WO2023040940A1 - Pvrig/tigit结合蛋白联合免疫检查点抑制剂用于治疗癌症 - Google Patents

Pvrig/tigit结合蛋白联合免疫检查点抑制剂用于治疗癌症 Download PDF

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WO2023040940A1
WO2023040940A1 PCT/CN2022/118959 CN2022118959W WO2023040940A1 WO 2023040940 A1 WO2023040940 A1 WO 2023040940A1 CN 2022118959 W CN2022118959 W CN 2022118959W WO 2023040940 A1 WO2023040940 A1 WO 2023040940A1
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pvrig
amino acid
cancer
seq
antibody
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French (fr)
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林侃
林�源
袁吉民
孙星
廖成
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Jiangsu Hengrui Pharmaceutical Co Ltd
Shanghai Shengdi Pharmaceutical Co Ltd
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Jiangsu Hengrui Pharmaceutical Co Ltd
Shanghai Shengdi Pharmaceutical Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure belongs to the field of medicine, and in particular, the present disclosure relates to the use of PVRIG/TIGIT binding protein combined with immune checkpoint inhibitors for treating cancer.
  • PVRIG Polyvirus receptor-related Ig domain containing protein
  • CD112R Polyvirus receptor-related Ig domain containing protein
  • 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.
  • TIGIT T cell immunoglobulin and ITIM domain
  • PVRL2 also known as CD112
  • the ligand of PVRIG binds to PVRIG, it will activate the ITIM domain in the intracellular region of PVRIG, making PVRIG play an immunosuppressive role.
  • PVRIG is mainly expressed on the surface of CD4 + T cells, CD8 + T cells and NK cells.
  • PVRIG and its ligand PVRL2 are highly expressed in a variety of solid tumors, including lung cancer, breast cancer, ovarian cancer, kidney cancer, gastric cancer, endometrial cancer, head and neck cancer, etc.
  • the expression of PVRIG in these cancers is highly correlated with TIGIT and PD-1. Similar to PD-1 and TIGIT, PVRIG-positive T cells were also Eomes-positive and Tbet-negative, suggesting that PVRIG is involved in the exhaustion of T cells. Therefore, PVRIG may represent a new immune checkpoint in addition to PD-1 and TIGIT, and play a role of redundancy.
  • TIGIT is highly expressed on lymphocytes, including cancer-infiltrating lymphocytes (TILs) and Tregs that infiltrate different types of cancer. Engagement of TIGIT with its cognate ligand PVR (aka CD155) has been demonstrated to directly inhibit NK cell cytotoxicity through its cytoplasmic ITIM domain. PVR is also widely expressed in cancer, suggesting that the TIGIT-PVR signaling axis may be a major immune escape mechanism in cancer.
  • Inhibitory receptors TIGIT, PVRIG and activating receptor DNAM-1 bind the same ligands CD155 and CD112, but the affinity of inhibitory receptors is higher (Y.Zhu et al. 2016, J Exp Med.213:167-176 ).
  • the down-regulation of DNAM-1 expression in NK cells isolated from tumor patients makes NK cells more susceptible to inhibition by TIGIT or PVRIG, blocking the combination of TIGIT and PVRIG can activate the cell killing function of NK cells (L.Martinet et al. 2015, Cell Reports. 11:85-97).
  • TIGIT and PVRIG are currently the most clinically successful immune checkpoint inhibitor monoclonal antibody, the up-regulation of other immune checkpoints on the surface of T cells will limit its efficacy.
  • TIGIT and PVRIG also regulate the activity of NK cells, suggesting that they may synergize with the function of PD-1/PD-L1 in NK cells. It has been reported in the literature that the high expression of TIGIT and PVRIG ligands CD155 and CD112 in lung cancer, ovarian cancer, colorectal cancer, and melanoma is significantly associated with poor prognosis after PD-1/PD-L1 treatment.
  • patients with high CD155 expression have a poor response to PD-1 antibodies, while patients with low CD155 expression have a good response to PD-1 (S. Whelan et al. 2019, Cancer Immunol Res. 7:257-268; A. Lepletier et al. 2020 , Clin Cancer Res. 26:3671-3681).
  • the combination of anti-PVRIG/TIGIT double antibody and PD-1/PD-L1 can potentially improve the effect of anti-tumor therapy by enhancing the activation of T cells and NK cells, overcoming PD-1/PD-L1 drug resistance and expanding the responding patient population .
  • the present disclosure aims to provide an anti-PVRIG/TIGIT bispecific antibody with high affinity, high selectivity, and high biological activity that inhibits tumor growth in vivo in combination with immune checkpoint inhibitors (such as PD-1 or PD-L1 antibodies) It is used for the treatment of cancer, and has shown good anti-tumor effect, and has the potential to provide better clinical efficacy than existing PD-1, TIGIT, PVRIG drugs or their combination.
  • the present disclosure provides a use of a PVRIG binding protein or a PVRIG/TIGIT binding protein in combination with an immune checkpoint inhibitor in the preparation of a medicament for treating a disease (eg, cancer), or a method for treating a disease (eg, cancer).
  • a disease eg, cancer
  • a method for treating a disease eg, cancer
  • the present disclosure provides a combination of a PVRIG-binding protein and a TIGIT-binding protein in the preparation of a drug for treating a disease (eg, cancer), or a method for treating a disease (eg, cancer).
  • a disease eg, cancer
  • immune checkpoint inhibitors are further included.
  • the present disclosure provides a combination of a PVRIG binding protein, a TIGIT binding protein and an immune checkpoint inhibitor in the preparation of a drug for treating a disease (eg, cancer), or a method for treating a disease (eg, cancer).
  • a disease eg, cancer
  • a method for treating a disease eg, cancer
  • a use of a PVRIG-binding protein for example, an anti-PVRIG antibody or an antigen-binding fragment thereof in combination with an immune checkpoint inhibitor in the preparation of a medicament for treating cancer
  • the PVRIG-binding protein comprising a single variable immunoglobulin domain, the immunoglobulin single variable domain comprising:
  • CDR1, CDR2 and CDR3 in any one of the amino acid sequences shown in SEQ ID NO: 2, 15-19, or
  • CDR1, CDR2 and CDR3 in any amino acid sequence shown in SEQ ID NO: 3, 21-25,
  • the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system;
  • the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are respectively
  • the immunoglobulin single variable domain of the aforementioned PVRIG binding protein is humanized, affinity matured, removed T cell epitopes, reduced antibody deamidation and/or modified to reduce antibody isomerization; some specific
  • the heavy chain framework region of the human germline template used in the humanization transformation process is IGHV3-7*01.
  • amino acid sequences of the immunoglobulin single variable domains of the aforementioned PVRIG binding proteins are as follows:
  • the aforementioned PVRIG binding protein further comprises a human immunoglobulin Fc region
  • the Fc region is the Fc region of human IgG1 or IgG4;
  • the Fc region of human IgG4 has S228P, F234A, L235A and/or K447A mutations.
  • PVRIG-binding protein and TIGIT-binding protein eg, anti-TIGIT antibody or antigen-binding fragment thereof
  • an immune checkpoint inhibitor in the preparation of a drug for treating cancer
  • the TIGIT binding protein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 32, 33 and 34 respectively, and the light chain variable region comprises HCDR1 shown in SEQ ID NO: 35, 36 and 37 respectively LCDR1, LCDR2, and LCDR3.
  • the CDR1, CDR2 and CDR3 are defined according to the Kabat numbering system.
  • the aforesaid binding protein specifically binding to TIGIT comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
  • the heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO: 38-40 or having at least 80%, at least 90% sequence identity thereto,
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 41 or 42 or having at least 80%, at least 90% sequence identity thereto;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 3 or having at least 80%, at least 90% sequence identity thereto,
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 42 or having at least 80%, at least 90% sequence identity thereto.
  • the aforementioned TIGIT-binding protein further comprises a human immunoglobulin Fc region
  • the Fc region is the Fc region of human IgG1 or IgG4;
  • the Fc region of human IgG4 has S228P, F234A, L235A and/or K447A mutations.
  • the aforementioned specifically binding TIGIT-binding protein comprises the full-length heavy chain sequence shown in SEQ ID NO: 26 or has at least 80%, at least 90% sequence identity therewith, and SEQ ID NO: 27
  • the full length sequence of the light chain shown is or has at least 80%, at least 90% sequence identity thereto.
  • PVRIG/TIGIT binding protein for example, anti-PVRIG/TIGIT bispecific antibody or antigen-binding fragment thereof
  • the binding protein comprises a first antigen-binding domain specifically binding to PVRIG and a second antigen-binding domain specifically binding to TIGIT, the first antigen-binding domain specifically binding to PVRIG comprising an immunoglobulin single variable domain
  • the immunoglobulin single variable domain comprises:
  • CDR1, CDR2 and CDR3 in any one of the amino acid sequences shown in SEQ ID NO: 2 and 15-19, or
  • the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system;
  • the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are respectively
  • the aforementioned first antigen-binding domain specifically binding to PVRIG is engineered by humanization, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation and/or reduction of antibody isomerization; some specific implementations
  • the heavy chain framework region of the human germline template used in the humanization transformation process is IGHV3-7*01.
  • the amino acid sequence of the immunoglobulin single variable domain in the first antigen-binding domain that specifically binds to PVRIG is shown in any of SEQ ID NO: 2 and 15-19; or as shown in SEQ ID NO: shown in any of 3 and 21-25; or have at least 80% sequence identity with any of the aforementioned sequences, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the aforementioned second antigen-binding domain that specifically binds to TIGIT comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 32, 33 and 34 respectively, and the light chain variable region comprises HCDR1 shown in SEQ ID NO: 35, 36 and 37 respectively LCDR1, LCDR2, and LCDR3.
  • the aforementioned second antigen-binding domain that specifically binds to TIGIT comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
  • the heavy chain variable region comprises at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, as shown in any of SEQ ID NO: 38-40 or at least 80% thereof , at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity of amino acid sequences,
  • the light chain variable region comprises at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% as shown in SEQ ID NO: 41 or 42 or at least 80% thereof.
  • the heavy chain variable region comprises at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% of SEQ ID NO: 38 or at least 80% thereof.
  • the light chain variable region comprises at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, as shown in or at least 80% of SEQ ID NO: 42, Amino acid sequences having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the aforementioned PVRIG/TIGIT binding protein further comprises a human immunoglobulin Fc region
  • the Fc region is the Fc region of human IgG1 or IgG4;
  • the Fc region of human IgG4 has S228P, F234A, L235A and/or K447A mutations.
  • the aforementioned first antigen-binding domain that specifically binds PVRIG and the second antigen-binding domain that specifically binds TIGIT are connected directly or through a linker;
  • the linker has an amino acid sequence shown as (G 4 S) x , wherein x is independently selected from an integer of 1-20;
  • the linker is an amino acid sequence represented by (G 4 S) 2 , (G 4 S) 3 , (G 4 S) 4 .
  • the immunoglobulin single variable domain that specifically binds the first antigen-binding domain of PVRIG is located at the N-terminal of the heavy chain variable region that specifically binds the second antigen-binding domain of TIGIT;
  • the immunoglobulin single variable domain that specifically binds the first antigen-binding domain of PVRIG is located at the C-terminus of the heavy chain variable region that specifically binds the second antigen-binding domain of TIGIT;
  • the immunoglobulin single variable domain that specifically binds the first antigen-binding domain of PVRIG is located at the N-terminus of the light chain variable region that specifically binds the second antigen-binding domain of TIGIT;
  • the immunoglobulin single variable domain that specifically binds the first antigen-binding domain of PVRIG is located at the C-terminus of the light chain variable region that specifically binds the second antigen-binding domain of TIGIT.
  • a PVRIG/TIGIT binding protein comprising a first polypeptide chain and a second polypeptide chain, wherein:
  • the first polypeptide chain comprises at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88% of any of SEQ ID NOS: 28-29 and 43-45, or at least 80% thereof.
  • Amino acid sequences having at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity
  • the second polypeptide chain comprises as shown in SEQ ID NO: 27, or has at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% , an amino acid sequence of at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or
  • the first polypeptide chain comprises SEQ ID NO: 26, or at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, An amino acid sequence of at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, the second polypeptide chain comprising As shown in SEQ ID NO: 30 or 31, or at least 80% thereof, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92 Amino acid sequences having %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the aforementioned PVRIG/TIGIT binding protein comprises two identical first polypeptide chains and two identical second polypeptide chains.
  • immune checkpoint inhibitors as described above include but are not limited to targeting PD-1/PD-L1, PD-L2, B7-1/CTLA-4, B7-2/CTLA-4, B7 -1/CD28, B7-2/CD28, B7-H2/ICOS, B7-H3, B7-H4, B7-H7, VISTA (PD-1H), BTNL2, TIM3, LAG3, TNFSF/TNFRSF (such as CD40-CD40L , CD27-CD70 or OX40-OX40L), leukocyte immunoglobulin-like receptor (LIR/LILR) or immunoglobulin-like transcript (ILT), cohesin or cohesin-like receptor protein (such as DNAM-1, CD226, Inhibitor of PTA1, TLISA1, CD96, TIGIT, Vstm3 and VSIG9)
  • the aforementioned immune checkpoint inhibitors include but are not limited to proteins or polypeptides, small molecules, and nucleic acid drugs.
  • the immune checkpoint inhibitor is PD-1 binding protein and/or PD-L1 binding protein.
  • the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an antigen-binding fragment of the antibody, or a fusion protein comprising the antibody or an antigen-binding fragment thereof.
  • the aforementioned PD-1 binding protein (for example, comprising or being an anti-PD-1 antibody or an antigen-binding fragment thereof) comprises:
  • HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NOs: 59, 60 and 61, respectively, and LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NOs: 62, 63 and 64, respectively; or
  • HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO:67, 68 and 69 respectively, and LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NO:70, 71 and 72 respectively.
  • the aforementioned PD-1 binding protein (for example, comprising or being an anti-PD-1 antibody or an antigen-binding fragment thereof) comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
  • the heavy chain variable region comprises at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, as shown in SEQ ID NO: 57 or at least 80% thereof.
  • the heavy chain variable region comprises at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, as shown in or at least 80% of SEQ ID NO: 73, An amino acid sequence of at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, said light chain variable region comprising At least 80% as shown in or related to SEQ ID NO: 74, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, Amino acid sequences having at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the aforementioned PD-1 binding protein further comprises a human immunoglobulin Fc region
  • the Fc region is the Fc region of human IgG1 or IgG4;
  • the Fc region of human IgG4 has S228P, F234A, L235A and/or K447A mutations.
  • the aforementioned PD-1 binding protein comprises a heavy chain (HC) and a light chain (LC), wherein:
  • the heavy chain comprises at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91% of SEQ ID NO: 65 or at least 80% thereof , at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity of the amino acid sequence, said light chain comprising such as SEQ ID NO: 66 Indicated or associated with at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% Amino acid sequences with %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or
  • the heavy chain comprises at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91% of SEQ ID NO: 75 or at least 80% thereof , at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity of the amino acid sequence, said light chain comprising such as SEQ ID NO: 76 Indicated or associated with at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% Amino acid sequences having %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the PD-1 binding protein is an anti-PD-1 antibody: Camrelizumab;
  • the Camrelizumab antibody has the CDRs shown below:
  • HCDR1 is SYMMS (SEQ ID NO: 67)
  • HCDR2 is TISGGGANTYYPDSVKG (SEQ ID NO: 68)
  • HCDR3 is QLYYFDY (SEQ ID NO: 69)
  • LCDR1 is LASQTIGTWLT (SEQ ID NO: 70)
  • LCDR2 is TATSLAD (SEQ ID NO: 71)
  • LCDR3 is QQVYSIPWT (SEQ ID NO: 72)
  • the underlined part is the CDR region, and the sequence of each sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the aforementioned PD-1 binding protein comprises or is an anti-PD-1 antibody or an antigen-binding fragment thereof, including but not limited to sintilimab, cemiplimab, JS-001, nivolumab, tislelizumab, pembrolizumab, AK-103, dostarlimab, PD1 -PIK, GLS-010, genolimzumab, BI-754091, spartalizumab, MGA-012, PF-06801591, XmAb-20717, CS-1003, Sym-021, AGEN-2034, MEDI-5752, MGD-013, AK-105 , AK-104, BCD-100, PF-06753512, HLX-10, AMP-224, LZM-009.
  • the aforementioned PD-L1 binding protein (eg, comprising or being an anti-PD-L1 antibody or an antigen-binding fragment thereof) comprises
  • HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO:48, 49 and 50 respectively, and LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NO:51, 52 and 53 respectively.
  • the aforementioned PD-L1 binding protein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
  • the heavy chain variable region comprises at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, as shown in SEQ ID NO: 46 or at least 80% thereof, An amino acid sequence of at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, said light chain variable region comprising At least 80% as shown in or related to SEQ ID NO: 47, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, Amino acid sequences having at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the aforementioned PD-L1 binding protein further comprises a human immunoglobulin Fc region
  • the Fc region is the Fc region of human IgG1 or IgG4;
  • the Fc region of the human IgG4 has S228P, F234A, L235A and/or K447A mutations.
  • the aforementioned PD-L1 binding protein comprises a heavy chain (HC) and a light chain (LC), wherein:
  • the heavy chain comprises at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91% of SEQ ID NO: 54 or at least 80% thereof , at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity of the amino acid sequence, said light chain comprising such as SEQ ID NO: 55 Indicated or associated with at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% Amino acid sequences having %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the aforementioned PD-L1 binding protein is an anti-PD-L1 antibody or an antigen-binding fragment thereof, including but not limited to avelumab, atezolizumab, durvalumab, CS-1001, M-7824, KL-A167, CX-072, BGB -A333, GNS-1480, CA-170, BMS-936559.
  • the anti-PD-L1 antibodies in WO2017084495 and WO2020094122 are incorporated herein in full.
  • the aforementioned PD-L1 binding protein is a fusion protein, and the fusion protein comprises an anti-PD-1 antibody or an antigen-binding fragment thereof and TGF- ⁇ trap or TGF- ⁇ RII ECD; in some specific embodiments, the TGF- The amino acid sequence of ⁇ RII ECD is shown in SEQ ID NO:56.
  • the TGF- ⁇ RII ECD is connected to the C-terminus of the full-length heavy chain through a linker, and the linker includes or is an amino acid sequence as shown in (G 4 S) x , wherein , x is an integer independently selected from 1-20; in some specific embodiments, the linker is (G 4 S) 4 G, (G 4 S) 3 G, (G 4 S) 2 G, (G 4 S) G.
  • the aforementioned fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein:
  • the first polypeptide chain comprises at least 80%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% of SEQ ID NO: 77 or 78 or at least 80% thereof. %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity of the amino acid sequence, the second polypeptide
  • the chain comprises at least 80% as shown in or with SEQ ID NO: 55, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92% Amino acid sequences having %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the aforementioned PD-L1 binding protein comprises two identical first polypeptide chains and two identical second polypeptide chains.
  • the aforementioned PD-L1 binding protein comprises or is a fusion protein of an anti-PD-L1 antibody or its antigen-binding fragment and TGF- ⁇ trap, including but not limited to M7824, GS-19, TS-1905, TST-005, HBM-7015, PM-8001, anti-PDL1-TGF ⁇ RIIecd.
  • the fusion of the anti-PD-L1 antibody or its antigen-binding protein fragment and TGF ⁇ RIIecd or TGF- ⁇ trap in WO2011109789, WO2015118175, WO2018218215, WO2020248926, WO2019222252, WO2021007428, WO2021063352, WO2021063350 is incorporated herein in its entirety.
  • the present disclosure provides a combination of a PVRIG binding protein and a TIGIT binding protein in the preparation of a drug for treating cancer.
  • the PVRIG binding protein comprises a single variable immunoglobulin as shown in SEQ ID NO: 16 domain
  • the TIGIT binding protein comprises HC shown in SEQ ID NO: 26 and LC shown in SEQ ID NO: 27.
  • the present disclosure provides a combination of PVRIG binding protein, TIGIT binding protein and immune checkpoint inhibitor in the preparation of a drug for treating cancer.
  • the PVRIG binding protein comprises SEQ ID NO: 16 an immunoglobulin single variable domain
  • the TIGIT binding protein comprises HC as shown in SEQ ID NO: 26 and LC as shown in SEQ ID NO: 27
  • the immune checkpoint inhibitor is selected from:
  • PD-1 binding protein which comprises an HC with an amino acid sequence as shown in SEQ ID NO: 65 and an LC with an amino acid sequence as shown in SEQ ID NO: 66;
  • PD-1 binding protein which comprises an HC with an amino acid sequence as shown in SEQ ID NO: 75 and an LC with an amino acid sequence as shown in SEQ ID NO: 76;
  • a PD-L1 binding protein comprising an HC with an amino acid sequence as shown in SEQ ID NO: 54 and an LC with an amino acid sequence as shown in SEQ ID NO: 55; or
  • a PD-L1 binding protein which comprises an HC with an amino acid sequence as shown in SEQ ID NO: 77 and an LC with an amino acid sequence as shown in SEQ ID NO: 55.
  • the present disclosure also provides a combination of a PVRIG/TIGIT binding protein and an immune checkpoint inhibitor in the preparation of a drug for treating cancer.
  • the PVRIG/TIGIT binding protein comprises as shown in SEQ ID NO: 43 HC (i.e. the first polypeptide chain) and LC (i.e. the second polypeptide chain) shown in SEQ ID NO: 27, and the immune checkpoint inhibitor is selected from:
  • a PD-1 binding protein comprising HC as shown in SEQ ID NO: 65 and LC as shown in SEQ ID NO: 66;
  • a PD-1 binding protein comprising HC as shown in SEQ ID NO: 75 and LC as shown in SEQ ID NO: 76;
  • a PD-L1 binding protein comprising HC as set forth in SEQ ID NO:54 and LC as set forth in SEQ ID NO:55; or
  • a PD-L1 binding protein comprising HC as shown in SEQ ID NO:77 and LC as shown in SEQ ID NO:55.
  • the cancer or tumor is selected from the following or a combination thereof: prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple-negative breast cancer, pancreatic cancer, gastric ( gastric/gastric) cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), glioma, renal carcinoma (RCC), lymphoma (NHL or HL), acute myelogenous leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma tumors, testicular germ cell tumors, mesothelioma, esophageal
  • the cancer or tumor is selected from the following cancers or combinations thereof: melanoma, triple negative breast cancer, gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), Merkel cell carcinoma, high MSI cancer, KRAS-mutated tumors, adult T-cell leukemia/lymphoma, and myelodysplastic syndrome (MDS).
  • the cancer or tumor is selected from the following cancers or combinations thereof: melanoma, triple-negative breast cancer, gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), Merkel cell carcinoma and high MSI cancer.
  • the cancer or tumor is selected from melanoma.
  • immune checkpoint inhibitors e.g., PD-1/PD-L1 inhibitors
  • the combined administration route is selected from oral administration, parenteral administration, and transdermal administration, and the parenteral administration includes but not limited to intravenous injection, subcutaneous injection, and intramuscular injection.
  • the present disclosure also provides a drug kit, or a drug packaging box, which contains the aforementioned PVRIG binding protein and TIGIT binding protein, or PVRIG binding protein, TIGIT binding protein and immune checkpoint inhibitor, or PVRIG/TIGIT combination Proteins or polynucleotides encoding them and immune checkpoint inhibitors (eg, PD-1/PD-L1 inhibitors).
  • a drug kit or a drug packaging box, which contains the aforementioned PVRIG binding protein and TIGIT binding protein, or PVRIG binding protein, TIGIT binding protein and immune checkpoint inhibitor, or PVRIG/TIGIT combination Proteins or polynucleotides encoding them and immune checkpoint inhibitors (eg, PD-1/PD-L1 inhibitors).
  • the present disclosure also provides a pharmaceutical composition, comprising the aforementioned effective amount of PVRIG-binding protein and TIGIT-binding protein, or PVRIG-binding protein, TIGIT-binding protein and immune checkpoint inhibitor, or PVRIG/TIGIT-binding protein or its encoded multinuclear glycosides and immune checkpoint inhibitors, and one or more pharmaceutically acceptable excipients, diluents or carriers.
  • the immune checkpoint inhibitors include but are not limited to targeting PD-1/PD-L1, PD-L2, B7-1/CTLA-4, B7-2/CTLA-4, B7-1/CD28, B7-2/CD28, B7-H2/ICOS, B7-H3, B7-H4, B7-H7, VISTA (PD-1H), BTNL2, TIM3, LAG3, TNFSF/TNFRSF (such as CD40-CD40L, CD27-CD70 or OX40-OX40L), leukocyte immunoglobulin-like receptor (LIR/LILR) or immunoglobulin-like transcript (ILT), cohesin or cohesin-like receptor protein (such as DNAM-1, CD226, PTA1, TLISA1, CD96 , TIGIT, Vstm3 and VSIG9), NKG2A, KIRs, NKG2A, Siglecs family (Siglec-7/9), CD47, CD94, CD200 signaling pathway inhibitors; more
  • the present disclosure provides a product comprising the aforementioned effective amount of PVRIG binding protein and TIGIT binding protein, or PVRIG binding protein, TIGIT binding protein and immune checkpoint inhibitor, or PVRIG/TIGIT binding protein and immune checkpoint inhibitor, or PVRIG-binding proteins and immune checkpoint inhibitors.
  • the present disclosure also provides a method for treating or alleviating cancer, comprising administering a treatment or alleviating effective amount of PVRIG-binding protein and TIGIT-binding protein, or PVRIG-binding protein, TIGIT-binding protein and immune A checkpoint inhibitor, or a PVRIG/TIGIT binding protein or polynucleotide encoding it and an immune checkpoint inhibitor.
  • the present disclosure provides a combination of PVRIG binding protein and TIGIT binding protein, or PVRIG binding protein, TIGIT binding protein and immune checkpoint inhibitor, or PVRIG/TIGIT combination, to a subject in need thereof.
  • the present disclosure provides a method for the aforementioned PVRIG/TIGIT-binding protein, or the aforementioned PVRIG-binding protein and TIGIT-binding protein, for treating or alleviating cancer, or the aforementioned PVRIG-binding protein, comprising administering an immune checkpoint inhibitor.
  • the present disclosure provides a method for treating or alleviating cancer with an immune checkpoint inhibitor, comprising administering the aforementioned PVRIG/TIGIT-binding protein, or administering the aforementioned PVRIG-binding protein and TIGIT-binding protein, or the aforementioned PVRIG-binding protein.
  • the present disclosure also provides a method for activating NK cells, ⁇ T cells and/or Th1 cells, comprising administering an effective amount of the foregoing pharmaceutical composition or the foregoing product to a subject in need.
  • the present disclosure also provides a method for increasing IFN- ⁇ production and/or secretion of pro-inflammatory cytokines in a subject, comprising administering an effective amount of the aforementioned pharmaceutical composition or the aforementioned product to a subject in need.
  • the present disclosure also provides a method for inhibiting PD-1 activity or promoting T cell proliferation or enabling a subject to benefit from immune response upregulation, comprising administering to the subject an effective amount of the aforementioned pharmaceutical composition or the aforementioned product; for example, the The expression of PD-L1 and/or PD-L2 in the subject is up-regulated; for another example, the subject suffers from cancer.
  • the aforementioned PVRIG/TIGIT binding protein and the aforementioned immune checkpoint inhibitor have a synergistic effect of inhibiting the growth of cancer cells, and can significantly improve the tumor suppressing effect of the immune checkpoint inhibitor compared with monotherapy.
  • the present disclosure incorporates the bispecific antibody structure and its preparation method in PCT/CN2021/080470 in its entirety, especially related to 1708-30H2. Additionally, the present disclosure provides polynucleotides and vectors:
  • the present disclosure provides polynucleotides encoding PVRIG binding proteins, PVRIG/TIGIT binding proteins, immune checkpoint inhibitors (eg, PD-1 binding proteins, PD-L1 binding proteins) of the present disclosure.
  • a polynucleotide of the present disclosure may be RNA, DNA or cDNA.
  • the polynucleotides of the present disclosure are substantially isolated polynucleotides.
  • a polynucleotide of the present disclosure may also be in the form of, may be present in, and/or may be part of a vector, such as a plasmid, cosmid, YAC, or viral vector.
  • the vector may especially be an expression vector, ie a vector providing for in vitro and/or in vivo (ie in a suitable host cell, host organism and/or expression system) expression of the PD-1 binding protein.
  • the expression vector typically comprises at least one polynucleotide of the present disclosure operably linked to one or more suitable expression control elements (eg, promoters, enhancers, terminators, etc.). The selection of said elements and their sequences for expression in a particular host is within the general knowledge of the person skilled in the art.
  • Regulatory elements and other elements useful or necessary for the expression of PVRIG binding proteins, PVRIG/TIGIT binding proteins, immune checkpoint inhibitors (e.g., PD-1 binding proteins, PD-L1 binding proteins) of the present disclosure are, for example, promoters, enhancers, promoters, terminators, integrators, selectable markers, leader sequences, reporter genes.
  • polynucleotides of the present disclosure may be prepared or obtained by known means (e.g., by automated DNA synthesis and/or recombinant DNA techniques) based on information on the amino acid sequence of the polypeptides of the present disclosure, and/or may be isolated from suitable natural sources . Additionally, the present disclosure provides host cells:
  • the present disclosure provides expressing or capable of expressing one or more PVRIG binding proteins of the present disclosure, PVRIG/TIGIT binding proteins, immune checkpoint inhibitors (e.g., PD-1 binding proteins, PD-L1 binding proteins) and/or containing the present disclosure
  • Recombinant host cells of the disclosed nucleic acids or vectors In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.
  • Bacterial cells include, for example, Gram-negative bacterial strains (such as Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus spp. (Bacillus) strains, Streptomyces (Streptomyces) strains, Staphylococcus (Staphylococcus) strains and Lactococcus (Lactococcus) strains) cells.
  • Gram-negative bacterial strains such as Escherichia coli strains, Proteus strains, and Pseudomonas strains
  • Gram-positive bacterial strains such as Bacillus spp. (Bacillus) strains, Streptomyces (Streptomyces) strains, Staphylococcus (Staphylococcus) strains and Lactococcus (Lactococcus) strains
  • Fungal cells include, for example, cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or Saccharomyces (such as Saccharomyces cerevisiae), fission yeast Schizosaccharomyces (such as Schizosaccharomyces pombe), Pichia (such as Pichia pastoris and Pichia methanolica) and Hansen A cell of a species of Saccharomyces (Hansenula).
  • Saccharomyces such as Saccharomyces cerevisiae
  • fission yeast Schizosaccharomyces such as Schizosaccharomyces pombe
  • Pichia such as Pichia pastoris and Pichia methanolica
  • Hansen A cell of a species of Saccharomyces Hansenula
  • Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.
  • the present disclosure can also be used with amphibian cells, insect cells, plant cells, and any other cell used in the art to express heterologous proteins.
  • the cells of the present disclosure do not develop into finished plants or individual animals.
  • the present disclosure provides production and preparation methods:
  • the present disclosure provides a method for preparing the PVRIG binding protein, PVRIG/TIGIT binding protein, and immune checkpoint inhibitor (for example, PD-1 binding protein, PD-L1 binding protein) target protein method of the present disclosure, and the method generally includes the following steps:
  • PVRIG-binding proteins, PVRIG/TIGIT-binding proteins, immune checkpoint inhibitors (e.g., PD-1-binding proteins, PD-L1-binding proteins) of the present disclosure can be expressed intracellularly (e.g., in the cytoplasm, Produced in the periplasm or in inclusion bodies), followed by isolation from the host cell and optionally further purified; or it can be produced extracellularly (for example, in the culture medium in which the host cell is grown), followed by isolation from the medium and optionally further purification .
  • polypeptides such as specific suitable expression vectors, transformation or transfection methods, selectable markers, methods of inducing protein expression, culture conditions, etc.
  • isolation and purification techniques suitable for producing the binding molecules or antibodies of the present disclosure, such as proteins of interest are well known to those skilled in the art.
  • Methods for producing and purifying antibodies are well known and can be found in the art, eg, Cold Spring Harbor's Antibody Laboratory Technique Guide (chapters 5-8 and 15).
  • Antibodies engineered in the present disclosure can also be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors.
  • Recombinant immunoglobulin expression vectors can stably transfect cells.
  • Mammalian-like expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free medium in bioreactors for antibody production.
  • the culture fluid that secretes the antibody can be purified and collected using conventional techniques.
  • Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and aggregates can also be removed by conventional methods such as molecular sieves and ion exchange.
  • the obtained product needs to be immediately frozen, such as -70°C, or freeze-dried.
  • PVRIG binding proteins, PVRIG/TIGIT binding proteins, immune checkpoint inhibitors (e.g., PD-1 binding proteins, PD-L1 binding proteins) of the present disclosure can also be obtained by other protein production methods known in the art, such as Chemical synthesis, including solid-phase or liquid-phase synthesis. Additionally, the present disclosure provides compositions or combinations of:
  • compositions or combinations comprising:
  • PVRIG/TIGIT binding proteins and immune checkpoint inhibitors eg, PD-1 binding protein or PD-L1 binding protein
  • immune checkpoint inhibitors eg, PD-1 binding protein or PD-L1 binding protein
  • PVRIG binding protein and immune checkpoint inhibitor eg, PD-1 binding protein or PD-L1 binding protein.
  • a pharmaceutical composition which contains an effective amount of the above-mentioned composition or combination for cancer treatment, alleviation or prevention, and at least one pharmaceutically acceptable excipient, diluent or carrier.
  • the unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of the aforementioned PVRIG/TIGIT binding protein and immune checkpoint inhibitor (for example, PD-1 binding protein or PD-L1 binding protein) , or the amount of the aforementioned PVRIG/TIGIT binding protein and immune checkpoint inhibitor (for example, PD-1 binding protein or PD-L1 binding protein) in the unit dose of the pharmaceutical composition is 0.1-2000mg, in some specific embodiments is 1-1000mg.
  • an article or product comprising a composition or combination.
  • the article comprises a container and a label.
  • Containers such as bottles, syringes and test tubes.
  • the container contains a composition or combination effective for treating a condition.
  • the label on or associated with the container indicates that the composition is used to treat the condition of choice.
  • compositions or combinations are provided, which contain:
  • the above-mentioned PVRIG/TIGIT binding protein comprises a first antigen-binding domain specifically binding to PVRIG and a second antigen-binding domain specifically binding to TIGIT, wherein:
  • the first antigen-binding domain that specifically binds to PVRIG comprises an immunoglobulin single variable domain, and the immunoglobulin single variable domain comprises: CDR1 shown in SEQ ID NO: 4, 5, 20, CDR2 and CDR3;
  • amino acid sequence as shown in any one of SEQ ID NO: 2, 15-19 or having at least 90% sequence identity thereto;
  • the second antigen-binding domain that specifically binds TIGIT comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the heavy chain variable region comprises as shown in SEQ ID NO: 38 or with An amino acid sequence having at least 90% sequence identity, said light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 42 or having at least 90% sequence identity thereto.
  • the above-mentioned PVRIG/TIGIT binding protein comprises a first polypeptide chain and a second polypeptide chain, wherein:
  • the first polypeptide chain comprises an amino acid sequence as shown in any one of SEQ ID NO: 28-29, 43-45, and the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 27; or
  • the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 26 and the second polypeptide chain comprises the amino acid sequences shown in SEQ ID NO: 30, 31.
  • the above-mentioned PD-1 binding protein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequence shown in SEQ ID NO: 59, 60, 61, and the light chain variable region comprises amino acids shown in SEQ ID NO: 62, 63, 64 Serial LCDR1, LCDR2 and LCDR3;
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequence shown in SEQ ID NO: 67, 68, 69, and the light chain variable region comprises amino acids shown in SEQ ID NO: 70, 71, 72 Serial LCDR1, LCDR2 and LCDR3;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 57 or at least 90% identical thereto, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 58 or at least 90% identical thereto identical amino acid sequences; or
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 73 or at least 90% identical thereto, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 74 or at least 90% identical thereto identical amino acid sequences.
  • the above-mentioned PD-1 binding protein comprises full-length heavy chain (HC) and full-length light chain (LC), wherein:
  • the full length of the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 65 or has at least 90% identity therewith
  • the full length of the light chain comprises the amino acid sequence shown in SEQ ID NO: 66 or has at least 90% identity therewith the amino acid sequence of; or
  • the full length of the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 75 or has at least 90% identity therewith
  • the full length of the light chain comprises the amino acid sequence shown in SEQ ID NO: 76 or has at least 90% identity therewith amino acid sequence.
  • the above PD-L1 binding protein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequence shown in SEQ ID NO: 48, 49, 50, and the light chain variable region comprises amino acids shown in SEQ ID NO: 51, 52, 53 Serial LCDR1, LCDR2 and LCDR3;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 46 or at least 90% identical thereto, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 47 or at least 90% identical thereto identical amino acid sequences.
  • the above-mentioned PD-L1 binding protein includes full-length heavy chain (HC) and full-length light chain (LC), wherein:
  • the full length of the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 54 or has at least 80% identity therewith
  • the full length of the light chain comprises the amino acid sequence shown in SEQ ID NO: 55 or has at least 80% identity therewith amino acid sequence.
  • the above PD-L1 binding protein also includes a TGF- ⁇ RII ECD domain, for example, the amino acid sequence of the TGF- ⁇ RII ECD domain is shown in SEQ ID NO:56.
  • the aforementioned PD-L1 binding protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising an amino acid sequence as shown in SEQ ID NO: 77 or 78 or having at least 90% identity thereto,
  • the second polypeptide chain comprises an amino acid sequence as set forth in, or at least 90% identical to, SEQ ID NO:55.
  • the present disclosure also provides the above-mentioned composition or combination therapy, a method for relieving the aforementioned tumor or a pharmaceutical use, a method for activating NK cells, ⁇ T cells and/or Th1 cells or a pharmaceutical use, increasing the production of IFN- ⁇ in a subject and/or A method or use of pro-inflammatory cytokine secretion, a method or use of inhibiting PD-1 activity or promoting T cell proliferation, or benefiting a subject from upregulation of an immune response, said subject's PD-L1 and/or PD The expression of -L2 may be upregulated.
  • At least 90% (sequence) identity encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99% or 100% (sequence) identity;
  • at least 80% (sequence) identity encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86 %, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, At least 99% or 100% (sequence) identity.
  • Figure 1 shows the mixed lymphocyte reaction (MLR) experimental results of anti-PVRIG nanobodies 30 and 151, using Tab5 as a positive control and hIgG4 as a negative control.
  • MLR mixed lymphocyte reaction
  • Figure 2 shows the results of the MLR experiment of the anti-PVRIG/TIGIT double antibody 1708-151H8, and simultaneously detected 151H8, 1708, hIgG4, Tab5, and Pembrolizumab as controls.
  • Figure 3A is the body weight diagram of the mice combined with anti-PVRIG/TIGIT double antibodies 1708-151, 1708 and 151 in the human melanoma A375 mixed with human PBMC subcutaneous transplanted tumor model in mice
  • Figure 3B is the corresponding mouse tumor volume picture.
  • Figure 4A is a diagram of mouse body weights of anti-PVRIG/TIGIT double antibodies 1708-151H7 and 1708-30H2 in a mouse subcutaneous xenograft tumor model of human melanoma A375 mixed with human PBMC
  • Figure 4B is a diagram of corresponding mouse tumor volume.
  • Figure 5A is a graph showing the effect of drugs A and B alone or in combination on the tumor volume of the human melanoma A375 mixed PBMC subcutaneous xenograft tumor model
  • Figure 5B is the corresponding tumor weight graph
  • Figure 5C is the corresponding mouse body weight graph
  • Fig. 5D is a graph of the relative change in body weight of the corresponding mice.
  • Figure 6A is a graph showing the effect of drugs A and C alone or in combination on the tumor volume of the human melanoma A375 mixed PBMC subcutaneous xenograft tumor model
  • Figure 6B is the corresponding tumor weight graph
  • Figure 6C is the corresponding mouse body weight graph
  • Fig. 6D is a graph of the relative change in body weight of the corresponding mice.
  • Figure 7A is a graph showing the effect of drugs A and D alone or in combination on the tumor volume of the human melanoma A375 mixed PBMC subcutaneous xenograft model
  • Figure 7B is the corresponding tumor weight graph
  • Figure 7C is the corresponding mouse body weight graph.
  • Programmed death 1 "Programmed death 1", “programmed cell death 1”, “protein PD-1”, “PD-1”, “PDCD1” and “hPD-1” are used interchangeably and include variants of human PD-1 Individuals, isotypes, species homologues, and analogs that share at least one common epitope with PD-1.
  • the complete PD-1 sequence can be found from GenBank accession number U64863.
  • “Programmed death ligand-1 (PD-L1)” is one of two cell surface glycoprotein ligands of PD-1 (the other being PD-L2), which downregulates T cells when bound to PD-1 Activation and cytokine secretion.
  • PD-L1 as used herein includes human PD-L1 (hPD-L1), variants, isoforms, and interspecies homologs of hPD-L1, and hPD-L1 having at least one common epitope analogues of .
  • the complete hPD-L1 sequence can be found with GenBank accession number Q9NZQ7.
  • PVRIG or “PVRIG protein” or “PVRIG polypeptide” may optionally include any such protein or variant, conjugate or fragment thereof, including but not limited to known or wild-type PVRIG as described herein, As well as any naturally occurring splice variants, amino acid variants or isoforms, and especially soluble extracellular domain (ECD) fragments of PVRIG.
  • ECD extracellular domain
  • TIGIT or "TIGIT protein” or “TIGIT polypeptide” may optionally include any such protein or variant, conjugate or fragment thereof, including but not limited to known or wild-type TIGIT as described herein, and any naturally occurring splice variants, amino acid variants or isoforms.
  • the complete TIGIT sequence can be found at GenBank accession number AAI01289.1.
  • Binding to PVRIG refers to being capable of interacting with PVRIG or its epitope, which may be of human origin.
  • Binding to TIGIT refers to being capable of interacting with TIGIT or its epitope, which may be of human origin.
  • Antigen-binding site refers to a discrete three-dimensional site on an antigen recognized by an antibody or antigen-binding fragment of the present disclosure.
  • Immuno checkpoint molecules include stimulatory and inhibitory immune checkpoint molecules, exemplary molecules include CD27, CD28, CD40, CD40L, CD122, OX40, OX40L, GITR, ICOS, A2AR, B7 -H3, B7-H4, B7-H7, BTLA, CTLA-4, IDO, KIR (Killer-cell Immunoglobulin-like Receptor), LAG3, PD-1, PD-L1, PD-L2, TIM-3, VISTA, TIGIT, NKG2A, etc.
  • Antibody is used in the broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (such as bispecific antibodies), full-length antibodies, and antibody fragments ( or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.
  • Antibody can refer to immunoglobulin, which is a tetrapeptide chain structure formed by two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five classes, or isotypes, of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are mu, delta, and gamma chains, respectively , ⁇ chain and ⁇ chain.
  • the same class of Ig can be divided into different subclasses according to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either kappa chains or lambda chains by difference in the constant region.
  • Each of the five Ig classes can have either a kappa chain or a lambda chain.
  • the sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region (C region).
  • the variable region includes 3 hypervariable regions (CDRs) and 4 framework regions (FRs) with relatively conserved sequences. Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDR).
  • Each light chain variable region (VL) and heavy chain variable region (VH) are composed of 3 CDR regions and 4 FR regions, and the sequence from the amino terminal to the carboxyl terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • Antigen-binding fragment encompasses single chain antibodies (i.e. full length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv , single domain antibody (e.g. VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibody, Bis-scFv, diabody, tribody, triabody, tetrabody and epitope binding fragments of any of the above (see e.g. Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217).
  • Antigen-binding fragments of the present disclosure also include Fab and Fab' fragments described in WO2005/003169, WO2005/003170 and WO2005/003171.
  • Multivalent antibodies may comprise multiple specificities such as bispecifics or may be monospecific (see e.g. WO 92/22583 and WO 05/113605), an example of the latter being the Tri-Fab described in WO 92/22583 (or TFM).
  • Bispecific antibody encompasses antibodies (including antibodies or antigen-binding fragments thereof such as single chain antibodies) that specifically bind to two different antigens or at least two different epitopes of the same antigen.
  • Bispecific antibodies of various structures have been disclosed in the prior art, which can be divided into IgG-like bispecific antibodies and antibody fragment bispecific antibodies according to the integrity of the IgG molecule, and can be divided into bivalent bispecific antibodies according to the number of antigen-binding regions.
  • trivalent, tetravalent or more valent bispecific antibodies according to whether the structure is symmetrical, can be divided into symmetrical structure bispecific antibodies and asymmetric structure bispecific antibodies.
  • bispecific antibodies based on antibody fragments such as Fab fragments lacking Fc fragments, which form bispecific antibodies by combining two or more Fab fragments in one molecule, which have lower immunogenicity, and Small molecular weight and high tumor tissue permeability
  • typical antibody structures of this type include F(ab)2, scFv-Fab, (scFv)2-Fab and other bispecific antibodies; IgG-like bispecific antibodies (such as Fc fragment), this type of antibody has a relatively large molecular weight, and the Fc fragment is helpful for the purification of the antibody in the later stage, and improves its solubility and stability.
  • the Fc part may also bind to the receptor FcRn to increase the serum half-life of the antibody.
  • Bispecific antibody structure models such as KiH, CrossMAb, Triomab quadroma, Fc ⁇ Adp, 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, F(ab)4-CrossMAb and other bispecific antibodies (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585–608 (2019); Chen S1 et al., J Immuno
  • antibodies of the present disclosure generally use the Kabat numbering system. EU numbering in Kabat is generally also used for constant domains and/or Fc domains.
  • deterministic delineation of the CDRs and identification of the residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and/or resolving the structure of the antibody-ligand complex. This can be achieved by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography.
  • Various analytical methods can be used to identify CDRs, including but not limited to Kabat numbering system, Chothia numbering system, AbM numbering system, IMGT numbering system, contact definition, conformation definition.
  • the Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see eg Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8).
  • the Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the location of certain structural ring regions. (See eg Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83).
  • the AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group for modeling antibody structures (see, e.g., 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 uses a combination of knowledge base and ab initio methods to model the tertiary structure of antibodies from basic sequences (see Samudrala et al., 1999, "AbM” in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198. Initio Protein Structure Prediction Using a Combined Hierarchical Approach” described).
  • a CDR may refer to a CDR defined by any method known in the art, including combinations of methods. The correspondence between various numbering systems is well known to those skilled in the art, and is exemplary, as shown in the following table.
  • the CDR amino acid residues of the VL and VH regions of the disclosed antibodies conform to the known Kabat numbering system in number and position.
  • Antibodies of the present disclosure may be polyclonal, monoclonal, xenogeneic, allogeneic, isogenic, or modified forms thereof, with monoclonal antibodies being particularly useful in various embodiments.
  • antibodies of the present disclosure are recombinant antibodies.
  • “recombinant” generally refers to products such as cells or nucleic acids, proteins or vectors, and means that the cells, nucleic acids, proteins or vectors have been modified by introducing heterologous nucleic acids or proteins or changing natural nucleic acids or proteins, or that the Said cells are derived from cells so modified.
  • a recombinant cell expresses a gene that is not present in the native (non-recombinant) form of the cell or expresses a native gene that would otherwise be aberrantly expressed, underexpressed, or not expressed at all.
  • a “domain” of a polypeptide or protein refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein.
  • a domain is responsible for a single functional property of a protein, and in many cases can be added, removed or transferred to other proteins without loss of the rest of the protein and/or the function of the domain.
  • Immunoglobulin domain refers to a globular region of an antibody chain (eg, a chain of a conventional tetrapeptide chain antibody or a chain of a heavy chain antibody), or a polypeptide consisting essentially of such a globular region. Immunoglobulin domains are characterized by their maintenance of the immunoglobulin fold characteristic of antibody molecules, consisting of a 2-layer sandwich of approximately seven antiparallel beta-sheet strands, optionally stabilized by conserved disulfide bonds, arranged in two beta-sheets .
  • Immunoglobulin variable domain refers to the term “framework region 1" or “FR1”, “framework region 2” or “FR2”, “framework region 3” or “FR3” essentially defined in the art and hereinafter as “FR1” or “FR1”, respectively. , and four “framework regions” of “framework region 4" or “FR4", wherein the framework regions are respectively referred to in the art and hereinafter as “complementarity determining region 1" or "CDR1", The three “complementarity determining regions” or “CDRs” of “complementarity determining region 2" or “CDR2”, and “complementarity determining region 3" or “CDR3” are spaced apart.
  • an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains are endowed with antigen-specificity by having an antigen-binding site.
  • Immunoglobulin variable domain refers to the term “framework region 1" or “FR1”, “framework region 2” or “FR2”, “framework region 3” or “FR3” essentially defined in the art and hereinafter as “FR1” or “FR1”, respectively. , and four “framework regions” of “framework region 4" or “FR4", wherein the framework regions are respectively referred to in the art and hereinafter as “complementarity determining region 1" or "CDR1", The three “complementarity determining regions” or “CDRs” of “complementarity determining region 2" or “CDR2”, and “complementarity determining region 3" or “CDR3” are spaced apart.
  • an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains are endowed with antigen-specificity by having an antigen-binding site.
  • Antibody framework (FR) refers to the portion of a variable domain that serves as a scaffold for the antigen-binding loops (CDRs) of the variable domain.
  • Immunoglobulin single variable domain is commonly used to refer to a variable domain that can be used without interaction with other variable domains (e.g. without the need for between the VH and VL domains of a conventional four-chain monoclonal antibody).
  • an immunoglobulin variable domain (which may be a heavy or light chain domain, including a VH, VHH or VL domain) that forms a functional antigen binding site.
  • immunoglobulin single variable domains include Nanobodies (including VHH, humanized VHH and/or camelized VH, such as camelized human VH), IgNAR, domains, as VH domains or derived from VH Domain (single domain) antibodies such as dAbsTM and (single domain) antibodies that are or are derived from VL domains such as dAbsTM.
  • Immunoglobulin single variable domains based on and/or derived from heavy chain variable domains, such as VH or VHH domains are generally preferred.
  • a specific example of an immunoglobulin single variable domain is a "VHH domain” (or simply "VHH") as defined below.
  • VHH domains also known as heavy chain single domain antibodies, VHH, VHH domains, VHH antibody fragments, VHH antibodies, nanobodies, are the subtype of antibodies known as “heavy chain antibodies” (i.e., “antibodies lacking light chains")
  • Antigen-binding variable domains of immunoglobulins Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature363, 446-448 (1993)).
  • VHH domain is used to distinguish the variable domain from the variable domain of the heavy chain (which is referred to in this disclosure as the "VH domain”) and the variable domain of the light chain present in antibodies of conventional tetrapeptide chain structure.
  • variable domains (which are referred to as “VL domains” in this disclosure).
  • the VHH domain specifically binds the epitope without the need for additional antigen-binding domains (in contrast to the VH or VL domains in conventional tetrapeptide chain antibodies, in which case the epitope is recognized by the VL domain together with the VH domain) .
  • the VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain.
  • VHH domain includes, but is not limited to, natural antibodies produced by camelids, humanized antibodies produced by camelids, or obtained by screening with phage display technology.
  • the total number of amino acid residues in the VHH domain will generally range from 110 to 120, often between 112 and 115. It should be noted, however, that smaller and longer sequences may also be suitable for the purposes described in this disclosure.
  • the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e. one according to Kabat numbering). or positions may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering).
  • Kabat numbering i.e. one according to Kabat numbering
  • numbers may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering.
  • numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.
  • Other numbering systems or coding conventions include Chothia, IMGT, AbM.
  • Humanized antibody also known as CDR-grafted antibody (CDR-grafted antibody) refers to an antibody produced by grafting a non-human CDR sequence into a human antibody variable region framework. It can overcome the strong immune response induced by chimeric antibodies due to carrying a large number of non-human protein components. In order to avoid a decrease in activity while reducing immunogenicity, minimal reverse mutations can be performed on the variable region of the fully human antibody to maintain activity.
  • Examples of "humanization” include that the VHH domain derived from Camelidae can be replaced by the amino acid sequence of the original VHH sequence with one or more amino acid residues present in the corresponding position in the VH domain of an antibody with a conventional tetrapeptide chain structure.
  • Humanization also referred to as “sequence optimization” in this disclosure, in addition to humanization, “sequence optimization” can also cover the improvement of properties by providing one or more Mutations are other modifications to the sequence such as removal of potential post-translational modification sites).
  • a humanized VHH domain can contain one or more fully human framework region sequences, and in some embodiments, can contain the human framework region sequences of IGHV3.
  • Another example of "humanization” includes grafting mouse CDR sequences into human antibody variable region frameworks, ie antibodies produced from different types of human germline antibody framework sequences. It can overcome the strong antibody variable antibody response induced by chimeric antibodies due to carrying a large number of mouse protein components.
  • Humanization methods such as protein surface amino acid humanization (resurfacing) and antibody humanization universal framework grafting method (CDR grafting to a universal framework), that is, CDR "grafting” to other "scaffolds” (including but not limited to human scaffolds or non-immunoglobulin scaffold).
  • CDR grafting to a universal framework that is, CDR "grafting” to other "scaffolds” (including but not limited to human scaffolds or non-immunoglobulin scaffold).
  • Scaffolds and techniques suitable for such CDR grafting are known in the art.
  • the germline DNA sequences of the human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database, and in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition.
  • the humanized antibody of the present disclosure also includes a humanized antibody that is further subjected to affinity maturation of CDRs by phage display.
  • a humanized antibody that is further subjected to affinity maturation of CDRs by phage display.
  • minimal reverse mutations or back mutations can be performed on the human antibody variable region framework sequence to maintain activity.
  • an “affinity matured” antibody is one that possesses one or more alterations in one or more hypervariable regions (HVRs) that result in the antibody's reactivity to the antigen as compared to a parental antibody that does not possess such alterations. Affinity improved.
  • an "affinity matured" PD-1 binding protein or PD-1 antibody has one or more changes in one or more CDRs that result in a different affinity for the antigen compared to its parental antibody Increase.
  • Affinity matured antibodies can be prepared by methods known in the art, for example, from Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc. Nat. Acad.
  • Fully human antibodies include antibodies having variable and constant regions of human germline immunoglobulin sequences. Fully human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). "Fully human antibody” does not include “humanized antibody”.
  • “competition” in the context of antigen binding proteins that compete for the same epitope (such as neutralizing antigen binding proteins or neutralizing antibodies), it means competition between antigen binding proteins, which is determined by the following assay:
  • the detected antigen binding protein eg, antibody or immunologically functional fragment thereof
  • RIA solid-phase direct or indirect radioimmunoassay
  • EIA solid-phase direct or indirect enzyme immunoassay
  • Sandwich competition assay see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253
  • solid-phase direct biotin-avidin EIA see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619
  • solid-phase Phase direct labeling assay solid phase direct labeling sandwich assay
  • Such assays involve the use of purified antigen (on a solid surface or cell surface) capable of binding to an unlabeled test antigen binding protein and a labeled reference antigen binding protein.
  • Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cells in the presence of the antigen binding protein to be tested.
  • the antigen binding protein to be tested is present in excess.
  • Antigen binding proteins identified by competition assays include: antigen binding proteins that bind to the same epitope as the reference antigen binding protein; and, epitopes that bind in sufficient proximity to the reference antigen binding protein.
  • a competing antigen binding protein when present in excess it will inhibit (e.g. reduce) by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70% - specific binding of 75% or 75% or more of the reference antigen binding protein to the common antigen. In certain instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
  • Antibodies can be competitively screened for binding to the same epitope using routine techniques known to those of skill in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete for antigen binding.
  • Specific binding and “selective binding” refer to the binding of an antibody to a predetermined epitope on an antigen.
  • an antibody when used as an analyte and an antibody is used as a ligand, when the surface plasmon resonance (SPR) technique is used in the instrument, the antibody is approximately below 10-7M or even smaller Equilibrium dissociation constant (KD) for binding to a predetermined antigen or its epitope, and its binding affinity to the predetermined antigen or its epitope is non-specific other than that of the predetermined antigen (or its epitope) or closely related antigens Antigen (such as BSA, etc.) at least twice the binding affinity.
  • An "antibody that recognizes an antigen” may be used interchangeably with a “specifically binding antibody” in this disclosure.
  • Binding affinity or “affinity” is used in the present disclosure as a measure of the strength of a non-covalent interaction between two molecules (eg, an antibody or portion thereof and an antigen).
  • the binding affinity between two molecules can be quantified by determining the dissociation constant (KD).
  • KD can be determined by measuring the kinetics of complex formation and dissociation using, for example, the Surface Plasmon Resonance (SPR) method (Biacore).
  • SPR Surface Plasmon Resonance
  • the rate constants corresponding to the association and dissociation of the monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively.
  • the value of the dissociation constant can be determined directly by well known methods and can even be calculated for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9:340-362).
  • KD can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432).
  • Other standard assays to assess the binding ability of an antibody against a target antigen are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays exemplified elsewhere in this disclosure.
  • Binding kinetics and binding affinity of antibodies can also be assessed by standard assays known in the art, such as surface plasmon resonance (SPR), for example by using the BiacoreTM system or KinExA. Binding affinities associated with different molecular interactions can be compared by comparing the KD values of individual antibody/antigen complexes, eg, a comparison of the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the KD value of an interaction of interest (e.g., a specific interaction between an antibody and an antigen) with an unintended interaction (e.g., a control antibody known not to bind PD-1) The KD value is evaluated.
  • SPR surface plasmon resonance
  • a “conservative substitution” refers to a substitution for another amino acid residue that has similar properties to the original amino acid residue.
  • lysine, arginine, and histidine have similar properties in that they have basic side chains
  • aspartic acid and glutamic acid have similar properties in that they have acidic side chains.
  • glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains
  • alanine , valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have nonpolar side chains.
  • “Homology”, “identity” or “sequence identity” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in two compared sequences is occupied by the same nucleotide or amino acid monomer, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position of.
  • the percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of compared positions x 100%. For example, two sequences are 60% homologous if there are 6 matches or homology at 10 positions in the two sequences when the sequences are optimally aligned. In general, comparisons are made when two sequences are aligned to yield the greatest percent homology.
  • Nucleic acid or “polynucleotide” are used interchangeably herein to refer to any molecule of DNA or RNA, single or double stranded and, in the case of single stranded, its complement, preferably double stranded DNA.
  • a nucleic acid is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.
  • a “host cell” includes any cell or cell culture that can be or has been a recipient of a vector for the incorporation of a polynucleotide insert.
  • a host cell includes the progeny of a single host cell, and the progeny may not necessarily be identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental or deliberate mutation.
  • Host cells include cells transfected and/or transformed in vivo with polynucleotides of the present disclosure.
  • Cell “cell line,” and “cell culture” are used interchangeably, and all such designations include progeny. It should also be understood that all progeny may not be precisely identical in DNA content due to deliberate or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cells are included.
  • Inhibition or “blocking” are used interchangeably and encompass both partial and complete inhibition/blocking. “Inhibition of growth” (e.g., in relation to cells) is intended to include any measurable decrease in cell growth.
  • Preventing the growth or “growth inhibiting” means inhibiting the growth or proliferation of cells.
  • Proliferative disease refers to a disorder associated with some degree of abnormal cell proliferation.
  • the proliferative disorder is cancer.
  • Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • Cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • Cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • Preventing cancer means delaying, inhibiting or preventing the onset of cancer in a subject in which the onset of cancer or tumorigenesis has not been proven, but has been determined, for example, by genetic screening or other methods, identified susceptibility to cancer. This also includes treating a subject with a precancerous condition to halt progression of the precancerous condition to a malignancy or to cause regression thereof.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid refers to the interaction of an exogenous drug, therapeutic, diagnostic, or composition with an animal. , humans, subjects, cells, tissues, organs or biological fluids, such as therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of cells includes contacting the reagents with the cells, and contacting the reagents with a fluid, wherein the fluid contacts the cells.
  • administering also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnostic, binding composition or by another cell. When applied to human, veterinary or research subjects, it refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent to a subject, for example, comprising any binding protein of the present disclosure or a pharmaceutical composition thereof as a therapeutic agent, the subject has suffered from, suspected of suffering from, prone to Suffering from one or more proliferative diseases, or symptoms thereof, for which the therapeutic agent is known to have a therapeutic effect.
  • a therapeutic agent is administered in a subject or population to be treated in an amount effective to alleviate one or more symptoms of a disease, either by inducing regression of such symptoms or inhibiting the progression of such symptoms to any clinically measurable extent.
  • the amount of a therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on factors such as the disease state, age, and weight of the subject, and the extent to which the drug produces the desired therapeutic effect in the subject. ability. Whether disease symptoms have been alleviated can be assessed by any of the clinical tests commonly used by a physician or other professional health care practitioner to assess the severity or progression of such symptoms.
  • any statistical test method known in the art such as Student's t-test, chi-square test, according to Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test determined that it should reduce the target disease symptoms in a statistically significant number of subjects.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a subject may vary depending on factors such as the condition being treated, the general health of the subject, the method, route and dose of administration, and the severity of side effects.
  • An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • a subject of the present disclosure can be an animal or a human subject.
  • Subject and “patient” in the present disclosure refer to mammals, especially primates, especially humans.
  • human PVRIG h-PVRIG-his recombinant protein with his tag
  • human PVRIG h-PVRIG-mIgG2a Fc
  • mouse PVRIG m-PVRIG-hIgG1Fc
  • His-tagged PVRL2 was purchased from AcroBiosystem (PV2-H52E2).
  • Example 1-1 Screening and preparation of anti-PVRIG nanobody
  • Example 1-1 the human PVRIG recombinant protein with his tag (h-PVRIG-his) was used to immunize alpacas.
  • PBMCs peripheral blood of camels on day 56, RNA was extracted, reverse transcribed into cDNA, and a phage library of anti-human PVRIG nanobody was constructed. After 3 rounds of screening, 400 clones were sequenced, and the variable region sequences of 2 strains are shown in Table 2, and the CDRs are shown in Table 3.
  • variable region of the above antibody was linked to the Fc region of the heavy chain of human IgG4.
  • the Fc region of the heavy chain includes the hinge region and carries S228P, F234A, L235A, K447A mutations (Eu nomenclature system) to construct a full-length antibody.
  • the anti-PVRIG antibody CPA.7.021 shown in WO2016134333 was screened from an antibody phage library, and its subtype is IgG1, which can bind well to human PVRIG but not to cynomolgus monkey PVRIG.
  • the heavy chain and light chain variable regions of CPA.7.021 were respectively connected with the human IgG4 heavy chain constant region (with S228P, F234A, L235A, K447A mutations) and the human Kappa light chain constant region to construct the positive antibody Tab5.
  • the PVRIG recombinant protein with his tag was directly coated, and after adding the antibody, the activity of the antibody binding to the antigen was detected by adding a secondary antibody (HRP-coupled anti-primary anti-Fc antibody) and HRP substrate TMB.
  • Human, cynomolgus or mouse PVRIG protein was coated on a 96-well plate, 100 ⁇ L per well at a concentration of 1 ⁇ g/mL, and incubated overnight at 4°C. Wash with lotion three times. Add 300 ⁇ L/well blocking solution (PBS+0.05% Tween20+1% BSA) and incubate at room temperature for 1 hour. Wash with lotion three times. Add 100 ⁇ L of anti-PVRIG antibody to be tested diluted with diluent to each well. Incubate at 37°C for 1 hour. Wash with lotion three times. 100 ⁇ L of HRP-labeled anti-human IgG secondary antibody (Sigma, A8667) was added to each well.
  • HEK293 stably transfected cell lines expressing human or cynomolgus PVRIG gene were prepared. Seed 2x105 cells per well in a 96-well plate. Centrifuge at 300g for 5 minutes, remove the supernatant, add 100 ⁇ L of the antibody to be tested, and incubate at 4°C for 1 hour. Remove the supernatant by centrifugation, wash 3 times with 200 ⁇ L washing solution (PBS+2% FBS), add 100 ⁇ L 1:500 diluted anti-human IgG secondary antibody (Invitrogen, A-11013) labeled with Alexa Fluor 488, and incubate at 4 °C for 1 Hour.
  • PBS+2% FBS 200 ⁇ L washing solution
  • Example 1-3 Function and activity verification of anti-PVRIG nanobody
  • Human PVRIG recombinant protein (h-PVRIG-mIgG2a Fc) was coated on a 96-well plate, 100 ⁇ L per well at a concentration of 1 ⁇ g/mL, and incubated overnight at 4°C. Wash with lotion three times. Add 300 ⁇ L/well blocking solution and incubate at room temperature for 1 hour. Wash with lotion three times. Add 50 ⁇ L of the diluted anti-PVRIG antibody to be tested and 50 ⁇ L of his-tagged ligand PVRL2 to each well, and incubate at 37°C for 1 hour. Wash with lotion three times. 100 ⁇ L of HRP-labeled anti-his-tag secondary antibody (Genscrpit) diluted 1:2000 times was added to each well.
  • HRP-labeled anti-his-tag secondary antibody (Genscrpit) diluted 1:2000 times was added to each well.
  • plvx-OS8 G418 resistance
  • transfect 293F cells G418 screening
  • flow cytometry to detect the expression of OS8 in the cloned cells and detect the activation of OS8 to Jurkat cells, select the clone with a moderate degree of activation, and obtain 293F - OS8 cell line
  • plvx-PVRL2 plasmid use it to infect 293F-OS8 cells, and screen out the clone with the highest PVRL2 expression by flow cytometry, thereby obtaining 293F-OS8-PVRL2 cell line.
  • plvx-NFAT-Luc Hygromycin resistance
  • plvx-NFAT-Luc Hygromycin resistance
  • infect Jurkat E6.1 cells add Hygromycin to screen out resistant clones, use OKT3 to stimulate clones, and screen out clones with medium Luciferase signal
  • Obtain the Jurkat-NFAT-Luc cell line construct the plvx-PVRIG (Puromycin resistance) vector, package it into a lentivirus, infect the Jurkat-NFAT-Luc cells, and select the clone with the highest expression of PVRIG by flow cytometry, thereby obtaining Jurkat - NFAT-Luc-PVRIG cell line.
  • plvx-NFAT-Luc Hygromycin resistance
  • 1E4 Jurkat-NFAT-Luc-PVRIG cells were incubated with the antibody to be tested at 37°C for 20 minutes. Add 1E5 293F-OS8-PVRL2 cells and incubate at 37°C for 5 hours. The supernatant was removed by centrifugation, the cells were lysed by adding Luciferase buffer (Promega, E6130), and the fluorescence value was detected. Calculate the EC 50 value to evaluate the in vitro cell activity of the anti-PVRIG antibody. The experimental results are shown in Table 8.
  • PVRIG is expressed on NK cells, while PVRL2 is expressed in many tumor cells, including K562 cells.
  • Anti-PVRIG antibodies can release the inhibitory effect of tumor cells on NK cell activity by blocking the combination of PVRL2 and PVRIG.
  • control group 1 a sample containing only culture medium
  • control group 2 a sample containing only NK92 cells
  • control group 3 a sample containing only K562 cells
  • the killing activity was calculated according to the following formula:
  • 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.
  • the fluorescence value of Glo is the fluorescence value of the control group three after adding the lysate
  • BGL is the fluorescence value of the control group after adding the lysate again.
  • PVRIG is expressed on T cells, whereas PVRL2 is expressed on DC cells.
  • Anti-PVRIG antibody can release the inhibition of DC cells on T cells and activate T cells by blocking the combination of PVRL2 and PVRIG.
  • PBMC peripheral blood of the first individual, and the cells were cultured in RPMI1640 medium containing 10% FBS with 50ng/mL GM-CSF (Peprotech, 300-03-100UG) and 50ng/mL IL-4 (Peprotech, 200-04-100UG) was added to the final concentration, and fresh medium containing cytokines was added every 2-3 days; after 6 days of culture, 1 ⁇ g/mL LPS (Sigma, L2880-25MG) was added to incubate for 24 hours, and the differentiation DC cells obtained from maturation.
  • GM-CSF 50ng/mL GM-CSF
  • IL-4 50ng/mL IL-4
  • PBMCs were isolated from peripheral blood from a second individual, from which CD3 + T cells were isolated using the EasySep Human CD3+ T Cell Isolation Kit (Stemcell, 17952). Adjust the density of CD3 + T cells and DC cells so that 1 ⁇ 10 5 CD3 + T cells and 2 ⁇ 10 4 DC cells are added to each well. Add the antibody to be tested, incubate at 37°C for 120 hours, take the supernatant, and detect the IFN ⁇ content in the supernatant with an ELISA kit (R&D, DY202).
  • a human germline template IGHV3-7*01 with high homology was obtained. Graft the CDRs into the corresponding human templates. The three-dimensional structure simulation and analysis of the transplanted single domain antibody were carried out again, and the buried residues, the residues that directly interact with the CDR region, and the residues that have an important impact on the conformation of the variable region were reverse mutated, and Optimize the chemically unstable amino acid residues in the CDR region to generate a series of humanized single domain antibodies.
  • the human germline template for each single domain antibody and the heavy chain variable region sequence of the humanized antibody are shown below.
  • the antibody 30H1-30H5 comprises CDR1 as shown in GDCMG (SEQ ID NO: 4), CDR2 as shown in TIDNAGRIKYADSVKG (SEQ ID NO: 5), and CDR3 as shown in GWTFGGQCSPAD (SEQ ID NO: 20).
  • the heavy chain variable region of the above humanized antibody was linked with the Fc region of the human IgG4 heavy chain to form a full-length anti-PVRIG antibody.
  • the Fc region of the heavy chain includes a hinge region, and carries S228P/F234A/L235A/K447A, or S228P/K447A mutations.
  • the antibody was expressed and purified according to conventional methods, and the target antibody was obtained after detection.
  • Example 1-5 Activity and function verification of humanized anti-PVRIG antibody
  • Example 11 FACS was used to detect the binding of anti-PVRIG antibody to human or cynomolgus PVRIG.
  • the experimental results are shown in Table 11.
  • Example 1-2 According to the Fortebio detection method in Example 1-2, the affinity of the humanized anti-PVRIG antibody to human PVRIG was detected. As shown in Table 12, all antibodies had high affinity to human PVRIG.
  • Example 1-3 According to the method of Example 1-3, the activity of the humanized anti-PVRIG antibody in the reporter gene cells was detected.
  • the experimental results are shown in Table 13.
  • the antibodies listed in the table all have the ability to activate Jurkat cells.
  • Example 1-3 the activation ability of the humanized anti-PVRIG antibody on NK cells was detected.
  • the experimental results are shown in Table 14-1 and Table 14-2. The results showed that all the antibodies tested had the obvious ability to activate NK cells and promote the killing of target cells K562 by NK cells.
  • the anti-PVRIG single domain antibody 151 was connected to the N-terminal or heavy chain or light chain of anti-TIGIT antibody 1708 through the GGGGSGGGGS (SEQ ID NO: 152) linker. C-terminal connected.
  • Four anti-PVRIG/TIGIT double antibodies were formed, named 1708-151-1, 1708-151-2, 1708-151-3, 1708-151-4, corresponding to 151 being connected to the N-terminal of the heavy chain of 1708, and the heavy chain C-terminus, light chain N-terminus and light chain C-terminus.
  • the anti-TIGIT antibody 1708 adopts the human IgG4 subtype with a mutation of S228P (Eu nomenclature system).
  • the anti-TIGIT antibody 1708 and the bispecific antibody sequences formed with 151 are shown in Table 15 below.
  • the sequence information of the anti-TIGIT antibody is shown in Table 16 and Table 17.
  • the TIGIT antibody in WO2019062832A is incorporated herein in its entirety.
  • Different humanized anti-PVRIG antibody variable regions (30H2, 151H7, 151H8) were linked to the heavy chain N-terminus of anti-TIGIT antibody 1708, that is, a bispecific antibody structure similar to 1708-151-1 was used to construct a double antibody.
  • the second polypeptide chains of 1708-30H2, 1708-151H7, 1708-151H8 were all identical to the light chain of 1708 (SEQ ID NO: 27).
  • Example 1-7 Activity and function verification of anti-PVRIG/TIGIT double antibody
  • Example 1-2 According to the method of Example 1-2 (the corresponding receptor and ligand were replaced by human TIGIT and human PVR), the experiment was carried out, and the results are shown in Table 18. The results showed that bispecific antibodies 1708-151-1, 1708-151-2, 1708-151-3, 1708-151-4 and anti-TIGIT antibody with different configurations were compatible with human TIGIT recombinant protein and overexpressed human TIGIT cells
  • the binding of TIGIT and the blocking of TIGIT binding to its ligand PVR are basically the same and there is no difference.
  • the anti-PVRIG antibody is connected to the N-terminal or C-terminal of the heavy and light chain of the anti-TIGIT antibody, it maintains the binding of PVRIG/TIGIT and the blocking of the ligand, and all show good expression and purity .
  • Example 1-2 According to the method of Example 1-2, the binding of the humanized anti-PVRIG/TIGIT double antibody to human and cynomolgus PVRIG, and the ligand blocking of human PVRIG were detected. The results are shown in Table 18. The results showed that all the double antibodies could bind to human PVRIG and block the binding of PVRIG to PVRL2.
  • Example 1-2 Similar to Example 1-2, the binding of the humanized anti-PVRIG/TIGIT double antibody to human and cynomolgus monkey TIGIT was detected, and the blocking of the binding of human TIGIT to the ligand was detected, wherein the PVRIG protein was replaced with TIGIT, and PVRL2 Replaced with PVRs.
  • the results are shown in Table 19. The results showed that all the double antibodies could bind to human and cynomolgus monkey TIGIT, and block the binding of TIGIT to PVR.
  • Biacore was used to detect the affinity of the double antibody to human PVRIG and human TIGIT.
  • the humanized bispecific antibody was captured on the Protein A biosensing chip (GE lifesciences, 29127557) of Biacore instrument (Biacore X100, GE), and then human PVRIG antigen (AcroBiosystem , PVG-H52H4) or human TIGIT antigen (AcroBiosystem, TIT-H52H3), the association and dissociation curves were obtained. The results are shown in Table 20.
  • the activation ability of the humanized anti-PVRIG/TIGIT double antibody on T cells was detected.
  • the experimental results are shown in Figure 2 and Table 21.
  • the results showed that the humanized anti-PVRIG/TIGIT double antibody 1708-151H8 had obvious ability to activate T cells and promote T cells to secrete IFN ⁇ .
  • the bispecific antibody was more active than anti-PVRIG antibody 151H8 alone and anti-TIGIT antibody 1708 alone.
  • Example 1-8 Anti-tumor effect evaluation of anti-PVRIG/TIGIT diabodies in human melanoma A375 mixed with human PBMC subcutaneous xenograft tumor model in mice
  • the full length of the heavy chain of 1708-IgG1 is the same as that of 1708, but the subtype of the constant region of the heavy chain is changed to IgG1; the full length of the light chain of 1708-IgG1 and the second polypeptide chain of 1708-151-IgG1 are the same Light chains are the same.
  • NCG mice female, 4-8 weeks old, weighing about 18-22 g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. All NCG mice were cultured according to the conditions of the IVC constant temperature and pressure system in the SPF animal room.
  • A375 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS). A375 cells in the exponential growth phase were collected and resuspended in HBSS to a suitable concentration for subcutaneous tumor inoculation in NCG mice. The A375 cells used for co-cultivation need to be treated with mitomycin C for 2 hours, and then washed three times with PBS. The peripheral blood of normal people was taken, and human PBMCs were separated by density gradient centrifugation and counted. Then PBMCs were resuspended to a concentration of 3 ⁇ 10 6 cells/mL in RPMI1640 medium (containing IL2 and 10% FBS), and co-cultured with A375 cells treated with mitomycin C.
  • FBS fetal bovine serum
  • mice After 6 days of co-culture, PBMCs were harvested, and freshly digested A375 cells were collected at the same time. Inoculate each mouse: 5 ⁇ 10 5 PBMCs, 4 ⁇ 10 6 A375 cells; inoculation volume: 0.2 mL/mouse (containing 50% Matrigel); inoculate subcutaneously on the right side of female NCG mice.
  • the mice were administered in groups randomly according to their body weight. The detailed administration methods, doses and routes of administration are shown in Table 22. The day of group administration was Day 0. Since the molecular weights of the anti-PVRIG antibody and the anti-TIGIT antibody are different, the dosage ensures that the anti-PVRIG antibody and the anti-TIGIT antibody have the same initial molar concentration.
  • N number of animals used; i.p.: intraperitoneal injection; Q2D: once every two days; administration volume: adjust the administration volume (0.1mL/10g) according to the body weight of tumor-bearing mice.)
  • mice After the administration started, the body weight and tumor volume of the mice were measured twice a week.
  • the experimental results are shown in Table 23 and Figures 3A and 3B, respectively.
  • mice were administered in groups randomly according to their body weight. The detailed administration methods, doses and routes of administration are shown in Table 24. The day of group administration was Day 0.
  • N number of animals used; i.p.: intraperitoneal injection; Q2D: once every two days; administration volume: adjust the administration volume (0.1mL/10g) according to the body weight of tumor-bearing mice.)
  • mice After the administration started, the body weight and tumor volume of the mice were measured twice a week. The experimental results are shown in Table 25 and Figures 4A-4B, respectively.
  • both the 1708-30H2 and 1708-151H7 double antibody groups could effectively inhibit tumor growth at low doses, and there was a significant difference between them (see Figure 4B).
  • Example 2 Evaluation of anti-tumor effect of anti-PVRIG/TIGIT bispecific antibody combined with anti-PD-L1 antibody on human melanoma A375 mixed PBMC subcutaneous xenograft model
  • Drug A 1708-30H2 (anti-PVRIG/TIGIT bispecific antibody), prepared by referring to the method of the aforementioned Example 1, using PBS to prepare a 3.58 mg/mL solution;
  • Drug B anti-PD-L1 antibody (Adebrelimab), prepared with reference to WO2017084495, using NS (normal saline) to prepare a 1.0 mg/mL solution;
  • Negative control isotype IgG4, provided by Shanghai Hengrui Pharmaceutical Co., Ltd., diluted with PBS.
  • NCG mice female, weighing about 18-22 g. Purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • Drug B (anti-PD-L1 antibody) antibody sequence is as follows:
  • the underlined part is the CDR region, and the sequence of each sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the CDR sequences in the light and heavy chains of each antibody are shown in Table 26.
  • A375 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS). A375 cells in the logarithmic growth phase were collected and resuspended in HBSS to a suitable concentration for subcutaneous tumor inoculation in NCG mice. The A375 cells used in the co-culture experiment were treated with mitomycin C for 2 h, and then washed three times with PBS. The revived PBMCs were added to A375 cells treated with mitomycin C, and PBMCs were co-cultured with A375 for 5 days in RPMI 1640 culture medium containing IL-2 and 10% FBS.
  • FBS fetal bovine serum
  • mice After co-cultivating PBMC and A375 for 5 days, collect PBMC and freshly digested A375 cells, PBMC 4 ⁇ 10 5 , A375 cells 4 ⁇ 10 6 , inoculate volume 0.2mL/one (containing 50% Matrigel), inoculate in NCG Subcutaneously on the right side of the mouse. After inoculation, the mice were randomly administered into groups according to their body weight. The detailed administration method, dosage and route of administration are shown in Table 27. The day of group administration was Day 0.
  • N number of animals used; i.p.: intraperitoneal injection; BIW*3: administered twice a week for 3 weeks, 6 times in total
  • TGI (%) (1-T/C) ⁇ 100%.
  • T/C% is the relative proliferation rate of the tumor
  • T/C% (Ti-T0)/(Vi-V0) ⁇ 100%, that is, at a certain point in time
  • Ti is the average tumor volume after the administration of the drug group
  • T0 is the average tumor volume of the treatment group at the first administration
  • V0 is the average tumor volume of the vehicle control group at the first administration
  • Vi is the average tumor volume of the vehicle control group after administration.
  • Body weight measurement of tumor-bearing mice the body weight of all tumor-bearing mice was measured twice a week.
  • the average tumor volume of the negative control group was 1033.50 ⁇ 83.02mm 3 ; (10mg/kg) + drug A (35.8mg/kg) administration group, the mean tumor volumes were 872.37 ⁇ 78.44mm 3 , 360.48 ⁇ 63.85mm 3 , 193.20 ⁇ 39.49mm 3 , and the tumor growth inhibition rate (TGI) was respectively 15.59%, 65.12%, and 81.31%.
  • the average tumor weights were 1.111 ⁇ 0.103g, 0.986 ⁇ 0.078g, 0.473 ⁇ 0.071g and 0.257 ⁇ 0.043g respectively.
  • mice in all the administration groups showed no abnormal behavior, indicating that the tumor-bearing mice had good tolerance to the drug at the dose tested, see Figure 5C, Figure 5D and Table 30.
  • Example 3 Evaluation of anti-tumor effect of anti-PVRIG/TIGIT bispecific antibody combined with fusion protein containing anti-PD-L1 antibody on human melanoma A375 mixed PBMC subcutaneous xenograft model
  • Drug A 1708-30H2 (anti-PVRIG/TIGIT bispecific antibody), prepared with reference to PCT/CN2021/080470, and prepared into a 3.58 mg/mL or 0.72 mg/mL solution with PBS;
  • Drug C a fusion protein (fusion protein 9) containing an anti-PD-L1 antibody, prepared with reference to WO20200941225, and prepared into a 0.1 mg/mL solution with PBS;
  • Negative control isotype IgG4, provided by Shanghai Hengrui Pharmaceutical Co., Ltd., diluted with PBS.
  • NCG mice female, weighing about 18-22 g. Purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • Drug C is a fusion protein in which an anti-PD-L1 antibody is linked to TGF- ⁇ RII ECD through a (G 4 S) 4 G linker, and the anti-PD-L1 antibody is the drug B antibody in Example 2, wherein TGF- ⁇ RII
  • the ECD sequence is a truncated or deleted 19 amino acids at the N-terminus, as follows:
  • the first polypeptide chain of fusion protein 9 (PD-L1 antibody and TGF- ⁇ RII ECD fusion protein)
  • the underline is the linker sequence, and the second polypeptide chain of fusion protein 9 (PD-L1 antibody and TGF- ⁇ RII ECD fusion protein) is shown in SEQ ID NO:55.
  • Fusion protein 9 is composed of two identical first polypeptide chains and two identical second polypeptide chains.
  • A375 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS). A375 cells in the logarithmic growth phase were collected and resuspended in HBSS to a suitable concentration for subcutaneous tumor inoculation in NCG mice. The A375 cells used in the co-culture experiment were treated with mitomycin C for 2 h, and then washed three times with PBS. The peripheral blood of normal people was taken, and human PBMCs were separated by density gradient centrifugation and counted. Then PBMCs were resuspended in RPMI1640 medium (containing IL-2 and 10% FBS), and co-cultured with A375 cells treated with mitomycin C.
  • FBS fetal bovine serum
  • mice After PBMC and A375 were co-cultured for 6 days, collect PBMC and freshly digested A375 cells, PBMC 5 ⁇ 10 5 cells, A375 cells 4 ⁇ 10 6 cells, inoculate volume 0.2mL/mouse (containing 50% Matrigel), inoculate in NCG mice Right subcutaneous. After inoculation, the mice were randomly administered into groups according to their body weight. The detailed administration methods, doses and routes of administration are shown in Table 31. The day of group administration was Day 0.
  • N number of animals used; i.p.: intraperitoneal injection; BIW*8: twice a week, administered 8 times)
  • Evaluation indicators refer to Example 2. All data were analyzed with Graphpad and expressed as mean ⁇ SEM. Use One way ANOVA LSD(L) test in Graphpad to compare the difference between the test drug group and the control group, and P ⁇ 0.05 is considered to have a significant difference.
  • TGI tumor growth inhibition rates
  • Example 4 Evaluation of anti-tumor effect of anti-PVRIG/TIGIT bispecific antibody combined with anti-PD-1 antibody on human melanoma A375 mixed PBMC subcutaneous xenograft model
  • Drug A 1708-30H2 (anti-PVRIG/TIGIT bispecific antibody), prepared with reference to PCT/CN2021/080470, and prepared into a 3.58 mg/mL or 0.72 mg/mL solution with PBS;
  • Drug D anti-PD-1 antibody (Hu23-11.IgG4AA), prepared with reference to WO2020156509, prepared into a 0.1 mg/mL solution with PBS;
  • Negative control isotype IgG4, provided by Shanghai Hengrui Pharmaceutical Co., Ltd., diluted with PBS.
  • NCG mice female, weighing about 18-22 g. Purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • Drug D (anti-PD-1 antibody) antibody sequence is as follows:
  • the underlined part is the CDR region, and the sequence of each sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the CDR sequences in the light and heavy chains of each antibody are shown in Table 34.
  • Example 2 For the steps of A375 cell culture, separation of PBMCs, co-cultivation and mixed inoculation of the two.
  • the mice were randomly administered in groups according to their body weight. The specific administration method, dosage and route of administration are shown in Table 35. The day of group administration was Day 0.
  • N number of animals used; i.p.: intraperitoneal injection; Q2D*18: once every two days, administered 18 times; BIW*5, twice a week, administered for 5 weeks, 10 times in total)
  • Evaluation indicators refer to Example 2. All data were analyzed with Graphpad and expressed as mean ⁇ SEM. Use the student's t test in Graphpad to compare the difference between the test drug group and the control group, and P ⁇ 0.05 is considered to have a significant difference.
  • the tumor volumes of the drug A group, the drug D group, and the drug A+drug D combination group all decreased, and the tumor growth inhibition rate (TGI ) were 33.75%, 82.58% and 94.80%, respectively, wherein the drug D group (p ⁇ 0.001) and the combination group (p ⁇ 0.001) showed significant tumor growth inhibition.
  • the tumor weight of the drug A+drug D combination group was significantly reduced (p ⁇ 0.05), indicating that the drug A and drug D combined group had a strong tumor growth inhibitory effect, and the combined drug administration showed A better anti-tumor effect was obtained (see Figure 7B and Table 37).

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Abstract

PVRIG/TIGIT结合蛋白联合免疫检查点抑制剂用于治疗癌症。具体地，提供PVRIG/TIGIT结合蛋白与免疫检查点抑制剂联合在制备治疗癌症的药物中的用途。

Description

PVRIG/TIGIT结合蛋白联合免疫检查点抑制剂用于治疗癌症
本公开要求2021年09月15日提交的中国专利申请(申请号CN202111078575.1)的优先权。
技术领域
本公开属于医药领域，具体地，本公开涉及PVRIG/TIGIT结合蛋白联合免疫检查点抑制剂用于治疗癌症的用途。
背景技术
PVRIG(Poliovirus receptor-related Ig domain containing protein)又名CD112R，与TIGIT(T cell immunoglobulin and ITIM domain)、CD96和CD226等同属于B7/CD28超家族，在免疫系统中起重要作用。当PVRIG的配体PVRL2(又名CD112)结合PVRIG时，会活化PVRIG胞内区的ITIM结构域，使PVRIG起到免疫抑制的作用。PVRIG主要表达在CD4 +T细胞，CD8 +T细胞和NK细胞的表面。PVRIG和其配体PVRL2在多种实体瘤中高表达，包括肺癌、乳腺癌、卵巢癌、肾癌、胃癌、子宫内膜癌、头颈癌等。PVRIG在这些癌症中的表达与TIGIT和PD-1有高度相关性。与PD-1和TIGIT相似，PVRIG阳性的T细胞也会呈现Eomes阳性和Tbet阴性，表明PVRIG与T细胞的耗竭有关。因此，PVRIG可能代表了除了PD-1和TIGIT之外的有一个新的免疫检查点，并起到冗余(redundancy)的作用。体外细胞实验和小鼠模型中显示，对小鼠PVRIG的敲除或抑制，可以有效抑制癌症的生长，并与PD-1和TIGIT抑制剂发生协调作用。TIGIT在淋巴细胞上高度表达，包括浸润不同类型癌症的癌症浸润淋巴细胞(TIL)和Treg。已经证明TIGIT与其同源配体PVR(又名CD155)的接合通过其细胞质ITIM结构域直接抑制NK细胞的细胞毒性。PVR也在癌症中广泛表达，表明TIGIT-PVR信号轴可能是癌症的主要免疫逃逸机制。抑制性受体TIGIT、PVRIG与激活型受体DNAM-1结合同样的配体CD155与CD112，但抑制性受体的亲和力更高(Y.Zhu等人2016，J Exp Med.213:167-176)。肿瘤病人体内分离的NK细胞的DNAM-1表达下调，导致NK细胞更易受到TIGIT或PVRIG的抑制，阻断TIGIT与PVRIG的结合，能够激活NK细胞的细胞杀伤功能(L.Martinet等人2015，Cell Reports.11:85-97)。
虽然PD-1/PD-L1是目前临床最成功的免疫检查点抑制剂单抗，但T细胞表面其他免疫检查点的表达上调会限制其疗效。TIGIT和PVRIG除了调控T细胞的功能，还调控NK细胞活性，提示与PD-1/PD-L1在NK细胞中的功能可能产生协同。文献报道TIGIT与PVRIG的配体CD155与CD112在肺癌、卵巢癌、结直肠癌、黑色素瘤中的高表达与PD-1/PD-L1治疗不良预后显著相关。如CD155表达高的患者对PD-1抗体响应差，而CD155表达低的患者对PD-1响应好(S.Whelan等人2019，Cancer Immunol Res.7:257-268；A.Lepletier等人2020，Clin Cancer Res. 26:3671-3681)。
联用抗PVRIG/TIGIT双抗与PD-1/PD-L1潜在能够从增强T细胞、NK细胞激活，克服PD-1/PD-L1耐药与拓展应答患者人群等方面提高抗肿瘤治疗的效果。本公开旨在提供具有抑制体内肿瘤生长的高亲和力、高选择性、高生物活性的抗PVRIG/TIGIT双特异性抗体与免疫检查点抑制剂(如PD-1或PD-L1抗体)联合用于治疗癌症的用途，并显示了良好的抑瘤效果，有潜力在临床提供比现有PD-1、TIGIT、PVRIG药物或其联用更佳的疗效。
发明内容
本公开提供PVRIG结合蛋白或PVRIG/TIGIT结合蛋白与免疫检查点抑制剂联合在制备治疗疾病(例如，癌症)的药物中的用途、或用于治疗疾病(例如，癌症)的方法。
本公开提供一种PVRIG结合蛋白和TIGIT结合蛋白联合在制备治疗疾病(例如，癌症)的药物中的用途、或用于治疗疾病(例如，癌症)的方法。一些实施方案中，在联合使用时，还进一步包括免疫检查点抑制剂。
本公开提供一种PVRIG结合蛋白、TIGIT结合蛋白和免疫检查点抑制剂联合在制备治疗疾病(例如，癌症)的药物中的用途、或用于治疗疾病(例如，癌症)的方法。
一些实施方案中，提供PVRIG结合蛋白(例如，抗PVRIG抗体或其抗原结合片段)与免疫检查点抑制剂联合在制备治疗癌症的药物中的用途，所述PVRIG结合蛋白包含免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含：
如SEQ ID NO：2、15-19任一所示氨基酸序列中的CDR1、CDR2和CDR3，或
如SEQ ID NO：3、21-25任一所示氨基酸序列中的CDR1、CDR2和CDR3，
所述CDR1、CDR2和CDR3是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的；
一些具体实施方案中，根据Kabat编号系统，所述免疫球蛋白单一可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别
如SEQ ID NO：4、5、20所示，
如SEQ ID NO：4、5、6所示，或
如SEQ ID NO：7、8、9所示。
一些实施方案中，前述PVRIG中的免疫球蛋白单一可变结构域为至少一个，例如，2、3、4、5、6、7、8个。
一些实施方案中，前述PVRIG结合蛋白的免疫球蛋白单一可变结构域是人源化、亲合力成熟、去除T细胞表位、降低抗体脱酰胺和/或降低抗体异构化改造的；一些具体实施方案中，人源化改造过程使用的人种系模板的重链框架区为 IGHV3-7*01。
一些实施方案中，前述PVRIG结合蛋白的免疫球蛋白单一可变结构域的氨基酸序列分别如
SEQ ID NO：2、15-19任一所示；或SEQ ID NO：3、21-25任一所示；或与前述序列任一具有至少80％、至少90％序列同一性。
一些实施方案中，前述PVRIG结合蛋白还包含人免疫球蛋白Fc区；
优选地，所述Fc区是人IgG1或IgG4的Fc区；
更优选地，所述人IgG4的Fc区具有S228P、F234A、L235A和/或K447A突变。
一些实施方案中，提供前述PVRIG结合蛋白和TIGIT结合蛋白(例如，抗TIGIT抗体或其抗原结合片段)与免疫检查点抑制剂联合在制备治疗癌症的药物中的用途。
一些实施方案中，所述TIGIT结合蛋白包含重链可变区(VH)和轻链可变区(VL)，其中：
所述重链可变区包含分别如SEQ ID NO：32、33和34所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO：35、36和37所示的LCDR1、LCDR2和LCDR3。所述CDR1、CDR2和CDR3是根据Kabat编号系统定义的。
一些实施方案中，前述特异性结合TIGIT结合蛋白包含重链可变区(VH)和轻链可变区(VL)，其中：
所述重链可变区包含如SEQ ID NO：38-40中任一所示或与之具有至少80％、至少90％序列同一性的氨基酸序列，
所述轻链可变区包含如SEQ ID NO：41或42所示或与之具有至少80％、至少90％序列同一性的氨基酸序列；
例如，所述重链可变区包含如SEQ ID NO：3所示或与之具有至少80％、至少90％序列同一性的氨基酸序列，
所述轻链可变区包含如SEQ ID NO：42所示或与之具有至少80％、至少90％序列同一性的氨基酸序列。
一些实施方案中，前述TIGIT结合蛋白还包含人免疫球蛋白Fc区；
优选地，所述Fc区是人IgG1或IgG4的Fc区；
更优选地，所述人IgG4的Fc区具有S228P、F234A、L235A和/或K447A突变。
一些具体实施方案中，前述特异性结合TIGIT结合蛋白包含如SEQ ID NO：26所示的重链全长序列或与之具有至少80％、至少90％序列同一性，和如SEQ ID NO：27所示的轻链全长序列或与之具有至少80％、至少90％序列同一性。
一些实施方案中，提供PVRIG/TIGIT结合蛋白(例如，抗PVRIG/TIGIT双特异性抗体或其抗原结合片段)与免疫检查点抑制剂联合在制备治疗癌症的药物中的 用途，所述PVRIG/TIGIT结合蛋白包含特异性结合PVRIG的第一抗原结合结构域和特异性结合TIGIT的第二抗原结合结构域，所述特异性结合PVRIG的第一抗原结合结构域包含免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含：
如SEQ ID NO：2和15-19中任一所示氨基酸序列中的CDR1、CDR2和CDR3，或
如SEQ ID NO：3和21-25中任一所示氨基酸序列中的CDR1、CDR2和CDR3，
所述CDR1、CDR2和CDR3是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的；
一些具体实施方案中，根据Kabat编号系统，所述免疫球蛋白单一可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别
如SEQ ID NO：4、5和20所示，
如SEQ ID NO：4、5和6所示，或
如SEQ ID NO：7、8和9所示。
一些实施方案中，前述特异性结合PVRIG的第一抗原结合结构域是人源化、亲合力成熟、去除T细胞表位、降低抗体脱酰胺和/或降低抗体异构化改造的；一些具体实施方案中，人源化改造过程使用的人种系模板的重链框架区为IGHV3-7*01。
一些实施方案中，前述特异性结合PVRIG的第一抗原结合结构域中免疫球蛋白单一可变结构域的氨基酸序列分别如SEQ ID NO：2和15-19中任一所示；或如SEQ ID NO：3和21-25中任一所示；或与前述序列任一具有至少80％序列同一性，例如，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
一些实施方案中，前述特异性结合TIGIT的第二抗原结合结构域包含重链可变区(VH)和轻链可变区(VL)，其中：
所述重链可变区包含分别如SEQ ID NO：32、33和34所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO：35、36和37所示的LCDR1、LCDR2和LCDR3。
一些实施方案中，前述特异性结合TIGIT的第二抗原结合结构域包含重链可变区(VH)和轻链可变区(VL)，其中：
所述重链可变区包含如SEQ ID NO：38-40中任一所示或与之具有至少80％，例如，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列，
所述轻链可变区包含如SEQ ID NO：41或42所示或与之具有至少80％，例 如，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列；
例如，所述重链可变区包含如SEQ ID NO：38所示或与之具有至少80％，例如，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列，
所述轻链可变区包含如SEQ ID NO：42所示或与之具有至少80％，例如，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列。
一些实施方案中，前述PVRIG/TIGIT结合蛋白还包含人免疫球蛋白Fc区；
优选地，所述Fc区是人IgG1或IgG4的Fc区；
更优选地，所述人IgG4的Fc区具有S228P、F234A、L235A和/或K447A突变。
一些实施方案中，前述特异性结合PVRIG的第一抗原结合结构域和特异性结合TIGIT的第二抗原结合结构域直接或通过连接子相连接；
例如，所述连接子为具有如(G 4S) x所示的氨基酸序列，其中，x独立地选自1-20的整数；
又例如，所述连接子为(G 4S) 2、(G 4S) 3、(G 4S) 4所示的氨基酸序列。
一些实施方案中，前述PVRIG/TIGIT结合蛋白中：
所述特异性结合PVRIG的第一抗原结合结构域的免疫球蛋白单一可变结构域位于特异性结合TIGIT的第二抗原结合结构域的重链可变区的N端；
所述特异性结合PVRIG的第一抗原结合结构域的免疫球蛋白单一可变结构域位于特异性结合TIGIT的第二抗原结合结构域的重链可变区的C端；
所述特异性结合PVRIG的第一抗原结合结构域的免疫球蛋白单一可变结构域位于特异性结合TIGIT的第二抗原结合结构域的轻链可变区的N端；和/或
所述特异性结合PVRIG的第一抗原结合结构域的免疫球蛋白单一可变结构域位于特异性结合TIGIT的第二抗原结合结构域的轻链可变区的C端。
一些实施方案中，提供PVRIG/TIGIT结合蛋白，其包含第一多肽链和第二多肽链，其中：
所述第一多肽链包含如SEQ ID NO：28-29和43-45中任一所示、或与其具有至少80％，例如，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列，所述第二多肽链包含如SEQ ID NO：27所示、或与其具有至少80％，例如，至少85％、至少86％、至少87％、 至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列；或者
所述第一多肽链包含如SEQ ID NO：26所示、或与其具有至少80％，例如，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列，所述第二多肽链包含如SEQ ID NO：30或31所示、或与其具有至少80％，例如，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列。
一些具体实施方案中，前述PVRIG/TIGIT结合蛋白包含两条相同的第一多肽链和两条相同的第二多肽链。一些实施方案中，如前所述的免疫检查点抑制剂包括但不限于靶向PD-1/PD-L1、PD-L2、B7-1/CTLA-4、B7-2/CTLA-4、B7-1/CD28、B7-2/CD28、B7-H2/ICOS、B7-H3、B7-H4、B7-H7、VISTA(PD-1H)、BTNL2、TIM3、LAG3、TNFSF/TNFRSF(如CD40-CD40L、CD27-CD70或OX40-OX40L)、白细胞免疫球蛋白样受体(LIR/LILR)或免疫球蛋白样转录子(ILT)、粘连蛋白或类粘连蛋白受体蛋白(如DNAM-1、CD226、PTA1、TLISA1、CD96、TIGIT、Vstm3和VSIG9)、NKG2A、KIRs、NKG2A、Siglecs family(Siglec-7/9)、CD47、CD94、CD200信号通路的抑制剂。
一些实施方案中，如前所述的免疫检查点抑制剂包括但不限于蛋白或多肽、小分子、核酸类药物。一些具体实施方案中，所述免疫检查点抑制剂为PD-1结合蛋白和/或PD-L1结合蛋白。在一些实施方案中，所述免疫检查点抑制剂为抗PD-1抗体、抗PD-L1抗体、所述抗体的抗原结合片段、或包含所述抗体或其抗原结合片段的融合蛋白。一些实施方案中，前述PD-1结合蛋白(例如，包含或为抗PD-1抗体或其抗原结合片段)包含：
分别如SEQ ID NO:59、60和61所示氨基酸序列的HCDR1、HCDR2和HCDR3，和分别如SEQ ID NO:62、63和64所示氨基酸序列的LCDR1、LCDR2和LCDR3；或
分别如SEQ ID NO:67、68和69所示氨基酸序列的HCDR1、HCDR2和HCDR3，和分别如SEQ ID NO:70、71和72所示氨基酸序列的LCDR1、LCDR2和LCDR3。
一些具体实施方案中，前述PD-1结合蛋白(例如，包含或为抗PD-1抗体或其抗原结合片段)包含重链可变区(VH)和轻链可变区(VL)，其中：
所述重链可变区包含如SEQ ID NO：57所示或与之具有至少80％，例如，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列，所述轻链可变区包含如SEQ ID NO：58所示或 与之具有至少80％，例如，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列；或
所述重链可变区包含如SEQ ID NO：73所示或与之具有至少80％，例如，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列，所述轻链可变区包含如SEQ ID NO：74所示或与之具有至少80％，例如，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列。
一些实施方案中，前述PD-1结合蛋白还包含人免疫球蛋白Fc区；
优选地，所述Fc区是人IgG1或IgG4的Fc区；
更优选地，所述人IgG4的Fc区具有S228P、F234A、L235A和/或K447A突变。
一些实施方案中，前述PD-1结合蛋白包含重链(HC)和轻链(LC)，其中：
所述重链包含如SEQ ID NO：65所示或与之具有至少80％，例如，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列，所述轻链包含如SEQ ID NO：66所示或与之具有至少80％，例如，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列；或
所述重链包含如SEQ ID NO：75所示或与之具有至少80％，例如，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列，所述轻链包含如SEQ ID NO：76所示或与之具有至少80％，例如，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列。
在一个实施方案中，所述PD-1结合蛋白为抗PD-1抗体：Camrelizumab；
所述Camrelizumab抗体具有如下所示的CDR：
HCDR1为SYMMS(SEQ ID NO：67)
HCDR2为TISGGGANTYYPDSVKG(SEQ ID NO：68)
HCDR3为QLYYFDY(SEQ ID NO：69)
LCDR1为LASQTIGTWLT(SEQ ID NO：70)
LCDR2为TATSLAD(SEQ ID NO：71)
LCDR3为QQVYSIPWT(SEQ ID NO：72)
>所述抗PD-1抗体Camrelizumab抗体的VH为
Figure PCTCN2022118959-appb-000001
>所述抗PD-1抗体Camrelizumab抗体的VL为
Figure PCTCN2022118959-appb-000002
上述抗PD-1抗体重轻链可变区序列中，下划线部分为CDR区，各序列依次顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
>所述抗PD-1抗体Camrelizumab的HC(Fc区具有S228P突变)为
Figure PCTCN2022118959-appb-000003
>所述抗PD-1抗体Camrelizumab的LC为
Figure PCTCN2022118959-appb-000004
一些实施方案中，前述PD-1结合蛋白包含或为抗PD-1抗体或其抗原结合片段，包括但不限于sintilimab、cemiplimab、JS-001、nivolumab、tislelizumab、pembrolizumab、AK-103、dostarlimab、PD1-PIK、GLS-010、genolimzumab、BI-754091、spartalizumab、MGA-012、PF-06801591、XmAb-20717、CS-1003、Sym-021、AGEN-2034、MEDI-5752、MGD-013、AK-105、AK-104、BCD-100、PF-06753512、HLX-10、AMP-224、LZM-009。此处全文引入WO2020156509、WO2017054646中的抗PD-1抗体或其抗原结合片段。一些实施方案中，前述PD-L1结合蛋白(例如，包含或为抗PD-L1抗体或其抗原结合片段)包含
分别如SEQ ID NO:48、49和50所示氨基酸序列的HCDR1、HCDR2和HCDR3，和分别如SEQ ID NO:51、52和53所示氨基酸序列的LCDR1、LCDR2和LCDR3。
一些具体实施方案中，前述PD-L1结合蛋白包含重链可变区(VH)和轻链可变 区(VL)，其中：
所述重链可变区包含如SEQ ID NO：46所示或与之具有至少80％，例如，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列，所述轻链可变区包含如SEQ ID NO：47所示或与之具有至少80％，例如，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列。
一些实施方案中，前述PD-L1结合蛋白还包含人免疫球蛋白Fc区；
例如，所述Fc区是人IgG1或IgG4的Fc区；
又例如，所述人IgG4的Fc区具有S228P、F234A、L235A和/或K447A突变。
一些实施方案中，前述PD-L1结合蛋白包含重链(HC)和轻链(LC)，其中：
所述重链包含如SEQ ID NO：54所示或与之具有至少80％，例如，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列，所述轻链包含如SEQ ID NO：55所示或与之具有至少80％，例如，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列。
一些实施方案中，前述PD-L1结合蛋白为抗PD-L1抗体或其抗原结合片段，包括但不限于avelumab、atezolizumab、durvalumab、CS-1001、M-7824、KL-A167、CX-072、BGB-A333、GNS-1480、CA-170、BMS-936559。此处全文引入WO2017084495、WO2020094122中的抗PD-L1抗体。一些实施方案中，前述PD-L1结合蛋白为融合蛋白，所述融合蛋白包含抗PD-1抗体或其抗原结合片段和TGF-βtrap或TGF-βRII ECD；一些具体实施方案中，所述TGF-βRII ECD的氨基酸序列如SEQ ID NO：56所示。
一些实施方案中，前述融合蛋白中，所述TGF-βRII ECD通过连接子与重链全长的C端连接，所述连接子包含或为如(G 4S) x所示的氨基酸序列，其中，x独立地选自1-20的整数；一些具体实施方案中，所述连接子为(G 4S) 4G、(G 4S) 3G、(G 4S) 2G、(G 4S)G。
一些实施方案中，前述融合蛋白包含第一多肽链和第二多肽链，其中：
所述第一多肽链包含如SEQ ID NO：77或78所示或与之具有至少80％，例如，至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列，所述第二多肽链包含如SEQ ID NO：55所示或与之具有至少80％，例如，至少85％、至少86％、至少87％、至少88％、至少 89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的氨基酸序列。
一些具体实施方案中，前述PD-L1结合蛋白包含两条相同的第一多肽链和两条相同的第二多肽链。
一些实施方案中，前述PD-L1结合蛋白包含或为抗PD-L1抗体或其抗原结合片段与TGF-βtrap的融合蛋白，包括但不限于M7824、GS-19、TS-1905、TST-005、HBM-7015、PM-8001、抗PDL1-TGFβRIIecd。此处全文引入WO2011109789、WO2015118175、WO2018218215、WO2020248926、WO2019222252、WO2021007428、WO2021063352、WO2021063350中的抗PD-L1抗体或其抗原结合片段与TGFβRIIecd或TGF-βtrap的融合蛋白。本公开提供了一种PVRIG结合蛋白和TIGIT结合蛋白联合在制备治疗癌症的药物中的用途，具体实施方案中，所述PVRIG结合蛋白包含如SEQ ID NO：16所示的免疫球蛋白单一可变结构域，以及所述TIGIT结合蛋白包含如SEQ ID NO：26所示的HC和如SEQ ID NO：27所示的LC。本公开提供了一种PVRIG结合蛋白、TIGIT结合蛋白和免疫检查点抑制剂联合在制备治疗癌症的药物中的用途，具体实施方案中，所述PVRIG结合蛋白包含如SEQ ID NO：16所示的免疫球蛋白单一可变结构域；所述TIGIT结合蛋白包含如SEQ ID NO：26所示的HC和如SEQ ID NO：27所示的LC；以及所述免疫检查点抑制剂选自：
PD-1结合蛋白，其包含氨基酸序列如SEQ ID NO：65所示HC和氨基酸序列如SEQ ID NO：66所示的LC；
PD-1结合蛋白，其包含氨基酸序列如SEQ ID NO：75所示HC和氨基酸序列如SEQ ID NO：76所示的LC；
PD-L1结合蛋白，其包含氨基酸序列如SEQ ID NO：54所示HC和氨基酸序列如SEQ ID NO：55所示的LC；或
PD-L1结合蛋白，其包含氨基酸序列如SEQ ID NO：77所示HC和氨基酸序列如SEQ ID NO：55所示的LC。本公开还提供了一种PVRIG/TIGIT结合蛋白和免疫检查点抑制剂联合在制备治疗癌症的药物中的用途，具体实施方案中，所述PVRIG/TIGIT结合蛋白包含如SEQ ID NO：43所示的HC(即第一多肽链)和如SEQ ID NO：27所示的LC(即第二多肽链)，以及所述免疫检查点抑制剂选自：
PD-1结合蛋白，其包含如SEQ ID NO：65所示HC和如SEQ ID NO：66所示的LC；
PD-1结合蛋白，其包含如SEQ ID NO：75所示HC和如SEQ ID NO：76所示的LC；
PD-L1结合蛋白，其包含如SEQ ID NO：54所示HC和如SEQ ID NO：55所示的LC；或
PD-L1结合蛋白，其包含如SEQ ID NO：77所示HC和如SEQ ID NO：55所 示的LC。一些实施方案中，所述癌症或肿瘤选自以下或其组合：前列腺癌、肝癌(HCC)、结直肠癌、卵巢癌、子宫内膜癌、乳腺癌、三阴性乳腺癌、胰腺癌、胃(stomach/gastric)癌、宫颈癌、头颈癌、甲状腺癌、睾丸癌、尿路上皮癌、肺癌(小细胞肺癌、非小细胞肺癌)、黑色素瘤、非黑素瘤皮肤癌(鳞状和基底细胞癌)、神经胶质瘤、肾癌(RCC)、淋巴瘤(NHL或HL)、急性骨髓性白血病(AML)、T细胞急性淋巴母细胞性白血病(T-ALL)、弥漫性大B细胞淋巴瘤、睾丸生殖细胞肿瘤、间皮瘤、食道癌、默克细胞癌(Merkel Cells cancer)、高MSI癌、KRAS突变肿瘤、成人T细胞白血病/淋巴瘤和骨髓增生异常综合征(MDS)。上述病症可以是与PVRIG和/或TIGIT异常表达相关的。一些具体实施方案中，所述癌症或肿瘤选自由以下癌症或其组合：黑色素瘤、三阴性乳腺癌、胃癌、肺癌(小细胞肺癌、非小细胞肺癌)、默克细胞癌、高MSI癌、KRAS突变肿瘤、成人T细胞白血病/淋巴瘤和骨髓增生异常综合症(MDS)。一些具体实施方案中，所述癌症或肿瘤选自由以下癌症或其组合：黑色素瘤、三阴性乳腺癌、胃癌、肺癌(小细胞肺癌、非小细胞肺癌)、默克细胞癌和高MSI癌。一些具体实施方案中，所述癌症或肿瘤选自黑色素瘤。
一些实施方案中，所述PVRIG结合蛋白(或其编码多核苷酸)和TIGIT结合蛋白(或其编码多核苷酸)，或PVRIG结合蛋白(或其编码多核苷酸)、TIGIT结合蛋白(或其编码多核苷酸)和免疫检查点抑制剂(例如，PD-1/PD-L1抑制剂)，或PVRIG/TIGIT结合蛋白(或其编码多核苷酸)和免疫检查点抑制剂(例如，PD-1/PD-L1抑制剂)联合用于癌症或肿瘤，给药次序可以同时给药、或者前后分开给药。
一些实施方案中，所述联合的给药途径选自经口给药、胃肠外给药、经皮给药，所述胃肠外给药包括但不限于静脉注射、皮下注射、肌肉注射。
本公开还提供了一种药物套组，或者一种药物包装盒，其中含有前述的PVRIG结合蛋白和TIGIT结合蛋白，或PVRIG结合蛋白、TIGIT结合蛋白和免疫检查点抑制剂，或PVRIG/TIGIT结合蛋白或其编码多核苷酸和免疫检查点抑制剂(例如，PD-1/PD-L1抑制剂)。
本公开还提供了一种药物组合物，包含前述的有效量的PVRIG结合蛋白和TIGIT结合蛋白，或PVRIG结合蛋白、TIGIT结合蛋白和免疫检查点抑制剂，或PVRIG/TIGIT结合蛋白或其编码多核苷酸和免疫检查点抑制剂，以及一种或多种可药用的赋形剂、稀释剂或载体。优选地，所述免疫检查点抑制剂包括但不限于靶向PD-1/PD-L1、PD-L2、B7-1/CTLA-4、B7-2/CTLA-4、B7-1/CD28、B7-2/CD28、B7-H2/ICOS、B7-H3、B7-H4、B7-H7、VISTA(PD-1H)、BTNL2、TIM3、LAG3、TNFSF/TNFRSF(如CD40-CD40L、CD27-CD70或OX40-OX40L)、白细胞免疫球蛋白样受体(LIR/LILR)或免疫球蛋白样转录子(ILT)、粘连蛋白或类粘连蛋白受体蛋白(如DNAM-1、CD226、PTA1、TLISA1、CD96、TIGIT、Vstm3和VSIG9)、NKG2A、 KIRs、NKG2A、Siglecs family(Siglec-7/9)、CD47、CD94、CD200信号通路的抑制剂；更优选地，所述免疫检查点抑制剂是PD-1/PD-L1信号通路抑制剂；最优选地，所述PD-1/PD-L1信号通路抑制剂选自前述的PD-1结合蛋白或其编码多核苷酸，和/或前述的PD-L1结合蛋白或其编码多核苷酸。
本公开提供一种产品，包含前述的有效量的PVRIG结合蛋白和TIGIT结合蛋白，或PVRIG结合蛋白、TIGIT结合蛋白和免疫检查点抑制剂，或PVRIG/TIGIT结合蛋白和免疫检查点抑制剂，或PVRIG结合蛋白和免疫检查点抑制剂。
另一方面，本公开还提供一种治疗或缓解癌症的方法，包括向有需要的受试者施用治疗或缓解有效量的PVRIG结合蛋白和TIGIT结合蛋白，或PVRIG结合蛋白、TIGIT结合蛋白和免疫检查点抑制剂，或PVRIG/TIGIT结合蛋白或其编码多核苷酸和免疫检查点抑制剂。
以及，本公开提供一种向有需要的受试者联合施用治疗或缓解有效量的PVRIG结合蛋白和TIGIT结合蛋白，或PVRIG结合蛋白、TIGIT结合蛋白和免疫检查点抑制剂，或PVRIG/TIGIT结合蛋白和免疫检查点抑制剂，或PVRIG结合蛋白和免疫检查点抑制剂用于治疗或缓解癌症的用途。
以及，本公开提供一种前述PVRIG/TIGIT结合蛋白，或前述PVRIG结合蛋白和TIGIT结合蛋白治疗或缓解癌症的方法，或前述PVRIG结合蛋白，包括施用免疫检查点抑制剂。
以及，本公开提供一种免疫检查点抑制剂治疗或缓解癌症的方法，包括施用前述PVRIG/TIGIT结合蛋白，或施用前述PVRIG结合蛋白和TIGIT结合蛋白，或前述PVRIG结合蛋白。
本公开还提供一种活化NK细胞、γδT细胞和/或Th1细胞的方法，包含向有需要的受试者施用有效量的前述药物组合物或前述产品。
本公开还提供一种增加受试者体内的IFN-γ产生和/或促炎性细胞因子分泌的方法，包含向有需要的受试者施用有效量的前述药物组合物或前述产品。
本公开还提供一种抑制PD-1活性或促进T细胞增殖或使受试者从免疫反应上调获益的方法，包括向受试者施用有效量的前述药物组合物或前述产品；例如，所述受试者的PD-L1和/或PD-L2的表达是上调的；又例如，所述受试者患有癌症。
一些实施方案中，前述PVRIG/TIGIT结合蛋白和前述免疫检查点抑制剂具有抑制癌细胞生长的协同作用，相较于单药疗法，能够显著改善免疫检查点抑制剂的抑癌效果。
本公开全文引入PCT/CN2021/080470中的双特异性抗体结构及其制备方法，特别是1708-30H2相关的。此外，本公开提供多核苷酸和载体：
本公开提供编码本公开的PVRIG结合蛋白、PVRIG/TIGIT结合蛋白、免疫检查点抑制剂(例如，PD-1结合蛋白、PD-L1结合蛋白)的多核苷酸。本公开的多核苷酸可为RNA、DNA或cDNA。根据本公开的一些实施方案，本公开的多核苷酸 是基本上分离的多核苷酸。
本公开的多核苷酸也可呈载体形式，可存在于载体中和/或可为载体的一部分，该载体例如质粒、粘端质粒、YAC或病毒载体。载体可尤其为表达载体，即可提供PD-1结合蛋白体外和/或体内(即在适合宿主细胞、宿主有机体和/或表达系统中)表达的载体。该表达载体通常包含至少一种本公开的多核苷酸，其可操作地连接至一个或多个适合的表达调控元件(例如启动子、增强子、终止子等)。针对在特定宿主中的表达对所述元件及其序列进行选择为本领域技术人员的常识。对本公开的PVRIG结合蛋白、PVRIG/TIGIT结合蛋白、免疫检查点抑制剂(例如，PD-1结合蛋白、PD-L1结合蛋白)的表达有用或必需的调控元件及其他元件例如为启动子、增强子、终止子、整合因子、选择标记物、前导序列、报告基因。
本公开的多核苷酸可基于本公开的多肽的氨基酸序列的信息通过已知的方式(例如通过自动DNA合成和/或重组DNA技术)制备或获得，和/或可从适合的天然来源加以分离。此外，本公开提供宿主细胞：
本公开提供表达或能够表达一种或多种本公开的PVRIG结合蛋白、PVRIG/TIGIT结合蛋白、免疫检查点抑制剂(例如，PD-1结合蛋白、PD-L1结合蛋白)和/或含有本公开的核酸或载体的重组宿主细胞。一些实施方案中，宿主细胞为细菌细胞、真菌细胞或哺乳动物细胞。
细菌细胞例如包括革兰氏阴性细菌菌株(例如大肠杆菌(Escherichia coli)菌株、变形杆菌属(Proteus)菌株及假单胞菌属(Pseudomonas)菌株)及革兰氏阳性细菌菌株(例如芽孢杆菌属(Bacillus)菌株、链霉菌属(Streptomyces)菌株、葡萄球菌属(Staphylococcus)菌株及乳球菌属(Lactococcus)菌株)的细胞。
真菌细胞例如包括木霉属(Trichoderma)、脉孢菌属(Neurospora)及曲菌属(Aspergillus)的物种的细胞；或者包括酵母属(Saccharomyces)(例如酿酒酵母(Saccharomyces cerevisiae))、裂殖酵母属(Schizosaccharomyces)(例如粟酒裂殖酵母(Schizosaccharomyces pombe))、毕赤酵母属(Pichia)(例如巴斯德毕赤酵母(Pichia pastoris)及嗜甲醇毕赤酵母(Pichia methanolica))及汉森酵母属(Hansenula)的物种的细胞。
哺乳动物细胞例如包括例如HEK293细胞、CHO细胞、BHK细胞、HeLa细胞、COS细胞等。
然而，本公开也可使用两栖类细胞、昆虫细胞、植物细胞及本领域中用于表达异源蛋白的任何其他细胞。
本公开的细胞不能发育成完成的植株或动物个体。此外，本公开提供生产、制备方法：
本公开提供制备本公开的PVRIG结合蛋白、PVRIG/TIGIT结合蛋白、免疫检查点抑制剂(例如，PD-1结合蛋白、PD-L1结合蛋白)的目的蛋白方法，所述方法通常包含以下步骤：
-在允许表达本公开的目的蛋白的条件下培养本公开的宿主细胞；及
-从培养物回收由所述宿主细胞表达的目的蛋白；及
-任选的，包括进一步纯化和/或修饰本公开的目的蛋白。
本公开的PVRIG结合蛋白、PVRIG/TIGIT结合蛋白、免疫检查点抑制剂(例如，PD-1结合蛋白、PD-L1结合蛋白)可在如上所述细胞中以细胞内方式(例如在细胞质中、在周质中或在包涵体中)产生，接着从宿主细胞分离且任选进一步纯化；或其可以细胞外方式(例如在培养宿主细胞的培养基中)产生，接着自培养基分离且任选进一步纯化。
用于重组产生多肽的方法及试剂，例如特定适合表达载体、转化或转染方法、选择标记物、诱导蛋白表达的方法、培养条件等在本领域中是已知的。类似地，适用于制造本公开的结合分子或抗体等目的蛋白的分离及纯化技术为本领域技术人员所公知。生产和纯化抗体的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南(5-8章和15章)。本公开工程化的抗体也可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至表达载体。重组的免疫球蛋白表达载体可以稳定地转染细胞。哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛，离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
然而，本公开PVRIG结合蛋白、PVRIG/TIGIT结合蛋白、免疫检查点抑制剂(例如，PD-1结合蛋白、PD-L1结合蛋白)也可以通过本领域已知的其它产生蛋白质的方法获得，例如化学合成，包括固相或液相合成。此外，本公开提供组合物或组合：
本公开提供组合物或组合，包含：
前述PVRIG/TIGIT结合蛋白和免疫检查点抑制剂(例如，PD-1结合蛋白或PD-L1结合蛋白)；或，
前述PVRIG结合蛋白和免疫检查点抑制剂(例如，PD-1结合蛋白或PD-L1结合蛋白)。
例如，提供药物组合物，其含有对癌症治疗、缓解或预防有效量的如上所述的组合物或组合，和至少一种可药用的赋形剂、稀释或载体。
在一些具体实施方式中，所述药物组合物单位计量中可含有0.01至99重量％的前述PVRIG/TIGIT结合蛋白和免疫检查点抑制剂(例如，PD-1结合蛋白或PD-L1结合蛋白)，或药物组合物单位剂量中含前述PVRIG/TIGIT结合蛋白和免疫检查点抑制剂(例如，PD-1结合蛋白或PD-L1结合蛋白)的量为0.1-2000mg，在一些具体实施方式中为1-1000mg。
一些实施方案中，提供制品或产品，包含组合物或组合。可选地，制品包含容器和标签。容器例如瓶、注射器和试管。容器容纳有效于治疗病症的组合物或组合。容器上或与容器相连的标签表明所述组合物用于治疗所选病症。示例性的，提供组合物或组合，其含有：
PVRIG/TIGIT结合蛋白和PD-1结合蛋白；或
PVRIG/TIGIT结合蛋白和PD-L1结合蛋白。
上述PVRIG/TIGIT结合蛋白包含特异性结合PVRIG的第一抗原结合结构域和特异性结合TIGIT的第二抗原结合结构域，其中：
所述特异性结合PVRIG的第一抗原结合结构域包含免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含：如SEQ ID NO：4、5、20所示的CDR1、CDR2和CDR3；
如SEQ ID NO：4、5、6所示的CDR1、CDR2和CDR3；
如SEQ ID NO：7、8、9所示的CDR1、CDR2和CDR3；
如SEQ ID NO：2、15-19任一所示或与之具有至少90％序列同一性的氨基酸序列；
如SEQ ID NO：3、21-25任一所示或与之具有至少90％序列同一性的氨基酸序列；
所述特异性结合TIGIT的第二抗原结合结构域包含重链可变区(VH)和轻链可变区(VL)，所述重链可变区包含如SEQ ID NO：38所示或与之具有至少90％序列同一性的氨基酸序列，所述轻链可变区包含如SEQ ID NO：42所示或与之具有至少90％序列同一性的氨基酸序列。
上述PVRIG/TIGIT结合蛋白包含第一多肽链和第二多肽链，其中：
所述第一多肽链包含如SEQ ID NO：28-29、43-45中任一所示的氨基酸序列，所述第二多肽链包含如SEQ ID NO：27所示的氨基酸序列；或者
所述第一多肽链包含如SEQ ID NO：26所示的氨基酸序列，所述第二多肽链包含如SEQ ID NO：30、31所示的氨基酸序列。
上述PD-1结合蛋白包含重链可变区(VH)和轻链可变区(VL)，其中：
所述重链可变区包含如SEQ ID NO:59、60、61所示氨基酸序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含如SEQ ID NO:62、63、64所示氨基酸序列的LCDR1、LCDR2和LCDR3；
所述重链可变区包含如SEQ ID NO:67、68、69所示氨基酸序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含如SEQ ID NO:70、71、72所示氨基酸序列的LCDR1、LCDR2和LCDR3；
所述重链可变区包含如SEQ ID NO：57所示或与之具有至少90％同一性的氨基酸序列，轻链可变区包含如SEQ ID NO：58所示或与之具有至少90％同一性的氨基酸序列；或
所述重链可变区包含如SEQ ID NO：73所示或与之具有至少90％同一性的氨基酸序列，轻链可变区包含如SEQ ID NO：74所示或与之具有至少90％同一性的氨基酸序列。
上述PD-1结合蛋白包含重链全长(HC)和轻链全长(LC)，其中：
所述重链全长包含如SEQ ID NO：65所示或与之具有至少90％同一性的氨基酸序列，轻链全长包含如SEQ ID NO：66所示或与之具有至少90％同一性的氨基酸序列；或
所述重链全长包含如SEQ ID NO：75所示或与之具有至少90％同一性的氨基酸序列，轻链全长包含如SEQ ID NO：76所示或与之具有至少90％同一性的氨基酸序列。
上述PD-L1结合蛋白包含包含重链可变区(VH)和轻链可变区(VL)，其中：
所述重链可变区包含如SEQ ID NO:48、49、50所示氨基酸序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含如SEQ ID NO:51、52、53所示氨基酸序列的LCDR1、LCDR2和LCDR3；
所述重链可变区包含如SEQ ID NO：46所示或与之具有至少90％同一性的氨基酸序列，轻链可变区包含如SEQ ID NO：47所示或与之具有至少90％同一性的氨基酸序列。
上述PD-L1结合蛋白包含重链全长(HC)和轻链全长(LC)，其中：
所述重链全长包含如SEQ ID NO：54所示或与之具有至少80％同一性的氨基酸序列，轻链全长包含如SEQ ID NO：55所示或与之具有至少80％同一性的氨基酸序列。
上述PD-L1结合蛋白中还包含TGF-βRII ECD结构域，例如，所述TGF-βRII ECD结构域的氨基酸序列如SEQ ID NO：56所示。
上述PD-L1结合蛋白包含第一多肽链和第二多肽链，所述第一多肽链包含如SEQ ID NO：77或78所示或与之具有至少90％同一性的氨基酸序列，所述第二多肽链包含如SEQ ID NO：55所示或与之具有至少90％同一性的氨基酸序列。
本公开同时提供上述组合物或组合治疗、缓解前述肿瘤的方法或制药用途，活化NK细胞、γδT细胞和/或Th1细胞的方法或制药用途，增加受试者体内的IFN-γ产生和/或促炎性细胞因子分泌的方法或用途，抑制PD-1活性或促进T细胞增殖或使受试者从免疫反应上调获益的方法或用途，所述受试者的PD-L1和/或PD-L2的表达可以是上调的。本公开中，“至少90％(序列)同一性”涵盖至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％(序列)同一性；“至少80％(序列)同一性”涵盖至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％(序列)同一性。
附图说明
图1为抗PVRIG纳米抗体30、151的混合淋巴细胞反应(MLR)实验结果，使用Tab5作为阳性对照，hIgG4作为阴性对照。
图2为抗PVRIG/TIGIT双抗1708-151H8的MLR实验结果，同时检测151H8、1708，hIgG4、Tab5、Pembrolizumab作为对照。
图3A为抗PVRIG/TIGIT双抗1708-151、1708和151的联用在人黑色素瘤A375混合人PBMC的小鼠皮下移植瘤模型中的小鼠体重图，图3B为对应的小鼠肿瘤体积图。
图4A为抗PVRIG/TIGIT双抗1708-151H7和1708-30H2在人黑色素瘤A375混合人PBMC的小鼠皮下移植瘤模型中的小鼠体重图，图4B为对应的小鼠肿瘤体积图。
图5A为药物A和B单独或联用时对人黑色素瘤A375混合PBMC皮下移植瘤模型的肿瘤体积的作用结果图，图5B为对应的肿瘤重量图，图5C为对应的小鼠体重图，图5D为对应的小鼠体重相对变化图。
图6A为药物A和C单独或联用时对人黑色素瘤A375混合PBMC皮下移植瘤模型的肿瘤体积的作用结果图，图6B为对应的肿瘤重量图，图6C为对应的小鼠体重图，图6D为对应的小鼠体重相对变化图。
图7A为药物A和D单独或联用时对人黑色素瘤A375混合PBMC皮下移植瘤模型的肿瘤体积的作用结果图，图7B为对应的肿瘤重量图，图7C为对应的小鼠体重图。
具体实施方式
术语定义
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除非在本公开中另有明确定义，本公开使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
“程序性死亡1”、“细胞程序性死亡1”、“蛋白PD-1”、“PD-1”、“PDCD1”和“hPD-1”可互换使用，且包括人PD-1的变体、同种型、物种同源物、以及与PD-1具有至少一个共同表位的类似物。完整的PD-1序列可以从GenBank登录号U64863找到。“程序性死亡配体-1(PD-L1)”是PD-1的两种细胞表面糖蛋白配体之一(另一种为PD-L2)，它在与PD-1结合时下调T细胞活化和细胞因子分泌。如本文中使用的“PD-L1”包括人PD-L1(hPD-L1)，hPD-L1的变体、同种型、和种间同源物，以及与hPD-L1具有至少一个共同表位的类似物。完整的hPD-L1序列 可以用GenBank登录号Q9NZQ7查到。
“PVRIG”或“PVRIG蛋白质”或“PVRIG多肽”可以任选地包括任何这类蛋白质或其变异体、结合物或片段，包括(但不限于)如本文所述的已知或野生型PVRIG，以及任何天然产生的剪接变异体、氨基酸变异体或同工型，并且尤其是PVRIG的可溶性胞外域(ECD)片段。此处ECD的定义如专利WO2016134333中的。完整的人类PVRIG序列可以GenBank登录号AAH73861.1找到。
“TIGIT”或“TIGIT蛋白质”或“TIGIT多肽”可以任选地包括任何这类蛋白质或其变异体、结合物或片段，包括(但不限于)如本文所述的已知或野生型TIGIT，以及任何天然产生的剪接变异体、氨基酸变异体或同工型。完整的TIGIT序列可以GenBank登录号AAI01289.1找到。
“与PVRIG结合”，指能与PVRIG或其表位相互作用，所述PVRIG或其表位可以是人源的。“与TIGIT结合”，指能与TIGIT或其表位相互作用，所述TIGIT或其表位可以是人源的。“抗原结合位点”指抗原上不连续的，由本公开抗体或抗原结合片段识别的三维空间位点。
“免疫检查点(immune checkpoint)分子”包括刺激性免疫检查点分子和抑制性免疫检查点分子，示例性分子包括CD27、CD28、CD40、CD40L、CD122、OX40、OX40L、GITR、ICOS、A2AR、B7-H3、B7-H4、B7-H7、BTLA、CTLA-4、IDO、KIR(Killer-cell Immunoglobulin-like Receptor)、LAG3、PD-1、PD-L1、PD-L2、TIM-3、VISTA、TIGIT、NKG2A等。
“抗体”以最广义使用，涵盖各种抗体结构，包括但不限于单克隆抗体，多克隆抗体；单特异性抗体，多特异性抗体(例如双特异性抗体)，全长抗体和抗体片段(或抗原结合片段，或抗原结合部分)，只要它们展现出期望的抗原结合活性。抗体可以指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(CDR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1，LCDR2，和LCDR3；重链的3个CDR区指HCDR1，HCDR2和HCDR3。
“抗原结合片段”涵盖单链抗体(即全长重链和轻链)；Fab、修饰的Fab、Fab’、修饰的Fab’、F(ab’)2、Fv、Fab-Fv、Fab-dsFv、单结构域抗体(例如VH或VL或VHH)、scFv、二价或三价或四价抗体、Bis-scFv、diabody、tribody、triabody、tetrabody和上述任意一种的表位结合片段(参见例如Holliger and Hudson,2005,Nature Biotech.23(9)：1126-1136；Adair and Lawson,2005,Drug Design Reviews-Online 2(3),209-217)。产生和制备这些抗原结合片段的方法在本领域是公知的(参见例如Verma等人,1998,Journal ofImmunological Methods,216,165-181)。Fab-Fv形式首先公开于WO2009/040562，其二硫键稳定化形式Fab-dsFv首先公开于WO2010/035012。本公开的抗原结合片段还包括描述于WO2005/003169、WO2005/003170和WO2005/003171中的Fab和Fab’片段。多价抗体可包含多特异性例如双特异性或可以是单特异性的(参见例如WO92/22583和WO05/113605)，后者的一个示例是描述于WO 92/22583中的Tri-Fab(或TFM)。
“双特异性抗体”涵盖对两个不同抗原或同一抗原的至少两个不同抗原表位特异性结合的抗体(包括抗体或其抗原结合片段，如单链抗体)。现有技术已公开了各种结构的双特异性抗体，根据IgG分子的完整性可分为IgG样双特异性抗体和抗体片段型双特异性抗体，根据抗原结合区域的数量可分为二价、三价、四价或更多价的双特异性抗体，根据结构左右是否对称可分为对称结构双特异性抗体和不对称结构双特异性抗体。其中，基于抗体片段的双特异性抗体，例如缺乏Fc片段的Fab片段，其通过将2个或多个Fab片段结合在一个分子中形成双特异性抗体，其具有较低的免疫原性，且分子量小，具有较高的肿瘤组织渗透性，该类型的典型的抗体结构如F(ab)2、scFv-Fab、(scFv)2-Fab等双特异性抗体；IgG样双特异性抗体(例如具有Fc片段)，这类抗体相对分子量较大，Fc片段有助于抗体后期的纯化，并提高其溶解性、稳定性，Fc部分还可能会与受体FcRn结合，增加抗体血清半衰期，典型的双特异性抗体结构模型如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、F(ab)4-CrossMAb等双特异性抗体(参见Aran F.Labrijn等，Nature Reviews Drug Discovery volume 18，pages585–608(2019)；Chen S1等，J Immunol Res.2019 Feb 11；2019：4516041)。
在未特殊指明的情况下，本公开的抗体通常使用Kabat编号系统。Kabat中的EU编号一般也用于恒定结构域和/或Fc结构域。
对于CDR的确定或定义，能够通过分辨抗体的结构和/或分辨抗体-配体复合物的结构来完成CDR的确定性描绘和包含抗体的结合位点的残基的鉴定。这可通过本领域技术人员已知的各种技术中的任一种，例如X射线晶体学来实现。多种分析方法可用于鉴定CDR，包括但不限于Kabat编号系统、Chothia编号系统、AbM 编号系统、IMGT编号系统、接触定义、构象定义。
Kabat编号系统是用于编号抗体中残基的标准并且通常用于鉴定CDR区域(参见例如Johnson&Wu，2000，Nucleic Acids Res.，28：214-8)。Chothia编号系统与Kabat编号系统类似，但Chothia编号系统考虑了某些结构环区域的位置。(参见例如Chothia等，1986，J.Mol.Biol.，196：901-17；Chothia等人，1989，Nature，342：877-83)。AbM编号系统使用建模抗体结构的由Oxford Molecular Group生产的计算机程序集成套件(参见例如Martin等，1989，ProcNatl Acad Sci(USA)，86：9268-9272；“AbMTM，A Computer Program for ModelingVariable Regions of Antibodies，”Oxford，UK；Oxford Molecular，Ltd)。AbM编号系统使用知识数据库和从头开始方法的组合，从基本序列建模抗体的三级结构(参见Samudrala等，1999，在PROTEINS，Structure，Function and Genetics Suppl.，3：194-198中的“Ab Initio Protein Structure Prediction Using a Combined HierarchicalApproach”描述的那些)。接触定义基于可用复杂晶体结构的分析(参见例如MacCallum等，1996，J.Mol.Biol.，5：732-45)。构象定义中，CDR的位置可鉴定为对抗原结合做出焓贡献的残基(参见例如Makabe等，2008，Journal ofBiological Chemistry，283：1156-1166)。另外其它的CDR边界定义可能不严格遵循上述方法之一，但仍然与Kabat CDR的至少一部分重叠，尽管根据特定残基或残基组不显著影响抗原结合的预测或实验结果，它们可缩短或延长。如本公开使用的，CDR可指通过本领域已知的任何方法(包括方法的组合)定义的CDR。各种编号系统之间的对应关系是本领域技术人员熟知的，示例性的，如下表中所示。
CDR IMGT Kabat AbM Chothia Contact
HCDR1 27-38 31-35 26-35 26-32 30-35
HCDR2 56-65 50-65 50-58 52-56 47-58
HCDR3 105-117 95-102 95-102 95-102 93-101
LCDR1 27-38 24-34 24-34 24-34 30-36
LCDR2 56-65 50-56 50-56 50-56 46-55
LCDR3 105-117 89-97 89-97 89-97 89-96
本公开的抗体的VL区和VH区的CDR氨基酸残基在数量和位置符合已知的Kabat编号系统。
本公开的抗体可以是多克隆的、单克隆的、异种的、同种异体的、同基因的或其经过修饰的形式，其中单克隆抗体尤其适用于多个实施例中。一般来说，本公开的抗体是重组抗体。如本文所用的“重组”泛指例如细胞或核酸、蛋白质或载体等产品，表示所述细胞、核酸、蛋白质或载体已经通过引入异源核酸或蛋白质或改变天然核酸或蛋白质而加以修饰，或所述细胞来源于如此修饰的细胞。例如，重组细胞表达天然(非重组)细胞形式内不存在的基因或表达原本异常表达、低表达或完全不表达的天然基因。
多肽或蛋白的“结构域”是指折叠蛋白结构，其能够独立于蛋白的其余部分维持其三级结构。一般而言，结构域负责蛋白的单个功能性质，且在许多情况下可添加、移除或转移至其它蛋白而不损失蛋白的其余部分和/或结构域的功能。
“免疫球蛋白结构域”是指抗体链(例如常规四肽链结构抗体的链或重链抗体的链)的球形区域，或是指基本上由这类球形区域组成的多肽。免疫球蛋白结构域的特征在于其维持抗体分子的免疫球蛋白折叠特征，其由排列在两个β折叠中任选由保守二硫键稳定的约7个反平行β折叠股的2层夹层组成。
“免疫球蛋白可变结构域”是指基本上由本领域及下文中分别称为“框架区1”或“FR1”、“框架区2”或“FR2”、“框架区3”或“FR3”、及“框架区4”或“FR4”的四个“框架区”组成的免疫球蛋白结构域，其中所述框架区由本领域及下文中分别称为“互补决定区1”或“CDR1”、“互补决定区2”或“CDR2”、及“互补决定区3”或“CDR3”的三个“互补决定区”或“CDR”间隔开。因此，免疫球蛋白可变结构域的一般结构或序列可如下表示为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。免疫球蛋白可变结构域因具有抗原结合位点而赋予其对抗原的特异性。
“免疫球蛋白可变结构域”是指基本上由本领域及下文中分别称为“框架区1”或“FR1”、“框架区2”或“FR2”、“框架区3”或“FR3”、及“框架区4”或“FR4”的四个“框架区”组成的免疫球蛋白结构域，其中所述框架区由本领域及下文中分别称为“互补决定区1”或“CDR1”、“互补决定区2”或“CDR2”、及“互补决定区3”或“CDR3”的三个“互补决定区”或“CDR”间隔开。因此，免疫球蛋白可变结构域的一般结构或序列可如下表示为:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。免疫球蛋白可变结构域因具有抗原结合位点而赋予其对抗原的特异性。
“抗体框架(FR)”，是指可变结构域的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。
“免疫球蛋白单一可变结构域”通常用于指可以在不与其他可变结构域相互作用的情况下(例如在没有如常规四链单克隆抗体的VH和VL结构域之间所需要的VH/VL相互作用的情况下)，形成功能性抗原结合位点的免疫球蛋白可变结构域(其可以是重链或轻链结构域，包括VH、VHH或VL结构域)。“免疫球蛋白单一可变结构域”的实例包括纳米抗体(包括VHH、人源化VHH和/或骆驼化VH，例如骆驼化人VH)、IgNAR、结构域、作为VH结构域或衍生自VH结构域的(单结构域)抗体(诸如dAbsTM)和作为VL结构域或衍生自VL结构域的(单结构域)抗体(诸如dAbsTM)。基于和/或衍生自重链可变结构域(诸如VH或VHH结构域)的免疫球蛋白单一可变结构域通常是优选地。免疫球蛋白单一可变结构域的一个具体实例为如下文定义的“VHH结构域”(或简称为“VHH”)。
“VHH结构域”，亦称为重链单域抗体、VHH、VHH结构域、VHH抗体片段、 VHH抗体、纳米抗体，是称为“重链抗体”(即“缺乏轻链的抗体”)的抗原结合免疫球蛋白的可变结构域(Hamers-Casterman C，Atarhouch T，Muyldermans S，Robinson G，Hamers C，Songa EB，Bendahman N，Hamers R.:“Naturally occurring antibodies devoid of light chains”；Nature363，446-448(1993))。使用术语“VHH结构域”以将所述可变结构域与存在于常规四肽链结构抗体中的重链可变结构域(其在本公开中称为“VH结构域”)以及轻链可变结构域(其在本公开中称为“VL结构域”)进行区分。VHH结构域特异性结合表位而无需其他抗原结合结构域(此与常规四肽链结构抗体中的VH或VL结构域相反，在该情况下表位由VL结构域与VH结构域一起识别)。VHH结构域为由单一免疫球蛋白结构域形成的小型稳定及高效的抗原识别单元。术语“重链单域抗体”、“VHH结构域”、“VHH”、“VHH结构域”、“VHH抗体片段”、“VHH抗体”、
Figure PCTCN2022118959-appb-000005
以及““
Figure PCTCN2022118959-appb-000006
结构域””(“Nanobody”为Ablynx N.V.公司，Ghent，Belgium的商标)可互换使用。“VHH结构域”包括但不限于经骆驼科动物产生的天然抗体，也可以是骆驼科动物产生的抗体后再经人源化的，也可以是经噬菌体体展示技术筛选获得的。VHH结构域中的氨基酸残基的总数将通常在110至120范围内，常常介于112与115之间。然而应注意较小及较长序列也可适于本公开所述的目的。获得结合特定抗原或表位的VHH的方法，先前已公开于以下文献中：R.van der Linden et al.，Journal of Immunological Methods，240(2000)185-195；Li et al.，J Biol Chem.，287(2012)13713-13721；Deffar et al.，African Journal of Biotechnology Vol.8(12)，pp.2645-2652，17June，2009和WO94/04678。
如本领域中对于VH结构域及VHH结构域所公知的，各CDR中的氨基酸残基的总数可能不同，且可能不对应于由Kabat编号指示的氨基酸残基的总数(即根据Kabat编号的一个或多个位置可能在实际序列中未被占据，或实际序列可能含有多于Kabat编号所允许数目的氨基酸残基)。这意味着一般而言，根据Kabat的编号可能对应或可能不对应于实际序列中氨基酸残基的实际编号。其它的编号系统或编码规则包括Chothia、IMGT、AbM。
“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将非人CDR序列移植到人的抗体可变区框架中产生的抗体。可以克服嵌合抗体由于携带大量非人蛋白成分，从而诱导的强烈的免疫应答反应。为避免在免疫原性下降的同时引起活性的下降，可对所述的全人抗体可变区可进行最少反向突变，以保持活性。“人源化”的例子包括可将源自骆驼科的VHH结构域通过以人常规四肽链结构抗体VH结构域中相应位置处存在的一个或多个氨基酸残基置换原始VHH序列的氨基酸序列中的一个或多个氨基酸残基而“人源化”(本公开中亦称为“序列优化”，除人源化外，“序列优化”也可涵盖通过提供VHH改良性质的一个或多个突变对序列进行的其它修饰，例如移除潜在的翻译后修饰位点)。人源化VHH结构域可含有一个或多个完全人框架区序列，且在一些具体 实施方案中，可含IGHV3的人框架区序列。“人源化”的又一例子包括将小鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体构架序列中产生的抗体。可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导的强烈的抗体可变抗体反应。人源化方法例如蛋白表面氨基酸人源化(resurfacing)及抗体人源化通用框架移植法(CDR grafting to a universal framework)，即将CDR“移植”于其它“支架”(包括但不限于人支架或非免疫球蛋白支架)上。适于所述CDR移植的支架及技术在本领域中是已知的。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库，以及在Kabat，E.A.等人，1991 Sequences of Proteins of Immunological Interest，第5版中找到。本公开的人源化抗体也包括进一步由噬菌体展示对CDR进行亲合力成熟后的人源化抗体。此外，为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。
“亲和力成熟的”抗体指与不拥有此类改变的亲本抗体相比，在一个或多个高变区(HVR)中具有一处或多处改变的抗体,此类改变导致该抗体对抗原的亲和力改善。例如，“亲合力成熟”的PD-1结合蛋白或PD-1抗体，在一个或多个CDR中具有一个或多个变化，所述变化导致对抗原的亲合力相比于其亲本抗体有所增加。亲合力成熟的抗体可通过例如由以下所述的本领域中已知的方法来制备：Marks等人，1992，Biotechnology 10：779-783或Barbas等人，1994，Proc.Nat.Acad.Sci，USA 91：3809-3813.；Shier等人，1995，Gene 169：147-155；Yelton等人，1995，Immunol.155：1994-2004；Jackson等人，1995，J.Immunol.154(7)：3310-9；及Hawkins等人，1992，J.MoI.Biol.226(3)：889896；KS Johnson及RE Hawkins，“Affinity maturation of antibodies using phage display”，Oxford University Press 1996。
“全人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本公开的全人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。“全人抗体”不包括“人源化抗体”。
当“竞争”用于竞争相同表位的抗原结合蛋白(例如中和抗原结合蛋白或中和抗体)的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定：待检测的抗原结合蛋白(例如抗体或其免疫学功能片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原(例如PVRIG抗原或其片段)的特异性结合。众多类型的竞争性结合测定可用于确定一种抗原结合蛋白是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methodsin Enzymology 9：242-253)；固相直接生物素-亲和素EIA(参见例如Kirkland等，1986，J.Immunol.137：3614-3619)、固相直接标记测定、固相直接标记夹心测定(参见例如Harlow和Lane，1988，Antibodies，A Laboratory Manual(抗体，实验室手册)，Cold Spring Harbor Press)； 用I-125标记物的固相直接标记RIA(参见例如Morel等，1988，Molec.Immunol.25：7-15)；固相直接生物素-亲和素EIA(参见例如Cheung，等，1990，Virology176：546-552)；和直接标记的RIA(Moldenhauer等，1990，Scand.J.Immunol.32：77-82)。通常所述测定法涉及使用能与带有未标记的检测抗原结合蛋白及标记的参考抗原结合蛋白结合的纯化抗原(所述抗原在固态表面或细胞表面上)。在待测抗原结合蛋白存在下，测量结合于固态表面或细胞的标记的量，来测量竞争性抑制。通常，待测抗原结合蛋白是过量存在的。由竞争性测定(竞争抗原结合蛋白)鉴定的抗原结合蛋白包括：与参考抗原结合蛋白相同的表位发生结合的抗原结合蛋白；以及，与充分接近参考抗原结合蛋白结合的表位所邻近的表位发生结合的抗原结合蛋白，所述两个表位在空间上互相妨碍结合的发生。在本公开实施例中提供关于用于测定竞争性结合的方法的其它详细资料。通常当竞争的抗原结合蛋白过量存在时，其将抑制(例如降低)至少40-45％、45-50％、50-55％、55-60％、60-65％、65-70％、70-75％或75％或更多参考抗原结合蛋白与共同抗原的特异性结合。在某些情况下，结合被抑制至少80-85％、85-90％、90-95％、95-97％或97％或更多。可使用本领域技术人员已知的常规技术，就与相同表位的结合竞争性筛选抗体。例如，可进行竞争和交叉竞争研究，以获得彼此竞争或交叉竞争与抗原结合的抗体。基于它们的交叉竞争来获得结合相同表位的抗体的高通量方法描述于国际专利公开WO03/48731中。因此，可使用本领域技术人员已知的常规技术，获得例如与本公开的抗体分子竞争结合PD-1上的相同表位的抗体。
“特异性结合”、“选择性结合”是指抗体与预定的抗原上的表位结合。例如，当使用人PD-1或其表位作为分析物并使用抗体作为配体，在仪器中通过表面等离子体共振(SPR)技术测定时，抗体以大约低于10-7M或甚至更小的平衡解离常数(KD)与预定的抗原或其表位结合，并且其与预定抗原或其表位结合的亲和力是其与预定抗原(或其表位)或紧密相关的抗原之外的非特异性抗原(如BSA等)结合的亲和力的至少两倍。“识别抗原的抗体”在本公开中可以与“特异性结合的抗体”互换使用。
“结合亲和力”或“亲和力”在本公开中用作两个分子(例如抗体或其部分与抗原)之间的非共价相互作用的强度量度。两个分子之间的结合亲和力可通过确定解离常数(KD)来量化。可通过使用例如表面等离子共振(SPR)方法(Biacore)测量复合物形成和解离的动力学来确定KD。对应于单价复合物的结合和解离的速率常数分别被称为结合速率常数ka(或kon)和解离速率常数kd(或koff)。KD通过方程KD＝kd/ka与ka和kd有关。解离常数的值可通过众所周知的方法直接确定，并且甚至可通过例如Caceci等人(1984，Byte 9：340-362)中所述的那些方法对于复杂混合物进行计算。例如，可使用双重过滤硝化纤维素滤器结合测定如Wong&Lohman(1993，Proc.Natl.Acad.Sci.USA 90：5428-5432)中公开的那种来确定KD。评估抗体针对靶抗原的结合能力的其它标准测定是本领域已知的，包括例如 ELISA、蛋白质印迹、RIA和流式细胞术分析、以及本公开其它地方例举的其它测定。抗体的结合动力学和结合亲和力也可通过本领域已知的标准测定，例如表面等离子共振(SPR)，例如通过使用BiacoreTM系统或KinExA来评价。可通过比较各个抗体/抗原复合物的KD值来比较与不同分子相互作用相关的结合亲和力，例如，不同抗体对于给定抗原的结合亲和力的比较。类似地，相互作用的特异性可通过确定和比较目的相互作用(例如抗体和抗原之间的特异性相互作用)的KD值与非目的相互作用(例如已知不结合PD-1的对照抗体)的KD值进行评价。
“保守性置换”指置换为具有与原始氨基酸残基相似的特性的另一个氨基酸残基。例如，赖氨酸、精氨酸和组氨酸具有相似的特性，在于它们具有碱性侧链，并且天冬氨酸和谷氨酸具有相似的特性，在于它们具有酸性侧链。此外，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸和色氨酸具有相似的特性，在于它们具有不带电荷极性侧链，并且丙氨酸、缬氨酸、亮氨酸、苏氨酸、异亮氨酸、脯氨酸、苯丙氨酸和甲硫氨酸具有相似的特性，在于它们具有非极性侧链。另外，酪氨酸、苯丙氨酸、色氨酸和组氨酸具有相似的特性，在于它们具有芳族侧链。因此，本领域技术人员将显而易见，甚至当置换如上文所述的显示相似特性的组中的氨基酸残基时，它将不显示特性的特定变化。
“同源性”、“同一性”或“序列同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同核苷酸或氨基酸单体占据时，例如如果两个DNA分子的每一个位置都被相同核苷酸占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100％的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
“核酸”或“多核苷酸”在本文可互换使用，指的是单链或双链的任何DNA分子或RNA分子以及在单链的情况下，它的互补序列的分子，优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
“宿主细胞”包括各个细胞或细胞培养物，其可为或已是用于掺入多核苷酸插入片段的载体的受体。宿主细胞包括单个宿主细胞的子代，并且由于天然、偶然或有意的突变，子代可不一定与原始亲本细胞完全相同(在形态学或基因组DNA互补体中)。宿主细胞包括用本公开的多核苷酸在体内转染和/或转化的细胞。“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括其后代。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。
“抑制”或“阻断”可互换使用，并涵盖部分和完全抑制/阻断这两者。“抑制 生长”(例如涉及细胞)旨在包括细胞生长任何可测量的降低。
“阻止...的生长”或“生长抑制”是指抑制细胞的生长或增殖。
“增殖性疾病”指与一定程度的异常细胞增殖有关的病症。在一个实施方案中，增殖性病症指癌症。“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。“癌症”、“癌性”、“增殖性病症”和“肿瘤”在本公开中提到时并不互相排斥。
“预防癌症”是指在受试者中延迟、抑制或防止癌症发作，所述受试者中癌症发生或肿瘤发生的起始尚未得到证实，但是通过例如遗传筛查或其它方法确定，已鉴定了癌症易感性。该还包括治疗具有癌变前病症的受试者以终止所述癌变前病症向恶性肿瘤的进展或导致其消退。
“给予”、“施用”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触，例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”、“施用”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予受试者内用或外用治疗剂，例如包含本公开的任一种结合蛋白或其药物组合物作为治疗剂，所述受试者已经患有、疑似患有、倾向于患有一种或多种增殖性疾病或其症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗受试者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床能测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如受试者的疾病状态、年龄和体重，以及药物在受试者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)在缓解某个受试者中目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的受试者中应当减轻目标疾病症状。
“有效量”包含足以改善或预防医学病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于受试者的有效量可依据以下因素而变化：如待治疗的病症、受试者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。本公开的受试者可以是动物或人类受试者。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该 说明包括该事件或环境发生或不发生的场合。“和/或”应视为特定揭示两种指定特征或组分中的每一者具有或不具有另一者。因此，诸如本公开中“A和/或B”的词组中所用的术语“和/或”包括“A及B”、“A或B”、“A”(单独)及“B”(单独)。除非上下文另外清楚要求，否则在整个说明书和权利要求书中，应将词语“包含”、“具有”、“包括”等理解为具有包含意义，而不是排他性或穷举性意义；也即，“包括但不仅限于”的意义。
本公开的“受试者”、“患者”意指哺乳动物，尤其灵长类动物，尤其是人。
发明的详细说明
以下结合实施例用于进一步描述，但这些实施例并非限制的范围。
实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，分子克隆，实验室手册，冷泉港实验室；当代分子生物学方法，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.PVRIG/TIGIT双特异性抗体
本实施例中，带his标签的人PVRIG(h-PVRIG-his)重组蛋白、带小鼠IgG2a的Fc标签的人PVRIG(h-PVRIG-mIgG2a Fc)重组蛋白、带人IgG1的Fc标签的小鼠PVRIG(m-PVRIG-hIgG1Fc)购自Acrobiosystems公司。带his标签的PVRL2购自AcroBiosystem(PV2-H52E2)。
实施例1-1.抗PVRIG纳米抗体的筛选和制备
1、免疫抗原、筛选抗原的序列和制备
表1.重组蛋白质的氨基酸序列
名称 氨基酸序列起止 Genbank登录号
h-PVRIG-his Thr41-Asp171 Q6DKI7-1
h-PVRIG-mIgG2a Fc Thr41-Asp171 Q6DKI7-1
m-PVRIG-hIgG1 Fc Ser35-Asp165 A0A1B0GS01-1
带his标签的食蟹猴PVRIG(cyno-PVRIG-his)重组蛋白序列如下:
Figure PCTCN2022118959-appb-000007
2、羊驼免疫、纳米抗体噬菌体展示文库构建和抗体筛选
使用实施例1-1的方法，使用带his标签的人PVRIG重组蛋白(h-PVRIG-his)免疫羊驼。从第56天的骆驼外周血中分离PBMC，提取RNA，反转录成cDNA，构建抗人PVRIG纳米抗体的噬菌体文库。经过3轮筛选，对400个克隆测序，其中2株的可变区序列如表2所示，CDR如表3所示。
表2.抗PVRIG纳米抗体的可变区
Figure PCTCN2022118959-appb-000008
表3.抗PVRIG纳米抗体的CDR(Kabat编号规则)
Figure PCTCN2022118959-appb-000009
将上述抗体可变区与人IgG4重链Fc区域连接，重链Fc区域包括铰链(hinge)区，并带有S228P，F234A，L235A，K447A突变(Eu命名系统)，构建全长抗体。
>hIgG4Fc(S228P/F234A/L235A/K447A)
Figure PCTCN2022118959-appb-000010
>30全长
Figure PCTCN2022118959-appb-000011
>151全长
Figure PCTCN2022118959-appb-000012
WO2016134333中所示的抗PVRIG抗体CPA.7.021筛选自抗体噬菌体库，其亚型为IgG1，能与人PVRIG较好结合，对食蟹猴PVRIG则无结合。将CPA.7.021的重链和轻链可变区，分别与人IgG4重链恒定区(带有S228P，F234A，L235A，K447A突变)和人Kappa轻链恒定区连接，构建阳性抗体Tab5。
>Tab5重链全长
Figure PCTCN2022118959-appb-000013
>Tab5轻链全长
Figure PCTCN2022118959-appb-000014
将表达上述氨基酸序列的核酸序列克隆到pcDNA3.1表达载体，按常规方法进行抗体表达和纯化，经检测，获得目的抗体。
实施例1-2.抗PVRIG纳米抗体与抗原PVRIG的亲合力鉴定
1、ELISA
用直接包被带his标签的PVRIG重组蛋白，加入抗体后，通过加入二抗(HRP偶联的抗一抗Fc的抗体)和HRP底物TMB检测抗体与抗原结合的活性。
人、食蟹猴或小鼠PVRIG蛋白包被96孔板，按1μg/mL浓度每孔100μL，4℃孵育过夜。洗液洗三遍。加入300μL/孔封闭液(PBS+0.05％Tween20+1％BSA)室温孵育1小时。洗液洗三遍。每孔加100μL用稀释液稀释好的抗PVRIG待测抗体。37℃孵育1小时。洗液洗三遍。每孔加入100μL HRP标记的抗人IgG二抗(Sigma，A8667)。37℃孵育1小时。洗液洗三遍。每孔加入100μL TMB，避光反应15分钟。加入50mL/孔的0.16M硫酸。Thermo MμLtiSkanFc酶标仪读取OD450，计算抗PVRIG抗体对PVRIG的结合EC 50值。所有抗体均对人或食蟹猴的PVRIG重组蛋白有较强的结合能力，但不结合小鼠PVRIG重组蛋白。
表4.抗PVRIG抗体对不同种系PVRIG重组蛋白的结合实验结果
抗体编号 人PVRIG-his ELISA EC 50(nM) 猴PVRIG-his ELISA EC 50(nM)
30 0.26 0.16
151 2.15 2.43
Tab5 2.86 无结合
hIgG4 无结合 无结合
2、FACS检测
制备获得表达人或食蟹猴PVRIG基因的HEK293稳转细胞株。在96孔板中每孔接种2x10 5个细胞。300g离心5分钟，去上清，加入100μL待测抗体，4℃孵育1小时。离心去除上清，用200μL洗液(PBS+2％FBS)洗涤3次，加入100μL 1:500稀释的用Alexa Fluor 488标记的抗人IgG二抗(Invitrogen，A-11013)，4℃孵育1小时。离心去除上清，用200μL洗液(PBS+2％FBS)洗涤3次。用100μL PBS重悬细胞，用流式细胞仪(BD FACS Calibur或BD FACS Canto_II)检测。所有抗体均对细胞表面表达的人或食蟹猴的PVRIG有较强的结合能力，明显强于阳性抗体Tab5，而Tab5甚至完全不结合食蟹猴PVRIG。
表5.抗PVRIG抗体对不同种系PVRIG的细胞结合实验结果
抗体编号 人PVRIG FACS EC 50(nM) 猴PVRIG FACS EC 50(nM)
30 N.A. 0.02
151 0.01 2.23
Tab5 2.13 无结合
hIgG4 无结合 无结合
(注：N.A.，not available，表示结合太强，在低浓度条件下抗体亦不发生解离，无法拟合得到准确的EC 50。)
3、Fortebio检测
将Protein A生物传感器(Fortebio，#18-5010)浸泡在200μL的KB缓冲液(PBS，pH 7.4，0.02％tween-20，0.1％BSA)中60秒，进行湿润处理。然后，用KB缓冲液将抗PVRIG抗体稀释到10μg/mL，将传感器置于200μL该溶液中，待读数为1.2nm时停止。将传感器浸泡于KB缓冲液中100秒，以洗脱多余的抗体。将带有his标签的人PVRIG用KB缓冲液以2倍梯度稀释至64nM-4nM之间。将传感器置于该溶液中结合300秒，再置于KB缓冲液中解离600秒。采用动态1：1结合方式拟合，则抗PVRIG抗体与人PVRIG的亲合力如表6所示。
结果显示，所有检测抗体均具有与人PVRIG的高亲合力。
表6.抗PVRIG抗体与人PVRIG的亲合力
抗体编号 Kon(1/Ms) Koff(1/s) K D(M)
30 2.84E+05 2.05E-04 7.23E-10
151 2.61E+05 5.22E-05 2.00E-10
Tab5 7.37E+05 1.61E-05 2.19E-10
实施例1-3.抗PVRIG纳米抗体的功能和活性验证
1、抗PVRIG纳米抗体阻断PVRIG和PVRL2结合实验
人PVRIG重组蛋白(h-PVRIG-mIgG2a Fc)包被96孔板，按1μg/mL浓度每孔100μL，4℃孵育过夜。洗液洗三遍。加入300μL/孔封闭液室温孵育1小时。洗液洗三遍。每孔加50μL稀释好的抗PVRIG待测抗体和50μL带his标签的配体 PVRL2，37℃孵育1小时。洗液洗三遍。每孔加入100μL按1:2000倍稀释的用HRP标记的抗his标签的二抗(Genscrpit)。37℃孵育1小时。洗液洗三遍。每孔加入100μL TMB，避光反应15分钟。加入50μL每孔的0.16M硫酸。Thermo MμLtiSkanFc酶标仪读取450nm OD值，计算抗PVRIG抗体对PVRIG与PVRL2结合阻断的IC 50值。
表7结果显示，所检测抗体均可以强烈抑制人PVRIG与人PVRL2的结合。
表7.抗体对人PVRIG/PVRL2结合的阻断实验
抗体编号 ELISA IC 50(nM)
30 1.11
151 0.37
Tab5 1.16
hIgG4 无阻断
2、抗PVRIG纳米抗体报告基因细胞活性实验
首先，构建plvx-OS8(G418抗性)质粒，转染293F细胞，G418筛选，用流式细胞仪检测克隆细胞OS8的表达同时检测OS8对Jurkat细胞的激活，选择激活程度中等的克隆，得到293F-OS8细胞株；构建plvx-PVRL2质粒，用它感染293F-OS8细胞，用流式细胞仪筛选出PVRL2表达量最高的克隆，从而得到293F-OS8-PVRL2细胞株。
其次，构建plvx-NFAT-Luc(Hygromycin抗性)，包装成慢病毒，感染Jurkat E6.1细胞，加Hygromycin筛选出有抗性的克隆，用OKT3去刺激克隆，筛选出Luciferase信号中等的克隆，得到Jurkat-NFAT-Luc细胞系；构建plvx-PVRIG(Puromycin抗性)载体，包装成慢病毒，感染Jurkat-NFAT-Luc细胞，经流式细胞仪筛选出PVRIG表达量最高的克隆，从而得到Jurkat-NFAT-Luc-PVRIG细胞株。
将1E4个Jurkat-NFAT-Luc-PVRIG细胞与待测抗体在37℃孵育20分钟。加入1E5个293F-OS8-PVRL2细胞，37℃孵育5小时。离心去除上清，加入Luciferase缓冲液(Promega，E6130)裂解细胞，检测荧光值。计算EC 50值评价抗PVRIG抗体的体外细胞活性。实验结果如表8所示。
结果显示，所检测抗体有较强的激活Jurkat细胞中Luciferase的能力，活性是阳性抗体的至少10倍以上，证明这些抗体可以结合PVRIG并阻断PVRL2与PVRIG的结合。
表8.抗PVRIG抗体报告基因细胞活性实验结果
抗体编号 PVRIG报告基因细胞活性实验EC 50(nM)
30 0.06
151 0.04
Tab5 0.74
hIgG4 无结合
3、抗PVRIG纳米抗体的NK细胞杀伤实验
PVRIG在NK细胞上表达，而PVRL2在很多肿瘤细胞(包括K562细胞)中表达。抗PVRIG抗体可以通过阻断PVRL2与PVRIG的结合，解除肿瘤细胞对NK细胞活性的抑制作用。
在96孔板中每孔加入50μL(总计1×10 5个)人恶性非霍奇金淋巴瘤NK92细胞。加入50μL 20nM或100nM待测抗体，37℃孵育30分钟。用洗液洗涤两次，重悬至2×10 5个/mL的密度。加入50μL(总计1×10 4个)的人慢性髓系白血病K562细胞，使得NK92细胞与K562细胞个数的比例为10:1。37℃孵育4小时。使用CytoTox-Glo细胞毒性系统(Promega，G9292)对杀伤活性进行测量。首先加入50μL AAF-Glo试剂，室温孵育15分钟，测量被NK92细胞杀死的K562细胞的荧光。再加入50μL裂解液，室温孵育15分钟，裂解细胞，测量所有细胞的荧光。准备三种对照组，分别是只包括培养液的样品(对照组一)，只包括NK92细胞的样品(对照组二)，只包括K562细胞的样品(对照组三)，进行同样的操作。
根据如下公式，计算杀伤活性：
杀伤活性(％)＝{[(R–BG)–(T–BG)–(E–BG)]/[(TL–BGL)–(T–BG)]}×100
其中，R为加入AAF-Glo后的荧光值，BG为对照组一在加入AAF-Glo的荧光值，E为对照组二在加入AAF-Glo的荧光值，T为对照组三在加入AAF-Glo的荧光值；TL为对照组三在加入裂解液后的荧光值，BGL为对照组一再加入裂解液后的荧光值。
实验结果如表9所示，表明所有检测的抗PVRIG抗体均可以明显的激活NK92细胞、杀伤K562细胞。
表9.抗PVRIG抗体的NK细胞杀伤实验
Figure PCTCN2022118959-appb-000015
4、抗PVRIG纳米抗体的混合淋巴细胞反应(MLR)实验
PVRIG在T细胞上表达，而PVRL2在DC细胞上表达。抗PVRIG抗体可以通过阻断PVRL2与PVRIG的结合，解除DC细胞对T细胞的抑制，活化T细胞。
从第一个体来源的外周血中分离PBMC，将细胞培养于含10％FBS的RPMI1640培养基中，以50ng/mL GM-CSF(Peprotech，300-03-100UG)和50ng/mL IL-4(Peprotech，200-04-100UG)的终浓度添加，每2-3天添加含细胞因子的新鲜培养基；培养6天后，加入1μg/mL LPS(Sigma，L2880-25MG)孵育24小时，收集分 化成熟得到的DC细胞。从第二个体来源的外周血中分离PBMC，使用EasySep人CD3 +T细胞分离试剂盒(Stemcell，17952)从中分离CD3 +T细胞。调整CD3 +T细胞和DC细胞的密度，使得每孔加入1×10 5个CD3 +T细胞和2×10 4个DC细胞。加入待测抗体，37℃孵育120小时，取上清，用ELISA试剂盒(R&D，DY202)检测上清中的IFNγ含量。
如表10和图1所示，相比对照抗体IgG4，所有检测抗PVRIG抗体均可以明显的激活T细胞分泌IFNγ。并且，在低剂量(如4nM、20nM)时，本公开的抗体30和151较之阳性对照Tab5的效果更优。
表10.抗PVRIG抗体混合淋巴细胞反应IFNγ分泌量
Figure PCTCN2022118959-appb-000016
实施例1-4.抗PVRIG纳米抗体的序列改造
通过对选定的抗PVRIG抗体分子进行三维结构同源建模，将抗PVRIG抗体序列与抗体GermLine数据库比较，获得同源性高的人种系模板IGHV3-7*01。将CDR移植到相应的人源模板中。对移植后的单域抗体再次进行三维结构模拟并分析，对包埋残基、与CDR区有直接相互作用的残基，以及对可变区的构象有重要影响的残基进行回复突变，并对CDR区化学不稳定氨基酸残基优化，产生一系列人源化单域抗体。各个单域抗体的人种系模板以及人源化抗体重链可变区序列如下所示。
>30H1可变区
Figure PCTCN2022118959-appb-000017
>30H2可变区
Figure PCTCN2022118959-appb-000018
>30H3可变区
Figure PCTCN2022118959-appb-000019
>30H4可变区
Figure PCTCN2022118959-appb-000020
Figure PCTCN2022118959-appb-000021
>30H5可变区
Figure PCTCN2022118959-appb-000022
可见，抗体30H1-30H5包含如GDCMG(SEQ ID NO：4)所示的CDR1，如TIDNAGRIKYADSVKG(SEQ ID NO：5)所示的CDR2，如GWTFGGQCSPAD(SEQ ID NO：20)所示的CDR3。
>151H2可变区
Figure PCTCN2022118959-appb-000023
>151H4可变区
Figure PCTCN2022118959-appb-000024
>151H7可变区
Figure PCTCN2022118959-appb-000025
>151H8可变区
Figure PCTCN2022118959-appb-000026
>151H9可变区
Figure PCTCN2022118959-appb-000027
将上述人源化抗体重链可变区与人IgG4重链Fc区域连接，构造形成全长抗PVRIG抗体。其中重链Fc区域包括铰链(hinge)区，并带有S228P/F234A/L235A/K447A，或S228P/K447A突变。按常规方法进行抗体的表达和纯化，经检测，得到目的抗体。
实施例1-5.人源化抗PVRIG抗体的活性和功能验证
1、人源化抗PVRIG抗体与表达PVRIG的细胞结合实验
依照实施例1-2的方法，FACS检测抗PVRIG抗体与人或食蟹猴PVRIG的结合。实验结果如表11所示。
表11.抗PVRIG单域抗体对不同种系PVRIG的FACS结合实验结果
抗体编号 人PVRIG FACS EC 50(nM) 猴PVRIG FACS EC 50(nM)
30H1 0.024 0.374
30H2 0.003 0.005
30H3 0.004 0.003
151H4 0.240 0.035
151H7 0.002 0.467
151H8 0.006 N.T.
151H9 0.004 3.942
Tab5 0.160 无结合
hIgG4 无结合 无结合
(注：N.T.，not tested，未测试。)
2、人源化抗PVRIG抗体与PVRIG的亲合力测定
依照实施例1-2的Fortebio检测方法，检测人源化抗PVRIG抗体与人PVRIG的亲合力。如表12所示，所有抗体均具有与人PVRIG的高亲合力。
表12.人源化抗PVRIG抗体与人PVRIG的亲合力
抗体编号 Kon(1/Ms) Koff(1/s) K D(M)
20H5 1.93E+05 1.35E-05 6.98E-11
30H2 1.69E+05 3.25E-04 1.92E-09
30H3 1.48E+05 3.58E-04 2.41E-09
151H7 1.57E+05 1.88E-04 1.20E-09
3、人源化抗PVRIG抗体报告基因细胞活性实验
依照实施例1-3的方法，检测人源化抗PVRIG抗体在报告基因细胞中的活性。实验结果如表13所示。表中列出的抗体均具有激活Jurkat细胞的能力。
表13.人源化抗PVRIG抗体报告基因细胞活性实验
Figure PCTCN2022118959-appb-000028
4、人源化抗PVRIG抗体的活化NK细胞杀伤能力实验
依照实施例1-3的方法，检测人源化抗PVRIG抗体对NK细胞的活化能力。 实验结果如表14-1和表14-2所示。结果显示，所测抗体都有明显的活化NK细胞的能力，促进NK细胞对于靶细胞K562的杀伤。
表14-1.人源化抗PVRIG抗体的NK细胞杀伤实验
Figure PCTCN2022118959-appb-000029
表14-2.人源化抗PVRIG抗体的NK细胞杀伤实验
Figure PCTCN2022118959-appb-000030
实施例1-6.抗PVRIG/TIGIT双抗的制备
为探索不同构造的抗PVRIG/TIGIT双抗对抗体功能的影响，将抗PVRIG单域抗体151通过GGGGSGGGGS(SEQ ID NO：152)连接子与抗TIGIT抗体1708的重链或轻链的N端或C端相连。形成4个抗PVRIG/TIGIT双抗，命名为1708-151-1，1708-151-2，1708-151-3，1708-151-4，分别对应151被连接在1708的重链N端，重链C端，轻链N端和轻链C端。抗TIGIT抗体1708采用人IgG4亚型，并带有S228P(Eu命名系统)的突变。抗TIGIT抗体1708和其与151形成的双特异性抗体序列如下表15所示。抗TIGIT抗体序列信息如表16和表17所示。此处全文引入WO2019062832A中的TIGIT抗体。
表15.抗PVRIG/TIGIT双抗的第一、第二多肽链序列
Figure PCTCN2022118959-appb-000031
Figure PCTCN2022118959-appb-000032
Figure PCTCN2022118959-appb-000033
表16.抗TIGIT抗体重链及轻链CDR区序列(Kabat编号规则)
Figure PCTCN2022118959-appb-000034
表17.抗TIGIT抗体重链VH及轻链VL序列
Figure PCTCN2022118959-appb-000035
将不同的人源化抗PVRIG抗体可变区(30H2，151H7，151H8)连接到抗TIGIT抗体1708的重链N端，即采用1708-151-1类似的双特异性抗体构造，构建双抗。
>1708-30H2第一多肽链
Figure PCTCN2022118959-appb-000036
>1708-151H7第一多肽链
Figure PCTCN2022118959-appb-000037
>1708-151H8第一多肽链
Figure PCTCN2022118959-appb-000038
1708-30H2、1708-151H7、1708-151H8的第二多肽链均与1708的轻链相同(SEQ ID NO：27)。
按常规方法进行抗体的瞬时转染、表达和纯化，经鉴定，得到本公开的全长抗PVRIG/TIGIT双抗。均显示了良好的表达量和纯度。
实施例1-7.抗PVRIG/TIGIT双抗的活性和功能验证
1、双特异性抗体与人PVRIG的结合及对配体PVRL2的阻断
依照实施例1-2的方法，进行实验。结果表明，不同构型的双特异性抗体1708-151-1、1708-151-2、1708-151-3、1708-151-4，与人PVRIG重组蛋白和过表达人PVRIG细胞的结合，以及对PVRL2结合PVRIG的阻断，基本一致、无差异。
2、双特异性抗体与人TIGIT的结合及对配体PVR的阻断
依照实施例1-2的方法(相应的受体和配体换为人TIGIT和人PVR)，进行实验，结果如表18所示。结果表明，不同构型的双特异性抗体1708-151-1、1708-151-2、1708-151-3、1708-151-4和抗TIGIT抗体，与人TIGIT重组蛋白和过表达人TIGIT细胞的结合，以及对TIGIT结合其配体PVR的阻断，基本一致、无差异。
可见，抗PVRIG抗体无论是连接到抗TIGIT抗体的重、轻链的N端或C端，都保持了对PVRIG/TIGIT的结合和配体的阻断，并且都显示了良好的表达量、纯度。
3、人源化抗PVRIG/TIGIT双抗与PVRIG/TIGIT的结合以及对相应配体的阻断
依照实施例1-2的方法，检测人源化抗PVRIG/TIGIT双抗对人和食蟹猴PVRIG的结合，对人PVRIG的配体阻断。结果如表18所示。结果表明，各双抗均可以结合人PVRIG，阻断PVRIG结合PVRL2。
表18.人源化双特异性抗体对PVRIG的结合和配体阻断
Figure PCTCN2022118959-appb-000039
与实施例1-2类似地，检测人源化抗PVRIG/TIGIT双抗对人和食蟹猴TIGIT的结合，对人TIGIT与配体结合的阻断，其中将PVRIG蛋白替换为TIGIT，并将PVRL2替换为PVR。结果如表19所示。结果表明，各双抗均可以结合人和食蟹猴TIGIT，阻断TIGIT结合PVR。
表19.人源化双特异性抗体对TIGIT的结合和配体阻断
Figure PCTCN2022118959-appb-000040
利用Biacore检测双抗与人PVRIG、与人TIGIT的亲合力。将人源化双特异性抗体捕获于Biacore仪器(Biacore X100，GE)的Protein A生物传感芯片(GE lifesciences，29127557)上，然后于芯片表面流经一系列浓度梯度下的人PVRIG抗原(AcroBiosystem，PVG-H52H4)或人TIGIT抗原(AcroBiosystem，TIT-H52H3)，获得结合和解离曲线。结果见表20。
表20.人源化双特异性抗体与人PVRIG、人TIGIT的亲合力
Figure PCTCN2022118959-appb-000041
4、抗PVRIG/TIGIT双抗的混合淋巴细胞反应(MLR)实验
依照实施例1-3第4部分的方法，检测人源化抗PVRIG/TIGIT双抗对T细胞的活化能力。实验结果如图2和表21所示。结果显示，人源化抗PVRIG/TIGIT双抗1708-151H8具有明显的活化T细胞的能力，促进T细胞分泌IFNγ。重要的是，双特异性抗体的活性强于单用抗PVRIG抗体151H8，单用抗TIGIT抗体1708。
表21.人源化双特异性抗体混合淋巴细胞反应IFNγ分泌量
Figure PCTCN2022118959-appb-000042
实施例1-8.抗PVRIG/TIGIT双抗体在人黑色素瘤A375混合人PBMC的小鼠皮下移植瘤模型中的抗肿瘤作用评估
为进一步探究双特异性抗体亚型在动物药效中的作用，进行动物药效试验。其中，1708-IgG1的重链全长与1708相同，只是重链恒定区亚型换为IgG1；1708-IgG1的轻链全长和1708-151-IgG1的第二多肽链均与与1708的轻链相同。
NCG小鼠，雌性，4-8周，体重约18-22g，购自江苏集萃药康生物科技有限公司。所有的NCG小鼠按照SPF级动物房IVC恒温恒压系统条件培养。
A375细胞培养在含10％胎牛血清(FBS)的DMEM培养液中。收集指数生长期的A375细胞，HBSS重悬至适合浓度用于NCG小鼠皮下肿瘤接种。共培养所用的A375细胞需经过丝裂霉素C处理2h后，PBS洗三次。取正常人外周血，用密度梯度离心法分离人PBMC，计数。然后用RPMI1640培养基(含IL2和10％FBS)将PBMC重悬至3×10 6个/mL的浓度，与丝裂霉素C处理后的A375细胞共培养。共培养6天后，收取PBMC，同时收取新鲜消化下来的A375细胞。每只小鼠接种：PBMC 5×10 5个，A375细胞4×10 6个；接种体积：0.2mL/只(含50％Matrigel)；接种于雌性NCG小鼠右侧皮下。根据小鼠体重随机进行分组给药，详细的给药方法、给药剂量和给药途径见表22，分组给药当天为第0天。由于抗PVRIG抗体和抗TIGIT抗体的分子量不同，该给药剂量保证了抗PVRIG抗体与抗TIGIT抗体拥有同样的起始摩尔浓度。
表22.给药方案
Figure PCTCN2022118959-appb-000043
(注：N：使用动物数量；i.p.：腹腔注射；Q2D：两天一次；给药体积：根据荷瘤鼠体重调整给药体积(0.1mL/10g)。)
给药开始后，小鼠每周2次测量体重及肿瘤体积。实验结果分别见表23和图3A、3B。
表23.抗PVRIG/TIGIT双抗在小鼠人源A375肿瘤模型中的抑瘤效果
Figure PCTCN2022118959-appb-000044
(注：与对照组(hIgG1)组比较，*P<0.05，**P<0.01，***P<0.001即认为具有显著性差异。)
实验结束时(给药后第26天)，与对照组相比，抗PVRIG抗体151-IgG4单药组没有明显差异。抗TIGIT抗体1708-IgG1单药组，抗PVRIG抗体151-IgG4与抗TIGIT抗体1708-IgG1联用组，肿瘤体积下降。而1708-151-IgG4双抗组甚至可以完全抑制肿瘤的生长，与其它组间具有显著性差异(见图3B)。
根据小鼠体重随机进行分组给药，详细的给药方法、给药剂量和给药途径见表24，分组给药当天为第0天。
表24.给药方案
Figure PCTCN2022118959-appb-000045
(注：N：使用动物数量；i.p.：腹腔注射；Q2D：两天一次；给药体积：根据荷瘤鼠体重调整给药体积(0.1mL/10g)。)
给药开始后，小鼠每周2次测量体重及肿瘤体积。实验结果分别见表25和图4A-4B。
表25.抗PVRIG/TIGIT双抗在小鼠人源A375肿瘤模型中的抑瘤效果
Figure PCTCN2022118959-appb-000046
(注：与对照组(hIgG1)组比较，*P<0.05，**P<0.01，***P<0.001即认为具有显著性差异。)
实验结束时(给药后第28天)，与对照组相比，1708-30H2与1708-151H7双抗 组均可以在低剂量下有效抑制肿瘤生长，与对照组间具有显著性差异(见图4B)。
实施例2.评价抗PVRIG/TIGIT双特异性抗体联合抗PD-L1抗体对人黑色素瘤A375混合PBMC皮下移植瘤模型的抗肿瘤作用
1、受试药物及材料
药物A：1708-30H2(抗PVRIG/TIGIT双特异性抗体)，参考前述实施例1方法制备，使用PBS配制3.58mg/mL溶液；
药物B：抗PD-L1抗体(Adebrelimab)，参考WO2017084495制备，使用NS(生理盐水)配制1.0mg/mL溶液；
阴性对照：同种型IgG4，上海恒瑞医药有限公司提供，用PBS稀释。
细胞：人黑色素瘤A375细胞，来源于中国科学院上海生命科学研究院。
实验动物：NCG小鼠，雌性，体重约18-22g。购自江苏集萃药康生物科技股份有限公司。
药物B(抗PD-L1抗体)抗体序列如下：
>抗PD-L1抗体重链可变区：
Figure PCTCN2022118959-appb-000047
>抗PD-L1抗体轻链可变区：
Figure PCTCN2022118959-appb-000048
上述抗体重轻链可变区序列中，下划线部分为CDR区，各序列依次顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。其中各抗体轻重链中CDR序列如表26所示。
表26.抗PD-L1抗体重链及轻链CDR区序列(Kabat编号规则)
Figure PCTCN2022118959-appb-000049
>抗PD-L1抗体重链全长：(Fc区带有S228P，F234A，L235A突变)
Figure PCTCN2022118959-appb-000050
Figure PCTCN2022118959-appb-000051
>抗PD-L1抗体轻链全长：
Figure PCTCN2022118959-appb-000052
2、实验方法
A375细胞培养在含10％胎牛血清(FBS)的DMEM培养液中。收集对数生长期的A375细胞，HBSS重悬至适合浓度用于NCG小鼠皮下肿瘤接种。共培养实验中用的A375细胞用丝裂霉素C处理2h，然后PBS洗三次。取复苏的PBMC加入经丝裂霉素C处理的A375细胞中，PBMC与A375共培养5天，培养液为含IL-2和10％FBS的RPMI 1640培养液。PBMC与A375共培养5天后，收取PBMC与新鲜消化下来的A375细胞，PBMC 4×10 5个，A375细胞4×10 6个，接种体积0.2mL/只(含50％基质胶)，接种于NCG小鼠右侧皮下。接种后，根据小鼠体重随机进行分组给药，详细的给药方法、给药剂量和给药途径见表27，分组给药当天为第0天。
表27.分组及给药方案
Figure PCTCN2022118959-appb-000053
(注：N：使用动物数量；i.p.：腹腔注射；BIW*3：每周两次，给药3周，共6次)
给药第14天，药物B改为每周一次；给药第21天，只给药物B每周一次和药物A每周两次；给药第28天，只给药物A每周两次。
3、评估指标
肿瘤体积测量：使用游标卡尺每周两次测量，肿瘤体积计算公式为V＝0.5×a×b 2，其中，a、b分别代表肿瘤的长径和宽径。
肿瘤生长抑制率TGI(％)＝(1-T/C)×100％。T/C％为肿瘤相对增殖率，T/C％＝(Ti-T0)/(Vi-V0)×100％，即在某一时间点，Ti为给药组开始给药后的平均肿瘤体积，T0为给药组首次给药时的平均肿瘤体积，V0为溶媒对照组首次给药时的平均肿瘤体积，Vi为溶媒对照组开始给药后的平均肿瘤体积。
荷瘤鼠体重测量：所有荷瘤鼠体重每周测量两次。体重变化用公式RCBW％＝(BWi–BW0)/BW0×100％，BWi是小鼠当前体重，BW0是分组当日的小 鼠体重。
数据均用Graphpad进行分析，采用平均值±SEM表示。用Graphpad中student’s t test比较受试药物组与对照组的组间差异，P<0.05认为具有显著性差异。
4、实验结果
如表28所示，给药第23天后，阴性对照组平均肿瘤体积为1033.50±83.02mm 3；药物A(35.8mg/kg)给药组、药物B(10mg/kg)给药组、药物B(10mg/kg)+药物A(35.8mg/kg)给药组的肿瘤平均体积分别为872.37±78.44mm 3、360.48±63.85mm 3、193.20±39.49mm 3，其肿瘤生长抑制率(TGI)分别为15.59％、65.12％、81.31％。
表28.各组小鼠肿瘤平均体积(平均值±SEM)
Figure PCTCN2022118959-appb-000054
(注：p<0.05即认为具有显著性差异。)
如表29所示，给药第24天时，结束阴性对照组、药物A(35.8mg/kg)给药组、药物B(10mg/kg)给药组和药物B(10mg/kg)+药物A(35.8mg/kg)给药组实验，其肿瘤平均重量分别为1.111±0.103g，0.986±0.078g、0.473±0.071g和0.257±0.043g。
表29.各组小鼠肿瘤平均重量(平均值±SEM)
Figure PCTCN2022118959-appb-000055
Figure PCTCN2022118959-appb-000056
(注：*p<0.05即认为具有显著性差异。)
参见图5A和图5B、表28和表29，与阴性对照组相比，药物B(10mg/kg)给药组和药物B(10mg/kg)+药物A(35.8mg/kg)给药组的肿瘤体积与肿瘤重量均存在统计学上的显著性差异(p＜0.05)，表明上述药物具有明显抑制肿瘤生长的作用。与药物B(10mg/kg)给药组相比，药物B(10mg/kg)+药物A(35.8mg/kg)联合给药组的肿瘤体积与肿瘤重量存在统计学上的显著性差异(p＜0.05)，表明药物B(10mg/kg)+药物A(35.8mg/kg)联合给药具有更好抑制肿瘤生长的作用。
此外，本次实验中，所有给药组的小鼠均无行为异常表现，表明荷瘤小鼠对该受试剂量下的药物具有良好的耐受性，参见图5C、图5D和表30。
表30.各组小鼠体重变化情况(平均值±SEM)
Figure PCTCN2022118959-appb-000057
实施例3.评价抗PVRIG/TIGIT双特异性抗体联合含抗PD-L1抗体的融合蛋白对人黑色素瘤A375混合PBMC皮下移植瘤模型的抗肿瘤作用
1、受试药物及材料
药物A：1708-30H2(抗PVRIG/TIGIT双特异性抗体)，参考PCT/CN2021/080470制备，使用PBS配制成3.58mg/mL或0.72mg/mL溶液；
药物C：含抗PD-L1抗体的融合蛋白(融合蛋白9)，参考WO20200941225中制备，使用PBS配制成0.1mg/mL溶液；
阴性对照：同种型IgG4，上海恒瑞医药有限公司提供，用PBS稀释。
细胞：人黑色素瘤A375细胞，来源于中国科学院上海生命科学研究院。
实验动物：NCG小鼠，雌性，体重约18-22g。购自江苏集萃药康生物科技股份有限公司。
药物C是抗PD-L1抗体通过(G 4S) 4G连接子与TGF-βRII ECD连接的融合蛋白，所述抗PD-L1抗体即是实施例2中的药物B抗体，其中TGF-βRII ECD序列是在N端有19个氨基酸的截短或缺失，如下所示：
>TGF-βRII ECD
Figure PCTCN2022118959-appb-000058
>抗PD-L1抗体重链全长(Fc区带有S228P,F234A,L235A,K447A突变)
Figure PCTCN2022118959-appb-000059
>抗PD-L1抗体轻链全长(SEQ ID NO:55)
>融合蛋白9(PD-L1抗体与TGF-βRII ECD融合蛋白)的第一多肽链
Figure PCTCN2022118959-appb-000060
下划线为连接子序列，融合蛋白9(PD-L1抗体与TGF-βRII ECD融合蛋白)的第二多肽链如SEQ ID NO:55所示。
融合蛋白9由两条相同的第一多肽链和两条相同的第二多肽链构成。
2、实验方法
A375细胞培养在含10％胎牛血清(FBS)的DMEM培养液中。收集对数生长期的A375细胞，HBSS重悬至适合浓度用于NCG小鼠皮下肿瘤接种。共培养实验中用的A375细胞用丝裂霉素C处理2h，然后PBS洗三次。取正常人外周血，用密度梯度离心法分离人PBMC，计数。然后用RPMI1640培养基(含IL-2和10％FBS)将PBMC重悬，与丝裂霉素C处理后的A375细胞共培养。PBMC与A375共培养6天后，收取PBMC与新鲜消化下来的A375细胞，PBMC5×10 5个，A375细胞4×10 6个，接种体积0.2mL/只(含50％Matrigel)，接种于NCG小鼠右侧皮下。接种后，根据小鼠体重随机进行分组给药，详细的给药方法、给药剂量和给药途径见表31，分组给药当天为第0天。
表31.分组及给药方案
Figure PCTCN2022118959-appb-000061
(注：N：使用动物数量；i.p.：腹腔注射；BIW*8：每周两次，给药8次)
评估指标参考实施例2。数据均用Graphpad进行分析，采用平均值±SEM表示。用Graphpad中One way ANOVA LSD(L)test比较受试药物组与对照组的组间差异，P<0.05即认为具有显著性差异。
3、实验结果
如表32所示，给药第22天后，给药组的肿瘤生长抑制率(TGI)分别为44.87％、50.54％、68.14％和78.93％。瘤重结果参见表33。
表32.各组小鼠肿瘤平均体积(平均值±SEM)
Figure PCTCN2022118959-appb-000062
(注：p<0.05即认为具有显著性差异。)
表33.各组小鼠肿瘤平均重量(平均值±SEM)
Figure PCTCN2022118959-appb-000063
(注：p<0.05即认为具有显著性差异。)
结果显示，与阴性对照组相比，药物A(7.2mg/kg)给药组、药物A(35.8mg/kg)给药组、药物C(1mg/kg)给药组和药物A(35.8mg/kg)+药物C(1mg/kg)给药组的肿瘤体积与肿瘤重量均存在统计学上的显著性差异(p＜0.05)，表明上述药物具有明显抑制肿瘤生长的作用(见图6A、图6B、表32和表33)。
本次实验过程中，各组小鼠的体重以及临床前的行为学均未出现明显异常变化(见图6C和图6D)，表明荷瘤小鼠对测试剂量下的各受试药物具有良好的耐受性。
实施例4.评价抗PVRIG/TIGIT双特异性抗体联合抗PD-1抗体对人黑色素瘤A375混合PBMC皮下移植瘤模型的抗肿瘤作用
1、受试药物及材料
药物A：1708-30H2(抗PVRIG/TIGIT双特异性抗体)，参考PCT/CN2021/080470制备，使用PBS配制成3.58mg/mL或0.72mg/mL溶液；
药物D：抗PD-1抗体(Hu23-11.IgG4AA)，参考WO2020156509制备，使用PBS配制成0.1mg/mL溶液；
阴性对照：同种型IgG4，上海恒瑞医药有限公司提供，用PBS稀释。
细胞：人黑色素瘤A375细胞，来源于中国科学院上海生命科学研究院。
实验动物：NCG小鼠，雌性，体重约18-22g。购自江苏集萃药康生物科技股份有限公司。
药物D(抗PD-1抗体)抗体序列如下：
>抗PD-1抗体重链可变区
Figure PCTCN2022118959-appb-000064
>抗PD-1抗体轻链可变区：
Figure PCTCN2022118959-appb-000065
上述抗体重轻链可变区序列中，下划线部分为CDR区，各序列依次顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。其中各抗体轻重链中CDR序列如表34所示。
表34.抗PD-1抗体重链及轻链CDR区序列
Figure PCTCN2022118959-appb-000066
Figure PCTCN2022118959-appb-000067
>抗PD-1抗体重链全长
Figure PCTCN2022118959-appb-000068
>抗PD-1抗体轻链全长
Figure PCTCN2022118959-appb-000069
2、实验方法
A375细胞培养、PBMC的分离以及二者的共培养和混合接种步骤参考实施例2。根据小鼠体重随机进行分组给药，具体给药方法、给药剂量和给药途径见表35，分组给药当天为第0天
表35.分组及给药
Figure PCTCN2022118959-appb-000070
(N：使用动物数量；i.p.：腹腔注射；Q2D*18：两天一次，给药18次；BIW*5，每周2次，给药5周，共10次)
评估指标参考实施例2。数据均用Graphpad进行分析，采用平均值±SEM表示。用Graphpad中student’s t test比较受试药物组与对照组的组间差异，P<0.05即认为具有显著性差异。
3、实验结果
如表36所示，给药第35天后，与阴性对照组相比，药物A组，药物D组，药物A+药物D联用组的肿瘤体积均减小，给药组肿瘤生长抑制率(TGI)分别为33.75％、82.58％和94.80％，其中药物D组(p<0.001)和联用组(p<0.001)表现出显著的肿瘤生长的抑制作用。与药物A组相比，联用组能显著抑制肿瘤的生长(33.75％vs 94.80％,p<0.05)；与药物D组相比，联合组虽未显示出显著性差异(p＝0.057)，但肿瘤体积减小，联用组的肿瘤生长抑制率更优(82.58％vs 94.80％)，表明药物A 与药物D联合组具有更好的抑瘤效果(见图7A和表36)。
表36.各组小鼠肿瘤平均体积(平均值±SEM)
Figure PCTCN2022118959-appb-000071
(注：*p<0.05即认为具有显著性差异。)
表37.各组小鼠肿瘤平均重量(平均值±SEM)
Figure PCTCN2022118959-appb-000072
(注：与药物D单药组相比，*p<0.05，即认为具有显著性差异。)
与药物D单药组相比，药物A+药物D联用组的肿瘤重量显著性减小(p<0.05)，表明药物A与药物D联合组具有的强的抑制肿瘤生长作用，联合给药表现出更好抑瘤效果(见图7B和表37)。
本次实验过程中，药物A单药组和药物A+药物D联用组在D35和D32各出现一只动物死亡，可能小鼠对PBMC引起的排斥反应导致的。其他小鼠的体重以及临床前的行为学均未出现明显异常变化，表明荷瘤小鼠对测试剂量下的各受试药物具有良好的耐受性(见图7C)。

Claims (28)

  1. PVRIG/TIGIT结合蛋白与免疫检查点抑制剂联合在制备治疗癌症的药物中的用途，其中，所述PVRIG/TIGIT结合蛋白包含特异性结合PVRIG的第一抗原结合结构域和特异性结合TIGIT的第二抗原结合结构域，所述特异性结合PVRIG的第一抗原结合结构域包含免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含：
    如SEQ ID NO：2和15-19中任一所示氨基酸序列中的CDR1、CDR2和CDR3，或
    如SEQ ID NO：3和21-25中任一所示氨基酸序列中的CDR1、CDR2和CDR3，
    所述CDR1、CDR2和CDR3是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的；
    优选地，根据Kabat编号系统，所述免疫球蛋白单一可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别
    如SEQ ID NO：4、5和20所示，
    如SEQ ID NO：4、5和6所示，或
    如SEQ ID NO：7、8和9所示；
    更优选地，所述PVRIG/TIGIT结合蛋白为抗PVRIG/TIGIT双特异性抗体或其抗原结合片段。
  2. 如权利要求1所述的用途，其中，所述特异性结合PVRIG的第一抗原结合结构域为人源化、亲合力成熟、去除T细胞表位、降低抗体脱酰胺和/或降低抗体异构化的，
    优选地，人源化改造过程使用的人种系模板的重链框架区为IGHV3-7*01。
  3. 如权利要求1或2所述的用途，其中，所述特异性结合PVRIG的第一抗原结合结构域中的免疫球蛋白单一可变结构域的氨基酸序列分别如
    SEQ ID NO：2和15-19中任一所示或与之具有至少90％序列同一性；或
    SEQ ID NO：3和21-25中任一所示或与之具有至少90％序列同一性。
  4. 如前述权利要求中任一项所述的用途，其中所述特异性结合TIGIT的第二抗原结合结构域包含重链可变区(VH)和轻链可变区(VL)，其中：
    所述重链可变区包含分别如SEQ ID NO：32、33和34所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含分别如SEQ ID NO：35、36和37所示的LCDR1、LCDR2和LCDR3。
  5. 如前述权利要求中任一项所述的用途，其中所述特异性结合TIGIT的第二 抗原结合结构域包含重链可变区(VH)和轻链可变区(VL)，其中：
    所述重链可变区包含如SEQ ID NO：38-40中任一所示或与之具有至少90％序列同一性的氨基酸序列，和
    所述轻链可变区包含如SEQ ID NO：41或42所示或与之具有至少90％序列同一性的氨基酸序列；
    优选地，所述重链可变区包含如SEQ ID NO：38所示或与之具有至少90％序列同一性的氨基酸序列，和
    所述轻链可变区包含如SEQ ID NO：42所示或与之具有至少90％序列同一性的氨基酸序列。
  6. 如前述权利要求中任一项所述的用途，其中所述PVRIG/TIGIT结合蛋白还包含人免疫球蛋白Fc区；
    优选地，所述Fc区是人IgG1或IgG4的Fc区；
    更优选地，所述人IgG4的Fc区具有S228P、F234A、L235A和/或K447A突变。
  7. 如前述权利要求中任一项所述的用途，其中所述特异性结合PVRIG的第一抗原结合结构域和特异性结合TIGIT的第二抗原结合结构域直接或通过连接子相连接；
    优选地，所述连接子为具有如(G 4S) x所示的氨基酸序列，其中，x独立地选自1-20的整数；
    更优选地，所述连接子为(G 4S) 2、(G 4S) 3、(G 4S) 4所示的氨基酸序列。
  8. 如前述权利要求中任一项所述的用途，其中所述特异性结合TIGIT的第二抗原结合结构域包含重链可变区(VH)和轻链可变区(VL)，其中：
    所述特异性结合PVRIG的第一抗原结合结构域的免疫球蛋白单一可变结构域位于特异性结合TIGIT的第二抗原结合结构域的重链可变区的N端；
    所述特异性结合PVRIG的第一抗原结合结构域的免疫球蛋白单一可变结构域位于特异性结合TIGIT的第二抗原结合结构域的重链可变区的C端；
    所述特异性结合PVRIG的第一抗原结合结构域的免疫球蛋白单一可变结构域位于特异性结合TIGIT的第二抗原结合结构域的轻链可变区的N端；或
    所述特异性结合PVRIG的第一抗原结合结构域的免疫球蛋白单一可变结构域位于特异性结合TIGIT的第二抗原结合结构域的轻链可变区的C端。
  9. 如前述权利要求中任一项所述的用途，其中所述PVRIG/TIGIT结合蛋白包含第一多肽链和第二多肽链，其中：
    所述第一多肽链包含如SEQ ID NO：28-29和43-45中任一所示或与其具有至 少90％序列同一性的氨基酸序列，所述第二多肽链包含如SEQ ID NO：27所示或与其具有至少90％序列同一性的氨基酸序列；或者
    所述第一多肽链包含如SEQ ID NO：26所示或与其具有至少90％序列同一性的氨基酸序列，所述第二多肽链包含如SEQ ID NO：30或31所示或与其具有至少90％序列同一性的氨基酸序列。
  10. 如权利要求9所述的用途，其中所述PVRIG/TIGIT结合蛋白包含两条相同的第一多肽链和两条相同的第二多肽链。
  11. 如前述权利要求中任一项所述的用途，其中所述免疫检查点抑制剂为PD-1/PD-L1信号通路抑制剂；
    优选地，所述PD-1/PD-L1信号通路抑制剂选自抗PD-1抗体、抗PD-L1抗体、所述抗体的抗原结合片段、或包含所述抗体或其抗原结合片段的融合蛋白；
    更优选地，所述抗PD-1抗体选自Camrelizumab、Sintilimab、Cemiplimab、JS-001、Nivolumab、Tislelizumab、Pembrolizumab、AK-103、Dostarlimab、PD1-PIK、GLS-010、Genolimzumab、BI-754091、Spartalizumab、MGA-012、PF-06801591、XmAb-20717、CS-1003、Sym-021、AGEN-2034、MEDI-5752、MGD-013、AK-105、AK-104、BCD-100、PF-06753512、HLX-10、AMP-224和LZM-009；所述抗PD-L1抗体选自Adebrelimab、Avelumab、Atezolizumab、Durvalumab、CS-1001、M-7824、KL-A167、CX-072、BGB-A333、GNS-1480、CA-170和BMS-936559；所述包含PD-L1抗体的融合蛋白选自M7824、GS-19、TS-1905、TST-005、HBM-7015、PM-8001和抗PDL1-TGFβRIIecd。
  12. 如权利要求11所述的用途，其中所述抗PD-1抗体或其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)，其中：
    所述重链可变区包含分别如SEQ ID NO:59、60和61所示氨基酸序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO:62、63和64所示氨基酸序列的LCDR1、LCDR2和LCDR3；或
    所述重链可变区包含分别如SEQ ID NO:67、68和69所示氨基酸序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO:70、71和72所示氨基酸序列的LCDR1、LCDR2和LCDR3；
    优选地，所述重链可变区包含如SEQ ID NO：57所示或与之具有至少90％同一性的氨基酸序列，所述轻链可变区包含如SEQ ID NO：58所示或与之具有至少90％同一性的氨基酸序列；或
    所述重链可变区包含如SEQ ID NO：73所示或与之具有至少90％同一性的氨基酸序列，轻链可变区包含如SEQ ID NO：74所示或与之具有至少90％同一性的 氨基酸序列。
  13. 如权利要求12所述的用途，其中所述抗PD-1抗体或其抗原结合片段还包含人免疫球蛋白Fc区；
    优选地，所述Fc区是人IgG1或IgG4的Fc区；
    更优选地，所述人IgG4的Fc区具有S228P、F234A、L235A和/或K447A突变。
  14. 如权利要求12或13所述的用途，其中所述抗PD-1抗体包含重链(HC)和轻链(LC)，其中：
    所述重链包含如SEQ ID NO：65所示或与之具有至少80％同一性的氨基酸序列，所述轻链包含如SEQ ID NO：66所示或与之具有至少80％同一性的氨基酸序列；或
    所述重链包含如SEQ ID NO：75所示或与之具有至少80％同一性的氨基酸序列，所述轻链包含如SEQ ID NO：76所示或与之具有至少80％同一性的氨基酸序列。
  15. 如权利要求11所述的用途，所述抗PD-L1抗体或其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)，其中：
    所述重链可变区包含分别如SEQ ID NO:48、49和50所示氨基酸序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO:51、52和53所示氨基酸序列的LCDR1、LCDR2和LCDR3；
    优选地，所述重链可变区包含如SEQ ID NO：46所示或与之具有至少90％同一性的氨基酸序列，所述轻链可变区包含如SEQ ID NO：47所示或与之具有至少90％同一性的氨基酸序列。
  16. 如权利要求15所述的用途，其中所述抗PD-L1抗体或其抗原结合片段还包含人免疫球蛋白Fc区；
    优选地，所述Fc区是人IgG1或IgG4的Fc区；
    更优选地，所述人IgG4的Fc区具有S228P、F234A、L235A和/或K447A突变。
  17. 如权利要求15或16所述的用途，其中所述抗PD-L1抗体包含重链(HC)和轻链(LC)，其中：
    所述重链包含如SEQ ID NO：54所示或与之具有至少80％同一性的氨基酸序列，所述轻链包含如SEQ ID NO：55所示或与之具有至少80％同一性的氨基酸序列。
  18. 如权利要求11、15至17任一项所述的用途，其中所述包含抗PD-L1抗体或其抗原结合片段的融合蛋白中还包含TGF-βRII ECD结构域，
    优选地，所述TGF-βRII ECD结构域的氨基酸序列如SEQ ID NO：56所示。
  19. 如权利要求18所述的用途，其中所述包含抗PD-L1抗体或其抗原结合片段的融合蛋白中，TGF-βRII ECD结构域通过连接子与重链全长的C端连接，所述连接子优选为(G 4S) 4G。
  20. 如权利要求19所述的用途，其中所述包含抗PD-L1抗体或其抗原结合片段的融合蛋白包含第一多肽链和第二多肽链，其中：
    所述第一多肽链包含如SEQ ID NO：77或78所示或与之具有至少90％同一性的氨基酸序列，所述第二多肽链包含如SEQ ID NO：55所示或与之具有至少90％同一性的氨基酸序列。
  21. 如前述权利要求任一项所述的用途，所述癌症选自肺癌、前列腺癌、乳腺癌、头颈部癌、食管癌、胃癌、结肠癌、结直肠癌、膀胱癌、宫颈癌、子宫癌、卵巢癌、肝癌、黑色素瘤、肾癌、鳞状细胞癌、血液系统癌症，或其任意组合；
    优选地，所述癌症为晚期或转移性的。
  22. 药物组合物，其含有PVRIG/TIGIT结合蛋白和免疫检查点抑制剂，其中所述PVRIG/TIGIT结合蛋白如权利要求1至10中任一项所定义，所述免疫检查点抑制剂如权利要求11-20中任一项所定义；优选地，所述药物组合物进一步包含至少一种可药用的赋形剂、稀释剂或载体。
  23. 一种治疗或缓解癌症的方法，所述方法包括向有需要的受试者同时、相继、或分开地施用治疗或缓解有效量的PVRIG/TIGIT结合蛋白和免疫检查点抑制剂；其中，所述PVRIG/TIGIT结合蛋白如权利要求1至10中任一项所定义，所述免疫检查点抑制剂如权利要求11-20中任一项所定义；
    优选地，所述癌症选自肺癌、前列腺癌、乳腺癌、头颈部癌、食管癌、胃癌、结肠癌、结直肠癌、膀胱癌、宫颈癌、子宫癌、卵巢癌、肝癌、黑色素瘤、肾癌、鳞状细胞癌、血液系统癌症，或其任意组合。
  24. 用于治疗或缓解癌症的药物组合，其包含PVRIG/TIGIT结合蛋白和免疫检查点抑制剂；其中，所述PVRIG/TIGIT结合蛋白如权利要求1至10中任一项所定义，所述免疫检查点抑制剂如权利要求11-20中任一项所定义；
    优选地，所述癌症选自肺癌、前列腺癌、乳腺癌、头颈部癌、食管癌、胃癌、 结肠癌、结直肠癌、膀胱癌、宫颈癌、子宫癌、卵巢癌、肝癌、黑色素瘤、肾癌、鳞状细胞癌、血液系统癌症，或其任意组合。
  25. 如权利要求24所述的用于治疗或缓解癌症的药物组合，其中所述PVRIG/TIGIT结合蛋白和免疫检查点抑制剂具有抑制癌细胞生长的协同作用，相较于单药疗法，能够显著改善免疫检查点抑制剂的抑癌效果。
  26. 一种活化NK细胞、γδT细胞和/或Th1细胞的方法，所述方法包含向有需要的受试者同时、相继、或分开地施用有效量的PVRIG/TIGIT结合蛋白和免疫检查点抑制剂；其中，所述PVRIG/TIGIT结合蛋白如权利要求1至10中任一项所定义，所述免疫检查点抑制剂如权利要求11-20中任一项所定义。
  27. 一种增加受试者体内的IFN-γ产生和/或促炎性细胞因子分泌的方法，所述方法包含向有需要的受试者同时、相继、或分开地施用有效量的PVRIG/TIGIT结合蛋白和免疫检查点抑制剂；其中，所述PVRIG/TIGIT结合蛋白如权利要求1至10中任一项所定义，所述免疫检查点抑制剂如权利要求11-20中任一项所定义。
  28. 一种抑制PD-1活性或促进T细胞增殖或使受试者从免疫反应上调获益的方法，所述方法包括向受试者同时、相继、或分开地施用有效量的PVRIG/TIGIT结合蛋白和免疫检查点抑制剂；其中，所述PVRIG/TIGIT结合蛋白如权利要求1至10中任一项所定义，所述免疫检查点抑制剂如权利要求11-20中任一项所定义。
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