Polypeptide Specific to Mucin 1 and Its Use

Abstract

The present invention relates to a polypeptide that binds to Mucin 1, an isolated polynucleotide encoding the same, a vector containing the polynucleotide, and a cell containing the vector. The present invention also relates to a chimeric antigen receptor comprising a polypeptide that binds to Mucin 1, an isolated polynucleotide encoding the chimeric antigen receptor, a vector containing the polynucleotide, an immune cell expressing the chimeric antigen receptor, and a composition and method for treating cancer comprising these.

Description

Technical Field

[0001] [Cross - reference to Related Applications] This application claims the benefit of priority based on Korean Patent Application No. 10 - 2020 - 0183768 filed on December 24, 2020 and Korean Patent Application No. 10 - 2021 - 0137757 filed on October 15, 2021, and all the contents disclosed in the documents of the Korean patent applications are included as part of this specification.

[0002] Relates to a polypeptide that binds to mucin 1 (Mucin1), an isolated polynucleotide encoding the same, a vector containing the polynucleotide, and a cell containing the vector. Also relates to a chimeric antigen receptor containing a polypeptide that binds to mucin 1, an isolated polynucleotide encoding the chimeric antigen receptor, a vector containing the polynucleotide, an immune cell expressing the chimeric antigen receptor, and a cancer treatment composition and treatment method containing these.

Background Art

[0003] Mucin 1 (Mucin1) is a membrane - bound glycoprotein present in epithelial cells of almost all organs in the body. Mucin 1 is a heterodimeric protein formed from the non - covalent interaction of an N - terminal subunit (MUC1 - N) and a C - terminal subunit (MUC1 - C). MUC1 - C forms an extracellular domain, a transmembrane domain, and a cytoplasmic tail, and MUC1 - N contains tandem repeats (TRs) of 20 amino acids (HGVTSAPDTRPAPGSTAPPA) including a glycosylation site, and interacts with the extracellular domain of MUC1 - C and is continuously linked to MUC1 - C.

[0004] In normal epithelial cells, mucin 1 is expressed at low levels and is limited to the apical membranes of the epithelium. However, it is overexpressed in solid cancer cells such as breast cancer, lung cancer, colorectal cancer, skin cancer, thyroid cancer, gastric cancer, pancreatic cancer, kidney cancer, ovarian cancer, and cervical cancer, randomly expressed throughout the cell surface, and abnormally glycosylated. Therefore, mucin 1 is known to be useful as a target molecule for detecting cancer lesions or as a target molecule for treating cancer.

Summary of the Invention

Problems to be Solved by the Invention

[0005] Currently, many anti-MUC1 antibodies for cancer treatment have been reported. However, in the case of anti-cancer therapy using antibodies, it is only applicable to cancer cells having an antigen specifically recognized by the antibody, the duration of effect is short, and resistance may occur during long-term administration. Therefore, there is a demand for the development of a technology that can activate the in vivo immune system and continuously exert a strong anti-cancer effect in various cancers.

Means for Solving the Problems

[0006] One embodiment provides a polypeptide that binds to mucin 1.

[0007] Another embodiment provides an isolated polynucleotide encoding the mucin 1-binding polypeptide.

[0008] Another embodiment provides a vector containing a polynucleotide encoding the mucin 1-binding polypeptide.

[0009] Another embodiment provides a cell containing a vector containing a polynucleotide encoding the mucin 1-binding polypeptide.

[0010] Another embodiment provides a chimeric antigen receptor containing the mucin 1-binding polypeptide.

[0011] Other embodiments provide an isolated polynucleotide encoding the chimeric antigen receptor.

[0012] Other embodiments provide a vector comprising a polynucleotide encoding the chimeric antigen receptor.

[0013] Other embodiments provide an immune cell that expresses the chimeric antigen receptor or comprises a polynucleotide encoding the chimeric antigen receptor.

[0014] Other embodiments provide a cancer therapeutic composition comprising the mucin 1-binding polypeptide; an isolated polynucleotide encoding the mucin 1-binding polypeptide; a vector comprising a polynucleotide encoding the mucin 1-binding polypeptide; a cell comprising a polynucleotide encoding the mucin 1-binding polypeptide; a chimeric antigen receptor comprising the mucin 1-binding polypeptide; an isolated polynucleotide encoding the chimeric antigen receptor; a vector comprising a polynucleotide encoding the chimeric antigen receptor; or an immune cell comprising a polynucleotide encoding the chimeric antigen receptor or expressing the chimeric antigen receptor. BRIEF DESCRIPTION OF THE DRAWINGS

[0015]

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Mode for Carrying Out the Invention

[0016] According to one aspect of the present invention, a polypeptide that binds to Mucin1 is provided. As a preferred example, the polypeptide that binds to Mucin1 in the present invention may be an antibody or an antigen-binding fragment thereof.

[0017] In one embodiment, a polypeptide that binds to Mucin1 is provided, which comprises a pair of heavy chain variable (VH) region and light chain variable (VL) region selected from the group consisting of: - A VH region comprising complementarity-determining regions (CDR) 1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and a VL region comprising CDR 1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; - A VH region comprising CDR 1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively, and a VL region comprising CDR 1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; - A VH region comprising CDR 1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, respectively, and a VL region comprising CDR 1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; - A VH region comprising CDR 1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively, and a VL region comprising CDR 1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively; - A VH region comprising CDR 1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively, and a VL region comprising CDR 1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively; - A VH region comprising CDR 1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63, respectively, and a VL region comprising CDR 1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, respectively; - A VH region containing CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO: 73, respectively, and a VL region containing CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76, respectively; - A VH region containing CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 81, SEQ ID NO: 82, and SEQ ID NO: 83, respectively, and a VL region containing CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 84, SEQ ID NO: 85, and SEQ ID NO: 86, respectively; and - A VH region containing CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 91, SEQ ID NO: 92, and SEQ ID NO: 93, respectively, and a VL region containing CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively.

[0018]

Table 1-1

Table 1-2

[0019] In another embodiment, a polypeptide that binds to mucin 1 is provided, comprising a pair of a heavy chain variable (VH) region and a light chain variable (VL) region selected from the group consisting of: - A VH region comprising the amino acid sequence of SEQ ID NO: 7 and a VL region comprising the amino acid sequence of SEQ ID NO: 8; - A VH region comprising the amino acid sequence of SEQ ID NO: 17 and a VL region comprising the amino acid sequence of SEQ ID NO: 18; - A VH region comprising the amino acid sequence of SEQ ID NO: 27 and a VL region comprising the amino acid sequence of SEQ ID NO: 28; - A VH region comprising the amino acid sequence of SEQ ID NO: 37 and a VL region comprising the amino acid sequence of SEQ ID NO: 38; - A VH region comprising the amino acid sequence of SEQ ID NO: 47 and a VL region comprising the amino acid sequence of SEQ ID NO: 48; - A VH region comprising the amino acid sequence of SEQ ID NO: 57 and a VL region comprising the amino acid sequence of SEQ ID NO: 58; - A VH region comprising the amino acid sequence of SEQ ID NO: 67 and a VL region comprising the amino acid sequence of SEQ ID NO: 68; - A VH region comprising the amino acid sequence of SEQ ID NO: 77 and a VL region comprising the amino acid sequence of SEQ ID NO: 78; - A VH region comprising the amino acid sequence of SEQ ID NO: 87 and a VL region comprising the amino acid sequence of SEQ ID NO: 88; and - A VH region comprising the amino acid sequence of SEQ ID NO: 97 and a VL region comprising the amino acid sequence of SEQ ID NO: 98.

[0020] [Table 2-1] [Table 2-2]

[0021] In the present invention, the term "antibody" is used in the broadest sense to generically refer to proteins that specifically bind to a particular antigen, and may be a protein produced by antigen stimulation within the immune system or a protein chemically synthesized or recombinantly produced, and the type thereof is not particularly limited. Specifically, as long as the desired biological activity is exhibited, it includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), synthetic antibodies (also referred to as antibody mimetics), chimeric antibodies, humanized antibodies, human antibodies, or antibody fusion proteins (also referred to as antibody conjugates).

[0022] A complete antibody (e.g., IgG type) has a structure with two full-length light chains and two full-length heavy chains, and each light chain is linked to the heavy chain by a disulfide bond. The constant region of an antibody is divided into a heavy-chain constant region and a light-chain constant region. The heavy-chain constant region has a gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) type, and as subclasses, it has gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), or alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.

[0023] The term "antigen-binding fragment" refers to a portion of an antibody that lacks at least some of the amino acids present in the full-length chain but can still specifically bind to an antigen. Such fragments are biologically active in that they bind to the target antigen and can compete with other antigen-binding molecules, including intact antibodies, for binding to a given epitope. Antigen-binding fragments may not contain the constant heavy-chain domains of the Fc region of the intact antibody (i.e., CH2, CH3, and CH4 depending on the antibody isotype). Examples of antigen-binding fragments include scFv (single chain variable fragment) (e.g., scFv, (scFv) 2 etc.), Fab (fragment antigen binding) (e.g., Fab, Fab’, F(ab’) 2 etc.), domain antibodies, peptibodies, minibodies, intrabodies, diabodies, triabodies, tetra-bodies, and single-chain antibodies, etc., but are not limited thereto. Also, the antigen-binding fragment may be an scFv, or a fusion polypeptide (scFv-Fc) in which the scFv is fused to the Fc site of an immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.) or a fusion polypeptide (scFv-Ck (kappa constant region) or scFv-Cλ (lambda constant region)) in which the scFv is fused to the constant region of the light chain (e.g., kappa or lambda), but is not limited thereto.

[0024] The term "heavy chain" is interpreted to include all full-length heavy chains and their fragments that contain a variable region domain V having an amino acid sequence with a variable region sequence sufficient to confer antigen specificity H and three constant region domains C H1 C H2 and C H3 and a hinge. Also, the term "light chain" is interpreted to include all full-length light chains and their fragments that contain a variable region domain V having an amino acid sequence with a variable region sequence sufficient to confer antigen specificity L and a constant region domain C L .

[0025] The term "Complementarity-determining regions (CDR)" refers to the sites in the variable regions of an antibody that confer binding specificity or affinity for an antigen. Generally, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in the heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in the light chain variable region. The CDRs can provide the main contact residues for the binding of an antibody or its fragment to an antigen or epitope. The "Framework region (FR)" refers to the non-CDR portions of the variable regions of the heavy and light chains. Generally, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in the heavy chain variable region and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in the light chain variable region. The exact amino acid sequence boundaries of a given CDR or FR can be readily determined using any one of a number of well-known systems such as the Kabat numbering system, the Chothia numbering system, the Contact numbering system, the IMGT numbering system, the Aho numbering system, the AbM numbering system, etc.

[0026] The term "variable region" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy chain variable (VH) region and the light chain variable (VL) region generally have a similar structure, and each domain contains four conserved framework regions (FRs) and three CDRs.

[0027] In certain embodiments, the mucin 1-binding polypeptide of the invention may be, but is not limited to, a scFv (single chain variable fragment), peptibody, Fab (fragment antigen binding), monoclonal antibody, bispecific antibody, minibody, domain antibody, synthetic antibody, chimeric antibody, humanized antibody, human antibody, or antibody fusion protein.

[0028] In a preferred embodiment, the mucin 1-binding polypeptide of the invention may be a scFv (single chain variable fragment).

[0029] The term "scFv (single chain variable fragment)" refers to a single-chain antibody fragment in which the heavy chain variable (VH) region and the light chain variable (VL) region of an antibody are covalently linked. The VH region and the VL region are either directly linked or linked by a linker.

[0030] The linker may be a water-soluble and / or flexible linker. For example, the N-terminus of VH and the C-terminus of VL are linked by the linker, or the C-terminus of VH and the N-terminus of VL are linked. The linker may be a peptide linker, for example, having a length of 10 to 30 amino acids, 10 to 25 amino acids, 10 to 20 amino acids, 10 to 15 amino acids, 15 to 30 amino acids, 15 to 25 amino acids, 15 to 20 amino acids, specifically, it may have a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids, but is not limited thereto.

[0031] Amino acid sequences suitable for the linker are known in the art. For example, the linker may be rich in glycine (Gly) for flexibility and may contain serine (Ser), asparagine (Asn), alanine (Ala) or threonine (Thr) for solubility. The linker may comprise 1 to 100, 2 to 50, or 5 to 25 in total of one or more selected from the group consisting of Gly, Asn, Ser, Thr and Ala. As a non-limiting example, the amino acid sequence of the linker may be a sequence having GGGS or GGGGS with various numbers of repetitions, for example, 2, 3, 4 and 5 repetitions. For example, the linker may be GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, or GGGGSGGGGSGGGAS, but is not limited thereto.

[0032] The scFv has no constant region and maintains the antigen specificity of the parental antibody despite the introduction of the linker. The scFv can be expressed from a polynucleotide encoding the VH region and the VL region, but is not limited thereto.

[0033] As a preferred embodiment, an scFv polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 69, SEQ ID NO: 79, SEQ ID NO: 89, and SEQ ID NO: 99 is provided.

[0034] In the present invention, an antibody or antigen-binding fragment thereof also includes mutants having equivalent activity, for example, mutants having about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% or more identity with the amino acid sequence described in the present invention and having equivalent biological activity are also included within the scope of the present invention.

[0035] According to another aspect of the present invention, an isolated polynucleotide encoding the mucin 1-binding polypeptide is provided.

[0036] As a preferred embodiment, the polynucleotide can include a nucleic acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 40, SEQ ID NO: 50, SEQ ID NO: 60, SEQ ID NO: 70, SEQ ID NO: 80, SEQ ID NO: 90, and SEQ ID NO: 100. Further, the present invention can include an isolated polynucleotide encoding a polypeptide having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% or more identity with the above sequence and having equivalent activity. In some embodiments, the polynucleotide is codon-optimizable for expression in humans.

[0037] According to another aspect of the present invention, a vector comprising the polynucleotide encoding the mucin 1-binding polypeptide is provided.

[0038] In the vector, the polynucleotide is operably linked to a promoter. The term "operatively linked" means a functional linkage between a nucleotide expression regulatory sequence (e.g., a promoter sequence) and another nucleotide sequence. By being operably linked, the regulatory sequence can regulate the transcription and / or translation of the other nucleotide sequence.

[0039] Vectors are typically constructed as vectors for cloning or vectors for expression. The expression vectors can use those commonly used in the art for expressing foreign proteins in plants, animals, or microorganisms. The vectors can be constructed by various methods known in the art.

[0040] Vectors are constructed using prokaryotic cells or eukaryotic cells as hosts. For example, when the vector used is an expression vector and a prokaryotic cell is used as the host, a strong promoter capable of allowing transcription to proceed (e.g., pL λIt is common to include promoters (such as CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), ribosome binding sites for the start of translation, and transcription / translation termination sequences. When using eukaryotic cells as hosts, origins of replication that function in eukaryotic cells contained in the vector include, but are not limited to, f1 origin of replication, SV40 origin of replication, pMB1 origin of replication, adenovirus origin of replication, AAV origin of replication, and BBV origin of replication. Also, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and HSV tk promoter) can be used and generally can have a polyadenylation sequence as the transcription termination sequence. Additionally, if necessary, enhancer sequences, 5'- and 3'-untranslated regions, secretion signal sequences, splicing junctions, selection markers (e.g., antibiotic resistance genes), etc. can be further included.

[0041] According to another aspect of the present invention, there is provided a cell comprising a vector containing a polynucleotide encoding the mucin 1-binding polypeptide.

[0042] The cells may be obtained by introducing the vector into an appropriate host cell. A host cell containing the vector may be useful for cloning or expressing the polynucleotide contained in the vector. The host cell is a cell that stably and continuously clones or expresses the vector, and any host cell known in the art can be used. For example, as prokaryotic cells, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus strains such as Bacillus thuringiensis, and enterobacteria and strains such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species. When transforming eukaryotic cells, as host cells, yeast (Saccharomyces cerevisiae), insect cells, plant cells, and animal cells, for example, Sp2 / 0, CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, MDCK cell lines, etc. are used, but not limited thereto.

[0043] Delivery (introduction) of the polynucleotide or the vector containing the same into a host cell can use delivery methods widely known in the art. The delivery method, for example, when the host cell is a prokaryotic cell, the CaCl 2 method or electroporation method, etc. can be used. When the host cell is a eukaryotic cell, microinjection method, calcium phosphate precipitation method, electroporation method, liposome-mediated transfection method, and gene bombardment, etc. can be used, but not limited thereto.

[0044] The method for selecting the transformed host cell can be easily carried out by a method widely known in the art using the phenotype expressed by the selection marker. For example, when the selection marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic. The transformed host cell can be cultured for a period sufficient for the polypeptide to be expressed or secreted, and separation and purification methods used for ordinary proteins, such as methods using solubility, such as salting out and solvent precipitation methods, dialysis, ultrafiltration, gel filtration, methods using differences in molecular weight such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis, methods using charge such as ion exchange chromatography and hydroxylapatite chromatography, methods using specific affinity such as affinity chromatography, methods using differences in hydrophobicity such as reverse phase high performance liquid chromatography, methods using differences in isoelectric point such as isoelectric focusing, etc. can be used to separate and purify the polypeptide.

[0045] According to another aspect of the present invention, a chimeric antigen receptor (CAR) comprising the mucin 1-binding polypeptide is provided. Since the mucin 1-binding polypeptide can be engineered to be expressed as part of a single chain together with other CAR components, it is suitable for use in chimeric antigen receptors.

[0046] A chimeric antigen receptor can typically include an extracellular domain that contains a mucin 1-binding polypeptide; a transmembrane domain; and an intracellular signaling domain (or T cell activation domain). Further, the extracellular domain can additionally include a spacer region (or hinge region) between the polypeptide and the transmembrane domain. Also, the chimeric antigen receptor can further include one or more co-stimulatory domains, preferably, the co-stimulatory domain can be located between the transmembrane domain and the intracellular signaling domain.

[0047] Thus, as a preferred example, a chimeric antigen receptor can include an extracellular domain that contains a mucin 1-binding polypeptide; a hinge domain; a transmembrane domain; one or more co-stimulatory domains; and an intracellular signaling domain. Each domain can be heterologous, i.e., composed of sequences derived from different protein chains. Each domain can be linked by a short oligo- or polypeptide linker, e.g., a linker 2 to 10 amino acids in length. Also, the chimeric antigen receptor consists of one polypeptide chain to which each domain is linked.

[0048] The extracellular domain contains a mucin 1-binding polypeptide as described above, which recognizes mucin 1 expressed on the surface of cancer cells.

[0049] The extracellular domain can further include a spacer region (or hinge region). The spacer region can be located between the mucin 1-binding polypeptide and the transmembrane domain. The spacer region allows the mucin 1-binding polypeptide to more flexibly recognize the target antigen at a certain distance from the cell membrane of the CAR-T cell. The spacer region can typically be a polypeptide and can have a length of 10 or more amino acids, for example, a length of 10 to 300 amino acids, 10 to 250 amino acids, 10 to 200 amino acids, 10 to 150 amino acids, 10 to 100 amino acids, 10 to 50 amino acids, but is not limited thereto.

[0050] Examples of the spacer region can include the hinge region of CD8α or CD28, or the constant region of immunoglobulin (IgG), etc., and mutations can be introduced to remove the off-target effect caused by these. For example, the constant region of immunoglobulin can be derived from IgG hinge alone, all or part of the CH2 and CH3 domains, and can be, for example, the Fc region. IgG can preferably be IgG2 or IgG4. In some embodiments, the spacer can be a chimeric polypeptide containing one or more of the hinge, CH2, and CH3 sequences derived from IgG2, IgG4, and / or IgG2 and IgG4. In some embodiments, the hinge domain can be represented by the sequence of SEQ ID NO: 112, but is not limited thereto.

[0051] The transmembrane domain serves to link the cell membrane domain and the signal transduction domain inside the cell membrane and is derived from natural or synthetic sources. When the source is natural, the domain is derived from any membrane-bound or membrane-crossing protein. For example, the transmembrane domain may be, but is not limited to, the transmembrane domain of the alpha, beta, or zeta chain of the T cell receptor, CD3 epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, or CD154. As a preferred example, the transmembrane domain may be, but is not limited to, the transmembrane domain of CD28 or CD8. When the source is synthetic, the synthetic transmembrane domain can contain hydrophobic residues such as leucine and valine, and can contain, but is not limited to, phenylalanine, tryptophan, and valine at each end. In some embodiments, the transmembrane domain may be represented by the sequence of SEQ ID NO: 113, but is not limited thereto.

[0052] The co-stimulatory domain is the site where the co-stimulatory signal is transmitted, and it is the site where CAR-T cells transmit signals to trigger an immune response and self-proliferate. This can be selectively introduced to improve the proliferation, cytotoxicity, sustained response, lifespan extension, etc. of CAR-T cells.The co-stimulatory domain is selected from one or more, for example, one, two or three, but not limited to, CD28, OX-40 (CD134), 4-1BB (CD137), CD2, CD7, CD27, CD30, CD40, PD-1, ICOS, LFA-1 (CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, SLAM (signaling lymphocytic activation molecule), activated NK cell receptor, BTLA, toll-like receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAMF1 (CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, or the signaling site of CD19a.Preferred embodiments include, but are not limited to, one or more, such as one, two, or three, selected from the signal transduction sites of CD28, OX-40 (CD134), 4-1BB (CD137), CD27, or ICOS. In some embodiments, the costimulatory domain may be represented by the sequence of SEQ ID NO: 114, but is not limited thereto.

[0053] The intracellular signal transduction domain is a site that activates a T cell immune response against an antigen bound to the mucin 1-binding polypeptide. The signal transduction domain can be a component of the T cell receptor (TCR) or contain a signal transduction motif known as an immunoreceptor tyrosine-based activation motif (ITAM). As an example, the signal transduction domain may be, but is not limited to, a signal transduction domain derived from TCR or CD3 zeta, FcR gamma, CD3 gamma, CD3 delta, and CD3 epsilon. A preferred example may be, but is not limited to, the signal transduction domain of CD3 zeta. The intracellular signal transduction domain can activate CAR-T cells when the mucin 1-binding polypeptide site of the extracellular domain binds to the target. For example, the CAR can stimulate T cell functions, such as cytolytic activity or T-helper activity, and induce the secretion of cytokines or other factors. In some embodiments, the intracellular signal transduction domain may be represented by the sequence of SEQ ID NO: 115, but is not limited thereto.

[0054] In some embodiments, the CAR can be expressed in a form that includes a signal sequence. The CAR can also be expressed together with an additional sequence useful for monitoring expression, such as a ribosome skip sequence, such as a 2A peptide, or a truncated cell surface polypeptide (such as tHER2 or tEGFR or truncated PSMA).

[0055] According to another aspect of the present invention, there is provided an isolated polynucleotide encoding the chimeric antigen receptor. In some embodiments, the polynucleotide is codon-optimizable for expression in humans.

[0056] According to another aspect of the present invention, there is provided a vector comprising a polynucleotide encoding the chimeric antigen receptor. The vector is constructed as a vector for gene cloning, a vector for protein expression, or a vector for gene delivery. In the case of a vector for cloning or expression, the details are as described above.

[0057] Any vector known in the art can be adapted to the present invention. For example, the vector may be a viral vector. For example, the vector may be, but is not limited to, a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adeno-associated virus (AAV) vector, or a lentiviral vector.

[0058] According to another aspect of the present invention, there are provided immune cells expressing a chimeric antigen receptor comprising the mucin 1-binding polypeptide.

[0059] Immune cells include, but are not limited to, T cells, tumor infiltrating lymphocytes (TIL), NK (Natural killer) cells, TCR-expressing cells, dendritic cells, or NK-T cells. Further, the immune cells may be derived from human induced pluripotent stem cells (iPSC). The immune cells are derived from any known source. For example, the immune cells are differentiated in vitro from a hematopoietic stem cell population or obtained from a patient. The immune cells can be obtained, for example, from peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. Also, the immune cells are derived from one or more immune cell lines available in the related art.

[0060] The immune cells may be autologous or allogenic. Autologous refers to those derived from the patient to be treated. Allogenic refers to those derived from another individual of the same species as the patient to be treated.

[0061] Further, the immune cells may be derived from human induced pluripotent stem cells (iPSC). Immune cells derived from iPSC have the advantages that they can self-proliferate, are easy to expand in large quantities compared to autologous or allogenic immune cells, can be used to produce a universal cell therapy agent applicable to all people, and enable homogeneous production and cost reduction.

[0062] The immune cells can be transfected or transduced by a vector using methods such as microinjection, electroporation, sonoporation, biolistic (e.g., gene gun), lipid transfection, polymer transfection, calcium phosphate precipitation, protoplast fusion, liposome-mediated transfection, nanoparticles, or polyplexes, but are not limited thereto.

[0063] According to another aspect of the present invention, there is provided a composition for preventing or treating cancer, comprising the mucin 1-binding polypeptide; an isolated polynucleotide encoding the mucin 1-binding polypeptide; a vector comprising a polynucleotide encoding the mucin 1-binding polypeptide; a cell comprising a polynucleotide encoding the mucin 1-binding polypeptide; a chimeric antigen receptor comprising the mucin 1-binding polypeptide; an isolated polynucleotide encoding the chimeric antigen receptor; a vector comprising a polynucleotide encoding the chimeric antigen receptor; or an immune cell comprising a polynucleotide encoding the chimeric antigen receptor or expressing the chimeric antigen receptor.

[0064] According to another aspect of the present invention, there is provided a method for preventing and / or treating cancer, comprising administering an effective amount of the mucin 1-binding polypeptide; an isolated polynucleotide encoding the mucin 1-binding polypeptide; a vector comprising a polynucleotide encoding the mucin 1-binding polypeptide; a cell comprising a polynucleotide encoding the mucin 1-binding polypeptide; a chimeric antigen receptor comprising the mucin 1-binding polypeptide; an isolated polynucleotide encoding the chimeric antigen receptor; a vector comprising a polynucleotide encoding the chimeric antigen receptor; or an immune cell comprising a polynucleotide encoding the chimeric antigen receptor or expressing the chimeric antigen receptor to a patient in need of prevention or treatment of cancer.

[0065] According to another aspect of the present invention, there is provided a composition for preventing or treating cancer, comprising a T cell comprising a chimeric antigen receptor comprising the mucin 1-binding polypeptide.

[0066] According to another aspect of the present invention, there is provided a method for preventing and / or treating cancer, comprising administering an effective amount of a T cell comprising a chimeric antigen receptor comprising the mucin 1-binding polypeptide to a patient in need of prevention or treatment of cancer.

[0067] For example, the cancer may be a solid cancer or a blood cancer, and non-limiting examples include breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, endometrial cancer, uterine cancer, colon cancer, colorectal cancer, rectal cancer, kidney cancer, nephroblastoma, skin cancer, oral squamous cell carcinoma, epidermoid carcinoma, nasopharyngeal carcinoma, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, lymphoma (e.g., Hodgkin's lymphoma or non-Hodgkin's lymphoma), gastric cancer, pancreatic cancer, testicular cancer, thyroid cancer, follicular thyroid carcinoma, melanoma, myeloma, multiple myeloma, mesothelioma, osteosarcoma, myelodysplastic syndrome, tumors of hepatic origin, soft tissue sarcoma, liposarcoma, gastrointestinal stromal sarcoma, malignant peripheral nerve sheath tumor (MPNST), Ewing's sarcoma, leiomyosarcoma, chondrosarcoma of hepatic origin, lymphoma, fibrosarcoma, rhabdomyosarcoma, teratocarcinoma, neuroblastoma, medulloblastoma, glioma, positive tumors of the skin, or leukemia. The lung cancer may be, for example, small cell lung carcinoma (SCLC) or non-small cell lung carcinoma (NSCLC). The leukemia may be, for example, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL) or chronic lymphocytic leukemia (CLL).

[0068] Preferably, the cancer is a cancer in which the MUC1 protein is expressed, and may be, but is not limited to, breast cancer, skin cancer, pancreatic cancer, prostate cancer, lung cancer, thyroid cancer, gastric cancer, ovarian cancer, colorectal cancer, liver cancer, gallbladder cancer, kidney cancer, cervical cancer, or bladder cancer. The cancer may be a primary cancer or a metastatic cancer.

[0069] The patient to be treated may be a patient receiving secondary anti-proliferative therapy. For example, the secondary anti-proliferative therapy may be chemotherapy, radiotherapy, immunotherapy, phototherapy, cryotherapy, toxin therapy, hormone therapy, or surgery.

[0070] The CAR-T anti-cancer treatment using T cells containing the chimeric antigen receptor containing the mucin 1-binding polypeptide of the present invention is carried out in a series of processes in which T cells are extracted from the blood of a healthy subject or a patient to be treated, genetically engineered to express the chimeric antigen receptor containing the mucin 1-binding polypeptide of the present invention, the engineered T cells are amplified and cultured, and the cultured engineered T cells are administered to the patient.

[0071] As a preferred embodiment, the step of extracting T cells from the blood of a healthy subject or a patient to be treated can be carried out, for example, by separating white blood cells from the blood using leukapheresis or aphresis to separate the white blood cell component, and then concentrating the T cells through a process to extract the T cells. The T cells are separated using specific antibody bead conjugates or markers at the CD4 / CD8 composition level. Alternatively, it is also possible to stably obtain a large amount of T cells by differentiation from stem cells.

[0072] The step of genetically engineering the extracted T cells to express the chimeric antigen receptor provided by the present invention can be carried out, for example, by injecting a nucleic acid molecule designed to express a chimeric antigen receptor (CAR) into the T cells using a vector, such as a viral vector (e.g., a lentiviral vector or a retroviral vector). The CAR may be introduced in DNA form, or after being introduced in RNA form, it may be reverse transcribed into DNA by reverse transcriptase and then integrated into the genome of the T cells. Also, in the case of an allogeneic cell therapy agent, genetic engineering can be additionally performed to remove or correct the gene that induces transplant rejection in donor T cells so as not to cause rejection reactions such as graft-versus-host disease (GvHD).

[0073] The step of amplifying and culturing the engineered T cells can be carried out by culturing, proliferating, and expanding the T cells using culture techniques known in the art. At this time, techniques for ensuring safety in the use of viruses and for selecting well-produced CAR-T cells are required.

[0074] Finally, the step of readministering the engineered T cells to the patient is carried out, for example, by infusion. As a preferred example, before the infusion of CAR-T cells, the patient can receive lymphodepleting conditioning chemotherapy using cyclophosphamide or fludarabine, etc., to reduce the white blood cell count. Also, cytokines such as IL-2 can be co-administered to improve the persistence of CAR-T cells.

[0075] The engineered T cells administered to a patient can mediate an immune response against tumor cells expressing mucin 1. Such immune responses include activation of T cells, secretion of cytokines such as IL-2 and IFN-gamma by T cells, proliferation and expansion of T cells that recognize tumor antigens, and T cell-mediated specific killing (tumor elimination) of target-positive cells. For example, if the CAR in CAR-T cells specifically binds to mucin 1, the T cells are activated by phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of CD3 zeta, and then proliferation, cytotoxicity, and / or cytokine secretion of the T cells are induced.

[0076] Thus, the anti-cancer therapy using CAR-T cells fundamentally activates the patient's immune system to exert a sustained anti-cancer effect, so it does not need to be continuously administered, and by using the patient's own T cells, there is an advantage that treatment tailored to the individual patient is possible.

[0077] Administration of the composition of the present invention can inhibit, arrest, delay, or prevent the onset or progression of a disease state, or can cause, induce, or promote an immune response.

[0078] The term "effective amount" means an amount sufficient to achieve a desired result when administered to an individual, including a human, for example, an amount effective to treat or prevent cancer. The effective amount varies depending on various factors such as the formulation method, administration method, age, weight, sex, disease severity, diet, administration time, administration route, excretion rate, and responsiveness of the patient. The dosage or treatment regimen can be adjusted to provide an optimal therapeutic response as understood by those skilled in the art.

[0079] A therapeutic usage using a therapeutically effective amount may consist of a single administration or, as another example, may include a series of applications. The duration of the treatment period is determined by various factors such as the formulation method, administration method, patient's age, weight, gender, disease severity, diet, administration time, administration route, excretion rate, and responsiveness. Also, it will be understood that the effective dosage of the anticancer composition used in the treatment can be increased or decreased during the course of an individual treatment usage. Variations in dosage may occur and will be clarified by standard diagnostic analyses known in the art. The composition of the present invention can be administered before, during, or after treatment using conventional anticancer agents, radiotherapy, hormone therapy, biotherapy, and / or surgical tumor resection in some aspects.

[0080] The composition of the present invention can be provided together with one or more additives selected from the group consisting of pharmaceutically acceptable carriers, diluents, excipients, and the like.

[0081] The pharmaceutically acceptable carrier is one commonly used in the formulation of antibodies and may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc., but is not limited thereto. The composition may further include one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., which are commonly used in the manufacture of pharmaceutical compositions, in addition to the above components.

[0082] The composition can be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration, and rectal administration. When administered orally, since proteins or peptides are digested, oral compositions are formulated to coat the active agent or to be protected from degradation in the stomach. Further, the composition can be administered by any device through which the active substance can move to the target cells.

[0083] The cells are administered using standard administration techniques, dosage forms, or devices. It is possible to appropriately use dosage forms and devices such as syringes and vials for the storage and administration of the composition. The administration of the cells may be autologous, allogenic, or xenogenic. Peripheral blood-derived immune cells or their progeny (e.g., derived in vivo, in vitro, or ex vivo) are administered by local injection including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When administering an immunocyte therapeutic agent, generally, it is formulated into an injectable unit dosage form (solution, suspension, emulsion). In some embodiments, the cell population is administered parenterally. Dosage forms include intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration, and rectal administration.

[0084] In some embodiments, the composition is a sterile liquid formulation, provided as, for example, an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which is buffered at a selected pH in some aspects. Liquid formulations are generally easier to manufacture than gels, other viscous compositions, and solid compositions. Also, liquid compositions are particularly convenient to administer, especially by injection. On the other hand, viscous compositions are formulated within an appropriate viscosity range to provide a longer contact period with a particular tissue. The liquid or viscous composition can include a carrier, which is a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0085] The sterile injection solution can be produced by integrating the binding molecule into a suitable carrier, diluent or solvent such as a mixing agent with excipients such as sterile water, physiological saline, glucose, dextrose, etc. The composition can also be lyophilized. The composition can contain auxiliary substances such as wetting agents, dispersing agents or emulsifying agents (e.g., methylcellulose), pH buffers, gelling or viscosity increasing additives, preservatives, flavoring agents, pigments, etc., depending on the route of administration and the desired manufacturing route.

[0086] A variety of additives can be added to improve the stability and sterility of the composition containing antibacterial preservatives, antioxidants, chelating agents and buffers. Prevention of the action of microorganisms can be ensured by a variety of antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. The long-term absorption of injectable pharmaceutical forms is brought about by the use of formulations that delay absorption, such as aluminum monostearate and gelatin.

Example

[0087] Hereinafter, the present invention will be described more specifically by way of examples. However, these are merely illustrative of the present invention, and the scope of the present invention is not limited by these examples.

[0088] Example 1. Production of scFv Binding to Mucin 1 Human mucin 1 (SEQ ID NO: 110) was conjugated with CD4 (SEQ ID NO: 111) to prepare a recombinant antigen, and clones secreting scFv specific to the antigen were screened from the scFv library, and 10 scFvs specifically binding to mucin 1 were selected. The sequence information of the 10 selected scFvs and the positive control group scFv G03 (a positive control group prepared using the amino acid sequence of the G3 antibody in Table 6 of WO2018 / 174544) used in the subsequent experiment is shown in Table 3.

[0089]

Table 3-1

Table 3-2

[0090]

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

[0091] The affinity of the scFv-Fc antibody containing the scFv for the mucin 1 antigen was measured using a Biacore T200 (GE healthcare). On a Series S Sensor Chip CM5 (GE healthcare, Cat.No.BR-1005-30), an anti-human IgG (Fc) antibody (GE healthcare, Cat.No.BR-1008-39, final concentration 25 μg / mL) was flowed at 5 μL / min for 360 seconds using an Amine Coupling Kit (GE healthcare, Cat.No.BR-1000-50) and immobilized to approximately 5000 - 7000 RU. Five different concentrations of the human MUC1 protein antigen within the concentration range from 25 nM to 400 nM were injected at a rate of 30 μL / min, and k a and k d values were determined, and from these, the K D value was calculated. As a result, the K D (M) value indicating the binding affinity for mucin 1 is shown in Table 5.

[0092]

Table 5

[0093] Example 2. Production of CAR-T Cells Using scFv Binding to Mucin 1 2-1. Generation of a Lentiviral Transfer Vector Expressing CAR A lentiviral vector (transfer vector or expression vector) expressing a chimeric antigen receptor (CAR) containing an extracellular domain (base sequence in Table 4) including an scFv polypeptide sequence, a hinge (CD8α) (SEQ ID NO: 112), a transmembrane domain (CD28) (SEQ ID NO: 113), a co-stimulatory domain (CD28) (SEQ ID NO: 114), and an intracellular signaling domain (CD3 zeta) (SEQ ID NO: 115) was produced. The scFv portion of the CAR was separated as a fragment by PCR from a plasmid supplied by the screening company, and the hinge, transmembrane protein, CD28, and CD3z signaling domains were separated as fragments by PCR from a previously synthesized plasmid. The two fragments were cloned into the Clonetech pLVX-EF1alpha vector using the Takara In-Fusion HD Cloning Kit. The lentiviral vector cloned in this way was transfected into the Takara Lenti-X 293T Cell Line (product code 632180) together with a packaging plasmid provided by the company's Lenti-X Expression System (EF1a Version) (product code 631253). After 24 hours, the soup was separated and the lentivirus therein was used for subsequent research.

[0094] 2-2. Production of Lentiviral Particles To generate lentivirus, the manufactured lentivirus transfer vector (transfer vector or expression vector) and the lentivirus packaging plasmid mix were transfected into Lenti-X 293T cells (Takara product number 632180) using the liposomal nucleic acid transfection reagent (lipofectamine, Gibco). One day before transduction, HEK293T cells were prepared to be present at about 6 - 70% of the culture plate area. The culture medium used was Dulbecco’s modified Eagle medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco), and 100 mm or 150 mm sized culture plates were used. The culture conditions were maintained at 37 °C, 5% CO 2 2, with humidified conditions. The transfer vector, the lentivirus packaging plasmid mix, and the liposomal nucleic acid transfection reagent were mixed at room temperature according to the manufacturer's instructions, and then HEK293T cells were transduced by treating the HEK293T cells on the culture plate in a dropwise manner. After transduction, the lentivirus produced in HEK293T cells was ejected extracellularly and existed in the cell culture medium in the form of virus particles. Forty-eight hours after transduction, only the cell culture medium was taken out from the culture plate and centrifuged to pellet the lentivirus particles. Thereafter, after dissolving the pellet using the solution, it was used immediately or stored in an ultra-low temperature freezer. The solution was the previously used DMEM medium or CTS optimizer T cell Expansion SFM 1L bottle (Gibco) used for culturing T cells, with Optimizer T-Cell Expansion Supplement (26 ml) added, CTS Innume Cell SR (Serum Replacement, Gibo) added to a final concentration of 2%, and CTS GlutaMAX-I Supplement 100X (Gibco) added to a final concentration of 1X.

[0095] 2-3. Production of CAR-T Cells Peripheral blood mononuclear cell (PBMC) samples were supplied under a research cooperation agreement with The Catholic University of Korea, and donor recruitment and blood collection were carried out with the approval of The Catholic University of Korea-Institutional Review Board. Blood from all healthy individuals was used. In the examples of the present invention, "Donor" refers to the donor of the PBMC sample used in the experiment, and the numbers after Donor mean that each donor is different (for example, represented by Donor03, Donor04, Donor05, Donor06, Donor07, Donor08, Donor18-09, Donor20-01, etc.). To produce CAR-T cells in which a chimeric antigen receptor (CAR) is expressed on the surface of T cells, frozen PBMC vials were thawed in a 37 °C constant temperature water bath. The day after thawing the frozen PBMCs, the cells were switched to a new medium after measuring the cell count. At the same time, T-cells were activated using T-cell specific amplification beads (Dynabeads Human T-expander CD3 / CD28, Gibco), and the chimeric antigen receptor (CAR) in lentiviral form was treated according to the number of activated cells and transduced into T-cells. To culture the transformed T-cells, an interleukin-2 (Interleukin-2, Norvatis) suspension dissolved in filtered sterilized distilled water was added to a concentration of 100 IU / ml based on the medium volume, and the medium was switched during the subsequent culture period or continuously added each time it was added. The cell culture period was set to a total of 10-11 days after activation, and the medium was switched every 1-2 days according to the cell count or newly added to proceed with the culture. When the culture period ended, the cell culture was terminated and the production of CAR-T cells was completed. The produced CAR-T cells were immediately used or mixed with a cell cryopreservation solution, injected into a cell cryopreservation vial, and stored in a liquid nitrogen tank. The confirmation of the expression level of the chimeric antigen receptor (CAR) and the analysis of the characteristics of the produced CAR-T cells were carried out by flow cytometry (FACS).

[0096] Example 3. Affinity Analysis of Soluble scFv for Mucin 1-Expressing Cells Unless otherwise specified, the information on the cell lines used in the examples of the present invention is as follows: -PANC-1; Korean Cell Line Bank, 21469 -HCC1954; Korean Cell Line Bank, 9S1954 -HCC70; Korean Cell Line Bank, 9S0070 -T47D; Korean Cell Line Bank, 30133 -HEK293T; ATCC, CRL-3216 -Huh7; Korean Cell Line Bank, 60104 -HepG2; ATCC, HB-8065 -SK-N-SH; Korean Cell Line Bank, 30011

[0097] A total of 4 cell lines were used in Example 3. Among them, HEK293 (ATCC) and PANC-1 (ATCC) cell lines were used as negative control groups, and HEK293T-v1 (transformed by ATCC and LG Chem) and T47D (Korean Cell Line Bank) cell lines were used as positive control groups. The HEK293T-v1 cell line used as the positive control group is a cell line in which the C subunit of MUC1 is expressed in the HEK293T cell line that does not express mucin 1. A total of 11 types of scFv were used, among which the positive control group is G03 (Table 3). The soluble scFv produced in Example 1 was diluted 5-fold each to prepare 8 concentrations in total. The concentrations at each step were 380 μg / mL, 76 μg / mL, 15.2 μg / mL, 3.04 μg / mL, 0.608 μg / mL, 0.1216 μg / mL, 0.02432 μg / mL, and 0.004864 μg / mL. The buffer used for soluble scFv dilution and cell washing is PBS containing 2% FBS and 10 mM EDTA. 4 types of cells were prepared at 1×10 6 / mL and placed in a 96-well U-bottom cell culture plate at 1×10 5It was placed at 100 μl / well and centrifuged at 1500 RPM for 5 minutes. The prepared soluble scFv was treated at 50 μl / well. It was left standing at 4°C for 1 hour. After adding 150 μl / well of the cell washing buffer and mixing, it was centrifuged at 1500 RPM for 5 minutes. After discarding the supernatant, 200 μl / well of the cell washing buffer was added and mixed, and then it was centrifuged at 1500 RPM for 5 minutes. Goat anti-human IgG Fc-biotin antibody (Invitrogen, A18821) was diluted 500-fold and treated at 50 μl / well. It was left standing at 4°C for 20 minutes. After adding 150 μl / well of the cell washing buffer and mixing, it was centrifuged at 1500 RPM for 5 minutes. After discarding the supernatant, 200 μl / well of the cell washing buffer was added and mixed, and then it was centrifuged at 1500 RPM for 5 minutes. PE-Streptavidin (BD Pharmigen, 554061) was diluted 1000-fold and treated at 50 μl / well. It was left standing at 4°C for 20 minutes. After adding 150 μl / well of the cell washing buffer and mixing, it was centrifuged at 1500 RPM for 5 minutes. After discarding the supernatant, 200 μl / well of the cell washing buffer was added and mixed, and then it was centrifuged at 1500 RPM for 5 minutes. 80 μl / well of the cell washing buffer was added and mixed, and then analyzed with a flow cytometer.

[0098] The affinity of the soluble scFv for mucin 1-expressing cells was confirmed by the PE MFI value among the result values of the flow cytometer. Without adding soluble scFv to each cell line, the value obtained by proceeding with staining using other fluorescent antibodies (Goat anti-human IgG Fc-biotin antibody, PE-Streptavidin) was used as the negative control group value, and the value when each cell-specific G03 was treated at a concentration of 380 μg / mL was used as the positive control group value. The MFI value was corrected as follows: (Experimental value - Negative control group value) / (Positive control group value - Negative control group value) × 100 = Corrected value of the experimental value Using the corrected value, EC 50 was determined, and the described result values are the result values of the two cell lines used as the positive control group (Table 6).

[0099]

Table 6

[0100] Example 4. Cytokine Secretion Analysis of CAR-T Cells The reactivity of immune cells (T cells) injected with anti-MUC1 scFv in CAR form against MUC1-positive tumor cells (T47D, HEK293T-V1) was evaluated. Ten types of immune cells 2445_1F09, 2447_3A01, 2447_3A08, 2447_3A09, 2447_3A12, 2447_3C08, 2447_3H02, 2447_3H08, 2447_2H08, 2447_3B07 (or abbreviated as 1F09, 3A01, 3A08, 3A09, 3A12, 3C08, 3H02, 3H08, 2H08, 3B07 respectively in some figures) injected with anti-MUC1 scFv in CAR form, which were produced according to Example 2, were co-cultured with T47D cells that naturally express a large amount of MUC1 and HEK293T-V1 cells overexpressing the C subunit of human MUC1 for 24 hours at a tumor cell:immune cell ratio of 1:1.5. The concentrations of two cytokines, interferon-gamma (IFNg) and interleukin-2 (IL-2), in the culture solution obtained therefrom were confirmed by ELISA. The ELISA analysis method is the same as the commonly used method. Briefly, it is as follows. First, a 96-well immunoplate was coated with a capture antibody that can capture interferon-gamma or interleukin-2. After one day, it was washed thoroughly with a washing buffer. A block buffer was added to each well and left standing at room temperature for 1 hour, and then washed thoroughly with the washing buffer again. To know the absolute value, a culture solution sample was placed in each well together with a standard solution of known concentration and left standing at room temperature for 2 hours to react. After washing thoroughly with the washing buffer, a detection antibody was added and reacted at room temperature for 2 hours. After washing thoroughly with the washing buffer, streptavidin labeled with HRP was added to each well and reacted at room temperature for 20 minutes. After washing thoroughly with the washing buffer, a substrate solution was added and reacted at room temperature for 20 minutes, and the reaction was stopped with a stop solution. The cytokine concentration in each well was calculated based on the absorbance corresponding to the reaction.

[0101] G03 in Figure 1 is the positive control group, and a group (NO LV) in which no CAR was injected was used as the negative control group. Also, Figure 1 is a result graph representing two independent experiments using T cell-derived cells, named Donor03 and Donor04 according to the origin of the T cells. In this example, "Donor" refers to the donor of the T cells used in the experiment, and the numbers after Donor mean that the donors are different from each other. Figure 1 is a graph in which the absorbance is normalized based on G03 of the control group, indicating that the tested immune cells secrete cytokines.

[0102] Figure 2 shows the results of cytokine secretion analysis evaluating the reactivity of immune cells injected with anti-MUC1 scFv in CAR form against MUC1-positive tumor cells (T47D), indicating that the tested immune cells secrete cytokines. The T cells are derived from Donor06. A group (Control T cell) in which no CAR was injected was used as the negative control group.

[0103] Figure 3 shows the results of cytokine secretion analysis evaluating the reactivity of immune cells injected with anti-MUC1 scFv in CAR form against mucin 1 non-expressing cells (HEK293T), indicating that interferon gamma cytokine is not secreted, and in the case of IL-2, the secretion amount does not increase significantly when compared with the negative control group (Control T cell). The T cells are derived from Donor06. A group (Control T cell) in which no CAR was injected was used as the negative control group.

[0104] Example 5. Cytotoxicity Analysis of CAR-T Cells Human breast cancer cell lines (T47D, HCC70, HCC1954), human liver cancer cell lines (Huh7, HepG2), and human neuroblastoma cell line (SK-N-SH) were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea), and human embryonic kidney cell line (HEK293T) was purchased from the American Type Culture Collection (ATCC, Manassas, VA). T47D, HCC70, and HCC1954 cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific), and HEK293T, Huh7, and HepG2 were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific), respectively. All media were supplemented with 10% (w / v) heat inactivated fetal bovine serum (FBS, Thermo Fisher Scientific). All cells were cultured at 37°C, 5% CO 2 conditions. The CAR-T cells produced according to Example 2 were cultured in CTS complete media (CTS® OpTmizer® T Cell Expansion SFM (Gibco, A1048501), 2% (w / v) CTS® Immune Cell SR (Gibco, A25961-01), 1X GlutaMAX® Supplement (Gibco, 35050-061)) at 37°C, 5% CO 2 conditions.

[0105] To analyze the mucin 1-specific cytotoxicity of CAR-T cells, the cell lysis against mucin 1-expressing cells (including T47D, HCC70, HCC1954, HEK293T-V1 (MUC1-C extracellular expression cells), etc.) and mucin 1-non-expressing cells (including HEK293T, HepG2, Huh7, SK-N-SH, etc.) was measured by the following method. First, mucin 1-expressing or non-expressing target cells stained with CellTrace® CFSE (invitrogen, C34554) were adjusted to 0.15~0.2×10 6Cells were dispensed at 150 μl / well and cultured for 1 day. On the same day, CAR-T cells cryopreserved at -196 °C were thawed and cultured for 1 day in CTS complete media containing 30 U / ml of IL-2 (Novartis, Proleukin) at a concentration of 2 × 10 6 cells / ml. The next day, the CAR expression rate of the CAR-T cells during culture was stained with Alexa Fluor 488-AffiniPure Goat Anti-Human IgG, F(ab’)2 Fragment Specific (Jackson laboratory, 109-545-097) and APC Mouse Anti-Human CD3 (BD Pharmingen®, 561811), and the ratio of both positive cells was determined using a flow cytometer (FACS). Based on the CAR expression rate, various types of CAR-T (E, effector cells) with the same number of CAR+ T cells were adjusted to a volume of 100 μl / well with the cell culture medium of each target cell, and added in proportion to the number of previously cultured CFSE-stained mucin 1-expressing or non-expressing cells (T, target cells). The cells were cultured with the target cells at 37 °C and 5% CO 2 2 for 5 hours to a maximum of 24 hours. Thereafter, both the CAR-T cells and the target cells were taken and stained with Fixable Viability Dye eFluor TM 450 (eBioscience TM , 65-0863-18) and APC Mouse Anti-Human CD3, and the number of living target cells was determined using a flow cytometer. At this time, the number of living target cells was defined as Fixable Viability Dye-negative and CFSE-positive cells. The titer calculation (killing activity, %) was determined by the following formula:

Number

[0106] The results are shown in FIGS. 4 to 8. In these drawings, G03 is the positive control group, and as the negative control group, a group (NO LV) into which no CAR was injected was used. These drawings are representative result graphs showing two independent experiments using T cells derived from two donors, which were named Donor03 and Donor04 according to the origin of the T cells. As shown in FIGS. 4 to 8, the tested immune cells showed cytotoxicity against mucin 1-expressing cells.

[0107] FIGS. 9 and 10 show the results of cytotoxicity experiments of CAR-T cells prepared using T cells derived from Donor05 and Donor06, respectively, against the human breast cancer cell line T47D. FIG. 11 shows the results of cytotoxicity experiments of CAR-T cells prepared using T cells derived from Donor07 against the human embryonic kidney cells HEK293T. FIG. 12 shows the results of cytotoxicity experiments of CAR-T cells prepared using T cells derived from Donor08 against the human breast cancer cell line HCC70. As shown in FIGS. 9 to 12, the tested immune cells showed cytotoxicity against mucin 1-expressing cells.

[0108] Example 6. Mucin 1-Specific Cytotoxicity Analysis of CAR-T Cells To examine whether CAR-T cells can distinguish normal cells from cancer cells based on the difference in antigen amount, the cell solubility against the breast cancer cell line (HCC70) and the human normal breast cell line (MCF10A) was measured by the following method: First, HCC70, which is a mucin 1-overexpressing cell, and MCF10A, which expresses less mucin 1, were stained with different concentrations of CellTrace TM CFSE (Invitrogen, C34554), and then mixed at different ratios. The ratios used were HCC70:MCF10A of 0:0.2, 0.025:0.175, 0.05:0.15, 0.075:0.125, 0.1:0.1, 0.125:0.075, 0.15:0.05, 0.175:0.025, 0.2:0. The cells mixed at a certain ratio were 0.2×10 6Cells were dispensed at 500 cells / 500 μl / well and cultured for 1 day. On the same day, CAR-T cells (T cells derived from Donor05 expressing the 2447_2H08 scFv-containing CAR) stored frozen at -196 °C were thawed and cultured for 1 day in CTS complete media containing 30 U / ml of IL-2 (Novartis, Proleukin) at a concentration of 2×10 6 cells / ml. The next day, the CAR expression rate of the CAR-T cells during culture (CAR having 2447_2H08 scFv) was stained with Alexa Fluor 488-AffiniPure Goat Anti-Human IgG, F(ab’)2 Fragment Specific (Jackson laboratory, 109-545-097) and APC Mouse Anti-Human CD3 (BD Pharmingen TM , 561811), and the ratio of both positive cells was determined by a flow cytometer (FACS). For each type of CAR-T (E, effector cells) adjusted to the same number of CAR+ T cells based on the CAR expression rate, the volume was adjusted to 100 μl / well with the cell culture medium of each target cell and added in proportion to the number of CFSE-stained mucin 1-expressing or non-expressing cells (T, target cells) cultured the previous day. The cells were cultured with the target cells for 24 hours at 37 °C and 5% CO 2 2. Then, both the CAR-T cells and the target cells were taken, stained with Fixable Viability Dye eFluor® 450 (eBioscience TM , 65-0863-18) and APC Mouse Anti-Human CD3, and the number of living target cells was determined by a flow cytometer. At this time, the number of living target cells was defined as cells that were Fixable Viability Dye negative and CFSE positive (strong CFSE signal in the case of HCC70, weak CFSE signal in the case of MCF10A). The titer calculation (killing activity, %) was performed in the same manner as in Example 5.

[0109] The results are shown in FIGS. 13 and 14. In these drawings, a group (mock T cell) in which no CAR was injected was used as a negative control group. As shown in FIGS. 13 and 14, CAR-T cells having 2447_2H08 scFv (represented as #2-CAR-T in the drawings) showed cell killing ability against breast cancer cells (HCC70) overexpressing mucin 1, whereas cytotoxicity against normal breast cell lines (MCF10A) weakly expressing mucin 1 did not appear or appeared in amounts that were significantly reduced.

[0110] Example 7. Anti-Cancer Efficacy Analysis of CAR-T Cells Using Animal Models 7-1. Anticancer efficacy analysis using a pancreatic cancer animal model Six-week-old female NOG mice were subcutaneously (s.c.) injected with 6×10 6 PANC1-v1 cells (PANC1 pancreatic cancer cells expressing MUC1), and when the tumor volume reached 500 mm 3 1×10 6 anti-MUC1 CAR-T cells (T cells derived from Donor 18-09 expressing the scFv-containing CAR according to the present invention) were intravenously (i.v.) administered, and then tumor growth was observed. Blood was collected 14 and 28 days after CAR-T cell administration to observe the number of human immune cells (hCD45+ cells) in the blood (n = 5 per group). As negative control groups, a PBS (Vehicle) injection group and T cells not expressing CAR (Control T cell) were used.

[0111] As a result, as shown in FIGS. 15 and 16, it was observed that tumors with a size of 500 mm 3 became smaller, and no tumors were observed visually about 20 days after T cell administration. However, in the case of the negative control group, it was observed that the size of the cancer continuously increased.

[0112] Next, in the same animal model as described above, when the tumor volume exceeded 1500 mm 3 (that is, 66 days after administration of PANC1-v1 cells), 1×10 6 or 0.5×10 6After intravenous (i.v.) administration of anti-MUC1 CAR-T cells, tumor growth was observed (n = 2 per group).

[0113] As a result, as shown in Fig. 17, the tumor size initially increased, but after about 10 days, the size gradually decreased and became difficult to measure after 30 days.

[0114] 7-2. Anticancer efficacy analysis using a breast cancer animal model 4×10 6 HCC1954 cells (a breast cancer cell line that naturally expresses MUC1) were subcutaneously (s.c.) injected into 9-week-old female NOG mice. When the tumor volume reached 500 mm 3 (23 days after cell administration), 0.5×10 6 or 0.1×10 6 of anti-MUC1 CAR-T cells (T cells derived from Donor20-01 expressing the scFv-containing CAR according to the present invention) were intravenously (i.v.) administered, and then tumor growth was observed (n = 7 per group). After T cell administration, blood was collected at regular intervals to observe the number of human immune cells (hCD45+ cells) in the blood (n = 3 per group). As a negative control group, a PBS (Vehicle) injection group and T cells that do not express CAR (Control T cell) were used.

[0115] As a result, as shown in Figs. 18 and 19, the tumor size, which was 500 mm 3 stagnated in size for 7 days after CAR-T cell administration, but then decreased rapidly and became too small to measure after 25 days. Also, the number of hCD45+ cells in the mouse blood showed an increasing trend and gradually decreased after about 25 days. This is considered to be because the CAR-T cells were activated after recognizing the antigen, proliferated, and the number of cells in the blood increased.

[0116] From the above description, those having ordinary knowledge in the technical field to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea and essential features. Therefore, it should be understood that all the above-described embodiments are illustrative in all respects and not restrictive. The scope of the present invention should be construed to include all changes or modified forms derived from the meaning and scope of the claims described below, which are derived from the claims and their equivalent concepts, rather than the above detailed description.

Claims

**Claim 1** A polypeptide that binds to Mucin1 and comprises a pair of heavy chain variable (VH) regions and light chain variable (VL) regions selected from the group consisting of: - A VH region comprising CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively, and a VL region comprising CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; - A VH region comprising CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, respectively, and a VL region comprising CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; - A VH region comprising CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively, and a VL region comprising CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively; - A VH region comprising CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, respectively, and a VL region comprising CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, respectively; - A VH region comprising CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively, and a VL region comprising CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively; - A VH region comprising CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63, respectively, and a VL region comprising CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66, respectively; - A VH region comprising CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO: 73, respectively, and a VL region comprising CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76, respectively; - A VH region comprising CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 81, SEQ ID NO: 82, and SEQ ID NO: 83, respectively, and a VL region comprising CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 84, SEQ ID NO: 85, and SEQ ID NO: 86, respectively; and - A VH region comprising CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 91, SEQ ID NO: 92, and SEQ ID NO: 93, respectively, and a VL region comprising CDR1, 2, and 3 represented by the amino acid sequences of SEQ ID NO: 94, SEQ ID NO: 95, and SEQ ID NO: 96, respectively. **Claim 2** The polypeptide according to claim 1, comprising a pair of a heavy chain variable (VH) region and a light chain variable (VL) region selected from the group consisting of: - A VH region comprising the amino acid sequence of SEQ ID NO: 17 and a VL region comprising the amino acid sequence of SEQ ID NO: 18; - A VH region comprising the amino acid sequence of SEQ ID NO: 27 and a VL region comprising the amino acid sequence of SEQ ID NO: 28; - A VH region comprising the amino acid sequence of SEQ ID NO: 37 and a VL region comprising the amino acid sequence of SEQ ID NO: 38; - A VH region comprising the amino acid sequence of SEQ ID NO: 47 and a VL region comprising the amino acid sequence of SEQ ID NO: 48; - A VH region comprising the amino acid sequence of SEQ ID NO: 57 and a VL region comprising the amino acid sequence of SEQ ID NO: 58; - A VH region comprising the amino acid sequence of SEQ ID NO: 67 and a VL region comprising the amino acid sequence of SEQ ID NO: 68; - A VH region comprising the amino acid sequence of SEQ ID NO: 77 and a VL region comprising the amino acid sequence of SEQ ID NO: 78; - A VH region comprising the amino acid sequence of SEQ ID NO: 87 and a VL region comprising the amino acid sequence of SEQ ID NO: 88; and - A VH region comprising the amino acid sequence of SEQ ID NO: 97 and a VL region comprising the amino acid sequence of SEQ ID NO:

98. **Claim 3** The polypeptide according to claim 1, which is an antibody or an antigen-binding fragment thereof. **Claim 4** The polypeptide according to claim 1, which is a scFv (single chain variable fragment), Fab (fragment antigen binding), monoclonal antibody, bispecific antibody, minibody, synthetic antibody, chimeric antibody, humanized antibody, or antibody fusion protein. **Claim 5** The polypeptide according to claim 1, wherein the VH region and the VL region are linked by a linker. **Claim 6** The polypeptide according to claim 5, wherein the amino acid sequence of the linker is GGGGSGGGGGSGGGA S.

7. The polypeptide according to claim 1, which is an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 69, SEQ ID NO: 79, SEQ ID NO: 89, and SEQ ID NO:

99.

8. An isolated polynucleotide encoding the polypeptide according to any one of claims 1 to 7.

9. A vector comprising the polynucleotide according to claim 8.

10. A cell comprising the vector according to claim 9.

11. A chimeric antigen receptor comprising the polypeptide according to any one of claims 1 to 7.

12. The chimeric antigen receptor according to claim 11, comprising an extracellular domain comprising the polypeptide; a transmembrane domain; and an intracellular signaling domain.

13. The chimeric antigen receptor according to claim 12, wherein the extracellular domain further comprises a spacer region between the polypeptide and the transmembrane domain.

14. The chimeric antigen receptor according to claim 13, wherein the spacer comprises the hinge region of CD8α or CD28, or all or part of the constant region of immunoglobulin (IgG).

15. The chimeric antigen receptor according to claim 12, wherein the transmembrane domain is the transmembrane domain of CD28 or CD8.

16. The chimeric antigen receptor according to claim 12, wherein the intracellular signaling domain is the CD3 zeta signaling domain.

17. The chimeric antigen receptor according to claim 12, further comprising one or more co-stimulatory domains.

18. The chimeric antigen receptor according to claim 17, wherein the co-stimulatory domain is located between the transmembrane domain and the intracellular signaling domain.

19. The chimeric antigen receptor according to claim 17, wherein the co-stimulatory domain is the signaling domain of CD28, OX-40, 4-1BB (CD137), CD27, or ICOS.

20. An isolated polynucleotide encoding the chimeric antigen receptor according to claim 11.

21. A vector comprising the polynucleotide according to claim 20.

22. An immune cell that expresses a chimeric antigen receptor comprising the polypeptide according to any one of claims 1 to 7, or comprises a polynucleotide encoding the chimeric antigen receptor.

23. The immune cell according to claim 22, which is a T cell, a tumor-infiltrating lymphocyte (TIL), a NK (Natural killer) cell, a TCR-expressing cell, a dendritic cell, or a NK-T cell.

24. The immune cell according to claim 22, which is an autologous T cell or an allogeneic T cell.

25. The polypeptide according to any one of claims 1 to 7; An isolated polynucleotide encoding the polypeptide; A vector comprising a polynucleotide encoding the polypeptide; A cell comprising a polynucleotide encoding the polypeptide; A chimeric antigen receptor comprising the polypeptide; An isolated polynucleotide encoding the chimeric antigen receptor; A vector comprising a polynucleotide encoding the chimeric antigen receptor; or A composition for preventing or treating cancer, comprising a polynucleotide encoding the chimeric antigen receptor or an immune cell expressing the chimeric antigen receptor.

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