Antibodies and their use in combating tumors - Patents.com

Abstract

Antibodies and their use in combating tumors are provided. Single-domain antibodies or antigen-binding fragments thereof capable of specifically binding to 4-1BB, and multispecific antibodies are provided. The antibodies have improved antitumor activity and reduced liver toxicity.

Description

Detailed Description of the Invention

[0001] This application is based on and claims priority to an application having CN application number 202211142594.0 and filing date September 20, 2022. The disclosure of the CN application is incorporated herein in its entirety.

[0002] Technical Field This application relates to the field of biotherapeutic technology, specifically to antibodies that target 4-1BB and HER2 and their use in tumor treatment.

[0003] Background technology 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 heterodimers with other ErbB family members to induce phosphorylation of tyrosine residues within the receptor's cytoplasmic domain, thereby activating multiple intracellular signaling pathways and promoting cancer cell proliferation and tumorigenesis. As one of the earliest targets used for tumor treatment, HER2-targeting therapeutics include monoclonal antibodies (mAbs such as trastuzumab and pertuzumab), tyrosine kinase inhibitors (TKIs such as neratinib and lapatinib), and antibody-drug conjugates (ADCs such as T-DM1). These drugs have been widely used in the treatment of breast or gastric cancers with HER2 overexpression and / or amplification. Despite the favorable clinical benefits of these HER2-targeted therapeutics, unmet medical needs persist, and new drugs must be continually developed to further improve clinical efficacy in patients with recurrent or metastatic disease.

[0004] The T cell costimulatory receptor TNFRSF9 (4-1BB) is a member of the TNF receptor family and is primarily expressed on activated T cells. When activated, it can enhance T cell effector activity and memory responses. Urelumab, a monoclonal antibody drug targeting 4-1BB, exerts its therapeutic effect by binding to and activating 4-1BB on T cells. Unfortunately, significant liver-related toxicity was observed during the clinical development of urelumab.

[0005] The development of antitumor drugs with better efficacy and higher safety remains an urgent issue to be solved in this field.

[0006] Contents of the invention This application discloses a single-domain antibody with high binding activity to 4-1BB, as well as a multispecific antibody targeting 4-1BB and HER2, developed based on this antibody. In particular, this application discloses a HER2xHER2x4-1BB trispecific antibody with unique antitumor activity and potent immune memory effects. The antibody is a 1+1 IgG1-like heterodimeric antibody composed of trastuzumab and pertuzumab, whose C-terminus is connected to an anti-4-1BB single-domain antibody (sdAb) via a G4S linker. The antibody retains Fc effector functions, which are important for the antitumor activity of trastuzumab and pertuzumab. Furthermore, the antibody has lower liver toxicity and improved safety.

[0007] Single Domain Antibodies In one aspect, the present application provides a single domain antibody or antigen-binding fragment thereof capable of specifically binding to 4-1BB, wherein the single domain antibody comprises: (a) a CDR1 having the sequence set forth in SEQ ID NO: 43 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to the sequence set forth in SEQ ID NO: 43; (b) a CDR2 having the sequence set forth in SEQ ID NO: 44 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to the sequence set forth in SEQ ID NO: 44; and (c) a CDR3 having the sequence set forth in SEQ ID NO: 45 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to the sequence set forth in SEQ ID NO: 45; The present invention provides a single domain antibody or antigen-binding fragment thereof, comprising:

[0008] In some embodiments, the single domain antibody or antigen-binding fragment thereof comprises a CDR1 set forth in SEQ ID NO:43, a CDR2 set forth in SEQ ID NO:44, and a CDR3 set forth in SEQ ID NO:45.

[0009] Single domain antibodies include: (i) the sequence set forth in SEQ ID NO: 7; (ii) a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequence set forth in SEQ ID NO: 7; or (iii) a sequence having 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 to the sequence set forth in SEQ ID NO: 7 The amino acid sequence is selected from the group consisting of:

[0010] In some embodiments, the substitutions are conservative substitutions.

[0011] The term "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 expressed upon activation of immune cells (both innate and adaptive immune cells). 4-1BB plays an important role in regulating the activity of various immune cells. As used herein, 4-1BB may be derived from a mammal, such as Homo sapiens (human) (NCBI accession number NP_001552.2).

[0012] In some embodiments, the single domain antibody specifically binds to the 4-1BB epitope CRD-4.

[0013] Polypeptide Constructs The present application also provides a polypeptide construct capable of specifically binding to 4-1BB, comprising the above-described single domain antibody or antigen-binding fragment thereof, and an immunoglobulin Fc domain.

[0014] In some embodiments, the immunoglobulin Fc domain is linked, directly or via a peptide linker, to the N-terminus and / or C-terminus (eg, the C-terminus) of the single domain antibody or antigen-binding fragment thereof.

[0015] In some embodiments, the peptide linker is m X n ) l G m’ wherein m, m', n, and l are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and X is selected from A and S; preferably, m is selected from 1, 2, 3, 4, and 5, n is selected from 1 and 2, l is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, m' is selected from 0 and 1, and X is selected from A and S.

[0016] In some embodiments, the peptide linker has the amino acid sequence set forth in SEQ ID NO: 17 or 18.

[0017] In some embodiments, the immunoglobulin Fc domain is an IgG Fc domain (eg, an IgG1 Fc domain including CH2 and CH3).

[0018] In some embodiments, the immunoglobulin Fc domain comprises the sequence set forth in SEQ ID NO: 19, or a sequence having 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 thereto, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) thereto.

[0019] In some embodiments, the immunoglobulin Fc domain comprises the sequence set forth in SEQ ID NO: 19 or 20.

[0020] In some embodiments, the polypeptide construct comprises or consists of the amino acid sequence set forth in SEQ ID NO:31 or 32.

[0021] multispecific antibodies The present application further provides a multispecific antibody that can activate 4-1BB signaling only when crosslinked with HER2-expressing tumor cells. Furthermore, the anti-4-1BB antibody or antigen-binding fragment thereof contained in the multispecific antibody may be characterized by localization and / or activation only in the tumor microenvironment (TME) and / or significantly lower liver toxicity compared to existing anti-4-1BB antibodies, while maintaining immune response enhancement and / or tumor treatment efficacy.

[0022] In particular, the present application provides multispecific antibodies, including single domain antibodies or antigen-binding fragments thereof, or polypeptide constructs as described above.

[0023] The present application further provides a multispecific antibody comprising a first antigen-binding domain specific for 4-1BB, wherein the first antigen-binding domain comprises the antibody or antigen-binding fragment thereof described above.

[0024] In some embodiments, the first antigen-binding domain comprises an antibody or antigen-binding fragment thereof described above.

[0025] In some embodiments, any of the above multispecific antibodies specifically binds to 4-1BB and also specifically binds to one or more other targets.

[0026] In some embodiments, the target is a tumor antigen.

[0027] In some embodiments, the tumor antigen is one or more selected from 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.

[0028] In some embodiments, the multispecific antibody is capable of binding to multiple identical or different tumor antigens.

[0029] In some embodiments, multispecific antibodies can bind to the same or different epitopes on the same tumor antigen.

[0030] In some embodiments, the multispecific antibody is capable of specifically binding to HER2.

[0031] "HER2 (human epidermal growth factor receptor 2)" is encoded by the ErbB2 gene and is a member of the epidermal growth factor receptor (EGFR / ErbB) family. HER2 is known to play a central role in regulating cell proliferation and differentiation. In particular, upon binding to extracellular growth factors, it exhibits a pronounced tendency to undergo homodimerization and / or heterodimerization with other HER receptors, leading to the activation of several forms of signal transduction pathways and the induction of apoptosis, survival, or cell proliferation. For example, the HER2 protein may be a polypeptide deposited under GenBank accession numbers NP_004439.2 or NP_001005862.1, etc., encoded by a nucleotide sequence (mRNA) deposited under GenBank accession numbers NM_004448.4, NM_001005862.3, etc., respectively.

[0032] The fragment that binds to HER2 and recognizes HER2 as an antigen can be selected from scFv, (scFv)2, Fab, Fab' and F(ab')2 of an anti-HER2 antibody.

[0033] In some embodiments, the multispecific antibody comprises at least one (e.g., one, two, or three) CDRs of the heavy chain variable region of an anti-HER2 antibody and / or at least one (e.g., one, two, or three) CDRs of the light chain variable region of an anti-HER2 antibody.

[0034] In some embodiments, the multispecific antibody comprises the heavy chain variable region of an anti-HER2 antibody and / or the light chain variable region of an anti-HER2 antibody.

[0035] In some embodiments, the anti-HER2 antibody is selected from trastuzumab, pertuzumab, and variants thereof.

[0036] In some embodiments, a 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, or additions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the wild-type sequence from which it is derived.

[0037] In some embodiments, the anti-HER2 antibody is a variant of trastuzumab. Specifically, the anti-HER2 antibody has the following three heavy chain variable region (VH) complementarity determining regions (CDRs): a VH CDR1 set forth in SEQ ID NO: 46, a VH CDR2 set forth in SEQ ID NO: 47, and a VH CDR3 set forth in SEQ ID NO: 48; and / or The following three light chain variable region (VL) complementarity-determining regions (CDRs): It comprises a VL CDR1 set forth in SEQ ID NO:49, a VL CDR2 set forth in SEQ ID NO:50, and a VL CDR3 set forth in SEQ ID NO:8.

[0038] In some embodiments, the multispecific antibody comprises a second antigen-binding domain specific for HER2.

[0039] In some embodiments, the first antigen-binding domain is a VHH; the second antigen-binding domain is a Fab, and the multispecific antibody comprises: (1) a peptide chain IA comprising a light chain variable region and a light chain constant region (CL) of a second antigen-binding domain; and (2) A peptide chain IB comprising the heavy chain variable region and heavy chain constant region of the second antigen-binding domain and the first antigen-binding domain; preferably, a peptide chain IB comprising, from the N-terminus to the C-terminus, the adjacent heavy chain variable region and heavy chain constant region of the second antigen-binding domain and the first antigen-binding domain. Includes.

[0040] In some embodiments, the heavy chain constant region can be selected from (e.g., Fc region): the heavy chain constant region of IgG1, IgG2, IgG3, and IgG4, particularly the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, e.g., the heavy chain constant region of human IgG1, e.g., CH1, CH2 and / or CH3 of human IgG1, and e.g., the Fc region of human IgG1.

[0041] In some embodiments, the heavy chain constant region is altered (e.g., mutated) to increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function.

[0042] In some embodiments, the heavy chain constant region comprises a LALA mutation, a CH3 knob mutation, a CH3 hole mutation, a CH1 / CL selective mutation CH SET1, a CH1 / CL selective mutation CH SET2CH SET2, and any combination thereof.

[0043] In some embodiments, the heavy chain constant region comprises: HC-1: amino acid sequence set forth in SEQ ID NO: 10; HC-2: amino acid sequence set forth in SEQ ID NO: 11; HC-3: amino acid sequence set forth in SEQ ID NO: 12; HC-4: amino acid sequence set forth in SEQ ID NO: 13; HC-5: amino acid sequence set forth in SEQ ID NO: 14; HC-6: amino acid sequence set forth in SEQ ID NO: 19; HC-7: amino acid sequence set forth in SEQ ID NO: 20; HC-8: the amino acid sequence set forth in SEQ ID NO: 37; and HC-9: amino acid sequence set forth in SEQ ID NO: 38 selected from the group consisting of: A 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, or additions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the wild-type sequence from which it is derived.

[0044] In some embodiments, the light chain constant region is selected from a kappa or a lambda light chain constant region or a variant thereof.

[0045] A 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, or additions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the wild-type sequence from which it is derived.

[0046] In some embodiments, the light chain constant region is LC-1: SEQ ID NO: 9; LC-2: SEQ ID NO: 15; and LC-3: SEQ ID NO: 16 selected from the group consisting of: A 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, or additions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the wild-type sequence from which it is derived.

[0047] In some embodiments, the CL of peptide chain IA can dimerize with the heavy chain constant region CH1 domain of peptide chain IB.

[0048] In some embodiments, the multispecific antibody comprises two peptide chains IA and two peptide chains IB; preferably, the heavy chain constant regions of the two peptide chains IB form a dimer.

[0049] In some embodiments, the domains are connected directly or via a peptide linker.

[0050] In some embodiments, the peptide linker is m X n ) l G m’ wherein m, m', n, and l are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and X is selected from A and S; preferably, m is selected from 1, 2, 3, 4, and 5, n is selected from 1 and 2, l is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, m' is selected from 0 and 1, and X is selected from A and S.

[0051] In some embodiments, the peptide linker has the amino acid sequence set forth in SEQ ID NO: 17 or 18.

[0052] In some embodiments, the multispecific antibody comprises: (i) the first antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7; (ii) the heavy chain variable region of the second antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, 3, or 5; (iii) the light chain variable region of the second antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, 4 or 6; characterized by one or more of the following:

[0053] In some embodiments, peptide chain IA comprises or consists of the amino acid sequence set forth in SEQ ID NO:22, 24, or 26.

[0054] In some embodiments, peptide chain IB comprises or consists of the amino acid sequence set forth in SEQ ID NO:21, 23 or 25.

[0055] In some embodiments, the multispecific antibody comprises: (1) A multispecific antibody comprising peptide chains IA and IB, wherein peptide chain IA comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 and peptide chain IB comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21; (2) A multispecific antibody comprising peptide chains IA and IB, wherein peptide chain IA comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 and peptide chain IB comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and (3) a multispecific antibody comprising peptide chains IA and IB, wherein peptide chain IA comprises or consists of the amino acid sequence set forth in SEQ ID NO: 26 and peptide chain IB comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25; Preferably, the multispecific antibody comprises two peptide chains IA and two peptide chains IB; Preferably, the heavy chain constant regions of the two peptide chains IB form a dimer.

[0056] In some embodiments, the multispecific antibody further comprises a third antigen-binding domain specific for HER2.

[0057] In some embodiments, 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 (1) peptide chain II-A, which includes the light chain variable region and the light chain constant region (CL) of the second antigen-binding domain; (2) Peptide chain II-B, which comprises the heavy chain variable region and heavy chain constant region of the second antigen-binding domain and the first antigen-binding domain; preferably, peptide chain II-B, which comprises, from the N-terminus to the C-terminus, the adjacent heavy chain variable region and heavy chain constant region of the second antigen-binding domain and the first antigen-binding domain; (3) peptide chain II-C, which includes the light chain variable region and light chain constant region (CL) of the third antigen-binding domain; and (4) Peptide chain II-D, comprising the heavy chain variable region, heavy chain constant region, and first antigen-binding domain of the third antigen-binding domain; preferably, peptide chain II-D, comprising, from the N-terminus to the C-terminus, the adjacent heavy chain variable region and heavy chain constant region of the third antigen-binding domain, and the first antigen-binding domain. Includes.

[0058] In some embodiments, the heavy chain constant region can be selected from (e.g., Fc region): the heavy chain constant region of IgG1, IgG2, IgG3, and IgG4, specifically the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, e.g., the heavy chain constant region of human IgG1, e.g., the CH1, CH2 and / or CH3 of human IgG1, and in another example, the Fc region of human IgG1.

[0059] In some embodiments, the heavy chain constant region is altered (e.g., mutated) to increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function.

[0060] In some embodiments, the heavy chain constant region comprises a LALA mutation, a CH3 knob mutation, a CH3 hole mutation, a CH1 / CL selective mutation CH SET1, a CH1 / CL selective mutation CH SET2, and any combination thereof.

[0061] In some embodiments, the light chain constant region is selected from a kappa or a lambda light chain constant region or a variant thereof.

[0062] A 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, or additions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the wild-type sequence from which it is derived.

[0063] In some embodiments, the CL of peptide chain II-A can form a dimer with the CH1 domain of the heavy chain constant region of peptide chain II-B; preferably, the CL of peptide chain II-C can form a dimer with the CH1 domain of the heavy chain constant region of peptide chain II-D.

[0064] In some embodiments, the multispecific antibody comprises one peptide chain II-A, one peptide chain II-B, one peptide chain II-C, and one peptide chain II-D. In some embodiments, the heavy chain constant regions of peptide chains II-B and II-D form a dimer. In some embodiments, the heavy chain constant region of peptide chain II-B comprises the amino acid sequence set forth in SEQ ID NO: 13, and the heavy chain constant region of peptide chain II-D comprises the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the heavy chain constant region of peptide chain II-B comprises the amino acid sequence set forth in SEQ ID NO: 37, and the heavy chain constant region of peptide chain II-D comprises the amino acid sequence set forth in SEQ ID NO: 38.

[0065] In some embodiments, the domains are connected directly or via a peptide linker.

[0066] In some embodiments, the peptide linker is m X n ) l G m’ wherein m, m', n, and l are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and X is selected from A and S.

[0067] In some embodiments, m is selected from 1, 2, 3, 4, and 5; n is selected from 1 and 2; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; m′ is selected from 0 and 1; and X is selected from A and S.

[0068] In some embodiments, the peptide linker has the amino acid sequence set forth in SEQ ID NO: 17 or 18.

[0069] In some embodiments, the multispecific antibody has the following characteristics: (i) the first antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7; (ii) the heavy chain variable region of the second antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, 3, or 5; (iii) the light chain variable region of the second antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, 4, or 6; (iv) the heavy chain variable region of the third antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, 3, or 5; (v) the light chain variable region of the third antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, 4, or 6; characterized by one or more of the following:

[0070] In some embodiments, peptide chain II-A comprises or consists of the amino acid sequence set forth in SEQ ID NO:28 or 40.

[0071] In some embodiments, peptide chain II-B comprises or consists of the amino acid sequence set forth in SEQ ID NO: 27 or 39.

[0072] In some embodiments, peptide chain II-C comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30 or 42.

[0073] In some embodiments, peptide chain II-D comprises or consists of the amino acid sequence set forth in SEQ ID NO:29 or 41.

[0074] In some embodiments, the multispecific antibody comprises: (1) A multispecific antibody comprising peptide chains II-A, II-B, II-C, and II-D, wherein peptide chain II-A comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28, peptide chain II-B comprises or consists of the amino acid sequence set forth in SEQ ID NO: 27, peptide chain II-C comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29, and peptide chain II-D comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30; and (2) A multispecific antibody comprising peptide chains II-A, II-B, II-C, and II-D, wherein peptide chain II-A comprises or consists of the amino acid sequence set forth in SEQ ID NO: 40, peptide chain II-B comprises or consists of the amino acid sequence set forth in SEQ ID NO: 39, peptide chain II-C comprises or consists of the amino acid sequence set forth in SEQ ID NO: 42, and peptide chain II-D comprises or consists of the amino acid sequence set forth in SEQ ID NO: 41. is selected from.

[0075] Peptide Linker The N-terminus or C-terminus (preferably the N-terminus) of the 4-1BB-binding moiety described herein is linked, either directly or via a peptide linker, to the C-terminus or N-terminus of the heavy chain constant region.

[0076] As used herein, the term "peptide linker" may refer to an oligopeptide containing 1 to 100 amino acids, particularly 2 to 50 amino acids, each of which may be any type of amino acid without any limitation. Any conventional peptide linker, with or without appropriate modifications, may be used to fulfill a particular purpose. In certain embodiments, the peptide linker may contain, for example, Gly, Asn, and / or Ser residues, and / or neutral amino acids such as Thr and / or Ala. Suitable amino acid sequences for peptide linkers may be known in the relevant technical field. The length of the peptide linker may be appropriately determined within limits that do not affect the function of the polypeptide and / or scFv. For example, a peptide linker can be formed 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 independently selected from the group consisting of Gly, Asn, Ser, Thr, and Ala.

[0077] In some embodiments, the peptide linker is m X n ) l G m’ wherein m, m', n and l are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; and X is selected from A and S.

[0078] In some embodiments, m is selected from 1, 2, 3, 4, and 5; n is selected from 1 and 2; l is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; m′ is selected from 0 and 1; and X is selected from A and S.

[0079] In some embodiments, the peptide linker has the amino acid sequence set forth in SEQ ID NO: 17 or 18.

[0080] Preparation of polynucleotides, recombinant vectors and antibodies In one aspect, the invention provides a polynucleotide encoding an antibody or antigen-binding fragment thereof according to any one of the preceding items.

[0081] In one aspect, the present invention provides a vector comprising the above-described polynucleotide.

[0082] In one aspect, the present invention provides a recombinant cell comprising the polynucleotide or vector described above. The recombinant cell may be a cell transfected with the recombinant vector.

[0083] In one aspect, the present invention provides a method for preparing an antibody or antigen-binding fragment thereof, comprising expressing a polynucleotide in a cell. The step of expressing the polynucleotide can be carried out by culturing cells containing the polynucleotide (e.g., the polynucleotide is 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 after the expression or culturing step.

[0084] Applicable The present invention further provides the use of an antibody or antigen-binding fragment thereof described herein in enhancing an immune response and / or treating a tumor.

[0085] Specifically, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, polynucleotide, vector or recombinant cell described in any one of the above items, and a pharmaceutically acceptable excipient.

[0086] In another aspect, the present invention further provides the use of the antibody or antigen-binding fragment thereof, polynucleotide, vector, recombinant cell or pharmaceutical composition according to any one of the preceding items in the manufacture of an anti-tumor medicament.

[0087] In another aspect, the present invention provides an anti-tumor method comprising the step of administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof, polynucleotide, vector, recombinant cell, or pharmaceutical composition described in any one of the preceding items.

[0088] In some embodiments, the tumor overexpresses HER2.

[0089] In some embodiments, 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, cutaneous or ocular melanoma, rectal cancer, perianal cancer, esophageal cancer, small intestine tumors, endocrine gland cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral 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 tumor, kidney cancer, cervical cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain tumor, bile duct cancer, and gallbladder cancer.

[0090] In some embodiments, the tumor is a primary or metastatic tumor.

[0091] Use in detection In another aspect, the present invention also provides a conjugate comprising the antibody or antigen-binding fragment thereof, or polypeptide construct according to any one of the above aspects, and a detectable label attached to the single domain antibody or antigen-binding fragment thereof, or polypeptide construct.

[0092] In some embodiments, the detectable label is selected from the group consisting of an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., an acridinium ester, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin.

[0093] On the other hand, the present invention also provides a kit comprising a single domain antibody or antigen-binding fragment thereof or polypeptide construct, or a conjugate according to any one of the above aspects.

[0094] In certain embodiments, the kit further comprises a second antibody capable of specifically recognizing the antigen specifically recognized by the antibody or antigen-binding fragment thereof, or 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, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin.

[0095] In some embodiments, the second antibody targets the same or a different antigenic epitope as the antibody or antigen-binding fragment thereof or polypeptide construct.

[0096] On the other hand, the present invention also provides a method for detecting the presence or level of 4-1BB in a sample, comprising using an antibody or antigen-binding fragment thereof or polypeptide construct, or conjugate described in any one of the above aspects.

[0097] In certain embodiments, the method is an immunoassay, such as immunoblotting, enzyme immunoassay (eg, ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.

[0098] In certain embodiments, the method comprises using the conjugate described above.

[0099] In certain embodiments, the method comprises using the antibody or antigen-binding fragment thereof, or polypeptide construct described in any one of the above aspects, and the method further comprises using a second antibody bearing a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., an acridinium ester, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin) to detect binding of the single domain antibody or antigen-binding fragment thereof, or polypeptide construct to the antigen.

[0100] In certain embodiments, the method includes (1) contacting a sample with an antibody or antigen-binding fragment thereof; and (2) detecting the formation of an antigen-antibody immune complex or the amount of the immune complex, wherein the formation of the immune complex indicates the presence of 4-1BB or cells expressing 4-1BB.

[0101] On the other hand, the present application also provides the use of a single domain antibody or antigen-binding fragment thereof, or a polypeptide construct, or a conjugate described in any one of the above aspects in the manufacture of a detection reagent used to detect the presence or level of 4-1BB in a sample.

[0102] In certain embodiments, the detection reagent detects the presence or level of 4-1BB in a sample by the methods for detecting the presence or level of 4-1BB in a sample described above.

[0103] In certain embodiments, the sample is a cell sample (eg, tumor cells) derived from a subject (eg, a mammal, preferably a human or monkey).

[0104] definition As used herein, unless otherwise specified, the scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology terms and related terms used herein, as well as experimental operation steps, are all terms and routine steps that are widely used in the corresponding fields.At the same time, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

[0105] As used herein, "at least one" or "one or more" may refer to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more.

[0106] The expression "comprising" or its synonymous or similar expressions "including," "containing," and "having" is open and does not exclude additional, unrecited elements, steps, or ingredients. The expression "consisting of..." excludes any unspecified elements, steps, or ingredients. The expression "consisting of..." means that its scope is limited to the specified elements, steps, or ingredients, and to such elements, steps, or ingredients as are necessary that do not materially affect the basic and novel characteristics of the subject matter sought to be protected. The expression "comprising" should be understood to encompass the expressions "consisting essentially of..." and "consisting of...".

[0107] The term "optionally" or "optional" means that the event or circumstance described thereafter may or may not occur, and that the description includes the occurrence of the event or circumstance and the non-occurrence of the event or circumstance.

[0108] As used herein, "peptide" and "polypeptide" refer to a polymer formed by two or more amino acids linked by peptide bonds. A "protein" can be formed by one or more polypeptides in a covalent or non-covalent manner. Unless otherwise specified, the terms "polypeptide" and "protein" are used interchangeably herein.

[0109] As used herein, "antibody" refers to an immunoglobulin or fragment thereof that specifically binds to an antigen epitope through at least one antigen-binding site. As used herein, the definition of antibody encompasses antigen-binding fragments. Thus, the term "antibody" includes multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, single-domain antibodies, and antigen-binding fragments. Antibodies can be synthetic (e.g., produced by chemical or biological coupling), enzymatically, or recombinantly produced. The antibodies provided herein include any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b). In one embodiment, an antibody of the present invention is a murine monoclonal antibody. In another embodiment, an antibody of the present invention is a humanized monoclonal antibody.

[0110] As used herein, a "traditional antibody" or "full-length antibody" generally comprises four polypeptides: two heavy chains (HC) and two light chains (LC). Each light chain comprises, from N- to C-terminus, a "light chain variable region (VL)" and a "light chain constant region (CL)." Each heavy chain comprises, from N- to C-terminus, a "heavy chain variable region (VH)" and a "heavy chain constant region (CH)." The heavy chain constant region may comprise, from N- to C-terminus, CH1, CH2, and CH3. In some immunoglobulin types (e.g., IgM and IgE), the heavy chain constant region may also comprise CH4. An "Fc" fragment refers to the fragment comprising CH2 and CH3, which provides a site for binding to an Fc receptor. A "hinge region" refers to the portion of an antibody connecting the Fab and Fc fragments of an immunoglobulin. In some cases, the hinge region refers to the portion of a T-cell receptor connecting the constant region and the transmembrane domain. As used herein, when used in reference to a chimeric antigen receptor, the term "hinge region" may also refer to any functional equivalent. Those skilled in the art can determine the locations of the VH, VL, CL, CH1, CH2, CH3, and hinge regions in an antibody according to known algorithms and software. Descriptions of applicable algorithms and software can be found, for example, in William R. Strohl, Lila M. Strohl, (2012), Antibody structure-function relationships, Woodhead Publishing Series in Biomedicine, Therapeutic Antibody Engineering, Woodhead Publishing, pages 37-56.

[0111] Typically, each VL and VH may comprise three highly variable "complementarity-determining regions (CDRs)" and four relatively conserved "framework regions (FRs)," connected from the N-terminus to the C-terminus by the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. As used herein, the light chain variable region CDRs (CDRLs) may be referred to as CDR-L1, CDR-L2, and CDR-L3, and the heavy chain variable region CDRs (CDRHs) may be referred to as CDR-H1, CDR-H2, and CDR-H3. It is generally believed that the six CDRs comprising a VH and VL pair determine the binding specificity of the antigen-binding site; however, in some cases, other fragments (e.g., single-domain antibodies, also known as nanobodies) comprising fewer than six CDRs (e.g., three, four, or five) also have the ability to bind to antigens. Those skilled in the art can identify CDRs using methods well known to those skilled in the art, such as using Kabat, Abm, or Chothia numbering systems.As used herein, multiple CDR numbering systems, such as Chothia, Abm, Kabat, and IMGT, can be used for the same variable region.Those skilled in the art can understand that although the CDRs defined by different numbering systems may be different, the CDRs corresponding to the same numbering system represent an effective antigen binding site that can bind to an antigen epitope.For descriptions of CDR numbering systems, see, e.g., Kabat numbering system: Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department 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. Dubel (ed.), Handbook of Therapeutic Antibodies. Weinheim: Wiley-VCH Verlag, pp. 95-118.

[0112] As used herein, the terms "framework region" and "framework" can be used interchangeably. As used herein, the terms "framework region," "framework," or "FR" residues refer to amino acid residues in the variable region of an antibody other than the CDR residues defined above.

[0113] An "Fv" fragment, composed of a VH and a VL through non-covalent interactions, is generally considered to be the smallest antigen-binding fragment containing an antigen-binding site. However, single-domain antibodies (also known as VHHs, sdAbs, or nanobodies) containing only the heavy chain variable region can also have antigen-binding ability. VH and VL can be connected by a peptide linker to obtain a "single-chain Fv (scFv)." By introducing a disulfide bond into Fv or scFv, a "disulfide-stabilized Fv (dsFv)" or a "single-chain disulfide-stabilized Fv (scdsFv or dsscFv)" can be obtained, respectively. In some embodiments, for example, the antigen-binding fragments in the single-domain antibodies, bispecific antibodies, and multispecific antibodies of the present invention preferably use scFvs or scdsFvs.

[0114] As used herein, "Fab" comprises one intact antibody light chain (VL-CL) and one antibody heavy chain variable region and one heavy chain constant region (VH-CH1, also referred to as Fd). A single-chain "Fab (scFab)" can be obtained by connecting the CL and CH1 in "Fab" with a peptide linker. "F(ab')2" essentially comprises two Fab fragments connected by a disulfide bond in the hinge region. "Fab'" is half of F(ab')2 and can be obtained by reducing the disulfide bond in the hinge region of F(ab')2.

[0115] Antibodies or antigen-binding fragments may be "monovalent," "bivalent," "trivalent," or "tetravalent" or "multivalent," meaning that they have multiple (e.g., 1, 2, 3, or more) antigen-binding sites.

[0116] If an antibody or antigen-binding fragment binds to two or more (e.g., 2, 3, 4, 5, or 6) antigens, it can also be referred to as a "multispecific antibody," such as a bispecific antibody, trispecific antibody, or tetraspecific antibody, which refer to multispecific antibodies capable of binding to two, three, or four antigens, respectively.

[0117] As used herein, a "diabody" refers to an antibody comprising two scFvs each having two antigen-binding sites (bivalent), in which the VH and VL chains in each scFv are connected by a short peptide linker (approximately 5 to 10 amino acid residues) and paired (i.e., the VH of a first scFv is paired with the VL of a second scFv, and the VL of the first scFv is paired with the VH of the second scFv) to form an antigen-binding site. Diabodies may be bispecific antibodies.

[0118] As used herein, an "antigen-binding fragment" of an antibody refers to a portion of a full-length antibody that is less than full-length but includes at least a portion of the variable region of the full-length antibody (e.g., including one or more CDRs and / or one or more antigen-binding sites), and thus retains at least some of the full-length antibody's ability to specifically bind to antigen. Antigen-binding fragments may include, for example, antibody derivatives produced by enzymatic treatment of a full-length antibody, synthetically produced derivatives, and recombinantly produced derivatives. Examples of antigen-binding fragments include, but are not limited to, Fv, scFv, dsFv, scdsFv, Fab, scFab, Fab', F(ab')2, diabody, Fd and Fd' fragments, and other fragments (e.g., fragments containing modifications). Antigen-binding fragments may include, for example, multiple peptide chains connected by disulfide bonds and / or peptide linkers and / or formed by noncovalent interactions.

[0119] As used herein, the term "monoclonal antibody" refers to a highly homogeneous antibody population in which the antibody molecules contained therein are substantially identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies generally specifically bind to a single antigen epitope. As used herein, monoclonal antibodies can be prepared by any method known to those skilled in the art, for example, produced from transgenic animals (e.g., transgenic mice), produced by immortalized B cells (e.g., B cell hybridomas), or prepared in bacterial, eukaryotic, or plant cells using recombinant DNA methods, or isolated from a phage antibody library. The antibody or antigen-binding fragment of the present invention is a monoclonal antibody. In one embodiment, the antibody or antigen-binding fragment of the present invention is a humanized monoclonal antibody.

[0120] A "chimeric antibody" refers to an antibody in which some portions (e.g., CDRs, FRs, variable regions, constant regions, or a combination thereof) are identical or homologous to corresponding sequences in antibodies derived from a particular species, and the remaining portions are identical or homologous to corresponding sequences in antibodies derived from another species. As used herein, "chimeric antibody" also encompasses antibodies containing portions belonging to different antibody types or subclasses. In certain embodiments, a chimeric antibody has a murine antibody variable region and a human antibody constant region.

[0121] As used herein, "humanized antibody" refers to an antibody containing non-human and human antibody sequences. Thus, a humanized antibody is a chimeric antibody containing minimal sequence derived from non-human immunoglobulin. A non-human antibody may be an antibody derived from any non-human species or an antibody containing portions of a non-human species (e.g., a chimeric antibody). Non-human species may include, for example, mouse, rat, rabbit, alpaca, or non-human primate. Techniques for obtaining a humanized antibody from a non-human antibody are well known to those skilled in the art. A humanized antibody can be produced from a non-human antibody (e.g., a murine antibody or a chimeric antibody), for example, by grafting the CDR sequence of the non-human antibody (e.g., a murine antibody) into a human antibody framework region. In some cases, to maintain the antigen-binding ability and / or stability of a humanized antibody, key amino acid residues from the framework sequences of a non-human antibody (e.g., a murine antibody) can be retained in the human antibody framework regions, i.e., "backmutations" can be performed (see, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. 81(21):6851-6855; Neuberger et al. (1984) Nature 312:604-608).

[0122] As used herein, "percent (%) sequence identity" and "sequence identity" of amino acid sequences have definitions well known to those of skill in the art and refer to the percentage of identity between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or by well-known algorithms). It can be determined using methods known to those of skill in the art, such as using publicly available computer software such as BLAST, BLAST-2, Clustal Omega, and FASTA software.

[0123] "Affinity" or "binding affinity" is used to measure the strength of mutual binding between an antibody and an antigen through non-covalent interactions. The measure of "affinity" is usually expressed as the equilibrium dissociation constant, K D or EC 50The measured equilibrium association constant (ka) and equilibrium dissociation constant (kd) can be reported as: K D = kd / ka, K D Affinity can be determined using conventional techniques known to those skilled in the art, such as biomembrane interference (e.g., the Octet Fortebio detection system can be used), radioimmunoassay, surface plasmon resonance, enzyme-linked immunosorbent assay, or flow cytometry.

[0124] As used herein, "specific binding" between an antibody and an antigen means that the antibody and antigen bind to each other with higher affinity. Typically, the K D The value should be at least about 10 -6 ~ at least about 10 -9 M or less, e.g., at least about 10 -6 , at least about 10 -7 , at least about 10 -8 , at least about 10 -9 It is below M.

[0125] As used herein, an amino acid sequence "derived from" or "derived from" a reference amino acid sequence is partially or completely identical or homologous to the reference amino acid sequence. For example, 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 compared to the wild-type sequence of the heavy chain constant region of the human immunoglobulin from which it is derived.

[0126] As used herein, 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 amino acid substitutions, deletions, or additions. Those skilled in the art will understand that amino acids in non-essential regions of a polypeptide or protein can be substituted with suitable conservative amino acids, and that such substitutions generally do not alter its biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th ed., 1987, The Benjamin / Cummings Pub. Co., p. 224). Suitable conservative substitutions are well known to those skilled in the art. The following table lists some non-limiting examples of common conservative substitutions of amino acid residues. In some cases, the amino acid substitutions are non-conservative substitutions. Those skilled in the art will understand that amino acid mutations or modifications can be made to antibodies or antibody fragments to alter their properties, for example, to alter the antibody glycosylation modification type and its ability to form interchain disulfide bonds. Antibodies or antigen-binding fragments thereof containing such amino acid mutations or modifications are also encompassed within the scope of the antibodies or antigen-binding fragments thereof of the present invention.

[0127] [Table A]

[0128] As used herein, the terms "polynucleotide," "nucleic acid," and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, and may generally include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0129] As used herein, "isolated" means that a material (e.g., a nucleic acid molecule or polypeptide) has been separated from its source or environment, i.e., it is substantially free of any other components.

[0130] As used herein, a "vector" is a vehicle for introducing exogenous nucleic acid into a host cell, and when the vector is transformed into a suitable host cell, the exogenous nucleic acid is amplified or expressed. A vector is usually free-standing, but can be designed so that a gene or a part thereof can be integrated into a chromosome of a genome. As used herein, the definition of a vector includes plasmids, linearized plasmids, virus vectors, cosmids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), etc.

[0131] As used herein, "expression vector" refers to a vector capable of expressing DNA, wherein the DNA is operably linked to regulatory sequences (e.g., promoter, ribosome binding site) that can affect DNA expression. Regulatory sequences may include promoter and terminator sequences, and may optionally include an origin of replication, a selection marker, an enhancer, a polyadenylation signal, etc. 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 free or that integrate into the host cell genome.

[0132] As used herein, a "recombinant cell" is a cell used to receive, maintain, replicate, or amplify a vector. Recombinant cells can also be used to express a polypeptide encoded by a nucleic acid or vector. Recombinant cells can be eukaryotic or prokaryotic.

[0133] As used herein, the term "treatment" refers to the amelioration of a disease / symptom, such as the reduction or elimination of the disease / symptom, the prevention or slowing of the onset, progression and / or worsening of the disease / symptom, etc. Thus, treatment includes prevention, treatment and / or cure.

[0134] An "effective amount" refers to a dose that is sufficient to reduce the severity of disease symptoms, increase the frequency and duration of disease-free intervals, or prevent damage or injury caused by the disease. An "effective amount" refers to the amount required to prevent, cure, ameliorate, block, or partially block a disease or symptom. For example, for tumor treatment, an "effective amount" of the antibody or antigen-binding fragment thereof, or pharmaceutical composition of the present invention preferably inhibits tumor cell proliferation or tumor growth by at least about 10%, 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%, and more preferably at least about 80%, compared to untreated subjects. The effectiveness of inhibiting tumor growth can be evaluated using conventional tumor animal models in the art, such as spontaneous tumors, induced tumors, and transplanted tumor animal models. Alternatively, the ability to inhibit cell proliferation can be examined using in vitro detection methods known to those skilled in the art. An effective amount of the antibody or antigen-binding fragment thereof, or pharmaceutical composition of the present invention can reduce tumor size or otherwise alleviate symptoms in a subject (e.g., prevent and / or treat metastasis or recurrence).Those skilled in the art can determine the effective amount based on factors such as the subject's age, physical condition, sex, severity of symptoms, and the specific composition or administration route.An effective amount can be administered in one or more doses.

[0135] As used herein, the term "pharmaceutically acceptable excipient" refers to an excipient that is pharmacologically and / or physiologically compatible with the subject and active ingredient, as known to those skilled 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 adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives. For example, pH adjusting agents 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 enhancing agents 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 for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and analogs thereof. Agents for delaying absorption include, but are not limited to, monostearate salts and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols, and polyols (e.g., glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc.The stabilizer has the meaning commonly understood by those skilled in the art and is capable of stabilizing the desired activity of the active ingredient in the drug, and includes, but is not limited to, sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein), or degradation products thereof (e.g., lactalbumin hydrolysate), etc.

[0136] As used herein, examples of mammals include, but are not limited to, humans, non-human primates, rats, mice, cows, horses, pigs, sheep, dogs, cats, etc. As used herein, the term "subject" refers to a mammal, such as a human. In some embodiments, the subject is a human. In some embodiments, the subject is a cancer patient, a human or animal suspected of having cancer, or at risk for cancer. As used herein, the terms "patient" and "subject" can be used interchangeably.

[0137] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The experimental examples of the present invention and their description are used to explain the present invention and do not constitute an undue limitation of the present invention. [Brief explanation of the drawings]

[0138] [Figure 1] FIG. 1 shows a schematic diagram of the structure of the multispecific antibodies described in this application. [Figure 2] FIG. 2 shows the binding of multispecific antibodies of the invention to human HER2 as detected on HER2-overexpressing cells N87 and CT26. [Figure 3] FIG. 3 shows the binding of a multispecific antibody of the invention to human 4-1BB, detected on cells CHO-h4-1BB overexpressing human 4-1BB. [Figure 4] FIG. 4 shows the binding of multispecific antibodies of the invention to FcR. [Figure 5] FIG. 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] FIG. 6 shows that multispecific antibodies of the invention mediate ADCC function via HER2. [Figure 7] FIG. 7 shows that HER2 mediates the release of the cytokines IL-2 and IFN-γ by primary T cells induced by the multispecific antibody of the invention. [Figure 8] FIG. 8 shows that FcR mediates the release of the cytokines IL-2 and IFN-γ by primary T cells induced by the multispecific antibodies of the invention. [Figure 9] FIG. 9 shows the results of SEC and LC / MS detection of the multispecific antibody HER2xHER2x4-1BB of the present invention. [Figure 10] Figure 10 shows the tumor inhibitory activity of multispecific antibodies of the invention in a tumor model of h-4-1BB KI BALB / c mice subcutaneously inoculated with CT26-h-HER2, where Figure 10A shows the effect on tumor volume 0-15 days post-administration and Figure 10B shows CD8+ T cell infiltration in mouse tumors. [Figure 11] FIG. 11 shows the tumor-inhibitory activity of multispecific antibodies of the invention in a tumor model in h-4-1BB KI BALB / c mice subcutaneously inoculated with CT26-h-HER2 (effect on tumor volume 11 to 27 days after administration). [Figure 12] FIG. 12 shows the antitumor activity of multispecific antibodies of the invention in a tumor model of h-4-1BB KI C57 mice subcutaneously inoculated with MC38-h-HER2.

[0139] Specific Models for Implementing the Invention The technical solutions of the present invention are clearly and completely described with examples and drawings of the present invention. Obviously, the described examples are only a part of the examples of the present invention, but not all of them. The following description of at least one experimental example is merely illustrative in nature and in no way a limitation on the present invention and its application or use. All other examples obtained by those skilled in the art based on the examples of the present invention without creative work are within the scope of the present invention.

[0140] [Table B-1] [Table B-2] [Table B-3]

[0141] SEQ ID NO: 1: Anti-trastuzumab mAb heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS SEQ ID NO: 2: Anti-trastuzumab mAb light chain variable region DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK SEQ ID NO: 3: Anti-Pertuzumab mAb heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS SEQ ID NO: 4: Anti-Pertuzumab mAb light chain variable region DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK SEQ ID NO: 5: Anti-HER2 mAb heavy chain variable region EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS

[0142] SEQ ID NO: 6: Anti-HER2 mAb light chain variable region DIQMTQSPSSLSASVGDRVTITCRASQSVQGAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHSTTPPTFGQGTKVEIK SEQ ID NO: 7: 4-1BB binding region of anti-4-1BB nanobody AB24ME QVQLVESGGGVVQPGRSLRLSCAASGSTFSIVAMGWYRQAPGKQRELVASIITGDGDTNYADSVKGRFTISRDNSKNTMYLQMNSLKPEDTAVYYCYARTGYGSSELEGHEYDYWGQGTQVTVSS SEQ ID NO: 8: Anti-HER2 mAb VLCDR3 QQHSTTPPT SEQ ID NO: 9: Amino acid sequence of human kappa light chain constant region (CL) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 10: Amino acid sequence of human IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0143] SEQ ID NO: 11: Amino acid sequence of human IgG1 (LALA mutation introduced to reduce Fc function) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 12: Amino acid sequence of human IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 13: CH1 / CL selectively mutated human IgG1 Fc amino acid sequence CH SET1 (knob mutations introduced) ASTKGPSVFPLAPSSKSTSGGTAALGCQVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYELSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 14: CH1 / CL selectively mutated human IgG1 Fc amino acid sequence CH SET2 (hole mutation introduced) ASTKGPSVFPRAPSSKSTSGGTAALGCLVRDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 15: CH1 / CL selectively mutated human kappa light chain constant region (CL) amino acid sequence CL SET1 RTVAAPSVFIFPPSDEQLKSGRASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSRLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0144] SEQ ID NO: 16: CH1 / CL selectively mutated human kappa light chain constant region (CL) amino acid sequence CL SET2 RTVAAPSVFIFPPSDEELKSGTASVQCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSELTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 17: (G4S)4G GGGGSGGGGSGGGGSGGGGSG SEQ ID NO: 18: (G4A)4 GGGGAGGGGAGGGGAGGGGA SEQ ID NO: 19: Amino acid sequence of human IgG1 Fc THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 20: Amino acid sequence of human IgG1 Fc (LALA mutation introduced to reduce Fc function) THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0145] SEQ ID NO: 21: HER2x4-1BB-1 heavy chain (HER2x4-1BB-1 peptide chain #1) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGGGGGAGGGGAGGGGAGGGGAQVQLVESGGGVVQPGRSLRLSCAASGSTFSIVAMGWYRQAPGKQRELVASIITGDGDTNYADSVKGRFTISRDNSKNTMYLQMNSLKPEDTAVYYCYARTGYGSSELEGHEYDYWGQGTQVTVSS SEQ ID NO: 22: HER2x4-1BB-1 light chain (HER2x4-1BB-1 peptide chain #2) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 23: HER2x4-1BB-2 heavy chain (HER2x4-1BB-2 peptide chain #1) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGGGGGAGGGGAGGGGAGGGGAQVQLVESGGGVVQPGRSLRLSCAASGSTFSIVAMGWYRQAPGKQRELVASIITGDGDTNYADSVKGRFTISRDNSKNTMYLQMNSLKPEDTAVYYCYARTGYGSSELEGHEYDYWGQGTQVTVSS SEQ ID NO: 24: HER2x4-1BB-2 light chain (HER2x4-1BB-2 peptide chain #2) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 25: HER2x4-1BB-3 heavy chain (HER2x4-1BB-3 peptide chain #1) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGGGGGAGGGGAGGGGAGGGGAQVQLVESGGGVVQPGRSLRLSCAASGSTFSIVAMGWYRQAPGKQRELVASIITGDGDTNYADSVKGRFTISRDNSKNTMYLQMNSLKPEDTAVYYCYARTGYGSSELEGHEYDYWGQGTQVTVSS

[0146] SEQ ID NO: 26: HER2x4-1BB-3 light chain (HER2x4-1BB-3 peptide chain #2) DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 27: HER2xHER2x4-1BB-1 heavy chain 1 (HER2xHER2x4-1BB-1 peptide chain #1) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTQGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCQ VEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYELSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGGGGGAGGGGAGGGGAGGGGAQVQLVESGGGVVQPGRSLRLSCAASGSTFSIVAMGWYRQAPGKQRELVASIITGDGDTNYADSVKGRFTISRDNSKNTMYLQMNSLKPEDTAVYYCYARTGYGSSELEGHEYDYWGQGTQVTVSS SEQ ID NO: 28: HER2xHER2x4-1BB-1 light chain 1 (HER2xHER2x4-1BB-1 peptide chain #2) DIQMTQSPSSLSASVGDRVTITCRASQSVQGAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHSTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGRASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSRLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 29: HER2xHER2x4-1BB-1 heavy chain 2 (HER2xHER2x4-1BB-1 peptide chain #3) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPRAPSSKSTSGGTAALGCLV RDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGGGGGAGGGGAGGGGAGGGGAQVQLVESGGGVVQPGRSLRLSCAASGSTFSIVAMGWYRQAPGKQRELVASIITGDGDTNYADSVKGRFTISRDNSKNTMYLQMNSLKPEDTAVYYCYARTGYGSSELEGHEYDYWGQGTQVTVSS SEQ ID NO: 30: HER2xHER2x4-1BB-1 light chain 2 (HER2xHER2x4-1BB-1 peptide chain #4) DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK RTVAAPSVFIFPPSDEELKSGTASVQCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSELTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0147] SEQ ID NO: 31: Anti-4-1BB VHH-1 THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGQVQLVESGGGVVQPGRSLRLSCAASGSTFSIVAMGWYRQAPGKQRELVASIITGDGDTNYADSVKGRFTISRDNSKNTMYLQMNSLKPEDTAVYYYCYARTGYGSSELEGHEYDYWGQGTQVTVSS SEQ ID NO: 32: Anti-4-1BB VHH-2 THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGQVQLVESGGGVVQPGRSLRLSCAASGSTFSIVAMGWYRQAPGKQRELVASIITGDGDTNYADSVKGRFTISRDNSKNTMYLQMNSLKPEDTAVYYYCYARTGYGSSELEGHEYDYWGQGTQVTVSS SEQ ID NO: 33: Urelumab heavy chain (Urelumab peptide chain #1) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLW GRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY GPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG SEQ ID NO: 34: Urelumab light chain (Urelumab peptide chain #2) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 35: PRS-343 heavy chain (PRS-343 peptide chain #1) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGGSQDSTSDLIPAPPLSKVPL QQNFQDNQFHGKWYVVGQAGNIRLREDKDPIKMMATIYELKEDKSYDVTMVKFDDKCCMYDIWTFVPGSQPGEFTLGKIKSFPGHTSSLVRVVSTNYNQHAMVFFKFVFQNREEFYITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG

[0148] SEQ ID NO: 36: PRS-343 light chain (PRS-343 peptide chain #2) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 37: Human IgG1 amino acid sequence HC-1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTEPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLLSVLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 38: Human IgG1 amino acid sequence HC-2 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLRSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 39: HER2xHER2x4-1B-2 heavy chain 1 (HER2xHER2x4-1B-2 peptide chain #1) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTEPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLLSVLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGGGGGAGGGGAGGGGAGGGGAQVQLVESGGGVVQPGRSLRLSCAASGSTFSIVAMGWYRQAPGKQRELVASIITGDGDTNYADSVKGRFTISRDNSKNTMYLQMNSLKPEDTAVYYCYARTGYGSSELEGHEYDYWGQGTQVTVSS SEQ ID NO: 40: HER2xHER2x4-1B-2 light chain 1 (HER2xHER2x4-1BB-2 peptide chain #2) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0149] SEQ ID NO: 41: HER2xHER2x4-1BB-2 heavy chain 2 (HER2xHER2x4-1BB-2 peptide chain #3) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLRSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGGGGGGAGGGGAGGGGAGGGGAQVQLVESGGGVVQPGRSLRLSCAASGSTFSIVAMGWYRQAPGKQRELVASIITGDGDTNYADSVKGRFTISRDNSKNTMYLQMNSLKPEDTAVYYYCYARTGYGSSELEGHEYDYWGQGTQVTVSS SEQ ID NO: 42: HER2xHER2x4-1BB-2 light chain 2 (HER2xHER2x4-1BB-2 peptide chain #4) DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 43: CDR1 of anti-4-1BB nanobody AB24ME GSTFSIVA SEQ ID NO: 44: CDR2 of anti-4-1BB nanobody AB24ME IITGDGDT SEQ ID NO: 45: CDR3 of anti-4-1BB nanobody AB24ME YARTGYGSSELEGHEYDY

[0150] SEQ ID NO: 46: Anti-HER2 mAb VHCDR1 GFNIKDTY SEQ ID NO: 47: Anti-HER2 mAb VHCDR2 IYPTQGYT SEQ ID NO: 48: Anti-HER2 mAb VHCDR3 SRWGGEGFYAMDY SEQ ID NO: 49: Anti-HER2 mAb VLCDR1 QSVQGA SEQ ID NO: 50: Anti-HER2 mAb VLCDR2 SAS [Example]

[0151] Example 1 Cloning and expression of multispecific antibodies In this example, three HER2x4-1BB bispecific antibodies, two anti-HER2xHER2x4-1BB trispecific antibodies, and two 4-1BB monoclonal antibodies were constructed, respectively: HER2x4-1BB-1: It consists of two polypeptide chains, the structure of which is shown in Figure 1. Peptide chain #1 has the amino acid sequence set forth in SEQ ID NO: 21 and contains the amino acid sequence of the heavy chain variable region of the anti-HER2 monoclonal antibody trastuzumab (SEQ ID NO: 1) (Patent Application No. WO1992022653A1) and the 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 AB24ME was linked to the C-terminus of the Fc via the 20-amino acid residue flexible peptide (G4A)4 (SEQ ID NO: 18). Peptide chain #2 had the amino acid sequence set forth in SEQ ID NO: 22 and contained the amino acid sequence of the light chain variable region of the anti-HER2 monoclonal antibody trastuzumab (SEQ ID NO: 2) (patent application number: WO1992022653A1) and the amino acid sequence of the human κ light chain constant region (CL) at the C-terminus of the VL amino acid sequence (SEQ ID NO: 9).

[0152] HER2x4-1BB-2: It consists of two polypeptide chains, the structure of which is shown in Figure 1. Peptide chain #1 has the amino acid sequence set forth in SEQ ID NO: 23 and contains the amino acid sequence of the heavy chain variable region of the anti-HER2 monoclonal antibody trastuzumab (SEQ ID NO: 1) (patent application number: WO1992022653A1) and a human IgG1 amino acid sequence (in which a LALA mutation was introduced to reduce Fc function; SEQ ID NO: 11). The N-terminus of the CD137-binding region amino acid sequence of the anti-CD137 single-domain antibody AB24ME (SEQ ID NO: 7) was linked to the C-terminus of the Fc via the 20-amino acid residue flexible peptide (G4A)4 (SEQ ID NO: 18). Peptide chain #2 had the amino acid sequence set forth in SEQ ID NO: 24, and contained the amino acid sequence of the light chain variable region of the anti-HER2 monoclonal antibody trastuzumab (SEQ ID NO: 2) (patent application number: WO1992022653A1) and the amino acid sequence of the human κ light chain constant region (CL) at the C-terminus of the VL amino acid sequence (SEQ ID NO: 9).

[0153] HER2x4-1BB-3: It consists of two polypeptide chains, the structure of which is shown in Figure 1. Peptide chain #1 had the amino acid sequence set forth in SEQ ID NO: 25 and contained the amino acid sequence of the heavy chain variable region of the anti-HER2 monoclonal antibody pertuzumab (SEQ ID NO: 3) (Patent Application No. US7449184) and the human IgG1 amino acid sequence (SEQ ID NO: 12). The N-terminus of the CD137-binding region amino acid sequence of the anti-CD137 single-domain antibody AB24ME (SEQ ID NO: 7) was linked to the C-terminus of the Fc via the 20-amino acid residue flexible peptide (G4A)4 (SEQ ID NO: 18). Peptide chain #2 had the amino acid sequence set forth in SEQ ID NO: 26 and contained the amino acid sequence of the light chain variable region of the anti-HER2 monoclonal antibody pertuzumab (SEQ ID NO: 4) (Patent Application No. 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).

[0154] HER2xHER2x4-1BB-1: It consists of four polypeptide chains, the structure of which is shown in Figure 1. Peptide chain #1 has the amino acid sequence set forth in SEQ ID NO: 27 and contains the amino acid sequence of the heavy chain variable region of an anti-HER2 monoclonal antibody (SEQ ID NO: 5) and the human IgG1 amino acid sequence (CH3 knob mutation and CH1 / CL selective mutation introduced) CHSET1 (patent application number: WO2021067404A2, SEQ ID NO: 13). The N-terminus of the CD137 binding region amino acid sequence (SEQ ID NO: 7) of the anti-CD137 single domain antibody AB24ME was linked to the C-terminus of the Fc via the 20-amino acid residue flexible peptide (G4A)4 (SEQ ID NO: 18). Peptide chain #2 had the amino acid sequence set forth in SEQ ID NO: 28, and contained the amino acid sequence of the light chain variable region of an anti-HER2 monoclonal antibody (SEQ ID NO: 6) and the human kappa light chain constant region (CL) amino acid sequence (CH1 / CL selective mutations introduced) CL SET1 (Patent Application No.: WO2021067404A2, SEQ ID NO: 15) at the C-terminus of the VL amino acid sequence. Peptide chain #3 had the amino acid sequence set forth in SEQ ID NO: 29, and contained the amino acid sequence of the heavy chain variable region of the anti-HER2 monoclonal antibody pertuzumab (SEQ ID NO: 3) (Patent Application No.: US7449184) and the human IgG1 amino acid sequence (CH3 hole mutations and CH1 / CL selective mutations introduced) CH SET2 (Patent Application No.: 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 AB24ME was linked to the C-terminus of Fc via the 20-amino acid residue flexible peptide (G4A)4 (SEQ ID NO: 18). Peptide chain #4 had the amino acid sequence set forth in SEQ ID NO: 30 and contained the amino acid sequence of the light chain variable region of the anti-HER2 monoclonal antibody pertuzumab (SEQ ID NO: 4) (Patent Application No.: US7449184) and the human kappa light chain constant region (CL) amino acid sequence (in which CH1 / CL selective mutations were introduced) CL SET2 (Patent Application No.: WO2021067404A2, SEQ ID NO: 16) at the C-terminus of the VL amino acid sequence.

[0155] HER2xHER2x4-1BB-2: It consists of four polypeptide chains, the structure of which is shown in Figure 1. Peptide chain #1 was synthesized according to the method described in the patent (patent application number: 201611016435.0) and contained the amino acid sequence of the heavy chain variable region of the anti-HER2 monoclonal antibody trastuzumab (SEQ ID NO: 1) (patent application number: WO1992022653A1) and the human IgG1 amino acid sequence HC-1 (SEQ ID NO: 37, patent application number: PCT / CN2017 / 111310), which has the amino acid sequence set forth in SEQ ID NO: 39 and can spontaneously form heterodimers. The N-terminus of the CD137-binding region amino acid sequence of the anti-CD137 single-domain antibody AB24ME (SEQ ID NO: 7) was linked to the C-terminus of the Fc via the 20-amino acid residue flexible peptide (G4A)4 (SEQ ID NO: 18). Peptide chain #2 had the amino acid sequence set forth in SEQ ID NO: 40 and contained the amino acid sequence of the light chain variable region of the anti-HER2 monoclonal antibody trastuzumab (SEQ ID NO: 2) (Patent Application No.: 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). Peptide chain #3 was synthesized by the method described in a patent (Patent Application No.: 201611016435.0) and had the amino acid sequence set forth in SEQ ID NO: 41 and contained the amino acid sequence of the heavy chain variable region of the anti-HER2 monoclonal antibody pertuzumab (SEQ ID NO: 3) (Patent Application No.: US7449184), which can spontaneously form heterodimers, and the human IgG1 amino acid sequence HC-2 (SEQ ID NO: 38, Patent Application No.: 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 AB24ME was linked to the C-terminus of Fc via the 20-amino acid residue flexible peptide (G4A)4 (SEQ ID NO: 18). Peptide chain #4 had the amino acid sequence set forth in SEQ ID NO: 42 and contained the amino acid sequence of the light chain variable region of the anti-HER2 monoclonal antibody pertuzumab (SEQ ID NO: 4) (Patent Application No.: 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).

[0156] Anti-4-1BB-VHH-1: It consists of two polypeptide chains and has the amino acid sequence set forth in SEQ ID NO: 31, containing the human IgG1 Fc amino acid sequence (SEQ ID NO: 19), with the N-terminus of the CD137-binding region amino acid sequence (SEQ ID NO: 7) of the anti-CD137 single domain antibody AB24ME linked to the C-terminus of the Fc via the 21 amino acid residue flexible peptide (G4S)4G (SEQ ID NO: 17).

[0157] Anti-4-1BB-VHH-2: It consists of two polypeptide chains and has the amino acid sequence set forth in SEQ ID NO: 32, containing the human IgG1 Fc amino acid sequence (in which a LALA mutation was introduced to reduce Fc function; SEQ ID NO: 20), with the N-terminus of the CD137-binding region amino acid sequence of the anti-CD137 single-domain antibody AB24ME (SEQ ID NO: 7) linked to the C-terminus of the Fc via the 21-amino acid residue flexible peptide (G4S)4G (SEQ ID NO: 17).

[0158] In this example, positive control molecules urelumab and PRS343 were constructed: Urelumab: The control molecule, urelumab (patent application number: WO2004010947), consisted of two polypeptide chains, peptide chain #1 having the amino acid sequence set forth in SEQ ID NO: 33 and peptide chain #2 having the amino acid sequence set forth in SEQ ID NO: 34.

[0159] PRS-343: The control molecule PRS-343 (patent application number: WO2016177802A1) consisted of two polypeptide chains, peptide chain #1 having the amino acid sequence set forth in SEQ ID NO: 35 and peptide chain #2 having the amino acid sequence set forth in SEQ ID NO: 36.

[0160] Example 2 Multispecific antibodies that bind to human HER2 In this experiment, expanded and cultured N87 cells (autologously expressing HER2) and CT26-hHER2 cells (overexpressing human HER2) were digested with 0.25% EDTA trypsin and washed once with culture medium. The cell density was adjusted to 2 × 10 6The resulting suspension was adjusted to 100 cells / ml. The resulting suspension was added to a 96-well flow plate at 100 μl / well and centrifuged for further use. Gradiently diluted antibodies were added to the 96-well flow plate with the cells at 100 μl / well and incubated at 4°C for 60 min. After washing twice with PBS, goat anti-human IgG-Fc (PE) (Abcam, ab98596) diluted 1:1000 in 2% BSA solution was added at 100 μl / well and incubated at 4°C for 60 min. After washing twice with PBS, the cells were finally resuspended in PBS at 100 μl / well and detected by a CytoFlex (Beckman) flow cytometer, and the corresponding mean fluorescence intensity (MFI) was calculated.

[0161] The experimental results are shown in Figure 2 and Tables 1 and 2. All of the HER2xHER2x4-1BB trispecific antibody and HER2x4-1BB bispecific antibody of the present invention were able to bind to HER2 expressed on human gastric cancer cells N87 (Figure 2A) and CT26 cells overexpressing human HER2 (Figure 2B), and their binding activity was comparable to that of HER2 monoclonal antibodies (trastuzumab or pertuzumab).

[0162] [Table 1]

[0163] [Table 2]

[0164] Example 3 Binding of multispecific antibodies to human 4-1BB In this experiment, expanded and cultured CHOS-h4-1BB (overexpressing human 4-1BB) cells were used in a volume of 2 × 10 6The cells were adjusted to a cell density of 100 cells / ml and added to a 96-well flow plate at 100 μl / well and centrifuged for further use. Serially diluted antibodies were added to the 96-well flow plate with the cells at 100 μl / well and incubated at 4°C for 60 min. After washing twice with PBS, goat anti-human IgG-Fc (PE) (Abcam, ab98596) diluted 1:1000 in 2% BSA solution was added at 100 μl / well and incubated at 4°C for 60 min. After washing twice with PBS, the cells were finally resuspended in PBS at 100 μl / well and detected using a CytoFlex (Beckman) flow cytometer, and the corresponding MFI was calculated.

[0165] The experimental results are shown in Figure 3 and Table 3. All of the HER2xHER2x4-1BB trispecific and HER2x4-1BB bispecific antibodies of the present invention were able to bind to CHOS-h4-1BB cells, with binding activity comparable to that of the 4-1BB monoclonal antibody (anti-4-1BB VHH) and weaker than that of the control antibodies urelumab and PRS-343.

[0166] [Table 3]

[0167] Example 4 Binding of multispecific antibodies to human FcR In this experiment, expanded and cultured THP-1 (which expresses FcR) and CHOS-hCD32b (which overexpresses human FcγRIIb) cells were used at 2 × 10 6The cells were adjusted to a cell density of 1000 cells / ml and added to a 96-well flow plate at 100 μl / well and centrifuged for further use. Serially diluted antibodies were added to the 96-well flow plate with the cells at 100 μl / well and incubated at 4°C for 60 min. After washing twice with PBS, goat anti-human IgG-Fc (PE) (Abcam, ab98596) diluted 1:1000 in 2% BSA solution was added at 100 μl / well and incubated at 4°C for 60 min. After washing twice with PBS, the cells were finally resuspended in PBS at 100 μl / well and detected using a CytoFlex (Beckman) flow cytometer, and the corresponding mean fluorescence intensity (MFI) was calculated.

[0168] The experimental results are shown in Figure 4 and Tables 4-5. The HER2xHER2x4-1BB trispecific antibody and HER2x4-1BB bispecific antibody of the present invention, which retain Fc effector function, were all able to bind to FcγRIIb expressed on THP-1 (FcR-autologous expression; Figure 4A) and CHOS-hCD32b (human FcR-overexpressing; Figure 4B) cells. The Fc of the HER2xHER2x4-1BB trispecific antibody employs a knob-in-hole mutation, and therefore its binding activity to FcR was slightly weaker than that of the HER2x4-1BB bispecific antibody and HER2 monoclonal antibody, which employ wild-type human IgG1 Fc. The Fc of urelumab and PRS-343 adopted wild-type human IgG4 Fc or human IgG4 Fc carrying the FALA mutation, respectively, and therefore their affinity with FcR was weaker than that of multispecific antibodies that retained Fc effector functions.

[0169] [Table 4]

[0170] [Table 5]

[0171] Example 5 Inhibition of HER2 signal-dependent cell proliferation by multispecific antibodies In this experiment, expanded and cultured N87 and SK-OV-3 (self-expressing HER2) cells were digested with 0.25% EDTA trypsin and washed once with culture medium. The cell density was 5 × 10 4 The antibody was added to the 96-well plate at 80 μl / well with the cells and incubated in a cell culture incubator for 3 to 5 days. Finally, the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7572) kit was used for signal generation, and the chemiluminescent signal was collected using a microplate reader.

[0172] As shown in Figures 5A and 5B, the multispecific antibodies significantly inhibited the proliferation of N87 or SK-OV-3 cells, with the HER2xHER2x4-1BB trispecific antibody having a significantly stronger inhibitory effect on cell proliferation than the HER2-4-1BB bispecific antibody or the HER2 monoclonal antibody. Furthermore, the inhibitory effect of the HER2 antibody on tumor cells was positively correlated with the HER2 expression level on the cells. The higher the HER2 expression level, the more pronounced the inhibitory effect of the antibody on cell proliferation.

[0173] Example 6 Induction of ADCC effect (reporter gene) by multispecific antibodies In this experiment, expanded and cultured N87 (self-expressing HER2) cells and CHOS-h4-1BB (overexpressing human 4-1BB) cells were used at 1.2 × 10 5 NFAT-luciferase / Jurkat CD16a (overexpressing CD16a and NFAT-Luc) effector cells at 10x10 / well and 3x10 4The antibodies were mixed at 1000p / well and inoculated into a 96-well white-bottom cell culture plate. Serially diluted polyspecific antibodies were then added to the 96-well plate, mixed, and incubated in a cell culture incubator for 6 hours. The Bio-Glo Luciferase Assay System (Promega, G7940) kit was used for signal generation, and the chemiluminescent signal was collected using an ELISA instrument.

[0174] The experimental results are shown in Figures 6A and 6C and Tables 6-7. The multispecific antibodies retaining Fc effector function mediated ADCC via HER2 expressed on N87 cells, thereby activating the CD16a-NFAT signaling pathway on Jurkat cells. In contrast, neither the HER2x4-1BB-2 bispecific antibody with the LALA mutation to disable Fc effector function nor the IgG4 Fc-based control molecule PRS-343 with the FALA mutation exhibited ADCC. Furthermore, when CHOS-h4-1BB cells were used as target cells, as shown in Figure 6B, neither multispecific antibody exhibited detectable ADCC against 4-1BB cells.

[0175] [Table 6]

[0176] [Table 7]

[0177] Example 7 Induction of primary T cells releasing cytokines (mediated by HER2+ cells) by multispecific antibodies Expanded and cultured target cells N87 (which express HER2) were digested with 0.25% EDTA trypsin and diluted to 1 × 10 410 cells / well were seeded into a 96-well culture plate (the culture plate was coated with 2 μg / ml anti-CD3 antibody at 100 μl / well one day before). PBMCs collected one day before were collected, and T cells in the PBMCs were separated using a T cell separation kit (Stemcell, 17951). 5 × 10 cells / well were seeded into a 96-well culture plate (the culture plate was coated with 2 μg / ml anti-CD3 antibody at 100 μl / well one day before). 4 The antibodies were added to the target cell wells at 1000p / well. Serially diluted multispecific antibodies were then added to the cell wells and incubated for 48 hours, after which the supernatants were collected. IL-2 and IFNγ levels in the supernatants were detected using a human IL-2 ELISA kit (Invitrogen, 88-7025-77) and a human IFNγ ELISA kit (Invitrogen, 88-7316-77).

[0178] The experimental results are shown in Figure 7. All of the multispecific antibodies of the present invention were able to induce primary T cells to release IL-2 (Figure 7A) or IFN-γ (Figure 7B) in the presence of N87 cells, demonstrating dose-dependence. Furthermore, the HER2xHER2x4-1BB trispecific antibody showed a significantly more potent ability to induce cytokine release compared to the HER2x4-1BB bispecific antibody and the control antibodies PRS-343 and urelumab.

[0179] Example 8 Induction of primary T cells releasing cytokines (mediated by FcR+ cells) by multispecific antibodies Expanded and cultured target cells CHOS-hCD32b (overexpressing human FcγRIIb) were centrifuged and counted to yield 1 × 10 4 10 cells / well were seeded into a 96-well culture plate (the culture plate was coated with 2 μg / ml anti-CD3 antibody at 100 μl / well one day before). PBMCs collected one day before were collected, and T cells in the PBMCs were separated using a T cell separation kit (Stemcell, 17951). 5 × 10 cells / well were seeded into a 96-well culture plate (the culture plate was coated with 2 μg / ml anti-CD3 antibody at 100 μl / well one day before). 4The cells / well were added to the target cell wells. Serially diluted multispecific antibodies were then added to the cell wells and incubated for 48 hours, after which the supernatants were collected. IL-2 and IFNγ levels in the supernatants were detected using a human IL-2 ELISA kit (Invitrogen, 88-7025-77) and a human IFNγ ELISA kit (Invitrogen, 88-7316-77).

[0180] The experimental results are shown in Figure 8. None of the multispecific antibodies of the present invention were able to induce the release of IL2 (Figure 8A) or IFN-γ (Figure 8B) by primary T cells in the presence of CHOS-hCD32b cells. In contrast, the positive control antibody, urelumab, successfully induced T cell activation and the release of pro-inflammatory factors upon FcR engagement. Furthermore, the negative control molecule, PRS-343, did not bind to CHOS-hCD32b and therefore was unable to induce cytokine release from primary T cells upon FcR interaction.

[0181] Example 9 Cell line screening In this example, using CH1 / CL selective mutation (patent number: WO2021 / 067404A2) and knob-in-hole technology, the heavy chain variable regions of anti-HER2 antibody and pertuzumab antibody were constructed in CH1 mutant CHSET1 (SEQ ID NO: 13) and CHSET2 (SEQ ID NO: 14), respectively. The N-terminus of the CD137 binding domain amino acid sequence (SEQ ID NO: 7) of the anti-CD137 single domain antibody AB24ME was linked to the C-terminus of the Fc via the 20-amino acid residue flexible peptide (G4A)4 (SEQ ID NO: 18). The light chain variable regions of anti-HER2 antibody and pertuzumab antibody were constructed in CLSET1 (SEQ ID NO: 15) and CLSET2 (SEQ ID NO: 16), respectively, on the CL mutant light chain constant region. A 1+1 asymmetric anti-HER2xHER2x4-1BB-1 trispecific antibody cell line was constructed.

[0182] Cell line construction The vector pCHO2.0-GS-Puro-H1-L1 carrying the heavy chain gene (SEQ ID NO: 27) and light chain gene (SEQ ID NO: 28) of the anti-HER2 antibody and the anti-4-1BB antibody, and the vector pCHO2.0-GS-Puro-H2-L2 carrying the heavy chain gene (SEQ ID NO: 29) and light chain gene (SEQ ID NO: 30) of the anti-pertuzumab anti-4-1BB antibody, were co-transfected into the host cell CHOS-ADP by electroporation. Puromycin and MSX screening pressure was applied to the transfected cells to perform pressure screening, resulting in the selection of high-yield minipools. Subsequently, after one round of limiting dilution, monoclonal identification was performed to isolate high-yield stable cell lines.

[0183] cell culture Dynamis AGT Medium was used as the basal medium, and cells were cultured at (1.0±0.2) × 10 6 The cells were inoculated at a density of 1000 cells / ml and subjected to fed-batch cultivation. On days 3, 5, 7, 9, and 11, Cell boost 7a powder supplement was added at 5.0±0.5% (w / w) of the initial culture weight, and Cell boost 7b powder supplement was added at 0.5±0.05% (w / w) of the initial culture weight. The dissolved oxygen was set at 40%, and the initial culture temperature was 36.5°C, reduced to 33.0°C on day 4. Based on glucose concentration measurements, 300 g / kg of glucose concentrate was added daily to reach a glucose concentration of 6.0 g / L in the cell culture, except on the harvest day. The culture was terminated on day 14 or when cell viability fell below 80%. Cell density and viability were monitored throughout the cultivation process using a Vicell analyzer (Beckman). Starting on day 7, antibody production was measured daily using a Cedex analyzer (Roche).

[0184] Quality assessment of stable cell line products The same one-step affinity purification method as used for the one-armed antibody in Example 1 was used. HPLC was used to obtain the purity of the protein. The HPLC method was as follows: mobile phase: 150 mM NaHPO·12H0, pH 7.0. Chromatography conditions: detection wavelength: 280 nm, column temperature: 25°C, flow rate: 0.5 ml / min, detection time: 30 min, TSK Gel G3000SWXL column. The SEC results are shown in Figure 9A, and the purity of the bispecific antibody obtained by one-step affinity purification was 98.3%.

[0185] The pairing of heavy and light chains of the protein was detected by high-performance liquid chromatography-mass spectrometry using a liquid-phase Vanquish UHPLC (Thermo), a mass spectrometer Q Exactive (Thermo), and a Waters ACQUITY UPLC BEH C4, 2.1 mm x 100 mm, chromatography column. A 50 μg sample was collected and diluted to 25 μl with ultrapure water. After centrifugation, 20 μl of the sample was collected and transferred to a sample vial. 5 μl was injected and analyzed for intact molecular weight using LC-MS. Chromatographic conditions were: column temperature: 80°C; UV detection wavelength: 280 nm; flow rate: 0.3 mL / min; mobile phase A: water containing 0.1% formic acid; mobile phase B: acetonitrile containing 0.1% formic acid. Mass spectrometry parameters were: ESI ion source; ion transport tube temperature: 320 °C; voltage: 3.8 kV; gas flow rate: 36 L / min; mode: positive ion Full MS; resolution: 17500; scan range: 600-4000 m / z. The results are shown in Figures 9B and 9C. The correct chain pairing rate of the purified trispecific antibody HER2xHER2x4-1BB-1 was greater than 98%.

[0186] Example 10 Studies on tumor-inhibitory activity of multispecific antibodies In this experiment, proof of concept of the antitumor activity of the multispecific antibodies of the invention was carried out in a tumor model using h-4-1BB KI BALB / c mice subcutaneously inoculated with CT26-h-HER2.

[0187] First, a tumor-bearing mouse model was established by subcutaneous inoculation of CT26-h-HER2 cells. 3 Once tumors reached a volume of 100 μg / mL, mice were randomized into groups and treated with G1: PBS, G2: 2.5 mg / kg PRS-343, G3: 2 mg / kg trastuzumab, G4: 2.3 mg / kg HER2x4-1BB-1, and G5: 2.3 mg / kg HER2x4-1BB-2 administered intraperitoneally (all groups were treated with equimolar doses). Changes in tumor volume and weight of mice in each group were monitored every 2–3 days for 2–3 weeks. The doses and administration schedules are shown in Table 8. After the experiment, tumors were harvested for immunohistochemistry to compare the infiltration of CD8+ T cells in tumors from different groups.

[0188] The experimental results are shown in Figure 10A. The bispecific antibody HER2x4-1BB-1, which retains Fc effector function, showed significantly better antitumor activity compared to the HER2x4-1BB-2 bispecific antibody with the LALA mutation to disable Fc effector function (TGI: 73.2% vs. 26.6%), as well as the control antibody PRS-343 (TGI: 6.5%) and the HER2 monoclonal antibody trastuzumab (TGI: 4.2%). Similarly, the results of immunohistochemistry detection (Figure 10B) also showed significantly stronger CD8+ T cell infiltration in tumors of mice treated with the HER2x4-1BB-1 bispecific antibody than in tumors in mice treated with the control antibody PRS-343, trastuzumab, and the HER2x4-1BB-2 bispecific antibody.

[0189] [Table 8]

[0190] Example 11 Studies on tumor-inhibitory activity of multispecific antibodies In this experiment, the anti-tumor activity of the multispecific antibodies of the invention was evaluated in a tumor model using h-4-1BB KI BALB / c mice subcutaneously inoculated with CT26-h-HER2.

[0191] First, a tumor-bearing mouse model was established by subcutaneous inoculation of h-HER2 CT26 cells. 3 Once tumors reached a volume of 100 μg, mice were randomized into groups and treated intraperitoneally with G1: PBS, G2: 3 mg / kg trastuzumab combined with 3 mg / kg pertuzumab, G3: 3.6 mg / kg PRS-343, G4: 3 mg / kg Enhertu (DS8201), G5: 3.6 mg / kg HER2x4-1BB-1, G6: 3.6 mg / kg HER2x4-1BB-3, G7: 1.8 mg / kg HER2x4-1BB-1 combined with 1.8 mg / kg HER2x4-1BB-3, and G8: 3.6 mg / kg HER2xHER2x4-1BB-2 (all groups were treated at equimolar doses). Changes in tumor volume and weight of mice within each group were monitored every 2–3 days for 2–3 weeks. The doses and administration schedules are shown in Table 9.

[0192] The experimental results are shown in Figure 11. The antitumor activity of the HER2xHER2x4-1BB trispecific antibody molecule (TGI: 85.7%) was significantly better than that of the equimolar control antibody PRS-343 (TGI: 37.7%), Enhertu (TGI: 47.7%), and the combination of the HER2 monoclonal antibodies trastuzumab and pertuzumab (TGI: 39.5%). Furthermore, the HER2xHER2x4-1BB trispecific antibody exhibited significantly more potent antitumor activity than the HER2x4-1BB bispecific antibodies HER2x4-1BB-1 (TGI: 66.9%) and HER2x4-1BB-3 (TGI: 59.7%), and had comparable efficacy to the group treated with the HER2x4-1BB bispecific antibody combination (HER2x4-1BB-1 + HER2x4-1BB-3, TGI: 83.7%).

[0193] [Table 9]

[0194] Example 12 Studies on tumor-inhibitory activity of multispecific antibodies In this experiment, the anti-tumor activity of the multispecific antibodies of the invention was evaluated in a tumor model using h-4-1BB KI C57 mice subcutaneously inoculated with MC38-h-HER2.

[0195] First, a tumor-bearing mouse model was established by subcutaneous inoculation of MC38-h-HER2 cells. 3 Once tumors had grown to a volume of 1000 mg / mL, mice were randomized into groups and treated by intraperitoneal injection with G1: PBS, G2: 3 mg / kg trastuzumab combined with 3 mg / kg pertuzumab, G3: 3.6 mg / kg PRS-343, G4: 3 mg / kg Enhertu (DS8201), G5: 1.8 mg / kg HER2x4-1BB-1 combined with 1.8 mg / kg HER2x4-1BB-3, and G6: 3.6 mg / kg HER2xHER2x4-1BB-1 (all groups were treated at equimolar doses). Changes in tumor volume and weight of mice within each group were monitored every 3–6 days for 2–3 weeks. The doses and administration schedules are outlined in Table 10.

[0196] The results are shown in Figure 12. All mice treated with the HER2xHER2x4-1BB trispecific antibody showed complete tumor regression, demonstrating significantly greater efficacy than both the equimolar control antibody PRS-343 (TGI: 54.9%) and the HER2 monoclonal antibody combination (trastuzumab + pertuzumab, TGI: 63.8%). Furthermore, in this tumor model, the groups treated with the HER2x4-1BB bispecific antibody combination (HER2x4-1BB-1 + HER2x4-1BB-3) and the group treated with Enhertu also achieved complete tumor regression.

[0197] [Table 10]

[0198] 1. Yarden, Y. and M. X. Sliwkowski, Untangling the ErbB signaling network. Nat Rev Mol Cell Biol, 2001, 2(2): 127-37. 2. Oh, DY and YJ Bang, HER2-targeted therapies - a role beyond breast cancer. Nat Rev Clin Oncol, 2020, 17(1):33-48. 3. Schaer, DA, D. Hirschhorn-Cymerman, and JD Wolchok, Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy. J Immunother Cancer, 2014, vol. 2:7. 4. Chen, S. et al., Combination of 4-1BB agonist and PD-1 antagonist promotes antitumor effector / memory CD8 T cells in a poorly immunogenic tumor model. Cancer Immunol Res, 2015, Vol. 3(2): 149-60.

[0199] Although specific embodiments of the present invention have been described in detail, it will be understood that those skilled in the art, based on all the teachings disclosed, can make various modifications and substitutions to these details and that these variations are within the scope of the present invention, the full scope of which is given by the appended claims and any equivalents thereof.

Claims

1. A single domain antibody or antigen-binding fragment thereof capable of specifically binding to 4-1BB, wherein the single domain antibody comprises: (a) a CDR1 having the sequence set forth in SEQ ID NO: 43, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to the sequence set forth in SEQ ID NO: 43; (b) a CDR2 having the sequence set forth in SEQ ID NO: 44, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to the sequence set forth in SEQ ID NO: 44; and (c) comprises a CDR3 having a sequence set forth in SEQ ID NO: 45, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to the sequence set forth in SEQ ID NO: 45; Preferably, the substitutions are conservative substitutions; Preferably, the single domain antibody or antigen-binding fragment thereof comprises a CDR1 set forth in SEQ ID NO: 43, a CDR2 set forth in SEQ ID NO: 44, and a CDR3 set forth in SEQ ID NO:

45.

2. The single domain antibody comprises: (i) the sequence set forth in SEQ ID NO: 7; (ii) a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequence set forth in SEQ ID NO: 7; or (iii) a sequence having 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 to the sequence set forth in SEQ ID NO:

7. comprising an amino acid sequence selected from:

2. The single domain antibody or antigen-binding fragment thereof according to claim 1, wherein the substitution is preferably a conservative substitution.

3. A polypeptide construct capable of specifically binding to 4-1BB, comprising the single domain antibody or antigen-binding fragment thereof according to claim 1 or 2, and an immunoglobulin Fc domain, Preferably, the immunoglobulin Fc domain is linked to the N-terminus and / or C-terminus (e.g., C-terminus) of the single domain antibody or antigen-binding fragment thereof directly or via a peptide linker; preferably, the peptide linker is ｍ X ｎ ) ｌ G ｍ’ wherein m, m', n, and l are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and X is selected from A and S; preferably, m is selected from 1, 2, 3, 4, and 5, n is selected from 1 and 2, l is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, m' is selected from 0 and 1, and X is selected from A and S); preferably, the peptide linker has the amino acid sequence set forth in SEQ ID NO: 17 or 18; preferably, the immunoglobulin Fc domain is an IgG Fc domain (e.g., an IgG1 Fc domain including CH2 and CH3); Preferably, the immunoglobulin Fc domain comprises the sequence set forth in SEQ ID NO: 19, or a sequence having 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 thereto, or a sequence having one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) thereto; Preferably, the immunoglobulin Fc domain comprises the sequence set forth in SEQ ID NO: 19 or 20; Preferably, said polypeptide construct comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31 or 32.

4. 10. A multispecific antibody comprising a single domain antibody or antigen-binding fragment thereof according to claim 1 or 2, or a polypeptide construct according to claim 3, Preferably, the multispecific antibody specifically binds to 4-1BB and is further capable of specifically binding to one or more other targets; Preferably, the target is a tumor antigen; Preferably, the tumor antigen is one or more selected from the group consisting of 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.

5. A multispecific antibody comprising a first antigen-binding domain specific for 4-1BB and a second antigen-binding domain specific for HER2, the first antigen-binding domain comprises the antibody or antigen-binding fragment thereof of claim 1 or 2; the second antigen-binding domain comprises at least one CDR of a heavy chain variable region of an anti-HER2 antibody and / or at least one CDR of a light chain variable region of an anti-HER2 antibody; Preferably, the second antigen-binding domain comprises the heavy chain variable region of an anti-HER2 antibody, and / or the light chain variable region of an anti-HER2 antibody; Preferably, the anti-HER2 antibody is selected from the group consisting of trastuzumab, pertuzumab, and variants thereof; A multispecific antibody, wherein 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 or additions; e.g. 1, 2, 3, 4, or 5 amino acid substitutions, deletions or additions) compared to the wild-type sequence from which it is derived.

6. A multispecific antibody comprising a first antigen-binding domain specific for 4-1BB and 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: (1) a peptide chain IA comprising a light chain variable region and a light chain constant region (CL) of the second antigen-binding domain; and (2) Peptide chain I-B comprising the heavy chain variable region and heavy chain constant region of the second antigen-binding domain and the first antigen-binding domain; preferably, peptide chain I-B comprising, from the N-terminus to the C-terminus, the adjacent heavy chain variable region and heavy chain constant region of the second antigen-binding domain and the first antigen-binding domain. Including; Preferably, the CL of the peptide chain IA can form a dimer with the CH1 domain of the heavy chain constant region of the peptide chain IB; Preferably, the multispecific antibody comprises two peptide chains IA and two peptide chains IB; preferably, the heavy chain constant regions of the two peptide chains IB form a dimer; Preferably, the domains are linked directly or via a peptide linker; preferably, the peptide linker is ｍ X ｎ ) ｌ G ｍ’ wherein m, m', n and l are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, and X is selected from A and S; preferably, m is selected from 1, 2, 3, 4 and 5, n is selected from 1 and 2, l is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, m' is selected from 0 and 1, and X is selected from A and S; and preferably, said peptide linker has the amino acid sequence set forth in SEQ ID NO: 17 or 18.

7. The following items: (i) the first antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7; (ii) the heavy chain variable region of the second antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, 3, or 5; (iii) the light chain variable region of the second antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, 4, or 6; characterized by one or more of: Preferably, said peptide chain IA comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22, 24 or 26; The multispecific antibody according to claim 5 or 6, wherein said peptide chain IB preferably comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21, 23 or 25.

8. further comprising a third antigen-binding domain specific for HER2; Preferably, 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: (1) a peptide chain II-A comprising a light chain variable region and a light chain constant region (CL) of the second antigen-binding domain; (2) Peptide chain II-B comprising the heavy chain variable region, heavy chain constant region, and the first antigen-binding domain of the second antigen-binding domain; preferably, peptide chain II-B comprising, from the N-terminus to the C-terminus, the adjacent heavy chain variable region and heavy chain constant region of the second antigen-binding domain, and the first antigen-binding domain; (3) a peptide chain II-C comprising a light chain variable region and a light chain constant region (CL) of the third antigen-binding domain; and (4) Peptide chain II-D comprising the heavy chain variable region, heavy chain constant region, and the first antigen-binding domain of the third antigen-binding domain; preferably, the peptide chain II-D comprising, from the N-terminus to the C-terminus, the adjacent heavy chain variable region and heavy chain constant region of the third antigen-binding domain, and the first antigen-binding domain. Including; Preferably, the CL of the peptide chain II-A can form a dimer with the CH1 domain of the heavy chain constant region of the peptide chain II-B; preferably, the CL of the peptide chain II-C can form a dimer with the CH1 domain of the heavy chain constant region of the peptide chain II-D; preferably, the heavy chain constant region of the peptide chain II-B comprises the amino acid sequence set forth in SEQ ID NO: 13, and the heavy chain constant region of the peptide chain II-D comprises the amino acid sequence set forth in SEQ ID NO: 14; preferably, the heavy chain constant region of the peptide chain II-B comprises the amino acid sequence set forth in SEQ ID NO: 37, and the heavy chain constant region of the peptide chain II-D comprises the amino acid sequence set forth in SEQ ID NO: 38; Preferably, the multispecific antibody comprises one peptide chain II-A, one peptide chain II-B, one peptide chain II-C, and one peptide chain II-D; preferably, the heavy chain constant regions of the peptide chains II-B and II-D form a dimer; Preferably, the domains are linked directly or via a peptide linker; preferably, the peptide linker is ｍ X ｎ ) ｌ G ｍ’ wherein m, m', n and l are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, and X is selected from A and S; preferably, m is selected from 1, 2, 3, 4 and 5, n is selected from 1 and 2, l is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, m' is selected from 0 and 1, and X is selected from A and S; and preferably, the peptide linker has the amino acid sequence set forth in SEQ ID NO: 17 or 18.

9. The following items: (i) the first antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7; (ii) the heavy chain variable region of the second antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, 3, or 5; (iii) the light chain variable region of the second antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, 4, or 6; (iv) the heavy chain variable region of the third antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, 3, or 5; (v) the light chain variable region of the third antigen-binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, 4, or 6; characterized by one or more of: Preferably, said peptide chain II-A comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28 or 40; Preferably, said peptide chain II-B comprises or consists of the amino acid sequence set forth in SEQ ID NO: 27 or 39; Preferably, said peptide chain II-C comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30 or 42; The multispecific antibody of claim 8, wherein the peptide chain II-D preferably comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29 or 41.

10. A polynucleotide encoding the antibody of any one of claims 1 to 9.

11. A vector comprising the polynucleotide of claim 10.

12. A recombinant cell comprising the polynucleotide of claim 10 or the vector of claim 11.

13. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described in any one of claims 1, 2, 4 to 9, a polypeptide construct described in claim 3, a polynucleotide described in claim 10, a vector described in claim 11 or a recombinant cell described in claim 12, and a pharmaceutically acceptable excipient.

14. 14. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1, 2, 4 to 9, the polypeptide construct according to claim 3, the polynucleotide according to claim 10, the vector according to claim 11, the recombinant cell according to claim 12 or the pharmaceutical composition according to claim 13 in the manufacture of a medicament for treating a tumor, comprising: Preferably, the tumor overexpresses HER2; Preferably, 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, cutaneous or ocular melanoma, rectal cancer, perianal cancer, esophageal cancer, small intestinal tumors, endocrine gland cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral 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 tumor, bile duct cancer and gallbladder cancer; Preferably, the tumor is a primary or metastatic tumor.

15. Used for tumor treatment; Preferably, the tumor overexpresses HER2; Preferably, 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, cutaneous or ocular melanoma, rectal cancer, perianal cancer, esophageal cancer, small intestinal tumors, endocrine gland cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral 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 tumor, bile duct cancer and gallbladder cancer; Preferably, the tumor is a primary or metastatic tumor, and the antibody or antigen-binding fragment thereof described in any one of claims 1, 2, 4 to 9, the polypeptide construct described in claim 3, the polynucleotide described in claim 10, the vector described in claim 11, the recombinant cell described in claim 12 or the pharmaceutical composition described in claim 13.

16. 14. A method for tumor treatment, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1, 2, 4 to 9, the polypeptide construct of claim 3, the polynucleotide of claim 10, the vector of claim 11, the recombinant cell of claim 12, or the pharmaceutical composition of claim 13, Preferably, the tumor overexpresses HER2; Preferably, 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, cutaneous or ocular melanoma, rectal cancer, perianal cancer, esophageal cancer, small intestinal tumors, endocrine gland cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral 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 tumor, bile duct cancer and gallbladder cancer; Preferably, the tumor is a primary or metastatic tumor.

17. A conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1, 2, 4 to 9, or the polypeptide construct of claim 3, and a detectable label attached to said antibody or antigen-binding fragment thereof or said polypeptide construct, Preferably, the detectable label is selected from the group consisting of an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin.

18. 18. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1, 2, 4 to 9, the polypeptide construct of claim 3, or the conjugate of claim 17, Preferably, the kit further comprises a second antibody capable of specifically recognizing the antigen specifically recognized by the antibody or antigen-binding fragment thereof, or the polypeptide construct; and 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, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin.

19. 18. A method for detecting the presence or level of 4-1BB in a sample, comprising using an antibody or antigen-binding fragment thereof according to any one of claims 1, 2, 4 to 9, a polypeptide construct according to claim 3, or a conjugate according to claim 17, Preferably, the method is an immunoassay such as immunoblotting, enzyme immunoassay (e.g., ELISA), chemiluminescence immunoassay, fluorescence immunoassay or radioimmunoassay; Preferably, the method comprises using the antibody or antigen-binding fragment thereof or the polypeptide construct, and the method further comprises using a second antibody bearing a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin) for detecting binding of the antibody or antigen-binding fragment thereof, or the polypeptide construct to an antigen; Preferably, the method comprises: (1) contacting the sample with the antibody or antigen-binding fragment thereof; and (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex, wherein the formation of the immune complex indicates the presence of 4-1BB or a cell expressing 4-1BB.

20. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1, 2, 4 to 9, the polypeptide construct according to claim 3, or the conjugate according to claim 17 in the manufacture of a detection reagent, wherein the detection reagent is used to detect the presence or level of 4-1BB in a sample; Preferably, the detection reagent detects the presence or level of 4-1BB in the sample by the method of claim 19; Preferably, the sample is a cell sample (e.g., tumor cells) derived from a subject (e.g., a mammal, preferably a human or a monkey).

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