Novel Anti-CD5 chimeric antigen receptor and use thereof

Abstract

The present invention relates to a novel anti-CD5 chimeric antigen receptor and use thereof. The chimeric antigen receptor of the present invention can specifically bind to the extracellular membrane-proximal domain of CD5, and immune cells expressing the chimeric antigen receptor exhibit reduced reactivity with respect to normal cells expressing CD5, and thus the present invention can be used as an efficient therapeutic agent for various CD5-mediated cancer diseases.

Description

Novel anti-CD5 chimeric antigen receptor and uses thereof

[0001] The present invention relates to a novel anti-CD5 chimeric antigen receptor and its use, and more particularly, to an antibody or antigen-binding fragment thereof that specifically binds to CD5, a chimeric antigen receptor comprising the same, and an immune cell expressing the chimeric antigen receptor.

[0002]

[0003] The global anticancer drug market has grown at an average annual rate of 10-13% over the past decade and is expected to reach a total of $200 billion by 2022. As of 2020, one in five men and one in six women worldwide will experience at least one case of cancer in their lifetime. The prevalence and mortality rates of cancer continue to rise, with one in eight men and one in 11 women dying from it.

[0004] The global anticancer drug market is undergoing a paradigm shift, starting with first-generation chemotherapy, and moving to second-generation targeted anticancer drugs and third-generation immunotherapy. Moving beyond the initial treatment methods that simply aimed to shrink and suppress cancer, active research is being conducted on targeted anticancer therapies that selectively attack cancer cells without damaging rapidly dividing cells. The development of these targeted anticancer therapies involves two stages: the selection of receptors specifically expressed in cancer cells, enabling selective targeting of cancer cells, and the development of targeting compounds that bind to these receptors.

[0005] Among these, interest is growing in cell therapy methods that use immune cells to strengthen them or genetically modify them and then infuse them back into the patient. For example, tumor-infiltrating lymphocytes (TIL), chimeric antigen receptors (CARs), and T-cell receptor (TCR) technologies are being studied. In particular, CAR-T cells, which are T cells introduced with chimeric antigen receptors (CARs), which are artificial receptors designed to convey antigen specificity, have been actively researched since CAR-T cells targeting CD19 received FDA approval as an anticancer agent in 2017. However, most of them only showed effects in vitro and had the problem of not showing significant therapeutic effects in vivo.

[0006] Meanwhile, while CAR-T cells are effective against tumors, they also have the side effect of nonspecifically attacking healthy tissue in some cases. CD5 is a type of differentiation cluster expressed in T and B cells. It is primarily found in bone marrow and lymphoid tissues and is overexpressed in T cells more than B cells, making it primarily used as a marker for T cells.

[0007] Additionally, most lymphomas that originate in T cells, such as peripheral T-cell lymphoma, anaplastic large cell lymphoma, and extranodal NK / T-cell lymphoma, express CD5; abnormal B cells also express CD5; and some lymphomas that originate in B cells, such as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma, also express CD5.

[0008] However, CAR-T therapy for treating T-cell tumors has problems because the cells into which the CAR gene is introduced are T cells, just like the tumor cells. When the T-cell-targeting CAR is transduced, the CAR-T cells exhibit a mutually killing effect (fratricide) during the culture process. As a result, there were problems such as low cell viability, low growth rate, cytokine release during the culture process, and cell exhaustion due to continuous stimulation.

[0009] Accordingly, there is a need to research and develop immune cells that can complement the above-mentioned problems of anti-CD5 CAR-T cells while maintaining anticancer activity against cancer cells expressing CD5.

[0010]

[0011] The technical problem to be achieved by the present invention is to provide an antibody or an antigen-binding fragment thereof that specifically binds to CD5.

[0012] In addition, the technical problem to be achieved by the present invention is to provide an anti-CD5 chimeric antigen receptor comprising the antibody or an antigen-binding fragment thereof.

[0013] In addition, a technical problem to be achieved by the present invention is to provide a nucleic acid molecule encoding the chimeric antigen receptor.

[0014] In addition, a technical problem to be achieved by the present invention is to provide an expression vector comprising the nucleic acid molecule.

[0015] In addition, the technical task to be achieved by the present invention is to provide an immune cell expressing the chimeric antigen receptor.

[0016] In addition, the technical task to be achieved by the present invention is to provide a pharmaceutical composition for preventing or treating cancer containing the immune cells.

[0017]

[0018] The technical problems to be solved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0019]

[0020] In order to achieve the above technical problem, one embodiment of the present invention provides a light chain variable region comprising a light chain CDR1 having an amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 10, SEQ ID NO: 19, and SEQ ID NO: 28; a light chain CDR2 having an amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 20, and SEQ ID NO: 29; and a light chain CDR3 having an amino acid sequence of any one of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 21, and SEQ ID NO: 30; and a heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence of any one of SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 22, and SEQ ID NO: 31; a heavy chain CDR2 having an amino acid sequence of any one of SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 23, and SEQ ID NO: 32; and a heavy chain CDR3 having an amino acid sequence of any one of SEQ ID NO: 6, SEQ ID NO: 15, SEQ ID NO: 24, and SEQ ID NO: 33; An antibody or antigen-binding fragment thereof that specifically binds to CD5 is provided.

[0021] In an embodiment of the present invention, the antibody or antigen-binding fragment thereof may comprise an amino acid sequence of any one of SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 27, and SEQ ID NO: 36.

[0022] In an embodiment of the present invention, the antibody or antigen-binding fragment thereof may bind to the CD5 extracellular membrane-proximal domain.

[0023] In an embodiment of the present invention, the CD5 extracellular membrane-proximal domain may comprise the amino acid sequence of SEQ ID NO: 56.

[0024] In an embodiment of the present invention, the antibody or antigen-binding fragment thereof may be a scFv, a Fab fragment, a F(ab') fragment, a F(ab')2 fragment, or an Fv fragment.

[0025] To achieve the above technical task, another embodiment of the present invention provides an anti-CD5 chimeric antigen receptor (CAR) comprising an extracellular binding domain comprising the antibody or an antigen-binding fragment thereof; a transmembrane domain; and an intracellular signaling domain.

[0026] In an embodiment of the present invention, the intracellular signaling domain is CD3 zeta, FcγR, ICOS (CD278), 4-1BB (CD137), OX40 (CD134), CD27, CD28, IL-2Rβ, IL-15R-α, CD40, MyD88, DAP10, DAP12, MHC class I molecule, TNF receptor protein, Immunoglobulin-like protein, cytokine receptor, integrin, SLAM protein, activating NK cell receptor, BTLA, Toll ligand receptor, CD2, CD7, CD30, CD40, CDS, ICAM-1, B7-H3, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83 specific ligands.

[0027] In an embodiment of the present invention, the transmembrane domain may be derived from any one selected from among T cell receptor (TCR) α chain, TCR β chain, CD3ζ, CD3ε, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and CD278.

[0028] In an embodiment of the present invention, the chimeric antigen receptor further comprises a hinge domain, wherein the extracellular binding domain is connected to the transmembrane domain by the hinge domain, and the hinge domain may be derived from any one selected from CD8, FcγRIIIα, and IgG1.

[0029] In an embodiment of the present invention, the chimeric antigen receptor may comprise any one of the amino acid sequences of SEQ ID NO: 51 to SEQ ID NO: 54.

[0030] To achieve the above technical task, another embodiment of the present invention provides a nucleic acid molecule encoding a chimeric antigen receptor.

[0031] In order to achieve the above technical task, another embodiment of the present invention provides an expression vector comprising the nucleic acid molecule.

[0032] To achieve the above technical task, another embodiment of the present invention provides an immune cell expressing the chimeric antigen receptor.

[0033] In an embodiment of the present invention, the immune cell may be at least one selected from among T cells, natural killer T cells, dendritic cells, killer dendritic cells, mast cells, natural killer cells, macrophages, and precursor cells thereof.

[0034] In order to achieve the above technical task, another embodiment of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the immune cells.

[0035]

[0036] The present invention relates to a novel anti-CD5 chimeric antigen receptor and its use. The chimeric antigen receptor of the present invention can specifically bind to the extracellular membrane-proximal domain of CD5, and immune cells expressing the chimeric antigen receptor exhibit reduced reactivity even toward normal cells expressing CD5, and thus can be utilized as an effective treatment for various CD5-mediated cancer diseases.

[0037]

[0038] The effects of the present invention are not limited to the above-described effects, and should be understood to include all effects that can be inferred from the composition of the invention described in the description or claims of the present invention.

[0039]

[0040] Figure 1 shows the results of measuring whether the scFv targeting CD5 discovered in the present invention binds to the CD5 protein.

[0041] Figure 2 is a drawing showing the configuration of a chimeric antigen receptor (CAR) used in the present invention, and shows the configuration of a CAR including H65, a positive control scFv, and A2, C7 scFv discovered in the present invention, respectively.

[0042] Figure 3 shows the results of transducing a CD5 CAR into a CD5 negative cell line and measuring the binding affinity to the recombinant CD5 protein.

[0043] Figure 4 shows a schematic diagram of the structure of a mutant CD5 prepared to confirm the binding site and the results of confirming the binding site for the CD5 protein of the positive control and A2, C7 scFv.

[0044] Figure 5 shows the results of measuring the CAR expression pattern of CD5 CAR transduced cells.

[0045] Figure 6 shows the results of measuring cell viability and growth of CD5 CAR transduced cells.

[0046] Figure 7 shows the results of measuring the secretion amounts of IFN-γ and TNF-α during the culture process of CD5 CAR transduced cells.

[0047] Figure 8 shows the results of measuring cell phenotype based on the expression pattern of each surface marker of CD5 CAR transduced cells.

[0048] Figure 9 shows the results of measuring the in vitro tumor cell killing ability of CD5 CAR transduced cells.

[0049] Figure 10 shows the results of measuring the in vivo tumor cell killing ability of CD5 CAR transduced cells.

[0050] Figure 11 shows the results of measuring the binding of scFv targeting the CD5 membrane-proximal domain discovered in the present invention to the CD5 protein.

[0051] Figure 12 is a diagram showing the configuration of a chimeric antigen receptor (CAR) used in the present invention, showing the configuration of a CAR using an scFv targeting the CD5 membrane-distal domain and an scFv targeting the CD5 membrane-proximal domain, including H65, a positive control scFv, respectively.

[0052] Figure 13 shows the results of transducing a CD5 CAR into a CD5 negative cell line and measuring the binding affinity to the recombinant CD5 protein.

[0053] Figure 14 shows the results of confirming the binding site for the CD5 protein of the positive control and scFv discovered in the present invention.

[0054] Figure 15 shows the results of measuring the CAR expression pattern of CD5 membrane-proximal domain targeting CAR transduced cells.

[0055] Figure 16 shows the results of measuring cell viability and growth of CD5 membrane-proximal domain-targeting CAR transduced cells.

[0056] Figure 17 shows the results of measuring cell phenotypes based on the expression patterns of each surface marker of CD5 membrane-proximal domain-targeting CAR transduced cells.

[0057] Figure 18 shows the results of measuring the in vitro tumor cell killing ability of CD5 membrane-proximal domain-targeting CAR transduced cells.

[0058] Figure 19 shows the results of measuring the in vivo tumor cell killing ability of CD5 membrane-proximal domain-targeting CAR transduced cells.

[0059]

[0060] Hereinafter, the present invention will be described in detail.

[0061]

[0062] The present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to CD5.

[0063] The term “antibody” used in the present invention refers to an immunoglobulin molecule that immunologically binds specifically to an epitope of an antigen and exhibits reactivity. The antibody may include a monoclonal antibody, a polyclonal antibody, an antibody having a full-length chain structure (full-length antibody), a functional fragment having at least an antigen-binding function (antigen-binding fragment), and a recombinant antibody.

[0064] The above monoclonal antibody refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, and such monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope. The full-length antibody has a structure having two full-length light chains and two full-length heavy chains, each light chain being capable of being linked to a heavy chain by a disulfide bond.

[0065] The above antibody comprises a heavy chain (HC) and a light chain (LC) polypeptide, and the heavy chain and light chain may include a variable region and a constant region.

[0066] The constant region is a site that mediates binding of the antibody to various types of cells of the immune system (such as T cells) and host tissues containing components of the complement system. The constant region has the same function regardless of the type of antigen as long as it is an antibody of the same type derived from the same species, and the amino acid sequence constituting the constant region is also the same or has a high degree of similarity between antibodies. The constant region can be divided into a heavy chain constant region (which may be abbreviated as CH) and a light chain constant region (which may be abbreviated as CL).

[0067] The variable region is an antibody portion having specificity for an antigen, and can be divided into a heavy chain variable region (which may be abbreviated as VH) and a light chain variable region (which may be abbreviated as VL). The variable region may include three CDRs (complementary-determining regions) and four FRs (framework regions). The CDRs may be ring-shaped portions involved in antigen recognition, and specificity for an antigen may be determined according to the amino acid sequence of the CDRs. The CDRs may be referred to as CDR1, CDR2, and CDR3 according to their order, and depending on which polypeptide among the heavy and light chains they are CDRs of, the heavy chain variable region may be referred to as CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region may be referred to as CDR-L1, CDR-L2, and CDR-L3. Likewise, FR can be referred to as FR-H1, FR-H2, FR-H3, FR-H4 for the heavy chain variable region, and FR-L1, FR-L2, FR-L3, FR-L4 for the light chain variable region.

[0068] The antigen-binding fragment of the present invention refers to any fragment of the antibody of the present invention that retains the antigen-binding function of the antibody. The antigen-binding fragment may be referred to interchangeably with terms such as "fragment", "antibody fragment", etc., and the antigen-binding fragment may be, for example, an scFv, a Fab fragment, an F(ab') fragment, an F(ab')2 fragment, or an Fv fragment.

[0069] The above scFv is known to have superior antigen binding affinity than VH or VL alone, as VH and VL are linked by a linker to form a continuous protein chain. Any linker commonly used in the art may be used as the linker connecting VH and VL.

[0070] The linker may be a peptide link and may have a length of about 10 to 25 amino acids. For example, the linker may include a hydrophilic amino acid such as glycine (G) and / or serine (S). More specifically, the linker may be GGSSRSSSSGGGGSGGGG.

[0071] The antibody of the present invention or an antigen-binding fragment thereof comprises a light chain variable region comprising a light chain CDR1 having an amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 10, SEQ ID NO: 19, and SEQ ID NO: 28; a light chain CDR2 having an amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 20, and SEQ ID NO: 29; and a light chain CDR3 having an amino acid sequence of any one of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 21, and SEQ ID NO: 30; and a heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence of any one of SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 22, and SEQ ID NO: 31; a heavy chain CDR2 having an amino acid sequence of any one of SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 23, and SEQ ID NO: 32; and a heavy chain CDR3 having an amino acid sequence of any one of SEQ ID NO: 6, SEQ ID NO: 15, SEQ ID NO: 24, and SEQ ID NO: 33.

[0072] The light chain variable region may include, for example, any one of the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 25, and SEQ ID NO: 34.

[0073] The heavy chain variable region may include, for example, any one of the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 26, and SEQ ID NO: 35.

[0074] For a more specific example, the antibody or antigen-binding fragment thereof may comprise an amino acid sequence of any one of SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 27, and SEQ ID NO: 36.

[0075] The antibody or antigen-binding fragment thereof may specifically bind to the CD5 extracellular membrane-proximal domain. The CD5 extracellular membrane-proximal domain may, for example, comprise the amino acid sequence of SEQ ID NO: 56.

[0076] The antibody or antigen-binding fragment thereof of the present invention may further include, for example, a heavy chain constant region and / or a light chain constant region of an antibody derived from a human, and, as long as the antibody or antigen-binding fragment thereof does not inhibit the property of specifically binding to CD5, the heavy chain constant region and / or the light chain constant region of the antibody derived from a human may be used without limitation in type or amino acid sequence.

[0077] The scope of the antibody or antigen-binding fragment of the present invention includes variants having conservative amino acid substitutions in the CDR region, and may include variants for the amino acid sequences listed above within the range capable of specifically recognizing CD5. For example, in addition to the binding affinity of the antibody, additional changes may be made to the amino acid sequence of the antibody to further improve its half-life, biocompatibility, and other biological properties. Considering such variants having biologically equivalent activity, the antibody of the present invention or the nucleic acid molecule encoding the same is interpreted to also include sequences that exhibit substantial identity with the described sequences. The substantial identity refers to a sequence that exhibits at least 61% homology, in one specific example, 70% homology, in another specific example, 80% homology, and in yet another specific example, 90% homology, when the sequence of the present invention and any other sequence are aligned to the greatest extent possible and the aligned sequences are analyzed using an algorithm commonly used in the art.

[0078] The present invention relates to an anti-CD5 chimeric antigen receptor.

[0079] The chimeric antigen receptor of the present invention comprises an extracellular binding domain comprising the antibody or an antigen-binding fragment thereof; a transmembrane domain; and an intracellular signaling domain.

[0080] The antibody or antigen-binding fragment thereof is as described above.

[0081] The intracellular signaling domain corresponds to a portion that transmits a signal generated upon binding of the chimeric antigen receptor to the antigen into the interior of the immune cell to induce a function of the immune cell (e.g., activation including release of cytotoxic (or cytolytic) factors against target cells to which the chimeric antigen receptor binds, cytokine production, proliferation and cytotoxicity or cytolytic activity, or other cellular responses induced by the antigen binding). The intracellular signaling domain may be a portion of a protein that transmits an effector function signal and instructs the cell to perform a specific function.

[0082] The intracellular signaling domain above may be an intracellular signaling domain that has been previously used in the development of a chimeric antigen receptor. For example, CD3 zeta, FcγR, ICOS (CD278), 4-1BB (CD137), OX40 (CD134), CD27, CD28, IL-2Rβ, IL-15R-α, CD40, MyD88, DAP10, DAP12, MHC class I molecule, TNF receptor protein, Immunoglobulin-like protein, cytokine receptor, integrin, SLAM protein, activating NK cell receptor, BTLA, Toll ligand receptor, CD2, CD7, CD30, CD40, CDS, ICAM-1, B7-H3, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, It may be derived from any one or more selected from IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83 specific ligands.More specifically, CD3 zeta and 4-1BB costimulatory signal sequences can be utilized.

[0083] The above transmembrane domain refers to a portion of the region that connects and fuses the extracellular domain and the intracellular signaling domain and plays a role in anchoring the chimeric antigen receptor to the plasma membrane of an immune cell. The transmembrane domain may be derived from a natural, synthetic, semi-synthetic, or recombinant source. For example, it may be derived from any one selected from the T cell receptor (TCR) α chain, TCR β chain, CD3ζ, CD3ε, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and CD278. More specifically, a CD8-derived transmembrane domain sequence may be used.

[0084] The chimeric antigen receptor further comprises a hinge domain, wherein the extracellular binding domain is connected to the transmembrane domain by the hinge domain, and the hinge domain may be derived from any one selected from, for example, CD8, FcγRIIIα, and IgG1. More specifically, it may comprise a CD8-derived hinge sequence.

[0085] The chimeric antigen receptor may further comprise a signal peptide for domain exposure. The signal peptide may be any secreted or transmembrane protein, which directs the transport of the chimeric antigen receptor to the cell membrane or cell surface and provides precise localization. The signal peptide may be derived from any one selected from CD8, an IgG1 heavy chain, an Igkappa light chain, and GMCSF. More specifically, it may be a CD8-derived signal peptide.

[0086] The present invention relates to a nucleic acid molecule encoding the chimeric antigen receptor.

[0087] The term “nucleic acid molecule” as used in the present invention has a comprehensive meaning including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base moieties are modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, (1990) 90:543-584). The sequence of the nucleic acid molecule encoding the heavy and light chain variable regions of the present invention may be modified. The modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

[0088] The present invention relates to an expression vector comprising the nucleic acid molecule.

[0089] The above expression vector may comprise an expression control sequence operably linked to the above nucleic acid.

[0090] The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Examples of such vectors include plasmids, i.e., circular double-stranded DNA fragments into which additional DNA segments can be ligated. Examples of such vectors include viral vectors, i.e., vectors into which additional DNA segments can be ligated within the viral genome. Such vectors are capable of autonomous replication within a host cell into which they are introduced. Examples include bacterial vectors having a bacterial origin of replication and episomal mammalian vectors. Furthermore, such vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of a host cell upon introduction into the host cell, thereby replicating together with the host genome. Furthermore, any vector can express a gene operably linked to the vector.

[0091] As used herein, the term "expression control sequence" refers to a polynucleotide sequence necessary for ligating a coding sequence to express and process the coding sequence. Expression control sequences include appropriate transcription initiation, termination, promoter, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation signals, sequences that stabilize cytoplasmic mRNA, sequences that increase translation efficiency (i.e., Kozak consensus sequences), and sequences that enhance protein stability, and, if desired, sequences that promote protein secretion. The nature of such expression control sequences varies depending on the host organism. In the case of prokaryotes, such expression control sequences generally include a promoter, a ribosome binding site, and a transcription termination sequence, and in the case of eukaryotes, such expression control sequences include a promoter and a transcription termination sequence. In the present invention, the expression control sequence includes at least all components whose presence is essential for the expression and processing process, and may include additional components whose presence is advantageous, such as a leader sequence and a fusion partner sequence.

[0092] The present invention relates to an immune cell expressing the chimeric antigen receptor.

[0093] The chimeric antigen receptor is as described above.

[0094] The above immune cells express the chimeric antigen receptor on their surface. The expression of the chimeric antigen receptor on the surface of the immune cells refers to any immune cell that has been engineered by the addition or modification of a nucleic acid encoding the chimeric antigen receptor. Therefore, in order to express the chimeric antigen receptor on the surface of the immune cell as described above, a polynucleotide encoding the chimeric antigen receptor or an expression vector containing the same can be transfected or transduced into the immune cell. The transfection can be performed by various methods known in the art, such as calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofectamine, and protoplast fusion. In addition, the transfection refers to the transfer of a gene into a cell using a virus or viral vector particle by means of infection. In this specification, transfection and transduction may be used interchangeably, but both are preferably interpreted in a broad sense as transformation of foreign gene transfer into a host cell, and a cell into which a foreign gene has been introduced through transfection or transduction is called a transformant.

[0095] The above immune cells can be used without limitation as long as they are cells that can induce immunity and induce the desired therapeutic effect, and can be obtained from peripheral blood, umbilical cord blood, bone marrow, tumor-infiltrating lymphocytes, lymph node tissue, or thymus tissue, and can be obtained by differentiation from placental cells, embryonic stem cells, induced pluripotent stem cells, or hematopoietic stem cells. In addition, the above immune cells can be obtained from humans, monkeys, chimpanzees, dogs, cats, mice, rats, and their genetically inserted species, as well as established cell lines.

[0096] The above-described method for obtaining immune cells may utilize any means known in the art, and may be obtained from autologous, allogeneic, or xenogeneic sources. "Autologous" refers to all cells derived from the same individual to be subsequently reintroduced into the individual, "allogeneic" refers to all cells derived from another animal of the same species as the individual into which the cells are being introduced, and "xenogeneic" refers to cells derived from an animal of a different species.

[0097] The above immune cell may be, for example, at least one selected from among T cells, natural killer T cells, dendritic cells, killer dendritic cells, mast cells, natural killer cells, macrophages, and precursor cells thereof.

[0098] The above immune cells have the advantage of exhibiting a lower rate of fratricide compared to immune cells expressing a chimeric antigen receptor comprising an antibody or an antigen-binding fragment thereof that binds specifically to the CD5 extracellular membrane-proximal domain.

[0099] The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising the above-described immune cells.

[0100] Regarding the above immune cells, it is as described above.

[0101] The term "cancer" is used interchangeably with "tumor" and refers to or means a physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation.

[0102] The cancer may include all cancers or carcinomas that can be treated with the pharmaceutical composition of the present invention, for example, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, follicular T-cell lymphoma, anaplastic large-cell lymphoma, adult T-cell leukemia / lymphoma, hepatosplenic T-cell lymphoma, mycosis fungoides, Sezary syndrome, extranodal NK / T-cell lymphoma, primary cutaneous gamma / delta T-cell lymphoma, cutaneous T-cell lymphoma, It may be mantle cell lymphoma, T-cell acute lymphobalstic lymphoma, chronic lymphoblastic leukemia, thymic carcinoma, non-Hodgkin lymphoma, diffuse large cell lymphoma, small lymphocytic lymphoma, or T-cell neoplasm.

[0103] “Treatment” as used in the present invention means an activity that improves or favorably changes symptoms caused by cancer.

[0104] As used herein, “prevention” means preventing the onset, recurrence, or transmission of a disease or disorder, or one or more symptoms caused by the disease / disorder, and may include prophylactic treatment for potential candidates.

[0105] The pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier, which is commonly used in formulations, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0106] The pharmaceutical composition of the present invention may further include, in addition to the above components, a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc. Suitable pharmaceutically acceptable carriers and preparations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, body weight, sex, degree of disease symptoms, food, administration time, administration route, excretion rate, and reaction sensitivity, and a generally skilled physician can easily determine and prescribe an effective dosage for the desired treatment. Meanwhile, the dosage of the pharmaceutical composition of the present invention is not limited thereto and may be 0.01-2000 mg / kg (body weight) per day.

[0107] The pharmaceutical composition of the present invention can be administered orally or parenterally. When administered parenterally, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. It is preferable that the route of administration of the pharmaceutical composition of the present invention be determined depending on the type of disease to which it is applied.

[0108] The pharmaceutical composition of the present invention can be manufactured in a unit dosage form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person of ordinary skill in the art to which the present invention pertains, and the method can be performed. In this case, the formulation 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, granules, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0109]

[0110] Hereinafter, the present invention will be described in detail by way of examples to specifically explain the present invention.

[0111]

[0112] Experimental methods and results

[0113] 1. Discovery of a novel scFv targeting CD5 and analysis of scFv characteristics

[0114] CD5 is a target cell surface protein used in CAR-T therapy to treat T-cell tumors. To develop a superior CAR structure for CAR-T therapy to treat T-cell tumors, two new CD5-targeting scFvs for the antigen-binding region of the CAR structure were discovered through phage display. To confirm that the two discovered scFvs actually bind to CD5, the scFv proteins were conjugated to the CD5-positive Jurkat cell line and the CD5-negative NLAM-6 cell line, which constitutively express CD5 on their cell surfaces, and binding was confirmed by flow cytometry (Fig. 1). Comparison with the negative control NALM-6 cell line and the isotype control confirmed binding of the two discovered scFvs to the Jurkat cell line.

[0115] The structure of the CD5 CAR comprised in the CD5 CAR-T therapeutic agent for T cell treatment of the present invention is shown in Fig. 2. The CD5 CAR has a structure in which a CD8 signal sequence domain; an anti-CD5 scFv sequence (VL-linker-VH domain); a c-myc-derived myc-tag domain; a CD8-derived hinge domain; a CD8-derived transmembrane domain; a 4-1BB-derived intracellular signaling domain; a CD3z-derived intracellular signaling domain and a stop codon, TAA, are sequentially linked. Three types of CAR structures are shown in Fig. 2 according to the type of anti-CD5 scFv sequence, among which H65 scFv is a positive control scFv.

[0116] To confirm the CD5 binding affinity of the developed scFvs, each CAR molecule composed of the developed scFvs was transduced into the CD5-negative K562 cell line. CAR molecule expression was confirmed in K562 cell lines expressing each CAR molecule using a myc-tag (Fig. 3a). Recombinant human CD5 protein was bound to the three manufactured cell lines and the untransduced K562 cell line, and the degree of binding according to the amount of recombinant human CD5 protein treated was analyzed by flow cytometry (Fig. 3b). It was confirmed that the CD5 binding affinity of the CAR molecules of each structure was no different.

[0117] To confirm the CD5 binding site of the discovered scFv, three mutant CD5 sequences were prepared. A mutant CD5 (DX23) was prepared by deleting the domain 1 (D1), which is the most distant from the membrane among the three extracellular domains of the CD5 protein, a mutant CD5 (DXX3) was prepared by deleting the extracellular domain 1 and the extracellular middle domain 2, and wild-type CD5. Each CD5 sequence was sequentially followed by a P2A self-cleaving peptide sequence linked to glycine (G)-serine (S)-glycine (G), an eGFP sequence, and a stop codon TAA (Fig. 4a). The three mutant CD5 sequences were transduced into K562 cells, a CD5-negative cell line, and the transduction efficiency was analyzed by flow cytometry (Fig. 4b). The CD5 CAR molecule shown in Fig. 2 was transduced into T cells. CD5 CAR-transduced T cells and the cell line shown in Fig. 4b were co-cultured at an E:T = 1:1 ratio for 48 hours, and the amount of INF-γ released into the culture medium during the co-culture was measured using a cytometric bead array method. The measured raw values ​​were normalized to the GFP expression level of the cell lines used in each co-culture (Fig. 4c). The positive control groups H65CAR5 and A2 CAR5 released INF-γ only in response to K562-CD5 (WT), and did not react with CD5 from which the CD5 membrane-distal domain was deleted. In addition, C7CAR5 released INF-γ even when only the CD5 membrane-proximal domain was present. Therefore, it was confirmed that the positive control H65 and A2 scFvs bound to the extracellular membrane-distal domain, and the C7 scFv bound to the extracellular membrane-proximal domain.

[0118]

[0119] 2. Production and characterization of CD5-targeted CAR-T cells

[0120] CD4-positive or CD8-positive T cells were isolated and purified from peripheral blood mononuclear cells (PBMCs) of healthy donors. The purified T cells were designated “TCs.” The frozen TCs were thawed, and T cell activation was induced for 48 hours using TransAct coated with CD3 and CD28 antibodies. The activated T cells were designated “ATs.” The ATs were treated with a lentiviral vector to transduce the CD5 CAR gene and cultured for 24 hours to transduce the CD5 CAR gene. The transduced T cells were harvested and cultured in CAR-T culture medium for an additional 10 days. The cultured T cells were designated “CTs.”

[0121] Mock cells without CAR gene transduction, H65CAR5 transduced with H65 CAR, A2CAR5 transduced with A2 CAR, and C7CAR5 transduced with C7 CAR were harvested, and CAR molecule expression was confirmed using myc-tag (Fig. 5). H65CAR5 and A2CAR5 showed similar CAR molecule positivity rates, while C7CAR5 showed a statistically significant lower CAR molecule positivity rate (*p<0.05, **p<0.01, ***<0.001, ns: not significant).

[0122] To confirm the degree of autophagy, cell viability at the end of culture was measured (Fig. 6a). H65CAR5 and A2CAR5 showed similar cell viability, while C7CAR5 showed a statistically significant higher cell viability (*p<0.05, **p<0.01, ***<0.001, ns: not significant). In addition, the growth of CD5 CAR-transduced T cells was measured. The number of CD5 CAR-transduced T cells after 10 days of additional culture was measured compared to the number of CD5 CAR-transduced T cells after 3 days of transduction and additional culture, and the growth rate of CD5 CAR-transduced T cells when cultured for 7 days was confirmed (Fig. 6b). H65CAR5 and A2CAR5 showed similar cell growth rates, while C7CAR5 showed a statistically significant higher cell growth rate (*p<0.05, **p<0.01, ***<0.001, ns: not significant). Through this, we were able to confirm that C7CAR5 among CD5 CAR transduced T cells had a low level of self-killing.

[0123] To determine the extent of the autophagy phenomenon, the concentration of cytokines released into the culture medium during the culture process was measured (Fig. 7). A portion of the culture supernatant was recovered and stored in a -80℃ ultra-low temperature freezer. After the culture procedure was completed, the stored culture supernatant was thawed, and the concentrations of INF-γ and TNF-α cytokines were measured using a cytometric bead array. It was confirmed that both cytokines were released at a statistically significantly lower level in C7CAR5 after 6 days of additional culture (*p<0.05, **p<0.01, ***<0.001, ns: not significant). This confirmed that C7CAR5 among CD5 CAR-transduced T cells exhibited a lower level of autophagy.

[0124] To determine the extent of autophagy, we examined the levels of cell surface markers and transcription factors associated with cell exhaustion at the end of culture. To confirm the persistent stimulation caused by autophagy, we analyzed the cell surface expression of CD69, a cell activation marker, using flow cytometry (Fig. 8a). Statistically significant lower levels of cell activation were observed in C7CAR5 cells. Furthermore, we confirmed the increased levels of immune checkpoint receptors induced by persistent stimulation (Fig. 8b). All three immune checkpoint receptors, PD1, TIGIT, and LAG3, were statistically significantly lower expressed in C7CAR5 cells. Furthermore, we analyzed the levels of transcription factors associated with persistent stimulation using intracellular staining (FACS) (Fig. 8c). We confirmed that the level of TOX transcription factor was statistically significantly lower in C7CAR5 cells (*p<0.05, **p<0.01, ***<0.001, ns: not significant). Through this, we were able to confirm that C7CAR5 among CD5 CAR transduced T cells had a low level of self-killing.

[0125]

[0126] 3. Analysis of the tumor cell killing ability of CD5 CAR-transduced T cells

[0127] 3-1) In vitro tumor cell killing ability analysis of CD5 CAR transduced T cells

[0128] CD5 CAR-transduced T cells were harvested and centrifuged at 600 x g for 3 minutes at room temperature. The supernatant was removed, suspended in assay medium, and the cell number was measured. Using the assay medium, 3 x 10 CAR-positive cells were counted. 3 The suspension was diluted to 100 μL and prepared. 1 x 10 GFP fluorescence-expressing target cell line was seeded in a 96-well plate. 4 cells / well(1x105 x cells / mL, 100 μL). 100 μL of CD5 CAR transduced T cells prepared so that the ratio of E (CD5 CAR transduced T cells): T (CD5 positive (Jurkat) or negative (NALM-6) target cell line) was 0.3:1 was added to each well. As a negative control, a Target Only group was also prepared by adding 100 μL of assay medium instead of CD5 CAR transduced T cells. The total amount of GFP fluorescence emitted from each well was calculated as the amount of target cell line by utilizing the fact that the GFP fluorescence disappears in the killed target cell line. The amount of GFP fluorescence was measured with the IncuCyte real-time cell analysis system and normalized by the following formula: (total GFP fluorescence per well at each measurement time point / total GFP fluorescence per well at the first measurement time point)*100 (Fig. 9). The tumor killing ability of CD5 CAR transduced T cells against the Jurkat cell line, a CD5 positive cell line, was observed.

[0129] 3-2) Analysis of in vivo tumor cell killing ability of CD5 CAR transduced T cells

[0130] For laboratory animals, specific pathogen free (SPF) NOD.Cg-Prkdc scid IL2γg tm1Sug / JicKoat (hereinafter NOG) mice (manufactured by Coretech Co., Ltd.) were used. Jurkat cells expressing firefly luciferase for mouse tumor transplantation were prepared by thawing the frozen cell line and subculturing once every 3 to 4 days. All cancer cells were harvested and centrifuged on the day of tumor transplantation into the mouse. The supernatant was removed, suspended using cell line culture medium, and the cell number was measured. Centrifugation was performed again, the supernatant was removed, and 5 x 10 6 A cell suspension was prepared at a concentration of 1 × 10 cells / mL. 0.2 mL of the prepared cell suspension was injected into the tail vein of each mouse in each experimental group to obtain a concentration of 1 × 10 6Cancer cells were transplanted into the mice at a concentration of 15 mg / ml per cell / head. Seven days after cancer cell transplantation, 200 μL of D-luciferin at a concentration of 15 mg / ml was intraperitoneally administered based on a body weight of 20–25 g, and the mice were anesthetized with inhalation of 2% isoflurane. Small animal bioimaging (Perkin Elmer, IVIS Spectrum Series) was performed 10–15 minutes after D-luciferin administration to confirm cancer cell transplantation. Eight days after cancer cell transplantation, the cultured CD5 CAR-transduced T cells were harvested and centrifuged at 600 × g for 3 minutes at room temperature. The supernatant was removed, suspended in assay medium, and the cell number was measured. Centrifugation was performed again, the supernatant was removed, and 5 × 10 CD5 CAR-positive T cells were counted using D-PBS. 6 A cell suspension was prepared at a concentration of 1 × 10 cells / mL. 0.2 mL of the prepared cell suspension was injected into the tail vein of each mouse in each experimental group to obtain 1 × 10 CD5 CAR transduced T cells. 6 cells / head were transplanted. Afterwards, small animal bioimaging was performed once or twice a week for all animals during the experimental period to monitor the amount of cancer cells. In addition, all animals were observed for death to evaluate the survival rate (Fig. 10). The small animal bioimaging results showed that C7CAR5 exhibited more sustained anticancer efficacy than the other groups (Fig. 10a). The graph of the small animal bioimaging results for each mouse also showed that C7CAR5 exhibited more sustained anticancer efficacy than the other groups (Fig. 10b). The survival rate results showed that C7CAR5 survived longer than the other groups (Fig. 10c). This confirmed that among CD5 CAR-transduced T cells, C7CAR5 has a higher in vivo tumor cell killing ability.

[0131]

[0132] 4. Discovery of a novel scFv targeting the membrane-proximal domain of CD5 and analysis of scFv characteristics.

[0133] As confirmed above, C7CAR5 has a lower degree of self-killing and superior in vivo tumor cell killing ability than H65CAR5 and A2CAR5, which utilize the existing positive control H65 scFv. In addition, it was confirmed that the target binding site of C7CAR5 is the membrane-proximal domain, unlike the other CAR5s. Therefore, in order to invent a superior CAR structure in the composition of CAR-T therapeutics for treating T cell tumors, the present inventors discovered a CD5 membrane-proximal domain-targeting scFv to be newly applied to the antigen-binding site in the CAR structure through the phage display method. To confirm whether the discovered scFv actually binds to the membrane-proximal domain of CD5, the scFv protein was bound to a CD5-negative K562 cell line, a K562 cell line transduced with wild-type CD5, and a K562 cell line transduced with a mutant CD5 (DXX3) that retains only the membrane-proximal domain, and the binding was confirmed through flow cytometry (Fig. 11). As a result of comparison with the negative control K562 cell line and isotype control, a number of scFvs binding to the K562-CD5 (DXX3) cell line were identified, and among them, three scFvs that also bind to K562-CD5 (WT) were finally selected.

[0134] The structure of the CD5 CAR comprised in the CD5 CAR-T therapeutic agent for T cell treatment of the present invention is shown in Fig. 12. The CD5 CAR has a structure in which a CD8 signal sequence domain; an anti-CD5 scFv sequence (VL-linker-VH domain); a c-myc-derived myc-tag domain; a CD8-derived hinge domain; a CD8-derived transmembrane domain; a 4-1BB-derived intracellular signaling domain; a CD3z-derived intracellular signaling domain and a stop codon, TAA, are sequentially linked. Six types of CAR structures are shown in Fig. 12 according to the type of anti-CD5 scFv sequence. Among the six types of CAR structures, two types of CARs including the positive control H65 scFv target the CD5 membrane-distal domain, and four types of CAR structures target the CD5 membrane-proximal domain.

[0135] To confirm the CD5 binding affinity of the developed scFvs, each CAR molecule composed of the developed scFvs was transduced into the CD5-negative K562 cell line. The expression of the CAR molecules in the K562 cell lines expressing each CAR molecule was confirmed using the myc-tag (Fig. 13a). Recombinant human CD5 protein was bound to the six manufactured cell lines and the untransduced K562 cell line, and the degree of binding was analyzed by flow cytometry (Fig. 13b). The crude results were normalized by dividing the binding affinity to the recombinant human CD5 protein by the expression level of the CAR molecule (Fig. 13c). It was confirmed that the binding affinity of the CAR molecules of each structure to CD5 did not differ (*p<0.05, **p<0.01, ***<0.001, ns: not significant).

[0136] To confirm the CD5 binding site of the identified scFv, the CD5 CAR molecule shown in Fig. 12 was transduced into T cells. The CD5 CAR-transduced T cells and the cell line shown in Fig. 4b were co-cultured at an E:T = 1:1 ratio for 48 hours, and the amount of INF-γ released into the culture medium during the co-culture was measured using a cytometric bead array method. The measured raw values ​​were normalized to the GFP expression level of the cell line used in each co-culture (Fig. 14). In addition, all three identified scFvs, C11CAR5, F8CAR5, and D9CAR5, released INF-γ even when only the CD5 membrane-proximal domain was present. Therefore, it was confirmed that the C11, F8, and D9 scFvs bind to the extracellular membrane-proximal domain.

[0137]

[0138] 5. Production and characterization of CD5 membrane-proximal domain-targeting CAR-transduced T cells.

[0139] CD5 CAR-transduced T cells were prepared in the same manner as above. NTD without CAR gene transduction, H65CAR5 transduced with H65 CAR, A2CAR5 transduced with A2 CAR, C7CAR5 transduced with C7 CAR, C11CAR5 transduced with C11 CAR, F8CAR5 transduced with F8 CAR, and D9CAR5 transduced with D9 CAR were harvested, and the expression of CAR molecules was confirmed at each time point using myc-tag (Fig. 15).

[0140] To determine the extent of autophagy, cell viability was measured at the end of culture (Fig. 16a). H65CAR5 and A2CAR5 showed similar cell viability, and CAR5 targeting the CD5 membrane-proximal domain showed a statistically significant higher cell viability (*p<0.05, **p<0.01, ***<0.001, ns: not significant). In addition, the growth potential of the entire cell was measured (Fig. 16b). The total number of cells after the end of culture was measured relative to the number of cells used for transduction to determine the growth potential of the entire cell during the entire culture period. H65CAR5 and A2CAR5 showed similar total cell growth potential, and CAR5 targeting the CD5 membrane-proximal domain showed a statistically significant higher total cell growth potential (*p<0.05, **p<0.01, ***<0.001, ns: not significant). In addition, the growth potential of CD5 CAR-transduced T was measured. The number of CD5 CAR-transduced T cells after 10 days of additional culture was measured compared to the number of CD5 CAR-transduced T cells after 3 days of transduction and additional culture, and the growth rate of CD5 CAR-transduced T cells when cultured for 7 days was confirmed (Fig. 16c). H65CAR5 and A2CAR5 showed similar cell growth rates, and D9CAR5 showed a statistically significant higher CD5 CAR-transduced T cell growth rate (*p<0.05, **p<0.01, ***<0.001, ns: not significant). This confirmed that among CD5 CAR-transduced T cells, CAR5 targeting the CD5 membrane-proximal domain had a low level of self-killing.

[0141] To determine the extent of autophagy, we examined the levels of cell surface markers and transcription factors associated with cell exhaustion at the end of culture. To confirm the persistent stimulation caused by autophagy, we analyzed the cell surface expression of CD69, a cell activation marker, using flow cytometry (Fig. 17a). Statistically significant lower levels of cell activation were observed in C7CAR5 and D9CAR5. Furthermore, we confirmed the increased levels of immune checkpoint receptors induced by persistent stimulation (Fig. 17b). We confirmed statistically significant lower expression of PD1 in C11CAR5 and D9CAR5. We confirmed statistically significant lower expression of LAG3 in all CD5 membrane-proximal domain targeting CAR5. Furthermore, we confirmed the levels of transcription factors associated with persistent stimulation using intracellular staining flow cytometry (Fig. 17c). We confirmed that the levels of TOX transcription factors were statistically significantly lower in CAR5 targeting all CD5 membrane-proximal domains (*p<0.05, **p<0.01, ***<0.001, ns: no significance). This confirmed that CAR5 targeting the CD5 membrane-proximal domain had a lower level of self-killing among CD5 CAR-transduced T cells.

[0142]

[0143] 6. Analysis of tumor cell killing ability of CD5 membrane-proximal domain-targeting CAR transduced T cells

[0144] In vitro tumor cell killing activity was analyzed in the same manner as described above (Fig. 18). As a result, tumor killing activity of CD5 CAR-transduced T cells against the Jurkat cell line, a CD5-positive cell line, was observed.

[0145] In addition, the in vivo tumor cell killing ability was analyzed in the same manner as above (Fig. 19). Small animal bioimaging results showed that the CD5 membrane-proximal domain-targeting CAR5 group exhibited stronger tumor cell killing ability than the CD5 membrane-distal domain-targeting CAR5 group. This confirmed that among CD5 CAR-transduced T cells, the CD5 membrane-proximal domain-targeting CAR5 group had a higher tumor cell killing ability in vivo.

Claims

1. A light chain variable region comprising a light chain CDR1 having an amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 10, SEQ ID NO: 19, and SEQ ID NO: 28; a light chain CDR2 having an amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 20, and SEQ ID NO: 29; and a light chain CDR3 having an amino acid sequence of any one of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 21, and SEQ ID NO: 30; and An antibody or antigen-binding fragment thereof that specifically binds to CD5, comprising a heavy chain variable region comprising a heavy chain CDR1 having an amino acid sequence selected from any one of SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 22, and SEQ ID NO: 31; a heavy chain CDR2 having an amino acid sequence selected from any one of SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 23, and SEQ ID NO: 32; and a heavy chain CDR3 having an amino acid sequence selected from any one of SEQ ID NO: 6, SEQ ID NO: 15, SEQ ID NO: 24, and SEQ ID NO:

33.

2. An antibody or antigen-binding fragment thereof comprising any one of the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 27, and SEQ ID NO: 36 in claim 1.

3. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof binds to the CD5 extracellular membrane-proximal domain.

4. An antibody or antigen-binding fragment thereof according to claim 3, wherein the CD5 extracellular membrane-proximal domain comprises the amino acid sequence of SEQ ID NO:

56.

5. In claim 1, the antibody or antigen-binding fragment thereof is an scFv, a Fab fragment, an F(ab') fragment, an F(ab')2 fragment, or an Fv fragment.

6. An anti-CD5 chimeric antigen receptor (CAR) comprising an extracellular binding domain comprising the antibody of claim 1 or an antigen-binding fragment thereof; a transmembrane domain; and an intracellular signaling domain.

7. In claim 6, the intracellular signaling domain is CD3 zeta, FcγR, ICOS (CD278), 4-1BB (CD137), OX40 (CD134), CD27, CD28, IL-2Rβ, IL-15R-α, CD40, MyD88, DAP10, DAP12, MHC class I molecule, TNF receptor protein, Immunoglobulin-like protein, cytokine receptor, integrin, SLAM protein, activated NK cell receptor, BTLA, Toll ligand receptor, CD2, CD7, CD30, CD40, CDS, ICAM-1, B7-H3, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), An anti-CD5 chimeric antigen receptor derived from at least one selected from SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83 specific ligands.

8. An anti-CD5 chimeric antigen receptor according to claim 6, wherein the transmembrane domain is derived from any one selected from among T cell receptor (TCR) α chain, TCR β chain, CD3ζ, CD3ε, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and CD278.

9. In claim 6, the chimeric antigen receptor further comprises a hinge domain, The extracellular binding domain is connected to the transmembrane domain by a hinge domain, An anti-CD5 chimeric antigen receptor, wherein the hinge domain is derived from any one selected from CD8, FcγRIIIα and IgG1.

10. An anti-CD5 chimeric antigen receptor according to claim 6, comprising any one of the amino acid sequences of SEQ ID NO: 51 to SEQ ID NO:

54.

11. A nucleic acid molecule encoding the chimeric antigen receptor of claim 6.

12. An expression vector comprising the nucleic acid molecule of claim 11.

13. An immune cell expressing the chimeric antigen receptor of claim 6.

14. In claim 13, the immune cell is at least one selected from among T cells, natural killer T cells, dendritic cells, killer dendritic cells, mast cells, natural killer cells, macrophages, and precursor cells thereof.

15. A pharmaceutical composition for preventing or treating cancer comprising the immune cell of claim 13.

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