Construct targeting 4-1BB and use thereof

Abstract

Provided are a construct targeting 4-1BB and a use thereof. The provided construct comprises an antigen-binding fragment targeting 4-1BB, wherein the amino acid sequence of the heavy chain variable region is as shown in positions 1-114 of SEQ ID No. 3 or positions 126-239 of SEQ ID No. 6, and the amino acid sequence of the light chain variable region is as shown in positions 1-107 of SEQ ID No. 4 or positions 1-107 of SEQ ID No. 6. The construct further comprises an antibody targeting MSLN, forming a bispecific antibody. The provided bispecific antibody has a good T cell activation effect, and also exhibits a good in vitro tumor cell killing effect and in vivo tumor inhibition effect. Thus, the provided bispecific antibody has important significance and application potential for preparing targeted antibody drugs.

Description

Targeting 4-1BB constructs and their applications Technical Field

[0001] The present invention belongs to the technical field of tumor treatment and immunology, relates to a 4-1BB-targeting construct and its application, and specifically relates to an anti-4-1BB humanized antibody or its antigen-binding fragment, a bispecific antibody thereof and its application. Background Art

[0002] The co-stimulatory molecule 4-1BB is an important member of the tumor necrosis factor receptor superfamily. It is a co-stimulatory signal that mediates T cell activation and is mainly expressed on the surface of activated T cells, NK cells, neutrophils and DC cells. The co-stimulatory signal mediated by 4-1BB can enhance the function of T cells, improve T cells' surveillance of tumor cells and immune defense against viral infection. The 4-1BB signaling pathway can also induce CD4 + T cell-mediated immune tolerance prevents the occurrence and development of autoimmune diseases. By intervening in the role of the 4-1BB pathway to regulate the immune function of lymphocytes, it is possible to become a new immunotherapy approach. In addition, 4-1BB is also expressed on the surface of Treg cells and is highly expressed on Treg in patients with various tumors, such as breast cancer, lung cancer, and colon cancer. Studies have shown that targeted clearance of 4-1BB+Treg can effectively inhibit tumor growth without affecting the function of CD8+T cells (Freeman ZT, Nirschl TR, Hovelson DH, et al. A conserved intratumoral regulatory T cell signature identifies 4-1BB as a pan-cancer target[J]. The Journal of Clinical Investigation, 2020, 130(3).).

[0003] Currently, a variety of drug forms targeting 4-1BB have entered the clinic, including but not limited to monoclonal antibodies, bispecific antibodies or multispecific antibodies, fusion proteins, etc. The mechanism of action of bispecific antibodies or multispecific antibodies is mostly to use tumor-associated antigens (TAAs) to specifically cross-link and activate 4-1BB signals in the tumor microenvironment, thereby avoiding systemic activation of the immune system and the toxic side effects it causes. TAA options include PD-L1, HER2, CD19, Claudin18.2, EGFR, ROR1, etc., but there are still few studies on the selection of mesothelin (MSLN) as a cross-linking target. Studies have shown that MSLN is not expressed or expressed at low levels in normal tissues, but is highly expressed in many cancers, including mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, etc. Therefore, the development of a drug that uses MSLN cross-linking to activate 4-1BB signals and thus enhance T cell killing ability has broad application prospects.

[0004] SUMMARY OF THE INVENTION

[0005] The technical problem to be solved by the present invention is to provide a 4-1BB-targeting construct and its application, and specifically provide a construct specifically targeting 4-1BB and its application in tumor treatment. In some embodiments, the 4-1BB-targeting construct comprises a monoclonal antibody that binds to 4-1BB. In some embodiments, the 4-1BB-targeting construct comprises a multispecific (e.g., bispecific) anti-4-1BB molecule that binds to 4-1BB and one or more other antigens, which comprises an antibody portion (e.g., scFv) that binds to 4-1BB, and a second antibody portion (e.g., VHH) that specifically recognizes a second antigen. In some embodiments, the second antigen is MSLN. The present invention further provides methods for preparing the antibodies and pharmaceutical compositions comprising these antibodies, as well as methods for using the antibodies and pharmaceutical compositions comprising these antibodies, for example, for treating cancer. The present invention is based in part on the discovery of anti-4-1BB monoclonal antibodies that bind to 4-1BB and multispecific antibodies that bind to 4-1BB and MSLN, which can increase the immune response of immune cells and have anti-tumor efficacy. Technical Solutions

[0006] In the first aspect, the present invention provides a construct comprising a 4-1BB-targeted antigen-binding fragment, comprising a 4-1BB-targeted antigen-binding fragment, wherein the 4-1BB-targeted antigen-binding fragment comprises a humanized heavy chain variable region (VH) and a humanized light chain variable region (VL), and the amino acid sequences of CDR1, CDR2, and CDR3 in the humanized heavy chain variable region are as follows: positions 31-35, 50-66, and 99-103, respectively; the amino acid sequences of CDR1, CDR2, and CDR3 in the humanized light chain variable region are as follows: positions 24-34, 50-56, and 89-97, respectively.

[0007] In the above, the construct may be an antibody or an antigen-binding fragment.

[0008] The CDR1, CDR2 and CDR3 of the humanized heavy chain variable region and the humanized light chain variable region were determined by the Kabat system.

[0009] In the construct comprising the 4-1BB-targeted antigen-binding fragment described above, in the 4-1BB-targeted antigen-binding fragment, the amino acid sequence of the humanized heavy chain variable region is such as SEQ ID No. 3, positions 1-114 or SEQ ID No. 6, positions 126-239, or has a consistency (identity) of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with SEQ ID No. 3, positions 1-114 or SEQ ID No. 6, positions 126-239; and / or

[0010] In the targeting 4-1BB antigen-binding fragment, the amino acid sequence of the humanized light chain variable region is SEQ ID No. 4, positions 1-107 or SEQ ID No. 6, positions 1-107, or has a consistency of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with SEQ ID No. 4, positions 1-107 or SEQ ID No. 6, positions 1-107. In the construct comprising the targeting 4-1BB antigen-binding fragment described above, the antigen-binding fragment may be at least one of the following: Fab, Fab' fragment, F(ab')2 fragment, nanobody, minimum recognition unit (MRU), Fv antibody, single-chain antibody and heavy chain antibody.

[0011] Furthermore, the 4-1BB-targeting antigen-binding fragment is in the form of Fab, Fab', Fv fragment, F(ab')2, scFv or di-scFv.

[0012] Furthermore, in the construct comprising the 4-1BB-targeting antigen-binding fragment described above, the 4-1BB-targeting antigen-binding fragment is in the form of scFv.

[0013] Furthermore, the scFv structure is specifically VH-connector peptide 1-VL or VL-connector peptide 1-VH. Further, the scFv structure is specifically VL-connector peptide 1-VH.

[0014] VL is a humanized light chain variable region, and VH is a humanized heavy chain variable region.

[0015] The amino acid sequence of VH-connecting peptide 1-VL mentioned above consists of VH amino acid sequence, connecting peptide 1 amino acid sequence and VL amino acid sequence in order from the N-terminus.

[0016] The VL-connector 1-VH mentioned above is an amino acid sequence consisting of a VL amino acid sequence, a connector 1 amino acid sequence and a VH amino acid sequence starting from the N-terminus.

[0017] In the construct comprising the 4-1BB-targeting antigen-binding fragment described above, a pair of electrostatic modifications are introduced between VL and VH in the scFv, wherein the charge modification is: VH39K-VL38D, VH38D-VL39K, VH39Y-VL38R, VH105D-VL43K or VH103D-VL44K; further, specifically VH39K-VL38D, wherein VH39K corresponds to the 164th K of SEQ ID No. 6, and VL38D corresponds to the 38th D of SEQ ID No. 6.

[0018] A pair of electrostatic modifications are introduced above, which is to form corresponding amino acid residues in corresponding positions in VL and VH of scFv, and the position numbering system is Kabat; for example, VH39K-VL38D means that the amino acid residue at position 39 of VH of scFv is K, and the amino acid residue at position 38 of VL is D, and the numbering system of positions 39 and 38 in the antibody is Kabat, and so on. In the construct comprising the 4-1BB-targeting antigen-binding fragment described above, a pair of disulfide bonds is introduced between VL and VH in the scFv, wherein the disulfide bond position is: VH44-VL100, VH100-VL50, VH100b-VL49, VH101-VL46 or VH105-VL43; specifically VH44-VL100 (specifically, C of VH44 and C of VL100 form a disulfide bond), wherein VH44 corresponds to C at position 169 of SEQ ID No. 6, and VL100 corresponds to C at position 100 of SEQ ID No. 6.

[0019] A pair of disulfide bonds is introduced above, that is, the amino acid residues at corresponding positions in VL and VH of the scFv are both cysteine ​​C (since cysteine ​​is the only amino acid that can form a disulfide bond, the description of the amino acid residues is omitted), and the position numbering system is Kabat; for example, VH44-VL100 means that the amino acid residue at position 44 of VH of the scFv is C, and forms a disulfide bond with the amino acid residue C at position 100 of VL, and the numbering system of positions 44 and 100 in the antibody is Kabat, and so on.

[0020] In the construct comprising the 4-1BB antigen-binding fragment described above, a pair of disulfide bonds is introduced between VL and connecting peptide 1, and between VH and connecting peptide 1 in the scFv, wherein the disulfide bond positions are VL42-connecting peptide 1 and VH105-connecting peptide 1, wherein VL42 corresponds to position 42 C of SEQ ID No. 6, and VH105 corresponds to position 231 C of SEQ ID No. 6.

[0021] A pair of disulfide bonds is introduced above, that is, the amino acid residues at the corresponding positions of the VL of the scFv and the connecting peptide 1 are both cysteine ​​C (since cysteine ​​is the only amino acid that can form a disulfide bond, the description of the amino acid residues is omitted), and the amino acid residues at the corresponding positions of the VH of the scFv and the connecting peptide 1 are both cysteine ​​C (since cysteine ​​is the only amino acid that can form a disulfide bond, the description of the amino acid residues is omitted), and the position numbering system is Kabat; for example, VL42-connecting peptide 1 means that the amino acid residue at position 42 of the VL of the scFv is C, and forms a disulfide bond with the C at position 10 of the connecting peptide 1 of the amino acid sequence of SEQ ID No. 19 (corresponding to the C at position 117 of SEQ ID No. 6), and the numbering system for position 42 in the antibody is Kabat; VH105-connecting peptide 1 means that the amino acid residue at position 105 of the VH of the scFv is C, and forms a disulfide bond with the C at position 13 of the connecting peptide 1 of the amino acid sequence of SEQ ID No. 19 (corresponding to the C at position 13 of SEQ ID No.6 The 120th position C) forms a disulfide bond, and the 105th position numbering system in the antibody is Kabat, and the others are numbered accordingly.

[0022] The amino acid positions in the above antibodies are based on Kabat counting (only antibody positions, not including the positions of connecting peptides), such as VH39K, VL38D, VH38D, VL39K, VH39Y, VL38R, VH105D, VL43K, VH103D, VL44K, VH44, VL100, VH100, VL50, VH100b, VL49, VH101, VL46, VH105, VL43, VL42, and VH105.

[0023] Further, in the construct comprising the 4-1BB-targeting antigen-binding fragment described above, the connecting peptide 1 is selected from any one or combination of the following: (G4S)3(GGGGSGGGGSGGGGS, SEQ ID No.13), (G4S)4(GGGGSGGGGSGGGGSGGGGS, SEQ ID No.14), GGSGGSGGCPPCGSGG (SEQ ID No.15), RGGGSGGSGGCPPCGGSGG (SEQ ID No.16), GGGSGGGSGCPPCGGGG (SEQ ID No.17), GGGSGGCPPCGGGSGG (SEQ ID No.18) or GGGSGGSGGCPPCGGSGG (SEQ ID No.19), preferably GGGSGGSGGCPPCGGSGG (SEQ ID No.19).

[0024] In the above, (G4S)3 represents an amino acid residue consisting of three repeats of G4S; (G4S)4 represents an amino acid residue consisting of four repeats of G4S.

[0025] Furthermore, the 4-1BB antigen-binding fragment targeting the 4-1BB comprises an amino acid sequence such as a fragment of SEQ ID NO. 6, or a fragment having an amino acid sequence that is more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more identical to SEQ ID No. 6.

[0026] In an embodiment of the present invention, the 4-1BB-targeting antigen-binding fragment is an anti-4-1BB scFv single-chain antibody, and its amino acid sequence is as shown in SEQ ID NO.6.

[0027] In the above, the construct may be a bispecific antibody.

[0028] In the construct comprising the targeting 4-1BB antigen binding fragment described above, the construct is a bispecific antibody comprising an antibody targeting another antigen. The target of the antibody targeting another antigen is PD-L1, MSLN, PSMA, B7-H3 or B7-H4, etc. In certain embodiments, the targeting 4-1BB construct provided by the present invention comprises an antigen binding fragment that binds to 4-1BB and a multispecific (e.g., bispecific) anti-4-1BB molecule that binds to one or more other antigens. The multispecific (e.g., bispecific) anti-4-1BB molecule comprises an antibody portion (e.g., scFv) that binds to 4-1BB, and a second antibody portion (e.g., VHH) that specifically recognizes a second antigen. The second antigen can be a tumor-associated antigen such as PD-L1, MSLN, PSMA, B7-H3 or B7-H4.

[0029] In the construct comprising the antigen-binding fragment targeting 4-1BB described above, the antibody targeting another antigen is a VHH nanobody targeting MSLN, wherein the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the VHH nanobody are as follows: positions 31-35, 50-66 and 99-101 of SEQ ID No. 5, respectively;

[0030] Furthermore, the amino acid sequence of the heavy chain variable region of the VHH nanobody is such as SEQ ID NO.5, or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with SEQ ID No.5.

[0031] Among them, the nanobody described in the present invention is also called single-domain antibody, sdAb, nanobody, etc. Compared with conventional monoclonal antibodies, in addition to lacking a light chain, there is no CH1 region between its heavy chain variable region and hinge region, and it only contains one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions.

[0032] In some embodiments, in the construct comprising the 4-1BB-targeting antigen-binding fragment described above, the structure of the construct is to fuse the heavy chain variable region of the MSLN-targeting VHH nanobody to the N-terminus of the Fc domain, and to connect the 4-1BB-targeting antigen-binding fragment to the C-terminus of the Fc domain via a connecting peptide 2, that is, the structure of the construct is VHH-Fc-connecting peptide 2-scFv. Further, the Fc domain is derived from human IgG, IgM, IgE, IgA or IgD; specifically, any one of IgG1, IgG2, IgG3 and IgG4; further, more specifically, IgG1.

[0033] The Fc domain herein (Fc region) is the C-terminal region of at least a portion of the constant region comprising an immunoglobulin heavy chain. The Fc domain includes a native sequence Fc domain and a variant Fc domain. The variant Fc domain may include a human variant Fc domain (e.g., an Fc domain of human IgG1, IgG2, IgG3, or IgG4) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions. In a specific embodiment of the present invention, the Fc domain is specifically composed of two constant domains (CH2 and CH3).

[0034] Further, the connecting peptide 2 is selected from the following: A(EAAAK)4ALE(AEAAAKEAAAKEAAAK EAAAKALE, SEQ ID No.22), KVDKKVEPKSCDKTHT (SEQ ID No.23), G4S, (G4S)n, wherein n is 1, 2, 3, 4, 5 or 6; further, more specifically (G4S)3(GGGGSGGGGSGGGGS, SEQ ID No.13).

[0035] In the construct comprising the 4-1BB-targeting antigen-binding fragment described above, the amino acid sequence of the bispecific antibody comprises an amino acid sequence as shown in SEQ ID NO: 7 or an amino acid sequence having 99% or more, 95% or more, 90% or more, 85% or more, 80% or more identity thereto.

[0036] In an embodiment of the present invention, the amino acid sequence of the bispecific antibody is SEQ ID NO: 7.

[0037] In a second aspect, the present invention provides a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises: (a1) the 4-1BB-targeting construct described in the first aspect; (a2) a pharmaceutically acceptable excipient, diluent, or carrier. Furthermore, the pharmaceutical composition may also include targets for other immune checkpoints such as PD-1, TIM-3, LAG-3, CTLA-4, OX40, or other TAAs such as MSLN, VEGF, EGFR, PD-L1, as well as small molecules such as peptides and chemical drugs.

[0038] In a third aspect, the present invention provides a nucleic acid encoding the 4-1BB-targeting construct described in the first aspect.

[0039] In a fourth aspect, the present invention provides a vector comprising the nucleic acid described in the third aspect.

[0040] In a fifth aspect, the present invention provides an expression cassette, a host bacterium or a host cell containing the nucleic acid described in the third aspect or the vector described in the fourth aspect.

[0041] In a sixth aspect, the present invention provides a method for preparing the 4-1BB-targeting construct described in the first aspect, the method comprising expressing the construct in the host bacteria or host cell described in the fifth aspect and isolating the construct from the host bacteria or host cell.

[0042] In a seventh aspect, the present invention provides use of the 4-1BB-targeting construct of the first aspect, the nucleic acid of the third aspect, the vector of the fourth aspect, or the expression cassette, host bacteria, or host cell of the fifth aspect in any of the following:

[0043] (B1) Preparation of antibody-targeted drugs;

[0044] (B2) preparing a product for activating T lymphocytes;

[0045] (B3) preparing a product that kills tumor cells;

[0046] (B4) preparing products that inhibit tumor cell growth;

[0047] (B5) Preparation of products for preventing and / or treating cancer.

[0048] In the above, further, in B2), the activation of T lymphocytes is the activation of T lymphocytes in the presence of tumor cells expressing MSLN, and in the embodiment of the present invention, specifically the activation of CD8+ T cells to secrete IFN-γ.

[0049] Further, in (B4), the cancer is selected from any one of the following: mesothelioma, ovarian cancer, lung cancer, esophageal cancer, pancreatic cancer, gastric cancer, bile duct cancer, endometrial cancer, thymic cancer, colon cancer and breast cancer.

[0050] The above products are medicines, compositions, health products, functional foods, foods for special medical purposes or other biological products.

[0051] Among the products mentioned above, the dosage form of the drug is injection, lyophilized powder for injection, aerosol, large infusion, dripping pills, pills, powder, granules, tablets, capsules, oral solution or emulsion.

[0052] The pharmaceutically acceptable carriers mentioned above may be excipients, stabilizers, suspending agents or diluents, etc., which are well known to those skilled in the art.

[0053] In the above, the pharmaceutically acceptable excipients, diluents or carriers include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). These materials can be used to make a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a conventional preparation, a sustained-release preparation, a controlled-release preparation, and various microparticle delivery systems. In order to make a unit dosage form into a tablet, various carriers well known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dried starch, alginate, agar powder, brown seaweed starch, sodium bicarbonate with citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption enhancers, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further prepared as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer tablets and multilayer tablets. To prepare the unit dosage form into a pill, a wide variety of carriers known in the art can be used. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginates, sodium lauryl sulfate, methylcellulose, and ethylcellulose. To prepare unit dosage forms as suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, higher alcohol esters, gelatin, and semi-synthetic glycerides. To prepare unit dosage forms as injectable preparations, such as solutions, emulsions, lyophilized powder injections, and suspensions, all diluents commonly used in the art can be used, including water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters.In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol may be added to the injection preparation. In addition, conventional cosolvents, buffers, pH adjusters, etc. may also be added. In addition, if necessary, colorants, preservatives, fragrances, flavorings, sweeteners or other materials may also be added to the pharmaceutical preparation. Beneficial effects

[0054] The bispecific antibodies provided by the present invention have good T cell activation effects, and on this basis, exhibit good in vitro tumor cell killing effects and in vivo tumor suppression effects. Therefore, the bispecific antibodies provided by the present invention have important significance and application potential for the preparation of antibody-targeted drugs.

[0055] Terms and Definitions

[0056] BsAb: bispecific antibody;

[0057] TAA: Tumor associated antibody;

[0058] VH: heavy chain variable domain;

[0059] VL: Light chain variable domain;

[0060] VHH: Nanobody, also known as single-domain antibody;

[0061] ELISA: Enzyme linked immunosorbent assay;

[0062] FACS: Fluorescence-activated cell sorting, also known as flow cytometry.

[0063] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0064] As used herein, when referring to the amino acid sequence of 4-1BB protein or 4-1BB protein (UniProt Q07011), it includes the full length of 4-1BB protein, or the extracellular fragment 4-1BB-ECD of 4-1BB or a fragment comprising 4-1BB-ECD; Also included is a fusion protein of 4-1BB-ECD, such as a fragment fused to the Fc protein fragment (mFc or hFc) of mouse or human IgG. The term "4-1BB protein" includes all such sequences, including natural or artificial variants thereof.

[0065] As used herein, the term EC50 refers to concentration for 50% of maximal effect, which refers to the concentration that can cause 50% of the maximal effect.

[0066] As used herein, the term "monoclonal antibody" or Antibody, unless otherwise specified, generally refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair having a "light" (L) chain and a "heavy" (H) chain). In a general sense, the heavy chain can be understood as the polypeptide chain with the larger molecular weight in the antibody, and the light chain refers to the polypeptide chain with the smaller molecular weight in the antibody. Light chains can be classified as kappa and lambda light chains. Heavy chains can generally be classified as μ, δ, γ, α or ε, and define the isotype of the antibody as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH 1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions with high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antibody binding site, respectively. The allocation of amino acids to each region or domain follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or the definitions of Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883. In particular, the heavy chain can also contain more than 3 CDRs, for example 6, 9, or 12. For example, in the bifunctional antibody of the present invention, the heavy chain can be the C-terminus of the heavy chain of an IgG antibody connected to the ScFv of another antibody, in which case the heavy chain contains 9 CDRs. The term "antibody" is not limited to any particular method for producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be an antibody of different types, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.

[0067] The CDRs, as used herein, stand for "complementarity determining regions," which are regions of the antibody variable domain that are highly variable in sequence and form structurally defined "hypervariable loops" and / or contain antigen-contacting residues called "antigen contact sites." CDRs are primarily responsible for binding to antigenic epitopes. A variable region typically contains three CDR regions: CDR1, CDR2, and CDR3, starting from the N-terminus.

[0068] As used herein, the term "nanobody" refers to an antibody fragment that contains only a single heavy chain variable region (VHH). "Nanobody", also known as single-domain antibody, is a special structure of VHH derived from heavy chain antibodies (such as camelid antibodies or shark antibodies), consisting of three highly variable regions (called complementary determining regions (CDRs)) and four framework regions (FRs), arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus, forming a binding domain that interacts with the antigen. Compared with traditional antibody heavy chains, nanobody CDR3 is longer, 13 to 18 amino acids, which to some extent compensates for the decreased antigen binding ability caused by the absence of light chains.

[0069] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen, and is also referred to as an "antigen-binding portion" or "antigen-binding domain". In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementary determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0070] The term "Fab" stands for antigen-binding fragment (Fab), which is composed of a complete antibody light chain and the VH (heavy chain variable region) and CH1 (heavy chain constant region 1) domains of the heavy chain. This refers to a heterodimer formed by the antibody's heavy chain Fd and a complete light chain bound by disulfide bonds. This refers to an antibody fragment consisting of the VL, VH, CL, and CH1 domains.

[0071] The term "Fab'" comprises a complete antibody light chain and a portion of an antibody heavy chain including the VH domain and the CH1 domain, as well as the region between the CH1 and CH2 domains, i.e., the hinge region. Thus, an interchain disulfide bond can form between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule.

[0072] The term "F(ab')2 fragment" consists of two Fab' fragments held together by a disulfide bond between the two heavy chains. It refers to an antibody fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region.

[0073] The term "Fv antibody" refers to a protein consisting solely of the heavy and light chain variable regions of an antibody. The heavy and light chain variable regions are linked by non-covalent bonds. This refers to an antibody fragment consisting of the VL and VH domains of a single antibody arm.

[0074] The term "single-chain antibody" (ScFv) refers to a protein composed of the heavy chain variable region and the light chain variable region of an antibody connected by a short peptide.

[0075] The term di-scFv refers to a protein in which two scFvs are linked by a short peptide.

[0076] The term "nanobody" refers to a heavy chain single-domain antibody VHH (variable domain of heavy chain of heavy-chain antibody), which is a protein composed of the variable region of the antibody heavy chain.

[0077] The term "minimum recognition unit (MRU)" refers to a structure containing only a single CDR in the variable region, with a molecular mass of only about 1% of that of a complete antibody, which can bind to the corresponding antigen.

[0078] The term "heavy chain antibody" (HCAb) refers to a protein consisting of only two heavy chains, without the presence of light chains, and without a CH1 region between the heavy chain variable region and the hinge region.

[0079] Antibody antigen-binding fragments (e.g., those described above) can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods) and screened for specificity in the same manner as for intact antibodies.

[0080] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0081] As used herein, the term "isolated" or "isolated" refers to something that is obtained artificially from its natural state. If a substance or component is "isolated" in nature, it may be that its natural environment has been altered, or that the substance has been separated from its natural environment, or both. For example, a polynucleotide or polypeptide that is naturally present in a living animal and has not been separated is considered isolated. The term "isolated" or "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0082] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0083] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0084] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) means that the antibody binds to the antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8M, 10 -9 M or 10 -10 The invention provides a method for binding an antigen with an affinity (KD) of M or less. In some embodiments of the invention, the term "targeting" refers to specific binding.

[0085] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0086] The term "identity" refers to sequence similarity to a natural nucleic acid sequence. Identity can be assessed visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to assess the identity between related sequences. The 90% or greater identity can be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] FIG1 is an ELISA test of the activity of anti-4-1BB chimeric antibody binding to recombinant human 4-1BB antigen.

[0088] FIG2 is a FACS assay showing the activity of anti-4-1BB chimeric antibodies binding to CHOK1-h4-1BB stably transfected cell lines.

[0089] FIG3 shows the activation of the 4-1BB signaling pathway by anti-4-1BB chimeric antibodies detected by reporter genes.

[0090] FIG4 shows the body weight of mice during the treatment of MC38 tumors with anti-4-1BB chimeric antibodies.

[0091] FIG5 shows the activity of anti-4-1BB chimeric antibodies in inhibiting the growth of mouse MC38 tumors.

[0092] FIG6 shows the ELISA assay for the activity of anti-4-1BB humanized antibodies binding to recombinant human 4-1BB antigen.

[0093] FIG7 shows the activation of the 4-1BB signaling pathway by anti-4-1BB humanized antibodies detected by reporter genes.

[0094] FIG8 is a schematic diagram of the structure of a BsAb molecule.

[0095] FIG9 is an ELISA test showing the activity of BsAb molecules binding to recombinant human MSLN antigen.

[0096] FIG10 is an ELISA test showing the activity of BsAb molecules binding to recombinant human 4-1BB antigen.

[0097] FIG11 is an ELISA test showing the activity of BsAb molecules simultaneously binding to recombinant human MSLN and human 4-1BB antigens.

[0098] FIG12 is a graph showing the activity of BsAb molecules binding to tumor cells expressing MSLN antigen detected by FACS.

[0099] FIG13 is a FACS assay showing the activity of BsAb molecules binding to CHOK1-h4-1BB stably transfected cell lines.

[0100] Figure 14 shows MSLN-mediated BsAb molecule activation of CD8 + T cells produce IFN-γ.

[0101] FIG15 shows the ADCC effect of BsAb molecules on tumor cells detected by reporter gene.

[0102] FIG16 shows the effect of BsAb molecules on mouse body weight during the treatment of mouse tumors.

[0103] FIG17 shows the inhibitory effect of BsAb molecules on tumor growth in the mouse MC38 / HuMSLN model. Modes for Carrying Out the Invention

[0104] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0105] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0106] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0107] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0108] The following examples do not include a detailed description of conventional methods, such as those used for gene amplification, recombinant plasmid construction, and introduction of plasmids into host cells. Such methods are described in many publications, including Sambrook, J., et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.

[0109] pcDNA3.4 vector: Invitrogen, Cat: A14697.

[0110] HEK293F cells: ATCC American Cell Bank.

[0111] H226 tumor cells: Nanjing Kebai Biotechnology Co., Ltd.

[0112] OVCAR3 cells: Nanjing Kebai Biotechnology Co., Ltd.

[0113] MC38 mouse colon cancer cells: Nanjing Kebai Biotechnology Co., Ltd.

[0114] Example 1. Production and Activity Detection of Anti-Human 4-1BB Monoclonal Antibodies

[0115] 1. Acquisition of anti-human 4-1BB monoclonal antibodies

[0116] 1) Immunization of mice to obtain anti-human 4-1BB monoclonal antibodies

[0117] Mice were immunized with an antigen containing the extracellular region of human 4-1BB to produce anti-4-1BB monoclonal antibodies. After three immunizations, blood was collected from the tail vein to measure antibody titers using ELISA. Splenocytes were harvested from mice with acceptable titers and fused using conventional methods to generate anti-human 4-1BB monoclonal antibody hybridoma cell lines. Positive hybridoma cell lines were screened using ELISA, and the resulting positive cell lines were expanded in stages. The supernatant was harvested and purified using a Protein A affinity chromatography column to obtain the antibody. Affinity characterization was then performed to select an anti-4-1BB monoclonal antibody with moderate affinity, namely, antibody B5.

[0118] A bivalent chimeric antibody (ChB5) was constructed by adding human heavy chain constant region (IgG1 subtype, with L234A and L235A modifications) and light chain constant region (kappa subtype) domains to the heavy chain variable region and light chain variable region of B5, respectively.

[0119] 2) Preparation of anti-human 4-1BB bivalent chimeric antibody ChB5

[0120] Full-length gene sequences encoding the corresponding heavy and light chains were synthesized. The heavy chain of the bivalent chimeric antibody ChB5 (SEQ ID NO.1) includes the heavy chain variable region of B5 (SEQ ID NO.1, positions 1-114) and the human heavy chain constant region (IgG1 subtype, with L234A and L235A modifications, SEQ ID NO.1, positions 115-444); the light chain of the bivalent chimeric antibody ChB5 (SEQ ID NO.2) includes the light chain variable region of B5 (SEQ ID NO.2, positions 1-107) and the light chain constant region (kappa subtype, SEQ ID NO.2, positions 108-214) constituting the structural domain.

[0121] The heavy chain was ligated between the XbaI and HindIII sites of the pcDNA3.4 vector to obtain the recombinant vector pChB5-H;

[0122] The light chain was ligated between the XbaI and HindIII sites of the pcDNA3.4 vector to obtain the recombinant vector pChB5-L;

[0123] The recombinant vectors pChB5-H and pChB5-L were introduced into the human embryonic kidney cell line HEK293F (ATCC American Cell Bank) to obtain recombinant cells, which were then cultured in a 37°C, 5% CO2 shaking incubator at a speed of 120 rpm.

[0124] The antibody protein was purified from the culture supernatant using a Protein A affinity chromatography column. The specific operation was as follows: first, a Protein A column (GE) was equilibrated with PBS, then the culture supernatant was passed through the column, and pre-eluted for 5 column volumes using Solution A (formula: solvent is water, solute and concentration are: 20mM sodium phosphate, 500mM NaCl, pH 5.0), then eluted for 5 column volumes using Solution B (formula: solvent is water, solute and concentration are: 20mM sodium acetate, 150mM NaCl, pH 3.5), and the eluted peak was collected and then concentrated using a 30KDa concentrator centrifuge tube to obtain the antibody, namely, the anti-human 4-1BB chimeric antibody ChB5. The antibody consists of a heavy chain and a light chain. The amino acid sequence of the heavy chain is shown in SEQ ID No. 1, and the amino acid sequence of the light chain is shown in SEQ ID No. 2. The heavy chain type of the ChB5 antibody is IgG1, and the light chain type is a kappa chain.

[0125] 2. Binding activity of chimeric antibody ChB5 to recombinant human 4-1BB antigen

[0126] The binding activity of the chimeric antibody to the recombinant human 4-1BB antigen Hu4-1BB-mFc was detected by ELISA. The details are as follows:

[0127] Hu4-1BB-mFc was diluted with NaHCO3 to 0.5 μg / ml, 100 μl was added to each well of the ELISA plate, incubated at 4°C overnight, and the plate was washed three times with PBST (PBS + 0.1% Tween 20); after blocking with PBST containing 5% milk at 37°C for 2 hours, the plate was washed three times with PBST; the test sample was then diluted with PBST containing 1% milk, 100 μl was added to each well of the ELISA plate, and incubated at room temperature for 1 hour; goat anti-human-HRP (Jackson, catalog number 109-035-088) was diluted with 1% milk in PBST, 100 μl was added to each well of the ELISA plate, and incubated at room temperature for 0.5 hour; TMB was added to each well, and color was developed at room temperature in the dark; H2SO4 was added to terminate the reaction; the absorbance (OD value) was measured at 450 nm using a SepctraMax Versa microplate reader, and the results were statistically analyzed using GraphPad Prism to calculate the EC50 value.

[0128] The sample to be tested is the chimeric antibody ChB5 or utomilumab (the antibody sequence was searched according to the IMGT database: https: / / www.imgt.org / mAb-DB / , IMGT / mAb-DB ID: 657; the nucleotide sequence encoding the antibody was gene synthesized and cloned into the pcDNA3.4 vector, and then introduced into HEK293F cells for expression and purification. For specific steps, see 1-2) in Example 1).

[0129] The amino acid sequence of the above-mentioned Hu4-1BB-mFc is as follows (SEQ ID No. 12):

[0130] The results are shown in Figure 1. Although the EC50 of the chimeric antibody ChB5 binding to the recombinant human 4-1BB antigen is slightly weaker than that of the control utomilumab, its upper platform is better than the latter.

[0131] 3. Binding of chimeric antibodies to CHOK1 cells stably expressing human 4-1BB

[0132] The binding affinity of the chimeric antibody ChB5 to CHOK1 cells transfected with human 4-1BB was determined by flow cytometry. For the construction of the CHOK1 cell line CHOK1-h4-1BB stably expressing human 4-1BB, see patent CN112794905A.

[0133] Specifically, the nucleotide sequence encoding the full-length human 4-1BB (Uniprot#Q07011) (SEQ ID No. 8) was inserted between the XbaⅠ and HindⅢ sites of the pCDNA3.4 vector. The resulting recombinant plasmid was verified to be correct by sequencing and then introduced into wild-type CHOK1 cells using Lipofectamine 3000 transfection reagent (Invitrogen) to obtain the CHO-K1 / 4-1BB cell line that highly expresses human 4-1BB.

[0134] CHOK1-h4-1BB cells were cultured to the logarithmic growth phase and digested with trypsin. The cells were pelleted by centrifugation at 1000 rpm for 5 min at 4°C and resuspended in PBS. The cell density was adjusted to 2×10 5 Each tube was washed once with 1% BSA (weight / volume percentage g / ml, dissolved in PBS) solution, and the supernatant was discarded. The antibody to be tested was diluted in 1% BSA to a starting concentration of 45 nM. A 2-fold serial dilution was performed in 6 steps. 100 μl of each antibody dilution was added to the EP tube containing the cells and incubated at 4°C in the dark for 1 hour. A 1% BSA dilution was used as a negative control. After incubation, 400 μl of PBS containing 2% BSA was added, centrifuged for 5 minutes, and the supernatant discarded. This procedure was repeated once. Each EP tube was added with 100 μl of a 200-fold diluted goat anti-human IgG-FITC (Jackson) and incubated at room temperature for 0.5 hour. 400 μl of 1× PBS containing 2% BSA was added, centrifuged at 2000 rpm for 5 minutes, and the supernatant discarded. This procedure was repeated once. After washing, the cells were resuspended in 400 μl of 1× PBS and analyzed by flow cytometry. Data were processed using GraphPad Prism statistical software.

[0135] The antibody to be tested is chimeric antibody ChB5 or Utomilumab or IgG (GenScript, Cat: A01006).

[0136] The results are shown in Figure 2 and Table 1. The binding ability of the anti-4-1BB chimeric antibody ChB5 to CHOK1-h4-1BB cells was similar to that of utomilumab.

[0137] Table 1 shows the EC50 of ChB5 binding to CHOK1-h4-1BB cells determined by FACS

[0138] 4. Activation of the 4-1BB signaling pathway by chimeric antibodies

[0139] The activation activity of the chimeric antibody on the 4-1BB signaling pathway was detected by reporter gene experiments. The details are as follows:

[0140] HEK293 / 4-1BB / NFkB-luc cells are HEK293 cells that express human 4-1BB and a stably integrated NFκB luciferase reporter gene.

[0141] The preparation method is as follows: the human 4-1BB sequence (SEQ ID No. 8) is inserted into the pCDNA3.4 vector as the target gene to obtain plasmid A. At the same time, the NFκB element sequence (SEQ ID No. 9) and the luciferase gene (SEQ ID No. 10) are inserted into different sites of the pGL4.10 vector (Ubao Bio) according to the general method in the field to obtain plasmid B. Then, the A and B plasmids are introduced into HEK293 cells (Shanghai Cell Bank, Chinese Academy of Sciences) using Lipofectamine 3000 transfection reagent (Invitrogen). G418 (purchased from Sangon Biotech (Shanghai) Co., Ltd.) is added for pressure screening, and finally HEK293 cells expressing human 4-1BB and stably integrated NFκB luciferase reporter genes are obtained.

[0142] HEK293 / 4-1BB / NFkB-luc cells were cultured to the logarithmic growth phase, digested, resuspended, counted, and added to a 96-well plate at 4 × 10 cells per well. 4 cells. The test samples were diluted to 12 μg / ml, followed by a 3-fold serial dilution into 7 wells and added to a 96-well plate. Crosslinker antibody Fab' goat anti-human IgG Fc (Jackson) was then added to the corresponding wells at a 2:1 ratio (denoted as ChB5+Crosslinker or utomilumab+Crosslinker in the figure). After incubation in a 37°C incubator for 18 hours, 100 μl of ONE-Glo Luciferase assay system reagent (Promega) was added to each well. Chemiluminescence was measured after incubation at room temperature for 10 minutes. A control without crosslinker antibody was used (denoted as ChB5 or utomilumab in the figure).

[0143] The samples to be tested are chimeric antibody ChB5 or utomilumab.

[0144] The results are shown in Figure 3 and Table 2. Similar to the positive control utomilumab, the anti-4-1BB chimeric antibody can effectively activate the 4-1BB signaling pathway only in the presence of cross-linking antibodies, and the activation intensity is significantly stronger than the latter.

[0145] Table 2 shows the EC50 and maximum fluorescence value of ChB5 activation of 4-1BB signaling pathway in reporter gene assay

[0146] 5. Inhibit the growth of tumor cells in vivo

[0147] MC38 cells were cultured in vitro and digested with trypsin, and the cells were plated at a rate of 2 × 10 6 6-8 week old C57BL / 6J-h4-1BB mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were subcutaneously inoculated and grouped and dosed 11 days after inoculation (the average tumor volume was about 58 mm 3 ), 6 mice per group, were given normal saline (denoted as Vehicle in the figure) or 10 mg / kg of monoclonal antibody, intraperitoneally, once. The body weight and tumor volume of the mice were measured twice a week. The volume was calculated as 1 / 2 × length × width × width (mm 3 ).

[0148] The above monoclonal antibody is a chimeric antibody ChB5 or Utomilumab.

[0149] The experimental results are shown in Figures 4 and 5. ChB5 has no significant effect on the body weight of mice during the treatment of mouse tumors and has good anti-tumor activity.

[0150] Example 2: Humanization and Activity Detection of Anti-Human 4-1BB Monoclonal Antibodies

[0151] 1. Humanization of anti-human 4-1BB monoclonal antibody

[0152] The sequences of the heavy and light chain variable regions of B5 were compared against databases to identify highly similar humanized framework regions. Subsequently, a series of analyses, including homology modeling and optimization of the antibody Fab, surface scanning to identify humanized mutation sites, virtual mutagenesis and molecular dynamics simulations to identify key amino acids, led to the design of a rationally designed humanized antibody. The sequences of the heavy and light chain variable regions of the humanized antibody are shown in SEQ ID No. 3, positions 1-114, and SEQ ID No. 4, positions 1-107, respectively.

[0153] A humanized antibody (HuB5) was constructed by adding the human heavy chain constant region (IgG1 subtype with L234A and L235A modifications) and light chain constant region (kappa subtype) domains to the heavy chain variable region and light chain variable region of the humanized antibody, respectively.

[0154] The details are as follows:

[0155] Full-length gene sequences encoding the corresponding antibody heavy and light chains were synthesized. The heavy chain of the humanized antibody HuB5 (SEQ ID No. 3) includes a humanized heavy chain variable region (SEQ ID NO. 3, positions 1-114) and a human heavy chain constant region (IgG1 subtype, with L234A and L235A modifications, SEQ ID NO. 3, positions 115-444). The light chain of the humanized antibody HuB5 (SEQ ID No. 4) includes a humanized light chain variable region (SEQ ID NO. 4, positions 1-107) and a light chain constant region (kappa subtype, SEQ ID NO. 4, positions 108-214).

[0156] The preparation method is the same as that of 1) 2) ChB5 in Example 1, except that the heavy chain of the ChB5 antibody is replaced by the heavy chain of the humanized antibody HuB5, and the light chain of the ChB5 antibody is replaced by the light chain of the humanized antibody HuB5.

[0157] The resulting humanized antibody HuB5 is a complete antibody consisting of a heavy chain and a light chain. The amino acid sequence of the heavy chain is shown in SEQ ID No. 3, and the amino acid sequence of the light chain is shown in SEQ ID No. 4. The heavy chain type of the HuB5 antibody is IgG1, and the light chain type is a kappa chain.

[0158] 2. Binding activity of humanized antibody HuB5 to recombinant human 4-1BB antigen

[0159] The effect of humanization on antibody affinity was tested by ELISA. The specific method is as described in Example 1, step 2, except that the sample to be tested is the humanized antibody HuB5 or the chimeric antibody ChB5.

[0160] The results are shown in FIG6 . Compared with the chimeric antibody, the humanized antibody HuB5 exhibited similar affinity to the recombinant human 4-1BB protein.

[0161] 3. Activation of the 4-1BB signaling pathway by humanized antibody HuB5

[0162] The activity of the humanized antibody HuB5 was further detected by a 4-1BB-activated reporter gene assay. The specific method is as described in Example 1, Section 4, except that the test samples were chimeric antibody ChB5 or humanized antibody HuB5.

[0163] The results are shown in Figure 7 and Table 3. Similar to the chimeric antibody, the humanized antibody HuB5 has a stronger activity in activating 4-1BB antigen in vitro than the positive control utomilumab.

[0164] Table 3 shows the EC50 and maximum fluorescence value of HuB5 activation of 4-1BB signaling pathway in reporter gene assay

[0165] Example 3. Anti-MSLN and 4-1BB bispecific antibodies and their applications

[0166] 1. Construction and Preparation of Anti-MSLN and 4-1BB Bispecific Antibodies

[0167] 1. Obtaining parental antibody VHH against MSLN

[0168] The parent antibody VHH of anti-MSLN comes from the anti-human MSLN nanoantibody anti-MSLN in Anwita's patent application WO2019246003A1. Analysis of the CDR region of anti-MSLN VHH identified a hotspot, namely aspartic acid in CDR2. Aspartic acid was mutated to glutamic acid, and the three CDR regions of the modified antibody VHH are shown in SEQ ID No. 5 at positions 31-35, 50-66, and 99-101, respectively. The variable region amino acid sequence is shown in SEQ ID No. 5 (hereinafter also referred to as anti-MSLN nanoantibody), which is used for the construction of anti-MSLN and 4-1BB bispecific antibodies.

[0169] 2. Construction of anti-MSLN and 4-1BB bispecific antibodies

[0170] The above-mentioned humanized B5 monoclonal antibody was transformed into an scFv format for the construction of an anti-MSLN and 4-1BB bispecific antibody: specifically, the heavy chain variable region VH (SEQ ID No. 3 positions 1-114) and the light chain variable region VL (SEQ ID No. 4 positions 1-107) were connected by a connecting peptide 1, i.e., VL-connecting peptide 1-VH; and a pair of electrostatic modifications were introduced between VL and VH in the scFv, wherein the charge modification can be (the position of the amino acid in the antibody is based on Kabat counting): VH39K-VL38D, VH38D-VL39K, VH39Y-VL38R, VH105D-VL43K or VH103D-VL44K; preferably VH39K-VL38D; and a pair of disulfide groups were introduced between VL and VH in the scFv. bond, wherein the disulfide bond position can be: VH44-VL100, VH100-VL50, VH100b-VL49, VH101-VL46 or VH105-VL43; preferably VH44-VL100; and a pair of disulfide bonds are introduced between VL and connecting peptide 1, and between VH and connecting peptide 1 in the scFv, wherein the disulfide bond positions are VL42-connecting peptide 1 and VH105-connecting peptide 1; wherein the connecting peptide 1 is selected from the following: (G4S)3(GGGGSGGGGSGGGGS, SEQ ID No. 13), (G4S) 4 (GGGGSGGGGSGGGGSGGGGS, SEQ ID No. 14), GGSGGSGGCPPCGSGG (SEQ ID No. 15), RGGGSGGSGGCPPCGGSGG (SEQ ID No. 16), GGGGSGGGSGCPPCGGGG (SEQ ID No. 17), GGGSGGCPPCGGGSGG (SEQ ID No. 17) No. 18) or GGGSGGSGGCPPCGGSGG (SEQ ID No. 19), preferably GGGSGGSGGCPPCGGSGG (SEQ ID No. 19).

[0171] The amino acid sequence of the modified anti-4-1BB scFv single-chain antibody is shown in SEQ ID No. 6, wherein positions 1-107 are humanized light chain variable regions, positions 108-125 are connecting peptide 1, positions 126-239 are humanized heavy chain variable regions, and a pair of electrostatic modifications VH39K-VL38D are introduced between VL and VH, a pair of disulfide bonds VH44-VL100 are introduced between VL and VH, and a pair of disulfide bonds are introduced between VL and connecting peptide 1 and between VH and connecting peptide 1: VL42-connecting peptide 1 and VH105-connecting peptide 1.

[0172] A bispecific antibody (BsAb) was constructed using the modified anti-MSLN VHH and anti-4-1BB scFv: the VHH variable region of anti-MSLN (SEQ ID No. 5) was fused to the N-terminus of the Fc domain, and the anti-4-1BB scFv (SEQ ID No. 6) was connected to the C-terminus of the Fc domain via a connecting peptide 2, i.e., the construct structure was VHH-Fc-connecting peptide 2-scFv; wherein the Fc domain was derived from human IgG, IgM, IgE, IgA or IgD, preferably any one of IgG1, IgG2, IgG3 and IgG4, more preferably IgG1; wherein the connecting peptide 2 was selected from the following: (G4S)n, wherein n is 1, 2, 3, 4, 5 or 6, preferably 3.

[0173] The structural schematic diagram of the bispecific antibody BsAb is shown in Figure 8, and the amino acid sequence is shown in SEQ ID NO: 7, wherein the amino acid sequence of the VHH variable region of anti-MSLN is SEQ ID No. 7 positions 1-112, the amino acid sequence of the Fc domain is SEQ ID No. 7 positions 113-343, the amino acid sequence of the connecting peptide 2 is SEQ ID No. 7 positions 344-358, and the amino acid sequence of the anti-4-1BB scFv single-chain antibody is SEQ ID No. 7 positions 359-597.

[0174] A full-length nucleotide sequence encoding a bispecific antibody BsAb was synthesized, and an XbaI restriction site (TCTAGA), a Kozak consensus recognition sequence (5'-GCCACC-3'), and a signal peptide sequence (5'-ATGGAGTTCGGCCTGTCCTGGCTGTTTCTGGTGGCCATCCTGAAGGGCGTGCAGTGC-3' (SEQ ID No. 20)) were introduced at the N-terminus. A stop codon and a HindIII restriction site (AAGCTT) were introduced at the C-terminus. The synthesized sequence was double-digested with XbaI and HindIII and then inserted into a similarly digested pcDNA3.4 vector. The recombinant vector of the bispecific antibody was obtained by sequencing verification.

[0175] The recombinant plasmid was introduced into the human embryonic kidney cell line HEK293F and cultured in a shaking incubator at 37°C, 5% CO2, and 120 rpm. The antibody protein was purified from the culture supernatant using a Protein A affinity chromatography column. The Protein A column (GE) was first equilibrated with PBS, and the culture supernatant was then passed through the column. The column was pre-eluted for 5 column volumes using Solution A (formulation: solvent: water, solute and concentration: 20 mM sodium phosphate, 500 mM NaCl, pH 5.0), followed by elution for 5 column volumes using Solution B (formulation: solvent: water, solute and concentration: 20 mM sodium acetate, 150 mM NaCl, pH 3.5). The eluted peak was collected and then concentrated using a 30 kDa concentrator to obtain the bispecific antibody BsAb.

[0176] The bispecific antibody BsAb was identified by SDS-PAGE electrophoresis. The results showed that the obtained bispecific antibody BsAb was a complete antibody, and its amino acid sequence was shown in SEQ ID No.7.

[0177] 2. Detecting the Binding Properties of BsAb to Two Antigens

[0178] 1. ELISA detection of BsAb binding characteristics to two antigens

[0179] Dilute HuMSLN-His to 1 μg / ml with NaHCO3, add 100 μl per well to the ELISA plate, and incubate at 4°C overnight; or dilute Hu4-1BB-mFc to 1 μg / ml with NaHCO3, add 100 μl per well to the ELISA plate, and incubate at 4°C overnight. The remaining steps are the same as those in step 2 of Example 1.

[0180] The amino acid sequence of HuMSLN-His is as follows (SEQ ID No. 21):

[0181] The sample to be tested is an anti-MSLN heavy chain antibody (comprising the variable region shown in SEQ ID No. 5 and the constant region of human IgG1), BsAb, or HuB5.

[0182] The result curves are shown in Figures 9 and 10. The EC50 of BsAb binding to recombinant human MSLN antigen was 0.1328 nM, which is about 1.6 times that of anti-MSLN nanobody (EC50 is 0.08357 nM); the EC50 of BsAb binding to recombinant human 4-1BB antigen was 15.30 nM, which is about 90 times that of HuB5 monoclonal antibody (EC50 is 0.1638 nM).

[0183] 2. ELISA detection of the simultaneous binding characteristics of BsAb to two antigens

[0184] HuMSLN-His was diluted with NaHCO3 to 1 μg / ml, 100 μl was added to each well of the ELISA plate, incubated at 4°C overnight, and washed 3 times with PBST (PBS + 0.1% Tween 20); after blocking with PBST containing 5% milk at 37°C for 2 hours, the plate was washed 3 times with PBST; the sample to be tested was diluted with PBST containing 1% milk, 100 μl was added to each well of the ELISA plate, incubated at room temperature for 1.5 hours, and washed 3 times with PBST; Hu4-1BB-mFc was diluted with 1% milk PBST to 1 ug / ml, 100 μl was added to each well of the ELISA plate, and incubated at room temperature for 1 hour; goat anti-mouse-HRP (Jackson) was diluted with 1% milk PBST, 100 μl was added to each well of the ELISA plate, and incubated at room temperature for 0.5 hour; TMB was added to each well, and the color was developed at room temperature in the dark; H2SO4 was added to terminate the reaction; SepctraMax was used. The absorbance (OD value) was measured at 450 nm using a Versa microplate reader, and the results were statistically analyzed using GraphPad Prism to calculate the EC50 value.

[0185] The above-mentioned samples to be tested are Anti-MSLN nanoantibodies or BsAb or HuB5.

[0186] The result curve is shown in Figure 11. Compared with Anti-MSLN nanobody and HuB5, BsAb can bind to human MSLN and 4-1BB antigens at the same time.

[0187] 3. FACS detection of the binding characteristics of BsAb to tumor cells expressing human MSLN and CHOK1 cells stably expressing human 4-1BB

[0188] H226 tumor cells or CHOK1-h4-1BB cells were cultured to the logarithmic growth phase, digested with trypsin, and centrifuged at 1000 rpm for 5 min at 4°C to precipitate the cells. The cells were resuspended in PBS and the cell density was adjusted to 2×10 5Each tube was washed once with 1% BSA (dissolved in PBS) and the supernatant was discarded. The antibody to be tested was diluted to the appropriate concentration with 1% BSA, and a 3-fold serial dilution was performed at 6 points. 100 μl of each antibody dilution was added to the EP tube containing the cells and incubated at 4°C in the dark for 1 hour. 1% BSA dilution was used as a negative control. After incubation, 400 μl of PBS containing 2% BSA was added, centrifuged for 5 minutes, and the supernatant was discarded. This operation was repeated once. 100 μl of 200-fold diluted goat anti-human IgG-APC (Jackson) was added to each EP tube and incubated at room temperature for 0.5 hour. 400 μl of 1×PBS containing 2% BSA was added, centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded. This operation was repeated once. After washing, 400 μl of 1×PBS was added to resuspend the cells and analyzed by flow cytometry. Data were processed using GraphPad Prism statistical software.

[0189] The antibody to be tested is anti-MSLN nanobody or BsAb or HuB5.

[0190] The results are shown in Figures 12, 13 and Table 4. The binding ability of BsAb to MSLN-expressing tumor cells H226 is close to that of the parental monoclonal antibody anti-MSLN, while the binding ability to CHOK1 stably transfected cell lines expressing 4-1BB is significantly lower than that of the parental monoclonal antibody HuB5.

[0191] The combination of ELISA and FACS showed that BsAb had a higher binding ability to MSLN antigen and a weaker binding ability to 4-1BB, thereby reducing the adverse effects of the Fc function in BsAb on CD8+ T cells expressing 4-1BB.

[0192] Table 4 shows the EC50 values ​​of BsAb binding to two antigens determined by FACS.

[0193] Example 4: In vitro efficacy testing of the biological function of BsAb

[0194] 1. BsAb activation effect on T cells

[0195] CD3 antibody (Biolegend, Cat. No. 317325) was diluted to 0.8 μg / ml with PBS and added to a 96-well plate. The plate was incubated at 37°C for 2 hours. The supernatant was removed and washed with PBS. CHOK1 / HuMSLN was added to the 96-well plate at 5×10 3 / well, cultured overnight in a 37 ° C incubator, and then 20 μg / ml of mitomycin (Annaiji, product number 50-07-7) was added and incubated for 3.5 hours. The BsAb prepared in Example 3 was diluted to an appropriate concentration and added to a 96-well plate. At the same time, HuMSLN-His 0.5 μg / ml (denoted as BsAb + HuMSLN-His in the figure) was added to the corresponding wells. Whole blood was taken from healthy people, and PBMCs were collected using lymphocyte separation fluid (Sigma) according to the instructions. Human CD8 magnetic beads (BD, product number 557941) were used to enrich human CD8 according to the instructions. + T cells were added to 96-well plates at 1 × 10 4 After incubation at 37°C for 3 days, the expression level of IFN-γ in the supernatant was detected.

[0196] The control was the one without the addition of BsAb (denoted as No Ab in the figure).

[0197] The control was made without adding HuMSLN-His (denoted as BsAb in the figure).

[0198] The CHOK1 / HuMSLN cell construction method is as follows: a nucleotide sequence (SEQ ID No. 11) encoding the full-length human MSLN (Uniprot#Q13421) was artificially synthesized. Then, according to common methods in the art, the target sequence (SEQ ID No. 11) was used to replace a small fragment between the XbaI and HindIII restriction enzyme recognition sites of the pCDNA3.4 vector to obtain the recombinant plasmid pCDNA3.4-MSLN. The recombinant plasmid pCDNA3.4-MSLN was introduced into CHOK1 cells using Lipofectamine 3000 transfection reagent (Invitrogen). After 48 hours, G418 (Shanghai Sangon Biotech Co., Ltd.) was added for selection, ultimately obtaining CHOK1 cells that highly express human MSLN (referred to as CHO-K1 / HuMSLN cells).

[0199] The results are shown in Figure 14. Compared with the No Ab group, BsAb can effectively activate CD8+ T cells to secrete IFN-γ in the presence of MSLN, and the activation ability is not affected by the presence of soluble MSLN.

[0200] 2. BsAb-mediated ADCC effect on tumor cells

[0201] Jurkat / FcγRIIIa(158V) / luc cells (Promega) were cultured to the logarithmic growth phase, digested, resuspended, counted, and added to 96-well plates at 3×10 cells per well. 4 OVCAR3 cells were cultured to the logarithmic growth phase, digested, resuspended, counted, and added to the corresponding 96-well plate, with 3×10 cells per well.4 cells. The test samples were diluted to 100 nM, and seven dilutions were added to a 96-well plate. HuMSLN-His 0.5 μg / ml was added to the corresponding wells. After incubation at 37°C for 18 hours, 100 μl of ONE-Glo Luciferase assay system reagent (Promega) was added to each well. Chemiluminescence was measured after incubation at room temperature for 10 minutes.

[0202] The control was made without adding HuMSLN-His (denoted as BsAb in the figure).

[0203] The test sample is a BsAb. As shown in Figure 15 and Table 5, the presence of soluble MSLN shifts the IC50 value of the BsAb-mediated ADCC effect to the right, but does not affect the upper plateau, that is, does not affect the maximum effect intensity.

[0204] Table 5 shows the EC50 and maximum fluorescence value of BsAb-mediated ADCC on tumor cells

[0205] Example 5: In vivo efficacy testing of BsAbs against tumor growth

[0206] Nanjing Kebai Biotechnology Co., Ltd. was commissioned to construct the MC38 / HuMSLN cell line and introduce the HuMSLN encoding gene (Uniprot#Q13421) into MC38 cells to express HuMSLN.

[0207] MC38 / HuMSLN cells were cultured in vitro and digested with trypsin. 6 6-8 week old C57BL / 6J-h4-1BB mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were subcutaneously inoculated with 50% matrix gel per mouse. The mice were divided into groups and dosed 5 days after inoculation (the average tumor volume was about 58 mm). 3 ), 6 mice per group, were given normal saline or 3.3 mg / kg BsAb, intraperitoneally, twice a week. Body weight and tumor volume were measured twice a week, and the volume was calculated using the formula 1 / 2 × length × width × width (mm 3 ).

[0208] The experimental results are shown in Figures 16 and 17. BsAb had no significant effect on the body weight of mice. Compared with the saline group, BsAb could effectively inhibit the growth of tumors in mice.

[0209] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

[0210] Industrial Applications

[0211] The bispecific antibodies provided by the present invention have important significance and application potential for preparing antibody-targeted drugs.

[0212] CROSS-REFERENCE TO RELATED APPLICATIONS

[0213] This application claims priority to the Chinese patent application (application number 202410135375.2) filed on January 31, 2024, the entire contents of which are incorporated herein by reference.

Claims

1. A construct comprising a 4-1BB-targeting antigen-binding fragment, comprising a 4-1BB-targeting antigen-binding fragment, wherein the 4-1BB-targeting antigen-binding fragment comprises a humanized heavy chain variable region and a humanized light chain variable region, wherein the amino acid sequences of CDR1, CDR2, and CDR3 in the humanized heavy chain variable region are as follows: positions 31-35, 50-66, and 99-103, respectively; and the amino acid sequences of CDR1, CDR2, and CDR3 in the humanized light chain variable region are as follows: positions 24-34, 50-56, and 89-97, respectively.

2. The construct according to claim 1, characterized in that: In the targeting 4-1BB antigen-binding fragment, the amino acid sequence of the humanized heavy chain variable region is such as SEQ ID No. 3, positions 1-114 or SEQ ID No. 6, positions 126-239, or has a consistency of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with SEQ ID No. 3, positions 1-114 or SEQ ID No. 6, positions 126-239.

3. The construct according to claim 1 or 2, characterized in that: In the targeting 4-1BB antigen-binding fragment, the amino acid sequence of the humanized light chain variable region is or SEQ ID No. 4, positions 1-107 or SEQ ID No. 6, positions 1-107, or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with SEQ ID No. 4, positions 1-107 or SEQ ID No. 6, positions 1-107.

4. The construct according to any one of claims 1 to 3, characterized in that: The 4-1BB-targeting antigen-binding fragment is in the form of Fab, Fab', Fv fragment, F(ab')2, scFv or di-scFv.

5. The construct according to any one of claims 1 to 4, characterized in that: The targeting 4-1BB antigen binding fragment is in the form of scFv; Furthermore, the scFv structure is specifically VH-connector peptide 1-VL or VL-connector peptide 1-VH; Furthermore, the scFv structure is specifically VL-connecting peptide 1-VH.

6. The construct according to claim 5, characterized in that: Introducing a pair of electrostatic modifications between VL and VH in the scFv, wherein the charge modifications are: VH39K-VL38D, VH38D-VL39K, VH39Y-VL38R, VH105D-VL43K or VH103D-VL44K; Introducing a pair of disulfide bonds between VL and VH in the scFv, wherein the disulfide bond positions are: VH44-VL100, VH100-VL50, VH100b-VL49, VH101-VL46 or VH105-VL43; A pair of disulfide bonds is introduced between VL and connecting peptide 1, and between VH and connecting peptide 1 in the scFv, wherein the disulfide bond positions are VL42-connecting peptide 1 and VH105-connecting peptide 1.

7. The construct according to claim 6, characterized in that: The charge modification is VH39K-VL38D; The position where a pair of disulfide bonds are introduced between VL and VH is VH44-VL100.

8. The construct according to any one of claims 5 to 7, characterized in that: The connecting peptide 1 is selected from the following: a fragment with an amino acid sequence of SEQ ID No. 13, a fragment with an amino acid sequence of SEQ ID No. 14, a fragment with an amino acid sequence of SEQ ID No. 15, a fragment with an amino acid sequence of SEQ ID No. 16, a fragment with an amino acid sequence of SEQ ID No. 17, a fragment with an amino acid sequence of SEQ ID No. 18 or a fragment with an amino acid sequence of SEQ ID No. 19, Furthermore, the connecting peptide 1 is specifically a fragment with an amino acid sequence of SEQ ID No.

19.

9. The construct according to any one of claims 1 to 8, characterized in that: The 4-1BB antigen-binding fragment targeting the 4-1BB antigen comprises an amino acid sequence such as a fragment of SEQ ID NO. 6, or a fragment having an amino acid sequence that is more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more identical to SEQ ID No.

6.

10. The construct according to claims 1-9, characterized in that: The construct is a bispecific antibody comprising an antibody targeting another antigen, wherein the target of the antibody targeting another antigen is PD-L1, MSLN, PSMA, B7-H3 or B7-H4.

11. The construct according to claim 10, characterized in that: The antibody targeting another antigen is a VHH nanobody targeting MSLN, wherein the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the VHH nanobody are as follows: positions 31-35, 50-66 and 99-101 of SEQ ID No. 5, respectively; Furthermore, the amino acid sequence of the heavy chain variable region of the VHH nanobody is such as SEQ ID NO.5, or has an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with SEQ ID No.

5.

12. The construct according to claim 11, characterized in that: The structural form of the construct is that the heavy chain variable region of the VHH nanobody targeting MSLN is fused to the N-terminus of the Fc domain, and the 4-1BB-targeting antigen-binding fragment is connected to the C-terminus of the Fc domain via a connecting peptide 2, that is, the structural form of the construct is VHH-Fc-connecting peptide 2-scFv; Furthermore, the Fc domain is derived from human IgG, IgM, IgE, IgA or IgD; specifically any one of IgG1, IgG2, IgG3 and IgG4; Furthermore, the Fc domain is specifically IgG1.

13. The construct according to claim 12, characterized in that: The connecting peptide 2 is selected from the following: A(EAAAK)4ALE, KVDKKVEPKSCDKTHT, G4S, (G4S)n, wherein n is 1, 2, 3, 4, 5 or 6; Furthermore, the connecting peptide 2 is specifically (G4S)3.

14. The 4-1BB-targeting construct according to any one of claims 10 to 13, wherein: The amino acid sequence of the bispecific antibody comprises SEQ ID NO: 7, or an amino acid sequence having 99% or greater, 95% or greater, 90% or greater, 85% or greater, 80% or greater, or 75% or greater identity thereto.

15. A pharmaceutical composition comprising: (a1) the 4-1BB-targeting construct according to any one of claims 1 to 14; and (a2) a pharmaceutically acceptable excipient, diluent, or carrier.

16. A nucleic acid encoding the 4-1BB targeting construct of any one of claims 1 to 14.

17. A vector comprising the nucleic acid according to claim 16.

18. An expression cassette, host bacteria or host cell comprising the nucleic acid of claim 16 or the vector of claim 17.

19. A method for preparing any one of claims 1-14 targeting 4-1BB constructs, the method comprising expressing the construct in a host bacteria or host cell as described in claim 18 and isolating the construct from the host bacteria or host cell.

20. Use of the 4-1BB-targeting construct according to any one of claims 1 to 14, the nucleic acid according to claim 16, the vector according to claim 17, or the expression cassette, host bacteria, or host cell according to claim 18 in any of the following: (B1) Preparation of antibody-targeted drugs; (B2) preparing a product for activating T lymphocytes; (B3) preparing a product that kills tumor cells; (B4) preparing products that inhibit tumor cell growth; (B5) Preparation of products for preventing and / or treating cancer.

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