WO2023020537A1 - 一种双特异性抗体及其用途 - Google Patents

一种双特异性抗体及其用途 Download PDF

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WO2023020537A1
WO2023020537A1 PCT/CN2022/113031 CN2022113031W WO2023020537A1 WO 2023020537 A1 WO2023020537 A1 WO 2023020537A1 CN 2022113031 W CN2022113031 W CN 2022113031W WO 2023020537 A1 WO2023020537 A1 WO 2023020537A1
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sequence
seq
amino acid
heavy chain
claudin
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PCT/CN2022/113031
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English (en)
French (fr)
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罗羿
陈俊颖
缪小牛
黄威峰
袁志军
彭绍岗
曾竣玮
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普米斯生物技术(珠海)有限公司
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Priority to AU2022330225A priority Critical patent/AU2022330225A1/en
Priority to CN202280054980.6A priority patent/CN117940461A/zh
Priority to KR1020247007803A priority patent/KR20240046224A/ko
Priority to CA3229014A priority patent/CA3229014A1/en
Publication of WO2023020537A1 publication Critical patent/WO2023020537A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • This application belongs to the field of biomedical technology, and more specifically, this application relates to a bispecific antibody, as well as an immunoconjugate and a pharmaceutical composition comprising it, and this application also relates to a bispecific antibody and an immunoconjugate comprising it Uses of substances and pharmaceutical compositions.
  • Gastric cancer is one of the most common cancers worldwide, with more than 1 million new diagnoses reported in 2018. my country is a country with a high incidence of gastric cancer. According to the latest data from the National Cancer Registry Center, it ranks second in the incidence of malignant tumors in the same period. The overall prognosis of gastric cancer is poor, with a 5-year survival rate of only 10% to 30%, and a 5-year survival rate of patients with advanced gastric cancer is less than 10%.
  • Claudin-18 isoform 2 (Claudin 18.2) is expressed only in gastric tissue cells and is a membrane-bound protein involved in the formation of the occlusive zone. However, Claudin 18.2 is upregulated in various types of tumor cells, including gastric and pancreatic cancers.
  • the monoclonal antibody drug Zolbetuximab (IMAB362) targeting Claudin 18.2 has shown certain curative effect and less toxicity in clinical studies.
  • Lymphocytes express a variety of co-stimulatory receptors, such as CD27, 4-1BB (CD137), OX40 (CD134) and GITR (CD357) in the tumor necrosis factor receptor superfamily. These receptors, when activated, enhance immune effects and memory responses. Targeting these receptors with agonist antibodies can effectively activate lymphocytes and enhance their antitumor effects.
  • 4-1BB is one of the important co-stimulatory receptors of T cells and NK cells, and its agonists combined with immune checkpoint inhibitors have shown good anticancer effects in clinical and preclinical studies, especially in immunogens. in less aggressive tumors.
  • severe target-related liver toxicity has been observed during the clinical development of 4-1BB monoclonal antibody agonists (eg, urelumab), proving that systemic activation of immune cells through the 4-1BB receptor has safety concerns.
  • the inventors of the present application have prepared a bispecific antibody through extensive experiments and repeated explorations. Mediated by Claudin 18.2 expressed by tumor cells, it specifically activates the 4-1BB receptor signaling pathway of immune cells, thereby effectively activating the anti-tumor effect of immune cells and simultaneously eliminating possible systemic activation toxicity.
  • the application provides a bispecific antibody comprising a Claudin 18.2 binding entity and a 4-1BB binding entity, wherein the Claudin 18.2 binding entity comprises two pairs of identical immunoglobulin chains, wherein , each pair of immunoglobulin chains has one light chain and one heavy chain, the heavy chain comprising a heavy chain variable region and a heavy chain constant region; the light chain comprising a light chain variable region and a light chain constant region;
  • the 4-1BB binding entity comprises a heavy chain variable region; and, the heavy chain variable region of the 4-1BB binding entity is connected to the C-terminus of the heavy chain of the Claudin 18.2 binding entity;
  • the Claudin 18.2 binding entity comprises: VH CDR1 or a variant thereof, VH CDR2 or a variant thereof, and VH CDR3 or a variant thereof contained in a heavy chain variable region (VH) as shown in SEQ ID NO: 1; and /or, VL CDR1 or variant thereof, VL CDR2 or variant thereof, and VL CDR3 or variant thereof contained in the light chain variable region (VL) as shown in SEQ ID NO:2;
  • the 4-1BB binding entity comprises: VH CDR1 or a variant thereof, VH CDR2 or a variant thereof, and VH CDR3 or a variant thereof contained in a heavy chain variable region (VH) as shown in SEQ ID NO: 3 or 25 body;
  • the variant has one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions, eg conservative substitutions) compared to the sequence from which it is derived.
  • amino acid substitutions, deletions or additions eg 1, 2 or 3 amino acid substitutions, deletions or additions, eg conservative substitutions
  • the substitutions are conservative substitutions.
  • the 3 CDRs contained in the VH and/or the 3 CDRs contained in the VL are defined by the Kabat, IMGT or Chothia numbering system.
  • the aforementioned antibody or antigen-binding fragment thereof has one or two characteristics selected from the following:
  • the Claudin 18.2 binding entity comprises:
  • a heavy chain variable region comprising the following three complementarity determining regions (CDRs): the sequence is VH CDR1 of SEQ ID NO:9, the sequence is VH CDR2 of SEQ ID NO:10, and the sequence is SEQ ID NO:11 and/or, the light chain variable region (VL) comprising the following 3 complementarity determining regions (CDRs): the sequence is VL CDR1 of SEQ ID NO:12, the sequence is VL CDR2 of SEQ ID NO:13 , the sequence is the VL CDR3 of SEQ ID NO: 14; wherein the CDR is defined by the IMGT numbering system; or
  • the 4-1BB binding entity comprises:
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • the application provides a bispecific antibody comprising a Claudin 18.2 binding entity and a 4-1BB binding entity, wherein the Claudin 18.2 binding entity comprises two pairs of identical immunoglobulin chains, wherein each A pair of immunoglobulin chains has a light chain and a heavy chain, the heavy chain comprising a heavy chain variable region and a heavy chain constant region; the light chain comprising a light chain variable region and a light chain constant region; the 4 - the 1BB binding entity comprises a heavy chain variable region; and, the heavy chain variable region of said 4-1BB binding entity is linked to the C-terminus of the heavy chain of said Claudin 18.2 binding entity;
  • the heavy chain variable region (VH) of the Claudin 18.2 binding entity comprises the following three complementarity determining regions (CDR):
  • VH CDR1 which consists of the following sequence: SEQ ID NO: 9, or has one or several amino acid substitutions, deletions or additions thereto (for example, 1, 2 or 3 amino acid substitutions, deletions) or add a sequence of ),
  • VH CDR2 which consists of the following sequence: SEQ ID NO: 10, or has one or several amino acid substitutions, deletions or additions thereto (such as 1, 2 or 3 amino acid substitutions, deletions) or add), and
  • VH CDR3 which consists of the following sequence: SEQ ID NO: 11, or has one or several amino acid substitutions, deletions or additions thereto (for example, 1, 2 or 3 amino acid substitutions, deletions) or add) sequence;
  • the light chain variable region (VL) of the Claudin 18.2 binding entity comprises the following 3 complementarity determining regions (CDRs):
  • VL CDR1 which consists of the following sequence: SEQ ID NO: 12, or has one or several amino acid substitutions, deletions or additions thereto (such as 1, 2 or 3 amino acid substitutions, deletions) or add a sequence of ),
  • VL CDR2 which consists of the following sequence: SEQ ID NO: 13, or has one or several amino acid substitutions, deletions or additions thereto (for example, 1, 2 or 3 amino acid substitutions, deletions) or add), and
  • VL CDR3 which consists of the following sequence: SEQ ID NO: 14, or has one or several amino acid substitutions, deletions or additions thereto (for example, 1, 2 or 3 amino acid substitutions, deletions) or add) sequence;
  • the heavy chain variable region of the 4-1BB binding entity comprises the following three complementarity determining regions (CDRs):
  • VH CDR1 which consists of the following sequence: SEQ ID NO: 15, or has one or several amino acid substitutions, deletions or additions thereto (for example, 1, 2 or 3 amino acid substitutions, deletions) or add a sequence of ),
  • VH CDR2 which consists of the following sequence: SEQ ID NO: 16, or has one or several amino acid substitutions, deletions or additions thereto (for example, 1, 2 or 3 amino acid substitutions, deletions) or add), and
  • VH CDR3 which consists of the following sequence: SEQ ID NO: 17, or has one or several amino acid substitutions, deletions or additions thereto (for example, 1, 2 or 3 amino acid substitutions, deletions) or add) sequence.
  • the CDRs described in any one of (i)-(ix) are defined according to the Kabat, IMGT or Chothia numbering system.
  • the Claudin 18.2 binding entity comprises: the following 3 heavy chain CDRs: the sequence is VH CDR1 of SEQ ID NO: 9, the sequence is VH CDR2 of SEQ ID NO: 10, and the sequence is SEQ ID NO: VH CDR3 of 11; and/or, the following 3 light chain CDRs: the sequence is VL CDR1 of SEQ ID NO: 12, the sequence is VL CDR2 of SEQ ID NO: 13, and the sequence is VL CDR3 of SEQ ID NO: 14 .
  • the heavy chain variable region (VH) of the 4-1BB binding entity comprises: the following three heavy chain CDRs: the sequence is the VH CDR1 of SEQ ID NO: 15, and the sequence is SEQ ID NO: 16
  • the VH CDR2 of the sequence is the VH CDR3 of SEQ ID NO:17.
  • the heavy chain variable region further comprises framework region sequences of human origin (eg, human immunoglobulin).
  • the Claudin 18.2 binding entity comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the Claudin 18.2 binding entity comprises: a VH having a sequence as shown in SEQ ID NO:1 and a VL having a sequence as shown in SEQ ID NO:2.
  • the 4-1BB binding entity comprises:
  • VH heavy chain variable region
  • (iii) have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% of the sequence shown in SEQ ID NO:3 or 25 %, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • substitutions described in (ii) are conservative substitutions.
  • the single domain antibody of the present application is not limited to a specific biological source or a specific preparation method.
  • the single domain antibodies of the present application can be obtained: (1) by "humanizing" a naturally occurring VHH domain or by expressing a nucleic acid encoding such a humanized VHH domain; (2) using a synthetic or semi- Preparation of proteins, polypeptides or other amino acid sequences by synthetic techniques; (3) preparation of nucleic acids encoding single domain antibodies by applying nucleic acid synthesis techniques, followed by expression of the nucleic acids thus obtained; and/or (4) by any combination of the foregoing.
  • the Claudin 18.2 binding entity further comprises:
  • the heavy chain constant region is an IgG heavy chain constant region, such as an IgGl, IgG2, IgG3 or IgG4 heavy chain constant region.
  • the light chain constant region is a kappa or lambda light chain constant region.
  • the Claudin 18.2 binding entity comprises the light chain constant region (CL) set forth in SEQ ID NO:8.
  • the bispecific antibody further comprises an Fc fragment that does not bind to an Fc receptor (FcR); alternatively, comprises an Fc fragment with reduced effector function that is antibody-dependent Cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) or antibody-dependent cellular phagocytosis (ADCP).
  • FcR Fc receptor
  • ADCC antibody-dependent Cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • the Fc fragment is selected from any one of the following mutations:
  • the Fc fragment is an IgGl Fc fragment with the L234A and L235A mutations.
  • the Fc fragment has the sequence shown in SEQ ID NO:4.
  • the bispecific antibody further comprises a peptide linker, and the Claudin 18.2 binding entity and the 4-1BB binding entity are linked by a peptide linker.
  • the C-terminus of the heavy chain constant region of the Claudin 18.2 binding entity is linked to the N-terminus of the heavy chain variable region of the 4-1BB binding entity by a peptide linker.
  • the peptide linker can comprise any amino acid, and in certain embodiments, the peptide linker comprises glycine (G) and serine (S). In certain embodiments, the peptide linker is 1-50 amino acids in length. The peptide linker is long enough to provide a sufficient degree of flexibility to allow the protein to fold properly. The length and amino acid composition of the peptide linker can be easily selected by those skilled in the art.
  • the peptide linker has a sequence as shown in SEQ ID NO: 5 or 26.
  • bispecific antibodies The preparation methods of various bispecific antibodies are disclosed in the prior art.
  • US5989830 discloses the preparation methods of bispecific antibodies.
  • the bispecific antibody fragments are respectively obtained by expressing bicistronic vectors encoding two polypeptide chains Yes, one of the polypeptide chains of the bispecific antibody has two VHs connected in series via a peptide linker (ie, VH1-peptide linker-VH2).
  • the heavy chain of the bispecific antibody has the amino acid sequence shown in SEQ ID NO: 6, 27, 28 or 29.
  • the light chain of the bispecific antibody has the amino acid sequence shown in SEQ ID NO:7.
  • the application provides an isolated nucleic acid molecule encoding the aforementioned bispecific antibody, or its heavy chain and/or light chain, or the heavy chain variable region and/or light chain of a Claudin 18.2 binding entity Either the light chain variable region, or the heavy chain variable region of the 4-1BB binding entity.
  • nucleic acid molecules are not limited by the method of their production, and can be obtained using genetic engineering recombination techniques or chemical synthesis methods.
  • the present application provides a vector comprising the isolated nucleic acid molecule as described above.
  • the vector is a cloning vector or an expression vector.
  • vectors of the invention are, for example, plasmids, cosmids, phage, and the like.
  • the present application provides a host cell comprising the aforementioned isolated nucleic acid molecule or the aforementioned vector.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).
  • the cells of the invention may also be cell lines, eg HEK293 cells.
  • the present application also provides a method for preparing the bispecific antibody of the present invention, which includes cultivating the host cell of the present invention under suitable conditions, and recovering the Claudin 18.2 binding entity of the present invention from the cell culture and/or 4-1BB binding entities.
  • the Claudin 18.2 binding entity and the 4-1BB binding entity are linked by a peptide linker.
  • the invention provides a kit comprising the bispecific antibody of the invention.
  • the bispecific antibodies of the invention further comprise a detectable label.
  • the kit further includes a second antibody that specifically recognizes the bispecific antibody of the invention.
  • the second antibody further comprises a detectable label.
  • detectable labels are well known to those skilled in the art and include, but are not limited to, radioactive isotopes, fluorescent substances, luminescent substances, colored substances and enzymes (such as horseradish peroxidase) and the like.
  • the present application provides an immunoconjugate comprising the aforementioned bispecific antibody and a therapeutic agent linked to the bispecific antibody.
  • the therapeutic agent is selected from cytotoxic agents.
  • the therapeutic agent is selected from the group consisting of alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, and random combination.
  • the immunoconjugate is an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the present application provides a pharmaceutical composition, which contains the aforementioned bispecific antibody or the aforementioned immunoconjugate, and a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutical compositions further comprise additional pharmaceutically active agents.
  • the additional pharmaceutically active agent is a drug having antineoplastic activity, such as alkylating agents, mitotic inhibitors, antineoplastic antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors, or oncolytic viruses.
  • antineoplastic activity such as alkylating agents, mitotic inhibitors, antineoplastic antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors, or oncolytic viruses.
  • the bispecific antibody or immunoconjugate and the additional pharmaceutically active agent are provided as separate components or as components of the same composition.
  • the present application provides a method for inhibiting the growth of tumor cells expressing Claudin 18.2 and/or killing the tumor cells, which comprises combining the tumor cells with an effective amount of the aforementioned bispecific antibody, Or the immunoconjugate as described above, or the pharmaceutical composition as described above.
  • the application provides the bispecific antibody as described above, or the use of the immunoconjugate as described above in the preparation of a medicament for use in a subject (such as a human) Prevention and/or treatment of tumors.
  • the medicament further comprises additional pharmaceutically active agents.
  • the additional pharmaceutically active agent is a drug having antineoplastic activity, such as alkylating agents, mitotic inhibitors, antineoplastic antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors, or oncolytic viruses.
  • antineoplastic activity such as alkylating agents, mitotic inhibitors, antineoplastic antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinases Inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors, or oncolytic viruses.
  • the tumor expresses Claudin 18.2.
  • the tumor involves tumor cells expressing Claudin 18.2.
  • said Claudin 18.2 is expressed on the surface of said tumor cells.
  • the tumor is selected from gastric cancer, liver cancer, biliary tract cancer, renal cell carcinoma, pancreatic cancer, non-small cell lung cancer, mesothelioma, ovarian cancer, testicular cancer, endometrial cancer, lung cancer, esophageal cancer , pancreatic cancer, bronchial cancer, breast cancer, ear nose throat (ENT) cancer, colon cancer, head and neck cancer, gallbladder cancer.
  • the subject is a mammal, such as a human.
  • the present application provides the use of the aforementioned bispecific antibody, or the aforementioned immunoconjugate, or the aforementioned pharmaceutical composition in the preparation of a kit, the reagent box for:
  • inducing cells for example, immune cells
  • Inducing cells eg, immune cells
  • cytokines eg, IFN ⁇ and IL-2
  • the immune cells are selected from B cells, T cells, NK cells.
  • Claudin-18 refers to a transmembrane protein located on the cell membrane.
  • CLDN18 a transmembrane protein located on the cell membrane.
  • Claudin 18.1 and Claudin 18.2 are very similar in structure, their expression in normal tissues and tumors is different.
  • Claudin 18.2 is only specifically expressed in gastric mucosal tissue, and the expression of Claudin 18.2 is up-regulated in gastric cancer, esophageal cancer, pancreatic cancer and other cancers.
  • the expression of Claudin 18.2 is not only detectable in primary tumors, but also abundantly expressed in tumor metastases.
  • Claudin 18.2 may also be referred to simply as "CLND18.2".
  • 4-1BB is also known as CD137, a member of the tumor necrosis factor receptor superfamily (TNFRSF9). Mainly expressed in activated T cells, it is a co-stimulatory molecule of T cells, and its ligand is 4-1BBL, the combination of the two can stimulate the activation and proliferation of T cells.
  • TNFRSF9 tumor necrosis factor receptor superfamily
  • antibody refers to an immunoglobulin molecule, usually composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC).
  • Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also comprising a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant domains are not directly involved in antibody-antigen binding, but exhibit a variety of effector functions, such as mediating immunoglobulin interactions with host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complement Binding of the first component (C1q) of the system.
  • VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL 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 (VH and VL) of each heavy chain/light chain pair form the antigen binding site, respectively.
  • the allocation of amino acids in each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and 1991)), or Chothia & Lesk (1987) J.Mol.Biol.196:901- 917; Definition by Chothia et al. (1989) Nature 342:878-883.
  • single domain antibody As used herein, the terms “single domain antibody (sdAb)", “domain antibody” and “nanobody” are used interchangeably and refer to antibodies composed of a single variable domain (eg heavy chain variable region) composed of antibody fragments.
  • a single domain antibody, domain antibody or nanobody consists of 4 framework regions and 3 complementarity determining regions, the 4 framework regions are respectively FR1-FR4, and the 3 complementarity determining regions are respectively CDR1 -CDR3.
  • the single domain antibody of the present application may have a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. These antibodies do not require light chain variable regions to bind antigens with high affinity and specificity.
  • nanobodies Compared with antibodies composed of heavy chain and light chain, nanobodies have high solubility, high stability to heat, pH, protease and other deforming agents, and only need single-chain expression to facilitate large-scale production.
  • bispecific antibody refers to a conjugate formed by a first antibody (or a fragment thereof) and a second antibody (or a fragment thereof) or an antibody analog through a conjugation arm,
  • the means of conjugation include but are not limited to chemical reaction, gene fusion and enzymatic.
  • Bispecific antibodies can be linked or produced by various methods, see, for example, the method of Songsivilai et al. (Clin. Exp. Immunol., 79:315-321 (1990)), and the method of Kostelny et al. (J. Immunol., 148:1547-1553 (1992)).
  • humanized antibody refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with a human antibody.
  • all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, FR) is derived from a human immunoglobulin (recipient antibody) .
  • Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune response, and the like.
  • the donor antibody can be a camelid antibody with desired properties (eg, antigen specificity, affinity, reactivity, ability to increase immune cell activity and/or ability to enhance immune response).
  • binding entity refers to any monomeric or multimeric protein or protein fragment that specifically binds a target antigen.
  • binding entity includes, but is not limited to, an antibody or binding portion thereof, such as an immunologically functional fragment.
  • peptide linker refers to a peptide adapted to be linked to an antibody binding entity.
  • immunoglobulin refers to a protein consisting of one or more polypeptides encoded by immunoglobulin genes. Immunoglobulins constitute the basic structural unit of antibodies. Antibodies are usually tetrameric and consist of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair of immunoglobulin chains, the light and heavy chain variable regions together are responsible for antigen binding, and the constant regions are responsible for antibody effector functions.
  • light chains of immunoglobulins can be classified as kappa (kappa) and lambda (lambda) light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • CDR complementarity determining region
  • the variable regions of the heavy and light chains each contain three CDRs, designated CDR1, CDR2 and CDR3.
  • CDR1, CDR2 and CDR3 The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al.
  • framework region or "FR” residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.
  • the terms "monoclonal antibody”, “mAb”, and “mAb” have the same meaning and are used interchangeably, which refers to one antibody molecule from a group of highly homogeneous antibody molecules.
  • Antibody or a fragment of an antibody that is, a population of antibody molecules that are identical except for natural mutations that may arise spontaneously.
  • mAbs are highly specific for a single epitope on an antigen.
  • polyclonal antibodies usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on antigens.
  • the modifier "monoclonal” only indicates that the antibody is characterized as being obtained from a highly homogeneous antibody population, and cannot be interpreted as requiring any specific method to prepare the antibody.
  • the monoclonal antibodies of the present invention can be prepared by various techniques, such as hybridoma technology (see, for example, Kohler et al. Nature, 256:495, 1975), recombinant DNA technology (see, for example, U.S. Patent Application No. 4,816,567), or bacteriophage Antibody library technology (see, eg, Clackson et al. Nature 352:624-628, 1991, or Marks et al. J. Mol. Biol. 222:581-597, 1991).
  • hybridoma technology see, for example, Kohler et al. Nature, 256:495, 1975
  • recombinant DNA technology see, for example, U.S. Patent Application No. 4,816,567
  • bacteriophage Antibody library technology see, eg, Clackson et al. Nature 352:624-628, 1991, or Marks et al. J. Mol. Biol. 222:581-597, 1991.
  • the term "subject” refers to a mammal, such as a primate mammal, such as a human.
  • the subject eg, a human
  • has a tumor eg, a tumor expressing CLDN6 and/or CLDN9, or is at risk of having the aforementioned diseases.
  • an effective amount refers to an amount sufficient to achieve, or at least partially achieve, the desired effect.
  • an effective amount for preventing a disease refers to an amount sufficient to prevent, arrest, or delay the occurrence of a disease (for example, a tumor);
  • an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent an existing disease The patient's disease and the amount of its complications. Determining such an effective amount is well within the capability of those skilled in the art.
  • amounts effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered concomitantly etc.
  • identity is used to refer to the match of sequences between two polypeptides or between two nucleic acids.
  • a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by an adenine, or both a position in each of the polypeptides is occupied by lysine)
  • Percent identity between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions being compared x 100. For example, two sequences are 60% identical if 6 out of 10 positions match.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
  • comparisons are made when two sequences are aligned for maximum identity.
  • Such alignments can be achieved using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453 which can be conveniently performed by computer programs such as the Align program (DNAstar, Inc.).
  • the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been integrated into the ALIGN program (version 2.0), can also be used, using the PAM120 weight residue table, A gap length penalty of 12 and a gap penalty of 4 were used to determine the percent identity between two amino acid sequences.
  • the algorithm of Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) in the GAP program that has been integrated into the GCG software package can be used, using the Blossum 62 matrix or the PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences.
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the expected properties of the protein/polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions for amino acid residues with amino acid residues that have similar side chains, e.g., are physically or functionally similar (e.g., have similar size, shape, charge, chemical properties, including Substitution of residues with the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art.
  • These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine) , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acid, proline, phenylalanine, methionine), beta branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g.
  • basic side chains e.g., lysine, arginine, and histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine
  • non-polar side chains such as
  • L234A mutation refers to the mutation of the 234th amino acid of the native Fc fragment from L to A.
  • L235A mutation refers to mutating the 235th amino acid of the natural Fc fragment from L to A.
  • the bispecific antibody prepared in the present application can recognize or bind to cells expressing Claudin 18.2 and/or recognize or bind to cells expressing 4-1BB. Moreover, it can activate the NF- ⁇ B signaling pathway of the cells, induce the cells to activate the activity of killing tumors, and can also induce the cells to secrete cytokines (for example, IFN ⁇ and IL-2), and can prevent and/or treat tumors in the subject . Moreover, the bispecific antibody has a more prominent effect of preventing and/or treating tumors than the commercially available finished monoclonal antibody or the monoclonal antibody used in combination.
  • the present application reduces the liver-related targeting effect.
  • Targeted antitumor efficacy without 4-1BB-related toxicity was achieved by using the tumor-associated antigen (TAA) Claudin 18.2 to cross-link the 4-1BB receptor lacking Fc receptor (FcR) binding properties.
  • TAA tumor-associated antigen
  • FcR Fc receptor
  • Figure 1 shows a schematic diagram of the bispecific antibody against Claudin 18.2 and 4-1BB of the present application.
  • Figure 2 shows the results of the binding of the bispecific antibody of the present application to Claudin 18.2.
  • Figure 2A shows the binding result of the bispecific antibody of the present application and Claudin 18.2 expressed by NUGC4 cells itself
  • Figure 2B shows the binding result of the bispecific antibody of the present application and human Claudin 18.2 overexpressed in MC38 cells
  • Claudin 18.2 ⁇ 4-1BB biAb is the bispecific antibody of this application
  • 4-1BB sdAb is 4-1BB single domain antibody
  • Claudin 18.2 mAb is Claudin 18.2 monoclonal antibody.
  • Figure 3 shows the binding results of the bispecific antibody of the present application and human 4-1BB overexpressed in CHO cells; wherein, Claudin 18.2 ⁇ 4-1BB biAb is the bispecific antibody of the present application, and 4-1BB sdAb is 4- 1BB single domain antibody, Claudin 18.2 mAb is Claudin 18.2 monoclonal antibody, Urelumab is 4-1BB monoclonal antibody.
  • Figure 4 shows the results of the bispecific antibody of the present application activating the NF- ⁇ B signaling pathway in Jurkat cells; among them, Claudin 18.2 ⁇ 4-1BB biAb is the bispecific antibody of the present application, and 4-1BB sdAb is the 4-1BB single domain Antibody, Claudin 18.2 mAb is Claudin 18.2 monoclonal antibody, Urelumab is 4-1BB monoclonal antibody.
  • Figure 5 shows the killing result of peripheral blood mononuclear cells (PBMC) induced by the bispecific antibody of the present application to Claudin 18.2 positive tumor cell NUGC4; wherein, Claudin 18.2 ⁇ 4-1BB biAb is the bispecific antibody of the present application, 4 -1BB sdAb is a 4-1BB single domain antibody, and Urelumab is a 4-1BB monoclonal antibody.
  • PBMC peripheral blood mononuclear cells
  • Figure 6 shows the results of the release of cytokines from PBMC induced by the bispecific antibody of the present application
  • Figure 6A is the result of the release of the cytokine IFN ⁇
  • Figure 6B is the result of the release of the cytokine IL-2; among them,
  • Claudin 18.2 ⁇ 4-1BB biAb is the bispecific antibody of this application
  • 4-1BB sdAb is the 4-1BB single domain antibody
  • Urelumab is the 4-1BB monoclonal antibody
  • Neg Ctrl is the negative control.
  • Figure 7 shows the test results of the bispecific antibody of the present application for tumor suppressive activity.
  • PBS is the negative control
  • Claudin 18.2 ⁇ 4-1BB biAb is the bispecific antibody of this application
  • IMAB362 is Claudin 18.2 monoclonal antibody
  • Claudin 18.2 mAb+4-1BB sdAb is the combination of two antibodies used to construct the bispecific antibody of this application medication.
  • Figure 8 shows the test results of the bispecific antibody of the present application on tumor suppressive memory effect.
  • PBS is the negative control
  • Claudin 18.2 ⁇ 4-1BB biAb is the bispecific antibody of different concentrations of this application
  • PBS in new 4-1BB KI mice is the negative control for re-inoculation of tumors.
  • an anti-Claudin 18.2 ⁇ 4-1BB bispecific antibody (biAb) was constructed.
  • Claudin 18.2x4-1BB biAb is composed of 2 polypeptide chains, and its structure is shown in Figure 1.
  • the heavy chain of the bispecific antibody has the amino acid sequence shown in SEQ ID NO: 6, which comprises the heavy chain variable region amino acid sequence of the monoclonal antibody anti-Claudin 18.2 mAb against claudin 18 isoform 2 (Claudin 18.2) (SEQ ID NO:1), amino acid sequence of human IgG1 Fc (introducing LALA mutation to reduce Fc function, SEQ ID NO:4), amino acid sequence of flexible peptide (SEQ ID NO:5) and 4-1BB single domain antibody Amino acid sequence of the 1BB binding region (SEQ ID NO:3).
  • the N-terminal of the 4-1BB heavy chain variable region amino acid sequence (SEQ ID NO:3) of the anti-4-1BB single domain antibody is connected to the C-terminal of the Fc through a flexible peptide (SEQ
  • the light chain of the bispecific antibody has the amino acid sequence shown in SEQ ID NO: 7, which comprises the light chain variable region amino acid sequence (SEQ ID NO: 2) of the monoclonal antibody anti-Claudin 18.2 mAb against claudin 18isoform 2, and The human kappa light chain constant region (CL) amino acid sequence (SEQ ID NO: 8) at the C-terminus of the VL amino acid sequence.
  • SEQ ID NO: 7 comprises the light chain variable region amino acid sequence (SEQ ID NO: 2) of the monoclonal antibody anti-Claudin 18.2 mAb against claudin 18isoform 2, and The human kappa light chain constant region (CL) amino acid sequence (SEQ ID NO: 8) at the C-terminus of the VL amino acid sequence.
  • the amino acid sequence containing the amino acid sequence encoding the heavy chain and light chain of the antibody (bispecific antibody Claudin 18.2x4-1BB biAb heavy chain amino acid sequence is shown in SEQ ID NO: 6, double The amino acid sequence of the specific antibody Claudin 18.2x4-1BB biAb light chain is shown in SEQ ID NO: 7) and the antibody expression plasmids of the nucleotide sequence were transferred into Expi-CHO cells respectively, and the transfection method was according to the product instructions, and the cell culture After 5 days, the supernatant was collected and the protein of interest was purified by sorting with protein A magnetic beads (purchased from GenScript).
  • the nucleotide sequences containing the heavy chain and light chain encoding antibody (the nucleotide sequences encoding the heavy chain and light chain of Urelumab monoclonal antibody are respectively shown as SEQ ID NO: 20 and Shown in SEQ ID NO: 21; the nucleotide sequence encoding the heavy chain of the 4-1BB single domain antibody is shown in SEQ ID NO: 22; the nucleotide sequences encoding the heavy chain and light chain of Claudin18.2 are shown in SEQ ID NO :23 and shown in SEQ ID NO:24) were transferred into Expi-CHO cells, the transfection method was according to the product instructions, and the supernatant was collected after 5 days of cell culture and sorted by protein A magnetic beads (purchased from GenScript) Purify the protein of interest.
  • GenScript protein A magnetic beads
  • Human Claudin 18.2 cDNA (from GeneKy, GCPLO162852) was coated with lentivirus, and MC38 was transfected to generate MC38 cells expressing human Claudin 18.2, which were named MC38-h Claudin 18.2 cells.
  • the bispecific antibody was diluted with PBS, and 100 ⁇ l/well of the above-mentioned diluted sample was added to the above-mentioned 96-well flow plate with cells, incubated at 4° C. for 60 min, and washed twice with PBS.
  • 100 ⁇ l/well of Goat anti-human IgG-Fc (PE) (purchased from Abcam) diluted 1000 times with PBS was added, incubated at 4° C. for 30 min, and washed twice with PBS.
  • 100 ⁇ l/well of PBS was added to resuspend the cells, detected on a CytoFlex (Beckman) flow cytometer and the corresponding mean fluorescence intensity (MFI) was calculated.
  • the bispecific antibody of the present invention binds to Claudin 18.2 expressed by human tumor cells themselves.
  • Figure 2B further shows that the bispecific antibody binds to human Claudin 18.2 overexpressed in murine tumor cell MC38, and has comparable binding activity to Claudin 18.2 monoclonal antibody.
  • CHO cells overexpressing human 4-1BB were generated by transfecting the PLVX vector of human 4-1BB cDNA cloned into the multiple cloning site (MCS) (synthetic autogen), and were named CHO-h4-1BB cells. Adjust the cell density of the expanded CHO-h4-1BB cells to 2 ⁇ 10 6 cells/ml, add 100 ⁇ l/well to a 96-well flow plate, and centrifuge for later use. The bispecific antibody was diluted with PBS, and 100 ⁇ l/well of the above-mentioned diluted sample was added to the above-mentioned 96-well flow plate with cells, incubated at 4° C. for 60 min, and washed twice with PBS.
  • MCS multiple cloning site
  • Target cells NUGC4 were co-transfected with 2 ⁇ 10 4 cells/well and 1.2 ⁇ 10 5 effector cells/well with 4-1BB NF- ⁇ B Luciferase/Jurkat (Jurkat (ATCC, TIB-152 TM ) cells expressing human 4-1BB carrier (synthesized by Jinweizhi) and luc2P/NF-kB-RE carrier (Promega, E8491), which is to obtain the cells) were mixed and inoculated into a 96-well cell culture white bottom plate, and the bispecific antibody after serial dilution After the addition of anti-CD3 at a final concentration of 1 ⁇ g/mL, co-incubate for 16 hours.
  • 4-1BB NF- ⁇ B Luciferase/Jurkat Jurkat (ATCC, TIB-152 TM ) cells expressing human 4-1BB carrier (synthesized by Jinweizhi) and luc2P/NF-kB-RE carrier (Promega,
  • Chemiluminescent signals were collected with a microplate reader after color development using the Bio-glo luciferase assay system (Promega G7940) kit. The results are shown in Figure 4, only the double antibody of the present invention activated the NF- ⁇ B signaling pathway of Jurkat cells.
  • the target cell NUGC4 was stained with the CellTrace TM Violet kit, and seeded into a 96-well transparent-bottomed black-edged cell culture plate at 1 ⁇ 10 4 per well, and cultured for 4 hours. Collect the PBMC recovered one day in advance and add 1 ⁇ 10 5 per well into the target cell well.
  • the serially diluted bispecific antibody was mixed with anti-CD3 at a final concentration of 0.5 ⁇ g/mL and added to the cell wells for incubation. After 48 hours, Cytation 5 was used to collect DAPI fluorescence signals and calculate the corresponding killing intensity. The results are shown in Figure 5, only the biantibody molecule induced PBMC to kill the Claudin 18.2 positive tumor cell NUGC4 (the values on the vertical axis in the figure are the results of deducting the background value).
  • the target cell NUGC4 cells were inoculated into 96-well transparent-bottomed black-edged cell culture plates at 1 ⁇ 10 4 per well, and cultured for 4 hours. Collect the PBMC recovered one day in advance and add 1 ⁇ 10 5 per well into the target cell well. The bispecific antibody after serial dilution was mixed with anti-CD3 at a final concentration of 0.5 ⁇ g/mL, added to the cell wells and incubated for 48 hours, and then the supernatant was collected.
  • the levels of IFN ⁇ and IL-2 in the supernatant were detected using human IL-2 ELISA kit (Invitrogen, 88-7025-77) and human IFN ⁇ (Invitrogen, 88-7316-77) ELISA kit and operated according to the supplier’s recommended steps.
  • human MC38-hClaudin 18.2 cells and h-4-1BB transgenic C57BL/6 mouse model were used to determine the anti-tumor effect of bispecific antibodies.
  • Sufficient MC38-hClaudin 18.2 cells were cultured and expanded in vitro (hClaudin 18.2 was overexpressed on the basis of MC38 cells). After trypsinization, the cells were collected, washed with PBS three times, counted, and inoculated at 1 ⁇ 10 6 cells/mouse 8-week-old female h-4-1BB transgenic C57BL/6 mice (purchased from Shanghai Southern Model Biotechnology Co., Ltd.) were subcutaneously subcutaneously on the right abdomen.
  • the subcutaneous tumor formation of tumor cells in the mice was observed daily, the maximum width W and the maximum long axis L of the subcutaneous tumors on the right abdomen of each animal were measured with a vernier caliper, and the weight of each mouse was weighed using an electronic balance.
  • human MC38-hClaudin 18.2 cells and h-4-1BB transgenic C57BL/6 mouse model were used to determine the dose-dependent anti-tumor effect of bispecific antibodies.
  • Sufficient MC38-hClaudin 18.2 cells were cultured and expanded in vitro (hClaudin 18.2 was overexpressed on the basis of MC38 cells). After trypsinization, the cells were collected, washed 3 times with PBS, counted, and inoculated at 1 ⁇ 10 6 cells/mouse 8-week-old female h-4-1BB transgenic C57BL/6 mice (purchased from Shanghai Southern Model Biotechnology Co., Ltd.) were subcutaneously subcutaneously on the right abdomen.
  • the subcutaneous tumor formation of tumor cells in the mice was observed daily, the maximum width W and the maximum long axis L of the subcutaneous tumors on the right abdomen of each animal were measured with a vernier caliper, and the weight of each mouse was weighed using an electronic balance.
  • the mice with too large and too small tumor volume were excluded, and the mice were divided into 5 groups according to the average tumor volume, with 6 mice in each group. According to the grouping regimen in Table 7, the groups were grouped and the corresponding doses of antibodies were injected.
  • mice were measured 2-3 times a week. The body weight and tumor volume of the mice were measured for the last time 29 days after tumor cell inoculation, and the mice were euthanized.
  • Claudin 18.2x4-1BB biAb significantly inhibited tumor growth in a dose-dependent manner within the dose range of 0.06mg/kg, 0.25mg/kg, 1mg/kg, and 4mg/kg , 4 mice in the 0.25 mg/kg group had tumor regression, 5 mice in the 1 mg/kg group had tumor regression, and all 6 mice in the 4 mg/kg group had tumor regression.
  • mice with tumor regression were re-inoculated with enough MC38-hClaudin18.2 tumor cells, and at the same time, 6 newly purchased human 4-1BB transgenic C57BL/6 mice were subcutaneously inoculated with the same amount of MC38-hClaudin 18.2 cells as the control group and the control group.
  • the tumors of the 6 mice in the group all grew rapidly, while the tumors of the mice that regressed after the administration of the double antibody all failed to grow, indicating that the mice in the double antibody group formed immune memory and could prevent the growth of the Rechallenge tumor.
  • Group 1 PBS 1 dose every 2 days, 4 times in total Group 2 Claudin 18.2x4-1BB biAb 0.06mg/kg 1 dose every 2 days, 4 times in total Group 3 Claudin 18.2x4-1BB biAb 0.25mg/kg 1 dose every 2 days, 4 times in total Group 4 Claudin 18.2x4-1BB biAb 1mg/kg 1 dose every 2 days, 4 times in total Group 5 Claudin 18.2x4-1BB biAb 4mg/kg 1 dose every 2 days, 4 times in total

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Abstract

本申请涉及一种双特异性抗体及其用途，还涉及包含其的免疫缀合物和药物组合物。本申请还涉及双特异性抗体以及包含其的免疫缀合物和药物组合物的用途。本申请制备的双特异性抗体能够识别或结合表达Claudin 18.2的细胞和/或识别或结合表达4-1BB的细胞。并且，能够在受试者中预防和/或治疗肿瘤。

Description

一种双特异性抗体及其用途 技术领域
本申请属于生物医药技术领域，更具体地，本申请涉及一种双特异性抗体，以及包含其的免疫缀合物和药物组合物，本申请还涉及双特异性抗体以及包含其的免疫缀合物和药物组合物的用途。
背景技术
胃癌是全球最常见的癌症之一，2018年报告的新诊断病例超过100万。我国是胃癌高发国家，据全国肿瘤登记中心最新数据估计，位居同期恶性肿瘤发病第2位。胃癌整体预后较差，5年生存率仅10％～30％，晚期胃癌患者5年生存率低于10％。在正常组织中，Claudin-18亚型2(Claudin 18.2)仅在胃组织细胞中表达，是一种参与形成封闭小带的膜结合蛋白。然而，Claudin 18.2在多种类型的肿瘤细胞中上调表达，包括胃癌和胰腺癌等。针对Claudin 18.2靶点的单克隆抗体药物Zolbetuximab(IMAB362)在临床研究中显示出了一定的疗效以及较小的毒性。
淋巴细胞表达多种共刺激受体，例如肿瘤坏死因子受体超家族中的CD27、4-1BB(CD137)、OX40(CD134)和GITR(CD357)等。这些受体在激活后可增强免疫效应和记忆反应。使用激动剂抗体靶向这些受体，可以有效地激活淋巴细胞，增强其抗肿瘤作用。4-1BB作为其中一种重要的T细胞和NK细胞共刺激受体，其激动剂联合免疫检查点抑制剂在临床上以及临床前研究中均表现出良好的抗癌作用，尤其是在免疫原性较差的肿瘤中。但是，4-1BB单抗激动剂(例如，Urelumab)的临床开发过程中观察到较为严重的靶向相关的肝脏毒性，证明了通过4-1BB受体系统性激活免疫细胞存在安全隐患。
因此，需要一种能够有效的激活免疫细胞抗肿瘤(例如，表达Claudin 18.2的肿瘤)作用同时能够消除可能存在的肝脏相关毒性的双特异性抗体。
发明内容
本申请的发明人经过大量实验和反复摸索，制备了一种双特异性抗体。通过肿瘤细胞表达的Claudin 18.2介导，特异性地激活免疫细胞4-1BB受体信号通路，从而既有效的激活了免疫细胞的抗肿瘤作用也同时消除了可能存在的系统性激活毒性。
因此，在第一方面，本申请提供了一种双特异性抗体，其包含Claudin 18.2结合实体和4-1BB结合实体，其中，所述Claudin 18.2结合实体包含两对相同的免疫球蛋白链，其中，每一对免疫球蛋白链具有一条轻链和一条重链，所述重链包含重链可变区和重链恒定区；所述轻链包含轻链可变区和轻链恒定区；所述4-1BB结合实体包含重链可变区；并且，所述4-1BB结合实体的重链可变区与所述Claudin 18.2结合实体的重链的C末端连接；其中，
所述Claudin 18.2结合实体包含：如SEQ ID NO:1所示的重链可变区(VH)中含有的VH CDR1或其变体、VH CDR2或其变体以及VH CDR3或其变体；和/或，如SEQ ID NO:2所示的轻链可变区(VL)中含有的VL CDR1或其变体、VL CDR2或其变体以及VL CDR3或其变体；
所述4-1BB结合实体包含：如SEQ ID NO:3或25所示的重链可变区(VH)中含有的VH CDR1或其变体、VH CDR2或其变体以及VH CDR3或其变体；
其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加，例如保守置换)。
在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述VH中含有的3个CDR和/或所述VL中含有的3个CDR由Kabat、IMGT或Chothia编号系统定义。
在某些实施方案中，如前所述的抗体或其抗原结合片段，其具有选自下列的一项或两项特征：
(1)所述Claudin 18.2结合实体包含：
包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:9的VH CDR1、序列为SEQ ID NO:10的VH CDR2、序列为SEQ ID NO:11的VH CDR3；和/或，包含下述3个互补决定区(CDRs)的轻链可变区(VL)：序列为SEQ ID NO:12的VL CDR1、序列为SEQ ID NO:13的VL CDR2、序列为SEQ ID NO:14的VL CDR3；其中，所述CDR由IMGT编号系统定义；或
(2)所述4-1BB结合实体包含：
包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:15的VH CDR1、序列为SEQ ID NO:16的VH CDR2、序列为SEQ ID NO:17的VH CDR3；其 中，所述CDR由IMGT编号系统定义。
在另一方面，本申请提供了一种双特异性抗体，其包含Claudin 18.2结合实体和4-1BB结合实体，其中，所述Claudin 18.2结合实体包含两对相同的免疫球蛋白链，其中，每一对免疫球蛋白链具有一条轻链和一条重链，所述重链包含重链可变区和重链恒定区；所述轻链包含轻链可变区和轻链恒定区；所述4-1BB结合实体包含重链可变区；并且，所述4-1BB结合实体的重链可变区与所述Claudin 18.2结合实体的重链的C末端连接；
其中，所述Claudin 18.2结合实体的重链可变区(VH)包含下述3个互补决定区(CDR)：
(i)VH CDR1，其由下述序列组成：SEQ ID NO:9，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
(ii)VH CDR2，其由下述序列组成：SEQ ID NO:10，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
(iii)VH CDR3，其由下述序列组成：SEQ ID NO:11，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
所述Claudin 18.2结合实体的轻链可变区(VL)包含下述3个互补决定区(CDR)：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO:12，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
(v)VL CDR2，其由下述序列组成：SEQ ID NO:13，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
(vi)VL CDR3，其由下述序列组成：SEQ ID NO:14，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
所述4-1BB结合实体的重链可变区包含下述3个互补决定区(CDR)：
(vii)VH CDR1，其由下述序列组成：SEQ ID NO:15，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
(viii)VH CDR2，其由下述序列组成：SEQ ID NO:16，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序 列，和
(ix)VH CDR3，其由下述序列组成：SEQ ID NO:17，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列。
在某些实施方案中，(i)-(ix)任一项中所述的CDR根据Kabat、IMGT或Chothia编号系统定义。
在某些实施方案中，所述Claudin 18.2结合实体，其包含：如下3个重链CDRs：序列为SEQ ID NO：9的VH CDR1，序列为SEQ ID NO：10的VH CDR2，序列为SEQ ID NO：11的VH CDR3；和/或，如下3个轻链CDRs：序列为SEQ ID NO：12的VL CDR1，序列为SEQ ID NO：13的VL CDR2，序列为SEQ ID NO：14的VL CDR3。
在某些实施方案中，所述4-1BB结合实体的重链可变区(VH)包含：如下3个重链CDRs：序列为SEQ ID NO：15的VH CDR1，序列为SEQ ID NO：16的VH CDR2，序列为SEQ ID NO：17的VH CDR3。
在某些实施方案中，所述重链可变区还包含来源于人的框架区序列(例如，人免疫球蛋白)。
在某些实施方案中，所述Claudin 18.2结合实体包含：
(a)重链可变区(VH)，其包含选自下列的氨基酸序列：
(i)SEQ ID NO:1所示的序列；
(ii)与SEQ ID NO:1所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO:1所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
和/或，
(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
(iv)SEQ ID NO:2所示的序列；
(v)与SEQ ID NO:2所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(vi)与SEQ ID NO:2所示的序列具有至少80％、至少85％、至少90％、至少91％、至 少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，所述Claudin 18.2结合实体包含：具有如SEQ ID NO:1所示的序列的VH和具有如SEQ ID NO:2所示的序列的VL。
在某些实施方案中，所述4-1BB结合实体包含：
(a)重链可变区(VH)，其包含选自下列的氨基酸序列：
(i)SEQ ID NO:3或25所示的序列；
(ii)与SEQ ID NO:3或25所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
(iii)与SEQ ID NO:3或25所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，(ii)中所述的置换是保守置换。
本申请的单域抗体不限于具体的生物来源或具体的制备方法。例如，本申请的单域抗体可以这样获得：(1)通过将天然存在的VHH结构域“人源化”或者通过表达编码这样的人源化VHH结构域的核酸；(2)应用合成或半合成技术制备蛋白、多肽或其它氨基酸序列；(3)通过应用核酸合成技术制备编码单域抗体的核酸，然后表达这样获得的核酸；和/或(4)通过前述的任何组合。
在某些实施方案中，所述Claudin 18.2结合实体还包含：
(a)人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；和
(b)人免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的野生型序列相比具有至多20个氨基酸的保守置换(例如至多15个、至多10个、或至多5个氨基酸的保守置换；例如1个，2个，3个，4个或5个氨基酸的保守置换)。
在某些实施方案中，所述重链恒定区是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区。
在某些实施方案中，所述轻链恒定区是κ或λ轻链恒定区。
在某些实施方案中，所述Claudin 18.2结合实体包含SEQ ID NO:8所示的轻链恒定区(CL)。
在某些实施方案中，所述双特异性抗体还包含不与Fc受体(FcR)结合的Fc片段；或者，包含具有降低的效应器功能的Fc片段，所述效应器功能为抗体依赖性细胞介导的细胞毒性(ADCC)、补体依赖的细胞毒性(CDC)或抗体依赖性细胞吞噬作用(ADCP)。
在某些实施方案中，所述Fc片段选自下列的任意一项突变：
(a)具有L235E突变的IgG1Fc片段；
(b)具有L234A和/或L235A突变的IgG1Fc片段；
(c)具有P329G或P329A突变的IgG1Fc片段；
(d)具有F234A和/或L235A或L235E突变的IgG4Fc片段；
(e)具有H268Q、V309L、A330S和/或P331S突变的IgG2Fc片段；或
(f)具有V234A、G237A、P238S、H268A、V309L、A330S和/或P331S突变的IgG2Fc片段。
在某些实施方案中，所述Fc片段为具有L234A和L235A突变的IgG1Fc片段。
在某些实施方案中，所述Fc片段具有SEQ ID NO:4所示的序列。
在某些实施方案中，所述双特异性抗体还包含肽接头，并且，所述Claudin 18.2结合实体和4-1BB结合实体通过肽接头连接。
在某些实施方案中，所述Claudin 18.2结合实体的重链恒定区的C端与4-1BB结合实体的重链可变区的N端通过肽接头连接。
肽接头可以包含任何氨基酸，在某些实施方案中，肽接头包含甘氨酸(G)和丝氨酸(S)。在某些实施方案中，肽接头的长度为1-50个氨基酸。肽连接体足够长以便能够提供足够的柔性程度，从而允许蛋白质适当地折叠。肽接头的长度、氨基酸组成可以很容易的由本领域技术人员选定。
在某些实施方案中，所述肽接头具有如SEQ ID NO:5或26所示的序列。
现有技术中公开了多种双特异性抗体的制备方法，例如，US5989830中公开了双特异 性抗体的制备方法，双特异性抗体片段分别由表达编码两个多肽链的双顺反子载体获得的，双特异性抗体的其中一个多肽链有两个通过肽接头串联的VH(即，VH1-肽接头-VH2)。
在某些实施方案中，所述双特异性抗体的重链具有如SEQ ID NO:6，27，28或29所示的氨基酸序列。
在某些实施方案中，所述双特异性抗体的轻链具有如SEQ ID NO:7所示的氨基酸序列。
在第二方面，本申请提供一种分离的核酸分子，其编码如前所述的双特异性抗体，或其重链和/或轻链，或Claudin 18.2结合实体的重链可变区和/或轻链可变区，或4-1BB结合实体的重链可变区。此类核酸分子不受限于其产生的方法，并且可以利用基因工程重组技术或化学合成方法获得。
在第三方面，本申请提供一种载体，其包含如前所述的分离的核酸分子。在某些实施方案中，所述载体为克隆载体或表达载体。在某些优选的实施方案中，本发明的载体是例如质粒，粘粒，噬菌体等等。
在第四方面，本申请提供一种宿主细胞，其包含如前所述的分离的核酸分子或如前所述的载体。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。本发明的细胞还可以是细胞系，例如HEK293细胞。
在另一个方面，本申请还提供了制备本发明的双特异性抗体的方法，其包括，在合适的条件下培养本发明的宿主细胞，和从细胞培养物中回收本发明的Claudin 18.2结合实体和/或4-1BB结合实体。
任选地，通过肽接头连接所述Claudin 18.2结合实体和4-1BB结合实体。
在另一个方面，本发明提供了一种试剂盒，其包括本发明的双特异性抗体。在某些优选的实施方案中，本发明的双特异性抗体还包括可检测的标记。在某些优选的实施方案 中，所述试剂盒还包括第二抗体，其特异性识别本发明的双特异性抗体。在某些优选的实施方案中，所述第二抗体还包括可检测的标记。此类可检测的标记是本领域技术人员熟知的，包括但不限于，放射性同位素，荧光物质，发光物质，有色物质和酶(例如辣根过氧化物酶)等。
在第五方面，本申请提供一种免疫缀合物，其包含如前所述的双特异性抗体以及连接于所述双特异性抗体的治疗剂。
在某些实施方案中，所述治疗剂选自细胞毒剂。
在某些实施方案中，所述治疗剂选自烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂，及其任意组合。
在某些实施方案中，所述免疫缀合物是抗体-药物偶联物(ADC)。
在第六方面，本申请提供一种药物组合物，其含有如前所述的双特异性抗体或者如前所述的免疫缀合物，以及药学上可接受的载体和/或赋形剂。
在某些实施方案中，药物组合物还包含另外的药学活性剂。
在某些实施方案中，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、分子靶向药物、免疫检查点抑制剂或溶瘤病毒。
在某些实施方案中，所述双特异性抗体或免疫缀合物与所述另外的药学活性剂作为分离的组分或作为同一组合物的组分提供。
在第七方面，本申请提供一种抑制表达Claudin 18.2的肿瘤细胞生长和/或杀伤所述肿瘤细胞的方法，其包括将所述肿瘤细胞与有效量的如前所述的双特异性抗体，或如前所述的免疫缀合物，或如前所述的药物组合物接触。
在第八方面，本申请提供了如前所述的双特异性抗体，或如前所述的免疫缀合物在制备药物中的用途，所述药物用于在受试者(例如人)中预防和/治疗肿瘤。
在某些实施方案中，药物还包含另外的药学活性剂。
在某些实施方案中，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、分子靶向药物、免疫检查点抑制剂或溶瘤病毒。
在某些实施方案中，所述肿瘤表达Claudin 18.2。
在某些实施方案中，所述肿瘤涉及表达Claudin 18.2的肿瘤细胞。在某些实施方案中，所述Claudin 18.2在所述肿瘤细胞表面上表达。
在某些实施方案中，所述肿瘤选自胃癌、肝癌、胆道癌、肾细胞癌、胰腺癌、非小细胞肺癌、间皮瘤、卵巢癌、睾丸癌、子宫内膜癌、肺癌、食管癌、胰腺癌、支气管癌、乳腺癌、耳鼻喉(ENT)癌、结肠癌、头颈癌、胆囊癌。
在某些实施方案中，所述受试者为哺乳动物，例如人。
在第九方面，本申请提供了如前所述的双特异性抗体，或如前所述的免疫缀合物，或如前所述的药物组合物在制备试剂盒中的用途，所述试剂盒用于：
(1)识别或结合表达Claudin 18.2的细胞；
(2)识别或结合表达4-1BB的细胞；
(3)激活细胞的NF-κB信号通路；
(4)诱导细胞(例如，免疫细胞)激活杀伤肿瘤的活性；
(5)诱导细胞(例如，免疫细胞)分泌细胞因子(例如，IFNγ和IL-2)；
(6)(1)-(5)的任意组合。
在某些实施方案中，所述免疫细胞选自B细胞，T细胞，NK细胞。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的分子遗传学、核酸化学、化学、分子生物学、生物化学、细胞培养、微生物学、细胞生物学、基因组学和重组DNA等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“Claudin-18(CLDN18)”是指位于细胞膜上的一个跨膜 蛋白。人类CLDN18基因的第一个外显子存在两个等位基因，可表达Claudin 18.1与Claudin 18.2两种不同的剪切突变体。Claudin 18.1与Claudin 18.2虽然结构极为相似，但其在正常组织和肿瘤中的表达均有差异。Claudin 18.2仅在胃黏膜组织中特异性表达，且Claudin 18.2在胃癌、食管癌、胰腺癌等癌种中表达发生上调。Claudin 18.2的表达不仅在原发肿瘤中可检测到，还在肿瘤转移灶中大量表达。本领域技术人员可以通过公共数据库(例如，NCBI)获知Claudin 18.2的序列，例如参见，NCBI数据库中的序列号NP_001002026.1。在本文中，“Claudin 18.2”也可简称为“CLND18.2”。
如本文中所使用的，术语“4-1BB”又称为CD137，属肿瘤坏死因子受体超家族成员(TNFRSF9)。主要表达于活化的T细胞，是T细胞协同刺激分子，其配体为4-1BBL，二者结合可刺激T细胞活化和增殖。本领域技术人员可以通过公共数据库(例如，NCBI)获知4-1BB的序列，例如参见，NCBI数据库中的序列号NP_001552.2。
如本文中所使用的，术语“抗体”是指，通常由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成的免疫球蛋白分子。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL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。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。
如本文中所使用的，术语“单域抗体(single domain antibody，sdAb)”、“结 构域抗体”与“纳米抗体(nanobody)”可互换使用，其是指由抗体中单个可变结构域(例如重链可变区)所组成的抗体片段。典型地，单域抗体、结构域抗体或纳米抗体由4个构架区和3个互补性决定区组成，所述4个构架区分别为FR1-FR4，所述3个互补性决定区分别为CDR1-CDR3。在某些实施方案中，本申请的单域抗体可以具有FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的结构。这些抗体不需要轻链可变区即可以高亲和力和特异性与抗原结合。与重链和轻链组成的抗体相比，纳米抗体可溶度高，对热、pH、蛋白酶和其它变形剂稳定度高，只需单链表达便于大规模生产。
如本文中所使用的，术语“双特异性抗体”是指其由第一抗体(或其片段)和第二抗体(或其片段)或抗体类似物通过偶联臂所形成的偶联物，偶联的方式包括但不限于化学反应、基因融合和酶促。双特异性抗体可通过各种方法连接或产生，例如参见Songsivilai等人的方法(Clin.Exp.Immunol.,79:315-321(1990))，以及Kostelny等人的方法(J.Immunol.,148:1547-1553(1992))。
如本文中所使用的，术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，FR)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力、增强免疫应答的能力等。供体抗体可以是有预期性质(例如，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力和/或增强免疫应答的能力)的驼源抗体。
如本文中所使用的，术语“结合实体”是指特异性结合靶抗原的任何单体或多聚体蛋白或蛋白片段。术语“结合实体”包括但不限于抗体或其结合部分，诸如免疫功能片段。
如本文中所使用的，术语“肽接头”是指经过调整能够连接抗体结合实体的肽。
如本文中所使用的，术语“免疫球蛋白”是指由一种或多种通过免疫球蛋白基因编码的多肽组成的蛋白质。免疫球蛋白构成抗体的基础结构单元。抗体通常为四聚体且由两对相同的免疫球蛋白链组成，每一对免疫球蛋白链具有一条轻链及一条重链。在每一对免疫球蛋白链中，轻链及重链可变区一起负责抗原结合，且恒定区负责抗体效应物功能。
如本文中所使用的，免疫球蛋白的“轻链”可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在重链和轻链的可变区中各含有三个CDR，命名为CDR1、CDR2和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)或IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)中的定义。对于给定的抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。
如本文中所使用的，术语“构架区(framework region)”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
如本文中所使用的，术语“单克隆抗体”、“单抗”、“mAb”具有相同的含义且可互换使用可互换，其是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即，除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。此外，修饰语“单克隆”仅表明该抗体的特征为从高度同源的抗体群中获得，不能理解为需要通过任何特定方法来制备所述抗体。
本发明的单克隆抗体可以通过多种技术进行制备，例如杂交瘤技术(参见，例如Kohler等人.Nature,256:495,1975)，重组DNA技术(参见，例如美国专利申请4,816,567)，或噬菌体抗体库技术(参见，例如Clackson等.Nature352：624-628,1991，或Marks等.J.Mol.Biol.222：581-597,1991)。
如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如人。在某些实施方式中，所述受试者(例如人)患有肿瘤(例如表达CLDN6和/或CLDN9的肿瘤)，或者，具有患有上述疾病的风险。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如，肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如，肿瘤)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾 病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.ApplBiosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoIBiol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、 非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.NatlAcad.Set USA 94:412-417(1997)，其通过引用并入本文)。
如本文中所使用的，术语“L234A突变”是指将天然的Fc片段的第234位氨基酸由L突变为A。同理的，“L235A突变”是指将天然的Fc片段的第235位氨基酸由L突变为A。
发明的有益效果
与现有技术相比，本申请制备的双特异性抗体能够识别或结合表达Claudin 18.2的细胞和/或识别或结合表达4-1BB的细胞。并且，能够激活细胞的NF-κB信号通路，诱导细胞激活杀伤肿瘤的活性，还能够诱导细胞分泌细胞因子(例如，IFNγ和IL-2)，并能够在受试者中预防和/或治疗肿瘤。并且，该双特异性抗体与市售成品单抗或联合使用的单抗相比，具有更突出的预防和/或治疗肿瘤的作用。
另外，本申请与4-1BB单抗相比，降低了肝脏相关靶向效应。通过使用肿瘤相关抗原(TAA)Claudin 18.2来交联缺乏Fc受体(FcR)结合特性的4-1BB受体，实现了无4-1BB相关毒性的靶向抗肿瘤功效。同时本申请设计了Fc域，能够有效去除或降低FcR结合，限制Fc介导的生物学活性。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了本申请抗Claudin 18.2和4-1BB的双特异性抗体的示意图。
图2显示了本申请的双特异性抗体与Claudin 18.2的结合结果。其中，图2A显示了本申请的双特异性抗体与NUGC4细胞自身表达的Claudin 18.2的结合结果；图2B显示了本申请的双特异性抗体与MC38细胞过表达的人Claudin 18.2的结合结果；其中，Claudin 18.2×4-1BB biAb为本申请的双特异性抗体，4-1BB sdAb为4-1BB单域抗体，Claudin 18.2 mAb为Claudin 18.2单抗。
图3显示了本申请的双特异性抗体与CHO细胞过表达的人4-1BB的结合结果；其中，Claudin 18.2×4-1BB biAb为本申请的双特异性抗体，4-1BB sdAb为4-1BB单域抗体，Claudin 18.2 mAb为Claudin 18.2单抗，Urelumab为4-1BB的单抗。
图4显示了本申请的双特异性抗体激活Jurkat细胞NF-κB信号通路的结果；其中，Claudin 18.2×4-1BB biAb为本申请的双特异性抗体，4-1BB sdAb为4-1BB单域抗体，Claudin 18.2 mAb为Claudin 18.2单抗，Urelumab为4-1BB的单抗。
图5显示了本申请的双特异性抗体诱导外周血单个核细胞(PBMC)对于Claudin 18.2阳性肿瘤细胞NUGC4的杀伤结果；其中，Claudin 18.2×4-1BB biAb为本申请的双特异性抗体，4-1BB sdAb为4-1BB单域抗体，Urelumab为4-1BB的单抗。
图6显示了本申请的双特异性抗体诱导PBMC的细胞因子释放的结果，图6A为细胞因子IFNγ释放的结果，图6B为细胞因子IL-2释放的结果；其中，Claudin 18.2×4-1BB biAb为本申请的双特异性抗体，4-1BB sdAb为4-1BB单域抗体，Urelumab为4-1BB的单抗，Neg Ctrl为阴性对照。
图7显示了本申请的双特异性抗体对肿瘤抑制活性的测试结果。其中，PBS为阴性对照，Claudin 18.2×4-1BB biAb为本申请的双特异性抗体，IMAB362为Claudin 18.2单抗，Claudin 18.2 mAb+4-1BB sdAb为构建本申请双抗的两种抗体的联合给药。
图8显示了本申请的双特异性抗体对肿瘤抑制记忆效应的测试结果。其中，PBS为阴性对照，Claudin 18.2×4-1BB biAb为本申请不同浓度的双特异性抗体，PBS in new 4-1BB KI mice为再次肿瘤接种时的阴性对照。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。
表1：序列的描述
Figure PCTCN2022113031-appb-000001
Figure PCTCN2022113031-appb-000002
Figure PCTCN2022113031-appb-000003
Figure PCTCN2022113031-appb-000004
Figure PCTCN2022113031-appb-000005
Figure PCTCN2022113031-appb-000006
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法。例如，本发明中所使用的免疫学、生物化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA等常规技术，可参见萨姆布鲁克(Sambrook)、弗里奇(Fritsch)和马尼亚蒂斯(Maniatis)，《分子克隆：实验室手册》(MOLECULAR CLONING:A LABORATORY MANUAL)，第2次编辑(1989)；《当代分子生物学实验手册》(CURRENT PROTOCOLS IN MOLECULAR BIOLOGY)(F.M.奥苏贝尔(F.M.Ausubel)等人编辑，(1987))；《酶学方法》(METHODS IN ENZYMOLOGY)系列(学术出版公司)：《PCR 2：实用方法》(PCR 2:A PRACTICAL APPROACH)(M.J.麦克弗森(M.J.MacPherson)、B.D.黑姆斯(B.D.Hames)和G.R.泰勒(G.R.Taylor)编辑(1995))，以及《动物细胞培养》(ANIMAL CELL CULTURE)(R.I.弗雷谢尼(R.I.Freshney)编辑(1987))。
另外，实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所 用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
实施例1.抗Claudin 18.2 x 4-1BB双特异性抗体的克隆和表达
在本实施例中，构建了抗Claudin 18.2×4-1BB双特异性抗体(biAb)。Claudin 18.2x4-1BB biAb由2条多肽链组成，其结构示意图如图1所示。该双特异性抗体的重链具有SEQ ID NO:6所示的氨基酸序列，其包含抗claudin18 isoform 2(Claudin 18.2)的单克隆抗体anti-Claudin 18.2 mAb的重链可变区氨基酸序列(SEQ ID NO:1)、人IgG1Fc的氨基酸序列(引入LALA突变以降低Fc功能，SEQ ID NO:4)、柔性肽的氨基酸序列(SEQ ID NO:5)以及抗4-1BB的单域抗体的4-1BB结合区域的氨基酸序列(SEQ ID NO:3)。其中，抗4-1BB的单域抗体的4-1BB重链可变区氨基酸序列(SEQ ID NO:3)的N端通过柔性肽(SEQ ID NO:5)连接于Fc的C端。
该双特异性抗体的轻链具有SEQ ID NO:7所示的氨基酸序列，其包含抗claudin18isoform2的单克隆抗体anti-Claudin 18.2 mAb的轻链可变区氨基酸序列(SEQ ID NO:2)，以及在所述VL氨基酸序列C端的人κ轻链恒定区(CL)氨基酸序列(SEQ ID NO:8)。
采用ExpiCHO TM表达系统试剂盒(购自Thermo)，将含有编码抗体重链和轻链氨基酸序列(双特异性抗体Claudin 18.2x4-1BB biAb重链的氨基酸序列如SEQ ID NO:6所示，双特异性抗体Claudin 18.2x4-1BB biAb轻链的氨基酸序列如SEQ ID NO:7所示)的核苷酸序列的抗体表达质粒分别转入Expi-CHO细胞中，转染方法按照商品说明书，细胞培养5天后收集上清利用蛋白A磁珠(购自金斯瑞)分选法纯化目的蛋白。将磁珠用适当体积的结合缓冲液(PBS+0.1％吐温20，pH 7.4)重悬(1-4倍磁珠体积)后加入至待纯化样品中，室温孵育1小时，期间温柔振荡。样品置于磁力架上(购自海狸)，弃去上清，磁珠用结合缓冲液清洗3遍。按照磁珠体积的3-5倍体积加入洗脱缓冲液(0.1M sodium citrate,pH 3.2)室温振荡5-10min，置回磁力架上，收集洗脱缓冲液，转移至已加入中和缓冲液(1M Tris，pH 8.54)的收集管中混匀。由此获得本申请的双特异性抗体。
采用ExpiCHO TM表达系统试剂盒(购自Thermo)，将含有编码抗体重链和轻链的核苷酸序列(编码Urelumab单抗重链和轻链的核苷酸序列分别如SEQ ID NO:20和SEQ ID NO: 21所示；编码4-1BB单域抗体重链的核苷酸序列如SEQ ID NO:22所示；编码Claudin18.2重链和轻链的核苷酸序列分别如SEQ ID NO:23和SEQ ID NO:24所示)的表达质粒转入Expi-CHO细胞中，转染方法按照商品说明书，细胞培养5天后收集上清利用蛋白A磁珠(购自金斯瑞)分选法纯化目的蛋白。将磁珠用适当体积的结合缓冲液(PBS+0.1％吐温20，pH 7.4)重悬(1-4倍磁珠体积)后加入至待纯化样品中，室温孵育1小时，期间温柔振荡。样品置于磁力架上(购自海狸)，弃去上清，磁珠用结合缓冲液清洗3遍。按照磁珠体积的3-5倍体积加入洗脱缓冲液(0.1M sodium citrate,pH 3.2)室温振荡5-10min，置回磁力架上，收集洗脱缓冲液，转移至已加入中和缓冲液(1M Tris，pH 8.54)的收集管中混匀。由此分别获得Urelumab单抗、4-1BB单域抗体和Claudin 18.2单抗。
实施例2.双特异性抗体与Claudin 18.2结合
通过慢病毒包被人Claudin 18.2 cDNA(来自吉凯基因，GCPLO162852)，转染MC38并产生表达人Claudin 18.2的MC38细胞，将其命名为MC38-h Claudin 18.2细胞。将扩大培养的细胞MC38-hClaudin 18.2和NUGC4(自身表达hClaudin 18.2)细胞用0.5mM EDTA消化，用培养基清洗一次后调整细胞密度至2×10 6细胞/ml，100μl/孔加入96孔流式板，离心备用。将双特异性抗体用PBS稀释，并将上述稀释好的样品100μl/孔加入上述带有细胞的96孔流式板中，4℃孵育60min，PBS清洗两次。100μl/孔加入用PBS稀释1000倍的Goat anti-human IgG-Fc(PE)(购自Abcam)，4℃孵育30min，PBS清洗两次。100μl/孔加入PBS重悬细胞，在CytoFlex(Beckman)流式细胞仪上进行检测并计算对应的平均荧光强度(MFI)。
在如上方法的测定实验中，实验结果如图2A所示，本发明的双特异性抗体与人肿瘤细胞自身表达的Claudin 18.2结合。图2B进一步显示，双特异性抗体与鼠肿瘤细胞MC38过表达的人的Claudin 18.2结合，且与Claudin 18.2单抗结合活性相当。
实施例3.双特异性抗体抗体与4-1BB结合
通过转染克隆到多克隆位点(MCS)的人4-1BB cDNA的PLVX载体(合成自擎科)，产生过表达人4-1BB的CHO细胞，将其命名为CHO-h4-1BB细胞。将扩大培养的CHO-h4-1BB细胞调整细胞密度至2×10 6细胞/ml，100μl/孔加入96孔流式板，离心备用。将双特异性抗体用PBS稀释，并将上述稀释好的样品100μl/孔加入上述带有细胞的96孔流式 板中，4℃孵育60min，PBS清洗两次。100μl/孔加入用PBS稀释100倍的Goat anti-human IgG-Fc(PE)(Abcam，ab98596)，4℃孵育30min，PBS清洗两次。100μl/孔加入PBS重悬细胞，在CytoFlex(Beckman)流式细胞仪上进行检测并计算对应的MFI。在如上方法的测定实验中，实验结果如图3所示，本发明的双特异性抗体和CHO-h4-1BB细胞有结合活性。
实施例4.双特异性抗体激活Jurkat细胞NF-κB信号通路
将靶细胞NUGC4按照2×10 4个/孔与1.2×10 5个/孔的效应细胞与4-1BB NF-κB Luciferase/Jurkat(将Jurkat(ATCC,TIB-152 TM)细胞共转染表达人4-1BB载体(金唯智合成)与luc2P/NF-kB-RE载体(Promega,E8491)，即获得该细胞)混合接种至96孔细胞培养白底板中，并将梯度稀释后的双特异性抗体与终浓度为1μg/mL的anti-CD3加入后共孵育16小时。使用Bio-glo luciferase assay system(Promega G7940)试剂盒显色后用酶标仪收集化学发光信号。结果如图4所示，只有本发明的双抗激活了Jurkat细胞的NF-κB信号通路。
实施例5.双特异性抗体诱导PBMC杀伤肿瘤细胞
将靶细胞NUGC4通过CellTrace TMViolet试剂盒进行细胞染色，并按照每孔1×10 4个/孔接种至96孔透明底黑边细胞培养板中，培养4小时。收集提前一天复苏的PBMC并按照每孔1×10 5个/孔加入靶细胞孔中。将梯度稀释后的双特异性抗体与终浓度为0.5μg/mL的anti-CD3混合并加入到细胞孔中孵育。48小时后使用Cytation 5收集DAPI荧光信号并计算对应的杀伤强度。结果如图5所示，只有双抗分子诱导了PBMC对于Claudin 18.2阳性肿瘤细胞NUGC4的杀伤(图中纵坐标的数值为扣除了背景值的结果)。
实施例6.双特异性抗体诱导PBMC的细胞因子释放
将靶细胞NUGC4细胞按照每孔1×10 4个/孔接种至96孔透明底黑边细胞培养板中，培养4小时。收集提前一天复苏的PBMC并按照每孔1×10 5个/孔加入靶细胞孔中。将梯度稀释后的双特异性抗体与终浓度为0.5μg/mL的anti-CD3混合并加入到细胞孔中孵育48小时后收集上清。上清中的IFNγ和IL-2水平使用human IL-2 ELISA kit (Invitrogen,88-7025-77)和human IFNγ(Invitrogen,88-7316-77)ELISA kit检测并按供应商推荐步骤操作。
结果如图6A和6B所示，只有双抗分子诱导了IFNγ和IL-2的释放。
实施例7.双特异性抗体的肿瘤抑制活性研究
本实验采用人MC38-hClaudin 18.2细胞和h-4-1BB转基因C57BL/6小鼠模型测定双特异性抗体的抗肿瘤作用。体外培养扩增足够的MC38-hClaudin 18.2细胞(在MC38细胞基础上过表达hClaudin 18.2)，胰酶消化后收集细胞，用PBS清洗3遍后计数，按1×10 6细胞/小鼠的量接种到雌性8周龄的h-4-1BB转基因C57BL/6小鼠(购自上海南方模式生物科技股份有限公司)右侧腹部皮下。每日观察肿瘤细胞在小鼠皮下成瘤情况，使用游标卡尺测量每只动物右侧腹部皮下肿瘤的最大宽轴W和最大长轴L，使用电子天平称量每只小鼠的体重。按肿瘤体积T＝1/2×W×W×L计算每只小鼠右侧腹部皮下肿瘤体积。剔除瘤体积过大和过小的小鼠，按平均瘤体积将小鼠平均分为4组，每组6只。按表6分组给药方案分组并注射相应剂量的抗体。
每周2-3次测量小鼠肿瘤体积与小鼠体重。于接种肿瘤细胞29天后最后一次测量小鼠体重与肿瘤体积，对小鼠执行安乐死。结果如图7所示，与PBS组相比，Claudin 18.2单抗IMAB362基本没有抑制肿瘤生长的效果，anti-Claudin 18.2单抗和anti-4-1BB单域抗体联合给药亦无抑瘤效果。而同摩尔剂量的Claudin 18.2x4-1BB biAb显著抑制了肿瘤的生长，该组6只小鼠全部肿瘤消退，与其他3组相比具有显著性差异。
表2.肿瘤抑制活性实验方案
Figure PCTCN2022113031-appb-000007
实施例8.双特异性抗体的肿瘤抑制活性研究(Rechallenge)
本实验采用人MC38-hClaudin 18.2细胞和h-4-1BB转基因C57BL/6小鼠模型测定双 特异性抗体的剂量依赖性地抗肿瘤作用。体外培养扩增足够的MC38-hClaudin 18.2细胞(在MC38细胞基础上过表达hClaudin 18.2)，胰酶消化后收集细胞，用PBS清洗3遍后计数，按1×10 6细胞/小鼠的量接种到雌性8周龄的h-4-1BB转基因C57BL/6小鼠(购自上海南方模式生物科技股份有限公司)右侧腹部皮下。每日观察肿瘤细胞在小鼠皮下成瘤情况，使用游标卡尺测量每只动物右侧腹部皮下肿瘤的最大宽轴W和最大长轴L，使用电子天平称量每只小鼠的体重。按肿瘤体积T＝1/2×W×W×L计算每只小鼠右侧腹部皮下肿瘤体积。剔除瘤体积过大和过小的小鼠，按平均瘤体积将小鼠平均分为5组，每组6只。按表7分组给药方案分组并注射相应剂量的抗体。
每周2-3次测量小鼠肿瘤体积与小鼠体重。于接种肿瘤细胞29天后最后一次测量小鼠体重与肿瘤体积，对小鼠执行安乐死。如图8所示，与PBS组相比，Claudin 18.2x4-1BB biAb在0.06mg/kg、0.25mg/kg、1mg/kg、4mg/kg剂量范围内可剂量依赖性地显著抑制了肿瘤的生长，0.25mg/kg组有4只小鼠肿瘤消退、1mg/kg组有5只小鼠肿瘤消退、4mg/kg组6只小鼠全部肿瘤消退。将肿瘤消退的15只再次接种足够的MC38-hClaudin18.2肿瘤细胞，同时在6只新采购的人4-1BB转基因C57BL/6小鼠皮下接种相同数量的MC38-hClaudin 18.2细胞作为对照组，对照组6只小鼠肿瘤均快速长大，而给予双抗后肿瘤消退的小鼠肿瘤全部无法生长，表明双抗组小鼠形成了免疫记忆，可阻止Rechallenge肿瘤的生长。
表3.剂量依赖性肿瘤抑制活性实验方案
组别 给药类别 给药剂量 给药频率
Group 1 PBS — 每两天给药1次，共4次
Group 2 Claudin 18.2x4-1BB biAb 0.06mg/kg 每两天给药1次，共4次
Group 3 Claudin 18.2x4-1BB biAb 0.25mg/kg 每两天给药1次，共4次
Group 4 Claudin 18.2x4-1BB biAb 1mg/kg 每两天给药1次，共4次
Group 5 Claudin 18.2x4-1BB biAb 4mg/kg 每两天给药1次，共4次
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (15)

  1. 一种双特异性抗体，其包含Claudin 18.2结合实体和4-1BB结合实体，其中，所述Claudin 18.2结合实体包含两对相同的免疫球蛋白链，其中，每一对免疫球蛋白链具有一条轻链和一条重链，所述重链包含重链可变区和重链恒定区；所述轻链包含轻链可变区和轻链恒定区；所述4-1BB结合实体包含重链可变区；并且，所述4-1BB结合实体的重链可变区与所述Claudin 18.2结合实体的重链的C末端连接；其中，
    所述Claudin 18.2结合实体包含：如SEQ ID NO:1所示的重链可变区(VH)中含有的VH CDR1或其变体、VH CDR2或其变体以及VH CDR3或其变体；和/或，如SEQ ID NO:2所示的轻链可变区(VL)中含有的VL CDR1或其变体、VL CDR2或其变体以及VL CDR3或其变体；
    所述4-1BB结合实体包含：如SEQ ID NO:3或25所示的重链可变区(VH)中含有的VH CDR1或其变体、VH CDR2或其变体以及VH CDR3或其变体；
    其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加，例如保守置换)；优选地，所述的置换是保守置换；
    优选地，所述VH中含有的3个CDR和/或所述VL中含有的3个CDR由Kabat、IMGT或Chothia编号系统定义。
  2. 权利要求1所述的抗体或其抗原结合片段，其具有选自下列的一项或两项特征：
    (1)所述Claudin 18.2结合实体包含：
    包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:9的VH CDR1、序列为SEQ ID NO:10的VH CDR2、序列为SEQ ID NO:11的VH CDR3；和/或，包含下述3个互补决定区(CDRs)的轻链可变区(VL)：序列为SEQ ID NO:12的VL CDR1、序列为SEQ ID NO:13的VL CDR2、序列为SEQ ID NO:14的VL CDR3；其中，所述CDR由IMGT编号系统定义；或
    (2)所述4-1BB结合实体包含：
    包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:15的VH CDR1、序列为SEQ ID NO:16的VH CDR2、序列为SEQ ID NO:17的VH CDR3；其 中，所述CDR由IMGT编号系统定义。
  3. 一种双特异性抗体，其包含Claudin 18.2结合实体和4-1BB结合实体，其中，所述Claudin 18.2结合实体包含两对相同的免疫球蛋白链，其中，每一对免疫球蛋白链具有一条轻链和一条重链，所述重链包含重链可变区和重链恒定区；所述轻链包含轻链可变区和轻链恒定区；所述4-1BB结合实体包含重链可变区；并且，所述4-1BB结合实体的重链可变区与所述Claudin 18.2结合实体的重链的C末端连接；
    其中，所述Claudin 18.2结合实体的重链可变区(VH)包含下述3个互补决定区(CDR)：
    (i)VH CDR1，其由下述序列组成：SEQ ID NO:9，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
    (ii)VH CDR2，其由下述序列组成：SEQ ID NO:10，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
    (iii)VH CDR3，其由下述序列组成：SEQ ID NO:11，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    所述Claudin 18.2结合实体的轻链可变区(VL)包含下述3个互补决定区(CDR)：
    (iv)VL CDR1，其由下述序列组成：SEQ ID NO:12，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
    (v)VL CDR2，其由下述序列组成：SEQ ID NO:13，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和(vi)VL CDR3，其由下述序列组成：SEQ ID NO:14，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    所述4-1BB结合实体的重链可变区包含下述3个互补决定区(CDR)：
    (vii)VH CDR1，其由下述序列组成：SEQ ID NO:15，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，
    (viii)VH CDR2，其由下述序列组成：SEQ ID NO:16，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，和
    (ix)VH CDR3，其由下述序列组成：SEQ ID NO:17，或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列；
    优选地，所述Claudin 18.2结合实体，其包含：如下3个重链CDRs：序列为SEQ ID NO：9的VH CDR1，序列为SEQ ID NO：10的VH CDR2，序列为SEQ ID NO：11的VH CDR3；和/或，如下3个轻链CDRs：序列为SEQ ID NO：12的VL CDR1，序列为SEQ ID NO：13的VL CDR2，序列为SEQ ID NO：14的VL CDR3；
    优选地，所述4-1BB结合实体的重链可变区(VH)包含：如下3个重链CDRs：序列为SEQ ID NO：15的VH CDR1，序列为SEQ ID NO：16的VH CDR2，序列为SEQ ID NO：17的VH CDR3；
    优选地，所述重链可变区还包含来源于人的框架区序列(例如，人免疫球蛋白)。
  4. 权利要求3所述的双特异性抗体，其中，所述Claudin 18.2结合实体包含：
    (a)重链可变区(VH)，其包含选自下列的氨基酸序列：
    (i)SEQ ID NO:1所示的序列；
    (ii)与SEQ ID NO:1所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (iii)与SEQ ID NO:1所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    和/或，
    (b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
    (iv)SEQ ID NO:2所示的序列；
    (v)与SEQ ID NO:2所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (vi)与SEQ ID NO:2所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，(ii)或(v)中所述的置换是保守置换；
    优选地，所述Claudin 18.2结合实体包含：具有如SEQ ID NO:1所示的序列的VH 和具有如SEQ ID NO:2所示的序列的VL。
  5. 权利要求3或4所述的双特异性抗体，其中，所述4-1BB结合实体包含：
    (a)重链可变区(VH)，其包含选自下列的氨基酸序列：
    (i)SEQ ID NO:3或25所示的序列；
    (ii)与SEQ ID NO:3或25所示的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)的序列；或
    (iii)与SEQ ID NO:3或25所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，(ii)中所述的置换是保守置换。
  6. 权利要求1-5任一项所述的双特异性抗体，其中，所述Claudin 18.2结合实体还包含：
    (a)人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；和
    (b)人免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的野生型序列相比具有至多20个氨基酸的保守置换(例如至多15个、至多10个、或至多5个氨基酸的保守置换；例如1个，2个，3个，4个或5个氨基酸的保守置换)；
    优选地，所述重链恒定区是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区；优选地，所述轻链恒定区是κ或λ轻链恒定区；
    优选地，所述Claudin 18.2结合实体包含SEQ ID NO:8所示的轻链恒定区(CL)；
    优选地，所述双特异性抗体还包含不与Fc受体(FcR)结合的Fc片段；或者，包含具有降低的效应器功能的Fc片段，所述效应器功能为抗体依赖性细胞介导的细胞毒性(ADCC)、补体依赖的细胞毒性(CDC)或抗体依赖性细胞吞噬作用(ADCP)；
    优选地，所述Fc片段选自下列的任意一项突变：
    (a)具有L235E突变的IgG1Fc片段；
    (b)具有L234A和/或L235A突变的IgG1Fc片段；
    (c)具有P329G或P329A突变的IgG1Fc片段；
    (d)具有F234A和/或L235A或L235E突变的IgG4Fc片段；
    (e)具有H268Q、V309L、A330S和/或P331S突变的IgG2Fc片段；或
    (f)具有V234A、G237A、P238S、H268A、V309L、A330S和/或P331S突变的IgG2Fc片段；
    更优选地，所述Fc片段为具有L234A和L235A突变的IgG1Fc片段；
    优选地，所述Fc片段具有SEQ ID NO:4所示的序列。
  7. 权利要求1-6任一项的双特异性抗体，其中，所述双特异性抗体还包含肽接头，并且，所述Claudin 18.2结合实体和4-1BB结合实体通过肽接头连接；
    优选地，所述Claudin 18.2结合实体的重链恒定区的C端与4-1BB结合实体的重链可变区的N端通过肽接头连接；
    优选地，所述肽接头具有如SEQ ID NO:5或26所示的序列；
    优选地，所述双特异性抗体的重链具有如SEQ ID NO:6，27，28或29所示的氨基酸序列；
    优选地，所述双特异性抗体的轻链具有如SEQ ID NO:7所示的氨基酸序列。
  8. 分离的核酸分子，其编码权利要求1-7任一项所述的双特异性抗体，或其重链和/或轻链，或Claudin 18.2结合实体的重链可变区和/或轻链可变区，或4-1BB结合实体的重链可变区。
  9. 载体，其包含权利要求8所述的分离的核酸分子；优选地，所述载体为克隆载体或表达载体。
  10. 宿主细胞，其包含权利要求8所述的分离的核酸分子或权利要求9所述的载体。
  11. 免疫缀合物，其包含权利要求1-7任一项所述的双特异性抗体以及连接于所述双特异性抗体的治疗剂；
    优选地，所述治疗剂选自细胞毒剂；
    优选地，所述治疗剂选自烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂，及其任意组合；
    优选地，所述免疫缀合物是抗体-药物偶联物(ADC)。
  12. 药物组合物，其含有权利要求1-7任一项所述的双特异性抗体或者权利要求11所述的免疫缀合物，以及药学上可接受的载体和/或赋形剂；
    优选地，药物组合物还包含另外的药学活性剂；
    优选地，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、分子靶向药物、免疫检查点抑制剂或溶瘤病毒；
    优选地，所述双特异性抗体或免疫缀合物与所述另外的药学活性剂作为分离的组分或作为同一组合物的组分提供。
  13. 一种抑制表达Claudin 18.2的肿瘤细胞生长和/或杀伤所述肿瘤细胞的方法，其包括将所述肿瘤细胞与有效量的权利要求1-7任一项所述的双特异性抗体，或权利要求11所述的免疫缀合物，或权利要求12所述的药物组合物接触。
  14. 权利要求1-7任一项所述的双特异性抗体，或权利要求11所述的免疫缀合物在制备药物中的用途，所述药物用于在受试者(例如人)中预防和/治疗肿瘤；
    优选地，药物还包含另外的药学活性剂；
    优选地，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如烷化剂、有丝分裂抑制剂、抗肿瘤抗生素、抗代谢物、拓扑异构酶抑制剂、酪氨酸激酶抑制剂、放射性核素剂、放射增敏剂、抗血管生成剂、细胞因子、分子靶向药物、免疫检查点抑制剂或溶瘤病毒；
    优选地，所述肿瘤表达Claudin 18.2；
    优选地，所述肿瘤涉及表达Claudin 18.2的肿瘤细胞；优选地，所述Claudin 18.2在所述肿瘤细胞表面上表达；
    优选地，所述肿瘤选自胃癌、肝癌、胆道癌、肾细胞癌、胰腺癌、非小细胞肺癌、间皮瘤、卵巢癌、睾丸癌、子宫内膜癌、肺癌、食管癌、胰腺癌、支气管癌、乳腺癌、耳鼻喉(ENT)癌、结肠癌、头颈癌、胆囊癌；
    优选地，所述受试者为哺乳动物，例如人。
  15. 权利要求1-7任一项所述的双特异性抗体，或权利要求11所述的免疫缀合物，或权利要求12所述的药物组合物在制备试剂盒中的用途，所述试剂盒用于：
    (1)识别或结合表达Claudin 18.2的细胞；
    (2)识别或结合表达4-1BB的细胞；
    (3)激活细胞的NF-κB信号通路；
    (4)诱导细胞(例如，免疫细胞)激活杀伤肿瘤的活性；
    (5)诱导细胞(例如，免疫细胞)分泌细胞因子(例如，IFNγ和IL-2)；
    (6)(1)-(5)的任意组合。
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