Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
• • 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
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
  • C07K2317/524—CH2 domain
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  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/71—Decreased effector function due to an Fc-modification
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • 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
  • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
  • G01N33/5759—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving compounds localised on the membrane of tumour or cancer cells

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.

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