Multi-specific antigen binding protein and application thereof

Abstract

The present invention provides a multispecific antigen binding protein comprising at least: (a) a first binding domain that specifically binds to a natural killer cell activated receptor (NKp46); and (b) a second binding domain that specifically binds to mesothelin (MSLN). The multispecific antigen binding protein provided by the invention can be bound with NKp46 and MSLN with high affinity, so that immune cells expressing the NKp46 are mediated to kill MSLN positive cells; and the multispecific antigen binding protein can have a full-length IgG configuration, so that the multispecific antigen binding protein has the advantages of a natural antibody in application. The invention also provides application of the multispecific antigen binding protein in tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a multispecific antigen binding protein capable of specifically binding to natural killer cell activating receptor NKp46 (NCR1 or CD335) and mesothelin (MSLN) and its application in disease treatment. Background Art

[0002] Natural Killer cell activating receptor NKp46, also known as NCR1 or CD335, is a 46kDa glycoprotein belonging to the immunoglobulin superfamily and consists of two extracellular Ig-like domains of the C2 type (LMoretta, C Bottino, D Pende et al., Seminars in Immunology 2006, 18, 151-158). Signaling is mediated by association with molecules CD3ξ and FcRγ containing immunoreceptor tyrosine activation motifs, which activate tyrosine phosphorylation after binding to ligands, causing NK cell activation (O Demaria, L Gauthier, G Debroas et al., Eur J Immunol 2021, 51, 1934-1942).

[0003] Studies have shown that NKp46 is expressed on resting and activated NK cells, innate lymphoid cells (ILCs), some T lymphocytes, and ILC3 subsets in the mucosa (EVivier, D Artis, M Colonna et al., Cell 2018, 174, 1054-1066). Several studies have analyzed the expression of activating receptors on the surface of NK cells infiltrating different tumor tissues. The results showed that various activating receptors such as NKG2D, NKp30, NKp44, and CD16 showed a down-regulation trend in tumor tissues, while the NKp46 receptor maintained a normal expression level (C Fauriat, S Just-Landi, F Mallet et al., Blood 2007, 109, 323-330; Mamessier, A Sylvain, ML Thibult et al., The Journal of Clinical Investigation 2011, 121, 3609-3622; S Platonova, J Cherfils-Vicini, D Damotte et al., Cancer Res 2011, 71, 5412-5422; L Gauthier, A Morel, N Anceriz et al., Cell 2019, 177, 1701-1713.e1716). This feature makes NKp46 a potential target for NK cell tumor immunotherapy.

[0004] Mesothelin (MSLN) is a cell surface glycoprotein encoded by the MSLN gene, which was first discovered by Chang et al. in 1992. The MSLN gene is located at 16p.13.3 of human chromosomes and contains 18 exons, encoding a 71kDa precursor protein, which is hydrolyzed to generate two protein products, 40kDa mesothelin and 31kDa megakaryocyte-potentiating factor (MPF) (K Chang and I Pastan. Proc Natl Acad Sci 1996, 93, 136-140). Mesothelin is anchored on the cell surface by glycosylphosphatidylinositol (GPI) and can regulate cell adhesion.

[0005] MSLN is only expressed in a small amount of normal tissues, including the pleura, pericardium, peritoneum and vaginal endometrium. Some researchers have studied MSLN knockout mice and found that compared with wild-type mice, MSLN knockout mice had no abnormalities in growth, reproduction and platelet count (TK Bera and IPastan. Molecular and Cellular Biology 2000, 20, 2902-2906). This shows that MSLN is not necessary for the normal growth, development and reproduction of mice. There are also experiments showing that MSLN may be involved in cell adhesion, differentiation and signal transduction (A Rump, Y Morikawa, M Tanaka et al., Journal of Biological Chemistry 2004, 279, 9190-9198). In short, the biological function of mesothelin under normal physiological conditions remains to be further studied. In contrast, several studies have found that MSLN is highly expressed in solid tumors such as ovarian cancer, pancreatic cancer, endometrial cancer, malignant mesothelioma, and lung adenocarcinoma (A Morello, M Sadelain, and PSAdusumilli. Cancer Discovery 2016, 6, 133-146). Overexpression of MSLN can activate multiple intracellular pathways, including NFκB, MAPK, and PI3K pathways, and can resist cell apoptosis or promote cell proliferation and metastasis by inducing the activation and expression of multiple matrix metalloproteinases (MMPs), causing cell resistance (SH Chen, WC Hung, P Wang, et al., Scientific Reports 2013, 3, 1870; UBharadwaj, C Marin-Muller, M Li, et al., Mol Cancer 2011, 10, 106; MC Chang, CA Chen, PJ Chen, et al., Biochemical Journal 2012, 442, 293-302). Studies have shown that the expression of MSLN differs between chemotherapy-sensitive and -resistant ovarian cancer patients, with MSLN expression significantly elevated in resistant patients. After downregulating MSLN expression, the chemotherapy sensitivity of ovarian cancer resistant cell lines was also significantly improved, reversing cell resistance to a certain extent.Rump et al. found in their study of ovarian cancer that MSLN can interact with the cancer antigen CA-125 (MUC16) on ovarian cancer with high affinity, leading to heterotypic adhesion between cells and promoting the metastasis of ovarian cancer cell lines (A Rump, Y Morikawa, M Tanaka, S Minami, N Umesaki, M Takeuchi and A Miyajima. Journal of Biological Chemistry 2004, 279, 9190-9198). In addition, the MSLN gene may be regulated by molecules in the Wnt signal transduction pathway. In tumors with persistent activation of the Wnt signal transduction pathway, such as ovarian cancer and pancreatic cancer, the expression level of MSLN increases (MG Prieve and RT Moon. BMC Developmental Biology 2003, 3, 2). According to clinical observations, increased expression of MSLN is often associated with tumor progression and decreased overall survival (SS Kachala, AJ Bograd, J Villena-Vargas et al., Clin Cancer Res 2014, 20, 1020-1028).

[0006] Compared with hematological tumors, the characteristics of solid tumors make it difficult for effector cells to infiltrate, and the expression of some solid tumor-related antigens such as human epidermal growth factor receptor 2 (HER2), epithelial cell adhesion molecule (EpCAM) and epidermal growth factor receptor (EGFR) in key tissues will cause unnecessary cytotoxicity of drugs targeting effector cells of such targets. Current studies have shown that MSLN is highly expressed in various solid tumors and only lowly expressed in a small number of mesothelial cells, and no abnormalities in growth and reproduction-related indicators were observed in mice with MSLN gene knockout. These characteristics make MSLN an important target for solid tumor immunotherapy.

[0007] Bi / multi-specific antibodies have two or more antigen binding sites, which can recognize different epitopes on the same or different antigens, have diverse structures, and different mechanisms of action. Among them, antibodies with full-length IgG structures containing the Fc region retain the excellent properties of natural IgG, and because of their large molecular weight and ability to bind to Fc receptors, such antibodies usually have a longer serum half-life, and can rely on the affinity between Fc and Protein A to capture antibodies in cell culture supernatants, making them easier to produce and purify (RE Kontermann. BioDrugs 2009, 23, 93-109). In the field of tumor treatment, the mechanism of action of bi / multi-specific antibodies can be roughly divided into effector cell redirection, immune regulation, and dual binding of targeted tumor cell receptors (A P Kufer, R Lutterbüse et al., Blood 2000, 95, 2098-2103; S Nie, Z Wang, M Moscoso-Castro et al., Antibody Therapeutics 2020, 3, 18-62; P Perez, RW Hoffman, S Shaw et al., Nature 1985, 316, 354-356; UD Staerz, O Kanagawa and MJ Bevan. Nature 1985, 314, 628-631). Among them, the development of effector cell redirection function antibodies accounts for a relatively high proportion, and this type of antibody is mainly T cell engagers that recruit T cells to the tumor site, which have shown good therapeutic effects in clinical treatment; however, the side effects caused by the drug, such as cytokine storm syndrome and neurotoxicity, should not be ignored.

[0008] After research and development in recent years, multiple bi / tri-specific binders targeting NK cells and tumor-associated antigens have been shown to have strong anti-tumor effects and high safety in preclinical and clinical studies. The structures of these molecules vary and can bind to specific tumor antigens and NK cell activating receptors (such as CD16, NKp30, NKp46, and NKG2D) to increase the cytotoxicity of NK cells against specific tumor targets; some of these configurations also integrate cytokines such as IL-15. They can be used as a monotherapy or in combination with other effective anticancer therapies, bringing new ideas to immunotherapy targeting NK cells (SK Phung, JS Miller and M Felices. Molecular Diagnosis & Therapy 2021, 25, 577-592). In the research of these NK cell binders, most of them are based on the NK cell activating receptor CD16 to achieve the recruitment of NK cells, and there are relatively few studies on the target of NKp46. In terms of antibody configuration design, most of them use non-IgG-like subtypes without Fc regions, and there are few studies on NK cell binders with full-length IgG configurations. Summary of the invention

[0009] In order to solve the above technical problems, an object of the present invention is to provide a multispecific antigen-binding protein, which comprises at least a portion targeting NKp46 and a portion targeting MSLN, and can bind NKp46 and MSLN with high affinity, thereby mediating the killing of MSLN-positive tumor cells by immune cells expressing NKp46. More particularly, the multispecific antigen-binding protein provided by the present invention can have a full-length IgG configuration, thereby having the advantages of natural antibodies in application, such as lower immunogenicity and longer in vivo half-life.

[0010] Another object of the present invention is to provide use of the multispecific antigen-binding protein in tumor treatment.

[0011] As used herein, the terms "multispecific antigen-binding protein", "natural killer cell engager", "NK cell engager", "NKCE", "bispecific antibody", "antibody" and the like are used interchangeably depending on the context.

[0012] The technical solution of the present invention is as follows.

[0013] In one aspect, the present invention provides a multispecific antigen-binding protein, comprising at least:

[0014] (a) a first binding domain, which specifically binds to natural killer cell activating receptor (NKp46);

[0015] (b) a second binding domain, which specifically binds to mesothelin (MSLN).

[0016] Optionally, in the multispecific antigen-binding protein provided by the present invention, the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody, and the heavy chain variable region comprises the following complementarity determining region (CDR) combination (H-CDR1-3): comprising the amino acid sequence shown in SEQ ID NO.7 ( DYVIN ) of H-CDR1; comprising the amino acid sequence shown in SEQ ID NO.8 ( EIYPGSGTNYYNEKFKA ) of H-CDR2; comprising the amino acid sequence shown in SEQ ID NO.9 ( RGRYGLYAMDY ) of H-CDR3; the light chain variable region comprises the following complementarity determining region (CDR) combination (L-CDR1-3): comprising the amino acid sequence shown in SEQ ID NO.12 ( RASQDISNYLN ) of L-CDR1; comprising the amino acid sequence shown in SEQ ID NO.13 ( YTSRLHS ) of L-CDR2; comprising the amino acid sequence shown in SEQ ID NO.14 ( QGNTRPWT )'s L-CDR3.

[0017] Furthermore, the heavy chain variable region in the first binding domain comprises the amino acid sequence shown in SEQ ID NO.10 or an amino acid sequence that is at least 75% identical to the amino acid sequence; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.15 or an amino acid sequence that is at least 75% identical to the amino acid sequence.

[0018] Optionally, in the multispecific antigen-binding protein provided by the present invention, the second binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody, wherein the heavy chain variable region comprises the following complementarity determining region (CDR) combination (H-CDR1-3): comprising the amino acid sequence shown in SEQ ID NO.17 ( SGSYYWS )'s H-CDR1; comprising the amino acid sequence shown in SEQ ID NO.18 ( YIYYSGSTNYNPSLKS ) of H-CDR2; comprising the amino acid sequence shown in SEQ ID NO.19 ( EDVLSGFDI ) of H-CDR3; the light chain variable region comprises the following complementarity determining region (CDR) combination (L-CDR1-3): comprising the amino acid sequence shown in SEQ ID NO.22 ( RASQSISSYLN ) of L-CDR1; comprising the amino acid sequence shown in SEQ ID NO.23 ( AASSLQS ) of L-CDR2; comprising the amino acid sequence shown in SEQ ID NO.24 ( QQSYDAFYT)'s L-CDR3.

[0019] Furthermore, the heavy chain variable region in the second binding domain comprises the amino acid sequence shown in SEQ ID NO.20 or an amino acid sequence that is at least 75% identical to the amino acid sequence; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.25 or an amino acid sequence that is at least 75% identical to the amino acid sequence.

[0020] In the context of the present invention, the phrase "at least 75% identity" used in relation to a sequence refers to any percentage number between 75% and 100% identity, such as 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% identity. In the multispecific antigen-binding proteins provided by the present invention, in particular in the first and second binding domains, the corresponding "heavy chain variable region" and "light chain variable region" both include the above CDR combination and the framework region between them, and the arrangement of each domain is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Alternatively, the "at least 75% identity" resulting in a maximum of 25% difference in amino acid sequence may exist in any framework region in the heavy chain variable region or the light chain variable region, or in any domain or sequence other than the heavy chain variable region and the light chain variable region. The differences may be caused by deletion, addition or substitution of amino acids at any position.

[0021] Preferably, in the multispecific antigen-binding protein provided by the present invention, the first binding domain or the second binding domain each has a structure selected from the following: Fab, Fab', F(ab')2, Fd, Fv, dAb, Fc, single-chain antibody, single-domain antibody or a combination thereof. In the case of a combination of different structures, for example, the first binding domain targeting NKp46 has a Fab-Fc structure, and the second binding domain targeting MSLN may have a Fab structure; or vice versa.

[0022] Furthermore, in the multispecific antigen-binding protein provided by the present invention, the first binding domain and the second binding domain further comprise a heavy chain constant region (CH) or a light chain constant region (CL) of an antibody, respectively. For example, preferably, the first binding domain and the second binding domain further comprise a heavy chain constant region selected from IgG, IgA, IgM, IgD or IgE and / or a κ or λ type light chain constant region, respectively.

[0023] Optionally, the multispecific antigen-binding protein provided by the present invention may also contain other binding domains, such as binding domains targeting other tumor antigens or immune cell receptors. In this case, the multispecific antigen-binding protein is, for example, a trispecific or higher-specific antigen-binding protein.

[0024] According to a specific embodiment of the present invention, the multispecific antigen-binding protein provided by the present invention is a bispecific antigen-binding protein, and the "bispecific" is directed against NKp46 and MSLN. NKp46 is expressed on resting and activated NK cells and other types of cells; therefore, the multispecific antigen-binding protein provided by the present invention can be a natural killer cell engager (NKCE).

[0025] Further, the multispecific antigen-binding protein provided by the present invention is an IgG-type antibody molecule. Depending on the target antigen binding properties, the IgG-type antibody molecule may be a bispecific antibody molecule. More preferably, the multispecific antigen-binding protein provided by the present invention is an IgG1 subtype antibody molecule.

[0026] According to a specific embodiment of the present invention, the multispecific antigen-binding protein provided by the present invention is a human IgG1 bispecific antibody molecule, which may comprise:

[0027] Heavy chain 1, comprising domains arranged according to VH-CH1-CH2-CH3 from N-terminus to C-terminus;

[0028] Light chain 1, comprising domains arranged in a VL-CL pattern from N-terminus to C-terminus;

[0029] Heavy chain 2, comprising domains arranged according to VH-CH1-CH2-CH3 from N-terminus to C-terminus;

[0030] Light chain 2, comprising domains arranged in a VL-CL pattern from N-terminus to C-terminus;

[0031] Among them, the VH and VL contained in heavy chain 1 and light chain 1 (or heavy chain 1 and light chain 1) are paired to form a first binding domain that binds to NKp46; the VH and VL contained in heavy chain 2 and light chain 2 (or heavy chain 2 and light chain 2) are paired to form a second binding domain that binds to MSLN.

[0032] Preferably, the multispecific antigen-binding protein provided by the present invention comprises a heavy chain (e.g., heavy chain 1 and heavy chain 2 as described above), which can be modified by the "Knobs-into-Holes" technique, for example, in the Fc CH3 portion to ensure its correct assembly; and / or, structural disulfide bonds can be introduced at one or more sites by amino acid replacement to improve its structural stability.

[0033] According to a specific embodiment of the present invention, in the multispecific antigen-binding protein provided by the present invention, heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 75% identity with the amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO.2 or an amino acid sequence having at least 75% identity with the amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO.3 or an amino acid sequence having at least 75% identity with the amino acid sequence; light chain 2 comprises the amino acid sequence shown in SEQ ID NO.4 or an amino acid sequence having at least 75% identity with the amino acid sequence. More specifically, the multispecific antigen-binding protein provided by the present invention can be constructed by the Bispecific Antibody by Protein Trans-Splicing (BAPTS) platform.

[0034] In another aspect, the present invention provides a nucleic acid molecule comprising nucleotide sequences each encoding an amino acid sequence comprised in a multispecific antigen-binding protein provided by the present invention.

[0035] In another aspect, the present invention provides a vector comprising the nucleic acid molecule described above. The vector may be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, etc. Depending on the application purpose of the vector, the vector may be an expression vector.

[0036] In another aspect, the present invention provides a host cell comprising the nucleic acid molecule or vector as described above, or a host cell transformed or transfected by the nucleic acid molecule or vector as described above. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterium or an insect, fungus, plant or animal cell.

[0037] In another aspect, the present invention further provides a composition comprising the multispecific antigen-binding protein provided by the present invention. Preferably, the composition is a pharmaceutical composition, which optionally further comprises a pharmaceutically acceptable carrier, adjuvant or excipient.

[0038] In another aspect, the present invention also provides the use of the multispecific antigen-binding protein, nucleic acid molecule, vector, host cell and / or composition in the preparation of a medicament for treating a tumor. Preferably, the tumor is a solid tumor, such as a solid tumor expressing MSLN or being MSLN-positive. According to a specific embodiment of the present invention, the tumor is, for example, gastric cancer, lung cancer, or ovarian cancer.

[0039] Accordingly, the present invention provides a method for treating a tumor, the method comprising administering the multispecific antigen-binding protein, nucleic acid molecule, vector, host cell and / or composition provided by the present invention to a subject in need thereof. The subject is a mammal, including a human or non-human primate, and a domestic, livestock or laboratory mammal such as a dog, cat, cow, pig, sheep, horse, mouse, rabbit, etc. Preferably, the subject is a human. Preferably, the tumor is a solid tumor, such as a solid tumor expressing MSLN or being MSLN positive. According to a specific embodiment of the present invention, the tumor is, for example, gastric cancer, lung cancer, or ovarian cancer.

[0040] As an important component of anti-tumor innate immunity, NK cells have attracted widespread attention in recent years. NK cells express a variety of receptors with activating or inhibitory functions on their surface, and the full activation of NK is regulated by the activation and inhibitory signals induced by these receptors. It is worth noting that many tumor cells downregulate the expression of MHC I molecules to escape the detection of cytotoxic CD8+ T cells. But at the same time, MHC I deficiency may fail to induce NK cell inhibitory receptor signals, thereby indirectly leading to increased activation signals and ultimately target cell lysis. Therefore, NK cells have therapeutic potential for situations where T cells cannot recognize targets due to MHC I downregulation. NKp46 is one of the activating receptors expressed by NK cells and is highly associated with NK cell-mediated natural cytotoxicity. Unlike other activating receptors, no significant downregulation of NKp46 was observed in the peripheral blood of patients with breast, liver, lung, kidney, and metastatic melanoma. Therefore, NKp46 is a suitable target for the development of NK cell engagers.

[0041] Based on the above reasons, the inventors of the present invention constructed a multispecific antigen-binding protein targeting NKp46 and MSLN, which can be used as an adapter of NK cells. In a specific embodiment of the present invention, a specific multispecific antigen-binding protein is named "NKp46 / MSLN NKCE", which is a bispecific antibody with a full-length IgG configuration. In the construction process of NKp46 / MSLNN NKCE, the inventors adopted the "BAPTS" platform technology proposed earlier, which solved the problem of light and heavy chain mismatch, greatly improved the expression efficiency of the antibody, and simplified the purification steps. At the same time, the full-length IgG configuration makes it have a half-life close to that of natural antibodies, and theoretically has lower immunogenicity. The purity and accurate molecular weight of NKp46 / MSLN NKCE were characterized by SEC-HPLC and LC / MS, and the results showed that the antibody had the correct molecular weight and high purity. In in vitro functional studies, the affinity of NKp46 / MSLN NKCE to the target antigen was determined by ELISA, SPR and cell binding experiments. Different methods were also used to evaluate its killing effect on different tumor cells. The results showed that NKp46 / MSLN NKCE showed excellent killing activity on a variety of tumor cell lines without producing off-target toxicity. In addition, the antibody-mediated NK cell killing mechanism of tumor cells was also studied, and the results showed that the antibody can effectively bridge NK cells and target cells, and promote NK cells to upregulate the expression of CD69, CD107A, TNF-α and IFN-γ. According to literature reports, bispecific antibodies based on T cell redirection will activate T cells, leading to the release of multiple cytokines, and will produce a strong cytokine release syndrome when used clinically. The inventors of the present invention evaluated the cytokine release level of the NK cells by detecting the cytokines in the supernatant of NK cells co-cultured with NKp46 / MSLN NKCE. The results showed that only extremely low TNF-α and IFN-γ were released, indicating that NKp46 / MSLN NKCE can effectively avoid the production of a large number of inflammatory cytokines and has high safety.

[0042] It can be seen that NKp46 / MSLN NKCE has efficient NK cell activation ability and better anti-tumor activity in vitro, while taking into account high safety, providing a new therapeutic approach for the treatment of tumors, especially MSLN-positive solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0044] Figure 1 A roadmap for the construction of NKp46 / MSLN NKCE targeting NKp46 and MSLN is shown.

[0045] Figure 2 Shown are the fragments included in the construction of the NKp46 / MSLN NKCE and the purification results of the parental monoclonal antibody.

[0046] Figure 3 The final purification result after separation of NKp46 / MSLN NKCE from unreacted fragments and byproducts is shown.

[0047] Figure 4 The purity results of NKp46 / MSLN NKCE obtained by SEC-HPLC analysis are shown.

[0048] Figure 5 The intact molecular weight of NKp46 / MSLN NKCE by LC / MS is shown.

[0049] Figure 6 The binding activity of NKp46 / MSLN NKCE to the target antigen is shown.

[0050] Figure 7 The binding activity of NKp46 / MSLN NKCE to simultaneously bind two target antigens is shown.

[0051] Figure 8 and Fig. 9 The affinity of NKp46 / MSLN NKCE and its parental mAb to the target antigen is shown.

[0052] Fig.10 The binding activity of NKp46 / MSLN NKCE to simultaneously bind two target antigens is shown.

[0053] Fig.11 The relative expression levels of MSLN on the surfaces of various target cells are shown.

[0054] Fig.12 The binding activity of NKp46 / MSLN NKCE to cell surface MSLN is shown.

[0055] Fig.13 The binding activity of NKp46 / MSLN NKCE to cell surface NKp46 is shown.

[0056] Fig.14 and Fig.15 The biological activity of NKp46 / MSLN NKCE in mediating NK cell activation is shown.

[0057] Fig.16 and Fig.17 NKp46 / MSLN NKCE-mediated killing of target cells is shown. DETAILED DESCRIPTION

[0058] The present invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0059] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

[0060] The variable region sequence targeting NKp46 is derived from the patent publication document WO2017114694A1; the variable region sequence targeting MSLN was obtained by the inventors through affinity maturation screening based on the monoclonal antibody sequence targeting MSLN. The anti-MSLN monoclonal antibody (Fc silent) with knockout ADCC function was prepared using conventional technology and was previously stored by the laboratory.

[0061] The antigen proteins used in the examples were all purchased commercially, wherein the NKp46 antigen used for ELISA and SPR contained a His tag (Catalog#NC1-H52H4, Beijing Biopsies Biotechnology Co., Ltd.); the MSLN antigen used for ELISA and SPR was tag-free (Catalog#13128-HNCH, Beijing Sino-Biological Technologies Co., Ltd.); and the MSLN antigen used for dual-binding SPR contained an Fc tag (Catalog#MSN-H82F6, Beijing Biopsies Biotechnology Co., Ltd.).

[0062] Example 1 Construction, expression and purification of NKp46 / MSLN NKCE

[0063] The multispecific antigen-binding protein NKp46 / MSLN NKCE of the present invention is assembled from a single-arm antibody fragment targeting NKp46 and a Fab fragment targeting MSLN.

[0064] 1.1 Gene synthesis and construction of expression vector

[0065] The construction route of NKp46 / MSLN NKCE is shown in Figure 1 .like Figure 1 As shown, the Fab-Fc fragment is named IntC / Fab NKp46 -Fc or "fragment A", involving three amino acid sequences: Anti-NKp46-LC, Anti-NKp46-HK, intC-CH2-CH3; Fab fragment named Fab MSLN -intN or "fragment B", involves two amino acid sequences: Anti-MSLN-LC, Anti-MSLN-HN.

[0066] The pcDNA3.4 vector containing CMV promoter and prokaryotic screening tag Ampicillin was used as the expression vector. The nucleotide sequence expressing the variable region (pcDNA3.4 vector contains the nucleotide sequence of the constant region) was obtained by gene synthesis. At the same time, the Kozak sequence and the nucleotide sequence encoding the signal peptide were introduced at the 5' end of the sequences encoding each light and heavy chain. Different insert fragments were amplified by PCR. The terminal sequence of the linearized vector was introduced at the 5' end of the forward / reverse PCR primer of the insert fragment, so that the 5' and 3' ends of the PCR product respectively carried sequences consistent with the two ends of the linearized vector. Q5 polymerase was used to amplify the pcDNA3.4 linearized vectors that were connected to the other functional regions of the antibody except the variable region. After each insert fragment and linearized vector were recovered, each vector fragment and the insert fragment were connected by homologous recombination. The connection product was transformed into DH5-α Escherichia coli competent cells, and the positive clones were selected after inverted culture at 37°C overnight, and plasmid extraction and sequencing verification were performed. Plasmids expressing IntC / FabNKp46-Fc (Anti-NKp46-LC, Anti-NKp46-HK, intC-CH2-CH3) and plasmids expressing FabMSLN-intN (Anti-MSLN-HN, Anti-MSLN-LC) were obtained.

[0067] (1) Anti-NKp46-LC, the amino acid sequence is shown in SEQ ID NO. 2, and the encoding nucleotide sequence is shown in

[0068] SEQ ID NO.26:

[0069] SEQ ID NO.2

[0070] DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTINNLEQEDIATYFCQQGNTRPWTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0071] SEQ ID NO.26

[0072] GATATCCAGATGACCCAGACCACCTCCAGCCTGAGCGCCAGCCTGGGCGACAGAG

[0073] TGACAATCAGCTGCAGGGCCAGCCAGGACATCTCCAATTACCTGAATTGGTATCA

[0074] ACAGAAGCCCGACGGCACAGTGAAGCTGCTGATCTACTACACCTCCAGGCTGCAC

[0075] AGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGAACAGATTACTCCCTGA

[0076] CCATCAACAACCTGGAGCAGGAGGATATCGCCACCTACTTCTGTCAGCAGGGCAA

[0077] CACCAGACCTTGGACATTTGGCGGCGGCACCAAGCTGGAGATCAAGCGTACGGTG

[0078] GCCGCCCCCAGCGTCTTCATCTTCCCGCCCAGCGACGAGCAGCTGAAGTCGGGCAC

[0079] GGCCAGCGTGGTGTGCCTCCTGAACAACTTCTACCCCCGCGAGGCGAAGGTCCAG

[0080] TGGAAGGTGGACAACGCCCTGCAGAGCGGGAACAGCCAGGAGAGCGTGACCGAG

[0081] CAGGACTCGAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAGG

[0082] CCGACTACGAGAAGCACAAGGTCTACGCCTGCGAGGTGACCCACCAGGGGCTCTC

[0083] GAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCTGA

[0084] (2) Anti-NKp46-HK, the amino acid sequence is shown in SEQ ID NO.1, and the coding nucleotide sequence is shown in SEQ ID NO.27:

[0085] SEQ ID NO.1

[0086] QVQLQQSGPELVKPGASVKMSCKASGYTFTDYVINWGKQRSGQGLEWIGEIYPGSGT

[0087] NYYNEKFKAKATLTADKSSNIAYMQLSSLTSEDSAVYFCARRGRYGLYAMDYWGQG

[0088] TSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT

[0089] FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP

[0090] CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN

[0091] AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP

[0092] REPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

[0093] GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO.27

[0094] CAGGTGCAGCTGCAGCAGTCCGGCCCTGAGCTGGTGAAGCCCGGCGCTTCCGTGA

[0095] AGATGTCCTGTAAGGCCAGCGGCTACACATTCACTGATTACGTGATCAACTGGGGC

[0096] AAGCAGAGATCCGGCCAGGGCCTGGAGTGGATCGGCGAGATCTACCCCGGCAGCG

[0097] GCACCAATTACTACAACGAGAAGTTTAAGGCCAAGGCCACCCTGACAGCCGACAA

[0098] GAGCTCCAACATCGCCTACATGCAGCTGTCCTCCCTGACCAGCGAGGACTCCGCCG

[0099] TGTACTTCTGCGCCAGAAGGGGCAGGTACGGCCTGTACGCTATGGATTACTGGGG

[0100] CCAGGGCACCTCCGTGACCGTGAGCAGCGCTAGCACGAAGGGGCCCAGCGTGTTC

[0101] CCGCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGGACCGCCGCCCTGGGCTGCC

[0102] TCGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCGTGGAACAGCGGCGCGCT

[0103] GACGAGCGGGGTCCACACCTTCCCGGCCGTGCTGCAGAGCAGCGGCCTCTACTCG

[0104] CTGAGCAGCGTGGTCACCGTGCCCAGCAGCAGCCTGGGGACCCAGACGTACATCT

[0105] GCAACGTGAACCACAAGCCCTCGAACACCAAGGTCGACAAGAAGGTGGAGCCCA

[0106] AGAGCTGCGACAAGACCCACACCTGCCCGCCCTGCCCCGCCCCCGAGCTCCTGGG

[0107] CGGGCCCAGCGTGTTCCTGTTCCCGCCCAAGCCCAAGGACACGCTCATGATCAGCC

[0108] GCACCCCCGAGGTCACCTGCGTGGTGGTCGACGTGAGCCACGAGGACCCCGAGGT

[0109] GAAGTTCAACTGGTACGTCGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCG

[0110] CGGGAGGAGCAGTACAACTCGACGTACCGCGTCGTGAGCGTGCTGACCGTCCTGC

[0111] ACCAGGACTGGCTCAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCC

[0112] TGCCCGCGCCCATCGAGAAGACCATCAGCAAGGCCAAGGGGCAGCCCCGGGAGCC

[0113] GCAGGTGTACACCCTGCCCCCCTGCCGCGACGAGCTCACGAAGAACCAGGTCAGC

[0114] CTGTGGTGCCTGGTGAAGGGCTTCTACCCCTCGGACATCGCCGTGGAGTGGGAGA

[0115] GCAACGGGCAGCCGGAGAACAACTACAAGACCACCCCGCCCGTCCTCGACAGCGA

[0116] CGGCAGCTTCTTCCTGTACAGCAAGCTGACGGTGGACAAGTCGCGGTGGCAGCAG

[0117] GGCAACGTGTTCAGCTGCAGCGTCATGCACGAGGCCCTCCACAACCACTACACCC

[0118] AGAAGAGCCTGAGCCTGAGCCCCGGGAAGTGA

[0119] (3) intC-CH2-CH3, the amino acid sequence is shown in SEQ ID NO.5, and the coding nucleotide sequence is shown in SEQ ID NO.28:

[0120] SEQ ID NO.5

[0121] DIQMTQSPSSMIKIATCKYLGKQDVYDIGVERDHNFALKNGFIASNCWECPAPELLGG

[0122] PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

[0123] YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPP

[0124] SRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLT

[0125] VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0126] SEQ ID NO.28

[0127] GACATCCAGATGACCCAGAGCCCGTCGAGCATGATCAAGATTGCAACCTGCAAGT

[0128] ACCTGGGCAAACAGGACGTGTATGACATCGGAGTCGAGCGGGATCACAACTTCGC

[0129] CCTGAAAAATGGGTTTATTGCTTCCAATTGCTGGGAGTGCCCCGCCCCCGAGCTCC

[0130] TGGGCGGGCCCAGCGTGTTCCTGTTCCCGCCCAAGCCCAAGGACACGCTCATGATC

[0131] AGCCGCACCCCCGAGGTCACCTGCGTGGTGGTCGACGTGAGCCACGAGGACCCCG

[0132] AGGTGAAGTTCAACTGGTACGTCGACGGCGTGGAGGTGCACAACGCCAAGACCAA

[0133] GCCGCGGGAGGAGCAGTACAACTCGACGTACCGCGTCGTGAGCGTGCTGACCGTC

[0134] CTGCACCAGGACTGGCTCAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAG

[0135] GCCCTGCCCGCGCCCATCGAGAAGACCATCAGCAAGGCCAAGGGGCAGCCCCGGG

[0136] AGCCGCAGGTGTGCACCCTGCCCCCCAGCCGCGACGAGCTCACGAAGAACCAGGT

[0137] CAGCCTGAGCTGCGCCGTGAAGGGCTTCTACCCCTCGGACATCGCCGTGGAGTGG

[0138] GAGAGCAACGGGCAGCCGGAGAACAACTACAAGACCACCCCGCCCGTCCTCGACA

[0139] GCGACGGCAGCTTCTTCCTGGTGAGCAAGCTGACGGTGGACAAGTCGCGGTGGCA

[0140] GCAGGGCAACGTGTTCAGCTGCAGCGTCATGCACGAGGCCCTCCACAACCACTAC

[0141] ACCCAGAAGAGCCTGAGCCTGAGCCCCGGGAAGTGA

[0142] (4) Anti-MSLN-LC, the amino acid sequence is shown in SEQ ID NO.4, and the coding nucleotide sequence is shown in SEQ ID NO.29:

[0143] SEQ ID NO.4

[0144] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVP

[0145] SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYDAFYTFGGGTKVEIKRTVAAPSVFIFP

[0146] PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS

[0147] STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0148] SEQ ID NO.29

[0149] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGT

[0150] CACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGC

[0151] AGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAG

[0152] TGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCA

[0153] TCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACGAC

[0154] GCGTTCTACACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGGACCGTGGCCG

[0155] CCCCCAGCGTCTTCATCTTCCCGCCCAGCGACGAGCAGCTGAAGTCGGGCACGGCC

[0156] AGCGTGGTGTGCCTCCTGAACAACTTCTACCCCCGCGAGGCGAAGGTCCAGTGGA

[0157] AGGTGGACAACGCCCTGCAGAGCGGGAACAGCCAGGAGAGCGTGACCGAGCAGG

[0158] ACTCGAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAGGCCGA

[0159] CTACGAGAAGCACAAGGTCTACGCCTGCGAGGTGACCCACCAGGGGCTCTCGAGC

[0160] CCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCTGA

[0161] (5) Anti-MSLN-HN, the amino acid sequence is shown in SEQ ID NO.6, and the coding nucleotide sequence is shown in SEQ ID NO.30:

[0162] SEQ ID NO.6

[0163] QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGST

[0164] NYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREDVLSGFDIWGQGTMVT

[0165] VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV

[0166] LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCLSYETE

[0167] ILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLI

[0168] RATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN

[0169] SEQ ID NO.30

[0170] CAGGTGCAGCTGCAGGAGTCCGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGT

[0171] CCCTCACCTGCACTGTCTCTGGTGGCTCCGTCAGCAGTGGTAGTTACTACTGGAGC

[0172] TGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGTATATCTATTACA

[0173] GTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGA

[0174] CACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACG

[0175] GCCGTGTATTACTGTGCGAGAGAGGACGTGTTGTCGGGGTTTGATATCTGGGGCCA

[0176] AGGGACAATGGTCACCGTCTCTTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCGC

[0177] TGGCCCCCAGCAGCAAGAGCACCAGCGGCGGGACCGCCGCCCTGGGCTGCCTCGT

[0178] CAAGGACTACTTCCCCGAGCCCGTGACCGTGTCGTGGAACAGCGGCGCGCTGACG

[0179] AGCGGGGTCCACACCTTCCCGGCCGTGCTGCAGAGCAGCGGCCTCTACTCGCTGA

[0180] GCAGCGTGGTCACCGTGCCCAGCAGCAGCCTGGGGACCCAGACGTACATCTGCAA

[0181] CGTGAACCACAAGCCCTCGAACACCAAGGTCGACAAGAAGGTGGAGCCCAAGAG

[0182] CTGCGACAAGACCCACACCTGCCTGTCCTACGAGACCGAAATCCTGACAGTGGAG

[0183] TATGGCCTGCTGCCAATCGGAAAGATTGTCGAGAAAAGGATCGAATGTACAGTGT

[0184] ACAGCGTCGATAACAATGGCAACATCTACACCCAGCCCGTGGCCCAGTGGCACGA

[0185] TAGAGGGGAGCAGGAAGTCTTCGAGTACTGCCTGGAAGACGGTTCTCTGATTAGG

[0186] GCTACTAAGGACCATAAATTCATGACCGTGGATGGACAGATGCTGCCCATCGACG

[0187] AGATTTTTGAGAGGGAACTGGACCTGATGCGGGTGGATAACCTGCCTAATTGA

[0188] The expression plasmid of the parent monoclonal antibody NKp46 mAb targeting NKp46 was constructed. The light chain expression plasmid of the parent monoclonal antibody NKp46 mAb was the same as the corresponding light chain expression plasmid of NKp46 / MSLN NKCE; the variable region of the heavy chain was cloned from the plasmid expressing Anti-NKp46-HK, and the obtained V region was inserted into the pcDNA3.4-ROR1-HC vector (IgG1 type antibody heavy chain expression vector with double enzyme digestion and replacement of V region stored in the laboratory) to obtain the heavy chain expression plasmid of NKp46 mAb.

[0189] In addition, it is necessary to prepare the parent monoclonal antibody MSLN mAb targeting MSLN. The light chain expression plasmid of the parent monoclonal antibody MSLNmAb is the same as the corresponding light chain plasmid of NKp46 / MSLN NKCE; the heavy chain expression plasmid used for MSLN mAb was previously stored in the laboratory and used after sequencing verification.

[0190] 2. Expression and purification of NKp46 / MSLN NKCE and parental monoclonal antibodies

[0191] 2.1 Antibody fragments and parental monoclonal antibody expression

[0192] Expi293F cells were used as expression cells. The day before transfection, Expi293F cells were resuspended in OPM-293CD05 medium to a concentration of 1.5 × 10 6 cells / mL, so that the cell density after overnight culture is about 3×10 6 cells / mL, ensuring that the cell viability is above 95%.

[0193] IntC / Fab NKp4 The 6-Fc antibody fragment (hereinafter referred to as fragment A) was expressed by transient co-transfection of three plasmids into Expi293F cells mediated by polyethylenimine (PEI). The three vectors were added at a total plasmid of 0.8 μg / 1 mL of cells, and the mass ratio of each plasmid was 1:1:1. PEI was added to the plasmid mixture at a PEI:DNA mass ratio of 3:1, mixed, and allowed to stand at room temperature for 20 minutes before adding the mixture to the cell suspension. On the first day after transfection, a cell feed of 5% of the volume of cell culture medium was added, and 3 g of glucose was added per 1 L of cell culture medium; on the third day, 4 g of glucose was added per 1 L of cell culture medium. Fab MSLN-intN antibody fragment (hereinafter referred to as fragment B) and parental monoclonal antibody expression were co-transfected into Expi293F cells with two vector plasmids (light chain and heavy chain) at a mass ratio of 1: 1 using the same operation process. After about 6 days of cell culture (when the cell viability dropped to 60-70%), the cell culture fluid was collected for purification.

[0194] 2.2 Antibody fragments and parent monoclonal antibody purification

[0195] Protein A (MabSelect SuRe LX affinity chromatography column) was used for affinity purification to obtain fragment A and parent monoclonal antibody. First, the column was equilibrated with a pH 7.2 equilibrium buffer (20mM NaH2PO4, 150mM NaCl) and then loaded with the sample. The impurities were removed with a pH 5.0 100mM citric acid buffer, eluted with a pH 3.0 100mM citric acid buffer, and then immediately neutralized with a pH 9.0 1M tris-HCl buffer. Fragment B was purified using a Capto L column. The purification scheme was the same as that of the Protein A column, except that the elution buffer was a pH 2.7 100mM citric acid buffer.

[0196] The obtained samples were subjected to SDS-PAGE analysis, and the results are shown in Figure 2 , wherein 2a is the SDS-PAGE image after the purification of fragment A, 2b is the SDS-PAGE image after the purification of fragment B; 2c is the SDS-PAGE image after the purification of NKp46mAb; 2d is the SDS-PAGE image after the purification of MSLNmAb. The successfully purified samples were dialyzed into PBS buffer.

[0197] 2.3 Trans-splicing reaction and NKp46 / MSLN NKCE purification

[0198] NKp46 / MSLN NKCE was synthesized by intein-mediated trans-cleavage reaction. The concentrations of fragment A and fragment B were adjusted to 5 μM each, and then TCEP was added to a final concentration of 0.5 mM and incubated at 37°C for 2 h. DHAA was added to a final concentration of 2 mM and incubated at room temperature for 0.5 h.

[0199] After SDS-PAGE verification, Protein A column was used to remove unreacted fragment B and byproduct intein, and the purification scheme was the same as that of fragment A. Capto TMThe unreacted fragment A was separated by MMC ImpRes column. The column was first equilibrated with pH 6.0 phosphate buffer (100 mM NaH2PO4) and then loaded with the sample. The unbound impurities were removed with the same buffer. Then, the column was gradient eluted with pH 6.0 phosphate buffer (100 mM NaH2PO4, 1 M NaCl) for 40 column volumes. The eluted fractions were collected. The obtained samples were analyzed by SDS-PAGE gel. Figure 3 As shown in Figures 3a and 3b, the target NKCE (lane 1) was generated by intein trans-splicing reaction, and the subsequent Protein A column purification successfully removed the unreacted Fab MSLN -intN fragment and byproduct intein (lane 2), but Capto TM The MMC ImpRes column failed to successfully separate the product NKCE from the unreacted fragment A (lanes 4-12).

[0200] Based on the difference in the number of kappa light chains in NKCE and fragment A, we attempted to use Capto L to separate the two components. The overall experimental steps were the same as the previous Capto L purification, but a pH 5.0-2.7 citric acid buffer gradient elution of 30 column volumes was set in the elution step. After collecting the eluted samples, the antibody purification effect was verified by SDS-PAGE. Figure 3 In 3c and 3d, the unreacted fragment A was successfully removed by this purification step (lane FT), and NKp46 / MSLN NKCE with higher purity was obtained.

[0201] NKp46 / MSLN NKCE contains the following four polypeptide chains (the CDRs shown below are defined according to the Kabat partitioning principle):

[0202] (1) Anti-NKp46-HK (SEQ ID NO. 1; the sequence in italics is VH, the underlined sequences are H-CDR1 to 3; the sequence in bold is CH1-CH2-CH3 knob)

[0203]

[0204]

[0205]

[0206] (2) Anti-NKp46-LC (SEQ ID NO. 2; the sequence in italics is VL, the underlined sequence is L-CDR1-3; the sequence in bold is CL)

[0207]

[0208]

[0209] (3) Anti-MSLN-HH (SEQ ID NO. 3; the sequence in italics is VH, the underlined sequence is H-CDR1-3; the sequence in bold is CH1-CH2-CH3 hole)

[0210]

[0211]

[0212] (4) Anti-MSLN-LC (SEQ ID NO. 4; the sequence in italics is VL, the underlined sequence is L-CDR1-3; the sequence in bold is CL)

[0213]

[0214]

[0215]

[0216] Example 2 Purity and molecular weight analysis of NKp46 / MSLN NKCE

[0217] The purity of NKp46 / MSLN NKCE was analyzed by size exclusion chromatography (SEC-HPLC). The specific method is as follows:

[0218] Dilute the sample appropriately with PBS to ensure that the sample concentration is >0.5 mg / mL, centrifuge at 12000 rpm for 5 min, and take the supernatant. The G3000SWXL HPLC Column was loaded on the HPLC instrument for automatic injection and isocratic elution at a flow rate of 0.9 mL / min for 20 min. The UV absorbance at 280 nm was analyzed using the chromatography software Waters GPC. Figure 4 As shown, the results show that the main peak accounts for about 91.96%.

[0219] The molecular weight of the antibody after removal of N-glycosylation was analyzed by liquid chromatography-mass spectrometry (LC / MS). The sample was placed in a 30kDa ultrafiltration centrifuge tube and centrifuged at 4°C and 4000rpm. After 200μL of liquid remained in the tube, N-sugar removal buffer (50mMNH4HCO3) was added and centrifuged for 3 times to complete the replacement. The antibody concentration after replacement solution was determined to be 0.8mg / mL. 2μL PNGaseF (500U / μL) was added and incubated at 37°C for 24h to obtain a sample with N-sugar removed. An appropriate amount of sample was taken and SDS-PAGE was used to verify the intact molecular weight of the antibody after N-sugar removal. Figure 5The results showed that the intact molecular weight of the product was 144956 Da.

[0220] Example 3 ELISA was used to measure the binding activity of NKp46 / MSLN NKCE with target antigen

[0221] Dilute the antigen to the target concentration (MSLN: 1μg / mL, NKp46: 2μg / mL), and coat the 96-well ELISA plate with 100μL per well at 4℃ overnight. Wash the plate three times with PBST (PBS+0.1% Tween 20), and then block it with PBS containing 3% BSA at room temperature for 1.5h. Dilute MSLN mAb and NKp46 / MSLN NKCE with 3% BSA to a concentration of 8μg / mL, and then perform 3-fold dilutions, with a total of 8 concentration gradients; dilute NKp46 mAb and NKp46 / MSLN NKCE with 3% BSA to a concentration of 10μg / mL, and then perform 9-fold dilutions, with a total of 6 concentration gradients, and set two replicates for each sample. After blocking, wash the plate three times with PBST, take the parental monoclonal antibody or NKCE to be tested and add it to the ELISA plate, and incubate at 37℃ for 1h. After incubation, wash the plate 3 times with PBST, add 1:10000 dilution of Anti-Human (H+L) HRP secondary antibody, and incubate at 37°C for 0.5h. After incubation, wash the plate 4 times with PBST, add TMB colorimetric solution and incubate in the dark for 5-15min, add ELISA stop solution (2M H2SO4) to stop the color reaction, and measure the absorbance at a wavelength of 450nm with an enzyme reader. GraphPad Prism8 software was used to analyze and process the data, and a four-parameter nonlinear regression model was used to fit the data.

[0222] like Figure 6 As shown in Figure 6a, NKp46 / MSLN NKCE and MSLN mAb can effectively bind to MSLN antigen, and the binding efficiency is dose-dependent. The EC values ​​of NKp46 / MSLN NKCE and MSLN mAb binding to MSLN antigen were obtained by curve simulation. 50 They are 0.3932μg / mL and 0.005843μg / mL respectively. Figure 6 As shown in Figure 6b, NKp46 / MSLN NKCE and NKp46 mAb can effectively bind to NKp46 antigen, and the binding efficiency is dose-dependent. The EC values ​​of NKp46 / MSLN NKCE and NKp46 mAb binding to NKp46 antigen were obtained by curve simulation. 50 They are 0.7785 μg / mL and 0.01462 μg / mL respectively.

[0223] The above experimental results show that under the same experimental conditions, the activity of NKp46 / MSLN NKCE in binding to human MSLN and NKp46 is weaker than that of the parental monoclonal antibody with the same target, which is presumably because the former binds monovalently to the antigen, while the parental monoclonal antibody binds bivalently to the antigen. The data suggest that NKp46 / MSLN NKCE has the activity of effectively binding to two target antigens.

[0224] Example 4 Sandwich ELISA was used to measure the simultaneous binding ability of NKp46 / MSLN NKCE to two target antigens

[0225] Sandwich ELISA was used to detect whether NKp46 / MSLN NKCE could bind to two antigens at the same time.

[0226] 1 μg / mL MSLN antigen (without His tag) was coated on a 96-well ELISA plate at 4°C overnight with 100 μL per well. The plate was washed three times with PBST and blocked with PBS containing 3% BSA at room temperature for 2 hours. After washing the plate four times with PBST, 10 μg / mL of the test antibody (MSLN mAb, NKp46 mAb, a mixture of two monoclonal antibodies (Combo), NKp46 / MSLN NKCE) was added and incubated at room temperature for 1 hour. The plate was washed four times with PBST, and 100 μL of His-tagged NKp46 antigen dilution (concentration: 2 μg / mL) was added to each well and incubated with shaking for 2 hours. The plate was washed four times with PBST again, and 100 μL of Anti-His tag HRP secondary antibody diluted 1:3000 was added to each well and incubated at 37°C for 1 hour. After washing the plate four times with PBST, 100 μL of TMB color development solution was added to each well and incubated at room temperature for 15 minutes. Add 50 μL of ELISA stop solution to each well and place it in an ELISA reader to detect the absorbance value at a wavelength of 450 nm. GraphPad Prism8 software was used to analyze and process the data. The results are shown in the figure. Figure 7 As shown, it was found that only the NKp46 / MSLN NKCE group presented a higher OD450nm value, proving that NKp46 / MSLN NKCE effectively binds to two different antigens simultaneously.

[0227] Example 5 Surface plasmon resonance was used to determine the binding kinetic parameters of NKp46 / MSLN NKCE and its parental monoclonal antibody to the target antigen

[0228] The binding affinity of NKp46 / MSLN NKCE to the antigen was determined by surface plasmon resonance assay using Biacore 8K.

[0229] The chip was activated for 7 to 10 minutes. The ligand proteins MSLN and NKp46 were diluted to 2 μg / mL and 1 μg / mL respectively with pH 5.0 acetic acid solution, and fixed on the activated CM5 chip. The coupling was performed for 600 to 1200 seconds, and then the chip was sealed for 7 minutes to complete the ligand fixation. The running buffer used was HBS-EP solution. MSLN mAb, MSLNmAb (Fc silent) in the concentration range of 1.56-50nM (isotropic dilution), and NKp46 / MSLN NKCE in the concentration range of 3.125-100nM (isotropic dilution) were used as analytes for MSLN antigen, and NKp46 mAb in the concentration range of 0.185-6nM (isotropic dilution), and NKp46 / MSLN NKCE in the concentration range of 0.625-20nM (isotropic dilution) were used as analytes for NKp46 antigen. They were injected into the sensor chip at a flow rate of 30μL / min, and the binding time was 120s. The proteins were dissociated in the running buffer for 240s (MSLN analyte) or 1000s (NKp46 analyte). After each binding cycle, the chip channels with fixed MSLN and NKp46 were regenerated with 10mM glycine at pH2.0 and pH1.5, respectively; Biacore TM Insight Evaluation Software 4.0 was used for data processing, and the kinetic curve was fitted with a 1:1 binding fitting model to obtain the binding rate constant (Ka), dissociation constant (Kd), and affinity constant (KD).

[0230] The results of the affinity constant determination of analyte NKp46 / MSLN NKCE, MSLN mAb, MSLN mAb (Fc silent), NKp46 mAb with MSLN and NKp46 are shown in Table 1. Figure 8 , Fig. 9 The affinity constants of NKp46 / MSLN NKCE to MSLN and NKp46 were 52.6 nM and 2.81 nM, respectively; the affinity constants of MSLN mAb and MSLN mAb (Fc silent) to MSLN were 7.02 nM and 8.17 nM, respectively; the affinity constant of NKp46 mAb to NKp46 was 0.145 nM.

[0231] The above results show that NKp46 / MSLN NKCE and its parental mAb have good target antigen binding ability. In MSLN binding, the KD value of NKp46 / MSLN NKCE is about one order of magnitude different from its parental mAb MSLN mAb. Similarly, in NKp46 binding, the difference between NKCE and the parental mAb is within one order of magnitude. Considering that the parental mAb is a divalent binding and NKCE is a monovalent binding, the detected KD value is within a reasonable range.

[0232] Table 1. Kinetic parameters of NKp46 / MSLN NKCE and its parental antibodies

[0233]

[0234] Example 6 Surface plasmon resonance assay to measure the simultaneous binding ability of NKp46 / MSLN NKCE to two antigens

[0235] The ligand protein NKp46 was diluted to 10 μg / mL with acetic acid solution at pH 5.0 and fixed to the CM5 sensor chip. The analytes NKp46 mAb and NKp46 / MSLN NKCE were diluted to 250 nM, and the MSLN-Fc ligand protein was diluted to 100 nM. The samples were injected into the sensor chip at a flow rate of 30 μL / min. The antibody binding time was 180 s, and the MSLN-Fc ligand protein binding time was 150 s. The chip was regenerated with 10 mM glycine at pH 1.5. Biacore TM Insight Evaluation Software 4.0 was used for data processing.

[0236] Test results such as Fig.10 As shown, it was found that only the NKp46 / MSLN NKCE-MSLN-Fc analyte group achieved two increases in response values, indicating that only in this group MSLN-Fc effectively bound to NKCE, indicating that NKp46 / MSLN NKCE can bind to two target antigens simultaneously.

[0237] Example 7 Flow cytometry was used to measure the binding activity of NKp46 / MSLN NKCE to MSLN on cells

[0238] Normal NCI-N87, NCI-H292, and Ovcar-3 cells in the logarithmic phase were used. After trypsin digestion, the enzyme activity was neutralized with complete medium. The cells were collected by centrifugation at 1000 rpm for 4 min at room temperature and resuspended in complete medium (RPMI 1640 medium containing 10% FBS). 5Transfer the cells to a 1.5 mL centrifuge tube and wash twice with staining buffer (PBS containing 2% FBS); add 100 μL of MSLN mAb or NKp46 / MSLN NKCE diluted in staining buffer (final concentrations of 25, 5, 1, 0.2, 0.04, 0.08, 0.0016, 0.00032 μg / mL), set up a blank control group Blank (no primary antibody and secondary antibody) and a negative control group NC (no primary antibody), mix gently and incubate at 4°C for 30 min; centrifuge at 1400 rpm, remove the supernatant, resuspend and wash the cells three times with staining buffer; resuspend the cells in 100 μL of staining buffer, add 1 μL to each sample The FITC-conjugated anti-human IgG (H+L) antibody was resuspended and mixed, and incubated at 4°C in the dark for 30 min; the cells were washed three times with staining buffer, and after removing the supernatant, 500 μL of staining buffer was added to resuspend the cells for flow cytometry analysis, and the FITC fluorescence signal intensity of each sample was recorded.

[0239] The relative expression levels of MSLN on the surface of various target cells have been identified by flow cytometry. Fig.11 11a and 11b. Combined with the experimental results, Fig.12 Figures 12a, 12b, and 12c show that NKp46 / MSLN NKCE and its parental mAb can bind to the MSLN antigen on different target cells in a dose-dependent manner. However, since the binding of the mAb is divalent and that of NKCE is monovalent, under low concentration conditions, NKCE is more easily dissociated during the washing process after antibody incubation. Therefore, it is predictable that the final values ​​reflect differences in the binding of the entire antibody molecule.

[0240] Example 8 Flow cytometry was used to measure the binding activity of NKp46 / MSLN NKCE to NKp46 on cells

[0241] The NKp46 positive cell line NCR1-293T was constructed based on the HEK293T cell line by lentiviral transfection and used as a model cell. NCR1-293T cells in the logarithmic growth phase were collected and cultured at 5×10 5Transfer the cells to a 1.5 mL centrifuge tube and wash twice with staining buffer (PBS containing 2% FBS); add 100 μL of NKp46 mAb or NKp46 / MSLN NKCE diluted in staining buffer (final concentrations of 25, 5, 1, 0.2, 0.04, 0.08, 0.0016, 0.00032 μg / mL), and set up a blank control group Blank (no primary antibody and secondary antibody) and a negative control group NC (no primary antibody), mix gently and incubate at 4°C for 30 min; centrifuge at 1400 rpm, remove the supernatant, resuspend and wash the cells three times with staining buffer; resuspend the cells in 100 μL of staining buffer, and add 1 μL to each sample. The FITC-conjugated anti-human IgG (H+L) antibody was resuspended and mixed, and incubated at 4°C in the dark for 30 min; the cells were washed three times with staining buffer, and after removing the supernatant, 500 μL of staining buffer was added to resuspend the cells for flow cytometry analysis, and the FITC fluorescence signal intensity of each sample was recorded.

[0242] Expression of NKp46 antigen on the surface of NCR1-293T cells Fig.13 As shown in Figure 13a, the binding experiment results are shown in Figure 13b, indicating that NKp46 / MSLN NKCE and its parental monoclonal antibody can bind to the NKp46 antigen on the cell in a dose-dependent manner. Although the binding ability is also affected by monovalent and bivalent, overall, NKCE and its parental monoclonal antibody have very similar binding curves. It is speculated that the affinity (KD) of the variable region of the antibody to the antigen is relatively high, and washing during the incubation process does not have a significant effect on the results. Under high antibody dose conditions, the antibody concentration is in a saturated state. When NKCE binds monovalently, more antibodies can be allowed to bind, and then indirectly labeled with anti-human IgG secondary antibodies, showing a higher offset.

[0243] Example 9 NKp46 / MSLN NKCE-mediated NK cell activation bioactivity assay

[0244] The activation activity of NKp46 / MSLN NKCE, MSLN mAb and NKp46 mAb on NK cells in the presence or absence of target cells was tested.

[0245] NCI-N87 cells in the logarithmic growth phase were collected and 50 μL, 2 × 10 5 10 cells were plated in 96-well plates; NK cells were sorted from normal human PBMCs and 50 μL, 2×10 5cells were added to the corresponding wells; the antibody (0.03-30 μg / mL, ten-fold dilution) was prepared using complete culture medium (RPMI 1640 culture medium containing 10% inactivated FBS), and 50 μL of the corresponding concentration of antibody was added to each well, so that the final concentration of the antibody was 1 / 3 of the prepared concentration; at the same time, a blank group (only NK cells were added) and a group without target cells (only 2×10 5 NK cells, antibodies were added at a final concentration of 10 μg / mL), background group (NK cells and target cells were added, no antibodies were added), and the total volume of the co-incubation system was 150 μL. BD GolgiStop TM (Add 4 μL to every 6 mL of cell culture medium), mix well and incubate at 37°C for 4 hours. Then transfer the cell mixture to a round-bottom 96-well plate, wash twice with staining buffer and resuspend in 100 μL staining buffer, add CD3-FITC, CD56-APC, CD69-PE, CD107A-BV421 antibodies, and incubate at 4°C in the dark for 30 minutes; add 250 μL staining buffer to each well and wash twice, centrifuge at 250×g for 4 minutes; gently resuspend the cells in 100 μL fixation / permeabilization solution (Fixation / Permeabilization solution, Cat: 554722, BD biosciences. Inc), and incubate at 4°C for 20 minutes. Add 250 μL 1×BD Perm / Wash to each well TM Buffer, wash twice. Gently resuspend the cells in 100 μL BD Perm / Wash TM TNF-α-BV650 and IFN-γ-PE-Cy7 antibodies were added to the buffer and incubated at 4°C in the dark for 30 min. 250 μL 1×BD Perm / Wash was added to each well. TM Buffer, washed twice, resuspended cells in staining buffer, and analyzed by flow cytometry.

[0246] The results are as follows Fig.14As shown, the results showed that for the group without target cells, CD69 had a certain background expression, which was considered to be related to the status of NK cell donors; after the addition of NKp46 / MSLN NKCE or MSLN mAb antibodies, the proportion of cells expressing CD69 increased (78.18% and 70.78%, respectively), which was considered to be caused by the binding of antibodies to CD16; but the proportion of cells expressing CD107A did not change significantly, indicating that these two antibodies had a certain activation effect on the status of NK cells, but could not cause degranulation; the proportion of cells expressing TNF-α and IFN-γ also did not change significantly. For the target cell and NK cell mixed groups, after adding NKp46 / MSLN NKCE or MSLN mAb antibodies, the proportion of cells expressing CD69, CD107A, TNF-α and IFN-γ increased significantly: under the action of NKp46 / MSLN NKCE, the proportion of CD69 positive cells was 90.45%, CD107A positive cells was 39.95%, TNF-α positive cells was 20.33%, and IFN-γ positive cells was 38.17%; under the action of MSLN mAb, the proportion of CD69 positive cells was 86.58%, CD107A cells was 20.02%, TNF-α positive cells was 4.68%, and IFN-γ positive cells was 11.17%, and the former had a more significant effect. This shows that NKp46 / MSLN NKCE can effectively activate NK cells.

[0247] In addition, NKp46 mAb was also tested, and it was observed that the antibody had no effect on NK cell activation regardless of the presence or absence of target cells, indicating that the binding of NKp46 / MSLN NKCE to NK cells before target cells does not cause NK cell activation, thus avoiding nonspecific killing caused by NK cell activation before reaching the tumor site. Fig.15 As shown, different concentrations of antibodies have different activation effects on NK cells, showing a certain degree of dose dependence.

[0248] Example 10 Luciferase reporter gene assay to detect NKp46 / MSLN NKCE-mediated target cell killing

[0249] The constructed NKp46 / MSLN NKCE, MSLN mAb, MSLN mAb (Fc silent) and NKp46 mAb were selected to detect their killing effects on MSLN-positive tumor cell lines NCI-N87 (hereinafter referred to as N87-luc), NCI-H292 (hereinafter referred to as H292-luc), Ovcar-3 (hereinafter referred to as Ovcar-3-luc) and MSLN-negative tumor cell line Raji (hereinafter referred to as Raji-luc) transformed with luciferase reporter gene.

[0250] Normal-shaped N87-luc, H292-luc, and Ovcar-3-luc cells in the logarithmic phase were used. After trypsin digestion, the enzyme activity was neutralized with complete medium, centrifuged at 1000 rpm for 4 min at room temperature, and resuspended in RPMI 1640 basal medium containing 2% FBS. 100 μL, 1×10 4 After discarding the culture medium in the original 96-well plate, resuspend the NK cells in RPMI 1640 basal medium containing 2% inactivated FBS, with 100 μL per well and 5×10 4 Each cell was added to the corresponding wells, so that the ratio of NK cells to N87-luc cells (E:T) was 5:1 or 2.5:1, and the ratio of NK to H292-luc and Ovcar-3-luc cells was 5:1. Each antibody was diluted to 300 μg / mL using RPMI 1640 basal medium (containing 2% inactivated FBS), and then diluted 10 times in a gradient, with a total of 9 concentration gradients. Two replicate wells were set up, and 50 μL of the corresponding concentration of antibody was added to each well, so that the final concentration of the antibody was 1 / 3 of the prepared concentration; at the same time, NK cell background killing wells (no antibody, 50 μL of culture medium), positive control wells (no antibody and NK cells, 150 μL of culture medium) and negative control wells (no antibody and NK cells, 100 μL of culture medium and 50 μL of 2% Triton X-100 solution) were set up. The 96-well plate was placed in a 5% CO2, 37°C carbon dioxide cell culture incubator and incubated for 20 hours. After the incubation, 100 μL of supernatant was discarded, and 100 μL of luciferin potassium salt solution (1.5 mg / mL) was added to each well, and the cells were allowed to stand for 5 min. The chemiluminescence value was detected using an enzyme-labeled instrument.

[0251] The killing detection method, cell number and effect-target ratio of Raji-luc cells are consistent with other MSLN-positive cells, but because they are suspension cells, they do not need to be digested and plated in advance. 50 μL Raji-luc cells are plated, 50 μL NK cell suspension and 50 μL antibodies of corresponding concentrations are added to each well, so that the final volume of each well system is 150 μL. When testing, 100 μL fluorescein potassium salt solution (1.5 mg / mL) is directly added, let stand for 5 minutes, and the chemiluminescence value is detected by an enzyme reader.

[0252] The cytotoxicity was calculated according to the following formula: Cytotoxicity (%) = [(fluorescence intensity positive control - fluorescence intensity negative control) - (fluorescence intensity experimental group - fluorescence intensity negative control)] / (fluorescence intensity positive control - fluorescence intensity negative control) * 100%, and the cytotoxicity % and concentration fitting data were plotted using GraphPad Prism8 software.

[0253] Depend on Fig.16As shown in Figures 16a to 16e, in each dosing group of N87-luc, H292-luc, and Ovcar-3-luc target cell lines, NKp46 / MSLN NKCE and MSLN mAb antibodies can effectively mediate NK cell killing of target cells, and the killing efficiency is dose-dependent, while MSLN mAb (Fc silent) has no obvious anti-tumor activity. Among them, the maximum killing efficiency of NKp46 / MSLN NKCE and MSLNmAb on N87-luc cells (minus the background killing of NK cells) at an effector-target ratio of 5:1 was 63.20% and 50.48%, respectively, and the killing EC 50 The maximum killing efficiency of H292-luc cells was 51.84% and 16.38%, respectively, and the killing efficiency of EC 50 The maximum killing efficiency of Ovcar-3-luc cells was 32.96% and 16.70%, respectively, and the killing efficiency of EC 50 When other conditions remained unchanged and the effector-target ratio was changed to 2.5:1, the antibody-mediated killing efficiency of N87-luc cells decreased (the maximum killing of NKCE and MSLN mAb was 40.99% and 32.27%, respectively), and there was no obvious killing effect on the MSLN negative cell line Raji-luc.

[0254] The above experimental results show that under the same experimental conditions, NKp46 / MSLN NKCE showed a stronger target cell killing effect than the parental monoclonal antibody MSLNmAb, especially for NCI-H292 cells and Ovcar-3 cells with relatively low MSLN expression. The results of the killing experiment on Raji cells showed that NKCE mediated NK cell-specific killing of MSLN-positive target cells.

[0255] Embodiment 11 Detection of NKp46 / MSLN NKCE-mediated target cell killing by double-staining flow cytometry with fluorescent dyes

[0256] To further explore the anti-tumor effect of the constructed NKp46 / MSLN NKCE, CFSE was used to label the target cells, and 7AAD dye was used to simultaneously label the late apoptotic cells and dead cells. The proportion of CFSE+7AAD+ cell population was used to evaluate the killing and apoptosis of MSLN-positive target cells mediated by NKp46 / MSLNN NKCE and the parental monoclonal antibody MSLN mAb.

[0257] The target cells NCI-N87, NCI-H292, and OVCAR-3 in the logarithmic growth phase were trypsinized and the enzyme activity was neutralized with complete medium. The cells were collected by centrifugation at 1000 rpm for 4 min at room temperature, and the supernatant was discarded and resuspended in staining buffer (PBS containing 2% inactivated FBS) to adjust the cell density to 1×10 6 cells / mL, add CFSE to a final concentration of 0.5μM, mix well and incubate at 37℃ in the dark for 30min. Add 5 times the volume of pre-cooled complete medium (RPMI1640 medium containing 10% inactivated FBS) to terminate the staining and place on ice for 5min. Centrifuge at 4℃ and 300×g for 3min, discard the supernatant, wash the cells 3 times with pre-cooled complete medium, resuspend in complete medium to complete cell staining. Incubate the target cells at 50μL and 5×10 4 cells were added to a 96-well plate; the NK cell density was adjusted to 50 μL per well, 2.5×10 5 Each cell was added to the corresponding well so that E:T=5:1; each antibody was diluted to 30 μg / mL using complete culture medium, and then diluted 10-fold gradient, with a total of 4 concentration gradients, and 50 μL of the corresponding concentration of antibody was added to each well, so that the final concentration of the antibody was 1 / 3 of the prepared concentration. At the same time, a blank group (unstained target cells), CFSE single staining group, 7AAD single staining group (target cells added with 50 μL 2% Triton X-100 solution), target cell background group (no antibody and NK cells, CFSE / 7AAD double staining), and NK background killing group (no antibody, CFSE / 7AAD double staining) were set up; mixed evenly, the well plate was placed in a 5% CO2, 37°C carbon dioxide cell culture incubator for 4 hours, 5 μL 7AAD was added to each well and incubated for 5 minutes, then the cell supernatant was directly transferred to the flow tube, 10 μL of trypsin was added to each well for digestion at room temperature for 3 minutes, transferred to the flow tube, a certain amount of staining buffer solution was added, and the machine was tested, and Flowjo was used to detect the staining buffer solution. 10.8.1 Software analysis results.

[0258] Depend on Fig.17 As shown in the figure, NKp46 / MSLN NKCE can significantly promote the apoptosis and death of NCI-N87, NCI-H292, and Ovcar-3 cells under 4h incubation conditions. In addition, although the parental antibody MSLN mAb with ADCC effect can also promote the lysis of target cells by NK cells, NKp46 / MSLN NKCE is significantly better than the parental monoclonal antibody MSLN mAb under various antibody administration concentration conditions.

[0259] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art may make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of the claims attached to the present invention.

Claims

1. A multispecific antigen-binding protein comprising at least: (a) a first binding domain, which specifically binds to natural killer cell activating receptor (NKp46); (b) a second binding domain, which specifically binds to mesothelin (MSLN).

2. The multispecific antigen-binding protein according to claim 1, characterized in that The first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody, wherein the heavy chain variable region comprises the following complementary determining region (CDR) combination (H-CDR1-3): H-CDR1 comprising the amino acid sequence shown in SEQ ID NO.7; H-CDR2 comprising the amino acid sequence shown in SEQ ID NO.8; H-CDR3 comprising the amino acid sequence shown in SEQ ID NO.9; the light chain variable region comprises the following complementary determining region (CDR) combination (L-CDR1-3): L-CDR1 comprising the amino acid sequence shown in SEQ ID NO.12; L-CDR2 comprising the amino acid sequence shown in SEQ ID NO.13; L-CDR3 comprising the amino acid sequence shown in SEQ ID NO.14; Preferably, the heavy chain variable region in the first binding domain comprises the amino acid sequence shown in SEQ ID NO. 10 or an amino acid sequence having at least 75% identity with said amino acid sequence; The light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 15 or an amino acid sequence having at least 75% identity thereto.

3. The multispecific antigen-binding protein according to claim 1 or 2, characterized in that The second binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody, wherein the heavy chain variable region comprises the following complementary determining region (CDR) combination (H-CDR1-3): H-CDR1 comprising the amino acid sequence shown in SEQ ID NO.17; H-CDR2 comprising the amino acid sequence shown in SEQ ID NO.18; H-CDR3 comprising the amino acid sequence shown in SEQ ID NO.19; the light chain variable region comprises the following complementary determining region (CDR) combination (L-CDR1-3): L-CDR1 comprising the amino acid sequence shown in SEQ ID NO.22; L-CDR2 comprising the amino acid sequence shown in SEQ ID NO.23; L-CDR3 comprising the amino acid sequence shown in SEQ ID NO.24; Preferably, the heavy chain variable region in the second binding domain comprises the amino acid sequence shown in SEQ ID NO. 20 or an amino acid sequence having at least 75% identity with said amino acid sequence; The light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 25 or an amino acid sequence having at least 75% identity thereto.

4. The multispecific antigen-binding protein according to any one of claims 1 to 3, characterized in that The first binding domain and the second binding domain further comprise a heavy chain constant region (CH) or a light chain constant region (CL) of an antibody, respectively, for example, a heavy chain constant region and / or a kappa or lambda type light chain constant region selected from IgG, IgA, IgM, IgD or IgE; Preferably, the multispecific antigen binding protein further comprises a binding domain that targets other tumor antigens or immune cell receptors; More preferably, the multispecific antigen binding protein is a bispecific binding protein, and even more preferably a natural killer cell engager (NKCE).

5. The multispecific antigen-binding protein according to any one of claims 1 to 4, characterized in that The multispecific antigen-binding protein is an IgG-type antibody molecule; Preferably, the multispecific antigen-binding protein is an antibody molecule of the IgG1 subtype.

6. The multispecific antigen-binding protein according to any one of claims 1 to 5, characterized in that The multispecific antigen-binding protein is a human IgG1 bispecific antibody molecule, comprising: 1) heavy chain 1, comprising domains arranged according to VH-CH1-CH2-CH3 from N-terminus to C-terminus; 2) light chain 1, which comprises domains arranged in a VL-CL pattern from N-terminus to C-terminus; 3) heavy chain 2, comprising domains arranged according to VH-CH1-CH2-CH3 from N-terminus to C-terminus; 4) light chain 2, comprising domains arranged in a VL-CL pattern from N-terminus to C-terminus; Among them, the VH and VL contained in heavy chain 1 and light chain 1 (or heavy chain 1 and light chain 1) are paired to form a first binding domain that binds to NKp46; the VH and VL contained in heavy chain 2 and light chain 2 (or heavy chain 2 and light chain 2) are paired to form a second binding domain that binds to MSLN.

7. The multispecific antigen-binding protein according to any one of claims 1 to 6, characterized in that Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence that is at least 75% identical to the amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO.2 or an amino acid sequence that is at least 75% identical to the amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO.3 or an amino acid sequence that is at least 75% identical to the amino acid sequence; light chain 2 comprises the amino acid sequence shown in SEQ ID NO.4 or an amino acid sequence that is at least 75% identical to the amino acid sequence.

8. A nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence comprised in the multispecific antigen-binding protein of any one of claims 1 to 7. A vector comprising the nucleic acid molecule according to claim 8. 10 . A host cell comprising the nucleic acid molecule of claim 8 or the vector of claim 9 , or transformed or transfected with the nucleic acid molecule of claim 8 or the vector of claim 9 .

11. A composition comprising the multispecific antigen-binding protein of any one of claims 1 to 7.

12. Use of the multispecific antigen-binding protein of any one of claims 1 to 7, the nucleic acid molecule of claim 8, the vector of claim 9, the host cell of claim 10 or the composition of claim 11 in the preparation of a medicament for treating a tumor; Preferably, the tumor is a solid tumor, such as a solid tumor expressing MSLN or being MSLN-positive.

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