Dual-antigen-targeting car for endoglin and mesothelin, and use thereof

Abstract

The present invention relates to CAR-expressing immune cells for improving cancer treatment and immunotherapy efficacy. More specifically, the present invention provides immune cells expressing a bispecific CAR (biCAR) that targets both endoglin and mesothelin, and thus is expected to be used as an immunotherapeutic agent capable of reducing immunosuppression by CAF in a tumor microenvironment and effectively killing cancer cells.

Description

Endoglin and mesothelin dual antigen-targeting CAR and uses thereof

[0001] The present invention relates to an immune cell expressing a chimeric antigen receptor for improving the efficacy of cancer treatment and immunotherapy, and more specifically, to a dual-targeting chimeric antigen receptor (biCAR) that simultaneously targets endoglin and mesotheliocytes and an immune cell expressing the same.

[0002] Natural Killer (NK) cells are cytotoxic effector cells that can reduce damaged, virus-infected, or malignant cells, regardless of antigen processing and presentation. NK cells play a crucial role in tumor immune surveillance and suppress metastasis through multiple killing mechanisms. Furthermore, allogeneic NK cells can be produced in vitro, clinically scalable, without additional gene editing. Meanwhile, carcinoma-associated fibroblasts (CAFs) contribute to the formation of an invasive tumor microenvironment. Despite the potential benefits of utilizing NK cells in the treatment of solid tumors, soluble mediators secreted by CAFs, such as IL-6, prostaglandin E2 (PGE2), indoleamine-pyrrole 2,3-dioxygenase (IDO), matrix metalloproteinases, and transforming growth factor-β (TGF-β), interfere with NK-mediated tumor killing, thereby contributing to the poor therapeutic response of NK cells to cancer. Thus, direct and indirect interactions between CAFs and NK cells within the tumor microenvironment (TME) exert major suppressive effects on the immune response.

[0003] Therefore, there is a need to develop a method that can evade the immune suppression response by CAFs in the tumor microenvironment and effectively target cancer cells.

[0004] The technical task to be achieved by the present invention is to provide a biCAR capable of targeting cancer cells while eliminating direct and indirect interactions between CAFs and NK cells in a tumor microenvironment, and an immune cell expressing the same, and to provide the same for use in cancer treatment.

[0005] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0006] To solve the above problem, the present invention provides a chimeric antigen receptor (CAR) targeting mesotheliocyte and endoglin.

[0007] The chimeric antigen receptor provided herein can be designated as a biCAR (bispecific CAR) as it simultaneously targets two antigens (mesopterin and endoglin).

[0008] The biCAR of the present invention may include an scFv targeting endoglin (ENG) and an scFv targeting mesothelin (MSLN) for targeting cancer cells.

[0009] As one embodiment of the present invention, the scFv of the endoglin target may include a heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 3, wherein each of the regions is connected by a linker, and the linker may include or consist of the amino acid sequence of SEQ ID NO: 4.

[0010] In another embodiment of the present invention, the scFv of the mesothelioma target may include a heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 8, wherein each of the regions is connected by a linker, and the linker may include or consist of the amino acid sequence of SEQ ID NO: 4.

[0011] In another embodiment of the present invention, the biCAR may further comprise one or more of (1) a signal peptide, (2) a hinge region, (3) a transmembrane domain, (4) a co-stimulatory signaling domain, and (5) an intracellular signaling domain, and may comprise each region in the following order: (a) or (b):

[0012] (a) N-terminus - [signal peptide] - [scFv of endoglin target] - [scFv of mesopterin target] - [hinge region] - [transmembrane domain] - [co-stimulatory signaling domain] - [intracellular signaling domain] - C-terminus

[0013] (b) N-terminal - [signal peptide] - [scFv of mesopterin target] - [scFv of endoglin target] - [hinge region] - [transmembrane domain] - [co-stimulatory signaling domain] - [intracellular signaling domain] - C-terminal

[0014] As another embodiment of the present invention, the scFv of the mesothelial target and the scFv of the endoglin target may be connected by a linker, and the linker may include or consist of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

[0015] As another embodiment of the present invention, the signal peptide may comprise or consist of the amino acid sequence of SEQ ID NO: 1.

[0016] In another embodiment of the present invention, the hinge region may comprise a hinge region of CD8, and the hinge region may comprise or consist of the amino acid sequence of SEQ ID NO: 9.

[0017] In another embodiment of the present invention, the transmembrane domain may comprise a transmembrane domain of CD8 or NKG2D, wherein the CD8 transmembrane domain may comprise or consist of the amino acid sequence of SEQ ID NO: 10, and the NKG2D transmembrane domain may comprise or consist of the amino acid sequence of SEQ ID NO: 13.

[0018] In another embodiment of the present invention, the co-stimulatory signaling domain may include one or more co-stimulatory signaling domains selected from the group consisting of 4-1BB, DAP10, 2B4, and combinations thereof. The 4-1BB co-stimulatory signaling domain may comprise or consist of the amino acid sequence of SEQ ID NO: 11, the DAP10 co-stimulatory signaling domain may comprise or consist of the amino acid sequence of SEQ ID NO: 14, and the 2B4 co-stimulatory signaling domain may comprise or consist of the amino acid sequence of SEQ ID NO: 17.

[0019] In another embodiment of the present invention, the intracellular signaling domain may be at least one intracellular signaling domain selected from the group consisting of CD3-ζ, DAP12, FcεRγ, and combinations thereof. The CD3-ζ intracellular signaling domain may comprise or consist of the amino acid sequence of SEQ ID NO: 13, the DAP12 intracellular signaling domain may comprise or consist of the amino acid sequence of SEQ ID NO: 15, and the FcεRγ intracellular signaling domain may comprise or consist of the amino acid sequence of SEQ ID NO: 16.

[0020] In addition, the present invention provides a nucleic acid encoding the above-described biCAR.

[0021] In addition, the present invention provides a vector expressing the above-described biCAR.

[0022] In addition, the present invention provides an immune cell transduced with a nucleic acid encoding the biCAR described above or a vector expressing the same. The immune cell expresses the biCAR described above.

[0023] As one embodiment of the present invention, the immune cell may be a natural killer (NK) cell or a T cell.

[0024] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises an immune cell expressing the above-described biCAR as an active ingredient.

[0025] In one embodiment of the present invention, the cancer may be a solid cancer.

[0026] The present invention provides a dual-targeting chimeric antigen receptor (biCAR) that simultaneously targets endoglin and mesoterin, exhibits anticancer activity by reacting with endoglin and / or mesoterin-expressing cells, reduces immunosuppression by CAFs in a microtumor environment, and exhibits cancer cell killing ability, and thus can be used as an immunotherapy agent for effective cancer treatment.

[0027] Figure 1 is a structural schematic diagram of a chimeric antigen receptor (biCAR) protein targeting endoglin and mesopterin of the present invention.

[0028] Figure 2 is the amino acid sequence of the biCAR protein of Figure 1.

[0029] Figure 3 is a schematic diagram of the structure of a modified biCAR protein in which the signal transduction domain is changed from the biCAR of Figure 1.

[0030] Figure 4 is the amino acid sequence of the modified biCAR protein of Figure 3.

[0031] Figure 5 shows the results of transducing NK cells with the biCAR of Figure 1 and confirming the efficiency using FACS.

[0032] Figure 6 shows the results of confirming the killing ability of biCAR-NK cells against cells overexpressing Endoglin or Mesothelin.

[0033] Figure 7 shows the results of measuring the cytokine secretion levels of biCAR-NK cells activated by Endoglin or Mesothelin.

[0034] Figure 8 shows the results of transducing the modified biCAR of Figure 3 into NK cells and confirming its efficiency using FACS.

[0035] Figure 9 shows the killing ability of modified biCAR-NK cells against cells overexpressing Endoglin or Mesothelin. Figure 9a is a schematic diagram of the experiment, Figure 9b is the result of confirming the cancer killing ability according to the antigen, and Figure 9c is the result of confirming the cancer killing ability according to each round.

[0036] Figure 10 shows the cross-sustained killing ability of modified biCAR-NK cells against cells overexpressing Endoglin or Mesothelin. Figure 10a is a schematic diagram of the experiment, Figure 10b is the result of confirming the cancer killing ability according to the antigen, and Figure 10c is the result of confirming the cancer killing ability according to each round.

[0037] The present inventors provide chimeric antigen receptor-expressing immune cells that can effectively target cancer cells while excluding the immunosuppressive activity of CAFs in a tumor microenvironment.

[0038] The chimeric antigen receptor (CAR) of the present invention targets mesothelial cells and endoglin simultaneously, and is a dual-specific chimeric antigen receptor in that it can target the two antigens simultaneously, and the dual-specific chimeric antigen receptor may be referred to herein as a biCAR.

[0039] The present inventors produced four types of biCARs as shown in Fig. 1 by varying the order of the two linkers forming the loop and the scFv targeting endoglin (ENG) and the scFv targeting mesothelin (MSLN), and transfected NK cells to produce biCAR-NK cells.

[0040] Specific information on each domain in the biCAR produced in the present invention is shown in Table 1 below.

[0041] Domain아미노산 서열서열번호signal peptideMALPVTALLLPLALLLHAARP19H10VLSYVLTQPPSVSVAPGQTARISCGGNNIGSKSVHWFQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVL2VHQVQLVESGGGVVQPGRSLRLSCAASGFAFINYGMHWVRQAPGKGLDWVAVISYDGSNKYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLMGATLFDNWGQGTLVTVSS3LinkerGGGGSGGGGSGGGGS4Linker1_loop1GGGGSGGGGSGGGGSGGGGS5Linker2_loop2GSTSGSGKPGSGEGSTKG6SSVLDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSGYPLTFGAGTKLEI7VHQVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSS8CD8 hingeTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD9CD8 TransmembraneIYIWAPLAGTCGVLLLSLVITLYC104-1BBKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL11CD3zRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR12

[0042]

[0043] In addition, the inventors of the present invention produced four modified biCARs as shown in Fig. 3 by variously changing and combining the co-stimulatory signaling domain and the intracellular signaling domain in the biCAR protein to increase the cancer killing efficacy, and transformed them into NK cells to produce biCAR-NK cells.

[0044] Specific information on the co-stimulatory signaling domain and intracellular signaling domain used in the modified biCAR is shown in Table 2 below.

[0045] Domain Amino acid sequence SEQ ID NO.NKG2D TransmembranePFFFCCFIAVAMGIRFIIMVT13DAP10LCARPRRSPAQEDGKVYINMPGRG14DAP12YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK15FcRγRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQ162B4ΔWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAY17

[0046]

[0047] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may optionally be conjugated to a moiety that is not composed of amino acids. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. A “fusion protein” refers to a chimeric protein encoding two or more individual protein sequences that are recombinantly expressed as a single moiety.

[0048] As used herein, "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof, and their complements, in single- or double-stranded form. The term "polynucleotide" refers to a linear sequence of nucleotides.

[0049] As used herein, "nucleotide" typically refers to a single unit, i.e., a monomer, of a polynucleotide. A nucleotide may be a ribonucleotide, a deoxyribonucleotide, or a modified version thereof. Examples of polynucleotides contemplated herein include single- and double-stranded DNA, single- and double-stranded RNA (including siRNA), and hybrid molecules comprising a mixture of single- and double-stranded DNA and RNA. As used herein, a nucleic acid also refers to a nucleic acid having the same basic chemical structure as a naturally occurring nucleic acid. Such analogs have modified sugars and / or modified ring substituents, but retain the same basic chemical structure as a naturally occurring nucleic acid. A nucleic acid mimetic refers to a chemical compound that has a structure different from the general chemical structure of a nucleic acid, but functions in a manner similar to a naturally occurring nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidites, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

[0050] As used herein, "expression" or "expressed" in relation to a gene refers to the transcription and / or translation product of that gene. The level of expression of a DNA molecule in a cell can be determined based on the amount of corresponding mRNA present in the cell or the amount of protein encoded by that DNA produced by the cell.

[0051] As used herein, "plasmid," "vector," or "expression vector" refers to a nucleic acid molecule encoding a gene and / or regulatory elements necessary for the expression of a gene. Expression of a gene from a plasmid can occur in cis or trans. When a gene is expressed in cis, the gene and regulatory elements are encoded by the same plasmid. Expression in trans refers to cases where the gene and regulatory elements are encoded by separate plasmids.

[0052] As used herein, the terms "transfection," "transduction," "transfecting," and "transducing" are used interchangeably and are defined as the process of introducing a nucleic acid molecule or protein into a cell. The nucleic acid is introduced into the cell using a non-viral or viral-based method. The nucleic acid molecule may be a genetic sequence encoding a complete protein or a functional portion thereof. Non-viral methods of transfection include any suitable transfection method that does not use viral DNA or viral particles as a delivery system to introduce a nucleic acid molecule into a cell.

[0053] As used herein, “antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragment thereof that specifically binds to and recognizes an antigen.

[0054] As used herein, a "chimeric antigen receptor" (CAR) refers to an engineered receptor capable of grafting desired antigen specificity into immune effector cells, such as T cells and NK cells. Typically, a CAR protein comprises an extracellular domain that introduces the desired specificity, a transmembrane domain, and an intracellular signaling domain that transmits a signal to an immune effector cell when the immune effector cell binds to the antigen. In certain embodiments, the extracellular domain may comprise a signal peptide, an antigen recognition domain, and a spacer region. In certain embodiments, the antigen recognition domain is derived from an antibody that specifically binds to the antigen. In certain embodiments, the antigen recognition domain is a single-chain variable fragment (scFv) derived from the antibody. In certain embodiments, the single-chain variable fragment (scFv) is derived from a humanized antibody (HuCAR scFv). In certain embodiments, the single-chain variable fragment comprises a heavy chain variable region fused to a light chain variable region via a flexible linker.

[0055] As used herein, the term "signal peptide" is used in its conventional sense in the art and refers to a peptide of about 5-30 amino acids in length. A signal peptide is present at the N-terminus of a newly synthesized protein that forms part of the secretory pathway. Proteins of the secretory pathway include, but are not limited to, proteins that reside within specific organelles (such as the endoplasmic reticulum, Golgi, or endosomes), are secreted from cells, or are inserted into the cell membrane. In some embodiments, the signal peptide forms part of a transmembrane domain of a protein.

[0056] The present invention provides a CAR protein (endoglin and / or mesotherin CAR protein) that binds to endoglin and / or mesotherin. Endoglin and / or mesotherin are antigens, and the chimeric antigen receptor (or CAR protein) is an antibody to endoglin and / or mesotherin, or a binding fragment that recognizes endoglin and / or mesotherin in the context of other membrane and intracellular components. In some embodiments, the anti-endoglin and / or mesotherin antibody or endoglin and / or mesotherin-binding fragment may be humanized.

[0057] The present invention provides a CAR protein capable of simultaneously targeting endoglin and mesoterin. In the present specification, a CAR protein that simultaneously targets two antigens, endoglin and mesoterin, is referred to as a biCAR.

[0058] In this specification, endoglin (ENG) is a 180 kDa homodimeric transmembrane protein expressed on the cell surface, also known as CD105 or edg-1. Endoglin promotes the growth of new blood vessels as well as cells that comprise and support existing blood vessels.

[0059] The CAR protein of the present invention can inhibit angiogenesis and small blood vessel expansion by targeting endoglin.

[0060] In this specification, mesothelin (MSLN) is a tumor-associated antigen known to be highly expressed in cancers including mesothelioma, as well as lung, ovarian, and pancreatic cancers. The CAR protein of the present invention specifically targets cancer cells expressing mesothelin on their surface, enabling the treatment of cancers expressing or overexpressing mesothelin.

[0061] In some embodiments, the endoglin and / or mesotherlin CAR protein may comprise, from N-terminus to C-terminus: a signal peptide; anti-endoglin heavy and light chain variable domains; a linker domain; anti-mesotherlin heavy and variable domains; a hinge region; a transmembrane domain; a co-stimulatory signaling domain, and an intracellular signaling domain.

[0062] In some embodiments, the transmembrane domain is derived from CD8, the co-stimulatory signaling domain is derived from 4-1BB, and the intracellular signaling domain is derived from CD3-ζ. In some embodiments, the transmembrane domain, the co-stimulatory signaling domain, and the intracellular signaling domain are modified, wherein the biCAR is referred to as a modified biCAR.

[0063] In some embodiments, the transmembrane domain in the modified biCAR can be from NKG2D, the co-stimulatory signaling domain can be from DAP10 or 2B4, and the intracellular signaling domain can be from DAP12 or FcεRγ.

[0064] In some embodiments, the nucleic acid encodes an antibody heavy chain variable domain and an antibody light chain variable domain from an antibody that binds endoglin and / or mesopterin.

[0065] In another aspect, an expression vector comprising a nucleic acid provided herein is provided, including embodiments thereof.

[0066] In another aspect, natural killer (NK) cells comprising an expression vector provided herein are provided, including embodiments thereof.

[0067] In this specification, NK cells comprising a vector expressing biCAR are referred to as biCAR-NK.

[0068] In another aspect, a mammalian cell comprising an expression vector provided herein is provided, including embodiments thereof.

[0069] In another aspect, a recombinant protein is provided. The recombinant protein comprises (i) an antibody region comprising a central cavity formed by a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the central cavity forms a peptide binding site comprising framework region amino acid residues; and (ii) a transmembrane domain.

[0070] In another aspect, a recombinant protein is provided. The recombinant protein comprises a first portion comprising an antibody heavy chain variable domain and a second portion comprising an antibody light chain variable domain and an antibody light chain constant domain. The first portion further comprises a transmembrane domain, wherein the antibody heavy chain variable domain, the antibody light chain variable domain, and the antibody (light chain or heavy chain) constant domain can together form an antibody region. The second portion further comprises a transmembrane domain, wherein the antibody heavy chain variable domain, the antibody light chain variable domain, and the antibody light chain constant domain can together form an antibody region, and the antibody heavy chain variable domain and the antibody light chain variable domain can together form an antibody region.

[0071] In another aspect, a mammalian cell comprising a recombinant protein provided herein is provided, including embodiments thereof, wherein the transmembrane domain is within the cell membrane of the mammalian cell.

[0072] In some embodiments, the transmembrane domain is a CD8 transmembrane domain. The term "CD8 transmembrane domain" as provided herein includes any recombinant or naturally occurring form of the transmembrane domain of CD8. In some aspects, the variants or homologues have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence compared to a naturally occurring CD8 transmembrane domain polypeptide.

[0073] In some embodiments, the intracellular T cell signaling domain is a CD3-ζ intracellular T cell signaling domain. In some embodiments, the intracellular T cell signaling domain comprises a signaling domain of the zeta (ζ) chain of the human CD3 complex.

[0074] In some embodiments, the isolated nucleic acid provided herein comprises an intracellular co-stimulatory signaling sequence encoding an intracellular co-stimulatory signaling domain. An "intracellular co-stimulatory signaling domain," as provided herein, comprises an amino acid sequence capable of providing co-stimulatory signaling in response to binding of an antigen to an antibody region provided herein, including embodiments thereof. In some embodiments, signal transduction by the co-stimulatory signaling domain results in the production of cytokines and the proliferation of T cells expressing the same.

[0075] In some embodiments, the isolated nucleic acids provided herein may comprise a spacer sequence encoding a spacer region. A "spacer region" as provided herein is a polypeptide that connects an antibody region to a transmembrane domain or connects various components of an antibody region. In some embodiments, the spacer region is between the antibody region and the transmembrane domain. In some embodiments, the spacer region connects a heavy chain variable region to a transmembrane domain. In some embodiments, the spacer region connects a heavy chain constant region to a transmembrane domain. In some embodiments, the spacer region connects a light chain variable region to a transmembrane domain. In some embodiments, the spacer region connects a light chain constant region to a transmembrane domain.

[0076] In some embodiments, the spacer region may comprise a hinge region.

[0077] In some embodiments, the spacer region may comprise an Fc region. Examples of spacer regions contemplated for the compositions and methods provided herein include, without limitation, immunoglobulin molecules or fragments thereof (e.g., IgG1, IgG2, IgG3, IgG4) and immunoglobulin molecules or fragments thereof (e.g., IgG1, IgG2, IgG3, IgG4) comprising a mutation that affects Fc receptor binding. In some embodiments, the spacer region is a fragment of IgG (e.g., IgG4), wherein the fragment comprises a deletion of the CH2 domain. The spacer region may be a peptide linker. In some embodiments, the nucleic acid may not comprise a spacer sequence encoding the spacer region.

[0078] A "heavy chain sequence" as provided herein refers to a nucleic acid sequence encoding a heavy chain domain as provided herein. A heavy chain domain as provided herein may comprise a heavy chain variable (VH) region and / or a heavy chain constant region (CH). A "light chain sequence" as provided herein refers to a nucleic acid sequence encoding a light chain domain as provided herein. A light chain domain as provided herein may comprise a light chain variable (VL) region and / or a light chain constant region (CL). The term "heavy chain domain" as used herein is used according to its ordinary meaning in the art and refers to a polypeptide comprising a heavy chain variable (VH) region and a heavy chain constant region (CH). The term "light chain domain" as used herein is used according to its ordinary meaning in the art and refers to a polypeptide comprising a light chain variable (VL) region and a light chain constant region (CL).

[0079] In some embodiments, the protein comprises an intracellular co-stimulatory signaling domain and a CD3-ζ intracellular T cell signaling domain.

[0080] In some embodiments, the protein comprises an intracellular co-stimulatory signaling domain from 4-1BB, DAP10, or 2B4 and an intracellular signaling domain from CD3-ζ, DAP12, or FcεRγ.

[0081] In some embodiments, the antibody heavy chain variable domain and the antibody light chain variable domain can be humanized.

[0082] Certain embodiments of the present disclosure relate to immune cells expressing the chimeric antigen receptor (CAR) described above. The immune cells may be T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or gamma-delta T cells), natural killer (NK) cells, invariant NK cells, or NKT cells. Also provided are methods for producing and manipulating the immune cells, as well as methods for using and administering the cells for adoptive cell therapy, wherein the cells may be autologous or allogeneic. Thus, the immune cells may be used in immunotherapy, such as targeting cancer cells.

[0083] Immune cells can be isolated from a subject, particularly a human subject. Immune cells can be obtained from a subject of interest, such as a subject suspected of having a particular disease or condition, a subject suspected of having a predisposition to a particular disease or condition, a subject undergoing therapy for a particular disease or condition, a subject who is a healthy volunteer or healthy donor, or a blood bank. Immune cells can be collected from any site present in the subject, including but not limited to blood, cord blood, spleen, thymus, lymph nodes, and bone marrow. Isolated immune cells can be used directly or stored for a period of time, such as by freezing.

[0084] Immune cells can be enriched / purified from any tissue, including but not limited to blood (including blood collected by a blood bank or cord blood bank), spleen, bone marrow, tissue removed and / or exposed during surgical procedures, and tissue obtained through biopsy procedures. The tissues / organs from which immune cells are enriched, isolated, and / or purified can be isolated from both living and non-living subjects, wherein the non-living subject is an organ donor. In certain embodiments, the immune cells are isolated from blood, such as peripheral blood or cord blood. In some aspects, immune cells isolated from cord blood have enhanced immunomodulatory capacity, as measured by CD4- or CD8-positive T cell suppression. In specific aspects, the immune cells are isolated from pooled blood, particularly pooled cord blood, to enhance immunomodulatory capacity. The pooled blood may be from two or more sources, for example, three, four, five, six, seven, eight, nine, ten or more sources (e.g., donor subjects).

[0085] The immune cell population can be obtained from a subject requiring therapy or suffering from a disease associated with reduced immune cell activity. Therefore, the cells will be autologous to the subject requiring therapy. Alternatively, the immune cell population can be obtained from a donor, preferably a histocompatibility-matched donor. The immune cell population can be harvested from peripheral blood, umbilical cord blood, bone marrow, spleen, or any other organ / tissue in which immune cells are present in the subject or donor. The immune cells can be isolated from a pool of subjects and / or donors, such as pooled umbilical cord blood.

[0086] When a population of immune cells is obtained from a donor distinct from the subject, the donor is preferably allogeneic, provided that the obtained cells are subject-compatible in that they can be introduced into the subject. Allogeneic donor cells may or may not be human leukocyte antigen (HLA) compatible. To render them subject-compatible, the allogeneic cells may be treated to reduce immunogenicity.

[0087] Immune cells (e.g., autologous or allogeneic T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or gamma-delta T cells), NK cells, invariant NK cells, or NKT cells can be genetically engineered to express antigen receptors such as engineered TCRs and / or chimeric antigen receptors (CARs). For example, host cells (e.g., autologous or allogeneic T-cells) are modified to express T cell receptors (TCRs) with antigen specificity for cancer antigens. In certain embodiments, NK cells are engineered to express TCRs. NK cells can be further engineered to express CARs. Multiple CARs and / or TCRs, for example, for different antigens, can be added to a single cell type, such as a T cell or NK cell.

[0088] In some embodiments, the cell comprises one or more nucleic acids introduced through genetic engineering that encode one or more antigen receptors, and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., not present in the cell or sample obtained from the cell, such as another organism or cell, and are not normally found in the cell being engineered and / or the organism from which the cell is derived. In some embodiments, the nucleic acids are non-naturally occurring, such as nucleic acids not found in nature (e.g., chimeric).

[0089] In some embodiments, the present disclosure provides a method of immunotherapy comprising administering an effective amount of immune cells of the present disclosure. In one embodiment, a medical condition or disorder is treated by transferring an immune cell population that elicits an immune response. In certain embodiments of the present disclosure, cancer or infection is treated by transferring an immune cell population that elicits an immune response. Provided herein is a method of treating or delaying the progression of cancer in a subject, comprising administering to the subject an effective amount of antigen-specific cell therapy. The method may be applied to the treatment of immune disorders, solid tumors, hematological malignancies, and viral infections.

[0090] Tumors for which the present treatment method is useful include any malignant cell type, such as those found in solid tumors or hematologic tumors. Exemplary solid tumors may include, but are not limited to, tumors of organs selected from the group consisting of the pancreas, colon, appendix, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematologic tumors include tumors of the bone marrow, T or B cell malignancies, leukemia, lymphoma, blastoma, and myeloma. Additional examples of cancers that can be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer (kidney or renal cancer), prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0091] Therapeutically effective amounts of immune cells can be administered by a number of routes, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intrasternal or intraarticular injection, or infusion.

[0092] Immune cell populations can be administered as a single or multiple doses over one to several days to improve the disease state or as periodic doses over an extended period of time to suppress disease progression or relapse, for example, in a treatment regimen consistent with the disease. The precise dosage used in the formulation will also vary depending on the route of administration and the severity of the disease or disorder, and should be determined by the physician's judgment and the individual patient's circumstances. The therapeutically effective number of immune cells will vary depending on the subject being treated, the severity and type of affliction, and the method of administration.

[0093] Also provided herein are pharmaceutical compositions and formulations comprising immune cells (e.g., T cells or NK cells) and a pharmaceutically acceptable carrier.

[0094]

[0095] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0096] [Example]

[0097] Example 1. Production of biCAR-NK cells

[0098] 1-1. Design of heterologous specific CAR (biCAR) proteins

[0099] Four types of biCARs were designed as shown in Fig. 1 by changing the order of the two linkers forming the loop and the scFv targeting endoglin (ENG) and the scFv targeting mesothelin (MSLN) (Figs. 1 and 2).

[0100]

[0101] 1-2. BiCAR gene transduction into NK92 cells

[0102] Polybrene (10 ug / ml) was added to the lentivirus containing the BiCAR gene designed in Example 1-1 and reacted for 5 minutes. After that, NK92 cells were seeded in a 24-well dish at a concentration of 1 X 10^6 / 500 uL media (+ IL2 cytokine 200 IU / ml). Then, the virus was added and centrifuged (400xg, 1 h 30 min, 32'C). After centrifugation, 1 ml of media was added and cultured in a CO2 incubator for 24 hours.

[0103] After 24 hours, all NK cells were collected, centrifuged, and cultured in new media containing IL2 cytokine (+IL2 cytokine 200 IU / ml), and sufficient NK92 cells were secured for FACS analysis.

[0104]

[0105] 1-3. Confirming transduction efficiency

[0106] NK92 cells (1 X 10^6 cells) were transferred to a round bottom tube. 2 ml of PBS was added, centrifuged (1,350 rpm, 3 min), and the supernatant was removed. The previous process was repeated once more. 100 ul of PBS + Biotinylated-Endoglin (1 ug) or Biotinylated-Mesothelin (1 ug) was added and resuspended. 20 min at 4°C. 2 ml of PBS was added, centrifuged (1,350 rpm, 3 min), and the supernatant was removed. The previous process was repeated once more. 100 ul of PBS + 0.4 ul of Streptavidin (Bdbioscience, 554061) was added and the supernatant was removed. 20 min at 4°C. After a 20-minute reaction, 2 mL of PBS was added, centrifuged (1,350 rpm, 3 minutes), and the supernatant was removed. The previous process was repeated once more. After resuspension in 200 μL of PBS, FACS analysis was performed. The results are shown in Figure 5.

[0107]

[0108] Example 2. K562 killing assay of biCAR-NK

[0109] WT K562, Endoglin, or Mesothelin-overexpressing cells were seeded at 0.2 x 10^6 cells in a 96-well dish. BiCAR-NK92 cells were harvested and counted. Then, the transduction efficiency was adjusted by mixing with WT NK92 cells, and then co-cultured with each K562 cell for 4 hours according to each E:T ratio.

[0110] After 4 hours, all cells in each well were harvested, 2 mL of PBS was added, centrifuged (1,350 rpm, 3 minutes), and the supernatant was removed. The previous process was repeated once more. Then, 100 μL of PE-conjugated-CD56 antibody (Biolegend, 318306) + live / dead fixable dye (eBioscience, 65-0866-14) solution for distinguishing NK92 cells was added and resuspended. Then, the cells were incubated at 4°C for 20 minutes. After 20 minutes of incubation, 2 mL of PBS was added, centrifuged (1,350 rpm, 3 minutes), and the supernatant was removed. The previous process was repeated once more. After resuspension in 200 μL of PBS, FACS analysis was performed. The results are shown in Figure 6 (A).

[0111]

[0112] Example 3. AsPc1 killing assay of biCAR-NK

[0113] One day before BiCAR-NK92 co-culture, 0.04 X 10^6 cells of AsPc1 Endgolin-overexpressing cells or Mesothelin-overexpressing cells were seeded in 96-well dishes with 100 uL of Media RPMI1640 (FBS 10%+Antibiotics 1%+Hepes 10mM) and cultured for 24 hours. After 24 hours of culture, all media was removed by suction and replaced with 100 uL of new Media RPMI1640 (FBS 10%+Antibiotics 1%+Hepes 10mM). After that, 0.1 X 10^6 cells of BiCAR NK92 were added to AsPc1 Endgolin-overexpressing cells or Mesothelin-overexpressing cells with 100 uL of Media (+IL2 cytokine 200IU / ml) and co-cultured for 24 hours.

[0114] After 24 hours of co-culture, all media containing dead AsPc1 cells and BiCAR NK92 cells were removed by suction. Then, 200 μL of PBS was added and removed by suction again. Then, 200 μL of RIPA Buffer was added to each well to lyse the remaining AsPc1 cells. Then, 200 μL of cell lysate dissolved in RIPA from each well was transferred to a new round-bottom 96-well dish. After centrifugation at 4000 rpm for 15 minutes, 50 μL of the supernatant excluding the cell pellet was transferred to a new white-bottom 96-well dish. After that, 50 μL of solution was treated to measure luminescence. [Solution: substrate 1 μL (Furimazine) + assay buffer 49 μL, Nano-Glo® Luciferase Assay System_CAT: N1120]. Luminescence was then measured using a microplate reader.

[0115] The measured value was calculated by calculating the signal of the remaining AsPc1 cells in the BiCAR-NK92 cell co-culture group based on the signal of the group containing only AsPc1 cells without BiCAR-NK92 co-culture. The result is as shown in Fig. 6 (B).

[0116]

[0117] Example 4. Comparison of the efficacy of BiCAR-NK92 cells in response to Endoglin or Mesothelin - Comparison of cytokines secreted after NK activation

[0118] K562 cells overexpressing endoglin or mesothelin were co-cultured with BiCAR-NK92 cells. After co-culture, all cells from each well were harvested and transferred to a round-bottom tube. The cells were then washed once with 2 mL of PBS. The supernatant was discarded, and 100 μL of IC fixation buffer (eBioscience, 00-8222) was added and incubated at 4°C for 20 minutes. After 20 minutes, the cells were resuspended in 2 mL of 1X permeabilization buffer (Invitrogen, 00-8333), centrifuged (1,350 rpm, 3 minutes), and the supernatant was removed. The previous step was repeated once more. The cells were then resuspended in 100 μL of 1X permeabilization buffer + CD56-PE (Biolegend, 318306) + IFNγ-APC (Biolegend, 502512). Afterwards, the reaction was performed at 4°C for 20 minutes. After 20 minutes of reaction, 2 mL of 1X permeabilization buffer was added, centrifuged (1,350 rpm, 3 minutes), and the supernatant was removed. The previous process was repeated once more. After resuspension in 200 μL of PBS, FACS analysis was performed. The results are shown in Figure 7.

[0119]

[0120] Example 5. Production of modified biCAR-NK

[0121] 5-1. Design and fabrication of a modified biCAR

[0122] To increase the cytotoxicity of BiCAR3, which has the best anticancer activity, we designed and produced a biCAR with altered co-stimulatory signaling domain and intracellular signaling domain (Figs. 3 and 4).

[0123]

[0124] 5-2. Transduction of the modified BiCAR gene into NK92 cells and confirmation of transduction efficiency.

[0125] The modified bi-CAR gene designed in Example 5-1 was transduced into NK cells using the same method as in Example 1-2 to produce modified biCAR-NK cells, and the expression of the introduced gene was confirmed using the same method as in Example 1-3. The results are shown in Fig. 8.

[0126]

[0127] Example 6. Confirmation of the sustained serial killing efficacy of modified biCAR-NK.

[0128] As shown in Figure 9a, the sustained efficacy of the modified biCAR-NK cell line was evaluated through repeated evaluations of the method for evaluating the mitochondrial efficacy against AsPC-1 in Example 3. The specific experimental procedures are as follows:

[0129] [DAY 0]

[0130] One day before BiCAR-NK92 co-culture, 0.04 X 10^6 cells of AsPc-1 Endgolin overexpressing cells or Mesothelin overexpressing cells were seeded in 100 uL of Media RPMI1640 (FBS 10% + Antibiotics 1% + Hepes 10mM) in a 96-well dish and cultured for 24 hours.

[0131] [DAY 1]

[0132] After culturing cancer cells for 24 hours, remove all media by suction and replace with 100 uL of new media RPMI1640 (FBS 10% + Antibiotics 1% + Hepes 10mM).

[0133] After that, BiCAR NK92 0.04 X 10^6 cells were co-cultured with 100 uL of Media (+IL2 cytokine 200IU / ml) each in AsPc1 Endgolin overexpressing cells or Mesothelin overexpressing cells for 24 hours.

[0134] [DAY 2]

[0135] After 24 hours, seed 0.04 X 10^6 cells of new AsPc-1 Endgolin overexpressing cells or Mesothelin overexpressing cells in 100 uL of Media RPMI1640 (FBS 10% + Antibiotics 1% + Hepes 10mM) in a 96-well dish and culture for 24 hours.

[0136] [DAY 3]

[0137] After 48 hours of co-culture, all media containing BiCAR NK92 cells were collected. Then, 200 uL of PBS was added and suction was used again to remove the media.

[0138] After that, add 200 uL of RIPA Buffer to each well to lyse the remaining AsPc1 cells. Then, transfer 200 uL of the cell lysate dissolved in RIPA from each well to a new round bottom 96-well dish. Centrifuge at 4000 rpm for 15 minutes. Then, transfer 50 uL of the supernatant excluding the cell pellet to a new white bottom 96-well dish. Then, process 50 uL of solution to measure luminescence. [Solution: substrate 1 uL (Furimazine) + assay buffer 49 uL, Nano-Glo® Luciferase Assay System_CAT: N1120]. After that, measure luminescence using a microplate reader.

[0139] The measured value is calculated by calculating the signal of the remaining AsPc1 cells in the BiCAR-NK92 cell co-culture group based on the signal of the group containing only AsPc1 cells without BiCAR-NK92 co-culture.

[0140] After removing all media from the AsPC-1 cells seeded on DAY 2 by suction, replace with 100 uL of new media RPMI1640 (FBS 10% + Antibiotics 1% + Hepes 10mM).

[0141] The BiCAR-NK92 cells collected above were seeded on DAY1 with AsPC-1_endoglin overexpressing or mesothelin overexpressing cells without cell counting, and the 2nd round of co-culture was started with new media with IL2 200IU / ML.

[0142] And after 48 hours of co-culture, luminescence activity is measured using the same method as above.

[0143] This co-culture was repeated 4 times in total. th Repeat until round.

[0144] The killing efficacy of the modified biCAR-NK cells for each antigen and round is shown in Figures 9b and 9c, respectively.

[0145]

[0146] Example 7. Confirmation of the reverse serial killing efficacy of modified biCAR-NK.

[0147] As shown in Figure 10a, the reverse serial killing efficacy of a modified biCAR-NK was evaluated similarly to the method for evaluating the sustained killing efficacy in AsPC-1 of Example 6. The specific experimental procedures are as follows:

[0148] [DAY 0]

[0149] One day before BiCAR-NK92 co-culture, 0.04 X 10^6 cells of AsPc-1 Endgolin overexpressing cells or Mesothelin overexpressing cells were seeded in 100 uL of Media RPMI1640 (FBS 10% + Antibiotics 1% + Hepes 10mM) in a 96-well dish and cultured for 24 hours.

[0150] [DAY 1]

[0151] After culturing cancer cells for 24 hours, remove all media by suction and replace with 100 uL of new media RPMI1640 (FBS 10% + Antibiotics 1% + Hepes 10mM).

[0152] After that, BiCAR NK92 0.04 X 10^6 cells were co-cultured with 100 uL of Media (+IL2 cytokine 200IU / ml) each in AsPc1 Endgolin overexpressing cells or Mesothelin overexpressing cells for 24 hours.

[0153] [DAY 2]

[0154] After 24 hours, seed 0.04 X 10^6 cells of new AsPc-1 Endgolin overexpressing cells or Mesothelin overexpressing cells in 100 uL of Media RPMI1640 (FBS 10% + Antibiotics 1% + Hepes 10mM) in a 96-well dish and culture for 24 hours.

[0155] [DAY 3]

[0156] After 48 hours of co-culture, all media containing BiCAR NK92 cells were collected. Then, 200 uL of PBS was added and suction was used again to remove the media.

[0157] After that, add 200 uL of RIPA Buffer to each well to lyse the remaining AsPc1 cells. Then, transfer 200 uL of the cell lysate dissolved in RIPA from each well to a new round bottom 96-well dish. Centrifuge at 4000 rpm for 15 minutes. Then, transfer 50 uL of the supernatant excluding the cell pellet to a new white bottom 96-well dish. Then, process 50 uL of solution to measure luminescence. [Solution: substrate 1 uL (Furimazine) + assay buffer 49 uL, Nano-Glo® Luciferase Assay System_CAT: N1120]. After that, measure luminescence using a microplate reader.

[0158] The measured value is calculated by calculating the signal of the remaining AsPc1 cells in the BiCAR-NK92 cell co-culture group based on the signal of the group containing only AsPc1 cells without BiCAR-NK92 co-culture.

[0159] After removing all media from the AsPC-1 cells seeded on DAY 2 by suction, replace with 100 uL of new media RPMI1640 (FBS 10% + Antibiotics 1% + Hepes 10mM).

[0160] The BiCAR-NK92 cells collected above were seeded on DAY1 with AsPC-1_endoglin overexpressing or mesothelin overexpressing cells without cell counting, and the 2nd round of co-culture was started with new media with IL2 200IU / ML.

[0161] At this time, BiCAR-NK92 cells co-cultured with AsPC-1 endoglin-overexpressing cells in the first round are cross-co-cultured with AsPC-1 mesothelin-overexpressing cells, and BiCAR-NK92 cells co-cultured with AsPC-1 mesothelin-overexpressing cells are cross-co-cultured with AsPC-1 endoglin-overexpressing cells.

[0162] And after 48 hours of co-culture, luminescence activity is measured using the same method as above.

[0163] This cross-culture was repeated four times in total. th Repeat until round.

[0164] The killing efficacy of the modified biCAR-NK cells for each antigen and round is shown in Figures 10b and 10c, respectively.

[0165]

[0166] Although the embodiments described above have been described with limited drawings, those skilled in the art will appreciate that various technical modifications and variations can be applied based on the above. For example, appropriate results can still be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents.

[0167] Therefore, other implementations, other embodiments, and equivalents to the claims also fall within the scope of the claims described below.

Claims

1. A dual-specific chimeric antigen receptor (biCAR) protein comprising an scFv targeting endoglin (ENG) and an scFv targeting mesothelin (MSLN).

2. In paragraph 1, A biCAR protein wherein the scFv of the endoglin target and the scFv of the mesopterin target are connected by a linker of SEQ ID NO: 5 or SEQ ID NO:

6.

3. In paragraph 1, The scFv of the above endoglin target comprises a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 2 and a light chain variable region consisting of an amino acid sequence of SEQ ID NO: 3, A biCAR protein, wherein the scFv of the above mesoterin target comprises a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 7 and a light chain variable region consisting of an amino acid sequence of SEQ ID NO:

8.

4. In paragraph 3, The heavy chain variable region and light chain variable region of the scFv of the above endoglin target are connected by a linker, A biCAR protein wherein the heavy chain variable region and the light chain variable region of the scFv of the above mesothelin target are connected by a linker.

5. In paragraph 4, A biCAR protein wherein the linker is composed of amino acids of sequence number 4.

6. In paragraph 1, A biCAR protein, wherein the biCAR further comprises at least one of (1) a signal peptide, (2) a hinge region, (3) a transmembrane domain, (4) a co-stimulatory signaling domain, and (5) an intracellular signaling domain.

7. In paragraph 6, The above biCAR includes regions (1) to (5), A biCAR protein in which the above (1) to (5) regions and the scFv of the endoglin target and the scFv of the mesopterin target are configured in one of the following orders: (a) N-terminus - [signal peptide] - [scFv of endoglin target] - [scFv of mesopterin target] - [hinge region] - [transmembrane domain] - [co-stimulatory signaling domain] - [intracellular signaling domain] - C-terminus (b) N-terminal - [signal peptide] - [scFv of mesopterin target] - [scFv of endoglin target] - [hinge region] - [transmembrane domain] - [co-stimulatory signaling domain] - [intracellular signaling domain] - C-terminal 8. In paragraph 6, A biCAR protein wherein the hinge region comprises the hinge region of CD8.

9. In paragraph 6, A biCAR protein wherein the transmembrane domain comprises a transmembrane domain of CD8 or NKG2D.

10. In paragraph 6, A biCAR protein, wherein the co-stimulatory signaling domain comprises at least one co-stimulatory signaling domain selected from the group consisting of 4-1BB, DAP10, 2B4, and combinations thereof.

11. In paragraph 6, A biCAR protein, wherein the intracellular signaling domain comprises at least one intracellular signaling domain selected from the group consisting of CD3-ζ, DAP12, FcεRγ, and combinations thereof.

12. Immune cells expressing the biCAR of paragraph 1.

13. In paragraph 12, The above immune cells are natural killer (NK) cells.

14. A nucleic acid encoding the biCAR of paragraph 1.

15. A vector containing the nucleic acid of Article 14.

16. A pharmaceutical composition for preventing or treating cancer, comprising the immune cells of Article 12 as an active ingredient.

17. In paragraph 16, A pharmaceutical composition wherein the cancer is a solid cancer.

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