WO2024061223A1 - 抗体及其在抗肿瘤中的应用 - Google Patents

抗体及其在抗肿瘤中的应用 Download PDF

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WO2024061223A1
WO2024061223A1 PCT/CN2023/119761 CN2023119761W WO2024061223A1 WO 2024061223 A1 WO2024061223 A1 WO 2024061223A1 CN 2023119761 W CN2023119761 W CN 2023119761W WO 2024061223 A1 WO2024061223 A1 WO 2024061223A1
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cancer
antigen
antibody
amino acid
seq
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PCT/CN2023/119761
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English (en)
French (fr)
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罗羿
陈连娣
黄威峰
缪小牛
闫尧
王超
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普米斯生物技术(珠海)有限公司
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Publication of WO2024061223A1 publication Critical patent/WO2024061223A1/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
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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 relates to the field of biotherapy technology, specifically to antibodies targeting 4-1BB and HER2 and their application in anti-tumor.
  • Human epidermal growth factor receptor 2 (HER2/ErbB2/Neu) is a member of the ERBB receptor tyrosine kinase family and is overexpressed in many solid tumors. It can form homodimers or interact with the ERBB family Other members form heterodimers that cause the phosphorylation of tyrosine residues in the cytoplasmic domain of the receptor, thereby initiating a variety of intracellular signaling pathways leading to cancer cell proliferation and tumorigenesis [1] .
  • therapeutic drugs targeting HER2 include monoclonal antibodies (mAbs, such as trastuzumab and pertuzumab), tyrosine kinase inhibitors (TKIs, such as naratinib and lapatinib), antibody-drug conjugates (ADCs, such as T-DM1), etc., these drugs have been widely used in the treatment of breast cancer or gastric cancer with HER2 overexpression and/or amplification [2 ] . Although these HER2-targeted therapeutic drugs have good clinical benefits, medical needs remain unmet, and new drugs need to be continuously developed to further improve the clinical efficacy in patients with recurrence or metastasis.
  • mAbs such as trastuzumab and pertuzumab
  • TKIs tyrosine kinase inhibitors
  • ADCs antibody-drug conjugates
  • T cell costimulatory receptor TNFRSF9 (4-1BB) is a member of the TNF receptor family and is mainly expressed on activated T cells. When activated, it can increase the effector activity and memory response of T cells [3,4] .
  • Urelumab is a monoclonal antibody drug targeting 4-1BB. It exerts its efficacy by binding and activating 4-1BB on T cells. Unfortunately, significant liver-related toxicities were observed during Urelumab's clinical development.
  • This application discloses a single domain antibody with high binding activity to 4-1BB, and a multispecific antibody targeting 4-1BB and HER2 developed on this basis.
  • this application discloses a HER2xHER2x4-1BB trispecific antibody, which has unique anti-tumor activity and a strong immune memory effect.
  • the antibody is a 1+1 IgG1-like heterodimer antibody composed of trastuzumab and pertuzumab, with an anti-4-1BB single domain antibody (sdAb) connected to its C-terminus through a G4S linker. ).
  • the antibody retains Fc effector functions, which are essential for the anti-tumor activity of trastuzumab and pertuzumab.
  • the antibody has lower liver toxicity and improved safety.
  • the application provides a single domain antibody or an antigen-binding fragment thereof capable of specifically binding to 4-1BB, the single domain antibody comprising:
  • CDR1 which has: a sequence as shown in SEQ ID NO:43, or one or several amino acid substitutions, deletions or additions (such as 1, 2) compared with the sequence shown in SEQ ID NO:43 Sequence with substitution, deletion or addition of one or three amino acids);
  • CDR2 which has: a sequence as shown in SEQ ID NO:44, or one or several amino acid substitutions, deletions or additions (e.g. 1, 2) compared to the sequence shown in SEQ ID NO:44 or 3 amino acid substitutions, deletions or additions); and
  • CDR3 which has: a sequence as shown in SEQ ID NO:45, or one or several amino acid substitutions, deletions or additions (such as 1, 2) compared with the sequence shown in SEQ ID NO:45 substitution, deletion or addition of one or three amino acids).
  • the single-domain antibody or its antigen-binding fragment comprises: CDR1 as shown in SEQ ID NO:43, CDR2 as shown in SEQ ID NO:44, and CDR3 as shown in SEQ ID NO:45.
  • the single domain antibody comprises an amino acid sequence selected from the following:
  • the substitutions are conservative substitutions.
  • 4-1BB also known as CD137 or TNFRSF9 (TNF receptor superfamily member 9), is a member of the TNF receptor superfamily (TNFRSF) and is a costimulatory molecule that plays an important role in immune cells (innate immunity cells and adaptive immune cells) are expressed upon activation. 4-1BB plays an important role in regulating the activity of various immune cells.
  • 4-1BB can be derived from a mammal, such as Homo sapiens (human) (NCBI Accession No. NP_001552.2).
  • the single domain antibody specifically binds the 4-1BB epitope CRD-4.
  • the present application also provides a polypeptide construct that specifically binds to 4-1BB, which includes the single domain antibody or antigen-binding fragment thereof as described above, and an immunoglobulin Fc domain.
  • the immunoglobulin Fc domain is linked to the N-terminus and/or C-terminus (eg, C-terminus) of the single domain antibody or antigen-binding fragment thereof, either directly or through a peptide linker.
  • the peptide linker has the structure shown in ( G m 6, 7, 8, 9 and 10, X is selected from A and S; preferably, m is selected from 1, 2, 3, 4 or 5, n is selected from 1 and 2, and l is selected from 1, 2, 3, 4 , 5, 6, 7, 8, 9 and 10, m' is selected from 0 and 1, X is selected from A and S.
  • the peptide linker has the amino acid sequence set forth in SEQ ID NO: 17 or 18.
  • the immunoglobulin Fc domain is an IgG Fc domain (eg, an IgG1 Fc domain, eg, comprising CH2 and CH3).
  • the immunoglobulin Fc domain comprises, or has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, A sequence that has at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, or has one or several amino acid substitutions thereto, Deletions or additions (eg, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) to the sequence.
  • the immunoglobulin Fc domain comprises the sequence set forth in SEQ ID NO: 19 or 20.
  • the polypeptide construct contains or consists of the amino acid sequence set forth in SEQ ID NO: 31 or 32.
  • the present application further provides a multispecific antibody, which can activate 4-1BB signaling only when cross-linked with tumor cells expressing HER2.
  • the anti-4-1BB antibody or its antigen-binding fragment contained in the multispecific antibody can be characterized by positioning and/or activating only in the tumor microenvironment (TME), and/or significantly reducing liver toxicity compared to existing anti-4-1BB antibodies, while maintaining the efficacy of immune response enhancement and/or tumor treatment.
  • TEE tumor microenvironment
  • the present application provides a multispecific antibody comprising the single domain antibody or antigen-binding fragment or polypeptide construct thereof as described above.
  • the application further provides a multispecific antibody comprising a first antigen-binding domain specific for 4-1BB,
  • the first antigen-binding domain includes the antibody or antigen-binding fragment thereof as described above.
  • the first antigen-binding domain comprises an antibody or antigen-binding fragment thereof as described above.
  • any of the multispecific antibodies described above specifically bind 4-1BB and additionally specifically bind one or more other targets.
  • the target is a tumor antigen.
  • the tumor antigen is selected from one or more of the following: CD19, CD20, CD22, CD23, CD38, CD40, CD49, CD52, CD56, CD74, CD80, CD95, CD138, CS1/SLAMF7, KiR, Thy-1, Ly-6, Fas, APO-1, EGFR, HER2, CXCR4, HLA, GM1 and DRD.
  • the multispecific antibodies can bind multiple same or different tumor antigens.
  • the multispecific antibodies can bind to the same or different epitopes on the same tumor antigen.
  • the multispecific antibody specifically binds HER2.
  • HER2 human epidermal growth factor receptor 2
  • EGFR/ErbB epidermal growth factor receptor 2
  • HER2 is known to play an important role in regulating cell proliferation and differentiation. In particular, when bound to extracellular growth factors, it has a strong tendency to assemble into homo- and/or heterodimers together with other HER receptors, which leads to the activation and induction of several forms of signaling pathways Apoptosis, survival or cell proliferation.
  • the HER2 protein can be a polypeptide deposited with GenBank accession numbers NP_004439.2, NP_001005862.1, etc., which are encoded by nucleotide sequences (mRNA) deposited with GenBank accession numbers NM 004448.4, NM_001005862.3, etc., respectively.
  • the portion that binds to HER2 and recognizes HER2 as an antigen may be selected from scFv, (scFv) 2 , Fab, Fab′, and F(ab′) 2 of an anti-HER2 antibody.
  • the multispecific antibody contains at least one (eg, 1, 2, or 3) CDRs of the heavy chain variable region of an anti-HER2 antibody, and/or at least one (eg, 1, 2, or 3) CDR of the light chain variable region of an anti-HER2 antibody.
  • the multispecific antibody contains the heavy chain variable region of an anti-HER2 antibody, and/or the light chain variable region of an anti-HER2 antibody.
  • the anti-HER2 antibody is selected from trastuzumab, pertuzumab, and variants thereof.
  • the variant has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids) compared to the wild-type sequence from which it is derived. deletion or addition; for example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids).
  • the anti-HER2 antibody is a variant of trastuzumab.
  • the anti-HER2 antibody contains the following three heavy chain variable regions (VH) complementarity determining regions (CDRs):
  • VH CDR1 shown in SEQ ID NO:46 VH CDR2 shown in SEQ ID NO:47, and VH CDR3 shown in SEQ ID NO:48; and/or,
  • VL variable region
  • CDR complementarity determining regions
  • VL CDR1 shown in SEQ ID NO:49
  • VL CDR2 shown in SEQ ID NO:50
  • VL CDR3 shown in SEQ ID NO:8.
  • the multispecific antibody contains a second antigen binding domain specific for HER2.
  • the first antigen binding domain is a VHH; the second antigen binding domain is a Fab, and the multispecific antibody comprises:
  • Peptide chain I-A which includes the light chain variable region and the light chain constant region (CL) of the second antigen-binding domain;
  • Peptide chain I-B which includes the heavy chain variable region of the second antigen-binding domain, the heavy chain constant region, and the first antigen-binding domain; preferably, the peptide chain I-B starts from the N-terminus
  • the C-terminus includes the heavy chain variable region and the heavy chain constant region adjacent to the second antigen binding domain and the first antigen binding domain.
  • the heavy chain constant region may be selected from (e.g., an Fc region): a heavy chain constant region of IgG1, IgG2, IgG3, and IgG4, specifically, a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4.
  • Chain constant region for example, the heavy chain constant region of human IgG1, for example, CH1, CH2 and/or CH3 of human IgG1, and for example, the Fc region of human IgG1.
  • the heavy chain constant region is altered (e.g., mutated) to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, number of cysteine residues , effector cell function, or complement function.
  • the heavy chain constant region contains a LALA mutation, a CH3 Knob mutation, a CH3 Hole mutation, a CH1/CL-preferring mutation CH SET1, a CH1/CL-preferring mutation CH SET2, CH SET2, and any combination thereof.
  • the heavy chain constant region is selected from:
  • HC-1 Amino acid sequence shown in SEQ ID NO:10;
  • HC-2 amino acid sequence shown in SEQ ID NO:11;
  • HC-3 Amino acid sequence shown in SEQ ID NO:12;
  • HC-4 Amino acid sequence shown in SEQ ID NO:13;
  • HC-5 Amino acid sequence shown in SEQ ID NO:14;
  • HC-6 Amino acid sequence shown in SEQ ID NO:19;
  • HC-7 Amino acid sequence shown in SEQ ID NO:20;
  • HC-8 the amino acid sequence shown in SEQ ID NO:37;
  • HC-9 Amino acid sequence shown in SEQ ID NO:38;
  • the variant has one or more amino acid substitutions, deletions, or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions) compared to the wild-type sequence from which it is derived. or addition; for example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids).
  • amino acid substitutions, deletions, or additions e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions
  • the light chain constant region is selected from the group consisting of kappa or lambda light chain constant regions or variants thereof;
  • the variant has one or more amino acid substitutions, deletions, or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions) compared to the wild-type sequence from which it is derived. or addition; for example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids).
  • amino acid substitutions, deletions, or additions e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions
  • the light chain constant region is selected from:
  • LC-2 SEQ ID NO:15;
  • the variant has one or more amino acid substitutions, deletions, or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions) compared to the wild-type sequence from which it is derived. or addition; for example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids).
  • amino acid substitutions, deletions, or additions e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions
  • the CL of the peptide chain I-A is capable of forming a dimer with the heavy chain constant region CH1 domain of the peptide chain I-B.
  • the multispecific antibody comprises two of the peptide chains I-A and two of the peptide chains I-B; preferably, the heavy chain constant regions of the two peptide chains I-B form a dimer.
  • domains are connected to each other directly or through peptide linkers.
  • the peptide linker has the structure shown in ( G m 6, 7, 8, 9 and 10, X is selected from A and S; preferably, m is selected from 1, 2, 3, 4 or 5, n is selected from 1 and 2, and l is selected from 1, 2, 3, 4 , 5, 6, 7, 8, 9 and 10, m' is selected from 0 and 1, X is selected from A and S.
  • the peptide linker has the amino acid sequence set forth in SEQ ID NO: 17 or 18.
  • the multispecific antibody is characterized by one or more of the following:
  • the first antigen-binding domain contains or consists of the amino acid sequence shown in SEQ ID NO:7;
  • the heavy chain variable region of the second antigen-binding domain contains or consists of the amino acid sequence shown in SEQ ID NO: 1, 3 or 5;
  • the light chain variable region of the second antigen-binding domain contains or consists of the amino acid sequence shown in SEQ ID NO: 2, 4 or 6.
  • the peptide chain I-A contains or consists of an amino acid sequence as shown in SEQ ID NO:22, 24 or 26.
  • the peptide chain I-B contains or consists of the amino acid sequence set forth in SEQ ID NO: 21, 23 or 25.
  • the multispecific antibody is selected from:
  • a multispecific antibody comprising the peptide chains I-A and I-B, wherein the peptide chain I-A contains or consists of the amino acid sequence shown in SEQ ID NO:22, and the peptide chain I-B contains the amino acid sequence shown in SEQ ID NO:22 The amino acid sequence shown in NO:21 or consisting of it;
  • a multispecific antibody comprising the peptide chains I-A and I-B, wherein the peptide chain I-A contains or consists of the amino acid sequence shown in SEQ ID NO:24, and the peptide chain I-B contains the amino acid sequence shown in SEQ ID NO:24 The amino acid sequence shown in NO: 23 or consisting of it;
  • a multispecific antibody comprising the peptide chains I-A and I-B, wherein the peptide chain I-A contains or consists of the amino acid sequence shown in SEQ ID NO:26, and the peptide chain I-B contains the amino acid sequence shown in SEQ ID NO:26 The amino acid sequence shown in NO: 25 or consisting of it;
  • the multispecific antibody comprises two of the peptide chains I-A and two of the peptide chains I-B;
  • the heavy chain constant regions of the two peptide chains IB form a dimer.
  • the multispecific antibody further contains a third antigen binding domain specific for HER2.
  • the first antigen binding domain is a VHH; the second antigen binding domain and the second antigen binding domain are Fab, and the multispecific antibody comprises:
  • Peptide chain II-A which includes the light chain variable region and the light chain constant region (CL) of the second antigen-binding domain;
  • Peptide chain II-B which includes the heavy chain variable region of the second antigen-binding domain, the heavy chain constant region, and the first antigen-binding domain; preferably, the peptide chain II- B includes the heavy chain variable region and the heavy chain constant region of the adjacent second antigen-binding domain and the first antigen-binding domain from the N-terminus to the C-terminus;
  • Peptide chain II-C which includes the light chain variable region and the light chain constant region (CL) of the third antigen-binding domain
  • Peptide chain II-D which includes the heavy chain variable region of the third antigen-binding domain, the heavy chain constant region, and the first antigen-binding domain; preferably, the peptide chain II- D includes the heavy chain variable region and the heavy chain constant region adjacent to the third antigen-binding domain and the first antigen-binding domain from the N-terminus to the C-terminus.
  • the heavy chain constant region may be selected from (e.g., an Fc region): a heavy chain constant region of IgG1, IgG2, IgG3, and IgG4, specifically, a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4.
  • Chain constant region for example, the heavy chain constant region of human IgG1, for example, CH1, CH2 and/or CH3 of human IgG1, and for example, the Fc region of human IgG1.
  • the heavy chain constant region is altered (e.g., mutated) to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, number of cysteine residues , effector cell function, or complement function.
  • the heavy chain constant region contains a LALA mutation, a CH3 Knob mutation, a CH3 Hole mutation, a CH1/CL-preferring mutation CH SET1, a CH1/CL-preferring mutation CH SET2, and any combination thereof.
  • the light chain constant region is selected from the group consisting of kappa or lambda light chain constant regions or variants thereof;
  • the variant has one or more amino acid substitutions, deletions, or additions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions) compared to the wild-type sequence from which it is derived. or addition; for example, substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids).
  • amino acid substitutions, deletions, or additions e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions
  • the CL of the peptide chain II-A is capable of forming a dimer with the heavy chain constant region CH1 domain of the peptide chain II-B; preferably, the CL of the peptide chain II-C is capable of forming a dimer. It forms a dimer with the CH1 domain of the heavy chain constant region of the peptide chain II-D.
  • the multispecific antibody comprises one of said peptide chain II-A, one of said peptide chain II-B, one of said peptide chain II-C, and one of said peptide chain II-D.
  • the heavy chain constant regions of peptide chains II-B and II-D form dimers.
  • the heavy chain constant region of the peptide chain II-B contains the amino acid sequence set forth in SEQ ID NO:13
  • the heavy chain constant region of the peptide chain II-D contains the amino acid sequence set forth in SEQ ID NO:14. Show the amino acid sequence.
  • the heavy chain constant region of the peptide chain II-B contains the amino acid sequence set forth in SEQ ID NO:37
  • the heavy chain constant region of the peptide chain II-D contains the amino acid sequence set forth in SEQ ID NO:38. Show the amino acid sequence.
  • domains are connected to each other directly or through peptide linkers.
  • the peptide linker has the structure shown in ( G m 6, 7, 8, 9 and 10, X is selected from A and S.
  • m is selected from 1, 2, 3, 4 or 5
  • n is selected from 1 and 2
  • l is selected from 1
  • m ' is chosen from 0 and 1
  • X is chosen from A and S.
  • the peptide linker has the amino acid sequence set forth in SEQ ID NO: 17 or 18.
  • the multispecific antibody is characterized by one or more of the following:
  • the first antigen-binding domain contains or consists of the amino acid sequence shown in SEQ ID NO:7;
  • the heavy chain variable region of the second antigen-binding domain contains or consists of the amino acid sequence shown in SEQ ID NO: 1, 3 or 5;
  • the light chain variable region of the second antigen-binding domain contains or consists of the amino acid sequence shown in SEQ ID NO: 2, 4 or 6;
  • the heavy chain variable region of the third antigen-binding domain contains or consists of the amino acid sequence shown in SEQ ID NO: 1, 3 or 5;
  • the light chain variable region of the third antigen-binding domain contains or consists of the amino acid sequence shown in SEQ ID NO: 2, 4 or 6.
  • the peptide chain II-A contains or consists of the amino acid sequence set forth in SEQ ID NO: 28 or 40.
  • the peptide chain II-B contains or consists of the amino acid sequence set forth in SEQ ID NO: 27 or 39.
  • the peptide chain II-C contains or consists of the amino acid sequence set forth in SEQ ID NO: 30 or 42.
  • the peptide chain II-D contains or consists of the amino acid sequence set forth in SEQ ID NO: 29 or 41.
  • the multispecific antibody is selected from:
  • a multispecific antibody comprising the peptide chains II-A, II-B, II-C and II-D, wherein the peptide chain II-A contains the amino acid sequence shown in SEQ ID NO: 28 Or consisting of it, the peptide chain II-B contains or consists of the amino acid sequence shown in SEQ ID NO:27, and the peptide chain II-C contains the amino acid sequence shown in SEQ ID NO:29 or consists of Its composition, the peptide chain II-D contains or consists of the amino acid sequence shown in SEQ ID NO:30;
  • a multispecific antibody comprising the peptide chains II-A, II-B, II-C and II-D, wherein the peptide chain II-A contains the amino acid sequence shown in SEQ ID NO: 40 Or consisting of it, the peptide chain II-B contains or consists of the amino acid sequence shown in SEQ ID NO:39, and the peptide chain II-C contains the amino acid sequence shown in SEQ ID NO:42 or consists of Its composition, the peptide chain II-D contains or consists of the amino acid sequence shown in SEQ ID NO:41.
  • N-terminus or C-terminus (preferably the N-terminus) of the 4-1BB binding moiety described herein is connected to the C-terminus or N-terminus of the heavy chain constant region directly or through a peptide linker.
  • peptide linker may refer to an oligopeptide including 1 to 100 amino acids, particularly 2 to 50 amino acids, and each amino acid may be any kind of amino acid without any limitation. Any conventional peptide linker may be used with or without appropriate modifications to suit the particular purpose.
  • the peptide linker may comprise, for example, Gly, Asn and/or Ser residues, and/or comprise neutral amino acids such as Thr and/or Ala. Suitable amino acid sequences for peptide linkers may be known in the relevant art.
  • the length of the peptide linker can be appropriately determined within a limit that does not affect the function of the polypeptide and/or scFv.
  • the peptide linker can be modified by including a total of about 1 to about 100 amino acids, about 2 to about 50 amino acids, or about 5 to about 25 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) amino acids, each amino acid is independently selected from Gly, Asn, Ser The group consisting of , Thr and Ala.
  • the peptide linker has the structure shown in ( G m 6, 7, 8, 9 and 10, X is selected from A and S.
  • m is selected from 1, 2, 3, 4 or 5
  • n is selected from 1 and 2
  • l is selected from 1
  • m ' is chosen from 0 and 1
  • X is chosen from A and S.
  • the peptide linker has the amino acid sequence set forth in SEQ ID NO: 17 or 18.
  • the invention provides a polynucleotide encoding the antibody or antigen-binding fragment thereof as described in any one of the preceding paragraphs.
  • the invention provides a vector comprising the polynucleotide described above.
  • the invention provides a recombinant cell comprising the polynucleotide or vector described above.
  • Recombinant cells may be cells transfected with a recombinant vector.
  • the invention provides methods of making said antibodies or antigen-binding fragments thereof, comprising expressing the polynucleotide in a cell.
  • the step of expressing the polynucleotide can be performed by culturing a cell containing the polynucleotide (eg, the polynucleotide in a recombinant vector) under conditions that allow expression of the polynucleotide.
  • the method may further comprise isolating and/or purifying the antibody or antigen-binding fragment thereof from the cell culture following the expression or culturing step.
  • the invention further provides the use of the antibodies described herein, or antigen-binding fragments thereof, in enhancing immune responses and/or treating tumors.
  • the present invention provides a pharmaceutical composition, which contains any of the aforementioned antibodies or antigen-binding fragments thereof, polynucleotides, vectors or recombinant cells, and pharmaceutically acceptable excipients.
  • the present invention further provides the use of any of the aforementioned antibodies or antigen-binding fragments thereof, polynucleotides, vectors, recombinant cells or pharmaceutical compositions in the preparation of anti-tumor drugs.
  • the present invention provides an anti-tumor method, which includes administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof, polynucleotide, vector, recombinant, or antibody as described in any one of the preceding paragraphs. Cell or pharmaceutical composition steps.
  • the tumor overexpresses HER2.
  • the tumor is selected from the group consisting of breast cancer, colon cancer, gastric cancer, lung cancer (e.g., lung squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma), peritoneal cancer, skin cancer, squamous cell carcinoma, melanoma of the skin or eyeball, rectal cancer, cancer near the anus, esophageal cancer, small bowel cancer, endocrine gland cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urinary tract cancer, chronic or acute leukemia, lymphocytic Lymphoma, liver cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular adenoma, colorectal cancer, endometrial or uterine cancer, salivary gland tumors, kidney Cancer, cervical cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, Brain cancer,
  • lung cancer
  • the tumor is a primary or metastatic tumor.
  • the present invention also provides a conjugate comprising the antibody or antigen-binding fragment thereof or polypeptide construct as described in any one of the above aspects, and a detectable label linked to the single domain antibody or antigen-binding fragment thereof or polypeptide construct.
  • the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent proteins), radionuclides or biotin.
  • an enzyme such as horseradish peroxidase or alkaline phosphatase
  • a chemiluminescent reagent such as acridinium esters, luminol and its derivatives, or ruthenium derivatives
  • fluorescent dyes such as fluorescein or fluorescent proteins
  • the present invention also provides a kit comprising the single domain antibody or antigen-binding fragment thereof or polypeptide construct, or conjugate as described in any one of the above aspects.
  • the kit further comprises a second antibody that specifically recognizes the antigen specifically recognized by the antibody or antigen-binding fragment thereof or the polypeptide construct; optionally, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide or biotin.
  • an enzyme e.g., horseradish peroxidase or alkaline phosphatase
  • a chemiluminescent agent e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives
  • a fluorescent dye e.g., fluorescein or fluorescent protein
  • the second antibody targets the same or a different antigenic epitope as the antibody, or antigen-binding fragment thereof, or the polypeptide construct.
  • the invention also provides a method for detecting the presence of 4-1BB in a sample or its level, which includes using an antibody or an antigen-binding fragment thereof or a polypeptide construct, or a conjugate as described in any one of the above aspects. compound.
  • the method is an immunological assay, such as a western blot, enzyme immunoassay (eg, ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.
  • immunological assay such as a western blot, enzyme immunoassay (eg, ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.
  • the methods include using a conjugate as described above.
  • the method includes using an antibody, or an antigen-binding fragment thereof, or a polypeptide construct as described in any one of the aspects above, and the method further includes using an antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish)). oxidase or alkaline phosphatase), chemiluminescent reagents (such as acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent protein), radionuclides or Biotin) secondary antibody to detect the single domain antibody or the binding of the polypeptide construct to an antigen.
  • a detectable label e.g., an enzyme (e.g., horseradish)
  • oxidase or alkaline phosphatase chemiluminescent reagents
  • chemiluminescent reagents such as acridinium esters, luminol and its derivatives, or ruthenium derivative
  • the method includes: (1) contacting the sample with the antibody or antigen-binding fragment thereof; (2) detecting the formation of an antigen-antibody immune complex or detecting the formation of the immune complex quantity.
  • the formation of immune complexes indicates the presence of 4-1BB or cells expressing 4-1BB.
  • the application also provides the use of the single domain antibody or antigen-binding fragment thereof, or polypeptide construct, or conjugate as described in any of the above aspects in the preparation of detection reagents for detection 4-The presence or level of 1BB in the sample.
  • the detection reagent detects the presence of 4-1BB in the sample or the level thereof by a method as described above for detecting the presence of 4-1BB in the sample or the level thereof.
  • the sample is a cell sample (eg, tumor cells) from a subject (eg, a mammal, preferably a human or a monkey).
  • a cell sample eg, tumor cells
  • a subject eg, a mammal, preferably a human or a monkey.
  • At least one or “one or more” can mean 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 (species) or more (species).
  • peptide and “polypeptide” refer to a polymer of two or more amino acids linked by peptide bonds.
  • a “protein” may be formed covalently or non-covalently from one or more polypeptides. Unless otherwise stated, the terms “polypeptide” and “egg” “White matter” is used interchangeably in this article.
  • antibody refers to an immunoglobulin or fragment thereof that specifically binds to an antigenic epitope through at least one antigen-binding site.
  • the definition of antibody encompasses antigen-binding fragments.
  • the term “antibody” includes multispecific antibodies (eg, bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies, and antigen-binding fragments.
  • Antibodies may be synthetic (eg, produced by chemical or biological conjugation), enzymatically produced, or recombinantly produced.
  • Antibodies provided herein include any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass (e.g., IgG2a and IgG2b).
  • the antibody of the invention is a murine monoclonal antibody.
  • the antibodies of the invention are humanized monoclonal antibodies.
  • a “traditional antibody” or “full-length antibody” generally contains four polypeptides: two heavy chains (HC) and two light chains (LC).
  • Each light chain includes a “light chain variable region (VL)” and a “light chain constant region (CL)” from the N-terminus to the C-terminus.
  • Each heavy chain includes a “heavy chain variable region (VH)” and a “heavy chain constant region (CH)” from the N-terminus to the C-terminus.
  • the heavy chain constant region may contain CH1, CH2 and CH3 from the N-terminus to the C-terminus.
  • the heavy chain constant region may also contain CH4.
  • Fc fragment refers to a fragment containing CH2 and CH3, which provides the binding site for the Fc receptor.
  • Hinge region refers to the portion of an antibody that connects the Fab and Fc fragments of the immunoglobulin.
  • the hinge region refers to the portion of the T cell receptor that connects the constant region and the transmembrane domain.
  • the hinge region may also refer to any functional equivalent.
  • Those skilled in the art can determine the positions of VH, VL, CL, CH1, CH2, CH3 and hinge regions in antibodies based on known algorithms and software. For a description of applicable algorithms and software, see, for example, William R. Strohl, Lila M.Strohl, (2012), Antibody structure–function relationships,In Woodhead Publishing Series in Biomedicine,Therapeutic Antibody Engineering,Woodhead Publishing,pp.37-56.
  • VL and VH each can contain three highly variable “complementarity determining regions (CDR)" and four relatively conserved “framework regions (FR)", and are organized in the order FR1-CDR1-FR2- from the N-terminus to the C-terminus.
  • the CDRs of the light chain variable region (CDRL) may be referred to as CDR-L1, CDR-L2 and CDR-L3
  • the CDRs of the heavy chain variable region (CDRH) may be referred to as CDR-H1, CDR-H2 and CDR-H3.
  • CDRs containing a pair of VH and VL determine the binding specificity of the antigen-binding site, but in some cases, other fragments (e.g., single Domain antibodies, also known as nanobodies) also have the ability to bind antigens.
  • CDRs can identify CDRs using methods well known in the art, such as Kabat, Abm or Chothia numbering. In this article, multiple CDR numbering systems can be used for the same variable region, such as Chothia, Abm, Kabat and IMGT.
  • CDRs defined by different numbering systems may be different, CDRs corresponding to the same numbering system represent effective antigen-binding sites capable of binding to antigenic epitopes.
  • CDR numbering system For a description of the CDR numbering system, see, for example, Kabat numbering system : Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242; Chothia numbering system : Chothia, C. et al. (1987) J. Mol. Biol. 196: 901-917; IMGT numbering system : Lefranc, M.-P., 2011 (6), IMGT, the International ImMunoGeneTics Information System Cold Spring Harb Protoc.; Abm numbering system : Martin, ACR and J. Allen (2007) "Bioinformatics tools for antibody engineering," in S. Dübel (ed.), Handbook of Therapeutic Antibodies. Weinheim: Wiley-VCH Verlag, pp. 95–118 .
  • framework area and “framework area” are used interchangeably.
  • framework region refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.
  • dsFv disulfide bond-stabilized Fv
  • scdsFv or dsscFv single-chain disulfide bond-stabilized Fv
  • scFv or scdsFv is preferably used as the antigen-binding fragment in the single domain antibody, bispecific antibody, or multispecific antibody of the present invention.
  • Fab includes an intact antibody light chain (VL-CL) and an antibody heavy chain variable region and a heavy chain constant region (VH-CH1, also known as Fd).
  • Single-chain “Fab (scFab)” can be obtained by connecting CL and CH1 in “Fab” using a peptide linker.
  • F(ab') 2 essentially consists of two Fab fragments linked by a disulfide bond in the hinge region.
  • Fab' is one half of F(ab') 2 , which can be obtained by reducing the disulfide bonds in the hinge region of F(ab') 2 .
  • An antibody or antigen-binding fragment may be "monovalent,” “bivalent,” “trivalent,” or “tetravalent” or more, meaning that it has multiple antigen-binding sites (e.g., 1, 2, 3, or 4 or more).
  • the antigens bound by the antibody or antigen-binding fragment are two or more (e.g., 2, 3, 4, 5 or 6), it can also be referred to as a "multispecific antibody", such as a bispecific antibody, a trispecific antibody or a tetraspecific antibody, which respectively represent multispecific antibodies that can bind to 2, 3 or 4 antigens.
  • a multispecific antibody such as a bispecific antibody, a trispecific antibody or a tetraspecific antibody, which respectively represent multispecific antibodies that can bind to 2, 3 or 4 antigens.
  • diabody refers to an antibody that contains two scFvs with two antigen-binding sites (bivalent), wherein the VH and VL in each scFv are connected by a short peptide linker ( Approximately 5-10 amino acid residues) are connected so that the VH and VL chains are paired (i.e., the VH of the first scFv is paired with the VL of the second scFv, and the VL of the first scFv is paired with the VH of the second scFv) to form an antigen-binding site. point.
  • Diabodies can be bispecific antibodies.
  • an "antigen-binding fragment" of an antibody refers to a portion of a full-length antibody that is less than the full length, but contains at least a portion of the variable region of the full-length antibody (e.g., contains one or more CDRs and/or one or more antigen-binding sites), and thus retains at least a portion of the full-length antibody's ability to specifically bind to an antigen.
  • An antigen-binding fragment may, for example, include antibody derivatives produced by enzymatic treatment of a full-length antibody, synthetically produced derivatives, and recombinantly produced derivatives.
  • antigen-binding fragments include, but are not limited to, Fv, scFv, dsFv, scdsFv, Fab, scFab, Fab', F(ab') 2 , diabodies, Fd and Fd' fragments, and other fragments (e.g., fragments containing modifications).
  • An antigen-binding fragment may contain multiple peptide chains, for example, connected by disulfide bonds and/or by peptide linkers and/or formed by non-covalent interactions.
  • the term "monoclonal antibody” refers to a population of highly homogeneous antibodies in which the antibody molecules contained are substantially identical to each other except for the possible presence of small amounts of naturally occurring mutations. Monoclonal antibodies usually bind specifically to a single antigenic epitope. Monoclonal antibodies described herein may be produced by any method known in the art, such as from transgenic animals (e.g., transgenic mice), from immortalized B cells (e.g., B cell hybridomas), or using recombinant DNA methods Prepared in bacterial, eukaryotic animal or plant cells, or isolated from phage antibody libraries. The antibodies or antigen-binding fragments of the invention are monoclonal antibodies. In one embodiment, the antibody or antigen-binding fragment thereof of the invention is a humanized monoclonal antibody.
  • chimeric antibody refers to an antibody in which a portion (e.g., CDRs, FRs, variable regions, constant regions, or combinations thereof) is identical or homologous to the corresponding sequence in an antibody derived from a particular species, and the remaining portion is identical to that in an antibody derived from a specific species. The corresponding sequence in an antibody from another species is identical or homologous.
  • chimeric antibody also encompasses antibodies containing portions belonging to different antibody types or subclasses. In a specific embodiment, the chimeric antibody has a murine antibody variable region and a human antibody constant region.
  • humanized antibody refers to an antibody that contains non-human antibody and human antibody sequences.
  • humanized antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins.
  • the non-human antibody may be an antibody derived from any non-human species or an antibody containing a portion derived from a non-human species (eg, a chimeric antibody).
  • Non-human species may include, for example, mice, rats, rabbits, alpacas, or non-human primates. Techniques for obtaining humanized antibodies from non-human antibodies are well known to those skilled in the art.
  • Humanized antibodies can be produced from non-human antibodies (eg, murine antibodies or chimeric antibodies), for example, by grafting the CDR sequences of the non-human antibody (eg, murine antibodies) into the human antibody framework regions.
  • the key amino acid residues of the framework sequence of the non-human antibody such as the mouse antibody
  • the key amino acid residues of the framework sequence of the non-human antibody can be retained in the human antibody framework region, that is, " "Reverse mutation” (see, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. 81 (21): 6851-6855; Neuberger et al. (1984) Nature 312: 604-608).
  • percent (%) sequence identity and “sequence identity” of amino acid sequences have the definition recognized in the art, which refers to the percentage of identity between two polypeptide sequences determined by sequence alignment (e.g., by manual inspection or a publicly available algorithm). It can be determined using methods known to those skilled in the art, such as using publicly available computer programs. Software such as BLAST, BLAST-2, Clustal Omega, and FASTA software.
  • “Avidity” or “binding affinity” is a measure of how strongly an antibody and antigen bind to each other through non-covalent interactions.
  • the magnitude of "affinity” can usually be reported as the equilibrium dissociation constant K D or EC 50 .
  • Affinity can be measured using conventional techniques known in the art, such as biofilm interference technology (for example, the Octet Fortebio detection system can be used), radioimmunoassay, surface plasmon resonance method, enzyme-linked immunoassay or flow cytometry, etc.
  • the KD value between two antibodies and antigens that specifically bind is at least about 10 -6 to at least about 10 -9 M or less, such as at least about 10 -6 , at least about 10 -7 , at least about 10 -8 , at least about 10 -9 M or lower.
  • an amino acid sequence "derived from” or “derived from” a reference amino acid sequence is partially or completely identical or homologous to said reference amino acid sequence.
  • an amino acid sequence derived from a heavy chain constant region of a human immunoglobulin has 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%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • a "variant" of a polypeptide or amino acid sequence has one or more amino acid mutations or modifications compared to the polypeptide or amino acid sequence from which it is derived.
  • Amino acid mutations include replacement, deletion or addition of amino acids. It will be appreciated by those skilled in the art that amino acids in non-essential regions of a polypeptide or protein can be replaced with suitable conservative amino acids and generally do not change their biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub.co., p.224). Suitable conservative substitutions are well known to those skilled in the art. Some common non-limiting examples of conservative substitutions of amino acid residues are listed in the table below.
  • amino acid substitutions are non-conservative substitutions. It will be appreciated by those skilled in the art that amino acid mutations or modifications can be made to antibodies or antibody fragments to change their performance, such as changing the type of antibody glycosylation modification and changing the ability to form interchain disulfide bonds. Antibodies or antigen-binding fragments thereof containing such amino acid mutations or modifications are also included within the scope of antibodies or antigen-binding fragments thereof of the present invention.
  • polynucleotide refers to an oligomer or polymer containing at least two linked nucleotides or nucleotide derivatives, which may generally include deoxyribonucleic acid ( DNA) and ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • isolated refers to a substance (eg, a nucleic acid molecule or polypeptide) that is separate from the source or environment in which it exists, ie, does not contain substantially any other components.
  • a "vector” is a vehicle used to introduce exogenous nucleic acid into a host cell so that when the vector is transformed into an appropriate host cell, the exogenous nucleic acid is amplified or expressed.
  • Vectors usually remain episomal, but can be designed to integrate the gene or part thereof into the chromosome of the genome.
  • the definition of vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (eg, yeast artificial chromosomes and mammalian artificial chromosomes), and the like.
  • expression vector refers to a vector capable of expressing DNA operably linked to regulatory sequences (eg, promoters, ribosome binding sites) capable of affecting DNA expression. Regulatory sequences may include promoter and terminator sequences, and optionally may include origins of replication, selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors may be plasmids, phage vectors, recombinant viruses, or other vectors that, when introduced into an appropriate host cell, result in expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic and/or prokaryotic cells as well as expression vectors that remain episomal or are integrated into the genome of the host cell.
  • a "recombinant cell” is a cell used to receive, maintain, replicate or amplify a vector. Recombinant cells can also be used to express polypeptides encoded by nucleic acids or vectors. Recombinant cells can be eukaryotic or prokaryotic cells.
  • treatment refers to the improvement of a disease/symptom, such as reducing or eliminating the disease/symptom, preventing or slowing the occurrence, progression and/or worsening of the disease/symptom. Treatment therefore includes prevention, treatment and/or cure.
  • an "effective amount” of an antibody or an antigen-binding fragment thereof, or a pharmaceutical composition of the invention preferably inhibits tumor cell growth or tumor growth by at least about 10% relative to an untreated subject, preferably At least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%.
  • the efficacy of inhibiting tumor growth can be evaluated using tumor animal models conventional in the art, such as spontaneous tumor, induced tumor, and transplanted tumor animal models. Alternatively, the ability to inhibit cell growth can also be examined using in vitro detection methods known in the art.
  • An effective amount of the antibody, antigen-binding fragment thereof, or pharmaceutical composition of the present invention can reduce tumor size, or otherwise alleviate the subject's symptoms (such as preventing and/or treating metastasis or recurrence).
  • One skilled in the art can determine the effective amount based on factors such as the subject's age, physical condition, gender, severity of symptoms, specific composition or route of administration.
  • An effective amount can be administered in one or more administrations.
  • the term "pharmaceutically acceptable excipient” refers to an excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives.
  • pH regulators include, but are not limited to, phosphate buffers.
  • Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol), and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc.
  • Stabilizers have the meanings commonly understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredients in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc.
  • sugars such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose
  • amino acids such as glutamic acid, glycine
  • proteins such as dried whey, albumin or casein
  • degradation products such as lactalbumin hydrolysate
  • mammals include, but are not limited to, humans, non-human primates, rats, mice, cattle, horses, pigs, sheep, dogs, cats, and the like.
  • subject refers to a mammal, such as a human.
  • the subject is a human.
  • the subject is a cancer patient, a human suspected of having or at risk of cancer, or an animal.
  • subject and subject are used interchangeably.
  • Figure 1 shows a schematic structural diagram of the multispecific antibody described in the present application.
  • Figure 2 shows the detection of the binding of the multispecific antibody of the present invention to human HER2 on HER2-overexpressing cells N87 and CT26.
  • Figure 3 shows the detection of the binding of the multispecific antibody of the present invention to human 4-1BB on CHO-h4-1BB, a cell overexpressing human 4-1BB.
  • Figure 4 shows the binding of multispecific antibodies of the present invention to FcR.
  • Figure 5 shows that the multispecific antibodies of the present invention block the proliferation of HER2 signal-dependent cells N87 or SK-OV-3.
  • Figure 6 shows that the multispecific antibodies of the present invention mediate ADCC effects through HER2.
  • Figure 7 shows that HER2 mediates the multispecific antibody of the present invention to induce Primary T cells to release cytokines IL-2 and IFN- ⁇ .
  • Figure 8 shows that FcR mediates the multispecific antibody of the present invention to induce primary T cells to release cytokines IL-2 and IFN- ⁇ .
  • Figure 9 shows the SEC and LC/MS detection results of the multispecific antibody HER2xHER2x4-1BB of the present invention.
  • Figure 10 shows the tumor inhibitory activity of the multispecific antibody of the present invention in the tumor model of h-4-1BB KI BALB/c mice subcutaneously inoculated with CT26-h-HER2.
  • Figure 10A 0-15 days after administration Effect of tumor volume
  • Figure 10B CD8+ T cell infiltration in mouse tumors.
  • Figure 11 shows the tumor inhibitory activity of the multispecific antibody of the present invention in the tumor model of h-4-1BB KI BALB/c mice subcutaneously inoculated with CT26-h-HER2 (effect on tumor volume 11-27 days after administration) ).
  • Figure 12 shows the subcutaneous inoculation of MC38-h-HER2 in h-4-1BB KI C57 mice with the multispecific antibody of the present invention. antitumor activity in tumor models.
  • HER2x4-1BB bispecific antibodies three kinds of HER2x4-1BB bispecific antibodies, two kinds of anti-HER2xHER2x4-1BB trispecific antibodies and two kinds of 4-1BB monoclonal antibodies were constructed, respectively:
  • HER2x4-1BB-1 consists of 2 polypeptide chains. Its structural diagram is shown in Figure 1.
  • Peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 21, which contains the anti-HER2 monoclonal antibody Trastuzumab (patent application No.: WO1992022653A1) heavy chain variable region amino acid sequence (SEQ ID NO: 1) and human IgG1 amino acid sequence (SEQ ID NO: 10).
  • the N-terminus of the CD137 binding region amino acid sequence (SEQ ID NO:7) of the anti-CD137 single domain antibody AB24 ME is connected to the C of Fc through a flexible peptide of 20 amino acid residues (G4A) 4 (SEQ ID NO:18) end.
  • Peptide chain #2 has the amino acid sequence shown in SEQ ID NO:22, which contains the amino acid sequence (SEQ ID NO:2) of the light chain variable region of the anti-HER2 monoclonal antibody Trastuzumab (patent application number: WO1992022653A1), and The amino acid sequence of the human kappa light chain constant region (CL) at the C-terminus of the VL amino acid sequence (SEQ ID NO: 9).
  • HER2x4-1BB-2 consists of 2 polypeptide chains. Its structural diagram is shown in Figure 1.
  • Peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 23, which contains the anti-HER2 monoclonal antibody Trastuzumab (patent application).
  • the N-terminus of the CD137 binding region amino acid sequence (SEQ ID NO:7) of the anti-CD137 single domain antibody AB24 ME is connected to the C of Fc through a flexible peptide of 20 amino acid residues (G4A) 4 (SEQ ID NO:18) end.
  • Peptide chain #2 has the amino acid sequence shown in SEQ ID NO:24, which contains the amino acid sequence (SEQ ID NO:2) of the light chain variable region of the anti-HER2 monoclonal antibody Trastuzumab (patent application number: WO1992022653A1), and The amino acid sequence of the human kappa light chain constant region (CL) at the C-terminus of the VL amino acid sequence (SEQ ID NO: 9).
  • HER2x4-1BB-3 consists of 2 polypeptide chains. Its structural diagram is shown in Figure 1.
  • Peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 25, which contains the anti-HER2 monoclonal antibody Pertuzumab (patent application No.: US7449184) heavy chain variable region amino acid sequence (SEQ ID NO:3) and human IgG1 amino acid sequence (SEQ ID NO:12).
  • SEQ ID NO:7 The N-terminus of the CD137 binding region amino acid sequence (SEQ ID NO:7) of the anti-CD137 single domain antibody AB24 ME is connected to the C of Fc through a flexible peptide of 20 amino acid residues (G4A) 4 (SEQ ID NO:18) end.
  • Peptide chain #2 has the amino acid sequence shown in SEQ ID NO:26, which includes the light chain variable region amino acid sequence (SEQ ID NO:4) of the anti-HER2 monoclonal antibody Pertuzumab (patent application number: US7449184), and The amino acid sequence of the human kappa light chain constant region (CL) at the C-terminus of the VL amino acid sequence (SEQ ID NO: 9).
  • HER2xHER2x4-1BB-1 consists of 4 polypeptide chains. Its structural diagram is shown in Figure 1.
  • Peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 27, which contains the heavy chain of the anti-HER2 monoclonal antibody.
  • Variable region amino acid sequence The CH3 Knob mutation and the CH1/CL-biased mutation CH SET1 (Patent Application Number: WO2021067404A2, SEQ ID NO: 13) were introduced into the human IgG1 amino acid sequence (SEQ ID NO: 5).
  • the N-terminus of the CD137 binding region amino acid sequence (SEQ ID NO:7) of the anti-CD137 single domain antibody AB24 ME is connected to the C of Fc through a flexible peptide of 20 amino acid residues (G4A) 4 (SEQ ID NO:18) end.
  • Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 28, which includes the light chain variable region amino acid sequence of the monoclonal antibody against HER2 (SEQ ID NO: 6), and the human amino acid sequence at the C-terminus of the VL amino acid sequence.
  • the CH1/CL-biased mutation CL SET1 was introduced into the kappa light chain constant region (CL) amino acid sequence (Patent application number: WO2021067404A2, SEQ ID NO: 15).
  • Peptide chain #3 has the amino acid sequence shown in SEQ ID NO:29, which includes the heavy chain variable region amino acid sequence (SEQ ID NO:3) of the anti-HER2 monoclonal antibody Pertuzumab (Patent Application No.: US7449184) and human IgG1
  • the amino acid sequence introduces CH3 Hole mutation and CH1/CL preference mutation CH SET2 (Patent application number: WO2021067404A2, SEQ ID NO: 14).
  • the N-terminus of the CD137 binding region amino acid sequence (SEQ ID NO:7) of the anti-CD137 single domain antibody AB24 ME is connected to the C of Fc through a flexible peptide of 20 amino acid residues (G4A) 4 (SEQ ID NO:18) end.
  • Peptide chain #4 has the amino acid sequence shown in SEQ ID NO:30, which includes the amino acid sequence (SEQ ID NO:4) of the light chain variable region of the anti-HER2 monoclonal antibody Pertuzumab (Patent Application No.: US7449184), and The CH1/CL-biased mutation CL SET2 was introduced into the human kappa light chain constant region (CL) amino acid sequence at the C-terminus of the VL amino acid sequence (Patent Application Number: WO2021067404A2, SEQ ID NO: 16).
  • HER2xHER2x4-1BB-2 consists of 4 polypeptide chains. Its structural diagram is shown in Figure 1.
  • Peptide chain #1 is synthesized according to the method described in the patent (Patent application number: 201611016435.0) with the amino acid shown in SEQ ID NO: 39
  • the sequence includes the heavy chain variable region amino acid sequence (SEQ ID NO: 1) of the anti-HER2 monoclonal antibody Trastuzumab (patent application number: WO1992022653A1) and the human IgG1 amino acid sequence HC-1 (which can spontaneously form heterodimers).
  • SEQ ID NO:37 patent application number: PCT/CN2017/111310).
  • the N-terminus of the CD137 binding region amino acid sequence (SEQ ID NO:7) of the anti-CD137 single domain antibody AB24 ME is connected to the C of Fc through a flexible peptide of 20 amino acid residues (G4A) 4 (SEQ ID NO:18) end.
  • Peptide chain #2 has the amino acid sequence shown in SEQ ID NO:40, which includes the amino acid sequence (SEQ ID NO:2) of the light chain variable region of the anti-HER2 monoclonal antibody Trastuzumab (Patent application number: WO1992022653A1), and The human kappa light chain constant region (CL) amino acid sequence at the C-terminus of the VL amino acid sequence (SEQ ID NO: 9).
  • Peptide chain #3 was synthesized according to the method described in the patent (Patent application number: 201611016435.0) and has the amino acid sequence shown in SEQ ID NO: 41, including the heavy chain variable of the anti-HER2 monoclonal antibody Pertuzumab (Patent application number: US7449184) region amino acid sequence (SEQ ID NO: 3) and the human IgG1 amino acid sequence HC-2 (SEQ ID NO: 38, patent application number: PCT/CN2017/111310) that can spontaneously form heterodimers.
  • the N-terminus of the CD137 binding region amino acid sequence (SEQ ID NO:7) of the anti-CD137 single domain antibody AB24 ME is passed through the 20 amino acid residues (G4A) 4 (SEQ ID NO:18)
  • the flexible peptide is attached to the C-terminus of Fc.
  • Peptide chain #4 has the amino acid sequence shown in SEQ ID NO:42, which contains the amino acid sequence (SEQ ID NO:4) of the light chain variable region of the anti-HER2 monoclonal antibody Pertuzumab (Patent Application No.: US7449184), and The human kappa light chain constant region (CL) amino acid sequence at the C-terminus of the VL amino acid sequence (SEQ ID NO: 9).
  • Anti-4-1BB-VHH-1 consists of 2 polypeptide chains, with the amino acid sequence shown in SEQ ID NO:31, which contains the human IgG1 Fc amino acid sequence (SEQ ID NO:19) and a single domain that will resist CD137
  • SEQ ID NO:7 The N-terminus of the CD137 binding region amino acid sequence (SEQ ID NO:7) of antibody AB24 ME is connected to the C-terminus of Fc through a flexible peptide of 21 amino acid residues (G4S) 4G (SEQ ID NO:17).
  • Anti-4-1BB-VHH-2 consists of 2 polypeptide chains and has the amino acid sequence shown in SEQ ID NO:32, which contains the human IgG1 Fc amino acid sequence (LALA mutation is introduced to reduce Fc function, SEQ ID NO:20 ) and the N-terminus of the CD137 binding region amino acid sequence (SEQ ID NO:7) of the anti-CD137 single domain antibody AB24 ME is connected to Fc through a flexible peptide of 21 amino acid residues (G4S) 4G (SEQ ID NO:17) C-side.
  • SEQ ID NO:32 which contains the human IgG1 Fc amino acid sequence (LALA mutation is introduced to reduce Fc function, SEQ ID NO:20 ) and the N-terminus of the CD137 binding region amino acid sequence (SEQ ID NO:7) of the anti-CD137 single domain antibody AB24 ME is connected to Fc through a flexible peptide of 21 amino acid residues (G4S) 4G (SEQ ID NO:17) C-side.
  • Urelumab The control molecule Urelumab (patent application number: WO2004010947) consists of two polypeptide chains, peptide chain #1 has the amino acid sequence shown in SEQ ID NO:33, and peptide chain #2 has the amino acid sequence shown in SEQ ID NO:34.
  • PRS-343 Control molecule PRS-343 (patent application number: WO2016177802A1) consists of 2 polypeptide chains.
  • Peptide chain #1 has the amino acid sequence shown in SEQ ID NO:35
  • peptide chain #2 has the amino acid sequence shown in SEQ ID NO:36. The amino acid sequence shown.
  • the expanded cultured N87 cells (self-expressing HER2) and CT26-hHER2 (overexpressing human HER2) cells were digested with 0.25% EDTA trypsin, washed once with culture medium, and then adjusted to a cell density of 2 ⁇ 10 6 cells/ml, 100 ⁇ l /well was added to a 96-well flow plate, centrifuged and set aside. Add the serially diluted antibody at 100 ⁇ l/well to the above-mentioned 96-well flow cytometry plate with cells, and incubate at 4°C for 60 minutes.
  • the experimental results are shown in Figure 2 and Table 1-2.
  • the HER2xHER2x4-1BB trispecific antibody and HER2x4-1BB bispecific antibody of the present invention have a strong affinity with human gastric cancer cell N87 (Figure 2A) and CT26 cells overexpressing human HER2 ( Figure 2A). 2B) binds to all HER2 expressed on it, and the binding activity is comparable to that of HER2 monoclonal antibodies (Trastuzumab or Pertuzumab).
  • Table 1 Binding of multispecific antibodies to HER2 overexpressed on human gastric cancer cell N87
  • CHOS-h4-1BB overexpressing human 4-1BB cells was adjusted to a cell density of 2 ⁇ 10 6 cells/ml, 100 ⁇ l/well was added to a 96-well flow plate, and centrifuged for later use.
  • PE Goat anti-human IgG-Fc
  • the experimental results are shown in Figure 3 and Table 3.
  • the HER2xHER2x4-1BB trispecific antibody and HER2x4-1BB bispecific antibody of the present invention have binding activity to CHOS-h4-1BB cells, and the binding activity is equivalent to that of the monoclonal antibody molecule of 4-1BB (anti-4-1BB VHH), and weaker than that of the control antibodies Urelumab and PRS-343.
  • the expanded cultured THP-1 (self-expressing FcRs) and CHOS-hCD32b (overexpressing human Fc ⁇ RIIb) cells were adjusted to a cell density of 2 ⁇ 10 6 cells/ml, 100 ⁇ l/well was added to a 96-well flow plate, and centrifuged for later use. . Add the serially diluted antibody at 100 ⁇ l/well to the above-mentioned 96-well flow cytometry plate with cells, and incubate at 4°C for 60 minutes.
  • the experimental results are shown in Figure 4 and Tables 4-5.
  • the HER2xHER2x4-1BB trispecific antibody and HER2x4-1BB bispecific antibody retaining the Fc effector function of the present invention bind to Fc ⁇ RIIb expressed on THP-1 (self-expressing FcRs, Figure 4A) and CHOS-hCD32b (overexpressing human FcR, Figure 4B) cells.
  • the Fc of the HER2xHER2x4-1BB trispecific antibody adopts a Knob in Hole mutation, so its binding activity to FcR is slightly weaker than that of the HER2x4-1BB bispecific antibody and HER2 monoclonal antibody using wild-type human IgG1 Fc.
  • Urelumab and PRS-343 The Fc used is wild-type human IgG4Fc or human IgG4Fc carrying FALA mutation, so its affinity to FcR is weaker than that of multispecific antibodies that retain the Fc effector function.
  • the expanded cultured N87 and SK-OV-3 (self-expressing HER2) cells were digested with 0.25% EDTA trypsin, washed once with culture medium, and then adjusted to a cell density of 5 ⁇ 10 4 cells/ml.
  • 80 ⁇ l/well was added to 96 wells. plate, set aside. Add 80 ⁇ l/well of the serially diluted antibody to the above-mentioned 96-well plate with cells, and place it in a cell culture incubator for incubation for 3-5 days. Use last After color development with Luminescent Cell Viability Assay (Promega, G7572) kit Chemiluminescent signals were collected using a microplate reader.
  • the multi-specific antibodies can significantly inhibit the proliferation of N87 or SK-OV-3 cells.
  • the HER2xHER2x4-1BB trispecific antibody has a significantly stronger inhibitory effect on cell proliferation than HER2-4-1BB.
  • Bispecific antibodies or HER2 monoclonal antibodies are shown in Figures 5A and 5B.
  • the inhibitory effect of HER2 antibodies on tumor cells is closely related to the expression level of HER2 on the cells. The higher the expression level of HER2, the more obvious the inhibitory effect of antibodies on cell proliferation.
  • the expanded culture of N87 (self-expressing HER2) cells and CHOS-h4-1BB (overexpressing human 4-1BB) cells were expanded according to NFAT Luciferase/Jurkat CD16a at 3 ⁇ 10 4 cells/well and 1.2 ⁇ 10 5 cells/well.
  • (overexpressing CD16a and NFAT-Luc) effector cells were mixed and inoculated into a 96-well white-bottom cell culture plate, and then serially diluted multispecific antibodies were added to the 96-well plate and mixed well, then placed in a cell culture incubator and incubated for 6 hours.
  • Use the Bio-Glo luciferase assay system Promega, G7940) kit to develop the color and collect the chemiluminescence signal with a microplate reader.
  • Multispecific antibodies that retain Fc effector function can mediate ADCC through HER2 expressed on N87 cells, thereby activating the CD16a-NFAT signaling pathway on Jurkat cells.
  • the HER2x4-1BB-2 dual antibody which removes the Fc effector function through LALA mutation
  • the control molecule PRS-343 which introduces FALA mutation on the basis of IgG4Fc
  • CHOS-h4-1BB cells were used as target cells, as shown in Figure 6B, no ADCC effect on 4-1BB cells was observed for all multispecific antibodies.
  • the expanded cultured target cells N87 (self-expressing HER2) were digested with 0.25% EDTA trypsin, and seeded into a 96-well culture plate at 1 ⁇ 10 4 cells/well (the culture plate was coated with 2 ⁇ g/ml anti one day in advance. -CD3 antibody, plated at 100 ⁇ l/well).
  • Collect the PBMC that were revived one day in advance use a T cell isolation kit (Stemcell, 17951) to isolate the T cells in the PBMC and add 5 ⁇ 10 4 cells/well into the target cell wells, and then gradually dilute the multispecific Antibodies were added to the cell wells and incubated for 48 hours, and then the supernatants were collected.
  • the levels of IL-2 and IFN ⁇ in the supernatant were detected using human IL-2 ELISA kit (Invitrogen, 88-7025-77) and human IFN ⁇ ELISA kit (Invitrogen, 88-7316-77).
  • the multispecific antibodies of the present invention can induce Primary T cells to release IL-2 (Figure 7A) or IFN- ⁇ ( Figure 7B) in the presence of N87 cells, and are dose-dependent.
  • the cytokine release ability induced by the HER2xHER2x4-1BB trispecific antibody is significantly stronger than that of the HER2x4-1BB bispecific antibody, and also stronger than the control antibodies PRS-343 and Urelumab.
  • Example 8 Multispecific antibodies induce primary T cells to release cytokines (FcR+ cell mediated)
  • the expanded cultured target cells CHOS-hCD32b (overexpressing human Fc ⁇ RIIb) were centrifuged and counted, and seeded into a 96-well culture plate at 1 ⁇ 10 4 cells/well (the culture plate was coated with 2 ⁇ g/ml anti- CD3 antibody, plated at 100 ⁇ l/well).
  • Collect the PBMC that were revived one day in advance use a T cell isolation kit (Stemcell, 17951) to isolate the T cells in the PBMC and add 5 ⁇ 10 4 cells/well into the target cell wells, and then gradually dilute the multispecific Antibodies were added to the cell wells and incubated for 48 hours, and then the supernatants were collected.
  • IL-2 and IFN ⁇ levels in the supernatant were determined using human IL-2 ELISA kit (Invitrogen, 88-7025-77) and human IFN ⁇ ELISA kit (Invitrogen, 88-7316-77) reagents. Box detection.
  • the multispecific antibody of the present invention cannot induce Primary T cells to release IL2 (Figure 8A) or IFN- ⁇ ( Figure 8B) in the presence of CHOS-hCD32b cells.
  • the positive control antibody Urelumab can induce T cell activation and release of pro-inflammatory factors through FcR.
  • the negative control molecule PRS-343 does not bind to CHOS-hCD32b cells, so it will not induce Primary T cells to release cytokines through FcR.
  • CH1/CL preference mutation (patent number: WO2021/067404A2) and Knob in hole technology are used to construct the heavy chain variable regions of anti-HER2 antibody and Pertuzumab antibody into CH SET1 (SEQ ID NO: 13) and CH SET2 (SEQ ID NO: 14) of CH1 mutation, and the N-terminus of the CD137 binding region amino acid sequence (SEQ ID NO: 7) of the anti-CD137 single domain antibody AB24 ME is connected to the C-terminus of Fc through a flexible peptide of 20 amino acid residues (G4A) 4 (SEQ ID NO: 18).
  • G4A 20 amino acid residues
  • the light chain variable regions of anti-HER2 antibody and Pertuzumab antibody are constructed into CL SET1 (SEQ ID NO: 15) and CL SET2 (SEQ ID NO: 16) of the CL mutant light chain constant region, respectively.
  • CL SET1 SEQ ID NO: 15
  • CL SET2 SEQ ID NO: 16
  • a 1+1 asymmetric anti-HER2xHER2x4-1BB-1 trispecific antibody cell line was constructed.
  • the vector pCHO2.0-GS-Puro-H1 containing the heavy chain gene (SEQ ID NO:27) and the light chain gene (SEQ ID NO:28) of the anti-HER2 antibody and the anti-4-1BB antibody was used by electroporation transfection.
  • Use Dynamis AGT Medium as the basic medium, inoculate at a density of (1.0 ⁇ 0.2) ⁇ 10 6 cells/ml, and culture until the 3rd, 5th, 7th, 9th, and 11th day with an initial culture of 5.0 ⁇ 0.5% (w/w).
  • the weight of 7a was fed, and 0.5 ⁇ 0.05% (w/w) of the initial culture weight of 7b was fed.
  • the dissolved oxygen was set to 40%, the initial culture temperature was 36.5°C, and the temperature was lowered to 33.0°C on the 4th day.
  • the culture was terminated on the 14th day or when the cell viability rate was lower than 80%.
  • cell density and viability were detected using Vicell (Beckman Company).
  • Cedex was used to detect antibody production every day.
  • the one-step purification method is the same as the single-arm antibody purification method in Example 1.
  • Use HPLC to detect protein purity The HPLC method is as follows, mobile phase: 150mM Na2HPO4 ⁇ 12H2O, pH7.0. Chromatographic conditions: detection wavelength: 280nm, column temperature: 25°C, flow rate: 0.5ml/min, detection time: 30min, TSKgel G3000SWXL chromatographic column.
  • the SEC results show in Figure 9A that the purity of the bispecific antibody obtained by one-step affinity purification was 98.3%.
  • High-performance liquid chromatography mass spectrometry was used to detect the pairing of heavy and light chains of the protein.
  • the instruments used were liquid chromatography system Vanquish UHPLC (Thermo), mass spectrometer Q Exactive (Thermo) and chromatographic column Waters ACQUITY UPLC BEH C4, 2.1mm ⁇ 100mm. Take 50 ⁇ g of sample, add ultrapure water to dilute to 25 ⁇ l, centrifuge and take 20 ⁇ l of sample into the injection bottle, inject 5 ⁇ l of sample, and use LC-MS to analyze the complete molecular weight.
  • the chromatographic conditions are: column temperature: 80°C; UV detection wavelength: 280nm; flow rate: 0.3mL/min; mobile phase A: aqueous solution (containing 0.1% formic acid); mobile phase B: acetonitrile solution (containing 0.1% formic acid).
  • the mass spectrometry parameters are: ESI ion source: ion transfer tube temperature 320°C, voltage 3.8kV, gas flow rate 36L/min; mode: positive ion Full MS; resolution: 17500; scanning range: 600-4000m/z. The results are shown in Figures 9B and 9C. The proportion of correctly paired products for the purified trispecific antibody HER2xHER2x4-1BB-1 was >98%.
  • the anti-tumor activity of the multispecific antibody of the present invention was demonstrated in a tumor model in which h-4-1BB KI BALB/c mice were subcutaneously inoculated with CT26-h-HER2.
  • a tumor-bearing mouse model was established by subcutaneous inoculation of CT26-h-HER2 cells.
  • the tumor volume grew to about 180 mm3
  • the mice were divided into groups and given intraperitoneal injections of G1: PBS, G2: 2.5 mg/kg of PRS-343, G3: 2 mg/kg of Trastuzumab, G4: 2.3 mg/kg of HER2x4-1BB-1, and G5: 2.3 mg/kg of HER2x4-1BB-2 (all groups were treated with equal molar doses).
  • the changes in tumor volume and body weight of mice in each group were monitored 2-3 days/time for 2-3 weeks.
  • the dosage and method of administration are shown in Table 8.
  • the tumors of the mice were taken for immunohistochemical detection to compare the infiltration of CD8+T cells in the tumors of different groups.
  • the experimental results are shown in Figure 10A.
  • the anti-tumor activity of the bispecific antibody HER2x4-1BB-1 that retains the Fc effector function is significantly better than the HER2x4-1BB-2 bispecific antibody that removes the Fc effector function by LALA mutation (TGI: 73.2% vs 26.6%), and is also stronger than the control antibody PRS-343 (TGI: 6.5%) and the HER2 monoclonal antibody Trastuzumab (TGI: 4.2%).
  • the anti-tumor activity of the multispecific antibody of the present invention was measured in a tumor model in which h-4-1BB KI BALB/c mice were subcutaneously inoculated with CT26-h-HER2.
  • tumor-bearing mouse models were established by inoculating h-HER2CT26 cells subcutaneously. When the tumor volume grew to about 180 mm, they were divided into groups. G1: PBS, G2: 3 mg/kg Trastuzumab combined with 3 mg/kg Pertuzumab were administered intraperitoneally.
  • G3 3.6mg/kg of PRS-343
  • G4 3mg/kg of Enhertu (DS8201)
  • G5 3.6mg/kg of HER2x4-1BB-1
  • G6 3.6mg/kg of HER2x4-1BB-3
  • G7 1.8mg/kg HER2x4-1BB-1 combined with 1.8mg/kg HER2x4-1BB-3
  • G8 3.6mg/kg HER2xHER2x4-1BB-2 (equal molar doses for all groups), monitor each group
  • the changes in tumor volume and weight of mice were monitored at a frequency of 2-3 days/time and continuously monitored for 2-3 weeks.
  • the dosage and method of administration are as shown in Table 9.
  • the anti-tumor activity of the HER2xHER2x4-1BB trispecific antibody is also significantly stronger than that of the HER2x4-1BB bispecific antibody HER2x4-1BB-1 (TGI: 66.9%) and HER2x4-1BB-3 (TGI: 59.7%).
  • the therapeutic effect of the -1BB bispecific antibody combination treatment group (HER2x4-1BB-1+HER2x4-1BB-3, TGI: 83.7%) was comparable.
  • the anti-tumor activity of the multispecific antibody of the present invention was measured in a tumor model in which h-4-1BB KI C57 mice were subcutaneously inoculated with MC38-h-HER2.
  • tumor-bearing mouse models were established by inoculating MC38-h-HER2 cells subcutaneously. When the tumor volume grew to about 100 mm, they were divided into groups.
  • G1 PBS
  • G2 3 mg/kg Trastuzumab combined with 3 mg/kg were administered intraperitoneally.
  • Pertuzumab G3: 3.6mg/kg PRS-343
  • G4 3mg/kg Enhertu (DS8201)
  • G5 1.8mg/kg HER2x4-1BB-1 combined with 1.8mg/kg HER2x4-1BB-3
  • G6 Treat with 3.6 mg/kg HER2xHER2x4-1BB-1 (equal molar doses in all groups).
  • Monitor the changes in tumor volume and weight of mice in each group. The monitoring frequency is 3-6 days/time, and continuous monitoring is performed for 2 to 3 days. Week, the dosage and method of administration are as shown in Table 10.
  • mice treated with the HER2xHER2x4-1BB trispecific antibody achieved complete tumor regression, which was significantly better than the control antibody PRS-343 (TGI: 54.9%) and HER2 monoclonal at equimolar doses.
  • Combination of antibodies (Trastuzumab+Pertuzumab, TGI: 63.8%).
  • the combination treatment group of HER2x4-1BB bispecific antibody (HER2x4-1BB-1+HER2x4-1BB-3) and Enhertu treatment also achieved complete tumor regression.

Abstract

提供抗体及其在抗肿瘤中的应用。提供能够特异性结合4-1BB的单域抗体或其抗原结合片段、以及多特异性抗体，抗体具有提高的抗肿瘤活性以及更低的肝脏毒性。

Description

抗体及其在抗肿瘤中的应用
本申请是以CN申请号为202211142594.0，申请日为2022年9月20日的申请为基础，并主张其优先权，该CN申请的公开内容在此作为整体引入本申请中。
技术领域
本申请涉及生物治疗技术领域，具体涉及靶向4-1BB以及HER2的抗体及其在抗肿瘤中的应用。
背景技术
人表皮生长因子受体2(HER2/ErbB2/Neu)是ERBB受体酪氨酸激酶家族的成员之一，在许多实体肿瘤中过表达，其能通过形成同源二聚体或与ERBB家族的其他成员形成异源二聚体引起受体胞质域内酪氨酸残基的磷酸化，从而引起细胞内多种信号通路被启动导致癌细胞增殖和肿瘤发生[1]。作为最早被用于肿瘤治疗的靶点之一，靶向HER2的治疗药物包括单克隆抗体(mAb，如曲妥珠单抗和帕妥珠单抗)，酪氨酸激酶抑制剂(TKIs，如来那替尼和拉帕替尼)，抗体-药物结合物(ADC，如T-DM1)等，这些药物在HER2过表达和/或扩增的乳腺癌或胃癌的治疗中已被广泛应用[2]。尽管这些HER2靶向治疗药有良好的临床效益，但是医疗需求仍未得到满足，需要持续开发新的药物以进一步提高对复发或转移患者的临床疗效。
T细胞共刺激受体TNFRSF9(4-1BB)是TNF受体家族的一员，主要表达在活化的T细胞上，当被激活时，可以提高T细胞的效应活性和记忆反应[3,4]。Urelumab是一款靶向4-1BB的单克隆抗体药，通过结合并激活T细胞上的4-1BB发挥疗效。但不幸的是，在Urelumab的临床开发过程中观察到了明显的肝脏相关毒性。
开发具有更好疗效以及更高安全性的抗肿瘤药物仍是本领域亟需解决的问题。
发明内容
本申请公开一种对4-1BB具有高结合活性的单域抗体，以及在此基础上开发得到的靶向4-1BB和HER2的多特异性抗体。特别的，本申请公开一种HER2xHER2x4-1BB三特异性抗体，其具有独特的抗肿瘤活性，且有很强的免疫记忆效应。该抗体是一种由曲妥珠单抗和帕妥珠单抗组成的1+1IgG1样异源二聚体抗体，其C端通过G4S连接子连接了一个抗4-1BB的单域抗体(sdAb)。该抗体保留了Fc效应功能，这对曲妥珠单抗和帕妥珠单抗发挥抗肿瘤活性至关重要。此外，该抗体具有更低的肝脏毒性，安全性得到提高。
单域抗体
在一个方面，本申请提供能够特异性结合4-1BB的单域抗体或其抗原结合片段，所述单域抗体包含：
(a)CDR1，其具有：如SEQ ID NO:43所示的序列，或与SEQ ID NO:43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个或3个氨基酸的置换、缺失或添加)的序列；
(b)CDR2，其具有：如SEQ ID NO:44所示的序列，或与SEQ ID NO:44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个或3个氨基酸的置换、缺失或添加)的序列；以及
(c)CDR3，其具有：如SEQ ID NO:45所示的序列，或与SEQ ID NO:45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个或3个氨基酸的置换、缺失或添加)的序列。
在一些实施方案中，所述单域抗体或其抗原结合片段包含：如SEQ ID NO:43所示的CDR1、如SEQ ID NO:44所示的CDR2、如SEQ ID NO:45所示的CDR3。
所述单域抗体包含选自下列的氨基酸序列：
(i)如SEQ ID NO:7所示的序列；
(ii)与SEQ ID NO:7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:7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。
在一些实施方案中，所述的置换是保守置换。
术语“4-1BB”，也称为CD137或TNFRSF9(TNF受体超家族成员9)，是TNF受体超家族(TNFRSF)的成员，并且是一种共刺激分子，其在免疫细胞(固有免疫细胞和适应性免疫细胞两者)活化后表达。4-1BB在调节各种免疫细胞的活性中起重要作用。如本文所用，4-1BB可以源自哺乳动物，例如智人(Homo sapiens)(人)(NCBI登录号NP_001552.2)。
在一些实施方案中，所述单域抗体特异性结合4-1BB表位CRD-4。
多肽构建体
本申请还提供特异性结合4-1BB的多肽构建体，其包含前文所述的单域抗体或其抗原结合片段，以及免疫球蛋白Fc结构域。
在一些实施方案中，所述免疫球蛋白Fc结构域直接或通过肽接头连接至所述单域抗体或其抗原结合片段的N端和/或C端(例如C端)。
在一些实施方案中，所述肽接头具有(GmXn)lGm’所示结构，m、m’、n和l各自独立地选自0、1、2、3、4、5、6、7、8、9和10，X选自A和S；优选地，m选自1、2、3、4或5，n选自1和2，l选自1、2、3、4、5、6、7、8、9和10，m’选自0和1，X选自A和S。
在一些实施方案中，所述肽接头具有SEQ ID NO:17或18所示氨基酸序列。
在一些实施方案中，所述免疫球蛋白Fc结构域是IgG的Fc结构域(例如IgG1的Fc结构域，例如包含CH2和CH3)。
在一些实施方案中，所述免疫球蛋白Fc结构域包含SEQ ID NO:19所示的序列，或与其相比具有至少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，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个、4个或5个氨基酸置换、缺失或添加)的序列。
在一些实施方案中，所述免疫球蛋白Fc结构域包含SEQ ID NO:19或20所示的序列。
在一些实施方案中，所述多肽构建体含有如SEQ ID NO:31或32所示的氨基酸序列或由其组成。
多特异性抗体
本申请进一步提供一种多特异性抗体，所述多特异性抗体可仅在与表达HER2的肿瘤细胞交联时活化4-1BB信号传导。此外，多特异性抗体中所包含的抗4-1BB抗体或其抗原结合片段的特征可以是，仅在肿瘤微环境(TME)中定位和/或激活，和/或与现有的抗4-1BB抗体相比显著地降低肝脏毒性，同时维持免疫应答增强和/或肿瘤治疗的功效。
具体地，本申请提供一种多特异性抗体，其包含前文所述的单域抗体或其抗原结合片段或多肽构建体。
本申请进一步提供一种多特异性抗体，其包含对4-1BB特异的第一抗原结合结构域， 所述第一抗原结合结构域包含前文所述的抗体或其抗原结合片段。
在一些实施方案中，所述第一抗原结合结构域包含前文所述的抗体或其抗原结合片段。
在一些实施方案中，上述任意多特异性抗体特异性结合4-1BB，并且额外地特异性结合一个或多个其它靶标。
在一些实施方案中，所述靶标为肿瘤抗原。
在一些实施方案中，所述肿瘤抗原选自下述的一个或多个：CD19、CD20、CD22、CD23、CD38、CD40、CD49、CD52、CD56、CD74、CD80、CD95、CD138、CS1/SLAMF7、KiR、Thy-1、Ly-6、Fas、APO-1、EGFR、HER2、CXCR4、HLA、GM1和DRD。
在一些实施方案中，所述多特异性抗体可以结合多个相同或不同的肿瘤抗原。
在一些实施方案中，所述多特异性抗体可以结合同一肿瘤抗原上的相同或不同表位。
在一些实施方案中，所述多特异性抗体特异性结合HER2。
“HER2(人表皮生长因子受体2)”由ERBB2基因编码，是表皮生长因子受体(EGFR/ErbB)的成员。已知HER2在调节细胞增殖和分化中起重要作用。特别是，当与细胞外生长因子结合时，它具有与其他HER受体一起组装成同源和/或异源二聚体的强烈趋势，这导致数种形式的信号转导通路的活化并诱导细胞凋亡、存活或细胞增殖。例如，HER2蛋白可以是以GenBank登录号NP_004439.2、NP_001005862.1等保藏的多肽，其分别由以GenBank登录号NM 004448.4、NM_001005862.3等保藏的核苷酸序列(mRNA)编码。
将HER2识别为抗原的与HER2结合的部分可以是选自抗HER2抗体的scFv、(scFv)2、Fab、Fab'和F(ab')2。
在一些实施方案中，所述多特异性抗体含有抗HER2抗体的重链可变区的至少一个(例如1、2或3个)CDR，和/或抗HER2抗体的轻链可变区的至少一个(例如1、2或3个)CDR。
在一些实施方案中，所述多特异性抗体含有抗HER2抗体的重链可变区，和/或抗HER2抗体的轻链可变区。
在一些实施方案中，所述抗HER2抗体选自曲妥珠单抗、帕妥珠单抗及其变体。
在一些实施方案中，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)。
在一些实施方案中，所述抗HER2抗体为曲妥珠单抗的变体。具体的，所述抗HER2抗体含有下述3个重链可变区(VH)互补决定区(CDR)：
SEQ ID NO:46所示的VH CDR1，SEQ ID NO:47所示的VH CDR2，和SEQ ID NO:48所示的VH CDR3；和/或，
下述3个轻链可变区(VL)互补决定区(CDR)：
SEQ ID NO:49所示的VL CDR1；SEQ ID NO:50所示的VL CDR2，和SEQ ID NO:8所示的VL CDR3。
在一些实施方案中，所述多特异性抗体含有对HER2特异的第二抗原结合结构域。
在一些实施方案中，所述第一抗原结合结构域是VHH；所述第二抗原结合结构域是Fab，所述多特异性抗体包含：
(1)肽链I-A，其包含所述第二抗原结合结构域的轻链可变区和轻链恒定区(CL)；
和，
(2)肽链I-B，其包含所述第二抗原结合结构域的重链可变区，重链恒定区，和所述第一抗原结合结构域；优选地，所述肽链I-B从N端至C端包含相邻的所述第二抗原结合结构域的重链可变区和重链恒定区和所述第一抗原结合结构域。
在一些实施方案中，所述重链恒定区可以选自(例如，Fc区)：IgG1、IgG2、IgG3、和IgG4的重链恒定区，具体地，人IgG1、IgG2、IgG3、或IgG4的重链恒定区，例如，人IgG1的重链恒定区，例如，人IgG1的CH1、CH2和/或CH3，再例如，人IgG1的Fc区。
在一些实施方案中，所述重链恒定区被改变(例如，突变)以增加或减少以下中的一种或多种：Fc受体结合、抗体糖基化、半胱氨酸残基的数量、效应细胞功能、或补体功能。
在一些实施方案中，所述重链恒定区含有LALA突变、CH3 Knob突变、CH3 Hole突变、CH1/CL偏好性突变CH SET1、CH1/CL偏好性突变CH SET2CH SET2、及其任意组合。
在一些实施方案中，所述重链恒定区选自：
HC-1：SEQ ID NO:10所示氨基酸序列；
HC-2：SEQ ID NO:11所示氨基酸序列；
HC-3：SEQ ID NO:12所示氨基酸序列；
HC-4：SEQ ID NO:13所示氨基酸序列；
HC-5：SEQ ID NO:14所示氨基酸序列；
HC-6：SEQ ID NO:19所示氨基酸序列；
HC-7：SEQ ID NO:20所示氨基酸序列；
HC-8：SEQ ID NO:37所示氨基酸序列；以及
HC-9：SEQ ID NO:38所示氨基酸序列；
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)。
在一些实施方案中，所述轻链恒定区选自κ或λ的轻链恒定区或其变体；
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)。
在一些实施方案中，所述轻链恒定区选自：
LC-1：SEQ ID NO:9；
LC-2：SEQ ID NO:15；以及，
LC-3：SEQ ID NO:16；
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)。
在一些实施方案中，所述肽链I-A的CL能够与所述肽链I-B的重链恒定区CH1结构域形成二聚体。
在一些实施方案中，所述多特异性抗体包含两条所述肽链I-A以及两条所述肽链I-B；优选地，两条所述肽链I-B的重链恒定区形成二聚体。
在一些实施方案中，各结构域之间直接或通过肽接头连接。
在一些实施方案中，所述肽接头具有(GmXn)lGm’所示结构，m、m’、n和l各自独立地选自0、1、2、3、4、5、6、7、8、9和10，X选自A和S；优选地，m选自1、2、3、4或5，n选自1和2，l选自1、2、3、4、5、6、7、8、9和10，m’选自0和1，X选自A和S。
在一些实施方案中，所述肽接头具有SEQ ID NO:17或18所示氨基酸序列。
在一些实施方案中，所述多特异性抗体特征在于下述的一项或多项：
(i)所述第一抗原结合结构域含有如SEQ ID NO:7所示的氨基酸序列或由其组成；
(ii)所述第二抗原结合结构域的重链可变区含有如SEQ ID NO:1、3或5所示的氨基酸序列或由其组成；
(iii)所述第二抗原结合结构域的轻链可变区含有如SEQ ID NO:2、4或6所示的氨基酸序列或由其组成。
在一些实施方案中，所述肽链I-A含有如SEQ ID NO:22、24或26所示的氨基酸序列或由其组成。
在一些实施方案中，所述肽链I-B含有如SEQ ID NO:21、23或25所示的氨基酸序列或由其组成。
在一些实施方案中，所述多特异性抗体选自：
(1)包含所述肽链I-A和I-B的多特异性抗体，其中，所述肽链I-A含有如SEQ ID NO:22所示的氨基酸序列或由其组成，所述肽链I-B含有如SEQ ID NO:21所示的氨基酸序列或由其组成；
(2)包含所述肽链I-A和I-B的多特异性抗体，其中，所述肽链I-A含有如SEQ ID NO:24所示的氨基酸序列或由其组成，所述肽链I-B含有如SEQ ID NO:23所示的氨基酸序列或由其组成；
和，
(3)包含所述肽链I-A和I-B的多特异性抗体，其中，所述肽链I-A含有如SEQ ID NO:26所示的氨基酸序列或由其组成，所述肽链I-B含有如SEQ ID NO:25所示的氨基酸序列或由其组成；
优选地，所述多特异性抗体包含两条所述肽链I-A以及两条所述肽链I-B；
优选地，两条所述肽链I-B的重链恒定区形成二聚体。
在一些实施方案中，所述多特异性抗体进一步含有对HER2特异的第三抗原结合结构域。
在一些实施方案中，所述第一抗原结合结构域是VHH；所述第二抗原结合结构域和第二抗原结合结构域是Fab，所述多特异性抗体包含：
(1)肽链II-A，其包含所述第二抗原结合结构域的轻链可变区和轻链恒定区(CL)；
(2)肽链II-B，其包含所述第二抗原结合结构域的重链可变区，重链恒定区，和所述第一抗原结合结构域；优选地，所述肽链II-B从N端至C端包含相邻的所述第二抗原结合结构域的重链可变区和重链恒定区和所述第一抗原结合结构域；
(3)肽链II-C，其包含所述第三抗原结合结构域的轻链可变区和轻链恒定区(CL)；
和，
(4)肽链II-D，其包含所述第三抗原结合结构域的重链可变区，重链恒定区，和所述第一抗原结合结构域；优选地，所述肽链II-D从N端至C端包含相邻的所述第三抗原结合结构域的重链可变区和重链恒定区和所述第一抗原结合结构域。
在一些实施方案中，所述重链恒定区可以选自(例如，Fc区)：IgG1、IgG2、IgG3、和IgG4的重链恒定区，具体地，人IgG1、IgG2、IgG3、或IgG4的重链恒定区，例如，人IgG1的重链恒定区，例如，人IgG1的CH1、CH2和/或CH3，再例如，人IgG1的Fc区。
在一些实施方案中，所述重链恒定区被改变(例如，突变)以增加或减少以下中的一种或多种：Fc受体结合、抗体糖基化、半胱氨酸残基的数量、效应细胞功能、或补体功能。
在一些实施方案中，所述重链恒定区含有LALA突变、CH3 Knob突变、CH3 Hole突变、CH1/CL偏好性突变CH SET1、CH1/CL偏好性突变CH SET2、及其任意组合。
在一些实施方案中，所述轻链恒定区选自κ或λ的轻链恒定区或其变体；
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)。
在一些实施方案中，所述肽链II-A的CL能够与所述肽链II-B的重链恒定区CH1结构域形成二聚体；优选地，所述肽链II-C的CL能够与所述肽链II-D的重链恒定区CH1结构域形成二聚体。
在一些实施方案中，所述多特异性抗体包含一条所述肽链II-A、一条所述肽链II-B、一条所述肽链II-C、以及一条所述肽链II-D。在一些实施方案中，所述肽链II-B和II-D的重链恒定区形成二聚体。在一些实施方案中，所述肽链II-B的重链恒定区含有如SEQ ID NO:13所示氨基酸序列，所述肽链II-D的重链恒定区含有如SEQ ID NO:14所示氨基酸序列。在一些实施方案中，所述肽链II-B的重链恒定区含有如SEQ ID NO:37所示氨基酸序列，所述肽链II-D的重链恒定区含有如SEQ ID NO:38所示氨基酸序列。
在一些实施方案中，各结构域之间直接或通过肽接头连接。
在一些实施方案中，所述肽接头具有(GmXn)lGm’所示结构，m、m’、n和l各自独立地选自0、1、2、3、4、5、6、7、8、9和10，X选自A和S。
在一些实施方案中，m选自1、2、3、4或5，n选自1和2，l选自1、2、3、4、5、6、7、8、9和10，m’选自0和1，X选自A和S。
在一些实施方案中，所述肽接头具有SEQ ID NO:17或18所示氨基酸序列。
在一些实施方案中，所述多特异性抗体的特征在于下述的一项或多项：
(i)所述第一抗原结合结构域含有如SEQ ID NO:7所示的氨基酸序列或由其组成；
(ii)所述第二抗原结合结构域的重链可变区含有如SEQ ID NO:1、3或5所示的氨基酸序列或由其组成；
(iii)所述第二抗原结合结构域的轻链可变区含有如SEQ ID NO:2、4或6所示的氨基酸序列或由其组成；
(iv)所述第三抗原结合结构域的重链可变区含有如SEQ ID NO:1、3或5所示的氨基酸序列或由其组成；
(v)所述第三抗原结合结构域的轻链可变区含有如SEQ ID NO:2、4或6所示的氨基酸序列或由其组成。
在一些实施方案中，所述肽链II-A含有如SEQ ID NO:28或40所示的氨基酸序列或由其组成。
在一些实施方案中，所述肽链II-B含有如SEQ ID NO:27或39所示的氨基酸序列或由其组成。
在一些实施方案中，所述肽链II-C含有如SEQ ID NO:30或42所示的氨基酸序列或由其组成。
在一些实施方案中，所述肽链II-D含有如SEQ ID NO:29或41所示的氨基酸序列或由其组成。
在一些实施方案中，所述多特异性抗体选自：
(1)包含所述肽链II-A、II-B、II-C和II-D的多特异性抗体，其中，所述肽链II-A含有如SEQ ID NO:28所示的氨基酸序列或由其组成，所述肽链II-B含有如SEQ ID NO:27所示的氨基酸序列或由其组成，所述肽链II-C含有如SEQ ID NO:29所示的氨基酸序列或由其组成，所述肽链II-D含有如SEQ ID NO:30所示的氨基酸序列或由其组成；
和，
(2)包含所述肽链II-A、II-B、II-C和II-D的多特异性抗体，其中，所述肽链II-A含有如SEQ ID NO:40所示的氨基酸序列或由其组成，所述肽链II-B含有如SEQ ID NO:39所示的氨基酸序列或由其组成，所述肽链II-C含有如SEQ ID NO:42所示的氨基酸序列或由其组成，所述肽链II-D含有如SEQ ID NO:41所示的氨基酸序列或由其组成。
肽接头
本文中所述与4-1BB结合的部分的N端或C端(优选N端)直接或通过肽接头与所述重链恒定区的C端或N端连接。
如本文所用，术语“肽接头”可以指包括1至100个氨基酸，特别是2至50个氨基酸的寡肽，每个氨基酸可以是任何种类的氨基酸，而没有任何限制。可以在有或没有适当修饰的情况下使用任何常规的肽接头以符合特定目的。在具体的实施方式中，肽接头可包含例如Gly、Asn和/或Ser残基，和/或包含中性氨基酸如Thr和/或Ala。适用于肽接头的氨基酸序列可以是相关领域已知的。肽接头的长度可以在不影响多肽和/或scFv功能的限度内适当确定。例如，肽接头可通过包括总共约1至约100个氨基酸、约2至约50个氨基酸或约5至约25个(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24或25个)氨基酸而形成，每个氨基酸独立地选自由Gly、Asn、Ser、Thr和Ala组成的组。
在一些实施方案中，所述肽接头具有(GmXn)lGm’所示结构，m、m’、n和l各自独立地选自0、1、2、3、4、5、6、7、8、9和10，X选自A和S。
在一些实施方案中，m选自1、2、3、4或5，n选自1和2，l选自1、2、3、4、5、6、7、8、9和10，m’选自0和1，X选自A和S。
在一些实施方案中，所述肽接头具有SEQ ID NO:17或18所示氨基酸序列。
多核苷酸、重组载体和抗体的制备
在一个方面，本发明提供一种多核苷酸，其编码前文任一项所述的抗体或其抗原结合片段。
在一个方面，本发明提供一种载体，其包括前文所述的多核苷酸。
在一个方面，本发明提供一种重组细胞，其包括前文所述的多核苷酸或载体。重组细胞可以是用重组载体转染的细胞。
在一个方面，本发明提供制备所述抗体或其抗原结合片段的方法，包括在细胞中表达多核苷酸。表达多核苷酸的步骤可以通过在允许多核苷酸表达的条件下培养包含多核苷酸(例如该多核苷酸在重组载体中)的细胞来进行。该方法可以进一步包括在表达或培养步骤之后，从细胞培养物中分离和/或纯化所述抗体或其抗原结合片段。
应用
本发明进一步提供本文所述抗体或其抗原结合片段在增强免疫应答和/或治疗肿瘤中的应用。
具体地，本发明提供一种药物组合物，其含有前文任一项所述的抗体或其抗原结合片段、多核苷酸、载体或重组细胞，以及药学上可接受的赋形剂。
在另一个方面，本发明进一个提供前文任一项所述的抗体或其抗原结合片段、多核苷酸、载体、重组细胞或药物组合物在制备抗肿瘤药物中的用途。
在另一个方面，本发明提供一种抗肿瘤方法，其包括向有此需要的受试者施用治疗有效量的前文任一项所述的抗体或其抗原结合片段、多核苷酸、载体、重组细胞或药物组合物的步骤。
在一些实施方案中，所述肿瘤过表达HER2。
在一些实施方案中，所述肿瘤选自乳腺癌、结肠癌、胃癌、肺癌(例如，肺鳞状细胞癌、小细胞肺癌、非小细胞肺癌、肺腺癌)、腹膜癌、皮肤癌、鳞状细胞癌、皮肤或眼球黑色素瘤、直肠癌、肛门附近的癌症、食道癌、小肠肿瘤、内分泌腺癌、甲状旁腺癌、肾上腺癌症、软组织肉瘤、尿道癌、慢性或急性白血病、淋巴细胞性淋巴瘤、肝癌、胃肠道癌、胰腺癌、胶质母细胞瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、肝细胞腺瘤、大肠癌、子宫内膜癌或子宫癌、唾液腺肿瘤、肾癌、宫颈癌、前列腺癌、外阴癌、甲状腺癌、头颈部癌、 脑癌、胆道癌和胆囊癌。
在一些实施方案中，所述肿瘤为原发性或转移性肿瘤。
检测用途
在另一个方面，本发明还提供了缀合物，其包含如上任一方面所述的抗体或其抗原结合片段或多肽构建体，以及与所述单域抗体或其抗原结合片段或多肽构建体连接的可检测的标记。
在一些实施方案中，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素。
在另一方面，本发明还提供了试剂盒，其包括如上任一方面所述的单域抗体或其抗原结合片段或多肽构建体、或缀合物。
在某些实施方案中，所述试剂盒还包含特异性识别所述抗体或其抗原结合片段或所述多肽构建体所特异性识别的抗原的第二抗体；任选地，所述第二抗体还包括可检测的标记，例如酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素。
在一些实施方案中，所述第二抗体与所述抗体或其抗原结合片段或所述多肽构建体靶向相同或不同的抗原表位。
在另一方面，本发明还提供了用于检测4-1BB在样品中的存在或其水平的方法，其包括使用如上任一方面所述的抗体或其抗原结合片段或多肽构建体、或缀合物。
在某些实施方案中，所述方法是免疫学检测，例如免疫印迹法、酶免疫测定法(例如ELISA)、化学发光免疫分析法、荧光免疫分析法或放射免疫测定法。
在某些实施方案中，所述方法包括使用如上所述的缀合物。
在某些实施方案中，所述方法包括使用如上任一方面所述的抗体或其抗原结合片段或多肽构建体，并且所述方法还包括使用携带可检测的标记(例如酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素)的第二抗体来检测所述单域抗 体或其抗原结合片段或所述多肽构建体与抗原的结合。
在某些实施方案中，所述方法包括：(1)将所述样品与所述抗体或其抗原结合片段接触；(2)检测抗原-抗体免疫复合物的形成或检测所述免疫复合物的量。所述免疫复合物的形成表明存在4-1BB或表达4-1BB的细胞。
在另一方面，本申请还提供了如上任一方面所述的单域抗体或其抗原结合片段，或多肽构建体，或缀合物在制备检测试剂中的用途，所述检测试剂用于检测4-1BB在样品中的存在或其水平。
在某些实施方案中，所述检测试剂通过如上所述的用于检测4-1BB在样品中的存在或其水平的方法来检测4-1BB在样品中的存在或其水平。
在某些实施方案中，所述样品为来自受试者(例如哺乳动物，优选人或猴)的细胞样品(例如，肿瘤细胞)。
定义
在本文中，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，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。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。
如本文所用，“至少一个(种)”或“一个(种)或多个(种)”可以表示1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20个(种)或更多个(种)。
表述“包含”或与其同义的类似表述“包括”、“含有”和“具有”等是开放性的，不排除额外的未列举的元素、步骤或成分。表述“由…组成”排除未指明的任何元素、步骤或成分。表述“基本上由…组成”指范围限制在指定的元素、步骤或成分，加上任选存在的不会实质上影响所要求保护的主题的基本和新的特征的元素、步骤或成分。应当理解，表述“包含”涵盖表述“基本上由…组成”和“由…组成”。
术语“任选”或“任选地”是指随后描述的事件或情况可能发生或可能不发生，该描述包括发生所述事件或情况和不发生所述事件或情况。
如本文所用，“肽”、“多肽”是指通过肽键连接的两个或更多个氨基酸形成的聚合物。“蛋白”可以由一条或多条多肽以共价或非共价方式形成。除非另有说明，否则术语“多肽”和“蛋 白质”在本文可互换使用。
如本文所用，“抗体”指免疫球蛋白或其片段，其通过至少一个抗原结合位点特异性结合抗原表位。在本文中，抗体的定义涵盖抗原结合片段。因此，术语“抗体”包括多特异性抗体(例如双特异性抗体)、人抗体、非人抗体、人源化抗体、嵌合抗体、单域抗体以及抗原结合片段。抗体可以是合成的(例如通过化学偶联或生物偶联产生的)、酶促处理得到的或重组产生的。本文所提供的抗体包括任何免疫球蛋白类型(例如，IgG、IgM、IgD、IgE、IgA和IgY)、任何类别(例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或亚类(例如，IgG2a和IgG2b)。在一实施方案中，本发明的抗体为鼠源单克隆抗体。在又一实施方案中，本发明的抗体为人源化单克隆抗体。
如本文所用，“传统抗体”或“全长抗体”通常包含四条多肽：两条重链(HC)和两条轻链(LC)。每条轻链从N末端到C末端包含“轻链可变区(VL)”和“轻链恒定区(CL)”。每条重链从N末端到C末端包含“重链可变区(VH)”以及“重链恒定区(CH)”。重链恒定区从N末端到C末端可以包含CH1、CH2和CH3。在某些免疫球蛋白类型(例如IgM和IgE)中，重链恒定区还可以包含CH4。“Fc”片段指包含CH2和CH3的片段，其提供与Fc受体结合的位点。“铰链区”指抗体中连接免疫球蛋白Fab和Fc片段的部分。在有些情况下，铰链区指T细胞受体中连接恒定区和跨膜结构域的部分。在本文中，当对于嵌合抗原受体使用时，铰链区还可以指任何功能等同物。本领域技术人员可以根据已知的算法和软件判断VH、VL、CL、CH1、CH2、CH3和铰链区在抗体中的位置，可以应用的算法和软件的描述可以参见例如William R.Strohl,Lila M.Strohl,(2012),Antibody structure–function relationships,In Woodhead Publishing Series in Biomedicine,Therapeutic Antibody Engineering,Woodhead Publishing,pp.37-56。
通常，VL和VH各自可以包含三个高度可变的“互补决定区(CDR)”和四个相对保守的“框架区(FR)”，并且从N末端到C末端以FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的次序连接。在本文中，轻链可变区的CDR(CDRL)可以称为CDR-L1、CDR-L2和CDR-L3，重链可变区的CDR(CDRH)可以称为CDR-H1、CDR-H2和CDR-H3。通常认为包含一对VH和VL的六个CDR决定抗原结合位点的结合特异性，但在某些情况下，包含少于六个CDR(例如三、四或五个)的其他片段(例如单域抗体，又称为纳米抗体)同样具有结合抗原的能力。本领域技术人员可以使用本领域熟知的方法识别CDR，例如使用Kabat、Abm或Chothia编号法。在本文中，对于同一个可变区可以使用多个CDR编号系统，例如Chothia、Abm、Kabat和IMGT。本领域技术人员应当理解，尽管由不同编号系统定义的CDR可能会不同，但是同一编号系统对应的CDR代表能够结合抗原表位的有效抗原结合位点。 有关CDR编号系统的描述可以参见例如，Kabat编号系统：Kabat,E.A.et al.(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242；Chothia编号系统：Chothia,C.et al.(1987)J.Mol.Biol.196:901-917；IMGT编号系统：Lefranc,M.-P.,2011(6),IMGT,the International ImMunoGeneTics Information System Cold Spring Harb Protoc.；Abm编号 系统：Martin,A.C.R.and J.Allen(2007)“Bioinformatics tools for antibody engineering,”in S.Dübel(ed.),Handbook of Therapeutic Antibodies.Weinheim:Wiley-VCH Verlag,pp.95–118。
在本文中，术语“框架区”和“构架区”可以互换使用。如本文中所使用的，术语“框架区”、“构架区”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。
通常认为，由一个VH和一个VL通过非共价作用构成的“Fv”片段为包含抗原结合位点的最小的抗原结合片段。但是只包含重链可变区的单域抗体(VHH，又称sdAb或纳米抗体)也可以有抗原结合能力。可以通过肽接头将VH和VL连接来获得“单链Fv(scFv)”。通过向Fv或scFv中引入二硫键可以分别获得“二硫键稳定的Fv(dsFv)”或“单链二硫键稳定的Fv(scdsFv或dsscFv)”。在一些实施方案中，例如本发明的单域抗体、双特异性抗体、多特异性抗体中的抗原结合片段优选使用scFv或scdsFv。
如本文所用，“Fab”包含一个完整的抗体轻链(VL-CL)和抗体重链可变区和一个重链恒定区(VH-CH1，也称为Fd)。用肽接头将“Fab”中的CL和CH1连接可以获得单链“Fab(scFab)”。“F(ab')2”基本上包含通过铰链区的二硫键连接的两个Fab片段。“Fab'”为F(ab')2的一半，其可以通过还原F(ab')2铰链区的二硫键获得。
抗体或抗原结合片段可以是“单价”、“二价”、“三价”或“四价”或更多价，是指其具有多个抗原结合位点(例如1、2、3或4个或者更多个)。
当抗体或抗原结合片段所结合的抗原为两种或更多种(例如2、3、4、5或6种)时，其又可以称为“多特异性抗体”，例如双特异性抗体、三特异性抗体或四特异性抗体，其分别表示能够结合2、3或4种抗原的多特异性抗体。
如本文所用，“双抗体(diabody)”指这样的抗体，其包含两个scFv，具有两个抗原结合位点(二价)，其中每个scFv中的VH和VL之间通过短肽接头(大约5-10个氨基酸残基)连接，使得VH和VL链间配对(即第一scFv的VH和第二scFv的VL配对，第一scFv的VL和第二scFv的VH配对)形成抗原结合位点。双抗体可以是双特异性抗体。
如本文所用，抗体的“抗原结合片段”指全长抗体的部分，其少于全长，但是至少包含全长抗体的部分可变区(例如包含一个或多个CDR和/或一个或多个抗原结合位点)，并因此保留全长抗体的至少部分特异性结合抗原的能力。抗原结合片段可以例如包括通过酶促处理全长抗体所产生的抗体衍生物、合成产生的衍生物、重组产生的衍生物。抗原结合片段的实例包括但不限于Fv、scFv、dsFv、scdsFv、Fab、scFab、Fab'、F(ab')2、双抗体、Fd和Fd'片段以及其他片段(例如包含修饰的片段)。抗原结合片段可以包含多条肽链，例如通过二硫键和/或通过肽接头连接和/或非共价作用构成。
如本文使用，术语“单克隆抗体”是指一群高度均质的抗体，其中所包含的抗体分子之间除了可能少量存在的可能天然发生的突变之外是基本相同的。单克隆抗体通常特异性结合单一的抗原表位。本文所述单克隆抗体可以通过本领域已知的任何方法制备，例如从转基因动物中产生(例如转基因小鼠)、由永生化的B细胞(例如B细胞杂交瘤)产生，或者使用重组DNA方法在细菌、真核动物或植物细胞中制备，或者从噬菌体抗体文库中分离。本发明的抗体或抗原结合片段为单克隆抗体。在一实施方案中，本发明的抗体或其抗原结合片段为人源化单克隆抗体。
“嵌合抗体”指这样的抗体，其中的一部分(例如CDR、FR、可变区、恒定区或其组合)与衍生自特定物种的抗体中相应序列相同或同源，剩余的部分与衍生自另一物种的抗体中相应序列相同或同源。在本文中，“嵌合抗体”还涵盖包含属于不同抗体类型或亚类的部分的抗体。在一具体实施方案中，所述嵌合抗体具有鼠源抗体可变区和人源抗体恒定区。
如本文所用，“人源化抗体”指含有非人源抗体和人源抗体序列的抗体。因此，人源化抗体是含有衍生自非人免疫球蛋白最小序列的嵌合抗体。非人抗体可以是来源于任何非人物种的抗体或其中包含非人物种来源的部分的抗体(例如嵌合抗体)。非人物种例如可以包括小鼠、大鼠、兔、羊驼或非人灵长类动物。由非人抗体获得人源化抗体的技术是本领域技术人员熟知的。人源化抗体可以由非人抗体(例如鼠源抗体或嵌合抗体)产生，例如，将非人抗体(例如鼠源抗体)的CDR序列移植到人抗体框架区中。在某些情况下，为了保持人源化抗体的抗原结合能力和/或稳定性，可以在人抗体框架区中保留非人抗体(例如鼠源抗体)框架序列的关键氨基酸残基，即进行“回复突变”(参见，例如Morrison et al.(1984)Proc.Natl.Acad.Sci.81(21):6851-6855；Neuberger et al.(1984)Nature 312:604-608)。
如本文所用，氨基酸序列的“百分比(％)序列同一性”、“序列同一性”具有本领域公认的定义，其指通过序列比对(例如通过人工检视或可公知的算法)确定的两个多肽序列之间相同的百分比。可以使用本领域技术人员已知的方法确定，例如使用可公开获得的计算机 软件如BLAST、BLAST-2、Clustal Omega、FASTA软件。
“亲和力”或“结合亲和力”用来衡量抗体和抗原之间通过非共价作用相互结合的强度。“亲和力”的大小通常可以报告为平衡解离常数KD或EC50。KD可以通过测量平衡缔合常数(ka)和平衡解离常数(kd)来计算：KD＝kd/ka。可以用本领域已知的常规技术测定亲和力，例如生物膜干涉技术(可以采用例如Octet Fortebio检测系统)、放射免疫法、表面等离子共振法、酶联免疫测定或流式细胞术等。
在本文中，抗体和抗原“特异性结合”是指抗体和抗原之间以较高的亲和力相互结合。通常，特异性结合的两个抗体和抗原之间的KD值为至少约10-6到至少约10-9M或更低，例如至少约10-6、至少约10-7、至少约10-8、至少约10-9M或更低。
在本文中，“源自”或“衍生自”参考氨基酸序列的氨基酸序列与所述参考氨基酸序列部分或者全部相同或同源。例如，衍生自人免疫球蛋白的重链恒定区的氨基酸序列与其所源自的人免疫球蛋白重链恒定区的野生型序列具有至少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。
在本文中，多肽或氨基酸序列的“变体”与其所源自的多肽或氨基酸序列相比，具有一个或多个氨基酸突变或修饰。氨基酸突变包括氨基酸的置换、缺失或添加。本领域技术人员应当理解多肽或蛋白中非必需区中的氨基酸可以用合适的保守氨基酸置换，并且一般不改变其生物活性(参见，例如Watson et al.,Molecular Biology of the Gene,4th Edition,1987,The Benjamin/Cummings Pub.co.,p.224)。合适的保守置换是本领域技术人员熟知的。在下表中列出一些常见的氨基酸残基保守置换的非限制性实例。在某些情况下，氨基酸置换是非保守置换。本领域即是人员应当理解，可以对抗体或抗体片段进行氨基酸突变或修饰来改变其性能，例如改变抗体糖基化修饰的类型，改变形成链间二硫键的能力。包含这类氨基酸突变或修饰的抗体或其抗原结合片段也涵盖在本发明的抗体或其抗原结合片段的范围之内。

在本文中，术语“多核苷酸”、“核酸”和“核酸分子”指包含至少两个连接的核苷酸或核苷酸衍生物的寡聚体或聚合物，通常可以包括脱氧核糖核酸(DNA)和核糖核酸(RNA)。
如本文所用，“分离的”是指物质(例如核酸分子或多肽)与其存在的来源或环境是分离的，即基本上不包含其他任何成分。
在本文中，“载体”是用于将外源核酸导入宿主细胞的媒介，当载体转化入适当的宿主细胞时，外源核酸得以扩增或表达。载体通常保持游离，但是可以设计为使基因或其部分整合入基因组的染色体。如本文所用，载体的定义涵盖质粒、线性化质粒、病毒载体、粘粒、噬菌体载体、噬菌粒、人工染色体(例如，酵母人工染色体和哺乳动物人工染色体)等。
如本文所用，“表达载体”指能够表达DNA的载体，所述DNA与能够影响DNA表达的调控序列(如启动子、核糖体结合位点)可操作地连接。调控序列可以包含启动子和终止子序列，并且任选地可以包含复制起点、选择标记、增强子、多腺苷酸化信号等。表达载体可以是质粒、噬菌体载体、重组病毒或其他载体，当引入适当的宿主细胞时，导致克隆DNA的表达。适当的表达载体是本领域技术人员公知的，并且包含在真核细胞和/或原核细胞中可复制的表达载体以及保持游离的表达载体或者整合入宿主细胞基因组的表达载体。
如本文所用，“重组细胞”是用于接受、保持、复制或扩增载体的细胞。重组细胞还可以用来表达核酸或载体所编码的多肽。重组细胞可以是真核细胞或原核细胞。
如本文所用，术语“治疗”指对疾病/症状的改善，例如使疾病/症状减轻或消失、防止或减缓疾病/症状的发生、进展和/或恶化。因此，治疗包括预防、治疗和/或治愈。
“有效量”是指这样的剂量，其足以使疾病症状的严重性降低，疾病无症状期的频率和持续时间增加，或者防止因疾病痛苦而引起的损伤或失能。“有效量”指防止、治愈、改善、阻滞或部分阻滞疾病或症状所需的量。例如，对于肿瘤的治疗，相对于未接受治疗的对象，“有效量”的本发明的抗体或其抗原结合片段、或药物组合物优选地将肿瘤细胞生长或肿瘤生长抑制至少约10％，优选至少约20％，更优选至少约30％，更优选至少约40％，更优选至少约50％，更优选至少约60％，更优选至少约70％，更优选至少约80％。抑制肿瘤生长的效力可以利用本领域常规的肿瘤动物模型评估，例如自发性肿瘤、诱发性肿瘤、移植性肿瘤动物模型。或者，也可以利用本领域公知的体外检测方法检查抑制细胞生长的能力。有效量的本发明的抗体或其抗原结合片段、或药物组合物能够减小肿瘤大小，或以其他方式缓解对象的症状(如预防和/或治疗转移或复发)。本领域技术人员可以根据例如对象的年龄、身体状况、性别、症状的严重程度、特定组合物或给药途径等因素来确定有效量。有效量可以在一次或多次施用中给予。
如本文中所使用的，术语“药学上可接受的赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂、表面活性剂、佐剂、离子强度增强剂、稀释剂、维持渗透压的试剂、延迟吸收的试剂、防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子、阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯、三氯叔丁醇、苯酚、山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水、水性缓冲液(如缓冲盐水)、醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞、2-苯氧乙醇、对羟苯甲酸酯、三氯叔丁醇、苯酚、山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠、明胶、SPGA、糖类(如山梨醇、甘露醇、淀粉、蔗糖、乳糖、葡聚糖、或葡萄糖)、氨基酸(如谷氨酸、甘氨酸)、蛋白质(如干燥乳清、白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。
如本文所用，哺乳动物的实例包括但不限于人、非人灵长类动物、大鼠、小鼠、牛、马、猪、羊、狗、猫等。在本文中，术语“对象”是指哺乳动物，例如人。在一些实施方案中，对象是人。在一些实施方案中，对象是癌症患者、怀疑患有癌症或处于患癌风险中的人或者动物。在本文中，术语“对象”和“受试者”可以互换使用。
附图说明
此处所说明的附图用来提供对本发明的进一步理解，构成本申请的一部分，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1显示本申请所述多特异性抗体结构示意图。
图2显示在过表达HER2的细胞N87和CT26上检测本发明所述多特异性抗体与人HER2的结合情况。
图3显示在过表达人4-1BB的细胞CHO-h4-1BB上检测本发明所述多特异性抗体与人4-1BB的结合情况。
图4显示本发明所述多特异性抗体与FcR的结合情况。
图5显示本发明所述多特异性抗体阻断HER2信号依赖的细胞N87或SK-OV-3的增殖。
图6显示本发明所述多特异性抗体通过HER2介导ADCC作用。
图7显示HER2介导本发明所述多特异性抗体诱导Primary T cell释放细胞因子IL-2、IFN-γ。
图8显示FcR介导本发明所述多特异性抗体诱导primary T细胞释放细胞因子IL-2、IFN-γ。
图9显示本发明所述多特异性抗体HER2xHER2x4-1BB的SEC以及LC/MS检测结果。
图10显示本发明所述多特异性抗体在h-4-1BB KI BALB/c小鼠皮下接种CT26-h-HER2的肿瘤模型中对肿瘤抑制活性，图10A：给药后0-15天对肿瘤体积影响，图10B：小鼠肿瘤内CD8+T细胞浸润情况。
图11显示本发明所述多特异性抗体在h-4-1BB KI BALB/c小鼠皮下接种CT26-h-HER2的肿瘤模型中对肿瘤抑制活性(给药后11-27天对肿瘤体积影响)。
图12显示本发明所述多特异性抗体在h-4-1BB KI C57小鼠皮下接种MC38-h-HER2 的肿瘤模型中的抗肿瘤活性。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。以下对至少一个示例性实施例的描述实际上仅仅是说明性的，绝不作为对本发明及其应用或使用的任何限制。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
序列信息简表













实施例1.多特异性抗体的克隆和表达
在本实施例中，构建了3种HER2x4-1BB双特异性抗体，2种抗HER2xHER2x4-1BB三特异性抗体和2种4-1BB单克隆抗体，分别为：
HER2x4-1BB-1:由2条多肽链组成，其结构示意图如图1所示，肽链#1具有SEQ ID NO:21所示的氨基酸序列，其包含抗HER2的单克隆抗体Trastuzumab(专利申请号：WO1992022653A1)的重链可变区氨基酸序列(SEQ ID NO:1)以及人IgG1氨基酸序列(SEQ ID NO:10)。将抗CD137的单域抗体AB24 ME的CD137结合区域氨基酸序列(SEQ ID NO:7)的N端通过20个氨基酸残基(G4A)4(SEQ ID NO:18)的柔性肽连接于Fc的C端。肽链#2具有SEQ ID NO:22所示的氨基酸序列，其包含抗HER2的单克隆抗体Trastuzumab(专利申请号：WO1992022653A1)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:9)。
HER2x4-1BB-2:由2条多肽链组成，其结构示意图如图1所示，肽链#1具有SEQ ID NO:23所示的氨基酸序列，其包含抗HER2的单克隆抗体Trastuzumab(专利申请号：WO1992022653A1)的重链可变区氨基酸序列(SEQ ID NO:1)以及人IgG1氨基酸序列(引入LALA突变以降低Fc功能，SEQ ID NO:11)。将抗CD137的单域抗体AB24 ME的CD137结合区域氨基酸序列(SEQ ID NO:7)的N端通过20个氨基酸残基(G4A)4(SEQ ID NO:18)的柔性肽连接于Fc的C端。肽链#2具有SEQ ID NO:24所示的氨基酸序列，其包含抗HER2的单克隆抗体Trastuzumab(专利申请号：WO1992022653A1)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:9)。
HER2x4-1BB-3:由2条多肽链组成，其结构示意图如图1所示，肽链#1具有SEQ ID NO:25所示的氨基酸序列，其包含抗HER2的单克隆抗体Pertuzumab(专利申请号：US7449184)的重链可变区氨基酸序列(SEQ ID NO:3)以及人IgG1氨基酸序列(SEQ ID NO:12)。将抗CD137的单域抗体AB24 ME的CD137结合区域氨基酸序列(SEQ ID NO:7)的N端通过20个氨基酸残基(G4A)4(SEQ ID NO:18)的柔性肽连接于Fc的C端。肽链#2具有SEQ ID NO:26所示的氨基酸序列，其包含抗HER2的单克隆抗体Pertuzumab(专利申请号：US7449184)的轻链可变区氨基酸序列(SEQ ID NO:4)，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:9)。
HER2xHER2x4-1BB-1:由4条多肽链组成，其结构示意图如图1所示，肽链#1具有SEQ ID NO:27所示的氨基酸序列，其包含抗HER2的单克隆抗体的重链可变区氨基酸序 列(SEQ ID NO:5)以及人IgG1氨基酸序列引入CH3 Knob突变和CH1/CL偏好性突变CH SET1(专利申请号：WO2021067404A2，SEQ ID NO:13)。将抗CD137的单域抗体AB24 ME的CD137结合区域氨基酸序列(SEQ ID NO:7)的N端通过20个氨基酸残基(G4A)4(SEQ ID NO:18)的柔性肽连接于Fc的C端。肽链#2具有SEQ ID NO:28所示的氨基酸序列，其包含抗HER2的单克隆抗体的轻链可变区氨基酸序列(SEQ ID NO:6)，以及在所述VL氨基酸序列C端的人κ轻链恒定区(CL)氨基酸序列引入CH1/CL偏好性突变CL SET1(专利申请号：WO2021067404A2，SEQ ID NO:15)。肽链#3具有SEQ ID NO:29所示的氨基酸序列，其包含抗HER2的单克隆抗体Pertuzumab(专利申请号：US7449184)的重链可变区氨基酸序列(SEQ ID NO:3)以及人IgG1氨基酸序列引入CH3 Hole突变和CH1/CL偏好性突变CH SET2(专利申请号：WO2021067404A2，SEQ ID NO:14)。将抗CD137的单域抗体AB24 ME的CD137结合区域氨基酸序列(SEQ ID NO:7)的N端通过20个氨基酸残基(G4A)4(SEQ ID NO:18)的柔性肽连接于Fc的C端。肽链#4具有SEQ ID NO:30所示的氨基酸序列，其包含抗HER2的单克隆抗体Pertuzumab(专利申请号：US7449184)的轻链可变区氨基酸序列(SEQ ID NO:4)，以及在所述VL氨基酸序列C端的人κ轻链恒定区(CL)氨基酸序列引入CH1/CL偏好性突变CL SET2(专利申请号：WO2021067404A2，SEQ ID NO:16)。
HER2xHER2x4-1BB-2:由4条多肽链组成，其结构示意图如图1所示，肽链#1按照专利(专利申请号：201611016435.0)中描述的方法合成具有SEQ ID NO:39所示的氨基酸序列，包含抗HER2的单克隆抗体Trastuzumab(专利申请号：WO1992022653A1)的重链可变区氨基酸序列(SEQ ID NO:1)及可以自发形成异源二聚体的人IgG1氨基酸序列HC-1(SEQ ID NO:37，专利申请号：PCT/CN2017/111310)。将抗CD137的单域抗体AB24 ME的CD137结合区域氨基酸序列(SEQ ID NO:7)的N端通过20个氨基酸残基(G4A)4(SEQ ID NO:18)的柔性肽连接于Fc的C端。肽链#2具有SEQ ID NO:40所示的氨基酸序列，其包含抗HER2的单克隆抗体Trastuzumab(专利申请号：WO1992022653A1)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:9)。肽链#3按照专利(专利申请号：201611016435.0)中描述的方法合成具有SEQ ID NO:41所示的氨基酸序列，包含抗HER2的单克隆抗体Pertuzumab(专利申请号：US7449184)的重链可变区氨基酸序列(SEQ ID NO:3)及可以自发形成异源二聚体的人IgG1氨基酸序列HC-2(SEQ ID NO:38，专利申请号：PCT/CN2017/111310)。将抗CD137的单域抗体AB24 ME的CD137结合区域氨基酸序列(SEQ ID NO:7)的N端通过20个氨基酸残基(G4A)4(SEQ ID NO:18)的 柔性肽连接于Fc的C端。肽链#4具有SEQ ID NO:42所示的氨基酸序列，其包含抗HER2的单克隆抗体Pertuzumab(专利申请号：US7449184)的轻链可变区氨基酸序列(SEQ ID NO:4)，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:9)。
Anti-4-1BB-VHH-1:由2条多肽链组成，具有SEQ ID NO:31所示的氨基酸序列，其包含人IgG1 Fc氨基酸序列(SEQ ID NO:19)以及将抗CD137的单域抗体AB24 ME的CD137结合区域氨基酸序列(SEQ ID NO:7)的N端通过21个氨基酸残基(G4S)4G(SEQ ID NO:17)的柔性肽连接于Fc的C端。
Anti-4-1BB-VHH-2:由2条多肽链组成，具有SEQ ID NO:32所示的氨基酸序列，其包含人IgG1 Fc氨基酸序列(引入LALA突变以降低Fc功能，SEQ ID NO:20)以及将抗CD137的单域抗体AB24 ME的CD137结合区域氨基酸序列(SEQ ID NO:7)的N端通过21个氨基酸残基(G4S)4G(SEQ ID NO:17)的柔性肽连接于Fc的C端。
在本实施例中，构建了阳性对照分子Urelumab和PRS343：
Urelumab:对照分子Urelumab(专利申请号：WO2004010947)由2条多肽链组成，肽链#1具有SEQ ID NO:33所示的氨基酸序列，肽链#2具有SEQ ID NO:34所示的氨基酸序列。
PRS-343:对照分子PRS-343(专利申请号：WO2016177802A1)由2条多肽链组成，肽链#1具有SEQ ID NO:35所示的氨基酸序列，肽链#2具有SEQ ID NO:36所示的氨基酸序列。
实施例2.多特异性抗体与人HER2结合
本实验将扩大培养的N87细胞(自身表达HER2)和CT26-hHER2(过表达人HER2)细胞用0.25％EDTA trypsin消化，用培养基清洗一次后调整细胞密度至2×106细胞/ml，100μl/孔加入96孔流式板，离心备用。将梯度稀释后的抗体按100μl/孔加入上述带有细胞的96孔流式板中，4℃孵育60min。PBS清洗两次后，100μl/孔加入用2％BSA溶液稀释1000倍的Goat anti-human IgG-Fc(PE)(Abcam,ab98596)，4℃孵育60min。PBS清洗两次，最后按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)。
实验结果如图2和表1-2所示，本发明的HER2xHER2x4-1BB三特异性抗体和HER2x4-1BB双特异性抗体与人胃癌细胞N87(图2A)和过表达人HER2的CT26细胞(图 2B)上表达的HER2均结合，且结合活性与HER2单抗(Trastuzumab或Pertuzumab)相当。
表1：多特异性抗体与人胃癌细胞N87上过表达的HER2的结合情况
表2：多特异性抗体与CT26上过表达的人HER2的结合情况
实施例3.多特异性抗体与人4-1BB结合
本实验将扩大培养的CHOS-h4-1BB(过表达人4-1BB)细胞调整细胞密度至2×106细胞/ml，100μl/孔加入96孔流式板，离心备用。将梯度稀释后的抗体按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2％BSA 溶液稀释1000倍的Goat anti-human IgG-Fc(PE)(Abcam,ab98596)，4℃孵育60min。PBS清洗两次，最后按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。
实验结果如图3和表3所示，本发明的HER2xHER2x4-1BB三特异性抗体及HER2x4-1BB双特异性抗体与CHOS-h4-1BB细胞均有结合活性，且结合活性与4-1BB的单抗分子(anti-4-1BB VHH)相当，弱于对照抗体Urelumab和PRS-343。
表3：多特异性抗体与CHOS细胞上过表达的人4-1BB的结合情况
实施例4.多特异性抗体与人FcR结合
本实验将扩大培养的细胞THP-1(自身表达FcRs)和CHOS-hCD32b(过表达人FcγRIIb)细胞调整细胞密度至2×106细胞/ml，100μl/孔加入96孔流式板，离心备用。将梯度稀释后的抗体按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2％BSA溶液稀释1000倍的Goat anti-human IgG-Fc(PE)(Abcam,ab98596)，4℃孵育60min。PBS清洗两次，最后按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)。
实验结果如图4和表4-5所示，本发明保留Fc effector功能的HER2xHER2x4-1BB三特异性抗体及HER2x4-1BB双特异性抗体与THP-1(自身表达FcRs,图4A)和CHOS-hCD32b(过表达人FcR,图4B)细胞上表达的FcγRIIb均结合。HER2xHER2x4-1BB三特异性抗体的Fc采用了Knob in Hole突变，因此其与FcR的结合活性略弱于采用了野生型人IgG1 Fc的HER2x4-1BB双特异性抗体和HER2单克隆抗体。Urelumab和PRS-343 的Fc分别采用的是野生型人IgG4Fc或人IgG4Fc携带FALA突变，因此其与FcR的亲和力要弱于保留了Fc effector功能的多特异性抗体。
表4：多特异性抗体与FcR的结合情况(THP-1)
表5：多特异性抗体与FcR的结合情况(CHOS-hCD32b)
实施例5.多特异性抗体阻断HER2信号依赖的细胞增殖
本实验将扩大培养的N87和SK-OV-3(自身表达HER2)细胞用0.25％EDTA trypsin消化，用培养基清洗一次后调整细胞密度至5×104细胞/ml，80μl/孔加入96孔板中，备用。将梯度稀释后的抗体按80μl/孔加入上述带有细胞的96孔板中，置于细胞培养箱中孵育3-5天。最后用Luminescent Cell Viability Assay(Promega,G7572)试剂盒显色后 用酶标仪收集化学发光信号。
结果如图5A和5B所示，多特异性抗体均能够明显抑制N87或SK-OV-3细胞的增殖，其中HER2xHER2x4-1BB三特异性抗体对细胞增殖抑制作用要明显强于HER2-4-1BB双特异性抗体或者HER2单克隆抗体。此外，HER2抗体对肿瘤细胞的抑制作用与细胞上HER2的表达量紧密相关，HER2表达量越高，抗体对细胞的增殖抑制作用更加明显。
实施例6.多特异性抗体诱导ADCC效应(报告基因)
本实验将扩大培养的N87(自身表达HER2)细胞和CHOS-h4-1BB(过表达人4-1BB)细胞按照3×104个/孔与1.2×105个/孔的NFAT Luciferase/Jurkat CD16a(过表达CD16a和NFAT-Luc)效应细胞混合接种至96孔细胞培养白底板中，随后将梯度稀释后的多特异性抗体加入96孔板中并混匀，置于细胞培养箱中孵育6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。
实验结果如图6A和6C和表6-7所示，保留Fc effector功能的多特异性抗体都能通过N87细胞上表达的HER2介导ADCC作用，从而激活Jurkat细胞上的CD16a-NFAT信号通路。相反地，通过LALA突变去除Fc effector功能的HER2x4-1BB-2双抗及在IgG4Fc基础上引入FALA突变的对照分子PRS-343均不能诱导ADCC效应。另外，当使用CHOS-h4-1BB细胞作为靶细胞时，如图6B结果表明，所有的多特异性抗体均没有观察到针对4-1BB细胞的ADCC效应。
表6：多特异性抗体诱导的ADCC效应(对应图6A)

表7：多特异性抗体诱导的ADCC效应(对应图6C)
实施例7.多特异性抗体诱导primary T细胞释放细胞因子(HER2+细胞介导)
将扩大培养的靶细胞N87(自身表达HER2)用0.25％EDTA trypsin消化，并按照每孔1×104个/孔接种至96孔培养板中(该培养板提前一天包被2μg/ml的anti-CD3抗体，按100μl/孔铺板)。收集提前一天复苏的PBMC，用T细胞分离试剂盒(Stemcell,17951)分离PBMC中的T细胞并按照每孔5×104个/孔加入靶细胞孔中，随后将梯度稀释后的多特异性抗体加入到细胞孔中共孵育48小时后收集上清液。上清中的IL-2和IFNγ水平用human IL-2 ELISA kit(Invitrogen,88-7025-77)和human IFNγELISA kit(Invitrogen,88-7316-77)试剂盒检测。
实验结果如图7所示，本发明的多特异性抗体均可在N87细胞存在的情况下诱导Primary T cell释放IL-2(图7A)或者IFN-γ(图7B),并且呈现剂量依赖性。此外，HER2xHER2x4-1BB三特异性抗体诱导的细胞因子释放能力要明显强于HER2x4-1BB双特异性抗体，也强于对照抗体PRS-343和Urelumab。
实施例8.多特异性抗体诱导primary T细胞释放细胞因子(FcR+细胞介导)
将扩大培养的靶细胞CHOS-hCD32b(过表达人FcγRIIb)离心计数，并按照每孔1×104个/孔接种至96孔培养板中(该培养板提前一天包被2μg/ml的anti-CD3抗体，按100μl/孔铺板)。收集提前一天复苏的PBMC，用T细胞分离试剂盒(Stemcell,17951)分离PBMC中的T细胞并按照每孔5×104个/孔加入靶细胞孔中，随后将梯度稀释后的多特异性抗体加入到细胞孔中共孵育48小时后收集上清液。上清中的IL-2和IFNγ水平用human IL-2 ELISA kit(Invitrogen,88-7025-77)和human IFNγELISA kit(Invitrogen,88-7316-77)试剂 盒检测。
实验结果如图8所示，本发明的多特异性抗体在CHOS-hCD32b细胞存在的情况下均不能诱导Primary T cell释放IL2(图8A)或者IFN-γ(图8B)。相反地，阳性对照抗体Urelumab能够通过FcR诱导T细胞活化并释放促炎因子。另外，阴性对照分子PRS-343由于与CHOS-hCD32b细胞不结合，因此同样不会通过FcR诱导Primary T cell释放细胞因子。
实施例9.细胞株的筛选
本实施例使用CH1/CL偏好性突变(专利号：WO2021/067404A2)以及Knob in hole技术，将抗HER2抗体和Pertuzumab抗体的重链可变区分别构建入CH1突变的CH SET1(SEQ ID NO:13)和CH SET2(SEQ ID NO:14)上且将抗CD137的单域抗体AB24 ME的CD137结合区域氨基酸序列(SEQ ID NO:7)的N端通过20个氨基酸残基(G4A)4(SEQ ID NO:18)的柔性肽连接于Fc的C端。抗HER2抗体和Pertuzumab抗体的轻链可变区分别构建入CL突变型的轻链恒定区上CL SET1(SEQ ID NO:15)和CL SET2(SEQ ID NO:16)上。构建了1+1非对称式抗HER2xHER2x4-1BB-1三特异性抗体细胞株。
细胞株构建
采用电击转染的方法将含有抗HER2抗体和抗4-1BB抗体的重链基因(SEQ ID NO:27)以及轻链基因(SEQ ID NO:28)的载体pCHO2.0-GS-Puro-H1-L1以及含有抗Pertuzuamb抗4-1BB抗体的重链基因(SEQ ID NO:29)以及轻链基因(SEQ ID NO:30)的载体pCHO2.0-GS-Puro-H2-L2共转入宿主细胞CHOS-ADP，利用Puromycin和MSX筛选压力对细胞进行加压筛选获得高产minipool，然后通过一轮有限稀释和单克隆鉴定，得到高产稳定的克隆细胞株。
细胞培养
以Dynamis AGT Medium为基础培养基，接种密度(1.0±0.2)×106cells/ml，培养至第3、5、7、9、11天分别流加5.0±0.5％(w/w)初始培养重量的7a补料，流加0.5±0.05％(w/w)初始培养重量的7b。溶氧设置40％，初始培养温度36.5℃，第4天降温至33.0℃。每天根据葡萄糖浓度检测结果，补加300g/kg葡萄糖浓缩液使细胞液中葡萄糖浓度达到6.0g/L，收获当天除外。培养至第14天或细胞活率低于80％时结束培养。在培养过程中，使用Vicell(Beckman公司)对细胞密度及活率进行检测，从第7天开始，每天使用Cedex(Roche公司)对抗体产量进行检测。
稳定细胞株产物质量鉴定
一步纯化方法同实施例1中单臂抗体的纯化方法。利用HPLC检测获得蛋白的纯度。HPLC方法如下，流动相：150mM Na2HPO4·12H2O，pH7.0。色谱条件：检测波长：280nm，柱温：25℃，流速：0.5ml/min，检测时间：30min，TSKgel G3000SWXL色谱柱。SEC结果如图9A显示，一步亲和纯化获得的双特异性抗体纯度为98.3％。
利用高效液相质谱检测获得蛋白的重轻链配对情况，使用仪器为液相系统Vanquish UHPLC(Thermo)、质谱仪Q Exactive(Thermo)及色谱柱Waters ACQUITY UPLC BEH C4,2.1mm×100mm。取样品50μg，加入超纯水稀释至25μl，离心取20μl样品至进样瓶，进样5μl，采用LC-MS分析完整分子量。色谱条件为：柱温：80℃；紫外检测波长：280nm；流速：0.3mL/min；流动相A：水溶液(含0.1％甲酸)；流动相B：乙腈溶液(含0.1％甲酸)。质谱参数为：ESI离子源：离子传输管温度320℃，电压3.8kV，气体流速36L/min；模式：正离子Full MS；分辨率：17500；扫描范围：600-4000m/z。结果如图9B和9C所示，纯化的三特异性抗体HER2xHER2x4-1BB-1的正确配对产物比例为>98％。
实施例10.多特异性抗体的肿瘤抑制活性研究
本实验在h-4-1BB KI BALB/c小鼠皮下接种CT26-h-HER2的肿瘤模型中对本发明的多特异性抗体的抗肿瘤活性进行概念验证。
首先采用皮下接种CT26-h-HER2细胞的方式建立荷瘤小鼠模型，待肿瘤体积长至约180mm3左右时进行分组，腹腔注射分别给予G1:PBS、G2:2.5mg/kg的PRS-343、G3:2mg/kg的Trastuzumab、G4:2.3mg/kg的HER2x4-1BB-1和G5:2.3mg/kg的HER2x4-1BB-2(所有组别采用相等的摩尔剂量)治疗，监测各组小鼠肿瘤体积和体重变化，监测频率为2-3天/次，连续监测2～3周，给药剂量和方式如表8。实验结束后，取小鼠的肿瘤进行免疫组化检测，比较不同组别的肿瘤内CD8+T细胞的浸润情况。
实验结果如图10A所示，保留Fc effector功能的双特异性抗体HER2x4-1BB-1的抗肿瘤活性明显优于LALA突变去除Fc effector功能的HER2x4-1BB-2双抗(TGI：73.2％vs26.6％),同时也强于对照抗体PRS-343(TGI：6.5％)和HER2单克隆抗体Trastuzumab(TGI：4.2％)。相同地，免疫组化检测的结果(图10B)也显示采用HER2x4-1BB-1双特异性抗体治疗后的小鼠肿瘤内的CD8+T细胞浸润明显强于对照抗体PRS-343，Trastuzumab以及HER2x4-1BB-2双抗。
表8：多特异性抗体的肿瘤抑制活性研究的给药方案
实施例11.多特异性抗体的肿瘤抑制活性研究
本实验在h-4-1BB KI BALB/c小鼠皮下接种CT26-h-HER2的肿瘤模型中测定本发明的多特异性抗体的抗肿瘤活性。
首先采用皮下接种h-HER2CT26细胞的方式建立荷瘤小鼠模型，待肿瘤体积长至约180mm3左右时进行分组，腹腔注射给予G1：PBS、G2：3mg/kg的Trastuzumab联合3mg/kg的Pertuzumab、G3：3.6mg/kg的PRS-343、G4：3mg/kg的Enhertu(DS8201)、G5：3.6mg/kg的HER2x4-1BB-1、G6：3.6mg/kg的HER2x4-1BB-3、G7：1.8mg/kg的HER2x4-1BB-1联合1.8mg/kg的HER2x4-1BB-3、G8：3.6mg/kg的HER2xHER2x4-1BB-2(所有组别采用相等的摩尔剂量)治疗，监测各组小鼠瘤体积和体重变化，监测频率均为2-3天/次，连续监测2～3周，给药剂量和方式如表9。
实验结果如图11所示，HER2xHER2x4-1BB三特异性抗体分子的抗肿瘤活性(TGI：85.7％)明显优于等摩尔剂量的对照抗体PRS-343(TGI：37.7％)、Enhertu(TGI：47.7％)及HER2单克隆抗体Trastuzumab和Pertuzumab的组合(TGI：39.5％)。此外，HER2xHER2x4-1BB三特异性抗体的抗肿瘤活性也明显强于HER2x4-1BB双特异性抗体HER2x4-1BB-1(TGI：66.9％)和HER2x4-1BB-3(TGI：59.7％)，与HER2x4-1BB双特异性抗体的联合治疗组(HER2x4-1BB-1+HER2x4-1BB-3，TGI：83.7％)的治疗效果相当。
表9：多特异性抗体的肿瘤抑制活性研究的给药方案

实施例12.多特异性抗体的肿瘤抑制活性研究
本实验在h-4-1BB KI C57小鼠皮下接种MC38-h-HER2的肿瘤模型中测定本发明的多特异性抗体的抗肿瘤活性。
首先采用皮下接种MC38-h-HER2细胞的方式建立荷瘤小鼠模型，待肿瘤体积长至约100mm3左右时进行分组，腹腔注射给予G1：PBS、G2：3mg/kg的Trastuzumab联合3mg/kg的Pertuzumab、G3：3.6mg/kg的PRS-343、G4：3mg/kg的Enhertu(DS8201)、G5：1.8mg/kg的HER2x4-1BB-1联合1.8mg/kg的HER2x4-1BB-3、G6：3.6mg/kg的HER2xHER2x4-1BB-1(所有组别采用相等的摩尔剂量)治疗，监测各组小鼠瘤体积和体重变化，监测频率均为3-6天/次，连续监测2～3周，给药剂量和方式如表10。
结果如图12所示，经HER2xHER2x4-1BB三特异性抗体治疗后的所有小鼠都达到了肿瘤完全消退，明显优于等摩尔剂量的对照抗体PRS-343(TGI：54.9％)及HER2单克隆抗体的联合用药(Trastuzumab+Pertuzumab，TGI：63.8％)。另外，在此肿瘤模型中，HER2x4-1BB双特异性抗体的联合治疗组(HER2x4-1BB-1+HER2x4-1BB-3)和Enhertu的治疗也都达到了肿瘤完全消退。
表10：多特异性抗体的肿瘤抑制活性研究的给药方案
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尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解，根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (20)

  1. 能够特异性结合4-1BB的单域抗体或其抗原结合片段，所述单域抗体包含：
    (a)CDR1，其具有：SEQ ID NO:43所示的序列，或与SEQ ID NO:43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个或3个氨基酸的置换、缺失或添加)的序列；
    (b)CDR2，其具有：如SEQ ID NO:44所示的序列，或与SEQ ID NO:44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个或3个氨基酸的置换、缺失或添加)的序列；以及
    (c)CDR3，其具有：如SEQ ID NO:45所示的序列，或与SEQ ID NO:45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个或3个氨基酸的置换、缺失或添加)的序列；
    优选地，所述的置换是保守置换；
    优选地，所述单域抗体或其抗原结合片段包含：如SEQ ID NO:43所示的CDR1、如SEQ ID NO:44所示的CDR2、以及如SEQ ID NO:45所示的CDR3。
  2. 权利要求1所述的单域抗体或其抗原结合片段，所述单域抗体包含选自下列的氨基酸序列：
    (i)如SEQ ID NO:7所示的序列；
    (ii)与SEQ ID NO:7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:7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，所述的置换是保守置换。
  3. 特异性结合4-1BB的多肽构建体，其包含权利要求1或2所述的单域抗体或其抗 原结合片段，以及免疫球蛋白Fc结构域；
    优选地，所述免疫球蛋白Fc结构域直接或通过肽接头连接至所述单域抗体或其抗原结合片段的N端和/或C端(例如C端)；优选地，所述肽接头具有(GmXn)lGm’所示结构，m、m’、n和l各自独立地选自0、1、2、3、4、5、6、7、8、9和10，X选自A和S；优选地，m选自1、2、3、4或5，n选自1和2，l选自1、2、3、4、5、6、7、8、9和10，m’选自0和1，X选自A和S；优选地，所述肽接头具有SEQ ID NO:17或18所示氨基酸序列；优选地，所述免疫球蛋白Fc结构域是IgG的Fc结构域(例如IgG1的Fc结构域，例如包含CH2和CH3)；
    优选地，所述免疫球蛋白Fc结构域包含SEQ ID NO:19所示的序列，或与其相比具有至少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，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个、4个或5个氨基酸置换、缺失或添加)的序列；
    优选地，所述免疫球蛋白Fc结构域包含SEQ ID NO:19或20所示的序列；
    优选地，所述多肽构建体含有如SEQ ID NO:31或32所示的氨基酸序列或由其组成。
  4. 一种多特异性抗体，其包含权利要求1或2所述的单域抗体或其抗原结合片段、或权利要求3所述的多肽构建体；
    优选地，所述多特异性抗体特异性结合4-1BB，并且额外地特异性结合一个或多个其它靶标；
    优选地，所述靶标为肿瘤抗原；
    优选地，所述肿瘤抗原选自下述的一个或多个：CD19、CD20、CD22、CD23、CD38、CD40、CD49、CD52、CD56、CD74、CD80、CD95、CD138、CS1/SLAMF7、KiR、Thy-1、Ly-6、Fas、APO-1、EGFR、HER2、CXCR4、HLA、GM1和DRD。
  5. 一种多特异性抗体，其包含对4-1BB特异的第一抗原结合结构域，和对HER2特异的第二抗原结合结构域，其中，
    所述第一抗原结合结构域包含权利要求1或2所述的抗体或其抗原结合片段；
    所述第二抗原结合结构域含有抗HER2抗体的重链可变区的至少一个CDR，和/或抗HER2抗体的轻链可变区的至少一个CDR；
    优选地，所述第二抗原结合结构域含有抗HER2抗体的重链可变区，和/或抗HER2抗体的轻链可变区；
    优选地，所述抗HER2抗体选自曲妥珠单抗、帕妥珠单抗及其变体；
    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)。
  6. 一种多特异性抗体，其包含对4-1BB特异的第一抗原结合结构域，和对HER2特异的第二抗原结合结构域；其中，
    所述第一抗原结合结构域是VHH；所述第二抗原结合结构域是Fab，所述多特异性抗体包含：
    (1)肽链I-A，其包含所述第二抗原结合结构域的轻链可变区和轻链恒定区(CL)；
    和，
    (2)肽链I-B，其包含所述第二抗原结合结构域的重链可变区，重链恒定区，和所述第一抗原结合结构域；优选地，所述肽链I-B从N端至C端包含相邻的所述第二抗原结合结构域的重链可变区和重链恒定区和所述第一抗原结合结构域；
    优选地，所述肽链I-A的CL能够与所述肽链I-B的重链恒定区CH1结构域形成二聚体；
    优选地，所述多特异性抗体包含两条所述肽链I-A以及两条所述肽链I-B；优选地，两条所述肽链I-B的重链恒定区形成二聚体；
    优选地，各结构域之间直接或通过肽接头连接；优选的，所述肽接头具有(GmXn)lGm’所示结构，m、m’、n和l各自独立地选自0、1、2、3、4、5、6、7、8、9和10，X选自A和S；优选地，m选自1、2、3、4或5，n选自1和2，l选自1、2、3、4、5、6、7、8、9和10，m’选自0和1，X选自A和S；优选地，所述肽接头具有SEQ ID NO:17或18所示氨基酸序列。
  7. 权利要求5或6所述的多特异性抗体，其特征在于下述的一项或多项：
    (i)所述第一抗原结合结构域含有如SEQ ID NO:7所示的氨基酸序列或由其组成；
    (ii)所述第二抗原结合结构域的重链可变区含有如SEQ ID NO:1、3或5所示的氨基酸序列或由其组成；
    (iii)所述第二抗原结合结构域的轻链可变区含有如SEQ ID NO:2、4或6所示的氨基酸序列或由其组成；
    优选地，所述肽链I-A含有如SEQ ID NO:22、24或26所示的氨基酸序列或由其组成；
    优选地，所述肽链I-B含有如SEQ ID NO:21、23或25所示的氨基酸序列或由其组成。
  8. 权利要求4-7任一项所述的多特异性抗体，其进一步含有对HER2特异的第三抗原结合结构域；
    优选地，所述第一抗原结合结构域是VHH；所述第二抗原结合结构域和第二抗原结合结构域是Fab，所述多特异性抗体包含：
    (1)肽链II-A，其包含所述第二抗原结合结构域的轻链可变区和轻链恒定区(CL)；
    (2)肽链II-B，其包含所述第二抗原结合结构域的重链可变区，重链恒定区，和所述第一抗原结合结构域；优选地，所述肽链II-B从N端至C端包含相邻的所述第二抗原结合结构域的重链可变区和重链恒定区和所述第一抗原结合结构域；
    (3)肽链II-C，其包含所述第三抗原结合结构域的轻链可变区和轻链恒定区(CL)；
    和，
    (4)肽链II-D，其包含所述第三抗原结合结构域的重链可变区，重链恒定区，和所述第一抗原结合结构域；优选地，所述肽链II-D从N端至C端包含相邻的所述第三抗原结合结构域的重链可变区和重链恒定区和所述第一抗原结合结构域；
    优选地，所述肽链II-A的CL能够与所述肽链II-B的重链恒定区CH1结构域形成二聚体；优选地，所述肽链II-C的CL能够与所述肽链II-D的重链恒定区CH1结构域形成二聚体；优选地，所述肽链II-B的重链恒定区含有如SEQ ID NO:13所示氨基酸序列，所述肽链II-D的重链恒定区含有如SEQ ID NO:14所示氨基酸序列；优选地，所述肽链 II-B的重链恒定区含有如SEQ ID NO:37所示氨基酸序列，所述肽链II-D的重链恒定区含有如SEQ ID NO:38所示氨基酸序列；
    优选地，所述多特异性抗体包含一条所述肽链II-A、一条所述肽链II-B、一条所述肽链II-C、以及一条所述肽链II-D；优选地，所述肽链II-B和II-D的重链恒定区形成二聚体；
    优选地，各结构域之间直接或通过肽接头连接；优选的，所述肽接头具有(GmXn)lGm’所示结构，m、m’、n和l各自独立地选自0、1、2、3、4、5、6、7、8、9和10，X选自A和S；优选地，m选自1、2、3、4或5，n选自1和2，l选自1、2、3、4、5、6、7、8、9和10，m’选自0和1，X选自A和S；优选地，所述肽接头具有SEQ ID NO:17或18所示氨基酸序列。
  9. 权利要求8所述的多特异性抗体，其特征在于下述的一项或多项：
    (i)所述第一抗原结合结构域含有如SEQ ID NO:7所示的氨基酸序列或由其组成；
    (ii)所述第二抗原结合结构域的重链可变区含有如SEQ ID NO:1、3或5所示的氨基酸序列或由其组成；
    (iii)所述第二抗原结合结构域的轻链可变区含有如SEQ ID NO:2、4或6所示的氨基酸序列或由其组成；
    (iv)所述第三抗原结合结构域的重链可变区含有如SEQ ID NO:1、3或5所示的氨基酸序列或由其组成；
    (v)所述第三抗原结合结构域的轻链可变区含有如SEQ ID NO:2、4或6所示的氨基酸序列或由其组成；
    优选地，所述肽链II-A含有如SEQ ID NO:28或40所示的氨基酸序列或由其组成；
    优选地，所述肽链II-B含有如SEQ ID NO:27或39所示的氨基酸序列或由其组成；
    优选地，所述肽链II-C含有如SEQ ID NO:30或42所示的氨基酸序列或由其组成；
    优选地，所述肽链II-D含有如SEQ ID NO:29或41所示的氨基酸序列或由其组成。
  10. 一种多核苷酸，其编码权利要求1-9任一项所述的抗体。
  11. 一种载体，其包括权利要求10所述的多核苷酸。
  12. 一种重组细胞，其包括权利要求10所述的多核苷酸或权利要求11所述的载体。
  13. 一种药物组合物，其含有权利要求1、2、4-9任一项所述的抗体或其抗原结合片段、权利要求3所述的多肽构建体、权利要求10所述的多核苷酸、权利要求11所述的载体或权利要求12所述的重组细胞，以及药学上可接受的赋形剂。
  14. 权利要求1、2、4-9任一项所述的抗体或其抗原结合片段、权利要求3所述的多肽构建体、权利要求10所述的多核苷酸、权利要求11所述的载体、权利要求12所述的重组细胞或权利要求13所述药物组合物在制备抗肿瘤药物中的用途；
    优选地，所述肿瘤过表达HER2；
    优选地，所述肿瘤选自乳腺癌、结肠癌、胃癌、肺癌(例如，肺鳞状细胞癌、小细胞肺癌、非小细胞肺癌、肺腺癌)、腹膜癌、皮肤癌、鳞状细胞癌、皮肤或眼球黑色素瘤、直肠癌、肛门附近的癌症、食道癌、小肠肿瘤、内分泌腺癌、甲状旁腺癌、肾上腺癌症、软组织肉瘤、尿道癌、慢性或急性白血病、淋巴细胞性淋巴瘤、肝癌、胃肠道癌、胰腺癌、胶质母细胞瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、肝细胞腺瘤、大肠癌、子宫内膜癌或子宫癌、唾液腺肿瘤、肾癌、宫颈癌、前列腺癌、外阴癌、甲状腺癌、头颈部癌、脑癌、胆道癌和胆囊癌；
    优选地，所述肿瘤为原发性或转移性肿瘤。
  15. 权利要求1、2、4-9任一项所述的抗体或其抗原结合片段、权利要求3所述的多肽构建体、权利要求10所述的多核苷酸、权利要求11所述的载体、权利要求12所述的重组细胞或权利要求13所述药物组合物，其用于抗肿瘤；
    优选地，所述肿瘤过表达HER2；
    优选地，所述肿瘤选自乳腺癌、结肠癌、胃癌、肺癌(例如，肺鳞状细胞癌、小细胞肺癌、非小细胞肺癌、肺腺癌)、腹膜癌、皮肤癌、鳞状细胞癌、皮肤或眼球黑色素瘤、直肠癌、肛门附近的癌症、食道癌、小肠肿瘤、内分泌腺癌、甲状旁腺癌、肾上腺癌症、 软组织肉瘤、尿道癌、慢性或急性白血病、淋巴细胞性淋巴瘤、肝癌、胃肠道癌、胰腺癌、胶质母细胞瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、肝细胞腺瘤、大肠癌、子宫内膜癌或子宫癌、唾液腺肿瘤、肾癌、宫颈癌、前列腺癌、外阴癌、甲状腺癌、头颈部癌、脑癌、胆道癌和胆囊癌；
    优选地，所述肿瘤为原发性或转移性肿瘤。
  16. 一种抗肿瘤方法，其包括向有此需要的受试者施用治疗有效量的权利要求1、2、4-9任一项所述的抗体或其抗原结合片段、权利要求3所述的多肽构建体、权利要求10所述的多核苷酸、权利要求11所述的载体、权利要求12所述的重组细胞或权利要求13所述药物组合物；
    优选地，所述肿瘤过表达HER2；
    优选地，所述肿瘤选自乳腺癌、结肠癌、胃癌、肺癌(例如，肺鳞状细胞癌、小细胞肺癌、非小细胞肺癌、肺腺癌)、腹膜癌、皮肤癌、鳞状细胞癌、皮肤或眼球黑色素瘤、直肠癌、肛门附近的癌症、食道癌、小肠肿瘤、内分泌腺癌、甲状旁腺癌、肾上腺癌症、软组织肉瘤、尿道癌、慢性或急性白血病、淋巴细胞性淋巴瘤、肝癌、胃肠道癌、胰腺癌、胶质母细胞瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、肝细胞腺瘤、大肠癌、子宫内膜癌或子宫癌、唾液腺肿瘤、肾癌、宫颈癌、前列腺癌、外阴癌、甲状腺癌、头颈部癌、脑癌、胆道癌和胆囊癌；
    优选地，所述肿瘤为原发性或转移性肿瘤。
  17. 一种缀合物，其包含权利要求1、2、4-9任一项所述的抗体或其抗原结合片段、或权利要求3所述的多肽构建体，以及与所述抗体或其抗原结合片段或多肽构建体连接的可检测的标记；
    优选地，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素。
  18. 一种试剂盒，其包括权利要求1、2、4-9任一项所述的抗体或其抗原结合片段、权利要求3所述的多肽构建体、或权利要求17所述缀合物；
    优选地，所述试剂盒还包含特异性识别所述抗体或其抗原结合片段或所述多肽构建体所特异性识别的抗原的第二抗体；任选地，所述第二抗体还包括可检测的标记，例如酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素。
  19. 用于检测4-1BB在样品中的存在或其水平的方法，其包括使用权利要求1、2、4-9任一项所述的抗体或其抗原结合片段、权利要求3所述的多肽构建体、或权利要求17所述缀合物；
    优选地，所述方法是免疫学检测，例如免疫印迹法、酶免疫测定法(例如ELISA)、化学发光免疫分析法、荧光免疫分析法或放射免疫测定法；
    优选地，所述方法包括使用所述抗体或其抗原结合片段或多肽构建体，并且所述方法还包括使用携带可检测的标记(例如酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素)的第二抗体来检测所述抗体或其抗原结合片段或所述多肽构建体与抗原的结合；
    优选地，所述方法包括：(1)将所述样品与所述抗体或其抗原结合片段接触；(2)检测抗原-抗体免疫复合物的形成或检测所述免疫复合物的量，所述免疫复合物的形成表明存在4-1BB或表达4-1BB的细胞。
  20. 权利要求1、2、4-9任一项所述的抗体或其抗原结合片段、权利要求3所述的多肽构建体、或权利要求17所述缀合物在制备检测试剂中的用途，所述检测试剂用于检测4-1BB在样品中的存在或其水平；
    优选地，所述检测试剂通过权利要求19所述的方法来检测4-1BB在样品中的存在或其水平；
    优选地，所述样品为来自受试者(例如哺乳动物，优选人或猴)的细胞样品(例如，肿瘤细胞)。
PCT/CN2023/119761 2022-09-20 2023-09-19 抗体及其在抗肿瘤中的应用 WO2024061223A1 (zh)

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