Antibodies targeting mesothelin and their uses

Abstract

This invention provides antibodies targeting MSLN, as well as multispecific antibodies, chimeric antigen receptors, antibody-drug conjugates, pharmaceutical compositions and kits comprising them, and their use in the diagnosis / treatment / prevention of diseases associated with MSLN expression.

Description

Technical Field

[0001] This invention belongs to the field of immunotherapy. More specifically, this invention relates to antibodies targeting mesothelin, and their use in the prevention and / or treatment and / or diagnosis of diseases. Background Technology

[0002] Mesothelin, also known as MSLN, is a cell surface glycoprotein encoded by the MSLN gene, which is anchored to the cell surface via glycosylphosphatidylinositol.

[0003] Mesothelin is a differentiation antigen found on normal mesothelial cells. It is rarely expressed in normal tissues, but is highly expressed in tumors such as mesothelioma, lung cancer, pancreatic cancer, breast cancer, and ovarian cancer. Therefore, mesothelin has the potential to become an important target for cancer treatment.

[0004] Researchers have studied MSLN gene knockout mice and found that these knockout mice exhibited normal development, reproduction, and blood cell counts. This indicates that mesothelin is not essential for normal growth, development, and reproduction in mice. In tumors, MSLN overexpression can activate the NF-κB, MAPK, and PI3K pathways, and induce apoptosis or promote cell proliferation, migration, and metastasis by inducing the activation and expression of MMP7 and MMP9. Abnormal MSLN expression plays an important role in tumor cell proliferation, adhesion, and drug resistance.

[0005] Therefore, this invention has developed a set of antibodies that specifically recognize MSLN and constructed corresponding chimeric antigen receptor immune cells, providing more options for the treatment of various tumors. Summary of the Invention

[0006] In a first aspect, the present invention provides an antibody or antigen-binding fragment thereof targeting MSLN, comprising:

[0007] (1) The heavy chain variable region comprising H-CDR1 selected from SEQ ID NO: 1-4, H-CDR2 selected from SEQ ID NO: 5-9, and H-CDR3 selected from SEQ ID NO: 10-13; and

[0008] (2) Light chain variable region, which includes L-CDR1 selected from SEQ ID NO: 14-18, L-CDR2 selected from SEQ ID NO: 19-22 and L-CDR3 selected from SEQ ID NO: 23-27.

[0009] Preferably, the antibody targeting MSLN or its antigen-binding fragment comprises:

[0010] (1) A heavy chain variable region comprising H-CDR1, H-CDR2, H-CDR3 as shown in SEQ ID NO: 1, 5, 10, or H-CDR1, H-CDR2, H-CDR3 as shown in SEQ ID NO: 2, 6, 11, or H-CDR1, H-CDR2, H-CDR3 as shown in SEQ ID NO: 3, 7, 12, or H-CDR1, H-CDR2, H-CDR3 as shown in SEQ ID NO: 4, 8, 13, or H-CDR1, H-CDR2, H-CDR3 as shown in SEQ ID NO: 1, 9, 10; and

[0011] (2) Light chain variable region, which includes L-CDR1, L-CDR2, L-CDR3 as shown in SEQ ID NO: 14, 19, 23, or L-CDR1, L-CDR2, L-CDR3 as shown in SEQ ID NO: 15, 20, 24, or L-CDR1, L-CDR2, L-CDR3 as shown in SEQ ID NO: 16, 21, 25, or L-CDR1, L-CDR2, L-CDR3 as shown in SEQ ID NO: 17, 22, 26, or L-CDR1, L-CDR2, L-CDR3 as shown in SEQ ID NO: 18, 19, 27.

[0012] In one embodiment, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with an amino acid sequence selected from SEQ ID NO: 28, 30, 32, 34, 36, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, and 124, or is selected from SEQ ID NO: 28, 30, 32, 34, 36, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, and 124. NO: 28, 30, 32, 34, 36, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, and 124 have one or more amino acid modifications (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), preferably conservative modifications of up to 10 amino acids; the light chain variable region is selected from SEQ ID. The amino acid sequences NO: 29, 31, 33, 35, 37, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, and 125 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, or are selected from SEQ ID NO: IDNO: 29, 31, 33, 35, 37, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, and 125 have one or more amino acid modifications (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), preferably up to 10 conserved modifications. Preferably, the modifications are conserved modifications, such as conserved substitutions, additions, and deletions of amino acids. In a preferred embodiment, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region selected from SEQ ID NO: 28, 30, 32, 34, 36, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121 and 124 and a light chain variable region selected from SEQ ID NO: 29, 31, 33, 35, 37, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122 and 125.

[0013] In one embodiment, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region selected from the following:

[0014] (a) The heavy chain variable region as shown in SEQ ID NO: 28 and the light chain variable region as shown in SEQ ID NO: 29;

[0015] (b) The heavy chain variable region as shown in SEQ ID NO: 30 and the light chain variable region as shown in SEQ ID NO: 31;

[0016] (c) The heavy chain variable region as shown in SEQ ID NO: 32 and the light chain variable region as shown in SEQ ID NO: 33;

[0017] (d) the heavy chain variable region as shown in SEQ ID NO: 34 and the light chain variable region as shown in SEQ ID NO: 35; or

[0018] (e) Heavy chain variable regions as shown in SEQ ID NO: 36 and light chain variable regions as shown in SEQ ID NO: 37;

[0019] (f) The heavy chain variable region as shown in SEQ ID NO: 73 and the light chain variable region as shown in SEQ ID NO: 74;

[0020] (g) Heavy chain variable region as shown in SEQ ID NO: 76 and light chain variable region as shown in SEQ ID NO: 77

[0021] (h) the heavy chain variable region as shown in SEQ ID NO: 79 and the light chain variable region as shown in SEQ ID NO: 80;

[0022] (i) the heavy chain variable region as shown in SEQ ID NO: 82 and the light chain variable region as shown in SEQ ID NO: 83;

[0023] (j) the heavy chain variable region as shown in SEQ ID NO: 85 and the light chain variable region as shown in SEQ ID NO: 86;

[0024] (k) the heavy chain variable region as shown in SEQ ID NO: 88 and the light chain variable region as shown in SEQ ID NO: 89; or

[0025] (l) the heavy chain variable region as shown in SEQ ID NO: 91 and the light chain variable region as shown in SEQ ID NO: 92;

[0026] (m) the heavy chain variable region as shown in SEQ ID NO: 94 and the light chain variable region as shown in SEQ ID NO: 95;

[0027] (n) Heavy chain variable region as shown in SEQ ID NO: 97 and light chain variable region as shown in SEQ ID NO: 98

[0028] (o) The heavy chain variable region as shown in SEQ ID NO: 100 and the light chain variable region as shown in SEQ ID NO: 101;

[0029] (p) Heavy chain variable region as shown in SEQ ID NO: 103 and light chain variable region as shown in SEQ ID NO: 83;

[0030] (q) The heavy chain variable region as shown in SEQ ID NO: 85 and the light chain variable region as shown in SEQ ID NO: 104;

[0031] (r) the heavy chain variable region as shown in SEQ ID NO: 106 and the light chain variable region as shown in SEQ ID NO: 107; or

[0032] (s) the heavy chain variable region as shown in SEQ ID NO: 109 and the light chain variable region as shown in SEQ ID NO: 110;

[0033] (t) the heavy chain variable region as shown in SEQ ID NO: 112 and the light chain variable region as shown in SEQ ID NO: 113;

[0034] (u) the heavy chain variable region as shown in SEQ ID NO: 115 and the light chain variable region as shown in SEQ ID NO: 116;

[0035] (v) The heavy chain variable region as shown in SEQ ID NO: 118 and the light chain variable region as shown in SEQ ID NO: 119;

[0036] (w) the heavy chain variable region as shown in SEQ ID NO: 121 and the light chain variable region as shown in SEQ ID NO: 122;

[0037] (x) The heavy chain variable region as shown in SEQ ID NO: 124 and the light chain variable region as shown in SEQ ID NO: 125.

[0038] In one embodiment, the antibody or antigen-binding fragment of the present invention has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, 102, 105, 108, 111, 114, 117, 120, 123, and 126 amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with an amino acid sequence selected from SEQ ID NO: 38-42, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, and 126, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with an amino acid sequence selected from SEQ ID NO: 38-42, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, and 126. Compared to the amino acid sequences of SEQ ID NOs 38-42, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, and 126, the amino acid sequences of these sequences have one or more amino acid modifications (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids). Preferably, the modifications are conservative modifications, such as conservative substitutions, additions, and deletions of amino acids. Preferably, the amino acid sequences of the antibodies or antigen-binding fragments of the present invention are selected from SEQ ID NOs 38-42, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, and 126.

[0039] In one embodiment, the antibody or antigen-binding fragment of the present invention is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody, preferably a humanized antibody.

[0040] The present invention also provides nucleic acid molecules encoding antibodies or antigen-binding fragments thereof. Therefore, in one embodiment, the nucleic acid molecule encoding said antibody or antigen-binding fragment has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a nucleotide sequence selected from SEQ ID NO: 43-47, and the antibody or antigen-binding fragment encoded therein is capable of specifically binding to the MSLN antigen. Preferably, the nucleic acid molecule encoding said antibody or antigen-binding fragment is selected from SEQ ID NO: 43-47.

[0041] In one embodiment, the antibody or antigen-binding fragment of the present invention is a multispecific antibody (preferably a bispecific or trispecific antibody) comprising an anti-MSLN antibody or antigen-binding fragment as described above, and one or more second antibodies or antigen-binding portions thereof that specifically bind to other tumor antigens.

[0042] In one embodiment, the second antibody or its antigen-binding portion may be in any antibody or antibody fragment form, such as a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, scFv-scFv, microantibody, biantibody, or sdAb.

[0043] The present invention also provides a vector comprising a nucleic acid molecule encoding the above-mentioned anti-MSLN antibody or its antigen-binding fragment, and a host cell expressing the anti-MSLN antibody or its antigen-binding fragment.

[0044] In another aspect, the present invention also provides a recombinant receptor (e.g., a recombinant TCR receptor, a chimeric antigen receptor, a T-cell fusion protein, or a T-cell antigen coupler) comprising the anti-MSLN antibody or its antigen-binding fragment as described herein. Preferably, the recombinant receptor is a chimeric antigen receptor, further comprising a transmembrane domain and an intracellular domain, the intracellular domain comprising one or more co-stimulatory domains and / or primary signaling domains. More preferably, the chimeric antigen receptor comprises the anti-MSLN antibody or its antigen-binding fragment as provided herein, a CD8α or CD28 transmembrane domain, a CD28 and / or 4-1BB co-stimulatory domain, and a CD3ζ primary signaling domain.

[0045] The present invention also provides a nucleic acid molecule encoding a recombinant receptor for targeting MSLN as defined above, and a vector comprising said nucleic acid molecule.

[0046] The present invention also provides cells comprising recombinant receptors targeting MSLNs as defined above, preferably immune cells, such as T cells, NK cells, NKT cells, macrophages, and dendritic cells. In a preferred embodiment, the engineered immune cells further comprise a second recombinant receptor targeting other tumor antigens, such as a second chimeric antigen receptor or a recombinant TCR receptor.

[0047] In another aspect, the present invention also provides an antibody conjugate comprising an anti-MSLN antibody as defined in the present invention or an antigen-binding fragment thereof and a second functional structure, wherein the second functional structure is selected from Fc, radioisotopes, structural portions with extended half-life, detectable markers, and drugs.

[0048] In one embodiment, the structural portion for extending the half-life is selected from: albumin-binding structures, transferrin-binding structures, polyethylene glycol molecules, recombinant polyethylene glycol molecules, human serum albumin, fragments of human serum albumin, and albumin polypeptides (including antibodies) bound to human serum albumin. In one embodiment, the detectable marker is selected from fluorophores, chemiluminescent compounds, bioluminescent compounds, enzymes, antibiotic resistance genes, and contrast agents. In one embodiment, the drug is selected from cytotoxins and immunomodulators.

[0049] In another aspect, the present invention also provides a detection kit comprising the anti-MSLN antibody or its antigen-binding fragment described in the present invention, an antibody-drug conjugate, engineered immune cells, or a recombinant receptor.

[0050] In another aspect, the present invention also provides a pharmaceutical composition comprising the anti-MSLN antibody or its antigen-binding fragment thereof as described herein, a recombinant receptor, engineered immune cells or antibody conjugates, and one or more pharmaceutically acceptable excipients.

[0051] In another aspect, the present invention also provides a method for treating and / or preventing and / or diagnosing diseases associated with MSLN expression, comprising administering to a subject an anti-MSLN antibody or an antigen-binding fragment thereof as described above, a chimeric antigen receptor, an antibody-drug conjugate, engineered immune cells, or a pharmaceutical composition. Invention Details

[0053] Unless otherwise stated, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0054] Anti-MSLN antibody or its antigen-binding fragment

[0055] As used herein, the term "antibody" has the broadest meaning as understood by those skilled in the art and includes monoclonal antibodies (comprising complete antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments or synthetic polypeptides carrying one or more CDR sequences capable of exhibiting desired biological activity. The antibodies described in this invention can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, etc.) or subclass (e.g., IgG1, IgG2, IgG2a, IgG3, IgG4, IgA1, IgA2, etc.).

[0056] As used herein, the term "antigen-binding fragment" or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of antibody fragments in this invention include, but are not limited to: Fab, Fab', F(ab')2, Fd, Fd', Fv, single-chain antibodies (scFv), disulfide-linked Fv (sdFv), linear antibodies, "dimeric" antibodies having two antigen-binding sites, natural ligands of said antigens, or functional fragments thereof. Therefore, unless the context clearly indicates otherwise, the term "antibody" in this invention encompasses antibody fragments or antigen-binding fragments as defined above. Accordingly, in one embodiment, the antibody of this invention is selected from full-length antibodies, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibodies, and dimeric antibodies.

[0057] Typically, a complete antibody consists of two heavy chains and two light chains linked together by disulfide bonds, each light chain being connected to its respective heavy chain via disulfide bonds, forming a "Y"-shaped structure. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain variable region contains three complementation-determining regions (CDRs): H-CDR1, H-CDR2, and H-CDR3, and the heavy chain constant region contains three constant structural domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (VL) and a light chain constant region. The light chain variable region contains three CDRs: L-CDR1, L-CDR2, and L-CDR3, and the light chain constant region contains one constant structural domain CL. Within the heavy / light chain variable regions, the CDRs are separated by more conserved frame regions (FRs). The variable regions of the heavy / light chains are responsible for the recognition and binding of antigens, while the constant regions mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system.

[0058] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using many numbering schemes well-known in the art, including: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (“Kabat” numbering scheme); Al-Lazikani et al. (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding sitetopography,” J. Mol. Biol. 262, 732-745 (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp. Immunol, January 2003; 27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” JMol Biol, June 8, 2001; 309(3):657-70 (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989, 86(23):9268-9272 (“AbM” numbering scheme).

[0059] The boundaries of a given CDR or FR can vary depending on the protocol used for identification. For example, the Kabat protocol is based on structure alignment, while the Chothia protocol is based on structural information. Both the Kabat and Chothia protocols number antibodies based on the length of the most common antibody region sequences, where insertions are indicated by insert letters (e.g., "30a") and deletions occur in some antibodies. These two protocols place certain insertions and deletions (indels) in different positions, resulting in different numbering schemes. The Contact protocol is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many ways. The AbM protocol is a compromise between the Kabat and Chothia definitions, based on the scheme used by the Oxford Molecular AbM antibody modeling software.

[0060] Therefore, unless otherwise specified, it should be understood that the “CDR” of a given antibody or its region (such as its variable region) encompasses the CDRs defined by any of the above-described schemes or other known schemes. For example, in specifying that a particular CDR (e.g., CDR3) contains a given amino acid sequence, it should be understood that such a CDR may also have the sequence of the corresponding CDR (e.g., CDR3) as defined by any of the above-described schemes or other known schemes. Similarly, unless otherwise specified, it should be understood that the FR of a given antibody or its region (such as its variable region) encompasses the FRs defined by any of the above-described schemes or other known schemes. Unless specifically indicated, the numbering scheme used herein to delineate the boundaries between CDRs and FRs adopts the Kabat scheme.

[0061] The terms “single-chain antibody” and “scFv” are used interchangeably herein and refer to antibodies composed of variable regions (VH) of the heavy chain and variable regions (VL) of the light chain linked by a linker. An optimal linker length and / or amino acid composition can be selected. The linker length significantly affects the folding and interactions of the variable regions of the scFv. In fact, using shorter linkers (e.g., between 5 and 10 amino acids) can prevent intra-chain folding. For information on the selection of linker size and composition, see, for example, Hollinger et al., 1993 Proc Natl Acad. Sci. USA 90:6444-6448; U.S. Patent Application Publications 2005 / 0100543, 2005 / 0175606, 2007 / 0014794; and PCT Publications WO2006 / 020258 and WO2007 / 024715, the entire contents of which are incorporated herein by reference. Commonly used connectors include GSTGSGSGKPGSGEGSTKG (SEQ ID NO: 71) and GGGGSGGGGSGGGGS (SEQ ID NO: 72). scFv can contain VH and VL connected in any order, such as VH-connector-VL or VL-connector-VH.

[0062] In one embodiment, the antibody or antigen-binding fragment of the present invention is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody, preferably a humanized antibody.

[0063] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the amino acid sequence of each heavy and light chain is homologous to a corresponding sequence from an antibody of a particular species or class, while the remaining segments of the chain are homologous to a corresponding sequence from another species or class. Generally, the variable regions of both the light and heavy chains are derived from the variable regions of antibodies from one species, while the constant regions are homologous to sequences from antibodies from another species. A significant advantage of this chimeric form is that the variable regions can be conveniently generated from currently known sources using readily available B cells or hybridomas from non-human hosts, while the combined constant regions are derived from, for example, human cells. The variable regions are easy to prepare and their specificity is unaffected by their source, while because the constant regions are derived from humans, the antibody is less likely to elicit a human immune response upon injection than when the constant regions are derived from non-human sources.

[0064] As used herein, a "humanized" antibody refers to an antibody in which all or substantially all of the CDR amino acid residues are derived from a non-human CDR and all or substantially all of the FR amino acid residues are derived from a human FR. A "humanized form" of a non-human antibody refers to a variant of the non-human antibody that has undergone humanization to generally reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are replaced by corresponding residues from a non-human antibody (e.g., an antibody derived from CDR residues) for example, to restore or improve antibody specificity or affinity.

[0065] Humanized antibodies and their preparation methods are well known to those skilled in the art, see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008). Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a "best fit" method; frame regions of common sequences of human antibodies derived from specific subgroups of light or heavy chain variable regions; human mature (somatic mutation) frame regions or human germline frame regions; and frame regions obtained by screening FR libraries.

[0066] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of this invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis or by in vivo somatic mutations).

[0067] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof targeting MSLN, comprising:

[0068] (1) The heavy chain variable region comprising H-CDR1 selected from SEQ ID NO: 1-4, H-CDR2 selected from SEQ ID NO: 5-9, and H-CDR3 selected from SEQ ID NO: 10-13; and

[0069] (2) A light chain variable region comprising L-CDR1 selected from SEQ ID NO: 14-18, L-CDR2 selected from SEQ ID NO: 19-22, and L-CDR3 selected from SEQ ID NO: 23-27. Preferably, the antibody targeting MSLN or its antigen-binding fragment comprises:

[0070] (1) A heavy chain variable region comprising H-CDR1, H-CDR2, H-CDR3 as shown in SEQ ID NO: 1, 5, 10, or H-CDR1, H-CDR2, H-CDR3 as shown in SEQ ID NO: 2, 6, 11, or H-CDR1, H-CDR2, H-CDR3 as shown in SEQ ID NO: 3, 7, 12, or H-CDR1, H-CDR2, H-CDR3 as shown in SEQ ID NO: 4, 8, 13, or H-CDR1, H-CDR2, H-CDR3 as shown in SEQ ID NO: 1, 9, 10; and

[0071] (2) Light chain variable region, which includes L-CDR1, L-CDR2, L-CDR3 as shown in SEQ ID NO: 14, 19, 23, or L-CDR1, L-CDR2, L-CDR3 as shown in SEQ ID NO: 15, 20, 24, or L-CDR1, L-CDR2, L-CDR3 as shown in SEQ ID NO: 16, 21, 25, or L-CDR1, L-CDR2, L-CDR3 as shown in SEQ ID NO: 17, 22, 26, or L-CDR1, L-CDR2, L-CDR3 as shown in SEQ ID NO: 18, 19, 27.

[0072] In one embodiment, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with an amino acid sequence selected from SEQ ID NO: 28, 30, 32, 34, 36, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, and 124, or is selected from SEQ ID NO: 28, 30, 32, 34, 36, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, and 124. NO: 28, 30, 32, 34, 36, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, and 124 have one or more amino acid modifications (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), preferably conservative modifications of up to 10 amino acids; the light chain variable region is selected from SEQ ID. The amino acid sequences NO: 29, 31, 33, 35, 37, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, and 125 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, or are selected from SEQ ID NO: IDNO: 29, 31, 33, 35, 37, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, and 125 have one or more amino acid modifications (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), preferably up to 10 conserved modifications. Preferably, the modifications are conserved modifications, such as conserved substitutions, additions, and deletions of amino acids. In a preferred embodiment, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region selected from SEQ ID NO: 28, 30, 32, 34, 36, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121 and 124 and a light chain variable region selected from SEQ ID NO: 29, 31, 33, 35, 37, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122 and 125.

[0073] In one embodiment, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region selected from the following:

[0074] (a) The heavy chain variable region as shown in SEQ ID NO: 28 and the light chain variable region as shown in SEQ ID NO: 29;

[0075] (b) The heavy chain variable region as shown in SEQ ID NO: 30 and the light chain variable region as shown in SEQ ID NO: 31;

[0076] (c) The heavy chain variable region as shown in SEQ ID NO: 32 and the light chain variable region as shown in SEQ ID NO: 33;

[0077] (d) the heavy chain variable region as shown in SEQ ID NO: 34 and the light chain variable region as shown in SEQ ID NO: 35; or

[0078] (e) Heavy chain variable regions as shown in SEQ ID NO: 36 and light chain variable regions as shown in SEQ ID NO: 37;

[0079] (f) The heavy chain variable region as shown in SEQ ID NO: 73 and the light chain variable region as shown in SEQ ID NO: 74;

[0080] (g) Heavy chain variable region as shown in SEQ ID NO: 76 and light chain variable region as shown in SEQ ID NO: 77

[0081] (h) the heavy chain variable region as shown in SEQ ID NO: 79 and the light chain variable region as shown in SEQ ID NO: 80;

[0082] (i) the heavy chain variable region as shown in SEQ ID NO: 82 and the light chain variable region as shown in SEQ ID NO: 83;

[0083] (j) the heavy chain variable region as shown in SEQ ID NO: 85 and the light chain variable region as shown in SEQ ID NO: 86;

[0084] (k) Heavy chain variable region as shown in SEQ ID NO: 88 and light chain variable region as shown in SEQ ID NO: 89;

[0085] (l) the heavy chain variable region as shown in SEQ ID NO: 91 and the light chain variable region as shown in SEQ ID NO: 92;

[0086] (m) the heavy chain variable region as shown in SEQ ID NO: 94 and the light chain variable region as shown in SEQ ID NO: 95;

[0087] (n) Heavy chain variable region as shown in SEQ ID NO: 97 and light chain variable region as shown in SEQ ID NO: 98

[0088] (o) The heavy chain variable region as shown in SEQ ID NO: 100 and the light chain variable region as shown in SEQ ID NO: 101;

[0089] (p) Heavy chain variable region as shown in SEQ ID NO: 103 and light chain variable region as shown in SEQ ID NO: 83;

[0090] (q) The heavy chain variable region as shown in SEQ ID NO: 85 and the light chain variable region as shown in SEQ ID NO: 104;

[0091] (r) the heavy chain variable region as shown in SEQ ID NO: 106 and the light chain variable region as shown in SEQ ID NO: 107; or

[0092] (s) the heavy chain variable region as shown in SEQ ID NO: 109 and the light chain variable region as shown in SEQ ID NO: 110;

[0093] (t) the heavy chain variable region as shown in SEQ ID NO: 112 and the light chain variable region as shown in SEQ ID NO: 113;

[0094] (u) the heavy chain variable region as shown in SEQ ID NO: 115 and the light chain variable region as shown in SEQ ID NO: 116;

[0095] (v) The heavy chain variable region as shown in SEQ ID NO: 118 and the light chain variable region as shown in SEQ ID NO: 119;

[0096] (w) the heavy chain variable region as shown in SEQ ID NO: 121 and the light chain variable region as shown in SEQ ID NO: 122;

[0097] (x) The heavy chain variable region as shown in SEQ ID NO: 124 and the light chain variable region as shown in SEQ ID NO: 125.

[0098] In one embodiment, the antibody or antigen-binding fragment of the present invention has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 99%, 102, 105, 108, 111, 114, 117, 120, 123, and 126 amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with an amino acid sequence selected from SEQ ID NO: 38-42, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, and 126, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with an amino acid sequence selected from SEQ ID NO: 38-42, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, and 126. Compared to the amino acid sequences of SEQ ID NOs 38-42, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, and 126, the amino acid sequences of these sequences have one or more amino acid modifications (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids). Preferably, the modifications are conservative modifications, such as conservative substitutions, additions, and deletions of amino acids. Preferably, the amino acid sequences of the antibodies or antigen-binding fragments of the present invention are selected from SEQ ID NOs 38-42, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, and 126.

[0099] In one embodiment, the antibody or antigen-binding fragment of the present invention is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody, preferably a humanized antibody.

[0100] The present invention also provides nucleic acid molecules encoding antibodies or antigen-binding fragments thereof. Therefore, in one embodiment, the nucleic acid molecule encoding said antibody or antigen-binding fragment has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a nucleotide sequence selected from SEQ ID NO: 43-47, and the antibody or antigen-binding fragment encoded therein is capable of specifically binding to the MSLN antigen. Preferably, the nucleic acid molecule encoding said antibody or antigen-binding fragment is selected from SEQ ID NO: 43-47.

[0101] As used herein, the term "conservative modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing that amino acid sequence. These conserved modifications include conserved substitutions, additions, and deletions of amino acids. Modifications can be introduced into the chimeric antigen receptor of the present invention using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved amino acid substitution is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications can be selected, for example, based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphiphilic properties of the residues involved.

[0102] As used herein, the term sequence “identity” refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is typically expressed as a percentage. Preferably, identity is determined over the overall length of the sequences being compared. Thus, two copies having completely identical sequences have 100% identity. Those skilled in the art will recognize that several algorithms can be used to determine sequence identity, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197), and Clustal W.

[0103] In one embodiment, the anti-MSLN antibody or its antigen-binding fragment is multispecific (preferably bispecific or trispecific antibody), and further comprises one or more second antibodies that specifically bind to other antigens.

[0104] As used herein, the term "multispecific" refers to an antigen-binding protein having multiple epitope specificities (i.e., the ability to specifically bind to two, three, or more different epitopes on a single biomolecule or the ability to specifically bind to epitopes on two, three, or more different biomolecules). As used herein, the term "bispecific" indicates that an antigen-binding protein has two different antigen-binding specificities.

[0105] In one implementation, the second antibody may be in any antibody or antibody fragment form, such as a full-length antibody, Fab, Fab', (Fab')2, Fv, scFv, scFv-scFv, microantibody, biantibody, or sdAb.

[0106] Therefore, in one embodiment, the second antibody targets antigens selected from the following: CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD30, CD33, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD126, CD138, CD171, CD 179a, DR4, DR5, TAC, TEM1 / CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CD133, IFNAR1, DLL3, kappa light chain, TIM3, TSHR, CD19, BAFF-R, CLL-1, EGFRvIII, GPRC5D, tEGFR, GD2, GD3, BCMA, Tn antigen, PSMA, ROR1, FLT3, FAP, TAG72 CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-llRa, IL-22Ra, IL-2, mesothelin, PSCA, PRSS21, VEGFR2, LewisY, PDG FR-β, SSEA-4, AFP, Folate receptor α, ErbB2 (Her2 / neu), ErbB3, ErbB4, MUC1, MUC16, EGFR, CS1, NCAM, Claudin18.2. c-Met, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gpl00, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM7R, CLDN6, MSLN, CXORF61, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, MAGE-A3, MAGE-A6, pod protein, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie 2. MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, PSA, survival protein and telomerase, PCTA-1 / Galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, TMPRSS2ETS fusion gene, NA17, PAX3, androgen receptor, progesterone receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGFβ, APRIL, NKG2D, NKG2D ligands, and / or pathogen-specific antigens, biotinylated molecules, molecules expressed by HIV, HCV, HBV, and / or other pathogens; and / or novel epitopes or novel antigens.

[0107] Nucleic acid, vector, host cell

[0108] In another aspect, the present invention relates to a nucleic acid molecule encoding the anti-MSLN antibody of the present invention. The nucleic acid of the present invention may be RNA, DNA, or cDNA. According to one embodiment of the present invention, the nucleic acid of the present invention is a substantially isolated nucleic acid.

[0109] In one embodiment, the nucleic acid molecule encoding the anti-MSLN antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequences selected from SEQ ID NO: 43-47, and the encoded anti-MSLN antibody is capable of specifically binding to MSLN (i.e., hardly binding to non-target antigens). Preferably, the nucleic acid molecule encoding the anti-MSLN antibody is as shown in SEQ ID NO: 43-47.

[0110] The nucleic acids of the present invention may also be in the form of a vector, which may be present in and / or part of a vector, such as a plasmid, a sticky-terminal plasmid, or a YAC. The vector may be, in particular, an expression vector, providing a means for expressing MSLN antibodies in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). This expression vector typically contains at least one nucleic acid molecule of the present invention operably linked to one or more suitable expression regulatory elements (e.g., promoters, enhancers, terminators, etc.). Selection of said regulatory elements and their sequences for expression in a particular host is well known to those skilled in the art. Specific examples of regulatory elements and other elements useful or necessary for the expression of the MSLN antibodies of the present invention include, but are not limited to, promoters, enhancers, terminators, integrators, selection markers, leader sequences, and reporter genes.

[0111] In another aspect, the present invention also provides host cells expressing the MSLN antibody, multispecific antibody, and / or containing the nucleic acid or vector of the present invention. Preferred host cells of the present invention are bacterial cells, fungal cells, or mammalian cells.

[0112] Suitable bacterial cells include Gram-negative bacterial strains (such as Escherichia coli, Proteus, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus, Streptomyces, Staphylococcus, and Lactococcus strains).

[0113] Suitable fungal cells include cells from species of the genera *Trichoderma*, *Neurospora*, and *Aspergillus*; or cells from species of the genera *Saccharomyces* (e.g., *Saccharomyces cerevisiae*), *Schizosaccharomyces* (e.g., *Schizosaccharomyces pombe*), *Pichia* (e.g., *Pichiapastoris* and *Pichia methanolica*), and *Hansenula*.

[0114] Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, etc.

[0115] However, the present invention may also use amphibian cells, insect cells, plant cells, and any other cells in the art used for expressing heterologous proteins.

[0116] Recombinant receptor

[0117] In another aspect, the present invention also provides a recombinant receptor comprising the anti-MSLN antibody as described above, such as a recombinant TCR receptor, a chimeric antigen receptor, a T-cell fusion protein, or a T-cell antigen coupler. Preferably, the present invention also provides a chimeric antigen receptor comprising the anti-MSLN antibody as described above.

[0118] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide that typically includes a ligand-binding domain (e.g., the antigen-binding portion of an antibody), a transmembrane domain, an optional co-stimulatory domain, and an intracellular signal transduction domain, all connected by a linker. CARs can utilize the antigen-binding properties of antibodies to specifically and reactively redirect T cells and other immune cells to a selected target in a non-MHC-restricted manner.

[0119] In one embodiment, the present invention provides a chimeric antigen receptor comprising an anti-MSLN antibody as described above or an antigen-binding fragment thereof, or containing the anti-MSLN antibody, a transmembrane domain, and an intracellular domain, wherein the intracellular domain comprises one or more co-stimulatory domains and / or primary signaling domains.

[0120] As used herein, the term "transmembrane domain" refers to a polypeptide structure that enables the expression of a chimeric antigen receptor on the surface of immune cells (e.g., lymphocytes, NK cells, or NKT cells) and guides the cellular response of immune cells against target cells. Transmembrane domains can be natural or synthetic and can be derived from any membrane-binding or transmembrane protein. When a chimeric antigen receptor binds to a target antigen, the transmembrane domain enables signal transduction. Transmembrane domains particularly suitable for use in this invention can be derived from, for example, the TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and functional fragments thereof. Alternatively, transmembrane domains can be synthetic and may primarily contain hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from the CD8α chain or CD28, and has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:49 or 51, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO:50 or 52.

[0121] In one embodiment, the costimulatory domain may be an intracellular functional signaling domain derived from the costimulatory molecule, comprising the entire intracellular portion of the costimulatory molecule or a functional fragment thereof. A “costimulatory molecule” refers to a homologous binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., proliferation) of the T cell. Costimulatory molecules include, but are not limited to, class 1 MHC molecules, BTLA, and Toll ligand receptors. Non-limiting embodiments of the co-stimulatory domains of the present invention include, but are not limited to, intracellular regions derived from the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, MSLN, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, and ZAP70. Preferably, the co-stimulatory domain of the CAR of the present invention is derived from 4-1BB, CD28, or 4-1BB+CD28. In one embodiment, the 4-1BB co-stimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:55, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO:56. In one embodiment, the CD28 co-stimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:53, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO:54.

[0122] As used herein, the term "primary signal transduction domain" refers to a protein portion that transduces effector functional signals and directs the cell to perform a specified function. In one embodiment, the intracellular signal transduction domain of the chimeric antigen receptor of the present invention may be an intracellular region sequence of a T-cell receptor and a co-receptor that, upon antigen receptor binding, act together to initiate signal transduction, as well as any derivatives or variants of these sequences and any synthetic sequences having the same or similar function. The intracellular signal transduction domain may contain a number of immunoreceptor tyrosine-based activation motifs (ITAMs). Non-limiting embodiments of the intracellular signal transduction domain of the present invention include, but are not limited to, intracellular regions of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, MSLN9a, MSLN9b, and CD66d. In a preferred embodiment, the signal transduction domain of the CAR of the present invention may include an intracellular CD3ζ region having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 57 or 59, or its coding sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO: 58 or 60.

[0123] In one embodiment, the chimeric antigen receptor of the present invention may further comprise a hinge region located between the antibody and the transmembrane domain. As used herein, the term "hinge region" generally refers to any oligopeptide or polypeptide that functions to connect the transmembrane domain to the antibody. Specifically, the hinge region is used to provide greater flexibility and accessibility to the antibody. The hinge region may contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. The hinge region may be wholly or partially derived from natural molecules, such as the extracellular regions of CD8, CD4, or CD28, or wholly or partially derived from the antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or it may be a fully synthetic hinge sequence. In a preferred embodiment, the hinge region comprises a hinge region portion of CD8α, CD28, FcγRIIIα receptor, IgG4, or IgG1, more preferably a CD8α, CD28, or IgG4 hinge, having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 65, 67, or 69, or its coding sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 66, 68, or 70.

[0124] In one embodiment, the CAR of the present invention may further comprise a signal peptide such that, when expressed in cells such as T cells, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface. The core of the signal peptide may contain a long, hydrophobic amino acid segment with a tendency to form a single α-helix. At the terminal end of the signal peptide, there is typically an amino acid segment that is recognized and cleaved by a signal peptidase. The signal peptidase may cleave the peptide during or after translocation to produce a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease. Signal peptides that can be used in the present invention are well known to those skilled in the art, such as signal peptides derived from B2M, CD8α, IgG1, GM-CSFRα, etc. In one embodiment, the signal peptide used in this invention is derived from B2M or CD8α, having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 61 or 63, or its coding sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO: 62 or 64.

[0125] In one embodiment, the CAR contains an anti-MSLN antibody or its antigen-binding fragment as provided herein, a CD8α or CD28 transmembrane region, a CD28 and / or 4-1BB co-stimulatory domain, and a CD3ζ intracellular signal transduction domain. In this embodiment, the CAR may further contain a signal peptide from B2M, CD8α, IgG1, or GM-CSFRα.

[0126] The present invention also provides a nucleic acid molecule encoding a chimeric antigen receptor targeting MSLN as defined above, and a vector comprising said nucleic acid molecule.

[0127] As used herein, the term "vector" is a medium nucleic acid molecule used to transfer (exogenous) genetic material into a host cell, in which the nucleic acid molecule may, for example, be replicated and / or expressed. Vectors generally include targeting vectors and expression vectors. A "targeting vector" is a medium for delivering isolated nucleic acids into the cell interior by, for example, homologous recombination or using a hybrid recombinase with a specific target site sequence. An "expression vector" is a vector used for the transcription of heterologous nucleic acid sequences (e.g., those encoding the chimeric antigen receptor polypeptide of the present invention) in a suitable host cell and for the translation of their mRNA. Suitable vectors for use in the present invention are known in the art and many are commercially available. In one embodiment, the vectors of the present invention include, but are not limited to, plasmids, viruses (e.g., retroviruses, lentiviruses, adenoviruses, vaccinia virus, Raul's sarcoma virus (RSV, polyomavirus, and adeno-associated virus (AAV) etc.), bacteriophages, phage particles, granules, and artificial chromosomes (including BAC and YAC). The vector itself is typically a nucleic acid molecule, usually consisting of a DNA sequence containing an insert (transgenic) and a larger sequence serving as the vector's "backbone." Engineered vectors typically also include an origin of autonomous replication in the host cell (if stable expression of the polynucleotide is desired), a selection marker, and a restriction enzyme cleavage site (e.g., a multiple cloning site, MCS). The vector may additionally include elements such as a promoter, polyA tail, 3'UTR, enhancer, terminator, insulator, operon, selection marker, reporter gene, target sequence, and / or protein purification tag. In one specific embodiment, the vector is an in vitro transcription vector.

[0128] Engineered immune cells

[0129] In one aspect, the present invention also provides engineered immune cells expressing the recombinant receptors (e.g., chimeric antigen receptors) described herein.

[0130] As used herein, the term "immune cell" refers to any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell-killing activity, secretion of cytokines, induction of ADCC and / or CDC). For example, immune cells can be T cells, macrophages, dendritic cells, monocytes, NK cells, and / or NKT cells. In one embodiment, the immune cell is derived from stem cells, such as adult stem cells, embryonic stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells. Preferably, the immune cell is a T cell. The T cell can be any T cell, such as cultured T cells, such as primary T cells, or T cells derived from cultured T cell lines such as Jurkat, SupT1, etc., or T cells obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species. T cells can be obtained from a variety of sources, including peripheral blood monocytes, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be concentrated or purified. T cells can be at any developmental stage, including but not limited to CD4+CD8+ T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), CD4-CD8- T cells, tumor-infiltrating cells, memory T cells, naive T cells, γδ-T cells, αβ-T cells, etc. In a preferred embodiment, the immune cells are human T cells. Various techniques known to those skilled in the art, such as Ficoll isolation, can be used to obtain T cells from the subject's blood.

[0131] In one embodiment, to reduce the risk of graft-versus-host disease, the engineered immune cells further comprise at least one gene selected from the following whose expression is suppressed or silenced: CD52, GR, dCK, TCR / CD3 genes (e.g., TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ), MHC-related genes (HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA), and immune checkpoint genes such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTP. N22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFBRRI, SMAD2, SMAD 3. SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3. Preferably, the engineered immune cells further comprise at least one gene selected from the following whose expression is suppressed or silenced: TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, PD1, LAG3, TIM3, CTLA4, more preferably TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA.

[0132] Methods for suppressing gene expression or silencing genes are well known to those skilled in the art. For example, gene expression can be suppressed using antisense RNA, RNA decoys, RNA aptamers, siRNA, shRNA / miRNA, trans dominant-negative proteins (TNPs), chimeric / antibody conjugates, chemokine ligands, anti-infective cellular proteins, intracellular antibodies (sFv), nucleoside analogs (NRTIs), non-nucleoside analogs (NNRTIs), integrase inhibitors (oligonucleotides, dinucleotides, and chemical agents), and protease inhibitors. Alternatively, gene silencing can also be achieved by mediating DNA breaks through, for example, broad-spectrum nucleases, zinc finger nucleases, TALE nucleases, or Cas enzymes in the CRISPR system.

[0133] In one embodiment, the engineered immune cells further comprise a second recombinant receptor targeting other tumor antigens, such as a recombinant TCR receptor or a chimeric antigen receptor. The other tumor antigens targeted by the second recombinant receptor may be selected from, for example, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD30, CD33, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD126, CD138, CD171, CD40L ... 179a, DR4, DR5, TAC, TEM1 / CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CD133, IFNAR1, DLL3, kappa light chain, TIM3, TSHR, CD19, GPRC5D, BAFF-R, CLL-1, EGFRvIII, tEGFR, GD2, GD3, BCMA, Tn antigen, PSMA, ROR1, FLT3, FAP, TAG72 CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-llRa, IL-22Ra, IL-2, mesothelin, PSCA, PRSS21, VEGFR2, LewisY, PDG FR-β, SSEA-4, AFP, Folate receptor α, ErbB2 (Her2 / neu), ErbB3, ErbB4, MUC1, MUC16, EGFR, CS1, NCAM, Claudin18.2. c-Met, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gpl00, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM7R, CLDN6, MSLN, CXORF61, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, MAGE-A3, MAGE-A6, pod protein, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie 2. MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, PSA, survival protein and telomerase, PCTA-1 / Galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, TMPRSS2ETS fusion gene, NA17, PAX3, androgen receptor, progesterone receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES 1. LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, muthsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGFβ, APRIL, NKG2D, NKG2D ligands, and / or pathogen-specific antigens, biotinylated molecules, and molecules expressed by HIV, HCV, HBV, and / or other pathogens.

[0134] In one embodiment, a plurality of immune cells are provided, each of which is engineered to express one or more chimeric antigen receptors. For example, in some embodiments, one immune cell is engineered to express a chimeric antigen receptor that binds to and / or targets MSLN (e.g., a CAR comprising the anti-MSLN antibody described in this invention), and another cell is engineered to express a chimeric antigen receptor that binds to and / or targets other antigens. In one embodiment, the immune cells may also express a multispecific chimeric antigen receptor that targets one or more antigens, including MSLN. For example, such a multispecific chimeric antigen receptor may comprise a multispecific antibody targeting MSLN, or simultaneously comprise the anti-MSLN antibody described in this invention and an antibody targeting other antigens. In such embodiments, the plurality of engineered immune cells may be administered together or separately. In one embodiment, the plurality of immune cells may be in the same composition or in different compositions. Exemplary compositions of cells include those described in the following sections of this application.

[0135] Antibody conjugates

[0136] In one aspect, the present invention provides an antibody conjugate comprising an anti-MSLN antibody as defined in the present invention and a second functional structure, wherein the second functional structure is selected from Fc, radioisotopes, structural portions with extended half-life, detectable markers, and drugs.

[0137] In one embodiment, the present invention provides an antibody conjugate comprising an anti-MSLN antibody as defined herein and an Fc. As used herein, the term "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain, including native Fc and variant Fc. "Native Fc" refers to a molecule or sequence comprising a non-antigen-binding fragment, whether in monomeric or multimeric form, generated by digesting an intact antibody. The immunoglobulin source that generates native Fc is preferably derived from humans. Native Fc fragments consist of monomeric polypeptides that can be linked covalently (e.g., disulfide bonds) and non-covalently into dimer or multimer forms. Depending on the class (e.g., IgG, IgA, IgE, IgD, IgM) or subtype (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2), the native Fc molecule has 1-4 intermolecular disulfide bonds between its monomeric subunits. An example of a natural Fc is a disulfide-linked dimer produced by digesting IgG with papain (see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). As used herein, the term “natural Fc” generally refers to monomeric, dimeric, and polymeric forms. A “variant Fc” is an amino acid sequence that differs from the amino acid sequence of a “natural” or “wild-type” Fc due to at least one “amino acid modification” as defined herein, also referred to as an “Fc variant.” Therefore, “Fc” also includes single-chain Fc (scFc), i.e., a single-chain Fc consisting of two Fc monomers linked by a polypeptide linker, capable of naturally folding into a functional dimer Fc region. In one embodiment, the Fc is preferably a human immunoglobulin Fc, more preferably a human IgG1 Fc.

[0138] In one embodiment, the present invention provides an antibody conjugate comprising an anti-MSLN antibody as defined herein and a radioactive isotope. Examples of radioactive isotopes that can be used in the present invention include, but are not limited to, At. 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32 Pb 212 99mTc 123 I, 18 F and 68 Ga.

[0139] In one embodiment, the present invention provides an antibody conjugate comprising an anti-MSLN antibody as defined in the present invention and a structural portion for extending half-life, wherein the structural portion for extending half-life is selected from albumin-binding structures, transferrin-binding structures, polyethylene glycol molecules, recombinant polyethylene glycol molecules, human serum albumin, fragments of human serum albumin, and albumin polypeptides (including antibodies) that bind to human serum albumin.

[0140] In one embodiment, the present invention provides an antibody conjugate comprising an anti-MSLN antibody as defined herein and a detectable marker. The term "detectable marker" herein refers to a compound that generates a detectable signal. For example, a detectable marker may be an MRI contrast agent, a scintillation scanning contrast agent, an X-ray imaging contrast agent, an ultrasound contrast agent, or an optical imaging contrast agent. Examples of detectable markers include fluorophores (such as fluorescein, Alexa, or anthocyanins), chemiluminescent compounds (such as luminol), bioluminescent compounds (such as luciferase or alkaline phosphatase), enzymes (such as horseradish peroxidase, glucose-6-phosphatase, β-galactosidase), antibiotic resistance genes (such as kanamycin, ampicillin, chloramphenicol, tetracycline, etc.), and contrast agents (such as nanoparticles or gadolinium). Those skilled in the art can select appropriate detectable markers based on the detection system used.

[0141] In one embodiment, the present invention provides an antibody-drug conjugate comprising an anti-MSLN antibody as defined herein and a drug, such as a cytotoxin or immunomodulator, conjugated to said anti-MSLN antibody (i.e., an antibody-drug conjugate). Typically, the drug is covalently linked to the antibody and is usually dependent on a linker. In one embodiment, the drug is a cytotoxin. In another embodiment, the drug is an immunomodulator. Examples of cytotoxins include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine, nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), 1-methylnitrosourea, cyclophosphamide, nitrogen mustard, busulfan, dibromomannitol, streptozotocin, mitomycin, cis-dichlorodiamineplatin(II) (DDP), cisplatin, carboplatin, zolrubicin, doxorubicin, detoxin, carminoxetine, idarubicin, epirubicin, mitoxantrone, and actinomycin. Bleomycin D, bleomycin, salinomycin, sclerosomycin, atrazocin (AMC), vincristine, vinblastine, paclitaxel, ricin, Pseudomonas exotoxin, gemcitabine, cytochalasin B, bacitracin D, ethidium bromide, emetine, etoposide, teniposide, colchicine, dihydroxyanthradinone, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (O,P'-(DDD)), interferon, and combinations thereof. Examples of immunomodulators include, but are not limited to, ganciclovir, etanercept, tacrolimus, sirolimus, vorciclosporine, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, glucocorticoids and their analogues, cytokines, stem cell growth factors, lymphotoxins, tumor necrosis factor (TNF), hematopoietic factors, interleukins (e.g., IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony-stimulating factors (e.g., G-CSF and (GM-CSF), interferons (e.g., interferon-α, interferon-β, and interferon-γ), stem cell growth factors named "S1 factor", erythropoietin, and thrombopoietin, or combinations thereof.

[0142] reagent kits and pharmaceutical compositions

[0143] In another aspect, the present invention also provides a detection kit comprising the antibodies, antibody-drug conjugates, or chimeric antigen receptors described herein.

[0144] In another aspect, the present invention also provides a pharmaceutical composition comprising the antibody described herein, a recombinant receptor such as a chimeric antigen receptor, engineered immune cells or antibody-drug conjugates, and one or more pharmaceutically acceptable excipients.

[0145] As used herein, the term "pharmaceuticalally acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient (i.e., capable of eliciting the desired therapeutic effect without causing any undesirable local or systemic effects), which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coatings, adsorbents, anti-adhesion agents, flow aids, antioxidants, flavoring agents, coloring agents, sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delay agents, stabilizers, and tension modifiers. Those skilled in the art know how to select suitable excipients to prepare the desired pharmaceutical compositions of the present invention. Exemplary excipients used in the pharmaceutical compositions of the present invention include saline, buffered saline, glucose, and water. Typically, the selection of a suitable excipient depends in particular on the active agent used, the disease to be treated, and the desired dosage form of the pharmaceutical composition.

[0146] The pharmaceutical compositions according to the invention are suitable for administration via a variety of routes. Typically, administration is performed via parenteral delivery. Parenteral delivery methods include local, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.

[0147] The pharmaceutical compositions according to the invention can also be prepared in various forms, such as solid, liquid, gaseous, or lyophilized forms, particularly as ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures, or fluid extracts, or in forms particularly suitable for the desired method of administration. Processes known in this invention for manufacturing pharmaceuticals may include, for example, conventional mixing, dissolving, granulation, coating, grinding, emulsification, encapsulation, embedding, or lyophilization processes. Pharmaceutical compositions containing, for example, immune cells as described herein, are generally provided in solution form and preferably contain pharmaceutically acceptable buffers.

[0148] The pharmaceutical compositions according to the invention can also be administered in combination with one or more other pharmaceutical agents suitable for treating and / or preventing the disease to be treated. Preferred examples of pharmaceutical agents suitable for combination include known anticancer drugs, such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, and orlistatine E. E) vincristine and doxorubicin; peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, DNases and RNases; radionuclides, such as iodine-131, rhenium-186, indium-111, iridium-90, bismuth-210 and 213, actinium-225 and astatine-213; prodrugs, such as antibody-directed enzyme prodrugs; immunostimulants, such as platelet factor 4, melanoma growth stimulating protein, etc.; antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral / bacterial protein domains, and viral / bacterial peptides. Furthermore, the pharmaceutical compositions of the present invention can also be used in combination with one or more other treatment methods, such as chemotherapy and radiotherapy.

[0149] Therapeutic / Preventive / Diagnostic Uses

[0150] In another aspect, the present invention also provides a method for treating and / or preventing and / or diagnosing diseases associated with MSLN expression, comprising administering to a subject an antibody, chimeric antigen receptor, antibody-drug conjugate, engineered immune cell, or pharmaceutical composition as described above.

[0151] In one implementation, diseases associated with MSLN expression include, but are not limited to, MSLN-positive adenocarcinoma, mesothelioma, lung cancer, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, bile duct cancer, gallbladder cancer, esophageal cancer, melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, rectal cancer, Hodgkin's lymphoma, pancreatic cancer, or prostate cancer.

[0152] The present invention will now be described in detail with reference to the accompanying drawings and examples. It should be noted that those skilled in the art should understand that the drawings and embodiments of the present invention are merely illustrative and do not constitute any limitation on the present invention. Where there is no contradiction, the embodiments and features described in this application can be combined with each other. Attached Figure Description

[0153] Figure 1The study showed that five mouse-derived MSLN antibodies specifically bound to CHO-MSLN cells.

[0154] Figure 2 This shows the CAR expression level in murine CAR T cells targeting MSLN.

[0155] Figure 3 This study demonstrates the killing effect of mouse CAR T cells targeting MSLN on target cells Huh7-Meso and non-target cells A549-CBG at various effector-to-target ratios.

[0156] Figure 4 This study demonstrates the degranulation effect of mouse CAR T cells targeting MSLN co-cultured with target cells A549-CBG-Meso, Hela-luci, Huh7-Meso-luci, and non-target cells A549-CBG.

[0157] Figure 5 The results show the IL2 release levels of mouse CAR T cells targeting MSLN after co-culturing with target cells A549-CBG-Meso, Hela-luci, Huh7-Meso-luci and non-target cells A549-CBG.

[0158] Figure 6 The study showed the IFNγ release levels of mouse CAR T cells targeting MSLN after co-culturing with target cells A549-CBG-Meso, Hela-luci, Huh7-Meso-luci, and non-target cells A549-CBG.

[0159] Figure 7 This study demonstrates the tumor-suppressive effect of murine CAR T cells targeting MSLN in mice.

[0160] Figure 8 This study demonstrates the CAR expression level in humanized CAR T cells targeting MSLN.

[0161] Figure 9 This study demonstrates the killing effect of humanized CAR T cells targeting MSLN on target cells HeLa cells, Huh7-Meso cells, and non-target cells Huh7 cells at various effector-to-target ratios.

[0162] Figure 10 This study demonstrates the degranulation effect of co-cultured humanized CAR T cells targeting MSLN with target cells HeLa cells, A549-CBG-Meso cells, Huh7-Meso cells, and non-target cells A549-CBG cells and Huh7 cells.

[0163] Figure 11The study showed the IL2 release levels after co-culturing humanized CAR T cells targeting MSLN with target cells HeLa cells, A549-CBG-Meso cells, Huh7-Meso cells, and non-target cells A549-CBG cells and Huh7 cells.

[0164] Figure 12 The study showed the IFNγ release levels after co-culturing humanized CAR T cells targeting MSLN with target cells HeLa cells, A549-CBG-Meso cells, Huh7-Meso cells, and non-target cells A549-CBG cells and Huh7 cells. Detailed Implementation

[0165] Example 1. Screening for anti-MSLN antibodies

[0166] Age-appropriate Balb / c mice were immunized with hMSLN-Fc protein (Acrobiosystems, catalog number MSN-H526x, where the amino acid sequence of hMSLN is shown in SEQ ID NO: 48), followed by repeated immunizations every 2–3 weeks for a total of 4 times. Then, mouse spleen lymphocytes were collected, mixed with SP2 / 0 myeloma cells, and PEG-mediated cell fusion was added to prepare hybridoma cells. MSLN-overexpressing cell lines (CHO-MSLN cells) were screened for hybridoma clones binding to MSLN using ELISA or flow cytometry. After multiple rounds of screening, five antibody clones specifically binding to MSLN were obtained. The amino acid and nucleic acid sequences of the five clones were sequenced. The heavy chain variable region (VH) and light chain variable region (VL) of each clone were ligated using adapters to obtain five single-chain antibodies, named 001, 002, 003, 004, and 005, with sequences shown in Table 1 below.

[0167] Table 1. Amino acid and nucleic acid sequences of the five scFvs

[0168]

[0169]

[0170] The five scFvs were transiently transfected into 293 cells to express antibodies, and the resulting antibodies were used to stain CHO-MSLN cells. The results were then analyzed by flow cytometry. Figure 1 As shown in the figure, all the antibodies detected strongly bound to CHO-MSLN cells but not to CHO cells, indicating that their binding to MSLN is specific.

[0171] Example 2. Preparation of CAR-T cells targeting MSLN and verification of their function

[0172] 2.1 Preparation of CAR-T cells

[0173] The sequences encoding the following proteins were synthesized and cloned into the pLVX vector (Public Protein / Plasmid Library (PPL), catalog number: PPL00157-4a): CD8α signal peptide (SEQ ID No: 63), anti-MSLN single-chain antibody (one of SEQ ID No: 38-42), CD8α hinge region (SEQ ID No: 65), CD8α transmembrane region (SEQ ID No: 49), 4-1BB intracellular region (SEQ ID No: 55), and CD3ζ intracellular region (SEQ ID No: 57). The correct insertion of the target sequences was confirmed by sequencing.

[0174] Add 3 ml of Opti-MEM (Gibco, catalog number 31985-070) to a sterile tube to dilute the plasmid. Then, add the packaging vector psPAX2 (Addgene, catalog number 12260) and the envelope vector pMD2.G (Addgene, catalog number 12259) in a plasmid:viral packaging vector:viral envelope vector ratio of 4:2:1. Next, add 120 μL of X-treme GENE HP DNA transfection reagent (Roche, catalog number 06366236001), mix immediately, and incubate at room temperature for 15 min. Then, add the plasmid / vector / transfection reagent mixture dropwise to a culture flask of 293T cells. Collect the virus at 24 and 48 hours, combine them, and then ultracentrifuge (25000g, 4℃, 2.5 h) to obtain concentrated lentivirus.

[0175] T cells were activated using DynaBeads CD3 / CD28 CTSTM (Gibco, catalog number 40203D) and cultured at 37°C and 5% CO2 for 1 day. Then, concentrated lentivirus was added, and after 3 days of continuous culture, five CAR-T cell lines targeting MSLN were obtained, named BH28-001, BH28-002, BH28-003, BH28-004, and BH28-005 CAR-T cells. Unmodified wild-type T cells (NT) served as a control.

[0176] After culturing at 37°C and 5% CO2 for 11 days, the expression level of anti-MSLN single-chain antibody on CAR-T cells was detected by flow cytometry using FITC-Rabbit anti-mouse IgG, F(ab')specific (Jackson Immunoresearch, catalog number 315-095-006). The results are as follows: Figure 2 As shown.

[0177] It can be seen that the anti-MSLN single-chain antibody prepared in this invention can be effectively expressed in CAR-T cells.

[0178] 2.2 Detection of the killing effect of CAR-T cells on target cells

[0179] With 1x10 4 The target cells (Huh7-Meso cells) or non-target cells (A549-CBG cells) were seeded into 96-well plates at a concentration of cells per well. Then, NT cells and CAR-T cells were seeded into 96-well plates at effector-to-target ratios (8:1, 4:1, 2:1, 1:1, and 0.5:1) for co-culture. Fluorescence values ​​were measured using a microplate reader after 16-18 hours. The killing efficiency was calculated using the formula: (mean fluorescence value of target cells - mean fluorescence value of sample cells) / mean fluorescence value of target cells × 100%. The results are shown below. Figure 3 As shown.

[0180] It can be seen that, under various effector-to-target ratios, the CAR T cells of the present invention exhibit strong killing effects on target cells Huh7-Meso, while the killing effect on non-target cells A549-CBG is relatively weak, indicating that each CAR-T cell only exhibits specific killing effects on cells expressing MSLN.

[0181] 2.3 Detection of CAR-T cell degranulation

[0182] With 1×10 5 Cell / well concentration: Target cells (A549-CBG-Meso cells, HeLa cells, Huh7-Meso cells) and non-target cells (A549-CBG cells) were seeded separately in 96-well plates. Various CAR T cells or NT cells (negative controls) were added at a 1:1 ratio. Then, 10 μL of PE Mouse anti-human CD107a antibody (BD, catalog number 555801) was added to each well, and the plates were incubated at 37°C and 5% CO2 in the dark. After 1 h, 20 μL of Golgi Stop (BD, catalog number 51-2092K2) was added to each well, and the plates were incubated at 37°C and 5% CO2 in the dark for 2.5 h. Then, 10 μL of APC anti-human CD8 (BD, catalog number 555369) was added to each well, and the plates were incubated at 37°C and 5% CO2 in the dark for 0.5 h. Cell samples from each well were analyzed by flow cytometry, and the proportion of CD107a+CD8+ cells to T cells was determined. Results Figure 4 As shown.

[0183] As can be seen, compared with NT cells, the CAR-T cells prepared in this invention showed significantly increased specific degranulation effects on the three target cell types: A549-CBG-Meso cells, HeLa cells, and Huh7-Meso cells.

[0184] 2.4 Detection of cytokine release levels in CAR-T cells

[0185] With 1x10 5 The concentration of cells / well was determined by seeding target cells (A549-CBG-Meso cells, HeLa cells, Huh7-Meso cells) and non-target cells (A549-CBG cells) in 96-well plates. BH395-CAR T cells, BH398-CAR T cells, or NT cells (negative control) were added at a 1:1 ratio. After co-culturing for 18-24 hours, the cell co-culture supernatant was collected.

[0186] As recommended by the manufacturer, the levels of IL2 and IFN-γ in the co-culture supernatant were detected using the Human IL-2 DuoSet ELISA Kit (R&D systems, catalog number DY202) and the Human IFN-gamma DuoSet ELISA Kit (R&D systems, catalog number DY285), respectively. The results are as follows: Figure 5 and Figure 6 As shown.

[0187] It can be seen that, compared with NT cells, the various CAR T cells of the present invention, after co-culturing with target cells, showed an increase in the cytokine IL2 (… Figure 5 ) and IFN-γ Figure 6 The release levels of ) were significantly increased, and this cytokine release was specific.

[0188] Example 3. Verification of the tumor-suppressive effect of CAR-T cells

[0189] Eighteen healthy female NPI mice aged 7 weeks were randomly divided into three groups of 6 mice each: NT group (negative control), BH28-003 group, and BH28-004 group. On day 0 (D0), each mouse was injected intravenously with 6 × 10⁶ NPI solution. 6 100 HeLa cells. Six days later (D6), each mouse was injected via tail vein with 2 x 10 cells, according to the group assignment. 6 NT cells or corresponding CAR-T cells. Tumor burden in mice was assessed weekly, and results were as follows: Figure 7 As shown.

[0190] As can be seen, in the NT group, the tumor burden in mice progressed rapidly throughout the experiment. Conversely, treatment with the CAR-T cells prepared according to this invention significantly inhibited tumor growth in tumor-bearing mice. Notably, in the BH28-004 group, the tumor burden decreased rapidly within days of CAR-T cell treatment, eventually disappearing, and remained at a very low level throughout the experiment without recurrence. This indicates that the CAR-T cells of this invention can effectively target and kill tumor cells, demonstrating a significant therapeutic effect on tumors.

[0191] Example 4. Humanization of mouse anti-human MSLN antibody

[0192] The murine antibodies BH28-003 and BH28-004 were humanized using the following method: First, human antibody sequences with high similarity were searched using the IGBLAST database (https: / / www.ncbi.nlm.nih.gov / igblast / ). Then, the FR region in the single-chain antibody was replaced with the corresponding human sequence. Next, individual amino acid residues were replaced according to their different physicochemical properties, and finally, multiple humanized single-chain antibodies were obtained. Their amino acid sequences are shown in Table 2.

[0193] Table 2. Sequences of MSLN humanized antibodies

[0194]

[0195]

[0196] Example 5. Preparation of humanized CAR-T cells and verification of their function

[0197] 5.1 Preparation of CAR-T cells

[0198] CAR-T cells were prepared using humanized antibodies according to the method described in 3.1, resulting in CAR-T cells containing anti-MSLN humanized antibodies: BH28-3V0, BH28-3V1, BH28-3V2, BH28-3V3, BH28-3V4, BH28-3V5, BH28-3V6, BH28-3V7, BH28-3V8, BH28-4V0, BH28-4V1, BH28-4V2, BH28-4V3, BH28-4V4, BH28-4V5, BH28-4V6, BH28-4V7, and BH28-4V8. Mouse CAR-T cells BH28-003 and BH28-004, as well as unmodified wild-type T cells (NT), were used as controls.

[0199] After culturing at 37℃ and 5% CO2 for 10 days, the expression level of anti-MSLN humanized antibody on CAR-T cells was detected by flow cytometry using FITC-Rabbit anti-mouse IgG, F(ab')specific (Jackson Immunoresearch, catalog number 315-095-006). The results are as follows: Figure 8 As shown.

[0200] It can be seen that the anti-MSLN humanized scFv in each humanized CAR-T cell prepared in this invention can be effectively expressed.

[0201] 5.2 Detection of the killing effect of CAR-T cells on target cells

[0202] With 1x10 4 The concentration of cells / well was determined by seeding target cells (HeLa cells, Huh7-Meso cells) or non-target cells (Huh7 cells) expressing the luciferase gene into 96-well plates. Then, NT cells and CAR T cells were seeded into 96-well plates at effector-to-target ratios of 16:1, 8:1, 4:1, and 2:1 for co-culture. Fluorescence values ​​were measured using a microplate reader after 16-18 hours. The killing efficiency was calculated using the formula: (mean fluorescence value of target cells - mean fluorescence value of sample cells) / mean fluorescence value of target cells × 100%. The results are shown below. Figure 9 As shown.

[0203] It can be seen that, under various effector-target ratios, the CAR T cells of the present invention exhibit strong killing effects on target cells HeLa cells and Huh7-Meso cells, while the killing effect on non-target cells Huh7 cells is weaker, indicating that each CAR T cell only exhibits specific killing effect on cells expressing MSLN.

[0204] 5.3 Detection of CAR-T cell degranulation

[0205] With 1×10 5To determine the cell / well concentration, target cells (HeLa cells, A549-CBG-Meso cells, Huh7-Meso cells) and non-target cells (A549-CBG cells, Huh7 cells) were seeded separately in 96-well plates. CAR T cells and NT cells (negative controls) were added at a 1:1 ratio. Then, 10 μL of PE Mouse anti-human CD107a antibody (BD, catalog number 555801) was added to each well, and the plates were incubated at 37°C and 5% CO2 in the dark. After 1 h, 20 μL of Golgi Stop (BD, catalog number 51-2092K2) was added to each well, and the plates were incubated at 37°C and 5% CO2 in the dark for 2.5 h. Finally, 10 μL of APC anti-human CD8 (BD, catalog number 555369) was added to each well, and the plates were incubated at 37°C and 5% CO2 in the dark for 0.5 h. Cell samples from each well were analyzed by flow cytometry, and the proportion of CD107a-positive cells and CD107a-CD8 double-positive cells among T cells was analyzed. The results are as follows: Figure 10 As shown.

[0206] As can be seen, compared with NT cells, the humanized CAR T cells prepared in this invention showed significantly increased specific degranulation activity on target cells (A549-CBG-Meso cells, HeLa cells, and Huh7-Meso cells), while no significantly increased degranulation activity was observed on non-target cells (A549-CBG cells and Huh7 cells).

[0207] 5.4 Detection of cytokine release levels in CAR-T cells

[0208] With 1x10 5 The concentration of cells / well was determined by seeding target cells (HeLa cells, A549-CBG-Meso cells, Huh7-Meso cells) and non-target cells (A549-CBG cells, Huh7 cells) into 96-well plates. CAR T cells and NT cells (negative control) were added at a 1:1 ratio. After co-culturing for 18-24 hours, the cell co-culture supernatant was collected.

[0209] As recommended by the manufacturer, the levels of IL2 and IFN-γ in the co-culture supernatant were detected using the Human IL-2 DuoSet ELISA Kit (R&D systems, catalog number DY202) and the Human IFN-gamma DuoSet ELISA Kit (R&D systems, catalog number DY285), respectively. The results are as follows: Figure 11 and Figure 12 As shown.

[0210] It can be seen that, compared with NT cells, the CAR T cells of the present invention, after co-culturing with target cells, showed an increase in the cytokine IL2 ( Figure 11 ) and IFN-γ Figure 12 The release levels of ) were significantly increased, and this cytokine release was specific.

[0211] The above results indicate that the anti-MSLN murine antibody and humanized antibody of the present invention can specifically bind to the MSLN protein, and the CAR T cells prepared using the above-mentioned anti-MSLN murine antibody and humanized antibody can effectively kill tumor target cells in vitro and in vivo.

[0212] It should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. It is understood by those skilled in the art that any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody targeting MSLN, comprising: (1) Heavy chain variable regions, comprising H-CDR1, H-CDR2, and H-CDR3 as shown in SEQ ID NO: 4, 8, and 13, respectively; and (2) Light chain variable region, which includes L-CDR1, L-CDR2 and L-CDR3 as shown in SEQ ID NO: 17, 22 and 26 respectively.

2. The antibody of claim 1, wherein the heavy chain variable region has at least 90% identity with an amino acid sequence selected from SEQ ID NO: 34, 100, 103, 106, 109, 112, 115, 118, 121 and 124, or has one or more conserved modifications of amino acids compared to an amino acid sequence selected from SEQ ID NO: 34, 100, 103, 106, 109, 112, 115, 118, 121 and 124; and the light chain variable region has at least 90% identity with an amino acid sequence selected from SEQ ID NO: 35, 101, 104, 107, 110, 113, 116, 119, 122 and 125, or has one or more conserved modifications of amino acids compared to an amino acid sequence selected from SEQ ID NO: 35, 101, 104, 107, 110, 113, 116, 119, 122 and 125.

3. The antibody of claim 1, wherein the antibody is selected from intact antibodies, Fab, Fab', F(ab')2 and scFv.

4. The antibody according to any one of claims 1-3, wherein the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

5. A nucleic acid molecule encoding the antibody according to any one of claims 1-4.

6. The nucleic acid molecule of claim 5, wherein it has at least 90% sequence identity with the nucleotide sequences selected from SEQ ID NO: 43-47, and the antibody encoded therefrom is capable of specifically binding to MSLN.

7. The antibody according to any one of claims 1-4, wherein the antibody is a multispecific antibody, the multispecific antibody comprising a second antibody or its antigen-binding portion that specifically binds to other antigens.

8. The antibody of claim 7, wherein the second antibody or its antigen-binding portion is selected from full-length antibodies, Fab, Fab', F(ab')2, Fv, scFv, scFv-scFv, microantibodies, biantibodies, or sdAb.

9. A vector comprising a nucleic acid molecule encoding an antibody according to any one of claims 1-4 and 7-8.

10. A host cell expressing the antibody according to any one of claims 1-4 and 7-8, wherein the host cell is not a plant cell.

11. A chimeric antigen receptor comprising the antibody according to any one of claims 1-4 and 7-8.

12. The chimeric antigen receptor of claim 11, wherein the chimeric antigen receptor further comprises a transmembrane domain and an intracellular domain, the intracellular domain comprising one or more co-stimulatory domains and / or primary signal transduction domains.

13. The chimeric antigen receptor of claim 12, wherein the transmembrane domain is selected from the transmembrane domains of the following proteins: TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

14. The chimeric antigen receptor of claim 12, wherein the primary signal transduction domain is selected from the intracellular regions of the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d.

15. The chimeric antigen receptor of claim 12, wherein the co-stimulatory domain is selected from the intracellular regions of the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, MSLN, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134, CD137, CD270, CD272, CD276, CD278, CD357, DAP10, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, and ZAP70.

16. An engineered immune cell comprising the chimeric antigen receptor as described in any one of claims 11-15.

17. The engineered immune cells of claim 16, wherein the cells are selected from T cells, NK cells, NKT cells, macrophages, and dendritic cells.

18. The engineered immune cell of claim 16, further comprising a second chimeric antigen receptor that targets other tumor antigens.

19. The engineered immune cells according to any one of claims 16-18 further comprise that the expression of at least one gene selected from the group consisting of: TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, PD1, LAG3, TIM3, and CTLA4 is suppressed or silenced.

20. An antibody conjugate comprising the antibody according to any one of claims 1-4 and 7-8, and a second functional structure, wherein the second functional structure is selected from radioisotopes and detectable markers.

21. A detection kit comprising the antibody according to any one of claims 1-4 and 7-8, the chimeric antigen receptor according to any one of claims 11-15, or the antibody conjugate according to claim 20.

22. A pharmaceutical composition comprising an antibody according to any one of claims 1-4 and 7-8, a chimeric antigen receptor according to any one of claims 11-15, an engineered immune cell according to any one of claims 16-19, or an antibody conjugate according to claim 20, and one or more pharmaceutically acceptable excipients.

23. Use of the antibody of any one of claims 1-4 and 7-8, the chimeric antigen receptor of any one of claims 11-15, the engineered immune cell of any one of claims 16-19, the antibody-drug conjugate of claim 20, or the pharmaceutical composition of claim 22 in the preparation of a medicament for treating and / or diagnosing diseases associated with MSLN expression, said diseases being selected from MSLN-positive mesothelioma, lung cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, bile duct cancer, gallbladder cancer, esophageal cancer, melanoma, renal cell carcinoma, head and neck squamous cell carcinoma, Hodgkin's lymphoma, pancreatic cancer, or prostate cancer.

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