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

Abstract

This disclosure relates to pharmaceutical compositions containing PD-1 / PVRIG / TIGIT binding proteins and their medical use. Specifically, this disclosure relates to pharmaceutical compositions containing PD-1 / PVRIG / TIGIT binding proteins and buffers. The pharmaceutical compositions of this disclosure have good biological activity and stability.

Description

Technical Field

[0001] This application claims the priority of Chinese Patent Application No. 202310250609.3, filed on March 15, 2023.

[0002] The present disclosure relates to the field of pharmaceutical preparations, and specifically, to a pharmaceutical composition containing a PD-1 / PVRIG / TIGIT binding protein and its pharmaceutical use.

Background Art

[0003] Immunotherapy is a hot spot in the field of cancer treatment, and immunotherapy drugs represented by anti-PD-1 antibodies have shown high clinical effectiveness and high safety.

[0004] PD-1 (Programmed Cell death-1) belongs to the CD28 receptor family and is an immune inhibitory receptor (Riley et al., 2009, Immunol. Rev. 29: 114-25). This family further includes CD28, CTLA-4, ICOS, PD-1 and BTLA. PD-1 is a type I transmembrane protein and is very similar in structure to CTLA-4, but PD-1 does not have the MYPPPY sequence that binds to B7-1 and B7-2. PD-1 is mainly expressed in 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, namely PD ligand 1 (also called PD-L1, CD274, B7-H1) and PD ligand 2 (also called PD-L2, B7-DC). Neither PD-L1 nor PD-L2 binds to other CD28 receptor family members. PD-L1 is expressed in lymphocytes (e.g., CD4 + T cells and CD8 +It is widely expressed in, for example, peripheral tissues, various tumor cells, virus-infected cells, etc., as well as T cells, macrophages, etc. PD-L2 is mainly expressed in activated dendritic cells and macrophages (Dong et al., 1999, Nat. Med. 5: 1365-9). When PD-1 binds to its ligand PD-L1 or PD-L2, it downregulates T cell functions including activation, differentiation and proliferation of T cells, and reduction of cytokine secretion.

[0006] PVRIG (Poliovirus receptor-related Ig domain containing protein), also called CD112R, belongs to the B7 / CD28 superfamily like TIGIT (T cell immunoglobulin and ITIM domain), CD96 and CD226, and plays an important role in the immune system. When PVRL2 (also called CD112), the ligand of PVRIG, binds to PVRIG, the ITIM domain in the intracellular region of PVRIG is activated, and PVRIG plays an immunosuppressive role. PVRIG is mainly CD4 + T cells, CD8 +PVRIG is expressed on the surface of T cells and NK cells. PVRIG and its ligand PVRL2 are highly expressed in various solid tumors, including lung cancer, breast cancer, ovarian cancer, renal cancer, gastric cancer, endometrial cancer, and head and neck cancer. PVRIG expression in these cancers is highly correlated with TIGIT and PD-1. Similar to PD-1 and TIGIT, PVRIG-positive T cells also exhibit Eomes positivity and Tbet negativity, indicating that PVRIG is associated with T cell depletion. Therefore, PVRIG may represent a novel immune checkpoint other than PD-1 and TIGIT and play a role in redundancy. As is evident from in vitro cell experiments and mouse models, knocking out or inhibiting mouse PVRIG can effectively inhibit cancer growth and produce synergistic effects with PD-1 and TIGIT inhibitors. TIGIT is highly expressed in lymphocytes and includes cancer-invading lymphocytes (TILs) and Tregs that invade different types of cancer. The binding of TIGIT to its homologous ligand PVR (also known as CD155) has been shown to directly inhibit the cytotoxicity of NK cells via its cytoplasmic ITIM domain. Since PVR is also widely expressed in cancer, the TIGIT-PVR signaling axis may be a major immune evasion mechanism in cancer. The inhibitory receptors TIGIT and PVRIG, and the activating receptor DNAM-1, bind to the same ligands, CD155 and CD112, but the inhibitory receptor has a higher affinity (Y. Zhu et al., 2016, J Exp Med. 213:167-176). Downregulation of DNAM-1 expression in NK cells isolated in vivo from tumor patients allows NK cells to be more readily inhibited by TIGIT or PVRIG, blocking TIGIT's binding to PVRIG and activating the cytotoxic function of NK cells (L. Martinet et al., 2015, Cell Reports. 11:85-97).

[0007] PD-1 / PD-L1 antibodies are currently the most clinically successful monoclonal immune checkpoint inhibitors, but their therapeutic efficacy is limited by the upregulation of the expression of other immune checkpoints on the surface of T cells. TIGIT and PVRIG not only modulate T cell function but also NK cell activity, suggesting a potential synergistic effect with PD-1 / PD-L1 function in NK cells. The literature has reported that high expression of CD155 and CD112, ligands for TIGIT and PVRIG, in lung cancer, ovarian cancer, colorectal cancer, and melanoma is significantly associated with poor prognosis in PD-1 / PD-L1 therapy. For example, patients with high CD155 expression tend to have a poor response to anti-PD-1 antibodies, while patients with low CD155 expression tend to 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] PVRIG antibodies and anti-PVRIG / TIGIT bispecific antibody drugs are not yet commercially available. Compugen's COM701 is the first humanized anti-PVRIG antibody approved for clinical use by the FDA worldwide and is currently in Phase I clinical trials to treat cancer. Surface Oncology is also developing the anti-PVRIG antibody SRF-813. Anti-TIGIT antibodies include Genentech's tiragolumab, BMS-986207 (jointly developed by Ono Pharmaceutical and BMS), Merck Sharp & Dohme's MK-7684, iTeos Therapeutics' EOS-884448, and Arcus Biosciences' AB-154, all of which are in Phase II clinical trials.

[0009] WO2023040945 provides a novel anti-PD-1 antibody and related anti-PD-1 / PVRIG / TIGIT trispecific antibodies, which are expected to enhance the activation of T cells and NK cells, overcome PD-1 / PD-L1 drug resistance, and expand the responsive patient population, thereby improving the efficacy of cancer immunotherapy. Furthermore, the anti-PD-1 / PVRIG / TIGIT trispecific antibody can simultaneously bind to multiple immune checkpoints on the same cell surface and has a potential affinity effect, potentially providing superior therapeutic efficacy clinically compared to conventional PD-1, TIGIT, and PVRIG antibody drugs or their combinations.

[0010] Antibody drugs, due to their large molecular weight and complex structure, are prone to instability through degradation, polymerization, or undesirable chemical modifications. Therefore, research into stabilized antibody drug formulations is particularly important to ensure that antibodies are suitable for administration, maintain stability during storage and subsequent use, and achieve better efficacy. The development of appropriate pharmaceutical compositions for new PD-1 / PVRIG / TIGIT binding proteins remains necessary. [Overview of the Initiative]

[0011] This disclosure provides a pharmaceutical composition containing a PD-1 / PVRIG / TIGIT binding protein that exhibits excellent stability.

[0012] This disclosure provides a pharmaceutical composition comprising a PD-1 / PVRIG / TIGIT binding protein and a buffer, wherein the buffer is selected from citrate buffers and histidine salt buffers. In some embodiments, the buffer is a citrate-sodium citrate buffer. In some embodiments, the buffer is a citrate-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.

[0013] 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 bind to PD-1, PVRIG, and TIGIT simultaneously or individually.

[0014] In some embodiments, the PD-1 / PVRIG / TIGIT binding protein comprises or includes an anti-PD-1 / PVRIG / TIGIT trispecific antibody.

[0015] In some embodiments, each antigen-binding domain in the PD-1 / PVRIG / TIGIT binding protein is selected from Fab, Fv, sFv, Fab', F(ab')2, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptide antibodies, domain antibodies, or any combination thereof. In specific embodiments, the first antigen-binding domain that specifically binds to PD-1 contains or is VHH, the second antigen-binding domain that specifically binds to PVRIG contains or is VHH, and the third antigen-binding domain that specifically binds to TIGIT contains or is Fab or F(ab')2.

[0016] In some embodiments, the PD-1 / PVRIG / TIGIT binding protein is modified by humanization, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation and / or reduction of antibody isomerization.

[0017] In some embodiments, the 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.

[0018] In some embodiments, the PD-1 / PVRIG / TIGIT binding protein has a first antigen-binding domain that specifically binds to PD-1, comprising (at least one) immunoglobulin monovariate domain, the immunoglobulin monovariate domain is It contains CDR1, CDR2, and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 2, 8-10, or 11-29, The above CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system.

[0019] In some embodiments, according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin monovariate domain are, respectively,

[0020] This is shown in sequence numbers 3, 30, and 31.

[0021] In some specific embodiments, the amino acid sequence of CDR1 of the above immunoglobulin monovariate 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, or 79, and the amino acid sequence of CDR3 is shown in any one of SEQ ID NOs: 5, 35, or 36.

[0022] In some specific embodiments, the amino acid sequences of the above-mentioned immunoglobulin monovariate domains CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 3, 32, and 35, respectively.

[0023] In some embodiments, the PD-1 / PVRIG / TIGIT binding protein has a second antigen-binding domain that specifically binds to PVRIG, comprising (at least one) immunoglobulin monovariate domain, It contains CDR1, CDR2, and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 38, 46-50, or 74-75.

[0024] In some specific embodiments, according to the Kabat numbering system, the amino acid sequences of the above immunoglobulin monovariate domains CDR1, CDR2, and CDR3 are shown in SEQ ID NOs. 39, 40, and 41, respectively.

[0025] In some embodiments, the PD-1 / PVRIG / TIGIT binding protein has a third antigen-binding domain that specifically binds to TIGIT, comprising a heavy chain variable region (VH) and a light chain variable region (VL), of which, The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NOs. 57, 58, and 59, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in SEQ ID NOs. 60, 61, and 62, respectively.

[0026] In some embodiments, the PD-1 / PVRIG / TIGIT binding protein has a first antigen-binding domain that specifically binds to PD-1, It includes an amino acid sequence that is represented in any one of sequence numbers 2, 8-10, or 11-29, or has at least 80% to at least 90% sequence identity with them.

[0027] In some embodiments, the PD-1 / PVRIG / TIGIT binding protein contains an amino acid sequence represented by any one of SEQ ID NOs: 38, 46-50, or 74-75, or an amino acid sequence having at least 80% to at least 90% sequence identity thereto.

[0028] In some embodiments, the PD-1 / PVRIG / TIGIT binding protein has a third antigen-binding domain that specifically binds to TIGIT, comprising a heavy chain variable region (VH) and a light chain variable region (VL), of which, The above heavy chain variable region includes an amino acid sequence shown in any one of sequence numbers 63-65, or having at least 80% to at least 90% identity thereto. The above light chain variable region includes an amino acid sequence shown in SEQ ID NO: 66 or 67, or having at least 80% to at least 90% identity thereto.

[0029] In some embodiments, the PD-1 / PVRIG / TIGIT binding protein has a third antigen-binding domain that specifically binds to TIGIT, comprising a heavy chain variable region (VH) and a light chain variable region (VL), of which, The above heavy chain variable region includes an amino acid sequence shown in Sequence ID No. 63, or one that is at least 80% to at least 90% identical thereto. The above light chain variable region includes an amino acid sequence shown in Sequence ID No. 67, or one that is at least 80% to at least 90% identical thereto.

[0030] In some specific embodiments, the third antigen-binding domain that specifically binds to TIGIT includes a full-length heavy chain (HC) and a full-length light chain (LC).

[0031] For example, the full-length heavy chain is an IgG1, IgG2, or IgG4 isotype, and the full-length light chain is a Kappa isotype. Furthermore, for example, the heavy chain sequence is an amino acid sequence shown in SEQ ID NO: 51, or having at least 80% to at least 90% sequence identity thereto, and the light chain sequence is an amino acid sequence shown in SEQ ID NO: 52, or having at least 80% to at least 90% sequence identity thereto.

[0032] In some embodiments, the PD-1 / PVRIG / TIGIT binding protein has 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, which are linked directly or via a linker. For example, the linker is (G4S) x The linker has the amino acid sequence shown in (G4S)2, (G4S)3, and (G4S)4, where x is independently selected from an integer between 1 and 20.

[0033] In some embodiments, in the 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 PVRIG.

[0034] In some embodiments, in the 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.

[0035] In some embodiments, the PD-1 / PVRIG / TIGIT binding protein described above has an antigen-binding domain that specifically binds to PVRIG located at the N-terminus or C-terminus of the antigen-binding domain that specifically binds to TIGIT.

[0036] In some embodiments, the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain are located on a single polypeptide chain or not located on a single polypeptide chain.

[0037] In some embodiments, the PD-1 / PVRIG / TIGIT binding protein is (I) 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 of the third antigen-binding domain that specifically binds to TIGIT]-CH1-Fc, and second polypeptide chain: [VL of the third antigen-binding domain that specifically binds to TIGIT]-Cκ, or (II) First polypeptide chain: [Second antigen-binding domain that specifically binds to PVRIG]-[linker 2]b-[VH of the third antigen-binding domain that specifically binds to TIGIT]-CH1-Fc, and second polypeptide chain: [First antigen-binding domain that specifically binds to PD-1]-[linker 3]c-[VL of the third antigen-binding domain that specifically binds to TIGIT]-Cκ, or (III) First polypeptide chain: [Second antigen-binding domain that specifically binds to PVRIG]-[Linker 2]b-[VH of the third antigen-binding domain that specifically binds to TIGIT]-CH1-Fc-[Linker 4]d-[First antigen-binding domain that specifically binds to PD-1], and second polypeptide chain: [VL of the third antigen-binding domain that specifically binds to TIGIT]-Cκ, or (IV) First polypeptide chain: [Second antigen-binding domain that specifically binds to PVRIG]-[linker 2]b-[VH of the third antigen-binding domain that specifically binds to TIGIT]-CH1-Fc, and second polypeptide chain: [VL of the third antigen-binding domain that specifically binds to TIGIT]-Cκ-[linker 5]e-[First antigen-binding domain that specifically binds to PD-1], It comprises a first polypeptide chain and a second polypeptide chain, as shown in the structure selected from.

[0038] The first and second polypeptide chains described above are both arranged in the order from the N-terminus to the C-terminus.

[0039] Among them, - represents a peptide bond, the linker is a polypeptide capable of realizing a linking function, and Linker 1, Linker 2, Linker 3, Linker 4, and Linker 5 may be the same or different, and a, b, c, d, and e may optionally and independently be 0 or 1.

[0040] For example, each linker is independently EPKSS or (G x S) y is a linker, where x is selected from integers from 1 to 5 (for example, 1, 2, 3, 4, 5), and y is selected from integers from 1 to 6 (for example, 1, 2, 3, 4, 5, 6).

[0041] When the linker is (G x S) y is a linker, for example, it is a linker shown by any one of (G4S)2, (G4S)3, and (G4S)4.

[0042] In some embodiments, a PD-1 / PVRIG / TIGIT binding protein is provided, which includes a first polypeptide chain and a second polypeptide chain, among which the first and second polypeptide chains respectively include the amino acid sequences shown in SEQ ID NO: 70 and 52, the first and second polypeptide chains respectively include the amino acid sequences shown in SEQ ID NO: 68 and 71, the first and second polypeptide chains respectively include the amino acid sequences shown in SEQ ID NO: 72 and 52, the first and second polypeptide chains respectively include the amino acid sequences shown in SEQ ID NO: 68 and 73, the first and second polypeptide chains respectively include the amino acid sequences shown in SEQ ID NO: 76 and 52, the first and second polypeptide chains respectively include the amino acid sequences shown in SEQ ID NO: 7, or, a combination of amino acid sequences having at least 80% or at least 90% sequence identity with any one of the above first and second polypeptide chains, is included.

[0043] In some embodiments, the 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.

[0044] In some specific embodiments, the PD-1 / PVRIG / TIGIT binding protein comprises two identical first polypeptide chains and two identical second polypeptide chains.

[0045] In some embodiments, the PD-1 / PVRIG / TIGIT binding proteins of the Disclosure contain an antigen-binding domain that specifically binds to PD-1 of the Disclosure, and therefore possess all or any of its properties and functions.

[0046] PD-1 / PVRIG / TIGIT binding proteins contain a PVRIG / TIGIT binding domain, (a) 1 × 10 -7 Less than M K D To bind to human PVRIG by value, (b) Blocking the interaction between PVRIG and its ligand (e.g., PVRL2), (c) To release the inhibitory effect of dendritic cells (DCs) on T cells and activate T cells, (d) To release the inhibitory effect of tumor cells on NK cells, It further possesses at least one of the following characteristics.

[0047] In some embodiments, the PD-1 / PVRIG / TIGIT binding proteins of this disclosure can inhibit 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%, and about 90%.

[0048] In some embodiments, the PD-1 / PVRIG / TIGIT binding protein of the present disclosure covers variants, which have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations compared to any one combination of the first and second polypeptide chains, and which 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., the CDR region and / or the FR region).

[0049] In some embodiments, the present invention provides a PD-1 / PVRIG / TIGIT-binding protein that binds to or competitively binds to the same PD-1, PVRIG, and / or TIGIT or its epitope as the PD-1 / PVRIG / TIGIT-binding protein of the present disclosure, or that blocks the binding of the PD-1 / PVRIG / TIGIT-binding protein of the present disclosure to PD-1, PVRIG, and / or TIGIT, or whose binding to PD-1, PVRIG, and / or TIGIT is blocked by the PD-1 / PVRIG / TIGIT-binding protein of the present disclosure.

[0050] This disclosure relates to a PVRIG-binding protein or an antigen-binding domain that specifically binds to PVRIG, each of which contains or may be selected from the PVRIG antibodies 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). Herein, the above patent is incorporated as a whole.

[0051] This disclosure relates to a TIGIT-binding protein or an antigen-binding domain that specifically binds to TIGIT, each of which includes or may 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 may 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). Herein, the above patent is incorporated as a whole.

[0052] In some embodiments, the concentration of the PD-1 / PVRIG / TIGIT binding protein in the pharmaceutical composition is 0.01 mg / mL to 500 mg / mL, for example, 0.05 mg / mL to 450 mg / mL, 0.05 mg / mL to 400 mg / mL, 0.05 mg / mL to 350 mg / mL, 0.05 mg / mL to 300 mg / mL, 0.05 mg / mL to 250 mg / mL, 0.05 mg / mL to 200 mg / mL, 0.05 mg / mL to 150 mg / mL, 0.05 mg / mL to 140 mg / mL, 0.05 mg / mL to 130 mg / mL, 0.05 mg / mL to 120 mg / mL, 0.05 mg / mL to 110 mg / mL, 0.05 mg / mL to 100 mg / mL, 0.1 mg / mL to 400 mg / mL, 0.1 mg / mL to 350 mg / mL, 0.1 mg / mL to 300 mg / mL, 0.1 mg / mL to 250 mg / mL, 0.1 mg / mL to 200 mg / mL, 0.1 mg / mL to 150 mg / mL, 0.1 mg / mL to 140 mg / mL, 0.1 mg / mL to 130 mg / mL, 0.1 mg / mL to 120 mg / mL, 0.1 mg / mL to 110 mg / mL, 0.1 mg / mL to 100 mg / mL, 0.5 mg / mL to 350 mg / mL, 0.5 mg / mL to 300 mg / mL, 0.5 mg / mL to 250 mg / mL, 0.5 mg / mL to 200 mg / mL, 0.5 mg / mL to 150 mg / mL, 0.5 mg / mL to 140 mg / mL, 0.5 mg / mL to 130 mg / mL, 0.5 mg / mL to 120 mg / mL, 0.5 mg / mL to 110 mg / mL, 0.5 mg / mL to 100 mg / mL, 1 mg / mL to 300 mg / mL, 1 mg / mL to 250 mg / mL, 1 mg / mL to 200 mg / mL, 1 mg / mL to 150 mg / mL, 1 mg / mL to 140 mg / mL, 1 mg / mL to 130 mg / mL, 1 mg / mL to 120 mg / mL, 1 mg / mL to 110 mg / mL, 1 mg / mL to 100 mg / mL, 1 mg / mL to 95 mg / mL, 1 mg / mL to 90 mg / mL, 1 mg / mL to 85 mg / mL, 1 mg / mL to 80 mg / mL, 1 mg / mL to 75 mg / mL, 1 mg / mL to 70 mg / mL, 1 mg / mL to 65 mg / mL, 1 mg / mL to 60 mg / mL, 1 mg / mL to 55 mg / mL, 1 mg / mL to 50 mg / mL, 1 mg / mL to 45 mg / mL,1 mg / mL to 40 mg / mL, 1 mg / mL to 35 mg / mL, 1 mg / mL to 30 mg / mL, 1 mg / mL to 25 mg / mL, 1 mg / mL to 20 mg / mL, 1 mg / mL to 15 mg / mL, 5 mg / mL to 90 mg / mL, 5 mg / mL to 85 mg / mL, 5 mg / mL to 80 mg / mL, 5 mg / mL to 75 mg / mL, 5 mg / mL to 70 mg / mL, 5 mg / mL to 65 mg / mL, 5 mg / mL to 60 mg / mL, 5 mg / mL to 55 mg / mL, 5 mg / mL to 50 mg / mL, 5 mg / mL to 45 mg / mL, 5 mg / mL to 40 mg / mL, 5 mg / mL to 35 mg / mL, 5 mg / mL to 30 mg / mL, 5 mg / mL to 25 mg / mL, 5 mg / mL to 20 mg / mL, 5 mg / mL to 15 mg / mL, 8 mg / mL to 12 mg / mL, 30 mg / mL to 250 mg / mL, 30 mg / mL to 200 mg / mL, 30 mg / mL to 190 mg / mL, 30 mg / mL to 180 mg / mL, 30 mg / mL to 170 mg / mL, 30 mg / mL to 160 mg / mL, 30 mg / mL to 150 mg / mL, 30 mg / mL to 140 mg / mL, 30 mg / mL to 130 mg / mL, 30 mg / mL to 120 mg / mL, 30 mg / mL to 110 mg / mL, 30 mg / mL to 100 mg / mL, 50 mg / mL to 200 mg / mL, 50 mg / mL to 190 mg / mL, 50 mg / mL to 180 mg / mL, 50 mg / mL to 170 mg / mL, 50 mg / mL to 160 mg / mL, 50 mg / mL to 150 mg / mL, 50 mg / mL to 140 mg / mL, 50 mg / mL to 130 mg / mL, 50 mg / mL to 120 mg / mL, 50 mg / mL to 110 mg / mL, 50 mg / mL to 100 mg / mL, 70 mg / mL to 180 mg / mL, 70 mg / mL to 170 mg / mL, 70 mg / mL to 160 mg / mL, 70 mg / mL to 150 mg / mL, 70 mg / mL to 140 mg / mL, 70 mg / mL to 130 mg / mL, 70 mg / mL to 120 mg / mL, 70 mg / mL to 110 mg / mL, 70 mg / mL to 100 mg / mL, 90 mg / mL to 110 mg / mL, 95 mg / mL to 105 mg / mL. In some embodiments, the concentration of the PD-1 / PVRIG / TIGIT binding protein in the pharmaceutical composition is,The concentration ranges from 0.1 mg / mL to 400 mg / mL. In some embodiments, the concentration of PD-1 / PVRIG / TIGIT binding protein in the pharmaceutical composition is 0.5 mg / mL to 200 mg / mL. In some embodiments, the concentration of PD-1 / PVRIG / TIGIT binding protein in the pharmaceutical composition is 1 mg / mL to 150 mg / mL. In some embodiments, the concentration of PD-1 / PVRIG / TIGIT binding protein in the pharmaceutical composition is 5 mg / mL to 45 mg / mL. In some embodiments, the concentration of PD-1 / PVRIG / TIGIT binding protein in the pharmaceutical composition is 50 mg / mL to 110 mg / mL. In some embodiments, the concentration of PD-1 / PVRIG / TIGIT binding protein in the pharmaceutical composition is approximately 1 mg / mL, approximately 5 mg / mL, approximately 10 mg / mL, approximately 15 mg / mL, approximately 20 mg / mL, approximately 25 mg / mL, approximately 30 mg / mL, approximately 40 mg / mL, approximately 45 mg / mL, approximately 50 mg / mL, approximately 55 mg / mL, approximately 60 mg / mL, approximately 70 mg / mL, approximately 75 mg / mL, approximately 80 mg / mL, approximately 85 mg / mL, approximately 90 mg / mL, approximately 95 mg / mL, approximately 100 mg / mL, approximately 110 mg / mL, approximately 120 mg / mL, approximately 130 mg / mL, or approximately 150 mg / mL. In some embodiments, the concentration of PD-1 / PVRIG / TIGIT binding protein in the pharmaceutical composition is approximately 10 mg / mL, approximately 20 mg / mL, approximately 25 mg / mL, approximately 50 mg / mL, approximately 80 mg / mL, approximately 100 mg / mL, approximately 120 mg / mL, approximately 130 mg / mL, or approximately 150 mg / mL. In some embodiments, the concentration of PD-1 / PVRIG / TIGIT binding protein in the pharmaceutical composition is approximately 10 mg / mL, approximately 20 mg / mL, approximately 25 mg / mL, approximately 50 mg / mL, approximately 80 mg / mL, or approximately 100 mg / mL. In some embodiments, the concentration of PD-1 / PVRIG / TIGIT binding protein in the pharmaceutical composition is approximately 120 mg / mL, approximately 130 mg / mL, or approximately 150 mg / mL. In some embodiments, the concentration of 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.It is at least 400 mg / mL or at least 500 mg / mL.

[0053] In some embodiments, the concentration of the buffer in the pharmaceutical composition is 0.1 mM to 50 mM, for example, 0.1 mM to 45 mM, 0.1 mM to 40 mM, 0.1 mM to 35 mM, 0.1 mM to 30 mM, 0.1 mM to 25 mM, 0.1 mM to 20 mM, 0.1 mM to 15 mM, 0.1 mM to 10 mM, 0.5 mM to 45 mM, 0.5 mM to 40 mM, 0.5 mM to 35 mM, 0.5 mM to 30 mM, 0.5 mM to 25 mM, 0.5 The concentrations are approximately 0.5 mM to 20 mM, 0.5 mM to 15 mM, 0.5 mM to 10 mM, 1 mM to 40 mM, 1 mM to 35 mM, 1 mM to 30 mM, 1 mM to 25 mM, 1 mM to 20 mM, 1 mM to 15 mM, 1 mM to 10 mM, 5 mM to 35 mM, 5 mM to 30 mM, 5 mM to 25 mM, 5 mM to 20 mM, 5 mM to 15 mM, 5 mM to 10 mM, 8 mM to 30 mM, 8 mM to 25 mM, 8 mM to 20 mM, 8 mM to 15 mM, and 8 mM to 12 mM. In some embodiments, the concentration of the buffering agent in the pharmaceutical composition is approximately 0.5 mM to 40 mM. In some embodiments, the concentration of the buffering agent in the pharmaceutical composition is approximately 1 mM to 30 mM. In some embodiments, the concentration of the buffer in the pharmaceutical composition is about 5 mM to 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.

[0054] 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 polysorbate, poloxamer, Triton, sodium dodecylsulfonate, sodium laurylsulfonate, sodium octylglucoside, lauryl sulfobetaine, myristyl sulfobetaine, linoleum sulfobetaine, stearin sulfobetaine, lauryl sarcosine, myristyl sarcosine, linoleum sarcosine, stearin sarcosine, linoleum betaine, myristyl betaine, cetyl betaine, lauramidopropyl betaine, The surfactant is one or more selected from cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmitamidopropyl betaine, isostearamidopropyl betaine, myristamidopropyl dimethylamine, palmitamidopropyl dimethylamine, isostearamidopropyl dimethylamine, sodium methyl cocoyl, sodium methyl oleyl taurate, polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol. In some embodiments, the surfactant is selected from polysorbates. In some embodiments, the surfactant is selected from polysorbate 20 and / or polysorbate 80. In some embodiments, the surfactant is selected from polysorbate 80.

[0055] In some embodiments, the concentration of the surfactant in the pharmaceutical composition is 0.01 mg / mL to 10 mg / mL, for example, 0.01 mg / mL to 9 mg / mL, 0.01 mg / mL to 8 mg / mL, 0.01 mg / mL to 7 mg / mL, 0.01 mg / mL to 6 mg / mL, 0.01 mg / mL to 5 mg / mL, 0.01 mg / mL to 4 mg / mL, 0.01 mg / mL to 3 mg / mL, 0.01 mg / mL to 2 mg / mL, 0.01 mg / mL to 1 mg / mL, 0.01 mg / mL to 0.5 mg / mL, 0.05 mg / mL mL~8mg / mL, 0.05mg / mL~7mg / mL, 0.05mg / mL~6mg / mL, 0.05mg / 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~4mg / 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 The concentrations range from mL to 3.5 mg / mL, 0.3 mg / mL to 3 mg / mL, 0.3 mg / mL to 2.5 mg / mL, 0.3 mg / mL to 2 mg / mL, 0.3 mg / mL to 1.5 mg / mL, 0.3 mg / mL to 1 mg / mL, 0.3 mg / mL to 0.5 mg / mL, 0.7 mg / mL to 3.5 mg / mL, 0.7 mg / mL to 3 mg / mL, 0.7 mg / mL to 2.5 mg / mL, 0.7 mg / mL to 2 mg / mL, 0.7 mg / mL to 1.5 mg / mL, 0.7 mg / mL to 1 mg / mL, and 0.7 mg / mL to 0.9 mg / 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.

[0056] In some embodiments, the pharmaceutical composition further comprises sugars. In some embodiments, the sugars are one or more selected from sucrose, glucose, trehalose, and maltose. In some embodiments, the sugar is sucrose.

[0057] In some embodiments, the concentration of sugars in the pharmaceutical composition is 0.1% w / v to 20% w / v, for example, 0.1% w / v to 15% w / v, 0.1% w / v to 14% w / v, 0.1% w / v to 13% w / v, 0.1% w / v to 12% w / v, 0.1% w / v to 11% w / v, 0.1% w / v to 10% w / v, 0.1% w / v to 9.5% w / v, 0.1% w / v to 9% w / v, and 0.1% w / v to 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 / The concentrations are ~10% w / v, 5% w / v ~ 9.5% w / v, 5% w / v ~ 9% w / v, 5% w / v ~ 8.5% w / v, 5% w / v ~ 8% w / v, 6% w / v ~ 10% w / v, 6% w / v ~ 9.5% w / v, 6% w / v ~ 9% w / v, 6% w / v ~ 8.5% w / v, 6% w / v ~ 8% w / v, 7% w / v ~ 9.5% w / v, 7% w / v ~ 9% w / v, 7% w / v ~ 8.5% w / v, or 7% w / v ~ 8% w / v. In some embodiments, the sugar concentration in the pharmaceutical composition is 1% w / v ~ 15% w / v. In some embodiments, the sugar concentration in the pharmaceutical composition is 3% w / v ~ 12% w / v. In some embodiments, the concentration of sugars in the pharmaceutical composition is 5% w / v to 10% w / v.In some embodiments, the concentration of sugars 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 sugars in the pharmaceutical composition is about 8% w / v. In some embodiments, the concentration of sugars 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.

[0058] In some embodiments, the pharmaceutical composition further comprises another amino acid or a pharmaceutically acceptable salt thereof. In some embodiments, the other amino acid or pharmaceutically acceptable salt thereof is arginine or a pharmaceutically acceptable salt thereof. In some embodiments, the other amino acid or pharmaceutically acceptable salt thereof is arginine hydrochloride.

[0059] In some embodiments, the concentration of the other amino acids or their medicinal salts in the pharmaceutical composition is 1 mM to 200 mM, for example, 1 mM to 190 mM, 1 mM to 180 mM, 1 mM to 170 mM, 1 mM to 160 mM, 1 mM to 150 mM, 1 mM to 140 mM, 1 mM to 130 mM, 1 mM to 120 mM, 1 mM to 110 mM, 1 mM to 100 mM, 1 mM to 90 mM, 1 mM to 80 mM, 1 mM to 70 mM, 1 mM to 60 mM, 1 mM to 50 mM, 1 mM to 40 mM, 1 mM to 30 mM, 1 mM to 20 mM, 5 mM to 180 mM, 5 mM to 170mM, 5mM~160mM, 5mM~150mM, 5mM~140mM, 5mM~130mM, 5mM~120mM, 5mM~11 0mM, 5mM~100mM, 5mM~90mM, 5mM~80mM, 5mM~70mM, 5mM~60mM, 5mM~50mM, 5mM ~40mM, 5mM~30mM, 5mM~20mM, 10mM~170mM, 10mM~160mM, 10mM~150mM, 10mM~ 140mM, 10mM~130mM, 10mM~120mM, 10mM~110mM, 10mM~100mM, 10mM~90mM, 10m M~80mM, 10mM~70mM, 10mM~60mM, 10mM~50mM, 10mM~40mM, 10mM~30mM, 10mM~ 20mM, 15mM~160mM, 15mM~150mM, 15mM~140mM, 15mM~130mM, 15mM~120mM, 15 mM~110mM, 15mM~100mM, 15mM~90mM, 15mM~80mM, 15mM~70mM, 15mM~60mM, 15 mM~50mM, 15mM~40mM, 15mM~30mM, 15mM~20mM, 20mM~150mM, 20mM~140mM, 20 mM~130mM, 20mM~120mM, 20mM~110mM, 20mM~100mM, 20mM~90mM, 20mM~80mM, 20mM~70mM, 20mM~60mM, 20mM~50mM, 20mM~40mM, 20mM~30mM, 25mM~140mM, 2 5mM~130mM, 25mM~120mM, 25mM~110mM, 25mM~100mM, 25mM~90mM, 25mM~80mM , 25mM~70mM, 25mM~60mM, 25mM~50mM, 25mM~40mM, 30mM~140mM, 30mM~130mM,The ranges are 30mM~120mM, 30mM~110mM, 30mM~100mM, 30mM~90mM, 30mM~80mM, 30mM~70mM, 30mM~60mM, 30mM~50mM, 30mM~40mM, 40mM~120mM, 40mM~110mM, 40mM~100mM, 40mM~90mM, 40mM~80mM, 40mM~70mM, 40mM~60mM, 40mM~50mM, 50mM~110mM, 50mM~100mM, 50mM~90mM, 50mM~80mM, 50mM~70mM, and 50mM~60mM. In some embodiments, the concentration of other amino acids or their medicinal salts in the pharmaceutical composition is 10 mM to 80 mM. In some embodiments, the concentration of other amino acids or their medicinal salts in the pharmaceutical composition is 20 mM to 70 mM. In some embodiments, the concentration of other amino acids or their medicinal salts in the pharmaceutical composition is 40 mM to 60 mM. In some embodiments, the concentration of other amino acids or their medicinal salts in the pharmaceutical composition is 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, or about 70 mM. In some embodiments, the concentration of the buffer in the pharmaceutical composition is about 50 mM. In some embodiments, the concentration of other amino acids or their medicinal salts in the pharmaceutical composition is 5 mM to 170 mM. In some embodiments, the concentration of other amino acids or their medicinal salts in the pharmaceutical composition is 10 mM to 150 mM. In some embodiments, the concentration of other amino acids or their medicinal salts in the pharmaceutical composition is 15 mM to 120 mM. In some embodiments, the concentration of other amino acids or their medicinal salts 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 other amino acids or their pharmaceutically acceptable salts in the pharmaceutical composition is approximately 20 mM, approximately 50 mM, or approximately 100 mM.

[0060] In some embodiments, the pH of the buffer in the pharmaceutical composition is 3.5 to 9, for example, 3.5 to 8.5, 3.5 to 8, 3.5 to 7.5, 3.5 to 7, 3.5 to 6.9, 3.5 to 6.8, 3.5 to 6.7, 3.5 to 6.6, 3.5 to 6.5, 3.5 to 6.4, 3.5 to 6.3, 3.5 to 6.2, 3.5 to 6.1, 3.5 to 6, 3.5 to 5.9, 3.5 to 5.8, 3.5 to 5.7, 3.5 to 5.6, 3.5 to 5.5, 4 to 5.4, 4 to 5.3, 4 to 5.2, 4 to 5.1, 4 to 5.0, 4 to 8.5, 4 to 8, 4 to 7.5, 4 to 7, 4 to 6.9, and 4 to 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 0.1, 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.The pH ranges are 6, 4.8-5.5, 5-6.5, 5-6.4, 5-6.3, 5-6.2, 5-6.1, 5-6, 5-5.9, 5-5.8, 5-5.7, 5-5.6, or 5-5.5. 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.

[0061] In some embodiments, the pharmaceutical composition further comprises a pH adjuster such as sodium hydroxide and / or hydrochloric acid.

[0062] In some embodiments, the pH of the pharmaceutical composition differs from that of the buffering agent contained therein by no more than ±0.5. In some embodiments, the pH of the pharmaceutical composition is 3.5 to 9, for example, 3.5 to 8.5, 3.5 to 8, 3.5 to 7.5, 3.5 to 7, 3.5 to 6.9, 3.5 to 6.8, 3.5 to 6.7, 3.5 to 6.6, 3.5 to 6.5, 3.5 to 6.4, 3.5 to 6.3, 3.5 to 6.2, 3.5 to 6.1, 3.5 to 6, 3.5 to 5.9, 3.5 to 5.8, 3.5 to 5.7, 3.5 to 5.6, 3.5 to 5.5, 4 to 5.4, 4 to 5.3, 4 to 5.2, 4 to 5.1, 4 to 5.0, 4 to 8.5, 4 to 8, 4 to 7.5, 4 to 7, 4 to 6 0.9, 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.The pH ranges are 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, 5-6.5, 5-6.4, 5-6.3, 5-6.2, 5-6.1, 5-6, 5-5.9, 5-5.8, 5-5.7, 5-5.6, or 5-5.5. 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.

[0063] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a PD-1 / PVRIG / TIGIT binding protein (for example, the first polypeptide chain sequence and the second polypeptide chain sequence are A17m0902-1708-151H7-01-C of SEQ ID NOs. 76 and 52, respectively, or the first polypeptide chain sequence and the second polypeptide chain sequence are A17m0902-1708-151H7-C of SEQ ID NOs. 72 and 52, respectively), which comprises any one of the following 1) to 6): 1) PD-1 / PVRIG / TIGIT binding protein, Histidine salt buffers, for example, histidine hydrochloride buffers or histidine acetate buffers, histidine-histidine hydrochloride buffers or histidine-acetate buffers, 2) PD-1 / PVRIG / TIGIT binding protein, Histidine salt buffer, Polysorbate, It is sucrose, Optionally, the above composition further comprises arginine or a medicinal salt thereof (e.g., arginine hydrochloride), 3) PD-1 / PVRIG / TIGIT binding protein, Histidine hydrochloride buffer, for example, histidine hydrochloride buffer, Polysorbate, It is sucrose, Optionally, the above composition further comprises arginine or a medicinal salt thereof (e.g., arginine hydrochloride), 4) PD-1 / PVRIG / TIGIT binding protein, Histidine acetate buffer, for example, histidine-acetate buffer, Polysorbate, It is sucrose, Optionally, the above composition further comprises arginine or a medicinal salt thereof (e.g., arginine hydrochloride), 5) PD-1 / PVRIG / TIGIT binding protein, Citrate buffers, for example, citrate-sodium citrate buffers, citrate-disodium citrate buffers, 6) PD-1 / PVRIG / TIGIT binding protein, Citrate buffers, for example, citrate-disodium citrate buffers, Polysorbate, It is sucrose, Optionally, the composition further comprises arginine or a medicinal salt thereof (e.g., arginine hydrochloride).

[0064] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a PD-1 / PVRIG / TIGIT binding protein (for example, the first polypeptide chain sequence and the second polypeptide chain sequence are A17m0902-1708-151H7-01-C of SEQ ID NOs. 76 and 52, respectively, or the first polypeptide chain sequence and the second polypeptide chain sequence are A17m0902-1708-151H7-C of SEQ ID NOs. 72 and 52, respectively), which comprises or is one of the following 1) to 6): 1) PD-1 / PVRIG / TIGIT binding protein, histidine salt buffer, polysorbate, sucrose, water for injection, optional arginine or its pharmaceutically acceptable salt (e.g., arginine hydrochloride), and optional sodium hydroxide and / or hydrochloric acid. 2) PD-1 / PVRIG / TIGIT binding protein, histidine hydrochloride buffer, polysorbate, sucrose, water for injection, optional arginine or its pharmaceutically acceptable salt (e.g., arginine hydrochloride), and optional sodium hydroxide and / or hydrochloric acid. 3) PD-1 / PVRIG / TIGIT binding protein, histidine-histidine hydrochloride buffer, polysorbate, sucrose, water for injection, optional arginine or its pharmaceutically acceptable salt (e.g., arginine hydrochloride), and optional sodium hydroxide and / or hydrochloric acid. 4) PD-1 / PVRIG / TIGIT binding protein, histidine-acetic acid buffer, polysorbate, sucrose, water for injection, optional arginine or its pharmaceutically acceptable salt (e.g., arginine hydrochloride), and optional sodium hydroxide and / or hydrochloric acid. 5) PD-1 / PVRIG / TIGIT binding protein, citrate buffer, polysorbate, sucrose, water for injection, optional arginine or its pharmaceutically acceptable salt (e.g., arginine hydrochloride), and optional sodium hydroxide and / or hydrochloric acid. 6) PD-1 / PVRIG / TIGIT binding protein, citrate-disodium citrate buffer, polysorbate, sucrose, water for injection, optional arginine or its pharmaceutically acceptable salt (e.g., arginine hydrochloride), and optional sodium hydroxide and / or hydrochloric acid.

[0065] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a PD-1 / PVRIG / TIGIT binding protein (for example, the first polypeptide chain sequence and the second polypeptide chain sequence are A17m0902-1708-151H7-01-C of SEQ ID NOs. 76 and 52, respectively, or the first polypeptide chain sequence and the second polypeptide chain sequence are A17m0902-1708-151H7-C of SEQ ID NOs. 72 and 52, respectively), which comprises any one of the following 1) to 10): 1) PD-1 / PVRIG / TIGIT binding protein in concentrations of 0.01 mg / mL to 500 mg / mL Histidine hydrochloride buffers of 0.1 mM to 50 mM, for example, histidine hydrochloride buffers or histidine acetate buffers, histidine-histidine hydrochloride buffers or histidine-acetate buffers, Polysorbates ranging from 0.01 mg / mL to 10 mg / mL It is sucrose with a concentration of 0.1 w / v to 20% w / v. Optionally, the above pharmaceutical composition further comprises 1 mM to 200 mM arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride), Furthermore, the pH of the above pharmaceutical composition is 3.5 to 7. 2) PD-1 / PVRIG / TIGIT binding protein in concentrations of 0.1 mg / mL to 400 mg / mL 0.5 mM to 40 mM histidine hydrochloride buffer, for example, histidine hydrochloride buffer or histidine acetate buffer, histidine-histidine hydrochloride buffer or histidine-acetate buffer, Polysorbate in concentrations of 0.05 mg / mL to 5 mg / mL, It is sucrose with a concentration of 1% w / v to 15% w / v. Optionally, the above pharmaceutical composition further comprises 5 mM to 170 mM arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride), Furthermore, the pH of the above pharmaceutical composition is 4 to 6.5. 3) PD-1 / PVRIG / TIGIT binding protein in concentrations of 0.5 mg / mL to 200 mg / mL Histidine hydrochloride buffers in concentrations of 1 mM to 30 mM, for example, histidine hydrochloride buffers or histidine acetate buffers, histidine-histidine hydrochloride buffers or histidine-acetate buffers. Polysorbate in concentrations of 0.1 mg / mL to 3 mg / mL, It is sucrose with a concentration of 3% w / v to 12% w / v. Optionally, the above pharmaceutical composition further comprises 10 mM to 150 mM arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride), Furthermore, the pH of the above pharmaceutical composition is 4.2 to 6.2. 4) PD-1 / PVRIG / TIGIT binding protein in concentrations of 1 mg / mL to 150 mg / mL 5 mM to 20 mM histidine hydrochloride buffer, for example, histidine hydrochloride buffer or histidine acetate buffer, histidine-histidine hydrochloride buffer or histidine-acetate buffer, Polysorbate in concentrations of 0.2 mg / mL to 2 mg / mL, It is sucrose with a concentration of 5% w / v to 10% w / v. Optionally, the above pharmaceutical composition further comprises 15 mM to 120 mM arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride), Furthermore, the pH of the above pharmaceutical composition is 4.5 to 6. 5) PD-1 / PVRIG / TIGIT binding protein in concentrations of 0.01 mg / mL to 500 mg / mL 0.1 mM to 50 mM citrate buffers, for example, citrate-disodium citrate buffer, Polysorbates ranging from 0.01 mg / mL to 10 mg / mL It is sucrose with a concentration of 0.1 w / v to 20% w / v. Optionally, the above pharmaceutical composition further comprises 1 mM to 200 mM arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride), Furthermore, the pH of the above pharmaceutical composition is 3.5 to 7. 6) PD-1 / PVRIG / TIGIT binding protein in concentrations of 0.1 mg / mL to 400 mg / mL 0.5 mM to 40 mM citrate buffers, for example, citrate-disodium citrate buffer, Polysorbate in concentrations of 0.05 mg / mL to 5 mg / mL, It is sucrose with a concentration of 1% w / v to 15% w / v. Optionally, the above pharmaceutical composition further comprises 5 mM to 170 mM arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride), Furthermore, the pH of the above pharmaceutical composition is 4 to 6.5. 7) PD-1 / PVRIG / TIGIT binding protein in concentrations of 0.5 mg / mL to 200 mg / mL 1 mM to 30 mM citrate buffers, for example, citrate-disodium citrate buffers, Polysorbate in concentrations of 0.1 mg / mL to 3 mg / mL, It is sucrose with a concentration of 3% w / v to 12% w / v. Optionally, the above pharmaceutical composition further comprises 10 mM to 150 mM arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride), Furthermore, the pH of the above pharmaceutical composition is 4.2 to 6.2. 8) PD-1 / PVRIG / TIGIT binding protein in concentrations of 1 mg / mL to 150 mg / mL 5 mM to 20 mM citrate buffers, for example, citrate-disodium citrate buffer, Polysorbate in concentrations of 0.2 mg / mL to 2 mg / mL, It is sucrose with a concentration of 5% w / v to 10% w / v. Optionally, the above pharmaceutical composition further comprises 15 mM to 120 mM arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride), Furthermore, the pH of the above pharmaceutical composition is 4.5 to 6. 9) A pharmaceutical composition which is any one of 1) to 8) in which the polysorbate is polysorbate 80. 10) A pharmaceutical composition comprising any one of 1) to 9), further comprising water for injection.

[0066] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a PD-1 / PVRIG / TIGIT binding protein (for example, the first polypeptide chain sequence and the second polypeptide chain sequence are A17m0902-1708-151H7-01-C of SEQ ID NOs. 76 and 52, respectively, or the first polypeptide chain sequence and the second polypeptide chain sequence are A17m0902-1708-151H7-C of SEQ ID NOs. 72 and 52, respectively), which comprises or is one of the following 1) to 16): 1) PD-1 / PVRIG / TIGIT binding protein in concentrations of 1-150 mg / mL, Approximately 10 mM histidine hydrochloride buffer or histidine acetate buffer, for example, histidine-histidine hydrochloride buffer or histidine acetate buffer. Polysorbate 80 in concentrations of 0.2 mg / mL to 2 mg / mL. It is sucrose with a concentration of approximately 8% w / v. Furthermore, the pH of the above pharmaceutical composition is 4.5 to 6. 2) PD-1 / PVRIG / TIGIT binding protein in concentrations of approximately 10 mg / mL, 20 mg / mL, 25 mg / mL, 50 mg / mL, 80 mg / mL, 100 mg / mL, 120 mg / mL, 130 mg / mL, or 150 mg / mL, for example, approximately 10 mg / mL, 50 mg / mL, 100 mg / mL, 130 mg / mL, or 150 mg / mL. Approximately 10 mM histidine hydrochloride buffer, for example, histidine-histidine hydrochloride buffer, Polysorbate 80 at approximately 0.4 mg / mL, It is sucrose with a concentration of approximately 8% w / v. Furthermore, the pH of the above pharmaceutical composition is approximately 5. 3) PD-1 / PVRIG / TIGIT binding protein in concentrations of approximately 10 mg / mL, 20 mg / mL, 25 mg / mL, 50 mg / mL, 80 mg / mL, 100 mg / mL, 120 mg / mL, 130 mg / mL, or 150 mg / mL, for example, approximately 10 mg / mL, 50 mg / mL, 100 mg / mL, 130 mg / mL, or 150 mg / mL. Approximately 10 mM histidine hydrochloride buffer, for example, histidine-histidine hydrochloride buffer, Polysorbate 80 at approximately 0.4 mg / mL, It is sucrose with a concentration of approximately 8% w / v. Furthermore, the pH of the above pharmaceutical composition is approximately 5.5. 4) PD-1 / PVRIG / TIGIT binding protein in concentrations of approximately 10 mg / mL, 20 mg / mL, 25 mg / mL, 50 mg / mL, 80 mg / mL, 100 mg / mL, 120 mg / mL, 130 mg / mL, or 150 mg / mL, for example, approximately 10 mg / mL, 50 mg / mL, 100 mg / mL, 130 mg / mL, or 150 mg / mL. Approximately 10 mM histidine hydrochloride buffer, for example, histidine-histidine hydrochloride buffer, Polysorbate 80 at approximately 0.8 mg / mL, It is sucrose with a concentration of approximately 8% w / v. Furthermore, the pH of the above pharmaceutical composition is approximately 5. 5) PD-1 / PVRIG / TIGIT binding protein in concentrations of approximately 10 mg / mL, 20 mg / mL, 25 mg / mL, 50 mg / mL, 80 mg / mL, 100 mg / mL, 120 mg / mL, 130 mg / mL, or 150 mg / mL, for example, approximately 10 mg / mL, 50 mg / mL, 100 mg / mL, 130 mg / mL, or 150 mg / mL. Approximately 10 mM histidine hydrochloride buffer, for example, histidine-histidine hydrochloride buffer, Polysorbate 80 at approximately 0.8 mg / mL, It is sucrose with a concentration of approximately 8% w / v. Furthermore, the pH of the above pharmaceutical composition is approximately 5.5. 6) PD-1 / PVRIG / TIGIT binding protein in concentrations of approximately 10 mg / mL, 20 mg / mL, 25 mg / mL, 50 mg / mL, 80 mg / mL, 100 mg / mL, 120 mg / mL, 130 mg / mL, or 150 mg / mL, for example, approximately 10 mg / mL, 50 mg / mL, 100 mg / mL, 130 mg / mL, or 150 mg / mL. Approximately 10 mM histidine acetate buffer, Polysorbate 80 at approximately 0.4 mg / mL, It is sucrose with a concentration of approximately 8% w / v. Furthermore, the pH of the above pharmaceutical composition is approximately 5. 7) PD-1 / PVRIG / TIGIT binding protein in concentrations of approximately 10 mg / mL, 20 mg / mL, 25 mg / mL, 50 mg / mL, 80 mg / mL, 100 mg / mL, 120 mg / mL, 130 mg / mL, or 150 mg / mL, for example, approximately 10 mg / mL, 50 mg / mL, 100 mg / mL, 130 mg / mL, or 150 mg / mL. Approximately 10 mM histidine acetate buffer, Polysorbate 80 at approximately 0.4 mg / mL, It is sucrose with a concentration of approximately 8% w / v. Furthermore, the pH of the above pharmaceutical composition is approximately 5.5. 8) PD-1 / PVRIG / TIGIT binding protein in concentrations of approximately 10 mg / mL, 20 mg / mL, 25 mg / mL, 50 mg / mL, 80 mg / mL, 100 mg / mL, 120 mg / mL, 130 mg / mL, or 150 mg / mL, for example, approximately 10 mg / mL, 50 mg / mL, 100 mg / mL, 130 mg / mL, or 150 mg / mL. Approximately 10 mM histidine acetate buffer, Polysorbate 80 at approximately 0.8 mg / mL, It is sucrose with a concentration of approximately 8% w / v. Furthermore, the pH of the above pharmaceutical composition is approximately 5. 9) PD-1 / PVRIG / TIGIT binding protein in concentrations of approximately 10 mg / mL, 20 mg / mL, 25 mg / mL, 50 mg / mL, 80 mg / mL, 100 mg / mL, 120 mg / mL, 130 mg / mL, or 150 mg / mL, for example, approximately 10 mg / mL, 50 mg / mL, 100 mg / mL, 130 mg / mL, or 150 mg / mL. Approximately 10 mM histidine acetate buffer, Polysorbate 80 at approximately 0.8 mg / mL, It is sucrose with a concentration of approximately 8% w / v. Furthermore, the pH of the above pharmaceutical composition is approximately 5.5. 10) PD-1 / PVRIG / TIGIT binding protein in concentrations of approximately 10 mg / mL, 20 mg / mL, 25 mg / mL, 50 mg / mL, 80 mg / mL, 100 mg / mL, 120 mg / mL, 130 mg / mL, or 150 mg / mL, for example, approximately 10 mg / mL, 50 mg / mL, 100 mg / mL, 130 mg / mL, or 150 mg / mL. A citrate buffer of approximately 10 mM, for example, citrate-disodium citrate buffer, Polysorbate 80 at approximately 0.4 mg / mL, It is sucrose with a concentration of approximately 8% w / v. Furthermore, the pH of the above pharmaceutical composition is approximately 5. 11) PD-1 / PVRIG / TIGIT binding protein in concentrations of approximately 10 mg / mL, 20 mg / mL, 25 mg / mL, 50 mg / mL, 80 mg / mL, 100 mg / mL, 120 mg / mL, 130 mg / mL, or 150 mg / mL, for example, approximately 10 mg / mL, 50 mg / mL, 100 mg / mL, 130 mg / mL, or 150 mg / mL. A citrate buffer of approximately 10 mM, for example, citrate-disodium citrate buffer, Polysorbate 80 at approximately 0.4 mg / mL, It is sucrose with a concentration of approximately 8% w / v. Furthermore, the pH of the above pharmaceutical composition is approximately 5.5. 12) PD-1 / PVRIG / TIGIT binding protein in concentrations of approximately 10 mg / mL, 20 mg / mL, 25 mg / mL, 50 mg / mL, 80 mg / mL, 100 mg / mL, 120 mg / mL, 130 mg / mL, or 150 mg / mL, for example, approximately 10 mg / mL, 50 mg / mL, 100 mg / mL, 130 mg / mL, or 150 mg / mL. A citrate buffer of approximately 10 mM, for example, citrate-disodium citrate buffer, Polysorbate 80 at approximately 0.8 mg / mL, It is sucrose with a concentration of approximately 8% w / v. Furthermore, the pH of the above pharmaceutical composition is approximately 5. 13) PD-1 / PVRIG / TIGIT binding protein in concentrations of approximately 10 mg / mL, 20 mg / mL, 25 mg / mL, 50 mg / mL, 80 mg / mL, 100 mg / mL, 120 mg / mL, 130 mg / mL, or 150 mg / mL, for example, approximately 10 mg / mL, 50 mg / mL, 100 mg / mL, 130 mg / mL, or 150 mg / mL. A citrate buffer of approximately 10 mM, for example, citrate-disodium citrate buffer, Polysorbate 80 at approximately 0.8 mg / mL, It is sucrose with a concentration of approximately 8% w / v. Furthermore, the pH of the above pharmaceutical composition is approximately 5.5. 14) A pharmaceutical composition according to 1) to 13), further comprising 20 mM, 50 mM, or 100 mM arginine or its hydrochloride salt, 15) A pharmaceutical composition according to 1) to 14), further comprising hydrochloric acid and / or sodium hydroxide. 16) When a fixed volume is required, sterile water for injection is used to make a fixed volume of 1 mL, and the final volume is 1 mL, as is the case with the pharmaceutical compositions of 1) to 15).

[0067] The pharmaceutical compositions disclosed herein already possess sufficient drug discovery stability and can be stored stably for a long period of time.

[0068] 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.

[0069] This disclosure provides a method for preparing the pharmaceutical composition, comprising the step of lysing the PD-1 / PVRIG / TIGIT binding protein.

[0070] To facilitate the transport of the pharmaceuticals, the pharmaceutical compositions of this disclosure may be further prepared as lyophilized formulations.

[0071] In some embodiments, the pharmaceutical composition is a liquid formulation, as described in any one of the above-mentioned pharmaceutical compositions. In some embodiments, the solvent of the liquid formulation is water, saline solution, or glucose solution.

[0072] This disclosure further provides a lyophilized formulation characterized in that, after being redissolved, it can form the pharmaceutical composition described in any one of the above.

[0073] This disclosure further provides lyophilized formulations obtained by lyophilizing any one of the pharmaceutical compositions described above.

[0074] This disclosure further provides a redissolved solution obtained by redissolving the above-mentioned lyophilized formulation. In some embodiments, the redissolved solution is selected from, but is not limited to, water for injection, physiological saline, or glucose solution.

[0075] This disclosure further provides a product comprising a container containing the above-mentioned pharmaceutical composition, the above-mentioned lyophilized formulation, or the above-mentioned redissolving solution. In one embodiment, the container is an injectable vial made of neutral borosilicate glass. In one embodiment, the product comprises a drug information leaflet.

[0076] This disclosure further provides pharmaceutical compositions or lyophilized preparations or redissolving solutions for lyophilized preparations that are agents for treating or alleviating diseases or medical conditions.

[0077] Methods of treating diseases and pharmaceutical uses This disclosure provides a method for preparing a pharmaceutical composition or lyophilized preparation or a redissolving solution for a lyophilized preparation for treating, alleviating, preventing, or diagnosing a disease or medical condition.

[0078] In some embodiments, a method is provided for improving, alleviating, treating or preventing a disease, comprising administering to a subject an effective amount of the above-mentioned pharmaceutical composition or lyophilized preparation or a redissolved solution of the lyophilized preparation for improvement, alleviation, treatment or prevention.

[0079] In some embodiments, the use of the above-mentioned pharmaceutical compositions or lyophilized formulations or redissolving solutions of lyophilized formulations of this disclosure is provided in the preparation of agents for improving, alleviating, treating or preventing diseases.

[0080] In some embodiments, the disease is a proliferative disorder or any other disease or condition characterized by uncontrolled cell growth (e.g., cancer, where cancer and tumor are interchangeable in this disclosure), such as related diseases (e.g., cancer) associated with overexpression of PD-L1 or abnormal expression of PVRIG, TIGIT.

[0081] In some embodiments, the cancer is a solid tumor or a hematological malignancy.

[0082] In some embodiments, the cancer is progressive or metastatic.

[0083] In some embodiments, the cancers are selected from or are a combination of prostate cancer, hepatic cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple-negative breast cancer, pancreatic cancer, stomach / gastric cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial carcinoma, lung cancer (small cell lung cancer, non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous cell carcinoma and basal cell carcinoma), glioma, renal cancer (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 carcinoma, high MSI cancer, KRAS mutation tumor, 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.

[0084] In some embodiments, the subject area has a pathological condition related to PVRIG and / or TIGIT. In some specific forms, the subject area includes cancers that express or do not express PVRIG, and further includes non-metastatic or non-invasive and invasive or metastatic cancers, in which PVRIG expression in immune cells, stromal cells or lesioned cells inhibits antitumor and anti-invasive immune responses. The methods of this disclosure are applicable, for example, to the treatment of angiogenic carcinoma.

[0085] In some embodiments, the present invention provides a method for treating or preventing an infection or sepsis in a subject, comprising administering to the subject a therapeutic or prophylactic effective amount of the pharmaceutical composition or lyophilized preparation or a redissolved solution of the lyophilized preparation. In some specific forms, the infection is a pathogen infection characterized by varying degrees of dysfunction of the virus-specific T cell response, such as HIV, HCV, or HBV. In some specific forms, the sepsis includes severe sepsis, septic shock, systemic inflammatory response syndrome (SIRS), bacteremia, sepsis, toxemia, and septic syndrome.

[0086] In some embodiments, methods are provided for activating target cytotoxic T cells (CTLs), activating target NK cells, activating target γδ T cells, activating target Th1 cells, activating, reducing, or removing the number and / or activity of at least one type of cell in a target regulatory T cell (Treg) in vivo, increasing the production of IFN-γ and / or the secretion of pro-inflammatory cytokines in vivo, inhibiting the interaction between PVRIG and PVRL2 in vivo, and blocking or inhibiting the interaction between PD-1 and PD-L1 / PD-L2 in vivo, all of which involve administering an effective amount of the above-mentioned pharmaceutical composition or lyophilized formulation or a re-dissolved solution of the lyophilized formulation to the subject.

[0087] The pharmaceutical compositions comprising the PD-1 / PVRIG / TIGIT binding protein of this disclosure can be used to treat patients requiring such treatment by parenteral administration. The parenteral administration route can be selected from subcutaneous injection, intramuscular injection, or intravenous injection. [Brief explanation of the drawing]

[0088] [Figure 1] These are the activity results for detecting anti-PD-1 nanobodies using the PD-1 / PD-L1 NFAT reporter gene system. Pembrolizumab was used as a positive control and hIgG4 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. [Figure 2] This study detected the degree of activation of A17m09_hIgG4 into T cells by cytokine release during a mixed lymphocyte reaction between DC cells and T cells. Pembrolizumab was used as a positive control and hIgG4 as a negative control. [Figure 3] Figure 3A shows the results of an in vivo efficacy study in which A17m09_hIgG4 inhibited tumor growth in mouse MC38 colon cancer. Figure 3B shows the results for mouse tumor volume and mouse body weight. Pembrolizumab was used as a positive control and PBS as a negative control. [Figure 4] These are the results of mixed lymphocyte reaction (MLR) experiments with anti-PVRIG nanobodies 30 and 151, using Tab5 as a positive control and hIgG4 as a negative control. [Figure 5] These are the results of an MLR experiment with the anti-PVRIG / TIGIT bispecific antibody 1708-151H8, simultaneously detecting 151H8, 1708, hIgG4, Tab5, and Pembrolizumab as controls. [Figure 6] This shows the antitumor results in a mouse subcutaneous tumor model of human melanoma A375 mixed with human PBMCs, using a combination of anti-PVRIG / TIGIT bispecific antibodies 1708-151, 1708, and 151. Figure 6B shows the corresponding mouse weight figures. [Figure 7] This shows the antitumor results of the anti-PVRIG / TIGIT bispecific antibodies 1708-151H7 and 1708-30H2 in a mouse subcutaneous tumor model of human melanoma A375 mixed with human PBMCs. Figure 7B shows the corresponding mouse body weight diagrams. [Figure 8] This is a schematic diagram showing the arrangement of the four antibodies, which are trispecific anti-PD-1 / PVRIG / TIGIT antibodies. [Figure 9] This shows the detection results of the binding of antibodies in four configurations, A17h1-1708-151H7, to the TIGIT antigen. [Figure 10] This shows the results of detecting the binding of antibodies in four configurations, A17h1-1708-151H7, to the PVRIG antigen. [Figure 11] These represent the binding ability of the anti-PD-1 / PVRIG / TIGIT trispecific antibody to cells overexpressing PVRIG, TIGIT, and PD-1 antigens, respectively, after PVRIG sequence optimization. [Figure 12]This involves the blockage of PD-L1 / PD-1 binding in the PD-1 / PD-L1 NFAT reporter gene system by antibodies in four configurations: A17h1-1708-151H7. [Figure 13] This shows the results of MLR detection using antibodies in four configurations: A17h1-1708-151H7. [Figure 14] These are experimental results regarding the NK cell killing function of antibodies in four configurations: A17h1-1708-151H7. [Figure 15] This study evaluates the antitumor activity of an anti-PD-1 / PVRIG / TIGIT trispecific antibody in a mouse subcutaneous tumor model in which human melanoma A375 responsive to PD-1 monoclonal antibodies was mixed with human PBMCs. [Figure 16] This study evaluates the antitumor activity of an anti-PD-1 / PVRIG / TIGIT trispecific antibody in a mouse subcutaneous tumor model in which human melanoma A375, which is unresponsive to PD-1 monoclonal antibody, is mixed with human PBMCs. [Modes for carrying out the invention]

[0089] 1. Terminology To make this disclosure more easily understood, several technical and scientific terms are defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have meanings that are generally understood by those skilled in the art.

[0090] The three-letter and one-letter amino acid codes used in this disclosure are as described in J.biol.chem, 243, p3558 (1968).

[0091] "Programmed death 1," "Programmed cell death 1," "Protein PD-1," "PD-1," "PDCD1," and "hPD-1" may be used interchangeably and include variants, isotypes, 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 registry number U64863. "Programmed death ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands of PD-1 (the other being PD-L2), and when it binds to PD-1, it downregulates T cell activation and cytokine secretion. "PD-L1" as used herein includes human PD-L1 (hPD-L1), variants, isotypes, and interspecies 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 registry number Q9NZQ7.

[0092] "PVRIG" or "PVRIG protein" or "PVRIG polypeptide" may optionally include known or wild-type PVRIG as described herein, and any naturally occurring splice variants, amino acid variants or isoforms, in particular fragments of the soluble extracellular domain (ECD) of PVRIG, including (but not limited to), any such protein or its variants, conjugates or fragments. Hereinafter, the definition of ECD is as described in patent WO2016134333. The complete human PVRIG sequence can be found at GenBank registration number AAH73861.1.

[0093] "TIGIT" or "TIGIT protein" or "TIGIT polypeptide" may optionally include, but not limited to, known or wild-type TIGIT as described herein, and any naturally occurring splice variants, amino acid variants, or isoforms of such proteins or their variants, conjugates, or fragments. The complete TIGIT sequence can be found at GenBank registration number AAI01289.1.

[0094] "Binding to PD-1" means being able to interact with PD-1 or its epitope, and the PD-1 or its epitope may be of human origin. "Binding to PVRIG" means being able to interact with PVRIG or its epitope, and the PVRIG or its epitope may be of human origin. "Binding to TIGIT" means being able to interact with TIGIT or its epitope, and the TIGIT or its epitope may be of human origin. "Antigen binding site" means a discontinuous three-dimensional spatial site in an antigen that is recognized by the antibody or antigen-binding fragment of this disclosure.

[0095] A “PD-1 binding protein” covers any molecule capable of specifically binding to the PD-1 protein or its epitope. A PD-1 binding protein may include an antibody against PD-1 as defined in this disclosure, its antigen-binding fragment, or its complex. A PD-1 binding protein further covers an immunoglobulin superfamily antibody (IgSF) or a CDR transplantation molecule. A “PD-1 binding protein” of this disclosure may include at least one immunoglobulin monovariate domain (e.g., VHH) that binds to PD-1. In some embodiments, a “PD-1 binding protein” may include two, three, four or more immunoglobulin monovariate domains (e.g., VHH) that bind to PD-1. The PD-1 binding proteins of this disclosure include the immunoglobulin monovariable domain of PD-1, as well as part of a part having effector function such as a linker and / or a half-life extension portion (e.g., an immunoglobulin monovariable domain that binds to serum albumin), and / or a fusion partner (e.g., serum albumin) and / or a compounded polymer (e.g., PEG) and / or an Fc region. In some embodiments, the “PD-1 binding protein” of this disclosure further covers a bispecific / multispecific antibody, which includes an immunoglobulin that binds to different antigens (e.g., a first antibody that binds to a first antigen (e.g., PD-1) and a second antibody that binds to a second antigen (e.g., PVRIG), optionally including a third specific antibody that binds to a third antigen (e.g., TIGIT), and further selectively including a fourth antibody that binds to a fourth antigen). The “PD-1 binding protein” covers “PD-1 / PVRIG binding protein”, “PD-1 / TIGIT binding protein”, and “PD-1 / PVRIG / TIGIT binding protein”.

[0096] The “PD-1 binding protein” or “anti-PD-1 antibody” of this disclosure may include, for example, one or more effector molecules in a complex manner. The “effector molecules” include, for example, antitumor agents, drugs, toxins, bioactive proteins (e.g., enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and their fragments (e.g., DNA, RNA and their fragments), radionuclides, particularly radioactive iodides, radioisotopes, chelated metals, nanoparticles, and reporter groups such as fluorescent compounds or compounds detectable by NMR or ESR spectroscopy. If the effector molecule is a polymer, it may typically be, for example, a synthetic or naturally occurring polymer such as a linear or branched polyalkylene group, polyalkene group or polyoxyalkylene polymer with optionally substituted linear or branched polyalkylene groups, or branched or unbranched polysaccharides such as homo or heteropolysaccharides. Specific optional substituents that may be present in the synthetic polymer include one or more hydroxyl groups, methyl groups or methoxy groups. Specific examples of synthetic polymers include selectively substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, and particularly selectively substituted poly(ethylene glycol) such as methoxypoly(ethylene glycol) or derivatives thereof. Specific naturally occurring polymers include lactose, amylose, glucan, glycogen or derivatives thereof. In one embodiment, the polymer is albumin or a fragment thereof, for example, human serum albumin or a fragment thereof. The combination of the polymer with a PD-1 binding protein or anti-PD-1 antibody can be achieved by conventional methods.

[0097] "Cytokines" is a general term for proteins released from a population of cells that act on other cells as intercellular substances. 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.

[0098] The term "antibody" encompasses a variety of antibody structures, as long as they exhibit the desired antigen-binding activity. This includes, but is 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). The term "antibody" may also refer to immunoglobulins, which are tetrapeptide chain structures consisting of two heavy chains and two light chains linked by interchain disulfide bonds. Immunoglobulins differ in their antigenicity due to variations in the amino acid composition and sequence order of the heavy chain constant region. This allows immunoglobulins to be divided into five types, or immunoglobulin isotypes, IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. Ig of the same type can be further divided into different subclasses based on differences in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains can be divided into κ chains or λ chains depending on the difference in their constant region. Each of the five types of Ig may have either a κ chain or a λ chain.

[0099] In antibody heavy and light chains, the sequence of approximately 110 amino acids near the N-terminus is significantly altered, forming a variable region (V region), while the remaining amino acid sequence near the C-terminus is relatively stable, forming a constant region (C region). The variable region includes three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). The three hypervariable regions determine the specificity of the antibody and are also called 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 from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3, while the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0100] "Antigen-binding fragments" cover 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, VL, or VHH), scFv, bivalent, trivalent, or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and any one of the above epitope-binding fragments (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136, and Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217).

[0101] Methods for producing and preparing these antigen-binding fragments are known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The Fab-Fv form was first disclosed in WO2009 / 040562, and its disulfide bond-stabilized form, Fab-dsFv, was first disclosed in WO2010 / 035012. The antigen-binding fragments of this disclosure further include the Fab and Fab' fragments described in WO2005 / 003169, WO2005 / 003170, and WO2005 / 003171. The polyvalent antibodies may include multispecific antibodies, such as bispecific antibodies, or monospecific antibodies (see, for example, WO92 / 22583 and WO05 / 113605), an example of the latter being Tri-Fab (or TFM) as described in WO92 / 22583.

[0102] "Bispecific antibodies" cover antibodies (including antibodies or their antigen-binding fragments, e.g., single-chain antibodies) that specifically bind to two different antigens or two or at least two different antigenic epitopes of the same antigen. Conventional technology has disclosed bispecific antibodies of various structures. Based on the integrity of the IgG molecule, they can be divided into IgG-like bispecific antibodies and antibody-fragment type bispecific antibodies. Based on the number of antigen-binding regions, they can be divided into bivalent, trivalent, quadrivalent, or more bispecific antibodies. Based on whether the structure is bilaterally symmetrical, they can be divided into symmetrical bispecific antibodies and asymmetrical bispecific antibodies. Among these, bispecific antibodies based on antibody fragments, for example, Fab fragments with Fc fragment deletion, form bispecific antibodies with relatively low immunogenicity, small molecular weight, and relatively high tumor tissue permeability by binding two or more Fab fragments within a single molecule. Typical antibody structures of this type include, for example, bispecific antibodies such as F(ab)2, scFv-Fab, and (scFv)2-Fab. Antibodies such as IgG-like bispecific antibodies (e.g., those containing an Fc fragment) have a relatively large molecular weight. The Fc fragment contributes to the later stages of antibody purification, improving its solubility and stability. Furthermore, the Fc portion may bind to the receptor FcRn, potentially increasing the serum half-life of the antibody.Typical bispecific antibody structural models include, for example, bispecific antibodies such as KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, 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, and F(ab)4-CrossMAb (Aran F. Labrijn et al., Nature Reviews Drug Discovery volume). See 18, pages 585-608 (2019), Chen S1 et al., J Immunol Res. 2019 Feb 11, 2019:4516041.

[0103] A "triply specific antibody" refers to an antibody that can specifically bind to three or at least three different epitopes, which may be epitopes of different antigens or different epitopes of the same antigen.

[0104] The definitive determination or definition of a CDR can be achieved by elucidating the structure of the antibody and / or the structure of the antibody-ligand complex, thereby achieving a reliable description of the CDR and identification of the binding site residues. This can be achieved by any one of the various techniques known to those skilled in the art, for example, by X-ray crystallography. Various analytical methods can be used for the identification of CDRs, including, but not limited to, the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definition, and conformational definition.

[0105] The Kabat numbering system is a standard for numbering residues in antibodies and is typically 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 location 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 from Oxford Molecular Group to model antibody structures (see, for example, Martin et al., 1989, ProcNatl Acad Sci (USA), 86:9268-9272, "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK, Oxford Molecular, Ltd). The AbM numbering system models the tertiary structure of antibodies from their basic sequences using a combination of knowledge databases and the ab initio method (see "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" in Samudrala et al., 1999, PROTEINS, Structure, Function and Genetics Suppl., 3:194-198). Contact definitions are based on the analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J.Mol.Biol., 5:732-45). In conformational definitions, the position of the CDR can be identified as a residue that makes an enthalpy contribution to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166).Furthermore, other definitions of CDR boundaries may not strictly adhere to one of the methods described above, but still overlap with at least some of the Kabat CDRs. CDR boundaries can be shortened or extended based on predictions or experimental results indicating that certain residues or groups of residues do not significantly affect antigen binding. As used in this disclosure, CDR can refer to a CDR defined by any known method (including combinations of methods) in the art. Correspondences between various numbering systems are well known to those skilled in the art and are, exemplarily, shown in Table 1 below.

[0106] [Table 1]

[0107] The CDR amino acid residues in the VL and VH regions of the antibodies disclosed herein correspond in number and position to the known Kabat numbering system.

[0108] The antibodies of this disclosure may be polyclonal, monoclonal, heterologous, homologous, or modified thereof, of which monoclonal antibodies are particularly applicable to multiple examples. Generally, the antibodies of this disclosure are recombinant antibodies.

[0109] As used herein, “recombinant” broadly refers to products such as cells, nucleic acids, proteins, or vectors, and indicates that such cells, nucleic acids, proteins, or vectors have already been modified by introducing heteronucleotides or proteins, or by altering native nucleic acids or proteins. For example, recombinant cells express genes that are not present in the natural (non-recombinant) cell form, or express native genes that are originally abnormally expressed, underexpressed, or not expressed at all.

[0110] A polypeptide or protein "domain" refers to a folded protein structure that can maintain its tertiary structure independently of other parts of the protein. Generally, a domain is responsible for a single functional property of the protein and, in many cases, may be added, removed, or transferred to another protein without losing the function of other parts of the protein and / or the domain itself.

[0111] The "immunoglobulin domain" refers to the spherical region of the antibody chain. The immunoglobulin domain is characterized by its ability to maintain the folding properties of the antibody molecule.

[0112] An "immunoglobulin variable domain" basically refers to an immunoglobulin domain consisting 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" or "CDRs"—"complementarity-determining region 1" or "CDR1," "complementarity-determining region 2" or "CDR2," and "complementarity-determining region 3" or "CDR3." Therefore, the general structure or sequence of an immunoglobulin variable domain may be shown as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Because the immunoglobulin variable domain has an antigen-binding site, it confers specificity to the antigen.

[0113] The term "antibody framework (FR)" refers to a portion of the variable domains used as stents in the antigen-binding loop (CDR) of the variable domain.

[0114] The term "immunoglobulin monovariate domain" is typically used to refer to an immunoglobulin monovariate domain (which may be a heavy or light chain domain and may include the VH, VHH, or VL domain) that can form a functional antigen-binding site when it does not interact with other monovariate domains (for example, when there is no VH / VL interaction required between the VH and VL domains of a conventional quadruple-chain monoclonal antibody). Examples of "immunoglobulin monovariate domains" include nanobodies (including camelid VH such as VHH, humanized VHH, and / or camelid human VH), IgNARs, domains, and (monodomain) antibodies (e.g., dAbs) as or derived from the VH domain. TM ) and antibodies (single domain) as or derived from VL domains (e.g., dAbs TM ) are included. Immunoglobulin monovariate domains based on and / or derived from heavy chain variable domains (e.g., VH or VHH domains) are generally preferred. One specific example of an immunoglobulin monovariate domain is the "VHH domain" (or abbreviated as "VHH") as defined below.

[0115] "VHH domain" refers to heavy chain single-domain antibodies, VHH, V HAlso known as the H domain, VHH antibody fragment, VHH antibody, or nanobody, it is a variable domain of antigen-binding immunoglobulin called a "heavy chain antibody" (i.e., a "light chain deletion antibody") (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 above VHH from the VH and VL present in antibodies with conventional tetrapeptide chain structures. The VHH domain specifically binds to an epitope without requiring other antigen-binding domains (however, in conventional tetrapeptide chain domain antibodies, the epitope is recognized by both the VH and VL domains). The VHH domain is a small, stable, and efficient antigen-recognition unit formed from a single immunoglobulin domain. "Heavy chain single-domain antibody", "VHH domain", "VHH", "V HHThe terms “domain,” “VHH antibody fragment,” “VHH antibody,” “Nanobody®,” and “Nanobody® domain” (“Nanobody” is a trademark of Ablynx NV, Ghent, Belgium) may be used interchangeably. “VHH domain” includes, but is not limited to, naturally occurring antibodies produced by camelids, and may also include antibodies that have been humanized after antibody production by camelids, or those obtained by screening using phage display technology. The total number of amino acid residues in a VHH domain is typically in the range of 110–120, and often between 112–115. However, it should be noted that relatively small and relatively long sequences may also be suitable for the purposes described herein. Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the following publications: R. van der Linden et al., Journal of Immunological Methods, 240(2000)185-195; Li et al., J Biol Chem., 287(2012)13713-13721; Deffer et al., African Journal of Biotechnology Vol.8(12), pp.2645-2652, 17 June, 2009; and WO94 / 04678.

[0116] As is known in this field regarding VH domains and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions based on the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat number). This generally means that the numbers based on Kabat may or may not correspond to the actual numbers of amino acid residues in the actual sequence. Other numbering systems or rules for VHH include Chothia, IMGT, and AbM.

[0117] A "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by transplanting a non-human CDR sequence into a human antibody variable region framework. The presence of a large amount of non-human protein components in chimeric antibodies can overcome the strong immune response induced by such antibodies. To avoid both decreased immunogenicity and decreased activity, activity can be maintained by performing the fewest possible reverse mutations on the fully human antibody variable region. An example of "humanization" includes replacing one or more amino acid residues in the original VHH sequence with one or more amino acid residues in the VH domain of a conventional tetrapeptide chain antibody at a corresponding position (in this disclosure, this is also called "sequence optimization," which may cover other modifications to the sequence, such as the removal of potential post-translational modification sites, in addition to humanization, thus providing improved characteristics of the VHH). The humanized VHH domain may contain one or more fully human framework region sequences. In some specific embodiments, the human framework region sequence of IGHV3 may be included. Further examples of "humanization" include antibodies produced by transplanting a mouse CDR sequence onto a variable region framework of a human antibody, i.e., a framework sequence of a different type of human germline antibody. The presence of a large amount of heterologous protein components in the chimeric antibody can overcome the induced strong antibody-variable antibody reaction. Humanization methods include, for example, resurfacing of protein surface amino acids and antibody-humanized universal framework grafting (CDR grafting to a universal framework) which involves "transplanting" the CDR into another "stent" (including, but not limited to, a human stent or a non-immunoglobulin stent). Stents and techniques suitable for the above CDR grafting are known in the art.For example, germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database and in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies disclosed herein also include humanized antibodies that have undergone affinity maturation to CDRs presented by phages. Furthermore, to avoid a decrease in immunogenicity and activity simultaneously, activity can be maintained by introducing the fewest possible reverse or reverse mutations into the above-mentioned human antibody variable region framework sequence.

[0118] An "affinity-mature" antibody is defined as an antibody that has one or more modifications in one or more hypervariable regions (HVRs) compared to the parent antibody, and such modifications improve the antibody's affinity for the antigen. For example, an "affinity-mature" PD-1 binding protein or PD-1 antibody has one or more changes in one or more CDRs, and these changes increase its affinity for the antigen compared to its parent antibody. Affinity-matured antibodies can be prepared by methods known in this field, such as 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):889896, KS Johnson & RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press 1996.

[0119] "Fully human antibodies" include antibodies having variable and constant regions of human germline immunoglobulin sequences. Fully human antibodies of this disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis, or in vivo somatic mutation). "Fully human antibodies" do not include "humanized antibodies."

[0120] Typically, "specifically binding" or "selectively binding" refers to the binding of a binding protein to an epitope in the antigen. The PD-1 binding protein, PD-1 / PVRIG binding protein, PD-1 / TIGIT binding protein, and PD-1 / PVRIG / TIGIT binding protein of this disclosure are measured in a Biacore, KinExA, or Fortibio assay, preferably 10 -7 ~10 -10 moles / liter (M), more preferably 10 -8 ~10 -10 moles / liter, more preferably 10 -9 ~10 -10 or a dissociation constant (K) less than or equal to that (K D ), and / or at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M, more preferably at least 10 -10 It binds to the antigen (i.e., PD-1, PVRIG and / or TIGIT) or its epitope with an association constant (KA) of M. -4 K is larger than M. D The values ​​are also generally considered to indicate nonspecific binding. Specific binding of a binding protein to an antigen or epitope can be measured by any known suitable method, including, for example, the surface plasmon resonance (SPR) assay, Scatchard assay and / or competitive binding assay (e.g., radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assay) described herein.

[0121] An "epitope" refers to a site in an antigen that binds to an immunoglobulin or antibody. Epitopes may be formed from adjacent amino acids or from non-adjacent amino acids arranged in parallel by tertiary folding of the protein. Epitopes formed from adjacent amino acids are usually retained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with a denaturing solvent. Epitopes typically have a unique spatial conformation and contain at least 3 to 15 amino acids. Methods for determining epitopes that bind to a given antibody are well known in the art and include Western blotting and immunoprecipitation detection analysis. Methods for determining the spatial conformation of epitopes include techniques in the art and techniques described in this disclosure, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0122] In this disclosure, "binding affinity" or "affinity" is used as an indicator of the strength of the non-covalent interaction between two molecules (e.g., an antibody or a part thereof and an antigen). The binding affinity between two molecules is expressed by the dissociation constant (K). D This can be quantified by determining the K. For example, by measuring the dynamics of composite formation and dissociation using the surface plasmon resonance (SPR) method (Biacore). D This can be determined. The rate constants corresponding to the bonding and dissociation of monovalent complexes are called the bonding rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. D is, K D The dissociation constant is related to ka and kd by the equation =kd / ka. The value of the dissociation constant can be directly determined by well known methods, and furthermore, it can be calculated for complex mixtures by methods such as those described by Caceci et al. (1984, Byte 9:340-362). For example, by a double-filtration nitrocellulose filter binding assay disclosed by Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432), K DThis can be determined. Other standard assays for evaluating the binding ability of antibodies to target antigens are known in the art and include, for example, ELISA, Western blotting, RIA and flow cytometry analysis, and other assays mentioned herein. Antibody binding kinetics and binding affinity can be determined using standard assays known in the art, such as surface plasmon resonance (SPR), for example, Biacore. TM The K of each antibody / antigen complex can be evaluated by the system or KinExA. D By comparing values, we can compare binding affinities related to interactions with different molecules, for example, comparing the binding affinities of different antibodies to a given antigen. Similarly, the specificity of the interaction is the K of the target interaction (e.g., a specific interaction between an antibody and an antigen). D K values ​​and non-targeted interactions (e.g., control antibodies known not to bind to PD-1) D It can be evaluated by determining and comparing values.

[0123] A "conservative substitution" refers to the substitution of an original amino acid residue with another amino acid residue that has similar properties. 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. Also, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have polar side chains with no charge, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have nonpolar side chains. Furthermore, 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 substitution of amino acid residues in the group exhibiting the above-mentioned similar properties does not result in any specific change in properties.

[0124] "Homologousity," "identity," or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. Two sequences being compared are homologous at a given position if all positions in those sequences are occupied by the same nucleotide or amino acid monomer. For example, two DNA molecules are homologous if all positions in those sequences are occupied by the same nucleotide. The percentage of homology between two sequences is a function of dividing the number of matching or homologous positions shared by the two sequences by the number of positions being compared and multiplying by 100%. For example, if the sequences are optimally aligned, and 6 out of 10 positions in the two sequences are matching or homologous, then the two sequences are 60% homologous. Generally, two sequences are compared when they are aligned to obtain the highest possible percentage of homology.

[0125] "Inhibition" and "blockage" may be used interchangeably and cover both partial and complete inhibition / blockage. "Inhibition of growth" (e.g., for cells) is intended to also include any measurable decrease in cell growth.

[0126] "To inhibit the growth of..." or "to inhibit growth" refers to inhibiting the growth or proliferation of cells.

[0127] "Proliferative disorders" refer to medical conditions associated with a certain degree of abnormal cell proliferation. In one embodiment, a proliferative disorder refers to cancer.

[0128] "Tumor" refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, and all precancerous and cancerous cells and tissues. "Cancer," "cancerous," "proliferative condition," and "tumor" are not mutually exclusive as they appear in this disclosure.

[0129] "Cancer prevention" means delaying, inhibiting, or preventing the onset of cancer in a subject, where the onset of cancer or the initiation of tumor formation in the subject has not been confirmed, but cancer susceptibility has been identified, for example, by genetic screening or other methods. It further includes halting the progression of the precancerous condition to a malignant tumor or inducing its regression by treating the subject with a precancerous condition.

[0130] "Optional" or "optionally" means that the event or situation described thereafter may occur, but does not necessarily have to occur, and the description includes both cases in which the event or situation occurs and cases in which it does not. "And / or" should be considered as specifically indicating whether each of the two designated features or components has or does not have the other. Accordingly, the term "and / or" as used in the phrase "A and / or B" in this disclosure includes "A and B," "A or B," "A" (alone) and "B" (alone). Unless otherwise specified in the context, throughout the specification and claims, words such as "contains," "has," and "includes" should be understood to have a comprehensive meaning, i.e., "includes, but not limited to," rather than an exclusive or exhaustive meaning.

[0131] As used herein, the terms “about” or “approximately” mean that a numerical value is within the acceptable margin of error of a specific value measured by a person skilled in the art, and the numerical portion is determined by how it is measured or measured (i.e., the limits of the measuring system). For example, in each execution in the art, “about” may mean a standard difference of 1 or more than 1. Alternatively, “about” or “basically including” may mean a range of at most ±30%, for example, pH about 5.5 means pH 5.5 ± 1.65. Furthermore, particularly with respect to biological systems or processes, the terms may mean at most one order of magnitude or at most five times the numerical value. Unless otherwise specified, where specific values ​​appear in this application and claims, the meaning of “about” or “basically including” should be assumed to be within the acceptable margin of error of the specific value.

[0132] A "buffering agent" refers to a buffering agent that can withstand changes in pH due to the action of its acid-base conjugated components. Examples of buffering agents that control pH within an appropriate range include trishydroxymethylaminomethane (Tris), acetate, succinate, gluconate, histidine salt, oxalate, lactate, phosphate, goji tartrate, fumarate, glycylglycine, and other organic acid buffering agents.

[0133] A "histidine salt buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include buffers such as histidine hydrochloride, histidine acetate, histidine phosphate, and histidine sulfate, and preferably 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 from histidine and hydrochloric acid.

[0134] A "citrate buffer" is a buffer containing citrate ions. Examples of citrate buffers include citrate-sodium citrate, citrate-potassium citrate, citrate-calcium citrate, and citrate-magnesium citrate. The preferred citrate buffer is citrate-sodium citrate buffer.

[0135] "Pharmaceutical composition" refers to a mixture comprising one or more antibodies described herein and other chemical components, the other components being, for example, physiologically / pharmaceutically acceptable carriers and excipients. The pharmaceutical composition is intended to maintain the stability of the active ingredient, facilitate administration to the body, and contribute to the absorption of the active ingredient in order to exert further biological activity.

[0136] In this disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0137] Unless otherwise specified, the solvent in the solutions of the pharmaceutical compositions described herein is water.

[0138] "Lyophilized formulation" refers to a pharmaceutical composition in liquid or solution form, or a formulation or pharmaceutical composition obtained after a vacuum lyophilization step has been performed on a liquid or solution formulation.

[0139] The pharmaceutical compositions described herein can achieve stable effects, and the PD-1 / PVRIG / TIGIT binding protein among them essentially retains its physical and / or chemical stability and / or biological activity after storage. For example, the pharmaceutical compositions essentially retain their physical and chemical stability and biological activity after storage. The storage period is generally selected based on a predetermined shelf life of the pharmaceutical composition. Currently, there are several analytical techniques for measuring protein stability, which can measure stability after storage at a predetermined temperature for a predetermined period.

[0140] A stable drug antibody formulation is one that shows no significant changes when stored at refrigerated temperatures (2-8°C) for at least 3 months, at least 6 months, at least 1 year, at least 2 years, and up to 2 years. Furthermore, a stable liquid formulation includes a liquid formulation that exhibits the desired characteristics after storage for periods including 1 month, 3 months, 6 months at temperatures including 25°C, or 1 month at 40°C. A typical acceptable standard of stability, measured by SEC-HPLC, is that the antibody monomer is degraded by no more than about 10%, preferably no more than about 5%. Visual analysis reveals that the drug antibody formulation is colorless or transparent to slightly milky white. The concentration, pH, and molar osmotic pressure of the above formulations have variations not exceeding ±10%. Typically, cleavage is observed by no more than about 10%, preferably no more than about 5%, and typically, aggregation is formed by no more than about 10%, preferably no more than about 5%.

[0141] If, upon visual inspection of color and / or transparency, or measurement by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS), the antibody does not show a significant increase in aggregation, precipitation, and / or denaturation, then the antibody is considered to "maintain its physical stability" in the drug formulation. Changes in protein conformation can be evaluated by fluorescence spectroscopy (which determines the tertiary structure of the protein) and FTIR spectroscopy (which determines the secondary structure of the protein).

[0142] If an antibody does not show significant chemical changes, it is considered to "maintain its chemical stability" in a drug formulation. Chemical stability can be evaluated by detecting and quantifying the chemically altered form of the protein. Degradation processes that constantly change the chemical structure of a protein include hydrolysis or cleavage (evaluated by methods such as size exclusion chromatography and SDS-PAGE), oxidation (evaluated by methods such as mass spectrometry or peptide mapping combined with MALDI / TOF / MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, and isoaspartic acid measurement), and isomerization (evaluated by measuring the isoaspartic acid content, peptide mapping, etc.).

[0143] If the biological activity of an antibody over a predetermined period falls within a predetermined range of the biological activity shown at the time of preparation of the drug formulation, the antibody is said to "retain its biological activity" in the drug formulation. The biological activity of an antibody can be determined, for example, by an antigen-binding assay.

[0144] "Administration," "giving," and "processing" refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biofluid when used with an animal, human, experimental subject, cell, tissue, organ, or biofluid. "Administration," "giving," and "processing" may also refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Processing of cells includes contact of the reagent with the cells and contact of the reagent with a fluid, of which the fluid comes into contact with the cells. "Administration," "giving," and "processing" further refer to processing cells, for example, in vitro and ex vivo, with a reagent, diagnostic, conjugate composition, or through another type of cell. "Processing" refers to therapeutic processing, prophylactic or precautionary measures, research, and diagnostic use when used with a human, veterinary, or research subject.

[0145] "Treatment" means administering an oral or topical therapeutic agent containing any one of the antibodies or pharmaceutical compositions thereof of this disclosure to a subject who already has, is at risk of having, or is prone to having, one or more proliferative disorders or symptoms thereof, and the known therapeutic agents have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered to the subject or population being treated in a dose that effectively alleviates one or more disease symptoms, whether by inducing regression of such symptoms or by inhibiting such symptoms from progressing to any clinically measurable degree. The dose of the therapeutic agent that effectively alleviates any specific disease symptom (also called the "therapeutic dose") can vary depending on several factors, including the subject's disease state, age and weight, and the agent's ability to produce the therapeutic effect required by the subject. Reduction in disease symptoms can be assessed by any clinical detection method commonly used by physicians or other professional healthcare providers to assess the severity and progression of the symptoms. Embodiments of the present disclosure (e.g., a treatment method or product) may be ineffective in alleviating a target disease symptom in a given subject, but any statistical test known in the art, such as the Student t-test, chi-squared test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test, may determine that they should alleviate a statistically significant number of subjects.

[0146] An "effective dose" includes an amount sufficient to improve or prevent the symptoms or condition of a medical condition. An effective dose further refers to an amount sufficient to allow or promote a diagnosis. The effective dose used for a subject may vary depending on factors such as the medical condition being treated, the subject's overall health, the method and route of administration and the dosage, and the severity of side effects. An effective dose may also be the maximum dose or administration plan to avoid significant side effects or toxic effects. The subjects of this disclosure may be animals or humans.

[0147] In this disclosure, “subjects” and “patients” refer to mammals, especially primates, and especially humans.

[0148] The equipment and methods used in the detection process are as follows:

[0149] exterior: Using a visual inspection method, the sample bottles were thoroughly wiped clean, and the color, transparency, and visible foreign matter of the samples were observed under an illumination of 1000-1500 lx, respectively, using a transparency meter with both a white and a black background.

[0150] Appearance detection equipment: Precision-made YB-2A transparency detector.

[0151] SEC molecular exclusion chromatography: This analytical method isolates solutes based on the correlation between the pore size of the gel pores and the coil size of the polymer sample molecules.

[0152] Percentage of SEC monomer content = 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.) SEC measurement equipment: Agilent 1260-Bio, Column: Waters, XBrige BEH 200Å SEC (300 × 7.8 mm, 3.5 μm) NR-CE capillary gel electrophoresis: This is an electrophoresis method in which a gel is moved using a capillary as a support medium, and it is a method of isolation based on the molecular weight of the sample under a constant voltage.

[0153] The purity percentage of non-reduced CE = A major peak / A total × 100% (A major peak is the peak area of ​​the major peak in the sample, and A total is the sum of all peak areas).

[0154] CE measuring instrument: Sciex model PA800 plus icIEF imaging capillary isoelectric focusing electrophoresis: This is a technique for isolating proteins based on differences in their isoelectric points (pI).

[0155] icIEF main peak content percentage = Main peak area / Total area × 100% (Total area is the sum of the areas of the acidic peak, main peak, and basic peak).

[0156] Protein Simple, manufacturer of icIEF measuring instruments, model number Muarice.

[0157] Measurement of protein concentration: Protein concentration measuring instrument: UV-Vis spectrophotometer, model number: Nano Drop 2000, optical path 1 mm.

[0158] Preparation process of exemplary antibody drug composition (formulation) Step 1: The PD-1 / PVRIG / TIGIT binding protein was mixed with the following prescribed amounts of additives to prepare a stock solution containing the PD-1 / PVRIG / TIGIT binding protein. After filtration, the solution was sampled using a central console to detect sterility. The stock solution was filtered through a 0.22 μm filter cartridge, and the filtrate was collected.

[0159] Step 2: The filling volume was adjusted to 1.15 mL, the filtrate was filled into a 2 mL vial, and the vial was sealed. Samples were taken at the start, during, and end of filling, and the difference in filling volume was detected using the center console.

[0160] Step 3: Start the capping machine, place the aluminum lid on, and cap the machine.

[0161] Step 4: Visually inspect the product for any defects, such as inaccurate filling amounts. Print and attach labels to the vials, print labels to the cartons, fold the cartons, place the contents inside, and attach the carton labels.

[0162] II. Examples and Test Examples The present disclosure will be further illustrated by the following embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0163] Experimental methods in the examples of this disclosure that do not specify concrete conditions generally follow conventional conditions or conditions recommended by the raw material or product manufacturers. Reagents whose specific sources are not specified are commercially available conventional reagents.

[0164] Examples The method for preparing and purifying the PD-1 / PVRIG / TIGIT binding protein in this application is described in the patent document with application number WO2023040945, and all of its contents are incorporated into this disclosure.

[0165] Example 1. Screening, preparation, and functional verification of anti-PD-1 nanobodies. In this example, His-tagged human PD-1 was used as the immunoantigen, and biotin-labeled human PD-1 and cynomolgus monkey PD-1 were used as screening antigens, all of which were purchased from AcroBiosystems.

[0166] Example 1-1. Screening and preparation of anti-PD-1 nanobodies 1. Sequences and preparation of immunoantigens and screening antigens [Table 2]

[0167] The sequence of human PD-1 protein (NP_005009.2) is as follows, where the underlined portion is the extracellular region sequence, and the amino acid start and end points are Leu25-Gln 167.

[0168] Human PD-1 amino acid sequence [ka]

[0169] 2. Alpaca immunization, construction of nanobody yeast display libraries, and antibody screening. 1) Alpaca immunity Two healthy alpacas were immunized. The first immunization was administered on day 0 by subcutaneous injection of 0.25 mg of human PD-1 antigen homogeneously mixed with 1 mL of complete Freund's adjuvant (CFA). On days 21, 42, and 63, 0.125 mg of human PD-1 antigen homogeneously mixed with 1 mL of incomplete Freund's adjuvant (IFA) was administered by subcutaneous injection. Blood samples of 10 mL, 50 mL, and 50 mL were collected on days 28, 49, and 70, respectively. Serum titers were detected, and alpaca peripheral lymphocytes meeting the library construction criteria were used to construct a nanobody yeast display library.

[0170] 2) Construction of a yeast library Peripheral blood samples of two alpacas were collected in 50 mL each after the third and fourth immunizations. PBMCs were isolated from the peripheral blood, RNA was extracted from the PBMCs, reverse transcribed, and total cDNA was obtained. The cDNA was used as the reaction template for the first round of nested PCR. Two rounds of nested PCR amplification were performed, and the nested PCR product fragment from the second round was ligated to a yeast library vector (yeast library vector pYDN3). The ligated product was electroporated to enter yeast competent cells. The insertion rate and library diversity were verified using colony PCR. As is evident from the sequencing results obtained by analyzing the number of library transformants, library insertion rate, and library diversity, the library volume of one alpaca library was 9.28 × 10⁶. 7 The other alpaca library has a library capacity of 1.54 × 10⁻¹⁴. 8 That was the case.

[0171] 3) Screening of nanobodies (VHH) Antibody screening was performed on this yeast display system using flow cytometry (FACS) sorting, library screening, and identification. The sorting process employed a two-glug sorting step: the first glug was enriched with biotin-labeled human PD-1 protein (Avitag-His Tag) magnetic beads, and the second glug was sorted using a biotin-labeled monkey PD-1 protein (His, Avitag) flow cytometer (flow antibodies: Mouse anti-HA tag Dylight 488, streptavidin Dylight 650). After the two glug sorting was complete, monoclonal identification, sequencing, and sequence analysis yielded 18 unique VHH sequences cross-bound to human and monkey PD-1 antigens. The A17 sequence is shown below.

[0172] >A17 Variable region [ka]

[0173] [Table 3]

[0174] The above sequence was ligated to a human IgG4 Fc fragment (containing the hinge region and having S228P according to the Eu numbering system) to construct a VHH-Fc antibody. Plasmids were constructed, HEK293 cells were transiently transfected and expressed, purified using a protein A column, the column was washed with PBS, and eluted with 0.1 M glycine buffer at pH 2.5. Dialysis was performed with PBS pH 7.4 buffer. The VHH-Fc antibody sequences based on human IgG4 Fc (containing the hinge region) and A17 are as follows: >hIgG4 [ka] >A17_ hIgG4 [ka]

[0175] Examples 1-2. Identification of the affinity of anti-PD-1 nanobodies for the antigen PD-1. 1. ELISA Human PD-1 protein (his tag) was dissolved in PBS at a concentration of 1 μg / mL and added to a 96-well plate at 100 μL / well. The antigen was coated overnight at 4°C. Washed three times with PBST (PBS + 0.05% tween20) solution. Added PBST / 1% BSA solution and incubated at 37°C for 1 hour, then blocked. After washing three times with PBST solution, candidate PD-1 VHH-Fc antibodies of different concentrations (diluted with PBST / 1% BSA solution) were added and incubated at 37°C for 1.5 hours. Washed three times with PBST solution. Added 100 μL of secondary antibody (Thermo) solution of anti-human IgG4 Fc coupled to HRP and incubated at 37°C for 1 hour. After washing three times with PBST solution, added TMB solution at 100 μL / well, reacted at room temperature for 5 minutes, then added 50 μL of stop solution, read the 450 nM value with a plate reader, and measured the EC. 50 Calculate, The results, as shown in Table 4, indicate that A17_hIgG4 has a relatively strong binding affinity to the PD-1 antigen.

[0176] [Table 4]

[0177] 2. FACS To detect the binding ability of anti-PD-1 nanobodies to PD-1 expressed on the cell membrane, candidate anti-PD-1 VHH-Fc antibodies at different concentrations were applied to 10 cells overexpressing human PD-1. 5In addition to Jurkat cells (Cat: J1250, Promega) in the wells, after incubating at room temperature for 20 min, they were washed twice with PBS, then 100 μL of fluorescent secondary antibody against human IgG Fc (Biolegend) was added, incubated at room temperature for 20 min, washed twice with PBS, resuspended in 250 μL of PBS, and the fluorescence signal was detected by FACS, and EC 50 was calculated.

[0178] Pembrolizumab (purchased from Baiying Bio) was used as a control, and the results are as shown in Table 5. And the EC 50 value of A17_hIgG4 is 0.3568 nM, which is significantly better than other antibodies obtained by synchronous screening (results not shown).

[0179]

Table 5

[0180] Example 1-3. Sequence modification of anti-PD-1 nanobody 1. Humanization and revertant mutations of anti-PD-1 nanobody Humanization of the antibody was performed by the CDR-grafting method. By using the IMGT or NCBI website, the parental anti-PD-1 antibody was aligned with the complete human germline genes in the IMGT or NCBI / igblast database, and the human germline genes IGHV3-23 (IGHV3-23*01, IGHV3-23*04) with high homology to the PD-1 nanobody were selected as humanization templates. The CDRs were transplanted, and four crucial residues (Y37, E44, R45, L47) in the FR2 of the nanobody were retained, and the crucial core residues close to the CDR region and the residues at the Vernier positions interacting with the CDRs were subjected to revertant mutations to obtain humanized antibodies A17h1, A17h2, A17h3, A6h1.

[0181] >Variable region of A17h1

Chemical formula

[0182] 2. Modification by removal of TCE (T cell epitope), reduction of antibody deamide, and reduction of antibody isomerization. To reduce the potential immunogenicity risk of the antibody, modification was performed by removing the TCE. To improve the drug potential of the antibody, antibody deamidation and modification of the antibody isomerization site were performed. The following sequences were obtained: >A17m01 Variable Region [ka] >A17m02 Variable Region [ka] >A17m03 Variable Region [ka] >A17m04 Variable Region [ka] >A17m05 Variable Region [ka] >A17m06 Variable Region [ka] >A17m07 Variable Region [ka] >A17m08 Variable Region [ka] >A17m09 Variable Region

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0183] And in order to improve the degree of humanization, A17m09 was further selected and the antibody sequence was modified for germline conversion, and the following sequences were obtained: >Variable region A17m0901

Chem.

Chem.

Chem.

Chem.

[0184] That is, A17 of this disclosure has the following general formula sequence: CDR1:DYSMS (Sequence ID 3) CDR2:IISGSGX1X2X3HYVDSVKG, where X1 is selected from V or G, X2 is selected from I or S, and X3 is selected from T or A (Sequence ID 30) In CDR3:VSDWX4X5Y, X4 is selected from D or E, and X5 is selected from D or E (Sequence ID 31).

[0185] Specifically, CDR2 is, [ka] Even if Specifically, CDR3 is, [ka] That's fine.

[0186] Examples 1-4. Detection of the blockage of anti-PD-1 nanobodies from binding to PD-L1 and PD-1 using a PD-1 / PD-L1 reporter gene system. The PD-1 / PD-L1 NFAT gene reporter system (Cat: J1250, Promega) allows for the detection of the biological activity of anti-PD-1 antibodies at the cellular level. This system consists of two genetically modified cell lines: Jurkat effector cells (containing the NFAT reporter gene luciferase) that overexpress PD-1, and CHO-K1 antigen-presenting cells that overexpress PD-L1. When these two cell lines are co-cultured, the PD-1 / PD-L1 interaction inhibits TCR-mediated NFAT activation and further inhibits the expression of the NFAT reporter gene luciferase. When the PD-1 / PD-L1 interaction is disrupted, TCR activation induces luciferase expression via the NFAT pathway. The degree of effector cell activation and the biological activity of the antibody can then be measured by adding Bio-Glo reagent and performing fluorescence quantitative detection using a photometer.

[0187] 4 × 10⁶ CHO-K1 cells overexpressing PD-L1 5 Diluted to 1 / mL, 100 μL was added to each well in a 96-well plate, and incubated overnight. After aspirating the medium, 1.25 × 10⁴ 6 40 μL of PD-1 Jurkat cells at 1 / mL and 40 μL of anti-PD-1 nanobody solutions at different concentrations (diluted with 1640+2% FBS solution) were rapidly added. Incubated at 37°C for 6 hours, then returned to room temperature. 40 μL of Bright-glo reagent (Cat: E2620, promega) was added to each well, shaken at 350 rpm in the dark for 5 minutes, and allowed to stand in the dark at room temperature for 5 minutes. Fluorescence values ​​were then read using a plate reader. IC50 50 The result was calculated.

[0188] Pembrolizumab and hIgG4 isotype were used as controls. The biological functional activity IC of A17_hIgG4 is shown in Figure 1A and Table 6. 50 The value was 1.199 nM, which is significantly better than other antibodies screened synchronously in this disclosure (for example, A3_hIgG4 was 7.62, A13_hIgG4 was 3.208, and the sequences of A3_hIgG4 and A13_hIgG4 are not shown). Of these, A6_hIgG4 is another antibody obtained in the screening of this application.

[0189] [Table 6]

[0190] Using the above method, the function of the humanized anti-PD-1 nanobody was verified, and as shown in Figure 1B and Table 7, the IC of A17h1_hIgG4 50 The activity was similar to that of the positive antibody Pembrolizumab. The biological activity IC of the A17m0901_hIgG4 and A17m0902_hIgG4 antibodies in the modified candidate molecule was observed. 50 The values ​​are 0.5307 nM and 0.4352 nM, respectively, which is superior to Pembrolizumab.

[0191] [Table 7]

[0192] Examples 1-5. Identification of the binding affinity of modified anti-PD-1 nanobodies to the antigen PD-1. Using a Biacore 8k (GE Healthcare) instrument, surface plasmon resonance (SPR) technology was employed to detect the affinity of anti-PD-1 nanobodies for the PD-1 antigen. For the experiment, a CM5 sensor chip was selected, and HBS-EP+ buffer solution (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) was used as the mobile phase. Anti-human IgG(Fc) antibody was prepared in 10 mM sodium acetate buffer (pH 5.0) at a concentration of 30 μg / mL, and the Immobilization program was selected for automated amino-coupling fixation of the anti-human IgG(Fc) antibody channel. Each antibody awaiting measurement was prepared as a ligand using HBS-EP+ buffer solution and captured by the anti-human IgG(Fc) antibody in the chip channel. Human or cynomolgus monkey PD-1 antigen protein (Sino Biological, 10377-H08H, 90311-C08H) was prepared as the analyte in HBS-EP+ buffer solution. The analyte was then gradient diluted 2-fold, and the experimental and reference channels were run at a flow rate of 30 μL / min for 1 minute of binding and 15 minutes of dissociation. A 10 mM regeneration buffer, Glycine pH 1.5 (GE Healthcare, BR-1003-54), was run at a flow rate of 10 μL / min for 30 seconds to determine the binding rate Ka, dissociation rate Kd, and dissociation constant (i.e., affinity K). D The following was calculated. See Tables 8 and 9 for the results.

[0193] [Table 8] [Table 9]

[0194] As shown by the results of binding to the human PD-1 antigen, the humanized antibody A17h1_hIgG4 and the subsequently modified and optimized antibodies A17m09_hIgG4, A17m0901_hIgG4, and A17m0902_hIgG4 are similar to Pembrolizumab in terms of K D The antibodies A17h1_hIgG4, A17m09_hIgG4, A17m0901_hIgG4, and A17m0902_hIgG4 all exhibit slower dissociation rates than Pembrolizumab. The A17h1_hIgG4, A17m09_hIgG4, A17m0901_hIgG4, and A17m0902_hIgG4 antibodies cross-bind with monkey PD-1, and the antibodies have similar affinity to both human and monkey PD-1.

[0195] Examples 1-6. In vitro efficacy verification of modified anti-PD-1 nanobodies using DC:T mixed lymphocyte reaction (MLR). By co-culturing mature dendritic cells (DCs) with heterogeneous T cells, T cells can be activated, and cytokine secretion by T cells can be promoted. This process is inhibited by the PD-1 / PD-L1 signaling pathway, and this inhibition can be reversed by using an anti-PD-1 antibody, thereby promoting T cell activation. Therefore, the in vitro efficacy of anti-PD-1 antibodies can be verified using DC:T mixed lymphocyte reaction experiments.

[0196] Monocytes were sorted from fresh peripheral blood (PBMC) of one healthy human using a sorting reagent kit (cat:19359, stemcell). After culturing for 7 days using a DC cell induction reagent kit (cat:10985, stemcell), the monocytes were induced to differentiate into mature DC cells. Subsequently, heterogeneous T cells were sorted from fresh PBMC of another healthy human using a sorting reagent kit (cat:17951, stemcell). 5,000 mature DC cells were co-cultured with 50,000 heterogeneous T cells, and candidate antibodies at corresponding concentrations were added. After co-culture for 6 days, the cell supernatant was aspirated, and the concentration of IFN-γ was detected using a Cisbio-HTRF IFN-γ detection reagent kit (Cat:62HIFNGPEH, Perkinelmer). 50 The result was calculated.

[0197] As shown in Figure 2, A17m09 hIgG4 showed in vitro efficacy in MLR experiments that was essentially consistent with that of Pembrolizumab. Among them, the EC of A17m09_hIgG4 50 It is 0.2931 nM, and the EC of Pembrolizumab 50 It is 0.3271 nM.

[0198] Examples 1-7. Inhibition of tumor growth in a mouse colon cancer model by anti-PD-1 nanobodies. This example investigated the in vivo antitumor efficacy of an anti-PD-1 antibody in a humanized PD-1 C57BL / 6 mouse MC38 tumor model.

[0199] MC38 mouse colon cancer cell line was cultured in DMEM medium (10% FBS) and 5 × 10⁶ cells were cultured. 5 Individual MC38 cells were subcutaneously inoculated into humanized PD-1 C57BL / 6 female mice (Jucui Yaokang), and the average tumor volume of the mice was 80 mm². 3At this stage, the animals were randomly divided into groups of eight, administered either antibody or control via intraperitoneal injection, and tumor volume and body weight were measured twice a week, with data recorded. Refer to Table 10 for the experimental grouping and administration plan. Considering that the molecular weight of A17m09_hIgG4 is approximately 75 kDa, which is only half the molecular weight of normal IgG, the dose was adjusted to half that of Pembrolizumab to achieve an equimolar dose.

[0200] As shown in Figures 3A and 3B, the in vivo antitumor efficacy of A17m09_hIgG4 at equimolar doses is consistent with that of pembrolizumab. See Table 11 for tumor volume and tumor inhibition rates.

[0201] [Table 10] [Table 11]

[0202] Example 2. Anti-PVRIG / TIGIT bispecific antibody In this example, the his-tagged human PVRIG (h-PVRIG-his) recombinant protein, the mouse IgG2a Fc-tagged human PVRIG (h-PVRIG-mIgG2a Fc) recombinant protein, and the human IgG1 Fc-tagged mouse PVRIG (m-PVRIG-hIgG1 Fc) were purchased from Acrobiosystems. The his-tagged PVRL2 was purchased from AcroBiosystem (PV2-H52E2).

[0203] Example 2-1. Screening and preparation of anti-PVRIG nanobodies 1. Immunogenetic antigens, sequences and preparation of screened antigens [Table 12]

[0204] The sequence of the his-tagged cynomolgus monkey PVRIG (cyno-PVRIG-his) recombinant protein is as follows: [ka]

[0205] 2. Alpaca immunization, construction of nanobody phage display libraries, and antibody screening. Alpacas were immunized with his-tagged human PVRIG recombinant protein (h-PVRIG-his) referring to the method of Example 1-1. PBMCs were isolated from the peripheral blood of 56-day-old camels, 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.

[0206] >151 Variable Region [ka]

[0207] [Table 13]

[0208] The above antibody variable region was ligated to the human IgG4 heavy chain Fc region, and the heavy chain Fc region included a hinge region and contained S228P, F234A, L235A, and K447A mutations (Eu naming system), and a full-length antibody was constructed.

[0209] >hIgG4 Fc(S228P / F234A / L235A / K447A) [ka]

[0210] >151 total length [ka]

[0211] The anti-PVRIG antibody CPA.7.021, described in WO2016134333, was screened from an antibody phage library. Its subtype is IgG1, and it binds relatively well to human PVRIG but not to cynomolgus monkey PVRIG. The heavy chain and light chain variable regions of CPA.7.021 were ligated to the human IgG4 heavy chain constant region (containing S228P, F234A, L235A, and K447A mutations) and the human Kappa light chain constant region, respectively, to construct the positive antibody Tab5.

[0212] >Tab5 double chain length [ka] >Tab5 Light Chain Length [ka]

[0213] The nucleic acid sequence expressing the above amino acid sequence was cloned into a pcDNA3.1 expression vector, and antibody expression and purification were performed according to conventional methods, followed by detection to obtain the target antibody.

[0214] Example 2-2. Identification of the affinity of anti-PVRIG nanobodies for the antigen PVRIG. 1. ELISA The PVRIG recombinant protein was directly coated with a his tag, and after adding the antibody, the antibody's binding activity to the antigen was detected by adding a secondary antibody (antibody of the anti-primary antibody Fc coupled with HRP) and the HRP substrate TMB.

[0215] 96-well plates were coated with 100 μL of human, cynomolgus monkey, or mouse PVRIG protein at a concentration of 1 μg / mL in each well and incubated overnight at 4°C. Washed three times with washing solution. 300 μL / well of blocking solution (PBS + 0.05% Tween 20 + 1% BSA) was added and incubated at room temperature for 1 hour. Washed three times with washing solution. 100 μL of diluted, awaiting-to-measure anti-PVRIG antibody was added to each well. Incubated at 37°C for 1 hour. Washed three times with washing solution. 100 μL of HRP-labeled anti-human IgG secondary antibody (Sigma, A8667) was added to each well. Incubated at 37°C for 1 hour. Washed three times with washing solution. 100 μL of TMB was added to each well and reacted in the dark for 15 minutes. 0.16 M sulfuric acid was added at 50 mL / well. The OD450 was read using a Thermo MultiSkan Fc plate reader, and the EC of the anti-PVRIG antibody that binds to the PVRIG recombinant protein was determined. 50 The values ​​were calculated. All antibodies in Table 14 have a relatively strong binding ability to human or cynomolgus monkey PVRIG recombinant protein, but do not bind to mouse PVRIG recombinant protein.

[0216] [Table 14]

[0217] 2. FACS detection A stable HEK293 transformed cell line expressing the human or cynomolgus monkey PVRIG gene was prepared. In a 96-well plate, 2 × 10⁶ cells were placed in each well. 5Cells were inoculated. They were centrifuged at 300g for 5 minutes, the supernatant was removed, 100 μL of antibody awaiting measurement 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 anti-human IgG secondary antibody (Invitrogen, A-11013) labeled with Alexa Fluor 488 diluted at 1:500 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 detected using a flow cytometer (BD FACS Calibur or BD FACS Canto II). All antibodies exhibit relatively strong binding ability to human or cynomolgus monkey PVRIG expressed on the cell surface, and are clearly stronger than the positive antibody Tab5. However, Tab5 does not bind to cynomolgus monkey PVRIG at all.

[0218] [Table 15]

[0219] 3. Fortebio detection A Protein A biosensor (Fortebio, #18-5010) was immersed in 200 μL of KB buffer (PBS, pH 7.4, 0.02% tween-20, 0.1% BSA) for 60 seconds for wetting treatment. Then, anti-PVRIG antibody was diluted to 10 μg / mL in KB buffer, and the sensor was placed in 200 μL of this solution, stopping until the reading reached 1.2 nm. Excess antibody was eluted by immersing the sensor in KB buffer for 100 seconds. His-tagged human PVRIG was diluted in KB buffer with a 2x gradient between 64 nM and 4 nM. The sensor was placed in this solution for 300 seconds to bind, and then placed in KB buffer for 600 seconds to dissociate. Fitting using a dynamic 1:1 binding method revealed the affinity of the anti-PVRIG antibody to human PVRIG, as shown in Table 16.

[0220] As the results show, all detected antibodies exhibit high affinity for human PVRIG.

[0221] [Table 16]

[0222] Examples 2-3. Verification of the function and activity of anti-PVRIG nanobodies. 1. Experiment to block the binding of PVRIG to PVRL2 using anti-PVRIG nanobodies. A 96-well plate was coated with human PVRIG recombinant protein (h-PVRIG-mIgG2a Fc) at a concentration of 1 μg / mL, with 100 μL per well, and incubated overnight at 4°C. Washed three times with washing solution. Blocking solution was added at 300 μL / well and incubated at room temperature for 1 hour. Washed three times with washing solution. 50 μL of diluted, awaiting-measurement anti-PVRIG antibody and 50 μL of his-tagged ligand PVRL2 were added to each well and incubated at 37°C for 1 hour. Washed three times with washing solution. 100 μL of HRP-labeled anti-his-tagged secondary antibody (Genscrpit), diluted 1:2000-fold, was added to each well. Incubated at 37°C for 1 hour. Washed three times with washing solution. 100 μL of TMB was added to each well and 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 Multiskan Fc plate reader, and the IC of the blockade of the anti-PVRIG antibody to the binding of PVRIG to PVRL2 was determined. 50 The value was calculated.

[0223] As shown in the results in Table 17, all of the detected antibodies were able to strongly inhibit the binding of human PVRIG to human PVRL2.

[0224] [Table 17]

[0225] 2. Cell activity experiment of the anti-PVRIG nanobody reporter gene First, we constructed the plvx-OS8 (G418 resistant) plasmid, transfected 293F cells with it, screened them with G418, detected the expression of OS8 in clonal cells by flow cytometry, and simultaneously detected the activation of Jurkat cells by OS8. We selected clones with moderate activation to obtain the 293F-OS8 cell line, constructed the plvx-PVRL2 plasmid, used it to infect 293F-OS8 cells, and screened the clones with the highest PVRL2 expression level by flow cytometry to obtain the 293F-OS8-PVRL2 cell line.

[0226] Next, we constructed plvx-NFAT-Luc (Hygromycin-resistant), packaged it into a lentivirus, infected Jurkat E6.1 cells, added hygromycin to screen for resistant clones, stimulated the clones with OKT3, and screened for clones with moderate luciferase signaling to obtain the Jurkat-NFAT-Luc cell line. Then we constructed the plvx-PVRIG (Puromycin-resistant) vector, packaged it into a lentivirus, infected Jurkat-NFAT-Luc cells, and screened for clones with the highest PVRIG expression level using a flow cytometer to obtain the Jurkat-NFAT-Luc-PVRIG cell line.

[0227] Four Jurkat-NFAT-Luc-PVRIG cells (1E4) and antibodies awaiting measurement were incubated at 37°C for 20 minutes. Five 293F-OS8-PVRL2 cells (1E5) were added and incubated at 37°C for 5 hours. The supernatant was removed by centrifugation, and the cells were lysed with Luciferase buffer (Promega, E6130). Fluorescence values ​​were detected. EC 50 The values ​​were calculated to evaluate the in vitro cell activity of the anti-PVRIG antibody. The experimental results are shown in Table 18.

[0228] As the results show, the detected antibodies had a relatively strong ability to activate Luciferase in Jurkat cells, with activity at least 10 times greater than that of the positive antibody. These antibodies were proven to bind to PVRIG and to block the binding of PVRL2 to PVRIG.

[0229] [Table 18]

[0230] 3. Experiments in killing NK cells with anti-PVRIG nanobodies PVRIG is expressed in NK cells, while PVRL2 is expressed in many tumor cells (including K562 cells). Anti-PVRIG antibodies can block the binding of PVRL2 to PVRIG, thereby reversing the inhibitory effect of tumor cells on NK cell activity.

[0231] In a 96-well plate, 50 μL (total 1 × 10⁶) is placed in each well. 5 Human malignant non-Hodgkin lymphoma NK92 cells were added. 50 μL of 20 nM or 100 nM antibody awaiting measurement was added and incubated at 37°C for 30 minutes. Washed twice with washing solution, 2 × 10⁶ cells were collected. 5 Resuspended to a density of 1 x 10 particles / mL. 50 μL (total 1 x 10 4 Human chronic myeloid leukemia K562 cells were added to adjust the ratio of NK92 cells to K562 cells to 10:1. The cells were incubated at 37°C for 4 hours. The cytotoxic activity was measured using the CytoTox-Glo cytotoxic system (Promega, G9292). First, 50 μL of AAF-Glo reagent was added and 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 and incubated at room temperature for 15 minutes to lyse the cells, and the fluorescence of all cells was measured. Three control groups were prepared, each containing only the culture medium (control group 1), only NK92 cells (control group 2), and only K562 cells (control group 3), and the same procedure was performed on each group.

[0232] The lethal activity was calculated based on the following formula: Killing activity (%) = {[(R-BG)-(T-BG)-(E-BG)] / [(TL-BGL)-(T-BG)]}×100, Of these, 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, T is the fluorescence value of control group 3 after adding AAF-Glo, TL is the fluorescence value of control group 3 after adding the dissolution, and BGL is the fluorescence value of control group 1 after adding the dissolution.

[0233] As shown in Table 19, the experimental results clearly demonstrated that all detected anti-PVRIG antibodies were capable of activating NK92 cells and killing K562 cells.

[0234] [Table 19]

[0235] 4. Experiments on mixed lymphocyte reaction (MLR) with anti-PVRIG nanobodies PVRIG is expressed in T cells, while PVRL2 is expressed in DC cells. Anti-PVRIG antibodies can block the binding of PVRL2 to PVRIG, thereby releasing the inhibition of DC cells against T cells and activating T cells.

[0236] PBMCs were isolated from peripheral blood of the first individual, and the cells were cultured in RPMI 1640 medium containing 10% FBS. Final concentrations of 50 ng / mL GM-CSF (Peprotech, 300-03-100UG) and 50 ng / mL IL-4 (Peprotech, 200-04-100UG) were added, and fresh medium containing cytokines was added every 2-3 days. After 6 days of incubation, 1 μg / mL LPS (Sigma, L2880-25MG) was added and incubated for 24 hours, and the resulting DC cells were collected after differentiation and maturation. PBMCs were isolated from peripheral blood of the second individual, and EasySep human CD3 +Using the T cell isolation reagent kit (Stemcell, 17952), select CD3 cells from among them. + T cells were isolated in each well. 5 individual CD3 + T cells and 2 × 10 4 To add individual DC cells, CD3 + T cell and DC cell densities were adjusted. Antibodies awaiting measurement were added, incubated at 37°C for 120 hours, and the supernatant was collected. The IFNγ content in the supernatant was detected using an ELISA reagent kit (R&D, DY202).

[0237] As shown in Table 20 and Figure 4, all detected anti-PVRIG antibodies clearly activated T cells and induced IFNγ secretion compared to the control antibody IgG4. Furthermore, at low doses (e.g., 4 nM, 20 nM), antibody 151 of this disclosure was more effective than the positive control Tab5. Of these, 30 antibodies were other antibodies obtained in the screening of this application.

[0238] [Table 20]

[0239] Examples 2-4. Realignment of anti-PVRIG nanobodies By performing three-dimensional structural homology modeling on selected anti-PVRIG antibody molecules, we compared the anti-PVRIG antibody sequence with the antibody GermLine database to obtain a highly homologous human germline template IGHV3-7*01. The CDR was transplanted into the corresponding human-derived template. Further three-dimensional structural simulations and analyses were performed on the transplanted nanobodies, and a series of humanized nanobodies were produced by introducing reverse mutations into embedded residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of the variable region, as well as optimizing chemically unstable amino acid residues in the CDR region. The sequences of the human germline template and the humanized antibody heavy chain variable region for each nanobodies are as follows.

[0240] >151H2 variable region [ka] >151H4 variable region [ka] >151H7 Variable Region [ka] >151H8 variable region [ka] >151H9 variable region [ka]

[0241] The humanized antibody heavy chain variable region described above was ligated to the human IgG4 heavy chain Fc region to construct a full-length anti-PVRIG antibody. The heavy chain Fc region contained a hinge region and possessed either the S228P / F234A / L235A / K447A or S228P / K447A mutation. Antibody expression and purification were performed according to conventional methods, and the target antibody was obtained through detection.

[0242] Examples 2-5. Verification of the activity and function of humanized anti-PVRIG antibodies. 1. Binding experiment of humanized anti-PVRIG antibody to PVRIG-expressing cells The binding of anti-PVRIG antibodies to human or cynomolgus monkey PVRIG was detected by FACS according to the method of Example 2-2. The experimental results are shown in Table 21.

[0243] [Table 21]

[0244] 2. Measurement of the affinity of humanized anti-PVRIG antibodies to PVRIG The affinity of humanized anti-PVRIG antibodies to human PVRIG protein was detected according to the Fortebio detection method of Example 2-2. As shown in Table 22, all antibodies exhibited high affinity for human PVRIG protein.

[0245] [Table 22]

[0246] 3. Activity experiments of humanized anti-PVRIG antibody in reporter genes in cells. The activity of humanized anti-PVRIG antibodies in reporter gene cells was detected according to the methods of Examples 2-3. The experimental results are shown in Table 23. All of the antibodies listed in the table have the ability to activate Jurkat cells.

[0247] [Table 23]

[0248] 4. Experiments to activate the NK cell-killing ability of humanized anti-PVRIG antibodies. The activating ability of humanized anti-PVRIG antibodies to NK cells was detected according to the methods of Examples 2-3. The experimental results are shown in Table 24. As the results show, all detected antibodies had clear NK cell activating ability and promoted the killing of NK cells against their target cell K562.

[0249] [Table 24-1] [Table 24-2]

[0250] Examples 2-6. Preparation of anti-PVRIG / TIGIT bispecific antibodies To investigate the effect of anti-PVRIG / TIGIT bispecific antibodies with different structures on antibody function, anti-PVRIG nanobody 151 was ligated to the N-terminus or C-terminus of the heavy or light chain of anti-TIGIT antibody 1708 using the GGGGSGGGGS (SEQ ID NO: 78) linker. Four anti-PVRIG / TIGIT bispecific antibodies were formed, named 1708-151-1, 1708-151-2, 1708-151-3, and 1708-151-4, respectively, with each ligated to the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain, and the C-terminus of the light chain of 1708, corresponding to 151. Anti-TIGIT antibody 1708 employs the human IgG4 subtype and has the S228P (Eu naming system) mutation. The sequences of anti-TIGIT antibody 1708 and the bispecific antibodies formed with it and 151 are shown in Table 25 below. The sequence information for the anti-TIGIT antibody is shown in Tables 26 and 27. The TIGIT antibody is disclosed in WO2019062832A, which is incorporated herein by reference in its entirety.

[0251] [Table 25-1] [Table 25-2] [Table 25-3] [Table 26] [Table 27-1] [Table 27-2]

[0252] Different humanized anti-PVRIG antibody variable regions (151H7, 151H8) were ligated to the N-terminus of the heavy chain of anti-TIGIT antibody 1708, thereby constructing a bispecific antibody structure similar to 1708-151-1. The double-underlined portion in the following sequence is the linker sequence.

[0253] >1708-151H7's first polypeptide chain [ka] >1708-151H8's first polypeptide chain [ka]

[0254] The second polypeptide chains of 1708-30H2, 1708-151H7, and 1708-151H8 are all the same as the light chain of 1708 (SEQ ID NO: 52).

[0255] Following conventional methods, transient transfection, expression, and purification of the antibody were performed, and the antibody was identified to obtain the full-length anti-PVRIG / TIGIT bispecific antibody of this disclosure, which showed good expression levels and purity in all cases.

[0256] Examples 2-7. Verification of the activity and function of anti-PVRIG / TIGIT bispecific antibodies. 1. Binding of bispecific antibodies to human PVRIG and blockade of ligand PVRL2. The experiment was conducted according to the methods of Examples 2-2 and 2-3. As is clear from the results, there was no statistically significant difference in the binding of the bispecific antibodies 1708-151-1, 1708-151-2, 1708-151-3, and 1708-151-4 in different configurations to human PVRIG recombinant protein and human PVRIG overexpressing cells, nor in their blocking of the binding of PVRL2 to PVRIG.

[0257] 2. Binding of bispecific antibodies to human TIGIT and blockage of ligand PVR. 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. As is clear from the results, there was no statistically significant difference in the binding of the bispecific antibodies 1708-151-1, 1708-151-2, 1708-151-3, 1708-151-4 and the anti-TIGIT antibody to human TIGIT recombinant protein and human TIGIT overexpressing cells, nor in the blocking of the binding of TIGIT and its ligand PVR.

[0258] This revealed that the anti-PVRIG antibody, regardless of whether it is ligated to the N-terminus or C-terminus of the heavy or light chain of the anti-TIGIT antibody, retains binding to PVRIG and TIGIT and blocks ligands, while also exhibiting good expression levels and purity.

[0259] 3. Binding of humanized anti-PVRIG / TIGIT bispecific antibodies to PVRIG and TIGIT, and blockage of corresponding ligands. The binding of humanized anti-PVRIG / TIGIT bispecific antibodies to human and cynomolgus monkey PVRIG, and the blockade of human PVRIG to its ligand were detected according to the methods of Examples 2-2 and 2-3. The results are shown in Table 28. As is clear from the results, each bispecific antibody can bind to human PVRIG and block the binding of PVRIG to PVRL2.

[0260] [Table 28]

[0261] Similar to Examples 2-2 and 2-3, the binding of humanized anti-PVRIG / TIGIT bispecific antibodies to human and cynomolgus monkey TIGIT, and the blocking of binding between human TIGIT and its ligand were detected. In these cases, the PVRIG protein was replaced with TIGIT, and PVRL2 was replaced with PVR. The results are shown in Table 29. As is clear from the results, each bispecific antibody can bind to human TIGIT and block the binding of TIGIT to PVR.

[0262] [Table 29]

[0263] The affinity of bispecific antibodies to human PVRIG and human TIGIT was detected using Biacore. Humanized bispecific antibodies were captured on a Protein A biosensor chip (GE lifesciences, 29127557) using a Biacore instrument (Biacore X100, GE). Then, human PVRIG antigen (AcroBiosystem, PVG-H52H4) or human TIGIT antigen (AcroBiosystem, TIT-H52H3) was passed over the chip surface under a series of concentration gradients to obtain binding and dissociation curves. The results are shown in Table 30.

[0264] [Table 30]

[0265] 4. Mixed lymphocyte reaction (MLR) experiment with anti-PVRIG / TIGIT bispecific antibodies The activating ability of humanized anti-PVRIG / TIGIT bispecific antibodies to T cells was detected according to the method of Part 4 of Example 2-3. The experimental results are shown in Figure 5 and Table 31. As shown in the results, the humanized anti-PVRIG / TIGIT bispecific antibody 1708-151H8 had a clear ability to activate T cells and promoted IFNγ secretion by T cells. Importantly, the activity of the bispecific antibody was stronger when anti-PVRIG antibody 151H8 was used alone than when anti-TIGIT antibody 1708 was used alone.

[0266] [Table 31]

[0267] Examples 2-8. Evaluation of antitumor activity in a mouse subcutaneous tumor model using human melanoma A375 mixed with human PBMCs, containing an anti-PVRIG / TIGIT bispecific antibody. To further investigate the effects of bispecific antibody subtypes on animal pharmacokinetics, animal pharmacokinetic studies were conducted. Of these, the heavy chain variable region of 1708-IgG1 was the same as that of 1708, except that the heavy chain constant region subtype was substituted with IgG1. The full-length light chain of 1708-IgG1 and the second polypeptide chain of 1708-151-IgG1 were both the same as that of 1708.

[0268] Female NCG mice, 4-8 weeks old and weighing approximately 18-22g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. All NCG mice were cultured in an IVC constant temperature and pressure system in an SPF-grade animal rearing room.

[0269] A375 cells were cultured in DMEM culture medium containing 10% fetal bovine serum (FBS). A375 cells in the exponential growth phase were collected, and HBSS was resuspended to an appropriate concentration for subcutaneous tumor inoculation of NCG mice. A375 cells used for co-culture needed to be treated with Mitomycin C for 2 hours, followed by three washes with PBS. Normal human peripheral blood was collected, and human PBMCs were isolated and counted by density gradient centrifugation. Subsequently, PBMCs were 3 × 10⁶ in RPMI1640 medium (containing IL2 and 10% FBS). 6 The PBMCs were resuspended at a concentration of cells / mL and co-cultured with A375 cells treated with Mitomycin C. After co-culture for 6 days, the PBMCs were harvested, and the freshly digested A375 cells were also harvested. Each mouse received 5 × 10⁶ cells. 5 Individual PBMC, 4 x 10 6Individual A375 cells were inoculated at an inoculation volume of 0.2 mL / mouse (containing 50% Matrigel) and administered subcutaneously to the right side of female NCG mice. Mice were randomly divided into groups according to their body weight and administered accordingly. Detailed administration methods, dosages, and routes are shown in Table 32, with the day of administration designated as day 0. Due to the different molecular weights of the anti-PVRIG antibody and anti-TIGIT antibody, the dosage was determined to ensure that both antibodies had similar initial molar concentrations.

[0270] [Table 32]

[0271] After administration began, 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.

[0272] [Table 33]

[0273] At the end of the experiment (26 days after administration), the anti-PVRIG antibody 151-IgG4 monotherapy group showed no significant difference compared to the control group. Tumor volume was reduced in the anti-TIGIT antibody 1708-IgG1 monotherapy group and the combination therapy group of anti-PVRIG antibody 151-IgG4 and anti-TIGIT antibody 1708-IgG1. On the other hand, the 1708-151-IgG4 bispecific antibody group was even able to completely inhibit tumor growth, showing a significant difference compared to the other groups (see Figure 6A).

[0274] Mice were randomly divided into groups based on their body weight and administered the drug. Detailed administration methods, dosages, and routes are shown in Table 34, with the day of administration being designated as day 0.

[0275] [Table 34]

[0276] After administration began, 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 to 7B, respectively.

[0277] [Table 35]

[0278] 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, and there was a significant difference between the two groups (see Figures 7A and 7B). Among these, 1708-30H2 is another antibody obtained in the screening described in this invention.

[0279] Example 3. Anti-PD-1 / PVRIG / TIGIT trispecific antibody Example 3-1. Design and preparation of an anti-PD-1 / PVRIG / TIGIT trispecific antibody The four types of triplicate antibodies are divided into four types, A, B, C, and D, as shown in Figure 8.

[0280] Type A: The anti-PD-1 nanobody is linked to the anti-PVRIG nanobody via a linker (e.g., GGGGSGGGGS), and then linked to the N-terminus of the heavy chain of the anti-TIGIT antibody via the linker (Figure 8A).

[0281] Type B: Anti-PVRIG antibodies are linked to the N-terminus of the heavy chain of the TIGIT IgG molecule via a linker, while anti-PD-1 antibodies are linked to the N-terminus of the light chain of the anti-TIGIT antibody via a linker (Figure 8B).

[0282] Type C: 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 heavy chain of the anti-TIGIT antibody via a linker (GGGGSGGGGS) (Figure 8C).

[0283] 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).

[0284] The triplicate antibodies in this disclosure are named as shown in Table 36. For example, A17m0902-1708-151H7-A indicates that the clones of the anti-PD-1, TIGIT, and PVRIG antibodies used are A17m0902, 1708, and 151H7, respectively, and that the triplicate antibody type is type A.

[0285] [Table 36]

[0286] 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.

[0287] The sequence is as follows: (Underlined parts are linkers, italicized parts are Fc or light chain Cκ): >First polypeptide chain of A17m0902-1708-151H7-A [ka]

[0288] The second polypeptide chain of A17m0902-1708-151H7-A is shown in SEQ ID NO: 52.

[0289] The first polypeptide chain of A17m0902-1708-151H7-B is shown in SEQ ID NO: 68.

[0290] >Second polypeptide chain of A17m0902-1708-151H7-B [ka]

[0291] >First polypeptide chain of A17m0902-1708-151H7-C [ka]

[0292] (Sequence ID 72) The second polypeptide chain of A17m0902-1708-151H7-C is shown in Sequence ID No. 52.

[0293] The first polypeptide chain of A17m0902-1708-151H7-D is shown in SEQ ID NO: 68.

[0294] >Second polypeptide chain of A17m0902-1708-151H7-D [ka]

[0295] Using conventional methods, cells can be transfected, expressed, purified, and detected to obtain the target antibody.

[0296] Example 3-2. Binding affinity of anti-PD-1 / PVRIG / TIGIT trispecific antibody to antigen. Detection was performed using ELISA. Human TIGIT or PVRIG protein (his tag, Acrobiosystems, PVG-H52H4) was dissolved at 1 μg / mL in PBS, and the solution was added to a 96-well plate at 100 μL / well. The antigen was coated overnight at 4°C. Washed three times with PBST solution. PBST / 1% BSA solution was added, and the plate was incubated at 37°C for 1 hour and blocked. After washing three times with PBST solution, trispecific antibodies of different concentrations (diluted with PBST / 1% BSA solution) were added and incubated at 37°C for 1.5 hours. Washed three times with PBST solution. 100 μL of secondary antibody (Thermo) solution of anti-human IgG4 Fc coupled with HRP was added and the plate was incubated at 37°C for 1 hour. After washing three times with PBST solution, add 100 μL / well of TMB solution and react at room temperature for 5 minutes, then add 50 μL of stop solution, read the OD450 value with a plate reader, and EC 50 The result was calculated.

[0297] 1. Integration with TIGIT All four configurations of the A17h1-1708-151H7 triplicate antibodies bound relatively strongly to TIGIT, with relatively small differences in binding ability and similarity to the binding ability of the 1708-151H7 bispecific antibody to TIGIT. See Figure 9 and Table 37 for results. Therefore, the configuration of all triplicate antibodies does not affect their binding to TIGIT.

[0298] [Table 37]

[0299] 2. Integration into PVRIG All four configurations of the A17h1-1708-151H7 triplicate antibodies bind relatively strongly to PVRIG, with relatively small differences in binding ability and similarity to the binding ability of the 1708-151H7 bispecific antibody to PVRIG. See Table 38 and Figure 10. Therefore, the configuration of all triplicate antibodies does not affect their binding to PVRIG.

[0300] [Table 38]

[0301] Example 3-3. PVRIG sequence optimization in anti-PD-1 / PVRIG / TIGIT trispecific antibody 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 (abbreviated as H). Since histidine, located at the N-terminus of the trispecific antibody molecular sequence, can undergo pyridoxal phosphate modification and subsequent pyridoxal modification during the actual antibody production process, considering this issue, it was necessary to mutate the first histidine in the original PVRIG sequence. Glutamic acid (abbreviated as E) and glutamine (abbreviated as Q) are the two most frequently occurring first amino acids in the nanobody sequence; therefore, H was mutated to E or Q. The mutated sequence of the PVRIG antibody 151H7 is as follows: >151H7 H1E [ka]

[0302] >151H7 H1Q [ka]

[0303] The triplicate antibody molecule that changes the first amino acid of the PVRIG(151H7) sequence from H to E was named A17m0902-1708-151H7-01-C, and the triplicate antibody molecule that changes the first amino acid of the PVRIG(151H7) sequence from H to Q was named A17m0902-1708-151H7-02-C.

[0304] The molecular type of A17m0902-1708-151H7-01-C and A17m0902-1708-151H7-02-C is type C, meaning that 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 (C in Figure 8).

[0305] The polypeptide chain sequences of A17m0902-1708-151H7-01-C and A17m0902-1708-151H7-02-C are as follows: >First polypeptide chain of A17m0902-1708-151H7-01-C [ka]

[0306] The second polypeptide chain of A17m0902-1708-151H7-01-C is shown in Sequence ID No. 52.

[0307] >First polypeptide chain of A17m0902-1708-151H7-02-C [ka]

[0308] The second polypeptide chain of A17m0902-1708-151H7-02-C is shown in SEQ ID NO: 52.

[0309] Examples 3-4. Antigen binding affinity after PVRIG sequence optimization in anti-PD-1 / PVRIG / TIGIT trispecific antibodies. We used HEK293F stable transformed cell lines expressing the human PVRIG gene, CHO-K1 stable transformed cell lines expressing the human TIGIT gene, and Jurkat cells expressing human PD-1, respectively. In a 96-well plate, 2 × 10⁶ cells were placed in each well. 5Cells were inoculated. They were centrifuged at 300g for 5 minutes, the supernatant was removed, 100 μL of antibody awaiting measurement 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 anti-human IgG secondary antibody (Invitrogen, A-11013) labeled with Alexa Fluor 488 diluted at 1:500 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 detected using a flow cytometer (BD FACS Calibur or BD FACS Canto II).

[0310] 1. Integration into PVRIG Two triplicate antibody molecules, A17m0902-1708-151H7-01-C and A17m0902-1708-151H7-02-C, in which the first amino acid of the PVRIG antibody was mutated, showed relatively similar binding activity to human PVRIG expressed on the cell surface and similar EC50 values ​​compared to the pre-mutation triplicate antibody molecule A17m0902-1708-151H7-C and the pre-mutation bispecificate antibody molecule 1708-151H7. This revealed that the first amino acid of the PVRIG antibody does not affect the binding of the triplicate antibody to PVRIG after mutation. See Table 39 and Figure 11A for the results.

[0311] [Table 39]

[0312] 2. Integration with TIGIT The two triplicate antibody molecules, A17m0902-1708-151H7-01-C and A17m0902-1708-151H7-02-C, showed relatively similar binding activity to human TIGIT expressed on the cell surface compared to the pre-mutation triplicate antibody molecule A17m0902-1708-151H7-C. Their similar EC50 values ​​indicated that the first amino acid of the PVRIG antibody did not affect the binding of the triplicate antibody to TIGIT after mutation. See Table 40 and Figure 11B for results.

[0313] [Table 40]

[0314] 3. Binding to PD-1 The two triplicate antibody molecules, A17m0902-1708-151H7-01-C and A17m0902-1708-151H7-02-C, exhibited similar binding activity to human PD-1 expressed on the cell surface and similar EC50 values ​​compared to the pre-mutation A17m0902-1708-151H7-C antibody. This revealed that the first amino acid of the PVRIG antibody does not affect the binding of the triplicate antibody to PD-1 after mutation. See Table 41 and Figure 11C for results.

[0315] [Table 41]

[0316] In summary, the results of the above binding activity experiments show that the two triplicate antibody molecules A17m0902-1708-151H7-01-C and A17m0902-1708-151H7-02-C, which have mutations in the first amino acid of the PVRIG antibody, have affinity for PVRIG, TIGIT, and PD-1 antigens expressed on the cell surface that corresponds to the parent A17m0902-1708-151H7-C antibody, and these mutations do not affect the binding of the triplicate antibody to PVRIG, TIGIT, and PD-1.

[0317] Examples 3-5. Detection of PD-1 blockade by anti-PD-1 / PVRIG / TIGIT trispecific antibodies using a PD-1 / PD-L1 reporter gene system. Detection was performed using the methods described in Examples 1-4. As shown in Figure 12, all four triplicate antibodies with A17h1 as the PD-1 terminus were able to block PD-1, but the triplicate antibodies of types B and C exhibited relatively better reporter gene activity.

[0318] Examples 3-6. Detection of activation of anti-PD-1 / PVRIG / TIGIT trispecific antibodies in T cells by MLR. DC:T mixed lymphocyte reaction experiments were performed on cells from human subjects using the methods of Examples 1-6. The results are shown in Figure 13 and Table 42. As shown in the results, all four configurations of the trispecific antibodies effectively promote T cell activation, release PD-1 / PD-L1 inhibition, and promote cytokine secretion by T cells, and all have relatively strong in vitro pharmacokinetic activity.

[0319] [Table 42]

[0320] Examples 3-7. Detection of activation of anti-PD-1 / PVRIG / TIGIT trispecific antibodies in NK cells by NK92 sterilization. The in vitro efficacy of anti-PD-1 / PVRIG / TIGIT trispecific antibodies can be validated using NK cell killing experiments. Cell killing experiments were performed using NK92 cells as effector cells and K562 cells as target cells. First, NK92 cells were co-incubated with different concentrations of candidate antibodies at 37°C for 30 minutes. Then, the antibody-pre-incubated NK92 cells were mixed with K562 cells labeled with CTV (Cat: C34557, Thermo) according to an effector / target ratio of 10:1. The culture conditions were set to 1640 + 10% FBS + 10 ng / mL IL-2 and the cells were co-incubated at 37°C for 4 hours. After removing the supernatant by centrifugation, the cells were resuspended using 10 μg / mL propidium iodide solution and dead cells were stained and labeled. After labeling for 10 minutes at room temperature, the percentage of dead cells (PI positive) in the CTV-labeled K562 cells was detected by FACS and the killing activity was calculated.

[0321] As shown in Figure 14, the triplicate antibody retains the biological function of the anti-PVRIG / TIGIT bispecific antibody that promotes NK cell killing, and the IC of each configuration 50 The values ​​are similar; see Table 43.

[0322] [Table 43]

[0323] Example 3-8. Evaluation of the antitumor activity of an anti-PD-1 / PVRIG / TIGIT trispecific antibody in a mouse subcutaneous tumor model of human melanoma A375 mixed with human PBMCs that responds to a PD-1 monoclonal antibody. This example investigated the in vivo efficacy of a PD-1 / PVRIG / TIGIT trispecific antibody in an in vivo tumor model of human melanoma A375 mixed with human PBMCs that respond to a PD-1 monoclonal antibody.

[0324] These were female NCG mice, 4-8 weeks old and weighing approximately 18-22g. (The mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) The mice were reared in an SPF-grade animal housing room under constant temperature and pressure conditions in an IVC (Incubation Vacuum) system.

[0325] Donor PBMCs responsive to PD-1 monoclonal antibodies in an in vivo drug efficacy model were screened from cryopreserved PBMCs and counted. PBMCs were resuspended to a constant concentration in RPMI1640 culture medium containing IL-2 and 10% FBS. PBMCs were added to A375 cells treated with Mitomycin C for 2 hours. After co-culturing with A375 for 5 days, the 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 given 5 × 10⁶ cells. 5 Individual PBMC, 4 x 10 6 Individual A375 cells were inoculated into the right subcutaneous tissue of NCG mice at a dose of 0.2 mL / mice (containing 50% Matrigel). Day 0 was designated as day 0, and on day 1, mice were randomly divided into groups based on their body weight and administered the solution.

[0326] Considering that the molecular weights of A17m0902_hIgG4 are 76 kDa, 1708-151H7 is 173.5 kDa, and A17m0902-1708-151H7-C is 200 kDa, an equimolar dosing plan was designed. The detailed dosing plan, dosage, and route of administration are shown in Table 44.

[0327] [Table 44]

[0328] After the start of administration, the body weight and tumor volume of the mice were measured twice a week.

[0329] Twenty-one days after administration, compared to the control group, the tumor growth inhibition rates (TGI) were 7.05%, 79.91%, 91.63%, and 88.25% 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, respectively. The group treated with the triple-specific antibody A17m0902-1708-151H7-C showed significantly superior efficacy compared to the group treated with the anti-PD-1 monoclonal antibody A17m0902_hIgG4, and also superior to the group receiving the PVRIG / TIGIT bispecific antibody + PD-1 monoclonal antibody combination.

[0330] The experiment was terminated 21 days after administration, and all mice were euthanized. The tumors were removed, weighed, and photographed to calculate the tumor weight TGI. The results were consistent with the trend of tumor volume TGI, and the efficacy of the group administered with the trispecific antibody A17m0902-1708-151H7-C was clearly superior to that of the group administered with the PD-1 monoclonal antibody A17m0902_hIgG4, and also superior to the group receiving the PVRIG / TIGIT bispecific antibody + PD-1 monoclonal antibody combination. The experimental results are shown in Table 45 and Figures 15A and 15B, respectively.

[0331] [Table 45]

[0332] Example 3-9. Evaluation of the antitumor activity of an anti-PD-1 / PVRIG / TIGIT trispecific antibody in a mouse subcutaneous tumor model of human melanoma A375 mixed with human PBMCs that did not respond to PD-1 monoclonal antibody. This example investigated the in vivo efficacy of an anti-PD-1 / PVRIG / TIGIT trispecific antibody in an in vivo tumor model of human melanoma A375 mixed with human PBMCs that did not respond to anti-PD-1 monoclonal antibodies.

[0333] Female NCG mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) weighing approximately 18-22g and aged 4-8 weeks were reared in an IVC constant temperature and pressure system in an SPF-grade animal rearing room.

[0334] Frozen PBMCs were screened to obtain donor PBMCs that did not respond to anti-PD-1 monoclonal antibodies in the in vivo drug efficacy model. The experimental design was the same as that described in Examples 3-8. The grouping and administration plan for the mouse experiments are shown in Table 46.

[0335] [Table 46]

[0336] After the start of administration, the body weight and tumor volume of the mice were measured twice a week.

[0337] Seventeen days after administration, the mean tumor volume of mice in each group was measured, and the tumor growth inhibition rates (TGI%) for 1708-151H7, A17m0902_hIgG4, A17m0902-1708-151H7-C, and 1708-151H7+A17m0902_hIgG4 in the experimental groups were 4.53%, 20.75%, 36.70%, and 31.95%, respectively, compared to the IgG control group.

[0338] The experiment was terminated 17 days after administration, and all mice were euthanized. Their tumors were removed, weighed, and photographed. By measuring the tumor weight of each group of mice, the experimental groups, compared to the IgG control group, showed tumor growth inhibition rates (TGI) and tumor weight TGI for 1708-151H7, A17m0902_hIgG4, A17m0902-1708-151H7-C, and 1708-151H7+A17m0902_hIgG4 to 0%, 16.8%, 38.2%, and 33.4%, respectively. The tumor weight TGI was consistent with the trend of tumor volume TGI. The experimental results are shown in Table 47 and Figures 16A and 16B, respectively.

[0339] Compared to the IgG control group, the A17m0902-1708-151H7-C administered group and the 1708-151H7+A17m0902_hIgG4 administered group showed a significant reduction in both tumor volume and weight (Figure 16A, Figure 16B, P<0.05), demonstrating a remarkable tumor-inhibiting effect. Since no significant weight loss or behavioral abnormalities were observed in any of the mice during this experiment, it was revealed that the mice possessed relatively good tolerance to the test drug under these experimental conditions.

[0340] [Table 47]

[0341] Example 4. Screening of PD-1 / PVRIG / TIGIT trispecific antibody preparation buffer system, pH value, and additives. The PD-1 / PVRIG / TIGIT trispecific antibody used was A17m0902-1708-151H7-C, and the amino acid sequences of its first and second polypeptide chains are SEQ ID NOs. 72 and 52, respectively. The following buffer solutions were prepared, and the corresponding additives and surfactants were added to prepare an antibody preparation with a PD-1 / PVRIG / TIGIT trispecific antibody concentration of 50 mg / mL. Stability studies were then conducted. The experimental results are shown in Table 48.

[0342] 1) F1: Add 8% w / v sucrose and 0.04 mg / mL polysorbate 80 to 10 mM citrate-disodium citrate buffer pH 5.0. 2) F2: Add 8% w / v sucrose and 0.04 mg / mL polysorbate 80 to 10 mM histidine-histidine hydrochloride buffer pH 5.5. 3) F3: Add 8% w / v sucrose and 0.04 mg / mL polysorbate 80 to 10 mM histidine-acetate buffer pH 5.0. 4) F4: Add 8% w / v sucrose and 0.04 mg / mL polysorbate 80 to 10 mM histidine-acetate buffer pH 5.5. 5) Add 8% w / v sucrose and 0.04 mg / mL polysorbate 80 to F5: 10 mM histidine-acetate buffer pH 6.0. 6) F6: Add 8% w / v sucrose, 0.04 mg / mL polysorbate 80, and 50 mM arginine to 10 mM histidine-acetate buffer pH 5.0. 7) F7: 10 mM histidine-acetate buffer pH 5.5 was mixed with 8% w / v sucrose, 0.04 mg / mL polysorbate 80, and 50 mM arginine.

[0343] [Table 48-1] [Table 48-2] [Table 49] [Table 50]

[0344] Experimental results: Formula F1 was clearly cloudy, and the protein was citrate-based and unstable.

[0345] As shown in the results of the freeze-thaw and shaking experiments (Table 48), after 3 days of shaking, formulations F3 (histidine-acetate buffer pH 5.0) and F4 (histidine-acetate buffer pH 5.5) were clear and free of emulsion, while the other formulations developed emulsion from T0. After repeated freeze-thaw cycles, all formulations showed good appearance stability. In the detection results of SEC, CEX, and NR-CE after shaking or repeated freeze-thaw cycles, formulations F2 to F7 showed no significant differences and exhibited relatively good stability.

[0346] As shown in the results of the high-temperature experiments (Tables 49-50), As shown by the visual results, after storage at 40°C for 4 weeks, none of the formulations F2 to F7 showed any significant changes in appearance.

[0347] As shown by the SEC results, after high-temperature storage at 40°C for 4 weeks, the purity of all formulations decreased, but the decrease in 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 smaller than that of formulations F5-F7.

[0348] As shown by the CEX results, after storage at 40°C for 4 weeks, the main CEX peak for each formulation showed a decreasing trend, and there were no significant differences between formulations.

[0349] As shown by the NR-CE results, after storage at 40°C for 4 weeks, the purity of the main peak of each formulation remained above 90%, and there was no significant difference between formulations.

[0350] In summary, formulations F3-F4 showed slightly better stability than the other formulations in terms of appearance, SEC, CEX, and NR-CE detection results. Therefore, F3 (histidine-acetate buffer pH 5.0) and F4 (histidine-acetate buffer pH 5.5) were selected for the next protein concentration screening.

[0351] Example 5. Screening of PD-1 / PVRIG / TIGIT trispecific antibody concentrations The PD-1 / PVRIG / TIGIT trispecific antibody used was A17m0902-1708-151H7-C, and the amino acid sequences of its first and second polypeptide chains were SEQ ID NOs. 72 and 52, respectively. Buffer systems of 10 mM histidine acetate at pH 5.0 and pH 5.5 were selected, and 8% w / v sucrose and 0.4 mg / mL polysorbate 80 were prepared. 1) F8: 10 mM histidine acetate pH 5.0, 50 mg / mL antibody, 2) F9: 10 mM histidine acetate pH 5.5, 50 mg / mL antibody, 3) F10: 10 mM histidine acetate pH 5.0, 100 mg / mL antibody, Stability studies were conducted on PD-1 / PVRIG / TIGIT trispecific antibody preparations with different antibody concentrations.

[0352] [Table 51-1] [Table 51-2] [Table 52-1] [Table 52-2]

[0353] Experimental results As shown in the freeze-thaw and shaking test results (Table 51), formulations F8 to F10 showed good appearance stability after shaking or freeze-thaw, and there were no significant differences in the detection results for SEC, CEX, and NR-CE among the formulations, indicating relatively good stability.

[0354] As shown in the results of the high-temperature experiment (Table 52), As is evident from the visual results, formulations with different antibody concentrations all exhibited a pale yellow appearance with no visible particles after being left at 40°C for 4 weeks, and there was no change in appearance compared to T0 when left at 2-8°C and 25°C for 4 weeks.

[0355] As is clear from the SEC results, after being left at 40°C for 4 weeks, the monomer content of each formulation clearly decreased. In the acetate-histidine buffer system, under the same antibody concentration (50 mg / mL), F8 (pH 5.0) had slightly better monomer and polymer content than F9 (pH 5.5). As the formulation concentration increased, the polymer 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, supporting the development of high-concentration formulations.

[0356] As is clear from the CEX results, after being left at 40°C for 4 weeks, the main peaks of the F8-F10 formulations showed a clear decrease, and the trends for each formulation were consistent. In the 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, indicating that antibody concentration did not have a significant impact on the decrease in the main peaks of the CEX.

[0357] As is clear from the NR-CE results, after storage at 40°C for 4 weeks, formulation F10 (100 mg / mL) showed the greatest decrease, but its main peak was still 91.49%. The other formulations also showed a slight decrease in purity, but the decrease was almost the same. The three formulations did not show a significant difference in their main peak at point T0 even after being left at 2-8°C and 25°C for 4 weeks.

[0358] As can be seen from combining shaking, repeated freeze-thaw cycles, and high-temperature experiments, the antibody exhibited good stability in the acetate-histidine buffer system, and its stability at pH 5.0 was also relatively good when developing concentrations up to 100 mg / mL.

[0359] Example 6. Screening of pH and concentration of PD-1 / PVRIG / TIGIT trispecific antibodies The PD-1 / PVRIG / TIGIT trispecific antibody used was A17m0902-1708-151H7-01-C, and the amino acid sequences of its first and second polypeptide chains were SEQ ID NOs. 76 and 52, respectively.

[0360] Select buffer systems of 10 mM histidine-acetic acid at pH 5.0 and pH 5.5, and prepare 8% w / v sucrose and 0.8 mg / mL polysorbate 80. 1) F11: 10 mM histidine acetate, pH 5.0, 50 mg / mL antibody, 2) F12: 10 mM histidine acetate pH 5.0, 80 mg / mL antibody, 3) F13: 10 mM histidine acetate pH 5.0, 100 mg / mL antibody, 4) F14: 10 mM histidine acetate pH 5.5, 50 mg / mL antibody, 5) F15: 10 mM histidine acetate pH 5.5, 100 mg / mL antibody, Stability studies were conducted on PD-1 / PVRIG / TIGIT trispecific antibody preparations with different antibody concentrations, including freeze-thaw cycles and shaking at high temperatures (40°C), 25°C, and 2-8°C.

[0361] [Table 53-1] [Table 53-2] [Table 54-1] [Table 54-2]

[0362] Experimental Results: As shown in the results of the freeze-thaw and shaking experiments (Table 53), formulations F11 to F15 showed good appearance stability after shaking or freeze-thaw. In the detection results for SEC, CEX, and NR-CE, there were no significant differences among the formulations, indicating relatively good stability.

[0363] As shown in the results of the high-temperature experiment (Table 54), As is evident from the visual results, none of the formulations with different antibody concentrations showed visible granules after being left at 40°C for two weeks, and the emulsion of F12-F15 increased. Furthermore, the appearance of each formulation remained unchanged compared to T0 even after being left at 2-8°C and 25°C for two weeks.

[0364] As is clear from the SEC results, after being left at 40°C for two weeks, the monomer content of each formulation clearly decreased. In the acetate-histidine buffer system, under the same antibody concentration (50 mg / mL), F11 (pH 5.0) had slightly better monomer and polymer content than F14 (pH 5.5), and the polymer content increased with increasing formulation concentration.

[0365] As is clear from the CEX results, after being left at 40°C for two weeks, the main peaks of formulations F11 to F15 showed a clear decrease, and the trends for each formulation were consistent. In the 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, indicating that antibody concentration did not have a significant effect on the decrease in the main peaks of the CEX.

[0366] As is evident from the NR-CE results, after storage at 40°C for two weeks, the main peak of each formulation decreased, but the degree of decrease was almost the same. The five formulations did not show a significant difference from the main peak at T0 point even after being left at 2-8°C and 25°C for two weeks.

[0367] As can be seen from combining 2W data from shaking, repeated freeze-thaw cycles, and high-temperature experiments, the antibody exhibits good stability in the acetate-histidine buffer system, with purity data at pH 5.0 and pH 5.5 all within acceptable limits. Polymerization increases slightly with increasing concentration, but remains within acceptable limits.

Claims

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

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

3. (B) The second antigen-binding domain comprises an immunoglobulin monovariate domain, the immunoglobulin monovariate domain comprising CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs. 74-75, 38, 46-50, wherein CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system. Preferably, the amino acid sequences of 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), of which the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NOs. 57, 58, and 59, respectively, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in SEQ ID NOs. 60, 61, and 62, respectively. The PD-1 / PVRIG / TIGIT binding protein according to claim 1 or 2.

4. (A) The first antigen-binding domain comprises an amino acid sequence represented by 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 the amino acid sequence of Sequence ID No. 27 or an amino acid sequence having at least 90% sequence identity therewith. and / or, (B) The second antigen-binding domain comprises an amino acid sequence represented by any one of SEQ ID NOs. 74-75, 38, 46-50, or having at least 90% sequence identity thereto. Preferably, the amino acid sequence is one of sequence numbers 74 and 48, or has at least 90% sequence identity with it. and / or, (C) The third antigen-binding domain includes VH and VL, of which, The VH comprises an amino acid sequence shown in any one of sequence numbers 63 to 65, or having at least 90% sequence identity thereto, and the VL comprises an amino acid sequence shown in sequence number 67 or 66, or having at least 90% sequence identity thereto. Preferably, VH includes SEQ ID NO: 63 or an amino acid sequence having at least 90% sequence identity thereto, and VL includes SEQ ID NO: 67 or an amino acid sequence having at least 90% sequence identity thereto. The PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 3.

5. 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. The PD-1 / PVRIG / TIGIT binding protein according to claim 4.

6. The first polypeptide chain, and A second polypeptide chain, Then, sequentially from the N-terminus to the C-terminus, (I) The first polypeptide chain is shown in the 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 shown in the structure [VL of the third antigen-binding domain]-Cκ, or (II) The first polypeptide chain is shown in the structure [second antigen-binding domain]-[linker 2]b-[VH of the third antigen-binding domain]-CH1-Fc, and The second polypeptide chain is shown in the structure [first antigen-binding domain]-[linker 3]c-[VL of the third antigen-binding domain]-Cκ, or (III) The first polypeptide chain is shown in the structure [second antigen-binding domain] - [linker 2] b - [VH of the third antigen-binding domain] - CH1 - Fc - [linker 4] d - [first antigen-binding domain], and The second polypeptide chain is shown in the structure [VL of the third antigen-binding domain]-Cκ, or (IV) The first polypeptide chain is shown in the structure [second antigen-binding domain]-[linker 2]b-[VH of the third antigen-binding domain]-CH1-Fc, and The second polypeptide chain is shown in the structure [VL of the third antigen-binding domain]-Cκ-[linker 5]e-[first antigen-binding domain], Among them, the hyphen indicates a peptide bond. Linkers 1, 2, 3, 4, and 5 may be the same or different, and a, b, c, d, and e are arbitrarily and independently 0 or 1. Preferably, linker 1, linker 2, linker 3, linker 4, linker 5 are independently (G ｘ S) ｙ And of these, x is selected from integers 1 to 5, and y is selected from integers 1 to 6, more preferably independently (G ４ S) ２ , (G ４ S) ３ , (G ４ S) ４ It is a linker that is one of the following: The PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 5.

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

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

9. It further contains sugars, preferably the sugars are sucrose. A pharmaceutical composition according to any one of claims 1 to 8.

10. It further contains other amino acids or medicinal salts thereof, preferably arginine or a medicinal salt thereof, and more preferably arginine-hydrochloride. A pharmaceutical composition according to any one of claims 1 to 9.

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

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

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

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

15. The sugar concentration is 0.1% w / v to 20% w / v, preferably 1% w / v to 15% w / v, more preferably 3% w / v to 12% w / v, and most preferably 5% w / v to 10% w / v. A pharmaceutical composition according to any one of claims 9 to 14.

16. The concentration of the other amino acids or their medicinal salts is 1 mM to 200 mM, preferably 5 mM to 170 mM, more preferably 10 mM to 150 mM, and most preferably 15 mM to 120 mM. A pharmaceutical composition according to any one of claims 10 to 15.

17. A pharmaceutical composition, A PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 7, Preferably a histidine hydrochloride buffer or a histidine acetate buffer, more preferably a histidine-histidine hydrochloride buffer or a histidine-acetic acid buffer, a histidine salt buffer, Polysorbate and, Including sucrose, Optionally, the composition further comprises arginine or a medicinal salt thereof. Pharmaceutical composition.

18. Includes one of the following groups 1) to 13): 1) PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 7, in a concentration of 0.01 mg / mL to 500 mg / mL A histidine salt buffer of 0.1 mM to 50 mM, preferably a histidine hydrochloride buffer or a histidine acetate buffer, more preferably a histidine-histidine hydrochloride buffer or a histidine-acetic acid buffer. Polysorbate in concentrations of 0.01 mg / mL to 10 mg / mL, It is sucrose with a concentration of 0.1% w / v to 20% w / v. Optionally, the pharmaceutical composition further comprises 1 mM to 200 mM arginine or a pharmaceutically acceptable salt thereof. Furthermore, the pH of the pharmaceutical composition is 3.5 to 7. 2) PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 7, in a concentration of 0.1 mg / mL to 400 mg / mL A 0.5 mM to 40 mM histidine salt buffer, preferably a histidine hydrochloride buffer or a histidine acetate buffer, more preferably a histidine-histidine hydrochloride buffer or a histidine-acetic acid buffer. Polysorbate in concentrations of 0.05 mg / mL to 5 mg / mL, It is sucrose with a concentration of 1% w / v to 15% w / v. Optionally, the pharmaceutical composition further comprises 5 mM to 170 mM arginine or a pharmaceutically acceptable salt thereof. Furthermore, the pH of the pharmaceutical composition is 4 to 6.

5. 3) PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 7, in a concentration of 0.5 mg / mL to 200 mg / mL A histidine salt buffer of 1 mM to 30 mM, preferably a histidine hydrochloride buffer or a histidine acetate buffer, more preferably a histidine-histidine hydrochloride buffer or a histidine-acetic acid buffer. Polysorbate in concentrations of 0.1 mg / mL to 3 mg / mL, It is sucrose with a concentration of 3% w / v to 12% w / v. Optionally, the pharmaceutical composition further comprises 10 mM to 150 mM arginine or a pharmaceutically acceptable salt thereof. Furthermore, the pH of the pharmaceutical composition is 4.2 to 6.

2. 4) PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 7, in a concentration of 1 mg / mL to 150 mg / mL A 5 mM to 20 mM histidine salt buffer, preferably a histidine hydrochloride buffer or a histidine acetate buffer, more preferably a histidine-histidine hydrochloride buffer or a histidine-acetic acid buffer. Polysorbate in concentrations of 0.2 mg / mL to 2 mg / mL, It is sucrose with a concentration of 5% w / v to 10% w / v. Optionally, the pharmaceutical composition further comprises 15 mM to 120 mM arginine or a pharmaceutically acceptable salt thereof. Furthermore, the pH of the pharmaceutical composition is 4.5 to 6. 5) PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 7, in an amount of 1 to 150 mg / mL Approximately 10 mM histidine hydrochloride buffer or histidine acetate buffer, preferably histidine-histidine hydrochloride buffer or histidine-acetic acid buffer. Polysorbate 80 in concentrations of 0.2 mg / mL to 1 mg / mL. It is approximately 8% w / v sucrose, Furthermore, the pH of the pharmaceutical composition is 4.5 to 6. 6) PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 7, in a concentration of approximately 10 mg / mL, approximately 20 mg / mL, approximately 25 mg / mL, approximately 50 mg / mL, approximately 80 mg / mL, approximately 100 mg / mL, approximately 120 mg / mL, approximately 130 mg / mL, or approximately 150 mg / mL. Approximately 10 mM histidine hydrochloride buffer, preferably histidine-histidine hydrochloride buffer. Polysorbate 80 at approximately 0.4 mg / mL, It is approximately 8% w / v sucrose, Furthermore, the pH of the pharmaceutical composition is approximately 5. 7) PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 7, in a concentration of approximately 10 mg / mL, approximately 20 mg / mL, approximately 25 mg / mL, approximately 50 mg / mL, approximately 80 mg / mL, approximately 100 mg / mL, approximately 120 mg / mL, 130 mg / mL, or approximately 150 mg / mL. Approximately 10 mM histidine hydrochloride buffer, preferably histidine-histidine hydrochloride buffer. Polysorbate 80 at approximately 0.4 mg / mL, It is approximately 8% w / v sucrose, Furthermore, the pH of the pharmaceutical composition is approximately 5.

5. 8) PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 7, in a concentration of approximately 10 mg / mL, approximately 20 mg / mL, approximately 25 mg / mL, approximately 50 mg / mL, approximately 80 mg / mL, approximately 100 mg / mL, approximately 120 mg / mL, approximately 130 mg / mL, or approximately 150 mg / mL. Approximately 10 mM histidine hydrochloride buffer, preferably histidine-histidine hydrochloride buffer. Polysorbate 80 at approximately 0.8 mg / mL, It is approximately 8% w / v sucrose, Furthermore, the pH of the pharmaceutical composition is approximately 5. 9) PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 7, in a concentration of approximately 10 mg / mL, approximately 20 mg / mL, approximately 25 mg / mL, approximately 50 mg / mL, approximately 80 mg / mL, approximately 100 mg / mL, approximately 120 mg / mL, approximately 130 mg / mL, or approximately 150 mg / mL. Approximately 10 mM histidine hydrochloride buffer, preferably histidine-histidine hydrochloride buffer. Polysorbate 80 at approximately 0.8 mg / mL, It is approximately 8% w / v sucrose, Furthermore, the pH of the pharmaceutical composition is approximately 5.

5. 10) PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 7, in a concentration of approximately 10 mg / mL, approximately 20 mg / mL, approximately 25 mg / mL, approximately 50 mg / mL, approximately 80 mg / mL, approximately 100 mg / mL, approximately 120 mg / mL, approximately 130 mg / mL, or approximately 150 mg / mL. Approximately 10 mM histidine-acetic acid buffer, Polysorbate 80 at approximately 0.4 mg / mL, It is approximately 8% w / v sucrose, Furthermore, the pH of the pharmaceutical composition is approximately 5. 11) PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 7, in a concentration of approximately 10 mg / mL, approximately 20 mg / mL, approximately 25 mg / mL, approximately 50 mg / mL, approximately 80 mg / mL, approximately 100 mg / mL, approximately 120 mg / mL, approximately 130 mg / mL, or approximately 150 mg / mL. Approximately 10 mM histidine-acetic acid buffer, Polysorbate 80 at approximately 0.4 mg / mL, It is approximately 8% w / v sucrose, Furthermore, the pH of the pharmaceutical composition is approximately 5.

5. 12) PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 7, in a concentration of approximately 10 mg / mL, approximately 20 mg / mL, approximately 25 mg / mL, approximately 50 mg / mL, approximately 80 mg / mL, approximately 100 mg / mL, approximately 120 mg / mL, approximately 130 mg / mL, or approximately 150 mg / mL. Approximately 10 mM histidine-acetic acid buffer, Polysorbate 80 at approximately 0.8 mg / mL, It is approximately 8% w / v sucrose, Furthermore, the pH of the pharmaceutical composition is approximately 5. 13) PD-1 / PVRIG / TIGIT binding protein according to any one of claims 1 to 7, in a concentration of approximately 10 mg / mL, approximately 20 mg / mL, approximately 25 mg / mL, approximately 50 mg / mL, approximately 80 mg / mL, approximately 100 mg / mL, approximately 120 mg / mL, approximately 130 mg / mL, or approximately 150 mg / mL. Approximately 10 mM histidine-acetic acid buffer, Polysorbate 80 at approximately 0.8 mg / mL, It is approximately 8% w / v sucrose, Furthermore, the pH of the pharmaceutical composition is approximately 5.

5. The pharmaceutical composition according to claim 17.

19. A lyophilized preparation, wherein the lyophilized preparation can be redissolved to form the pharmaceutical composition described in any one of claims 1 to 18, or the lyophilized preparation is obtained by lyophilizing the pharmaceutical composition described in any one of claims 1 to 18. Freeze-dried preparation.

20. The freeze-dried preparation described in claim 19 is prepared by redissolving it, Reconstitution solution.

21. A container comprising a pharmaceutical composition according to any one of claims 1 to 18, a lyophilized preparation according to claim 19, or a re-dissolving solution according to claim 20, product.

22. The use of a pharmaceutical composition according to any one of claims 1 to 19, a lyophilized preparation according to claim 19, or a redissolving solution according to claim 20 in the preparation of a drug for treating cancer, wherein the cancer is preferably 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 hematological cancers. use.

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