Antibody numbering	VH	HCDR1	HCDR2	HCDR3	VL	LCDR1	LCDR2	LCDR3
									MES-02-2C9-1A11	1	2	3	4	5	6	7	8
MES-02-4C4-1A7/1E3	11	12	3	13	14	15	7	16
									MES-02-4D5-1E6/1A3	19	20	21	22	23	24	7	25
MES-10-4G8-1A11/1E5	28	29	30	31	32	33	34	35

Note that: each numerical value in the table indicates a sequence number, i.e. "1" indicates "SEQ ID NO:1", and the sequence numbers of VH, HCDR1, HCDR2, HCDR3, VL, LCDR1, LCDR2, LCDR3 shown in the table are the numbers of the amino acid sequences thereof.

Preferably, the MES-02-2C9-1A11 antibody of the present invention has a heavy chain sequence as shown in SEQ ID NO. 1 and a light chain sequence as shown in SEQ ID NO. 5; the MES-02-4C4-1A7/1E3 antibody has a heavy chain sequence shown as SEQ ID NO. 11 and a light chain sequence shown as SEQ ID NO. 14; the MES-02-4D5-1E6/1A3 antibody has a heavy chain sequence shown in SEQ ID NO. 19 and a light chain sequence shown in SEQ ID NO. 23; the MES-10-4G8-1A11/1E5 antibody has a heavy chain sequence shown as SEQ ID NO. 28 and a light chain sequence shown as SEQ ID NO. 32.

In another preferred embodiment, the antibody is a murine or human chimeric monoclonal antibody against MSLN protein, and its heavy chain constant region and/or light chain constant region may be a humanized heavy chain constant region or light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is a heavy chain constant region or light chain constant region of human IgG1, igG2, or the like.

The invention also provides other proteins or fusion expression products having the antibodies of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain and a light chain comprising a variable region, provided that the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the variable regions of the heavy chain and light chain of the antibodies of the invention.

In general, the antigen binding properties of antibodies can be described by 3 specific regions located in the heavy and light chain variable regions, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, the β -sheets formed by the FR therebetween are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody.

The variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest because they are involved, at least in part, in binding to an antigen. Thus, the invention includes those molecules having monoclonal antibody light and heavy chain variable regions with CDRs, so long as the CDRs are 90% or more (preferably 95% or more, most preferably 98% or more) homologous to the CDRs identified herein.

The invention includes not only intact monoclonal antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.

As used herein, the term "heavy chain variable region" is used interchangeably with "VH".

As used herein, the term "variable region" is used interchangeably with "complementarity determining region (complementarity determining region, CDR)".

As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.

The antibody of the present invention refers to a polypeptide having MSLN protein binding activity and comprising the CDR regions described above. The term also includes variants of polypeptides comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.

The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes under high or low stringency conditions with the encoding DNA of an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.

The invention also provides other polypeptides, such as fusion proteins comprising a human antibody or fragment thereof. In addition to nearly full length polypeptides, the invention also includes fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the antibody of the invention.

In the present invention, a "conservative variant of an antibody of the present invention" refers to a polypeptide in which at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids of similar or similar nature, as compared to the amino acid sequence of the antibody of the present invention. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table 2.

TABLE 2

The invention also provides polynucleotide molecules encoding the antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The coding region sequences encoding the mature polypeptide may be identical to or degenerate variants of the coding region sequences set forth in SEQ ID NOs 9-10, 17-18, 26-27, 36-37, 39, 41, 43, 45, 47, 49, 51, 53 and 55-58. As used herein, "degenerate variant" refers in the present invention to a nucleotide sequence encoding a polypeptide having the same amino acid sequence as the polypeptide of the present invention, but differing from the coding region sequences set forth in SEQ ID NOS 9-10, 17-18, 26-27, 36-37, 39, 41, 43, 45, 47, 49, 51, 53 and 55-58.

Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; a coding sequence for a mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) of the mature polypeptide, and non-coding sequences.

The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding the polypeptide, or may include additional coding and/or non-coding sequences.

The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42℃and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NOs 1-8, 11-16, 19-25, 28-35, 38, 40, 42, 44, 46, 48, 50, 52 and 54.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleotides, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form.

At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; animal cells of CHO, COS7, 293 cells, and the like.

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, which are capable of absorbing DNA, can be obtained after an exponential growth phase and treated by the CaCl₂ method using procedures well known in the art. Another approach is to use MgCl₂. Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.

The antibodies of the invention may be used alone or in combination or coupling with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.

Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or enzymes capable of producing a detectable product.

Therapeutic agents that may be conjugated or coupled to an antibody of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, cancer metastasis reviews (CANCER METASTASIS REVIEWS) 24, 539); 2. biotoxicity (Chaudhary et al, 1989, nature 339, 394; epel et al, 2002, cancer immunology and immunotherapy (Cancer Immunology and Immunotherapy) 51, 565); 3. cytokines such as IL-2 et al (Gillies et al, 1992, proc. Natl. Acad. Sci. USA (PNAS) 89, 1428; card et al, 2004, cancer immunology and immunotherapy (Cancer Immunology and Immunotherapy) 53, 345; halin et al, 2003, cancer research (CANCER RESEARCH) 63, 3202); 4. gold nanoparticles/nanorods (Lapotko et al, 2005, cancer communication (CANCER LETTERS) 239, 36; huang et al, 2006, journal of the american Society of chemistry (Journal of THE AMERICAN CHEMICAL Society) 128, 2115); 5. viral particles (Peng et al, 2004, gene therapy (GENE THERAPY) 11, 1234); 6. liposomes (Mamot et al, 2005, cancer research (CANCER RESEARCH) 65, 11631); 7. nano magnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticle, and the like.

Antibodies to

As used herein, the term "antibody" refers to an immunoglobulin that is a tetrapeptide chain structure formed from two identical heavy chains and two identical light chains joined by an interchain disulfide bond. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be assigned to five classes, or different types of immunoglobulins, i.e., igM, igD, igG, igA and IgE, and the heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. IgG represents the most important class of immunoglobulins, which can be divided into 4 subclasses again due to differences in chemical structure and biological function: igG1, igG2, igG3 and IgG4. Light chains are classified as either kappa or lambda chains by the difference in constant regions. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

The sequences of the heavy and light chains of the antibody near the N-terminus vary widely, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable region includes 3 hypervariable regions (HVRs) and 4 FR Regions (FR) that are relatively conserved in sequence. The amino acid sequences of the 4 FRs are relatively conserved and do not directly participate in the binding reaction. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) consists of 3 CDR regions and 4 FR regions, arranged in sequence from amino-to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain, namely the light chain hypervariable region (LCDR), refer to LCDR1, LCDR2 and LCDR3; the 3 CDR regions of the heavy chain, namely heavy chain hypervariable regions (HCDR), refer to HCDR1, HCDR2 and HCDR3. The number and positions of CDR amino acid residues in the LCVR and HCVR regions of an antibody or antigen binding fragment according to the invention are in accordance with the known Kabat numbering convention or in accordance with the Chothia and IMGT numbering convention. The four FR regions in the natural heavy and light chain variable regions are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and form together with the CDRs of the other chain an antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody. The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.

As used herein, the term "antigen binding fragment" refers to a Fab fragment, fab 'fragment, F (ab')₂ fragment, or a single Fv fragment having antigen binding activity. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and have a minimal antibody fragment of the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structures required for antigen binding. Non-limiting examples of antigen binding fragments include: (i) Fab fragments; (ii) A F (ab')₂ fragment; (iii) Fd fragment; (iv) Fv fragments; (v) an scFv molecule; (vi) a dAb fragment; and (vii) a minimal recognition unit consisting of amino acid residues mimicking an antibody hypervariable region (e.g., an independent Complementarity Determining Region (CDR) such as a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. As used herein, the expression "antigen binding fragment" also encompasses within other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small Modular Immunopharmaceuticals (SMIPs), and shark variable IgNAR domains.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring any particular method for producing the antibody.

The invention also includes monoclonal antibodies having the corresponding amino acid sequences of the anti-MSLN protein monoclonal antibodies, monoclonal antibodies having the variable region chains of the anti-MSLN protein monoclonal antibodies, and other proteins or protein conjugates and fusion expression products having these chains. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a light chain and a heavy chain comprising a hypervariable region (complementarity determining region, CDR), provided that the hypervariable region is identical or at least 90% homologous, preferably at least 95% homologous, to the hypervariable regions of the light chain and heavy chain of the invention.

Immunoconjugates and fusion expression products include, as known to those of skill in the art: conjugates of drugs, toxins, cytokines (cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules in combination with said anti-MSLN protein monoclonal antibodies or fragments thereof. The invention also includes cell surface markers or antigens that bind to the anti-MSLN monoclonal antibodies or fragments thereof.

The invention includes not only intact monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab')₂ fragments; antibody heavy chain; an antibody light chain.

As used herein, the term "chimeric antibody" is an antibody molecule expressed by a host cell by splicing the V region gene of a murine antibody to the C region gene of a human antibody into a chimeric gene, followed by insertion into a vector. The high specificity and affinity of the parent mouse antibody are maintained, and the human Fc segment of the parent mouse antibody can effectively mediate biological effect functions.

As used herein, the term "humanized antibody", a variable region engineered version of the murine antibody of the present invention, has CDR regions derived (or substantially derived) from a non-human antibody (preferably a mouse monoclonal antibody), and FR regions and constant regions substantially derived from human antibody sequences; i.e., grafting murine-resistant CDR region sequences onto different types of human germline antibody framework sequences. Because CDR sequences are responsible for most of the antibody-antigen interactions, recombinant antibodies that mimic the properties of a particular naturally occurring antibody can be expressed by constructing expression vectors.

In the present invention, antibodies may be monospecific, bispecific, trispecific, or more multispecific.

Single chain antibody

As used herein, the terms "single chain antibody", "scFv" are antibodies in which the antibody heavy and light chain variable regions are linked by a short peptide (linker) of 15 to 20 amino acids. The scFv can better retain the affinity activity of the scFv to the antigen, and has the characteristics of small molecular weight, strong penetrating power, weak antigenicity and the like.

The chimeric antigen receptor for targeting cells expressing MSLN comprises a single-chain antibody for targeting MSLN, wherein the amino acid sequence of the single-chain antibody for targeting MSLN comprises a sequence selected from the group consisting of: SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54.

And, the single chain antibody comprises an antibody heavy chain variable region and an antibody light chain variable region selected from the group consisting of:

Hybridoma cell strain

The invention also provides a hybridoma cell strain capable of producing the MSLN protein monoclonal antibody; preferably, the present invention provides hybridoma cell lines with high titers against MSLN protein monoclonal antibodies.

After obtaining the hybridoma producing the MSLN protein monoclonal antibody of the invention, the person skilled in the art can conveniently use the hybridoma cell line to prepare the antibody. Furthermore, the structure of the antibodies of the invention (e.g., the heavy and light chain variable regions of the antibodies) can be readily known to those skilled in the art, and then the monoclonal antibodies of the invention can be prepared by recombinant methods.

Preparation of monoclonal antibodies

Antibodies of the invention may be prepared by various techniques known to those skilled in the art. For example, the antigens of the invention may be administered to animals to induce monoclonal antibody production. For monoclonal antibodies, hybridoma technology can be used to prepare or can be prepared using recombinant DNA methods.

Representative myeloma cells are those that fuse efficiently, support stable high levels of antibody production by the antibody-producing cell of choice, and are sensitive to the medium (HAT medium matrix), including myeloma cell lines, e.g., murine myeloma cell lines, including those derived from MOPC-21 and MPC-11 mouse tumors (available from Salk Institute Cell Distribution Center, san diego, california, usa) and SP-2, NZ0 or X63-Ag8-653 cells (available from AMERICAN TYPE Culture Collection, rocyvale, maryland, usa). Human myeloma and mouse-human hybrid myeloma cell lines have also been described for the production of human monoclonal antibodies.

The culture medium in which the hybridoma cells are grown is analyzed to detect the production of monoclonal antibodies having the desired specificity, such as by an in vitro binding assay, e.g., an enzyme-linked immunosorbent assay (ELISA) or a Radioimmunoassay (RIA). The location of cells expressing the antibody can be detected by FACS. The hybridoma clones can then be subcloned by limiting dilution steps (subcloned) and grown by standard methods (Goding, monoclonal antibody (Monoclonal Antibodies): principles and practices (PRINCIPLES AND PRACTICE), ACADEMIC PRESS (1986) pages 59-103). Suitable media for this purpose include, for example, DMEM or RPMI-1640 medium. In addition, hybridoma cells can grow as ascites tumors in animals.

Monoclonal antibodies secreted by the subclones are suitably isolated from culture medium, ascites fluid or serum by conventional immunoglobulin purification procedures such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

The invention provides a monoclonal antibody against MSLN protein, in particular a monoclonal antibody against MSLN protein. In a preferred embodiment of the invention, the monoclonal antibodies are prepared by culturing hybridoma cells. Taking supernatant of hybridoma cell culture, roughly extracting IgG by a saturated ammonium sulfate precipitation method, and purifying the roughly extracted antibody by an affinity chromatography column (Protein G-Sephrose).

In a preferred embodiment of the present invention, the monoclonal antibody is prepared by the method of producing a monoclonal antibody by using Balb/C mouse ascites. Approximately hybridoma cells were inoculated into the abdominal cavity of sensitized mice, and obvious abdominal distension was seen around 10 days. Extracting ascites, coarse extracting with saturated ammonium sulfate precipitation, and purifying the coarse extracted antibody with affinity chromatographic column (Protein G-Sephrose).

Methods and samples

The present invention relates to a method for detecting tumors in a sample lysed in cells and/or tissues. The method comprises the following steps: obtaining a cell and/or tissue sample; dissolving a sample in a medium; detecting the level of MSLN protein in the solubilized sample. The sample used in the method of the invention may be any sample comprising cells present in a cell preservation fluid, as used in liquid-based cell assays.

Kit for detecting a substance in a sample

The invention also provides a kit comprising the antibody (or fragment thereof) of the invention or the detection plate of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like.

The invention further relates to a detection kit for detecting the MSLN level, which comprises an antibody for recognizing MSLN protein, a lysis medium for dissolving a sample, and general reagents and buffers required for detection, such as various buffers, detection markers, detection substrates, etc. The detection kit may be an in vitro diagnostic device.

Chimeric Antigen Receptor (CAR)

The Chimeric Antigen Receptor (CAR) of the invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. Extracellular domains include target-specific binding elements (also referred to as antigen binding domains). The intracellular domain includes a costimulatory signaling region and a zeta chain moiety. A costimulatory signaling region refers to a portion of an intracellular domain that comprises a costimulatory molecule. Costimulatory molecules are cell surface molecules that are required for the efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands.

Between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR

Between domains, a linker may be incorporated. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to connect a transmembrane domain to an extracellular domain or cytoplasmic domain of a polypeptide chain. The linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

In a preferred embodiment of the invention, the extracellular domain of the CAR provided by the invention comprises an antigen binding domain that targets MSLN. The CARs of the invention, when expressed in T cells, are capable of antigen recognition based on antigen binding specificity. When it binds to its cognate antigen, affects tumor cells, causes tumor cells to not grow, to be caused to die or to be otherwise affected, and causes the patient's tumor burden to shrink or eliminate. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of the combination of the 4-1BB signaling domain, and the CD3 zeta signaling domain.

As used herein, "antigen binding domain," "single chain antibody fragment" refers to Fab fragments, fab 'fragments, F (ab') 2 fragments, or single Fv fragments having antigen binding activity. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and have a minimal antibody fragment of the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structures required for antigen binding. The antigen binding domain is typically an scFv (single-chain variable fragment). The size of scFv is typically 1/6 of that of an intact antibody. The single chain antibody is preferably an amino acid sequence encoded by a single nucleotide chain. As a preferred mode of the invention, the antigen binding domain comprises an antibody, preferably a single chain antibody, which specifically recognizes MSLN.

For hinge and transmembrane regions (transmembrane domains), the CAR may be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain is used that naturally associates with one of the domains in the CAR. In some examples, the transmembrane domain may be selected, or modified by amino acid substitutions, to avoid binding such domain to the transmembrane domain of the same or a different surface membrane protein, thereby minimizing interactions with other members of the receptor complex.

The intracellular domain in the CAR of the invention comprises the signaling domain of 4-1BB and the signaling domain of CD3 zeta.

Polynucleotide molecules and vectors

The invention also provides polynucleotide molecules encoding the antibodies or fragments thereof or fusion proteins thereof as described above and chimeric antigen receptors as described above. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to SEQ ID No.:9-10, 17-18, 26-27, 36-37, 39, 41, 43, 45, 47, 49, 51, 53 and 55-58, or a degenerate variant. As used herein, "degenerate variant" refers in the present invention to a polypeptide that encodes a polypeptide having the same amino acid sequence as the polypeptide of the present invention, but is identical to SEQ ID No.:9-10, 17-18, 26-27, 36-37, 39, 41, 43, 45, 47, 49, 51, 53 and 55-58.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleotides, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form. At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cells, T cells, NK cells, etc.

Chimeric antigen receptor T cells (CAR-T cells)

As used herein, the terms "CAR-T cell", "CAR-T cell of the invention" include CAR-T cells comprised in the fourth aspect of the invention.

CAR-T cells have the following advantages over other T cell-based therapies: (1) the course of action of CAR-T cells is not restricted by MHC; (2) In view of the fact that many tumor cells express the same tumor antigen, CAR gene construction for a certain tumor antigen can be widely utilized once completed; (3) The CAR can utilize not only tumor protein antigens but also glycolipid non-protein antigens, so that the target range of the tumor antigens is enlarged; (4) The use of autologous patient cells reduces the risk of rejection; (5) The CAR-T cells have an immunological memory function and can survive in vivo for a long time.

As used herein, the terms "MSLN-CART cells", "MSLN CAR-T cells" refer to CAR-T cells that target MSLN, i.e., T cells that express single chain antibodies that bind MSLN on the cell surface.

The invention provides a CAR-T cell comprising a MSLN-targeted CAR as set forth in the sixth and seventh aspects of the invention.

In a preferred embodiment of the invention, the invention utilizes CAR-T cells constructed from humanized MSLN scFv to further increase its killing effect and tumor-clearing capacity.

Formulations (or pharmaceutical compositions)

The invention also provides a pharmaceutical composition (or formulation) comprising an antibody according to the first aspect of the invention, a chimeric antigen receptor according to the second aspect of the invention, a polynucleotide molecule according to the third aspect of the invention, a recombinant expression vector according to the fourth aspect of the invention or an engineered immune cell according to the sixth aspect of the invention, and a pharmaceutically acceptable carrier, excipient or diluent. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.

The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the antibodies (or conjugates thereof) of the invention described above, chimeric antigen receptor, or chimeric antigen receptor T cells, and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 1 microgram per kilogram of body weight to about 10 milligrams per kilogram of body weight per day. In addition, the pharmaceutical compositions of the present invention may also be used with other therapeutic agents.

When a pharmaceutical composition is used, a safe and effective amount of the immunizing agent is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight and in most cases no more than about 8 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

Therapeutic applications

The invention also provides the use of an antibody according to the first aspect of the invention, a chimeric antigen receptor according to the second aspect of the invention, a polynucleotide molecule according to the third aspect of the invention, a recombinant expression vector according to the fourth aspect of the invention, an engineered immune cell according to the sixth aspect of the invention or a formulation according to the eighth aspect of the invention for the prevention and/or treatment of cancer comprising tumor cells expressing MSLN.

The cancer is a solid tumor, the cancer selected from the group consisting of: lung cancer, colorectal cancer, breast cancer, gastric cancer, ovarian cancer, liver cancer, pancreatic cancer, bladder cancer, cervical cancer, endometrial cancer, prostate cancer, small intestine adenocarcinoma, oral cancer, or nasopharyngeal cancer.

For ex vivo immune cell preparation, at least one of the following occurs in vitro prior to administration of the cells into a mammal: i) Expanding the cells, ii) introducing a nucleotide encoding the CAR into the cells, and/or iii) cryopreserving the cells.

Ex vivo cell processing procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably a human) and genetically modified (i.e., transduced or transfected in vitro) with vectors expressing the CARs disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient may be human, and the CAR-modified cells may be autologous or allogeneic, syngeneic with respect to the recipient (syngeneic).

The invention provides a method of treating a tumor comprising administering to a subject in need thereof an effective amount of a universal CAR-T cell of the invention.

The universal CAR-T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-15, IL-17 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present invention may comprise a target cell as described herein in combination with one or more pharmaceutically or clinically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The compositions of the present invention are preferably formulated for intravenous administration.

The pharmaceutical composition of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by such factors as the nature of the condition of the patient, the type and severity of the disease, although appropriate dosages may be determined by clinical trials.

When referring to an "immunologically effective amount", "antitumor effective amount", "tumor-inhibiting effective amount" or "therapeutic amount", the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, degree of infection or metastasis and individual differences of the condition of the patient (subject). It can be generally stated that: the pharmaceutical compositions comprising T cells described herein may be administered at a dose of 10⁴ to 10⁹ cells/kg body weight, preferably at a dose of 10⁵ to 10⁷ cells/kg body weight (including all whole values within those ranges). T cell compositions may also be administered at these doses multiple times. Cells can be administered by using injection techniques well known in immunotherapy (see, e.g., rosenberg et al, new Eng. J. Of Med.319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be determined by one skilled in the medical arts by monitoring the patient for signs of disease.

Administration of the subject compositions may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intradesmally, intraspinal, intramuscularly, by intravenous (i.v.) injection or intraperitoneally, intrapleurally. In another embodiment, the T cell composition of the invention is preferably administered by i.v. intravenous injection. The composition of T cells can be injected directly into the tumor, lymph node or site of infection. (CART cell products are mainly intravenous infusion, and can be directly infused into tumor, lymph node or infection site)

In certain embodiments of the invention, the cells are activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels, in combination with (e.g., prior to, concurrent with, or subsequent to) administration to a patient of any number of relevant therapeutic modalities, including, but not limited to, treatment with: other cytotoxic chemotherapeutic agents such as antiviral therapies, cidofovir, interleukin-2, IFN- γ, cytarabine (also known as ARA-C), checkpoint inhibitors such as PD-1 antibodies, anti-CTLA-4 antibodies and agents that inhibit cytokine storms, other therapies such as anti-IL-6 receptor microbead antibodies. In a further embodiment, the T cells of the invention may be used in combination with: chemotherapy, radiation, immunosuppressives such as cyclosporine, azathioprine, methotrexate, mycophenolate and antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the invention are administered to a patient in combination (e.g., before, simultaneously or after) with bone marrow transplantation, using a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide. For example, in one embodiment, the subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives injection of the expanded immune cells of the invention after transplantation. In an additional embodiment, the expanded cells are administered pre-operatively or post-operatively.

The dose of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio administered to humans may be carried out according to accepted practices in the art. Typically, from 1 x 10⁶ to 1 x 10¹⁰ universal CAR-DNT cells of the invention can be administered to a patient by, for example, intravenous infusion, per treatment or per course of treatment.

The main advantages of the invention include:

(1) The antibody of the invention has high specificity and strong affinity, can be prepared in large quantity, and has easy control of monoclonal antibody quality.

(2) The antibody of the invention can be used for targeting drugs, antibody drug conjugates or multifunctional antibodies which specifically target MSLN positive tumor cells.

(3) The MSLN CAR-T cell provided by the invention has stronger killing and cytokine release functions than a positive control CAR-T cell, which indicates that the CAR-T cell has more effective effect in vivo

(4) The antibody of the invention can be used as a secretory antibody which is expressed by chimeric antigen receptor immune cells together and acts on local tumor.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Example 1

Preparation of anti-Mesothelin (MES or MSLN) antibodies

The main technical scheme of the embodiment is as follows:

1. the main technical scheme of the embodiment is as follows:

1. mice were immunized with 3 DNA shots +1 cell shots, two weeks apart. Wherein, the DNA refers to human MESDNA, and the cell used is CHO-MES (i.e. CHO cell expressing MES).

2. FACS screening was performed using CHO-MES (CHO cells expressing MES) cells.

The method comprises the following experimental steps:

1. Immunized mice

Experimental mice were immunized according to the protocol of table 3:

Table 3 immunization time protocol for mice

Mouse ID	Exempt from	Two-way valve	Three-free	Impact immunity
					MES-A-01	2020/09/25	2020/10/10	2020/10/23
MES-A-02	2020/09/25	2020/10/10	2020/10/23	2020/11/09
					MES-A-03	2020/09/25	2020/10/10	2020/10/23
MES-A-04	2020/09/25	2020/10/10	2020/10/23
					MES-A-05	2020/09/25	2020/10/10	2020/10/23
MES-A-06	2020/09/25	2020/10/10	2020/10/23
					MES-A-07	2020/09/25	2020/10/10	2020/10/23
MES-A-08	2020/09/25	2020/10/10	2020/10/23
					MES-A-09	2020/09/25	2020/10/10	2020/10/23
MES-A-10	2020/09/25	2020/10/10	2020/10/23	2020/11/10

Wherein, one exempts from: each mouse of MES-A-01-10 was dnA immunized (60 μg, intramuscular injection); and (2) avoiding: each mouse of MES-A-01-10 was DNA immunized; three-way: each mouse of MES-A-01-10 was DNA immunized; impact immunization: MES-A-01, 10 were pulsed with CHO-MES cells.

2. Serum detection

(1) And (3) flow detection:

1) Appropriate amounts of CHO-MES cells were dispensed into 1.5mL Ep tubes, 4000rpm,5min, and the supernatant was aspirated.

2) 50. Mu.L of serum diluted with PBS1:100 resuspended the above cells and stood at 4℃for 15min.

3) 4000Rpm,5min, the supernatant was pipetted off.

4) Mu.L of goat anti-mouse (Goat anti-mouse) IgGFc-FITC diluted with PBS1:500 resuspended the above cells and stood at 4℃for 15min.

5) And 3, the same step as the step.

6) Cells were resuspended in 200 μl PBS and FACS analyzed.

Pre-and post-three-immune mouse sera were tested for binding to CHO-MES by FACS as shown in FIG. 1. The results show that 10 mice have better immune response after three-way, and two mice of MES-A-02,10 are selected to be fused.

3. Fusion of

After 3 or 4 days of impact immunization, B lymphocytes of the mice are respectively fused with myeloma cells to obtain corresponding hybridoma cells.

4. Post-fusion screening

(1) FACS detection screening: each mouse was plated with 5 96-well plates (12×8 wells, 1-8 for row number, a-H for column number, as in table 1), numbered 1,2,3,4, 5, respectively, for FACS detection.

The results of MES-A-02 screening are shown in FIG. 2, and MES-02-2C9,4C4,4D5,4D7,5G2 was finally selected for subcloning.

The results of MES-A-10 screening are shown in FIG. 3, and MES-10-3D2,4G8,4D5,5A12,5B10 was finally selected for subcloning.

(2) Subclone screening

As shown in FIG. 4, the subclone detection results of MES-02-2C9,4C4,4D5,4D7,5G2 by FACS detection are shown, and the MES-02-2C9,4C4,4D5 subclone was finally selected for expansion culture.

The subclone detection results of MES-10-3D2,4G8,4D5,5A12,5B10 are shown in FIG. 5, and the MES-10-3D2,4G8,5B10 subclone was finally selected for expansion culture.

5. Cell strain

Preferred positive subclones are summarized in Table 6 as determined by FACS as described above.

TABLE 6 preferred Positive subclone summary results

Clone number	Subcloning number	Subtype type
			MES-02-2C9	1A11，1E3，1E8	IgG1
MES-02-4C4	1A7，1E3	IgG1
			MES-02-4D5	1E6，1E11，1A2，1A3	IgG1
MES-10-3D2	1A4，1A9，1E5	IgG1
			MES-10-4G8	1A11，1E5	IgG1
MES-10-5B10	1C9，1C2，1B12，1C11

Example 2

Construction of recombinant MES antibodies

The preferred positive subclones selected from example 1 were: MES-02-2C9, MES-02-4C4, MES-02-4D5 and MES-10-4G8, and these subclones were selected based on the flow positive results of the hybridoma cell supernatants on CHO-MES cells after fusion.

And subjected to antibody sequence and subtype analysis. The specific experimental steps are as follows:

(1) CHO-MES cells were aliquoted into 1.5mL EP tubes.

(2) Centrifuge at 4000rpm for 5min and discard supernatant.

(3) 50. Mu.L of hybridoma supernatant was taken and resuspended, and left to stand at 4℃for 15min.

(4) Centrifuge at 4000rpm for 5min and discard supernatant.

(5) The cells were resuspended in 50. Mu.L of Goat anti-mouse IgG1-FITC, goat anti-mouse IgG1a-FITC and Goat anti-mouse IgG1b-FITC diluted 1:200 and allowed to stand at 4℃for 15min.

(6) Centrifuge at 4000rpm for 5min and discard supernatant.

(7) Cells were resuspended in 300 μl PBS and flow analyzed.

The results of the flow analysis are shown in Table 7.

The results show that: the MES-02-2C9 antibody subtype was IgG1. Antibody gene sequencing was performed using the Sanger method of capillary electrophoresis.

Details concerning the MES-02-2C9, MES-02-4C4, MES-02-4D5, and MES-10-4G8 antibodies are summarized in Table 7.

TABLE 7 subtype and gene sequence of antibodies

Clone number	Heavy chain	Light chain
			MES-02-2C9-1A11	IGHV5-9-3	IGKV12-46
MES-02-4C4-1A7、1E3	IGHV5-9-3	IGKV12-46
			MES-02-4D5-1E6、1A3	IGHV5-9-3	IGKV12-46
MES-10-4G8-1A11、1E5	IGHV1SS22	IGKV6-15

The heavy chain IGHV5-9-3 sequence of MES-02-2C9-1A11 is shown in SEQ ID NO. 9 (DNA) and SEQ ID NO. 1 (amino acid).

The light chain IGKV12-46 sequence of MES-02-2C9-1A11 is shown as SEQ ID NO. 10 (DNA) and SEQ ID NO. 5 (amino acid).

The heavy chain IGHV5-9-3 sequence of MES-02-4C4-1A7/1E3 is shown in SEQ ID NO. 17 (DNA) and SEQ ID NO. 11 (amino acid).

The light chain IGKV12-46 sequence of the MES-02-4C4-1A7/1E3 is shown as SEQ ID NO. 18 (DNA) and SEQ ID NO. 14 (amino acid).

The heavy chain IGHV5-9-3 sequence of MES-02-4D5-1E6/1A3 is shown in SEQ ID NO. 26 (DNA) and SEQ ID NO. 19 (amino acid).

The light chain IGKV12-46 sequence of MES-02-4D5-1E6/1A3 is shown as SEQ ID NO. 27 (DNA) and SEQ ID NO. 23 (amino acid).

The heavy chain IGHV1SS22 sequence of MES-10-4G8-1A11/1E5 is shown in SEQ ID NO:36 (DNA) and SEQ ID NO:28 (amino acid).

The light chain IGKV6-15 sequence of the MES-10-4G8-1A11/1E5 is shown as SEQ ID NO. 37 (DNA) and SEQ ID NO. 32 (amino acid).

Expression verification of recombinant antibodies:

The method comprises the following specific steps: the heavy chain variable region and the light chain variable region nucleotide sequences of the MES-02-2C9, MES-02-4C4, MES-02-4D5 and MES-10-4G8 antibodies are cloned into pCAG eukaryotic expression vectors respectively to obtain recombinant expression vectors for respectively expressing the heavy chain variable region and the light chain variable region of the antibodies.

10 Μg of light and heavy chain expression vector was co-transferred into 293T cells in 10cm dishes using the calcium phosphate method, and the supernatant was collected three days later and FACS verified in CHO-MES cells.

The FACS validation experiment includes the following steps:

(1) CHO-MES cells were aliquoted into 1.5mL EP tubes.

(2) Centrifuge at 4000rpm for 5min and discard supernatant.

(3) 50. Mu.L of 293T cells were transfected into the supernatant and resuspended, and allowed to stand at 4℃for 15min.

(4) Centrifuge at 4000rpm for 5min and discard supernatant.

(5) The cells were resuspended in 50. Mu.L of Goat anti-mouse IgG Fc-FITC diluted 1:200 and allowed to stand at 4℃for 15min.

(6) Centrifuge at 4000rpm for 5min and discard supernatant.

(7) Cells were resuspended in 300 μl PBS and flow analyzed.

The results of the FACS validation are shown in FIGS. 6-9. The results showed that the results of the transfection of MES-02-2C9 (MES-02-2C 9-1A 11), MES-02-4C4 (MES-02-4C 4-1A7/1E 3), MES-02-4D5 (MES-02-4D 5-1E6/1A 3) and MES-10-4G8 (MES-10-4G 8-1A11/1E 5) gave better signals and correct sequences.

Example 3

Construction of chimeric antigen receptor expression plasmids containing scFv targeting MSLN

By artificial synthesis of a DNA fragment containing the following partial nucleotides: nucleotides encoding leader, nucleotides encoding MSLN scFv (carcinoembryonic antigen-targeting scFv), nucleotides encoding the CD8 hinge region, nucleotides encoding the CD8 transmembrane region, nucleotides encoding 4-1BB, nucleotides encoding CD3 ζ.

Wherein the MSLN scFv are each single chain antibodies of the invention selected from the group consisting of preferred antibodies of: MES-10-4G8, MES-02-4D5, MES-02-2C9.

Wherein, the structural schematic of the chimeric antigen receptor containing scFv targeting MSLN is shown in FIG. 10.

The above DNA fragment was inserted downstream of EF1alpha promoter of lentiviral expression vector to obtain MSLN-targeted chimeric antigen receptor expression plasmid (LV-MESO-scFv-BB-z), as shown in FIG. 12.

Example 4

Chimeric antigen receptor expression plasmid transfection T cell of targeting MSLN

(1) Packaging preparation of lentiviruses

Chimeric antigen receptor expression and construction plasmids, envelope plasmids were transfected into 293T/17 cells at a ratio of 3:2:1 using calcium phosphate transfection. 12 hours after transfection, fresh DMEM medium containing 10% FBS was changed, and sodium butyrate was added at a final concentration of 5 mM. After 48 hours of transfection, the cell culture supernatant containing the virus was aspirated into a centrifuge tube, centrifuged at 1500rpm at 4℃for 10min, and the supernatant was transferred to a fresh centrifuge tube, filtered through a 0.45 μm filter and stored at-80 ℃.

(2) Preparation of T cells

10Ml of fresh blood of healthy people is taken, and peripheral blood mononuclear cells are separated by lymphocyte separation liquid (rhyme flyblow) and the specific method is shown in the specification. Cell density was adjusted to 2X10⁶/ml with T551 medium containing 4% autologous serum and induction culture was performed with IL-2 at a final concentration of 300U/ml, CD3 mab at 100ng/ml for 24h to give T cells.

(3) Expanded culture of lentivirus-infected T cells and post-infection T cells

The slow virus solution was added at MOI 10 to 1 well of a 6-well plate containing 2X10⁶ of the above-described induced-cultured T cells, and co-cultured in a 5% CO₂ incubator at 37 ℃. After three days, the cells were washed by centrifugation, fresh T551 medium containing 300IU/ml IL-2, 100ng/ml CD3 mab and 4% human autologous serum was added, and the cell density was adjusted to 2X10⁶/ml for continued culture. Cell density was measured every 2-3 days, centrifuged and cell density was adjusted with fresh medium for continuous expansion culture. This is repeated until the cells have been expanded to a sufficient amount.

After testing for efficient expression of the chimeric antigen receptor in T cells, CAR-T cells against MSLN were obtained.

Example 5

Specific killing Activity of CAR-T cells on MSLN-positive malignant cells

5X 10⁴ K562 cells (MSLN positive, expressed as MSLN-K562) with high expression of MSLN and control K562 cells (MSLN negative, expressed as K562) were seeded in 96-well plates and the cells were cultured in two cell culture wells as effector cells (Effector): target cells (Target) =10:1, 3:1, 1:1 three ratios CAR-T cells (4G8 MSLN-CART, 4D5 MSLN-CART, 2C9 MSLN-CART), CAR-T positive control cells (MSLN-CART positive control) and unmodified T cells (T) were added. After 18h incubation, specific killing activity of CAR-T cells against high MSLN expressing K562 cells was detected, operating according to LDH cytotoxicity assay kit (bi yun day) instructions.

The results show that the CAR-T cell group (group 1) has a stronger killing effect on K562 cells that highly express MSLN than the CAR-T positive control cell group (group 2) and the unmodified T cell group (group 3), and that this difference has significance (p < 0.05) (fig. 13); there was no significant difference in killing of control K562 cells (p > 0.05) (fig. 14).

Example 6

ELISA for detecting IL-2, IFN-g expression

5X 10⁴ K562 target cells (MSLN positive) and control K562 target cells (MSLN negative) with high expression of MSLN were inoculated in 96-well plates and effector cells (Effector) were added to each of the two cell culture wells: target cells (Target) =3:1 CAR-T cells (4 g8 MSLN-CART, 4D5MSLN-CART, 2c9 MSLN-CART), MSLN-CART positive control cells (MSLN-CART positive control) and unmodified T cell (T) equivalent cells were added. After 18h incubation, the supernatants were assayed for IL-2 and IFN-g content, following the procedure of the Human IL-2ELISA Kit (Davidae).

As shown in fig. 15 and 16, the results show that the CAR-T cell group produced large amounts of IL-2 and IFN-g for K562 cells that highly expressed MSLN at an effective target ratio of 3:1 compared to the CAR-T positive control cell group and T cell group, and this difference was significant (p < 0.05); the cells of control K562 had no induction of cytokines and no significant differences (p > 0.05). Thus CAR-T cells have specific cytokine-induced effects on tumor cells that highly express MSLN.

Example 7

Flow cytometry detects CAR expression at the surface of CAR-T cells

On the seventh day of lentiviral infection of T cells, 3×10⁵ MSLN CAR-T cells, positive control cells, and negative control cells were taken, respectively, and expression of CAR at the CAR-T cell surface was detected on a flow cytometer using FITC-labeled goat anti-mouse-F (ab)₂ antibody.

FIG. 11 shows flow cell results of CAR-T cell positive rates of MES-02-2C9 (MES-02-2C 9-1A 11), MES-02-4C4 (MES-02-4C 4-1A7/1E 3) and MES-02-4D5 (MES-02-4D 5-1E6/1A 3) and MES-10-4G8 (MES-10-4G 8-1A11/1E 5) after lentiviral transfection of activated T cells and seven days of culture.

The results showed that CAR-T cells consistently maintained constant high expression, indicating that CAR-T cells consistently maintained a constant high proportion.

In summary, the chimeric antigen receptor of the present invention, which targets MSLN, can effectively and specifically target malignant cells expressing MSLN surface antigen, thereby providing a more efficient and less adverse and adverse effect method for treating some tumors expressing MSLN surface antigen.

The sequence information for the antibodies and chimeric antigen receptors of the invention is shown in table 8 below:

TABLE 8

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.