Monobody-based chimeric antigen receptor and immune cells comprising the same

Abstract

The present invention relates to a monobody-based chimeric antigen receptor. Such a monobody-based chimeric antigen receptor has an extracellular ligand-binding domain that comprises a monobody. The present invention also relates to an immune cell expressing the monobody-based chimeric antigen receptor on the cell surface membrane.

Description

Technical Field

[0001] The present invention relates to a monobody-based chimeric antigen receptor. The monobody-based chimeric antigen receptor has an extracellular ligand-binding domain containing a monobody. The present invention also relates to an immune cell expressing the monobody-based chimeric antigen receptor on the cell surface membrane.

Background Art

[0002] In the treatment of solid cancer, it is difficult to completely remove cancer stem cells, remaining cancer cells, or metastatic cancer cells only by surgical resection. Therefore, a combined treatment method combining surgical resection with radiation therapy, chemotherapy, target therapy, immunotherapy, etc. is used. In particular, immunotherapy uses the immune system in the body and thus has almost no side effects seen in radiation therapy and chemotherapy.

[0003] T cells are immune cells that mediate the adaptive immune response and account for 75% of all lymphocytes. T cells mostly exist in an inactive state in the blood, but when stimulated by an antigen, they change to an active form and can remove cells having a specific antigen. The action of T cells is activated by a receptor (T cell receptor; TCR) that can recognize an antigen, co-stimulatory molecules, cytokines, etc.

[0004] However, although the TCR of T cells binds to MHC (major histocompatibility complex) molecules bound to tumor-associated antigens, cancer cells suppress the expression of such MHC or alter the functions of immune checkpoints such as CTLA-4 and PD-1 expressed on the surface of T cells, suppressing the functions of T cells. Therefore, T cells (Chimeric Antigen Receptor T cell; CAR-T) that express a chimeric antigen receptor capable of directly recognizing tumor-associated antigens rather than recognizing MHC molecules intracellularly, or T cells that express a specific antibody sequence against the immune checkpoint receptor of cancer cells intracellularly, have begun to be developed.

[0005] The structure of the currently developed chimeric antigen receptor is divided into an scFv (single chain variable fragment) portion that recognizes an antigen, a transmembrane domain, and a signaling domain that transmits signals intracellularly. Pharmaceutical companies and clinicians such as Novartis, Gilead, Celgene, and AbbVie have developed CAR-T using scFv that specifically binds to the CD19 antigen of blood cancer cells as a treatment for blood cancer (Patent Documents 10 to 14).

[0006] CAR-based immunotherapy that combines various gene manipulation techniques is also eagerly awaited for solid cancers. However, in the case of solid cancers, there has been no progress due to heterogeneity in which different antigens are expressed for each patient, complex characteristics of the tumor microenvironment such as hypoxia, and restrictions on the migration and activity of T cells.

[0007] The Ephrin receptor is a membrane receptor-type tyrosine kinase (RTK) with a size of 108 kDa and is divided into three parts. On the outer side of the cell membrane, there are a ligand-binding domain, a cysteine-rich region, and two fibronectin type III repeat structures. In the center, there is a transmembrane part, and on the cytoplasmic side, it has a kinase activity domain (Non-Patent Document 4). The Ephrin receptor is a protein that binds to Ephrin, a ligand, and transmits signals from the outside of the cell to the inside of the cell through the phosphorylation process, and is involved in cell proliferation, cell migration, differentiation, synaptic transmission of nerve cells, tissue remodeling, osteoblast differentiation, and angiogenesis (Non-Patent Documents 5 and 6). Depending on the structure of the Ephrin receptor and the ligand-receptor specificity, it is divided into nine types of group A (EphA1-8 and EphA10) and five types of group B (EphB1-4 and EphB6) (Non-Patent Documents 7-10). Among these, Ephrin receptor A2 (EphA2) is expressed only in very small amounts in normal cells, but is non-specifically overexpressed in almost all cancer cells such as liver cancer, prostate cancer, pancreatic cancer, head and neck cancer, gastric cancer, colorectal cancer, lung cancer, cervical cancer, and ovarian cancer, and promotes carcinogenesis, metastasis, angiogenesis, and resistance of tumors. EphA2 has about 65% protein homology with EphA1 and is found at approximately the same position in the lungs of rats. It is overexpressed together with EphA1 and its ligand Ephrin-A1 in several malignant tumors including non-small cell lung cancer, and is involved in the progression of carcinogenesis and malignancy through the interaction of EphA2 / A1-Ephrin-A1 (Non-Patent Documents 11 and 12).

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

[0009] [Non-Patent Document 1] Park SH, Park S, Kim DY, Pyo A, Kimura RH, Sathirachinda A, Choy HE, Min JJ, Gambhir SS, Hong Y. Isolation and Characterization of a Monobody with a Fibronectin Domain III Scaffold That Specifically Binds EphA2. PLoS One. 2015 Jul 15;10(7):e0132976. doi:10.1371 / journal.pone.0132976. eCollection 2015. PMID:26177208 [Non-Patent Document 2] Kim MA, Yoon HS, Park SH, Kim DY, Pyo A, Kim HS, Min JJ, Hong Y. Engineering of monobody conjugates for human EphA2-specific optical imaging. PLoS One. 2017 Jul 7;12(7):e0180786. doi:10.1371 / journal.pone.0180786. eCollection 2017. PMID:28686661 [Non-Patent Document 3] Pyo A, You SH, Sik Kim H, Young Kim J, Min JJ, Kim DY, Hong Y. Production of (64)Cu-labeled monobody for imaging of human EphA2-expressing tumors. Bioorg Med Chem Lett. 2020 Jul 15;30(14):127262. doi:10.1016 / j.bmcl.2020.127262. Epub 2020 May 15. PMID:32527560 [Non-Patent Document 4] Labrador et al., 1997; Pasquale, 1997 [Non-Patent Document 5] Pasquale EB. Cell. 133:38 - 52, 2008 [Non-Patent Document 6] Barquilla A et al., Annual Review of Pharmacology and Toxicology 55:465-487, 2015 [Non-Patent Document 7] Ieguchi K. Endocrine, Metabolic & Immune Disorders - Drug Targets 15:119-128, 2015 [Non-Patent Document 8] Eph Nomenclature Committee. Cell 90:403-404, 1997 [Non-Patent Document 9] Lindberg RA et al., Molecular and Cellular Biology 10(6):6324, 1990 [Non-Patent Document 10] Davis S et al., Science 266(5186):816-819, 1994 [Non-Patent Document 11] Naj AC et al., Nature Genetics 43:436-441, 2011 [Non-Patent Document 12] Finney AC et al., Circulation 136:566-582, 2017 [Summary of the Invention] [Problems to be Solved by the Invention]

[0010] In conventional CAR immune cells (e.g., CAR-T cells) using a single-chain variable fragment (scFv) of an antibody as an extracellular ligand-binding domain, the following problems existed.

[0011] 1) Since it is an animal-derived protein obtained from a mouse monoclonal antibody, the binding to the cancer antigen is strong. Therefore, severe side effects such as cytokine release syndrome (CRS), graft-versus-host disease, and tumor lysis syndrome occur, and rapid immune system contraction occurs due to the excessive activity of the immune system, resulting in a decrease in the tumor suppression effect in the long term.

[0012] 2) Different from blood cancers with CD19 as a single antigen, solid cancers have various different antigens (heterogeneity) for each patient. To construct diverse libraries against these cancer antigens, it is necessary to use antibody information derived from animals, which requires high costs, long time, and great effort.

[0013] 3) Antibodies, bispecific antibodies, and scFv are large in size. When used for the treatment of solid cancers, they have low penetrability into tissues and are less likely to exert sufficient therapeutic effects.

[0014] 4) When bicistronic scFv, that is, two antigen recognition sites are expressed in T cells or NK cells, the affinity of scFv for the antigen decreases, the therapeutic effect decreases, or side effects due to non-specific binding may occur.

[0015] 5) Even when an scFv-based CAR immune cell therapy agent is administered, mutations occur in the cancer cell epitopes targeted by CAR, and the incidence of antigen avoidance is high.

[0016] To solve these problems of scFv, nanobodies have been developed. Nanobodies are similar to scFv, can recognize antigens, and have the advantage of high penetrability into tissues because they are smaller in size than scFv. However, there are the following problems when used for the treatment of cancer cells.

[0017] 1) Compared with antibodies and scFv, the binding affinity to antigens is relatively low.

[0018] 2) Since nanobodies themselves have no toxicity to cancer cells, various operations (for example, binding an anticancer agent or fusing with T cells or NK cells) are required to use them as cancer therapeutics.

[0019] 3) The single body itself has low stability in serum. For example, in order to use the single body as a contrast agent, a relatively large amount must be administered (for example, more than about 60 μg).

[0020] 4) Since the single body has a short half-life in the body (<24 h), it must be injected frequently.

[0021] In order to solve the above problems, the present inventors have completed a novel single body-based CAR immune cell that can selectively recognize and bind to cancer cells, has excellent in vivo penetrability, high efficacy, is harmless even when administered in a large amount, and can induce immune memory while remaining persistently in tissues.

[0022] An object of the present invention is to provide a single body-based chimeric antigen receptor and an immune cell expressing the same on the cell surface membrane in order to solve the problems of the above-mentioned prior art.

Means for Solving the Problems

[0023] The present invention relates to a single body-based chimeric antigen receptor having an extracellular ligand-binding domain containing a single body.

[0024] The single body may specifically bind to a protein selected from the group consisting of CRL1, ephrin receptor, EphA2, β-galactosidase, RAS, Abl kinase, VEGFR2, MBP, SARS-CoV-2, Bcr-Abl kinase, STAT3, EGFR, VEGFR2, SH3 domain of human Lyn tyrosine kinase, MLKL, Fluc homologues, mitogen-activated protein kinase (MAPK), ERK2, MAPK14, kinase SH2 domain, SUMO, AurA, WDR5, Fluc family, GFP, receptor-binding domain of SARS-CoV-2, SH3 domain of FYN, SH2 domain of ABL, SUMO1, Bcr-Abl, GPR56 ECR, PD-L1, glypican-3, PCSK9, Gp41, CD4, and IL-23.

[0025] The single body may specifically bind to an ephrin receptor, EphA2, or human EphA2.

[0026] The single body may contain the amino acid sequence of SEQ ID NO: 8.

[0027] The single body-based chimeric antigen receptor may further contain a transmembrane domain and an intracellular signaling domain.

[0028] The transmembrane domain may be at least one selected from the group consisting of the alpha chain of the T cell receptor, the beta chain of the T cell receptor, the zeta chain of the T cell receptor, the alpha chain of CD8, the beta chain of CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, a part thereof, and a combination thereof.

[0029] The intracellular signaling domain may contain at least one signaling domain selected from the group consisting of TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD5, CD22, CD79a, CD79b, and CD66d.

[0030] The intracellular signaling domain may contain at least one selected from the group consisting of the OX40 domain, the CD2 domain, the CD27 domain, the CD28 domain, the CDS domain, the ICAM-1 domain, the LFA-1 domain, and the 4-1BB domain.

[0031] The single body-based chimeric antigen receptor of the present invention may further contain a hinge.

[0032] It may relate to a polynucleotide containing a nucleic acid sequence encoding the single body-based chimeric antigen receptor of the present invention.

[0033] The present invention may also relate to an expression vector containing the polynucleotide.

[0034] The present invention may also relate to an engineered immune cell that expresses the monobody-based chimeric antigen receptor on the cell surface membrane.

[0035] The immune cell may be a leukocyte, neutrophil, eosinophil, basophil, monocyte, lymphocyte, T cell, cytotoxic T cell, natural killer T cell, dendritic cell, or a combination thereof.

[0036] The present invention may also relate to a pharmaceutical composition for treating cancer, comprising the immune cell.

Advantages of the Invention

[0037] The monobody-based chimeric antigen receptor according to the present invention, the immune cell expressing the same on the cell surface membrane, and the composition for preventing or treating cancer using the same exhibit the following effects.

[0038] 1) In conventional radiotherapy or chemotherapy treatments such as DNA nucleotide analogs (cisplatin-based and 5-FU-based) that only suppress rapid cell growth, side effects (e.g., hair loss and digestive disorders) caused by suppressing the growth of normal cells can be reduced.

[0039] 2) Since a small-sized monobody based on human FN3 is used, side effects of autoimmune diseases and metastasis of cancer cells due to anti-cancer agent resistance that occur in target antibody therapy used as an animal-derived antibody-based anti-cancer treatment method and chimeric T cell therapy using scFv obtained from a library of mouse monoclonal antibodies can be minimized.

[0040] 3) Beyond the limitations of monobodies that have no killing ability against cancer, are unstable, and have a short lifespan and are only used for diagnosis, solid cancers expressing cancer antigens (e.g., EphA2) can be effectively suppressed or removed, enabling simultaneous cancer diagnosis and treatment.

[0041] 4) Monobody-based CAR immune cells have excellent anti-cancer effects compared to the conventional solid cancer treatment that administers T cells alone. For example, they can be used as an immuno-oncology agent for treating or preventing solid cancers such as pancreatic cancer, prostate cancer, and ovarian cancer that express EphA2 as an antigen.

[0042] 5) FN3 monobodies have the advantage of being able to produce various types of monobodies against target antigens. Thereby, monobody-based CAR immune cells specific to various antigens can be produced, which is very effective for cancer treatment.

[0043] 6) Monobody-based CAR immune cells are smaller in size compared to other CAR immune cells (for example, the size of a monobody is about 1 / 3 of the scFv size), and are more easily penetrable into cancer tissues than scFv-based CARs. When trying to produce a multivalent CAR in a viral vector, considering viral packing, up to four monobody sequences that recognize cancer antigens can be incorporated into the vector, which is an advantage in overcoming the heterogeneity of cancer antigens, a characteristic of solid cancers. Also, because they have sufficient selectivity and specificity for cancer antigens, they can block the avoidance of cancer immune cells and effectively prevent or treat cancer.

Brief Description of Drawings

[0044]

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Embodiments for Carrying Out the Invention

[0045] Unless otherwise defined in the present invention, all technical terms and scientific terms used in the present invention have the same meaning as generally understood by those skilled in the fields of gene therapy, biochemistry, genetics, and molecular biology.

[0046] Unless otherwise specified, when implementing the present invention, conventional techniques in cell biology, cell culture, molecular biology, microbiology, recombinant DNA, and immunology can be utilized, which belong to the techniques in the relevant field.

[0047] Monobody-based chimeric antigen receptor Relates to the single - body - based chimeric antigen receptor (CAR) of the present invention.

[0048] The single - body - based chimeric antigen receptor of the present invention may have an extracellular ligand - binding domain.

[0049] The term "extracellular ligand - binding domain" used in the present invention means an oligopeptide or polypeptide capable of binding to a ligand. Preferably, the domain can interact with cell - surface molecules. For example, the extracellular ligand - binding domain may be selected to recognize a ligand that acts as a cell - surface marker on target cells associated with any disease.

[0050] The term "monobody" used in the present invention specifically binds to a ligand or antigen present on the surface of a target cell. The monobody can be variously selected according to the desired target. For example, the monobody may specifically bind to a protein selected from the group consisting of CRL1, ephrin receptor, EphA2, β-galactosidase, RAS, Abl kinase, VEGFR2, MBP, SARS-CoV-2, Bcr-Abl kinase, STAT3, EGFR, VEGFR2, the SH3 domain of human Lyn tyrosine kinase, MLKL, Fluc homologues, mitogen-activated protein kinase (MAPK), ERK2, MAPK14, kinase SH2 domain, SUMO, AurA, WDR5, Fluc family, GFP, the receptor-binding domain of SARS-CoV-2, the SH3 domain of FYN, the SH2 domain of ABL, SUMO1, Bcr-Abl, GPR56 ECR, PD-L1, glypican-3, PCSK9, Gp41, CD4, and IL-23, but is not limited thereto.

[0051] Furthermore, the monobody may be a known one.

[0052] The monobody may specifically bind to an ephrin receptor, EphA2, or human EphA2.

[0053] The monobody may have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 8.

[0054] The monobody-based CAR may further include at least one selected from the group consisting of a hinge, a transmembrane domain, and an intracellular signaling domain.

[0055] Furthermore, the single - body - based CAR is expressed on the cell surface membrane. Thus, the single - body - based CAR may include a transmembrane domain. A prominent feature of a suitable transmembrane domain is the ability to be expressed on the surface of a cell, preferably an immune cell (particularly a lymphocyte cell or an NK cell in the present invention), and to interact to induce a cellular response of the immune cell against a given target cell. The transmembrane domain may be derived from a natural source or a synthetic source. The transmembrane domain may be derived from any membrane - bound or transmembrane protein.

[0056] The transmembrane domain may be at least one selected from the group consisting of the T - cell receptor alpha chain, the T - cell receptor beta chain, the T - cell receptor zeta chain, the CD8 alpha chain, the CD8 beta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, a part thereof, and combinations thereof.

[0057] The hinge and transmembrane domain may include the human CD8 alpha chain or a part thereof. The hinge and transmembrane domain may include the amino acid sequence of SEQ ID NO: 9, preferably an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 9.

[0058] The intracellular signaling domain of the single - body - based CAR can cause intracellular signaling after binding the extracellular ligand - binding domain to a target that causes activation of an immune cell and an immune response. The signaling domain can cause activation of at least one of the normal effector functions of the immune cell expressing the single - body - based CAR. For example, the effector function of a T cell may be cytotoxic activity or helper activity including cytokine secretion.

[0059] The term "signal transduction domain" used in the present invention refers to a part of a protein that converts effector function signals in immune cells and induces cells to perform special functions.

[0060] The intracellular signal transduction domain may include at least one selected from the group consisting of TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD5, CD22, CD79a, CD79b, and CD66d.

[0061] The intracellular signal transduction domain may include at least one selected from the group consisting of OX40 domain, CD2 domain, CD27 domain, CD28 domain, CDS domain, ICAM-1 domain, LFA-1 domain, and 4-1BB domain.

[0062] The intracellular signal transduction domain may include at least one selected from the group consisting of CD28 domain, 4-1BB domain, and CD3 zeta domain, preferably including CD28 domain and CD3 zeta domain, or 4-1BB domain and CD3 zeta domain.

[0063] The CD28 domain may include the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity.

[0064] The 4-1BB domain may include the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity.

[0065] The CD3 zeta domain may comprise the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity thereto.

[0066] Polynucleotide and vector The present invention relates to a polynucleotide comprising a nucleic acid sequence encoding a single-chain antibody-based CAR, or a vector comprising such a nucleic acid sequence or polynucleotide. Here, the vector may mean an expression vector.

[0067] The polynucleotide may comprise the nucleic acid sequence of SEQ ID NO: 2. The polynucleotide may comprise a nucleic acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 2.

[0068] The polynucleotide may comprise at least one nucleic acid sequence among SEQ ID NOs: 1 to 6, or may comprise at least one nucleic acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity to the nucleic acid sequences of SEQ ID NOs: 1 to 6.

[0069] The nucleic acid sequence encoding the single-chain antibody-based CAR may be codon-optimized for expression in human cells.

[0070] As used herein, "vector" may mean a construct capable of delivering at least one gene or sequence of interest into a host cell, preferably capable of expressing it. Examples include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors complexed with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0071] Viral vectors may include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses, such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses, such as picornaviruses and alphaviruses, and double-stranded DNA viruses, such as adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses that may be used as vectors may be, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis-sarcoma, mammalian type C, type B virus, type D virus, HTLV-BLV group, lentivirus, and spumavirus.

[0072] Method for manipulating immune cells The present invention relates to a method for producing immune cells for immunotherapy, comprising introducing a single-chain antibody-based CAR into immune cells and expanding the cells.

[0073] The present invention relates to a method for manipulating immune cells, comprising providing immune cells and introducing a polynucleotide into the immune cells.

[0074] The present invention relates to a method for manipulating immune cells, comprising providing immune cells and expressing a single-chain antibody-based CAR on the surface of the cells.

[0075] The present invention may involve modifying immune cells with at least one polynucleotide encoding a single-body-based CAR and expressing the polynucleotide in the cells. The method for producing or manipulating the immune cells of the present invention may be an in vitro or ex vivo method. The polynucleotide may be contained in a lentiviral vector for stable expression in cells.

[0076] Manipulated immune cells The present invention relates to an isolated cell or cell line obtained by the method for producing or manipulating the immune cells. Here, the isolated cell may contain a single-body-based CAR.

[0077] The isolated cells of the present invention may contain a population of CARs having different extracellular ligand-binding domains. In particular, the isolated cells may contain an exogenous polynucleotide sequence encoding a CAR.

[0078] The immune cells of the present invention may mean cells of hematopoietic origin that are functionally involved in the initiation and / or execution of innate and / or adaptive immune responses. The immune cells of the present invention may be derived from stem cells. The stem cells may be adult stem cells, non-human embryonic stem cells, non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, pluripotent stem cells, or hematopoietic stem cells.

[0079] The immune cells of the present invention may be human CD3+ T cells. In one embodiment, the isolated cells may be T cells selected from the group consisting of dendritic cells, killer dendritic cells, mast cells, NK cells, B cells or inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes or helper T lymphocytes. In one embodiment, the cells may be derived from the group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes.

[0080] Cells can be obtained from a subject by various non-limiting methods. Cells can be obtained from a plurality of non-limiting sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors.

[0081] Any available T cell line known to those skilled in the art may be used. The cells may be derived from a healthy donor, a patient who has received a cancer diagnosis, or a patient who has received a diagnosis of an infectious disease. The cells may be part of a mixed population of cells exhibiting different phenotypic characteristics. A cell line obtained from T cells manipulated according to the above method can be used, and the immune cells according to the present invention may be resistant to immunosuppressive treatment.

[0082] The immune cells may be, but are not limited to, leukocytes, neutrophils, eosinophils, basophils, monocytes, lymphocytes, T cells, cytotoxic T cells, natural killer T cells, dendritic cells, or combinations thereof.

[0083] Treatment using monobody-based CAR immune cells Monobody-based CAR immune cells, engineered immune cells, or populations of those cells may be used as a pharmaceutical composition for preventing or treating cancer.

[0084] The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising monobody-based CAR immune cells or engineered immune cells.

[0085] The present invention relates to a method for preventing or treating cancer in a subject in need thereof, comprising the step of administering monobody-based CAR immune cells to the subject in need thereof.

[0086] The present invention relates to the use of monobody-based CAR immune cells for the manufacture of a medicament for preventing or treating cancer.

[0087] The present invention may be a single - body - based CAR immune cell for use in preventing or treating cancer.

[0088] The term "subject" used in the present invention is a mammal, more preferably a human. Mammals include, but are not limited to, livestock, pets, primates, horses, dogs, cats, mice, and rats.

[0089] In the present invention, the treatment may be part of autologous immunotherapy or part of allogeneic immunotherapy. Autologous may mean that the cells, cell lines or cell populations used to treat a subject are obtained from the subject or from a donor compatible with human leukocyte antigen (HLA). Allogeneic may mean that the cells or cell populations used to treat a subject are obtained from a donor rather than from the subject.

[0090] Cancer may be melanoma, squamous cell carcinoma, breast cancer, head and neck cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, testicular cancer, prostate cancer, ovarian cancer, bladder cancer, skin cancer, brain cancer, angiosarcoma, mastocytoma, leukemia, lymphoma, liver cancer, lung cancer, pancreatic cancer, gastric cancer, kidney cancer, colorectal cancer, hematopoietic tumors or metastatic cancers thereof, and is not limited thereto. Furthermore, cancer may be a non - solid tumor or a solid cancer.

[0091] Non - solid tumors or non - solid cancers may be myeloma, lymphoma, or leukemia, and are not limited thereto.

[0092] Solid cancers may be at least one selected from the group consisting of pancreatic cancer, prostate cancer, ovarian cancer, breast cancer, cervical cancer, skin cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, and lung cancer, and are not limited thereto.

[0093] The following examples are provided for illustrative purposes only and do not limit the scope of the present invention in any way. In fact, in addition to what has been presented and described in the present invention, various modifications of the present invention will be apparent to those skilled in the relevant technical fields from the foregoing description, and they are included within the scope of the appended claims.

[0094] Conventional anti-cancer immunotherapy is known as a blood cancer treatment method in which a human cancer antigen is recognized and bound by a mouse-based antibody molecule, specifically an antibody fragment (scFv), and then cancer cells are removed by various cytokine secretions or attacks by peripheral immune cells. However, there are problems such as side effects caused by animal-derived antibodies, low tissue penetration due to the size of scFv, and in the case of solid cancers, the need to simultaneously recognize at least two or more antigens due to the characteristic (Heterogeneity) of expressing different antigens for each patient.

[0095] In the present invention, in order to compensate for such drawbacks of scFv, it was confirmed that using a monobody based on human fibronectin FN3 as the extracellular ligand-binding site of the chimeric antigen receptor has excellent preventive and therapeutic effects on solid cancers.

Example

[0096] Preparation of a vector expressing a monobody-based chimeric antigen receptor 1. Preparation for the preparation of a recombinant lentiviral vector For the preparation of a recombinant plasmid vector expressing a monobody-based chimeric antigen receptor, the following strains and vectors were prepared.

[0097] (1) Vector to be replicated The pETh-E1GSE1 vector containing the nucleic acid sequence (SEQ ID NO: 2) expressing a monobody (hereinafter, E1 monobody) that specifically binds to human EphA2 was obtained by license from the research team of Professor Hong Yong-jin belonging to the Industry-University Cooperation Group of Jeonnam National University, and a vector expressing a chimeric antigen receptor was prepared. Specifically, the expression vector is pLenti7.3 / V5-DEST from Invitrogen TMUsing the Gateway (registered trademark) Vector, a nucleic acid sequence encoding a chimeric antigen receptor consisting of a CD8α leader sequence - MSLNscFv - CD8 H&TM - CD28 - CD3ζ or a CD8α leader sequence - MSLN scFv - CD8 H&TM - 4 - 1BB - CD3ζ was included between the 5’UTR and 3’UTR of the pLenti7.3 / V5 - DEST vector. Two such lentiviral vectors were prepared (pLenti7.3::CD8α leader sequence - MSLNscFv - CD8 H&T - CD28 - CD3ζ vector and pLenti7.3::CD8α leader sequence - MSLNscFv - CD8 H&T - 4 - 1BB - CD3ζ vector).

[0098] The nucleic acid sequences constituting the single - body - based chimeric antigen receptor in the recombinant plasmid vector are shown in Table 1 below.

[0099]

Table 1

[0100] The nucleic acid sequences in Table 1 can be expressed in immune cells to express a single - body - based chimeric antigen receptor consisting of the amino acid sequences in Table 2 below on the cell surface membrane.

[0101]

Table 2

[0102] (2) Preparation of cells for vector replication Host cells for the pETh-E1GSE1 vector: E. coli DH5α (F-endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG purB20 φ80d lacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK-mK+), λ-) Host cells for the p lentiviral vector: E. coli Stbl3 (F-mcrB mrr hsdS20(rB-, mB-) recA13 supE44 ara-14 galK2 lacY1 proA2 rpsL20(Str R ) xyl-5 λ leumtl-1), Invitrogen (registered trademark)

[0103] The host cells were cultured using LB (Luria-Bertani) solid medium (Difco Laboratories, USA) at 37°C under the condition of 200 rpm, and 100 μg / mL of kanamycin or ampicillin was added to all the media to prepare the host cells. Then, a single colony of each of the cultured host cells was inoculated into LB liquid medium (Difco Laboratories, USA) for preculture, and 1% of it was inoculated into SOB (Super Optimal Broth) medium and cultured at 18°C for 24 hours, and then washed with Inoue TB buffer for preparation.

[0104] (3) Preparation of transformed cells for large-scale vector production Each of the prepared vectors was mixed with the host cells, and then left standing on ice for 10 minutes, treated at 42°C for 90 seconds and on ice for 3 minutes to prepare transformed cells into which each vector was incorporated. After the prepared transformed cells were smeared on LB solid medium containing antibiotics, they were cultured at 37°C. Thereby, each vector was prepared in a large amount.

[0105] 2. Preparation of recombinant lentiviral vector expressing E1 single-chain antibody-chimeric antigen receptor From the two lentiviral vectors (pLenti7.3::CD8α leader sequence - MSLNscFv - CD8 H&T - CD28 - CD3ζ vector and pLenti7.3::CD8α leader sequence - MSLNscFv - CD8 H&T - 4 - 1BB - CD3ζ vector) prepared above, lentiviral vectors excluding the scFv sequence were prepared. First, using the two vectors as templates, inverse PCR was performed using the CPL - F primer, CPL - R primer in Table 3 below, and CloneAmp HiFi PCR Premix (Takara). The vectors amplified in this way were separated and purified to prepare two types of MSLNscFv - free chimeric antigen receptor vectors.

[0106] The E1 single - body sequence was amplified by performing PCR on the pETh - E1GSE1 vector using the E1 - F primer and E1 - R primer in Table 3 below, and then separated and purified.

[0107]

Table 3

[0108] The prepared vectors and the E1 single - body sequence were inserted with the E1 single - body sequence at the site where the scFv sequence was present by the homologous recombination method using the Overlap cloner DNA cloning kit (Elpisbio) to prepare a recombinant lentiviral vector (pLenti7.3 - EphA2 single - body chimeric antigen receptor vector) that expresses a chimeric antigen receptor containing the E1 single - body. The prepared recombinant lentiviral vectors were named EphA2 mCAR - 1 and EphA2 mCAR - 2 respectively. Its structure is shown in Figure 1. Schematic diagrams of the methods for preparing the above vectors are shown in Figures 2A and 2B.

Example

[0109] Preparation (VEC - 1 and VEC - 2) and evaluation of immune cells expressing the E1 single - body - chimeric antigen receptor on the cell membrane surface 1. Generation of Lentivirus Containing Recombinant Lentiviral Vector Lentiviruses containing EphA2 mCAR-1 and EphA2 mCAR-2 prepared in Example 1 were prepared.

[0110] 2. Preparation of Immune Cells and Transduction by Viral Infection (1) Isolation of CD3+ T Cells from PBMC Peripheral blood mononuclear cells (PBMC) were isolated from human donor blood using Lymphoprep TM , STEMCELL). Subsequently, human CD3+ T cells were isolated from the isolated PBMC by positive selection using CD3 microbeads (Miltenyi Biotech).

[0111] (2) Activation of CD3+ T Cells Anti-CD3 / anti-CD28 magnetic beads (anti-CD3 / CD28 Dynabead; Gibco) were mixed with the isolated human CD3+ T cells at a ratio of 1:1. After 24 hours of mixing, the activated magnetic beads were removed using a MACSiMAG separator mechanism. Subsequently, the cells were washed with RPMI-1640.

[0112] (3) Transduction with Lentivirus T cells infected with lentivirus were prepared by viral infection using polybrene (Sigma Aldrich). Specifically, activated human CD3+ T cells were infected with lentiviruses containing EphA2 mCAR-1 and EphA2 mCAR-2 respectively, and the viral medium was replaced 24 hours later. Cell growth began 48 hours after infection. The engineered immune cells thus prepared were named VEC-1 and VEC-2 respectively (containing EphA2 mCAR-1 and EphA2 mCAR-2 respectively). The structure in which the antibody-based chimeric antigen receptor was expressed on the cell membrane surface is shown in Figure 3.

[0113] 3. Evaluation of Engineered Immune Cells Whether the EphA2 mCAR-1 and EphA2 mCAR-2 vectors introduced from the previously prepared VEC-1 and VEC-2 immune cells are correctly expressed or not, and the activation state of such immune cells were confirmed using FACS (Fluorescence-activated cell sorting).

[0114] In the experiment, Attune NxT flow cytometry (Thermo Fisher Scientific) was used. For each immune cell, the presence or absence of the expression of the introduced vector was confirmed using an anti-GFP (Green Fluorescent Protein) antibody. Since the GFP expression site exists together in the vector, in immune cells that express such a vector in large quantities, the analysis result by the anti-GFP antibody should be measured highly. Also, the presence or absence of activation of T cells was confirmed using an anti-CD25-PE antibody (PE Mouse Anti-Human CD25, BD Pharmingen TM ) and an anti-CD69-APC antibody (APC Mouse Anti-Human CD69, BD Pharmingen (registered trademark)).

[0115] When the expression rates of the introduced vectors (that is, E1 monoclonal antibody-chimeric antigen receptor sequences) were comparatively analyzed by FACS, compared with the control group without the introduction of the vector, VEC-1 immune cells showed an expression rate of about 40%, and VEC-2 immune cells showed an expression rate of about 30% (see Figure 4). From these results, it was confirmed that the vectors introduced into the immune cells were well-expressed, and that the E1 monoclonal antibody-chimeric antigen receptor was appropriately expressed in the engineered immune cells VEC-1 and VEC-2.

[0116] Also, in the engineered immune cells VEC-1 and VEC-2, it was confirmed that CD25, a cell proliferation activity marker, was expressed in the same manner as in the control group, and that CD69, an activation marker of cytotoxic T cells, was expressed in a larger amount compared with the control group (see Figure 5). Therefore, it was confirmed that both VEC-1 and VEC-2 were in an activated state.

Example

[0117] In vitro evaluation of cancer cell killing ability To evaluate the anti-cancer ability of engineered immune cells VEC-1 and VEC-2 expressing E1 single-chain antibody-chimeric antigen receptor against EphA2-specific cancer antigens, cancer cells expressing EphA2 (ovarian cancer cell line OVCAR-3; pancreatic cancer cell lines CAPAN-2 and AsPC-1; prostate cancer cell line PC-3, MSI-H gastric cancer cell line SNU638 and MSI-H colorectal cancer cell lines DLD1, Lovo, HCT8, HCT15, see Figure 6) and E1 single-chain antibody CAR-T cells (i.e., VEC-1 and VEC-2 cells) were co-cultured, and the cancer cell killing ability of the engineered immune cells was measured (see Figure 7 and Figure 8).

[0118] (1) The ovarian cancer cell line OVCAR-3; pancreatic cancer cell lines Capan-2 and AsPC-1; and prostate cancer cell line PC-3; gastric cancer cell line SNU638, colorectal cancer cell lines DLD1, LoVo, HCT-8, HCT-15 were purchased from the Korean Cell Line Bank and used.

[0119] In these cell lines, to confirm whether EphA2 is expressed, the presence and degree of expression of the EphA2 antigen were analyzed by FACS using an anti-EphA2 antibody. As a result, it was confirmed that all of these cancer cell lines express EphA2 at a high level (see Figure 6), and it should be possible to confirm the cancer cell killing ability of E1 single-chain antibody CAR-T cells targeting EphA2 using them.

[0120] (2) The cancer cell lines in which the expression of the EphA2 antigen was confirmed above were seeded at 1×10 4 cells per well in a 96-well plate, placed in a BSC for 30 minutes, and then the cancer cells were cultured in a cell incubator at 37°C and 5% CO 2 for 24 hours. Then, 4×10 4Individual E1 single - body CAR - T cells (i.e., VEC - 1 and VEC - 2 cells) were treated and co - cultured for 72 - 90 hours. While co - culturing, the viability and cell motility activity of the cells were analyzed by RTCA as follows.

[0121] (3) Kinematic analysis by RTCA (xCELLigence, ACEA Biosciences, Inc.) Gold microelectrodes attached to the bottom surface of the wells of a microtiter plate (E - Plate) were used as biosensors. When immersed in a conductive solution (e.g., buffer or standard tissue culture medium) and a potential was applied to such electrodes (both ends of the biosensor), electrons exited from the cathode terminal. These electrons passed through the bulk solution and were deposited on the anode terminal, completing the circuit. When adherent cells were present at the electrode - solution interface, the flow of electrons was obstructed, which was because the signal changed according to the interaction between the bulk solution and the biosensor. Therefore, by measuring the resistance value of impedance generated while the cells were growing, various cell reactions (number of cells, growth rate, cell size, cell shape, changes in substrate attachment state) can be automatically measured kinetically. The resistance value detected by the cells is represented by an index called Cell Index (CI), and the change in the CI value indicates a change in the cell shape.

[0122] (4) When the killing ability of the transduced E1 single - body CAR - T cells (VEC - 1 or VEC - 2) against cancer cells was compared and analyzed with that of non - transduced human T cells (control group), it was confirmed by RTCA analysis that the killing ability of VEC - 1 and VEC - 2 increased significantly (20 - 100%) compared to the control group (see Figure 7). From these results, it can be seen that the engineered immune cells according to the present invention have excellent cancer cell killing ability.

[0123] (5) The cancer cell killing ability of E1 single - body CAR - T (VEC - 1) immune cells using the 3D co - culture method was evaluated in comparison with the control group.

[0124] 1×103 The prostate cancer cells (PC-3) were seeded in a 96-well plate at a cell count, and after culturing for 3 days, 2×10 3 VEC-1 at a cell count was added to the medium for co-culture. Then, using the Sartorius IncuCyte® S3 Live-Cell Analysis System, the cancer cell killing ability of the cytotoxic-T cell control group (Control-T) and VEC-1 was compared and analyzed. It was observed that VEC-1 invaded the prostate cancer cell line (PC-3) spherical structure and destroyed the spherical structure, and killing of the cell line occurred earlier than the control group, starting 58 minutes after co-culture. At 14 hours and 58 minutes of co-culture, it was observed that VEC-1 completely eliminated the spherical (sphere) structure of the prostate cancer cells (PC-3), while the cytotoxic-T cell control group remained spherical without destroying the morphology of the spherical structure and was observed in the center of the spherical form (see Figure 8).

Example

[0125] Evaluation of the anti-tumor ability of immune cells in a xenograft mouse model All animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC, Jeonnam National University, Republic of Korea). Four-week-old female NSG (NOD SCID gamma) mice were supplied from the SPF animal laboratory of the Hwasun Vaccine Center.

[0126] The pancreatic cancer cells AsPC-1 / luc were suspended in a solution prepared by mixing PBS (Welgene) and high-concentration Matrigel (Corning) at a ratio of 1:1 at a concentration of 4×10 6 cells / 200 uL to 8×10 6 cells / 200 uL. The PC-3 cells were suspended in a solution prepared by mixing PBS (Welgene) and high-concentration Matrigel (Corning) at a ratio of 1:1 at a concentration of 3×10 6 cells / 100 uL to 1×10 7 cells / 100 uL. In the experiment, AsPC-1 / luc (2×10 6 cells) and PC-3 (3×10 6Subcutaneously injected the cells).

[0127] When the tumor size reached 100 - 150 mm 3 Once the tumor size reached 100 - 150 mm, 1×10 of the prepared PBS, control group T cells, VEC - 1, and VEC - 2 immune cells in 200 uL were intravenously injected once. 5 - 10 mice were used for each group, and the tumor size and mouse body weight were measured twice a week. 7 / 200uL was intravenously injected once. Five to ten mice were used for each group, and the tumor size and mouse body weight were measured twice a week.

[0128] 1. Cell thawing and cell culture Experimental materials: RPMI 1640 (Gibco), FBS (Gibco), P / S (Gibco), L - glutamine 200 mM (Gibco), 100φ cell culture flasks, baskets, tumor cell lines (AsPC - 1 and PC - 3), Solution 18 AO·DAPI (ChemoMetec), PBS (Welgene), TrypLE TM (Gibco). The human pancreatic cancer cell line AsPC - 1 and the human prostate cancer cell line PC - 3 were obtained from ATCC (American Type Culture Collection). The human pancreatic cancer cell line AsPC - 1 / luc expressing luciferase was obtained from JCRB (Cell bank, Australia).

[0129] Cell thawing: Take the tumor cell line from the liquid nitrogen tank and quickly thaw it in a 37°C constant temperature water bath. Then, transfer it to a clean bench when there is floating thin ice and put it into a 15 mL conical tube. Add 9 mL of cell culture medium (RPMI 1640 + 10% FBS + 1% P / S + 2 mM L - glutamine) to the 15 mL tube and centrifuge at 350 g for 3 minutes. Remove the supernatant, add 1 mL of cell culture solution to completely resuspend it, then add 9 mL of cell culture solution to make it up to 10 mL and resuspend again. Calculate the cell number using the cell number counting solution (PBS:Solution 18 = 90:5), and adjust the cell density to 0.5×10 6 / 10 mL and 1×10 6Cells were seeded in 100φ flasks according to 10mL (for PC-3 and AsPC-1 respectively). Then, subcultures were performed at 3-day intervals (when the cell density reached 70 - 80%).

[0130] Subculture: The supernatant in the flask was removed. Then, 10mL of PBS was added and the cells were washed to remove impurities such as cell residues, and then the supernatant was discarded. To detach the cells from the flask, TrypLE TM (2mL or 4mL) was added to 100φ and 150φ flasks respectively. Observed under a microscope, when the cells became round and floating, 10mL or 30mL of cell culture medium (RPMI 1640 + 10% FBS + 1% P / S + 2mM L-glutamine) was added, and the cells were transferred to a 50mL conical tube and centrifuged at 500g for 3 - 5 minutes. The supernatant was removed, then 1mL of cell culture medium was added to resuspend the cells, and then 9mL of cell culture medium was added. After measuring the cell count using a cell count solution (PBS:Solution 18 = 90:5), cancer cells (e.g., pancreatic cancer cells AsPC-1: 1×10 6 / 10mL) were placed in a culture container for culture.

[0131] 2. Preparation of mice Experimental materials: PICO 5053 (Orient Bio), g-irradiated 18% beta-chip (Orient Bio), sterilized tap water and water bottle, autoclave, clipper (John B&P), wet tissue, Ifran solution (Hana Pharmaceutical), depilatory (veet), respiratory anesthesia machine and chamber Experimental method: Feed (PICO 5053), bedding (r-irradiated 18% beta-chip), sterilized tap water, and cages were changed twice a week. Hair was removed 3 days before tumor implantation and removed during the experiment if the tumor grew so much that it was not visible well. Mice were anesthetized by respiratory anesthesia with isoflurane at a concentration of 2 - 3%. The hair on the right flank of the mouse was removed with a clipper, a depilatory was applied and left for about 1 minute, and then the depilatory was wiped off with a wet tissue.

[0132] 3. Establishment of a human tumor xenograft mouse model Experimental materials: cells, 1 mL syringe (Korea Vaccine), 25 1 / 2 G needle (Korea Vaccine), high-concentration Matrigel (Corning), ice box, EP tube, respiratory anesthetic machine and chamber, Ifran solution (Hana Pharmaceutical). In the case of Matrigel, it was left overnight in the refrigerator the day before xenotransplantation. Since Matrigel gels at room temperature, the experiment was always carried out with it stabbed in ice. Experimental method: A 100 μL solution in which AsPC-1 / luc (2×10 6 cells) and PC-3 (3×10 6 cells) were mixed with Matrigel at a ratio of 1:1 was subcutaneously injected into the right flank of mice anesthetized with 2-3% isoflurane. Also, 100 μL of a solution in which each tumor cell line (4×10 6 cells / 100 μL of PBS) was mixed with Matrigel at a ratio of 1:1 was subcutaneously injected into the mouse abdominal cavity.

[0133] 4. Injection of chimeric antigen receptor T cells Experimental materials: 27 1 / 2 G needle (Chonglim), 1 mL syringe (Korea Vaccine), PBS, normal T cells (control group), E1 monoclonal body CAR-T cells (VEC-1 and VEC-2), mouse corrector (John B&P), heat irradiator (John B&P), 70% alcohol swab, calipers (John B&P), weighing scale, camera. Experimental method: The tumor size and body weight of the mice were measured, and the tumor size and body weight of the mice were adjusted in the same way for each group. When the tumor size reached 100 - 150 mm 3 , 200 uL of PBS and cells (1×10 7 / 200 μL of control group normal T cells and E1 monoclonal body CAR-T cells) were intravenously injected into the tail vein of the mice.

[0134] 5. Mouse monitoring (tumor size and body weight measurement) Experimental materials: respiratory anesthetic machine and chamber, Ifran solution (Hana Pharmaceutical), calipers (John B&P), weighing scale, camera. Experimental method: After anesthetizing the mice with 2 - 3% isoflurane by respiration, the tumor size and body weight were measured twice a week. The size of the mouse tumor was measured by using calipers to measure the long axis (L), short axis (W), and height (H), and then multiplying by 0.52. The body weight of the mice was measured using an electronic balance.

[0135] 6. Experimental results (1) Experiments using prostate cancer cell lines Prostate cancer cell line (PC - 3) 1×10 7 Cells / 100 μL and 1×PBS were xenografted into the subcutaneous tissue of the right mid - back of mice to establish a mouse model. 12 days after tumor formation, for each group, PBS, control group T cells (activated control group normal T cells not transduced with virus; Cont - T), VEC - 1 cells, and VEC - 2 cells were injected intravenously, and then the tumor size was measured with a scale bar. The results are shown in Figure 9.

[0136] As shown in Figure 9, in the mice injected with VEC - 1 or VEC - 2, after 22 days of tumor formation, the tumor size was significantly reduced compared with the mice injected with only control group normal T cells or PBS.

[0137] E1 monoclonal body CAR - T cells were administered intravenously. Then, to grasp the recovery of the mice's physical condition and the presence or absence of side effects caused by the administration, the body weight of the mice was measured. The results are shown in Figure 10.

[0138] As shown in Figure 10, the mice administered with VEC - 1 or VEC - 2 recovered their health earlier than the mice administered with control group normal T cells. After that, the normal T cells began to show a tendency to recover 30 days later. The mice given only PBS had a deteriorating health condition while their body weight decreased in proportion to the tumor size.

[0139] At the end point of the experiment (50 days), the survival rate of the prostate cancer xenograft mouse model was evaluated using the Kaplan - Meier method. The results are shown in Figure 11. As shown in Figure 11, both groups of mice administered with VEC - 1 and VEC - 2 showed a high survival rate, which was significantly superior to that of the control group normal T cells.

[0140] Forty-four days after transplanting tumors into the right mid-back of the mice, E1 monoclonal body CAR-T cells (VEC-1 or VEC-2) were intravenously injected, and the tumor size was measured on the 28th day thereafter. The results are shown in Figure 12. As shown in Figure 12, in the mouse groups administered VEC-1 or VEC-2, it was confirmed that the tumors were significantly smaller compared to the mice administered normal T cells and the mice group administered only PBS.

[0141] (2) Experiments using pancreatic cancer cell lines Pancreatic cancer cells (AsPC-1, 2×10 6 cells) were transplanted into the mouse abdominal cavity, and 14 days later, PBS, normal T cells, VEC-1, and VEC-2 were intraperitoneally administered. Four weeks after administration, the degree of metastasis of pancreatic cancer cells was photographed using an in vivo imaging system (In Vivo Imaging System; IVIS). The results are shown in Figure 13. As shown in Figure 13, it was confirmed that in the mice administered VEC-1 and VEC-2, the tumors hardly grew or metastasized compared to the mice administered only normal T cells and PBS.

[0142] 2×10 6 pancreatic cancer cells were transplanted into the mouse abdominal cavity, and 14 days later, PBS, normal T cells (Con T), VEC-1, and VEC-2 were intraperitoneally administered, and the tumor size was measured by bioluminescence imaging (Bioluminescence Imaging; BIO). The results are shown in Figure 14. As shown in Figure 14, in the mice administered VEC-1 or VEC-2, the tumors hardly grew compared to the mice administered normal T cells and the mice administered only PBS, and this persisted up to 4 weeks after transplantation of pancreatic cancer cells.

[0143] The single-body-based chimeric antigen receptor according to the present invention and immune cells (for example, cytotoxic T cells) expressing the same on the cell membrane surface are found to have an excellent anti-cancer effect of significantly suppressing the growth of prostate cancer and pancreatic cancer cells and significantly increasing the survival rate of mice. Therefore, various cancers including solid cancers can be prevented or treated by using immune cells containing the single-body-based chimeric antigen receptor.

Claims

**Claim 1** A monobody-based chimeric antigen receptor having an extracellular ligand-binding domain comprising a monobody, wherein the monobody comprises the amino acid sequence of SEQ ID NO: 8, the monobody-based chimeric antigen receptor comprises the hinge and transmembrane domain of the CD8 alpha chain, and the intracellular signaling domain of CD3 zeta, the monobody-based chimeric antigen receptor further comprises the intracellular signaling domain of CD28 or 4-1BB, a monobody-based chimeric antigen receptor. **Claim 2** The monobody-based chimeric antigen receptor according to claim 1, wherein the hinge and transmembrane domain of the CD8 alpha chain consists of the amino acid sequence of SEQ ID NO:

9. **Claim 3** The monobody-based chimeric antigen receptor according to claim 1, wherein the intracellular signaling domain of CD3 zeta consists of the amino acid sequence of SEQ ID NO:

12. **Claim 4** The monobody-based chimeric antigen receptor according to claim 1, wherein the intracellular signaling domain of CD28 consists of the amino acid sequence of SEQ ID NO:

10. **Claim 5** The monobody-based chimeric antigen receptor according to claim 1, wherein the intracellular signaling domain of 4-1BB consists of the amino acid sequence of SEQ ID NO:

11. **Claim 6** A polynucleotide comprising a nucleic acid sequence encoding the monobody-based chimeric antigen receptor according to any one of claims 1 to 5. **Claim 7** An expression vector comprising the polynucleotide according to claim 6. **Claim 8** An immune cell expressing the monobody-based chimeric antigen receptor according to any one of claims 1 to 5 on the cell surface membrane. **Claim 9** The immune cell according to claim 8, wherein the immune cell is a leukocyte, neutrophil, eosinophil, basophil, monocyte, lymphocyte, T cell, cytotoxic T cell, natural killer T cell, dendritic cell, or a combination thereof. **Claim 10** A pharmaceutical composition for preventing or treating cancer, comprising the immune cell according to claim 8. **Claim 11** The pharmaceutical composition for preventing or treating cancer according to claim 10, wherein the cancer is at least one selected from the group consisting of melanoma, squamous cell carcinoma, breast cancer, head and neck cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, testicular cancer, prostate cancer, ovarian cancer, bladder cancer, skin cancer, brain cancer, angiosarcoma, mastocytoma, leukemia, lymphoma, liver cancer, lung cancer, pancreatic cancer, gastric cancer, kidney cancer, colorectal cancer, hematopoietic tumors, and metastatic cancers thereof.

12. The pharmaceutical composition for preventing or treating cancer according to claim 11, wherein the cancer is pancreatic cancer, prostate cancer, or ovarian cancer.

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