WO2023006040A1 - 抗pvrig/抗tigit双特异性抗体和应用 - Google Patents

抗pvrig/抗tigit双特异性抗体和应用 Download PDF

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WO2023006040A1
WO2023006040A1 PCT/CN2022/108648 CN2022108648W WO2023006040A1 WO 2023006040 A1 WO2023006040 A1 WO 2023006040A1 CN 2022108648 W CN2022108648 W CN 2022108648W WO 2023006040 A1 WO2023006040 A1 WO 2023006040A1
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seq
antibody
tigit
binding
antigen
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PCT/CN2022/108648
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English (en)
French (fr)
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赵晓峰
刘雷
刘杨
付雅媛
曹卓晓
唐任宏
任晋生
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江苏先声药业有限公司
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Priority to EP22848645.2A priority Critical patent/EP4378954A4/en
Priority to US18/292,480 priority patent/US20240343803A1/en
Priority to AU2022320667A priority patent/AU2022320667A1/en
Priority to CA3227972A priority patent/CA3227972A1/en
Priority to CN202280053417.7A priority patent/CN117751143A/zh
Priority to JP2024505449A priority patent/JP2024530451A/ja
Publication of WO2023006040A1 publication Critical patent/WO2023006040A1/zh

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    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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Definitions

  • the present invention relates to the technical field, in particular, to an anti-PVRIG/anti-TIGIT bispecific antibody.
  • Immunotherapy is based on manipulating and/or modulating the immune system, including both innate and acquired immune responses.
  • the goal of immunotherapy is to treat disease by controlling the immune response to "foreign agents" such as pathogens or tumor cells.
  • the immune system is a highly complex system made up of many cell types with complex and nuanced systems controlling those interactions and responses.
  • the concept of cancer immunosurveillance is based on the theory that the immune system can identify tumor cells, initiate an immune response and inhibit tumor development and/or progression. However, it is clear that many cancer cells have developed mechanisms to evade the immune system, which can allow unchecked tumor growth.
  • Cancer/tumor immunotherapy focuses on the development of new and novel agonists and/or antagonists that can activate and/or activate the immune system to achieve a more effective anti-tumor response, enhance the killing of tumor cells and/or inhibit tumor growth.
  • PVRIG is expressed on NK cells and T cells and shares several similarities with other known immune checkpoints.
  • the identification and methods used to demonstrate that PVRIG is an immune checkpoint receptor are discussed in WO2016/134333, which is expressly incorporated herein by reference.
  • PVRIG binds to its ligand (PVRL2)
  • PVRL2 its ligand
  • Blocking the binding of PVRL2 to PVRIG cuts off this inhibitory signal from PVRIG and thus modulates NK and T cell immune responses.
  • Utilizing PVRIG antibodies that block binding to PVRL2 is a therapeutic approach that enhances NK cell and T cell killing of cancer cells. Blocking antibodies that bind PVRIG and block the binding of its ligand PVRL2 have been generated.
  • TIGIT is another target of interest, as binding to its cognate ligand PVR has been shown to directly inhibit NK and T cell cytotoxicity through its intracellular ITIM domain. Knockout of the TIGIT gene or blocking antibodies to the TIGIT/PVR interaction have been shown to enhance NK cell killing in vitro, or exacerbate autoimmune disease in vivo.
  • TIGIT induces PVR-mediated signaling in dendritic cells or tumor cells, leading to increased production of anti-inflammatory cytokines such as IL10.
  • TIGIT expression was closely correlated with that of another important co-inhibitory receptor, PD-1. TIGIT and PD-1 are coexpressed on many human and murine tumor infiltrating lymphocytes (TILs).
  • TIGIT and PVRIG belong to the DNAM superfamily and have been shown to co-express in a variety of tumor-infiltrating lymphocytes to play an immunosuppressive role.
  • tumor-infiltrating effector T cells co-expressed with TIGIT, PVRIG and PD-1 are considered to be infiltrating T cells The most dominant group of effector T cells in the population.
  • bispecific antibodies capable of simultaneously targeting PVRIG and TIGIT have potentially synergistic effects and are an attractive therapeutic modality for single antibody therapy. Such bispecific antibodies will allow simultaneous targeting of two immune checkpoint receptors while potentially further synergizing with existing anti-PD-1/L-1 antibody therapies, providing novel therapeutic approaches in cancer therapy means play an important role.
  • the present invention provides an anti-PVRIG/anti-TIGIT antibody, a nucleic acid encoding it, a method for preparing the antibody, a pharmaceutical composition containing the antibody, and related uses of the pharmaceutical composition for treating tumors.
  • the present invention provides an anti-PVRIG/anti-TIGIT bispecific antibody comprising:
  • a first antigen binding portion comprising a heavy chain variable region (VH) and a light chain variable region (VL), said VH and VL forming an anti-TIGIT antigen binding domain;
  • said TIGIT VH comprises SEQ ID NO: HCDR1, HCDR2 and HCDR3 of the VH described in any one of 72 or 87;
  • the TIGIT VL comprises LCDR1, LCDR2 and LCDR3 of the VL described in any one of SEQ ID NO: 68 or 91;
  • a second antigen-binding portion comprising a VHH specifically binding to PVRIG, said VHH comprising CDR1, CDR2 and CDR3 of any one of SEQ ID NO: 200 or 211.
  • HCDR1 of the first antigen-binding portion comprises the sequence described in any one of SEQ ID NO: 21 or 33;
  • HCDR2 comprises the sequence described in any one of SEQ ID NO: 22 or 34 ;
  • HCDR3 comprises the sequence described in any one of SEQ ID NO: 23 or 35;
  • LCDR1 of the first antigen-binding portion comprises the sequence described in any one of SEQ ID NO: 18 or 96;
  • LCDR2 comprises the sequence described in any one of SEQ ID NO: 19 or 31;
  • LCDR3 comprises the sequence described in any one of SEQ ID NO : the sequence described in any one of 20 or 32;
  • CDR1 of the second antigen-binding portion comprises the sequence described in any one of SEQ ID NO: 168 or 147;
  • CDR2 comprises the sequence described in any one of SEQ ID NO: 207 or 148;
  • CDR3 comprises the sequence described in any one of SEQ ID NO: 207 or 148; : The sequence described in any one of 208 or 149.
  • the first antigen binding moiety comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the following sequence:
  • substitutions are Conservative amino acid substitutions.
  • the second antigen binding portion comprises CDR1, CDR2 and CDR3 of the following sequence:
  • substitutions are Conservative amino acid substitutions.
  • the VH of the first antigen-binding portion comprises a sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 72 or 87; the VL of the first antigen-binding portion comprises SEQ ID NO: A sequence having at least 90% identity to the amino acid sequence shown in 68 or 91.
  • the second antigen binding moiety comprises a sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 200 or 211.
  • the first antigen-binding portion is a full-length antibody comprising two heavy chains and two light chains; the C-terminus of the second antigen-binding portion is fused to at least N-terminus of a heavy chain.
  • the heavy chain fusion polypeptide includes PVRIG VHH-(G4S)4 Linker-TIGIT VH-CH1-hinge-CH2-CH3 from N-terminus to C-terminus, and the light chain polypeptide includes TIGIT VL- cl.
  • the heavy chain fusion polypeptide comprises a sequence at least 80% identical to the amino acid sequence shown in SEQ ID NO: 227, 229, 231 or 233
  • the light chain polypeptide comprises a sequence with SEQ ID NO: 226, 228, A sequence having at least 80% identity to the amino acid sequence shown in 230 or 232.
  • the bispecific antibody is a humanized antibody.
  • the bispecific antibody specifically binds to human or monkey PRVIG or TIGIT protein; preferably, its KD for binding to human or monkey TIGIT is better than 1.00E-7M, and its KD for binding to human or monkey PRVIG is better than 1.00E-7M. KD is better than 1.00E-8M; more preferably, it can be combined with TIGIT and PVRIG at the same time.
  • the invention provides an antibody or antigen-binding fragment that specifically binds TIGIT, comprising:
  • Heavy chain variable region wherein said heavy chain variable region comprises three complementarity determining regions (HCDR): HCDR1, HCDR2 and HCDR3, wherein, numbered according to the Kabat numbering system, said HCDR1 comprises SEQ ID
  • described HCDR1 comprises the aminoacid sequence shown in SEQ ID NO:45,51,57,63
  • described HCDR2 comprises SEQ ID NO:46,52,58,64
  • the HCDR3 comprises SEQ ID NO: 47, 53, 59, 65; and,
  • a light chain variable region wherein the light chain variable region comprises three complementarity determining regions (LCDRs): LCDR1, LCDR2 and LCDR3, wherein, numbered according to the Kabat numbering system, the LCDR1 comprises SEQ ID NO: 18 , 24, 30, 36, 93, 94, 95, and 96 amino acid sequences, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 19, 25, 31, 37, and the LCDR3 comprises SEQ ID NO: 20 , 26, 32, and 38 amino acid sequences; according to the IMGT numbering system numbering, the LCDR1 comprises the amino acid sequences shown in SEQ ID NO: 42, 48, 54, 60, and the LCDR2 comprises SEQ ID NO: 43, 49 The amino acid sequence shown in , 55, 61, the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 44, 50, 56, 62.
  • LCDRs complementarity determining regions
  • the antibody or antigen-binding fragment comprises LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of the following sequence:
  • the antibody or antigen-binding fragment comprises:
  • the heavy chain variable region which comprises the same sequence as SEQ ID NO: 10, 11, 12, 13, 69, 70, 71, 72, 81, 82, 83, 84, 85, 87, 101, 102 or 103 Amino acid sequences of at least 80% identity; or/and
  • a light chain variable region comprising the same sequence as SEQ ID NO: 14, 15, 16, 17, 66, 67, 68, 78, 79, 80, 86, 88, 89, 90, 91, 98, 99 or 100 amino acid sequences having at least 80% identity.
  • the antibody or antigen-binding fragment :
  • a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 102 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 98, 99 or 100
  • a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 103 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 98, 99 or 100; or
  • the antibody or antigen-binding fragment comprises a heavy chain variable region, wherein, compared with the VH shown in SEQ ID NO.10, the heavy chain variable region has at least a mutation selected from the group consisting of: Natural sequence numbering, S30T, G44K, W47Y, I48M, V67I or V71R; preferably, at least S30T and V71R mutations; more preferably, at least S30T, G44K and V71R mutations; more preferably, at least S30T, G44K, I48M , V67I and V71R mutations; more preferably, at least S30T, G44K, W47Y and V71R mutations;
  • the VH shown in SEQ ID NO.11 has at least a mutation selected from the group: numbered in natural order, T28A, R72A, T74K or A76S; preferably, at least T28A, R72A, T74K and A76S;
  • the VH shown in SEQ ID NO.12 it has at least a mutation selected from the group: numbered in natural order, I29M, S30T, G44K, W47Y, I48M, V67I or V71R; preferably, at least has S30T and V71R mutations; More preferably, at least I29M, S30T and V71R mutations; more preferably, at least I29M, S30T, G44K and V71R mutations; more preferably, at least I29M, S30T, G44K, I48M, V67I and V71R mutations; more preferably , with at least I29M, S30T, G44K, W47Y and V71R;
  • mutations selected from the group: numbering in natural order, R44G, R72V, T74K, S75L or A76S; preferably, at least R72V and T74K mutations; more preferably, At least R72V, T74K, S75L and A76S mutations; more preferably, at least R44G, R72V, T74K, S75L and A76S mutations.
  • the antibody or antigen-binding fragment comprises a light chain variable region, wherein, compared with the VL shown in SEQ ID NO.14, the light chain variable region has at least a mutation selected from the group consisting of: Natural sequence numbering, L37Q, P43S or L47M; preferably, at least the L47M mutation; more preferably, at least the L37Q and L47M mutation; more preferably, at least the P43S and L47M mutation;
  • mutations selected from the group: numbering in natural order, N31Q, N31T, N31D, G32A, Q38H or P43S; preferably, at least Q38H and P43S mutations; more preferably Preferably, at least N31Q, Q38H and P43S mutations; more preferably, at least N31T, Q38H and P43S mutations; more preferably, at least N31D, Q38H and P43S mutations; more preferably, at least G32A, Q38H and P43S mutations;
  • mutations selected from the group: numbering in natural order, L37Q, P43S or Q45K; preferably, at least L37Q and Q45K mutations; more preferably, at least P43S mutations ;
  • the antibody or antigen-binding fragment specifically binds to human or monkey TIGIT protein; preferably, its KD for binding to human or monkey TIGIT is better than 1.00E-8M.
  • the antibody or antigen-binding fragment is a murine antibody, a humanized antibody, a fully human antibody, or a chimeric antibody.
  • the antibody or antigen-binding fragment is selected from monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific molecules (e.g., bispecific antibodies), monovalent antibodies, Multivalent Antibody, Whole Antibody, Fragment of Whole Antibody, Naked Antibody, Conjugated Antibody, Chimeric Antibody, Humanized Antibody, Fully Human Antibody, Fab, Fab', Fab'-SH, F(ab')2, Fd , Fv, scFv, diabody or single domain antibody.
  • the present invention provides a nanobody or antigen-binding fragment specifically binding to PVRIG, which comprises the VH described in any one of SEQ ID NO.107-119, 198-204, 211-216, 219-225 HCDR1, HCDR2 and HCDR3.
  • said HCDR1, HCDR2 and HCDR3 of said Nanobody or antigen-binding fragment are identified according to the IMGT numbering system, e.g. selected from Table 21; said HCDR1, HCDR2 and HCDR3 are identified according to the Kabat numbering system, e.g. selected from Table 22, Table 29.
  • the Nanobody or the antigen-binding fragment, HCDR1-3 of the VH shown in SEQ ID NO.107, according to the IMGT or Kabat numbering system, has such as SEQ ID NO: 120-122 or SEQ ID NO: 159- the sequence shown in 161;
  • HCDR1-3 of the VH shown in SEQ ID NO.108 has the sequence shown in SEQ ID NO: 123-125 or SEQ ID NO: 162-164 according to the IMGT or Kabat numbering system;
  • HCDR1-3 of the VH shown in SEQ ID NO.109 has a sequence as shown in SEQ ID NO: 126-128 or SEQ ID NO: 165-167 according to the IMGT or Kabat numbering system;
  • HCDR1-3 of the VH shown in SEQ ID NO.110 has the sequence shown in SEQ ID NO:129-131 or SEQ ID NO:168-170 according to the IMGT or Kabat numbering system;
  • HCDR1-3 of the VH shown in SEQ ID NO.111 has the sequences shown in SEQ ID NO:132-134 and SEQ ID NO:171-173 according to the IMGT or Kabat numbering system;
  • HCDR1-3 of the VH shown in SEQ ID NO.112 has a sequence as shown in SEQ ID NO:135-137 or SEQ ID NO:174-176 according to the IMGT or Kabat numbering system;
  • HCDR1-3 of the VH shown in SEQ ID NO.113 has the sequence shown in SEQ ID NO:138-140 or SEQ ID NO:177-179 according to the IMGT or Kabat numbering system;
  • HCDR1-3 of the VH shown in SEQ ID NO.114 has the sequence shown in SEQ ID NO:141-143 or SEQ ID NO:180-182 according to the IMGT or Kabat numbering system;
  • HCDR1-3 of the VH shown in SEQ ID NO.115 has a sequence as shown in SEQ ID NO:144-146 or SEQ ID NO:183-185 according to the IMGT or Kabat numbering system;
  • HCDR1-3 of the VH shown in SEQ ID NO.116 has a sequence as shown in SEQ ID NO:147-149 or SEQ ID NO:186-188 according to the IMGT or Kabat numbering system;
  • HCDR1-3 of the VH shown in SEQ ID NO.117 has the sequence shown in SEQ ID NO:150-152 or SEQ ID NO:189-191 according to the IMGT or Kabat numbering system;
  • HCDR1-3 of the VH shown in SEQ ID NO.118 has the sequence shown in SEQ ID NO:153-155 or SEQ ID NO:192-194 according to the IMGT or Kabat numbering system;
  • HCDR1-3 of the VH shown in SEQ ID NO.119 has a sequence as shown in SEQ ID NO:156-158 or SEQ ID NO:195-197 according to the IMGT or Kabat numbering system;
  • HCDR1-3 of the VH shown in SEQ ID NO.198 has the sequence shown in SEQ ID NO:168-170 according to the Kabat numbering system;
  • HCDR1-3 of the VH shown in SEQ ID NO.199 has the sequences shown in SEQ ID NO:168, 207 and 170 according to the Kabat numbering system;
  • HCDR1-3 of the VH shown in SEQ ID NO.200 has the sequences shown in SEQ ID NO: 168, 207 and 208 according to the Kabat numbering system;
  • HCDR1-3 of the VH shown in SEQ ID NO.201 has the sequences shown in SEQ ID NO: 168, 207 and 209 according to the Kabat numbering system;
  • HCDR1-3 of the VH shown in SEQ ID NO.202 has the sequences shown in SEQ ID NO:168, 169 and 208 according to the Kabat numbering system;
  • HCDR1-3 of the VH shown in SEQ ID NO.203 and 204 have the sequences shown in SEQ ID NO:168, 210 and 208 according to the Kabat numbering system;
  • HCDR1-3 of the VH shown in SEQ ID NO.211-215 has the sequence shown in SEQ ID NO:147-149 according to the IMGT numbering system;
  • HCDR1-3 of the VH shown in SEQ ID NO.216 has the sequences shown in SEQ ID NO:147, 148 and 218 according to the IMGT numbering system;
  • HCDR1-3 of the VH shown in SEQ ID NO.219-225 has the sequence shown in SEQ ID NO:156-158 according to the IMGT numbering system.
  • said Nanobody or antigen-binding fragment comprises at least 80% identity or 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to said HCDR1, HCDR2 and HCDR3 CDRs sequence, preferably, the replacement is a conservative amino acid replacement.
  • the Nanobody or antigen-binding fragment comprises the VH shown in any one of SEQ ID NO.107-119, 198-204, 211-216, 219-225, or in combination with SEQ ID NO.
  • the VH shown in any one of 119, 198-204, 211-216, 219-225 has at least 80% identity or a sequence of at most 20 mutations; the mutation can be selected from insertion, deletion and/or replacement, and the replacement Conservative amino acid substitutions are preferred.
  • the Nanobody or antigen-binding fragment comprises at least a mutation sequence selected from the group consisting of: A97V, K98E, N54D, N108S, S110A, numbered in natural order, compared to the VH shown in SEQ ID NO.
  • G55A or S75T more preferably, at least A97V and K98E mutations; more preferably, at least A97V, K98E and N54D mutations; more preferably, at least A97V, K98E, N54D and N108S mutations; more preferably, at least A97V, K98E, N54D, and S110A mutations; more preferably, at least A97V, K98E, and N108S mutations; more preferably, at least A97V, K98E, G55A, and N108S mutations; more preferably, at least S75T, A97V, K98E, G55A and N108S mutation;
  • VH shown in SEQ ID NO: 116 have at least a mutation sequence selected from the group consisting of: numbered in natural order, S35T, V37F, G44E, L45R, W47F, N50T, L79V, V61S, D62H, T122I or M123Q more preferably, at least V37F, G44E, L45R, W47F and N50T mutations; more preferably, at least S35T, V37F, G44E, L45R, W47F and N50T mutations; more preferably, at least S35T, V37F, G44E, L45R , W47F, N50T, and L79V mutations; more preferably, at least S35T, V37F, G44E, L45R, W47F, N50T, V61S, and D62H mutations; more preferably, at least S35T, V37F, G44E, L45R, W47F, N50T, T122I and
  • SEQ ID NO: 119 At least have a mutation sequence selected from the group: numbered in natural order, S35G, V37Y, G44D, L45R, W47L, N50T, Y58K, Y59I, D72G, N73D, Y79S , L78V or Y94F; more preferably, at least S35G, V37Y, G44D, L45R, W47L and N50T mutations; more preferably, at least S35G, V37Y, G44D, L45R, W47L, N50T and Y58K mutations; more preferably, at least Have S35G, V37Y, G44D, L45R, W47L, N50T, Y58K, D72G and N73D mutations; more preferably, have at least S35G, V37Y, G44D, L45R, W47L, N50T, Y58K, D72G and N73D mutations; more preferably, have at least S35G,
  • the Nanobody or antigen-binding fragment is: (1) a chimeric Nanobody or fragment thereof; (2) a humanized Nanobody or fragment thereof; or (3) a fully human Nanobody or fragment thereof .
  • the Nanobody or antigen-binding fragment comprises or does not comprise an antibody heavy chain constant region; optionally, the antibody heavy chain constant region may be selected from human, alpaca, mouse, rat, rabbit or sheep; alternatively, the heavy chain constant region of the antibody may be selected from IgG, IgM, IgA, IgE or IgD, and the IgG may be selected from IgG1, IgG2, IgG3 or IgG4; alternatively, the heavy chain is constant
  • the region may be selected from an Fc region, a CH3 region or a complete heavy chain constant region, preferably, the heavy chain constant region is a human Fc region; preferably, the Nanobody or antigen-binding fragment is a heavy chain antibody.
  • the anti-PVRIG/anti-TIGIT bispecific antibody of the present invention is also coupled with a therapeutic agent or tracer; preferably, the therapeutic agent is selected from the group consisting of drugs, toxins, radioisotopes, chemotherapeutics or immunomodulators, and the tracer is selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, Chemiluminescent labels, ultrasound contrast agents and photosensitizers.
  • the present invention provides a multispecific molecule comprising the anti-PVRIG/anti-TIGIT bispecific antibody, the antibody or antigen-binding fragment that specifically binds to TIGIT; or the antibody that specifically binds to PVRIG Nanobodies or antigen-binding fragments; preferably, the multispecific molecule can be bispecific, trispecific or tetraspecific, more preferably, the multispecific molecule can be bivalent, tetravalent or hexavalent price.
  • the multispecific molecule is a tandem scFv, diabody (Db), single chain diabody (scDb), dual affinity retargeting (DART) antibody, F(ab')2, dual Variable Domain (DVD) Antibodies, KiH Antibodies, Docking and Locking (DNL) Antibodies, Chemically Cross-Linked Antibodies, Heteropolymeric Nanobodies or Heteroconjugate Antibodies.
  • the present invention provides a chimeric antigen receptor (CAR) comprising at least an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, the extracellular antigen binding domain comprising said antibody or antigen-binding fragment specifically binding to TIGIT; or comprising said Nanobody or antigen-binding fragment specifically binding to PVRIG.
  • CAR chimeric antigen receptor
  • the present invention provides an immune effector cell expressing the chimeric antigen receptor, or comprising a nucleic acid fragment encoding the chimeric antigen receptor; preferably, the immune effector cell is selected from T Cell, NK cell (natural killer cell), NKT cell (natural killer T cell), DNT cell (double negative T cell), monocyte, macrophage, dendritic cell or mast cell, said T cell is preferably selected from Cytotoxic T cells, regulatory T cells or helper T cells; preferably, the immune effector cells are autoimmune effector cells or allogeneic immune effector cells.
  • the present invention provides an isolated nucleic acid fragment encoding any of the above-mentioned bispecific antibodies, any of the above-mentioned antibodies or antigen-binding fragments that specifically bind to TIGIT; any of the above-mentioned specific binding to PVRIG Nanobodies or antigen-binding fragments of any of the above-mentioned multispecific molecules, or any of the above-mentioned chimeric antigen receptors.
  • the present invention provides a vector comprising the above-mentioned nucleic acid fragment.
  • the present invention provides a host cell comprising the above-mentioned vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungus (yeast), insect cells or mammalian cells Cells (CHO cell line or 293T cell line).
  • the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungus (yeast), insect cells or mammalian cells Cells (CHO cell line or 293T cell line).
  • the present invention provides a method for preparing any of the above-mentioned bispecific antibodies, any of the above-mentioned antibodies or antigen-binding fragments that specifically bind to TIGIT; any of the above-mentioned Nanobodies that specifically bind to PVRIG or An antigen-binding fragment, or any of the above-mentioned multispecific molecules, comprising culturing the above-mentioned host cells, and isolating the antibodies or molecules expressed by said cells.
  • the present invention provides a method for preparing the immune effector cells, which includes introducing a nucleic acid fragment encoding any one of the CARs described above into the immune effector cells, and optionally, also including initiating the immune effector cells.
  • the cells express any one of the above CARs.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising any of the above-mentioned bispecific antibodies, any of the above-mentioned antibodies or antigen-binding fragments that specifically bind to TIGIT; any of the above-mentioned antibodies that specifically bind to PVRIG Nanobodies or antigen-binding fragments, any of the above-mentioned multispecific molecules, the above-mentioned immune effector cells, nucleic acid fragments, vectors, host cells, or products prepared by the method, and pharmaceutically acceptable carriers.
  • the pharmaceutical composition further comprises an additional therapeutic agent; preferably, the additional therapeutic agent is an antineoplastic agent; more preferably, the antineoplastic agent is a PD-1 axis binding antagonist.
  • any of the above-mentioned bispecific antibodies disclosed in the present invention any of the above-mentioned antibodies or antigen-binding fragments that specifically bind to TIGIT; any of the above-mentioned nanobodies or antigen-binding fragments that specifically bind to PVRIG Fragments, any of the above-mentioned multispecific molecules, the above-mentioned immune effector cells, nucleic acid fragments, vectors, host cells, products prepared by the method, or pharmaceutical compositions used in the preparation of drugs for the treatment of cancer or infectious diseases purposes; wherein the cancer is selected from solid tumors and blood tumors, preferably, the tumor is selected from leukemia, multiple myeloma, lymphoma, myelodysplastic syndrome, prostate cancer, liver cancer, colorectal cancer, anal cancer, Ovarian cancer, endometrial cancer, cervical cancer, abdominal cancer, breast cancer, pancreatic cancer, stomach cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial
  • the drug is used in combination with an additional therapeutic agent or with surgery; wherein the additional therapeutic agent or the surgery is selected from radiation therapy, chemotherapy, oncolytic drugs, cytotoxic agents, cytokines, Surgery, immunostimulatory antibodies, immunomodulatory drugs, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, vaccines or cellular immunotherapy.
  • additional therapeutic agent or the surgery is selected from radiation therapy, chemotherapy, oncolytic drugs, cytotoxic agents, cytokines, Surgery, immunostimulatory antibodies, immunomodulatory drugs, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, vaccines or cellular immunotherapy.
  • the additional therapeutic agent is administered before, after, or concurrently with the drug.
  • the medicament is used in combination with a PD-1 axis binding antagonist.
  • the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist; preferably, the PD-1 The binding antagonist is an anti-PD-1 antibody; more preferably, the PD-1 binding antagonist is selected from MDX 1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP- 514), PDR001, REGN2810, and the group consisting of BGB-108; preferably, the PD-L1 binding antagonist is an anti-PD-L1 antibody; more preferably, the PD-L1 binding antagonist is selected from MPDL3280A (atezolizumab ), YW243.55.S70, MDX-1105, MEDI4736 (durvalumab), Tecentriq and MSB0010718C (avelumab); preferably, the PD-L2
  • the present invention also provides a method for treating cancer or infectious diseases, comprising administering an effective amount of any of the above-mentioned bispecific antibodies, any of the above-mentioned bispecific antibodies that specifically bind to TIGIT, to a patient in need thereof.
  • the cancer is selected from solid tumors and blood tumors, preferably, the tumor is selected from leukemia, multiple myeloma, lymphoma, myelodysplastic syndrome, prostate cancer, liver cancer, colorectal cancer Cancer, Anal Cancer, Ovarian Cancer, Endometrial Cancer, Cervical Cancer, Abdominal Cancer, Breast Cancer, Pancreatic Cancer, Stomach Cancer, Head and Neck Cancer, Thyroid Cancer, Testicular Cancer, Urothelial Cancer, Lung Cancer, Melanoma, Non-Melanoma Skin cancer, glioma, kidney cancer, mesothelioma, esophageal cancer, non-small cell lung cancer,
  • the method further comprises administering to a patient in need thereof an effective amount of a PD-1 axis binding antagonist, wherein the PD-1 axis binding antagonist is selected from the group consisting of PD-1 binding antagonists, PD - the group consisting of an L1 binding antagonist, and a PD-L2 binding antagonist; preferably, the PD-1 binding antagonist is an anti-PD-1 antibody; more preferably, the PD-1 binding antagonist is selected from the group consisting of MDX 1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and the group consisting of BGB-108; preferably, the PD-L1 binding antagonist
  • the agent is an anti-PD-L1 antibody; more preferably, the PD-L1 binding antagonist is selected from the group consisting of MPDL3280A (atezolizumab), YW243.55.S70, MDX
  • the present invention also provides any of the above-mentioned bispecific antibodies, any of the above-mentioned antibodies or antigen-binding fragments that specifically bind to TIGIT; any of the above-mentioned nanobodies or antigen-binding fragments that specifically bind to PVRIG, Any of the above-mentioned multispecific molecules, the above-mentioned immune effector cells, nucleic acid fragments, vectors, host cells, products prepared by the method, or pharmaceutical compositions are used for the pretreatment of cancer or infectious diseases; wherein the cancer is selected from From solid tumors and blood tumors, preferably, the tumor is selected from leukemia, multiple myeloma, lymphoma, myelodysplastic syndrome, prostate cancer, liver cancer, colorectal cancer, anal cancer, ovarian cancer, endometrial cancer , cervical cancer, abdominal cancer, breast cancer, pancreatic cancer, stomach cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer,
  • the anti-PVRIGxTIGIT humanized bispecific antibody of the present invention can specifically target tumor cells, effectively mediate the killing effect on tumor cell lines, and has good safety while achieving excellent tumor suppression effect.
  • compositions including A and B should be understood as the following technical scheme: a composition composed of A and B, and a composition containing other components in addition to A and B, all fall into Into the scope of the aforementioned "a composition”.
  • T cell immune receptor with Ig and ITIM domains are used interchangeably and include various mammalian isotypes such as human Tigit, Orthologs of human Tigit, and analogs comprising at least one epitope within Tigit, and analogs having at least one epitope shared with TIGIT.
  • the amino acid sequence of TIGIT eg, human TIGIT
  • nucleotide sequence encoding it are known in the art.
  • PVRIG or “PVRIG protein” herein may optionally include any such protein or variants, conjugates or fragments thereof, including but not limited to known or wild-type PVRIG as described herein, as well as any natural The resulting splice variants, amino acid variants or isoforms, and especially the ECD fragments of PVRIG.
  • Anti-PVRIG antibodies (including antigen-binding fragments) that bind to PVRIG and prevent activation by PVRL2 (e.g., most commonly by blocking the interaction of PVRIG and PVLR2) are used to enhance T cell and/or NK cell activation and for therapeutic Diseases such as cancer and pathogenic infections.
  • anti-PVRIG/anti-TIGIT antibody and "bispecific PVRIG/TIGIT antibody” and “anti-PVRIG/anti-TIGIT bispecific antibody” are used interchangeably herein, and the anti-PVRIG/anti-TIGIT bispecific antibody of the present invention Specifically binds to human TIGIT, and preferably to the ECD of human TIGIT, and to PVRIG, and more preferably to the ECD of human PVRIG.
  • an antigen-binding molecule eg, an antibody
  • an antigen-binding molecule specifically binds an antigen and substantially the same antigen with high affinity, typically, but does not bind an unrelated antigen with high affinity.
  • Affinity is usually reflected in an equilibrium dissociation constant (KD), where a lower KD indicates a higher affinity.
  • KD equilibrium dissociation constant
  • high affinity generally refers to having about 1 ⁇ 10 -7 M or lower, about 1 ⁇ 10 -8 M or lower, about 1 ⁇ 10 -9 M or lower, about 1 ⁇ 10 -10 M or lower, 1 ⁇ 10 -11 M or lower, or 1 ⁇ 10 -12 M or lower KD.
  • KD KD/Ka, where Kd represents the dissociation rate and Ka represents the on-rate.
  • the equilibrium dissociation constant KD can be measured by methods known in the art, such as surface plasmon resonance (eg Biacore) or equilibrium dialysis.
  • antigen binding molecule is used herein in the broadest sense to refer to a molecule that specifically binds an antigen.
  • antigen binding molecules include, but are not limited to, antibodies or antibody mimetics.
  • Antibody mimic refers to an organic compound or binding domain that can specifically bind to an antigen, but has nothing to do with the structure of an antibody.
  • antibody mimics include but are not limited to affibody, affitin, affilin, designed ankyrin repeat proteins (DARPins), aptamers or Kunitz-type domain peptides.
  • antibody is used herein in the broadest sense to refer to a polypeptide comprising sufficient sequence from the variable region of an immunoglobulin heavy chain and/or sufficient sequence from the variable region of an immunoglobulin light chain to be capable of specifically binding to an antigen or peptide combinations.
  • Antibody herein encompasses various forms and various structures as long as they exhibit the desired antigen-binding activity.
  • Antibody herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antibody, as well as fully synthetic scaffolds comprising, eg, biocompatible polymers.
  • Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.
  • Antibody herein includes a typical "four-chain antibody”, which belongs to the immunoglobulins composed of two heavy chains (HC) and two light chains (LC); In the N-terminal to C-terminal direction, it consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain; and, When the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain; the light chain is composed of a light chain variable region (VL) and a light chain constant in the N-terminal to C-terminal direction.
  • VH heavy chain variable region
  • CH1 domain a heavy chain constant region
  • HR hinge region
  • CH2 domain a heavy chain constant region CH2 domain
  • CH3 domain heavy chain constant region
  • the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain
  • the light chain is
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are respectively the ⁇ chain and the delta chain , ⁇ chain, ⁇ chain and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3, IgG4, and IgA can be divided into IgA1 and IgA.
  • IgA2 Light chains are classified as either kappa chains or lambda chains by difference in the constant region.
  • Each of the five Ig classes can have either a kappa chain or a lambda chain.
  • Antibody herein also includes antibodies that do not comprise light chains, for example, antibodies produced from Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and alpaca ( Heavy-chain antibodies (HCAbs) produced by camelids such as Vicugna pacos) and immunoglobulin new antigen receptors (Ig new antigen receptors, IgNAR) found in cartilaginous fishes such as sharks.
  • HCAbs Heavy-chain antibodies
  • Ig new antigen receptors Ig new antigen receptors, IgNAR
  • heavy chain antibody refers to an antibody that lacks the light chains of conventional antibodies.
  • the term specifically includes, but is not limited to, homodimeric antibodies comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.
  • the term “nanobody” refers to the natural heavy chain antibody that lacks the light chain in camels, and its variable region can be cloned to obtain a single domain antibody consisting of only the variable region of the heavy chain, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.
  • VHH Very domain of heavy chain of heavy chain antibody
  • single domain antibody single domain antibody, sdAb
  • CH1 light chain and heavy chain constant region 1
  • an “antibody” herein may be derived from any animal, including but not limited to humans and non-human animals selected from primates, mammals, rodents and vertebrates, such as camelids, llamas , proto-ostrich, alpaca, sheep, rabbit, mouse, rat or cartilaginous fishes (eg sharks).
  • Antibody herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies , conjugated antibody, chimeric antibody, humanized antibody or fully human antibody.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., except for possible variants (such as containing naturally occurring mutations or arising during the manufacture of a formulation, such variants typically appear as In addition to being present in small amounts), the individual antibodies comprising the population are identical and/or bind the same epitope. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), in monoclonal antibody preparations each monoclonal antibody is directed against a single determinant on the antigen.
  • monoclonal antibodies can be produced by a variety of techniques including, but not limited to, hybridoma technology, recombinant DNA methods, phage library display technology and the use of transgenic animals containing all or part of the human immunoglobulin loci methods and other methods known in the art.
  • the term "monospecific” herein refers to having one or more binding sites, wherein each binding site binds the same epitope of the same antigen.
  • multispecific herein refers to having at least two antigen binding sites, each of which is associated with a different epitope of the same antigen or with a different epitope of a different antigen. combined.
  • terms such as “bispecific”, “trispecific”, “tetraspecific” and the like refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.
  • valence herein refers to the presence of a defined number of binding sites in an antibody/antigen binding molecule. Accordingly, the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” denote one binding site, two binding sites, four binding sites and six binding sites in an antibody/antigen binding molecule, respectively. point of existence.
  • full-length antibody intact antibody
  • intact antibody intact antibody
  • Antigen-binding fragment and “antibody fragment” are used interchangeably herein, and do not possess the full structure of an intact antibody, but only include partial or partial variants of an intact antibody that possess the ability to bind Antigen capacity.
  • Antigen-binding fragment or “antibody fragment” herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab') 2 , Fd, Fv, scFv, diabody and single domain antibody.
  • Papain digestion of intact antibodies yields two identical antigen-binding fragments, termed "Fab” fragments, each containing the variable domains of the heavy and light chains, as well as the constant domain of the light chain and the first constant domain of the heavy chain (CH1 ).
  • Fab fragment herein refers to a light chain fragment comprising the VL domain and the constant domain (CL) of the light chain, and an antibody fragment comprising the VH domain and the first constant domain (CH1) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy-terminus of the CH1 domain of the heavy chain, including one or more cysteines from the antibody hinge region.
  • Fab'-SH is a Fab' fragment in which the cysteine residues of the constant domains bear a free thiol group. Pepsin treatment yields an F(ab') 2 fragment with two antigen-combining sites (two Fab fragments) and part of the Fc region.
  • Fd refers to an antibody consisting of VH and CH1 domains.
  • Fv refers to an antibody fragment consisting of a single-armed VL and VH domain.
  • the Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a variable region (such as the Fd fragment, which contains only three CDRs specific for an antigen) is able to recognize and bind antigen, although perhaps with a lower affinity than the full binding site.
  • scFv single-chain variable fragment
  • linker see, e.g., Bird et al., Science 242:423 -426 (1988); Huston et al, Proc. New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of the repeated GGGGS amino acid sequence or variants thereof.
  • a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur.J. Immunol.31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001 ), Cancer Immunol.
  • diabody herein, whose VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains of the same chain, thus forcing the domains to The complementary domains of the other chain pair and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2:1121-1123 (1994)).
  • naked antibody refers to an antibody that is not conjugated to a therapeutic agent or tracer
  • conjugated antibody refers to an antibody conjugated to a therapeutic agent or tracer
  • the therapeutic agent is selected from Drugs, toxins, radioisotopes, chemotherapeutics or immunomodulators
  • the tracer is selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers.
  • Chimeric antibody herein refers to an antibody whose light chain and/or heavy chain are partly derived from an antibody (which may be derived from a specific species or belong to a specific class or subclass of antibodies). class), and the other part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains the Binding activity to target antigen (U.S.P 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:68516855 (1984)).
  • chimeric antibody may include antibodies (e.g., human-mouse chimeric antibodies) in which the antibody's heavy and light chain variable regions are derived from a primary antibody (e.g., a murine antibody), and the antibody's heavy and light chains are The light chain constant region is from a second antibody (eg, a human antibody).
  • a primary antibody e.g., a murine antibody
  • the light chain constant region is from a second antibody (eg, a human antibody).
  • humanized antibody herein refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with a human antibody.
  • all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) is derived from a human Immunoglobulin (receptor antibody).
  • Humanized antibodies usually retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune response, etc.
  • Fully human antibody refers to antibodies having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region also is derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "fully human antibodies” herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.
  • another mammalian species eg, mouse
  • variable region herein refers to the region in the heavy or light chain of an antibody that is involved in making the antibody bind to an antigen
  • “heavy chain variable region” is used interchangeably with “VH” and “HCVR”
  • “light chain variable region” can be used interchangeably with “VL” and “LCVR”.
  • the variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies generally have similar structures, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). See, eg, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p.91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity.
  • complementarity determining region and “CDR” are used interchangeably herein, and generally refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or the light chain variable region (VL). It can form a precise complementarity with the antigen epitope, so it is also called complementarity determining region.
  • the CDR of the variable region of the heavy chain can be abbreviated as HCDR
  • the CDR of the variable region of the light chain can be abbreviated as LCDR.
  • framework region or “FR region” are used interchangeably and refer to those amino acid residues in an antibody heavy chain variable region or light chain variable region other than the CDRs.
  • CDR CDR
  • Kabat et al. J.Biol.Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest” (1991); People such as Chothia, J.Mol.Biol.196:901-917 (1987); People such as Al-Lazikani B., J.Mol.Biol., 273:927-948 (1997); People such as MacCallum, J.Mol .Biol.262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc M.P.
  • CDR herein can be marked and defined by methods known in the art, including but not limited to Kabat numbering system, Chothia numbering system or IMGT numbering system, and the tool websites used include but not limited to AbRSA website (http://cao.labshare.
  • CDRs herein include overlaps and subsets of amino acid residues defined in different ways.
  • Kabat numbering system herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
  • IMGT numbering system herein generally refers to the numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev.Comparat.Immunol. 27:55-77, 2003.
  • IMGT ImMunoGeneTics information system
  • heavy chain constant region herein refers to the carboxy-terminal portion of the heavy chain of an antibody that is not directly involved in binding the antibody to an antigen, but exhibits effector functions, such as interaction with Fc receptors, which are relative to the antibody's available Variable domains have more conserved amino acid sequences.
  • the "heavy chain constant region” at least includes: CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof.
  • Heavy chain constant region includes "full-length heavy chain constant region” and “heavy chain constant region fragment", the former has a structure substantially similar to that of a natural antibody constant region, while the latter only includes “full-length heavy chain constant region” part".
  • a typical "full-length antibody heavy chain constant region” consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain In the case of an antibody, it does not include a CH1 domain.
  • a typical "heavy chain constant region segment" can be selected from CH1, Fc or CH3 domains.
  • light chain constant region refers to the carboxy-terminal part of the antibody light chain, which is not directly involved in the binding of the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.
  • Fc refers to the carboxy-terminal part of the antibody obtained by papain hydrolysis of the whole antibody, which typically includes the CH3 and CH2 domains of the antibody.
  • Fc regions include, for example, native sequence Fc regions, recombinant Fc regions and variant Fc regions.
  • the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus.
  • the C-terminal lysine of the Fc region (residue 447 according to the Kabat numbering system) can be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody, thus the Fc region can comprise or excluding Lys447.
  • amino acid herein generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
  • amino acids in each of the following groups belong to each other's conservative amino acid residues, and the substitution of amino acid residues in the group belongs to the conservative amino acid substitution:
  • identity can be calculated by aligning the sequences for optimal comparison purposes in order to determine the percentage "identity" of two amino acid sequences or two nucleic acid sequences (for example, can be optimal alignment to introduce gaps in one or both of the first and second amino acid sequences or nucleic acid sequences or non-homologous sequences may be discarded for comparison purposes).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between two sequences will vary with the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to optimally align the two sequences.
  • the comparison of sequences and the calculation of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm in the GAP program that has been integrated into the GCG software package (available at www.gcg.com), using the Blossum 62 matrix or The PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6 determine the percent identity between two amino acid sequences.
  • the GAP program in the GCG software package (available at www.gcg.com), using the NWSgapdna.CMP matrix with gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5 or 6, determining the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • nucleic acid and protein sequences described herein may further be used as "query sequences" to perform searches against public databases, eg to identify other family member sequences or related sequences.
  • search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.
  • the default parameters of the respective programs eg, XBLAST and NBLAST
  • XBLAST and NBLAST can be used. See www.ncbi.nlm.nih.gov.
  • chimeric antigen receptor herein refers to an artificial cell surface receptor engineered to be expressed on immune effector cells and to specifically bind an antigen, comprising at least (1) an extracellular antigen-binding domain, such as an antibody The variable heavy or light chain, (2) the transmembrane domain that anchors the CAR into immune effector cells, and (3) the intracellular signaling domain.
  • CARs are able to redirect T cells and other immune effector cells to a target of choice, such as cancer cells, in a non-MHC-restricted manner using an extracellular antigen-binding domain.
  • Antagonistic antibodies targeting inhibitory immune checkpoints include anti-CTLA4 mAbs (such as ipilimumab, tremelimumab), anti-PD-1 (such as nivolumab BMS -936558/MDX-1106/ONO-4538, CT-011, lambrozilumab MK-3475, MEDI-0680 (AMP-514), PDR001, REGN2810,, BGB-108), anti-PDL-1 antagonists (such as BMS-936559 /MDX-1105, MEDI4736, RG-7446/MPDL3280A, MSB0010718C, YW243.55.S70); anti-LAG-3 (such as IMP-321), anti-TIM-3, anti-BTLA, anti-B7-H4, anti-B7-H3 , Anti-VISTA.
  • CTLA4 mAbs such as ipilimumab, tremelimumab
  • anti-PD-1 such as nivolu
  • Agonistic antibodies that enhance immunostimulatory proteins include anti-CD40 mAb (such as CP-870,893, lucatumumab, dacilizumab), anti-CD137 mAb (such as BMS-663513 urelumab, PF-05082566) , anti-OX40 mAb (such as anti-OX40), anti-GITR mAb (such as TRX518), anti-CD27 mAb (such as CDX-1127) and anti-ICOS mAb.
  • anti-CD40 mAb such as CP-870,893, lucatumumab, dacilizumab
  • anti-CD137 mAb such as BMS-663513 urelumab, PF-05082566
  • anti-OX40 mAb such as anti-OX40
  • anti-GITR mAb such as TRX5128
  • anti-CD27 mAb such as CDX-1127
  • anti-ICOS mAb anti-ICOS mAb
  • immunomodulatory drug herein may be, for example, thymosin al. Rationale: Thymosin alpha 1 (T ⁇ 1) is a naturally occurring thymosin peptide that acts as an endogenous regulator of the innate and adaptive immune systems. It is used worldwide to treat diseases associated with immune dysfunction, including viral infections such as hepatitis B and C, certain cancers, and for vaccine boosting. In particular, recent advances in immunomodulatory research point to a beneficial role of Ta1 treatment in septic patients (Wu et al. Critical Care 2013, 17:R8).
  • nucleic acid includes any compound and/or substance comprising a polymer of nucleotides.
  • Each nucleotide consists of a base, especially a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose) and phosphate groups.
  • cytosine C
  • G guanine
  • A adenine
  • T thymine
  • U uracil
  • nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule.
  • the sequence of bases is usually expressed 5' to 3'.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including for example complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, and synthetic forms of DNA or RNA comprising both Mixed polymers of one or more of these molecules.
  • Nucleic acid molecules can be linear or circular.
  • nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms.
  • nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides.
  • Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of antibodies of the invention in vitro and/or in vivo, for example in a host or patient.
  • DNA eg cDNA
  • RNA eg mRNA
  • Such DNA (eg cDNA) or RNA (eg mRNA) vectors may be unmodified or modified.
  • mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to generate antibodies in vivo (see e.g.
  • An "isolated" nucleic acid herein refers to a nucleic acid molecule that has been separated from components of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.
  • vector refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it has been linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
  • host cell herein refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical to the parental cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • the term "pharmaceutical composition” refers to a preparation that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain substances that are unacceptably toxic to the subject to which the pharmaceutical composition is administered. additional ingredients.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) an undesired physiological change or pathology, such as the progression of cancer, in the subject being treated.
  • beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, diminished extent of disease, stable disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of disease state, and remission (whether partial response or complete response), whether detectable or undetectable.
  • Those in need of treatment include those already with the condition or disease as well as those prone to have the condition or disease or those in which the condition or disease is to be prevented.
  • slow down lessen, weaken, moderate, alleviate, etc., the meaning of eliminate, disappear, not occur, etc. is also included.
  • subject refers to an organism receiving treatment for a particular disease or condition as described herein.
  • subjects and patients include mammals, such as humans, primate (e.g., monkeys) or non-primate mammals, receiving treatment for a disease or disorder.
  • an effective amount herein refers to an amount of a therapeutic agent effective to prevent or alleviate a disease condition or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue or subject.
  • Effective amount also refers to an amount of a compound sufficient to relieve symptoms, eg, treat, cure, prevent or alleviate the associated medical condition, or to increase the rate of treatment, cure, prevent or alleviate such condition.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially or simultaneously.
  • cancer refers to or describes the physiological condition in mammals typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition.
  • tumor or “neoplastic” herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.
  • cancer and “tumor” are not mutually exclusive when referred to herein.
  • EC50 refers to the half-maximal effective concentration, which includes the concentration of antibody that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the 50% concentration of antibody at which a maximal effect is observed and can be measured by methods known in the art.
  • G4S linking peptide herein refers to a GS combination of glycine (G) and serine (S), used to link multiple proteins together to form a fusion protein.
  • G glycine
  • S serine
  • the commonly used GS combination is (GGGGS)n, and the length of the linker sequence can be changed by changing the size of n.
  • glycine and serine can also be combined to generate different linker sequences, such as the (G4S)4Linker used in the present invention, and the combination of GS is GGGGS.
  • Figure 1 shows the binding activity of anti-TIGIT human-mouse chimeric antibody to human TIGIT ECD-mFc fusion protein.
  • Figure 2 shows the binding activity of anti-TIGIT human-mouse chimeric antibody to cynomolgus TIGIT ECD-mFc fusion protein.
  • Figure 3 shows the binding activity of anti-TIGIT human-mouse chimeric antibody to CHO-K1 human TIGIT high-expressing strain cells.
  • Figure 4 shows the binding activity of anti-TIGIT human-mouse chimeric antibody to cells expressing CHO-K1 human TIGIT.
  • Figure 5 shows the binding activity of anti-TIGIT human-mouse chimeric antibody to CHO-K1 human TIGIT low-expressing strain cells.
  • Fig. 6 shows the binding activity of anti-TIGIT human-mouse chimeric antibody to CHO-K1 cynomolgus monkey TIGIT cells.
  • Figure 7 shows the effect of anti-TIGIT human-mouse chimeric antibody blocking the interaction between Bio-CD155-His and CHO-K1 TIGIT.
  • Figure 8 shows the effect of anti-TIGIT human-mouse chimeric antibody blocking the interaction between TIGIT ECD-mFc and CHO-K1 CD155.
  • Figure 9 FACS detection of the expression levels of PVRIG, TIGIT on the surface of NK cells from different donor sources (donor-010 and donor-050), and the expression levels of PVR and PVRL2 on the surface of the tumor cell line WIDR.
  • A The black hollow peaks in the figure refer to the expression of PVRIG/TIGIT on the surface of NK cells, and the gray solid peaks refer to the isotype controls corresponding to the two detection antibodies.
  • B The black hollow peaks in the figure refer to the expression of PVR/PVRL2 on the surface of WIDR cells, and the gray solid peaks refer to the isotype controls corresponding to the two detection antibodies.
  • Figure 10 shows the effect of co-incubation of anti-TIGIT human-mouse chimeric antibody with WIDR cells and NK cells on NK cell degranulation (CD107a).
  • the abscissa is the concentration of the tested antibody, and the ordinate is the percentage of positive cells for CD107a.
  • RG6058-hIgG1 is a positive control antibody
  • anti-HEL-hIgG1 is a negative isotype control antibody.
  • Figure 11 shows the effect of anti-TIGIT human-mouse chimeric antibody on NK cells killing WIDR target cells.
  • the abscissa is the concentration of the tested antibody, the ordinate is the death rate of the target cells, RG6058-hIgG1 is the positive control antibody, and anti-HEL-hIgG1 is the negative isotype control antibody.
  • CMV antigen-recall assay detects the effect of anti-TIGIT human-mouse chimeric antibody on the functional activity of antigen-specific CD8 T cells.
  • A CMV IgG-positive donor 128 PBMCs were induced by CMV pp65 (495-503) for 11 days, the proportion of CD8 T cells in PBMCs, the proportion of CMV pp65-specific CD8 T cells and FMO control;
  • B PVRIG and TIGIT on CMV pp65-specific CD8 and PD-1 expression;
  • C the expression of PVRL2 and PVR on Colo205;
  • D the secretion level of IFN- ⁇ in the cell supernatant after co-incubation for 18 hours.
  • the positive control is RG6058-hIgG1, and the negative control is no treatment (without any drug treatment).
  • the percentage on the histogram is the percentage of IFN- ⁇ secretion increase of the tested antibody compared with the no treatment group.
  • Figure 13 shows the binding activity of anti-TIGIT humanized antibody to human TIGIT ECD-mFc fusion protein.
  • Figure 14 shows the binding activity of the anti-TIGIT humanized antibody to the cynomolgus monkey TIGIT ECD-mFc fusion protein.
  • Figure 15 shows the binding activity of the humanized anti-TIGIT antibody to CHO-K1 human TIGIT high-expressing strain cells.
  • Figure 16 shows the binding activity of the humanized anti-TIGIT antibody to cells expressing CHO-K1 human TIGIT.
  • Figure 17 shows the binding activity of the humanized anti-TIGIT antibody to CHO-K1 human TIGIT low-expressing strain cells.
  • Fig. 18 shows the binding activity of anti-TIGIT humanized antibody to CHO-K1 cynomolgus TIGIT cells.
  • Figure 19 shows the effect of humanized anti-TIGIT antibodies blocking the interaction of Bio-CD155-His with CHO-K1 human TIGIT.
  • Figure 20 shows the effect of humanized antibodies against TIGIT blocking the interaction of TIGIT ECD-mFc with CHO-K1 CD155.
  • Figure 21 shows the effect of anti-TIGIT humanized antibodies blocking the interaction of TIGIT ECD-mFc with CHO-K1 CD112.
  • Fig. 22 shows the binding activity of anti-TIGIT humanized antibody to human PBMC.
  • FIG. 23 shows the effect of anti-TIGIT humanized antibody on NK cells killing WIDR target cells.
  • the abscissa in the figure is the concentration of the tested antibody, and the ordinate is the death rate of the target cells.
  • TIGIT-CHI-002, TIGIT-CHI-005, TIGIT-CHI-006 and TIGIT-CHI-070 are the chimeric cells before humanization Antibody, RG6058-hIgG1 is positive control antibody, anti-HA HcAb-hIgG1 is negative isotype control antibody.
  • CMV antigen-recall assay detects the effect of anti-TIGIT humanized antibody on the functional activity of antigen-specific CD8 T cells.
  • A CMV IgG-positive donor 622 PBMCs were induced by CMV pp65(495-503) for 11 days, the proportion of CD8 T cells in PBMCs, the proportion of CMV pp65-specific CD8 T cells, FMO control;
  • B CMV pp65-specific CD8 T cells The expression of PVRIG, TIGIT, PD-1, CD226;
  • C the secretion level of IFN- ⁇ in the cell supernatant after plating for 18 hours.
  • Positive controls are RG6058-hIgG1 and TIGIT-CHI-002, and negative controls are no treatment (without any drug treatment).
  • the percentage on the bar graph is the percentage of IFN- ⁇ secretion increase of the tested antibody compared with the no treatment group.
  • FIG. 25 FACS detection of human TIGIT overexpression cell line CHO-K1 human TIGIT
  • Fig. 26A The binding ability of PVRIG test antibody and human PVRIG recombinant protein.
  • the figure shows the binding ability of the tested antibodies PVRIG-A11, A15, A30, A35, A43, A50, A60, A75, A104, A105, A113, A117, A118 to human PVRIG protein.
  • COM701-hIgG1, COM701-hIgG4 and SRF813-hIgG1 are the positive controls of this experiment; anti-HA HcAb-hIgG1 and anti-CD38 HcAb-hIgG1 are the negative controls of this experiment.
  • FIG. 26B The binding ability of PVRIG test antibody and cynomolgus monkey PVRIG recombinant protein.
  • the figure shows the binding ability of the tested antibodies PVRIG-A11, A15, A30, A35, A43, A50, A60, A75, A104, A105, A113, A117, A118 to the cynomolgus monkey PVRIG protein.
  • COM701-hIgG1, COM701-hIgG4 and SRF813-hIgG1 are the positive controls of this experiment; anti-HA HcAb-hIgG1, anti-CD38 HcAb-hIgG1 and anti-Fluorescein-hIgG1 are the negative controls of this experiment.
  • Fig. 27A The binding activity of PVRIG test antibody to human PVRIG on the surface of Flpin CHO-PVRIG cells.
  • the figure shows the binding ability of the tested antibodies PVRIG-A11, A15, A30, A35, A43, A50, A60, A75, A104, A105, A113, A117, A118 to human PVRIG on the surface of FlpinCHO-PVRIG cells.
  • COM701-hIgG1 and SRF813-hIgG1 are positive control antibodies
  • anti-CD38 HcAb-hIgG1 is a negative isotype control antibody.
  • Fig. 27B The binding activity of PVRIG test antibody to FlpinCHO-cyno PVRIG cell surface cynomolgus PVRIG.
  • the figure shows the binding ability of the tested antibodies PVRIG-A11, A15, A30, A50, A60, A105, A117, A118 to FlpinCHO-cyno PVRIG cell surface cynomolgus PVRIG.
  • COM701-hIgG1 and SRF813-hIgG1 are positive control antibodies
  • anti-CD38 HcAb-hIgG1 is a negative isotype control antibody.
  • FIG. 28 PVRIG test antibody blocks the interaction between human PVRIG and human PVRL2 recombinant protein.
  • the figure shows the binding blocking effect of tested antibodies PVRIG-A11, A15, A30, A35, A43, A50, A60, A75, A104, A105, A113, A117, A118 on PVRIG and PVRL2, in which COM701-hIgG4 and SRF813 -hIgG1 is the positive control of this experiment; anti-HA HcAb-hIgG1 and anti-CD38 HcAb-hIgG1 are the negative controls of this experiment.
  • FIG. 29 PVRIG test antibody blocks the binding of CHO-K1-CD112 cells to human PVRIG-mFc protein.
  • the figure shows that the tested antibodies PVRIG-A11, A15, A30, A35, A43, A50, A60, A75, A104, A105, A113, A117, A118 compete for the ability of CHO-K1-CD112 cells to bind to human PVRIG-mFc protein.
  • COM701-hIgG4 and SRF813-hIgG1 are positive control antibodies
  • anti-CD38 HcAb-hIgG1 is negative isotype control antibody.
  • Figure 30 The expression levels of PVR and PVRL2 on the cell surface of the tumor cell line Reh.
  • the black hollow peak in the figure refers to the expression of PVR/PVRL2 on the surface of Reh cells, and the gray solid peak represents the isotype control corresponding to the detection antibody.
  • Figure 31 Effect of PVRIG test antibody on NK cell degranulation.
  • Figure A refers to the effect of tested antibodies PVRIG-A11, A15, A30 on the expression of CD107a in NK cells when NK cells (donor-010) were incubated with target cells Reh;
  • Figure B refers to NK (donor-010) and target cells WIDR During incubation, the effects of tested antibodies PVRIG-A60, A75, A43, A35 on the expression of NK cells CD107a;
  • Figure C refers to the effects of tested antibodies PVRIG-A104, A105, Effects of A118, A113, and A117 on the expression of CD107a in NK cells; the abscissa is the concentration of the tested antibody, and the ordinate is the proportion of strong positive cells for CD107a.
  • COM701-hIgG1 and SRF813-hIgG1 are positive control antibodies
  • anti-HA HcAb - hIgG1 is a negative is
  • Figure 32 The effect of PVRIG test antibody on the killing effect of NK cells on target cells.
  • Panel A refers to the promoting effect of the tested antibodies PVRIG-A11, A15, and A30 on NK cells (donor-010) killing target cell WIDR at a concentration of 6.87nM.
  • each test antibody has statistical difference (**p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001, One-Way ANOVA Analysis); Fig.
  • B refers to the promoting effect of the tested antibody PVRIG-A50 on NK cells (donor-010) killing target cell WIDR at different concentrations; A43, A104, A105, A113, A117, A118 promote NK cells (donor-050) killing target cell WIDR.
  • the abscissa of Figures B and C is the concentration of the tested antibody, and the ordinate is the death rate of the target cells.
  • COM701-hIgG1 and SRF813-hIgG1 are positive control antibodies, and anti-HA HcAb-hIgG1 is a negative isotype control antibody.
  • FIG. 33 CMV antigen-recall assay detects the function improvement effect of PVRIG antibody on antigen-specific CD8 T cells.
  • Panel A CMV IgG-positive donor 021 PBMCs were induced by CMV pp65 (495-503) for 11 days, and the ratio of CD8 in PBMCs, CMV pp65-specific CD8 ratio;
  • Panel B CMV pp65-specific CD8 (donor 021) on PVRIG, TIGIT , expression of PD-1, CD226;
  • Panel C expression of PVRL2, PVR and HLA-A2 on colo205.
  • Panel D IFN- ⁇ secretion levels in cell supernatants after plating and co-incubation for 18 hours.
  • the positive control in this experimental system is COM701-hIgG4, SRF813-hIgG1, the negative control is no treatment (without any drug treatment), and the final concentration of the antibody is 70nM.
  • the secretion of IFN- ⁇ in the cell supernatant was significantly increased (*p ⁇ 0.05,**p ⁇ 0.01,***p ⁇ 0.001, ****p ⁇ 0.0001, one-way ANOVA Analysis).
  • Fig. 34A The binding ability of PVRIG humanized antibody to human PVRIG recombinant protein.
  • the figure shows the binding activity of the tested humanized molecule and its parental antibodies PVRIG-A50, A105, A118 to human PVRIG protein; among them, anti-HA HcAb-hIgG1, anti-CD38 HcAb-hIgG1 and anti-Fluorescein-hIgG1 are Negative control for this experiment.
  • Fig. 34B The binding ability of PVRIG humanized antibody to cynomolgus monkey PVRIG recombinant protein.
  • the figure shows the binding activity of the tested humanized molecule and its parental antibodies PVRIG-A50, A105, A118 to the cynomolgus monkey PVRIG protein; among them, anti-HA HcAb-hIgG1, anti-CD38 HcAb-hIgG1 and anti-Fluorescein- hIgG1 was the negative control for this experiment.
  • Fig. 35A The binding activity of PVRIG humanized antibody to human PVRIG on the surface of FlpinCHO-PVRIG cells.
  • the figure shows the binding ability of the tested humanized molecule and its control parental antibodies PVRIG-A50, A105, A118 to human PVRIG on the surface of FlpinCHO-PVRIG cells.
  • COM701-hIgG1 and SRF813-hIgG1 are positive control antibodies
  • anti-CD38 HcAb-hIgG1 and anti-Fluorescein-hIgG1 are negative isotype control antibodies.
  • FIG. 35B Binding of PVRIG humanized antibody to FlpinCHO-cyno PVRIG cell surface cynomolgus PVRIG.
  • the figure shows the binding ability of the tested humanized molecule and its control parental antibodies PVRIG-A50, A105, A118 and FlpinCHO-cyno PVRIG cell surface cynomolgus monkey PVRIG.
  • COM701-hIgG1 and SRF813-hIgG1 are positive control antibodies
  • anti-CD38 HcAb-hIgG1 and anti-Fluorescein-hIgG1 are negative isotype control antibodies.
  • FIG. 36 PVRIG humanized antibody blocks the binding of human PVRIG and human PVRL2.
  • the figure shows the blocking effect of the tested humanized molecule and its parental antibodies PVRIG-A50, A105, A118 on the binding of human PVRIG protein and human PVRL2 protein; anti-HA HcAb-hIgG1 and anti-Fluorescein-hIgG1 are the test negative control.
  • FIG. 37 PVRIG humanized antibody blocks the binding of CHO-K1-CD112 cells to human PVRIG-mFc protein.
  • the figure shows the ability of the tested humanized molecule and its control parental antibodies PVRIG-A50, A105, A118 to compete with CHO-K1-CD112 cells for binding to human PVRIG-mFc protein.
  • COM701-hIgG4 and SRF813-hIgG1 are positive control antibodies
  • anti-CD38 HcAb-hIgG1 and anti-Fluorescein-hIgG1 are negative isotype control antibodies.
  • Figure 38 Effect of PVRIG humanized antibody on NK cell killing.
  • the test humanized molecule and its control parental antibody PVRIG-A50 (A), A118 (B), A105 (C) promote NK (donor-050) killing target cell WIDR.
  • the abscissa in the figure is the concentration of the tested antibody, and the ordinate is the death rate of the target cells.
  • COM701-hIgG1 and SRF813-hIgG1 are positive control antibodies, and anti-HA HcAb-hIgG1 and anti-Fluorescein-hIgG1 are negative isotype control antibodies.
  • FIG. 39 CMV antigen-recall assay detects the function improvement effect of PVRIG humanized antibody on antigen-specific CD8 T cells.
  • the positive control in this experimental system is the PVRIG parental antibody before humanization, the negative control is no treatment (without any drug treatment), and the final concentration of the antibody is 70nM.
  • PVRIG-A50-H1a, H1b, H1d, H2a The four humanized antibodies of PVRIG-A50 (PVRIG-A50-H1a, H1b, H1d, H2a) had no significant difference in effect compared with PVRIG-A50 (One-way ANOVA Analysis); the two humanized antibodies of PVRIG-A118 (PVRIG-A118-H3, H5) had no significant difference with PVRIG-A118; the effect of PVRIG-A105-H2 was significantly weaker than that of PVRIG-A105 (*p ⁇ 0.05, one-way ANOVA Analysis), and the remaining 2 human sources Antibodies (PVRIG-A105-H1, H3) had no significant difference with PVRIG-A105 (One-way ANOVA Analysis).
  • FIG. 40 Schematic diagram of the composition and structure of four humanized bispecific antibodies
  • Figure 44 ELISA detection of the binding of four humanized double antibodies to cynomolgus monkey TIGIT-ECD-mFc protein
  • Figure 47 FACS detection of binding activity of four humanized double antibodies to CHO-K1-human TIGIT high expression cell line
  • Figure 48 FACS detection of binding activity of four humanized double antibodies to cell lines expressing in CHO-K1-human TIGIT
  • Figure 49 FACS detection of binding activity of four humanized double antibodies to CHO-K1-human TIGIT low expression cell line
  • Figure 50 FACS detection of binding activity of four humanized double antibodies to CHO-K1-cynomolgus monkey TIGIT cell line
  • Figure 52 FACS detection of four humanized double antibodies blocking the binding activity of PVRIG-ECD-mFc and CHO-K1-CD112
  • Figure 53 ELISA detection of four humanized double antibodies blocking the binding activity of TIGIT-ECD-mFc and CHO-K1-CD155
  • Figure 54 FACS detection of four humanized double antibodies blocking the binding activity of Bio-CD155-His protein to CHO-K1 human TIGIT
  • Figure 55 FACS detection of binding activity of four humanized double antibodies to human PBMC
  • Figure 56 BIAcore detection of co-binding of humanized bispecific antibody with PVRIG and TIGIT. Binding curves of LC-BsAb-002 humanized double antibody (A) and LC-BsAb-006 humanized double antibody (B) to human TIGIT protein and human PVRIG protein, and the antibodies of human TIGIT and human PVRIG protein were continuously injected respectively Antigen binding curve, black triangle represents the time point of corresponding protein injection.
  • NK cell degranulation assay to detect the function-promoting effect of anti-PVRIGxTIGIT humanized bispecific antibody on NK cells.
  • NK cell degranulation experiment process A
  • FACS detection of NK cell PVRIG, TIGIT and WIDR cell PVR, PVRL2 expression levels B
  • humanized bispecific antibodies LC-BsAb-002 and LC-BsAb-006 for NK
  • CD107a expression on cells EC50, AUC, WIDR of target cells, C
  • C the effect of humanized bispecific antibody LC-BsAb-002 on the expression of CD107a in NK cells
  • TF-1 of target cells D
  • Figure 58 NK cell killing assay to detect the killing effect of NK cells on WIDR cells mediated by anti-PVRIGxTIGIT humanized bispecific antibody.
  • Expression levels of PVRIG and TIGIT in NK cells from different donors (Donor-050, 831, 715) (A), expression levels of PVR and PVRL2 on WIDR of target cells (B), NK cytotoxicity experiment procedure (C), LC-BsAb -002 and LC-BsAb-006 on NK cell-mediated killing of WIDR cells from three different donors (EC50, AUC, D), and LC-BsAb-002 on NK cell-mediated killing of TF-1 cells (E )
  • NK cell ADCC assay to detect the direct killing effect on human Treg cells mediated by anti-PVRIGxTIGIT humanized bispecific antibody.
  • NK cell-mediated ADCC killing experiment process A
  • expression levels of PVRIG and TIGIT in human Treg cells B
  • comparison of ADCC killing of human Treg by LC-BsAb-002 and LC-BsAb-006 with different IgG Fc EC50, AUC, C
  • Figure 60 ADCP activity on human Treg cells mediated by anti-PVRIGxTIGIT humanized bispecific antibody.
  • CMV antigen-recall assay detects the function promoting effect of anti-PVRIGxTIGIT humanized bispecific antibody on antigen-specific CD8 T cells.
  • CMV antigen-recall experimental procedure A
  • the control in this experimental system is anti-TIGIT antibody: RG6058, TIGIT-002-H4L3, TIGIT-005-H2L1d, anti-PVRIG antibody: COM701, PVRIG-A50-H1b, negative control is no treatment (without any drug treatment) ( C), the secretion level of IFN- ⁇ in the cell supernatant after the humanized double antibody, single-drug combination and its combination with anti-PD-L1 antibody were co-incubated for 18 hours.
  • control in this experimental system is anti-TIGIT antibody: RG6058, TIGIT-002-H4L3, TIGIT-005-H2L1d, anti-PVRIG antibody: COM701, PVRIG-A50-H1b, anti-PD-L1 antibody: Tecentriq, negative control is no treatment (without any drug treatment) (D)
  • Figure 62 CMV antigen-recall assay detects the function promoting effect of anti-PVRIG ⁇ TIGIT humanized bispecific antibody combined with Tecentriq on antigen-specific CD8 T cells.
  • the control in this experimental system is anti-TIGIT antibody: RG6058, anti-PVRIG antibody: COM701, anti-PD-L1 antibody: Tecentriq(B)
  • Figure 63 Tumor growth curves of each test group in A375 hPBMC humanized animal model
  • Figure 64 The tumor growth curves of individual mice in each test group in the A375 hPBMC humanized animal model
  • Figure 65 Changes in body weight of mice after administration in each test group in the A375 hPBMC humanized animal model
  • Figure 66 Tumor growth curves of different doses of bispecific antibody alone and in combination with Tecentriq in the A375 hPBMC humanized animal model
  • Figure 67 The tumor growth curves of individual mice in each test group in different doses of bispecific antibody alone and in combination with Tecentriq in the A375 hPBMC humanized animal model
  • Figure 68 Changes in body weight of mice in each test group after administration of different doses of bispecific antibody alone and in combination with Tecentriq in the A375 hPBMC humanized animal model
  • TIGIT protein (NCBI sequence number: XP_024309156.1) and cynomolgus monkey TIGIT protein (NCBI: XP_015300911.1) as the templates of TIGIT in this disclosure, design the amino acid sequences of antigens and detection proteins involved in this disclosure, optional Different tags were fused on the basis of the TIGIT protein, respectively cloned into the PTT5 vector (Invitrogen), transiently expressed in 293 cells or stably expressed and purified in CHO cells, and the proteins encoding the disclosed antigens and detection proteins were obtained.
  • PTT5 vector Invitrogen
  • Fusion protein of human TIGIT extracellular region and mouse IgG2a Fc fragment Human TIGIT ECD-mFc, for detection.
  • the underlined part is the signal peptide, and the italic part is mFc.
  • Fusion protein of human TIGIT extracellular region and human IgG1 Fc segment human TIGIT ECD-hFc, used for detection.
  • the underlined part is the signal peptide, and the italic part is Fc.
  • Fusion protein of human TIGIT extracellular domain and His tag human TIGIT ECD-his, for detection.
  • the underlined part is the signal peptide, and the italic part is the His tag.
  • Fusion protein of cynomolgus monkey TIGIT extracellular region and mouse IgG2a Fc segment cynomolgus monkey TIGIT ECD-mFc, used for detection.
  • the underlined part is the signal peptide, and the italic part is mFc.
  • Fusion protein of cynomolgus monkey TIGIT extracellular region and human IgG1 Fc segment cynomolgus monkey TIGIT ECD-hFc, used for detection.
  • the underlined part is the signal peptide, and the italic part is hFc
  • Plasmid transfection was carried out on the CHO-K1 cell line (purchased from ATCC) ( 3000 Transfection Kit (purchased from Invitrogen, Cat. No.: L3000-015), cultured selectively in DMEM/F12 medium containing 10% (w/w) fetal bovine serum containing 10 ⁇ g/ml puromycin for 2 weeks, and then spread monoclonal The cells were placed in a 96-well plate and cultured at 37° C. with 5% (v/v) CO 2 . After about 2 weeks, some monoclonal wells were selected for expansion. The amplified clones were screened by flow cytometry. Cell lines with better growth, higher fluorescence intensity, and monoclonality were selected to continue to be expanded and cultured and frozen in liquid nitrogen.
  • Full-length human TIGIT (NCBI: XP_024309156.1): used to construct the human TIGIT overexpression cell line CHO-K1 human TIGIT, through FACS detection, the high expression strain 1D2, the medium expression strain 2D10 and the low expression strain 1C6 were selected ( Figure 25 )
  • cynomolgus TIGIT (NCBI serial number: XP_015300911.1): used to construct the cynomolgus TIGIT overexpression cell line CHO-K1 cynomolgus TIGIT
  • Full-length human CD155 (NCBI serial number: NP_006496.4): used to construct the human CD155 overexpression cell line CHO-K1CD155.
  • CD112 Full-length human CD112 (NCBI sequence number: NP_001036189.1/NCBI RefSeq: Q92692): used to construct CD112 overexpression cell line CHO-K1 CD112
  • the anti-human TIGIT antibody is obtained by hybridoma technology, the human TIGIT-ECD-mFc fusion protein is used to immunize mice, the splenocytes of the immunized mice are isolated, and the cells are fused with mouse myeloma cells by electrofusion, cultured in HAT selective medium, Take the culture supernatant for identification, select the clone that secretes the target antibody for subcloning, and finally obtain the mouse monoclonal antibody through production and purification.
  • the detailed description is as follows:
  • mice Female, 6-8 weeks old (Shanghai Slack Experimental Animal Co., Ltd., animal production license number: SCXK (Shanghai) 2017-0005). Breeding environment: SPF grade. After the mice were purchased, they were kept in the experimental animal room (Heyuan Biotechnology (Shanghai) Co., Ltd.) for 1 week, with 12/12 hour light/dark cycle adjustment, temperature 20-25°C; humidity 40°C. -60%. The acclimatized mice were immunized according to the following scheme.
  • Human TIGIT-ECD (Acro Cat No. TIT-H5253) with mFc tag was used for immunization.
  • Immunization regimen with Gold Adjuvant (Sigma Cat No.T2684) and Thermo Alum (Thermo Cat No.77161) adjuvant cross-immunization.
  • Gold Adjuvant Gold Adjuvant
  • Vaccination was carried out after antigen emulsification, at the 1st, 8th, 15th, 22nd, 29th, 36th, 43rd, and 50th.
  • the emulsified antigen was injected intraperitoneally (IP) at 50 ⁇ g/mouse.
  • IP intraperitoneally
  • SC multiple points
  • the intraperitoneal injection and the subcutaneous injection were alternately performed every other week, blood was collected on the 20th, 27th, 34th, and 48th day, and the antibody Titer of the mouse serum was determined by ELISA method.
  • the spleen and lymph node cells of the mice with high antibody Titer in the serum were selected for fusion, and 3 days before the fusion, shock immunization was performed, and 50 ⁇ g/mouse of the antigen prepared by normal saline was injected intraperitoneally (IP) solution.
  • IP intraperitoneally
  • Spleen and lymph node cells were fused with myeloma SP2/0 cells ( CRL-1581) were fused to obtain hybridoma cells.
  • the fused hybridoma cells were used at a density of 5 ⁇ 10 5 /ml with complete medium containing 20% FBS (Excell Cat No.FND500), 1 ⁇ HAT (Sigma Cat No.H0262-10VL, DMEM of 1 ⁇ NEAA (Gibco Cat No.10569044) medium resuspended, 100 ⁇ L/well was inoculated in a 96-well flat bottom plate, incubated at 37°C, 5% CO 2 for 6-7 days, removed the supernatant, and added 200 ⁇ l of HT complete medium containing 10% per well FBS, 1 ⁇ HT (Sigma Cat No.H0137-10VL), 1 ⁇ NEAA in DMEM medium, 37 ° C, 5% CO 2 After culturing overnight, ELISA detection was performed.
  • the 116 monoclonal antibodies obtained above were identified by ELISA, FACS, BIAcore, etc., and 13 candidate antibodies were obtained, namely TIGIT-F2-002, TIGIT-F2-005, TIGIT-F2-006, TIGIT-F2-011 , TIGIT-F3-034, TIGIT-F4-044, TIGIT-F5-057, TIGIT-F5-067, TIGIT-F5-070, TIGIT-F5-072, TIGIT-F6-084, TIGIT-F6-088, TIGIT -F6-104.
  • the experimental results show that, compared with the control anti-TIGIT antibody Roche RG6058, the anti-TIGIT mouse monoclonal antibody can effectively bind human TIGIT ECD-hFc and cynomolgus monkey TIGIT ECD-hFc.
  • amino acid sequence corresponding to RG6058 is as follows:
  • Collect the cells adjust the concentration to 5 ⁇ 10 5 /mL with 10% FBS-DMEM/F12 medium (Excell, CAT#FSP500; Gibco, CAT#11330), add to 96-well cell culture plate (corning, CAT#3599), 100 ⁇ L/well, culture overnight at 37°C with 5% CO 2 , throw off the culture supernatant, add cell fixation solution (Beiyuntian, CAT#P0098), 50 ⁇ L/well, fix at room temperature for 1 hour, wash with 0.05% Tween20- Wash once with PBS, add 5% skimmed milk powder-PBS, 250 ⁇ L/well, incubate at 37°C for 2-4 hours, wash three times with 0.05% Tween20-PBS on a plate washer, add purified mouse monoclonal antibody diluent with 1% BSA Dilute to 13nM, serially dilute 12 concentration points by 3 times, 50 ⁇ L/well, incubate at 37°C for 1.5-2 hours, wash
  • the experimental results show that compared with the control anti-TIGIT antibody Roche RG6058, the purified mouse anti-TIGIT monoclonal antibody can effectively bind to the CHO-K1 human TIGIT high-expressing cell line, the CHO-K1 human TIGIT medium-expressing cell line, and the CHO-K1 human TIGIT low-expressing cell line.
  • Collect cells wash once with PBS (Hyclone, CAT#SH30256), resuspend to 2 ⁇ 10 5 /40 ⁇ L in 1% BSA-PBS, dilute antibody to 210 nM with 1% BSA-PBS, serially dilute 12 concentration points by 3 times, 1 Dilute Bio-CD155-His (Shenzhou, 10109-H08H) with %BSA-PBS to 3 ⁇ g/mL, then mix 40 ⁇ L of cells with 40 ⁇ L of antibody diluent and 40 ⁇ L of Bio-CD155-His diluent, and incubate at 4°C for 60 minutes.
  • PBS Hyclone, CAT#SH30256
  • Collect CHO-K1 CD155 cells adjust the concentration to 5 ⁇ 10 5 /mL with 10% FBS-DMEM/F12 medium (Excell, CAT#FSP500; Gibco, CAT#11330), add to 96-well cell culture plate (corning, CAT #3599), 100 ⁇ L/well, culture overnight at 37°C in 5% CO 2 , throw off the culture supernatant, add cell fixative (Beyond, CAT#P0098), 50 ⁇ L/well, fix at room temperature for 1 hour, use on a plate washer Wash once with 0.05% Tween20-PBS, add 5% skimmed milk powder-PBS, 250 ⁇ L/well, incubate at 37°C for 2-4 hours, wash three times with 0.05% Tween20-PBS on a plate washer; mix mouse monoclonal antibody with human TIGIT ECD-hFc (working concentration 30ng/mL) was mixed and incubated for half an hour; then the antigen-antibody mixture was added to
  • Table 1 below shows that all 13 antibodies exhibited good TIGIT binding ability and the effect of blocking the interaction between TIGIT and CD155, and reached or exceeded the anti-TIGIT control antibody Roche RG6058.
  • TIGIT-CHI-002 The above murine antibodies were identified by constructing human-mouse chimeric antibodies, and four chimeric antibodies were identified: TIGIT-CHI-002, TIGIT-CHI-005, TIGIT-CHI-006 and TIGIT-CHI-070.
  • Table 2 shows the VH/VL sequences of the chimeric antibodies
  • Table 3 shows the KABAT analysis results of the chimeric antibodies
  • Table 4 shows the IMGT analysis results of the chimeric antibodies.
  • Collect cells wash once with PBS (Hyclone, CAT#SH30256), resuspend to 2 ⁇ 10 5 /40 ⁇ L in 1% BSA-PBS, dilute antibody to 210 nM with 1% BSA-PBS, serially dilute 12 concentration points by 3 times, 1 Dilute Bio-CD155-His (Shenzhou, 10109-H08H) with %BSA-PBS to 3 ⁇ g/mL, then mix 40 ⁇ L of cells with 40 ⁇ L of antibody diluent and 40 ⁇ L of Bio-CD155-His diluent, and incubate at 4°C for 60 minutes.
  • PBS Hyclone, CAT#SH30256
  • Collect CHO-K1 CD155 cells wash once with PBS (Hyclone, CAT#SH30256), resuspend to 2 ⁇ 10 5 /40 ⁇ L with 1% BSA-PBS, dilute the antibody to be tested to 600nM with 1% BSA-PBS, and serially dilute 2.5 times 12 concentration points, dilute TIGIT ECD-mFc to 6 ⁇ g/mL with 1% BSA-PBS, then mix 40 ⁇ L cells with 40 ⁇ L antibody diluent and 40 ⁇ L TIGIT ECD-mFc diluent, incubate at 4°C for 60 minutes, wash twice with PBS, Join Alexa 647 fluorescein-labeled secondary antibody (working dilution 1:800, Jackson, CAT#115-605-003), resuspended cells in 100 ⁇ L/well, incubated at 4°C for 40 minutes, washed twice with PBS, and washed with 1% BSA-PBS , res
  • Table 5 shows a summary of TIGIT chimeric antibody characterization.
  • NK cells Natural killer cells
  • FACS Fluorescence Activated Cell Sorting
  • the expression of PVR and PVRL2 on WIDR cells was detected by FACS.
  • the WIDR cells were digested with trypsin to form a cell suspension, and the cells were counted using a cell counter (Beckman Coulter, Vi-CELL). Three flow tubes were taken, and 1E+5 cells were added to each flow tube. Cells were washed twice with PBS. After centrifugation, remove the supernatant, take one tube and add 300ul Staining buffer (PBS+2%FBS) as an unstained tube, and add 100ul staining solution (PBS+1*Zombie Violet (Biolegend, 423114) to each of the other two tubes ) and incubate at room temperature for 15 minutes.
  • PBS+2%FBS 300ul Staining buffer
  • 100ul staining solution PBS+1*Zombie Violet (Biolegend, 423114
  • Staining buffer to wash the cells twice, remove the supernatant, and add staining solution to each tube respectively: add 100 ⁇ l of staining solution to the first tube (Staining buffer+PerCP-Cy5.5 Mouse anti-hPVR detection antibody+APC Mouse anti-hPVRL2 detection Antibody, PVR detection antibody: Biolegend 337612, PVRL2 detection antibody: Biolegend 337412), add 100 ⁇ l isotype control staining solution (Staining buffer+PerCP-Cy5.5 Mouse IgG1 ⁇ isotype control antibody+APC ⁇ Mouse IgG1 isotype control antibody, PerCP-Cy5 .5 mIgG1 ⁇ isotype control antibody: Biolegend 400150, APC mIgG1 ⁇ isotype control antibody: Biolegend 400122), mix well and incubate at 4°C for 30 minutes.
  • NK cells Natural killer cells
  • FACS FACS was used to detect the CD107a signal of NK cells (Natural killer cells) to indicate the effect of the tested antibody on the degranulation process of NK cells. Resuscitate PBMC one day in advance, sort NK cells (Stemcell, 17955), add 200IU/ml h-IL2 (RD, 202-IL) and 10ng/ml h-IL12 (Peprotech, 200-12-50UG) to stimulate overnight, The next day the planking experiment was carried out.
  • NK cells Natural killer cells
  • NK cells use a cell counter (Beckman Coulter, Vi-CELL) to count NK cells, take a certain number of NK cells, centrifuge at a speed of 350g for 5 minutes, discard the supernatant and resuspend to 0.5E+6 cells with assay buffer /ml density, protein transport inhibitor (Invitrogen, 00498093) and APC mouse anti-human detection antibody (Biolegend, 328620) were added to the cell suspension. Add the treated NK cell suspension to the well-coated 96-well U-bottom plate, 50 ⁇ l per well, mix well and incubate at room temperature for 15 minutes.
  • a cell counter Beckman Coulter, Vi-CELL
  • the target cell WIDR was digested with trypsin into a cell suspension, and the target cells were counted using a cell counter (Beckman Coulter, Vi-CELL), and an appropriate number of cells were taken out, centrifuged at 200g for 5 minutes, and the supernatant was discarded Then resuspend the cells with assay buffer to a density of 0.25E+6 cells/ml. After incubation, add target cell suspension to the well plate, 100 ⁇ l per well, at this time, each well contains 25,000 NK cells, 25,000 target cells and different concentrations of test antibodies, and the wells containing only NK cells are used as resting cells.
  • wells containing NK cells and target cells were used as no drug control. Each well was mixed evenly and placed in a 37°C incubator for 16 hours. FACS staining to detect changes in CD107a: transfer the cells in the well plate to a 96-well V-bottom plate in parallel, wash twice with PBS, discard the supernatant, and add staining solution (PBS+2% FBS+1*concentration zombie violet (Biolegend, 423114) + PE mouse anti-CD56 detection antibody (Biolegend, 318306)) were mixed evenly and incubated at 4°C for 30 minutes.
  • staining solution PBS+2% FBS+1*concentration zombie violet (Biolegend, 423114) + PE mouse anti-CD56 detection antibody (Biolegend, 318306)
  • NK cell killing assay to detect the killing effect of NK cells on target cells by anti-TIGIT chimeric antibody
  • FACS FACS was used to detect the lysis level of target cells (WIDR) to infer the effect of the tested antibody on the killing function of NK (Natural killer cell) cells. Resuscitate PBMC one day in advance, sort NK cells (Stemcell, 17955), add 200IU/ml h-IL2 (RD, 202-IL) and 10ng/ml h-IL12 (Peprotech, 200-12-50UG) overnight stimulation, The next day the planking experiment was carried out.
  • WIDR lysis level of target cells
  • Digest the target cell WIDR with trypsin to form a cell suspension use a cell counter (Beckman Coulter, Vi-CELL) to count the WIDR cells, take out an appropriate number of cells, put them in a centrifuge and centrifuge at a speed of 200g for 5 minutes, discard After cleaning, resuspend with an appropriate amount of PBS and add CellTrace Violet (Invitrogen, C34557A) staining solution to make the final concentration of CellTrace Violet 5 ⁇ M.
  • CellTrace Violet Invitrogen, C34557A
  • NK cells use a cell counter to count NK cells, take a certain number of NK cells, centrifuge at 350g for 5 minutes, discard the supernatant and resuspend to a density of 0.5E+6 cells/ml with assay buffer. Add the treated NK cell suspension to the well-coated 96-well U-bottom plate, 50 ⁇ l per well, mix well and incubate at room temperature for 15 minutes.
  • WIDR cell staining After WIDR cell staining, add 5 times the volume of complete medium (MEM+10% FBS+1*P/S+1*non-essential amino acid+1*sodium glutamate) to the cell suspension to terminate the reaction, and use 200g Centrifuge at a high speed for 5 minutes, discard the supernatant and resuspend the cells to a density of 0.25E+6 cells/ml with assay buffer. After NK and drug incubation, add WIDR cell suspension to the well plate, 100 ⁇ l per well, at this time, each well contains 25,000 NK cells, 25,000 WIDR cells and different concentrations of test antibodies, and the wells only contain WIDR cells As a resting control, wells containing NK and WIDR served as no drug control.
  • Figure 11 shows that the negative control anti-HEL-hIgG1 has no significant effect on NK killing, the four chimeric antibodies tested can effectively promote the killing of NK cells on the target cell WIDR, and the killing of NK cells by the four chimeric antibodies The promoting effect was not weaker than that of the positive control RG6058-hIgG1.
  • CMV antigen-recall assay detects the function improvement effect of anti-TIGIT chimeric antibody on antigen-specific CD8 T cells
  • CMV pp65 (495-503) polypeptide-induced CMV pp65-specific CD8 T in PBMCs of Anti-CMV IgG-positive donors was used as effector cells, and the colo205 tumor cell line after pp65 pulsed was used as target cells. Functional improvement of antigen-specific CD8 T cells.
  • PBMCs were revived, they were treated with 1mg/mL CMV pp65(495-503) polypeptide (Anaspec, product number AS-28328), 2ng/mL human IL-2 (R&D, product number IL-202), 10ng/mL human IL-7 ( Peprotech, Cat. No. 200-07) complete medium (RPMI1640-Glutamax+5%AB serum+1%P/S+(1 ⁇ )2- ⁇ -mercaptoethanol) resuspended to 2 ⁇ 10 6 /mL, 5mL/well inoculated Incubate in 6-well plates at 37 °C 5% CO 2 for 6 days.
  • 1mg/mL CMV pp65(495-503) polypeptide Anaspec, product number AS-28328
  • 2ng/mL human IL-2 R&D, product number IL-202
  • 10ng/mL human IL-7 Peprotech, Cat. No. 200-07
  • complete medium RPMI1640-Glutamax+5%AB serum
  • Flow detection antibodies are as follows: Livedead Near IR (Invitrogen, catalog number L34976), CD8-PerCp Cy5.5 (BD, catalog number 565310), CD3-PE-Cy7 (Biolegend, catalog number 300316), T-select HLA-A*0201 CMV pp65 Tetramer-PE (MBL, Cat. No.
  • TS-0010-1C TS-0010-1C
  • PVRIG-AF488 R&D, Cat. No. FAB93651G-100UG
  • TIGIT-APC Biolegend, Cat. No. 372706
  • PD-1-BV421 BD, Cat. No. 562516
  • CD8 T was isolated as effector cells from the induced PBMCs using a CD8 T sorting kit (Stemcell, Cat. No. 17953), resuspended with AIM-V and adjusted to a cell density of 0.4 ⁇ 10 6 /mL.
  • Colo205 was used as target cells, digested with TrypLE TM Express Enzyme (Gibco, product number 12605010), resuspended in AIM-V (Gibco, product number 31035-025) containing 50ng/mL pp65, and adjusted the cell density to 1 ⁇ 10 6 /ml, Treat at 37°C with 5% CO 2 for 1-2 hours, then centrifuge at 250g for 5 minutes, and discard the supernatant.
  • TIGIT antibody or positive control was diluted to 280 nM with AIM-V.
  • the final drug concentration in this system is 70nM, CD8 T is 20000/well, colo205 is 20000/well. After the incubation, centrifuge at 400 g to take the supernatant, and use an ELISA kit (Daktronics, Cat. No. 1110003) to detect the level of human IFN- ⁇ in the supernatant.
  • the positive control in this system is RG6058-hIgG1, and the negative control is no treatment.
  • the flow cytometric detection antibodies of PVRL2 and PVR expression on Colo205 are as follows: liveddead-BV421 (Invitrogen, product number L34964), PVRL2-APC (Biolegend, product number 337412), PVR-PerCp Cy5.5 (Biolegend, product number 337612), PD-L1- PE-Cy7 (BD, Cat. No. 558017).
  • Embodiment 5 Humanization of anti-human TIGIT monoclonal antibody
  • the CDRs of the murine antibody were grafted into corresponding human templates to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the key amino acids in the backbone sequence were back-mutated to the corresponding amino acids of the mouse antibody to ensure the original affinity, that is, a humanized anti-TIGIT monoclonal antibody was obtained.
  • the CDR amino acid residues of antibodies are usually determined and annotated by the Kabat numbering system.
  • the humanized light chain templates of the mouse antibody TIGIT-002 are IGKV2-29*02/IGKV4-1*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV4-38-2*01 and IGHJ6*01.
  • the CDRs of the murine antibody TIGIT-002 were grafted into its human template respectively to obtain the corresponding humanized version.
  • the key amino acids in the FR region sequence of the humanized antibody of TIGIT-002 were back-mutated to the corresponding amino acids of the mouse antibody to ensure the original affinity.
  • the specific back-mutation design is shown in Table 6.
  • Grafted means that the mouse antibody CDR is implanted into the human germline IGKV2-29*02 FR region sequence; L47M means that the 47th position of Grafted is mutated back to M, and so on.
  • the numbering of backmutated amino acids is the natural sequence numbering.
  • the specific sequence of the variable region of TIGIT-002 humanized antibody is as follows:
  • TIGIT-002.VL1 The amino acid sequence of TIGIT-002.VL1 is shown in SEQ ID NO: 66:
  • TIGIT-002.VL2 The amino acid sequence of TIGIT-002.VL2 is shown in SEQ ID NO: 67:
  • TIGIT-002.VL3 The amino acid sequence of TIGIT-002.VL3 is shown in SEQ ID NO: 68:
  • TIGIT-002.VH1 The amino acid sequence of TIGIT-002.VH1 is shown in SEQ ID NO: 69:
  • TIGIT-002.VH2 The amino acid sequence of TIGIT-002.VH2 is shown in SEQ ID NO: 70:
  • TIGIT-002.VH3 The amino acid sequence of TIGIT-002.VH3 is shown in SEQ ID NO: 71:
  • TIGIT-002.VH4 The amino acid sequence of TIGIT-002.VH4 is shown in SEQ ID NO: 72:
  • amino acid sequence of the humanized light chain template IGKV2-29*02 is shown in SEQ ID NO: 73:
  • amino acid sequence of the humanized light chain template IGKV4-1*01 is shown in SEQ ID NO: 74:
  • amino acid sequence of the humanized light chain template IGKJ4*01 is shown in SEQ ID NO: 75:
  • amino acid sequence of the humanized heavy chain template IGHV4-38-2*01 is shown in SEQ ID NO: 76:
  • amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 77:
  • the present invention respectively selects different light chain and heavy chain sequences from the reverse mutation design of the light chain and heavy chain variable regions of the above-mentioned 002 humanized antibody for cross-combination, and finally obtains a variety of 002 humanized antibodies, each antibody
  • the amino acid sequence of the variable region is as follows:
  • TIGIT-002-H1L1 indicates that the heavy chain is selected from TIGIT-002.VH1 and the light chain is selected from TIGIT-002.VL1. The same below.
  • the humanized light chain templates of murine antibody TIGIT-006 are IGKV2-29*02/IGKV4-1*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV4-38-2*01 and IGHJ6*01.
  • the CDRs of the murine antibody TIGIT-006 were grafted into its human template respectively to obtain the corresponding humanized version.
  • the key amino acids in the FR region sequence of the humanized antibody of TIGIT-006 were back-mutated to the corresponding amino acids of the mouse antibody to ensure the original affinity.
  • the specific back-mutation design is shown in Table 9.
  • Grafted means that the mouse antibody CDR is implanted into the human germline IGKV2-29*02 FR region sequence; L37Q means that the 37th L of Grafted is mutated back to Q, and so on.
  • the numbering of backmutated amino acids is the natural sequence numbering.
  • variable region of TIGIT-006 humanized antibody is as follows:
  • TIGIT-006.VL1 The amino acid sequence of TIGIT-006.VL1 is shown in SEQ ID NO: 78:
  • TIGIT-006.VL2 The amino acid sequence of TIGIT-006.VL2 is shown in SEQ ID NO: 79:
  • TIGIT-006.VL3 The amino acid sequence of TIGIT-006.VL3 is shown in SEQ ID NO: 80:
  • TIGIT-006.VH1 The amino acid sequence of TIGIT-006.VH1 is shown in SEQ ID NO: 81:
  • TIGIT-006.VH2 The amino acid sequence of TIGIT-006.VH2 is shown in SEQ ID NO: 82:
  • TIGIT-006.VH3 The amino acid sequence of TIGIT-006.VH3 is shown in SEQ ID NO: 83:
  • TIGIT-006.VH4 The amino acid sequence of TIGIT-006.VH4 is shown in SEQ ID NO: 84:
  • TIGIT-006.VH5 The amino acid sequence of TIGIT-006.VH5 is shown in SEQ ID NO: 85:
  • amino acid sequence of the humanized light chain template IGKV2-29*02 is shown in SEQ ID NO: 73:
  • amino acid sequence of the humanized light chain template IGKV4-1*01 is shown in SEQ ID NO: 74:
  • amino acid sequence of the humanized light chain template IGKJ4*01 is shown in SEQ ID NO: 75:
  • amino acid sequence of the humanized heavy chain template IGHV4-38-2*01 is shown in SEQ ID NO: 76:
  • amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 77:
  • the present invention respectively selects different light chain and heavy chain sequences from the reverse mutation design of the humanized antibody light chain and heavy chain variable regions of TIGIT-006 for cross-combination, and finally obtains a variety of humanized TIGIT-006 Antibodies, the amino acid sequences of the variable regions of each antibody are as follows:
  • TIGIT-006-H1L1 indicates that the heavy chain is selected from TIGIT-006.VH1, and the light chain is selected from TIGIT-006.VL1. The same below.
  • the humanized light chain templates of the murine antibody TIGIT-005 are IGKV4-1*01 and IGKJ4*01, the humanized heavy chain templates are IGHV1-3*01 and IGHJ6*01, and the CDRs of the murine antibody TIGIT-005 Transplant them into their human source templates respectively to obtain the corresponding humanized versions.
  • the key amino acids in the FR region sequence of the humanized antibody of TIGIT-005 were back-mutated to the corresponding amino acids of the mouse antibody to ensure the original affinity.
  • the specific back-mutation design is shown in Table 12.
  • Grafted (IGKV4-1*01) means that the mouse antibody CDR is implanted into the human germline IGKV4-1*01 FR region sequence, Q38H means that the 38th Q of Grafted is mutated back to H, and so on.
  • the numbering of backmutated amino acids is the natural sequence numbering.
  • variable region of TIGIT-005 humanized antibody is as follows:
  • TIGIT-005.VL1 The amino acid sequence of TIGIT-005.VL1 is shown in SEQ ID NO: 86:
  • TIGIT-005.VH2 The amino acid sequence of TIGIT-005.VH2 is shown in SEQ ID NO: 87:
  • amino acid sequence of the humanized light chain template IGKV4-1*01 is shown in SEQ ID NO: 74:
  • amino acid sequence of the humanized light chain template IGKJ4*01 is shown in SEQ ID NO: 75:
  • amino acid sequence of the humanized heavy chain template IGHV1-3*01 is shown in SEQ ID NO: 106:
  • amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 77:
  • the TIGIT-005 antibody has a site NG that is prone to chemical modification, and we performed point mutations on NG to eliminate the risk of modification.
  • TIGIT-005.VL1a The amino acid sequence of TIGIT-005.VL1a is shown in SEQ ID NO: 88:
  • TIGIT-005.VL1b The amino acid sequence of TIGIT-005.VL1b is shown in SEQ ID NO: 89:
  • TIGIT-005.VL1c The amino acid sequence of TIGIT-005.VL1c is shown in SEQ ID NO: 90:
  • TIGIT-005.VL1d The amino acid sequence of TIGIT-005.VL1d is shown in SEQ ID NO: 91:
  • TIGIT-005.VL1e The amino acid sequence of TIGIT-005.VL1e is shown in SEQ ID NO: 92:
  • the present invention respectively selects different light chain and heavy chain sequences from the reverse mutation design of the humanized antibody light chain and heavy chain variable regions of TIGIT-005 for cross-combination, and finally obtains six humanized TIGIT-005 Antibodies, the amino acid sequences of the variable regions of each antibody are as follows:
  • TIGIT-005-H2L1 indicates that the heavy chain is selected from TIGIT-005.VH2, and the light chain is selected from TIGIT-005.VL1. The same below.
  • the humanized light chain templates of the mouse antibody TIGIT-070 are IGKV1-39*01/IGKV4-1*01 and IGKJ3*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ6*01.
  • the CDRs of the antibody TIGIT-070 were grafted into its human template respectively, that is, the corresponding humanized version was obtained.
  • the key amino acids in the FR region sequence of the humanized antibody of TIGIT-070 were back-mutated to the corresponding amino acids of the mouse antibody to ensure the original affinity.
  • the specific back-mutation design is shown in Table 15.
  • Grafted (IGKV1-39*01) means that the mouse antibody CDR is implanted into the human germline IGKV1-39*01 FR region sequence;
  • A43S means that the 43rd A of Grafted is mutated back to S, and so on.
  • the numbering of backmutated amino acids is the natural sequence numbering.
  • variable region of TIGIT-070 humanized antibody is as follows:
  • TIGIT-070.VL1 The amino acid sequence of TIGIT-070.VL1 is shown in SEQ ID NO: 98:
  • TIGIT-070.VL2 The amino acid sequence of TIGIT-070.VL2 is shown in SEQ ID NO: 99:
  • TIGIT-070.VL3 The amino acid sequence of TIGIT-070.VL3 is shown in SEQ ID NO: 100:
  • TIGIT-070.VH1 The amino acid sequence of TIGIT-070.VH1 is shown in SEQ ID NO: 101:
  • TIGIT-070.VH2 The amino acid sequence of TIGIT-070.VH2 is shown in SEQ ID NO: 102:
  • TIGIT-070.VH3 The amino acid sequence of TIGIT-070.VH3 is shown in SEQ ID NO: 103:
  • amino acid sequence of the humanized light chain template IGKV1-39*01 is shown in SEQ ID NO: 104:
  • amino acid sequence of the humanized light chain template IGKV4-1*01 is shown in SEQ ID NO: 74:
  • amino acid sequence of the humanized light chain template IGKJ3*01 is shown in SEQ ID NO: 105:
  • amino acid sequence of the humanized heavy chain template IGHV1-3*01 is shown in SEQ ID NO: 106:
  • amino acid sequence of the humanized heavy chain template IGHJ6*01 is shown in SEQ ID NO: 77:
  • the present invention respectively selects different light chain and heavy chain sequences from the reverse mutation design of the humanized antibody light chain and heavy chain variable regions of TIGIT-070 above, and performs cross-combination, and finally obtains a variety of humanized TIGIT-070 Antibodies, the amino acid sequences of the variable regions of each antibody are as follows:
  • TIGIT-070-H1L1 indicates that the heavy chain is selected from TIGIT-070.VH1 and the light chain is selected from TIGIT-070.VL1. The same below.
  • BIAcore was used to detect the affinity of the above-mentioned humanized antibody to human TIGIT ECD-His, and the binding activity of the above-mentioned humanized antibody to human TIGIT ECD-mFc was detected with reference to the method of Example 4 (a), (b), (d). , the binding activity to CHO-K1 human TIGIT, the binding activity to CHO-K1 cynomolgus TIGIT, the binding activity to cynomolgus TIGIT ECD-mFc, and the ability to block the binding of human TIGIT ECD-mFc to CHO-K1 CD155 The results are shown in Table 18.
  • TIGIT-002-H4L3, TIGIT-005-H2L1d, TIGIT-006-H5L3, and TIGIT-070-H1L1 maintained the same affinity, binding activity and blocking effect as the chimeric antibody, and were not affected by Humanization is obviously affected, and the overall performance is better than RG6058.
  • Embodiment 6 Identification of anti-TIGIT humanized antibody
  • Example 4(a) For the experimental method, see Example 4(a).
  • the experimental results show that, consistent with the control antibody Roche RG6058, the anti-TIGIT humanized antibody can effectively bind to human TIGIT ECD-mFc ( Figure 13) and cynomolgus monkey TIGIT ECD-mFc ( Figure 14)
  • Example 4(b) For the experimental method, see Example 4(b).
  • the experimental results show that, consistent with the control antibody Roche RG6058, the anti-TIGIT humanized antibody can effectively bind to the cell line with high expression of CHO-K1-TIGIT ( Figure 15), the cell line expressing CHO-K1-TIGIT ( Figure 16), and the cell line of CHO-K1 - TIGIT low expression cell line ( Figure 17) and CHO-K1-cynomolgus monkey TIGIT cell line ( Figure 18).
  • Example 4(c) For the experimental method, see Example 4(c). As shown in Figure 19, both the anti-TIGIT humanized antibody and the control antibody RG6058 can effectively block the binding of Bio-CD155-His protein to CHO-K1-human TIGIT cells.
  • both the anti-TIGIT humanized antibody and the control antibody RG6058 can effectively block the binding of human TIGIT protein to CHO-K1-CD155 cells.
  • Collect CHO-K1 CD112 cells wash once with PBS (Hyclone, CAT#SH30256), resuspend to 2 ⁇ 10 5 /40 ⁇ L with 1% BSA-PBS, dilute humanized antibody to 600nM with 1% BSA-PBS, 2.5-fold continuous gradient Dilute 12 concentration points, dilute TIGIT ECD-mFc to 6 ⁇ g/mL with 1% BSA-PBS, then mix 40 ⁇ L cells with 40 ⁇ L antibody diluent and 40 ⁇ L TIGIT ECD-mFc diluent, incubate at 4°C for 60 minutes, wash twice with PBS , join Alexa 647 fluorescein-labeled secondary antibody (working dilution 1:800, Jackson, CAT#115-605-003), resuspended cells in 100 ⁇ L/well, incubated at 4°C for 40 minutes, washed twice with PBS, and washed with 1% BSA-PBS , res
  • Table 19 summarizes the characteristics of these four humanized antibody strains.
  • the four humanized antibodies TIGIT-002-H4L3, TIGIT-005-H2L1d, TIGIT-006-H5L3, and TIGIT-070-H1L1 have the same binding tendency with PBMCs and stable cells with high, medium and low expression levels of human TIGIT.
  • TIGIT-002-H4L3 and TIGIT-005-H2L1d are stronger than TIGIT-006-H5L3 and TIGIT-070-H1L1, both stronger than RG6058, and the monovalent affinity with human TIGIT ECD-His is 0.0994nM, 0.0852nM, 0.1145nM, 0.2505 nM, RG6058 is 0.1560nM, and all have strong cross-reactivity with TIGIT in cynomolgus monkeys; in terms of blocking characterization in vitro, the four humanized antibodies all showed significant blocking effects on TIGIT-CD155 and TIGIT- Capacity of CD112 to interact.
  • FIG. 23 shows that: the negative control anti-HA HcAb-hIgG1 has no significant effect on NK killing, and four humanized antibodies TIGIT-002-H4L3, TIGIT-005-H2L1d, TIGIT-006-H5L3 and TIGIT-070-H1L1 can inhibit NK killing in different degrees.
  • NK can effectively promote the killing of target cells, among which TIGIT-002-H4L3, TIGIT-005-H2L1d and their humanized pre-chimeric antibodies TIGIT-CHI-002 and TIGIT-CHI-005 have comparable NK killing promotion ability.
  • CMV antigen-recall assay detects the function improvement effect of anti-TIGIT humanized antibody on antigen-specific CD8 T cells
  • PBMCs were revived, they were treated with 1mg/mL CMV pp65(495-503) polypeptide (Anaspec, product number AS-28328), 2ng/mL human IL-2 (R&D, product number IL-202), 10ng/mL human IL-7 ( Peprotech, Cat. No. 200-07) complete medium (RPMI1640-Glutamax+5%AB serum+1%P/S+1 ⁇ 2- ⁇ mercaptoethanol) resuspended to 2 ⁇ 10 6 /mL, 5mL/well inoculated in Incubate in 6-well plates at 37 °C 5% CO 2 for 6 days.
  • 1mg/mL CMV pp65(495-503) polypeptide Anaspec, product number AS-28328
  • 2ng/mL human IL-2 R&D, product number IL-202
  • 10ng/mL human IL-7 Peprotech, Cat. No. 200-07
  • complete medium RPMI1640-Glutamax+5%AB serum+1%P
  • Flow detection antibodies are as follows: Livedead Near IR (Invitrogen, catalog number L34976), CD8-PerCp Cy5.5 (BD, catalog number 565310), CD3-PE-Cy7 (Biolegend, catalog number 300316), T-select HLA-A*0201 CMV pp65 Tetramer-PE (MBL, Cat. No. TS-0010-1C), PVRIG-AF488 (R&D, Cat. No. FAB93651G-100UG), TIGIT-APC (Biolegend, Cat. No. 372706), PD-1-BV421 (BD, Cat. No. 562516)
  • CD8 T was isolated as effector cells from the induced PBMCs using a CD8 T sorting kit (Stemcell, Cat. No. 17953), resuspended with AIM-V and adjusted to a cell density of 0.4 ⁇ 10 6 /mL. After sorting, the purity of CD8 and the expression of CD226 were detected.
  • Colo205 was used as target cells, digested with TrypLE TM Express Enzyme (Gibco, product number 12605010), resuspended in AIM-V (Gibco, product number 31035-025) containing 20ng/mLpp65 and adjusted the cell density to 1 ⁇ 10 6 /mL, 37 Centrifuge at 5% CO 2 for 3 hours, then centrifuge at 250 g for 5 minutes, and discard the supernatant. Then the cells were resuspended to 0.5 ⁇ 10 6 /mL with AIM-V.
  • Anti-TIGIT humanized antibody or positive control was diluted to 280nM with AIM-V.
  • the positive control in this system is RG6058-hIgG1 and the antibody before TIGIT humanization (TIGIT-CHI-002), and the negative control is no treatment.
  • TIGIT-CHI-002 The positive control in this system is RG6058-hIgG1 and the antibody before TIGIT humanization
  • TIGIT-CHI-002 The positive control in this system is RG6058-hIgG1 and the antibody before TIGIT humanization
  • TIGIT-CHI-002 the negative control is no treatment.
  • the CD8 T purity and CD226 expression flow detection antibodies are as follows: livedead-BV421 (Invitrogen, product number L34964), CD8-FITC (BD, product number 555366), CD226-PE-Cy7 (Biolegend, product number 338316).
  • the flow detection antibodies of PVRL2, PVR, PD-L1, and HLA-A2 expressed on Colo205 are as follows: livedead-BV421 (Invitrogen, product number L34964), PVRL2-APC (Biolegend, product number 337412), PVR-PerCp Cy5.5 (Biolegend, Product No. 337612), PD-L1-PE-Cy7 (BD, Product No. 558017), HLA-A2-PE (Biolegend, Product No. 343306).
  • RNA iso Plus (Takara, catalog number: 9109), aliquoted into 1.5mL EP tubes and stored at -80°C. Total RNA extracted from frozen lymphocytes was reverse-transcribed into cDNA, and then two rounds of VHH PCR amplification were performed using cDNA as a template. The VHH product amplified by the second round of PCR was digested with restriction enzymes to construct a phage library. The established bacterial library was collected, and the library insertion rate and diversity were sequenced and analyzed.
  • Tables 20 to 22 The CDRs of their sequences were analyzed by IMGT and KABAT software respectively.
  • the corresponding sequence information is shown in Tables 20 to 22 below, and Table 20 shows the candidate antibody molecules.
  • Table 21 shows the results of IMGT analysis of candidate antibody molecules
  • Table 22 shows the results of KABAT analysis of candidate antibody molecules.
  • Example 8 ELISA detection of specific binding of anti-PVRIG antibodies to human and cynomolgus PVRIG proteins
  • Example 7 The VHH is connected with human IgG1-Fc) for serial dilution (initial concentration 20nM, 3.33-fold serial dilution or 3nM, 3-fold serial dilution), 100 ⁇ L/well was added, and incubated with shaking at room temperature for 1.5 hours; after washing the plate, add mouse antibody Human (mouse anti-human) IgG Fc-HRP (Jackson ImmunoResearch, Cat.
  • Positive control antibodies include COM701-hIgG1 (Patent No.: US20180244774A1), COM701-hIgG4 (patent No.: US20180244774A1) and SRF813-hIgG1 (Patent No.: US20200040081A1); negative control antibodies include anti-HA HcAb-hIgG1 (Chengdu A Parker, Cat. No. NBR022), anti-CD38 HcAb-hIgG1 (in-house) and anti-Fluorescein-hIgG1 (in-house).
  • amino acid sequence corresponding to COM701 is as follows:
  • FIG. 26A and Table 23 The binding results of 13 anti-PVRIG antibodies to human PVRIG protein are shown in FIG. 26A and Table 23, and the binding results to cynomolgus monkey PVRIG are shown in FIG. 26B and Table 23. The data showed that all tested antibodies could specifically bind to human or cynomolgus PVRIG protein.
  • staining buffer 2% FBS+PBS
  • centrifuge 350 ⁇ g to remove the supernatant
  • wash the cells twice and resuspend the cells to 2 ⁇ 106 cells/ml in staining buffer Density, spread into a 96-well plate, add 50 ⁇ l of cell suspension to each well, and set aside.
  • Use the staining buffer to dilute the antibody 3.3 times from the highest concentration of 46nM (twice the concentration), add the diluted antibody to the well containing 50 ⁇ l of cell suspension, and place it on a microplate shaker at 400rpm for 1 Minutes to fully mix the antibody and cells, and incubate at 4°C for 30 minutes.
  • PE goat anti-Human IgG Fc antibody (ebioscience, 12-4998-82) was diluted 250 times with staining buffer, added to the washed cell wells at a volume of 100 ⁇ l per well, mixed evenly, and stained at 4°C for 30 minutes. After staining, wash twice with staining buffer, and finally resuspend cells with 200 ⁇ l of staining buffer, and detect signals on a flow cytometer (BD CantoII). The stronger the fluorescent signal, the stronger the binding ability of the antibody to PVRIG.
  • the binding activity of the antibody was normalized to the percentage of positive molecule COM701-hIgG1 and SRF813-hIgG1 antibody, the higher the percentage value, the stronger the binding activity of the antibody.
  • the results in Table 24 show that the tested antibodies PVRIG-A11, A35, A43, A105, A117, A118 have stronger binding activity to human PVRIG than COM701-hIgG1 and SRF813-hIgG1. Among them, the binding activity of PVRIG-A105 and A117 to cynomolgus monkey PVRIG was also stronger than that of COM701-hIgG1 and SRF813-hIgG1.
  • Example 10 BIAcore detects the affinity of anti-PVRIG antibody and human PVRIG protein
  • Biacore was used to detect the specific binding between the tested anti-PVRIG antibody and human PVRIG protein.
  • the ProteinA chip was used, and the time required for the chip to capture the diluted antibody was determined by manual operation (manual run), so that the saturated binding antigen Rmax was 50RU.
  • Human PVRIG protein Human PVRIG-His, Acro C227P1-9ARF1-T4
  • the affinity of the antibody to the antigen was measured using multi-cycle kinetics. In each cycle, after the injection of the antibody, a gradient concentration of PVRIG protein is injected to make the binding and dissociation process of the antigen and the antibody occur.
  • Example 11 ELISA detection of anti-PVRIG antibody blocking the combination of PVRIG and PVRL2
  • Example 12 FACS detection of anti-PVRIG antibodies blocking the binding of CHO-K1-CD112 cells to human PVRIG-mFc protein
  • CHO-K1 stable cells (named CHO-K1-CD112) transfected with a high-expression plasmid of human CD112 were obtained. Experiment when over 80%. Discard the cell culture medium, rinse with PBS and add 1ml of trypsin (Gibico, 25200-72) to digest for 2 minutes, stop the digestion with Ham's F12 (Gibico, 21127-022) complete medium containing 10% FBS and make cells Suspension.
  • Human PVRIG-mFc protein (Acro, PVG-H5253) working solution was prepared with staining buffer at a concentration of 1 ⁇ g/ml (quadruple concentration), added to a 96-well plate, and 50 ⁇ l of PVRIG-mFc working solution was added to each well.
  • Use the staining buffer to dilute the antibody 3-fold from the highest concentration of 275nM (four-fold concentration) add the diluted antibody to the well containing 50 ⁇ l PVRIG-mFc, and place it on a microplate shaker at 400rpm for 1 Minutes, fully mix the antibody and PVRIG-mFc protein, and incubate at 4°C for 30 minutes.
  • all tested antibodies could block the binding of CHO-K1-CD112 cells to human PVRIG-mFc protein.
  • the competitive blocking activity of the tested antibody was normalized to the percentage of the reference molecule COM701-hIgG1 and SRF813-hIgG1 antibody, the smaller the percentage value, the stronger the blocking activity of the antibody.
  • the results in Table 27 show that the blocking activity of the tested antibodies PVRIG-A11, A15, A30, and A50 is stronger than that of COM701-hIgG1 and SRF813-hIgG1.
  • Anti-PVRIG antibodies compete for CHO-K1-CD112 cells to bind to human PVRIG-mFc protein
  • NK cells Natural killer cells
  • FACS was used to detect the expression of PVRIG and TIGIT on NK cells (Natural killer cells).
  • NK cells Natural killer cells
  • the results are shown in FIG. 9A .
  • the NK cells from different donors donor-010 and donor-050 all expressed PVRIG and TIGTI on the surface.
  • Example 14 NK cell degranulation assay to detect the function-promoting effect of anti-PVRIG antibody on NK cells
  • NK cell Natural killer cell
  • FACS was used to detect the signal of NK cell (Natural killer cell) CD107a to indicate the effect of the tested antibody on the degranulation process of NK cells.
  • NK cell Natural killer cell
  • FIG 31 shows that the negative control anti-HA HcAb-hIgG1 has no effect on the CD107a of NK cells, and the 12 PVRIG test antibodies and control antibodies COM701-hIgG1 and SRF813-hIgG1 can increase the expression of CD107a in NK cells to varying degrees, which shows that Both the test antibody and the control antibody can effectively promote the activation of NK cells.
  • Example 15 NK cell killing assay detects the killing effect of PVRIG antibody-mediated NK cells on tumor cell lines
  • FIG. 32 shows that the negative control anti-HA HcAb-hIgG1 has no effect on NK killing, and all 13 tested antibodies can effectively promote NK's killing of target cell WIDR, and except for PVRIG-A35 and A43, the remaining 11 tested antibodies The killing promotion functions of WIDR cells were stronger than or equivalent to the control antibody COM701-hIgG1.
  • Example 16 CMV antigen-recall assay detects the function improvement effect of anti-PVRIG antibody on antigen-specific CD8 T cells
  • Anti-CMV IgG-positive donor PBMCs were induced by CMV pp65(495-503) polypeptide, CMV pp65-specific CD8T was used as effector cells, and the colo205 tumor cell line after pp65 pulsed was used as target cells. Functional improvement of antigen-specific CD8 T cells.
  • CMV IgG+PBMC Resuscitate CMV IgG+PBMC with 1mg/mL CMV pp65(495-503) polypeptide (Anaspec, Cat. No. AS-28328), 2ng/mL human IL-2 (R&D, Cat. No. IL-202), 10ng/mL human IL- 7 (Peprotech, Cat. No. 200-07) complete medium (RPMI1640-Glutamax+5%AB serum+1%P/S+(1 ⁇ )2- ⁇ -mercaptoethanol) resuspended to 2 ⁇ 10 6 /mL, 5mL/ Wells were seeded in 6-well plates and cultured at 37°C in 5% CO for 6 days.
  • complete medium RPMI1640-Glutamax+5%AB serum+1%P/S+(1 ⁇ )2- ⁇ -mercaptoethanol
  • Flow detection antibodies are as follows: Live/deadNear IR (Invitrogen, catalog number L34976), CD8-PerCp Cy5.5 (BD, catalog number 565310), CD3-PE-Cy7 (Biolegend, catalog number 300316), T-select HLA-A*0201 CMV pp65 Tetramer-PE (MBL, Cat. No. TS-0010-1C), PVRIG-AF488 (R&D, Cat. No. FAB93651G-100UG), TIGIT-APC (Biolegend, Cat. No. 372706), PD-1-BV421 (BD, Cat. No. 562516).
  • CD8 T was isolated as effector cells from the induced PBMCs using a CD8 T sorting kit (Stemcell, Cat. No. 17953), resuspended with AIM-V and adjusted to a cell density of 0.4 ⁇ 10 6 /mL. The purity of CD8 T and the expression of CD226 after sorting were detected.
  • Colo205 was used as target cells, digested with TrypLE TM Express Enzyme (Gibco, product number 12605010), resuspended in AIM-V (Gibco, product number 31035-025) containing 20ng/mL pp65, and adjusted the cell density to 1 ⁇ 10 6 /ml, Treat at 37°C with 5% CO 2 for 3 hours, then centrifuge at 250g for 5 minutes, and discard the supernatant. The cells were resuspended with AIM-V to 0.5 ⁇ 10 6 /mL, and flow cytometric detection showed that Colo205 cells highly expressed PVRL2, PVR and HLA-A2, as shown in Figure 33 C.
  • Anti-PVRIG antibody or negative control was diluted to 280 nM with AIM-V.
  • the final drug concentration in this system is 70nM
  • CD8 T is 20000/well
  • colo205 cells is 50000/well.
  • centrifuge at 400 g to take the supernatant, and use an ELISA kit (Daktronics, Cat. No.
  • the positive control in this system is COM701-hIgG4, SRF813-hIgG1, and the negative control is no treatment.
  • the secretion of IFN- ⁇ in the cell supernatant was significantly increased after the treatment of most of the tested PVRIG antibodies.
  • CD8 T purity and CD226 expression flow detection antibodies were as follows: livedead-BV421 (Invitrogen, product number L34964), CD8-FITC (BD, product number 555366), CD226-PE-Cy7 (Biolegend, product number 338316).
  • Colo205 cell PVRL2, PVR, PD-L1, HLA-A2 expression flow detection antibodies are as follows: livedead-BV421 (Invitrogen, product number L34964), PVRL2-APC (Biolegend, product number 337412), PVR-PerCp Cy5.5 (Biolegend, Product No. 337612), PD-L1-PE-Cy7 (BD, Product No. 558017), HLA-A2-PE (Biolegend, Product No. 343306).
  • the heavy chain variable region germline genes with high homology to alpaca antibodies were selected as templates,
  • the CDRs of the alpaca antibody were transplanted into the corresponding human templates to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the key amino acids in the backbone sequence were back-mutated to the corresponding amino acids of the alpaca antibody to ensure the original affinity, that is, the humanized anti-PVRIG monoclonal antibody was obtained.
  • the humanized heavy chain templates of the alpaca antibody PVRIG-A50 are IGHV3-23*04 and IGHJ3*01, and the CDRs of the alpaca antibody PVRIG-A50 are grafted into their human templates to obtain the corresponding humanized versions , where antibody CDR amino acids are determined and annotated by the Kabat numbering system.
  • the key amino acids in the FR region sequence of the humanized antibody of PVRIG-A50 were back-mutated to the corresponding amino acids of the alpaca antibody to ensure the original affinity.
  • Grafted (IGHV3-23*04) means that the CDR of the target antibody is implanted into the human germline IGHV3-23*04 FR region sequence; the first + followed by A97V means that the 97th A of Grafted is mutated back to V; the second + followed by N54D represents a point mutation at the NG site, and so on.
  • the numbering of mutated amino acids is the natural sequence numbering, the same below.
  • variable region of the PVRIG-A50 humanized antibody is as follows:
  • amino acid sequence of A50.VH1 (PVRIG-A50-H1) is shown in SEQ ID NO: 198:
  • amino acid sequence of A50.VH1a (PVRIG-A50-H1a) is shown in SEQ ID NO: 199:
  • amino acid sequence of A50.VH1b (PVRIG-A50-H1b) is shown in SEQ ID NO: 200:
  • amino acid sequence of A50.VH1c (PVRIG-A50-H1c) is shown in SEQ ID NO: 201:
  • amino acid sequence of A50.VH1d (PVRIG-A50-H1d) is shown in SEQ ID NO: 202:
  • amino acid sequence of A50.VH1e (PVRIG-A50-H1e) is shown in SEQ ID NO: 203:
  • amino acid sequence of A50.VH2a (PVRIG-A50-H2a) is shown in SEQ ID NO: 204:
  • amino acid sequence of the humanized heavy chain template IGHV3-23*04 is shown in SEQ ID NO: 205:
  • amino acid sequence of the humanized heavy chain template IGHJ3*01 is shown in SEQ ID NO: 206:
  • the humanized heavy chain templates of the alpaca antibody PVRIG-A105 are IGHV3-7*01 and IGHJ3*01, and the CDRs of the alpaca antibody PVRIG-A105 are grafted into their human templates respectively to obtain the corresponding humanized versions , where antibody CDR amino acids are determined and annotated by the IMGT numbering system.
  • the key amino acids in the FR region sequence of the humanized antibody of PVRIG-A105 were back-mutated to the corresponding amino acids of the alpaca antibody to ensure the original affinity.
  • Grafted(IGHV3-7*01) means that the CDR of the target antibody is implanted into the human germline IGHV3-7*01 FR region sequence; the first + followed by V37F means that the 37th V of Grafted is mutated back to F; the second + The latter C103S represents a point mutation at the Cys site, and so on.
  • the numbering of mutated amino acids is the natural sequence numbering, the same below.
  • variable region of the PVRIG-A105 humanized antibody is as follows:
  • amino acid sequence of A105.VH1 (PVRIG-A105-H1) is shown in SEQ ID NO: 211:
  • amino acid sequence of A105.VH2 (PVRIG-A105-H2) is shown in SEQ ID NO: 212:
  • amino acid sequence of A105.VH3 (PVRIG-A105-H3) is shown in SEQ ID NO: 213:
  • amino acid sequence of A105.VH4 (PVRIG-A105-H4) is shown in SEQ ID NO: 214:
  • amino acid sequence of A105.VH5 (PVRIG-A105-H5) is shown in SEQ ID NO: 215:
  • amino acid sequence of A105.VH3a (PVRIG-A105-H3a) is shown in SEQ ID NO: 216:
  • amino acid sequence of the humanized heavy chain template IGHV3-7*01 is shown in SEQ ID NO: 217:
  • amino acid sequence of the humanized heavy chain template IGHJ3*01 is shown in SEQ ID NO: 206:
  • the humanized heavy chain templates of the alpaca antibody PVRIG-A118 are IGHV3-7*01 and IGHJ3*01, and the CDRs of the alpaca antibody PVRIG-A118 are grafted into their human templates respectively to obtain the corresponding humanized versions , where antibody CDR amino acids are determined and annotated by the IMGT numbering system.
  • the key amino acids in the FR region sequence of the humanized antibody of PVRIG-A118 were back-mutated to the corresponding amino acids of the alpaca antibody to ensure the original affinity.
  • the specific design is shown in Table 32.
  • Grafted (IGHV3-7*01) means that the CDR of the target antibody is implanted into the human germline IGHV3-7*01 FR region sequence; the first + followed by S35G means that the 35th S of Grafted is mutated back to G; others are analogous .
  • the numbering of back-mutated amino acids is the natural sequence numbering, the same below.
  • variable region of the PVRIG-A118 humanized antibody is as follows:
  • amino acid sequence of A118.VH1 (PVRIG-A118-H1) is shown in SEQ ID NO: 219:
  • amino acid sequence of A118.VH2 (PVRIG-A118-H2) is shown in SEQ ID NO: 220:
  • amino acid sequence of A118.VH3 (PVRIG-A118-H3) is shown in SEQ ID NO: 221:
  • amino acid sequence of A118.VH4 (PVRIG-A118-H4) is shown in SEQ ID NO: 222:
  • amino acid sequence of A118.VH5 (PVRIG-A118-H5) is shown in SEQ ID NO: 223:
  • amino acid sequence of A118.VH6 (PVRIG-A118-H6) is shown in SEQ ID NO: 224:
  • amino acid sequence of A118.VH7 (PVRIG-A118-H7) is shown in SEQ ID NO: 225:
  • amino acid sequence of the humanized heavy chain template IGHV3-7*01 is shown in SEQ ID NO: 217:
  • amino acid sequence of the humanized heavy chain template IGHJ3*01 is shown in SEQ ID NO: 206:
  • the binding effect of the humanized antibody was normalized to its corresponding pre-humanized parental antibody, and the percentage value was higher than 100%, indicating that the binding effect of the humanized antibody was better than that of the parental antibody.
  • most of the humanized antibodies of PVRIG-A50, PVRIG-A105, and PVRIG-A118 specifically bind to human/cynomolgus PVRIG protein.
  • Example 19 FACS detection of the binding of PVRIG humanized antibody to human PVRIG on the surface of FlpinCHO-PVRIG cells and cynomolgus monkey PVRIG on the surface of FlpinCHO-cyno PVRIG cells
  • Example 9 For the experimental method, see Example 9. The larger the calculated AUC value, the stronger the binding ability of the humanized antibody to FlpinCHO-human/cyno PVRIG cells.
  • Figure 35A most of the humanized molecules of the tested antibodies PVRIG-A50, A105 and A118 bind to human PVRIG on the surface of FlpinCHO-PVRIG cells at the same level as their parental antibodies.
  • Figure 35B shows that, except for PVRIG-A118 humanized molecules, the binding of PVRIG in cynomolgus monkeys was significantly weaker than that of the corresponding parental antibodies, and the binding of most of the humanized molecules of PVRIG-A50 and A105 to PVRIG in cynomolgus monkeys was significantly weaker.
  • the binding activity of the humanized antibody was normalized to the percentage of the reference molecule COM701-hIgG1 and SRF813-hIgG1 antibody, as shown in Table 35, the higher the percentage value, the stronger the binding activity of the antibody.
  • Example 20 BIAcore detects the affinity of PVRIG humanized antibody and human PVRIG protein
  • the inhibitory effect of the humanized antibody was normalized to its corresponding pre-humanized parental antibody, and the percentage value was higher than 100%, indicating that the inhibitory effect of the humanized antibody was better than that of the parental antibody.
  • the blocking curve of the humanized antibody is shown in Figure 36, and the inhibitory activity is shown in Table 37. As shown in Figure 36 and Table 37, most of the humanized antibodies of PVRIG-A50, PVRIG-A105 and PVRIG-A118 can significantly inhibit the binding of human PVRIG to human PVRL2.
  • Example 22 FACS detection of PVRIG humanized antibody blocking the binding of CHO-K1-CD112 cells to human PVRIG-mFc protein
  • the experimental method refers to Example 12.
  • the experimental results are shown in Figure 37, most of the humanized antibodies can block the binding of CHO-K1-CD112 cells to human PVRIG-mFc protein.
  • the blocking activity of the humanized antibody was normalized to the percentage of the reference molecule COM701-hIgG1 and SRF813-hIgG1 antibody, the smaller the percentage value, the better the blocking effect of the antibody.
  • Anti-PVRIG humanized antibodies block CHO-K1-CD112 cells binding to human PVRIG-mFc protein
  • Example 23 NK cell killing assay to detect the killing effect of NK cells on tumor cell lines mediated by PVRIG humanized antibody
  • Example 4(g) For the experimental method, refer to Example 4(g).
  • the experimental results show that all the humanized antibodies in Figure 38 can promote the killing of target cells by NK cells to varying degrees, and A in Figure 38 shows that among the seven humanized antibodies, PVRIG-A50-H1b, PVRIG-A50-H2a
  • the promotion effect on NK cells killing target cells is equivalent to that of the parental antibody PVRIG-A50 before humanization;
  • B in Figure 38 shows that PVRIG-A118-H3, H4, H5 and H6 among the seven humanized antibodies can kill target cells on NK cells
  • the promotion effect of PVRIG-A118 is equivalent to that of the parental antibody PVRIG-A118 before humanization;
  • C in Figure 38 shows that the promotion effect of PVRIG-A105-H1, H2, and H3 on NK cells killing target cells among the five humanized antibodies is similar to that of humanization
  • the former parental antibody PVRIG-A105 was comparable.
  • Example 24 CMV antigen-recall assay detects the function improvement effect of anti-PVRIG humanized antibody on antigen-specific CD8 T cells
  • PBMC Resuscitate PBMC, with 1mg/mL CMV pp65 (495-503) polypeptide (Anaspec, product number AS-28328), 2ng/mL human IL-2 (R&D, product number IL-202), 10ng/mL human IL-7 (Peprotech , Cat. No. 200-07) complete medium (RPMI1640-Glutamax+5%AB serum+1%P/S+1 ⁇ 2- ⁇ mercaptoethanol) was resuspended to 2 ⁇ 10 6 /mL, 5mL/well was inoculated in 6 Incubate in a well plate at 37 °C 5% CO 2 for 6 days.
  • Flow detection antibodies are as follows: Livedead Near IR (Invitrogen, catalog number L34976), CD8-PerCp Cy5.5 (BD, catalog number 565310), CD3-PE-Cy7 (Biolegend, catalog number 300316), T-select HLA-A*0201 CMV pp65 Tetramer-PE (MBL, Cat. No. TS-0010-1C), PVRIG-AF488 (R&D, Cat. No. FAB93651G-100UG), TIGIT-APC (Biolegend, Cat. No. 372706), PD-1-BV421 (BD, Cat. No. 562516)
  • CD8 T cells were isolated as effector cells from the induced PBMCs using a CD8 T cell sorting kit (Stemcell, Cat. No. 17953), resuspended with AIM-V and adjusted to a cell density of 0.4 ⁇ 10 6 /mL. The purity of CD8 T cells and the expression of CD226 after sorting were detected. Colo205 was used as target cells, digested with TrypLE TM Express Enzyme (Gibco, Cat. No. 12605010), resuspended in AIM-V medium (Gibco, Cat. No.
  • the remaining humanized antibodies were compared with their respective parental antibodies before humanization. There was no statistically significant difference (One-way ANOVA Analysis).
  • the CD8 T purity and CD226 expression flow detection antibody information is as follows: live/dead-BV421 (Invitrogen, product number L34964), CD8-FITC (BD, product number 555366), CD226-PE-Cy7 (Biolegend, product number 338316) .
  • the flow cytometric detection antibody information of PVRL2, PVR, PD-L1, HLA-A2 expression on Colo205 is as follows: live/dead-BV421 (Invitrogen, catalog number L34964), PVRL2-APC (Biolegend, catalog number 337412), PVR-PerCp Cy5.5 (Biolegend, Cat. No. 337612), PD-L1-PE-Cy7 (BD, Cat. No. 558017), HLA-A2-PE (Biolegend, Cat. No. 343306).
  • Two anti-PVRIG humanized VHH antibodies PVRIG-A50-H1b, PVRIG-A105-H1
  • two anti-TIGIT humanized monoclonal antibodies TIGIT-002-H4L3, TIGIT-005-H2L1d
  • G4S Peptides link the anti-PVRIG humanized VHH antibody to the N-terminal of the anti-TIGIT humanized antibody heavy chain to generate anti-PVRIGxTIGIT humanized bispecific antibodies (Figure 40), named LC-BsAb-002 and LC-BsAb respectively -006, LC-BsAb-009 and LC-BsAb-010.
  • Table 39 shows the sequences of heavy chain fusion polypeptides (HC) and light chain polypeptides (LC) of four bispecific antibodies.
  • the anti-TIGIT positive control antibody is Roche’s RG6058-hIgG1
  • the anti-PVRIG positive control antibody is Compugen’s COM701-hIgG4.
  • Example 26 ELISA detection of specific binding of anti-PVRIGxTIGIT humanized bispecific antibody to human and cynomolgus PVRIG proteins
  • Positive control antibodies include COM701-hIgG1, PVRIG-A50-H1b, PVRIG-A105-H1; negative control antibodies include anti-Fluorescein-hIgG1 (inhouse).
  • the binding results of the 4 humanized double antibodies to human PVRIG protein are shown in Figure 41 and Tables 40 and 41, and the binding results to cynomolgus monkey PVRIG are shown in Figure 42 and Tables 40 and 41. The data showed that all four humanized double antibodies could specifically bind to human or cynomolgus PVRIG protein.
  • LC-BsAb-002 and LC-BsAb-006 The binding of LC-BsAb-002 and LC-BsAb-006 to human PVRIG protein was slightly weaker than its control monoclonal antibody PVRIG-A50-H1b and positive control COM701-hIgG1; LC-BsAb-009 and LC-BsAb-010 combined with human or
  • the binding of PVRIG protein in cynomolgus monkeys was basically equivalent to its relative monoclonal antibody PVRIG-A105-H1, and was better than that of the positive control COM701-hIgG1.
  • Example 27 ELISA detection of specific binding of anti-PVRIGxTIGIT humanized bispecific antibody to human and cynomolgus TIGIT proteins
  • the positive control antibodies include RG6058-hIgG1, TIGIT-002-H4L3 and TIGIT-005-H2L1d; the negative control antibody is anti-Fluorescein-hIgG1 (in house).
  • the binding results of the 4 humanized double antibodies to human TIGIT protein are shown in Figure 43 and Tables 42 and 43, and the binding results to cynomolgus monkey TIGIT are shown in Figure 44 and Tables 42 and 43. The data showed that all four humanized double antibodies could specifically bind to human or cynomolgus TIGIT protein.
  • the binding of LC-BsAb-002 and 009 to human TIGIT protein was better than that of the positive control RG6058-hIgG1, which was equivalent to that of the corresponding monoclonal antibody TIGIT-002-H4L3; the binding of LC-BsAb-009 to its corresponding monoclonal antibody TIGIT protein Anti-TIGIT-002-H4L3 and the positive control RG6058-hIgG1 are basically equivalent, and LC-BsAb-002 is slightly weaker than the corresponding monoclonal antibody and the positive control.
  • LC-BsAb-006 and 010 to human TIGIT protein/cynomolgus monkey TIGIT protein is better than that of their corresponding monoclonal antibody TIGIT-005-H2L1d and the positive control RG6058-hIgG1.
  • Example 28 FACS detection of binding activity of anti-PVRIGxTIGIT humanized bispecific antibody to FlpinCHO human PVRIG and FlpinCHO cynomolgus monkey PVRIG
  • CHO-K1 stable cells transfected with human or cynomolgus PVRIG high-expression plasmids CCL-61 TM ), named FlpinCHO-hPVRIG, FlpinCHO-cyno PVRIG, human PVRIG full-length plasmid (NCBI RefSeq: NP_076975), and cynomolgus monkey PVRIG full-length plasmid (NCBI Ref Seq: XP_014989941) were all synthesized by General Biotechnology , experiments were performed when the cell density did not exceed 80%.
  • staining buffer 2% FBS+PBS
  • centrifuge 350 ⁇ g to remove the supernatant
  • wash the cells twice and resuspend the cells to 2 ⁇ 106 cells/ml in staining buffer Density, spread into a 96-well plate, add 50 ⁇ l of cell suspension to each well, and set aside.
  • Use the staining buffer to dilute the antibody 3.3 times from the highest concentration of 46nM (twice the concentration), add the diluted antibody to the well containing 50 ⁇ l of cell suspension, and place it on a microplate shaker at 400rpm for 1 Minutes to fully mix the antibody and cells, and incubate at 4°C for 30 minutes.
  • PE goat anti-Human IgG Fc antibody (ebioscience, 12-4998-82) was diluted 250 times with staining buffer, added to the washed cell wells at a volume of 100 ⁇ l per well, mixed evenly, and stained at 4°C for 30 minutes. After staining, wash twice with staining buffer, and finally resuspend the cells with 200 ⁇ l staining buffer, and detect the signal (BD CantoII) on a flow cytometer. The stronger the signal, the stronger the binding ability of the antibody to PVRIG.
  • Figure 45 shows that all four humanized double antibodies have good binding activity to human PVRIG, and can also bind cynomolgus monkey PVRIG well ( Figure 46), and all of them are better than their corresponding anti-PVRIG humanized monoclonal antibodies.
  • Example 29 FACS detection of binding activity of anti-PVRIGxTIGIT humanized bispecific antibody to CHO-K1 human TIGIT (high/medium/low expression strain) and CHO-K1 cynomolgus monkey TIGIT cells
  • Example 30 HTRF method to detect anti-PVRIGxTIGIT humanized bispecific antibody blocking the interaction between PVRIG protein and PVRL2 protein
  • Ratio Signal665nm/Signal 620nm ⁇ 10 4 .
  • IC50 the molar concentration of the antibody
  • the positive controls in this experiment are COM701-hIgG1, PVRIG-A50-H1b, PVRIG-A105-H1; the negative control antibodies are anti-Fluorescein-hIgG1 (in house).
  • the effect of 4 humanized double antibodies blocking PVRIG-PVRL2 binding is shown in Figure 51 and Tables 44 and 45.
  • Example 31 FACS detection of anti-PVRIGxTIGIT humanized bispecific antibody blocking CHO-K1 human CD112 cells binding to human PVRIG-mFc protein
  • CHO-K1 stable cells (named CHO-K1-CD112) transfected with a high-expression plasmid of human CD112 were taken.
  • the full-length human CD112 plasmid was synthesized by General Biosynthesis (NP_001036189.1/NCBI RefSeq:Q92692), and the cell density did not exceed Experiment at 80%. Discard the cell culture medium, rinse with PBS and add 1ml of trypsin (Gibico, 25200-72) to digest for 2 minutes, stop the digestion with Ham's F12 (Gibico, 21127-022) complete medium containing 10% FBS and make cells Suspension.
  • Human PVRIG-mFc protein (Acro, PVG-H5253) working solution was prepared with staining buffer at a concentration of 1 ⁇ g/ml (quadruple concentration), added to a 96-well plate, and 50 ⁇ l of PVRIG-mFc working solution was added to each well.
  • Use the staining buffer to dilute the antibody 3-fold from the highest concentration of 275nM (four-fold concentration) add the diluted antibody to the well containing 50 ⁇ l PVRIG-mFc, and place it on a microplate shaker at 400rpm for 1 Minutes, fully mix the antibody and PVRIG-mFc protein, and incubate at 4°C for 30 minutes.
  • Example 32 ELISA method to detect the activity of anti-PVRIGxTIGIT humanized bispecific antibody blocking the binding of human TIGIT to CHO-K1 CD155
  • the CHO-K1 CD155 cells constructed in Example 2 were collected, adjusted to 5 ⁇ 10 5 /mL with 10% FBS-DMEM/F12 medium (Excell, FSP500; Gibco, 11330), and added to a 96-well cell culture plate (corning, 3599), 100 ⁇ L/well, cultured overnight at 37°C in 5% CO 2 , discarded the culture supernatant, added cell fixative (Beyond, P0098), 50 ⁇ L/well, fixed at room temperature for 1 hour, and washed with 0.05% Tween20 on a plate washer -Wash once with PBS, add 5% skimmed milk powder-PBS, 250 ⁇ L/well, incubate at 37°C for 2-4 hours, wash three times with 0.05% Tween20-PBS on a plate washer; human TIGIT ECD-mFc (working concentration 100ng/mL ) mixed with the sample and incubated for half an hour; then the antigen-antibody mixture was added to the cell plate,
  • Example 33 FACS method to detect the activity of anti-PVRIGxTIGIT humanized bispecific antibody blocking the binding of Bio-CD155-His to CHO-K1 human TIGIT
  • Collect cells wash once with PBS (Hyclone, SH30256), resuspend to 2 ⁇ 10 5 /40 ⁇ L in 1% BSA-PBS, dilute antibody to 210 nM with 1% BSA-PBS, serially dilute 12 concentration points by 3 times, 1% BSA - Dilute Bio-CD155-His (Shenzhou, 10109-H08H) in PBS to 3 ⁇ g/mL, then mix 40 ⁇ L cells with 40 ⁇ L antibody diluent and 40 ⁇ L Bio-CD155-His diluent, incubate at 4°C for 60 minutes, wash with PBS Twice, add APC-labeled streptavidin (working dilution 1:1700, Biolegend, 405243), resuspend the cells in 100 ⁇ L/well, incubate at 4°C for 40 minutes, wash twice with PBS, wash with 1% BSA-PBS, 100 ⁇ L/well was resuspended, and the
  • Example 34 FACS method to detect the binding activity of anti-PVRIGxTIGIT humanized bispecific antibody to human PBMC
  • Example 35 BIAcore detects the affinity of anti-PVRIGxTIGIT humanized bispecific antibody to human, cynomolgus monkey and mouse TIGIT and PVRIG proteins
  • the ProteinA chip was used, and the time required for the chip to capture the diluted antibody was determined by manual operation (manual run), so that the saturated binding antigen Rmax was 50RU.
  • Human, cynomolgus and mouse TIGIT and PVRIG proteins were diluted to 20, 10, 5, 2.5, 1.25nM.
  • the affinity of the antibody to the antigen was measured using multi-cycle kinetics. In each cycle, after antibody injection, gradient concentrations of human, cynomolgus monkey and mouse TIGIT and PVRIG proteins were injected to make the combination and dissociation process of antigen and antibody occur. After each cycle, use Glycine pH1.5 to regenerate the Protein A chip (remove the protein on the chip).
  • Example 36 BIAcore detection of co-binding of anti-PVRIGxTIGIT humanized bispecific antibody with human TIGIT and PVRIG proteins
  • BIAcore was used to characterize the simultaneous binding properties of bispecific antibodies to two antigens.
  • HBS-EP+ 10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P
  • the following four-step detection was carried out: only binding to hTIGIT single antigen, only binding to hPVRIG single antigen, first binding to hTIGIT and then binding to hPVRIG, first binding to hPVRIG and then binding to hTIGIT, and All antigens reached saturation.
  • the binding curve of the antibody antigen was collected, the capture levels of the two bispecific antibodies, and the binding signals of TIGIT and PVIRIG in each experiment were recorded, and the stoichiometric ratio of the antigen-antibody molecules was calculated, as shown in Table 47 and in Figure 56 A and B are the antibody-antigen binding curves of LC-BsAb-002 and LC-BsAb-006 combined with TIGIT and PVIRG, respectively, and continuous injection of TIGIT and PVRIG, respectively.
  • the binding signals produced by continuous injection of TIGIT and PVIRG are almost consistent with those produced by individual injections of TIGIT and PVIRG; It shows that LC-BsAb-002 and LC-BsAb-006 can combine with hTIGIT and hPVRIG at the same time, and there is no interaction between the two antigens; comprehensively consider the molecular weight of the antibody and antigen, as well as the capture level of the antibody and the binding level of the antigen , the stoichiometric ratio of TIGIT to LC-BsAb-002 was preliminarily estimated to be 1.76; the stoichiometric ratio of TIGIT to LC-BsAb-006 was 1.86; The stoichiometric ratio of BsAb-006 is 2.18, and the stoichiometric ratio of the two antigens and antibodies is close to 2. Considering the error caused by the detection method, we speculate that one LC
  • Example 37 NK cell degranulation assay to detect the function-promoting effect of anti-PVRIGxTIGIT humanized bispecific antibody on NK cells
  • FACS was used to detect the expression level of CD107a in NK cells, indicating the effect of the tested antibody on the activation of NK cells (A in Figure 57 shows the experimental procedure).
  • FACS was used to detect the expression of PVRIG, TIGIT and WIDR cell surface PVR and PVRL2 on NK cells (Natural killer cells).
  • NK cells use a cell counter (Beckman Coulter, Vi-CELL) to count NK cells, take three flow tubes, add 1e+5 NK cells to each flow tube, add PBS to wash the cells twice, remove the supernatant, Take one tube and add 300 ⁇ l Staining buffer (PBS+2% FBS) as an unstained tube, and add 100 ⁇ l of staining solution (PBS+1*Zombie Violet (Biolegend, 423114)) to each of the other two tubes, mix well and incubate at room temperature 15 minutes.
  • PBS+2% FBS Staining buffer
  • staining solution PBS+1*Zombie Violet (Biolegend, 423114)
  • PBS+2% FBS 300 ⁇ l Staining buffer
  • staining solution PBS+1* Zombie Violet (Biolegend, 423114)
  • Staining buffer to wash the cells twice, remove the supernatant, and add staining solution to each tube respectively: add 100 ⁇ l of staining solution to the first tube (Staining buffer+PerCP-Cy5.5 Mouse anti-hPVR detection antibody+APC Mouse anti-hPVRL2 detection Antibody, PVR detection antibody: Biolegend 337612, PVRL2 detection antibody: Biolegend 337412), add 100 ⁇ l isotype control staining solution (Staining buffer+PerCP-Cy5.5 Mouse IgG1 ⁇ isotype control antibody+APC ⁇ Mouse IgG1 isotype control antibody, PerCP-Cy5 .5 mIgG1 ⁇ isotype control antibody: Biolegend 400150, APC mIgG1 ⁇ isotype control antibody: Biolegend 400122), mix well and incubate at 4°C for 30 minutes.
  • human PBMCs were revived, human NK cells were sorted with a sorting kit (Stemcell, 17955), and 200 IU/ml of h-IL2 (R&D, 202-IL) and 10 ng/ml of h-IL12 (Peprotech , 200-12-50UG) stimulated overnight, and carried out the plating experiment the next day.
  • a sorting kit Stemcell, 17955
  • 200 IU/ml of h-IL2 R&D, 202-IL
  • 10 ng/ml of h-IL12 Peprotech , 200-12-50UG
  • NK cells use a cell counter (Beckman Coulter, Vi-CELL) to count NK cells, take a certain number of NK cells, centrifuge at a speed of 350g for 5 minutes, discard the supernatant and resuspend to 0.5E+6 cells with assay buffer /ml density, protein transport inhibitor (Invitrogen, 00498093) and APC mouse anti-human detection antibody (Biolegend, 328620) were added to the cell suspension. Add the treated NK cell suspension to the well-coated 96-well U-bottom plate, 50 ⁇ l per well, mix well and incubate at room temperature for 15 minutes.
  • a cell counter Beckman Coulter, Vi-CELL
  • the target cell WIDR was digested with trypsin into a cell suspension (Reh cells were directly mixed evenly), and the target cells were counted using a cell counter (Beckman Coulter, Vi-CELL), and an appropriate number of cells was taken out, and the cells were mixed with 200 g Centrifuge at high speed for 5 minutes, discard the supernatant and resuspend the cells to a density of 0.25e+6 cells/ml with assay buffer. After incubation, add target cell suspension to the well plate, 100 ⁇ l per well, at this time, each well contains 25,000 NK cells, 25,000 target cells and different concentrations of test antibodies, and the wells containing only NK cells are used as resting cells.
  • NK and WIDR were used as no drug control.
  • Each well was mixed evenly and placed in a 37°C incubator for 16 hours.
  • FACS staining was carried out: the cells in the well plate were transferred to a 96-well V-bottom plate in parallel, washed twice with PBS, the supernatant was discarded, and a staining solution (PBS+2% FBS+1* concentration of zombie violet (Biolegend, Biolegend, 423114)+PE mouse anti-CD56 detection antibody (Biolegend, 318306)) were mixed evenly and incubated at 4°C for 30 minutes.
  • NK cell degranulation experiment (using TF-1 as the target cell).
  • the experimental method refers to Example 37B.
  • the experimental results are shown in Figure 57 D, indicating that the humanized double antibody can degranulate NK cells, and the effect is better than PVRIG positive control antibody COM701-hIgG4 and TIGIT positive control antibody RG6058-hIgG1, which is equivalent to COM701 - hIgG4 and RG6058-hIgG1 combination group.
  • the promotion effect of the humanized double antibody on NK cell degranulation is also superior to its PVRIG arm antibody PVRIG-A50-H1b and TIGIT arm antibody TIGIT-002-H4L3, combined with PVRIG-A50-H1b and TIGIT-002-H4L3 We correspond by team.
  • Example 38 NK cell killing assay to detect the killing effect of NK cells on tumor cell lines mediated by anti-PVRIGxTIGIT humanized bispecific antibody
  • FACS was used to detect the lysis level of target cells (WIDR) to indicate the effect of the tested antibody on the killing function of NK cells on target cells.
  • PBMCs were revived, and the NK cells were sorted out with a sorting kit (Stemcell, 17955), and 200 IU/ml of h-IL2 (RD, 202-IL) and 10 ng/ml of h-IL12 (Peprotech , 200-12-50UG) stimulated overnight, and carried out the plating experiment the next day.
  • a sorting kit Stemcell, 17955
  • 200 IU/ml of h-IL2 RD, 202-IL
  • 10 ng/ml of h-IL12 Peprotech , 200-12-50UG
  • the target cell WIDR was digested with trypsin into a cell suspension, and the WIDR cells were counted using a cell counter (Beckman Coulter, Vi-CELL), and an appropriate number of cells were taken out, and placed in a centrifuge at a speed of 200g for 5 minutes. After discarding the supernatant, resuspend with an appropriate amount of PBS, and then add CellTrace Violet (Invitrogen, C34557A) staining solution, so that the final concentration of CellTrace Violet is 5 ⁇ M.
  • CellTrace Violet Invitrogen, C34557A
  • the WIDR suspension added with the dye solution was mixed evenly and incubated in a 37°C incubator for 10 minutes, shaking and mixing during the period, and a part of WIDR cells were taken out to detect the expression levels of PVR and PVRL2 on WIDR by the method described in Example 13.
  • use a cell counter to count NK cells take a certain number of NK cells, centrifuge at 350g for 5 minutes, discard the supernatant and resuspend to a density of 0.5e+6 cells/ml with assay buffer.
  • Add the treated NK cell suspension to the well-coated 96-well U-bottom plate, 50 ⁇ l per well, mix well and incubate at room temperature for 15 minutes.
  • WIDR cell staining After WIDR cell staining, add 5 times the volume of complete culture (MEM+10%FBS+1*P/S+1*non-essential amino acid+1*sodium glutamate) base to the cell suspension to terminate the reaction, and use 200g Centrifuge at a high speed for 5 minutes, discard the supernatant and resuspend the cells to a density of 0.25e+6 cells/ml with assay buffer. After NK and drug incubation, add WIDR cell suspension to the well plate, 100 ⁇ l per well, at this time, each well contains 25,000 NK cells, 25,000 WIDR cells and different concentrations of test antibodies, and the wells only contain WIDR cells As a resting control, wells containing NK cells and WIDR served as no drug control.
  • a in Figure 58 shows that the three NK donors (Donor-050, Donor-831, Donor-715) all express a certain level of PVRIG and TIGIT
  • B in Figure 58 shows that PVR and PVRL2 are highly expressed on the target cell WIDR
  • C is a brief experimental process of NK cells killing WIDR cells.
  • D in Figure 58 shows that the negative control anti-Fluorescein-hIgG1 has no obvious effect on NK killing, and the two humanized double antibodies tested can effectively promote NK ( 3 donor sources) on the killing of target cell WIDR, the EC50 and the area under the curve (AUC) of two humanized double antibodies on killing WIDR cells under different NK donors are indicated in the table.
  • TF-1 Using TF-1 as the target cell, the killing effect of NK cells on TF-1 mediated by anti-PVRIGxTIGIT humanized bispecific antibody was detected. Killing tumor cells, the activity is better than PVRIG positive control antibody COM701-hIgG4, TIGIT positive control antibody RG6058-hIgG1 and the combination of COM701-hIgG4 and RG6058-hIgG1.
  • the activity of the humanized double antibody was also better than that of its PVRIG arm antibody PVRIG-A50-H1b, TIGIT arm antibody TIGIT-002-H4L3, and the combination of PVRIG-A50-H1b and TIGIT-002-H4L3.
  • Example 39 NK cell ADCC assay to detect the direct killing effect on human Treg cells mediated by anti-PVRIGxTIGIT humanized bispecific antibody
  • the lysis level of target cells was detected by FACS to indicate the direct ADCC killing effect of the tested antibody on NK cells on target cells (A in FIG. 59 ).
  • PBMC peripheral blood mononuclear cells
  • NK cells were sorted out as effector cells with a sorting kit (Stemcell, 17955), and 200 IU/ml of h-IL2 (RD, 202-IL) and 10 ng/ml of h-IL2 were added.
  • IL12 (Peprotech, 200-12-50UG) was stimulated overnight, and the plating experiment was carried out the next day.
  • Treg regulatory T cells isolated from PBMC were used as target cells (Stemcell, 18063) to be expanded in vitro by Dynabeads (Gibco, 11129D) for 12 days to obtain expanded Treg cells for experiments, which were mentioned in Example 13 before the experiment Methods and reagents for detecting the expression of TIGIT and PVRIG on Treg cells.
  • Effector cells and target cells were co-incubated at a ratio of 5:1, and serially diluted test humanized bispecific antibodies or isotype control anti-Fluorescein-hIgG1, anti-Fluorescein-hIgG4 antibodies were added in a 37°C incubator After incubation for 4 hours, add PI staining, and finally read the proportion of PI-positive Treg cells to evaluate the ADCC (antibody-dependent cell-mediated cytotoxicity) killing effect of the tested bispecific antibody on target cell Treg .
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Monocytes were isolated from donor PBMCs, induced with 75ng/mL GM-CSF for seven days to differentiate into macrophages, and labeled with CellTrace Violet as effector cells.
  • Human Treg cells sorted from human PBMC by Regulatory T Cell Isolation Kit were expanded in vitro with Dynabeads Human Treg Expander and activated for 13 days as target cells, and then the target cells were labeled with CFSE dye. Effector cells were co-incubated with target cells at a ratio of 4:1.
  • test antibody diluted test antibody, negative control Hel hIgG1 antibody, its single-arm antibody (PVRIG-A50-H1b and TIGIT-002-H4L3) or a combination of two monoclonal antibodies, and incubate at 37°C for 4 hours. After the incubation, the cell dye PI was added, and flow cytometry was used to detect the proportion of CellTrace Violet positive cells among CFSE positive Treg cells, and evaluate the ADCP effect of the antibody to be tested.
  • PVRIG-A50-H1b and TIGIT-002-H4L3 single-arm antibody
  • the antibody to be tested activated the ADCP function of Treg cells in a dose-dependent manner.
  • PVRIG-A50-H1b or COM701-hIgG4 had little ADCP activity.
  • TIGIT-002-H4L3 or RG6058-hIgG1 exhibited dose-dependent ADCP activity.
  • AUC area under the curve
  • the ADCP activity of the antibody to be tested is slightly weaker than that of TIGIT-002-H4L3 and the combination of two single-arm antibodies (PVRIG-A50-H1b+TIGIT-002-H4L3).
  • the ADCP effect of the tested antibody was comparable to that of RG6058-hIgG1 and the combination of two positive antibodies (COM701-hIgG4+RG6058-hIgG1).
  • the ADCP activity of the antibody to be tested is equivalent to that of the two single-arm antibody combinations (PVRIG-A50-H1b+TIGIT-002-H4L3) and the two positive antibody combinations (COM701-hIgG4+RG6058-hIgG1) ( B) in Figure 60.
  • Example 41 Effect of anti-PVRIGxTIGIT humanized bispecific antibody on cytokine release in human PBMC from healthy donors
  • Lipopolysaccharide and CD3 monoclonal antibody were used as positive controls, Anti-Hel hIgG1 antibody was used as negative control, and RG6058-hlgG1 and COM701-hlgG4 were used as monoclonal antibody controls for TIGIT and PVRIG, respectively.
  • the secretion levels of IFN- ⁇ , IL-2 and IL-10 in PBMCs were comparable to the negative control or below the detection limit; under solid phase conditions After the antibody to be tested at a high concentration point (2850nM), the secretion levels of TNF- ⁇ and IL-6 were significantly higher than the negative control or comparable to the negative control; but compared with RG6058-hlgG1 and COM701-hlgG4 under the same conditions, The antibody to be tested will not additionally increase the secretion of five cytokines, IFN- ⁇ , IL-2, IL-6, IL-10 and TNF- ⁇ , in unstimulated PBMC of healthy people in vitro.
  • the antibody to be tested under the same conditions will not increase the IFN- ⁇ , IL-2, IL- 6. Secretion of IL-10 and TNF- ⁇ 5 cytokines.
  • Example 42 CMV antigen-recall assay detects the function promoting effect of anti-PVRIGxTIGIT humanized bispecific antibody on antigen-specific CD8 T cells
  • CMV pp65-specific CD8T induced by CMV pp65 (495-503) polypeptide was used as effector cells in CMV IgG-positive donor PBMCs, and colo205 after pp65 pulsed was used as target cells. Functional promotion of pp65-specific CD8 T cells (A in FIG. 61 ).
  • PBMCs were revived, they were treated with 1 ⁇ g/mL CMV pp65(495-503) polypeptide (Anaspec, catalog number AS-28328), 2ng/mL human IL-2 (R&D, catalog number IL-202), 10ng/mL human IL-7 ( Peprotech, Cat. No. 200-07) complete medium (RPMI1640-Glutamax+5%AB serum+1%P/S+(1 ⁇ )2- ⁇ -mercaptoethanol) resuspended to 2 ⁇ 10 6 /mL, 5mL/well inoculated Incubate in 6-well plates at 37 °C 5% CO 2 for 6 days.
  • complete medium RPMI1640-Glutamax+5%AB serum+1%P/S+(1 ⁇ )2- ⁇ -mercaptoethanol
  • Flow detection antibodies are as follows: Livedead Near IR (Invitrogen, catalog number L34976), CD8-PerCp Cy5.5 (BD, catalog number 565310), CD3-PE-Cy7 (Biolegend, catalog number 300316), T-select HLA-A*0201 CMV pp65 Tetramer-PE (MBL, Cat. No. TS-0010-1C), PVRIG-AF488 (R&D, Cat. No. FAB93651G-100UG), TIGIT-APC (Biolegend, Cat. No. 372706), PD-1-BV421 (BD, Cat. No. 562516)
  • CD8 was isolated as effector cells by a CD8 sorting kit (Stemcell, product number 17953), resuspended in AIM-V medium and adjusted to a cell density of 0.4 ⁇ 10 6 /mL (if CD8 was induced and frozen If it is not stored, the cell number needs to be increased to 0.7 ⁇ 10 6 /mL). After sorting, the purity of CD8 and the expression of CD226 were detected.
  • Colo205 was used as target cells, digested with TrypLE TM Express Enzyme (Gibco, product number 12605010), resuspended in AIM-V (Gibco, product number 31035-025) containing 20ng/mL pp65, and adjusted the cell density to 1 ⁇ 10 6 /ml, Treat at 37°C with 5% CO 2 for 3 hours, then centrifuge at 250g for 5 minutes, and discard the supernatant. Afterwards, the cells were resuspended to 0.5 ⁇ 10 6 /mL with AIM-V, and the expressions of PVRL2, PVR and PD-L1 were detected by flow cytometry (B in Figure 61).
  • the antibody to be tested (humanized double antibody and Tecentriq) was diluted to 280nM with AIM-V medium.
  • the final drug concentration in this system is 70nM
  • CD8 is 20000/well
  • colo205 is 50000/well.
  • centrifuge at 400 g to take the supernatant, and use an ELISA kit (Daktronics, Cat. No.
  • the flow cytometric detection antibodies for the purity of CD8T after sorting are as follows: livedead-BV421 (Invitrogen, product number L34964), CD8-FITC (BD, product number 555366).
  • Example 43 CMV antigen-recall assay detects the function promoting effect of anti-PVRIG ⁇ TIGIT humanized bispecific antibody combined with Tecentriq on antigen-specific CD8 T cells
  • the induced PBMCs were separated by CD8 T sorting kit ( Stemcell , Cat. The ratio of T cells, adjusted for the number of CD8 T cells in the microplate).
  • Colo205 pretreated overnight with a final concentration of 100ng/mL IFN- ⁇ in the complete medium was used as target cells, digested with TrypLE TM Express Enzyme (Gibco, Cat. No. 12605010), washed twice and resuspended in AIM-V containing 20ng/mL pp65 (Gibco, Cat. No. 31035-025) and adjust the cell density to 1 ⁇ 10 6 /ml, treat with 5% CO 2 at 37°C for 3 hours, then centrifuge at 250g for 5 minutes, and discard the supernatant.
  • the cells were resuspended to 0.5 ⁇ 10 6 /mL with AIM-V, and the expressions of PVRL2, PVR and PD-L1 were detected by flow cytometry (A in Figure 62).
  • Antibodies to be tested humanized double antibody, Tecentriq, combination of double antibody and Tecentriq, combination of two positive control monoclonal antibodies (COM701-hIgG4 and RG6058-hIgG1), three monoclonal antibodies (COM701-hIgG4, RG6058 - the combination of hIgG1 and Tecentriq) was diluted with AIM-V medium to 280nM (4 ⁇ ) as the initial concentration, followed by 10-fold serial dilution, with a total of 6 concentration points.
  • A375 cells were inoculated subcutaneously on the right side of female NPG mice (strain: NPG; Beijing Weitongda Biotechnology Co., Ltd.) at a concentration of 5 ⁇ 10 6 cells/0.1 mL.
  • Hu PBMC cells were injected into the tail vein of mice at a concentration of 5 ⁇ 10 6 cells/0.2 mL.
  • the tumor grew to about 82 mm 3 , 56 mice were randomly divided into groups according to the tumor volume, with 8 mice in each group.
  • b Statistical comparison between the tumor volume of the administration group and the tumor volume of the Vehicle control group on the 13th day of group administration, Two-way ANOVA analysis, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001,* ***P ⁇ 0.0001.
  • Body weight results As shown in Figure 65 and Table 51, except for the control molecule Tecentriq, which had a significant weight loss and showed toxic and side effects, the body weight change trends of the other control and candidate molecules LC-BsAb-002 and LC-BsAb-006 were basically the same as those of Consistent with PBS, subsequent weight loss was a phenomenon of GVHD induced by PBMC reconstitution.
  • the anti-PVRIGxTIGIT humanized bispecific antibody molecules LC-BsAb-002 and LC-BsAb-006 had a significant inhibitory effect on the growth of A375 subcutaneous transplanted tumors, and the tumor inhibitory effect was better than the positive drugs RG6058-hIgG1 and COM701-hIgG4
  • RG6058-hIgG1 and COM701-hIgG4 As a single drug, it is equivalent to the combination of positive control antibody Tecentriq and RG6058-hIgG1+COM701-hIgG4.
  • no toxic side effects of the candidate molecules were observed during the administration observation process, indicating that the candidate molecules were safe and tolerable under this model.
  • Example 45 In vivo drug efficacy evaluation of anti-PVRIGxTIGIT humanized bispecific antibody combined with Tecentriq in mice
  • A375 cells were inoculated subcutaneously on the right side of female NPG mice (strain: NPG; Beijing Weitongda Biotechnology Co., Ltd.) at a concentration of 5 ⁇ 10 6 cells/0.1 mL.
  • Hu PBMC cells were injected into the tail vein of mice at a concentration of 5.5 ⁇ 106/0.2mL.
  • mice were randomly selected according to the tumor volume and divided into 5 groups.
  • LC-BsAb-002 (11.7mg/kg, 9 rats), LC-BsAb-002 (5.9mg/kg, 9 rats), Tecentriq (3mg/kg, 10 rats; lotNO .HK65567, Roche), LC-BsAb-002+Tecentriq (5.9mg/kg+3mg/kg, 8 rats).
  • the route of administration in all groups was intraperitoneal injection, administered twice a week, 5 times in a row, and the experiment ended 3 days after the last administration.
  • b Statistical comparison between the tumor volume of the administration group and the tumor volume of the Vehicle control group on the 13th day of group administration, Two-way ANOVA analysis, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001,* ***P ⁇ 0.0001.
  • Body weight results As shown in Figure 68 and Table 53, the body weight of the mice in the LC-BsAb-002+Tecentriq combination group decreased to a certain extent, but did not show obvious side effects.
  • the trend of body weight change of Tecentriq group, LC-BsAb-002 (11.7mpk) and LC-BsAb-002 (5.9mpk) administration group was basically the same as that of PBS group.
  • the body weight of the administration group was statistically compared with the body weight of the Vehicle control group on the 13th day of group administration, and analyzed by Two-way ANOVA.
  • LC-BsAb-002 had a significant inhibitory effect on the growth of A375 subcutaneous xenograft tumors, and the inhibitory effect showed an obvious dose-dependent relationship with the increase of the dose.
  • the combined use of LC-BsAb-002 and Tecentriq has significantly better antitumor effects than their respective single use, and has a significant combined effect.
  • the following candidate molecules showed safety and tolerance.

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Abstract

提供一种能够特异性地结合PVRIG和TIGIT的双特异性抗体，所述双特异性抗体能够调节免疫细胞的功能，可作为药物治疗与免疫异常有关的疾病，例如肿瘤。

Description

抗PVRIG/抗TIGIT双特异性抗体和应用 技术领域
本发明涉及领域领域，具体而言，涉及抗PVRIG/抗TIGIT双特异性抗体。
背景技术
免疫疗法的基础在于操控及/或调节免疫系统，包括先天免疫反应及后天免疫反应二者。免疫疗法的目标为藉由控制对“外来剂”(例如病原体或肿瘤细胞)的免疫反应来治疗疾病。免疫系统是由众多细胞类型所组成的高度复杂系统，这些细胞具有控制那些交互作用及反应的复杂且细微的系统。癌症免疫监测的概念是基于免疫系统可辨识肿瘤细胞、启动免疫反应及抑制肿瘤发展及/或进展的理论。然而，明确的是许多癌细胞已发展出逃避免疫系统的机制，其可容许不受抑制的肿瘤生长。癌/肿瘤免疫疗法着重于发展可活化及/或激活免疫系统的新型且新颖的激动和/或拮抗剂，以达成更有效的抗肿瘤反应，增强对肿瘤细胞的杀伤及/或抑制肿瘤生长。
PVRIG在NK细胞和T细胞的细胞上表达，并且与其它已知的免疫检查点具有若干相似之处。用于展示PVRIG是免疫检查点受体的鉴定和方法在WO2016/134333中进行了论述，所述文献通过引用明确地并入本文。当PVRIG与其配体(PVRL2)结合时，引发抑制信号，其起到减弱NK细胞和T细胞针对靶细胞的免疫反应(即类似于PD-1/PD-L1)的作用。阻断PVRL2与PVRIG的结合会切断PVRIG的这种抑制信号，并因此调节NK细胞和T细胞的免疫反应。利用阻断与PVRL2结合的PVRIG抗体是一种增强NK细胞和T细胞杀伤癌细胞的治疗方法。已经产生了结合PVRIG并阻断其配体PVRL2的结合的阻断抗体。
类似地，TIGIT是另一个所关注的标靶，已经证明与其同源配体PVR的结合通过其细胞内ITIM域直接抑制NK细胞和T细胞的细胞毒性。TIGIT基因的敲除或TIGIT/PVR相互作用的阻断抗体已展示在体外可以增强NK细胞杀伤，或者加剧体内自身免疫疾病。除了对T细胞和NK细胞的直接作用外，TIGIT还可在树突状细胞或肿瘤细胞中诱导PVR介导的信号传导，从而引起抑炎细胞因子(例如IL10)的产生增加。值得注意的是，TIGIT表达与另一种重要的共抑制受体PD-1的表达密切相关。TIGIT和PD-1在许多人类和鼠类的肿瘤浸润淋巴细胞(TIL)上共表达。
TIGIT和PVRIG同属于DNAM超家族，并被被证明在多种肿瘤浸润淋巴细胞中共表达发挥免疫抑制作用，同时，TIGIT、PVRIG和PD-1共表达的肿瘤浸润效应T细胞被认为是浸润T细胞群体中最主要的一群效应T细胞。因此，能够同时靶向PVRIG和TIGIT的双特异性抗体具有有潜在的协同效果，是用于单一抗体疗法的有吸引力的治疗方式。此类双特异性抗体将允许同时靶向两个免疫检查点受体同时能与现有的抗PD-1/L-1抗体疗法有潜在的进一步协同效果，在癌症治疗中提供新到的治疗手段发挥重要作用。
发明内容
鉴于双特异性抗体潜在的协同效果，为了提高免疫抑制作用，解决免疫检查点抑制剂应答效率不佳的问题，特提出本发明。
本发明提供抗PVRIG/抗TIGIT抗体，编码其的核酸，抗体制备方法，含有所述抗体的药 物组合物，以及药物组合物用于治疗肿瘤的相关用途。
在第一个方面，本发明提供了一种抗PVRIG/抗TIGIT双特异性抗体，其包含：
(a)第一抗原结合部分，其包括重链可变区(VH)和轻链可变区(VL)，所述VH和VL形成抗TIGIT抗原结合域；其中，所述TIGIT VH包含SEQ ID NO：72或87任一项所述VH的HCDR1、HCDR2和HCDR3；所述TIGIT VL包含SEQ ID NO：68或91任一项所述VL的LCDR1、LCDR2和LCDR3；
(b)第二抗原结合部分，其包括特异性结合PVRIG的VHH，所述VHH包含SEQ ID NO：200或211任一项所述序列的CDR1、CDR2和CDR3。
在一些实施例中，(a)所述第一抗原结合部分的HCDR1包含SEQ ID NO：21或33任一项所述的序列；HCDR2包含SEQ ID NO：22或34任一项所述的序列；HCDR3包含SEQ ID NO：23或35任一项所述的序列；
(b)所述第一抗原结合部分的LCDR1包含SEQ ID NO：18或96任一项所述的序列；LCDR2包含SEQ ID NO：19或31任一项所述的序列；LCDR3包含SEQ ID NO：20或32任一项所述的序列；
(c)所述第二抗原结合部分的CDR1包含SEQ ID NO：168或147任一项所述的序列；CDR2包含SEQ ID NO：207或148任一项所述的序列；CDR3包含SEQ ID NO：208或149任一项所述的序列。
在一些实施例中，所述第一抗原结合部分包含以下序列的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3：
(1)分别为SEQ ID NO：21、22、23、18、19和20；或
(2)分别为SEQ ID NO：33、34、35、96、31和32；或
(3)与上述(1)至(2)所示序列具有至少90％同一性或具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列，优选地，所述替换为保守氨基酸的替换。
在一些实施例中，所述第二抗原结合部分包含以下序列的CDR1、CDR2和CDR3：
(1)分别为SEQ ID NO：168、207和208；或
(2)分别为SEQ ID NO：147、148和149；或
(3)与上述(1)至(2)所示序列具有至少90％同一性或具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列，优选地，所述替换为保守氨基酸的替换。
在一些实施例中，所述第一抗原结合部分的VH包含与SEQ ID NO：72或87所示氨基酸序列至少90％同一性的序列；所述第一抗原结合部分的VL包含SEQ ID NO：68或91所示氨基酸序列至少90％同一性的序列。
在一些实施例中，第二抗原结合部分包含SEQ ID NO：200或211所示氨基酸序列至少90％同一性的序列。
在一些实施例中，所述第一抗原结合部分是一种全长抗体，包括两条重链和两条轻链；所述第二抗原结合部分的C端融合到第一抗原结合部分的至少一条重链的N端。
在一些实施例中，重链融合多肽从N端到C端包括PVRIG VHH-(G4S)4 Linker-TIGIT  VH-CH1-铰链-CH2-CH3，轻链多肽从N端到C端包括TIGIT VL-CL。
在一些实施例中，所述重链融合多肽包括与SEQ ID NO：227、229、231或233所示氨基酸序列至少80％同一性的序列，轻链多肽包括与SEQ ID NO：226、228、230或232所示氨基酸序列至少80％同一性的序列。
在一些实施例中，所述双特异性抗体为人源化抗体。
在一些实施例中，所述双特异性抗体与人、猴PRVIG或TIGIT蛋白特异性结合；优选地，其与人、猴TIGIT结合的KD优于1.00E-7M，与人、猴PRVIG结合的KD优于1.00E-8M；更优选地，可同时与TIGIT和PVRIG相结合。
在另一方面，本发明提供了一种特异性结合TIGIT的抗体或抗原结合片段，其包含：
(1)重链可变区(VH)，其中所述重链可变区包含三个互补决定区(HCDR)：HCDR1、HCDR2和HCDR3，其中，按照Kabat编号系统编号，所述HCDR1包含SEQ ID NO:21、27、33、39所示的氨基酸序列，所述HCDR2包含SEQ ID NO:22、28、34、40所示的氨基酸序列，所述HCDR3包含SEQ ID NO:23、29、35、41所示的氨基酸序列；按照IMGT编号系统编号，所述HCDR1包含SEQ ID NO:45、51、57、63所示的氨基酸序列，所述HCDR2包含SEQ ID NO:46、52、58、64，所述HCDR3包含SEQ ID NO:47、53、59、65；以及，
轻链可变区(VL)，其中所述轻链可变区包含三个互补决定区(LCDR)：LCDR1、LCDR2和LCDR3，其中，按照Kabat编号系统编号，所述LCDR1包含SEQ ID NO:18、24、30、36、93、94、95、96所示的氨基酸序列，所述LCDR2包含SEQ ID NO:19、25、31、37所示的氨基酸序列，所述LCDR3包含SEQ ID NO:20、26、32、38所示的氨基酸序列；按照IMGT编号系统编号，所述LCDR1包含SEQ ID NO:42、48、54、60所示的氨基酸序列，所述LCDR2包含SEQ ID NO:43、49、55、61所示的氨基酸序列，所述LCDR3包含SEQ ID NO:44、50、56、62所示的氨基酸序列。
在一些实施例中，所述抗体或抗原结合片段包含以下序列的LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3：
(1)分别为SEQ ID NO:18、19、20、21、22和23；或
(2)分别为SEQ ID NO:24、25、26、27、28和29；或
(3)分别为SEQ ID NO:30、31、32、33、34和35；或
(4)分别为SEQ ID NO:36、37、38、39、40和41；或
(5)分别为SEQ ID NO:42、43、44、45、46和47；或
(6)分别为SEQ ID NO:48、49、50、51、52和53；或
(7)分别为SEQ ID NO:54、55、56、57、58和59；或
(8)分别为SEQ ID NO:60、61、62、63、64和65；或
(9)分别为SEQ ID NO:93、31、32、33、34和35；或
(10)分别为SEQ ID NO:94、31、32、33、34和35；或
(11)分别为SEQ ID NO:95、31、32、33、34和35；或
(12)分别为SEQ ID NO:96、31、32、33、34和35；或
(13)与上述(1)至(12)所示序列具有至少80％同一性或具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列，优选地，所述替换为保守氨基酸的替换。
在一些实施例中，所述抗体或抗原结合片段包含：
(1)重链可变区，其包含与SEQ ID NO:10、11、12、13、69、70、71、72、81、82、83、84、85、87、101、102或103具有至少80％同一性的氨基酸序列；或/和
(2)轻链可变区，其包含与SEQ ID NO:14、15、16、17、66、67、68、78、79、80、86、88、89、90、91、98、99或100具有至少80％同一性的氨基酸序列。
在一些实施例中，所述抗体或抗原结合片段：
(1)包含SEQ ID NO:10所示氨基酸序列的重链可变区及包含SEQ ID NO:14所示氨基酸序列的轻链可变区；或
(2)包含SEQ ID NO:11所示氨基酸序列的重链可变区及包含SEQ ID NO:15所示氨基酸序列的轻链可变区；或
(3)包含SEQ ID NO:12所示氨基酸序列的重链可变区及包含SEQ ID NO:16所示氨基酸序列的轻链可变区；或
(4)包含SEQ ID NO:13所示氨基酸序列的重链可变区及包含SEQ ID NO:17所示氨基酸序列的轻链可变区；或
(5)包含SEQ ID NO:69所示氨基酸序列的重链可变区及包含SEQ ID NO:66、67或68所示氨基酸序列的轻链可变区；或
(6)包含SEQ ID NO:70所示氨基酸序列的重链可变区及包含SEQ ID NO:66、67或68所示氨基酸序列的轻链可变区；或
(7)包含SEQ ID NO:71所示重链可变区及包含SEQ ID NO:66、67或68所示氨基酸序列的轻链可变区；或
(8)包含SEQ ID NO:72所示氨基酸序列的重链可变区及包含SEQ ID NO:66、67或68所示氨基酸序列的轻链可变区；或
(9)包含SEQ ID NO:81、82、83、84或85所示氨基酸序列的重链可变区及包含SEQ ID NO:78所示氨基酸序列的轻链可变区；或
(10)包含SEQ ID NO:81、82、83、84或85所示氨基酸序列的重链可变区及包含SEQ ID NO:79所示氨基酸序列的轻链可变区；或
(11)包含SEQ ID NO:81、82、83、84或85所示氨基酸序列的重链可变区及包含SEQ ID NO:80所示氨基酸序列的轻链可变区；或
(12)包含SEQ ID NO:87所示氨基酸序列的重链可变区及包含SEQ ID NO:86、88、89、90或91所示氨基酸序列的轻链可变区；或
(13)包含SEQ ID NO:101所示氨基酸序列的重链可变区及包含SEQ ID NO:98、99或100所示氨基酸序列的轻链可变区；或
(14)包含SEQ ID NO:102所示氨基酸序列的重链可变区及包含SEQ ID NO:98、99或100所示氨基酸序列的轻链可变区
(15)包含SEQ ID NO:103所示氨基酸序列的重链可变区及包含SEQ ID NO:98、99或100所示氨基酸序列的轻链可变区；或
与上述(1)至(15)所示序列具有至少80％同一性或至多20个突变的序列；所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
在一些实施例中，所述抗体或抗原结合片段包含重链可变区，其中，所述重链可变区与SEQ ID NO.10所示VH相比至少具有选自下组的突变：按自然顺序编号，S30T，G44K，W47Y，I48M，V67I或V71R；优选地，至少具有S30T和V71R突变；更优选地，至少具有S30T，G44K和V71R突变；更优选地，至少具有S30T，G44K，I48M，V67I和V71R突变；更优选地，至少具有S30T，G44K，W47Y和V71R突变；
或与SEQ ID NO.11所示VH相比至少具有选自下组的突变：按自然顺序编号，T28A，R72A，T74K或A76S；优选地，至少具有T28A，R72A，T74K和A76S；
或与SEQ ID NO.12所示VH相比至少具有选自下组的突变：按自然顺序编号，I29M，S30T，G44K，W47Y，I48M，V67I或V71R；优选地，至少具有S30T和V71R突变；更优选地，至少具有I29M，S30T和V71R突变；更优选地，至少具有I29M，S30T，G44K和V71R突变；更优选地，至少具有I29M，S30T，G44K，I48M，V67I和V71R突变；更优选地，至少具有I29M，S30T，G44K，W47Y和V71R；
或与SEQ ID NO.13所示VH相比至少具有选自下组的突变：按自然顺序编号，R44G，R72V，T74K，S75L或A76S；优选地，至少具有R72V和T74K突变；更优选地，至少具有R72V，T74K，S75L和A76S突变；更优选地，至少具有R44G，R72V，T74K，S75L和A76S突变。
在一些实施例中，所述抗体或抗原结合片段包含轻链可变区，其中，所述轻链可变区与SEQ ID NO.14所示VL相比至少具有选自下组的突变：按自然顺序编号，L37Q，P43S或L47M；优选地，至少具有L47M突变；更优选地，至少具有L37Q和L47M突变；更优选地，至少具有P43S和L47M突变；
或与SEQ ID NO.15所示VL相比至少具有选自下组的突变：按自然顺序编号，N31Q，N31T，N31D，G32A，Q38H或P43S；优选地，至少具有Q38H和P43S突变；更优选地，至少具有N31Q，Q38H和P43S突变；更优选地，至少具有N31T，Q38H和P43S突变；更优选地，至少具有N31D，Q38H和P43S突变；更优选地，至少具有G32A，Q38H和P43S突变；
或与SEQ ID NO.16所示VL相比至少具有选自下组的突变：按自然顺序编号，L37Q，P43S或Q45K；优选地，至少具有L37Q和Q45K突变；更优选地，至少具有P43S突变；
或与SEQ ID NO.17所示VL相比至少具有选自下组的突变：按自然顺序编号，A43S，P43S或I48V；优选地，至少具有A43S突变；更优选地，至少具有A43S和I48V突变；更优选地，至少具有P43S和I48V突变。
在一些实施例中，所述抗体或抗原结合片段与人、猴TIGIT蛋白特异性结合；优选地， 其与人、猴TIGIT结合的KD优于1.00E-8M。
在一些实施例中，所述抗体或抗原结合片段为鼠抗体、人源化抗体、全人抗体或嵌合抗体。
在一些实施例中，所述抗体或抗原结合片段选自单克隆抗体、多克隆抗体、天然抗体、工程化抗体、单特异性抗体、多特异性分子(例如双特异性抗体)、单价抗体、多价抗体、完整抗体、完整抗体的片段、裸抗体、缀合抗体、嵌合抗体、人源化抗体、全人抗体、Fab、Fab’、Fab’-SH、F(ab’)2、Fd、Fv、scFv、双抗体(diabody)或单域抗体。
在另一方面，本发明提供了一种特异性结合PVRIG的纳米抗体或抗原结合片段，其包含SEQ ID NO.107-119、198-204、211-216、219-225任一项所述VH的HCDR1、HCDR2和HCDR3。
在一些实施例中，所述纳米抗体或抗原结合片段的所述HCDR1、HCDR2和HCDR3根据IMGT编号系统确定，例如选自表21；所述HCDR1、HCDR2和HCDR3根据Kabat编号系统确定，例如选自表22、表29。
在一些实施例中，所述纳米抗体或抗原结合片段，SEQ ID NO.107所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：120～122或SEQ ID NO：159～161所示的序列；
SEQ ID NO.108所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：123～125或SEQ ID NO：162～164所示的序列；
SEQ ID NO.109所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：126～128或SEQ ID NO：165～167所示的序列；
SEQ ID NO.110所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：129～131或SEQ ID NO：168～170所示的序列；
SEQ ID NO.111所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：132～134、SEQ ID NO：171～173所示的序列；
SEQ ID NO.112所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：135～137或SEQ ID NO：174～176所示的序列；
SEQ ID NO.113所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：138～140或SEQ ID NO：177～179所示的序列；
SEQ ID NO.114所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：141～143或SEQ ID NO：180～182所示的序列；
SEQ ID NO.115所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：144～146或SEQ ID NO：183～185所示的序列；
SEQ ID NO.116所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：147～149或SEQ ID NO：186～188所示的序列；
SEQ ID NO.117所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：150～152或SEQ ID NO：189～191所示的序列；
SEQ ID NO.118所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：153～155或SEQ ID NO：192～194所示的序列；
SEQ ID NO.119所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：156～158或SEQ ID NO：195～197所示的序列；
SEQ ID NO.198所示VH的HCDR1～3按照Kabat编号系统，具有如SEQ ID NO：168～170所示的序列；
SEQ ID NO.199所示VH的HCDR1～3按照Kabat编号系统，具有如SEQ ID NO：168、207和170所示的序列；
SEQ ID NO.200所示VH的HCDR1～3按照Kabat编号系统，具有如SEQ ID NO：168、207和208所示的序列；
SEQ ID NO.201所示VH的HCDR1～3按照Kabat编号系统，具有如SEQ ID NO：168、207和209所示的序列；
SEQ ID NO.202所示VH的HCDR1～3按照Kabat编号系统，具有如SEQ ID NO：168、169和208所示的序列；
SEQ ID NO.203、204所示VH的HCDR1～3按照Kabat编号系统，具有如SEQ ID NO：168、210和208所示的序列；
SEQ ID NO.211～215所示VH的HCDR1～3按照IMGT编号系统，具有如SEQ ID NO：147～149所示的序列；
SEQ ID NO.216所示VH的HCDR1～3按照IMGT编号系统，具有如SEQ ID NO：147、148和218所示的序列；
SEQ ID NO.219-225所示VH的HCDR1～3按照IMGT编号系统，具有如SEQ ID NO：156～158所示的序列。
在一些实施例中，所述纳米抗体或抗原结合片段包含与所述HCDR1、HCDR2和HCDR3相比具有至少80％同一性或具有1、2、3或更多个氨基酸插入、缺失和/或替换的CDRs序列，优选地，所述替换为保守氨基酸的替换。
在一些实施例中，所述纳米抗体或抗原结合片段包含SEQ ID NO.107-119、198-204、211-216、219-225任一项所示的VH，或者与SEQ ID NO.107-119、198-204、211-216、219-225任一项所示VH具有至少80％同一性或至多20个突变的序列；所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
在一些实施例中，所述纳米抗体或抗原结合片段包含与SEQ ID NO.110所示VH相比至少具有选自下组的突变序列：按自然顺序编号，A97V、K98E、N54D、N108S、S110A、G55A或S75T；更优选地，至少具有A97V和K98E突变；更优选地，至少具有A97V、K98E和N54D突变；更优选地，至少具有A97V、K98E、N54D和N108S突变；更优选地，至少具有A97V、K98E、N54D和S110A突变；更优选地，至少具有A97V、K98E和N108S突变；更优选地，至少具有A97V、K98E、G55A和N108S突变；更优选地，至少具有S75T、A97V、K98E、G55A和N108S突变；
或包含与SEQ ID NO：116所示VH相比，至少具有选自下组的突变序列：按自然顺序编号，S35T、V37F、G44E、L45R、W47F、N50T、L79V、V61S、D62H、T122I或M123Q；更优选地，至少具有V37F、G44E、L45R、W47F和N50T突变；更优选地，至少具有S35T、V37F、G44E、L45R、W47F和N50T突变；更优选地，至少具有S35T、V37F、G44E、L45R、W47F、N50T和L79V突变；更优选地，至少具有S35T、V37F、G44E、L45R、W47F、N50T、V61S和D62H突变；更优选地，至少具有S35T、V37F、G44E、L45R、W47F、N50T、T122I和M123Q突变；
或包含与SEQ ID NO：119所示VH相比，至少具有选自下组的突变序列：按自然顺序编号，S35G、V37Y、G44D、L45R、W47L、N50T、Y58K、Y59I、D72G、N73D、Y79S、L78V或Y94F；更优选地，至少具有S35G、V37Y、G44D、L45R、W47L和N50T突变；更优选地，至少具有S35G、V37Y、G44D、L45R、W47L、N50T和Y58K突变；更优选地，至少具有S35G、V37Y、G44D、L45R、W47L、N50T、Y58K、D72G和N73D突变；更优选地，至少具有S35G、V37Y、G44D、L45R、W47L、N50T、Y58K、D72G、N73D和Y79S突变；更优选地，至少具有S35G、V37Y、G44D、L45R、W47L、N50T、Y58K、D72G、N73D和L78V突变；更优选地，至少具有S35G、V37Y、G44D、L45R、W47L、N50T、Y58K、Y59I、D72G和N73D突变；更优选地，至少具有S35G、V37Y、G44D、L45R、W47L、N50T、Y58K、D72G、N73D和Y94F突变。
在一些实施例中，所述纳米抗体或抗原结合片段为：(1)嵌合纳米抗体或其片段；(2)人源化纳米抗体或其片段；或(3)全人纳米抗体或其片段。
在一些实施例中，所述纳米抗体或抗原结合片段包含或不包含抗体重链恒定区；可选的，所述抗体重链恒定区可选自人、羊驼、小鼠、大鼠、兔或羊；可选地，所述抗体重链恒定区可选自IgG、IgM、IgA、IgE或IgD，所述IgG可选自IgG1，IgG2，IgG3或IgG4；可选地，所述重链恒定区可选自Fc区、CH3区或完整重链恒定区，优选地，所述重链恒定区为人Fc区；优选地，所述纳米抗体或抗原结合片段为重链抗体。
在另一方面，本发明所述抗PVRIG/抗TIGIT双特异性抗体，所述特异性结合TIGIT的抗体或抗原结合片段，所述特异性结合PVRIG的纳米抗体或抗原结合片段还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自药物、毒素、放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂。
在另一方面，本发明提供了一种多特异性分子，其包含所述抗PVRIG/抗TIGIT双特异性抗体，所述特异性结合TIGIT的抗体或抗原结合片段；或所述特异性结合PVRIG的纳米抗体或抗原结合片段；优选地，所述多特异性分子可为双特异性、三特异性或四特异性，更优选地，所述多特异性分子可为二价、四价或六价。
在一些实施例中，所述多特异性分子为串联scFv、双功能抗体(Db)、单链双功能抗体(scDb)、双重亲和力再靶向(DART)抗体、F(ab')2、双重可变域(DVD)抗体、臼包杵(KiH)抗体、对接及锁定(DNL)抗体、化学交联抗体、杂多聚纳米抗体或异结合物抗体。
在另一方面，本发明提供了一种嵌合抗原受体(CAR)，其至少包含细胞外抗原结合结 构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含所述特异性结合TIGIT的抗体或抗原结合片段；或包含所述特异性结合PVRIG的纳米抗体或抗原结合片段。
在另一方面，本发明提供了一种免疫效应细胞，其表达所述嵌合抗原受体，或包含编码所述嵌合抗原受体的核酸片段；优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、DNT细胞(double negative T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞，所述T细胞优选自细胞毒性T细胞、调节性T细胞或辅助性T细胞；优选地，所述免疫效应细胞为自体免疫效应细胞或同种异体免疫效应细胞。
在另一方面，本发明提供了一种分离的核酸片段，其编码上述任一种双特异性抗体，上述任一种特异性结合TIGIT的抗体或抗原结合片段；上述任一种特异性结合PVRIG的纳米抗体或抗原结合片段，上述任一种多特异性分子，或上述任一种嵌合抗原受体。
在另一方面，本发明提供了一种载体(vector)，其包含上述核酸片段。
在另一方面，本发明提供了一种宿主细胞，其包含上述载体；优选地，所述细胞为原核细胞或真核细胞，例如细菌(大肠杆菌)、真菌(酵母)、昆虫细胞或哺乳动物细胞(CHO细胞系或293T细胞系)。
在另一方面，本发明提供了一种制备上述任一种双特异性抗体的方法，上述任一种特异性结合TIGIT的抗体或抗原结合片段；上述任一种特异性结合PVRIG的纳米抗体或抗原结合片段，或上述任一种多特异性分子，其包括培养上述宿主细胞，以及分离所述细胞表达的抗体或分子。
在另一方面，本发明提供了一种制备所述免疫效应细胞的方法，其包括将编码上述任一种CAR的核酸片段导入所述免疫效应细胞，可选地，还包括启动所述免疫效应细胞表达上述任一种CAR。
在另一方面，本发明提供了一种药物组合物，其包含上述任一种双特异性抗体，上述任一种特异性结合TIGIT的抗体或抗原结合片段；上述任一种特异性结合PVRIG的纳米抗体或抗原结合片段，上述任一种多特异性分子，上述免疫效应细胞，核酸片段，载体，宿主细胞，或所述方法制备获得的产品，和药学上可接受的载体。
在一些实施例中，所述药物组合物进一步包含另外的治疗剂；优选地，所述另外的治疗剂是抗肿瘤剂；更优选地，所述抗肿瘤剂是PD-1轴结合拮抗剂。
在另一方面，还提供了本发明公开的上述任一种双特异性抗体，上述任一种特异性结合TIGIT的抗体或抗原结合片段；上述任一种特异性结合PVRIG的纳米抗体或抗原结合片段，上述任一种多特异性分子，上述免疫效应细胞，核酸片段，载体，宿主细胞，所述方法制备获得的产品，或药物组合物在制备用于治疗癌症或感染性疾病的药物中的用途；其中所述癌症选自实体肿瘤和血液肿瘤，优选地，所述肿瘤选自白血病，多发性骨髓瘤，淋巴瘤，骨髓增生异常综合征，前列腺癌，肝癌，结直肠癌，肛门癌、卵巢癌，子宫内膜癌，宫颈癌，腹腔癌、乳腺癌，胰腺癌，胃癌，头颈癌，甲状腺癌，睾丸癌，泌尿道上皮癌，肺癌，黑色素瘤，非黑素瘤皮肤癌，神经胶质瘤，肾癌，间皮瘤，食道癌，非小细胞肺癌，小细胞肺癌，膀胱癌，肉瘤，成胶质细胞瘤，胸腺癌，蕈样肉芽肿，默克尔细胞癌，高MSI癌和KRAS突变型肿瘤。
在一些实施例中，所述药物与另外的治疗剂或与手术组合使用；其中所述另外的治疗剂或所述手术选自放射疗法、化学疗法、溶瘤药物、细胞毒性剂、细胞因子、外科手术、免疫刺激性抗体、免疫调节药物、共刺激分子的激活剂、抑制性分子的抑制剂、疫苗或细胞免疫疗法。
在一些实施例中，所述另外的治疗剂在所述药物之前或之后施用，或与所述药物并行施用。
在一些实施例中，所述药物与PD-1轴结合拮抗剂组合使用。
在一些实施例中，所述PD-1轴结合拮抗剂选自由PD-1结合拮抗剂，PD-L1结合拮抗剂，和PD-L2结合拮抗剂组成的组；优选地，所述PD-1结合拮抗剂是抗PD-1抗体；更优选地，所述PD-1结合拮抗剂选自由MDX 1106(nivolumab)，MK-3475(pembrolizumab)，CT-011(pidilizumab)，MEDI-0680(AMP-514)，PDR001，REGN2810，和BGB-108组成的组；优选地，所述PD-L1结合拮抗剂是抗PD-L1抗体；更优选地，所述PD-L1结合拮抗剂选自由MPDL3280A(atezolizumab)，YW243.55.S70，MDX-1105，MEDI4736(durvalumab)，Tecentriq和MSB0010718C(avelumab)组成的组；优选地，所述PD-L2结合拮抗剂是抗PD-L2抗体；更优选地，所述PD-L2结合拮抗剂是免疫粘附素。
在另一方面，本发明还提供了一种治疗癌症或感染性疾病的方法，包含向有此需要的患者施用有效量的上述任一种双特异性抗体，上述任一种特异性结合TIGIT的抗体或抗原结合片段；上述任一种特异性结合PVRIG的纳米抗体或抗原结合片段，上述任一种多特异性分子，上述免疫效应细胞，核酸片段，载体，宿主细胞，所述方法制备获得的产品，或药物组合物；其中所述癌症选自实体肿瘤和血液肿瘤，优选地，所述肿瘤选自白血病，多发性骨髓瘤，淋巴瘤，骨髓增生异常综合征，前列腺癌，肝癌，结直肠癌，肛门癌、卵巢癌，子宫内膜癌，宫颈癌，腹腔癌、乳腺癌，胰腺癌，胃癌，头颈癌，甲状腺癌，睾丸癌，泌尿道上皮癌，肺癌，黑色素瘤，非黑素瘤皮肤癌，神经胶质瘤，肾癌，间皮瘤，食道癌，非小细胞肺癌，小细胞肺癌，膀胱癌，肉瘤，成胶质细胞瘤，胸腺癌，蕈样肉芽肿，默克尔细胞癌，高MSI癌和KRAS突变型肿瘤。
在一些实施例中，所述方法还包括向有此需要的患者施用有效量的PD-1轴结合拮抗剂，其中，所述PD-1轴结合拮抗剂选自由PD-1结合拮抗剂，PD-L1结合拮抗剂，和PD-L2结合拮抗剂组成的组；优选地，所述PD-1结合拮抗剂是抗PD-1抗体；更优选地，所述PD-1结合拮抗剂选自由MDX 1106(nivolumab)，MK-3475(pembrolizumab)，CT-011(pidilizumab)，MEDI-0680(AMP-514)，PDR001，REGN2810，和BGB-108组成的组；优选地，所述PD-L1结合拮抗剂是抗PD-L1抗体；更优选地，所述PD-L1结合拮抗剂选自由MPDL3280A(atezolizumab)，YW243.55.S70，MDX-1105，MEDI4736(durvalumab)，Tecentriq和MSB0010718C(avelumab)组成的组；优选地，所述PD-L2结合拮抗剂是抗PD-L2抗体；更优选地，所述PD-L2结合拮抗剂是免疫粘附素。
在另一方面，本发明还提供了上述任一种双特异性抗体，上述任一种特异性结合TIGIT的抗体或抗原结合片段；上述任一种特异性结合PVRIG的纳米抗体或抗原结合片段，上述任一种多特异性分子，上述免疫效应细胞，核酸片段，载体，宿主细胞，所述方法制备获得的产品，或药物组合物，用于预治疗癌症或感染性疾病；其中所述癌症选自实体肿瘤和血液肿 瘤，优选地，所述肿瘤选自白血病，多发性骨髓瘤，淋巴瘤，骨髓增生异常综合征，前列腺癌，肝癌，结直肠癌，肛门癌、卵巢癌，子宫内膜癌，宫颈癌，腹腔癌、乳腺癌，胰腺癌，胃癌，头颈癌，甲状腺癌，睾丸癌，泌尿道上皮癌，肺癌，黑色素瘤，非黑素瘤皮肤癌，神经胶质瘤，肾癌，间皮瘤，食道癌，非小细胞肺癌，小细胞肺癌，膀胱癌，肉瘤，成胶质细胞瘤，胸腺癌，蕈样肉芽肿，默克尔细胞癌，高MSI癌和KRAS突变型肿瘤。
本发明的抗PVRIGxTIGIT人源化双特异性抗体可特异性靶向肿瘤细胞，有效介导对肿瘤细胞系的杀伤作用，在取得优异的肿瘤抑制效果的同时，具备良好的安全性。
术语定义和说明
除非本发明另外定义，与本发明相关的科学和技术术语应具有本领域普通技术人员所理解的含义。
此外，除非本文另有说明，本文单数形式的术语应包括复数形式，复数形式的术语应包括单数形式。更具体地，如在本说明书和所附权利要求中所使用的，除非另外明确指出，否则单数形式“一种”和“这种”包括复数指示物。
本文术语“包括”、“包含”和“具有”之间可互换使用，旨在表示方案的包含性，意味着所述方案可存在除所列出的元素之外的其他元素。同时应当理解，在本文中使用“包括”、“包含”和“具有”描述，也提供“由……组成”方案。示例性地，“一种组合物，包括A和B”，应当理解为以下技术方案：由A和B组成的组合物，以及除A和B外，还含有其他组分的组合物，均落入前述“一种组合物”的范围内。
术语“和/或”在本文使用时，包括“和”、“或”和“由所属术语链接的要素的全部或任何其他组合”的含义。
术语“具有Ig和ITIM域的T细胞免疫受体”、“TIGIT”、“TIGIT抗原”、“Vstm3”和“WUCAM”可互换使用，并且包括各种哺乳动物同种型，例如人Tigit、人Tigit的直系同源物、和包含Tigit内的至少一个表位的类似物，以及具有至少一个与TIGIT共有的表位的类似物。TIGIT(例如人TIGIT)的氨基酸序列、以及编码其的核苷酸序列是本领域已知的。
本文术语“PVRIG”或“PVRIG蛋白质”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ECD片段。结合到PVRIG并且防止被PVRL2活化(例如，最常通过阻断PVRIG和PVLR2的相互作用)的“抗PVRIG抗体”(包括抗原结合片段)用于增强T细胞和/或NK细胞活化并且用于治疗疾病，如癌症和病原体感染。
本文术语“抗PVRIG/抗TIGIT抗体”和“双特异性PVRIG/TIGIT抗体”和“抗PVRIG/抗TIGIT双特异性抗体”可互换地使用，本发明的抗PVRIG/抗TIGIT双特异性抗体特异性地结合于人类TIGIT，并且优选是人类TIGIT的ECD，以及PVRIG，并且再优选是人类PVRIG的ECD。
本文术语“特异性结合”是指抗原结合分子(例如抗体)通常以高亲和力特异性结合抗原和实质上相同的抗原，但不以高亲和力结合不相关抗原。亲和力通常以平衡解离常数(equilibrium dissociation constant，KD)来反映，其中较低KD表示较高亲和力。以抗体为 例，高亲和力通常指具有约1×10 -7M或更低、约1×10 -8M或更低、约1×10 -9M或更低、约1×10 -10M或更低、1×10 -11M或更低或1×10 -12M或更低的KD。KD计算方式如下：KD＝Kd/Ka，其中Kd表示解离速率，Ka表示结合速率。可采用本领域周知的方法测量平衡解离常数KD，如表面等离子共振(例如Biacore)或平衡透析法测定。
本文术语“抗原结合分子”按最广义使用，是指特异性结合抗原的分子。示例性地，抗原结合分子包括但不限于抗体或抗体模拟物。“抗体模拟物”是指能够与抗原特异性结合，但与抗体结构无关的有机化合物或结合域，示例性地，抗体模拟物包括但不限于affibody、affitin、affilin、经设计的锚蛋白重复蛋白(DARPin)、核酸适体或Kunitz型结构域肽。
本文术语“抗体”按最广义使用，是指包含来自免疫球蛋白重链可变区的足够序列和/或来自免疫球蛋白轻链可变区的足够序列，从而能够特异性结合至抗原的多肽或多肽组合。本文“抗体”涵盖各种形式和各种结构，只要它们展现出期望的抗原结合活性。本文“抗体”包括具有移植的互补决定区(CDR)或CDR衍生物的替代蛋白质支架或人工支架。此类支架包括抗体衍生的支架(其包含引入以例如稳定化抗体三维结构的突变)以及包含例如生物相容性聚合物的全合成支架。参见，例如Korndorfer et al.,2003,Proteins:Structure,Function,and Bioinformatics,53(1):121-129(2003)；Roque et al.,Biotechnol.Prog.20:639-654(2004)。此类支架还可以包括非抗体衍生的支架，例如本领域已知可用于移植CDR的支架蛋白，包括但不限于肌腱蛋白、纤连蛋白、肽适体等。
本文“抗体”包括一种典型的“四链抗体”，其属于由两条重链(HC)和两条轻链(LC)组成的免疫球蛋白；重链是指这样的多肽链，其在N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成；并且，当所述全长抗体为IgE同种型时，任选地还包括重链恒定区CH4结构域；轻链是在N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链；重链与重链之间、重链与轻链之间通过二硫键连接，形成“Y”字型结构。由于免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将本文“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，IgA可分为IgA1和IgA2。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链或λ链。
本文“抗体”还包括不包含轻链的抗体，例如，由单峰驼(Camelus dromedarius)、双峰驼(Camelus bactrianus)、大羊驼(Lama glama)、原驼(Lama guanicoe)和羊驼(Vicugna pacos)等骆驼科动物产生的重链抗体(heavy-chain antibodies,HCAbs)以及在鲨等软骨鱼纲中发现的免疫球蛋白新抗原受体(Ig new antigen receptor,IgNAR)。
如本文所用，术语“重链抗体”是指缺乏常规抗体的轻链的抗体。该术语具体包括但不限于在不存在CH1结构域的情况下包含VH抗原结合结构域以及CH2和CH3恒定结构域的同型二聚体抗体。
如本文所用，术语“纳米抗体”是指骆驼体内存在天然的缺失轻链的重链抗体，克隆其可变区可以得到只有重链可变区组成的单域抗体，也称为VHH(Variable domain of heavy chain of heavy chain antibody)，它是最小的功能性抗原结合片段。
本文术语“纳米抗体(nanobody)”、“单域抗体”(single domain antibody,sdAb)具有相同的含义并可互换使用，是指克隆重链抗体的可变区，构建仅由一个重链可变区组成的单域抗体，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的重链抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体。
关于“重链抗体”和“纳米抗体”的进一步描述可参见：Hamers-Casterman等，Nature.1993；363；446-8；Muyldermans的综述文章(Reviews inMolecular Biotechnology 74：277-302，2001)；以及以下专利申请，其被作为一般背景技术提及：WO 94/04678，WO 95/04079和WO 96/34103；WO94/25591，WO 99/37681，WO 00/40968，WO 00/43507，WO 00/65057，WO 01/40310，WO 01/44301，EP 1134231和WO 02/48193；WO97/49805，WO 01/21817，WO 03/035694，WO 03/054016和WO 03/055527；WO 03/050531；WO 01/90190；WO03/025020；以及WO 04/041867，WO 04/041862，WO 04/041865，WO 04/041863，WO 04/062551，WO 05/044858，WO 06/40153，WO 06/079372，WO 06/122786，WO 06/122787和WO 06/122825以及这些申请中提到的其他现有技术。
本文“抗体”可以来源于任何动物，包括但不限于人和非人动物，所述非人动物可选自灵长类动物、哺乳动物、啮齿动物和脊椎动物，例如骆驼科动物、大羊驼、原鸵、羊驼、羊、兔、小鼠、大鼠或软骨鱼纲(例如鲨)。
本文“抗体”包括但不限于单克隆抗体、多克隆抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、完整抗体、完整抗体的片段、裸抗体、缀合抗体、嵌合抗体、人源化抗体或全人抗体。
本文术语“单克隆抗体”是指从基本上同质的抗体群体获得的抗体，即，除了可能的变异体(例如含有天然存在的突变或在制剂的生产过程中产生，此类变体通常以少量存在)之外，包含所述群体的各个抗体是相同的和/或结合相同的表位。与通常包括针对不同决定簇(表位)的不同抗体的多克隆抗体制剂相反，单克隆抗体制剂中的每种单克隆抗体针对抗原上的单一决定簇。本文修饰语“单克隆”不应解释为需要通过任何特定方法产生所述抗体或抗原结合分子。举例来说，单克隆抗体可通过多种技术制得，包括(但不限于)杂交瘤技术、重组DNA方法、噬菌体库展示技术和利用含有全部或部分人免疫球蛋白基因座的转殖基因动物的方法和其它本领域已知的方法。
本文术语“单特异性”是指表示具有一个或多个结合位点，其中每个结合位点结合相同抗原的相同表位。
本文术语“多特异性”是指具有至少两个抗原结合位点，所述至少两个抗原结合位点中的每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。因此，诸如“双特异性”、“三特异性”、“四特异性”等术语是指抗体/抗原结合分子可以结合的不同表位的数目。
本文术语“价”表示抗体/抗原结合分子中规定数目的结合位点的存在。因此,术语“单价”、“二价”、“四价”和“六价”分别表示抗体/抗原结合分子中一个结合位点、两个结合位点、四个结合位点和六个结合位点的存在。
本文“全长抗体”、“完好抗体”和“完整抗体”在本文中可互换使用，是指具有基本上与天然抗体结构相似的结构。
本文“抗原结合片段”和“抗体片段”在本文中可互换使用，其不具备完整抗体的全部结构，仅包含完整抗体的局部或局部的变体，所述局部或局部的变体具备结合抗原的能力。本文“抗原结合片段”或“抗体片段”包括但不限于Fab、Fab’、Fab’-SH、F(ab’) 2、Fd、Fv、scFv、双抗体(diabody)和单域抗体。
完整抗体的木瓜蛋白酶消化生成两个同一的抗原结合片段，称作“Fab”片段，每个含有重和轻链可变域，还有轻链的恒定域和重链的第一恒定域(CH1)。如此,本文术语“Fab片段”指包含轻链的VL域和恒定域(CL)的轻链片段,和重链的VH域和第一恒定域(CH1)的抗体片段。Fab'片段因在重链CH1域的羧基末端增加少数残基而与Fab片段不同,包括来自抗体铰链区的一个或多个半胱氨酸。Fab’-SH是其中恒定域的半胱氨酸残基携带游离硫醇基团的Fab’片段。胃蛋白酶处理产生具有两个抗原结合位点(两个Fab片段)和Fc区的一部分的F(ab’) 2片段。
本文术语“Fd”是指由VH和CH1结构域组成的抗体。本文术语“Fv”是指由单臂VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为，六个CDR赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDR)也能够识别并结合抗原，尽管其亲和力可能低于完整的结合位点。
本文术语“scFv”(single-chain variable fragment)是指包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键，形成二硫键连接的Fv(dsFv)。
本文术语“双抗体(diabody)”，其VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Holliger P.等人,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)，和Poljak R.J.等人,Structure 2:1121-1123(1994))。
本文术语“裸抗体”是指不与治疗剂或示踪剂缀合的抗体；术语“缀合抗体”是指与治疗剂或示踪剂缀合的抗体，优选地，所述治疗剂选自药物、毒素、放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂。
本文术语“嵌合抗体(Chimeric antibody)”是指，这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚 类)，但无论如何，其仍保留对目标抗原的结合活性(U.S.P 4,816,567 to Cabilly et al.；Morrison et al.,Proc.Natl.Acad.Sci.USA,81:68516855(1984))。例如，术语“嵌合抗体”可包括这样的抗体(例如人鼠嵌合抗体)，其中抗体的重链和轻链可变区来自第一抗体(例如鼠源抗体)，而抗体的重链和轻链恒定区来自第二抗体(例如人抗体)。
本文术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留或部分保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力、增强免疫应答的能力等。
本文术语“全人抗体”是指具有其中FR和CDR二者都源自人种系免疫球蛋白序列的可变区的抗体。此外，如果抗体包含恒定区，则恒定区也源自人种系免疫球蛋白序列。本文全人抗体可以包括不由人种系免疫球蛋白序列编码的氨基酸残基(例如，通过体外随机或位点特异性诱变或通过体内体细胞突变引入的突变)。然而，本文“全人抗体”不包括其中来源于另一个哺乳动物物种(例如小鼠)的种系的CDR序列已被移植到人框架序列上的抗体。
本文术语“可变区”是指抗体重链或轻链中牵涉使抗体结合抗原的区域，“重链可变区”与“VH”、“HCVR”可互换使用，“轻链可变区”与“VL”、“LCVR”可互换使用。天然抗体的重链和轻链的可变域(分别是VH和VL)一般具有相似的结构,每个域包含四个保守的框架区(FR)和三个高变区(HVR)。参见例如Kindt et al.,Kuby Immunology,6th ed.,W.H.Freeman and Co.,p.91(2007)。单个VH或VL域可足以赋予抗原结合特异性。本文术语“互补决定区”与“CDR”可互换使用，通常指重链可变区(VH)或轻链可变区(VL)的高变区(HVR)，该部位因在空间结构上可与抗原表位形成精密的互补，故又称为互补决定区，其中，重链可变区CDR可缩写为HCDR，轻链可变区CDR可缩写为LCDR。本术语“构架区”或“FR区”可互换，是指抗体重链可变区或轻链可变区中除CDR以外的那些氨基酸残基。通常典型的抗体可变区由4个FR区和3个CDR区按以下顺序组成：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
对于CDR的进一步描述，参考Kabat等人,J.Biol.Chem.,252:6609-6616(1977)；Kabat等人,美国卫生与公共服务部，“Sequences of proteins of immunological interest”(1991)；Chothia等人,J.Mol.Biol.196:901-917(1987)；Al-Lazikani B.等人,J.Mol.Biol.,273:927-948(1997)；MacCallum等人,J.Mol.Biol.262:732-745(1996)；Abhinandan和Martin,Mol.Immunol.,45:3832-3839(2008)；Lefranc M.P.等人,Dev.Comp.Immunol.,27:55-77(2003)；以及Honegger和Plückthun,J.Mol.Biol.,309:657-670(2001)。本文“CDR”可由本领域公知的方式加以标注和定义，包括但不限于Kabat编号系统、Chothia编号系统或IMGT编号系统，使用的工具网站包括但不限于AbRSA网站(http://cao.labshare.cn/AbRSA/cdrs.php)、abYsis网站(www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)和IMGT网站(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results)。本文CDR包括不同定义方式的氨基酸残基的重叠(overlap)和子集。
本文术语“Kabat编号系统”通常是指由Elvin A.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。
本文术语“IMGT编号系统”通常是指基于由Lefranc等人发起的国际免疫遗传学信息系统(The international ImMunoGeneTics information system(IMGT))的编号系统，可参阅Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003。
本文术语“重链恒定区”是指抗体重链的羧基端部分，其不直接参与抗体与抗原的结合，但是表现出效应子功能，诸如与Fc受体的相互作用，其相对于抗体的可变结构域具有更保守的氨基酸序列。“重链恒定区”至少包含：CH1结构域，铰链区，CH2结构域，CH3结构域，或其变体或片段。“重链恒定区”包括“全长重链恒定区”和“重链恒定区片段”，前者具有基本上与天然抗体恒定区基本相似的结构，而后者仅包括“全长重链恒定区的一部分”。示例性地，典型的“全长抗体重链恒定区”由CH1结构域-铰链区-CH2结构域-CH3结构域组成；当抗体为IgE时，其还包括CH4结构域；当抗体为重链抗体时，则其不包括CH1结构域。示例性地，典型的“重链恒定区片段”可选自CH1、Fc或CH3结构域。
本文术语“轻链恒定区”是指抗体轻链的羧基端部分，其不直接参与抗体与抗原的结合，所述轻链恒定区可选自恒定κ结构域或恒定λ结构域。
本文术语“Fc”是指完整抗体经木瓜蛋白酶水解而成的抗体羧基端部分，典型地，其包含抗体的CH3和CH2结构域。Fc区包括例如天然序列Fc区、重组Fc区和变体Fc区。尽管免疫球蛋白重链的Fc区的边界可以略微变化，但是人IgG重链的Fc区通常被定义为从Cys226位置的氨基酸残基或从Pro230延伸至其羧基末端。Fc区的C末端赖氨酸(根据Kabat编号系统的残基447)可以例如在抗体的产生或纯化过程中，或通过对编码抗体重链的核酸重组工程化而除去，因此，Fc区可包括或不包括Lys447。
本文术语“保守氨基酸”通常是指属于同一类或具有类似特征(例如电荷、侧链大小、疏水性、亲水性、主链构象和刚性)的氨基酸。示例性地，下述每组内的氨基酸属于彼此的保守氨基酸残基，组内氨基酸残基的替换属于保守氨基酸的替换：
示例性地，以下六组是被认为是互为保守性置换的氨基酸的实例：
1)丙氨酸(A)、丝氨酸(S)、苏氨酸(T)；
2)天冬氨酸(D)、谷氨酸(E)；
3)天冬酰胺(N)、谷氨酰胺(Q)；
4)精氨酸(R)、赖氨酸(K)、组氨酸(H)；
5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；和
6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。
本文术语“同一性”可通过以下方式计算获得：为确定两个氨基酸序列或两个核酸序列的“同一性”百分数，将所述序列出于最佳比较目的比对(例如，可以为最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
考虑到为最佳比对这两个序列而需要引入的空位的数目和每个空位的长度，两个序列之间的同一性百分数随所述序列共有的相同位置变化而变化。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。例如，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。又例如，使用GCG软件包中的GAP程序(在www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
额外地或备选地，可以进一步使用本发明所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。例如，可以使用Altschul等人,(1990)J.Mol.Biol.215:403-10的NBLAST及XBLAST程序(版本2.0)执行此类检索。BLAST核苷酸检索可以用NBLAST程序，评分＝100、字长度＝12执行，以获得与本发明核酸分子同源的核苷酸序列。BLAST蛋白质检索可以用XBLAST程序、评分＝50、字长度＝3执行，以获得与本发明蛋白质分子同源的氨基酸序列。为了出于比较目的获得带空位的比对结果，可以如Altschul等人,(1997)Nucleic Acids Res.25:3389-3402中所述那样使用空位BLAST。当使用BLAST和空位BLAST程序时，可以使用相应程序(例如，XBLAST和NBLAST)的默认参数。参见www.ncbi.nlm.nih.gov。
本文术语“嵌合抗原受体(CAR)”是指经改造以在免疫效应细胞上表达并且特异性结合抗原的人工细胞表面受体，其包含至少(1)细胞外抗原结合结构域，例如抗体的可变重链或轻链，(2)锚定CAR进入免疫效应细胞的跨膜结构域，和(3)胞内信号传导结构域。CAR能够利用细胞外抗原结合结构域以非MHC限制性的方式将T细胞和其它免疫效应细胞重定向至所选择的靶标，例如癌细胞。
本文术语“免疫刺激性抗体”包括：1)靶向抑制性免疫检查点的拮抗性抗体包括抗CTLA4mAb(如伊匹单抗、曲美单抗)、抗PD-1(如纳武单抗BMS-936558/MDX-1106/ONO-4538、CT-011、lambrozilumab MK-3475、MEDI-0680(AMP-514)，PDR001，REGN2810，、BGB-108)、抗PDL-1拮抗剂(如BMS-936559/MDX-1105、MEDI4736、RG-7446/MPDL3280A、MSB0010718C、YW243.55.S70)；抗LAG-3(如IMP-321)、抗TIM-3、抗BTLA、抗B7-H4、抗B7-H3、抗VISTA。2)增强免疫刺激性蛋白的激动性抗体包括抗CD40mAb(如CP-870,893、鲁卡木单抗、达西珠单抗)、抗CD137mAb(如BMS-663513乌瑞鲁单抗、PF-05082566)、抗OX40mAb(如抗OX40)、抗GITR mAb(如TRX518)、抗CD27mAb(如CDX-1127)以及抗ICOS mAb。“免疫刺激性抗体”可通过直接调节免疫功能，即阻断其它抑制性靶标或增强免疫刺激性蛋白质，促进抗肿瘤免疫。
本文术语“免疫调节药物”可以为，如胸腺素α1。原理：胸腺素α1(Tα1)是天然产生的胸腺肽，充当先天性和适应性免疫系统的内源性调节因子。它在世界范围内用于治疗与免疫功能障碍相关的疾病，包括病毒感染，如B型和C型肝炎、某些癌症，并且用于疫苗增强。尤 其，免疫调节研究的最新进展指出了Ta1处理在脓毒性患者中的有利作用(Wu等人《危急护理》2013，17：R8)。
本文术语“核酸”包括包含核苷酸的聚合物的任何化合物和/或物质。每个核苷酸由碱基，特别是嘌呤或嘧啶碱基(即胞嘧啶(C)、鸟嘌呤(G)、腺嘌呤(A)、胸腺嘧啶(T)或尿嘧啶(U))、糖(即脱氧核糖或核糖)和磷酸基团组成。通常，核酸分子由碱基的序列描述，由此所述碱基代表核酸分子的一级结构(线性结构)。碱基的序列通常表示为5′至3′。在本文中，术语核酸分子涵盖脱氧核糖核酸(DNA)，包括例如互补DNA(cDNA)和基因组DNA、核糖核酸(RNA)，特别是信使RNA(mRNA)、DNA或RNA的合成形式，以及包含两种或更多种这些分子的混合的聚合物。核酸分子可以是线性的或环状的。此外，术语核酸分子包括有义链和反义链二者，以及单链和双链形式。而且，本文所述的核酸分子可含有天然存在的或非天然存在的核苷酸。非天然存在的核苷酸的例子包括具有衍生的糖或磷酸骨架键合或化学修饰的残基的修饰的核苷酸碱基。核酸分子还涵盖DNA和RNA分子，其适合作为载体用于在体外和/或体内，例如在宿主或患者中，直接表达本发明的抗体。此类DNA(例如cDNA)或RNA(例如mRNA)载体可以是未修饰的或修饰的。例如，可以对mRNA进行化学修饰以增强RNA载体的稳定性和/或被编码分子的表达，从而可以将mRNA注入到受试者内以在体内产生抗体(参见例如Stadler等人，Nature Medicine 2017，published online 2017年6月12日，doi：10.1038/nm.4356或EP2101823B1)。本文“分离的”核酸指已经与其天然环境的组分分开的核酸分子。分离的核酸包括在下述细胞中含有的核酸分子，所述细胞通常含有该核酸分子，但该核酸分子存在于染色体外或存在于不同于其天然染色体位置的染色体位置处。
本文术语“载体”是指能够扩增与其连接的另一个核酸的核酸分子。该术语包括作为自我复制型核酸结构的载体以及整合入已引入该载体的宿主细胞的基因组中的载体。某些载体能够指导与它们可操作连接的核酸的表达。这样的载体在本文中称为“表达载体”。
本文术语“宿主细胞”是指细胞中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞和来源于其的后代，而不考虑传代的次数。后代在核酸内容物上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括具有与在初始转化的细胞中筛选或选择的相同功能或生物学活性的突变体后代。
本文术语“药物组合物”是指这样的制剂，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不含有对施用所述药物组合物的受试者具有不可接受的毒性的另外的成分。
本文术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如癌症的进展。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
本文术语“受试者”是指接受对如本发明所述的特定疾病或病症的治疗的生物体。对象和患者的实例包括接受疾病或病症治疗的哺乳动物，如人、灵长类动物(例如，猴)或非灵 长类哺乳动物。
本文术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。当将活性成分单独给予个体时，治疗有效剂量单指该成分。当应用某一组合时，治疗有效剂量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给予。
本文术语“癌症”指向或描述哺乳动物中典型地以不受调节的细胞生长为特征的生理状况。此定义中包括良性和恶性癌症。本文术语“肿瘤”或“瘤”是指所有赘生性(neoplastic)细胞生长和增殖,无论是恶性的还是良性的,及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”和“肿瘤”在本文中提到时并不互相排斥。
本文术语“EC50”是指半最大有效浓度，其包括在指定暴露时间之后诱导基线与最大值之间的半途响应的抗体浓度。EC50本质上代表其中观察到起最大作用的50％的抗体浓度，可通过本领域已知方法测量。
本文术语“G4S连接肽”是指甘氨酸(G)和丝氨酸(S)的GS组合，用于将多个蛋白连接在一起形成融合蛋白。常用的GS组合是(GGGGS)n，通过改变n的大小来改变接头序列的长度。同时，甘氨酸和丝氨酸还可以通过其他组合产生不同的接头序列，比如在本发明中使用的(G4S)4Linker，GS组合为GGGGS。
附图说明
图1显示了抗TIGIT的人-鼠嵌合抗体与人TIGIT ECD-mFc融合蛋白的结合活性。
图2显示了抗TIGIT的人-鼠嵌合抗体与食蟹猴TIGIT ECD-mFc融合蛋白的结合活性。
图3显示了抗TIGIT的人-鼠嵌合抗体与CHO-K1人TIGIT高表达株细胞的结合活性。
图4显示了抗TIGIT的人-鼠嵌合抗体与CHO-K1人TIGIT中表达株细胞的结合活性。
图5显示了抗TIGIT的人-鼠嵌合抗体与CHO-K1人TIGIT低表达株细胞的结合活性。
图6显示了抗TIGIT的人-鼠嵌合抗体与CHO-K1食蟹猴TIGIT细胞的结合活性。
图7显示了抗TIGIT的人-鼠嵌合抗体阻断Bio-CD155-His与CHO-K1 TIGIT相互作用的效应。
图8显示了抗TIGIT的人-鼠嵌合抗体阻断TIGIT ECD-mFc与CHO-K1 CD155相互作用的效应。
图9 FACS检测不同供体来源(donor-010和donor-050)的NK细胞表面PVRIG，TIGIT及肿瘤细胞系WIDR细胞表面PVR及PVRL2表达水平。A，图中黑色空心峰图是指NK细胞表面PVRIG/TIGIT的表达，灰色实心峰图是指两个检测抗体对应的同型对照。B，图中黑色空心峰图是指WIDR细胞表面PVR/PVRL2的表达，灰色实心峰图是指两个检测抗体对应的同型对照。
图10显示了抗TIGIT的人-鼠嵌合抗体与WIDR细胞和NK细胞共孵育对NK细胞脱颗粒(CD107a)作用的影响。横坐标为受试抗体浓度，纵坐标为CD107a的阳性细胞占比。其中，RG6058-hIgG1为阳性对照抗体，anti-HEL-hIgG1为阴性同型对照抗体。
图11显示了抗TIGIT的人-鼠嵌合抗体对NK细胞杀伤WIDR靶细胞作用的影响。横坐标为受试抗体的浓度，纵坐标为靶细胞的死亡率，RG6058-hIgG1为阳性对照抗体， anti-HEL-hIgG1为阴性同型对照抗体。
图12 CMV antigen-recall assay检测抗TIGIT的人-鼠嵌合抗体对抗原特异性CD8 T细胞功能活性的影响。A，CMV IgG阳性donor 128 PBMC经CMV pp65(495-503)诱导11天后的PBMC中CD8T细胞的比例，CMV pp65特异性CD8 T细胞比例及FMO对照；B，CMV pp65特异性CD8上PVRIG、TIGIT和PD-1的表达；C，Colo205上PVRL2和PVR的表达；D，铺板共孵育18小时后细胞上清中IFN-γ的分泌水平。阳性对照为RG6058-hIgG1,阴性对照为no treatment(不加任何药物处理)，柱状图上的百分比为受试抗体相较于no treatment组IFN-γ分泌提升的百分比。
图13显示了抗TIGIT的人源化抗体与人TIGIT ECD-mFc融合蛋白的结合活性。
图14显示了抗TIGIT的人源化抗体与食蟹猴TIGIT ECD-mFc融合蛋白的结合活性。
图15显示了抗TIGIT的人源化抗体与CHO-K1人TIGIT高表达株细胞的结合活性。
图16显示了抗TIGIT的人源化抗体与CHO-K1人TIGIT中表达株细胞的结合活性。
图17显示了抗TIGIT的人源化抗体与CHO-K1人TIGIT低表达株细胞的结合活性。
图18显示了抗TIGIT的人源化抗体与CHO-K1食蟹猴TIGIT细胞的结合活性。
图19显示了抗TIGIT的人源化抗体阻断Bio-CD155-His与CHO-K1人TIGIT相互作用的效应。
图20显示了抗TIGIT的人源化抗体阻断TIGIT ECD-mFc与CHO-K1 CD155相互作用的效应。
图21显示了抗TIGIT的人源化抗体阻断TIGIT ECD-mFc与CHO-K1 CD112相互作用的效应。
图22显示了抗TIGIT的人源化抗体与人PBMC的结合活性。
图23显示了抗TIGIT的人源化抗体对NK细胞杀伤WIDR靶细胞作用的影响。图中横坐标为受试抗体的浓度，纵坐标为靶细胞的死亡率，TIGIT-CHI-002、TIGIT-CHI-005、TIGIT-CHI-006和TIGIT-CHI-070为人源化前的嵌合抗体，RG6058-hIgG1为阳性对照抗体，anti-HA HcAb-hIgG1为阴性同型对照抗体。
图24 CMV antigen-recall assay检测抗TIGIT的人源化抗体对抗原特异性CD8 T细胞功能活性的影响。A，CMV IgG阳性donor 622 PBMC经CMV pp65(495-503)诱导11天后的PBMC中CD8 T细胞的比例，CMV pp65特异性CD8 T细胞比例，FMO对照；B，CMV pp65特异性CD8 T细胞上PVRIG、TIGIT、PD-1、CD226的表达；C，铺板共孵育18小时后细胞上清中IFN-γ的分泌水平。阳性对照为RG6058-hIgG1和TIGIT-CHI-002,阴性对照为no treatment(不加任何药物处理)。柱状图上的百分比为受试抗体相较于no treatment组IFN-γ分泌提升的百分比。
图25 FACS检测人TIGIT过表达细胞株CHO-K1人TIGIT
图26A PVRIG受试抗体和人PVRIG重组蛋白的结合能力。图中显示了受试抗体PVRIG-A11、A15、A30、A35、A43、A50、A60、A75、A104、A105、A113、A117、A118与人PVRIG蛋白的结合能力。其中COM701-hIgG1、COM701-hIgG4和SRF813-hIgG1为该实验的阳性对照；anti-HA HcAb-hIgG1和anti-CD38 HcAb-hIgG1为该实验的阴性对照。
图26B PVRIG受试抗体和食蟹猴PVRIG重组蛋白的结合能力。图中显示了受试抗体 PVRIG-A11、A15、A30、A35、A43、A50、A60、A75、A104、A105、A113、A117、A118与食蟹猴PVRIG蛋白的结合能力。其中COM701-hIgG1、COM701-hIgG4和SRF813-hIgG1为该实验的阳性对照；anti-HA HcAb-hIgG1、anti-CD38 HcAb-hIgG1和anti-Fluorescein-hIgG1为该实验的阴性对照。
图27A PVRIG受试抗体与Flpin CHO-PVRIG细胞表面人PVRIG的结合活性。图中显示了受试抗体PVRIG-A11、A15、A30、A35、A43、A50、A60、A75、A104、A105、A113、A117、A118与FlpinCHO-PVRIG细胞表面人PVRIG的结合能力。其中，COM701-hIgG1及SRF813-hIgG1为阳性对照抗体，anti-CD38 HcAb-hIgG1为阴性同型对照抗体。
图27B PVRIG受试抗体与FlpinCHO-cyno PVRIG细胞表面食蟹猴PVRIG的结合活性。图中显示了受试抗体PVRIG-A11、A15、A30、A50、A60、A105、A117、A118与FlpinCHO-cyno PVRIG细胞表面食蟹猴PVRIG的结合能力。其中，COM701-hIgG1及SRF813-hIgG1为阳性对照抗体，anti-CD38 HcAb-hIgG1为阴性同型对照抗体。
图28 PVRIG受试抗体阻断人PVRIG与人PVRL2重组蛋白间的相互作用。图中显示了受试抗体PVRIG-A11、A15、A30、A35、A43、A50、A60、A75、A104、A105、A113、A117、A118对PVRIG和PVRL2的结合阻断作用，其中COM701-hIgG4和SRF813-hIgG1为该实验的阳性对照；anti-HA HcAb-hIgG1和anti-CD38 HcAb-hIgG1为该实验的阴性对照。
图29 PVRIG受试抗体阻断CHO-K1-CD112细胞与人PVRIG-mFc蛋白的结合。图中显示了受试抗体PVRIG-A11、A15、A30、A35、A43、A50、A60、A75、A104、A105、A113、A117、A118竞争CHO-K1-CD112细胞结合人PVRIG-mFc蛋白的能力。其中，COM701-hIgG4及SRF813-hIgG1为阳性对照抗体，anti-CD38 HcAb-hIgG1为阴性同型对照抗体。
图30肿瘤细胞系Reh细胞表面PVR及PVRL2表达水平。图中黑色空心峰图是指Reh细胞表面PVR/PVRL2的表达，灰色实心峰图是指检测抗体对应的同型对照。
图31 PVRIG受试抗体对NK细胞脱颗粒作用的影响。图A是指NK细胞(donor-010)与靶细胞Reh孵育时，受试抗体PVRIG-A11、A15、A30对NK细胞CD107a表达的影响；图B是指NK(donor-010)与靶细胞WIDR孵育时，受试抗体PVRIG-A60、A75、A43、A35对NK细胞CD107a表达的影响；图C是指NK细胞(donor-050)与靶细胞WIDR孵育时，受试抗体PVRIG-A104、A105、A118、A113、A117对NK细胞CD107a表达的影响；横坐标为受试抗体浓度，纵坐标为CD107a的强阳性细胞占比，其中，COM701-hIgG1及SRF813-hIgG1为阳性对照抗体，anti-HA HcAb-hIgG1为阴性同型对照抗体。
图32 PVRIG受试抗体对NK细胞对靶细胞的杀伤作用的影响。图A是指在6.87nM浓度时，受试抗体PVRIG-A11、A15、A30对NK细胞(donor-010)杀伤靶细胞WIDR的促进作用。各受试抗体与阴性同型对照anti-HA HcAb-hIgG1相比具有统计学差异(**p<0.01,***p<0.001,****p<0.0001,One-Way ANOVA Analysis)；图B是指在不同浓度下，受试抗体PVRIG-A50对NK细胞(donor-010)杀伤靶细胞WIDR的促进作用；图C是指在不同浓度下，受试抗体PVRIG-A60、A75、A35、A43、A104、A105、A113、A117、A118对NK细胞(donor-050)杀伤靶细胞WIDR的促进作用。图B和C的横坐标为受试抗体的浓度，纵坐标为靶细胞的死亡率，COM701-hIgG1及SRF813-hIgG1为阳性对照抗体，anti-HA HcAb-hIgG1为阴性同型对照抗体。
图33 CMV antigen-recall assay检测PVRIG抗体对抗原特异性CD8 T细胞的功能改善作用。图A，CMV IgG阳性donor 021 PBMC经CMV pp65(495-503)诱导11天后的PBMC中CD8的比例，CMV pp65特异性CD8比例；图B，CMV pp65特异性CD8(donor 021)上PVRIG、TIGIT、PD-1、CD226的表达；图C，colo205上PVRL2、PVR和HLA-A2的表达。图D，铺板共孵育18小时后细胞上清中IFN-γ的分泌水平。此实验体系中的阳性对照为COM701-hIgG4,SRF813-hIgG1,阴性对照为no treatment(不加任何药物处理)，抗体的作用终浓度均为70nM。和no treatment组相比，抗体PVRIG-A15,A30,A60,75,105,117和118作用后，细胞上清中IFN-γ的分泌显著增加(*p<0.05,**p<0.01,***p<0.001,****p<0.0001,one-way ANOVA Analysis)。
图34A PVRIG人源化抗体与人PVRIG重组蛋白的结合能力。图中分别为受试人源化分子与其对照parental抗体PVRIG-A50、A105、A118与人PVRIG蛋白的结合活性；其中anti-HA HcAb-hIgG1、anti-CD38 HcAb-hIgG1和anti-Fluorescein-hIgG1为该实验的阴性对照。
图34B PVRIG人源化抗体与食蟹猴PVRIG重组蛋白的结合能力。图中分别为受试人源化分子与其对照parental抗体PVRIG-A50、A105、A118与食蟹猴PVRIG蛋白的结合活性；其中anti-HA HcAb-hIgG1、anti-CD38 HcAb-hIgG1和anti-Fluorescein-hIgG1为该实验的阴性对照。
图35A PVRIG人源化抗体与FlpinCHO-PVRIG细胞表面人PVRIG的结合活性。图中分别为受试人源化分子与其对照parental抗体PVRIG-A50、A105、A118与FlpinCHO-PVRIG细胞表面人PVRIG的结合能力。其中，COM701-hIgG1及SRF813-hIgG1为阳性对照抗体，anti-CD38 HcAb-hIgG1及anti-Fluorescein-hIgG1为阴性同型对照抗体。
图35B PVRIG人源化抗体与FlpinCHO-cyno PVRIG细胞表面食蟹猴PVRIG的结合。图中分别为受试人源化分子与其对照parental抗体PVRIG-A50、A105、A118与FlpinCHO-cyno PVRIG细胞表面食蟹猴PVRIG的结合能力。其中，COM701-hIgG1及SRF813-hIgG1为阳性对照抗体，anti-CD38 HcAb-hIgG1及anti-Fluorescein-hIgG1为阴性同型对照抗体。
图36 PVRIG人源化抗体阻断人PVRIG与人PVRL2结合。图中分别为受试人源化分子与其对照parental抗体PVRIG-A50、A105、A118对人PVRIG蛋白和人PVRL2蛋白结合的阻断作用；anti-HA HcAb-hIgG1和anti-Fluorescein-hIgG1为该实验的阴性对照。
图37 PVRIG人源化抗体阻断CHO-K1-CD112细胞与人PVRIG-mFc蛋白的结合。图中分别为受试人源化分子与其对照parental抗体PVRIG-A50、A105、A118竞争CHO-K1-CD112细胞结合人PVRIG-mFc蛋白的能力。其中，COM701-hIgG4及SRF813-hIgG1为阳性对照抗体，anti-CD38 HcAb-hIgG1及anti-Fluorescein-hIgG1为阴性同型对照抗体。
图38 PVRIG人源化抗体对NK细胞杀伤作用的影响。在不同浓度下，受试人源化分子与其对照parental抗体PVRIG-A50(A)，A118(B)，A105(C)对NK(donor-050)杀伤靶细胞WIDR的促进作用。图中横坐标为受试抗体的浓度，纵坐标为靶细胞的死亡率，COM701-hIgG1及SRF813-hIgG1为阳性对照抗体，anti-HA HcAb-hIgG1和anti-Fluorescein-hIgG1为阴性同型对照抗体。
图39 CMV antigen-recall assay检测PVRIG人源化抗体对抗原特异性CD8 T细胞的功能改善作用。铺板共孵育18小时后细胞上清中IFN-γ的分泌水平。此实验体系中的阳性对照为 人源化前的PVRIG parental抗体,阴性对照为no treatment(不加任何药物处理)，抗体的作用终浓度均为70nM。*p<0.05,**p<0.01,***p<0.001,****p<0.0001vs.no treatment,one-way ANOVA Analysis。PVRIG-A50的4个人源化抗体(PVRIG-A50-H1a,H1b,H1d,H2a)的作用效果与PVRIG-A50无显著性差异(One-way ANOVA Analysis)；PVRIG-A118的2个人源化抗体(PVRIG-A118-H3,H5)与PVRIG-A118无显著性差异；PVRIG-A105-H2的作用效果显著弱于PVRIG-A105(*p<0.05,one-way ANOVA Analysis)，其余的2个人源化抗体(PVRIG-A105-H1,H3)与PVRIG-A105无显著性差异(One-way ANOVA Analysis)。
图40四个人源化双特异性抗体的组成及结构示意图
图41 ELISA检测四个人源化双抗与人PVRIG-ECD-mFc蛋白的结合
图42 ELISA检测四个人源化双抗与食蟹猴PVRIG-ECD-mFc蛋白的结合
图43 ELISA检测四个人源化双抗与人TIGIT-ECD-mFc蛋白的结合
图44 ELISA检测四个人源化双抗与食蟹猴TIGIT-ECD-mFc蛋白的结合
图45 FACS检测四个人源化双抗与FlpinCHO-人PVRIG细胞的结合活性
图46 FACS检测四个人源化双抗与FlpinCHO-食蟹猴PVRIG细胞的结合活性
图47 FACS检测四个人源化双抗与CHO-K1-人TIGIT高表达细胞株的结合活性
图48 FACS检测四个人源化双抗与CHO-K1-人TIGIT中表达细胞株的结合活性
图49 FACS检测四个人源化双抗与CHO-K1-人TIGIT低表达细胞株的结合活性
图50 FACS检测四个人源化双抗与CHO-K1-食蟹猴TIGIT细胞株的结合活性
图51 HTRF检测四个人源化双抗阻断PVRIG蛋白与PVRL2蛋白的结合
图52 FACS检测四个人源化双抗阻断PVRIG-ECD-mFc与CHO-K1-CD112的结合活性
图53 ELISA检测四个人源化双抗阻断TIGIT-ECD-mFc与CHO-K1-CD155的结合活性
图54 FACS检测四个人源化双抗阻断Bio-CD155-His蛋白与CHO-K1人TIGIT的结合活性
图55 FACS检测四个人源化双抗与人PBMC的结合活性
图56 BIAcore检测人源化双特异性抗体与PVRIG和TIGIT的共结合。LC-BsAb-002人源化双抗(A)和LC-BsAb-006人源化双抗(B)与人TIGIT蛋白，人PVRIG蛋白的结合曲线及分别连续注射人TIGIT和人PVRIG蛋白的抗体抗原结合曲线，黑色三角代表对应蛋白注射的时间点。
图57 NK细胞脱粒实验检测抗PVRIGxTIGIT人源化双特异性抗体对NK细胞的功能促进作用。NK细胞脱颗粒实验流程(A)，FACS检测NK细胞PVRIG，TIGIT和WIDR细胞PVR，PVRL2的表达水平(B)，人源化双特异性抗体LC-BsAb-002和LC-BsAb-006对于NK细胞CD107a表达水平的影响(EC50，AUC，靶细胞WIDR，C)，人源化双特异性抗体LC-BsAb-002对于NK细胞CD107a表达水平的影响(靶细胞TF-1，D)
图58 NK细胞杀伤实验检测抗PVRIGxTIGIT人源化双特异性抗体介导的NK细胞对WIDR细胞的杀伤作用。不同供体(Donor-050，831，715)NK细胞PVRIG和TIGIT的表达水平(A)，靶细胞WIDR上PVR和PVRL2的表达水平(B)，NK细胞毒实验流程(C)，LC-BsAb-002和LC-BsAb-006对三个不同供体NK细胞介导的WIDR细胞的杀伤(EC50，AUC，D)，LC-BsAb-002对NK细胞介导的TF-1细胞的杀伤(E)
图59 NK细胞ADCC实验检测抗PVRIGxTIGIT人源化双特异性抗体介导的对人Treg细胞的直接杀伤作用。NK细胞介导的ADCC杀伤实验流程(A)，人Treg细胞PVRIG和TIGIT表达水平(B)，不同IgG Fc的LC-BsAb-002和LC-BsAb-006对人Treg的ADCC杀伤对比(EC50，AUC，C)
图60抗PVRIGxTIGIT人源化双特异性抗体介导的对人Treg细胞的ADCP活性。
图61 CMV antigen-recall assay检测抗PVRIGxTIGIT人源化双特异性抗体对抗原特异性CD8 T细胞的功能促进作用。CMV antigen-recall实验流程(A)，pp65特异性CD8 T细胞PVRIG、TIGIT、PD-1表达水平及IFN-γ预处理后Colo205细胞PVRL2，PVR及PD-L1的表达水平(B)，铺板共孵育18小时后细胞上清中IFN-γ的分泌水平。此实验体系中的对照为抗TIGIT抗体：RG6058，TIGIT-002-H4L3，TIGIT-005-H2L1d，抗PVRIG抗体：COM701，PVRIG-A50-H1b，阴性对照为no treatment(不加任何药物处理)(C)，人源化双抗，单药联用及其分别与抗PD-L1抗体联用铺板共孵育18小时后细胞上清中IFN-γ的分泌水平。此实验体系中的对照为抗TIGIT抗体：RG6058，TIGIT-002-H4L3，TIGIT-005-H2L1d，抗PVRIG抗体：COM701，PVRIG-A50-H1b，抗PD-L1抗体：Tecentriq，阴性对照为no treatment(不加任何药物处理)(D)
图62 CMV antigen-recall assay检测抗PVRIG×TIGIT人源化双特异性抗体与Tecentriq联用后对抗原特异性CD8 T细胞的功能促进作用。pp65特异性CD8 T细胞PVRIG、TIGIT、PD-1表达水平及IFN-γ预处理后Colo205细胞PVRL2，PVR及PD-L1的表达水平(A),人源化双抗，联用及其分别与抗PD-L1抗体联用铺板共孵育18小时后细胞上清中IFN-γ的分泌水平。此实验体系中的对照为抗TIGIT抗体：RG6058，抗PVRIG抗体：COM701，抗PD-L1抗体：Tecentriq(B)
图63 A375 hPBMC人源化动物模型中各受试组肿瘤生长曲线
图64 A375 hPBMC人源化动物模型中各受试组单只小鼠肿瘤生长曲线
图65 A375 hPBMC人源化动物模型中各受试组给药后小鼠体重变化情况
图66 A375 hPBMC人源化动物模型中不同剂量双特异性抗体单药及与Tecentriq联用各受试组肿瘤生长曲线
图67 A375 hPBMC人源化动物模型中不同剂量双特异性抗体单药及与Tecentriq联用各受试组单只小鼠肿瘤生长曲线
图68 A375 hPBMC人源化动物模型中不同剂量双特异性抗体单药及与Tecentriq联用各受试组给药后小鼠体重变化情况
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本发明实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1 TIGIT抗原的制备
以人TIGIT蛋白(NCBI序列号：XP_024309156.1)、食蟹猴TIGIT蛋白(NCBI：XP_015300911.1)作为本公开TIGIT的模板，设计本公开涉及的抗原及检测用蛋白的氨基酸序列，可选的在TIGIT蛋白基础上融合不同的标签，分别克隆到PTT5载体上(Invitrogen)，在293细胞瞬转表达或CHO细胞稳定表达纯化，获得编码本公开抗原及检测用蛋白。
人TIGIT胞外区与小鼠IgG2a Fc段的融合蛋白：人TIGIT ECD-mFc，用于检测。
Figure PCTCN2022108648-appb-000001
注释：划横线部分为信号肽，斜体部分为mFc。
人TIGIT胞外区与人IgG1 Fc段的融合蛋白：人TIGIT ECD-hFc，用于检测。
Figure PCTCN2022108648-appb-000002
注释：划横线部分为信号肽，斜体部分为Fc。
人TIGIT胞外区与His标签的融合蛋白：人TIGIT ECD-his，用于检测。
Figure PCTCN2022108648-appb-000003
注释：划横线部分为信号肽，斜体部分为His标签。
食蟹猴TIGIT胞外区与小鼠IgG2a Fc段的融合蛋白：食蟹猴TIGIT ECD-mFc，用于检测。
Figure PCTCN2022108648-appb-000004
注释：划横线部分为信号肽，斜体部分为mFc。
食蟹猴TIGIT胞外区与人IgG1 Fc段的融合蛋白：食蟹猴TIGIT ECD-hFc，用于检测。
Figure PCTCN2022108648-appb-000005
Figure PCTCN2022108648-appb-000006
注释：划横线部分为信号肽，斜体部分为hFc
实施例2 CHO-K1工程细胞株的构建
编码人TIGIT全长氨基酸序列(NCBI：XP_024309156.1)，食蟹猴TIGIT全长氨基酸序列(NCBI：XP_015300911.1)，人CD155全长氨基酸序列(NCBI：NP_006496.4)，人CD112全长氨基酸序列(NCBI：NP_001036189.1)的核苷酸序列被克隆到pcDNA3.1载体(购自Clontech)并制备质粒。对CHO-K1细胞系(购自ATCC)进行质粒转染(
Figure PCTCN2022108648-appb-000007
3000 Transfection Kit，购自Invitrogen，货号：L3000-015)后，在含10μg/ml puromycin的含10％(w/w)胎牛血清的DMEM/F12培养基中选择性培养2周后铺单克隆细胞到96孔板，并置于37℃，5％(v/v)CO 2培养，大约2周后选择部分单克隆孔进行扩增。对扩增后的克隆经流式细胞分析法进行筛选。选择长势较好、荧光强度较高、单克隆的细胞系继续扩大培养并液氮冻存。
全长人TIGIT(NCBI：XP_024309156.1)：用于构建人TIGIT过表达细胞株CHO-K1人TIGIT，通过FACS检测，挑选出高表达株1D2，中表达株2D10和低表达株1C6(图25)
Figure PCTCN2022108648-appb-000008
注释：信号肽(单下划线)+胞外区+跨膜区(双下划线)+胞内区(斜体部分)
全长食蟹猴TIGIT(NCBI序列号：XP_015300911.1)：用于构建食蟹猴TIGIT过表达细胞株CHO-K1食蟹猴TIGIT
Figure PCTCN2022108648-appb-000009
注释：信号肽(单下划线)+胞外区+跨膜区(双下划线)+胞内区(斜体部分)
全长人CD155(NCBI序列号：NP_006496.4)：用于构建人CD155过表达细胞株CHO-K1CD155.
Figure PCTCN2022108648-appb-000010
注释：信号肽(单下划线)+胞外区+跨膜区(双下划线)+胞内区(斜体部分)
全长人CD112(NCBI序列号：NP_001036189.1/NCBI RefSeq:Q92692)：用于构建CD112过表达细胞株CHO-K1 CD112
Figure PCTCN2022108648-appb-000011
注释：信号肽(单下划线)+胞外区+跨膜区(双下划线)+胞内区(斜体部分)
实施例3抗人TIGIT鼠源单克隆抗体的生成
抗人TIGIT抗体通过杂交瘤技术得到，用人TIGIT-ECD-mFc融合蛋白免疫小鼠，分离免疫小鼠脾细胞，通过电融合的方式与小鼠骨髓瘤细胞进行细胞融合，HAT选择培养基培养，取培养上清进行鉴定，挑选分泌目的抗体的克隆进行亚克隆，最终经生产纯化获得鼠源单克隆抗体。详细描述如下：
A.动物免疫
实验用SJL白小鼠，雌性，6～8周龄(上海斯莱克实验动物有限责任公司，动物生产许可证号：SCXK(沪)2017-0005)。饲养环境：SPF级。小鼠购进后，在实验动物房(和元生物技术(上海)股份有限公司)，实验室环境适应性饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。将已适应环境的小鼠按以下方案免疫。
免疫抗原带mFc标签的人TIGIT-ECD(Acro Cat No.TIT-H5253)。免疫方案：用
Figure PCTCN2022108648-appb-000012
Gold Adjuvant(Sigma Cat No.T2684)与Thermo
Figure PCTCN2022108648-appb-000013
Alum(Thermo Cat No.77161)佐剂交叉免疫。抗原与佐剂(
Figure PCTCN2022108648-appb-000014
Gold Adjuvant)比例为1:1，抗原与佐剂(Thermo
Figure PCTCN2022108648-appb-000015
Alum)比例为2:1，50μg/只/次(首次免疫)，25μg/只/次(加强免疫)。抗原乳化后进行接种，时间为第1、8、15、22、29、36、43、50。第1天腹腔注射(I.P.)50μg/只的乳化后抗原。第8天皮下多点注射(S.C.，一般背部和腹股沟4点)25μg/只。腹腔注射和皮下注射隔周交叉进行，第20，27，34，48天取血，用ELISA方法确定小鼠血清的抗体Titer。在第6～8次免疫以后，选择血清中抗体Titer高的小鼠的脾脏和淋巴结细胞进行融合，在融合前3天进行冲击免疫，腹腔内(I.P.)注射50μg/只的生理盐水配制的抗原溶液。
B.B细胞融合
采用优化的电融合(BTX ECM2001+)步骤将脾和淋巴结细胞与骨髓瘤细胞SP2/0细胞(
Figure PCTCN2022108648-appb-000016
CRL-1581)进行融合得到杂交瘤细胞。融合后的杂交瘤细胞以5×10 5/ml的密度用完全培养基含20％FBS(Excell CatNo.FND500)，1×ΗΑΤ(Sigma Cat No.H0262-10VL，1×NEAA的DMEM(Gibco Cat No.10569044)培养基重悬，100μL/孔接种于96孔平底板中，37℃，5％CO 2孵育6-7天后，去除上清，加入200μ1每孔的含有ΗΤ完全培养基含10％FBS，l× HT(Sigma Cat No.H0137-10VL)，1×NEAA的DMEM培养基，37℃，5％CO 2培养过夜后，进行ELISA检测。
C.杂交瘤细胞的筛选
融合7～10天后，取杂交瘤细胞上清液，通过ELISA方法检测与人TIGIT ECD-hFc(内部生产)的结合活性，挑选出阳性克隆；第二天检测阳性克隆上清液与CHO-K1人TIGIT(内部构建)的结合活性、与CHO-K1食蟹猴TIGIT(内部构建)的结合活性、以及阻断人TIGIT ECD-hFc与CHO-K1 CD155(内部构建)结合的效应，挑选目标克隆进行亚克隆。
亚克隆细胞培养7～10天后，通过ELISA方法筛选，挑选目标杂交瘤单克隆细胞扩大培养至24孔，2～3天后，检测培养上清与人TIGIT ECD-hFc的结合活性、与CHO-K1人TIGIT(内部构建)的结合活性、与CHO-K1食蟹猴TIGIT(内部构建)的结合活性、与食蟹猴TIGIT ECD-hFc(内部生产)的结合活性、阻断Bio-CD155-His(义翘神州，10109-H08H)与CHO-K1人TIGIT(内部构建)相互作用的效应、以及阻断人TIGIT ECD-hFc与CHO-K1 CD155(内部构建)相互作用的效应，选择目标克隆进行生产并纯化获得116株单克隆抗体。
D.鼠源单克隆抗体的鉴定
对以上获得的116株单克隆抗体通过ELISA、FACS、BIAcore等进行鉴定，获得13株候选抗体，分别是TIGIT-F2-002、TIGIT-F2-005、TIGIT-F2-006、TIGIT-F2-011、TIGIT-F3-034、TIGIT-F4-044、TIGIT-F5-057、TIGIT-F5-067、TIGIT-F5-070、TIGIT-F5-072、TIGIT-F6-084、TIGIT-F6-088、TIGIT-F6-104。
(a)抗TIGIT鼠源单克隆抗体与人TIGIT ECD-hFc和食蟹猴TIGIT ECD-hFc的结合活性检测
用PBS pH7.4(Hyclone，CAT#SH30256)稀释羊抗人IgG抗体(Jackson，CAT:109-006-098)至4μg/mL,加入酶标板(corning,CAT#9018)，50μL/孔，4℃过夜，甩掉包被液，加入5％脱脂奶粉(生工生物，CAT#A600669-0250)-PBS，250μL/孔，37℃孵育2～4小时，在洗板机(Biotek,CAT#405TUS)上用0.05％Tween20-PBS(生工生物，CAT#A100777-0500，B548117-0500)洗三次，加入人TIGIT ECD-hFc(内部构建，工作浓度30ng/mL)和食蟹猴TIGIT ECD-hFc(内部构建，工作浓度30ng/mL)，50μL/孔，4℃孵育过夜，在洗板机上用0.05％Tween20-PBS洗三次，加入纯化鼠源单克隆抗体(1％BSA稀释至13nM，经3倍连续梯度稀释12个浓度点)，50μL/孔，37℃孵育1.5～2小时，在洗板机上用0.05％Tween20-PBS洗三次，以1:5000稀释比用1％BSA(生工生物，CAT#A500023-0100)-PBS稀释HRP酶标抗体(Jackson,CAT#115-035-003)，加入酶标板中，50μL/孔，37℃孵育1小时，在洗板机上用0.05％Tween20-PBS洗三次，加入TMB显色液(KPL,CAT#52-00-03),50μL/孔，室温孵育7～10分钟，加入1M HCL，50μL/孔，终止反应，酶标仪(Biotek,Powerwave HT)读取OD450nm。实验结果表明，和对照抗TIGIT抗体罗氏RG6058相比，抗TIGIT鼠源单克隆抗体能有效结合人TIGIT ECD-hFc和食蟹猴TIGIT ECD-hFc。
RG6058对应的氨基酸序列如下所示：
RG6058 VH SEQ ID NO 234:
Figure PCTCN2022108648-appb-000017
RG6058 VL SEQ ID NO 235:
Figure PCTCN2022108648-appb-000018
(b)ELISA方法检测抗TIGIT鼠源单克隆抗体与CHO-K1人TIGIT和CHO-K1食蟹猴TIGIT的结合活性
收集细胞，用10％FBS-DMEM/F12培养基(Excell,CAT#FSP500；Gibco,CAT#11330)调整浓度至5×10 5/mL，加入96孔细胞培养板(corning,CAT#3599)，100μL/孔，37℃5％CO 2培养过夜，甩掉培养上清，加入细胞固定液(碧云天，CAT#P0098)，50μL/孔，室温固定1小时，在洗板机上用0.05％Tween20-PBS洗一次，加入5％脱脂奶粉-PBS，250μL/孔，37℃孵育2～4小时，在洗板机上用0.05％Tween20-PBS洗三次，加入纯化鼠源单克隆抗体稀释液用1％BSA稀释至13nM，经3倍连续梯度稀释12个浓度点，50μL/孔，37℃孵育1.5～2小时，在洗板机上用0.05％Tween20-PBS洗三次，以1:5000稀释比用1％BSA(生工生物，CAT#A500023-0100)-PBS稀释HRP酶标抗体(Jackson,CAT#115-035-003)，加入细胞板中，50μL/孔，37℃孵育1小时，在洗板机上用0.05％Tween20-PBS洗三次，加入TMB显色液(KPL,CAT#52-00-03)，50μL/孔，37℃孵育10分钟，加入1M HCL，50μL/孔，终止反应，酶标仪(Biotek,Powerwave HT)读取OD450nm。实验结果表明和对照抗TIGIT抗体罗氏RG6058相比，纯化鼠源抗TIGIT单克隆抗体可以有效结合CHO-K1人TIGIT高表达细胞株，CHO-K1人TIGIT中表达细胞株，CHO-K1人TIGIT低表达细胞株及CHO-K1-食蟹猴TIGIT细胞株。
(c)FACS方法检测抗TIGIT鼠源单克隆抗体阻断Bio-CD155-His与CHO-K1人TIGIT的相互作用
收集细胞，PBS(Hyclone，CAT#SH30256)洗一次，1％BSA-PBS重悬至2×10 5/40μL，1％BSA-PBS稀释抗体至210nM，3倍连续梯度稀释12个浓度点，1％BSA-PBS稀释Bio-CD155-His(义翘神州，10109-H08H)至3μg/mL，随后将40μL细胞与40μL抗体稀释液以及40μL Bio-CD155-His稀释液混合，4℃孵育60分钟，PBS洗两次，加入APC标记的链霉亲和素(工作稀释度1:1700，Biolegend，405243)，100μL/孔重悬细胞，4℃孵育40分钟，PBS洗两次，用1％BSA-PBS，100μL/孔重悬，在流式细胞仪(BD,CantoⅡ)上分析细胞样品。实验结果表明，纯化的鼠源单克隆抗体与对照抗体RG6058都可以有效阻断Bio-CD155-His蛋白与CHO-K1-人TIGIT高表达细胞株的结合。
(d)ELISA方法检测抗TIGIT鼠源单克隆抗体抑制人TIGIT与CHO-K1 CD155的相互作用
收集CHO-K1 CD155细胞，用10％FBS-DMEM/F12培养基(Excell,CAT#FSP500；Gibco,CAT#11330)调整浓度至5×10 5/mL，加入96孔细胞培养板(corning,CAT#3599),100μL/孔，37℃5％CO 2培养过夜，甩掉培养上清，加入细胞固定液(碧云天，CAT#P0098)，50μL/孔，室温固定1小时，在洗板机上用0.05％Tween20-PBS洗一次，加入5％脱脂奶粉-PBS，250μL/孔，37℃孵育2～4小时，在洗板机上用0.05％Tween20-PBS洗三次；将鼠源单克隆抗体与人TIGIT ECD-hFc(工作浓度30ng/mL)混合孵育半小时；随后将抗原抗体混合液加入细胞板中，50μL/孔，37℃孵育1.5～2小时，在洗板机上用0.05％Tween20-PBS洗三次，以1:5000稀释比用1％BSA(生工生物，CAT#A500023-0100)-PBS稀释HRP酶标抗体(Merck，CAT#AP113P)，加入细胞板中，50μL/孔，37℃孵育1小时，在洗板机上用0.05％Tween20-PBS 洗三次，加入TMB显色液(KPL,CAT#52-00-03),50μL/孔，37℃孵育10分钟，加入1M HCL，50μL/孔，终止反应，酶标仪(Biotek,Powerwave HT)读取OD450nm。实验结果表明，纯化的鼠源单克隆抗体与对照抗体RG6058都可以有效阻断人TIGIT蛋白与CHO-K1-CD155细胞的结合。
下表1显示，13株抗体都展现了良好的TIGIT结合能力、以及阻断TIGIT与CD155相互作用的效应，且达到或超过抗TIGIT对照抗体罗氏RG6058。
表1 TIGIT鼠源单克隆抗体的鉴定
Figure PCTCN2022108648-appb-000019
实施例4抗人TIGIT嵌合抗体的鉴定
上述鼠源抗体经构建人-鼠嵌合抗体鉴定确认了4株嵌合抗体：TIGIT-CHI-002、TIGIT-CHI-005、TIGIT-CHI-006和TIGIT-CHI-070。表2示出嵌合抗体的VH/VL序列，表3示出嵌合抗体的KABAT分析结果，表4示出嵌合抗体的IMGT分析结果。
表2 TIGIT嵌合抗体VH/VL序列
Figure PCTCN2022108648-appb-000020
Figure PCTCN2022108648-appb-000021
表3 TIGIT嵌合抗体的Kabat分析结果
Figure PCTCN2022108648-appb-000022
表4 TIGIT嵌合抗体的IMGT分析结果
Figure PCTCN2022108648-appb-000023
Figure PCTCN2022108648-appb-000024
(a)抗TIGIT嵌合抗体与人TIGIT ECD-mFc和食蟹猴TIGIT ECD-mFc的结合活性检测
用PBS pH7.4(Hyclone，CAT#SH30256)稀释羊抗鼠IgG抗体(Jackson，CAT:115-006-071)至4μg/mL,加入酶标板(corning,CAT#9018)，50μL/孔，4℃过夜，甩掉包被液，加入5％脱脂奶粉(生工生物，CAT#A600669-0250)-PBS，250μL/孔，37℃孵育2～4小时，在洗板机(Biotek,CAT#405TUS)上用0.05％Tween20-PBS(生工生物，CAT#A100777-0500，B548117-0500)洗三次，加入人TIGIT ECD-mFc(内部构建，工作浓度30ng/mL)和食蟹猴TIGIT ECD-mFc(内部构建，工作浓度30ng/mL)，50μL/孔，4℃孵育过夜，在洗板机上用0.05％Tween20-PBS洗三次，加入待测抗体(1％BSA稀释至13nM,经3倍连续梯度稀释12个浓度点)，50μL/孔，37℃孵育1.5～2小时，在洗板机上用0.05％Tween20-PBS洗三次，以1:5000稀释比用1％BSA(生工生物，CAT#A500023-0100)-PBS稀释HRP酶标抗体(Merck，CAT#AP113P)，加入酶标板中，50μL/孔，37℃孵育1小时，在洗板机上用0.05％Tween20-PBS洗三次，加入TMB显色液(KPL,CAT#52-00-03)，50μL/孔，室温孵育7～10分钟，加入1M HCL，50μL/孔，终止反应，酶标仪(Biotek,Powerwave HT)读取OD450nm。实验结果表明和对照抗体罗氏RG6058相一致，抗TIGIT嵌合抗体能有效结合人TIGIT ECD-mFc(图1)和食蟹猴TIGIT ECD-mFc(图2)
(b)FACS检测抗TIGIT嵌合抗体与CHO-K1人TIGIT高中低表达水平的细胞和CHO-K1食蟹猴TIGIT的结合活性
收集细胞，PBS(Hyclone，CAT#SH30256)洗一次，1％BSA-PBS重悬至2×10 5/50μL，1％BSA-PBS稀释抗体至80nM，3倍连续梯度稀释12个浓度点，将50μL细胞与50μL待测抗体稀释液混合，4℃孵育60分钟，PBS洗两次，加入Alexa
Figure PCTCN2022108648-appb-000025
647荧光素标记的二抗(1:800)(Jackson，CAT#109-605-088)，100μL/孔重悬细胞，4℃孵育40分钟，PBS洗两次，用1％BSA-PBS，100μL/孔重悬，在流式细胞仪(BD,CantoⅡ)上分析细胞样品。实验结果表明和对照抗体罗氏RG6058一致，抗TIGIT嵌合抗体可以有效结合CHO-K1-TIGIT高表达细胞株(图3)，CHO-K1-TIGIT中表达细胞株(图4)，CHO-K1-TIGIT低表达细胞株(图5)及CHO-K1-食蟹猴TIGIT细胞株(图6)。
(c)FACS方法检测抗TIGIT嵌合抗体阻断Bio-CD155-His与CHO-K1人TIGIT高表达株的相互作用
收集细胞，PBS(Hyclone，CAT#SH30256)洗一次，1％BSA-PBS重悬至2×10 5/40μL，1％BSA-PBS稀释抗体至210nM，3倍连续梯度稀释12个浓度点，1％BSA-PBS稀释Bio-CD155-His(义翘神州，10109-H08H)至3μg/mL，随后将40μL细胞与40μL抗体稀释液以及40μL Bio-CD155-His稀释液混合，4℃孵育60分钟，PBS洗两次，加入APC标记的链霉亲和素(工作稀释度1:1700，Biolegend，405243)，100μL/孔重悬细胞，4℃孵育40分钟，PBS洗两次，用1％BSA-PBS，100μL/孔重悬，在流式细胞仪(BD,CantoⅡ)上分析细胞样品。如图7所示，抗TIGIT嵌合抗体与对照抗体RG6058都可以有效阻断Bio-CD155-His蛋白与CHO-K1-人TIGIT细胞的结合。
(d)FACS方法检测抗TIGIT嵌合抗体阻断TIGIT ECD-mFc与CHO-K1 CD155的相互作用
收集CHO-K1 CD155细胞，PBS(Hyclone，CAT#SH30256)洗一次，1％BSA-PBS重悬至2×10 5/40μL，1％BSA-PBS稀释待测抗体至600nM，2.5倍连续梯度稀释12个浓度点，1％BSA-PBS稀释TIGIT ECD-mFc至6μg/mL，随后将40μL细胞与40μL抗体稀释液以及40μL TIGIT ECD-mFc稀释液混合，4℃孵育60分钟，PBS洗两次，加入Alexa
Figure PCTCN2022108648-appb-000026
647荧光素标记的二抗(工作稀释度1：800，Jackson,CAT#115-605-003)，100μL/孔重悬细胞，4℃孵育40分钟，PBS洗两次，用1％BSA-PBS，100μL/孔重悬，在流式细胞仪(BD,CantoⅡ)上分析细胞样品。如图8所示，抗TIGIT嵌合抗体与对照抗体RG6058都可以有效阻断人TIGIT蛋白与CHO-K1-CD155细胞的结合。
表5显示TIGIT嵌合抗体鉴定汇总。
表5 TIGIT嵌合抗体鉴定汇总
Figure PCTCN2022108648-appb-000027
(e)NK细胞表面PVRIG及TIGIT的表达及WIDR细胞表面PVR和PVRL2的表达检测
利用FACS检测NK细胞(Natural killer cell)上PVRIG，TIGIT的表达情况。首先，使 用细胞计数仪(Beckman Coulter，Vi-CELL)将NK细胞计数，取三个流式管，每个流式管加入1E+5个NK细胞，加PBS洗涤细胞两次，去上清，取一管加入300μl Staining buffer(PBS+2％FBS)做为未染色管待用，另外两管每管加入染液100μl(PBS+1*的Zombie Violet(Biolegend，423114))混匀后室温孵育15分钟。随后加Staining buffer洗涤细胞两次，去上清，每管加入Fc阻断剂50μl(Staining buffer+Fcx blocker(Biolegend，422302))混匀后4℃孵育15分钟。接着分别向每管加入染液，第一管加入50ul的2*染液(Staining buffer+PE-Cy7 Mouse anti-hCD3检测抗体+PE Mouse anti-hCD56检测抗体+APC Mouse anti-hTIGIT检测抗体+AF488 Rabbit anti-hPVRIG检测抗体，CD3检测抗体：Biolegend 300316，CD56检测抗体：Biolegend 318306，TIGIT检测抗体：Biolegend 372706，PVRIG检测抗体：RD FAB93651G)，第二管加入50μl的2*同型对照染液(Staining buffer+PE-Cy7 Mouse anti-hCD3检测抗体+PE Mouse anti-hCD56检测抗体+APC Mouse IgG2aκ同型对照抗体+AF488 Rabbit IgGκ同型对照抗体，APC mIgG2aκ同型对照抗体：Biolegend 400222，AF488 Rabbit IgGκ同型对照抗体:RD IC1051G)，混匀后4℃孵育30分钟。时间到后用Staining buffer洗涤两次，离心后加300μl Staining buffer混匀。随后上机检测(Thermo Attune NxT)，最终读取Zombie Violet阴性细胞群中CD56阳性CD3阴性的细胞群的占比和Zombie Violet阴性细胞群中CD56阳性CD3阴性的细胞群的APC、AF488信号。图9中A表明，不同供体donor-010及donor-050的NK细胞表面均有PVRIG及TIGIT的表达。
利用FACS检测WIDR细胞上PVR和PVRL2的表达。首先，将WIDR细胞用胰酶消化成细胞悬液，使用细胞计数仪(Beckman Coulter，Vi-CELL)将细胞计数，取三个流式管，每个流式管加入1E+5个细胞，加PBS洗涤细胞两次。离心后去上清，取一管加入300ul Staining buffer(PBS+2％FBS)做为未染色管待用，另外两管每管加入染液100ul(PBS+1*的Zombie Violet(Biolegend，423114))混匀后室温孵育15分钟。随后加Staining buffer洗涤细胞两次，去上清，分别向每管加入染液：第一管加入100μl染液(Staining buffer+PerCP-Cy5.5 Mouse anti-hPVR检测抗体+APC Mouse anti-hPVRL2检测抗体，PVR检测抗体：Biolegend 337612，PVRL2检测抗体：Biolegend 337412)，第二管加入100μl同型对照染液(Staining buffer+PerCP-Cy5.5 Mouse IgG1κ同型对照抗体+APCκMouse IgG1同型对照抗体，PerCP-Cy5.5 mIgG1κ同型对照抗体：Biolegend 400150，APC mIgG1κ同型对照抗体：Biolegend 400122)，混匀后4℃孵育30分钟。时间到后用Staining buffer洗涤两次，离心后加300μl Staining buffer混匀。随后上机检测(Thermo Attune NxT)，最终读取Zombie Violet阴性细胞群的PerCP-Cy5.5、APC信号。图9中B显示，WIDR细胞表面的高表达PVR及PVRL2。
(f)NK细胞脱粒实验检测抗TIGIT嵌合抗体对NK细胞功能的促进作用
利用FACS检测NK细胞(Natural killer cell)的CD107a信号来指示受试抗体对NK细胞脱颗粒过程的影响。提前一天复苏PBMC，分选NK细胞(Stemcell，17955)，加入200IU/ml的h-IL2(RD,202-IL)和10ng/ml的h-IL12(Peprotech，200-12-50UG)刺激过夜，第二天进行铺板实验。首先，用assay buffer(RPMI1640-Glutamax+10％FBS+1×P/S)将抗体稀释到最高浓度275nM(四倍浓度)，然后继续用assay buffer开始进行10倍梯度稀释，将稀释好的抗体加至超低吸附96孔U底板(Costar，7007)中，每孔50μl，待用。其次，使用细胞计数仪(Beckman Coulter，Vi-CELL)将NK细胞计数，取一定数目的NK细胞，以350g的 速度离心5分钟，弃上清后用assay buffer重悬至0.5E+6个细胞/ml的密度，向细胞悬液中加入蛋白转运抑制剂(Invitrogen，00498093)和APC mouse anti-human检测抗体(Biolegend，328620)。向铺好药的96孔U底板中加入处理好的NK细胞悬液，每孔50μl，混合均匀后室温孵育15分钟。孵育期间，将靶细胞WIDR用胰酶消化成细胞悬液，使用细胞计数仪(Beckman Coulter，Vi-CELL)将靶细胞计数，取出适量数目的细胞，以200g的速度离心5分钟，弃上清后用assay buffer将细胞重悬至0.25E+6个细胞/ml的密度。孵育结束后，向孔板中加入靶细胞悬液，每孔100μl，此时每孔中含有25000个NK细胞，25000个靶细胞以及不同浓度的受试抗体，只含有NK细胞的孔作为静息对照，含有NK细胞及靶细胞的孔作为无药物对照。将每个孔混合均匀后放入37℃培养箱孵育16h。FACS染色检测CD107a的变化情况：将孔板中的细胞平行转移至96孔V底板，用PBS洗涤两次，弃上清，每孔加入染液(PBS+2％FBS+1*浓度的zombie violet(Biolegend，423114)+PE mouse anti-CD56检测抗体(Biolegend，318306))混合均匀后4℃孵育30分钟。时间到后，staining buffer洗涤两次，弃上清，每孔加入150μl的staining buffer重悬上机检测(Thermo Attune NxT)。最终读取CD56阳性细胞中CD107a强阳性细胞群的占比，CD107a强阳性细胞占比越高，代表NK细胞的脱粒作用越强，NK细胞的激活程度越高。图10显示，阴性对照anti-HEL-hIgG1对NK表面的CD107a没有影响，三个受试抗体均能不同程度的提高NK细胞CD107a的表达，这表明三个受试抗体均可以有效促进NK的激活。
(g)NK细胞杀伤实验检测抗TIGIT嵌合抗体的NK细胞对靶细胞杀伤促进作用
利用FACS检测靶细胞(WIDR)的裂解水平来推测受试抗体对NK(Natural killer cell)细胞杀伤功能的影响。提前一天复苏PBMC，分选NK细胞(Stemcell，17955)，加入200IU/ml的h-IL2(RD,202-IL)和10ng/ml的h-IL12(Peprotech，200-12-50UG)过夜刺激，第二天进行铺板实验。首先，用assay buffer(RPMI1640-Glutamax+10％FBS+1×P/S)将抗体稀释到最高浓度275nM(四倍浓度)，随后用assay buffer开始进行10倍梯度稀释，将稀释好的抗体加至超低吸附96孔U底板(Costar，7007)中，每孔50μl，待用。将靶细胞WIDR用胰酶消化成细胞悬液，使用细胞计数仪(Beckman Coulter，Vi-CELL)将WIDR细胞计数，取出适量数目的细胞，置于离心机以200g的速度离心5分钟，弃上清后用适量PBS重悬后加入CellTrace Violet(Invitrogen，C34557A)染液，使CellTrace Violet终浓度为5μM。将加入染液的WIDR悬液混合均匀置于37℃培养箱孵育10分钟，期间摇晃混合。与此同时，使用细胞计数仪将NK细胞计数，取一定数目的NK细胞，以350g的速度离心5分钟，弃上清后用assay buffer重悬至0.5E+6个细胞/ml的密度。向铺好药的96孔U底板中加入处理好的NK细胞悬液，每孔50μl，混合均匀后室温孵育15分钟。WIDR细胞染色结束后，向细胞悬液中加入5倍体积完全培养基(MEM+10％FBS+1*P/S+1*非必须氨基酸+1*谷氨酸钠)进行终止反应，以200g的速度离心5分钟，弃上清后用assay buffer将细胞重悬至0.25E+6个细胞/ml的密度。NK与药物孵育结束后，向孔板中加入WIDR细胞悬液，每孔100μl，此时每孔中含有25000个NK细胞，25000个WIDR细胞以及不同浓度的受试抗体，只含WIDR细胞的孔作为静息对照，含有NK及WIDR的孔作为无药物对照。将每个孔混合均匀后放入37℃培养箱孵育4h。FACS染色检测WIDR细胞裂解水平：每孔加入染液(PBS+PI(Propidium Iodide，Invitrogen，P3566))混合均匀后室温孵育20分钟。时间到后，上机检测(Thermo Attune  NxT)，最终读取CTV阳性细胞中PI阳性细胞群的占比，PI阳性细胞占比越多，代表NK细胞的杀伤作用越强。图11显示，阴性对照anti-HEL-hIgG1对NK杀伤没有显著影响，受试的4个嵌合抗体均能有效促进NK细胞对靶细胞WIDR的杀伤，并且4个嵌合抗体对NK细胞的杀伤促进作用不弱于阳性对照RG6058-hIgG1。
(h)CMV antigen-recall assay检测抗TIGIT嵌合抗体对抗原特异性CD8 T细胞的功能改善作用
Anti-CMV IgG阳性donor的PBMC经CMV pp65(495-503)多肽诱导的CMV pp65特异性CD8 T作为效应细胞，经pp65 pulsed后的colo205肿瘤细胞系作为靶细胞的实验体系中，检测TIGIT抗体对抗原特异性CD8 T细胞的功能改善作用。
PBMC复苏后，用含有1mg/mL CMV pp65(495-503)多肽(Anaspec,货号AS-28328)、2ng/mL human IL-2(R&D,货号IL-202)、10ng/mL human IL-7(Peprotech,货号200-07)的完全培养基(RPMI1640-Glutamax+5％AB serum+1％P/S+(1×)2-β巯基乙醇)重悬至2×10 6/mL，5mL/孔接种于6孔板中，37℃5％CO 2培养6天。第6天，收集所有细胞，撤去培养基中的pp65和IL-7，将细胞一分为二，并重悬于含有100IU/mL human IL-2的完全培养基中，继续培养2天。第8天，收集所有细胞，重悬于含有100IU/mL human IL-2的完全培养基中、并调整细胞密度为2×10 6/mL，继续培养。第11天，收集所有细胞，流式检测PBMC中CD8 T细胞的比例，CMV pp65(495-503)特异性CD8T的比例(图12中A)及该细胞上PVRIG、TIGIT、PD-1的表达(图12中B)。流式检测抗体如下：Livedead Near IR(Invitrogen，货号L34976)，CD8-PerCp Cy5.5(BD，货号565310)，CD3-PE-Cy7(Biolegend,货号300316)，T-select HLA-A*0201 CMV pp65 Tetramer-PE(MBL，货号TS-0010-1C)，PVRIG-AF488(R&D，货号FAB93651G-100UG)，TIGIT-APC(Biolegend,货号372706)，PD-1-BV421(BD,货号562516)。
上述诱导后的PBMC经CD8 T分选试剂盒(Stemcell，货号17953)分离出CD8 T作为效应细胞，用AIM-V重悬并调整细胞密度至0.4×10 6/mL。Colo205作为靶细胞，经TrypLE TM Express Enzyme(Gibco,货号12605010)消化，重悬于含有50ng/mL pp65的AIM-V(Gibco,货号31035-025)并调整细胞密度至1×10 6/ml，37℃5％CO 2处理1-2小时，之后250g离心5分钟，弃上清。之后细胞用AIM-V重悬至0.2×10 6/mL，流式检测Colo205细胞高表达PVRL2，PVR如图12中C所示。TIGIT抗体或阳性对照用AIM-V稀释至280nM。在低吸附96孔U底板(Corning,货号7007)中依次加入50μL抗体，50μL CD8 T，100μL pp65处理过的colo205，并用排枪轻轻混匀，37℃5％CO 2孵育18小时。此体系中药物终浓度为70nM，CD8 T为20000/孔，colo205为20000/孔。孵育结束后400g离心取上清，用ELISA试剂盒(达科为，货号1110003)检测上清中human IFN-γ的水平。此体系中的阳性对照为RG6058-hIgG1，阴性对照为no treatment。如图12中D所示，相较于RG6058-hIgG1，3个TIGIT受试抗体作用后，细胞上清中IFN-γ的分泌在统计学上均无显著性差异但均显著高于no treatment组，每个柱状图上的百分比意为相较于no treatment组IFN-γ分泌提升的百分比。
Colo205上PVRL2、PVR表达的流式检测抗体如下：livedead-BV421(Invitrogen,货号L34964)，PVRL2-APC(Biolegend，货号337412)，PVR-PerCp Cy5.5(Biolegend，货号337612),PD-L1-PE-Cy7(BD，货号558017)。
实施例5抗人TIGIT单克隆抗体的人源化
通过比对IMGT(http://imgt.cines.fr)人类抗体重轻链可变区种系基因数据库，分别挑选与鼠源抗体同源性高的重链和轻链可变区种系基因作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。根据需要，将骨架序列中关键氨基酸回复突变为鼠源抗体对应的氨基酸，以保证原有的亲和力，即得到人源化抗TIGIT单克隆抗体。其中抗体的CDR氨基酸残基通常由Kabat编号系统确定并注释。
1、TIGIT-002的人源化
鼠源抗体TIGIT-002的人源化轻链模板为IGKV2-29*02/IGKV4-1*01和IGKJ4*01，人源化重链模板为IGHV4-38-2*01和IGHJ6*01，将鼠源抗体TIGIT-002的CDR分别移植到其人源模板中，即获得对应的人源化版本。根据需要，将TIGIT-002的人源化抗体的FR区序列中关键氨基酸进行回复突变为鼠源抗体对应的氨基酸，以保证原有的亲和力，具体回复突变设计见表6。
表6 TIGIT-002的人源化抗体回复突变设计
Figure PCTCN2022108648-appb-000028
注：Grafted(IGKV2-29*02)代表将鼠抗体CDR植入人种系IGKV2-29*02FR区序列；L47M表示将Grafted第47位L突变回M，其它依此类推。回复突变氨基酸的编号为自然顺序编号。TIGIT-002人源化抗体可变区具体序列如下：
TIGIT-002.VL1氨基酸序列如SEQ ID NO：66所示：
Figure PCTCN2022108648-appb-000029
TIGIT-002.VL2氨基酸序列如SEQ ID NO：67所示：
Figure PCTCN2022108648-appb-000030
TIGIT-002.VL3氨基酸序列如SEQ ID NO：68所示：
Figure PCTCN2022108648-appb-000031
TIGIT-002.VH1氨基酸序列如SEQ ID NO：69所示：
Figure PCTCN2022108648-appb-000032
TIGIT-002.VH2氨基酸序列如SEQ ID NO：70所示：
Figure PCTCN2022108648-appb-000033
TIGIT-002.VH3氨基酸序列如SEQ ID NO：71所示：
Figure PCTCN2022108648-appb-000034
TIGIT-002.VH4氨基酸序列如SEQ ID NO：72所示：
Figure PCTCN2022108648-appb-000035
人源化轻链模板IGKV2-29*02氨基酸序列如SEQ ID NO：73所示：
Figure PCTCN2022108648-appb-000036
人源化轻链模板IGKV4-1*01氨基酸序列如SEQ ID NO：74所示：
Figure PCTCN2022108648-appb-000037
人源化轻链模板IGKJ4*01氨基酸序列如SEQ ID NO：75所示：
Figure PCTCN2022108648-appb-000038
人源化重链模板IGHV4-38-2*01氨基酸序列如SEQ ID NO：76所示：
Figure PCTCN2022108648-appb-000039
人源化重链模板IGHJ6*01氨基酸序列如SEQ ID NO：77所示：
Figure PCTCN2022108648-appb-000040
本发明分别从上述002的人源化抗体轻链和重链可变区的回复突变设计中，选择不同的轻链和重链序列进行交叉组合，最终获得多种002人源化抗体，各抗体的可变区氨基酸序列如下：
表7 TIGIT-002人源化抗体可变区对应氨基酸序列
Fv 002.VH1 002.VH2 002.VH3 002.VH4
002.VL1 TIGIT-002-H1L1* TIGIT-002-H2L1 TIGIT-002-H3L1 TIGIT-002-H4L1
002.VL2 TIGIT-002-H1L2 TIGIT-002-H2L2 TIGIT-002-H3L2 TIGIT-002-H4L2
002.VL3 TIGIT-002-H1L3 TIGIT-002-H2L3 TIGIT-002-H3L3 TIGIT-002-H4L3
*TIGIT-002-H1L1表示重链选自TIGIT-002.VH1，轻链选自TIGIT-002.VL1。下同。
根据Kabat编号系统，上述12个人源化抗体VH和VL序列分析结果如表8所示。
表8 TIGIT-002人源化抗体VH和VL序列的Kabat分析结果
Figure PCTCN2022108648-appb-000041
2、TIGIT-006的人源化
鼠源抗体TIGIT-006的人源化轻链模板为IGKV2-29*02/IGKV4-1*01和IGKJ4*01，人源化重链模板为IGHV4-38-2*01和IGHJ6*01，将鼠源抗体TIGIT-006的CDR分别移植到其人源模板中，即获得对应的人源化版本。根据需要，将TIGIT-006的人源化抗体的FR区序列中关键氨基酸进行回复突变为鼠源抗体对应的氨基酸，以保证原有的亲和力，具体回复突变设 计见表9。
表9 TIGIT-006的人源化抗体回复突变设计
Figure PCTCN2022108648-appb-000042
注：Grafted(IGKV2-29*02)代表将鼠抗体CDR植入人种系IGKV2-29*02 FR区序列；L37Q表示将Grafted第37位L突变回Q，其它依此类推。回复突变氨基酸的编号为自然顺序编号。
TIGIT-006人源化抗体可变区具体序列如下：
TIGIT-006.VL1氨基酸序列如SEQ ID NO：78所示：
Figure PCTCN2022108648-appb-000043
TIGIT-006.VL2氨基酸序列如SEQ ID NO：79所示：
Figure PCTCN2022108648-appb-000044
TIGIT-006.VL3氨基酸序列如SEQ ID NO：80所示：
Figure PCTCN2022108648-appb-000045
TIGIT-006.VH1氨基酸序列如SEQ ID NO：81所示：
Figure PCTCN2022108648-appb-000046
TIGIT-006.VH2氨基酸序列如SEQ ID NO：82所示：
Figure PCTCN2022108648-appb-000047
TIGIT-006.VH3氨基酸序列如SEQ ID NO：83所示：
Figure PCTCN2022108648-appb-000048
TIGIT-006.VH4氨基酸序列如SEQ ID NO：84所示：
Figure PCTCN2022108648-appb-000049
TIGIT-006.VH5氨基酸序列如SEQ ID NO：85所示：
Figure PCTCN2022108648-appb-000050
人源化轻链模板IGKV2-29*02氨基酸序列如SEQ ID NO：73所示：
Figure PCTCN2022108648-appb-000051
Figure PCTCN2022108648-appb-000052
人源化轻链模板IGKV4-1*01氨基酸序列如SEQ ID NO：74所示：
Figure PCTCN2022108648-appb-000053
人源化轻链模板IGKJ4*01氨基酸序列如SEQ ID NO：75所示：
Figure PCTCN2022108648-appb-000054
人源化重链模板IGHV4-38-2*01氨基酸序列如SEQ ID NO：76所示：
Figure PCTCN2022108648-appb-000055
人源化重链模板IGHJ6*01氨基酸序列如SEQ ID NO：77所示：
Figure PCTCN2022108648-appb-000056
本发明分别从上述TIGIT-006的人源化抗体轻链和重链可变区的回复突变设计中，选择不同的轻链和重链序列进行交叉组合，最终获得多种TIGIT-006人源化抗体，各抗体的可变区氨基酸序列如下：
表10 TIGIT-006人源化抗体可变区对应氨基酸序列
Fv 006.VH1 006.VH2 006.VH3 006.VH4 006.VH5
006.VL1 TIGIT-006-H1L1* TIGIT-006-H2L1 TIGIT-006-H3L1 TIGIT-006-H4L1 TIGIT-006-H5L1
006.VL2 TIGIT-006-H1L2 TIGIT-006-H2L2 TIGIT-006-H3L2 TIGIT-006-H4L2 TIGIT-006-H5L2
006.VL3 TIGIT-006-H1L3 TIGIT-006-H2L3 TIGIT-006-H3L3 TIGIT-006-H4L3 TIGIT-006-H5L3
*TIGIT-006-H1L1表示重链选自TIGIT-006.VH1，轻链选自TIGIT-006.VL1。下同。
根据Kabat编号系统，上述15个人源化抗体VH和VL序列分析结果如表11所示。
表11 TIGIT-006人源化抗体VH和VL序列的Kabat分析结果
Figure PCTCN2022108648-appb-000057
3、TIGIT-005的人源化
鼠源抗体TIGIT-005的人源化轻链模板为IGKV4-1*01和IGKJ4*01，人源化重链模板为IGHV1-3*01和IGHJ6*01，将鼠源抗体TIGIT-005的CDR分别移植到其人源模板中，即获得对应的人源化版本。根据需要，将TIGIT-005的人源化抗体的FR区序列中关键氨基酸进行回复突变为鼠源抗体对应的氨基酸，以保证原有的亲和力，具体回复突变设计见表12。
表12 TIGIT-005的人源化抗体回复突变设计
Figure PCTCN2022108648-appb-000058
注：Grafted(IGKV4-1*01)代表将鼠抗体CDR植入人种系IGKV4-1*01 FR区序列，Q38H表示将Grafted第38位Q突变回H，其它依此类推。回复突变氨基酸的编号为自然顺序编号。
TIGIT-005人源化抗体可变区具体序列如下：
TIGIT-005.VL1氨基酸序列如SEQ ID NO：86所示：
Figure PCTCN2022108648-appb-000059
TIGIT-005.VH2氨基酸序列如SEQ ID NO：87所示：
Figure PCTCN2022108648-appb-000060
人源化轻链模板IGKV4-1*01氨基酸序列如SEQ ID NO：74所示：
Figure PCTCN2022108648-appb-000061
人源化轻链模板IGKJ4*01氨基酸序列如SEQ ID NO：75所示：
Figure PCTCN2022108648-appb-000062
人源化重链模板IGHV1-3*01氨基酸序列如SEQ ID NO：106所示：
Figure PCTCN2022108648-appb-000063
人源化重链模板IGHJ6*01氨基酸序列如SEQ ID NO：77所示：
Figure PCTCN2022108648-appb-000064
TIGIT-005抗体存在易发生化学修饰的位点NG，我们对NG进行点突变以消除修饰风险。在其中五个实施例中，我们分别对005.VL1的NG进行突变，加粗所示为突变位点，突变后的序列如下：
TIGIT-005.VL1a氨基酸序列如SEQ ID NO：88所示：
Figure PCTCN2022108648-appb-000065
TIGIT-005.VL1b氨基酸序列如SEQ ID NO：89所示：
Figure PCTCN2022108648-appb-000066
TIGIT-005.VL1c氨基酸序列如SEQ ID NO：90所示：
Figure PCTCN2022108648-appb-000067
TIGIT-005.VL1d氨基酸序列如SEQ ID NO：91所示：
Figure PCTCN2022108648-appb-000068
TIGIT-005.VL1e氨基酸序列如SEQ ID NO：92所示：
Figure PCTCN2022108648-appb-000069
本发明分别从上述TIGIT-005的人源化抗体轻链和重链可变区的回复突变设计中，选择不同的轻链和重链序列进行交叉组合，最终获得6种TIGIT-005人源化抗体，各抗体的可变区氨基酸序列如下：
表13 TIGIT-005人源化抗体可变区对应氨基酸序列
Figure PCTCN2022108648-appb-000070
*TIGIT-005-H2L1表示重链选自TIGIT-005.VH2，轻链选自TIGIT-005.VL1。下同。
根据Kabat编号系统，上述6个人源化抗体VH和VL序列分析结果如表14所示。
表14 TIGIT-005人源化抗体VH和VL序列的Kabat分析结果
Figure PCTCN2022108648-appb-000071
4、TIGIT-070的人源化
鼠源抗体TIGIT-070的人源化轻链模板为IGKV1-39*01/IGKV4-1*01和IGKJ3*01，人源化重链模板为IGHV1-3*01和IGHJ6*01，将鼠源抗体TIGIT-070的CDR分别移植到其人源模板中，即获得对应的人源化版本。根据需要，将TIGIT-070的人源化抗体的FR区序列中关键氨基酸进行回复突变为鼠源抗体对应的氨基酸，以保证原有的亲和力，具体回复突变设计见表15。
表15 TIGIT-070的人源化抗体回复突变设计
Figure PCTCN2022108648-appb-000072
注：Grafted(IGKV1-39*01)代表将鼠抗体CDR植入人种系IGKV1-39*01 FR区序列；A43S表示将Grafted第43位A突变回S，其它依此类推。回复突变氨基酸的编号为自然顺序编号。
TIGIT-070人源化抗体可变区具体序列如下：
TIGIT-070.VL1氨基酸序列如SEQ ID NO：98所示：
Figure PCTCN2022108648-appb-000073
TIGIT-070.VL2氨基酸序列如SEQ ID NO：99所示：
Figure PCTCN2022108648-appb-000074
TIGIT-070.VL3氨基酸序列如SEQ ID NO：100所示：
Figure PCTCN2022108648-appb-000075
TIGIT-070.VH1氨基酸序列如SEQ ID NO：101所示：
Figure PCTCN2022108648-appb-000076
TIGIT-070.VH2氨基酸序列如SEQ ID NO：102所示：
Figure PCTCN2022108648-appb-000077
TIGIT-070.VH3氨基酸序列如SEQ ID NO：103所示：
Figure PCTCN2022108648-appb-000078
人源化轻链模板IGKV1-39*01氨基酸序列如SEQ ID NO：104所示：
Figure PCTCN2022108648-appb-000079
人源化轻链模板IGKV4-1*01氨基酸序列如SEQ ID NO：74所示：
Figure PCTCN2022108648-appb-000080
人源化轻链模板IGKJ3*01氨基酸序列如SEQ ID NO：105所示：
Figure PCTCN2022108648-appb-000081
人源化重链模板IGHV1-3*01氨基酸序列如SEQ ID NO：106所示：
Figure PCTCN2022108648-appb-000082
人源化重链模板IGHJ6*01氨基酸序列如SEQ ID NO：77所示：
Figure PCTCN2022108648-appb-000083
本发明分别从上述TIGIT-070的人源化抗体轻链和重链可变区的回复突变设计中，选择不同的轻链和重链序列进行交叉组合，最终获得多种TIGIT-070人源化抗体，各抗体的可变区氨基酸序列如下：
表16 TIGIT-070人源化抗体可变区对应氨基酸序列
Fv 070.VH1 070.VH2 070.VH3
070.VL1 TIGIT-070-H1L1* TIGIT-070-H2L1 TIGIT-070-H3L1
070.VL2 TIGIT-070-H1L2 TIGIT-070-H2L2 TIGIT-070-H3L2
070.VL3 TIGIT-070-H1L3 TIGIT-070-H2L3 TIGIT-070-H3L3
*TIGIT-070-H1L1表示重链选自TIGIT-070.VH1，轻链选自TIGIT-070.VL1。下同。
根据Kabat编号系统，上述9个人源化抗体VH和VL序列分析结果如表17所示。
表17 TIGIT-070人源化抗体VH和VL序列的Kabat分析结果
Figure PCTCN2022108648-appb-000084
5、抗人TIGIT单克隆抗体的人源化变体鉴定
采用BIAcore检测上述人源化抗体与人TIGIT ECD-His的亲和力，并参考实施例4(a)、(b)、(d)的方法检测上述人源化抗体与人TIGIT ECD-mFc的结合活性、与CHO-K1人TIGIT的结合活性、与CHO-K1食蟹猴TIGIT的结合活性、与食蟹猴TIGIT ECD-mFc的结合活性、以及阻断人TIGIT ECD-mFc与CHO-K1 CD155结合的效应，结果如表18所示。
可见，TIGIT-002-H4L3、TIGIT-005-H2L1d、TIGIT-006-H5L3、TIGIT-070-H1L1四株人源化抗体保持了和嵌合抗体相当的亲和力、结合活性和阻断效应，未受人源化明显影响，且表现总体优于RG6058。
表18 SJL小鼠衍生的抗人TIGIT单克隆抗体的人源化变体鉴定汇总
Figure PCTCN2022108648-appb-000085
Figure PCTCN2022108648-appb-000086
无数据：未检测
实施例6抗TIGIT人源化抗体的鉴定
(a)抗TIGIT人源化抗体与人TIGIT ECD-mFc和食蟹猴TIGIT ECD-mFc的结合活性检测
实验方法参见实施例4(a)。实验结果表明和对照抗体罗氏RG6058相一致，抗TIGIT人源化抗体能有效结合人TIGIT ECD-mFc(图13)和食蟹猴TIGIT ECD-mFc(图14)
(b)FACS检测抗TIGIT人源化抗体与CHO-K1人TIGIT高中低表达水平的细胞和CHO-K1食蟹猴TIGIT的结合活性
实验方法参见实施例4(b)。实验结果表明和对照抗体罗氏RG6058一致，抗TIGIT人源化抗体可以有效结合CHO-K1-TIGIT高表达细胞株(图15)，CHO-K1-TIGIT中表达细胞株(图16)，CHO-K1-TIGIT低表达细胞株(图17)及CHO-K1-食蟹猴TIGIT细胞株(图18)。
(c)FACS方法检测抗TIGIT人源化抗体阻断Bio-CD155-His与CHO-K1人TIGIT的相互作用
实验方法参见实施例4(c)。如图19所示，抗TIGIT人源化抗体与对照抗体RG6058都可以有效阻断Bio-CD155-His蛋白与CHO-K1-人TIGIT细胞的结合。
(d)FACS方法检测抗TIGIT人源化抗体阻断TIGIT ECD-mFc与CHO-K1 CD155的相互作用
实验方法参见实施例4(d)。如图20所示，抗TIGIT人源化抗体与对照抗体RG6058都可以有效阻断人TIGIT蛋白与CHO-K1-CD155细胞的结合。
(e)FACS方法检测抗TIGIT人源化抗体阻断TIGIT ECD-mFc与CHO-K1 CD112相互作用的效应
收集CHO-K1 CD112细胞，PBS(Hyclone，CAT#SH30256)洗一次，1％BSA-PBS重悬 至2×10 5/40μL，1％BSA-PBS稀释人源化抗体至600nM，2.5倍连续梯度稀释12个浓度点，1％BSA-PBS稀释TIGIT ECD-mFc至6μg/mL，随后将40μL细胞与40μL抗体稀释液以及40μL TIGIT ECD-mFc稀释液混合，4℃孵育60分钟，PBS洗两次，加入Alexa
Figure PCTCN2022108648-appb-000087
647荧光素标记的二抗(工作稀释度1：800，Jackson,CAT#115-605-003)，100μL/孔重悬细胞，4℃孵育40分钟，PBS洗两次，用1％BSA-PBS，100μL/孔重悬，在流式细胞仪(BD,CantoⅡ)上分析细胞样品。如图21所示，抗TIGIT人源化抗体与对照抗体RG6058都可以有效阻断人TIGIT蛋白与CHO-K1-CD112细胞的结合。
(f)FACS方法检测抗TIGIT人源化抗体与人PBMC的结合活性
取新鲜的人PBMC(AllCells，PB004-C)，将细胞调整至5×10 5/mL，同时加入SEA(Toxin Technology,Inc.，CAT：AT101)至100ng/mL，37℃5％CO 2培养三天；三天后收集细胞，PBS(Hyclone，CAT#SH30256)洗一次,加入Fc Block(BD，564220)，4℃孵育10分钟，PBS洗两次，1％BSA-PBS重悬至2×10 5/50μL，1％BSA-PBS稀释人源化抗体至80nM，3倍连续梯度稀释12个浓度点，将50μL细胞与50μL抗体稀释液混合，4℃孵育60分钟，PBS洗两次，加入Alexa
Figure PCTCN2022108648-appb-000088
647荧光素标记的二抗(工作稀释度1：800，Jackson，CAT#109-605-088)，100μL/孔重悬细胞，4℃孵育60分钟，PBS洗两次，用1％BSA-PBS，100μL/孔重悬，在流式细胞仪(BD,CantoⅡ)上分析细胞样品。如图22所示，和对照抗体罗氏RG6058相一致，抗TIGIT人源化抗体能有效结合SEA刺激后的人PBMC。
表19汇总了这四株人源化抗体的特性。TIGIT-002-H4L3、TIGIT-005-H2L1d、TIGIT-006-H5L3、TIGIT-070-H1L1四株人源化抗体与PBMC、以及与人TIGIT高中低表达水平的稳转株细胞的结合趋势一致，TIGIT-002-H4L3和TIGIT-005-H2L1d强于TIGIT-006-H5L3和TIGIT-070-H1L1，都强于RG6058，与人TIGIT ECD-His单价亲和力依次为0.0994nM、0.0852nM、0.1145nM、0.2505nM，RG6058为0.1560nM，且都与食蟹猴TIGIT有较强的交叉反应性；在体外阻断表征方面，四株人源化抗体都表现出了较显著的阻断TIGIT-CD155以及TIGIT-CD112相互作用的能力。
表19 TIGIT-002-H4L3、TIGIT-005-H2L1d、TIGIT-006-H5L3、TIGIT-070-H1L1四株人源化抗体的生物活性
Figure PCTCN2022108648-appb-000089
Figure PCTCN2022108648-appb-000090
(g)NK细胞杀伤实验检测抗TIGIT人源化抗体对NK杀伤靶细胞的促进作用
实验方法参见实施例4(g)。图23表明：阴性对照anti-HA HcAb-hIgG1对NK杀伤没有显著影响，四个人源化抗体TIGIT-002-H4L3、TIGIT-005-H2L1d、TIGIT-006-H5L3和TIGIT-070-H1L1能不同程度地促进NK对靶细胞的杀伤，其中TIGIT-002-H4L3、TIGIT-005-H2L1d与其人源化前嵌合抗体TIGIT-CHI-002、TIGIT-CHI-005的NK杀伤促进能力相当。
(h)CMV antigen-recall assay检测抗TIGIT人源化抗体对抗原特异性CD8 T细胞的功能改善作用
PBMC复苏后，用含有1mg/mL CMV pp65(495-503)多肽(Anaspec,货号AS-28328)、2ng/mL human IL-2(R&D,货号IL-202)、10ng/mL human IL-7(Peprotech,货号200-07)的完全培养基(RPMI1640-Glutamax+5％AB serum+1％P/S+1×2-β巯基乙醇)重悬至2×10 6/mL，5mL/孔接种于6孔板中，37℃5％CO 2培养6天。第6天，收集所有细胞，撤去培养基中的pp65和IL-7，将细胞一分为二，并重悬于含有100IU/mL human IL-2的完全培养基中，继续培养2天。第8天，收集所有细胞，重悬于含有100IU/mL human IL-2的完全培养基中、并调整细胞密度为2×10 6/mL，继续培养。第11天，收集所有细胞，流式检测PBMC中CD8T细胞的比例，CMV pp65(495-503)特异性CD8T的比例(图24中A)及该细胞上PVRIG、TIGIT、PD-1和CD226的表达(图24中B)。流式检测抗体如下：Livedead Near IR(Invitrogen，货号L34976)，CD8-PerCp Cy5.5(BD，货号565310)，CD3-PE-Cy7(Biolegend,货号300316)，T-select HLA-A*0201 CMV pp65 Tetramer-PE(MBL，货号TS-0010-1C)，PVRIG-AF488(R&D，货号FAB93651G-100UG)，TIGIT-APC(Biolegend,货号372706)，PD-1-BV421(BD,货号562516)
上述诱导后的PBMC经CD8 T分选试剂盒(Stemcell，货号17953)分离出CD8 T作为效应细胞，用AIM-V重悬并调整细胞密度至0.4×10 6/mL。分选后的CD8检测纯度及CD226的表达。Colo205作为靶细胞，经TrypLE TMExpress Enzyme(Gibco,货号12605010)消化，重悬于含有20ng/mLpp65的AIM-V(Gibco,货号31035-025)并调整细胞密度至1×10 6/mL，37℃5％CO 2处理3小时，之后250g离心5分钟，弃上清。之后细胞用AIM-V重悬至0.5×10 6/mL。抗TIGIT人源化抗体或阳性对照用AIM-V稀释至280nM。在低吸附96孔U底板(Corning,货号7007)中依次加入50μL抗体，50μL CD8 T，100μL pp65处理过的colo205，并用排枪轻轻混匀，37℃5％CO2孵育18小时。此体系中药物终浓度为70nM，CD8 T为20000/孔，colo205为50000/孔。孵育结束后400g离心取上清，用ELISA试剂盒(达科为，货号1110003)检测上清中human IFN-γ的水平。此体系中的阳性对照为RG6058-hIgG1和TIGIT人源化前的抗体(TIGIT-CHI-002)，阴性对照为no treatment。如图24中C所示，相较于RG6058-hIgG1，4个TIGIT人源化抗体(TIGIT-002-H4L3、TIGIT-005-H2L1d、TIGIT-006-H5L3、TIGIT-070-H1L1)作用后，细胞上清中的IFN-γ的分泌在统计学上均无显著性差异(One-way ANOVA Analysis)；相较于TIGIT人源化前的嵌合抗体(TIGIT-CHI-002)，4个抗TIGIT人源化抗体作用后，细胞上清中的IFN-γ的分泌在统计学上均无显著性差异(One-way ANOVA Analysis)但均显著高于no treatment组，每个柱状图上的百分比意为相较于no treatment组IFN-γ分泌提升的百分 比。
分选后CD8 T纯度及其上CD226表达的流式检测抗体如下：livedead-BV421(Invitrogen,货号L34964)，CD8-FITC(BD,货号555366)，CD226-PE-Cy7(Biolegend，货号338316)。Colo205上PVRL2、PVR、PD-L1、HLA-A2表达的流式检测抗体如下：livedead-BV421(Invitrogen,货号L34964)，PVRL2-APC(Biolegend，货号337412)，PVR-PerCp Cy5.5(Biolegend，货号337612),PD-L1-PE-Cy7(BD，货号558017),HLA-A2-PE(Biolegend,货号343306)。
实施例7 PVRIG羊驼VHH抗体的制备
选取两只成年健康羊驼(Alpaca)(委托成都阿帕克公司购买)在第一次免疫注射用弗氏完全佐剂(complete freund'sadjuvant，CFA，购自SIGMA，货号：F5881)混合人PVRIG重组蛋白(Acro Biosystems,货号：PVG H5257)，后三次免疫注射的佐剂则用弗氏不完全佐剂(incomplete freund's adjuvant，IFA，购自SIGMA，货号：F5506)混合与第一次相同的人PVRIG重组蛋白，4次免疫均采用皮下注射。免疫前取血10mL，留为阴性血清对照，在第二次免疫后取血10mL检测血清抗体滴度，在第三和四次免疫后分别取外周血50mL分离淋巴细胞，根据淋巴细胞量加入5mL RNA iso Plus(Takara，货号：9109)，分装于1.5mL EP管中于-80℃保存。从冻存的淋巴细胞中提取总RNA反转录成cDNA，然后以cDNA为模板分别进行两轮VHH PCR扩增。酶切第二轮PCR扩增的VHH产物进行噬菌体文库的构建。收集建立的细菌库，并对库插入率和多样性进行测序分析。
对噬菌体进行两轮亲和淘选，对特异性结合靶蛋白人PVRIG-his(AcroBiosystems，货号PVG-H52H4)的噬菌体克隆进行鉴定。将淘选中和人PVRIG-His蛋白结合较好的优选克隆测序后通过同源重组方法克隆到表达载体中，其中CH2和CH3恒定区都来自于人IgG1，完整表达序列是信号肽-VHH-铰链区-CH2-CH3。经过一系列理化和功能筛选后共获得13个阳性候选抗体分子，其序列的CDRs分别用IMGT和KABAT软件分析，对应的序列信息如下表20至22所示，其中表20示出候选抗体分子的VHH序列，表21示出候选抗体分子的IMGT分析结果，表22示出候选抗体分子的KABAT分析结果。
表20候选抗体分子VHH序列
Figure PCTCN2022108648-appb-000091
Figure PCTCN2022108648-appb-000092
表21候选抗体分子的IMGT分析结果
Figure PCTCN2022108648-appb-000093
Figure PCTCN2022108648-appb-000094
表22候选抗体分子的KABAT分析结果
Figure PCTCN2022108648-appb-000095
实施例8 ELISA检测抗PVRIG抗体与人和食蟹猴PVRIG蛋白的特异性结合
酶标板中预先包被100μL/孔的0.5μg/mL人PVRIG-his(AcroBiosystems，货号PVG-H52H4)或食蟹猴PVRIG蛋白(Novoprotein，货号C09B)；将受试抗PVRIG抗体(实施例7所述VHH连接人IgG1-Fc构成)进行梯度稀释(起始浓度20nM、3.33倍梯度稀释或3nM、3倍梯度稀释)，100μL/孔加样，室温振荡孵育1.5小时；洗板后加入鼠抗人(mouse anti-human)IgG Fc-HRP(Jackson ImmunoResearch，货号209-035-098)工作液(1:10000稀释)，100μL/孔加样，室温振荡孵育1.0小时；再次洗板，加入HRP的底物TMB(Thermo，货号34029)进行显色，加入终止液终止反应后用酶标仪(MD i3x)读取吸光值。以抗体浓度为横 坐标，对应的OD值为纵坐标绘制抗体的结合曲线，四参数拟合(GraphPad Prism9)，计算EC50值。EC50值越小，抗体与人/食蟹猴PVRIG结合的能力越强。阳性对照抗体有COM701-hIgG1(Patent No.:US20180244774A1)、COM701-hIgG4(patent No.:US20180244774A1)和SRF813-hIgG1(Patent No.:US20200040081A1)；阴性对照抗体有anti-HA HcAb-hIgG1(成都阿帕克，货号NBR022)、anti-CD38 HcAb-hIgG1(in-house)和anti-Fluorescein-hIgG1(in-house)。
COM701对应的氨基酸序列如下所示：
COM701 VH SEQ ID NO 236:
Figure PCTCN2022108648-appb-000096
COM701 VL SEQ ID NO 237:
Figure PCTCN2022108648-appb-000097
SRF813 VH SEQ ID NO 238:
Figure PCTCN2022108648-appb-000098
SRF813 VL SEQ ID NO 239:
Figure PCTCN2022108648-appb-000099
13个抗PVRIG抗体与人PVRIG蛋白的结合结果见图26A和表23，与食蟹猴PVRIG的结合结果见图26B和表23。数据表明，所有受试抗体均能和人或食蟹猴PVRIG蛋白特异性结合。
表23抗PVRIG抗体与人或食蟹猴PVRIG蛋白特异性结合的ELISA结果
Figure PCTCN2022108648-appb-000100
实施例9 FACS检测抗PVRIG抗体与FlpinCHO-PVRIG细胞及FlpinCHO-cyno PVRIG细胞的结合
取转染了人或食蟹猴PVRIG高表达质粒的CHO-K1稳定细胞(分别命名为FlpinCHO-PVRIG，FlpinCHO-cyno PVRIG)，人PVRIG全长质粒(NCBI Ref Seq:NP_076975),以及食蟹猴PVRIG全长质粒(NCBI Ref Seq:XP_014989941)均由通用生物合成，在细胞密度不超过80％时进行实验。弃去细胞培养基，用PBS润洗并加入1ml Versene(Gibico，15040-066)消化8-10分钟，用含10％FBS的Ham’s F12(Gibico，21127-022)完全培养基终止消化后制成细胞悬液。细胞计数仪(Beckman Coulter，Vi-CELL)计数后，取适量细胞悬液，350×g离心去上清，PBS洗细胞两遍后，用死活染料Zombie violet(Biolegend，423114)进行染色，室温孵育20分钟。孵育结束后，用染色缓冲液(2％FBS+PBS)终止染色，350×g离心去上清，洗细胞两遍后，用染色缓冲液将细胞重悬为2×10 6个细胞/ml的密度，铺入96孔板，每孔加入50μl的细胞悬液，待用。用染色缓冲液将抗体从最高浓度46nM(两倍浓度)开始进行3.3倍梯度稀释，将稀释好的抗体加至已含有50μl细胞悬液的孔中，置于微孔板振荡器上400rpm震荡1分钟，使抗体与细胞充分混合，4℃孵育30分钟。孵育结束后用染色缓冲液清洗细胞两次，每孔200μl，350×g离心5分钟弃上清。用染色缓冲液将PE goat anti-Human IgG Fc抗体(ebioscience，12-4998-82)稀释250倍，以每孔100μl的体积加至洗完的细胞孔中，混合均匀，4℃染色30分钟。染色结束后同样用染色缓冲液清洗两次，最后用200μl染色缓冲液重悬细胞，流式上机检测信号(BD CantoII)。荧光信号越强，表示抗体与PVRIG的结合能力越强。以抗体浓度为横坐标，对应的平均荧光强度MFI倍数为纵坐标绘制抗体的结合曲线，四参数拟合(GraphPad Prism9)，计算结合曲线的AUC值。AUC值越大，受试抗体与FlpinCHO-PVRIG、FlpinCHO-cyno PVRIG细胞结合的能力越强。如图27A、图27B所示，所有受试抗体均可与过表达细胞表面人/食蟹猴PVRIG结合。将抗体的结合活性归一化到与阳性分子COM701-hIgG1以及SRF813-hIgG1抗体的百分比，百分比值越高，说明抗体结合的活性越强。表24结果表明，受试抗体PVRIG-A11、A35、A43、A105、A117、A118与人PVRIG的结合活性强于COM701-hIgG1及SRF813-hIgG1。其中，PVRIG-A105、A117与食蟹猴PVRIG的结合活性也强于COM701-hIgG1及SRF813-hIgG1。
表24抗PVRIG抗体与过表达细胞表面人/食蟹猴PVRIG的结合
Figure PCTCN2022108648-appb-000101
Figure PCTCN2022108648-appb-000102
实施例10 BIAcore检测抗PVRIG抗体与人PVRIG蛋白的亲和力
利用Biacore检测受试抗PVRIG抗体与人PVRIG蛋白之间的特异性结合。该实验采用ProteinA芯片，通过手工操作(manual run)测定出芯片捕获稀释后的抗体所需要的时间，以使得能饱和结合抗原Rmax为50RU。将人PVRIG蛋白(Human PVRIG-His,Acro C227P1-9ARF1-T4)梯度稀释至20，10，5，2.5，1.25nM。采用多循环动力学测得抗体与抗原的亲和力。在每一个循环中，注射抗体后再注入梯度浓度的PVRIG蛋白，使抗原与抗体发生结合与解离过程。每个循环后用Glycine pH1.5进行ProteinA芯片的再生(去除芯片上的蛋白)。应用BIAcore T200分析软件拟合抗体抗原的亲和力KD。表25结果可知，所有受试抗PVRIG抗体与人PVRIG蛋白之间存在特异性结合，且亲和力水平较高。
表25抗PVRIG抗体与人PVRIG蛋白特异性结合的BIAcore结果
Figure PCTCN2022108648-appb-000103
实施例11 ELISA检测抗PVRIG抗体阻断PVRIG与PVRL2的结合
酶标板中预先包被0.5μg/mL 100μL/孔的人PVRIG-his蛋白(AcroBiosystems，货号PVG-H52H4)，将所有受试抗PVRIG抗体从最高浓度16nM进行2倍梯度稀释，稀释后的抗体分别与18ng/mL人PVRL2-mFc(AcroBiosystems,货号CD2-H5257)等体积混合，按100μL/孔加入酶标板中，室温振荡孵育2.0小时；洗板后加入羊抗鼠IgG Fc-HRP(Jackson ImmunoResearch，货号115-035-071)工作液(1:10000稀释)，100μL/孔，室温振荡孵育1.0小时；再次洗板，加入HRP的底物TMB(Thermo,货号34029)进行显色，加入终止液终止反应后用酶标仪(MD i3X)读取吸光值。以抗体浓度为横坐标，对应的OD值为纵坐标绘制抗体的抑制曲线，四参数拟合(GraphPad Prism9)，计算IC50值。IC50值越小，抗体抑制人PVRIG与人PVRL2结合的能力越强。阳性对照抗体及阴性对照抗体，同实施例8。13个受 试抗体的阻断曲线见图28，抑制活性见表26。从图28及表26可知，所有受试抗体均能显著地抑制人PVRIG与人PVRL2蛋白的结合。
表26受试抗体阻断PVRIG和PVRL2的结合
样品编号 IC50(pM)
PVRIG-A11 121.40
PVRIG-A15 207.70
PVRIG-A30 254.70
PVRIG-A50 195.70
PVRIG-A60 55.66
PVRIG-A75 62.11
PVRIG-A35 54.31
PVRIG-A43 53.65
PVRIG-A104 65.93
PVRIG-A105 57.07
PVRIG-A113 92.00
PVRIG-A117 52.84
PVRIG-A118 51.35
实施例12 FACS检测抗PVRIG抗体阻断CHO-K1-CD112细胞与人PVRIG-mFc蛋白的结合
取转染了人CD112高表达质粒的CHO-K1稳定细胞(命名为CHO-K1-CD112)，人CD112全长质粒由通用生物合成(NP_001036189.1/NCBI Ref Seq:Q92692)，在细胞密度不超过80％时进行实验。弃去细胞培养基，用PBS润洗并加入1ml胰酶(Gibico，25200-72)消化2分钟，用含10％FBS的Ham’s F12(Gibico，21127-022)完全培养基终止消化后制成细胞悬液。细胞计数仪(Beckman Coulter，Vi-CELL)计数后，取适量细胞悬液，350×g离心去上清，PBS洗细胞两遍后，用死活染料Zombie violet(Biolegend，423114)进行染色，室温孵育20分钟。孵育结束后，用染色缓冲液(2％FBS+PBS)终止染色，350×g离心去上清，洗细胞两遍后，用染色缓冲液将细胞重悬为1×10 6个细胞/ml的密度，待用。铺入96孔板，每孔加入50μl的细胞悬液，待用。用染色缓冲液配制人PVRIG-mFc蛋白(Acro，PVG-H5253)工作液，浓度为1μg/ml(四倍浓度)，加入96孔板，每孔加入50μl的PVRIG-mFc工作液。用染色缓冲液将抗体从最高浓度275nM(四倍浓度)开始进行3倍梯度稀释，将稀释好的抗体加至已含有50μl PVRIG-mFc的孔中，置于微孔板振荡器上400rpm震荡1分钟，使抗体与PVRIG-mFc蛋白充分混合，4℃孵育30分钟。孵育结束后，直接加入上述准备的细胞悬液，每孔加入100μl，枪头轻柔混匀后，4℃孵育30分钟。孵育结束后用染色缓冲液清洗细胞两次，每孔200μl，350×g离心5分钟弃上清。用染色缓冲液将PE goat anti-mouse IgG Fc抗体(Biolegend，405337)稀释250倍，以每孔100μl的体积加至洗完的细胞孔中，混合均匀，4℃染色30分钟。染色结束后同样用染色缓冲液清洗两次，最后用200μl染色缓冲液重悬细胞，流式上机检测信号(BD CantoII)。荧光信号越弱，表示抗体与竞争CHO-K1-CD112细胞结合PVRIG-mFc蛋白的能力越强。以抗体浓度为横坐标，对应的平均荧光强度MFI倍数为纵 坐标绘制抗体的结合曲线，四参数拟合(GraphPad Prism9)，计算抗体的IC50值以及结合曲线AUC值。IC50值及AUC值越小，表示抗体竞争CHO-K1-CD112细胞结合PVRIG-mFc蛋白的能力越强，即抗体的阻断效果越好。如图29所示，所有受试抗体均可阻断CHO-K1-CD112细胞结合人PVRIG-mFc蛋白。将受试抗体的竞争阻断活性归一化到与对照分子COM701-hIgG1以及SRF813-hIgG1抗体的百分比，百分比值越小，说明抗体的阻断活性越强。表27结果表明，受试抗体PVRIG-A11、A15、A30、A50的阻断活性均强于COM701-hIgG1以及SRF813-hIgG1。
表27抗PVRIG抗体竞争CHO-K1-CD112细胞结合人PVRIG-mFc蛋白
Figure PCTCN2022108648-appb-000104
实施例13 NK细胞表面PVRIG和TIGIT及肿瘤细胞系Reh和WIDR细胞表面PVR和PVRL2的表达检测
利用FACS检测NK细胞(Natural killer cell)上PVRIG，TIGIT的表达情况。实验方法参考实施例4(e)，结果如图9A所示，不同供体donor-010及donor-050的NK细胞表面均有PVRIG及TIGTI的表达。
利用FACS检测Reh/WIDR细胞上PVR和PVRL2的表达情况。实验方法参考实施例4(e)，其中直接吹匀Reh制备细胞悬液。WIDR细胞表面的PVR及PVRL2的表达如图9B所述。图30显示，Reh细胞表面的PVR表达为阴性，PVRL2的表达为阳性。
实施例14 NK细胞脱粒实验检测抗PVRIG抗体对NK细胞的功能促进作用
利用FACS检测NK细胞(Natural killer cell)CD107a的信号来指示受试抗体对NK细胞脱颗粒过程的影响。实验方法参考实施例4(f)，其中Reh细胞直接混合均匀制备细胞悬液。图31显示，阴性对照anti-HA HcAb-hIgG1对NK细胞的CD107a没有影响，12个PVRIG受试抗体及对照抗体COM701-hIgG1和SRF813-hIgG1均能不同程度的提高NK细胞CD107a的表达，这表明受试抗体和对照抗体均可以有效促进NK细胞的激活。
实施例15 NK细胞杀伤实验检测PVRIG抗体介导的NK细胞对肿瘤细胞系的杀伤作用
利用FACS检测靶细胞(WIDR)的被裂解水平来反映受试抗体对NK细胞杀伤功能的影响。实验方法参考实施例4(g)。图32显示，阴性对照anti-HA HcAb-hIgG1对NK杀伤没有影响，13个受试抗体均能有效促进NK对靶细胞WIDR的杀伤，并且除了PVRIG-A35和A43外，剩余11个受试抗体的对WIDR细胞的杀伤促进功能均强于或与对照抗体COM701-hIgG1相当。
实施例16 CMV antigen-recall assay检测抗PVRIG抗体对抗原特异性CD8 T细胞的功能改善作用
Anti-CMV IgG阳性donor的PBMC经CMV pp65(495-503)多肽诱导的CMV pp65特异性CD8T作为效应细胞，经pp65 pulsed后的colo205肿瘤细胞系作为靶细胞的实验体系中，检测抗PVRIG抗体对抗原特异性CD8 T细胞的功能改善作用。
复苏CMV IgG+PBMC，用含有1mg/mL CMV pp65(495-503)多肽(Anaspec,货号AS-28328)、2ng/mL human IL-2(R&D,货号IL-202)、10ng/mL human IL-7(Peprotech,货号200-07)的完全培养基(RPMI1640-Glutamax+5％AB serum+1％P/S+(1×)2-β巯基乙醇)重悬至2×10 6/mL，5mL/孔接种于6孔板中，37℃5％CO 2培养6天。第6天，收集所有细胞，撤去培养基中的pp65多肽和IL-7，将细胞一分为二，并重悬于含有100IU/mL human IL-2的完全培养基中，继续培养2天。第8天，收集所有细胞，重悬于含有100IU/mL human IL-2的完全培养基中、并调整细胞密度为2×10 6/mL，继续培养。第11天，收集所有细胞，流式检测PBMC中CD8 T细胞的比例，CMV pp65(495-503)特异性CD8 T的比例及该细胞上PVRIG、TIGIT、PD-1的表达如图33中A、B所示，经pp65诱导后，CMV pp65(495-503)特异性CD8 T的比例超过80％，图33中B所示pp65+CD8+T(donor021)表达不同水平的PVRIG，TIGIT，PD-1及CD226。流式检测抗体如下：Live/deadNear IR(Invitrogen，货号L34976)，CD8-PerCp Cy5.5(BD，货号565310)，CD3-PE-Cy7(Biolegend,货号300316)，T-select HLA-A*0201 CMV pp65 Tetramer-PE(MBL，货号TS-0010-1C)，PVRIG-AF488(R&D，货号FAB93651G-100UG)，TIGIT-APC(Biolegend,货号372706)，PD-1-BV421(BD,货号562516)。
上述诱导后的PBMC经CD8 T分选试剂盒(Stemcell，货号17953)分离出CD8 T作为效应细胞，用AIM-V重悬并调整细胞密度至0.4×10 6/mL。检测分选后的CD8 T纯度及CD226的表达。Colo205作为靶细胞，经TrypLE TMExpress Enzyme(Gibco,货号12605010)消化，重悬于含有20ng/mL pp65的AIM-V(Gibco,货号31035-025)并调整细胞密度至1×10 6/ml，37℃5％CO 2处理3小时，之后250g离心5分钟，弃上清。细胞用AIM-V重悬至0.5×10 6/mL，流式检测Colo205细胞高表达PVRL2，PVR及HLA-A2如图33中C所示。抗PVRIG抗体或阴性对照用AIM-V稀释至280nM。在低吸附96孔U底板(Corning,货号7007)中依次加入50μL抗体，50μL CD8 T，100μL pp65处理过的colo205细胞，并用排枪轻轻混匀，37℃5％CO 2孵育18小时。此体系中药物终浓度为70nM,CD8 T为20000/孔，colo205细胞为50000/孔。孵育结束后400g离心取上清，用ELISA试剂盒(达科为，货号1110003)检测上清中human IFN-γ的水平。此体系中的阳性对照为COM701-hIgG4,SRF813-hIgG1,阴性对照为no treatment。如图33中D所示，相较于no treatment组，绝大部分受试PVRIG抗体作用后，细胞上清中IFN-γ的分泌明显增加。
分选后CD8 T纯度及其CD226表达的流式检测抗体如下：livedead-BV421(Invitrogen,货号L34964)，CD8-FITC(BD,货号555366)，CD226-PE-Cy7(Biolegend，货号338316)。Colo205细胞PVRL2、PVR、PD-L1、HLA-A2表达的流式检测抗体如下：livedead-BV421(Invitrogen,货号L34964)，PVRL2-APC(Biolegend，货号337412)，PVR-PerCp Cy5.5(Biolegend，货号337612),PD-L1-PE-Cy7(BD，货号558017),HLA-A2-PE(Biolegend,货号343306)。
实施例17羊驼抗人PVRIG抗体的人源化
通过比对IMGT(http://imgt.cines.fr)人类抗体重轻链可变区种系基因数据库，分别挑选与羊驼抗体同源性高的重链可变区种系基因作为模板，将羊驼抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。根据需要，将骨架序列中关键氨基酸回复突变为羊驼抗体对应的氨基酸，以保证原有的亲和力，即得到人源化抗PVRIG单克隆抗体。
1、PVRIG-A50的人源化
羊驼抗体PVRIG-A50的人源化重链模板为IGHV3-23*04和IGHJ3*01，将羊驼抗体PVRIG-A50的CDR分别移植到其人源模板中，即获得对应的人源化版本，此处抗体CDR氨基酸是由Kabat编号系统确定并注释。根据需要，将PVRIG-A50的人源化抗体的FR区序列中关键氨基酸进行回复突变为羊驼抗体对应的氨基酸，以保证原有的亲和力。对于PVRIG-A50抗体中存在易发生化学修饰的位点NG和糖基化位点NLS，我们对NG/NLS进行点突变以消除修饰风险。具体设计见表28。
表28 PVRIG-A50的人源化抗体设计
Figure PCTCN2022108648-appb-000105
注：Grafted(IGHV3-23*04)代表将目标抗体CDR植入人种系IGHV3-23*04FR区序列；第一个+后面A97V表示将Grafted第97位A突变回V；第二个+后面N54D表示对NG位点进行的点突变，其它依此类推。突变氨基酸的编号为自然顺序编号，下同。
PVRIG-A50人源化抗体可变区具体序列如下：
A50.VH1(PVRIG-A50-H1)氨基酸序列如SEQ ID NO：198所示：
Figure PCTCN2022108648-appb-000106
A50.VH1a(PVRIG-A50-H1a)氨基酸序列如SEQ ID NO：199所示：
Figure PCTCN2022108648-appb-000107
A50.VH1b(PVRIG-A50-H1b)氨基酸序列如SEQ ID NO：200所示：
Figure PCTCN2022108648-appb-000108
A50.VH1c(PVRIG-A50-H1c)氨基酸序列如SEQ ID NO：201所示：
Figure PCTCN2022108648-appb-000109
A50.VH1d(PVRIG-A50-H1d)氨基酸序列如SEQ ID NO：202所示：
Figure PCTCN2022108648-appb-000110
A50.VH1e(PVRIG-A50-H1e)氨基酸序列如SEQ ID NO：203所示：
Figure PCTCN2022108648-appb-000111
A50.VH2a(PVRIG-A50-H2a)氨基酸序列如SEQ ID NO：204所示：
Figure PCTCN2022108648-appb-000112
人源化重链模板IGHV3-23*04氨基酸序列如SEQ ID NO：205所示：
Figure PCTCN2022108648-appb-000113
人源化重链模板IGHJ3*01氨基酸序列如SEQ ID NO：206所示：
Figure PCTCN2022108648-appb-000114
根据Kabat编号系统，上述7个人源化抗体VH序列分析结果如表29所示。
表29 PVRIG-A50人源化抗体VH序列的Kabat分析结果
Figure PCTCN2022108648-appb-000115
2、PVRIG-A105的人源化
羊驼抗体PVRIG-A105的人源化重链模板为IGHV3-7*01和IGHJ3*01，将羊驼抗体PVRIG-A105的CDR分别移植到其人源模板中，即获得对应的人源化版本，此处抗体CDR氨基酸是由IMGT编号系统确定并注释。根据需要，将PVRIG-A105的人源化抗体的FR区序列中关键氨基酸进行回复突变为羊驼抗体对应的氨基酸，以保证原有的亲和力。PVRIG-A105抗体存在两个游离的半胱氨酸，为了提高抗体的稳定性，我们对Cys进行点突变。具体设计见表30。
表30 PVRIG-A105的人源化抗体设计
Figure PCTCN2022108648-appb-000116
注：Grafted(IGHV3-7*01)代表将目标抗体CDR植入人种系IGHV3-7*01 FR区序列；第一个+后面V37F表示将Grafted第37位V突变回F；第二个+后面C103S表示对Cys位点进行的点突变，其它依此类推。突变氨基酸的编号为自然顺序编号，下同。
PVRIG-A105人源化抗体可变区具体序列如下：
A105.VH1(PVRIG-A105-H1)氨基酸序列如SEQ ID NO：211所示：
Figure PCTCN2022108648-appb-000117
A105.VH2(PVRIG-A105-H2)氨基酸序列如SEQ ID NO：212所示：
Figure PCTCN2022108648-appb-000118
A105.VH3(PVRIG-A105-H3)氨基酸序列如SEQ ID NO：213所示：
Figure PCTCN2022108648-appb-000119
A105.VH4(PVRIG-A105-H4)氨基酸序列如SEQ ID NO：214所示：
Figure PCTCN2022108648-appb-000120
A105.VH5(PVRIG-A105-H5)氨基酸序列如SEQ ID NO：215所示：
Figure PCTCN2022108648-appb-000121
A105.VH3a(PVRIG-A105-H3a)氨基酸序列如SEQ ID NO：216所示：
Figure PCTCN2022108648-appb-000122
人源化重链模板IGHV3-7*01氨基酸序列如SEQ ID NO：217所示：
Figure PCTCN2022108648-appb-000123
人源化重链模板IGHJ3*01氨基酸序列如SEQ ID NO：206所示：
Figure PCTCN2022108648-appb-000124
根据IMGT编号系统，上述6个人源化抗体VH序列分析结果如表31所示。
表31 PVRIG-A105人源化抗体VH序列的IMGT分析结果
Figure PCTCN2022108648-appb-000125
3、PVRIG-A118的人源化
羊驼抗体PVRIG-A118的人源化重链模板为IGHV3-7*01和IGHJ3*01，将羊驼抗体PVRIG-A118的CDR分别移植到其人源模板中，即获得对应的人源化版本，此处抗体CDR氨基酸是由IMGT编号系统确定并注释。根据需要，将PVRIG-A118的人源化抗体的FR区序列中关键氨基酸进行回复突变为羊驼抗体对应的氨基酸，以保证原有的亲和力。具体设计见表32。
表32 PVRIG-A118的人源化抗体设计
Figure PCTCN2022108648-appb-000126
注：Grafted(IGHV3-7*01)代表将目标抗体CDR植入人种系IGHV3-7*01 FR区序列；第一个+后面S35G表示将Grafted第35位S突变回G；其它依此类推。回复突变氨基酸的编号为自然顺序编号，下同。
PVRIG-A118人源化抗体可变区具体序列如下：
A118.VH1(PVRIG-A118-H1)氨基酸序列如SEQ ID NO：219所示：
Figure PCTCN2022108648-appb-000127
A118.VH2(PVRIG-A118-H2)氨基酸序列如SEQ ID NO：220所示：
Figure PCTCN2022108648-appb-000128
A118.VH3(PVRIG-A118-H3)氨基酸序列如SEQ ID NO：221所示：
Figure PCTCN2022108648-appb-000129
A118.VH4(PVRIG-A118-H4)氨基酸序列如SEQ ID NO：222所示：
Figure PCTCN2022108648-appb-000130
A118.VH5(PVRIG-A118-H5)氨基酸序列如SEQ ID NO：223所示：
Figure PCTCN2022108648-appb-000131
A118.VH6(PVRIG-A118-H6)氨基酸序列如SEQ ID NO：224所示：
Figure PCTCN2022108648-appb-000132
A118.VH7(PVRIG-A118-H7)氨基酸序列如SEQ ID NO：225所示：
Figure PCTCN2022108648-appb-000133
人源化重链模板IGHV3-7*01氨基酸序列如SEQ ID NO：217所示：
Figure PCTCN2022108648-appb-000134
人源化重链模板IGHJ3*01氨基酸序列如SEQ ID NO：206所示：
Figure PCTCN2022108648-appb-000135
根据IMGT编号系统，上述7个人源化抗体VH序列分析结果如表33所示。
表33 PVRIG-A118人源化抗体VH序列的IMGT分析结果
Figure PCTCN2022108648-appb-000136
实施例18 ELISA检测PVRIG人源化抗体与人和食蟹猴PVRIG蛋白的特异性结合
酶标板中预先包被100μL/孔的0.5μg/ml人PVRIG-his(AcroBiosystems，货号PVG-H52H4)或食蟹猴PVRIG蛋白(Novoprotein，货号C09B)；将受试抗PVRIG人源化抗体进行梯度稀释(起始浓度3nM，3倍梯度稀释)，100μL/孔加样，室温振荡孵育1.5小时；洗板后加入鼠抗人(mouse anti-human)IgG Fc-HRP(Jackson ImmunoResearch，货号209-035-098)工作液(1:10000稀释)，100μL/孔加样，室温振荡孵育1.0小时；再次洗板，加入HRP的底物TMB(Thermo，货号34029)进行显色，加入终止液终止反应后用酶标仪(MD i3x)读取吸光值。以抗体浓度为横坐标，对应的OD值为纵坐标绘制抗体的结合曲线，四参数拟合(GraphPad Prism9)，计算EC50值。EC50值越小，抗体与人/食蟹猴PVRIG结合的能力越强。将人源化抗体的结合效果均归一化到其对应的人源化前parental抗体，百分比值高于100％，说明人源化抗体结合的效果比parental抗体好。如图34A、图34B和表34所示，PVRIG-A50、PVRIG-A105、和PVRIG-A118的绝大部分人源化抗体均与人/食蟹猴PVRIG蛋 白特异性地结合。
表34抗PVRIG人源化抗体与人或食蟹猴PVRIG蛋白特异性结合ELISA结果
Figure PCTCN2022108648-appb-000137
*parental抗体:各自对应的人源化前的母抗体
实施例19 FACS检测PVRIG人源化抗体与FlpinCHO-PVRIG细胞表面人PVRIG、以及FlpinCHO-cyno PVRIG细胞表面食蟹猴PVRIG的结合
实验方法参见实施例9。计算出的AUC值越大，表示人源化抗体与FlpinCHO-human/cyno PVRIG细胞结合的能力越强。如图35A所示，受试抗体PVRIG-A50、A105和A118的绝大部分人源化分子与FlpinCHO-PVRIG细胞表面人PVRIG的结合与其parental抗体结合能力相当。图35B表明，受试抗体除PVRIG-A118人源化分子与食蟹猴PVRIG结合显著弱于对应parental抗体外，PVRIG-A50、A105的绝大部分人源化分子与食蟹猴PVRIG的结合较好，且与其parental抗体结合能力相当。将人源化抗体的结合活性归一化到与对照分子COM701-hIgG1以及SRF813-hIgG1抗体的百分比，如表35所示，百分比值越高，说明抗体结合的活性越强。
表35抗PVRIG人源化抗体与过表达细胞表面人/食蟹猴PVRIG的结合
Figure PCTCN2022108648-appb-000138
Figure PCTCN2022108648-appb-000139
*parentalAb:各自对应的人源化前的母抗体
实施例20 BIAcore检测PVRIG人源化抗体与人PVRIG蛋白的亲和力
实验方法参见实施例10。表36结果可知，所有受试PVRIG人源化抗体与人PVRIG蛋白之间存在特异性结合，且亲和力水平较高。
表36抗PVRIG人源化抗体与人PVRIG蛋白特异性结合的BIAcore结果
Figure PCTCN2022108648-appb-000140
Figure PCTCN2022108648-appb-000141
实施例21 ELISA检测抗PVRIG人源化抗体阻断PVRIG与PVRL2的结合
实验方法参见实施例11。将人源化抗体的抑制效果均归一化到其对应的人源化前parental抗体，百分比值高于100％，说明人源化抗体抑制的效果比parental抗体好。人源化抗体的阻断曲线见图36，抑制活性见表37。如图36和表37所示，PVRIG-A50、PVRIG-A105和PVRIG-A118的绝大部分人源化抗体均能显著地抑制人PVRIG与人PVRL2结合。
表37抗PVRIG人源化抗体阻断PVRIG和PVRL2的结合
Figure PCTCN2022108648-appb-000142
*parental抗体:各自对应的人源化前的母抗体
实施例22 FACS检测PVRIG人源化抗体阻断CHO-K1-CD112细胞与人PVRIG-mFc蛋白的结合
实验方法参照实施例12。实验结果如图37所示，大部分人源化抗体可阻断CHO-K1-CD112细胞结合人PVRIG-mFc蛋白。将人源化抗体的阻断活性归一化到与对照分子COM701-hIgG1以及SRF813-hIgG1抗体的百分比，百分比值越小，说明抗体阻断效果越好。
表38抗PVRIG人源化抗体阻断CHO-K1-CD112细胞结合人PVRIG-mFc蛋白
Figure PCTCN2022108648-appb-000143
实施例23 NK细胞杀伤实验检测PVRIG人源化抗体介导的NK细胞对肿瘤细胞系的杀伤作用
实验方法参考实施例4(g)。实验结果显示，图38中的所有人源化抗体均能不同程度地促进NK细胞对靶细胞的杀伤，其中图38中A显示七个人源化抗体中PVRIG-A50-H1b、PVRIG-A50-H2a对NK细胞杀伤靶细胞的促进作用与人源化前的parental抗体PVRIG-A50相当；图38中B表明七个人源化抗体中PVRIG-A118-H3、H4、H5及H6对NK细胞杀伤靶细胞的促进作用与人源化前的parental抗体PVRIG-A118相当；图38中C表明：五个人源化抗体中PVRIG-A105-H1、H2、H3对NK细胞杀伤靶细胞的促进作用与人源化前的parental抗体PVRIG-A105相当。
实施例24 CMV antigen-recall assay检测抗PVRIG人源化抗体对抗原特异性CD8 T细胞的功能改善作用
复苏PBMC，用含有1mg/mL CMV pp65(495-503)多肽(Anaspec,货号AS-28328)、2ng/mL human IL-2(R&D,货号IL-202)、10ng/mL human IL-7(Peprotech,货号200-07)的完全培养基(RPMI1640-Glutamax+5％AB serum+1％P/S+1×2-β巯基乙醇)重悬至2×10 6/mL，5mL/孔接种于6孔板中，37℃5％CO 2培养6天。第6天，收集所有细胞，撤去培养基中的pp65和IL-7，将细胞一分为二，并重悬于含有100IU/mL human IL-2的完全培养基中，继续培养2 天。第8天，收集所有细胞，重悬于含有100IU/mL human IL-2的完全培养基中、并调整细胞密度为2×10 6/mL。第11天，收集所有细胞，流式检测PBMC中CD8 T细胞的比例，CMV pp65(495-503)特异性CD8 T的比例(图24中A)及该细胞上PVRIG、TIGIT、PD-1的表达水平(图24中B)。流式检测抗体如下：Livedead Near IR(Invitrogen，货号L34976)，CD8-PerCp Cy5.5(BD，货号565310)，CD3-PE-Cy7(Biolegend,货号300316)，T-select HLA-A*0201 CMV pp65 Tetramer-PE(MBL，货号TS-0010-1C)，PVRIG-AF488(R&D，货号FAB93651G-100UG)，TIGIT-APC(Biolegend,货号372706)，PD-1-BV421(BD,货号562516)
上述诱导后的PBMC经CD8 T细胞分选试剂盒(Stemcell，货号17953)分离出CD8 T细胞作为效应细胞，用AIM-V重悬并调整细胞密度至0.4×10 6/mL。检测分选后CD8 T细胞纯度及CD226的表达。Colo205作为靶细胞，经TrypLE TM Express Enzyme(Gibco,货号12605010)消化，重悬于含有20ng/mLpp65的AIM-V培养基(Gibco,货号31035-025)并调整细胞密度至1×10 6/mL，37℃5％CO 2处理3小时，250g离心5分钟，弃上清。细胞用AIM-V培养基重悬至0.5×10 6/mL。PVRIG人源化抗体或阴性对照抗体用AIM-V培养基稀释至280nM。在低吸附96孔U底板(Corning,货号7007)中依次加入50μL抗体，50μL CD8 T，100μL pp65处理过的colo205细胞，混匀后37℃5％CO2孵育18小时。此体系中药物终浓度为70nM,CD8 T为20000/孔，colo205为50000/孔。孵育结束后400g离心取上清，用ELISA试剂盒(达科为，货号1110003)检测上清中human IFN-γ的水平。此体系中的阳性对照为PVRIG人源化前的parental抗体，阴性对照为no treatment。如图39所示，相较于no treatment组，PVRIG人源化抗体可显著增加细胞上清中的IFN-γ的水平。受试抗体中，除PVRIG-A105-H2的作用效果显著弱于PVRIG-A105外(*p<0.05,one-way ANOVA Analysis)，其余的人源化抗体与各自的人源化前parental抗体在统计学上无显著性差异(One-way ANOVA Analysis)。分选后CD8 T纯度及CD226表达的流式检测抗体信息如下：live/dead-BV421(Invitrogen,货号L34964)，CD8-FITC(BD,货号555366)，CD226-PE-Cy7(Biolegend，货号338316)。Colo205上PVRL2、PVR、PD-L1、HLA-A2表达的流式检测抗体信息如下：live/dead-BV421(Invitrogen,货号L34964)，PVRL2-APC(Biolegend，货号337412)，PVR-PerCp Cy5.5(Biolegend，货号337612)，PD-L1-PE-Cy7(BD，货号558017)，HLA-A2-PE(Biolegend,货号343306)。
实施例25抗PVRIGxTIGIT人源化双特异性抗体构建体的设计
两个抗PVRIG人源化VHH抗体(PVRIG-A50-H1b,PVRIG-A105-H1)及两个抗TIGIT人源化单克隆抗体(TIGIT-002-H4L3,TIGIT-005-H2L1d)，用G4S连接肽将抗PVRIG人源化VHH抗体连接到抗TIGIT人源化抗体重链的N端，产生抗PVRIGxTIGIT人源化双特异性抗体(图40)，分别命名为LC-BsAb-002、LC-BsAb-006、LC-BsAb-009和LC-BsAb-010。表39所示为4种双特异性抗体重链融合多肽(HC)和轻链多肽(LC)的序列。
表39双特异抗体融合多肽序列
Figure PCTCN2022108648-appb-000144
Figure PCTCN2022108648-appb-000145
Figure PCTCN2022108648-appb-000146
在构建双抗的同时，构建阳性对照抗体，此处抗TIGIT阳性对照抗体为罗氏的RG6058-hIgG1，抗PVRIG阳性对照抗体为Compugen的COM701-hIgG4，对应的氨基酸序列参见前文。
实施例26 ELISA检测抗PVRIGxTIGIT人源化双特异性抗体与人和食蟹猴PVRIG蛋白的特异性结合
酶标板中预先包被50μL/孔的1.0μg/mL人PVRIG(AcroBiosystems，货号PVG-H52H4)或50μL/孔的0.5μg/mL食蟹猴PVRIG(Novoprotein，货号C09B)；将受试抗体进行梯度稀释(起始浓度13nM、3倍梯度稀释，12个浓度点)，50μL/孔加样，37℃孵育2.0小时；洗板后加入羊抗人(Goat anti-human IgG Fc-HRP(Merck，货号AP113P))工作液(1:5000稀释)，50μL/孔加样，37℃孵育1.0小时；再次洗板，加入HRP的底物TMB(KPL，货号5120-0077)37℃显色10min，加入终止液终止反应后用酶标仪(PE，Ensight-HH3400)读取吸光值。以抗体摩尔浓度为横坐标，对应的OD值为纵坐标绘制抗体的结合曲线，四参数拟合(GraphPad Prism9)，计算EC50值。EC50值越小，抗体与人或食蟹猴PVRIG结合的能力越强。阳性对照抗体有COM701-hIgG1、PVRIG-A50-H1b、PVRIG-A105-H1；阴性对照抗体有 anti-Fluorescein-hIgG1(inhouse)。4个人源化双抗与人PVRIG蛋白的结合结果见图41和表40、41，与食蟹猴PVRIG的结合结果见图42和表40、41。数据表明，4个人源化双抗均能和人或食蟹猴PVRIG蛋白特异性结合。LC-BsAb-002和LC-BsAb-006与人PVRIG蛋白的结合相对其对照单抗PVRIG-A50-H1b及阳性对照COM701-hIgG1稍弱；LC-BsAb-009和LC-BsAb-010与人或食蟹猴PVRIG蛋白的结合，均与其相对照单抗PVRIG-A105-H1基本相当、并优于阳性对照COM701-hIgG1。
表40人源化双抗与人或食蟹猴PVRIG蛋白特异性结合的ELISA结果
Figure PCTCN2022108648-appb-000147
表41人源化双抗与人或食蟹猴PVRIG蛋白特异性结合的ELISA结果
Figure PCTCN2022108648-appb-000148
实施例27 ELISA检测抗PVRIGxTIGIT人源化双特异性抗体与人和食蟹猴TIGIT蛋白的特异性结合
酶标板中预先包被50μL/孔的4.0μg/mL羊抗鼠IgG Fc(Jackson，货号115-006-071)；封闭洗涤后，加入50μL/孔30ng/ml人TIGIT ECD-mFc(in house)或食蟹猴TIGIT ECD-mFc(in house)，37℃孵育2小时；洗板后，加入50μl/孔梯度稀释的受试抗体(起始浓度13nM、3倍梯度稀释，12个浓度点)，37℃孵育2.0小时；洗板后加入羊抗人IgG Fc-HRP(Merck，货号AP113P))工作液(1:5000稀释)，50μL/孔加样，37℃孵育1.0小时；再次洗板，加入HRP的底物TMB(KPL，货号5120-0077)进行显色，加入终止液终止反应后用酶标仪(PE，Ensight-HH3400)读取吸光值。以抗体摩尔浓度为横坐标，对应的OD值为纵坐标绘制抗体的结合曲线，四参数拟合(GraphPad Prism9)，计算EC50值。EC50值越小，抗体与人或食蟹猴TIGIT结合的能力越强。阳性对照抗体有RG6058-hIgG1、TIGIT-002-H4L3和TIGIT-005-H2L1d；阴性对照抗体为anti-Fluorescein-hIgG1(in house)。4个人源化双抗与人TIGIT蛋白的结合结果见图43和表42、43，与食蟹猴TIGIT的结合结果见图44和表42、43。数据表明，4个人源化双抗均能和人或食蟹猴TIGIT蛋白特异性结合。其中LC-BsAb-002和009与人TIGIT蛋白的结合优于阳性对照RG6058-hIgG1，与其对应单抗TIGIT-002-H4L3相 当；与食蟹猴TIGIT蛋白的结合，LC-BsAb-009与其对应单抗TIGIT-002-H4L3及阳性对照RG6058-hIgG1基本相当，LC-BsAb-002则比对应单抗及阳性对照稍弱。LC-BsAb-006和010与人TIGIT蛋白/食蟹猴TIGIT蛋白的结合，均优于其对应的单抗TIGIT-005-H2L1d及阳性对照RG6058-hIgG1。
表42人源化双抗与人或食蟹猴TIGIT蛋白特异性结合的ELISA结果
Figure PCTCN2022108648-appb-000149
表43人源化双抗与人或食蟹猴TIGIT蛋白特异性结合的ELISA结果
Figure PCTCN2022108648-appb-000150
实施例28 FACS检测抗PVRIGxTIGIT人源化双特异性抗体与FlpinCHO人PVRIG和FlpinCHO食蟹猴PVRIG的结合活性
取转染了人或食蟹猴PVRIG高表达质粒的CHO-K1稳定细胞(
Figure PCTCN2022108648-appb-000151
CCL-61 TM)，分别命名为FlpinCHO-hPVRIG，FlpinCHO-cyno PVRIG，人PVRIG全长质粒(NCBI RefSeq:NP_076975),以及食蟹猴PVRIG全长质粒(NCBI Ref Seq:XP_014989941)均由通用生物合成，在细胞密度不超过80％时进行实验。弃去细胞培养基，用PBS润洗并加入1ml Versene(Gibico，15040-066)消化8-10分钟，用含10％FBS的Ham’s F12(Gibico，21127-022)完全培养基终止消化后制成细胞悬液。计数后，取适量细胞悬液，350×g离心去上清，PBS洗细胞两遍后，用死活染料Zombie violet(Biolegend，423114)进行染色，室温孵育20分钟。孵育结束后，用染色缓冲液(2％FBS+PBS)终止染色，350×g离心去上清，洗细胞两遍后，用染色缓冲液将细胞重悬为2×10 6个细胞/ml的密度，铺入96孔板，每孔加入50μl的细胞悬液，待用。用染色缓冲液将抗体从最高浓度46nM(两倍浓度)开始进行3.3倍梯度稀释，将稀释好的抗体加至已含有50μl细胞悬液的孔中，置于微孔板振荡器上400rpm震荡1分钟，使抗体与细胞充分混合，4℃孵育30分钟。孵育结束后用染色缓冲液清洗细胞两次，每孔200μl，350×g离心5分钟弃上清。用染色缓冲液将PE goat anti-Human IgG Fc抗体(ebioscience，12-4998-82)稀释250倍，以每孔100μl的体积加至洗完的细胞孔中，混合均匀，4℃染色30 分钟。染色结束后同样用染色缓冲液清洗两次，最后用200μl染色缓冲液重悬细胞，流式上机检测信号(BD CantoII)，信号越强，表示抗体与PVRIG的结合能力越强。图45显示，4个人源化双抗均有较好的人PVRIG结合活性，亦能较好结合食蟹猴PVRIG(图46)，且均优于其对应的抗PVRIG人源化单抗。
实施例29 FACS检测抗PVRIGxTIGIT人源化双特异性抗体与CHO-K1人TIGIT(高/中/低表达株)和CHO-K1食蟹猴TIGIT细胞的结合活性
收集细胞，PBS(Hyclone，SH30256)洗一次，1％BSA-PBS重悬至2×10 5/50μL，1％BSA-PBS稀释抗体至80nM，3倍连续梯度稀释12个浓度点，将50μL细胞与50μL抗体稀释液混合，4℃孵育60分钟，PBS洗两次，加入Alexa
Figure PCTCN2022108648-appb-000152
647荧光素标记的二抗(1:800)(Jackson，109-605-088)，100μL/孔重悬细胞，4℃孵育40分钟，PBS洗两次，用1％BSA-PBS，100μL/孔重悬，在流式细胞仪(BD,CantoⅡ)上分析细胞样品。4个人源化双抗与CHO-K1人TIGIT(高/中/低表达株，图47，48，49)和CHO-K1食蟹猴TIGIT细胞均有较好的结合活性(图50)。
实施例30 HTRF方法检测抗PVRIGxTIGIT人源化双特异性抗体阻断PVRIG蛋白与PVRL2蛋白的相互作用
分别将PVRIG-mFc(ACRO Biosystems,货号PVG-H5253)和Bio-CD112-His(Sino Biological,货号10005-H08H)稀释至0.5μg/ml；分别将Streptavidin-Tb cryptate(Cisbio,货号)和PAb anti mouse IgG-XL665(Cisbio,货号)稀释至20μg/ml和0.8μg/ml。待测抗体起始浓度为120nM，3倍梯度稀释，共12个浓度点。将上述稀释好的PVRIG-mFc、Bio-CD112-His、Streptavidin-Tb Crytate和PAb anti mouse IgG-XL665按1:1:1:1混合，10μl/孔加入384孔板中(PE,货号6007299)；之后10μl/孔加入梯度稀释的待测抗体,1500rpm离心30s，37℃孵育1h；酶标仪上读板(PE,Envision2105)，波长选用665nm和620nm。根据公式Ratio＝Signal665nm/Signal 620nm×10 4进行数据换算。以抗体的摩尔浓度为横坐标、Ratio为纵坐标，进行4参数拟合，并计算IC50。IC50越小，说明抗体阻断PVRIG与PVRL2结合的效果越好。本实验中的阳性对照为COM701-hIgG1、PVRIG-A50-H1b，PVRIG-A105-H1；阴性对照抗体有anti-Fluorescein-hIgG1(in house)。4个人源化双抗阻断PVRIG-PVRL2结合的效果见图51和表44、45。数据表明，4个人源化双抗均能阻断人PVRIG与PVRL2蛋白的结合。其中LC-BsAb-002的阻断效果均优于其对应单抗PVRIG-A50-H1b及阳性对照COM701-hIgG1，LC-BsAb-006的阻断效果优于其对应单抗PVRIG-A50-H1b，但稍弱于阳性对照COM701-hIgG1；LC-BsAb-009和LC-BsAb-010的阻断效果均不如其对应单抗PVRIG-A105-H1及阳性对照COM701-hIgG1。
表44人源化双抗阻断PVRIG和PVRL2的结合
Figure PCTCN2022108648-appb-000153
Figure PCTCN2022108648-appb-000154
表45人源化双抗阻断PVRIG和PVRL2的结合
Figure PCTCN2022108648-appb-000155
实施例31 FACS检测抗PVRIGxTIGIT人源化双特异性抗体阻断CHO-K1人CD112细胞结合人PVRIG-mFc蛋白
取转染了人CD112高表达质粒的CHO-K1稳定细胞(命名为CHO-K1-CD112)，人CD112全长质粒由通用生物合成(NP_001036189.1/NCBI RefSeq:Q92692)，在细胞密度不超过80％时进行实验。弃去细胞培养基，用PBS润洗并加入1ml胰酶(Gibico，25200-72)消化2分钟，用含10％FBS的Ham’s F12(Gibico，21127-022)完全培养基终止消化后制成细胞悬液。细胞计数仪(Beckman Coulter，Vi-CELL)计数后，取适量细胞悬液，350×g离心去上清，PBS洗细胞两遍后，用死活染料Zombie violet(Biolegend，423114)进行染色，室温孵育20分钟。孵育结束后，用染色缓冲液(2％FBS+PBS)终止染色，350×g离心去上清，洗细胞两遍后，用染色缓冲液将细胞重悬为1×10 6个细胞/ml的密度，待用。铺入96孔板，每孔加入50μl的细胞悬液，待用。用染色缓冲液配制人PVRIG-mFc蛋白(Acro，PVG-H5253)工作液，浓度为1μg/ml(四倍浓度)，加入96孔板，每孔加入50μl的PVRIG-mFc工作液。用染色缓冲液将抗体从最高浓度275nM(四倍浓度)开始进行3倍梯度稀释，将稀释好的抗体加至已含有50μl PVRIG-mFc的孔中，置于微孔板振荡器上400rpm震荡1分钟，使抗体与PVRIG-mFc蛋白充分混合，4℃孵育30分钟。孵育结束后，直接加入上述准备的细胞悬液，每孔加入100μl，枪头轻柔混匀后，4℃孵育30分钟。孵育结束后用染色缓冲液清洗细胞两次，每孔200μl，350×g离心5分钟弃上清。用染色缓冲液将PE goat anti-mouse IgG Fc抗体(Biolegend，405337)稀释250倍，以每孔100μl的体积加至洗完的细胞孔中，混合均匀，4℃染色30分钟。染色结束后同样用染色缓冲液清洗两次，最后用200μl染色缓冲液重悬细胞，流式上机检测信号(BD CantoII)。荧光信号越弱，表示抗体阻断CHO-K1人CD112细胞结合PVRIG-mFc蛋白的能力越强。图52显示，4个人源化双抗均可阻断CHO-K1人CD112细胞结合人PVRIG-mFc蛋白。
实施例32 ELISA方法检测抗PVRIGxTIGIT人源化双特异性抗体阻断人TIGIT与CHO-K1 CD155结合的活性
收集实施例2构建的CHO-K1 CD155细胞，用10％FBS-DMEM/F12培养基(Excell,FSP500；Gibco,11330)调整浓度至5×10 5/mL，加入96孔细胞培养板(corning,3599)，100μL/孔，37℃5％CO 2培养过夜，甩掉培养上清，加入细胞固定液(碧云天，P0098)，50μL/孔， 室温固定1小时，在洗板机上用0.05％Tween20-PBS洗一次，加入5％脱脂奶粉-PBS，250μL/孔，37℃孵育2～4小时，在洗板机上用0.05％Tween20-PBS洗三次；将人TIGIT ECD-mFc(工作浓度100ng/mL)与样品混合孵育半小时；随后将抗原抗体混合液加入细胞板中，50μL/孔，37℃孵育1.5～2小时，在洗板机上用0.05％Tween20-PBS洗三次，以1:5000稀释比用1％BSA(生工生物，A500023-0100)-PBS稀释HRP酶标抗体(Jackson,115-035-003)，加入细胞板中，50μL/孔，37℃孵育1小时，在洗板机上用0.05％Tween20-PBS洗三次，加入TMB显色液(KPL,52-00-03),50μL/孔，37℃孵育10分钟，加入1M HCL，50μL/孔，终止反应，酶标仪(Biotek,Powerwave HT)读取OD450nm。图53表明，4个人源化双抗均可阻断人TIGIT与CHO-K1CD155结合。
实施例33 FACS方法检测抗PVRIGxTIGIT人源化双特异性抗体阻断Bio-CD155-His与CHO-K1人TIGIT结合的活性
收集细胞，PBS(Hyclone，SH30256)洗一次,1％BSA-PBS重悬至2×10 5/40μL，1％BSA-PBS稀释抗体至210nM，3倍连续梯度稀释12个浓度点，1％BSA-PBS稀释Bio-CD155-His(义翘神州，10109-H08H)至3μg/mL，随后将40μL细胞与40μL抗体稀释液以及40μL Bio-CD155-His稀释液混合，4℃孵育60分钟，PBS洗两次，加入APC标记的链霉亲和素(工作稀释度1:1700，Biolegend，405243)，100μL/孔重悬细胞，4℃孵育40分钟，PBS洗两次，用1％BSA-PBS，100μL/孔重悬，在流式细胞仪(BD,CantoⅡ)上分析细胞样品。图54表明，4个人源化双抗均可阻断Bio-CD155-His与CHO-K1人TIGIT结合。
实施例34 FACS方法检测抗PVRIGxTIGIT人源化双特异性抗体与人PBMC的结合活性
取新鲜的人PBMC(AllCells，PB004-C)，将细胞调整至5×10 5/mL，同时加入SEA(Toxin Technology,Inc.，AT101)至100ng/mL，37℃5％CO 2培养三天；三天后收集细胞，PBS(Hyclone，SH30256)洗一次,加入Fc Block(BD，564220)，4℃孵育10分钟，PBS洗两次，1％BSA-PBS重悬至2×10 5/50μL，1％BSA-PBS稀释人源化抗体至80nM，3倍连续梯度稀释12个浓度点，将50μL细胞与50μL抗体稀释液混合，4℃孵育60分钟，PBS洗两次，加入Alexa
Figure PCTCN2022108648-appb-000156
647荧光素标记的二抗(工作稀释度1：800，Jackson，109-605-088)，100μL/孔重悬细胞，4℃孵育60分钟，PBS洗两次，用1％BSA-PBS，100μL/孔重悬，在流式细胞仪(BD,CantoⅡ)上分析细胞样品。图55显示，4个人源化双抗与人PBMC均有较好的结合活性。
实施例35 BIAcore检测抗PVRIGxTIGIT人源化双特异性抗体与人，食蟹猴和小鼠TIGIT及PVRIG蛋白的亲和力
该实验采用ProteinA芯片，通过手工操作(manual run)测定出芯片捕获稀释后的抗体所需要的时间，以使得能饱和结合抗原Rmax为50RU。将人、食蟹猴和小鼠TIGIT及PVRIG蛋白梯度稀释至20，10，5，2.5，1.25nM。采用多循环动力学测得抗体与抗原的亲和力。在每一个循环中，注射抗体后再注入梯度浓度的人、食蟹猴和小鼠TIGIT及PVRIG蛋白，使抗原与抗体发生结合与解离过程。每个循环后用Glycine pH1.5进行Protein A芯片的再生(去 除芯片上的蛋白)。应用BIAcore T200分析软件拟合抗体抗原的亲和力KD。表46结果可知，2个人源化双抗与人和食蟹猴TIGIT及PVRIG蛋白之间存在特异性结合，且亲和力水平较高，但与小鼠TIGIT及PVRIG蛋白不结合。
表46人源化双抗与不同种属TIGIT及PVRIG蛋白的亲和力
Figure PCTCN2022108648-appb-000157
实施例36 BIAcore检测抗PVRIGxTIGIT人源化双特异性抗体与人TIGIT及PVRIG蛋白的共结合
应用BIAcore表征双特异性抗体与两抗原同时结合特性。首先用ProteinA芯片捕获抗体LC-BsAb-002和LC-BsAb-006，然后分别注射TIGIT和PVRIG his标签蛋白，以及分别连续注射TIGIT和PVIRIG、PVRIG和TIGIT，记录抗体和抗原的结合信号，最后用Glycine pH1.5完成芯片再生，其中流动相为HBS-EP+(10mM HEPES,150mM NaCl,3mM EDTA,0.05％surfactant P20)，流速为30μL/min，与不同抗原结合时间均为300s，再生时间为30s，检测温度为25℃，hTIGIT分析浓度为20nM，hPVRIG分析浓度为50nM。应用BIAcore 8K分析软件(版本号2.0)对数据进行分析，记录抗体捕获水平Capture Level、不同抗原的结合信号Binding Responses(RU)，并根据抗原抗体分子量计算抗原抗体分子化学计量比，初步估计一个抗体分子可以结合几个抗原。为了确认抗体LC-BsAb-002与抗原TIGIT&PVRIG的相互作用关系，开展了以下四步检测：只结合hTIGIT单个抗原、只结合hPVRIG单个抗原、先结合hTIGIT后结合hPVRIG、先结合hPVRIG后结合hTIGIT，并且各抗原均达到饱和状态。收集抗体抗原的结合曲线，记录2个双特异性抗体的捕获水平、以及各试验中TIGIT和PVIRIG的结合信号，并以此计算了抗原抗体分子化学计量比，如表47所示，图56中A、B分别为LC-BsAb-002和LC-BsAb-006分别与TIGIT和PVIRG结合、以及分别连续注射TIGIT和PVRIG的抗体抗原结合曲线。如表47和图56A和56B所示，连续注射TIGIT和PVIRG产生的结合信号，与单独注射TIGIT和PVIRG产生的结合信号几乎一致；并且正反连续TIGIT 和PVIRG产生的结合信号也几乎一致，这说明LC-BsAb-002和LC-BsAb-006可同时与hTIGIT和hPVRIG相结合，并且两个抗原之间不存在互相影响；综合考虑抗体和抗原的分子量、以及抗体的捕获水平和抗原的结合水平，初步估算出TIGIT与LC-BsAb-002的化学计量比为1.76；TIGIT与LC-BsAb-006的化学计量比为1.86；PVIRIG与LC-BsAb-002的化学计量比为2.14，PVIRIG与LC-BsAb-006的化学计量比为2.18，两抗原抗体化学计量比都接近于2，考虑到检测方法带来的误差，我们推测一个LC-BsAb-002或一个LC-BsAb-006双抗分子可以同时结合两个TIGIT分子和两个PVRIG分子。
表47人源化双抗与TIGIT及PVRIG蛋白的BIAcore结合情况
Figure PCTCN2022108648-appb-000158
实施例37 NK细胞脱粒实验检测抗PVRIGxTIGIT人源化双特异性抗体对NK细胞的功能促进作用
利用FACS检测NK细胞CD107a的表达水平指示受试抗体对NK细胞激活的影响(图57中A展示了实验流程)。
A.利用FACS检测NK细胞(Natural killer cell)上PVRIG，TIGIT及WIDR细胞表面PVR和PVRL2的表达情况。
首先，使用细胞计数仪(Beckman Coulter，Vi-CELL)将NK细胞计数，取三个流式管，每个流式管加入1e+5个NK细胞，加PBS洗涤细胞两次，去上清，取一管加入300μl Staining buffer(PBS+2％FBS)做为未染色管待用，另外两管每管加入染液100μl(PBS+1*的Zombie Violet(Biolegend，423114))混匀后室温孵育15分钟。随后加Staining buffer洗涤细胞两次，去上清，每管加入Fc阻断剂50μl(Staining buffer+Fcx blocker(Biolegend，422302))混匀后4℃孵育15分钟。接着分别向每管加入染液，第一管加入50μl的2*染液(Staining buffer+PE-Cy7 Mouse anti-hCD3检测抗体+PE Mouse anti-hCD56检测抗体+APC Mouse anti-hTIGIT检测抗体+AF488 Rabbit anti-hPVRIG检测抗体，CD3检测抗体：Biolegend 300316，CD56检测抗体：Biolegend 318306，TIGIT检测抗体：Biolegend 372706，PVRIG检测抗体：RD FAB93651G)，第二管加入50μl的2*同型对照染液(Staining buffer+PE-Cy7 Mouse anti-hCD3检测抗体+PE Mouse anti-hCD56检测抗体+APC Mouse IgG2aκ同型对照抗体+AF488 Rabbit IgGκ同型对照抗体，APC mIgG2aκ同型对照抗体：Biolegend 400222，AF488 Rabbit IgGκ同型对照抗体:RD IC1051G)，混匀后4℃孵育30分钟。时间到后用Staining buffer 洗涤两次，离心后加300μl Staining buffer混匀。随后上机检测(Thermo Attune NxT)，最终读取Zombie Violet阴性细胞群中CD56阳性CD3阴性的细胞群的占比和Zombie Violet阴性细胞群中CD56阳性CD3阴性的细胞群的APC、AF488信号。
将WIDR细胞用胰酶消化成细胞悬液，使用细胞计数仪(Beckman Coulter，Vi-CELL)将细胞计数，取三个流式管，每个流式管加入1e+5个细胞，加PBS洗涤细胞两次。离心后去上清，取一管加入300μl Staining buffer(PBS+2％FBS)做为未染色管待用，另外两管每管加入染液100μl(PBS+1*的Zombie Violet(Biolegend，423114))混匀后室温孵育15分钟。随后加Staining buffer洗涤细胞两次，去上清，分别向每管加入染液：第一管加入100μl染液(Staining buffer+PerCP-Cy5.5 Mouse anti-hPVR检测抗体+APC Mouse anti-hPVRL2检测抗体，PVR检测抗体：Biolegend 337612，PVRL2检测抗体：Biolegend 337412)，第二管加入100μl同型对照染液(Staining buffer+PerCP-Cy5.5 Mouse IgG1κ同型对照抗体+APCκMouse IgG1同型对照抗体，PerCP-Cy5.5 mIgG1κ同型对照抗体：Biolegend 400150，APC mIgG1κ同型对照抗体：Biolegend 400122)，混匀后4℃孵育30分钟。时间到后用Staining buffer洗涤两次，离心后加300μl Staining buffer混匀。随后上机检测(Thermo Attune NxT)，最终读取Zombie Violet阴性细胞群的PerCP-Cy5.5、APC信号。图57中B显示实验所使用的NK细胞表达一定水平的PVRIG和TIGIT，同时靶细胞WIDR高表达配体PVR和PVRL2。
B.NK细胞脱颗粒实验(以WIDR为靶细胞)。
实验的前一天将人PBMC复苏，用分选试剂盒(Stemcell，17955)分选人NK细胞，加入200IU/ml的h-IL2(R&D,202-IL)和10ng/ml的h-IL12(Peprotech，200-12-50UG)过夜刺激，第二天进行铺板实验。首先，用assay buffer(RPMI1640-Glutamax+10％FBS+1×P/S)将抗体稀释到最高浓度275nM(四倍浓度)，然后继续用assay buffer开始进行10倍梯度稀释，将稀释好的抗体加至超低吸附96孔U底板(Costar，7007)中，每孔50μl，待用。其次，使用细胞计数仪(Beckman Coulter，Vi-CELL)将NK细胞计数，取一定数目的NK细胞，以350g的速度离心5分钟，弃上清后用assay buffer重悬至0.5E+6个细胞/ml的密度，向细胞悬液中加入蛋白转运抑制剂(Invitrogen，00498093)和APC mouse anti-human检测抗体(Biolegend，328620)。向铺好药的96孔U底板中加入处理好的NK细胞悬液，每孔50μl，混合均匀后室温孵育15分钟。孵育期间，将靶细胞WIDR用胰酶消化成细胞悬液(Reh细胞直接混合均匀)，使用细胞计数仪(Beckman Coulter，Vi-CELL)将靶细胞细胞计数，取出适量数目的细胞，以200g的速度离心5分钟，弃上清后用assay buffer将细胞重悬至0.25e+6个细胞/ml的密度。孵育结束后，向孔板中加入靶细胞悬液，每孔100μl，此时每孔中含有25000个NK细胞，25000个靶细胞以及不同浓度的受试抗体，只含有NK细胞的孔作为静息对照，只含NK及WIDR作为无药物对照。将每个孔混合均匀后放入37℃培养箱孵育16h。最后进行FACS染色：将孔板中的细胞平行转移至96孔V底板，用PBS洗涤两次，弃上清，每孔加入染液(PBS+2％FBS+1*浓度的zombie violet(Biolegend，423114)+PE mouse anti-CD56检测抗体(Biolegend，318306))混合均匀后4℃孵育30分钟。时间到后，staining buffer洗涤两次，弃上清，每孔加入150μl的staining buffer重悬上机检测(Thermo Attune NxT)。最终读取CD56阳性细胞中CD107a强阳性细胞群的占比，CD107a强阳性细胞占比越高，代表NK的脱粒作用越强，NK的激活程度越高。图57中C显示，阴性对照anti-Fluorescein-hIgG1 对NK细胞的CD107a表达没有影响，候选人源化双抗均能不同程度的提高NK细胞CD107a的表达，这表明受试抗体可以有效促进NK细胞的激活。
C.NK细胞脱颗粒实验(以TF-1为靶细胞)。
实验方法参考实施例37B，实验结果如图57中D显示，表明人源化双抗能NK细胞脱颗粒，作用优于PVRIG阳性对照抗体COM701-hIgG4和TIGIT阳性对照抗体RG6058-hIgG1，等同于COM701-hIgG4和RG6058-hIgG1联用组。同时，人源化双抗对NK细胞脱颗粒的促进作用也优于其PVRIG臂抗体PVRIG-A50-H1b和TIGIT臂抗体TIGIT-002-H4L3，与PVRIG-A50-H1b和TIGIT-002-H4L3联用组相当。
实施例38 NK细胞杀伤实验检测抗PVRIGxTIGIT人源化双特异性抗体介导的NK细胞对肿瘤细胞系的杀伤作用
利用FACS检测靶细胞(WIDR)的裂解水平来指示受试抗体对NK细胞对靶细胞杀伤功能的影响。
实验的前一天将PBMC复苏，用分选试剂盒(Stemcell，17955)将NK细胞分选出来，加入200IU/ml的h-IL2(RD,202-IL)和10ng/ml的h-IL12(Peprotech，200-12-50UG)过夜刺激，第二天进行铺板实验。用实施例13中提到的方法检测3个NK供体(Donor-050，Donor-831，Donor-715)上PVRIG和TIGIT的表达水平，同时，用assay buffer(RPMI1640-Glutamax+10％FBS+1×P/S)将待测抗体稀释到最高浓度275nM(四倍浓度)，随后用assay buffer开始进行10倍梯度稀释，将稀释好的抗体加至超低吸附96孔U底板(Costar，7007)中，每孔50μl，待用。其次，将靶细胞WIDR用胰酶消化成细胞悬液，使用细胞计数仪(Beckman Coulter，Vi-CELL)将WIDR细胞计数，取出适量数目的细胞，置于离心机以200g的速度离心5分钟，弃上清后用适量PBS重悬后加入CellTrace Violet(Invitrogen，C34557A)染液，使CellTrace Violet终浓度为5μM。将加入染液的WIDR悬液混合均匀置于37℃培养箱孵育10分钟，期间摇晃混合，取出一部分WIDR细胞用实施例13中描述的方法检测WIDR上PVR和PVRL2的表达水平。与此同时，使用细胞计数仪将NK细胞计数，取一定数目的NK细胞，以350g的速度离心5分钟，弃上清后用assay buffer重悬至0.5e+6个细胞/ml的密度。向铺好药的96孔U底板中加入处理好的NK细胞悬液，每孔50μl，混合均匀后室温孵育15分钟。WIDR细胞染色结束后，向细胞悬液中加入5倍体积完全培养(MEM+10％FBS+1*P/S+1*非必须氨基酸+1*谷氨酸钠)基进行终止反应，以200g的速度离心5分钟，弃上清后用assay buffer将细胞重悬至0.25e+6个细胞/ml的密度。NK与药物孵育结束后，向孔板中加入WIDR细胞悬液，每孔100μl，此时每孔中含有25000个NK细胞，25000个WIDR细胞以及不同浓度的受试抗体，只含WIDR细胞的孔作为静息对照，含有NK细胞及WIDR的孔作为无药物对照。将每个孔混合均匀后放入37℃培养箱孵育4h。最后，做FACS染色：每孔加入染液(PBS+PI(Propidium Iodide，Invitrogen，P3566))混合均匀后室温孵育20分钟。时间到后，上机检测(Thermo Attune NxT)，最终读取CTV阳性细胞中PI阳性细胞群的占比，PI阳性细胞占比越多，代表NK的杀伤作用越强。图58中A示3个NK供体(Donor-050，Donor-831，Donor-715)都表达一定水平的PVRIG和TIGIT，图58中B示靶细胞WIDR上高表达PVR和PVRL2，图58中C为NK细胞对WIDR细胞的杀伤实验的简要实验流程，图58中D显 示，阴性对照anti-Fluorescein-hIgG1对NK杀伤没有明显影响，受试的2个人源化双抗均能有效促进NK(3个donor来源)对靶细胞WIDR的杀伤，表中标示了不同NK供体下两个人源化双抗对WIDR细胞杀伤的EC50和曲线下面积(AUC)。
以TF-1为靶细胞，检测抗PVRIGxTIGIT人源化双特异性抗体介导的NK细胞对TF-1的杀伤作用，结果如图58中E所示，表明人源化双抗能促进NK细胞杀伤肿瘤细胞，活性优于PVRIG阳性对照抗体COM701-hIgG4、TIGIT阳性对照抗体RG6058-hIgG1和COM701-hIgG4与RG6058-hIgG1联用组。同时，人源化双抗的活性也优于其PVRIG臂抗体PVRIG-A50-H1b、TIGIT臂抗体TIGIT-002-H4L3和PVRIG-A50-H1b与TIGIT-002-H4L3联用组。
实施例39 NK细胞ADCC实验检测抗PVRIGxTIGIT人源化双特异性抗体介导的对人Treg细胞的直接杀伤作用
利用FACS检测靶细胞(Treg)的裂解水平来指示受试抗体对NK细胞对靶细胞的直接ADCC杀伤效应(图59中A)。
实验的前一天将PBMC复苏，用分选试剂盒(Stemcell，17955)将NK细胞分选出来作为效应细胞，加入200IU/ml的h-IL2(RD,202-IL)和10ng/ml的h-IL12(Peprotech，200-12-50UG)过夜刺激，第二天进行铺板实验。用PBMC中分离Treg(调节性T细胞)作为靶细胞(Stemcell，18063)经Dynabeads(Gibco，11129D)体外扩增12天后获得扩增的Treg细胞用于实验，实验前用实施例13中提到的方法和试剂检测Treg细胞上TIGIT和PVRIG的表达。效应细胞与靶细胞以5：1的比例共孵育，并加入系列梯度稀释的受试人源化双特异性抗体或同型对照anti-Fluorescein-hIgG1，anti-Fluorescein-hIgG4抗体，37℃培养箱中孵育4小时后，加入PI染色，最终读取PI阳性的Treg细胞占比，以此来评估受试双特异性抗体对靶细胞Treg的ADCC(抗体依赖型细胞介导的细胞毒性作用)杀伤效应。结果显示，分离扩增的人Treg细胞高表达TIGIT和PVRIG(图59中B)，受试人源化双抗只有在hIgG1的Fc下才能显示出对Treg细胞的浓度依赖性ADCC杀伤，而对应的hIgG4 Fc形式没有表现出明显的对Treg细胞的ADCC效应，与两个阴性对照抗体anti-Fluorescein-hIgG1，anti-Fluorescein-hIgG4相当(图59中C)，表中列出了两个受试人源化抗体在hIgG1 Fc形式下的对Treg细胞ADCC杀伤效应的EC50和AUC。
实施例40抗PVRIGxTIGIT人源化双特异性抗体的ADCP活性
从供体的PBMC中分离单核细胞，并用75ng/mL GM-CSF诱导七天分化成巨噬细胞，用CellTrace Violet标记后作为效应细胞。通过调节性T细胞分离试剂盒从人PBMC中分选的人Treg细胞，用Dynabeads Human Treg Expander体外扩增并活化13天作为靶细胞，然后用CFSE染料标记靶细胞。将效应细胞与靶细胞以4:1的比例共孵育。加入连续梯度稀释的待测抗体、阴性对照Hel hIgG1抗体、其单臂抗体(PVRIG-A50-H1b和TIGIT-002-H4L3)或两个单抗的联用组合，在37℃下孵育4小时。孵育结束后，加入细胞染料PI，采用流式细胞术检测CFSE阳性Treg细胞中CellTrace Violet阳性细胞比例，评价待测抗体的ADCP作用。
如图60中A显示，待测抗体以剂量依赖的方式激活Treg细胞的ADCP作用。 PVRIG-A50-H1b或COM701-hIgG4几乎没有ADCP活性。TIGIT-002-H4L3或RG6058-hIgG1表现出剂量依赖性ADCP活性。根据ADCP曲线的曲线下面积(AUC)，待测抗体的ADCP活性略弱于TIGIT-002-H4L3以及两种单臂抗体组合(PVRIG-A50-H1b+TIGIT-002-H4L3)。待测抗体的ADCP效应与RG6058-hIgG1和两种阳性抗体组合(COM701-hIgG4+RG6058-hIgG1)相当。根据ADCP曲线的E max，待测抗体的ADCP活性与两种单臂抗体组合(PVRIG-A50-H1b+TIGIT-002-H4L3)和两种阳性抗体组合(COM701-hIgG4+RG6058-hIgG1)相当(图60中B)。
实施例41抗PVRIGxTIGIT人源化双特异性抗体对来自健康供体的人PBMC中细胞因子释放的影响
本实验研究待测抗体对健康人未刺激的PBMC中细胞因子分泌的影响。采用3个健康志愿者的PBMC，在液相或固相条件下与待测抗体孵育24小时，然后运用流式方法检测PBMC上清中IFN-γ，IL-2，IL-6，IL-10和TNF-α5种细胞因子的分泌水平。脂多糖和CD3单克隆抗体作为阳性对照，Anti-Hel hIgG1抗体作为阴性对照，同时RG6058-hlgG1和COM701-hlgG4分别作为TIGIT端和PVRIG端的单抗对照。
结果显示，阳性对照CD3单克隆抗体在液相或固相条件下，LPS在液相条件下与3个健康志愿者的未刺激PBMC孵育24小时后，PBMC中IFN-γ，IL-2，IL-6，IL-10和TNF-α5种细胞因子的分泌水平有不同程度的升高。在液相条件下，不同浓度的待测抗体与未刺激PBMC孵育24小时后，PBMC中IFN-γ，IL-2，IL-6，IL-10和TNF-α的分泌水平与阴性对照相当或在检测限以下。固相条件下，不同浓度的待测抗体与未刺激PBMC孵育24小时后，PBMC中IFN-γ、IL-2和IL-10的分泌水平与阴性对照相当或在检测限以下；固相条件下高浓度点(2850nM)的待测抗体作用后，TNF-α和IL-6的分泌水平显著高于阴性对照或与阴性对照相当；但与相同条件下的RG6058-hlgG1和COM701-hlgG4相比，待测抗体在体外不会额外增加健康人未刺激PBMC中IFN-γ，IL-2，IL-6，IL-10和TNF-α5种细胞因子的分泌。
综上，相较于TIGIT端单抗RG6058-hIgG1和PVRIG端单抗COM701-hIgG4，相同条件下的待测抗体在体外不会额外增加健康人未刺激PBMC IFN-γ，IL-2，IL-6，IL-10和TNF-α5种细胞因子的分泌。
实施例42 CMV antigen-recall assay检测抗PVRIGxTIGIT人源化双特异性抗体对抗原特异性CD8 T细胞的功能促进作用
本实验原理：CMV IgG阳性donor的PBMC经CMV pp65(495-503)多肽诱导的CMV pp65特异性CD8T作为效应细胞，经pp65 pulsed后的colo205作为靶细胞的实验体系中，考察抗PVRIG&TIGIT双抗对pp65特异性CD8 T细胞的功能促进作用(图61中A)。
PBMC复苏后，用含有1μg/mL CMV pp65(495-503)多肽(Anaspec,货号AS-28328)、2ng/mL human IL-2(R&D,货号IL-202)、10ng/mL human IL-7(Peprotech,货号200-07)的完全培养基(RPMI1640-Glutamax+5％AB serum+1％P/S+(1×)2-β巯基乙醇)重悬至2×10 6/mL，5mL/孔接种于6孔板中，37℃5％CO 2培养6天。第6天，收集所有细胞，撤去培养基中的pp65和IL-7，将细胞一分为二，并重悬于含有100IU/mL human IL-2的完全培养基中，继续培养2天。第8天，收集所有细胞，重悬于含有100IU/mL human IL-2的完全培养基中、并 调整细胞密度为2×10 6/mL，继续培养。第11天，收集所有细胞，流式检测CMV pp65(495-503)特异性CD8 T该细胞上PVRIG、TIGIT、PD-1的表达(图61中B)。流式检测抗体如下：Livedead Near IR(Invitrogen，货号L34976)，CD8-PerCp Cy5.5(BD，货号565310)，CD3-PE-Cy7(Biolegend,货号300316)，T-select HLA-A*0201 CMV pp65 Tetramer-PE(MBL，货号TS-0010-1C)，PVRIG-AF488(R&D，货号FAB93651G-100UG)，TIGIT-APC(Biolegend,货号372706)，PD-1-BV421(BD,货号562516)
上述诱导后的PBMC经CD8分选试剂盒(Stemcell，货号17953)分离出CD8作为效应细胞，用AIM-V培养基重悬并调整细胞密度至0.4×10 6/mL(若CD8诱导后经冻存，则需将细胞数加大至0.7×10 6/mL)。分选后的CD8检测纯度及CD226的表达。Colo205作为靶细胞，经TrypLE TM Express Enzyme(Gibco,货号12605010)消化，重悬于含有20ng/mL pp65的AIM-V(Gibco,货号31035-025)并调整细胞密度至1×10 6/ml，37℃5％CO 2处理3小时，之后250g离心5分钟，弃上清。之后细胞用AIM-V重悬至0.5×10 6/mL，并用流式检测其PVRL2，PVR及PD-L1的表达(图61中B)。将待测抗体(人源化双抗及Tecentriq)用AIM-V培养基稀释至280nM。在低吸附96孔U底板(Corning,货号7007)中依次加入50μL抗体，50uL CD8，100μL pp65处理过的colo205，并用排枪轻轻混匀，37℃5％CO 2孵育18小时。此体系中药物终浓度为70nM，CD8为20000/孔，colo205为50000/孔。孵育结束后400g离心取上清，用ELISA试剂盒(达科为，货号1110003)检测上清中human IFN-γ的水平。此体系中的阳性对照为COM701-hIgG4和RG6058-hIgG1,阴性对照为no treatment。分选后CD8T纯度的流式检测抗体如下：livedead-BV421(Invitrogen,货号L34964)，CD8-FITC(BD,货号555366)。
如图61中C和表48所示，2个双抗分子LC-BsAb-002和LC-BsAb-006在各浓度点上，IFN-γ的释放水平在统计学上无显著性差异，双抗分子在高浓度点(LC-BsAb-002在70nM,LC-BsAb-006在70和7nM)的因子释放显著多于相同条件下的联用组合，2个候选分子对应的联用组合与阳性对照抗体RG605-hIgG1和COM701-hIgG4的联用组合在各浓度点无显著性差异，整体趋势来看2个双抗分子相较于阳性对照抗体RG605-hIgG1和COM701-hIgG4的联用有更好的促进CD8 T释放IFN-γ的作用。
如图61中D，2个双抗分子，LC-BsAb-002和LC-BsAb-006与Tencentriq联用后，相较于双抗本身Human IFN-γ的分泌均显著增加(t-test,**P<0.01)，PVRIG单抗、TIGIT单抗和PD-L1单抗三个药物的联用，相较于PVRIG单抗和TIGIT单抗二个药物的联用，Human IFN-γ的分泌均显著增加(t-test,*P<0.05)，图中柱状图上的百分比为相较于抗TIGIT阳性对照抗体RG6058-hIgG1 IFN-γ提高的百分比。
表48细胞上清中human IFN-γ的检测结果的统计学分析数据(two-way ANOVA)
Figure PCTCN2022108648-appb-000159
Figure PCTCN2022108648-appb-000160
实施例43 CMV antigen-recall assay检测抗PVRIG×TIGIT人源化双特异性抗体与Tecentriq联用后对抗原特异性CD8 T细胞的功能促进作用
实验原理：同实施例40(图61中A)。
抗原特异性CD8 T细胞的诱导：同实施例40。铺板当天用流式检测其上PVRIG、TIGIT和PD-1的表达(图62中A)。
诱导后的PBMC经CD8 T分选试剂盒(Stemcell，货号17953)分离出CD8 T作为效应细胞，用AIM-V培养基重悬并调整细胞密度至0.8×10 6/mL(根据抗原特异性CD8 T细胞的比例，对微孔板中CD8 T细胞数进行调整)。完全培养基添加终浓度100ng/mL IFN-γ预处理过夜的Colo205作为靶细胞，经TrypLE TMExpress Enzyme(Gibco,货号12605010)消化，洗涤2遍 后重悬于含有20ng/mL pp65的AIM-V(Gibco,货号31035-025)并调整细胞密度至1×10 6/ml，37℃5％CO 2处理3小时，之后250g离心5分钟，弃上清。之后细胞用AIM-V重悬至0.5×10 6/mL，并用流式检测其PVRL2，PVR及PD-L1的表达(图62中A)。将待测抗体(人源化双抗、Tecentriq、双抗与Tecentriq的联用，2个阳性对照单抗(COM701-hIgG4和RG6058-hIgG1)的联用、三个单抗(COM701-hIgG4、RG6058-hIgG1和Tecentriq)的联用)用AIM-V培养基稀释至280nM(4×)作为起始浓度，后续10倍梯度稀释，共6个浓度点。在低吸附96孔U底板(Corning,货号7007)中依次加入50μL抗体，50μL CD8，100μL pp65处理过的colo205，并用排枪轻轻混匀，37℃5％CO 2共孵育18小时。此体系中药物终浓度分别为70nM、7nM、0.7nM、0.07nM、0.007nM和0.0007nM，CD8为40000/孔，colo205为50000/孔。共孵育结束后400g离心取上清，用ELISA试剂盒(达科为，货号1110003)检测上清中human IFN-γ的水平。
如图62中B及表49所示，根据IFN-γ拟合曲线的AUC进行排序：LC-BsAb-002+Tecentriq>RG6058-hIgG1+COM701-hIgG4+Tecentriq>LC-BsAb-002>RG6058-hI gG1+COM701-hIgG4>Tecentriq。AUC越大，说明效力越强，LC-BsAb-002与Tencentriq联用后，相较于双抗LC-BsAb-002本身Human IFN-γ的分泌明显增加；COM701-hIgG4、RG6058-hIgG1和Tecentriq三个药物的联用，相较于COM701-hIgG4和RG6058-hIgG1二个药物的联用，Human IFN-γ的分泌明显增加。
表49细胞上清中human IFN-γ检测结果的曲线拟合情况
Figure PCTCN2022108648-appb-000161
*数据曲线无法拟合
实施例44抗PVRIGxTIGIT人源化双特异性抗体的小鼠体内药效评估
A375细胞以5×10 6个/0.1mL浓度接种于雌性5-6周NPG小鼠(品系：NPG；北京维通达生物技术有限公司)的右侧皮下。A375细胞接种后一天，Hu PBMC细胞以5×10 6个/0.2mL浓度尾静脉注射于小鼠体内，待肿瘤生长到大约82mm 3时按肿瘤体积挑选56只随机分组，每组8只，共7组，分别为：Vehicle(PBS)、RG6058-hIgG1(10mg/kg)、COM701-hIgG4(10mg/kg)、RG6058-hIgG1+COM701-hIgG4(10mg/kg+10mg/kg)、Tecentriq(5mg/kg；lotNO.HK65567，Roche)、LC-BsAb-002(11.7mg/kg)、LC-BsAb-006(11.7mg/kg)。所有组给药途径均为腹腔注射，每周给药2次，连续给药4次，末次给药3天后结束实验。给药和观察期间每周测量3次小鼠体重和肿瘤体积，并记录测量值，计算肿瘤体积(长径×短径 2/2)和生长抑制率(TGI TV(％)＝(1-(Tn-T0)/(Vn-V0))×100％
药效结果：如图63所示，受试候选分子LC-BsAb-002、LC-BsAb-006给药后对A375肿瘤生长有明显抑制作用，抑制水平与阳性分子RG6058-hIgG1+COM701-hIgG4联用、Tecentriq在同一水平。在分组给药第13天，通过各受试药组与阴性对照PBS组的肿瘤抑制率(TGI) 及差异性分析(表11)，LC-BsAb-002和LC-BsAb-006的TGI分别为82.16％和78.59％且较PBS有显著性(P<0.005)，TGI水平好于RG6058-hIgG1(TGI＝42.55)、COM701-hIgG4(TGI＝0.23％)单药，与RG6058-hIgG1+COM701-hIgG4联用(TGI＝83.32％)相当。单只小鼠肿瘤生长曲线如图64所示显示出和图63相同的趋势。
表50受试物对A375细胞移植HuPBMC NPG小鼠肿瘤体积的影响
Figure PCTCN2022108648-appb-000162
注：a：平均数±标准差；
b：给药组肿瘤体积与Vehicle对照组肿瘤体积在分组给药第13天进行统计学比较，Two-way ANOVA分析，*P<0.05,**P<0.01，***P<0.001,****P<0.0001。
体重结果：如图65及表51所示，除对照分子Tecentriq有显著体重下降，表现出毒副作用外，其余对照及候选分子LC-BsAb-002、LC-BsAb-006给药体重变化趋势基本和PBS一致，后续的体重下降为PBMC重建导致的GVHD现象。
表51受试物对A375细胞移植HuPBMC NPG小鼠体重的影响
Figure PCTCN2022108648-appb-000163
注：a：平均数±标准差；
b：给药组体重与Vehicle对照组体重在分组给药第13天进行统计学比较，Two-way ANOVA分析。
最终结果表明，抗PVRIGxTIGIT人源化双特异性抗体分子LC-BsAb-002和LC-BsAb-006对A375皮下移植瘤生长有显著抑制作用，抑瘤效果优于阳性药RG6058-hIgG1和COM701-hIgG4单药，与阳性对照抗体Tecentriq和RG6058-hIgG1+COM701-hIgG4联用相当。同时，在给药观察过程中未观察到候选分子毒副作用，表明在该模型下候选分子表现安全耐受。
实施例45抗PVRIGxTIGIT人源化双特异性抗体与Tecentriq联用的小鼠体内药效评估
A375细胞以5×10 6个/0.1mL浓度接种于雌性5-6周NPG小鼠(品系：NPG；北京维通达生物技术有限公司)的右侧皮下。A375细胞接种后一天，Hu PBMC细胞以5.5×10 6个/0.2mL浓度尾静脉注射于小鼠体内，待肿瘤生长到大约82.78mm 3时按肿瘤体积挑选45只随机分组，共5组，分别为：Vehicle(PBS，9只)、LC-BsAb-002(11.7mg/kg，9只)、LC-BsAb-002(5.9mg/kg，9只)、Tecentriq(3mg/kg，10只；lotNO.HK65567，Roche)、LC-BsAb-002+Tecentriq(5.9mg/kg+3mg/kg，8只)。所有组给药途径均为腹腔注射，每周给药2次，连续给药5次，末次给药3天后结束实验。给药和观察期间每周测量3次小鼠体重和肿瘤体积，并记录测量值，计算肿瘤体积(长径×短径 2/2)和生长抑制率(TGI TV(％)＝(1-(Tn-T0)/(Vn-V0))×100％。
药效结果：如图66所示，受试候选分子LC-BsAb-002给药后对A375肿瘤生长有明显抑制作用，且给药剂量越高，对A375肿瘤生长的抑制作用越强。而LC-BsAb-002与Tecentriq联用对A375肿瘤生长的抑制水平显著优于LC-BsAb-002和Tecentriq的单药。在分组给药第17天，通过各受试药组与阴性对照PBS组的肿瘤抑制率(TGI)及差异性分析(表52)，发现LC-BsAb-002(11.7mg/kg)、LC-BsAb-002(5.9mg/kg)和Tecentriq(3mpk)的TGI分别为66.56％、60.51％和41.53，与PBS组相比有显著差异(P<0.0001、P＝0.0003和P＝0.0015)；LC-BsAb-002与Tecentriq联用组的TGI为80.44％，与PBS组相比有显著差异(P<0.0001)，且优于LC-BsAb-002(5.9mg/kg)和Tecentriq(3mpk)的单用TGI。单只小鼠肿瘤生长曲线如图67所示，各组肿瘤生长趋势与图66相同。
表52受试物对A375细胞移植HuPBMC NPG小鼠肿瘤体积的影响
Figure PCTCN2022108648-appb-000164
注：a：平均数±标准差；
b：给药组肿瘤体积与Vehicle对照组肿瘤体积在分组给药第13天进行统计学比较，Two-way ANOVA分析，*P<0.05,**P<0.01，***P<0.001,****P<0.0001。
体重结果：如图68及表53所示，LC-BsAb-002+Tecentriq联用组的小鼠体重有一定下降，但并没有表现出明显的毒副作用。Tecentriq组、LC-BsAb-002(11.7mpk)和LC-BsAb-002(5.9mpk)给药组小鼠体重变化趋势与PBS组基本一致。
表53受试物对A375细胞移植HuPBMC NPG小鼠体重的影响
Figure PCTCN2022108648-appb-000165
注：a：平均数±标准差；
b：给药组体重与Vehicle对照组体重在分组给药第13天进行统计学比较，Two-way ANOVA分析分析。
最终结果表明，抗PVRIGxTIGIT人源化双特异性抗体分子LC-BsAb-002对A375皮下移植瘤生长有显著抑制作用，且随给药剂量的提升，抑制作用呈明显的剂量依赖关系。而LC-BsAb-002和Tecentriq联用的抑瘤效果显著优于各自的单用，有显著的联用效果，同时，在给药观察过程中未观察到候选分子的毒副作用，表明在该模型下候选分子表现安全耐受。

Claims (49)

  1. 一种抗PVRIG/抗TIGIT双特异性抗体，其包含：
    (a)第一抗原结合部分，其包括重链可变区(VH)和轻链可变区(VL)，所述VH和VL形成抗TIGIT抗原结合域；其中，所述TIGIT VH包含SEQ ID NO：72或87任一项所述VH的HCDR1、HCDR2和HCDR3；所述TIGIT VL包含SEQ ID NO：68或91任一项所述VL的LCDR1、LCDR2和LCDR3；
    (b)第二抗原结合部分，其包括特异性结合PVRIG的VHH，所述VHH包含SEQ ID NO：200或211任一项所述序列的CDR1、CDR2和CDR3。
  2. 权利要求1的双特异性抗体，其中：
    (a)所述第一抗原结合部分的HCDR1包含SEQ ID NO：21或33任一项所述的序列；HCDR2包含SEQ ID NO：22或34任一项所述的序列；HCDR3包含SEQ ID NO：23或35任一项所述的序列；
    (b)所述第一抗原结合部分的LCDR1包含SEQ ID NO：18或96任一项所述的序列；LCDR2包含SEQ ID NO：19或31任一项所述的序列；LCDR3包含SEQ ID NO：20或32任一项所述的序列；
    (c)所述第二抗原结合部分的CDR1包含SEQ ID NO：168或147任一项所述的序列；CDR2包含SEQ ID NO：207或148任一项所述的序列；CDR3包含SEQ ID NO：208或149任一项所述的序列。
  3. 权利要求2的双特异性抗体，其中，所述第一抗原结合部分包含以下序列的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3：
    (1)分别为SEQ ID NO：21、22、23、18、19和20；或
    (2)分别为SEQ ID NO：33、34、35、96、31和32；或
    (3)与上述(1)至(2)所示序列具有至少90％同一性或具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列，优选地，所述替换为保守氨基酸的替换。
  4. 权利要求2～3的双特异性抗体，其中，所述第二抗原结合部分包含以下序列的CDR1、CDR2和CDR3：
    (1)分别为SEQ ID NO：168、207和208；或
    (2)分别为SEQ ID NO：147、148和149；或
    (3)与上述(1)至(2)所示序列具有至少90％同一性或具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列，优选地，所述替换为保守氨基酸的替换。
  5. 权利要求1～4的双特异性抗体，其中，所述第一抗原结合部分的VH包含与SEQ ID NO：72或87所示氨基酸序列至少90％同一性的序列；所述第一抗原结合部分的VL包含SEQ  ID NO：68或91所示氨基酸序列至少90％同一性的序列。
  6. 权利要求1～5的双特异性抗体，其中，第二抗原结合部分包含SEQ ID NO：200或211所示氨基酸序列至少90％同一性的序列。
  7. 权利要求1～6的双特异性抗体，其中，所述第一抗原结合部分是一种全长抗体，包括两条重链和两条轻链；所述第二抗原结合部分的C端融合到第一抗原结合部分的至少一条重链的N端。
  8. 权利要求7的双特异性抗体，其中，重链融合多肽从N端到C端包括PVRIG VHH-(G4S)4 Linker-TIGIT VH-CH1-铰链-CH2-CH3，轻链多肽从N端到C端包括TIGIT VL-CL。
  9. 权利要求7～8的双特异性抗体，其中，所述重链融合多肽包括与SEQ ID NO：227、229、231或233所示氨基酸序列至少80％同一性的序列，轻链多肽包括与SEQ ID NO：226、228、230或232所示氨基酸序列至少80％同一性的序列。
  10. 权利要求1～9的双特异性抗体，其为人源化抗体。
  11. 权利要求1～10的双特异性抗体，其与人、猴PRVIG或TIGIT蛋白特异性结合；优选地，其与人、猴TIGIT结合的KD优于1.00E-7M，与人、猴PRVIG结合的KD优于1.00E-8M；更优选地，其可同时与TIGIT和PVRIG相结合。
  12. 一种特异性结合TIGIT的抗体或抗原结合片段，其包含：
    (1)重链可变区(VH)，其中所述重链可变区包含三个互补决定区(HCDR)：HCDR1、HCDR2和HCDR3，其中，按照Kabat编号系统编号，所述HCDR1包含SEQ ID NO:21、27、33、39所示的氨基酸序列，所述HCDR2包含SEQ ID NO:22、28、34、40所示的氨基酸序列，所述HCDR3包含SEQ ID NO:23、29、35、41所示的氨基酸序列；按照IMGT编号系统编号，所述HCDR1包含SEQ ID NO:45、51、57、63所示的氨基酸序列，所述HCDR2包含SEQ ID NO:46、52、58、64，所述HCDR3包含SEQ ID NO:47、53、59、65；以及，
    (2)轻链可变区(VL)，其中所述轻链可变区包含三个互补决定区(LCDR)：LCDR1、LCDR2和LCDR3，其中，按照Kabat编号系统编号，所述LCDR1包含SEQ ID NO:18、24、30、36、93、94、95、96所示的氨基酸序列，所述LCDR2包含SEQ ID NO:19、25、31、37所示的氨基酸序列，所述LCDR3包含SEQ ID NO:20、26、32、38所示的氨基酸序列；按照IMGT编号系统编号，所述LCDR1包含SEQ ID NO:42、48、54、60所示的氨基酸序列，所述LCDR2包含SEQ ID NO:43、49、55、61所示的氨基酸序列，所述LCDR3包含SEQ ID NO: 44、50、56、62所示的氨基酸序列。
  13. 权利要求12的抗体或抗原结合片段，其包含以下序列的LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3：
    (1)分别为SEQ ID NO:18、19、20、21、22和23；或
    (2)分别为SEQ ID NO:24、25、26、27、28和29；或
    (3)分别为SEQ ID NO:30、31、32、33、34和35；或
    (4)分别为SEQ ID NO:36、37、38、39、40和41；或
    (5)分别为SEQ ID NO:42、43、44、45、46和47；或
    (6)分别为SEQ ID NO:48、49、50、51、52和53；或
    (7)分别为SEQ ID NO:54、55、56、57、58和59；或
    (8)分别为SEQ ID NO:60、61、62、63、64和65；或
    (9)分别为SEQ ID NO:93、31、32、33、34和35；或
    (10)分别为SEQ ID NO:94、31、32、33、34和35；或
    (11)分别为SEQ ID NO:95、31、32、33、34和35；或
    (12)分别为SEQ ID NO:96、31、32、33、34和35；或
    (13)与上述(1)至(12)所示序列具有至少80％同一性或具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列，优选地，所述替换为保守氨基酸的替换。
  14. 权利要求12或13的抗体或抗原结合片段，其包含：
    (1)重链可变区，其包含与SEQ ID NO:10、11、12、13、69、70、71、72、81、82、83、84、85、87、101、102或103具有至少80％同一性的氨基酸序列；或/和
    (2)轻链可变区，其包含与SEQ ID NO:14、15、16、17、66、67、68、78、79、80、86、88、89、90、91、98、99或100具有至少80％同一性的氨基酸序列。
  15. 权利要求12～14的抗体或抗原结合片段，其：
    (1)包含SEQ ID NO:10所示氨基酸序列的重链可变区及包含SEQ ID NO:14所示氨基酸序列的轻链可变区；或
    (2)包含SEQ ID NO:11所示氨基酸序列的重链可变区及包含SEQ ID NO:15所示氨基酸序列的轻链可变区；或
    (3)包含SEQ ID NO:12所示氨基酸序列的重链可变区及包含SEQ ID NO:16所示氨基酸序列的轻链可变区；或
    (4)包含SEQ ID NO:13所示氨基酸序列的重链可变区及包含SEQ ID NO:17所示氨基酸序列的轻链可变区；或
    (5)包含SEQ ID NO:69所示氨基酸序列的重链可变区及包含SEQ ID NO:66、67或68所示氨基酸序列的轻链可变区；或
    (6)包含SEQ ID NO:70所示氨基酸序列的重链可变区及包含SEQ ID NO:66、67或68所示氨基酸序列的轻链可变区；或
    (7)包含SEQ ID NO:71所示重链可变区及包含SEQ ID NO:66、67或68所示氨基酸序列的轻链可变区；或
    (8)包含SEQ ID NO:72所示氨基酸序列的重链可变区及包含SEQ ID NO:66、67或68所示氨基酸序列的轻链可变区；或
    (9)包含SEQ ID NO:81、82、83、84或85所示氨基酸序列的重链可变区及包含SEQ ID NO:78所示氨基酸序列的轻链可变区；或
    (10)包含SEQ ID NO:81、82、83、84或85所示氨基酸序列的重链可变区及包含SEQ ID NO:79所示氨基酸序列的轻链可变区；或
    (11)包含SEQ ID NO:81、82、83、84或85所示氨基酸序列的重链可变区及包含SEQ ID NO:80所示氨基酸序列的轻链可变区；或
    (12)包含SEQ ID NO:87所示氨基酸序列的重链可变区及包含SEQ ID NO:86、88、89、90或91所示氨基酸序列的轻链可变区；或
    (13)包含SEQ ID NO:101所示氨基酸序列的重链可变区及包含SEQ ID NO:98、99或100所示氨基酸序列的轻链可变区；或
    (14)包含SEQ ID NO:102所示氨基酸序列的重链可变区及包含SEQ ID NO:98、99或100所示氨基酸序列的轻链可变区
    (15)包含SEQ ID NO:103所示氨基酸序列的重链可变区及包含SEQ ID NO:98、99或100所示氨基酸序列的轻链可变区；或
    (16)与上述(1)至(15)所示序列具有至少80％同一性或至多20个突变的序列；所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
  16. 权利要求12～15的抗体或抗原结合片段，其包含重链可变区，其中，所述重链可变区与SEQ ID NO.10所示VH相比至少具有选自下组的突变：按自然顺序编号，S30T，G44K，W47Y，I48M，V67I或V71R；优选地，至少具有S30T和V71R突变；更优选地，至少具有S30T，G44K和V71R突变；更优选地，至少具有S30T，G44K，I48M，V67I和V71R突变；更优选地，至少具有S30T，G44K，W47Y和V71R突变；
    或与SEQ ID NO.11所示VH相比至少具有选自下组的突变：按自然顺序编号，T28A，R72A，T74K或A76S；优选地，至少具有T28A，R72A，T74K和A76S；
    或与SEQ ID NO.12所示VH相比至少具有选自下组的突变：按自然顺序编号，I29M，S30T，G44K，W47Y，I48M，V67I或V71R；优选地，至少具有S30T和V71R突变；更优选地，至少具有I29M，S30T和V71R突变；更优选地，至少具有I29M，S30T，G44K和V71R突变；更优选地，至少具有I29M，S30T，G44K，I48M，V67I和V71R突变；更优选地，至少具有I29M，S30T，G44K，W47Y和V71R；
    或与SEQ ID NO.13所示VH相比至少具有选自下组的突变：按自然顺序编号，R44G，R72V，T74K，S75L或A76S；优选地，至少具有R72V和T74K突变；更优选地，至少具有R72V，T74K，S75L和A76S突变；更优选地，至少具有R44G，R72V，T74K，S75L和A76S突变。
  17. 权利要求12～16的抗体或抗原结合片段，其包含轻链可变区，其中，所述轻链可变区与SEQ ID NO.14所示VL相比至少具有选自下组的突变：按自然顺序编号，L37Q，P43S或L47M；优选地，至少具有L47M突变；更优选地，至少具有L37Q和L47M突变；更优选地，至少具有P43S和L47M突变；
    或与SEQ ID NO.15所示VL相比至少具有选自下组的突变：按自然顺序编号，N31Q，N31T，N31D，G32A，Q38H或P43S；优选地，至少具有Q38H和P43S突变；更优选地，至少具有N31Q，Q38H和P43S突变；更优选地，至少具有N31T，Q38H和P43S突变；更优选地，至少具有N31D，Q38H和P43S突变；更优选地，至少具有G32A，Q38H和P43S突变；
    或与SEQ ID NO.16所示VL相比至少具有选自下组的突变：按自然顺序编号，L37Q，P43S或Q45K；优选地，至少具有L37Q和Q45K突变；更优选地，至少具有P43S突变；
    或与SEQ ID NO.17所示VL相比至少具有选自下组的突变：按自然顺序编号，A43S，P43S或I48V；优选地，至少具有A43S突变；更优选地，至少具有A43S和I48V突变；更优选地，至少具有P43S和I48V突变。
  18. 权利要求12～17的抗体或抗原结合片段，其与人、猴TIGIT蛋白特异性结合；优选地，其与人、猴TIGIT结合的KD优于1.00E-8M。
  19. 权利要求12～18的抗体或抗原结合部分，其为鼠抗体、人源化抗体、全人抗体或嵌合抗体。
  20. 权利要求12～19的抗体或抗原结合片段，其选自单克隆抗体、多克隆抗体、天然抗体、工程化抗体、单特异性抗体、多特异性分子(例如双特异性抗体)、单价抗体、多价抗体、完整抗体、完整抗体的片段、裸抗体、缀合抗体、嵌合抗体、人源化抗体、全人抗体、Fab、Fab’、Fab’-SH、F(ab’)2、Fd、Fv、scFv、双抗体(diabody)或单域抗体。
  21. 一种特异性结合PVRIG的纳米抗体或抗原结合片段，其包含SEQ ID NO.107-119、198-204、211-216、219-225任一项所述VH的HCDR1、HCDR2和HCDR3。
  22. 权利要求21的纳米抗体或抗原结合片段，其中，所述HCDR1、HCDR2和HCDR3根据IMGT编号系统确定，例如选自表21；所述HCDR1、HCDR2和HCDR3根据Kabat编号系统确定，例如选自表22、表29。
  23. 权利要求21或22任一项的纳米抗体或抗原结合片段，其中，SEQ ID NO.107所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：120～122或SEQ ID NO：159～161所示的序列；
    SEQ ID NO.108所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：123～125或SEQ ID NO：162～164所示的序列；
    SEQ ID NO.109所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：126～128或SEQ ID NO：165～167所示的序列；
    SEQ ID NO.110所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：129～131或SEQ ID NO：168～170所示的序列；
    SEQ ID NO.111所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：132～134、SEQ ID NO：171～173所示的序列；
    SEQ ID NO.112所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：135～137或SEQ ID NO：174～176所示的序列；
    SEQ ID NO.113所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：138～140或SEQ ID NO：177～179所示的序列；
    SEQ ID NO.114所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：141～143或SEQ ID NO：180～182所示的序列；
    SEQ ID NO.115所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：144～146或SEQ ID NO：183～185所示的序列；
    SEQ ID NO.116所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：147～149或SEQ ID NO：186～188所示的序列；
    SEQ ID NO.117所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：150～152或SEQ ID NO：189～191所示的序列；
    SEQ ID NO.118所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：153～155或SEQ ID NO：192～194所示的序列；
    SEQ ID NO.119所示VH的HCDR1～3按照IMGT或Kabat编号系统，具有如SEQ ID NO：156～158或SEQ ID NO：195～197所示的序列；
    SEQ ID NO.198所示VH的HCDR1～3按照Kabat编号系统，具有如SEQ ID NO：168～170所示的序列；
    SEQ ID NO.199所示VH的HCDR1～3按照Kabat编号系统，具有如SEQ ID NO：168、207和170所示的序列；
    SEQ ID NO.200所示VH的HCDR1～3按照Kabat编号系统，具有如SEQ ID NO：168、207和208所示的序列；
    SEQ ID NO.201所示VH的HCDR1～3按照Kabat编号系统，具有如SEQ ID NO：168、207和209所示的序列；
    SEQ ID NO.202所示VH的HCDR1～3按照Kabat编号系统，具有如SEQ ID NO：168、169和208所示的序列；
    SEQ ID NO.203、204所示VH的HCDR1～3按照Kabat编号系统，具有如SEQ ID NO：168、210和208所示的序列；
    SEQ ID NO.211～215所示VH的HCDR1～3按照IMGT编号系统，具有如SEQ ID NO：147～149所示的序列；
    SEQ ID NO.216所示VH的HCDR1～3按照IMGT编号系统，具有如SEQ ID NO：147、148和218所示的序列；
    SEQ ID NO.219-225所示VH的HCDR1～3按照IMGT编号系统，具有如SEQ ID NO：156～158所示的序列。
  24. 权利要求21-23任一项的纳米抗体或抗原结合片段，其包含与所述HCDR1、HCDR2和HCDR3相比具有至少80％同一性或具有1、2、3或更多个氨基酸插入、缺失和/或替换的CDRs序列，优选地，所述替换为保守氨基酸的替换。
  25. 权利要求21-24任一项的纳米抗体或抗原结合片段，其包含SEQ ID NO.107-119、198-204、211-216、219-225任一项所示的VH，或者与SEQ ID NO.107-119、198-204、211-216、219-225任一项所示VH具有至少80％同一性或至多20个突变的序列；所述突变可选自插入、缺失和/或替换，所述替换优选为保守氨基酸的替换。
  26. 权利要求25所述的纳米抗体或抗原结合片段，其包含与SEQ ID NO.110所示VH相比至少具有选自下组的突变序列：按自然顺序编号，A97V、K98E、N54D、N108S、S110A、G55A或S75T；更优选地，至少具有A97V和K98E突变；更优选地，至少具有A97V、K98E和N54D突变；更优选地，至少具有A97V、K98E、N54D和N108S突变；更优选地，至少具有A97V、K98E、N54D和S110A突变；更优选地，至少具有A97V、K98E和N108S突变；更优选地，至少具有A97V、K98E、G55A和N108S突变；更优选地，至少具有S75T、A97V、K98E、G55A和N108S突变；
    或包含与SEQ ID NO：116所示VH相比，至少具有选自下组的突变序列：按自然顺序编号，S35T、V37F、G44E、L45R、W47F、N50T、L79V、V61S、D62H、T122I或M123Q；更优选地，至少具有V37F、G44E、L45R、W47F和N50T突变；更优选地，至少具有S35T、V37F、G44E、L45R、W47F和N50T突变；更优选地，至少具有S35T、V37F、G44E、L45R、W47F、N50T和L79V突变；更优选地，至少具有S35T、V37F、G44E、L45R、W47F、N50T、V61S和D62H突变；更优选地，至少具有S35T、V37F、G44E、L45R、W47F、N50T、T122I和M123Q突变；
    或包含与SEQ ID NO：119所示VH相比，至少具有选自下组的突变序列：按自然顺序编号，S35G、V37Y、G44D、L45R、W47L、N50T、Y58K、Y59I、D72G、N73D、Y79S、L78V或Y94F；更优选地，至少具有S35G、V37Y、G44D、L45R、W47L和N50T突变；更优选地，至少具有S35G、V37Y、G44D、L45R、W47L、N50T和Y58K突变；更优选地，至少具有S35G、V37Y、G44D、L45R、W47L、N50T、Y58K、D72G和N73D突变；更优选地，至少具有S35G、V37Y、G44D、L45R、W47L、N50T、Y58K、D72G、N73D和Y79S突变；更优选地，至少具有S35G、V37Y、G44D、L45R、W47L、N50T、Y58K、D72G、N73D和L78V突变；更优选地，至少具有S35G、V37Y、G44D、L45R、W47L、N50T、Y58K、Y59I、D72G和N73D突变；更优选地，至少具有S35G、V37Y、G44D、L45R、W47L、N50T、Y58K、D72G、N73D和Y94F突变。
  27. 权利要求21～26任一项的纳米抗体或抗原结合片段，其与人、猴PVRIG蛋白特异性结合；优选地，其与人、猴PVRIG结合的KD优于2.00E-9 M。
  28. 权利要求21～27任一项的纳米抗体或抗原结合片段，其为：(1)嵌合纳米抗体或其片段；(2)人源化纳米抗体或其片段；或(3)全人纳米抗体或其片段。
  29. 权利要求21～28任一项的纳米抗体或抗原结合片段，其包含或不包含抗体重链恒定区；可选的，所述抗体重链恒定区可选自人、羊驼、小鼠、大鼠、兔或羊；可选地，所述抗体重链恒定区可选自IgG、IgM、IgA、IgE或IgD，所述IgG可选自IgG1，IgG2，IgG3或IgG4；可选地，所述重链恒定区可选自Fc区、CH3区或完整重链恒定区，优选地，所述重链恒定区为人Fc区；优选地，所述纳米抗体或抗原结合片段为重链抗体。
  30. 权利要求1～11任一项的抗PVRIG/抗TIGIT双特异性抗体，权利要求12～20任一项的特异性结合TIGIT的抗体或抗原结合片段，权利要求21～29任一项的特异性结合PVRIG的纳米抗体或抗原结合片段，其还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自药物、毒素、放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂。
  31. 一种多特异性分子，其包含权利要求1～11任一项的抗PVRIG/抗TIGIT双特异性抗体，权利要求12～20任一项的特异性结合TIGIT的抗体或抗原结合片段；或包含权利要求21～29任一项的特异性结合PVRIG的纳米抗体或抗原结合片段；优选地，所述多特异性分子可为双特异性、三特异性或四特异性，更优选地，所述多特异性分子可为二价、四价或六价。
  32. 权利要求31的多特异性分子，其为串联scFv、双功能抗体(Db)、单链双功能抗体(scDb)、双重亲和力再靶向(DART)抗体、F(ab')2、双重可变域(DVD)抗体、臼包杵(KiH)抗体、对接及锁定(DNL)抗体、化学交联抗体、杂多聚纳米抗体或异结合物抗体。
  33. 一种嵌合抗原受体(CAR)，其至少包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含权利要求12～20任一项的抗体或抗原结合片段；或包含权利要求21～29任一项的纳米抗体或抗原结合片段。
  34. 一种免疫效应细胞，其表达权利要求33所述的嵌合抗原受体，或包含编码权利要求33所述嵌合抗原受体的核酸片段；优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、DNT细胞(double negative T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞，所述T细胞优选自细胞毒性T细胞、调节性T细胞或辅助性T细胞；优选地，所述免疫效应细胞为自体免疫效应细胞或同种异体免疫效应细胞。
  35. 一种分离的核酸片段，其编码权利要求1～11任一项所述的双特异性抗体，权利要求 12～20任一项的抗体或抗原结合片段，权利要求21～29任一项的纳米抗体或抗原结合片段，权利要求31～32任一项所述多特异性分子，或权利要求33所述的嵌合抗原受体。
  36. 一种载体(vector)，其包含权利要求35所述的核酸片段。
  37. 一种宿主细胞，其包含权利要求36所述的载体；优选地，所述细胞为原核细胞或真核细胞，例如细菌(大肠杆菌)、真菌(酵母)、昆虫细胞或哺乳动物细胞(CHO细胞系或293T细胞系)。
  38. 一种制备权利要求1～11任一项所述的双特异性抗体，权利要求12～20任一项所述的抗体或抗原结合片段，权利要求21～29任一项所述的纳米抗体或抗原结合片段，或权利要求31～32任一项所述多特异性分子的方法，其包括培养权利要求37的细胞，以及分离所述细胞表达的抗体或分子。
  39. 一种制备权利要求34所述免疫效应细胞的方法，其包括将编码权利要求33所述CAR的核酸片段导入所述免疫效应细胞，可选地，还包括启动所述免疫效应细胞表达权利要求34所述CAR。
  40. 一种药物组合物，其包含权利要求1～11任一项所述的双特异性抗体，或权利要求12～20任一项所述的抗体或抗原结合片段，或权利要求21～29任一项所述的纳米抗体或抗原结合片段，或权利要求31～32任一项所述多特异性分子，或权利要求34的免疫效应细胞，或权利要求35所述的核酸片段，或权利要求36所述载体，或权利要求37所述宿主细胞，或权利要求38～39所述方法制备获得的产品，和药学上可接受的载体。
  41. 权利要求40的药物组合物，其进一步包含另外的治疗剂；优选地，所述另外的治疗剂是抗肿瘤剂；更优选地，所述抗肿瘤剂是PD-1轴结合拮抗剂。
  42. 权利要求1～11任一项所述的双特异性抗体，或权利要求12～20任一项所述的抗体或抗原结合片段，或权利要求21～29任一项所述的纳米抗体或抗原结合片段，或权利要求31～32任一项所述多特异性分子，或权利要求34的免疫效应细胞，或权利要求35所述的核酸片段，或权利要求36所述载体，或权利要求37所述宿主细胞，或权利要求38～39所述方法制备获得的产品，或权利要求40～41所述药物组合物在制备用于治疗癌症或感染性疾病的药物中的用途；其中所述癌症选自实体肿瘤和血液肿瘤，优选地，所述肿瘤选自白血病，多发性骨髓瘤，淋巴瘤，骨髓增生异常综合征，前列腺癌，肝癌，结直肠癌，肛门癌、卵巢癌，子宫内膜癌，宫颈癌，腹腔癌、乳腺癌，胰腺癌，胃癌，头颈癌，甲状腺癌，睾丸癌，泌尿道上皮癌，肺癌，黑色素瘤，非黑素瘤皮肤癌，神经胶质瘤，肾癌，间皮瘤，食道癌，非小细胞肺 癌，小细胞肺癌，膀胱癌，肉瘤，成胶质细胞瘤，胸腺癌，蕈样肉芽肿，默克尔细胞癌，高MSI癌和KRAS突变型肿瘤。
  43. 权利要求42所述的用途，其中，所述药物与另外的治疗剂或与手术组合使用；其中所述另外的治疗剂或所述手术选自放射疗法、化学疗法、溶瘤药物、细胞毒性剂、细胞因子、外科手术、免疫刺激性抗体、免疫调节药物、共刺激分子的激活剂、抑制性分子的抑制剂、疫苗或细胞免疫疗法。
  44. 权利要求43所述的用途，其中，所述另外的治疗剂在所述药物之前或之后施用，或与所述药物并行施用。
  45. 权利要求42～44所述的用途，其中，所述药物与PD-1轴结合拮抗剂组合使用。
  46. 权利要求45所述的用途，其中，所述PD-1轴结合拮抗剂选自由PD-1结合拮抗剂，PD-L1结合拮抗剂，和PD-L2结合拮抗剂组成的组；优选地，所述PD-1结合拮抗剂是抗PD-1抗体；更优选地，所述PD-1结合拮抗剂选自由MDX 1106(nivolumab)，MK-3475(pembrolizumab)，CT-011(pidilizumab)，MEDI-0680(AMP-514)，PDR001，REGN2810，和BGB-108组成的组；优选地，所述PD-L1结合拮抗剂是抗PD-L1抗体；更优选地，所述PD-L1结合拮抗剂选自由MPDL3280A(atezolizumab)，YW243.55.S70，MDX-1105，MEDI4736(durvalumab)，Tecentriq和MSB0010718C(avelumab)组成的组；优选地，所述PD-L2结合拮抗剂是抗PD-L2抗体；更优选地，所述PD-L2结合拮抗剂是免疫粘附素。
  47. 一种治疗癌症或感染性疾病的方法，包含向有此需要的患者施用有效量的权利要求1～11任一项所述的双特异性抗体，或权利要求12～20任一项所述的抗体或抗原结合片段，或权利要求21～29任一项所述的纳米抗体或抗原结合片段，或权利要求31～32任一项所述多特异性分子，或权利要求34的免疫效应细胞，或权利要求35所述的核酸片段，或权利要求36所述载体，或权利要求37所述宿主细胞，或权利要求38～39所述方法制备获得的产品，或权利要求40～41所述药物组合物；其中所述癌症选自实体肿瘤和血液肿瘤，优选地，所述肿瘤选自白血病，多发性骨髓瘤，淋巴瘤，骨髓增生异常综合征，前列腺癌，肝癌，结直肠癌，肛门癌、卵巢癌，子宫内膜癌，宫颈癌，腹腔癌、乳腺癌，胰腺癌，胃癌，头颈癌，甲状腺癌，睾丸癌，泌尿道上皮癌，肺癌，黑色素瘤，非黑素瘤皮肤癌，神经胶质瘤，肾癌，间皮瘤，食道癌，非小细胞肺癌，小细胞肺癌，膀胱癌，肉瘤，成胶质细胞瘤，胸腺癌，蕈样肉芽肿，默克尔细胞癌，高MSI癌和KRAS突变型肿瘤。
  48. 权利要求47的方法，其中还包括向有此需要的患者施用有效量的PD-1轴结合拮抗剂，其中，所述PD-1轴结合拮抗剂选自由PD-1结合拮抗剂，PD-L1结合拮抗剂，和PD-L2结合拮抗剂组成的组；优选地，所述PD-1结合拮抗剂是抗PD-1抗体；更优选地，所述PD-1 结合拮抗剂选自由MDX 1106(nivolumab)，MK-3475(pembrolizumab)，CT-011(pidilizumab)，MEDI-0680(AMP-514)，PDR001，REGN2810，和BGB-108组成的组；优选地，所述PD-L1结合拮抗剂是抗PD-L1抗体；更优选地，所述PD-L1结合拮抗剂选自由MPDL3280A(atezolizumab)，YW243.55.S70，MDX-1105，MEDI4736(durvalumab)，Tecentriq和MSB0010718C(avelumab)组成的组；优选地，所述PD-L2结合拮抗剂是抗PD-L2抗体；更优选地，所述PD-L2结合拮抗剂是免疫粘附素。
  49. 权利要求1～11任一项所述的双特异性抗体，或权利要求12～20任一项所述的抗体或抗原结合片段，或权利要求21～29任一项所述的纳米抗体或抗原结合片段，或权利要求31～32任一项所述多特异性分子，或权利要求34的免疫效应细胞，或权利要求35所述的核酸片段，或权利要求36所述载体，或权利要求37所述宿主细胞，或权利要求38～39所述方法制备获得的产品，或权利要求40～41所述药物组合物，用于预治疗癌症或感染性疾病；其中所述癌症选自实体肿瘤和血液肿瘤，优选地，所述肿瘤选自白血病，多发性骨髓瘤，淋巴瘤，骨髓增生异常综合征，前列腺癌，肝癌，结直肠癌，肛门癌、卵巢癌，子宫内膜癌，宫颈癌，腹腔癌、乳腺癌，胰腺癌，胃癌，头颈癌，甲状腺癌，睾丸癌，泌尿道上皮癌，肺癌，黑色素瘤，非黑素瘤皮肤癌，神经胶质瘤，肾癌，间皮瘤，食道癌，非小细胞肺癌，小细胞肺癌，膀胱癌，肉瘤，成胶质细胞瘤，胸腺癌，蕈样肉芽肿，默克尔细胞癌，高MSI癌和KRAS突变型肿瘤。
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Publication number Priority date Publication date Assignee Title
WO2023236980A1 (zh) * 2022-06-08 2023-12-14 山东先声生物制药有限公司 一种pvrig/tigit双特异性抗体药物组合物及其用途
WO2024153182A1 (zh) * 2023-01-19 2024-07-25 山东先声生物制药有限公司 抗pvrig/抗tigit双特异性抗体在治疗恶性肿瘤中的用途
WO2024213097A1 (zh) * 2023-04-14 2024-10-17 山东先声生物制药有限公司 一种靶向人cd16和肿瘤抗原的多特异性抗体
WO2024251160A1 (en) * 2023-06-06 2024-12-12 Wuxi Biologics (Shanghai) Co., Ltd. Anti-pvrig antibodies and uses thereof
WO2024251161A1 (en) * 2023-06-06 2024-12-12 Wuxi Biologics (Shanghai) Co., Ltd. Anti-tigit x pvrig antibodies and uses thereof
WO2025033553A1 (ja) * 2023-08-10 2025-02-13 国立大学法人 東京大学 新規なｈｖｅｍ結合タンパク質およびそれを含む医薬組成物
WO2025061165A1 (en) * 2023-09-20 2025-03-27 Laekna Therapeutics Shanghai Co., Ltd. Antibodies against pvrig or tigit, bispecific antibodies constructed therefrom, methods for their preparation and uses thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021180205A1 (zh) * 2020-03-13 2021-09-16 江苏恒瑞医药股份有限公司 Pvrig结合蛋白及其医药用途

Citations (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
WO1994004678A1 (en) 1992-08-21 1994-03-03 Casterman Cecile Immunoglobulins devoid of light chains
WO1994025591A1 (en) 1993-04-29 1994-11-10 Unilever N.V. PRODUCTION OF ANTIBODIES OR (FUNCTIONALIZED) FRAGMENTS THEREOF DERIVED FROM HEAVY CHAIN IMMUNOGLOBULINS OF $i(CAMELIDAE)
WO1995004079A1 (fr) 1993-08-02 1995-02-09 Raymond Hamers Vecteur recombinant contenant une sequence d'un gene de lipoproteine pour l'expression de sequences de nucleotides
WO1996034103A1 (en) 1995-04-25 1996-10-31 Vrije Universiteit Brussel Variable fragments of immunoglobulins - use for therapeutic or veterinary purposes
WO1997049805A2 (en) 1996-06-27 1997-12-31 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Recognition molecules interacting specifically with the active site or cleft of a target molecule
WO1999037681A2 (en) 1998-01-26 1999-07-29 Unilever Plc Method for producing antibody fragments
WO2000040968A1 (en) 1999-01-05 2000-07-13 Unilever Plc Binding of antibody fragments to solid supports
WO2000043507A1 (en) 1999-01-19 2000-07-27 Unilever Plc Method for producing antibody fragments
WO2000065057A1 (en) 1999-04-22 2000-11-02 Unilever Plc Inhibition of viral infection using monovalent antigen-binding proteins
WO2001021817A1 (en) 1999-09-24 2001-03-29 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Recombinant phages capable of entering host cells via specific interaction with an artificial receptor
WO2001040310A2 (en) 1999-11-29 2001-06-07 Unilever Plc Immobilisation of proteins using a polypeptide segment
WO2001044301A1 (en) 1999-11-29 2001-06-21 Unilever Plc Immobilized single domain antigen-binding molecules
EP1134231A1 (en) 2000-03-14 2001-09-19 Unilever N.V. Antibody heavy chain variable domains against human dietary enzymes, and their uses
WO2001090190A2 (en) 2000-05-26 2001-11-29 National Research Council Of Canada Single-domain antigen-binding antibody fragments derived from llama antibodies
WO2002048193A2 (en) 2000-12-13 2002-06-20 Unilever N.V. Camelidae antibody arrays
WO2003025020A1 (fr) 2001-09-13 2003-03-27 Institute For Antibodies Co., Ltd. Procede pour creer une banque d'anticorps de chameaux
WO2003035694A2 (en) 2001-10-24 2003-05-01 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Functional heavy chain antibodies, fragments thereof, library thereof and methods of production thereof
WO2003050531A2 (en) 2001-12-11 2003-06-19 Algonomics N.V. Method for displaying loops from immunoglobulin domains in different contexts
WO2003054016A2 (en) 2001-12-21 2003-07-03 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Method for cloning of variable domain sequences
WO2003055527A2 (en) 2002-01-03 2003-07-10 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Immunoconjugates useful for treatment of tumours
WO2004041865A2 (en) 2002-11-08 2004-05-21 Ablynx N.V. Stabilized single domain antibodies
WO2004041867A2 (en) 2002-11-08 2004-05-21 Ablynx N.V. Camelidae antibodies against imminoglobulin e and use thereof for the treatment of allergic disorders
WO2004062551A2 (en) 2003-01-10 2004-07-29 Ablynx N.V. RECOMBINANT VHH SINGLE DOMAIN ANTIBODY FROM CAMELIDAE AGAINST VON WILLEBRAND FACTOR (vWF) OR AGAINST COLLAGEN
WO2005044858A1 (en) 2003-11-07 2005-05-19 Ablynx N.V. Camelidae single domain antibodies vhh directed against epidermal growth factor receptor and uses therefor
WO2006040153A2 (en) 2004-10-13 2006-04-20 Ablynx N.V. Single domain camelide anti -amyloid beta antibodies and polypeptides comprising the same for the treatment and diagnosis of degenarative neural diseases such as alzheimer's disease
WO2006079372A1 (en) 2005-01-31 2006-08-03 Ablynx N.V. Method for generating variable domain sequences of heavy chain antibodies
WO2006122825A2 (en) 2005-05-20 2006-11-23 Ablynx Nv Single domain vhh antibodies against von willebrand factor
WO2006122786A2 (en) 2005-05-18 2006-11-23 Ablynx Nv Improved nanobodies™ against tumor necrosis factor-alpha
WO2016134333A1 (en) 2015-02-19 2016-08-25 Compugen Ltd. Anti-pvrig antibodies and methods of use
EP2101823B1 (en) 2007-01-09 2016-11-23 CureVac AG Rna-coded antibody
WO2018017864A2 (en) * 2016-07-20 2018-01-25 Oncomed Pharmaceuticals, Inc. Pvrig-binding agents and uses thereof
CN110088132A (zh) * 2016-08-17 2019-08-02 康姆普根有限公司 抗tigit抗体，抗pvrig抗体及其组合
WO2020018879A1 (en) * 2018-07-20 2020-01-23 Surface Oncology, Inc. Anti-cd112r compositions and methods
CN111995681A (zh) * 2020-05-09 2020-11-27 华博生物医药技术（上海）有限公司 抗tigit的抗体、其制备方法和应用
CN113039202A (zh) * 2018-06-01 2021-06-25 康姆普根有限公司 抗pvrig/抗tigit双特异性抗体和使用方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119487079A (zh) * 2022-06-08 2025-02-18 山东先声生物制药有限公司 一种pvrig/tigit双特异性抗体药物组合物及其用途

Patent Citations (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
WO1994004678A1 (en) 1992-08-21 1994-03-03 Casterman Cecile Immunoglobulins devoid of light chains
WO1994025591A1 (en) 1993-04-29 1994-11-10 Unilever N.V. PRODUCTION OF ANTIBODIES OR (FUNCTIONALIZED) FRAGMENTS THEREOF DERIVED FROM HEAVY CHAIN IMMUNOGLOBULINS OF $i(CAMELIDAE)
WO1995004079A1 (fr) 1993-08-02 1995-02-09 Raymond Hamers Vecteur recombinant contenant une sequence d'un gene de lipoproteine pour l'expression de sequences de nucleotides
WO1996034103A1 (en) 1995-04-25 1996-10-31 Vrije Universiteit Brussel Variable fragments of immunoglobulins - use for therapeutic or veterinary purposes
WO1997049805A2 (en) 1996-06-27 1997-12-31 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Recognition molecules interacting specifically with the active site or cleft of a target molecule
WO1999037681A2 (en) 1998-01-26 1999-07-29 Unilever Plc Method for producing antibody fragments
WO2000040968A1 (en) 1999-01-05 2000-07-13 Unilever Plc Binding of antibody fragments to solid supports
WO2000043507A1 (en) 1999-01-19 2000-07-27 Unilever Plc Method for producing antibody fragments
WO2000065057A1 (en) 1999-04-22 2000-11-02 Unilever Plc Inhibition of viral infection using monovalent antigen-binding proteins
WO2001021817A1 (en) 1999-09-24 2001-03-29 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Recombinant phages capable of entering host cells via specific interaction with an artificial receptor
WO2001040310A2 (en) 1999-11-29 2001-06-07 Unilever Plc Immobilisation of proteins using a polypeptide segment
WO2001044301A1 (en) 1999-11-29 2001-06-21 Unilever Plc Immobilized single domain antigen-binding molecules
EP1134231A1 (en) 2000-03-14 2001-09-19 Unilever N.V. Antibody heavy chain variable domains against human dietary enzymes, and their uses
WO2001090190A2 (en) 2000-05-26 2001-11-29 National Research Council Of Canada Single-domain antigen-binding antibody fragments derived from llama antibodies
WO2002048193A2 (en) 2000-12-13 2002-06-20 Unilever N.V. Camelidae antibody arrays
WO2003025020A1 (fr) 2001-09-13 2003-03-27 Institute For Antibodies Co., Ltd. Procede pour creer une banque d'anticorps de chameaux
WO2003035694A2 (en) 2001-10-24 2003-05-01 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Functional heavy chain antibodies, fragments thereof, library thereof and methods of production thereof
WO2003050531A2 (en) 2001-12-11 2003-06-19 Algonomics N.V. Method for displaying loops from immunoglobulin domains in different contexts
WO2003054016A2 (en) 2001-12-21 2003-07-03 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Method for cloning of variable domain sequences
WO2003055527A2 (en) 2002-01-03 2003-07-10 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Immunoconjugates useful for treatment of tumours
WO2004041865A2 (en) 2002-11-08 2004-05-21 Ablynx N.V. Stabilized single domain antibodies
WO2004041862A2 (en) 2002-11-08 2004-05-21 Ablynx N.V. Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor
WO2004041863A2 (en) 2002-11-08 2004-05-21 Ablynx N.V. Single domain antibodies directed against interferon- gamma and uses therefor
WO2004041867A2 (en) 2002-11-08 2004-05-21 Ablynx N.V. Camelidae antibodies against imminoglobulin e and use thereof for the treatment of allergic disorders
WO2004062551A2 (en) 2003-01-10 2004-07-29 Ablynx N.V. RECOMBINANT VHH SINGLE DOMAIN ANTIBODY FROM CAMELIDAE AGAINST VON WILLEBRAND FACTOR (vWF) OR AGAINST COLLAGEN
WO2005044858A1 (en) 2003-11-07 2005-05-19 Ablynx N.V. Camelidae single domain antibodies vhh directed against epidermal growth factor receptor and uses therefor
WO2006040153A2 (en) 2004-10-13 2006-04-20 Ablynx N.V. Single domain camelide anti -amyloid beta antibodies and polypeptides comprising the same for the treatment and diagnosis of degenarative neural diseases such as alzheimer's disease
WO2006079372A1 (en) 2005-01-31 2006-08-03 Ablynx N.V. Method for generating variable domain sequences of heavy chain antibodies
WO2006122787A1 (en) 2005-05-18 2006-11-23 Ablynx Nv Serum albumin binding proteins
WO2006122786A2 (en) 2005-05-18 2006-11-23 Ablynx Nv Improved nanobodies™ against tumor necrosis factor-alpha
WO2006122825A2 (en) 2005-05-20 2006-11-23 Ablynx Nv Single domain vhh antibodies against von willebrand factor
EP2101823B1 (en) 2007-01-09 2016-11-23 CureVac AG Rna-coded antibody
WO2016134333A1 (en) 2015-02-19 2016-08-25 Compugen Ltd. Anti-pvrig antibodies and methods of use
US20180244774A1 (en) 2015-02-19 2018-08-30 Compugen Ltd. Anti-pvrig antibodies and methods of use
WO2018017864A2 (en) * 2016-07-20 2018-01-25 Oncomed Pharmaceuticals, Inc. Pvrig-binding agents and uses thereof
CN110088132A (zh) * 2016-08-17 2019-08-02 康姆普根有限公司 抗tigit抗体，抗pvrig抗体及其组合
CN112274637A (zh) * 2016-08-17 2021-01-29 康姆普根有限公司 抗tigit抗体、抗pvrig抗体及其组合
CN113039202A (zh) * 2018-06-01 2021-06-25 康姆普根有限公司 抗pvrig/抗tigit双特异性抗体和使用方法
WO2020018879A1 (en) * 2018-07-20 2020-01-23 Surface Oncology, Inc. Anti-cd112r compositions and methods
US20200040081A1 (en) 2018-07-20 2020-02-06 Surface Oncology, Inc. Anti-CD112R Compositions and Methods
CN111995681A (zh) * 2020-05-09 2020-11-27 华博生物医药技术（上海）有限公司 抗tigit的抗体、其制备方法和应用

Non-Patent Citations (31)

* Cited by examiner, † Cited by third party
Title
"NCBI", Database accession no. NP_001036189.1
ABHINANDANMARTIN, MOL. IMMUNOL, vol. 45, 2008, pages 3832 - 3839
ALFTHAN, PROTEIN ENG, vol. 8, 1995, pages 725 - 731
AL-LAZIKANI B. ET AL., J. MOL. BIOL., vol. 273, 1997, pages 927 - 948
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 10
ALTSCHUL ET AL.: "25", NUCLEIC ACIDS RES., 1997, pages 3389 - 3402
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426
CHOI ET AL., EUR. J. IMMUNOL., vol. 31, 2001, pages 94 - 106
E. MEYERSW. MILLER, CABIOS, vol. 4, 1989, pages 11 - 17
HAMERS-CASTERMAN ET AL., NATURE, vol. 363, 1993, pages 446 - 8
HOLLIGER P ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 6444 - 6448
HONEGGERPLUCKTHUN, J. MOL. BIOL., vol. 309, 2001, pages 657 - 670
HU ET AL., CANCER RES, vol. 56, 1996, pages 3055 - 3061
HUSTON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 5879 - 5883
KABAT ET AL., J. BIOL. CHEM., vol. 252, 1977, pages 6609 - 6616
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
KINDT ET AL.: "Kuby Immunology", 2007, W.H. FREEMAN AND CO, pages: 91
KIPRIYANOV ET AL., J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
KORNDORFER ET AL., PROTEINS: STRUCTURE, FUNCTION, AND BIOINFORMATICS, vol. 53, no. 1, 2003, pages 121 - 129
LEFRANC ET AL., DEV.COMPARAT.IMMUNOL, vol. 27, 2003, pages 55 - 77
LEFRANC M.P. ET AL., DEV. COMP. IMMUNOL., vol. 27, 2003, pages 55 - 77
MACCALLUM ET AL., J. MOL BIOL., vol. 262, 1996, pages 732 - 745
MORRISON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 6851 6855
MUYLDERMANS, REVIEWS IN MOLECULAR BIOTECHNOLOGY, vol. 74, 2001, pages 277 - 302
NEEDLEMAWUNSCH, J.MOL.BIOL., vol. 48, 1970, pages 444 - 453
POLJAK R. J. ET AL., STRUCTURE, vol. 113, 1994, pages 1121 - 1123
ROQUE ET AL., BIOTECHNOL. PROG, vol. 20, 2004, pages 639 - 654
See also references of EP4378954A4
STADLER ET AL., NATURE MEDICINE, 12 June 2017 (2017-06-12)
WUL, CARE, vol. 17, 2013, pages R8
XU FENG; SUNDERLAND ALEXANDER; ZHOU YUE; SCHULICK RICHARD D.; EDIL BARISH H.; ZHU YUWEN: "Blockade of CD112R and TIGIT signaling sensitizes human natural killer cell functions", CANCER IMMUNOLOGY IMMUNOTHERAPY, SPRINGER, BERLIN/HEIDELBERG, vol. 66, no. 10, 16 June 2017 (2017-06-16), Berlin/Heidelberg , pages 1367 - 1375, XP036330843, ISSN: 0340-7004, DOI: 10.1007/s00262-017-2031-x *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023236980A1 (zh) * 2022-06-08 2023-12-14 山东先声生物制药有限公司 一种pvrig/tigit双特异性抗体药物组合物及其用途
WO2024153182A1 (zh) * 2023-01-19 2024-07-25 山东先声生物制药有限公司 抗pvrig/抗tigit双特异性抗体在治疗恶性肿瘤中的用途
WO2024213097A1 (zh) * 2023-04-14 2024-10-17 山东先声生物制药有限公司 一种靶向人cd16和肿瘤抗原的多特异性抗体
WO2024251160A1 (en) * 2023-06-06 2024-12-12 Wuxi Biologics (Shanghai) Co., Ltd. Anti-pvrig antibodies and uses thereof
WO2024251161A1 (en) * 2023-06-06 2024-12-12 Wuxi Biologics (Shanghai) Co., Ltd. Anti-tigit x pvrig antibodies and uses thereof
WO2025033553A1 (ja) * 2023-08-10 2025-02-13 国立大学法人 東京大学 新規なｈｖｅｍ結合タンパク質およびそれを含む医薬組成物
WO2025061165A1 (en) * 2023-09-20 2025-03-27 Laekna Therapeutics Shanghai Co., Ltd. Antibodies against pvrig or tigit, bispecific antibodies constructed therefrom, methods for their preparation and uses thereof

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