Humanized Anti-MSLN antibody and use thereof

Abstract

The present invention provides a humanized anti-MSLN antibody and a use thereof. The humanized MSLN antibody of the present invention is modified to improve affinity, and the affinity thereof for binding to a human MSLN antigen is equal to or better than that of a commercially available humanized anti-MSLN antibody.

Description

A humanized anti-MSLN antibody and its application Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a humanized MSLN antibody and its applications. Background Technology

[0002] Mesothelin (MSLN) is a glycoprotein found on cell surfaces and in serum. Its gene encodes a 69 kDa precursor protein, which is enzymatically hydrolyzed during maturation into a membrane-bound protein of approximately 40 kDa, retaining its C-terminus. This mature mesothelin is the precursor. The approximately 30 kDa N-terminal fragment of the hydrolyzed protein is a fragment of megakaryocyte-promoting factor (MPF) that is detached and released extracellularly into blood and urine. Under normal circumstances, MSLN expression is limited to mesothelial cells (peritoneum, pericardium, and pleural cavity) at low levels, and is essentially absent in other tissues and cells. However, MSLN has been found to be significantly overexpressed in various tumor types, including mesothelioma, pancreatic cancer, non-small cell lung cancer, lung adenocarcinoma, fallopian tube cancer, head and neck cancer, cervical cancer, and ovarian cancer. Studies have shown that abnormal MSLN expression promotes tumor cell proliferation, invasion, and metastasis. The fact that MSLNs are not expressed or are expressed at low levels in normal cells gives them a potential advantage as a specific target for targeted cancer therapy. Clinical studies have shown that MSLN expression is correlated with tumor severity and survival. Summary of the Invention

[0003] In view of this, the present invention provides a humanized antibody or antigen-binding fragment comprising VH and VL, wherein;

[0004] VH includes one or more of the following:

[0005] The amino acid sequence of CDR-H1 is shown in SEQ ID NO:39: X1YTMN, where X1 is Y, G or N;

[0006] The amino acid sequence of CDR-H2 is shown in SEQ ID NO:4: LITPYNGASSYNQKFRG; and / or,

[0007] The amino acid sequence of CDR-H3 is shown in SEQ ID NO:6: GGYDGRGFDY;

[0008] And / or,

[0009] VL includes one or more of the following:

[0010] The amino acid sequence of CDR-L1 is shown in SEQ ID NO:40: SASSX2VSYMH, where X2 is S or Y;

[0011] The amino acid sequence of CDR-L2 is shown in SEQ ID NO:11: DTSKLAS; and / or,

[0012] The amino acid sequence of CDR-L3 is shown in SEQ ID NO:13: QQWSKHPLT.

[0013] Preferably, X1 is G.

[0014] Preferably, X2 is S.

[0015] The aforementioned antibody or antigen-binding fragment, which targets MSLN, is humanized and has more than 80% sequence homology with any one of the following amino acid sequences:

[0016] a. The VH region is selected from one of the following:

[0017] SEQ ID NO:21: EVQLVQSGAEVKKPGASVKVSCKASGYSFTYYTMNWVRQAPGQSLEWIGLITPYNGASSYNQKFRGRATLTVDKSASTAYMELSSLRSEDMAVYFCARGGYDGRGFDYWGQGTTVTVSS;

[0018] SEQ ID NO:23: EVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQSLEWIGLITPYNGASSYNQKFRGRATLTVDKSASTAYMELSSLRSEDMAVYFCARGGYDGRGFDYWGQGTTVTVSS;

[0019] SEQ ID NO:25: EVQLVQSGAEVKKPGASVKVSCKASGYSFTNYTMNWVRQAPGQSLEWIGLITPYNGASSYNQKFRGRATLTVDKSASTAYMELSSLRSEDMAVYFCARGGYDGRGFDYWGQGTTVTVSS;

[0020] b. The VL zone is selected from one of the following:

[0021] SEQ ID NO:22: DIELTQSPSAMSSVGDRVTITCSASSSVSYMHWYQQKPGTSPKRWIYDTSKLASGVPSRFSGSGSGNSYTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKVEIK;

[0022] SEQ ID NO:24: DIELTQSPSAMSSASVGDRVTITCSASSYVSYMHWYQQKPGTSPKRWIYDTSKLASGVPSRFSGSGSGNSYTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKVEIK;

[0023] SEQ ID NO:26: DIQLTQSPSSLSASPGDRVTITCSASSYVSYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGNDYTLTISSVQPEDFATYYCQQWSKHPLTFGGGTKVEIK.

[0024] The antibody or antigen-binding fragment of any of the above, wherein the VH includes one or more of the following:

[0025] The amino acid sequence of FR-H1 is shown in SEQ ID NO:1: EVQLVQSGAEVKKPGASVKVSCKASGYSFT;

[0026] The amino acid sequence of FR-H2 is shown in SEQ ID NO:3: WVRQAPGQSLEWIG;

[0027] The amino acid sequence of FR-H3 is shown in SEQ ID NO:5: RATLTVDKSASTAYMELSSLRSEDMAVYFCAR; and / or,

[0028] The amino acid sequence of FR-H4 is shown in SEQ ID NO:7: WGQGTTVTVSS.

[0029] The antibody or antigen-binding fragment described in any of the above-mentioned embodiments, wherein the VL comprises one or more of the following:

[0030] The amino acid sequence of FR-L1 is shown in SEQ ID NO:41: DIX3LTQSPSX4X5SASX6GDRVTITC, where X3 is E or Q, X4 is A or S, X5 is M or L, and X6 is V or P;

[0031] The amino acid sequence of FR-L2 is shown in SEQ ID NO:42: WYQQKPGX7X8PKRWIY, where X7 is T or K and X8 is S or A;

[0032] The amino acid sequence of FR-L3 is shown in SEQ ID NO:43: GVPSRFSGSGSGNX9YTLTISSX 10 QPEDFATYY, where X9 is S or D, X 10 It is L or V; and / or,

[0033] The amino acid sequence of FR-L4 is shown in SEQ ID NO:44: FGX 11 GTKVEIK, where X 11 It's either Q or G.

[0034] The present invention also provides an scFv targeting MSLN, comprising any of the above-mentioned antibody or antigen-binding fragments;

[0035] The VH and VL are connected by a flexible linker peptide.

[0036] More preferably, the flexible linker peptide is selected from (G4S)n linker peptide and linker peptide 1 with an amino acid sequence as shown in SEQ ID NO:33; wherein n=1 to 4.

[0037] Also provided is an isolated polynucleotide that encodes an antibody or antigen-binding fragment of any one of the above options or the above-described scFv.

[0038] This invention provides the use of antibodies or antigen-binding fragments, scFvs, or polynucleotides in the preparation of targeting vectors.

[0039] The present invention provides a targeting vector, the surface of which contains a recombinant targeting molecule, the recombinant targeting molecule containing a targeting binding region, the targeting binding region containing any one of the above-mentioned antibody or antigen-binding fragments or scFv.

[0040] Preferably, the vector is selected from one or more of lipid nanoparticles, virus-like particles, extracellular vesicles, adenovirus, adeno-associated virus, pseudolentiviral vectors, and retroviral vectors.

[0041] More preferably, the vector is a pseudolentiviral vector or a retroviral vector.

[0042] In some embodiments, the targeting carrier, the recombinant targeting molecule further includes a transmembrane region, the targeting binding region being directly or indirectly connected to the transmembrane region and displayed on the surface of the targeting carrier.

[0043] In some embodiments, the transmembrane region is selected from the transmembrane regions of the following proteins: CD2, CD3, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1.

[0044] More preferably, the transmembrane region is the transmembrane region of CD8α, and the amino acid sequence of the transmembrane region of CD8α has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with the amino acid sequence of SEQ ID NO:34.

[0045] In some embodiments, the recombinant targeting molecule further includes a linker domain, through which the targeting binding region is indirectly connected to the transmembrane region; the linker domain is selected from:

[0046] (a) Immunoglobulin hinge region, wherein the immunoglobulin hinge region is selected from wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA and IgD hinge regions;

[0047] (b) Hinge region, wherein the hinge region is selected from the wild-type or modified hinge regions of the following proteins: CD28, CD7, CD8, CD8α, CD8β, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154;

[0048] (c) All or part of the Fc domains, wherein the Fc domains are selected from one or more of the CH1, CH2, and CH3 domains; and

[0049] (d) Stem regions of type II C-lectins, wherein the type II C-lectins are selected from the stem regions of CD23, CD69, CD72, CD94, NKG2A and NKG2D.

[0050] More preferably, the connecting domain is the hinge region of CD8α, and the amino acid sequence of the hinge region of CD8α has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with the amino acid sequence of SEQ ID NO:35.

[0051] The present invention also provides the use of any of the above-mentioned antibodies or antigen-binding fragments, scFvs or polynucleotides in the preparation of chimeric antigen receptors.

[0052] This invention provides a chimeric antigen receptor comprising:

[0053] (a) Extracellular antigen-binding region;

[0054] (b) Transmembrane region; and

[0055] (c) Intracellular signal transduction domains;

[0056] The extracellular antigen-binding region contains any one of the above-mentioned antibodies or antigen-binding fragments or scFv.

[0057] In some embodiments, the transmembrane region is selected from the transmembrane regions of the following proteins: CD2, CD3, TCR, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, O. X40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcεRIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1.

[0058] Preferably, the transmembrane region is the transmembrane region of CD8α.

[0059] In some embodiments, the intracellular signal transduction domain is selected from the intracellular signal transduction domains of the following proteins: CD3ε, CD3γ, CD3δ, CD3ζ, CD79a, CD79b, FcεRIγ, FcεRβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP12, and other intracellular signal transduction domains of proteins whose intracellular signal transduction domains contain at least one ITAM.

[0060] Preferably, the intracellular signal transduction domain is the intracellular signal transduction domain of CD3ζ, and the amino acid sequence of the intracellular signal transduction domain of CD3ζ of the CAR has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:36.

[0061] In some embodiments, the chimeric antigen receptor further includes a hinge region connecting the extracellular antigen-binding region and the transmembrane region;

[0062] The hinge region is selected from the hinge regions of the following proteins: CD28, CD8, CD8α, CD8β, CD3, CD45, Ig4, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154.

[0063] More preferably, the hinge region is the hinge region of CD8α.

[0064] In some embodiments, the chimeric antigen receptor further includes a co-stimulatory signal transduction domain.

[0065] In some embodiments, the co-stimulatory signal transduction domain is selected from one or more of the co-stimulatory signal transduction domains of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, CD8α, CD8β, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcαRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40, and MyD88.

[0066] More preferably, the costimulatory signal transduction domain is selected from one or more of the costimulatory signal transduction domains of 4-1BB and CD28.

[0067] In some embodiments of the present invention, the amino acid sequence of the co-stimulatory signal transduction domain of the 4-1BB of the CAR has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:37.

[0068] In some embodiments of the present invention, the chimeric antigen receptor further comprises a signal peptide located at the N-terminus of the chimeric antigen receptor;

[0069] The signal peptide is selected from the signal peptides of the following proteins: HLA-A, CD8α, CD33, Igκ, IL-2, and GM-CSFRα.

[0070] More preferably, the signal peptide is a CD8α signal peptide. The amino acid sequence of the CD8α signal peptide has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:38.

[0071] In another aspect, the present invention also provides an isolated polynucleotide that encodes one of the chimeric antigen receptors described above.

[0072] In another aspect, the present invention also provides an engineered immune cell that expresses any of the aforementioned chimeric antigen receptors or contains the polynucleotide encoding any of the aforementioned chimeric antigen receptors.

[0073] Preferably, the immune cells are selected from one or more of T cells, NK cells, NK / T cells, neutrophils, monocytes, dendritic cells, and macrophages.

[0074] More preferably, the immune cell is a T cell.

[0075] A composition comprising an antibody or antigen-binding fragment, a vector, or an engineered immune cell, any one of the above options;

[0076] Preferably, the composition may further comprise a pharmaceutically acceptable excipient or carrier.

[0077] The application of the above composition in the preparation of anticancer drugs.

[0078] Preferably, the cancer is a solid tumor;

[0079] The solid cancers are selected from mesothelioma, pancreatic cancer, lung adenocarcinoma, fallopian tube cancer, head and neck cancer, cervical cancer, ovarian cancer, non-small cell lung cancer, and gastric cancer.

[0080] The present invention also provides a reagent for detecting MSLN protein or functional domains containing its antigenic determinants, said reagent comprising an antibody or antigen-binding fragment of any one of the above options;

[0081] Preferably, the reagent further comprises a modification portion linked to the antibody or antigen-binding fragment, the modification portion comprising a detectable label or therapeutic agent;

[0082] More preferably, the detectable marker includes one or more of enzymes, radionuclides, fluorescent dyes, luminescent substances, and biotin.

[0083] On the other hand, a method for detecting MSLN protein or functional domains containing its antigenic determinants for non-diagnostic purposes includes obtaining a sample suspected of containing MSLN protein or functional domains containing its antigenic determinants, contacting the sample with an antibody or antigen-binding fragment or the reagent described above to form an antibody-antigen complex, and detecting the presence of the antibody-antigen complex. Beneficial effects

[0084] Through extensive and in-depth research, the inventors of this invention unexpectedly obtained a high-affinity humanized MSLN antibody. Compared with widely used commercially available humanized antibodies such as MORAb-009, the humanized MSLN antibody provided by this invention has significantly higher affinity.

[0085] In this article:

[0086] As used in this invention, the term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies), biantibodies, single-chain variable fragments (scFv, also known as "single-chain antibodies"), single-domain antibodies (VHH or nanobodies), and antibody fragments, particularly antigen-binding fragments such as Fab', F(ab'), and Fv2. In some embodiments of this invention, the terms "immunoglobulin (Ig)" and "antibody" are used interchangeably.

[0087] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domains of the heavy and light chains are referred to as "VH" and "VL," respectively. These domains are typically the most variable parts of the antibody (relative to other antibodies of the same type) and contain antigen-binding sites.

[0088] A basic tetrameric antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). IgM antibodies consist of five basic heterotetrameric units and an additional polypeptide called the J chain, containing 10 antigen-binding sites; while IgA antibodies contain 2-5 basic tetrameric units, which can combine with the J chain to form multivalent assemblies. In the case of IgG, a tetrameric unit is typically about 150,000 Daltons. Each light chain is linked to the heavy chain by a covalent disulfide bond, while two heavy chains are linked to each other by one or more disulfide bonds, the number of which depends on the isoform of the heavy chains. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at its N-terminus, followed by three (CH1, CH2, and CH3 for each α and γ chain) and four (CH1, CH2, CH3, and CH4 for the μ and ε isoforms) constant domains (CH), and a hinge region located between the CH1 and CH2 domains. Each light chain has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at its other end. VL is aligned with VH, while CL is aligned with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are thought to form interfaces between the variable domains of the light and heavy chains. Pairs of VH and VL together form an antigen-binding site. For information on the structure and properties of different classes of antibodies, see Basic and Clinical Immunology, Eighth Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw, Appleton & Lange, Norwalk, CT. 1994, p. 71 and Chapter 6. Light chains from any vertebrate species can be classified into one of two distinct types, called κ and λ, based on the amino acid sequence of their constant domain. Immunoglobulins can be classified into different classes or isotypes based on the amino acid sequence of their heavy chain constant domain (CH). There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each with heavy chains called α, δ, ε, γ, and μ, respectively. Based on relatively minor differences in CH sequence and function, the γ and α classes can be further subdivided into subclasses; for example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0089] Without substantially affecting antibody activity, those skilled in the art can modify the sequence of the present invention by one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) to obtain variants of the antibody or its functional fragment sequence. These variants include, but are not limited to: deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) to the C-terminus and / or N-terminus. In the art, conservative substitution with amino acids of similar or comparable properties generally does not alter protein function. For example, substitution of amino acids with similar properties in the FR and / or CDR regions. Amino acid residues that can be conservatively substituted are well known in the art, and the amino acid residues substituted by the aforementioned methods may or may not be encoded by the genetic code. For example, adding one or more amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein. These are all considered to be included within the scope of protection of this invention.

[0090] "BLI technology": As a label-free optical biosensing technology, BLI is commonly used for real-time monitoring and analysis of biomolecular interactions (such as antigen-antibody interactions) and for quantifying their binding strength and kinetics. Unlike SPR technology, BLI does not use a metal chip. Instead, it utilizes a biosensor to couple ligands to the end of the sensor, which is then immersed in the sample to capture the analyte. BLI technology is based on the principle of optical interference. A thin, biocompatible fiber optic film is coated on the tip of the biosensor probe. When visible light passes through the fiber optic film, it is reflected from two surfaces within the film. The resulting two reflected beams interfere, forming a primary interference pattern. When ligands are coupled to the film through different forces, the film thickness changes, and new interference patterns are formed when visible light passes through. The shift in the new pattern compared to the previous one reflects the change in film thickness. At this point, the BLI instrument can be used to monitor the relative displacement changes in real time and convert them into system-specific parameters and spectra, including the binding constant (ka or kon) and dissociation constant (kd or koff), as well as the initial binding rate. Affinity (KD) and concentration information can be obtained through fitting calculation analysis.

[0091] "Sequence identity": Generally speaking, "sequence identity" or "sequence homology" refers to the exact correspondence between nucleotides or amino acids of two polynucleotide or polypeptide sequences. Typically, techniques for determining sequence identity involve identifying the nucleotide sequence of a polynucleotide and / or the amino acid sequence it encodes, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percentage of identity." Whether it's a nucleic acid or amino acid sequence, the percentage of identity between two sequences is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence, and then multiplied by 100. For example, the advanced BLAST computer program available from the National Institutes of Health can also be used to compare sequence information to determine the percentage of identity. The BLAST procedure is based on the following alignment methods: Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990) and discussed in Altschul et al., J. Mol. Biol. 215: 403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997). In short, the BLAST procedure defines identity as the number of identical alignment symbols (usually nucleotides or amino acids) divided by the total number of shorter symbols in both sequences. The procedure can be used to determine the percentage of identity over the entire length of the compared protein.

[0092] In some embodiments of the invention, the sequence of the “variant” used herein may have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with its source sequence. The sequence identity described herein can be measured using sequence analysis software, such as the computer program BLAST with default parameters, particularly BLASTP or TBLASTN. The invention also includes molecules having variable regions of antibody heavy chains with CDR regions, provided that their CDR regions have more than 90% (preferably more than 95%, most preferably more than 98%) homology with the CDR regions identified herein. The antibodies of the invention can be prepared using methods conventional in the art, such as single-chain antibodies, for example, phage display techniques well known in the art; or, the various antibodies of the invention can be expressed in other cell lines. Sequences encoding the various antibodies of the invention can be used to transform suitable mammalian host cells. Transformation can be performed using any known method, including packaging polynucleotides in a virus (or viral vector) and transducing host cells with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Host mammalian cell lines suitable for expression are well known in the art, such as various immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), and human hepatocellular carcinoma cells (e.g., HepG2). Particularly preferred cell lines are selected by identifying which cell lines have high expression levels and produce antibodies with basic CD79B binding properties.

[0093] “Specific binding”: As used herein, the term “specific binding” refers to binding that occurs between paired molecular species (e.g., receptor and ligand). When the interaction of two species produces a non-covalently bound complex, the binding that occurs is typically the result of electrostatic, hydrogen bonding, or lipophilic interactions. In various embodiments, specific binding between one or more species is direct. In some embodiments of the invention, the affinity of specific binding is about 2 times that of background binding, about 5 times that of background binding, about 10 times that of background binding, about 20 times that of background binding, about 50 times that of background binding, about 100 times that of background binding, or about 1000 times or more that of background binding.

[0094] As used in this invention, the term "chimeric antigen receptor (CAR)" includes an extracellular antigen-binding region comprising any of the humanized MSLN antibodies disclosed herein. In some embodiments of this invention, the CAR disclosed herein comprises: (a) an extracellular antigen-binding region; (b) a transmembrane region; and (c) an intracellular signal transduction domain; wherein the extracellular antigen-binding region comprises the humanized MSLN antibody disclosed herein.

[0095] This invention discloses a CAR comprising an extracellular antigen-binding region containing one or more humanized MSLN antibodies. The humanized MSLN antibodies may have the same or different origins and may have the same or different sizes. Any humanized MSLN antibody known in the art or disclosed in this invention, including the humanized MSLN antibody disclosed in this invention, can be used to construct the CAR described herein. The humanized MSLN antibody may be derived from any species, including but not limited to mice, rats, camels, llamas, lampreys, sharks, goats, rabbits, and cattle. The single-domain antibodies contemplated in this invention also include naturally occurring single-domain antibody molecules from species other than camelids and sharks.

[0096] In addition to the extracellular antigen-binding region disclosed in this invention, the chimeric antigen receptor disclosed in this invention may also include one or more of the following structures: linkers (e.g., peptide linkers, including flexible and rigid linkers), signal peptides, hinge regions, transmembrane domains, co-stimulatory signal transduction domains, and intracellular signal transduction domains. In some embodiments of this invention, different domains of the chimeric antigen receptor may also be fused together via peptide linkers. Depending on the structural and / or functional characteristics of the single-domain antibody and / or various domains, each peptide linker in the chimeric antigen receptor may have the same or different lengths and / or sequences. Those skilled in the art can independently select and optimize each peptide linker. In some embodiments of this invention, the peptide linkers contain flexible residues (e.g., glycine and serine) such that adjacent domains can move freely relative to each other. For example, a glycine-serine duplex may be a suitable peptide linker.

[0097] The peptide linker can have any suitable length. In some embodiments of the invention, the peptide linker is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more amino acid lengths.

[0098] In some embodiments of the present invention, the peptide linker is no more than 100, 90, 80, 75, 70, 60, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acid lengths. In some embodiments of the present invention, the peptide linker length is about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

[0099] Peptide linkers can have naturally occurring or non-natural sequences. For example, sequences derived from the hinge region of heavy-chain-only antibodies can be used as linkers. See, for example, WO1996 / 34103.

[0100] In some embodiments of the present invention, the peptide linker is a flexible linker. Exemplary flexible linkers include, but are not limited to, glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GSG)n, (GGGS)n, and (GGGGS)n / (G4S)n, where n is an integer at least 1, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.

[0101] Flexible linkers are typically used when the linked domains require a certain degree of movement or interaction (Chen X, Zaro JL, Shen WC., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct;65(10):1357-69.). Flexible linkers are usually composed of small, nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids (Argos P. An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J Mol Biol. 1990;211:943–958.). These small amino acids provide flexibility and also allow for the movement of the linked functional domains. For commonly used flexible linkers, see Chen X, Zaro JL, Shen WC., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct;65(10):1357-69, which is incorporated herein by reference in its entirety.

[0102] This invention also discloses nucleic acids encoding any of the aforementioned humanized MSLN antibodies or chimeric antigen receptors. The nucleic acids / polynucleotides of this invention can be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0103] “Nucleic acid” refers to any compound and / or substance, such as a polynucleotide, that comprises polymers containing nucleotides. Each nucleotide consists of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented as 5' to 3'. In this document, the term “nucleic acid” encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers containing mixtures of two or more of these molecules. “Nucleic acid” can be linear or circular. Furthermore, “nucleic acid” includes both sense strands (coding strands) and antisense strands (template strands), as well as single-stranded and double-stranded forms. Moreover, the “nucleic acid” described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derived sugar or phosphate backbones or chemically modified residues.

[0104] As is well known to those skilled in the art, due to the degeneracy of the genetic code, a vast quantity of nucleic acids can be produced, all of which encode the antibodies or chimeric antigen receptors of the present invention. Therefore, given the identification of specific amino acid sequences, those skilled in the art can produce any number of different nucleic acids by simply modifying the sequence of one or more codons without altering the amino acid sequence encoding the protein. Thus, the present invention also relates to polynucleotides that hybridize with the aforementioned polynucleotide sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that are hybridizable with the polynucleotides described in the present invention under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides.

[0105] The full-length nucleic acid sequences or fragments of various antibodies or chimeric antigen receptors of this invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequences artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0106] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, polypeptides, etc.) involved in this invention include biomolecules existing in isolated forms. Currently, the DNA sequence encoding the polypeptide (or fragment thereof, or derivative thereof) of this invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the polypeptide sequence of this invention through chemical synthesis.

[0107] The present invention also relates to nucleic acid constructs, such as expression vectors and recombinant vectors, comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins. Vectors typically contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. These sequences (collectively referred to in some embodiments as "flanking sequences") typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splicing sites, a leader sequence encoding a polypeptide secretion, a ribosome binding site, a polyadenylated sequence, a multi-linker region for inserting a nucleic acid encoding an antibody to be expressed, and optional marker elements.

[0108] The host cells involved in this invention can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include bacterial cells of Escherichia coli, Streptomyces, and Salmonella typhimurium; fungal cells of yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0109] In some embodiments of the present invention, the host cell can be various functional cells well known in the art, such as various cytotoxic cells, including but not limited to cytokine-induced killer (CIK) cells, dendritic cell-stimulated cytokine-induced killer (DC-CIK) cells, cytotoxic T lymphocytes (CTL), γδT21 cells, natural killer (NK) cells, tumor-infiltrating lymphocytes (TIL), lymphokine-activated killer (LAK) cells, CD3AK cells (cytotoxic cells with anti-CD3 monoclonal antibodies), and CAR-T / TCR-T cells. In some embodiments of the present invention, the cytotoxic cell is a T cell or an NK cell. Exemplary NK cells include, but are not limited to, primary NK cells, NK cell lines (such as NK92), and NKT cells. In some embodiments of the present invention, the NK cell is a primary NK cell. Exemplary T cells include, but are not limited to, T cells from mixed populations such as peripheral blood T lymphocytes, umbilical cord blood T lymphocytes, cytotoxic cytotoxic T cells (CTLs), helper T cells, suppressor / regulatory T cells, γδ T cells, cytokine-induced killer cells (CIKs), and tumor-infiltrating lymphocytes (TILs). In some embodiments, the T cells are peripheral blood T lymphocytes or umbilical cord blood T lymphocytes.

[0110] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. Furthermore, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0111] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media, such as serum-containing or serum-free media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0112] The peptides used in the above methods can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0113] This invention also discloses vectors for cloning and expressing any of the chimeric antigen receptors of this invention. In some embodiments of the invention, the vectors are adapted to replicate and integrate in eukaryotic cells, such as mammalian cells. In some embodiments of the invention, the vectors are viral vectors. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof. Viral vector technology is well known in the art and has been described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. (2001)) and other virology and molecular biology manuals.

[0114] Numerous virus-based systems have been developed in the prior art for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged into retroviral particles using techniques known in the art. Recombinant viruses can then be isolated in vitro or ex vivo and delivered to engineered mammalian cells. Many retroviral systems are known in the art. In some embodiments of the present invention, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments of the present invention, lentiviral vectors are used. In some embodiments of the present invention, self-inactivated lentiviral vectors are used. For example, self-inactivated lentiviral vectors carrying sequences encoding immunomodulators (e.g., immune checkpoint inhibitors) and / or self-inactivated lentiviral vectors carrying chimeric antigen receptors can be packaged using methods known in the art. Using methods known in the art, the resulting lentiviral vectors can be used to transduce mammalian cells (e.g., primary human T cells). Vectors derived from retroviruses (such as lentiviruses) are suitable tools for achieving long-term gene transfer because they allow for long-term, stable integration of transgenes and their proliferation in progeny cells. Lentiviral vectors also have the advantages of low immunogenicity and the ability to transduce non-proliferating cells.

[0115] In some embodiments of the present invention, the vector comprises any nucleic acid encoding the CAR described herein. The nucleic acid can be cloned into the vector using any molecular cloning method known in the art, including, for example, using restriction endonuclease sites and one or more selection markers. In some embodiments of the present invention, the nucleic acid is operatively linked to a promoter. A variety of promoters have been explored for gene expression in mammalian cells, and any promoter known in the art can be used in this invention. Promoters can be further classified as constitutive promoters or regulatory promoters, such as inducible promoters.

[0116] In some embodiments of the present invention, the nucleic acid encoding the CAR is operatively linked to a constitutive promoter. A constitutive promoter allows a heterologous gene (also known as a transgene) to be constitutively expressed in a host cell. Exemplary constitutive promoters contemplated by the present invention include, but are not limited to, the cytomegalovirus (CMV) promoter, the human elongation factor-1α (hEF1α) promoter, the ubiquitin C (UbiC) promoter, the glycerol phosphokinase (PGK) promoter, the simian virus 40 (SV40) early promoter, and the chicken β-actin-CMV early enhancer-coupled (CAGG) promoter. The efficiency of such constitutive promoters in driving transgene expression has been extensively compared in numerous studies. For example, Michael C. Milone et al. (Molecular Therapy, 17(8):1453-1464(2009)) compared the efficiency of CMV, hEF1α, UbiC, and PGK in driving chimeric antigen receptor expression in primary human T cells and concluded that the hEF1α promoter not only induces the highest levels of transgene expression but is also optimal in CD4+ and CD8+ human T cells. In some embodiments of the present invention, the nucleic acid encoding the CAR is operatively linked to the hEF1α promoter.

[0117] In some embodiments of the present invention, the nucleic acid encoding CAR is operatively linked to an inducible promoter. An inducible promoter is a type of regulatory promoter. An inducible promoter can be induced by one or more conditions, such as physical conditions, the microenvironment of engineered immune cells or the physiological state of engineered immune cells, or an inducer.

[0118] In some embodiments of the present invention, the vector further comprises a selectable marker gene or a reporter gene to select cells expressing CAR from a host cell population transfected via a lentiviral vector. Both the selectable marker and the reporter gene may have suitable regulatory sequences flanking them for expression in the host cells. For example, the vector may contain transcription and translation terminators, a start sequence, and a promoter for regulating nucleic acid sequence expression.

[0119] "MOI" stands for "Multiplicity of Infection" and refers to the number of viral particles added to each cell during the infection process. For example, when one million viral particles are added to one million cells, MOI = 1. Attached Figure Description

[0120] Figure 1: Antibody structure modeling;

[0121] Figure 2: A, B, C, D, and E are the affinity (Kd value) test results of the modified humanized anti-MSLN full-length antibodies 3D3, 3D10, 3E3, and 3G2 and the humanized full-length antibody MORAb-009 binding to the human MSLN antigen in Example 1, respectively.

[0122] Figure 3: CAR-MLSN plasmid;

[0123] Figure 4: Tumor cell killing experiment. Detailed Implementation

[0124] The present invention and its technical effects will be clearly and completely described below with reference to embodiments, so as to fully understand the technical solution, the technical problem solved, and the beneficial effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments; other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0125] Experimental methods not specifically described in the following examples were performed according to conventional methods and conditions known in the art, or as selected according to the product instructions. Reagents and raw materials not specifically described in this invention are all commercially available.

[0126] Example 1

[0127] Design and expression of human anti-MSLN monoclonal antibodies:

[0128] A. Obtaining the VH and VL region sequences of the humanized antibody.

[0129] Based on MORAb-009, a new humanized MSLN antibody was developed. Computer algorithms were used to simulate antigen-antibody binding and select saturation mutation sites to assist antibody development. As shown in the figure below, structural modeling of the antibody heavy and light chains highlights the importance of specific positions for antigen-binding affinity, yielding a series of humanized VH regions (huVH1, huVH2, and huVH3) and humanized VL regions (huVL1, huVL2, and huVL3) for the anti-MSLN antibody; the relevant sequences are shown below:

[0130] huVH1 region:

[0131] EVQLVQSGAEVKKPGASVKVSCKASGYSFTYYTMNWVRQAPGQSLEWIGLITPYNGASSYNQKFRGRATLTVDKSASTAYMELSSLRSEDMAVYFCARGGYDGRGFDYWGQGTTVTVSS (SEQ ID NO:21)

[0132] huVH2 region:

[0133] EVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQSLEWIGLITPYNGASSYNQKFRGRATLTVDKSASTAYMELSSLRSEDMAVYFCARGGYDGRGFDYWGQGTTVTVSS (SEQ ID NO:23)

[0134] huVH3 region:

[0135] EVQLVQSGAEVKKPGASVKVSCKASGYSFTNYTMNWVRQAPGQSLEWIGLITPYNGASSYNQKFRGRATLTVDKSASTAYMELSSLRSEDMAVYFCARGGYDGRGFDYWGQGTTVTVSS (SEQ ID NO:25)

[0136] huVL1 region:

[0137] DIELTQSPSAMSASVGDRVTITCSASSSVSYMHWYQQKPGTSPKRWIYDTSKLASGVPSRFSGSGSGNSYTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKVEIK (SEQ ID NO:22)

[0138] huVL2 region:

[0139] DIELTQSPSAMSASVGDRVTITCSASSYVSYMHWYQQKPGTSPKRWIYDTSKLASGVPSRFSGSGSGNSYTLTISSLQPEDFATYYCQQWSKHPLTFGQGTKVEIK (SEQ ID NO:24)

[0140] huVL3 region:

[0141] DIQLTQSPSSLSASPGDRVTITCSASSYVSYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGNDYTLTISSVQPEDFATYYCQQWSKHPLTFGGGTKVEIK (SEQ ID NO: 26)

[0142] According to the Kabat method, the CDR regions of the antibody are defined as follows: the CDR-H2 and CDR-H3 regions of the huVH1, huVH2, and huVH3 regions have the same sequence, while the CDR-H1 region has a different sequence; the CDR-L2 and CDR-L3 regions of the huVL1, huVL2, and huVL3 regions have the same sequence, while the CDR-L1 region has a different sequence.

[0143] The sequences of FR1, 2, 3, and 4 regions in huVH1, huVH2, and huVH3 are identical;

[0144] The sequences of FR1, 2, 3 and 4 in huVL1 and huVL2 are the same, while the sequences of FR1, 2, 3 and 4 in huVL3 are different from those in huVL1 and huVL2.

[0145] The specific amino acid sequences of each CDR and FR region are shown below:

[0146] huVH1 / 2 / 3-FR1:EVQLVQSGAEVKKPGASVKVSCKASGYSFT (SEQ ID NO:1)

[0147] huVH1-CDR-H1:YYTMN (SEQ ID NO:2)

[0148] huVH1 / 2 / 3-FR2:WVRQAPGQSLEWIG (SEQ ID NO:3)

[0149] huVH1 / 2 / 3-CDR-H2: LITPYNGASSYNQKFRG (SEQ ID NO:4)

[0150] huVH1 / 2 / 3-FR3:RATLTVDKSASTAYMELSSLRSEDMAVYFCAR (SEQ ID NO:5)

[0151] huVH1 / 2 / 3-CDR-H3:GGYDGRGFDY (SEQ ID NO:6)

[0152] huVH1 / 2 / 3-FR4: WGQGTTVTVSS (SEQ ID NO:7)

[0153] huVL1 / 2-FR1: DIELTQSPSAMSASVGDRVTITC (SEQ ID NO:8)

[0154] huVL1-CDR-L1: SASSSVSYMH (SEQ ID NO:9)

[0155] huVL1 / 2-FR2: WYQQKPGTSPKRWIY (SEQ ID NO:10)

[0156] huVL1 / 2 / 3-CDR-L2: DTSKLAS (SEQ ID NO:11)

[0157] huVL1 / 2-FR3: GVPSRFSGSGSGNSYTLTISSLQPEDFATYYC (SEQ ID NO:12)

[0158] huVL1 / 2 / 3-CDR-L3: QQWSKHPLT (SEQ ID NO:13)

[0159] huVL1 / 2-FR4: FGQGTKVEIK (SEQ ID NO:14)

[0160] huVH2-CDR-H1: GYTMN (SEQ ID NO:15)

[0161] huVL2 / 3-CDR-L1: SASSYVSYMH (SEQ ID NO:16)

[0162] huVH3-CDR-H1: NYTMN (SEQ ID NO:17)

[0163] huVL3-FR1: DIQLTQSPSSLSASPGDRVTITC (SEQ ID NO:18)

[0164] huVL3-FR2: WYQQKPGKAPKRWIY (SEQ ID NO:19)

[0165] huVL3-FR3: GVPSRFSGSGSGNDYTLTISSVQPEDFATYYC (SEQ ID NO:20)

[0166] huVL3-FR4: FGGGTKVEIK (SEQ ID NO:27)

[0167] Human MSLN antigen (C-his):

[0168] EVEKTACPSGKKAREIDESLIFYKKWELEEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRGQLDKDTLDTLT AFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDHHHHHH (SEQ ID NO:28)

[0169] B. Constructing full-length antibodies

[0170] Humanized full-length anti-MSLN antibodies were constructed by fusing the obtained huVH1, huVH2, and huVH3 regions with the constant region of the human IgG1 heavy chain, and fusing the huVL1, huVL2, and huVL3 regions with the constant region of the human kappa light chain, respectively, to obtain humanized full-length anti-MSLN antibodies 3D3 (containing the huVH1 and huVL1 regions), 3D10 (containing the huVH2 and huVL1 regions), 3E3 (containing the huVH1 and huVL2 regions), and 3G2 (containing the huVH3 and huVL3 regions), respectively. The commonly used humanized full-length anti-MSLN antibody MORAb-009 was selected, and its VH and VL regions were fused with the constant regions of the human IgG1 heavy chain and the human kappa light chain, respectively. The relevant sequences are shown below:

[0171] The heavy chain constant region of human IgG1:

[0172] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:29)

[0173] Human kappa light chain constant region:

[0174] VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:30)

[0175] MORAb - 009 / 4F3F - VH region:

[0176] QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS (SEQ ID NO:31)

[0177] MORAb - 009 / 4F3F - VL region:

[0178] DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK (SEQ ID NO:32)

[0179] C. Full - length antibody affinity detection

[0180] Using BLI (Biolayer Interferometry) technology and Octet® Anti-HIS (HIS2) Biosensors (Satorius, #18-5114), the affinity parameters (Kd values) of the humanized anti-MSLN full-length antibodies 3D3, 3D10, 3E3, 3G2, and MORAb-009 for binding to the human MSLN antigen were detected. The results are shown in Figures 1A, 1B, 1C, 1D, and 1E, respectively.

[0181] The Kd, ka, and kdis values ​​of each full-length antibody binding to the human MSLN antigen are shown in Table 1 below:

[0182] Table 1

[0183] Antibody ID KD(M)Ka(1 / Ms)Kdis(1 / s)MORAb-0095.86×10 -10 4.31×10 5 2.53×10 -4 3D3(huVH1-huVL1) 5.86×10 -10 4.29×10 5 2.51×10 -4 3D10(huVH2-huVL1) 3.76×10 -10 5.49×10 5 2.06×10 -4 3G2(huVH3-huVL3)4.87×10 -10 5.21×10 5 2.53×10 -4 3E3(huVH1-huVL2)5.74×10 -10 4.69×10 5 2.70×10 -4

[0184] The calculated affinity of the humanized full-length anti-MSLN antibody 3D3 is 5.86 × 10⁻⁶. -10 The affinity of M;3D10 is 3.76×10. -10 The affinity of M;3E3 is 5.74 × 10⁻⁶. -10 The affinity of M;3G2 is 4.87 × 10⁻⁶. -10 M; while the affinity of MORAb-009 is 5.86 × 10⁻⁶. -10M. It can be seen that the humanized anti-MSLN full-length antibody 3D3 provided by the present invention has a similar affinity to the commercially available antibody MORAb-009 commonly used in the field; moreover, the humanized anti-MSLN full-length antibodies 3D10, 3E3 and 3G2 provided by the present invention have significantly higher affinity than MORAb-009.

[0185] Example 2

[0186] Preparation of lentiviral vectors

[0187] A. Constructing the envelope plasmid:

[0188] Using molecular cloning techniques, a lentiviral envelope plasmid was constructed based on the pMD2.G plasmid as the backbone, which included: wild-type VSV-G; membrane-type expression of anti-CD3 antibody: the structure from the N-terminus to the C-terminus is as follows: CD8α signal peptide, anti-CD3 antibody (scFv derived from the anti-CD3 monoclonal antibody UCHT1, scFv-UCHT1), CD8α hinge region, and CD8α transmembrane region.

[0189] Among them, the amino acid sequence of CD3 antibody (scFv) UCHT1 is as SEQ ID NO:45:DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKGGGGSGGGGSGGGGS GGGGSEVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS;

[0190] B. Constructing CAR plasmids:

[0191] A chimeric antigen receptor targeting MSLN was constructed. The structure of CAR-MSLN from the N-terminus to the C-terminus is as follows: CD8α signal peptide, anti-MSLN, CD8α hinge region, CD8α transmembrane region, 4-1BB co-stimulatory signal transduction domain and CD3ζ intracellular signal transduction domain (plasmid map shown in Figure 3 below).

[0192] C. Packaging of lentiviruses:

[0193] Add 9 μg of CAR plasmid, 4 μg of pMDLg / pRRE packaging plasmid, 2 μg of pRSV-REV packaging plasmid, and 2 μg of envelope plasmid to 1 mL of Opti-MEM medium. After shaking well, add 64 μL of PEI reagent, mix well by pipetting, and let stand for 10 minutes. Then add to the culture medium of HEK-293T cells. Replace the medium after 6 hours. Collect the supernatant of the medium 48 hours after transfection, filter it through a 0.45 μm filter membrane, centrifuge at 50,000 g for 2.5 h, discard the supernatant, resuspend the obtained lentiviral vector in 200 μL of F12 medium, and freeze at -80℃.

[0194] Example 3

[0195] Tumor killing experiment

[0196] Day 0: Resuscitate 5 groups of 1×10⁻⁶ PBMCs from cryopreserved inactive PBMCs of Donor 1 (healthy person). 5 Individual inactive PBMCs were prepared by mixing each group of individual inactive PBMCs with target cells (PANC-1-Luci+ breast cancer cells) at ratios of 0:1, 1:1, 1:2, 1:5, and 1:10, respectively. The mixed cells were resuspended in 200 μL of 1640 medium containing 10% FBS, and IL-7 and IL-15 were added (only on Day 0, not subsequently).

[0197] On Day 0, the lentivirus prepared in Example 1 was added to the mixed cells according to MOI=1 and co-incubated under the culture conditions of 37°C and 5% CO2.

[0198] On Day 5, chemiluminescence was used to detect the fluorescence values ​​in the mixed cells of each group, and the killing efficiency was calculated as: = (control group fluorescence - experimental group fluorescence) / control group fluorescence * 100%. The results are shown in Figure 4.

Claims

1. A humanized antibody or antigen-binding fragment, characterized in that, Including VH and VL, of which; VH includes one or more of the following: The amino acid sequence of CDR-H1 is shown in SEQ ID NO:39: X1YTMN, where X1 is Y, G or N; The amino acid sequence of CDR-H2 is shown in SEQ ID NO:4: LITPYNGASSYNQKFRG; and / or, The amino acid sequence of CDR-H3 is shown in SEQ ID NO:6: GGYDGRGFDY; And / or, VL includes one or more of the following: The amino acid sequence of CDR-L1 is shown in SEQ ID NO:40: SASSX2VSYMH, where X2 is S or Y; The amino acid sequence of CDR-L2 is shown in SEQ ID NO:11: DTSKLAS; and / or, The amino acid sequence of CDR-L3 is shown in SEQ ID NO:13: QQWSKHPLT.

2. The antibody or antigen-binding fragment as described in claim 1, characterized in that, X1 is G.

3. The antibody or antigen-binding fragment as described in claim 1, characterized in that, X2 is S.

4. The antibody or antigen-binding fragment according to any one of claims 1 to 3, characterized in that... , The antibody or antigen-binding fragment targets MSLN, is humanized, and has more than 80% sequence homology with any one of the following amino acid sequences: The VH region is selected from one of the following: SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; and / or, The VL region is selected from one of the following: SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:

26.

5. The antibody or antigen-binding fragment according to any one of claims 1 to 3, characterized in that, The VH includes one or more of the following: The amino acid sequence of FR-H1 is shown in SEQ ID NO:1; the amino acid sequence of FR-H2 is shown in SEQ ID NO:3; the amino acid sequence of FR-H3 is shown in SEQ ID NO:5; and / or, the amino acid sequence of FR-H4 is shown in SEQ ID NO:

7.

6. The antibody or antigen-binding fragment according to any one of claims 1 to 3, characterized in that, The VL includes one or more of the following: The amino acid sequence of FR-L1 is shown in SEQ ID NO:41: DIX3LTQSPSX4X5SASX6GDRVTITC, where X3 is E or Q, X4 is A or S, X5 is M or L, and X6 is V or P; The amino acid sequence of FR-L2 is shown in SEQ ID NO:42: WYQQKPGX7X8PKRWIY, where X7 is T or K and X8 is S or A; The amino acid sequence of FR-L3 is shown in SEQ ID NO:43: GVPSRFSGSGSGNX9YTLTISSX 10 QPEDFATYY, where X9 is S or D, X 10 It is L or V; and / or, The amino acid sequence of FR-L4 is shown in SEQ ID NO:44: FGX 11 GTKVEIK, where X 11 It's either Q or G.

7. A scFv targeting MSLN, characterized in that, Includes an antibody or antigen-binding fragment as described in any one of claims 1 to 6; The VH and VL are connected by a flexible linker peptide.

8. An isolated polynucleotide, characterized in that, The polynucleotide encodes an antibody or antigen-binding fragment of any one of claims 1 to 6 or the scFv of claim 7.

9. The use of an antibody or antigen-binding fragment of any one of claims 1 to 6, the scFv of claim 7, or the polynucleotide of claim 8 in the preparation of a targeting vector.

10. A targeting vector, characterized in that, The surface of the targeting vector contains a recombinant targeting molecule, the recombinant targeting molecule containing a targeting binding region, the targeting binding region containing an antibody or antigen binding fragment of any one of claims 1 to 6 or the scFv of claim 7.

11. The targeting vector as described in claim 10, characterized in that, The vector is selected from one or more of lipid nanoparticles, virus-like particles, extracellular vesicles, adenovirus, adeno-associated virus, pseudolentiviral vectors, and retroviral vectors.

12. The targeting carrier according to claim 11, characterized in that, The vectors are pseudolentiviral vectors and retroviral vectors.

13. The targeting vector according to any one of claims 10 to 12, characterized in that, The recombinant targeting molecule further includes a transmembrane region, and the targeting binding region is directly or indirectly connected to the transmembrane region and is displayed on the surface of the targeting carrier.

14. The targeting vector as described in claim 13, characterized in that, The transmembrane region is selected from the transmembrane regions of the following proteins: CD2, CD3, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1.

15. The targeting carrier according to claim 13, characterized in that, The recombinant targeting molecule further includes a linker domain, through which the targeting binding region is indirectly connected to the transmembrane region; the linker domain is selected from: (a) Immunoglobulin hinge region, wherein the immunoglobulin hinge region is selected from wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA and IgD hinge regions; (b) Hinge region, wherein the hinge region is selected from the wild-type or modified hinge regions of the following proteins: CD28, CD7, CD8, CD8α, CD8β, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154; (c) All or part of the Fc domains, wherein the Fc domains are selected from one or more of the CH1, CH2, and CH3 domains; and (d) Stem regions of type II C-lectins, wherein the type II C-lectins are selected from the stem regions of CD23, CD69, CD72, CD94, NKG2A and NKG2D.

16. The use of an antibody or antigen-binding fragment of any one of claims 1 to 6, the scFv of claim 7, or the polynucleotide of claim 8 in the preparation of a chimeric antigen receptor.

17. A chimeric antigen receptor, characterized in that, Include: (a) Extracellular antigen-binding region; (b) Transmembrane region; and (c) Intracellular signal transduction domains; The extracellular antigen-binding region comprises an antibody or antigen-binding fragment of any one of claims 1 to 6 or the scFv of claim 7.

18. The chimeric antigen receptor as described in claim 17, characterized in that, The transmembrane region is selected from the transmembrane regions of the following proteins: CD2, CD3, TCR, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcεRIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1.

19. The chimeric antigen receptor as described in claim 17, characterized in that, The intracellular signal transduction domain is selected from the intracellular signal transduction domains of the following proteins: CD3ε, CD3γ, CD3δ, CD3ζ, CD79a, CD79b, FcεRIγ, FcεRβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP12, and other intracellular signal transduction domains of proteins containing at least one ITAM.

20. The chimeric antigen receptor according to any one of claims 17 to 19, characterized in that, The chimeric antigen receptor further includes a hinge region connecting the extracellular antigen-binding region and the transmembrane region; The hinge region is selected from the hinge regions of the following proteins: CD28, CD8, CD8α, CD8β, CD3, CD45, Ig4, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154.

21. The chimeric antigen receptor according to claim 20, characterized in that, The chimeric antigen receptor also includes a co-stimulatory signal transduction domain.

22. The chimeric antigen receptor according to claim 21, characterized in that, The co-stimulatory signal transduction domain is selected from one or more of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, CD8α, CD8β, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcαRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40, and MyD88.

23. The chimeric antigen receptor according to claim 22, characterized in that, The chimeric antigen receptor further includes a signal peptide located at the N-terminus of the chimeric antigen receptor; The signal peptide is selected from the signal peptides of the following proteins: HLA-A, CD8α, CD33, Igκ, IL-2, and GM-CSFRα.

24. An isolated polynucleotide, characterized in that, The polynucleotide encodes a chimeric antigen receptor according to any one of claims 17 to 23.

25. The use of an antibody or antigen-binding fragment of any one of claims 1 to 6, the scFv of claim 7, the polynucleotide of claim 8 or 24, the targeting vector of any one of claims 9 to 15, or the chimeric antigen receptor of any one of claims 17 to 23 in the preparation of engineered immune cells.

26. An engineered immune cell, characterized in that, The engineered immune cells express a chimeric antigen receptor of any one of claims 17 to 23 or comprise the polynucleotide of claim 24.

27. The engineered immune cells as described in claim 26, characterized in that, The immune cells are selected from one or more of T cells, NK cells, NK / T cells, neutrophils, monocytes, dendritic cells, and macrophages.

28. A composition, characterized in that, The composition comprises an antibody or antigen-binding fragment of any one of claims 1 to 6, a vector of any one of claims 10 to 15, or the engineered immune cell of claim 26; Preferably, the composition may further comprise a pharmaceutically acceptable excipient or carrier.

29. Use of the composition of claim 28 in the preparation of an anticancer drug.

30. The application as described in claim 29, characterized in that, The cancer in question is a solid tumor; The solid cancers are selected from mesothelioma, pancreatic cancer, lung adenocarcinoma, fallopian tube cancer, head and neck cancer, cervical cancer, ovarian cancer, non-small cell lung cancer, and gastric cancer.

31. A reagent for detecting MSLN protein or functional domains containing its antigenic determinants, characterized in that, The reagent comprises an antibody or antigen-binding fragment according to any one of claims 1 to 6; Preferably, the reagent further comprises a modification portion linked to the antibody or antigen-binding fragment, the modification portion comprising a detectable label or therapeutic agent; More preferably, the detectable marker includes one or more of enzymes, radionuclides, fluorescent dyes, luminescent substances, and biotin.

32. A method for detecting MSLN protein or functional domains containing its antigenic determinants for non-diagnostic purposes, characterized in that, The method includes obtaining a sample suspected of containing MSLN protein or containing its antigenic determinant functional domain, contacting the sample with an antibody or antigen-binding fragment of any one of claims 1 to 6 or a reagent of claim 31 to form an antibody-antigen complex, and detecting the presence of the antibody-antigen complex.

Images