Targeting 4-1BB constructs and uses thereof

Abstract

The invention discloses a targeted 4-1BB construct and an application of the targeted 4-1BB construct. The construction body provided by the invention comprises an antigen binding fragment targeting 4-1BB, wherein the amino acid sequence of a heavy chain variable region is as shown in the 1st-114 sites of SEQ ID No.3 or the 126-239 sites of SEQ ID No.6; and the amino acid sequence of the light chain variable region is as shown in the 1st-107th sites of SEQ ID No.4 or the 1st-107th sites of SEQ ID No.6. The construct further comprises an antibody targeting MSLN to form a bispecific antibody, and the bispecific antibody provided by the invention has a good T cell activation effect, and shows a good in-vitro tumor cell killing effect and an in-vivo tumor inhibition effect on the basis, so that the MSLN-targeted MSLN-targeted MSLN-targeted MSLN-targeted MSLN-targeted MSLN-targeted MSLN-targeted MSLN-targeted MSLN-targeted The bispecific antibody provided by the invention has important significance and application potential when being used for preparing antibody targeted drugs.

Description

Technical Field

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

[0002] The co-stimulatory molecule 4-1BB, an important member of the tumor necrosis factor receptor superfamily, mediates co-stimulatory signals for T cell activation and is mainly expressed on the surfaces of activated T cells, NK cells, neutrophils and DC cells. The co-stimulatory signals mediated by 4-1BB can enhance the function of T cells, improve the surveillance of T cells against tumor cells and the immune defense against viral infections. The 4-1BB signaling pathway can also induce CD4 + T cell-mediated immune tolerance and prevent the occurrence and development of autoimmune diseases. Modulating the immune function of lymphocytes by intervening in the action of the 4-1BB pathway may become a new immune therapy approach. In addition, 4-1BB is also expressed on the surface of Treg cells and is highly expressed on Tregs in many cancer patients, such as breast cancer, lung cancer, colon cancer, etc. Studies have shown that targeted elimination of 4-1BB+ Tregs can effectively inhibit tumor growth without affecting the function of CD8+ T cells (Freeman Z T, Nirschl T R, Hovelson D H, 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, various 4-1BB targeting drug forms have entered clinical trials, including but not limited to monoclonal antibodies, bispecific antibodies or multispecific antibodies, fusion proteins, etc. The action mechanisms of bispecific antibodies or multispecific antibodies mostly utilize tumor-associated antigens (TAAs) to specifically crosslink and activate 4-1BB signals in the tumor microenvironment, aiming to avoid systemic activation of the immune system and its associated toxic and side effects. The selection of TAAs includes PD-L1, HER2, CD19, Claudin18.2, EGFR, ROR1, etc., but there are still few studies on choosing mesothelin (MSLN) as the crosslinking target. Studies have shown that MSLN is not expressed or is lowly expressed in normal tissues and is highly expressed in many cancers, including mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, etc. Therefore, developing a drug that utilizes MSLN to crosslink and activate 4-1BB signals to enhance the killing ability of T cells has broad application prospects. Summary of the Invention

[0004] The object of the present invention is to provide a 4-1BB-targeting construct and its application, specifically to 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 additional 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 of using the antibodies and pharmaceutical compositions comprising these antibodies, for example, for treating cancer. The present invention is partly based 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 enhance the immune response of immune cells and have antitumor efficacy.

[0005] In a first aspect, this aspect provides a construct comprising a 4-1BB-targeting antigen-binding fragment, which comprises a 4-1BB-targeting antigen-binding fragment, and the 4-1BB-targeting antigen-binding fragment comprises a humanized heavy-chain variable region and a humanized light-chain variable region. The amino acid sequences of CDR1, CDR2, and CDR3 in the humanized heavy-chain variable region are successively shown as positions 31-35, 50-66, and 99-103 of SEQ ID No. 3; the amino acid sequences of CDR1, CDR2, and CDR3 in the humanized light-chain variable region are successively shown as positions 24-34, 50-56, and 89-97 of SEQ ID No. 4.

[0006] In the construct comprising a 4-1BB-targeting antigen-binding fragment described above, in the 4-1BB-targeting antigen-binding fragment, the amino acid sequence of the humanized heavy-chain variable region is shown as positions 1-114 of SEQ ID No. 3 or positions 126-239 of SEQ ID No. 6, 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 positions 1-114 of SEQ ID No. 3 or positions 126-239 of SEQ ID No. 6; and / or

[0007] In the targeting 4-1BB antigen-binding fragment, the amino acid sequence of the humanized light chain variable region is shown as positions 1-107 of SEQ ID No. 4 or positions 1-107 of SEQ ID No. 6, 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 positions 1-107 of SEQ ID No. 4 or positions 1-107 of SEQ ID No. 6.

[0008] In the construct containing the targeting 4-1BB antigen-binding fragment described above, the form of the targeting 4-1BB antigen-binding fragment is Fab, Fab’, Fv fragment, F(ab’)2, scFv or di-scFv;

[0009] Further, the form of the targeting 4-1BB antigen-binding fragment is scFv;

[0010] Further, the specific structure of the scFv is VH-linker 1-VL or VL-linker 1-VH, specifically VL-linker 1-VH;

[0011] Further, a pair of electrostatic modifications are introduced between VL and VH in the scFv, and the charge modifications are: VH39K-VL38D, VH38D-VL39K, VH39Y-VL38R, VH105D-VL43K or VH103D-VL44K; specifically VH39K-VL38D;

[0012] Further, a pair of disulfide bonds are introduced between VL and VH in the scFv, and the positions of the disulfide bonds are: VH44-VL100, VH100-VL50, VH100b-VL49, VH101-VL46 or VH105-VL43; specifically VH44-VL100;

[0013] Further, a pair of disulfide bonds are introduced between VL and linker 1 and between VH and linker 1 in the scFv, and the positions of the disulfide bonds are VL42-linker 1 and VH105-linker 1;

[0014] Further, linker 1 is selected from the following: (G4S)3, (G4S)4, GGSGGSGGCPPCGSGG, RGGGSGGSGGCPPCGGSGG, GGGSGGGSGCPPCGGGG, GGGSGGCPPCGGGSGG or GGGSGGSGGCPPCGGSGG, preferably GGGSGGSGGCPPCGGSGG;

[0015] Furthermore, the antigen-binding fragment targeting 4-1BB comprises the amino acid sequence shown in SEQ ID NO.6, or has a sequence 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.6.

[0016] In the construct comprising the antigen-binding fragment targeting 4-1BB described above, the construct is a bispecific antibody comprising an antibody targeting another antigen, wherein the other target is PD-L1, MSLN, PSMA, B7-H3 or B7-H4; in some embodiments, the 4-1BB targeting construct provided by the present invention comprises a multispecific (e.g., bispecific) anti-4-1BB molecule that binds 4-1BB and one or more additional antigens, which comprises an antibody portion (e.g., scFv) that binds 4-1BB and a second antibody portion (e.g., VHH) that specifically recognizes a second antigen. The second antigen may be a tumor-associated antigen such as PD-L1, MSLN, PSMA, B7-H3 or B7-H4.

[0017] Furthermore, 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 sequentially shown in positions 31-35, 50-66 and 99-101 of SEQ ID No.5;

[0018] Furthermore, the amino acid sequence of the heavy chain variable region of the VHH nanobody is as shown in SEQ ID NO.5, or has a sequence 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.

[0019] Among them, the nanobody of 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 the hinge region, and it only contains one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions.

[0020] In some embodiments, in the construct comprising the antigen-binding fragment targeting 4-1BB described above, the structural form of the construct is to fuse the heavy chain variable region of the VHH nanobody targeting MSLN to the N-terminus of the Fc domain, and connect the antigen-binding fragment targeting 4-1BB to the C-terminus of the Fc domain through Linker 2, that is, the structural form of the construct is VHH-Fc-Linker 2-scFv;

[0021] Further, the Fc domain is derived from human IgG, IgM, IgE, IgA or IgD; specifically any one of IgG1, IgG2, IgG3 and IgG4; more specifically IgG1;

[0022] Further, the linker peptide 2 is selected from the following: A(EAAAK)4ALE, KVDKKVEPKSCDKTHT, G4S, (G4S)n, where n is 1, 2, 3, 4, 5 or 6; more specifically (G4S)3;

[0023] Further, the bispecific antibody comprises an amino acid sequence as shown in SEQ ID NO: 7 or having an identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% thereto.

[0024] In a second aspect, the present invention provides a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises: (a1) the 4-1BB targeting construct of the first aspect; (a2) a pharmaceutically acceptable excipient, diluent or carrier. Further, the pharmaceutical composition may further include targeting other immune checkpoints such as PD-1, TIM-3, LAG-3, CTLA-4, OX40, etc., or targeting other TAAs such as MSLN, VEGF, EGFR, PD-L1, etc., as well as small molecules such as polypeptides and chemical drugs.

[0025] In a third aspect, the present invention provides a nucleic acid encoding the 4-1BB targeting construct of the first aspect.

[0026] In a fourth aspect, the present invention provides a vector containing the nucleic acid of the third aspect.

[0027] In a fifth aspect, the present invention provides an expression cassette, host bacterium or host cell containing the nucleic acid of the third aspect or the vector of the fourth aspect.

[0028] In a sixth aspect, the present invention provides a method for preparing the 4-1BB targeting construct of the first aspect, the method comprising expressing the construct in a host bacterium or host cell as described in the fifth aspect and isolating the construct from the host bacterium or host cell.

[0029] In a seventh aspect, the present invention provides the 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 bacterium or host cell of the fifth aspect in any of the following:

[0030] (B1) Preparing an antibody-targeted drug;

[0031] (B2) Preparing a product for activating T lymphocytes;

[0032] (B3) Products for killing tumor cells;

[0033] (B4) Products for inhibiting the growth of tumor cells;

[0034] (B5) Products for preventing and / or treating cancer.

[0035] Further, in B2), the activated T lymphocytes are activated in the presence of tumor cells expressing MSLN, and specifically in the embodiments of the present invention, CD8+ T cells are activated to secrete IFN-γ.

[0036] Further, in (B4), the cancer is selected from any one of the following: mesothelioma, ovarian cancer, lung cancer, esophageal cancer, pancreatic cancer, gastric cancer, cholangiocarcinoma, endometrial cancer, thymic cancer, colon cancer, and breast cancer.

[0037] Wherein, the CDR in the present invention is the "complementary determining region", which is the region in the variable domain of the antibody that is highly variable in sequence and forms a structurally defined "hypervariable loop" and / or contains antigen contact residues "antigen contact points". The CDR is mainly responsible for binding to the epitope. A variable region usually contains 3 CDR regions, which are CDR1, CDR2, and CDR3 in sequence from the N-terminus.

[0038] Experimental results show that the bispecific antibody provided by the present invention has a good T cell activation effect, and on this basis, shows good in vitro tumor cell killing effect and in vivo tumor suppression effect. Therefore, the bispecific antibody provided by the present invention has important significance and application potential for the preparation of antibody-targeted drugs.

[0039] Terms and Definitions

[0040] BsAb: Bispecific antibody;

[0041] TAA: Tumor associated antigen;

[0042] VH: Heavy chain variable domain;

[0043] VL: Light chain variable domain;

[0044] VHH: Nanobody, also known as single-domain antibody;

[0045] ELISA: Enzyme linked immunosorbent assay;

[0046] FACS: Fluorescence-activated cell sorting, also known as flow cytometry.

[0047] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all conventional procedures widely used in the respective fields. At the same time, for a better understanding of the present invention, the definitions and explanations of relevant terms are provided below.

[0048] As used herein, when referring to the amino acid sequence of the 4-1BB protein or the 4-1BB protein (UniProt Q07011), it includes the full length of the 4-1BB protein, or the extracellular fragment 4-1BB-ECD of 4-1BB or a fragment containing 4-1BB-ECD; it also includes fusion proteins of 4-1BB-ECD, such as fragments fused with the Fc protein fragment (mFc or hFc) of mouse or human IgG. The term "4-1BB protein" includes all such sequences, including their natural or artificial variants.

[0049] As used herein, the term EC50 refers to the concentration for 50% of maximal effect, which is the concentration that can cause 50% of the maximal effect.

[0050] As used herein, the term "monoclonal antibody" or "Antibody", unless otherwise indicated, generally refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair consisting of one "light" (L) chain and one "heavy" (H) chain. In general, the heavy chain can be understood as the polypeptide chain with a larger molecular weight in the antibody, and the light chain is the polypeptide chain with a smaller molecular weight in the antibody. The light chain can be classified into κ and λ light chains. The heavy chain is usually classified into μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable region and the constant region are joined 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 (CH1, 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 the antibody can mediate the binding of the immunoglobulin 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 further divided 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 in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form the antibody binding site. The assignment of amino acids to each region or domain follows the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or 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 three CDRs, such as 6, 9, or 12. For example, in the bispecific antibodies of the present invention, the heavy chain can be the C-terminus of the heavy chain of an IgG antibody linked to the ScFv of another antibody, in which case the heavy chain contains 9 CDRs. The term "antibody" is not limited by any particular method of producing the antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0051] As used herein, the term "nanobody" refers to a polypeptide that contains only one variable heavy chain region (VHH) and two conventional CH2 and CH3 regions. "Nanobody" is a special structure of VHH single-domain antibody based on the heavy chain antibody of alpaca, consisting of 3 CDRs and 4 FRs. Compared with the heavy chain of traditional antibody, the CDR3 of nanobody is longer, with 13 - 18 amino acids, which compensates to a certain extent for the decrease in antigen-binding ability caused by the lack of light chain.

[0052] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide that contains a fragment of a full-length antibody and retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and / or competes with the full-length antibody for specific binding to the antigen, and is also referred to as "antigen-binding portion", "antigen-binding domain". In some cases, the antigen-binding fragment includes Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and such polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0053] As used herein, the term "Fv fragment" means an antibody fragment composed of the VL and VH domains of a single arm of an antibody; the term "Fab fragment" means an antibody fragment composed of the VL, VH, CL, and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment that contains two Fab fragments linked by a disulfide bridge on the hinge region.

[0054] Antigen-binding fragments of an antibody (e.g., the antibody fragments described above) can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of the antibody can be screened for specificity in the same manner as for intact antibodies.

[0055] In this document, 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.

[0056] As used herein, the term "isolated" or "separated" refers to being obtained by artificial means from its natural state. If a "separated" substance or component occurs in nature, it may be that its natural environment has changed, or the substance has been separated from its natural environment, or both. For example, a certain polynucleotide or polypeptide that naturally exists in a living animal body in an unseparated state, and the highly purified same polynucleotide or polypeptide separated from this natural state is called isolated. The term "isolated" or "separated" does not exclude the admixture of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0057] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector enables the expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic element carried by it can 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); phages such as λ 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, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, polyomaviruses (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. In addition, the vector can also contain an origin of replication.

[0058] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, and it includes, but is 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 fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0059] 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 the antigen it targets. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) means that the antibody binds with a dissociation constant 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 Bind the antigen with an affinity (KD) of M or less. In some embodiments of the present invention, the term "targeting" refers to specific binding.

[0060] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "pAb" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are usually represented by single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala. Brief Description of the Drawings

[0061] Figure 1 For ELISA to detect the activity of anti-4-1BB chimeric antibody binding to recombinant human 4-1BB antigen.

[0062] Figure 2 For FACS to detect the activity of anti-4-1BB chimeric antibody binding to CHOK1-h4-1BB stable cell line.

[0063] Figure 3 For reporter gene assay to detect the activation of 4-1BB signaling pathway by anti-4-1BB chimeric antibody.

[0064] Figure 4 For the body weight of mice during the treatment of mouse MC38 tumors with anti-4-1BB chimeric antibody.

[0065] Figure 5 For the activity of anti-4-1BB chimeric antibody in inhibiting the growth of mouse MC38 tumors.

[0066] Figure 6 For ELISA to detect the activity of anti-4-1BB humanized antibody binding to recombinant human 4-1BB antigen.

[0067] Figure 7 For reporter gene assay to detect the activation of 4-1BB signaling pathway by anti-4-1BB humanized antibody.

[0068] Figure 8 For the structural schematic diagram of BsAb molecule.

[0069] Figure 9 For ELISA to detect the activity of BsAb molecule binding to recombinant human MSLN antigen.

[0070] Figure 10 For ELISA to detect the activity of BsAb molecule binding to recombinant human 4-1BB antigen.

[0071] Figure 11To detect the activity of BsAb molecules binding to recombinant human MSLN and human 4-1BB antigens simultaneously by ELISA.

[0072] Figure 12 To detect the activity of BsAb molecules binding to tumor cells expressing MSLN antigen by FACS.

[0073] Figure 13 To detect the activity of BsAb molecules binding to CHOK1-h4-1BB stable transfected cell line by FACS.

[0074] Figure 14 For MSLN-mediated activation of CD8 + T cells to produce IFN-γ.

[0075] Figure 15 To detect the ADCC effect of BsAb molecules on tumor cells by reporter gene assay.

[0076] Figure 16 To detect the effect of BsAb molecules on the body weight of mice during the treatment of mouse tumors.

[0077] Figure 17 To detect the inhibitory effect of BsAb molecules on tumor growth in the mouse MC38 / HuMSLN model. Detailed implementation manners

[0078] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0079] The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0080] The present invention will be further described in detail below in conjunction with the specific implementation manners. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements and do not limit the present invention in any way.

[0081] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0082] The following examples do not include a detailed description of traditional methods, such as those for gene amplification, recombinant plasmid construction, and introducing plasmids into host cells. Such methods have been described in many publications, including Sambrook, J., et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.

[0083] pcDNA3.4 vector: Invitrogen, Cat: A14697.

[0084] HEK293F cells: ATCC American Type Culture Collection.

[0085] H226 tumor cells: Nanjing Kebai Biotechnology Co., Ltd.

[0086] OVCAR3 cells: Nanjing Kebai Biotechnology Co., Ltd.

[0087] MC38 murine colon cancer cells: Nanjing Kebai Biotechnology Co., Ltd.

[0088] Example 1. Generation and Activity Detection of Anti-human 4-1BB Monoclonal Antibody

[0089] 1. Obtaining Anti-human 4-1BB Monoclonal Antibody

[0090] 1) Immunizing Mice to Obtain Anti-human 4-1BB Monoclonal Antibody

[0091] Mice were immunized with an antigen containing the extracellular region of human 4-1BB to generate monoclonal antibodies against 4-1BB. After 3 immunizations, blood was collected from the tail vein and the antibody titer was detected by ELISA. Mice with qualified titers were selected, and their spleen cells were removed. Hybridoma cell lines producing anti-human 4-1BB monoclonal antibodies were prepared by conventional cell fusion methods. Positive hybridoma cell lines were screened by ELISA, and the selected positive cell lines were cultured step by step in an expanded manner. The supernatant was harvested and the antibody was purified through a Protein A affinity chromatography column. Further, a monoclonal antibody against 4-1BB with medium affinity, namely B5 monoclonal antibody, was selected through affinity characterization.

[0092] A bivalent chimeric antibody (ChB5) was constructed by adding the human heavy chain constant region (IgG1 subtype, with L234A and L235A modifications) and the light chain constant region (Kappa subtype) domains to the heavy chain variable region and the light chain variable region of B5, respectively.

[0093] 2) Preparation of Anti-human 4-1BB Bivalent Chimeric Antibody ChB5

[0094] Synthesize the full-length gene sequences encoding the heavy and light chains. The heavy chain of the bivalent chimeric antibody ChB5 (SEQ ID NO.1) includes the heavy chain variable region of B5 (positions 1-114 of SEQ ID NO.1) and the human heavy chain constant region (IgG1 subtype, with L234A and L235A modifications, positions 115-444 of SEQ ID NO.1); the light chain of the bivalent chimeric antibody ChB5 (SEQ ID NO.2) includes the light chain variable region of B5 (positions 1-107 of SEQ ID NO.2) and the light chain constant region (Kappa subtype, positions 108-214 of SEQ ID NO.2) to form the domain.

[0095] Ligate the heavy chain between the XbaI and HindIII sites of the pcDNA3.4 vector to obtain the recombinant vector pChB5-H;

[0096] Ligate the light chain between the XbaI and HindIII sites of the pcDNA3.4 vector to obtain the recombinant vector pChB5-L;

[0097] Introduce the recombinant vectors pChB5-H and pChB5-L into the human embryonic kidney cell line HEK293F (ATCC American Type Culture Collection) to obtain recombinant cells, and then culture the recombinant cells in a 37°C, 5% CO2 shaking incubator at a rotation speed of 120 rpm.

[0098] Purify the antibody protein from the culture supernatant using a Protein A affinity chromatography column. The specific operation is as follows: First, equilibrate the Protein A column (GE Healthcare) with PBS, then pass the culture supernatant through the column. First, pre-elute with Solution A (formula: solvent is water, solute and concentration are: 20 mM sodium phosphate, 500 mM NaCl, pH 5.0) for 5 column volumes, and then elute with Solution B (formula: solvent is water, solute and concentration are: 20 mM sodium acetate, 150 mM NaCl, pH 3.5) for 5 column volumes. Collect the elution peak, and then concentrate it using a 30KDa concentrator centrifugal tube to obtain the antibody, that is, the anti-human 4-1BB chimeric antibody ChB5. This antibody is composed of a heavy chain and a light chain. The amino acid sequence of the heavy chain is as shown in SEQ ID No.1, and the amino acid sequence of the light chain is as shown in SEQ ID No.2. The heavy chain type of the ChB5 antibody is IgG1, and the light chain type is κ chain.

[0099] 2. Binding activity of the chimeric antibody ChB5 to the recombinant human 4-1BB antigen

[0100] Detect the binding activity of the chimeric antibody to the recombinant human 4-1BB antigen Hu4-1BB-mFc by ELISA. The specific steps are as follows:

[0101] Dilute Hu4-1BB-mFc with NaHCO3 to 0.5 μg / ml, add 100 μl per well to the ELISA plate, and incubate overnight at 4°C. Wash the plate 3 times with PBST (PBS + 0.1% Tween 20); after incubating and blocking with PBST containing 5% milk at 37°C for 2 hours, wash the plate 3 times with PBST; then dilute the sample to be tested with PBST containing 1% milk, add 100 μl per well to the ELISA plate, and incubate at room temperature for 1 hour; dilute goat anti-human-HRP (Jackson, catalog number 109-035-088) with PBST containing 1% milk, add 100 μl per well to the ELISA plate, and incubate at room temperature for 0.5 hour; add TMB to each well and develop color in the dark at room temperature; add H2SO4 to terminate; use a SepctraMax Versa microplate reader to measure the absorbance value (OD value) at 450 nm, and use GraphPad Prism to statistically analyze the results and calculate the EC50 value.

[0102] The above sample to be tested is the chimeric antibody ChB5 or utomilumab (search for the antibody sequence according to the IMGT database: https: / / www.imgt.org / mAb-DB / , IMGT / mAb-DB ID: 657; synthesize the nucleotide sequence encoding the antibody by gene synthesis and clone it into the pcDNA3.4 vector, and introduce it into HEK293F cells for expression and purification. For the specific steps, see 2) of 1 in Example 1).

[0103] The amino acid sequence of the above Hu4-1BB-mFc is as follows: MGNSCYNIVATLLLVLNFERTRS LQDPCSNCPAGTF CDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELT KKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQ PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0104] The results are as Figure 1 shown. Although the EC50 of the chimeric antibody ChB5 for binding to the recombinant human 4-1BB antigen is slightly weaker than that of the control utomilumab, the upper platform is better than the latter.

[0105] 3. Binding of the chimeric antibody to CHOK1 cells stably expressing human 4-1BB

[0106] The binding affinity of the chimeric antibody ChB5 to human 4-1BB transfected CHOK1 cells was determined by flow cytometry. For the construction method of the CHOK1 cell line CHOK1-h4-1BB stably expressing human 4-1BB, refer to Patent CN112794905A.

[0107] Specifically: The nucleotide sequence (SEQ ID No.8) encoding the full length of human 4-1BB (Uniprot#Q07011) was inserted between the XbaⅠ and HindⅢ sites of the pCDNA3.4 vector. After the obtained recombinant plasmid was verified to be correct by sequencing, it was transfected into wild-type CHOK1 cells using Lipofectamine 3000 transfection reagent (Invitrogen), and the cell line CHO-K1 / 4-1BB with high expression of human 4-1BB was obtained.

[0108] CHOK1-h4-1BB cells were cultured to the logarithmic growth phase, digested with trypsin, centrifuged at 4°C and 1000 rpm for 5 min to precipitate the cells, resuspended with PBS, and the cells were adjusted to 2×10 5 cells / tube, washed once with 1% BSA (mass-volume percentage g:ml, dissolved in PBS) solution, and the supernatant was removed. The antibody to be tested was diluted to 45 nM with 1% BSA as the starting concentration, and serially diluted 6 points with a 2-fold gradient. 100 μl of the antibody dilution at each concentration was added to the EP tubes containing the cells, and incubated at 4°C in the dark for 1 hour, using 1% BSA dilution as the negative control. After incubation, 400 μl of PBS containing 2% BSA was added and centrifuged for 5 minutes to discard the supernatant, and this operation was repeated once. 100 μl of goat anti-human IgG-FITC (Jackson) diluted 200-fold was added to each EP tube, incubated at room temperature for 0.5 hour, 400 μl of 1×PBS containing 2% BSA was added and centrifuged at 2000 rpm for 5 minutes to discard the supernatant, and this operation was repeated once. After washing, 400 μl of 1×PBS was added to resuspend the cells, and then detected by flow cytometry. The data was processed using GraphPad Prism statistical software.

[0109] The above-mentioned antibody to be tested was the chimeric antibody ChB5 or Utomilumab or IgG (GenScript, Cat: A01006).

[0110] The results are as Figure 2 shown in Table 1, and the binding ability of the anti-4-1BB chimeric antibody ChB5 to CHOK1-h4-1BB cells is similar to that of utomilumab.

[0111] Table 1 shows the EC50 of the binding of ChB5 to CHOK1-h4-1BB cells determined by the FACS method

[0112] ChB5 Utomilumab IgG EC50 (nM) 2.109 2.134 /

[0113] 4. Activation of the 4-1BB signaling pathway by chimeric antibodies

[0114] The activation activity of chimeric antibodies on the 4-1BB signaling pathway was detected by reporter gene experiments. The specific steps are as follows:

[0115] HEK293 / 4-1BB / NFkB-luc cells are HEK293 cells that express human 4-1BB and stably integrate the NFκB luciferase reporter gene.

[0116] The preparation method is as follows: The human 4-1BB sequence (SEQ ID No.8) was 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) were inserted into different sites of the pGL4.10 vector (Youbao Biotech) as target genes according to the general methods in the art to obtain plasmid B. Then, plasmids A and B were co-transfected into HEK293 cells (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) using Lipofectamine 3000 transfection reagent (Invitrogen). G418 (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was added for pressure screening, and finally HEK293 cells expressing human 4-1BB and stably integrating the NFκB luciferase reporter gene were obtained.

[0117] The HEK293 / 4-1BB / NFkB-luc cells were cultured to the logarithmic growth phase, digested, resuspended, counted, and then added to a 96-well plate, with 4×10 4 cells per well. The test samples were diluted to 12 μg / ml respectively, and then serially diluted 7-fold in 3-fold increments and added to the 96-well plate. Then, the cross-linking antibody Fab’ goat anti-human IgG Fc (Jackson) was added to the corresponding wells at a ratio of 2:1 (denoted as ChB5+Crosslinker or utomilumab+Crosslinker in the figure). After incubation in a 37 °C incubator for 18 h, 100 μl of ONE-Glo Luciferase assay system reagent (Promega) was added to each well. After incubation at room temperature for 10 min, the chemiluminescence value was measured. The addition of no cross-linking antibody was used as a control (denoted as ChB5 or utomilumab in the figure).

[0118] The test samples were chimeric antibody ChB5 or utomilumab respectively.

[0119] The results are as Figure 3As shown in 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 the cross-linked antibody, and the activation intensity is significantly stronger than the latter.

[0120] Table 2 shows the EC50 and maximum fluorescence value of the activation of the 4-1BB signaling pathway by ChB5 in the reporter gene assay

[0121]

[0122] 5. Inhibiting the growth of tumor cells in vivo

[0123] MC38 cells were cultured in vitro, digested with trypsin, and then subcutaneously inoculated into 6-8-week-old C57BL / 6J-h4-1BB mice (Jiangsu Jicui Yakang Biotech Co., Ltd.) at a density of 2×10 6 / mouse. Eleven days after inoculation, the mice were grouped and administered drugs (the average tumor volume was about 58 mm 3 ). Each group had 6 mice, and they were respectively given normal saline (recorded as Vehicle in the figure) or 10 mg / kg of monoclonal antibody by intraperitoneal injection once. After that, the body weight and tumor volume of the mice were measured twice a week, and the volume formula was 1 / 2×length×width×width (mm 3 ).

[0124] The above monoclonal antibody was the chimeric antibody ChB5 or Utomilumab.

[0125] The experimental results are as Figure 4 and Figure 5 shown. ChB5 had no obvious effect on the body weight of mice during the treatment of mouse tumors and had good anti-tumor activity.

[0126] Example 2. Humanization and activity detection of anti-human 4-1BB monoclonal antibody

[0127] 1. Humanization of anti-human 4-1BB monoclonal antibody

[0128] The heavy chain variable region and light chain variable region sequences of B5 were compared with the database to obtain a highly similar humanized framework region. Subsequently, through homology modeling and optimization of the antibody Fab, surface scanning was used to determine the humanized mutation sites, virtual mutation and molecular dynamics simulation were carried out to determine the key amino acids, and a series of analyses were used to design a reasonable humanized antibody. The heavy chain variable region and light chain variable region sequences of the antibody obtained by humanization are shown in positions 1-114 of SEQ ID No. 3 and positions 1-107 of SEQ ID No. 4, respectively.

[0129] 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.

[0130] The details are as follows:

[0131] 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).

[0132] 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.

[0133] 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.

[0134] 2. Binding activity of humanized antibody HuB5 to recombinant human 4-1BB antigen

[0135] 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.

[0136] The results are as follows Figure 6 As shown, the humanized antibody HuB5 exhibited similar affinity to the recombinant human 4-1BB protein compared to the chimeric antibody.

[0137] 3. Activation of the 4-1BB signaling pathway by humanized antibody HuB5

[0138] 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.

[0139] The results are as Figure 7 shown in Table 3. Similar to the chimeric antibody, the humanized antibody HuB5 has stronger activity in activating the 4-1BB antigen in vitro than the positive control utomilumab.

[0140] Table 3 shows the EC50 and maximum fluorescence value of the reporter gene assay for the activation of the 4-1BB signaling pathway by HuB5

[0141]

[0142] Example 3, Anti-MSLN and 4-1BB Bispecific Antibody and Its Application

[0143] I. Construction and Preparation of Anti-MSLN and 4-1BB Bispecific Antibody

[0144] 1. Obtaining the parental antibody VHH against MSLN

[0145] The parental antibody VHH against MSLN is the anti-human MSLN nanobody anti-MSLN from the patent application WO2019246003A1 of Anwita Company. Analysis of the CDR regions of anti-MSLN VHH identified a hot spot, i.e., aspartic acid in CDR2. Aspartic acid was mutated to glutamic acid. The three CDR regions of the modified antibody VHH are shown in positions 31-35, 50-66, and 99-101 of SEQ ID No. 5 in sequence, and the variable region amino acid sequence is shown in SEQ ID No. 5 (hereinafter also referred to as anti-MSLN nanobody), which is used for the construction of the anti-MSLN and 4-1BB bispecific antibody.

[0146] 2. Construction of Anti-MSLN and 4-1BB Bispecific Antibody

[0147] The above humanized B5 monoclonal antibody was modified into scFv form for the construction of anti-MSLN and 4-1BB bispecific antibody: specifically, the heavy chain variable region VH (positions 1-114 of SEQ ID No. 3) and the light chain variable region VL (positions 1-107 of SEQ ID No. 4) were connected by linker peptide 1, that is, VL-linker peptide 1-VH; and a pair of electrostatic modifications were introduced between VL and VH in the scFv, where the charge modifications can be (the positions of amino acids in the antibody are based on kabat counting): VH39K-VL38D, VH38D-VL39K, VH39Y-VL38R, VH105D-VL43K or VH103D-VL44K; preferably VH39K-VL38D; and a pair of disulfide bonds were introduced between VL and VH in the scFv, where the disulfide bond positions can be: VH44-VL100, VH100-VL50, VH100b-VL49, VH101-VL46 or VH105-VL43; preferably VH44-VL100; and a pair of disulfide bonds were introduced between VL and linker peptide 1, and between VH and linker peptide 1 in the scFv, where the disulfide bond positions are VL42-linker peptide 1 and VH105-linker peptide 1; where the linker peptide 1 is selected from the following: (G4S)3, (G4S)4, GGSGGSGGCPPCGSGG, RGGGSGGSGGCPPCGGSGG, GGGSGGGSGCPPCGGGG, GGGSGGCPPCGGGSGG or GGGSGGSGGCPPCGGSGG, preferably GGGSGGSGGCPPCGGSGG.

[0148] The amino acid sequence of the modified anti-4-1BB scFv single-chain antibody is shown in SEQ ID No. 6, where positions 1-107 are the humanized light chain variable region, positions 108-125 are linker peptide 1, positions 126-239 are the humanized heavy chain variable region, 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: VL42-linker peptide 1 and VH105-linker peptide 1 are introduced between VL and linker peptide 1, and between VH and linker peptide 1.

[0149] Construct a bispecific antibody (BsAb) using the above-modified anti-MSLN VHH and anti-4-1BB scFv: fuse the VHH variable region of anti-MSLN (SEQ ID No. 5) to the N-terminus of the Fc domain, and connect the above anti-4-1BB scFv (SEQ ID No. 6) to the C-terminus of the Fc domain through linker 2, that is, the construct structure is VHH-Fc-linker 2-scFv; wherein the Fc domain is derived from human IgG, IgM, IgE, IgA or IgD, preferably any one of IgG1, IgG2, IgG3 and IgG4, more preferably IgG1; wherein the linker 2 is selected from the following: (G4S)n, where n is 1, 2, 3, 4, 5 or 6, preferably 3.

[0150] The schematic structural diagram of the bispecific antibody BsAb is as Figure 8 shown, and the amino acid sequence is as shown in SEQ ID NO: 7, wherein the amino acid sequence of the VHH variable region of anti-MSLN is positions 1-112 of SEQ ID No. 7, the amino acid sequence of the Fc domain is positions 113-343 of SEQ ID No. 7, the amino acid sequence of linker 2 is positions 344-358 of SEQ ID No. 7, and the amino acid sequence of the anti-4-1BB scFv single-chain antibody is positions 359-597 of SEQ ID No. 7.

[0151] Synthesize the full-length nucleotide sequence encoding the bispecific antibody BsAb and introduce an XbaI restriction site (TCTAGA), a kozak consensus sequence (5’-GCCACC-3’), and a signal peptide sequence (5’-ATGGAGTTCGGCCTGTCCTGGCTGTTTCTGGTGGCCATCCTGAAGGGCGTGCAGTGC-3’) at the N-terminus, and introduce a stop codon and a HindIII restriction site (AAGCTT) at the C-terminus. After double digestion with XbaI and HindIII, the synthesized sequence is ligated into the pcDNA3.4 vector that has also been digested with enzymes, and the recombinant vector of the bispecific antibody is obtained after sequencing verification.

[0152] The recombinant plasmid was introduced into the human embryonic kidney cell line HEK293F and cultured in a shaking incubator at 37 °C and 5% CO2 with a rotation speed of 120 rpm. The antibody protein was purified from the culture supernatant using a Protein A affinity chromatography column. First, the Protein A column (GE) was equilibrated with PBS, and then the culture supernatant was passed through the column. First, it was pre-eluted with Solution A (formula: solvent is water, solute and concentration are: 20 mM sodium phosphate, 500 mM NaCl, pH 5.0) for 5 column volumes, and then eluted with Solution B (formula: solvent is water, solute and concentration are: 20 mM sodium acetate, 150 mM NaCl, pH 3.5) for 5 column volumes. The elution peak was collected, and then concentrated using a 30KDa concentrator centrifugal tube to obtain the bispecific antibody BsAb.

[0153] 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 as shown in SEQ ID No.7.

[0154] II. Detection of the binding characteristics of BsAb to two antigens

[0155] 1. ELISA detection of the binding characteristics of BsAb to two antigens

[0156] HuMSLN-His was diluted to 1 μg / ml with NaHCO3, and 100 μl per well was added to the ELISA plate and incubated overnight at 4 °C; or Hu4-1BB-mFc was diluted to 1 μg / ml with NaHCO3, and 100 μl per well was added to the ELISA plate and incubated overnight at 4 °C. The remaining steps were the same as 2 in Example 1.

[0157] The amino acid sequence of the above HuMSLN-His is as follows:

[0158] METDTLLLWVLLLWVPGSTG EVEKTACPSGKKAPEIDESLIFYKKWELEACVDAALLATQMDRVNAIPF TYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQVATLIDRFVKG RGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQS FLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLG LGLQGGIPNGYLVLDLSMQEALS LEGGGHHHHHH

[0159] The above sample to be tested is an anti-MSLN nanobody (including the variable region shown in SEQ ID No.5 and the constant region of human IgG1) or BsAb or HuB5.

[0160] The result curve is as Figure 9 and Figure 10As shown, the EC50 of the BsAb for the binding to recombinant human MSLN antigen was 0.1328 nM, about 1.6 times that of the anti-MSLN nanobody (EC50 was 0.08357 nM); the EC50 of the BsAb for the binding to recombinant human 4-1BB antigen was 15.30 nM, more than 90 times that of the HuB5 monoclonal antibody (EC50 was 0.1638 nM).

[0161] 2. Detection of the simultaneous binding characteristics of BsAb to two antigens by ELISA

[0162] Dilute HuMSLN-His to 1 μg / ml with NaHCO3, add 100 μl per well to the ELISA plate, and incubate overnight at 4°C. Wash the plate 3 times with PBST (PBS + 0.1% Tween 20); after incubating and blocking with PBST containing 5% milk at 37°C for 2 hours, wash the plate 3 times with PBST; then dilute the sample to be tested with PBST containing 1% milk, add 100 μl per well to the ELISA plate, and incubate at room temperature for 1.5 hours. Wash the plate 3 times with PBST; then dilute Hu4-1BB-mFc to 1 μg / ml with PBST containing 1% milk, add 100 μl per well to the ELISA plate, and incubate at room temperature for 1 hour; dilute goat anti-mouse-HRP (Jackson) with PBST containing 1% milk, add 100 μl per well to the ELISA plate, and incubate at room temperature for 0.5 hour; add TMB to each well and develop color in the dark at room temperature; add H2SO4 to terminate; use a SepctraMax Versa microplate reader to measure the absorbance value (OD value) at 450 nm, and use GraphPad Prism to statistically analyze the results and calculate the EC50 value.

[0163] The sample to be tested above was Anti-MSLN nanobody or BsAb or HuB5.

[0164] The result curve is as Figure 11 shown. Compared with the Anti-MSLN nanobody and HuB5, BsAb can simultaneously bind to human MSLN and 4-1BB antigens.

[0165] 3. Detection of the binding characteristics of BsAb to tumor cells expressing human MSLN and CHOK1 cells stably expressing human 4-1BB by FACS

[0166] Culture H226 tumor cells or CHOK1-h4-1BB cells to the logarithmic growth phase. After digestion with trypsin, centrifuge at 4°C and 1000 rpm for 5 min to precipitate the cells, resuspend them with PBS, and adjust the cells to 2×10 5Per tube, wash once with 1% BSA (dissolved in PBS) solution, and discard the supernatant. Dilute the antibody to be tested with 1% BSA to an appropriate concentration, perform 6-point 3-fold serial dilution, take 100 μl of the antibody diluent at each concentration, and add them separately to the EP tubes containing cells. Incubate at 4°C in the dark for 1 hour, using 1% BSA diluent as the negative control. After incubation, add 400 μl of PBS containing 2% BSA and centrifuge for 5 minutes to discard the supernatant, and repeat this operation once. Add 100 μl of goat anti-human IgG-APC (Jackson) diluted 200-fold to each EP tube, incubate at room temperature for 0.5 hour, add 400 μl of 1×PBS containing 2% BSA, centrifuge at 2000 rpm for 5 minutes to discard the supernatant, and repeat this operation once. After washing, add 400 μl of 1×PBS to resuspend the cells, then detect them on a flow cytometer, and process the data with GraphPad Prism statistical software.

[0167] The antibody to be tested described above is an anti-MSLN nanobody or BsAb or HuB5.

[0168] The results are as Figure 12 、 Figure 13 and shown in Table 4. The binding ability of BsAb to the tumor cell H226 expressing MSLN is close to that of the parental monoclonal antibody anti-MSLN, while the binding ability to the stable transfected cell line CHOK1 expressing 4-1BB is significantly lower than that of the parental monoclonal antibody HuB5.

[0169] Combining ELISA and FACS, it can be seen that BsAb has a high binding ability to the MSLN antigen, while the binding ability to 4-1BB is weak, thus reducing the adverse effects of the Fc function in BsAb on CD8+ T cells expressing 4-1BB.

[0170] Table 4 shows the binding EC50 of BsAb to the two antigens determined by the FACS method.

[0171] EC50 (nM) BsAb Anti-MSLN HuB5 H226 1.827 2.628 / CHOK1-h4-1BB 9.438 / 2.202

[0172] Example 4. Detect the biological function of BsAb in vitro

[0173] 1. Activation effect of BsAb on T cells

[0174] Dilute the CD3 antibody (Biolegend, catalog number 317325) with PBS to 0.8 μg / ml, add it to a 96-well plate, and incubate at 37°C for 2 hours. Discard the supernatant, wash with PBS, then add CHOK1 / HuMSLN to the 96-well plate, 5×10 3wells, incubated overnight in a 37°C incubator and then added with 20 μg / ml mitomycin (Aunejie, catalog number 50-07-7) and incubated for 3.5 h. Dilute the BsAb prepared in Example 3 to an appropriate concentration and add it to a 96-well plate. At the same time, add 0.5 μg / ml of HuMSLN-His (denoted as BsAb+HuMSLN-His in the figure) to the corresponding wells. Take healthy human whole blood, collect PBMCs using lymphocyte separation medium (Sigma) according to the instructions, and use human CD8 magnetic beads (BD, catalog number 557941) to enrich human CD8 + T cells, add them to a 96-well plate, 1×10 4 cells / well, and detect the expression level of IFN-γ in the supernatant after incubating at 37°C for 3 days.

[0175] Use no addition of BsAb as a control (denoted as No Ab in the figure).

[0176] Use no addition of HuMSLN-His as a control (denoted as BsAb in the figure)

[0177] The construction method of CHOK1 / HuMSLN cells is as follows: Artificially synthesize the nucleotide sequence (SEQ ID No.11) encoding the full length of human MSLN (Uniprot#Q13421). Then, according to the general method in the art, replace the small fragment between the XbaI and HindIII restriction enzyme recognition sites of the pCDNA3.4 vector with the target sequence (SEQ ID No.11) to obtain the recombinant plasmid pCDNA3.4-MSLN. Use Lipofectamine 3000 transfection reagent (Invitrogen) to introduce the recombinant plasmid pCDNA3.4-MSLN into CHOK1 cells. After 48 h, add G418 (Sangon Biotech (Shanghai) Co., Ltd.) for screening, and finally obtain CHOK1 cells with high expression of human MSLN (abbreviation: CHO-K1 / HuMSLN cells).

[0178] The results are as Figure 14 shown. Compared with the No Ab group, in the presence of MSLN, BsAb can effectively activate CD8+ T cells to secrete IFN-γ, and the activation ability is not affected by the presence of soluble MSLN.

[0179] 2. ADCC effect of BsAb on tumor cells

[0180] Culture Jurkat / FcγRIIIa(158V) / luc cells (Promega) to the logarithmic growth phase, digest, resuspend, count, and add them to a 96-well plate, 3×10 4cells; The OVCAR3 cells were cultured to the logarithmic growth phase, digested, resuspended, counted, and then added to the corresponding 96-well plates, with 3×10 4 cells per well. The test samples were respectively diluted to 100 nM and serially diluted 7-fold in 4-fold increments and added to the 96-well plates. Meanwhile, 0.5 μg / ml of HuMSLN-His was added to the corresponding wells. After incubation in a 37 °C incubator for 18 h, 100 μl of ONE-Glo Luciferase assay system reagent (Promega) was added to each well. After incubation at room temperature for 10 min, the chemiluminescence value was measured.

[0181] Taking no addition of HuMSLN-His as the control (denoted as BsAb in the figure)

[0182] The above-mentioned test sample was BsAb. The results are as Figure 15 shown in and Table 5. The presence of soluble MSLN would shift the IC50 value of the ADCC effect mediated by BsAb to the right, but would not affect the upper plateau, that is, would not affect the maximum effect intensity.

[0183] Table 5 shows the EC50 and maximum fluorescence value of the ADCC of BsAb against tumor cells

[0184] BsAb BsAb+HuMSLN-His EC50 (nM) 0.08471 3.729 <![CDATA[Maximum fluorescence value (×10 3 )]]> 10.61 11.04

[0185] Example 5. In vivo efficacy detection of the inhibitory effect of BsAb on tumor growth

[0186] Nanjing Kebai Biotechnology Co., Ltd. was commissioned to construct the MC38 / HuMSLN cell line. The HuMSLN-encoding gene (Uniprot#Q13421) was introduced into MC38 cells to enable the expression of HuMSLN.

[0187] MC38 / HuMSLN cells were cultured in vitro. After digestion with trypsin, they were subcutaneously inoculated into 6-8-week-old C57BL / 6J-h4-1BB mice (Jiangsu Jicui Yakang Biotechnology Co., Ltd.) at 1×10 6 / mouse (containing 50% Matrigel). Five days after inoculation, grouping and drug administration were started (the average tumor volume was about 58 mm 3 ). There were 6 mice in each group, and they were respectively given normal saline or 3.3 mg / kg of BsAb by intraperitoneal injection, twice a week. The body weight and tumor volume were measured twice a week, and the volume formula was 1 / 2 × length × width × width (mm 3 ).

[0188] The experimental results are as Figure 16 and Figure 17 shown. BsAb had no obvious effect on the body weight of mice. Compared with the normal saline group, BsAb could effectively inhibit the tumor growth of mice.

[0189] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses or improvements of the present invention, including those that depart from the scope disclosed in this application but are made by conventional techniques known in the art. Applications of some basic features can be made within the scope of the appended claims below.

Claims

1. A construct comprising a targeting 4-1BB antigen-binding fragment, which includes a targeting 4-1BB antigen-binding fragment. The targeting 4-1BB antigen-binding fragment includes a humanized heavy-chain variable region and a humanized light-chain variable region. The amino acid sequences of CDR1, CDR2, and CDR3 in the humanized heavy-chain variable region are successively shown as positions 31-35, 50-66, and 99-103 of SEQ ID No. 3; the amino acid sequences of CDR1, CDR2, and CDR3 in the humanized light-chain variable region are successively shown as positions 24-34, 50-56, and 89-97 of SEQ ID No.

4.

2. The construct according to claim 1, comprising a targeting 4-1BB antigen-binding fragment, characterized in that: In the targeting 4-1BB antigen-binding fragment, the amino acid sequence of the humanized heavy-chain variable region is shown as positions 1-114 of SEQ ID No. 3 or positions 126-239 of SEQ ID No. 6, 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 positions 1-114 of SEQ ID No. 3 or positions 126-239 of SEQ ID No. 6; and / or In the targeting 4-1BB antigen-binding fragment, the amino acid sequence of the humanized light-chain variable region is shown as positions 1-107 of SEQ ID No. 4 or positions 1-107 of SEQ ID No. 6, 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 positions 1-107 of SEQ ID No. 4 or positions 1-107 of SEQ ID No.

6.

3. The construct comprising a targeting 4-1BB antigen-binding fragment according to claim 1 or 2, characterized in that: The form of the targeting 4-1BB antigen-binding fragment is Fab, Fab’, Fv fragment, F(ab’)2, scFv, or di-scFv; Further, the form of the targeting 4-1BB antigen-binding fragment is scFv; Further, the specific structure of the scFv is VH-linker 1-VL or VL-linker 1-VH, specifically VL-linker 1-VH; Further, a pair of electrostatic modifications is introduced between VL and VH in the scFv, and the charge modifications are: VH39K-VL38D, VH38D-VL39K, VH39Y-VL38R, VH105D-VL43K, or VH103D-VL44K; specifically VH39K-VL38D; Further, a pair of disulfide bonds is introduced between VL and VH in the scFv, and the positions of the disulfide bonds are: VH44-VL100, VH100-VL50, VH100b-VL49, VH101-VL46, or VH105-VL43; specifically VH44-VL100; Further, a pair of disulfide bonds is introduced between VL and linker 1, and between VH and linker 1 in the scFv, and the positions of the disulfide bonds are VL42-linker 1 and VH105-linker 1; Further, the linker peptide 1 is selected from the following: (G4S)3, (G4S)4, GGSGGSGGCPPCGSGG, RGGGSGGSGGCPPCGGSGG, GGGSGGGSGCPPCGGGG, GGGSGGCPPCGGGSGG or GGGSGGSGGCPPCGGSGG, preferably GGGSGGSGGCPPCGGSGG; Further, the antigen-binding fragment targeting 4-1BB comprises the amino acid sequence shown in SEQ ID NO.6, or has a sequence 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.

6.

4. The targeting 4-1BB construct according to claims 1-3, characterized in that: The construct is a bispecific antibody comprising an antibody targeting another antigen, wherein the other target is PD-L1, MSLN, PSMA, B7-H3 or B7-H4; Further, 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 shown in positions 31-35, 50-66 and 99-101 of SEQ ID No.5 in sequence; Further, the amino acid sequence of the heavy chain variable region of the VHH nanobody is shown in SEQ ID NO.5, or has a sequence identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with SEQID No.

5.

5. The targeting 4-1BB construct according to claim 4, characterized in that: The structural form of the construct is to fuse the heavy chain variable region of the VHH nanobody targeting MSLN to the N-terminus of the Fc domain, and connect the antigen-binding fragment targeting 4-1BB to the C-terminus of the Fc domain through linker peptide 2, that is, the structural form of the construct is VHH-Fc-linker 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; more specifically IgG1; Further, the linker peptide 2 is selected from the following: A(EAAAK)4ALE, KVDKKVEPKSCDKTHT, G4S, (G4S)n, where n is 1, 2, 3, 4, 5 or 6; more specifically (G4S)3; Further, the bispecific antibody comprises the amino acid sequence shown in SEQ ID NO:7 or has a sequence identity of more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% with it.

6. A pharmaceutical composition, which comprises: (a1) any one of the targeting 4-1BB constructs according to claims 1-5; (a2) a pharmaceutically acceptable excipient, diluent or carrier.

7. A nucleic acid encoding any one of the targeting 4-1BB constructs according to claims 1-5.

8. A vector containing the nucleic acid according to claim 7.

9. An expression cassette, host bacterium or host cell comprising the nucleic acid according to claim 7 or the vector according to claim 8.

10. A method for preparing the targeting 4-1BB construct according to any one of claims 1-5, the method comprising expressing the construct in the host bacterium or host cell as described in claim 9 and isolating the construct from the host bacterium or host cell.

11. Use of the targeting 4-1BB construct according to any one of claims 1-5, the nucleic acid according to claim 7, the vector according to claim 8, or the expression cassette, host bacterium or host cell according to claim 9 in any of the following: (B1) Preparing an antibody-targeted drug; (B2) Preparing a product for activating T lymphocytes; (B3) Preparing a product for killing tumor cells; (B4) Preparing a product for inhibiting the growth of tumor cells; (B5) Preparing a product for preventing and / or treating cancer.

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