Chimeric antigen receptor (CAR) expressing cells that recognize CEA

Abstract

The present invention relates to genetically engineered cells comprising a recombinant nucleic acid expression construct, the recombinant nucleic acid expression construct comprising a first nucleic acid sequence region encoding a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that recognizes carcinoembryonic antigen (CEA) protein, a second nucleic acid sequence region encoding a checkpoint inhibitor molecule, and a third nucleic acid sequence region encoding an immunostimulatory cytokine. In a further aspect, the present invention relates to genetically engineered cells, which are preferably T cells or NK cells, preferably cytotoxic T lymphocytes. The present invention further relates to anti-CEA CAR-T cells or anti-CEA CAR-NK cells of the present invention, which preferentially recognize membrane-bound CEA protein and express checkpoint inhibitor molecules and / or immunostimulatory interleukins in the vicinity of tumor tissue. The present invention further includes the medical use of such cells in the treatment of medical disorders associated with the presence of CEA-expressing pathogenic cells, preferably cancer cells, more preferably cancer cells of solid malignant tumors.

Description

[Technical Field]

[0001] The present invention relates to genetically modified cells comprising a recombinant nucleic acid expression construct, the recombinant nucleic acid expression construct comprising a first nucleic acid sequence region encoding a chimeric antigen receptor (CAR) containing an extracellular antigen-binding domain that recognizes carcinoembryonic antigen (CEA) protein, a second nucleic acid sequence region encoding a checkpoint inhibitor molecule, and a third nucleic acid sequence region encoding an immunostimulatory cytokine. In a further embodiment, the present invention relates to genetically modified cells, which are preferably T cells or NK cells, preferably cytotoxic T lymphocytes. The present invention further relates to anti-CEA CAR-T cells or anti-CEA CAR-NK cells of the present invention that preferentially recognize membrane-bound CEA protein and express checkpoint inhibitor molecules and / or immunostimulatory interleukins in the vicinity of tumor tissue. The present invention further includes the medical use of such cells in the treatment of medical disorders associated with the presence of CEA-expressing pathogenic cells, preferably cancer cells, more preferably cancer cells of solid malignant tumors. [Background technology]

[0002] Targeting the vulnerability of cancer cells is the goal of personalized cancer treatment, also known as "personalized medicine" or "targeted therapy." Adoptive T-cell immunotherapy is considered a promising antitumor treatment. Genetically modified T cells expressing chimeric antigen receptors (CARs) eliminate tumor cells by binding to tumor antigens through antigen-antibody recognition. These chimeric antigen receptor T (CAR-T) cells directly recognize tumor cells without going through the antigen presentation process and without being restricted by the major histocompatibility complex (MHC). The use of CAR-T cell therapies has introduced the promising concept of gene therapy into hematology and oncology.

[0003] CAR-T therapy offers promising antitumor effects and hematological CD19 +The researchers demonstrated a high complete remission rate for malignant tumors. They have applied CAR-T therapy to solid tumors by targeting multiple tumor-associated antigens, such as human epidermal growth factor receptor 2 (HER2), carboxylic acid anhydrase IX (CAIX), carcinoembryonic antigen (CEA), disialoganglioside (GD2), and interleukin (IL)-13 receptor alpha-2 (IL-13Rα2).

[0004] The advantage of CAR technology lies in the fact that, in ex vivo settings within research facilities, patient autologous T cells or donor allogeneic T cells can be genetically modified using recombinant surface molecules that, on the one hand, possess specificity for tumor cells, and on the other hand, mediate T cell activation when tumor cells are recognized. The prototype of this surface molecule has an extracellular portion containing an antibody for tumor cell antigen recognition, preferably an scFv single-chain antibody, and an intracellular portion containing a signaling chain for T cell activation, preferably the CD3ζ chain of the T cell receptor (TCR). A transmembrane domain anchors the molecule to the cell membrane. Because the surface molecule is composed of an antibody (fragment) on one side and a T cell signaling unit on the other, it is called a "chimeric antigen receptor" (CAR). Therefore, in contrast to the innate T cell receptor (TCR), CARs are recombinant surface receptors that recognize their target antigen via antibodies and are independent of the HLA (human leukocyte antigen) complex. The genetic information for each CAR has so far been introduced into patient or donor T cells ex vivo by transduction using retroviral or lentiviral vectors, and these T cells then express the CAR on their surface. CAR-T cells are retransfused to the patient and bind to specific antigens formed on the surface of cancer cells. "First-generation" CARs have a signaling chain for primary T cell activation, usually a CD3ζ signaling chain, while "second-generation" CARs use the CD3ζ chain together with a costimulatory unit, preferably derived from the CD28 family, to induce sustained T cell activation.

[0005] While some studies using these generations of CARs have observed some efficacy, including complete regression of a glioblastoma treated with multiple infusions of IL-13Rα2 CAR-T cells, the overall outcomes of CAR-T cell therapy have been insufficient in numerous studies, particularly in the case of solid tumors. Other significant concerns with CAR-T therapy include the adverse events of cytokine release syndrome and on-target / off-tumor effects. CAR-T therapy can also cause neurological side effects, such as speech disorders, tremors, delirium, and epileptic seizures. Therefore, redesigning CARs with the aim of creating safer and more effective treatments is necessary to improve the safety and efficacy of CAR-T cell therapy.

[0006] Several important aspects exist for effective CAR-T cell strategies in the treatment of solid tumors. Among the solid tumor antigens targeted by CAR-T cell therapy are CEA, EGFR, EGFRvIII, GD2, HER2, IL13Rα2, PSCA, CEA, Tn-MUC1, and PSMA. While all of these antigens are overexpressed and / or amplified in tumors compared to normal tissues, their protein expression is not clearly limited to tumor cells. An exception is EGFRvIII, which is a common oncogenic rearrangement in glioblastoma characterized by the deletion of exons 2-7 of EGFR. Therefore, in contrast to CD19, a B-cell limited antigen expressed in many B-cell malignancies, targeting solid tumor antigens raises considerable toxicity concerns, which may limit their usefulness in CAR-T cell therapy. In other words, tumor specificity of selected antigens is one important aspect in CAR design for effective and safe CAR-T cell strategies.

[0007] Another well-established aspect is tumor resistance to individual therapeutic agents, because the majority of tumors are heterogeneous. Long-term targeting of a single drug susceptibility pathway can ultimately lead to relapse and escape variants of drug-resistant tumors. Acquired or endogenous resistance patterns have been observed after CAR-T cell therapy. While CD19 CAR-T cell therapy has shown sustained clinical remission in 70-90% of patients with B-cell malignancies, including acute lymphoblastic leukemia (ALL), recent clinical trial follow-up data have shown common resistance mechanisms, with loss and / or downregulation of the CD19 antigen, which is involved in such resistance, occurring in 70% of patients who relapsed after treatment. Similar resistance mechanisms of antigen leakage have been observed in early clinical findings using CAR-T cells in solid tumors. There is strong evidence that a rational design combining tumor-specific antigen targeting with other antitumor strategies is necessary for effective disease management.

[0008] The immunosuppressive tumor microenvironment presents another challenge for effective CAR-T cell therapy. Unlike most hematological malignancies, there are no local immunosuppressive pathways that interfere with antitumor immunity and limit adoptive T-cell therapy. Solid tumors can be highly infiltrative and control therapeutic responses through multiple cell types that support tumor growth, angiogenesis, and metastasis. In addition to tumor cells themselves, immune cells, such as regulatory T cells and myeloid suppressor cells, induce the production of local cytokines, chemokines, and growth factors, including IL-4, IL-10, VEGF, and TGFβ, in solid tumors. Similarly, immune checkpoint pathways, including PD-1 and CTLA-4, may be highly activated in tumors, leading to attenuation of antitumor immunity. There is strong evidence that the tumor microenvironment may control response to and resistance to immunotherapy and limit the effectiveness of CAR-T cell therapy.

[0009] Carcinoembryonic antigen (CEA) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. CEA is soluble in the blood and is also known to bind to the cell membrane, primarily in tumor cells. As a cell surface glycoprotein, this protein is involved in cell adhesion, intracellular signaling, and tumor progression. Soluble CEA is used as a clinical biomarker for solid tumors, particularly malignant tumors, such as gastrointestinal cancer, and may promote tumor development through its role as a cell adhesion molecule. CEA also acts as an oncogene, promoting tumor progression and inducing treatment resistance in colorectal cancer cells. Furthermore, the encoded protein can regulate differentiation, apoptosis, and cell polarity. Currently, there is no curative treatment for highly CEA-positive tumor stem cells.

[0010] Currently, alternative treatment options for solid tumors generally include surgical tumor removal, chemotherapy, interleukins, checkpoint inhibitors, and antibodies specific to surface proteins. Several clinical trials are underway for CEA-positive tumor diseases. There are currently no CAR-T therapies for solid tumors.

[0011] Many research institutions have long attempted to make such CAR combinations functional and overcome the aforementioned technical difficulties, but without success. To the best of our knowledge, no equivalent functional anti-CEA CAR constructs combined with effective immunostimulatory cytokines and checkpoint inhibitor molecules have been previously described, and none are currently available in anti-CEA antibody research relevant to the medical indications of our invention.

[0012] The main concern with CAR-T therapy is the risk of a "cytokine storm" associated with a violent antitumor response mediated by a large number of activated T cells (Non-Patent Literature 1). Side effects may include high fever, low blood pressure, and / or organ failure, which can lead to death. Cytokines produced by CAR-NK cells, unlike those produced by CAR-T cells, reduce the risk of harmful cytokine-mediated responses.

[0013] Therefore, to improve CAR immunotherapy, it is necessary to overcome complex technical and biological challenges. The present invention addresses three of the most difficult areas that require attention in CAR vector design and the development of next-generation CARs for solid tumors, namely (1) targeting tumor-specific antigens, (2) tumor antigen heterogeneity and escape, and (3) dealing with the immunosuppressive tumor microenvironment. The present invention described below relates to solutions to this problem.

Prior Art Documents

Non-Patent Documents

[0014]

Non-Patent Document 1

Summary of the Invention

[0015] In light of the prior art, the technical problem underlying the present invention is to provide a novel strategy for cancer immunotherapy. Specifically, the problem underlying the present invention is to provide an appropriate means for locally stimulating a targeted immune response directed against tumor tissue in a subject while avoiding tumor escape from immunotherapy. A further problem underlying the present invention is to provide means for checkpoint inhibitors and for stimulating an immune response, which means provide an effective local effect in the target tumor tissue while reducing the level of unwanted side effects.

[0016] This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

[0017] Therefore, the present invention provides a genetically modified cell comprising a recombinant nucleic acid expression construct encoding a CAR, wherein the construct comprises the following: (a) A first nucleic acid sequence region encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular antigen-binding domain that recognizes a carcinoembryonic antigen (CEA) protein, the first nucleic acid sequence region, and (b) A second nucleic acid sequence region encoding a checkpoint inhibitor molecule, (c) A third nucleic acid sequence region encoding an immunostimulatory cytokine, This includes genetically modified cells.

[0018] Accordingly, the present invention relates to genetically modified cells expressing a recombinant nucleic acid construct, the recombinant nucleic acid construct comprising a first nucleic acid sequence region encoding a chimeric antigen receptor (CAR) that recognizes a carcinoembryonic antigen (CEA) protein.

[0019] The genetically modified cells are preferably CAR-T cell products or CAR-NK cell products, which confer to human T cells or NK cells high cytotoxic activity against well-defined tumors, solid tumors, and liquid tumors, while preserving non-pathogenic cells in the tissue surrounding the tumor, such as mammary cells, pancreatic cells, lung cells, colon cells, or hepatocytes, as well as hematopoietic cells.

[0020] In preferred embodiments, all T cells, B cells, and NK cells are preserved. This is because the CAR-T cell product of the present invention shows little to no activity towards these cells.

[0021] The present invention further relates to a novel chimeric antigen receptor (CAR) that recognizes (preferably specifically recognizes) the antigen CEA on cancer cells. In a preferred embodiment, genetically modified cells expressing an anti-CEA CAR are immune cells that recognize the CEA antigen on cancer cells and lyse cancer cells possessing the CEA antigen. Accordingly, the present invention relates to cell products such as highly therapeutic pharmaceuticals designed and manufactured for medical use, and these cell products include immune cells transduced with a next-generation CEA-CAR that can be used for cancer treatment.

[0022] The present invention provides an efficient and specific means for treating malignant diseases.

[0023] In a preferred embodiment, the immune cells are T cells. In another embodiment, the immune cells are T cells (CAR-T) containing an artificial T cell receptor, e.g., a chimeric antigen receptor, which specifically binds to tumor antigens (Lee, DW et al., Clin Cancer Res; 2012; 18(10); 2780-90). In a preferred embodiment, the immune cells are NK cells. In another embodiment, the immune cells are NK cells (CAR-NK) containing an artificial NK cell receptor, e.g., a chimeric antigen receptor, which specifically binds to tumor antigens.

[0024] The present invention enables the stimulation of cells involved in antitumor immune responses, thereby locally activating, supporting, and / or enhancing antitumor immune responses. The present invention enables the delivery of one or more immunostimulatory cytokines in effective and therapeutically appropriate doses, while avoiding the significant side effects inherent in systemic administration of cytokines without appropriate targeted agents, through the expression of recombinant nucleic acid expression constructs in transplanted CAR-T cells or CAR-NK cells. Accordingly, the present invention relates to the use of CAR-T cells or CAR-NK cells as targeted agents and / or vehicles for locally delivering immunomodulatory signals, preferably immunostimulatory signals, to inflammatory areas, preferably in and near tumor tissue.

[0025] A critical limiting factor in the successful development and medical use of immunotherapies is the tumor's ability to evade and / or suppress the innate immune response against tumor cells by establishing an immunosuppressive tumor microenvironment. This phenomenon is known as tumor-mediated immunosuppression and is largely mediated by the secretion of anti-inflammatory cytokines by immune cells and checkpoint proteins (e.g., regulatory T cells and monocyte-derived suppressor cells) present in tumors exhibiting a regulatory phenotype. Therefore, the present invention provides means for modifying the tumor microenvironment, making it pro-inflammatory, thereby promoting the activation of immune cells present in the tumor and the recruitment and activation of external immune cells, thereby promoting broad activation of the immune system against the tumor and / or improving the therapeutic efficacy of antitumor immunotherapy.

[0026] In one embodiment, the recombinant nucleic acid expression construct described herein can be administered to human cells for the purpose of modifying the tumor microenvironment to be advantageous and beneficial to immunotherapy.

[0027] The present invention utilizes CAR-T cells or CAR-NK cells as a cell vehicle for delivering immunomodulatory effectors that stimulate an immune response, thereby leveraging their unique tumor antigen targeting effect to induce CAR-T cells or CAR-NK cells in tumor regions, thereby exerting local therapeutic effects based on the stimulation of an appropriate immune response and inhibition of checkpoint proteins, the immune response in this case preferably relating to the host's (target's) innate immune response, thereby improving the potency and therapeutic effect of immunotherapies, such as chimeric antibodies, adoptive immunotherapies, antitumor vaccines, and / or checkpoint inhibitors. The present invention further provides means for selectively switching off genetically modified cell products expressing CAR, as described herein, i.e., for the safe clinical use of the above cells. This safety feature can be achieved in some embodiments by inducible suicide genes.

[0028] Surprisingly, CAR-T cells modified with recombinant nucleic acid expression constructs containing nucleic acid sequence regions encoding CARs, one or more immunostimulatory cytokines, and / or checkpoint inhibitor molecules, as described herein, exhibit unexpectedly good expression and secretion of the cytokines and checkpoint inhibitor molecules both in vitro and in vivo. Those skilled in the art would not have anticipated that these specific cytokines and checkpoint inhibitor molecules could be expressed in sufficient quantities, effluxed from cells in sufficient quantities, and potentially induce or enhance a desired local immune response based on either a spontaneous response or immunotherapy.

[0029] The present invention also includes the expression of combinations of immune-activating cytokines and / or checkpoint inhibitor molecules in tumors via CAR-T cells or CAR-NK cells as described herein, for the purpose of attracting immune effector and helper cells, inducing immune activation, promoting the maturation of immune memory cells, and / or suppressing the emergence and persistence of suppressive and / or regulatory immune cells.

[0030] In one embodiment, a combination of tumor antigen-targeting CARs, immunostimulatory cytokines, and / or checkpoint inhibitor molecules is used to promote the activation of various segments of the immune response, including innate and adaptive immune responses, effectors, helpers, and / or antigen-presenting cells.

[0031] On the other hand, cytokines, such as IL-2, IL-7, IL-15, and IL-21, specifically activate cytotoxic lymphocytes, such as T cells and NK cells, which initiate a specific response against tumor cells. Similarly, IL-15 activates cytotoxic lymphocytes, but also monocytes and helper cells.

[0032] More specifically, the fundamental problem of the present invention is to provide appropriate means for locally stimulating a targeted immune response directed towards tumor tissue in a target while reducing tumors that evade immunotherapy. A further fundamental problem of the present invention is to provide checkpoint inhibitors and means for stimulating immune responses that provide effective local effects in target tumor tissue while reducing the level of unwanted side effects.

[0033] Therefore, the combination of tumor-specific CARs, immunostimulatory cytokines, and checkpoint inhibitor molecules results in synergistic effects, an immune-tolerant tumor microenvironment, and / or a low tumor escape rate, similar to or exceeding those observed in innate immune responses. This invention enhances therapeutic efficacy and tumor regression rates.

[0034] It was a surprising finding that the innate immune response can be virtually accurately reflected or act similarly, in an enhanced manner, using a combined CAR-T cell or CAR-NK cell approach. Therefore, the present invention is based on the surprising finding that a locally more effective and safer antitumor response can be obtained by providing CAR-T cells with a combination of transgenes encoding tumor-specific CARs, immunostimulatory cytokines (or more), and checkpoint inhibitor molecules. This combination leads to unique local expression and secretion of immunostimulatory factors, which in turn induce a local antitumor response, including a multi-segment immune response, without inducing the systemic toxicity often observed when cytokines are applied systemically to tumor patients.

[0035] In one embodiment, the recombinant nucleic acid expression construct of the present invention includes an additional sequence region, also known as the fourth sequence region, which encodes a chemokine receptor, for example, the chemokine receptor CCR4. Preferably, the chemokine receptor enables cell migration to tumor cells.

[0036] Chemokine receptors are cytokine receptors expressed and presented on the surface of cells that interact with a certain type of cytokine called chemokines. Chemokines are a family of small cytokines, i.e., cell-secreted signaling proteins, that typically induce targeted chemotaxis of reactive cells, causing them to move toward or away from chemokine-producing cells. They are often referred to as chemotactic cytokines. Therefore, the expression of chemokine receptors is associated with the further benefit of improving the cell migration or motility of CAR-expressing cells toward target cells, as described in the present invention.

[0037] CCR4 (also known as CC chemokine receptor type 4 or CD194) belongs to the G protein-binding receptor family and is a receptor for the CC chemokines CCL2 (MCP-1), CCL4 (MIP-1), CCL5 (RANTES), CCL17 (TARC), and CCL22 (macrophage-derived chemokine). For example, CCL2 recruits monocytes, T cells, and dendritic cells to inflammatory sites; CCL4 is a chemotactic factor for natural killer cells, monocytes, and various other immune cells; and CCL5 is chemotactic for T cells, eosinophils, and basophils, as well as recruiting leukocytes to inflammatory sites. Therefore, expression of CCR4 enables improved recruitment and / or migration of genetically modified cells expressing the CAR of the present invention to tumor tissue.

[0038] Those skilled in the art may also consider other chemokine receptors (e.g., one or more of CCR1 to CCR11), which can be selected based on the tumor type and the antigen specificity of the CAR in order to improve the specific migration characteristics of the modified cells.

[0039] Furthermore, as demonstrated in the examples, the unique properties of this particular CAR-T cell combination allow it to be induced into tumor tissue and engrafted within it, thereby maintaining the expression of therapeutic tumor-specific CARs, immune stimulator cytokine factors, and checkpoint inhibitor molecules, for the purpose of maintaining the immune response necessary for therapeutic effect.

[0040] In one embodiment, the first nucleic acid sequence region encoding the CAR is as follows: (d) A nucleic acid sequence encoding an extracellular antigen-binding domain that recognizes the CEA protein, wherein the antigen-binding domain comprises an antibody or antibody fragment, (e) A nucleic acid sequence encoding a transmembrane domain, (f) A nucleic acid sequence encoding an intracellular co-stimulatory domain, Includes.

[0041] In alternative embodiments, the present invention envisions directing the cells and constructs of the present invention to alternative antigens, for example, by including a nucleic acid sequence in any of the first nucleic acid sequence regions encoding a CAR that encodes an extracellular antigen-binding domain, in which case the antigen-binding domain binds to cancer-related antigens selected from the group consisting of: folate receptor alpha (FRa), ERBB2 (Her2 / neu), EphA2, IL-13Ra2, epidermal growth factor receptor (EGFR), mesothelin, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, TnAg, prostate-specific membrane antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, interleukin-11 receptor a(I L-11Ra), PSCA, PRSS21, VEGFR2, LewisY, CD24, Platelet-derived growth factor receptor beta (PDGFR-beta), SSEA-4, CD20, MUC1, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, Tyrosinase, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o- Acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, Regmine, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 variant, prostein, survivorbin, telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras variant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2)ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1.

[0042] In a preferred embodiment, the antigen-binding domain of the CAR construct recognizes CEA, and the product of the corresponding immune cells expressing the construct, preferably CAR-T cells or CAR-NK cells, confers high cytotoxic activity to human T cells or human NK cells against CEA-positive pathogenic cells.

[0043] Further embodiments of the first nucleic acid sequence region encoding the CAR are provided below. In any of the nucleic acids encoding the antigen-binding domain of the CAR described herein, the antigen-binding domain of the CAR is linked to a transmembrane domain by a hinge region. In any of the nucleic acids encoding the transmembrane domain described herein, the transmembrane domain comprises a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.In any nucleic acid encoding an intracellular signaling domain of a CAR as described herein, the intracellular signaling domain comprises a co-stimulatory signaling domain, the co-stimulatory signaling domain comprises a functional signaling domain, the functional signaling domain comprises an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphoid activator molecule (SLAM protein), an activated NK cell receptor, BTLA, T Oll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta IL2R Gamma, IL7R Alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (C It is obtained from proteins selected from the group consisting of D244, 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, and CD19a.

[0044] In a preferred embodiment of the immunotherapy approach of the present invention, patient-derived T cells or NK cells are transduced, preferably by a retrovirus, to express an artificial immune receptor described herein, which consists of an extracellular antibody-derived antigen-recognition portion fused with a transmembrane portion followed by an intracellular signaling domain. Thus, the construct described herein provides transduced T cells or NK cells with antitumor cell-lysing ability.

[0045] For the first time, anti-CEA CAR-T cells enable the targeting of tumor cells in the tumor microenvironment, as demonstrated in the examples described herein. Surprisingly and unexpectedly to those skilled in the art, anti-CEA CAR-T cells preferentially recognize solid CEA bound to the tumor cell membrane rather than soluble CEA.

[0046] The tumor antigen-specific CAR-T cells described herein, such as anti-CEA CAR-T cells or CAR-NK cells, can be applied in preferred embodiments to the treatment of patients with solid tumors who are not eligible for other therapies. More specifically, embodiments of the present invention relate to the treatment of the following patient populations: i) Patients with multidrug resistance, ii) Patients who are not eligible for allogeneic stem cell transplantation, iii) Patients with complications who are not eligible for further chemotherapy, iv) Patients suffering from solid tumors and / or liquid tumors, v) Elderly patients who cannot tolerate chemotherapy, vi) Although it is a progressive disease, CAR is applicable as salvage therapy even after multiple other standard treatment plans have failed. vii) This is applicable even when the antigen density on target tumor cells is low and the antibody is likely to fail, and / or viii) This can be applied as monotherapy in cases where antibodies are not present. ix) This can be used in combination with one or more anticancer therapies, anticancer drugs, or transplants.

[0047] The anti-CEA CARs described herein impart high binding activity to T cells or NK cells, which is necessary for antitumor efficacy. The present invention exhibits unparalleled low off-target reactivity to other tissues.

[0048] As demonstrated in the following examples, in an in vitro co-culture system, anti-CEA CAR-T cells are activated by contact with CEA-expressing human tumor cell lines. These T cells then produce an effector phenotype with high levels of cytotoxic activity.

[0049] Furthermore, cytotoxic assays on selected target cell lines that are CEA-negative demonstrate that selective cytotoxicity is obtained only in CEA-positive cell lines.

[0050] Surprisingly, the combined expression of anti-CEA CAR, immunostimulatory cytokine IL-15, and PD-1 checkpoint inhibitors in Jurcut cells exhibits a synergistic effect (see Examples). While those skilled in the art may anticipate the additive effect of the combinations described herein, they would not have anticipated the synergistic effect shown in the Examples. This unexpected synergistic effect represents a special technical feature of the present invention.

[0051] Therefore, the CAR of the present invention represents a remarkable and beneficial approach to the treatment of the conditions described herein. The use of anti-CEACAR has never been previously attempted or described as a promising approach to the treatment of solid or liquid tumors, more preferably CEA-positive tumors. The minimal (if any) unwanted side effects, due to the selectivity of the markers, also represent a beneficial and remarkable aspect of the present invention. In particular, in patients who have developed resistance to other solid or liquid tumor treatments, the present invention represents a very promising approach to eradicating malignant tumors.

[0052] The cancers described herein refer to all types of cancer, neoplasms, or malignant tumors found in humans. In some embodiments, it is advantageous that the CAR constructs of the present invention can be used to treat solid tumors and / or liquid tumors, i.e., leukemia or lymphoma. In the current state of the art, CAR constructs can be used to treat either solid tumors or liquid tumors. In some embodiments, genetically modified cells are used as pharmaceuticals, in which case the disorder to be treated by the immune cells is selected from the group of cancers described herein, preferably consisting of breast cancer, pancreatic tumors, colon cancer, and acute myeloid leukemia.

[0053] In one embodiment, the extracellular antigen-binding domain of the CAR encoded by the first nucleic acid sequence region specifically recognizes CEA.

[0054] Examples of CEA-expressing cells are known to those skilled in the art and can be identified by further screening for cancer or other pathogenic cells. Cell lines expressing CEA are preferably MC-38, HaCaT, CAPAN-2, SCLC-21H, and / or cells isolated from the gastrointestinal tract.

[0055] Examples of CEA-recognizing domains suitable for use in CAR are further described herein. In one embodiment, the CEA-recognizing domain is provided in the sequence listing.

[0056] In one embodiment, the first nucleic acid sequence region encoding the CAR is constitutively expressed by at least a promoter or a promoter / enhancer combination, the promoter or promoter / enhancer is preferably selected from the group consisting of the spleen focus-forming virus (SFFV) promoter, the EF1 alpha promoter (e.g., the EF1 alpha S promoter), the PGK promoter, the CMV promoter, the SV40 promoter, the GAG ​​promoter, and the UBC promoter, more preferably the spleen focus-forming virus (SFFV) promoter.

[0057] In preferred embodiments, the promoter region includes a constitutive promoter region, such as the elongation factor 1-alpha (EF1a) promoter region described herein. The CAR is operably linked to the promoter region containing the constitutive promoter region. Due to the beneficial tumor antigen specificity of CAR-T cells or CAR-NK cells against tumors in the target body, it is preferable to use a constitutive promoter for the expression of one or more immune response-stimulating cytokines after systemic or local administration.

[0058] In one embodiment, the first nucleic acid sequence region encoding the CAR and the second nucleic acid sequence region encoding the checkpoint inhibitor molecule are configured to encode a polycistronic mRNA that includes coding regions for the polypeptide sequences of the CAR and the checkpoint inhibitor molecule, and an amino acid sequence containing a polypeptide cleavage site is positioned between the CAR polypeptide and the checkpoint inhibitor molecule polypeptide.

[0059] In some embodiments, polypeptide cleavage sites are selected from the group consisting of P2A, T2A, E2A, and F2A.

[0060] In one embodiment, the first nucleic acid sequence region encoding the CAR and the second nucleic acid sequence region encoding the checkpoint inhibitor molecule are configured to encode a polycistronic mRNA that includes coding regions for at least the polypeptide sequences of the CAR and the checkpoint inhibitor molecule, and an amino acid sequence containing a polypeptide cleavage site P2A is positioned between the CAR polypeptide and the checkpoint inhibitor molecule polypeptide.

[0061] In a preferred embodiment, the checkpoint inhibitor molecule encoded by the second nucleic acid sequence region is a dominant-negative polypeptide and / or antibody that inhibits and / or blocks an immune checkpoint protein, and this checkpoint inhibitor molecule is preferably a dominant-negative cleavage type PD1 polypeptide or a PD1 antibody.

[0062] Normally, potentially cancerous cells are destroyed by the immune system. All cancer cells undergo changes that distinguish them from surrounding cells, the most obvious of which is their ability to proliferate without inhibition. Cancer cells utilize mechanisms to evade normal immune system regulation. Checkpoint proteins have been shown to function by communicating with the immune system, thereby preventing potentially cancerous cells from being destroyed. While other molecules may exist that signal that a cell is cancerous, if there are enough checkpoint proteins on the cell surface, the immune system may overlook the cancer signal.

[0063] Ligand-receptor interactions, which have been studied as targets for cancer therapy, are the interactions between the transmembrane programmed cell death 1 protein (PD-1, also known as CD279) and its ligand, PD-1 ligand 1 (PD-L1). In normal physiological function, PD-L1 on the cell surface binds to PD-1 on the surface of immune cells, which inhibits the activity of immune cells. Upregulation of PD-L1 on the surface of cancer cells appears to allow cancer cells to evade the host immune system by inhibiting T cells, which T cells might otherwise be able to attack tumor cells. Antibodies that bind to either PD-1 or PD-L1 and thus block the interaction may allow T cells to attack tumors.

[0064] Checkpoint inhibitors (also known as immune checkpoint modulators, or CPMs) are designed to reduce the effectiveness of checkpoint proteins. While checkpoint inhibitors can have various mechanisms of action, when effective, they cause the immune system to recognize other molecules on the surface of cancer cells.

[0065] In a preferred embodiment, the medical use of genetically modified CAR-T cells or CAR-NK cells described herein is characterized by the gene transfer and expression of a checkpoint inhibitor, preferably a PD-L1 and / or PD-1 inhibitor, in combination with the CAR and immunostimulatory cytokine.

[0066] In a preferred embodiment, the checkpoint inhibitor molecule is a dominant-negative cleavage type of PD-1 (dnPD1opt). DnPD1opt binds to native PD-1 and therefore blocks the PD-1 protein.

[0067] In one embodiment, a third nucleic acid sequence region encoding an immunostimulatory cytokine (also referred to as an immune response stimulating cytokine) comprises nucleic acid sequences encoding one or more immune response stimulating cytokines operatively linked to one or more promoters, wherein at least one of the cytokines is selected from the group consisting of IL-15, IL-15RA, IL-2, IL-7, IL-12, IL-21, IFN-gamma, and IFN-beta. Cytokines that stimulate immune responses are known to those skilled in the art and can be evaluated and / or identified by established, commonly used assays.

[0068] In some embodiments, the present invention encompasses combinations of cytokines, checkpoint inhibitor molecules, and tumor-specific CAR transgenes in cells as described herein, and in particular encompasses any given specific combination of the cytokines disclosed herein, preferably any given specific combination of one or more of IL-15, IL-15RA, IL-2, IL-7, IL-12, IL-21, IFN-gamma, IFN-beta, and CD28.

[0069] A tumor treatment method comprising the ex vivo production of CAR-T cells or CAR-NK cells as described herein, defined by at least one immunostimulatory cytokine, and allogeneic CAR-T cell or CAR-NK cell constructs as described herein, and the transfer of allogeneic CAR-T cell or CAR-NK cell products into a patient, preferably in combination with other antitumor immunotherapies, leads to the remarkable and beneficial concept of stimulating the local immune system in an antitumor immune response, either by the innate immune system or by combination immunotherapy. It was unexpected that CAR-T cells encoding transgenic immunostimulatory cytokines, as shown in the examples, could be used as effective antitumor adjuvants in stimulating the antitumor response. However, boosting the local antitumor immune response using CAR-T cells or CAR-NK cells further encoding transgenic immunostimulatory cytokines and checkpoint inhibitor molecules is a special technical feature of the present invention.

[0070] In a preferred embodiment, the genetically modified mesenchymal stem cells described herein are characterized in that the immune response stimulating cytokine is IL-15.

[0071] Interleukin-15 (IL-15) is a cytokine that has structural similarity to IL-2. Like IL-2, IL-15 binds and signals through a complex composed of the IL-2 / IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). IL-15 induces the proliferation of natural killer cells. Natural killer cells are cells of the innate immune system, and their main role is to kill virus-infected or cancerous cells. In preclinical models, IL-15 is associated with CD8 + It has been shown to enhance T-cell anti-tumor immunity (Klebanoff CA, et al. Proc. Natl. Acad. Sci. USA 101 (7): 1969-74).

[0072] The present invention enables remarkable and advantageous antitumor effects through the expression of immunostimulatory cytokines in CAR-T cells as described herein. The expression of immunostimulatory cytokines by CAR-T cells as described herein supports an antitumor immune response, leading to a reduction in tumor size and / or growth, and demonstrably reducing and / or avoiding the side effects that occur with systemic administration of such cytokines known in the art. Side effects, such as nausea and vomiting, pain of the mouth or tongue, diarrhea, drowsiness, allergic reactions, fever or chills, urticaria, itching, headache, cough, shortness of breath, or swelling of the face, tongue, or throat, may be avoided by therapies utilizing CAR-T cells or CAR-NK cells as described herein.

[0073] Accordingly, the present invention provides means for reducing the side effects of cytokine therapy and a combination of cytokines and immunotherapy, which are preferably achieved by systemic administration of cells, but are exerted in a tissue-specific manner through cell therapy using CAR-T cells or CAR-NK cells expressing the above cytokines and checkpoint inhibitors under appropriate tissue-specific conditions.

[0074] Accordingly, the present invention relates to genetically modified cells used as pharmaceuticals as described herein, wherein the exogenous nucleic acid comprises a region encoding one or more immunostimulatory cytokines operatively linked to one or more promoters or promoter / enhancer combinations, wherein the cytokines are selected from the group consisting of IL-15, IL-7, IL-12, IL-2, IL-21, IFN-gamma, and IFN-beta.

[0075] The present invention also relates to genetically modified cells containing exogenous nucleic acid molecules, which include a region encoding a CAR, an immunostimulatory molecule (which may include more) that induces T cell proliferation and / or differentiation (and / or maturation into memory cells and avoidance of tumor-mediated immunosuppression), and a checkpoint inhibitor molecule that inhibits the immunosuppressive environment, both of which are operably linked to a promoter or promoter / enhancer combination.

[0076] The immunostimulatory molecules that induce T cell proliferation and / or differentiation may be cytokines as described herein, or a combination of cytokines and checkpoint inhibitor molecules. A combination may be preferable to ensure that solid or liquid tumor cells are appropriately attracted by the cytokines and directed toward a memory phenotype.

[0077] Surprisingly, CAR-T cells modified with the gene-transfected immunostimulatory cytokines and checkpoint inhibitor molecules described herein exhibit unexpectedly favorable expression and secretion of the cytokines and checkpoint inhibitors. Those skilled in the art could not have foreseen that these specific cytokines and checkpoint inhibitors could be expressed and effluxed from cells in sufficient quantities to induce or enhance a desired local immune response, either through innate response or immunotherapy.

[0078] In one preferred embodiment, a third nucleic acid sequence region encoding an immunostimulatory cytokine is operably linked to one or more constitutive promoters, preferably human promoters, and more preferably NFAT promoters.

[0079] In one preferred embodiment, the human promoter is an NF-κB promoter.

[0080] The use of "tumor-specific" promoters, i.e., promoters that are preferentially expressed or induced under inflammatory or "cancer-like" conditions, can be synergistically effective in combination with CAR-T cell or CAR-NK cell recruitment signals in reducing unwanted systemic effects. The CAR-T cells or CAR-NK cells of the present invention migrate toward inflammatory tissue, particularly tumor tissue, thereby providing an effective means of avoiding systemic expression of encoded cytokines or checkpoint inhibitors in the patient's body. The use of promoters for cytokine expression that are preferentially expressed under inflammatory conditions or are present in tumor tissue further enhances the reduction of systemic expression in a synergistic manner, thereby bringing about the remarkable benefits of the T-cell or NK-cell-based administration modes of cytokines described herein.

[0081] In one preferred embodiment, the genetically modified cells described herein comprise a recombinant nucleic acid expression construct, which further comprises one or more nucleic acid sequences encoding an immune response stimulating cytokine described herein, wherein the immune response stimulating cytokine is as follows: (a) A signal sequence, preferably the sequence described in Sequence ID No. 29, or a sequence having at least 80% sequence identity with Sequence ID No. 29, (b) N-terminal IL15RA polypeptide, preferably the sequence described in SEQ ID NO: 30, or a sequence having at least 80% sequence identity with SEQ ID NO: 30, (c) A linking loop sequence, preferably the sequence described in sequence number 31, or a sequence having at least 80% sequence identity with sequence number 31, (d) IL-15 polypeptide, preferably the sequence described in SEQ ID NO: 32, or a sequence having at least 80% sequence identity with SEQ ID NO: 32, Includes.

[0082] In any of the nucleic acids described herein, the promoter region includes a nucleotide sequence that induces the expression of (a) upon activation of immune effector cells. In one embodiment, the constitutive promoter region includes a promoter of a gene that is induced upon activation of immune effector cells. In one embodiment, the constitutive promoter region includes an activated T cell nuclear factor (NFAT) promoter, an NF-κB promoter, an IL-15 promoter, or an IL-15 receptor (IL-15R) promoter. In one embodiment, the activation-conditional regulatory region includes one or more binding sites for a transcription modulator, such as a transcription factor that induces gene expression upon activation of immune effector cells. In one embodiment, the activation-conditional promoter region includes one or more NFAT binding sites.

[0083] The immune response-stimulating cytokines or portions thereof described herein also include sequences having at least 70%, 80%, preferably 90%, or 95% sequence identity with humanized sequences explicitly disclosed or disclosed through sequence formulas.

[0084] In preferred embodiments, sequence variants having 70% or more sequence identity with the immune response stimulating cytokine sequences listed herein maintain IL-15 agonist activity and possess essentially the same or similar functional properties as the immune response stimuli that bind to the specific sequences referred to herein; that is, the PD-1 binding is essentially the same or similar with respect to affinity, specificity, and mode of binding.

[0085] In one preferred embodiment, the recombinant nucleic acid expression construct encoding the CAR is as follows: A CAR signal sequence, preferably the sequence described in SEQ ID NO: 14, or a sequence having at least 80% sequence identity with SEQ ID NO: 14, An antigen-binding domain of a CAR that specifically recognizes CEA, preferably the sequence described in SEQ ID NO: 15 and SEQ ID NO: 19, or a sequence having at least 80% sequence identity with SEQ ID NOs: 15 and 19, The extracellular constant region of the immunoglobulin heavy chain of CAR, preferably the sequence described in SEQ ID NO: 23, or a sequence having at least 80% sequence identity with SEQ ID NO: 23, A CD28 signaling domain, preferably the sequence described in SEQ ID NO: 24, or a sequence having at least 80% sequence identity with SEQ ID NO: 24, wherein the CD28 signaling domain includes a transmembrane domain, preferably the sequence described in SEQ ID NO: 25, or a sequence having at least 80% sequence identity with SEQ ID NO: 25, A CD3 zeta signaling domain, preferably the sequence described in SEQ ID NO: 26, or a sequence having at least 80% sequence identity with SEQ ID NO: 26, Includes.

[0086] In a preferred embodiment, sequence variants having 70% or more sequence identity with the specific CAR sequences of SEQ ID NOs. 15 to 23 maintain CEA recognition and have essentially the same or similar functional properties as the VH and VL domains having the specific CAR sequences of SEQ ID NOs. 15 to 22; that is, the CEA recognition is essentially the same or similar with respect to affinity, specificity, and epitope binding mode.

[0087] Furthermore, the order of the light chain and heavy chain fragments can be reversed according to the desired stereochemistry of the antigen-binding fragment.

[0088] Furthermore, in some embodiments, the linker arrangement between the heavy and light chains can be modified using standard techniques.

[0089] Furthermore, the nucleic acid sequences encoding CAR have been codon-optimized to improve CAR expression. These modifications allow for sufficient surface expression on T cells or NK cells while maintaining appropriate antigen binding or recognition. High affinity and high binding activity enable CAR-T cells and CAR-NK cells to i) recognize tumor target cells with high, intermediate, or low CEA surface expression, and ii) be activated against such cells and kill them.

[0090] The anti-CEA CAR-T cell products and anti-CEA CAR-NK cell products described herein are characterized by unique properties.

[0091] The anti-CEA CARs described herein possess high affinity and confer high specificity and binding activity to T cells and / or NK cells. These properties enable CAR-T cells or CAR-NK cells to i) recognize, ii) be activated against, and iii) kill tumor target cells with high and low CEA surface expression.

[0092] The number of CEA antigens expressed on the surface of tumor cells can be quantified by using a fluorescently coupled anti-CEA antibody in combination with Quantibrite beads (manufactured by BD). A preferred method for quantifying CEA antigens expressed on the surface of tumor cells is "fluorescence-activated cell sorting / cell analysis" (FACS). The fluorescence intensity of the beads correlates precisely with the number of fluorescent antibodies bound to the cells, and this is a measure of the number of CEA molecules on the cells.

[0093] The VH and VL fragments described herein can be arranged in multiple configurations within the CAR while still maintaining high specificity and affinity for the target epitope. In some embodiments, the CAR can be configured in a VH-VL configuration or a VL-VH configuration, with diversity in linkers, hinges, transmembrane domains, co-stimulatory domains, and / or activating domains, while still maintaining its effectiveness. This remarkable feature of the present invention provides greater flexibility in the design of CEA-directed CARs, thereby allowing for further modification and / or optimization of the CAR structure based on the VH and VL domains described herein if any further development is needed or desired.

[0094] The CARs or portions thereof described herein also include sequences having at least 70%, 80%, preferably 90%, sequence identity with humanized sequences that are explicitly disclosed or disclosed through sequence formulas.

[0095] In preferred embodiments, sequence variants having 70% or more sequence identity with specific VH and VL sequences listed herein maintain CEA recognition and have essentially the same or similar functional properties as the VH and VL domains having the specific sequences referred to herein; that is, the CEA recognition is essentially the same or similar with respect to affinity, specificity, and epitope binding mode.

[0096] In further embodiments, the present invention relates to a chimeric antigen receptor (CAR) polypeptide comprising one or more linker domains, spacer domains, transmembrane domains, and signal transduction domains. In one embodiment, the CAR comprises an intracellular domain, which comprises a co-stimulatory domain and a signal transduction (activation) domain.

[0097] The exchange of signaling domains addresses the requirement for either a potent and rapid effector phase (CD28 co-stimulatory domain) or long-term relapse control as ensured by the T cell memory population (4-1BB signaling domain). As demonstrated herein, various signaling domains can be exchanged in multiple configurations, thereby providing CARs with design flexibility without losing favorable binding properties.

[0098] Thanks to variants (by adding alternative components) adopted as linker domains, spacer domains, transmembrane domains, and intracellular domains, it becomes clear that various components can be replaced according to the requirements of those skilled in the art, while maintaining CEA recognition properties, and thus maintaining the desired biological effect.

[0099] In some embodiments, particularly in the context of immune cells lacking CD3 zeta expression, the absence of CD3 zeta is possible.

[0100] In one preferred embodiment, a genetically modified cell comprising a recombinant nucleic acid expression construct further comprises one or more nucleic acid sequences encoding a checkpoint inhibitor molecule described in any one of the prior claims, wherein the checkpoint inhibitor molecule includes: A dominant-negative cleavage type checkpoint protein, preferably the dominant-negative cleavage type PD1 described in SEQ ID NO: 28, or a sequence having at least 80% sequence identity with SEQ ID NO: 28. The above checkpoint protein is located adjacent to the polypeptide cleavage site that cleaves the checkpoint inhibitor molecule from the CAR polypeptide, and the cleavage site is preferably selected from the group consisting of P2A, T2A, E2A, and F2A.

[0101] In a preferred embodiment, the recombinant nucleic acid expression construct further comprises one or more nucleic acid sequences encoding checkpoint inhibitor molecules described herein, wherein the checkpoint inhibitor molecules are A dominant-negative cleavage type PD1 as described in Sequence ID No. 28, or a sequence having at least 80% sequence identity with Sequence ID No. 28, Includes, The above checkpoint protein is located adjacent to the polypeptide cleavage site P2A, which cleaves the checkpoint inhibitor molecule from the CAR polypeptide.

[0102] In a preferred embodiment, the recombinant nucleic acid expression construct includes a nucleic acid sequence region encoding the following: CARs that specifically recognize human CEA, Checkpoint inhibitor molecule, dominant-negative cleavage type PD1 polypeptide, An immunostimulatory cytokine comprising a signal sequence, an N-terminal IL15RA polypeptide, a linking loop sequence, and an IL-15 polypeptide, wherein the immunostimulatory cytokine is operatively linked to one or more promoters, and Polypeptide cleavage site P2A.

[0103] In one embodiment, the immunostimulatory cytokine includes an immunostimulatory cytokine described herein, which comprises an IL-15 peptide and / or an IL-15RA peptide, a signal sequence, and a linking loop sequence. Preferred sequences are disclosed herein.

[0104] In one embodiment, the immunostimulatory cytokine comprises an immunostimulatory cytokine described herein, which comprises an IL-15 peptide and / or an IL-15RA peptide. Preferred sequences are disclosed herein.

[0105] Normally, potentially cancerous cells are destroyed by the immune system. All cancer cells undergo changes that distinguish them from surrounding cells, the most obvious of which is their ability to proliferate without inhibition. Cancer cells utilize mechanisms to evade normal immune system regulation. Checkpoint proteins have been shown to function by communicating with the immune system, thereby preventing potentially cancerous cells from being destroyed. While other molecules may exist that signal that a cell is cancerous, if there are enough checkpoint proteins on the cell surface, the immune system may overlook the cancer signal.

[0106] The checkpoint inhibitor molecules or portions thereof described herein also include sequences having at least 70%, 80%, preferably 90%, sequence identity with humanized sequences that are explicitly disclosed or disclosed through sequence formulas.

[0107] In preferred embodiments, sequence variants having 70% or more sequence identity with dominant-negative cleavage PD1 sequences listed herein maintain PD-1 binding and possess essentially the same or similar functional properties as PD-1 proteins that bind to specific sequences referred to herein; that is, the PD-1 binding is essentially the same or similar with respect to affinity, specificity, and mode of binding.

[0108] Ligand-receptor interactions, which have been studied as targets for cancer therapy, are the interactions between transmembrane programmed cell death 1 protein (PD-1, also known as CD279) and its ligand, PD-1 ligand 1 (PD-L1). In normal physiological function, PD-L1 on the cell surface binds to PD-1 on the surface of immune cells, thereby inhibiting the activity of immune cells. Upregulation of PD-L1 on the surface of cancer cells appears to allow cancer cells to evade the host immune system by inhibiting T cells, which T cells might otherwise be able to attack tumor cells. Antibodies that block the interaction by binding to either PD-1 or PD-L1 may allow T cells to attack tumors. In a preferred embodiment, the checkpoint inhibitor molecule is a dominant-negative cleavage type PD-1 polypeptide that blocks PD-1 by binding to it.

[0109] Checkpoint inhibitors (also known as immune checkpoint modulators, or CPMs) are designed to reduce the effectiveness of checkpoint proteins. While checkpoint inhibitors may have various mechanisms of action, when effective, they allow the immune system to recognize other molecules on the surface of cancer cells.

[0110] When CAR-T cells expressing immunostimulatory cytokines that induce T cell proliferation and / or differentiation are administered in combination with checkpoint inhibitors, a synergistic effect is achieved regarding the desired anticancer effect. The cytokines or other immunostimulatory factors lead to a localized improvement in the T cell response against cancerous tissue, while the checkpoint inhibitors also enable T cells to more effectively attack and destroy cancerous tissue. The effects of these two agents combine in a synergistic manner, resulting in a greater technical effect than the sum of the effects of each agent considered individually.

[0111] Regarding the use of "ready-to-deliver" allogeneic or autologous CAR-T cells or CAR-NK cells, preferably CAR-NK cells, the inventors have developed a method for manipulating CEA-expressing CAR-T cells or CAR-NK cells that have lower allogeneity than conventional CAR-expressing cells.

[0112] In a preferred embodiment, the genetically modified cells are of the same type with respect to the patient to whom the cells are delivered.

[0113] In a preferred embodiment, the genetically modified cells are homogeneous T cells, NK cells, or Treg cells with respect to the patient to whom the cells are delivered.

[0114] In a preferred embodiment, the genetically modified cells are autologous cells with respect to the patient to whom the cells are delivered.

[0115] In a preferred embodiment, the genetically modified cells are autologous NK cells with respect to the patient to whom the cells are delivered.

[0116] In another embodiment, CAR-T cells or CAR-NK cells further include one or more immunosuppressive defeating proteins and / or inducible suicide genes that induce the death of those cells, enabling the selective destruction of CAR-T cells or CAR-NK cells. In some cases, since the manipulated T cells can propagate and persist for many years after administration, it may be desirable to provide a safety mechanism that enables the selective removal of administered T cells. Therefore, in some embodiments of the present invention, the method may include transforming T cells with recombinant suicide genes. These recombinant suicide genes are used to reduce the risk of direct toxicity and / or uncontrolled proliferation of such T cells after administration to a target. Suicide genes enable the selective removal of transformed cells in vivo. In more detail, suicide genes have the ability to convert a non-toxic prodrug into a cytotoxic drug or to express a toxic gene expression product. In other words, a “suicide gene” is preferably a nucleic acid encoding a product that causes cell death, either by itself or in the presence of other compounds. In one embodiment, the suicide gene is herpes simplex virus thymidine kinase.

[0117] In one preferred embodiment, the chimeric antigen receptor (CAR) preferably recognizes membrane-bound CEA rather than soluble CEA.

[0118] CEA proteins exist in mammals, preferably humans, in both soluble and solid forms. While only the solid form is found in tumor cell membranes, the soluble form plays a role in endothelial cell activation and angiogenesis. Many researchers have attempted to create CARs with antigen-binding domains that recognize only CEA bound to tumor cell membranes, but have so far been unsuccessful. Those skilled in the art would not have anticipated that this particular anti-CEA CAR would preferably target CEA on the tumor membrane while missing soluble CEA. Furthermore, those skilled in the art would not have anticipated that this particular anti-CEA CAR, when combined with immunostimulatory cytokines and / or checkpoint inhibitor molecules, could be expressed in sufficient quantities, released from cells in sufficient quantities, and, based on immunotherapy, induce or enhance a desired local immune response, targeting only CEA-positive pathogenic cells in solid and liquid tumors, preferably solid tumor cells. The development of the anti-CEA CARs described herein, which distinguish between these two types of CEA, represents a uniquely technically complex solution of the present invention.

[0119] In one embodiment, the cells are immune cells, preferably selected from the group consisting of induced pluripotent stem cells (iPSCs), preferably iPSC strain ND50039, immortalized immune cells including NK-92 cells and YT cells, primary immune cells including natural killer (NK) cells, cytokine-induced killer cells (CIK), and T lymphocytes, wherein the T lymphocytes are preferably CD4 or CD8 T cells, more preferably cytotoxic T lymphocytes, helper T cells, or tumor-infiltrating lymphocytes (TILs).

[0120] A further aspect of the present invention relates to genetically modified cells comprising recombinant nucleic acid expression constructs or CARs as described herein, wherein the cells are iPSC strain ND50039.

[0121] Immune cells are preferably T cells, CD4 + T cells, CD8 +Selected from T cells, B cells, dendritic cells, granulocytes, innate lymphoid cells (ILCs), megakaryocytes, monocytes / macrophages, natural killer (NK) cells, NK-92, YT cells, MCF-7, Jurcut cells, MC32A cells, HEK293 cells, platelets, red blood cells (RBCs), and / or thymocytes.

[0122] Adoptive cell transfer attacks cancer cells using a T-cell-based cytotoxic response. T cells that are naturally or genetically engineered to be reactive to the patient's cancer are generated in vitro and then returned to the cancer patient. Autologous tumor-infiltrating lymphocytes have been used as an effective treatment for patients with metastatic melanoma. This is achievable by collecting T cells found with the patient's tumor and training them to attack cancer cells. These T cells can be called tumor-infiltrating lymphocytes (TILs). Such T cells can be stimulated and proliferated in vitro using high concentrations of IL-2, anti-CD3, and alloreactive feeder cells. Traditionally, these T cells are then returned to the patient, and IL-2 is administered externally to further enhance their anticancer activity.

[0123] Accordingly, the present invention encompasses adoptive cell transfer in combination with the administration of CAR-T cells as described herein. The present invention encompasses the fact that by administering genetically modified T cells prior to immune cells (e.g., CAR-T cells), appropriate chemokines are expressed, thereby enhancing the chemotaxis of CAR-T and other immune effector cells administered during adoptive cell transfer. The expression of stimulating cytokines enhances T cell activation locally, preferably within or near the tumor, subsequently inducing a memory effector cell phenotype, thereby extending the therapeutic effect of the treatment.

[0124] The present invention further includes adoptive cell transfer in combination with the administration of CAR-T cells as described herein. The present invention encompasses the fact that the administration of genetically modified CAR-NK cells induces the lysis of tumor-transformed cells in a manner independent of major histocompatibility class I or II. NK cells can directly lyse tumor-transformed cells and can also enhance tumor recognition and destruction by adaptive immune cells by acting as a bridge between innate and adaptive immune responses. Unlike the mechanism by which T cells lyse tumor cells, which requires the recognition of tumor antigens presented in major histocompatibility class I or II contexts by specific T cell receptors, NK cells can kill tumor cells without prior sensitization to tumor antigens. NK cells act as the first line of defense against novel transformed cells. NK cells kill tumor targets through receptor-mediated cytotoxicity. This process depends on the presence of tumor-specific antibodies bound to tumor surface antigens.

[0125] This characteristic of CAR-NK cell products makes it possible to reduce the risk of graft-versus-host disease in allogeneic cell settings. CAR-NK cell products can preferably be used as allogeneic, "ready-to-ship" products. In the significance of the present invention, CAR constructs lacking antigen-binding domains are particularly suitable as platform technologies that allow for flexible exchange of antigen-recognition regions.

[0126] However, cells can be obtained from subjects other than the patient of interest and are therefore considered homogeneous. As used herein, cells or their progenitor cells are "homogeneous" with respect to the subject if they originate from another subject of the same species. As used herein, cells or their progenitor cells are "autologous" with respect to the subject if they originate from the same subject. In preferred embodiments, immune cells are autologous to the subject of medical treatment.

[0127] In a preferred embodiment, genetically modified immune cells comprising and / or expressing a nucleic acid molecule or vector described herein are CD4+ T cells and / or CD8 + T cells, preferably CD4 + T cells and CD8 + characterized by being a mixture with T cells. Such a T cell population, preferably CD4 + transformed cells and CD8 + Compositions containing both transformed cells exhibit particularly effective cytolytic activity against various solid tumors and liquid tumors, such as colorectal cancer, preferably against those cells and / or the related pathological conditions described herein.

[0128] In a preferred embodiment, the genetically modified immune cells containing the nucleic acid molecules or vectors described herein and / or expressing the CARs described herein are CD4 + T cells and CD8 + T cells, preferably in a ratio of 1:10 to 10:1, more preferably 5:1 to 1:5, 2:1 to 1:2 or 1:1. The above ratio, preferably a 1:1 CD4 + / CD8 + Administration of CEA-directed modified CAR-T cells expressing the CARs described herein in a ratio of 1:1 results in beneficial features during the treatment of the diseases mentioned herein, for example, such a ratio leads to improved treatment response and reduced toxicity.

[0129] A further surprising aspect of the present invention is the improved stability of the CARs disclosed herein. The CAR polypeptide can be easily stored for a long time under appropriate conditions without any loss of binding affinity.

[0130] In one embodiment, the immune cells are derived from human peripheral blood, human umbilical cord blood, or induced pluripotent stem cells (iPSCs).

[0131] In one embodiment, the immune cells are derived from the iPS cell line ND50039.

[0132] In one embodiment, the genetically modified immune cells are derived from iPSCs that have been genetically modified with recombinant nucleic acid constructs described herein before differentiating into immune cells.

[0133] In one embodiment, the genetically modified immune cells are derived from iPSC cells or iPSC strain ND50039 that have been genetically modified by electroporation or chemical transduction using the recombinant nucleic acid expression construct described herein, and these cells are as follows: (a) A CAR described herein that specifically recognizes human CEA, (b) Checkpoint inhibitor molecules dominant-negative cleavage type PD1 polypeptide as described herein, (c) an immunostimulatory cytokine as described herein, comprising IL15 peptide and / or IL-15RA peptide, a signal sequence, and a linking loop sequence, (d) polypeptide cleavage site P2A, Includes, (e) The above-mentioned genetically modified iPSC strain ND50039 or genetically modified iPS cells differentiate into NK cells or T cells.

[0134] Transplantation of autologous or allogeneic cells to which the CAR transgenes described herein have been added is feasible. For example, when reintroduced into a patient after autologous cell transplantation, T cells modified with the CARs of the present invention as described herein can recognize and kill tumor cells. CIK cells can have enhanced cytotoxic activity compared to other T cells and therefore become immune cells of preferred embodiments of the present invention. As will be obvious to those skilled in the art, other cells can also be used as immune effector cells possessing the CARs described herein. In particular, immune effector cells also include NK cells, NK T cells, neutrophils, and macrophages. Immune effector cells also include precursor cells of effector cells, in which case such precursor cells can be induced to differentiate into CAR-T effector cells in vivo or in vitro. The precursor cells can be iPS cells that become immune effector cells under defined culture conditions.

[0135] In this embodiment, the precursor iPS cell line ND50039 is cultured under defined culture conditions to become immunoeffector cells.

[0136] In one embodiment, the immune response stimulating cytokine maintains or enhances the activity, survival, and / or number of immune cells within and / or near the tumor tissue.

[0137] The present invention encompasses adoptive cell transfer in combination with the administration of CAR-T cells or CAR-NK cells as described herein. The present invention includes the fact that administering genetically modified CAR-T cells or CAR-NK cells prior to immune cells (e.g., CAR-T cells) enhances the chemotaxis of CAR-T and other immune effector cells administered during adoptive cell transfer by expressing appropriate chemokines. The expression of stimulating cytokines enhances T cell activation locally, preferably within or near the tumor, subsequently inducing a memory effector cell phenotype, thereby extending the therapeutic effect of the treatment.

[0138] In one embodiment, the genetically modified cells are to be used as a pharmaceutical in the treatment of medical disorders associated with the presence of pathogenic cells expressing CEA, preferably cancer cells, more preferably cancer cells of solid and / or liquid malignant tumors, preferably solid tumors, more preferably cancer cells of colon cancer, rectal cancer, lung cancer, breast cancer, liver cancer, pancreatic cancer, gastric cancer, and ovarian cancer, more preferably CEA-positive metastatic tumor cells.

[0139] Therapy using genetically modified cells according to the present invention can be combined with one or more anticancer therapies or pharmaceuticals in some embodiments, and such anticancer therapies or pharmaceuticals are preferably selected from the group consisting of antibody therapy, vaccines, oncolytic virus therapy, chemotherapy, radiotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser phototherapy, immunosuppression, or transplantation.

[0140] A further aspect of the present invention relates to a chimeric antigen receptor (CAR) polypeptide encoded by a recombinant nucleic acid expression construct.

[0141] In preferred embodiments, the CAR polypeptide is encoded by a recombinant nucleic acid expression construct described herein, the construct comprising a CAR signaling sequence, an antigen-binding domain of CAR that specifically recognizes CEA, an extracellular constant region of the immunoglobulin heavy chain of CAR, a CD28 signaling domain, and a CD3 zeta signaling domain.

[0142] In preferred embodiments, the CD3 zeta signaling domain may be absent from the CAR polypeptide, particularly in the context of immune cells lacking CD3 zeta expression.

[0143] In a preferred embodiment, the recombinant nucleic acid expression construct encoding the CAR further comprises a polypeptide, the polypeptide being: An immunostimulatory cytokine IL-15 polypeptide comprising a signal sequence, an N-terminal IL-15RA polypeptide, a linking loop sequence, and an IL-15 polypeptide, A checkpoint inhibitor molecule dominant-negative cleavage type PD1 polypeptide located adjacent to the polypeptide cleavage site P2A for cleaving checkpoint inhibitor molecules from a CAR polypeptide, Includes.

[0144] A further aspect of the present invention is a recombinant nucleic acid expression construct encoding a chimeric antigen receptor (CAR), wherein the construct is as follows: (a) A first nucleic acid sequence region encoding a chimeric antigen receptor (CAR), wherein the CAR includes a first nucleic acid sequence region comprising an extracellular antigen-binding domain that recognizes carcinoembryonic antigen (CEA) protein, (b) A second nucleic acid sequence region encoding a checkpoint inhibitor molecule, (c) A third nucleic acid sequence region encoding an immunostimulatory cytokine, This relates to recombinant nucleic acid expression constructs, including [specific examples].

[0145] In one embodiment, a recombinant nucleic acid expression construct is, or may be, referred to as a nucleic acid construct. In some embodiments, the construct may be provided with or without a promoter. Those skilled in the art can identify a suitable promoter and / or generate a construct with a suitable promoter, depending on the intended use.

[0146] In a preferred embodiment, the recombinant nucleic acid expression construct is as follows: A first nucleic acid sequence region encoding a chimeric antigen receptor (CAR) as described herein, wherein the CAR comprises a first nucleic acid sequence region including an extracellular antigen-binding domain that recognizes a carcinoembryonic antigen (CEA) protein, A second nucleic acid sequence region encoding a checkpoint inhibitor molecule as described herein, wherein the checkpoint inhibitor molecule comprises a second nucleic acid sequence region located adjacent to a polypeptide cleavage site as described herein, The third nucleic acid sequence region encoding an immunostimulatory cytokine as described herein, Includes.

[0147] In a preferred embodiment, the recombinant nucleic acid expression construct is as follows: A first nucleic acid sequence region encoding a chimeric antigen receptor (CAR as described herein), wherein the CAR includes an extracellular antigen-binding domain that recognizes carcinoembryonic antigen (CEA) protein, A checkpoint inhibitor molecule described herein comprises a second nucleic acid sequence region encoding a dominant-negative cleavage type PD1 polypeptide, wherein the checkpoint inhibitor molecule is located adjacent to the polypeptide cleavage site P2A, and A third nucleic acid sequence region encoding an immunostimulatory cytokine as described herein, comprising IL-15RA peptide and / or IL-15 peptide, a signal sequence, and a linking loop sequence, wherein the immunostimulatory cytokine is operably linked to one or more promoters as described herein, Includes.

[0148] In one embodiment, the immunostimulatory cytokine includes an immunostimulatory cytokine described herein, which comprises an IL-15 peptide and / or an IL-15RA peptide, a signal sequence, and a linking loop sequence. Preferred sequences are disclosed herein.

[0149] In one embodiment, the immunostimulatory cytokine comprises an immunostimulatory cytokine described herein, which comprises an IL-15 peptide and / or an IL-15RA peptide. Preferred sequences are disclosed herein.

[0150] In one embodiment, the recombinant nucleic acid expression construct described herein can be administered to human cells. Accordingly, the present invention relates to a recombinant nucleic acid expression construct comprising a first nucleic acid sequence region encoding a chimeric antigen receptor (CAR) and corresponding immune cells expressing the construct, preferably CAR-T cells or CAR-NK cells, which confer to human T cells or NK cells high cytotoxic activity against well-defined tumors, solid tumors, or liquid tumors, while overlooking non-pathogenic cells in the surrounding tissue, such as pancreatic cells, lung cells, colon cells, or hepatocytes.

[0151] The present invention also includes expressing a combination of immune-activating cytokines and / or checkpoint inhibitor molecules in tumors via CAR-T cells or CAR-NK cells as described herein, for the purpose of attracting immune effector and helper cells, inducing immune activation, promoting the maturation of memory immune cells, and / or suppressing the emergence and persistence of suppressive and / or regulatory immune cells.

[0152] In one embodiment, the present invention provides allogeneic anti-CEA CAR-expressing T cells or NK cells expressing two or more immunostimulatory and / or immunosuppressive suicide genes and / or inducible suicide genes that enable the destruction of the cells. Suicide genes include, but are not limited to, thymidine kinases of alphaherpesviruses (HHV1-3), bacterial gene cytosine deaminases (which can convert 5-fluorocytosine to the highly toxic compound 5-fluorouracil), and inducible caspase-9 or caspase-8. Inducible caspase-9 can be activated by a special dimerizing chemoinducer (CID). Suicide genes can also be polypeptides that are expressed on the cell surface and can make the cells sensitive to therapeutic monoclonal antibodies. Suicide gene expression can be induced, for example, by doxycycline adapted for human cells.

[0153] In preferred embodiments, the expression system is preferably in the form of a vector, such as a viral vector, plasmid, or transposon vector, preferably a sleeping beauty vector, which can achieve exceptionally high transduction rates of human T cells. Transduction systems vary depending on the modular design of the CAR construct, i.e., lentiviruses, adeno-associated virus vectors, and transposons are available depending on the requirements and priorities of those skilled in the art when implementing the present invention. In preferred embodiments, the adeno-associated virus vector or lentivirus vector of the present invention is used for gene transfer into cells, preferably proliferating immune cells and dormant immune cells, and for gene therapy applications.

[0154] In a further embodiment of the present invention, the present invention relates to isolated nucleic acid molecules, preferably in the form of vectors, such as viral vectors or transposon vectors, preferably in the form of sleeping beauty vectors, wherein the nucleic acid molecules are selected from the group consisting of: a) Nucleic acid molecules containing the following nucleotide sequences, Encoding a chimeric antigen receptor (CAR) polypeptide according to any embodiment of the CAR described herein. Encodings of extracellular antigen-binding domains, transmembrane domains, and intracellular domains. However, the extracellular antigen-binding domain is encoded by at least one sequence from SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 13, and / or b) Nucleic acid molecules containing the following nucleotide sequences, Encoding a checkpoint inhibitor molecule according to any embodiment of the CAR described herein, wherein the extracellular checkpoint inhibitor molecule is encoded by at least one sequence of SEQ ID NO: 7, SEQ ID NO: 13, and / or Sequences encoding immunostimulatory cytokines, wherein immunostimulatory cytokines are encoded by at least one sequence from SEQ ID NOs. 10 to 12, or SEQ ID NO. 13. c) Nucleic acid molecules that are complementary to nucleotide sequences according to a) and b); d) A nucleic acid molecule comprising a nucleotide sequence having sufficient sequence identity to be functionally similar / equivalent to a nucleotide sequence according to a) or b) or c), preferably having at least 70% sequence identity to a nucleotide sequence according to a) or b) or c); e) Nucleic acid molecules that, as a result of the genetic code, degenerate into nucleotide sequences following a) to d); and / or A nucleic acid molecule that follows the nucleotide sequence of f)a)~e), which has been modified by deletion, addition, substitution, rearrangement, inversion, and / or insertion, and is functionally similar / equivalent to the nucleotide sequence following a)~e).

[0155] The term "degenerate" (or "degenerate into") refers to a situation where the nucleotide sequences of nucleic acid molecules differ, but according to the genetic code, this does not result in any difference in the amino acid protein products of the translated nucleotide sequences.

[0156] The present invention further relates to a method for generating genetically modified cells, the method comprising delivering or transferring a nucleic acid construct encoding a CAR as described herein, the method can be used in conjunction with one or more gene transfer techniques, such as lentiviral vectors, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors, chemical translocation, electroporation, and mRNA translocation, preferably adeno-associated virus vectors.

[0157] Adeno-associated virus (AAV) vectors are currently recognized as gene transfer vectors that offer the safest and most efficient profile for in vivo gene transfer. Several AAV serotypes, including AAV2 and AAV8, have been used to efficiently target human cells, and long-term expression of therapeutic transgenes has been recorded. The AAV serotype group includes, in particular, serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and RhlO.

[0158] The transfer of genetic information / nucleic acid molecules for CEACAR includes insertion into target cell lines via CRISPR / Cas and TALEN, where the target cells are preferably T lymphocytes, natural killer cells, and induced pluripotent stem cells (iPS). In one embodiment, the iPS cell line is ND50039. All appropriate methods for transferring genetic information / nucleic acid molecules for CEA CAR into cells expressing the CAR are encompassed in the present invention, and those skilled in the art can select an appropriate method when carrying out the invention. For example, several methods for transforming T cells are known in the art, such as any given viral gene transfer method, e.g., based on modified retroviridae, and non-viral methods, e.g., DNA transposons, and direct transfer of DNA or RNA by electroporation. Appropriate methods for transferring genetic information / nucleic acid molecules into any cell type using chemical transposition are also known to those skilled in the art. In one embodiment, genetic information / nucleic acid molecules are transferred to iPS cells, preferably iPS cell line ND50039, by electroporation.

[0159] Furthermore, the signaling components of the CAR constructs are replaced in a three-step cloning procedure, which enables those skilled in the art to create modular and tailor-made constructs of clinically applicable anti-CEA CARs.

[0160] The present invention further relates to a method for treating the conditions described herein, which typically involves administering a therapeutically effective dose of CAR or immune cells expressing the CAR to a patient in need of such treatment.

[0161] The present invention provides technical solutions for efficient solid or liquid tumor therapy using CAR constructs described herein, which possess antigen-binding domains that selectively recognize tumor-specific antigens, actively stimulate immune cells, particularly T cells and NK cells, and activate an immune-stimulated tumor microenvironment through efficient checkpoint inhibitors.

[0162] The present invention further relates to a pharmaceutical composition comprising genetically modified cells according to the present invention as described herein and a pharmaceutically acceptable carrier. The compositions described herein may be administered to a patient by subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous or intralymphatic injection, or intraperitoneal injection.

[0163] In some embodiments, the pharmaceutically acceptable carrier is prepared, for example, in the form of a therapeutic cell product.

[0164] In one embodiment, the therapeutic cell product of the pharmaceutical composition is intended for use in the treatment and / or prevention of the medical disorders described herein.

[0165] In one embodiment, the pharmaceutical composition can be administered to a patient before, after, and / or in combination with one or more anticancer therapies or pharmaceuticals, such therapies or pharmaceuticals include antibody therapy, vaccines, oncolytic virus therapy, chemotherapy, radiotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser phototherapy, B-cell ablative therapy, T-cell ablative therapy, immunosuppression, peripheral blood stem cell transplantation, or bone marrow stem cell transplantation.

[0166] In preferred embodiments, genetically modified cells or cell lines derived from genetically modified cells according to the present invention can be used to treat, modify, or prevent cancer, cancer metastasis and / or autoimmune disorders, preferably disorders associated with the presence of pathogenic cells expressing CEA.

[0167] Detailed description of the invention A crucial function of the immune system is to recognize and eliminate tumors. Tumor antigens are either specifically expressed on tumor cells and not found on non-pathogenic cells, or they are abnormally expressed, for example, at levels at least twice as high as those found on non-pathogenic cells. Antigens specifically found on tumor cells may appear foreign to the immune system, and the presence of such antigens can trigger an attack by immune cells on transformed tumor cells. Some antigens originate from oncogenic viruses, such as human papillomavirus (HPV), which causes cervical cancer. One example of an abnormally expressed protein is an enzyme called tyrosinase, which, when expressed in large quantities, can convert certain skin cells (e.g., melanocytes) into tumors called melanoma. Another possible source of tumor antigens is a protein that is normally important in regulating cell proliferation and survival but mutates into a molecule called an oncogene, which often causes cancer. However, the innate immune response often fails to eliminate tumors, and therapeutic intervention is urgently needed.

[0168] The primary immune response to tumors is to destroy abnormal cells with the help of killer T cells, and sometimes helper T cells. Tumor antigens are presented on MHC class I molecules in a similar manner to viral antigens. This allows killer T cells to recognize tumor cells as abnormal. NK cells also kill tumor cells in a similar manner, especially when tumor cells have fewer MHC class I molecules on their surface than normal cells. This is a common phenomenon in tumors. Some tumor cells also release products that inhibit the immune response, such as the cytokine TGF-β. TGF-β suppresses the activity of macrophages and lymphocytes. Cytokine-induced killer cells (CIKs) are a group of immune effector cells that exhibit a hybrid of T-like and NK-like phenotypes. These cells are produced by incubating mononuclear cells derived from human peripheral blood (PBMC) or umbilical cord blood with interferon-gamma (IFN-γ), anti-CD3 antibody, recombinant human interleukin (IL-)1, and recombinant human interleukin (IL)-2 in ex vivo.

[0169] Accordingly, the present invention provides means for enabling and / or enhancing an antitumor immune response by co-expressing a tumor antigen, such as a CEA-directed chimeric antigen receptor (CAR), a checkpoint inhibitor molecule, and an immunostimulatory cytokine, such as IL-15, in genetically modified immune cells, such as T lymphocytes, cytokine-induced killer cells (CIKs), and NK cells, as described herein.

[0170] Immunotherapy, in the context of this invention, should be understood to encompass any therapeutic agent that utilizes the immune system to treat cancer. Immunotherapy takes advantage of the fact that cancer cells have subtly different molecules on their surface that can be detected by the immune system. These molecules are known as cancer antigens and are most commonly proteins, but also include molecules such as carbohydrates, lipids, and lipoproteins. By using these antigens as targets, immunotherapy induces or enhances the immune system in attacking tumor cells. Furthermore, this invention exceptionally uses the CARs described herein in combination with immunostimulatory cytokines to induce activation and proliferation of T cells and / or natural killer (NK) cells, and in combination with checkpoint inhibitor molecules to enhance the endogenous antitumor activity of the immune system.

[0171] Immunotherapy encompasses cell therapy and antibody therapy without particular limitations. Cell therapy typically involves the administration of immune cells isolated from the patient's blood or tumor. Immune cells targeting the tumor to be treated are activated, cultured, and returned to the patient, where they attack the cancer. Cell types that can be used in this way include natural killer cells, lymphokine-activated killer cells, cytotoxic T cells, and dendritic cells, without particular limitations. Dendritic cell therapy induces an antitumor response by causing dendritic cells to present tumor antigens. Dendritic cells present antigens to lymphocytes, which activates the lymphocytes and stimulates them to kill other cells that present antigens.

[0172] Antibodies are proteins produced by the immune system that bind to target antigens on the cell surface. Antibodies that bind to cancer antigens can be used in cancer treatment. Cell surface receptors are common targets of antibody therapy, and examples of such receptors include CD19, CD44, CD20, CD274, and CD279. Once bound to a cancer antigen, antibodies can induce antibody-dependent cell-mediated cytotoxicity, activate the complement system, or prevent the receptor from interacting with its ligand, all of which can lead to cell death. Several antibodies have been approved for cancer treatment, including alemtuzumab, ipilimumab, nivolumab, ofatumumab, and rituximab.

[0173] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune system attack mechanism that requires antibodies to bind to the surface of target cells. Antibodies are composed of a binding region (Fab) and an Fc region, and immune cells can detect the Fc region via Fc surface receptors. Fc receptors are found on many immune system cells, including natural killer cells. When natural killer cells encounter antibody-coated cells, the antibody's Fc region interacts with the natural killer cell's Fc receptor, leading to the release of perforin and granzyme B. These two chemicals induce programmed cell death (apoptosis) in tumor cells. Effective antibodies include rituximab, ofatumumab, and alemtuzumab.

[0174] The complement system contains blood proteins that can induce cell death after antibodies bind to the cell surface. Generally, the complement system deals with exogenous pathogens, but in cancer, it can be activated by therapeutic antibodies. As long as the antibody has the Fc region of IgG1, chimeric antibodies, humanized antibodies, or human antibodies can trigger the complement system. Complement can induce cell death through activation of membrane invasion complexes (known as complement-dependent cytotoxicity); enhanced antibody-dependent cytotoxicity; and CR3-dependent cytotoxicity. Complement-dependent cytotoxicity occurs when antibodies bind to the surface of cancer cells, C1 complexes bind to these antibodies, and protein pores are subsequently formed in the cancer cell membrane.

[0175] The CARs of the present invention can enable and / or enhance the immunotherapies described herein through their unique properties derived from combinations with immunostimulatory transgene cytokines and endogenous antitumor activity-boosting checkpoint inhibitor molecules.

[0176] Chimeric antigen receptor According to the present invention, a chimeric antigen receptor (CAR) comprises an extracellular antigen-binding domain containing an antibody or antibody fragment that binds to a target antigen, a transmembrane domain, and an intracellular domain. A CAR is typically described as comprising an extracellular ectodomain (antigen-binding domain) derived from an antibody and an endodomain containing a signal transduction module derived from a T cell signaling protein.

[0177] In one preferred embodiment, the ectodomain preferably includes a variable region composed of the heavy and light chains of an immunoglobulin, which is configured as a single-chain variable fragment (scFv). The scFv is preferably bound to a hinge region that provides flexibility and transmits signals to an intracellular signaling domain via an anchored transmembrane domain. The transmembrane domain is preferably derived from CD8a or CD28. In the first generation CAR, the signaling domain consists of the zeta chain of the TCR complex. The term “generation” refers to the structure of the intracellular signaling domain. The second generation CAR comprises a single costimulatory domain derived from CD28 or 4-1BB. The third generation CAR already contains two costimulatory domains, e.g., CD28, 4-1BB, ICOS or OX40, CD3ζ. The present invention preferably relates to the second generation or third generation CAR.

[0178] In various embodiments, genetically engineered receptors are provided that redirect the cytotoxicity of immune effector cells to B cells. These genetically engineered receptors are referred to herein as chimeric antigen receptors (CARs). CARs are molecules that combine antibody-based specificity for a desired antigen (e.g., CEA) with a T-cell receptor-activating intracellular domain, resulting in the production of chimeric proteins that exhibit specific anti-CEA cell immune activity. As used herein, the term "chimera" refers to a molecule composed of different protein or DNA portions from different origins.

[0179] The CARs contemplated herein comprise a CEA, a transmembrane domain, and an extracellular domain (also called a binding domain or antigen-binding domain) that binds to an intracellular domain or an intracellular signaling domain. The anti-CEA antigen-binding domain of the CAR interlocks with the CEA on the surface of the target cell, resulting in CAR clustering and the transmission of an activation stimulus to the CAR-containing cell. A key characteristic of CARs is their ability to redirect the specificity of immune effector cells, thereby inducing the production of molecules that can mediate proliferation, cytokine production, phagocytosis, or cell death of target antigen-expressing cells independently of the major histocompatibility complex (MHC), thus leveraging the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific co-receptors.

[0180] In various embodiments, the CAR comprises an extracellular binding domain containing a humanized CEA-specific binding domain, a transmembrane domain, and one or more intracellular signaling domains. In specific embodiments, the CAR comprises an extracellular binding domain containing a humanized anti-CEA antibody or its antigen-binding fragment, one or more spacer domains, a transmembrane domain, and one or more intracellular signaling domains. The “extracellular antigen-binding domain” or “extracellular binding domain” is used interchangeably and confers the ability of the CAR to specifically bind to the target antigen of interest, i.e., CEA. The binding domain may be derived from a natural, synthetic, semi-synthetic, or recombinant source. Preferably, it is an scFv domain.

[0181] "Specific binding" should be interpreted by those skilled in the art as referring to the various experimental procedures that can be used to test binding and binding specificity, which are readily apparent to those skilled in the art. Methods for measuring equilibrium association constants or equilibrium dissociation constants are known in the art. Some cross-reactivity or background binding may be unavoidable in many protein-protein interactions, and this should not diminish the "specificity" of binding between CARs and epitopes. "Specific binding" describes the binding of an anti-CEA antibody or its antigen-binding fragment (or a CAR containing it) to CEA with a binding affinity greater than background binding. The term "directed against" can also be applied in understanding the interaction between antibodies and epitopes when considering the term "specificity."

[0182] An "antigen (Ag)" refers to a compound, composition, or substance that can stimulate antibody production or a T-cell response in an animal. In certain embodiments, the target antigen is an epitope of a CEA polypeptide. An "epitope" refers to a region of the antigen to which a binding agent binds. An epitope can be formed from both adjacent or non-adjacent amino acids juxtaposed by the tertiary folding of the protein.

[0183] A "single-chain Fv" or "scFv" antibody fragment comprises a VH domain and a VL domain of the antibody, where these domains exist in one orientation (e.g., VL-VH or VH-VL) on a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain, enabling the scFv to form a structure desirable for antigen binding. In a preferred embodiment, the CAR contemplated herein is an scFv and includes an antigen-specific binding domain which may be mouse, human, or humanized scFv. Single-chain antibodies can be cloned from the V region gene of a hybridoma specific to the desired target.

[0184] In certain embodiments, the antigen-specific binding domain is a humanized scFv that binds to a human CEA polypeptide. Examples of suitable variable heavy chains for constructing the anti-CEA CARs envisioned herein include, but are not limited to, the amino acid sequence described in SEQ ID NO: 19. Examples of suitable variable light chains for constructing the anti-CEA CARs envisioned herein include, but are not limited to, the amino acid sequence described in SEQ ID NO: 15.

[0185] Antibodies and antibody fragments The CAR preferably includes an extracellular antigen-binding domain containing an antibody or antibody fragment that binds to a CEA polypeptide. Therefore, the antibodies or antibody fragments of the present invention include, but are not limited to, polyclonal antibodies, monoclonal antibodies, bispecific antibodies, human antibodies, humanized antibodies, or chimeric antibodies, single-chain fragments (scFv), single-chain variable fragments (ssFv), single-domain antibodies (e.g., VHH fragments derived from nanobodies), Fab fragments, F(ab')2 fragments, fragments generated by Fab expression libraries, anti-idiotype antibodies, and any of the above epitope-binding fragments or combinations thereof, provided that they preferably retain binding properties comparable to those of the corresponding CDR or CAR described herein, which include the VH and VL regions. Mini-antibodies and multivalent antibodies such as diabodies, triabodies, tetravalent antibodies, and peptabodies may also be used in the methods of the present invention. The immunoglobulin molecules of the present invention may be globulin molecules of any class (i.e., IgG, IgE, IgM, IgD, and IgA) or subclass immune systems. Therefore, as used herein, the term antibody encompasses antibodies and antibody fragments included in the CAR of the present invention, which are either produced by modification of full-length antibodies or newly synthesized using recombinant DNA methods.

[0186] As used herein, “antibody” generally refers to a protein consisting of one or more polypeptides substantially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene. When the term “antibody” is used, it may also be considered to refer to the term “antibody fragment.” Known immunoglobulin genes include the κ, λ, α, γ, δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which also define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. Basic immunoglobulin (antibody) structural units are known to include tetramers or dimers. Each tetramer consists of two pairs of identical polypeptide chains, each pair having one “light” (L) chain (approximately 25 kD) and one “heavy” (H) chain (approximately 50 kD–70 kD). The N-terminus of each chain defines a variable region of approximately 100 to 110 amino acids or more, primarily responsible for antigen recognition. The terms "variable light chain" and "variable heavy chain" refer to these variable regions of the light and heavy chains, respectively. Optionally, an antibody or the immunological portion of an antibody may be chemically conjugated with other proteins or expressed as a fusion protein with other proteins.

[0187] The CAR of the present invention is intended to bind to mammalian, particularly human, protein targets. The protein names used may correspond to either mouse or human versions of the protein.

[0188] The affinity of the binding domain polypeptide and CAR protein according to this disclosure can be easily determined using prior art, for example, by competitive ELISA (enzyme-linked immunosorbent assay), or by binding-association assays or substitution assays using labeled ligands or surface plasmon resonance devices such as Biacore.

[0189] Humanized antibodies comprising one or more CDRs of the antibody of the present invention or one or more CDRs derived from said antibody can be prepared using any method known in the art. For example, a monoclonal antibody can be humanized using four general steps: (1) determining the nucleotide and predicted amino acid sequences of the light chain and heavy chain variable domains of the starting antibody; (2) designing the humanized antibody, i.e., determining which antibody framework regions to use in the humanization process; (3) the actual humanization methodology / technique; and (4) translocation and expression of the humanized antibody. See, for example, U.S. Patent Nos. 4,816,567, 5,807,715, 5,866,692, 6,331,415, 5,530,101, 5,693,761, 5,693,762, 5,585,089, 6,180,370, 5,225,539, and 6,548,640.

[0190] The term "humanized antibody" means that at least a portion of the framework region of the immunoglobulin and optionally a portion of the CDR region or other regions involved in binding are derived from or adapted to a human immunoglobulin sequence. Humanized, chimeric, or partially humanized versions of mouse monoclonal antibodies can be produced, for example, using recombinant DNA technology, by deviating from mouse and / or human genomic DNA sequences encoding H and L chains, or from cDNA clones encoding H and L chains. Humanized forms of mouse antibodies can be generated by ligating the CDR region of a non-human antibody to a human constant region using recombinant DNA methods (Queen et al., 1989, International Publication No. 90 / 07861). Alternatively, the monoclonal antibody used in the method of the present invention may be a human monoclonal antibody. Human antibodies can be obtained, for example, by phage display (International Publication No. 91 / 17271, International Publication No. 92 / 01047).

[0191] As used herein, humanized antibody also refers to non-human (e.g., mouse, camel, llama, shark) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody) containing minimal sequences derived from non-human immunoglobulins.

[0192] As used herein, a human antibody or humanized antibody, or a human antibody fragment or humanized antibody fragment, means an antibody having an amino acid sequence corresponding to an antibody produced by a human, and / or an antibody prepared using any technique for producing human antibodies known in the art or disclosed herein. A human antibody or fragment thereof can be selected to have the same epitope specificity as a particular mouse antibody by competitive binding experiments or otherwise. Surprisingly, the humanized antibodies of the present invention have useful functional properties common to a considerable extent with mouse antibodies. Human polyclonal antibodies can also be obtained in serum form from humans immunized with an immunogenic agent. Optionally, such polyclonal antibodies can be enriched by affinity purification using amyloid fibrils and / or non-fibrillary polypeptides or fragments thereof as affinity reagents. Monoclonal antibodies can be obtained from serum according to the technique described in International Publication No. 99 / 60846.

[0193] Variable area and CDR The variable region of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either individually or in combination. Each of the heavy and light chain variable regions consists of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are closely linked by the FRs and, together with the CDRs of other chains, contribute to the formation of the antibody's antigen-binding site.

[0194] Numerous techniques are available for determining CDR, such as approaches based on interspecific sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)) and approaches based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al. (1997) J. Molec. Biol. 273:927-948). An alternative method is the IMGT (International Immunogenetics) information system (Marie-Paule Lefranc). The Kabat definition is based on sequence diversity and is the most commonly used method. The Chothia definition is based on the location of structural loop regions, while the AbM definition is a compromise between the two and is used by Oxford Molecular's AbM antibody modeling software (see Dr. Andrew CR Martin's group: www.bioinf.org.uk). As used herein, CDR may refer to a CDR defined by one or more methods, or a combination thereof.

[0195] In some embodiments, the present invention provides an antibody or fragment thereof incorporated into a CAR, the antibody or fragment thereof comprising at least one CDR, at least two, at least three, or more CDRs substantially identical to at least one CDR, at least two, at least three, or more CDRs of the antibody of the present invention. Other embodiments include an antibody having at least two, three, four, five, or six CDRs substantially identical to at least two, three, four, five, or six CDRs of or derived from the antibody of the present invention. In some embodiments, at least one, two, three, four, five, or six CDRs are at least about 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, or 99% identical to at least one, two, or three CDRs of the antibody of the present invention. For the purposes of this invention, the degree of activity may differ (greater or less) compared to the above-mentioned antibodies, but it is understood that the binding specificity and / or overall activity are generally maintained.

[0196] Additional components of CAR In certain embodiments, the CAR contemplated herein may include linkers containing amino acid sequences that link the VH and VL domains and provide a spacer function compatible with the interaction of the two subbinding domains, for example, linker residues added between various domains for appropriate spacing and conformation of the molecule, so that the resulting polypeptide retains specific binding affinity to the same target molecule as an antibody containing the same light and heavy chain variable regions. The CAR contemplated herein may include one, two, three, four, or five or more linkers. In certain embodiments, the length of the linkers is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intermediate length of amino acids. Examples of linkers include glycine polymers, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other mobile linkers known in the art, such as Whitlow linkers. Glycine polymers and glycine-serine polymers are relatively amorphous and can therefore function as neutral tethers between domains of fusion proteins such as CARs described herein. In certain embodiments, one or more “spacers” or “spacer polypeptides” follow the CAR binding domain, referring to regions that move the antigen-binding domain away from the effector cell surface, enabling proper intercellular contact, antigen binding, and activation. In certain embodiments, the spacer domain is part of an immunoglobulin, but is not limited to one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. In one embodiment, the spacer domain comprises the CH2 and CH3 domains of IgG1 or IgG4. In one embodiment, the Fc-binding domain of such a spacer / hinge region is mutated to prevent the binding of CAR to an Fc receptor expressed on macrophages and other innate immune cells.

[0197] In some embodiments, the binding domain of the CAR is followed by one or more "hinge domains," which are involved in positioning the antigen-binding domain away from the effector cell surface to enable proper intercellular contact, antigen binding, and activation. The CAR may contain one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived from a natural, synthetic, semi-synthetic, or recombinant source. The hinge domain may contain the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. Suitable exemplary hinge domains for use in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins, such as CD8α, CD4, CD28, PD1, CD152, and CD7, which may be wild-type hinge regions derived from these molecules or may be modified. In another embodiment, the hinge domain includes the hinge region of PD1, CD152, or CD8α.

[0198] The "transmembrane domain" is a part of the CAR that fuses the extracellular binding portion and the intracellular signaling domain, fixing the CAR to the plasma membrane of an immunoeffector cell. The TM domain may be derived from a natural, synthetic, semi-synthetic, or recombinant source. The TM domain may be derived from the T cell receptors CD3s, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and the α, β, or ζ chain of PD1. In one embodiment, the CAR envisioned herein includes a TM domain derived from CD8α or CD28.

[0199] In certain embodiments, the CAR as intended herein includes an intracellular signaling domain. “Intracellular signaling domain” refers to a portion of the CAR that transmits information of effective anti-CEA CAR binding to human CEA polypeptides into the interior of immune effector cells, and is involved in the induction of effector cell function, such as activation, cytokine production, proliferation, and release of cytotoxic factors to CAR-bound target cells, or other cytotoxic activity, including antigen binding to the extracellular domain of the CAR. The term “effector function” refers to the specific function of immune effector cells. The effector function of T cells may, for example, assist in activity including cytolytic activity or cytokine secretion. Therefore, the term “intracellular signaling domain” refers to a portion of a protein that transmits effector function signals and instructs cells to perform specific functions. The CAR as intended herein includes one or more costimulatory signaling domains that enhance the potency, proliferation, and / or memory formation of T cells expressing the CAR receptor. As used herein, the term “costimulatory signaling domain” refers to the intracellular signaling domain of a costimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal necessary for the efficient activation and function of T lymphocytes when binding to an antigen. Furthermore, co-stimulatory molecules are cell surface molecules that are not antigen receptors or their ligands that contribute to an effective immune response. Examples of co-stimulatory molecules include MHC class I molecules, BTLA and Toll ligand receptors, OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).Further examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, L These ligands specifically bind to, but are not limited to, FA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD26), 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.

[0200] The co-stimulatory intracellular signaling domain can be the intracellular portion of the co-stimulatory molecule. In one embodiment, the CAR includes an intracellular domain comprising a co-stimulatory domain and a signaling (activation) domain. Thus, a CAR construct may include the intracellular signaling domain (CD3ζ) of the innate T cell receptor complex and one or more co-stimulatory domains that provide a second signal to stimulate full T cell activation. The co-stimulatory domain is thought to increase cytokine production in CAR-T cells and promote T cell replication and T cell persistence. The co-stimulatory domain has also been shown to potentially prevent CAR-T cell depletion, increase T cell antitumor activity, and enhance CAR-T cell survival in patients. As a non-limiting example, CAR constructs with a 4-1BB co-stimulatory domain have been associated in preclinical studies with slow, persistent proliferation and effector function, increased persistence, and abundant memory center cells (TCMs) in the T cell subset composition. 4-1BB belongs to the tumor necrosis factor (TNF) superfamily and is an in vivo, inducible glycoprotein receptor primarily expressed on antigen-activated CD4 and CD8 T cells. CD28, a non-limiting example, belongs to the immunoglobulin (Ig) superfamily. It is constitutively expressed on resting and activated CD4 and CD8 T cells and plays a crucial role in T cell activation by stimulating the PI3K-AKT signaling pathway. In one embodiment, the intracellular domain includes both the 4-1BB and CD28 co-stimulatory domains. Other co-stimulatory domains include ICOS and OX40, which can be combined with the CD3ζ signaling (activation) domain.

[0201] The cytokines described herein may relate to any mammalian cytokines corresponding to the cytokines designated herein. Preferably, the cytokines relate to human cytokines or mouse cytokines. Cancer immunotherapy attempts to stimulate the immune system to reject and destroy tumors. Initially, immunotherapy treatments involved the administration of cytokines such as "interleukins," as described herein.

[0202] Checkpoint inhibitors, also known as immune checkpoint modulators, are designed to reduce the effectiveness of checkpoint proteins. Checkpoint inhibitors may have various mechanisms of action, but when effective, they cause the immune system to recognize other molecules on the surface of cancer cells. Checkpoint inhibitors of the immune response are selected from the group consisting of: PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR beta.

[0203] polypeptide The terms "peptide," "polypeptide," "polypeptide fragment," and "protein" are used interchangeably unless otherwise specified, and are used in their usual sense, i.e., as sequences of amino acids. Polypeptides are not limited to a specific length and, for example, may include full-length protein sequences or fragments of full-length proteins, and may include post-translational modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications known in the art that are both naturally occurring and those that are not naturally occurring.

[0204] In various embodiments, the CAR polypeptides envisioned herein include a signal (or leader) sequence at the N-terminus of the protein that directs the transport of the protein during or after translation. The polypeptides may be prepared using any of the various well-known recombinant and / or synthetic techniques. Specifically, the polypeptides envisioned herein include sequences having a CAR of the Disclosure, or a deletion from a CAR disclosed herein, an addition thereto, and / or a substitution of one or more amino acids therein.

[0205] As used herein, “isolated peptide” or “isolated polypeptide,” etc., refers to the in vitro isolation and / or purification of a peptide or polypeptide molecule from the cellular environment and from association with other components of the cell, i.e., it is not significantly associated with the substance in vivo. Similarly, “isolated cell” refers to a cell obtained in vivo from a tissue or organ and substantially free of the extracellular matrix.

[0206] nucleic acid As used herein, the terms “polynucleotide” or “nucleic acid molecule” refer to messenger RNA (mRNA), RNA, genomic RNA (gRNA), positive-strand RNA (RNA(+)), negative-strand RNA (RNA(-)), genomic DNA (gDNA), complementary DNA (cDNA), or recombinant DNA. Polynucleotides include single-stranded and double-stranded polynucleotides. Preferably, the polynucleotides of the present invention include polynucleotides or variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein, wherein the variant typically retains at least one biological activity of the reference sequence. In various exemplary embodiments, the present invention partially envisions polynucleotides comprising expression vectors, viral vectors, and transducer plasmids, as well as compositions and cells comprising them. Polynucleotides can be prepared, manipulated, and / or expressed using any of the various established techniques known and available in the art. To express a desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into a suitable vector. Examples of vectors include plasmids, self-replicating sequences, and transposable elements. Further exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phages or M13 phages, and animal viruses. Examples of animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40).Examples of expression vectors include the pClneo vector (Promega) for expression in mammalian cells; and pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In certain embodiments, the coding sequences of chimeric proteins disclosed herein may be ligated into such expression vectors for the expression of chimeric proteins in mammalian cells. The “regulatory elements” or “control sequences” present in the expression vectors are the untranslated regions of the vector, i.e., replication start sites, selection cassettes, promoters, enhancers, translation initiation signals (Schein-Dalgarno sequences or Kozak sequences), introns, polyadenylated sequences, and 5' and 3' untranslated regions, which interact with host cell proteins to perform transcription and translation. Such elements may differ in their strength and specificity. Depending on the vector system and host used, any number of suitable transcription and translation elements (including ubiquitous promoters and inducible promoters) may be used.

[0207] vector In certain embodiments, cells (e.g., immune effector cells such as T cells) are transduced with an adeno-associated virus vector, retroviral vector, or lentiviral vector encoding a CAR. For example, immune effector cells are transduced with a CAR-encoding vector that contains a humanized anti-CEA antibody or antigen-binding fragment that binds to a CEA polypeptide, along with transmembrane and intracellular signaling domains, so that the transduced cells can induce a CAR-mediated cytotoxic response.

[0208] In some embodiments, a particular advantage of the present invention is the use of AAV for gene transfer of the recombinant nucleic acid constructs of the present invention, due to the high safety and transduction efficiency of AAV in vivo. Variants of AAV, e.g., AAV and capsid variants, can provide or transfer polynucleotides and / or proteins that provide desired, i.e., therapeutic effects and thereby treat a variety of diseases. For example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8 and their variants, as well as AAV capsid variants (e.g., 4-1), are useful vectors for providing therapeutic genes that treat cells, tissues, and organs. The recombinant virus and AAV vectors of the present invention, containing a vector genome (virus or AAV) (capsid-forming (encapside and encapsidate)), contain additional factors that function in cis or trans. The AAV vector is selected from the group containing AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8 AAV capsid sequences, or contains AAV1, AAV2, AAV3 capsid variants, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8.

[0209] Retroviruses are a common tool for gene delivery. In certain embodiments, retroviruses are used to deliver polynucleotides encoding chimeric antigen receptors (CARs) to cells. As used herein, the term “retrovirus” refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates that genomic DNA into the host genome. Once integrated into the host genome, the virus is referred to as a “provirus.” The provirus acts as a template for RNA polymerase II, directing the expression of RNA molecules encoding structural proteins and enzymes required to generate new viral particles.

[0210] Examples of retroviruses suitable for use in specific embodiments include, but are not limited to, Moloney's mouse leukemia virus (M-MuLV), Moloney's mouse sarcoma virus (MoMSV), Harvey's mouse sarcoma virus (HaMuSV), mouse mammary cancer virus (MuMTV), gibbon leukemia virus (GaLV), feline leukemia virus (FLV), Supumavirus, Friend's mouse leukemia virus, mouse stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentiviruses. As used herein, the term "lentivirus" refers to a group (or genus) of compound retroviruses. Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV types 1 and 2), Visna-Maedi virus (VMV), canine arthritis encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV). In one embodiment, an HIV-based vector skeleton (i.e., an HIV cis-acting sequence element) is preferred. In certain embodiments, a lentivirus is used to deliver polynucleotides containing CARs to cells.

[0211] As used herein, the term "vector" refers to a nucleic acid molecule capable of introducing or carrying another nucleic acid molecule. The nucleic acid to be introduced is generally ligated, for example, inserted, into the vector nucleic acid molecule. The vector may contain a sequence that directs autonomous replication in a cell, or a sequence sufficient to enable integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-deficient retroviruses and lentiviruses. In further embodiments of the present invention, CrispR / Cas and TALEN-mediated insertion of nucleic acids encoding a CEA CAR may be used. Suitable vectors for CrispR / Cas and TALEN-mediated insertion are known to those skilled in the art.

[0212] As will be apparent to those skilled in the art, the term “viral vector” is broadly used to refer to either a nucleic acid molecule (e.g., a transplasmid) containing a viral nucleic acid element that facilitates the introduction or integration of a nucleic acid molecule into a cell’s genome, or a viral particle that mediates the introduction of nucleic acids. Viral particles typically include various viral components and, if applicable, host cell components in addition to nucleic acids.

[0213] The term "viral vector" can refer to a virus or viral particle capable of introducing nucleic acids into cells, or to the introduced nucleic acid itself. Viral vectors and introduced plasmids contain structural and / or functional genetic elements primarily derived from viruses. The term "retroviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements, or parts thereof, primarily derived from retroviruses.

[0214] Therefore, in a preferred embodiment, the present invention relates to a method for transposing cells with an expression vector encoding a CAR. For example, in some embodiments, the vector includes additional sequences, such as sequences that promote CAR expression, such as promoters, enhancers, polyA signals, and / or one or more introns. In a preferred embodiment, the sequence encoding the CAR is adjacent to a transposon sequence, and as a result, the presence of a transposase allows the encoding sequence to be incorporated into the genome of the transposed cell.

[0215] In a preferred embodiment, the present invention relates to a method for translocating cells with an expression vector encoding a CAR using electroporation. Electroporation is a physical method that creates pores in the cell membrane by applying an electric shock to the cell. These pores allow for increased diffusion of substances into the cell. This increased permeability allows for easier translocation.

[0216] Sonoporation is similar to electroporation, except that it uses ultrasound to stimulate the cell membrane. The ultrasound also creates turbulence in the fluid surrounding the cell, which increases the diffusivity across the membrane.

[0217] In a preferred embodiment, the present invention relates, therefore, to a method for transfecting cells with an expression vector encoding a CAR using chemical transfection. Chemical transfection refers to calcium phosphate transfection, which is known to those skilled in the art. Calcium phosphate transfection uses calcium phosphate bound to DNA (A Watson and D. Latchman, "Gene Delivery into Neuronal Cells by Calcium Phosphate-Mediated Transfection," Methods, Volume 10, Issue 3, December 1996, Pages 289-291). In gene therapy, the use of calcium phosphate particles as transfection agents for therapeutic polynucleotides has been suggested. See U.S. Patent No. 5,460,831. By binding DNA or RNA to the particle nucleus and delivering it to target cells, the expression of a therapeutic protein is brought about.

[0218] In some embodiments, genetically transformed cells are further transposed by a transposase that facilitates the integration of the CAR-coding sequence into the genome of the transposed cells. In some embodiments, the transposase is provided as a DNA expression vector. However, in preferred embodiments, the transposase is provided as an expressible RNA or protein so that long-term expression of the transposase does not occur in the transgenic cells. For example, in some embodiments, the transposase is provided as mRNA (e.g., mRNA including a cap and a poly-A tail). Any transposase system may be used according to embodiments of the present invention. However, in some embodiments, the transposase is a salmonid-type Tel-like transposase (SB). For example, the transposase may be a so-called "sleeping beauty" transposase (see, for example, U.S. Patent No. 6,489,458, which is part of this specification by reference). In some embodiments, the transposase is a genetically engineered enzyme having increased enzymatic activity. Some specific examples of transposases include, but are not limited to, SB 10, SB 11, or SB100X transposases (see, for example, Mates et al, 2009, Nat Genet. 41(6):753-61 or U.S. Patent No. 9,228,180, which are incorporated herein by reference). For example, the method may involve electroporating cells with mRNA encoding SB 10, SB 11, or SB 100X transposase.

[0219] Array variant: For example, sequence variants of claimed nucleic acids, proteins, antibodies, antibody fragments, and / or CARs, defined by % sequence identity and maintaining similar binding properties of the present invention, are also included within the scope of the invention. Such variants, which show alternative sequences but maintain essentially the same binding properties, such as target specificity, as the specific sequence presented, are known as functional analogues or functionally similar.

[0220] Sequence identity refers to the percentage of identical nucleotides or amino acids when sequence alignment is performed.

[0221] As used herein, “sequence identity” refers to the degree to which sequences are identical across a comparison window on a nucleotide-level or amino acid-level basis. Therefore, “percentage of sequence identity” can be calculated by comparing two optimally aligned sequences across a comparison window, determining the number of positions in which identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) are found in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. This specification includes nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein, wherein the polypeptide variant typically retains at least one biological activity of the reference polypeptide.

[0222] Those skilled in the art will understand that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode the polypeptides described herein. Some of these polynucleotides have minimal homology or sequence identity to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage frequency are specifically contemplated in the present invention. Deletions, substitutions, and other changes in the sequence that constitute the described sequence identity are also encompassed in the present invention.

[0223] Protein sequence modifications that can result from substitutions are also within the scope of the present invention. Substitutions as defined herein are modifications made to the amino acid sequence of a protein, where one or more amino acids are replaced by the same number of (different) amino acids, resulting in a protein with a different amino acid sequence than the primary protein. Preferably, substitutions can be made without significantly altering the function of the protein. Like additions, substitutions can be natural or artificial. It is known in the art that amino acid substitutions can be made without significantly altering the function of a protein. This is especially true when relating to “conservative” amino acid substitutions, where the modification is the substitution of one amino acid for another amino acid with similar properties. Such “conservative” amino acids can be natural or synthetic amino acids that, due to their size, charge, polarity, and conformation, can be substituted without significantly affecting the structure and function of the protein. Many amino acids can often be substituted with conservative amino acids without adversely affecting the function of the protein.

[0224] Generally, the conserved amino acids are the nonpolar amino acids Gly, Ala, Val, Ile, and Leu; the nonpolar aromatic amino acids Phe, Trp, and Tyr; the neutral polar amino acids Ser, Thr, Cys, Gln, Asn, and Met; the positively charged amino acids Lys, Arg, and His; and the negatively charged amino acids Asp and Glu. This list is not exhaustive. For example, it is known that Ala, Gly, Ser, and sometimes Cys can be substituted for each other despite belonging to different groups.

[0225] In substitutional variants, at least one amino acid residue is removed from the antibody molecule and a different residue is inserted in its place. While hypervariable regions are among the most interesting sites for introducing substitutional mutations, changes to the framework are also attempted. If such substitutions result in changes to biological activity, they are referred to as "exemplary substitutions" in the table below, or larger changes related to amino acid classes, as further described below, may be introduced, and the products are screened.

[0226] Potential amino acid substitutions: [Table 1]

[0227] Substantial modification of the biological properties of an antibody is achieved by selecting substitutions that have a significantly different effect on (a) the structure of the polypeptide backbone in the substitution region, for example, as a sheet or helical structure; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the maintenance of the side chain size.

[0228] Conservative amino acid substitutions are not limited to naturally occurring amino acids but also include synthetic amino acids. Commonly used synthetic amino acids include omega-amino acids of various chain lengths and cyclohexylalanine, which are neutral, nonpolar analogs; citrulline and methionine sulfoxides, which are neutral, nonpolar analogs; phenylglycine, which is an aromatic neutral analog; cysteic acid, which is a negatively charged analog; and ornithine, which is a positively charged amino acid analog. As with naturally occurring amino acids, this list is not exhaustive and is merely an example of substitutions known in the art.

[0229] Genetically modified cells and immune cells In certain embodiments, the present invention envisions genetically modified cells to express a CAR contemplated herein, for use in the treatment of a disease. As used herein, the terms “genetically modified” or “genetically modified” refer to the addition of external gene material in the form of DNA or RNA to the entire genetic material of a cell. The terms “genetically modified cell,” “genetically modified immune cell,” “modified cell,” and “redirected cell” are used synonymously. As used herein, the term “gene therapy” refers to the permanent or temporary introduction of external gene material in the form of DNA or RNA to the entire genetic material of a cell for the purpose of restoring, correcting, or altering gene expression, or for the expression of a therapeutic polypeptide, such as a CAR. In certain embodiments, the CAR contemplated herein is introduced into and expressed in immune effector cells to redirect the specificity of immune effector cells to a target antigen of interest, such as a CEA polypeptide.

[0230] "Immune cells" or "immune effector cells" are any cells of the immune system that possess one or more effector functions (e.g., cytotoxic cell-killing activity, cytokine secretion, ADCC and / or CDC induction). Immune effector cells can be differentiated from iPSCs (induced pluripotent stem cells) or derived from human peripheral blood and / or human umbilical cord blood.

[0231] iPSC cell lines can also be obtained from public supply and cell preservation institutions, such as the NINDS Human Cell and Data Repository (https: / / stemcells.nindsgenetics.org / ). For example, publicly available iPSC cells can be selected from the group of iPS cell lines NDS00159;NDS00249;NDS00250, NDS00251, NDS00252, NDS00253, NDS00254, NDS00255, NDS00256, NDS00257, NDS00258, NDS00259, NDS00260, NDS00261, NDS00262, NDS00263, and ND50039. In one embodiment, the iPS cell line NDS00159 is used.

[0232] The immune effector cells of the present invention may be autogeneic ("self") or non-autogeneic ("non-self," e.g., allogeneic, syngeneic, or heterogeneic). As used herein, "autogeneic" refers to cells derived from the same subject and is a preferred embodiment of the present invention. As used herein, "allogeneic" refers to cells of the same species that are genetically different from the cells being compared.

[0233] As used herein, “syngeneic” refers to cells of a different subject that are genetically identical to the cells being compared. As used herein, “heterogeneic” refers to cells of a different species from the cells being compared. In preferred embodiments, the cells of the present invention are autologous or allogeneic. Exemplary immune effector cells used with CARs as intended herein include T lymphocytes. The terms “T cell” or “T lymphocyte” are recognized in the art and are intended to encompass thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, cytokine-induced killer cells (CIK cells), or activated T lymphocytes. Cytokine-induced killer (CIK) cells are typically CD3 and CD56-positive non-major histocompatibility complex (MHC)-restricted natural killer (NK)-like T lymphocytes. T cells are T helper (Th;CD4 +T cells may be, for example, T helper 1 (Th1) or T helper 2 (Th2) cells. T cells are cytotoxic T cells (CTLs; CD8 + T cells), CD4 + CD8 + These may be T cells, CD4-CD8 T cells, or any other subset of T cells.

[0234] As used herein, “immortalized” refers to immortalized cells as a population of cells that are not normally expected to regenerate through an infinite number of cell cycles. Through mutation, immortalized cells evade normal cellular senescence and instead continue to proliferate. This mutation can occur spontaneously, or it can be induced by UV light, genetic manipulation, or by the entry of viruses, toxins, or bacteria into the cells. Thus, these cells can be cultured in vitro for extended periods for experimental, therapeutic, or medical purposes. Examples of immortalized immune cells include K13, αβT cells, γδT cells, NK cells, NK-92 and YT cells, stem cells, or stem cell-derived cells, where stem cell-derived cells include the immune system cells mentioned above, preferably NK T cells, NK-92 cells, YT cells, and NK cells. Immortalized T cell lines may retain their lytic function.

[0235] Other exemplary populations of T cells suitable for use in specific embodiments include naive T cells and memory T cells, as well as stem cell-like memory cells (TSCMs).

[0236] For example, when reintroduced into a patient after autologous cell transplantation, CAR-modified T cells of the present invention as described herein can recognize and kill tumor cells. CIK cells may have enhanced cytotoxic activity compared to other T cells and therefore represent a preferred embodiment of the immune cells of the present invention.

[0237] As will be understood by those skilled in the art, other cells may also be used as immune effector cells in conjunction with the CARs described herein. In particular, immune effector cells include NK cells, NK T cells, neutrophils, and macrophages. Effector cell precursors may also be included, where such precursors can be induced to differentiate into immune effector cells in vivo or in vitro. The precursors may be iPSCs that become immune effector cells under defined culture conditions. The present invention provides a method for producing CAR-expressing immune effector cells as intended herein. In one embodiment, the method involves transfecting or transducing immune effector cells isolated from an organism so that the immune effector cells express one or more CARs described herein. In a particular embodiment, the immune effector cells are isolated from an organism and genetically modified without further manipulation in vitro. Such cells can then be directly re-administered to an organism. In further embodiments, immunoeffector cells are first activated and stimulated to proliferate in vitro, and then genetically modified to express the CAR. In this regard, immunoeffector cells may be cultured before and / or after genetic modification (i.e., transduction or transtransfer to express the CAR as intended herein).

[0238] In certain embodiments, the cell source is obtained from a subject prior to the in vitro manipulation or genetic modification of the immunoeffector cells described herein. In certain embodiments, the CAR-modified immunoeffector cells include T cells. T cells can be obtained from a number of sources, but are not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from infection sites, ascites, pleural fluid, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from blood units taken from a subject using any number of techniques known to those skilled in the art, e.g., sedimentation, e.g., FICOLL® separation, antibody-conjugate bead-based methods, e.g., MACS® separation (Miltenyi). In one embodiment, cells derived from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains T cells, lymphocytes including monocytes, granulocytes, and B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in a suitable buffer or culture medium for subsequent processing. The cells may be washed with PBS or another suitable solution lacking calcium, magnesium, and most, but not all, other divalent cations. As will be recognized by those skilled in the art, the washing step may be achieved by methods known to those skilled in the art, for example, by using a semi-automatic flow-through centrifuge, such as the Cobe 2991 cell processing device or Baxter CytoMate. After washing, the cells may be resuspended in various biocompatible buffers or other physiological salines, with or without buffers. In a particular embodiment, undesirable components of the apheresis sample may be removed in the culture medium to which the cells are directly resuspended.

[0239] In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and removing monocytes, for example, by PERCOLL® gradient centrifugation. Specific subpopulations of T cells may be further isolated by positive or negative selection techniques. One method used herein is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a monoclonal antibody cocktail directed against cell surface markers present on negatively selected cells.

[0240] PBMCs can be directly genetically modified to express CAR using the method intended herein. In certain embodiments, T lymphocytes may be further isolated after isolation of PBMCs, and in certain embodiments, both cytotoxic and helper T lymphocytes may be sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and / or proliferation. CD8 + Cells can be obtained by using standard methods. In some embodiments, CD8 + Cells are naive, central memory, and effector cells, and these types of CD8 + Further selection is achieved by identifying the cell surface antigens associated with each individual cell.

[0241] In some embodiments, the immune cells of the present invention, for example, T cells or NK cells as described herein, can be obtained from induced pluripotent stem cells (iPSCs) using methods known to those skilled in the art. The accepted approach to generating CAR-T cells relies on the genetic modification and proliferation of mature circulating T cells. Such processes utilize autologous T cells and reduce the risk of allogeneic graft-versus-host (GvHD) disease through endogenous TCR expression and rejection via MHC incompatibility. As an alternative, direct in vitro differentiation of modified T cells derived from pluripotent stem cells, such as induced pluripotent stem cells, provides an essentially unlimited supply of cells that can be genetically modified to express the CARs of the present invention. In some embodiments, a so-called master iPSC line can be maintained, which serves as a renewable source for consistently and repeatedly producing homogeneous cell products. In some embodiments, the master iPSC cell line is intended to be transformed with CAR-coding nucleic acids before proliferation and differentiation into the desired immune cells, preferably T cells or NK cells.

[0242] In one embodiment, the master iPSC cell line ND50039 is intended to be transformed with a CAR-coding nucleic acid construct before proliferation and differentiation into desired immune cells, preferably T cells or NK cells. T lymphocytes can be generated, for example, from iPSCs or from previously genetically modified iPSCs, so that iPSCs can be modified with CAR-coding nucleic acids and subsequently proliferated and differentiated into T cells for administration to a patient.

[0243] Just as iPSCs can be modified with CAR-coding nucleic acid constructs and subsequently proliferated and differentiated into NK cells for administration to patients, NK cells can also be generated from previously genetically modified iPSCs. Differentiation into appropriate immune cells, such as T cells or NK cells, can also be performed from iPSCs before transformation with CAR-coding nucleic acids and proliferation, and therefore before transformation with CAR-coding nucleic acid constructs and proliferation of appropriate immune cells, such as T cells and NK cells. All possible combinations of iPSC proliferation, genetic modification, and proliferation of cells to an appropriate number for administration are intended in this invention.

[0244] Immune effector cells, such as T cells or NK cells, may be genetically modified after isolation using known methods, or they may be activated and proliferated in vitro (or differentiated in the case of progenitor cells) before genetic modification. In certain embodiments, immune effector cells, such as T cells or NK cells, are genetically modified with a chimeric antigen receptor as intended herein (e.g., transduced with a viral vector containing nucleic acid encoding a CAR), and then activated and proliferated in vitro. In various embodiments, T cells are, for example, U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, and 7,172,869 CAR can be activated and amplified before or after genetic modification to express CAR using the methods described in Patent Nos. 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and U.S. Patent Application Publication No. 20060121005.

[0245] In further embodiments, for example, a mixture of one, two, three, four, or five or more different expression vectors can be used to genetically modify a donor immunoeffector cell population, where each vector encodes a different chimeric antigen receptor protein as intended herein. The resulting modified immunoeffector cells form a mixed population of modified cells, where some of the modified cells express two or more different CAR proteins.

[0246] In one embodiment, the present invention provides a method for storing immunoeffector cells expressing genetically modified mouse, human, or humanized CAR proteins that target CEA proteins, the method comprising cryopreserving immunoeffector cells so that they remain viable upon thawing. Fractions of immunoeffector cells expressing CAR proteins can be cryopreserved by methods known in the art to provide a permanent source of such cells for future treatment of patients suffering from B cell, T cell, NK cell, dendritic cell (DC), or cytotoxicity-induced killer cell (CIK) related conditions. When needed, the cryopreserved transformed immunoeffector cells can be thawed, grown, and proliferated to increase the number of such cells.

[0247] Compositions and Formulations The compositions envisioned herein may include one or more polypeptides, polynucleotides, vectors containing these, genetically modified immune effector cells, etc. Examples of compositions, but not limited to, pharmaceutical compositions. “Pharmaceutical composition” means a composition formulated in a pharmaceutically acceptable or physiologically acceptable solution for administration to cells or animals, either alone or in combination with one or more other therapeutic modalities. It will also be understood that, if necessary, the compositions of the present invention may be administered in combination with other active agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharmaceutically active agents. There are substantially no limitations on other components that may be included in a composition, provided that additional active agents do not adversely affect the composition's ability to deliver the intended treatment.

[0248] The term "pharmaceutically acceptable" is used herein to mean a compound, substance, composition, and / or dosage form that is suitable for use in contact with human and animal tissues without causing excessive toxicity, irritation, allergic response, or other problems or complications, within the bounds of sound medical judgment, and that is commensurate with a reasonable benefit / risk ratio.

[0249] As used herein, “pharmaceutically acceptable carriers, diluents, or excipients” include, but are not limited to, any adjuvants, carriers, excipients, flow enhancers, sweeteners, diluents, preservatives, colorants, flavorings, surfactants, humectants, dispersants, suspending agents, stabilizers, isotonic agents, solvents, surfactants, or emulsifiers that are deemed acceptable for use in humans or domesticated animals by the United States Food and Drug Administration. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars, e.g., lactose, glucose, and sucrose; starches, e.g., corn starch and potato starch; cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin, silicone, bentonite, silicic acid, zinc oxide; oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, e.g., propylene glycol; polyols, e.g., glycerin, sorbitol, mannitol, and polyethylene glycol; esters, e.g., ethyl oleate and ethyl laurate; agar; buffers, e.g., magnesium hydroxide and aluminum hydroxide; alginic acid; water free of pyrogens; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; and any other suitable substances used in pharmaceutical formulations.

[0250] AAV vectors, lentiviral vectors, and / or other components, agonists, drugs, biologics (proteins), such as pharmaceutically acceptable carriers or excipients, can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are particularly useful for administration and delivery to subjects in vivo or ex vivo.

[0251] In certain embodiments, the compositions of the present invention include the amount of CAR-expressing immune effector cells intended herein. As used herein, the term “amount” means an “effective amount” or “effective dose” of genetically modified therapeutic cells, such as T cells, NK cells, or CIK cells, to achieve a beneficial or desired preventive or therapeutic outcome, including clinical outcomes.

[0252] The “prophylactic effective dose” refers to the amount of genetically modified therapeutic cells effective in achieving the desired preventive outcome. Because prophylactic doses are administered to subjects before or during the early stages of a disease, the prophylactic effective dose is typically less than, but not necessarily less than, the therapeutic effective dose. The term "prophylactic" does not necessarily mean the complete prevention or cessation of a particular medical disorder. The term "prophylactic" can also mean a reduction in the risk of developing a particular medical disorder or the risk of its symptoms worsening.

[0253] The "therapeutic dose" of genetically modified immune cells may vary depending on factors such as the individual's condition, age, sex, and weight, as well as the ability of stem cells and progenitor cells to induce the desired response in the individual. The therapeutic dose is also such that the therapeutically beneficial effect outweighs any toxic or adverse effects of the virus or transduced therapeutic cells. The term "therapeutic dose" encompasses the amount effective in "treating" a subject (e.g., a patient). Where a therapeutic dose is indicated, the exact amount of the composition of the present invention administered may be determined by a physician, taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and the patient's (subject's) condition. Generally, the pharmaceutical compositions comprising T cells, CIK cells, or NK cells described herein are 10 2 Individual cells / kg body weight ~10 10 Cells per kg of body weight, preferably 10 5 Individual cells / kg body weight ~10 7It can be stated that the dosage can be administered in individual cells / kg body weight (including all integer values ​​within that range). The number of cells depends on the final intended use of the composition and the type of cells contained therein. In the uses provided herein, the volume of cells is generally 1 liter or less, and may be 500 mL or less, and even 250 mL or 100 mL or less. Therefore, the desired cell density is typically 10 6 Higher than individual cells / ml, generally 10 7 Higher than individual cells / mL, generally 10 8 The number of cells / mL is greater than 10. A clinically relevant number of immune cells is cumulatively 10 5 Individual cells, 10 6 Individual cells, 10 7 Individual cells, 10 8 Individual cells, 10 9 Individual cells, 10 10 Individual cells, 10 11 Individual cells, or 10 12 The cells may be divided into multiple injections equal to or greater than a single cell. In some embodiments of the present invention, fewer cells may be administered, particularly when all injected cells are redirected to a specific target antigen. The CAR-expressing cell composition may be administered multiple times in doses within these ranges. The cells may be allogeneic, syngeneic, heterogeneic, or autologous to the patient being treated.

[0254] Generally, the compositions containing activated and expanded cells as described herein can be used in the treatment and prevention of diseases occurring in individuals in an immunocompromised state. Specifically, the compositions containing CAR-modified T cells contemplated herein are used for the treatment of cancer, more preferably solid malignancies and liquid malignancies, more preferably colorectal cancer, lung cancer, breast cancer, liver cancer, pancreatic cancer, gastric cancer, and ovarian cancer, more preferably CEA-positive metastatic tumor cells. The CAR-modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a carrier, diluent, excipient, and / or other components, and the other components can be, for example, interleukin or other immune response-stimulating cytokines, such as IL-15 and / or checkpoint inhibitory molecules, such as PD1 polypeptide or PD1 antibody, or a cell population. In certain embodiments, the pharmaceutical compositions contemplated herein contain some amount of genetically modified T cells combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0255] The pharmaceutical compositions of the present invention containing a population of CAR-expressing immune effector cells such as T cells or NK cells or CIK cells may contain buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (such as aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration.

[0256] Liquid pharmaceutical compositions (regardless of whether they are in the form of solutions, suspensions, etc.) may include one or more of the following: sterile diluents such as water for injection, saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride, synthetic monoglycerides or diglycerides such as non-volatile oils that can act as solvents or suspending agents, polyethylene glycol, glycerin, propylene glycol, or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate, and tonicity agents such as sodium chloride or dextrose. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multi-dose vials. Pharmaceutical compositions for injection are preferably sterile.

[0257] In certain embodiments, the compositions contemplated herein include an effective amount of CAR-expressing immune effector cells, either alone or in combination with one or more therapeutic agents. Thus, CAR-expressing immune effector cell compositions can be administered alone or in combination with other known cancer treatments such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy. The compositions can also be administered in combination with antibiotics. Such therapeutic agents may be recognized in the art as standard treatments for certain pathologies described herein, e.g., certain cancers. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiation therapy, therapeutic antibodies, or other active and adjuvant substances.

[0258] Mixed immunotherapy encompasses concurrent, simultaneous, or combined therapies and involves administering genetically modified immune cells expressing CAR-encoding nucleic acid constructs in combination with immunotherapeutic drugs, such as checkpoint inhibitors and / or immunostimulatory cytokines, so that the therapies may be administered within minutes of each other, at the same time, on the same day, in the same week, or in the same month. Combinations containing one or more of the above-mentioned genetically modified immune cells and other immunotherapeutic drugs can also be used for the purpose of simultaneously administering various components in single doses or dosages.

[0259] As used herein, the term “tumor microenvironment” refers to the cellular environment in which any given tumor exists, which includes the tumor stroma, surrounding blood vessels, immune cells, fibroblasts, other cells, signaling molecules, and the extracellular matrix (ECM).

[0260] treatment method The genetically modified immune effector cells contemplated herein provide an improved method of adoptive immunotherapy used to treat medical disorders associated with the presence of pathogenic cells expressing CEA, including, but not limited to, immunomodulatory conditions, hematological malignancies, and solid malignancies.

[0261] As used herein, “medical disorder associated with the presence of pathogenic cells expressing CEA” refers to a condition in which cells are involved in the pathophysiology of a disease that exhibits CEA expression, preferably CEA presentation on the cell surface, such as cancer or autoimmune diseases. CEA expression can be identified by various methods known to those skilled in the art, for example, by isolating cells from a patient and evaluating the cells by PCR using primers that point to CEA transcripts, by immunostaining with anti-CEA antibodies, or by flow cytometry analysis. Such pathogenic cells can typically be pathogenic mature B cells and / or memory B cells and / or pathogenic T cells and / or T follicular helper cells and / or tumor stem cells and / or solid tumor cells and / or liquid tumor cells and / or metastatic cancer cells and / or NK cells and / or CIK cells and / or dendritic cells.

[0262] As used herein, “cancer” refers to a disease characterized by the uncontrolled growth of abnormal cells. Cancer refers to any type of malignant growth or carcinogenic process, metastatic tissue or malignant transformed cells, tissue, or organ, regardless of histopathological type or stage of invasion. Cancer cells may spread locally or to other parts of the body via the bloodstream or lymphatic system. Cancer cells that spread to other parts of the body are called “metastatic cells” or “metastatic tumor cells.” As used herein, the terms “tumor” and “cancer” are used synonymously, and for example, both terms include solid and liquid tumors, for example, systemic or circulating tumors, pre-malignant and malignant cancers and tumors. Examples of liquid cancers include acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and acute myeloid leukemia (ACLL). Examples of treatable breast cancers include, but are not limited to, ductal carcinoma (AML), chronic myeloid leukemia (CML), Hodgkin lymphoma, non-Hodgkin lymphoma, and myeloma. Examples of solid tumors include malignant organ systems such as the liver, lungs, breast, lymphatic system, digestive system (e.g., colon), genitourinary system (e.g., kidneys, urothelial cells), prostate, and throat, including sarcomas, adenocarcinomas, and carcinomas. Examples of treatable breast cancers include ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), and invasive tubal carcinoma (IDC). Invasive tubular carcinoma includes tubular carcinoma, medullary carcinoma, mucinous carcinoma, papillary carcinoma, and cribriform carcinoma), invasive lobular carcinoma (ILC), inflammatory breast cancer, male breast cancer, Paget's disease of the nipple, phyllodes tumor of the breast, and recurrent and / or metastatic breast cancer. Among adenocarcinomas are the most malignant tumors, such as colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, and esophageal cancer. In some forms, cancer is melanoma, for example, advanced melanoma. Metastatic lesions of cancer can also be treated or prevented with the methods and compositions of the present invention.Other treatable cancers include bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, acute myeloid leukemia, chronic myeloid leukemia, and acute phosphorus This includes paoblastic leukemia, chronic or acute leukemia including chronic lymphocytic leukemia, solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axial tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, asbestos-induced T-cell lymphoma including environmental cancers, and cancer. Metastatic cancer, for example, treatment of metastatic cancer expressing PD-L1 (Iwai et al. (2005) Int. Immunol. 17: 133-144) can be carried out using the inhibitory molecules described in this invention.

[0263] A “cancer-associated antigen” or “tumor antigen” is a molecule (usually a protein, carbohydrate, or lipid) that is intercommutably expressed on the surface of cancer cells, either completely or as fragments (e.g., MHC / peptide), and the drug preferentially targets cancer cells. Tumor antigens refer to antigens commonly found in certain hyperproliferative diseases. In one embodiment, the hyperproliferative disease antigens of the present invention are derived from primary or metastatic cancers such as melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney and breast cancer, and adenocarcinomas such as prostate cancer, ovarian cancer, and pancreatic cancer. In certain embodiments, the tumor antigen is a marker expressed in both normal and cancer cells, such as a cell lineage marker like CD19 on B cells. In certain embodiments, the tumor antigen is overexpressed in cancer cells compared to normal cells, for example, by 1x, 2x, 3x, or more than 1x overexpression compared to normal cells. In certain forms, the tumor antigen is a cell surface molecule that is improperly synthesized in cancer cells, for example, a molecule that has deletions, additions, or mutations compared to molecules expressed in normal cells. In certain embodiments, the tumor antigen is exclusively expressed on the cell surface of cancer cells, either as a whole or as a fragment (e.g., MHC / peptide), and is neither synthesized nor expressed on the surface of normal cells. In certain embodiments, the CAR of the present invention comprises a CAR containing an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to an MHC-presenting peptide. Typically, peptides derived from endogenous proteins are housed in the pocket of a major histocompatibility complex (MHC) class I molecule, and CD8 +They are recognized by the T cell receptor (TCR) of T lymphocytes. Class I MHC complexes are constitutively expressed in all nucleated cells. In cancer, viral and / or tumor-specific peptide / MHC complexes have become a unique class of cell surface targets for immunotherapy. TCR-like antibodies are described as targeting peptides of viruses or tumor antigens associated with human leukocyte antigen (HLA)-A1 or HLA-A2 (see, for example, Sastry et al., J Virol. 2011 85 (5): 1935-1942, Sergeeva et al, Blood, 2011 117 (16): 4262-4272, Verma et al., JImmunol 2010 184 (4): 2156-2165, Willemsen et al., Gene Ther 2001 8 (21): 1601-1608, Dao et al., Sci Transl Med 2013 5 (176): 176ra33, Tassev et al., Cancer Gene Ther 2012 19 (2): 84-100). For example, TCR-like antibodies can be identified by searching libraries such as the human scFv phage display library.

[0264] In certain embodiments, compositions comprising CAR-modified T cells as intended herein are used to treat cancer, including, but are not limited to, solid malignancies such as rectal cancer, lung cancer, breast cancer, liver cancer, pancreatic cancer, gastric cancer, ovarian cancer, and CEA-positive metastatic tumor cells, or hematological malignancies such as acute myeloid leukemia, non-Hodgkin lymphoma (NHL), such as B-cell NHL or T-cell non-Hodgkin lymphoma, with or without leukemic tumor cell metastasis.

[0265] As used herein, “treatment” or “to treat” includes any beneficial or desired effect on the symptoms or pathology of a disease or condition, and may include even a slight decrease in one or more measurable markers of the disease or condition being treated. Treatment may optionally include reduction or improvement of the symptoms of a disease or condition, or delay in the progression of a disease or condition. “Treatment” does not necessarily mean the complete eradication or cure of a disease or condition or its associated symptoms.

[0266] As used herein, “prevention” and similar terms, such as “prevented,” “preventing,” or “preventively,” mean methods of preventing, inhibiting, or reducing the likelihood of the onset or recurrence of a disease or condition. This also includes delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, “prevention” and similar terms also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition before its onset or recurrence.

[0267] In one embodiment, a treatment for a cancer-related condition in a subject requiring treatment of a cancer-related condition comprises administering a composition comprising genetically modified immune effector cells as intended herein in an effective amount, e.g., a therapeutically effective amount. The amount and frequency of administration are determined by factors such as the patient's condition and the type and severity of the patient's disease, although the appropriate dose may be determined by clinical trials.

[0268] The administration of the compositions contemplated herein can be effected by any convenient method, including aerosol inhalation, injection, ingestion, infusion, implant, or transplantation. In a preferred embodiment, the composition is administered parenterally. As used herein, the terms "parenteral administration" and "administered parenterally" refer to a mode of administration other than enteral and topical administration and include, but are not limited to, administration by injection, typically by intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, intraspinal, and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

[0269] Sequence Preferred nucleic acid sequences of the present invention: [Table 2] [Table 2-1]

[0270] Preferred nucleic acid sequences of the present invention: [Table 3]

[0271] Drawings The present invention is illustrated by way of example through the drawings disclosed herein. The provided drawings represent specific non-limiting embodiments and are not intended to limit the scope of the present invention. [Brief Description of the Drawings]

[0272] [Figure 1] It is a diagram showing the cytotoxicity of CEA CAR-transduced YT cells against MCF-7 cells. [Figure 2] It is a diagram showing that checkpoint inhibition by dominant negative PD1 (dnPD1opt) leads to improvement of NFAT promoter activity. [Figure 3] This figure compares the activity of the IL-15 superagonist (15R15) with that of IL-2 or IL-15. [Figure 4] This figure shows how the NF-κB promoter activity of anti-CEA CAR-expressing Jurcut cells stimulates target cells (MCF-7) with and without checkpoint inhibition (dnPD1opt) or IL-15 superagonist (15R15). [Figure 5] This figure shows the cytotoxicity of CEA CAR-induced YT cells against MC32A cells. [Figure 6] This figure shows the specific release of IL-15 superagonist when stimulated in the CEA-expressing tumor cell line MCF-7. [Modes for carrying out the invention]

[0273] Detailed description of the drawing: Figure 1: Cytotoxicity of CEA CAR-transduced YT cells against MCF-7 cells: YT cells were transduced with a lentivirus encoding the CEA CAR construct along with dnPD1opt and 15R15 (YT CEA CAR 15R15) or an empty vector (YT control construct). Subsequently, YT CEA CAR 15R15 cells or the YT control construct were added to a pair of wells in a 96-well plate containing MCF-7 cells, and a nonspecific cytotoxic signal (etoposide 10 μg / ml) was added to an additional well. Cytotoxicity was identified after 18 hours.

[0274] Figure 2: Checkpoint inhibition by dominant-negative PD1 (dnPD1opt) leads to improved NFAT promoter activity: Jurcut cells expressing GFP under NFAT promoter control were transduced with a dentPD1opt-expressing lentiviral vector or a control vector. Then, one day earlier, the cells were exposed to a cell line expressing high levels of PD-L1 (U251-PD-L1) in a pair of wells of a 96-well plate. The 96-well plate was supplemented with increasing levels of TCR-linked proliferation signaling (phytohemagglutinin: PHA). The number of GFP-expressing cells was identified by flow cytometry.

[0275] Figure 3: Comparison of IL-15 superagonist (15R15) activity with IL-2 or IL-15: The IL-15 transgene 15R15 or empty plasmid was expressed in HEK293 cells after transient transduction, and collected 2 days after transduction. The supernatant was double-tested for IL-2 / IL-15 specific activity using a bioassay with the Hek-Blue IL-2 reporter cell line, as instructed by the manufacturer (Invivogen).

[0276] Figure 4: Shows how NF-κB promoter activity in anti-CEA CAR-expressing Jurkat cells stimulates target cells (MCF-7) with and without checkpoint inhibition (dnPD1opt) or IL-15 superagonist (15R15): Jurkat cells expressing GFP under NF-κB promoter control were transduced with lentiviral vectors encoding the following: (1) CEA-CAR construct combined with dnPD1opt and 15R15 (Jurkat-CEA-CAR-dnPD1opt-IL15R15), (2) CEA-CAR construct combined with dnPD1opt (Jurkat-CEA-CAR-dnPD1opt), (3) CEA-CAR construct (Jurkat CEA-CAR), or (4) empty lentiviral vector (empty vector). Transduced Jurkat cells were transferred to MCF-7 target cells. After 1 day, positive cells were analyzed by flow cytometry.

[0277] Figure 5: Cytotoxicity of CEA CAR-induced YT cells against MC32A cells: The cytotoxicity of CEA CAR-transduced scBW431 / 26-hFcz YT cells against CEA-positive MC32A cells was analyzed by a 6-hour chromium release assay in the absence or presence of soluble CEA protein in the culture medium. Cell lysis of target cells was determined along with the standard deviation.

[0278] Figure 6: Specific release of IL-15 superagonist upon stimulation with CEA-expressing tumor cell line MCF-7: CEA-expressing cells were mixed with responder cells in various ratios, namely (A) CEA CARYTs for MCF-7 cells and (B) CEA CARs for MCF7 cells, or an equal number of responder cells expressing a control vector (empty CARs). After 18 hours, the supernatant was collected, and the amount of IL15 activity was identified using an IL-2 / IL-15 reporter cell line. [Examples]

[0279] The present invention will be demonstrated through the examples disclosed herein. The examples provided represent specific embodiments and are not intended to limit the scope of the invention. The examples should be construed as non-limiting illustrations and technical assistance for carrying out the invention.

[0280] Using known lentiviral gene transfer methods, lentiviral vectors encoding anti-CEA CAR, checkpoint inhibitors, dominant-negative cleavage-type PD-1 protein, immunostimulatory cytokines, IL-15 gene transdermal 15R15, or combinations thereof, are introduced into immune cell lines. The cell lines thus modified lyse the CEA-positive cancer cell line MCF-7, while the immune cell line YT, which is not modified with anti-CEA CAR, cannot adequately lyse the cancer cell line MCF-7 (Example 1, Figure 1). Further examples are shown below: Successful PD-1 checkpoint inhibition (Example 2, Figure 2), High activity of IL-15 superagonist (Example 3, Figure 3), Synergistic effect of a combination of anti-CEA CAR, PD-1 checkpoint inhibitor, and IL-15 superagonist (Example 4, Figure 4). Binding of anti-CEA CAR to cell membrane-bound CEA protein, independent of the presence of soluble CEA protein (Example 5, Figure 5).

[0281] Following Dull et al. (1998), lentiviruses were packaged, generated, and transposed into cells using the described high-safety third-generation plasmid system. Cell transposition was performed using polyethyleneimine, following the manufacturer's (Polyplus) instructions.

[0282] Hek293T cells and MC32A cells were cultured in DMEM containing 10% thermo-inactivated FCS and penicillin / streptomycin. Jarcut cells were cultured in RPMI containing 10% thermo-inactivated FCS and penicillin / streptomycin. YT cells were cultured in RPMI containing 10% thermo-inactivated FCS, penicillin / streptomycin, and 10 IU / ml of IL-2. GFP was measured by flow cytometry using FACS-Calibur. Cytotoxicity was identified according to standard procedures using the crystal violet assay or chromium release assay.

[0283] Example 1: Cytotoxicity of YT cells against MCF-7 cells when transduced with an anti-CEA CAR construct. This example shows experimental results using YT cells transduced with an empty lentiviral vector (YT control construct) or with a lentiviral vector containing a nucleic acid sequence region encoding a CEA-binding chimeric antigen receptor (CAR), the checkpoint inhibitor dnPD1opt, and / or the immunostimulatory cytokine 15R15 (YT CEA CAR15R15) (Figure 1). YT CEA CAR 15R15 cells or YT control construct cells were then added in pairs to a 96-well plate containing MCF-7 cells, and a nonspecific cytotoxic signal (etoposide 10 μg / ml) was added to additional wells. Cytotoxicity was identified after 18 hours. The CAR DNA sequence was transferred to the YT immune cell line using a known lentiviral gene transfer method. The thus modified cell line lysates the CEA-positive cancer cell line MCF-7. The YT immune cell line, not modified with CAR, does not sufficiently lyse the cancer cell line MCF-7 (Figure 1).

[0284] Example 2: Checkpoint inhibition by dominant-negative PD1 (dnPD1opt) leads to improved NFAT promoter activity. This example describes an experiment demonstrating successful inhibition of the checkpoint protein PD-1 via dntPD1opt, a dominant-negative cleavage type of PD-1 (Figure 2). Jurcut cells express GFP under the control of the NFAT promoter. Jurcut cells were transduced with either a control lentiviral vector or a lentiviral vector encoding the checkpoint inhibitor (dntPD1opt). One day prior, these cells were exposed in pairs in a 96-well plate to a cell line expressing high concentrations of PD-L1 (U251-PD-L1), and to increasing concentrations of TCR-mediated T cell stimulation and phytohemagglutinin (PHA). Flow cytometry identified the number of GFP-expressing cells, and found that the number was higher in dntPD1opt-expressing Jurcut cells compared to the negative control. This demonstrates successful inhibition of the checkpoint protein PD-1 at a biologically relevant level.

[0285] Example 3: The activity of IL-15 superagonist (15R15) is compared with that of IL-2 or IL-15. This example describes an experiment demonstrating the superagonist activity of the IL-15 transgene 15R15 (Figure 3). The IL-15 transgene 15R15 or an empty plasmid was expressed in HEK293 cells after transient transduction and collected 2 days after transduction. The supernatant was tested twice for IL-2 / IL-15 specific activity using a bioassay with the Hek-Blue IL-2 reporter cell line, as described by the manufacturer (Invivogen). OD260 was measured. This successfully demonstrates that the superagonist activity of the IL-15 transgene 15R15 is higher than that of the negative control, IL-2, and IL-15 at a biologically relevant level.

[0286] Example 4: Demonstrates how the NF-κB promoter activity of anti-CEA CAR-expressing Jurcut cells stimulates target cells (MCF-7) with and without checkpoint inhibition (dnPD1opt) or IL-15 superagonist (15R15). This example describes an experiment demonstrating that Jurkat cells expressing anti-CEA CAR, the checkpoint inhibitor dnPD1opt, and the IL-15 transgene 15R15 successfully exhibited NF-κB promoter activity when exposed to MCF-7 target cells (Figure 4). GFP expression under NF-κB promoter control was identified in Jurkat cells transduced with the following lentiviral vectors: (1) an anti-CEA CAR construct combined with the checkpoint inhibitor dntPD1opt and the IL-15 transgene 15R15 (Jurkat-CEA-CAR-dnPD1opt), (2) an anti-CEA CAR construct combined with the checkpoint inhibitor dntPD1opt (Jurkat-CEA-CAR-dnPD1opt), (3) anti-CEA-CAR (Jurkat CEA-CAR), or (4) an empty lentiviral vector (empty vector). Transduced Jurkat cells were transferred to MCF-7 target cells. One day later, GFP-positive cells were identified by flow cytometry. The Jurkat-CEA-CAR-dnPD1opt-IL15R15 combination showed very high levels of GFP expression, and higher than Jurkat-CEA-CAR-dnPD1opt, Jurkat CEA-CAR, and the empty vector sample. The GFP expression level of Jurkat-CEA-CAR-dnPD1opt-IL15R15 was higher than the additive effect, thus demonstrating a synergistic effect in the combination described in this invention.

[0287] Example 5: Cytotoxicity of anti-CEA CAR-induced YT cells against MC32A cells This example demonstrates that the recognition of cell membrane-bound CEA protein by anti-CEA CAR is independent of the presence of soluble CEA protein (Figure 5). The cytotoxicity of anti-CEA CAR transducible scBW431 / 26-hFcz YT cells against CEA-positive MC32A cells was analyzed by a 6-hour chromium release assay in the absence or presence of soluble CEA protein in the culture medium. Cell lysis of target cells was determined along with its standard deviation. The cytotoxic activity of scBW431 / 26-hFczYT cells transduced with an anti-CEA CAR lentiviral vector against MC32A cells remained at a similar level regardless of whether soluble CEA protein was added to the culture medium.

[0288] Example 6: Specific release of IL-15 superagonist upon stimulation of CEA-expressing tumor cell line MCF-7 CEA-expressing cells were mixed with responder cells in various ratios, namely (A) CEA CARYT for MCF-7 cells and (B) CEA CAR for MCF7 cells, or an equal number of responder cells expressing a control vector (empty CAR). After 18 hours, the supernatant was collected, and the amount of IL-15 activity was identified using an IL-2 / IL-15 reporter cell line. As can be seen from the figure, CEA-CAR-expressing cells induce a dose-dependent response after incubation with CEA-expressing MCF7 cells.

[0289] References Kreye, J., et al Humancerebrospinal fluid monoclonal N-methyl-D-aspartate receptor autoantibodies aresufficient for encephalitis pathogenesis. Brain 139, 2641-2652 (2016).

[0290] Drawing translation Figure 1 % MaximumCytotoxicity (Etoposide) YT measureconstruct YT control structure Figure 2 dnPD1opt transduction Control Transduction Figure 3 Undiluted 1:4 dilution Control Supernatant 15R15 Supernatant 15R15 supernatant Figure 4 % activaed (GFP) Empty vector Figure 5 Lyse w / o soluble CEAprotein Soluble CEA protein Figure 6 CAR NK on MCF-7 CAR NK for MCF-7 U / ml IL2 equivalent U / ml of IL2 equivalent Ratio Effector: Target Ratio Control (Empty CAR)

Claims

1. Genetically modified cells comprising a recombinant nucleic acid expression construct encoding a CAR, wherein the construct is as follows: (a) A first nucleic acid sequence region encoding a chimeric antigen receptor (CAR), wherein the CAR includes an extracellular antigen-binding domain that recognizes an insoluble carcinoembryonic antigen (CEA) protein, and the extracellular antigen-binding domain includes a variable heavy chain (VH) and a variable light chain (VL), wherein the VH includes a heavy chain complementarity-determining region 1 (H-CDR1) described in SEQ ID NO: 20, a heavy chain complementarity-determining region 2 (H-CDR2) described in SEQ ID NO: 21, and a heavy chain complementarity-determining region 3 (H-CDR3) described in SEQ ID NO: 22, and the VL includes a light chain complementarity-determining region 1 (L-CDR1) described in SEQ ID NO: 16, a light chain complementarity-determining region 2 (L-CDR2) described in SEQ ID NO: 17, and a light chain complementarity-determining region 3 (L-CDR3) described in SEQ ID NO: 18, and (b) A second nucleic acid sequence region encoding a checkpoint inhibitor molecule, wherein the checkpoint inhibitor molecule is a dominant-negative cleavage type PD1 polypeptide, (c) A third nucleic acid sequence region encoding an immunostimulatory cytokine, wherein the immunostimulatory cytokine includes IL-15 peptide and IL-15RA peptide, Genetically modified cells, including those containing this technology.

2. (a) The second nucleic acid sequence region encodes a dominant-negative cleavage type PD1 of sequence number 28 or a sequence having 90% sequence identity therewith, wherein the dominant-negative cleavage type PD1 binds to the PD-1 protein and blocks the PD-1 protein, and (b) The third nucleic acid sequence region encodes an immunostimulatory cytokine of sequence number 35 or a sequence having 90% sequence identity thereto, the third nucleic acid sequence region is operably linked to one or more constitutive promoters, the immunostimulatory cytokine maintains or enhances the activity, survival and / or number of immune cells within and / or near tumor tissue. Genetically modified cells as described in claim 1.

3. (a) The second nucleic acid sequence region encodes the dominant-negative cleavage type PD1 described in Sequence ID No. 28, and (b) The third nucleic acid sequence region encodes the immunostimulatory cytokine described in SEQ ID NO: 35 Genetically modified cells according to claim 1 or 2.

4. The genetically modified cell according to any one of claims 1 to 3, wherein the chimeric antigen receptor (CAR) preferentially binds to membrane-bound CEA rather than soluble CEA.

5. The genetically modified cell according to any one of claims 1 to 4, wherein the first nucleic acid sequence region encoding the CAR and the second nucleic acid sequence region encoding the checkpoint inhibitor molecule are configured to encode a polycistronic mRNA including coding regions for the polypeptide sequences of the CAR and the checkpoint inhibitor molecule, and the amino acid sequence including a polypeptide cleavage site is located between the CAR polypeptide and the checkpoint inhibitor molecule polypeptide.

6. The genetically modified cell according to any one of claims 1 to 5, wherein the polypeptide cleavage site is selected from the group consisting of P2A, T2A, E2A, and F2A.

7. The genetically modified cell according to any one of claims 1 to 6, wherein the immunostimulatory cytokine is an IL-15 superagonist.

8. The invention includes a recombinant nucleic acid expression construct encoding a CAR, wherein the CAR is as follows: CAR signal array and The antigen-binding domain of the CAR that specifically recognizes CEA, The extracellular constant region of the immunoglobulin heavy chain of CAR, CD28 signaling domain including transmembrane domain, CD3 zeta signaling domain and A genetically modified cell according to any one of claims 1 to 7, including the above.

9. The invention includes a recombinant nucleic acid expression construct encoding a CAR, wherein the CAR is as follows: The CAR signal sequence described in Sequence ID No. 14, The antigen-binding domain of a CAR that specifically recognizes CEA, as described in Sequence ID No. 15 and Sequence ID No. 19, The extracellular constant region of the immunoglobulin heavy chain of CAR, as described in Sequence ID No. 23, The CD28 signaling domain described in Sequence ID No. 24, wherein the CD28 signaling domain includes the transmembrane domain described in Sequence ID No. 25, The CD3 zeta signaling domain described in Sequence ID No. 26, A genetically modified cell according to any one of claims 1 to 8, including the above.

10. The aforementioned checkpoint inhibitor molecules are as follows: (a) Contains a dominant-negative cleavage type checkpoint protein, (b) The checkpoint protein is located adjacent to the polypeptide cleavage site for cleaving the checkpoint inhibitor molecule from the CAR polypeptide, Genetically modified cells according to any one of claims 1 to 9.

11. The genetically modified cell according to any one of claims 1 to 10, wherein the dominant-negative cleavage type checkpoint protein is the dominant-negative cleavage type PD1 described in Sequence ID No. 28, and the cleavage site is selected from the group consisting of P2A, T2A, E2A, and F2A.

12. The aforementioned immunostimulatory cytokines are as follows: (a) Signal sequence and, (b) N-terminal IL15RA polypeptide and (c) Linked loop array and (d) IL-15 polypeptide and A genetically modified cell according to any one of claims 1 to 11, including the above.

13. The aforementioned immunostimulatory cytokines are as follows: (a) The signal sequence described in Sequence ID No. 29, (b) The N-terminal IL15RA polypeptide described in SEQ ID NO: 30, (c) The linked loop sequence described in Sequence ID No. 31, and, (d) The IL-15 polypeptide described in Sequence ID No. 32, A genetically modified cell according to any one of claims 1 to 12, including the above.

14. The recombinant nucleic acid expression constructs are as follows: A CAR containing an extracellular antigen-binding domain that specifically recognizes carcinoembryonic antigen (CEA) protein, Checkpoint inhibitor molecule, dominant-negative cleavage type PD1 polypeptide, An immunostimulatory cytokine comprising a signal sequence, an N-terminal IL15RA polypeptide, a linked loop sequence, and an IL-15 polypeptide, A genetically modified cell according to any one of claims 1 to 13, comprising a nucleic acid sequence region encoding a

15. The genetically modified cells according to any one of claims 1 to 14, wherein the cells are selected from immune cells, induced pluripotent stem cells (iPSCs), immortalized immune cells including iPSC line ND50039, NK-92 cells and YT cells, natural killer (NK) cells, NK T cells, cytokine-induced killer cells (CIK), and T lymphocytes, wherein the T lymphocytes are CD4 or CD8 T cells, or cytotoxic T lymphocytes, helper T cells, or tumor-infiltrating lymphocytes (TILs).

16. A pharmaceutical product comprising genetically modified cells according to any one of claims 1 to 15, used for the treatment of a medical disorder associated with the presence of pathogenic cells expressing CEA.

17. The pharmaceutically acceptable agent according to claim 16, wherein the aforementioned medical impairment includes cancer cells of breast cancer, pancreatic cancer, colon cancer, rectal cancer, lung cancer, liver cancer, gastric cancer, and ovarian cancer, solid malignant tumors expressing CEA, or CEA-positive metastatic tumor cells.

18. The pharmaceutical product according to claim 16 or 17, wherein the genetically modified cells are alloimmune cells or autoimmune cells with respect to the patient to whom the cells are delivered.

19. A recombinant nucleic acid expression construct encoding a chimeric antigen receptor (CAR), wherein the construct is as follows: (a) A first nucleic acid sequence region encoding a chimeric antigen receptor (CAR), wherein the CAR includes an extracellular antigen-binding domain that recognizes an insoluble carcinoembryonic antigen (CEA) protein, and the extracellular antigen-binding domain includes a variable heavy chain (VH) and a variable light chain (VL), wherein the VH includes a heavy chain complementarity-determining region 1 (H-CDR1) described in SEQ ID NO: 20, a heavy chain complementarity-determining region 2 (H-CDR2) described in SEQ ID NO: 21, and a heavy chain complementarity-determining region 3 (H-CDR3) described in SEQ ID NO: 22, and the VL includes a light chain complementarity-determining region 1 (L-CDR1) described in SEQ ID NO: 16, a light chain complementarity-determining region 2 (L-CDR2) described in SEQ ID NO: 17, and a light chain complementarity-determining region 3 (L-CDR3) described in SEQ ID NO: 18, and (b) A second nucleic acid sequence region encoding a checkpoint inhibitor molecule, wherein the checkpoint inhibitor molecule is a dominant-negative cleavage type PD1 polypeptide, (c) A third nucleic acid sequence region encoding an immunostimulatory cytokine, wherein the immunostimulatory cytokine includes IL-15 peptide and IL-15RA peptide, Recombinant nucleic acid expression constructs, including those mentioned above.

20. The aforementioned structure is as follows: (a) A first nucleic acid sequence region encoding a chimeric antigen receptor (CAR), wherein the CAR includes an extracellular antigen-binding domain that recognizes an insoluble carcinoembryonic antigen (CEA) protein, (b) A checkpoint inhibitor molecule comprising a second nucleic acid sequence region encoding a dominant-negative cleavage type PD1 polypeptide, wherein the checkpoint inhibitor molecule is located adjacent to the polypeptide cleavage site P2A, (c) A third nucleic acid sequence region encoding an immunostimulatory cytokine, comprising IL-15RA peptide and IL-15 peptide, a signal sequence, and a linking loop sequence, wherein the immunostimulatory cytokine is operably linked to one or more promoters, A recombinant nucleic acid expression construct according to claim 19, comprising:

21. A method for generating genetically modified cells according to any one of claims 1 to 15, comprising delivering or transferring a recombinant nucleic acid expression construct according to claim 19 or 20 into cells in vitro.

22. A chimeric antigen receptor (CAR) polypeptide encoded by the recombinant nucleic acid expression construct according to claim 19 or 20.

23. A pharmaceutical composition comprising a genetically modified cell according to any one of claims 1 to 15, prepared in the form of a therapeutic cell product, and a pharmaceutically acceptable carrier.

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