WO2021063336A1 - 靶向cldn18.2的抗体及其制备方法和应用 - Google Patents

靶向cldn18.2的抗体及其制备方法和应用 Download PDF

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WO2021063336A1
WO2021063336A1 PCT/CN2020/118650 CN2020118650W WO2021063336A1 WO 2021063336 A1 WO2021063336 A1 WO 2021063336A1 CN 2020118650 W CN2020118650 W CN 2020118650W WO 2021063336 A1 WO2021063336 A1 WO 2021063336A1
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amino acid
seq
acid sequence
antibody
variant
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PCT/CN2020/118650
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French (fr)
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郑明晋
张伟
王杰利
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和铂医药(苏州)有限公司
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Priority to CN202080068747.4A priority Critical patent/CN114502592A/zh
Priority to JP2022520345A priority patent/JP7461469B2/ja
Priority to KR1020227014720A priority patent/KR20220075396A/ko
Priority to US17/764,338 priority patent/US20220332814A1/en
Priority to EP20872423.7A priority patent/EP4039706A4/en
Publication of WO2021063336A1 publication Critical patent/WO2021063336A1/zh

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Definitions

  • the present invention relates to the field of biomedicine, in particular to an antibody targeting CLDN 18.2 and a preparation method and application thereof.
  • Cancer is one of the most deadly diseases in humans today. According to the 2018 World Health Organization (WHO) report, approximately 18.07 million new cancer patients occur every year. There are approximately 9.55 million deaths from cancer each year. According to WHO estimates, gastric cancer is the fifth most commonly diagnosed cancer in the world. Gastric cancer is the third (for men) and fourth (for women) cause of cancer-related deaths. There are 1 million newly diagnosed gastric cancer patients worldwide each year, and about 35% of the first diagnosed gastric cancer patients in the United States are metastatic gastric cancer. The 5-year survival rate of diagnosed advanced gastric cancer is 5%, and the median survival time is about 6 months.
  • the first-line medication for the treatment of patients with metastatic/recurrent gastric cancer is divided into two situations: 1Patients with positive HER2-neu test are treated with trastuzumab (Transtuzumab) combined with chemotherapeutics; 2Patients with negative HER2-neu test are only available Chemotherapy has poor therapeutic effect (Front Pharmacol. 2018 Sep 13; 9:404).
  • Genbank registration number of CLDN18 (Claudin 18, Claudin18) molecular splice variant 1 (CLD18A1, CLDN18.1) is NP_057453, NM016369
  • Genbank registration number of splice variant 2 (CLD18A2, CLDN18.2) is NM_001002026, NP_001002026 )
  • Claudin is an internal membrane protein located in the tight junction of epithelium and endothelium.
  • the other two main tight binding family proteins are occludin and junctional adhesion molecule (JAM).
  • Claudin is an essential component for tight binding, and plays an important role in maintaining the polarity of epithelial cells, controlling paracellular proliferation, and regulating cell growth and differentiation. It is speculated that claudin is almost inaccessible to antibodies in well-structured epithelium, but becomes exposed in tumor cells. The claudin molecule crosses the cell membrane four times, and both the N-terminal and C-terminal ends in the cytoplasm.
  • the human CLDN18.2 (claudin 18.2, Claudin 18.2) protein is a transmembrane protein with a total length of 261 amino acids, of which 1-23 are signal peptides; it has two extramembrane regions which are approximately 55 behind the signal peptide.
  • Extracellular loop 1 (ECL1) of amino acids and ECL2 of 23 amino acids CLDN18.1 (claudin 18.1, Claudin 18.1) and CLDN18.2 have differences in the first 21 amino acids including the first TM and loop 1 (ie ECL1) in the N-terminal, while the C-terminal primary protein sequence is the same.
  • the ECL1 regions of human CLDN18.2 and human CLDN18.1 are very similar, and the ECL2 regions of human CLDN18.2 and human CLDN18.1 are exactly the same. Therefore, the development of antibodies against human CLDN18.2 protein targets requires looking for antibodies against the ECL1 region or spatial structure of human CLDN18.2 protein. This makes this work more difficult.
  • CLDN18.1 is selectively expressed in the epithelium of normal lung and stomach (Mol Cell Biol. 2001 Nov; 21(21): 7380-90.).
  • the expression of CLDN18.2 in normal tissues is highly restricted, limited to differentiated cells of the gastric epithelium, and not in the gastric stem cell area. But it is strongly expressed in several cancer types, including stomach, esophagus, pancreas, and lung tumors, as well as human cancer cell lines.
  • the molecular weight of this protein differs in some cancers and adjacent normal tissues.
  • the higher molecular weight proteins observed in healthy tissues can be converted to the same molecular weight as observed in cancer by treating the tissue lysate with the deglycosylated compound PNGaseF.
  • claudins are less N-glycosylated in cancer than their normal tissue counterparts. This structural difference is likely to produce altered epitopes.
  • the classic N-glycosylation motif is in the 116th amino acid of the D3 domain of the molecule's loop (CN103509110B).
  • IMAB362 Claudiximab (IMAB362) antibody (see WO 2014/146672) is in clinical trials for human CLDN18.2 antibody research.
  • IMAB362 can induce ADCC (antibody-dependent cell-mediated cytotoxicity) effects and CDC (complement dependent cytotoxicity) effects, and mediate tumor killing.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement dependent cytotoxicity
  • IMAB362 has shown encouraging efficacy in phase I and phase II clinical trials for the treatment of advanced gastroesophageal cancer (Eur J Cancer. 2018 Sep; 100:17-26).
  • IMAB362 is a chimeric antibody of human and mouse origin, which has the risk of immunogenicity, and its affinity is not very high.
  • the technical problem to be solved by the present invention is to overcome the deficiency of the lack of antibodies targeting CLDN 18.2 in the art, and provides an antibody targeting CLDN 18.2 (human claudin 18.2) and a preparation method and application thereof.
  • the first aspect of the present invention provides an antibody or antigen-binding fragment thereof targeting CLDN18.2, which comprises a light chain variable region (VL) and/or a heavy chain variable region (VH)
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3; wherein: the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 8 or its variants.
  • the HCDR2 comprises an amino acid sequence selected from SEQ ID NO: 16 and its variant 2, SEQ ID NO: 18 and its variant 3, and the HCDR3 comprises any one of SEQ ID NO: 26-29
  • the LCDR1 comprises the sequence shown in SEQ ID NO: 42 or its variant 4
  • the LCDR2 comprises the sequence shown in SEQ ID NO: 47 or its variant 5
  • the LCDR3 comprises SEQ ID The sequence shown in NO: 55 or its variant 6;
  • the variant is an antibody or antigen-binding fragment containing the above variants with 1, 2, or 3 amino acid substitutions, deletions or additions based on the original sequence Maintain the ability to combine with CLDN 18.2.
  • amino acid mutations Similar to "have 3, 2 or 1 amino acid mutations" in “amino acid mutations” means that compared with the original amino acid sequence, the variant sequence has amino acid mutations, including amino acid insertions based on the original amino acid sequence, Missing or replacement.
  • the CDR mutations can include 3, 2 or 1 amino acid mutations, and the same or different numbers of amino acid residues can optionally be selected for mutations between these CDRs.
  • CDR1 can be mutated. There are no amino acid mutations for CDR2 and CDR3.
  • the mutations may include those currently known to those skilled in the art, for example, some mutations that may be made to the antibody during the production or application of the antibody, for example, the transcription of the CDR regions that may exist.
  • Potential post-translational modifications (PTMs) sites are mutated, including antibody aggregation, deamidation sensitivity (asparaginedeamidation, site (NG, NS, NH, etc.), aspartic acid isomerization (DG, DP) ) Sensitive sites, N- ⁇ P ⁇ S/T (N- ⁇ P ⁇ S/T) sensitive sites, and oxidation-sensitive sites and other related mutations.
  • PTMs post-translational modifications
  • the mutation of the variant 1 preferably occurs at least at position 6 and/or 7 of the amino acid sequence shown in SEQ ID NO: 8.
  • the mutation of the variant 2 preferably occurs at least at position 5 of the amino acid sequence shown in SEQ ID NO: 16.
  • the mutation of the variant 3 preferably occurs at least at position 3 of the amino acid sequence shown in SEQ ID NO: 18.
  • the mutation of the variant 4 preferably occurs at least at the 8th and/or 9th position of the amino acid sequence shown in SEQ ID NO:42.
  • the mutation of the variant 5 preferably occurs at least at position 1 and/or position 4 of the amino acid sequence shown in SEQ ID NO: 47.
  • the mutation of the variant 6 preferably occurs at least in one or more of the 3 to 5 positions of the amino acid sequence shown in SEQ ID NO: 55.
  • the variant 1 contains the mutation S6G and/or Y7F
  • the variant 2 contains the mutation G5R
  • the variant 3 contains the mutation D3E
  • the variant 4 contains the mutation S8R and/or N9Y
  • Variant 5 contains the mutations G1D and/or T4N
  • the variant 6 contains one or more of the mutations Y3R/N, N4S, and N5Y.
  • amino acid sequence of the variant 1 is shown in SEQ ID NO: 6 or 7.
  • amino acid sequence of the variant 2 is shown in SEQ ID NO: 17;
  • amino acid sequence of the variant 3 is shown in SEQ ID NO: 19;
  • amino acid sequence of the variant 4 is shown in SEQ ID NO: 40 or 41;
  • amino acid sequence of the variant 5 is shown in SEQ ID NO: 48;
  • amino acid sequence of the variant 6 is shown in any one of SEQ ID NO: 56-58.
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 7
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 17, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO:
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 8, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 18, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 28;
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 8, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 16, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 29; or, the HCDR1
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 8, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 19, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 28;
  • the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 41, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 48, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 56;
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO: 42, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 47, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 57;
  • the amino acid sequence of the LCDR2 is shown in NO: 42, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 47, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 58.
  • the VH further includes a heavy chain variable region framework region (VH FWR), and/or, the VL also includes a light chain variable region framework region (VL FWR);
  • VH FWR heavy chain variable region framework region
  • VL FWR light chain variable region framework region
  • the VH FWR is a heavy chain variable region framework region of a human antibody
  • the VL FWR is a light chain variable region framework region of a human antibody. among them:
  • the gene encoding the heavy chain variable region framework region is preferably derived from the germline V gene IGHV3-23; preferably, in the heavy chain variable region framework region, HFR1 includes any one of SEQ ID NO: 2 to 4
  • HFR2 includes the amino acid sequence shown in any one of SEQ ID NO: 10-14 or the variant thereof
  • HFR3 includes the amino acid sequence shown in any one of SEQ ID NO: 21-24 or A variant thereof
  • HFR4 includes the amino acid sequence shown in any one of SEQ ID NO: 31 to 33 or a variant thereof.
  • the gene encoding the framework region of the light chain variable region is preferably derived from the germline V gene IGKV3-11 or IGKV3-15;
  • LFR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 35 to 38 or a variant thereof
  • LFR2 comprises the amino acid sequence shown in SEQ ID NO: 44 or 45
  • An amino acid sequence or a variant thereof LFR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 50 to 53 or a variant thereof
  • LFR4 comprises the amino acid sequence shown in SEQ ID NO: 60 or 61 or a variant thereof.
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 7
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 17
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO. : 27
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO: 41
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO: 48
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO: 56;
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 8
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 18
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 28
  • the amino acid sequence of the LCDR1 The sequence is shown in SEQ ID NO: 42
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO: 47
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO: 57;
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 8
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 16
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 29, and the amino acid sequence of the LCDR1
  • the sequence is shown in SEQ ID NO: 42
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO: 47
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO: 55;
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 8
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 16
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 29, and the amino acid sequence of the LCDR1
  • the sequence is shown in SEQ ID NO: 42
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO: 47
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO: 58;
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 8
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 19
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 28
  • the LCDR1 The amino acid sequence of is shown in SEQ ID NO: 42
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO: 47
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO: 57.
  • the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 64 or a variant thereof, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 71 The amino acid sequence of or its variants;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 67 or a variant thereof, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 73 or a variant thereof;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 65 or a variant thereof, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 72 or a variant thereof;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 68 or a variant thereof, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 74 or a variant thereof;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 66 or a variant thereof
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 72 or a variant thereof ;
  • the variant retains at least the function of the pre-mutation sequence, and the identity of the variant and the pre-mutation sequence is at least 85%, preferably at least 90%, more preferably at least 95%, and still more preferably at least 99%.
  • the amino acid sequences of the CDRs listed above are all shown in accordance with Chothia's definition rules (the claims of the present invention are also shown in accordance with Chothia's definition rules).
  • the CDR of an antibody can be defined in a variety of ways in the art, such as the Kabat definition rule based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, U.S. National Institute of Health, Bethesda, Maryland (1991)) and Chothia definition rules based on the location of structural loop regions (see JMol Biol 273:927-48, 1997).
  • the combined definition rule including the Kabat definition and Chothia definition can also be used to determine the amino acid residues in the variable domain sequence.
  • the combined definition rule is to combine the Kabat definition and the Chothia definition. Based on this, a larger scope is selected, as shown in Table 1-1. It should be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or its region (e.g., variable region) should be understood to encompass the above-mentioned already described by the present invention. Complementary decision area defined by any one of the known schemes. Although the scope of protection claimed in the claims of the present invention is based on the sequence shown in Chothia's definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of the present invention.
  • Laa-Lbb can refer to the amino acid sequence from the N-terminus of the antibody light chain, from the aa to the bb position
  • Haa-Hbb can refer to the amino acid sequence from the N-terminus of the antibody heavy chain, from the aa to the bb sequence.
  • L24-L34 can refer to the amino acid sequence from the 24th to the 34th starting from the N-terminus of the antibody light chain according to the Chothia coding rules
  • H26-H32 can refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain and following the Chothia coding rules The amino acid sequence from position 26 to position 32.
  • the antibody targeting CLDN18.2 further includes an antibody heavy chain constant region and an antibody light chain constant region;
  • the heavy chain constant region is selected from hIgG1, hIgG2, hIgG3, hIgG4 or mutations thereof, and the light chain constant region is selected from human antibody light chain kappa chain or lambda chain or mutations thereof;
  • the heavy chain constant region is hIgG1
  • the light chain constant region is the light chain kappa chain of a human antibody.
  • the antibody targeting CLDN18.2 is a full-length antibody, Fab, Fab', F(ab') 2 , Fv, scFv (single chain antibody fragment, single chain antibody), bispecific antibody, multispecific Sex antibodies, heavy chain antibodies or single domain antibodies, or monoclonal antibodies or polyclonal antibodies made from the above antibodies.
  • the monoclonal antibody can be developed by a variety of approaches and technologies, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.
  • the antibody targeting CLDN18.2 when the antibody targeting CLDN18.2 is a bispecific antibody, it may include a first protein functional region and a second protein functional region.
  • the first protein functional region can be the above-mentioned protein, which targets to bind CLDN 18.2; the second protein functional region is a protein that does not target to bind CLDN 18.2 or is a protein that also targets to bind CLDN 18.2 but is not the original The antibody targeting CLDN18.2 of the invention.
  • the first protein functional domain may be an immunoglobulin
  • the second protein functional domain may be one or more scFv; or, the second protein functional domain may be an immunoglobulin, and the first protein The functional area can be one or more scFvs.
  • the antibody targeting CLDN18.2 is a full-length antibody
  • the full-length antibody includes a heavy chain and a light chain
  • the heavy chain comprises an amino acid sequence as shown in any of SEQ ID NOs: 77 to 90
  • the light chain includes an amino acid sequence as shown in any one of SEQ ID NO: 93-96.
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 77, the light chain comprises the amino acid sequence shown in SEQ ID NO: 93; and the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 93; ID NO: 78, the light chain includes the amino acid sequence shown in SEQ ID NO: 94; the heavy chain includes the amino acid sequence shown in SEQ ID NO: 79, and the light chain includes The amino acid sequence shown in SEQ ID NO: 93; the heavy chain includes the amino acid sequence shown in SEQ ID NO: 85, the light chain includes the amino acid sequence shown in SEQ ID NO: 93; the heavy chain includes The amino acid sequence shown in SEQ ID NO: 83, the light chain includes the amino acid sequence shown in SEQ ID NO: 93; the heavy chain includes the amino acid sequence shown in SEQ ID NO: 84, the light chain It includes the amino acid sequence shown in SEQ ID NO: 93; the heavy chain includes the amino acid sequence shown in SEQ ID NO: 81, and the light chain includes the
  • the mutation means that one or more amino acid residues have been deleted, substituted or added in the amino acid sequence of the VL and/or VH, and the amino acid sequence of the mutation has the same amino acid sequence as the amino acid sequence of the VL and/or VH.
  • the "Fab fragment” consists of the CH1 and variable regions of one light chain and one heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • the "Fc" region contains two heavy chain fragments containing the CH1 and CH2 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic effect of the CH3 domain.
  • the "Fab'fragment” contains a light chain and a portion of a heavy chain that contains the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, so that it can be between the two heavy chains of the two Fab' fragments
  • the "F(ab') 2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Therefore the F(ab') 2 fragment consists of two Fab' fragments held together by the disulfide bond between the two heavy chains.
  • the term "Fv” means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody, but lacks a constant region.
  • the scFv single chain antibody fragment
  • the scFv single chain antibody fragment
  • the VL and VH domains pair to form a monovalent molecule by making it possible to produce a linker that is a single polypeptide chain [see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad Sci. USA 85:5879-5883 (1988)].
  • Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of a repetitive G 4 S amino acid sequence or variants thereof.
  • linkers having the amino acid sequence (G 4 S) 4 or (G 4 S) 3 can be used, but variants thereof can also be used.
  • multispecific antibody is used in its broadest sense to encompass antibodies with polyepitope specificity.
  • These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has polyepitope specificity; having two or more VL and VH region antibodies, each VH-VL unit binds to a different target or a different epitope of the same target; an antibody with two or more single variable regions, each single variable region is Binding of different targets or different epitopes of the same target; full-length antibodies, antibody fragments, bispecific antibodies (diabodies), and triabodies (triabodies), antibody fragments covalently or non-covalently linked together Wait.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibodies of the present invention include monoclonal antibodies.
  • the monoclonal antibody or mAb or Ab in the present invention refers to an antibody obtained from a single cloned cell line, and the cell line is not limited to eukaryotic, prokaryotic or phage cloned cell lines.
  • the "heavy chain antibody” refers to an antibody that contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, also known as HCAbs.
  • the "single domain antibody”, also known as “nanobody”, refers to the VHH structure cloned from the heavy chain antibody, which is the smallest unit known to bind the target antigen.
  • the second aspect of the present invention provides an isolated nucleic acid, which encodes the antibody targeting CLDN 18.2 as described in the first aspect of the present invention.
  • the preparation method of the nucleic acid is a conventional preparation method in the art, and preferably includes the following steps: obtain the nucleic acid molecule encoding the above-mentioned antibody through gene cloning technology, or obtain the nucleic acid molecule encoding the above-mentioned antibody through the method of artificial full-sequence synthesis .
  • polynucleotide homologues of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the antibody sequence within the scope of maintaining the activity of the antibody.
  • the third aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid as described in the second aspect of the present invention.
  • the recombinant expression vector can be obtained by conventional methods in the art, that is, the nucleic acid molecule of the present invention is connected to various expression vectors to be constructed.
  • the expression vector is a variety of conventional vectors in the field, as long as it can hold the aforementioned nucleic acid molecule.
  • the recombinant expression vector is a plasmid, a cosmid, a phage or a viral vector
  • the viral vector is preferably a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.
  • the fourth aspect of the present invention provides a transformant, which contains the recombinant expression vector as described in the third aspect of the present invention in a host cell.
  • the preparation method of the recombinant expression transformant may be a conventional preparation method in the art, for example: the recombinant expression vector is transformed into a host cell.
  • the host cell is a variety of conventional host cells in the art, as long as the recombinant expression vector can replicate itself stably and the nucleic acid carried can be effectively expressed.
  • the host cell is E. coli TG1 or BL21 cell (expressing single-chain antibody or Fab antibody), or CHO-K1 cell (expressing full-length IgG antibody).
  • the aforementioned recombinant expression plasmid is transformed into a host cell to obtain the preferred recombinant expression transformant of the present invention.
  • the transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a heat shock method or an electrotransformation method.
  • the antibody targeting CLDN18.2 can be used to prepare a chimeric antigen receptor (CAR) and so on to modify it on cells such as T cells or NK cells.
  • CAR chimeric antigen receptor
  • the present invention therefore provides an antibody or antigen-binding fragment thereof comprising the CLDN18.2-targeting antibody as described in the first aspect of the present invention.
  • it is a chimeric antigen receptor that uses the scFv of the aforementioned CLDN18.2-targeting antibody as an extracellular antigen-binding domain. Therefore, in order to solve the above technical problems, the fifth aspect of the present invention provides a genetically modified cell comprising the antibody targeting CLDN 18.2 as described in the first aspect of the present invention.
  • the genetically modified cells are eukaryotic cells, preferably isolated human cells; more preferably immune cells such as T cells (for example in the form of CAR-T), or NK cells.
  • immune cells such as T cells (for example in the form of CAR-T), or NK cells.
  • the sixth aspect of the present invention provides a method for preparing an antibody targeting CLDN18.2, which comprises culturing the transformant as described in the fourth aspect of the present invention, and obtaining the target CLDN18 from the culture. .2 antibodies.
  • the seventh aspect of the present invention provides an antibody drug conjugate (ADC), which comprises a cytotoxic agent and the antibody targeting CLDN 18.2 as described in the first aspect of the present invention.
  • ADC antibody drug conjugate
  • the cytotoxic agent is preferably a cytotoxin, a chemotherapeutic agent, a radioisotope, a therapeutic nucleic acid, an immunomodulator, an anti-angiogenesis agent, an anti-proliferative pro-apoptotic agent, or a cytolytic enzyme; more preferably, the cytotoxic agent is a microtubule Protein synthase inhibitor-methyl auristatin F (MMAF), or methyl auristatin E (MMAE).
  • MMAF microtubule Protein synthase inhibitor-methyl auristatin F
  • MMAE methyl auristatin E
  • the preparation method of the antibody-drug conjugate can be conventional in the art, preferably the preparation method described in Doronina, 2006, Bioconjugate Chem. 17, 114-124.
  • the preparation method produces antibody-drug conjugates with a minimum low conjugation fraction (LCF) of less than 10%.
  • the antibody-drug conjugate can exist in any physical form known in the art, preferably a clear solution.
  • the eighth aspect of the present invention provides a pharmaceutical composition comprising the antibody targeting CLDN 18.2 as described in the first aspect of the present invention and/or the antibody as described in the seventh aspect of the present invention Antibody drug conjugate, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition preferably also includes other anti-tumor antibodies as active ingredients, and/or contains hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutics, and oncolytic drugs. , Cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and one or more of the group consisting of vaccines.
  • the pharmaceutically acceptable carrier may be a conventional carrier in the art, and the carrier may be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient.
  • the pharmaceutical excipients are conventional pharmaceutical excipients in the field, and preferably include pharmaceutically acceptable excipients, fillers, stabilizers or diluents. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the above-mentioned protein and/or the above-mentioned antibody drug conjugate, and 0.01-99.99% of the pharmaceutical carrier, and the percentage is based on the percentage of the pharmaceutical composition. Mass percentage.
  • the pharmaceutical composition is an anti-tumor drug. More preferably for the treatment of gastric cancer, esophageal cancer, lung cancer, ovarian cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, head and neck cancer, bronchial cancer, glioma and/or Drugs for leukemia.
  • the administration route of the pharmaceutical composition of the present invention is preferably parenteral administration, injection administration or oral administration.
  • the injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection.
  • the pharmaceutical composition is a variety of conventional dosage forms in the art, preferably in the form of solid, semi-solid or liquid, that is, in the form of an aqueous solution, non-aqueous solution or suspension, and more preferably in the form of tablets, capsules, and granules. Medicine, injection or infusion, etc. More preferably, it is administered via intravascular, subcutaneous, intraperitoneal or intramuscular administration.
  • the pharmaceutical composition can also be administered as an aerosol or coarse spray, that is, nasal administration; or, intrathecal, intramedullary or intraventricular administration. More preferably, the pharmaceutical composition can also be administered transdermally, transdermally, topically, enterally, intravaginally, sublingually, or rectally.
  • the dosage level of the pharmaceutical composition of the present invention can be adjusted according to the amount of the composition to achieve the desired diagnosis or treatment result.
  • the administration schedule can also be a single injection or multiple injections, or be adjusted.
  • the selected dosage level and regimen depend on the activity and stability (ie, half-life) of the pharmaceutical composition, formulation, route of administration, combination with other drugs or treatments, diseases or disorders to be detected and/or treated, And various factors such as the health status and previous medical history of the subject to be treated can be reasonably adjusted.
  • the therapeutically effective dose of the pharmaceutical composition of the present invention can be estimated initially in cell culture experiments or animal models such as rodents, rabbits, dogs, pigs and/or primates. Animal models can also be used to determine the appropriate concentration range and route of administration. It can then be used to determine useful dosages and routes of administration in humans. Generally, the determination and adjustment of an effective amount or dosage for administration and the evaluation of when and how to make such adjustments are known to those skilled in the art.
  • the above-mentioned antibody targeting CLDN18.2 the above-mentioned antibody-drug conjugate and/or another therapeutic or diagnostic agent can each be used as a single agent and used within any time frame suitable for performing the intended treatment or diagnosis. Therefore, these single agents can be administered substantially simultaneously (ie, as a single formulation or within minutes or hours) or sequentially and consecutively. For example, these single agents can be administered within one year, or within 10, 8, 6, 4, or 2 months, or within 4, 3, 2, or 1 week, or within 5, 4, 3, 2, or 1 day.
  • the ninth aspect of the present invention provides the antibody targeting CLDN 18.2 as described in the first aspect of the present invention, the antibody-drug conjugate according to the seventh aspect of the present invention, and/or the first aspect of the present invention.
  • the tumor is a CLDN18.2 positive tumor; more preferably, the tumor is gastric cancer, esophageal cancer, lung cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, Glioma and/or leukemia.
  • the present invention also provides the antibody targeting CLDN18.2 according to the first aspect of the present invention, the antibody-drug conjugate according to the seventh aspect of the present invention, and/or the antibody-drug conjugate according to the seventh aspect of the present invention.
  • the tumor is as described in the ninth aspect of the present invention.
  • the tenth aspect of the present invention provides a kit of medicines, which comprises a medicine box A and a medicine box B, the medicine box A is the first aspect of the present invention targeting CLDN 18.2 Antibody, and/or the antibody-drug conjugate according to the seventh aspect of the present invention, and/or the pharmaceutical composition according to the eighth aspect of the present invention;
  • the kit B contains other anti-tumor antibodies or contains the other Anti-tumor antibody pharmaceutical composition
  • the kit B may also contain chemotherapeutics, oncolytic drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, vaccines, imaging agents, diagnostics Drugs, hormone preparations, targeted small molecule preparations, proteasome inhibitors, etc., or the kit B also contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, as well as hormone preparations, targeted small molecules Molecular preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutics
  • the antibody or antigen-binding fragment thereof targeting CLDN18.2 as described in the first aspect of the present invention, the chimeric antigen receptor as described above in the present invention, and the antibody as described in the fifth aspect of the present invention The genetically modified cells, the antibody-drug conjugate according to the seventh aspect of the present invention and/or the pharmaceutical composition according to the eighth aspect of the present invention can also be used in combination with other drugs, such as hormone preparations, targeted drugs Small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutics, oncolytic drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, vaccines, etc.
  • drugs such as hormone preparations, targeted drugs Small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutics, oncolytic drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, vaccines, etc.
  • CLDN18.2 positive cells described in the present invention are cells that overexpress CLDN18.2 protein, such as the NUGC4_D8 cell line; on the contrary, they are called “CLDN18.2 negative” cells.
  • the eleventh aspect of the present invention provides a method for diagnosing, treating and/or preventing a disease or condition mediated by CLDN 18.2, the method comprising administering to a patient in need a therapeutically effective amount as described in the first aspect
  • the disease or condition mediated by CLDN18.2 may be a tumor, preferably a CLDN18.2 positive tumor, more preferably gastric cancer, esophageal cancer, lung cancer, ovarian cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer , Pancreatic cancer, bladder cancer, head and neck cancer, bronchial cancer, glioma and/or leukemia.
  • the twelfth aspect of the present invention provides a method for immunological detection or determination of CLDN 18.2, which comprises using the antibody or antigen-binding fragment thereof targeting CLDN 18.2 as described in the first aspect, and the antibody drug as described in the seventh aspect Conjugate or pharmaceutical composition as described in the eighth aspect.
  • the thirteenth aspect of the present invention provides a combination therapy, which includes respectively administering the antibody or antigen-binding fragment thereof targeting CLDN18.2 as described in the first aspect, and the antibody-drug conjugate of the seventh aspect to patients in need Or the pharmaceutical composition according to the eighth aspect, and a second therapeutic agent;
  • the second therapeutic agent preferably comprises other anti-tumor antibodies or a pharmaceutical composition comprising the other anti-tumor antibodies, and/or is composed of Hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutics, oncolytic drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccine composition One or more of the groups.
  • variable generally refers to the fact that certain parts of the sequence of the variable domain of an antibody change strongly, which forms the binding and specificity of various specific antibodies to their specific antigens.
  • variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the light chain and heavy chain variable regions, which are called complementarity determining regions (CDR) or hypervariable regions (HVR).
  • CDR complementarity determining regions
  • HVR hypervariable regions
  • the more highly conserved part of the variable domain is called the framework (FWR).
  • the variable domains of the natural heavy chain and light chain each contain four FWR regions, most of which adopt a ⁇ -sheet configuration, are connected by three CDRs to form a loop connection, and in some cases form part of a ⁇ -sheet structure.
  • the CDRs in each chain are close together through the FWR region, and together with the CDRs from the other chain form the antigen binding site of the antibody.
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions. , Such as participating in the antibody-dependent cytotoxicity of the antibody.
  • compositions and methods are intended to mean that the composition and method include the described elements but do not exclude other elements, but based on the understanding of the context, it also includes the case of "consisting of”.
  • CLDN18.2 includes isotypes, mammalian (e.g., human) CLDN18.2, species homologs of human CLDN18.2, and analogs containing at least one common epitope with CLDN18.2.
  • the amino acid sequence of CLDN18.2 (for example, human CLDN18.2) is known in the art, as shown in the NCBI database.
  • CLDN18.1 includes isotypes, mammalian (for example, human) CLDN18.1, species homologs of human CLDN18.1, and analogs containing at least one common epitope with CLDN18.1.
  • the amino acid sequence of CLDN18.1 e.g., human CLDN18.1 is known in the art, as shown in the NCBI database.
  • epitopope refers to a part of an antigen (for example, human CLDN 18.2) that specifically interacts with an antibody molecule.
  • the term “competition” in the present invention refers to the ability of an antibody molecule to interfere with the binding of an anti-CLDN18.2 antibody molecule to a target (for example, human CLDN18.2). Interference with binding can be direct or indirect (for example, through allosteric modulation of antibody molecules or targets).
  • a competitive binding assay eg, FACS assay, ELISA, or BIACORE assay
  • FACS assay e.g, FACS assay, ELISA, or BIACORE assay
  • antibody in the present invention includes immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE.
  • the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain. , ⁇ chain and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3 and IgG4.
  • the light chain is divided into ⁇ chain or ⁇ chain by the difference of the constant region.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the antibody light chain variable region of the present invention may further include a light chain constant region, and the light chain constant region includes a human kappa, lambda chain or a variant thereof.
  • the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region, and the heavy chain constant region comprises human IgG1, 2, 3, 4 or variants thereof.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region).
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FWR) with relatively conserved sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • CDR complementarity determining regions
  • Each light chain variable region (VL) and heavy chain variable region (VH) is composed of 3 CDR regions and 4 FWR regions.
  • the sequence from the amino terminus to the carboxy terminus is: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region is composed of 3 domains (CH1, CH2, and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of a domain CL.
  • the constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (for example, effector cells) and the first component (C1q) of the classical complement system.
  • the VH and VL regions can also be subdivided into hyperdenaturation regions [called complementarity determining regions (CDR)], interspersed with more conservative regions called framework regions (FWR).
  • CDR complementarity determining regions
  • FWR framework regions
  • Each VH and VL is composed of 3 CDRs and 4 FWRs arranged in the following order: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4 from the amino terminal to the carboxyl terminal.
  • the variable regions (VH and VL) corresponding to each heavy chain/light chain respectively form the antibody binding site.
  • the heavy chain may also include more than 3 CDRs, such as 6, 9 or 12 CDRs.
  • the heavy chain may be the N-terminus of the heavy chain of an IgG antibody connected to the ScFv of another antibody. In this case, the heavy chain contains 9 CDRs.
  • human antibody includes antibodies having variable and constant regions of human germline immunoglobulin sequences.
  • the human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the term “human antibody” does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences (ie, "humanized antibodies”) .
  • the term "specificity" with respect to antibodies means antibodies that recognize specific antigens but do not substantially recognize or bind to other molecules in the sample.
  • an antibody that specifically binds to an antigen from one species can also bind to that antigen from one or more species.
  • this interspecies cross-reactivity does not change the classification of antibodies based on specificity.
  • antibodies that specifically bind to an antigen can also bind to different allelic forms of the antigen.
  • this cross-reactivity itself does not change the classification of antibodies according to specificity.
  • the term "specific” or “specific binding” can be used to refer to the interaction of an antibody, protein, or peptide with a second chemical substance, meaning that the interaction depends on a specific structure on the chemical substance (e.g., antigenic determination).
  • Clusters or epitopes for example, antibodies generally recognize and bind to specific protein structures, not proteins. If the antibody has specificity for epitope "A”, the presence of molecules containing epitope A (or free, unlabeled A) in the reaction containing labeled "A” and antibody will reduce binding to the antibody The amount of labeled A.
  • chimeric antigen receptor refers to: an extracellular domain (extracellular binding domain), a hinge domain, a transmembrane domain (transmembrane region) and a cytoplasmic domain capable of binding antigen
  • the signal is transmitted to the polypeptide of the structural domain (ie, the intracellular signal domain).
  • the hinge domain can be considered as a part for providing flexibility to the extracellular antigen binding region.
  • the intracellular signal domain refers to a protein that transmits information into the cell to regulate cell activity by generating a second messenger through a certain signal transduction pathway, or a protein that functions as an effector by corresponding to such a messenger, and produces a protein that can promote CAR Signals of immune effector function of cells (e.g. CART cells).
  • the intracellular signal domain includes a signal transduction domain, and may also include a costimulatory intracellular domain derived from a costimulatory molecule.
  • Identity refers to the sequence similarity between two polynucleotide sequences or between two polypeptides.
  • positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position .
  • the percent identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100. For example, in an optimal sequence alignment, if 6 of the 10 positions in the two sequences match or are homologous, then the two sequences will be 60% homologous.
  • Optimization refers to a mutation that maintains or improves the binding of the antibody to an antigen. In the present invention, it refers to a mutation that maintains, maintains, or improves the binding of CLDN 18.2.
  • polypeptide if single chain
  • protein if single chain
  • nucleic acid nucleic acid sequence
  • nucleotide sequence nucleotide sequence
  • polynucleotide sequence nucleotide sequence
  • polynucleotide sequence nucleotide sequence
  • mutation includes substitutions, additions, and/or deletions of amino acids or nucleotides.
  • amino acid substitutions and “conservative amino acid substitutions” refer to substitutions of amino acid residues with another amino acid residue and with similar side chains The amino acid residues are replaced.
  • Lentivirus refers to the genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they can deliver a significant amount of genetic information into the host cell's DNA, so they are one of the most effective methods for gene delivery vehicles. HIV, SIV and FIV are all examples of lentiviruses. Vectors from lentiviruses provide a means to achieve significant horizontal gene transfer in vivo.
  • vector is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell.
  • Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides related to ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term “vector” includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and the like.
  • host cell refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, or fibroblasts, CHO cells, COS cells, and NSO cells. , HeLa cells, BHK cells, HEK 293 cells or human cells and other animal cells.
  • transfection refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, Liposome fusion, lipofection, protoplast fusion, retroviral infection and biolistics.
  • immunode refers to a cell that can trigger an immune response
  • immunodet cells include, for example, white blood cells (leukocytes) derived from hematopoietic stem cells (HSC) produced in bone marrow, lymphocytes (T cells, B cells, natural killer (NK) cells, and bone marrow-derived cells (neutrophils). , Eosinophils, basophils, monocytes, macrophages, dendritic cells).
  • HSC hematopoietic stem cells
  • T cells lymphocytes
  • B cells natural killer cells
  • dendritic cells dendritic cells
  • T cell refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity, and are different from other lymphocytes (such as B cells) in that there are T cell receptors on the cell surface.
  • T cells include all types of immune cells that express CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, T regulatory cells (Treg), and ⁇ - ⁇ T cells.
  • CD4+ cells T helper cells
  • CD8+ cells cytotoxic T cells
  • Tug T regulatory cells
  • ⁇ - ⁇ T cells ⁇ - ⁇ T cells.
  • Cytotoxic cells include CD8+ T cells, natural killer (NK) cells and neutrophils, which can mediate cytotoxic reactions.
  • NK cells refers to a type of lymphocytes that originate in the bone marrow and play an important role in the innate immune system. NK cells provide a rapid immune response against virus-infected cells, tumor cells or other stressed cells, even if antibodies and major histocompatibility complexes are absent on the cell surface.
  • immune cells can be blood-derived, such as autologous T cells, allogeneic T cells, autologous NK cells, allogeneic NK cells, or cell lines, such as using EBV virus infection to prepare NK cell lines, from embryonic stem cells and iPSC induces differentiated NK cells and NK92 cell lines.
  • blood-derived such as autologous T cells, allogeneic T cells, autologous NK cells, allogeneic NK cells, or cell lines, such as using EBV virus infection to prepare NK cell lines, from embryonic stem cells and iPSC induces differentiated NK cells and NK92 cell lines.
  • Optional, “any”, “any” or “any” means that the event or environment described later can but need not occur, and the description includes the occasion where the event or environment occurs or does not occur.
  • “optionally comprising 1 antibody heavy chain variable region” means that an antibody heavy chain variable region of a specific sequence may but does not have to be present.
  • “a” and “a” are used in the present invention to refer to one or more grammatical objects. Unless the content clearly indicates otherwise, the term “or” is used in the present invention to mean and use the term “and/or” interchangeably.
  • “About” and “approximately” should generally mean the acceptable degree of error in the measured quantity in view of the nature or accuracy of the measurement.
  • the exemplary degree of error is generally within its 10% range and more generally within its 5% range.
  • the methods and compositions disclosed in the present invention encompass polypeptides and nucleic acids that have specified sequences, variant sequences, or sequences substantially identical or similar to them, for example, at least 85%, 90%, 95%, 99% of the specified sequence. Or more of the same sequence.
  • amino acid sequences the term "substantially identical" is used in the present invention to refer to the first amino acid sequence.
  • EC 50 refers to the concentration for 50% of maximal effect (concentration for 50% of maximal effect), that is, the concentration that can cause 50% of the maximal effect.
  • the pharmaceutical composition of the present invention can be made into various dosage forms according to needs, and the doctor can determine the beneficial dosage for the patient according to factors such as the patient's type, age, weight, general disease condition, and administration mode.
  • the mode of administration can be, for example, injection or other treatment methods.
  • antibody drug conjugate As used in the present invention, the terms “antibody drug conjugate” or “ADC” are used interchangeably.
  • Auristatin is a fully synthetic drug, and its chemical structure is relatively easy to modify in order to optimize its physical properties and drug properties.
  • the auristatin derivatives used for coupling with antibodies mainly include monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).
  • the former is made of natural tubulin polymerase inhibitors.
  • the synthetic pentapeptide derived from dolastatin-10 is synthesized by adding a 2-amino-1-phenylpropyl-1-ol to the C-terminus.
  • the inhibitory activity of MMAE on a variety of human tumor cell lines is less than one nanomolar.
  • MMAF In order to reduce the cytotoxic activity of MMAE itself, MMAF adds a phenylalanine to the C-terminus of octopusin 10. Because of the introduction of a carboxyl group in the structure, MMAF has poor cell membrane permeability, and therefore has significant biological activity on cells. Reduced, but the inhibitory activity on cells is greatly improved after coupling with the antibody (US7750116).
  • the antibody cytotoxic drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof comprises an antibody of the invention conjugated with one or more maytansinoid molecules.
  • Maytansinoids are mitotic inhibitors that have no effect by inhibiting tubulin multimerization. Maytansine was originally isolated from the East African shrub Maytenus serrata (US Patent No. 3,896,111). It was subsequently discovered that certain microorganisms also produce maytansinoid, such as maytansinol and C-3 maytansinol (US Patent No. 4,151,042).
  • Maytansinoid drug modules are attractive drug modules in antibody-drug conjugates because they are: (i) relatively easy to prepare by fermentation or chemical modification or derivatization of fermentation products; (ii) easy Derivatization with functional groups suitable for coupling to antibodies via non-disulfide linkers; (iii) stable in plasma; and (iv) effective against a variety of tumor cell lines.
  • Maytansinoids suitable for use as maytansinoids drug modules are well known in the art, and can be isolated from natural sources according to known methods or produced using genetic engineering techniques (see Yu et al. (2002) PNAS99: 7968-7973). Maytansinol and maytansinol analogues can also be prepared synthetically according to known methods.
  • Exemplary embodiments of the maytansinoid drug module include: DM1, DM3, and DM4, as disclosed herein.
  • the method, composition, and combination therapy of the present invention can be combined with other active agents or treatment methods.
  • the method includes administering the anti-CLDN18 of the present invention to a subject in an amount effective to treat or prevent a disease (for example, cancer).
  • a disease for example, cancer
  • .2 Antibody molecule optionally, with PD-1, PD-L1, PD-L2, LAG-3, CTLA-4, Tim-3 antibody (immunotherapy) or other tumor treatment antibodies, Her-2, EGFR, Combinations of one or more inhibitors of VEGF, VEGFR antibodies, etc., as well as ADC (such as T-DM1), bispecific antibodies, chemotherapeutic drugs, etc., and also include administration of anti-CLDN18.2 antibody molecules, additional active agents or all It is administered in an amount or dose that is higher, lower than, or equal to the amount or dose of each active agent used alone (e.g., as a monotherapy).
  • the additional active agent or the total amount or dose administered is lower than the amount or dose of each active agent used alone (for example, as a monotherapy) (for example, at least 20%, at least 30%, at least 40%). % Or at least 50%).
  • the anti-CLDN18.2 antibody and the drug conjugate of the CLDN18.2 antibody can bind to CLDN18.2 to induce apoptosis of target cells (tumor cells), inhibit tumor cell growth, and increase the body Effector cells can kill tumor cells ADCC and CDC to achieve the purpose of treating cancer patients.
  • the anti-CLDN18.2 antibody and the drug conjugate of the CLDN18.2 antibody described in the present invention demonstrate the anti-tumor effect of the antibody of the present invention through these mechanisms, as well as methods for inhibiting the growth of tumor cells, including A therapeutically effective amount of the anti-CLDN18.2 antibody and the drug conjugate of the CLDN18.2 antibody described in the present invention is administered to the subject. This method is suitable for in vivo treatment of cancer. In order to obtain a targeted specific therapeutic effect, the anti-CLDN18.2 antibody molecule can be administered together with other antibodies.
  • the combination can be administered in any order or at the same time for cancer types, especially tumor patients with high expression of CLDN18.2.
  • it is provided to treat (e.g., reduce or alleviate) a hyperproliferative condition or disease (e.g., cancer) in the subject in the subject.
  • the method includes administering one or more anti-CLDN18.2 antibodies or drug conjugates of CLDN18.2 antibodies of the present invention to the subject alone or in combination with other active agents or treatment modalities.
  • an anti-CLDN18.2 antibody molecule alone or in combination with another immunomodulator (for example, anti-LAG-3, anti-Tim-3, anti-PD-1 or anti-PD-L1, anti-CTLA-4 antibody molecule) for treatment Gastric cancer, pancreatic cancer, lung cancer, esophageal cancer, ovarian cancer, etc.
  • another immunomodulator for example, anti-LAG-3, anti-Tim-3, anti-PD-1 or anti-PD-L1, anti-CTLA-4 antibody molecule
  • Anti-CLDN18.2 antibody molecules can be administered in combination with one or more of the following: immune-based strategies, targeted drugs (for example, VEGF inhibitors such as monoclonal antibodies against VEGF); VEGF tyrosine kinase inhibitors such as Sunil Tinib, Sorafenib, Apatinib; RNAi inhibitors or inhibitors of downstream mediators of VEGF signaling, for example, inhibitors of the mammalian target of rapamycin (mTOR).
  • targeted drugs for example, VEGF inhibitors such as monoclonal antibodies against VEGF
  • VEGF tyrosine kinase inhibitors such as Sunil Tinib, Sorafenib, Apatinib
  • RNAi inhibitors or inhibitors of downstream mediators of VEGF signaling for example, inhibitors of the mammalian target of rapamycin (mTOR).
  • cancer As used in the present invention, the terms “cancer”, “cancer”, and “cancer patient” are intended to include all types of cancerous growths or tumorigenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of their tissue disease. Physical type or aggressiveness stage. Examples include, but are not limited to, solid tumors, hematological cancers, soft tissue tumors, and metastatic lesions.
  • Non-limiting examples of cancers that can be suitably treated with the antibody targeting CLDN18.2 disclosed in the present invention include gastric cancer, esophageal cancer, lung cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreas Cancer, bladder cancer, glioma and/or leukemia, etc., or their metastatic lesions.
  • the antibody of the present invention has binding affinity, ADCC (antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity), CDC (complement dependent cytotoxicity, complement dependent cytotoxicity), growth Inhibitory effect, endocytosis activity, etc. have obvious advantages, which have great potential for the treatment of tumors.
  • HBM1029 antibody, PR003197, PR003340, PR003292, PR003293, PR003240, PR003291, PR003289, PR003890, PR003891, PR003894, PR003897, and PR003898 are compared with IMAB362 analogs for the endogenous expression of CLDN18.2.
  • the HBM1029, PR003197, PR003340, PR003240, PR003894 antibodies of the present invention can specifically induce a stronger ADCC effect than the IMAB362 analogs in a dose-dependent manner; the HBM1029, PR003197, PR003340 antibodies can In a dose-dependent manner, HEK293 hCLDN 18.2 induces a stronger CDC effect than the IMAB362 analog; HBM1029 antibody can induce a stronger growth inhibitory effect than the IMAB362 analog in a dose-dependent manner; HBM1029 antibody can in a dose-dependent manner than NUGC4_D8 Induces stronger endocytosis activity than IMAB362 analogs; when HBM1029 antibody is co-cultured with MMAF-coupled anti-human IgG antibody, it can produce stronger cytotoxic effects on NUGC4_D8 cells and HEK293 hCLDN18.2 cells in a dose-dependent manner than IMAB362 analogs .
  • A, B, and C respectively show the binding affinity of HBM1029 and PR002727 antibodies to NUGC4_D8, HEK293 hCLDN18.2, and HEK293 hCLDN18.1 cells.
  • Figure 2 A and B show the binding affinity of PR003197, PR003292, PR003293, and PR003340 antibodies to NUGC4_D8, HEK293 hCLDN18.2 cells;
  • Figure 2C shows the binding affinity of PR003197, PR003292, PR003293, PR003340, PR002725 antibodies to HEK293 hCLDN18.1 cells Binding affinity.
  • Figure 3 A and B respectively show the binding affinity of PR003240, PR003291, PR003289 and HBM1029 antibodies to NUGC4_D8, HEK293 hCLDN18.1 cells.
  • Figure 4 A and B respectively show the binding affinity of PR003890, PR003891, PR003894, PR003897, and PR003898 antibodies to NUGC4_D8, HEK293 hCLDN18.2 cells; C shows PR003890, PR003891, PR003894, PR003897, PR003898, PR002725 and HBM1029 antibodies to HEK293 The binding affinity of hCLDN18.1 cells.
  • Figure 5 shows that the HBM1029 antibody exhibits ADCC activity on NUGC4_D8, HEK293 hCLDN 18.1 cells through human PBMC.
  • Figure 6 shows that PR003894, PR003240, PR003340, PR003197 and HBM1029 exhibit ADCC activity on NUGC4_D8 cells by human PBMC.
  • Figure 7 shows that HBM1029, PR003894, PR003240, PR003340, PR003197, PR003891, and PR003898 exhibit ADCC activity on NUGC4_D8 cells by reporter cells.
  • Figure 8 shows that the HBM1029 antibody triggers the CDC effect on HEK293 hCLDN18.2 cells, HEK293 hCLDN18.1 cells and NUGC4_D8 cells.
  • Figure 9 shows the CDC effect of HBM1029, PR003197, and PR003340 in HEK293 hCLDN18.2 cells.
  • Figure 10 shows the growth inhibitory activity of HBM1029 antibody in HEK293 hCLDN18.1 and HEK293 hCLDN18.2.
  • Figure 11 shows the endocytosis activity of HBM1029 antibody in NUGC4_D8 cells.
  • A is the mixed incubation of NUGC4_D8 cells and 200 nM antibody for different time periods
  • B is the mixed incubation of NUGC4_D8 cells and different concentrations of antibody for 1 hour.
  • Figure 12 shows the survival rate of target cells when the HBM1029 antibody and the anti-human IgG antibody coupled to MMAF are co-cultured.
  • Figure 13 shows the competitive binding affinity of HBM1029 antibody to IMAB362-FITC analog in HEK293 hCLDN18.2 cells. Different concentrations of HBM1029 antibody, 20nM IMAB362-FITC analog, HEK293 hCLDN18.2 cells were mixed and incubated.
  • Figure 14 shows the pharmacokinetic profile of IMAB362 analog, HBM1029.
  • Figure 15 shows the in vivo pharmacodynamic study of IMAB362 analogue and HBM1029.
  • the preparation method of the human CLDN18.2 expression vector for immunizing fully humanized transgenic mice is: synthesize the cDNA sequence encoding human CLDN18.2 (Uniprot ID P56856-iso2), and clone the coding sequence of the above gene into the pCAGGS plasmid by enzyme digestion (YOUBIO, VT1076).
  • the construction of the HEK293 (ATCC, Cat#: CRL-1573) cell line stably expressing human CLDN18.1 or CLDN18.2 is specifically: the plasmid encoding human CLDN18.1 (GenScript, OHu29174D) or CLDN18.2 (GenScript, OHu03374D) Transfected into HEK293 cells to generate stable cell lines overexpressing human CLDN18.1 or CLDN18.2.
  • the expression of CLDN18.1 and CLDN18.2 was detected by fluorescence activated cell sorting (FACS).
  • 20,000 transfected cells were plated in each well of a 96-well plate, and then a commercially available rabbit anti-human CLDN18 antibody (LifeSpan Bio, Cat#: LS-C168812-400) was added. After incubating for 1 hour at 4°C, the plate was washed twice with PBS, and then AF-680 coupled goat anti-rabbit IgG secondary antibody (Invitrogen, Cat#: A21109) was added. After incubating for 1 hour at 4°C, the plate was washed 3 times with PBS, and then the cell fluorescence was monitored using a FACS instrument (IntelliCytiQuePlus BR).
  • the human CLDN18.2 expression vector prepared above and the HEK293 cells (HEK293 hCLDN18.2 cells) expressing human CLDN18.2 prepared above were used to immunize fully humanized transgenic mice (HarbourH2L2 mice, which are commercially available). Rat, purchased from Hebo Bio).
  • the human CLDN18.2 vector and gold powder were used to make the bullets of the gene gun, and the mice were immunized at multiple points with the gene gun.
  • the expression vector DNA was immunized with 50 ⁇ g each time, with an interval of 2 weeks. After three immunizations, then HEK293 hCLDN18.2 cells were used for immunization.
  • mice were immunized with 4 ⁇ 10 6 cells each time with an interval of 2 weeks. After 2 times, the mice were taken blood to measure the titer.
  • the binding affinity of mouse serum was used by FACS using CHO K1 cells expressing human CLDN18.2 (CHOK1 hCLDN18.2, purchased from kyinno (KC-1180) or CHO K1 cells of CLDN18.1 (CHOK1 hCLDN18.1, purchased from kyinno ( KC-1181)) was tested.
  • mice were selected for hybridoma fusion, and the mice were sprinted 3 days before fusion.
  • the immunogen was HEK293 hCLDN18.2 cells, and the dose was 4 ⁇ 10 6 cells.
  • the preliminary screening is performed by Mirrorball using CHO K1 cells expressing human CLDN18.2.
  • Re-screening was tested by FACS using CHOK1hCLDN18.1 and CHOK1hCLDN18.2 cells.
  • the cells were centrifuged at 300g for 5 minutes, and then FACS buffer (containing Resuspend in 2% FBS in PBS). Adjust the cell density to 106 cells/ml, add 50 ⁇ l of cell suspension to each well of a 96-well plate. Add 50 ⁇ l of supernatant to each well of a 96-well plate. At 4 After 2 hours of incubation at 4°C, the plate was washed twice with FACS buffer. Then, FACS buffer (BiolegendCat#: 405407) containing APC-coupled goat anti-rat IgG secondary antibody was added.
  • the plate was used Wash twice with FACS buffer. Cells are resuspended in fixative, and then use FACS instrument (ACEA NovoCyte) to monitor cell fluorescence. Specific hybridomas are subcloned by limiting dilution method and cultured in a carbon dioxide incubator at 37°C7 After days, the subcloning screening will be carried out. The subcloning screening will use Mirrorball to express human CLDN 18.2 CHO K1 cells were tested.
  • the combined definition rule including the Kabat definition and Chothia definition can also be used to determine the amino acid residues in the variable domain sequence.
  • the combined definition rule is to combine the Kabat definition and the Chothia definition. Based on this, a larger scope is taken. For details, see Table 1-1 in the Summary of the Invention. The germline gene analysis and PTM site analysis obtained after sequencing in this example are shown in Table 2 below. The design information of antigen binding protein mutation sites is shown in Table 3 below. The sequence numbering information of the antigen binding protein is shown in Table 4 below.
  • PR002726C mentioned in the above table is HBM1029, and the two refer to the same antibody in the present invention
  • PR000400 is the IMAB362 analog, and the two refer to the same antibody in the present invention.
  • VH antibody heavy chain variable domain sequence
  • the antibody light chain variable domain sequence (VL) is genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding the human antibody Ig ⁇ light chain constant domain sequence to encode the full-length light chain of the antibody.
  • VL antibody light chain variable domain sequence
  • the plasmid encoding the antibody heavy chain (Genscript US) and the plasmid encoding the antibody light chain (Genscript US) are simultaneously transfected into mammalian host cells (such as human embryonic kidney cells HEK293), using conventional recombinant protein expression and purification techniques to obtain Purified recombinant antibody with correct pairing and assembly of light and heavy chains.
  • mammalian host cells such as human embryonic kidney cells HEK293
  • HEK293 cells were expanded and cultured in FreeStyle TM F17 Expression Medium medium (Thermo, Cat#: A1383504). Before the start of transient transfection, adjust the cell concentration to 6-8 ⁇ 10 5 cells/ml and incubate for 24 hours in a 37°C 8% CO 2 shaker. The cell concentration is 1.2 ⁇ 10 6 cells/ml.
  • DSF Differential Scanning Fluorimetry
  • the binding affinity of the antibody was obtained by FACS using HEK293 cells expressing human CLDN18.2 or CLDN18.1 and NUGC4_D8 cells endogenously expressing human CLDN18.2 (NUGC cell line was purchased from JCRB (Cat#: JCRB0834), and obtained by limiting dilution screening NUGC4_D8 subcloned cells) for detection, specifically: the cells were centrifuged at 300g for 5 minutes, and then resuspended in FACS buffer (PBS containing 2% FBS). The cell density was adjusted to 106 cells/ml, and 50 ⁇ l of cell suspension was added to each well of the 96-well plate.
  • FACS buffer PBS containing 2% FBS
  • the antibody was diluted with FACS buffer to different concentrations, and 50 ⁇ l of antibody diluent was added to each well of the 96-well plate. After incubating for 2 hours at 4°C, the plate was washed twice with FACS buffer. After that, FACS buffer containing APC-coupled goat anti-human IgG secondary antibody (final concentration 1.5 ⁇ g/ml, JacksonCat#: 109-605-098) was added. After incubating for 1 hour at 4°C, the plate was washed twice with FACS buffer. The cells were resuspended in fixative, and then the cell fluorescence was monitored using a FACS instrument (ACEA NovoCyte).
  • IMAB362 analog (IMAB362 analog is self-made, the heavy chain amino acid sequence is shown in SEQ ID NO: 75, and the light chain amino acid sequence is shown in SEQ ID NO: 91 (synthesized by Genscript Biology), which is variable with IMAB362 The regions are identical, only a few amino acids are different in the constant region, and the activities of the two are similar) as a positive control, and human Iso IgG1 (CrownBio, Cat#: C0001-4) antibody as a negative control.
  • Figure 1 shows the binding affinity of HBM1029 antibody to NUGC4_D8 cells endogenously expressing CLDN18.2 and HEK293 cells overexpressing human CLDN18.2 (HEK293 hCLDN 18.2) or HEK293 cells overexpressing human CLDN18.1 (HEK293 hCLDN18.1) .
  • PR002727 antibody can bind to NUGC4_D8 cells in a dose-dependent manner.
  • HBM1029 antibody can bind to HEK293 hCLDN18.2 and NUGC4_D8 cells in a dose-dependent manner.
  • HBM1029 antibody to HEK293 hCLDN18.2 cells is comparable to IMAB362 analog; HBM1029 antibody has higher binding affinity to NUGC4_D8 cells that endogenously express CLDN18.2 than IMAB362 analog.
  • the EC 50 value of HBM1029 is shown in Table 7. The EC 50 value of the binding affinity of HBM1029 to NUGC4_D8 cells endogenously expressing CLDN18.2 is lower than that of IMAB362 analogs. HBM1029 has low binding affinity to HEK293 hCLDN18.1 cells. At the same time, it can be inferred that HBM1029 binds to ECL1 (Extracellular loop 1) of human CLDN18.2 protein instead of ECL2.
  • ECL1 Extracellular loop 1
  • Figure 2 Figure 3, and Figure 4 show the CLDN18.2 antibody against NUGC4_D8 cells endogenously expressing CLDN18.2 or HEK293 cells overexpressing human CLDN18.2 (HEK293 hCLDN 18.2) or HEK293 cells overexpressing human CLDN18.1 ( HEK293 hCLDN18.1) binding affinity.
  • HBM1029, PR003197, PR003340, PR003292, PR003293, PR003240, PR003291, PR003289, PR003890, PR003891, PR003894, PR003897, PR003898 antibodies compared with IMAB362 analogs showed higher binding to NUGC4_D8 cells endogenously expressing CLDN18.2
  • the affinity is low for HEK293 hCLDN18.1 cells, while PR002725 has high affinity for HEK293 hCLDN18.1.
  • the non-radioactive cytotoxicity analysis kit detects the activity of CLDN18.2 antibody against NUGC4_D8 cells endogenously expressing human CLDN18.2 and HEK293 hCLDN 18.1 to induce ADCC effect.
  • Human PBMC (Miaotong) was centrifuged at 300g for 5 minutes and cultured in medium (RPMI1640+10% FBS) overnight. The target cells and human PBMC were centrifuged at 300g for 5 minutes, and then resuspended in medium (RPMI1640+2% FBS).
  • the antibody to be tested was diluted with culture medium (RPMI1640+2% FBS) to different concentrations and added to each well. Incubate the sample at 37°C for at least 4 hours, then add 10 times Triton-X 100 lysate (RPMI1640+2%FBS+10%Triton-X 100) to the target cells’ maximum LDH release control wells and volume correction control wells (Volume correction control).
  • Results First calculate the correction readings, subtract the readings of the medium background control wells from the readings of the experimental wells, the target cell spontaneous release of LDH control wells, and the effector cell spontaneous release of LDH control wells, and then subtract the maximum LDH release control wells of the target cells. Volume correction control well readings.
  • ADCC activity (%) (experimental hole correction reading-effector cell spontaneous release of LDH control hole correction reading-target cell spontaneous release of LDH control hole correction reading) / (target cell maximum LDH release control hole correction reading-target cell spontaneous Release LDH (corrected readings for control wells) ⁇ 100.
  • Figure 5 shows the ADCC activity of HBM1029 antibody on NUGC4_D8 cells endogenously expressing CLDN18.2 and HEK293 hCLDN18.1.
  • the HBM1029 antibody can specifically induce a stronger ADCC effect on NUGC4_D8 than the IMAB362 analog in a dose-dependent manner, but no cytotoxic effect was observed on HEK-293 cells overexpressing CLDN18.1.
  • EC HBM1029 50 values are shown in Table 8, HBM1029 50 values induced in EC ADCC NUGC4_D8 lower than IMAB362 like.
  • Figure 6 shows the ADCC activity of HBM1029, PR003197, PR003340, PR003240, and PR003894 on NUGC4_D8 cells that endogenously express CLDN18.2.
  • HBM1029, PR003197, PR003340, PR003240, and PR003894 can specifically induce NUGC4_D8 similar to IMAB362 in a dose-dependent manner. Material strong ADCC effect.
  • NUGC4_D8 and HEK293 hCLDN18.1 were centrifuged at 300g for 5 minutes, and then resuspended in RPMI1640+4% FBS serum medium. Adjust the cell density to 6 ⁇ 10 5 cells/ml, add 50 ⁇ l of cell suspension into each well of a 96-well plate, and incubate overnight at 37°C.
  • Jurkat Fc ⁇ RIIIa-V158/NFAT-Luc cells were centrifuged at 400g for 4 minutes, and then resuspended in RPMI1640+4% FBS serum medium. The cell density was adjusted to 3 ⁇ 10 6 cells/ml, and 50 ⁇ l of cell suspension was added to each well of the 96-well plate. The antibody was diluted with RPMI1640+4% FBS medium to different concentrations, and 50 ⁇ l of antibody diluent was added to each well of a 96-well plate. The cells were incubated with the antibody at 37°C for 5 hours. Leave the 96-well plate at room temperature for 30 minutes, and add 60 ⁇ l/well of room temperature One-Glo Chromogenic Solution (Promega).
  • FIG. 7 shows the ADCC activity of CLDN18.2 antibody on NUGC4_D8 cells that endogenously express CLDN18.2.
  • HBM1029, PR003197, PR003340, PR003240, PR003894, PR003891, PR003898 can specifically induce NUGC4_D8 in a dose-dependent manner compared to IMAB362 analogs Strong ADCC effect.
  • CellTiter-Glo luminescence cell viability detection kit (Promega, Cat#: G7573) was used to detect the ability of CLDN18.2 antibody to trigger CDC effect against HEK293 hCLDN18.1, HEK293 hCLDN18.2 and NUGC4_D8 cells.
  • the target cells HEK293 hCLDN18.1 and HEK293 hCLDN18.2 were centrifuged at 300g for 5 minutes, and then resuspended in DMEM serum-free medium.
  • the target cells NUGC4_D8 were centrifuged at 300g for 5 minutes, and then resuspended in RPMI1640 serum-free medium.
  • the density of the target cells was adjusted to 4 ⁇ 10 5 cells/ml, and 25 ⁇ l of cell suspension was added to each well of the 96-well plate.
  • the antibody was diluted with serum-free medium to different concentrations, and 25 ⁇ l of antibody diluent was added to each well of a 96-well plate.
  • 50 ⁇ l of normal human serum (GemCell, Cat#: 100-512) was added, the final concentration was 50%, and the obtained mixture was incubated at 37°C for 24 hours. Leave the 96-well plate at room temperature for 30 minutes, and add 100 ⁇ l/well of room temperature CellTiter-Glo color developing solution. After that, the sample was incubated at room temperature for 10 minutes in the dark. Read the plate with PE Enspire.
  • CDC activity (%) [1-(luminescent sample)/(luminescent mock control)] ⁇ 100.
  • IMAB362 analog was used as a positive control
  • human Iso IgG1 (CrownBio, Cat#: C0001-4) antibody was used as a negative control.
  • Figure 8 shows the CDC activity of HBM1029 antibody on NUGC4_D8 cells endogenously expressing CLDN18.2, HEK293 cells overexpressing human CLDN18.1, and HEK293 cells overexpressing human CLDN18.2.
  • HBM1029 antibody can induce a stronger CDC effect on HEK293 hCLDN18.2 in a dose-dependent manner than IMAB362 analogues, but CDC activity was not observed on NUGC4_D8 cells and HEK293 cells overexpressing human CLDN18.1.
  • the EC 50 value of HBM1029 is shown in Table 9. The EC 50 value of HBM1029 induced CDC in HEK293 hCLDN18.2 is lower than that of IMAB362 analogs.
  • Figure 9 shows the CDC activity of PR003197 and PR003340 on HEK293 cells overexpressing human CLDN18.2.
  • PR003197 and PR003340 antibodies can induce a stronger CDC effect on HEK293 hCLDN18.2 than IMAB362 analogs in a dose-dependent manner.
  • CellTiter-Glo luminescence cell viability detection kit (Promega, Cat#: G7573) was used to detect the ability of CLDN18.2 antibody to induce growth inhibition against HEK293 hCLDN18.1 and HEK293 hCLDN18.2.
  • the cells HEK293 hCLDN18.1 and HEK293 hCLDN18.2 were centrifuged at 300g for 5 minutes, and then resuspended in DMEM+0.5% FBS serum medium. Adjust the cell density to 1.2 ⁇ 10 5 cells/ml, add 50 ⁇ l of cell suspension into each well of a 96-well plate, and incubate overnight at 37°C.
  • the antibody is diluted with culture medium to different concentrations, and 50 ⁇ l of antibody diluent is added to each well of the 96-well plate.
  • IMAB362 analog was used as a positive control, and human Iso IgG1 (CrownBio, Cat#: C0001-4) antibody was used as a negative control.
  • Figure 10 shows the growth inhibitory activity of the HBM1029 antibody against HEK293 hCLDN18.1 and HEK293 hCLDN18.2.
  • the HBM1029 antibody can induce a stronger growth inhibitory effect on HEK293 hCLDN 18.2 than the IMAB362 analog in a dose-dependent manner.
  • the EC 50 value of HBM1029 is shown in Table 10.
  • the endocytosis activity of the antibody was detected by FACS using NUGC4_D8 cells.
  • the cells were trypsinized and washed once with FACS buffer (PBS containing 2% FBS). The cells were centrifuged at 300g for 5 minutes, and then resuspended in FACS buffer. Adjust the cell density to 4 ⁇ 10 6 cells/ml, and place on ice to pre-cool for 30 minutes.
  • the antibody was diluted with FACS buffer to different concentrations and placed on ice to pre-cool for 30 minutes. Add 700 ⁇ l of cell suspension and 700 ⁇ l of antibody dilution into the wells of the pre-chilled deep well plate.
  • the plate was washed 3 times with pre-chilled FACS buffer. Resuspend the cells with 250 ⁇ l of pre-cooled FACS buffer, add 100 ⁇ l of cell suspension and 1.1ml of 37°C pre-warmed FACS buffer to the wells of a deep well plate pre-warmed at 37°C, deep wells pre-cooled at 4°C Add 100 ⁇ l of cell suspension and 1.1ml of FACS buffer pre-cooled at 4°C into the wells of the plate. The obtained mixture was taken at 0, 30, 60, 120, and 240 minutes to take 50 ⁇ L of cell suspension (10 5 /well) and place in a pre-chilled deep well plate (containing 1.2 mL of FACS buffer).
  • IMAB362 analog was used as a positive control, and human Iso IgG1 (CrownBio, Cat#: C0001-4) antibody was used as a negative control.
  • Figure 11 shows the endocytosis activity of HBM1029 antibody incubated for different times on NUGC4_D8 cells. The HBM1029 antibody induced about 50% endocytosis activity after 30 minutes of incubation. Figure 11 also shows that HBM1029 antibody can induce stronger endocytosis activity in NUGC4_D8 than IMAB362 analogs in a dose-dependent manner.
  • EC HBM1029 50 values are shown in Table 11, EC 50 values HBM1029 induced endocytosis in NUGC4_D8 lower than IMAB362 like.
  • Cells NUGC4_D8 were centrifuged at 300g for 5 minutes, and then resuspended in RPMI1640+10% FBS serum medium. The cell density was adjusted to 2 ⁇ 10 4 cells/ml, and 50 ⁇ l of cell suspension was added to each well of a 96-well plate. Incubate overnight at 37°C.
  • the HBM1029 antibody was diluted with culture medium to different concentrations, and 25 ⁇ l of antibody diluent was added to each well of a 96-well plate.
  • the MMAF-coupled anti-human IgG antibody was diluted with culture medium, and 25 ⁇ l of antibody diluent was added to each well of a 96-well plate to a final concentration of 6.6 nM.
  • HBM1029 antibody When HBM1029 antibody is co-cultured with MMAF-coupled anti-human IgG antibody, NUGC4_D8 cells and HEK293hCLDN18.2 cells can produce stronger cytotoxic effects than IMAB362 analogs in a dose-dependent manner, but HEK293 hCLDN18.1 cells do not produce cells. Toxic effect.
  • IMAB362 analogs were coupled to IMAB362-FITC analogs using FITC fluorescence coupling kit (Abcam, Cat#: ab188285).
  • the cells were centrifuged at 300g for 5 minutes, and then resuspended in FACS buffer (PBS containing 2% FBS).
  • FACS buffer PBS containing 2% FBS.
  • the cell density was adjusted to 106 cells/ml, and 50 ⁇ l of cell suspension was added to each well of the 96-well plate.
  • FITC coupling antibody was diluted with FACS buffer, and 50 ⁇ l coupling antibody diluent was added to each well of a 96-well plate.
  • the antibody to be competitively bound is diluted with FACS buffer to different concentrations, and 50 ⁇ l of antibody diluent is added to each well of a 96-well plate. After incubating for 2 hours at 4°C, the plate was washed twice with FACS buffer. The cells were resuspended in fixative, and then the fluorescence of the cells was monitored using a FACS instrument (ACEA NovoCyte). Human Iso IgG1 (CrownBio, Cat#: C0001-4) antibody was used as a negative control.
  • Figure 13 shows the competitive binding affinity of the HBM1029 antibody to the IMAB362-FITC analog in HEK293 hCLDN 18.2 cells.
  • HBM1029 antibody can compete to bind to HEK293 hCLDN18.2 cells in a dose-dependent manner. It is speculated that the binding epitope of HBM1029 antibody and IMAB362 analog is similar. HBM1029 can compete with IMAB362 analogs and inhibit its binding to cells expressing CLDN18.2; while IMAB362 analogs are known to only bind CLDN18.2 and not CLDN18.1. Therefore, it is speculated that HBM1029 binds to ECL1 (Extracellular loop 1) of human CLDN18.2 protein instead of ECL2.
  • ECL1 Extracellular loop 1
  • the pharmacokinetics of CLDN18.2 antibody is as follows: select 6 female BALB/c nude mice weighing 18-22 grams, and administer the antibody drug by intravenous injection at a dose of 5 mg/kg; a group of 3 mice Whole blood was collected before the drug and 15 minutes, 24 hours (1 day), 4, and 10 days after the drug. The other group of 3 rats was only before the drug and 5 hours after the drug, on the second day. Whole blood was collected on the 7th and 14th days. The whole blood was allowed to stand for 30 minutes to coagulate, then centrifuged at 2,000 rpm at 4°C for 5 minutes and the separated serum samples were frozen at -80°C until analysis. In this example, the ELISA method was used to quantitatively determine the drug concentration in the mouse serum.
  • a goat anti-human Fc polyclonal antibody coated on a 96-well plate is used to capture the human Fc-containing antibody in the mouse serum, and then an HRP-labeled goat anti-human Fc secondary antibody is added for detection.
  • HRP-labeled goat anti-human Fc secondary antibody is added for detection.
  • NCA non-compartmental model
  • Figure 14 and Table 12 show the pharmacokinetic parameters of IMAB362 analogue, HBM1029. The results showed that the half-lives of IMAB362 analog and HBM1029 in mice were 248 and 282 hours, respectively.
  • each NCG mouse is subcutaneously inoculated with 5 ⁇ 10 6 NUGC4_D8 tumor cells.
  • the tumor cells are first resuspended in a mixture of PBS and Matrigel (1:1) (0.1ml), then mixed with PBMC (resuspended in 0.05mL PBS) and inoculated subcutaneously.
  • PBMC resuspended in 0.05mL PBS
  • the mice were administered in groups. 18 mice were divided into 3 groups. After grouping, the administration was started. The administration cycle was twice a week.
  • the method of medicine is tail vein injection. After starting the administration, weigh the body weight and tumor volume twice a week.
  • tumor volume (mm 3 ) 0.5 ⁇ tumor long diameter ⁇ tumor short diameter2.
  • the experimental observation was ended on the 21st day after the administration, and then all mice were euthanized. Data analysis adopts t-test.
  • Figure 15 shows the results of the in vivo pharmacodynamic study of IMAB362 analogue, HBM1029.
  • the average tumor volume of mice in the control group was 1526 mm 3 .
  • the average tumor volume of the test drug IMAB362 analog (50mg/kg) treatment group on the 21st day after administration was 728mm 3 , which was significantly different from the vehicle control group (p value 0.0052), and the tumor inhibition rate TGI (%) was 52.29%.
  • the average tumor volume of the test drug HBM1029 (50mg/kg) treatment group was 618mm 3 on the 21st day after administration. Compared with the vehicle control group, it has a significant tumor suppression effect (p value is 0.0009), and the tumor suppression rate TGI (%) is 59.47%. Throughout the treatment and administration period, the animals showed good drug tolerance, and there was no serious weight loss or animal death.

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Abstract

提供了靶向CLDN18.2的抗体或其抗原结合片段及其制备方法和应用。所述抗体包含轻链可变区和/或重链可变区，所述重链可变区包含HCDR1、HCDR2和HCDR3，所述的轻链可变区包含LCDR1、LCDR2和LCDR3。与现有技术相比，所述抗体的结合亲和力、ADCC、CDC、生长抑制效应、内吞活性等都有明显的优势。

Description

靶向CLDN18.2的抗体及其制备方法和应用
本申请要求申请日为2019/9/30的中国专利申请2019109413163的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及生物医药领域，尤其涉及一种靶向CLDN18.2的抗体及其制备方法和应用。
背景技术
癌症是现今人类头号致命疾病之一。据2018世界卫生组织(WHO)报告称，每年约新发生1807万例癌症病患。癌症每年约有955万例死亡。根据WHO的估计，胃癌是全世界排名第五大最普遍被诊断的癌症。胃癌是癌症相关死亡的第三(对于男性而言)和第四大(对于女性而言)病因。全球每年有100万新诊断罹患胃癌的患者，美国约35％初次诊断罹患胃癌的患者为转移性胃癌。确诊晚期胃癌5年存活率为5％，中位数生存期约为6个月。治疗转移/复发性胃癌患者的第一线用药分做两种情况：①HER2-neu试验阳性病患，使用曲妥珠单抗(Transtuzumab)合并化疗药治疗；②HER2-neu试验阴性病患，仅可以化疗药治疗，其治疗效果不佳(Front Pharmacol.2018 Sep 13；9:404)。
CLDN18(密蛋白18，Claudin18)分子剪接变体1(CLD18A1，即CLDN18.1)的Genbank登记号为NP_057453、NM016369，剪接变体2(CLD18A2，即CLDN18.2)的Genbank登记号为NM_001002026、NP_001002026)是分子量约27.9/27.72kD的内在跨膜蛋白。密蛋白是位于上皮和内皮的紧密连接中的内在膜蛋白。另两个主要的紧密结合家族蛋白是闭合蛋白(occludin)和连接粘合分子(JAM)。密蛋白是紧密结合的必需成分，在保持上皮细胞极性、控制细胞旁扩散、以及调控细胞生长分化方面起到重要作用。据推测，密蛋白在构造良好的上皮中几乎无法接近抗体，但在肿瘤细胞中则变得暴露出来。密蛋白分子跨细胞膜四次，N端和C端均落在细胞质中。其中，人CLDN18.2(密蛋白18.2，Claudin 18.2)蛋白是一个跨膜蛋白，全长261个氨基酸，其中1-23为信号肽；其有两个膜外区域分别为信号肽后面大约55个氨基酸的胞外区1(Extracellular loop 1，ECL1)和23个氨基酸ECL2。CLDN18.1(密蛋白18.1，Claudin 18.1)和CLDN18.2在包括第一个TM和环1(即ECL1)的N端前21个氨基酸中存在差异，而C端的一级蛋白质序列相同。人CLDN18.2和人CLDN18.1的ECL1区域非常相似，而且人CLDN18.2 和人CLDN18.1的ECL2区域则完全相同。因而针对人CLDN18.2蛋白靶点抗体的开发，需要寻找针对人CLDN18.2蛋白的ECL1区域或空间结构的抗体。这使得这方面的工作变得更加困难。CLDN18.1在正常肺和胃的上皮中选择性表达(Mol Cell Biol.2001Nov；21(21):7380-90.)。CLDN18.2在正常组织中的表达高度受限，仅限于胃上皮的分化细胞中，而不存在于胃干细胞区。但在若干癌症类型中强烈表达，包括胃、食管、胰腺和肺肿瘤以及人癌症细胞系。该蛋白质的分子量在一些癌症和邻近的正常组织中存在差异。在健康组织中观察到的较高分子量的蛋白质可以通过用去糖基化化合物PNGaseF处理组织裂解液而转变成与癌症中所观察到相同的分子量。这提示与其正常组织对应物相比，密蛋白在癌症中N-糖基化较少。这种结构差异很可能产生改变的表位。经典的N-糖基化基序在该分子环D3结构域的116位氨基酸中(CN103509110B)。
目前针对人CLDN18.2抗体研究只有Claudiximab(IMAB362)抗体(参见WO 2014/146672)在临床试验阶段。IMAB362能够诱导ADCC(antibody-dependentcell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)效应和CDC(complement dependent cytotoxicity，补体依赖细胞毒作用)效应，及介导肿瘤杀伤。IMAB362在治疗胃食道癌晚期的I期和II期临床试验中表现出鼓舞人心的效力(Eur J Cancer.2018 Sep；100:17-26)。但IMAB362是人、鼠源嵌合抗体，存在免疫原性风险，亲和力不是很高。细胞学实验证明其仅有微弱的内吞活性，不适合做ADC开发，且治疗效果极为有限。由于大量恶性肿瘤的未被满足的医学需求，对具有更多可取的药学特征的其他CLDN18.2抗体存在需求。因此，本领域缺乏有效的靶向人CLDN18.2蛋白的抗体特别是全人源的抗体，以及细胞结合活性、ADCC活性、CDC活性、生长抑制效应、内吞活性等更好的抗体。
发明内容
本发明所要解决的技术问题是为了克服本领域缺乏靶向CLDN18.2的抗体的缺陷，提供了一种靶向CLDN18.2(人密蛋白18.2)的抗体及其制备方法和应用。
为解决上述技术问题，本发明的第一方面提供了一种靶向CLDN18.2的抗体或其抗原结合片段，其包含轻链可变区(VL)和/或重链可变区(VH)，所述重链可变区包含HCDR1、HCDR2和HCDR3，所述的轻链可变区包含LCDR1、LCDR2和LCDR3；其中：所述HCDR1包含如SEQ ID NO:8所示的氨基酸序列或其变体1，所述HCDR2包含选自SEQ ID NO:16及其变体2、SEQ ID NO:18及其变体3中的氨基酸序列，所述HCDR3包含SEQ ID NO:26～29中的任一个所示的氨基酸序列，所述LCDR1包含SEQ ID NO: 42所示的序列或其变体4，所述LCDR2包含SEQ ID NO:47所示的序列或其变体5，所述LCDR3包含SEQ ID NO:55所示的序列或其变体6；所述变体为在原始序列基础上进行1个、2个或3个氨基酸的取代、缺失或添加，包含上述变体的抗体或抗原结合片段保持与CLDN18.2结合能力。
在类似“具有3、2或1个氨基酸突变”中“氨基酸突变”是指相较于原氨基酸序列而言，变体的序列存在氨基酸的突变，包括在原氨基酸序列的基础上发生氨基酸的插入、缺失或替换。示例性的解释是对CDR的突变可以包含3个、2个或1个氨基酸的突变，这些CDR之间可以任选地选择相同或不同数目的氨基酸残基进行突变，例如可以是对CDR1进行1个氨基酸的突变，对CDR2和CDR3不进行氨基酸突变。
本发明中，所述突变可以包括目前如本领域技术人员公知的突变，例如在抗体的生产或者应用过程中，可能会对抗体进行的一些突变，例如对可能存在的，特别是CDR区的转录后修饰(Potential post-translational modifications，PTMs)的位点进行突变，包括抗体的聚集、脱酰胺基敏感(asparaginedeamidation，位点(NG，NS，NH等)、天冬氨酸异构(DG，DP)敏感位点、N糖基化(N-{P}S/T)敏感位点及氧化敏感位点等相关突变。
关于如上所述的“变体”，其中：
所述变体1的突变优选至少发生于SEQ ID NO:8所示氨基酸序列的第6位和/或第7位。
所述变体2的突变优选至少发生于SEQ ID NO:16所示氨基酸序列的第5位。
所述变体3的突变优选至少发生于SEQ ID NO:18所示氨基酸序列的第3位。
所述变体4的突变优选至少发生于SEQ ID NO:42所示氨基酸序列的第8位和/或第9位。
所述变体5的突变优选至少发生于SEQ ID NO:47所示氨基酸序列的第1位和/或第4位。
所述变体6的突变优选至少发生于SEQ ID NO:55所示氨基酸序列的第3～5位中的一位或者多位。
较佳地，所述变体1含有突变S6G和/或Y7F，所述变体2含有突变G5R，所述变体3含有突变D3E，所述变体4含有突变S8R和/或N9Y，所述变体5含有突变G1D和/或T4N，所述变体6含有突变Y3R/N、N4S以及N5Y中的一个或多个。
在本发明一较佳实施方案中，所述变体1的氨基酸序列如SEQ ID NO:6或7所示。
在本发明一较佳实施方案中，所述变体2的氨基酸序列如SEQ ID NO:17所示；
在本发明一较佳实施方案中，所述变体3的氨基酸序列如SEQ ID NO:19所示；
在本发明一较佳实施方案中，所述变体4的氨基酸序列如SEQ ID NO:40或41所示；
在本发明一较佳实施方案中，所述变体5的氨基酸序列如SEQ ID NO:48所示；
在本发明一较佳实施方案中，所述变体6的氨基酸序列如SEQ ID NO:56～58任一所示。
在本发明一更佳实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:7所示、所述HCDR2的氨基酸序列如SEQ ID NO:17所示且所述HCDR3的氨基酸序列如SEQ ID NO:27所示；所述HCDR1的氨基酸序列如SEQ ID NO:8所示、所述HCDR2的氨基酸序列如SEQ ID NO:18所示且所述HCDR3的氨基酸序列如SEQ ID NO:28所示；所述HCDR1的氨基酸序列如SEQ ID NO:8所示、所述HCDR2的氨基酸序列如SEQ ID NO:16所示且所述HCDR3的氨基酸序列如SEQ ID NO:29所示；或者，所述HCDR1的氨基酸序列如SEQ ID NO:8所示、所述HCDR2的氨基酸序列如SEQ ID NO:19所示且所述HCDR3的氨基酸序列如SEQ ID NO:28所示；
和/或，所述LCDR1的氨基酸序列如SEQ ID NO:41所示、所述LCDR2的氨基酸序列如SEQ ID NO:48所示且所述LCDR3的氨基酸序列如SEQ ID NO:56所示；所述LCDR1的氨基酸序列如SEQ ID NO:42所示、所述LCDR2的氨基酸序列如SEQ ID NO:47所示且所述LCDR3的氨基酸序列如SEQ ID NO:57所示；所述LCDR1的氨基酸序列如SEQ ID NO:42所示、所述LCDR2的氨基酸序列如SEQ ID NO:47所示且所述LCDR3的氨基酸序列如SEQ ID NO:55所示；或者，所述LCDR1的氨基酸序列如SEQ ID NO:42所示、所述LCDR2的氨基酸序列如SEQ ID NO:47所示且所述LCDR3的氨基酸序列如SEQ ID NO:58所示。
优选地，所述VH还包括重链可变区框架区(VH FWR)，和/或，所述VL还包括轻链可变区框架区(VL FWR)；
更优选地，所述VH FWR为人抗体的重链可变区框架区，所述VL FWR为人抗体的轻链可变区框架区。其中：
编码所述重链可变区框架区的基因优选来源于胚系V基因IGHV3-23；较佳地，所述重链可变区框架区中，HFR1包含如SEQ ID NO:2～4任一所示的氨基酸序列或其变体，HFR2包含如SEQ ID NO:10～14任一所示的氨基酸序列或其变体，HFR3包含如SEQ ID NO:21～24任一所示的氨基酸序列或其变体，HFR4包含如SEQ ID NO:31～33任一所示的氨基酸序列或其变体。
编码所述轻链可变区框架区的基因优选来源于胚系V基因IGKV3-11或者IGKV3- 15；
较佳地，所述轻链可变区框架区中，LFR1包含如SEQ ID NO:35～38任一所示的氨基酸序列或其变体，LFR2包含如SEQ ID NO:44或45所示的氨基酸序列或其变体，LFR3包含如SEQ ID NO:50～53任一所示的氨基酸序列或其变体，LFR4包含如SEQ ID NO:60或61所示的氨基酸序列或其变体。
在本发明一最佳实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:7所示、所述HCDR2的氨基酸序列如SEQ ID NO:17所示、所述HCDR3的氨基酸序列如SEQ ID NO:27所示、所述LCDR1的氨基酸序列如SEQ ID NO:41所示、所述LCDR2的氨基酸序列如SEQ ID NO:48所示且所述LCDR3的氨基酸序列如SEQ ID NO:56所示；
所述HCDR1的氨基酸序列如SEQ ID NO:8所示、所述HCDR2的氨基酸序列如SEQ ID NO:18所示、所述HCDR3的氨基酸序列如SEQ ID NO:28所示、所述LCDR1的氨基酸序列如SEQ ID NO:42所示、所述LCDR2的氨基酸序列如SEQ ID NO:47所示且所述LCDR3的氨基酸序列如SEQ ID NO:57所示；
所述HCDR1的氨基酸序列如SEQ ID NO:8所示、所述HCDR2的氨基酸序列如SEQ ID NO:16所示、所述HCDR3的氨基酸序列如SEQ ID NO:29所示、所述LCDR1的氨基酸序列如SEQ ID NO:42所示、所述LCDR2的氨基酸序列如SEQ ID NO:47所示且所述LCDR3的氨基酸序列如SEQ ID NO:55所示；
所述HCDR1的氨基酸序列如SEQ ID NO:8所示、所述HCDR2的氨基酸序列如SEQ ID NO:16所示、所述HCDR3的氨基酸序列如SEQ ID NO:29所示、所述LCDR1的氨基酸序列如SEQ ID NO:42所示、所述LCDR2的氨基酸序列如SEQ ID NO:47所示且所述LCDR3的氨基酸序列如SEQ ID NO:58所示；
或者，所述HCDR1的氨基酸序列如SEQ ID NO:8所示、所述HCDR2的氨基酸序列如SEQ ID NO:19所示、所述HCDR3的氨基酸序列如SEQ ID NO:28所示、所述LCDR1的氨基酸序列如SEQ ID NO:42所示、所述LCDR2的氨基酸序列如SEQ ID NO:47所示且所述LCDR3的氨基酸序列如SEQ ID NO:57所示。
在本发明一具体实施方案中，所述重链可变区包含如SEQ ID NO:64所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:71所示的氨基酸序列或其变体；
所述重链可变区包含如SEQ ID NO:67所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:73所示的氨基酸序列或其变体；
所述重链可变区包含如SEQ ID NO:65所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:72所示的氨基酸序列或其变体；
所述重链可变区包含如SEQ ID NO:68所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:74所示的氨基酸序列或其变体；
或者，所述重链可变区包含如SEQ ID NO:66所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:72所示的氨基酸序列或其变体；
其中，所述变体至少保留突变前序列的功能，且所述变体与突变前序列的同一性至少为85％，优选至少90％，更优选至少95％，进一步更优选至少99％。
在本申请中，上述所列CDR的氨基酸序列均是按照Chothia定义规则所示出的(本发明的权利要求中也是按照Chothia定义规则所示出的序列)。但是，本领域人员公知，在本领域中可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991))和基于结构环区域位置的Chothia定义规则(参见JMol Biol 273:927-48,1997)。在本申请中，还可以使用包含了Kabat定义和Chothia定义的Combined定义规则确定可变结构域序列中的氨基酸残基。其中Combined定义规则即是将Kabat定义和Chothia定义的范围相结合，基于此取了一个更大的范围，详见表1-1。本领域技术人员应当理解的是，除非另有规定，否则术语给定抗体或其区(例如可变区)的“CDR”及“互补决定区”应了解为涵盖如通过本发明描述的上述已知方案中的任何一种界定的互补决定区。虽然本发明的权利要求中请求保护的范围是基于Chothia定义规则所示出的序列，但是根据其他CDR的定义规则所对应的氨基酸序列也应当落在本发明的保护范围中。
表1-1本申请抗体CDR定义方法(可参见http://bioinf.org.uk/abs/)
  Kabat Chothia Combined
VL CDR1 L24--L34 L24--L34 L24-L34
VL CDR2 L50--L56 L50--L56 L50-L56
VL CDR3 L89--L97 L89--L97 L89-L97
VH CDR1 H31--H35 H26--H32 H26-H35
VH CDR2 H50--H65 H52--H56 H50-H65
VH CDR3 H95--H102 H95--H102 H95-H102
其中，Laa-Lbb可以指从抗体轻链的N端开始，第aa位至第bb位的氨基酸序列；Haa-Hbb可以指从抗体重链的N端开始，第aa位至第bb位的氨基酸序列。例如，L24-L34可以指从抗体轻链N端开始，按照Chothia编码规则的从第24位至第34位的氨基酸序列；H26-H32可以指从抗体重链N端开始，按照Chothia编码规则的从第26位至第32位的氨基酸序列。本领域技术人员公知，在用Chothia编码CDR时，有些位置会有插入位点的情况，例如以本发明中如SEQ ID NO:17所示的VH CDR2氨基酸序列为例，其 在第52位后有52A插入的情况，如下表1-2中所示。
表1-2
氨基酸位置编号 52 52A 53 54 55 56
VH CDR2(SEQ ID NO:17) S G S G R S
优选地，所述靶向CLDN18.2的抗体还包括抗体重链恒定区和抗体轻链恒定区；
更优选地，所述重链恒定区选自hIgG1、hIgG2、hIgG3或hIgG4或其突变，所述轻链恒定区选自人源抗体轻链κ链或者λ链或其突变；
进一步更优选地，所述重链恒定区为hIgG1，且所述轻链恒定区为人源抗体的轻链κ链。
优选地，所述靶向CLDN18.2的抗体为全长抗体、Fab、Fab’、F(ab’) 2、Fv、scFv(single chain antibody fragment，单链抗体)、双特异性抗体、多特异性抗体、重链抗体或单域抗体，或由上述抗体制得的单克隆抗体或多克隆抗体。所述单克隆抗体可以由多种途径和技术进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，主流是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。
当所述靶向CLDN18.2的抗体为双特异性抗体时，其可以包括第一蛋白功能区和第二蛋白功能区。所述第一蛋白功能区可以为上述的蛋白质，其靶向结合CLDN18.2；所述第二蛋白功能区为非靶向结合CLDN18.2的蛋白质或者为同样靶向结合CLDN18.2但非本发明所述的靶向CLDN18.2的抗体。其中，所述第一蛋白功能区可以为免疫球蛋白，所述第二蛋白功能区可以为一个或多个scFv；或者，所述第二蛋白功能区可以为免疫球蛋白，所述第一蛋白功能区可以为一个或多个scFv。
优选地，所述靶向CLDN18.2的抗体是全长抗体，所述全长抗体包括重链和轻链，所述重链包含如SEQ ID NO:77～90中任一所示的氨基酸序列，所述轻链包含如SEQ ID NO:93～96任一所示的氨基酸序列。
在一更具体的实施方案中，所述重链包含如SEQ ID NO:77所示的氨基酸序列，所述轻链包含如SEQ ID NO:93所示的氨基酸序列；所述重链包含如SEQ ID NO:78所示的氨基酸序列，所述轻链包含如SEQ ID NO:94所示的氨基酸序列；所述重链包含如SEQ ID NO:79所示的氨基酸序列，所述轻链包含如SEQ ID NO:93所示的氨基酸序列；所述重链包含如SEQ ID NO:85所示的氨基酸序列，所述轻链包含如SEQ ID NO:93所示的氨基酸序列；所述重链包含如SEQ ID NO:83所示的氨基酸序列，所述轻链包含如SEQ ID NO:93所示的氨基酸序列；所述重链包含如SEQ ID NO:84所示的氨基酸序列，所述轻链包含如SEQ ID NO:93所示的氨基酸序列；所述重链包含如SEQ ID NO:81所示的氨基酸序列，所述轻链包含如SEQ ID NO:95所示的氨基酸序列；所述重链包含如SEQ  ID NO:82所示的氨基酸序列，所述轻链包含如SEQ ID NO:96所示的氨基酸序列；所述重链包含如SEQ ID NO:80所示的氨基酸序列，所述轻链包含如SEQ ID NO:94所示的氨基酸序列；所述重链包含如SEQ ID NO:86所示的氨基酸序列，所述轻链包含如SEQ ID NO:95所示的氨基酸序列；所述重链包含如SEQ ID NO:87所示的氨基酸序列，所述轻链包含如SEQ ID NO:96所示的氨基酸序列；所述重链包含如SEQ ID NO:88所示的氨基酸序列，所述轻链包含如SEQ ID NO:94所示的氨基酸序列；所述重链包含如SEQ ID NO:89所示的氨基酸序列，所述轻链包含如SEQ ID NO:95所示的氨基酸序列；或者，所述重链包含如SEQ ID NO:90所示的氨基酸序列，所述轻链包含如SEQ ID NO:96所示的氨基酸序列。以下使用表1-3汇总示例抗体的序列编号。
表1-3
抗体名称 轻重链类型 链 Fv FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4
PR000400 HC 75 62 1 5 9 15 20 25 30
PR000400 LC 91 69 34 39 43 46 49 54 59
PR002725 HC 76 63 2 6 10 16 21 26 31
PR002725 LC 92 70 35 40 44 47 50 55 60
PR002726 HC 77 64 2 7 11 17 22 27 31
PR002726 LC 93 71 36 41 45 48 51 56 60
PR002727 HC 78 65 3 8 12 18 21 28 32
PR002727 LC 94 72 37 42 45 47 50 57 60
PR003197 HC 79 64 2 7 11 17 22 27 31
PR003197 LC 93 71 36 41 45 48 51 56 60
PR003340 HC 85 64 2 7 11 17 22 27 31
PR003340 LC 93 71 36 41 45 48 51 56 60
PR003292 HC 83 64 2 7 11 17 22 27 31
PR003292 LC 93 71 36 41 45 48 51 56 60
PR003293 HC 84 64 2 7 11 17 22 27 31
PR003293 LC 93 71 36 41 45 48 51 56 60
PR003289 HC 81 67 4 8 13 16 23 29 31
PR003289 LC 95 73 38 42 45 47 52 55 61
PR003291 HC 82 68 2 8 14 16 24 29 33
PR003291 LC 96 74 38 42 45 47 53 58 60
PR003240 HC 80 66 3 8 12 19 21 28 32
PR003240 LC 94 72 37 42 45 47 50 57 60
PR003890 HC 86 67 4 8 13 16 23 29 31
PR003890 LC 95 73 38 42 45 47 52 55 61
PR003891 HC 87 68 2 8 14 16 24 29 33
PR003891 LC 96 74 38 42 45 47 53 58 60
PR003894 HC 88 66 3 8 12 19 21 28 32
PR003894 LC 94 72 37 42 45 47 50 57 60
PR003897 HC 89 67 4 8 13 16 23 29 31
PR003897 LC 95 73 38 42 45 47 52 55 61
PR003898 HC 90 68 2 8 14 16 24 29 33
PR003898 LC 96 74 38 42 45 47 53 58 60
注：上表中数字代表各抗体或其功能片段在序列表中的SEQ ID NO:，抗体名称不对抗体结构起任何限定作用。
所述突变为所述VL和/或VH的氨基酸序列上发生了一个或多个氨基酸残基的缺失、取代或添加，且所述突变的氨基酸序列与所述VL和/或VH的氨基酸序列具有至少85％序列同一性，并保持或改善了所述抗体与CLDN18.2的结合；所述至少85％序列同一性优选为至少90％序列同一性；更优选为至少95％序列同一性；最优选为至少99％序列同一性。
本发明中，“Fab片段”由一条轻链和一条重链的CH1及可变区组成。Fab分子的重链不能与另一个重链分子形成二硫键。“Fc”区含有包含抗体的CH1和CH2结构域的两个重链片段。两个重链片段由两个或多个二硫键并通过CH3结构域的疏水作用保持在一起。“Fab’片段”含有一条轻链和包含VH结构域和CH1结构域以及CH1和CH2结构域之间区域的一条重链的部分，由此可在两个Fab’片段的两条重链之间形成链间二硫键以形成F(ab’) 2分子。“F(ab’) 2片段”含有两条轻链和两条包含CH1和CH2结构域之间的恒定区的部分的重链，由此在两条重链间形成链间二硫键。因此F(ab’) 2片段由通过两条重链间的二硫键保持在一起的两个Fab’片段组成。术语“Fv”意指向抗体的单臂的VL和VH结构域组成的抗体片段，但缺少恒定区。
本发明中，所述的scFv(single chain antibody fragment，单链抗体)可为本领域常规的单链抗体，其包括重链可变区、轻链可变区和15～20个氨基酸的短肽。其中VL和VH结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子[参见，例如，Bird等人，Science 242:423-426(1988)和Huston等人，Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)]。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的G 4S氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(G 4S) 4或(G 4S) 3接头，但也可使用其变体。
术语“多特异性抗体”按其最广义使用，涵盖具有多表位特异性的抗体。这些多特异性抗体包括但不限于：包含重链可变区(VH)和轻链可变区(VL)的抗体，其中该VH-VL单元具有多表位特异性；具有两个或多个VL和VH区的抗体，每个VH-VL单元与不同的靶点或同一个靶点的不同表位结合；具有两个或更多个单可变区的抗体，每个单可变区与不同的靶点或同一个靶点的不同的表位结合；全长抗体、抗体片段、双特异性抗体(diabodies)、和三抗体(triabodies)、共价或非共价连接在一起的抗体片段等。
本发明的抗体包括单克隆抗体。本发明所述的单克隆抗体或mAb或Ab，指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的，原核的或噬菌体的克隆细胞株。
本发明中，所述的“重链抗体”指的是只包含一个重链可变区(VHH)和两个常规的CH2与CH3区的抗体，又称为HCAbs。
本发明中，所述的“单域抗体”，又称为“纳米抗体”，指的是从重链抗体中克隆出来的VHH结构，是已知的可结合目标抗原的最小单位。
为解决上述技术问题，本发明的第二方面提供了一种分离的核酸，其编码如本发明第一方面所述的靶向CLDN18.2的抗体。
所述核酸的制备方法为本领域常规的制备方法，较佳地，包括以下的步骤：通过基因克隆技术获得编码上述抗体的核酸分子，或者通过人工全序列合成的方法得到编码上述抗体的核酸分子。
本领域技术人员知晓，编码上述抗体的氨基酸序列的碱基序列可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对编码该抗体序列基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。
为解决上述技术问题，本发明的第三方面提供了一种重组表达载体，其包含如本发明第二方面所述的分离的核酸。
所述重组表达载体可通过本领域常规方法获得，即：将本发明所述的核酸分子连接于各种表达载体上构建而成。所述的表达载体为本领域常规的各种载体，只要其能够容载前述核酸分子即可。
优选地，所述重组表达载体为质粒、粘粒、噬菌体或病毒载体，所述病毒载体优选逆转录病毒载体、慢病毒载体、腺病毒载体或腺相关病毒载体。
为解决上述技术问题，本发明的第四方面提供了一种转化体，其在宿主细胞中包含如本发明第三方面所述的重组表达载体。
所述重组表达转化体的制备方法可为本领域常规的制备方法，例如为：将上述重组表达载体转化至宿主细胞中制得。所述的宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核酸可被有效表达即可。优选地，所述宿主细胞为E.coli TG1或BL21细胞(表达单链抗体或Fab抗体)，或者CHO-K1细胞(表达全长IgG抗体)。将前述重组表达质粒转化至宿主细胞中，即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。
本发明中，所述的靶向CLDN18.2的抗体可以用于制备成嵌合抗原受体(CAR)等从而将其修饰在例如T细胞或NK细胞等细胞上面。本发明因此提供一种包含如本发明第一方面所述的靶向CLDN18.2的抗体或其抗原结合片段。例如其为利用上述靶向CLDN18.2的抗体的scFv作为胞外抗原结合结构域的嵌合抗原受体。因此，为解决上述 技术问题，本发明的第五方面提供了一种基因修饰的细胞，其包含如本发明第一方面所述的靶向CLDN18.2的抗体。
优选地，所述基因修饰的细胞为真核细胞，优选分离的人细胞；更优选免疫细胞如T细胞(例如以CAR-T的形式)，或NK细胞。
为解决上述技术问题，本发明的第六方面提供了一种靶向CLDN18.2的抗体的制备方法，其包含培养如本发明第四方面所述的转化体，从培养物中获得靶向CLDN18.2的抗体。
为解决上述技术问题，本发明的第七方面提供了一种抗体药物偶联物(ADC)，其包含细胞毒剂，以及如本发明第一方面所述的靶向CLDN18.2的抗体。
所述细胞毒剂优选细胞毒素、化学治疗剂、放射性同位素、治疗性核酸、免疫调节剂、抗血管生成剂、抗增殖促凋亡剂或细胞溶解酶；更优选地，所述细胞毒剂为微管蛋白合成酶抑制剂——甲基奥瑞他汀F(MMAF)，或者为甲基奥瑞他汀E(MMAE)。
所述的抗体药物偶联物的制备方法可为本领域常规，较佳地采用Doronina，2006，Bioconjugate Chem.17,114-124所记载的制备方法。优选地，所述的制备方法产生具有最低限度的低偶联级分(LCF)小于10％的抗体药物偶联物。
所述的抗体药物偶联物能够以本领域所知的任何物理形态而存在，较佳地为澄清溶液。
为解决上述技术问题，本发明的第八方面提供了一种药物组合物，其包含如本发明第一方面所述的靶向CLDN18.2的抗体和/或如本发明第七方面所述的抗体药物偶联物，和药学上可接受的载体。
所述的药物组合物较佳地还包括其他抗肿瘤抗体作为活性成分，和/或含有由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
所述的药学上可接受的载体可为本领域常规的载体，所述的载体可以为任意合适的生理学或药学上可接受的药物辅料。所述的药物辅料为本领域常规的药物辅料，较佳地包括药学上可接受的赋形剂、填充剂、稳定剂或稀释剂等。更佳地，所述的药物组合物包括0.01～99.99％的上述蛋白质和/或上述的抗体药物偶联物，和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
较佳地，所述的药物组合物是抗肿瘤的药物。更佳地为治疗胃癌、食管癌、肺癌、卵巢癌、黑素瘤、肾癌、乳腺癌、结肠直肠癌、肝癌、胰腺癌、膀胱癌、头颈癌、支气管癌、 神经胶质瘤和/或白血病的药物。
本发明所述的药物组合物的给药途径较佳地为肠胃外施用、注射给药或口服给药。所述注射给药较佳地包括静脉注射、肌肉注射、腹腔注射、皮内注射或皮下注射等途径。所述的药物组合物为本领域常规的各种剂型，较佳地为固体、半固体或液体的形式，即可以为水溶液、非水溶液或混悬液，更佳的为片剂、胶囊、颗粒剂、注射剂或输注剂等。更佳地为经由血管内、皮下、腹膜内或肌内施用。较佳地，所述药物组合物还可以作为气雾剂或粗喷雾剂施用，即经鼻施用；或者，鞘内、髓内或心室内施用。更佳地，所述的药物组合物还可以透皮、经皮、局部、肠内、阴道内、舌下或经直肠施用。
本发明所述的药物组合物的给药剂量水平可以根据达到所需诊断或治疗结果的组合物量而调整。施用方案也可以为单次注射或多次注射，或进行调整。所选择的剂量水平和方案依赖于包括所述药物组合物的活性和稳定性(即，半衰期)、制剂、施用途径、与其他药物或治疗的组合、待检测和/或治疗的疾病或病症、以及待治疗的受试者的健康状况和先前医疗史等各种因素而进行合理地调整。
对于本发明的所述药物组合物的治疗有效剂量可以最初在细胞培养实验或动物模型例如啮齿类动物、兔、犬、猪和/或灵长类动物中进行估计。动物模型也可以用于测定合适的施用浓度范围和途径。随后可以用于确定在人中施用的有用剂量和途径。一般地，施用有效量或剂量的确定和调整以及何时和如何进行此类调整的评估为本领域技术人员已知。
对于组合疗法，上述靶向CLDN18.2的抗体、上述抗体药物偶联物和/或另外的治疗或诊断剂可以各自作为单一药剂，在适合于执行预期治疗或诊断的任何时间范围内进行使用。因此，这些单一药剂可以基本上同时(即作为单一制剂或在数分钟或数小时内)或以按顺序连续施用。例如，这些单一药剂可以在一年内，或10、8、6、4或2个月内，或4、3、2、或1周内，或5、4、3、2或1天内施用。
关于制剂、剂量、施用方案和可测量的治疗结果的另外指导，参见Berkow等人(2000)The Merck Manual of Medical Information(Merck医学信息手册)和Merck&Co.Inc.,Whitehouse Station,New Jersey；Ebadi(1998)CRC Desk Reference of Clinical Pharmacology(临床药理学手册)等著作。
为解决上述技术问题，本发明的第九方面提供了如本发明第一方面所述的靶向CLDN18.2的抗体、本发明第七方面所述的抗体药物偶联物和/或本发明第八方面所述的药物组合物在制备诊断、预防和/或治疗肿瘤的药物中的应用。
优选地，所述肿瘤为CLDN18.2阳性肿瘤；更优选地，所述肿瘤为胃癌、食管癌、 肺癌、黑素瘤、肾癌、乳腺癌、结肠直肠癌、肝癌、胰腺癌、膀胱癌、神经胶质瘤和/或白血病。
为解决上述技术问题，本发明还提供了如本发明第一方面所述的靶向CLDN18.2的抗体、本发明第七方面所述的抗体药物偶联物和/或本发明第八方面所述的药物组合物在诊断、预防和/或治疗肿瘤中的应用。优选地，所述肿瘤如本发明第九方面所述。
为解决上述技术问题，本发明的第十方面提供一种套装药盒，其包含药盒A和药盒B，所述的药盒A为本发明第一方面所述的靶向CLDN18.2的抗体、和/或本发明第七方面所述的抗体药物偶联物、和/或本发明第八方面所述的药物组合物；所述的药盒B含有其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，所述药盒B也可含化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂、疫苗、成像剂、诊断剂、激素制剂、靶向小分子制剂、蛋白酶体抑制剂等等，或者所述药盒B同时含其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，以及激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂、疫苗等等。所述的药盒A和药盒B可以同时使用，也可以先使用药盒A再使用药盒B，还可以先使用药盒B再使用药盒A，可以根据具体应用时的实际需求而定。
为了解决上述技术问题，如本发明第一方面所述的靶向CLDN18.2的抗体或其抗原结合片段、如本发明如上所述的嵌合抗原受体、如本发明第五方面所述的基因修饰的细胞、本发明第七方面所述的抗体药物偶联物和/或如本发明第八方面所述的药物组合物还可以与其他药物进行联合用药，例如可以与激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂、疫苗等和/或其他抗肿瘤抗体(或者包含所述其他抗肿瘤抗体的药物组合物)进行联合用药。本发明所述的“CLDN18.2阳性”的细胞即为过表达CLDN18.2蛋白的细胞，如NUGC4_D8细胞株；反之，则称为“CLDN18.2阴性”的细胞。
本发明的第十一方面提供一种诊断、治疗和/或预防CLDN18.2介导的疾病或病症的方法，所述方法包括向有需要的患者施用治疗有效量的如第一方面所述的靶向CLDN18.2的抗体或其抗原结合片段、如第七方面所述的抗体药物偶联物或如第八方面所述的药物组合物，或者使用如第十方面所述的套装药盒治疗有需要的患者。
其中所述CLDN18.2介导的疾病或病症可为肿瘤，优选CLDN18.2阳性肿瘤，更优选胃癌、食管癌、肺癌、卵巢癌、黑素瘤、肾癌、乳腺癌、结肠直肠癌、肝癌、胰腺癌、膀胱癌、头颈癌、支气管癌、神经胶质瘤和/或白血病。
本发明的第十二方面提供免疫检测或者测定CLDN18.2的方法，其包括使用如第一方面所述的靶向CLDN18.2的抗体或其抗原结合片段、如第七方面所述的抗体药物偶联物或如第八方面所述的药物组合物。
本发明的第十三方面提供种联合疗法，其包括分别向有需要的患者施用如第一方面所述的靶向CLDN18.2的抗体或其抗原结合片段、如第七方面的抗体药物偶联物或如第八方面所述的药物组合物，和第二治疗剂；所述第二治疗剂较佳地包含其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
在本发明中，术语“可变”通常是指这样的事实，即抗体的可变结构域的序列的某些部分变化强烈，它形成各种特定抗体对其特定抗原的结合和特异性。然而，变异性并非均匀地分布在抗体的整个可变区中。它集中在轻链和重链可变区中的三个区段，被称为互补决定区(CDR)或高变区(HVR)。可变域中更高度保守的部分被称为框架(FWR)。天然重链和轻链的可变结构域各自包含四个FWR区，大部分采用β-折叠构型，通过三个CDRs连接，形成环连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的CDRs通过FWR区紧密靠近在一起，并与来自另一条链的CDR一起形成抗体的抗原结合位点，恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
本发明所用氨基酸三字母代码和单字母代码如本领域技术人员知晓，或J.Biol.Chem,243,p3558(1968)中所述。
如本文使用的，术语“包括”或“包含”旨在表示组合物和方法包括所述的元素但不排除其他元素，但根据上下文的理解，也包括“由……组成”的情况。
术语“CLDN18.2”包括同种型、哺乳动物(例如人)的CLDN18.2、人CLDN18.2的物种同源物和包含至少一个与CLDN18.2的共同表位的类似物。CLDN18.2(例如人CLDN18.2)的氨基酸序列是本领域中己知的，如NCBI数据库显示。
术语“CLDN18.1”包括同种型、哺乳动物(例如人)的CLDN18.1、人CLDN18.1的物种同源物和包含至少一个与CLDN18.1的共同表位的类似物。CLDN18.1(例如人 CLDN18.1)的氨基酸序列是本领域中己知的，如NCBI数据库显示。
术语“表位”指抗原(例如，人CLDN18.2)中与抗体分子特异性相互作用的部分。术语“竞争”在本发明中指抗体分子干扰抗CLDN18.2抗体分子与靶(例如，人CLDN18.2)结合的能力。对结合作用的干扰可以是直接或间接的(例如，通过抗体分子或靶的变构调节作用)。可以使用竞争结合测定法(例如，FACS测定法、ELISA或BIACORE测定法)确定抗体分子是否能够干扰另一种抗体分子与其靶结合的程度。
本发明所述的术语“抗体”包括免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3和IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。
在本发明中，本发明所述的抗体轻链可变区可进一步包含轻链恒定区，所述的轻链恒定区包含人源的κ、λ链或其变体。在本发明中，本发明所述的抗体重链可变区可进一步包含重链恒定区，所述的重链恒定区包含人源的IgG1、2、3、4或其变体。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FWR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FWR区组成，从氨基端到羧基端依次排列的顺序为：FWR1、CDR1、FWR2、CDR2、FWR3、CDR3、FWR4。轻链的3个CDR区指LCDR1、LCDR2和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。
在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(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)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域[称为互补决定区(CDR)]，其间散布有较保守的称为构架区(FWR)的区域。各VH和VL由按下列顺序：FWR1、CDR1、FWR2、CDR2、FWR3、CDR3、FWR4从氨基末端至羧基末排列的3个 CDR和4个FWR组成。各重链/轻链对应的可变区(VH和VL)分别形成抗体结合部位。特别地，重链还可以包含3个以上CDR，例如6、9或12个。例如在本发明的双特异性抗体中，重链可以是IgG抗体的重链的N端连接另一个抗体的ScFv，这种情况下重链含有9个CDR。
术语“人源抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本发明的人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。然而，术语“人抗体”不包括这样的抗体，即其中已将衍生自另一种哺乳动物物种(诸如小鼠)种系的CDR序列移植到人骨架序列上(即“人源化抗体”)。
如本发明所用，关于抗体的术语“特异性”意指识别特异性抗原但基本上不识别或结合样品中的其他分子的抗体。例如，特异性结合来自一个物种的抗原的抗体也可以结合来自一个或更多个物种的该抗原。但是，这种种间交叉反应性本身不改变抗体根据特异性的分类。在另一个实例中，特异性结合抗原的抗体也可以结合该抗原的不同等位基因形式。然而，这种交叉反应性本身不改变抗体根据特异性的分类。在一些情况下，术语“特异性”或“特异性结合”可用于指抗体、蛋白质或肽与第二化学物质的相互作用，意味着该相互作用取决于化学物质上特定结构(例如，抗原决定簇或表位)的存在；例如，抗体一般识别并结合特定的蛋白质结构，而不是蛋白质。如果抗体对表位“A”具有特异性，则在含有经标记的“A”和抗体的反应中，含有表位A的分子(或游离的，未标记的A)的存在将减少结合于抗体的标记的A的量。
本文中使用的术语“嵌合抗原受体”或“CAR”指：包含能够结合抗原的胞外域(胞外结合结构域)、铰链结构域、跨膜结构域(跨膜区)和使胞质信号传到结构域的多肽(即胞内信号域)。铰链结构域可以被认为是用于向细胞外抗原结合区提供柔性的一部分。胞内信号域指经由确定的信号传导途径通过产生第二信使而将信息传递到细胞内以调节细胞活性的蛋白质、或通过相应于此类信使而作为效应子发挥作用的蛋白质，产生可以促进CAR的细胞(例如CART细胞)的免疫效应子功能的信号。胞内信号域包含信号传导结构域，还可以包括源自共刺激分子的共刺激胞内结构域。
“同一性”、“变异序列”、“突变”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同一性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序 列为60％同源。一般而言，当比对两个序列而得到最大的同一性百分率时进行比较。“优化”指保持或改善了所述抗体与抗原结合的突变，在本发明中，指保持、维持或改善了与CLDN18.2的结合的突变。
术语“多肽”、“肽”和“蛋白质”(如果为单链)在本发明中互换地使用。术语“核酸”、“核酸序列”，“核苷酸序列”或“多核苷酸序列”和“多核苷酸”互换使用。
术语“突变”包括氨基酸或核苷酸的取代、添加和/或缺失，“氨基酸取代”和“保守性氨基酸取代”分别是其中氨基酸残基以另一种氨基酸残基置换和以具有相似侧链的氨基酸残基置换。
本文所使用的“慢病毒”是指逆转录病毒科(Retroviridae family)的属。慢病毒在逆转录病毒中是独特的，其能够感染非分裂细胞；它们可以将显著量的遗传信息递送到宿主细胞的DNA中，因此它们是基因递送载体的一种最有效方法。HIV、SIV和FIV都是慢病毒的实例。来自慢病毒的载体提供了在体内实现显著水平基因转移的手段。
本文使用的术语“载体”是包含分离的核酸并可用于将分离的核酸递送至细胞内部的组合物。在本领域中已知许多载体，包括但不限于线性多核苷酸、与离子或两亲化合物相关的多核苷酸、质粒和病毒。因此，术语“载体”包括自主复制的质粒或病毒。该术语还应被解释为包括促进核酸转移到细胞中的非质粒和非病毒化合物，例如聚赖氨酸化合物、脂质体等。病毒载体的实例包括但不限于腺病毒载体、腺相关病毒载体、逆转录病毒载体等。
本发明使用的表述“细胞”、“细胞系”可互换使用，并且所有这类名称都包括后代。术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌等原核细胞，如酵母细胞等的真菌细胞，或者如纤维原细胞、CHO细胞、COS细胞、NSO细胞、HeLa细胞、BHK细胞、HEK 293细胞或人细胞等的动物细胞。
术语“转染”是指将外源核酸引入真核细胞。转染可以通过本领域已知的各种手段来实现，包括磷酸钙-DNA共沉淀、DEAE-葡聚糖介导的转染、聚凝胺介导的转染、电穿孔、显微注射、脂质体融合、脂质转染、原生质体融合、逆转录病毒感染和生物弹道技术(biolistics)。
术语“免疫细胞”指可以引发免疫应答的细胞，“免疫细胞”及其语法上的其他形式可以指任何来源的免疫细胞。“免疫细胞”包括例如衍生自在骨髓中产生的造血干细胞(HSC)的白血细胞(白细胞)、淋巴细胞(T细胞、B细胞、自然杀伤(NK)细胞和骨髓来源的细胞(嗜中性粒细胞、嗜酸性粒细胞、嗜碱性粒细胞、单核细胞、巨噬细胞、树突状细胞)。术语“免疫细胞”也可以是人或非人的。
如本文使用的，术语“T细胞”是指在胸腺中成熟的一类淋巴细胞。T细胞在细胞介导的免疫中起重要作用，并且与与其他淋巴细胞(例如B细胞)的不同点在于细胞表面上存在T细胞受体。“T细胞”包括表达CD3的所有类型的免疫细胞，包括T辅助细胞(CD4+细胞)、细胞毒性T细胞(CD8+细胞)、自然杀伤T细胞、T调节细胞(Treg)和γ-δT细胞。“细胞毒性细胞”包括CD8+T细胞、自然杀伤(NK)细胞和嗜中性粒细胞，这些细胞能够介导细胞毒性反应。如本文使用的，术语“NK细胞”是指起源于骨髓并且在先天免疫系统中起重要作用的一类淋巴细胞。NK细胞提供针对病毒感染的细胞、肿瘤细胞或其他应激细胞的快速免疫反应，即使是细胞表面上不存在抗体和主要组织相容性复合体。
例如，免疫细胞可以是来自血液的，如自体的T细胞、异体T细胞、自体NK细胞、异体NK细胞，也可以来源自细胞系，如利用EBV病毒感染来制备NK细胞系，从胚胎干细胞和iPSC诱导分化来的NK细胞以及NK92细胞系等。
“任选”、“任一”、“任意”或“任一项”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选包含1个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。本发明所用，“一个”和“一种”在本发明中用来指的一个或多于一个的语法对象。除非内容明确提示，否则术语“或”在本发明中用来意指术语“和/或”并且与之互换使用。“约”和“大约”应当通常意指鉴于测量的性质或精度，所测量的量的可接受误差程度。示例性误差程度一般在其10％范围内和更一般在其5％范围内。本发明公开的方法和组合物涵盖这样的多肽和核酸，它们具有指定的序列，变异序列或与其基本上相同或相似的序列，例如，与序列指定至少85％、90％、95％、99％或更多相同的序列。在氨基酸序列的情况下，术语“基本上相同”在本发明中用来指第一氨基酸序列。
如本文中所使用的，术语EC 50是指半最大效应浓度(concentration for 50％of maximal effect)，即能引起50％最大效应的浓度。
本发明的药物组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。给药方式例如可以采用注射或其它治疗方式。
如本发明所用，术语“抗体药物偶联物”或“ADC”可互换使用。
澳瑞他汀是全合成药物，化学结构式相对容易改造，以便优化其物理性质和成药特性。用于和抗体偶联的澳瑞他汀衍生物主要包括单甲基澳瑞他汀E(MMAE)和单甲基澳瑞他汀F(MMAF)，前者是由天然微管蛋白聚合酶抑制剂尾海兔素10(dolastatin-10)衍生出的合成五肽，在C-端加上一个2-氨基-1-苯基丙基-1-醇而合成。MMAE对多种人 类肿瘤细胞株的抑制活性小于一纳摩尔。为了降低MMAE自身细胞毒活性，MMAF在尾海兔素10的C-端加上一个苯丙氨酸，因为在结构上引入一个羧基，MMAF的细胞膜通过性较差，因此对细胞的生物活性显著降低，但是和抗体偶联后对细胞的抑制活性大幅度提高(US7750116)。
在有些实施方案中，抗体细胞毒性药物偶联物或其可药用盐或溶剂化合物包含偶联有一个或多个美登木素生物碱分子的本发明抗体。美登木素生物碱是通过抑制微管蛋白多聚化未发挥作用的有丝分裂抑制剂。美登素最初从东非灌木齿叶美登木(Maytenus serrata)分离得到(美国专利No.3,896,111)。随后发现某些微生物也生成美登木素生物碱，诸如美登醇和C-3美登醇醋(美国专利No.4,151，042)。美登木素生物碱药物模块在抗体-药物偶联物中是有吸引力的药物模块，因为它们：(i)相对易于通过发酵或发酵产物的化学修饰或衍生化来制备；(ii)易于用适于通过非二硫化物接头偶联至抗体的官能团衍生化；(iii)在血浆中稳定；且(iv)有效针对多种肿瘤细胞系。适于用作美登木素生物碱药物模块的美登素化合物是本领域公知的，而且可以依照已知方法从天然来源分离，或是使用遗传工程技术生产(参见Yu等(2002)PNAS99：7968-7973)。美登醇和美登醇类似物也可以依照已知方法合成制备。美登木素生物碱药物模块的例示性实施方案包括：DM1、DM3和DM4，正如本文中所公开的。
本发明所述的方法、组合物、联合治疗可以与其他活性剂或治疗方式，所述的方法包括向对象以有效治疗或预防疾病(例如，癌症)的量，施用本发明所述的抗CLDN18.2抗体分子，任选地，与PD-1、PD-L1、PD-L2、LAG-3、CTLA-4、Tim-3抗体(免疫治疗)或其它肿瘤治疗抗体，Her-2、EGFR、VEGF、VEGFR抗体等，以及ADC(如T-DM1)，双特异抗体，化疗药物等的一种或多种抑制剂的组合，还包括施用抗CLDN18.2抗体分子、额外的活性剂或全部可以按这样的量或剂量施用，所述量或剂量高于、低于或等于单独(例如，作为单一疗法)使用的每种活性剂的量或剂量。抗CLDN18.2抗体，额外的活性剂或全部的施用量或剂量比单独(例如，作为单一疗法)使用的每种活性剂的量或剂量低(例如，至少20％、至少30％、至少40％或至少50％)。
此外，正如本发明的实施例中描述的那样，抗CLDN18.2抗体以及CLDN18.2抗体的药物偶联物可以结合CLDN18.2诱导靶细胞(肿瘤细胞)凋亡，抑制肿瘤细胞生长，增加体内效应细胞对肿瘤细胞ADCC，CDC杀伤作用来达到治疗癌症患者的目的。因此，在某些实施方案中，本发明所描述的抗CLDN18.2抗体以及CLDN18.2抗体的药物偶联物通过这些机理显示本发明抗体抗肿瘤效应，以及抑制肿瘤细胞生长的方法，包括将治疗有效量的本发明中所述的抗CLDN18.2抗体以及CLDN18.2抗体的药物偶联物施用于 受试者。该方法适用于癌症的体内治疗。为了获得靶向特异治疗效果，抗CLDN18.2抗体分子可以与其它抗体一起施用。在将CLDN18.2抗体以及CLDN18.2抗体的药物偶联物组合一种或多种活性剂施用时，该组合可以以任何顺序或同时施用癌症类型，特别是CLDN18.2高表达的肿瘤患者。在某些方面中，提供在对象中治疗对象(例如，减少或缓解)过度增生性病状或疾病(例如，癌症)。该方法包括向对象单独或与其他活性剂或治疗方式组合地施用本发明所述的一种或多种抗CLDN18.2抗体或CLDN18.2抗体的药物偶联物。
使用单独的或与另一种免疫调节剂(例如抗LAG-3，抗Tim-3,抗PD-l或抗PD-L1、抗CTLA-4抗体分子)组合的抗CLDN18.2抗体分子来治疗胃癌、胰腺癌、肺癌、食管道癌、卵巢癌等。抗CLDN18.2抗体分子可以与以下一者或多者组合施用：基于免疫的策略、靶向药物(例如，VEGF抑制剂如针对VEGF的单克隆抗体)；VEGF酪氨酸激酶抑制剂如舒尼替尼、索拉非尼、阿帕替尼；RNAi抑制剂或VEGF信号传导的下游介导物的抑制剂，例如，雷帕霉素哺乳动物靶(mTOR)的抑制剂。
如本发明所用，术语“癌”、“癌症”、“癌症病人”意在包括全部类型的癌性生长物或致瘤过程、转移性组织或恶性转化的细胞、组织或器官，无论其组织病理学类型或侵袭力阶段。例子包括但不限于实体瘤、血液学癌、软组织肿瘤和转移性病灶。
可以利用本发明中公开的靶向CLDN18.2的抗体适合治疗的癌症的非限定性的实例包括，胃癌、食管癌、肺癌、黑素瘤、肾癌、乳腺癌、结肠直肠癌、肝癌、胰腺癌、膀胱癌、神经胶质瘤和/或白血病等，或其转移性病灶。
需知：本发明中提及“变体1”、“变体2”等时，术语后的阿拉伯数字“1”、“2”没有实际意义，仅为指代相同术语。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明的抗体与现有技术相比，其结合亲和力、ADCC(antibody-dependentcell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)、CDC(complement dependent cytotoxicity，补体依赖细胞毒作用)、生长抑制效应、内吞活性等都有明显的优势，从而具有极大的治疗肿瘤的潜力。在本发明某一较佳实施例中，HBM1029抗体、PR003197、PR003340、PR003292、PR003293、PR003240、PR003291、PR003289、PR003890、PR003891、PR003894、PR003897以及PR003898与IMAB362类似物相比对内生性表达CLDN18.2的NUGC4_D8细胞呈现更高的结合亲和力；本发明的HBM1029、PR003197、PR003340、 PR003240、PR003894抗体能以剂量依赖方式特异性地于NUGC4_D8诱导较IMAB362类似物强的ADCC效应；HBM1029、PR003197、PR003340抗体能以剂量依赖方式于HEK293 hCLDN18.2诱导较IMAB362类似物强的CDC效应；HBM1029抗体能以剂量依赖方式于HEK293 hCLDN 18.2诱导较IMAB362类似物强的生长抑制效应；HBM1029抗体能以剂量依赖方式于NUGC4_D8诱导较IMAB362类似物强的内吞活性；HBM1029抗体与MMAF耦合的抗人IgG抗体共培养时，能以剂量依赖方式于NUGC4_D8细胞和HEK293 hCLDN18.2细胞产生较IMAB362类似物强的细胞毒杀效应。
附图说明
图1中A、B、C分别示出HBM1029以及PR002727抗体对NUGC4_D8、HEK293 hCLDN18.2、HEK293 hCLDN18.1细胞的结合亲和力。
图2中A、B示出PR003197、PR003292、PR003293、PR003340抗体对NUGC4_D8、HEK293 hCLDN18.2细胞的结合亲和力；图2C示出了PR003197、PR003292、PR003293、PR003340、PR002725抗体对HEK293 hCLDN18.1细胞的结合亲和力。
图3的A、B分别示出PR003240、PR003291、PR003289以及HBM1029抗体对NUGC4_D8、HEK293 hCLDN18.1细胞的结合亲和力。
图4的A、B分别示出PR003890、PR003891、PR003894、PR003897、PR003898抗体对NUGC4_D8、HEK293 hCLDN18.2细胞的结合亲和力；C示出PR003890、PR003891、PR003894、PR003897、PR003898、PR002725以及HBM1029抗体对HEK293 hCLDN18.1细胞的结合亲和力。
图5示出HBM1029抗体通过人PBMC表现出对NUGC4_D8、HEK293 hCLDN 18.1细胞的ADCC活性。
图6示出PR003894、PR003240、PR003340、PR003197以及HBM1029通过人PBMC表现出对NUGC4_D8细胞的ADCC活性。
图7示出HBM1029、PR003894、PR003240、PR003340、PR003197、PR003891、PR003898通过reporter细胞表现出对NUGC4_D8细胞的ADCC活性。
图8为HBM1029抗体于HEK293 hCLDN18.2细胞、HEK293 hCLDN18.1细胞及NUGC4_D8细胞引发CDC效应。
图9为HBM1029、PR003197、PR003340于HEK293 hCLDN18.2细胞引发CDC效应。
图10为HBM1029抗体于HEK293 hCLDN18.1及HEK293 hCLDN18.2引发的生长 抑制活性。
图11为HBM1029抗体于NUGC4_D8细胞的内吞活性。其中，(A)为NUGC4_D8细胞与200nM抗体混合孵育不同时间段，(B)为NUGC4_D8细胞与不同浓度抗体混合孵育1小时。
图12为HBM1029抗体与MMAF耦合的抗人IgG抗体共培养时靶细胞的存活率。
图13为HBM1029抗体对IMAB362-FITC类似物于HEK293 hCLDN18.2细胞的竞争结合亲和力。不同浓度的HBM1029抗体、20nM IMAB362-FITC类似物、HEK293 hCLDN18.2细胞混合孵育。
图14为IMAB362类似物、HBM1029药代动力学轮廓。
图15为IMAB362类似物、HBM1029的体内药效学研究。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1抗原制备、小鼠免疫及杂交瘤制备
a.免疫小鼠的表达载体制备
免疫全人源化的转基因小鼠的人CLDN18.2表达载体制备方法为：合成编码人CLDN18.2(Uniprot ID P56856-iso2)的cDNA序列，通过酶切将上述基因的编码序列克隆到pCAGGS质粒(YOUBIO，VT1076)中。
b.稳转细胞株的制备
稳定表达人CLDN18.1或CLDN18.2的HEK293(ATCC,Cat#：CRL-1573)细胞株的构建具体为：将编码人CLDN18.1(GenScript,OHu29174D)或CLDN18.2(GenScript,OHu03374D)质粒转染至HEK293细胞，生成过表达人CLDN18.1或CLDN18.2的稳定细胞株。CLDN18.1和CLDN18.2的表达经荧光激活细胞分类术(FACS)检测。具体而言，20,000个转染细胞在96孔板的各孔中铺板，并在之后加入市售兔抗人CLDN18抗体(LifeSpan Bio，Cat#：LS-C168812-400)。4℃孵育1小时后，板用PBS洗涤2遍，再加入AF-680耦联的山羊抗兔IgG二抗(Invitrogen，Cat#：A21109)。4℃孵育1小时后，板用PBS洗涤3遍，之后使用FACS仪器(IntelliCytiQue Plus BR)监测细胞荧光度。
实施例2 CLDN18.2单克隆抗体的生成和筛选
用上述制得的人CLDN18.2表达载体和上述制得的表达人CLDN18.2的HEK293细胞(HEK293 hCLDN18.2细胞)免疫全人源化的转基因小鼠(HarbourH2L2小鼠，为商购化小鼠，购于和铂生物)。将人CLDN18.2载体与金粉做成基因枪的子弹，用基因枪对小鼠腹部进行多点免疫。表达载体DNA每次免疫50μg，每次间隔2周，免疫三次后，接着用HEK293 hCLDN18.2细胞进行免疫，每只小鼠每次免疫4×10 6个细胞，每次间隔2周，细胞免疫2次后小鼠采血测效价。小鼠血清的结合亲和力通过FACS使用表达人CLDN18.2的CHO K1细胞(CHOK1 hCLDN18.2，购买自kyinno(KC-1180)或CLDN18.1的CHO K1细胞(CHOK1 hCLDN18.1，购买自kyinno(KC-1181))进行检测。根据免疫小鼠血清效价的检测结果，挑选小鼠进行杂交瘤融合，融合前3天对小鼠进行冲刺免疫，免疫原为HEK293 hCLDN18.2细胞，剂量为4×10 6个细胞。取小鼠的脾细胞和淋巴结细胞与小鼠的骨髓瘤细胞SP2/0按2:1(细胞数量比)的比例混合，混合后的细胞用电融合仪(BTX ECM2001)进行细胞融合，将融合后的细胞铺在96孔细胞培养板，在二氧化碳培养箱中37℃培养10天后进行杂交瘤的初筛。初筛通过Mirrorball使用表达人CLDN18.2的CHO K1细胞进行检测，具体为：细胞用含培养基(F12K 10％FBS)重悬，将细胞的密度调整到5×10 4细胞/ml，在384孔板的各孔内加入40μl细胞悬液，于二氧化碳培养箱37℃培养过夜。在孔中加入核染料DRAQ5进行染色，然后弃掉孔板的上清，取出杂交瘤培养板的上清50μl加入到384微孔板中，4℃孵育2个小时后，加入AF488偶联的荧光二抗(invitrogen，Cat#：A11006)4℃孵育过夜，然后将384微孔板进行Mirrorball的上机检测。挑选出阳性的杂交瘤，从96孔培养板中转到24孔培养板扩大培养，5天后对24孔培养板孔中的上清进行复筛。复筛通过FACS使用CHOK1hCLDN18.1和CHOK1hCLDN18.2细胞进行检测。细胞于300g离心5分钟，然后用FACS缓冲液(含2％FBS的PBS)重悬。将细胞的密度调整到10 6细胞/ml，在96孔板的各孔内加入50μl细胞悬液。在96孔板的各孔内加入50μl上清。于4℃孵育2小时后，板用FACS缓冲液洗涤2遍。之后，加入含APC耦合的山羊抗大鼠IgG二抗的FACS缓冲液(BiolegendCat#：405407)。在4℃孵育1小时后，板用FACS缓冲液洗涤2遍。细胞用固定液重悬，之后使用FACS仪(ACEA NovoCyte)监测细胞荧光。对特异性佳的杂交瘤采用有限稀释法进行亚克隆，在二氧化碳培养箱中37℃培养7天后进行亚克隆的初筛。亚克隆初筛通过Mirrorball使用表达人CLDN18.2的CHO K1细胞进行检测。根据检测结果和在显微镜下观察，挑出既是单克隆又是与CHOK1/CLDN18.2阳性结合的克隆，扩大到24孔细胞培养板中，在二氧化碳培养箱中37℃培养3天后再对孔中的上清进行复筛。复筛通过FACS (同上述复筛步骤)使用CHOK1hCLDN18.1和CHOK1hCLDN18.2细胞系进行检测。特异性结合的单克隆，用亚型鉴定试剂盒(invitrogen，Cat#：88-50640-88)进行亚型鉴定。挑选抗体亚型为IgG2b的细胞进行测序(测序公司为金唯智生物科技有限公司)。
本领域人员公知，在本领域中可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991))和基于结构环区域位置的Chothia定义规则(参见JMol Biol 273:927-48,1997)。在本申请中，还可以使用包含了Kabat定义和Chothia定义的Combined定义规则确定可变结构域序列中的氨基酸残基。其中Combined定义规则即是将Kabat定义和Chothia定义的范围相结合，基于此取了一个更大的范围，详见发明内容部分的表1-1。该实施例经测序后所得的胚系基因分析和PTM位点分析如下表2所示。抗原结合蛋白突变位点设计信息如下表3所示。抗原结合蛋白序列编号表信息如下表4所示。
表2抗体的胚系基因分析和PTM位点分析
克隆号 VH胚系V基因 VL胚系V基因 VH PTM VL PTM 重组抗体 重组抗体亚型
11C12-13C2 VH3-23 VK3-15 无 无 PR002725 人IgG1
13E6F4 VH3-23 VK3-11 无 无 PR002726 人IgG1
31H3E2 VH3-30 VK3-15 DG(HCDR2),DG(HCDR3) 无 PR002727 人IgG1
205A7F1D3 VH3-23 VK3-15 无 无 PR003289 人IgG1
214C4G11 VH3-23 VK3-15 无 无 PR003291 人IgG1
表3抗原结合蛋白突变位点设计
初始抗体 变体 可变区突变 重组抗体亚型 Fc突变
PR002726 PR003197 无 人IgG1 S239D,I332E
PR002726 PR003292 无 人IgG1 M252Y,S254T,T256E
PR002726 PR003293 无 人IgG1 S239D,I332E,M252Y,S254T,T256E
PR002726 PR003340 无 人IgG1 S239D,I332E,K274Q,Y300F,L309V,Y296F,A339T,V397M
PR003289 PR003890 无 人IgG1 S239D,I332E
PR003289 PR003897 无 人IgG1 S239D,I332E,K274Q,Y300F,L309V,Y296F,A339T,V397M
PR003291 PR003891 无 人IgG1 S239D,I332E
PR003291 PR003898 无 人IgG1 S239D,I332E,K274Q,Y300F,L309V,Y296F,A339T,V397M
PR002727 PR003240 VH:D54E 人IgG1 S239D,I332E
PR002727 PR003894 VH:D54E 人IgG1 S239D,I332E,K274Q,Y300F,L309V,Y296F,A339T,V397M
表4抗原结合蛋白序列编号表
抗体编号 轻链 重链 VL VH LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3
PR000400 91 75 69 62 39 46 54 5 15 25
PR002725 92 76 70 63 40 47 55 6 16 26
PR002726 93 77 71 64 41 48 56 7 17 27
PR002727 94 78 72 65 42 47 57 8 18 28
PR003197 93 79 71 64 41 48 56 7 17 27
PR003340 93 85 71 64 41 48 56 7 17 27
PR003292 93 83 71 64 41 48 56 7 17 27
PR003293 93 84 71 64 41 48 56 7 17 27
PR003289 95 81 73 67 42 47 55 8 16 29
PR003291 96 82 74 68 42 47 58 8 16 29
PR003240 94 80 72 66 42 47 57 8 19 28
PR003890 95 86 73 67 42 47 55 8 16 29
PR003891 96 87 74 68 42 47 58 8 16 29
PR003894 94 88 72 66 42 47 57 8 19 28
PR003897 95 89 73 67 42 47 55 8 16 29
PR003898 96 90 74 68 42 47 58 8 16 29
注：以上表格中提及的PR002726C即为HBM1029，本发明中两者指代相同的抗体；PR000400即为IMAB362类似物，本发明中两者指代相同的抗体。
实施例3全长CLDN18.2单克隆抗体的表达、纯化和表征
在得到编码抗体分子的轻、重链可变结构域序列以后，可以采用常规的重组DNA技术，将轻、重链可变结构域序列和相应的人的抗体轻、重链恒定结构域序列进行融合表达，得到重组抗体分子。在本实施例中，抗体重链可变结构域序列(VH)通过基因合成并克隆到编码人IgG1抗体重链恒定结构域序列的哺乳动物细胞表达质粒载体中，以编码产生IgG1抗体的全长重链。抗体轻链可变结构域序列(VL)通过基因合成并克隆到编码人抗体Igκ轻链恒定结构域序列的哺乳动物细胞表达质粒载体中，以编码产生抗体的全长轻链。在本实施例中，由于从免疫的Harbour H2L2小鼠得到的单克隆抗体分子可变结构域的序列是人源抗体序列，因而本实施例也得到全人源的抗CLDN18.2重组IgG1抗体。
将编码抗体重链的质粒(Genscript US)和编码抗体轻链的质粒(Genscript US)同时转染哺乳动物宿主细胞(如人胚肾细胞HEK293)，利用常规的重组蛋白表达和纯化技术，可以得到具有轻重链正确配对组装的纯化的重组抗体。具体来说，将HEK293细胞在FreeStyle TMF17 Expression Medium培养基(Thermo，Cat#：A1383504)中扩充培养。瞬时转染开始之前，调节细胞浓度至6～8×10 5细胞/ml，于37℃ 8％CO 2摇床中培养24小 时，细胞浓度在1.2×10 6细胞/ml。准备30ml培养的细胞。将上述编码抗体重链的质粒和编码抗体轻链的质粒以2:3(质量比)的比例混合共计30μg质粒溶解于1.5ml Opti-MEM减血清培养基(Thermo，Cat#：31985088)，并用0.22μm滤膜过滤除菌。再取1.5ml Opti-MEM溶入1mg/ml PEI(Polysciences，Cat#：23966-2)120μl，静置5分钟。把PEI缓慢加入质粒中，室温孵育10分钟，边摇晃培养瓶边缓慢滴入质粒PEI混合溶液，于37℃ 8％CO 2摇床中培养5天。5天后观测细胞活率。收集培养物，以3300g转速离心10分钟后取上清；然后将上清高速离心去除杂质。用PBS(pH7.4)平衡含有MabSelect TM(GE Healthcare Life Science，Cat#：71-5020-91 AE)的重力柱(Bio-Rad，Cat#：7311550)，2-5倍柱体积冲洗。将上清样品过柱；用5-10倍柱体积的PBS冲洗柱子，再用pH3.5的0.1M甘氨酸洗脱目的蛋白，后用pH 8.0的Tris-HCl调节至中性，最后用超滤管(Millipore，Cat#：UFC901024)浓缩换液至PBS缓冲液，得到纯化的抗体溶液。最后用NanoDrop(Thermo Scientific TMNanoDrop TMOne)测定浓度，分装、存储备用。
取上述纯化的样品适量分别上样至分析型SEC柱TSKgel G3000SWxl(HPLC仪器型号:安捷伦1260 Infinity II)，检测样品的纯度，保证均一样品的纯度在95％以上。该方法流动相为1×PBS，pH7.4(生工，Cat#：E607016)，室温，流速1.0ml/min，样品浓度1mg/ml，进样体积20μl，检测波长280nm。采集后用ChemStation软件对色谱图进行积分并计算相关数据。取上述纯化的样品适量分别上样至分析型HIC柱TSKge1Buty1-NPR 4.6*35(HPLC仪器型号:安捷伦1260 Infinity II)，检测样品的纯度及疏水性。该方法由16分钟内从100％流动相A(20mM PB，1.8M(NH4) 2SO 4,pH6.0)至100％流动相B(20mM PB，pH6.0)的线性梯度组成。流速设定为0.7ml/min，样品浓度1mg/ml，进样体积20μl，检测波长280nm。采集后用ChemStation软件对色谱图进行积分并计算相关数据。差示扫描荧光法(Differential Scanning Fluorimetry,DSF)是一种常用的高通量的用来测定蛋白质热稳定性的方法。它使用实时荧光定量PCR仪器通过监测与去折叠的蛋白分子结合的染料的荧光强度的变化，来反映蛋白质的变性的过程，从而反映出蛋白分子的热稳定性。本实施例利用DSF方法来测定蛋白分子热变性温度(Tm)。10μg蛋白加入96-孔PCR板(Thermo，Cat#：AB-0700/W)，接着加入2μl 100×稀释的染料SYPROTM(Invitrogen，2008138)，然后加入缓冲液使得终体积为40μl每孔。将PCR板密封，放置于实时荧光定量PCR仪器(Bio-Rad CFX96 PCR System)，先于25℃孵育5min，然后以0.2℃/0.2min的梯度逐渐从25℃升温至95℃，在测试结束时将温度降至25℃。使用FRET扫描模式并使用Bio-Rad CFX Maestro软件进行数据分析并计算出样品的Tm。所得结果如下表5所示。
表5
Figure PCTCN2020118650-appb-000001
实施例4 CLDN18.2单克隆抗体的结合亲和力
抗体的结合亲和力通过FACS使用表达人CLDN18.2或CLDN18.1的HEK293细胞及内生性表达人CLDN18.2的NUGC4_D8细胞(NUGC细胞系购买自JCRB(Cat#：JCRB0834)，用有限稀释法筛选得到NUGC4_D8亚克隆细胞)进行检测，具体是：细胞于300g离心5分钟，然后用FACS缓冲液(含2％FBS的PBS)重悬。将细胞的密度调整到10 6细胞/ml，在96孔板的各孔内加入50μl细胞悬液。抗体以FACS缓冲液稀释成不同浓度，在96孔板的各孔内加入50μl抗体稀释液。于4℃孵育2小时后，板用FACS缓冲液洗涤2遍。之后，加入含APC耦合的山羊抗人IgG二抗的FACS缓冲液(终浓度为1.5μg/ml，JacksonCat#：109-605-098)。在4℃孵育1小时后，板用FACS缓冲液洗涤2遍。细胞用固定液重悬，之后使用FACS仪(ACEA NovoCyte)监测细胞荧光。IMAB362类似物(IMAB362类似物为自制所得，重链氨基酸序列如SEQ ID NO:75所示，轻链氨基酸序列如SEQ ID NO:91所示(由Genscript生物公司合成)，其与IMAB362的可变区完全相同，恒定区上仅有个别氨基酸不同，两者活性类似)作为阳性对照，human Iso IgG1(CrownBio，Cat#：C0001-4)抗体作为阴性对照。图1示出HBM1029抗体对内生性表达CLDN18.2的NUGC4_D8细胞及过表达人CLDN18.2的HEK293细胞(HEK293 hCLDN 18.2)或过表达人CLDN18.1的HEK293细胞(HEK293 hCLDN18.1)的结合亲和力。PR002727抗体能以剂量依赖方式结合于NUGC4_D8细胞。HBM1029抗体能以剂量依赖方式结合于HEK293 hCLDN18.2及NUGC4_D8细胞。HBM1029抗体对HEK293 hCLDN18.2细胞的结合亲和力与IMAB362类似物相当；HBM1029抗体与IMAB362类似物相比对内生性表达CLDN18.2的NUGC4_D8细胞呈现更高的结合亲和力。HBM1029的EC 50值显示在表7中，HBM1029对内生性表达CLDN18.2的NUGC4_D8细胞结合亲和力的EC 50值较IMAB362类似物低。HBM1029与HEK293 hCLDN18.1细胞结合亲低。同时可推知HBM1029结合于人CLDN18.2蛋白的ECL1(胞外区1，Extracellular loop 1)而非ECL2。图2、图3和图4示出CLDN18.2抗体对内生性表达CLDN18.2的NUGC4_D8细胞或过表达人CLDN18.2的HEK293细胞(HEK293 hCLDN 18.2)或过表达人CLDN18.1的HEK293细胞(HEK293 hCLDN18.1)的结合亲和力。结果显示：HBM1029、PR003197、PR003340、PR003292、PR003293、PR003240、PR003291、PR003289、PR003890、PR003891、 PR003894、PR003897、PR003898抗体与IMAB362类似物相比对内生性表达CLDN18.2的NUGC4_D8细胞呈现更高的结合亲和力，与HEK293 hCLDN18.1细胞结合亲和力低，而PR002725与HEK293 hCLDN18.1亲和力高。
表6 IMAB362类似物的氨基酸序列(由Genscript生物公司合成)
重链氨基酸序列 SEQ ID NO:75
轻链氨基酸序列 SEQ ID NO:91
表7 HBM1029抗体的结合亲和力EC 50
Figure PCTCN2020118650-appb-000002
实施例5 CLDN18.2抗体的ADCC活性
使用CytoTox
Figure PCTCN2020118650-appb-000003
非放射性细胞毒性分析试剂盒(Promega，Cat#：G1780)，检测CLDN18.2抗体针对内生性表达人CLDN18.2的NUGC4_D8细胞及HEK293 hCLDN 18.1引发ADCC效应的活性。人PBMC(Miaotong)以300g离心5分钟，并在培养基(RPMI1640+10％FBS)中培养过夜。靶细胞及人PBMC于300g离心5分钟，然后用培养基(RPMI1640+2％FBS)重悬。将靶细胞的密度调整到2×10 5细胞/ml，PBMC的细胞密度调整至少为6×10 6/ml，两种细胞各加50μl到96孔板的孔中(效靶比至少是30∶1)。将待测抗体以培养基(RPMI1640+2％FBS)稀释成不同浓度加入各孔。样本在37℃孵育至少4小时，然后于靶细胞最大LDH释放对照孔和体积校正对照孔(Volume correction control)加入10乘Triton-X 100裂解液(RPMI1640+2％FBS+10％Triton-X 100)，混合均匀在37℃孵育0.5小时。96孔板于300g离心5分钟，取出50μl上清液，然后以50μl/孔的浓度加入LDH显色液。混合物避光常温放置20分钟后，板在MD StakMax上进行读数(OD 490)。IMAB362类似物作为阳性对照，human Iso IgG1(CrownBio，Cat#：C0001-4)抗体作为阴性对照。结果先计算校正读数，将实验孔、靶细胞自发性释放LDH对照孔、效应细胞自发性释放LDH对照孔的读数减去培养基背景对照孔读数，其次将靶细胞最大LDH释放对照孔读数减去体积校正对照孔读数。ADCC活性(％)＝(实验孔校正读数-效应细胞自发性释放LDH对照孔校正读数-靶细胞自发性释放LDH对照孔校正读数)/(靶细胞最大 LDH释放对照孔校正读数-靶细胞自发性释放LDH对照孔校正读数)×100。图5示出HBM1029抗体对内生性表达CLDN18.2的NUGC4_D8细胞及HEK293 hCLDN18.1的ADCC活性。HBM1029抗体能以剂量依赖方式特异性地于NUGC4_D8诱导较IMAB362类似物强ADCC效应，而在过表达CLDN18.1的HEK-293细胞上没有观察到细胞毒性效果。HBM1029的EC 50值显示在表8中，HBM1029于NUGC4_D8诱导ADCC的EC 50值较IMAB362类似物低。
表8 HBM1029的ADCC活性
Figure PCTCN2020118650-appb-000004
图6示出HBM1029、PR003197、PR003340、PR003240、PR003894对内生性表达CLDN18.2的NUGC4_D8细胞的ADCC活性，HBM1029、PR003197、PR003340、PR003240、PR003894能以剂量依赖方式特异性地于NUGC4_D8诱导较IMAB362类似物强ADCC效应。
使用Jurkat FcγRIIIa-V158/NFAT-Luc细胞，检测CLDN18.2抗体针对NUGC4_D8及HEK293 hCLDN18.1引发ADCC效应的活性。NUGC4_D8及HEK293 hCLDN18.1于300g离心5分钟，然后用RPMI1640+4％FBS血清培养基重悬。将细胞的密度调整到6×10 5细胞/ml，在96孔板的各孔内加入50μl细胞悬液，于37℃孵育过夜。Jurkat FcγRIIIa-V158/NFAT-Luc细胞于400g离心4分钟，然后用RPMI1640+4％FBS血清培养基重悬。将细胞的密度调整到3×10 6细胞/ml，在96孔板的各孔内加入50μl细胞悬液。抗体以RPMI1640+4％FBS培养基稀释成不同浓度，在96孔板的各孔内加入50μl抗体稀释液。细胞与抗体于37℃孵育5小时。将96孔板于常温静置30分钟，添加60μl/孔之常温One-Glo显色液(Promega)。之后样品避光常温孵育10分钟。用PE Enspire读板。IMAB362类似物作为阳性对照，human Iso IgG1(CrownBio，Cat#：C0001-4)抗体作为阴性对照。图7示出CLDN18.2抗体对内生性表达CLDN18.2的NUGC4_D8细胞的ADCC活性，HBM1029、PR003197、PR003340、PR003240、PR003894、PR003891、PR003898能以剂量依赖方式特异性地于NUGC4_D8诱导较IMAB362类似物强ADCC效应。
实施例6 CLDN18.2抗体的CDC活性
使用CellTiter-Glo发光法细胞活力检测试剂盒(Promega，Cat#：G7573)，检测CLDN18.2抗体针对HEK293 hCLDN18.1，HEK293 hCLDN18.2及NUGC4_D8细胞引发CDC效应的能力。靶细胞HEK293 hCLDN18.1及HEK293 hCLDN18.2于300g离心5分 钟，然后用DMEM无血清培养基重悬。靶细胞NUGC4_D8于300g离心5分钟，然后用RPMI1640无血清培养基重悬。将靶细胞的密度调整到4×10 5细胞/ml，在96孔板的各孔内加入25μl细胞悬液。抗体以无血清培养基稀释成不同浓度，在96孔板的各孔内加入25μl抗体稀释液。加入50μl的正常人血清(GemCell，Cat#：100-512)，终浓度为50％，获得的混合物于37℃孵育24小时。将96孔板于常温静置30分钟，添加100μl/孔之常温CellTiter-Glo显色液。之后样品避光常温孵育10分钟。用PE Enspire读板。CDC活性(％)＝[1-(luminescent sample)/(luminescent mock control)]×100。IMAB362类似物作为阳性对照，human Iso IgG1(CrownBio，Cat#：C0001-4)抗体作为阴性对照。图8示出HBM1029抗体对内生性表达CLDN18.2的NUGC4_D8细胞、过表达人CLDN18.1的HEK293细胞及过表达人CLDN18.2的HEK293细胞的CDC活性。HBM1029抗体能以剂量依赖方式于HEK293 hCLDN18.2诱导较IMAB362类似物强CDC效应，而在NUGC4_D8细胞和过表达人CLDN18.1的HEK293细胞上没有观察到CDC活性。HBM1029的EC 50值显示在表9中，HBM1029于HEK293 hCLDN18.2诱导CDC的EC 50值较IMAB362类似物低。
表9 HBM1029的CDC活性
Figure PCTCN2020118650-appb-000005
图9示出PR003197、PR003340对过表达人CLDN18.2的HEK293细胞的CDC活性。PR003197、PR003340抗体能以剂量依赖方式于HEK293 hCLDN18.2诱导较IMAB362类似物强的CDC效应。
实施例7 CLDN18.2抗体的生长抑制活性
使用CellTiter-Glo发光法细胞活力检测试剂盒(Promega，Cat#：G7573)，检测CLDN18.2抗体针对HEK293 hCLDN18.1及HEK293 hCLDN18.2引发生长抑制的能力。细胞HEK293 hCLDN18.1及HEK293 hCLDN18.2于300g离心5分钟，然后用DMEM+0.5％FBS血清培养基重悬。将细胞的密度调整到1.2×10 5细胞/ml，在96孔板的各孔内加入50μl细胞悬液，于37℃孵育过夜。抗体以培养基稀释成不同浓度，在96孔板的各孔内加入50μl抗体稀释液。HEK293 hCLDN18.1及HEK293 hCLDN18.2细胞与抗体于37℃孵育3天。将96孔板于常温静置30分钟，添加100μl/孔之常温CellTiter-Glo显色液。之后样品避光常温孵育10分钟。用PE Enspire读板。生长抑制活性(％)＝[1-(luminescent sample)/(luminescent mock control)]×100。IMAB362类似物作为阳性对照， human Iso IgG1(CrownBio，Cat#：C0001-4)抗体作为阴性对照。图10示出HBM1029抗体对HEK293 hCLDN18.1及HEK293 hCLDN18.2引发的生长抑制活性。HBM1029抗体能以剂量依赖方式于HEK293 hCLDN 18.2诱导较IMAB362类似物强生长抑制效应。HBM1029的EC 50值显示在表10中。
表10 HBM1029的生长抑制活性。
Figure PCTCN2020118650-appb-000006
实施例8 CLDN18.2抗体的内吞(internalization)活性(FACS-based assay)
抗体的内吞活性通过FACS使用NUGC4_D8细胞进行检测。以胰酶消化细胞，并用FACS缓冲液(含2％FBS的PBS)洗涤1次。细胞于300g离心5分钟，然后用FACS缓冲液重悬。将细胞的密度调整到4×10 6细胞/ml，并置于冰上预冷30分钟。抗体以FACS缓冲液稀释成不同浓度，并置于冰上预冷30分钟。在预冷的深孔板的孔内加入700μl细胞悬浮液，及700μl抗体稀释液。于4℃孵育2小时后，板用预冷的FACS缓冲液洗涤3遍。用250μl预冷的FACS缓冲液重悬细胞，在37℃预热的深孔板的孔内加入100μl细胞悬浮液，及1.1ml 37℃预热的FACS缓冲液，在4℃预冷的深孔板的孔内加入100μl细胞悬浮液，及1.1ml 4℃预冷的FACS缓冲液。获得的混合物分别在0，30，60，120和240分钟取50μL细胞悬浮液(10 5/孔)置于提前预冷的深孔板(含1.2mL FACS缓冲液)中。细胞于300g离心5分钟，之后加入含AF647耦合的山羊抗人IgG二抗的FACS预冷缓冲液(终浓度为1.5μg/ml，Jackson，Cat#：109-605-088)。在4℃孵育1小时后，板用FACS预冷缓冲液洗涤2遍。细胞用固定液重悬，之后使用FACS仪(BD Canto II)捡测细胞荧光。内吞活性(％)＝(1-MFI 37℃/MFI 4℃)×100。IMAB362类似物作为阳性对照，human Iso IgG1(CrownBio，Cat#：C0001-4)抗体作为阴性对照。图11示出孵育不同时间的HBM1029抗体对NUGC4_D8细胞的内吞活性。HBM1029抗体孵育30分钟后诱导约50％内吞活性。图11还示出HBM1029抗体能以剂量依赖方式于NUGC4_D8诱导较IMAB362类似物强的内吞活性。HBM1029的EC 50值显示在表11中，HBM1029于NUGC4_D8诱导内吞的EC 50值较IMAB362类似物低。
表11 HBM1029的内吞活性。
Figure PCTCN2020118650-appb-000007
实施例9 CLDN18.2抗体的内吞活性(cytotoxicity-based method)
使用CellTiter-Glo发光法细胞活力检测试剂盒(Promega，Cat#：G7573)，针对HEK293hCLDN18.1，HEK293 hCLDN18.2及NUGC4_D8细胞，检测HBM1029抗体与MMAF耦合的抗人IgG抗体(Moradec，Cat#：AH-102-AF)共培养引发细胞毒杀的能力。细胞HEK293hCLDN18.1及HEK293 hCLDN18.2于300g离心5分钟，然后用DMEM+10％FBS血清培养基重悬，将细胞的密度调整到4×10 4细胞/ml。细胞NUGC4_D8于300g离心5分钟，然后用RPMI1640+10％FBS血清培养基重悬，将细胞的密度调整到2×10 4细胞/ml，在96孔板的各孔内加入50μl细胞悬液，于37℃孵育过夜。HBM1029抗体以培养基稀释成不同浓度，在96孔板的各孔内加入25μl抗体稀释液。MMAF耦合的抗人IgG抗体以培养基稀释，在96孔板的各孔内加入25μl抗体稀释液，终浓度为6.6nM。细胞与抗体于37℃孵育3天。将96孔板于常温静置30分钟，添加100μl/孔之常温CellTiter-Glo显色液。之后样品避光常温孵育10分钟。用PE Enspire读板。细胞存活率(％)＝[(luminescent sample)/(luminescent mock control)]×100。IMAB362类似物作为阳性对照，human Iso IgG1(CrownBio，Cat#：C0001-4)抗体作为阴性对照。图12示出HBM1029抗体与MMAF耦合的抗人IgG抗体共培养时靶细胞的存活率。HBM1029抗体与MMAF耦合的抗人IgG抗体共培养时，能以剂量依赖方式于NUGC4_D8细胞和HEK293hCLDN18.2细胞产生较IMAB362类似物强的细胞毒杀效应，但于HEK293 hCLDN18.1细胞则未产生细胞毒杀效应。
实施例10 HBM1029抗体的竞争结合活性
抗体的竞争结合亲和力通过FACS使用表达人CLDN18.2的HEK293细胞进行检测。IMAB362类似物使用FITC荧光耦联试剂盒(Abcam，Cat#：ab188285)耦合为IMAB362-FITC类似物。细胞于300g离心5分钟，然后用FACS缓冲液(含2％FBS的PBS)重悬。将细胞的密度调整到10 6细胞/ml，在96孔板的各孔内加入50μl细胞悬液。FITC耦联抗体以FACS缓冲液稀释，在96孔板的各孔内加入50μl耦联抗体稀释液。欲进行竞争结合的抗体以FACS缓冲液稀释成不同浓度，在96孔板的各孔内加入50μl抗体稀释液。于4℃孵育2小时后，板用FACS缓冲液洗涤2遍。细胞用固定液重悬，之后使用FACS仪(ACEA NovoCyte)监测细胞荧光。human Iso IgG1(CrownBio，Cat#：C0001-4)抗体作为阴性对照。图13示出HBM1029抗体对IMAB362-FITC类似物于HEK293 hCLDN 18.2细胞的竞争结合亲和力。HBM1029抗体能以剂量依赖方式竞争结合于HEK293 hCLDN18.2细胞，推测HBM1029抗体与IMAB362类似物结合表位相似。HBM1029可与IMAB362类似物竞争，抑制其结合于CLDN18.2表达的细胞；而已知IMAB362类似物只结合CLDN18.2，不结合CLDN18.1。故推测HBM1029结合于人CLDN18.2蛋白的ECL1(胞外区1，Extracellular loop 1)而非ECL2。
实施例11 CLDN18.2抗体的药代动力学研究
CLDN18.2抗体的药代动力学，方法如下，选取体重18～22克的雌性BALB/c nude小鼠6只，按5mg/kg的剂量通过静脉注射给与抗体药物；一组3只于给药前以及给药后15分钟、24小时(1天)、第4天、和第10天采集全血，另一组3只于只于给药前以及给药后5小时、第2天、第7天、和第14天采集全血。将全血静置30分钟使其凝固，随后在4℃下以2,000rpm离心5分钟并将分离的血清样品在-80℃下冻存直至分析。本实施例采用ELISA方法来定量测定小鼠血清中的药物浓度。ELISA方法，通过包被于96孔板的山羊抗人Fc多克隆抗体来捕获小鼠血清中的含有人Fc的抗体，然后加入HRP标记的山羊抗人Fc第二抗体来检测。使用Phoenix WinNonlin软件8.2版，选用非房室模型(NCA)对血药浓度数据进行分析以评价其药代动力学参数。
图14和表12显示的是IMAB362类似物、HBM1029的药代动力学参数。结果表明，IMAB362类似物、HBM1029在小鼠体内的半衰期分别为248、282小时。
表12 IMAB362类似物、HBM1029药代动力学参数
Figure PCTCN2020118650-appb-000008
实施例11 CLDN18.2抗体的体内药效学研究
CLDN18.2抗体的体内药效学研究，方法如下，细胞接种当天每只NCG小鼠皮下接种5×10 6NUGC4_D8肿瘤细胞，肿瘤细胞首先重悬在PBS与Matrigel(1:1)的混合液中(0.1ml)，然后与PBMC(重悬在0.05mL的PBS)混合，皮下接种。当各组小鼠平均瘤体积在90mm 3时进行分组给药，18只小鼠被分为3组，分组后开始给药，给药周期为一周两次，共进行了6次给药，给药方式为尾静脉注射。开始给药后，每周称量体重及 瘤体积两次，瘤体积计算方式为:肿瘤体积(mm 3)＝0.5×肿瘤长径×肿瘤短径2。给药后第21天结束实验观察，随后所有小鼠进行安乐处理。数据分析采用t-test。图15显示的是IMAB362类似物、HBM1029的体内药效学研究结果。给药后第21天，对照组小鼠的平均肿瘤体积为1526mm 3。测试药IMAB362类似物(50mg/kg)治疗组在给药后第21天平均肿瘤体积为728mm 3，相较溶媒对照组有显著性差异(p值为0.0052)，肿瘤抑制率TGI(％)为52.29％。测试药HBM1029(50mg/kg)治疗组在给药后第21天平均肿瘤体积为618mm 3，相较溶媒对照组有显著的肿瘤抑制作用(p值为0.0009)，肿瘤抑制率TGI(％)为59.47％。整个治疗给药期间，动物均表现出良好的药物耐受，未出现严重的体重下降及动物死亡现象。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (24)

  1. 一种靶向CLDN18.2的抗体或其抗原结合片段，其特征在于，其包含轻链可变区和/或重链可变区，所述重链可变区包含HCDR1、HCDR2和HCDR3，所述的轻链可变区包含LCDR1、LCDR2和LCDR3；其中：所述HCDR1包含如SEQ ID NO:8所示的氨基酸序列或其变体1，所述HCDR2包含选自SEQ ID NO:16及其变体2、SEQ ID NO:18及其变体3中的氨基酸序列，所述HCDR3包含SEQ ID NO:26～29中的任一个所示的氨基酸序列，所述LCDR1包含SEQ ID NO:42所示的序列或其变体4，所述LCDR2包含SEQ ID NO:47所示的序列或其变体5，所述LCDR3包含SEQ ID NO:55所示的序列或其变体6；所述变体为在原始序列基础上进行1个、2个或3个氨基酸的取代、缺失或添加，包含上述变体的抗体或抗原结合片段保持与CLDN18.2的结合能力。
  2. 如权利要求1所述的抗体或其抗原结合片段，其特征在于，
    所述变体1的突变至少发生于SEQ ID NO:8所示氨基酸序列的第6位和/或第7位；
    所述变体2的突变至少发生于SEQ ID NO:16所示氨基酸序列的第5位；
    所述变体3的突变至少发生于SEQ ID NO:18所示氨基酸序列的第3位；
    所述变体4的突变至少发生于SEQ ID NO:42所示氨基酸序列的第8位和/或第9位；
    所述变体5的突变至少发生于SEQ ID NO:47所示氨基酸序列的第1位和/或第4位；
    所述变体6的突变至少发生于SEQ ID NO:55所示氨基酸序列的第3～5位中的一位或者多位；较佳地：
    所述变体1含有突变S6G和/或Y7F，所述变体2含有突变G5R，所述变体3含有突变D3E，所述变体4含有突变S8R和/或N9Y，所述变体5含有突变G1D和/或T4N，所述变体6含有突变Y3R/N、N4S以及N5Y中的一个或多个；更佳地：
    所述变体1的氨基酸序列如SEQ ID NO:6或7所示；
    所述变体2的氨基酸序列如SEQ ID NO:17所示；
    所述变体3的氨基酸序列如SEQ ID NO:19所示；
    所述变体4的氨基酸序列如SEQ ID NO:40或41所示；
    所述变体5的氨基酸序列如SEQ ID NO:48所示；
    所述变体6的氨基酸序列如SEQ ID NO:56～58任一所示。
  3. 如权利要求2所述的靶向CLDN18.2的抗体或其抗原结合片段，其特征在于，
    所述HCDR1的氨基酸序列如SEQ ID NO:7所示、所述HCDR2的氨基酸序列如SEQ ID NO:17所示且所述HCDR3的氨基酸序列如SEQ ID NO:27所示；
    所述HCDR1的氨基酸序列如SEQ ID NO:8所示、所述HCDR2的氨基酸序列如SEQ ID NO:18所示且所述HCDR3的氨基酸序列如SEQ ID NO:28所示；
    所述HCDR1的氨基酸序列如SEQ ID NO:8所示、所述HCDR2的氨基酸序列如SEQ ID NO:16所示且所述HCDR3的氨基酸序列如SEQ ID NO:29所示；
    或者，所述HCDR1的氨基酸序列如SEQ ID NO:8所示、所述HCDR2的氨基酸序列如SEQ ID NO:19所示且所述HCDR3的氨基酸序列如SEQ ID NO:28所示；
    较佳地，所述重链可变区框架区为人抗体的重链可变区框架区，编码所述重链可变区框架区的基因优选来源于胚系V基因IGHV3-23；
    更佳地，所述重链可变区框架区中，HFR1包含如SEQ ID NO:2～4任一所示的氨基酸序列或其变体，HFR2包含如SEQ ID NO:10～14任一所示的氨基酸序列或其变体，HFR3包含如SEQ ID NO:21～24任一所示的氨基酸序列或其变体，HFR4包含如SEQ ID NO:31～33任一所示的氨基酸序列或其变体。
  4. 如权利要求2或3所述的靶向CLDN18.2的抗体或其抗原结合片段，其特征在于，
    所述LCDR1的氨基酸序列如SEQ ID NO:41所示、所述LCDR2的氨基酸序列如SEQ ID NO:48所示且所述LCDR3的氨基酸序列如SEQ ID NO:56所示；
    所述LCDR1的氨基酸序列如SEQ ID NO:42所示、所述LCDR2的氨基酸序列如SEQ ID NO:47所示且所述LCDR3的氨基酸序列如SEQ ID NO:57所示；
    所述LCDR1的氨基酸序列如SEQ ID NO:42所示、所述LCDR2的氨基酸序列如SEQ ID NO:47所示且所述LCDR3的氨基酸序列如SEQ ID NO:55所示；
    或者，所述LCDR1的氨基酸序列如SEQ ID NO:42所示、所述LCDR2的氨基酸序列如SEQ ID NO:47所示且所述LCDR3的氨基酸序列如SEQ ID NO:58所示；
    较佳地，所述轻链可变区框架区为人抗体的轻链可变区框架区，编码所述轻链可变区框架区的基因优选来源于胚系V基因IGKV3-11或者IGKV3-15；
    更佳地，所述轻链可变区框架区中，LFR1包含如SEQ ID NO:35～38任一所示的氨基酸序列或其变体，LFR2包含如SEQ ID NO:44或45所示的氨基酸序列或其变体，LFR3包含如SEQ ID NO:50～53任一所示的氨基酸序列或其变体，LFR4包含如SEQ ID NO:60或61所示的氨基酸序列或其变体。
  5. 如权利要求4所述的靶向CLDN18.2的抗体或其抗原结合片段，其特征在于，
    所述HCDR1的氨基酸序列如SEQ ID NO:7所示、所述HCDR2的氨基酸序列如SEQ ID NO:17所示、所述HCDR3的氨基酸序列如SEQ ID NO:27所示、所述LCDR1的氨基酸序列如SEQ ID NO:41所示、所述LCDR2的氨基酸序列如SEQ ID NO:48所示且所 述LCDR3的氨基酸序列如SEQ ID NO:56所示；
    所述HCDR1的氨基酸序列如SEQ ID NO:8所示、所述HCDR2的氨基酸序列如SEQ ID NO:18所示、所述HCDR3的氨基酸序列如SEQ ID NO:28所示、所述LCDR1的氨基酸序列如SEQ ID NO:42所示、所述LCDR2的氨基酸序列如SEQ ID NO:47所示且所述LCDR3的氨基酸序列如SEQ ID NO:57所示；
    所述HCDR1的氨基酸序列如SEQ ID NO:8所示、所述HCDR2的氨基酸序列如SEQ ID NO:16所示、所述HCDR3的氨基酸序列如SEQ ID NO:29所示、所述LCDR1的氨基酸序列如SEQ ID NO:42所示、所述LCDR2的氨基酸序列如SEQ ID NO:47所示且所述LCDR3的氨基酸序列如SEQ ID NO:55所示；
    所述HCDR1的氨基酸序列如SEQ ID NO:8所示、所述HCDR2的氨基酸序列如SEQ ID NO:16所示、所述HCDR3的氨基酸序列如SEQ ID NO:29所示、所述LCDR1的氨基酸序列如SEQ ID NO:42所示、所述LCDR2的氨基酸序列如SEQ ID NO:47所示且所述LCDR3的氨基酸序列如SEQ ID NO:58所示；
    或者，所述HCDR1的氨基酸序列如SEQ ID NO:8所示、所述HCDR2的氨基酸序列如SEQ ID NO:19所示、所述HCDR3的氨基酸序列如SEQ ID NO:28所示、所述LCDR1的氨基酸序列如SEQ ID NO:42所示、所述LCDR2的氨基酸序列如SEQ ID NO:47所示且所述LCDR3的氨基酸序列如SEQ ID NO:57所示。
  6. 如权利要求5所述的靶向CLDN18.2的抗体或其抗原结合片段，其特征在于，所述重链可变区包含如SEQ ID NO:64所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:71所示的氨基酸序列或其变体；
    所述重链可变区包含如SEQ ID NO:67所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:73所示的氨基酸序列或其变体；
    所述重链可变区包含如SEQ ID NO:65所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:72所示的氨基酸序列或其变体；
    所述重链可变区包含如SEQ ID NO:68所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:74所示的氨基酸序列或其变体；
    或者，所述重链可变区包含如SEQ ID NO:66所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:72所示的氨基酸序列或其变体；
    其中，所述变体至少保留突变前序列的功能，且所述变体与突变前序列的同一性至少为85％，优选至少90％，更优选至少95％，进一步更优选至少99％。
  7. 如权利要求1～6任一项所述的靶向CLDN18.2的抗体或其抗原结合片段，其特征 在于，所述靶向CLDN18.2的抗体还包含抗体重链恒定区和抗体轻链恒定区；较佳地，所述重链恒定区选自hIgG1、hIgG2、hIgG3或hIgG4或其变体，所述轻链恒定区选自人源抗体的κ链或者λ链或其变体；更佳地，所述重链恒定区为hIgG1，且所述轻链恒定区为人源抗体的κ链。
  8. 如权利要求1～7任一项所述的靶向CLDN18.2的抗体或其抗原结合片段，其特征在于，所述靶向CLDN18.2的抗体为全长抗体、Fab、Fab’、F(ab’) 2、Fv、scFv、双特异性抗体、多特异性抗体、重链抗体或单域抗体，或由上述抗体制得的单克隆抗体或多克隆抗体。
  9. 如权利要求8所述的靶向CLDN18.2的抗体或其抗原结合片段，其特征在于，其是全长抗体，所述全长抗体包括重链和轻链；
    所述重链包含如SEQ ID NO:77～90中任一所示的氨基酸序列，所述轻链包含如SEQ ID NO:93～96任一所示的氨基酸序列；较佳地：
    所述重链包含如SEQ ID NO:77所示的氨基酸序列，所述轻链包含如SEQ ID NO:93所示的氨基酸序列；
    所述重链包含如SEQ ID NO:78所示的氨基酸序列，所述轻链包含如SEQ ID NO:94所示的氨基酸序列；
    所述重链包含如SEQ ID NO:79所示的氨基酸序列，所述轻链包含如SEQ ID NO:93所示的氨基酸序列；
    所述重链包含如SEQ ID NO:85所示的氨基酸序列，所述轻链包含如SEQ ID NO:93所示的氨基酸序列；
    所述重链包含如SEQ ID NO:83所示的氨基酸序列，所述轻链包含如SEQ ID NO:93所示的氨基酸序列；
    所述重链包含如SEQ ID NO:84所示的氨基酸序列，所述轻链包含如SEQ ID NO:93所示的氨基酸序列；
    所述重链包含如SEQ ID NO:81所示的氨基酸序列，所述轻链包含如SEQ ID NO:95所示的氨基酸序列；
    所述重链包含如SEQ ID NO:82所示的氨基酸序列，所述轻链包含如SEQ ID NO:96所示的氨基酸序列；
    所述重链包含如SEQ ID NO:80所示的氨基酸序列，所述轻链包含如SEQ ID NO:94所示的氨基酸序列；
    所述重链包含如SEQ ID NO:86所示的氨基酸序列，所述轻链包含如SEQ ID NO:95 所示的氨基酸序列；
    所述重链包含如SEQ ID NO:87所示的氨基酸序列，所述轻链包含如SEQ ID NO:96所示的氨基酸序列；
    所述重链包含如SEQ ID NO:88所示的氨基酸序列，所述轻链包含如SEQ ID NO:94所示的氨基酸序列；
    所述重链包含如SEQ ID NO:89所示的氨基酸序列，所述轻链包含如SEQ ID NO:95所示的氨基酸序列；
    或者，所述重链包含如SEQ ID NO:90所示的氨基酸序列，所述轻链包含如SEQ ID NO:96所示的氨基酸序列。
  10. 一种分离的核酸，其编码如权利要求1～9任一项所述的靶向CLDN18.2的抗体或其抗原结合片段。
  11. 一种重组表达载体，其包含如权利要求10所述的分离的核酸；优选地，所述重组表达载体为质粒、粘粒、噬菌体或病毒载体，所述病毒载体优选逆转录病毒载体、慢病毒载体、腺病毒载体或腺相关病毒载体。
  12. 一种转化体，其在宿主细胞中包含如权利要求11所述的重组表达载体；优选地，所述宿主细胞为E.coli TG1、BL21细胞，或者CHO-K1细胞。
  13. 一种嵌合抗原受体，其包含如权利要求1～9任一项所述的靶向CLDN18.2的抗体或其抗原结合片段。
  14. 一种基因修饰的细胞，其特征在于，其包含如权利要求1～9任一项所述的靶向CLDN18.2的抗体或其抗原结合片段；优选地，所述基因修饰的细胞为真核细胞，优选分离的人细胞；更优选免疫细胞如T细胞，或NK细胞。
  15. 一种靶向CLDN18.2的抗体或其抗原结合片段的制备方法，其包含培养如权利要求12所述的转化体，从培养物中获得靶向CLDN18.2的抗体或其抗原结合片段。
  16. 一种抗体药物偶联物，其包含细胞毒性剂，以及如权利要求1～9任一项所述的靶向CLDN18.2的抗体或其抗原结合片段；优选地，所述细胞毒性剂为MMAF或MMAE。
  17. 一种药物组合物，其包含如权利要求1～9任一项所述的靶向CLDN18.2的抗体或其抗原结合片段和/或如权利要求16所述的抗体药物偶联物，以及药学上可接受的载体；
    较佳地，所述药物组合物还含有由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
  18. 如权利要求1～9任一项所述的靶向CLDN18.2的抗体或其抗原结合片段、权利要求16所述的抗体药物偶联物和/或权利要求17所述的药物组合物在制备诊断、预防和/或治疗肿瘤的药物中的应用；优选地，所述肿瘤为CLDN18.2阳性肿瘤；更优选地，所述肿瘤为胃癌、食管癌、肺癌、卵巢癌、黑素瘤、肾癌、乳腺癌、结肠直肠癌、肝癌、胰腺癌、膀胱癌、头颈癌、支气管癌、神经胶质瘤和/或白血病。
  19. 试剂盒，其包括如权利要求1～9任一项所述的靶向CLDN18.2的抗体或其抗原结合片段、如权利要求13所述的嵌合抗原受体、如权利要求14所述的基因修饰的细胞、或如权利要求16中所述的抗体药物偶联物或如权利要求17所述的药物组合物；
    优选地，所述试剂盒还包括(i)施用抗体或其抗原结合片段或抗体药物偶联物或药物组合物的装置；和/或(ii)使用说明。
  20. 一种套装药盒，其包含药盒A和药盒B，其中：
    所述药盒A含有如权利要求1～9任一项所述的靶向CLDN18.2的抗体或其抗原结合片段、如权利要求13所述的嵌合抗原受体、如权利要求14所述的基因修饰的细胞、如权利要求16所述的抗体药物偶联物和/或如权利要求17所述的药物组合物；
    所述药盒B含有其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
  21. 一种诊断、治疗和/或预防CLDN18.2介导的疾病或病症的方法，所述方法包括向有需要的患者施用治疗有效量的如权利要求1～9任一项所述的靶向CLDN18.2的抗体或其抗原结合片段、如权利要求13所述的嵌合抗原受体、如权利要求16所述的抗体药物偶联物或如权利要求17所述的药物组合物，或者使用如权利要求20所述的套装药盒治疗有需要的患者。
  22. 如权利要求21所述的方法，其特征在于，所述的疾病或病症为肿瘤，优选CLDN18.2阳性肿瘤，更优选胃癌、食管癌、肺癌、卵巢癌、黑素瘤、肾癌、乳腺癌、结肠直肠癌、肝癌、胰腺癌、膀胱癌、头颈癌、支气管癌、神经胶质瘤和/或白血病。
  23. 一种免疫检测或者测定CLDN18.2的方法，其包括使用如权利要求1～9任一项所述的靶向CLDN18.2的抗体或其抗原结合片段、如权利要求13所述的嵌合抗原受体、如权利要求16所述的抗体药物偶联物或如权利要求17所述的药物组合物；优选地，所述检测为非诊断和/或治疗目的的。
  24. 一种联合疗法，其包括分别向有需要的患者施用如权利要求1～9任一项所述的 靶向CLDN18.2的抗体或其抗原结合片段、如权利要求13所述的嵌合抗原受体、如权利要求16所述的抗体药物偶联物或如权利要求17所述的药物组合物，和第二治疗剂；所述第二治疗剂较佳地包含其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
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