Antibody for recognizing MSLN and application

Abstract

The invention relates to the field of biological medicine, in particular to an antibody for recognizing MSLN and application. According to the invention, the ScFv for identifying MSLN is obtained through screening, and the ScFv can be used for detecting expression of MSLN in immunohistochemistry, flow cytometry and the like; meanwhile, compared with the control and the prior art, the ScFv and the immune cells constructed on the basis of taking the ScFv as the extracellular recognition domain have the advantages that the tumor cell killing capability is obviously improved, and the application prospect is wide.

Description

Technical Field

[0001] This application relates to the biomedical field, and in particular to antibodies that identify MSLN and their uses. Background Technology

[0002] MSLN (Mesothelin) is a glycoprotein encoded by the MSLN gene and anchored to the cell surface via glycosylphosphatidylinositol. MSLN is a differentiation antigen present on normal mesothelial cells, but its biological function in normal tissues is not well understood. Increasing evidence from preclinical and clinical studies suggests that aberrant expression of MSLN plays a positive role in tumor malignant transformation and tumor invasiveness by promoting cancer cell proliferation, local invasion and metastasis, and resisting cytotoxic agent-induced apoptosis. MSLN is rarely expressed in normal tissues but is highly expressed in tumors such as ovarian cancer, endometrial cancer, pancreatic cancer, and mesothelioma, making it a potential target for cell therapy. Currently, there is still significant need for treatment of tumors such as ovarian cancer, endometrial cancer, pancreatic cancer, and mesothelioma; therefore, developing new antibody resources that recognize MSLN with stronger recognition capabilities or better anti-tumor effects is of great importance. Summary of the Invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide an antibody for recognizing MSLN and its use.

[0004] This invention provides an antibody that recognizes MSLN.

[0005] This invention provides an antibody that recognizes MSLN.

[0006] The amino acid sequence of its light chain variable region CDR1 is QX L1 X L2 X L3 X L4 X L5 ; where X L1 Selected from D, G, T, or S; X L2 Selected from L, S, V, or I; X L3 Selected from G or S; X L4 Selected from S or T; X L5 Selected from R or Y;

[0007] The amino acid sequence of CDR2 in the light chain variable region is X. L6 AS; where X L6 Choose from A or G;

[0008] The amino acid sequence of its light chain variable region CDR3 is CCX. L7 X L8 SX L9 PX L10 X L11X L12 ; where X L7 Selected from L or S; X L8 Selected from N or Y; X L9 Selected from Y, T, or none; X L10 Selected from L, I, F, or S; X L11 Selected from T or W; X L11 Selected from none or T;

[0009] The amino acid sequence of CDR1 in the heavy chain variable region is GX. H1 TFX H2 SYX H3 , where X H1 Selected from Y or F; X H2 Selected from T or S; X H2 Choose from Y, F, W, or D;

[0010] The amino acid sequence of CDR2 in the heavy chain variable region is X H4 X H5 X H6 X H7 X H8 GX H9 T, where X H4 Selected from I or M; X H5 Selected from N or S; X H6 Selected from P or S; X H7 Selected from S or N; X H8 Selected from G or S; X H9 Selected from S or N;

[0011] The amino acid sequence of CDR3 in the heavy chain variable region is ARX. H10 X H11 X H12 X H13 X H14 X H15 X H16 X H17 X H18 X H19 X H20 X H21 DX H22； X H10 For D, Y, or none; X H11 R or none; X H12 For P, R, D, or none; X H13 For F, A, or G; X H14 For S, A, or Q; X H15 For S or W; X H16 For D or G; X H17 For Y, A, or P; X H18 For Y or F; X H19 Y or none; X H20G or none; X H21 M or none; X H22 It can be V or I.

[0012] Furthermore, in the antibody described in this invention,

[0013] The amino acid sequence of CDR1 in the light chain variable region is at least one of the following: SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:19 and / or SEQ ID NO:58;

[0014] The amino acid sequence of CDR2 in the light chain variable region is at least one of the following: SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:20 and / or SEQ ID NO:59;

[0015] The amino acid sequence of CDR3 in the light chain variable region is at least one of the following: SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:21 and / or SEQ ID NO:60;

[0016] The amino acid sequence of CDR1 in the heavy chain variable region is at least one of the following: SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:22 and / or SEQ ID NO:61;

[0017] The amino acid sequence of CDR2 in the heavy chain variable region is at least one of the following: SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:23 and / or SEQ ID NO:62;

[0018] The amino acid sequence of CDR3 in the heavy chain variable region is at least one of the following: SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24 and / or SEQ ID NO:63.

[0019] Furthermore,

[0020] The variable region of the light chain includes the amino acid sequence CDR1 as shown in SEQ ID NO:3, the amino acid sequence CDR2 as shown in SEQ ID NO:4, and the amino acid sequence CDR3 as shown in SEQ ID NO:5; and / or

[0021] The variable region of the light chain includes the amino acid sequence CDR1 as shown in SEQ ID NO:11, the amino acid sequence CDR2 as shown in SEQ ID NO:12, and the amino acid sequence CDR3 as shown in SEQ ID NO:13; and / or

[0022] The variable region of the light chain includes an amino acid sequence as shown in SEQ ID NO:19 (CDR1), an amino acid sequence as shown in SEQ ID NO:20 (CDR2), and an amino acid sequence as shown in SEQ ID NO:21 (CDR3); and / or

[0023] The variable region of the light chain includes CDR1, an amino acid sequence as shown in SEQ ID NO:58, CDR2, an amino acid sequence as shown in SEQ ID NO:59, and CDR3, an amino acid sequence as shown in SEQ ID NO:60;

[0024] The variable region of the heavy chain includes the amino acid sequence CDR1 as shown in SEQ ID NO:6, the amino acid sequence CDR2 as shown in SEQ ID NO:7, and the amino acid sequence CDR3 as shown in SEQ ID NO:8; and / or

[0025] The variable region of the heavy chain includes the amino acid sequence CDR1 as shown in SEQ ID NO:14, the amino acid sequence CDR2 as shown in SEQ ID NO:15, and the amino acid sequence CDR3 as shown in SEQ ID NO:16; and / or

[0026] The variable region of the heavy chain includes the amino acid sequence CDR1 as shown in SEQ ID NO:22, the amino acid sequence CDR2 as shown in SEQ ID NO:23, and the amino acid sequence CDR3 as shown in SEQ ID NO:24; and / or

[0027] The variable region of the heavy chain includes CDR1 with an amino acid sequence as shown in SEQ ID NO:61, CDR2 with an amino acid sequence as shown in SEQ ID NO:62, and CDR3 with an amino acid sequence as shown in SEQ ID NO:63.

[0028] In this invention, the antibody CDR definition systems are different, and their sequences also differ. Specifically, the CDR definition systems include the Kabat system, Chothia system, MacCallum system, IMGT system, and / or AHo system. In specific embodiments of this invention, the IMGT system definition is used.

[0029] In the antibody described in this invention,

[0030] The amino acid sequence of the light chain variable region is at least one of the following: SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:17 and / or SEQ ID NO:64;

[0031] The amino acid sequence of the heavy chain variable region is at least one of SEQ ID NO:2, SEQ ID NO:10 or SEQ ID NO:18 and / or SEQ ID NO:65.

[0032] Specifically,

[0033] The amino acid sequence of its light chain is shown in SEQ ID NO:1, and the amino acid sequence of its heavy chain is shown in SEQ ID NO:2;

[0034] The amino acid sequence of its light chain is shown in SEQ ID NO:9, and the amino acid sequence of its heavy chain is shown in SEQ ID NO:10.

[0035] The amino acid sequence of its light chain is shown in SEQ ID NO:17, and the amino acid sequence of its heavy chain is shown in SEQ ID NO:18.

[0036] The amino acid sequence of its light chain is shown in SEQ ID NO:64, and the amino acid sequence of its heavy chain is shown in SEQ ID NO:65.

[0037] The antibody of the present invention has its light chain and heavy chain linked by a linker, the amino acid sequence of which is shown in SEQ ID NO:25.

[0038] In specific embodiments of the present invention, the amino acid sequences of the antibodies are shown in SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 and / or SEQ ID NO:54.

[0039] In this invention, the antibodies include RK-05-D10, RK-08-G03, RK-05-E06, and / or RK-05-C07;

[0040] Specifically,

[0041] The amino acid sequences of LCDR1 of RK-05-D10 are shown in SEQ ID NO:3; the amino acid sequences of LCDR2 are shown in SEQ ID NO:4; the amino acid sequences of LCDR3 are shown in SEQ ID NO:5; the amino acid sequences of HCDR1 are shown in SEQ ID NO:6; the amino acid sequences of HCDR2 are shown in SEQ ID NO:7; the amino acid sequences of HCDR3 are shown in SEQ ID NO:8; the amino acid sequences of VL are shown in SEQ ID NO:1; the amino acid sequences of VH are shown in SEQ ID NO:2; and the amino acid sequences of RK-05-D10 are shown in SEQ ID NO:48.

[0042] The amino acid sequences of LCDR1 of RK-08-G03 are shown in SEQ ID NO:11; the amino acid sequences of LCDR2 are shown in SEQ ID NO:12; the amino acid sequences of LCDR3 are shown in SEQ ID NO:13; the amino acid sequences of HCDR1 are shown in SEQ ID NO:14; the amino acid sequences of HCDR2 are shown in SEQ ID NO:15; the amino acid sequences of HCDR3 are shown in SEQ ID NO:16; the amino acid sequences of VL are shown in SEQ ID NO:9; the amino acid sequences of VH are shown in SEQ ID NO:10; and the amino acid sequences of RK-08-G03 are shown in SEQ ID NO:49.

[0043] The amino acid sequences of LCDR1 of RK-05-E06 are shown in SEQ ID NO:19; the amino acid sequences of LCDR2 are shown in SEQ ID NO:20; the amino acid sequences of LCDR3 are shown in SEQ ID NO:21; the amino acid sequences of HCDR1 are shown in SEQ ID NO:22; the amino acid sequences of HCDR2 are shown in SEQ ID NO:23; the amino acid sequences of HCDR3 are shown in SEQ ID NO:24; the amino acid sequences of VL are shown in SEQ ID NO:17; the amino acid sequences of VH are shown in SEQ ID NO:18; and the amino acid sequences of RK-05-E06 are shown in SEQ ID NO:50.

[0044] The amino acid sequences of LCDR1 of RK-05-C07 are shown in SEQ ID NO:58; the amino acid sequences of LCDR2 are shown in SEQ ID NO:59; the amino acid sequences of LCDR3 are shown in SEQ ID NO:60; the amino acid sequences of HCDR1 are shown in SEQ ID NO:61; the amino acid sequences of HCDR2 are shown in SEQ ID NO:62; the amino acid sequences of HCDR3 are shown in SEQ ID NO:63; the amino acid sequences of VL are shown in SEQ ID NO:64; the amino acid sequences of VH are shown in SEQ ID NO:2; and the amino acid sequences of RK-05-C07 are shown in SEQ ID NO:65.

[0045] The antibody described in this invention can be a single-chain antibody (scFv), a double-chain antibody, or an antibody containing only the F(ab) region; this invention does not limit the specific antibody.

[0046] The antibody described in this invention can be of the type IgG, IgA, IgM, IgE and / or IgD, and this invention does not limit it.

[0047] The antibodies described in this invention can be derived from rats, mice, rabbits, cattle, sheep, etc., and this invention does not limit the source.

[0048] This invention, through screening, obtained ScFvs capable of recognizing MSLNs, specifically including (RK-05-A09 (SEQ ID NO:52), RK-05-D10 (SEQ ID NO:48), RK-07-F11 (SEQ ID NO:53), RK-05-C07 (SEQ ID NO:54), RK-08-A05 (SEQ ID NO:55), RK-08-G03 (SEQ ID NO:49), RK-05-G03 (SEQ ID NO:56), and RK-05-E06 (SEQ ID NO:50). Further screening and testing showed that RK-05-C07 and RK-05-E06 have better MSLN recognition capabilities, capable of recognizing MSLN expression in HeLa cervical cancer cells. They can be used as reagents or reagent combinations for detecting MSLN expression in diagnostic kits, and can be used for detecting MSLN expression using immunohistochemistry, flow cytometry, and other techniques.

[0049] Furthermore, this invention constructed a chimeric antigen receptor (CAR structure) containing the aforementioned antibody. Experimental results showed that the chimeric antigen receptors containing RK-05-D10, RK-08-G03, and RK-05-E06 exhibited good anti-tumor effects, with a significant improvement in anti-tumor efficacy compared to the control. Among them, the chimeric antigen receptor containing RK-08-G03 (MLSN(RK-08-G03)-8h-8TM-BBZ, SEQ ID NO:31) was the best, followed by the chimeric antigen receptor containing RK-05-E06 (MLSN(RK-05-E06)-8h-8TM-BBZ, SEQ ID NO:32).

[0050] Furthermore, in this invention, RK-05-E06 can not only be used for MLSN detection, but also for the construction of immune cells. Experimental results show that RK-05-E06 can be used for the construction of immune cells or immune structures without being limited by various components. In specific embodiments of this invention, the selection of CAR structures, fusion sequences and / or promoter expression elements were examined. All of the above can be recombined with RK-05-E06 to achieve good anti-tumor effects.

[0051] This invention provides a biological material comprising at least one of the following: A) to D):

[0052] A) The nucleic acid of the antibody described in this invention;

[0053] B) A recombinant vector containing nucleic acids as shown in A);

[0054] C) Transform or transfect host cells with the recombinant vector shown in B);

[0055] D) Cells expressing the antibodies described in this invention;

[0056] E) Solids labeled, fixed, connected, or attached to or adhered with the antibodies described in this invention;

[0057] F) Biomarked or chemically labeled antibodies of the present invention;

[0058] F) Cultures obtained by culturing host cells as shown in C).

[0059] The nucleic acid described in this invention can be DNA, RNA, cDNA, or PNA. In embodiments of this invention, the nucleic acid is in the form of DNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). The nucleic acid can be topologically linear or circular. The nucleic acid can be part of a vector (e.g., an expression or cloning vector) or a fragment thereof. The nucleic acid can be obtained directly from natural sources or can be prepared with the assistance of recombinant, enzymatic, or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0060] Furthermore, the nucleic acids described in this invention can be optimized or unoptimized. The optimization includes, but is not limited to: codon usage bias, elimination of secondary structures that are unfavorable to expression (such as hairpin structures), alteration of GC content, CpG dinucleotide content, mRNA secondary structure, hidden splicing sites, early polyadenylation sites, internal ribosome entry and binding sites, negative CpG islands, RNA unstable regions, repetitive sequences (direct repeats, inverted repeats, etc.), and restriction sites that may affect cloning.

[0061] The present invention also provides a transcription unit for the recombinant antibody, wherein the transcription unit refers to a DNA sequence from the start of a promoter to the end of a terminator. Regulatory fragments may also be included on either side of or between the promoter and terminator, and these regulatory fragments may include a promoter operatively linked to a nucleic acid sequence, an enhancer, a transcription termination signal, a polyadenylation sequence, a replication origin, a nucleic acid restriction site, and a homologous recombination site, such as an enhancer of the promoter, a poly(A) signal, etc.

[0062] The present invention provides a recombinant vector comprising a vector backbone and the nucleic acid described herein.

[0063] Furthermore, the source of the carrier skeleton described in this invention includes vectors for plants, animals, bacteria, fungi, bacteriophages, or viruses, and this invention does not limit this.

[0064] The recombinant vector described in this invention refers to a recombinant nucleic acid vector, a recombinant DNA molecule containing the desired coding sequence and suitable nucleic acid sequences or elements essential for the expression of an operatively linked coding gene in a specific host organism. Nucleic acid sequences or elements essential for expression in model organisms or mammalian cells include promoters, ribosome binding sites, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and terminators. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In this specification, "plasmid" and "vector" are sometimes used interchangeably because plasmids are currently the most commonly used form of vector. However, this invention intends to include other forms of expression vectors that perform equivalent functions, which are known in the art or will become known, including but not limited to: plasmids, phage particles, viral vectors, and / or simply potential genomic inserts.

[0065] The host cell provided by this invention has its genome integrated with nucleic acids as described in this invention, or transfected or transformed with recombinant vectors as described in this invention.

[0066] Furthermore, the transformation methods include chemical transformation and electrotransformation; the transfection methods include calcium phosphate co-precipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0067] The host cells provided by this invention can be derived from plants, animals, bacteria, fungi, bacteriophages, or viruses, and this invention does not limit the source. This invention uses vectors constructed using recombinant DNA technology to transform or transfect host cells. These transformed host cells are capable of replicating the protein-encoding vector, expressing the desired protein, or performing a corresponding function.

[0068] The present invention provides a method for preparing the antibody, which involves culturing host cells as described in the present invention to obtain a mixture containing the antibody as described in the present invention.

[0069] The present invention provides cells expressing the antibodies described herein, wherein;

[0070] Solids labeled, fixed, connected, or attached to or adhered with the antibodies described in this invention;

[0071] The antibodies described in this application can be labeled, fixed, linked, or attached to or adhered to a solid material, which can be a solid-phase carrier such as a well plate, magnetic beads, or nanomaterials; the linking can be direct or indirect coupling, such as covalent coupling to the solid material, and this invention does not limit this.

[0072] This invention provides antibodies with the aforementioned biomarkers or chemical markers. In this invention, the biomarkers or chemical markers include, but are not limited to, enzymes, biotinylate, fluorescein, chemiluminescence, isotopes, colloids, latex microspheres, and / or magnetic beads. The enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-antiperoxidase bridges, alkaline phosphatase-antialkaline phosphatase bridges, and β-galactosidase-antiβ-galactosidase bridges. The biotinylate class includes, but is not limited to, biotin and its derivatives. The fluorescein class includes, but is not limited to, AF350, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, Oregon Green 488, Pacific Blue dye, Pacific Orange dye, and Texas... Red and PerCP dyes; the chemiluminescent types include, but are not limited to, isoluminol and its derivatives, acridine esters and their derivatives, ruthenium terpyridine and its derivatives, etc.; the isotope types include, but are not limited to, iodine labeling; the colloidal labels include, but are not limited to, colloidal gold, colloidal carbon, colloidal selenium, etc.

[0073] This invention provides the application of at least one of the following (a) to (b) in the preparation of MSLN detection products:

[0074] a) The antibody described in this invention;

[0075] b) The biomaterials described in this invention.

[0076] The present invention provides MSLN detection reagents and / or kits, which include at least one of the antibodies and / or biological materials described in the present invention and excipients.

[0077] This invention provides a chimeric antigen receptor that targets MSLN-positive cells, using the antibody described in this invention as the antigen recognition domain.

[0078] Furthermore, the chimeric antigen receptor described in this invention further includes a hinge, a transmembrane region, and an intracellular signal transduction region:

[0079] The hinge is selected from at least one of CD8α hinge (8h), CD7 (7h) hinge and / or IgG4 (G4h) hinge.

[0080] The transmembrane region is selected from at least one of the CD8α (8TM) transmembrane region and / or the CD28 transmembrane region (28TM);

[0081] The intracellular signal transduction region is selected from at least one of the CD137 intracellular region, CD3Z and / or CD28 intracellular region.

[0082] Furthermore, the chimeric antigen receptor of the present invention includes:

[0083] Antibody, CD8 hinge (8h), CD8α transmembrane region (8TM) and intracellular signal transduction region composed of CD137 and CD3z (BBZ intracellular signal transduction region);

[0084] Antibody, CD8 hinge (8h), CD28α transmembrane region (28TM), and intracellular signal transduction region composed of CD28 and CD3z (28Z intracellular signal transduction region);

[0085] Antibody, IgG4 hinge (G4h), CD28α transmembrane region (28TM) and intracellular signal transduction region composed of CD28 and CD3z (28Z intracellular signal transduction region);

[0086] Antibody, CD7 hinge (7h), CD28α transmembrane region (28TM) and intracellular signal transduction region (28BBZ intracellular signal transduction region) composed of CD28, CD137 and CD3z.

[0087] In a specific embodiment of the present invention, the chimeric antigen receptor includes at least one of the chimeric antigen receptors with amino acid sequences such as SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:43, SEQ ID NO:44 and / or SEQ ID NO:45.

[0088] In this invention, the chimeric antigen receptor contains:

[0089] MSLN(RK-05-D10)-8h-8TM-BBZ comprises an RK-05-D10 antibody, a CD8 hinge (8h), a CD8α transmembrane region (8TM), and an intracellular signal transduction region composed of CD137 and CD3z (BBZ intracellular signal transduction region); its amino acid sequence is shown in SEQ ID NO:30.

[0090] MLSN(RK-08-G03)-8h-8TM-BBZ comprises an RK-08-G03 antibody, a CD8 hinge (8h), a CD8α transmembrane region (8TM), and an intracellular signal transduction region composed of CD137 and CD3z (BBZ intracellular signal transduction region); its amino acid sequence is shown in SEQ ID NO:31.

[0091] MLSN(RK-05-E06)-8h-8TM-BBZ comprises an RK-05-E06 antibody, a CD8 hinge (8h), a CD8α transmembrane region (8TM), and an intracellular signal transduction region composed of CD137 and CD3z (BBZ intracellular signal transduction region); its amino acid sequence is shown in SEQ ID NO:32.

[0092] MSLN(RK-05-E06)-8h-28TM-28Z comprises an RK-05-E06 antibody, a CD8 hinge (8h), a CD28α transmembrane region (28TM), and an intracellular signal transduction region composed of CD28 and CD3z (28Z intracellular signal transduction region); its amino acid sequence is shown in SEQ ID NO:43.

[0093] MSLN(RK-05-E06)-G4h-28TM-28Z (SEQ ID NO:44) comprises an RK-05-E06 antibody, an IgG4 hinge (G4h), a CD28α transmembrane region (28TM), and an intracellular signal transduction region composed of CD28 and CD3z (28Z intracellular signal transduction region); its amino acid sequence is shown in SEQ ID NO:44.

[0094] MSLN(RK-05-E06)-7h-28TM-28BBZ comprises an RK-05-E06 antibody, a CD7 hinge (7h), a CD28α transmembrane region (28TM), and an intracellular signal transduction region composed of CD28, CD137, and CD3z (28BBZ intracellular signal transduction region); its amino acid sequence is shown in SEQ ID NO:45.

[0095] In specific embodiments of the present invention, it has been demonstrated that chimeric antigen receptors containing RK-05-D10, RK-08-G03, and RK-05-E06 have good anti-tumor effects, and the anti-tumor effect is significantly improved compared with the control and other combinations; among them, the chimeric antigen receptor containing RK-08-G03 (MLSN(RK-08-G03)-8h-8TM-BBZ, SEQ ID NO:31) is the best, followed by the chimeric antigen receptor containing RK-05-E06 (MLSN(RK-05-E06)-8h-8TM-BBZ, SEQ ID NO:32).

[0096] This invention constructs a fusion protein containing the chimeric antigen receptor described above. Immune cells constructed by fusing the chimeric antigen receptor containing RK-05-E06 with the fusion protein exhibit significantly enhanced cytotoxicity compared to control and existing immune cells. Furthermore, RK-05-E06 can be used for MSLN detection and as a tumor prevention and treatment drug for MSLN-positive expression. In this invention, RK-05-E06, as a tumor prevention and treatment drug, can be combined with various cytotoxic elements, such as CAR structures. Even with different compositions of expression elements in the CAR structure of RK-05-E06 (e.g., hypoxia promoter), it still maintains a high cytotoxic effect.

[0097] This invention provides a nucleic acid encoding the chimeric antigen receptor described above;

[0098] This invention provides a vector comprising the nucleic acid described herein;

[0099] This invention provides a virus comprising the vector described herein.

[0100] This invention provides the application of the chimeric antigen receptor in the preparation of immune cells.

[0101] The present invention provides immune cells comprising chimeric antigen receptors as described in the present invention, or derived from virus-transduced immune cells as described in the present invention.

[0102] Furthermore, the immune cells mentioned include, but are not limited to, T cells, T cell precursors, NK cells, or macrophages, etc., and the present invention does not limit them;

[0103] In this invention, the chimeric antigen receptor can be used to modify immune cells. It can be directly modified or expressed on the surface of immune cells such as T cells, NK cells, and macrophages to form CAR-T, CAR-NK, CAR-macrophage, and other modified immune cells.

[0104] Furthermore, the chimeric antigen receptor-modified immune cells (such as CAR-T cells) can also express fusion proteins that target one or more different targets, wherein the one or more targets are selected from tumor antigens and / or immune checkpoint proteins.

[0105] The tumor antigens are selected from: prostate stem cell antigen (PSCA), carcinoembryonic antigen (CEA), CAM5, CD123, thyroid-stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD30; CD70; CD123; CD 138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); Interleukin-13 receptor subunit α (IL-13Rα); Interleukin-11 receptor α (IL-11Rα); Prostate-specific membrane antigen (PSMA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; Tyrosinase; Mesothelin; EpCAM; Protease serine 21 (PRSS21); Vascular endothelial growth factor receptor; Lewis (Y) antigen; CD24; Platelet-derived growth factor receptor β (PDGFR-β); Stage-specific embryonic antigen-4 (SSEA-4); Cell surface-associated mucin 1 (MUC1), MUC6; Epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EG... FRvIII); Neural cell adhesion molecule (NCAM); Carbonic anhydrase IX (CAIX); LMP2; Hepatic ligand A receptor 2 (EphA2); Fucosyl GM1; Sialyl Lewis adhesion molecule (sLe); Ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TGS5; High molecular weight melanoma-associated antigen (HMWMAA); O-acetyl GD2 ganglioside (OAcGD2); Folate receptor; Tumor vascular endothelial marker 1 (TEM1 / CD248); Tumor vascular endothelial marker 7-associated (TEM7R); Claudin6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B cell maturation antigen (BCMA); CA9; κ light chain (kappa Light chain); CSPG4; EGP2, EGP40; FAP; FAR; FBP; Embryonic AchR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; fibronectin; tendinin; carcinoembryonic variant of tumor necrosis zone; G protein-coupled receptor class C5-member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1);The following are gene sequences associated with globoH glycoceramide: globoH (hexose moiety); breast differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenaline receptor β3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternating reading frame protein (TARP); nephroblastoma protein (WT1); ETS translocation variant gene 6 (ETV6-AML); spermin 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-C). T-2); Fos-associated antigen 1; p53 mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosamine transferase V (NA17); pairing box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myeloma virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like protein (BORIS); Squamous cell carcinoma antigen 3 recognized by T cells (SART3); Pax-5 (PAX5) pairing box protein; proacrosin-binding protein sp32 (OYTES1); Lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchoring protein 4 (AKAP-4); Synovial sarcoma, X-breakpoint 2 (SSX2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc slice of IgA receptor The following are included: FCAR; Leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); Bone marrow stromal cell antigen 2 (BST2); EGF-like module mucin-like hormone receptor-like 2 (EMR2); Lymphocyte antigen 75 (LY75); Phosphatidylinositol proteoglycan-3 (GPC3); Fc receptor-like 5 (FCRL5); Immunoglobulin λ-like polypeptide 1 (IGLL1); and CD155, etc., but this invention does not limit these components.

[0106] The immune checkpoint proteins are selected from at least one of the following: 2B4, 4-1BB, 4-1BB ligand, B7-1, B7-2, B7H2, B7H3, B7H4, B7H6, BTLA, CD155, CD160, CD19, CD200, CD27, CD27 ligand, CD28, CD40, CD40 ligand, CD47, CD48, CTLA-4, DNAM-1, Galectin-9, GITR, GITR ligand, HVEM, ICOS, ICOS ligand, IDOI, KIR, 3DL3, LAG-3, OX40, OX40 ligand, PD-L1, PD-1, PD-L2, LAG3, PGK, SIRPα, TIM-3, TIGIT, and / or VSIG8.

[0107] In this invention, the fusion protein may be a protein targeting different targets, and the protein targeting different targets may be modified on the surface of immune cells by covalent linking or coupling. The fusion protein of this invention may also be a fusion protein composed of proteins targeting different targets and various elements for expression in cells. The elements include, but are not limited to, transmembrane regions and / or intracellular signal transduction regions. This invention does not limit the specific elements. The fusion-expressed product modifies immune cells in vitro or in vivo.

[0108] In a specific embodiment of the present invention, the fusion protein includes a fusion protein that recognizes CD47 or CD155, wherein the fusion protein that recognizes CD47 is a SIRPγ fusion protein (SEQ ID NO:33), and the fusion protein that recognizes CD155 is a CD226 fusion protein (SEQ ID NO:36) or a TIGIT fusion protein (SEQ ID NO:37).

[0109] In this invention, the fusion protein and the chimeric antigen receptor can be individually modified on the surface of immune cells using chemical or biological methods, or they can be modified together using a combination of chemical and biological methods. Alternatively, they can be independently modified on the surface of immune cells using biological methods, or recombinant sequences can be constructed and combined to modify the surface of immune cells using biological methods. The fusion protein and chimeric antigen receptor in the recombinant sequence can be linked by a cleaving peptide, which is selected from any one of P2A, F2A, T2A, and / or E2A; this invention does not limit this selection. In this invention, the biological method refers to in vivo self-assembly. Those skilled in the art can recombine the sequences containing the fusion protein and chimeric antigen receptor with suitable elements and transfect cells to express the fusion protein and chimeric antigen receptor. These elements include promoters, terminators, enhancers, replicons, and other suitable elements essential for expression in a specific host organism; these will not be elaborated upon in this invention.

[0110] In a specific embodiment of the present invention, the recombinant sequences of the fusion protein and the chimeric antigen receptor specifically include:

[0111] MSLN(RK-05-D10)-8h-8TM-BBZ-P2A-SIRP-28TM-28, whose amino acid sequence is shown in SEQ ID NO:34;

[0112] MSLN(RK-08-G03)-8h-8TM-BBZ-P2A-SIRP-28TM-28, whose amino acid sequence is shown in SEQ ID NO:35;

[0113] MSLN(RK-05-E06)-8h-8TM-BBZ-P2A-SIRP-28TM-28, whose amino acid sequence is shown in SEQ ID NO:40;

[0114] MSLN(RK-05-E06)-8h-8TM-BBZ-P2A-CD226-28TM-28, whose amino acid sequence is shown in SEQ ID NO:41;

[0115] MSLN(RK-05-E06)-8h-8TM-BBZ-P2A-TIGIT-28TM-28, the amino acid sequence of which is shown in SEQ ID NO:42.

[0116] This invention, through screening, obtained a ScFv that recognizes MSLN. The ScFv can recognize MSLN and can be used for MSLN detection, specifically including but not limited to detection of MSLN expression using immunohistochemistry, flow cytometry, etc. Simultaneously, the ScFv of this invention can be used as a drug or drug combination component for treating MSLN. Furthermore, Fc structures or bispecific antibodies can be designed based on this ScFv for the treatment of tumors with positive MSLN expression. Alternatively, the ScFv can be used as an extracellular recognition domain, combined with extracellular segments, intracellular T cell co-stimulatory signals, etc., to form a chimeric antigen receptor (CAR) or an extracellular recognition portion similar to a fusion protein structure. Different designed CAR structures can be expressed on T cells, including αβT cells or γδT cells, to form CAR-T cells; they can also be expressed on genetically modified T cells, such as universal T cells, to form universal or allogeneic CAR-T cells (UCAR-T); they can also be expressed on NK cells to form CAR-NK cells; or they can be expressed on macrophages or other immune cells such as Tregs.

[0117] The present invention provides a method for preparing the aforementioned immune cells, which includes obtaining the immune cells by transducing them with the aforementioned virus and then culturing them.

[0118] This invention provides the application of any one of the following (I) to (VII) in the preparation of anti-tumor drugs:

[0119] I) The antibody described in this invention;

[0120] II) The biomaterials described in this invention;

[0121] III) The chimeric antigen receptor described in this invention;

[0122] IV) The nucleic acid described in this invention;

[0123] V), the carrier described in this invention;

[0124] VI) The virus described in this invention;

[0125] VII) The immune cells described in this invention.

[0126] Furthermore, in the applications described in this invention, the tumor includes at least one of rectal cancer, pancreatic cancer, endometrial cancer, uterine cancer, ovarian cancer, kidney cancer, and / or bladder cancer.

[0127] The drug for preventing and treating tumors of the present invention comprises at least one of the following raw materials: i) to vii).

[0128] i) The antibody described in this invention;

[0129] ii) The biomaterials described in this invention;

[0130] iii) The chimeric antigen receptor described in this invention;

[0131] iv) The nucleic acid described in this invention;

[0132] v) The carrier described in this invention;

[0133] vi) The virus described in this invention;

[0134] vii), the immune cells described in this invention.

[0135] The present invention provides pharmaceutical compositions comprising the pharmaceutical ingredients described herein and pharmaceutically acceptable excipients.

[0136] The pharmaceutical compositions provided by this invention also include combinations of the pharmaceuticals described in this invention and other pharmaceuticals with anticancer effects, but this invention does not limit the scope of these combinations.

[0137] The drugs described in this invention include, but are not limited to, ADC drugs, CAR-T drugs, CAR-NK drugs, bispecific antibody drugs, etc.

[0138] This invention provides a method for preventing and treating tumors, which involves applying the drug described in this invention.

[0139] This invention obtains a ScFv that recognizes MSLN through screening, which can be used to detect MSLN expression by immunohistochemistry, flow cytometry and other techniques. At the same time, the ScFv itself, as well as immune cells constructed based on the ScFv as an extracellular recognition domain, significantly improves the tumor cell killing ability compared with controls and existing technologies, and has broad application prospects. Attached Figure Description

[0140] Figure 1 Show ScFv filter;

[0141] Figure 2 Detection of MSLN expression in HeLa cells;

[0142] Figure 3 The screening of ScFv for MSLN expression in cervical cancer cells is shown;

[0143] Figure 4 The results of CAR-T cells expressing CAR constructed from selected ScFv cells killing MSLN-positive cervical cancer cells are shown.

[0144] Figure 5 The results of CAR-T cells expressing CAR constructed from selected ScFv cells killing MSLN-positive cervical cancer cells are shown.

[0145] Figure 6 The results of CAR-T cells expressing CAR constructed from selected ScFv cells killing MSLN-positive cervical cancer cells are shown.

[0146] Figure 7 The study showed the killing effect of CAR-T cells expressing different CAR structures on MSLN-positive cervical cancer cells.

[0147] Figure 8 The results of CAR-T cells with different CAR structures killing MSLN-positive cervical cancer cells are shown. Among them, A represents the killing results of CAR11-CAR13; B represents the killing results of CAR14-CAR17.

[0148] Figure 9 This demonstrates the in vivo efficacy of CAR-T cells expressing CAR constructed from screened ScFv.

[0149] Figure 10 The persistence of CAR-T cells expressing CAR constructed from the selected ScFv in vivo was shown to be 0.0122.

[0150] Figure 11 MSLN expression detection in tumor tissue. Detailed Implementation

[0151] This invention provides antibodies for identifying MSLNs and their uses. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0152] the term:

[0153] An antibody is a protein or polypeptide sequence that binds to a specific antigen, which is a protein or polypeptide sequence expressed on the cell membrane or secreted extracellularly that can be recognized by the antibody. Antibodies can be antibodies containing an Fc structure, or they can be single-chain antibodies (ScFv), single- or double-domain (VH or VHH) antibodies.

[0154] Adoptive cell therapy (ACT) refers to a treatment method that uses cells from the body's own immune system, which are cultured and modified externally before being reinfused into the body to eliminate diseases.

[0155] Chimeric antigen receptor:

[0156] "Chimeric antigen receptor" or "CAR" refers to a group of engineered peptides or proteins that, when in immune effector cells, bind to specific antigens contained on target cells and generate intracellular signals upon recognition of the specific antigens, activating downstream pathways in the cell where the receptor is located, thereby initiating the killing effect of the immune effector cells on the target cells.

[0157] A CAR typically comprises at least one extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain. The extracellular antigen-binding domain specifically recognizes antigens, and non-limiting examples include single-chain variable fragments (scFv) derived from antibodies, fragment antigen-binding regions (Fab) selected from libraries, single-domain fragments or natural ligands that bind to their homologous receptors, artificially designed target-specific recognition domains such as fibronectin type III (FN3) domain combinations, and designed ankyrin repeat proteins (DARPins) that recognize specific targets. The signaling domain typically contains an immune-receptor tyrosine-based activation motif (ITAM), whose basic composition is YXXL / V, where Y is tyrosine, L / V refers to leucine or valine, and X can be any amino acid. When the receptor binds to its corresponding ligand, the tyrosine residue in the ITMA linked to it can be phosphorylated by a type of protein tyrosine kinase PTK that is connected to the cell membrane. This recruits other free intracellular protein kinases or adaptor proteins to transmit activation signals into the cell.

[0158] In some implementations, the "signal transduction domain" is selected as an intracellular signal transduction domain of TCRζ (CD3ζ, also written as CD3Z or CD3z) or FcεRIγ. As used herein, the "co-stimulatory domain" is also referred to as the "co-stimulatory signaling domain" and is primarily used to provide co-stimulatory signals to enhance the capabilities of immune cells, including, for example, enhancing the proliferation, survival, and / or development of memory cells.

[0159] In some implementations, the "co-stimulatory domain" is selected from intracellular domains of CD28, 4-1BB (CD137), OX40 (CD134), etc. As used herein, the "transmembrane domain," also known as a "transmembrane region," refers to a thermodynamically stable protein structural region anchored within the cell membrane. Transmembrane domains can be obtained from natural proteins, such as the transmembrane domain derived from the T cell receptor (TCR).

[0160] In some embodiments, the transmembrane domain is selected from the transmembrane domains of CD4, CD8α, CD28, and CD3ζ.

[0161] "Chimeric antigen receptors" or "CARs" can have various structures, such as those containing secretible or membrane-expressed cytokines or antibody gene sequences; and those containing structures that can be regulated for activation or inactivation, including: suicide switches such as inducible caspase-9 (iCasp9), thymidine kinase (HSV-TK) and suicide epitopes in herpes simplex virus, truncated EGFR (EGFRt), and Fas-FasL apoptosis structures; and induced CAR structures such as: peptide neo-epitope (PNE), fluorescein (FITC), 10 amino acids (5B9 tag), FITC-HM-3 bifunctional molecule (FHBM) and scFv, leucine ZipFv linked to antibody, streptavidin 2 (mSA2) biotin-binding domain, VIPER CAR inducible structures, and biotin-biding immune receptors. The receptor (BBIR) system; the "logic gate" regulatory system that binds to the SynNotch receptor, etc.

[0162] In some embodiments, chimeric antigen receptors can be used to modify immune cells, such as expressing them on the surface of T, NK, and macrophages to form CAR-T, CAR-NK, and CAR-macrophage modified immune cells. In some embodiments, the chimeric antigen receptor-modified immune cells, such as CAR-T cells, can also express fusion proteins targeting one or more different targets, where the target molecules are selected from tumor antigens and / or immune checkpoint proteins.

[0163] The tumor antigens are selected from: prostate stem cell antigen (PSCA), carcinoembryonic antigen (CEA), CAM5, CD123, thyroid-stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD30; CD70; CD123; CD 138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); Interleukin-13 receptor subunit α (IL-13Rα); Interleukin-11 receptor α (IL-11Rα); Prostate-specific membrane antigen (PSMA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; Tyrosinase; Mesothelin; EpCAM; Protease serine 21 (PRSS21); Vascular endothelial growth factor receptor; Lewis (Y) antigen; CD24; Platelet-derived growth factor receptor β (PDGFR-β); Stage-specific embryonic antigen-4 (SSEA-4); Cell surface-associated mucin 1 (MUC1), MUC6; Epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EG... FRvIII); Neural cell adhesion molecule (NCAM); Carbonic anhydrase IX (CAIX); LMP2; Hepatic ligand A receptor 2 (EphA2); Fucosyl GM1; Sialyl Lewis adhesion molecule (sLe); Ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TGS5; High molecular weight melanoma-associated antigen (HMWMAA); O-acetyl GD2 ganglioside (OAcGD2); Folate receptor; Tumor vascular endothelial marker 1 (TEM1 / CD248); Tumor vascular endothelial marker 7-associated (TEM7R); Claudin6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B cell maturation antigen (BCMA); CA9; κ light chain (kappa Light chain); CSPG4; EGP2, EGP40; FAP; FAR; FBP; Embryonic AchR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; fibronectin; tendinin; carcinoembryonic variant of tumor necrosis zone; G protein-coupled receptor class C5-member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1);The following are gene sequences associated with globoH glycoceramide: globoH (hexose moiety); breast differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenaline receptor β3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternating reading frame protein (TARP); nephroblastoma protein (WT1); ETS translocation variant gene 6 (ETV6-AML); spermin 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-C). T-2); Fos-associated antigen 1; p53 mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosamine transferase V (NA17); pairing box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myeloma virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like protein (BORIS); Squamous cell carcinoma antigen 3 recognized by T cells (SART3); Pax-5 (PAX5); proacrosin-binding protein sp32 (OYTES1); Lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchoring protein 4 (AKAP-4); Synovial sarcoma, X-breakpoint 2 (SSX2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); IgA receptor The Fc fragment of the body (FCAR); member of the leukocyte immunoglobulin-like receptor subfamily 2 (LILRA2); member of the CD300 molecule-like family f (CD300LF); member of the C-type lectin domain family 12 A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); mucin-like hormone receptor-like 2 containing an EGF-like module (EMR2); lymphocyte antigen 75 (LY75); phosphatidylinositol proteoglycan-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin λ-like polypeptide 1 (IGLL1) and CD155;

[0164] The immune checkpoint proteins are selected from: 2B4, 4-1BB, 4-1BB ligand, B7-1, B7-2, B7H2, B7H3, B7H4, B7H6, BTLA, CD155, CD160, CD19, CD200, CD27, CD27 ligand, CD28, CD40, CD40 ligand, CD47, CD48, CTLA-4, DNAM-1, Galectin-9, GITR, GITR ligand, HVEM, ICOS, ICOS ligand, IDOI, KIR, 3DL3, LAG-3, OX40, OX40 ligand, PD-L1, PD-1, PD-L2, LAG3, PGK, SIRPα, TIM-3, TIGIT, and VSIG8.

[0165] Specifically, the chimeric antigen receptor-modified T cells of this application also express fusion proteins that recognize CD47 or CD155. The fusion protein recognizing CD47 is a SIRPγ fusion protein, and the fusion protein recognizing CD155 is a CD226 fusion protein or a TIGIT fusion protein. The expression of these fusion proteins can be prepared by linking a self-cleaving peptide (2A peptide) or a ribosomal cleaving peptide to a CAR gene sequence to form a multi-expression vector, or by transducing the CAR vector and target cells separately. The amino acid sequence of RK-05-D10(VL) is as follows:

[0166]

[0167] The amino acid sequence of RK-05-D10(VH):

[0168] EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPS GGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDRPESSDYYYGMDV WGQGTTVTVSS(SEQ ID NO:2);

[0169] The amino acid sequence of RK-05-D10(LCDR1): QDLGSR (SEQ ID NO:3);

[0170] The amino acid sequence of RK-05-D10(LCDR2): AAS (SEQ ID NO:4);

[0171] The amino acid sequence of RK-05-D10(LCDR3): QQLNSYPLT (SEQ ID NO:5);

[0172] The amino acid sequence of RK-05-D10(HCDR1): GYTFTSYY (SEQ ID NO:6);

[0173] The amino acid sequence of RK-05-D10(HCDR2): INPSGGST (SEQ ID NO:7);

[0174] The amino acid sequence of RK-05-D10(HCDR3) is: ARDRPESSDYYYGMDV (SEQ ID NO:8);

[0175] Amino acid sequence of RK-08-G03 (VL): DIELTQSPSTLSASSVGDRVTITCRASQGSSSYLAWYQQKPGKAPKLLIHAASTLQSGVPSR FSGSGSGTEFTLTISSLQSEDFATYYCQQLNSYPITFGQGTRLEIKR (SEQ IDNO: 9);

[0176] The amino acid sequence of RK-08-G03(VH) is: QIQLVQSGAEVKKPGASVKVSCKASGYTFTSYFMHWVRQAPGQGPEWMGVINPSGGST SYAQKFQGRVTMTRDTSTSTVYMELSSLRSDDTAVYYCARYRAASDAFDIWGQGTTVTVSS (SEQ ID NO:10);

[0177] The amino acid sequence of RK-08-G03(LCDR1): QGSSSY (SEQ ID NO:11);

[0178] The amino acid sequence of RK-08-G03(LCDR2): AAS (SEQ ID NO:12);

[0179] The amino acid sequence of RK-08-G03(LCDR3): QQLNSYPIT (SEQ ID NO:13);

[0180] The amino acid sequence of RK-08-G03(HCDR1): GYTFTSYF (SEQ ID NO:14);

[0181] The amino acid sequence of RK-08-G03(HCDR2) is: INPSGGST (SEQ ID NO:15);

[0182] The amino acid sequence of RK-08-G03(HCDR3) is: ARYRAASDAFDI (SEQ ID NO:16);

[0183] Amino acid sequence of RK-05-E06 (VL): AIRLTQSPSSSLSAAVGDRVTITCRASQTVSTYLNWYQQKLGKAPKLLIYGASNLQSGVPS RFSGSGSGTDFSLTISNLQTEDVAIYYCQQSYSPFWTFGQGTKLEIKG (SEQ IDNO: 17);

[0184] Amino acid sequence of RK-05-E06 (VH): EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYWMHWVRQAPGKGLEWVSAISGSGGST YYADSVKGRFTISRDNSKNTLYLEMNSLRPEDTAVYYCARFSWDPYYGMDVWGQGTMVTVSS (SEQ ID NO: 18);

[0185] The amino acid sequence of RK-05-E06(LCDR1): QTVSTY (SEQ ID NO:19);

[0186] The amino acid sequence of RK-05-E06(LCDR2): GAS (SEQ ID NO:20);

[0187] The amino acid sequence of RK-05-E06(LCDR3): QQSYSPFWT (SEQ ID NO:21);

[0188] The amino acid sequence of RK-05-E06(HCDR1): GFTFSSYW (SEQ ID NO:22);

[0189] The amino acid sequence of RK-05-E06(HCDR2): ISSSGGST (SEQ ID NO:23);

[0190] The amino acid sequence of RK-05-E06(HCDR3) is: ARFSWDPYYGMDV (SEQ ID NO:24);

[0191] The amino acid sequence of the linker is: GSTGSGSGKPGSGEGSTKG (SEQ ID NO:25);

[0192] The amino acid sequence of 8h is: TTTAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 26);

[0193] The amino acid sequence of 8TM is: IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:27);

[0194] The amino acid sequence of BB(CD137) is: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:28);

[0195] Amino acid sequence of CD3ζ (CD3Z): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ IDNO: 29);

[0196] The amino acid sequence of MSLN(RK-05-D10)-8h-8TM-BBZ:

[0197]

[0198]

[0199] The amino acid sequence of MLSN(RK-08-G03)-8h-8TM-BBZ:

[0200] The amino acid sequence of MLSN(RK-05-E06)-8h-8TM-BBZ:

[0201] Amino acid sequence of SIRPγ fusion protein: MPVPASWPHPPGPFLLLTLLLGLTEVAGEEELQMIQPEKLLLVTVGKTATLHCTVTSLLPVGPVLWFRGVGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPENVEFKSGPGTEMALGAKPSAPVVLGPAARTTPEHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPTGQSVAYSIRSTARVVLDPWDVRSQVICEVAHVTLQGDPLRGTANLSEAIRVPPTLEVTQQPMRVGNQVNVTCQVRKFYPQSLQLTWSENGNVCQRETASTLTENKDGTYNWTSWFLVNISDQRDDVVLTCQVKHDGQLAVSKRLALEVTVHQKDQSSDATPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:33);

[0202] Amino acid sequence of MSLN(RK-05-D10)-8h-8TM-BBZ-PM-2A-SIRP-28TM-28:

[0203]

[0204]

[0205] Amino acid sequence of MSLN(RK-08-G03)-8h-8TM-BBZ-PM-2A-SIRP-28TM-28:

[0206] Amino acid sequence of CD226 fusion protein: EEVLWHTSVPFAENMSLECVYPSMGILTQVEWFKIGTQQDSIAIFSPTHGMVIRKPYAERVYFLNSTMASNNMTLFFRNASEDDVGYYSCSLYTYPQGTWQKVIQVVQSDSFEAAVPSNSHIVSEPGKNVTLTCQPQMTWPVQAVRW EKIQPRQIDLLTYCNLVHGRNFTSKFPRQIVSNCSHGRWSVIVIPDVTVSDSGLYRCYLQASAGENETFVMRLTVAEGKTDNQYTLFVATRFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:36);

[0207] Amino acid sequence of TIGIT fusion protein::MTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAIWNADLGWHISPSFKDRVAPGPGLDLTLQSLTVNDTGEYFCV YHTYPDGTYTGRIFLEVLESSVAEHGARFQIPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ IDNO:37);

[0208] The amino acid sequence of 28TM is: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:38);

[0209] The amino acid sequence of 28 is: RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:39);

[0210] The amino acid sequence of MSLN(RK-05-E06)-8h-8TM-BBZ-P2A-SIRP-28TM-28 is as follows:

[0211]

[0212]

[0213] The amino acid sequence of MSLN(RK-05-E06)-8h-8TM-BBZ-P2A-CD226-28TM-28 is as follows:

[0214]

[0215] The amino acid sequence of MSLN(RK-05-E06)-8h-8TM-BBZ-P2A-TIGIT-28TM-28 is as follows:

[0216]

[0217] The amino acid sequence of MSLN(RK-05-E06)-8h-28TM-28Z:

[0218]

[0219] The amino acid sequence of SLN(RK-05-E06)-G4h-28TM-28Z:

[0220]

[0221]

[0222] The amino acid sequence of MSLN(RK-05-E06)-7h-28TM-28BBZ:

[0223]

[0224] Amino acid sequence of G4h: ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLP SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:46);

[0225] The amino acid sequence of 7h is: APPRASALPAPPTGSALPDPQTASALPDPPAASALP (SEQ ID NO:47);

[0226] The amino acid sequence of RK-05-D10:

[0227]

[0228] The amino acid sequence 3 of RK-08-G03:

[0229]

[0230] The amino acid sequence of RK-05-E06

[0231]

[0232] Nucleotide sequence of the hypoxic promoter atcccacagtgcatacgtgggctccaacaggtcctcttgtcgagccacagtgcatacgtgggctccaacaggtcctcttgtcgagccacagtgcatacgtgggctccaacaggtcctcttgtcgagccacagtgc atacgtgggctccaacaggtcctcttgtcgagccacagtgcatacgtgggctccaacaggtcctcttgtcgagatctggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcactag(SEQ ID NO:51);

[0233] The amino acid sequence of RK-05-A09:

[0234]

[0235]

[0236] The amino acid sequence of RK-07-F11:

[0237]

[0238] The amino acid sequence of RK-05-C07:

[0239]

[0240] The amino acid sequence of RK-08-A05:

[0241]

[0242] The amino acid sequence of RK-05-G03 is as follows:

[0243]

[0244] The amino acid sequence of P2Z is: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:57);

[0245] The amino acid sequence of LCDR1 in RK-05-C07 is: QSISSY (SEQ ID NO:58);

[0246] The amino acid sequence of LCDR2 in RK-05-C07 is: AAS (SEQ ID NO:59);

[0247] The amino acid sequence of LCDR3 in RK-05-C07 is: QQSYSTPSWT (SEQ ID NO:60);

[0248] The amino acid sequence of HCDR1 in RK-05-C07 is: GYTFTSYD (SEQ ID NO:61);

[0249] The amino acid sequence of HCDR2 in RK-05-C07 is: MNPNSGNT (SEQ ID NO:62);

[0250] The amino acid sequence of HCDR3 in RK-05-C07 is: ARDGQWGAFDI (SEQ ID NO:63);

[0251] The amino acid sequence of VL of RK-05-C07 is EIVMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPSWTFGQGTKVDIKR (SEQ ID NO: 64);

[0252] The amino acid sequence of VH in RK-05-C07 is: EVQLVESGAEVKKPGASVKVSCKASGYTFTSYDIHWVRQATGQGLEWMGWMNPNSGN TGYARKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDGQWGAFDIWGQGTSLTVSS (SEQ ID NO:65);

[0253] The light chain amino acid sequence in CAR18 is: QPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQ GSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGT (SEQ ID NO: 66);

[0254] The heavy chain amino acid sequence in CAR18 is: QVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKW YNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSS (SEQ ID NO:67);

[0255] VL represents the light chain of ScFv, VH represents the heavy chain sequence of ScFv, CDR represents the complementarity-determining region of the antibody, and the light and heavy chains each have three subregions: CDR1, CDR2, and CDR3. The CDR region recognizes and binds to specific antigenic determinants by interacting with the antigen. Its sequence diversity determines the specificity and affinity of the antibody. Linker represents the linking peptides in the heavy and light chains.

[0256] Those skilled in the art should know that there are multiple ways to define a Complementary Determination Region (CDR). The LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 described in this application are defined by the IMGT system. LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 are collectively referred to as Complementary Determination Regions (CDRs). Besides the IMGT system definition, different CDR definition methods can also be combined (CDR1 and CDR2 are defined by one method, and CDR3 by another). Specific different CDR definition methods are shown in Table 1.

[0257] Table 1. Different CDR definition methods

[0258] Kabat Chothia MacCallum IMGT AHo VH CDR1 31-35 26-32 30-35 27-38 25-40 VH CDR2 50-65 53-55 47-58 56-65 58-77 VH CDR3 95-102 96-101 93-101 105-117 109-137 VL CDR1 24-34 26-32 30-36 27-38 25-40 VL CDR2 50-56 50-52 46-55 56-65 58-77 VL CDR3 89-97 91-96 89-96 105-117 109-137

[0259] in:

[0260] The amino acid numbering in the Kabat system definition follows the Kabat numbering system (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0261] The amino acid numbering in the Chothia system definition follows the Chothia numbering system (see, for example, Chothia and Lesk J.Mol.Biol.196:901-917(1987)).

[0262] The amino acid numbering in the MacCallum system definition follows the MacCallum numbering system (see MacCallumetal., J.Mol.Biol.262:732-745(1996); Abhinandan and Martin, Mol.Immunol.,45:3832-3839(2008)).

[0263] The amino acid numbering in the IMGT system definition follows the IMGT numbering system (see, for example, Lefranc MP. (2013) IMGT Unique Numbering. In: Dubitzky W., Wolkenhauer O., Cho KH., Yokota H. (eds) Encyclopedia of Systems Biology. Springer, New York, NY; https: / / doi.org / 10.1007 / 978-1-4419-9863-7_127).

[0264] The amino acid numbering in the AHo system definition (see, for example, Honegger and Plückthun, J.Mol.Biol., 309:657-670 (2001)) is based on the AHo numbering system.

[0265] Because CDRs have different definitions, those skilled in the art can define CDRs according to different methods used in LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of this application, thereby obtaining CDRs that are sequence-differentiated from the same combination of heavy chain variable regions and light chain variable regions as in this application. Those skilled in the art should understand that even if the sequences of CDR combinations obtained from the antibodies of this application through different definitions are different from those of LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of this application, antibodies containing such CDR combinations are still undoubtedly covered within the scope of this application.

[0266] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0267] Example 1: ScFv selection

[0268] 1. Construction of a fully human single-chain antibody library

[0269] PBMCs were isolated using Ficoll separation buffer, and total RNA was extracted and reverse transcribed into cDNA using conventional methods. Based on the similarity of the heavy and light chain germline gene sequences, degenerate primers were designed at both ends of the variable region, respectively. PCR was used to obtain the heavy chain and light chain variable region gene fragments of the antibody. The scFv nucleic acid fragment was amplified using conventional overlap PCR (PCR method referenced from *Molecular Cloning: A Laboratory Manual* (3rd edition), Joe Sambrook and David Russell, Science Press, USA). The scFv nucleic acid fragment was ligated into the phage vector pComb3xss, and the product was transformed into TGI strains using an electroporator to obtain a fully human single-chain antibody library.

[0270] 2. Preparation of a phage-display fully human single-chain antibody library

[0271] Add the bacterial culture to fresh SOC liquid medium with ampicillin at a concentration of 100 μg / mL, and resuscitate at 37°C and 250 rpm. Incubate until OD600 ≈ 0.5, then add VSCM13 helper phage at a VCSM13:bacteria ratio of 50:1, mix thoroughly, incubate for 30 min, and then continue incubation on a shaker. Centrifuge the culture and discard the supernatant. Resuspend the precipitate in SOB medium containing 50 μg / mL ampicillin and 50 μg / mL kanamycin, and incubate overnight at 30°C and 250 rpm. Centrifuge the bacterial culture at 4°C and 8000 rpm, collect the supernatant, add 1 / 5 volume of 20% PEG 8000 2.5 mmol / L NaCl solution, incubate on ice, then centrifuge at 4°C and 12000 rpm. Resuspend the precipitated phage in PBS, centrifuge at 4°C and 15000 rpm, and collect the supernatant.

[0272] 3. Antigen selection

[0273] MSLN protein with an Fc tag was co-incubated with proteinG magnetic beads to prepare MSLN-proteinG-coupled magnetic beads. The coupled magnetic beads were then extracted into the prepared phage display library. After three rounds of co-incubation, washing, and elution, specific monoclonal antibodies against the MSLN antigen were enriched.

[0274] 4. Antigen detection

[0275] After the initial screening, the final batch of single-clone plaques were selected for chemiluminescence detection and screening. Positive phages were then used to detect MSLN-positive HeLa cells. Several groups of positive phages were selected for protein expression, resulting in protein-expressing clones (RK-05-A09 (SEQ ID NO:52), RK-05-D10 (SEQ ID NO:48), RK-07-F11 (SEQ ID NO:53), RK-05-C07 (SEQ ID NO:54), RK-08-A05 (SEQ ID NO:55), RK-08-G03 (SEQ ID NO:49), RK-05-G03 (SEQ ID NO:56), and RK-05-E06 (SEQ ID NO:50)) which were then applied to downstream research.

[0276] The results are as follows Figure 1 As shown, A-22-1 and A-22-0 are positive controls for commercially available detection antibodies. The results indicate that RK-05-A09, RK-05-D10, RK-07-F11, RK-05-C07, RK-08-A05, RK-08-G03, RK-05-G03, and RK-05-E06 can all recognize the MSLN antigen expressed on HeLa cells.

[0277] Example 2: ScFv for MSLN identification and detection

[0278] 1. Expression and purification of ScFv

[0279] The plasmid of the positive phage clone was mixed with competent bacteria and incubated on ice for 5 min, followed by heat shock at 42°C for 90 s. After another ice incubation, the mixture was spread onto LB agar plates containing ampicillin and incubated overnight at 37°C. Single colonies were picked and incubated in LB medium at 37°C. When the OD of the bacterial culture reached 0.5–1.0, an inducer was added to induce expression. The bacterial cells were collected by centrifugation, and the precipitate was resuspended in PBS. After sonication and lysis buffer treatment, the precipitate was discarded, and the supernatant was collected. The target protein was obtained after filtration through 0.22 μm.

[0280] 2. HeLa is an MSLN-positive cell line. MSLN on the surface of HeLa cells was detected by flow cytometry using a commercially available antibody (R&D, FAB32652P). The results are as follows: Figure 2 As shown:

[0281] MSLN-positive HeLa cells were divided into 7 groups, and each group was aliquoted into 1.5 mL Eppendorf tubes, with 5 × 10 cells per tube. 5Cells were used as target cells. After centrifugation at 400g for 5 min, the supernatant was discarded. Cells were labeled with RK-08-A05, RK-05-C07, RK-05-E06, RK-07-F11, and RK-08-G03scFv solutions to a concentration of 30 μg / mL. 100 μL of each cell group was resuspended in the target cells and incubated at 4℃ for 1 h. The cells were then resuspended in 1 mL of PBS, centrifuged at 400g for 5 min, and the supernatant was discarded. 30 μL of Anti-His-647 fluorescent secondary antibody was added, and the cells were resuspended and incubated at 4℃ in the dark for 30 min. The cells were then resuspended in 1 mL of PBS, washed twice, centrifuged at 400g for 5 min, and the supernatant was discarded. The cells were then resuspended in 100 μL of PBS and transferred to detection tubes for analysis.

[0282] The positive rate and mean fluorescence intensity (MFI) of flow cytometry staining were detected, and the results are as follows: Figure 3 As shown in the figure; the results show that RK-05-C07 and RK-05-E06 can recognize MSLN expression in HeLa cervical cancer cells, indicating that RK-05-C07 and RK-05-E06 ScFv can be used as reagents or reagent combinations for detecting MSLN expression in detection kits, immunohistochemistry, flow cytometry and other techniques to detect MSLN expression.

[0283] In some embodiments, the antibodies described in this application can be labeled with biotin or chemical chromogenic agents, including but not limited to enzymes, biotinylate, fluorescein, chemiluminescence, isotopes, colloids, latex microspheres, and magnetic beads; the enzymes include but are not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-antiperoxidase bridges, alkaline phosphatase-antialkaline phosphatase bridges, and β-galactosidase-antiβ-galactosidase bridges; the biotinylate class includes but is not limited to biotin and its derivatives; the fluorescein class includes but is not limited to AF350, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, Oregon Green 488, Pacific Blue dye, Pacific Orange dye, and Texas... Red and PerCP dyes; the chemiluminescent types include, but are not limited to, isoluminol and its derivatives, acridine esters and their derivatives, ruthenium terpyridine and its derivatives, etc.; the isotope types include, but are not limited to, iodine labeling; the colloidal labels include, but are not limited to, colloidal gold, colloidal carbon, colloidal selenium, etc.

[0284] In some embodiments, the antibody described in this application can also be labeled, fixed, linked, or attached to a solid object. The fixed object can be a solid-phase carrier such as a well plate, magnetic beads, or nanomaterials. The linking can be direct or indirect coupling, such as linking to the solid object through covalent coupling.

[0285] Example 3: Constructing a CAR structure using ScFv for CAR-T

[0286] The selected RK-05-A09, RK-05-D10, RK-07-F11, RK-05-C07, RK-08-A05, RK-08-G03, RK-05-G03, and RK-05-E06 ScFv were introduced into a viral expression system containing a CD8 hinge transmembrane domain, a 4-1BBZ co-activation domain, and a CD3ζ signaling domain for CAR structure design, as shown in Table 2.

[0287] Table 2. Composition of CAR structures constructed by ScFv

[0288] name CAR structure / structural element sequence CAR1 MSLN(RK-05-A09)-8h-8TM-BBZ CAR2 MSLN(RK-05-D10)-8h-8TM-BBZ(SEQ ID NO:30) CAR3 MSLN(RK-07-F11)-8h-8TM-BBZ CAR4 MLSN(RK-05-C07)-8h-8TM-BBZ CAR5 MLSN(RK-08-A05)-8h-8TM-BBZ CAR6 MLSN(RK-08-G03)-8h-8TM-BBZ(SEQ ID NO:31) CAR7 MLSN(RK-05-G03)-8h-8TM-BBZ CAR8 MLSN(RK-05-E06)-8h-8TM-BBZ(SEQ ID NO:32)

[0289] 8h is a sequence derived from the hinge region of human CD8α; 8TM is a sequence derived from the transmembrane region of human CD8α; BB is derived from 4-1BB, also known as the intracellular region sequence of CD137; CD3ζ is sometimes written as CD3Z, which is the intracellular region sequence derived from human CD3ζ; BBZ is an abbreviation for the combination of BB and CD3ζ.

[0290] Lentiviral structures were constructed using lentiviral expression vectors to prepare lentiviruses. PBMCs (in some embodiments, these can be apheresis blood, cryopreserved and thawed PBMCs, or freshly isolated PBMCs) or CD3-positive T lymphocytes obtained from PBMCs (in some embodiments, these CD3-positive T lymphocytes can be αβ T cells, γδ T cells, or a mixed cell population) were conjugated with CD3 / CD28 antibodies using Dynabeads. TM CD3 / CD28, 40203D) activates T cells, and after activation, MSLN CAR-T cells (CAR-T cells are effector cells) are prepared by transducing lentiviral vectors targeting MSLN. The cells are cultured for 7-21 days and then harvested.

[0291] HeLa (MSLN+), SK-OV-3 (not detected by commercial antibody), and DLD-1 (MSLN-) were used as target cells. All of these cells stably expressed Luc-GFP through a lentiviral system. Multiple batches were randomly plated at a ratio of 2:1 between CAR-T effector cells and target cells. Control T or CT cells expressing CAR served as negative controls. The Medium group consisted of cells plated only with the corresponding target cells. After 24 hours of co-incubation, the Luc intensity of the Medium group with only target cells was used as the basis for calculation. The CAR-T cell killing rate was calculated by measuring the Luc intensity after co-incubation with effector cells. The specific conversion formula is: Cell killing rate (%) = (Luc value of Medium group - Luc value of experimental group) / Luc value of Medium group × 100%.

[0292] The killing effect of CAR-T cells expressing CAR structures constructed from screened ScFv on MSLN-positive HeLa cervical cancer cells is shown in Tables 3-5. Figures 4-6 As shown. Among them. Figures 4-6 CAR-T cells prepared from PBMCs from different donors Figure 4 For donor #152, Figure 5 For donor #125, Figure 6 For the supplier #948.

[0293] According to Tables 3-5 Figures 4-6 The results show that CAR-T cells prepared by constructing CAR structures using RK-05-D10, RK-08-G03, and RK-05-E06 can effectively kill MSLN-positive tumor cells, while other ScFv cells do not have this function.

[0294] Table 3. Comparison of cell killing rates of CAR1 to CAR3

[0295] lethality % Hela DLD-1 CAR1 50.44 32.14 CAR2 86.39 37.93 CAR3 45.35 34.54 Control T 38.31 22.14 Medium 0 0

[0296] Table 4. Comparison of cell killing rates of CAR4 to CAR6

[0297] lethality % Hela SK-OV-3 CAR4 66.06 60.12 CAR3 47.52 58.81 CAR5 44.25 57.71 CAR6 92.66 59.11 CT 42.37 58.19 Medium 0 0

[0298] Table 5. Comparison of cell killing rates between CAR7 and CAR8

[0299] lethality % Hela DLD-1 CAR7 37.56 32.26 CAR8 72.23 40.49 CT 40.39 36.11 Medium 0 0

[0300] Example 4: Applicability Verification of Different CAR Structures Based on ScFv Screening

[0301] A new CAR structure was designed for RK-05-D10 and RK-08-G03 according to Example 3, and its lethality was verified. The CAR structure is shown in Table 6:

[0302] Table 6. Applicability verification of different CAR structures constructed using the selected ScFv

[0303] name CAR structure / structural element sequence SIRPγ fusion protein SIRP-28TM-28 (SEQ ID NO:33) CAR9 MSLN(RK-05-D10)-8h-8TM-BBZ-P2A-SIRP-28TM-28(SEQ ID NO:34) CAR10 MSLN(RK-08-G03)-8h-8TM-BBZ-P2A-SIRP-28TM-28(SEQ ID NO:35)

[0304] The results of the damage are as follows Figure 7 As shown in the results, RK-05-D10 and RK-08-G03 can function in different CAR structures, and the expressed CAR-T cells have the ability to kill MSLN-positive tumor cells.

[0305] A new CAR structure was designed for RK-05-E06 according to Example 3, and its lethality was verified. The CAR structure is shown in Table 7 below:

[0306] Table 7. CAR structure designed for RK-05-E06

[0307]

[0308] 7h is a sequence derived from the hinge region of human CD7; G4h is a sequence derived from the hinge region of human IgG4; 28TM is a sequence derived from the transmembrane region of human CD28; 28 is an intracellular region sequence derived from human CD28; CD3ζ is sometimes written as CD3Z, which is an intracellular region sequence derived from human CD3ζ; 28Z is an abbreviation for the combination of 28 and CD3ζ; P2A is a cleavage peptide; 28BBZ is an abbreviation for the combination of 28, CD137, and CD3ζ.

[0309] The result of the killing is as follows Figure 8 As shown in A and B in the figure; the results show that RK-05-E06 can function in different CAR structures, and the expressed CAR-T cells have the ability to kill MSLN-positive tumor cells.

[0310] Example 5 Comparison of the effectiveness of CAR-T constructed using ScFv screened with existing ScFv

[0311] The efficacy of the CAR11 structure constructed using the screened RK-05-E06 will be compared with that of the best existing CAR structure, MSLN(P4)-8h-28TM-28Z (named CAR18, whose light chain amino acid sequence is shown in SEQ ID NO:66 and heavy chain amino acid sequence is shown in SEQ ID NO:67), through in vivo experiments.

[0312] Immunodeficient mice (NCG) aged 6-8 weeks were selected and intraperitoneally inoculated with HeLa (MSLN+-Luc-GFP) cells at a rate of 1.0E+06 cells / mouse to bear tumors. Seven days after tumor inoculation, mice were randomly assigned to groups for CAR-T infusion, with CAR-T cells (5E5 CAR+ cells / mouse) infused intraperitoneally. Small animal in vivo imaging and tumor measurement were used to regularly monitor changes in mouse fluorescence values ​​and the persistence of CAR-T cells in mouse blood, thereby conducting in vivo pharmacodynamic evaluation. Results are as follows: Figure 9 and Figure 10 As shown.

[0313] The results showed that the CAR-T cells constructed from the CAR11 structure of the RK-05-E06 we selected had an effective ability to clear MSLN-positive tumors and had good persistence in vivo.

[0314] MSLN is highly expressed in epithelial malignancies, especially ovarian cancer, pancreatic cancer, gastric cancer, and cervical cancer. We used an anti-MSLN antibody (Abcam, ab93620) as the primary antibody and Zhongshan Jinqiao's goat anti-rabbit IgG (205080712) as the secondary antibody to detect MSLN on tissue microarrays: HDgs-C140PT01-T-301, HRPS-C060PT01-T-129, and HDRS-C060PT01-T-100. The results are as follows. Figure 11 As shown.

[0315] The results showed that MSLN was highly expressed in rectal cancer, pancreatic cancer, endometrial cancer, uterine cancer, ovarian cancer, renal cancer, and bladder cancer. Based on the above analysis, the ScFv we screened can be used to detect MSLN expression in the above tumors, and RK-05-E06 can be used for drugs, drug combinations, or drug components, or the effective component composition of drugs such as ADC drugs, CAR-T drugs, CAR-NK drugs, and bispecific antibody drugs for solid tumors with high MSLN expression, such as rectal cancer, pancreatic cancer, endometrial cancer, uterine cancer, ovarian cancer, renal cancer, and bladder cancer.

[0316] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antibody recognizing MSLN, characterized in that The amino acid sequence of CDR1 of the heavy chain variable region is X L1 X L2 X L3 X L4 X L5 ; wherein X L1 is selected from D, G, T, or S; X L2 is selected from L, S, V or I; X L3 is selected from G or S; X L4 is selected from S or T; X L5 is selected from R or Y; The amino acid sequence of CDR2 of the heavy chain variable region is X L6 AS; wherein X L6 is selected from A or G; the amino acid sequence of CDR3 of the light chain variable region thereof is CCX L7 X L8 SX L9 PX L10 X L11 X L12 ; wherein X L7 is selected from L or S; X L8 is selected from N or Y; X L9 is selected from Y, T or null; X L10 is selected from L, I, F or S; X L11 is selected from T or W; X L11 is selected from null or T; The amino acid sequence of CDR1 of the heavy chain variable region is GX H1 TFX H2 SYX H3 wherein X H1 is selected from Y or F; X H2 is selected from T or S; X H2 is selected from Y, F, W or D; the amino acid sequence of CDR2 of the heavy chain variable region is X H4 X H5 X H6 X H7 X H8 GX H9 T, wherein X H4 is selected from I or M; X H5 is selected from N or S; X H6 is selected from P or S; X H7 is selected from S or N; X H8 is selected from G or S; X H9 is selected from S or N; The amino acid sequence of CDR3 of the heavy chain variable region is ARX H10 X H11 X H12 X H13 X H14 X H15 X H16 X H17 X H18 X H19 X H20 X H21 DX H22； X H10 is D, Y or null; X H11 is R or null; X H12 is P, R, D or null; X H13 is F, A or G; X H14 is S, A or Q; X H15 is S or W; X H16 is D or G; X H17 is Y, A or P; X H18 is Y or F; X H19 is Y or null; X H20 is G or null; X H21 is M or null; X H22 is V or I.

2. The antibody according to claim 1, characterized in that the variable region of the light chain comprises a CDR1 of the amino acid sequence as shown in SEQ ID NO: 3, a CDR2 of the amino acid sequence as shown in SEQ ID NO: 4 and a CDR3 of the amino acid sequence as shown in SEQ ID NO: 5; and / or the variable region of the light chain comprises a CDR1 of the amino acid sequence as shown in SEQ ID NO: 11, a CDR2 of the amino acid sequence as shown in SEQ ID NO: 12 and a CDR3 of the amino acid sequence as shown in SEQ ID NO: 13; and / or the variable region of the light chain comprises a CDR1 of the amino acid sequence as shown in SEQ ID NO: 19, a CDR2 of the amino acid sequence as shown in SEQ ID NO: 20 and a CDR3 of the amino acid sequence as shown in SEQ ID NO: 21; and / or the variable region of the light chain comprises a CDR1 of the amino acid sequence as shown in SEQ ID NO: 58, a CDR2 of the amino acid sequence as shown in SEQ ID NO: 59 and a CDR3 of the amino acid sequence as shown in SEQ ID NO: 60; the variable region of the heavy chain comprises a CDR1 of the amino acid sequence as shown in SEQ ID NO: 6, a CDR2 of the amino acid sequence as shown in SEQ ID NO: 7 and a CDR3 of the amino acid sequence as shown in SEQ ID NO: 8; and / or the variable region of the heavy chain comprises a CDR1 of the amino acid sequence as shown in SEQ ID NO: 14, a CDR2 of the amino acid sequence as shown in SEQ ID NO: 15 and a CDR3 of the amino acid sequence as shown in SEQ ID NO: 16; and / or the variable region of the heavy chain comprises a CDR1 of the amino acid sequence as shown in SEQ ID NO: 22, a CDR2 of the amino acid sequence as shown in SEQ ID NO: 23 and a CDR3 of the amino acid sequence as shown in SEQ ID NO: 24; and / or the variable region of the heavy chain comprises a CDR1 of the amino acid sequence as shown in SEQ ID NO: 61, a CDR2 of the amino acid sequence as shown in SEQ ID NO: 62 and a CDR3 of the amino acid sequence as shown in SEQ ID NO:

63.

3. The antibody according to claim 1 or 2, characterized in that the amino acid sequence of the variable region of the light chain is at least one of SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17 and / or SEQ ID NO: 64; the amino acid sequence of the variable region of the heavy chain is at least one of SEQ ID NO: 2, SEQ ID NO: 10 or SEQ ID NO: 18 and / or SEQ ID NO:

65.

4. Biomaterials characterized in that, at least one of the following A) to F): A) a nucleic acid encoding the antibody according to any one of claims 1 to 3; B) a recombinant vector comprising the nucleic acid according to A); C) a host cell transformed or transfected with the recombinant vector according to B); D) a cell expressing the antibody according to any one of claims 1 to 3; E) a solid object having the antibody according to any one of claims 1 to 3 labeled, immobilized, linked or attached, adhered; F) the antibody according to any one of claims 1 to 3, which is biotagged or chemically tagged; F) a culture obtained by culturing the host cell according to C).

5. Use of at least one of a) to b) in the manufacture of a MSLN detection product: a) the antibody according to any one of claims 1 to 3; b) the biological material according to claim 4.

6. A MSLN detection reagent and / or kit characterized by, A pharmaceutical composition comprising at least one of the antibody according to any one of claims 1 to 3 and / or the biological material according to claim 4 and an excipient.

7. A chimeric antigen receptor targeting MSLN positive cells, characterized in that, The antibody according to any one of claims 1 to 3 as an antigen recognition domain.

8. The chimeric antigen receptor of claim 7, wherein, Further comprising a hinge, a transmembrane region and an intracellular signal transduction region; The hinge is selected from at least one of CD8a, CD7 and / or IgG4; The transmembrane region is selected from at least one of CD8a and / or CD28; The intracellular signal transduction region is selected from at least one of CD137, CD3Z and / or CD28.

9. The chimeric antigen receptor of claim 8, wherein, The chimeric antigen receptor comprises at least one of the amino acid sequences according to SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 43, SEQ ID NO: 44 and / or SEQ ID NO:

45.

10. A nucleic acid encoding the chimeric antigen receptor according to any one of claims 7 to 9.

11. Vector, characterized in that, A pharmaceutical composition comprising the nucleic acid according to claim 10.

12. A virus, characterized in that, A pharmaceutical composition comprising the vector according to claim 11.

13. An immune cell, characterized in that, A pharmaceutical composition comprising the chimeric antigen receptor according to any one of claims 7 to 9 or the immune cell prepared by transducing the immune cell with the virus according to claim 12.

14. The immune cell of claim 13, wherein, The immune cell further comprises a fusion protein targeting one or more different targets selected from tumor antigens and / or immune checkpoint proteins.

15. Use of any one of I) to VII) in the manufacture of a medicament for preventing and treating a tumor: I) the antibody according to any one of claims 1 to 3; II) the biological material according to claim 4; III) the chimeric antigen receptor according to any one of claims 7 to 9; IV) the nucleic acid according to claim 10; V) the vector according to claim 11; VI) the virus according to claim 12; VII) the immune cell according to claim 13 or 14.

16. The use according to claim 15, characterized in that, The tumor comprises at least one of colorectal cancer, pancreatic cancer, endometrial cancer, uterine cancer, ovarian cancer, kidney cancer and / or bladder cancer.

17. A medicament for preventing and treating a tumor, characterized by, The raw material comprises at least one of i) to vii): i) the antibody according to any one of claims 1 to 3; ii) the biological material according to claim 4; iii) the chimeric antigen receptor according to any one of claims 7 to 9; iv) the nucleic acid according to claim 10; v) the vector according to claim 11; vi) the virus according to claim 12; vii) the immune cell according to claim 13 or 14.

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