WO2023221935A1 - 结合pd-l1和cldn18.2的抗体及其用途 - Google Patents

结合pd-l1和cldn18.2的抗体及其用途 Download PDF

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WO2023221935A1
WO2023221935A1 PCT/CN2023/094266 CN2023094266W WO2023221935A1 WO 2023221935 A1 WO2023221935 A1 WO 2023221935A1 CN 2023094266 W CN2023094266 W CN 2023094266W WO 2023221935 A1 WO2023221935 A1 WO 2023221935A1
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amino acid
acid sequence
seq
antigen
heavy chain
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PCT/CN2023/094266
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French (fr)
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钱雪明
顾怡
滕菲
李红俊
郭欢欢
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苏州创胜医药集团有限公司
创胜集团医药有限公司
创胜医药有限公司
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Publication of WO2023221935A1 publication Critical patent/WO2023221935A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts

Definitions

  • the invention provides an antibody. This antibody can specifically bind to human CLDN18.2 and human PD-L1.
  • the invention further provides nucleic acid molecules encoding the antibodies, expression vectors for expressing the antibodies, host cells and preparation methods thereof.
  • the invention also provides diagnostic and therapeutic methods using the antibodies of the invention.
  • PD-1 Programmed death 1
  • PD-L1 and PD-L2 are key co-inhibitory molecules in the regulation of T cell-mediated immune responses.
  • PD-1 is a type I membrane protein with a single extracellular immunoglobulin superfamily (IgSF) V-set domain expressed on the surface of activated T cells in peripheral tissues (Zhang X et al., "Immunity", 2004, 20(3): 337-347).
  • IgSF immunoglobulin superfamily
  • PD-L1 and PD-L2 are commonly expressed on dendritic cells and macrophages, and their extracellular domains consist of a membrane-distal IgSF V-set domain and a membrane-proximal IgSF C-set domain (Latchman Y et al., "Nature Immunology", 2001, 2(3):261-268)
  • PD-L1 also negatively regulates T cell function by interacting with another receptor, B7.1 (also known as B7-1 or CD80).
  • B7.1 also known as B7-1 or CD80.
  • PD-L1 is frequently overexpressed in different tumors and its interaction with PD-1 on T cells enables cancer cells to evade T cell-mediated immune responses. Therefore, blocking the PD-1/PD-L1 interaction can restore T cell activation and anti-tumor responses.
  • an association has been shown between PD-L1 expression by tumor cells and/or infiltrating immune cells and clinical response to PD-1/PD-L1 targeted therapies, this association is not perfect. Only a small number of PD-L1-positive tumors respond to these treatments, while some PD-L1-negative tumors also respond to these treatments. Therefore, anti-PD-L1 antibodies still face great challenges in having high affinity, high therapeutic efficacy and low side effects at the same time.
  • Claudin is a key structural and functional component of epithelial tight junctions, where it regulates cell-cell permeability, maintains ion homeostasis, and supports cell adhesion and polarity.
  • Tight junction proteins are 22-27 kDa tetraspan transmembrane proteins that multimerize within or across cell membranes to form a protective barrier.
  • the 24 tight junction proteins that have been reported differ due to their tissue localization specificity and due to their interactions with other proteins.
  • Claudin 18 was originally identified as a target gene of the transcription factor T/EBP/NKX2.1. Consistent with its homology to other tight junction protein family members, CLDN18 was confirmed to localize to tight junctions in mouse and human cells. CLDN18 has been shown to encode two isoforms resulting from alternative splicing: CLDN18.1, which is expressed specifically in normal lungs, and CLDN18.2, which is expressed in differentiated cells of the gastric mucosa.
  • CLDN18.2 is a 261 amino acid protein with two extracellular loops and shares 92% sequence identity with CLDN18.1. Unlike the second extracellular loop, CLDN18.2's The first extracellular loop differs from CLDN18.1 by eight amino acids. CLDN18.2 has more limited homology with other family members, sharing 29%-34% overall identity with CLDN1, CLDN6, and CLDN7.
  • CLDN18.2 is expressed in several tumor types, including gastric, pancreatic, esophageal, mucinous ovarian, and non-small cell lung cancer.
  • CLDN18.2 expression in gastric cancer includes the invasive front and metastatic sites, but the absolute levels of CLDN18 have been reported to be reduced in these settings.
  • the expression of CLDN18.2 in multiple tumor types, in which normal tissue expression is primarily restricted to differentiated cells in the stomach, has led to the consideration of CLDN18.2 as a therapeutic target in gastric cancer and other indications.
  • the invention provides a novel anti-PD-L1 antibody and a bispecific antibody constructed using the novel anti-PD-L1 antibody and a CLDN18.2 antibody.
  • the anti-PD-L1 antibody of the present invention has a high binding affinity to human PD-L1 and is able to recognize human PD-L1.
  • anti-PD-L1 antibodies can specifically bind PD-L1 (eg, human PD-L1) with high affinity.
  • PD-L1 eg, human PD-L1
  • the anti-CLDN18.2/PD-L1 bispecific antibodies of the invention have one or more of the following properties and therefore have improved therapeutic efficacy:
  • Binding to (human) PD-L1, such as in PD-1 or PD-L1 positive cells e.g., tumor cells expressing PD-1 or PD-L1, e.g., high expression or low expression, especially low expression of PD -1 or PD-L1
  • PD-L1 positive cells e.g., tumor cells expressing PD-1 or PD-L1, e.g., high expression or low expression, especially low expression of PD -1 or PD-L1
  • ADCC effects ability to produce ADCC effects on target cells, such as inducing ADCC effects through binding to CLDN18.2 and/or binding to PD-L1;
  • T cells such as activating T cells to secrete interferons, such as IFN- ⁇ , such as inhibiting the activation of T cells by blocking PD-1;
  • the tumor inhibition rate is above 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%.
  • Bispecific antibody which contains a first binding specificity for (human) PD-L1 and a first binding specificity for (human) The second binding specificity of CLDN18.2, wherein the first antigen-binding region that specifically binds PD-L1 includes the 3 CDRs of the heavy chain variable region VH, HCDR1, HCDR2 and HCDR3, and the light chain variable region VL 3 CDRs, LCDR1, LCDR2 and LCDR3, among which
  • the HCDR1, HCDR2 and HCDR3 are the three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:17; and the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO:22
  • the VL contains three complementary determining regions LCDR1, LCDR2 and LCDR3.
  • the HCDR1, HCDR2 and HCDR3 are respectively HCDR1 as shown in SEQ ID NO:18, HCDR2 as shown in SEQ ID NO:19, and HCDR3 as shown in SEQ ID NO:20; and the LCDR1, LCDR2 and LCDR3 It is LCDR1 as shown in SEQ ID NO:23, LCDR2 as shown in SEQ ID NO:24 and LCDR3 as shown in SEQ ID NO:25.
  • the first antigen-binding region that specifically binds to PD-L1 includes a light chain variable region VL, wherein the light chain variable region
  • the first antigen-binding region that specifically binds PD-L1 includes a heavy chain variable region VH and a light chain variable region VL, wherein
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17 or 30 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identical thereto. , an amino acid sequence with 98% or 99% identity or consisting of the amino acid sequence; and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 22 or 31 or has at least 90%, 91%, or consist of an amino acid sequence that is 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.
  • the first antigen-binding region that specifically binds to PD-L1 includes a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL respectively comprises or consists of the amino acid sequence shown below:
  • the bispecific antibody of any one of technical solutions 1-6 which specifically binds to the second CLDN18.2
  • the antigen-binding region includes three CDRs of the heavy chain variable region VH, HCDR1, HCDR2 and HCDR3, and three CDRs of the light chain variable region VL, LCDR1, LCDR2 and LCDR3, among which
  • the HCDR1, HCDR2 and HCDR3 are the three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:1; and the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO:6
  • the VL contains three complementary determining regions LCDR1, LCDR2 and LCDR3.
  • HCDR1 shown in SEQ ID NO:2 Such as HCDR1 shown in SEQ ID NO:2, HCDR2 shown in SEQ ID NO:3, HCDR3 shown in SEQ ID NO:4; LCDR1 shown in SEQ ID NO:7, such as SEQ ID NO:8 LCDR2 shown and LCDR3 shown as SEQ ID NO:9.
  • the second antigen-binding region that specifically binds CLDN18.2 comprises a light chain variable region VL, wherein the light chain variable region
  • the second antigen-binding region that specifically binds to CLDN18.2 includes a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL respectively comprises or consists of the amino acid sequences shown below: SEQ ID NO: 1 and SEQ ID NO: 6.
  • the bispecific antibody according to any one of technical solutions 1-11, wherein the first antigen-binding region that specifically binds to PD-L1 and/or the second antigen-binding region that specifically binds to CLDN18.2 further comprises The heavy chain constant region HC or the Fc region derived from the HC, for example, the antibody heavy chain constant region HC is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1.
  • (i) Comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 13 or consisting of said amino acid sequence; or
  • (i) Comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 14 or consisting of said amino acid sequence; or
  • (i) Comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 16 or consisting of said amino acid sequence; or
  • the first antigen-binding region that specifically binds PD-L1 includes a heavy chain, wherein the heavy chain includes SEQ ID NO: 21 an amino acid sequence, or comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence, Or consisting of the amino acid sequence.
  • the first antigen-binding region that specifically binds to PD-L1 includes a light chain, wherein the light chain includes SEQ ID NO: 26 an amino acid sequence, or comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence, Or consisting of the amino acid sequence.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 21, or contains at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, An amino acid sequence that is 97%, 98% or 99% identical, or consists of said amino acid sequence; and the light chain includes the amino acid sequence of SEQ ID NO: 26, or includes an amino acid sequence that is at least 85%, 90% identical to said amino acid sequence. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences, or consisting of said amino acid sequences.
  • the second antigen-binding region that specifically binds CLDN18.2 includes a heavy chain, wherein the heavy chain includes SEQ ID NO:5 an amino acid sequence, or comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence, Or consisting of the amino acid sequence.
  • the second antigen-binding region that specifically binds CLDN18.2 comprises a light chain, wherein the light chain comprises SEQ ID NO: 10 ammonia amino acid sequence, or comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to said amino acid sequence , or consisting of the amino acid sequence.
  • the bispecific antibody of technical solution 21 or 22, wherein the second antigen-binding region that specifically binds CLDN18.2 includes a heavy chain and a light chain, wherein
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 5, or contains at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, An amino acid sequence that is 97%, 98% or 99% identical, or consists of said amino acid sequence; and the light chain includes the amino acid sequence of SEQ ID NO: 10, or includes an amino acid sequence that is at least 85%, 90% identical to said amino acid sequence. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences, or consisting of said amino acid sequences.
  • the bispecific antibody of technical solution 23, wherein the second antigen-binding region specifically binding to CLDN18.2 includes a heavy chain and a light chain, wherein the heavy chain includes the amino acid shown in SEQ ID NO: 5 sequence, or consisting of the amino acid sequence, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 10.
  • the bispecific antibody according to any one of technical solutions 1-24, wherein the first antigen-binding region that specifically binds to PD-L1 and/or the second antigen-binding region that specifically binds to CLDN18.2 is derived from Humanized or chimeric antibodies.
  • the bispecific antibody according to any one of technical solutions 1-25, wherein the first antigen-binding region that specifically binds to PD-L1 and/or the second antigen-binding region that specifically binds to CLDN18.2 is Antibody fragments selected from Fab, Fab', Fab'-SH, Fv, single chain antibodies (eg scFv), (Fab') 2 , single domain antibodies such as VHH, dAb (domain antibody) or linear antibodies, e.g.
  • the scFv comprises a disulfide bond stabilizing mutation, for example wherein the heavy chain variable region and/or the light chain variable region comprises a mutation that introduces a disulfide bond, for example the heavy chain variable region is comprised in A mutation in which the amino acid at position 44 is substituted with cysteine (Kabat numbering), and/or the light chain variable region includes a mutation in which the amino acid at position 100 is substituted with cysteine (Kabat numbering).
  • the scFv includes the amino acid sequence of SEQ ID NO: 27 or 29, or Comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence, or consists of said Amino acid sequence composition.
  • the bispecific antibody of technical solution 26, wherein the second antigen-binding region that specifically binds to CLDN18.2 is scV.
  • the scFv includes the amino acid sequence of SEQ ID NO: 11, or includes The amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence, or is composed of the amino acid sequence composition.
  • G4S Amino acid sequence
  • the bispecific antibody according to any one of technical solutions 1-29, wherein the first antigen-binding region that specifically binds to PD-L1 is a full-length antibody, and the first antigen-binding region that specifically binds to CLDN18.2
  • the second antigen-binding region is scFv; or the second antigen-binding region that specifically binds CLDN18.2 is a full-length antibody, and the The first antigen-binding region that specifically binds to PD-L1 is scFv.
  • the light chain contains VL-A-CL from N-terminus to C-terminus;
  • the heavy chain contains VH-A-CH-scFv from N-terminus to C-terminus;
  • scFv contains VH-B and VL-B;
  • VL-A is the light chain variable region VL that specifically binds to the first antigen-binding region of PD-L1
  • VH-A is the heavy chain variable region VH that specifically binds to the first antigen-binding region of PD-L1
  • VH -B is the heavy chain variable region VH that specifically binds to the second antigen-binding region of CLDN18.2
  • VL-B is the light chain variable region VL that specifically binds to the second antigen-binding region of CLDN18.2
  • VL- A is the light chain variable region VL that specifically binds to the second antigen-binding region of CLDN18.2
  • VH-A is the heavy chain variable region VH that specifically binds to the second antigen-binding region of CLDN18.2
  • VH-B is The heavy chain variable region VH that specifically binds to the first antigen-binding region of PD-L1
  • VL-B is the light chain variable region VL that specifically
  • CL is the light chain constant region
  • CH is the heavy chain constant region
  • scFv contains a linker or no linker between VH-B and VL-B;
  • CH and scFv contain a linker or do not contain a linker.
  • the bispecific antibody of technical solution 33 which contains two heavy chains and two light chains, for example, the heavy chains are the same and/or the light chains are the same.
  • the bispecific antibody according to any one of technical solutions 33-37, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 10 or 26, or has at least 85% or 90% similarity with the amino acid sequence. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences, or consisting of said amino acid sequences.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 32 or 34, or contains at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% similarity to the amino acid sequence. %, 97%, 98% or 99% identity to an amino acid sequence, or consisting of said amino acid sequence;
  • the light chain comprises the amino acid sequence of SEQ ID NO: 10, or contains at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence , an amino acid sequence that is 98% or 99% identical, or consists of said amino acid sequence;
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 33, or contains at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, An amino acid sequence that is 97%, 98% or 99% identical, or consists of said amino acid sequence;
  • the light chain includes the amino acid sequence of SEQ ID NO: 26, or includes at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence. , or consisting of an amino acid sequence that is 98% or 99% identical.
  • the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 32 or 34; and the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 10;
  • the heavy chain includes or consists of the amino acid sequence of SEQ ID NO: 33;
  • the light chain includes or consists of the amino acid sequence of SEQ ID NO: 26.
  • Expression vector which contains the nucleic acid molecule of technical solution 41.
  • the expression vector is pCDNA, such as pCDNA3.1.
  • Host cell which contains the nucleic acid molecule described in technical solution 41 or the expression vector described in technical solution 42.
  • the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as 293F cells. Or 293T cells or Expi-CHO cells.
  • a method for preparing the bispecific antibody according to any one of technical solutions 1 to 40 which method includes culturing the nucleic acid molecule comprising technical solution 41 or under conditions suitable for chain expression of the antibody.
  • Immunoconjugate which contains the bispecific antibody described in any one of technical solutions 1-40;
  • the immunoconjugate of technical solution 45 which also contains chemotherapeutic agents, cytokines, cytotoxic agents or small molecule drugs, purification parts such as magnetic beads, biotin, clearance modifiers (for example, polymers that extend half-life, such as PEG), chemotherapeutic agents, toxins, radioisotopes, lanthanides, detectable labels (e.g., luminescent labels, fluorescent labels, enzyme substrate labels), DNA alkylating agents, topoisomerase inhibitors, tubulin binding agents or other anticancer drugs.
  • chemotherapeutic agents cytokines, cytotoxic agents or small molecule drugs
  • purification parts such as magnetic beads, biotin, clearance modifiers (for example, polymers that extend half-life, such as PEG), chemotherapeutic agents, toxins, radioisotopes, lanthanides, detectable labels (e.g., luminescent labels, fluorescent labels, enzyme substrate labels), DNA alkylating agents, topoisomerase inhibitors, tubulin binding
  • compositions or drugs or preparations which comprise the bispecific antibody described in any one of technical solutions 1-40, or the immunoconjugate of technical solutions 45 or 46, and optionally pharmaceutical excipients.
  • Pharmaceutical combination product which contains the bispecific antibody according to any one of technical solutions 1-40, or the immunoconjugate of technical solutions 45 or 46, and one or more other therapeutic agents (such as chemotherapy Agents, anticancer drugs, immunotherapeutic agents, anti-angiogenic agents, targeted therapy therapeutic agents, cell therapy therapeutic agents, gene therapy therapeutic agents, hormone therapy therapeutic agents, antiviral agents, antibiotics, analgesics, antioxidants, metals chelating agents or cytokines).
  • therapeutic agents such as chemotherapy Agents, anticancer drugs, immunotherapeutic agents, anti-angiogenic agents, targeted therapy therapeutic agents, cell therapy therapeutic agents, gene therapy therapeutic agents, hormone therapy therapeutic agents, antiviral agents, antibiotics, analgesics, antioxidants, metals chelating agents or cytokines.
  • a method for preventing or treating cancer in a subject comprising administering to the subject an effective amount of the bispecific antibody according to any one of technical solutions 1-40, or the immunoconjugation of technical solutions 45 or 46 or the pharmaceutical composition or drug or preparation of technical solution 47; or the pharmaceutical combination product of technical solution 48.
  • PD-L1 protein is present on the cell surface of said cancer cell or tumor cell
  • PD-L1 protein is present on the cell surface of the cancer cell or tumor cell, and compared with a cancer or tumor that highly expresses PD-L1, there is a low level of PD on the cell surface of the cancer or tumor cell.
  • the cancer is a solid tumor, such as gastrointestinal tract tumor, biliary tract tumor or lung cancer, such as gastric cancer, esophageal cancer, pancreatic cancer, colorectal cancer, cholangiocarcinoma, gallbladder cancer cancer or lung cancer.
  • a solid tumor such as gastrointestinal tract tumor, biliary tract tumor or lung cancer, such as gastric cancer, esophageal cancer, pancreatic cancer, colorectal cancer, cholangiocarcinoma, gallbladder cancer cancer or lung cancer.
  • therapies such as treatment modalities (such as surgery or radiotherapy) and/or other therapeutic agents (such as chemotherapeutic agents, anti-cancer drugs, immunotherapy agent, anti-angiogenic agent, targeted therapy therapeutic agent, cell therapy therapeutic agent, gene therapy therapeutic agent, hormonal therapy therapeutic agent, antiviral agent, antibiotic, analgesic, antioxidant, metal chelator or cytokine) combined administration .
  • therapies such as treatment modalities (such as surgery or radiotherapy) and/or other therapeutic agents (such as chemotherapeutic agents, anti-cancer drugs, immunotherapy agent,
  • a method for detecting the presence of PD-L1 and/or CLDN18.2 in biological samples which includes
  • Figure 1 shows that the ADCC effect mediated by CLDN18.2 antibody in different cells can upregulate the expression of PD-L1 in cells;
  • Figure 2 shows the structural schematic diagram of three CLDN18.2 ⁇ PD-L1 bispecific antibodies.
  • Figure 3 shows the binding activity of three CLDN18.2 ⁇ PD-L1 bispecific antibodies and 18B10 antibodies to NUGC-4 cells;
  • Figure 4 shows the binding activities of three CLDN18.2 ⁇ PD-L1 bispecific antibodies and AM4B6 antibodies to PD-L1 positive cells;
  • Figure 5 shows the ADCC effect of CLDN18.2 ⁇ PD-L1 bispecific antibody and 18B10 antibody on NUGC-4;
  • Figure 6 shows the ADCC effect of CLDN18.2 ⁇ PD-L1 bispecific antibody and AM4B6 antibody on PD-L1 positive cells
  • Figure 7 shows the blocking of PD-1 and PD-L1 binding by CLDN18.2 ⁇ PD-L1 bispecific antibody and AM4B6 antibody;
  • Figure 8 shows the activation effect of CLDN18.2 ⁇ PD-L1 bispecific antibody, AM4B6 and 18B10 on PBMC;
  • Figure 9 shows the inhibitory effects of CLDN18.2 ⁇ PD-L1 bispecific antibodies, AM4B6 and 18B10 on tumor models.
  • the term “comprises” or “includes” means the inclusion of the stated element, integer or step, but not the exclusion of any other element, integer or step.
  • the term “comprises” or “includes” is used herein, it also encompasses a combination of the stated elements, integers, or steps unless otherwise indicated.
  • reference is made to an antibody variable region that "comprises” a particular sequence it is also intended to encompass antibody variable regions that consist of that particular sequence.
  • Claudin protein is the most important skeleton protein that determines the structure of tight junctions between cells, participates in adherens junctions, and plays an important role in the metastasis and invasion of tumor cells.
  • Claudin protein widely exists in mammalian epithelial and endothelial cells, and its distribution is mainly on the sides of epithelial cells and on the plasma membrane of basal cells. Different Claudin proteins have their own specific expression in different tissues.
  • the Claudin18 (CLDN18) gene is located at 3q22.3, has a molecular weight of 24kDa, contains 261 amino acid residues, and is a member of the Claudins superfamily.
  • CLDN18.2 has completely identical sequences in different species such as humans, mice, and macaques, while the CLDN18.2 protein homology between humans and mice reaches 84%, indicating that the CLDN18.2 protein sequence is extremely conserved.
  • CLDN18.2, or any variants and isoforms thereof can be isolated from cells or tissues in which they are naturally expressed, or recombinantly produced using techniques well known in the art and/or those described herein.
  • CLDN18.2 described herein is human CLDN18.2.
  • the term "anti-CLDN18.2 antibody”, “anti-CLDN18.2”, “CLDN18.2 antibody” or “antibody that binds CLDN18.2” or “antibody that specifically binds CLDN18.2” refers to such an antibody , the antibody is capable of binding (human) CLDN18.2 with sufficient affinity.
  • the (human) CLDN18.2 antibody binds (human) CLDN18.2 with high affinity in vitro or in vivo.
  • the (human) CLDN18.2 antibody does not bind CLDN18.1.
  • the (human) CLDN18.2 antibody binds to cells expressing CLDN18.2 but not to cells expressing CLDN18.1.
  • the binding is measured, for example, by radioimmunoassay (RIA), biofilm thin layer interferometry (BLI), MSD assay, or surface plasmon resonance (SPR), or flow cytometry.
  • RIA radioimmunoassay
  • BLI biofilm thin layer interferometry
  • the terms "programmed cell death 1 ligand 1", “PD-L1”, “programmed death ligand 1", “cluster of differentiation 274", “CD274" or “B7 homolog 1” refer to Any native PD-L1 from any vertebrate source, including mammals, such as primates (eg, humans) and rodents (eg, mice and rats).
  • the term encompasses "full-length", unprocessed PD-L1 as well as any form of PD-L1 resulting from processing in the cell.
  • PD-L1 can exist as a transmembrane protein or as a soluble protein.
  • the term also encompasses naturally occurring variants of PD-L1, such as splice variants or allelic variants.
  • the basic structure of PD-L1 includes four domains: extracellular Ig-like V-type domain and Ig-like C2-type domain, transmembrane domain and cytoplasmic domain.
  • the PD-L1 described herein is human PD-L1. Additional information on the human PD-L1 gene, including the genomic DNA sequence, can be found under NCBI Gene ID No. 29126. The amino acid sequence of an exemplary full-length human PD-L1 protein can be found, for example, under NCBI accession number NP_001254653 or UniProt accession number Q9NZQ7.
  • the terms “anti-PD-L1 antibody”, “anti-PD-L1”, “PD-L1 antibody” or “antibody that binds PD-L1” or “antibody that specifically binds to PD-L1” refer to such antibodies , the antibody can bind to PD-L1 protein or fragments thereof with sufficient affinity.
  • the (human) PD-L1 antibody binds (human) PD-L1 with high affinity in vitro or in vivo.
  • the (human) PD-L1 antibody does not bind non-PD-L1, such as PD-L2.
  • the (human) PD-L1 antibody Binds to cells expressing PD-L1.
  • the binding is measured, for example, by radioimmunoassay (RIA), biofilm thin layer interferometry (BLI), MSD assay, or surface plasmon resonance (SPR), or flow cytometry.
  • the terms “whole antibody”, “full-length antibody”, “complete antibody” and “intact antibody” are used interchangeably herein to refer to a naturally occurring antibody containing at least two heavy chains (H) interconnected by disulfide bonds and two light chains (L) glycoproteins.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (herein abbreviated as VL) and a light chain constant region (herein abbreviated as CL).
  • the light chain constant region consists of one domain, CL.
  • VH and VL regions can be further divided into hypervariable regions (called complementarity determining regions (CDR)), with more conservative regions (called framework regions (FR)) interposed.
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL consists of three CDRs and four
  • the FR composition is arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the constant region does not directly participate in the binding of antibodies to antigens, but shows a variety of effector functions.
  • the antibody heavy chain constant region includes CH1, CL and Fc domains, among which CH2, CH3 and optional CH4 domains constitute the Fc domain, which can be selected according to the expected function of the antibody molecule.
  • the constant region may be an immunoglobulin constant domain of IgA, IgD, IgE, IgG or IgM, especially a human IgG, for example, a constant domain of a human IgG1, IgG2, IgG3 or IgG4, preferably a constant domain of a human IgG1 .
  • the Fc region of an antibody may comprise CH2 and CH3 domains from IgG1. Immunoglobulin constant regions may have native or variant sequences.
  • the antibody heavy chain constant region HC of the invention is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1. In some preferred embodiments, the antibody heavy chain constant region HC of the invention
  • (iii) Comprises one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence of SEQ ID NO: 13 ( Preferably amino acid substitutions, more preferably amino acid conservative substitutions) amino acid sequences or consist of said amino acid sequences.
  • the antibody light chain constant region LC of the invention is a Lambda or Kappa light chain constant region. In some embodiments, the antibody light chain constant region LC of the invention
  • (iii) Comprises one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence of SEQ ID NO: 16 ( Preferably amino acid substitution, more preferably amino acid conservative substitution) amino acid sequence or by the amino acid sequence composition.
  • antibody fragment includes a portion of an intact antibody.
  • the antibody fragment is an antigen-binding fragment.
  • antigen-binding fragment is a portion or segment of an intact or full-length antibody that has fewer amino acid residues than an intact or full-length antibody and is capable of binding to an antigen or interacting with an intact antibody (i.e., an intact antibody from which the antigen-binding fragment is derived). ) competes for binding to the antigen.
  • Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab') 2 , Fd, Fv fragments, disulfide bond stabilized Fv fragments (dsFv), (dsFv) 2 , bispecific dsFv (dsFv- dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies, camelized single domain antibodies , Nanobodies, domain antibodies and bivalent domain antibodies.
  • the "Fab” of an antibody refers to a part of an antibody composed of a single light chain (including variable region and constant region) and a single heavy chain variable region and the first constant region bound by a disulfide bond.
  • Fab' refers to a Fab fragment that includes a portion of the hinge region.
  • F(ab') 2 refers to the dimer of Fab'.
  • the Fv fragment consists of the VL and VH domains of a single arm of the antibody.
  • the two domains of the Fv fragment, VL and VH are encoded by independent genes, using recombinant methods, they can be linked by a synthetic linker peptide that allows the two domains to be produced as a single protein chain, as described
  • the VL and VH regions in a single protein chain are paired to form a single-chain Fv (scFv).
  • the antibody fragments can be obtained by chemical methods, recombinant DNA methods or protease digestion methods.
  • scFv is used herein to refer to a single chain antibody fragment comprising a heavy chain variable domain VH and a light chain variable domain VL connected by a linker, where VH and VL pair to form an antigen binding site.
  • a “complementarity determining region” or “CDR region” or “CDR” is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen-contacting residues ( "antigen contact point”) region.
  • CDRs are mainly responsible for binding to antigenic epitopes.
  • the CDRs of the heavy and light chains are often referred to as CDR1, CDR2, and CDR3 and are numbered sequentially starting from the N-terminus.
  • the CDRs located within the variable domain of the antibody heavy chain are termed HCDR1, HCDR2 and HCDR3, while the CDRs located within the variable domain of the antibody light chain are termed LCDR1, LCDR2 and LCDR3.
  • each CDR in a given light chain variable region or heavy chain variable region amino acid sequence can be determined using any one or a combination of many well-known antibody CDR assignment schemes, including For example: Chothia based on the three-dimensional structure of antibodies and the topology of CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., U.S.
  • CDR or “CDR sequence” encompasses CDR sequences determined in any of the above ways. CDRs can also be determined based on having the same Kabat number position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention). Unless otherwise stated, in the present invention, when referring to residue positions in an antibody variable region (including heavy chain variable region residues and light chain variable region residues), it means that according to Kabat et al., Numbering positions in the Kabat numbering system in Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).
  • HCDR and LCDR in the antibodies of the invention are determined according to the Kabat protocol.
  • the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment schemes may be different. That is, the CDR sequences of the same antibody variable region defined under different assignment schemes are different. Therefore, when referring to an antibody defined by a specific CDR sequence as defined in the invention, the scope of said antibody also encompasses antibodies whose variable region sequences comprise said specific CDR sequence but which differ due to the application of different protocols (e.g. Different assignment scheme rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.
  • Fc domain or "Fc region” or “Fc fragment” is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a native immunoglobulin "Fc domain” contains two or three constant domains, namely a CH2 domain, a CH3 domain and an optional CH4 domain.
  • the immunoglobulin Fc domain contains the second and third constant domains (CH2 domain and CH3 domain) derived from the heavy chain of antibodies of the IgG, IgA and IgD classes; or contains derived from IgM and the second, third and fourth constant domains (CH2 domain, CH3 domain and CH4 domain) of the heavy chain of IgE class antibodies.
  • amino acid residue numbering in the Fc region or heavy chain constant region is according to, e.g., Edelman, GM et al., Proc. Natl. Acad.
  • Fc domain or “Fc region” or “Fc fragment” does not include the heavy chain variable region VH and the light chain variable region VL as well as the heavy chain constant region CH1 and the light chain constant region of an immunoglobulin. CL, but in some cases may include a hinge region N-terminal to the heavy chain constant region.
  • the heavy chain constant region Fc suitable for use in the present invention is from an antibody heavy chain constant region, such as a constant region from human IgG1, IgG2, IgG3 or IgG4, preferably from an IgG1 constant region.
  • Fc comprises the amino acid sequence set forth in SEQ ID NO: 14 or 15, or comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, An amino acid sequence that is 97%, 98% or 99% identical, or consists of said amino acid sequence.
  • Fc fragments dimerize to form Fc dimers.
  • the two Fc fragments in the Fc dimer are identical.
  • Fc fragments heterodimerize into Fc heterodimers.
  • the Fc fragment may contain mutations for heterodimerization, such as knot-in mutations.
  • effector function refers to cell-mediated or complement-mediated cellular functions resulting from interactions between the Fc region of an antibody and the C1q complement protein or Fc receptor (FcR) on immune cells.
  • Toxic effects include, but are not limited to, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) effects.
  • ADCC is a cell-mediated immune defense mechanism whereby effector cells of the immune system actively lyse target cells whose membrane surface antigens have been bound by specific antibodies.
  • ADCC requires immune effector cells, which are generally known to be natural killer (NK) cells that typically interact with IgG antibodies.
  • ADCC can also be mediated by macrophages, neutrophils, and eosinophils.
  • ADCC involves activation of effector cells expressing Fc receptors by antibodies expressing the Fc portion.
  • Fc receptors For example, the most common Fc receptor on the surface of NK cells is called CD16 or Fc ⁇ RIII. Once the Fc receptor binds to the Fc region of IgG, NK cells release cytotoxic factors that cause target cell death.
  • the eosinophil Fc receptor FceRI
  • both ADCC and CDC can be modulated by Fc isoform engineering, Fc gene mutation, or Fc glycosylation profile modification.
  • target refers to the object to which the binding molecule is bound.
  • the target can be an antigen, a ligand, or a receptor.
  • antigen refers to a molecule that triggers an immune response. This immune response may involve antibody production or activation of specific immune cells, or both.
  • antigens can be derived from recombinant or genomic DNA.
  • epipe refers to the portion of an antigen that specifically interacts with an antibody molecule.
  • target binding region refers to the portion of a multispecific binding molecule, such as a bispecific binding molecule, that binds a specific target or antigen. In the case of multispecific antibodies or bispecific antibodies, the "target binding region” is also called the “antigen binding region”.
  • the term “monospecific” antibody refers to an antibody having one or more binding sites, each of which binds to the same epitope of the same antigen.
  • the term “multispecific” antibody refers to an antibody having at least two antigen-binding sites, wherein at least one antigen-binding site binds a different antigenic epitope relative to the remaining antigen-binding sites, e.g., the same Different epitopes on an antigen or different epitopes on different antigens.
  • the invention provides bispecific antibodies against PD-L1 and CLDN18.2.
  • bispecific antibody refers to an antibody with two binding specificities, which includes a first antigen binding region and a second antigen-binding region, wherein the first antigen-binding region binds one antigen or epitope and the second antigen-binding region binds another antigen or another epitope. Therefore, bispecific antibodies according to the invention comprise specificity for two different antigens, or for two different epitopes of one antigen.
  • Bispecific antibody formats include IgG-like bispecific antibodies.
  • an "IgG-like bispecific antibody” refers to a bispecific antibody that contains an Fc dimer.
  • an IgG-like bispecific antibody type contains two Fab regions and two Fc regions, and the heavy and light chains of each Fab can be derived from a separate monoclonal antibody.
  • IgG-like bispecific antibodies include full-length antibodies as well as other antibody fragments, such as scFv, linked to the full-length antibody.
  • Bispecific antibodies of the invention can be prepared using bispecific antibody formats or techniques known in the art.
  • bivalent, trivalent, and tetravalent indicate the presence of two, three, or four binding sites, respectively, in the antibody construct.
  • Bispecific antibodies according to the invention are at least bivalent and may be multivalent, for example bivalent, trivalent, tetravalent or hexavalent. In some embodiments, the bispecific antibodies of the invention are tetravalent.
  • chimeric refers to an antibody or antigen binding having a portion of a heavy chain and/or light chain derived from one species and the remainder of the heavy chain and/or light chain derived from a different species. fragment.
  • a chimeric antibody may include a constant region derived from a human and a variable region derived from a non-human animal (eg, derived from a mouse).
  • the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.
  • humanized as used herein is meant to include antibodies or antigen-binding fragments that include CDRs derived from non-human animals, FR regions derived from humans, and constant regions derived from humans, when applicable.
  • affinity refers to the strength of the non-covalent interaction between an immunoglobulin molecule (ie, an antibody) or a fragment thereof and an antigen.
  • the term "anti,” “binding,” or “specifically binds” means that the binding is selective for the target or antigen and can be distinguished from undesired or nonspecific interactions.
  • the ability of a binding site to bind to a specific target or antigen can be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or biofilm thin layers Interferometry or MSD measurement or surface plasmon resonance (SPR) measurement.
  • Percent identity (%) refers to a candidate sequence after aligning it with the specific amino acid sequence shown in this specification and introducing gaps if necessary to achieve the maximum percent sequence identity, and without taking into account any When conservative substitutions are included as part of sequence identity, the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of the specific amino acid sequence shown in this specification.
  • the invention contemplates variants of the antibody molecules of the invention that have a substantial degree of identity, e.g., at least 80% identity, with respect to the antibody molecules and their sequences specifically disclosed herein. , 85%, 90%, 95%, 97%, 98% or 99% or higher.
  • the variants may comprise conservative changes, or be conservatively modified variants.
  • conservative changes include substitutions, deletions, or additions to the polypeptide sequence that do not substantially alter the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in the replacement of an amino acid with a chemically similar amino acid. It is well known in the art to provide conservative substitution tables for functionally similar amino acids.
  • the term "conservative sequence change" is used to refer to amino acid modifications that do not significantly affect or alter the target antigen-binding characteristics of the antibody molecule or binding protein molecule of the invention containing the amino acid sequence.
  • the conservatively modified variant maintains a binding affinity for the target antigen of at least 80%, 85%, 90%, 95%, 98%, 99% or higher, such as 100-110% or higher, relative to the parent antibody or binding protein.
  • linker refers to any molecule that enables direct connection of different portions of a bispecific binding molecule.
  • linkers that establish covalent connections between different molecular moieties include peptide linkers and non-protein polymers including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene or polyethylene glycol, polypropylene glycol. copolymer.
  • the linker is a peptide linker (also known as a "linker peptide"), which refers to a short sequence of amino acids, such as glycine (G) and/or serine (S), alone or in combination and/or a threonine residue (T), or from the hinge region of an immunoglobulin, used to link the amino acid sequence of the first part of the binding molecule to the second part of the binding molecule.
  • a peptide linker can connect a first target binding region of a binding molecule to a second target binding region.
  • a peptide linker can also connect one part of the antibody to another part of the antibody, such as a light chain variable region to a heavy chain variable region.
  • the peptide linker is of a length sufficient to connect the two entities in a manner such that they maintain their conformation relative to each other so as not to interfere with the desired activity.
  • the linking peptide is 5-50 amino acids in length, for example, 10, 15, 20, 25, 30 amino acids in length.
  • the linking peptide comprises the amino acid sequences (GS)n, (GGS)n, (GSGGS)n, (GGGGS)n, (GGGS)n, and (GGGGS)nG, where n is an integer equal to or greater than 1 , for example, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.
  • the linker is, for example, as shown in SEQ ID NO: 28.
  • host cell refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include primary transformed cells and progeny derived therefrom.
  • a host cell is any type of cell system that can be used to produce the antibody molecules of the invention, including eukaryotic cells, eg, mammalian cells, insect cells, yeast cells; and prokaryotic cells, eg, E. coli cells.
  • Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissue or animal tissue.
  • vector when used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced.
  • expression vector refers to a vector comprising a recombinant polynucleotide containing expression control sequences operably linked to the nucleotide sequence to be expressed.
  • the expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, viruses and adeno-associated viruses).
  • cosmids e.g., naked or contained in liposomes
  • viruses e.g., lentiviruses, retroviruses, adenoviruses, viruses and adeno-associated viruses.
  • composition refers to a composition that is in a form effective to permit the biological activity of the active ingredients contained therein and does not contain additional ingredients that would be unacceptable toxicities to the subject to whom the composition is administered. ingredients.
  • pharmaceutically acceptable excipient refers to a diluent, adjuvant (e.g. Freund's adjuvant (complete and incomplete)), excipients, carriers or stabilizers, etc.
  • adjuvant e.g. Freund's adjuvant (complete and incomplete)
  • excipients e.g., carriers or stabilizers, etc.
  • therapeutic agent encompasses any substance that is effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents (e.g., immunosuppressive agents ), chemotherapeutic agents, anticancer agents, immunotherapeutic agents, anti-angiogenic agents, targeted therapies, cell therapy therapeutic agents, gene therapy therapeutic agents, hormone therapy therapeutic agents, antiviral agents, antibiotics, analgesics, antioxidants , metal chelators or cytokines.
  • chemotherapeutic agents e.g., cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents (e.g., immunosuppressive agents ), chemotherapeutic agents, anticancer agents, immunotherapeutic agents, anti-angiogenic agents, targeted therapies, cell therapy therapeutic agents, gene therapy therapeutic agents, hormone therapy therapeutic agents, antiviral agents, antibiotics, analgesics, antioxidants , metal chelators or cytokines.
  • small molecule drugs refers to low molecular weight organic compounds capable of modulating biological processes.
  • Small molecules are defined as molecules with a molecular weight of less than 10 kD, usually less than 2 kD and preferably less than 1 kD.
  • Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimetics. As therapeutic agents, small molecules can be more permeable to cells, less susceptible to degradation, and less likely to elicit an immune response than larger molecules.
  • immunomodulator refers to natural or synthetic agents or drugs that inhibit or modulate immune responses.
  • the immune response can be a humoral response or a cellular response.
  • Immunomodulators include immunosuppressants.
  • immune modulators of the invention include immune checkpoint inhibitors or immune checkpoint agonists.
  • immunotherapy refers to a type of therapy that stimulates the immune system to fight diseases such as cancer or generally strengthens the immune system.
  • immunotherapy therapeutics include, but are not limited to, checkpoint modulators, adoptive cell transfer, cytokines, oncolytic viruses, and therapeutic vaccines.
  • Targeted therapy is a type of therapy that acts on specific molecules associated with cancer, such as specific proteins that are present in cancer cells but not in normal cells or are more abundant in cancer cells, or that help Target molecules in the cancer microenvironment for cancer growth and survival.
  • Targeted therapy targets therapeutic agents to tumors, thereby sparing normal tissue from the effects of the therapeutic agent.
  • drug combination or combination product refers to non-fixed combination products or fixed combination products, including but not limited to pharmaceutical kits and pharmaceutical compositions.
  • non-fixed combination means that the active ingredients (e.g., (i) the immunoconjugates of the invention, and (ii) other therapeutic agents) are administered in separate entities simultaneously, without specific time limits, or at the same or different times. Administration to the patient is spaced, sequentially, wherein such administration provides prophylactically or therapeutically effective levels of two or more active agents in the patient.
  • fixed combination means that two or more active agents are administered simultaneously to a patient in the form of a single entity.
  • the dosages and/or time intervals of two or more active agents are preferably selected so that the combined use of the components produces an effect in treating the disease or condition that is greater than that achieved by either component alone.
  • Each component may be in a separate formulation form, and the formulation forms may be the same or different.
  • neoplastic refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
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  • cancer cancer
  • anti-tumor effect or “tumor inhibitory effect” or “tumor inhibitory effect” refers to a biological effect that can be demonstrated by a variety of means, including but not limited to, for example, reduction in tumor volume, reduction in tumor cell number, reduction in tumor cell proliferation, or Tumor cell survival is reduced.
  • treating means slowing, interrupting, retarding, alleviating, stopping, reducing, or reversing the onset of symptoms, complications, or biochemical signs of a disease, relieving symptoms, or preventing or inhibiting further progression of the disease, condition, or condition.
  • prevention includes the inhibition of the occurrence or progression of a disease or condition or symptoms of a particular disease or condition.
  • subjects with a family history of cancer are candidates for a preventive regimen.
  • prevention refers to the administration of a drug before signs or symptoms of cancer occur, particularly in a subject at risk for cancer.
  • an effective amount refers to an amount or dose of an antibody or fragment or composition or combination of the invention that produces the desired effect in a patient in need of treatment or prophylaxis when administered to the patient in single or multiple doses.
  • a “therapeutically effective amount” means an amount effective to achieve the desired therapeutic result, at the required doses and for the required period of time.
  • a therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably inhibits a measurable parameter or improves a measurable parameter by at least about 40%, even more preferably at least about 50%, 55%, 60%, 65%, 70%, 75%, relative to an untreated subject %, 80%, 85%, 90% or even 100%.
  • prophylactically effective amount means an amount effective to achieve the desired prophylactic result, at the required dosage and for the required period of time. Generally, the prophylactically effective amount will be less than the therapeutically effective amount because the prophylactic dose is administered in the subject before or at an earlier stage of the disease.
  • combination therapy refers to the administration of two or more therapeutic agents or treatment modalities (eg, radiation therapy or surgery) to treat a disease described herein.
  • administration involves co-administration of the therapeutic agents in a substantially simultaneous manner, for example, in a single capsule having a fixed ratio of the active ingredients.
  • administration involves co-administration of the individual active ingredients in multiple or separate containers (eg tablets, capsules, powders and liquids). Powders and/or liquids can be reconstituted or diluted to the desired dosage prior to administration.
  • administration includes administering each type of therapeutic agent at approximately the same time or at different times in a sequential manner. In either case, the treatment regimen will provide for the beneficial effects of the drug combination in treating the disorder or condition described herein.
  • mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). mouse). In particular, individuals are people.
  • tissue sample refers to a collection of cells or fluid obtained from a patient or subject.
  • the source of the tissue or cell sample may be solid tissue, like from fresh, frozen and/or preserved organs or tissue samples or biopsy samples or aspiration samples; blood or any blood component; body fluids, such as tears, vitreous humor, cerebrospinal fluid , amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from a subject at any time during pregnancy or development.
  • the tissue sample is tumor tissue.
  • Tissue samples may contain compounds that are not naturally associated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.
  • the antibodies of the invention are bispecific antibodies or multispecific antibodies, which, for example, comprise a first binding specificity for PD-L1 and a second binding specificity for CLDN18.2, and optionally One or more additional binding specificities.
  • one aspect of the invention relates to a bispecific antibody comprising
  • a first antigen-binding region and a second antigen-binding region wherein the first antigen-binding region specifically binds PD-L1, and the second antigen-binding region specifically binds CLDN18.2.
  • PD-L1 is human PD-L1 and/or CLDN18.2 is human CLDN18.2.
  • the first antigen-binding region of the bispecific antibody suitable for use in the present invention may comprise or consist of a full-length antibody that specifically binds PD-L1 of the present invention or an antigen-binding fragment thereof, as long as it is capable of specifically binding PD-L1. That is, including but not limited to, for example, full-length antibodies that specifically bind PD-L1, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibodies, VHH or heavy chain antibodies, etc.
  • the second antigen-binding region of the bispecific antibody suitable for use in the present invention may comprise or consist of a full-length antibody that specifically binds CLDN18.2 or an antigen-binding fragment thereof, as long as it can specifically bind CLDN18.2, Including but not limited to, for example, full-length antibodies that specifically bind to CLDN18.2, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibodies, VHH or heavy chain antibodies, etc.
  • the bispecific antibodies of the invention are IgG-like bispecific antibodies. Accordingly, in some embodiments, the bispecific antibodies of the invention comprise Fc dimers.
  • the bispecific antibodies of the invention comprise a full-length antibody as the first antigen-binding region, and a scFv as the second antigen-binding region. In some embodiments, the bispecific antibodies of the invention comprise a full-length antibody as the second antigen-binding region, and a scFv as the first antigen-binding region.
  • the first antigen-binding region that specifically binds PD-L1 is a full-length antibody
  • the second antigen-binding region that specifically binds CLDN18.2 is scFv
  • the second antigen-binding region that specifically binds to CLDN18.2 is a full-length antibody
  • the first antigen-binding region that specifically binds to PD-L1 is scFv.
  • the scFv is linked (via or not via a linker) to the C-terminus of the heavy chain constant region of a full-length antibody.
  • two scFvs are each linked (via or not via a linker) to the C-terminus of two heavy chain constant regions of a fully anti-antibody.
  • the bispecific antibody includes a full-length antibody and two scFvs, wherein the full-length antibody includes two heavy chains and two light chains, and the two scFvs are respectively connected to the C-termini of the two heavy chains. Connect (via or not via connectors).
  • the first antigen-binding region that specifically binds PD-L1 as the bispecific antibody of the present invention can be derived from PCT/CN2021/131389.
  • the first antigen-binding region of the invention that specifically binds PD-L1 includes three complementarity determining regions (HCDR) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3.
  • HCDR complementarity determining regions
  • the first antigen-binding region of the invention that specifically binds PD-L1 includes three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.
  • LCDRs complementarity determining regions
  • the first antigen-binding region of the invention that specifically binds PD-L1 includes 3 complementarity-determining regions (HCDRs) from the heavy chain variable region and 3 HCDRs from the light chain variable region. (LCDR).
  • HCDRs complementarity-determining regions
  • the first antigen-binding region of the invention that specifically binds PD-L1 comprises a heavy chain variable region (VH). In some aspects, the first antigen-binding region of the invention that specifically binds PD-L1 comprises a light chain variable region (VL). In some aspects, the first antigen-binding region of the invention that specifically binds PD-L1 includes a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementarity determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementarity determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.
  • the first antigen-binding region of the invention that specifically binds PD-L1 further comprises an antibody heavy chain constant region HC. In some embodiments, the first antigen-binding region of the present invention that specifically binds PD-L1 further includes the antibody light chain constant region LC. In some embodiments, the first antigen-binding region of the present invention that specifically binds to PD-L1 further includes a heavy chain constant region HC and a light chain constant region LC.
  • the heavy chain variable region of the first antigen-binding region that specifically binds to PD-L1 of the present invention is the heavy chain of the antibody that specifically binds to PD-L1 or its antigen-binding fragment in PCT/CN2021/131389 Variable area.
  • amino acid (iii) Comprises one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably) compared to the amino acid sequence of SEQ ID NO: 17 or 30
  • the amino acid sequence (amino acid substitution, more preferably amino acid conservative substitution) consists of the amino acid sequence.
  • the amino acid change does not occur in the CDR region.
  • the light chain variable region of the first antigen-binding region that specifically binds to PD-L1 is the light chain of the antibody or antigen-binding fragment thereof that specifically binds to PD-L1 in PCT/CN2021/131389 Variable area.
  • (iii) Comprises one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably) compared to the amino acid sequence of SEQ ID NO: 22 or 31 amino acid substitution, More preferably, the amino acid sequence (conservative substitution of amino acids) consists of the amino acid sequence. Preferably, the amino acid changes do not occur in the CDR region.
  • the three complementarity determining regions (HCDR) of the first antigen-binding region of the present invention that specifically bind to PD-L1 are from the heavy chain variable region, and HCDR1, HCDR2 and HCDR3 are from PCT/CN2021/131389
  • the three HCDR regions comprise at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) )the sequence of,
  • the CDR is determined by the Kabat protocol.
  • the anti-PD-L1 antibody of the present invention has three complementarity determining regions (LCDRs) from the light chain variable region, and LCDR1, LCDR2 and LCDR3 are from PCT/CN2021/131389 that specifically bind PD-L1.
  • the three LCDR regions comprise at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) )the sequence of,
  • the CDR is determined by the Kabat protocol.
  • HCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO:18, or HCDR1 contains one, two or three changes compared to the amino acid sequence of SEQ ID NO:18 (preferably Amino acid substitutions, preferably conservative substitutions) of the amino acid sequence.
  • HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 19, or HCDR2 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO: 19 (preferably Amino acid substitutions, preferably conservative substitutions) of the amino acid sequence.
  • the HCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO:20, or the HCDR3 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO:20 (preferably Amino acid substitutions, preferably conservative substitutions) of the amino acid sequence.
  • LCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO:23, or LCDR1 contains one, two or three changes compared to the amino acid sequence of SEQ ID NO:23 (preferably Amino acid substitutions, preferably conservative substitutions) of the amino acid sequence.
  • LCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 24, or LCDR2 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO: 24 (preferably Amino acid substitutions, preferably conservative substitutions) of the amino acid sequence.
  • LCDR3 comprises the amino acid sequence of SEQ ID NO:25, or consists of the amino acid sequence
  • the amino acid sequence composition, or LCDR3 includes an amino acid sequence with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:25.
  • the first antigen-binding region of the invention that specifically binds PD-L1 includes VH and VL, wherein
  • the VH contains the amino acid sequence shown in SEQ ID NO: 17 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto
  • the VL contains the amino acid sequence shown in SEQ ID NO: 22 or has at least 90%, 91%, 92%, 93%, 94%, 95%, or consists of an amino acid sequence that is 96%, 97%, 98% or 99% identical; or
  • the VH contains the amino acid sequence shown in SEQ ID NO:30 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.
  • the identity of the amino acid sequence or consists of the amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO: 31 or has at least 90%, 91%, 92%, 93%, 94%, 95%, An amino acid sequence that is 96%, 97%, 98% or 99% identical or consists of said amino acid sequence.
  • the first antigen-binding region that specifically binds PD-L1 of the present invention includes HCDR1 as shown in SEQ ID NO:18, HCDR2 as shown in SEQ ID NO:19, as shown in SEQ ID NO:19 HCDR3 as shown in SEQ ID NO:20; LCDR1 as shown in SEQ ID NO:23, LCDR2 as shown in SEQ ID NO:24 and LCDR3 as shown in SEQ ID NO:25.
  • the first antigen-binding region that specifically binds PD-L1 of the invention includes VH and VL, wherein the first antigen-binding region that specifically binds PD-L1 includes a heavy chain variable Region VH and light chain variable region VL, wherein VH and VL respectively comprise or consist of the amino acid sequence shown below:
  • the first antigen-binding region of the invention that specifically binds PD-L1 comprises a heavy chain. In some embodiments of the invention, the first antigen-binding region of the invention that specifically binds PD-L1 comprises a light chain. In some embodiments of the invention, the first antigen-binding region of the invention that specifically binds PD-L1 includes a heavy chain and a light chain.
  • the heavy chain of the first antigen-binding region that specifically binds to PD-L1 includes the amino acid sequence of SEQ ID NO: 21, or includes an amino acid sequence that is at least 85%, 90%, or 91% identical to that of SEQ ID NO: 21. , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences, or consisting of said amino acid sequences.
  • the light chain of the first antigen-binding region that specifically binds to PD-L1 comprises the amino acid sequence of SEQ ID NO: 26, or has an amino acid sequence that is at least 85%, 90%, or 91% identical to that of SEQ ID NO: 26. , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences, or consisting of said amino acid sequences.
  • the first antigen-binding region that specifically binds PD-L1 includes a heavy chain and a light chain, wherein the heavy chain includes the amino acid sequence shown in SEQ ID NO: 21, or is identical to the amino acid sequence Have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% Or an amino acid sequence with 99% identity, or consisting of the amino acid sequence, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 26, or has at least 85%, 90%, or 91% with the amino acid sequence. , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences, or consisting of said amino acid sequences.
  • the first antigen-binding region that specifically binds PD-L1 includes a heavy chain and a light chain, wherein the heavy chain includes the amino acid sequence shown in SEQ ID NO: 21, or consists of the amino acid sequence Composed, and the light chain includes or consists of the amino acid sequence shown in SEQ ID NO: 26.
  • the amino acid changes in the first antigen-binding region that specifically binds PD-L1 as described herein include amino acid substitutions, insertions, or deletions.
  • the amino acid changes described in the present invention occur in regions outside the CDRs (eg in the FR). More preferably, the amino acid changes described in the present invention occur in regions outside the heavy chain variable region and/or outside the light chain variable region.
  • the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
  • the amino acid of the first antigen-binding region that specifically binds to PD-L1 according to the invention is changed to introduce a disulfide bond in the variable region (such as the FR region) to obtain disulfide bond stabilization.
  • the heavy chain variable region includes a mutation in which the amino acid at position 44 is replaced with cysteine (Kabat numbering), and/or the light chain variable region includes a substitution of the amino acid at position 100 with cysteine (Kabat numbering) amino acid mutations (Kabat numbering).
  • the mutation is introduced when the first antigen binding region is a scFv that specifically binds PD-L1.
  • the heavy chain constant region of the first antigen-binding region that specifically binds to PD-L1 is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1.
  • the light chain constant region of an anti-PD-L1 antibody of the invention is a lambda or kappa light chain constant region, such as a kappa light chain constant region.
  • the anti-PD-L1 antibody from which the first antigen-binding region that specifically binds PD-L1 is derived is humanized.
  • the anti-PD-L1 antibody from which the first antigen-binding region that specifically binds PD-L1 is derived is a chimeric antibody.
  • the first antigen-binding region of the present invention that specifically binds to PD-L1 is an antibody fragment (eg, an antigen-binding fragment), preferably an antibody fragment selected from the following: Fab, Fab', Fab'-SH, Fv, single chain antibody (eg scFv), (Fab') 2 , single domain antibody such as VHH, dAb (domain antibody) or linear antibody.
  • the first antigen-binding region that specifically binds PD-L1 is scFv.
  • Second antigen-binding region that specifically binds CLDN18.2
  • the second antigen-binding region that specifically binds CLDN18.2 as the bispecific antibody of the present invention can be derived from WO2021032157A1.
  • the second antigen-binding region of the invention that specifically binds CLDN18.2 includes three complementarity determining regions (HCDR) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3.
  • HCDR complementarity determining regions
  • the second antigen-binding region of the invention that specifically binds CLDN18.2 includes three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.
  • LCDRs complementarity determining regions
  • the second antigen-binding region of the invention that specifically binds CLDN18.2 comprises 3 complementarity-determining regions (HCDRs) from the heavy chain variable region and 3 HCDRs from the light chain variable region. (LCDR).
  • HCDRs complementarity-determining regions
  • the second antigen-binding region of the invention that specifically binds CLDN18.2 comprises a heavy chain variable region (VH). In some aspects, the second antigen-binding region of the invention that specifically binds CLDN18.2 comprises a light chain variable region (VL). In some aspects, the second antigen-binding region of the invention that specifically binds CLDN18.2 includes a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementarity determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementarity determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.
  • the second antigen-binding region of the invention that specifically binds CLDN18.2 further comprises an antibody heavy chain constant region HC. In some embodiments, the second antigen-binding region of the present invention that specifically binds CLDN18.2 further comprises an antibody light chain constant region LC. In some embodiments, the second antigen-binding region of the present invention that specifically binds CLDN18.2 further includes a heavy chain constant region HC and a light chain constant region LC.
  • the heavy chain variable region of the second antigen-binding region that specifically binds to CLDN18.2 is the heavy chain variable region of the antibody that specifically binds to CLDN18.2 or its antigen-binding fragment disclosed in WO2021032157A1. district.
  • the heavy chain variable region of the invention specifically binds to the second antigen binding region of CLDN18.2
  • (iii) Comprises one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:1 , more preferably amino acid conservative substitution) amino acid sequence consists of the amino acid sequence, preferably, the amino acid changes do not occur in the CDR region.
  • the light chain variable region of the second antigen-binding region that specifically binds to CLDN18.2 of the present invention is the light chain variable region of the antibody that specifically binds to CLDN18.2 or its antigen-binding fragment disclosed in WO2021032157A1. district.
  • the light chain variable region of the invention specifically binds to the second antigen binding region of CLDN18.2
  • (i) Comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:5; Consisting of the amino acid sequence; or
  • (iii) Comprises one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 5 , more preferably The amino acid sequence (conservative substitution of amino acids) consists of the amino acid sequence. Preferably, the amino acid changes do not occur in the CDR region.
  • the three complementarity-determining regions (HCDRs) of the second antigen-binding region of the present invention that specifically bind to CLDN18.2 are from the heavy chain variable region.
  • HCDR1, HCDR2, and HCDR3 are from the specific complementarity-determining regions (HCDR) disclosed in WO2021032157A1.
  • the three HCDR regions comprise at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) )the sequence of,
  • the CDR is determined by the Kabat protocol.
  • the anti-CLDN18.2 antibody of the present invention has three complementarity determining regions (LCDRs) from the light chain variable region, and LCDR1, LCDR2 and LCDR3 are from the antibodies specifically binding to CLDN18.2 disclosed in WO2021032157A1 or The complementarity determining region (LCDR) of its antigen-binding fragment, LCDR1, LCDR2 and LCDR3, or selected from
  • the three LCDR regions comprise at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) )the sequence of,
  • the CDR is determined by the Kabat protocol.
  • the HCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO:2, or the HCDR1 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO:2 (preferably Amino acid substitutions, preferably conservative substitutions) of the amino acid sequence.
  • HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:3, or HCDR2 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO:3 (preferably Amino acid substitutions, preferably conservative substitutions) of the amino acid sequence.
  • the HCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO:4, or the HCDR3 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO:4 (preferably Amino acid substitutions, preferably conservative substitutions) of the amino acid sequence.
  • LCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO:7, or LCDR1 contains one, two or three changes compared to the amino acid sequence of SEQ ID NO:7 (preferably Amino acid substitutions, preferably conservative substitutions) of the amino acid sequence.
  • LCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO:8, or LCDR2 comprises one, two or three changes compared to the amino acid sequence of SEQ ID NO:8 (preferably Amino acid substitutions, preferably conservative substitutions) of the amino acid sequence.
  • LCDR3 comprises the amino acid sequence of SEQ ID NO:9, or consists of the amino acid sequence
  • the amino acid sequence composition, or LCDR3 includes an amino acid sequence with one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 9.
  • the second antigen-binding region of the invention that specifically binds CLDN18.2 includes VH and VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO: 1 or has at least 90% difference therewith. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to or consisting of an amino acid sequence, and the VL contains SEQ ID NO: The amino acid sequence shown in 6 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto or consisting of the amino acid sequence composition.
  • the second antigen-binding region of the invention that specifically binds CLDN18.2 includes HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:3, as shown in SEQ ID NO:3 HCDR3 as shown in SEQ ID NO:4; LCDR1 as shown in SEQ ID NO:7, LCDR2 as shown in SEQ ID NO:8 and LCDR3 as shown in SEQ ID NO:9.
  • the second antigen-binding region that specifically binds CLDN18.2 of the invention includes VH and VL, wherein the second antigen-binding region that specifically binds CLDN18.2 includes a heavy chain variable Region VH and light chain variable region VL, wherein VH and VL respectively comprise or consist of the amino acid sequences shown below: SEQ ID NO: 1 and SEQ ID NO: 6.
  • the second antigen-binding region of the invention that specifically binds CLDN18.2 comprises a heavy chain. In some embodiments of the invention, the second antigen-binding region of the invention that specifically binds CLDN18.2 comprises a light chain. In some embodiments of the invention, the second antigen-binding region of the invention that specifically binds CLDN18.2 includes a heavy chain and a light chain.
  • the heavy chain of the second antigen-binding region that specifically binds to CLDN18.2 comprises the amino acid sequence of SEQ ID NO: 5, or comprises an amino acid sequence that is at least 85%, 90%, or 91% identical to that of SEQ ID NO: 5. , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences, or consisting of said amino acid sequences.
  • the light chain of the second antigen-binding region that specifically binds to CLDN18.2 includes the amino acid sequence of SEQ ID NO: 10, or includes an amino acid sequence that is at least 85%, 90%, or 91% identical to that of SEQ ID NO: 10. , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences, or consisting of said amino acid sequences.
  • the second antigen-binding region that specifically binds CLDN18.2 includes a heavy chain and a light chain, wherein the heavy chain includes the amino acid sequence shown in SEQ ID NO: 5, or is identical to the amino acid sequence. or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, and
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 10, or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, An amino acid sequence that is 98% or 99% identical, or consists of said amino acid sequence.
  • the second antigen-binding region that specifically binds CLDN18.2 includes a heavy chain and a light chain, wherein the heavy chain includes the amino acid sequence shown in SEQ ID NO: 5, or consists of the amino acid sequence Composed, and the light chain includes or consists of the amino acid sequence shown in SEQ ID NO: 10.
  • Amino acid changes in the binding region include amino acid substitutions, insertions or deletions.
  • the amino acid changes described in the present invention occur in regions outside the CDRs (eg in the FR). More preferably, the amino acid changes described in the present invention occur in regions outside the heavy chain variable region and/or outside the light chain variable region.
  • the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
  • the amino acid of the second antigen-binding region that specifically binds CLDN18.2 is changed to introduce a disulfide bond in the variable region (such as the FR region) to obtain disulfide bond stabilization.
  • the heavy chain variable region includes a mutation in which the amino acid at position 44 is replaced with cysteine (Kabat numbering), and/or the light chain variable region includes a substitution of the amino acid at position 100 with cysteine (Kabat numbering) amino acid mutations (Kabat numbering).
  • the mutation is introduced when the first antigen binding region is a scFv that specifically binds PD-L1.
  • the heavy chain constant region of the second antigen-binding region that specifically binds to CLDN18.2 is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1.
  • the light chain constant region of an anti-CLDN18.2 antibody of the invention is a lambda or kappa light chain constant region, such as a kappa light chain constant region.
  • the anti-CLDN18.2 antibody from which the second antigen-binding region that specifically binds CLDN18.2 is derived is humanized.
  • the anti-CLDN18.2 antibody from which the second antigen-binding region that specifically binds CLDN18.2 is derived is a chimeric antibody.
  • the second antigen-binding region of the invention that specifically binds CLDN18.2 is an antibody fragment (eg, an antigen-binding fragment), preferably an antibody fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single chain antibody (eg scFv), (Fab') 2 , single domain antibody such as VHH, dAb (domain antibody) or linear antibody.
  • the second antigen-binding region that specifically binds CLDN18.2 is scFv.
  • one of the antigen-binding regions of a bispecific antibody can be a full-length antibody, which contains two heavy chains and two light chains, wherein each heavy chain consists of a heavy chain variable region VH and a heavy chain constant Area CH consists of.
  • the heavy chain constant region consists of 3 domains CH1, CH2 and CH3 (and optionally CH4).
  • Each light chain consists of a light chain variable region VL and a light chain constant region CL.
  • the light chain constant region consists of one domain, CL.
  • Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • Full-length antibodies of the invention comprise an Fc region.
  • the Fc region may comprise two or three constant domains, namely a CH2 domain, a CH3 domain and optionally a CH4 domain.
  • the antibody Fc region may also bear an IgG hinge region or a portion of an IgG hinge region at the N-terminus, for example, an IgG1 hinge region or a portion of an IgG1 hinge region.
  • mutations can be introduced into the full-length antibody, for example, mutations that increase the effector function of the Fc region therein. Suitable mutations targeting various properties of full-length antibodies are known in the art.
  • the scFv fragment that is one of the bispecific antibody binding regions consists of one polypeptide chain comprising the VH and VL domains of the antibody, wherein the VH and VL are linked (e.g., via a linker) to pair to form the antigen binding site point.
  • the scFv is in a trans configuration, comprising from N-terminus to C-terminus: VH, linker, and VL (VH-linker-VL). In other embodiments, the scFv is in a cis configuration, comprising from N-terminus to C-terminus: VL, linker, and VH (VL-linker-VH).
  • the scFv antigen-binding site comprised in an antibody molecule of the invention is a disulfide-stabilized (ie, introduced disulfide mutation) scFv.
  • a disulfide-stabilized (ie, introduced disulfide mutation) scFv for example, the amino acid at position 100 in the VH region of scFv is mutated to cysteine (kabat), and the amino acid at position 44 in the VL region of scFv is mutated to cysteine (kabat).
  • the scFv is fused at the N-terminus of the VH chain to the C-terminus of another antigen-binding region (eg, the C-terminus of the heavy chain constant region).
  • the scFv contained in the bispecific antibody of the present invention specifically binds to CLDN18.2, that is, it is the second antigen-binding region that specifically binds to CLDN18.2.
  • the scFv comprises the amino acid sequence of SEQ ID NO: 11, or comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% identity to an amino acid sequence, or consisting of said amino acid sequence.
  • the scFv contained in the bispecific antibody of the present invention specifically binds to PD-L1, that is, it is the first antigen-binding region that specifically binds to PD-L1.
  • the scFv comprises the amino acid sequence of SEQ ID NO: 27, or comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% identity to an amino acid sequence, or consisting of said amino acid sequence.
  • the scFv comprises a disulfide bond stabilizing mutation comprising, having, or consisting of the amino acid sequence of SEQ ID NO: 29.
  • the scFv comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, An amino acid sequence with 98% or 99% identity, and the amino acid at position 100 in the VH region of scFv is mutated to cysteine (kabat), and the amino acid at position 44 in the VL region of scFv is mutated to Cysteine (kabat).
  • two Fc regions in a bispecific antibody of the invention dimerize to form a dimeric Fc.
  • first and second Fc regions are the same. In other embodiments, the first Fc region and the second Fc region are different, pair and heterodimerize.
  • Fc region fragments suitable for use in the antibody molecules of the invention can be any antibody Fc region.
  • the Fc area may include Native sequence Fc regions and variant Fc regions.
  • the native sequence Fc domain covers various naturally occurring immunoglobulin Fc sequences, such as the Fc regions of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389/fimmu.2014.00520.).
  • the Fc region of an antibody of the invention may comprise two or three constant domains, namely a CH2 domain, a CH3 domain and optionally a CH4 domain.
  • the antibody Fc region may also bear an IgG hinge region or a portion of an IgG hinge region at the N-terminus, for example, an IgG1 hinge region or a portion of an IgG1 hinge region. Mutations may be contained in the hinge region.
  • the Fc region of the antibody of the present invention includes: CH2-CH3 from N terminus to C terminus, or includes: hinge region-CH2-CH3 from N terminus to C terminus.
  • a suitable Fc region for use in the antibodies or bispecific antibodies of the invention is a human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3, or human IgG4 Fc.
  • the Fc region comprises or consists of the amino acid sequence SEQ ID NO: 14 or an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or higher identity thereto. .
  • the Fc region in the antibodies or bispecific antibodies of the invention can be mutated to obtain desired properties. Mutations in the Fc region are known in the art. For example, if the two Fc regions are different, knob-into-hole technology can be introduced to promote heterodimerization of the Fc regions. This technique can be found, for example, in Merchant, A.M., et al. (1998). "An efficient route to human bispecific IgG.” Nat Biotechnol 16(7):677-681.
  • antibody molecules of the invention may also comprise modifications in the Fc domain that alter binding affinity to one or more Fc receptors, depending on the intended use of the antibody molecule.
  • the Fc region is modified in properties of its effector function (eg, ADCC of the Fc region).
  • the effector function has been reduced or eliminated relative to the wild isotype Fc region, or has been increased.
  • effector function is reduced or eliminated by using an Fc isotype or Fc region modification that naturally has reduced or eliminated effector function, such as introducing an Fc region modification on L234, L235, and/or P331,
  • an Fc region modification such as introducing an Fc region modification on L234, L235, and/or P331,
  • the mutated Fc region comprises the amino acid sequence SEQ ID NO: 15 or an amino acid sequence having at least 90% identity thereto, such as 95%, 96%, 97%, 99% or higher identity or consisting of its composition.
  • the Fc region includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 15, such as 95%, 96%, 97%, 99% or higher and includes L234F, L235E and P331S mutation.
  • the invention provides a bispecific antibody having a structure as shown in Figure 2A, 2B or 2C.
  • the invention provides a bispecific antibody comprising a light chain and a heavy chain, wherein the light chain comprises VL-A-CL from the N-terminus to the C-terminus;
  • the heavy chain contains VH-A-CH-scFv from N-terminus to C-terminus;
  • scFv contains VH-B and VL-B;
  • VL-A is the light chain variable region VL that specifically binds to the first antigen-binding region of PD-L1
  • VH-A is the heavy chain variable region VH that specifically binds to the first antigen-binding region of PD-L1;
  • VH-B is the heavy chain variable region VH that specifically binds to the second antigen-binding region of CLDN18.2, and VL-B is the light chain variable region VL that specifically binds to the second antigen-binding region of CLDN18.2;
  • VL-A is the light chain variable region VL that specifically binds to the second antigen-binding region of CLDN18.2, and VH-A is the heavy chain variable region VH that specifically binds to the second antigen-binding region of CLDN18.2;
  • VH-B is the heavy chain variable region VH that specifically binds to the first antigen-binding region of PD-L1, and VL-B is the light chain variable region VL that specifically binds to the first antigen-binding region of PD-L1;
  • CL is the light chain constant region
  • CH is the heavy chain constant region
  • scFv contains a linker or no linker between VH-B and VL-B;
  • CH and scFv contain a linker or do not contain a linker.
  • bispecific antibodies of the invention comprise two heavy chains and two light chains, eg, two identical heavy chains and two identical light chains.
  • the scFv contains VH-B-VL-B from N-terminus to C-terminus, and VH-B is connected at its N-terminus to the C-terminus of CH with or without a linker.
  • the bispecific antibodies of the invention comprise a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 32, 33 or 34, or has an amino acid sequence that is at least or consist of an amino acid sequence that is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.
  • the bispecific antibodies of the invention comprise a heavy chain and a light chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 10 or 26, or has an amino acid sequence that is at least 85% identical to that of SEQ ID NO: 10 or 26. , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences, or consisting of said amino acid sequences.
  • the bispecific antibodies of the invention comprise a heavy chain and a light chain, wherein
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 32 or 34, or contains at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% similarity to the amino acid sequence. %, 97%, 98% or 99% identity to an amino acid sequence, or consisting of said amino acid sequence;
  • the light chain comprises the amino acid sequence of SEQ ID NO: 10, or contains at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence , or consisting of an amino acid sequence that is 98% or 99% identical; or
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 33, or contains at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, An amino acid sequence that is 97%, 98% or 99% identical, or consists of said amino acid sequence;
  • the light chain includes the amino acid sequence of SEQ ID NO: 26, or includes at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence. , or consisting of an amino acid sequence that is 98% or 99% identical.
  • the bispecific antibodies of the invention comprise a heavy chain and a light chain, wherein
  • the heavy chain includes or consists of the amino acid sequence of SEQ ID NO: 32 or 34;
  • the light chain includes or consists of the amino acid sequence of SEQ ID NO: 10; or
  • the heavy chain includes or consists of the amino acid sequence of SEQ ID NO: 33;
  • the light chain includes or consists of the amino acid sequence of SEQ ID NO: 26.
  • the bispecific antibodies of the invention comprise 2 heavy chains and 2 light chains, wherein
  • Each of the heavy chains comprises the amino acid sequence of SEQ ID NO: 32 or 34, or contains at least 85%, 90%, 91%, 92%, 93%, 94%, 95% of the same amino acid sequence as the described amino acid sequence. , or consisting of an amino acid sequence that is 96%, 97%, 98% or 99% identical;
  • each of the light chains includes the amino acid sequence of SEQ ID NO: 10, or includes at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, An amino acid sequence that is 97%, 98% or 99% identical, or consists of said amino acid sequence;
  • Each of the heavy chains includes the amino acid sequence of SEQ ID NO: 33, or contains at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% similarity to the amino acid sequence. %, 97%, 98% or 99% identity to an amino acid sequence, or consisting of said amino acid sequence;
  • each of the light chains includes the amino acid sequence of SEQ ID NO: 26, or includes at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, An amino acid sequence that is 97%, 98% or 99% identical, or consists of said amino acid sequence.
  • the bispecific antibodies of the invention comprise 2 heavy chains and 2 light chains, wherein
  • Each of the heavy chains includes or consists of the amino acid sequence of SEQ ID NO: 32 or 34;
  • each of the light chains includes or consists of the amino acid sequence of SEQ ID NO: 10;
  • Each of the heavy chains includes or consists of the amino acid sequence of SEQ ID NO: 33;
  • the light chain includes or consists of the amino acid sequence of SEQ ID NO: 26.
  • the invention provides nucleic acids encoding any of the above anti-CLDN18.2 antibodies or anti-PD-L1 antibodies or bispecific antibodies or any chain thereof.
  • the nucleic acid of the present invention includes a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO: 17, 21, 22, 26, 27, 29, 30, 31, 32, 33, and 34, or encoding and selecting
  • the amino acid sequence shown in any one of SEQ ID NO: 17, 21, 22, 26, 27, 29, 30, 31, 32, 33, 34 has at least 85%, 90%, 91%, 92%, 93 Nucleic acids with amino acid sequences that are %, 94%, 95%, 96%, 97%, 98% or 99% identical.
  • each antibody or polypeptide amino acid sequence may be encoded by multiple nucleic acid sequences because of codon degeneracy.
  • Nucleic acid sequences encoding molecules of the invention can be generated using methods well known in the art, such as by de novo solid-phase DNA synthesis, or by PCR amplification.
  • the DNA encoding the antibody can be readily isolated and sequenced using conventional procedures (eg, by using oligonucleotide probes capable of binding specifically to the genes encoding the heavy and light chains of the antibody). Coding DNA can also be obtained synthetically.
  • the invention provides nucleic acids encoding any of the above antibodies or any antibody chain.
  • the polypeptide encoded by the nucleic acid can display (human) CLDN18.2 and/or (human) PD-L1 antigen binding ability.
  • the invention provides nucleic acids encoding any of the above bispecific antibodies.
  • the polypeptide encoded by the nucleic acid When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid is capable of displaying (human) CLDN18.2 and (human) PD-L1 antigen binding capabilities.
  • the nucleic acids encoding each chain of the bispecific antibody can be in the same vector or in different vectors.
  • nucleic acids encoding each chain of a bispecific antibody can be introduced into the same or different host cells for expression.
  • a method for producing a bispecific antibody of the invention includes the steps of culturing a host cell comprising a nucleic acid encoding each chain of the molecule under conditions suitable for expression of each chain of the molecule, Bispecific antibodies of the invention are produced.
  • vectors are available that provide vectors comprising the nucleic acids.
  • Vector components typically include, but are not limited to, one or more of the following: signal sequence, origin of replication, one or more marker genes, enhancer elements, promoter (e.g., SV40, CMV, EF-1 ⁇ ), and transcription Terminate sequence.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YAC).
  • vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., papopaviruses) , SV40), lambda phage and M13 phage, plasmid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI , pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT,
  • a host cell comprising said nucleic acid or said vector, eg a vector for cloning or expressing a bispecific antibody.
  • the host cell is eukaryotic.
  • the host cell is selected from filamentous fungi, yeast cells (eg Saccharomyces cerevisiae), plant cells, insect cells, mammalian cells such as CHO cells (eg CHO-S, such as ExpiCHO-S) or 293 cells ( For example, 293F or HEK293 cells)) or other cells suitable for preparing antibodies or fragments thereof.
  • the host cell is prokaryotic, such as a bacterium, such as E. coli.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparation of antibodies or fragments thereof.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies.
  • fungal and yeast strains in which glycosylation pathways have been “humanized” result in the production of partially or fully human glycosylation Antibodies with tylated pattern.
  • Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts.
  • mammalian cell lines engineered to be suitable for suspension growth may be used.
  • useful mammalian host cell lines are the monkey kidney CV1 line (COS-7) transformed with SV40; the human embryonic kidney line (HEK293, 293F or 293T cells), and the like.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, ExpiCHO, and others; and myeloma cell lines such as Y0, NSO, and Sp2/0.
  • CHO Chinese hamster ovary
  • a method for preparing a bispecific antibody of the present invention comprises culturing a cell containing a protein encoding the bispecific antibody (e.g., any a polypeptide chain and/or polypeptide chains) or a host cell comprising an expression vector of said nucleic acid, as provided above, and optionally recovering said host cell (or host cell culture medium) Bispecific antibodies.
  • a protein encoding the bispecific antibody e.g., any a polypeptide chain and/or polypeptide chains
  • a host cell comprising an expression vector of said nucleic acid, as provided above, and optionally recovering said host cell (or host cell culture medium) Bispecific antibodies.
  • Polynucleotides encoding the polypeptide chains of the bispecific antibodies of the invention can be inserted into one or more vectors for further cloning and/or expression in host cells.
  • Expression vectors can be constructed using methods well known to those skilled in the art. Once an expression vector containing one or more nucleic acid molecules of the invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques.
  • bispecific antibodies When using recombinant techniques, bispecific antibodies can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the bispecific antibody is produced intracellularly, particulate debris of the host cells or solubilized fragments can be removed as a first step, for example by centrifugation or ultrafiltration. When bispecific antibodies are secreted into the culture medium, the supernatant from such expression systems is typically first concentrated using commercially available protein concentration filters.
  • Bispecific antibodies prepared as described herein can be purified by known state-of-the-art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like.
  • the actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those skilled in the art.
  • Protein A immobilized on a solid phase is used for immunoaffinity purification of antibodies and antigen-binding fragments thereof.
  • the matrix to which affinity ligands are attached is most commonly agarose, but other matrices are available.
  • Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than with agarose.
  • Bakerbond ABXTM resin can be used for purification.
  • the purity of the antibody molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
  • bispecific antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art.
  • the bispecific antibodies of the invention are tested for their target (eg antigen, eg free antigen or antigen expressed on cells, eg PD-L1 and/or CLDN18.2) binding activity, eg by known methods such as Radioimmunoassay (RIA)/biofilm thin-layer interference technology, ELISA, flow cytometry, electrochemiluminescence (ECL) or surface plasmon resonance (SPR), etc. Binding to CLDN18.2 and/or PD-L1 expressed on a cell can be determined using methods known in the art, and exemplary methods are disclosed herein. In some embodiments, flow cytometry (FACS) measurements are used.
  • target eg antigen, eg free antigen or antigen expressed on cells, eg PD-L1 and/or CLDN18.2
  • binding activity eg by known methods such as Radioimmunoassay (RIA)/biofilm thin-layer interference technology, ELISA, flow cytometry, electrochemiluminescence (EC
  • the present invention also provides assays for identifying the biological activity of bispecific antibodies.
  • Biological activity is selected from the properties of the bispecific antibodies of the invention.
  • the binding activity of the antibody molecule of the present invention to cells expressing CLDN18.2 and/or PD-L1 can be determined by methods known in the art, such as fluorescent reporters and flow cytometry, or methods disclosed in the examples herein. Exemplary methods are used to determine, for example, the binding of the antibody molecule of the present invention to CLDN18.2 and/or PD-L1 expressed on the cell by the method shown in Example 3-1 or 3-2.
  • the induction of ADCC activity by the antibody molecule of the present invention can be determined by methods known in the art, such as the method described in Example 3-3 or 3-4.
  • blocking the binding activity of the antibody molecule of the present invention to PD-L1/PD-1 can be accomplished by methods known in the art, such as the transcription and expression of reporter genes, such as the NFAT-luc reporter gene system, etc., or implemented herein Measured by exemplary methods disclosed in Examples, for example, measured by the method shown in Examples 3-5.
  • the activation activity of the antibody molecule of the present invention on T cells and/or the inhibition of T cell activation by blocking PD-1 methods known in the art can be used, such as by detecting cytokines after activating T cells (for example, The release of interferons, such as IFN ⁇ , is measured, for example, by the exemplary method disclosed in Example 3-6 herein, for example, by the method shown in Example 3-1 or 3-2.
  • the tumor inhibitory activity or structural safety of the antibody molecule of the present invention can be determined by methods known in the art, such as performing tumor inhibition experiments on mouse tumor models, for example, by the method shown in Example 4.
  • Cells for use in any of the above in vitro assays are primary cells or cell lines, including cells that naturally express or overexpress CLDN18.2 (e.g., human) or PD-L1 (e.g., human), e.g., overexpress CLDN18.2 or PD-L1 L1 cells, such as gastric cancer cell lines NUGC-4, KATOII, SNU-620 or HEK293T-PD-L1 cells overexpressing PD-L1.
  • CLDN18.2 e.g., human
  • PD-L1 e.g., human
  • overexpress CLDN18.2 or PD-L1 L1 cells such as gastric cancer cell lines NUGC-4, KATOII, SNU-620 or HEK293T-PD-L1 cells overexpressing PD-L1.
  • the invention provides immunoconjugates comprising any of the bispecific antibodies described herein.
  • the immunoconjugate comprises a bispecific antibody of the invention, and one or more conjugate moieties, such as a chemotherapeutic agent, a cytokine, a cytotoxic agent or a small molecule drug, such as a purification moiety such as a magnetic Beads, biotin, clearance modifiers (e.g., half-life extending polymers such as PEG), chemotherapeutic agents, toxins, radioisotopes, lanthanides, detectable labels (e.g., luminescent labels, fluorescent labels, enzyme substrate labels ), DNA alkylating agents, topoisomerase inhibitors, tubulin binding agents or other anticancer drugs.
  • conjugate moieties such as a chemotherapeutic agent, a cytokine, a cytotoxic agent or a small molecule drug, such as a purification moiety such as a magnetic Beads, biotin,
  • the invention provides a composition or medicament or formulation comprising any bispecific antibody described herein, preferably the composition is a pharmaceutical composition.
  • the composition or drug or preparation of the present invention may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers, such as pharmaceutically acceptable liquids, gels, etc. Glue or solid carrier, aqueous vehicle, non-aqueous vehicle, antimicrobial agent, isotonic agent, buffer, antioxidant, anesthetic, suspending/distributing agent, sequestering agent or chelating agent, diluent, adjuvant, Excipients or nontoxic auxiliary substances, other components known in the art, or various combinations thereof.
  • suitable pharmaceutical excipients such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers, such as pharmaceutically acceptable liquids, gels, etc.
  • Glue or solid carrier aqueous vehicle, non-aqueous vehicle, antimicrobial agent, isotonic agent, buffer, antioxidant, anesthetic
  • compositions or medicaments or preparations of the invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions, powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.
  • compositions are formulated as injectable compositions.
  • injectable pharmaceutical compositions may be prepared in any conventional form, such as liquid solutions, suspensions, emulsions, or solid forms suitable for the production of liquid solutions, suspensions, or emulsions.
  • Injectable preparations may include sterile and/or pyrogen-free solutions prepared for injection, sterile dry soluble products (such as lyophilized powders, including subcutaneous tablets) prepared for combination with solvents before use, sterile suspensions prepared for injection , sterile dry insoluble products and sterile and/or pyrogen-free emulsions prepared for combination with a vehicle prior to use. Solutions can be aqueous or non-aqueous.
  • Medicaments or formulations containing the bispecific antibodies described herein can be prepared by mixing the bispecific antibodies of the invention with the desired purity and one or more optional pharmaceutical excipients, for example in a lyophilized formulation or form of aqueous solution.
  • compositions or medicaments or preparations of the present invention may also contain more than one active ingredient required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, it may be desirable to also provide other therapeutic agents.
  • the other therapeutic agent is selected from the group consisting of chemotherapeutic agents, anti-cancer drugs, immunotherapeutic agents, anti-angiogenic agents, targeted therapy therapeutic agents, cell therapy therapeutic agents, gene therapy therapeutic agents, hormone therapy therapeutic agents , antiviral agents, antibiotics, analgesics, antioxidants, metal chelators or cytokines.
  • the present invention also provides a complete pharmaceutical kit containing the pharmaceutical combination, for example, the complete pharmaceutical kit includes in the same package:
  • the other therapeutic agent such as a chemotherapeutic agent, an anti-cancer drug, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapy therapeutic agent, a cell therapy therapeutic agent, a gene therapy therapeutic agent, a hormone therapy therapeutic agent, Antiviral agents, antibiotics, analgesics, antioxidants, metal chelators or cytokines.
  • One aspect of the invention provides a method of preventing or treating a disease in a subject, comprising administering to the subject a bispecific antibody of the invention, or an immunoconjugate, composition or medicament or formulation comprising the same.
  • the invention provides a method of specifically activating T cells in a subject, comprising administering to the subject a bispecific antibody of the invention, or an immunoconjugate, composition or medicament comprising the same or preparations.
  • the disease is, for example, a tumor, such as cancer.
  • Cancer can be early, intermediate or late or metastatic.
  • the cancer can be a solid tumor or a hematological tumor.
  • the cancer is a gastrointestinal tract tumor, a biliary tract tumor, or a lung cancer, such as gastric cancer, esophageal cancer, pancreatic cancer, colorectal cancer, cholangiocarcinoma, gallbladder cancer, or lung cancer, and the like.
  • the cancer will benefit from blockade of PD-L1 binding to PD-1, or from inhibition of PD-L1 and/or CLDN18.2.
  • the cancer is a cancer characterized by having protein expression of PD-L1 or having elevated protein levels and/or nucleic acid levels (e.g., elevated expression) of PD-L1, e.g., of the cancer
  • the tumor cells have protein expression of PD-L1 or have increased protein levels and/or nucleic acid levels of PD-L1 (e.g., increased expression, e.g., compared to non-cancerous or non-tumor cells or normal cells of the same tissue type).
  • the cancer is a cancer characterized by having protein expression of CLDN18.2 or having elevated protein levels and/or nucleic acid levels (e.g., elevated expression) of CLDN18.2, e.g., the cancer Tumor cells that have protein expression of CLDN18.2 or have elevated protein levels and/or nucleic acid levels of CLDN18.2 (e.g., increased expression, e.g., compared to non-cancerous or non-tumor cells or normal cells of the same tissue type) .
  • the cancer is characterized by having protein expression of PD-L1 and CLDN18.2 or having elevated protein levels and/or nucleic acid levels (eg, elevated expression) of PD-L1 and CLDN18.2 Cancer, for example, having protein expression of PD-L1 and CLDN18.2 or having elevated protein and/or nucleic acid levels of PD-L1 and CLDN18.2 (e.g., elevated expression, e.g., phase than non-cancerous or non-tumor cells or normal cells of the same tissue type).
  • elevated expression e.g., phase than non-cancerous or non-tumor cells or normal cells of the same tissue type.
  • the cancer is characterized by having protein expression of CLDN18.2 or having elevated protein levels and/or nucleic acid levels (eg, elevated expression) of CLDN18.2; and
  • the cancer refers to a cancer in which PD-L1 protein is present on the cell surface of cancer cells or tumor cells, or in which the PD-L1 protein is present on the cell surface of cancer or tumor cells compared to non-cancerous cells of the same tissue type. Cancers with higher levels of PD-L1.
  • the cancer refers to a cancer in which CLDN18.2 protein is present on the cell surface of cancer cells or tumor cells, or in which the CLDN18.2 protein is present on the cell surface of cancer or tumor cells compared to non-cancer cells of the same tissue type. Cancers with higher levels of CLDN18.2.
  • the cancer refers to a cancer in which cancer cells or tumor cells have CLDN18.2 protein on their cell surface, or in which the CLDN18.2 protein is present on the cancer or tumor cells compared to non-cancerous cells of the same tissue type. have higher levels of CLDN18.2 on their cell surface, and
  • PD-L1 protein is present on the cell surface of said cancer cell or tumor cell
  • PD-L1 protein is present on the cell surface of the cancer cell or tumor cell, and compared with a cancer or tumor that highly expresses PD-L1, there is a low level of PD on the cell surface of the cancer or tumor cell.
  • PD-L1 and/or CLDN18.2 expression or increased expression can be determined in a diagnostic or prognostic assay by assessing increased levels of PD-L1 and/or CLDN18.2 present on the cell surface (e.g., by immunohistochemistry assay; IHC).
  • cells encoding PD-L1 and/or may be measured, for example, by fluorescence in situ hybridization (FISH), southern blotting, or polymerase chain reaction (PCR) techniques, such as real-time quantitative PCR (RT-PCR).
  • FISH fluorescence in situ hybridization
  • PCR polymerase chain reaction
  • RT-PCR real-time quantitative PCR
  • Expression of PD-L1 and/or CLDN18.2 can also be studied by measuring shed antigens (eg, extracellular domains or soluble proteins of PD-L1 and/or CLDN18.2) in biological fluids such as serum.
  • shed antigens eg, extracellular domains or soluble proteins of PD-L1 and/or CLDN18.2
  • biological fluids such as serum.
  • various in vivo assays are also available.
  • cells in a patient can be exposed to anti-PD-L1 and/or CLDN18.2 antibodies, optionally labeled with a detectable label (e.g., a radioisotope), and can be e.g., by external scanning radioactivity or by Analyzing biopsies taken from patients who were previously exposed to the antibodies evaluates the binding of the antibodies to the patient's cells.
  • a detectable label e.g., a radioisotope
  • the subject has been identified as likely to respond to PD-L1 and/or CLDN18.2 antagonists.
  • the presence or levels of PD-L1 and/or CLDN18.2 on a biological sample of interest may indicate whether the subject from which the biological sample is derived is likely to respond to a PD-L1 and/or CLDN18.2 antagonist.
  • the test sample is derived from cancer cells or tissue, or tumor-infiltrating immune cells.
  • the presence or upregulated levels of PD-L1 and/or CLDN18.2 in a test biological sample is indicative of the likelihood of a response.
  • the term "upregulation" refers to the level of PD-L1 and/or CLDN18.2 protein in a test sample compared to the level of PD-L1 and/or CLDN18.2 protein in a reference sample detected using the same antibody.
  • the overall level increase is not less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or larger.
  • the reference sample may be a control sample obtained from a healthy or non-diseased individual, or a healthy or non-diseased sample obtained from the same individual from which the test sample was obtained.
  • a reference sample may be a non-diseased sample adjacent to or in the vicinity of a test sample (eg, a tumor).
  • PD-L1 expression as defined herein can be measured by TPS or CPS score.
  • TPS refers to the percentage of tumor cells showing partial or complete PD-L1 membrane staining at any intensity under IHC immunohistochemistry to the total number of tumor cells.
  • CPS refers to the sum of the number of PD-L1 stained cells (including tumor cells, lymphocytes, macrophages) divided by the total number of tumor cells, and multiplied by 100 to obtain the result.
  • High PD-L1 expression refers to a CPS score of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% in the tumor or cancer tissue , 9%, 10%, 15% or more than 20%. Therefore, "cancer or tumor that highly expresses PD-L1" means that in the cancer or tumor tissue, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9 %, 10%, 15% or more of the cells are positive for PD-L1 expression.
  • the bispecific antibodies of the invention may be administered by any suitable method, including parenteral administration, intrapulmonary administration, and Administer intranasally and, if required for local treatment, intralesional administration.
  • Parenteral injection or infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous injection or infusion.
  • the administration may be by any suitable route, such as by injection, for example intravenously or subcutaneously.
  • Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • bispecific antibodies of the invention and immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) of the invention (either alone or with one or more other (when used in combination with therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether it is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and the sensitivity to the antibody. response, and the attending physician’s judgment.
  • the antibody is suitably administered to the patient in a single treatment or over a series of treatments.
  • the invention provides the use of a bispecific antibody of the invention or an immunoconjugate or composition or combination product comprising the same in the production or preparation of a medicament for use as described herein, e.g. For the prevention or treatment of related diseases or conditions mentioned herein.
  • bispecific antibodies and immunoconjugates, compositions, pharmaceutical compositions, formulations, etc. comprising the same
  • Administration for the purposes described herein, for example for the prevention and/or treatment of the relevant diseases or conditions mentioned herein.
  • the treatment modality is, for example, surgery or radiation therapy.
  • the other therapeutic agents such as chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulatory agents (e.g., immunosuppressive agents), chemotherapeutic agents, anti-cancer drugs, immunotherapeutic agents , anti-angiogenic agent, targeted therapy therapeutic agent, cell therapy therapeutic agent, gene therapy therapeutic agent, hormonal therapy therapeutic agent, antiviral agent, antibiotic, analgesic, antioxidant, metal chelator or cytokine.
  • immunosuppressive agents e.g., immunosuppressive agents
  • chemotherapeutic agents e.g., anti-cancer drugs
  • immunotherapeutic agents e.g., anti-cancer drugs
  • anti-angiogenic agent e.g., targeted therapy therapeutic agent
  • targeted therapy therapeutic agent e.g., cell therapy therapeutic agent, gene therapy therapeutic agent, hormonal therapy therapeutic agent, antiviral agent, antibiotic, analgesic, antioxidant, metal chelator or cytokine.
  • the anti-bispecific antibodies provided herein can be used to detect the presence of CLDN18.2 and/or PD-L1 in a biological sample.
  • the term "detection" when used herein includes quantitative or qualitative detection. Exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), antibody molecule complexing Magnetic beads, ELISA assays, PCR-techniques (eg RT-PCR).
  • the biological sample is a body fluid, such as blood, serum, or plasma.
  • the methods include contacting a biological sample with a bispecific antibody as described herein under conditions that allow it to bind to PD-L1 and/or CLDN18.2, and detecting the presence of the bispecific antibody as described herein. Whether complexes form between antibodies and PD-L1 and/or CLDN18.2. Complex formation indicates the presence of PD-L1.
  • the method can be an in vitro or in vivo method.
  • the sample is obtained prior to treatment with the bispecific antibodies of the invention. In some embodiments, the sample is obtained prior to treatment with other therapies (eg, anti-PD-L1 antibody treatment or anti-CLDN18.2 antibody treatment). In some embodiments, the sample is obtained during or after treatment with other therapies.
  • other therapies eg, anti-PD-L1 antibody treatment or anti-CLDN18.2 antibody treatment.
  • the sample is obtained during or after treatment with other therapies.
  • PD-L1 and/or CLDN18.2 is detected prior to treatment, eg, prior to initiating treatment or prior to a session following a treatment interval.
  • Example 1 Changes in PD-L1 expression of tumor cells after CLDN18.2 (Claudin18.2) antibody mediates ADCC (antibody-dependent cell-mediated cytotoxicity) effect.
  • S1 represents the supernatant of target cells alone without adding antibodies
  • S2 represents the supernatant of target cells plus human PBMC plus isotype control hIgG1
  • S3 represents the supernatant of target cells plus human PBMC plus CLDN18.2 antibody 18B10
  • S4 represents the supernatant of target cells plus IFN- ⁇ .
  • target cells in group S1 expressed a background level of PD-L1.
  • the expression of PD-L1 increased in groups S3 and S4, while group S2 only changed slightly.
  • the antibody-mediated ADCC effect can upregulate the expression of PD-L1 in target cells, and the mechanism may be mediated by IFN- ⁇ produced by the ADCC effect.
  • Chaganty BKR et al. (Chaganty BKR et al., Cancer Letters, 2018: S0304383518303288) also found that IFN- ⁇ produced by the HER2 antibody trastuzumab through ADCC can upregulate the expression of PD-L1 in HER2-positive tumor cells.
  • IgG(H)scfv-18B10-AM4B6V1 is a bispecific antibody against CLDN18.2 and anti-PD-L1. Its light chain is derived from the light chain of anti-CLDN18.2 antibody 18B10. The heavy chain is composed of the heavy chain of the anti-CLDN18.2 antibody 18B10 and the scfv of the PD-L1 antibody AM4B6 (PCT/CN2021/131389). The two are connected together with a (Gly4Ser)4 linker, as shown in Figure 2A and Table 1 shown. IgG(H)scfv-AM4B6-18B10V1 is also a bispecific antibody against PD-L1 and anti-CLDN18.2.
  • the heavy chain is composed of the heavy chain of the anti-PD-L1 antibody AM4B6 and the scfv of the CLDN18.2 antibody 18B10, which are connected together with a (Gly4Ser)4 linker, as shown in Figure 2B and Table 1.
  • a (Gly4Ser)4 linker As shown in Figure 2B and Table 1.
  • the C-terminal lysine residue of the antibody heavy chain was mutated to alanine to reduce proteolytic cleavage.
  • Two disulfide bonds (H44C-L100C) are introduced into the scfv of the anti-PD-L1 antibody AM4B6 of the V1 molecule, thereby generating the IgG(H) scfv-18B10-AM4B6V2 molecule.
  • the light chain of IgG(H)scfv-18B10-AM4B6V2 is identical to the light chain of the IgG(H)scfv-18B10-AM4B6V1 molecule.
  • the heavy chain of anti-CLDN18.2 antibody 18B10 and the scfv of anti-PD-L1 antibody AM4B6 are also connected using a (Gly4Ser)4 linker, as shown in Figure 2C and Table 1.
  • the C-terminal lysine residue of the constant region of the heavy chain of antibody 18B10 was mutated to alanine to reduce proteolytic cleavage.
  • the CLDN18.2 ⁇ PD-L1 bispecific antibody was expressed by transfecting Expi-CHO cells with the vector pCDNA3.1(+) containing DNA encoding the light chain and DNA encoding IgG-scfv, using ExpiFectamine according to the method provided by Invitrogen. TM CHO reagent. The supernatant was collected when the cell viability exceeded 60% and filtered through a 0.22 ⁇ m filter membrane to remove cell debris. The filtered supernatant was then loaded onto a pre-equilibrated Protein A affinity column. Protein A resin was washed with equilibration buffer (PBS) and antibodies were eluted with 25mM citrate (pH 3.5).
  • PBS equilibration buffer
  • the purified antibody solution was adjusted to pH 6.0-7.0 using 1M Tris base (pH 9.0).
  • the sample was further purified using Superdex chromatography to remove aggregates. Finally, the purified antibodies were characterized by SDS-PAGE and SEC-HPLC analyses.
  • PD-L1 monoclonal antibody AM4B6 (PCT/CN2021/131389, the heavy chain is SEQ ID NO: 40, which has the L234F/L235E/P331S mutation in the Fc region that weakens ADCC activity, the light chain is SEQ ID NO: 26). Its expression and purification methods were prepared as described for bispecific antibody methods.
  • the binding activity of IgG(H)scfv-18B10-AM4B6V1 to NUGC-4 cells is similar to that of monoclonal antibody 18B10, except that the maximum signal value is slightly lower than 18B10. In contrast, binding of IgG(H)scfv-AM4B6-18B10V1 was greatly affected.
  • the binding activity of IgG(H)scfv-18B10-AM4B6V2 to NUGC-4 cells is also similar to that of IgG(H)scfv-18B10-AM4B6V1 and 18B10, indicating that its binding activity to CLDN18.2 is not affected by disulfide. The impact of key introduction.
  • HEK293T-PD-L1 cells were purchased from Sino-US Crown Biotechnology Co., Ltd., product number 2005.
  • the detection method of CLDN18.2 ⁇ PD-L1 bispecific antibody binding to cells is the same as above.
  • ADCC activity was detected using Jurkat-NFAT-luc-Fc ⁇ RIIIA-V176 cells as effector cells and NUGC-4 cells as target cells.
  • Jurkat-NFAT-luc-Fc ⁇ RIIIA-V176 cells were constructed in-house by Suzhou Chuangsheng Pharmaceutical Group Co., Ltd. Simply, pGL4.30-luc/NFAT-RE/Hygro plasmid (Promega) was transferred to Jurkat cells (Shanghai Institutes for Biological Sciences, Cat#SCSP-513), and then screened with hygromycin to obtain cells with stable expression.
  • strain Jurkat-NFAT-luc was transferred to Jurkat cells (Shanghai Institutes for Biological Sciences, Cat#SCSP-513), and then screened with hygromycin to obtain cells with stable expression.
  • the Fc ⁇ RIIIA-V176 (SEQ ID NO:37) sequence was constructed into the vector pVitro-neo (InvivoGen) to obtain the pVitro-neo-pcDNA3.1-Fc ⁇ RIIIA-V176 plasmid.
  • the obtained cell line was further transfected with pVitro-neo-Fc ⁇ RIIIA-V176 plasmid, and the stable expression cell line Jurkat-NFAT-luc-Fc ⁇ RIIIA-V176 was obtained by screening with antibiotic G418.
  • NUGC-4 cells in the logarithmic growth phase were resuspended in RPMI1640+10% FBS, then plated at 1 ⁇ 10 4 cells per well, and incubated at 37°C for 30 minutes to obtain a culture plate containing target cells.
  • Antibody and isotype control hIgG1 were diluted with RPMI1640 + 10% FBS (starting concentration 20 nM, 5-fold dilution), and then added to the target cell culture plate at a final concentration of 20 to 0.001 nM.
  • Jurkat-NFAT-luc-Fc ⁇ RIIIA-V176 cells in logarithmic growth phase were added to the above plate at 6 ⁇ 10 4 cells per well.
  • CLDN18.2 ⁇ PD-L1 bispecific antibody IgG(H)scfv-18B10-AM4B6V1 is similar to monoclonal antibody 18B10 and can produce an ADCC effect on the target cell NUGC-4.
  • the ADCC of the two The activity is similar.
  • the ADCC activity of IgG(H)scfv-AM4B6-18B10V1 was greatly affected.
  • the ADCC activity of IgG(H)scfv-18B10-AM4B6V2 was basically the same as that of IgG(H)scfv-18B10-AM4B6V1.
  • IgG(H)scfv-18B10-AM4B6V1 can also produce ADCC effect on the target cell HEK293T-PD-L1.
  • IgG(H)scfv-AM4B6-18B10V1 can also induce the ADCC effect through the binding of PD-L1.
  • This experiment used 293T-PD-L1-CD3L cells that express both PD-L1 and anti-CD3scFv, and Jurkat-NFAT-Luc-PD-1 cells that express both PD-1 and NFAT reporter gene. Activation of NFAT can cause luciferase gene transcription and expression, which can be detected by adding its substrate.
  • the two cell lines were constructed as follows:
  • the above HEK293T-PD-L1 cells were transfected with the plasmid pcDNA3.1-hygro-CD3L containing anti-CD3 scfv (obtained by constructing the anti-CD3 scfv (SEQ ID NO:38) sequence into the vector pcDNA3.1(+) ), and then selected and cultured in DMEM+10% FBS medium containing 3 ⁇ g/mL puromycin and 10 ⁇ g/mL hygromycin B.
  • the selected cells are 293T-PD-L1-CD3L cells.
  • Jurkat cells (Shanghai Cell Institute of the Chinese Academy of Sciences, Catalog No. SCSP-513) were transfected with a plasmid containing PD-1 (the plasmid containing PD-1 was constructed by constructing the human PD-1 sequence (SEQ ID NO: 39) into pcDNA3.1 (+), obtain human PD-1-pcDNA3.1(+)) and plasmid pGL4.30-luc/NFAT-RE/Hygro (Promega) containing NFAT-luciferase gene, and incubate them in G418 containing 500 ⁇ g/mL Jurkat-NFAT-Luc-PD-1 cells were cultured in RPMI1640+10% FBS selection medium with 200 ⁇ g/mL hygromycin B.
  • 293T-PD-L1-CD3L cells were resuspended at 2 ⁇ 10 6 cells/ml.
  • the above-constructed CLDN18.2 ⁇ PD-L1 bispecific antibody, monoclonal antibody AM4B6 and isotype control antibody hIgG1 were respectively diluted gradiently with RPMI medium + 2% FBS (initial concentration 33 nm, 3-fold gradient).
  • Jurkat-NFAT-Luc-PD1 cells were resuspended in RPMI medium+2% FBS at 4 ⁇ 10 ⁇ 6 cells/ml. Then add 20 ⁇ l of Jurkat-NFAT-Luc-PD1 cells per well into the half-well plate and incubate at 37°C, 5% CO for 5 hours. Finally, add 60 ⁇ l of OneGlo detection reagent (Promega, E6120) to each well and incubate at room temperature for 5 minutes. Read the chemiluminescence value with a microplate reader. Data were analyzed using GraphPad Prism software.
  • the blocking activity of IgG(H)scfv-18B10-AM4B6V1 and IgG(H)scfv-AM4B6-18B10V1 on PD-L1/PD-1 is similar to that of PD-L1 monoclonal antibody.
  • AM4B6 is similar, which indicates that in addition to the ADCC killing effect on tumor cells, the two bispecific antibodies can also block the immunosuppressive effect of PD-L1/PD-1 in the tumor microenvironment.
  • the blocking activity of IgG(H)scfv-18B10-AM4B6V2 was similar to that of IgG(H)scfv-18B10-AM4B6V1, as shown in Figure 7B .
  • Human PBMC (Miaoshun, TPCS PB025C) were stimulated with CD3 antibody (Dynabeads, Gibco, Cat. No. 11131D) for 24 hours in advance to obtain pre-stimulated effector cells. Then NCI-H460-CLDN18.2 cells (Kangyuan Bochuang (Cat#KC1450)) (target cells) in the logarithmic growth phase were mixed with pre-stimulated PBMC Co-incubate with an effector/target ratio of 15:1.
  • the isotype control group also had significant IFN- ⁇ .
  • Both AM4B6 and 18B10 induced higher IFN- ⁇ production than the isotype control antibody, indicating that the blocking effect of PD-L1 and the ADCC activity mediated by CLDN18.2 coexist in this experimental system.
  • IgG(H)scfv-18B10-AM4B6V1 produced higher IFN- ⁇ , indicating that the bispecific antibody effectively combines the effects of two targets.
  • NCI-H460-CLDN18.2 cells are human large cell lung cancer cells transfected with CLDN18.2. They endogenously express PD-L1 and were purchased from Kangyuan Bochuang (Cat#KC1450). Digest and subculture using trypsin-EDTA (Hyclone) and subculture twice a week. Cells in the logarithmic growth phase were used for tumor inoculation into NOD-SCID mice, which were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. and were 5-6 weeks old at the time of inoculation.
  • mice Each SPF female NOD-SCID mouse was subcutaneously inoculated with 3 ⁇ 10 ⁇ 6 NCI-H460-CLDN18.2 cells and 1.5 ⁇ 10 ⁇ 6 human PBMC, as well as 50% matrigel (Corning). Four hours after inoculation, the mice were randomly divided into 5 groups, with 10 mice in each group. 30mg/kg isotype control hIgG1, 15mg/kg 18B10, 15mg/kg AM4B6, 15mg/kg 18B10+15mg/kg AM4B6 and 20mg/kg IgG(H)scfv-18B10-AM4B6V1 were administered i.p. twice a week, respectively. Administration was given for a total of 5 weeks.
  • a caliper (INSIZE) to measure the tumor diameter 2 or 3 times a week.
  • the results were analyzed using Prism GraphPad and expressed as mean ⁇ S.E.M.
  • Use T test to compare the differences between the two groups. If the p value is ⁇ 0.05(*) or ⁇ 0.01(**), the difference is significant.
  • CLDN18.2 antibody 18B10 or PD-L1 antibody AM4B6 alone had only very weak tumor inhibitory activity, with a tumor inhibition rate (TGI) of 33% ( Figure 9, Table 2).
  • TGI tumor inhibition rate
  • the TGI of the CLDN18.2 ⁇ PD-L1 bispecific antibody IgG(H)scfv-18B10-AM4B6V1 administration group further increased to 70%, indicating that in this tumor model that expresses both CLDN18.2 and PD-L1, Bispecific antibodies have better efficacy.

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Abstract

本发明提供了一种抗体。该抗体可以特异性结合人CLDN18.2和人PD-L1。本发明进一步提供了编码所述抗体的核酸分子、用于表达所述抗体的表达载体、宿主细胞及其制备方法。本发明还提供了使用本发明的抗体的诊断和治疗方法。

Description

结合PD-L1和CLDN18.2的抗体及其用途 技术领域
本发明提供了一种抗体。该抗体可以特异性结合人CLDN18.2和人PD-L1。本发明进一步提供了编码所述抗体的核酸分子、用于表达所述抗体的表达载体、宿主细胞及其制备方法。本发明还提供了使用本发明的抗体的诊断和治疗方法。
背景技术
程序性死亡1(PD-1)及其配体PD-L1和PD-L2是T细胞介导的免疫应答的调节中的关键共抑制分子。PD-1是一种在外周组织中的激活的T细胞的表面上表达的具有单个胞外免疫球蛋白超家族(IgSF)V-集结构域(V-set domain)的I型膜蛋白(Zhang X等人,《免疫力(Immunity)》,2004,20(3):337-347)。PD-L1和PD-L2通常在树突状细胞和巨噬细胞上表达，并且它们的胞外结构域由膜远端IgSF V-集结构域和膜近端IgSF C-set结构域构成(Latchman Y等人,《自然免疫学(Nature Immunology)》,2001,2(3):261-268)
PD-L1还通过与另一种受体B7.1(也称为B7-1或CD80)相互作用来负调节T细胞功能。PD-L1/PD-1和PD-L1/B7.1复合物的形成负调节T细胞受体信号传导，从而导致随后下调T细胞激活并抑制抗肿瘤免疫活性。
PD-L1在不同的肿瘤中经常过度表达并且其与T细胞上的PD-1相互作用使癌细胞能够避开T细胞介导的免疫应答。因此，阻断PD-1/PD-L1相互作用可以恢复T细胞激活和抗肿瘤应答。尽管已显示肿瘤细胞和/或浸润性免疫细胞表达PD-L1与对PD-1/PD-L1靶向疗法的临床应答之间存在关联，但这种关联并非完美无缺。只有少数PD-L1阳性肿瘤对这些治疗有应答，而同时某些PD-L1阴性肿瘤对这些治疗也有应答。因此，抗PD-L1抗体在同时具有高亲和力，高治疗功效和低毒副作用上，还是面临着很大的挑战。
Claudin是上皮紧密连接的关键结构和功能组分，其作用是调节细胞-细胞通透性、维持离子稳态、并且支持细胞粘附和极性。紧密连接蛋白是22-27kDa的四分子交联体(tetraspan)跨膜蛋白，其在细胞膜内或跨细胞膜进行多聚化以形成保护屏障。已经报道的24种紧密连接蛋白由于它们的组织定位特异性和由于它们与其他蛋白质的相互作用而不同。
紧密连接蛋白18(CLDN18)最初被鉴定为转录因子T/EBP/NKX2.1的靶基因。符合其与其他紧密连接蛋白家族成员的同源性，CLDN18被证实定位于小鼠和人的细胞紧密连接处。已显示CLDN18编码由可变剪接产生的两种同种型：在正常肺中特异性表达的CLDN18.1、和在胃粘膜的分化细胞中表达的CLDN18.2。
CLDN18.2是具有两个细胞外环的261个氨基酸的蛋白质，并且与CLDN18.1具有92％的序列同一性。与第二个细胞外环不同，CLDN18.2的 第一个细胞外环与CLDN18.1具有八个氨基酸差异。CLDN18.2与其他家族成员的同源性更为有限，与CLDN1、CLDN6和CLDN7具有29％-34％的总体同一性。
CLDN18.2在几种肿瘤类型(包括胃癌、胰腺癌、食管癌、粘液性卵巢癌和非小细胞肺癌)中表达。CLDN18.2在胃癌中的表达包括浸润性前沿和转移部位，但是据报道CLDN18的绝对水平在这些环境中降低。CLDN18.2在多种肿瘤类型中的表达(其中正常组织表达主要限于胃中的分化细胞)已经导致将CLDN18.2视为胃癌和其他适应症的治疗靶标。
因此，通过CLDN18.2抗体的特异性将PD-L1抗体的作用局限在CLDN18.2表达的肿瘤部位，这样可以在提高PD-L1抗体局部浓度的同时，也减少了PD-L1抗体在正常组织的分布和毒副作用。这样就需要能够特异性结合PD-L1和CLDN18.2的双特异性抗体。
发明内容
本发明提供了一种新型的抗PD-L1抗体，以及应用其构建的与CLDN18.2抗体的双特异性抗体。在一些实施方案中，本发明的抗PD-L1抗体与人PD-L1的结合亲和力很高，能够识别人PD-L1。
在一些实施方案中，抗PD-L1抗体可以以高亲和力特异性结合PD-L1(例如人PD-L1)。
本发明的抗CLDN18.2/PD-L1双特异性抗体具有以下一种或多种特性，因此具有改善的治疗功效：
(1)与(人)CLDN18.2结合，例如在CLDN18.2阳性细胞(例如表达CLDN18.2的肿瘤细胞)中，与细胞上表达的CLDN18.2结合，例如以高亲和力与CLDN18.2结合；
(2)与(人)PD-L1结合，例如在PD-1或PD-L1阳性细胞(例如，表达PD-1或PD-L1的肿瘤细胞，例如高表达或低表达，尤其是低表达PD-1或PD-L1)中，与细胞上表达的PD-L1结合，例如以高亲和力与PD-L1结合；
(3)能够产生对靶细胞的ADCC效应，例如通过对CLDN18.2的结合和/或对PD-L1的结合诱导ADCC效应；
(4)能够通过ADCC效应诱导CLDN18.2的阳性细胞中PD-L1的表达升高，例如在不表达PD-L1的CLDN18.2的阳性细胞中诱导PD-L1的表达；
(5)有效阻断PD-1与PD-L1的结合；
(6)对T细胞具有有效的激活作用，例如激活T细胞分泌干扰素，例如IFN-γ，例如通过阻断PD-1对T细胞的激活的抑制；
(7)具有更好的肿瘤抑制作用，例如肿瘤抑制率在35％、40％、45％、50％、55％、60％、65％、70％以上。
本发明进一步涉及如下技术方案：
1、双特异性抗体，其包含针对(人)PD-L1的第一结合特异性和针对(人) CLDN18.2的第二结合特异性，其中所述特异性结合PD-L1的第一抗原结合区包含重链可变区VH的3个CDR，HCDR1、HCDR2和HCDR3，以及轻链可变区VL的3个CDR，LCDR1、LCDR2和LCDR3，其中
所述HCDR1、HCDR2和HCDR3为如SEQ ID NO:17所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3；且所述LCDR1、LCDR2和LCDR3为如SEQ ID NO:22所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3。
2、技术方案1的双特异性抗体，其中
所述HCDR1、HCDR2和HCDR3分别为如SEQ ID NO:18所示的HCDR1、如SEQ ID NO：19所示的HCDR2、如SEQ ID NO:20所示的HCDR3；且所述LCDR1、LCDR2和LCDR3为如SEQ ID NO:23所示的LCDR1、如SEQ ID NO:24所示的LCDR2和如SEQ ID NO:25所示的LCDR3。
3、技术方案1或2的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区包含重链可变区VH，其中所述重链可变区
(i)包含与SEQ ID NO:17或30所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:17或30所示的氨基酸序列或由所述氨基酸序列组成。
4、技术方案1-3中任一项的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区包含轻链可变区VL，其中所述轻链可变区
(i)包含与SEQ ID NO:22或31所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:22或31的氨基酸序列或由所述氨基酸序列组成。
5、技术方案1-4中任一项的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区包含重链可变区VH和轻链可变区VL，其中
(i)所述重链可变区包含SEQ ID NO:17或30所示的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；且所述轻链可变区包含SEQ ID NO:22或31所示的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
6、技术方案1-5中任一项的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区包含重链可变区VH和轻链可变区VL，其中VH和VL分别包含如下所示的氨基酸序列或由所述氨基酸序列组成：
(i).SEQ ID NO:17和SEQ ID NO:30；或
(ii).SEQ ID NO:22和SEQ ID NO:31。
7、技术方案1-6中任一项的双特异性抗体，所述特异性结合CLDN18.2的第二 抗原结合区包含重链可变区VH的3个CDR，HCDR1、HCDR2和HCDR3，以及轻链可变区VL的3个CDR，LCDR1、LCDR2和LCDR3，其中
所述HCDR1、HCDR2和HCDR3为如SEQ ID NO:1所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3；且所述LCDR1、LCDR2和LCDR3为如SEQ ID NO:6所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3。
8、技术方案7的双特异性抗体，其中所述特异性结合CLDN18.2的第二抗原结合区包含
如SEQ ID NO:2所示的HCDR1、如SEQ ID NO：3所示的HCDR2、如SEQ ID NO:4所示的HCDR3；如SEQ ID NO:7所示的LCDR1、如SEQ ID NO:8所示的LCDR2和如SEQ ID NO:9所示的LCDR3。
9、技术方案7或8的双特异性抗体，其中所述特异性结合CLDN18.2的第二抗原结合区包含重链可变区VH，其中所述重链可变区
(i)包含与SEQ ID NO1所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:1所示的氨基酸序列或由所述氨基酸序列组成。
10、技术方案7-9中任一项的双特异性抗体，其中所述特异性结合CLDN18.2的第二抗原结合区包含轻链可变区VL，其中所述轻链可变区
(i)包含与SEQ ID NO:6所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:6的氨基酸序列或由所述氨基酸序列组成。
11、技术方案7-10中任一项的双特异性抗体，其中所述特异性结合CLDN18.2的第二抗原结合区包含重链可变区VH和轻链可变区VL，其中VH和VL分别包含如下所示的氨基酸序列或由所述氨基酸序列组成：SEQ ID NO:1和SEQ ID NO:6。
12、技术方案1-11中任一项所述双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区和/或特异性结合CLDN18.2的第二抗原结合区还包含重链恒定区HC或来自所述HC的Fc区，例如，所述抗体重链恒定区HC为IgG1、IgG2、IgG3或IgG4的重链恒定区，优选的IgG1的重链恒定区。
13、技术方案12所述的双特异性抗体，其中所述重链恒定区
(i)包含与选自SEQ ID NO:13的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或
(ii)包含选自SEQ ID NO:13的氨基酸序列或由所述氨基酸序列组成。
14、技术方案12所述的双特异性抗体，其中所述Fc区
(i)包含与选自SEQ ID NO:14的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或
(ii)包含选自SEQ ID NO:14的氨基酸序列或由所述氨基酸序列组成。
15、技术方案1-14中任一项所述的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区和/或特异性结合CLDN18.2的第二抗原结合区还包含轻链恒定区，例如所述轻链恒定区为lambda或kappa轻链恒定区。
16、技术方案15的双特异性抗体，其中所述轻链恒定区
(i)包含与选自SEQ ID NO:16的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或
(ii)包含选自SEQ ID NO:16的氨基酸序列或由所述氨基酸序列组成。
17、技术方案1-16中任一项所述的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区包含重链，其中所述重链包含SEQ ID NO:21的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
18、技术方案1-17中任一项所述的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区包含轻链，其中所述轻链包含SEQ ID NO:26的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
19、技术方案12或13所述的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区包含重链和轻链，其中
(i)所述重链包含SEQ ID NO:21的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成；且所述轻链包含SEQ ID NO:26的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
20、技术方案19所述的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区包含重链和轻链，其中所述重链包含SEQ ID NO：21所示的氨基酸序列，或由所述氨基酸序列组成，且所述轻链包含SEQ ID NO:26所示的氨基酸序列或由所述氨基酸序列组成。
21、技术方案1-20中任一项所述的双特异性抗体，其中所述特异性结合CLDN18.2的第二抗原结合区包含重链，其中所述重链包含SEQ ID NO:5的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
22、技术方案1-21中任一项所述的双特异性抗体，其中所述特异性结合CLDN18.2的第二抗原结合区包含轻链，其中所述轻链包含SEQ ID NO:10的氨 基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
23、技术方案21或22所述的双特异性抗体，其中所述特异性结合CLDN18.2的第二抗原结合区包含重链和轻链，其中
(i)所述重链包含SEQ ID NO:5的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成；且所述轻链包含SEQ ID NO:10的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
24、技术方案23所述的双特异性抗体，其中所述特异性结合CLDN18.2的第二抗原结合区包含重链和轻链，其中所述重链包含SEQ ID NO：5所示的氨基酸序列，或由所述氨基酸序列组成，且所述轻链包含SEQ ID NO:10所示的氨基酸序列或由所述氨基酸序列组成。
25、技术方案1-24任一项所述的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区和/或特异性结合CLDN18.2的第二抗原结合区来源于人源化抗体或嵌合抗体。
26、技术方案1-25中任一项所述的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区和/或特异性结合CLDN18.2的第二抗原结合区为选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体(例如scFv)、(Fab’)2、单结构域抗体例如VHH、dAb(domain antibody)或线性抗体，例如所述scFv包含二硫键稳定的突变，所述突变例如其中所述重链可变区和/或轻链可变区包含引入二硫键的突变，例如所述重链可变区包含在第44位氨基酸取代为半胱氨酸的突变(Kabat编号)，和/或所述轻链可变区包含在第100位氨基酸取代为半胱氨酸的突变(Kabat编号)。
27、技术方案26所述的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区为scV，优选地，所述scFv包含SEQ ID NO:27或29的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
28、技术方案26所述的双特异性抗体，其中所述特异性结合CLDN18.2的第二抗原结合区为scV，优选地，所述scFv包含SEQ ID NO:11的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
29、技术方案1-28中任一项所述的双特异性抗体，其中所述双特异性抗体中的第一抗原结合区和第二抗原结合区之间通过接头连接，所述接头例如包含氨基酸序列(G4S)n,其中n＝1,2,3,4或5，优选地，n＝3或4，更优选地，n＝4。
30、技术方案1-29中任一项所述的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区为全长抗体，且所述特异性结合CLDN18.2的第二抗原结合区为scFv；或者所述特异性结合CLDN18.2的第二抗原结合区为全长抗体，且所 述特异性结合PD-L1的第一抗原结合区为scFv。
31、技术方案30的双特异性抗体，其中scFv与全长抗体的重链恒定区的C末端连接。
32、技术方案30的双特异性抗体，其中两个scFv分别与全抗抗体的两个重链恒定区的C末端连接。
33、技术方案1-29中任一项所述的双特异性抗体，其包含轻链和重链，
其中轻链从N端到C端包含VL-A-CL；
重链从N端到C端包含VH-A-CH-scFv；
其中scFv包含VH-B和VL-B；
其中VL-A为特异性结合PD-L1的第一抗原结合区的轻链可变区VL，VH-A为特异性结合PD-L1的第一抗原结合区的重链可变区VH；VH-B为特异性结合CLDN18.2的第二抗原结合区的重链可变区VH，VL-B为特异性结合CLDN18.2的第二抗原结合区的轻链可变区VL；或者VL-A为特异性结合CLDN18.2的第二抗原结合区的轻链可变区VL，VH-A为特异性结合CLDN18.2的第二抗原结合区的重链可变区VH；VH-B为特异性结合PD-L1的第一抗原结合区的重链可变区VH，VL-B为特异性结合PD-L1的第一抗原结合区的轻链可变区VL；
CL为轻链恒定区；
CH为重链恒定区；
scFv的VH-B和VL-B之间包含接头或不包含接头；
其中所述CH与scFv之间包含接头或不包含接头。
34、技术方案33的双特异性抗体，其包含两条重链和两条轻链，例如所述重链相同和/或所述轻链相同。
35、技术方案33或34所述的双特异性抗体，其中所述scFv从N端到C端包含VH-B-VL-B，且VH-B在其N末端与CH的C末端通过或不通过接头连接。
36、技术方案33-35中任一项所述的双特异性抗体，其中所述接头例如包含氨基酸序列(G4S)n,其中n＝1,2,3,4或5，优选地，n＝3或4，更优选地，n＝4。
37、技术方案33-36中任一项所述的双特异性抗体，其中所述重链包含SEQ ID NO:32、33或34的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
38、技术方案33-37中任一项所述的双特异性抗体，其中所述轻链包含SEQ ID NO:10或26的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
39、技术方案33或34所述的双特异性抗体，其中
(i)所述重链包含SEQ ID NO:32或34的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成；
且所述轻链包含SEQ ID NO:10的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成；
(ii)所述重链包含SEQ ID NO:33的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成；
且所述轻链包含SEQ ID NO:26的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
40、技术方案39所述的双特异性抗体，其中
(i)所述重链包含SEQ ID NO:32或34的氨基酸序列或由所述氨基酸序列组成；且所述轻链包含SEQ ID NO:10的氨基酸序列或由所述氨基酸序列组成；
(ii)所述重链包含SEQ ID NO:33的氨基酸序列或由所述氨基酸序列组成；
且所述轻链包含SEQ ID NO:26的氨基酸序列或由所述氨基酸序列组成。
41、核酸分子，其编码技术方案1-40中任一项的所述的双特异性抗体的任一条链。
42、表达载体，其包含技术方案41的核酸分子，优选地，所述表达载体为pCDNA，例如pCDNA3.1。
43、宿主细胞，其包含技术方案41所述的核酸分子或技术方案42所述的表达载体，优选地，所述宿主细胞是原核的或真核的，例如293细胞或CHO细胞，例如293F细胞或293T细胞或Expi-CHO细胞。
44、制备技术方案1-40中任一项所述的双特异性抗体的方法，所述方法包括，在适合所述抗体的链表达的条件下，培养包含技术方案41所述的核酸分子或技术方案42所述的表达载体的宿主细胞，或技术方案43所述的宿主细胞，和任选地从所述宿主细胞(或宿主细胞培养基)回收所述抗体。
45、免疫缀合物，其包含技术方案1-40中任一项所述的双特异性抗体；
46、技术方案45的免疫缀合物，其还包含化疗剂、细胞因子、细胞毒性剂或小分子药物、纯化部分如磁珠、生物素、清除修饰剂(例如，延长半衰期的聚合物，如PEG)、化学治疗剂、毒素、放射性同位素、镧系元素、可检测标记(例如，发光标记、荧光标记、酶底物标记)、DNA烷基化剂、拓扑异构酶抑制剂、微管蛋白结合剂或其它抗癌药物。
47、药物组合物或药物或制剂，其包含技术方案1-40中任一项的所述的双特异性抗体，或技术方案45或46的免疫缀合物以及任选地药用辅料。
48、药物组合产品，其包含技术方案1-40中任一项所述的双特异性抗体，或技术方案45或46的免疫缀合物，以及一种或多种其它治疗剂(例如化学治疗剂、抗癌药物、免疫治疗剂、抗血管生成剂、靶向疗法治疗剂、细胞疗法治疗剂、基因疗法治疗剂、激素疗法治疗剂、抗病毒剂、抗生素、镇痛剂、抗氧化剂、金属螯合剂或细胞因子)。
49、在受试者中预防或治疗癌症的方法，包括向受试者施用有效量的技术方案1-40中任一项所述的双特异性抗体，或技术方案45或46的免疫缀合物，或技术方案47的药物组合物或药物或制剂；或技术方案48的药物组合产品。
50、技术方案49的方法，其中所述癌症的肿瘤细胞表面存在CLDN18.2的表达，例如与相同组织的非癌细胞相比，所述肿瘤细胞表面表达的CLDN18.2高。
51、技术方案50的方法，其中
(i)在所述癌细胞或肿瘤细胞在其细胞表面存在PD-L1蛋白，
(ii)与相同组织类型的非癌细胞相比，在所述癌症或肿瘤细胞的细胞表面具有更高水平的PD-L1；
(iii)在所述癌细胞或肿瘤细胞在其细胞表面不存在PD-L1蛋白的表达，或者
(iv)在所述癌细胞或肿瘤细胞在其细胞表面存在PD-L1蛋白，且与高表达PD-L1的癌症或肿瘤相比，在所述癌症或肿瘤细胞的细胞表面具有低水平的PD-L1；或
(v)与相同组织类型的非癌细胞相比，在所述癌症或肿瘤细胞的细胞表面具有更高水平的PD-L1，且与高表达PD-L1的癌症或肿瘤相比，在所述癌症或肿瘤细胞的细胞表面具有低水平的PD-L1。
52、技术方案49-51中任一项的方法，其中所述癌症是实体肿瘤，例如胃肠道肿瘤、胆道肿瘤或肺癌，例如胃癌、食管癌、胰腺癌、结直肠癌、胆管癌、胆囊癌或肺癌。
53、技术方案49-52中任一项的方法，其中所述方法还包括与其它疗法例如治疗方式(例如手术或放疗)和/或其它治疗剂(例如化学治疗剂、抗癌药物、免疫治疗剂、抗血管生成剂、靶向疗法治疗剂、细胞疗法治疗剂、基因疗法治疗剂、激素疗法治疗剂、抗病毒剂、抗生素、镇痛剂、抗氧化剂、金属螯合剂或细胞因子)组合施用。
54、检测PD-L1和/或CLDN18.2在生物样品中的存在的方法，其包括
(i)将生物样品与技术方案1-40中任一项所述的双特异性抗体在允许其与PD-L1和/或CLDN18.2结合的条件下接触，
(ii)检测在该抗体或双特异性抗体和PD-L1和/或CLDN18.2之间是否形成复合物，
其中复合物的形成表示存在PD-L1和/或CLDN18.2。
附图说明
图1显示了CLDN18.2抗体在不同的细胞中介导的ADCC效应能上调细胞中PD-L1的表达；
图2显示了三种CLDN18.2×PD-L1双特异性抗体的结构示意图。
图3显示了三种CLDN18.2×PD-L1双特异性抗体和18B10抗体与NUGC-4细胞的结合活性；
图4显示了三种CLDN18.2×PD-L1双特异性抗体和AM4B6抗体与PD-L1阳性细胞的结合活性；
图5显示了CLDN18.2×PD-L1双特异性抗体和18B10抗体对NUGC-4的ADCC效应；
图6显示了CLDN18.2×PD-L1双特异性抗体和AM4B6抗体对PD-L1阳性细胞的ADCC效应；
图7显示了CLDN18.2×PD-L1双特异性抗体和AM4B6抗体对PD-1与PD-L1结合的阻断；
图8显示了CLDN18.2×PD-L1双特异性抗体、AM4B6和18B10对PBMC的激活作用；
图9显示了CLDN18.2×PD-L1双特异性抗体、AM4B6和18B10对肿瘤模型的抑制作用。
发明详述：
应理解本发明不限于本文中描述的特定方法学、方案和试剂，因为这些可以变化。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本发明的范围，其仅会由所附权利要求书限制。
I.定义
为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。除非另外定义，本文中使用的所有技术和科学术语与本发明所属领域中普通技术人员通常的理解具有相同的含义。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
如本文所用，术语“和/或”意指可选项中的任一项或可选项的两项或多项或全部。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
在本文中当提及“第一”“第二”、后缀“A”和后缀“B”时，仅为了区分两个结构 域或两条链，而不以任何方式表明两个结构域的位置。
本文所用的术语“CLAUDIN”或“CLDN”是决定细胞间紧密连接结构的最重要的骨架蛋白，其参与粘附连接，并且在肿瘤细胞的转移和侵袭中起到重要作用。Claudin蛋白广泛存在于哺乳动物上皮和内皮细胞中，其分布主要是上皮细胞侧面以及基底细胞质膜上。不同的Claudin蛋白在不同组织中具有各自特异性表达，其中Claudin18(CLDN18)基因定位于3q22.3，分子量24kDa，包含261个氨基酸残基，属Claudins超家族成员，其蛋白结构包含2个细胞外环和4个穿膜区。人CLDN18或Claudin18蛋白的两个亚型分别是Claudin18.1或CLDN18.1(UniProt ID:P56856-1)，和Claudin18.2或CLDN18.2(UniProt ID:P56856-2)，在二者蛋白的一级结构序列中，仅N端信号肽至细胞外环1(Loop1)结构的某些位置的氨基酸残基上有所区别，尤其是在细胞外环1上，CLDN18.1和CLDN18.2仅8个氨基酸不同。CLDN18的两种亚型蛋白的种属间序列同源性也非常高。其中CLDN18.2的细胞外环1在人、小鼠、猕猴等不同物种上序列完全一致，而人与小鼠的CLDN18.2蛋白同源性达到84％，表明CLDN18.2蛋白序列极端保守性(O.Tureci.等人.,Gene 481:83-92,2011)。CLDN18.2或其任何变体和同种型可以从天然表达它们的细胞或组织中分离，或者使用本领域熟知的技术和/或本文所述的那些技术重组产生。在一个实施方案中，本文所述的CLDN18.2是人CLDN18.2。
本文所用的术语“抗CLDN18.2抗体”、“抗CLDN18.2”、“CLDN18.2抗体”或“结合CLDN18.2的抗体”或“特异性结合CLDN18.2的抗体”是指这样的抗体，所述抗体能够以足够的亲和力结合(人)CLDN18.2。在一个实施方案中，所述(人)CLDN18.2抗体在体外或体内以高亲和力结合(人)CLDN18.2。在一个实施方案中，所述(人)CLDN18.2抗体不结合CLDN18.1。在一个实施方案中，所述(人)CLDN18.2抗体与表达CLDN18.2的细胞结合而不结合表达CLDN18.1的细胞。在一些实施方案中，所述结合例如通过放射性免疫测定(RIA)、生物膜薄层干涉测定法(BLI)、MSD测定法或表面等离子体共振法(SPR)或流式细胞术测量的。
如本文所用的术语“程序性细胞死亡1配体1”、“PD-L1”、“程序性死亡配体1”、“分化簇274”、“CD274”或“B7同系物1”是指来自任何脊椎动物来源的任何天然PD-L1，所述任何脊椎动物来源包括哺乳动物，诸如灵长类(例如，人)和啮齿类(例如，小鼠和大鼠)。所述术语涵盖“全长”、未加工的PD-L1以及由细胞中的加工所产生的任何形式的PD-L1。PD-L1可作为跨膜蛋白或作为可溶性蛋白存在。所述术语还涵盖天然存在的PD-L1的变体，例如剪接变体或等位基因变体。PD-L1的基本结构包括4个结构域：胞外Ig样V型结构域和Ig样C2型结构域、跨膜结构域以及细胞质结构域。在一个实施方案中，本文所述的PD-L1是人PD-L1。可在NCBI Gene ID No.29126下找到关于人PD-L1基因(包括基因组DNA序列)的另外信息。示例性全长人PD-L1蛋白的氨基酸序列可例如在NCBI登录号NP_001254653或UniProt登录号Q9NZQ7下找到。
本文所用的术语“抗PD-L1抗体”、“抗PD-L1”、“PD-L1抗体”或“结合PD-L1的抗体”或“特异性结合PD-L1的抗体”是指这样的抗体，所述抗体能够以足够的亲和力结合PD-L1蛋白或其片段。在一个实施方案中，所述(人)PD-L1抗体在体外或体内以高亲和力结合(人)PD-L1。在一个实施方案中，所述(人)PD-L1抗体不结合非PD-L1例如PD-L2。在一个实施方案中，所述(人)PD-L1抗体 与表达PD-L1的细胞结合。在一些实施方案中，所述结合例如通过放射性免疫测定(RIA)、生物膜薄层干涉测定法(BLI)、MSD测定法或表面等离子体共振法(SPR)或流式细胞术测量的。
术语“全抗体”、“全长抗体”、“完全抗体”和“完整抗体”在本文中可互换地用来指天然存在的包含由二硫键相互连接的至少两条重链(H)和两条轻链(L)的糖蛋白。每条重链由重链可变区(本文中缩写为VH)和重链恒定区(在本文中缩写为CH)组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(本文中缩写为VL)和轻链恒定区(在本文中缩写为CL)组成。轻链恒定区由一个结构域CL组成。VH区和VL区可以进一步再划分为超变区(为互补决定区(CDR)，其间插有较保守的区域(为构架区(FR))。每个VH和VL由三个CDR和4个FR组成，从氨基端到羧基端以如下顺序排列：FR1,CDR1,FR2,CDR2,FR3,CDR3,FR4。恒定区不直接参与抗体与抗原的结合，但是显示出多种效应子功能。
在本文中，抗体重链恒定区包括CH1，CL和Fc结构域，其中CH2,CH3和可选的CH4结构域构成Fc结构域，可以根据抗体分子的预期功能进行选择。例如，恒定区可以是IgA,IgD,IgE,IgG或IgM，尤其是人IgG的免疫球蛋白恒定结构域，例如，人IgG1，IgG2,IgG3或IgG4的恒定结构域，优选人IgG1的恒定结构域。例如，抗体的Fc区可以包含来自IgG1的CH2和CH3结构域。免疫球蛋白恒定区可以具有天然序列或变体序列。
在一些实施方案中，本发明的抗体重链恒定区HC为IgG1、IgG2、IgG3或IgG4的重链恒定区，优选的IgG1的重链恒定区。在一些优选的实施方案中，本发明的抗体重链恒定区HC
(i)包含与SEQ ID NO:13的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；
(ii)包含SEQ ID NO:13的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:13的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列组成。
在一些实施方案中，本发明的抗体轻链恒定区LC为Lambda或Kappa轻链恒定区。在一些实施方案中，本发明的抗体轻链恒定区LC
(i)包含与SEQ ID NO:16的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；
(ii)包含SEQ ID NO:16的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:16的氨基酸序列相比具有1个或多个(优选不超过20个或10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由所述氨基酸序列 组成。
术语“抗体片段”包括完整抗体的一部分。在优选的实施方案中，抗体片段为抗原结合片段。
术语“抗原结合片段”是比完整或全长抗体的氨基酸残基数要少的完整或全长抗体的一部分或一段，其能结合抗原或与完整抗体(即与抗原结合片段所来源的完整抗体)竞争结合抗原。可以通过重组DNA技术、或通过酶或化学切割完整的抗体制备抗原结合片段。抗原结合片段包括但不限于双抗体、Fab、Fab'、F(ab')2、Fd、Fv片段、二硫键稳定的Fv片段(dsFv)、(dsFv)2、双特异性dsFv(dsFv-dsFv')、二硫键稳定的双抗体(ds双抗体)、单链抗体分子(scFv)、scFv二聚体(二价双抗体)、多特异性抗体、骆驼化(camelized)单结构域抗体、纳米抗体、结构域抗体和二价结构域抗体。
抗体的“Fab”是指由单条轻链(包括可变区和恒定区)和单条重链的可变区和第一恒定区经二硫键结合起来组成的抗体的一部分。
“Fab'”是指包括铰链区的一部分的Fab片段。
“F(ab')2”是指Fab'的二聚体。
Fv片段由抗体单臂的VL和VH结构域组成。另外，虽然Fv片段的两个结构域VL和VH由独立的基因编码，但是使用重组方法，可以将它们通过能够使这两个结构域作为单条蛋白链产生的合成性连接肽连接，在所述单条蛋白链中VL区和VH区配对以形成单链Fv(scFv)。可以通过化学方法、重组DNA方法或蛋白酶消化法获得所述抗体片段。“scFv”在本文中用于指，包含由接头连接的重链可变结构域VH和轻链可变结构域VL的单链抗体片段，其中VH和VL配对形成抗原结合位点。
“互补决定区”或“CDR区”或“CDR”是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派方案的任一种或其组合确定，所述指派方案包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(在万维网上imgt.cines.fr/上)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。
以下为采用kabat、AbM、Chothia、Contact和IMGT方案定义的CDR的区域范围。
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。CDR也可以基于与参考CDR序列(例如本发明示例性CDR之任一)具有相同的Kabat编号位置而确定。除非另有说明，否则在本发明中，当提及抗体可变区中的残基位置(包括重链可变区残基和轻链可变区残基)时，是指根据Kabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991)中的Kabat编号系统的编号位置。
在一个实施方案中，本发明抗体中的HCDR和LCDR按照Kabat方案确定。
应该注意，基于不同的指派方案获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派方案下定义的同一抗体可变区的CDR序列有所不同。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派方案规则或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
术语“Fc结构域”或“Fc区”或“Fc片段”在本文中用来定义免疫球蛋白重链的含有至少一部分恒定区的C端区域。该术语包括天然序列Fc区和变体Fc区。天然的免疫球蛋白“Fc结构域”包含两个或三个恒定结构域，即CH2结构域、CH3结构域和可选的CH4结构域。例如，在天然抗体中，免疫球蛋白Fc结构域包含源自IgG、IgA和IgD类抗体的重链的第二和第三恒定结构域(CH2结构域和CH3结构域)；或者包含源自IgM和IgE类抗体的重链的第二、第三和第四恒定结构域(CH2结构域、CH3结构域和CH4结构域)。除非本文中另外说明，否则Fc区或重链恒定区中的氨基酸残基编号根据如Edelman,G.M.et al.,Proc.Natl.Acad. USA,63,78-85(1969)(https://pubmed.ncbi.nlm.nih.gov/5257969/)中所述的EU编号体系(也称作EU索引)进行编号。在本文中，术语“Fc结构域”或“Fc区”或“Fc片段”不包括免疫球蛋白的重链可变区VH和轻链可变区VL以及重链恒定区CH1和轻链恒定区CL，但在一些情况下可以包括在重链恒定区N端的铰链区。在一些实施方案中，适用于本发明的重链恒定区Fc来自抗体重链恒定区，例如人IgG1、IgG2、IgG3或IgG4的恒定区，优选来自IgG1的恒定区。在一些实施方案中，Fc包含SEQ ID NO:14或15所示的氨基酸序列，或包含与其具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。在本发明的一些实施方案中，Fc片段二聚化构成Fc二聚体。在一些实施方案中，Fc二聚体中的的两个Fc片段相同。在一些实施方案中，Fc片段异二聚化为Fc异二聚体。在Fc片段异二聚化为异二聚体的情况下，Fc片段可以包含用于异二聚化的突变，例如结入扣突变。
如本文所使用的，术语“效应子功能”是指由抗体的Fc区与免疫细胞上的C1q补体蛋白或Fc受体(FcR)之间的相互作用引起的细胞介导或补体介导的细胞毒性作用。示例性效应子功能包括但不限于抗体依赖性细胞毒性(ADCC)、抗体依赖性细胞介导的吞噬作用(ADCP)和补体依赖性细胞毒性(CDC)效应。ADCC是细胞介导的免疫防御机制，由此免疫系统的效应细胞主动裂解靶细胞，该靶细胞的膜表面抗原已被特异性抗体结合。ADCC需要免疫效应细胞，其通常已知是典型地与IgG抗体相互作用的天然杀伤(NK)细胞。然而，ADCC也可以由巨噬细胞、中性粒细胞和嗜酸性粒细胞介导。ADCC涉及通过表达Fc部分的抗体激活表达Fc受体的效应细胞。例如，NK细胞表面上最常见的Fc受体被称为CD16或FcγRIII。一旦Fc受体结合IgG的Fc区，NK细胞就释放导致靶细胞死亡的细胞毒性因子。同样，嗜酸性粒细胞的Fc受体(FceRI)将识别IgE。相比之下，在CDC中，由于经典途径补体激活，补体系统的分子“C1q”与抗体Fc区结合，并且这种结合触发补体级联，其导致在靶细胞表面形成膜攻击复合物(MAC)。在治疗性抗体或抗体构建体中，ADCC和CDC两者都可以通过Fc同种型工程、Fc基因突变或Fc糖基化谱修饰来调节。
术语“靶标”是指结合分子所针对的被结合物。靶标可以是抗原，也可以是配体或受体。术语“抗原”是指引发免疫应答的分子。这种免疫应答可能涉及抗体产生或特异性免疫细胞的活化，或两者兼有。技术人员将理解，任何大分子，包括基本上所有的蛋白质或肽，都可以用作抗原。此外，抗原可以衍生自重组或基因组DNA。如本文所用，术语“表位”指抗原中与抗体分子特异性相互作用的部分。
如本文所用的术语“靶标结合区”是指多特异性结合分子，例如双特异性结合分子的结合特定靶标或抗原的部分。在多特异性抗体或双特异性抗体时，也将“靶标结合区”称为“抗原结合区”。
如本文所用，术语“单特异性”抗体指具有一个或多个结合位点的抗体，所述位点中的每一个位点与相同抗原的相同表位结合。如本文所用，术语“多特异性”抗体指具有至少两个抗原结合位点的抗体，其中至少一个抗原结合位点，相对于其余的抗原结合位点，结合不同的抗原表位，例如，相同抗原上的不同表位或不同抗原上的不同表位。例如，本发明提供了针对PD-L1和CLDN18.2的双特异性抗体。
术语“双特异性抗体”是指具有两个结合特异性的抗体，其包含第一抗原结合 区和第二抗原结合区，其中所述第一抗原结合区结合一种抗原或表位且所述第二抗原结合区结合另一抗原或另一表位。因此，根据本发明的双特异性抗体包含对于两种不同的抗原，或对一种抗原的两个不同的表位的特异性。
双特异性抗体形式包括IgG样双特异性抗体。本文所述的“IgG样双特异性抗体”是指包含Fc二聚体的双特异性抗体。在一些实施方案中，IgG样双特异性抗体类型包含两个Fab区域和两个Fc区域,每个Fab的重链和轻链可以来自单独的单克隆抗体。在一些实施方案中，IgG样双特异性抗体包括全长抗体以及与全长抗体相连的其他抗体片段，例如scFv。可以使用本领域已知的双特异性抗体形式或技术来制备本发明的双特异性抗体。
根据本发明的术语“价”表示在抗体分子中存在指定数目的结合位点。因此，术语二价、三价、四价分别表示在抗体构建物中存在两个、三个或四个结合位点。根据本发明的双特异性抗体是至少二价的并且可以是多价的，例如二价、三价、四价或六价的。在一些实施方案中，本发明的双特异性抗体是四价的。
本文使用的术语“嵌合”是指具有来源于一种物种的重链和/或轻链的一部分并且所述重链和/或轻链的其余部分来源于另一不同物种的抗体或抗原结合片段。在说明性实例中，嵌合抗体可以包括来源于人的恒定区和来源于非人动物(如来源于小鼠)的可变区。在一些实施例中，所述非人动物是哺乳动物，例如小鼠、大鼠、兔、山羊、绵羊、豚鼠或仓鼠。
本文使用的术语“人源化”是指包括来源于非人动物的CDR、来源于人的FR区以及来源于人的恒定区(当适用时)的抗体或抗原结合片段。
本文使用的术语“亲和力”是指免疫球蛋白分子(即，抗体)或其片段与抗原之间非共价相互作用的强度。
如本文所用，术语“抗”、“结合”或“特异性结合”意指结合作用对靶标或抗原是选择性的并且可以与不想要的或非特异的相互作用区别。结合位点与特定靶标或抗原结合的能力可以通过流式细胞术或酶联免疫吸附测定法(ELISA)或本领域已知的常规结合测定法如通过放射性免疫测定(RIA)或生物膜薄层干涉测定法或MSD测定法或表面等离子体共振法(SPR)测定。
氨基酸序列的“同一性百分数(％)”是指将候选序列与本说明书中所示的具体氨基酸序列进行比对并且如有必要的话为达到最大序列同一性百分数而引入空位后，并且不考虑任何保守置换作为序列同一性的一部分时，候选序列中与本说明书中所示的具体氨基酸序列的氨基酸残基相同的氨基酸残基百分数。在一些实施方案中，本发明考虑本发明抗体分子的变体，所述变体相对于在本文中具体公开的抗体分子及其序列而言具有相当程度的同一性，例如同一性为至少80％,85％,90％,95％,97％,98％或99％或更高。所述变体可以包含保守性改变，或为保守性修饰变体。
对于多肽序列，“保守性改变”包括对多肽序列的置换、缺失或添加，但不实质性改变多肽序列的期望功能活性。例如，保守性置换常常导致某个氨基酸置换为化学上相似的氨基酸。提供功能上相似氨基酸的保守性置换表是本领域熟知的。以下列出了8组含有互为保守替换的氨基酸：1)丙氨酸(A)、甘氨酸(G)；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)； 5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)；7)丝氨酸(S)、苏氨酸(T)；和8)半胱氨酸(C)、甲硫氨酸(M)。在一些实施方案中，术语“保守序列改变”用于指不显著影响或改变含有氨基酸序列的本发明抗体分子或结合蛋白分子的目的抗原结合特征的氨基酸修饰。例如保守修改变体相对于亲本抗体或结合蛋白保持对目的抗原至少80％,85％,90％,95％,98％,99％或更高，例如100-110％或更高的结合亲和力。
如本文所用的术语“接头”是指使得能够直接连接双特异性结合分子的不同部分的任何分子。在不同分子部分之间建立共价连接的接头的实例包括肽接头和非蛋白质聚合物，包括但不限于聚乙二醇(PEG)、聚丙二醇、聚氧化烯或聚乙二醇、聚丙二醇的共聚物。在一些实施方案中，接头是肽接头(又称为“连接肽”)，其是指是指由氨基酸组成的短氨基酸序列，例如单独或组合使用的甘氨酸(G)和/或丝氨酸(S)和/或苏氨酸残基(T)，或来自免疫球蛋白的铰链区，用于将结合分子的第一部分的氨基酸序列连接至结合分子的第二部分。例如，肽接头可以将结合分子的第一靶标结合区连接至第二靶标结合区。例如，肽接头也可以将抗体的一部分连接至抗体的另一部分，诸如将轻链可变区连接至重链可变区。优选地，所述肽接头具有这样的长度，其足以连接两个实体，其方式使得它们维持它们相对于彼此的构象，使得不妨碍期望的活性。在一个实施方案中，连接肽具有5-50个氨基酸长度，例如，10,15,20,25,30个氨基酸长度。在一个实施方案中，连接肽包含氨基酸序列(GS)n、(GGS)n、(GSGGS)n、(GGGGS)n、(GGGS)n和(GGGGS)nG，其中n是等于或大于1的整数，例如，n是2、3、4、5、6、7、8、9、10的整数。有用的接头还包括甘氨酸-丙氨酸聚合物、丙氨酸-丝氨酸聚合物和其他柔性接头。在一些实施方案中，所述肽接头是(GGGGS)n，其中n＝1-10，例如1、2、3或4，例如SEQ ID NO:12所示的序列。在一些实施方案中，所述接头例如如SEQ ID NO:28所示。
术语“宿主细胞”指已经向其中引入外源多核苷酸的细胞，包括这类细胞的子代。宿主细胞包括“转化体”和“转化的细胞”，这包括原代转化的细胞和从其衍生的子代。宿主细胞是可以用来产生本发明抗体分子的任何类型的细胞系统，包括真核细胞，例如，哺乳动物细胞、昆虫细胞、酵母细胞；和原核细胞，例如，大肠杆菌细胞。宿主细胞包括培养的细胞，也包括转基因动物、转基因植物或培养的植物组织或动物组织内部的细胞。
术语“载体”当在本文中使用时是指能够增殖与其相连的另一个核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及结合到已经引入其的宿主细胞的基因组中的载体。术语“表达载体”是指包含重组多核苷酸的载体，其包含有效连接要表达的核苷酸序列的表达控制序列。表达载体包含足够的用于表达的顺式作用元件；用于表达的其它元件可以由宿主细胞提供或在体外表达系统中。表达载体包括本领域已知的所有那些，包括被掺入重组多核苷酸的粘粒、质粒(例如，裸的或包含在脂质体中)和病毒(例如，慢病毒、逆转录病毒、腺病毒和腺伴随病毒)。
术语“药物组合物”指这样的组合物，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，并且不包含对施用所述组合物的受试者具有不可接受的毒性的另外的成分。
术语“药用辅料”指与活性物质一起施用的稀释剂、佐剂(例如弗氏佐剂(完全 和不完全的))、赋形剂、载体或稳定剂等。
本文所述的术语“治疗剂”涵盖在预防或治疗肿瘤，例如癌症中有效的任何物质，包括化疗剂、细胞因子、细胞毒性剂、其它抗体、小分子药物或免疫调节剂(例如免疫抑制剂)、化学治疗剂、抗癌药物、免疫治疗剂、抗血管生成剂、靶向疗法、细胞疗法治疗剂、基因疗法治疗剂、激素疗法治疗剂、抗病毒剂、抗生素、镇痛剂、抗氧化剂、金属螯合剂或细胞因子。
术语“小分子药物”是指低分子量的能够调节生物过程的有机化合物。“小分子”被定义为分子量小于10kD、通常小于2kD和优选小于1kD的分子。小分子包括但不限于无机分子、有机分子、含无机组分的有机分子、含放射性原子的分子、合成分子、肽模拟物和抗体模拟物。作为治疗剂，小分子可以比大分子更能透过细胞、对降解更不易感和更不易于引发免疫应答。
本文使用的术语“免疫调节剂”指抑制或调节免疫应答的天然或合成活性剂或者药物。免疫应答可以是体液应答或细胞应答。免疫调节剂包含免疫抑制剂。在一些实施方案中，本发明的免疫调节剂包括免疫检查点抑制剂或免疫检查点激动剂。
如本文所使用的，术语“免疫疗法”是指刺激免疫系统对抗如癌症等疾病或以一般方式增强免疫系统的疗法类型。免疫疗法治疗剂的实例包括但不限于检查点调节剂、过继细胞转移、细胞因子、溶瘤病毒和治疗性疫苗。
“靶向疗法”是作用于与癌症相关的特定分子的疗法类型，所述特定分子如存在于癌细胞中但不存在于正常细胞中或在癌细胞中更丰富的特定蛋白质，或有助于癌症生长和存活的癌症微环境中的靶分子。靶向疗法将治疗剂靶向肿瘤，从而使正常组织免受治疗剂的影响。
术语“药物组合或组合产品”是指非固定组合产品或固定组合产品，包括但不限于药盒、药物组合物。术语“非固定组合”意指活性成分(例如，(i)本发明的免疫缀合物、以及(ii)其他治疗剂)以分开的实体被同时、无特定时间限制或以相同或不同的时间间隔、依次地施用于患者，其中这类施用在患者体内提供预防或治疗有效水平的两种或更多种活性剂。术语“固定组合”意指两种或更多种活性剂以单个实体的形式被同时施用于患者。优选对两种或更多种活性剂的剂量和/或时间间隔进行选择，从而使各部分的联合使用能够在治疗疾病或病症时产生大于单独使用任何一种成分所能达到的效果。各成分可以各自呈单独的制剂形式，其制剂形式可以相同也可以不同。
术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”、“癌性”和“肿瘤”在本文中提到时并不互相排斥。术语“肿瘤”涵盖实体瘤和液体肿瘤。
术语“抗肿瘤作用”或“抑瘤作用”或“肿瘤抑制作用”指可以通过多种手段展示的生物学效果，包括但不限于例如，肿瘤体积减少、肿瘤细胞数目减少、肿瘤细胞增殖减少或肿瘤细胞存活减少。
术语“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转疾病的症状、并发症、或生化指征的发作、缓解症状或阻止或抑制疾病、病状或病症的进一步发展。
术语“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。在一些实施方式中，具有癌症家族病史的受试者是预防性方案的候选。通常，在癌症的背景中，术语“预防”是指在癌症的病征或症状发生前，特别是在具有癌症风险的受试者中发生前的药物施用。
术语“有效量”指本发明的抗体或片段或组合物或组合的这样的量或剂量，其以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。
“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。治疗有效量也是这样的一个量，其中抗体或抗体片段或组合物或组合的任何有毒或有害作用不及治疗有益作用。相对于未治疗的对象，“治疗有效量”优选地抑制可度量参数或改善可度量参数至少约40％、甚至更优选地至少约50％、55％、60％、65％、70％、75％、80％、85％、90％甚至100％。
“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在对象中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量将小于治疗有效量。
术语“组合疗法”是指施用两种或更多种治疗剂或治疗方式(例如放射疗法或手术)以治疗本文所述疾病。这种施用包括以基本上同时的方式共同施用这些治疗剂，例如以具有固定比例的活性成分的单一胶囊。或者，这种施用包括对于各个活性成分在多种或在分开的容器(例如片剂、胶囊、粉末和液体)中的共同施用。粉末和/或液体可以在施用前重构或稀释至所需剂量。此外，这种施用还包括以大致相同的时间或在不同的时间以顺序的方式使用每种类型的治疗剂。在任一情况下，治疗方案将提供药物组合在治疗本文所述的病症或病状中的有益作用。
术语“个体”或“受试者”可互换地使用，是指哺乳动物。哺乳动物包括但不限于驯化动物(例如，奶牛、绵羊、猫、犬和马)、灵长类(例如，人和非人灵长类如猴)、兔和啮齿类(例如，小鼠和大鼠)。特别地，个体是人。
“受试者/患者/个体样品”指从患者或受试者得到的细胞或流体的集合。组织或细胞样品的来源可以是实体组织，像来自新鲜的、冷冻的和/或保存的器官或组织样品或活检样品或穿刺样品；血液或任何血液组分；体液，诸如泪液、玻璃体液、脑脊液、羊膜液(羊水)、腹膜液(腹水)、或间隙液；来自受试者的妊娠或发育任何时间的细胞。在一些实施方案中，组织样品是肿瘤组织。组织样品可能包含在自然界中天然不与组织混杂的化合物，诸如防腐剂、抗凝剂、缓冲剂、固定剂、营养物、抗生素、等等。
II.双特异性抗体
在一些实施方案中，本发明的抗体是双特异性抗体或多特异性抗体，其例如包含针对PD-L1的第一结合特异性和针对CLDN18.2的第二结合特异性，以及任选地一种或多种的其他结合特异性。
因此，本发明的一个方面涉及一种双特异性抗体，其包含
第一抗原结合区和第二抗原结合区，其中第一抗原结合区特异性结合PD-L1，和第二抗原结合区特异性结合CLDN18.2。
具体地，PD-L1是人PD-L1和/或CLDN18.2是人CLDN18.2。
适用于本发明的双特异性抗体的第一抗原结合区可以包含本发明的特异性结合PD-L1的全长抗体或其抗原结合片段，或由其组成，只要其能够特异性结合PD-L1即可，包括但不限于，例如特异性结合PD-L1的全长抗体、单链Fv、Fab、Fab'、(Fab)2、单域抗体、VHH或重链抗体等。
适用于本发明的双特异性抗体的第二抗原结合区可以包含特异性结合CLDN18.2的全长抗体或其抗原结合片段，或由其组成，只要其能够特异性结合CLDN18.2即可，包括但不限于，例如特异性结合CLDN18.2的全长抗体、单链Fv、Fab、Fab'、(Fab)2、单域抗体、VHH或重链抗体等。
在一些实施方案中，本发明的双特异性抗体是IgG样双特异性抗体。因此，在一些实施方案中，本发明的双特异性抗体包含Fc二聚体。
在本发明的一些实施方案中，所述双特异性抗体中的第一抗原结合区和第二抗原结合区之间通过接头连接，所述接头例如包含氨基酸序列(G4S)n,其中n＝1,2,3,4或5，优选地，n＝3或4，更优选地，n＝4。
在一些实施方案中，本发明的双特异性抗体包含全长抗体作为第一抗原结合区，以及scFv作为第二抗原结合区。在一些实施方案中，本发明的双特异性抗体包含全长抗体作为第二抗原结合区，以及scFv作为第一抗原结合区。
在一些实施方案中，在本发明的双特异性抗体中，所述特异性结合PD-L1的第一抗原结合区为全长抗体，且所述特异性结合CLDN18.2的第二抗原结合区为scFv；或者所述特异性结合CLDN18.2的第二抗原结合区为全长抗体，且所述特异性结合PD-L1的第一抗原结合区为scFv。
在一些具体的实施方案中，scFv与全长抗体的重链恒定区的C末端连接(经由或不经由接头)。
在一些具体的实施方案中，两个scFv分别与全抗抗体的两个重链恒定区的C末端连接(经由或不经由接头)。
在一些具体的实施方案中，所述双特异性抗体包含全长抗体和两个scFv，其中全长抗体包含两条重链和两条轻链，两个scFv分别与两条重链的C末端连接(经由或不经由接头)。
特异性结合PD-L1的第一抗原结合区
作为本发明的双特异性抗体的特异性结合PD-L1的第一抗原结合区可以来自于PCT/CN2021/131389。
在一些实施方案中，本发明的特异性结合PD-L1的第一抗原结合区包含3个来自重链可变区的互补决定区(HCDR)，HCDR1、HCDR2和HCDR3。
在一些实施方案中，本发明的特异性结合PD-L1的第一抗原结合区包含3个来自轻链可变区的互补决定区(LCDR)，LCDR1、LCDR2和LCDR3。
在一些实施方案中，本发明的特异性结合PD-L1的第一抗原结合区包含3个来自重链可变区的互补决定区(HCDR)和3个来自轻链可变区的互补决定区 (LCDR)。
在一些方面中，本发明的特异性结合PD-L1的第一抗原结合区包含重链可变区(VH)。在一些方面中，本发明的特异性结合PD-L1的第一抗原结合区包含轻链可变区(VL)。在一些方面中，本发明的特异性结合PD-L1的第一抗原结合区包含重链可变区(VH)和轻链可变区(VL)。在一些实施方案中，所述重链可变区包含3个来自重链可变区的互补决定区(CDR)，HCDR1、HCDR2和HCDR3。在一些实施方案中，所述轻链可变区包含3个来自轻链可变区的互补决定区(CDR)，LCDR1、LCDR2和LCDR3。
在一些实施方案中，本发明的特异性结合PD-L1的第一抗原结合区还包含抗体重链恒定区HC。在一些实施方案中，本发明特异性结合PD-L1的第一抗原结合区还包含抗体轻链恒定区LC。在一些实施方案中，本发明特异性结合PD-L1的第一抗原结合区还包含重链恒定区HC和轻链恒定区LC。
在一些实施方案中，本发明的特异性结合PD-L1的第一抗原结合区的重链可变区为PCT/CN2021/131389中特异性结合PD-L1的抗体或其抗原结合片段的重链可变区。
在一些实施方案中，本发明的特异性结合PD-L1的第一抗原结合区的重链可变区
(i)包含与SEQ ID NO:17或30的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:17或30的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:17或30的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列由所述氨基酸序列组成，优选地，所述氨基酸改变不发生在CDR区中。
在一些实施方案中，本发明的特异性结合PD-L1的第一抗原结合区的轻链可变区为PCT/CN2021/131389中特异性结合PD-L1的抗体或其抗原结合片段的轻链可变区。
在一些实施方案中，本发明的特异性结合PD-L1的第一抗原结合区的轻链可变区
(i)包含与SEQ ID NO:22或31的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:22或31的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:22或31的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换， 更优选氨基酸保守置换)的氨基酸序列由所述氨基酸序列组成，优选地，所述氨基酸改变不发生在CDR区中。
在一些实施方案中，本发明的特异性结合PD-L1的第一抗原结合区的3个来自重链可变区的互补决定区(HCDR)，HCDR1、HCDR2和HCDR3为来自PCT/CN2021/131389中特异性结合PD-L1的抗体或其抗原结合片段的重链互补决定区(HCDR)，HCDR1、HCDR2和HCDR3，或选自
(i)如SEQ ID NO:17或30中任一项所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，或
(ii)相对于(i)中任一项的序列，在所述三个HCDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列，
例如所述CDR通过Kabat方案确定。
在一些实施方案中，本发明的抗PD-L1抗体3个来自轻链可变区的互补决定区(LCDR)，LCDR1、LCDR2和LCDR3为来自PCT/CN2021/131389中特异性结合PD-L1的抗体或其抗原结合片段的轻链互补决定区(LCDR)，LCDR1、LCDR2和LCDR3，或选自
(i)如SEQ ID NO:22或31中任一项所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3，或
(ii)相对于(i)中任一项的序列，在所述三个LCDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列，
例如所述CDR通过Kabat方案确定。
在一些实施方案中，HCDR1包含SEQ ID NO:18的氨基酸序列，或由所述氨基酸序列组成，或者HCDR1包含与SEQ ID NO:18的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，HCDR2包含SEQ ID NO:19的氨基酸序列，或由所述氨基酸序列组成，或者HCDR2包含与SEQ ID NO:19的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，HCDR3包含SEQ ID NO:20的氨基酸序列，或由所述氨基酸序列组成，或者HCDR3包含与SEQ ID NO:20的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，LCDR1包含SEQ ID NO:23的氨基酸序列，或由所述氨基酸序列组成，或者LCDR1包含与SEQ ID NO:23的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，LCDR2包含SEQ ID NO:24的氨基酸序列，或由所述氨基酸序列组成，或者LCDR2包含与SEQ ID NO:24的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，LCDR3包含SEQ ID NO:25的氨基酸序列，或由所述氨 基酸序列组成，或者LCDR3包含与SEQ ID NO:25的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在本发明的一些具体实施方案中，本发明的特异性结合PD-L1的第一抗原结合区包含VH和VL，其中
(i)所述VH含有SEQ ID NO:17所示的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，和所述VL含有SEQ ID NO:22所示的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或
(ii)所述VH含有SEQ ID NO:30所示的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，和所述VL含有SEQ ID NO:31所示的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
在本发明的一些具体实施方案中，本发明的特异性结合PD-L1的第一抗原结合区包含如SEQ ID NO:18所示的HCDR1、如SEQ ID NO:19所示的HCDR2、如SEQ ID NO:20所示的HCDR3；如SEQ ID NO:23所示的LCDR1、如SEQ ID NO:24所示的LCDR2和如SEQ ID NO:25所示的LCDR3。
在本发明的一些具体实施方案中，本发明的特异性结合PD-L1的第一抗原结合区包含VH和VL，其中所述特异性结合PD-L1的第一抗原结合区包含重链可变区VH和轻链可变区VL，其中VH和VL分别包含如下所示的氨基酸序列或由所述氨基酸序列组成：
(i).SEQ ID NO:17和SEQ ID NO:30；或
(ii).SEQ ID NO:22和SEQ ID NO:31。
在本发明的一些实施方案中，本发明的特异性结合PD-L1的第一抗原结合区包含重链。在本发明的一些实施方案中，本发明的特异性结合PD-L1的第一抗原结合区包含轻链。在本发明的一些实施方案中，本发明的特异性结合PD-L1的第一抗原结合区包含重链和轻链。
在一些具体的实施方案中，特异性结合PD-L1的第一抗原结合区的重链包含SEQ ID NO:21的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
在一些具体的实施方案中，特异性结合PD-L1的第一抗原结合区的轻链包含SEQ ID NO:26的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
在一些具体的实施方案中，特异性结合PD-L1的第一抗原结合区包含重链和轻链，其中所述重链包含SEQ ID NO：21所示的氨基酸序列，或与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％ 或99％同一性的氨基酸序列，或由所述氨基酸序列组成，且所述轻链包含SEQ ID NO:26所示的氨基酸序列，或与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
在一些具体的实施方案中，特异性结合PD-L1的第一抗原结合区包含重链和轻链，其中所述重链包含SEQ ID NO：21所示的氨基酸序列，或由所述氨基酸序列组成，且所述轻链包含SEQ ID NO:26所示的氨基酸序列或由所述氨基酸序列组成。
在本发明的一个实施方案中，本文所述的特异性结合PD-L1的第一抗原结合区的氨基酸改变包括氨基酸的置换、插入或缺失。在优选的实施方案中，本发明所述的氨基酸改变发生在CDR外的区域(例如在FR中)。更优选地，本发明所述的氨基酸改变发生在重链可变区外和/或轻链可变区外的区域。优选的，本文所述的氨基酸改变为氨基酸置换，优选地保守置换。
在本发明的一个实施方案中，本发明所述的特异性结合PD-L1的第一抗原结合区的氨基酸改变为在可变区(例如FR区)中引入二硫键从而获得二硫键稳定的突变，例如所述重链可变区包含在第44位氨基酸取代为半胱氨酸的突变(Kabat编号)，和/或所述轻链可变区包含在第100位氨基酸取代为半胱氨酸的突变(Kabat编号)。在一些实施方案中，在第一抗原结合区为特异性结合PD-L1的scFv时引入所述突变。
在一些实施方案中，本发明的特异性结合PD-L1的第一抗原结合区的重链恒定区为IgG1、IgG2、IgG3或IgG4的重链恒定区，优选的IgG1的重链恒定区。在一些实施方案中，本发明的抗PD-L1抗体的轻链恒定区为lambda或kappa轻链恒定区，例如Kappa轻链恒定区。
在一些实施方案中，特异性结合PD-L1的第一抗原结合区所来源抗PD-L1抗体是人源化的。
在一些实施方案中，特异性结合PD-L1的第一抗原结合区所来源抗PD-L1抗体是嵌合抗体。
在一个实施方案中，本发明的特异性结合PD-L1的第一抗原结合区为抗体片段(例如抗原结合片段)，优选地选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体(例如scFv)、(Fab’)2、单结构域抗体例如VHH、dAb(domain antibody)或线性抗体。优选地，特异性结合PD-L1的第一抗原结合区为scFv。
特异性结合CLDN18.2的第二抗原结合区
作为本发明的双特异性抗体的特异性结合CLDN18.2的第二抗原结合区可以来自于WO2021032157A1。
在一些实施方案中，本发明的特异性结合CLDN18.2的第二抗原结合区包含3个来自重链可变区的互补决定区(HCDR)，HCDR1、HCDR2和HCDR3。
在一些实施方案中，本发明的特异性结合CLDN18.2的第二抗原结合区包含3个来自轻链可变区的互补决定区(LCDR)，LCDR1、LCDR2和LCDR3。
在一些实施方案中，本发明的特异性结合CLDN18.2的第二抗原结合区包含3个来自重链可变区的互补决定区(HCDR)和3个来自轻链可变区的互补决定区(LCDR)。
在一些方面中，本发明的特异性结合CLDN18.2的第二抗原结合区包含重链可变区(VH)。在一些方面中，本发明的特异性结合CLDN18.2的第二抗原结合区包含轻链可变区(VL)。在一些方面中，本发明的特异性结合CLDN18.2的第二抗原结合区包含重链可变区(VH)和轻链可变区(VL)。在一些实施方案中，所述重链可变区包含3个来自重链可变区的互补决定区(CDR)，HCDR1、HCDR2和HCDR3。在一些实施方案中，所述轻链可变区包含3个来自轻链可变区的互补决定区(CDR)，LCDR1、LCDR2和LCDR3。
在一些实施方案中，本发明的特异性结合CLDN18.2的第二抗原结合区还包含抗体重链恒定区HC。在一些实施方案中，本发明特异性结合CLDN18.2的第二抗原结合区还包含抗体轻链恒定区LC。在一些实施方案中，本发明特异性结合CLDN18.2的第二抗原结合区还包含重链恒定区HC和轻链恒定区LC。
在一些实施方案中，本发明的特异性结合CLDN18.2的第二抗原结合区的重链可变区为WO2021032157A1中公开的特异性结合CLDN18.2的抗体或其抗原结合片段的重链可变区。
在一些实施方案中，本发明的特异性结合CLDN18.2的第二抗原结合区的重链可变区
(i)包含与SEQ ID NO:1的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:1的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:1的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列由所述氨基酸序列组成，优选地，所述氨基酸改变不发生在CDR区中。
在一些实施方案中，本发明的特异性结合CLDN18.2的第二抗原结合区的轻链可变区为WO2021032157A1中公开的特异性结合CLDN18.2的抗体或其抗原结合片段的轻链可变区。
在一些实施方案中，本发明的特异性结合CLDN18.2的第二抗原结合区的轻链可变区
(i)包含与SEQ ID NO:5的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
(ii)包含SEQ ID NO:5的氨基酸序列或由所述氨基酸序列组成；或者
(iii)包含与SEQ ID NO:5的氨基酸序列相比具有1个或多个(优选不超过10个，更优选不超过5、4、3、2、1个)的氨基酸改变(优选氨基酸置换，更优选 氨基酸保守置换)的氨基酸序列由所述氨基酸序列组成，优选地，所述氨基酸改变不发生在CDR区中。
在一些实施方案中，本发明的特异性结合CLDN18.2的第二抗原结合区的3个来自重链可变区的互补决定区(HCDR)，HCDR1、HCDR2和HCDR3为来自WO2021032157A1中公开的特异性结合CLDN18.2的抗体或其抗原结合片段的互补决定区(HCDR)，HCDR1、HCDR2和HCDR3，或选自
(i)如SEQ ID NO:1所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3，或
(ii)相对于(i)中任一项的序列，在所述三个HCDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列，
例如所述CDR通过Kabat方案确定。
在一些实施方案中，本发明的抗CLDN18.2抗体3个来自轻链可变区的互补决定区(LCDR)，LCDR1、LCDR2和LCDR3为来自WO2021032157A1中公开的特异性结合CLDN18.2的抗体或其抗原结合片段的互补决定区(LCDR)，LCDR1、LCDR2和LCDR3，或选自
(i)如SEQ ID NO:5所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3，或
(ii)相对于(i)中任一项的序列，在所述三个LCDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸置换，优选保守置换)的序列，
例如所述CDR通过Kabat方案确定。
在一些实施方案中，HCDR1包含SEQ ID NO:2的氨基酸序列，或由所述氨基酸序列组成，或者HCDR1包含与SEQ ID NO:2的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，HCDR2包含SEQ ID NO:3的氨基酸序列，或由所述氨基酸序列组成，或者HCDR2包含与SEQ ID NO:3的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，HCDR3包含SEQ ID NO:4的氨基酸序列，或由所述氨基酸序列组成，或者HCDR3包含与SEQ ID NO:4的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，LCDR1包含SEQ ID NO:7的氨基酸序列，或由所述氨基酸序列组成，或者LCDR1包含与SEQ ID NO:7的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，LCDR2包含SEQ ID NO:8的氨基酸序列，或由所述氨基酸序列组成，或者LCDR2包含与SEQ ID NO:8的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在一些实施方案中，LCDR3包含SEQ ID NO:9的氨基酸序列，或由所述氨 基酸序列组成，或者LCDR3包含与SEQ ID NO:9的氨基酸序列相比具有一个、两个或三个改变(优选氨基酸置换，优选保守置换)的氨基酸序列。
在本发明的一些具体实施方案中，本发明的特异性结合CLDN18.2的第二抗原结合区包含VH和VL，其中所述VH含有SEQ ID NO:1所示的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成，和所述VL含有SEQ ID NO:6所示的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
在本发明的一些具体实施方案中，本发明的特异性结合CLDN18.2的第二抗原结合区包含如SEQ ID NO:2所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:4所示的HCDR3；如SEQ ID NO:7所示的LCDR1、如SEQ ID NO:8所示的LCDR2和如SEQ ID NO:9所示的LCDR3。
在本发明的一些具体实施方案中，本发明的特异性结合CLDN18.2的第二抗原结合区包含VH和VL，其中所述特异性结合CLDN18.2的第二抗原结合区包含重链可变区VH和轻链可变区VL，其中VH和VL分别包含如下所示的氨基酸序列或由所述氨基酸序列组成：SEQ ID NO:1和SEQ ID NO:6。
在本发明的一些实施方案中，本发明的特异性结合CLDN18.2的第二抗原结合区包含重链。在本发明的一些实施方案中，本发明的特异性结合CLDN18.2的第二抗原结合区包含轻链。在本发明的一些实施方案中，本发明的特异性结合CLDN18.2的第二抗原结合区包含重链和轻链。
在一些具体的实施方案中，特异性结合CLDN18.2的第二抗原结合区的重链包含SEQ ID NO:5的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
在一些具体的实施方案中，特异性结合CLDN18.2的第二抗原结合区的轻链包含SEQ ID NO:10的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
在一些具体的实施方案中，特异性结合CLDN18.2的第二抗原结合区包含重链和轻链，其中所述重链包含SEQ ID NO：5所示的氨基酸序列，或与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成，且所述轻链包含SEQ ID NO:10所示的氨基酸序列，或与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
在一些具体的实施方案中，特异性结合CLDN18.2的第二抗原结合区包含重链和轻链，其中所述重链包含SEQ ID NO：5所示的氨基酸序列，或由所述氨基酸序列组成，且所述轻链包含SEQ ID NO:10所示的氨基酸序列或由所述氨基酸序列组成。
在本发明的一个实施方案中，本文所述的特异性结合CLDN18.2的第二抗原 结合区的氨基酸改变包括氨基酸的置换、插入或缺失。在优选的实施方案中，本发明所述的氨基酸改变发生在CDR外的区域(例如在FR中)。更优选地，本发明所述的氨基酸改变发生在重链可变区外和/或轻链可变区外的区域。优选的，本文所述的氨基酸改变为氨基酸置换，优选地保守置换。
在本发明的一个实施方案中，本发明所述的特异性结合CLDN18.2的第二抗原结合区的氨基酸改变为在可变区(例如FR区)中引入二硫键从而获得二硫键稳定的突变，例如所述重链可变区包含在第44位氨基酸取代为半胱氨酸的突变(Kabat编号)，和/或所述轻链可变区包含在第100位氨基酸取代为半胱氨酸的突变(Kabat编号)。在一些实施方案中，在第一抗原结合区为特异性结合PD-L1的scFv时引入所述突变。
在一些实施方案中，本发明的特异性结合CLDN18.2的第二抗原结合区的重链恒定区为IgG1、IgG2、IgG3或IgG4的重链恒定区，优选的IgG1的重链恒定区。在一些实施方案中，本发明的抗CLDN18.2抗体的轻链恒定区为lambda或kappa轻链恒定区，例如Kappa轻链恒定区。
在一些实施方案中，特异性结合CLDN18.2的第二抗原结合区所来源抗CLDN18.2抗体是人源化的。
在一些实施方案中，特异性结合CLDN18.2的第二抗原结合区所来源抗CLDN18.2抗体是嵌合抗体。
在一个实施方案中，本发明的特异性结合CLDN18.2的第二抗原结合区为抗体片段(例如抗原结合片段)，优选地选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体(例如scFv)、(Fab’)2、单结构域抗体例如VHH、dAb(domain antibody)或线性抗体。优选地，特异性结合CLDN18.2的第二抗原结合区为scFv。
适用于本发明的双特异性抗体的全长抗体
在一些实施方案中，双特异性抗体的抗原结合区之一可以为全长抗体，其包含两条重链和两条轻链，其中每条重链由重链可变区VH和重链恒定区CH组成。重链恒定区由3个结构域CH1、CH2和CH3(和可选的CH4)组成。每条轻链由轻链可变区VL和轻链恒定区CL组成。轻链恒定区由一个结构域CL组成。每个VH和VL由三个CDR和4个FR组成，从氨基端到羧基端以如下顺序排列：FR1,CDR1,FR2,CDR2,FR3,CDR3,FR4。
本发明的全长抗体包含Fc区。所述Fc区可以包含两个或三个恒定结构域，即CH2结构域、CH3结构域和可选的CH4结构域。在一些实施方案中，抗体Fc区还可以在N端带有IgG铰链区或部分IgG铰链区，例如，IgG1铰链区或部分IgG1铰链区。
在一些实施方案中，可以在全长抗体中引入突变，例如增加其中的Fc区的效应子功能的突变。本领域已知针对全长抗体的各个性质的适用的突变。
适用于本发明的双特异性抗体的scFv
在一些实施方案中，作为双特异性抗体结合区之一的scFv片段由包含抗体VH和VL结构域的一条多肽链组成，其中所述VH和VL连接(例如通过接头)以配对形成抗原结合位点。
在一些实施方案中，所述scFv为反式构型，从N端到C端包含：VH、接头、和VL(VH-接头-VL)。在另一些实施方案中，所述scFv为顺式构型，从N端到C端包含：VL、接头、和VH(VL-接头-VH)。在一些实施方案中，接头为由氨基酸残基组成的肽接头。适宜的肽接头是本领域技术人员已知的。在一个实施方案中，接头为5-50个氨基酸长度，例如5-30个氨基酸长，例如15个氨基酸或20个氨基酸长度。在一个实施方案中，接头包含氨基酸序列(G4S)n,其中n＝1,2,3,4或5，优选地，n＝3或4，更优选地，n＝4。
在一些实施方案中，包含在本发明抗体分子中的scFv抗原结合位点为二硫键稳定(即引入二硫键突变)的scFv。例如，在scFv中的VH区中的第100位的氨基酸突变为半胱氨酸(kabat)，且在scFv的VL区中的第44位的氨基酸突变为半胱氨酸(kabat)。
在一些实施方案中，在所述双特异性抗体中，scFv在VH链的N端融合在另一抗原结合区的C端(例如重链恒定区的C端)。
在一些实施方案中，本发明的双特异性抗体中包含的scFv特异性结合CLDN18.2，即为特异性结合CLDN18.2的第二抗原结合区。在一些实施方案中，所述scFv包含SEQ ID NO:11的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
在一些实施方案中，本发明的双特异性抗体中包含的scFv特异性结合PD-L1，即为特异性结合PD-L1的第一抗原结合区。在一些实施方案中，所述scFv包含SEQ ID NO:27的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。在一些实施方案中，所述scFv包含二硫键稳定的突变，其包含SEQ ID NO:29的氨基酸序列，或包含与所述氨基酸序列具有，或由所述氨基酸序列组成。在一些实施方案中，所述scFv包含与SEQ ID NO:29所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，且在scFv中的VH区中的第100位的氨基酸突变为半胱氨酸(kabat)，且在scFv的VL区中的第44位的氨基酸突变为半胱氨酸(kabat)。
适用于本发明双特异性抗体的Fc二聚体
在一个实施方案中，本发明双特异性抗体中的两个Fc区二聚化形成二聚Fc。
在一些实施方案中，第一和第二Fc区相同。在另一些实施方案中，第一Fc区和第二Fc区不同，两者配对并异二聚化。
适用于本发明抗体分子的Fc区片段可以是任何抗体Fc区。Fc区可以包括 天然序列Fc区和变体Fc区。天然序列Fc结构域涵盖天然存在的各种免疫球蛋白Fc序列，例如各种Ig亚型以及其同种异型的Fc区(Gestur Vidarsson等,IgG subclasses and allotypes:from structure to effector functions，20October 2014,doi:10.3389/fimmu.2014.00520.)。例如，本发明抗体的Fc区可以包含两个或三个恒定结构域，即CH2结构域、CH3结构域和可选的CH4结构域。在一些实施方案中，抗体Fc区还可以在N端带有IgG铰链区或部分IgG铰链区，例如，IgG1铰链区或部分IgG1铰链区。在所述铰链区中可以含有突变。
优选地，本发明抗体的Fc区从N端到C端包含:CH2-CH3，或从N端到C端包含：铰链区-CH2-CH3。在一些实施方案中，适用于本发明抗体或双特异性抗体的Fc区为人的IgG Fc，例如，人IgG1Fc,人IgG2Fc,人IgG3或人IgG4Fc。在一个实施方案中，Fc区包含氨基酸序列SEQ ID NO:14或与其具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列或由其组成。
可以将本发明的抗体或双特异性抗体中的Fc区进行突变以获得所需的特性。本领域已知对Fc区的突变。例如，如果两个Fc区不同，则可以引入knob-into-hole技术来促进Fc区的异二聚化。此技术参见例如Merchant,A.M.,et al.(1998)."An efficient route to human bispecific IgG."Nat Biotechnol 16(7):677-681。
如本领域技术人员理解的，根据本发明抗体分子的预期用途，本发明抗体分子也可以在Fc结构域中包含改变对一种或多种Fc受体的结合亲和力的修饰。在一个实施方案中，在Fc区的效应子功能(例如Fc区的ADCC)的特性上修饰Fc区。例如，在一个实施方案中，所述效应子功能相对于野生同种型Fc区已经被降低或消除，或者已经被增加。在一个实施方案中，通过使用天然具有降低或消除的效应子功能的Fc同种型或Fc区修饰来降低或消除效应子功能，例如在L234、L235、和/或P331上引入Fc区修饰，例如引入L234F、L235E或P331S，或引入L234F、L235E和P331S(EU编号)。在一些实施方案中，突变的Fc区包含氨基酸序列SEQ ID NO:15或与其具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列或由其组成。在一些实施方案中，所述Fc区包含与SEQ ID NO:15具有至少90％同一性，例如95％，96％，97％，99％或更高的同一性的氨基酸序列且包含L234F、L235E和P331S突变。
示例性的双特异性抗体分子
在一些优选的实施方案中，本发明提供了一种双特异性抗体，其具有如图2A、2B或2C的结构。
在一些优选的实施方案中，本发明提供了一种双特异性抗体，其包含轻链和重链，其中轻链从N端到C端包含VL-A-CL；
重链从N端到C端包含VH-A-CH-scFv；
其中scFv包含VH-B和VL-B；
其中VL-A为特异性结合PD-L1的第一抗原结合区的轻链可变区VL，VH-A为特异性结合PD-L1的第一抗原结合区的重链可变区VH；
VH-B为特异性结合CLDN18.2的第二抗原结合区的重链可变区VH，VL-B为特异性结合CLDN18.2的第二抗原结合区的轻链可变区VL；
或者VL-A为特异性结合CLDN18.2的第二抗原结合区的轻链可变区VL，VH-A为特异性结合CLDN18.2的第二抗原结合区的重链可变区VH；
VH-B为特异性结合PD-L1的第一抗原结合区的重链可变区VH，VL-B为特异性结合PD-L1的第一抗原结合区的轻链可变区VL；
CL为轻链恒定区；
CH为重链恒定区；
scFv的VH-B和VL-B之间包含接头或不包含接头；
其中所述CH与scFv之间包含接头或不包含接头。
在一些具体的实施方案中，本发明的双特异性抗体包含两条重链和两条轻链，例如两条相同的重链和两条相同的轻链。
在一些具体的实施方案中，scFv从N端到C端包含VH-B-VL-B，且VH-B在其N末端与CH的C末端通过或不通过接头连接。
在一些具体的实施方案中，所述接头例如包含氨基酸序列(G4S)n,其中n＝1,2,3,4或5，优选地，n＝3或4，更优选地，n＝4。
在一些具体的实施方案中，本发明的双特异性抗体包含重链和轻链，其中所述重链包含SEQ ID NO:32、33或34的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
在一些具体的实施方案中，本发明的双特异性抗体包含重链和轻链，其中所述轻链包含SEQ ID NO:10或26的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
在一些具体的实施方案中，本发明的双特异性抗体包含重链和轻链，其中
(i)所述重链包含SEQ ID NO:32或34的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成；
且所述轻链包含SEQ ID NO:10的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成；或者
(ii)所述重链包含SEQ ID NO:33的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成；
且所述轻链包含SEQ ID NO:26的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
在一些具体的实施方案中，本发明的双特异性抗体包含重链和轻链，其中
(i)所述重链包含SEQ ID NO:32或34的氨基酸序列或由所述氨基酸序列组成；
且所述轻链包含SEQ ID NO:10的氨基酸序列或由所述氨基酸序列组成；或者
(ii)所述重链包含SEQ ID NO:33的氨基酸序列或由所述氨基酸序列组成；
且所述轻链包含SEQ ID NO:26的氨基酸序列或由所述氨基酸序列组成。
在一些具体的实施方案中，本发明的双特异性抗体包含2条重链和2条轻链，其中
(i)所述每条重链包含SEQ ID NO:32或34的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成；
且所述每条轻链包含SEQ ID NO:10的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成；
(ii)所述每条重链包含SEQ ID NO:33的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成；
且所述每条轻链包含SEQ ID NO:26的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
在一些具体的实施方案中，本发明的双特异性抗体包含2条重链和2条轻链，其中
(i)所述每条重链包含SEQ ID NO:32或34的氨基酸序列或由所述氨基酸序列组成；
且所述每条轻链包含SEQ ID NO:10的氨基酸序列或由所述氨基酸序列组成；
(ii)所述每条重链包含SEQ ID NO:33的氨基酸序列或由所述氨基酸序列组成；
且所述轻链包含SEQ ID NO:26的氨基酸序列或由所述氨基酸序列组成。
III.编码抗体的核酸以及包含其的宿主细胞
在一方面，本发明提供了编码以上任何抗CLDN18.2抗体或抗PD-L1抗体或双特异性抗体或其任一条链的核酸。
例如，本发明的核酸包含编码选自SEQ ID NO:17、21、22、26、27、29、30、31、32、33、34中任一项所示氨基酸序列的核酸，或编码与选自SEQ ID NO:17、21、22、26、27、29、30、31、32、33、34中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列的核酸。
如本领域技术人员明了的，因为密码子简并性，每一个抗体或多肽氨基酸序列可以由多种核酸序列编码。编码本发明分子的核酸序列可以采用本领域熟知的方法，例如通过从头固相DNA合成，或通过PCR扩增而产生。
对抗体进行编码的DNA可使用常规程序(例如通过使用能够与对抗体的重链和轻链进行编码的基因特异性地结合的寡核苷酸探针)容易地分离和测序。编码DNA也可以通过合成方法获得。
在一方面，本发明提供编码以上任何抗体或任何抗体链的核酸。当从适宜的表达载体表达时，由所述核酸编码的多肽能够显示(人)CLDN18.2和/或(人)PD-L1抗原结合能力。
在再一方面，本发明提供编码以上任何双特异性抗体的核酸。当从适宜的表达载体表达时，由所述核酸编码的多肽能够显示(人)CLDN18.2和(人)PD-L1抗原结合能力。在一个实施方案中，编码双特异性抗体的各条链的核酸可以在相同的载体中或在不同的载体中。在再一个实施方案中，编码双特异性抗体的各条链的核酸可以引入相同或不同的宿主细胞以表达。
因此，在一些实施方案中，本发明的双特异性抗体的生产方法包括步骤：在适于表达所述分子的各条链的条件下，培养包含编码所述各条链的核酸的宿主细胞，产生本发明双特异性抗体。
在一个实施方案中，提供包含所述核酸的载体许多载体可用。载体组件通常包括但不限于以下中的一种或多种：信号序列、复制起点、一种或多种标志物基因、增强子元件、启动子(例如，SV40、CMV、EF-1α)和转录终止序列。在一个实施方案中，载体是表达载体，例如真核表达载体。载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。载体的实例包括但不限于逆转录病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(例如，单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳多空病毒(例如，SV40)、λ噬菌体和M13噬菌体、质粒pcDNA3.3、pMD18-T、pOptivec、pCMV、pEGFP、pIRES、pQD-Hyg-GSeu、pALTER、pBAD、pcDNA、pCal、pL、pET、pGEMEX、pGEX、pCI、pEGFT、pSV2、pFUSE、pVITRO、pVIVO、pMAL、pMONO、pSELECT、pUNO、pDUO、Psg5L、pBABE、pWPXL、pBI、p15TV-L、pPro18、pTD、pRS10、pLexA、pACT2.2、pCMV-SCRIPT.RTM.、pCDM8、pCDNA1.1/amp、pcDNA3.1、pRc/RSV、PCR 2.1、pEF-1、pFB、pSG5、pXT1、pCDEF3、pSVSPORT、pEF-Bos等。在一个实施方案中，载体是例如pcDNA载体，例如pcDNA3.1。
在一个实施方案中，提供包含所述核酸或所述载体的宿主细胞，例如用于克隆或表达双特异性抗体的载体。在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自丝状真菌、酵母细胞(例如酿酒酵母)、植物细胞、昆虫细胞、哺乳动物细胞例如CHO细胞(例如CHO-S，如ExpiCHO-S)或293细胞(例如293F或HEK293细胞))或适用于制备抗体或其片段的其它细胞。在一个实施方案中，宿主细胞是原核的，例如是细菌，例如大肠杆菌。
在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞或适用于制备抗体或其片段的其它细胞。例如，真核微生物诸如丝状真菌或酵母是关于编码抗体的载体的合适克隆或表达宿主。例如，糖基化途径已经进行“人源化”的真菌和酵母菌株导致产生具有部分或完全人糖 基化模式的抗体。适于表达糖基化抗体的宿主细胞也衍生自多细胞生物体(无脊椎动物和脊椎动物)。也可以将脊椎动物细胞用作宿主。例如，可以使用被改造以适合于悬浮生长的哺乳动物细胞系。有用的哺乳动物宿主细胞系的其它实例是用SV40转化的猴肾CV1系(COS-7)；人胚肾系(HEK293、293F或293T细胞)等。其它有用的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞，包括DHFR-CHO细胞、CHO-S细胞、ExpiCHO等；以及骨髓瘤细胞系如Y0，NS0和Sp2/0。本领域已知适合产生抗体的哺乳动物宿主细胞系。
IV.本发明的抗CLDN18.2抗体或抗PD-L1抗体或双特异性抗体的生产和纯化
在一个实施方案中，提供了制备本发明双特异性抗体的方法，其中所述方法包括，在适合双特异性抗体或其链表达的条件下，培养包含编码所述双特异性抗体(例如任意一条多肽链和/或多条多肽链)的核酸或包含所述核酸的表达载体的宿主细胞，如上文所提供的，和任选地从所述宿主细胞(或宿主细胞培养基)回收所述双特异性抗体。
可以将编码本发明双特异性抗体的多肽链的多核苷酸插入一个或多个载体中以便进一步在宿主细胞中克隆和/或表达。可以使用本领域技术人员熟知的方法来构建表达载体。一旦已经制备了用于表达的包含本发明的一种或多种核酸分子的表达载体，则可以将表达载体转染或引入适宜的宿主细胞中。多种技术可以用来实现这个目的，例如，原生质体融合、磷酸钙沉淀、电穿孔、逆转录病毒的转导、病毒转染、基因枪、基于脂质体的转染或其他常规技术。
当使用重组技术时，双特异性抗体可以在细胞内、周质间隙中产生，或者直接分泌到培养基中。如果在细胞内产生双特异性抗体，那么作为第一步骤，可以例如通过离心或超滤来去除宿主细胞或溶解的片段的微粒状碎片。当双特异性抗体被分泌到培养基中时，通常首先使用可商购获得的蛋白质浓缩过滤器对来自此类表达系统的上清液进行浓缩。
如本文所述制备的双特异性抗体可以通过已知的现有技术如高效液相色谱、离子交换层析、凝胶电泳、亲和层析、尺寸排阻层析等纯化。用来纯化特定蛋白质的实际条件还取决于净电荷、疏水性、亲水性等因素，并且这些对本领域技术人员是显而易见的。
在某些实施方案中，固定在固相上的蛋白A用于抗体和其抗原结合片段的免疫亲和纯化。亲和配体附着的基质最常为琼脂糖，但其它基质也是可用的。机械力稳定的基质如可控孔度玻璃或聚(苯乙烯二乙烯)苯与用琼脂糖相比可实现更快的流速和更短的处理时间。在抗体包括CH3结构域的情况下，Bakerbond ABXTM树脂可用于纯化。根据待回收的抗体，用于蛋白质纯化的其它技术也是可用的，如在离子交换柱上进行分级、乙醇沉淀、反相HPLC、在二氧化硅上进行色谱法、在肝素SEPHAROSETM上进行色谱法、在阴离子或阳离子交换树脂(如聚天冬氨酸柱)上进行色谱法、色谱聚焦、SDS-PAGE、以及硫酸铵沉淀。
可以通过多种熟知分析方法中的任一种方法确定本发明的抗体分子的纯度，所述熟知分析方法包括尺寸排阻层析、凝胶电泳、高效液相色谱等。
V.对双特异性抗体的测定法。
可以通过本领域中已知的多种测定法对本文中提供的双特异性抗体进行鉴定，筛选，或表征其物理/化学特性和/或生物学活性。
一方面，对本发明的双特异性抗体测试其靶标(例如抗原，例如游离的抗原或在细胞上表达的抗原，例如PD-L1和/或CLDN18.2)结合活性，例如通过已知的方法诸如放射性免疫测定(RIA)/生物膜薄层干涉技术、ELISA、流式细胞术电化学发光(ECL)或表面等离子体共振法(SPR)等来进行。可使用本领域已知方法来测定对细胞上表达的CLDN18.2和/或PD-L1的结合，本文中公开了例示性方法。在一些实施方案中，使用流式细胞术(FACS)测量。
本发明还提供了用于鉴定双特异性抗体的生物学活性的测定法。生物学活性选自本发明的双特异性抗体的性质。
例如，对于本发明的抗体分子对表达CLDN18.2和/或PD-L1的细胞的结合活性，可以通过本领域已知的方法，例如荧光报道分子和流式细胞术，或本文实施例公开的示例性方法测定，例如通过如实施例3-1或3-2所示的方法测定本发明的抗体分子与细胞上表达的CLDN18.2和/或PD-L1的结合。
例如，对于本发明的抗体分子对ADCC活性的诱导，可以通过本领域已知的方法，例如实施例3-3或3-4所述的方法测定。
例如，对于本发明抗体分子对PD-L1/PD-1的结合活性的阻断，可以通过本领域已知的方法，例如报告基因的转录和表达例如NFAT-luc报告基因系统等，或本文实施例公开的示例性方法测定，例如通过如实施例3-5所示的方法测定。
例如，对于本发明的抗体分子对T细胞的激活活性和/或对阻断PD-1对T细胞激活的抑制，可以通过本领域已知的方法，例如通过检测激活T细胞后细胞因子(例如干扰素，如IFNγ)的释放来测定，例如本文实施例3-6公开的示例性方法测定，例如通过如实施例3-1或3-2所示的方法测定
例如，对于本发明抗体分子对肿瘤的抑制活性或结构安全性，可以通过本领域已知的方法，例如针对小鼠肿瘤模型进行肿瘤抑制实验，例如通过实施例4所示的方法来测定。
供任何上述体外测定法使用的细胞为原代细胞或细胞系，包括天然表达或过表达CLDN18.2(例如人)或PD-L1(例如人)的细胞，例如过表达CLDN18.2或PD-L1的细胞，例如胃癌细胞系NUGC-4、KATOII、SNU-620或过表达PD-L1的HEK293T-PD-L1细胞。
可以理解的是，能够使用本发明的抗体和别的活性剂的组合来进行任何上述测定法。
VI、本发明双特异性抗体的免疫缀合物、药物组合物、药物组合产品和试剂盒
在一些实施方案，本发明提供了包含本文所述的任何双特异性抗体的免疫缀合物。优选地，所述免疫缀合物包含本发明的双特异性抗体，以及一种或多种缀合物部分，例如化疗剂、细胞因子、细胞毒性剂或小分子药物、如纯化部分如磁 珠、生物素、清除修饰剂(例如，延长半衰期的聚合物，如PEG)、化学治疗剂、毒素、放射性同位素、镧系元素、可检测标记(例如，发光标记、荧光标记、酶底物标记)、DNA烷基化剂、拓扑异构酶抑制剂、微管蛋白结合剂或其它抗癌药物。
在一些实施方案中，本发明提供包含本文所述的任何双特异性抗体的组合物或药物或制剂，优选地组合物为药物组合物。本发明的所述组合物或药物或制剂还可以包含合适的药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂，例如药学上可接受的液体、凝胶或固体载体、水性媒剂、非水性媒剂、抗微生物剂、等渗剂、缓冲剂、抗氧化剂、麻醉剂、悬浮剂/分配剂、多价螯合剂或螯合剂、稀释剂、佐剂、赋形剂或无毒辅助物质、本领域已知的其它组分或其各种组合。对于药用辅料的使用及其用途，亦参见“Handbook of Pharmaceutical Excipients”,第八版,R.C.Rowe,P.J.Seskey和S.C.Owen,Pharmaceutical Press,London,Chicago。
本发明的组合物或药物或制剂可以处于多种形式。这些形式例如包括液体、半固体和固体剂型，如液态溶液剂、散剂或混悬剂、脂质体剂和栓剂。优选的形式取决于预期的施用模式和治疗用途。
在某些实施例中，将药物组合物调配成可注射组合物。可注射药物组合物可以以任何常规形式制备，所述常规形式例如液体溶液、悬浮液、乳液或适用于产生液体溶液、悬浮液或乳液的固体形式。注射制剂可以包括准备注射的无菌和/或无热原溶液、准备在使用前与溶剂组合的无菌干燥可溶性产品(如冻干粉末，包括皮下注射片剂)、准备注射的无菌悬浮液、准备在使用前与媒剂组合的无菌干燥的不溶性产品以及无菌和/或无热原乳液。溶液可以是水性的或非水性的。可以通过将具有所需纯度的本发明的双特异性抗体与一种或多种任选的药用辅料混合来制备包含本文所述的双特异性抗体的药物或制剂，例如以冻干制剂或水溶液的形式。
本发明的组合物或药物或制剂还可以包含超过一种活性成分，所述活性成分是被治疗的特定适应证所需的，优选具有不会不利地彼此影响的互补活性的那些活性成分。例如，理想的是还提供其它治疗剂。
在一些实施方案中，所述其他治疗剂选自化学治疗剂、抗癌药物、免疫治疗剂、抗血管生成剂、靶向疗法治疗剂、细胞疗法治疗剂、基因疗法治疗剂、激素疗法治疗剂、抗病毒剂、抗生素、镇痛剂、抗氧化剂、金属螯合剂或细胞因子。
本发明还提供了包含所述药物组合的成套药盒，例如所述成套药盒在同一包装内包含：
-含有包含本发明的双特异性抗体的药物组合物的第一容器；
-含有包含其它治疗剂的药物组合物的第二容器。
在一些实施方案中，所述其他治疗剂例如化学治疗剂、抗癌药物、免疫治疗剂、抗血管生成剂、靶向疗法治疗剂、细胞疗法治疗剂、基因疗法治疗剂、激素疗法治疗剂、抗病毒剂、抗生素、镇痛剂、抗氧化剂、金属螯合剂或细胞因子。
VII、抗CLDN18.2抗体或抗PD-L1抗体或双特异性抗体的用途和应用其的方法。
本发明一方面提供了在受试者中预防或治疗疾病的方法，包括向受试者施用本发明的双特异性抗体，或包含其的免疫缀合物、组合物或药物或制剂。在一些实施方案中，本发明提供了在受试者中特异性激活T细胞的方法，包括向受试者施用本发明的双特异性抗体，或包含其的免疫缀合物、组合物或药物或制剂。
在一些实施方案中，所述疾病例如肿瘤，例如癌症。癌症可以处于早期、中期或晚期或是转移性癌。在一些实施方案中，癌症可以是实体肿瘤或血液肿瘤。在一些实施方案中，所述癌症是胃肠道肿瘤、胆道肿瘤或肺癌，例如胃癌、食管癌、胰腺癌、结直肠癌、胆管癌、胆囊癌或肺癌等。
在一些实施方案中，所述癌症将受益于PD-L1与PD-1结合的阻断，或受益于PD-L1和/或CLDN18.2的抑制。
在一些实施方案中，所述癌症是表征为具有PD-L1的蛋白质表达或具有升高的PD-L1的蛋白质水平和/或核酸水平(例如表达升高)的癌症，例如，所述癌症的肿瘤细胞中具有PD-L1的蛋白质表达或具有升高的PD-L1的蛋白质水平和/或核酸水平(例如表达升高，例如相比相同组织类型的非癌或非肿瘤细胞或正常细胞)。
在一些实施方案中，所述癌症是表征为具有CLDN18.2的蛋白质表达或具有升高的CLDN18.2的蛋白质水平和/或核酸水平(例如表达升高)的癌症，例如，所述癌症的肿瘤细胞中具有CLDN18.2的蛋白质表达或具有升高的或CLDN18.2的蛋白质水平和/或核酸水平(例如表达升高，例如相比相同组织类型的非癌或非肿瘤细胞或正常细胞)。
在一些实施方案中，所述癌症是表征为具有PD-L1和CLDN18.2的蛋白质表达或具有升高的PD-L1和CLDN18.2的蛋白质水平和/或核酸水平(例如表达升高)的癌症，例如，所述癌症的肿瘤细胞中具有PD-L1和CLDN18.2的蛋白质表达或具有升高的PD-L1和CLDN18.2的蛋白质水平和/或核酸水平(例如表达升高，例如相比相同组织类型的非癌或非肿瘤细胞或正常细胞)。
在一些实施方案中，所述癌症表征为具有CLDN18.2的蛋白质表达或具有升高的CLDN18.2的蛋白质水平和/或核酸水平(例如表达升高)；且
(i)不具有PD-L1蛋白质表达；
(ii)相比非癌症具有升高的PD-L1的蛋白质水平和/或核酸水平(例如表达升高)，但是其蛋白质表达、蛋白质水平和/或核酸水平低于高表达PD-L1的癌症；且的癌症；或
(iii)具有升高的PD-L1的蛋白质水平和/或核酸水平(例如表达升高，例如相比相同组织类型的非癌或非肿瘤细胞或正常细胞)。
在一些具体的实施方案中，所述癌症是指癌细胞或肿瘤细胞在其细胞表面存在PD-L1蛋白的癌症，或与相同组织类型的非癌细胞相比，在癌症或肿瘤细胞的细胞表面具有更高水平的PD-L1的癌症。
在一些具体的实施方案中，所述癌症是指癌细胞或肿瘤细胞在其细胞表面存在CLDN18.2蛋白的癌症，或与相同组织类型的非癌细胞相比，在癌症或肿瘤细胞的细胞表面具有更高水平的CLDN18.2的癌症。
在一些具体的实施方案中，所述癌症是指这样的癌症，其中癌细胞或肿瘤细胞在其细胞表面存在CLDN18.2蛋白，或与相同组织类型的非癌细胞相比，在癌症或肿瘤细胞的细胞表面具有更高水平的CLDN18.2，且
(i)在所述癌细胞或肿瘤细胞在其细胞表面存在PD-L1蛋白，
(ii)与相同组织类型的非癌细胞相比，在所述癌症或肿瘤细胞的细胞表面具有更高水平的PD-L1；
(iii)在所述癌细胞或肿瘤细胞在其细胞表面不存在PD-L1蛋白的表达，或者
(iv)在所述癌细胞或肿瘤细胞在其细胞表面存在PD-L1蛋白，且与高表达PD-L1的癌症或肿瘤相比，在所述癌症或肿瘤细胞的细胞表面具有低水平的PD-L1；或
(v)与相同组织类型的非癌细胞相比，在所述癌症或肿瘤细胞的细胞表面具有更高水平的PD-L1，且与高表达PD-L1的癌症或肿瘤相比，在所述癌症或肿瘤细胞的细胞表面具有低水平的PD-L1。
PD-L1和/或CLDN18.2表达或升高的表达可以在诊断或预后测定中通过评估存在于细胞表面上的PD-L1和/或CLDN18.2水平增加来确定(例如，通过免疫组织化学测定；IHC)。可替代地或另外地，可以例如通过荧光原位杂交(FISH)、southern印迹或聚合酶链反应(PCR)技术，如实时定量PCR(RT-PCR)来测量细胞中编码PD-L1和/或CLDN18.2的核酸的水平。还可以通过测量如血清等生物流体中的脱落抗原(例如，PD-L1和/或CLDN18.2的胞外结构域或可溶性蛋白)来研究PD-L1和/或CLDN18.2的表达。除了上述测定之外，各种体内测定也是可获得的。例如，可以将患者体内的细胞暴露于抗PD-L1和/或CLDN18.2抗体，所述抗体任选地用可检测标记(例如，放射性同位素)进行标记，并且可以例如通过外部扫描放射性或通过分析取自先前暴露于抗体的患者的活组织检查评估抗体与患者细胞的结合。
在一些实施例中，受试者已被鉴定为可能对PD-L1和/或CLDN18.2拮抗剂有应答。所关注生物样品上PD-L1和/或CLDN18.2的存在或水平可以指示生物样品所源自的受试者是否可能对PD-L1和/或CLDN18.2拮抗剂有应答。在一些实施例中，测试样品源自癌细胞或组织、或肿瘤浸润免疫细胞。在某些实施例中，测试生物样品中PD-L1和/或CLDN18.2的存在或上调水平指示应答的可能性。如本文所使用的，术语“上调”是指与使用相同抗体检测的参考样品中的PD-L1和/或CLDN18.2蛋白水平相比，测试样品中PD-L1和/或CLDN18.2的蛋白水平总体增加不少于10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％或更大。参考样品可以是从健康或非患病个体获得的对照样品，或者是从测试样品所获自的同一个体获得的健康或非患病样品。例如，参考样品可以是与测试样品(例如，肿瘤)相邻或在测试样品附近的非患病样品。
如本文所定义的“PD-L1的表达”可以通过TPS或CPS评分来进行衡量。本文所用的术语“TPS”是指IHC免疫组化法下，肿瘤细胞在任何强度下显示部分或完整的PD-L1膜染色的细胞数占总肿瘤细胞数的百分比。本文所用的术语“CPS”是指PD-L1染色细胞(包括肿瘤细胞、淋巴细胞、巨噬细胞)的数目之和除以肿瘤细胞总数，乘以100得出结果。
如本文所定义的“PD-L1高表达”是指在所述肿瘤或癌症组织中，其CPS评分至少1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、15％或20％以上。因此，“高表达PD-L1的癌症或肿瘤”是指在所述癌症或肿瘤组织中，至少1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、15％或20％以上的细胞对PD-L1的表达是阳性的。
本发明的双特异性抗体(以及包含其的免疫缀合物、组合物、药物组合物、制剂、组合产品等)可以通过任何合适的方法给药，包括肠胃外给药，肺内给药和鼻内给药，并且，如果局部治疗需要，病灶内给药。肠胃外注射或输注包括肌内、静脉内、动脉内、腹膜内或皮下注射或输注。在一定程度上根据用药是短期或长期性而定，可通过任何适合途径，例如通过注射，例如静脉内或皮下注射用药。本文中涵盖各种用药时程，包括，但不限于，单次给药或在多个时间点多次给药、推注给药及脉冲输注。
为了预防或治疗疾病，本发明的双特异性抗体(以及包含其的免疫缀合物、组合物、药物组合物、制剂、组合产品等)的合适剂量(当单独或与一种或多种其他的治疗剂组合使用时)将取决于待治疗疾病的类型、抗体的类型、疾病的严重性和进程、以预防目的施用还是以治疗目的施用、以前的治疗、患者的临床病史和对所述抗体的应答，和主治医师的判断力。所述抗体以一次治疗或经过一系列治疗合适地施用于患者。在其他方面，本发明提供本发明的双特异性抗体或包含其的免疫缀合物或组合物或组合产品在生产或制备药物中的用途，所述药物用于本文所述的用途，例如用于预防或治疗本文提及的相关疾病或病症。
在一些实施方案中，双特异性抗体(以及包含其的免疫缀合物、组合物、药物组合物、制剂等)还能与一种或多种其它疗法例如治疗方式和/或其它治疗剂组合施用，用于本文所述的用途，例如用于预防和/或治疗本文提及的相关疾病或病症。
在一些实施方案中，所述治疗方式例如手术或放疗。
在一些实施方案中，所述其他治疗剂例如化疗剂、细胞因子、细胞毒性剂、其它抗体、小分子药物或免疫调节剂(例如免疫抑制剂)、化学治疗剂、抗癌药物、免疫治疗剂、抗血管生成剂、靶向疗法治疗剂、细胞疗法治疗剂、基因疗法治疗剂、激素疗法治疗剂、抗病毒剂、抗生素、镇痛剂、抗氧化剂、金属螯合剂或细胞因子。
VII、诊断和检测
在一些实施方案中，本文中提供的抗双特异性抗体可以用于检测CLDN18.2和/或PD-L1在生物样品中的存在。
术语“检测”用于本文中时，包括定量或定性检测，示例性的检测方法可以涉及免疫组织化学、免疫细胞化学、流式细胞术(例如，FACS)、抗体分子复合的 磁珠、ELISA测定法、PCR-技术(例如，RT-PCR)。在某些实施方案中，生物样品是体液，例如血液、血清或血浆。
在某些实施方案中，所述方法包括将生物样品与如本文所述的双特异性抗体在允许其与PD-L1和/或CLDN18.2结合的条件下接触，并检测在该双特异性抗体和PD-L1和/或CLDN18.2之间是否形成复合物。复合物的形成表示存在PD-L1。该方法可以是体外或体内方法。
在本文中提供的一些实施方案中，样品是在用本发明的双特异性抗体治疗之前获得的。在一些实施方案中，样品是在用其他疗法(例如抗PD-L1抗体治疗或抗CLDN18.2抗体治疗)之前获得的。在一些实施方案中，样品是在用其他疗法治疗过程中，或者用其他疗法治疗后获得的。
在一些实施方案中，在治疗之前，例如，在起始治疗之前或在治疗间隔后的某次治疗之前检测PD-L1和/或CLDN18.2。
实施例
虽然本发明已经参考特定实施例(其中一些是优选实施例)进行了详细的展示和描述，但本领域技术人员应该理解，在不脱离本文所公开的本发明的精神和范围的情况下，可以在形式和细节上进行各种改变，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1：肿瘤细胞在CLDN18.2(Claudin18.2)抗体介导ADCC(抗体依赖性细胞介导的细胞毒性)效应后，其PD-L1表达的变化。
本申请中CLDN18.2抗体的产生和纯化方法已在申请号为WO2021032157A1的专利文件中描述。在本试验中，三种不同的CLDN18.2表达的胃癌细胞系NUGC-4(JCRB，Cat#JCRBB0834)、KATOIII(ATCC，Cat#HTB-103)和SNU-620(Cobioer，Cat#CBP6058)作为靶细胞并加到细胞培养板中。培养基分别为NUGC-4:DMEM+20％FBS,KATOIII:IMDM+20％FBS,SNU-620:RPMI-1640+10％FBS。
无抗体，30μg/ml抗CLDN18.2抗体18B10，30μg/ml同型对照hIgG1(重链为SEQ ID NO:35，轻链为SEQ ID NO0:36)或5ng/ml IFN-γ(R&D，货号285-IF)与5×104靶细胞一起加入相应的孔中。将培养板在37℃培养30分钟后，加入1×106人外周血单个核细胞(PBMC)(妙顺，TPCS PB025C)作为效应细胞，其E/T(效应细胞/靶细胞)比为20:1。37℃培养72小时后，将每个孔的100μl细胞培养上清液转移到另一个细胞培养板上，并加入相同的5×104靶细胞。在第二个培养板在37℃培养72小时后，收集靶细胞，并使用小鼠抗人PD-L1抗体(BD Pharmingen，Cat#557924)通过流式细胞术分析PD-L1表达。S1代表未加抗体的单独靶细胞的上清液；S2代表靶细胞加人PBMC加上同型对照hIgG1的上清液；S3代表靶细胞加人PBMC加上CLDN18.2抗体18B10的上清液；S4代表加IFN-γ的靶细胞的上清液。
如图1A-C所示，S1组靶细胞表达一个本底水平的PD-L1，与之相比，S3和S4组的PD-L1表达增加，而S2组仅有轻微变化。这一结果表明CLDN18.2 抗体介导的ADCC效应可上调靶细胞PD-L1的表达，其机制可能是通过ADCC效应产生的IFN-γ介导的。Chaganty BKR等人(Chaganty BKR等人，《癌症快报》，2018:S0304383518303288)也发现HER2抗体曲妥珠单抗通过ADCC作用产生的IFN-γ可以上调HER2阳性的肿瘤细胞的PD-L1的表达。
实施例2：CLDN18.2×PD-L1双特异抗体的产生
IgG(H)scfv-18B10-AM4B6V1是一种抗CLDN18.2和抗PD-L1的双特异抗体。它的轻链来自于抗CLDN18.2抗体18B10的轻链。重链是由抗CLDN18.2抗体18B10的重链和PD-L1抗体AM4B6(PCT/CN2021/131389)的scfv组成，二者用一个(Gly4Ser)4连接子连接在一起，如图2A和表1所示。IgG(H)scfv-AM4B6-18B10V1也是一种抗PD-L1和抗CLDN18.2的双特异抗体。它的轻链来自于抗PD-L1抗体AM4B6的轻链。重链是由抗PD-L1抗体AM4B6的重链和CLDN18.2抗体18B10的scfv组成，二者用一个(Gly4Ser)4连接子连接在一起，如图2B和表1所示。在连接处，抗体重链的C端赖氨酸残基突变为丙氨酸，以减少蛋白水解裂解。
将两个二硫键(H44C-L100C)引入V1分子的抗PD-L1抗体AM4B6的scfv中，即生成IgG(H)scfv-18B10-AM4B6V2分子。IgG(H)scfv-18B10-AM4B6V2的轻链与IgG(H)scfv-18B10-AM4B6V1分子的轻链相同。抗CLDN18.2抗体18B10的重链和抗PD-L1抗体AM4B6的scfv之间也是用(Gly4Ser)4连接子连接，如图2C和表1所示。在连接处，抗体18B10重链恒定区的C端赖氨酸残基突变为丙氨酸，以减少蛋白水解裂解。
CLDN18.2×PD-L1双特异抗体的表达是用含有编码轻链的DNA和编码IgG-scfv的DNA的载体pCDNA3.1(+)转染Expi-CHO细胞，按照Invitrogen提供的方法，使用ExpiFectamineTM CHO试剂。当细胞存活率超过60％时收集上清液，并通过0.22μm过滤膜过滤以去除细胞碎片。随后将过滤的上清液装载到预平衡的蛋白质A亲和柱上。蛋白A树脂用平衡缓冲液(PBS)洗涤，然后用25mM柠檬酸盐(pH3.5)洗脱抗体。使用1M Tris碱基(pH 9.0)将纯化抗体溶液调节至pH 6.0-7.0。用Superdex色谱法进一步纯化样品，以去除聚集物。最后，通过SDS-PAGE和SEC-HPLC分析对纯化的抗体进行了表征。
表1.CLDN18.2×PD-L1双特异抗体的序列
PD-L1单克隆抗体AM4B6(PCT/CN2021/131389，重链为SEQ ID NO:40，其在Fc区具有减弱ADCC活性的L234F/L235E/P331S突变，轻链为SEQ ID NO:26)。其表达和纯化方法如双特异性抗体方法所述制备。
实施例3:CLDN18.2×PD-L1双特异抗体的体外研究
1.与CLDN18.2阳性细胞的结合
收集对数生长期的NUGC-4细胞，并将其重新悬浮在PBS中。使用FACS洗涤缓冲液(PBS+2％FBS)进行3次洗涤后，每孔加入100μl梯度稀释好的CLDN18.2×PD-L1双特异性抗体或PD-L1抗体AM4B6或同型对照抗体hIgG1(起始浓度400nM，4倍稀释)，浓度范围为400nM至0.006nM，在4℃下孵育30分钟。然后用FACS洗涤缓冲液洗涤细胞3次，然后在4℃下与100μl/孔山羊抗人IgG-FITC(Abcam，货号98623)(1:400稀释)再共孵育30分钟。最后使用FACS洗涤缓冲液洗涤3次后，流式细胞仪(Beckman，CytoFLEX)分析细胞。
如图3A所示，IgG(H)scfv-18B10-AM4B6V1与NUGC-4细胞的结合活性与单抗18B10类似，只是最大信号值略低于18B10。相比之下，IgG(H)scfv-AM4B6-18B10V1的结合受到很大影响。图3B所示，IgG(H)scfv-18B10-AM4B6V2与NUGC-4细胞的结合活性也与IgG(H)scfv-18B10-AM4B6V1和18B10相似，表明其与CLDN18.2的结合活性未受二硫键引入的影响。
2.与PD-L1阳性细胞的结合
HEK293T-PD-L1细胞是购买自中美冠科生物技术有限公司，货号2005。CLDN18.2×PD-L1双特异抗体与细胞结合的检测方法同上。
如图4A所示，IgG(H)scfv-18B10-AM4B6V1和IgG(H)scfv-AM4B6-18B10V1与PD-L1阳性细胞的结合类似于单抗AM4B6。IgG(H)scfv-18B10-AM4B6V1的EC50比AM4B6略高，但最大信号值高于AM4B6。IgG(H)scfv-18B10-AM4B6V2与PD-L1的结合未受二硫键引入的影响(Figure 4B)。
3.CLDN18.2抗体介导的ADCC活性
ADCC活性是通过Jurkat-NFAT-luc-FcγRIIIA-V176细胞作为效应细胞，而NUGC-4细胞作为靶细胞来检测。Jurkat-NFAT-luc-FcγRIIIA-V176细胞是由苏州创胜医药集团有限公司内部构建的。简单地，向Jurkat细胞(Shanghai Institutes for Biological Sciences,Cat#SCSP-513)转入pGL4.30-luc/NFAT-RE/Hygro质粒(Promega)，然后用潮霉素进行筛选，得到稳定表达的细胞株Jurkat-NFAT-luc。将FcγRIIIA-V176(SEQ ID NO:37)序列构建到载体pVitro-neo(InvivoGen)中获得pVitro-neo-pcDNA3.1-FcγRIIIA-V176质粒。在得到的细胞株中继续转染pVitro-neo-FcγRIIIA-V176质粒，用抗生素G418筛选得到稳定表达细胞株Jurkat-NFAT-luc-FcγRIIIA-V176。
接下来，将对数生长期的NUGC-4细胞重悬在RPMI1640+10％FBS，然后以每孔1×104细胞铺板，37℃孵育30分钟，获得包含把靶细胞的培养板。抗体和同型对照hIgG1用RPMI1640+10％FBS稀释(起始浓度为20nM，5倍稀释)，然后以终浓度20到0.001nM加入靶细胞培养板中。取对数生长期的Jurkat-NFAT-luc-FcγRIIIA-V176细胞以每孔6×104细胞加入到上述板中。在37℃孵育6小时后，拿到室温平衡30分钟。用Cell Titer-Glo Luminescent Cell Viability Assay Kit检测活细胞数，通过酶标仪(Thermo VARIOSKAN FLASH 3001)读荧光值。
如图5A所示，CLDN18.2×PD-L1双特异抗体IgG(H)scfv-18B10-AM4B6V1与单抗18B10类似，可以产生对靶细胞NUGC-4的ADCC效应，二者的ADCC 活性相近。IgG(H)scfv-AM4B6-18B10V1的ADCC活性受到了很大影响。如图5B所示，IgG(H)scfv-18B10-AM4B6V2的ADCC活性与IgG(H)scfv-18B10-AM4B6V1基本相同。
4.抗PD-L1抗体介导的ADCC活性
ADCC报告基因实验的方法同上述。
如图6所示，IgG(H)scfv-18B10-AM4B6V1也可以对靶细胞HEK293T-PD-L1产生ADCC效应。IgG(H)scfv-AM4B6-18B10V1也可以通过PD-L1的结合诱导ADCC效应。
5.阻断PD-L1/PD-1结合的活性
本实验使用既表达PD-L1又表达anti-CD3scFv的293T-PD-L1-CD3L细胞，以及既表达PD-1又带有NFAT报告基因的Jurkat-NFAT-Luc-PD-1细胞。NFAT的激活可以引起荧光素酶基因转录和表达，通过加入其底物进行检测。两株细胞的构建如下：
在上述HEK293T-PD-L1细胞中转染含有抗CD3的scfv的质粒pcDNA3.1-hygro-CD3L(将抗CD3的scfv(SEQ ID NO:38)序列构建到载体pcDNA3.1(+)中获得)，然后用含有3μg/mL的嘌呤霉素和10μg/mL的潮霉素B的DMEM+10％FBS培养基进行筛选培养，筛选出的细胞即为293T-PD-L1-CD3L细胞。
在Jurkat细胞(上海中科院细胞所，货号SCSP-513)转染含有PD-1的质粒(其中含有PD-1的质粒是将编码人PD-1序列(SEQ ID NO:39)构建到pcDNA3.1(+)，获得人PD-1-pcDNA3.1(+))和含有NFAT-荧光素酶基因的质粒pGL4.30-luc/NFAT-RE/Hygro(Promega)，并在含有500μg/mL的G418和200μg/mL潮霉素B的RPMI1640+10％FBS筛选培养基中培养，即得到Jurkat-NFAT-Luc-PD-1细胞。
简单的，将293T-PD-L1-CD3L细胞以2×106细胞/ml重悬。每孔加入20μl细胞到半孔板中。分别用RPMI medium+2％FBS梯度稀释(起始浓度33nm，3倍梯度)上述构建的CLDN18.2×PD-L1双特异抗体、单抗AM4B6以及同型对照抗体hIgG1。每孔加入20μl抗体到半孔板中，37℃，5％CO2孵育30分钟。将Jurkat-NFAT-Luc-PD1细胞用RPMI medium+2％FBS以4×10^6细胞/ml重悬。然后每孔加入20μl Jurkat-NFAT-Luc-PD1细胞到半孔板中，并于37℃，5％CO2孵育5小时。最后，每孔加入60μl的OneGlo detection reagent(Promega,E6120)，室温孵育5分钟。用酶标仪读取化学发光值。数据用GraphPad Prism软件进行分析。
如图7A所示，与结合的结果相似的，IgG(H)scfv-18B10-AM4B6V1和IgG(H)scfv-AM4B6-18B10V1对PD-L1/PD-1的阻断活性与PD-L1单抗AM4B6相近，这表明除了对肿瘤细胞的ADCC杀伤作用以外，两个双特异抗体还可以阻断肿瘤微环境中PD-L1/PD-1的免疫抑制作用。IgG(H)scfv-18B10-AM4B6V2的阻断活性与IgG(H)scfv-18B10-AM4B6V1相似，如图7B所示。
6.CLDN18.2×PD-L1双特异抗体对PBMC的激活作用
预先用CD3抗体(Dynabeads,Gibco,货号11131D)对人PBMC(妙顺，TPCS PB025C)刺激24小时，获得预刺激的效应细胞。然后将对数生长期的NCI-H460-CLDN18.2细胞(康源博创(Cat#KC1450))(靶细胞)与预刺激的PBMC 以效/靶比为15：1的比例共孵育。同时加入抗体，包括同型对照hIgG1，AM4B6，18B10以及IgG(H)scfv-18B10-AM4B6V1,在37℃，5％CO2孵育48小时。培养结束时，用Human IFN-gamma DuoSet ELISA试剂盒(R&D,DY285B)检测细胞上清中的IFN-γ。
如图8所示，在CD3抗体的刺激作用下，同型对照组也有显著的IFN-γ。AM4B6和18B10均比同型对照抗体诱导产生更高的IFN-γ，说明PD-L1的阻断效应和CLDN18.2介导的ADCC活性同时存在在该实验体系中。与两个单抗相比，IgG(H)scfv-18B10-AM4B6V1产生更高的IFN-γ，说明该双特异抗体有效的结合了两个靶点的作用。
实施例4:CLDN18.2×PD-L1双特异抗体的体内研究
在NCI-H460-CLDN18.2和人PBMC共接种的异种移植肿瘤模型上的药效学研究
NCI-H460-CLDN18.2细胞是转染了CLDN18.2的人大细胞肺癌，其内源性表达PD-L1，购买自康源博创(Cat#KC1450)。用胰酶-EDTA(Hyclone)消化传代培养，每周传代两次。取对数生长期的细胞用于肿瘤接种NOD-SCID小鼠，所述小鼠购自杭州子源实验动物科技有限公司，接种时每周为5-6周龄。
每只SPF级雌性NOD-SCID小鼠皮下接种3×10^6的NCI-H460-CLDN18.2细胞和1.5×10^6的人PBMC，以及50％matrigel(Corning)。接种4小时后，随机分为5组，每组10只小鼠。分别i.p.给予30mg/kg同型对照hIgG1，15mg/kg 18B10，15mg/kg AM4B6，15mg/kg 18B10+15mg/kg AM4B6以及20mg/kg IgG(H)scfv-18B10-AM4B6V1，每周给药2次，共给药5周。
用卡尺(INSIZE)测量肿瘤直径，每周测2或3次，肿瘤体积用公式V＝0.5a×b^2计算，a为长直径，b为短直径，单位为mm^3。结果用Prism GraphPad分析，用平均值±S.E.M来表示。用T检验比较两组差异，p值<0.05(*)或<0.01(**)则差异显著。
单独CLDN18.2抗体18B10或PD-L1抗体AM4B6都仅有非常弱的肿瘤抑制活性，其肿瘤抑制率(TGI)为33％(图9，表2)。当两个抗体联合给药时，其TGI增加至60％。而CLDN18.2×PD-L1双特异抗体IgG(H)scfv-18B10-AM4B6V1给药组的TGI进一步增加至70％，说明在这种既表达CLDN18.2又表达PD-L1的肿瘤模型上，双特异抗体有更好的疗效。
表2.IgG(H)scfv-18B10-AM4B6V1在NCI-H460-CLDN18.2和人hPBMC共接种的异种移植肿瘤模型上第36天的肿瘤抑制率
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Claims (54)

  1. 双特异性抗体，其包含针对(人)PD-L1的第一结合特异性和针对(人)CLDN18.2的第二结合特异性，其中所述特异性结合PD-L1的第一抗原结合区包含重链可变区VH的3个CDR，HCDR1、HCDR2和HCDR3，以及轻链可变区VL的3个CDR，LCDR1、LCDR2和LCDR3，其中
    所述HCDR1、HCDR2和HCDR3为如SEQ ID NO:17所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3；且所述LCDR1、LCDR2和LCDR3为如SEQ ID NO:22所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3。
  2. 权利要求1的双特异性抗体，其中
    所述HCDR1、HCDR2和HCDR3分别为如SEQ ID NO:18所示的HCDR1、如SEQ ID NO：19所示的HCDR2、如SEQ ID NO:20所示的HCDR3；且所述LCDR1、LCDR2和LCDR3为如SEQ ID NO:23所示的LCDR1、如SEQ ID NO:24所示的LCDR2和如SEQ ID NO:25所示的LCDR3。
  3. 权利要求1或2的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区包含重链可变区VH，其中所述重链可变区
    (i)包含与SEQ ID NO:17或30所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
    (ii)包含SEQ ID NO:17或30所示的氨基酸序列或由所述氨基酸序列组成。
  4. 权利要求1-3中任一项的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区包含轻链可变区VL，其中所述轻链可变区
    (i)包含与SEQ ID NO:22或31所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
    (ii)包含SEQ ID NO:22或31的氨基酸序列或由所述氨基酸序列组成。
  5. 权利要求1-4中任一项的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区包含重链可变区VH和轻链可变区VL，其中
    (i)所述重链可变区包含SEQ ID NO:17或30所示的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；且所述轻链可变区包含SEQ ID NO:22或31所示的氨基酸序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成。
  6. 权利要求1-5中任一项的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区包含重链可变区VH和轻链可变区VL，其中VH和VL分别包含如下所示的氨基酸序列或由所述氨基酸序列组成：
    (iii).SEQ ID NO:17和SEQ ID NO:30；或
    (iv).SEQ ID NO:22和SEQ ID NO:31。
  7. 权利要求1-6中任一项的双特异性抗体，所述特异性结合CLDN18.2的第二抗原结合区包含重链可变区VH的3个CDR，HCDR1、HCDR2和HCDR3，以及轻链可变区VL的3个CDR，LCDR1、LCDR2和LCDR3，其中
    所述HCDR1、HCDR2和HCDR3为如SEQ ID NO:1所示的VH中所含的三个互补决定区域HCDR1、HCDR2和HCDR3；且所述LCDR1、LCDR2和LCDR3为如SEQ ID NO:6所示的VL中所含的三个互补决定区域LCDR1、LCDR2和LCDR3。
  8. 权利要求7的双特异性抗体，其中所述特异性结合CLDN18.2的第二抗原结合区包含
    如SEQ ID NO:2所示的HCDR1、如SEQ ID NO：3所示的HCDR2、如SEQ ID NO:4所示的HCDR3；如SEQ ID NO:7所示的LCDR1、如SEQ ID NO:8所示的LCDR2和如SEQ ID NO:9所示的LCDR3。
  9. 权利要求7或8的双特异性抗体，其中所述特异性结合CLDN18.2的第二抗原结合区包含重链可变区VH，其中所述重链可变区
    (i)包含与SEQ ID NO：1所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
    (ii)包含SEQ ID NO:1所示的氨基酸序列或由所述氨基酸序列组成。
  10. 权利要求7-9中任一项的双特异性抗体，其中所述特异性结合CLDN18.2的第二抗原结合区包含轻链可变区VL，其中所述轻链可变区
    (i)包含与SEQ ID NO:6所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或者
    (ii)包含SEQ ID NO:6的氨基酸序列或由所述氨基酸序列组成。
  11. 权利要求7-10中任一项的双特异性抗体，其中所述特异性结合CLDN18.2的第二抗原结合区包含重链可变区VH和轻链可变区VL，其中VH和VL分别包含如下所示的氨基酸序列或由所述氨基酸序列组成：SEQ ID NO:1和SEQ ID NO:6。
  12. 权利要求1-11中任一项所述双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区和/或特异性结合CLDN18.2的第二抗原结合区还包含重链恒定区HC或来自所述HC的Fc区，例如，所述抗体重链恒定区HC为IgG1、IgG2、IgG3或IgG4的重链恒定区，优选的IgG1的重链恒定区。
  13. 权利要求12所述的双特异性抗体，其中所述重链恒定区
    (i)包含与选自SEQ ID NO:13的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或
    (ii)包含选自SEQ ID NO:13的氨基酸序列或由所述氨基酸序列组成。
  14. 权利要求12所述的双特异性抗体，其中所述Fc区
    (i)包含与选自SEQ ID NO:14的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或
    (ii)包含选自SEQ ID NO:14的氨基酸序列或由所述氨基酸序列组成。
  15. 权利要求1-14中任一项所述的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区和/或特异性结合CLDN18.2的第二抗原结合区还包含轻链恒定区，例如所述轻链恒定区为lambda或kappa轻链恒定区。
  16. 权利要求15的双特异性抗体，其中所述轻链恒定区
    (i)包含与选自SEQ ID NO:16的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列或由所述氨基酸序列组成；或
    (ii)包含选自SEQ ID NO:16的氨基酸序列或由所述氨基酸序列组成。
  17. 权利要求1-16中任一项所述的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区包含重链，其中所述重链包含SEQ ID NO:21的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
  18. 权利要求1-17中任一项所述的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区包含轻链，其中所述轻链包含SEQ ID NO:26的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
  19. 权利要求12或13所述的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区包含重链和轻链，其中
    (i)所述重链包含SEQ ID NO:21的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成；且所述轻链包含SEQ ID NO:26的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
  20. 权利要求19所述的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区包含重链和轻链，其中所述重链包含SEQ ID NO：21所示的氨基酸序列，或由所述氨基酸序列组成，且所述轻链包含SEQ ID NO:26所示的氨基酸序列，或由所述氨基酸序列组成。
  21. 权利要求1-20中任一项所述的双特异性抗体，其中所述特异性结合CLDN18.2的第二抗原结合区包含重链，其中所述重链包含SEQ ID NO:5的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
  22. 权利要求1-21中任一项所述的双特异性抗体，其中所述特异性结合CLDN18.2的第二抗原结合区包含轻链，其中所述轻链包含SEQ ID NO:10的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
  23. 权利要求21或22所述的双特异性抗体，其中所述特异性结合CLDN18.2的第二抗原结合区包含重链和轻链，其中
    (i)所述重链包含SEQ ID NO:5的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成；且所述轻链包含SEQ ID NO:10的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
  24. 权利要求23所述的双特异性抗体，其中所述特异性结合CLDN18.2的第二抗原结合区包含重链和轻链，其中所述重链包含SEQ ID NO：5所示的氨基酸序列，或由所述氨基酸序列组成，且所述轻链包含SEQ ID NO:10所示的氨基酸序列，或由所述氨基酸序列组成。
  25. 权利要求1-24任一项所述的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区和/或特异性结合CLDN18.2的第二抗原结合区来源于人源化抗体或嵌合抗体。
  26. 权利要求1-25中任一项所述的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区和/或特异性结合CLDN18.2的第二抗原结合区为选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体(例如scFv)、(Fab’)2、单结构域抗体例如VHH、dAb(domain antibody)或线性抗体，例如所述scFv包含二硫键稳定的突变，所述突变例如其中所述重链可变区和/或轻链可变区包含引入二硫键的突变，例如所述重链可变区包含在第44位氨基酸取代为半胱氨酸的突变(Kabat编号)，和/或所述轻链可变区包含在第100位氨基酸取代为半胱氨酸的突变(Kabat编号)。
  27. 权利要求26所述的双特异性抗体，其中所述特异性结合PD-L1的第一抗原结合区为scFv，优选地，所述scFv包含SEQ ID NO:27或29的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
  28. 权利要求26所述的双特异性抗体，其中所述特异性结合CLDN18.2的第二抗原结合区为scFv，优选地，所述scFv包含SEQ ID NO:11的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
  29. 权利要求1-28中任一项所述的双特异性抗体，其中所述双特异性抗体中的第一抗原结合区和第二抗原结合区之间通过接头连接，所述接头例如包含氨基酸序列(G4S)n,其中n＝1,2,3,4或5，优选地，n＝3或4，更优选地，n＝4。
  30. 权利要求1-29中任一项所述的双特异性抗体，其中所述特异性结合PD-L1 的第一抗原结合区为全长抗体，且所述特异性结合CLDN18.2的第二抗原结合区为scFv；或者所述特异性结合CLDN18.2的第二抗原结合区为全长抗体，且所述特异性结合PD-L1的第一抗原结合区为scFv。
  31. 权利要求30的双特异性抗体，其中scFv与全长抗体的重链恒定区的C末端连接。
  32. 权利要求30的双特异性抗体，其中两个scFv分别与全抗抗体的两个重链恒定区的C末端连接。
  33. 权利要求1-29中任一项所述的双特异性抗体，其包含轻链和重链，
    其中轻链从N端到C端包含VL-A-CL；
    重链从N端到C端包含VH-A-CH-scFv；
    其中scFv包含VH-B和VL-B；
    其中VL-A为特异性结合PD-L1的第一抗原结合区的轻链可变区VL，VH-A为特异性结合PD-L1的第一抗原结合区的重链可变区VH；VH-B为特异性结合CLDN18.2的第二抗原结合区的重链可变区VH，VL-B为特异性结合CLDN18.2的第二抗原结合区的轻链可变区VL；或者VL-A为特异性结合CLDN18.2的第二抗原结合区的轻链可变区VL，VH-A为特异性结合CLDN18.2的第二抗原结合区的重链可变区VH；VH-B为特异性结合PD-L1的第一抗原结合区的重链可变区VH，VL-B为特异性结合PD-L1的第一抗原结合区的轻链可变区VL；
    CL为轻链恒定区；
    CH为重链恒定区；
    scFv的VH-B和VL-B之间包含接头或不包含接头；
    其中所述CH与scFv之间包含接头或不包含接头。
  34. 权利要求33的双特异性抗体，其包含两条重链和两条轻链，例如所述重链相同和/或所述轻链相同。
  35. 权利要求33或34所述的双特异性抗体，其中所述scFv从N端到C端包含VH-B-VL-B，且VH-B在其N末端与CH的C末端通过或不通过接头连接。
  36. 权利要求33-35中任一项所述的双特异性抗体，其中所述接头例如包含氨基酸序列(G4S)n,其中n＝1,2,3,4或5，优选地，n＝3或4，更优选地，n＝4。
  37. 权利要求33-36中任一项所述的双特异性抗体，其中所述重链包含SEQ ID NO:32、33或34的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
  38. 权利要求33-37中任一项所述的双特异性抗体，其中所述轻链包含SEQ ID NO:10或26的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
  39. 权利要求33或34所述的双特异性抗体，其中
    (i)所述重链包含SEQ ID NO:32或34的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成；
    且所述轻链包含SEQ ID NO:10的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成；
    (ii)所述重链包含SEQ ID NO:33的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成；
    且所述轻链包含SEQ ID NO:26的氨基酸序列，或包含与所述氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由所述氨基酸序列组成。
  40. 权利要求39所述的双特异性抗体，其中
    (i)所述重链包含SEQ ID NO:32或34的氨基酸序列或由所述氨基酸序列组成；且所述轻链包含SEQ ID NO:10的氨基酸序列或由所述氨基酸序列组成；
    (ii)所述重链包含SEQ ID NO:33的氨基酸序列或由所述氨基酸序列组成；且所述轻链包含SEQ ID NO:26的氨基酸序列或由所述氨基酸序列组成。
  41. 核酸分子，其编码权利要求1-40中任一项的所述的双特异性抗体的任一条链。
  42. 表达载体，其包含权利要求41的核酸分子，优选地，所述表达载体为pCDNA，例如pCDNA3.1。
  43. 宿主细胞，其包含权利要求41所述的核酸分子或权利要求42所述的表达载体，优选地，所述宿主细胞是原核的或真核的，例如293细胞或CHO细胞，例如293F细胞或293T细胞或Expi-CHO细胞。
  44. 制备权利要求1-40中任一项所述的双特异性抗体的方法，所述方法包括，在适合所述抗体的链表达的条件下，培养包含权利要求41所述的核酸分子或权利要求42所述的表达载体的宿主细胞，或权利要求43所述的宿主细胞，和任选地从所述宿主细胞(或宿主细胞培养基)回收所述抗体。
  45. 免疫缀合物，其包含权利要求1-40中任一项所述的双特异性抗体；
  46. 权利要求45的免疫缀合物，其还包含化疗剂、细胞因子、细胞毒性剂或小分子药物、纯化部分如磁珠、生物素、清除修饰剂(例如，延长半衰期的聚合物，如PEG)、化学治疗剂、毒素、放射性同位素、镧系元素、可检测标记(例如，发光标记、荧光标记、酶底物标记)、DNA烷基化剂、拓扑异构酶抑制剂、微管蛋白结合剂或其它抗癌药物。
  47. 药物组合物或药物或制剂，其包含权利要求1-40中任一项的所述的双特异 性抗体，或权利要求45或46的免疫缀合物以及任选地药用辅料。
  48. 药物组合产品，其包含权利要求1-40中任一项所述的双特异性抗体，或权利要求45或46的免疫缀合物，以及一种或多种其它治疗剂(例如化学治疗剂、抗癌药物、免疫治疗剂、抗血管生成剂、靶向疗法治疗剂、细胞疗法治疗剂、基因疗法治疗剂、激素疗法治疗剂、抗病毒剂、抗生素、镇痛剂、抗氧化剂、金属螯合剂或细胞因子)。
  49. 在受试者中预防或治疗癌症的方法，包括向受试者施用有效量的权利要求1-40中任一项所述的双特异性抗体，或权利要求45或46的免疫缀合物，或权利要求47的药物组合物或药物或制剂；或权利要求48的药物组合产品。
  50. 权利要求49的方法，其中所述癌症的肿瘤细胞表面存在CLDN18.2的表达，例如与相同组织的非癌细胞相比，所述肿瘤细胞表面表达的CLDN18.2高。
  51. 权利要求50的方法，其中
    (i)在所述癌细胞或肿瘤细胞在其细胞表面存在PD-L1蛋白，
    (ii)与相同组织类型的非癌细胞相比，在所述癌症或肿瘤细胞的细胞表面具有更高水平的PD-L1；
    (iii)在所述癌细胞或肿瘤细胞在其细胞表面不存在PD-L1蛋白的表达，或者
    (iv)在所述癌细胞或肿瘤细胞在其细胞表面存在PD-L1蛋白，且与高表达PD-L1的癌症或肿瘤相比，在所述癌症或肿瘤细胞的细胞表面具有低水平的PD-L1；或
    (v)与相同组织类型的非癌细胞相比，在所述癌症或肿瘤细胞的细胞表面具有更高水平的PD-L1，且与高表达PD-L1的癌症或肿瘤相比，在所述癌症或肿瘤细胞的细胞表面具有低水平的PD-L1。
  52. 权利要求49-51中任一项的方法，其中所述癌症是实体肿瘤，例如胃肠道肿瘤、胆道肿瘤或肺癌，例如胃癌、食管癌、胰腺癌、结直肠癌、胆管癌、胆囊癌或肺癌。
  53. 权利要求49-52中任一项的方法，其中所述方法还包括与其它疗法例如治疗方式(例如手术或放疗)和/或其它治疗剂(例如化学治疗剂、抗癌药物、免疫治疗剂、抗血管生成剂、靶向疗法治疗剂、细胞疗法治疗剂、基因疗法治疗剂、激素疗法治疗剂、抗病毒剂、抗生素、镇痛剂、抗氧化剂、金属螯合剂或细胞因子)组合施用。
  54. 检测PD-L1和/或CLDN18.2在生物样品中的存在的方法，其包括
    (i)将生物样品与权利要求1-40中任一项所述的双特异性抗体在允许其与PD-L1和/或CLDN18.2结合的条件下接触，
    (ii)检测在该抗体或双特异性抗体和PD-L1和/或CLDN18.2之间是否形成复合物，
    其中复合物的形成表示存在PD-L1和/或CLDN18.2。
PCT/CN2023/094266 2022-05-17 2023-05-15 结合pd-l1和cldn18.2的抗体及其用途 WO2023221935A1 (zh)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021032157A1 (en) * 2019-08-20 2021-02-25 Mabspace Biosciences (Suzhou) Co., Limited Novel anti-cldn18.2 antibodies
WO2021218874A1 (zh) * 2020-04-27 2021-11-04 启愈生物技术(上海)有限公司 一种靶向人claudin和人PDL1蛋白的双特异抗体及其应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021032157A1 (en) * 2019-08-20 2021-02-25 Mabspace Biosciences (Suzhou) Co., Limited Novel anti-cldn18.2 antibodies
WO2021218874A1 (zh) * 2020-04-27 2021-11-04 启愈生物技术(上海)有限公司 一种靶向人claudin和人PDL1蛋白的双特异抗体及其应用

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