WO2022012559A1 - 抗cldn-18.2抗体及其用途 - Google Patents

抗cldn-18.2抗体及其用途 Download PDF

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Publication number
WO2022012559A1
WO2022012559A1 PCT/CN2021/106125 CN2021106125W WO2022012559A1 WO 2022012559 A1 WO2022012559 A1 WO 2022012559A1 CN 2021106125 W CN2021106125 W CN 2021106125W WO 2022012559 A1 WO2022012559 A1 WO 2022012559A1
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Prior art keywords
seq
amino acid
acid sequence
variable region
chain variable
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PCT/CN2021/106125
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English (en)
French (fr)
Inventor
周岳华
张静
刘辉
刘洪川
武海
姚剑
黄兰青
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Shanghai Junshi Biosciences Co Ltd
Suzhou Junmeng Biosciences Co Ltd
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Shanghai Junshi Biosciences Co Ltd
Suzhou Junmeng Biosciences Co Ltd
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Application filed by Shanghai Junshi Biosciences Co Ltd, Suzhou Junmeng Biosciences Co Ltd filed Critical Shanghai Junshi Biosciences Co Ltd
Priority to KR1020237001805A priority Critical patent/KR102916382B1/ko
Priority to JP2023501905A priority patent/JP7726978B2/ja
Priority to CA3186099A priority patent/CA3186099A1/en
Priority to CN202180061297.0A priority patent/CN116096416B/zh
Priority to PH1/2023/550089A priority patent/PH12023550089A1/en
Priority to US18/016,279 priority patent/US20240059771A1/en
Priority to AU2021307516A priority patent/AU2021307516B2/en
Priority to BR112023000657A priority patent/BR112023000657A2/pt
Priority to EP21842171.7A priority patent/EP4180457A4/en
Publication of WO2022012559A1 publication Critical patent/WO2022012559A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention provides antibodies or antigen-binding fragments thereof that specifically bind to CLDN-18.2 and compositions comprising the same. Also provided are nucleic acid molecules encoding the antibodies of the present invention or antigen-binding fragments thereof, expression vectors and host cells for expressing the antibodies of the present invention or antigen-binding fragments thereof, and therapeutic or diagnostic methods and uses for the antibodies of the present invention or antigen-binding fragments thereof .
  • Gastric cancer is one of the most common cancers worldwide. According to the statistics of the World Health Organization's Cancer Control Program, there are as many as 7 million deaths from cancer in the world every year, of which 700,000 patients die from gastric cancer. Compared with conventional gastric cancer treatment regimens, antibody-based treatment regimens have far-reaching application prospects due to their high specificity and low side effects.
  • Claudin also known as CLDN
  • CLDN is a family of cell surface proteins that build paracellular barriers and control the flow of molecules between cells, and at least 26 species have been identified so far.
  • Members of the Claudin protein family are important structural components of tight junctions and play an important role in maintaining epithelial cell polarity, controlling paracellular spreading, and regulating cell growth and differentiation.
  • the Claudin molecule crosses the cell membrane four times and both the N- and C-termini land in the cytoplasm.
  • Different Claudin members are expressed in different tissues, and alterations in their function are associated with cancer development. Changes in the expression levels of Claudin 1, Claudin 18 and Claudin 10 were associated with colorectal cancer, gastric cancer and hepatocellular carcinoma, respectively.
  • Claudin 18 has two alternative splicing variants, CLDN-18.1 and CLDN-18.2.
  • Claudin 18.1 (CLDN-18.1) is selectively expressed in normal lung and gastric epithelium.
  • Claudin 18.2 (CLDN-18.2) is slightly expressed in normal gastric epithelial short-lived cells, but in tumor cells, Claudin 18.2 is strongly expressed in a variety of cancer types, such as 75% of gastric cancer patients highly expressed Claudin 18.2,50 % of pancreatic cancer patients highly expressed Claudin 18.2, 30% of esophageal cancer patients highly expressed Claudin 18.2, and it was also highly expressed in lung cancer and so on.
  • the present invention provides an anti-CLDN-18.2 antibody or an antigen-binding fragment thereof, which has the advantages of high affinity and high specificity against human CLDN-18.2.
  • the anti-CLDN-18.2 antibodies or antigen-binding fragments thereof provided by the present invention can be used as stand-alone therapy or in combination with other therapies/or other anti-cancer agents, such as in the treatment of cancer.
  • the present invention provides an anti-CLDN-18.2 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein,
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein
  • the sequence of the HCDR1 is selected from the amino acid sequences shown in SEQ ID NOs: 1, 10, 16, 22, 28, 34 and 37;
  • the sequence of the HCDR2 is selected from the amino acid sequences shown in SEQ ID NO: 62, 63, 17, 23, 29, 35, 38, 72 and 74, and X 1 in the HCDR2 shown in SEQ ID NO: 62 is C or S, X 2 is T or S, and X 3 is D or G, and X 4 is K or T in the HCDR2 shown in SEQ ID NO: 63; and
  • the sequence of the HCDR3 is selected from the amino acid sequences shown in SEQ ID NOs: 3, 12, 18, 24, 30, 36, 39, 73 and 75;
  • the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein
  • the sequence of the LCDR1 is selected from the amino acid sequences shown in SEQ ID NOs: 4, 7, 13, 19, 25 and 31;
  • the sequence of the LCDR2 is selected from the amino acid sequences shown in SEQ ID NOs: 5, 8, 14, 20, 26 and 32;
  • the sequence of the LCDR3 is selected from the amino acid sequences shown in SEQ ID NOs: 6, 9, 15, 21, 27 and 33.
  • the heavy chain variable region of the present invention comprises:
  • HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively;
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 28, SEQ ID NO: 74 and SEQ ID NO: 75, respectively; or
  • the light chain variable region comprises:
  • LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively;
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively; or
  • VI a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33, respectively; or
  • VI a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively; or
  • VIII a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33, respectively; or
  • (IX) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively; or
  • (x) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively; or
  • XI a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively; or
  • XII a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 10, SEQ ID NO: 40 and SEQ ID NO: 12, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively; or
  • XIII a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:1, SEQ ID NO:41 and SEQ ID NO:3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively; or
  • XIV a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:1, SEQ ID NO:72 and SEQ ID NO:73, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively; or
  • XV a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NO:1, SEQ ID NO:72 and SEQ ID NO:3, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively; or
  • XVI a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 28, SEQ ID NO: 74 and SEQ ID NO: 30, respectively; and a light chain variable region comprising The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively; or
  • XVII a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NO: 28, SEQ ID NO: 74 and SEQ ID NO: 75, respectively; and a light chain variable region comprising The amino acid sequences are LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively.
  • the amino acid sequence of the heavy chain variable region of the present invention comprises a sequence selected from the group consisting of SEQ ID NOs: 42, 45, 47, 49, 51, 53, 54, 76, 78, 82 and 83 , or with at least 95%, 96%, 97%, 98% or 99% of any of the sequences shown in SEQ ID NOs: 42, 45, 47, 49, 51, 53, 54, 76, 78, 82 and 83 % identical amino acid sequences; and
  • the amino acid sequence of the light chain variable region comprises a sequence selected from the group consisting of sequences shown in SEQ ID NOs: 43, 44, 46, 48, 50 and 52, or a Any of the sequences shown has an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • VI a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:51 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:52; or
  • VII a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:53 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:50; or
  • VIII a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:54 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:52; or
  • (X) a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:51 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:46; or
  • the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, wherein
  • the amino acid sequence of the heavy chain variable region comprises a sequence selected from the group consisting of sequences shown in SEQ ID NOs: 55, 57, 58, 59, 60, 76, 78, 79, 82 and 83, or with SEQ ID NOs: 55, 57 , 58, 59, 60, 76, 78, 79, 82, and 83 have amino acid sequences that are at least 95%, 96%, 97%, 98%, or 99% identical to any of the sequences; and
  • the amino acid sequence of the light chain variable region comprises a sequence selected from the group consisting of the sequences shown in SEQ ID NOs: 56, 61, 77, 80 and 81, or has Amino acid sequences of at least 95%, 96%, 97%, 98% or 99% identity.
  • the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises, for example, SEQ ID NOs: 55, 57, 60, 76 The amino acid sequence shown in any one of , 78, 79, 82 or 83; and the light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 56, 61, 77, 80 or 81 .
  • the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises, for example, SEQ ID NO: 55, 57, 82 or 83 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:56.
  • the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises as set forth in SEQ ID NO: 60, 76 or 78 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:61.
  • the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, wherein
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:55; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:56; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:57; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:56; or
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:58; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:56; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:59; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:56; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:60; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:61; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:76; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:77; or
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:79; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:80; or
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:55; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:81.
  • the antibody or antigen-binding fragment thereof of the present invention comprises: a heavy chain, the amino acid sequence of which is shown in SEQ ID NO:64 or a variant thereof or SEQ ID NO:68 or a variant thereof , and a light chain, the amino acid sequence of which is shown in SEQ ID NO: 65 or a variant thereof or SEQ ID NO: 69 or a variant thereof, wherein the variant comprises 1, 2, 3, 4 or 5 amino acid changes;
  • the variant of SEQ ID NO:64 comprises an amino acid change at position 50 or 63 or a combination thereof; and/or the variant of SEQ ID NO:68 comprises an amino acid at position 63 or 65 or a combination thereof Variety;
  • the variant of SEQ ID NO:64 comprises S50C or S63T or a combination thereof; and/or the variant of SEQ ID NO:68 comprises G63D or T65K or a combination thereof.
  • the antibody or antigen-binding fragment thereof of the invention comprises: a heavy chain and a light chain, wherein
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:64, or has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% with the amino acid sequence shown in SEQ ID NO:64 an amino acid sequence of % or 99% sequence identity; and the light chain comprises the amino acid sequence set forth in SEQ ID NO:65, or has at least 90%, 92%, 94%, or at least 90%, 92%, 94%, or the amino acid sequences of %, 95%, 96%, 97%, 98% or 99% sequence identity; or
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:68, or has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% with the amino acid sequence shown in SEQ ID NO:68 an amino acid sequence of % or 99% sequence identity; and the light chain comprises the amino acid sequence shown in SEQ ID NO:69, or has at least 90%, 92%, 94%, or at least 90%, 92%, 94%, or the amino acid sequences of %, 95%, 96%, 97%, 98% or 99% sequence identity; or
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:84, or has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% with the amino acid sequence shown in SEQ ID NO:84 an amino acid sequence of % or 99% sequence identity; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 85, or has at least 90%, 92%, 94%, or at least 90%, 92%, 94%, or the amino acid sequences of %, 95%, 96%, 97%, 98% or 99% sequence identity; or
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 86, or has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% with the amino acid sequence shown in SEQ ID NO: 86 an amino acid sequence of % or 99% sequence identity; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 87, or has at least 90%, 92%, 94%, or at least 90%, 92%, 94%, or the amino acid sequences of %, 95%, 96%, 97%, 98% or 99% sequence identity; or
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 88, or has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% with the amino acid sequence shown in SEQ ID NO: 88 An amino acid sequence of % or 99% sequence identity; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 89, or has at least 90%, 92%, 94% with the amino acid sequence shown in SEQ ID NO: 89 amino acid sequences of %, 95%, 96%, 97%, 98% or 99% sequence identity; or
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:90, or has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% with the amino acid sequence shown in SEQ ID NO:90 an amino acid sequence of % or 99% sequence identity; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 91, or has at least 90%, 92%, 94%, or at least 90%, 92%, 94%, or the amino acid sequences of %, 95%, 96%, 97%, 98% or 99% sequence identity; or
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:92, or has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% with the amino acid sequence shown in SEQ ID NO:92 an amino acid sequence of % or 99% sequence identity; and the light chain comprises the amino acid sequence shown in SEQ ID NO:93, or has at least 90%, 92%, 94% with the amino acid sequence shown in SEQ ID NO:93 amino acid sequences of %, 95%, 96%, 97%, 98% or 99% sequence identity; or
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:94, or has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% with the amino acid sequence shown in SEQ ID NO:94 an amino acid sequence of % or 99% sequence identity; and the light chain comprises the amino acid sequence set forth in SEQ ID NO:95, or has at least 90%, 92%, 94%, or at least 90%, 92%, 94%, or the amino acid sequences of %, 95%, 96%, 97%, 98% or 99% sequence identity; or
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:96, or has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% with the amino acid sequence shown in SEQ ID NO:96 an amino acid sequence of % or 99% sequence identity; and the light chain comprises the amino acid sequence shown in SEQ ID NO:97, or has at least 90%, 92%, 94%, or at least 90%, 92%, 94%, or the amino acid sequences of %, 95%, 96%, 97%, 98% or 99% sequence identity; or
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO:98, or has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% with the amino acid sequence shown in SEQ ID NO:98 an amino acid sequence of % or 99% sequence identity; and the light chain comprises the amino acid sequence set forth in SEQ ID NO:99, or has at least 90%, 92%, 94%, or at least 90%, 92%, 94%, or the amino acid sequences of %, 95%, 96%, 97%, 98% or 99% sequence identity; or
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 100, or has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% with the amino acid sequence shown in SEQ ID NO: 100 an amino acid sequence of % or 99% sequence identity; and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 101, or has at least 90%, 92%, 94%, or at least 90%, 92%, 94 Amino acid sequences of %, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • the antibodies of the invention are murine antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.
  • the antigen-binding fragment of the present invention is a Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv or sdAb or diabody.
  • the antibody of the invention is of any IgG subtype, such as IgGl, IgG2, IgG3 or IgG4; preferably; the antibody is low or afucosylated.
  • the antibodies of the invention are hypofucosylated.
  • the antibodies of the invention are afucosylated.
  • the invention provides an isolated anti-CLDN-18.2 antibody or antigen-binding fragment thereof having one or more of the following properties:
  • the present invention provides a polynucleotide encoding an anti-CLDN-18.2 antibody or antigen-binding fragment thereof as described herein.
  • the present invention provides an expression vector comprising the polynucleotide as described herein, preferably, the vector is a eukaryotic expression vector.
  • the present invention provides a host cell comprising a polynucleotide as described herein or an expression vector as described herein, or expressing an anti-CLDN-18.2 antibody or antigen binding thereof as described herein Fragments, preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.
  • the present invention provides a method for preparing an anti-CLDN-18.2 antibody or antigen-binding fragment thereof as described herein, the method comprising under conditions suitable for expression of the antibody or antigen-binding fragment thereof
  • the host cells described herein are cultured and the expressed antibodies or antigen-binding fragments thereof are recovered from the host cells.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-CLDN-18.2 antibody or antigen-binding fragment thereof as described herein, a polynucleotide as described herein, an expression vector as described herein, or an antigen-binding fragment thereof as described herein
  • the host cell and a pharmaceutically acceptable carrier or excipient.
  • the present invention provides an antibody or antigen-binding fragment thereof, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical combination as described herein
  • a drug in the manufacture of a medicament for the treatment and/or prevention of a disease or condition mediated by CLDN-18.2, preferably, the disease or condition is cancer.
  • the present invention provides an antibody or antigen-binding fragment thereof, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical combination as described herein
  • the present invention provides a method of treating and/or preventing a disease or disorder mediated by CLDN-18.2, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof as described herein,
  • the disease or disorder is cancer as described in the polynucleotides described herein, the expression vectors described herein, the host cells described herein, or the pharmaceutical compositions described herein.
  • the cancer is selected from the group consisting of gastric cancer, esophageal cancer, gastroesophageal cancer, pancreatic cancer, bile duct cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, bowel cancer and bladder cancer.
  • the present invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical composition described herein, and one or more additional therapeutic agents.
  • the present invention provides a kit comprising an antibody or antigen-binding fragment thereof as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical composition as described herein, preferably it further comprises a delivery device.
  • the present invention provides a method of detecting the presence of CLDN-18.2 in a sample using an antibody or antigen-binding fragment thereof as described herein.
  • Figure 1 Cell-level affinity of chimeric anti-CLDN-18.2 antibodies determined by flow cytometry.
  • Figures 2a and 2b ADCC activity of chimeric anti-CLDN-18.2 antibodies as determined by reporter gene assay.
  • Figure 3 CDC activity of chimeric anti-CLDN-18.2 antibodies determined by flow cytometry.
  • Figures 4a, 4b and 4c Cell-level affinity of humanized anti-CLDN-18.2 antibodies determined by flow cytometry.
  • FIGS 5a, 5b and 5c ADCC activity of humanized anti-CLDN-18.2 antibodies as determined by reporter gene assay.
  • Figures 6a, 6b and 6c CDC activity of humanized anti-CLDN-18.2 antibodies determined by flow cytometry.
  • Figure 7 Inhibitory effect of humanized anti-CLDN-18.2 antibody on tumor growth of M-NSG mice transplanted with human gastric cancer MKN45 hClaudin18.2 Mixeno.
  • Figure 8 Inhibitory effect of humanized anti-CLDN-18.2 antibody on tumor growth of human pancreatic cancer hCLDN18.2 MIA PaCa-2 transplanted in CB-17 SCID mice.
  • CLDN-18.2 or "Claudin18.2” is one of two splice variants of Claudin 18.
  • the term refers to any native CLDN-18.2 from any vertebrate (including mammals such as primates (eg, humans)) and rodents (eg, mice and rats), unless otherwise stated.
  • the term encompasses "full-length” unprocessed CLDN-18.2 as well as any form of CLDN-18.2 or any fragment thereof produced by intracellular processing.
  • the term also includes naturally-occurring variants of CLDN-18.2, eg, splice variants or allelic variants.
  • CLDN-18.2 refers to the full length of CLDN-18.2 from human and cynomolgus monkeys or a fragment thereof (such as a mature fragment thereof lacking a signal peptide).
  • percent (%) amino acid sequence identity or simply “identity” is defined as the maximum percent sequence identity obtained when amino acid sequences are aligned (and where necessary gaps are introduced), and no conservative substitutions are considered to be Following the portion of sequence identity, the percentage of amino acid residues in the candidate amino acid sequence that are identical to those in the reference amino acid sequence.
  • Sequence alignments to determine percent amino acid sequence identity can be performed using various methods in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to obtain maximal alignment over the full length of the sequences being compared.
  • immune response refers to the action by, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and the production of soluble macromolecules (including antibodies, cytokines and complement) by these cells or by the liver, which results in selective Damage, destroy or eliminate invading pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation.
  • signal transduction pathway or “signal transduction activity” refers to a biochemical causal relationship, typically initiated by protein-protein interactions such as the binding of growth factors to receptors, that results in the transmission of signals from one part of a cell to another of the cell. part.
  • delivery involves specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions leading to signal transduction.
  • the penultimate process usually involves nuclear events that lead to changes in gene expression.
  • activity or “biological activity”, or the terms “biological property” or “biological signature” are used interchangeably herein and include, but are not limited to, epitope/antigen affinity and specificity, neutralization or antagonism of CLDN in vivo or in vitro -18.2 ability activity, IC 50, in vivo stability of the antibody and the immunogenic properties of the antibody.
  • Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (ie, generally with non-human homologues of the targeting peptide, or with other proteins or tissues), and retention of The ability of proteins to be expressed at high levels in mammalian cells.
  • antibody refers to any form of antibody that possesses the desired biological activity. Accordingly, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), humanized antibodies, fully human antibodies, Chimeric and camelized single domain antibodies.
  • isolated antibody refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules such as nucleic acids, proteins, lipids, sugars or other substances such as cell debris and growth media .
  • isolated does not mean the complete absence of such materials or the absence of water, buffers or salts unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the binding compound described herein.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include large numbers of antibodies directed against (or specific for) different epitopes.
  • the modifier "monoclonal” indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method.
  • full-length antibody refers to an immunoglobulin molecule comprising four peptide chains in nature: two heavy (H) chains (about 50-70 kDa in full length) and two light (L) chains (full length) 25kDa) are connected to each other by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) and spaced by more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH or VL region consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • Fc region is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region extends from Cys226 of the heavy chain or from Pro230 to the carboxy terminus.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, such as Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service National As described in Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.
  • antibody-binding fragment of an antibody (“parent antibody”) includes fragments or derivatives of an antibody, typically including at least one fragment of the antigen-binding or variable region (eg, one or more CDRs) of the parent antibody that retains the parental At least some binding specificity of an antibody.
  • antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab') 2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments and multispecific antibodies.
  • a binding fragment or derivative typically retains at least 10% of its antigen-binding activity when the antigen-binding activity is expressed on a molar basis.
  • the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody.
  • antigen-binding fragments of antibodies may include conservative or non-conservative amino acid substitutions that do not significantly alter their biological activity (referred to as “conservative variants” or “functionally conservative variants” of an antibody).
  • binding compound refers to both antibodies and binding fragments thereof.
  • single-chain Fv or "scFv” antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • Fv polypeptides typically also comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
  • domain antibody is an immunologically functional immunoglobulin fragment containing only the heavy chain variable region or the light chain variable region.
  • two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody.
  • the two VH regions of a bivalent domain antibody can target the same or different antigens.
  • bivalent antibody contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, bivalent antibodies can be bispecific.
  • diabody refers to a small antibody fragment with two antigen-binding sites comprising a heavy chain linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH) Variable domain (VH).
  • VL light chain variable domain
  • VH-VL or VL-VH Variable domain
  • chimeric antibody is an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first antibody and the second antibody are from different species.
  • the variable domains are obtained from rodent or the like antibodies (“parental antibodies”), while the constant domain sequences are obtained from human antibodies, such that the resulting chimeric antibody induces induction in human subjects as compared to the parental rodent antibody The likelihood of an adverse immune response is low.
  • humanized antibody refers to a form of antibody that contains sequences from human and non-human (eg, mouse, rat) antibodies.
  • humanized antibodies comprise substantially all of at least one, usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the hypervariable loops Framework (FR) regions are the framework regions of human immunoglobulin sequences.
  • FR hypervariable loops Framework
  • a humanized antibody optionally may comprise at least a portion of a human immunoglobulin constant region (Fc).
  • Fully human antibody refers to an antibody comprising only human immunoglobulin protein sequences. Fully human antibodies may contain murine sugar chains if produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Likewise, “mouse antibody” refers to an antibody comprising only mouse immunoglobulin sequences. Alternatively, fully human antibodies may contain rat sugar chains if produced in rats, in rat cells, or in hybridomas derived from rat cells. Likewise, “rat antibody” refers to an antibody comprising only rat immunoglobulin sequences.
  • an “isotype” antibody refers to the class of antibody provided by the heavy chain constant region genes (eg, IgM, IgE, IgG such as IgGl, IgG2, or IgG4). Isotypes also include modified forms of one of these species, wherein modifications have been made to alter Fc function, eg, to enhance or reduce effector function or binding to Fc receptors.
  • heavy chain constant region genes eg, IgM, IgE, IgG such as IgGl, IgG2, or IgG4
  • epitope refers to the region of an antigen to which an antibody binds. Epitopes can be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of the protein.
  • affinity or "binding affinity” refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair.
  • affinity molecule X for its partner Y can generally dissociation constant (K D) is represented by equilibrium, the equilibrium dissociation constant and the dissociation rate constant is the rate constant (k dis, respectively and k on) ratio.
  • K D dissociation constant
  • k dis rate constant
  • k on rate constant
  • Affinity can be measured by common methods known in the art. One specific method used to measure affinity is the ForteBio kinetic binding assay herein.
  • does not bind to a protein or cell means, does not bind to the protein or cell, or does not bind to it with high affinity, ie binds the protein or cell with a K D of 1.0 x 10 -6 M or higher, more preferably 1.0 x 10 -5 M or higher, more preferably 1.0 ⁇ 10 -4 M or higher, 1.0 ⁇ 10 -3 M or higher, more preferably 1.0 ⁇ 10 -2 M or higher.
  • high affinity for an IgG antibody, the antigen refers to K D of 1.0 ⁇ 10 -6 M or less, preferably 5.0 ⁇ 10 -8 M or less, more preferably 1.0 ⁇ 10 -8 M or lower, 5.0 ⁇ 10 -9 M or lower, more preferably 1.0 ⁇ 10 -9 M or lower.
  • high affinity binding may vary.
  • IgM subtype "high affinity” means a binding K D of 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8 M or less.
  • antibody-dependent cytotoxicity refers to cell-mediated immune defense in which immune system effector cells actively associate cell membrane surface antigens with antibodies, such as Claudin18. 2 Antibodies that bind to target cells such as cancer cells are lysed.
  • CDC complement-dependent cytotoxicity
  • IgG and IgM antibodies that, when bound to surface antigens, initiate the canonical complement pathway, including formation of membrane attack complexes and lysis of target cells.
  • the antibodies of the present invention when bound to Claudin 18.2, trigger CDC against cancer cells.
  • nucleic acid or “polynucleotide” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless expressly limited, the term includes nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides (see, in Kariko et al. Human US Patent No. 8,278,036, which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods of synthesizing such mRNA molecules, and methods for delivering therapeutic proteins in vivo).
  • nucleic acid sequence also implicitly includes conservatively modified variants thereof (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complements thereof, as well as sequences explicitly indicated.
  • degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • Construct refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single- or double-stranded DNA or RNA polynucleotide molecule), derived from Any source, capable of integrating with the genome or replicating autonomously, constitutes a polynucleotide molecule in which one or more polynucleotide molecules have been functionally linked (ie, operably linked).
  • the recombinant construct will typically comprise a polynucleotide of the invention operably linked to transcription initiation regulatory sequences that direct transcription of the polynucleotide in a host cell. Expression of the nucleic acids of the invention can be directed using both heterologous and non-heterologous (ie, endogenous) promoters.
  • Vector refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (ie, the introduction of heterologous DNA into a host cell).
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector in which additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors).
  • vectors After introduction into the host cell, other vectors (eg, non-episomal mammalian vectors) integrate into the genome of the host cell and thus replicate together with the host genome. In addition, certain vectors are capable of directing the expression of operably linked genes. Such vectors are referred to herein as "expression vectors".
  • expression vector refers to a nucleic acid molecule capable of replicating and expressing a gene of interest when transformed, transfected or transduced into a host cell.
  • Expression vectors contain one or more phenotypic selectable markers and origins of replication to ensure maintenance of the vector and to provide for amplification within the host if desired.
  • Activation can have the same meaning, eg, activation, stimulation, or treatment of a cell or receptor with a ligand, unless the context otherwise or clearly dictates.
  • Ligand includes natural and synthetic ligands, such as cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies.
  • Ligand also includes small molecules, such as peptidomimetics of cytokines and peptidomimetics of antibodies.
  • Activation can refer to cellular activation regulated by internal mechanisms as well as external or environmental factors.
  • a “response/response”, eg, the response of a cell, tissue, organ, or organism, includes changes in biochemical or physiological behavior (eg, concentration, density, adhesion or migration, gene expression rate, or differentiation state within a biological compartment), wherein changes Related to activation, stimulation, or processing, or to internal mechanisms such as genetic programming.
  • the terms “treating” or “treating” of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (ie, slowing or arresting or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, “treating” or “treating” refers to alleviating or ameliorating at least one physical parameter, including those physical parameters that may not be discernible by a patient. In another embodiment, “treating” or “treating” refers to modulating a disease or disorder physically (eg, stabilization of discernible symptoms), physiologically (eg, stabilization of physical parameters), or both. Unless explicitly described herein, methods for assessing treatment and/or prevention of disease are generally known in the art.
  • Subject includes any human or non-human animal.
  • non-human animal includes all vertebrates, eg, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, and the like.
  • cyno or “cynomolgus monkey” refers to a cynomolgus monkey.
  • Administration "in combination with” one or more other therapeutic agents includes simultaneous (co) administration and sequential administration in any order.
  • “Therapeutically effective amount”, “therapeutically effective dose” and “effective amount” mean that the CLDN-18.2 antibody or antigen-binding fragment thereof of the present invention, when administered alone or in combination with other therapeutic agents, is effective in preventing Or an amount that ameliorates the symptoms of one or more diseases or conditions or the progression of that disease or condition.
  • a therapeutically effective dose also refers to an amount of the antibody or antigen-binding fragment thereof sufficient to cause amelioration of symptoms, eg, an amount that treats, cures, prevents or ameliorates a related medical condition or increases the rate of treatment, cure, prevention or amelioration of such a condition.
  • the therapeutically effective dose refers to that ingredient only.
  • a therapeutically effective dose refers to the combined amount of active ingredients that elicits a therapeutic effect, whether administered in combination, sequentially or simultaneously.
  • An effective amount of the therapeutic agent will result in an improvement in the diagnostic criterion or parameter by at least 10%; usually by at least 20%; preferably by at least about 30%; more preferably by at least 40%, and most preferably by at least 50%.
  • Cancer and “cancerous” refer to or describe a physiological disorder in mammals that is often characterized by unregulated cell growth. Benign and malignant cancers as well as dormant tumors or micrometastases are included in this definition. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, cancer of the stomach or gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, Cancer of salivary gland, kidney or kidney, liver, prostate, vulva, thyroid, liver, and various types of head and neck cancer, and B-cell lymphomas (including low-grade/follicular non-HoJ King's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate/follicular NHL, intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small anucleate NHL, bulky disease (NHL), small lymph
  • the invention provides anti-CLDN-18.2 antibodies or antigen-binding fragments thereof.
  • anti-CLDN-18.2 antibody refers to the ability to bind CLDN-18.2 protein or fragment thereof with sufficient affinity such that the Antibodies can be used as diagnostic and/or therapeutic agents targeting CLDN-18.2.
  • Antibodies of the invention can be produced using any suitable method for producing antibodies. Any suitable form of CLDN-18.2 can be used as an immunogen (antigen) for generating antibodies. By way of example and not limitation, any CLDN-18.2 variant or fragment thereof can be used as an immunogen. In some embodiments, hybridoma cells producing murine monoclonal anti-human CLDN-18.2 antibodies can be produced by methods well known in the art. Antibodies derived from rodents (eg, mice) may cause unwanted antibody immunogenicity when used in vivo as therapeutics, and repeated use results in an immune response against the therapeutic antibody that at least results in loss of therapeutic efficacy , and in severe cases lead to a potentially fatal allergic reaction.
  • rodents eg, mice
  • the chimeric or humanized antibodies of the invention can be prepared based on the sequences of the prepared murine monoclonal hybridoma antibodies.
  • DNA encoding heavy and light chain immunoglobulins can be obtained from murine hybridomas of interest and engineered to contain non-murine (eg, human) immunoglobulin sequences using standard molecular biology techniques.
  • the chimeric CLDN-18.2 antibody of the present invention can use methods known in the art to operably link hybridoma-derived immunoglobulin heavy and light chain variable regions to human IgG constant regions ( See, eg, US Patent No. 4,816,567 to Cabilly et al.) for obtaining chimeric heavy chains and chimeric light chains for preparation.
  • the chimeric antibodies of the invention comprise constant regions that may be selected from any human IgG subtype, such as IgGl, IgG2, IgG3, IgG4, preferably IgG4.
  • the chimeric CLDN-18.2 antibody of the present invention can be obtained by "mixing and matching" transfected expression cells with chimeric light chain and chimeric heavy chain expression plasmids. CLDN-18.2 binding can be tested using the binding assays described above and other conventional binding assays (eg, ELISA).
  • variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known protocols, including Chothia based on the three-dimensional structure of the antibody and topology of the CDR loops (Chothia et al.
  • the CDR boundaries of the variable region of the same antibody obtained based on different assignment systems may vary. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different.
  • the scope of said antibodies also covers antibodies whose variable region sequences comprise said specific CDR sequences, but due to the application of different schemes (e.g. different assignment systems or combinations), resulting in their claimed CDR boundaries being different from the specific CDR boundaries defined by the present invention.
  • Antibodies with different specificities have different CDRs.
  • CDRs vary from antibody to antibody, only a limited number of amino acid positions within CDRs are directly involved in antigen binding.
  • the minimal binding unit can be a sub-portion of a CDR.
  • the residues of the remainder of the CDR sequence can be determined by the structure and protein folding of the antibody, as will be apparent to those skilled in the art. Accordingly, the present invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be replaced by conservative amino acid residues.
  • murine CDR regions can be inserted into human germline framework regions using methods known in the art. See US Patent Nos. 5,225,539 to Winter et al. and US Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.
  • amino acid changes include amino acid deletions, insertions or substitutions.
  • the anti-CLDN-18.2 antibodies or antigen-binding fragments thereof of the invention include those that have been mutated by amino acid deletions, insertions, or substitutions, but which are still identical to the antibodies described above (particularly in the CDR regions depicted in the sequences above) Those antibodies that have amino acid sequences that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.
  • the antibodies of the invention have no more than 1, 2, 3, 4, or 5 amino acid mutations in the CDR regions that have been mutated by amino acid deletions, insertions, or substitutions when compared to the CDR regions depicted in a particular sequence.
  • the polynucleotides encoding the antibodies of the invention include those that have been mutated by nucleotide deletions, insertions, or substitutions, but still have at least about 60, 70, or 60, 70 or more of the coding region corresponding to the CDRs depicted in the sequences described above. , 80, 90, 95 or 100% identical polynucleotides.
  • one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants.
  • An Fc region variant may comprise a human Fc region sequence (eg, a human IgGl, IgG2, IgG3, or IgG4 Fc region) comprising amino acid modifications (eg, substitutions) at one or more amino acid positions.
  • cysteine-engineered antibodies such as "thioMAbs,” in which one or more residues of the antibody are replaced with cysteine residues.
  • the antibody can be altered to increase or decrease its degree of glycosylation and/or to alter its glycosylation pattern. Addition or deletion of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence so as to create or remove one or more glycosylation sites. For example, one or more amino acid substitutions can be made to eliminate one or more sites of glycosylation, thereby eliminating glycosylation at that site.
  • Antibodies can be prepared with altered types of glycosylation, eg, low or no-fucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC capacity of antibodies.
  • the present invention provides antibodies that are low or afucosylated such that the binding affinity of the antibody to a receptor expressed on effector cells can be significantly increased, thereby resulting in an antibody with enhanced Antibody-dependent cell-mediated cytotoxicity (ADCC) activity.
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • the average fucose within the sugar chain at Asn 297 can be calculated relative to the sum of all sugar structures (eg complex, hybrid and high mannose structures) linked to Asn 297 as measured by MALDI-TOF mass spectrometry amount, thereby determining the amount of fucose, for example as described in WO2008/077546.
  • Asn297 refers to the aspartate residue located in the Fc region at approximately position 297 (EU numbering of Fc region residues); however, due to minor sequence changes in the antibody, Asn297 may also be located approximately ⁇ 3 upstream or downstream of position 297 amino acid positions, that is, between positions 294 and 300. See eg US2003/0157108; US2004/0093621.
  • Such antibody variants can be produced in cell lines capable of producing defucosylated or hypofucosylated antibodies. Examples of such cells include Lecl3 CHO cells deficient in protein fucosylation (Ripka, J. et al., Arch. Biochem. Biophys. 249(1986):533-545; US2003/0157108.
  • using Fucosidase cleaves the fucose residues of the antibody.
  • a glycoform modulator is used to control the fucose residue of the antibody.
  • the glycoform modulator can be CDFS01, which is commercially available from Shanghai Opmax Biotechnology Ltd. Defucosylation can be performed by conventional methods well known in the art.
  • the level of fucosylation of an antibody can be structurally defined.
  • “afucosylated” or “afucosylated” means that the content of fucose in an antibody is less than 5%, eg, about 0%, less than 1%, less than 2%, less than 3%, less than 4%.
  • the term “low fucosylation” means that the content of fucose in the antibody is about 5% or more and less than 30%; for example, the content of fucose in the antibody is about 5%-10%, 10% %-15%, 15%-20%, 20%-25%, 25%-30%, 10%-20%, 20%-30%, 10%-30%.
  • the term “low or afucosylated” means that the content of fucose in the antibody is less than 30%.
  • the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available.
  • Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers.
  • Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl - 1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol,
  • the present invention provides a polynucleotide encoding an anti-CLDN-18.2 antibody or antigen-binding fragment thereof as described herein.
  • the polynucleotide may comprise a polynucleotide encoding the amino acid sequence of the light chain variable region and/or heavy chain variable region of an antibody, or a polynucleotide comprising the amino acid sequence encoding the light chain and/or heavy chain of an antibody .
  • the present invention provides an expression vector comprising the polynucleotide as described herein, preferably, the vector is a eukaryotic expression vector.
  • the polynucleotides as described herein are contained in one or more expression vectors.
  • the present invention provides a host cell comprising a polynucleotide as described herein or an expression vector as described herein, preferably the host cell is a eukaryotic cell, more preferably a mammalian cell .
  • the present invention provides a method for preparing an anti-CLDN-18.2 antibody or antigen-binding fragment thereof as described herein, the method comprising under conditions suitable for expression of the antibody or antigen-binding fragment thereof
  • the antibody or antigen-binding fragment thereof is expressed in a host cell as described herein, and the expressed antibody or antigen-binding fragment thereof is recovered from the host cell.
  • the invention provides mammalian host cells for expressing the recombinant antibodies of the invention, including a number of immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese Hamster Ovary (CHO) cells, NSO, SP2/0 cells, HeLa cells, Baby Hamster Kidney (BHK) cells, Monkey Kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many others cell line. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels.
  • ATCC American Type Culture Collection
  • the present invention provides a method of producing an anti-CLDN-18.2 antibody, wherein the method comprises, when introducing an expression vector into a mammalian host cell, by culturing the host cell for a period of time sufficient to allow the antibody to grow in the host.
  • the antibody is produced by expression in the cell, or more preferably by secretion of the antibody into the medium in which the host cell is grown.
  • Antibodies can be recovered from the culture medium using standard protein purification methods.
  • afucosylated antibodies are advantageous because they generally have more potent potency than their fucosylated counterparts in vitro and in vivo, and are unlikely to be immunogenic , because their carbohydrate structure is a normal component of native human serum IgG.
  • compositions and pharmaceutical preparations are provided.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-CLDN-18.2 antibody or antigen-binding fragment thereof as described herein, a polynucleotide as described herein, an expression vector as described herein, the host cell described above, and a pharmaceutically acceptable carrier or excipient.
  • anti-CLDN-18.2 antibodies or pharmaceutical compositions thereof provided by the present invention may incorporate suitable carriers, excipients and other agents in formulations for combined administration, thereby providing improved transfer, delivery, tolerance, and the like.
  • composition refers to a formulation that allows the active ingredients contained therein to exist in a biologically effective form and does not contain additional ingredients that would be unacceptably toxic to the subject to whom the formulation is administered.
  • the compounds containing The pharmaceutical formulations of the anti-CLDN-18.2 antibodies described herein are preferably in the form of aqueous solutions or lyophilized formulations.
  • compositions or formulations of the present invention may also contain one or more other active ingredients required for the particular indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other .
  • the other active ingredient is a chemotherapeutic agent, an immune checkpoint inhibitor, a growth inhibitory agent, an antibiotic, or various anti-tumor or anti-cancer agents known, in a suitable amount effective for the intended use exist in combination.
  • the pharmaceutical compositions of the present invention further comprise compositions of polynucleotides encoding anti-CLDN-18.2 antibodies.
  • the present invention provides a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical composition described herein, and one or more additional therapeutic agents.
  • the present invention provides a kit comprising an antibody or antigen-binding fragment thereof as described herein, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical composition as described herein, preferably it further comprises a delivery device.
  • the present invention provides an antibody or antigen-binding fragment thereof, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical combination as described herein
  • a drug in the manufacture of a medicament for the treatment and/or prevention of a disease or condition mediated by CLDN-18.2, preferably, the disease or condition is cancer.
  • the present invention provides an antibody or antigen-binding fragment thereof, a polynucleotide as described herein, an expression vector as described herein, a host cell as described herein, or a pharmaceutical combination as described herein
  • the present invention provides a method of treating and/or preventing a disease or disorder mediated by CLDN-18.2, comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof as described herein,
  • the disease or disorder is cancer as described in the polynucleotides described herein, the expression vectors described herein, the host cells described herein, or the pharmaceutical compositions described herein.
  • the cancer is selected from the group consisting of gastric cancer, esophageal cancer, gastroesophageal cancer, pancreatic cancer, bile duct cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, bowel cancer and bladder cancer.
  • modes of administration of the present invention include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (eg, in arthritic joints), by inhalation, aerosol delivery, or intratumoral administration Wait.
  • the present invention provides for co-administration of a therapeutically effective amount of one or more therapies (eg, therapeutic modalities and/or other therapeutic agents) to a subject.
  • the therapy includes surgery and/or radiation therapy.
  • the methods or uses provided herein further comprise administering to an individual one or more therapies (eg, therapeutic modalities and/or other therapeutic agents).
  • therapies eg, therapeutic modalities and/or other therapeutic agents.
  • Antibodies of the invention can be used alone or in combination with other therapeutic agents in therapy. For example, it can be co-administered with at least one additional therapeutic agent.
  • PD-1 antibody, PD-L1 antibody, LAG-3 antibody and/or CTLA-4 antibody for example, PD-1 antibody, PD-L1 antibody, LAG-3 antibody and/or CTLA-4 antibody.
  • the cancer disease treatment method of the present invention further comprises administering a CLDN-18.2 expression stabilizing or increasing agent.
  • the expression of CLDN-18.2 is preferably on the cell surface of cancer cells.
  • the CLDN-18.2 expression stabilizing or increasing agent may be oxaliplatin and/or 5-FU.
  • the present invention provides a method of detecting the presence of CLDN-18.2 in a sample using an antibody or antigen-binding fragment thereof as described herein.
  • detection includes quantitative or qualitative detection.
  • the sample is a biological sample.
  • the biological sample is blood, serum, or other fluid sample of biological origin.
  • the biological sample comprises cells or tissues.
  • the plasmid HG20047-U containing the cDNA sequence of the human CLDN-18 gene was purchased from Yiqiao Shenzhou, and the human CLDN-18 part was amplified by PCR using the forward primer 5'-GTACgctagccaccTgaagagcggatccgctcttctTGCCCTGAAATGCATCCGCATTGGCAGC-3' and the reverse primer 5'-GATCgcggccgccctgcaggTTACACATAGTCGTGCTTGGAAGGATAAG-3' Gene fragment.
  • HXP-CLDN18P eukaryotic expression plasmid system
  • the HXP-hCLDN-18.2 obtained in Example 1 was mixed with an equal amount of immune adjuvant (Freund's adjuvant), and five 8-week-old female BALB/c mice were taken for intramuscular immunization. 100 ⁇ g of DNA plasmid was used per mouse for the primary immunization. Thereafter, booster immunizations were performed every 2 or 3 weeks for a total of 5 times, each time using 50 ⁇ g of DNA plasmid per mouse. In the last booster immunization, stable CHO cells overexpressing CLDN-18.2 were used superimposed, and each mouse was injected with 1e+7 cells intraperitoneally.
  • the inguinal lymph nodes, popliteal lymph nodes and spleen of the mice were taken, and the lymphocyte-rich suspension was obtained after grinding in DMEM medium.
  • Sp2/0 (ATCC) fusion The fusion products were cultured in DMEM complete medium containing 1:50 HAT (hypoxanthine, methotrexate and thymidine) for 5 days to screen for successfully fused cells (ie, hybridomas). It was then changed to DMEM complete medium containing 1:50 HT (hypoxanthine and thymidine) until the end of the selection.
  • HAT hyperxanthine, methotrexate and thymidine
  • the ratio of DMEM complete medium is: 15% FBS (fetal bovine serum)+1:50L-glutamine+100U/mL penicillin-streptomycin+1:100 OPI (oxaloacetate, pyruvate and insulin), incubator with the proviso that 8% CO 2, 37 °C.
  • FBS fetal bovine serum
  • OPI oxaloacetate, pyruvate and insulin
  • the DNA coding sequence corresponding to the variable region of the anti-CLDN-18.2 murine antibody was determined by a method based on degenerate primer PCR.
  • the candidate hybridoma cells were cultured, cells were collected by centrifugation at 1000 rpm, and total RNA was extracted with Trizol. Using this as a template to synthesize the first-strand cDNA, and then using the first-strand cDNA as a subsequent template to amplify the corresponding variable region DNA coding sequence by PCR.
  • the primer sequences used in the amplification reaction were complementary to the first framework and constant regions of the variable region of the antibody (Larrick, JW et al., 1990, Scand. J.
  • the reaction conditions were denaturation at 94 °C for 1 min, annealing at 58 °C for 1 min, extension at 72 °C for 15 s, a total of 32 cycles, and then incubated at 72 °C for 10 min.
  • the PCR product was recovered and purified.
  • the amplified product was sequenced to obtain the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-CLDN-18.2 murine antibody.
  • the germline and rearranged Ig variable region sequence databases were searched for consensus sequences using NCBI Ig-Blast (http://www.ncbi.nlm.nih.gov/projects/igblast/). Based on Kabat (Wu, TT and Kabat, EA 1970 J. Exp. Med., 132: 211-250) and the IMGT system (Lefranc M.-P.
  • amino acid sequences of the light chain and heavy chain variable regions and CDRs of the anti-CLDN-18.2 murine antibody are shown in the following table:
  • the coding sequences of the heavy chain constant region Fc and the light chain constant region ⁇ were cloned from human blood cells (from Beijing Institute of Blood) and introduced into the pCDNA3.1 plasmid.
  • the coding sequences of the heavy chain and light chain variable regions of the aforementioned anti-CLDN-18.2 murine antibody were synthesized by Genscript Company. After the heavy chain variable region coding sequences and light chain variable region coding sequences of various anti-CLDN-18.2 murine antibodies were digested with Bspq I, various combinations as shown in Table 2 were used, and the constant region coding sequences that had been introduced were introduced.
  • the pCDNA3.1 plasmid was sequenced to confirm the correct clone.
  • the gastric cancer cell line NUGC4-CLDN-18.2 cells overexpressing human CLDN-18.2 were respectively diluted with the aforementioned anti-CLDN-18.2 chimeric antibodies Chi-JS012-2, Chi-JS012-9, Chi-JS012-10 in a series of concentrations.
  • Chi-JS012-21 and Chi-JS012-27 were incubated at 4°C for 30 min, then washed and incubated with fluorescently labeled secondary antibodies. Finally, the fluorescence intensity was detected by BD CantoII flow cytometer. The stronger the fluorescent signal, the higher the affinity of the antibody to the target. Fitting by GraphPad dose-dependent antibody binding curves (FIG. 1) and calculate the EC 50 (Table 3), the positive and negative controls, respectively IMAB362 and anti-KLH hu-IgG1 antibody.
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • the aforementioned anti-CLDN-18.2 chimeric antibodies Chi-JS012-2, Chi-JS012-9, Chi-JS012-10, Chi-JS012-21 and Chi-JS012-27 were diluted to serial concentrations with overexpressing human CLDN, respectively.
  • -18.2 target cells CHO-CLDN-18.2 (50,000 cells), and effector cells Jurkat ADCC (100,000 cells) expressing NFAT-Luc and Fc ⁇ RIIIaR were incubated at 37°C for 6h, then the substrate one-glo was added and labeled with enzyme The luciferase signal was detected by the instrument. The higher the fluorescence reading, the stronger the ADCC effect.
  • Antibody dose-dependent ADCC effect curves were fitted by GraphPad ( Figures 2a and 2b), positive and negative controls were IMAB362 and anti-KLH hu-IgG1 antibody, respectively.
  • FIG. 2a and 2b EC 50 values of Chi-JS012-2, Chi-JS012-9, Chi-JS012-10, Chi-JS012-21 and Chi-JS012-27 mediate ADCC effector were 56.08ng / mL, 43.6 ng/mL, 49.51 ng/mL, 63.09 ng/mL, and 43.21 ng/mL, comparable to the positive control IMAB362 (Fig. 2a: 35.98; Fig. 2b: 34.41 ng/mL).
  • the target cells CHO-CLDN-18.2 cells (100,000 cells) overexpressing human CLDN-18.2 were diluted into a series of concentrations of the aforementioned anti-CLDN-18.2 chimeric antibodies Chi-JS012-2, Chi-JS012-9, Chi -JS012-10 and Chi-JS012-27 were mixed, and ten-fold diluted complement serum (Quidel, cat#A113) was added, followed by incubation at 37°C for 1 h. Finally, the cells were resuspended in PBS and PI dye was added. After incubation at 4°C for 5 min, the fluorescence intensity was detected by BD Canto II flow cytometer. Antibody dose-dependent complement killing curves were fitted by GraphPad ( Figure 3). Positive and negative controls were IMAB362 and anti-KLH hu-IgG1 antibody, respectively.
  • EC 50 values shown in Figure 3 Chi-JS012-2, Chi-JS012-9, Chi-JS012-10 and Chi-JS012-27 CDC mediated effects were 461.2ng / mL, 316ng / mL, 358.3ng /mL and 264.5ng/mL, comparable to the positive control IMAB362 (353.6ng/mL).
  • the antibody variable region was optimized by humanization.
  • the optimization results are as follows:
  • HCDR2 CISSGSGTIYYADTVKG (SEQ ID NO: 2) optimization of S ISSGSGTIYYAD S VKG (SEQ ID NO : 41) or YISSGSGTIYYADSVKG (SEQ ID NO: 72) ;
  • HCDR2 WINTYTGESTYADDFKG (SEQ ID NO: 11) optimized to WINTYTGESTYAD G F T G (SEQ ID NO: 40),
  • TISGGDSYTYYPDSVRG (SEQ ID NO: 29) was optimized TISGGDSYTYYPDSV K G (SEQ ID NO: 74);
  • HCDR3 AYYGNGFSF (SEQ ID NO:3) optimized to AYYGN A FSF (SEQ ID NO:73);
  • HCDR3 SYNGNSLPY (SEQ ID NO:30) optimized to SYN A NSLPY (SEQ ID NO:75);
  • Humanized anti-CLDN-18.2 antibodies JS012-Chi9-hu7, JS012-Chi9-hu7-v2, JS012-Chi27-hu3-v2, JS012-Chi27-hu6-v3-2 and JS012-Chi2-hu2-v3-2 were performed follow-up experiments.
  • the CDR/variable region sequences of the above antibodies are shown in Tables 4-1 and 4-2, and the amino acid sequences and nucleotide coding sequences of the heavy/light chains are shown in Table 5.
  • the heavy chain and light chain variable region coding sequences of humanized anti-CLDN-18.2 antibodies were synthesized by Genscript Company, and the heavy chain variable region coding sequences and light chain variable regions of various synthetic humanized anti-CLDN-18.2 antibodies.
  • the coding sequence was digested with Bspq I and introduced into the pCDNA3.1 plasmid that already contained the coding sequence of the constant region, and the correct clone was confirmed by sequencing.
  • the humanized antibody JS012-Chi2-hu2-v3-2 was optimized by adding a glycoform regulator (CDFS01, Shanghai Opmax Biotechnology Co., Ltd.) in cell culture to obtain a defucosylated antibody JS012-Chi2- hu2-v3-2a.
  • Antibody JS012-Chi2-hu2-v3-2a is a humanized anti-CLDN-18.2 antibody that is less than 30% fucosylated.
  • the fluorescence signal is detected by FACS; the stronger the detected fluorescence signal, the higher the antibody affinity, that is, the higher the target binding activity.
  • Antibody binding curves were fitted with GraphPad ( Figures 4a, 4b and 4c), positive and negative controls were IMAB362 and anti-KLH hu-IgG1 antibodies, respectively.
  • JS012-Chi9-hu7-v2, JS012-Chi9-hu7, JS012-Chi27-hu3-v2, JS012-Chi2-hu2-v3-2 and JS012-Chi27-hu6-v3- 2 can bind to human CLDN-18.2 highly expressed on the surface of gastric cancer cell line NUGC4 cells, among which JS012-Chi9-hu7-v2, JS012-Chi9-hu7, JS012-Chi27-hu3-v2 and JS012-Chi2-hu2-v3 -2 EC 50 comparable to the positive control.
  • the humanized anti-CLDN-18.2 antibody is of human IgG1 subtype, which can mediate ADCC effect.
  • the Fc end of the antibody binds to the Fc ⁇ IIIaR receptor on the surface of NK cells to activate NK cells to kill target cells.
  • the ADCC effect in vivo was simulated by a reporter gene system.
  • the humanized anti-CLDN-18.2 antibody was co-incubated with the target cells CHO-CLDN-18.2 cells and Jurkat ADCC effector cells, and the signal was detected by a microplate reader after adding the substrate one-glo. Data were analyzed with GraphPad to compare dose-dependent antibody ADCC effects ( Figures 5a, 5b and 5c). Positive and negative controls were IMAB362 and anti-KLH hu-IgG1 antibody, respectively.
  • JS012-Chi9-hu7-v2 As shown in Figures 5a, 5b and 5c, JS012-Chi9-hu7-v2, JS012-Chi9-hu7, JS012-Chi27-hu3-v2, JS012-Chi2-hu2-v3-2 and JS012-Chi27-hu6-v3- 2
  • the EC 50 values of ADCC-mediated effects were 22.5ng/mL, 23.63ng/mL, 35.75ng/mL, 14.18ng/mL and 74.24ng/mL, which were significantly better than the positive control IMAB362.
  • Humanized anti-CLDN-18.2 antibody can also mediate the CDC effect, killing target cells by forming a membrane attack complex.
  • Complement serum (1:10 dilution) was incubated with humanized anti-CLDN-18.2 antibody and target cells CHO-CLDN-18.2 (100,000 cells) for 1 h in a 37°C incubator, and the target was detected by propidium iodide (PI) staining Cell survival and killing situation, calculate the relative killing rate.
  • PI propidium iodide
  • the dose-dependent CDC effect of the humanized anti-CLDN-18.2 antibody was reflected in relative killing rates ( Figures 6a, 6b and 6c). Positive and negative controls were IMAB362 and anti-KLH hu-IgG1 antibody, respectively.
  • JS012-Chi9-hu7-v2 JS012-Chi9-hu7, JS012-Chi27-hu3-v2, JS012-Chi2-hu2-v3-2 and JS012-Chi27-hu6-v3- 2
  • the EC 50 values for mediating CDC effects were 134.2 ng/mL, 86.04 ng/mL, 168.9 ng/mL, 166.5 ng/mL and 714.9 ng/mL, in which JS012-Chi9-hu7-v2, JS012-Chi9-hu7 , JS012-Chi27-hu3-v2 and JS012-Chi2-hu2-v3-2 were significantly better than the positive control IMAB362.
  • Example 11 Human Gastric Transplantation of Humanized Anti-CLDN-18.2 Antibody to M-NSG Mice Inhibitory effect of cancer MKN45 hClaudin18.2 Mixeno tumor growth
  • Anti-KLH hIgG1 negative control group 10mg/kg
  • IMAB362 positive control group 10mg/kg.
  • TGI% tumor inhibition rate
  • the mean tumor volume of the anti-KLH hIgG1 negative control group was 1276 ⁇ 228 mm 3 .
  • the mean tumor volumes of JS012-Chi2-hu2a v3-2 at doses of 1, 3 and 10 mg/kg were 235 ⁇ 25 mm 3 , 243 ⁇ 33 mm 3 and 343 ⁇ 63 mm 3 , respectively, with TGI scores of 81.6%, 81.0% and 73.1 %, showing a significant tumor suppressor effect.
  • the mean tumor volume of IMAB362 at the 10 mg/kg dose was 361 ⁇ 73 mm 3 with a TGI of 71.7%. Under the same dose condition (10mg/kg), the inhibitory effect of JS012-Chi2-hu2 v3-2a was slightly better than that of IMAB362.
  • Example 12 Humanized anti-CLDN-18.2 antibody to CB-17 SCID mice transplanted with human pancreas Inhibitory effect of adenocarcinoma hCLDN18.2 MIA PaCa-2 tumor growth
  • Anti-KLH hIgG1 negative control group 3mg/kg
  • IMAB362 positive control group 3mg/kg
  • JS012-Chi2-hu2 v3-2a treatment group 3mg/kg.
  • the groups were administered on the same day.
  • the route of administration in all groups was intraperitoneal injection, twice a week for 6 consecutive administrations, and the experiment was terminated 4 days after the last administration.
  • the tumor volume and body weight were measured 3 times a week, and the body weight and tumor volume of the mice were recorded.
  • TGI% tumor inhibition rate
  • T0 the mean tumor volume of the treatment group or the positive control group on the 0th day of administration
  • Vi the negative control group on the ith day of administration
  • the mean tumor volume of , V0 the mean tumor volume of the negative control group on the 0th day of administration
  • the mean tumor volume in the anti-KLH hIgG1 group was 2235 ⁇ 145 mm3.
  • the mean tumor volume of IMAB362 at the 3 mg/kg dose was 465 ⁇ 74 mm3 with a TGI of 82.5%.
  • the mean tumor volumes of JS012-Chi2-hu2 v3-2a at doses of 0.3, 1 and 3 mg/kg were 1455 ⁇ 142mm3, 673 ⁇ 153mm3 and 74 ⁇ 19mm3, respectively, and the TGI was 36.3%, 72.8% and 100.6%, respectively.

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Abstract

提供了与CLDN-18.2特异性结合的抗体或其抗原结合片段和包含其的组合物。还提供了编码所述抗体或其抗原结合片段的核酸分子，用于表达所述抗体或其抗原结合片段的表达载体和宿主细胞，以及所述抗体或其抗原结合片段的治疗或诊断方法和用途。

Description

抗CLDN-18.2抗体及其用途 技术领域
本发明提供了与CLDN-18.2特异性结合的抗体或其抗原结合片段和包含其的组合物。还提供了编码本发明抗体或其抗原结合片段的核酸分子，用于表达本发明抗体或其抗原结合片段的表达载体和宿主细胞，以及本发明抗体或其抗原结合片段的治疗或诊断方法和用途。
背景技术
胃癌是全世界范围内发病率最高的癌症之一。据世界卫生组织癌控项目的统计数据，全球每年死于癌症的患者高达700万，其中死于胃癌的患者占70万例。与常规的胃癌治疗方案相比，基于抗体的治疗方案由于具有高特异性和低副作用，具有深远的应用前景。
Claudin，又称CLDN，是一类建立细胞旁屏障并控制细胞间分子流动的细胞表面蛋白家族，目前已发现至少有26种。Claudin蛋白家族成员是紧密连接的重要结构成分，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。Claudin分子跨细胞膜四次，N端和C端均落在细胞质中。不同的Claudin成员在不同组织中表达，且其功能的改变与癌症形成相关联。Claudin 1、Claudin 18和Claudin 10的表达水平变化分别与肠癌、胃癌和肝细胞癌相关。
Claudin 18(CLDN18)有两种选择性剪接变异体CLDN-18.1和CLDN-18.2。Claudin 18.1(CLDN-18.1)选择性地在正常肺和胃上皮中表达。Claudin 18.2(CLDN-18.2)在正常胃上皮短寿细胞中呈现微量表达，但在肿瘤细胞中，Claudin 18.2在多种癌症类型中均呈现强烈表达，如75％的胃癌患者高表达Claudin 18.2，50％的胰腺癌患 者高表达Claudin 18.2，30％的食管癌患者高表达Claudin 18.2，在肺癌等中也有高表达。
尽管目前已有Ganymed公司开发的Claudiximab(IMAB362)在胃食道癌晚期的II期临床试验中(WO2007059997)，仍需要在亲和力、特异性等方面相比于已知抗体改善的新型抗CLDN18.2抗体。
发明内容
本发明提供了抗CLDN-18.2抗体或其抗原结合片段，其具有针对人CLDN-18.2的高亲和力和高特异性等优势。本发明提供的抗CLDN-18.2抗体或其抗原结合片段可作为独立的疗法或与其它疗法/或其他抗癌药剂联合，用于诸如癌症的治疗。
在一个方面，本发明提供了一种抗CLDN-18.2抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中，
所述重链可变区包含HCDR1、HCDR2和HCDR3，其中
所述HCDR1的序列选自如SEQ ID NO:1、10、16、22、28、34和37所示的氨基酸序列；
所述HCDR2的序列选自如SEQ ID NO:62、63、17、23、29、35、38、72和74所示的氨基酸序列，所述SEQID NO:62所示的HCDR2中X 1为C或S，X 2为T或S，所述SEQ ID NO:63所示的HCDR2中X 3为D或G，X 4为K或T；和
所述HCDR3的序列选自如SEQ ID NO:3、12、18、24、30、36、39、73和75所示的氨基酸序列；和
所述轻链可变区包含LCDR1、LCDR2和LCDR3，其中
所述LCDR1的序列选自如SEQ ID NO:4、7、13、19、25和31所示的氨基酸序列；
所述LCDR2的序列选自如SEQ ID NO:5、8、14、20、26和32所示的氨基酸序列；和
所述LCDR3的序列选自如SEQ ID NO:6、9、15、21、27 和33所示的氨基酸序列。
在一些实施方式中，本发明所述重链可变区包含：
(Ⅰ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或
(Ⅱ)氨基酸序列分别如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的HCDR1、HCDR2和HCDR3；或
(Ⅲ)氨基酸序列分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的HCDR1、HCDR2和HCDR3；或
(Ⅳ)氨基酸序列分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的HCDR1、HCDR2和HCDR3；或
(Ⅴ)氨基酸序列分别如SEQ ID NO:28、SEQ ID NO:29和SEQ ID NO:30所示的HCDR1、HCDR2和HCDR3；或
(Ⅵ)氨基酸序列分别如SEQ ID NO:34、SEQ ID NO:35和SEQ ID NO:36所示的HCDR1、HCDR2和HCDR3；或
(Ⅶ)氨基酸序列分别如SEQ ID NO:37、SEQ ID NO:38和SEQ ID NO:39所示的HCDR1、HCDR2和HCDR3；或
(Ⅷ)氨基酸序列分别如SEQ ID NO:10、SEQ ID NO:40和SEQ ID NO:12所示的HCDR1、HCDR2和HCDR3；或
(Ⅸ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:41和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或
(Ⅹ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:72和SEQ ID NO:73所示的HCDR1、HCDR2和HCDR3；或
(Ⅺ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:72和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或
(Ⅻ)氨基酸序列分别如SEQ ID NO:28、SEQ ID NO:74和SEQ ID NO:75所示的HCDR1、HCDR2和HCDR3；或
(XIII)氨基酸序列分别如SEQ ID NO:28、SEQ ID NO:74和SEQ ID NO:30所示的HCDR1、HCDR2和HCDR3；和
所述轻链可变区包含：
(Ⅰ)氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；或
(Ⅱ)氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的LCDR1、LCDR2和LCDR3；或
(Ⅲ)氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的LCDR1、LCDR2和LCDR3；或
(Ⅳ)氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的LCDR1、LCDR2和LCDR3；或
(Ⅴ)氨基酸序列分别如SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的LCDR1、LCDR2和LCDR3；或
(Ⅵ)氨基酸序列分别如SEQ ID NO:31、SEQ ID NO:32和SEQ ID NO:33所示的LCDR1、LCDR2和LCDR3。
在一些实施方式中，本发明所述抗体或其抗原结合片段包含：
(Ⅰ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；或
(Ⅱ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的LCDR1、LCDR2和LCDR3；或
(Ⅲ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的LCDR1、LCDR2和LCDR3；或
(Ⅳ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的LCDR1、LCDR2和LCDR3；或
(Ⅴ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的LCDR1、LCDR2和LCDR3；或
(Ⅵ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:28、SEQ ID NO:29和SEQ ID NO:30所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:31、SEQ ID NO:32和SEQ ID NO:33所示的LCDR1、LCDR2和LCDR3；或
(Ⅶ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:34、SEQ ID NO:35和SEQ ID NO:36所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的LCDR1、LCDR2和LCDR3；或
(Ⅷ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:37、SEQ ID NO:38和SEQ ID NO:39所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:31、SEQ ID NO:32和SEQ ID NO:33所示的LCDR1、LCDR2和LCDR3；或
(Ⅸ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的LCDR1、LCDR2和LCDR3；或
(Ⅹ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:28、SEQ ID NO:29和SEQ ID NO:30所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的LCDR1、LCDR2和LCDR3；或
(Ⅺ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的LCDR1、LCDR2和LCDR3；或
(Ⅻ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:10、SEQ  ID NO:40和SEQ ID NO:12所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的LCDR1、LCDR2和LCDR3；或
(XIII)重链可变区，其包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:41和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的LCDR1、LCDR2和LCDR3；或
(XⅣ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:72和SEQ ID NO:73所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的LCDR1、LCDR2和LCDR3；或
(XⅤ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:72和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的LCDR1、LCDR2和LCDR3；或
(XⅥ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:28、SEQ ID NO:74和SEQ ID NO:30所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的LCDR1、LCDR2和LCDR3；或
(XⅦ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:28、SEQ ID NO:74和SEQ ID NO:75所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的LCDR1、LCDR2和LCDR3。
在一些实施方式中，本发明所述重链可变区的氨基酸序列包含选自如SEQ ID NO:42、45、47、49、51、53、54、76、78、82和83所示的序列，或与SEQ ID NO:42、45、47、49、51、53、54、76、78、82和83所示序列中任一者具有至少95％、96％、97％、98％或99％同一性的氨基酸序列；和
所述轻链可变区的氨基酸序列包含选自如SEQ ID NO:43、44、 46、48、50和52所示的序列，或与SEQ ID NO:43、44、46、48、50和52所示序列中任一者具有至少95％、96％、97％、98％或99％同一性的氨基酸序列。
在一些实施方式中，本发明所述抗体或其抗原结合片段包含：
(Ⅰ)氨基酸序列如SEQ ID NO:42所示的重链可变区和氨基酸序列如SEQ ID NO:43所示的轻链可变区；或
(Ⅱ)氨基酸序列如SEQ ID NO:42所示的重链可变区和氨基酸序列如SEQ ID NO:44所示的轻链可变区；或
(Ⅲ)氨基酸序列如SEQ ID NO:45所示的重链可变区和氨基酸序列如SEQ ID NO:46所示的轻链可变区；或
(Ⅳ)氨基酸序列如SEQ ID NO:47所示的重链可变区和氨基酸序列如SEQ ID NO:48所示的轻链可变区；或
(Ⅴ)氨基酸序列如SEQ ID NO:49所示的重链可变区和氨基酸序列如SEQ ID NO:50所示的轻链可变区；或
(Ⅵ)氨基酸序列如SEQ ID NO:51所示的重链可变区和氨基酸序列如SEQ ID NO:52所示的轻链可变区；或
(Ⅶ)氨基酸序列如SEQ ID NO:53所示的重链可变区和氨基酸序列如SEQ ID NO:50所示的轻链可变区；或
(Ⅷ)氨基酸序列如SEQ ID NO:54所示的重链可变区和氨基酸序列如SEQ ID NO:52所示的轻链可变区；或
(Ⅸ)氨基酸序列如SEQ ID NO:45所示的重链可变区和氨基酸序列如SEQ ID NO:48所示的轻链可变区；或
(Ⅹ)氨基酸序列如SEQ ID NO:51所示的重链可变区和氨基酸序列如SEQ ID NO:46所示的轻链可变区；或
(Ⅺ)氨基酸序列如SEQ ID NO:49所示的重链可变区和氨基酸序列如SEQ ID NO:46所示的轻链可变区。
在一些实施方式中，本发明所述抗体或其抗原结合片段包含重链可变区和轻链可变区，其中
所述重链可变区的氨基酸序列包含选自如SEQ ID NO:55、57、 58、59、60、76、78、79、82和83所示的序列，或与SEQ ID NO:55、57、58、59、60、76、78、79、82和83所示序列中任一者具有至少95％、96％、97％、98％或99％同一性的氨基酸序列；和
所述轻链可变区的氨基酸序列包含选自如SEQ ID NO:56、61、77、80和81所示的序列，或与SEQ ID NO:56、61、77、80和81所示序列具有至少95％、96％、97％、98％或99％同一性的氨基酸序列。
在一些实施方式中，本发明所述抗体或其抗原结合片段包含重链可变区和轻链可变区，其中所述重链可变区包含如SEQ ID NO:55、57、60、76、78、79、82或83中任一项所示的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:56、61、77、80或81中任一项所示的氨基酸序列。
在一些实施方式中，本发明所述抗体或其抗原结合片段包含重链可变区和轻链可变区，其中所述重链可变区包含如SEQ ID NO:55、57、82或83所示的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:56所示的氨基酸序列。
在一些实施方式中，本发明所述抗体或其抗原结合片段包含重链可变区和轻链可变区，其中所述重链可变区包含如SEQ ID NO:60、76或78所示的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:61所示的氨基酸序列。
在一些实施方式中，本发明所述抗体或其抗原结合片段包含重链可变区和轻链可变区，其中
所述重链可变区包含如SEQ ID NO:55所示的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:56所示的氨基酸序列；或
所述重链可变区包含如SEQ ID NO:57所示的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:56所示的氨基酸序列；或
所述重链可变区包含如SEQ ID NO:58所示的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:56所示的氨基酸序列；或
所述重链可变区包含如SEQ ID NO:59所示的氨基酸序列；和所 述轻链可变区包含如SEQ ID NO:56所示的氨基酸序列；或
所述重链可变区包含如SEQ ID NO:60所示的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:61所示的氨基酸序列；或
所述重链可变区包含如SEQ ID NO:76所示的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:77所示的氨基酸序列；或
所述重链可变区包含如SEQ ID NO:79所示的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:80所示的氨基酸序列；或
所述重链可变区包含如SEQ ID NO:55所示的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:81所示的氨基酸序列。在一些实施方式中，本发明所述抗体或其抗原结合片段包含：重链，所述重链的氨基酸序列如SEQ ID NO:64或其变体或者SEQ ID NO:68或其变体所示，和轻链，所述轻链的氨基酸序列如SEQ ID NO:65或其变体或者SEQ ID NO:69或其变体所示，其中所述变体在其可变区包含1、2、3、4或5个氨基酸变化；
优选地，SEQ ID NO:64的变体包含在第50或63位或其组合处的氨基酸变化；和/或SEQ ID NO:68的变体包含在第63或65位或其组合处的氨基酸变化；
优选地，SEQ ID NO:64的变体包含S50C或S63T或其组合；和/或SEQ ID NO:68的变体包含G63D或T65K或其组合。
在一些实施方式中，本发明所述抗体或其抗原结合片段包含：重链和轻链，其中
所述重链包含如SEQ ID NO:64所示的氨基酸序列，或与SEQ ID NO:64所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:65所示的氨基酸序列，或与SEQ ID NO:65所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
所述重链包含如SEQ ID NO:68所示的氨基酸序列，或与SEQ ID NO:68所示的氨基酸序列具有至少90％、92％、94％、95％、96％、 97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:69所示的氨基酸序列，或与SEQ ID NO:69所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
所述重链包含如SEQ ID NO:84所示的氨基酸序列，或与SEQ ID NO:84所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:85所示的氨基酸序列，或与SEQ ID NO:85所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
所述重链包含如SEQ ID NO:86所示的氨基酸序列，或与SEQ ID NO:86所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:87所示的氨基酸序列，或与SEQ ID NO:87所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
所述重链包含如SEQ ID NO:88所示的氨基酸序列，或与SEQ ID NO:88所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:89所示的氨基酸序列，或与SEQ ID NO:89所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
所述重链包含如SEQ ID NO:90所示的氨基酸序列，或与SEQ ID NO:90所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:91所示的氨基酸序列，或与SEQ ID NO:91所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
所述重链包含如SEQ ID NO:92所示的氨基酸序列，或与SEQ  ID NO:92所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:93所示的氨基酸序列，或与SEQ ID NO:93所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
所述重链包含如SEQ ID NO:94所示的氨基酸序列，或与SEQ ID NO:94所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:95所示的氨基酸序列，或与SEQ ID NO:95所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
所述重链包含如SEQ ID NO:96所示的氨基酸序列，或与SEQ ID NO:96所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:97所示的氨基酸序列，或与SEQ ID NO:97所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
所述重链包含如SEQ ID NO:98所示的氨基酸序列，或与SEQ ID NO:98所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:99所示的氨基酸序列，或与SEQ ID NO:99所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
所述重链包含如SEQ ID NO:100所示的氨基酸序列，或与SEQ ID NO:100所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:101所示的氨基酸序列，或与SEQ ID NO:101所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
在一些实施方式中，本发明所述抗体或其抗原结合片段包含：
(Ⅰ)氨基酸序列如SEQ ID NO:64所示的重链和氨基酸序列如SEQ ID NO:65所示的轻链；或
(Ⅱ)氨基酸序列如SEQ ID NO:68所示的重链和氨基酸序列如SEQ ID NO:69所示的轻链。
在一些实施方式中，本发明所述抗体为鼠源抗体、嵌合抗体、人源化抗体、或全人抗体。
在一些实施方式中，本发明所述抗原结合片段为Fab、Fab'、Fab'-SH、F(ab')2、Fv、scFv或sdAb或双抗体。
在一些实施方式中，本发明所述抗体为任何IgG亚型，如IgG1、IgG2、IgG3或IgG4；优选地；所述抗体是低或无岩藻糖基化的。
在一些实施方式中，本发明所述抗体是低岩藻糖基化的。
在一些实施方式中，本发明所述抗体是无岩藻糖基化的。
在另一个方面，本发明提供了一种分离的抗CLDN-18.2抗体或其抗原结合片段，其具有以下特性中的一种或多种：
(1)与本文所述的抗CLDN-18.2抗体或其抗原结合片段结合相同或者完全或部分重叠的人CLDN-18.2蛋白的表位；
(2)与本文所述的抗CLDN-18.2抗体或其抗原结合片段竞争结合人CLDN-18.2蛋白的表位；
(3)与人CLDN-18.2蛋白结合，但是不结合人CLDN-18.1蛋白；
(4)诱导表达人CLDN-18.2蛋白的细胞的ADCC效应；和
(5)诱导表达人CLDN-18.2蛋白的细胞的CDC效应。
在又一个方面，本发明提供了一种多核苷酸，其编码如本文所述的抗CLDN-18.2抗体或其抗原结合片段。
在又一个方面，本发明提供了一种表达载体，其包含如本文所述的多核苷酸，优选地，所述载体为真核表达载体。
在又一个方面，本发明提供了一种宿主细胞，其包含如本文所述的多核苷酸或如本文所述的表达载体，或表达如本发明所述的抗CLDN-18.2抗体或其抗原结合片段，优选地，所述宿主细胞是真核 细胞，更优选哺乳动物细胞。
在又一个方面，本发明提供了一种用于制备如本文所述的抗CLDN-18.2抗体或其抗原结合片段的方法，所述方法包括在适合于所述抗体或其抗原结合片段表达的条件下培养本文所述的宿主细胞，并从所述宿主细胞回收所表达的抗体或其抗原结合片段。
在又一个方面，本发明提供了一种药物组合物，其包含如本文所述的抗CLDN-18.2抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、或本文所述的宿主细胞，和药学上可接受的载体或赋形剂。
在又一个方面，本发明提供了如本文所述的抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物在制备用于治疗和/或预防CLDN-18.2介导的疾病或病症的药物中的用途，优选地，所述疾病或病症为癌症。
在又一个方面，本发明提供了如本文所述的抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物，其用于治疗和/或预防CLDN-18.2介导的疾病或病症，优选地，所述疾病或病症为癌症。
在又一个方面，本发明提供了一种治疗和/或预防CLDN-18.2介导的疾病或病症的方法，其包括向有需要的受试者施用如本文所述的抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物，优选地，所述疾病或病症为癌症。
在一些实施方式中，所述癌症选自胃癌、食道癌、胃食管癌、胰腺癌、胆管癌、肺癌、卵巢癌、结肠癌、肝癌、头颈癌、胆囊癌、肠癌和膀胱癌药物。
在又一个方面，本发明提供了一种药物组合，其包含如本文所述的抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物，以及一 种或多种另外的治疗剂。
在又一个方面，本发明提供了一种试剂盒，其包括如本文所述的抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物，优选其进一步包括给药装置。
在又一个方面，本发明提供了一种使用如本文所述的抗体或其抗原结合片段检测CLDN-18.2在样品中的存在的方法。
附图说明
图1：流式细胞术测定的嵌合抗CLDN-18.2抗体的细胞水平亲和力。
图2a和2b：报告基因法测定的嵌合抗CLDN-18.2抗体的ADCC活性。
图3：流式细胞术测定的嵌合抗CLDN-18.2抗体的CDC活性。
图4a、4b和4c：流式细胞术测定的人源化抗CLDN-18.2抗体的细胞水平亲和力。
图5a、5b和5c：报告基因法测定的人源化抗CLDN-18.2抗体的ADCC活性。
图6a、6b和6c：流式细胞术测定的人源化抗CLDN-18.2抗体的CDC活性。
图7：人源化抗CLDN-18.2抗体对M-NSG小鼠移植人胃癌MKN45 hClaudin18.2 Mixeno肿瘤生长的抑制作用。
图8：人源化抗CLDN-18.2抗体对CB-17 SCID小鼠移植人胰腺癌hCLDN18.2 MIA PaCa-2肿瘤生长的抑制作用。
具体实施方式
定义
除非另有说明，本发明的实施将采用分子生物学(包括重组技术)、微生物学、细胞生物学、生物化学和免疫学的常规技术，这些 都在本领域的技术范围内。
为了可以更容易地理解本发明，某些科技术语具体定义如下。除非本文其它部分另有明确定义，否则本文所用的科技术语都具有本发明所属领域普通技术人员通常理解的含义。关于本领域的定义及术语，专业人员具体可参考Current Protocolsin Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。本文(包括权利要求书)所用单数形式包括其相应的复数形式，除非文中另有明确规定。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
术语“和/或”应理解为意指可选项中的任一项或可选项中的任意两项或更多项的组合。
术语“CLDN-18.2”或“Claudin18.2”，是Claudin 18的两个剪接变异体之一。该术语指来自任何脊椎动物(包括哺乳动物如灵长类动物(例如人))和啮齿类动物(例如，小鼠和大鼠)的任何天然CLDN-18.2，除非另有说明。该术语涵盖“全长”未加工的CLDN-18.2以及由细胞内加工产生的任何形式的CLDN-18.2或其任何片段。该术语还包括天然存在的CLDN-18.2的变体，例如，剪接变体或等位变体。在一个优选实施方式中，CLDN-18.2是指来自人和食蟹猴CLDN-18.2全长或其片段(诸如其缺乏信号肽的成熟片段)。
术语“百分比(％)氨基酸序列同一性”或简称“同一性”定义为在将氨基酸序列进行比对(并在必要时导入空位)以获取最大百分比序列同一性，且不将任何保守取代视为序列同一性的部分之后，候选氨基酸序列中的氨基酸残基与参比氨基酸序列中的相同氨基酸残基的百分比。可使用本领域各种方法进行序列比对以便测定百分比氨基酸序列同一性，例如，使用公众可得到的计算机软件如BLAST、BLAST-2、ALIGN或MEGALIGN(DNASTAR)软件。本领域技术人员可以决定测量比对的适宜参数，包括对所比较的序列全长获得最 大比对所需的任何算法。
术语“免疫应答”是指由例如淋巴细胞、抗原呈递细胞、吞噬细胞、粒细胞和由上述细胞或肝产生可溶性大分子(包括抗体、细胞因子和补体)的作用，该作用导致从人体选择性损害、破坏或清除侵入的病原体、感染病原体的细胞或组织、癌细胞或者在自体免疫或病理性炎症的情况下的正常人细胞或组织。
术语“信号转导途径”或“信号转导活性”是指通常由蛋白质间相互作用诸如生长因子对受体的结合启动的生化因果关系，所述关系导致信号从细胞的一部分传递至细胞的另一部分。一般地，传递包括引起信号转导的系列反应中的一种或多种蛋白质上的一个或多个酪氨酸、丝氨酸或苏氨酸残基的特定磷酸化。倒数第二过程通常包括细胞核事件，从而导致基因表达的变化。
术语“活性”或“生物活性”，或术语“生物性质”或“生物特征”此处可互换使用，包括但不限于表位/抗原亲和力和特异性、在体内或体外中和或拮抗CLDN-18.2活性的能力、IC 50、抗体的体内稳定性和抗体的免疫原性质。本领域公知的抗体的其它可鉴定的生物学性质或特征包括，例如，交叉反应性(即通常与靶定肽的非人同源物，或与其它蛋白质或组织的交叉反应性)，和保持哺乳动物细胞中蛋白质高表达水平的能力。使用本领域公知的技术观察、测定或评估前面提及的性质或特征，所述技术包括但不局限于ELISA、FACS或BIACORE等离子体共振分析、不受限制的体外或体内中和测定、受体结合、细胞因子或生长因子的产生和/或分泌、信号转导和不同来源(包括人类、灵长类或任何其它来源)的组织切片的免疫组织化学。
术语“抗体”是指具有所需生物活性的任何形式的抗体。因此，其以最广义使用，具体包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、全人抗体、嵌合抗体和骆驼源化单结构域抗体。
术语“分离的抗体”是指结合化合物的纯化状态，且在这种情况下意指该分子基本不含其它生物分子，例如核酸、蛋白质、脂质、 糖或其它物质例如细胞碎片和生长培养基。术语“分离(的)”并非意指完全不存在这类物质或不存在水、缓冲液或盐，除非它们以明显干扰本文所述结合化合物的实验或治疗应用的量存在。
术语“单克隆抗体”是指获自基本均质抗体群的抗体，即组成该群的各个抗体除可少量存在的可能天然存在的突变之外是相同的。单克隆抗体是高度特异性的，针对单一抗原表位。相比之下，常规(多克隆)抗体制备物通常包括大量针对不同表位(或对不同表位有特异性)的抗体。修饰语“单克隆”表明获自基本均质抗体群的抗体的特征，且不得解释为需要通过任何特定方法产生抗体。
术语“全长抗体”，是指在天然存在时包含四条肽链的免疫球蛋白分子：两条重(H)链(全长时约50-70kDa)和两条轻(L)链(全长时约25kDa)通过二硫键互相连接。每一条重链由重链可变区(在本文中缩写为VH)和重链恒定区(在本文中缩写为CH)组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每一条轻链由轻链可变区(在本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可被进一步细分为具有高可变性的互补决定区(CDR)和其间隔以更保守的称为框架区(FR)的区域。每一个VH或VL区由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白对宿主组织或因子(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(Clq))的结合。
术语“Fc区”用于定义含有至少一部分的恒定区的免疫球蛋白重链的C-末端区域。该术语包括天然序列Fc区和变体Fc区。在一个实施方式中，人IgG重链Fc区从重链的Cys226或从Pro230延伸至羧基端。然而，Fc区的C-端赖氨酸(Lys447)可以存在或可以不存在。除非本文中另外指出，Fc区或恒定区中的氨基酸残基的编号根据EU编号系统，也称为EU索引，如Kabat,E.A.等，Sequences of Proteins of Immunological Interest，第5版，Public Health Service  National Institutes of Health,Bethesda,MD(1991),NIH Publication 91-3242中所述。
术语抗体(“亲代抗体”)的“抗原结合片段”包括抗体的片段或衍生物，通常包括亲代抗体的抗原结合区或可变区(例如一个或多个CDR)的至少一个片段，其保持亲代抗体的至少一些结合特异性。抗体结合片段的实例包括但不限于Fab，Fab'，F(ab') 2和Fv片段；双抗体；线性抗体；单链抗体分子，例如sc-Fv；由抗体片段形成的纳米抗体(nanobody)和多特异性抗体。当抗原的结合活性在摩尔浓度基础上表示时，结合片段或衍生物通常保持其抗原结合活性的至少10％。优选结合片段或衍生物保持亲代抗体的抗原结合亲和力的至少20％、50％、70％、80％、90％、95％或100％或更高。还预期抗体的抗原结合片段可包括不明显改变其生物活性的保守或非保守氨基酸取代(称为抗体的“保守变体”或“功能保守变体”)。术语“结合化合物”是指抗体及其结合片段两者。
术语“单链Fv”或“scFv”抗体是指包含抗体的VH和VL结构域的抗体片段，其中这些结构域存在于单条多肽链中。Fv多肽一般还包含VH和VL结构域之间的多肽接头，其使scFv能够形成用于抗原结合的所需结构。
术语“结构域抗体”是只含有重链可变区或轻链可变区的免疫功能性免疫球蛋白片段。在某些情况下，两个或更多个VH区与肽接头共价连接形成二价结构域抗体。二价结构域抗体的2个VH区可靶向相同或不同的抗原。
术语“二价抗体”包含2个抗原结合部位。在某些情况下，2个结合部位具有相同的抗原特异性。然而，二价抗体可以是双特异性的。
术语“双抗体”是指具有两个抗原结合部位的小抗体片段，所述片段包含在同一多肽链(VH-VL或VL-VH)中与轻链可变结构域(VL)连接的重链可变结构域(VH)。通过使用短得不允许在同一链的两个结构域之间配对的接头，迫使该结构域与另一链的互补结构域配对并产生两个抗原结合部位。
术语“嵌合抗体”是具有第一抗体的可变结构域和第二抗体的恒定结构域的抗体，其中第一抗体和第二抗体来自不同物种。通常，可变结构域获自啮齿动物等的抗体(“亲代抗体”)，而恒定结构域序列获自人抗体，使得与亲代啮齿动物抗体相比，所得嵌合抗体在人受试者中诱导不良免疫应答的可能性较低。
术语“人源化抗体”是指含有来自人和非人(例如小鼠、大鼠)抗体的序列的抗体形式。一般而言，人源化抗体包含基本所有的至少一个、通常两个可变结构域，其中所有或基本所有的超变环相当于非人免疫球蛋白的超变环，而所有或基本所有的构架(FR)区是人免疫球蛋白序列的构架区。人源化抗体任选可包含至少一部分的人免疫球蛋白恒定区(Fc)。
术语“全人抗体”是指只包含人免疫球蛋白蛋白质序列的抗体。如在小鼠中、在小鼠细胞中或在来源于小鼠细胞的杂交瘤中产生，则全人抗体可含有鼠糖链。同样，“小鼠抗体”是指仅包含小鼠免疫球蛋白序列的抗体。或者，如果在大鼠中、在大鼠细胞中或在来源于大鼠细胞的杂交瘤中产生，则全人抗体可含有大鼠糖链。同样，“大鼠抗体”是指仅包含大鼠免疫球蛋白序列的抗体。
“同种型”抗体是指由重链恒定区基因提供的抗体种类(例如，IgM、IgE、IgG诸如IgGl、IgG2或IgG4)。同种型还包括这些种类之一的修饰形式，其中修饰已被产生来改变Fc功能，例如以增强或减弱效应子功能或对Fc受体的结合。
术语“表位”是指抗体所结合的抗原区域。表位可以由连续的氨基酸形成或者通过蛋白的三级折叠而并置的非连续氨基酸形成。
“亲和力”或“结合亲和力”指反映结合对子的成员之间相互作用的固有结合亲和力。分子X对其配偶物Y的亲和力可以通常由平衡解离常数(K D)代表，平衡解离常数是解离速率常数和结合速率常数(分别是k dis和k on)的比值。亲和力可以由本领域已知的常见方法测量。用于测量亲和力的一个具体方法是本文中的ForteBio动力学结合测定法。
术语“不结合”蛋白或细胞是指，不与蛋白或细胞结合，或者不以高亲和力与其结合，即结合蛋白或细胞的K D为1.0×10 -6M或更高，更优选1.0×10 -5M或更高，更优选1.0×10 -4M或更高、1.0×10 -3M或更高，更优选1.0×10 -2M或更高。
术语“高亲和性”对于IgG抗体而言，是指对于抗原的K D为1.0×10 -6M或更低，优选5.0×10 -8M或更低，更优选1.0×10 -8M或更低、5.0×10 -9M或更低，更优选1.0×10 -9M或更低。对于其他抗体亚型，“高亲和性”结合可能会变化。例如，IgM亚型的“高亲和性”结合是指K D为10 -6M或更低，优选10 -7M或更低，更优选10 -8M或更低。
术语“抗体依赖的细胞毒性”、“抗体依赖的细胞介导的细胞毒性”或“ADCC”是指细胞介导的免疫防御，其中免疫系统效应细胞主动地将细胞膜表面抗原与抗体，例如Claudin18.2抗体，结合的靶细胞例如癌细胞裂解。
术语“补体依赖的细胞毒性”或“CDC”是指IgG和IgM抗体的效应功能，当与表面抗原结合时引发典型的补体途径，包括形成膜攻击复合体以及靶细胞裂解。本发明的抗体，与Claudin 18.2结合时，引发对癌细胞的CDC。
术语“核酸”或“多核苷酸”是指脱氧核糖核酸(DNA)或核糖核酸(RNA)及其呈单链或双链形式的聚合物。除非明确地限制，否则术语包括具有与参照核酸相似的结合性质并且以与天然存在的核苷酸相似的方式被代谢的含有已知的天然核苷酸的类似物的核酸(参见，属于Kariko等人的美国专利No.8,278,036，其公开了尿苷被假尿苷替代的mRNA分子，合成所述mRNA分子的方法以及用于在体内递送治疗性蛋白的方法)。除非另有所指，否则特定核酸序列还隐含地包括其保守修饰的变体(例如，简并密码子取代)、等位基因、直系同源物、SNP和互补序列以及明确指出的序列。具体地，简并密码子取代可通过生成其中一个或多个选择的(或全部)密码子的第三位被混合碱基和/或脱氧肌苷残基取代的序列来实现(Batzer等 人，Nucleic Acid Res.19:5081(1991)；Ohtsuka等人，J.Biol.Chem.260:2605-2608(1985)；和Rossolini等人，Mol.Cell.Probes 8:91-98(1994))。
“构建体”是指任何重组多核苷酸分子(诸如质粒、粘粒、病毒、自主复制多核苷酸分子、噬菌体或线性或环状单链或双链DNA或RNA多核苷酸分子)，衍生自任何来源，能够与基因组整合或自主复制，构成如下多核苷酸分子，其中已经以功能操作的方式连接(即，可操作地连接)一或多个多核苷酸分子。重组构建体通常会包含可操作地连接至转录起始调节序列的本发明的多核苷酸，这些序列会导引多核苷酸在宿主细胞中的转录。可使用异源及非异源(即，内源)启动子两者导引本发明的核酸的表达。
“载体”是指任何重组多核苷酸构建体，该构建体可用于转化的目的(即将异源DNA引入到宿主细胞中)。一种类型的载体为“质粒”，是指环状双链DNA环，可将额外DNA区段连接至该环中。另一类型的载体为病毒载体，其中可将额外DNA区段连接至病毒基因组中。某些载体能够在被引入到的宿主细胞中自主复制(例如，具有细菌复制起点的细菌载体及游离型哺乳动物载体)。在引入到宿主细胞中后，其他载体(例如，非游离型哺乳动物载体)整合至宿主细胞的基因组中，且因此与宿主基因组一起复制。此外，某些载体能够导引被操作性连接的基因的表达。本文将此类载体称为“表达载体”。
本文所用术语“表达载体”是指能够在转化、转染或转导至宿主细胞中时复制及表达目的基因的核酸分子。表达载体包含一或多个表型选择标记及复制起点，以确保维护载体及以在需要的情况下于宿主内提供扩增。
用于细胞或受体的“活化”、“刺激”和“处理”可具有相同含义，例如细胞或受体用配体活化、刺激或处理，除非上下文另外或明确规定。“配体”包括天然和合成配体，例如细胞因子、细胞因子变体、类似物、突变蛋白和来源于抗体的结合化合物。“配体”还包括小分 子，例如细胞因子的肽模拟物和抗体的肽模拟物。“活化”可指通过内部机制以及外部或环境因素调节的细胞活化。“应答/反应”，例如细胞、组织、器官或生物体的应答，包括生化或生理行为(例如生物区室内的浓度、密度、粘附或迁移、基因表达速率或分化状态)的改变，其中改变与活化、刺激或处理有关，或者与例如遗传编程等内部机制有关。
如本文中所用，术语任何疾病或病症的“治疗”或“医治”在一个实施方式中是指改善疾病或病症(即，减缓或阻止或减少疾病的进展或其临床症状的至少一个)。在另一个实施方式中，“治疗”或“医治”是指缓解或改善至少一个身体参数，包括可能不能被患者辨别出的那些物理参数。在另一个实施方式中，“治疗”或“医治”是指在身体上(例如，可辨别的症状的稳定)、生理上(例如，身体参数的稳定)或在这两方面调节疾病或病症。除非在本文中明确描述，否则用于评估疾病的治疗和/或预防的方法在本领域中通常是已知的。
“受试者”包括任何人或非人动物。术语“非人动物”包括所有脊椎动物，例如哺乳动物和非哺乳动物，诸如非人灵长类动物、绵羊、狗、猫、马、牛、鸡、两栖动物、爬行动物等。如本文中所用，术语“cyno”或“食蟹猴”是指食蟹猴。
“联合”一种或多种其它治疗剂的施用包括同时(共同)施用和任意次序的连续施用。
“治疗有效量”、“治疗有效剂量”和“有效量”是指本发明的CLDN-18.2抗体或其抗原结合片段当单独或与其它治疗药物组合给予细胞、组织或受试者时，有效预防或改善一种或多种疾病或病况的症状或该疾病或病况的发展的量。治疗有效剂量还指足以导致症状改善的抗体或其抗原结合片段的量，例如治疗、治愈、预防或改善相关医学病况或者提高这类病况的治疗、治愈、预防或改善的速度的量。当对个体施用单独给予的活性成分时，治疗有效剂量仅是指该成分。当组合施用时，治疗有效剂量是指引起治疗效果的活性成分的综合量，不论是组合、依次给予还是同时给予。治疗剂的有 效量将导致诊断标准或参数提高至少10％；通常至少20％；优选至少约30％；更优选至少40％，最优选至少50％。
“癌症”和“癌性”指或描述哺乳动物中特征通常为细胞生长不受调控的生理疾患。此定义中包括良性和恶性癌症以及休眠肿瘤或微转移。癌症的例子包括但不限于癌，淋巴瘤，母细胞瘤，肉瘤，和白血病。此类癌症的更具体例子包括鳞状细胞癌，肺癌(包括小细胞肺癌，非小细胞肺癌，肺的腺癌，和肺的鳞癌)，腹膜癌，肝细胞癌，胃的癌或胃癌(包括胃肠癌)，胰腺癌，成胶质细胞瘤，宫颈癌，卵巢癌，肝癌，膀胱癌，肝瘤(hepatoma)，乳腺癌，结肠癌，结肠直肠癌，子宫内膜癌或子宫癌，唾液腺癌，肾癌或肾的癌，肝癌，前列腺癌，外阴癌，甲状腺癌，肝的癌，及各种类型的头和颈癌，以及B细胞淋巴瘤(包括低级/滤泡性非何杰金氏淋巴瘤(NHL)，小淋巴细胞性(SL)NHL，中级/滤泡性NHL，中级弥漫性NHL，高级成免疫细胞性NHL，高级成淋巴细胞性NHL，高级小无核裂细胞性NHL，贮积病(bulky disease)NHL，套细胞淋巴瘤，AIDS相关淋巴瘤，和瓦尔登斯特伦氏(Waldenstrom)巨球蛋白血症)，慢性淋巴细胞性白血病(CLL)，急性成淋巴细胞性白血病(ALL)，毛细胞性白血病，慢性成髓细胞性白血病，和移植后淋巴增殖性病症(PTLD)，以及与瘢痣病(phakomatoses)，水肿(诸如与脑瘤有关的)和梅格斯氏(Meigs)综合征有关的异常血管增殖。
抗CLDN-18.2抗体
在一个方面，本发明提供了抗CLDN-18.2抗体或其抗原结合片段。术语“抗CLDN-18.2抗体”、“抗CLDN-18.2”、“CLDN-18.2抗体”或“结合CLDN-18.2的抗体”是指能够以足够的亲合力结合CLDN-18.2蛋白或其片段以致所述抗体可以用作靶向CLDN-18.2中的诊断剂和/或治疗剂。
可采用用于产生抗体的任何合适方法来产生本发明的抗体。任何合适形式的CLDN-18.2都可用作产生抗体的免疫原(抗原)。通 过举例而非限制，任何CLDN-18.2变体或其片段都可用作免疫原。在一些实施方式中，产生鼠源的单克隆抗人CLDN-18.2抗体的杂交瘤细胞可通过本领域公知的方法产生。来源于啮齿动物(如小鼠)的抗体在体内用作治疗药物时可能引起不需要的抗体免疫原性，重复使用导致人体产生针对治疗性抗体的免疫应答，这类免疫应答至少导致丧失治疗功效，而严重的则导致潜在致死过敏反应。降低啮齿动物抗体的免疫原性的一种方法包括嵌合抗体的产生，其中将小鼠可变区与人恒定区融合(Liu等(1987)Proc.Natl.Acad.Sci.USA84:3439-43)。然而，嵌合抗体中的完整啮齿动物可变区的保留仍可能在患者中引起有害的免疫原性。将啮齿动物可变结构域的互补决定区(CDR)环移植到人构架上(即人源化)已被用于进一步将啮齿动物序列减至最低(Jones等(1986)Nature 321:522；Verhoeyen等(1988)Science 239:1534)。
在一些实施方式中，本发明的嵌合或人源化抗体可基于所制备的鼠单克隆杂交瘤抗体的序列来制备。编码重链和轻链免疫球蛋白的DNA可以从目标鼠杂交瘤中获得，并且使用标准分子生物学技术进行工程改造以包含非鼠(例如人)免疫球蛋白序列。
在一些实施方式中，本发明所述的嵌合CLDN-18.2抗体，可使用本领域已知的方法将杂交瘤来源的免疫球蛋白重链和轻链可变区与人IgG恒定区有效连接(参见例如属于Cabilly等人的美国专利No.4,816,567)，获得嵌合型重链和嵌合型轻链来制备。在一些实施方式中，本发明的嵌合抗体包含的恒定区可选自任何人IgG亚型，如IgG1、IgG2、IgG3、IgG4，优选IgG4。
在一些实施方式中，本发明的嵌合CLDN-18.2抗体可由嵌合型轻链与嵌合型重链表达质粒“混合和匹配”转染表达细胞获得，此类“混合和匹配”的抗体的CLDN-18.2结合可使用上述结合测定和其它常规结合测定(例如，ELISA)来进行测试。
本发明的所述抗体的可变区CDR的精确氨基酸序列边界可使用许多公知的方案的任何方案来确定，包括基于抗体的三维结构和 CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883；Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest，第4版，U.S.Department of Health and Human Services,National Institutes of Health(1987))，AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(1999 Nucleic Acids Research,27,209-212)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。本发明抗体的CDR可以由本领域的技术人员根据本领域的任何方案(例如不同的指派系统或组合)确定边界。
应该注意，基于不同的指派系统获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派系统下定义的同一抗体可变区的CDR序列有所不同。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
具有不同特异性(即，针对不同抗原的不同结合位点)的抗体具有不同的CDR。然而，尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat,Chothia、AbM、Contact和North方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia定义的其余CDR残基可以被保守氨基酸残基替代。
本发明所述的人源化抗体，可以使用本领域已知的方法将鼠源 CDR区插入人种系框架区。参见Winter等人的美国专利No.5,225,539及Queen等人的美国专利No.5,530,101；5,585,089；5,693,762和6,180,370。
在一些实施方式中，氨基酸变化包括氨基酸缺失、插入或置换。在一些实施方式中，本发明的抗CLDN-18.2抗体或其抗原结合片段包括具有已通过氨基酸缺失、插入或置换突变的，但仍与上述抗体(特别地在上述序列中描绘的CDR区中)有至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列的那些抗体。在一些实施方式中，本发明的抗体与具体序列中描绘的CDR区相比较时，在CDR区中已通过氨基酸缺失、插入或置换的氨基酸突变不超过1、2、3、4或5个。
在一些实施方式中，编码本发明抗体的多核苷酸包括已通过核苷酸缺失、插入或置换突变的，但仍然与上文中所述的序列中描绘的CDR对应编码区具有至少约60、70、80、90、95或100％同一性的多核苷酸。
在一些实施方式中，可在本文中所提供抗体的Fc区中引入一个或多个氨基酸修饰，以此产生Fc区变体。Fc区变体可包含在一或多个氨基酸位置处包含氨基酸修饰(例如置换)的人Fc区序列(例如人IgG1、IgG2、IgG3或IgG4Fc区)。
在一些实施方式中，可能需要产生经半胱氨酸工程改造的抗体，例如“硫代MAb”，其中抗体的一或多个残基经半胱氨酸残基置换。
在一些实施方式中，可以对抗体进行改变以增加或降低其糖基化程度和/或改变其糖基化模式。对抗体的糖基化位点的添加或缺失可通过改变氨基酸序列以便产生或移除一或多个糖基化位点而方便地实现。举例而言，可实施一或多种氨基酸取代以消除或多个糖基化位点，由此消除该位点处的糖基化。可制备具有改变类型的糖基化的抗体，例如具有减小量的岩藻糖基残基的低或无岩藻糖化抗体或具有增加的等分GlcNac结构的抗体。这类改变的糖基化模式已显示可增加抗体的ADCC能力。
在一些优选实施方式中，本发明提供这样的抗体，该抗体是低或无岩藻糖基化的，从而可以显著地增加抗体与效应细胞上表达受体的结合亲和力，由此导致抗体具有增强的抗体依赖性细胞介导的细胞毒性(ADCC)活性。可以通过MALDI-TOF质谱法测量，相对于与Asn 297连接的所有糖结构(例如复合型、杂合型及高甘露糖型结构)的总和，计算在Asn297处糖链内的岩藻糖的平均量，由此确定岩藻糖的量，例如WO2008/077546中所述。Asn297是指位于Fc区中大约位置297处(Fc区残基的EU编号)的天冬氨酸残基；然而，由于抗体中微小的序列变化，Asn297也可能位于位置297上游或下游约±3个氨基酸位置，即位置294与300之间。参见例如US2003/0157108；US2004/0093621。可以在能够产生去岩藻糖基化或低岩藻糖基化抗体的细胞系中产生此类抗体变体。此类细胞的实例包括蛋白质岩藻糖基化缺陷的Lecl3 CHO细胞(Ripka,J.等，Arch.Biochem.Biophys.249(1986):533-545；US2003/0157108。在一些实施方式中，使用岩藻糖苷酶切除抗体的岩藻糖残基。在一些实施方式中，使用糖型调节剂来控制抗体的岩藻糖残基。糖型调节剂可以是CDFS01，可商购自上海奥浦迈生物科技有限公司。去岩藻糖基化可通过本领域公知的常规方法进行。
抗体的岩藻糖基化的水平可在结构上加以界定。如本文所述，“非岩藻糖基化”或“无岩藻糖基化”是指在抗体中，岩藻糖的含量低于5％，例如约0％、低于1％、低于2％、低于3％、低于4％。术语“低岩藻糖基化”是指在抗体中，岩藻糖的含量是约为大于等于5％且低于30％；例如抗体中岩藻糖的含量约为5％-10％、10％-15％、15％-20％、20％-25％、25％-30％、10％-20％、20％-30％、10％-30％。术语“低岩藻糖基化或无岩藻糖基化”是指在抗体中岩藻糖的含量低于30％。
在一些实施方式中，本文中所提供的抗体可进一步经修饰为含有本领域中已知且轻易获得的其他非蛋白质部分。适合抗体衍生作用的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制 性实例包括，但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1，3-二烷、聚-1，3，6-三烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或无规共聚物)、及葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、聚环氧丙烷/氧化乙烯共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇、及其混合物。
抗体表达
在又一个方面，本发明提供了一种多核苷酸，其编码如本文所述的抗CLDN-18.2抗体或其抗原结合片段。所述多核苷酸可以包含编码抗体的轻链可变区和/或重链可变区的氨基酸序列的多核苷酸，或包含编码抗体的轻链和/或重链的氨基酸序列的多核苷酸。
在又一个方面，本发明提供了一种表达载体，其包含如本文所述的多核苷酸，优选地，所述载体为真核表达载体。在一些实施方式中，如本文所述的多核苷酸包含在一个或多个表达载体中。
在又一个方面，本发明提供了一种宿主细胞，其包含如本文所述的多核苷酸或如本文所述的表达载体，优选地，所述宿主细胞是真核细胞，更优选哺乳动物细胞。
在又一个方面，本发明提供了一种用于制备如本文所述的抗CLDN-18.2抗体或其抗原结合片段的方法，所述方法包括在适合于所述抗体或其抗原结合片段表达的条件下在本文所述的宿主细胞中表达所述抗体或其抗原结合片段，并从所述宿主细胞回收所表达的抗体或其抗原结合片段。
本发明提供用于表达本发明的重组抗体的哺乳动物宿主细胞，包括可获自美国典型培养物保藏中心(ATCC)的许多永生化细胞系。这些尤其包括中国仓鼠卵巢(CHO)细胞、NS0、SP2/0细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞、A549细胞、293T细胞和许多其它细胞系。哺乳动物宿主细胞包括人、小鼠、大鼠、狗、猴、猪、山羊、牛、马和仓鼠细胞。通过测定哪 种细胞系具有高表达水平来选择特别优选的细胞系。
在一个实施方式中，本发明提供制备抗CLDN-18.2抗体的方法，其中所述方法包括，将表达载体导入哺乳动物宿主细胞中时，通过将宿主细胞培养足够的一段时间，以允许抗体在宿主细胞中表达，或者更优选抗体分泌到宿主细胞生长的培养基中,来产生抗体。可采用标准蛋白质纯化方法从培养基中回收抗体。
很可能由不同细胞系表达或在转基因动物中表达的抗体彼此具有不同的糖基化。然而，由本文提供的核酸分子编码的或包含本文提供的氨基酸序列的所有抗体是本发明的组成部分，而不论抗体的糖基化如何。同样，在某些实施方式中，非岩藻糖基化抗体是有利的，因为它们通常在体外和体内具有比其岩藻糖基化对应物更强力的功效，并且不可能是免疫原性的，因为它们的糖结构是天然人血清IgG的正常组分。
药物组合物和药物制剂
在又一个方面，本发明提供了一种药物组合物，其包含如本文所述的抗CLDN-18.2抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞，和药学上可接受的载体或赋形剂。
应理解，本发明提供的抗CLDN-18.2抗体或其药物组合物可以整合制剂中合适的运载体、赋形剂和其他试剂以联合给药，从而提供改善的转移、递送、耐受等。
术语“药物组合物”指这样的制剂，其允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述制剂的受试者具有不可接受的毒性的另外的成分。
可以通过将具有所需纯度的本发明的抗CLDN-18.2抗体与一种或多种任选的药用辅料(Remington's Pharmaceutical Sciences，第16版，Osol,A.编辑(1980))混合来制备包含本文所述的抗CLDN-18.2抗体的药物制剂，优选地以水溶液或冻干制剂的形式。
本发明的药物组合物或制剂还可以包含一种或多种其它活性成分，所述活性成分是被治疗的特定适应证所需的，优选具有不会不利地影响彼此的互补活性的那些活性成分。在一些实施方式中，其它的活性成分为化疗剂、免疫检查点抑制剂、生长抑制剂、抗生素或已知的各种抗肿瘤或抗癌剂，所述活性成分以对于目的用途有效的量合适地组合存在。在一些实施方式中，本发明的药物组合物还包含编码抗CLDN-18.2抗体的多核苷酸的组合物。
在又一个方面，本发明提供了一种药物组合，其包含如本文所述的抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物，以及一种或多种另外的治疗剂。
在又一个方面，本发明提供了一种试剂盒，其包括如本文所述的抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物，优选其进一步包括给药装置。
医药用途
在又一个方面，本发明提供了如本文所述的抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物在制备用于治疗和/或预防CLDN-18.2介导的疾病或病症的药物中的用途，优选地，所述疾病或病症为癌症。
在又一个方面，本发明提供了如本文所述的抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达载体、本文所述的宿主细胞、或本文所述的药物组合物，其用于治疗和/或预防CLDN-18.2介导的疾病或病症，优选地，所述疾病或病症为癌症。
在又一个方面，本发明提供了一种治疗和/或预防CLDN-18.2介导的疾病或病症的方法，其包括向有需要的受试者施用如本文所述的抗体或其抗原结合片段、本文所述的多核苷酸、本文所述的表达 载体、本文所述的宿主细胞、或本文所述的药物组合物，优选地，所述疾病或病症为癌症。
在一些实施方式中，所述癌症选自胃癌、食道癌、胃食管癌、胰腺癌、胆管癌、肺癌、卵巢癌、结肠癌、肝癌、头颈癌、胆囊癌、肠癌和膀胱癌药物。
在一些实施方式中，本发明给药方式包括但不限于口服、静脉内、皮下、肌内、动脉内、关节内(例如在关节炎关节中)、通过吸入、气雾剂递送或肿瘤内给予等。
在一些实施方式中，本发明提供了向受试者联合施用治疗有效量的一种或多种疗法(例如治疗方式和/或其它治疗剂)。在一些实施方式中，所述疗法包括手术治疗和/或放射疗法。
在一些实施方式中，本发明提供的方法或用途还包括向个体施用一种或多种疗法(例如治疗方式和/或其它治疗剂)。可以单独或与疗法中的其它治疗剂组合使用本发明的抗体。例如，可以与至少一种另外的治疗剂共施用。例如，PD-1抗体、PD-L1抗体、LAG-3抗体和/或CTLA-4抗体。
在一个实施方式中，本发明的癌症疾病治疗方法还包括施用CLDN-18.2表达稳定或增加剂。CLDN-18.2的表达优选在癌细胞的细胞表面。CLDN-18.2表达稳定或增加剂可以是奥沙利铂和/或5-FU。
用于诊断和检测的方法
在又一个方面，本发明提供了一种使用如本文所述的抗体或其抗原结合片段检测CLDN-18.2在样品中的存在的方法。术语“检测”用于本文中时，包括定量或定性检测。在一些实施方式中，所述样品是生物样品。在某些实施方式中，生物样品是血、血清或生物来源的其他液体样品。在某些实施方式中，生物样品包含细胞或组织。
本发明包括所叙述特定实施方式的所有组合。本发明的进一步 实施方式及可应用性的完整范畴将自下文所提供的详细描述变得显而易见。然而，应理解，尽管详细描述及特定实施例指示本发明的优选实施方式，但仅以说明的方式提供这些描述及实施例，因为本发明的精神及范畴内的各种改变及修改将自此详细描述对熟悉此项技术者变得显而易见。出于所有目的，包括引文在内的本文所引用的所有公开物、专利及专利申请将以引用的方式全部并入本文。
实施例
提供以下实施例以证明并进一步解释本发明的一些优选的实施方式和方面，不应被解释为限制其范围。
实施例1、用于抗CLDN-18.2抗体制备的重组蛋白人CLDN-18.2 (hCLDN-18.2)的制备
从义翘神州购买含有人CLDN-18基因cDNA序列的质粒HG20047-U,利用正向引物5’-GTACgctagccaccTgaagagcggatccgctcttctTGCCCTGAAATGCATCCGCATTGGCAGC-3’和反向引物5’-GATCgcggccgccctgcaggTTACACATAGTCGTGCTTGGAAGGATAAG-3’，PCR扩增人CLDN-18部分基因片段。扩增片段经NheI和SbfI双酶切后，克隆到真核表达质粒系统(HXP)中，得到构建体HXP-CLDN18P。送南京金斯瑞基因合成hCLDN-18.2部分基因序列，通过BSPQI酶切将基因序列克隆到HXP-CLDN18P中，最终得到构建体HXP-hCLDN-18.2。
实施例2、小鼠杂交瘤细胞的制备
2.1、免疫动物
将实施例1获得的HXP-hCLDN-18.2与等量免疫佐剂(福氏佐剂)混合，取5只8周大雌性BALB/c小鼠进行肌内注射免疫。初次免疫每只小鼠使用100μg DNA质粒。之后每隔2周或3周进行加强 免疫，共进行5次，每次每只小鼠使用50μg DNA质粒。最后一次加强免疫中，叠加使用过表达CLDN-18.2的稳定CHO细胞，每只小鼠腹腔注射1e+7细胞。
2.2、细胞融合
在最后一针加强免疫4天后，取小鼠腹股沟淋巴结、腘窝淋巴结和脾脏，在DMEM培养基中碾磨后取富含淋巴细胞的悬浮液，按常规电转方法将其与小鼠骨髓瘤细胞Sp2/0(ATCC)融合。将融合产物在含1:50HAT(次黄嘌呤、氨甲蝶呤和胸腺嘧啶核苷)的DMEM完全培养基中培养5天以筛选成功融合的细胞(即杂交瘤细胞)。然后换成含1:50HT(次黄嘌呤和胸腺嘧啶核苷)的DMEM完全培养基直至筛选结束。
DMEM完全培养基配比是：15％FBS(胎牛血清)+1:50L-谷氨酰胺+100U/mL青链霉素+1:100 OPI(草酰乙酸、丙酮酸和胰岛素)，培养箱条件是8％CO 2，37℃。
实施例3、小鼠杂交瘤细胞的筛选和所得抗CLDN-18.2鼠源抗 体的性能检测
在3840株不同多克隆杂交瘤细胞株中，根据细胞水平的结合反应，筛选出可结合人CLDN-18.2蛋白且不结合人CLDN18.1蛋白的抗体的杂交瘤细胞株。经亚克隆得到单克隆杂交瘤细胞株之后，根据体外ADCC和CDC进行功能性筛选，最终得到7株单克隆杂交瘤细胞株，对应表达的抗体1H17、2B19、4G3、9J24、9O24、10L8和10N10特异性结合人CLDN-18.2重组蛋白且不结合人CLDN-18.1重组蛋白，并有强ADCC和CDC活性。
实施例4、抗CLDN-18.2鼠源抗体的可变区序列的测定(根据 Kabat或IMGT表示)
用基于简并引物PCR的方法，测定抗CLDN-18.2鼠源抗体的可变区对应的DNA编码序列。培养候选杂交瘤细胞，1000rpm离心收 集细胞，并以Trizol提取总RNA。以此为模板合成第一链cDNA，之后以第一链cDNA作为后续模板，PCR扩增对应的可变区DNA编码序列。扩增反应中所使用的引物序列与抗体的可变区第一框架区和恒定区互补(Larrick，J.W.等,1990,Scand.J.Immunol.,32,121-128和Coloma,J.J.等,(1991)BioTechniques,11,152-156)。在50μl反应体系中，分别加入cDNA 1μl，10×PCR缓冲液5μl，上游及下游引物各1μl(25pmol)，dNTP 1μl，25mmol PL MgCl 2 1μl，H 2O 39μl，95℃预变性10min，加Taq酶1μl，进入温度循环，进行PCR扩增。反应条件为94℃变性1min，58℃退火1min，72℃延伸15s，共32个循环，然后72℃保温10min。回收并纯化PCR产物。对扩增产物测序，得到抗CLDN-18.2鼠源抗体的重链可变区和轻链可变区氨基酸序列。
用NCBI Ig-Blast(http://www.ncbi.nlm.nih.gov/projects/igblast/)在种系和重排Ig可变区序列数据库中搜索共有序列。基于Kabat(Wu,T.T及Kabat,E.A.1970 J.Exp.Med.,132:211-250)及IMGT系统(Lefranc M.-P.等,1999 Nucleic Acids Research,27,209-212)，藉由序列批注及藉由基于因特网的序列分析(http://www.Imgt.org/IMGT_vquest/share/textes/index.html与http://www.ncbi.nlm.nih.gov/igblast/)确定互补决定区(CDR)氨基酸序列。
抗CLDN-18.2鼠源抗体的轻链和重链可变区及CDR的氨基酸序列如下表所示：
表1.抗CLDN-18.2鼠源抗体的CDR和可变区氨基酸序列(KABAT方案)
Figure PCTCN2021106125-appb-000001
Figure PCTCN2021106125-appb-000002
实施例5、抗CLDN-18.2嵌合抗体的构建
考虑杂交瘤细胞表达抗体的表达量、活性、类型等，选择抗CLDN-18.2鼠源抗体1H17、1H17-2、2B19、4G3、9J24和9O24继续向下进行。
从人血细胞(来自北京血液研究所)中克隆重链恒定区Fc和轻链恒定区κ的编码序列，引入pCDNA3.1质粒中。前述抗CLDN-18.2鼠源抗体的重链和轻链可变区编码序列由Genscript公司合成。各种抗CLDN-18.2鼠源抗体的重链可变区编码序列和轻链可变区编码序列经Bspq I酶切后，采用如表2所示的各种组合，引入已经引入恒定区编码序列的pCDNA3.1质粒中，并经测序确定正确克隆。将各种嵌合型重链和轻链表达质粒混合配对转染表达细胞，获得5种抗CLDN-18.2嵌合抗体，其编号和相应可变区氨基酸序列具体见表2。后续的实验材料均由此系列质粒转染细胞后提取获得。
表2.抗CLDN-18.2嵌合抗体的编号及其重链和轻链可变区的来源
嵌合抗体编号 VH VL
Chi-JS012-2 1H17 VH 1H17-2 VL
Chi-JS012-9 2B19 VH 2B19 VL
Chi-JS012-10 2B19 VH 4G3 VL
Chi-JS012-21 9J24 VH 2B19 VL
Chi-JS012-27 9O24 VH 2B19 VL
实施例6、嵌合抗体的检测
6.1抗体结合亲和力：
将过表达人CLDN-18.2的胃癌细胞株NUGC4-CLDN-18.2细胞 分别与梯度稀释成系列浓度的前述抗CLDN-18.2嵌合抗体Chi-JS012-2、Chi-JS012-9、Chi-JS012-10、Chi-JS012-21和Chi-JS012-27在4℃孵育30min，然后洗涤并与荧光标记的二抗一起孵育。最后用BD CantoⅡ流式细胞仪检测荧光强度。荧光信号越强，表示抗体与靶标亲和力越高。通过GraphPad拟合抗体剂量依赖性的结合曲线(图1)并计算EC 50(表3)，阳性对照和阴性对照分别是IMAB362和抗KLH hu-IgG1抗体。
如图1和表3所示，Chi-JS012-2、Chi-JS012-9、Chi-JS012-10、Chi-JS012-21和Chi-JS012-27均能够结合在胃癌细胞株NUGC4-CLDN-18.2细胞表面上高表达的人CLDN-18.2。
表3.抗CLDN-18.2嵌合抗体的细胞水平亲和力(EC 50)
嵌合抗体 EC 50(ng/mL)
Chi-JS012-2 4023
Chi-JS012-9 3218
Chi-JS012-10 3195
Chi-JS012-21 2373
Chi-JS012-27 2920
6.2抗体依赖的细胞介导细胞毒性作用(ADCC)：
将梯度稀释成系列浓度的前述抗CLDN-18.2嵌合抗体Chi-JS012-2、Chi-JS012-9、Chi-JS012-10、Chi-JS012-21和Chi-JS012-27分别与过表达人CLDN-18.2的靶细胞CHO-CLDN-18.2(50000个细胞)，以及表达NFAT-Luc和FcγRIIIaR的效应细胞Jurkat ADCC(100000个细胞)在37℃共孵育6h，然后加入底物one-glo并用酶标仪检测荧光素酶信号。荧光读值越高，表示ADCC效应越强。通过GraphPad拟合抗体剂量依赖性的ADCC效应曲线(图2a和2b)，阳性对照和阴性对照分别是IMAB362和抗KLH hu-IgG1抗体。
如图2a和2b所示，Chi-JS012-2、Chi-JS012-9、Chi-JS012-10、Chi-JS012-21和Chi-JS012-27介导ADCC效应的EC 50值分别为56.08ng/mL、43.6ng/mL、49.51ng/mL、63.09ng/mL和43.21ng/mL，与 阳性对照IMAB362(图2a：35.98；图2b：34.41ng/mL)相当。
6.3补体依赖性细胞毒性(CDC)：
将过表达人CLDN-18.2的靶细胞CHO-CLDN-18.2细胞(100000个细胞)分别与梯度稀释成系列浓度的前述抗CLDN-18.2嵌合抗体Chi-JS012-2、Chi-JS012-9、Chi-JS012-10和Chi-JS012-27混合，加入十倍稀释的补体血清(Quidel，cat#A113),然后在37℃孵育1h。最后用PBS重悬细胞并加入PI染料，于4℃孵育5min后用BD CantoⅡ流式细胞仪检测荧光强度。通过GraphPad拟合抗体剂量依赖性的补体杀伤曲线(图3)。阳性对照和阴性对照分别是IMAB362和抗KLH hu-IgG1抗体。
如图3所示，Chi-JS012-2、Chi-JS012-9、Chi-JS012-10和Chi-JS012-27介导CDC效应的EC 50值分别为461.2ng/mL，316ng/mL，358.3ng/mL和264.5ng/mL，与阳性对照IMAB362(353.6ng/mL)相当。
实施例7、抗体可变区的人源化改造
出于降低抗体的免疫原性等目的，对抗体可变区进行人源化改造优化，优化结果如下：
HCDR2：CISSGSGTIYYADTVKG(SEQ ID NO:2)优化为 SISSGSGTIYYAD SVKG(SEQ ID NO:41)或YISSGSGTIYYADSVKG(SEQ ID NO:72)；
HCDR2：WINTYTGESTYADDFKG(SEQ ID NO:11)优化为WINTYTGESTYAD GF TG(SEQ ID NO:40)，
HCDR2：TISGGDSYTYYPDSVRG(SEQ ID NO:29)优化为TISGGDSYTYYPDSV KG(SEQ ID NO:74)；
HCDR3：AYYGNGFSF(SEQ ID NO:3)优化为AYYGN AFSF(SEQ ID NO:73)；
HCDR3：SYNGNSLPY(SEQ ID NO:30)优化为SYN ANSLPY (SEQ ID NO:75)；
从而得到了一系列人源化抗CLDN-18.2抗体。
人源化抗CLDN-18.2抗体JS012-Chi9-hu7、JS012-Chi9-hu7-v2、JS012-Chi27-hu3-v2、JS012-Chi27-hu6-v3-2和JS012-Chi2-hu2-v3-2进行后续实验。上述抗体的CDR/可变区序列如表4-1和4-2所示，重/轻链的氨基酸序列和核苷酸编码序列如表5所示。
人源化抗CLDN-18.2抗体的重链和轻链可变区编码序列由Genscript公司合成，各种合成的人源化抗CLDN-18.2抗体的重链可变区编码序列和轻链可变区编码序列经Bspq I酶切后引入已经含有恒定区编码序列的pCDNA3.1质粒中，并经测序确定正确克隆。将各种人源化型重链和轻链表达质粒混合配对转染表达细胞(CHOK118,苏州君盟)，表达的抗体通过离心回收后，按常规方法进行抗体纯化，得到表4-1及4-2中人源化抗CLDN-18.2抗体。
其中，人源化抗体JS012-Chi2-hu2-v3-2经过在细胞培养中添加糖型调节剂(CDFS01，上海奥浦迈生物科技有限公司)进行工艺优化，得到去岩藻糖化抗体JS012-Chi2-hu2-v3-2a。抗体JS012-Chi2-hu2-v3-2a为岩藻糖化低于30％的人源化抗CLDN-18.2抗体。
表4-1.人源化抗CLDN-18.2抗体可变区和CDR序列(KABAT方案)
Figure PCTCN2021106125-appb-000003
表4-2.人源化抗CLDN-18.2抗体可变区和CDR序列(KABAT方案)
Figure PCTCN2021106125-appb-000004
表5.人源化抗CLDN-18.2抗体的重链和轻链的氨基酸和核苷酸编码序列
Figure PCTCN2021106125-appb-000005
实施例8、人源化抗CLDN-18.2抗体的检测
通过FACS检测抗体与细胞表面表达的CLDN-18.2结合的活性 来检测5种人源化抗CLDN-18.2抗体JS012-Chi9-hu7、JS012-Chi9-hu7-v2、JS012-Chi27-hu3-v2、JS012-Chi27-hu6-v3-2和JS012-Chi2-hu2-v3-2的结合活性。将NUGC4-CLDN-18.2细胞与梯度稀释的不同浓度的前述人源化抗CLDN-18.2抗体一起孵育，然后洗涤并与荧光标记二抗一起孵育。通过FACS检测荧光信号；检测的荧光信号越强，则抗体亲和力越高，即靶标结合活性越高。用GraphPad拟合抗体结合曲线(图4a、4b和4c)，阳性对照和阴性对照分别是IMAB362和抗KLH hu-IgG1抗体。
如图4a、4b和4c所示，JS012-Chi9-hu7-v2、JS012-Chi9-hu7、JS012-Chi27-hu3-v2、JS012-Chi2-hu2-v3-2和JS012-Chi27-hu6-v3-2都能够结合在胃癌细胞株NUGC4细胞表面上高表达的人CLDN-18.2，其中JS012-Chi9-hu7-v2、JS012-Chi9-hu7、JS012-Chi27-hu3-v2和JS012-Chi2-hu2-v3-2的EC 50与阳性对照相当。
实施例9、人源化抗CLDN-18.2抗体的ADCC
人源化抗CLDN-18.2抗体为人IgG1亚型，能够介导ADCC效应，抗体Fc端与NK细胞表面FcγIIIaR受体结合，激活NK细胞杀伤靶细胞。利用表达FcγIIIaR的NFAT通路荧光素酶系统，通过报告基因系统模拟体内ADCC效应。将人源化抗CLDN-18.2抗体与靶细胞CHO-CLDN-18.2细胞以及Jurkat ADCC效应细胞共孵育，在加入底物one-glo后用酶标仪检测信号。用GraphPad分析数据，比较剂量依赖性的抗体ADCC效应(图5a、5b和5c)。阳性对照和阴性对照分别是IMAB362和抗KLH hu-IgG1抗体。
如图5a、5b和5c所示，JS012-Chi9-hu7-v2、JS012-Chi9-hu7、JS012-Chi27-hu3-v2、JS012-Chi2-hu2-v3-2和JS012-Chi27-hu6-v3-2介导ADCC效应的EC 50值分别为22.5ng/mL，23.63ng/mL，35.75ng/mL，14.18ng/mL和74.24ng/mL，均明显优于阳性对照IMAB362。
实施例10、人源化抗CLDN-18.2抗体的CDC
人源化抗CLDN-18.2抗体还可以介导CDC效应，通过形成攻膜复合物的方式杀伤靶细胞。补体血清(1:10稀释)与人源化抗CLDN-18.2抗体以及靶细胞CHO-CLDN-18.2(100000个细胞)于37℃培养箱共孵育1h，通过碘化丙啶(PI)染色检测靶细胞存活杀伤情况，计算相对杀伤率。人源化抗CLDN-18.2抗体的剂量依赖性CDC效应用相对杀伤率来反映(图6a、6b和6c)。阳性对照和阴性对照分别是IMAB362和抗KLH hu-IgG1抗体。
如图6a、6b和6c所示，JS012-Chi9-hu7-v2、JS012-Chi9-hu7、JS012-Chi27-hu3-v2、JS012-Chi2-hu2-v3-2和JS012-Chi27-hu6-v3-2介导CDC效应的EC 50值分别为134.2ng/mL、86.04ng/mL、168.9ng/mL、166.5ng/mL和714.9ng/mL，其中JS012-Chi9-hu7-v2、JS012-Chi9-hu7、JS012-Chi27-hu3-v2和JS012-Chi2-hu2-v3-2明显优于阳性对照IMAB362。
实施例11：人源化抗CLDN-18.2抗体对M-NSG小鼠移植人胃 癌MKN45 hClaudin18.2 Mixeno肿瘤生长的抑制作用
1、测试目的
评价本发明JS012-Chi2-hu2 v3-2a在人胃癌MKN45 hClaudin18.2 Mixeno皮下移植模型中的抗肿瘤作用。
2、测试过程
将5×10 6个MKN45 hClaudin18.2肿瘤细胞(上海诺百生物)混合5×10 6个PBMC细胞(上海澳能生物)，0.2ml/只(含细胞和50％基质胶的培养基RPMI1640(Gibco))，接种于M-NSG小鼠(上海南方模式生物科技股份有限公司)的右后背皮下，根据体重挑选50只随机分组，每组10只，共5组，分别为
Anti-KLH hIgG1阴性对照组，10mg/kg；
JS012-Chi2-hu2 v3-2a治疗组，1mg/kg；
JS012-Chi2-hu2 v3-2a治疗组，3mg/kg；
JS012-Chi2-hu2 v3-2a治疗组，10mg/kg；
IMAB362阳性对照组，10mg/kg。
分组当天接种肿瘤细胞后4h给药，所有组的给药途径均为腹腔注射，每周给药2次，连续给药9次，末次给药3天后结束实验。每周测量肿瘤体积及体重2次，记录小鼠体重和肿瘤体积。实验结束时，将小鼠安乐死，计算肿瘤抑制率TGI％(TGI％＝[1-T/C]×100％)。(T：治疗组或阳性对照组在实验终点的肿瘤体积均值，C：阴性对照组在实验终点的肿瘤体积均值)。
如图7所示，在实验终点，anti-KLH hIgG1阴性对照组平均肿瘤体积为1276±228mm 3。JS012-Chi2-hu2a v3-2在1、3和10mg/kg剂量条件下的平均肿瘤体积分别为235±25mm 3、243±33mm 3和343±63mm 3，TGI分为81.6％、81.0％和73.1％，表现显著的抑瘤作用。IMAB362在10mg/kg剂量下的平均肿瘤体积为361±73mm 3，TGI为71.7％。同剂量条件下(10mg/kg)，JS012-Chi2-hu2 v3-2a抑瘤作用略优于IMAB362。
实施例12：人源化抗CLDN-18.2抗体对CB-17 SCID小鼠移植人胰 腺癌hCLDN18.2 MIA PaCa-2肿瘤生长的抑制作用
1、测试目的
评价本发明JS012-Chi2-hu2 v3-2a在人胰腺癌hCLDN18.2 MIA PaCa-2皮下移植模型中的抗肿瘤作用。
2、测试过程
将5×10 6个hCLDN18.2 MIA PaCa-2细胞(Accurusbio-C3002)，0.2ml/只(含细胞和50％基质胶的培养基DMEM(Gibco))，接种于CB-17SCID小鼠(上海吉辉实验动物饲养有限公司)的右后背皮下，待平均肿瘤体积约为89mm 3时，根据肿瘤体积挑选40只动物随机分组，每组8只，共5组，分别为
Anti-KLH hIgG1阴性对照组，3mg/kg；
IMAB362阳性对照组，3mg/kg；
JS012-Chi2-hu2 v3-2a治疗组，0.3mg/kg；
JS012-Chi2-hu2 v3-2a治疗组，1mg/kg；
JS012-Chi2-hu2 v3-2a治疗组，3mg/kg。
分组当天给药，所有组的给药途径均为腹腔注射，每周给药2次，连续给药6次，末次给药4天后结束实验。每周测量肿瘤体积及体重3次，记录小鼠体重和肿瘤体积。实验结束时，将小鼠安乐死，计算肿瘤抑制率TGI％(TGI％＝[1-(Ti-T0)/(Vi-V0)]×100％)。(Ti：治疗组或阳性对照组在给药第i天的肿瘤体积均值，T0：治疗组或阳性对照组在给药第0天的肿瘤体积均值；Vi：阴性对照组在给药第i天的肿瘤体积均值，V0：阴性对照组在给药第0天的肿瘤体积均值)。
如图8所示，在实验终点，anti-KLH hIgG1组平均肿瘤体积为2235±145mm3。IMAB362在3mg/kg剂量下的平均肿瘤体积为465±74mm3，TGI为82.5％。JS012-Chi2-hu2 v3-2a在0.3、1和3mg/kg剂量条件下的平均肿瘤体积分别为1455±142mm3、673±153mm3和74±19mm3，TGI分为36.3％、72.8％和100.6％，表现显著的抑瘤作用。同剂量条件下(3mg/kg)，JS012-Chi2-hu2 v3-2a抑瘤作用显著优于IMAB362。
序列表
Figure PCTCN2021106125-appb-000006
Figure PCTCN2021106125-appb-000007
Figure PCTCN2021106125-appb-000008
Figure PCTCN2021106125-appb-000009
Figure PCTCN2021106125-appb-000010
Figure PCTCN2021106125-appb-000011
Figure PCTCN2021106125-appb-000012
Figure PCTCN2021106125-appb-000013
Figure PCTCN2021106125-appb-000014
Figure PCTCN2021106125-appb-000015

Claims (16)

  1. 一种抗CLDN-18.2抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中，
    所述重链可变区包含HCDR1、HCDR2和HCDR3，其中
    所述HCDR1的序列选自如SEQ ID NO:1、10、16、22、28、34和37所示的氨基酸序列；
    所述HCDR2的序列选自如SEQ ID NO:62、63、17、23、29、35、38、72和74所示的氨基酸序列，所述SEQ ID NO:62所示的HCDR2中X 1为C或S，X 2为T或S，所述SEQ ID NO:63所示的HCDR2中X 3为D或G，X 4为K或T；和
    所述HCDR3的序列选自如SEQ ID NO:3、12、18、24、30、36、39、73和75所示的氨基酸序列；和
    所述轻链可变区包含LCDR1、LCDR2和LCDR3，其中
    所述LCDR1的序列选自如SEQ ID NO:4、7、13、19、25和31所示的氨基酸序列；
    所述LCDR2的序列选自如SEQ ID NO:5、8、14、20、26和32所示的氨基酸序列；和
    所述LCDR3的序列选自如SEQ ID NO:6、9、15、21、27和33所示的氨基酸序列。
  2. 如权利要求1所述的抗体或其抗原结合片段，其中
    所述重链可变区包含：
    (Ⅰ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或
    (Ⅱ)氨基酸序列分别如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的HCDR1、HCDR2和HCDR3；或
    (Ⅲ)氨基酸序列分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的HCDR1、HCDR2和HCDR3；或
    (Ⅳ)氨基酸序列分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的HCDR1、HCDR2和HCDR3；或
    (Ⅴ)氨基酸序列分别如SEQ ID NO:28、SEQ ID NO:29和SEQ ID NO:30所示的HCDR1、HCDR2和HCDR3；或
    (Ⅵ)氨基酸序列分别如SEQ ID NO:34、SEQ ID NO:35和SEQ ID NO:36所示的HCDR1、HCDR2和HCDR3；或
    (Ⅶ)氨基酸序列分别如SEQ ID NO:37、SEQ ID NO:38和SEQ ID NO:39所示的HCDR1、HCDR2和HCDR3；或
    (Ⅷ)氨基酸序列分别如SEQ ID NO:10、SEQ ID NO:40和SEQ ID NO:12所示的HCDR1、HCDR2和HCDR3；或
    (Ⅸ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:41和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或
    (Ⅹ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:72和SEQ ID NO:73所示的HCDR1、HCDR2和HCDR3；或
    (Ⅺ)氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:72和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；或
    (Ⅻ)氨基酸序列分别如SEQ ID NO:28、SEQ ID NO:74和SEQ ID NO:75所示的HCDR1、HCDR2和HCDR3；或
    (XIII)氨基酸序列分别如SEQ ID NO:28、SEQ ID NO:74和SEQ ID NO:30所示的HCDR1、HCDR2和HCDR3；和
    所述轻链可变区包含：
    (Ⅰ)氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；或
    (Ⅱ)氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的LCDR1、LCDR2和LCDR3；或
    (Ⅲ)氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的LCDR1、LCDR2和LCDR3；或
    (Ⅳ)氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的LCDR1、LCDR2和LCDR3；或
    (Ⅴ)氨基酸序列分别如SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的LCDR1、LCDR2和LCDR3；或
    (Ⅵ)氨基酸序列分别如SEQ ID NO:31、SEQ ID NO:32和SEQ ID NO:33所示的LCDR1、LCDR2和LCDR3。
  3. 如权利要求2所述的抗体或其抗原结合片段，其包含：
    (Ⅰ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；或
    (Ⅱ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的LCDR1、LCDR2和LCDR3；或
    (Ⅲ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的LCDR1、LCDR2和LCDR3；或
    (Ⅳ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的LCDR1、LCDR2和LCDR3；或
    (Ⅴ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的LCDR1、LCDR2和LCDR3；或
    (Ⅵ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:28、SEQ ID NO:29和SEQ ID NO:30所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:31、SEQ ID NO:32和SEQ ID NO:33所示的LCDR1、LCDR2和LCDR3；或
    (Ⅶ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:34、SEQ ID NO:35和SEQ ID NO:36所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示的LCDR1、LCDR2和LCDR3；或
    (Ⅷ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:37、SEQ ID NO:38和SEQ ID NO:39所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:31、SEQ ID NO:32和SEQ ID NO:33所示的LCDR1、LCDR2和LCDR3；或
    (Ⅸ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:20和SEQ ID NO:21所示的LCDR1、LCDR2和LCDR3；或
    (Ⅹ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:28、SEQ ID NO:29和SEQ ID NO:30所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的LCDR1、LCDR2和LCDR3；或
    (Ⅺ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的LCDR1、LCDR2和LCDR3；或
    (Ⅻ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:10、SEQ ID NO:40和SEQ ID NO:12所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的LCDR1、LCDR2和LCDR3；或
    (XIII)重链可变区，其包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:41和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的LCDR1、LCDR2和LCDR3；或
    (XⅣ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:1、SEQ  ID NO:72和SEQ ID NO:73所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的LCDR1、LCDR2和LCDR3；或
    (XⅤ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:72和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示的LCDR1、LCDR2和LCDR3；或
    (XⅥ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:28、SEQ ID NO:74和SEQ ID NO:30所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的LCDR1、LCDR2和LCDR3；或
    (XⅦ)重链可变区，其包含氨基酸序列分别如SEQ ID NO:28、SEQ ID NO:74和SEQ ID NO:75所示的HCDR1、HCDR2和HCDR3；和轻链可变区，其包含氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15所示的LCDR1、LCDR2和LCDR3。
  4. 如权利要求1-3中任一项所述的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中
    (1)所述重链可变区的氨基酸序列包含选自如SEQ ID NO:42、45、47、49、51、53、54、76、78、82和83所示的序列，或与SEQ ID NO:42、45、47、49、51、53、54、76、78、82和83所示序列中任一者具有至少95％、96％、97％、98％或99％同一性的氨基酸序列；和
    所述轻链可变区的氨基酸序列包含选自如SEQ ID NO:43、44、46、48、50和52所示的序列，或与SEQ ID NO:43、44、46、48、50和52所示序列中任一者具有至少95％、96％、97％、98％或99％同一性的氨基酸序列；或
    (2)所述抗体或其抗原结合片段包含：
    氨基酸序列如SEQ ID NO:42所示的重链可变区和氨基酸序列如SEQ ID NO:43所示的轻链可变区；或
    氨基酸序列如SEQ ID NO:42所示的重链可变区和氨基酸序列如SEQ ID NO:44所示的轻链可变区；或
    氨基酸序列如SEQ ID NO:45所示的重链可变区和氨基酸序列如SEQ ID NO:46所示的轻链可变区；或
    氨基酸序列如SEQ ID NO:47所示的重链可变区和氨基酸序列如SEQ ID NO:48所示的轻链可变区；或
    氨基酸序列如SEQ ID NO:49所示的重链可变区和氨基酸序列如SEQ ID NO:50所示的轻链可变区；或
    氨基酸序列如SEQ ID NO:51所示的重链可变区和氨基酸序列如SEQ ID NO:52所示的轻链可变区；或
    氨基酸序列如SEQ ID NO:53所示的重链可变区和氨基酸序列如SEQ ID NO:50所示的轻链可变区；或
    氨基酸序列如SEQ ID NO:54所示的重链可变区和氨基酸序列如SEQ ID NO:52所示的轻链可变区；或
    氨基酸序列如SEQ ID NO:45所示的重链可变区和氨基酸序列如SEQ ID NO:48所示的轻链可变区；或
    氨基酸序列如SEQ ID NO:51所示的重链可变区和氨基酸序列如SEQ ID NO:46所示的轻链可变区；或
    氨基酸序列如SEQ ID NO:49所示的重链可变区和氨基酸序列如SEQ ID NO:46所示的轻链可变区。
  5. 如权利要求1-3中任一项所述的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中
    (1)所述重链可变区的氨基酸序列包含选自如SEQ ID NO:55、57、58、59、60、76、78、79、82和83所示的序列，或与SEQ ID NO:55、57、58、59、60、76、78、79、82和83所示序列中任一者具有至少95％、96％、97％、98％或99％同一性的氨基酸序列；和
    所述轻链可变区的氨基酸序列包含选自如SEQ ID NO:56、61、77、80和81所示的序列，或与SEQ ID NO:56、61、77、80和81所示序列中任一者具有至少95％、96％、97％、98％或99％同一性的 氨基酸序列；或
    (2)所述重链可变区包含如SEQ ID NO:55、57、60、76、78、79、82或83中任一项所示的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:56、61、77、80或81中任一项所示的氨基酸序列；或
    (3)所述重链可变区包含如SEQ ID NO:55、57、82或83所示的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:56所示的氨基酸序列；或
    (4)所述重链可变区包含如SEQ ID NO:60、76或78所示的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:61所示的氨基酸序列；或
    (5)所述重链可变区包含如SEQ ID NO:55所示的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:56所示的氨基酸序列；或
    (6)所述重链可变区包含如SEQ ID NO:57所示的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:56所示的氨基酸序列；或
    (7)所述重链可变区包含如SEQ ID NO:58所示的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:56所示的氨基酸序列；或
    (8)所述重链可变区包含如SEQ ID NO:59所示的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:56所示的氨基酸序列；或
    (9)所述重链可变区包含如SEQ ID NO:60所示的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:61所示的氨基酸序列；或
    (10)所述重链可变区包含如SEQ ID NO:76所示的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:77所示的氨基酸序列；或
    (11)所述重链可变区包含如SEQ ID NO:79所示的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:80所示的氨基酸序列；或
    (12)所述重链可变区包含如SEQ ID NO:55所示的氨基酸序列；和所述轻链可变区包含如SEQ ID NO:81所示的氨基酸序列。
  6. 如权利要求1-3任一项所述的抗体或其抗原结合片段，其包含重链和轻链，其中
    (1)所述重链的氨基酸序列如SEQ ID NO:64或其变体或者SEQ  ID NO:68或其变体所示，和轻链，所述轻链的氨基酸序列如SEQ ID NO:65或其变体或者SEQ ID NO:69或其变体所示，其中所述变体在其可变区包含1、2、3、4或5个氨基酸变化；优选地，SEQ ID NO:64的变体包含在第50或63位或其组合处的氨基酸变化；和/或SEQ ID NO:68的变体包含在第63或65位或其组合处的氨基酸变化；优选地，SEQ ID NO:64的变体包含S50C或S63T或其组合；和/或SEQ ID NO:68的变体包含G63D或T65K或其组合；或
    (2)所述重链包含如SEQ ID NO:64所示的氨基酸序列，或与SEQ ID NO:64所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:65所示的氨基酸序列，或与SEQ ID NO:65所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
    (3)所述重链包含如SEQ ID NO:68所示的氨基酸序列，或与SEQ ID NO:68所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:69所示的氨基酸序列，或与SEQ ID NO:69所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
    (4)所述重链包含如SEQ ID NO:84所示的氨基酸序列，或与SEQ ID NO:84所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:85所示的氨基酸序列，或与SEQ ID NO:85所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
    (5)所述重链包含如SEQ ID NO:86所示的氨基酸序列，或与SEQ ID NO:86所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:87所示的氨基酸序列，或与SEQ ID NO:87所示的氨 基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
    (6)所述重链包含如SEQ ID NO:88所示的氨基酸序列，或与SEQ ID NO:88所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:89所示的氨基酸序列，或与SEQ ID NO:89所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
    (7)所述重链包含如SEQ ID NO:90所示的氨基酸序列，或与SEQ ID NO:90所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:91所示的氨基酸序列，或与SEQ ID NO:91所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
    (8)所述重链包含如SEQ ID NO:92所示的氨基酸序列，或与SEQ ID NO:92所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:93所示的氨基酸序列，或与SEQ ID NO:93所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
    (9)所述重链包含如SEQ ID NO:94所示的氨基酸序列，或与SEQ ID NO:94所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:95所示的氨基酸序列，或与SEQ ID NO:95所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
    (10)所述重链包含如SEQ ID NO:96所示的氨基酸序列，或与SEQ ID NO:96所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含 如SEQ ID NO:97所示的氨基酸序列，或与SEQ ID NO:97所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
    (11)所述重链包含如SEQ ID NO:98所示的氨基酸序列，或与SEQ ID NO:98所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:99所示的氨基酸序列，或与SEQ ID NO:99所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或
    (12)所述重链包含如SEQ ID NO:100所示的氨基酸序列，或与SEQ ID NO:100所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；和所述轻链包含如SEQ ID NO:101所示的氨基酸序列，或与SEQ ID NO:101所示的氨基酸序列具有至少90％、92％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
  7. 如权利要求1-6中任一项所述的抗体或其抗原结合片段，其中所述抗体为鼠源抗体、嵌合抗体、人源化抗体、或全人抗体；所述抗原结合片段为Fab、Fab'、Fab'-SH、Fv、scFv、F(ab')2、sdAb或双抗体。
  8. 如权利要求1-6中任一项所述的抗体或其抗原结合片段，其中所述抗体为任何IgG亚型，如IgG1、IgG2、IgG3或IgG4；优选地；所述抗体是低或无岩藻糖基化的。
  9. 一种分离的抗CLDN-18.2抗体或其抗原结合片段，其具有以下特性中的一种或多种：
    (1)与权利要求1-8中任一项所述的抗CLDN-18.2抗体或其抗原结合片段结合相同或者完全或部分重叠的人CLDN-18.2蛋白的表位；
    (2)与权利要求1-8中任一项所述的抗CLDN-18.2抗体或其抗原结合片段竞争结合人CLDN-18.2蛋白的表位；
    (3)与人CLDN-18.2蛋白结合，但是不结合人CLDN-18.1蛋白；
    (4)诱导表达人CLDN-18.2蛋白的细胞的ADCC效应；和
    (5)诱导表达人CLDN-18.2蛋白的细胞的CDC效应。
  10. 一种多核苷酸，其编码如权利要求1-9中任一项所述的抗CLDN-18.2抗体或其抗原结合片段。
  11. 一种表达载体，其包含如权利要求10所述的多核苷酸，优选地，所述载体为真核表达载体。
  12. 一种宿主细胞，其包含如权利要求10所述的多核苷酸，或如权利要求11所述的表达载体，或表达如权利要求1-9中任一项所述的抗CLDN-18.2抗体或其抗原结合片段，优选地，所述宿主细胞是真核细胞，更优选哺乳动物细胞。
  13. 一种用于制备如权利要求1-6中任一项所述的抗CLDN-18.2抗体或其抗原结合片段的方法，所述方法包括在适合于所述抗体或其抗原结合片段表达的条件下培养权利要求12所述的宿主细胞，并从所述宿主细胞回收所表达的所述抗体或其抗原结合片段。
  14. 一种药物组合物，其包含如权利要求1-9中任一项所述的抗CLDN-18.2抗体或其抗原结合片段、权利要求10所述的多核苷酸、权利要求11所述的表达载体、权利要求12所述的宿主细胞，和药学上可接受的载体或赋形剂。
  15. 如权利要求1-9中任一项所述的抗体或其抗原结合片段、权利要求10所述的多核苷酸、权利要求11所述的表达载体、权利要求12所述的宿主细胞、或权利要求14所述的药物组合物在制备用于治疗和/或预防CLDN-18.2介导的疾病或病症的药物中的用途，优选地，所述疾病或病症为癌症；
    更优选地，所述癌症选自胃癌、食道癌、胃食管癌、胰腺癌、胆管癌、肺癌、卵巢癌、结肠癌、肝癌、头颈癌、胆囊癌、肠癌和膀胱癌药物。
  16. 一种药物组合，其包含如权利要求1-9中任一项所述的抗体或其抗原结合片段、权利要求10所述的多核苷酸、权利要求11所 述的表达载体、权利要求12所述的宿主细胞、或权利要求14所述的药物组合物，以及一种或多种另外的治疗剂。
PCT/CN2021/106125 2020-07-13 2021-07-13 抗cldn-18.2抗体及其用途 Ceased WO2022012559A1 (zh)

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KR1020237001805A KR102916382B1 (ko) 2020-07-13 2021-07-13 항-cldn-18.2 항체 및 그 용도
JP2023501905A JP7726978B2 (ja) 2020-07-13 2021-07-13 抗ｃｌｄｎ－１８．２抗体及びその用途
CA3186099A CA3186099A1 (en) 2020-07-13 2021-07-13 Anti-cldn-18.2 antibody and use thereof
CN202180061297.0A CN116096416B (zh) 2020-07-13 2021-07-13 抗cldn-18.2抗体及其用途
PH1/2023/550089A PH12023550089A1 (en) 2020-07-13 2021-07-13 Anti-cldn-18.2 antibody and use thereof
US18/016,279 US20240059771A1 (en) 2020-07-13 2021-07-13 Anti-cldn-18.2 antibody and use thereof
AU2021307516A AU2021307516B2 (en) 2020-07-13 2021-07-13 Anti-CLDN-18.2 antibody and use thereof
BR112023000657A BR112023000657A2 (pt) 2020-07-13 2021-07-13 Anticorpo anti-cldn-18.2 e uso do mesmo
EP21842171.7A EP4180457A4 (en) 2020-07-13 2021-07-13 ANTI-CLDN-18.2 ANTIBODIES AND USE THEREOF

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