Antibody and application thereof in tumor resistance

Abstract

The invention provides an antibody and an application thereof in tumor resistance. Provided are a single-domain antibody or an antigen-binding fragment thereof capable of specifically binding 4-1BB, and a multispecific antibody, the antibody having improved antitumor activity and lower hepatotoxicity.

Description

Antibodies and their applications in anti-tumor

[0001] This application is based on the application with CN application number 202211142594.0 and application date September 20, 2022, and claims its priority. The disclosed content of the CN application is hereby introduced as a whole into this application. Technical Field

[0002] The present application relates to the field of biotherapy technology, and specifically to antibodies targeting 4-1BB and HER2 and their application in anti-tumor treatment. Background Art

[0003] 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 members of the ERBB family, causing phosphorylation of tyrosine residues in the receptor cytoplasmic domain, thereby activating multiple signaling pathways in the cell, leading to cancer cell proliferation and tumorigenesis. [1] As one of the earliest targets used for tumor treatment, HER2-targeted therapeutic drugs include monoclonal antibodies (mAbs, such as trastuzumab and pertuzumab), tyrosine kinase inhibitors (TKIs, such as neratinib and lapatinib), antibody-drug conjugates (ADCs, such as T-DM1), etc. These drugs have been widely used in the treatment of breast cancer or gastric cancer with HER2 overexpression and / or amplification. [2] Although these HER2-targeted therapeutics have good clinical benefits, medical needs remain unmet, and new drugs need to be continuously developed to further improve the clinical efficacy of patients with recurrent or metastatic disease.

[0004] The T cell co-stimulatory 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 the effector activity and memory response of T cells. [3,4] Urelumab is a monoclonal antibody drug that targets 4-1BB and 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 anti-tumor drugs with better efficacy and higher safety remains an urgent problem to be solved in this field.

[0006] Summary of the Invention

[0007] The present application discloses a single domain antibody with high binding activity to 4-1BB, and a multispecific antibody targeting 4-1BB and HER2 developed on this basis. In particular, the present application discloses a HER2xHER2x4-1BB trispecific antibody, which has unique anti-tumor activity and a strong immune memory effect. The antibody is a 1+1 IgG1-like heterodimer antibody composed of trastuzumab and pertuzumab, and its C-terminus is connected to an anti-4-1BB single domain antibody (sdAb) through a G4S linker. The antibody retains the Fc effector function, which is crucial for trastuzumab and pertuzumab to exert anti-tumor activity. In addition, the antibody has lower liver toxicity and improved safety.

[0008] Single domain antibodies

[0009] In one aspect, the present application provides a single-domain antibody or an antigen-binding fragment thereof that can specifically bind to 4-1BB, wherein the single-domain antibody comprises:

[0010] (a) CDR1 having: a sequence as shown in SEQ ID NO: 43, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 43;

[0011] (b) CDR2 having: a sequence as shown in SEQ ID NO: 44, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 44; and

[0012] (c) CDR3 having: a sequence as shown in SEQ ID NO: 45, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 45.

[0013] In some embodiments, the single-domain antibody or antigen-binding fragment thereof comprises: a CDR1 as shown in SEQ ID NO:43, a CDR2 as shown in SEQ ID NO:44, and a CDR3 as shown in SEQ ID NO:45.

[0014] The single domain antibody comprises an amino acid sequence selected from the group consisting of:

[0015] (i) the sequence shown in SEQ ID NO: 7;

[0016] (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 shown in SEQ ID NO: 7; or

[0017] (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.

[0018] In some embodiments, the substitutions are conservative substitutions.

[0019] The term "4-1BB", also referred to as CD137 or TNFRSF9 (TNF receptor superfamily member 9), is a member of the TNF receptor superfamily (TNFRSF), and is a co-stimulatory molecule that is expressed after immune cells (both innate immune cells and adaptive immune cells) are activated. 4-1BB plays an important role in regulating the activity of various immune cells. As used herein, 4-1BB can be derived from mammals, such as Homo sapiens (people) (NCBI accession number NP_001552.2).

[0020] In some embodiments, the single domain antibody specifically binds to the 4-1BB epitope CRD-4.

[0021] polypeptide constructs

[0022] The present application also provides a polypeptide construct that specifically binds to 4-1BB, which comprises the single-domain antibody or antigen-binding fragment thereof described above, and an immunoglobulin Fc domain.

[0023] 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.

[0024] In some embodiments, the peptide linker has (G m X n ) l G m’ In the structure shown, 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 or 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.

[0025] In some embodiments, the peptide linker has the amino acid sequence shown in SEQ ID NO: 17 or 18.

[0026] In some embodiments, the immunoglobulin Fc domain is an Fc domain of IgG (eg, an Fc domain of IgG1, eg, comprising CH2 and CH3).

[0027] In some embodiments, the immunoglobulin Fc domain comprises the sequence shown 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.

[0028] In some embodiments, the immunoglobulin Fc domain comprises the sequence shown in SEQ ID NO: 19 or 20.

[0029] In some embodiments, the polypeptide construct comprises or consists of the amino acid sequence shown in SEQ ID NO: 31 or 32.

[0030] Multispecific antibodies

[0031] The present application further provides a multispecific antibody that can activate 4-1BB signaling only when cross-linked with tumor cells expressing HER2. In addition, the anti-4-1BB antibody or its antigen-binding fragment contained in the multispecific antibody can be characterized by positioning and / or activating only in the tumor microenvironment (TME), and / or significantly reducing liver toxicity compared to existing anti-4-1BB antibodies, while maintaining the efficacy of immune response enhancement and / or tumor treatment.

[0032] Specifically, the present application provides a multispecific antibody comprising the single-domain antibody or antigen-binding fragment or polypeptide construct described above.

[0033] 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.

[0034] In some embodiments, the first antigen binding domain comprises an antibody or antigen binding fragment thereof as described above.

[0035] In some embodiments, any of the multispecific antibodies described above specifically binds 4-1BB and additionally specifically binds one or more other targets.

[0036] In some embodiments, the target is a tumor antigen.

[0037] In some embodiments, the tumor antigen is selected from one or more of the following: CD19, CD20, CD22, CD23, CD38, CD40, CD49, CD52, CD56, CD74, CD80, CD95, CD138, CS1 / SLAMF7, KiR, Thy-1, Ly-6, Fas, APO-1, EGFR, HER2, CXCR4, HLA, GM1 and DRD.

[0038] In some embodiments, the multispecific antibody can bind to multiple identical or different tumor antigens.

[0039] In some embodiments, the multispecific antibodies can bind to the same or different epitopes on the same tumor antigen.

[0040] In some embodiments, the multispecific antibody specifically binds HER2.

[0041] "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). HER2 is known to play an important role in regulating cell proliferation and differentiation. In particular, when bound to extracellular growth factors, it has a strong tendency to assemble into homo- and / or heterodimers with other HER receptors, which leads to the activation of several forms of signal transduction pathways and induces apoptosis, survival or cell proliferation. For example, the HER2 protein can be a polypeptide deposited with GenBank accession numbers NP_004439.2, NP_001005862.1, etc., which is encoded by nucleotide sequences (mRNA) deposited with GenBank accession numbers NM 004448.4, NM_001005862.3, etc.

[0042] The portion that binds to HER2 and recognizes HER2 as an antigen may be selected from scFv, (scFv) 2 , Fab, Fab′, and F(ab′) 2 of an anti-HER2 antibody.

[0043] In some embodiments, the multispecific antibody contains at least one (e.g., 1, 2, or 3) CDRs of the heavy chain variable region of an anti-HER2 antibody, and / or at least one (e.g., 1, 2, or 3) CDRs of the light chain variable region of an anti-HER2 antibody.

[0044] In some embodiments, the multispecific antibody contains the heavy chain variable region of an anti-HER2 antibody, and / or the light chain variable region of an anti-HER2 antibody.

[0045] In some embodiments, the anti-HER2 antibody is selected from trastuzumab, pertuzumab, and variants thereof.

[0046] In some embodiments, the variant has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions).

[0047] In some embodiments, the anti-HER2 antibody is a variant of trastuzumab. Specifically, the anti-HER2 antibody contains the following three heavy chain variable region (VH) complementarity determining regions (CDRs):

[0048] VH CDR1 shown in SEQ ID NO:46, VH CDR2 shown in SEQ ID NO:47, and VH CDR3 shown in SEQ ID NO:48; and / or,

[0049] The following three light chain variable region (VL) complementarity determining regions (CDRs):

[0050] VL CDR1 shown in SEQ ID NO:49; VL CDR2 shown in SEQ ID NO:50, and VL CDR3 shown in SEQ ID NO:8.

[0051] In some embodiments, the multispecific antibody contains a second antigen binding domain specific for HER2.

[0052] In some embodiments, the first antigen-binding domain is a VHH; the second antigen-binding domain is a Fab, and the multispecific antibody comprises:

[0053] (1) peptide chain IA, which comprises the light chain variable region and light chain constant region (CL) of the second antigen-binding domain;

[0054] and,

[0055] (2) a peptide chain IB comprising the heavy chain variable region, the heavy chain constant region, and the first antigen-binding domain of the second antigen-binding domain; preferably, the peptide chain IB comprises the adjacent heavy chain variable region and heavy chain constant region of the second antigen-binding domain and the first antigen-binding domain from the N-terminus to the C-terminus.

[0056] 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, for example, the heavy chain constant region of human IgG1, for example, CH1, CH2 and / or CH3 of human IgG1, for example, the Fc region of human IgG1.

[0057] In some embodiments, the heavy chain constant region is altered (e.g., mutated) to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function.

[0058] In some embodiments, the heavy chain constant region contains a LALA mutation, a CH3 Knob mutation, a CH3 Hole mutation, a CH1 / CL preferred mutation CH SET1, a CH1 / CL preferred mutation CH SET2CH SET2, and any combination thereof.

[0059] In some embodiments, the heavy chain constant region is selected from:

[0060] HC-1: amino acid sequence shown in SEQ ID NO: 10;

[0061] HC-2: amino acid sequence shown in SEQ ID NO: 11;

[0062] HC-3: amino acid sequence shown in SEQ ID NO: 12;

[0063] HC-4: amino acid sequence shown in SEQ ID NO: 13;

[0064] HC-5: amino acid sequence shown in SEQ ID NO: 14;

[0065] HC-6: amino acid sequence shown in SEQ ID NO: 19;

[0066] HC-7: amino acid sequence shown in SEQ ID NO: 20;

[0067] HC-8: amino acid sequence shown in SEQ ID NO: 37; and

[0068] HC-9: amino acid sequence shown in SEQ ID NO: 38;

[0069] The variant has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions).

[0070] In some embodiments, the light chain constant region is selected from a light chain constant region of kappa or lambda, or a variant thereof;

[0071] The variant has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions).

[0072] In some embodiments, the light chain constant region is selected from:

[0073] LC-1: SEQ ID NO: 9;

[0074] LC-2: SEQ ID NO: 15; and

[0075] LC-3: SEQ ID NO: 16;

[0076] The variant has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions).

[0077] In some embodiments, the CL of the peptide chain IA is capable of forming a dimer with the heavy chain constant region CH1 domain of the peptide chain IB.

[0078] 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.

[0079] In some embodiments, the domains are connected directly or through a peptide linker.

[0080] In some embodiments, the peptide linker has (G m X n ) l G m’In the structure shown, 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 or 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.

[0081] In some embodiments, the peptide linker has the amino acid sequence shown in SEQ ID NO: 17 or 18.

[0082] In some embodiments, the multispecific antibody is characterized by one or more of the following:

[0083] (i) the first antigen-binding domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 7;

[0084] (ii) the heavy chain variable region of the second antigen-binding domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, 3 or 5;

[0085] (iii) the light chain variable region of the second antigen-binding domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 2, 4 or 6.

[0086] In some embodiments, the peptide chain IA contains or consists of the amino acid sequence shown in SEQ ID NO: 22, 24 or 26.

[0087] In some embodiments, the peptide chain IB contains or consists of the amino acid sequence shown in SEQ ID NO: 21, 23 or 25.

[0088] In some embodiments, the multispecific antibody is selected from:

[0089] (1) A multispecific antibody comprising peptide chains IA and IB, wherein the peptide chain IA comprises or consists of the amino acid sequence shown in SEQ ID NO: 22, and the peptide chain IB comprises or consists of the amino acid sequence shown in SEQ ID NO: 21;

[0090] (2) a multispecific antibody comprising peptide chains IA and IB, wherein the peptide chain IA comprises or consists of the amino acid sequence shown in SEQ ID NO: 24, and the peptide chain IB comprises or consists of the amino acid sequence shown in SEQ ID NO: 23;

[0091] and,

[0092] (3) a multispecific antibody comprising peptide chains IA and IB, wherein the peptide chain IA comprises or consists of the amino acid sequence shown in SEQ ID NO: 26, and the peptide chain IB comprises or consists of the amino acid sequence shown in SEQ ID NO: 25;

[0093] Preferably, the multispecific antibody comprises two peptide chains IA and two peptide chains IB;

[0094] Preferably, the heavy chain constant regions of the two peptide chains IB form a dimer.

[0095] In some embodiments, the multispecific antibody further contains a third antigen-binding domain specific for HER2.

[0096] 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:

[0097] (1) peptide chain II-A, which comprises the light chain variable region and light chain constant region (CL) of the second antigen-binding domain;

[0098] (2) peptide chain II-B, which comprises the heavy chain variable region, heavy chain constant region, and the first antigen-binding domain of the second antigen-binding domain; preferably, the peptide chain II-B comprises the adjacent heavy chain variable region and heavy chain constant region of the second antigen-binding domain and the first antigen-binding domain from the N-terminus to the C-terminus;

[0099] (3) peptide chain II-C, which comprises the light chain variable region and light chain constant region (CL) of the third antigen-binding domain;

[0100] and,

[0101] (4) Peptide chain II-D, which comprises 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 comprises the adjacent heavy chain variable region and heavy chain constant region of the third antigen-binding domain and the first antigen-binding domain from the N-terminus to the C-terminus.

[0102] 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, for example, the heavy chain constant region of human IgG1, for example, CH1, CH2 and / or CH3 of human IgG1, for example, the Fc region of human IgG1.

[0103] In some embodiments, the heavy chain constant region is altered (e.g., mutated) to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function.

[0104] In some embodiments, the heavy chain constant region contains a LALA mutation, a CH3 Knob mutation, a CH3 Hole mutation, a CH1 / CL preferred mutation CH SET1, a CH1 / CL preferred mutation CH SET2, and any combination thereof.

[0105] In some embodiments, the light chain constant region is selected from a light chain constant region of kappa or lambda, or a variant thereof;

[0106] The variant has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions).

[0107] In some embodiments, the CL of the peptide chain II-A can form a dimer with the heavy chain constant region CH1 domain of the peptide chain II-B; preferably, the CL of the peptide chain II-C can form a dimer with the heavy chain constant region CH1 domain of the peptide chain II-D.

[0108] 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.

[0109] In some embodiments, the domains are connected directly or through a peptide linker.

[0110] In some embodiments, the peptide linker has (G m X n ) l G m’ In the structure shown, 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.

[0111] In some embodiments, m is selected from 1, 2, 3, 4, or 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.

[0112] In some embodiments, the peptide linker has the amino acid sequence shown in SEQ ID NO: 17 or 18.

[0113] In some embodiments, the multispecific antibody is characterized by one or more of the following:

[0114] (i) the first antigen-binding domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 7;

[0115] (ii) the heavy chain variable region of the second antigen-binding domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, 3 or 5;

[0116] (iii) the light chain variable region of the second antigen-binding domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 2, 4 or 6;

[0117] (iv) the heavy chain variable region of the third antigen-binding domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, 3 or 5;

[0118] (v) The light chain variable region of the third antigen-binding domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 2, 4 or 6.

[0119] In some embodiments, the peptide chain II-A comprises or consists of the amino acid sequence shown in SEQ ID NO: 28 or 40.

[0120] In some embodiments, the peptide chain II-B comprises or consists of the amino acid sequence shown in SEQ ID NO: 27 or 39.

[0121] In some embodiments, the peptide chain II-C comprises or consists of the amino acid sequence shown in SEQ ID NO: 30 or 42.

[0122] In some embodiments, the peptide chain II-D comprises or consists of the amino acid sequence shown in SEQ ID NO: 29 or 41.

[0123] In some embodiments, the multispecific antibody is selected from:

[0124] (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 shown in SEQ ID NO: 28, peptide chain II-B comprises or consists of the amino acid sequence shown in SEQ ID NO: 27, peptide chain II-C comprises or consists of the amino acid sequence shown in SEQ ID NO: 29, and peptide chain II-D comprises or consists of the amino acid sequence shown in SEQ ID NO: 30;

[0125] and,

[0126] (2) A multispecific antibody comprising peptide chains II-A, II-B, II-C and II-D, wherein the peptide chain II-A contains or consists of the amino acid sequence shown in SEQ ID NO:40, the peptide chain II-B contains or consists of the amino acid sequence shown in SEQ ID NO:39, the peptide chain II-C contains or consists of the amino acid sequence shown in SEQ ID NO:42, and the peptide chain II-D contains or consists of the amino acid sequence shown in SEQ ID NO:41.

[0127] Peptide linker

[0128] The N-terminus or C-terminus (preferably the N-terminus) of the portion binding to 4-1BB described herein is directly or via a peptide linker linked to the C-terminus or N-terminus of the heavy chain constant region.

[0129] As used herein, the term "peptide linker" may refer to an oligopeptide comprising 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 may be used with or without appropriate modification to meet specific purposes. In a specific embodiment, the peptide linker may comprise, for example, Gly, Asn and / or Ser residues, and / or neutral amino acids such as Thr and / or Ala. The amino acid sequence suitable for a peptide linker may be known in the relevant art. The length of the peptide linker may be appropriately determined within the limits of not affecting the polypeptide and / or scFv function. 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.

[0130] In some embodiments, the peptide linker has (G m X n )l G m’ In the structure shown, 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.

[0131] In some embodiments, m is selected from 1, 2, 3, 4, or 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.

[0132] In some embodiments, the peptide linker has the amino acid sequence shown in SEQ ID NO: 17 or 18.

[0133] Preparation of polynucleotides, recombinant vectors and antibodies

[0134] In one aspect, the present invention provides a polynucleotide encoding the antibody or antigen-binding fragment thereof described in any one of the preceding claims.

[0135] In one aspect, the present invention provides a vector comprising the polynucleotide described above.

[0136] 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 a recombinant vector.

[0137] In one aspect, the present invention provides a method for preparing the antibody or its antigen-binding fragment, comprising expressing a polynucleotide in a cell. The step of expressing the polynucleotide can be performed by culturing a cell comprising the polynucleotide (e.g., the polynucleotide in a recombinant vector) under conditions that allow expression of the polynucleotide. The method can further comprise, after the expression or culturing step, isolating and / or purifying the antibody or its antigen-binding fragment from the cell culture.

[0138] application

[0139] The present invention further provides use of the antibodies or antigen-binding fragments thereof described herein in enhancing immune response and / or treating tumors.

[0140] 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 foregoing items, and a pharmaceutically acceptable excipient.

[0141] In another aspect, the present invention further provides use of any of the above-mentioned antibodies or antigen-binding fragments thereof, polynucleotides, vectors, recombinant cells or pharmaceutical compositions in the preparation of anti-tumor drugs.

[0142] In another aspect, the present invention provides an anti-tumor method, comprising the step of administering a therapeutically effective amount of any of the above antibodies or antigen-binding fragments thereof, polynucleotides, vectors, recombinant cells or pharmaceutical compositions to a subject in need thereof.

[0143] In some embodiments, the tumor overexpresses HER2.

[0144] In some embodiments, the tumor is selected from breast cancer, colon cancer, stomach cancer, lung cancer (e.g., squamous cell lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma), peritoneal cancer, skin cancer, squamous cell carcinoma, melanoma of the skin or eye, rectal cancer, cancer near the anus, esophageal cancer, small intestine tumor, 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 cancer or uterine cancer, salivary gland tumor, kidney cancer, cervical cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, biliary tract cancer, and gallbladder cancer.

[0145] In some embodiments, the tumor is a primary or metastatic tumor.

[0146] Detection purpose

[0147] In another aspect, the present invention also provides a conjugate comprising the antibody or antigen-binding fragment thereof or polypeptide construct as described in any one of the above aspects, and a detectable label linked to the single domain antibody or antigen-binding fragment thereof or polypeptide construct.

[0148] In some embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0149] In another aspect, the present invention also provides a kit comprising the single domain antibody or antigen-binding fragment thereof or polypeptide construct, or conjugate as described in any one of the above aspects.

[0150] In certain embodiments, the kit further comprises a second antibody that specifically recognizes the antigen specifically recognized by the antibody or antigen-binding fragment thereof or the polypeptide construct; optionally, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0151] In some embodiments, the second antibody targets the same or a different antigenic epitope as the antibody or antigen-binding fragment thereof or the polypeptide construct.

[0152] In another aspect, the present invention also provides a method for detecting the presence or level of 4-1BB in a sample, comprising using the antibody or antigen-binding fragment thereof or polypeptide construct, or conjugate as described in any one of the above aspects.

[0153] In certain embodiments, the method is an immunological assay, such as immunoblotting, an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0154] In certain embodiments, the methods comprise use of a conjugate as described above.

[0155] In certain embodiments, the method comprises using the antibody or antigen-binding fragment thereof or polypeptide construct as described in any one of the above aspects, and the method further comprises using a second antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide or biotin) to detect binding of the single domain antibody or antigen-binding fragment thereof or the polypeptide construct to the antigen.

[0156] In certain embodiments, 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. The formation of the immune complex indicates the presence of 4-1BB or cells expressing 4-1BB.

[0157] 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 as described in any of the above aspects in the preparation of a detection reagent for detecting the presence or level of 4-1BB in a sample.

[0158] In certain embodiments, the detection reagent detects the presence or level of 4-1BB in a sample by a method as described above for detecting the presence or level of 4-1BB in a sample.

[0159] In certain embodiments, the sample is a cell sample (eg, tumor cell) from a subject (eg, a mammal, preferably a human or a monkey).

[0160] definition

[0161] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are standard procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0162] As used herein, "at least one" or "one or more" can mean 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more.

[0163] The expression "comprising" or its synonyms "including," "containing," and "having" are open-ended and do not exclude additional unrecited elements, steps, or ingredients. The expression "consisting of excludes any elements, steps, or ingredients not specified. The expression "consisting essentially of" means that the scope is limited to the specified elements, steps, or ingredients, plus any optional elements, steps, or ingredients that do not materially affect the basic and novel characteristics of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of."

[0164] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes both occurring and not occurring.

[0165] 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 from one or more polypeptides in a covalent or non-covalent manner. Unless otherwise indicated, the terms "polypeptide" and "protein" are used interchangeably herein.

[0166] As used herein, "antibody" refers to an immunoglobulin or a fragment thereof that specifically binds to an antigenic epitope through at least one antigen binding site. In this article, the definition of an antibody encompasses an antigen binding fragment. Therefore, 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 coupling or biological coupling), enzymatically treated, 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, the antibody of the present invention is a mouse monoclonal antibody. In another embodiment, the antibody of the present invention is a humanized monoclonal antibody.

[0167] As used herein, "traditional antibodies" or "full-length antibodies" typically comprise four polypeptides: two heavy chains (HC) and two light chains (LC). Each light chain comprises a "light chain variable region (VL)" and a "light chain constant region (CL)" from N-terminus to C-terminus. Each heavy chain comprises a "heavy chain variable region (VH)" and a "heavy chain constant region (CH)" from N-terminus to C-terminus. The heavy chain constant region may comprise CH1, CH2, and CH3 from N-terminus to C-terminus. In certain immunoglobulin types (e.g., IgM and IgE), the heavy chain constant region may also comprise CH4. The "Fc" fragment refers to a fragment comprising CH2 and CH3, which provides a site for binding to the Fc receptor. The "hinge region" refers to the portion of an antibody that connects the immunoglobulin Fab and Fc fragments. In some cases, the hinge region refers to the portion of a T cell receptor that connects the constant region and the transmembrane domain. Herein, when used for a chimeric antigen receptor, the hinge region may also refer to any functional equivalent. Those skilled in the art can determine the positions of VH, VL, CL, CH1, CH2, CH3 and hinge regions in an antibody based on known algorithms and software. For descriptions of applicable algorithms and software, see, for example, William R. Strohl, Lila M. Strohl, (2012), Antibody structure–function relationships, In Woodhead Publishing Series in Biomedicine, Therapeutic Antibody Engineering, Woodhead Publishing, pp. 37-56.

[0168] Typically, VL and VH each comprise three highly variable "complementarity determining regions (CDRs)" and four relatively conserved "framework regions (FRs)", and are connected in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus. Herein, the CDRs of the light chain variable region (CDRL) may be referred to as CDR-L1, CDR-L2, and CDR-L3, and the CDRs of the heavy chain variable region (CDRH) may be referred to as CDR-H1, CDR-H2, and CDR-H3. It is generally believed that the six CDRs comprising a pair of VH and VL determine the binding specificity of the antigen binding site, but in some cases, other fragments (e.g., single domain antibodies, also known as nanobodies) comprising less 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 in the art, such as using the Kabat, Abm, or Chothia numbering systems. Herein, multiple CDR numbering systems, such as Chothia, Abm, Kabat, and IMGT, may be used for the same variable region. Those skilled in the art will understand that although the CDRs defined by different numbering systems may be different, the CDRs corresponding to the same numbering system represent effective antigen binding sites capable of binding to antigen epitopes. For a description of the CDR numbering system, see, for example, the Kabat numbering system: Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; the Chothia numbering system: Chothia, C. et al. (1987) J. Mol. Biol. 196: 901-917; the IMGT numbering system: Lefranc, M.-P., 2011 (6), IMGT, the International ImMunoGeneTics Information System Cold Spring Harb Protoc.; the Abm numbering system: Martin, ACR and J. Allen (2007) “Bioinformatics tools for antibody engineering,” in S. Dübel (ed.), Handbook of Therapeutic Antibodies. Weinheim: Wiley-VCH Verlag, pp. 95–118.

[0169] In this article, the terms "framework region" and "framework region" can be used interchangeably. As used herein, the terms "framework region", "framework region" or "FR" residues refer to those amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0170] It is generally believed that the "Fv" fragment composed of a VH and a VL through non-covalent interaction is the smallest antigen-binding fragment containing an antigen-binding site. However, single-domain antibodies (VHH, also known as sdAb or nanoantibodies) that only contain 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 disulfide bonds into Fv or scFv, "disulfide-stabilized Fv (dsFv)" or "single-chain disulfide-stabilized Fv (scdsFv or dsscFv)" can be obtained respectively. In some embodiments, for example, the antigen-binding fragment in the single-domain antibody, bispecific antibody, and multispecific antibody of the present invention preferably uses scFv or scdsFv.

[0171] As used herein, "Fab" comprises a complete antibody light chain (VL-CL) and an antibody heavy chain variable region and a heavy chain constant region (VH-CH1, also referred to as Fd). A single-chain "Fab (scFab)" can be obtained by linking the CL and CH1 in "Fab" with a peptide linker. "F(ab')2" essentially comprises two Fab fragments linked by a disulfide bond in the hinge region. "Fab'" is half of F(ab')2, which can be obtained by reducing the disulfide bond in the hinge region of F(ab')2.

[0172] An antibody or antigen-binding fragment may be "monovalent," "bivalent," "trivalent," or "tetravalent," or more, meaning that it has multiple antigen-binding sites (e.g., 1, 2, 3, or 4 or more).

[0173] When the antibody or antigen-binding fragment binds to two or more antigens (e.g., 2, 3, 4, 5, or 6), it can also be referred to as a "multispecific antibody", such as a bispecific antibody, a trispecific antibody, or a tetraspecific antibody, which refers to a multispecific antibody that can bind to 2, 3, or 4 antigens, respectively.

[0174] As used herein, "diabody" refers to an antibody comprising two scFvs, having two antigen-binding sites (divalent), wherein the VH and VL in each scFv are connected by a short peptide linker (about 5-10 amino acid residues), such that the VH and VL chains pair (i.e., the VH of the first scFv and the VL of the second scFv pair, and the VL of the first scFv and the VH of the second scFv pair) to form an antigen-binding site. The diabody can be a bispecific antibody.

[0175] As used herein, an "antigen-binding fragment" of an antibody refers to a portion of a full-length antibody that is less than the full length but comprises at least a portion of the variable region of the full-length antibody (e.g., comprising one or more CDRs and / or one or more antigen-binding sites), and thus retains at least a portion of the full-length antibody's ability to specifically bind to an antigen. An antigen-binding fragment may, for example, include antibody derivatives produced by enzymatic treatment of a full-length antibody, synthetically produced derivatives, and recombinantly produced derivatives. Examples of antigen-binding fragments include, but are not limited to, Fv, scFv, dsFv, scdsFv, Fab, scFab, Fab', F(ab')2, diabodies, Fd and Fd' fragments, and other fragments (e.g., fragments comprising modifications). An antigen-binding fragment may comprise multiple peptide chains, for example, connected by disulfide bonds and / or by peptide linkers and / or constituted by non-covalent interactions.

[0176] As used herein, the term "monoclonal antibody" refers to a group of highly homogeneous antibodies that are essentially identical to one another except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies typically specifically bind to a single antigenic epitope. The monoclonal antibodies described herein can be prepared by any method known in the art, such as by production from transgenic animals (e.g., transgenic mice), by production from immortalized B cells (e.g., B cell hybridomas), or by production using recombinant DNA methods in bacteria, eukaryotic animals, or plant cells, or by isolation from phage antibody libraries. The antibodies or antigen-binding fragments of the present invention are monoclonal antibodies. In one embodiment, the antibodies or antigen-binding fragments thereof of the present invention are humanized monoclonal antibodies.

[0177] A "chimeric antibody" refers to an antibody in which a portion (e.g., CDR, FR, variable region, constant region, or a combination thereof) is identical or homologous to the corresponding sequence in an antibody derived from a particular species, and the remaining portion is identical or homologous to the corresponding sequence in an antibody derived from another species. As used herein, "chimeric antibody" also encompasses antibodies that contain portions belonging to different antibody types or subclasses. In a specific embodiment, the chimeric antibody has a murine antibody variable region and a human antibody constant region.

[0178] As used herein, "humanized antibody" refers to an antibody containing a non-human antibody and a human antibody sequence. Therefore, a humanized antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. A non-human antibody can be an antibody derived from any non-human species or an antibody (e.g., a chimeric antibody) comprising a portion of a non-human species source. Non-human species can, for example, include mice, rats, rabbits, alpacas, or non-human primates. The technology for obtaining humanized antibodies from non-human antibodies is well known to those skilled in the art. Humanized antibodies can be produced by non-human antibodies (e.g., mouse antibodies or chimeric antibodies), for example, by transplanting the CDR sequences of non-human antibodies (e.g., mouse antibodies) into human antibody framework regions. In some cases, in order to maintain the antigen-binding ability and / or stability of the humanized antibody, key amino acid residues of the non-human antibody (e.g., murine antibody) framework sequence can be retained in the human antibody framework region, i.e., "back mutations" are performed (see, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. 81(21): 6851-6855; Neuberger et al. (1984) Nature 312: 604-608).

[0179] As used herein, "percent (%) sequence identity" or "sequence identity" of amino acid sequences has a generally accepted definition in the art, which refers to the percentage of identity between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or a publicly known algorithm). This can be determined using methods known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal Omega, or FASTA software.

[0180] "Affinity" or "binding affinity" is a measure of the strength of the non-covalent binding between an antibody and an antigen. The magnitude of "affinity" is usually reported as the equilibrium dissociation constant, K. D or EC 50 . K D The equilibrium association constant (ka) and the equilibrium dissociation constant (kd) can be calculated: D = kd / ka. Affinity can be determined using conventional techniques known in the art, such as biofilm interferometry (e.g., Octet Fortebio detection system), radioimmunoassay, surface plasmon resonance, enzyme-linked immunosorbent assay, or flow cytometry.

[0181] In this article, the "specific binding" between an antibody and an antigen refers to the binding between the antibody and the antigen with a high affinity. D A value of at least about 10 -6 to at least about 10 -9M or less, such as at least about 10 -6 , at least about 10 -7 , at least about 10 -8 , at least about 10 -9 M or lower.

[0182] As used herein, an amino acid sequence that is "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 the 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 to the wild-type sequence of the heavy chain constant region of the human immunoglobulin from which it is derived.

[0183] 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 appreciate that amino acids in non-essential regions of a polypeptide or protein can be substituted with suitable conservative amino acids, generally without altering their biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224). Suitable conservative substitutions are well known to those skilled in the art. The following table lists some non-limiting examples of common conservative substitutions of amino acid residues. In some cases, an amino acid substitution is a non-conservative substitution. Those skilled in the art will appreciate that amino acid mutations or modifications can be made to antibodies or antibody fragments to alter their properties, such as by changing the type of antibody glycosylation or the 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.

[0184] 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 generally can include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0185] As used herein, "isolated" means that a substance (eg, a nucleic acid molecule or a polypeptide) is separated from its source or environment, ie, does not substantially contain any other components.

[0186] As used herein, a "vector" is a medium for introducing exogenous nucleic acid into a host cell, which, when transformed into an appropriate host cell, is amplified or expressed. The vector typically remains episomal, but can be designed to integrate a gene or part thereof into a chromosome of the genome. As used herein, the definition of vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), and the like.

[0187] As used herein, "expression vector" refers to a vector capable of expressing DNA, which is operably linked to regulatory sequences (such as promoters, ribosome binding sites) that can affect DNA expression. Regulatory sequences can include promoter and terminator sequences, and optionally can include an origin of replication, a selective marker, an enhancer, a polyadenylation signal, etc. The expression vector can be a plasmid, a phage vector, a recombinant virus, or other vector that, when introduced into an appropriate host cell, results in the 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 cells and / or prokaryotic cells, as well as expression vectors that remain free or that are integrated into the host cell genome.

[0188] As used herein, a "recombinant cell" is a cell that is used to receive, maintain, replicate, or amplify a vector. A recombinant cell can also be used to express a nucleic acid or polypeptide encoded by a vector. A recombinant cell can be a eukaryotic cell or a prokaryotic cell.

[0189] As used herein, the term "treatment" refers to the improvement of a disease / symptom, such as alleviating or eliminating the disease / symptom, preventing or slowing the occurrence, progression and / or worsening of the disease / symptom. Therefore, treatment includes prevention, therapy and / or cure.

[0190] An "effective amount" refers to a dose that is sufficient to reduce the severity of disease symptoms, increase the frequency and duration of asymptomatic periods of the disease, or prevent damage or disability caused by the pain of the disease. An "effective amount" refers to the amount required to prevent, cure, improve, block, or partially block a disease or symptom. For example, for the treatment of tumors, an "effective amount" of an antibody or antigen-binding fragment thereof, or pharmaceutical composition of the present invention preferably inhibits tumor cell growth 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%, relative to an untreated subject. The efficacy of inhibiting tumor growth can be assessed 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 growth can be examined using in vitro assays known in the art. An effective amount of an antibody or antigen-binding fragment thereof, or pharmaceutical composition of the present invention can reduce tumor size, or otherwise alleviate the symptoms of the subject (such as preventing and / or treating metastasis or recurrence). One skilled in the art can determine the effective amount based on factors such as the subject's age, physical condition, sex, severity of symptoms, specific composition or route of administration, etc. The effective amount can be administered in one or more administrations.

[0191] As used herein, the term "pharmaceutically acceptable excipient" refers to an excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, carbohydrates (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein), or degradation products thereof (such as lactalbumin hydrolysate), and the like.

[0192] As used herein, examples of mammals include, but are not limited to, humans, non-human primates, rats, mice, cattle, horses, pigs, sheep, dogs, cats, and the like. 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 of developing cancer. As used herein, the terms "subject" and "subject" are used interchangeably. BRIEF DESCRIPTION OF THE DRAWINGS

[0193] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0194] FIG1 shows a schematic diagram of the structure of the multispecific antibody described in the present application.

[0195] FIG2 shows the binding of the multispecific antibody of the present invention to human HER2 detected on HER2-overexpressing cells N87 and CT26.

[0196] FIG3 shows the binding of the multispecific antibody of the present invention to human 4-1BB detected on CHO-h4-1BB cells overexpressing human 4-1BB.

[0197] FIG4 shows the binding of the multispecific antibody of the present invention to FcR.

[0198] FIG5 shows that the multispecific antibodies of the present invention block the proliferation of HER2 signaling-dependent cells N87 or SK-OV-3.

[0199] FIG6 shows that the multispecific antibody of the present invention mediates ADCC via HER2.

[0200] FIG7 shows that HER2 mediates the multispecific antibody of the present invention to induce Primary T cells to release cytokines IL-2 and IFN-γ.

[0201] FIG8 shows that FcR mediates the multispecific antibody of the present invention to induce primary T cells to release cytokines IL-2 and IFN-γ.

[0202] FIG9 shows the SEC and LC / MS detection results of the multispecific antibody HER2xHER2x4-1BB of the present invention.

[0203] Figure 10 shows the tumor inhibitory activity of the multispecific antibody of the present invention in a CT26-h-HER2 tumor model inoculated subcutaneously in h-4-1BB KI BALB / c mice. Figure 10A shows the effect on tumor volume 0-15 days after administration. Figure 10B shows the infiltration of CD8+ T cells in mouse tumors.

[0204] FIG11 shows the tumor inhibitory activity of the multispecific antibody of the present invention in a tumor model in which h-4-1BB KI BALB / c mice were subcutaneously inoculated with CT26-h-HER2 (effect on tumor volume 11-27 days after administration).

[0205] FIG12 shows the anti-tumor activity of the multispecific antibody of the present invention in a tumor model in which h-4-1BB KI C57 mice were subcutaneously inoculated with MC38-h-HER2. DETAILED DESCRIPTION

[0206] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and in no way constitutes any limitation on the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0207] Sequence Information Table

[0208] Example 1. Cloning and expression of multispecific antibodies

[0209] In this example, three HER2x4-1BB bispecific antibodies, two anti-HER2xHER2x4-1BB trispecific antibodies, and two 4-1BB monoclonal antibodies were constructed, namely:

[0210] 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 shown in SEQ ID NO: 21, which contains the heavy chain variable region amino acid sequence (SEQ ID NO: 1) of the anti-HER2 monoclonal antibody Trastuzumab (patent application number: WO1992022653A1) and the human IgG1 amino acid sequence (SEQ ID NO: 10). The N-terminus of the CD137 binding region amino acid sequence (SEQ ID NO: 7) of the anti-CD137 single-domain antibody AB24 ME is connected to the C-terminus of the Fc through a flexible peptide of 20 amino acid residues (G4A)4 (SEQ ID NO: 18). Peptide chain #2 has the amino acid sequence shown in SEQ ID NO:22, which comprises the light chain variable region amino acid sequence (SEQ ID NO:2) of the anti-HER2 monoclonal antibody Trastuzumab (patent application number: WO1992022653A1), and the human κ light chain constant region (CL) amino acid sequence (SEQ ID NO:9) at the C-terminus of the VL amino acid sequence.

[0211] HER2x4-1BB-2: It consists of two polypeptide chains, the schematic structure of which is shown in Figure 1. Peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 23, which contains the heavy chain variable region amino acid sequence (SEQ ID NO: 1) of the anti-HER2 monoclonal antibody Trastuzumab (patent application number: WO1992022653A1) and the human IgG1 amino acid sequence (introducing the LALA mutation to reduce Fc function, SEQ ID NO: 11). The N-terminus of the CD137 binding region amino acid sequence (SEQ ID NO: 7) of the anti-CD137 single-domain antibody AB24 ME is connected to the C-terminus of the Fc through a flexible peptide of 20 amino acid residues (G4A)4 (SEQ ID NO: 18). Peptide chain #2 has the amino acid sequence shown in SEQ ID NO:24, which comprises the light chain variable region amino acid sequence (SEQ ID NO:2) of the anti-HER2 monoclonal antibody Trastuzumab (patent application number: WO1992022653A1), and the human κ light chain constant region (CL) amino acid sequence (SEQ ID NO:9) at the C-terminus of the VL amino acid sequence.

[0212] HER2x4-1BB-3: It consists of two polypeptide chains, the structure of which is shown in Figure 1. Peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 25, which contains the heavy chain variable region amino acid sequence (SEQ ID NO: 3) of the anti-HER2 monoclonal antibody Pertuzumab (patent application number: US7449184) and the human IgG1 amino acid sequence (SEQ ID NO: 12). The N-terminus of the CD137 binding region amino acid sequence (SEQ ID NO: 7) of the anti-CD137 single-domain antibody AB24 ME is connected to the C-terminus of the Fc through a flexible peptide of 20 amino acid residues (G4A)4 (SEQ ID NO: 18). Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 26, which contains the light chain variable region amino acid sequence (SEQ ID NO: 4) of the anti-HER2 monoclonal antibody Pertuzumab (patent application number: US7449184) and the human kappa light chain constant region (CL) amino acid sequence (SEQ ID NO: 9) at the C-terminus of the VL amino acid sequence.

[0213] HER2xHER2x4-1BB-1: It consists of four polypeptide chains, the schematic diagram of which is shown in Figure 1. Peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 27, which contains the heavy chain variable region amino acid sequence of the anti-HER2 monoclonal antibody (SEQ ID NO: 5) and the human IgG1 amino acid sequence introduced with the CH3 Knob mutation and the CH1 / CL preference mutation CH SET1 (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 AB24 ME is connected to the C-terminus of the Fc through a flexible peptide of 20 amino acid residues (G4A)4 (SEQ ID NO: 18). Peptide chain #2 has the amino acid sequence set forth in SEQ ID NO:28, comprising 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 introduced at the C-terminus of the VL amino acid sequence, the CH1 / CL-preferring mutation CL SET1 (patent application number: WO2021067404A2, SEQ ID NO:15). Peptide chain #3 has the amino acid sequence set forth in SEQ ID NO:29, comprising the amino acid sequence of the heavy chain variable region of the anti-HER2 monoclonal antibody Pertuzumab (patent application number: US7449184) (SEQ ID NO:3), and the human IgG1 amino acid sequence introduced with the CH3 Hole mutation and the CH1 / CL-preferring mutation CH SET2 (patent application number: WO2021067404A2, SEQ ID NO:14). The N-terminus of the CD137 binding region amino acid sequence (SEQ ID NO: 7) of the anti-CD137 single-domain antibody AB24 ME is connected to the C-terminus of the Fc via a flexible peptide of 20 amino acid residues (G4A)4 (SEQ ID NO: 18). Peptide chain #4 has the amino acid sequence shown in SEQ ID NO: 30, which comprises the light chain variable region amino acid sequence (SEQ ID NO: 4) of the anti-HER2 monoclonal antibody Pertuzumab (patent application number: US7449184), and the human kappa light chain constant region (CL) amino acid sequence at the C-terminus of the VL amino acid sequence. The CH1 / CL preferential mutation CL SET2 (patent application number: WO2021067404A2, SEQ ID NO: 16) is introduced.

[0214] 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 has the amino acid sequence shown in SEQ ID NO: 39. It contains the amino acid sequence of the heavy chain variable region of the anti-HER2 monoclonal antibody trastuzumab (patent application number: WO1992022653A1) (SEQ ID NO: 1) and the human IgG1 amino acid sequence HC-1 (SEQ ID NO: 37, patent application number: PCT / CN2017 / 111310) that can spontaneously form heterodimers. The N-terminus of the CD137 binding region amino acid sequence (SEQ ID NO: 7) of the anti-CD137 single-domain antibody AB24 ME is linked to the C-terminus of the Fc via a flexible peptide of 20 amino acid residues (G4A)4 (SEQ ID NO: 18). Peptide chain #2 has the amino acid sequence set forth in SEQ ID NO:40 and comprises the light chain variable region amino acid sequence (SEQ ID NO:2) of the anti-HER2 monoclonal antibody trastuzumab (patent application number: WO1992022653A1), and the human kappa light chain constant region (CL) amino acid sequence (SEQ ID NO:9) at the C-terminus of the VL amino acid sequence. Peptide chain #3 has the amino acid sequence set forth in SEQ ID NO:41 and comprises the heavy chain variable region amino acid sequence (SEQ ID NO:3) of the anti-HER2 monoclonal antibody pertuzumab (patent application number: US7449184), and the human IgG1 amino acid sequence HC-2 (SEQ ID NO:38, patent application number: PCT / CN2017 / 111310), which can spontaneously form heterodimers, according to the method described in patent application number: 201611016435.0. The N-terminus of the CD137 binding region amino acid sequence (SEQ ID NO: 7) of the anti-CD137 single-domain antibody AB24 ME was linked to the C-terminus of the Fc via a flexible peptide of 20 amino acid residues (G4A)4 (SEQ ID NO: 18). Peptide chain #4 has the amino acid sequence shown in SEQ ID NO: 42, which comprises the light chain variable region amino acid sequence (SEQ ID NO: 4) of the anti-HER2 monoclonal antibody Pertuzumab (patent application number: US7449184), and the human kappa light chain constant region (CL) amino acid sequence (SEQ ID NO: 9) at the C-terminus of the VL amino acid sequence.

[0215] Anti-4-1BB-VHH-1: consists of two polypeptide chains and has the amino acid sequence shown in SEQ ID NO: 31, which includes the human IgG1 Fc amino acid sequence (SEQ ID NO: 19) and the N-terminus of the CD137 binding region amino acid sequence (SEQ ID NO: 7) of the anti-CD137 single-domain antibody AB24 ME is connected to the C-terminus of Fc through a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO: 17).

[0216] Anti-4-1BB-VHH-2: consists of 2 polypeptide chains and has the amino acid sequence shown in SEQ ID NO: 32, which includes the human IgG1 Fc amino acid sequence (LALA mutation introduced to reduce Fc function, SEQ ID NO: 20) and the N-terminus of the CD137 binding region amino acid sequence (SEQ ID NO: 7) of the anti-CD137 single-domain antibody AB24 ME is connected to the C-terminus of Fc through a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO: 17).

[0217] In this example, positive control molecules Urelumab and PRS343 were constructed:

[0218] Urelumab: The control molecule Urelumab (patent application number: WO2004010947) consists of two polypeptide chains, peptide chain #1 has the amino acid sequence shown in SEQ ID NO:33, and peptide chain #2 has the amino acid sequence shown in SEQ ID NO:34.

[0219] PRS-343: The control molecule PRS-343 (patent application number: WO2016177802A1) consists of two polypeptide chains, peptide chain #1 has the amino acid sequence shown in SEQ ID NO:35, and peptide chain #2 has the amino acid sequence shown in SEQ ID NO:36.

[0220] Example 2. Multispecific Antibodies Binding to Human HER2

[0221] In this experiment, N87 cells (self-expressing HER2) and CT26-hHER2 cells (overexpressing human HER2) were digested with 0.25% EDTA trypsin, washed once with culture medium, and then the cell density was adjusted to 2×10 6Cells were diluted 100 μl / well in a 96-well flow cytometry plate and centrifuged for later use. Serially diluted antibodies were added to the 96-well flow cytometry plate at 100 μl / well and incubated at 4°C for 60 min. After washing twice with PBS, 100 μl / well of Goat anti-human IgG-Fc (PE) (Abcam, ab98596) diluted 1000-fold with 2% BSA solution was added and incubated at 4°C for 60 min. After washing twice with PBS, cells were resuspended in 100 μl / well of PBS and analyzed on a CytoFlex (Beckman) flow cytometer, and the corresponding mean fluorescence intensity (MFI) was calculated.

[0222] The experimental results are shown in Figure 2 and Table 1-2. The HER2xHER2x4-1BB trispecific antibody and HER2x4-1BB bispecific antibody of the present invention bind to HER2 expressed on human gastric cancer cells N87 (Figure 2A) and CT26 cells overexpressing human HER2 (Figure 2B), and the binding activity is comparable to that of HER2 monoclonal antibodies (Trastuzumab or Pertuzumab).

[0223] Table 1: Binding of multispecific antibodies to HER2 overexpressed on human gastric cancer cell N87

[0224] Table 2: Binding of multispecific antibodies to human HER2 overexpressed on CT26

[0225] Example 3. Multispecific Antibodies Binding to Human 4-1BB

[0226] In this experiment, the cell density of CHOS-h4-1BB (overexpressing human 4-1BB) cells was adjusted to 2×10 6 Cells were diluted 100 μl / well in a 96-well flow cytometer and centrifuged for later use. Serially diluted antibodies were added to the 96-well flow cytometer at 100 μl / well and incubated at 4°C for 60 min. After washing twice with PBS, 100 μl / well of Goat anti-human IgG-Fc (PE) (Abcam, ab98596) diluted 1000-fold in 2% BSA solution was added and incubated at 4°C for 60 min. After washing twice with PBS, cells were resuspended in 100 μl / well of PBS and analyzed on a CytoFlex (Beckman) flow cytometer, and the corresponding MFI was calculated.

[0227] The experimental results are shown in Figure 3 and Table 3. The HER2xHER2x4-1BB trispecific antibody and HER2x4-1BB bispecific antibody of the present invention both have binding activity to CHOS-h4-1BB cells, and the binding activity is comparable to that of the 4-1BB monoclonal antibody molecule (anti-4-1BB VHH), but weaker than that of the control antibodies Urelumab and PRS-343.

[0228] Table 3: Binding of multispecific antibodies to human 4-1BB overexpressed on CHOS cells

[0229] Example 4. Binding of multispecific antibodies to human FcR

[0230] In this experiment, the cell density of THP-1 (self-expressing FcRs) and CHOS-hCD32b (overexpressing human FcγRIIb) cells was adjusted to 2×10 6 Cells were diluted 100 μl / well in a 96-well flow cytometer and centrifuged for later use. Serially diluted antibodies were added to the 96-well flow cytometer containing cells at 100 μl / well and incubated at 4°C for 60 min. The cells were washed twice with PBS, and Goat anti-human IgG-Fc (PE) (Abcam, ab98596) diluted 1000-fold in 2% BSA solution was added at 100 μl / well and incubated at 4°C for 60 min. The cells were washed twice with PBS, and finally, 100 μl / well of PBS was added to resuspend the cells. The cells were analyzed on a CytoFlex (Beckman) flow cytometer, and the corresponding mean fluorescence intensity (MFI) was calculated.

[0231] As shown in Figure 4 and Tables 4-5, the experimental results show that the HER2xHER2x4-1BB trispecific antibody and HER2x4-1BB bispecific antibody of the present invention, which retain Fc effector function, bind to FcγRIIb expressed on both THP-1 (self-expressing FcRs, Figure 4A) and CHOS-hCD32b (overexpressing human FcRs, Figure 4B) cells. The HER2xHER2x4-1BB trispecific antibody incorporates a Knob in Hole mutation in its Fcc, resulting in slightly weaker FcR binding activity than the HER2x4-1BB bispecific antibody and HER2 monoclonal antibody, which utilize wild-type human IgG1 Fc. Urelumab and PRS-343 utilize wild-type human IgG4 Fc or human IgG4 Fc with the FALA mutation, respectively, resulting in weaker FcR binding affinity than multispecific antibodies that retain Fc effector function.

[0232] Table 4: Binding of multispecific antibodies to FcR (THP-1)

[0233] Table 5: Binding of multispecific antibodies to FcR (CHOS-hCD32b)

[0234] Example 5. Multispecific antibodies block HER2 signaling-dependent cell proliferation

[0235] In this experiment, N87 and SK-OV-3 cells (which express HER2) were digested with 0.25% EDTA trypsin, washed once with culture medium, and then the cell density was adjusted to 5×10 4 cells / ml, 80μl / well was added to the 96-well plate and set aside. The gradient diluted antibody was added to the 96-well plate with cells at 80μl / well and placed in a cell culture incubator for 3-5 days. The chemiluminescent signal was collected using a microplate reader after color development using the Luminescent Cell Viability Assay (Promega, G7572) kit.

[0236] The results, as shown in Figures 5A and 5B, show that all multispecific antibodies significantly inhibited the proliferation of N87 or SK-OV-3 cells. The HER2xHER2x4-1BB trispecific antibody exhibited significantly stronger inhibitory effects on cell proliferation than the HER2-4-1BB bispecific antibody or the HER2 monoclonal antibody. Furthermore, the inhibitory effect of HER2 antibodies on tumor cells was closely correlated with the level of HER2 expression on the cells; higher HER2 expression levels resulted in a more pronounced inhibitory effect on cell proliferation.

[0237] Example 6. Multispecific Antibody-Induced ADCC Effect (Reporter Gene)

[0238] In this experiment, N87 cells (which express HER2) and CHOS-h4-1BB cells (which overexpress human 4-1BB) were cultured and cultured at a rate of 3×10 4 / hole and 1.2×10 5 A mixture of NFAT Luciferase / Jurkat CD16a (overexpressing CD16a and NFAT-Luc) effector cells was seeded into a 96-well white-bottom cell culture plate. A serially diluted multispecific antibody was then added to the 96-well plate and mixed thoroughly. The plate was then incubated in a cell culture incubator for 6 hours. The color was developed using the Bio-Glo luciferase assay system (Promega, G7940), and the chemiluminescent signal was collected using a microplate reader.

[0239] The experimental results, as shown in Figures 6A and 6C and Tables 6-7, show that all multispecific antibodies that retain Fc effector function can mediate ADCC through HER2 expressed on N87 cells, thereby activating the CD16a-NFAT signaling pathway on Jurkat cells. In contrast, the HER2x4-1BB-2 bispecific antibody, which eliminates the Fc effector function through the LALA mutation, and the control molecule PRS-343, which introduces the FALA mutation on the IgG4 Fc, failed to induce ADCC. Furthermore, when CHOS-h4-1BB cells were used as target cells, as shown in Figure 6B, no ADCC effect was observed against 4-1BB cells for any of the multispecific antibodies.

[0240] Table 6: ADCC effect induced by multispecific antibodies (corresponding to Figure 6A)

[0241] Table 7: ADCC effect induced by multispecific antibodies (corresponding to Figure 6C)

[0242] Example 7. Multispecific Antibodies Induce Cytokine Release from Primary T Cells (Mediated by HER2+ Cells)

[0243] The expanded target cells N87 (which express HER2) were digested with 0.25% EDTA trypsin and 1×10 4 10 cells / well were seeded into 96-well culture plates (the culture plates were coated with 2 μg / ml anti-CD3 antibody one day in advance, and 100 μl / well was plated). PBMCs that had been revived one day in advance were collected, and T cells in PBMCs were isolated using a T cell isolation kit (Stemcell, 17951) and plated at 5×10 cells / well. 4 Target cells were added to the target wells, followed by serially diluted multispecific antibodies. After incubation for 48 hours, the supernatant was collected. IL-2 and IFNγ levels in the supernatant were measured using a human IL-2 ELISA kit (Invitrogen, 88-7025-77) and a human IFNγ ELISA kit (Invitrogen, 88-7316-77).

[0244] As shown in Figure 7, the multispecific antibodies of the present invention all induced primary T cells to release IL-2 (Figure 7A) or IFN-γ (Figure 7B) in the presence of N87 cells in a dose-dependent manner. Furthermore, the HER2xHER2x4-1BB trispecific antibody induced significantly stronger cytokine release than the HER2x4-1BB bispecific antibody and the control antibodies PRS-343 and Urelumab.

[0245] Example 8. Multispecific Antibodies Induce Cytokine Release from Primary T Cells (FcR+ Cell-Mediated)

[0246] The expanded target cells CHOS-hCD32b (overexpressing human FcγRIIb) were centrifuged and counted, and 1×10 4 10 cells / well were seeded into 96-well culture plates (the culture plates were coated with 2 μg / ml anti-CD3 antibody one day in advance, and 100 μl / well was plated). PBMCs that had been revived one day in advance were collected, and T cells in PBMCs were isolated using a T cell isolation kit (Stemcell, 17951) and plated at 5×10 cells / well. 4 Target cells were added to the target wells, followed by serially diluted multispecific antibodies. After incubation for 48 hours, the supernatant was collected. IL-2 and IFNγ levels in the supernatant were measured using a human IL-2 ELISA kit (Invitrogen, 88-7025-77) and a human IFNγ ELISA kit (Invitrogen, 88-7316-77).

[0247] As shown in Figure 8 , the multispecific antibodies of the present invention failed to induce primary T cells to release IL2 (Figure 8A) or IFN-γ (Figure 8B) in the presence of CHOS-hCD32b cells. In contrast, the positive control antibody, urelumab, was able to induce T cell activation and release of proinflammatory cytokines via FcRs. Furthermore, the negative control molecule, PRS-343, also did not induce primary T cells to release cytokines via FcRs, as it does not bind to CHOS-hCD32b cells.

[0248] Example 9. Screening of cell lines

[0249] This example uses CH1 / CL preference mutation (patent number: WO2021 / 067404A2) and Knob in hole technology to construct the heavy chain variable regions of the anti-HER2 antibody and the Pertuzumab antibody into the CH1 mutant CH SET1 (SEQ ID NO: 13) and CH SET2 (SEQ ID NO: 14), respectively, and the N-terminus of the CD137 binding region amino acid sequence (SEQ ID NO: 7) of the anti-CD137 single-domain antibody AB24 ME is connected to the C-terminus of Fc through a flexible peptide of 20 amino acid residues (G4A) 4 (SEQ ID NO: 18). The light chain variable regions of the anti-HER2 antibody and the Pertuzumab antibody are respectively constructed into CL SET1 (SEQ ID NO: 15) and CL SET2 (SEQ ID NO: 16) on the CL mutant light chain constant region. A 1+1 asymmetric anti-HER2xHER2x4-1BB-1 trispecific antibody cell line was constructed.

[0250] Cell line construction

[0251] The vector pCHO2.0-GS-Puro-H1-L1 containing the heavy chain gene (SEQ ID NO: 27) and light chain gene (SEQ ID NO: 28) of the anti-HER2 antibody and anti-4-1BB antibody, and the vector pCHO2.0-GS-Puro-H2-L2 containing the heavy chain gene (SEQ ID NO: 29) and light chain gene (SEQ ID NO: 30) of the anti-Pertuzuamb anti-4-1BB antibody were co-transfected into the host cell CHOS-ADP by electroporation. The cells were pressure-screened using Puromycin and MSX screening pressure to obtain a high-yield minipool. Then, through a round of limiting dilution and monoclonal identification, a high-yield and stable clonal cell line was obtained.

[0252] Cell culture

[0253] Dynamis AGT Medium was used as the basal medium, and the inoculation density was (1.0±0.2)×10 6cells / ml, and 5.0±0.5% (w / w) of the initial culture weight of 7a was added as feed on the 3rd, 5th, 7th, 9th and 11th days, and 0.5±0.05% (w / w) of the initial culture weight of 7b was added as feed. The dissolved oxygen was set at 40%, the initial culture temperature was 36.5°C, and the temperature was lowered to 33.0°C on the 4th day. Every day, according to the glucose concentration test results, 300g / kg of glucose concentrate was added to make the glucose concentration in the cell fluid reach 6.0g / L, except on the day of harvest. The culture was terminated on the 14th day or when the cell viability was lower than 80%. During the culture process, the cell density and viability were detected using Vicell (Beckman Company), and the antibody production was detected every day starting from the 7th day using Cedex (Roche Company).

[0254] Stable cell line product quality identification

[0255] The one-step purification method was the same as that used for the one-armed antibody in Example 1. Protein purity was determined by HPLC. The HPLC method was as follows: mobile phase: 150 mM Na₂HPO₄·12H₂O, pH 7.0. Chromatographic conditions: detection wavelength: 280 nm, column temperature: 25°C, flow rate: 0.5 ml / min, detection time: 30 min, TSKgel G3000SWXL column. SEC results, as shown in Figure 9A, indicate a 98.3% purity of the bispecific antibody obtained by one-step affinity purification.

[0256] The heavy and light chain pairing of the protein was determined by high-performance liquid chromatography-mass spectrometry using a Vanquish UHPLC system (Thermo), a Q Exactive mass spectrometer (Thermo), and a Waters ACQUITY UPLC BEH C4 column, 2.1 mm × 100 mm. A 50 μg sample was diluted to 25 μl with ultrapure water. After centrifugation, 20 μl of the sample was transferred to an injection vial, and 5 μl was injected for intact molecular weight analysis using LC-MS. Chromatographic conditions were: column temperature: 80°C; UV detection wavelength: 280 nm; flow rate: 0.3 mL / min; mobile phase A: aqueous solution (containing 0.1% formic acid); mobile phase B: acetonitrile solution (containing 0.1% formic acid). Mass spectrometry parameters were: ESI source: ion transfer 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, as shown in Figures 9B and 9C, showed that the purified trispecific antibody HER2xHER2x4-1BB-1 had a corrected product ratio of >98%.

[0257] Example 10. Study on tumor inhibitory activity of multispecific antibodies

[0258] This experiment conducted proof-of-concept testing of the anti-tumor activity of the multispecific antibody of the present invention in a CT26-h-HER2 tumor model in which h-4-1BB KI BALB / c mice were subcutaneously inoculated.

[0259] First, a tumor-bearing mouse model was established by subcutaneously inoculating CT26-h-HER2 cells. 3 Around 6:00 p.m., mice were divided into groups and given intraperitoneal injections of 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 (all groups received equal molar doses). Tumor volume and body weight changes in each group were monitored every 2-3 days for 2-3 weeks. The dosage and method of administration are shown in Table 8. After the experiment, tumors were harvested from the mice for immunohistochemistry to compare the infiltration of CD8+ T cells in the tumors of the different groups.

[0260] As shown in Figure 10A, the anti-tumor activity of the bispecific antibody HER2x4-1BB-1, which retains Fc effector function, was significantly superior to that of the HER2x4-1BB-2 bispecific antibody, which has the LALA mutation and lacks Fc effector function (TGI: 73.2% vs. 26.6%). It was also stronger than the control antibody PRS-343 (TGI: 6.5%) and the HER2 monoclonal antibody trastuzumab (TGI: 4.2%). Similarly, immunohistochemistry (Figure 10B) showed that CD8+ T cell infiltration in tumors of mice treated with the HER2x4-1BB-1 bispecific antibody was significantly stronger than that of the control antibody PRS-343, trastuzumab, and the HER2x4-1BB-2 bispecific antibody.

[0261] Table 8: Dosing regimen for tumor inhibitory activity studies of multispecific antibodies

[0262] Example 11. Study on tumor inhibitory activity of multispecific antibodies

[0263] In this experiment, the anti-tumor activity of the multispecific antibody of the present invention was determined in a tumor model in which h-4-1BB KI BALB / c mice were subcutaneously inoculated with CT26-h-HER2.

[0264] First, a tumor-bearing mouse model was established by subcutaneously inoculating h-HER2CT26 cells. 3The mice were divided into groups at around 3 pm and given intraperitoneal injection of 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, G8: 3.6 mg / kg HER2xHER2x4-1BB-2 (all groups received equal molar doses). The changes in tumor volume and body weight of the mice in each group were monitored at a frequency of 2-3 days / time for 2-3 weeks. The dosage and method of administration are shown in Table 9.

[0265] As shown in Figure 11, the anti-tumor activity of the HER2xHER2x4-1BB trispecific antibody molecule (TGI: 85.7%) was significantly superior to that of equimolar doses of the control antibodies PRS-343 (TGI: 37.7%), Enhertu (TGI: 47.7%), and the combination of the HER2 monoclonal antibodies trastuzumab and pertuzumab (TGI: 39.5%). In addition, the anti-tumor activity of the HER2xHER2x4-1BB trispecific antibody was significantly stronger than that of the HER2x4-1BB bispecific antibodies HER2x4-1BB-1 (TGI: 66.9%) and HER2x4-1BB-3 (TGI: 59.7%), and was comparable to the therapeutic effect of the HER2x4-1BB bispecific antibody combination treatment group (HER2x4-1BB-1 + HER2x4-1BB-3, TGI: 83.7%).

[0266] Table 9: Dosing regimen for tumor inhibitory activity studies of multispecific antibodies

[0267] Example 12. Study on tumor inhibitory activity of multispecific antibodies

[0268] In this experiment, the anti-tumor activity of the multispecific antibody of the present invention was determined in a tumor model in which h-4-1BB KI C57 mice were subcutaneously inoculated with MC38-h-HER2.

[0269] First, a tumor-bearing mouse model was established by subcutaneously inoculating MC38-h-HER2 cells. 3The mice were divided into groups at around 3 pm and given intraperitoneal injection of 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, G6: 3.6 mg / kg HER2xHER2x4-1BB-1 (all groups received equal molar doses). The tumor volume and body weight changes of the mice in each group were monitored at a frequency of 3-6 days / time for 2-3 weeks. The dosage and method of administration are shown in Table 10.

[0270] As shown in Figure 12, all mice treated with the HER2xHER2x4-1BB trispecific antibody achieved complete tumor regression, significantly superior to an equimolar dose of the control antibody PRS-343 (TGI: 54.9%) and the combination of HER2 monoclonal antibodies (Trastuzumab + Pertuzumab, TGI: 63.8%). Furthermore, in this tumor model, the combination of the HER2x4-1BB bispecific antibody (HER2x4-1BB-1 + HER2x4-1BB-3) and Enhertu also achieved complete tumor regression.

[0271] Table 10: Dosing regimen for tumor inhibitory activity studies of multispecific antibodies

[0272] 1. Yarden, Y. and MXSliwkowski, Untangling the ErbB signaling network. Nat Rev Mol Cell Biol, 2001.2(2):p.127-37.

[0273] 2.Oh, DYand YJBang, HER2-targeted therapies-a role beyond breast cancer. Nat Rev Clin Oncol, 2020.17(1):p.33-48.

[0274] 3. Schaer, DA, D. Hirschhorn-Cymerman, and JD Wolchok, Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy. J Immunother Cancer, 2014.2: p.7.

[0275] 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, 2015.3(2):p.149-60.

[0276] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details based on all the teachings disclosed, and these changes are all within the scope of protection of the present invention. The full scope of the present invention 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, the single-domain antibody comprising: (a) CDR1 having: the sequence shown in SEQ ID NO: 43, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 43; (b) CDR2 having: a sequence as shown in SEQ ID NO: 44, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 44; and (c) CDR3 having: a sequence as shown in SEQ ID NO: 45, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 45; Preferably, the substitution is a conservative substitution; Preferably, the single-domain antibody or antigen-binding fragment thereof comprises: CDR1 as shown in SEQ ID NO:43, CDR2 as shown in SEQ ID NO:44, and CDR3 as shown in SEQ ID NO:

45.

2. The single-domain antibody or antigen-binding fragment thereof according to claim 1, wherein the single-domain antibody comprises an amino acid sequence selected from the group consisting of: (i) the sequence shown 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 shown 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; Preferably, the substitutions are conservative substitutions.

3. A polypeptide construct that specifically binds 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 connected 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 has (G 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 or 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 shown in SEQ ID NO: 17 or 18; preferably, the immunoglobulin Fc domain is an IgG Fc domain (e.g., an IgG1 Fc domain, e.g., comprising CH2 and CH3); Preferably, the immunoglobulin Fc domain comprises the sequence shown 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 shown in SEQ ID NO: 19 or 20; Preferably, the polypeptide construct comprises or consists of the amino acid sequence shown in SEQ ID NO: 31 or 32.

4. A multispecific antibody comprising the single domain antibody or antigen-binding fragment thereof according to claim 1 or 2, or the polypeptide construct according to claim 3; Preferably, the multispecific antibody specifically binds to 4-1BB and additionally specifically binds to one or more other targets; Preferably, the target is a tumor antigen; Preferably, the tumor antigen is selected from one or more of the following: CD19, CD20, CD22, CD23, CD38, CD40, CD49, CD52, CD56, CD74, CD80, CD95, CD138, CS1 / SLAMF7, KiR, Thy-1, Ly-6, Fas, APO-1, EGFR, HER2, CXCR4, HLA, GM1 and DRD.

5. A multispecific antibody comprising a first antigen-binding domain specific for 4-1BB and a second antigen-binding domain specific for HER2, wherein: The first antigen-binding domain comprises the antibody or antigen-binding fragment thereof according to claim 1 or 2; The second antigen-binding domain contains at least one CDR of the heavy chain variable region of an anti-HER2 antibody, and / or at least one CDR of the light chain variable region of an anti-HER2 antibody; Preferably, the second antigen-binding domain contains 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 trastuzumab, pertuzumab and variants thereof; The variant has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions, deletions or additions; such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions).

6. A multispecific antibody comprising a first antigen-binding domain specific for 4-1BB and a second antigen-binding domain specific for HER2; wherein, The first antigen-binding domain is VHH; the second antigen-binding domain is Fab, and the multispecific antibody comprises: (1) peptide chain IA, which comprises the light chain variable region and light chain constant region (CL) of the second antigen-binding domain; and, (2) a peptide chain IB comprising the heavy chain variable region, the heavy chain constant region, and the first antigen-binding domain of the second antigen-binding domain; preferably, the peptide chain IB comprises the adjacent heavy chain variable region and heavy chain constant region of the second antigen-binding domain and the first antigen-binding domain from the N-terminus to the C-terminus; Preferably, the CL of the peptide chain IA is capable of forming a dimer with the heavy chain constant region CH1 domain 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 connected directly or via a peptide linker; preferably, the peptide linker has (G m X n ) l G m’ The structure shown in FIG5 , 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 or 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 shown in SEQ ID NO: 17 or 18.

7. The multispecific antibody according to claim 5 or 6, characterized in that One or more of the following: (i) the first antigen-binding domain comprises or consists of the amino acid sequence shown 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 shown 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 shown in SEQ ID NO: 2, 4 or 6; Preferably, the peptide chain IA comprises or consists of the amino acid sequence shown in SEQ ID NO: 22, 24 or 26; Preferably, the peptide chain IB contains or consists of the amino acid sequence shown in SEQ ID NO: 21, 23 or 25.

8. The multispecific antibody of any one of claims 4 to 7, 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) peptide chain II-A, which comprises the light chain variable region and light chain constant region (CL) of the second antigen-binding domain; (2) peptide chain II-B, which comprises the heavy chain variable region, heavy chain constant region, and the first antigen-binding domain of the second antigen-binding domain; preferably, the peptide chain II-B comprises the adjacent heavy chain variable region and heavy chain constant region of the second antigen-binding domain and the first antigen-binding domain from the N-terminus to the C-terminus; (3) peptide chain II-C, which comprises the light chain variable region and light chain constant region (CL) of the third antigen-binding domain; and, (4) peptide chain II-D, which comprises 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 comprises the adjacent heavy chain variable region and heavy chain constant region of the third antigen-binding domain and the first antigen-binding domain from the N-terminus to the C-terminus; Preferably, the CL of the peptide chain II-A can form a dimer with the heavy chain constant region CH1 domain of the peptide chain II-B; preferably, the CL of the peptide chain II-C can form a dimer with the heavy chain constant region CH1 domain of the peptide chain II-D; preferably, the heavy chain constant region of the peptide chain II-B contains the amino acid sequence as shown in SEQ ID NO: 13, and the heavy chain constant region of the peptide chain II-D contains the amino acid sequence as shown in SEQ ID NO: 14; preferably, the peptide chain The heavy chain constant region of peptide chain II-B comprises the amino acid sequence shown in SEQ ID NO: 37, and the heavy chain constant region of peptide chain II-D comprises the amino acid sequence shown 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 connected directly or via a peptide linker; preferably, the peptide linker has (G m X n ) l G m’ The structure shown in FIG5 , 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 or 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 shown in SEQ ID NO: 17 or 18.

9. The multispecific antibody according to claim 8, characterized in that One or more of the following: (i) the first antigen-binding domain comprises or consists of the amino acid sequence shown 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 shown 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 shown 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 shown 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 shown in SEQ ID NO: 2, 4 or 6; Preferably, the peptide chain II-A comprises or consists of the amino acid sequence shown in SEQ ID NO: 28 or 40; Preferably, the peptide chain II-B comprises or consists of the amino acid sequence shown in SEQ ID NO: 27 or 39; Preferably, the peptide chain II-C comprises or consists of the amino acid sequence shown in SEQ ID NO: 30 or 42; Preferably, the peptide chain II-D contains or consists of the amino acid sequence shown in SEQ ID NO: 29 or 41.

10. A polynucleotide encoding the antibody according to any one of claims 1 to 9. A vector comprising the polynucleotide according to claim 10 . 12 . A recombinant cell comprising the polynucleotide of claim 10 or the vector of claim 11 .

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

14. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1, 2, 4-9, 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 preparation of an anti-tumor drug; Preferably, the tumor overexpresses HER2; Preferably, the tumor is selected from breast cancer, colon cancer, stomach cancer, lung cancer (e.g., squamous cell lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma), peritoneal cancer, skin cancer, squamous cell carcinoma, melanoma of the skin or eye, rectal cancer, cancer near the anus, esophageal cancer, small intestine tumor, 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 cancer or uterine cancer, salivary gland tumor, kidney cancer, cervical cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, bile duct cancer and gallbladder cancer; Preferably, the tumor is a primary or metastatic tumor.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1, 2, 4-9, 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, for use in anti-tumor treatment; Preferably, the tumor overexpresses HER2; Preferably, the tumor is selected from breast cancer, colon cancer, gastric cancer, lung cancer (e.g., squamous cell lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma), peritoneal cancer, skin cancer, squamous cell carcinoma, melanoma of the skin or eye, rectal cancer, cancer near the anus, esophageal cancer, small intestinal tumor, endocrine gland cancer, parathyroid cancer, adrenal 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 cancer or uterine cancer, salivary gland tumors, kidney cancer, cervical cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, biliary tract cancer, and gallbladder cancer; Preferably, the tumor is a primary or metastatic tumor.

16. An anti-tumor method comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1, 2, 4-9, 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; Preferably, the tumor overexpresses HER2; Preferably, the tumor is selected from breast cancer, colon cancer, stomach cancer, lung cancer (e.g., squamous cell lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma), peritoneal cancer, skin cancer, squamous cell carcinoma, melanoma of the skin or eye, rectal cancer, cancer near the anus, esophageal cancer, small intestine tumor, 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 cancer or uterine cancer, salivary gland tumor, kidney cancer, cervical cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, 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 according to any one of claims 1, 2, 4-9, or the polypeptide construct according to claim 3, and a detectable label linked to the antibody or antigen-binding fragment thereof or polypeptide construct; Preferably, the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent protein), a radionuclide or biotin.

18. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1, 2, 4-9, the polypeptide construct according to claim 3, or the conjugate according to claim 17; Preferably, the kit further comprises a second antibody that specifically recognizes the antigen specifically recognized by the antibody or antigen-binding fragment thereof or the polypeptide construct; optionally, the second antibody further comprises a detectable label, such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent protein), a radionuclide or biotin.

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

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

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