Cotinine-targeting antibody or antigen-binding fragment thereof, chimeric antigen receptor containing same, and uses thereof

Abstract

The present invention relates to an antibody or an antigen-binding fragment thereof targeting cotinine, a chimeric antibody receptor comprising the same, and uses thereof. The antibody of the present invention is an antibody that specifically binds to cotinine, and in particular, is an antibody that more specifically binds to the S-isomer of cotinine than the R-isomer. In addition, the sequence is unique because it has very low homology compared to the CDR sequence of conventional cotinine-targeting antibodies. Cells expressing the chimeric antigen receptor comprising the anti-cotinine antibody or antigen-binding fragment of the present invention bind to a switch bound with cotinine and react to the target cell line, thereby inducing immune cell activity. Therefore, the antibody or antigen-binding fragment of the present invention can be used as a CAR immune cell therapy agent, in addition to suppressing immune side effects caused by overactivity of T cells by regulating the activation of cotinine-mediated chimeric antigen receptor effector cells.

Description

[Technical field]

[0001] This invention was made under the support of the Ministry of Trade, Industry and Energy of the Republic of Korea under project specific number 1415175509 and detailed project number 20002893. The research management specialist for this project is the Korea Institute for Industrial Technology Assessment and Management, the research project name is "Bioindustry Core Technology Development Project", the research topic name is "Development of HER2-targeted ovarian cancer therapeutics using innovative switchable CART technology", the sponsor is AbClone Co., Ltd., and the research period is from 2018.10.01 to 2022.06.30.

[0002] This patent application claims priority to Korean Patent Application No. 10-2022-0031019 filed with the Korean Intellectual Property Office on March 11, 2022, and Korean Patent Application No. 10-2022-0113082 filed with the Korean Intellectual Property Office on September 6, 2022, the disclosures of which are incorporated herein by reference.

[0003] The present invention relates to an antibody or an antigen-binding fragment thereof that targets cotinine, a chimeric antibody receptor comprising the same, and uses thereof. [Background technology]

[0004] Numerous publications and patent documents are referenced and cited throughout this specification, the disclosures of which are incorporated herein by reference in their entirety to more clearly describe the level of the art to which the present invention pertains and the content of the present invention.

[0005] T cells, one of the immune cells, are well known to play an important role in cell-mediated immunity in humans and animals. CAR-T therapeutic agents are genetically modified T cells that are introduced with a receptor gene that specifically recognizes cells expressing target molecules (such as cancer cells) to activate the cytotoxicity of T cells, thereby inducing the death of the target cells. In particular, CAR-T therapeutic agents have been considered a dream anticancer drug, as they have shown a cure rate of about 80% in early clinical trials targeting leukemia patients who were refractory to all existing treatments by activating the weakened immune system. Kymriah, manufactured by Novartis, and Yescarta, manufactured by Kite Pharma, a subsidiary of Gilead, were approved by the FDA in August 2017 and commercialized in October.

[0006] However, despite these initial successful clinical efficacies, the stability of existing CAR-T cells has been questioned because their activation and expansion in vivo cannot be controlled. For example, CAR-T cells may persist in the body for a long time, which may pose potential risks and lead to serious symptoms such as cytokine release syndrome (CRS). Therefore, a new next-generation CAR-T platform called switchable CAR-T has been developed that overcomes the problems of existing CAR-T therapeutics by providing a switch function that can regulate the activity of CAR-T cells.

[0007] In the Switchable CAR-T system, the mediator that acts as the switch is a substance that can be recognized by the chimeric antigen receptor of CAR-T and can recognize the target molecule at the same time. For example, in a recent study, cotinine was bound as an adaptor to an antibody used in existing cancer immunotherapy, and used as a switch molecule to construct a switchable CAR-T system that utilizes a conventional antibody that specifically binds to cotinine. This makes it possible to use the ON / OFF mechanism of the switch molecule to regulate the activation of CAR-T cells, which had been raised as a problem in the past. In addition, by binding cotinine to an antibody used in existing cancer immunotherapy, it has become possible to create a multi-targeting CAR-T system using various switch molecules with cotinine bound to them.

[0008] Cotinine is a metabolite of nicotine, and is not biosynthesized in the body and does not induce physiological activity, making it a useful component of switch molecules. However, in order to improve the switchable CAR system that uses cotinine as an adaptor for switch molecules, it is necessary to develop improved cotinine-specific antibodies that can induce better cytotoxic activity. Summary of the Invention [Problem to be solved by the invention]

[0009] The present inventors have attempted to develop a novel antibody capable of inducing a more excellent cytotoxic activity than existing cotinine target antibodies. They have also made intensive research efforts to develop a chimeric antigen receptor utilizing the novel antibody of the present invention. As a result, they have developed a novel antibody or an antigen-binding fragment thereof that has a very low homology compared to the CDR sequence of a conventional cotinine target antibody. They have also produced a chimeric antigen receptor utilizing the humanized antibody and an effector cell expressing the chimeric antigen receptor, and found that effective immune cell activity is induced at a higher concentration than conventional cotinine target antibodies, thus completing the present invention.

[0010] It is therefore an object of the present invention to provide an antibody or antigen-binding fragment thereof that targets cotinine and specifically binds via the S-isomer of cotinine.

[0011] Another object of the present invention is to provide a chimeric antigen receptor comprising said antibody or an antigen-binding fragment thereof and an effector cell thereof.

[0012] It is yet another object of the present invention to provide a pharmaceutical composition for immunotherapy comprising a switch molecule for regulating activation of the effector cell and the effector cell, and a pharmaceutical kit thereof. [Means for solving the problem]

[0013] The present invention provides the following inventions 1 to 27.

[0014] 1. An antibody or antigen-binding fragment thereof that binds more specifically to the S-isomer of cotinine compared to the R-isomer.

[0015] In 2.1, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO:1, an HCDR2 comprising the amino acid sequence of SEQ ID NO:2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:3; and

[0016] An antibody or antigen-binding fragment thereof that more specifically binds to the S-isomer of cotinine, comprising: a light chain variable region comprising LCDR1 having the amino acid sequence of SEQ ID NO: 4, LCDR2 having the amino acid sequence of SEQ ID NO: 5, and LCDR3 having the amino acid sequence of SEQ ID NO: 6.

[0017] 3.1, the antibody or antigen-binding fragment thereof

[0018] (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:8; or

[0019] (ii) An antibody or antigen-binding fragment thereof that specifically binds to the S-isomer of cotinine, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:9 and a light chain variable region having the amino acid sequence of SEQ ID NO:10.

[0020] In 4.1, the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a scFv, Fab, F(ab), and F(ab)2, each of which comprises a heavy chain variable region and a light chain variable region.

[0021] 5. A nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof according to any one of 1 to 4.

[0022] In 6.5, the nucleic acid molecule is

[0023] (i) comprising the nucleotide sequence of SEQ ID NO: 12 encoding a heavy chain variable region, and the nucleotide sequence of SEQ ID NO: 13 encoding a light chain variable region; or

[0024] (ii) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 14 encoding a heavy chain variable region, and the nucleotide sequence of SEQ ID NO: 15 encoding a light chain variable region.

[0025] A recombinant vector comprising the nucleic acid molecule of 7.5.

[0026] A host cell containing the recombinant vector of 8.7.

[0027] 9. A chimeric antigen receptor comprising an antibody or an antigen-binding fragment thereof that more specifically binds to the S-isomer of cotinine according to any one of 1 to 4.

[0028] 10. A chimeric antigen receptor effector cell expressing a chimeric antigen receptor comprising an antibody or an antigen-binding fragment thereof that more specifically binds to the S-isomer of cotinine according to any one of 1 to 4.

[0029] In 11.10, the chimeric antigen receptor effector cell is activated and regulated by a switch molecule comprising: (a) the S-isomer of cotinine, and (b) a targeting moiety that binds to a cell surface molecule on a target cell.

[0030] 12. The effector cell of claim 11, wherein the cell surface molecule on the target cell is selected from the group consisting of HER2, CD19, EGFR, VEGFR2, CD80, CD86, mesothelin, prostate specific membrane antigen (PSMA), CD20, MUC1, and combinations thereof.

[0031] 13. The chimeric antigen receptor effector cell in accordance with 11, wherein the targeting moiety constituting the switch molecule for regulating activation is an antibody, an antigen-binding fragment of an antibody, or a target binding polypeptide.

[0032] 14. The chimeric antigen receptor effector cell according to 13, wherein the switch molecule for regulating activation is a cotinine-conjugated affibody to which S-isomer cotinine is conjugated.

[0033] A pharmaceutical composition for immunotherapy comprising the chimeric antigen receptor effector cells of 15.10 as an active ingredient.

[0034] 16. A pharmaceutical kit for immunotherapy comprising, as an active ingredient, a chimeric antigen receptor effector cell according to 10 to 14.

[0035] 17. A method comprising administering a chimeric antigen receptor effector cell according to 10 to 14, and a switch molecule that binds to the chimeric antigen receptor, to a subject in need of treatment.

[0036] In 18.17, the method is a method for treating a disease or condition associated with a tumor or cancer.

[0037] In 19.17, the method is a method for treating a disease or condition associated with autoimmunity.

[0038] 20. Administering the chimeric antigen receptor effector cells of 10 to 14 and a switch molecule that binds to the chimeric antigen receptor to a subject in need of treatment; and

[0039] A method comprising the step of inhibiting the activity of a chimeric antigen receptor effector cell.

[0040] 21. The method of 20, wherein the method suppresses side effects caused by administration of chimeric antigen receptor effector cells.

[0041] 22. The method of claim 20, wherein the step of suppressing the activity of the chimeric antigen receptor effector cell is adding a substance that binds to the chimeric antigen receptor.

[0042] 23. The method of 22, wherein the substance that binds to the chimeric antigen receptor is cotinine, preferably S-cotinine.

[0043] 24. The method of claim 20, wherein the step of suppressing the activity of the chimeric antigen receptor effector cell is adding a substance that binds to the switch molecule.

[0044] 25. The method of claim 24, wherein the substance that binds to the switch molecule is an isolated antibody or antigen-binding fragment that binds cotinine.

[0045] 26. The method of 24, wherein the substance that binds to the switch molecule is a surface molecule of an isolated target cell that binds to a targeting moiety of the switch molecule.

[0046] 27. A chimeric antigen receptor-effector cell therapeutic system comprising a chimeric antigen receptor effector cell according to 10 to 14, and a switch molecule that binds to the chimeric antigen receptor.

[0047] The present inventors have developed an antibody or an antigen-binding fragment thereof that is capable of inducing superior cytotoxic activity compared to conventional cotinine-targeting antibodies.

[0048] In particular, it has been confirmed that chimeric antigen receptor effector cells comprising the novel cotinine-targeting antibody or its antigen-binding fragment of the present invention induce effective immune cell activity at high concentrations depending on the type of cotinine isomer, compared to conventional cotinine-targeting antibodies.

[0049] According to one aspect of the invention, the invention provides an antibody or antigen-binding fragment thereof that more specifically binds to the S-isomer of cotinine compared to the R-isomer.

[0050] As used herein, the term "cotinine" refers to a major metabolic product of nicotine, a substance having the structure of Chemical Formula 1 below.

[0051] [ka]

[0052] Cotinine is not biosynthesized in the body and does not induce physiological activity, so it does not change the properties of the conjugated substance, and can be bound to an antibody that specifically recognizes cotinine. In addition, it is easily produced in a form bound to a carboxyl group, so it can be bound to a conjugated substance.

[0053] Cotinine has stereoisomers classified into S-isomer and R-isomer.

[0054] In this specification, the term "isomer" refers to a molecule that has the same molecular formula but differs in the connection method or spatial arrangement of constituent atoms. Isomers can be broadly classified into constitutional isomers (or structural isomers) and stereoisomers. Structural isomers refer to compounds that have the same molecular formula but different connection states of atoms in the molecule (such as the type and number of bonds, and the type and position of functional groups). Stereoisomers refer to compounds that have the same connection states of atoms but different spatial arrangements between atoms and do not overlap with each other. The stereoisomers are further classified into enantiomers or optical isomers, and partial stereoisomers. The enantiomers refer to isomers that do not overlap with the original compound in the mirror image relationship with each other. Stereoisomers other than enantiomers, i.e., isomers that are not mirror images but do not overlap, are all classified as partial stereoisomers, which include geometric isomers and conformational isomers.

[0055] Therefore, even if isomers have the same molecular formula, they may be classified as different compounds having different physical / chemical properties. For example, even if the physical properties are the same in the living body, only one optical isomer may have physiological activity due to the specific environment for the optical isomer due to optical activity. Therefore, the importance of each isomer having different physical / chemical properties is emphasized in terms of its biological or pharmaceutical role.

[0056] Cotinine has stereoisomers, which are represented by substances having the structure of the following chemical formula 2. These have the same name except for the prefix "cis-trans" or "RS", and are therefore hereinafter referred to as the S-isomer and R-isomer of cotinine in this specification.

[0057] [ka]

[0058] In one embodiment of the present invention, cotinine, which is a mixture of two isomers, was separated into two single isomers using a chiral column, and it was confirmed that the antibodies and antigen-binding fragments thereof of the present invention bind more specifically to the S-isomer of cotinine than to the R-isomer.

[0059] In one embodiment of the present invention, the antibody or antigen-binding fragment thereof that more specifically binds to the S-isomer of cotinine of the present invention is an antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising HCDR1 having the amino acid sequence of SEQ ID NO: 1, HCDR2 having the amino acid sequence of SEQ ID NO: 2, and HCDR3 having the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising LCDR1 having the amino acid sequence of SEQ ID NO: 4, LCDR2 having the amino acid sequence of SEQ ID NO: 5, and LCDR3 having the amino acid sequence of SEQ ID NO: 6.

[0060] In a specific embodiment of the present invention, the antibody or antigen-binding fragment thereof that more specifically binds to the S-isomer of cotinine of the present invention is an antibody or antigen-binding fragment thereof that comprises: (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:8; or (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:9, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:10.

[0061] As used herein, the term "antibody" refers to an antibody that specifically binds to a specific antigen, and includes complete antibody forms as well as antigen binding fragments of antibody molecules. A complete antibody has two full-length light chains and two full-length heavy chains, and each light chain is linked to a 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, and the heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, with gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2) subclasses. The light chain constant region has kappa (κ) and lambda (λ) types (Cellular and Molecular Immunology, Wonsiewicz, MJ, Ed., Chapter 45, pp. 41-50, WB Saunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, Sinauer Associates, Inc., Sunderland, MA (1984)).

[0062] As used herein, the term "antibody" refers to an antibody that specifically binds to cotinine, including both the complete antibody form and the antigen binding fragment of the antibody molecule. Specifically, as used herein, the term "antibody" refers to an antibody that more specifically binds to the S-isomer of cotinine than the R-isomer, including both the complete antibody form and the antigen binding fragment of the antibody molecule.

[0063] As used herein, the term "antigen binding fragment" refers to a fragment that retains antigen-binding function, and includes Fab, F(ab'), F(ab')2, chemically linked F(ab')2, and Fv. Among antibody fragments, Fab (fragment antigen binding) has a structure that includes light and heavy chain variable regions, a light chain constant region, and the first heavy chain constant region (CH1), and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibody is produced by disulfide bonding of the cysteine ​​residues in the hinge region of Fab'. Fv is the smallest antibody fragment having only a heavy chain variable region and a light chain variable region, and recombinant techniques for producing the fragments are disclosed in PCT International Patent Publications WO88 / 10649, WO88 / 106630, WO88 / 07085, WO88 / 07086, and WO88 / 09344. In a two-chain Fv, the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, and in a single-chain Fv (single-chain variable fragment, scFv), the heavy chain variable region and the single chain variable region are generally linked by a covalent bond via a peptide linker or directly at the C-terminus, and can form a dimer-like structure like the two-chain Fv. Such antibody fragments can be obtained using proteolytic enzymes (for example, whole antibodies can be restricted with papain to obtain Fab fragments, or pepsin to obtain F(ab')2 fragments) or produced by genetic recombination techniques.

[0064] The antibody or antigen-binding fragment thereof of the present invention is specifically in the form of an scFv or in the form of a complete antibody. The heavy chain constant region may be selected from any one of the isotypes: gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε). Preferably, the heavy chain constant region is gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3) or gamma 4 (IgG4), most specifically gamma 4 (IgG4) isotype. The light chain constant region may be of the kappa (κ) or lambda (λ) type, preferably of the kappa (κ) type. Thus, a specific antibody of the present invention is in the form of an scFv or IgG4 having a kappa (κ) light chain and a gamma 4 (IgG4) heavy chain, but is not necessarily limited thereto.

[0065] As used herein, the term "heavy chain" refers to a full-length heavy chain or a fragment thereof that includes an antibody variable region domain VH and three constant region domains CH1, CH2, and CH3, as well as a hinge, and includes an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. Additionally, as used herein, the term "light chain" refers to a full-length light chain or a fragment thereof that includes an antibody variable region domain VL and constant region domain CL, as well as an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen.

[0066] As used herein, the term "CDR (complementarity determining region)" refers to the amino acid sequence of the hypervariable region of an immunoglobulin heavy chain and a light chain (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., USDepartment of Health and Human Services, National Institutes of Health (1987)). Each of the heavy chain (HCDR1, HCDR2, and HCDR3) and the light chain (LCDR1, LCDR2, and LCDR3) contains three CDRs, and a framework region (FR) exists between these CDRs and plays a role in supporting the CDR ring. The CDR is a cyclic portion involved in antigen recognition, provides the main contact residues when an antibody binds to an antigen or an antigenic determining site (epitope), and determines the specificity of the antibody to the antigen.

[0067] The term "Framework" or "FR" refers to variable domain residues other than the hypervariable region residues. The FR of a variable domain generally consists of four FR domains, FR1, FR2, FR3 and FR4.

[0068] As used herein, the term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of native antibodies generally have a similar structure, with each domain containing four conserved framework regions and three hypervariable regions (HVR) (Kindt et al., Kuby Immunology, 6th Edition, WH Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Antibodies that bind to a particular antigen may also be isolated using the VH or VL domain from an antibody that binds to the antigen and screening a library of complementary VH or VL domains, respectively.

[0069] As used herein, the term "specifically binds" or the like means that an antibody or antigen-binding fragment thereof, or other construct such as an scFv, specifically interacts with and immunologically reacts with an antigen or a corresponding substance. Specific binding is at least about 1×10 -6 M or less (e.g., 9×10 -7 M, 8×10 -7 M, 7×10 -7 M, 6×10 -7 M, 5×10 -7 M, 4×10 -7 M, 3×10 -7 M, 2×10 -7 M, or 1 x 10 -7 M), preferably 1 × 10 -7 M or less (e.g., 9×10 -8 M, 8×10 -8 M, 7×10 -8 M, 6×10 -8 M, 5×10 -8 M, 4×10 -8 M, 3×10 -8 M, 2×10 -8 M, or 1 x 10 -8 M), more preferably 1×10 -8 M or less (e.g., 9×10-9 M, 8×10 -9 M, 7×10 -9 M, 6×10 -9 M, 5×10 -9 M, 4×10 -9 M, 3×10 -9 M, 2×10 -9 M, or 1 x 10 -9 M) equilibrium dissociation constant (e.g., K D indicates more specific binding.) Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

[0070] The antibodies or antigen-binding fragments thereof of the present invention include full-length or intact polyclonal- or monoclonal-antibodies, as well as antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fab3, Fv and variants thereof), fusion proteins comprising one or more antibody moieties, human antibodies, humanized antibodies, chimeric antibodies, minibodies, diabodies, triabodies, tetrabodies, linear antibodies, single chain antibodies (scFv), scFv-Fc, bispecific antibodies, multispecific antibodies, glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies as other modified sequence forms of immunoglobulin molecules containing an antigen recognition site of the required specificity. Specific examples of modified antibodies and antigen-binding fragments thereof include nanobodies, AlbudAbs, DARTs (dual affinity re-targeting), BiTEs (bispecific T-cell engagers), TandAbs (tandem diabodies), DAFs (dual acting Fab), two-in-one antibodies, SMIPs (small modular immunopharmaceuticals), FynomAbs (fynomers fused to antibodies), DVD-Igs (dual variable domain immunoglobulin), CovX-bodies (peptide modified antibodies), duobodies, and triomAbs. The list of such antibodies and antigen-binding fragments thereof is not limited to the above.

[0071] In a specific embodiment of the present invention, the antibody or antigen-binding fragment thereof of the present invention is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, scFv, Fab, F(ab), and F(ab)2, each of which comprises the above-mentioned heavy chain variable region and light chain variable region, but is not limited thereto.

[0072] Preferably, the antibody or antigen-binding fragment thereof may be a humanized antibody.

[0073] The term "humanized antibody", as used herein, is a chimeric immunoglobulin that contains minimal sequence derived from a non-human immunoglobulin of a non-human (e.g., murine) antibody, immunoglobulin chain or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequence of an antibody). In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the complementarity determining regions (CDRs) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are not found in the CDR or framework sequences of the recipient or donor antibody. Such modifications are made to further improve and optimize antibody performance. In general, the humanized antibody may comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions have the sequences of human immunoglobulin FR regions. The humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc region), or substantially a human immunoglobulin constant region (Fc region) sequence.

[0074] According to another aspect of the present invention, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof that binds more specifically to the S-isomer of cotinine as described above.

[0075] In one embodiment of the present invention, it is sufficient for a nucleotide sequence to encode the amino acid sequence constituting the antibody or antigen-binding fragment thereof of the present invention, and it is clear to one skilled in the art that the nucleotide sequence is not limited to a specific nucleotide sequence. This is because even if a mutation occurs in the nucleotide sequence, when the mutated nucleotide sequence is expressed as a protein, it may not result in a change in the protein sequence. This is called codon degeneracy. Thus, the nucleotide sequence includes nucleotide sequences containing functionally equivalent codons or codons encoding the same amino acid (for example, due to codon degeneracy, there are six codons encoding arginine or serine), or codons encoding biologically equivalent amino acids.

[0076] In a specific embodiment of the present invention, the nucleic acid molecule of the present invention is a nucleic acid molecule comprising: (i) a nucleotide sequence of SEQ ID NO: 12 encoding a heavy chain variable region and a nucleotide sequence of SEQ ID NO: 13 encoding a light chain variable region; or (ii) a nucleotide sequence of SEQ ID NO: 14 encoding a heavy chain variable region and a nucleotide sequence of SEQ ID NO: 15 encoding a light chain variable region.

[0077] As used herein, the term "nucleic acids" refers to a term that encompasses DNA (gDNA and cDNA) and RNA molecules. Nucleotides, which are the basic building blocks of nucleic acid molecules, include natural nucleotides as well as analogues in which the sugar or base moiety is modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

[0078] Considering the above-mentioned mutations having biologically equivalent activity, the nucleic acid molecule of the present invention encoding the amino acid sequence constituting the antibody and its antigen fragment is also interpreted as including a sequence showing substantial identity thereto. The substantial identity means a sequence showing at least 60% or more homology, more specifically 70% or more homology, even more specifically 80% or more homology, particularly specifically 90% or more homology, and most specifically 95% or more homology, when the above-mentioned sequence of the present invention is aligned with any other sequence so as to correspond as closely as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. The alignment method for such sequence comparison is well known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math. 2:482 (1981); Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24:307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc. Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992) and Pearson et al. al., Meth. Mol. Biol. 24:307-31 (1994), but is not limited thereto.

[0079] In another aspect, the present invention provides a recombinant vector comprising the nucleic acid molecule.

[0080] As used herein, the term "vector" refers to a means for expressing a gene of interest in a host cell, and includes plasmid vectors; cosmid vectors; and viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors.

[0081] According to a specific embodiment of the present invention, in the vector of the present invention, the nucleic acid molecule encoding the anti-CD19 antibody or antigen-binding fragment thereof is operatively linked to a promoter.

[0082] As used herein, the term "operably linked" refers to the functional association of a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) with another nucleic acid sequence, whereby the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0083] The recombinant vector system of the present invention may be constructed by various methods known in the art, specific methods for which are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

[0084] Typically, the vector of the present invention may be constructed as a vector for cloning or as a vector for expression, and may be constructed in a prokaryotic or eukaryotic host cell.

[0085] For example, when the vector of the present invention is an expression vector and a eukaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) may be used, which generally has a polyadenylation sequence as a transcription termination sequence.

[0086] The vectors of the present invention may be fused to other sequences to facilitate purification of the antibodies or antigen-binding fragments expressed therefrom, such as glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA).

[0087] On the other hand, the expression vector of the present invention contains an antibiotic resistance gene commonly used in the art as a selection marker, such as resistance genes to ampicillin, gentamicin, cavenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0088] According to yet another aspect of the present invention, there is provided a host cell transformed with said recombinant vector.

[0089] Any host cell known in the art can be used as a host cell in which the vector of the present invention can be stably and continuously cloned and expressed. For example, suitable eukaryotic host cells for the vector include, but are not limited to, monkey kidney cells 7 (COS7), NSO cells, SP2 / 0, Chinese Hamster Ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT78 cells, and HEK-293 cells.

[0090] As used herein, the terms "transformed," "transduced," or "transfected" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transformed," "transduced," or "transfected" cell is a cell that has been transformed, transduced, or transfected with exogenous nucleic acid, including the cell itself and its progeny by subculture.

[0091] According to yet another aspect, the present invention provides a chimeric antigen receptor comprising the above-mentioned antibody or antigen-binding fragment thereof that specifically binds to cotinine.

[0092] The chimeric antigen receptor antibody or antigen-binding fragment thereof is an anti-cotinine antibody or antigen-binding fragment thereof.

[0093] In an embodiment of the present invention, there is provided a chimeric antigen receptor linked to an anti-cotinine antibody or an antigen-binding fragment thereof, the chimeric antigen receptor comprising an anti-cotinine antibody, a hinge domain, a transmembrane domain, and a signal transduction domain.

[0094] As used herein, the term "chimeric antigen receptor (CAR)" refers to an artificially engineered hybrid protein or polypeptide that includes a target binding domain linked to an effector cell signaling domain or an effector cell activation domain (e.g., a T cell signaling domain or a T cell activation domain). Chimeric antigen receptors (CARs) generally have the ability to redirect T cell specificity and reactivity to selected targets in a non-MHC restricted manner using the antigen-binding properties of monoclonal antibodies. Non-MHC restricted antigen recognition provides T cells expressing chimeric antigen receptors with the ability to recognize antigens independent of antigen processing, circumventing a major mechanism of tumor escape. Additionally, chimeric antigen receptors are advantageously not dimerized with endogenous T cell receptor (TCR) alpha and beta chains when expressed in T cells.

[0095] Specifically, the chimeric antigen receptor of the present invention refers to a "switchable chimeric antigen receptor (sCAR)." The extracellular domain of a conventional, general classical chimeric antigen receptor contains an antibody or an antigen-binding fragment of an antibody that directly targets a specific antigen on the cell surface of a target cell. However, the extracellular domain of the chimeric antigen receptor of the present invention does not directly target a cell surface antigen of a target cell, but contains the above-mentioned antibody or an antigen-binding fragment thereof that specifically binds to cotinine, thereby targeting cotinine (specifically, cotinine conjugated with a switch molecule).

[0096] The "hinge domain" refers to a domain that links an anti-cotinine antibody or a fragment thereof to a transmembrane domain. It may contain a cysteine ​​residue, and the cysteine ​​residue may be involved in the binding of the hinge domain to an antibody. For example, the hinge domain may be, but is not limited to, a hinge domain derived from CD8 or CD28, a hinge domain derived from IgG1, IgG2, IgG4, or IgGD, a KIR (killer immunoglobulin-like receptor) extracellular domain, and combinations thereof.

[0097] The "transmembrane domain" refers to a site that can link the hinge domain and the signal transduction domain of the chimeric antigen receptor. The transmembrane domain can penetrate the cell membrane of a cell so that the anti-cotinine antibody or antigen-binding fragment thereof of the chimeric antigen receptor is located on the cell surface and the signal transduction domain is located inside the cell. The transmembrane domain may be selected from the group consisting of, but is not limited to, the alpha, beta or zeta chain of the T cell receptor, CD27, CD28, CD3 epsilon (ε), CD45, CD4, CD5, CD8 (CD8α), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

[0098] The "intracellular signaling domain" refers to a portion that is located inside the cell membrane of an immune cell, i.e., in the cytoplasm, and transmits a signal into the cell to activate the immune response of the immune cell when the antibody or its antigen-binding fragment contained in the extracellular domain binds to a target antigen.

[0099] The intracellular signaling domain of the present invention may be one or more intracellular signaling domains selected from the group consisting of CD3 zeta (ζ), CD3 gamma (γ), CD3 delta (δ), CD3 epsilon (ε), FcR gamma, FcR beta, CD5, CD22, CD79a, CD79b and CD66d, but is not limited thereto, and may preferably be CD3 zeta (ζ).

[0100] Furthermore, the intracellular signaling domain of the present invention may further comprise a costimulatory domain, which may be one or more costimulatory domains selected from the group consisting of CD2, CD7, CD27, CD28, CD30, CD40, 4-1BB (CD137), OX40 (CD134), ICOS, LFA-1, GITR, MyD88, DAP1, PD-1, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83, but is not limited thereto.

[0101] Additionally, the chimeric antigen receptors of the present invention may include modified forms of the antibodies and domains described above. In this case, the modifications may be made by substituting, deleting, or adding one or more amino acids in the amino acid sequences of the wild-type antibodies and domains without altering the function of the antibodies and domains. Typically, the substitutions are alanine or may be made by conservative amino acid substitutions that do not affect the charge, polarity, or hydrophobicity of the overall protein.

[0102] According to yet another aspect, the present invention provides a chimeric antigen receptor effector cell expressing a chimeric antigen receptor comprising the above-mentioned antibody or antigen-binding fragment thereof that specifically binds to cotinine.

[0103] As used herein, the term "effector cell" refers to a cell that is transformed with a vector containing a gene encoding a chimeric antigen receptor polypeptide and expresses the introduced chimeric antigen receptor on the cell membrane.

[0104] In one embodiment of the present invention, an effector cell refers to an immune cell of hematopoietic origin that is responsible for initiating and / or executing an innate and / or acquired immune response.

[0105] The immune cells of the present invention may be immune cells such as dendritic cells, killer dendritic cells, mast cells, T cells, NKT cells, regulator T cells, NK cells (natural killer cells), or B cells, and may be obtained from bone marrow, peripheral blood, peripheral blood mononuclear cells, or umbilical cord blood.

[0106] In one embodiment of the present invention, the cells may be allogeneic or autologous cells to the subject to which they are administered.

[0107] In a specific embodiment of the present invention, the effector cell may be a T cell, which is an immune cell that expresses the chimeric antigen receptor on its surface.

[0108] In one embodiment of the present invention, an effector cell is linked with the antibody or antigen-binding fragment thereof of the present invention and expresses the above-mentioned chimeric antigen receptor to target the cotinine-labeled conjugate. In this case, the binding between the effector cell and the cotinine-labeled conjugate may be an antigen-antibody binding between the anti-cotinine antibody or antigen-binding fragment thereof of the chimeric antigen receptor and cotinine.

[0109] Furthermore, effector cells may be bound to the cotinine-labeled conjugate to induce their activity. Here, the cotinine-labeled conjugate plays a role of a switch molecule that controls the activation of the effector cells. The activation of the effector cells includes exerting a cytotoxic function against target cells, secreting cytokines, and a combination thereof.

[0110] In a specific embodiment of the invention, the activation of the chimeric antigen receptor effector cells is regulated by a switch molecule comprising: (a) the S-isomer of cotinine, and (b) a targeting moiety that binds to a cell surface molecule on a target cell.

[0111] In a more specific embodiment, the targeting moiety of the switch molecule is an antibody, an antigen-binding fragment of an antibody, or a target binding polypeptide.

[0112] In a more specific embodiment of the present invention, the switch molecule is a cotinine-conjugated affibody to which S-isomer cotinine is conjugated.

[0113] In the present specification, the term "switch molecule" refers to an adaptor molecule that separates and mediates the target recognition domain of the chimeric antigen receptor and the cell signaling domain of the chimeric antigen receptor in an effector cell using the chimeric antigen receptor. The switch molecule switches the target of the chimeric antigen receptor to a heterogeneous target or a resistant tumor, or when side effects occur due to excessive activation of cells expressing the chimeric antigen receptor, the administration of the switch molecule can be adjusted to reduce the activity of the chimeric antigen receptor effector cell (Cao et al., Angew Chem Int Ed Engl. 2016 June 20; 55(26): 7520-7524.).

[0114] As used herein, the term "target binding polypeptide" refers to a non-immunoglobulin polypeptide molecule that has binding affinity for a target antigen like an antibody, but is structurally unrelated to antibodies. The target binding polypeptides are also called antibody-like molecules or antibody mimetics, and generally have a molecular weight of 3 KDa to 20 KDa, unlike antibodies, which have a molecular weight of about 150 KDa. The target-binding polypeptides include affibodies derived from the Z-domain of protein A, affilins advantageously in gamma-B crystallin or ubiquitin, affimers derived from cystatins, alphabodies, anticalins derived from lipocalins, avimers derived from cell membrane receptor domains, DARPins derived from ankyrin repeat motifs, fynomers derived from the SH3 domain of Fyn, Kunits domain peptides derived from the Kunits domain of protease inhibitors, and the 10th type 3 domain of fibronectin (10 th These include, but are not limited to, monobodies derived from the type III domain of fibronectin, and nanoCLAMPs derived from the carbohydrate binding module 32-2 of NagH from Clostridium perfringens.

[0115] The target-binding polypeptide may be engineered to have binding affinity for any target antigen by various screening methods known in the art, such as phage display, ribosome display, etc.

[0116] As used herein, the term "affibody" refers to the Z domain of Protein A of Staphylococcus aureus, which is a site that has affinity for IgG. An affibody is a small protein consisting of 58 amino acid residues, and the 13 amino acids that form the binding surface with IgG in the protein sequence of such an affibody molecule can bind to various target antigens depending on the amino acid sequence, and a library can be constructed because random sequences are possible. Similar to antibodies, affibody molecules capable of binding to various target antigens can be selected from a library by screening methods such as phage libraries and yeast two hybrid (Y2H). Using the characteristics of affibody molecules that can bind to antigens, affibodies that specifically bind to HER2 and amyloid-β have been developed in recent years (Orlova et al. 2006, Cancer Res., Gronwall et al., 2007, J. Biotechnol.). In addition, since affibodies have a very small molecular weight of 6Kda, they are generally diffused throughout the body when administered to the human body and are quickly removed by renal filtration, compared to IgG-type antibodies, which generally have a molecular weight of 150Kda. Therefore, affibodies are mainly applied to diagnostic sample research and development (Goldstein R et al., 2013, Expert Rev Anticancer Ther.).

[0117] In one embodiment of the present invention, the affibody constituting the switch molecule and cotinine may be bound by chemical conjugation.

[0118] The chemical conjugation may be performed using a chemical crosslinker, which may include, but is not limited to, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), dicyclohexyl carbodiimide (DCC), N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (Sulfo-NHS), imidoester-based crosslinkers, and sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). Any chemical crosslinker commonly used in the field of the present invention may be used without limitation.

[0119] Furthermore, the affibody and cotinine constituting the switch molecule may be indirectly linked via a linker.

[0120] Those of ordinary skill in the art will know that the creation of fusion proteins may generally involve the use of linkers between the functional moieties to be fused, and there are different types of linkers with different properties, such as flexible amino acid linkers, inflexible linkers, and cleavable amino acid linkers. Linkers have been used to increase the expression level, improve the biological activity, enable targeting, modify the pharmacokinetics, or increase the stability and improve the folding of fusion proteins.

[0121] According to yet another aspect of the present invention, there is provided a pharmaceutical composition for immunotherapy comprising the above-mentioned chimeric antigen receptor effector cell as an active ingredient.

[0122] In one embodiment of the present invention, the pharmaceutical composition for immunotherapy may further comprise the above-mentioned switch molecule for regulating the activity of chimeric antigen receptor effector cells.

[0123] In the present specification, the term "immunotherapy" refers to a cancer treatment method that helps the immune system to eliminate cancer. Immunotherapy is divided into active immunotherapy and passive immunotherapy. Active immunotherapy includes i) cancer vaccine therapy, which activates the immune system by injecting cancer cells or substances produced by cancer cells into the human body, and ii) immune-modulating therapy, which activates specific white blood cells by administering immune-modulating agents such as cytokines (interferon, interleukin, etc.) and growth factors. Passive immunotherapy includes therapeutic antibodies that bind to specific cancer cells and immune cell therapy. Immune cell therapy specifically includes, but is not limited to, dendritic cell vaccine therapy, CAR-T (chimeric antigen receptor T cell) therapy, NK cell therapy (natural killer cell therapy), CTL therapy (cytotoxic T lymphocyte therapy), and adoptive cell transfer. In the present invention, immunotherapy primarily means the immune cell therapy described above.

[0124] The term "cancer" refers to a disease characterized by the rapid and unregulated growth and proliferation of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Cancer includes solid and non-solid tumors (e.g., hematological tumors such as leukemia and lymphoma).

[0125] The term "tumor" refers to an abnormal growth of tissue that may be benign, precancerous, malignant, or metastatic. In particular, a solid tumor is usually a mass of abnormal tissue that does not contain cysts or liquid areas, and may be benign or malignant. Different types of solid tumors are named for the type of cells that form the tumor (e.g., sarcoma, carcinoma, and lymphoma).

[0126] Preferably, the disease or disorder targeted by the immunotherapy of the present invention may be cancer, and the cancer may be a solid cancer or a blood cancer. Here, the solid cancer may be lung cancer, colon cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, melanoma, retinoblastoma, thymus cancer, gastric cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, or pancreatic cancer. The blood cancer may be a cancer of the blood or bone marrow, for example, lymphoma, leukemia, or multiple myeloma.

[0127] Therefore, the therapeutic system of chimeric antigen receptor effector cells according to the present invention can be used to treat cancer by administering a pharmaceutical composition containing effector cells expressing the chimeric antigen receptor, and can also provide an efficient treatment by further administering a switch molecule to regulate the activity of the effector cells.

[0128] In particular, as demonstrated by the examples of the present invention, since the switch molecule of the present invention includes an affibody that specifically binds to a HER2 antigen, diseases that can be treated using the above-mentioned pharmaceutical composition may be human and mammalian diseases associated with cells expressing HER2.

[0129] Specifically, the switch molecule comprising the anti-HER2 affibody bound to cotinine of the present invention and the effector cell expressing a chimeric antigen receptor targeting the cotinine of the switch molecule effectively kills SKOV3, a cancer cell line overexpressing HER2 on the surface, and OVCAR3, a cancer cell line expressing HER2. Therefore, the effector cell expressing a chimeric antigen receptor comprising an antibody or an antigen-binding fragment thereof that more specifically binds to the S-isomer of cotinine of the present invention can be usefully used as an active ingredient of a pharmaceutical composition for treating carcinomas expressing HER2.

[0130] More specifically, the disease to be treated may be, but is not limited to, breast cancer, gastric cancer, ovarian cancer, colon cancer, and endometrial cancer.

[0131] Furthermore, the pharmaceutical composition of the present invention may further comprise a switch molecule comprising a targeting moiety that binds to a cell surface molecule of a target cell, thereby enabling manipulation of the target antigen of the targeting moiety and allowing it to be effectively used in the prevention or treatment of a wider variety of diseases.

[0132] The pharma- ceutically acceptable carriers contained in the pharmaceutical composition of the present invention are those commonly used in formulation, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0133] In addition to the above ingredients, the pharmaceutical composition of the present invention may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharma- ceutically acceptable carriers and formulations are described in Remington's Pharmaceutical Sciences (1999). thThis is described in detail in “The Journal of the American Academy of Sciences, Vol. 1, No. 1, 1995.”

[0134] The pharmaceutical compositions of the present invention can be administered orally or parenterally, for example, by intravenous injection, subcutaneous injection, intravascular injection, intramuscular injection, intraperitoneal injection, intrasternal injection, intratumoral injection, local administration, intranasal administration, intracerebral administration, intracranial administration, intrapulmonary administration, and intrarectal administration, but are not limited to these.

[0135] The appropriate dosage of the pharmaceutical composition of the present invention may be adjusted depending on various factors including the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the pharmaceutical composition, the type of dosage form, the administration method, and the age, weight, sex and diet of the patient, the administration time, the administration route, the treatment period and drugs used concomitantly, and an ordinary physician skilled in the art can easily determine and prescribe an effective dosage for the desired treatment. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.1-9×10 5 ~0.1-9×10 9 The term "pharmaceutical effective amount" as used herein means an amount sufficient to treat the above-mentioned diseases.

[0136] The pharmaceutical composition of the present invention may be prepared in the form of a unit dose or in a multi-dose container by formulating it with pharma- ceutically acceptable carriers and / or excipients in a manner that can be easily carried out by a person having ordinary skill in the art to which the invention pertains. In this case, the dosage form may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may further include a dispersant or stabilizer.

[0137] The pharmaceutical compositions of the present invention may also further comprise, in addition to the switch molecules described above, other pharma- ceutical active agents or drugs, such as, but not limited to, chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and the like.

[0138] According to yet another aspect of the present invention, there is provided a pharmaceutical kit for immunotherapy comprising the above-mentioned chimeric antigen receptor effector cells as an active ingredient.

[0139] In one embodiment of the present invention, the pharmaceutical kit may further include the above-mentioned switch molecule for regulating activation of chimeric antigen receptor effector cells.

[0140] Since the pharmaceutical kit for immunotherapy of the present invention includes the chimeric antigen receptor effector cell and switch molecule, which are another embodiment of the present invention described above, in order to avoid excessive complexity in the description of this specification, the overlapping content will be incorporated by reference and the description thereof will be omitted. Effect of the Invention

[0141] The features and advantages of the present invention can be summarized as follows:

[0142] (a) The present invention provides cotinine-targeting antibodies, or antigen-binding fragments thereof, that bind more specifically to the S-isomer of cotinine than to the R-isomer.

[0143] (b) The present invention provides a chimeric antigen receptor comprising an antibody or an antigen-binding fragment thereof that targets cotinine and more specifically binds to the S-isomer of cotinine, and an effector cell expressing the chimeric antigen receptor.

[0144] (c) The present invention provides a switch molecule for activating an effector cell expressing the chimeric antigen receptor.

[0145] (d) The present invention provides a pharmaceutical composition for immunotherapy and a pharmaceutical kit for immunotherapy, which comprise the chimeric antigen receptor and the switch molecule.

[0146] (e) When using the cotinine-targeting antibody or its antigen-binding fragment that binds more specifically to the S-isomer of cotinine of the present invention, it is possible to induce superior cytotoxic activity compared to conventional antibodies. In addition, since the specificity of the binding between the antibody and the switch molecule is further improved, the action of chimeric antigen receptor effector cells such as CAR-T can be more finely regulated, and thus the side effects caused by the persistence of effector cells in the body can be effectively regulated. [Brief description of the drawings]

[0147] [Figure 1] FIG. 1 shows quantitative confirmation of cotinine-specific antibody binding by ELISA analysis. [Diagram 2] This figure shows a graph of MFI (mean fluorescence intensity) values ​​obtained by confirming the cell binding ability of a HER2-binding switch (trastuzumab conjugated with cotinine) in NCI-N87 cells, which are HER2-expressing cells. [Diagram 3] FIG. 1 shows a quantitative comparison of the binding of the hz33B2 antibody using ELISA analysis. [Figure 4] FIG. 1 shows the binding of the hz33B2 antibody confirmed by flow cytometry. [Diagram 5] Schematic diagram of a CAR structure containing the hz33B2 antibody and the SUN10 antibody used as a control. [Figure 6] This figure shows the cytotoxic effect of CAR-T containing a cotinine-conjugated affibody and an anti-cotinine antibody against SKOV-3, a HER2-overexpressing cell line, and OVCAR-3, a HER2-expressing cell line. [Figure 7] FIG. 1 shows the binding of the hz33B2 antibody to the switches bound to each cotinine isomer, confirmed by ELISA analysis. [Figure 8] This figure shows the binding of switches bound to each cotinine isomer to CAR-T containing the hz33B2 antibody, confirmed by flow cytometry. [Figure 9] This is a graph showing the cytotoxic effects of CAR-T containing switches bound to each cotinine isomer and anti-cotinine antibody against the SKOV-3 cell line, a HER2-overexpressing cell line. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0148] The present invention will be described in more detail below using examples. These examples are merely for the purpose of more specifically illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples according to the gist of the present invention.

[0149] Throughout this specification, "%" used to indicate the concentration of a particular substance is, unless otherwise specified, % (wt / wt) for solid / solid, % (wt / vol) for solid / liquid, and % (vol / vol) for liquid / liquid.

[0150] Working Example Example 1. Development of an antibody that binds to cotinine 1.1. Development of antibody-producing cells that specifically bind to cotinine 1.1.1. Selection of antibody-producing cells that specifically bind to cotinine In order to develop an antibody that specifically binds to cotinine, cells that produce antibodies that specifically bind to cotinine were generated and selected.

[0151] First, cotinine was bound to KLH (Thermo, 77600) using SMCC (Thermo, 22122) as a linker. KLH and SMCC were mixed in a weight ratio of 2:1, reacted at RT for 1 hour, and then the remaining SMCC was removed using an ultra-centrifugal filter. Cotinine was mixed with KLH bound to SMCC in a ratio of 1:1, and then bound overnight at 4°C. 100μg of KLH-cotinine was mixed with Freund's adjuvant (Sigma, F5506) and injected into the abdominal cavity of BALB / c mice (Daehan Bio Co., Ltd.). Two weeks later, 100μg of KLH-cotinine was diluted in PBS and injected again, and three days later, the spleen of the mouse was removed and lymphocytes were isolated. The isolated lymphocytes were mixed with myeloma cell line SP2 / 0-Ag14 (ATCC, CRL-1581) at a ratio of 5:1 and fused using PEG-1500 (Roche, 10783641001). The fused cells were cultured in a medium containing HAT supplement (Sigma, H0262), and the fused cells (hybridomas) were selectively selected and cultured.

[0152] 1.1.2. Confirmation of antibody production ability of cells that specifically bind to cotinine An ELISA method was used to confirm whether the selected hybridoma cells produced antibodies that bind to the antigen. First, BSA-cotinine was prepared using the same method as the KLH-cotinine preparation method, which can detect antibodies that specifically bind to cotinine. BSA-cotinine was attached to a Costar 96-well plate (Corning, 3590) at a concentration of 1 μg / mL for 1 hour at room temperature. After washing three times with TBS-T (0.05% Triton X-100), the plate was blocked with 300 μl of TBS-T / SM (2% skim milk) for 30 minutes at room temperature. The blocked plate was washed three times with TBS-T, and the selected hybridoma culture solution was added to perform an antigen-antibody reaction at 37° C. for 1 hour. Here, SUN10 antibody (US2008-0226650 A1) was used as a positive control group. After washing three times with TBS-T, anti-mIgG-HRP (Pierce, 31439) was added as a secondary antibody diluted 1:5,000 in TBS-T / SM and reacted at 37°C for 1 hour. Next, the cells were washed three times with TBS-T, TMB (Surmodics, TMBC-1000-01) was added to develop color at room temperature for 5 minutes, and 1N sulfuric acid (Duksan, 254) was added to stop the color development. The absorbance was measured at 450 nm using Victor X3 (PerkinElmer, 2030-0030). As a result, it was confirmed that the selected hybridoma cells produced antibodies that specifically bind to cotinine.

[0153] As a result, as shown in FIG. 1, 20E12, 30G5, and 33B2 were selected as antibodies that specifically bind to BSA-cotinine.

[0154] 1.2. Confirmation of cell binding ability of antibodies that specifically bind to cotinine To confirm the cell binding ability of the selected antibodies that specifically bind to BSA-cotinine, the cell binding ability was evaluated by performing a cell binding affinity assay using NCI-N87 (ATCC, CRL5822), a cancer cell expressing HER2, and anti-HER2 switch (trastuzumab bound to cotinine).

[0155] Fabrication of cotinine-linked HER2-targeting switch molecules First, trans-4-cotinine carboxylic acid (Sigma, 33224-01-0) was bound to human IgG trastuzumab by EDC reaction to prepare anti-HER2 switch. The anti-HER2 switch was quantified using Protein assay dye (Bio-Rad, Cat. No. 500-0006), and the concentration and purity were confirmed by SDS-PAGE and Coomassie blue staining.

[0156] Confirmation of cell binding ability between cotinine-specific antibody and targeting switch Then, 5 × 10 NCI-N87 cells were 5 The cells were prepared in a tube and centrifuged at 1,200 rpm for 3 minutes to collect the cells. After washing with PBS containing 5% FBS, the cells were treated with anti-HER2 switch and incubated at 4°C for 1 hour. The cells were washed three times with 200 μl of PBS containing 5% FBS by centrifugation at 1,200 rpm for 3 minutes. The hybridoma culture medium was then treated and incubated at 4°C for 1 hour. The cells were washed three times in the same manner as above. Next, the cells were treated with anti-mouse IgG-FITC (Jackson, 715-095-150) at 1 μg / mL and incubated at 4°C for 45 minutes in the dark. The cells were washed three times in the same manner as above. The presence or absence of binding of the selected antibodies to the cells was determined using a FACS instrument.

[0157] As a result, it was confirmed that the 33B2 antibody had the best binding ability to the targeting switch, as shown in Figure 2. Table 1 below shows sequence information for the selected 33B2 antibody.

[0158] [Table 1]

[0159] Example 2. Creation and production of humanized antibodies In order to make the selected antibodies more pharmacologic, humanized antibodies were produced.

[0160] 2.1. Generation of humanized antibodies Using the CDR grafting method, a humanized antibody of the mouse 33B2 antibody developed in Example 1 was developed. For the human antibody to which the CDR of the developed antibody is grafted, the V and J genes of human germline antibody genes with high similarity were selected based on the base sequence using IMGT / V-QUEST (Brochet, X. et al., Nucl Acids Res. 36: 503-508 (2008)). The heavy chain V and J genes were selected from IGHV1-46*01 and IGHJ4*03, respectively, with similarities of 74.65% and 81.25%, respectively. The light chain V and J genes were selected from IGLV7-46*01 and IGLJ3*02, respectively, with similarities of 63.54% and 88.24%, respectively. During synthesis, a ClaI restriction enzyme site was inserted in the forward direction, an NheI restriction enzyme site was inserted in the heavy chain in the reverse direction, and a BsiWI restriction enzyme site was inserted in the light chain in the reverse direction. Furthermore, a signal sequence shown in Table 2 below was added in the forward direction of the variable region to enable secretion of the humanized antibody into cell culture medium.

[0161] [Table 2]

[0162] Each of the synthesized variable regions was synthesized using human constant region Cκ and C. H The heavy chain portion was TA cloned into the pcDNA3.3-TOPO (Invitrogen, K8300-01) vector, and the light chain portion was TA cloned into the pOptiVEC-TOPO (Invitrogen, 12744-017) vector. Table 3 below shows the sequence information of the humanized antibody (hz33B2) that specifically binds to cotinine.

[0163] [Table 3]

[0164] 2.2. Production and purification of humanized antibodies FreeStyle TM The cloned vector was transiently transfected into 293F (Invitrogen, R790-07) animal cells using polyethyleneimine (Polyscience Inc., 23966), and the humanized antibody was purified from the cell culture medium using Protein-A Ceramic HyperD F resin (PALL, 20078-028).

[0165] The purified humanized antibodies were quantified using BCA analysis (Pierce, 23227) and subjected to SDS-PAGE to confirm concentration and purity by Coomassie Blue staining.

[0166] Example 3. Confirmation of binding ability of humanized antibody (hz33B2) 3.1. Confirmation of cotinine binding ability of humanized antibody (hz33B2) To confirm the cotinine-binding ability of the humanized antibody (hz33B2) developed in Example 2, ELISA and flow cytometry were performed using the cotinine-conjugated anti-HER2 affibody.

[0167] Plates coated with hHER2-ECD-His protein at a concentration of 2 μg / mL were washed three times with TBS-T (0.05% Triton X-100) and then blocked with 200 μl of TBS-T / SM (3% skim milk) at room temperature for 120 minutes. After washing the blocked plate three times with TBS-T, cotinine-conjugated anti-HER2 affibody (ZQAA1-Cot) was immobilized at a concentration of 2 μg / mL for 1 hour at room temperature. After washing the plate three times with TBS-T, humanized antibodies were treated at 9 points starting from 20 μg / mL and diluted 1 / 10, and incubated at room temperature for 1 hour. Here, SUN10 antibody (US2008-0226650A1) was used as a positive control group. Then, anti-hIgG-Fc-HRP (Invitrogen, H10007) was added as a secondary antibody at room temperature for 1 hour. Finally, a color reaction was carried out using TMB, and the OD450 value was measured using an ELISA reader.

[0168] As a result, as shown in FIG. 3, it was confirmed that the humanized antibody (hz33B2) has high binding ability to cotinine.

[0169] 3.2. Confirmation of cell binding ability of humanized antibody (hz33B2) To confirm the degree of cell binding of the humanized antibody, the cell binding ability was evaluated by performing a cell binding affinity assay using SKOV-3 expressing HER2 and an anti-HER2 affibody conjugated with cotinine.

[0170] HER2-expressing SKOV-3 (Korea Cell Line Bank, 30077) was cultured at 2 × 10 5After preparing cells / tube, the cells were centrifuged at 1,200 rpm for 3 minutes to collect the cells. After washing three times with 5% FBS-containing PBS, cotinine-conjugated anti-HER2 affibody (ZQAA1-Cot) was incubated at 4°C for 1 hour at a concentration of 2 μg / mL. After washing three times with 5% FBS-containing PBS, humanized antibodies were treated at 1 / 10 dilutions starting from 20 μg / mL for 6 points and incubated at 4°C for 1 hour. Here, SUN10 antibody (US2008-0226650A1) was used as a positive control. Next, the cells were washed three times using 200 μl of 5% FBS-containing PBS and centrifuged at 1,200 rpm for 3 minutes. After that, the cells were treated with 1 μg / mL of anti-human IgG-BV421 (BD, 562581) as a secondary antibody and incubated at 4°C for 30 minutes in the dark. After washing in the same manner as above, the fluorescence intensity was measured using a FACS machine.

[0171] As a result, as shown in Figure 4, it was confirmed that the humanized antibody (hz33B2) has high cell binding ability by binding to the target switch bound to cotinine.

[0172] Example 4. Development of CAR-T containing humanized anti-cotinine antibody and confirmation of its activity 4.1. Construction of lentivirus containing chimeric antigen receptor linked to anti-cotinine antibody fragment A chimeric antigen receptor was developed using an anti-cotinine antibody fragment. The chimeric antigen receptor was composed of a CD8 leader, anti-cotinine in the form of an scFV, CD8 hinge and transmembrane domains, CD137 cytoplasmic domain, and CD3 zeta cytoplasmic domain. After codon optimization for the chimeric antigen receptor, it was cut with SpeI / XhoI and ligated to pLenti6-V5 / DEST lentiviral vector (Invitrogen, V53306) modified with the EF-1 alpha promoter in Table 4 below.

[0173] [Table 4]

[0174] The prepared construct was confirmed by nucleotide sequence analysis, and the sequence information thereof is shown in Table 5 below.

[0175] [Table 5]

[0176] The prepared lentivirus construct was transduced into Lenti-X293T (Takara Bio Inc., 632180) cell line together with a nucleic acid encoding the viral envelope protein VSV-G (vesicular stomatitis indiana virus G protein) and a plasmid pCMV-dR8.91 containing the gag, pol, and rev genes. Transduction was performed using Lipofectamine 2000 (Invitrogen, 11668019) according to the manufacturer's protocol. After 72 hours, the culture medium containing the lentivirus was concentrated 10-fold using a centrifugal filter device (Millipore, UFC910024) and stored frozen at -80°C.

[0177] 4.2. Production of cytotoxic T cells displaying chimeric antigen receptors containing anti-cotinine antibody fragments on their surface Using the lentivirus produced in Example 4.1, cytotoxic T cells were produced in which a chimeric antigen receptor containing an anti-cotinine antibody fragment was displayed on the surface.

[0178] First, human naive T cells were isolated and transfected with Dynabeads. TMThe cells were stimulated with Human T-Activator CD3 / CD28 (Thermofisher Scientific, 11131D) for 24 hours, and polybrene (Sigma-Aldrich, H9268) and the lentivirus were added to the cells, and the cells were transduced while being cultured for 24 hours. Then, the medium was replaced with IL-2 (Gibco, CTP0021)-containing medium, and the cells were cultured under conditions of 5% CO2 and 37°C. The T cells that displayed the chimeric antigen receptor containing the anti-cotinine antibody fragment on the surface were used for the following experiments within 24 hours of production.

[0179] 4.3. Confirmation of cytotoxicity effect using CAR-T containing anti-cotinine antibody Using the CAR-T prepared in Example 4.2 above and an anti-HER2 affibody conjugated with cotinine, it was confirmed whether these complexes induce the activation of chimeric antigen receptor cells by recognizing HER2 on the cell surface.

[0180] Specifically, SKOV3-Luc and OVCAR3-Luc cell lines, which are gene-transduced cell lines, were constructed by introducing a lentivirus (Biosettia, GlowCell-16p-1) that expresses GFP luciferase into SKOV-3, a HER2-overexpressing cell line, and OVCAR-3, a HER2-expressing cell line, and used in the experiment. First, the SKOV3-Luc cell line and the OVCAR3-Luc cell line were dispensed into a 96-well plate at 1 x 104 cells per well. The prepared cytotoxic T cells were added to the plate to which the Luc cell line was dispensed so that the ratio of SKOV3-Luc or OVCAR3-Luc:cytotoxic T cells per well was 2:1 (cell number ratio). Cotinine-conjugated anti-HER2 affibody was added to the test group treated with Luc cell line and cytotoxic T cells at appropriate concentrations (0.1, 1, 10, 100 nM) and cultured for 24 hours at 5% CO2 and 37°C. SUN10-CAR-T cells (US2008-0226650A1) were used as a positive control. After culture, the toxic effect of the cytotoxic T cells was confirmed by luciferase measurement (Bio-Glo Luciferase assay system, Promega, G7941). This was confirmed by disrupting the remaining SKOV3-Luc or OVCAR3-Luc cell lines after incubation of cytotoxic T cells, cotinine-conjugated affibody, and the Luc cell line with 3X lysis buffer (75 mM Tris (pH 8.0), 30% glycerol, 3% Triton X100) and reacting the eluted luciferase with the substrate. The lysis ratio was determined based on the signal from the well in which only the Luc cell line was cultured (100%).

[0181] As shown in Figure 6, it was confirmed that the cytotoxic effect increased as the treatment concentration of the cotinine-conjugated affibody increased for all cell lines. In particular, it was confirmed that the cytotoxic effect was comparable to that of SUN10-CAR-T (US2008-0226650A1), whose cytotoxic effect has already been reported, or that the cytotoxic effect was relatively superior at high concentrations.

[0182] Example 5. Confirmation of CAR-T binding ability and cytotoxic activity by cotinine isomers Since cotinine exists in a mixed form of two isomers, we sought to determine whether the developed anti-cotinine binding antibodies have different binding properties and cytotoxic activity for each cotinine isomer.

[0183] Anti-HER2 affibody production with cotinine isomers Cotinine, which is a mixture of two isomers, was separated into two single isomers using a chiral column. The separated isomers were determined to be R- and S-isomers by crystal structure analysis. Two switches were synthesized by binding the separated isomers to ZQAA1, an anti-HER2 affibody: ZQAA1-Cot(R) and ZQAA1-Cot(S).

[0184] 5.1. Confirmation of CAR-T binding ability using cotinine isomers To confirm the binding ability of CAR-T to the anti-HER2 switches having each of the different cotinine isomers prepared above, ELISA and flow cytometry were used.

[0185] 5.1.1. Confirmation of CAR-T binding ability by ELISA analysis First, ELISA analysis was used to confirm the binding ability of CAR-T to the two types of anti-HER2 switches.

[0186] A plate coated with hHER2-ECD-His protein at a concentration of 2 μg / mL was washed three times with TBS-T (0.05% Triton X-100) and then blocked with 200 μl of TBS-T / SM (3% skim milk) at room temperature for 120 minutes. After washing the blocked plate three times with TBS-T, two anti-HER2 switches, each bound to a different cotinine isomer, were immobilized at a concentration of 2 μg / mL for 1 hour at room temperature. After washing the plate three times with TBS-T, humanized antibodies were added at 9 points starting from 20 μg / mL and diluted 1 / 10, and incubated at room temperature for 1 hour. The secondary antibody, anti-hIgG-Fc-HRP (Invitrogen, H10007), was treated at room temperature for 1 hour, and TMB was added to perform a color reaction. The OD450 value was then measured using an ELISA reader.

[0187] As a result, as shown in Figure 7, it was confirmed that CAR-T containing the anti-cotinine antibody of the present invention has high binding ability to the anti-HER2 switch (ZQAA1-Cot(S)) bound to the S-isomer of cotinine. This showed the same or higher level of binding ability compared to the results for the anti-HER2 switch (ZQAA1-Cot) bound to the mixed form of cotinine, and furthermore, it was found that the binding ability to the S-type cotinine isomer was slightly higher at high concentrations.

[0188] On the other hand, it was confirmed that the R-isomer of cotinine hardly binds to the anti-HER2 switch (ZQAA1-Cot(R)). From this, it was confirmed that CAR-T containing the anti-cotinine antibody of the present invention specifically binds to the S-isomer of cotinine.

[0189] 5.1.2. Confirmation of CAR-T binding ability by flow cytometry Next, flow cytometry was used to confirm whether the two types of anti-HER2 switches bind to CAR-T containing an anti-cotinine antibody.

[0190] The h33B2-CART prepared in Example 4.2 was added to 2 × 105 After preparing cells / tube, the cells were collected by centrifugation at 1,200 rpm for 3 minutes. After washing with 5% FBS-containing PBS, two types of anti-HER2 switches were added at a concentration of 2 μg / mL and incubated at 4 °C for 1 hour. After washing the cells in the same manner as above, HER2-ECD-Fc was added at a concentration of 2 μg / mL and incubated at 4 °C for 1 hour. The cells were then washed three times with 200 μl of 5% FBS-containing PBS by centrifugation at 1,200 rpm for 3 minutes. Next, the cells were treated with 2 μl of anti-CD3-APC-H7 (BD, 560176), 2 μl of an-Human-Fc-PE (BioLegend, 410708), and 5 μl of 7-ADD (BD, 559925), and incubated at 4 °C for 1 hour in the dark. The cells were then washed three times with 200 μl of 5% FBS-containing PBS by centrifugation at 1,200 rpm for 3 minutes, and the fluorescence intensity was measured using a FACS instrument.

[0191] As a result, as shown in Figure 8, it was confirmed that the CAR-T containing the anti-cotinine of the present invention has high binding ability to the anti-HER2 switch (ZQAA1-Cot(S)) bound to the S-isomer of cotinine. In particular, it showed the same or higher level of high binding ability compared to the anti-HER2 switch (ZQAA1-Cot) bound to the mixed form of cotinine, while it was found to have almost no binding ability to the R-isomer of cotinine. This was confirmed in the same way as the above ELISA analysis results.

[0192] 5.2. Confirmation of CAR-T cytotoxic activity by cotinine isomers Using CAR-T containing the anti-cotinine antibody of the present invention, we attempted to confirm the activation of cytotoxic activity of CAR-T by the two types of anti-HER2 switches. Therefore, using the hz33B2-CART prepared in Example 4.2, the cytotoxic activity was evaluated in the same manner as in Example 4.3.

[0193] Specifically, the SKOV3-Luc cell line was plated in a 96-well plate at 1 × 10 cells per well. 4The cytotoxic T cells were added to the plate containing the Luc cell line at a ratio of 1:2 per well (SKOV3-Luc:cytotoxic T cells). The two anti-HER2 affibodies were added to the test group containing the Luc cell line and the cytotoxic T cells at the appropriate concentrations (0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 nM) and cultured for 24 hours under conditions of 5% CO2 and 37°C. After culture, the cytotoxic T cells and cytotoxicity were confirmed by luciferase measurement (Bio-Glo Luciferase assay system, Promega, G7941). The cytotoxic effect was confirmed by disrupting the remaining SKOV3-Luc cell line after incubation of the cytotoxic T cells, the cotinine-conjugated affibody, and the Luc cell line with 3X lysis buffer (75 mM Tris (pH 8.0), 30% glycerol, 3% Triton X100), and reacting the eluted luciferase with the substrate. The lysis ratio was determined based on the signal from the well where only the Luc cell line was cultured (100%).

[0194] As a result, excellent cytotoxic activity of the anti-HER2 switch bound to the S-isomer of cotinine (ZQAA1-Cot(S)) was confirmed in Figure 9. In particular, this showed the same level of high cytotoxic activity as that of the anti-HER2 switch bound to the mixed form of cotinine (ZQAA1-Cot). On the other hand, the cytotoxic activity of the R-isomer of cotinine was only slightly increased only at high concentration treatment, and was found to have a significantly lower level of activity.

[0195] Therefore, it was confirmed that the anti-cotinine antibodies of the present invention show differences in cotinine binding ability and cytotoxic activity of CAR-T using the same depending on the type of cotinine isomer.

Claims

1. An antibody or antigen-binding fragment thereof that more specifically binds to the S-isomer of cotinine compared to the R-isomer.

2. The antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO:1, an HCDR2 comprising the amino acid sequence of SEQ ID NO:2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:3; and The antibody or antigen-binding fragment thereof of claim 1, which more specifically binds to the S-isomer of cotinine, comprising a light chain variable region comprising an LCDR1 having the amino acid sequence of SEQ ID NO:4, an LCDR2 having the amino acid sequence of SEQ ID NO:5, and an LCDR3 having the amino acid sequence of SEQ ID NO:

6.

3. The antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:8; or (ii) The antibody or antigen-binding fragment thereof of claim 1, which specifically binds to the S-isomer of cotinine, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

10.

4. The antibody or antigen-binding fragment may be a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a scFv, a Fab, a F(ab), or a F(ab) comprising a heavy chain variable region and a light chain variable region. ２ The antibody or antigen-binding fragment thereof of claim 1, which is selected from the group consisting of:

5. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. The nucleic acid molecule comprises: (i) comprising the nucleotide sequence of SEQ ID NO: 12 encoding a heavy chain variable region, and the nucleotide sequence of SEQ ID NO: 13 encoding a light chain variable region; or (ii) the nucleic acid molecule of claim 5, comprising a nucleotide sequence of SEQ ID NO: 14 encoding a heavy chain variable region and a nucleotide sequence of SEQ ID NO: 15 encoding a light chain variable region.

7. A recombinant vector comprising the nucleic acid molecule of claim 5.

8. A host cell comprising the recombinant vector of claim 7.

9. A chimeric antigen receptor comprising an antibody or an antigen-binding fragment thereof that more specifically binds to the S-isomer of cotinine of any one of claims 1 to 4.

10. A chimeric antigen receptor effector cell expressing a chimeric antigen receptor comprising an antibody or an antigen-binding fragment thereof that more specifically binds to the S-isomer of cotinine according to any one of claims 1 to 4.

11. The chimeric antigen receptor effector cell according to claim 10, wherein the activation of the chimeric antigen receptor effector cell is regulated by a switch molecule comprising: (a) the S-isomer of cotinine, and (b) a targeting moiety that binds to a cell surface molecule on a target cell.

12. The chimeric antigen receptor effector cell according to claim 11, wherein the targeting moiety constituting the switch molecule for regulating activation is an antibody, an antigen-binding fragment of an antibody, or a target binding polypeptide.

13. The chimeric antigen receptor effector cell according to claim 12, wherein the switch molecule for regulating activation is a cotinine-conjugated affibody to which S-isomer cotinine is conjugated.

14. A pharmaceutical composition for immunotherapy comprising the chimeric antigen receptor effector cell of claim 10 as an active ingredient.

15. A pharmaceutical kit for immunotherapy comprising the chimeric antigen receptor effector cell of claim 10 as an active ingredient.

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