TIGIT inhibitors. TIGIT is highly expressed on lymphocytes, including cancer-infiltrating lymphocytes (TILs) and Tregs that infiltrate various cancer types. It has been shown that the binding of TIGIT to its cognate ligand PVR (also known as CD155) directly inhibits the cytotoxicity of NK cells through its cytoplasmic ITIM domain. PVR is also widely expressed in cancer, indicating that the TIGIT-PVR signaling axis may be the main immune escape mechanism of cancer. The inhibitory receptors TIGIT and PVRIG bind to the same ligands CD155 and CD112 as the activating receptor DNAM-1, but the inhibitory receptor has a higher affinity (Y. Zhu et al. 2016, J Exp Med. 213: 167-176). DNAM-1 expression is downregulated in NK cells isolated from tumor patients, making NK cells more susceptible to inhibition by TIGIT or PVRIG. Blocking the binding of TIGIT and PVRIG can activate the cell killing function of NK cells (L. Martinet et al. 2015, Cell Reports. 11: 85-97).

[0007] Although PD-1 / PD-L1 antibodies are currently the most successful monoclonal immune checkpoint inhibitors in clinical practice, upregulation of other immune checkpoints on the surface of T cells limits their efficacy. In addition to regulating T cell function, TIGIT and PVRIG also regulate NK cell activity, suggesting that they may synergize with the function of PD-1 / PD-L1 in NK cells. Literature reports that high expression of CD155 and CD112, the ligands for TIGIT and PVRIG, in lung cancer, ovarian cancer, colorectal cancer, and melanoma is significantly associated with poor prognosis after PD-1 / PD-L1 treatment. For example, patients with high CD155 expression have a poor response to anti-PD-1 antibodies, while patients with low CD155 expression have a good response to PD-1 (S. Whelan et al. 2019, Cancer Immunol Res. 7:257-268; A. Lepletier et al. 2020, Clin Cancer Res. 26:3671-3681).

[0008] No PVRIG antibodies or anti-PVRIG / TIGIT bispecific antibody drugs have yet to be marketed. Compugen's COM701 is the world's first FDA-approved humanized anti-PVRIG antibody for clinical trials and is currently in Phase I clinical trials for the treatment of cancer. Surface Oncology is also developing the anti-PVRIG antibody SRF-813. Anti-TIGIT antibodies include Genentech's tiragolumab, BMS-986207, developed in collaboration between Ono Pharmaceutical and BMS, Merck's MK-7684, iTeos Therapeutics' EOS-884448, and Arcus Biosciences' AB-154, all in Phase II clinical trials.

[0009] WO2023040945 provides novel anti-PD-1 antibodies and related anti-PD-1 / PVRIG / TIGIT trispecific antibodies, which are expected to enhance T cell and NK cell activation, overcome PD-1 / PD-L1 resistance, and expand the patient population that responds, thereby improving the effectiveness of cancer immunotherapy. Furthermore, the anti-PD-1 / PVRIG / TIGIT trispecific antibodies can simultaneously bind to multiple immune checkpoints on the same cell surface, potentially exhibiting an avidity effect. Therefore, they have the potential to provide better clinical efficacy than existing PD-1, TIGIT, PVRIG antibody drugs, or their combination.

[0010] Antibody drugs have large molecular weights and complex structures, making them susceptible to degradation, aggregation, and undesirable chemical modifications, leading to instability. To ensure that antibodies are suitable for administration and maintain stability during storage and subsequent use, leading to optimal efficacy, research into stable formulations of antibody drugs is crucial. For new PD-1 / PVRIG / TIGIT binding proteins, the development of suitable pharmaceutical compositions remains a challenge.

[0011] Summary of the Invention

[0012] The present disclosure provides a pharmaceutical composition containing a PD-1 / PVRIG / TIGIT binding protein, which has excellent stability.

[0013] The present disclosure provides a pharmaceutical composition comprising a PD-1 / PVRIG / TIGIT binding protein and a buffer, wherein the buffer is selected from a citrate buffer and a histidine salt buffer. In some embodiments, the buffer is a citric acid-sodium citrate buffer. In some embodiments, the buffer is a citric acid-disodium citrate buffer. In some embodiments, the buffer is a histidine salt buffer. In some embodiments, the buffer is a histidine-hydrochloride buffer. In some embodiments, the buffer is a histidine-acetate buffer. In some embodiments, the buffer is a histidine-histidine hydrochloride buffer. In some embodiments, the buffer is a histidine-acetate buffer.

[0014] In some embodiments, the PD-1 / PVRIG / TIGIT binding protein comprises a first antigen binding domain that specifically binds to PD-1, a second antigen binding domain that specifically binds to PVRIG, and a third antigen binding domain that specifically binds to TIGIT, and can specifically bind to PD-1, PVRIG, and TIGIT simultaneously or separately.

[0015] In some embodiments, the aforementioned PD-1 / PVRIG / TIGIT binding protein comprises or is an anti-PD-1 / PVRIG / TIGIT trispecific antibody.

[0016] In some embodiments, each antigen-binding domain in the aforementioned PD-1 / PVRIG / TIGIT binding protein is selected from Fab, Fv, sFv, Fab', F(ab')2, linear antibody, single-chain antibody, scFv, sdAb, sdFv, nanobody, peptibody, domain antibody, or any combination thereof. In specific embodiments, the first antigen-binding domain that specifically binds to PD-1 comprises or is a VHH, the second antigen-binding domain that specifically binds to PVRIG comprises or is a VHH, and the third antigen-binding domain that specifically binds to TIGIT is Fab or F(ab')2.

[0017] In some embodiments, the aforementioned PD-1 / PVRIG / TIGIT binding protein is humanized, affinity matured, T cell epitope removed, antibody deamidation reduced, and / or antibody isomerization reduced.

[0018] In some embodiments, the aforementioned PD-1 / PVRIG / TIGIT binding protein is a recombinant antibody, a camel antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof.

[0019] In some embodiments, in the aforementioned PD-1 / PVRIG / TIGIT binding protein, the first antigen-binding domain that specifically binds to PD-1 comprises (at least one) immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises:

[0020] CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 2, 8-10, 11-29,

[0021] The CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems.

[0022] In some embodiments, according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are:

[0023] As shown in SEQ ID NO: 3, 30, 31.

[0024] In some specific embodiments, the amino acid sequence of CDR1 of the immunoglobulin single variable domain is shown in SEQ ID NO: 3, the amino acid sequence of CDR2 is shown in any one of SEQ ID NOs: 4, 32-34, 79, and the amino acid sequence of CDR3 is shown in any one of SEQ ID NOs: 5, 35, 36.

[0025] In some specific embodiments, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NOs: 3, 32, and 35, respectively.

[0026] In some embodiments, in the aforementioned PD-1 / PVRIG / TIGIT binding protein, the second antigen-binding domain that specifically binds to PVRIG comprises (at least one) immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises:

[0027] CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 38, 46-50, 74-75.

[0028] In some specific embodiments, according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are as shown in SEQ ID NO: 39, 40 and 41, respectively.

[0029] In some embodiments, in the aforementioned PD-1 / PVRIG / TIGIT binding protein, the third antigen-binding domain that specifically binds to TIGIT comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0030] The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NOs: 57, 58 and 59, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID NOs: 60, 61 and 62, respectively.

[0031] In some embodiments, in the aforementioned PD-1 / PVRIG / TIGIT binding protein, the first antigen binding domain that specifically binds to PD-1 comprises:

[0032] An amino acid sequence as shown in any one of SEQ ID NOs: 2, 8-10, 11-29, or having at least 80% or at least 90% sequence identity thereto.

[0033] In some embodiments, in the aforementioned PD-1 / PVRIG / TIGIT binding protein, the second antigen binding domain that specifically binds to PVRIG comprises an amino acid sequence as shown in any one of SEQ ID NOs: 38, 46-50, 74-75, or a sequence having at least 80% or at least 90% sequence identity therewith.

[0034] In some embodiments, in the aforementioned PD-1 / PVRIG / TIGIT binding protein, the third antigen-binding domain that specifically binds to TIGIT comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0035] The heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 63-65, or a sequence having at least 80% or at least 90% identity thereto,

[0036] The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 66 or 67, or an amino acid sequence at least 80% or at least 90% identical thereto.

[0037] In some embodiments, in the aforementioned PD-1 / PVRIG / TIGIT binding protein, the third antigen-binding domain that specifically binds to TIGIT comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0038] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 63 or a sequence having at least 80% or at least 90% identity thereto,

[0039] The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 67 or an amino acid sequence at least 80% or at least 90% identical thereto.

[0040] In some embodiments, the third antigen binding domain that specifically binds to TIGIT comprises a full-length heavy chain (HC) and a full-length light chain (LC).

[0041] For example, the full-length heavy chain is of IgG1, IgG2 or IgG4 isotype, and the full-length light chain is of Kappa isotype;

[0042] For another example, the heavy chain sequence is shown in SEQ ID NO: 51, or an amino acid sequence having at least 80% or at least 90% sequence identity thereto, and the light chain sequence is shown in SEQ ID NO: 52, or an amino acid sequence having at least 80% or at least 90% sequence identity thereto.

[0043] In some embodiments, in the aforementioned PD-1 / PVRIG / TIGIT binding protein, the first antigen binding domain that specifically binds to PD-1, the second antigen binding domain that specifically binds to PVRIG, and the third antigen binding domain that specifically binds to TIGIT are connected directly or through a linker. For example, the linker has a structure such as (G4S) x The amino acid sequence shown, wherein x is independently selected from an integer of 1-20; for another example, the linker is the amino acid sequence shown by (G4S)2, (G4S)3, (G4S)4.

[0044] In some embodiments, the aforementioned PD-1 / PVRIG / TIGIT binding protein has an antigen binding domain that specifically binds to PD-1 located at the N-terminus or C-terminus of the antigen binding domain that specifically binds to PVRIG.

[0045] In some embodiments, the aforementioned PD-1 / PVRIG / TIGIT binding protein, the antigen binding domain that specifically binds to PD-1 is located at the N-terminus or C-terminus of the antigen binding domain that specifically binds to TIGIT.

[0046] In some embodiments, in the aforementioned PD-1 / PVRIG / TIGIT binding protein, the antigen binding domain that specifically binds to PVRIG is located at the N-terminus or C-terminus of the antigen binding domain that specifically binds to TIGIT.

[0047] In some embodiments, the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain are on the same polypeptide chain, or are not on the same polypeptide chain.

[0048] In some embodiments, the aforementioned PD-1 / PVRIG / TIGIT binding protein comprises a first polypeptide chain and a second polypeptide chain, wherein the structure is selected from the following:

[0049] (I) a first polypeptide chain: [first antigen-binding domain that specifically binds to PD-1]-[Linker 1]a-[second antigen-binding domain that specifically binds to PVRIG]-[Linker 2]b-[VH that specifically binds to the third antigen-binding domain of TIGIT]-CH1-Fc, and a second polypeptide chain: [VL that specifically binds to the third antigen-binding domain of TIGIT]-Cκ; or

[0050] (II) a first polypeptide chain: [the second antigen-binding domain that specifically binds to PVRIG]-[Linker 2]b-[VH that specifically binds to the third antigen-binding domain of TIGIT]-CH1-Fc, and a second polypeptide chain: [the first antigen-binding domain that specifically binds to PD-1]-[Linker 3]c-[VL that specifically binds to the third antigen-binding domain of TIGIT]-Cκ; or

[0051] (III) a first polypeptide chain: [the second antigen-binding domain that specifically binds to PVRIG]-[Linker 2]b-[VH that specifically binds to the third antigen-binding domain of TIGIT]-CH1-Fc-[Linker 4]d-[the first antigen-binding domain that specifically binds to PD-1], and a second polypeptide chain: [VL that specifically binds to the third antigen-binding domain of TIGIT]-Cκ; or

[0052] (IV) A first polypeptide chain: [the second antigen-binding domain that specifically binds to PVRIG]-[Linker 2]b-[VH that specifically binds to the third antigen-binding domain of TIGIT]-CH1-Fc, and a second polypeptide chain: [VL that specifically binds to the third antigen-binding domain of TIGIT]-Cκ-[Linker 5]e-[the first antigen-binding domain that specifically binds to PD-1].

[0053] The first and second polypeptide chains are arranged in order from N-terminus to C-terminus.

[0054] Wherein, - represents a peptide bond, the linker is a polypeptide capable of realizing the linking function, linker 1, linker 2, linker 3, linker 4, and linker 5 may be the same or different; a, b, c, d, and e may be optionally and independently 0 or 1.

[0055] For example, each linker is independently EPKSS or (G x S) y A linker, wherein x is selected from an integer of 1-5 (e.g., 1, 2, 3, 4, 5), and y is selected from an integer of 1-6 (e.g., 1, 2, 3, 4, 5, 6).

[0056] When the linker is (G x S) y In the case of a linker, for example, it is any one of (G4S)2, (G4S)3, and (G4S)4.

[0057] In some embodiments, a PD-1 / PVRIG / TIGIT binding protein is provided, comprising a first polypeptide chain and a second polypeptide chain, wherein:

[0058] The first and second polypeptide chains comprise the amino acid sequences shown in SEQ ID NOs: 70 and 52, respectively;

[0059] The first and second polypeptide chains comprise the amino acid sequences shown in SEQ ID NOs: 68 and 71, respectively;

[0060] The first and second polypeptide chains comprise the amino acid sequences shown in SEQ ID NOs: 72 and 52, respectively;

[0061] The first and second polypeptide chains comprise the amino acid sequences shown in SEQ ID NOs: 68 and 73, respectively;

[0062] The first and second polypeptide chains comprise the amino acid sequences shown in SEQ ID NOs: 76 and 52, respectively;

[0063] The first and second polypeptide chains comprise the amino acid sequences shown in SEQ ID NOs: 77 and 52, respectively;

[0064] Or an amino acid sequence combination having at least 80% or at least 90% sequence identity with either the first or second polypeptide chain.

[0065] In some embodiments, the aforementioned PD-1 / PVRIG / TIGIT binding protein of the present disclosure comprises two identical or different first polypeptide chains, and two identical or different second polypeptide chains.

[0066] In some embodiments, the PD-1 / PVRIG / TIGIT binding protein comprises two identical first polypeptide chains and two identical second polypeptide chains.

[0067] In some embodiments, the PD-1 / PVRIG / TIGIT binding protein of the present disclosure has all or any of the properties and functions of the PD-1 / PVRIG / TIGIT binding protein because it comprises the antigen-binding domain of the present disclosure that specifically binds to PD-1.

[0068] The PD-1 / PVRIG / TIGIT binding protein comprises a PVRIG / TIGIT binding domain and has at least one of the following characteristics:

[0069] (a) With a value less than 1×10 -7 M's K D Values ​​bind to human PVRIG;

[0070] (b) blocking the interaction between PVRIG and its ligand (e.g., PVRL2);

[0071] (c) Relieve the inhibitory effect of dendritic cells (DCs) on T cells and activate T cells;

[0072] (d) Eliminate the inhibitory effect of tumor cells on NK cells.

[0073] In some embodiments, the aforementioned PD-1 / PVRIG / TIGIT binding protein disclosed herein is capable of inhibiting tumor growth by at least about 10%, for example, at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.

[0074] In some embodiments, the aforementioned PD-1 / PVRIG / TIGIT binding proteins disclosed herein encompass variants, which have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations compared to any combination of the aforementioned first and second polypeptide chains; the amino acid mutations may be conservative replacements, substitutions, or modifications, and / or deletions or additions that do not affect function. The mutations may occur in the antigen-binding domain that specifically binds to PD-1, the antigen-binding domain that specifically binds to PVRIG, or the antigen-binding domain that specifically binds to TIGIT (e.g., CDR regions and / or FR regions).

[0075] In some embodiments, a PD-1 / PVRIG / TIGIT binding protein is provided, which binds to or competes for binding to the same PD-1, PVRIG and / or TIGIT or an epitope thereof as the aforementioned PD-1 / PVRIG / TIGIT binding protein of the present disclosure; or blocks the binding of the aforementioned PD-1 / PVRIG / TIGIT binding protein of the present disclosure to PD-1, PVRIG and / or TIGIT; or its binding to PD-1, PVRIG and / or TIGIT is blocked by the aforementioned PD-1 / PVRIG / TIGIT binding protein of the present disclosure.

[0076] The present disclosure relates to PVRIG-binding proteins or antigen-binding domains that specifically bind to PVRIG, which may comprise or be selected from the PVRIG antibodies described in WO2016134333, WO2016134335, WO2018033798, WO2018220446, WO2019079777, WO2019232484, WO2020018879, WO2021021837, WO2021097294, WO2021091605, and WO2021113831. For example, the PVRIG antibody may be any one of CPA.7.002, CPA.7.005, CPA.7.021, and CPA.7.050 (see WO2016134333). The aforementioned patents are incorporated herein in their entirety.

[0077] In the present disclosure, the TIGIT binding protein or the antigen binding domain that specifically binds to TIGIT may comprise or be selected from the TIGIT antibodies in WO2019062832, WO2009126688, WO2014089113, WO2015009856, WO2015143343, WO2015174439, WO2016028656, WO2016106302, WO2017053748, WO2017030823, US20160176963, US20130251720, WO2019232484, and WO2019062832. For example, the TIGIT antibody can be any one of CPA.9.083.H4 (S241P), CPA.9.086.H4 (S241P), CHA.9.547.7.H4 (S241P), and CHA.9.547.13.H4 (S241P) (see WO2019232484). The above patents are incorporated herein in their entirety.

[0078] In some embodiments, the concentration of the PD-1 / PVRIG / TIGIT binding protein in the pharmaceutical composition is 0.01 mg / mL - 500 mg / mL, such as 0.05 mg / mL - 450 mg / mL, 0.05 mg / mL - 400 mg / mL, 0.05 mg / mL - 350 mg / mL, 0.05 mg / mL - 300 mg / mL, 0.05 mg / mL - 250 mg / mL, 0.05 mg / mL - 200 mg / mL, 0.05 mg / mL - 150 mg / mL, 0.05 mg / mL - 140 mg / mL, 0.05 mg / mL - 130 mg / mL, 0.05 mg / mL - 120 mg / mL, 0.05 mg / mL - 110 mg / mL, 0.05 mg / mL - 100 mg / mL, 0.1 mg / mL - 400 mg / mL, 0.1 mg / mL - 350 mg / mL, 0.1 mg / mL - 300 mg / mL, 0.1 mg / mL - 250 mg / mL, 0.1 mg / mL - 200 mg / mL, 0.1 mg / mL - 150 mg / mL, 0.1 mg / mL - 140 mg / mL, 0.1 mg / mL - 130 mg / mL, 0.1 mg / mL - 120 mg / mL, 0.1 mg / mL - 110 mg / mL, 0.1 mg / mL - 100 mg / mL, 0.5 mg / mL - 350 mg / mL, 0.5 mg / mL - 300 mg / mL, 0.5 mg / mL - 250 mg / mL, 0.5 mg / mL - 200 mg / mL, 0.5 mg / mL - 150 mg / mL, 0.5 mg / mL - 140 mg / mL, 0.5 mg / mL - 130 mg / mL, 0.5 mg / mL - 120 mg / mL, 0.5 mg / mL - 110 mg / mL, 0.5 mg / mL - 100 mg / mL, 1 mg / mL - 300 mg / mL, 1 mg / mL - 250 mg / mL, 1 mg / mL - 200 mg / mL, 1 mg / mL - 150 mg / mL, 1 mg / mL - 140 mg / mL, 1 mg / mL - 130 mg / mL, 1 mg / mL - 120 mg / mL, 1 mg / mL - 110 mg / mL, 1 mg / mL - 100 mg / mL, 1 mg / mL - 95 mg / mL, 1 mg / mL - 90 mg / mL, 1 mg / mL - 85 mg / mL, 1 mg / mL - 80 mg / mL, 1 mg / mL - 75 mg / mL, 1 mg / mL - 70 mg / mL, 1 mg / mL - 65 mg / mL, 1 mg / mL - 60 mg / mL, 1 mg / mL - 55 mg / mL, 1 mg / mL - 50 mg / mL, 1 mg / mL - 45 mg / mL, 1 mg / mL - 40 mg / mL,1mg / mL-35mg / mL、1mg / mL-30mg / mL、1mg / mL-25mg / mL、1mg / mL-20mg / mL、1mg / mL-15mg / mL、5mg / mL-90mg / mL、5mg / mL-85mg / mL、5mg / mL-80mg / mL、5mg / mL-75mg / mL、5mg / mL-70mg / mL、5mg / mL-65mg / mL、5mg / mL-60mg / mL、5mg / mL-55mg / mL、5mg / mL-50mg / mL、 5mg / mL-45mg / mL、5mg / mL-40mg / mL、5mg / mL-35mg / mL、5mg / mL-30mg / mL、5mg / mL-25mg / mL、5mg / mL-20mg / mL、5mg / mL-15mg / mL、8mg / mL-12mg / mL、30mg / mL-250mg / mL、30mg / mL-200mg / mL、30mg / mL-190mg / mL、30mg / mL-180mg / mL、30mg / mL-170mg / mL、30mg / mL-160mg / mL、30mg / mL-150mg / mL、30mg / mL-140mg / mL、30mg / mL-130mg / mL、30mg / mL-120mg / mL、30mg / mL-110mg / mL、30mg / mL-100mg / mL、50mg / mL-200mg / mL、50mg / mL-190mg / mL、50mg / mL-180mg / mL、50mg / mL-170mg / mL、50mg / mL-160mg / mL、50mg / mL-150mg / mL、50mg / mL-140mg / mL、50mg / mL-130mg / mL、50mg / mL-120mg / mL、50mg / mL-110mg / mL、50mg / mL-100mg / mL、70mg / mL-180mg / mL、70mg / mL-170mg / mL、70mg / mL-160mg / mL、70mg / mL-150mg / mL、70mg / mL-140mg / mL、70mg / mL-130mg / mL、70mg / mL-120mg / mL、70mg / mL-110mg / mL、70mg / mL-100mg / mL、90mg / mL-110mg / mL、95 mg / mL-105 mg / mL. In some embodiments, the concentration of the PD-1 / PVRIG / TIGIT binding protein in the pharmaceutical composition is 0.1 mg / mL-400 mg / mL. In some embodiments, the concentration of the PD-1 / PVRIG / TIGIT binding protein in the pharmaceutical composition is 0.5 mg / mL-200 mg / mL. In some embodiments, the concentration of the PD-1 / PVRIG / TIGIT binding protein in the pharmaceutical composition is 1 mg / mL-150 mg / mL. In some embodiments, the concentration of the PD-1 / PVRIG / TIGIT binding protein in the pharmaceutical composition is 5 mg / mL-45 mg / mL. In some embodiments, the concentration of the PD-1 / PVRIG / TIGIT binding protein in the pharmaceutical composition is 50 mg / mL-110 mg / mL. In some embodiments, the concentration of the PD-1 / PVRIG / TIGIT binding protein in the pharmaceutical composition is about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, or about 150 mg / mL. In some embodiments, the concentration of the PD-1 / PVRIG / TIGIT binding protein in the pharmaceutical composition is about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, about 130 mg / mL, or about 150 mg / mL. In some embodiments, the concentration of the PD-1 / PVRIG / TIGIT binding protein in the pharmaceutical composition is 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, or about 100 mg / mL. In some embodiments, the concentration of the PD-1 / PVRIG / TIGIT binding protein in the pharmaceutical composition is about 120 mg / mL, about 130 mg / mL, or about 150 mg / mL. In some embodiments, the concentration of the PD-1 / PVRIG / TIGIT binding protein in the pharmaceutical composition is at least 50 mg / mL, at least 100 mg / mL, at least 200 mg / mL, at least 400 mg / mL, or at least 500 mg / mL.

[0079] In some embodiments, the concentration of the buffer in the pharmaceutical composition is 0.1 mM-50 mM, e.g., 0.1 mM-45 mM, 0.1 mM-40 mM, 0.1 mM-35 mM, 0.1 mM-30 mM, 0.1 mM-25 mM, 0.1 mM-20 mM, 0.1 mM-15 mM, 0.1 mM-10 mM, 0.5 mM-45 mM, 0.5 mM-40 mM, 0.5mM-35mM, 0.5mM-30mM, 0.5mM-25mM, 0.5mM-20mM, 0.5mM-15mM, 0.5mM-10mM, 1mM-40mM, 1mM-35mM, 1mM-30mM, 1mM-25mM, 1mM-20mM, 1mM -15mM, 1mM-10mM, 5mM-35mM, 5mM-30mM, 5mM-25mM, 5mM-20mM, 5mM-15mM, 5mM-10mM, 8mM-30mM, 8mM-25mM, 8mM-20mM, 8mM-15mM, 8mM-12mM. In some embodiments, the concentration of the buffer in the pharmaceutical composition is 0.5 mM-40 mM. In some embodiments, the concentration of the buffer in the pharmaceutical composition is 1 mM-30 mM. In some embodiments, the concentration of the buffer in the pharmaceutical composition is 5 mM-20 mM. In some embodiments, the concentration of the buffer in the pharmaceutical composition is about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM. In some embodiments, the concentration of the buffer in the pharmaceutical composition is about 10 mM. In some embodiments, the concentration of the buffer in the pharmaceutical composition is at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, or at least 50 mM.

[0080] In some embodiments, the pharmaceutical composition further comprises a surfactant. In some embodiments, the surfactant is an ionic or nonionic surfactant. In some embodiments, the surfactant is selected from polysorbate, polyhydroxyalkylene, Triton, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauroyl One or more of the copolymer of aminopropyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitamidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, methyl cocoyl sodium, methyl oleyl taurate sodium, polyethylene glycol, polypropylene glycol and ethylene and propylene glycol. In some embodiments, the surfactant is selected from polysorbate. In some embodiments, the surfactant is selected from polysorbate 20 and / or polysorbate 80. In some embodiments, the surfactant is polysorbate 80.

[0081] In some embodiments, the concentration of the surfactant in the pharmaceutical composition is 0.01 mg / mL-10 mg / mL, for example, 0.01 mg / mL-9 mg / mL, 0.01 mg / mL-8 mg / mL, 0.01 mg / mL-7 mg / mL, 0.01 mg / mL-6 mg / mL, 0.01 mg / mL-5 mg / mL, 0.01 mg / mL-4 mg / mL, 0.01 mg / mL-3 mg / mL, 0.01 mg / mL-2 mg / mL, 0.01 mg / mL-1 mg / mL, 0.01 mg / mL-0.5 mg / mL, 0.05 mg / mL-8 mg / mL, 0.05 mg / mL-7 mg / mL, 0.05 mg / mL-6 mg / mL, 0.05 mg / mL -5mg / mL, 0.05mg / mL-4mg / mL, 0.05mg / mL-3mg / mL, 0.05mg / mL-2mg / mL, 0.05mg / mL-1mg / mL, 0.05mg / mL-0.5mg / mL, 0.1mg / mL-6mg / mL, 0.1mg / mL-5mg / mL, 0.1mg / mL-4m g / mL, 0.1mg / mL-3mg / mL, 0.1mg / mL-2mg / mL, 0.1mg / mL-1mg / mL, 0.1mg / mL-0.5mg / mL, 0.2mg / mL-5mg / mL, 0.2mg / mL-4.5mg / mL, 0.2mg / mL-4mg / mL, 0.2mg / mL-3.5mg / mL, 0.2mg / mL-3mg / mL, 0.2mg / mL-2.5mg / mL, 0.2mg / mL-2mg / mL, 0.2mg / mL-1.5mg / mL, 0.2mg / mL-1mg / mL, 0.2mg / mL-0 .5mg / mL, 0.3mg / mL-4.5mg / mL, 0.3mg / mL-4mg / mL, 0.3mg / mL-3.5mg / mL, 0.3mg / mL-3mg / mL, 0.3mg / mL-2.5mg / mL, In some embodiments, the concentration of the surfactant in the pharmaceutical composition is 0.05 mg / mL to 5 mg / mL.In some embodiments, the concentration of the surfactant in the pharmaceutical composition is 0.1 mg / mL to 3 mg / mL. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is 0.2 mg / mL to 2 mg / mL. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1 mg / mL, about 1.1 mg / mL, about 1.2 mg / mL, about 1.3 mg / mL, about 1.4 mg / mL, about 1.5 mg / mL, about 1.6 mg / mL, about 1.7 mg / mL, about 1.8 mg / mL, about 1.9 mg / mL, or about 2 mg / mL. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is about 0.2 mg / mL, about 0.4 mg / mL, about 0.6 mg / mL, or about 0.8 mg / mL. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is about 0.4 mg / mL or about 0.8 mg / mL. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is at least 0.4 mg / mL, at least 0.8 mg / mL, at least 2 mg / mL, at least 3 mg / mL, at least 5 mg / mL, or at least 10 mg / mL.

[0082] In some embodiments, the pharmaceutical composition further comprises a carbohydrate. In some embodiments, the carbohydrate is selected from one or more of sucrose, glucose, trehalose, and maltose. In some embodiments, the carbohydrate is sucrose.

[0083] In some embodiments, the concentration of carbohydrates in the pharmaceutical composition is 0.1% w / v-20% w / v, for example, 0.1% w / v-15% w / v, 0.1% w / v-14% w / v, 0.1% w / v-13% w / v, 0.1% w / v-12% w / v, 0.1% w / v-11% w / v, 0.1% w / v-10% w / v, 0.1% w / v-9.5% w / v, 0.1% w / v-9% w / v, 0.1% w / v-8.5% w / v, 0.1 %w / v-8%w / v, 1%w / v-15%w / v, 1%w / v-14%w / v, 1%w / v-13%w / v, 1%w / v-12%w / v, 1%w / v-11%w / v, 1%w / v-10%w / v , 1%w / v-9.5%w / v, 1%w / v-9%w / v, 1%w / v-8.5%w / v, 1%w / v-8%w / v, 2%w / v-13%w / v, 2%w / v-12%w / v, 2%w / v-11%w / v, 2% w / v-10% w / v, 2% w / v-9.5% w / v, 2% w / v-9% w / v, 2% w / v-8.5% w / v, 2% w / v-8% w / v, 3% w / v-12% w / v, 3% w / v-11% w / v, 3% w / v-10% w / v, 3% w / v-9.5% w / v, 3% w / v-9% w / v, 3% w / v-8.5% w / v, 3% w / v-8% w / v, 5% w / v-11% w / v, 5% w / v In some embodiments, the concentration of carbohydrates in the pharmaceutical composition is between 1% w / v and 15% w / v. In some embodiments, the concentration of carbohydrates in the pharmaceutical composition is between 3% w / v and 12% w / v. In some embodiments, the concentration of carbohydrates in the pharmaceutical composition is 5% w / v to 10% w / v. In some embodiments, the concentration of carbohydrates in the pharmaceutical composition is about 3% w / v, about 3.5% w / v, about 4% w / v, about 4.5% w / v, about 5% w / v, about 5.5% w / v, about 6% w / v, about 6.5% w / v, about 7% w / v, about 7.5% w / v, about 8% w / v, about 8.5% w / v, about 9% w / v, about 9.5% w / v, about 10% w / v, about 10.5% w / v, about 11% w / v, about 11.5% w / v, or about 12% w / v.In some embodiments, the concentration of carbohydrates in the pharmaceutical composition is about 8% w / v. In some embodiments, the concentration of carbohydrates in the pharmaceutical composition is at least 8% w / v, at least 10% w / v, at least 12% w / v, at least 15% w / v, or at least 20% w / v.

[0084] In some embodiments, the pharmaceutical composition further comprises other amino acids or pharmaceutically acceptable salts thereof. In some embodiments, the other amino acids or pharmaceutically acceptable salts thereof are arginine or pharmaceutically acceptable salts thereof. In some embodiments, the other amino acids or pharmaceutically acceptable salts thereof are arginine-HCl.

[0085] In some embodiments, the concentration of the other amino acids or pharmaceutically acceptable salts thereof in the pharmaceutical composition is 1mM-200mM, for example, 1mM-190mM, 1mM-180mM, 1mM-170mM, 1mM-160mM, 1mM-150mM, 1mM-140mM, 1mM-130mM, 1mM-120mM, 1mM-110mM, 1mM-100mM, 1mM-90mM, 1mM-80mM, 1mM-70mM, 1mM-60mM, 1mM-50mM, 1mM-40mM, 1mM-30mM, 1mM-20mM, 5mM-180mM, 5mM-170mM, 5mM-160mM, 5mM-150mM, 1mM-140mM, 1mM-130mM, 1mM-120mM, 1mM-110mM, 1mM-100mM, 1mM-90mM, 1mM-80mM, 1mM-70mM, 1mM-60mM, 1mM-50mM, 1mM-40mM, 1mM-30mM, 1mM-20mM, -150mM, 5mM-140mM, 5mM-130mM, 5mM-120mM, 5mM-110mM, 5mM-100mM, 5mM-9 0mM, 5mM-80mM, 5mM-70mM, 5mM-60mM, 5mM-50mM, 5mM-40mM, 5mM-30mM, 5mM-2 0mM, 10mM-170mM, 10mM-160mM, 10mM-150mM, 10mM-140mM, 10mM-130mM, 10m M-120mM, 10mM-110mM, 10mM-100mM, 10mM-90mM, 10mM-80mM, 10mM-70mM, 10m M-60mM, 10mM-50mM, 10mM-40mM, 10mM-30mM, 10mM-20mM, 15mM-160mM, 15mM -150mM, 15mM-140mM, 15mM-130mM, 15mM-120mM, 15mM-110mM, 15mM-100mM, 15mM-90mM, 15mM-80mM, 15mM-70mM, 15mM-60mM, 15mM-50mM, 15mM-40mM, 15 mM-30mM, 15mM-20mM, 20mM-150mM, 20mM-140mM, 20mM-130mM, 20mM-120mM, 2 0mM-110mM, 20mM-100mM, 20mM-90mM, 20mM-80mM, 20mM-70mM, 20mM-60mM, 2 0mM-50mM, 20mM-40mM, 20mM-30mM, 25mM-140mM, 25mM-130mM, 25mM-120mM, 2 5mM-110mM, 25mM-100mM, 25mM-90mM, 25mM-80mM, 25mM-70mM, 25mM-60mM, 2 5mM-50mM, 25mM-40mM, 30mM-140mM, 30mM-130mM, 30mM-120mM, 30mM-110mM,In some embodiments, the concentration of other amino acids or pharmaceutically acceptable salts thereof in the pharmaceutical composition is 10 mM to 80 mM. In some embodiments, the concentration of the other amino acids or their pharmaceutically acceptable salts in the pharmaceutical composition is 20mM-70mM. In some embodiments, the concentration of the other amino acids or their pharmaceutically acceptable salts in the pharmaceutical composition is 40mM-60mM. In some embodiments, the concentration of the other amino acids or their pharmaceutically acceptable salts in the pharmaceutical composition is about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, about 50mM, about 55mM, about 60mM, about 65mM, or about 70mM. In some embodiments, the concentration of the buffer in the pharmaceutical composition is about 50mM. In some embodiments, the concentration of the other amino acids or their pharmaceutically acceptable salts in the pharmaceutical composition is 5mM-170mM. In some embodiments, the concentration of the other amino acids or their pharmaceutically acceptable salts in the pharmaceutical composition is 10mM-150mM. In some embodiments, the concentration of the other amino acids or their pharmaceutically acceptable salts in the pharmaceutical composition is 15mM-120mM. In some embodiments, the concentration of the other amino acid or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, or about 120 mM. In some embodiments, the concentration of the other amino acid or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is about 20 mM, about 50 mM, or about 100 mM.

[0086] In some embodiments, the pH of the buffer in the pharmaceutical composition is 3.5-9, e.g., 3.5-8.5, 3.5-8, 3.5-7.5, 3.5-7, 3.5-6.9, 3.5-6.8, 3.5-6.7, 3.5-6.6, 3.5-6.5, 3.5-6.4, 3.5-6.3, 3.5-6.2, 3.5-6.1, 3.5-6, 3.5-5.9, 3.5-5.8, 3.5-5.7, 3.5-5.6, 3.5-5.5, 4-5.4, 4-5.3, 4-5.2, 4-5.1, 4-5.0, 4-8.5, 4-8, 4-7.5, 4-7, 4-6.9, 4-6.8, 4-6.7, 4-6.6, 4-6.5, 4-6.4, 4-6.3, 4-6.2, 4-6.1, 4-6, 4-5.9, 4-5.8, 4-5.7, 4-5.6, 4-5.5, 4-5.4, 4-5.3, 4-5.2, 4-5.1, 4-5.0, 4.2-8, 4.2-7.5, 4.2-7, 4.2-6.9, 4.2 -6.8, 4.2-6.7, 4.2-6.6, 4.2-6.5, 4.2-6.4, 4.2-6.3, 4.2-6.2, 4.2-6.1, 4.2-6, 4.2-5.9, 4.2-5.8, 4.2-5.7, 4.2-5.6, 4.2-5.5, 4.2-5.4, 4.2-5.3, 4. 2-5.2, 4.2-5.1, 4.2-5.0, 4.5-8, 4.5-7.5, 4.5-7, 4.5-6.9, 4.5-6.8, 4.5-6.7, 4.5-6.6, 4.5-6.5, 4.5-6.4, 4.5-6.3, 4.5-6.2, 4.5-6.1, 4.5-6, 4.5-5 .9, 4.5-5.8, 4.5-5.7, 4.5-5.6, 4.5-5.5, 4.5-5.4, 4.5-5.3, 4.5-5.2, 4.5-5.1, 4.5-5.0, 4.6-7.5, 4.6-7, 4.6-6.9, 4.6-6.8, 4.6-6.7, 4.6-6.6, 4.6- 6.5, 4.6-6.4, 4.6-6.3, 4.6-6.2, 4.6-6.1, 4.6-6, 4.6-5.9, 4.6-5.8, 4.6-5.7, 4.6-5.6, 4.6-5.5, 4.6-5.4, 4.6-5.3, 4.6-5.2, 4.6-5.1, 4.6-5.0, 4.8 -7, 4.8-6.9, 4.8-6.8, 4.8-6.7, 4.8-6.6, 4.8-6.5, 4.8-6.4, 4.8-6.3, 4.8-6.2, 4.8-6.1, 4.8-6, 4.8-5.9, 4.8-5.8, 4.8-5.7, 4.8-5.6, 4.8-5.5, 5-6.In some embodiments, the pH of the buffer in the pharmaceutical composition is 3.5-7. In some embodiments, the pH of the buffer in the pharmaceutical composition is 4.0-6.5. In some embodiments, the pH of the buffer in the pharmaceutical composition is 4.2-6.2. In some embodiments, the pH of the buffer in the pharmaceutical composition is 4.5-6.0. In some embodiments, the pH of the buffer in the pharmaceutical composition is about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5. In some embodiments, the pH of the buffer in the pharmaceutical composition is about 5.0, about 5.2, about 5.3, about 5.5, about 5.6, about 5.8, or about 6.0. In some embodiments, the pH of the buffer in the pharmaceutical composition is about 5.0 or about 5.5. In some embodiments, the pH of the buffer in the pharmaceutical composition is at least 5.0, at least 5.5, at least 6.0, at least 6.5, at least 7, or at least 9.

[0087] In some embodiments, the pharmaceutical composition further comprises a pH adjusting agent, such as sodium hydroxide and / or hydrochloric acid.

[0088] In some embodiments, the pH of the pharmaceutical composition differs by no more than ±0.5 from the pH of the buffer it contains. In some embodiments, the pH of the pharmaceutical composition is 3.5-9, e.g., 3.5-8.5, 3.5-8, 3.5-7.5, 3.5-7, 3.5-6.9, 3.5-6.8, 3.5-6.7, 3.5-6.6, 3.5-6.5, 3.5-6.4, 3.5-6.3, 3.5-6.2, 3.5-6.1, 3.5-6, 3.5-5.9, 3.5-5.8, 3.5-5.7, 3.5-5.6, 3.5-5.5, 4-5.4, 4-5.3, 4-5.2, 4-5.1, 4-5.0, 4-8.5, 4-8, 4-7.5, 4-7, 4-6.9, 4-6.8, 4-6. 7, 4-6.6, 4-6.5, 4-6.4, 4-6.3, 4-6.2, 4-6.1, 4-6, 4-5.9, 4-5.8, 4-5.7, 4-5.6, 4-5.5, 4-5.4, 4-5.3, 4-5.2, 4-5.1, 4-5.0, 4.2-8, 4.2-7.5, 4.2-7, 4.2-6.9, 4.2-6.8, 4.2-6.7, 4.2-6.6, 4.2-6.5, 4.2-6.4, 4.2-6.3, 4.2-6.2, 4.2-6.1, 4.2-6, 4.2-5.9, 4.2-5.8, 4.2-5.7, 4.2-5.6, 4.2-5.5 , 4.2-5.4, 4.2-5.3, 4.2-5.2, 4.2-5.1, 4.2-5.0, 4.5-8, 4.5-7.5, 4.5-7, 4.5-6.9, 4.5-6.8, 4.5-6.7, 4.5-6.6, 4.5-6.5, 4.5-6.4, 4.5-6.3, 4.5-6.2, 4.5-6.1, 4.5-6, 4.5-5.9, 4.5-5.8, 4.5-5.7, 4.5-5.6, 4.5-5.5, 4.5-5.4, 4.5-5.3, 4.5-5.2, 4.5-5.1, 4.5-5.0, 4.6-7.5, 4.6-7, 4.6-6 .9, 4.6-6.8, 4.6-6.7, 4.6-6.6, 4.6-6.5, 4.6-6.4, 4.6-6.3, 4.6-6.2, 4.6-6.1, 4.6-6, 4.6-5.9, 4.6-5.8, 4.6-5.7, 4.6-5.6, 4.6-5.5, 4.6-5.4, 4.6-5.3, 4.6-5.2, 4.6-5.1, 4.6-5.0, 4.8-7, 4.8-6.9, 4.8-6.8, 4.8-6.7, 4.8-6.6, 4.8-6.5, 4.8-6.4, 4.8-6.3, 4.8-6.2, 4.8-6.1, 4.8-6, 4.8.In some embodiments, the pH of the pharmaceutical composition is 3.5-7. In some embodiments, the pH of the pharmaceutical composition is 4.0-6.5. In some embodiments, the pH of the pharmaceutical composition is 4.2-6.2. In some embodiments, the pH of the pharmaceutical composition is 4.5-6.0. In some embodiments, the pH of the pharmaceutical composition is about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5. In some embodiments, the pH of the pharmaceutical composition is about 5.0, about 5.2, about 5.3, about 5.5, about 5.6, about 5.8, or about 6.0. In some embodiments, the pH of the pharmaceutical composition is about 5.0 or about 5.5. In some embodiments, the pH of the pharmaceutical composition is at least 5.0, at least 5.5, at least 6.0, at least 6.5, at least 7, or at least 9.

[0089] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a PD-1 / PVRIG / TIGIT binding protein (e.g., A17m0902-1708-151H7-01-C whose first polypeptide chain and second polypeptide chain sequences are SEQ ID NOs: 76 and 52, respectively, or A17m0902-1708-151H7-C whose first polypeptide chain and second polypeptide chain sequences are SEQ ID NOs: 72 and 52, respectively), comprising any one of the following groups 1) to 6):

[0090] 1) PD-1 / PVRIG / TIGIT binding protein;

[0091] Histidine salt buffers, such as histidine hydrochloride buffer or histidine acetate buffer, histidine-histidine hydrochloride buffer or histidine-acetate buffer;

[0092] 2) PD-1 / PVRIG / TIGIT binding protein;

[0093] Histidine buffer;

[0094] Polysorbate;

[0095] sucrose;

[0096] Optionally, the composition further comprises arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride);

[0097] 3) PD-1 / PVRIG / TIGIT binding protein;

[0098] Histidine hydrochloride buffer, such as histidine-histidine hydrochloride buffer;

[0099] Polysorbate;

[0100] sucrose;

[0101] Optionally, the composition further comprises arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride);

[0102] 4) PD-1 / PVRIG / TIGIT binding protein;

[0103] Histidine acetate buffers, such as histidine-acetate buffer;

[0104] Polysorbate;

[0105] sucrose;

[0106] Optionally, the composition further comprises arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride);

[0107] 5) PD-1 / PVRIG / TIGIT binding protein;

[0108] Citrate buffers, such as citric acid-sodium citrate buffer, citric acid-disodium citrate buffer;

[0109] 6) PD-1 / PVRIG / TIGIT binding protein;

[0110] Citrate buffers, such as citric acid-disodium citrate buffer;

[0111] Polysorbate;

[0112] sucrose;

[0113] Optionally, the composition further comprises arginine or a pharmaceutically acceptable salt thereof (eg, arginine hydrochloride).

[0114] In some embodiments, the present disclosure provides a pharmaceutical composition containing a PD-1 / PVRIG / TIGIT binding protein (e.g., A17m0902-1708-151H7-01-C whose first polypeptide chain and second polypeptide chain sequences are SEQ ID NOs: 76 and 52, respectively, or A17m0902-1708-151H7-C whose first polypeptide chain and second polypeptide chain sequences are SEQ ID NOs: 72 and 52, respectively), which comprises or is any one of the following groups 1) to 6):

[0115] 1) PD-1 / PVRIG / TIGIT binding protein; histidine salt buffer; polysorbate; sucrose; water for injection, optionally arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride) and optionally sodium hydroxide and / or hydrochloric acid;

[0116] 2) PD-1 / PVRIG / TIGIT binding protein; histidine hydrochloride buffer; polysorbate; sucrose; water for injection, optionally arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride) and optionally sodium hydroxide and / or hydrochloric acid;

[0117] 3) PD-1 / PVRIG / TIGIT binding protein; histidine-histidine hydrochloride buffer; polysorbate; sucrose; water for injection, optionally arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride) and optionally sodium hydroxide and / or hydrochloric acid;

[0118] 4) PD-1 / PVRIG / TIGIT binding protein; histidine-acetate buffer; polysorbate; sucrose; water for injection, optionally arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride) and optionally sodium hydroxide and / or hydrochloric acid;

[0119] 5) PD-1 / PVRIG / TIGIT binding protein; citrate buffer; polysorbate; sucrose; water for injection, optionally arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride) and optionally sodium hydroxide and / or hydrochloric acid;

[0120] 6) PD-1 / PVRIG / TIGIT binding protein; citric acid-disodium citrate buffer; polysorbate; sucrose; water for injection, optionally arginine or a pharmaceutically acceptable salt thereof (eg, arginine hydrochloride) and optionally sodium hydroxide and / or hydrochloric acid.

[0121] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a PD-1 / PVRIG / TIGIT binding protein (e.g., A17m0902-1708-151H7-01-C whose first polypeptide chain and second polypeptide chain sequences are SEQ ID NOs: 76 and 52, respectively, or A17m0902-1708-151H7-C whose first polypeptide chain and second polypeptide chain sequences are SEQ ID NOs: 72 and 52, respectively), comprising any one of the following groups 1) to 10):

[0122] 1) 0.01mg / mL-500mg / mL PD-1 / PVRIG / TIGIT binding protein;

[0123] 0.1 mM-50 mM histidine hydrochloride buffer, such as histidine hydrochloride buffer or histidine acetate buffer, histidine-histidine hydrochloride buffer or histidine-acetate buffer;

[0124] 0.01mg / mL-10mg / mL polysorbate;

[0125] 0.1 w / v-20% w / v sucrose;

[0126] Optionally, the pharmaceutical composition further comprises 1 mM-200 mM arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride);

[0127] and the pH of the pharmaceutical composition is 3.5-7;

[0128] 2) 0.1mg / mL-400mg / mL PD-1 / PVRIG / TIGIT binding protein;

[0129] 0.5 mM-40 mM histidine hydrochloride buffer, such as histidine hydrochloride buffer or histidine acetate buffer, histidine-histidine hydrochloride buffer or histidine-acetate buffer;

[0130] 0.05mg / mL-5mg / mL polysorbate;

[0131] 1% w / v-15% w / v sucrose;

[0132] Optionally, the pharmaceutical composition further comprises 5 mM-170 mM arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride);

[0133] and the pH of the pharmaceutical composition is 4-6.5;

[0134] 3) 0.5mg / mL-200mg / mL PD-1 / PVRIG / TIGIT binding protein;

[0135] 1 mM-30 mM histidine hydrochloride buffer, such as histidine hydrochloride buffer or histidine acetate buffer, histidine-histidine hydrochloride buffer or histidine-acetate buffer;

[0136] 0.1mg / mL-3mg / mL polysorbate;

[0137] 3% w / v-12% w / v sucrose;

[0138] Optionally, the pharmaceutical composition further comprises 10 mM-150 mM arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride);

[0139] and the pH of the pharmaceutical composition is 4.2-6.2;

[0140] 4) 1mg / mL-150mg / mL PD-1 / PVRIG / TIGIT binding protein;

[0141] 5 mM-20 mM histidine hydrochloride buffer, such as histidine hydrochloride buffer or histidine acetate buffer, histidine-histidine hydrochloride buffer or histidine-acetate buffer;

[0142] 0.2mg / mL-2mg / mL polysorbate;

[0143] 5% w / v-10% w / v sucrose;

[0144] Optionally, the pharmaceutical composition further comprises 15 mM-120 mM arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride);

[0145] and the pH of the pharmaceutical composition is 4.5-6;

[0146] 5) 0.01mg / mL-500mg / mL PD-1 / PVRIG / TIGIT binding protein;

[0147] 0.1 mM-50 mM citrate buffer, such as citric acid-disodium citrate buffer;

[0148] 0.01mg / mL-10mg / mL polysorbate;

[0149] 0.1 w / v-20% w / v sucrose;

[0150] Optionally, the pharmaceutical composition further comprises 1 mM-200 mM arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride);

[0151] and the pH of the pharmaceutical composition is 3.5-7;

[0152] 6) 0.1mg / mL-400mg / mL PD-1 / PVRIG / TIGIT binding protein;

[0153] 0.5 mM-40 mM citrate buffer, such as citric acid-disodium citrate buffer;

[0154] 0.05mg / mL-5mg / mL polysorbate;

[0155] 1% w / v-15% w / v sucrose;

[0156] Optionally, the pharmaceutical composition further comprises 5 mM-170 mM arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride);

[0157] and the pH of the pharmaceutical composition is 4-6.5;

[0158] 7) 0.5mg / mL-200mg / mL PD-1 / PVRIG / TIGIT binding protein;

[0159] 1 mM-30 mM citrate buffer, such as citric acid-disodium citrate buffer;

[0160] 0.1mg / mL-3mg / mL polysorbate;

[0161] 3% w / v-12% w / v sucrose;

[0162] Optionally, the pharmaceutical composition further comprises 10 mM-150 mM arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride);

[0163] and the pH of the pharmaceutical composition is 4.2-6.2;

[0164] 8) 1mg / mL-150mg / mL PD-1 / PVRIG / TIGIT binding protein;

[0165] 5 mM-20 mM citrate buffer, such as citric acid-disodium citrate buffer;

[0166] 0.2mg / mL-2mg / mL polysorbate;

[0167] 5% w / v-10% w / v sucrose;

[0168] Optionally, the pharmaceutical composition further comprises 15 mM-120 mM arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride);

[0169] and the pH of the pharmaceutical composition is 4.5-6;

[0170] 9) is any one of the pharmaceutical compositions of 1) to 8), wherein the polysorbate is polysorbate 80

[0171] 10) is the pharmaceutical composition according to any one of 1) to 9), further comprising water for injection.

[0172] In some embodiments, the present disclosure provides a pharmaceutical composition containing a PD-1 / PVRIG / TIGIT binding protein (e.g., A17m0902-1708-151H7-01-C whose first polypeptide chain and second polypeptide chain sequences are SEQ ID NOs: 76 and 52, respectively, or A17m0902-1708-151H7-C whose first polypeptide chain and second polypeptide chain sequences are SEQ ID NOs: 72 and 52, respectively), which comprises or is any one of the following groups 1) to 16):

[0173] 1) 1-150 mg / mL PD-1 / PVRIG / TIGIT binding protein;

[0174] about 10 mM histidine hydrochloride buffer or histidine acetate buffer, such as histidine-histidine hydrochloride buffer or histidine-acetate buffer;

[0175] 0.2mg / mL-2mg / mL polysorbate 80;

[0176] About 8% w / v sucrose;

[0177] and the pH of the pharmaceutical composition is 4.5-6;

[0178] 2) about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, about 130 mg / mL, or about 150 mg / mL of PD-1 / PVRIG / TIGIT binding protein, such as about 10 mg / mL, about 50 mg / mL, about 100 mg / mL, about 130 mg / mL, or about 150 mg / mL;

[0179] About 10 mM histidine hydrochloride buffer, such as histidine-histidine hydrochloride buffer;

[0180] About 0.4 mg / mL polysorbate 80;

[0181] About 8% w / v sucrose;

[0182] and the pH of the pharmaceutical composition is about 5;

[0183] 3) about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, about 130 mg / mL, or about 150 mg / mL of PD-1 / PVRIG / TIGIT binding protein, such as about 10 mg / mL, about 50 mg / mL, about 100 mg / mL, about 130 mg / mL, or about 150 mg / mL;

[0184] About 10 mM histidine hydrochloride buffer, such as histidine-histidine hydrochloride buffer;

[0185] About 0.4 mg / mL polysorbate 80;

[0186] About 8% w / v sucrose;

[0187] and the pH of the pharmaceutical composition is about 5.5;

[0188] 4) about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, about 130 mg / mL, or about 150 mg / mL of PD-1 / PVRIG / TIGIT binding protein, such as about 10 mg / mL, about 50 mg / mL, about 100 mg / mL, about 130 mg / mL, or about 150 mg / mL;

[0189] About 10 mM histidine hydrochloride buffer, such as histidine-histidine hydrochloride buffer;

[0190] About 0.8 mg / mL polysorbate 80;

[0191] About 8% w / v sucrose;

[0192] and the pH of the pharmaceutical composition is about 5;

[0193] 5) about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, about 130 mg / mL, or about 150 mg / mL of PD-1 / PVRIG / TIGIT binding protein, such as about 10 mg / mL, about 50 mg / mL, about 100 mg / mL, about 130 mg / mL, or about 150 mg / mL;

[0194] About 10 mM histidine hydrochloride buffer, such as histidine-histidine hydrochloride buffer;

[0195] About 0.8 mg / mL polysorbate 80;

[0196] About 8% w / v sucrose;

[0197] and the pH of the pharmaceutical composition is about 5.5;

[0198] 6) about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, about 130 mg / mL, or about 150 mg / mL of PD-1 / PVRIG / TIGIT binding protein, such as about 10 mg / mL, about 50 mg / mL, about 100 mg / mL, about 130 mg / mL, or about 150 mg / mL;

[0199] approximately 10 mM histidine-acetate buffer;

[0200] About 0.4 mg / mL polysorbate 80;

[0201] About 8% w / v sucrose;

[0202] and the pH of the pharmaceutical composition is about 5;

[0203] 7) about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, about 130 mg / mL, or about 150 mg / mL of PD-1 / PVRIG / TIGIT binding protein, such as about 10 mg / mL, about 50 mg / mL, about 100 mg / mL, about 130 mg / mL, or about 150 mg / mL;

[0204] approximately 10 mM histidine-acetate buffer;

[0205] About 0.4 mg / mL polysorbate 80;

[0206] About 8% w / v sucrose;

[0207] and the pH of the pharmaceutical composition is about 5.5;

[0208] 8) about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, about 130 mg / mL, or about 150 mg / mL of PD-1 / PVRIG / TIGIT binding protein, such as about 10 mg / mL, about 50 mg / mL, about 100 mg / mL, about 130 mg / mL, or about 150 mg / mL;

[0209] approximately 10 mM histidine-acetate buffer;

[0210] About 0.8 mg / mL polysorbate 80;

[0211] About 8% w / v sucrose;

[0212] and the pH of the pharmaceutical composition is about 5;

[0213] 9) about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, about 130 mg / mL, or about 150 mg / mL of PD-1 / PVRIG / TIGIT binding protein, such as about 10 mg / mL, about 50 mg / mL, about 100 mg / mL, about 130 mg / mL, or about 150 mg / mL;

[0214] approximately 10 mM histidine-acetate buffer;

[0215] About 0.8 mg / mL polysorbate 80;

[0216] About 8% w / v sucrose;

[0217] and the pH of the pharmaceutical composition is about 5.5;

[0218] 10) about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, about 130 mg / mL, or about 150 mg / mL of PD-1 / PVRIG / TIGIT binding protein, such as about 10 mg / mL, about 50 mg / mL, about 100 mg / mL, about 130 mg / mL, or about 150 mg / mL;

[0219] about 10 mM citrate buffer, such as citric acid-disodium citrate buffer;

[0220] About 0.4 mg / mL polysorbate 80;

[0221] About 8% w / v sucrose;

[0222] and the pH of the pharmaceutical composition is about 5;

[0223] 11) about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, about 130 mg / mL, or about 150 mg / mL of PD-1 / PVRIG / TIGIT binding protein, such as about 10 mg / mL, about 50 mg / mL, about 100 mg / mL, about 130 mg / mL, or about 150 mg / mL;

[0224] about 10 mM citrate buffer, such as citric acid-disodium citrate buffer;

[0225] About 0.4 mg / mL polysorbate 80;

[0226] About 8% w / v sucrose;

[0227] and the pH of the pharmaceutical composition is about 5.5;

[0228] 12) about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, about 130 mg / mL, or about 150 mg / mL of PD-1 / PVRIG / TIGIT binding protein, such as about 10 mg / mL, about 50 mg / mL, about 100 mg / mL, about 130 mg / mL, or about 150 mg / mL;

[0229] about 10 mM citrate buffer, such as citric acid-disodium citrate buffer;

[0230] About 0.8 mg / mL polysorbate 80;

[0231] About 8% w / v sucrose;

[0232] and the pH of the pharmaceutical composition is about 5;

[0233] 13) about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL about 120 mg / mL, about 130 mg / mL, or about 150 mg / mL of PD-1 / PVRIG / TIGIT binding protein, for example, about 10 mg / mL, about 50 mg / mL, about 100 mg / mL, about 130 mg / mL, or about 150 mg / mL;

[0234] about 10 mM citrate buffer, such as citric acid-disodium citrate buffer;

[0235] About 0.8 mg / mL polysorbate 80;

[0236] About 8% w / v sucrose;

[0237] and the pH of the pharmaceutical composition is about 5.5;

[0238] 14) The pharmaceutical composition of 1)-13), further comprising 20 mM, 50 mM or 100 mM arginine or its hydrochloride;

[0239] 15) The pharmaceutical composition of 1)-14), further comprising hydrochloric acid and / or sodium hydroxide;

[0240] 16) is the pharmaceutical composition of 1)-15), wherein the final volume is 1 mL; when the volume needs to be fixed, the volume is fixed to 1 mL with water for injection.

[0241] The pharmaceutical composition disclosed herein has sufficient drug stability and can be stored stably for a long time.

[0242] In some embodiments, the pharmaceutical composition is stable at 2-8° C. for at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, or at least 36 months. In some embodiments, the pharmaceutical composition is stable at 25° C. for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the pharmaceutical composition is stable at 40° C. for at least 7 days, at least 14 days, at least 28 days, at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months.

[0243] The present disclosure provides a method for preparing the aforementioned pharmaceutical composition, comprising the step of dissolving the PD-1 / PVRIG / TIGIT binding protein.

[0244] To facilitate drug transportation, the pharmaceutical composition of the present disclosure can be further prepared into a lyophilized preparation.

[0245] In certain embodiments, the pharmaceutical composition as described in any one of the above items is a liquid preparation. In some embodiments, the solvent of the liquid preparation is water, normal saline or glucose solution.

[0246] The present disclosure also provides a lyophilized preparation, characterized in that the lyophilized preparation can form any of the above pharmaceutical compositions after reconstitution.

[0247] The present disclosure also provides a lyophilized preparation, which is obtained by freeze-drying the pharmaceutical composition as described above.

[0248] The present disclosure provides a reconstitution solution, wherein the reconstitution solution is prepared by reconstitution of the aforementioned lyophilized preparation. In certain embodiments, the reconstitution solution is selected from but not limited to water for injection, physiological saline or glucose solution.

[0249] The present disclosure also provides a product, comprising a container containing the aforementioned pharmaceutical composition, the aforementioned lyophilized formulation, or the aforementioned reconstituted solution. In certain embodiments, the container is a neutral borosilicate glass injection vial. In certain embodiments, the product includes a package insert.

[0250] The present disclosure also provides a pharmaceutical composition or a lyophilized formulation or a reconstituted solution of a lyophilized formulation, which is used as a medicine for treating or alleviating a disease or condition.

[0251] Methods for treating diseases and pharmaceutical uses

[0252] The present disclosure provides a method for using the aforementioned pharmaceutical composition or lyophilized preparation or a reconstituted solution of the lyophilized preparation for treating, alleviating, preventing, or diagnosing a disease or condition.

[0253] In some embodiments, a method for improving, alleviating, treating or preventing a disease is provided, comprising administering to a subject an effective amount of the aforementioned pharmaceutical composition or lyophilized preparation or a reconstituted solution of the lyophilized preparation for improving, alleviating, treating or preventing the disease.

[0254] In some embodiments, there is provided use of the aforementioned pharmaceutical composition or lyophilized preparation or reconstituted solution of the lyophilized preparation disclosed herein in the preparation of a drug for improving, alleviating, treating or preventing a disease.

[0255] In some embodiments, the aforementioned disease is a proliferative disease or any other disease or condition characterized by uncontrolled cell growth (such as cancer. In the present disclosure, cancer and tumor can be used interchangeably), such as a disease (such as cancer) associated with overexpression of PD-L1 or abnormal expression of PVRIG or TIGIT.

[0256] In some embodiments, the aforementioned cancer is a solid tumor or a hematological tumor.

[0257] In some embodiments, the aforementioned cancer is advanced or metastatic.

[0258] In some embodiments, the aforementioned cancer is selected from the following or a combination thereof: prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple-negative breast cancer, pancreatic cancer, stomach (stomach / gastric) cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), glioma, renal cell carcinoma (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumor, mesothelioma, esophageal cancer, Merkel cell cancer, MSI-high cancer, KRAS mutant tumors, adult T-cell leukemia / lymphoma and myelodysplastic syndrome (MDS). For example, selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, stomach cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, hematological cancer, or any other disease or condition characterized by uncontrolled cell growth.

[0259] In some embodiments, the subject has a condition associated with PVRIG and / or TIGIT. In some embodiments, the subject's condition includes cancer that expresses or does not express PVRIG, and further includes non-metastatic or non-invasive cancers as well as invasive or metastatic cancers, wherein PVRIG expression by immune cells, stromal cells, or diseased cells suppresses anti-tumor and anti-invasive immune responses. The methods of the present disclosure are suitable, for example, for treating vascularized cancers.

[0260] In some embodiments, a method for treating or preventing infection or sepsis in a subject is provided, comprising administering to the subject a therapeutically or prophylactically effective amount of the aforementioned pharmaceutical composition or lyophilized formulation or a reconstituted solution of the lyophilized formulation. In some embodiments, the infection is a pathogen infection characterized by varying degrees of functional impairment of virus-specific T cell responses, such as HIV, HCV, and HBV. In some embodiments, the sepsis includes severe sepsis, septic shock, systemic inflammatory response syndrome (SIRS), bacteremia, sepsis, toxemia, and septic syndrome.

[0261] In some embodiments, a method for activating cytotoxic T cells (CTLs) in a subject is provided, a method for activating NK cells in a subject is provided, a method for activating γδ T cells in a subject is provided, a method for activating Th1 cells in a subject is provided, a method for activating, reducing or eliminating the cell number and / or activity of at least one of regulatory T cells (Treg) in a subject is provided, a method for increasing IFN-γ production and / or proinflammatory cytokine secretion in a subject is provided, a method for inhibiting the interaction between PVRIG and PVRL2 in a subject is provided, and a method for blocking or inhibiting the interaction between PD-1 and PD-L1 / PD-L2 in a subject is provided, all of which comprise administering to the subject an effective amount of the aforementioned pharmaceutical composition or lyophilized formulation or a reconstituted solution of the lyophilized formulation.

[0262] The pharmaceutical composition containing the PD-1 / PVRIG / TIGIT binding protein disclosed herein can be used to treat patients in need of such treatment by parenteral administration, which can be administered subcutaneously, intramuscularly, or intravenously. BRIEF DESCRIPTION OF THE DRAWINGS

[0263] Figures 1A and 1B show the activity of anti-PD-1 nanobodies detected using the PD-1 / PD-L1 NFAT reporter gene system. Pembrolizumab was used as a positive control, and hIgG4 was used as a negative control. Figure 1A shows the results for A6_IgG4 and A17_IgG4. Figure 1B shows the results for A17h1_IgG4, A17m09_hIgG4, A17m0901_hIgG4, A17m0902_hIgG4, A17m0903_hIgG4, and A17m0905_hIgG4.

[0264] Figure 2 shows the degree of T cell activation by A17m09_hIgG4 detected by cytokine release in DC cell:T cell mixed lymphocyte reaction, using Pembrolizumab as a positive control and hIgG4 as a negative control.

[0265] Figures 3A and 3B show the in vivo efficacy test results of A17m09_hIgG4 in inhibiting the growth of MC38 colon cancer tumors in mice. Figure 3A shows the mouse tumor volume results, and Figure 3B shows the mouse weight results. Pembrolizumab was used as a positive control and PBS was used as a negative control.

[0266] FIG4 shows the mixed lymphocyte reaction (MLR) experimental results of anti-PVRIG nanobodies 30 and 151, using Tab5 as a positive control and hIgG4 as a negative control.

[0267] Figure 5 shows the MLR experimental results of the anti-PVRIG / TIGIT bispecific antibody 1708-151H8, and 151H8, 1708, hIgG4, Tab5, and Pembrolizumab were tested as controls.

[0268] Figures 6A and 6B show the anti-tumor results of the combination of anti-PVRIG / TIGIT bispecific antibodies 1708-151 and 1708 and 151 in a subcutaneous transplant tumor model of human melanoma A375 mixed with human PBMC in mice. Figure 6B shows the corresponding mouse body weight.

[0269] Figures 7A and 7B show the anti-tumor effects of the anti-PVRIG / TIGIT bispecific antibodies 1708-151H7 and 1708-30H2 in a subcutaneous transplant model of human melanoma A375 mixed with human PBMCs. Figure 7B shows the corresponding mouse body weight.

[0270] Figure 8 is a schematic diagram of four antibody configurations of anti-PD-1 / PVRIG / TIGIT trispecific antibodies.

[0271] FIG9 shows the detection results of antibodies with four configurations of A17h1-1708-151H7 binding to TIGIT antigen.

[0272] FIG10 shows the detection results of antibodies with four configurations of A17h1-1708-151H7 binding to PVRIG antigen.

[0273] Figures 11A, 11B, and 11C respectively show the binding abilities of the PVRIG sequence optimized anti-PD-1 / PVRIG / TIGIT trispecific antibody to cells overexpressing PVRIG, TIGIT, and PD-1 antigens.

[0274] Figure 12 shows the blocking effect of 4 antibodies with different configurations, A17h1-1708-151H7, on PD-L1 / PD-1 binding in the PD-1 / PD-L1 NFAT reporter gene system.

[0275] FIG13 shows the MLR test results of antibodies with four configurations of A17h1-1708-151H7.

[0276] FIG14 shows the NK cell killing function test results of antibodies with four configurations of A17h1-1708-151H7.

[0277] Figures 15A and 15B are evaluations of the anti-tumor effects of the anti-PD-1 / PVRIG / TIGIT trispecific antibody in a PD-1 monoclonal antibody-responsive human melanoma A375 mixed with human PBMC subcutaneous xenograft mouse model.

[0278] Figures 16A and 16B are evaluations of the anti-tumor effect of the anti-PD-1 / PVRIG / TIGIT trispecific antibody in a mouse subcutaneous transplant tumor model of human melanoma A375 mixed with human PBMCs that is unresponsive to PD-1 monoclonal antibody. DETAILED DESCRIPTION

[0279] 1. Terminology

[0280] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0281] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0282] "Programmed death 1," "programmed cell death 1," "protein PD-1," "PD-1," "PDCD1," and "hPD-1" are used interchangeably and include variants, isoforms, species homologs, and analogs of human PD-1 that share at least one common epitope with PD-1. The complete PD-1 sequence can be found at GenBank Accession No. U64863. "Programmed death ligand-1 (PD-L1)" is one of the two cell surface glycoprotein ligands of PD-1 (the other being PD-L2), which downregulates T cell activation and cytokine secretion when bound to PD-1. "PD-L1," as used herein, includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, and analogs that share at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found at GenBank Accession No. Q9NZQ7.

[0283] "PVRIG" or "PVRIG protein" or "PVRIG polypeptide" may optionally include any such protein or variant, conjugate or fragment thereof, including but not limited to known or wild-type PVRIG as described herein, as well as any naturally occurring splice variants, amino acid variants or isoforms, and in particular, soluble extracellular domain (ECD) fragments of PVRIG. The definition of ECD herein is as described in patent WO2016134333. The complete human PVRIG sequence can be found at GenBank accession number AAH73861.1.

[0284] "TIGIT" or "TIGIT protein" or "TIGIT polypeptide" may optionally include any such protein or variant, conjugate, or fragment thereof, including but not limited to known or wild-type TIGIT as described herein, as well as any naturally occurring splice variant, amino acid variant, or isoform. The complete TIGIT sequence can be found at GenBank Accession No. AAI01289.1.

[0285] "Binding to PD-1" means being able to interact with PD-1 or its epitope, which may be of human origin. "Binding to PVRIG" means being able to interact with PVRIG or its epitope, which may be of human origin. "Binding to TIGIT" means being able to interact with TIGIT or its epitope, which may be of human origin. "Antigen binding site" refers to a discrete three-dimensional site on an antigen that is recognized by the antibodies or antigen-binding fragments of the present disclosure.

[0286] "PD-1 binding protein" encompasses any protein or any molecule that can specifically bind to PD-1 or its epitope. PD-1 binding proteins may include antibodies, antigen-binding fragments thereof, or conjugates thereof as defined in the present disclosure directed against PD-1. PD-1 binding proteins also encompass immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules. The "PD-1 binding protein" of the present disclosure may comprise at least one immunoglobulin single variable domain (such as VHH) that binds to PD-1. In some embodiments, the "PD-1 binding protein" may comprise 2, 3, 4 or more immunoglobulin single variable domains (such as VHH) that bind to PD-1. In addition to comprising an immunoglobulin single variable domain of PD-1, the PD-1 binding protein of the present disclosure may also comprise a linker and / or a portion having effector function, such as a half-life extending portion (such as an immunoglobulin single variable domain that binds to serum albumin), and / or a fusion partner (such as serum albumin) and / or a conjugated polymer (such as PEG) and / or an Fc region. In some embodiments, the “PD-1 binding protein” of the present disclosure also encompasses bi / multispecific antibodies, which contain immunoglobulins that bind to different antigens (such as a first antibody that binds to a first antigen (such as PD-1) and a second antibody that binds to a second antigen (such as PVRIG), optionally including a third specific antibody that binds to a third antigen (such as TIGIT), and further optionally, including a fourth antibody that binds to a fourth antigen. “PD-1 binding protein” encompasses “PD-1 / PVRIG binding protein”, “PD-1 / TIGIT binding protein”, and “PD-1 / PVRIG / TIGIT binding protein”.

[0287] The “PD-1 binding protein” or “anti-PD-1 antibody” of the present disclosure may comprise one or more effector molecules, for example, in a conjugated manner. The “effector molecules” include, for example, anti-tumor agents, drugs, toxins, biologically active proteins (e.g., enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof (e.g., DNA, RNA and fragments thereof), radionuclides, in particular radioiodides, radioisotopes, chelated metals, nanoparticles, and reporter groups such as fluorescent compounds or compounds detectable by NMR or ESR spectroscopy. When the effector molecule is a polymer, it may generally be a synthetic or naturally occurring polymer, such as an optionally substituted linear or branched polyalkylene, polyalkenylene or polyoxyalkylene polymer or a branched polysaccharide or an unbranched polysaccharide, such as a homopolymer or heteropolymer. Specific optional substituents that may be present on the above-mentioned synthetic polymers include one or more hydroxyl, methyl or methoxy groups. Specific examples of synthetic polymers include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, particularly optionally substituted poly(ethylene glycol) such as methoxypoly(ethylene glycol) or derivatives thereof. Specific naturally occurring polymers include lactose, amylose, dextran, glycogen or derivatives thereof. In one embodiment, the polymer is albumin or a fragment thereof, such as human serum albumin or a fragment thereof. Conjugation of the polymer to the PD-1 binding protein or anti-PD-1 antibody can be achieved by conventional methods.

[0288] "Cytokine" is a general term for proteins released by one cell population that act as intercellular mediators on other cells. Examples of such cytokines include lymphokines, monokines, chemokines, and traditional polypeptide hormones. Exemplary cytokines include human IL-2, IFN-γ, IL-6, TNFα, IL-17, and IL-5.

[0289] "Antibodies" encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. Antibodies may refer to immunoglobulins, which are tetrapeptide chains composed of two heavy chains and two light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant regions of immunoglobulins' heavy chains differ, resulting in different antigenicity. Based on this, immunoglobulins can be divided into five classes, or so-called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Igs of the same class can be further divided into different subclasses based on differences in the amino acid composition of their hinge regions and the number and position of heavy chain disulfide bonds, such as IgG, which can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either κ or λ chains based on differences in their constant regions. Each of the five Ig classes can have either kappa or lambda chains.

[0290] The approximately 110 amino acids near the N-terminus of an antibody's heavy and light chains vary greatly in sequence and constitute the variable region (V region). The remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region comprises three hypervariable regions (HVRs) and four framework regions (FRs), whose sequences are relatively conserved. These three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from amino to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0291] "Antigen-binding fragment" encompasses single-chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g., VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3)209-217).

[0292] Methods for producing and preparing these antigen-binding fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The Fab-Fv format was first disclosed in WO2009 / 040562, and its disulfide-stabilized form, Fab-dsFv, was first disclosed in WO2010 / 035012. Antigen-binding fragments of the present disclosure also include Fab and Fab' fragments described in WO2005 / 003169, WO2005 / 003170, and WO2005 / 003171. Multivalent antibodies may comprise multispecifics, such as bispecifics, or may be monospecifics (see, for example, WO92 / 22583 and WO05 / 113605), an example of which is Tri-Fab (or TFM) described in WO 92 / 22583.

[0293] "Bispecific antibodies" cover antibodies (including antibodies or antigen-binding fragments thereof, such as single-chain antibodies) that specifically bind to two different antigens or two or at least two different antigenic epitopes of the same antigen. The prior art has disclosed bispecific antibodies of various structures. According to the integrity of the IgG molecule, it can be divided into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies. According to the number of antigen-binding regions, it can be divided into bispecific antibodies of divalent, trivalent, tetravalent or more valence. According to whether the structure is symmetrical on the left and right, it can be divided into bispecific antibodies of symmetrical structure and bispecific antibodies of asymmetrical structure. Among them, bispecific antibodies based on antibody fragments, such as Fab fragments lacking Fc fragments, form bispecific antibodies by combining two or more Fab fragments into one molecule. They have low immunogenicity, small molecular weight, and high tumor tissue permeability. Typical antibody structures of this type include bispecific antibodies such as F(ab)2, scFv-Fab, (scFv)2-Fab, etc. IgG-like bispecific antibodies (e.g., with an Fc fragment) have a relatively large molecular weight. The Fc fragment facilitates the subsequent purification of the antibody and improves its solubility and stability. The Fc portion may also bind to the receptor FcRn, increasing the antibody serum half-life. Typical bispecific antibody structural models include KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1TCBs, 1Fab-IgG TDB, FynomAb, two-in-one / DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb and other bispecific antibodies (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585–608 (2019); Chen S1 et al., J Immunol Res. 2019 Feb 11;2019:4516041).

[0294] A "trispecific antibody" refers to an antibody that can specifically bind to three or at least three different antigenic epitopes, which may be epitopes of different antigens or different epitopes of the same antigen.

[0295] For the determination or definition of CDRs, the definitive delineation of CDRs and the identification of residues in the binding site can be accomplished by resolving the structure of the antibody and / or the structure of the antibody-ligand complex. This can be accomplished by any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions.

[0296] The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8). The Chothia numbering system is similar to the Kabat numbering system, but takes into account the positions of certain structural loop regions. (See, e.g., Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83). The AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group for modeling antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd). The AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from primary sequences (see Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," PROTEINS, Structure, Function and Genetics Suppl., 3: 194-198). Contact definitions are based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5: 732-45). In conformational definitions, CDR positions can be identified as residues that make enthalpic contributions to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166). Other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs. CDR boundaries may be shortened or extended based on predictions or experimental results that a particular residue or group of residues does not significantly affect antigen binding. As used in this disclosure, CDR may refer to CDR defined by any method known in the art (including a combination of methods). The correspondence between various numbering systems is well known to those skilled in the art, and is exemplified by the following Table 1.

[0297] Table 1. Relationships between CDR numbering systems

[0298] The CDR amino acid residues of the VL and VH regions of the antibodies of the present disclosure conform in number and position to the well-known Kabat numbering system.

[0299] The antibodies of the present disclosure may be polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic, or modified forms thereof, with monoclonal antibodies being particularly suitable for use in various embodiments. Generally, the antibodies of the present disclosure are recombinant antibodies.

[0300] As used herein, "recombinant" refers broadly to products such as cells, nucleic acids, proteins, or vectors, indicating that the cells, nucleic acids, proteins, or vectors have been modified by the introduction of heterologous nucleic acids or proteins or by altering native nucleic acids or proteins. For example, recombinant cells express genes that are not present in the native (non-recombinant) cell form or express native genes that are abnormally expressed, underexpressed, or not expressed at all.

[0301] A "domain" of a polypeptide or protein refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein and in many cases can be added, removed, or transferred to other proteins without loss of function of the rest of the protein and / or the domain.

[0302] "Immunoglobulin domain" refers to the globular region of an antibody chain. An immunoglobulin domain is characterized in that it maintains the characteristic folding of the antibody molecule.

[0303] An "immunoglobulin variable domain" refers to an immunoglobulin domain consisting essentially of four "framework regions" (Framework Region 1, or FR1), "Framework Region 2, or FR2," "Framework Region 3, or FR3," and "Framework Region 4, or FR4), and three "complementarity determining regions" (Complementarity Determining Region 1, or CDR1), "Complementarity Determining Region 2, or CDR2," and "Complementarity Determining Region 3, or CDR3." Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An immunoglobulin variable domain confers specificity for an antigen by having an antigen-binding site.

[0304] "Antibody framework (FR)" refers to the portion of a variable domain that serves as a scaffold for the antigen binding loops (CDRs) of that variable domain.

[0305] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which may be a heavy or light chain domain, including a VH, VHH or VL domain) that can form a functional antigen binding site without interacting with other variable domains (e.g., without the VH / VL interactions required between the VH and VL domains of conventional four-chain monoclonal antibodies). Examples of "immunoglobulin single variable domains" include nanobodies (including VHH, humanized VHH and / or camelized VH, e.g., camelized human VH), IgNAR, domains, (single domain) antibodies that are VH domains or derived from VH domains (such as dAbs), and antibodies that are VH domains or derived from VH domains. TM ) and (single domain) antibodies (such as dAbs) as the VL domain or derived from the VL domain TM ). Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. A specific example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") as defined below.

[0306] "VHH domain", also known as heavy chain single domain antibody, VHH, V H H domain, VHH antibody fragment, VHH antibody, nanobody, is the variable domain of the antigen-binding immunoglobulin called "heavy chain antibody" (i.e., "antibody lacking light chain") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH domain" is used to distinguish the VHH from the VH and VL present in conventional tetrapeptide chain structure antibodies. The VHH domain specifically binds to the epitope without the need for other antigen-binding domains (however, in conventional tetrapeptide chain structure antibodies, the epitope is recognized by the VL domain together with the VH domain). The VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain. The terms "heavy chain single domain antibody", "VHH domain", "VHH", "V HH domain", "VHH antibody fragment", "VHH antibody", as well as" "VHH domain" ("Nanobody" is a trademark of Ablynx NV, Ghent, Belgium) can be used interchangeably. "VHH domain" includes but is not limited to natural antibodies produced by camelids, antibodies produced by camelids that have been humanized, or antibodies obtained by phage display technology. The total number of amino acid residues in a VHH domain will generally be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in the present disclosure. Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185-195; Li et al., J Biol Chem., 287 (2012) 13713-13721; Deffar et al., African Journal of Biotechnology Vol.8(12), pp.2645-2652, 17 June, 2009 and WO94 / 04678.

[0307] As is well known in the art for VH and VHH domains, the total number of amino acid residues in each CDR may be different and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions numbered according to Kabat may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. Other numbering systems or coding conventions for VHH include Chothia, IMGT, and AbM.

[0308] "Humanized antibodies," also known as CDR-grafted antibodies, are antibodies produced by transplanting non-human CDR sequences into a human antibody variable region framework. This can overcome the strong immune response induced by chimeric antibodies due to the presence of a large number of non-human protein components. To avoid a decrease in immunogenicity and activity, minimal reverse mutations can be performed on the fully human antibody variable region to maintain activity. Examples of "humanization" include: replacing one or more amino acid residues in a Camelidae-derived VHH domain with those in the original VHH sequence at positions corresponding to those in a conventional tetrapeptide-structured antibody VH domain (also referred to as "sequence optimization" in this disclosure; in addition to humanization, "sequence optimization" may also encompass other modifications to the sequence by one or more mutations that provide improved VHH properties, such as removal of potential post-translational modification sites). A humanized VHH domain may contain one or more fully human framework region sequences. In some embodiments, it may contain human framework region sequences from IGHV3. Another example of "humanization" includes transplanting the CDR sequence of a mouse to the antibody variable region framework of a person, i.e., antibodies produced in different types of human germline antibody framework sequences. The strong antibody variable antibody reaction induced by carrying a large amount of heterologous protein components can be overcome in chimeric antibodies. Humanization methods such as protein surface amino acid humanization (resurfacing) and antibody humanization universal framework transplantation (CDR grafting to a universal framework) are about to "transplant" CDRs on other "supports" (including but not limited to human supports or non-immunoglobulin supports). Supports and technologies suitable for the CDR transplantation are known in the art. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database, as well as in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies of the present disclosure also include humanized antibodies further subjected to affinity maturation of CDRs by phage display. In addition, in order to avoid a decrease in activity caused by a decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity.

[0309] An "affinity matured" antibody is one that has one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody, such alterations resulting in an improvement in the affinity of the antibody for the antigen. For example, an "affinity matured" PD-1 binding protein or PD-1 antibody has one or more alterations in one or more CDRs that result in an increase in affinity for the antigen compared to its parent antibody. Affinity matured antibodies can be prepared by methods known in the art, for example, as described in Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813; Shier et al., 1995, Gene 169:147-155; Yelton et al., 1995, Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al., 1992, J. MoI. Biol. 226(3):889-896; KS Johnson and RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press 1996.

[0310] "Fully human antibodies" include antibodies with variable and constant regions of human germline immunoglobulin sequences. Fully human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). "Fully human antibodies" do not include "humanized antibodies."

[0311] Generally, "specific binding" or "selective binding" refers to binding of a binding protein to an epitope on an antigen. The PD-1 binding protein, PD-1 / PVRIG binding protein, PD-1 / TIGIT binding protein, PD-1 / PVRIG / TIGIT binding protein of the present disclosure will be measured as in Biacore or KinExA or Fortibio assays, preferably 10 -7 to 10 -10 Mole / liter (M), more preferably 10 -8 to 10 -10 mol / L, even more preferably 10 -9 to 10 -10 or lower dissociation constant (K D ), and / or with at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10-9 M, more preferably at least 10 -10 The association constant (KA) of M binds to the antigen (i.e., PD-1, PVRIG and / or TIGIT) or its epitope. Any -4 M's K D Values ​​are generally considered to indicate nonspecific binding. Specific binding of a binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competitive assays) as described herein.

[0312] "Epitope" refers to a site on an antigen that binds to an immunoglobulin or antibody. An epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost after treatment with denaturing solvents. An epitope generally comprises at least 3-15 amino acids in a unique spatial conformation. Methods for determining the binding of an epitope to a given antibody are well known in the art and include immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and the techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0313] "Binding affinity" or "affinity" is used in this disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or portion thereof and an antigen). The binding affinity between two molecules can be determined by determining the dissociation constant (K D K can be determined by measuring the kinetics of complex formation and dissociation using, for example, surface plasmon resonance (SPR) methods (Biacore). D The rate constants corresponding to the association and dissociation of a monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. D Through equation K D = kd / ka is related to ka and kd. The value of the dissociation constant can be determined directly by well-known methods and can even be calculated for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9: 340-362). For example, K can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). DOther standard assays for assessing the binding ability of an antibody to a target antigen are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays exemplified in this disclosure. The binding kinetics and binding affinity of an antibody can also be determined by standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using Biacore TM The K of each antibody / antigen complex can be compared by comparing the K D The K values ​​can be used to compare the binding affinities associated with different molecular interactions, for example, the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the K values ​​of the interactions of interest (e.g., the specific interaction between an antibody and an antigen). D The K values ​​are compared with those of non-target interactions (e.g., control antibodies known not to bind PD-1). D The value is evaluated.

[0314] A "conservative substitution" refers to a substitution with another amino acid residue having properties similar to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have non-polar side chains. In addition, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when substituting an amino acid residue in a group that exhibits similar properties as described above, it will not exhibit specific changes in properties.

[0315] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same nucleotide or amino acid monomer, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0316] "Inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blocking. "Inhibit growth" (eg, involving cells) is intended to include any measurable decrease in cell growth.

[0317] "Arresting the growth of" or "growth inhibition" refers to inhibiting the growth or proliferation of cells.

[0318] "Proliferative disease" refers to a disorder associated with some degree of abnormal cell proliferation. In one embodiment, the proliferative disorder is cancer.

[0319] "Tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. "Cancer," "cancerous," "proliferative disorders," and "tumor" are not mutually exclusive when referred to in this disclosure.

[0320] "Preventing cancer" refers to delaying, inhibiting, or preventing the onset of cancer in a subject in whom the onset of cancer or tumorigenesis has not been demonstrated, but in whom a predisposition to cancer has been identified, for example, by genetic screening or other methods. This also includes treating a subject with a precancerous condition to halt the progression of the precancerous condition to a malignant tumor or to cause its regression.

[0321] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. "And / or" should be taken as specifically disclosing that each of the two specified features or components has or does not have the other. Thus, the term "and / or" as used in phrases such as "A and / or B" in this disclosure includes "A and B," "A or B," "A" (alone), and "B" (alone). Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc. should be understood to have an inclusive sense, rather than an exclusive or exhaustive sense; that is, the sense of "including but not limited to."

[0322] As used herein, the term "about" or "approximately" refers to a value that is within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which value depends in part on how the value is measured or determined (i.e., the limitations of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation per practice in the art. Alternatively, "about" or "substantially comprising" can mean a range of up to ±30%, for example, a pH of about 5.5 means pH 5.5 ± 1.65. In addition, particularly for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times a value. Unless otherwise indicated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0323] "Buffer" refers to a buffer that tolerates changes in pH through the action of its acid-base conjugate components. Examples of buffers that control pH within an appropriate range include tris, acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0324] A "histidine salt buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, and the like. Preferred are histidine hydrochloride buffers or histidine acetate buffers. Histidine acetate buffers are prepared from histidine and acetic acid, and histidine hydrochloride buffers are prepared from histidine and histidine hydrochloride, or histidine and hydrochloric acid.

[0325] "Citrate buffer" is a buffer comprising citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. A preferred citrate buffer is citric acid-sodium citrate buffer.

[0326] A "pharmaceutical composition" refers to a mixture containing one or more antibodies described herein with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to maintain the stability of the active ingredient, facilitate administration to an organism, and promote absorption of the active ingredient to exert its biological activity.

[0327] In the present disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0328] Unless otherwise specified, the solvent in the solution form of the pharmaceutical composition described in the present disclosure is water.

[0329] "Lyophilized formulation" refers to a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a liquid or solution formulation has been subjected to a vacuum freeze-drying step.

[0330] The pharmaceutical compositions disclosed herein can achieve a stable effect: the PD-1 / PVRIG / TIGIT binding protein substantially retains its physical stability and / or chemical stability and / or biological activity after storage. For example, the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, there are various analytical techniques for measuring protein stability after storage at a selected temperature for a selected period of time.

[0331] A stable pharmaceutical antibody formulation is one in which no significant change is observed after storage at refrigerated temperatures (2-8°C) for at least 3 months, at least 6 months, at least 1 year, at least 2 years, or up to 2 years. In addition, a stable liquid formulation includes a liquid formulation that exhibits desired characteristics after storage at 25°C for a period of 1 month, 3 months, 6 months, or 1 month at 40°C. Typical acceptable standards for stability are as follows: typically no more than about 10%, preferably no more than about 5%, of the antibody monomers degrade as measured by SEC-HPLC. By visual analysis, the pharmaceutical antibody formulation is colorless or clear to slightly opalescent. The concentration, pH, and weight-gram molecular osmotic pressure concentration of the formulation vary by no more than ±10%. Typically, no more than about 10%, preferably no more than about 5%, of truncation is observed, and typically no more than about 10%, preferably no more than about 5%, of aggregation is formed.

[0332] An antibody "retains its physical stability" in a pharmaceutical formulation if it shows no significant increase in aggregation, precipitation, and / or denaturation as measured by visual inspection of color and / or clarity, or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).

[0333] An antibody "retains its chemical stability" in a pharmaceutical formulation if it shows no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide mapping in combination with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.).

[0334] An antibody "retains its biological activity" in a pharmaceutical formulation if its biological activity at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared. The biological activity of an antibody can be determined, for example, by antigen binding assays.

[0335] "Administer," "give," and "treat," as they apply to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administer," "give," and "treat" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "give," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. "Treatment," as it applies to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0336] "Treatment" means administering an internal or external therapeutic agent, such as any of the antibodies disclosed herein or pharmaceutical compositions thereof, to a subject who has, is suspected of having, or is predisposed to having one or more proliferative diseases or symptoms thereof, and for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the treated subject or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of the therapeutic agent that effectively alleviates any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other health care professionals to evaluate the severity or progression of the symptoms. Although an embodiment of the present disclosure (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the symptoms of the target disease in a certain subject, it should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0337] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount for a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or a human subject.

[0338] The "subject" and "patient" of the present disclosure refer to mammals, especially primates, and especially humans.

[0339] The equipment and methods used in the testing process are as follows:

[0340] Appearance:

[0341] Using the visual method, wipe the sample bottle clean and observe the sample color, clarity and visible foreign matter on a clarity tester with a white background and a black background under a light intensity of 1000-1500lx.

[0342] Appearance inspection instrument: Jingtuo Instrument YB-2A clarity tester.

[0343] SEC size exclusion chromatography:

[0344] An analytical method that separates solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecules.

[0345] SEC monomer content percentage = A monomer / A total * 100% (A monomer is the peak area of ​​the main peak monomer in the sample, and A total is the sum of all peak areas.)

[0346] SEC instrument: Agilent 1260-Bio; chromatographic columns: Waters, XBrige SEC (300×7.8mm 3.5μm)

[0347] NR-CE capillary gel electrophoresis:

[0348] A method of electrophoresis in which gel is transferred to a capillary tube as a supporting medium and samples are separated according to their molecular weight at a certain voltage.

[0349] Non-reduced CE purity percentage = A main peak / A total * 100% (A main peak is the peak area of ​​the main peak in the sample, and A total is the sum of all peak areas.

[0350] CE test instrument: Sciex model PA800 plus

[0351] icIEF imaging capillary isoelectric focusing electrophoresis:

[0352] A technique for separating proteins based on their isoelectric points (pI).

[0353] icIEF main peak content percentage = main peak area / total area*100% (total area is the sum of the areas of acidic peak, main peak and basic peak).

[0354] The instrument used for icIEF determination was manufactured by Protein Simple, model number: Muarice.

[0355] Protein concentration determination:

[0356] Protein concentration was determined using a UV-visible spectrophotometer (Nano Drop 2000) with a pathlength of 1 mm.

[0357] Exemplary Antibody Pharmaceutical Composition (Formulation) Preparation Process

[0358] Step 1: Mix the PD-1 / PVRIG / TIGIT binding protein with the following excipients in the prescribed amounts to prepare a stock solution containing the PD-1 / PVRIG / TIGIT binding protein. After filtration, sample the solution for sterility testing. Filter the stock solution through a 0.22 μm filter cartridge and collect the filtrate.

[0359] Step 2: Adjust the filling volume to 1.15 mL, fill the filtrate into a 2 mL vial, add a stopper, and take samples at the beginning, middle, and end of filling to detect the difference in filling volume.

[0360] Step 3: Turn on the capping machine, add aluminum caps, and start capping.

[0361] Step 4: Visual inspection to confirm that the product has no defects such as inaccurate filling quantity. Print and apply vial labels; print carton labels, folding cartons, packing, and affixing box labels.

[0362] 2. Implementation and Test Examples

[0363] The present disclosure is further described in detail by the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0364] Experimental methods in the examples disclosed herein that do not specify specific conditions are generally performed under conventional conditions or according to the conditions recommended by the raw material or product manufacturers. Reagents without specific sources are commercially available.

[0365] Example

[0366] The preparation and purification methods of the PD-1 / PVRIG / TIGIT binding protein in this application have been described in patent document No. WO2023040945, the entire content of which is incorporated into the present disclosure.

[0367] Example 1. Screening, preparation and functional verification of anti-PD-1 nanobodies

[0368] In this example, human PD-1 with a His tag was used as the immune antigen, and human PD-1 and cynomolgus monkey PD-1 labeled with biotin were used as screening antigens, all of which were purchased from AcroBiosystems.

[0369] Example 1-1. Screening and preparation of anti-PD-1 nanobodies

[0370] 1. Immunization of antigens, screening of antigen sequences and preparation

[0371] Table 2. Information on immunized alpacas and PD-1 antigen used for screening

[0372] The sequence of human PD-1 protein (NP_005009.2) is as follows, where the underlined portion is the extracellular region sequence, with amino acids starting and ending at Leu25-Gln 167.

[0373] >Human PD-1 amino acid sequence

[0374] 2. Alpaca Immunization, Nanobody Yeast Display Library Construction, and Antibody Screening

[0375] 1) Alpaca Immunization

[0376] Two healthy alpacas were immunized. For the initial immunization on day 0, 0.25 mg of human PD-1 antigen was mixed with 1 mL of Freund's complete adjuvant (CFA) and injected subcutaneously. On days 21, 42, and 63, 0.125 mg of human PD-1 antigen was mixed with 1 mL of Freund's incomplete adjuvant (IFA) and injected subcutaneously. 10 mL, 50 mL, and 50 mL of blood were collected on days 28, 49, and 70, respectively. Serum titers were tested, and alpaca peripheral lymphocytes that met the library construction standards were used to construct a nanoantibody yeast display library.

[0377] 2) Yeast library construction

[0378] 50 mL of peripheral blood was collected from two alpacas after the third and fourth immunizations, and PBMCs were isolated from the peripheral blood. RNA was extracted from the PBMCs and reverse transcribed to obtain total cDNA. The cDNA was used as the template for the first round of nested PCR reaction. After two rounds of nested PCR amplification, the second round of nested PCR product fragment was ligated with a yeast library vector (yeast library vector pYDN3), and the ligated product was electroporated into yeast competent cells. The insertion rate and library diversity were verified by colony PCR. Based on the number of library transformants, library insertion rate, and library diversity, the sequencing results showed that the library capacity of one alpaca was 9.28×10 7 , the other alpaca library has a capacity of 1.54×10 8 .

[0379] 3) Nanobody (VHH) screening

[0380] For this yeast display system, flow cytometry (FACS) sorting, library screening, and identification were used to screen antibodies. Two rounds of sorting were used: the first round used biotinylated human PD-1 protein (Avitag-His Tag) magnetic beads for enrichment, and the second round used a flow cytometer to sort biotinylated monkey PD-1 protein (His, Avitag) (the flow cytometric antibodies were: Mouse anti-HA tag Dylight 488; streptavidin Dylight 650). After the two rounds of sorting, monoclonal identification, sequencing, and sequence analysis were performed to obtain 18 unique VHH sequences that cross-bind between human and monkey PD-1 antigens. The A17 sequence is shown below.

[0381] >A17 variable region

[0382] Table 3. CDRs of anti-PD-1 nanobodies (Kabat numbering convention)

[0383] The above sequence was linked to a human IgG4 Fc fragment (including the hinge region and with S228P, according to the Eu numbering system) to construct a VHH-Fc antibody. The plasmid was constructed, transiently transfected into HEK293 cells, expressed, and purified using a protein A column. The column was washed with PBS and eluted with 0.1M glycine buffer, pH 2.5. The column was dialyzed into PBS buffer, pH 7.4. The sequences of the human IgG4 Fc (including the hinge region) and A17-based VHH-Fc antibody are as follows:

[0384] >hIgG4

[0385] >A17_hIgG4

[0386] Example 1-2. Affinity identification of anti-PD-1 nanobody and antigen PD-1

[0387] 1.ELISA

[0388] Use PBS to dissolve human PD-1 protein (his tag) to 1μg / mL, add 100uL / well to a 96-well plate, and incubate at 4°C overnight to coat the antigen. Wash three times with PBST (PBS+0.05% tween20) solution. Add PBST / 1% BSA solution and incubate at 37°C for 1h for blocking. After washing three times with PBST solution, add different concentrations of candidate PD-1VHH-Fc antibodies (diluted with PBST / 1% BSA solution) and incubate at 37°C for 1.5 hours. Wash three times with PBST solution. Add 100μL HRP-conjugated anti-human IgG4 Fc secondary antibody (Thermo) solution and incubate at 37°C for 1 hour. After washing three times with PBST solution, add 100μL / well TMB solution, react at room temperature for 5min, add 50μL stop solution, and then use an enzyme reader to read the 450nM value and calculate the EC 50 ,

[0389] The results are shown in Table 4, which show that A17_hIgG4 has a strong binding affinity with PD-1 antigen.

[0390] Table 4. EC of anti-PD-1 nanobody detected by ELISA 50 value

[0391] 2.FACS

[0392] To detect the binding ability of anti-PD-1 nanobody to PD-1 expressed on cell membrane, different concentrations of candidate anti-PD-1 VHH-Fc antibody were added to 10 5100 μL of anti-human IgG Fc fluorescent secondary antibody (Biolegend) was added to Jurkat cells (Cat: J1250, Promega) overexpressing human PD-1 at room temperature for 20 minutes and washed twice with PBS. The cells were then incubated at room temperature for 20 minutes and washed twice with PBS. The cells were resuspended in 250 μL of PBS and the fluorescence signal was detected by FACS. EC 50 .

[0393] Pembrolizumab (purchased from Bio-Bio) was used as a control, and the results are shown in Table 5. In addition, the EC 50 The value was 0.3568 nM, which was significantly better than other antibodies obtained in the simultaneous screening (results not shown).

[0394] Table 5. ECs of anti-PD-1 nanobody binding to cell surface PD-1 detected by FACS 50 value

[0395] Example 1-3. Sequence modification of anti-PD-1 nanobody

[0396] 1. Humanization and reversion mutation of anti-PD-1 nanobodies

[0397] Antibody humanization was performed using the CDR-grafting method. The parental anti-PD-1 antibody was aligned with fully human germline genes in the IMGT or NCBI / igblast databases. Human germline genes IGHV 3-23 (IGHV3-23*01; IGHV3-23*04), which are highly homologous to the PD-1 nanobody, were selected as humanization templates. The CDRs were grafted, retaining four key residues in FR2 of the nanobody (Y37, E44, R45, and L47). Key core residues near the CDR regions and Vernier position residues interacting with the CDRs were backmutated to generate humanized antibodies A17h1, A17h2, A17h3, and A6h1.

[0398] >A17h1 variable region

[0399] >A17h2 variable region

[0400] >A17h3 variable region

[0401] 2. Remove TCE (T cell epitope), reduce antibody deamidation, and reduce antibody isomerization modification

[0402] To reduce the potential immunogenicity risk of the antibody, TCE removal was performed. To improve the drugability of the antibody, antibody deamidation and antibody isomerization site modifications were performed. The following sequence was obtained:

[0403] >A17m01 variable region

[0404] >A17m02 variable region

[0405] >A17m03 variable region

[0406] >A17m04 variable region

[0407] >A17m05 variable region

[0408] >A17m06 variable region

[0409] >A17m07 variable region

[0410] >A17m08 variable region

[0411] >A17m09 variable region

[0412] >A17m10 variable region

[0413] >A17m11 variable region

[0414] >A17m12 variable region

[0415] >A17m13 variable region

[0416] >A17m14 variable region

[0417] >A17m15 variable region

[0418] Furthermore, A17m09 was further selected to germline the antibody sequence to improve the degree of humanization, and the following sequence was obtained:

[0419] >A17m0901 variable region

[0420] >A17m0902 variable region

[0421] >A17m0903 variable region

[0422] >A17m0905 variable region

[0423] That is, A17 of the present disclosure has the following general sequence:

[0424] CDR1: DYSMS (SEQ ID NO: 3)

[0425] CDR2: IISGSGX1X2X3HYVDSVKG, wherein X1 is selected from V or G, X2 is selected from I or S, and X3 is selected from T or A (SEQ ID NO: 30)

[0426] CDR3: VSDWX4X5Y, wherein X4 is selected from D or E, and X5 is selected from D or E (SEQ ID NO: 31).

[0427] Specifically, CDR2 can be:

[0428] IISGSGVIAHYVDSVKG (SEQ ID NO: 4)

[0429] IISGSGVITHYVDSVKG (SEQ ID NO: 32)

[0430] IISGSGGIAHYVDSVKG (SEQ ID NO: 33)

[0431] IISGSGVSAHYVDSVKG (SEQ ID NO: 34)

[0432] IISGSGGITHYVDSVKG (SEQ ID NO: 79)

[0433] Specifically, CDR3 can be:

[0434] VSDWDDY (SEQ ID NO: 5)

[0435] VSDWEDY (SEQ ID NO: 35)

[0436] VSDWDEY (SEQ ID NO: 36).

[0437] Example 1-4. Detection of the blocking effect of anti-PD-1 nanoantibodies on PD-L1 binding to PD-1 by PD-1 / PD-L1 reporter gene system

[0438] The PD-1 / PD-L1 NFAT gene reporter system (Cat: J1250, Promega) can be used to detect the biological activity of anti-PD-1 antibodies at the cellular level. The system consists of two genetically engineered cell lines: Jurkat effector cells that overexpress PD-1 (containing the NFAT reporter gene luciferase) and CHO-K1 antigen-presenting cells that overexpress PD-L1. When these two cells are co-cultured, the PD-1 / PD-L1 interaction inhibits TCR-mediated NFAT activation, thereby inhibiting the expression of the NFAT reporter gene luciferase. When the PD-1 / PD-L1 interaction is disrupted, TCR activation induces luciferase expression through the NFAT pathway. The degree of effector cell activation and the biological activity of the antibody can be measured by adding Bio-Glo reagent and using a luminometer for quantitative fluorescence detection.

[0439] CHO-K1 cells overexpressing PD-L1 were diluted to 4 × 10 5 / mL, add 100 μL to each well of a 96-well plate and culture overnight. After aspirating the culture medium, quickly add 40 μL of 1.25×10 6 / mL PD-1 Jurkat cells and 40μL anti-PD-1 nanobody solution (1640+2% FBS solution dilution) of different concentrations were incubated at 37°C for 6 hours, returned to room temperature, and 40μL Bright-glo reagent (Cat: E2620, Promega) was added to each well. The cells were shaken at 350rpm in the dark for 5 minutes. After standing at room temperature in the dark for 5 minutes, the fluorescence value was read using a microplate reader and the IC was calculated. 50 .

[0440] Pembrolizumab and hIgG4 isotype were used as controls. As shown in Figure 1A and Table 6, the biological functional activity IC of A17_hIgG4 50 The value was 1.199 nM, which was significantly better than other antibodies screened simultaneously in the present disclosure (e.g., A3_hIgG4 was 7.62, A13_hIgG4 was 3.208; the sequences of A3_hIgG4 and A13_hIgG4 are not shown). Among them, A6_hIgG4 is another antibody screened in the present application.

[0441] Table 6. IC values ​​of anti-PD-1 nanoantibodies detected using the PD-1 / PD-L1 NFAT reporter gene system 50 value

[0442] The above method was used to functionally verify the humanized anti-PD-1 nanobody. See Figure 1B and Table 7. The IC of A17h1_hIgG4 50The activity is similar to that of the positive antibody Pembrolizumab. Among the candidate molecules after sequence modification, the biological activity IC 50 The values ​​were 0.5307nM and 0.4352nM, respectively, which were better than Pembrolizumab.

[0443] Table 7. IC values ​​of modified anti-PD-1 nanobodies detected by PD-1 / PD-L1 NFAT reporter gene system 50

[0444] Example 1-5. Identification of the binding affinity of the modified anti-PD-1 nanobody to the antigen PD-1

[0445] A Biacore 8k (GE Healthcare) instrument was used to detect the affinity of anti-PD-1 nanoantibodies to the PD-1 antigen using surface plasmon resonance (SPR). A CM5 sensor chip was used for the experiment, and the mobile phase was HBS-EP+ buffer solution (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20). Anti-human IgG (Fc) antibody was prepared into a 30μg / mL solution using 10mM sodium acetate buffer (pH 5.0), and the Immobilization program was selected to automatically couple the anti-human IgG (Fc) antibody channel to amino groups. Each antibody to be tested was prepared as a ligand using HBS-EP+ buffer solution and captured using the anti-human IgG (Fc) antibody on the chip channel. Human or cynomolgus monkey PD-1 antigen protein (Sino Biological, 10377-H08H; 90311-C08H) was used as the analyte and prepared with HBS-EP+ buffer solution. The analyte was diluted 2-fold and flowed through the experimental channel and reference channel at a flow rate of 30 μL / min. The binding time was 1 min and the dissociation time was 15 min. The regeneration buffer 10 mM Glycine pH 1.5 (GE Healthcare, BR-1003-54) was run at a flow rate of 10 μl / min for 30 s. The association rate Ka and dissociation rate Kd, as well as the dissociation constant (i.e., affinity K) were calculated. D ). The results are shown in Tables 8 and 9.

[0446] Table 8. SPR affinity data of anti-PD-1 nanobody binding to human PD-1 antigen

[0447] Table 9. SPR affinity data of anti-PD-1 nanobody binding to cynomolgus monkey PD-1 antigen

[0448] The results of binding to human PD-1 antigen showed that the humanized antibody A17h1_hIgG4 and the subsequently modified and optimized antibodies A17m09_hIgG4, A17m0901_hIgG4, and A17m0902_hIgG4 had similar K values ​​to Pembrolizumab. D The dissociation rates of A17h1_hIgG4, A17m09_hIgG4, A17m0901_hIgG4, and A17m0902_hIgG4 were all slower than those of pembrolizumab. The antibodies A17h1_hIgG4, A17m09_hIgG4, A17m0901_hIgG4, and A17m0902_hIgG4 cross-bind to monkey PD-1 and have similar affinities to human and monkey PD-1.

[0449] Example 1-6. DC: T mixed lymphocyte reaction (MLR) test of the in vitro efficacy of the modified anti-PD-1 nanobody

[0450] Co-culturing mature dendritic cells (DCs) with allogeneic T cells can activate T cells and promote their cytokine secretion. This process is inhibited by PD-1 / PD-L1 signaling, and anti-PD-1 antibodies can relieve this inhibition and promote T cell activation. Therefore, the DC:T mixed lymphocyte reaction assay can be used to test the in vitro efficacy of anti-PD-1 antibodies.

[0451] Monocytes were isolated from fresh peripheral blood (PBMC) of a healthy individual using a sorting kit (cat: 19359, stemcell). Monocytes were induced to differentiate into mature DC cells after seven days of culture using a DC cell induction kit (cat: 10985, stemcell). Allogeneic T cells were then isolated from fresh PBMC of another healthy individual using a sorting kit (cat: 17951, stemcell). 5,000 mature DC cells were co-cultured with 50,000 allogeneic T cells, and candidate antibodies of corresponding concentrations were added. After six days of co-culture, the cell supernatant was aspirated, and the IFN-γ concentration was detected using a Cisbio-HTRF IFN-γ detection kit (Cat: 62HIFNGPEH, PerkinElmer), and the EC 50 .

[0452] As shown in Figure 2, A17m09_hIgG4 showed in vitro efficacy that was basically consistent with Pembrolizumab in the MLR experiment. 50The EC of pembrolizumab is 0.2931 nM. 50 It is 0.3271nM.

[0453] Example 1-7. Anti-PD-1 Nanobody Inhibits Tumor Growth in Mouse Colon Cancer Model

[0454] This example verifies the in vivo anti-tumor efficacy of anti-PD-1 antibodies in the MC38 tumor model of humanized PD-1 C57BL / 6 mice.

[0455] MC38 mouse colon cancer cell line was cultured in DMEM medium (10% FBS) and 5×10 5 MC38 cells were inoculated subcutaneously in humanized PD-1 C57BL / 6 female mice (Jicui Yaokang) until the average tumor volume of the mice reached 80 mm 3 Around 60 days, the mice were randomly divided into 8 groups and given intraperitoneal injections of the antibody or control. Tumor volume was measured and body weight was measured twice weekly, and data were recorded. The experimental grouping and dosing schedule are shown in Table 10. Considering that the molecular weight of A17m09_hIgG4 is approximately 75 kDa, only half that of normal IgG, the dose was adjusted to half that of pembrolizumab to achieve an equimolar dose.

[0456] As shown in Figures 3A and 3B, at equivalent molar doses, the in vivo antitumor efficacy of A17m09_hIgG4 was consistent with that of pembrolizumab. Tumor volume and tumor inhibition rate are shown in Table 11.

[0457] Table 10. Mouse experimental groups and dosing regimens

[0458] Table 11. Mouse tumor volume and tumor inhibition rate

[0459] (Note: ip: intraperitoneal injection; BIW*7: twice a week, a total of 7 times; compared with the control group, *P<0.05, **P<0.01, ***P<0.001 were considered to be significantly different)

[0460] Example 2. Anti-PVRIG / TIGIT bispecific antibody

[0461] In this example, recombinant human PVRIG with a histogram tag (h-PVRIG-his), recombinant human PVRIG with a mouse IgG2a Fc tag (h-PVRIG-mIgG2a Fc), and recombinant mouse PVRIG with a human IgG1 Fc tag (m-PVRIG-hIgG1 Fc) were purchased from Acrobiosystems. His-tagged PVRL2 was purchased from AcroBiosystems (PV2-H52E2).

[0462] Example 2-1. Screening and preparation of anti-PVRIG nanobodies

[0463] 1. Sequence and preparation of immune antigens and screening antigens

[0464] Table 12. Amino acid sequence of recombinant protein

[0465] The sequence of the his-tagged cynomolgus monkey PVRIG (cyno-PVRIG-his) recombinant protein is as follows:

[0466] 2. Alpaca Immunization, Nanobody Phage Display Library Construction, and Antibody Screening

[0467] Following the method of Example 1-1, alpacas were immunized with his-tagged human PVRIG recombinant protein (h-PVRIG-his). PBMCs were isolated from peripheral blood on day 56 of the camel, RNA was extracted, and reverse transcribed into cDNA to construct a phage library of anti-human PVRIG nanobodies. After three rounds of screening, 400 clones were sequenced, and the variable region sequences of two of these clones are shown below, with the CDRs shown in Table 13.

[0468] >151 variable regions

[0469] Table 13. CDRs of anti-PVRIG nanobodies (Kabat numbering convention)

[0470] The above antibody variable regions were connected to the human IgG4 heavy chain Fc region, which includes the hinge region and carries S228P, F234A, L235A, and K447A mutations (Eu nomenclature system) to construct a full-length antibody.

[0471] >hIgG4 Fc(S228P / F234A / L235A / K447A)

[0472] >151 full length

[0473] The anti-PVRIG antibody CPA.7.021, described in WO2016134333, was screened from an antibody phage library. Its subtype is IgG1, and it binds well to human PVRIG but not to cynomolgus monkey PVRIG. The heavy and light chain variable regions of CPA.7.021 were ligated to the human IgG4 heavy chain constant region (with S228P, F234A, L235A, and K447A mutations) and the human kappa light chain constant region, respectively, to construct the positive antibody Tab5.

[0474] >Tab5 heavy chain full length

[0475] >Tab5 light chain full length

[0476] The nucleic acid sequence expressing the above amino acid sequence was cloned into the pcDNA3.1 expression vector, and the antibody was expressed and purified according to conventional methods. After detection, the target antibody was obtained.

[0477] Example 2-2. Identification of affinity between anti-PVRIG nanobodies and antigen PVRIG

[0478] 1.ELISA

[0479] The his-tagged PVRIG recombinant protein was directly coated and the antibody was added. The binding activity of the antibody to the antigen was detected by adding the secondary antibody (HRP-conjugated anti-primary antibody Fc antibody) and the HRP substrate TMB.

[0480] Human, cynomolgus monkey or mouse PVRIG protein was coated on a 96-well plate, 100 μL was added to each well at a concentration of 1 μg / mL, and incubated overnight at 4°C. Wash three times with the washing solution. Add 300 μL / well of blocking solution (PBS + 0.05% Tween20 + 1% BSA) and incubate at room temperature for 1 hour. Wash three times with the washing solution. Add 100 μL of anti-PVRIG test antibody diluted in diluent to each well. Incubate at 37°C for 1 hour. Wash three times with the washing solution. Add 100 μL of HRP-labeled anti-human IgG secondary antibody (Sigma, A8667) to each well. Incubate at 37°C for 1 hour. Wash three times with the washing solution. Add 100 μL of TMB to each well and react in the dark for 15 minutes. Add 50 mL / well of 0.16 M sulfuric acid. Read the OD450 using a Thermo MμLtiSkanFc microplate reader to calculate the binding EC value of the anti-PVRIG antibody to the PVRIG recombinant protein. 50 All antibodies in Table 14 have strong binding ability to human or cynomolgus monkey PVRIG recombinant protein, but do not bind to mouse PVRIG recombinant protein.

[0481] Table 14. Binding assay results of anti-PVRIG antibodies to PVRIG recombinant proteins from different species

[0482] 2. FACS detection

[0483] Prepare HEK293 stable cell lines expressing human or cynomolgus monkey PVRIG gene. Inoculate 2x10 cells per well in a 96-well plate. 5 Cells were centrifuged at 300 g for 5 minutes, the supernatant was removed, 100 μL of the test antibody was added, and the cells were incubated at 4°C for 1 hour. The supernatant was removed by centrifugation, and the cells were washed three times with 200 μL of washing solution (PBS + 2% FBS). 100 μL of a 1:500 dilution of an anti-human IgG secondary antibody labeled with Alexa Fluor 488 (Invitrogen, A-11013) was added and the cells were incubated at 4°C for 1 hour. The supernatant was removed by centrifugation, and the cells were washed three times with 200 μL of washing solution (PBS + 2% FBS). The cells were resuspended in 100 μL of PBS and analyzed by flow cytometry (BD FACS Calibur or BD FACS Canto_II). All antibodies showed strong binding to human or cynomolgus monkey PVRIG expressed on the cell surface, significantly stronger than the positive antibody Tab5, which did not even bind to cynomolgus monkey PVRIG at all.

[0484] Table 15. Cell binding assay results of anti-PVRIG antibodies against PVRIG from different species

[0485] 3. Fortebio test

[0486] A Protein A biosensor (Fortebio, #18-5010) was soaked in 200 μL of KB buffer (PBS, pH 7.4, 0.02% tween-20, 0.1% BSA) for 60 seconds to wet the sensor. Anti-PVRIG antibody was then diluted to 10 μg / mL in KB buffer, and the sensor was placed in 200 μL of this solution. The sensor was then immersed in KB buffer for 100 seconds to elute excess antibody. His-tagged human PVRIG was diluted in KB buffer in a two-fold gradient to concentrations ranging from 64 nM to 4 nM. The sensor was allowed to bind in this solution for 300 seconds and then dissociate in KB buffer for 600 seconds. Using a dynamic 1:1 binding model, the affinity of the anti-PVRIG antibody for human PVRIG is shown in Table 16.

[0487] The results showed that all tested antibodies had high affinity to human PVRIG.

[0488] Table 16. Affinity of anti-PVRIG antibodies to human PVRIG

[0489] Example 2-3. Function and activity verification of anti-PVRIG nanobody

[0490] 1. Anti-PVRIG Nanobody Blocking PVRIG and PVRL2 Binding Experiment

[0491] Human PVRIG recombinant protein (h-PVRIG-mIgG2a Fc) was coated onto a 96-well plate at a concentration of 1 μg / mL, with 100 μL per well added. The plates were incubated overnight at 4°C. The plates were washed three times with the wash buffer. 300 μL / well of blocking buffer was added and incubated at room temperature for 1 hour. The plates were washed three times with the wash buffer. 50 μL of diluted anti-PVRIG test antibody and 50 μL of the his-tagged ligand PVRL2 were added to each well and incubated at 37°C for 1 hour. The plates were washed three times with the wash buffer. 100 μL of a 1:2000 diluted HRP-conjugated anti-his-tag secondary antibody (Genscrpit) was added to each well. The plates were incubated at 37°C for 1 hour. The plates were washed three times with the wash buffer. 100 μL of TMB was added to each well and the plates were reacted in the dark for 15 minutes. 50 μL of 0.16 M sulfuric acid was added to each well. The OD value at 450 nm was read using a Thermo MμLtiSkanFc microplate reader, and the IC value of the anti-PVRIG antibody blocking the binding of PVRIG to PVRL2 was calculated. 50 value.

[0492] The results in Table 17 show that all the antibodies tested can strongly inhibit the binding of human PVRIG to human PVRL2.

[0493] Table 17. Blocking experiments of antibodies against human PVRIG / PVRL2 binding

[0494] 2. Anti-PVRIG Nanobody Reporter Gene Cell Activity Experiment

[0495] First, a plvx-OS8 (G418-resistant) plasmid was constructed and transfected into 293F cells. G418 screening was performed, and the expression of OS8 in cloned cells was detected by flow cytometry. At the same time, the activation of Jurkat cells by OS8 was detected. Clones with moderate activation were selected to obtain the 293F-OS8 cell line. A plvx-PVRL2 plasmid was constructed and used to infect 293F-OS8 cells. The clones with the highest PVRL2 expression were screened by flow cytometry to obtain the 293F-OS8-PVRL2 cell line.

[0496] Secondly, plvx-NFAT-Luc (Hygromycin resistance) was constructed, packaged into lentivirus, and infected Jurkat E6.1 cells. Hygromycin-resistant clones were screened out, and clones with moderate Luciferase signals were screened out with OKT3 to obtain the Jurkat-NFAT-Luc cell line. The plvx-PVRIG (Puromycin resistance) vector was constructed and packaged into lentivirus, which was used to infect Jurkat-NFAT-Luc cells. The clones with the highest PVRIG expression were screened out with flow cytometry to obtain the Jurkat-NFAT-Luc-PVRIG cell line.

[0497] 1E4 Jurkat-NFAT-Luc-PVRIG cells were incubated with the test antibody at 37°C for 20 minutes. 1E5 293F-OS8-PVRL2 cells were added and incubated at 37°C for 5 hours. The supernatant was removed by centrifugation, and Luciferase buffer (Promega, E6130) was added to lyse the cells. Fluorescence was measured. EC values ​​were calculated. 50 The in vitro cellular activity of anti-PVRIG antibodies was evaluated using the PCR amplification assay. The experimental results are shown in Table 18.

[0498] The results showed that the tested antibodies had a strong ability to activate Luciferase in Jurkat cells, and the activity was at least 10 times that of the positive antibodies, proving that these antibodies can bind to PVRIG and block the binding of PVRL2 to PVRIG.

[0499] Table 18. Results of anti-PVRIG antibody reporter gene cell activity assay

[0500] 3. NK cell killing experiment of anti-PVRIG nanobody

[0501] PVRIG is expressed on NK cells, while PVRL2 is expressed on many tumor cells (including K562 cells). Anti-PVRIG antibodies can block the binding of PVRL2 to PVRIG, thereby relieving the inhibitory effect of tumor cells on NK cell activity.

[0502] Add 50 μL (a total of 1 × 10 5 NK92 cells (human malignant non-Hodgkin's lymphoma) were cultured. 50 μL of 20 nM or 100 nM test antibody was added and incubated at 37°C for 30 minutes. Wash twice with washing buffer and resuspend to 2 × 10 5 Add 50 μL (a total of 1×10 4The human chronic myeloid leukemia K562 cells (NK92 cells) were added to make the ratio of NK92 cells to K562 cells 10:1. Incubate at 37°C for 4 hours. The killing activity was measured using the CytoTox-Glo cytotoxicity system (Promega, G9292). First, 50 μL of AAF-Glo reagent was added, incubated at room temperature for 15 minutes, and the fluorescence of K562 cells killed by NK92 cells was measured. Then 50 μL of lysis solution was added, incubated at room temperature for 15 minutes, the cells were lysed, and the fluorescence of all cells was measured. Three control groups were prepared, namely, samples including only culture medium (control group one), samples including only NK92 cells (control group two), and samples including only K562 cells (control group three), and the same operation was performed.

[0503] The killing activity was calculated according to the following formula:

[0504] Killing activity (%) = {[(R–BG)–(T–BG)–(E–BG)] / [(TL–BGL)–(T–BG)]} × 100;

[0505] Among them, R is the fluorescence value after adding AAF-Glo, BG is the fluorescence value of control group 1 after adding AAF-Glo, E is the fluorescence value of control group 2 after adding AAF-Glo, and T is the fluorescence value of control group 3 after adding AAF-Glo; TL is the fluorescence value of control group 3 after adding lysate, and BGL is the fluorescence value of control group 1 after adding lysate again.

[0506] The experimental results are shown in Table 19, indicating that all tested anti-PVRIG antibodies can significantly activate NK92 cells and kill K562 cells.

[0507] Table 19. NK cell killing experiment of anti-PVRIG antibody

[0508] 4. Mixed lymphocyte reaction (MLR) experiment of anti-PVRIG nanobody

[0509] PVRIG is expressed on T cells, while PVRL2 is expressed on DCs. Anti-PVRIG antibodies can block the binding of PVRL2 to PVRIG, relieving DC inhibition of T cells and activating T cells.

[0510] PBMCs were isolated from peripheral blood from the first individual and cultured in RPMI 1640 medium containing 10% FBS. GM-CSF (Peprotech, 300-03-100UG) and IL-4 (Peprotech, 200-04-100UG) were added at a final concentration of 50 ng / mL, and fresh medium containing cytokines was added every 2-3 days. After 6 days of culture, 1 μg / mL LPS (Sigma, L2880-25MG) was added and incubated for 24 hours. The differentiated and mature DCs were collected. PBMCs were isolated from peripheral blood from the second individual and cultured using EasySep human CD3 + CD3 T cell isolation kit (Stemcell, 17952) was used to isolate + T cells. Adjust CD3 + The density of T cells and DC cells was such that 1 × 10 5 CD3 + T cells and 2 × 10 4 Add the antibody to be tested, incubate at 37°C for 120 hours, collect the supernatant, and use ELISA kit (R&D, DY202) to detect the IFNγ content in the supernatant.

[0511] As shown in Table 20 and Figure 4 , all tested anti-PVRIG antibodies significantly activated T cells to secrete IFNγ compared to the control antibody IgG4. Furthermore, at low doses (e.g., 4 nM and 20 nM), the disclosed antibody 151 was more effective than the positive control Tab5. Antibody 30 is another antibody screened in this application.

[0512] Table 20. IFNγ secretion in mixed lymphocyte reaction with anti-PVRIG antibodies

[0513] Example 2-4. Sequence modification of anti-PVRIG nanobody

[0514] By performing three-dimensional homology modeling on selected anti-PVRIG antibody molecules and comparing the anti-PVRIG antibody sequence with the GermLine database, a highly homologous human germline template, IGHV3-7*01, was obtained. The CDRs were then transplanted into the corresponding human template. The transplanted nanobodies were subjected to further three-dimensional structural modeling and analysis. Backmutations were performed on buried residues, residues that directly interact with the CDR regions, and residues that significantly influence the conformation of the variable region. Chemically labile amino acid residues in the CDR regions were also optimized to generate a series of humanized nanobodies. The human germline templates and humanized antibody heavy chain variable region sequences for each nanobody are shown below.

[0515] >151H2 variable region

[0516] >151H4 variable region

[0517] >151H7 variable region

[0518] >151H8 variable region

[0519] >151H9 variable region

[0520] The humanized antibody heavy chain variable region was linked to the human IgG4 heavy chain Fc region to construct a full-length anti-PVRIG antibody. The heavy chain Fc region, including the hinge region, harbored either S228P / F234A / L235A / K447A or S228P / K447A mutations. The antibody was expressed and purified according to conventional methods, and the desired antibody was obtained after testing.

[0521] Example 2-5. Activity and Function Verification of Humanized Anti-PVRIG Antibodies

[0522] 1. Binding experiment of humanized anti-PVRIG antibody to PVRIG-expressing cells

[0523] According to the method of Example 2-2, the binding of anti-PVRIG antibodies to human or cynomolgus monkey PVRIG was detected by FACS. The experimental results are shown in Table 21.

[0524] Table 21. FACS binding assay results of anti-PVRIG nanobodies to PVRIG from different species

[0525] (Note: NT, not tested)

[0526] 2. Determination of the affinity of humanized anti-PVRIG antibody to PVRIG

[0527] The affinity of the humanized anti-PVRIG antibodies to the human PVRIG protein was tested using the Fortebio assay described in Example 2-2. As shown in Table 22, all antibodies had high affinity to the human PVRIG protein.

[0528] Table 22. Affinity of humanized anti-PVRIG antibodies to human PVRIG

[0529] 3. Humanized anti-PVRIG antibody reporter gene cell activity experiment

[0530] The activity of humanized anti-PVRIG antibodies in reporter cells was tested according to the method of Example 2-3. The experimental results are shown in Table 23. All antibodies listed in the table have the ability to activate Jurkat cells.

[0531] Table 23. Humanized anti-PVRIG antibody reporter gene cell activity experiment

[0532] 4. Activation of NK Cell Killing Ability of Humanized Anti-PVRIG Antibody

[0533] The NK cell activation ability of humanized anti-PVRIG antibodies was tested according to the method of Example 2-3. The experimental results are shown in Table 24. The results show that all tested antibodies have a significant ability to activate NK cells and promote NK cell killing of target cells K562.

[0534] Table 24. NK cell killing experiment of humanized anti-PVRIG antibodies

[0535] Example 2-6. Preparation of anti-PVRIG / TIGIT bispecific antibodies

[0536] To explore the effects of different anti-PVRIG / TIGIT bispecific antibody structures on antibody function, the anti-PVRIG nanobody 151 was linked to the N-terminus or C-terminus of the heavy or light chain of the anti-TIGIT antibody 1708 via a GGGGSGGGGS (SEQ ID NO: 78) linker. Four anti-PVRIG / TIGIT bispecific antibodies were formed, designated 1708-151-1, 1708-151-2, 1708-151-3, and 1708-151-4, corresponding to 151 linked to the N-terminus, C-terminus, N-terminus, and C-terminus of the heavy chain, heavy chain, light chain, and light chain, respectively. Anti-TIGIT antibody 1708 was of the human IgG4 subtype and harbored the S228P mutation (Eu nomenclature). The sequences of anti-TIGIT antibody 1708 and the bispecific antibody formed with 151 are shown in Table 25 below. The anti-TIGIT antibody sequence information is shown in Tables 26 and 27. TIGIT antibodies have been disclosed in WO2019062832A, which is incorporated by reference in its entirety.

[0537] Table 25. Sequences of the first and second polypeptide chains of anti-PVRIG / TIGIT bispecific antibodies

[0538] Table 26. Anti-TIGIT Antibody Heavy and Light Chain CDR Region Sequences (Kabat Numbering Convention)

[0539] Table 27 Anti-TIGIT Antibody Heavy Chain VH and Light Chain VL Sequences

[0540] The bispecific antibody was constructed by linking different humanized anti-PVRIG antibody variable regions (151H7, 151H8) to the N-terminus of the heavy chain of the anti-TIGIT antibody 1708, using a similar bispecific antibody structure as 1708-151-1. The double-underlined linker sequence in the following sequence is the linker sequence.

[0541] >1708-151H7 first polypeptide chain

[0542] >1708-151H8 first polypeptide chain

[0543] The second polypeptide chains of 1708-30H2, 1708-151H7, and 1708-151H8 are all identical to the light chain of 1708 (SEQ ID NO: 52).

[0544] The antibody was transiently transfected, expressed, and purified according to conventional methods, and the full-length anti-PVRIG / TIGIT bispecific antibody disclosed herein was obtained after identification, which showed good expression level and purity.

[0545] Example 2-7. Activity and Function Verification of Anti-PVRIG / TIGIT Bispecific Antibodies

[0546] 1. Binding of the bispecific antibody to human PVRIG and blocking its ligand PVRL2

[0547] Experiments were conducted according to the methods of Examples 2-2 and 2-3. The results showed that there were no statistically significant differences in the binding of bispecific antibodies 1708-151-1, 1708-151-2, 1708-151-3, and 1708-151-4 of different configurations to human PVRIG recombinant protein and cells overexpressing human PVRIG, as well as in their ability to block PVRL2 binding to PVRIG.

[0548] 2. Binding of bispecific antibodies to human TIGIT and blocking of ligand PVR

[0549] The experiments were conducted according to the methods of Examples 2-2 and 2-3 (with the corresponding receptors and ligands replaced with human TIGIT and human PVR), and the results are shown in Table 28. The results showed that there was no statistically significant difference in the binding of bispecific antibodies 1708-151-1, 1708-151-2, 1708-151-3, 1708-151-4 and anti-TIGIT antibodies of different configurations to human TIGIT recombinant protein and cells overexpressing human TIGIT, as well as in the blocking of TIGIT binding to its ligand PVR.

[0550] It can be seen that whether the anti-PVRIG antibody is connected to the N-terminus or C-terminus of the heavy and light chains of the anti-TIGIT antibody, it maintains the binding to PVRIG and TIGIT and the blocking of the ligand, and shows good expression level and purity.

[0551] 3. Binding of humanized anti-PVRIG / TIGIT bispecific antibodies to PVRIG and TIGIT and blocking of corresponding ligands

[0552] Using the methods of Examples 2-2 and 2-3, the humanized anti-PVRIG / TIGIT bispecific antibodies were tested for binding to human and cynomolgus monkey PVRIG and for ligand blocking of human PVRIG. The results are shown in Table 28. The results demonstrate that each bispecific antibody can bind to human PVRIG and block PVRIG binding to PVRL2.

[0553] Table 28. Binding and ligand blocking of PVRIG by humanized bispecific antibodies

[0554] Similar to Examples 2-2 and 2-3, humanized anti-PVRIG / TIGIT bispecific antibodies were tested for binding to human and cynomolgus macaque TIGIT and blocking ligand binding of human TIGIT, in which the PVRIG protein was replaced with TIGIT and PVRL2 was replaced with PVR. The results are shown in Table 29. The results demonstrate that each bispecific antibody can bind to human TIGIT and block TIGIT binding to PVR.

[0555] Table 29. Binding and ligand blocking of TIGIT by humanized bispecific antibodies

[0556] Biacore was used to test the affinity of the bispecific antibodies for human PVRIG and human TIGIT. The humanized bispecific antibodies were captured on a Protein A biosensor chip (GE Lifesciences, 29127557) on a Biacore instrument (Biacore X100, GE). A series of concentration gradients of human PVRIG antigen (AcroBiosystem, PVG-H52H4) or human TIGIT antigen (AcroBiosystem, TIT-H52H3) were then passed over the chip surface to generate association and dissociation curves. The results are shown in Table 30.

[0557] Table 30. Affinity of humanized bispecific antibodies to human PVRIG and human TIGIT

[0558] 4. Mixed Lymphocyte Reaction (MLR) Experiment with Anti-PVRIG / TIGIT Bispecific Antibodies

[0559] The T cell activation ability of the humanized anti-PVRIG / TIGIT bispecific antibody was tested according to the method in Section 4 of Example 2-3. The experimental results are shown in Figure 5 and Table 31. The results show that the humanized anti-PVRIG / TIGIT bispecific antibody 1708-151H8 has a significant ability to activate T cells and promote the secretion of IFNγ by T cells. Importantly, the activity of the bispecific antibody is stronger than that of the anti-PVRIG antibody 151H8 or the anti-TIGIT antibody 1708 alone.

[0560] Table 31. IFNγ secretion in mixed lymphocyte reaction of humanized bispecific antibodies

[0561] Example 2-8. Evaluation of the anti-tumor effect of anti-PVRIG / TIGIT bispecific antibody in a subcutaneous transplant tumor model of human melanoma A375 mixed with human PBMC in mice

[0562] To further explore the role of bispecific antibody subtype in animal efficacy, animal efficacy studies were conducted. The heavy chain variable region of 1708-IgG1 is identical to that of 1708, except that the heavy chain constant region is subtyped to IgG1. The full-length light chain of 1708-IgG1 and the second polypeptide chain of 1708-151-IgG1 are identical to the light chain of 1708.

[0563] NCG mice, female, 4-8 weeks old, weighing approximately 18-22 g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. All NCG mice were cultured in an SPF animal room under the constant temperature and pressure system of IVC.

[0564] A375 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). A375 cells were collected during the exponential growth phase and resuspended in HBSS to a suitable concentration for subcutaneous tumor inoculation in NCG mice. The A375 cells used in co-culture were treated with Mitomycin C for 2 hours and then washed three times with PBS. Human PBMCs were isolated from normal human peripheral blood by density gradient centrifugation and counted. PBMCs were then resuspended in RPMI1640 medium (containing IL-2 and 10% FBS) to a concentration of 3 × 10 6 The cells were co-cultured with A375 cells treated with Mitomycin C at a concentration of 5 × 10 cells / mL. After 6 days of co-culture, PBMCs were harvested and freshly digested A375 cells were harvested. Each mouse was inoculated with 5 × 10 PBMCs. 5 A375 cells 4×10 6 Inoculation volume: 0.2 mL / mouse (containing 50% Matrigel); inoculation was performed subcutaneously on the right flank of female NCG mice. Mice were randomly grouped and administered according to body weight. Detailed dosing method, dosage, and route of administration are shown in Table 32. The day of group administration was designated Day 0. Due to the different molecular weights of anti-PVRIG and anti-TIGIT antibodies, this dosing dose ensured that the anti-PVRIG and anti-TIGIT antibodies had the same starting molar concentration.

[0565] Table 32. Dosage regimen

[0566] (Note: N: number of animals used; ip: intraperitoneal injection; Q2D: once every two days; administration volume: adjusted according to the weight of the tumor-bearing mice (0.1 mL / 10 g))

[0567] After the start of drug administration, the tumor volume and body weight of the mice were measured twice a week. The experimental results are shown in Table 33 and Figures 6A and 6B, respectively.

[0568] Table 33. Anti-tumor effect of anti-PVRIG / TIGIT bispecific antibodies in the mouse humanized A375 tumor model

[0569] (Note: Compared with the control group (hIgG1), *P<0.05, **P<0.01, ***P<0.001 were considered to be significantly different)

[0570] At the end of the experiment (day 26 after dosing), there was no significant difference in the anti-PVRIG antibody 151-IgG4 monotherapy group compared to the control group. Tumor volume decreased in the anti-TIGIT antibody 1708-IgG1 monotherapy group and in the combination of the anti-PVRIG antibody 151-IgG4 and anti-TIGIT antibody 1708-IgG1 groups. The 1708-151-IgG4 bispecific antibody group even completely inhibited tumor growth, demonstrating significant differences compared to the other groups (see Figure 6A).

[0571] The mice were randomly divided into groups and administered according to their body weight. The detailed administration method, dosage and route of administration are shown in Table 34. The day of group administration was designated as Day 0.

[0572] Table 34. Dosage regimen

[0573] (Note: N: number of animals used; ip: intraperitoneal injection; Q2D: once every two days; administration volume: adjusted according to the weight of the tumor-bearing mice (0.1 mL / 10 g))

[0574] After the start of drug administration, the body weight and tumor volume of the mice were measured twice a week. The experimental results are shown in Table 35 and Figures 7A-7B, respectively.

[0575] Table 35. Anti-tumor effect of anti-PVRIG / TIGIT bispecific antibodies in the mouse humanized A375 tumor model

[0576] (Note: Compared with the control group (hIgG1), *P<0.05, **P<0.01, ***P<0.001 were considered to be significantly different)

[0577] At the end of the experiment (28 days after administration), the 1708-151H7 bispecific antibody group was able to effectively inhibit tumor growth at low doses compared to the control group, with significant differences compared to the control group (see Figures 7A and 7B). 1708-30H2 is another antibody screened in this application.

[0578] Example 3. Anti-PD-1 / PVRIG / TIGIT trispecific antibody

[0579] Example 3-1. Design and preparation of anti-PD-1 / PVRIG / TIGIT trispecific antibodies

[0580] The four types of trispecific antibodies are divided into four types: A, B, C, and D, as shown in Figure 8.

[0581] Type A: The anti-PD-1 nanobody is connected to the anti-PVRIG nanobody through a linker (such as GGGGSGGGGS), and then connected to the N-terminus of the heavy chain of the anti-TIGIT antibody through a linker (A in Figure 8).

[0582] Type B: The anti-PVRIG antibody is linked to the N-terminus of the heavy chain of the TIGIT IgG molecule via a linker, while the anti-PD-1 antibody is linked to the N-terminus of the light chain of the anti-TIGIT antibody via a linker ( FIG8B ).

[0583] Type C: Anti-PVRIG antibody is linked to the N-terminus of the heavy chain of TIGIT IgG via a linker. Anti-PD-1 antibody is linked to the C-terminus of the heavy chain of anti-TIGIT antibody via a linker (GGGGSGGGGS) ( Figure 8C ).

[0584] Type D: The anti-PVRIG antibody is linked to the N-terminus of the heavy chain of the TIGIT IgG molecule via a linker. The anti-PD-1 antibody is linked to the C-terminus of the light chain of the anti-TIGIT antibody via a linker (Figure 8D).

[0585] The trispecific antibodies in the present disclosure are named as shown in Table 36. For example, A17m0902-1708-151H7-A represents that the clones of the anti-PD-1, TIGIT and PVRIG antibodies used are A17m0902, 1708 and 151H7, respectively, and the trispecific antibody type is type A.

[0586] Table 36. Nomenclature of anti-PD-1 / PVRIG / TIGIT trispecific antibodies

[0587] The A17h1-related molecules in Table 36 are all based on the corresponding A17m0902-related molecules, with the A17m0902 (SEQ ID NO: 27) sequence replaced by the A17h1 (SEQ ID NO: 8) sequence.

[0588] The sequence is as follows (underlined is the linker, italics are Fc or light chain Cκ):

[0589] >A17m0902-1708-151H7-A first polypeptide chain

[0590] The second polypeptide chain of A17m0902-1708-151H7-A is shown in SEQ ID NO: 52.

[0591] The first polypeptide chain of A17m0902-1708-151H7-B is shown in SEQ ID NO: 68.

[0592] >A17m0902-1708-151H7-B second polypeptide chain

[0593] >A17m0902-1708-151H7-C first polypeptide chain

[0594] The second polypeptide chain of A17m0902-1708-151H7-C is shown in SEQ ID NO: 52.

[0595] The first polypeptide chain of A17m0902-1708-151H7-D is shown in SEQ ID NO: 68.

[0596] >A17m0902-1708-151H7-D second polypeptide chain

[0597] The target antibody can be obtained after cell transfection, expression, purification and detection using conventional methods.

[0598] Example 3-2. Binding affinity of anti-PD-1 / PVRIG / TIGIT trispecific antibodies to antigens

[0599] ELISA was used for detection. Human TIGIT or PVRIG protein (his tag, Acrobiosystems, PVG-H52H4) was dissolved in PBS to 1 μg / mL, and 100 μL / well of the solution was added to a 96-well plate and incubated at 4°C overnight to coat the antigen. Wash three times with PBST solution. Add PBST / 1% BSA solution and incubate at 37°C for 1 hour for blocking. After washing three times with PBST solution, add trispecific antibodies of different concentrations (diluted with PBST / 1% BSA solution) and incubate at 37°C for 1.5 hours. Wash three times with PBST solution. Add 100 μL of HRP-coupled anti-human IgG4 Fc secondary antibody (Thermo) solution and incubate at 37°C for 1 hour. After washing three times with PBST solution, add 100 μL / well TMB solution, react at room temperature for 5 minutes, add 50 μL of stop solution, read the OD450 value using an enzyme reader, and calculate the EC 50 .

[0600] 1. Integration with TIGIT

[0601] All four configurations of the A17h1-1708-151H7 trispecific antibody showed strong binding to TIGIT, with minimal differences in binding ability, and similar binding ability to the 1708-151H7 bispecific antibody for TIGIT. The results are shown in Figure 9 and Table 37. Therefore, all trispecific antibody configurations did not affect their binding to TIGIT.

[0602] Table 37. EC of trispecific antibodies binding to TIGIT antigen 50 value

[0603] 2. Combination with PVRIG

[0604] All four configurations of the A17h1-1708-151H7 trispecific antibody strongly bound to PVRIG, with minimal differences in binding ability. These binding abilities were similar to those of the 1708-151H7 bispecific antibody (see Table 38 and Figure 10 ). Therefore, all three trispecific antibody configurations did not affect their binding to PVRIG.

[0605] Table 38. EC of trispecific antibodies binding to PVRIG antigen 50 value

[0606] Example 3-3: PVRIG sequence optimization in anti-PD-1 / PVRIG / TIGIT trispecific antibody

[0607] In the design of the PD-1 / PVRIG / TIGIT trispecific antibody A17m0902-1708-151H7-C, the first amino acid of the original PVRIG antibody sequence (151H7) is histidine (single-letter abbreviation H). The histidine at the N-terminus of the trispecific antibody molecule sequence may undergo pyridoxal phosphate modification and pyridoxal modification after dephosphorylation during the actual antibody production process. Based on this issue, it is necessary to mutate the first histidine of the original PVRIG sequence. Since glutamic acid (single-letter abbreviation E) and glutamine (single-letter abbreviation Q) are the two most frequently occurring amino acids in the first position of the nanobody sequence, H is mutated to E or Q. The mutant sequence of the PVRIG antibody 151H7 is as follows:

[0608] >151H7 H1E

[0609] >151H7 H1Q

[0610] The trispecific antibody molecule in which the first amino acid of the PVRIG antibody (151H7) sequence was changed from H to E was named A17m0902-1708-151H7-01-C; the trispecific antibody molecule in which the first amino acid of the PVRIG (151H7) sequence was changed from H to Q was named A17m0902-1708-151H7-02-C.

[0611] The molecular type of A17m0902-1708-151H7-01-C and A17m0902-1708-151H7-02-C is type C: that is, the anti-PVRIG antibody is linked to the N-terminus of the heavy chain of the anti-TIGIT antibody via linker 1. The anti-PD-1 antibody is linked to the C-terminus of the heavy chain of the anti-TIGIT antibody via linker 2 ( Figure 8 , C).

[0612] The polypeptide chain sequences of A17m0902-1708-151H7-01-C and A17m0902-1708-151H7-02-C are as follows:

[0613] >A17m0902-1708-151H7-01-C first polypeptide chain

[0614] The second polypeptide chain of A17m0902-1708-151H7-01-C is shown in SEQ ID NO: 52.

[0615] >A17m0902-1708-151H7-02-C first polypeptide chain

[0616] The second polypeptide chain of A17m0902-1708-151H7-02-C is shown in SEQ ID NO: 52.

[0617] Example 3-4. Binding affinity of PVRIG sequence in anti-PD-1 / PVRIG / TIGIT trispecific antibody to antigen after optimization

[0618] HEK293F stably transfected cell lines expressing human PVRIG gene, CHO-K1 stably transfected cell lines expressing human TIGIT gene, and Jurkat cells expressing human PD-1 were used. 2x10 cells were seeded per well in a 96-well plate. 5 Cells were centrifuged at 300 g for 5 minutes, the supernatant was removed, 100 μL of the antibody to be tested was added, and the cells were incubated at 4°C for 1 hour. The supernatant was removed by centrifugation, and the cells were washed three times with 200 μL of washing buffer (PBS + 2% FBS). 100 μL of a 1:500 dilution of an anti-human IgG secondary antibody labeled with Alexa Fluor 488 (Invitrogen, A-11013) was added and the cells were incubated at 4°C for 1 hour. The supernatant was removed by centrifugation, and the cells were washed three times with 200 μL of washing buffer (PBS + 2% FBS). The cells were resuspended in 100 μL of PBS and analyzed using a flow cytometer (BD FACS Calibur or BD FACS Canto II).

[0619] 1. Combination with PVRIG

[0620] The two trispecific antibodies, A17m0902-1708-151H7-01-C and A17m0902-1708-151H7-02-C, after the first amino acid mutation of the PVRIG antibody, showed consistent binding activity to human PVRIG expressed on the cell surface, as did the pre-mutated trispecific antibody A17m0902-1708-151H7-C and the pre-mutated bispecific antibody 1708-151H7, with similar EC50 values. This indicates that the first amino acid mutation of the PVRIG antibody does not affect the binding of the trispecific antibodies to PVRIG. The results are shown in Table 39 and Figure 11A.

[0621] Table 39. Antibody binding assay for cells expressing human PVRIG MAX and EC50 results

[0622] 2. Integration with TIGIT

[0623] The two trispecific antibodies, A17m0902-1708-151H7-01-C and A17m0902-1708-151H7-02-C, showed consistent binding activity to human TIGIT expressed on the cell surface, with similar EC50 values, as did the pre-mutation trispecific antibody A17m0902-1708-151H7-C. This suggests that the mutation of the first amino acid of the PVRIG antibody does not affect the binding of the trispecific antibody to TIGIT. See Table 40 and Figure 11B for the results.

[0624] Table 40. Trispecific antibody binding assay for human TIGIT-expressing cells MAX and EC 50 result

[0625] 3. Binding to PD-1

[0626] The two trispecific antibody molecules, A17m0902-1708-151H7-01-C and A17m0902-1708-151H7-02-C, showed similar binding activity to human PD-1 expressed on the cell surface, with similar EC50 values, as the pre-mutation A17m0902-1708-151H7-C antibody. This indicates that the mutation of the first amino acid of the PVRIG antibody does not affect the binding of the trispecific antibody to PD-1. See Table 41 and Figure 11C for the results.

[0627] Table 41. EMAX and EC of the binding experiment of trispecific antibodies to cells expressing human PD-1 50 result

[0628] Summarizing the above binding activity experimental results, the two trispecific antibody molecules A17m0902-1708-151H7-01-C and A17m0902-1708-151H7-02-C after the first amino acid mutation of the PVRIG antibody have affinities with PVRIG, TIGIT, and PD-1 antigens expressed on the cell surface that are comparable to those of the parent A17m0902-1708-151H7-C antibody. These mutations do not affect the binding of the trispecific antibody to PVRIG, TIGIT, and PD-1.

[0629] Example 3-5. Detection of PD-1 Blockade by Anti-PD-1 / PVRIG / TIGIT Trispecific Antibodies Using a PD-1 / PD-L1 Reporter Gene System

[0630] Detection was performed using the methods described in Examples 1 to 4. As shown in Figure 12, all four trispecific antibodies with A17h1 as the PD-1 end structure were able to block PD-1, but the reporter gene activity of the B and C trispecific antibodies was relatively better.

[0631] Example 3-6. MLR detection of T cell activation by anti-PD-1 / PVRIG / TIGIT trispecific antibodies

[0632] According to the methods of Examples 1-6, cells from human subjects were subjected to DC:T mixed lymphocyte reaction experiments. The results are shown in Figure 13 and Table 42. The results show that all four trispecific antibodies can effectively promote T cell activation, relieve the inhibition of PD-1 / PD-L1, and promote T cell cytokine secretion, and all have strong in vitro pharmacodynamic activity.

[0633] Table 42. MLR results of four trispecific antibodies

[0634] Example 3-7. NK92 Killing Detection of NK Cell Activation by Anti-PD-1 / PVRIG / TIGIT Trispecific Antibodies

[0635] The in vitro efficacy of anti-PD-1 / PVRIG / TIGIT trispecific antibodies can be tested using an NK cell cytotoxicity assay. NK92 cells were used as effector cells and K562 cells as target cells for cytotoxicity assays. NK92 cells were incubated with various concentrations of candidate antibodies at 37°C for 30 minutes. The antibody-preincubated NK92 cells were then mixed with CTV (Cat: C34557, Thermo)-labeled K562 cells at a 10:1 effector-target ratio. The cells were cultured at 1640°C with 10% FBS and 10 ng / mL IL-2 for 4 hours at 37°C. After centrifugation and removal of the supernatant, the cells were resuspended in 10 μg / mL propidium iodide solution and stained for dead cells. After 10 minutes of labeling at room temperature, the proportion of dead cells (PI-positive) in the CTV-labeled K562 cells was determined by FACS, and the cytotoxicity was calculated.

[0636] As shown in Figure 14, the trispecific antibody retains the biological function of the anti-PVRIG / TIGIT bispecific antibody in promoting NK cell killing, and the IC 50 The values ​​are similar, see Table 43.

[0637] Table 43. NK cell killing function of four trispecific antibodies

[0638] Example 3-8. Evaluation of the anti-tumor effect of anti-PD-1 / PVRIG / TIGIT trispecific antibody in a subcutaneous transplant tumor model of human melanoma A375 mixed with human PBMCs responsive to PD-1 monoclonal antibody

[0639] This example verifies the in vivo efficacy of the PD-1 / PVRIG / TIGIT trispecific antibody in an in vivo human melanoma A375 mixed human PBMC tumor model that responds to PD-1 monoclonal antibody.

[0640] NCG mice, female, 4-8 weeks old, weighing approximately 18-22 g (mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were housed in an SPF animal room under constant temperature and pressure conditions in the IVC.

[0641] Donor PBMCs that respond to PD-1 monoclonal antibodies in an in vivo efficacy model were screened from frozen PBMCs and counted. PBMCs were resuspended in RPMI 1640 culture medium containing IL-2 and 10% FBS to a certain concentration. PBMCs were added to A375 cells treated with Mitomycin C for 2 hours. After co-culture with A375 for 5 days, PBMCs were harvested. A375 cells were cultured in DMEM culture medium containing 10% fetal bovine serum (FBS), and freshly digested A375 cells were harvested. Each mouse was inoculated with: 5×10 PBMCs 5A375 cells 4×10 6 The inoculation volume was 0.2 mL / mouse (containing 50% Matrigel) and the mice were subcutaneously inoculated on the right side of the NCG mice. The day of inoculation was designated as day 0, and mice were randomly divided into groups and given medication according to their body weight on day 1.

[0642] Considering the molecular weight of A17m0902_hIgG4 (76 kDa), 1708-151H7 (173.5 kDa), and A17m0902-1708-151H7-C (200 kDa), an equimolar dosing regimen was designed. The detailed dosing regimen, dosage, and route of administration are shown in Table 44.

[0643] Table 44. Mouse experimental groups and dosing regimen

[0644] (Note: N: number of animals used; ip: intraperitoneal injection; BIW*8: twice a week, a total of 8 times)

[0645] After the start of drug administration, the body weight and tumor volume of mice were measured twice a week.

[0646] After 21 days of dosing, compared with the control group, the tumor growth inhibition rates (TGI) in the 1708-151H7 (11.5 mg / kg) group, the A17m0902_hIgG4 (5 mg / kg) group, the A17m0902-1708-151H7-C (13.3 mg / kg) group, and the 1708-151H7 (11.5 mg / kg) + A17m0902_hIgG4 (5 mg / kg) group were 7.05%, 79.91%, 91.63%, and 88.25%, respectively. The trispecific antibody A17m0902-1708-151H7-C group showed significantly better efficacy than the anti-PD-1 monoclonal antibody A17m0902_hIgG4 group and the combination of the PVRIG / TIGIT bispecific antibody and the PD-1 monoclonal antibody.

[0647] The experiment was terminated 21 days after dosing. All mice were euthanized, their tumors removed, weighed, and photographed. Tumor weight TGI was calculated, showing a consistent trend with tumor volume TGI. The results also showed that the trispecific antibody A17m0902-1708-151H7–C group exhibited significantly superior efficacy compared to the PD-1 monoclonal antibody A17m0902_hIgG4 group and the combination of the PVRIG / TIGIT bispecific antibody and PD-1 monoclonal antibody. The results are shown in Table 45 and Figures 15A and 15B, respectively.

[0648] Table 45. Anti-tumor effect of anti-PD-1 / PVRIG / TIGIT trispecific antibodies in the PD-1 monoclonal antibody-responsive mouse humanized A375 tumor model

[0649] (Note: N: number of animals used; ip: intraperitoneal injection; BIW: twice a week)

[0650] Example 3-9. Evaluation of the anti-tumor effect of an anti-PD-1 / PVRIG / TIGIT trispecific antibody in a PD-1 monoclonal antibody-unresponsive human melanoma A375 mixed with human PBMC subcutaneous xenograft mouse model

[0651] This example verifies the in vivo efficacy of the anti-PD-1 / PVRIG / TIGIT trispecific antibody in an in vivo human melanoma A375 mixed human PBMC tumor model that is unresponsive to anti-PD-1 monoclonal antibodies.

[0652] NCG mice, female, 4-8 weeks old, weighing approximately 18-22 g (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), were housed in an SPF animal room under constant temperature and pressure conditions in the IVC system.

[0653] Donor PBMCs that do not respond to anti-PD-1 mAbs in in vivo efficacy models were screened from cryopreserved PBMCs. The experimental protocol was the same as described in Examples 3-8. The mouse experimental grouping and dosing schedule are shown in Table 46.

[0654] Table 46. Mouse experimental groups and dosing regimen

[0655] (Note: N: number of animals used; ip: intraperitoneal injection; BIW*8: twice a week, a total of 8 times)

[0656] After the start of drug administration, the body weight and tumor volume of mice were measured twice a week.

[0657] After 17 days of administration, the average tumor volume of each group of mice was measured. Compared with the IgG control group, the tumor growth inhibition rates (TGI%) of the experimental groups 1708-151H7, A17m0902_hIgG4, A17m0902-1708-151H7-C, and 1708-151H7+A17m0902_hIgG4 were 4.53%, 20.75%, 36.70%, and 31.95%, respectively.

[0658] The experiment was terminated 17 days after dosing, and all mice were euthanized, their tumors removed, weighed, and photographed. Tumor weights were measured in each group. Compared to the IgG control group, the tumor growth inhibition rates (TGI%) for the experimental groups (1708-151H7, A17m0902_hIgG4, A17m0902-1708-151H7-C, and 1708-151H7+A17m0902_hIgG4) were 0%, 16.8%, 38.2%, and 33.4%, respectively. The TGI for tumor weight was consistent with the TGI for tumor volume. The results are shown in Table 47 and Figures 16A and 16B, respectively.

[0659] Compared with the IgG control group, tumor volume and weight were significantly reduced in the A17m0902-1708-151H7-C and 1708-151H7 + A17m0902_hIgG4 groups (Figures 16A and 16B, P < 0.05), demonstrating a significant tumor-suppressing effect. During this experiment, none of the mice exhibited significant weight loss or behavioral abnormalities, indicating good tolerance to the test drug under these experimental conditions.

[0660] Table 47. Anti-tumor effect of anti-PD-1 / PVRIG / TIGIT trispecific antibodies in a human A375 tumor mouse model that does not respond to PD-1 monoclonal antibodies

[0661] (Note: Compared with the control group (hIgG1), *P<0.05, **P<0.01, ***P<0.001 were considered to be significantly different)

[0662] Example 4. Screening of buffer system, pH value and excipients for PD-1 / PVRIG / TIGIT trispecific antibody formulation

[0663] The PD-1 / PVRIG / TIGIT trispecific antibody used was the aforementioned A17m0902-1708-151H7-C, whose first and second polypeptide chain amino acid sequences are SEQ ID NOs: 72 and 52, respectively. The following buffer was prepared, and the corresponding excipients and surfactants were added to prepare a 50 mg / mL PD-1 / PVRIG / TIGIT trispecific antibody formulation for stability studies. The experimental results are shown in Table 48.

[0664] 1) F1 10 mM citric acid-disodium citrate buffer pH 5.0, 8% w / v sucrose, 0.04 mg / mL polysorbate 80;

[0665] 2) F2 10 mM histidine-histidine hydrochloride buffer pH 5.5, 8% w / v sucrose, 0.04 mg / mL polysorbate 80;

[0666] 3) F3 10 mM histidine-acetate buffer pH 5.0, 8% w / v sucrose, 0.04 mg / mL polysorbate 80;

[0667] 4) F4 10 mM histidine-acetate buffer pH 5.5, 8% w / v sucrose, 0.04 mg / mL polysorbate 80;

[0668] 5) F5 10 mM histidine-acetate buffer pH 6.0, 8% w / v sucrose, 0.04 mg / mL polysorbate 80;

[0669] 6) F6 10 mM histidine-acetate buffer pH 5.0, 8% w / v sucrose, 0.04 mg / mL polysorbate 80, 50 mM arginine;

[0670] 7) F7 10 mM histidine-acetate buffer pH 5.5, 8% w / v sucrose, 0.04 mg / mL polysorbate 80, 50 mM arginine.

[0671] Table 48. Screening results of influencing factors (freeze-thaw and shaking)

[0672] Table 49. Appearance results at 40℃-1

[0673] Table 50.40℃ Purity Screening Results-2

[0674] Experimental results:

[0675] Formulation F1 showed obvious turbidity, and the protein was unstable in the citric acid system.

[0676] The results of the freeze-thaw and shaking experiments showed (Table 48): After shaking for 3 days, formulations F3 (histidine-acetate buffer system pH 5.0) and F4 (histidine-acetate buffer system pH 5.5) showed no opalescence and were clear, while the other formulations showed opalescence from T0 onwards; all formulations showed good appearance stability after repeated freeze-thaw cycles; and there was no significant difference in the SEC, CEX, and NR-CE test results for formulations F2 to F7 after shaking or repeated freeze-thaw cycles, demonstrating good stability.

[0677] High temperature test results (Table 49-50):

[0678] The appearance results showed that after being stored at 40℃ for 4 weeks, the appearance of prescriptions F2 to F7 did not change significantly.

[0679] SEC results showed that after 4 weeks of storage at 40°C, the SEC purity of all formulations decreased, among which the degree of decrease of F2 (histidine hydrochloride system pH 5.5), F3 (histidine acetate buffer system pH 5.0) and F4 (histidine acetate buffer system pH 5.5) was less than that of formulations F5 to F7.

[0680] The CEX results showed that after being stored at 40°C for 4 weeks, the main CEX peaks of all prescriptions showed a downward trend, and there was little difference between the prescriptions.

[0681] NR-CE results showed that after storage at 40°C for 4 weeks, the purity of the main peak of each formulation remained above 90%, with little difference between the formulations.

[0682] In summary, formulations F3 and F4 showed slightly better stability than the other formulations in terms of formulation appearance, SEC, CEX, and NR-CE assays. Therefore, formulations F3 (histidine-acetate buffer, pH 5.0) and F4 (histidine-acetate buffer, pH 5.5) were selected for the next round of protein concentration screening.

[0683] Example 5. Screening of PD-1 / PVRIG / TIGIT trispecific antibody concentrations

[0684] The PD-1 / PVRIG / TIGIT trispecific antibody used was the aforementioned A17m0902-1708-151H7-C, whose first and second polypeptide chain amino acid sequences are SEQ ID NOs: 72 and 52, respectively. A 10 mM histidine-acetate buffer system at pH 5.0 and pH 5.5 was selected to prepare PD-1 / PVRIG / TIGIT trispecific antibody formulations at different antibody concentrations using 8% w / v sucrose and 0.4 mg / mL polysorbate 80:

[0685] 1) F8 10 mM histidine-acetate pH 5.0, 50 mg / mL antibody;

[0686] 2) F9 10 mM histidine-acetate pH 5.5, 50 mg / mL antibody;

[0687] 3) F10 10 mM histidine-acetate pH 5.0, 100 mg / mL antibody;

[0688] Conduct stability studies.

[0689] Table 51 Antibody concentration screening shaking and freeze-thaw results

[0690] Table 52. Antibody concentration screening high temperature test results

[0691] Note: “ / ” means there are not enough samples to observe the appearance, and the result is not available.

[0692] Experimental results

[0693] The results of the freeze-thaw and shaking experiments (Table 51) showed that the appearance stability of the F8-F10 formulations was good after shaking or freeze-thaw, and there was no significant difference in the SEC, CEX and NR-CE test results among the formulations, demonstrating good stability.

[0694] The high temperature test results (Table 52) show that:

[0695] The appearance results showed that after being placed at 40°C for 4 weeks, the formulations with different antibody concentrations all turned light yellow with no visible particles; when placed at 2-8°C and 25°C for 4 weeks, the appearance did not change compared to T0.

[0696] SEC results showed that after 4 weeks at 40°C, the monomer content of each formulation decreased significantly. In the acetate-histidine buffer system, at the same antibody concentration (50 mg / mL), the monomer and aggregate content of F8 (pH 5.0) were slightly better than those of F9 (pH 5.5). As the formulation concentration increased, the aggregate content increased, but the monomer content of F10 (pH 5.0, antibody concentration 100 mg / mL) was also around 91.23%. The long-term stability data at 2-8°C was good, which can support the development of high-concentration formulations.

[0697] CEX results showed that after 4 weeks at 40°C, the main peaks of formulations F8 and F10 decreased significantly, with the trends remaining consistent across formulations. In an acetate-histidine pH 5.0 buffer system, the main peaks of F8 (antibody concentration 50 mg / mL) and F10 (100 mg / mL) were 22.97% and 21.74%, respectively. Antibody concentration had little effect on the decrease in the CEX main peak.

[0698] NR-CE results showed that after four weeks of storage at 40°C, the purity of formulation F10 (100 mg / mL) decreased the most, but the main peak remained at 91.49%. The purity of the other formulations also decreased slightly, but the decline was comparable. The main peak at T0 remained similar for all three formulations after four weeks at 2-8°C and 25°C.

[0699] Combining shaking, repeated freeze-thaw and high temperature experiments, it was found that the antibody was stable in the acetate-histidine buffer system. When the concentration was developed to 100 mg / mL, the stability was also good at pH 5.0.

[0700] Example 6. Screening of pH and concentration of PD-1 / PVRIG / TIGIT trispecific antibodies

[0701] The PD-1 / PVRIG / TIGIT trispecific antibody used was the aforementioned A17m0902-1708-151H7-01-C, whose first polypeptide chain and second polypeptide chain amino acid sequences were SEQ ID NOs: 76 and 52, respectively.

[0702] 10 mM histidine-acetate pH 5.0 and pH 5.5 buffer systems were used to prepare 8% w / v sucrose, 0.8 mg / mL polysorbate 80, and PD-1 / PVRIG / TIGIT trispecific antibody formulations at different antibody concentrations:

[0703] 1) F11 10 mM histidine-acetate pH 5.0, 50 mg / mL antibody;

[0704] 2) F12 10 mM histidine-acetate pH 5.0, 80 mg / mL antibody;

[0705] 3) F13 10 mM histidine-acetate pH 5.0, 100 mg / mL antibody;

[0706] 4) F14 10 mM histidine-acetate pH 5.5, 50 mg / mL antibody;

[0707] 5) F15 10 mM histidine-acetate pH 5.5, 100 mg / mL antibody;

[0708] Stability studies were performed including high temperature (40°C), 25°C, 2-8°C, freeze-thaw and shaking.

[0709] Table 53. Antibody pH and concentration screening shaking and freeze-thaw results

[0710] Table 54. Antibody pH and concentration screening shaking and freeze-thaw results

[0711] Experimental results: The freeze-thaw and shaking test results (Table 53) showed that the appearance stability of F11-F15 formulations was good after shaking or freeze-thaw, and there was no significant difference in the SEC, CEX and NR-CE test results among the formulations, demonstrating good stability.

[0712] The high temperature test results (Table 54) show that:

[0713] The appearance results showed that after being placed at 40°C for 2 weeks, the formulations with different antibody concentrations had no visible particles and the opalescence of F12 to F15 was enhanced. When placed at 2-8°C and 25°C for 2 weeks, the appearance of each formulation did not change compared with T0.

[0714] SEC results showed that after being placed at 40°C for 2 weeks, the monomer content of each formulation decreased significantly. In the acetate-histidine buffer system, at the same antibody concentration (50 mg / mL), the monomer content and aggregate content of F11 (pH 5.0) were slightly better than those of F14 (pH 5.5); as the formulation concentration increased, the aggregate content increased.

[0715] CEX results showed that after 2 weeks at 40°C, the main peaks of formulations F11 to F15 decreased significantly, while the trends of all formulations remained consistent. In an acetate-histidine pH 5.0 buffer system, the main peaks of F11 (antibody concentration 50 mg / mL) and F13 (100 mg / mL) were 59.58% and 59.32%, respectively. The antibody concentration had little effect on the decrease in the CEX main peak.

[0716] NR-CE results showed that after 2 weeks of storage at 40℃, the main peaks of all formulations decreased, but the decrease was similar. The main peaks of the five formulations stored at 2-8℃ and 25℃ for 2 weeks were not much different from the main peaks at T0.

[0717] Combining the shaking and repeated freeze-thaw and high temperature experiment 2W data, it can be seen that the antibody has good stability in the acetate-histidine buffer system, and the purity data at pH 5.0 and pH 5.5 are within the acceptable range; with the increase of concentration, the aggregates increase slightly, but are within the acceptable range.

Claims

1. A pharmaceutical composition comprising a PD-1 / PVRIG / TIGIT binding protein and a buffer, wherein the PD-1 / PVRIG / TIGIT binding protein comprises: The first antigen-binding domain that specifically binds to PD-1, A second antigen binding domain that specifically binds to PVRIG, and Specifically binds to the third antigen-binding domain of TIGIT, The first antigen binding domain comprises or is an immunoglobulin single variable domain comprising: CDR1, CDR2, and CDR3 in any one of the amino acid sequences set forth in SEQ ID NOs: 27, 2, 8-10, 11-26, 28-29, wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems; Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are as shown in SEQ ID NOs: 3, 30, 31, or as shown in SEQ ID NOs: 3, 32, 35, respectively; The buffer is selected from citrate buffer and histidine buffer, preferably histidine buffer, more preferably histidine-hydrochloride buffer or histidine-acetate buffer, most preferably histidine-histidine hydrochloride buffer or histidine-acetate buffer.

2. The pharmaceutical composition according to claim 1, wherein the PD-1 / PVRIG / TIGIT binding protein is an anti-PD-1 / PVRIG / TIGIT trispecific antibody.

3. The PD-1 / PVRIG / TIGIT binding protein according to claim 1 or 2, wherein: (B) the second antigen-binding domain comprises an immunoglobulin single variable domain, comprising: CDR1, CDR2, and CDR3 of any one of the amino acid sequences represented by SEQ ID NOs: 74-75, 38, and 46-50, wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems; Preferably, the amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 39, 40 and 41, respectively; and / or, (C) The third antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: the VH comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NOs: 57, 58 and 59, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NOs: 60, 61 and 62, respectively.

4. The PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 3, wherein: (A) the first antigen-binding domain comprises: an amino acid sequence as shown in any one of SEQ ID NOs: 27, 2, 8-10, 11-26, 28-29, or having at least 90% sequence identity thereto; Preferably, it is SEQ ID NO: 27 or an amino acid sequence having at least 90% sequence identity thereto; and / or, (B) the second antigen-binding domain comprises: an amino acid sequence as shown in any one of SEQ ID NOs: 74-75, 38, 46-50, or having at least 90% sequence identity thereto; Preferably, it is an amino acid sequence shown in any one of SEQ ID NOs: 74 and 48, or a sequence having at least 90% sequence identity thereto; and / or, (C) The third antigen-binding domain comprises VH and VL, wherein: The VH comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 63-65, or having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence as set forth in SEQ ID NOs: 67 or 66, or having at least 90% sequence identity thereto; Preferably, the VH comprises SEQ ID NO: 63 or an amino acid sequence having at least 90% sequence identity thereto, and the VL comprises SEQ ID NO: 67 or an amino acid sequence having at least 90% sequence identity thereto.

5. The PD-1 / PVRIG / TIGIT binding protein of claim 4, wherein the third antigen binding domain comprises a heavy chain (HC) and a light chain (LC); The HC sequence is an amino acid sequence shown in SEQ ID NO: 51 or having at least 90% sequence identity thereto, and the LC sequence is an amino acid sequence shown in SEQ ID NO: 52 or having at least 90% sequence identity thereto.

6. The PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 5, comprising: The first polypeptide chain, and The second polypeptide chain, in, In order from N-terminus to C-terminus: (I) the first polypeptide chain is represented by the following structure: [first antigen-binding domain]-[linker 1]a-[second antigen-binding domain]-[linker 2]b-[VH of the third antigen-binding domain]-CH1-Fc, and The second polypeptide chain is represented by the following structure: [VL of the third antigen-binding domain]-Cκ; or (II) the first polypeptide chain is represented by the following structure: [Second antigen-binding domain]-[Linker 2]b-[VH of the third antigen-binding domain]-CH1-Fc, and The second polypeptide chain is represented by the following structure: [first antigen-binding domain]-[Linker 3]c-[VL of the third antigen-binding domain]-Cκ; or (III) the first polypeptide chain is represented by the following structure: [Second Antigen Binding Domain]-[Linker 2]b-[VH of Third Antigen Binding Domain]-CH1-Fc-[Linker 4]d-[First Antigen Binding Domain], and The second polypeptide chain is represented by the following structure: [VL of the third antigen-binding domain]-Cκ; or (IV) the first polypeptide chain is represented by the following structure: [Second antigen-binding domain]-[Linker 2]b-[VH of the third antigen-binding domain]-CH1-Fc, and The second polypeptide chain is represented by the following structure: [VL of the third antigen-binding domain]-Cκ-[Linker 5]e-[First antigen-binding domain]; Among them, - represents a peptide bond, The linker 1, linker 2, linker 3, linker 4, and linker 5 may be the same or different; a, b, c, d, and e are optionally and independently 0 or 1; Preferably, the linker 1, linker 2, linker 3, linker 4, and linker 5 are independently (G x S) y , wherein x is selected from an integer of 1-5, and y is selected from an integer of 1-6; more preferably, they are independently any one of the linkers shown in (G4S)2, (G4S)3, and (G4S)4.

7. The PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 6, comprising a first polypeptide chain and a second polypeptide chain selected from any one of the following groups: The first and second polypeptide chains comprise the amino acid sequences shown in SEQ ID NOs: 76 and 52, respectively; The first and second polypeptide chains comprise the amino acid sequences shown in SEQ ID NOs: 77 and 52, respectively; The first and second polypeptide chains comprise the amino acid sequences shown in SEQ ID NOs: 70 and 52, respectively; The first and second polypeptide chains comprise the amino acid sequences shown in SEQ ID NOs: 68 and 71, respectively; The first and second polypeptide chains comprise the amino acid sequences shown in SEQ ID NOs: 72 and 52, respectively; The first and second polypeptide chains comprise the amino acid sequences shown in SEQ ID NOs: 68 and 73, respectively; or, a combination of amino acid sequences having at least 90% sequence identity with each of the first and second polypeptide chains; Preferably, the PD-1 / PVRIG / TIGIT binding protein has two identical first polypeptide chains and two identical second polypeptide chains.

8. The pharmaceutical composition according to any one of claims 1 to 7, further comprising a surfactant, preferably, the surfactant is polysorbate, more preferably, the surfactant is polysorbate 80 or polysorbate 20, most preferably, the surfactant is polysorbate 80.

9. The pharmaceutical composition according to any one of claims 1 to 8, further comprising a sugar, preferably, the sugar is sucrose.

10. The pharmaceutical composition according to any one of claims 1 to 9, further comprising other amino acids or pharmaceutically acceptable salts thereof, preferably arginine or a pharmaceutically acceptable salt thereof, more preferably arginine-hydrochloride.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the pH of the pharmaceutical composition is 3.5-7, preferably 4-6.5, more preferably 4.2-6.2, most preferably 4.5-6.

12. The pharmaceutical composition of any one of claims 1 to 11, wherein the concentration of the PD-1 / PVRIG / TIGIT binding protein is 0.01 mg / mL-500 mg / mL, preferably 0.1 mg / mL-400 mg / mL, more preferably 0.5 mg / mL-200 mg / mL, and most preferably 1 mg / mL-150 mg / mL.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the concentration of the buffer is 0.1 mM-50 mM, preferably 0.5 mM-40 mM, more preferably 1 mM-30 mM, most preferably 5 mM-20 mM.

14. The pharmaceutical composition of any one of claims 8 to 13, wherein the concentration of the surfactant is 0.01 mg / mL-10 mg / mL, preferably 0.05 mg / mL-5 mg / mL, more preferably 0.1 mg / mL-3 mg / mL, most preferably 0.2 mg / mL-2 mg / mL.

15. The pharmaceutical composition of any one of claims 9 to 14, wherein the saccharide concentration is 0.1% w / v-20% w / v, preferably 1% w / v-15% w / v, more preferably 3% w / v-12% w / v, most preferably 5% w / v-10% w / v.

16. The pharmaceutical composition according to any one of claims 10 to 15, wherein the concentration of the other amino acid or a pharmaceutically acceptable salt thereof is 1 mM-200 mM, preferably 5 mM-170 mM, more preferably 10 mM-150 mM, most preferably 15 mM-120 mM.

17. A pharmaceutical composition comprising: The PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 7; Histidine salt buffer, preferably histidine hydrochloride buffer or histidine acetate buffer, more preferably histidine-histidine hydrochloride buffer or histidine-acetate buffer; Polysorbate; sucrose; Optionally, the composition further comprises arginine or a pharmaceutically acceptable salt thereof.

18. The pharmaceutical composition according to claim 17, comprising any one of the following 1) to 13): 1) 0.01 mg / mL-500 mg / mL of the PD-1 / PVRIG / TIGIT binding protein of any one of claims 1 to 7; 0.1 mM-50 mM histidine salt buffer, preferably histidine hydrochloride buffer or histidine acetate buffer, more preferably histidine-histidine hydrochloride buffer or histidine-acetate buffer; 0.01mg / mL-10mg / mL polysorbate; 0.1% w / v-20% w / v sucrose; Optionally, the pharmaceutical composition further comprises 1 mM-200 mM arginine or a pharmaceutically acceptable salt thereof; and the pH of the pharmaceutical composition is 3.5-7; 2) 0.1 mg / mL-400 mg / mL of the PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 7; 0.5 mM-40 mM histidine salt buffer, preferably histidine hydrochloride buffer or histidine acetate buffer, more preferably histidine-histidine hydrochloride buffer or histidine-acetate buffer; 0.05mg / mL-5mg / mL polysorbate; 1% w / v-15% w / v sucrose; Optionally, the pharmaceutical composition further comprises 5mM-170mM arginine or a pharmaceutically acceptable salt thereof; and the pH of the pharmaceutical composition is 4-6.5; 3) 0.5 mg / mL-200 mg / mL of the PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 7; 1 mM-30 mM histidine salt buffer, preferably histidine hydrochloride buffer or histidine acetate buffer, more preferably histidine-histidine hydrochloride buffer or histidine-acetate buffer; 0.1mg / mL-3mg / mL polysorbate; 3% w / v-12% w / v sucrose; Optionally, the pharmaceutical composition further comprises 10 mM-150 mM arginine or a pharmaceutically acceptable salt thereof; and the pH of the pharmaceutical composition is 4.2-6.2; 4) 1 mg / mL-150 mg / mL of the PD-1 / PVRIG / TIGIT binding protein of any one of claims 1 to 7; 5mM-20mM histidine salt buffer, preferably histidine hydrochloride buffer or histidine acetate buffer, more preferably histidine-histidine hydrochloride buffer or histidine-acetate buffer; 0.2mg / mL-2mg / mL polysorbate; 5% w / v-10% w / v sucrose; Optionally, the pharmaceutical composition further comprises 15mM-120mM arginine or a pharmaceutically acceptable salt thereof; and the pH of the pharmaceutical composition is 4.5-6; 5) 1-150 mg / mL of the PD-1 / PVRIG / TIGIT binding protein of any one of claims 1 to 7; About 10 mM histidine hydrochloride buffer or histidine acetate buffer, preferably histidine-histidine hydrochloride buffer or histidine-acetate buffer; 0.2mg / mL-1mg / mL polysorbate 80; About 8% w / v sucrose; and the pH of the pharmaceutical composition is 4.5-6; 6) about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, about 130 mg / mL, or about 150 mg / mL of the PD-1 / PVRIG / TIGIT binding protein of any one of claims 1 to 7; About 10 mM histidine hydrochloride buffer, preferably histidine-histidine hydrochloride buffer; About 0.4 mg / mL polysorbate 80; About 8% w / v sucrose; and the pH of the pharmaceutical composition is about 5; 7) about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, 130 mg / mL, or about 150 mg / mL of the PD-1 / PVRIG / TIGIT binding protein of any one of claims 1 to 7; About 10 mM histidine hydrochloride buffer, preferably histidine-histidine hydrochloride buffer; About 0.4 mg / mL polysorbate 80; About 8% w / v sucrose; and the pH of the pharmaceutical composition is about 5.5; 8) about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, about 130 mg / mL, or about 150 mg / mL of the PD-1 / PVRIG / TIGIT binding protein of any one of claims 1 to 7; About 10 mM histidine hydrochloride buffer, preferably histidine-histidine hydrochloride buffer; About 0.8 mg / mL polysorbate 80; About 8% w / v sucrose; and the pH of the pharmaceutical composition is about 5; 9) about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, about 130 mg / mL, or about 150 mg / mL of the PD-1 / PVRIG / TIGIT binding protein of any one of claims 1 to 7; About 10 mM histidine hydrochloride buffer, preferably histidine-histidine hydrochloride buffer; About 0.8 mg / mL polysorbate 80; About 8% w / v sucrose; and the pH of the pharmaceutical composition is about 5.5; 10) about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, about 130 mg / mL, or about 150 mg / mL of the PD-1 / PVRIG / TIGIT binding protein of any one of claims 1 to 7; approximately 10 mM histidine-acetate buffer; About 0.4 mg / mL polysorbate 80; About 8% w / v sucrose; and the pH of the pharmaceutical composition is about 5; 11) about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, about 130 mg / mL, or about 150 mg / mL of the PD-1 / PVRIG / TIGIT binding protein of any one of claims 1 to 7; approximately 10 mM histidine-acetate buffer; About 0.4 mg / mL polysorbate 80; About 8% w / v sucrose; and the pH of the pharmaceutical composition is about 5.5; 12) about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, about 130 mg / mL, or about 150 mg / mL of the PD-1 / PVRIG / TIGIT binding protein of any one of claims 1 to 7; approximately 10 mM histidine-acetate buffer; About 0.8 mg / mL polysorbate 80; About 8% w / v sucrose; and the pH of the pharmaceutical composition is about 5; 13) about 10 mg / mL, about 20 mg / mL, about 25 mg / mL, about 50 mg / mL, about 80 mg / mL, about 100 mg / mL, About 120 mg / mL, about 130 mg / mL, or about 150 mg / mL of the PD-1 / PVRIG / TIGIT binding protein of any one of claims 1 to 7; approximately 10 mM histidine-acetate buffer; About 0.8 mg / mL polysorbate 80; About 8% w / v sucrose; And the pH of the pharmaceutical composition is about 5.

5.

19. A lyophilized preparation, which can form the pharmaceutical composition of any one of claims 1 to 18 after being reconstituted, or which is obtained by freeze-drying the pharmaceutical composition of any one of claims 1 to 18.

20. A reconstituted solution, wherein the reconstituted solution is prepared by reconstituted the lyophilized preparation according to claim 19.

21. A product comprising a container containing the pharmaceutical composition of any one of claims 1 to 18, the lyophilized formulation of claim 19, or the reconstituted solution of claim 20.

22. Use of the pharmaceutical composition of any one of claims 1 to 19, the lyophilized preparation of claim 19, or the reconstituted solution of claim 20 in the preparation of a medicament for treating cancer; preferably, the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, and blood system cancer.

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