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WO2023040935A1 - 一种含抗pvrig/tigit双特异性抗体的药物组合物 - Google Patents

一种含抗pvrig/tigit双特异性抗体的药物组合物 Download PDF

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WO2023040935A1
WO2023040935A1 PCT/CN2022/118942 CN2022118942W WO2023040935A1 WO 2023040935 A1 WO2023040935 A1 WO 2023040935A1 CN 2022118942 W CN2022118942 W CN 2022118942W WO 2023040935 A1 WO2023040935 A1 WO 2023040935A1
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pvrig
antibody
pharmaceutical composition
seq
amino acid
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PCT/CN2022/118942
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English (en)
French (fr)
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杨健健
田晨敏
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江苏恒瑞医药股份有限公司
上海盛迪医药有限公司
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Priority to CA3231553A priority Critical patent/CA3231553A1/en
Priority to JP2024515316A priority patent/JP2024535768A/ja
Priority to CN202280057654.0A priority patent/CN117858903A/zh
Priority to EP22869304.0A priority patent/EP4403573A1/en
Publication of WO2023040935A1 publication Critical patent/WO2023040935A1/zh

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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • 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 pharmaceutical preparations, and in particular relates to a pharmaceutical composition comprising a bispecific antibody and its use as a medicine.
  • PVRIG also known as CD112R
  • CD112R is a protein expressed on the cell surface, which belongs to the B7/CD28 superfamily like TIGIT, CD96 and CD226, and plays an important role in the immune system.
  • PVRIG contains an extracellular domain, a transmembrane domain, and an intracellular domain.
  • PVRL2 also called CD112
  • 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 many 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 besides PD-1 and TIGIT and play a role of redundancy. In vitro cell experiments and mouse models have shown that knockout or inhibition of mouse PVRIG can effectively inhibit tumor growth and coordinate with PD-1 and TIGIT inhibitors.
  • TIGIT is highly expressed on lymphocytes, including tumor-infiltrating lymphocytes (TILs) and Tregs that infiltrate different types of tumors. Engagement of TIGIT signaling with its cognate ligand PVR (also known as CD155) has been demonstrated to directly inhibit NK cell cytotoxicity through its cytoplasmic ITIM domain. PVR is also widely expressed in tumors, suggesting that the TIGIT-PVR signaling axis may be a major immune escape mechanism in cancer.
  • TILs tumor-infiltrating lymphocytes
  • Tregs that infiltrate different types of tumors.
  • PVR also known as CD155
  • PVR is also widely expressed in tumors, suggesting that the TIGIT-PVR signaling axis may be a major immune escape mechanism in cancer.
  • the present disclosure provides a pharmaceutical composition containing an anti-PVRIG/TIGIT bispecific antibody, which composition has therapeutic activity.
  • the composition also has advantages such as good stability.
  • the present disclosure provides a pharmaceutical composition comprising an anti-PVRIG/TIGIT bispecific antibody and a buffer, wherein the anti-PVRIG/TIGIT bispecific antibody comprises a first antigen-binding structure that specifically binds PVRIG domain and a second antigen-binding domain that specifically binds to TIGIT, and the buffer is a histidine buffer or an acetate buffer.
  • the buffer is a histidine-histidine hydrochloride buffer or an acetic acid-sodium acetate buffer.
  • the buffer is a histidine-histidine hydrochloride buffer.
  • the pharmaceutical composition according to any one of the above, wherein the pH of the pharmaceutical composition is 5.0-6.5.
  • the pH of the pharmaceutical composition is 5.5 to 6.5; in some embodiments, the pH of the pharmaceutical composition is 5.5-6.0; in some embodiments, the pH of the pharmaceutical composition is About 6.0.
  • point values are referred to in this disclosure, it should be understood that such point values include a margin of error. This error range is due to factors such as laboratory environment, personnel operation, instruments, methodology, and measurement errors. Taking pH as an example, when the measured value is about 6.0, it should be understood that it includes a margin of error. As an example, "about 6.0" means 6.0 ⁇ 0.2 when measuring a formulation using an industrial pH meter.
  • the pH of the pharmaceutical composition is 5.0-5.5; in some embodiments, the pH of the pharmaceutical composition is about 5.5.
  • the pH of the pharmaceutical composition is 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5, or is at these points Any range between values.
  • the pH of the pharmaceutical composition obtained by exchanging the buffer almost coincides with the pH of the buffer.
  • the pH drift of pharmaceutical preparations is generally small (within the range of ⁇ 0.3). In some embodiments, the pH of the pharmaceutical formulation drifts within ⁇ 0.1.
  • the concentration of the anti-PVRIG/TIGIT bispecific antibody is 100 mg/mL. In some embodiments, the concentration of the anti-PVRIG/TIGIT bispecific antibody is 100 mg/mL ⁇ 8 mg/mL. In some embodiments, the concentration of the anti-PVRIG/TIGIT bispecific antibody is 50 mg/mL. In some embodiments, the concentration of the anti-PVRIG/TIGIT bispecific antibody is 50 mg/mL ⁇ 5 mg/mL.
  • the concentration of the anti-PVRIG/TIGIT bispecific antibody is 1 mg/mL, 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL, 45 mg/mL, 50 mg/mL, 55 mg/mL , 60mg/mL, 65mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL, 110mg/mL, 120mg/mL, 130mg/mL, 140mg/mL or 150mg/mL, or these point values any range in between.
  • the surfactant is a nonionic surfactant.
  • the surfactant is selected from the group consisting of poloxamers (e.g., Poloxamer 188), polysorbates (e.g., polysorbate 20, polysorbate 80), polyhydroxyalkylenes, Triton, Deca Sodium Dialkyl Sulfonate, Sodium Lauryl Sulfonate, Sodium Octyl Glycoside, Lauryl-Sultaine, Myristyl-Sultaine, Linoleyl-Sultaine, Stearyl-Sultaine betaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-sarcosine, cetyl Waxyl-
  • poloxamers e.g.
  • the surfactant is a polysorbate or a poloxamer. In some embodiments, the surfactant is polysorbate 80, polysorbate 20, or poloxamer 188. In some embodiments, the surfactant is polysorbate 80.
  • the pharmaceutical composition of any one of the above, wherein the concentration of the surfactant is 0.05 mg/mL to 1.0 mg/mL.
  • the surfactant concentration is 0.2 mg/mL to 0.6 mg/mL.
  • the surfactant concentration is 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg /mL, 0.8mg/mL, 0.9mg/mL, or 1.0mg/mL, or any range between these point values.
  • the surfactant concentration is 0.4 mg/mL.
  • the surfactant concentration is 0.4 mg/mL ⁇ 0.1 mg/mL.
  • the surfactant is 0.4 mg/mL polysorbate 80.
  • the pharmaceutical composition of any one of the above which includes an osmotic pressure regulator.
  • the osmotic pressure regulator is a sugar (including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc.), amino acids (including arginine, glycine, semi cystine, histidine, etc.) or salts (sodium chloride, potassium chloride, calcium chloride, etc.).
  • the osmotic pressure regulator is a sugar selected from the group consisting of: glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, erythritol, glycerol, arabic Sugar alcohol, xylitol, sorbitol (also known as sorbitol), mannitol, milibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, Maltitol, lactitol and iso-maltulose.
  • the osmotic pressure regulator is one or more selected from the group consisting of sucrose, trehalose, sorbitol, arginine, glycine and sodium chloride.
  • the osmotic pressure regulator is a non-reducing disaccharide; in some embodiments, the osmotic pressure regulator is trehalose or sucrose; in some embodiments, the osmotic pressure regulator is sucrose.
  • the concentration of the osmotic pressure regulator is 70 mg/mL to 100 mg/mL.
  • the osmolarity adjusting agent concentration is 75 mg/mL to 100 mg/mL; in some embodiments, the osmolarity adjusting agent concentration is 75 mg/mL to 90 mg/mL; in some embodiments, the osmolarity adjusting agent at a concentration of 70 mg/mL to 90 mg/mL; in some embodiments, the osmolarity regulator is at a concentration of 75 mg/mL to 85 mg/mL; in some embodiments, the osmolarity regulator is at a concentration of 75 mg/mL to 80 mg/mL; In some embodiments, the osmolarity regulator concentration is 80 mg/mL; in some embodiments, the osmolarity regulator concentration is 80 mg/mL ⁇ 5 mg/mL.
  • non-limiting examples of osmolarity regulator concentrations include 60 mg/mL, 65 mg/mL, 70 mg/mL, 75 mg/mL, 80 mg/mL, 85 mg/mL, 90 mg/mL, 95 mg/mL, 100 mg/mL mL, and any range between these point values.
  • the aforementioned pharmaceutical composition is an isotonic formulation.
  • the osmotic pressure regulator controls the osmotic pressure of the aforementioned pharmaceutical composition at 280-320 mOsm, and in some embodiments, the osmotic pressure is controlled at about 290-300 mOsm.
  • the osmolarity regulator controls the osmolarity at 280 mOsm, 290 mOsm, 295 mOsm, 300 mOsm, 305 mOsm, 310 mOsm, 320 mOsm, and any range between these point values.
  • the osmolarity regulator is 80 mg/mL sucrose.
  • the osmolarity regulator is 80 mg/mL ⁇ 5 mg/mL sucrose.
  • the concentration of the buffer is 5 mM to 100 mM. In some embodiments, the buffer is at a concentration of 10 mM to 30 mM. In some embodiments, the buffer is at a concentration of 5 mM to 15 mM. In some embodiments, the concentration of the buffer is 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM, and any range between these point values. In some embodiments, the buffer is 10 mM His-HCl. In some embodiments, the buffer is 10 mM ⁇ 5 mM His-HCl.
  • the pharmaceutical composition as described in any one of the above, wherein the anti-PVRIG/TIGIT bispecific antibody, the first antigen-binding domain that specifically binds PVRIG comprises at least one immunoglobulin single variable domain (such as VHH), said at least one immunoglobulin single variable domain (such as VHH) comprises three complementarity determining regions CDR1, CDR2 and CDR3, wherein: CDR1 is selected from SEQ ID NO:7,10,13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, or 3, 2, 1 or more amino acid differences, and/or CDR2 selected from SEQ ID NO: 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53 , 56, 59, 62, 65 any one of the amino acid sequences shown or have 3, 2, 1 or more amino acid sequences with amino acid differences, and/or CDR3 is selected from SEQ ID NO: 9, 12, 15, 18, 21, 24, 27, 30,
  • SEQ ID NO: 7-21 is based on Kabat coding rules
  • SEQ ID NO: 22-36 is based on Chothia coding rules
  • SEQ ID NO: 37-51 is based on IMGT coding rules
  • SEQ ID NO:52-66 are based on AbM coding rules.
  • the pharmaceutical composition as described in any one of the above, wherein the anti-PVRIG/TIGIT bispecific antibody, the first antigen-binding domain that specifically binds PVRIG comprises at least one immunoglobulin single variable Domains (such as VHH), said immunoglobulin single variable domain comprises CDR1, CDR2, CDR3 in any one of the sequences shown in SEQ ID NO: 2, 75-79; or comprises SEQ ID NO: 3, 80- CDR1, CDR2, CDR3 in any of the sequences shown in 84; or CDR1, CDR2, CDR3 in any of the sequences shown in SEQ ID NO: 4, 86-90; or in any of the sequences shown in SEQ ID NO: 5, 91-95 CDR1, CDR2, CDR3 in a shown sequence; or CDR1, CDR2, CDR3 in any one of the sequences shown in SEQ ID NO: 6, 96-100.
  • immunoglobulin single variable domain comprises CDR1, CDR2, CDR3 in any one of the sequences shown in SEQ ID NO
  • the immunoglobulin single variable domain comprises CDR1, CDR2, CDR3 in any one of SEQ ID NO: 3, 80-84. In some embodiments, the immunoglobulin single variable domain comprises CDR1, CDR2, CDR3 in SEQ ID NO:81. In some embodiments, the CDR1, CDR2, CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system. In some embodiments, the CDR1, CDR2, and CDR3 are determined according to the Kabat numbering rules.
  • the first antigen-binding domain (such as VHH) that specifically binds PVRIG comprises three complementarity determining regions CDR1, CDR2 and CDR3, wherein: the amino acid sequences of CDR1, CDR2 and CDR3 are as follows: The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 7, 8, and 150 respectively; the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 10, respectively.
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 10, 11, and 151; the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 13, 14, and 15, respectively;
  • the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 16, 17, and 18, respectively; or the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 19, 20, and 21, respectively.
  • the first antigen-binding domain (such as VHH) that specifically binds to PVRIG comprises three complementarity determining regions CDR1, CDR2 and CDR3, wherein: the amino acid sequences of CDR1, CDR2 and CDR3 are as follows: The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 25, 26, and 27 respectively; the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 28, respectively.
  • amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 31, 32 and 33 respectively; or the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 34, 35 and 36 respectively .
  • the first antigen-binding domain (such as VHH) that specifically binds to PVRIG comprises three complementary determining regions CDR1, CDR2, and CDR3, wherein: the amino acid sequences of CDR1, CDR2, and CDR3 are as follows: The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 40, 41, and 42 respectively; the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 43, respectively.
  • amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 46, 47 and 48 respectively; or the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 49, 50 and 51 respectively .
  • the first antigen-binding domain (such as VHH) that specifically binds to PVRIG comprises three complementarity determining regions CDR1, CDR2 and CDR3, wherein: the amino acid sequences of CDR1, CDR2 and CDR3 are as follows: The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 55, 56, and 57 respectively; the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 58, respectively.
  • amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 61, 62 and 63 respectively; or the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 64, 65 and 66 respectively .
  • the pharmaceutical composition as described in any one of the above, wherein the anti-PVRIG/TIGIT bispecific antibody, the first antigen-binding domain that specifically binds PVRIG comprises at least one immunoglobulin single variable domain (such as VHH), said immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3, said CDR1, CDR2 and CDR3 are defined according to the Kabat numbering system, wherein, CDR1 comprises the amino acid sequence of SEQ ID NO: 10, CDR2 comprises the amino acid sequence of SEQ ID NO: 11, and CDR3 comprises the amino acid sequence of SEQ ID NO: 151.
  • the pharmaceutical composition as described in any one of the above, wherein the anti-PVRIG/TIGIT bispecific antibody, which specifically binds to the first antigen-binding domain of PVRIG comprises SEQ ID NO: 2-6, 75-84, 86-100 any one of the amino acid sequences shown, or at least 70%, at least 80% of at least one sequence in SEQ ID NO: 2-6, 75-84, 86-100 , amino acid sequences that are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical.
  • the first antigen-binding domain of the anti-PVRIG/TIGIT bispecific antibody comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, wherein the first antigen binding domain of the anti-PVRIG/TIGIT bispecific antibody comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of SEQ ID NO:81 Amino acid sequences of sequences that are % or at least 99% identical.
  • the second antigen-binding domain that specifically binds TIGIT is a second antibody, and in some embodiments, the second antibody includes a heavy chain (HC) and a light chain (LC); the first antibody is located at the heavy chain of the second antibody N-terminal and/or C-terminal of the chain or light chain.
  • the pharmaceutical composition according to any one of the above, wherein the anti-PVRIG/TIGIT bispecific antibody comprises 1 second antibody that specifically binds to TIGIT and 2 primary antibodies that specifically bind to PVRIG
  • the second antibody includes two HCs and two LCs, the VH of one HC and the VL of one LC of the second antibody form an antigen-binding site, and the VH of the other HC forms an antigen-binding site with the VL of another LC.
  • one of the first antibodies is N-terminal to the heavy or light chain of the second antibody and the other first antibody is C-terminal to the heavy or light chain of the second antibody.
  • each first antibody is located at the N-terminus of the two heavy chains or the two light chains of the second antibody, respectively; C-terminus of the light chain. In some embodiments, each first antibody is respectively N-terminal to the two heavy chains of the second antibody. In some embodiments, each first antibody is respectively C-terminal to the two heavy chains of the second antibody. In some embodiments, the 2 primary antibodies are the same. In some embodiments, the 2 primary antibodies are not identical.
  • the N-terminus of the light chain of the second antibody is either connected to the C-terminus of the light chain of the second antibody, or connected to the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain or the C-terminus of the light chain or any of them combination.
  • the linker is selected from: an amino acid sequence such as (GmSn)x or (GGNGT)x or (YGNGT)x, wherein m and n are each independently selected from an integer of 1-8 (eg , 1, 2, 3, 4, 5, 6, 7 or 8), x is independently selected from an integer of 1-20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).
  • the linker is GS, GAP, ASGS (SEQ ID NO: 154), G 4 S (SEQ ID NO: 155), (G 4 S) 2 (SEQ ID NO: 152), (G 4 S) 3 (SEQ ID NO: 156), (G 4 S) 4 (SEQ ID NO: 157), (G 4 S) 5 (SEQ ID NO: 158), (G 4 S) 6 (SEQ ID NO: 159), YGNGT (SEQ ID NO: 160), (YGNGT) 2 (SEQ ID NO: 161), (YGNGT) 3 (SEQ ID NO: 162), (YGNGT) 4 (SEQ ID NO: 163), (YGNGT ) 5 (SEQ ID NO: 164) or the amino acid sequence shown in (YGNGT) 6 (SEQ ID NO: 165).
  • the second antibody is a full length antibody.
  • the heavy chain of the second antibody is of IgG isotype, such as IgG1, IgG2, IgG3 or IgG4, for example of IgG1 isotype; and/or the light chain of the second antibody is of Kappa isotype type.
  • the LCs contain the same CDRs.
  • the two HCs of the second antibody comprise the same VH and/or the two LCs of the second antibody comprise the same VL.
  • the two HCs of the second antibody have the same amino acid sequence and/or the two LCs of the second antibody have the same amino acid sequence.
  • the pharmaceutical composition according to any one of the above, wherein the anti-PVRIG/TIGIT bispecific antibody comprises two primary antibodies, wherein the two primary antibodies have the same or different amino acids sequence. In some embodiments, the two primary antibodies have the same amino acid sequence.
  • the pharmaceutical composition according to any one of the above, wherein the anti-PVRIG/TIGIT bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, wherein for each polypeptide chain:
  • the first polypeptide chain each independently comprises VHH as the heavy chain (HC) of the first antibody, and the second antibody;
  • the second polypeptide chains each independently comprise a light chain (LC) of a second antibody
  • each of the first polypeptide chains independently comprises a heavy chain (HC) of a second antibody; and ii) each of the second polypeptide chains independently comprises a light chain (LC) of a first antibody and a second antibody; wherein , the VHH is connected directly or via a linker to the N-terminal and/or C-terminal of the LC of the second antibody.
  • the anti-PVRIG/TIGIT bispecific antibody comprises two identical first polypeptide chains and two identical second polypeptide chains.
  • the pharmaceutical composition according to any one of the above, wherein the anti-PVRIG/TIGIT bispecific antibody, wherein the second antigen-binding domain that specifically binds to TIGIT is any anti-TIGIT antibody.
  • the TIGIT antibody can be any of CPA.9.083.H4(S241P), CPA.9.086.H4(S241P), CHA.9.547.7.H4(S241P) and CHA.9.547.13.H4(S241P) (See WO2019232484).
  • the pharmaceutical composition according to any one of the above, wherein the anti-PVRIG/TIGIT bispecific antibody, wherein the second antigen-binding domain that specifically binds to TIGIT is a second antibody.
  • the anti-TIGIT antibody in WO2019062832 is incorporated in its entirety as the second antibody.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 respectively as shown in SEQ ID NO: 115, 116 and 117, and the light chain variable region comprises respectively as shown in SEQ ID NO: 115, 116 and 117 LCDR1, LCDR2 and LCDR3 shown in NO: 118, 119 and 120; or heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 121, 122 and 123 respectively, and light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 124, 125 and 126, respectively; or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 127, 128 and 129, respectively, and the light chain
  • the variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 130, 131 and 132, respectively; or the heavy chain variable region comprises HCDR1, HCDR2 and
  • the pharmaceutical composition as described in any one of the above, wherein the anti-PVRIG/TIGIT bispecific antibody, wherein the second antigen-binding domain that specifically binds to TIGIT comprises a heavy chain variable region ( VH) and light chain variable region (VL), wherein: the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 121, and HCDR2 comprises the amino acid sequence of SEQ ID NO: 122 and HCDR3 comprises the amino acid sequence of SEQ ID NO: 123, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 124, LCDR2 comprises the amino acid sequence of SEQ ID NO: 125 and LCDR3 comprises the amino acid sequence of SEQ ID NO: 126.
  • VH heavy chain variable region
  • VL light chain variable region
  • the second antigen-binding domain that specifically binds TIGIT comprises the VH of the amino acid sequence shown in any one of SEQ ID NO: 145-147, or comprises the VH of the amino acid sequence shown in any one of SEQ ID NO: 145-147 At least one sequence has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity of amino acid sequence VH, and/or comprises SEQ The VL of the amino acid sequence shown in any one of ID NO:148-149, or comprises and at least 70%, at least 80%, at least 90%, at least 95%, at least one sequence in SEQ ID NO:148-149 VL of an amino acid sequence having 96%, at least 97%, at least 98%, at least 99% sequence identity.
  • the heavy chain variable region of the second antigen-binding domain specifically binding to TIGIT comprises the amino acid sequence of SEQ ID NO: 145, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 149.
  • the second antigen binding domain that specifically binds TIGIT comprises a heavy chain (HC) and a light chain (LC).
  • said second antigen-binding domain that specifically binds TIGIT comprises at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97% of SEQ ID NO: 102 %, at least 98% or at least 99% of the HC of the amino acid sequence of sequence identity, and at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97% with SEQ ID NO: 103 , LC of amino acid sequences having at least 98% or at least 99% sequence identity.
  • the second antigen-binding domain that specifically binds TIGIT has: HC comprising the amino acid sequence of SEQ ID NO: 102, and LC comprising the amino acid sequence of SEQ ID NO: 103.
  • the second antigen-binding domain that specifically binds TIGIT comprises a heavy chain variable region such as SEQ ID NO: 145, and a light chain variable region such as SEQ ID NO: 149.
  • the second antigen-binding domain that specifically binds TIGIT has: HC having an amino acid sequence such as SEQ ID NO: 102, and LC having an amino acid sequence such as SEQ ID NO: 103.
  • the heavy chain variable region of the binding domain or the second antibody comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 121, 122 and 123 respectively, and the light chain variable region comprises respectively SEQ ID NO: 124, 125, LCDR1, LCDR2, LCDR3 shown in 126.
  • the heavy chain variable region of the binding domain or the second antibody comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 121, 122 and 123 respectively, and the light chain variable region comprises respectively SEQ ID NO: 124, 125, LCDR1, LCDR2, LCDR3 shown in 126.
  • the pharmaceutical composition according to any one of the above, wherein the anti-PVRIG/TIGIT bispecific antibody, wherein the first antigen-binding domain or the first antibody that specifically binds PVRIG has: comprising VHH of the amino acid sequence shown as one of SEQ ID NO: 6, 79, 81, 92, 98, 99, or comprising at least 70% with one of SEQ ID NO: 6, 79, 81, 92, 98, 99 , a VHH of an amino acid sequence of at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; said second antigen-binding structure that specifically binds TIGIT
  • the domain or the second antibody has: VH comprising an amino acid sequence as shown in any one of SEQ ID NO: 145-147 or comprising at least 70%, at least 80%, at least 90% of one of SEQ ID NO: 145-147 , a VH of an amino acid sequence of at least 9
  • the anti-PVRIG/TIGIT bispecific antibody specifically binds to the first antigen-binding domain of PVRIG or the first antibody has a VHH as shown in SEQ ID NO: 81, which specifically binds to The second antigen-binding domain of TIGIT has a VH as shown in SEQ ID NO: 145 and a VL as shown in SEQ ID NO: 149.
  • the anti-PVRIG/TIGIT bispecific antibody specifically binds to the first antigen-binding domain of PVRIG or the first antibody has a VHH as shown in SEQ ID NO: 81, which specifically binds to The second antigen-binding domain of TIGIT has HC as shown in SEQ ID NO: 102 and LC as shown in SEQ ID NO: 103.
  • the Fc is an IgG isotype Fc, such as the Fc region of IgG1, IgG2, IgG3, or IgG4; in some embodiments, the Fc includes one or more amino acid mutations selected from the group consisting of (mutation Sites are indexed according to EU):
  • Mutations that result in enhanced binding to Fc ⁇ RIIIa to cause enhanced ADCC mutations that result in reduced binding to Fc ⁇ RIIb, for example 236A, 239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F , 236A/332E, 239D/332E/330Y, 239D, 332E/330L, 299T, 297N or any combination thereof;
  • v one or more amino acid mutations at positions 329, 331 and 322, or any combination thereof, such that the antibody has altered C1q binding and/or reduces or eliminates complement-dependent cytotoxicity (CDC);
  • the pharmaceutical composition as described in any one of the above, wherein the anti-PVRIG/TIGIT bispecific antibody comprises: the first polypeptide chain as shown in SEQ ID NO: 104, and as SEQ ID NO: The second polypeptide chain shown in 103; the first polypeptide chain shown in SEQ ID NO: 105, the second polypeptide chain shown in SEQ ID NO: 103; the first polypeptide chain shown in SEQ ID NO: 102 , the second polypeptide chain as shown in SEQ ID NO: 106; the first polypeptide chain as shown in SEQ ID NO: 102, the second polypeptide chain as shown in SEQ ID NO: 107; or as SEQ ID NO: 108 - the first polypeptide chain shown in any one of 112 and 114, the second polypeptide chain shown in SEQ ID NO: 103; or at least 70% with the above-mentioned first polypeptide chain and/or the second polypeptide chain , at least 80%, at least 90%, at least 95%,
  • the pharmaceutical composition according to any one of the above, wherein the anti-PVRIG/TIGIT bispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain Comprising the amino acid sequence of SEQ ID NO: 109, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 103.
  • the anti-PVRIG/TIGIT bispecific antibody comprises two identical first polypeptide chains and two identical second polypeptide chains.
  • the pharmaceutical composition according to any one of the above, wherein the anti-PVRIG/TIGIT bispecific antibody has at least one of the following characteristics:
  • the KD value of binding to human PVRIG is less than 1 ⁇ 10 -7 M;
  • the KD value of the anti-PVRIG/TIGIT bispecific antibody may be less than 1 ⁇ 10 -7 M, less than 1 ⁇ 10 -8 M, less than 1 ⁇ 10 -9 M, less than 1 ⁇ 10 - 10 M. In some embodiments, wherein the anti-PVRIG/TIGIT bispecific antibody is capable of inhibiting tumor growth by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60% , at least about 70%, at least about 80%.
  • composition according to any one of the above, which comprises the following components:
  • the pharmaceutical composition comprises the following components:
  • the pharmaceutical composition comprises the following components:
  • anti-PVRIG/TIGIT bispecific antibody comprising the first polypeptide chain of the amino acid sequence of SEQ ID NO: 109 and comprising the amino acid of SEQ ID NO: 103
  • the drug The pH of the composition is 6.0.
  • the pharmaceutical composition comprises the following components:
  • the pharmaceutical composition comprises the following components:
  • the pharmaceutical composition comprises the following components:
  • the pharmaceutical composition comprises the following components:
  • anti-PVRIG/TIGIT bispecific antibody of 50mg/mL ⁇ 5mg/mL described anti-PVRIG/TIGIT bispecific antibody comprises the first polypeptide chain of the amino acid sequence of SEQ ID NO: 109 and comprises SEQ ID NO : the second polypeptide chain of the amino acid sequence of 103,
  • the pharmaceutical composition comprises the following components:
  • the pharmaceutical composition comprises the following components:
  • the pharmaceutical composition comprises the following components:
  • the pharmaceutical composition comprises the following components:
  • the pharmaceutical composition comprises the following components:
  • the pharmaceutical composition comprises the following components:
  • the pharmaceutical composition comprises the following components:
  • the pharmaceutical composition comprises the following components:
  • the pharmaceutical composition comprises the following components:
  • the pharmaceutical composition comprises the following components:
  • the pharmaceutical composition comprises the following components:
  • the pharmaceutical composition comprises the following components:
  • the pharmaceutical composition comprises the following components:
  • the pharmaceutical composition comprises the following components:
  • the pharmaceutical composition comprises the following components:
  • the pharmaceutical composition comprises the following components:
  • the pharmaceutical composition comprises the following components:
  • the pharmaceutical composition as described above is a liquid formulation.
  • the solvent of the liquid formulation is water.
  • the present disclosure also provides a lyophilized preparation, which is characterized in that the lyophilized preparation can form the pharmaceutical composition as described above after reconstitution.
  • the present disclosure also provides a lyophilized preparation, which is a lyophilized preparation of the pharmaceutical composition as described in any one of the above.
  • the present disclosure also provides a method for preparing a freeze-dried preparation, which includes the step of freeze-drying the pharmaceutical composition as described above.
  • the freeze-drying described in any one of the above sequentially includes the steps of pre-freezing, primary drying and secondary drying.
  • the present disclosure also provides a freeze-dried preparation, which is obtained by freeze-drying the pharmaceutical composition as described in any one of the above.
  • the present disclosure also provides a reconstitution solution, which is characterized in that the reconstitution solution is prepared by reconstitution of the freeze-dried preparation as described in any one of the above items.
  • the present disclosure also provides a reconstitution solution, which is a reconstitution preparation of the lyophilized preparation described in any one of the above.
  • the components and content of the reconstitution solution described in any one of the above items are the same as those of the aforementioned pharmaceutical composition.
  • the reconstitution solution as described in any one of the above which comprises the following components:
  • the reconstitution solution as described in any one of the above which comprises the following components:
  • the reconstitution solution as described in any one of the above which comprises the following components:
  • the reconstitution solution as described in any one of the above which comprises the following components:
  • the reconstitution solution comprises the following components:
  • the reconstitution solution comprises the following components:
  • anti-PVRIG/TIGIT bispecific antibody comprising the first polypeptide chain of the amino acid sequence of SEQ ID NO: 109 and comprising the amino acid of SEQ ID NO: 103
  • the drug The pH of the composition is 6.0.
  • the pharmaceutical composition or reconstitution solution as described in any one of the above which is an intravenous injection preparation, a subcutaneous injection preparation, an intraperitoneal injection preparation or an intramuscular injection preparation; in some embodiments, as described in any one of the above The above pharmaceutical composition or reconstitution solution, which is an intravenous injection preparation.
  • the pharmaceutical composition or reconstitution solution according to any one of the above is suitable for intravenous injection, subcutaneous injection, intraperitoneal injection or intramuscular injection.
  • the pharmaceutical composition or reconstitution solution or freeze-dried formulation as described in any one of the above is used for the preparation of drugs for intravenous injection, subcutaneous injection, intraperitoneal injection or intramuscular injection.
  • kits which includes at least one container, and each container independently contains the pharmaceutical composition as described in any one of the above, the lyophilized preparation as described in any one of the above, or the compound composition as described in any one of the above. solution.
  • the present disclosure also provides methods for diagnosing, treating, and alleviating the symptoms of a subject, comprising administering to the subject an effective amount of the pharmaceutical composition as described in any one of the above, the frozen drug composition as described in any one of the above A dry preparation, a reconstitution solution as in any of the above, or a kit as in any of the above.
  • the present disclosure also provides a method for activating cytotoxic T cells (CTL) in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition as described in any one of the above, any of the above The lyophilized formulation of any one of the above, the reconstitution solution of any one of the above, or the kit of any one of the above, wherein said subgroup of CTLs in said subject is activated.
  • CTL cytotoxic T cells
  • the present disclosure also provides a method for activating NK cells in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition as described in any one of the above, or the lyophilized preparation as described in any one of the above .
  • the present disclosure also provides a method for activating ⁇ T cells in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition as described in any one of the above, or the lyophilized preparation as described in any one of the above .
  • the present disclosure also provides a method for activating Th1 cells in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition as described in any one of the above, or the lyophilized preparation as described in any one of the above .
  • the present disclosure also provides a method of activating, reducing or eliminating the cell number and/or activity of at least one regulatory T cell (Treg) in a subject, comprising administering to the subject An effective amount of the pharmaceutical composition as described in any one of the above, the freeze-dried preparation as described in any of the above, the reconstitution solution as described in any of the above or the kit as described in any of the above.
  • Treg regulatory T cell
  • the present disclosure also provides a method for increasing interferon- ⁇ production and/or secretion of pro-inflammatory cytokines in a subject, comprising administering to the subject an effective amount of any one of the above
  • the pharmaceutical composition of any of the above, the lyophilized preparation of any of the above, the reconstitution solution of any of the above, or the kit of any of the above comprising administering to the subject an effective amount of any one of the above.
  • the present disclosure also provides a method for inhibiting the interaction of PVRIG and PVLR2 in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition as described in any one of the above, any one of the above The lyophilized formulation of the above item, the reconstitution solution of any of the above items, or the kit of any of the above items.
  • the present disclosure also provides the pharmaceutical composition as described in any of the above, the lyophilized preparation as described in any of the above, the reconstitution solution as described in any of the above, or the kit as described in any of the above. Use in medicines for the treatment of diseases.
  • the present disclosure also provides a method for treating a disease, the method comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition as described in any one of the above, the lyophilized preparation as described in any one of the above, The reconstitution solution of any of the above or the kit of any of the above.
  • the present disclosure also provides the pharmaceutical composition as described in any of the above, the lyophilized preparation as described in any of the above, the reconstitution solution as described in any of the above, or the kit as described in any of the above, which For the treatment of diseases.
  • the disease of any one of the preceding is a proliferative disease (eg, a tumor).
  • the tumor is selected from the group consisting of prostate cancer, liver cancer (HCC), colorectal cancer (colon cancer, rectal cancer), ovarian cancer, endometrial cancer, breast cancer (e.g. triple negative cancer) or a combination thereof.
  • breast cancer pancreatic cancer
  • gastric gastric
  • cervical 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
  • AML acute myelogenous leukemia
  • T-ALL T-cell acute lymphoblastic leukemia
  • diffuse large B-cell lymphoma testicular germ cell tumor
  • mesothelioma mesothelioma
  • esophageal cancer also known as "esophageal cancer”
  • Merkel Cells cancer MSI-high cancer
  • KRAS-mutated tumors adult T Leukemia/Lymphoma and Myelodysplastic Syndrome (MDS).
  • MDS Myelodysplastic Syndrome
  • the above-mentioned disease may be a disease associated with abnormal expression of PVRIG and/or TIGIT.
  • the tumor is selected from the following cancers or combinations thereof: triple-negative breast cancer, gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), Merkel cell carcinoma, high MSI cancer, KRAS mutant tumors, Adult T-cell leukemia/lymphoma and myelodysplastic syndrome (MDS).
  • MDS myelodysplastic syndrome
  • the tumor is selected from the group consisting of triple negative breast cancer, gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), Merkel cell carcinoma, and high MSI cancer, or a combination thereof.
  • the above-mentioned disease patient has a condition related to PVRIG and/or TIGIT.
  • the subject's condition includes cancers that express or do not express PVRIG and further includes non-metastatic or non-invasive and invasive or metastatic cancers in which PVRIG is expressed by immune cells, stromal cells, or diseased cells Inhibits anti-tumor responses and anti-invasive immune responses.
  • the disease is a vascularized tumor.
  • the tumor is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colorectal cancer, bladder cancer, cervical cancer, endometrial cancer, ovarian cancer, liver cancer, melanoma, Kidney cancer, squamous cell carcinoma, hematologic cancer, or any other disease or condition characterized by uncontrolled cell growth.
  • the disease is infection or sepsis.
  • the infection is a pathogenic infection characterized by varying degrees of dysfunction of virus-specific T cell responses, such as HIV, HCV, HBV.
  • the sepsis is selected from severe sepsis, septic shock, systemic inflammatory response syndrome (SIRS), bacteremia, sepsis, toxaemia, and septic syndrome.
  • FIG 1 shows the results of PVRIG reporter gene cell activity detection with anti-PVRIG antibody.
  • Fig. 2 is the detection result of NK cell activity activated by anti-PVRIG antibody in NK cell killing experiment.
  • Figure 3 is the detection results of the activation of T cells by the anti-PVRIG antibody in the MLR experiment.
  • Figure 4A to Figure 4B are the detection results of the activity of the humanized anti-PVRIG antibody in PVRIG reporter gene cells.
  • Figure 5A to Figure 5B are the detection results of humanized anti-PVRIG antibody activation of NK cells in NK cell killing experiments.
  • Figure 6A to Figure 6E show the binding activity of the humanized anti-PVRIG/TIGIT bispecific antibody to human PVRIG recombinant protein, human PVRIG overexpression cells, cynomolgus monkey PVRIG recombinant protein, cynomolgus monkey PVRIG overexpression cells, and The activity test results of blocking the combination of human PVRIG and human PVRL2.
  • Figure 7A to Figure 7E show the binding activity of the humanized anti-PVRIG/TIGIT bispecific antibody to human TIGIT recombinant protein, human TIGIT overexpression cells, cynomolgus TIGIT recombinant protein, cynomolgus TIGIT overexpression cells, and The activity test results of blocking the combination of human TIGIT and human PVR.
  • Fig. 8 is the detection result of the activation of T cells by the humanized anti-PVRIG/TIGIT bispecific antibody in the MLR experiment.
  • Figures 9A to 9B respectively show the effects of anti-PVRIG/TIGIT bispecific antibodies on the body weight and tumor volume of mice in the subcutaneous xenograft tumor model of human melanoma A375 mixed with human PBMC.
  • Figures 10A to 10B respectively show the effects of anti-PVRIG/TIGIT bispecific antibody on the body weight and tumor volume of mice in a subcutaneous xenograft model of human melanoma A375 mixed with human PBMC.
  • 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-combining site refers to a continuous or discontinuous three-dimensional site on an antigen recognized by the antibody of the present disclosure.
  • Antibody or “immunoglobulin” is used in the broadest sense and encompasses a variety of antibody structures including, but not limited to, traditional antibodies (four Peptide chain structure antibody), and Fab, Fv, sFv, F(ab')2, linear antibody, single chain antibody, scFv, sdAb, sdFv, nanobody, peptibody, domain antibody (heavy chain (VH) antibody, light chain (VL) antibody) and multispecific antibodies (bispecific antibody, diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv).
  • antibody is intended to encompass a full-length antibody, individual chains thereof, and any portion, domain, or fragment thereof that has antigen-binding activity (i.e., an antigen-binding fragment); Multispecific antibodies having parts, domains or fragments of antigen binding activity (including but not limited to antigen binding domains or fragments such as VHH domains or VH/VL domains).
  • Traditional antibodies or immunoglobulins usually have a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chain They are ⁇ -chain, ⁇ -chain, ⁇ -chain, ⁇ -chain, and ⁇ -chain, respectively.
  • 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 (kappa) chain or a lambda (lambda) chain.
  • antibodies of the present disclosure specifically bind to PVRIG and/or TIGIT.
  • Antigen-binding fragments include, but are not limited to: (i) Fab fragments consisting of VL, VH, CL and CH1 domains; (ii) Fd fragments consisting of VH and CH1 domains; (iii) F( ab') 2 fragments, a bivalent fragment comprising two linked Fab fragments; (vii) single-chain Fv molecules (scFv), wherein the VH domain and the VL domain are connected by a peptide linker, the peptide link (Bird et al., 1988, Science 242: 423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A.
  • the 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.
  • bispecific antibody refers to an antibody capable of specifically binding to two different antigens or two different epitopes of the same antigen.
  • Bispecific antibodies of various structures have been disclosed in the prior art. According to the integrity of the IgG molecule, it can be divided into IgG-like bispecific antibodies and antibody fragment bispecific antibodies. According to the number of antigen-binding regions, bispecific antibodies can be divided into bivalent, trivalent, tetravalent or more valent. According to whether the structure is symmetrical, it can be divided into symmetrical structure bispecific antibody and asymmetric structure bispecific antibody.
  • 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, 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 (for example with Fc fragments).
  • 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 VHH-IgG, KiH, CrossMAb, Triomab quadroma, Fc ⁇ Adp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1TCBs, 1Fab-IgG TDB, FynomAb , two-in-one/DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, Bispecific antibodies such as DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages585–608 (2019); Chen S1
  • 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.
  • the 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.
  • Monoclonal antibody refers to a population of antibody molecules produced by a single B cell clone and capable of interacting with a specific antigenic epitope.
  • polyclonal antibody refers to a population of antibody molecules that are produced by multiple B cell clones and can interact with a specific antigen.
  • Monoclonal antibodies typically exhibit a single binding affinity for the particular antigen with which they interact.
  • antigen is defined more broadly and is generally intended to include target molecules specifically recognized by antibodies.
  • immunization procedures for the production of antibodies, or molecules or mimetics for use in antibody library screening are included.
  • “Homology” or “identity” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in both sequences being compared are occupied by the same base or amino acid residue, two DNA molecules are homologous if a position is occupied by adenine at that position. The percent identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of all positions being compared 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.
  • a “domain” of a polypeptide or protein refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, domains are 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 the globular region of an antibody chain, or to a polypeptide consisting essentially of such a globular region.
  • antigen-binding domains include: Fab fragments, Fd fragments, F(ab')2 fragments, scFv, full-length antibodies, and the like.
  • Immunoglobulin variable domain refers to an immunoglobulin domain consisting essentially of "framework region 1" or “FR1”, “framework region 2" or “FR2”, “framework region 3” or “ FR3”, and “framework region 4" or “FR4", “complementarity determining region 1” or “CDR1”, “complementarity determining region 2" or “CDR2”, and “complementarity determining region 3" or “CDR3".
  • the general structure or sequence of 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.
  • CDR the definitive delineation of the CDR or the identification of the residues of the binding site 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). Contact definitions are based on analysis of available complex crystal structures (see eg MacCallum et al., 1996, J. Mol. Biol., 5:732-45).
  • a CDR may refer to a CDR defined by any method known in the art, including combinations of methods. The methods used herein can utilize CDRs defined according to any of these methods. For any given embodiment comprising more than one CDR, the CDRs may be defined according to any of the Kabat, Chothia, extended, AbM, IMGT, contact and/or conformational definitions.
  • Immunoglobulin single variable domain is commonly used to refer to a VH/VL domain that can be used without interaction with other variable domains (e.g. without the VH/VL domains required between VH and VL domains in conventional quadruplex antibodies). interaction), an immunoglobulin variable domain (which may be a heavy chain domain or a light chain domain, including a VH, VHH or VL domain) that forms a functional antigen binding site.
  • immunoglobulin single variable domains include Nanobodies (including VHHs, such as humanized VHHs and/or camelized VHs, such as camelized human VHs), IgNARs, domains, as VH domains or derived from VH domain (single domain) antibodies such as dAbs TM and (single domain) antibodies that are or are derived from VL domains such as dAbs TM .
  • Immunoglobulin single variable domains based on and/or derived from heavy chain variable domains, such as VH or VHH domains, are generally preferred.
  • a specific example of an immunoglobulin single variable domain is a "VHH domain” (or simply "VHH") as defined below.
  • VHH domains also known as heavy chain single domain antibodies, VHH, VHH antibody fragments, VHH antibodies, nanobodies, are antigen-binding immunoglobulins known as “heavy chain antibodies” (ie, “antibodies lacking light chains") Protein variable domains (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 heavy chain variable domain (which is referred to in this disclosure as the "VH domain”) and the light chain variable domain present in conventional tetrapeptide chain antibodies.
  • VHH domains are small, stable antigen recognition units formed by a single immunoglobulin domain. Terms “heavy chain single domain antibody”, “VHH domain”, “VHH”, “VHH domain”, “VHH antibody fragment”, “VHH antibody” (“Nanobody” is a trademark of Ablynx N.V., Ghent, Belgium) Can be used interchangeably.
  • 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 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). This means that, in general, 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 applicable to VHH include Chothia, IMGT, AbM.
  • 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.
  • VHH domains (alone or as part of larger polypeptides) offer a number of significant advantages over the use of conventional VH and VL domains, scFv or conventional antibody fragments (e.g. Fab- or F(ab')2-fragments):
  • VHH domains can be expressed from a single gene and do not require post-translational folding or modification;
  • VHH domains can be engineered into multivalent and multispecific formats
  • -VHH domains are highly soluble and have no tendency to aggregate
  • -VHH domains are highly stable to heat, pH, proteases and denaturants, and therefore can reduce the dependence on refrigeration equipment during production, storage or transportation, thereby achieving cost, time and environmental savings;
  • VHH domain is relatively small compared with conventional tetrapeptide chain structure antibodies and their antigen-binding fragments (about 15kDa or 1/10 the size of conventional IgG), so compared with conventional tetrapeptide chain structure antibodies and their antigen-binding fragments , show higher tissue permeability and can be administered in higher doses;
  • VHH domains can display so-called cavity-binding properties (especially due to their extended CDR3 loop compared to conventional VH domains), allowing access to targets and epitopes inaccessible to conventional tetrapeptide chain structured antibodies and antigen-binding fragments thereof.
  • Fc variant or “variant Fc” means a protein comprising amino acid modifications in the Fc domain.
  • Fc variants of the present disclosure are defined by the amino acid modifications that make them up.
  • S228P or 228P is an Fc variant having a proline substitution at position 228 relative to the parental Fc polypeptide, where numbering is according to the EU index.
  • the WT amino acid may not be specified, in which case the aforementioned variant is referred to as 228P.
  • humanization examples include that the VHH domain derived from Camelidae can be replaced by one or more amino acid residues present in the corresponding position in the VH domain of an antibody with a conventional tetrapeptide chain structure in humans, replacing the original VHH sequence One or more amino acid residues in the amino acid sequence of the human, so as to realize "humanization”.
  • sequence optimization in addition to humanization, “sequence optimization” may also encompass other modifications of the sequence by one or more mutations that provide improved properties of the VHH, such as the removal of potential post-translational modifications location.
  • a humanized VHH domain may contain one or more fully human framework region sequences, and in some embodiments, may contain the human framework region sequences of IGHV3.
  • 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 heterologous 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 on a 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 the humanized antibody after further affinity maturation of CDR by phage display.
  • minimal reverse mutations or back mutations can be performed on the human antibody variable region framework sequence to maintain the activity.
  • An "affinity matured” antibody has one or more changes in one or more CDRs that result in an increase in affinity for the antigen compared to its respective parental antibody.
  • 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.
  • antibodies of the present disclosure will be present at preferably 10 ⁇ 7 to 10 ⁇ 10 moles/liter (M), more preferably 10 ⁇ 8 to 10 ⁇ 10 moles/liter, even more preferably as measured in a Biacore or KinExA or Fortibio assay. 10 ⁇ 9 to 10 ⁇ 10 or lower dissociation constant (KD), and/or at least 10 ⁇ 7 M, preferably at least 10 ⁇ 8 M, more preferably at least 10 ⁇ 9 M, more preferably at least 10 ⁇ 10 M
  • KD dissociation constant
  • the association constant (KA) of the binding antigen (ie PVRIG) to be bound Any KD value greater than 10 -4 M is generally considered to indicate non-specific binding.
  • Specific binding of an antibody to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and/or competitive binding assays (e.g., radioactive Immunoassay (RIA), Enzyme Immunoassay (EIA) and Sandwich Competitive Assay.
  • SPR surface plasmon resonance
  • RIA radioactive Immunoassay
  • EIA Enzyme Immunoassay
  • Sandwich Competitive Assay e.g., sandwich competitive Assay.
  • an “epitope” or “antigenic determinant” used interchangeably refers to any portion of an antigen to which an antibody binds.
  • Antigenic determinants usually contain chemically active surface groups, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which may be a "linear " epitope or "conformational” epitope.
  • a linear epitope all points of interaction between a protein and an interacting molecule (such as an antibody) exist linearly along the primary amino acid sequence of the protein.
  • Epitopes for a given antigen can be identified using a number of epitope mapping techniques well known in the art (e.g. Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996), US4708871).
  • 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 (for example see WO03/48731 for high throughput screening methods).
  • antibodies and antigen-binding fragments thereof that compete for binding to the same epitope on PVRIG as the antibody molecules of the present disclosure can be obtained using routine techniques known to those skilled in the art.
  • Specific binding and “selective binding” refer to the binding of an antibody to a predetermined epitope on an antigen.
  • the antibody is detected at approximately lower than 10 -7 M or even higher
  • a small equilibrium dissociation constant (K D ) binds to a predetermined antigen or its epitope, and its binding affinity to the predetermined antigen or its epitope is lower than that of the predetermined antigen (or its epitope) or closely related antigens At least twice the binding affinity of non-specific antigens (such as BSA, etc.).
  • An "antibody that recognizes an antigen” is used interchangeably herein with a “specifically binding antibody”.
  • Binding affinity is used herein as a measure of the strength of a non-covalent interaction between two molecules (eg, an antibody or portion thereof and an antigen) to describe a monovalent interaction (intrinsic activity).
  • 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 directly determined by well-known methods (see Caceci et al., 1984, Byte 9: 340-362; Wong & Lohman, 1993, PNAS 90: 5428-5432).
  • Other standard assays to assess the binding ability of an antibody against a target antigen include, for example, ELISA, Western blot, RIA, and flow cytometry analysis, among other assays exemplified elsewhere herein.
  • an anti-PVRIG antibody of the present disclosure is capable of binding its target with an affinity at least 2-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold greater than the affinity with which it binds another non-PVRIG molecule Times, 1,000 times, or 10,000 times are not limited herein.
  • Constant modifications apply to both amino acid and nucleotide sequences.
  • conservative modification refers to the mutual substitution of those nucleic acids encoding identical or essentially identical amino acid sequences, or where the nucleotides do not encode an amino acid sequence, to essentially identical nucleotide sequences .
  • amino acid sequences a conservative modification refers to the replacement of an amino acid in a protein by another amino acid with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, and rigidity, etc.) such that frequent changes can be made without changing protein biological activity.
  • amino acid mutations include amino acid substitutions, deletions, insertions, modifications, and any combination thereof to achieve a final construct such that the final construct possesses desired properties, such as enhanced stability, increased activity.
  • Amino acid sequence deletions and insertions include amino-terminal and/or carboxy-terminal deletions and insertions.
  • Preferred amino acid mutations are amino acid substitutions. To alter eg the binding properties of an anti-PVRIG antibody, non-conservative amino acid substitutions can be made, ie one amino acid is replaced with another amino acid having different structural and/or chemical properties.
  • Preferred amino acid substitutions include replacing hydrophobic amino acids with hydrophilic amino acids.
  • Amino acid substitutions include those made from non-naturally occurring amino acids or from naturally occurring amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine, Amino acid) replacement.
  • Amino acid mutations can be generated using genetic or chemical methods known in the art, including site-directed mutagenesis, PCR, gene synthesis, chemical modification, and the like. Amino acid mutations can occur in the CDR region, FR region or Fc region of an antibody.
  • Antibodies or antigen-binding fragments engineered in the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO 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.
  • “Pharmaceutical composition” means a mixture containing one or more antibodies described herein (such as anti-PVRIG/TIGIT bispecific antibody) or a physiologically/pharmaceutically acceptable salt or prodrug thereof and other chemical components, wherein Examples of other components are: eg physiologically/pharmaceutically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thus exert biological activity.
  • a “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate buffered saline, water, emulsions such as oil/water emulsions, and various types of wetting agents.
  • the diluent for parenteral administration is phosphate buffered saline (PBS) or physiological (0.9%) saline.
  • Compositions comprising such carriers are formulated by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18th ed., A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and R Remington, The Science and Practice of Pharmacy 20th ed. Mack Publishing, 2000).
  • Buffering agent refers to a buffering agent that regulates changes in pH through the action of its acid-base conjugated component.
  • buffers to control pH in an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate , glycylglycine and other organic acid buffers.
  • a “histidine buffer” is a buffer comprising histidine.
  • histidine buffers include histidine-histidine hydrochloride, histidine-acetate histidine, histidine-phosphohistidine, histidine-histidine sulfate buffers, preferably histidine Acid-histidine hydrochloride buffer.
  • Histidine-HCl histidine buffers can be formulated with histidine and hydrochloric acid, or with histidine and histidine hydrochloride.
  • Acetate buffer is a buffer that includes acetate ions.
  • acetate buffers include acetic acid-acetic acid sodium salt, histidine-acetic acid histidine, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, and the like.
  • a preferred acetate buffer is acetic acid-acetate sodium salt.
  • citrate buffer is a buffer comprising citrate ions.
  • citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like.
  • a preferred citrate buffer is citric acid-sodium citrate.
  • succinate buffer is a buffer that includes succinate ions.
  • succinate buffers include succinate-succinate sodium salt, succinate-succinate potassium salt, succinate-succinate calcium salt, and the like.
  • a preferred succinate buffer is succinic acid-succinic acid sodium salt.
  • the succinic acid-sodium succinate can be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.
  • a “phosphate buffer” is a buffer that includes phosphate ions.
  • the phosphate buffer include disodium hydrogenphosphate-sodium dihydrogenphosphate, disodium hydrogenphosphate-potassium dihydrogenphosphate, disodium hydrogenphosphate-citric acid, and the like.
  • a preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.
  • “Lyophilized formulation” means a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a liquid or solution formulation has been subjected to a freeze-drying step.
  • the pharmaceutical composition described in the present disclosure can achieve a stable effect: the antibody therein (such as anti-PVRIG/TIGIT bispecific antibody) substantially retains its physical stability and/or chemical stability and/or biological stability after storage. Active pharmaceutical composition.
  • a pharmaceutical composition substantially retains its physical and chemical stability and its biological activity upon storage.
  • the shelf life is generally selected based on the intended shelf life of the pharmaceutical composition.
  • Stable formulations include formulations in which no statistically significant changes are observed when stored at refrigerated temperatures (2-8° C.) for at least 3 months, preferably 6 months, more preferably 1 year, even more preferably up to 2 Year.
  • stable liquid formulations also include liquid formulations that exhibit desired characteristics after storage at a temperature including 25°C for a period of time including 1 month, 3 months, or 6 months.
  • Typical examples of stability Usually no more than about 10%, preferably no more than about 5%, of the anti-PVRIG/TIGIT bispecific antibody aggregates or degrades as measured by SEC-HPLC.
  • the formulation is a pale yellow to near colorless clear liquid or a colorless clear liquid, or clear to slightly milky white.
  • the concentration, pH and osmolality of the formulations had no more than ⁇ 10% variation.
  • no more than about 10%, preferably no more than about 5% reduction is observed.
  • no more than about 10%, preferably no more than about 5% aggregates are formed.
  • the anti-PVRIG/TIGIT bispecific antibody preparation of the present disclosure is stored at 40°C for one month, and the SEC monomer % is greater than or equal to 95% (such as greater than 95%, greater than 96%, greater than 97%) , greater than 98%, or greater than 99%) and/or CE-SDS(NR)% greater than or equal to 95% (eg, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%).
  • the anti-PVRIG/TIGIT bispecific antibody does not show significant aggregation after visual inspection for color and/or clarity, or as measured by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS) increased, precipitated and/or denatured, the anti-PVRIG/TIGIT bispecific antibody "retains its physical stability" in the pharmaceutical formulation. Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).
  • An anti-PVRIG/TIGIT bispecific antibody "retains its chemical stability" in a pharmaceutical formulation if the anti-PVRIG/TIGIT bispecific antibody exhibits no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often alter protein chemical structure include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and CE-SDS), oxidation (by methods such as peptide mapping combined with mass spectrometry or MALDI/TOF/MS, etc.
  • deamidation evaluationated by methods such as ion-exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartic acid measurement, etc.
  • isomerization by measuring isoaspartic acid content, Peptide mapping, etc. to evaluate).
  • An anti-PVRIG/TIGIT bispecific antibody "retains its biological activity".
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to an exogenous drug, therapeutic, diagnostic or composition Contact with animals, humans, subjects, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, 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, composition or by another cell.
  • Treatment as it applies to human, veterinary or research subjects means therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as a pharmaceutical composition comprising any of the present disclosure, to a subject having one or more symptoms of a disease for which the therapeutic agent is known to be effective These symptoms are therapeutic.
  • 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, to induce regression of such symptoms or to inhibit the progression of such symptoms to any clinically measured 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 t test, chi-square test, It should reduce the target disease symptoms in a statistically significant number of subjects as determined by Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent the symptoms 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.
  • Subjects of the present disclosure can be animal or human subjects.
  • compositions of the present disclosure may be administered by any suitable means, including parenteral, intrapulmonary, intranasal, and if desired for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection.
  • a variety of dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • the pharmaceutical compositions of the present disclosure are administered intravenously.
  • compositions of the present disclosure are to be formulated, dosed and administered in a manner consistent with good medical practice.
  • Factors to be considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual subject, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and what is known to the medical practitioner. other factors.
  • the pharmaceutical composition may also be formulated with one or more other agents for the prophylaxis or treatment of the disorder. The effective amount of such other agents depends on the amount of antigen-binding molecule present in the pharmaceutical composition, the type of disorder or treatment, and other factors.
  • the same dosages and routes of administration as described herein may be used, or at about 1 to 99% of the dosages described herein, or at any dosage, and any route empirically/clinically determined to be suitable.
  • PCT/CN2021/080470 (application date: 2021.3.13; priority patent application number: CN202010174835.4) is fully incorporated into this disclosure by reference.
  • Human PVRIG with his tag (h-PVRIG-his) recombinant protein
  • human PVRIG with mouse IgG2a Fc tag h-PVRIG-mIgG2a Fc
  • mouse PVRIG with human IgG1 Fc tag (m- PVRIG-hIgG1 Fc) is a purified commercial protein reagent purchased from Acrobiosystems, and its sequence is shown in Table 1.
  • the recombinant protein was expressed by transient transfection in HEK293 cells by conventional methods, and the supernatant was collected and purified by Ni-NTA. After testing, cyno-PVRIG-his was obtained.
  • Anti-human PVRIG monoclonal single domain antibody was produced by immunizing camels.
  • the immune antigen is human PVRIG recombinant protein with his tag (h-PVRIG-his).
  • Emulsification with Freund's adjuvant (Sigma, Lot No.: F5881/F5506): Complete Freund's adjuvant (CFA) CFA for the first time, and incomplete Freund's adjuvant (IFA) for the rest of booster immunization.
  • CFA Complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • the phage library was diluted and blocked with BSA, co-incubated with magnetic beads Dynabeads (M-280, invitrogen), and the phage after negative screening incubation were collected.
  • the Dynabeads were coated and blocked with biotin-labeled human PVRIG with his tag, the phage suspension collected after negative screening was incubated with the Dynabeads, and the phages were eluted with trypsin. After three rounds of screening, 400 clones obtained in the third round of screening were selected for sequencing.
  • the heavy chain sequences of five single domain antibodies are shown in Table 2, and the CDRs of different coding rules are shown in Table 3.
  • the heavy chain variable regions of the five antibodies in Example 2 were connected to the human IgG4 heavy chain Fc region to form a full-length anti-PVRIG antibody.
  • the Fc region of the heavy chain includes the hinge region, and carries S228P, F234A, L235A, K447A mutations (Eu nomenclature system).
  • the anti-PVRIG antibody CPA.7.021 shown in WO2016134333A1 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 gene sequence encoding the above sequence was synthesized, digested with BamHI and XhoI, and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20) through the BamHI/XhoI restriction site.
  • the expression vector and transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1:2, and incubated in a CO 2 incubator for 4- 5 days. After the expressed antibody was recovered by centrifugation, the antibody was purified according to conventional methods, and the target antibody was obtained after detection.
  • ELISA experiments were used to examine the binding properties of anti-PVRIG antibodies.
  • the PVRIG recombinant protein with his tag was directly coated, and after the antibody was added, 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 monkey or mouse PVRIG protein was coated with 96-well ELISA plate, 100 ⁇ L per well at a concentration of 1 ⁇ g/mL, and incubated overnight at 4°C. Wash three times with washing solution, 250 ⁇ L per well. Shake for 10 seconds per wash to ensure thorough cleaning. Add 300 ⁇ L/well blocking solution (PBS+0.05% Tween20+1% BSA) and incubate at room temperature for 1 hour. Wash three times with washing solution, 250 ⁇ L per well. Shake for 10 seconds per wash to ensure thorough cleaning. Add 100 ⁇ L of anti-PVRIG antibody to be tested diluted with diluent to each well. Incubate at 37°C for 1 hour.
  • the binding properties of anti-PVRIG antibodies were detected by flow cytometry (FACS).
  • FACS flow cytometry
  • a cell line overexpressing human or cynomolgus monkey PVRIG was constructed. After the antibody was added, the activity of the antibody binding to the antigen was detected by adding a secondary antibody.
  • the expression plasmid carrying human or cynomolgus monkey PVRIG gene sequence was transfected in HEK293 cells, and overexpressed and stably transfected monoclonal cell lines were obtained by antibiotic selection and infinite dilution. Seed 2 ⁇ 105 overexpressing 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.
  • the specific method is to coat human PVRIG recombinant protein (h-PVRIG-mIgG2a Fc) with mouse IgG2a Fc tag on a 96-well microtiter plate, add anti-PVRIG antibody to fully bind and occupy the epitope, and then add his tagged PVRL2 (PV2-H52E2, AcroBiosystem), by detecting the his tag, to calculate the binding amount of PVRIG and PVRL2, and calculate the IC 50 value of the anti-PVRIG antibody blocking the PVRIG active site.
  • human PVRIG recombinant protein h-PVRIG-mIgG2a Fc
  • mouse IgG2a Fc tag mouse IgG2a Fc tag
  • the h-PVRIG-mIgG2a Fc protein was coated on a 96-well ELISA plate, 100 ⁇ L per well at a concentration of 1 ⁇ g/mL, and incubated overnight at 4°C. Wash three times with washing solution, 250 ⁇ L per well. Shake for 10 seconds per wash to ensure thorough cleaning. Add 300 ⁇ L/well blocking solution and incubate at room temperature for 1 hour. Wash three times with washing solution, 250 ⁇ L per well. Shake for 10 seconds per wash to ensure thorough cleaning. 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.
  • plvx-OS8 G418 resistance
  • transfect 293F cells G418 screening
  • use 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
  • construct plvx-PVRL2 plasmid use it to infect 293F-OS8 cells, and use flow cytometry to screen out the clone with the highest expression of PVRL2, so as to obtain 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
  • 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.
  • the cultured NK92 cell line (NK cells from human malignant non-Hodgkin's lymphoma patients) was washed twice with washing solution (including RPMI 1640, 5% FBS, 10ng/mL IL-2) and resuspended to 2 ⁇ 10 6 unit/mL density.
  • washing solution including RPMI 1640, 5% FBS, 10ng/mL IL-2
  • 50 ⁇ L a total of 1 ⁇ 105
  • NK92 cells per well in a 96-well plate Add 50 ⁇ L 20nM or 100nM antibody to be tested, and incubate at 37°C for 30 minutes. Wash twice with washing solution and resuspend to a density of 2 ⁇ 105 /mL.
  • control group 1 Three kinds of control groups were prepared, namely a sample containing only culture medium (control group 1), a sample containing only NK92 cells (control group 2), and 150 ⁇ L of a sample containing only K562 cells (control group 3), and the same operation was performed.
  • the killing activity was calculated according to the following formula:
  • Killing activity (%) ⁇ [(R–BG)-(T-BG)-(E–BG)]/[(TL-BGL)–(T-BG)] ⁇ 100
  • 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 in dendritic cells (DC cells).
  • Anti-PVRIG antibody can release the inhibitory effect of dendritic cells on T cells and activate T cells by blocking the combination of PVRL2 and PVRIG.
  • PBMCs were isolated from the peripheral blood of the first individual, and the cells were cultured in RPMI 1640 medium containing 10% FBS with 50 ng/mL GM-CSF (Peprotech, 300-03-100UG) and 50 ng/mL IL- Add cytokines to the final concentration of 4 (Peprotech, 200-04-100UG), and add fresh medium containing cytokines every 2-3 days; after culturing for 6 days, add 1 ⁇ g/mL LPS (Sigma, L2880-25MG) and incubate for 24 hours , collect the differentiated and mature DC cells.
  • RPMI 1640 medium containing 10% FBS with 50 ng/mL GM-CSF (Peprotech, 300-03-100UG) and 50 ng/mL IL- Add cytokines to the final concentration of 4 (Peprotech, 200-04-100UG), and add fresh medium containing cytokines every 2-3 days; after culturing for 6 days, add 1 ⁇ g/m
  • PBMC peripheral blood from a second source, 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 120h, take the supernatant, and detect the IFN ⁇ content in the supernatant with an ELISA kit (R&D, DY202). The experimental results are shown in Figure 3 and Table 11.
  • the heavy chain variable region sequence was compared with the antibody GermLine database to obtain the human race with high homology department template.
  • the framework region of the camelid single domain antibody was replaced with the heavy chain framework region of the human germline template, the CDRs (according to the Kabat numbering system) were retained, and then combined with the Fc region of human IgG (IgG4Fc with S228P, F234A, L235A, K447A mutations) ) reorganization.
  • antibody 20H1-20H5 comprises CDR1 as shown in TDCMG (SEQ ID NO: 7), CDR2 as shown in HIDSDGIPRYVDSVKG (SEQ ID NO: 8), and CDR2 as shown in GFKFDEDYCAPND (SEQ ID NO: 150) CDR3.
  • antibodies 30H1-30H5 comprise CDR1 as shown in GDCMG (SEQ ID NO: 10), CDR2 as shown in TIDNAGRIKYADSVKG (SEQ ID NO: 11), and CDR3 as shown in GWTFGGQCSPAD (SEQ ID NO: 151) .
  • 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 mutations.
  • the antibody was expressed and purified according to conventional methods, and the target antibody was obtained after detection.
  • Example 8 the activity of the humanized anti-PVRIG antibody in reporter cells was detected.
  • the experimental results are shown in FIGS. 4A-4B and Table 19.
  • the antibodies listed in the table all have the ability to activate Jurkat cells.
  • Example 15 Experiment of activated NK cell killing ability of humanized anti-PVRIG antibody
  • Example 9 the activation ability of the humanized anti-PVRIG antibody on NK cells was detected.
  • the experimental results are shown in Figure 5A- Figure 5B and Table 20-Table 21.
  • the results show that all the humanized anti-PVRIG antibodies of the present disclosure have 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 of the heavy chain or light chain of the anti-TIGIT antibody 1708 through the GGGGSGGGGS (SEQ ID NO: 152) linker. terminal or C-terminal.
  • 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 22 below.
  • the sequence information of the anti-TIGIT antibody is shown in Table 23-24.
  • the TIGIT antibody in WO2019062832A1 is incorporated herein in its entirety.
  • Example 5 and Example 6 experiments were carried out, and the results are shown in Table 26.
  • the results showed that the binding of bispecific antibodies with different configurations to human PVRIG recombinant protein and overexpressed human PVRIG cells, as well as the blocking of PVRL2 binding to PVRIG, were basically the same and had no difference.
  • Example 5 and Example 6 the corresponding receptors and ligands were replaced by human TIGIT and human PVR
  • the experiment was carried out, and the results are shown in Table 27.
  • the results showed that the binding of bispecific antibodies and anti-TIGIT antibodies with different configurations to human TIGIT recombinant protein and overexpressed human TIGIT cells, as well as the blocking of TIGIT binding to its ligand PVR, were basically consistent and had no difference.
  • the connection mode of the anti-PVRIG antibody 151 basically has no effect on the binding of the anti-TIGIT antibody to TIGIT.
  • the affinities of humanized bispecific antibodies to human PVRIG, cynomolgus monkey PVRIG, and human TIGIT were tested using Biacore.
  • 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), cynomolgus monkey PVRIG antigen (SEQ ID NO: 1), or human TIGIT antigen (AcroBiosystem, TIT-H52H3).
  • Biacore instruments (Biacore X100, GE) were used to detect the reaction signals in real time to obtain association and dissociation curves. The data obtained from the experiment were fitted with the (1:1) Binding model using BiacoreX100 evaluation software2.0 GE software to obtain the affinity value, as shown in Table 31.
  • Example 20 Mixed lymphocyte reaction (MLR) experiment of humanized anti-PVRIG/TIGIT bispecific antibody
  • the activation ability of the humanized anti-PVRIG/TIGIT bispecific antibody on T cells was detected.
  • the experimental results are shown in Figure 8 and Table 32.
  • the results showed that the humanized anti-PVRIG/TIGIT bispecific antibody 1708-151H8 has the obvious ability to activate T cells and promote the secretion of IFN ⁇ by T cells.
  • the bispecific antibody was more active than anti-PVRIG antibody 151H8 alone and anti-TIGIT antibody 1708 alone.
  • Example 21 Evaluation of anti-tumor effect of anti-PVRIG/TIGIT bispecific antibody in human melanoma A375 mixed with human PBMC subcutaneous xenograft tumor model in mice
  • 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-culture need to be treated with Mitomycin C for 2 hours, and 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 method, dosage and route of administration are shown in Table 34. 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 Tables 35-36 and Figures 9A-9B respectively.
  • mice were administered in groups at random according to their body weight.
  • the detailed administration methods, doses and routes of administration are shown in Table 37.
  • the day of group administration was Day 0.
  • Dosing regimen Method of administration 1 hIgG4 7 35.8 Q2D i.p. 2 1708-30H2 IgG4 7 12 Q2D i.p. 3 1708-151H7 IgG4 7 12 Q2D i.p.
  • 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 Tables 38-39 and Figures 10A-10B respectively.
  • both the 1708-30H2IgG4 and 1708-151H7IgG4 double antibody groups could effectively inhibit tumor growth at low doses, and there was a significant difference between them (see Figure 10A and 10B).
  • SEC% SEC monomer content percentage
  • a monomer is the peak area of the main peak monomer in the sample, and A total is the sum of all peak areas).
  • NR-CE also known as CE-SDS (NR) capillary gel electrophoresis
  • Non-reducing CE purity percentage A main peak/A total*100% (A main peak is the peak area of the main peak in the sample, A total is the sum of all peak areas.)
  • CE determination instrument Beckman capillary electrophoresis instrument model plus800.
  • iCIEF neutral peak content percentage neutral peak area/total area*100% (the total area is the sum of the areas of acidic peak, neutral peak and basic peak).
  • the instrument used for iCIEF determination simple protein, model muarice.
  • the freezing point method is used to measure the osmotic pressure. Based on the proportional relationship between the freezing point depression value and the molar concentration of the solution, a high-sensitivity temperature-sensing element is used to measure the freezing point of the solution, which is converted into osmotic pressure by electricity.
  • the instrument used for the determination of osmotic pressure Loser, model OM815.
  • the anti-PVRIG/TIGIT bispecific antibody used in the following preparation examples is 1708-30H2.
  • the concentration of the bispecific antibody was measured using a protein concentration meter.
  • Protein concentration determination instrument UV-Vis spectrophotometer, model: Nano Drop oneC, optical path 1mm.
  • the following buffer system was used to prepare the anti-PVRIG/TIGIT bispecific antibody (1708-30H2) preparation with a protein concentration of about 50 mg/mL, wherein the buffer system was as follows:
  • AA 10mM acetic acid-sodium acetate
  • His-HCl 10mM histidine-histidine hydrochloride
  • M means month
  • 40 °C M1 means 40 °C constant temperature for 1 month
  • a 10mM His-HCl pH 6.0 buffer system was used to prepare preparations with a low concentration of 50mg/mL and a high concentration of 100mg/mL.
  • the prepared preparation is sterile filtered, filled, stoppered and capped.
  • the samples were forced to degrade under the high temperature condition of 40°C, and the stability of the formulation was investigated with SEC, CE-SDS (NR) and iCIEF as evaluation indicators.
  • the experimental results are shown in Table 41.
  • the experimental results show that: after the preparation samples were placed at a high temperature of 40°C for 1 month, in terms of the purity of the preparation groups with different concentrations, the SEC, CE-SDS(NR) and iCIEF values of the preparations with high protein concentration were relatively slightly lower .
  • anti-PVRIG/TIGIT antibody (1708-30H2) preparations containing 50mg/mL antibody, 80mg/mL sucrose, and different concentrations of surfactants, specific surfactant types and Concentrations are as follows:
  • the prepared preparation is sterile filtered, filled, stoppered and capped.
  • the samples were forced to degrade under the high temperature condition of 40°C, and the stability of the preparation was investigated by evaluating the appearance, SEC, CE-SDS (NR) and iCIEF.
  • a buffer system of 10mM His-HCl pH5.5 was used to prepare anti-PVRIG/TIGIT antibody (1708-30H2) preparations containing 50mg/mL antibody, 0.4mg/mL polysorbate 80 and different concentrations of sucrose.
  • the specific sucrose concentration was as follows:
  • buffer system to prepare anti-PVRIG/TIGIT antibody (1708-30H2) preparations containing 50 mg/mL antibody, 80 mg/mL sucrose, and 0.4 mg/mL polysorbate 80, wherein the buffer system is as follows:
  • the prepared preparation is sterile filtered, filled, stoppered and capped.
  • the samples were forced to degrade under the high temperature condition of 40°C, and the stability of the preparation was investigated by evaluating the appearance, SEC, CE-SDS (NR) and iCIEF.
  • buffer system to prepare anti-PVRIG/TIGIT antibody preparation (1708-30H2) containing 50 mg/mL antibody, 80 mg/mL sucrose, and 0.4 mg/mL polysorbate 80.
  • the buffer system is as follows:
  • the prepared preparation is sterile filtered, filled, stoppered and capped.
  • the samples were forced to degrade under the high temperature condition of 40°C, and the stability of the preparation was investigated by evaluating the appearance, SEC, CE-SDS (NR) and iCIEF.
  • the anti-PVRIG/TIGIT antibody (1708-30H2) preparation containing 50mg/mL antibody, 0.4mg/mL polysorbate 80, and 80mg/mL sucrose was prepared using 10mM His-HCl pH6.0 buffer system.
  • the prepared preparation is sterile filtered, filled, stoppered and capped.
  • the samples were placed at 25°C and 2 to 8°C for 6 months, and the stability of the preparation was investigated by evaluating the appearance, SEC, CE-SDS (NR) and iCIEF.
  • 10mM His-HCl pH6.0 buffer system was used to prepare 3 batches of anti-PVRIG/TIGIT antibody (1708-30H2) preparations containing 50mg/mL antibody, 0.4mg/mL polysorbate 80, and 80mg/mL sucrose.
  • the pH value of 3 batches of finished preparations was measured, compared with the pH value of the buffer solution, and the pH drift was investigated.
  • the prepared preparation is sterile filtered, filled, stoppered and capped.
  • the samples were placed under the condition of 2 to 8°C for 12 months, and the stability of the formulation was investigated by taking the appearance, SEC, and iCIEF as evaluation indicators.

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Abstract

一种含抗PVRIG/TIGIT双特异性抗体的药物组合物。

Description

一种含抗PVRIG/TIGIT双特异性抗体的药物组合物
本申请要求2021年09月15日提交的中国专利申请202111078172.7的优先权。
技术领域
本披露属于药物制剂领域，具体涉及一种包含双特异性抗体的药物组合物，以及其作为药物的用途。
背景技术
这里的陈述仅提供与本披露有关的背景信息，而不必然地构成现有技术。
PVRIG，又名CD112R，是细胞表面表达的蛋白，与TIGIT、CD96和CD226等同属于B7/CD28超家族，在免疫系统中起重要作用。
PVRIG包含一个胞外区，一个跨膜区，和一个胞内区。当其配体PVRL2(又叫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在很多实体瘤中都有高表达，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，可以有效抑制肿瘤的生长，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，包括浸润不同类型肿瘤的肿瘤浸润淋巴细胞(TIL)和Treg。已经证明TIGIT信号传导与其同源配体PVR(也称为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也在肿瘤中广泛表达，表明TIGIT-PVR信号轴可能是癌症的主要免疫逃逸机制。
但是，目前尚未有抗PVRIG/TIGIT双特异性抗体药物进入临床。现有技术中仍然缺乏能够抑制体内癌症或肿瘤生长的高亲合力(avidity)、高选择性、高生物活性的抗PVRIG/TIGIT双特异性抗体的制剂。
发明内容
本披露提供一种含抗PVRIG/TIGIT双特异性抗体的药物组合物，该组合物具有治疗活性。此外，该组合物还可具有稳定性好等优势。
在一些实施方案中，本披露提供一种药物组合物，包含抗PVRIG/TIGIT双特异性抗体和缓冲剂，其中所述抗PVRIG/TIGIT双特异性抗体包含特异性结合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，所述缓冲剂为组氨酸缓冲剂或醋酸盐缓冲剂。
在一些实施方案中，所述缓冲剂为组氨酸-盐酸组氨酸缓冲剂或醋酸-醋酸钠盐缓冲剂。
在一些具体的实施方案中，所述缓冲剂为组氨酸-盐酸组氨酸缓冲剂。
在一些实施方案中，如上任一项所述的药物组合物，其中所述药物组合物的pH为5.0-6.5。在一些实施方案中，所述药物组合物的pH为5.5至6.5；在一些实施方案中，所述药物组合物的pH为5.5-6.0；在一些实施方案中，所述药物组合物的pH为约6.0。当本披露中提及点值时，应当理解该点值包含了误差范围。这种误差范围是由于实验室环境、人员操作、仪器、方法学、测量误差等因素所致。以pH为例，当测值为约6.0时，应当理解其包含了误差范围。作为一个示例，采用工业用pH计测量制剂时，“约6.0”表示6.0±0.2。
在一些实施方案中，所述药物组合物的pH为5.0-5.5；在一些实施方案中，所述药物组合物的pH为约5.5。
在一些实施方案中，所述药物组合物的pH为5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4或6.5，或者为这些点值之间的任意范围。
通常，通过置换缓冲剂获得的药物组合物的pH与缓冲剂pH几乎一致。同时，本领域技术人员公知，在药物制剂的过程中，有时可能会存在pH飘移，但药物制剂的pH的飘移一般很小(±0.3范围内)。在一些实施方案中，药物制剂的pH的飘移在±0.1范围内。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体的浓度为1mg/mL至150mg/mL。在一些实施方案中，所述抗PVRIG/TIGIT双特异性抗体的浓度为1mg/mL至100mg/mL。在一些实施方案中，所述抗PVRIG/TIGIT双特异性抗体的浓度为40mg/mL至100mg/mL。在一些实施方案中，所述抗PVRIG/TIGIT双特异性抗体的浓度为50mg/mL至100mg/mL。在一些实施方案中，所述抗PVRIG/TIGIT双特异性抗体的浓度为40mg/mL至60mg/mL。在一些实施方案中，所述抗PVRIG/TIGIT双特异性抗体的浓度为100mg/mL。在一些实施方案中，所述抗PVRIG/TIGIT双特异性抗体的浓度为100mg/mL±8mg/mL。在一些实施方案中，所述抗PVRIG/TIGIT双特异性抗体的浓度为50mg/mL。在一些实施方案中，所述抗PVRIG/TIGIT双特异性抗体的浓度为50mg/mL±5mg/mL。在一些实施方案中，所述抗PVRIG/TIGIT双特异性抗体的浓度为1mg/mL、10mg/mL、20mg/mL、30mg/mL、40mg/mL、45mg/mL、50mg/mL、55mg/mL、60mg/mL、65mg/mL、70mg/mL、80mg/mL、90mg/mL、100mg/mL、 110mg/mL、120mg/mL、130mg/mL、140mg/mL或150mg/mL，或者为这些点值之间的任意范围。
在一些实施方案中，如上任一项所述的药物组合物，其中所述药物组合物包含表面活性剂。在一些实施方案中，所述表面活性剂是非离子表面活性剂。在一些实施方案中，所述表面活性剂选自泊洛沙姆(例如泊洛沙姆188)、聚山梨酯(例如聚山梨酯20、聚山梨酯80)、聚羟亚烃、Triton、十二烷基磺酸钠、月桂基磺酸钠、辛基糖甙钠、月桂基-磺基甜菜碱、肉豆蔻基-磺基甜菜碱、亚油基-磺基甜菜碱、硬脂基-磺基甜菜碱、月桂基-肌氨酸、肉豆蔻基-肌氨酸、亚油基-肌氨酸、硬脂基-肌氨酸、亚油基-甜菜碱、肉豆蔻基-甜菜碱、鲸蜡基-甜菜碱、月桂酰胺基丙基-甜菜碱、柯卡酰胺基丙基-甜菜碱、亚油酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-甜菜碱、棕榈酰胺基丙基-甜菜碱、异硬脂酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-二甲基胺、棕榈酰胺基丙基-二甲基胺、异硬脂酰胺基丙基-二甲基胺、甲基可可酰基钠、甲基油基牛磺酸钠、聚乙二醇、聚丙二醇、乙烯与丙烯二醇的共聚物等。在一些实施方案中，所述表面活性剂为聚山梨酯或泊洛沙姆。在一些实施方案中，所述表面活性剂为聚山梨酯80、聚山梨酯20或泊洛沙姆188。在一些实施方案中，所述表面活性剂为聚山梨酯80。
在一些实施方案中，如上任一项所述的药物组合物，其中所述表面活性剂浓度为0.05mg/mL至1.0mg/mL。在一些实施方案中，所述表面活性剂浓度为0.2mg/mL至0.6mg/mL。在一些实施方案中，所述表面活性剂浓度为0.05mg/mL、0.1mg/mL、0.2mg/mL、0.3mg/mL、0.4mg/mL、0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL或1.0mg/mL，或者为这些点值之间的任意范围。在一些实施方案中，所述表面活性剂浓度为0.4mg/mL。在一些实施方案中，所述表面活性剂浓度为0.4mg/mL±0.1mg/mL。
在一些实施方案中，所述表面活性剂为0.4mg/mL的聚山梨酯80。
在一些实施方案中，如上任一项所述的药物组合物，其包括渗透压调节剂。在一些实施方案中，渗透压调节剂为糖(包括单糖，二糖，三糖，多糖，糖醇，还原性糖，非还原性糖等等)、氨基酸(包括精氨酸、甘氨酸、半胱氨酸、组氨酸等等)或盐类(氯化钠、氯化钾、氯化钙等)。在一些实施方案中，所述渗透压调节剂为糖，所述糖选自：葡萄糖，蔗糖，海藻糖，乳糖，果糖，麦芽糖，右旋糖苷，甘油，赤藻糖醇，丙三醇，阿拉伯糖醇，木糖醇，山梨糖醇(也称山梨醇)，甘露醇，密里二糖，松三糖，蜜三糖，甘露三糖，水苏糖，麦芽糖，乳果糖，麦芽酮糖，麦芽糖醇，乳糖醇和异-麦芽酮糖。在一些实施方案中，所述渗透压调节剂选自由蔗糖、海藻糖、山梨糖醇、精氨酸、甘氨酸和氯化钠组成的组中的一种或多种。在一些实施方案中，所述渗透压调节剂为非还原性二糖；在一些实施方案中，所述渗透压调节剂为海藻糖或蔗糖；在一些实施方案中，所述渗透压调节剂为蔗糖。
在一些实施方案中，如上任一项所述的药物组合物，渗透压调节剂浓度为70mg/mL至100mg/mL。在一些实施方案中，渗透压调节剂浓度为75mg/mL至100mg/mL；在一些实施方案中，渗透压调节剂浓度为75mg/mL至90mg/mL；在一些实施方案中，渗透压调节剂浓度为70mg/mL至90mg/mL；在一些实施方案中，渗透压调节剂浓度为75mg/mL至85mg/mL；在一些实施方案中，渗透压调节剂浓度为75mg/mL至80mg/mL；在一些实施方案中，渗透压调节剂浓度为80mg/mL；在一些实施方案中，渗透压调节剂浓度为80mg/mL±5mg/mL。在一些实施方案中，渗透压调节剂浓度非限制性实施包括60mg/mL、65mg/mL、70mg/mL、75mg/mL、80mg/mL、85mg/mL、90mg/mL、95mg/mL、100mg/mL，以及这些点值之间的任一范围。
在一些实施方案中，前述药物组合物为等渗制剂。在一些实施方案中，所述渗透压调节剂使前述药物组合物渗透压控制在280-320mOsm，在一些实施方案中，渗透压控制在约290-300mOsm。在一些实施方案中，渗透压调节剂控制渗透压在280mOsm、290mOsm、295mOsm、300mOsm、305mOsm、310mOsm、320mOsm，以及这些点值之间的任一范围。
在一些实施方案中，所述渗透压调节剂为80mg/mL蔗糖。
在一些实施方案中，所述渗透压调节剂为80mg/mL±5mg/mL蔗糖。
在一些实施方案中，如上任一项所述的药物组合物，所述缓冲剂的浓度为5mM至100mM。在一些实施方案中，所述缓冲剂的浓度为10mM至30mM。在一些实施方案中，所述缓冲剂的浓度为5mM至15mM。在一些实施方案中，所述缓冲剂的浓度为5mM、10mM、15mM、20mM、25mM、30mM、40mM、50mM、60mM、70mM、80mM、90mM或100mM，以及这些点值之间的任一范围。在一些实施方案中，所述缓冲剂为10mM His-HCl。在一些实施方案中，所述缓冲剂为10mM±5mM His-HCl。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体，其特异性结合PVRIG的第一抗原结合结构域包含至少一个免疫球蛋白单一可变结构域(如VHH)，所述至少一个免疫球蛋白单一可变结构域(如VHH)包含三个互补决定区CDR1、CDR2和CDR3，其中：CDR1选自SEQ ID NO：7、10、13、16、19、22、25、28、31、34、37、40、43、46、49、52、55、58、61、64任一所示的氨基酸序列或与之具有3、2、1个或更多氨基酸差异的氨基酸序列，和/或CDR2选自SEQ ID NO：8、11、14、17、20、23、26、29、32、35、38、41、44、47、50、53、56、59、62、65任一所示的氨基酸序列或与之具有3、2、1个或更多氨基酸差异的氨基酸序列，和/或CDR3选自SEQ ID NO：9、12、15、18、21、24、27、30、33、36、39、42、45、48、51、54、57、60、63、66、150、151任一所示的氨基酸序列或与之具有3、2、1个或更多氨基酸差异的氨基酸序列；其中，SEQ ID NO：7-21是依据Kabat编码规则，SEQ ID NO：22-36是 依据Chothia编码规则，SEQ ID NO：37-51是依据IMGT编码规则，SEQ ID NO：52-66是依据AbM编码规则。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体，其特异性结合PVRIG的第一抗原结合结构域包含至少一个免疫球蛋白单一可变结构域(如VHH)，所述免疫球蛋白单一可变结构域包含SEQ ID NO：2、75-79任一所示序列中的CDR1、CDR2、CDR3；或包含SEQ ID NO：3、80-84任一所示序列中的CDR1、CDR2、CDR3；或包含SEQ ID NO：4、86-90任一所示序列中的CDR1、CDR2、CDR3；或包含SEQ ID NO：5、91-95任一所示序列中的CDR1、CDR2、CDR3；或包含SEQ ID NO：6、96-100任一所示序列中的CDR1、CDR2、CDR3。在一些实施方案中，所述免疫球蛋白单一可变结构域包含SEQ ID NO：3、80-84任一所示序列中的CDR1、CDR2、CDR3。在一些实施方案中，所述免疫球蛋白单一可变结构域包含SEQ ID NO：81中的CDR1、CDR2、CDR3。在一些实施方案中，所述CDR1、CDR2、CDR3是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的。在一些实施方案中，所述CDR1、CDR2、CDR3是根据Kabat编号规则确定的。在一些实施方案中，依据Kabat编码规则，特异性结合PVRIG的第一抗原结合结构域(如VHH)包含三个互补决定区CDR1、CDR2和CDR3，其中：CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：7、8、9所示；CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：7、8、150所示；CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：10、11、12所示；CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：10、11、151所示；CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：13、14、15所示；CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：16、17、18所示；或CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：19、20、21所示。在一些实施方案中，依据Chothia编码规则，特异性结合PVRIG的第一抗原结合结构域(如VHH)包含三个互补决定区CDR1、CDR2和CDR3，其中：CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：22、23、24所示；CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：25、26、27所示；CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：28、29、30所示；CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：31、32、33所示；或CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：34、35、36所示。在一些实施方案中，依据IMGT编码规则，特异性结合PVRIG的第一抗原结合结构域(如VHH)包含三个互补决定区CDR1、CDR2和CDR3，其中：CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：37、38、39所示；CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：40、41、42所示；CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：43、44、45所示；CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：46、47、48所示；或CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：49、50、51所示。在一些实施方案中，依据AbM 编码规则，特异性结合PVRIG的第一抗原结合结构域(如VHH)包含三个互补决定区CDR1、CDR2和CDR3，其中：CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：52、53、54所示；CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：55、56、57所示；CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：58、59、60所示；CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：61、62、63所示；或CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：64、65、66所示。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体，其特异性结合PVRIG的第一抗原结合结构域包含至少一个免疫球蛋白单一可变结构域(如VHH)，所述免疫球蛋白单一可变结构域包含CDR1、CDR2和CDR3，所述CDR1、CDR2和CDR3根据Kabat编号系统定义，其中，CDR1包含SEQ ID NO：10的氨基酸序列，CDR2包含SEQ ID NO：11的氨基酸序列，和CDR3包含SEQ ID NO：151的氨基酸序列。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体，其特异性结合PVRIG的第一抗原结合结构域(如VHH)包含SEQ ID NO：2-6、75-84、86-100任一所示的氨基酸序列，或包含与SEQ ID NO：2-6、75-84、86-100中的至少一条序列具有至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％同一性的氨基酸序列。在一些实施方案中，其中所述抗PVRIG/TIGIT双特异性抗体的第一抗原结合结构域包含SEQ ID NO：81的氨基酸序列。在一些实施方案中，其中所述抗PVRIG/TIGIT双特异性抗体的第一抗原结合结构域包含与SEQ ID NO：81具有至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的序列的氨基酸序列。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体，其中，特异性结合PVRIG的第一抗原结合结构域为第一抗体，其是VHH；特异性结合TIGIT的第二抗原结合结构域为第二抗体，在一些实施方案中，第二抗体包括重链(HC)和轻链(LC)；所述第一抗体位于第二抗体的重链或轻链的N端和/或C端。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体包含1个特异性结合TIGIT的第二抗体和2个特异性结合PVRIG的第一抗体；所述第二抗体包括两条HC和两条LC，第二抗体的一条HC的VH与一条LC的VL形成抗原结合部位，另一条HC的VH与另一条LC的VL形成抗原结合部位。在一些实施方案中，其中一个第一抗体位于第二抗体的重链或轻链的N端，另一个第一抗体位于第二抗体的重链或轻链的C端。在一些实施方案中，每个第一抗体分别位于第二抗体的两条重链或两条轻链的N端；或者，每个第一抗体分别位于第二抗体的两条重链或两条轻链的C端。在一些实施方案中，每个第一抗体分别位于第二抗体的两条重链的N端。在一些实施方案中，每个第一抗 体分别位于第二抗体的两条重链的C端。在一些实施方案中，所述2个第一抗体是相同的。在一些实施方案中，所述2个第一抗体是不相同的。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体，其中第二抗体可以连接有1、2、3、4、5、6、7、8个第一抗体，所述第一抗体可以是相同的或不同的，可以均连接在第二抗体的重链N端，或均连接在第二抗体的重链C端，或均连接在第二抗体的轻链N端，或均连接在第二抗体的轻链C端，或连接在第二抗体的重链N端、重链C端、轻链N端或轻链C端或其任意组合。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体，其中第一抗体直接或通过连接子与第二抗体的重链的N端或C端连接。在一些实施方案中，所述连接子选自：如(GmSn)x或(GGNGT)x或(YGNGT)x所示的氨基酸序列，其中m、n各自独立地选自1-8的整数(例如，1、2、3、4、5、6、7或8)，x独立地选自1-20的整数(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20)。在一些实施方案中，连接子为GS、GAP、ASGS(SEQ ID NO：154)、G 4S(SEQ ID NO：155)、(G 4S) 2(SEQ ID NO：152)、(G 4S) 3(SEQ ID NO：156)、(G 4S) 4(SEQ ID NO：157)、(G 4S) 5(SEQ ID NO：158)、(G 4S) 6(SEQ ID NO：159)、YGNGT(SEQ ID NO：160)、(YGNGT) 2(SEQ ID NO：161)、(YGNGT) 3(SEQ ID NO：162)、(YGNGT) 4(SEQ ID NO：163)、(YGNGT) 5(SEQ ID NO：164)或(YGNGT) 6(SEQ ID NO：165)所示的氨基酸序列。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体，其中所述第二抗体的重链包含重链可变区(VH)和重链恒定区(CH)，轻链包含轻链可变区(VL)和轻链恒定区(CL)。在一些实施方案中，第二抗体为全长抗体。在一些实施方案中，所述第二抗体的重链为IgG同种型，例如IgG1、IgG2、IgG3或IgG4，例如为IgG1同种型；和/或所述第二抗体的轻链为Kappa同种型。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体，其中第二抗体的两条HC包含相同的CDR，和/或第二抗体的两条LC包含相同的CDR。在一些实施方案中，所述第二抗体的两条HC包含相同的VH和/或第二抗体的两条LC包含相同的VL。在一些实施方案中，所述第二抗体的两条HC具有相同的氨基酸序列和/或第二抗体的两条LC具有相同的氨基酸序列。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体包含两个第一抗体，其中所述两个第一抗体具有相同或不相同的氨基酸序列。在一些实施方案中，两个第一抗体具有相同的氨基酸序列。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT 双特异性抗体包含两条第一多肽链和两条第二多肽链，其中对于每条多肽链：
a)第一多肽链各自独立地包含VHH作为第一抗体、和第二抗体的重链(HC)；和
b)第二多肽链各自独立地包含第二抗体的轻链(LC)；
其中，VHH直接或通过接头与第二抗体的HC的N端和/或C端相连。或者，i)第一多肽链各自独立地包含第二抗体的重链(HC)；和ii)第二多肽链各自独立地包含第一抗体和第二抗体的轻链(LC)；其中，VHH直接或通过接头与第二抗体的LC的N端和/或C端相连。在一些实施方案中，所述抗PVRIG/TIGIT双特异性抗体包含两条相同的第一多肽链和两条相同的第二多肽链。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体，其中所述特异性结合TIGIT的第二抗原结合结构域为任意的抗TIGIT抗体。此处全文引入WO2009126688、WO2014089113、WO2015009856、WO2015143343、WO2015174439、WO2016028656、WO2016106302、WO2017053748、WO2017030823、US20160176963、US20130251720、WO2019232484、WO2019062832中的TIGIT抗体。例如，TIGIT抗体可以为CPA.9.083.H4(S241P)、CPA.9.086.H4(S241P)、CHA.9.547.7.H4(S241P)和CHA.9.547.13.H4(S241P)中的任一种(参见WO2019232484)。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体，其中特异性结合TIGIT的第二抗原结合结构域为第二抗体。在一些实施方案中，全文引入WO2019062832中的抗TIGIT抗体作为第二抗体。在一些实施方案中，所述第二抗体中：重链可变区包含分别如SEQ ID NO：115、116和117所示的HCDR1、HCDR2和HCDR3，和轻链可变区包含分别如SEQ ID NO：118、119和120所示的LCDR1、LCDR2和LCDR3；或重链可变区包含分别如SEQ ID NO：121、122和123所示的HCDR1、HCDR2和HCDR3，和轻链可变区包含分别如SEQ ID NO：124、125和126所示的LCDR1、LCDR2和LCDR3；或重链可变区包含分别如SEQ ID NO：127、128和129所示的HCDR1、HCDR2和HCDR3，和轻链可变区包含分别如SEQ ID NO：130、131和132所示的LCDR1、LCDR2和LCDR3；或重链可变区包含分别如SEQ ID NO：133、134和135所示的HCDR1、HCDR2和HCDR3，和轻链可变区包含分别如SEQ ID NO：136、137和138所示的LCDR1、LCDR2和LCDR3；或重链可变区包含分别如SEQ ID NO：139、140和141所示的HCDR1、HCDR2和HCDR3，和轻链可变区包含分别如SEQ ID NO：142、143和144所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体，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HCDR1、HCDR2和HCDR3，其中HCDR1包含SEQ ID NO：121的氨基酸序列、HCDR2包含SEQ ID  NO：122的氨基酸序列和HCDR3包含SEQ ID NO：123的氨基酸序列，和所述轻链可变区包含LCDR1、LCDR2和LCDR3，其中LCDR1包含SEQ ID NO：124的氨基酸序列、LCDR2包含SEQ ID NO：125的氨基酸序列和LCDR3包含SEQ ID NO：126的氨基酸序列。在一些实施方案中，所述特异性结合TIGIT的第二抗原结合结构域包含SEQ ID NO：145-147任一项所示的氨基酸序列的VH，或包含与SEQ ID NO：145-147中的至少一条序列具有至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性的氨基酸序列的VH，和/或包含SEQ ID NO：148-149任一项所示的氨基酸序列的VL，或包含与SEQ ID NO：148-149中的至少一条序列具有至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性的氨基酸序列的VL。在一些实施方案中，所述特异性结合TIGIT的第二抗原结合结构域的重链可变区包含SEQ ID NO：145的氨基酸序列，轻链可变区包含SEQ ID NO：149的氨基酸序列。在一些实施方案中，其中所述特异性结合TIGIT的第二抗原结合结构域包含重链(HC)和轻链(LC)。在一些实施方案中，其中所述特异性结合TIGIT的第二抗原结合结构域包含与SEQ ID NO：102具有至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的氨基酸序列的HC，和与SEQ ID NO：103具有至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的氨基酸序列的LC。在一些实施方案中，其中所述特异性结合TIGIT的第二抗原结合结构域具有：包含SEQ ID NO：102的氨基酸序列的HC，和包含SEQ ID NO：103的氨基酸序列的LC。在一些实施方案中，所述特异性结合TIGIT的第二抗原结合结构域包含如SEQ ID NO：145的重链可变区，和如SEQ ID NO：149的轻链可变区。在一些实施方案中，其中所述特异性结合TIGIT的第二抗原结合结构域具有：如SEQ ID NO：102的氨基酸序列的HC，和如SEQ ID NO：103的氨基酸序列的LC。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体，其中所述特异性结合PVRIG的第一抗原结合结构域或第一抗体(如VHH)包含如SEQ ID NO：7、8、9所示的CDR1、CDR2、CDR3，或包含如SEQ ID NO：7、8、150所示的CDR1、CDR2、CDR3；特异性结合TIGIT的第二抗原结合结构域或第二抗体的重链可变区包含分别如SEQ ID NO：121、122、123所示的HCDR1、HCDR2、HCDR3，轻链可变区包含分别如SEQ ID NO：124、125、126所示的LCDR1、LCDR2、LCDR3。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体，其中所述特异性结合PVRIG的第一抗原结合结构域或第一抗体(如VHH)包含如SEQ ID NO：10、11、12所示的CDR1、CDR2、CDR3，或包含如SEQ ID NO：10、11、151所示的CDR1、CDR2、CDR3；特异性结合TIGIT的第二抗原结合结构域或第二抗体的重链可变区包含分别如SEQ ID NO：121、122、 123所示的HCDR1、HCDR2、HCDR3，轻链可变区包含分别如SEQ ID NO：124、125、126所示的LCDR1、LCDR2、LCDR3。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体，其中所述特异性结合PVRIG的第一抗原结合结构域或第一抗体具有：包含如SEQ ID NO：6、79、81、92、98、99之一所示的氨基酸序列的VHH，或包含与SEQ ID NO：6、79、81、92、98、99之一具有至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的氨基酸序列的VHH；所述特异性结合TIGIT的第二抗原结合结构域或第二抗体具有：包含如SEQ ID NO：145-147任一项所示的氨基酸序列的VH或包含与SEQ ID NO：145-147之一具有至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的氨基酸序列的VH，和包含如SEQ ID NO：148-149任一项所示的氨基酸序列的VL或包含与SEQ ID NO：148-149之一具有至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的氨基酸序列的VL；在一些实施方案中，所述特异性结合TIGIT的第二抗原结合结构域或第二抗体具有：包含SEQ ID NO：102或与SEQ ID NO：102具有至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的氨基酸序列的HC，和包含SEQ ID NO：103或与SEQ ID NO：103具有至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的氨基酸序列的LC。在一些实施方案中，其中所述抗PVRIG/TIGIT双特异性抗体，其特异性结合PVRIG的第一抗原结合结构域或第一抗体具有如SEQ ID NO：81所示的VHH，其特异性结合TIGIT的第二抗原结合结构域具有如SEQ ID NO：145所示的VH和如SEQ ID NO：149所示的VL。在一些实施方案中，其中所述抗PVRIG/TIGIT双特异性抗体，其特异性结合PVRIG的第一抗原结合结构域或第一抗体具有如SEQ ID NO：81所示的VHH，其特异性结合TIGIT的第二抗原结合结构域具有如SEQ ID NO：102所示的HC和如SEQ ID NO：103所示的LC。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体包含Fc区；在一些实施方案中，所述特异性结合TIGIT的第二抗原结合结构域或第二抗体包含Fc区。在一些实施方案中，所述Fc为IgG同种型Fc，例如IgG1、IgG2、IgG3或IgG4的Fc区；在一些实施方案中，所述Fc包括选自以下的一个或多个氨基酸突变(突变位点根据EU索引)：
i)改变CH1的铰链区中的半胱氨酸残基的数量，以便于轻链和重链的组装或提高或降低抗体的稳定性；
ii)使得与FcγRIIIa的结合增强的突变，以引起增强的ADCC，使得与FcγRIIb的结合减弱的突变，例如236A、239D、239E、332E、332D、239D/332E、267D、267E、328F、267E/328F、236A/332E、239D/332E/330Y、239D、332E/330L、299T、 297N或其任意组合；
iii)延长生物半衰期的突变，例如T252L、T254S、T256F、428L、434A、434S428L/434S或其任意组合；
iv)234、235、236、237、297、318、320和322位的一个或多个氨基酸突变或其任意组合，以改变抗体对效应配体的亲合力，同时保留亲本抗体的抗原结合能力；
v)329、331和322位的一个或多个氨基酸突变或其任意组合，使得抗体具有改变了的C1q结合和/或降低或消除补体依赖性细胞毒性(CDC)；
vi)231-239内的一个或多个氨基酸突变或其任意组合，使得抗体固定补体的能力改变；
vii)238、239、248、249、252、254、255、256、258、265、267、268、269、270、272、276、278、280、283、285、286、289、290、292、293、294、295、296、298、301、303、305、307、309、312、315、320、322、324、326、327、329、330、331、333、334、335、337、338、340、360、373、376、378、382、388、389、398、414、416、419、430、434、435、437、438或439中的一个或多个氨基酸突变或其任意组合，以提高ADCC的能力和/或提高抗体对Fcγ受体的亲合力；
viii)S228P、F234A、L235A和/或K447A的氨基酸突变；
ix)S354C，E356D，M358L和/或T366W的氨基酸突变。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体包含：如SEQ ID NO：104所示第一多肽链，和如SEQ ID NO：103所示第二多肽链；如SEQ ID NO：105所示第一多肽链，如SEQ ID NO：103所示第二多肽链；如SEQ ID NO：102所示第一多肽链，如SEQ ID NO：106所示第二多肽链；如SEQ ID NO：102所示第一多肽链，如SEQ ID NO：107所示第二多肽链；或如SEQ ID NO：108-112、114任一所示第一多肽链，如SEQ ID NO：103所示所示第二多肽链；或与上述第一多肽链和/或第二多肽链具有至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性的变体。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体包含第一多肽链和第二多肽链，其中：所述第一多肽链包含SEQ ID NO：109的氨基酸序列，所述第二多肽链包含SEQ ID NO：103的氨基酸序列。在一些实施方案中，其中所述抗PVRIG/TIGIT双特异性抗体包含两条相同的第一多肽链和两条相同的第二多肽链。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体为与如上任一项所述的双特异性抗体竞争性结合的抗PVRIG/TIGIT双特异性抗体。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体具有下述特征中的至少一个：
(a)结合人PVRIG的KD值小于1×10 -7M；
(b)阻断PVRIG与其配体(例如，PVRL2)的相互作用；
(c)解除树突状细胞对T细胞的抑制作用，活化T细胞；
(d)解除肿瘤细胞对NK细胞的抑制作用；
(e)抑制肿瘤生长。
在一些实施方案中，其中所述抗PVRIG/TIGIT双特异性抗体的KD值可以小于1×10 -7M，小于1×10 -8M，小于1×10 -9M，小于1×10 -10M。在一些实施方案中，其中所述抗PVRIG/TIGIT双特异性抗体能够抑制肿瘤生长至少约10％，至少约20％，至少约30％，至少约40％，至少约50％，至少约60％，至少约70％，至少约80％。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)1mg/mL至100mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体，
(b)0.2mg/mL至0.6mg/mL的聚山梨酯80，
(c)75mg/mL至90mg/mL的蔗糖，和
(d)10mM至30mM的组氨酸缓冲剂，药物组合物的pH为5.5至6.0。
在一些实施方案中，所述药物组合物包含如下组分：
(a)50mg/mL至100mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体，
(b)0.2mg/mL至0.6mg/mL的聚山梨酯80，
(c)80mg/mL的蔗糖，和(d)10mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为5.5至6.0；
在一些实施方案中，所述药物组合物包含如下组分：
(a)50mg/mL的抗PVRIG/TIGIT双特异性抗体，所述抗PVRIG/TIGIT双特异性抗体包含SEQ ID NO：109的氨基酸序列的第一多肽链和包含SEQ ID NO：103的氨基酸序列的第二多肽链，(b)0.4mg/mL的聚山梨酯80，(c)80mg/mL的蔗糖，和(d)10mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为6.0。
在一些实施方案中，所述药物组合物包含如下组分：
(a)50mg/mL至100mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体，
(b)0.2mg/mL至0.6mg/mL的聚山梨酯80，
(c)80mg/mL±5mg/mL的蔗糖，和
(d)10mM±5mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为5.5至6.0。
在一些实施方案中，所述药物组合物包含如下组分：
(a)1mg/mL至100mg/mL的所述抗PVRIG/TIGIT双特异性抗体，(b)0.2mg/mL至0.6mg/mL的聚山梨酯80，(c)70mg/mL至90mg/mL的蔗糖，和(d)10mM至30mM的组氨酸缓冲剂，所述药物组合物的pH为5.5至6.5。
在一些实施方案中，所述药物组合物包含如下组分：
(a)50mg/mL至100mg/mL的所述抗PVRIG/TIGIT双特异性抗体，(b)0.2mg/mL至0.6mg/mL的聚山梨酯80，(c)75mg/mL至80mg/mL的蔗糖，和(d)10mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为5.5至6.5。
在一些实施方案中，所述药物组合物包含如下组分：
(a)50mg/mL±5mg/mL的抗PVRIG/TIGIT双特异性抗体，所述抗PVRIG/TIGIT双特异性抗体包含SEQ ID NO：109的氨基酸序列的第一多肽链和包含SEQ ID NO：103的氨基酸序列的第二多肽链，
(b)0.4mg/mL±0.1mg/mL的聚山梨酯80，
(c)80mg/mL±5mg/mL的蔗糖，和
(d)10mM±5mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为6.0±0.2。
在一些实施方案中，所述的药物组合物，其包含如下组分：
(a)40mg/mL至60mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体(例如1708-30H2)，
(b)0.2mg/mL至0.6mg/mL的聚山梨酯80，
(c)75mg/mL至80mg/mL的蔗糖，和
(d)10mM至30mM的组氨酸缓冲剂，药物组合物的pH为5.5至6.0。
在一些实施方案中，所述的药物组合物，其包含如下组分：
(a)50mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体(例如1708-30H2)，(b)0.4mg/mL的聚山梨酯80，(c)75mg/mL至80mg/mL的蔗糖，和(d)10mM至30mM的醋酸盐缓冲剂，药物组合物的pH为5.0至5.5。
在一些实施方案中，所述的药物组合物，其包含如下组分：
(a)50mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体(例如1708-30H2)，(b)0.4mg/mL的聚山梨酯80，(c)80mg/mL的蔗糖，和(d)10mM醋酸-醋酸钠盐缓冲剂，pH为5.0。
在一些实施方案中，所述的药物组合物，其包含如下组分：
(a)50mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体(例如1708-30H2)，(b)0.4mg/mL的聚山梨酯80，(c)80mg/mL的蔗糖，和(d)10mM醋酸-醋酸钠盐缓冲剂，pH为5.5。
在一些实施方案中，所述的药物组合物，其包含如下组分：
(a)50mg/mL±5mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体 (例如1708-30H2)，
(b)0.4mg/mL±0.1mg/mL的聚山梨酯80，
(c)75mg/mL至80mg/mL的蔗糖，和
(d)10mM至30mM的醋酸盐缓冲剂，药物组合物的pH为5.0至5.5。
在一些实施方案中，所述的药物组合物，其包含如下组分：
(a)50mg/mL±5mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体(例如1708-30H2)，
(b)0.4mg/mL±0.1mg/mL的聚山梨酯80，
(c)80mg/mL±5mg/mL的蔗糖，和
(d)10mM±5mM醋酸-醋酸钠盐缓冲剂，pH为5.5±0.2。
在一些实施方案中，所述的药物组合物，其包含如下组分：
(a)50mg/mL±5mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体(例如1708-30H2)，(b)0.4mg/mL±0.1mg/mL的聚山梨酯80，(c)80mg/mL±5mg/mL的蔗糖，和(d)10mM±5mM醋酸-醋酸钠盐缓冲剂，pH为5.5。
在一些实施方案中，所述的药物组合物，其包含如下组分：
(a)50mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体(例如1708-30H2)，(b)0.4mg/mL的聚山梨酯80，(c)80mg/mL的蔗糖，和(d)10mM组氨酸-盐酸组氨酸缓冲剂，pH为5.5。
在一些实施方案中，所述的药物组合物，其包含如下组分：
(a)50mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体(例如1708-30H2)，(b)0.4mg/mL的聚山梨酯80，(c)80mg/mL的蔗糖，和(d)10mM组氨酸-盐酸组氨酸缓冲剂，pH为6.0。
在一些实施方案中，所述的药物组合物，其包含如下组分：
(a)50mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体(例如1708-30H2)，(b)0.4mg/mL的聚山梨酯80，(c)80mg/mL的蔗糖，和(d)10mM组氨酸-盐酸组氨酸缓冲剂，pH为6.5。
在一些实施方案中，所述的药物组合物，其包含如下组分：
(a)100mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体(例如1708-30H2)，(b)0.4mg/mL的聚山梨酯80，(c)80mg/mL的蔗糖，和(d)10mM组氨酸-盐酸组氨酸缓冲剂，pH为6.0。
在一些实施方案中，所述的药物组合物，其包含如下组分：
(a)50mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体(例如1708-30H2)，(b)0.2mg/mL的聚山梨酯80，(c)80mg/mL的蔗糖，和(d)10mM组氨酸-盐酸组氨酸缓冲剂，pH为6.5。
在一些实施方案中，所述的药物组合物，其包含如下组分：
(a)50mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体(例如 1708-30H2)，(b)0.6mg/mL的聚山梨酯80，(c)80mg/mL的蔗糖，和(d)10mM组氨酸-盐酸组氨酸缓冲剂，pH为6.5。
在一些实施方案中，所述的药物组合物，其包含如下组分：
(a)50mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体(例如1708-30H2)，(b)0.4mg/mL的聚山梨酯20，(c)80mg/mL的蔗糖，和(d)10mM组氨酸-盐酸组氨酸缓冲剂，pH为6.5。
在一些实施方案中，所述的药物组合物，其包含如下组分：
(a)50mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体(例如1708-30H2)，(b)0.4mg/mL的泊洛沙姆188，(c)80mg/mL的蔗糖，和(d)10mM组氨酸-盐酸组氨酸缓冲剂，pH为6.5。
在一些实施方案中，所述的药物组合物，其包含如下组分：
(a)50mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体(例如1708-30H2)，(b)0.4mg/mL的聚山梨酯20，(c)80mg/mL的蔗糖，和(d)10mM组氨酸-盐酸组氨酸缓冲剂，pH为5.5。
在一些实施方案中，所述的药物组合物，其包含如下组分：
(a)50mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体(例如1708-30H2)，(b)0.4mg/mL的聚山梨酯20，(c)75mg/mL的蔗糖，和(d)10mM组氨酸-盐酸组氨酸缓冲剂，pH为5.5。
在一些实施方案中，如上所述的药物组合物，所述药物组合物是液体制剂。在一些实施方案中，所述液体制剂的溶剂是水。
本披露还提供一种的冻干制剂，其特征在于所述冻干制剂复溶后可形成如上任一项所述的药物组合物。
本披露还提供一种冻干制剂，其为如上任一项所述的药物组合物的冻干形式制剂。
本披露还提供一种制备冻干制剂的方法，其中包括将如上任一项所述的药物组合物进行冷冻干燥的步骤。在一些实施方案中，如上任一项所述冷冻干燥依次包括预冻、一次干燥和二次干燥的步骤。
本披露还提供一种冻干制剂，所述制剂通过将如上任一项所述的药物组合物经冷冻干燥获得。
本披露还提供一种复溶溶液，其特征在于所述复溶溶液是通过将如上任一项所述的冻干制剂经复溶制备获得。
本披露还提供一种复溶溶液，其为如上任一项所述的冻干制剂的复溶形式制剂。
在一些实施方案中，如上任一项的所述的复溶溶液，其组分和含量与前述的药物组合物的组分和含量相同。
在一些实施方案中，如上任一项所述的复溶溶液，其包含如下组分：
(a)1mg/mL至150mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗 体，(b)0.05mg/mL至1.0mg/mL的聚山梨酯80，(c)70mg/mL至100mg/mL的蔗糖，和(d)5mM至100mM的组氨酸缓冲剂，药物组合物的pH为5.0至6.5。
在一些实施方案中，如上任一项所述的复溶溶液，其包含如下组分：
(a)1mg/mL至100mg/mL的所述抗PVRIG/TIGIT双特异性抗体，(b)0.2mg/mL至0.6mg/mL的聚山梨酯80，(c)70mg/mL至90mg/mL的蔗糖，和(d)10mM至30mM的组氨酸缓冲剂，所述药物组合物的pH为5.5至6.5。
在一些实施方案中，如上任一项所述的复溶溶液，其包含如下组分：
(a)50mg/mL至100mg/mL的所述抗PVRIG/TIGIT双特异性抗体，(b)0.2mg/mL至0.6mg/mL的聚山梨酯80，(c)75mg/mL至80mg/mL的蔗糖，和(d)10mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为5.5至6.5。
在一些实施方案中，如上任一项所述的复溶溶液，其包含如下组分：
(a)1mg/mL至100mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体，(b)0.2mg/mL至0.6mg/mL的聚山梨酯80，(c)75mg/mL至90mg/mL的蔗糖，和(d)10mM至30mM的组氨酸缓冲剂，药物组合物的pH为5.5至6.0。
在一些实施方案中，所述复溶溶液包含如下组分：
(a)50mg/mL至100mg/mL的如上任一项所述的抗PVRIG/TIGIT双特异性抗体，(b)0.2mg/mL至0.6mg/mL的聚山梨酯80，(c)80mg/mL的蔗糖，和(d)10mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为5.5至6.0。
在一些实施方案中，所述复溶溶液包含如下组分：
(a)50mg/mL的抗PVRIG/TIGIT双特异性抗体，所述抗PVRIG/TIGIT双特异性抗体包含SEQ ID NO：109的氨基酸序列的第一多肽链和包含SEQ ID NO：103的氨基酸序列的第二多肽链，(b)0.4mg/mL的聚山梨酯80，(c)80mg/mL的蔗糖，和(d)10mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为6.0。
在一些实施方案中，如上任一项所述的药物组合物或复溶溶液，其为静脉注射制剂、皮下注射制剂、腹腔注射制剂或肌肉注射制剂；在一些实施方案中，如上任一项所述的药物组合物或复溶溶液，其为静脉注射制剂。
在一些实施方案中，如上任一项所述的药物组合物或复溶溶液，其适于静脉注射、皮下注射、腹腔注射或肌肉注射。
在一些实施方案中，如上任一项所述的药物组合物或复溶溶液或冻干制剂，其用于制备静脉注射、皮下注射、腹腔注射或肌肉注射的药物。
本披露还提供一种药盒，其包括至少一个容器，各容器独立地包含如上任一项所述的药物组合物、如上任一项所述的冻干制剂或如上任一项所述的复溶溶液。
在一些实施方案中，本披露还提供诊断、治疗、缓解受试者病症的方法，包含向所述受试者施用有效量的如上任一项所述的药物组合物、如上任一项的冻干制剂、如上任一项的复溶溶液或如上任一项所述的药盒。
在一些实施方案中，本披露还提供活化受试者的细胞毒性T细胞(CTL)的 方法，包含向所述受试者施用有效量的如上任一项所述的药物组合物、如上任一项的冻干制剂、如上任一项的复溶溶液或如上任一项所述的药盒，其中所述受试者的所述CTL的亚组被活化。
在一些实施方案中，本披露还提供活化受试者的NK细胞的方法，包含向所述受试者施用有效量的如上任一项所述的药物组合物、如上任一项的冻干制剂、如上任一项的复溶溶液或如上任一项所述的药盒，其中所述受试者的所述NK细胞的亚组被活化。
在一些实施方案中，本披露还提供活化受试者的γδT细胞的方法，包含向所述受试者施用有效量的如上任一项所述的药物组合物、如上任一项的冻干制剂、如上任一项的复溶溶液或如上任一项所述的药盒，其中所述受试者的所述γδT细胞的亚组被活化。
在一些实施方案中，本披露还提供活化受试者的Th1细胞的方法，包含向所述受试者施用有效量的如上任一项所述的药物组合物、如上任一项的冻干制剂、如上任一项的复溶溶液或如上任一项所述的药盒，其中所述受试者的所述Th1细胞的亚组被活化。
在一些实施方案中，本披露还提供活化、减少或消除受试者体内的调节性T细胞(Treg)中的至少一种的细胞数量和/或活性的方法，包含向所述受试者施用有效量的如上任一项所述的药物组合物、如上任一项的冻干制剂、如上任一项的复溶溶液或如上任一项所述的药盒。
在一些实施方案中，本披露还提供增加受试者体内的干扰素-γ产生和/或促炎性细胞因子分泌的方法，包含向所述受试者施用有效量的如上任一项所述的药物组合物、如上任一项的冻干制剂、如上任一项的复溶溶液或如上任一项所述的药盒。
在一些实施方案中，本披露还提供抑制受试者体内的PVRIG和PVLR2的相互作用的方法，包含向所述受试者施用有效量的如上任一项所述的药物组合物、如上任一项的冻干制剂、如上任一项的复溶溶液或如上任一项所述的药盒。
在一些实施方案中，本披露还提供如上任一项所述的药物组合物、如上任一项的冻干制剂、如上任一项的复溶溶液或如上任一项所述的药盒在制备治疗疾病的药物中的用途。
在一些实施方案中，本披露还提供一种治疗疾病的方法，所述方法包括向受试者施用治疗有效量的如上任一项所述的药物组合物、如上任一项的冻干制剂、如上任一项的复溶溶液或如上任一项所述的药盒。
在一些实施方案中，本披露还提供如上任一项所述的药物组合物、如上任一项的冻干制剂、如上任一项的复溶溶液或如上任一项所述的药盒，其用于治疗疾病。
在一些具体实施方案中，前面任一项所述的疾病为增殖性疾病(例如肿瘤)。在一些具体实施方案中，所述肿瘤选自以下或其组合：前列腺癌、肝癌(HCC)、 结直肠癌(结肠癌、直肠癌)、卵巢癌、子宫内膜癌、乳腺癌(例如三阴性乳腺癌)、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、非黑色素瘤皮肤癌(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、食管癌(又称“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)。在一些具体实施方案中，上述疾病可以是与PVRIG和\或TIGIT异常表达相关的疾病。在一些具体实施方案中，所述肿瘤选自由以下癌症或其组合：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)。在一些具体实施方案中，所述肿瘤选自由以下癌症或其组合：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。在一些具体实施方案中，上述疾病患者具有与PVRIG和/或TIGIT相关的病况。在一些具体方案中，受试者病况包括表达或不表达PVRIG的癌症并且进一步包括非转移性或非浸润性以及浸润性或转移性癌症，其中免疫细胞、基质细胞或发生病变的细胞的PVRIG表达抑制抗肿瘤反应和抗浸润性免疫反应。在一些具体方案中，所述疾病为血管化肿瘤。在一些实施方案中，所述肿瘤选自肺癌、前列腺癌、乳腺癌、头颈癌、食管癌、胃癌、结直肠癌、膀胱癌、宫颈癌、子宫内膜癌、卵巢癌、肝癌、黑色素瘤、肾癌、鳞状细胞癌、血液系统癌症或者任何特征在于不受控细胞生长的其它疾病或病症。
在一些实施方案中，所述疾病为感染或脓毒症。在一些具体方案中，所述感染是病原体感染，以病毒特异性T细胞反应的不同程度的功能性障碍为特征，如HIV、HCV、HBV。在一些具体方案中，所述脓毒症选自重度脓毒症、脓毒性休克、全身炎症反应综合征(SIRS)、菌血症、败血症、毒血症和脓毒性综合征。
附图说明
图1为抗PVRIG抗体的PVRIG报告基因细胞活性检查结果。
图2为抗PVRIG抗体在NK细胞杀伤实验中激活NK细胞的活性检测结果。
图3为抗PVRIG抗体在MLR实验中激活T细胞的活性检测结果。
图4A至图4B为人源化的抗PVRIG抗体在PVRIG报告基因细胞活性检测结果。
图5A至图5B为人源化的抗PVRIG抗体在NK细胞杀伤实验中激活NK细胞的活性检测结果。
图6A至图6E分别显示人源化的抗PVRIG/TIGIT双特异性抗体对人PVRIG重组蛋白、人PVRIG过表达细胞、食蟹猴PVRIG重组蛋白、食蟹猴PVRIG过表 达细胞的结合活性、以及阻断人PVRIG与人PVRL2结合的活性检测结果。
图7A至图7E分别显示人源化的抗PVRIG/TIGIT双特异性抗体对人TIGIT重组蛋白、人TIGIT过表达细胞、食蟹猴TIGIT重组蛋白、食蟹猴TIGIT过表达细胞的结合活性、以及阻断人TIGIT与人PVR结合的活性检测结果。
图8为人源化的抗PVRIG/TIGIT双特异性抗体在MLR实验中激活T细胞的活性检测结果。
图9A至图9B分别显示抗PVRIG/TIGIT双特异性抗体在人黑色素瘤A375混合人PBMC的小鼠皮下移植瘤模型中对小鼠体重和肿瘤体积的影响。
图10A至图10B分别显示抗PVRIG/TIGIT双特异性抗体在人黑色素瘤A375混合人PBMC的小鼠皮下移植瘤模型中对小鼠体重和肿瘤体积的影响。
具体实施方式
术语
为了更容易理解本披露，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，否则本文使用的所有其它技术和科学术语都具有本披露所属领域的一般技术人员通常理解的含义。
本披露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。
“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”指抗原上连续或不连续的，由本披露抗体识别的三维空间位点。
“抗体”或“免疫球蛋白”以最广义使用，并且涵盖各种抗体结构，包括但不限于传统的抗体(由两条重链和两条轻链通过链间二硫键连接而成的四肽链结构抗体)，以及具有抗原结合活性的Fab、Fv、sFv、F(ab’)2、线性抗体、单链 抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体peptibody、结构域抗体(重链(VH)抗体、轻链(VL)抗体)和多特异性抗体(双特异性抗体、diabody、triabody和tetrabody、串联二-scFv、串联三-scFv)。
本披露中使用的术语“抗体”意图涵盖全长抗体、其单个的链、及其任意具有抗原结合活性的部分(也即抗原结合片段)、结构域或片段；以及包含其单个的链及任意具有抗原结合活性的部分、结构域或片段的多特异性抗体(包括但不限于抗原结合结构域或片段，例如VHH结构域或VH/VL结构域)。传统的抗体或免疫球蛋白通常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。根据重链恒定区的氨基酸组成和排列顺序不同，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中每类Ig都可以有κ(kappa)链或λ(lambda)链。在一些实施方案中，本披露的抗体特异性地结合到PVRIG和/或TIGIT。
本披露的“抗原结合片段”包括但不限于：(i)由VL、VH、CL和CH1结构域组成的Fab片段；(ii)由VH和CH1结构域组成的Fd片段；(iii)F(ab’)2片段，一种包含两个连接着的Fab片段的二价片段；(vii)单链Fv分子(scFv)，其中VH结构域和VL结构域通过肽连接子连接，所述肽连接子允许两个结构域结合形成抗原结合位点；(Bird等人，1988，Science 242：423-426；Huston等人，1988，Proc.Natl.Acad.Sci.U.S.A.85：5879-5883)242，通过引用完全并入本文中)；(iv)“双功能抗体”或“三功能抗体”，通过基因融合构造的多价或多特异性片段(Tomlinson等人，2000，Methods Enzymol.326：461-479；WO94/13804；Holliger等人，1993，PNAS 90：6444-6448，全部通过引用完全并入本文中)；(v)“结构域抗体”或“dAb”(有时称为“免疫球蛋白单一可变结构域”)，包括来自其它物种的免疫球蛋白单一可变结构域，如啮齿动物(例如，如WO00/29004中所公开)、护士鲨和骆驼科V-HH dAb；(vi)SMIP(小分子免疫药物)、骆驼抗体、纳米抗体以及IgNAR；(vii)上述(i)-(vi)的人源化抗体。
在未特殊指明的情况下，本披露的抗体通常使用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。
术语“双特异性抗体”指能够对两个不同抗原或同一抗原的两个不同抗原表位特异性结合的抗体。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。例如，典型的双特异性抗体结构模型如VHH-IgG、KiH、CrossMAb、Triomab quadroma、FcΔAdp、ART-Ig、BiMAb、Biclonics、BEAT、DuoBody、Azymetric、XmAb、2:1TCBs、1Fab-IgG TDB、FynomAb、two-in-one/DAF、scFv-Fab-IgG、DART-Fc、LP-DART、CODV-Fab-TL、HLE-BiTE、F(ab)2-CrossMAb、IgG-(scFv)2、Bs4Ab、DVD-Ig、Tetravalent-DART-Fc、(scFv)4-Fc、CODV-Ig、mAb2、F(ab)4-CrossMAb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.2019Feb 11；2019:4516041)。
本披露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，本披露的抗体是重组抗体。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。
“单克隆抗体”是指由单一B细胞克隆产生的并且能够与特定抗原表位发生相互作用的抗体分子群体。而“多克隆抗体”是指含有由多个B细胞克隆产生的能够与特定抗原相互作用的抗体分子群体。单克隆抗体典型地对与其发生相互作用的特定抗原展现出单一的结合亲合力。
在本文中，抗原被更广义地定义，并且一般预期包括由抗体特异性识别的靶分子。因此包括用于产生抗体的免疫接种过程、或用于抗体文库筛选中使用的分子或模拟物。
“同源性”或“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。当两个待比较序列中的位置均被相同碱基或氨基酸残基占据时，如果两个DNA分子的一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同一性百分比是两个序列共有的匹配或同源位置数除以待比较的全部位置数×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。
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、移除或转移至其他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”是指抗体链的球形区域，或是指基本上由这类球形区域组成的多肽。在一些实施方式中，“抗原结合结构域”的非限制性的实例包括：Fab片段、Fd片段、F(ab’)2片段、scFv、全长抗体等。
“免疫球蛋白可变结构域”是指基本上由以下组成的免疫球蛋白结构域：“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”、“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”。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。
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，来完成对CDR的确定性描绘或进行对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(enthalpy)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。如本文使用的，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。本文使用的方法可利用根据这些方法中的任一种定义的CDR。对于包含超过一个CDR的任何给定实施例，可根据Kabat、Chothia、延伸的、AbM、IMGT、接触和/或构象定义中的任一个来定义CDR。
“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(例如在没有常规四链抗体中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(其可以是重链结构域或轻链结构域，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，例如人源化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dAbs TM)和作为VL结构域或衍生自VL结构域的(单结构域)抗体(诸如dAbs TM)。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的。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区别于常规四肽链结构抗体中存在的重链可变结构域(其在本披露中称为“VH结构域”)以及轻链可变结构域(其在本披露中称为“VL结构域”)。VHH结构域特异性结合表位而无需其他抗原结合结构域(与常规四肽链结构抗体中的VH或VL结构域不同，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”(“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，也可以是经噬菌体展示技术筛选获得的。
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。其它适用于VHH的编号系统或编码规则包括Chothia、IMGT、AbM。
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披露所述的目的。
VHH结构域(单独或作为较大多肽的一部分)提供许多优于使用常规VH及VL结构域、scFv或常规抗体片段(例如Fab-或F(ab’)2-片段)的显著优势：
-仅需要单一结构域以高亲合力及高选择性结合抗原，从而使得既不需要存在 两个单独结构域，也不需要确保该两个结构域以适当空间构象及构型存在(例如scFv一般需要使用经特别设计的接头)；
-VHH结构域可由单一基因表达且不需要翻译后折叠或修饰；
-VHH结构域可改造成多价及多特异性格式；
-VHH结构域高度可溶且无聚集趋势；
-VHH结构域对热、pH、蛋白酶及变性剂高度稳定，且因此可在制备、储存或运输中，降低对冷冻设备的依赖性，从而达成节约成本、时间及环境；
-VHH结构域可制备且相对廉价，甚至在生产所需的规模上亦如此；
-VHH结构域与常规四肽链结构抗体及其抗原结合片段相比相对较小(大约15kDa或大小为常规IgG的1/10)，因此相比于常规四肽链结构抗体及其抗原结合片段，显示较高的组织渗透性且可以较高剂量给药；
-VHH结构域可显示所谓腔结合性质(尤其由于与常规VH结构域相比其延长的CDR3环)，从而可到达常规四肽链结构抗体及其抗原结合片段不可到达的靶及表位。
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。
“Fc变异体”或“变异体Fc”意指在Fc结构域中包含氨基酸修饰的蛋白质。本披露的Fc变异体根据构成其的氨基酸修饰来定义。因此，举例来说，S228P或228P是相对于亲本Fc多肽在位置228处具有脯氨酸取代的Fc变异体，其中编号是根据EU索引。WT氨基酸可以不指明，在此情况下前述变异体称为228P。
“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，从而实现“人源化”。本披露中亦称为“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他修饰，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异源蛋白成分，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”于其他“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，1991Sequences of  Proteins of Immunological Interest，第5版中找到。本披露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所引起的活性下降，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。
“亲合力成熟”的抗体，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各自的亲本抗体有所增加。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。
通常，本披露的抗体将以如于Biacore或KinExA或Fortibio测定中测量的优选10 -7至10 -10摩尔/升(M)、更优选10 -8至10 -10摩尔/升、甚至更优选10 -9至10 -10或更低的解离常数(KD)，和/或以至少10 -7M、优选至少10 -8M、更优选至少10 -9M，更优选至少10 -10M的缔合常数(KA)结合所要结合的抗原(即PVRIG)。任何大于10 -4M的KD值一般都视为指示非特异性结合。抗体对抗原或表位的特异性结合可以以已知的任何适合方式来测定，包括例如本披露所述的表面等离子体共振术(SPR)测定、Scatchard测定和/或竞争性结合测定(例如放射免疫测定(RIA)、酶免疫测定(EIA)及夹心式竞争性测定。
“表位”或可互换使用的“抗原决定簇”指抗体所结合的抗原上的任何部分。抗原决定簇通常包含化学活性表面基团，例如氨基酸或糖侧链，并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸，其可以是“线性”表位或“构象”表位。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的位置位于彼此分开的氨基酸残基。可使用本领域中熟知的许多表位定位技术鉴别给定抗原的表位(例如Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)，US4708871)。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(高通量筛选方法如参见WO03/48731)。因此，可使用本领域技术人员已知的常规技术，获得与本披露的抗体分子竞争结合PVRIG上的相同表位的抗体及其抗原结合片段。
“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。通常，当使用重组人PVRIG、TIGIT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(K D)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合力是其与预定抗原(或其表位)或紧密相关的抗原之外的非特异性抗原(如BSA等)结合的亲合力的至少两倍。“识别抗原的抗体”在本文中可以与“特异性结合的抗体”互换使用。
“结合亲合力”在本文中用作两个分子(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，用于描述单价相互作用(固有活性)。两个分子之间的结合亲合力可通过确定解离常数(K D)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K D。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)。K D通过方程K D＝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(参见Caceci等人，1984，Byte 9：340-362；Wong&Lohman，1993，PNAS 90：5428-5432)。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、以及本文其它地方例举的其它测定。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K D值与非目的相互作用(例如已知不结合PVRIG的对照抗体)的K D值进行评价。在一些实施方案中，本披露的抗PVRIG抗体能够与其靶结合的亲合力比它与另一种非PVRIG分子结合的亲合力大至少2倍、10倍、50倍、100倍、200倍、500倍、1,000倍或10,000倍，这里不属于限制性定义。
“保守修饰”适用于氨基酸和核苷酸序列。对于特定的核苷酸序列，保守修饰是指编码相同或基本相同的氨基酸序列的那些核酸的相互置换，或在核苷酸不编码氨基酸序列的情况下，是指基本上相同的核苷酸序列。对于氨基酸序列，保守修饰是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)MolecμLar Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224页，(第4版))。
“氨基酸突变”包括氨基酸取代、缺失、插入、修饰及其任意组合，以实现最终构建体，使得最终构建体拥有期望的特性，例如增强的稳定性、提高的活性。氨基酸序列缺失和插入包括在氨基端和/或羧基端的缺失和插入。优选的氨基酸突变是氨基酸取代。为了改变例如抗PVRIG抗体的结合特性，可以将非保守性的氨基酸取代，即将一个氨基酸用具有不同结构和/或化学特性的另一种氨基酸替换。优选的氨基酸取代包括用亲水性氨基酸替换疏水性氨基酸。氨基酸取代包括由非天然存在的氨基酸或由20种标准氨基酸的天然存在的氨基酸衍生物(例如4-羟脯氨酸、3-甲基组氨酸、鸟氨酸、高丝氨酸、5-羟赖氨酸)替换。可以使用本领域中 公知的遗传或化学方法生成氨基酸突变，包括定点诱变、PCR、基因合成、化学修饰等方法。氨基酸突变可以发生在抗体的CDR区、FR区或Fc区。
本披露工程化的抗体或抗原结合片段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CHO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。
“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地场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示含有一种或多种本文所述抗体(例如抗PVRIG/TIGIT双特异性抗体)或其生理学上/药学可接受的盐或前体药物与其他化学组分的混合物，其中其他组分的示例是：例如生理学/药学可接受的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
“药学可接受的载体”或“药学可接受的赋形剂”包括当与活性成分组合时，允许该成分保留生物学活性并且不与受试者的免疫系统反应的任何材料。例子包括但不限于任何标准药物载体，例如磷酸盐缓冲盐水溶液、水、乳剂如油/水乳剂、和各种类型的润湿剂。在一些实施例中，用于肠胃外施用的稀释剂是磷酸盐缓冲盐水(PBS)或生理(0.9％)盐水。包含此类载体的组合物通过众所周知的常规方法配制(参见例如Remington′s Pharmaceutical Sciences，第18版，A.Gennaro，编辑，Mack PublishingCo.，Easton，PA，1990；以及R Remington，The Science and Practice of Pharmacy第20版Mack Publishing，2000)。
“缓冲剂”指通过其酸-碱共轭组分的作用而调节pH变化的缓冲剂。将pH控制在适当范围中的缓冲剂的例子包括醋酸盐、琥珀酸盐、葡萄糖酸盐、组氨酸、草酸盐、乳酸盐、磷酸盐、枸橼酸盐、酒石酸盐、延胡索酸盐、甘氨酰甘氨酸和其它有机酸缓冲剂。
“组氨酸缓冲剂”是包含组氨酸的缓冲剂。组氨酸缓冲剂的实例包括组氨酸-盐酸组氨酸，组氨酸-醋酸组氨酸，组氨酸-磷酸组氨酸，组氨酸-硫酸组氨酸等缓冲剂，优选组氨酸-盐酸组氨酸缓冲剂。组氨酸-盐酸组氨酸缓冲剂可由组氨酸与盐酸配制而成，或者由组氨酸与盐酸组氨酸配制而成。
“醋酸盐缓冲剂”是包括醋酸根离子的缓冲剂。醋酸盐缓冲剂的实例包括醋酸-醋酸钠盐、组氨酸-醋酸组氨酸、醋酸-醋酸钾、醋酸-醋酸钙、醋酸-醋酸镁等。优选的醋酸盐缓冲剂是醋酸-醋酸钠盐。
“枸橼酸盐缓冲剂”是包括枸橼酸根离子的缓冲剂。枸橼酸盐缓冲剂的实例包括枸橼酸-枸橼酸钠、枸橼酸-枸橼酸钾、枸橼酸-枸橼酸钙、枸橼酸-枸橼酸镁等。优选的枸橼酸盐缓冲剂是枸橼酸-枸橼酸钠。
“琥珀酸盐缓冲剂”是包括琥珀酸根离子的缓冲剂。琥珀酸盐缓冲剂的实例包括琥珀酸-琥珀酸钠盐、琥珀酸-琥珀酸钾、琥珀酸-琥珀酸钙盐等。优选的琥珀酸盐缓冲剂是琥珀酸-琥珀酸钠盐。示例性的，所述的琥珀酸-琥珀酸钠可由琥铂酸与氢氧化钠配制而成，或由琥铂酸与琥珀酸钠盐配制而成。
“磷酸盐缓冲剂”是包括磷酸根离子的缓冲剂。磷酸盐缓冲剂的实例包括磷酸氢二钠-磷酸二氢钠、磷酸氢二钠-磷酸二氢钾、磷酸氢二钠-枸橼酸等。优选的磷酸盐缓冲剂是磷酸氢二钠-磷酸二氢钠。
“冻干制剂”表示液体或溶液形式的药物组合物或液体或溶液制剂经冷冻干燥步骤之后获得的制剂或药物组合物。
本披露所述的药物组合物能够达到一种稳定的效果：其中的抗体(例如抗PVRIG/TIGIT双特异性抗体)在贮藏后基本上保留其物理稳定性和/或化学稳定性和/或生物学活性的药物组合物。优选地，药物组合物在贮藏后基本上保留其物理和化学稳定性以及其生物学活性。贮藏期一般基于药物组合物的预定保存期来选择。目前有多种测量蛋白质稳定性的分析技术，可测量在选定温度贮藏选定时间段后的稳定性。
稳定的制剂包括在下述情况下没有观察到统计学显著变化的制剂：在冷藏温度(2-8℃)保存至少3个月、优选6个月、更优选1年，甚至更优选地多达2年。
另外，稳定的液体制剂也包括这样的液体制剂：其在包括25℃的温度保存包括1个月、3个月或6个月在内的时段后表现出期望的特征。稳定性的典型的例子：通过SEC-HPLC测得，通常不超过约10％、优选不超过约5％的抗PVRIG/TIGIT双特异性抗体发生聚集或降解。通过视觉分析，制剂是淡黄色近无色澄明液体或者无色澄明液体，或澄清至稍微乳白色。所述制剂的浓度、pH和重量克分子渗透压浓度具有不超过±10％变化。通常观察到不超过约10％、优选不超过约5％的减少。通常形成不超过约10％、优选不超过约5％的聚集。
在一些实施方案中，本披露的抗PVRIG/TIGIT双特异性抗体制剂在40℃高温条件下放置一个月，SEC单体％大于或等于95％(例如大于95％、大于96％、大于97％、大于98％或大于99％)和/或CE-SDS(NR)％大于或等于95％(例如大于95％、大于96％、大于97％、大于98％或大于99％)。
如果在目检颜色和/或澄清度后，或者通过UV光散射、尺寸排阻色谱法(SEC)和动态光散射(DLS)测得，抗PVRIG/TIGIT双特异性抗体没有显示出显著的聚集增加、沉淀和/或变性，那么所述抗PVRIG/TIGIT双特异性抗体在药物制剂中“保留它的物理稳定性”。蛋白构象的变化可以通过荧光光谱法(其确定蛋白三级结构)和通过FTIR光谱法(其确定蛋白二级结构)来评价。
如果抗PVRIG/TIGIT双特异性抗体没有显示出显著的化学改变，那么所述抗PVRIG/TIGIT双特异性抗体在药物制剂中“保留它的化学稳定性”。通过检测和定量化学上改变的形式的蛋白，可以评估化学稳定性。经常改变蛋白化学结构的降解过程包括水解或截短(通过诸如尺寸排阻色谱法和CE-SDS等方法来评价)、氧化(通过诸如与质谱法或MALDI/TOF/MS结合的肽谱法等方法来评价)、脱酰胺作用(通过诸如离子交换色谱法、毛细管等电聚焦、肽谱法、异天冬氨酸测量等方法来评价)和异构化(通过测量异天冬氨酸含量、肽谱法等来评价)。
如果抗PVRIG/TIGIT双特异性抗体在给定时间的生物活性是在制备药物制剂时表现出的生物活性的预定范围内，那么所述抗PVRIG/TIGIT双特异性抗体在药物制剂中“保留它的生物活性”。
“施用”、“给予”和“处理”，当其应用于动物、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。“施用”、“给予”和“处理”可以指例如治疗、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。“施用”、“给予”和“处理”还意指通过试剂、诊断、组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当其应用于人、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，例如包含本披露的任一种的药物组合物，所述受试者具有一种或多种疾病症状，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，以诱导这类症状退化或抑制这类症状发展到任何临床右测量的程度。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。尽管本披露的实施方案(例如治疗方法或药盒)在缓解每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。本披露的受试者可以是动物或人类受试者。
本披露的药物组合物可通过任何合适的手段施用，包括肠胃外、肺内和鼻内，并且如果需要局部治疗，则病灶内施用。肠胃外输注包括肌肉内、静脉内、动脉内、腹膜内或皮下施用。给药可以通过任何适当的途径，例如，通过注射，诸如静脉内或皮下注射。本文考虑多种给药时间方案，包括但不限于，单次或在多个时间点多次施用，推注施用和脉冲输注。在一些实施方案中，本披露的药物组合物通过静脉内施用。
本披露的药物组合物将以符合良好医疗实践的方式配制、给药和施用。在此背景下考虑的因素包括所治疗的具体病症、所治疗的具体哺乳动物、个体受试者的临床状况、病症的起因、试剂的递送部位、施用方法、施用时间安排以及医学从业者已知的其他因素。任选地，药物组合物还可以与用于预防或治疗所述病症的一种或更多种其它试剂一起配制。此类其它试剂的有效量取决于药物组合物中存在的抗原结合分子的量、病症或治疗的类型以及其它因素。可以与本文所述相同的剂量和施用路径使用，或以本文所述剂量的约1至99％使用，或以任何剂量使用，并通过经验/临床确定为合适的任何途径使用。
在以上说明书中提出了本披露一种或多种实施方式的细节。虽然可使用与本文所述类似或相同的任何方法和材料来实施或测试本披露，但是以下描述优选的方法和材料。通过说明书和权利要求书，本披露的其他特点、目的和优点将是显而易见的。在说明书和权利要求书中，除非上下文中有清楚的另外指明，单数形式包括复数指代物的情况。除非另有定义，本文使用的所有技术和科学术语都具有本披露所属领域普通技术人员所理解的一般含义。说明书中引用的所有专利和出版物都通过引用纳入。提出以下实施例是为了更全面地说明本披露的优选实施方式。这些实施例不应以任何方式理解为限制本披露的范围，本披露的范围由权利要求书限定。
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-抗PVRIG/TIGIT双特异性抗体制备与检测
PCT/CN2021/080470(申请日：2021.3.13；优先权专利申请号：CN202010174835.4)通过援引完整收入本披露。
实施例1.PVRIG蛋白质序列及制备
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-hIgG1 Fc)为购买自Acrobiosystems公司的纯化商业蛋白试剂，其序列见表1。
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 PCTCN2022118942-appb-000001
在HEK293细胞中通过常规方法进行瞬时转染表达重组蛋白，收集上清并通过Ni-NTA进行纯化。经检测，获得cyno-PVRIG-his。
实施例2.抗人PVRIG单域抗体的产生
抗人PVRIG单克隆单域抗体通过免疫骆驼产生。免疫抗原为带his标签的人PVRIG重组蛋白(h-PVRIG-his)。用弗氏佐剂(Sigma，Lot No.：F5881/F5506)乳化：首次用弗氏完全佐剂(CFA)CFA，其余加强免疫用弗氏不完全佐剂(IFA)。免疫注射时间为第0，14，28，42天。于第56天采血进行血检，用ELISA方法检测骆驼血清，确定骆驼血清中的抗体滴度。
取200mL骆驼外周血，分离其中的PBMC，用Trizol提取细胞中的RNA，并反转录成cDNA。用PCR的方法扩增单域抗体可变区的基因，克隆到噬菌体载体中，从而建立抗人PVRIG单域抗体的噬菌体文库。
将噬菌体文库用BSA稀释封闭，与磁珠Dynabeads(M-280，invitrogen)共孵育，收集负筛孵育后的噬菌体。用生物素标记的带有his标签的人PVRIG包被封闭Dynabeads，将负筛后收集的噬菌体悬液与所述Dynabeads孵育，用胰酶洗脱噬菌体。经过3轮筛选，挑选第3轮筛选获得的400个克隆进行测序，其中5株单域抗体的重链序列如表2所示，不同编码规则的CDR如表3所示。
表2.抗PVRIG抗体重链可变区(HCVR)序列
Figure PCTCN2022118942-appb-000002
Figure PCTCN2022118942-appb-000003
表3.抗PVRIG抗体的重链互补决定区(CDR)序列
Figure PCTCN2022118942-appb-000004
Figure PCTCN2022118942-appb-000005
实施例3.全长抗PVRIG抗体的制备
将实施例2中5个抗体的重链可变区与人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突变(Eu命名系统)。WO2016134333A1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。
5个抗体和阳性抗体的全长序列如表4所示。
表4.全人源抗PVRIG抗体的重轻链全长序列
Figure PCTCN2022118942-appb-000006
Figure PCTCN2022118942-appb-000007
(注：下划线为重链的Fc结构域)
合成编码上述序列的基因序列，用BamHI和XhoI消化后，通过BamHI/XhoI酶切位点插入到pcDNA3.1表达载体(Life Technologies Cat.No.V790-20)中。将表达载体和转染试剂PEI(Polysciences，Inc.Cat.No.23966)以1:2的比例转染HEK293细胞(Life Technologies Cat.No.11625019)，并置于CO 2孵育箱中孵育4-5天。表达的抗体通过离心回收后，按常规方法进行抗体纯化，经检测，获得目的抗体。
实施例4.抗PVRIG抗体与PVRIG重组蛋白的结合实验
ELISA实验被用来检测抗PVRIG抗体的结合特性。用直接包被带his标签的PVRIG重组蛋白，抗体加入后，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孔酶标板，按1μg/mL浓度每孔100μL，4℃孵育过夜。洗液洗三遍，每孔250μL。每次洗涤震荡10秒以保证充分清洗。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250μL。每次洗涤震荡10秒以保证充分清洗。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250μL。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250μL。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5.抗PVRIG抗体对不同种系PVRIG重组蛋白的结合实验结果
Figure PCTCN2022118942-appb-000008
实施例5.抗PVRIG抗体与表达PVRIG的细胞结合实验
用流式细胞仪(FACS)来检测抗PVRIG抗体的结合特性。构建过表达人或食蟹猴PVRIG的细胞株，抗体加入后，通过加入二抗检测抗体与抗原结合的活性。
将带有人或食蟹猴PVRIG基因序列的表达质粒在HEK293细胞中进行转染，并用抗生素筛选和无限稀释法获得过表达稳定转染单克隆细胞株。在96孔板中每孔接种2×10 5个过表达细胞。300g离心5分钟，去除上清，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6.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)
20 N.A. N.A.
30 N.A. 0.02
38 0.24 0.34
39 0.004 6.97
151 0.01 2.23
Tab5 2.13 无结合
IgG4 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。)
实施例6.抗PVRIG抗体阻断PVRIG和PVRL2结合实验
本实验中，通过体外阻断实验，检测所筛选出来的抗PVRIG抗体阻断人PVRIG和其配体人PVRL2的结合能力。具体方法是将带小鼠IgG2a Fc标签的人PVRIG重组蛋白(h-PVRIG-mIgG2a Fc)包被到96孔酶标板上，加入抗PVRIG抗体充分结合占据表位后，再加入带his标签的PVRL2(PV2-H52E2，AcroBiosystem)，通过检测his标签，来计算PVRIG与PVRL2的结合量，计算抗PVRIG抗体对PVRIG活性位点阻断的IC 50值。
h-PVRIG-mIgG2a Fc蛋白包被96孔酶标板，按1μg/mL浓度每孔100μL，4℃孵育过夜。洗液洗三遍，每孔250μL。每次洗涤震荡10秒以保证充分清洗。加入300μL/孔封闭液室温孵育1小时。洗液洗三遍，每孔250μL。每次洗涤震荡10秒以保证充分清洗。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250μL。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250μL。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。
结果显示，所有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)
20 1.18
30 1.11
38 0.93
39 0.76
151 0.37
Tab5 1.16
IgG4 无阻断
实施例7.抗PVRIG抗体与PVRIG的亲合力测定
将Protein A生物传感器(Fortebio，#18-5010)浸泡在200μL的KB缓冲剂(PBS，pH 7.4，0.02％tween-20，0.1％BSA)中60秒，进行湿润处理。然后，用KB缓冲剂将抗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缓冲剂中100秒，以洗脱多余的抗体。将带有his标签的人PVRIG用KB缓冲剂以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。将传感器置于KB缓冲剂中解离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8所示。
结果显示，所有检测抗体均具有与人PVRIG的高亲合力。
表8.抗PVRIG抗体与人PVRIG的亲合力
抗体编号 Kon(1/Ms) Koff(1/s) KD(M)
20 3.43E+05 8.07E-05 2.36E-10
30 2.84E+05 2.05E-04 7.23E-10
38 1.32E+05 2.87E-04 2.17E-09
39 2.42E+05 1.69E-04 6.96E-10
151 2.61E+05 5.22E-05 2.00E-10
Tab5 7.37E+05 1.61E-05 2.19E-10
实施例8.抗PVRIG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缓冲剂(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。实验结果如图1和表9所示。
结果显示，所有检测抗体均有较强的激活Jurkat细胞中Luciferase的能力，活性是阳性抗体的3.7-18.5倍，证明这些抗体可以结合PVRIG并阻断PVRL2与PVRIG的结合。
表9.抗PVRIG抗体报告基因细胞活性实验结果
抗体株编号 PVRIG报告基因细胞活性实验EC 50(nM)
20 0.04
30 0.06
38 0.20
39 0.06
151 0.04
Tab5 0.74
IgG4 无结合
实施例9.抗PVRIG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。
将培养的NK92细胞系(人恶性非霍奇金淋巴瘤患者的NK细胞)用洗液(包括RPMI 1640，5％FBS，10ng/mL IL-2)洗涤两次，并重悬至2×10 6个/mL的密度。在96孔板中每孔加入50μL(总计1×10 5个)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，裂解孔中所有细胞，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)，150μL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。
实验结果如图2和表10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10.抗PVRIG抗体的NK细胞杀伤实验
Figure PCTCN2022118942-appb-000009
实施例10.抗PVRIG抗体的混合淋巴细胞反应(MLR)实验
PVRIG在T细胞上表达，而PVRL2在树突状细胞(DC细胞)中表达。抗PVRIG抗体可以通过阻断PVRL2与PVRIG的结合，解除树突状细胞对T细胞的抑制作用，活化T细胞。
混合淋巴细胞反应，是指两个无关个体、功能正常的淋巴细胞在体外混合培养时，由于主要组织相容性抗原不同，可相互刺激对方的T细胞发生增殖。从第一个个体来源的外周血中分离PBMC，将细胞培养于含10％FBS的RPMI 1640培养基中，以50ng/mL GM-CSF(Peprotech，300-03-100UG)和50ng/mL IL-4(Peprotech，200-04-100UG)的终浓度添加细胞因子，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120h，取上清，用ELISA试剂盒(R&D，DY202)检测上清中的IFNγ含量。实验结果如图3和表11所示。
结果表明，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，本公开的抗体较之阳性对照Tab5的效果更优。
表11.抗PVRIG抗体混合淋巴细胞反应IFNγ分泌量
Figure PCTCN2022118942-appb-000010
实施例11.抗PVRIG抗体的人源化改造
在所获得的骆驼单域抗体20、抗体30、抗体38、抗体39和抗体151的VH 典型结构的基础上，将重链可变区序列与抗体GermLine数据库比较，获得同源性高的人种系模板。将骆驼单域抗体的框架区替换为人种系模板的重链框架区，CDR(根据Kabat编号系统)保留，再与人源IgG的Fc区域(带有S228P，F234A，L235A，K447A突变的IgG4Fc)重组。以骆驼单域抗体的三维结构为基础，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表12-16所示。
表12.抗体20的人种系模板和人源化抗体序列
Figure PCTCN2022118942-appb-000011
根据表12，抗体20H1-20H5包含如TDCMG(SEQ ID NO：7)所示的CDR1，如HIDSDGIPRYVDSVKG(SEQ ID NO：8)所示的CDR2，和如GFKFDEDYCAPND(SEQ ID NO：150)所示的CDR3。
表13.抗体30的人种系模板和人源化抗体序列
Figure PCTCN2022118942-appb-000012
Figure PCTCN2022118942-appb-000013
根据表13，抗体30H1-30H5包含如GDCMG(SEQ ID NO：10)所示的CDR1，如TIDNAGRIKYADSVKG(SEQ ID NO：11)所示的CDR2，如GWTFGGQCSPAD(SEQ ID NO：151)所示的CDR3。
表14.抗体38的人种系模板和人源化抗体序列
Figure PCTCN2022118942-appb-000014
表15.抗体39的人种系模板和人源化抗体序列
Figure PCTCN2022118942-appb-000015
Figure PCTCN2022118942-appb-000016
表16.抗体151的人种系模板和人源化抗体序列
Figure PCTCN2022118942-appb-000017
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突变。
>人IgG4重链Fc区域(S228P/F234A/L235A/K447A)
Figure PCTCN2022118942-appb-000018
Figure PCTCN2022118942-appb-000019
>人IgG4重链Fc区域(S228P/K447A)
Figure PCTCN2022118942-appb-000020
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12.人源化抗PVRIG抗体与表达PVRIG的细胞结合实验
依照实施例5的方法，用流式细胞仪检测人源化抗PVRIG抗体与人或食蟹猴PVRIG的结合。实验结果如表17所示。
表17.人源化抗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)
20H1 0.019 0.658
20H2 0.006 0.338
20H3 0.006 1.500
20H4 0.008 0.591
20H5 0.005 0.007
30H1 0.024 0.374
30H2 0.003 0.005
30H3 0.004 0.003
38H2 0.088 0.137
38H4 0.062 0.095
38H7 0.040 0.049
38H8 0.065 N.T.
38H9 0.068 N.T.
39H1 0.004 0.251
39H2 0.006 0.019
39H3 0.005 0.348
39H4 0.005 0.006
39H5 0.006 0.208
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
IgG4 无结合 无结合
(注：N.T.，not tested，未测试。)
实施例13.人源化抗PVRIG抗体与PVRIG的亲合力测定
依照实施例7的方法，检测人源化抗PVRIG抗体与人PVRIG的亲合力。结果如表18所示。表中列出的所有抗体均具有与人PVRIG的高亲合力。
表18.人源化抗PVRIG抗体与人PVRIG的亲合力
抗体编号 Kon(1/Ms) Koff(1/s) KD(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
39H1 2.64E+05 8.65E-04 3.28E-09
39H2 1.80E+05 1.24E-04 6.92E-10
39H4 1.89E+05 9.11E-05 4.82E-10
151H7 1.57E+05 1.88E-04 1.20E-09
实施例14.人源化抗PVRIG抗体报告基因细胞活性实验
依照实施例8的方法，检测人源化抗PVRIG抗体在报告基因细胞中的活性。实验结果如图4A-图4B和表19所示。表中列出的抗体均具有激活Jurkat细胞的能力。
表19.人源化抗PVRIG抗体报告基因细胞活性实验
抗体编号 PVRIG报告基因细胞活性实验EC 50(nM)
20H5 0.042
30H2 0.176
30H3 0.078
39H1 0.191
39H2 0.074
39H4 0.094
151H4 3.549
151H7 0.038
151H8 0.058
Tab5 1.380
IgG4 无激活
实施例15.人源化抗PVRIG抗体的活化NK细胞杀伤能力实验
依照实施例9的方法，检测人源化抗PVRIG抗体对NK细胞的活化能力。实验结果如图5A-图5B和表20-表21所示。结果显示，本公开人源化抗PVRIG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20.人源化抗PVRIG抗体的NK细胞杀伤实验
Figure PCTCN2022118942-appb-000021
Figure PCTCN2022118942-appb-000022
表21.人源化抗PVRIG抗体的NK细胞杀伤实验
Figure PCTCN2022118942-appb-000023
实施例16.抗PVRIG/TIGIT双特异性抗体的制备
为探索不同构造的抗PVRIG/TIGIT双特异性抗体对抗体功能的影响，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双特异性抗体，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22所示。抗TIGIT抗体序列信息如表23-24所示。此处全文引入WO2019062832A1中的TIGIT抗体。
表22.抗PVRIG/TIGIT双特异性抗体的第一、第二多肽链序列
Figure PCTCN2022118942-appb-000024
Figure PCTCN2022118942-appb-000025
Figure PCTCN2022118942-appb-000026
表23.抗TIGIT抗体重链及轻链CDR区序列(Kabat编号规则)
Figure PCTCN2022118942-appb-000027
表24.抗TIGIT抗体重链VH及轻链VL序列
Figure PCTCN2022118942-appb-000028
Figure PCTCN2022118942-appb-000029
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双特异性抗体。双特异性抗体的表达量和纯度如表25所示。将纳米抗体偶联到普通单克隆抗体上，无论是重、轻链的N端还是C端，均显示了良好的表达量和纯度。
表25.抗PVRIG/TIGIT双特异性抗体的表达量和纯度
抗体编号 表达量(mg/L) SEC纯度(％)
1708-151-1 140 97.6
1708-151-2 108 95.7
1708-151-3 160 94.7
1708-151-4 158 96.2
实施例17.抗PVRIG/TIGIT双特异性抗体与PVRIG和TIGIT的结合以及对相应配体的阻断实验
1.不同构型的双特异性抗体与人PVRIG的结合及对配体PVRL2的阻断
依照实施例4、实施例5和实施例6的方法，进行实验，结果如表26所示。结果表明，不同构型的双特异性抗体，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。
表26.双特异性抗体对PVRIG的结合和配体阻断
Figure PCTCN2022118942-appb-000030
2.不同构型的双特异性抗体与人TIGIT的结合及对配体PVR的阻断
依照实施例4、实施例5和实施例6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27所示。结果表明，不同构型的双特异性抗体和抗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。抗PVRIG抗体151的连接方式对于抗TIGIT抗体与TIGIT的结合基本无影响。
表27.双特异性抗体对TIGIT的结合和配体阻断
Figure PCTCN2022118942-appb-000031
综合表26-27的数据，我们发现抗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和TIGIT的结合和配体的阻断，并且都显示了良好的表达量、纯度。
实施例18.人源化抗PVRIG/TIGIT双特异性抗体的制备
将不同的人源化抗PVRIG抗体(20H5，30H2，39H2，151H7，151H8)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，序列如表28所示。
表28.人源化抗PVRIG/TIGIT双特异性抗体的第一和第二多肽链全长序列
Figure PCTCN2022118942-appb-000032
Figure PCTCN2022118942-appb-000033
Figure PCTCN2022118942-appb-000034
按常规方法进行抗体的瞬时转染、表达和纯化，经鉴定，得到目的双抗。
实施例19.人源化抗PVRIG/TIGIT双特异性抗体与PVRIG和TIGIT的结合以及对相应配体的阻断
依照实施例4、5、6的方法，检测人源化抗PVRIG/TIGIT双特异性抗体对人和食蟹猴PVRIG的结合，对人PVRIG的配体阻断。结果如表29和图6A-6E所示。结果表明，各个人源化双特异性抗体，均可以结合人PVRIG，阻断PVRIG结合PVRL2。1708-151H8对食蟹猴PVRIG结合较弱。
表29.人源化双特异性抗体对PVRIG的结合和配体阻断
Figure PCTCN2022118942-appb-000035
与实施例4、5、6类似地，检测人源化抗PVRIG/TIGIT双特异性抗体对人和食蟹猴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30和图7A-图7E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30.人源化双特异性抗体对TIGIT的结合和配体阻断
Figure PCTCN2022118942-appb-000036
利用Biacore检测人源化双特异性抗体与人PVRIG，食蟹猴PVRIG，和人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)，食蟹猴PVRIG抗原(SEQ ID NO：1)，或人TIGIT抗原(AcroBiosystem，TIT-H52H3)。利用Biacore仪器(Biacore X100，GE)实时检测反应信号从而获得结合和解离曲线。实验得到的数据用BiacoreX100 evaluation software2.0 GE软件以(1:1)Binding模型进行拟合，得出亲合力数值，见表31。
表31.人源化双特异性抗体与人PVRIG，食蟹猴PVRIG，和人TIGIT的亲合力
Figure PCTCN2022118942-appb-000037
实施例20.人源化抗PVRIG/TIGIT双特异性抗体的混合淋巴细胞反应(MLR)实验
依照实施例10的方法，检测人源化抗PVRIG/TIGIT双特异性抗体对T细胞的活化能力。实验结果如图8和表32所示。结果显示，人源化抗PVRIG/TIGIT双特异性抗体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32.人源化双特异性抗体混合淋巴细胞反应IFNγ分泌量
Figure PCTCN2022118942-appb-000038
实施例21.抗PVRIG/TIGIT双特异性抗体在人黑色素瘤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，除了上述IgG4亚型的双特异性抗体，我们也合成了相应的IgG1亚型的抗体，用于动物药效试验。在该实验中用到的其他之前未描述的抗体序列如表33。
表33.IgG1型人源化抗PVRIG/TIGIT双特异性抗体的第一和第二多肽链全长序列
Figure PCTCN2022118942-appb-000039
Figure PCTCN2022118942-appb-000040
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Mitomycin 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Mitomycin 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。每只老鼠接种：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34，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34.给药方案
Figure PCTCN2022118942-appb-000041
Figure PCTCN2022118942-appb-000042
(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35-36和图9A-图9B。
表35.抗PVRIG/TIGIT双特异性抗体对小鼠体重的影响
Figure PCTCN2022118942-appb-000043
表36.抗PVRIG/TIGIT双特异性抗体在小鼠人源A375肿瘤模型中的抑瘤效果
Figure PCTCN2022118942-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单药组没有明显差异。抗TIGIT抗体1708-IgG1单药组，抗PVRIG抗体151与抗TIGIT抗体1708-IgG1联用组，1708-151-IgG1双抗组，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9B)。
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37，分组给药当天为第0天。
表37.给药方案
组别 给药组 数量 剂量(mg/kg) 给药方案 给药方式
1 hIgG4 7 35.8 Q2D i.p.
2 1708-30H2 IgG4 7 12 Q2D i.p.
3 1708-151H7 IgG4 7 12 Q2D i.p.
(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38-39和图10A-图10B。
表38.抗P RIG/TIGIT双特异性抗体对小鼠体重的影响
Figure PCTCN2022118942-appb-000045
表39.抗PVRIG/TIGIT双特异性抗体在小鼠人源A375肿瘤模型中的抑瘤效果
Figure PCTCN2022118942-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，1708-30H2IgG4与1708-151H7IgG4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10A和图10B)。
制备例-抗PVRIG/TIGIT双特异性抗体制剂
制剂制备与检测过程中使用的设备、抗体及方法如下：
1.SEC分子排阻色谱法：
根据凝胶孔隙的孔径大小与高分子样品分子的线团尺寸间的相对关系，而对溶质进行分离的分析方法。
SEC％(SEC单体含量百分比)＝A单体/A总*100％(A单体为样品中主峰单体的峰面积，A总为所有峰面积之和)。
SEC测定用仪器：安捷伦HPLC 1260；柱子：waters，XBridge
Figure PCTCN2022118942-appb-000047
SEC(7.8×300mm 3.5μm)。
2.NR-CE(也称CE-SDS(NR))毛细管凝胶电泳：
将凝胶移到毛细管中作为支持介质进行的一种电泳，并在一定的电压下根据样品分子量的大小进行分离的方法。
非还原CE纯度百分比＝A主峰/A总*100％(A主峰为样品中主峰的峰面积，A 总为所有峰面积之和。)CE测定用仪器：Beckman毛细管电泳仪型号plus800。
3.iCIEF成像毛细管等点聚焦电泳：
根据蛋白质等电点pI的不同进行分离的技术。
iCIEF中性峰含量百分比＝中性峰面积/总面积*100％(总面积为酸性峰、中性峰和碱性峰面积之和)。
iCIEF测定用仪器：simple protein，型号muarice。
4.渗透压测定：
冰点法测定渗透压，以冰点下降值与溶液的摩尔浓度成正比例关系为基础，采用高灵敏度感温元件，测定溶液结冰点，通过电量转化为渗透压。
渗透压测定用仪器：罗泽Loser，型号OM815。
5.蛋白浓度测定：
以下制备例所采用的抗PVRIG/TIGIT双特异性抗体为1708-30H2。双特异性抗体的浓度采用蛋白浓度计。
蛋白浓度测定仪器：紫外可见分光光度计，型号：Nano Drop oneC，光程为1mm。
制备例1.抗PVRIG/TIGIT双特异性抗体制剂缓冲体系及pH值的筛选
采用下列缓冲体系，制备蛋白浓度约为50mg/mL的抗PVRIG/TIGIT双特异性抗体(1708-30H2)制剂，其中缓冲体系如下：
1)10mM醋酸-醋酸钠盐(简称AA)，pH 5.0
2)10mM AA，pH5.5
3)10mM琥珀酸-琥珀酸钠盐(简称SA)，pH6.0
4)10mM组氨酸-盐酸组氨酸(简称His-HCl)，pH5.5
5)10mM His-HCl，pH6.0
6)10mM His-HCl，pH6.5
7)10mM磷酸氢二钠-磷酸二氢钠盐(简称PB)，pH8.0
8)10mM氨基丁三醇-盐酸盐(简称Tris-HCl)，pH8.0
9)10mM Tris-HCl，pH8.5；
对制备完成的制剂进行无菌过滤，灌装，加塞，轧盖。制剂样品在40℃高温条件下强制降解，以外观、SEC、CE-SDS(NR)和iCIEF为评价指标，考察制剂稳定性。
实验结果见表40。结果显示，制剂样品在40℃高温条件下放置一个月后：
-外观方面，SA缓冲体系组制剂出现大量颗粒，差于其他缓冲体系组制剂；
-纯度方面，缓冲体系pH≥8.0的制剂样品，SEC单体和CE-SDS(NR)下降较多，下降范围分别为3.3％至5.6％和7.1％至14.9％。另外，缓冲体系pH＝5.0或pH≥8.0的制剂样品，其iCIEF中性峰纯度下降超过30％。
表40.抗PVRIG/TIGIT抗体制剂的缓冲体系及pH值筛选实验结果
Figure PCTCN2022118942-appb-000048
注：M表示月，40℃M1表示40℃恒温放置1个月
制备例2.抗PVRIG/TIGIT双特异性抗体制剂蛋白浓度的筛选
采用10mM His-HCl pH 6.0缓冲体系，制备蛋白浓度分别为低浓度50mg/mL和高浓度100mg/mL的制剂。制备完成的制剂无菌过滤，灌装，加塞，轧盖。样品在40℃高温条件下强制降解，以SEC、CE-SDS(NR)和iCIEF为评价指标，考察制剂稳定性。
实验结果见表41，实验结果表明：制剂样品在40℃高温条件下放置1个月后，不同浓度制剂组纯度方面，高蛋白浓度制剂的SEC、CE-SDS(NR)和iCIEF值相对略低。
表41.不同蛋白浓度制剂的稳定性实验结果
Figure PCTCN2022118942-appb-000049
制备例3.抗PVRIG/TIGIT双特异性抗体制剂中表面活性剂种类和浓度的筛选
采用10mM His-HCl pH6.5的缓冲体系，制备含50mg/mL抗体，80mg/mL蔗糖，以及不同浓度的表面活性剂的抗PVRIG/TIGIT抗体(1708-30H2)制剂，具体表面活性剂种类和浓度如下：
1)0.2mg/mL聚山梨酯80；
2)0.4mg/mL聚山梨酯80；
3)0.6mg/mL聚山梨酯80；
4)0.4mg/mL聚山梨酯20；
5)0.4mg/mL泊洛沙姆188；
制备完成的制剂无菌过滤，灌装，加塞，轧盖。样品在40℃高温条件下强制降解，以外观、SEC、CE-SDS(NR)和iCIEF为评价指标，考察制剂稳定性。
实验结果见表42。实验结果表明，各实验组制剂均具备良好的稳定性。
表42.不同表面活性剂制剂的稳定性实验结果
Figure PCTCN2022118942-appb-000050
制备例4.抗PVRIG/TIGIT双特异性抗体制剂糖浓度的筛选
采用10mM His-HCl pH5.5的缓冲体系，制备含50mg/mL抗体，0.4mg/mL聚山梨酯80以及不同浓度的蔗糖的抗PVRIG/TIGIT抗体(1708-30H2)制剂，具体蔗糖浓度如下：
1)75mg/mL蔗糖；
2)80mg/mL蔗糖；
制备完成的制剂，测定其渗透压。
实验结果见表43。结果表明，蔗糖浓度在75mg/mL-80mg/mL时渗透压均在等渗范围内(280-320mOsm)。
表43.不同蔗糖浓度的制剂的渗透压实验结果
蔗糖编号 蔗糖浓度(mg/mL) 制剂渗透压值(mOsm)
1 75 290
2 80 300
制备例5.抗PVRIG/TIGIT双特异性抗体在不同缓冲体系中的稳定性实验
使用下列缓冲体系，制备含50mg/mL抗体，80mg/mL蔗糖，0.4mg/mL聚山梨酯80的抗PVRIG/TIGIT抗体(1708-30H2)制剂，其中缓冲体系如下：
1)10mM AA pH5.5
2)10mM His-HCl pH5.5
制备完成的制剂无菌过滤，灌装，加塞，轧盖。样品在40℃高温条件下强制降解，以外观、SEC、CE-SDS(NR)和iCIEF为评价指标，考察制剂稳定性。
实验结果见表44，结果显示，制剂样品在40℃高温条件下放置1个月后，以His-HCl和AA为缓冲体系的制剂的外观、SEC、CE-SDS(NR)无显著差异，但iCIEF值显示His-HCl缓冲体系组的中性峰纯度高于AA。
表44.不同缓冲体系制剂的稳定性实验结果
Figure PCTCN2022118942-appb-000051
制备例6.抗PVRIG/TIGIT双特异性抗体制剂在不同pH值下的稳定性
使用下列缓冲体系，制备含50mg/mL抗体，80mg/mL蔗糖，0.4mg/mL聚山梨酯80的抗PVRIG/TIGIT抗体制剂(1708-30H2)，缓冲体系如下：
1)10mM His-HCl pH5.5
2)10mM His-HCl pH6.0
3)10mM His-HCl pH6.5
制备完成的制剂无菌过滤，灌装，加塞，轧盖。样品在40℃高温条件下强制降解，以外观、SEC、CE-SDS(NR)和iCIEF为评价指标，考察制剂稳定性。
实验结果见表45。实验结果表明，His-HCl缓冲剂的pH在5.5至6.5之间时，各组制剂均具有良好的稳定性。
表45.不同pH的缓冲体系制剂稳定性实验结果
Figure PCTCN2022118942-appb-000052
制备例7.抗PVRIG/TIGIT双特异性抗体制剂的稳定性实验
采用10mM His-HCl pH6.0的缓冲体系，制备含50mg/mL抗体，0.4mg/mL聚山梨酯80，80mg/mL蔗糖的抗PVRIG/TIGIT抗体(1708-30H2)制剂。制备完成的制剂无菌过滤，灌装，加塞，轧盖。样品放置在25℃及2至8℃条件下6个月，以外观、SEC、CE-SDS(NR)和iCIEF为评价指标，考察制剂稳定性。
实验结果见表46。结果表明，优选制剂具备良好的稳定性。
表46.制剂稳定性实验结果
Figure PCTCN2022118942-appb-000053
制备例8.抗PVRIG/TIGIT双特异性抗体制剂的pH漂移实验
采用10mM His-HCl pH6.0的缓冲体系，分别制备3批含50mg/mL抗体，0.4mg/mL聚山梨酯80，80mg/mL蔗糖的抗PVRIG/TIGIT抗体(1708-30H2)制剂。测定3批制剂成品的pH值，与缓冲液pH值对比，考察pH漂移情况。
实验结果见表47。结果表明，制剂pH漂移没有统计学上的显著变化。
表47.制剂pH测定的实验结果
批次号 制剂pH值 pH漂移值
1 6.07 0.07
2 6.07 0.07
3 6.07 0.07
制备例9.抗PVRIG/TIGIT双特异性抗体制剂中不同表面活性剂的稳定性实验
采用10mM His-HCl pH6.0的缓冲体系，制备含50mg/mL抗体，80mg/mL蔗糖，以及不同种类的表面活性剂的抗PVRIG/TIGIT抗体(1708-30H2)制剂，具体表面活性剂种类和浓度如下：
1)0.4mg/mL聚山梨酯80；
2)0.4mg/mL聚山梨酯20；
3)0.4mg/mL泊洛沙姆188；
制备完成的制剂无菌过滤，灌装，加塞，轧盖。样品放置在2至8℃条件下12个月，以外观、SEC、和iCIEF为评价指标，考察制剂稳定性。
实验结果见表48。结果表明，上述制剂具备良好的稳定性。
表48.不同表面活性剂对制剂稳定性影响的实验结果
Figure PCTCN2022118942-appb-000054
Figure PCTCN2022118942-appb-000055

Claims (16)

  1. 一种药物组合物，包含抗PVRIG/TIGIT双特异性抗体和缓冲剂，其中：
    所述抗PVRIG/TIGIT双特异性抗体包含特异性结合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；
    所述缓冲剂为组氨酸缓冲剂或醋酸盐缓冲剂；
    优选地，所述组氨酸缓冲剂为组氨酸-盐酸组氨酸缓冲剂，或
    优选地，所述醋酸盐缓冲剂为醋酸-醋酸钠盐缓冲剂。
  2. 根据权利要求1所述的药物组合物，其中所述药物组合物的pH为5.0至6.5；
    优选地，所述药物组合物的pH为5.5至6.5；
    更优选地，所述药物组合物的pH为6.0±0.2。
  3. 根据权利要求1或2所述的药物组合物，其中所述抗PVRIG/TIGIT双特异性抗体的浓度为1mg/mL至150mg/mL；
    优选地，所述抗PVRIG/TIGIT双特异性抗体的浓度为1mg/mL至100mg/mL；
    更优选地，所述抗PVRIG/TIGIT双特异性抗体的浓度为50mg/mL±5mg/mL。
  4. 根据权利要求1至3任一项所述的药物组合物，其中所述药物组合物包含表面活性剂；
    优选地，所述表面活性剂为聚山梨酯或泊洛沙姆；
    更优选地，所述表面活性剂为聚山梨酯80。
  5. 根据权利要求4所述的药物组合物，其中所述表面活性剂的浓度为0.05mg/mL至1.0mg/mL；
    优选地，所述表面活性剂的浓度为0.2mg/mL至0.6mg/mL；
    更优选地，所述表面活性剂的浓度为0.4mg/mL±0.1mg/mL。
  6. 根据权利要求1至5任一项所述的药物组合物，其中所述药物组合物包含渗透压调节剂；
    优选地，所述渗透压调节剂选自由蔗糖、海藻糖、山梨糖醇、精氨酸、甘氨酸和氯化钠组成的组中的一种或更多种；
    更优选地，所述渗透压调节剂为蔗糖。
  7. 根据权利要求6所述的药物组合物，其中所述渗透压调节剂的浓度为70mg/mL至100mg/mL；
    优选地，所述渗透压调节剂的浓度为70mg/mL至90mg/mL；
    更优选地，所述渗透压调节剂的浓度为80mg/mL±5mg/mL。
  8. 根据权利要求1至7任一项所述的药物组合物，其中所述缓冲剂的浓度为5mM至100mM；
    优选地，所述缓冲剂的浓度为10mM至30mM；
    更优选地，所述缓冲剂的浓度为10mM±5mM。
  9. 根据权利要求1至8任一项所述的药物组合物，其中：
    所述特异性结合PVRIG的第一抗原结合结构域包含至少一个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CDR1、CDR2和CDR3，所述CDR1、CDR2和CDR3分别包含SEQ ID NO：3、80-84任一所示序列中的CDR1、CDR2和CDR3的氨基酸序列；
    优选地，所述免疫球蛋白单一可变结构域的CDR1、CDR2和CDR3根据Kabat编号系统定义，其中，CDR1包含SEQ ID NO：10的氨基酸序列，CDR2包含SEQ ID NO：11的氨基酸序列，和CDR3包含SEQ ID NO：151的氨基酸序列；
    更优选地，所述免疫球蛋白单一可变结构域包含SEQ ID NO：81的氨基酸序列。
  10. 根据权利要求1至9任一项所述的药物组合物，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VH包含HCDR1、HCDR2和HCDR3，其中HCDR1包含SEQ ID NO：121的氨基酸序列、HCDR2包含SEQ ID NO：122的氨基酸序列和HCDR3包含SEQ ID NO：123的氨基酸序列，和
    所述VL包含LCDR1、LCDR2和LCDR3，其中LCDR1包含SEQ ID NO：124的氨基酸序列、LCDR2包含SEQ ID NO：125的氨基酸序列和LCDR3包含SEQ ID NO：126的氨基酸序列；
    优选地，所述VH包含SEQ ID NO：145的氨基酸序列，和所述VL包含SEQ ID NO：149的氨基酸序列；
    更优选地，所述特异性结合TIGIT的第二抗原结合结构域包含重链和轻链，所述重链包含SEQ ID NO：102的氨基酸序列，所述轻链包含SEQ ID NO：103的氨基酸序列；
    最优选地，所述抗PVRIG/TIGIT双特异性抗体包含第一多肽链和第二多肽链，其中：所述第一多肽链包含SEQ ID NO：109的氨基酸序列，所述第二多肽链包含SEQ ID NO：103的氨基酸序列。
  11. 根据权利要求1至10任一项所述的药物组合物，其包含如下组分：
    (a)1mg/mL至100mg/mL的所述抗PVRIG/TIGIT双特异性抗体，
    (b)0.2mg/mL至0.6mg/mL的聚山梨酯80，
    (c)70mg/mL至90mg/mL的蔗糖，和
    (d)10mM至30mM的组氨酸缓冲剂，所述药物组合物的pH为5.5至6.5；
    优选地，所述药物组合物包含如下组分：
    (a)50mg/mL至100mg/mL的所述抗PVRIG/TIGIT双特异性抗体，
    (b)0.2mg/mL至0.6mg/mL的聚山梨酯80，
    (c)75mg/mL至80mg/mL的蔗糖，和
    (d)10mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为5.5至6.5；
    更优选地，所述药物组合物包含如下组分：
    (a)50mg/mL±5mg/mL的抗PVRIG/TIGIT双特异性抗体，所述抗PVRIG/TIGIT双特异性抗体包含SEQ ID NO：109所示氨基酸序列的第一多肽链和包含SEQ ID NO：103所示氨基酸序列的第二多肽链，
    (b)0.4mg/mL±0.1mg/mL的聚山梨酯80，
    (c)80mg/mL±5mg/mL的蔗糖，和
    (d)10mM±5mM的组氨酸-盐酸组氨酸缓冲剂；所述药物组合物的pH为6.0±0.2。
  12. 一种冻干制剂，所述冻干制剂复溶后可形成权利要求1至11任一项所述的药物组合物。
  13. 一种制备冻干制剂的方法，其中包括将权利要求1至11中任一项所述的药物组合物进行冷冻干燥的步骤。
  14. 一种冻干制剂，所述制剂通过权利要求13所述的方法获得的。
  15. 根据权利要求1至11任一项所述的药物组合物，其为静脉注射制剂、皮下注射制剂、腹腔注射制剂或肌肉注射制剂；优选为静脉注射制剂。
  16. 一种治疗疾病的方法，所述方法包括：
    向受试者施用治疗有效量的权利要求1至11、15中任一项所述的药物组合物、权利要求12或14所述的冻干制剂；
    优选地，所述疾病为增殖性疾病、感染或脓毒症；
    更优选地，所述增殖性疾病为肿瘤；
    最优选地，所述肿瘤选自以下的任一项：肺癌、前列腺癌、乳腺癌、头颈癌、食管癌、胃癌、结直肠癌、膀胱癌、宫颈癌、子宫内膜癌、卵巢癌、肝癌、黑色素瘤、肾癌、鳞状细胞癌和血液系统癌症。
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