Mucosa-associated invariant T (MAIT) cells expressing chimeric antigen receptors

Abstract

This invention provides mucosa-associated invariant T (MAIT) cells expressing chimeric antigen receptors, and a method for producing the same. [Solution] A mucosa-associated invariant T cell expressing a chimeric antigen receptor is provided, wherein, in one embodiment, the chimeric antigen receptor includes an antigen-binding domain that specifically binds to or recognizes a target antigen, and specifically binds to any TAA expressed on the surface of tumor cells, preferably CD19, GD2, EGFR, CD20, CD22, CD33, CD138, CD52, CD30, ROR1, HER2, EpCAM, MUC-1, MUC5AC, BCMA, CD38, SLAMF7 / CS1, CD123, IL-13Ra2, HER2, LeY, MUC16, or PSMA.

Description

[Technical Field]

[0001] The present invention relates to immunotherapy, particularly immunotherapy for treating cancer, infectious diseases, or autoimmune diseases. This relates to therapy. More specifically, the present invention relates to chimeric antigen receptors (CARs) This concerns mucosal-associated invariant T (MAIT) cells that express this condition. [Background technology]

[0002] Immunotherapy using T cells expressing chimeric antigen receptors (CAR-T) is a noteworthy clinical development. The results are shown in the treatment of leukemia, as well as in the treatment of cancer and other diseases such as infections or autoimmune diseases. It is one of the most promising new strategies for doing so. Among these CARs, there are some that target CD19, and those that target CD19 This offers a prospect of complete remission in patients with relapsed or refractory hematopoietic malignancies. CAR-T cells Lymphocyte therapy involves genetically modifying lymphocytes to express CARs, which enable the recognition of specific antigens. This is a representative example of immunotherapy that involves substitution. The T cells are modified in this way during antigen recognition. It is activated by a signaling domain that converts it into a potent cell killer. The bed protocol primarily utilizes autologous T cells collected from patients by apheresis. The cells were genetically modified to express CAR and cultured in vitro to increase their number. And finally, it is reinjected into the same patient. This method requires the creation of patient-specific cells. Yes, and this can lead to variations in efficacy and safety in the final cell product, depending on the patient. This inevitably leads to fluctuations. This method, based on autologous cell transplantation, It also has other drawbacks, such as the production time (approximately 2 months between apheresis and reinjection). 、It is important when a patient is facing a life-threatening emergency. Moreover, the immune cells from the diseased patient may not be fully functional or may be significantly reduced in number. Therefore, such treatment using the patient's own lymphocytes is costly and time-consuming, and is not suitable for the widespread use of CAR T cells. [Prior Art Documents] [Patent Documents]

[0003] [Patent Document 1] International Publication No. 2014 / 031687 [Patent Document 2] U.S. Patent No. 8,822,647 [Patent Document 3] U.S. Patent Application Publication No. 2014 / 0271635 [Non-Patent Documents]

[0004] [Non-Patent Document 1] Qasim et al., Sci Transl Med., 2017 Jan 25, 9(374) [Non-Patent Document 2] Rotolo et al., Cancer Cell. 2018 Oct 8;34(4):596 - 610 [Non-Patent Document 3] Hudecek et al. (2013) Clin.Cancer Res., 19:3153 [Non-Patent Document 4] Brueggemann and Kotrova; Blood Advances 2017 1:2456 - 2466 [Summary of the Invention] [Problems to be Solved by the Invention]

[0005] As described above, allogeneic cells from a third party are pre-produced, characterized in detail, and rapidly administered to the patient It would be desirable to develop immunotherapies that can be used in conjunction with. However, there are certain difficulties in using allogeneic cells (i.e., cells obtained from other individuals of the same species). In particular, allogeneic cells are likely to be rejected by the recipient's immune cells in a process called host-versus-graft rejection (Hv G), which limits their effectiveness. Furthermore importantly, allogeneic cells may trigger graft-versus-host disease (GVHD). In fact, allogeneic T cells maintain an endogenous T cell receptor (T CR) that can recognize host tissues as foreign, which may lead to severe tissue damage and death-causing GVHD. Current approaches involve the generation of universal CA R-T cells in which the endogenous TCR is genetically inactivated (Qasim et al., Sci Transl Med., 2017 Jan 25, 9(374)). Other strategies are based on the use of immune cell populations with low alloreactivity, such as CAR-NK cells lacking endogenous TCR or CAR-NKT cells (Rotol o et al., Cancer Cell. 2018 Oct 8;34(4):596-610 ). However, the frequency of NKT cells in human blood is very low (0.01- 0.5%). Therefore, alternative and / or improved allogeneic CAR-T cell-based immunotherapies are

[0006] needed.

Means for Solving the Problems

[0007] The present invention relates to mucosal-associated invariant T (MAIT) cells expressing a chimeric antigen receptor (CAR) for use in treating cancer, immune diseases or infectious diseases in a subject, wherein M ... AIT cells are allogeneic mucosa-associated invariant T (MAIT) cells relative to the target organism. It provides cells.

[0008] In certain embodiments, MAIT cells expressing chimeric antigen receptors (CARs) are cancer It is used for the treatment of [condition].

[0009] The cancer may be a hematological malignancy. In another specific embodiment, the cancer is preferably, Breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer and It may be a solid tumor selected from the group consisting of lung cancer.

[0010] In one embodiment, CAR-MAIT cells are administered to immunocompromised subjects. In particular, CA R-MAIT cells are administered after pre-treatment to impair the target's immunodeficiency. Pre-treatment is chemotherapy. It may be an antibody for radiation therapy and / or lymphocyte depletion.

[0011] In a particular embodiment, the disease is a hematological malignancy. In this embodiment, CAR -MAIT cells are preferably used before hematopoietic stem cell (HSC) transplantation or before hematopoietic stem cell (HSC) It is administered immediately after transplantation.

[0012] In certain embodiments, cancer, at the minimal residual disease (MRD) stage, is leukemia, li It is a hematological malignancy, such as lymphoma or multiple myeloma.

[0013] Preferably, MAIT cells produce CARs that specifically bind to tumor-associated antigens (TAAs). To reveal.

[0014] TAA can be expressed on the surface of tumor cells. Preferably, TAA is CD19, GD2, EGFR, CD20, CD22, CD33, CD138, CD52, CD30, ROR1 , HER2, EpCAM, MUC-1, MUC5AC, BCMA, CD38, SLAMF 7 / CS1, CD123, IL-13Ra2, HER2, LeY, MUC16 or PSM A. More preferably, TAA is CD19, CD20, CD22, CD33, C D138, BCMA, CD38, SLAMF7 / CS1, IL-13Ra2, or HER2 That is the case.

[0015] In another embodiment, MAIT cells are intracellular tumor proteins or intracellular tumor-related proteins. Antigens, especially WT-1, NY-ESO-1, MAGE, PRAME, RAS, and mesothelin. c-Met, CEA, CSPG-4, EBNA3C, CA-125 or GPA7 are specifically It expresses CARs that target specific targets. [Brief explanation of the drawing]

[0016] [Figure 1]MAIT cells do not proliferate in vitro in response to allogeneic peripheral blood mononuclear cells (PBMCs). Responder PBMCs labeled with carboxyfluorescein succinimimidyl ester (CFSE) were cultured together with irradiated allogeneic PBMCs (in a 1:1 ratio). Responder T cells were identified at the end of 6 days of culture by gating for viable cells and then for CD3+ cells. The proliferation of normal CD3 T cells (Tconv) and Vα7.2+ CD161high MAIT cells was quantified by CFSE dilution (%) of CFSElow cells. A) The number of representative CFSE-stained quadrants in T cells (Tconv) (left panel) and MAIT cells (right panel) after 6 days of culture in the presence of allogeneic (upper panel) or autologous (lower panel) PBMCs indicates the percentage of proliferative (CFSELOW) cells (horizontal bars) in the shown population. B) Individual values ​​and mean ± SD values ​​(paired t-tests) for proliferative (CFSELOW)T (Tconv) and MAIT cells (n=6 experiments using various recipient / donor pairs) are shown. [Figure 2] MAIT cells are not involved in GVHD induction in humanized mouse models. Irradiated (1.3 Gy) immunodeficient NOD-Scid-IL-2Rγnull (NSG) mice (n=3) were infused with 3 × 10⁶ human PBMCs and monitored to assess the progression of GVHD. They were euthanized when they showed a weight loss of less than 15% (±45 days). The graph shows the proportion of CD3 T cells and MAIT cells in human (CD45+) leukocytes measured by flow cytometry at 45 days after initial inoculation (huPBMCs) and transplantation into peripheral blood (PB), spleen, bone marrow (BM), liver, colon, and lung of diseased mice. [Figure 3]In vitro increase in MAIT cells. PBMCs (10⁶ / ml) from healthy donors were cultured for 17 days in the presence of cytokines and synthetic MAIT cell ligands. The percentage and absolute number of MAIT cells were quantified by flow cytometry before the culture period (day 0), and at days 6, 10, and 17. A) The representative number of Vα7.2+ CD161high MAIT cells (gated in CD3 T cells) over the culture period indicates the percentage of MAIT cells among CD3 T cells. B) Increase in MAIT cells over the culture period (absolute number of magnification changes). [Figure 4] Detection of CAR expression in MAIT cells. Purified CD3 T cells were activated in vitro for 24 hours with anti-CD3 / CD28 beads and transduced with lentiviral vectors encoding CAR and fluorescent reporter protein (BFP) to allow monitoring of CAR+ T cells. Cell surface expression of BFP in transduced cells (upper panel) or control non-transduced cells (lower panel) was determined after 6 days in normal CD3 T cells and MAIT cells. The number in the quadrant (horizontal bar) indicates the percentage of BFP+ cells in the shown population. [Modes for carrying out the invention]

[0017] This invention relates to chimeric antigen receptors (CARs), which are referred to herein as CAR-MAIT cells. Mucosa-associated invariant T (MAIT) cells that express this cell type are being used in the field of allogeneic adoptive immunotherapy. To provide for use in [location / place].

[0018] CAR-MAIT cells did not show the potential for allochemical reactions and responded to allogeneic cells in an invaginal manner. They do not proliferate in the turf, are not involved in inducing graft-versus-host disease (GVHD), and are the opposite of normal T cells. According to the present invention, CAR-MAIT cells can be easily produced and used in large quantities in vitro. It increases.

[0019] CAR-MAIT cells can be stored, for example, in a cryogenic unit, and several cells can be used in recipes. It is "ready for use" for rapid administration to the target. This preparation is cost-effective. The risk is high, and despite HLA mismatch between subjects, the risk of inducing GVHD is low. It represents a universal treatment.

[0020] [Definition] As used herein, the terms “subject,” “host,” “individual,” or “patient” are used in this specification. , refers to mammals, preferably humans, and refers to males or females of any age who require treatment.

[0021] The terms "tumor-associated antigen," "TAA," "tumor antigen," and "cancer cell antigen" are defined in the original text. In the details, the terms are used interchangeably. In each case, the term refers to cancer cells, and also This refers to selectively expressed peptides, proteins, glycoproteins, or carbohydrates.

[0022] The term "graft-versus-host disease (GVHD)" refers to the transfer of immunocompetent lymphocytes from donated cells. GVHD refers to a common and serious complication involving a reaction in the tissues of the transplant recipient itself. This refers to any use or inclusion of hematopoietic stem cells from either related or unrelated donors. These are complications that can occur during transplantation. "Treating" a disease or "treating" a condition is the patient's responsibility. This refers to any action intended to restore a state of health. "Treatment" refers to one or more symptoms Alternatively, alleviation or recovery of the condition, reduction in the severity of the disease, or stabilization of the disease state (for example, remission of the patient). Maintaining a healthy state, preventing disease or preventing its spread, delaying disease progression or Slowing down, remission or mitigation of symptoms, reduced frequency of disease relapses and (whether partial or complete) Treatment may include, but is not limited to, remission. Treatment may include cure, reduction, or preventative effects. The term "prophylactic" refers to the severity or frequency of a particular condition. It can be considered to reduce [something]. "Preventive" also refers to having already been diagnosed with a specific condition. This includes preventing the recurrence of the condition in patients who are in a state of illness. c) may also reduce or delay the severity of the existing condition. The therapeutic effect is to slow the progression of the disease, restore or alleviate the symptoms, and achieve remission. This includes improved prognosis. Relief means that signs or symptoms of the disease or condition appear. It can occur before or after it appears. Therefore, "to treat" or "treatment" is a disease. Or it may include "preventing" or "blocking" an undesirable state. In one embodiment... Therefore, treating cancer involves inhibiting the growth or proliferation of cancer cells or killing cancer cells. This includes causing cancer to develop. In certain embodiments, treating cancer may increase the risk of metastasis or development This includes reducing the current state. In another specific embodiment, treating cancer is to prevent recurrence. It can sometimes refer to stopping the cancer. Treating cancer can also refer to maintaining the patient in a state of remission. There is a match.

[0023] As used herein, “disorder” or “disease” The term "sease" refers to errors in genes or expression, infection, toxins, nutrient intake deficiencies, or other related issues. Or a body vessel that does not function properly due to imbalance, toxicity, or the influence of unfavorable environmental factors. It refers to an organ, part, structure, or system. Preferably, such terms refer to a health disorder or This refers to a disease, for example, a disease that destroys normal physical or mental functions. Furthermore, the term "disorder" refers to immune and / or inflammatory diseases affecting animals and / or humans. Preferably, the terms "disorder" or "disease" refer to cancer, infectious disease, or immune disorder.

[0024] As used herein, the term “cancer” refers to the rapid and uncontrolled growth of abnormal cells. It is defined as a disease having the following characteristics: Cancer cells spread locally or through the bloodstream and lymphatic system. It can spread to other parts of the body.

[0025] The term "infectious disease" refers to a disease caused by organisms such as bacteria, viruses, fungi, or parasites. It refers to a disability.

[0026] The terms “immune disease” or “autoimmune disease” are used herein as It is characterized by damage to cells, tissues and / or organs, and by the immune response of the target, it is destroyed itself This refers to a condition in an object that is caused by cells, tissues, and / or organs.

[0027] [CAR-MAIT cells] The "CAR-MAIT cells" of the present invention are mucosal cells that express chimeric antigen receptors (CARs). This refers to tangent invariant T (MAIT) cells.

[0028] Mucosa-associated invariant T (MAIT) cells are a subset of T cells. In humans... MAIT cells, found in the blood, liver, and mucous membranes, play a role in preventing microbial activity and infection. To control. MAIT cells have semi-invariant T cell receptor α (TCRα) chains (in humans) Vα7.2-Jα33 / 20 / 12) has the characteristic of being able to bind to a limited number of TCRβ chains. This semi-invariant TCR is monomorphic, highly conserved MHC class I activity. Restricted by the MR1 molecule. The usual recognition of the classical MHC-peptide complex. In contrast to T cells, MAIT cells are presented by MR1, and are either 5-OP-RU or 5 - Recognizes microorganism-induced riboflavin precursor derivatives such as OE-RU in adult MAIT cells. CD3 + CD4 - Vα7.2 + CD161 hi gh (or IL-18a) high Or CD26 high ) and the corresponding stain It can be easily identified using this method. Upon recognizing the MR1 ligand, MAIT cells target inflammatory sites. It releases kine (IFNγ, TNFα, IL-17) and perforin-dependent cells of target cells. They mediate cytotoxicity. MAIT cells selectively localize to the liver and mucous membranes, including the lungs and intestines. Furthermore, it is abundant in adult peripheral blood (1-10% of T cells) and very scarce in umbilical cord blood (T <0.1% of cells.

[0029] This invention relates to the use of homogeneous, heterogeneous cells, i.e., cells intended for or ultimately intended for cell therapy. MAIT cells isolated from and / or otherwise prepared from subjects other than the target subject. Regarding use. As used herein, "allogenes" The term "c)" refers to various individuals of the same species, in this case, the donor and recipient are genetically related. This refers to cells or tissues obtained from individuals that are not identical to the second target. In some embodiments, the second target However, it expresses the same HLA genotype as the first subject. CAR-MAIT is associated with GVHD. It is shown not to mediate damage to host tissue. (A) potent effector cells and Therefore, CAR-MAITs are allogeneic sett It is useful as a universal tool for adoptive immunotherapy in [the context of the disease].

[0030] In relation to the present invention, MAIT cells are genetically modified to become chimeric antigen receptors. The body (CAR) is manifested.

[0031] "Chimera antigen receptors (CARs)" are artificial T as used herein. This refers to cell receptors, chimeric T cell receptors, or chimeric immune receptors, and specific immune effectors. Genetically modified receptors with artificial specificity to cells, specifically MAIT cells. It includes.

[0032] CARs are typically connected by a transmembrane domain, and their external domain (extracellular domain) Includes the in-domain and endodomain (cytoplasmic domain). The external domain expressed on the surface may consist of an antigen-binding domain or a receptor domain, and optionally, an antigen-binding domain. It includes a binding domain and a spacer (or hinge) region that connects the binding domain to the transmembrane domain. The domain is typically a hydrophobic α-helix spanning the lipid bilayer of the cell membrane. The endodomain of AR is an intracellular signal that induces activation of MAIT cells upon antigen binding. It consists of signaling modules. The endodomain is a signaling domain, as described below. It may include several elements.

[0033] (Antigen-binding domain) The extracellular domain of CAR is an antigen-binding domain that specifically binds to or recognizes the target antigen. Includes "in".

[0034] As used herein, "bind" or "binds" relates to peptides, polypeptides, proteins, fusion proteins, and antibodies (including antibody fragments) that recognize and contact an antigen. Preferably, it relates to antigen-antibody type interactions. By "specifically binds," it is meant that the antigen-binding domain of the CAR recognizes a specific antigen and does not substantially recognize or bind other molecules in a given sample. "Specifically binds" depends on the presence of a specific structure (e.g., an antigenic determinant or epitope). As used herein, the term "specifically binds" means contact with an affinity of at least 10 M between the antigen-binding domain of the CAR and the antigen. In some embodiments, the antigen-binding domain of the CAR binds with an affinity of at least about 10 M, preferably 10 M, 10 M, 10 M. The binding affinity can be measured by any method available to those skilled in the art, particularly surface plasmon resonance (SPR). peptides, proteins, fusion proteins, and antibodies (including antibody fragments) that recognize and contact an antigen. Preferably, it relates to antigen-antibody type interactions. By "specifically binds," it is meant that the antigen-binding domain of the CAR recognizes a specific antigen and does not substantially recognize or bind other molecules in a given sample. Preferably, it relates to antigen-antibody type interactions. "Specifically binds" depends on the presence of a specific structure (e.g., an antigenic determinant or epitope). As used herein, the term "specifically binds" means contact with an affinity of at least 10 M between the antigen-binding domain of the CAR and the antigen. Preferably, it relates to antigen-antibody type interactions. -6 Preferably, it relates to antigen-antibody type interactions. In some embodiments, the antigen-binding domain of the CAR binds with an affinity of at least about 10 -7 M, preferably -8 10 -9 M, -10 10 M. The binding affinity can be measured by any method available to those skilled in the art, particularly surface plasmon resonance (SPR). The binding affinity can be measured by any method available to those skilled in the art, particularly surface plasmon resonance (SPR).

[0035] In one embodiment, such an antigen-binding domain is an antibody, preferably a single-chain antibody. Preferably, the antibody is a humanized antibody. In particular, such an antigen-binding domain is a fragment antigen-binding (Fab) fragment, F(ab’)2 fragment, F ab’ fragment, Fv fragment, recombinant IgG (rlgG) fragment, single-chain antibody fragment, single-chain variable fragment (scFv), single-domain antibody (e.g., sdAb, sdFv, nanobody) fragment, bispecific antibody, and single-chain antibody fragment, single-chain variable fragment (scFv), single-domain antibody (e.g., sdAb, sdFv, nanobody) fragment, bispecific antibody, and This is an antibody fragment selected from a multispecific antibody formed from antibody fragments. In certain embodiments, the antibody has a variable heavy chain region and / or variable light chain region, such as scFv. It is a single-chain antibody fragment containing a region. In particular, such antigen-binding domains are Fab And selected from scFv.

[0036] In embodiments where the antigen-targeting domain is scFv, the scFv is a mobile linker From the variable heavy chain (VH) and variable light chain (VL) regions of the antigen-specific mAb linked by this, It can be induced. scFv retains the same specificity and similar affinity as the induced full antibody. The peptide linker connecting the VH domain and VL domain of scFv is one of the possible The carboxyl terminus of one variable domain is connected to the amino terminus of the other variable domain, VH-VL Without compromising the fidelity between the pairing and the antigen-binding site, They join. The peptide linker can vary in amino acid length from 10 to 30. One implementation form In this state, the scFv peptide linker is a Gly / Ser linker, and the amino acid composition Column Gly-Gly-Gly-Ser (Sequence ID: 1) or Gly-Gly-Gly-Gl Contains one or more repetitions of y-Ser(sequence number: 2).

[0037] The extracellular domain of a CAR may contain one or more antigen-binding domains.

[0038] In certain embodiments, CAR specifically binds to tumor-associated antigens (TAAs). CAR is any TAA expressed on the surface of tumor cells, preferably CD19, GD2 , EGFR, CD20, CD22, CD33, CD138, CD52, CD30, ROR 1, HER2, EpCAM, MUC-1, MUC5AC, BCMA, CD38, SLAM F7 / CS1, CD123, IL-13Ra2, HER2, LeY, MUC16, PSM TAA specifically binds to CD19, CD20, CD22, and C. D33, CD138, BCMA, CD38, SLAMF7 / CS1, IL-13Ra2 or It is HER2.

[0039] In certain embodiments, CAR specifically binds to CD19.

[0040] In another specific embodiment, CAR is an intracellular oncoprotein or intracellular tumor-related protein. Antigens, especially WT-1, NY-ESO-1, MAGE, PRAME, RAS, and mesothelin. Targeting c-Met, CEA, CSPG-4, EBNA3C, CA-125, and GPA7. In particular, the intracellular oncoplastic protein or tumor-associated antigen is treated on the cell surface. It is then expressed as a peptide that binds to the histocompatibility (HLA) module.

[0041] In another specific embodiment, CAR-MAIT cells are used for the treatment of infectious diseases. In this embodiment, the CAR can target pathogenic components expressed on the surface of infected cells. Preferably, CAR targets viral proteins expressed on the cell surface, and Preferably, the CAR targets the HIV envelope protein.

[0042] In another specific embodiment, CAR-MAIT cells are used for the treatment of autoimmune diseases. In this embodiment, CAR is preferably an autoimmune B cell expressed on the surface of an autoimmune B cell. By targeting responsive antibodies, for example, CAR targets desmoglein 3 in pemphigus vulgaris. It targets autoreactive antibodies.

[0043] (Spacer or hinge domain) CARs are optionally spacers that link the antigen-binding domain to the transmembrane domain or Includes hinge domains.

[0044] In some embodiments, the CAR is located between the antigen-binding domain and the transmembrane domain and / Alternatively, it includes a hinge sequence between the transmembrane domain and the intracytoplasmic domain. Those skilled in the art will know the hinge You will understand that the sequence is a short sequence of amino acids that promotes flexibility.

[0045] In particular, the spacer or hinge domain that links the antigen-binding domain to the transmembrane domain is The antigen-binding domain should be flexible enough to be oriented in a way that allows it to recognize the antigen. It is designed.

[0046] The hinge is located in the immunoglobulin Fc region, for example, the IgG1 Fc region, the IgG2 Fc region. region, IgG3 Fc region, IgG4 Fc region, IgE Fc region, IgM Fc region, Or it may be derived from or include at least a portion of the IgA Fc region. In the embodiment, the hinge domain is IgG1 within the range of CH2 and CH3 domains. A decrease in the Fc region of IgG2, IgG3, IgG4, IgE, IgM, or IgA immunoglobulins. It includes at least a part of it.

[0047] The hinges shown as examples are not limited to CD8a hinges, CD28 hinges, and lgG1 / lg hinges. G4 (hinge-Fc portion) sequence, IgG4 hinge alone, and linked to CH2 and CH3 domains. These include IgG4 hinges that have been modified or IgG4 hinges linked to the CH3 domain, and they , Hudecek et al. (2013) Clin. Cancer Res.,19:3153 International Publication No. 2014 / 031687, U.S. Patent No. 8,822,647 Or as described in U.S. Patent Application Publication No. 2014 / 0271635. Hinge As a domain, the present invention relates to the usable CD8a residues 118-178 (GenBan k Accession No. NP_001759.3), CD8 residues 135-19 5 (GenBank Accession No. AAA35664), residue 3 of CD4 15~396(GenBank Accession No.NP_000607.1) , or residues 137-152 of CD28 (GenBank Accession No. N P_006130.1) relates to all or part of it. Also, as a spacer domain, anti A portion of the constant region of the H chain or L chain (the CH1 region or CL region) can be used. Furthermore, The spacer domain may be an artificially synthesized sequence.

[0048] In some embodiments, for example, the hinge array is derived from CD8α molecules or CD28 molecules. To be led astray.

[0049] (Transmembrane domain) The transmembrane domain of CAR functions to fix receptors to the cell surface. Domain selection may be influenced by adjacent spacers and intracellular sequences.

[0050] In some embodiments, transmembrane domains are derived from natural or synthetic raw materials. When natural, the domain in some embodiments is any membrane-bound or transmembrane protein. It is derived from the cell. The transmembrane region consists of the α, β, or ζ chains of the T cell receptor, CD28, and CD3. ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, C D137, CD154, ICOS / CD278, GITR / CD357, NKG2D, and This includes those derived from DAP molecules (i.e., at least their transmembrane regions). (m). Alternatively, the transmembrane domains in some embodiments are synthetic. In this case, the synthetic transmembrane domain contains mainly hydrophobic residues such as leucine and valine. In some embodiments, a triplicate of phenylalanine, tryptophan, and valine is synthesized. You will find them at the ends of each transmembrane domain. Transmembrane domains are thermal in the membrane. It is mechanically stable. It has a single α-helix, a transmembrane β-barrel, and gramicidin A It may be a β-helix or any other structure.

[0051] Optionally, short oligo- or polypeptide linkers are preferably of an amino acid length Between 2 and 10, and between the transmembrane domain and the intracellular signaling domain of CAR. A linkage can be formed. A glycine-serine pair can provide a suitable linker.

[0052] (Intracellular domain) "Intracellular domain," "cytoplasmic domain," and "intracellular signal transduction domain" The terms used herein are interchangeable. The role of the intracellular domain of CAR is, As soon as the extracellular domain recognizes the antigen, it generates an activation signal to MAIT cells. In particular, the intracellular domain of CAR is essential for the normal effector function of MAIT cells. To trigger or elicit at least one activation.

[0053] Examples of intracellular domain sequences particularly used in the present invention include lymphocyte receptor chains and TCRs. / CD3 complex protein, Fc receptor subunit, IL-2 receptor subunit, C D3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD 79a, CD79b, CD66d, CD278(ICOS), FcsRI, DAP10, This includes those derived from the intracellular signaling domain of DAP12. The intracellular domain contains a cytoplasmic signaling sequence derived from CD3ζ, which is particularly important. It is preferable.

[0054] The intracellular domain of CAR is either alone or combined with a co-stimulatory domain (CD3ζ). It can be designed to include signal transduction domains (such as the Gnar signaling domain). These can be defined as cell surface molecules required for an effective response of lymphocytes to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), and OX40. (CD134), CD30, CD40, CD244 (2B4), ICOS, Lymphocyte function Related antigens-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and , CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, It includes ligands that specifically bind to MyD88, BTNL3, and NKG2D. The intracellular signaling portion of the co-stimulatory domain can function alone or in combination with other co-stimulatory domains. They can be used together. In particular, CAR is CD27, CD28, 4-1BB (CD137 ), OX40(CD134), CD30, CD40, CD244(2B4), ICOS, Two of the following groups are selected from the group consisting of LFA-1, CD2, CD7, LIGHT, NKG2C, and B7-H3. One or more co-stimulatory domains, as well as CD83, CD8, CD4, b2c, CD80, CD8 6. Specifically binds to DAP10, DAP12, MyD88, BTNL3, and NKG2D. This may include any combination of ligands.

[0055] Therefore, for example, CAR is a signaling domain such as the CD3ζ signaling domain. CD28 and CD40, CD28 and 4-1BB (CD137), CD28 and OX Two co-stimulatory domains selected from 40 (CD134) and CD28 and LFA-1. It can be designed to include the following.

[0056] "First-generation CARs" contain a single signaling domain. One additional co-stimulus CARs that include a signal transduction domain along with the domain are called "second generation," and On the other hand, those containing a signal transduction domain along with two additional co-stimulatory domains are "third This is referred to as a "generation." For example, first-generation CARs use only CD3ζ chains, a single signal transmission This includes the domain. Second and third generation CARs are CD28, CD27, OX-4 One or more additional co-stimulatory signals, such as 0 (CD134) and 4-1BB (CD137). It consists of Gnar transmission domains. For example, the second generation CAR has CD3ζ and CD28 sig It may include a primary transmission domain, while the third generation CAR includes CD3ζ, CD28 and OX- It may include either 40 (CD134) or 4-1BB (CD137).

[0057] As described above, the CAR of the present invention is a first-generation, second-generation, or third-generation CAR. Good. Preferably, the CAR is a second or third-generation CAR.

[0058] "TRUCKs" represent the recently developed "fourth generation" of cars. TRUCKs(T cells redirected for universal Cytokine killing generates gene transfer products that accumulate in target tissues. CAR-redirected T cells (CAR-red) are used as vehicles to release (irrected T cell). Products, such as pro-inflammatory cytokines, Once T cells are activated by CAR, they can be constitutively generated or induced. Enzymes or Other substances, such as immunomodulatory molecules, are similarly activated by CAR-redirected T cells in target lesions. It can be produced and deposited in this. This strategy involves two distinct transgenes, for example, (i) CA R and (ii) a cell activation response promoter linked to cytokines such as IL-12 It is necessary to express CAR. Therefore, immunostimulatory cytokines such as IL-12 are necessary. It is secreted during meetings.

[0059] In certain embodiments, the CAR is a fourth-generation CAR as defined above.

[0060] In a particular embodiment, multiple CARs, such as CARs that bind to various antigens, are a single It can be expressed by MAIT cells.

[0061] [Generation of CAR-MAIT cells] (Acquisition of MAIT cells) CAR-MAIT cells are generated from cell cultures or from individual subjects or blood banks. The procedure includes the step of preparing MAIT cells from a blood sample. MAIT cells are preferably This is derived from potential donors, particularly healthy donors.

[0062] Cells are extracted from blood samples (including peripheral blood and umbilical cord blood), and are separated, centrifuged, and genetically modified. Sub-operation, washing and / or incubation, etc., obtained from one or more processing steps. It can be obtained from a sample. In certain embodiments, a sample from a donor is Includes peripheral blood mononuclear cells (PBMCs).

[0063] In certain embodiments, MAIT cells are, but are not limited to, peripheral blood and peripheral blood mononuclear cells. (PBMCs), bone marrow, umbilical cord blood, and other samples are collected from any site present in the target.

[0064] In certain embodiments, MAIT cells are removed by apheresis, particularly leukocyte apheresis. They are collected.

[0065] (Isolation of MAIT cells) As is known to those skilled in the art, various methods exist for isolating immune cells from a target. It is readily available, or, for example, Life Technologies' Dyna beads(registered trademark) system; STEMcell Technologies Inc. EasySep®, RoboSep, RosetteSep, SepMat e(registered trademark); MACS(registered trademark) cell isolation technology of Miltenyi Biotec. Cell surface marker expression and other commercially available cell separation and isolation kits (e.g., IL, R) Using ISOCELL from Pierce, Inc. of Rockford, it is possible to make it conform to the present application. Yes, it is possible. MAIT cells are available in beads or kits, with a cell surface The marker may be isolated using other binders specific to the marker.

[0066] In some embodiments, isolation is affinity or immunoaffinity-based. In some aspects, isolation involves cell expression or one or more markers, typically fine This includes isolating MAIT cells based on the expression level of cell surface markers, and the MA Isolation of IT cells involves, for example, antibodies or binding parts that specifically bind to such markers. Incubate with the nare, then generally a washing step, and then antibody or Cells to which the binding partner is bound, or cells not bound to the antibody or binding partner. This is done by separating the reagent-bound MAIT cells. Positive selections are retained for further use, and / or antibodies or conjugation partners. - This is based on negative selection, which retains MAIT cells that are not bound to this condition. This can be done. In some embodiments, the separation step is repeated, and there the positive A fraction from one step in which a positive or negative choice was made is then used in subsequent positive or negative decisions. It undergoes another separation step, such as negative selection.

[0067] In some embodiments, populations of MAIT cells are collected via flow cytometry. The cells are then concentrated, and there, cells stained for multiple cell surface markers are in the fluid flow. This includes a fluorescent cell sorter (FACS) and / or a preparative scale (FACS). Microelectromechanical systems (MEMS) chips, for example, for flow cytometric detection. It is transported in combination with the system. In this way, multiple markers can be reached simultaneously. Based on this, a positive or negative choice can be made.

[0068] In some embodiments, the isolation of MAIT cells is performed using CD3, CD8, Vα7.2, and CD 161 CD26 and / or IL-18Ra (CD218a) positive or high Surface expression and / or negative expression of CD4, and / or optionally NKG2D or This is based on the presence of the NKp30 receptor.

[0069] In certain embodiments, MAIT cells are subjected to antibodies, particularly anti-Vα7.2 antibodies and anti--I antibodies. A vi coated with L18Rα, anti-CD161, or anti-CD26 antibody Positive isolation is performed using a sample.

[0070] Isolated MAIT cells can be used immediately or stored for a certain period of time by freezing, etc. It is possible.

[0071] (Activation and increase of MAIT cells) Whether before or after modifying the genes of MAIT cells to express the desired CAR The cells may be activated and / or increase in number. In some embodiments, MAIT cells are It is incubated under stimulating conditions or in the presence of an stimulant. Such conditions are recombinant For example, to induce the proliferation, increase, activation, and / or survival of MAIT cells by introducing antigen receptors. To mimic antigen exposure and / or stimulate cells for genetic engineering. , including those designed. Conditions include one or more specific culture media, temperature, oxygen content, carbon dioxide. Content, time, drugs, e.g., nutrients, amino acids, antibiotics, ions and / or cytokines Stimulatory factors such as chemokines, antigens, binding partners, fusion proteins, and recombinant soluble receptors. This may include any other drugs designed to activate MAIT cells.

[0072] For example, MAIT cells, along with anti-CD3 antibodies and / or anti-CD28 antibodies, It can be incubated under conditions that stimulate its growth.

[0073] In some embodiments, the MAIT cells of the present invention are co-cultured with tissue or cells. This allows for in vitro proliferation. In some embodiments, MAIT cells are non It is increased by co-culturing with feeder cells such as dividing PBMCs. In this case, non-dividing feeder cells are irradiated PBMC feeder cells, especially autologous or similar. This may include irradiated PBMCs from different types of systems.

[0074] In certain embodiments, MAIT cells are irradiated PBMCs from autologous or allogeneic strains. and IL-2, IL-7, IL-12, IL-18 and / or IL-15 cytokines CD3 / CD28 stimulation in the presence of [specific substance] increases these receptors in vitro.

[0075] In certain embodiments, MAIT cells are 5-OP-RU and / or 5-OE-RU MAIT cells are increased and / or activated in vitro in the presence of such cell-activating ligands.

[0076] In another specific embodiment, the method is used to obtain, in particular, cells from a donor cell sample from the donor The procedure includes a step of selectively increasing MAIT cells in vitro from PBMCs. In vitro MAIT cell increase was observed in the presence of synthetic 5-OP-RU and also selectively. This can be done by culturing PBMCs from a donor along with cytokines. In particular, PBMCs from donors are used to obtain 5-OP-RU and IL-2 such as rhuIL-2. Cultivate in the presence of [the substance].

[0077] In certain embodiments, MAIT cells are preferably administered to the patient ex vivo. Prior to that, for at least approximately 5 days, preferably approximately 10 days or more, more preferably approximately The increase is then allowed for 15 days or more, most preferably approximately 20 days or more.

[0078] In another embodiment, MAIT cells were less increased before administration to the patient compared to day 0. At most about 100 times, preferably at least about 200 times, more preferably at least about 400 times, preferably at least about 600 times, more preferably at least about 1000 times, More preferably, it was increased by at least about 1500 times.

[0079] In some embodiments, the preparation method includes a freezing step, for example, isolation, incubation This includes cryopreserving cells either before or after (genetic) manipulation.

[0080] (Transduction and selection of cells) In certain embodiments, MAIT cells are phenotyped to express one or more CARs. To implement.

[0081] Nucleic acid constructs encoding CARs are introduced into MAIT cells as Naked DNA or in a suitable manner. It is understood that it can be introduced into a vector.

[0082] Naked DNA is generally contained within plasmid expression vectors in an orientation suitable for expression. This refers to the DNA that codes for the chimeric receptor. It is a substance that introduces nucleic acid constructs into host cells. The rational method involves calcium phosphate precipitation, lipofection, particle bombardment, and microinjection. This includes methods such as injection, electroporation and similar processes. Macromolecular complexes, nanocapsules, Microspheres, beads, and oil-in-water emulsions, micelles, mixed micelles and lipo Other methods can be used, including lipid-based systems containing osomes.

[0083] In certain embodiments, nucleic acid constructs encoding CARs are retroviruses, adenoviruses, and other substances. Viral vectors such as viruses, adeno-associated viruses, herpesviruses, and lentiviruses. —to be introduced into MAIT cells. In particular, the vector is a lentiviral vector. ru.

[0084] Various assays were performed to confirm the presence of CAR sequences in MAIT cells. Such assays include, for example, Southern and Northern blotting, and RT-PCR. and well-known "molecular biological" assays such as quantitative PCR, and the presence or absence of specific peptides. Biochemical assays such as the detection of CA on the cell surface, and flow cytometry. This includes "immunological" assays such as the detection of R expression.

[0085] [Pharmaceutical composition] Further description is provided for pharmaceutical or veterinary compositions containing the aforementioned CAR-MAIT cells.

[0086] The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The “acceptable carriers” referred to are any known compounds or pharmaceutically or veterinarily controlled substances to those skilled in the art. This refers to combinations of compounds known to be useful in the formulation of scientific compositions. Specific implementations In terms of form, the term "pharmaceutically acceptable" is used by the federal or state government, that is, This is a regulatory authority listed in the United States or the European Pharmacopoeia, or is used in animals and humans. This means that it is approved for use by other generally recognized pharmacopoeias.

[0087] The term "carrier" is used along with the diluent to which CAR-MAIT cells are administered. This refers to an adjuvant, additive, or vehicle. Such pharmaceutical carriers include water and stone. Oils, animal, plant or synthetic derived, including peanut oil, soybean oil, mineral oil, sesame oil and similar It can be used as a sterilizing solution for oils, etc. Water is used when administering pharmaceutical compositions intravenously. It is a preferred carrier. Physiological saline and aqueous dextrose and glycerol solutions are also suitable. Furthermore, it can be used as a liquid carrier, particularly for injectable drug solutions. The additives include starch, glucose, lactose, sucrose, and sodium stearate. Glycerol monostearate, talc, sodium chloride, dried skim milk, glyceryl The composition includes rollol, propylene glycol, water, ethanol, and similar substances. If desired, a small amount of a humectant, emulsifier, or pH buffer may also be included.

[0088] In a further embodiment, the pharmaceutical or veterinary composition is such that each population has various CARs It may include one or more populations of MAIT-cells that express [specific expression].

[0089] Pharmaceutical or veterinary compositions include parenteral, intravenous, intramuscular, subcutaneous administration and similar methods. It can be formulated for any conventional route of administration.

[0090] In the case of parenteral administration, the pharmaceutical or veterinary composition is preferably administered intravenously. It can be done.

[0091] Ideally, the drug should not have any adverse effects on the desired immune-enhancing effect of CAR-MAIT cells. Academically acceptable forms are used.

[0092] Pharmaceutical or veterinary compositions, in amounts that are therapeutically or prophylactically effective, etc., for treating a disease or condition. Contains CAR-MAIT cells in an amount effective for treating or preventing the disease. A single dose of the composition in the desired amount. It can be delivered by administration, multiple doses of the composition, or continuous administration of the composition. CAR-MAIT The dosage of cells can be determined by standard procedures well known to those skilled in the art. The patient's physiological data... (For example, age, size, weight and health, as well as the recipient's weight, and any other medications being used.) The type of treatment, frequency of treatment, and nature of the desired effect, as well as the route of administration, and the appropriate dose for the patient. This needs to be considered when determining the effective therapeutic dose to administer.

[0093] [use] This invention relates to the use of MAIT cells, which are heterogeneous to the target, in the treatment of the target disease. Regarding CAR-MAIT for this purpose.

[0094] In a preferred embodiment, the CAR-MAIT cells of the present invention are used in therapeutic products, ideally It is used as a "commercially available" product.

[0095] In one embodiment, the disease or disorder to be treated is a proliferative disorder or disorder, preferably cancer, infection Infectious diseases or disorders, preferably infections caused by viruses, bacteria, or fungi, inflammatory diseases or disorders A pathological condition selected from harms and immune diseases or disorders, preferably autoimmune or autoimmune diseases. ru.

[0096] In certain embodiments, CAR-MAIT cells and / or pharmaceutical compositions are HIV-infected. Infection, hepatitis A, B, or C virus infection, herpesvirus infection (e.g., VZV, HSV-) 1. Infections of HSV-6, HSV-II, CMV, and EBV, and influenza viruses, etc. It is suitable for treating viral infections. Examples of non-viral infections include aspergillosis and cystitis. Associated with angidadian disease, coccidioidomycosis, and cryptococcosis and histoplasmosis. This may include chronic fungal diseases such as [mention specific diseases]. Non-specific examples of chronic bacterial infections include: It can be Chlamydia pneumoniae, Listeria monocytogenes, and Mycobacterium tuberculosis. Infections are also sexually transmitted infections. For example, this includes chlamydia, gonorrhea, diphtheria, or cholera.

[0097] In certain embodiments, CAR-MAIT cells and / or pharmaceutical compositions are autoimmune It is suitable for treating abnormalities. Autoimmune abnormalities affect almost all organ systems in the subject, not limited to... However, diseases of the nervous system, gastrointestinal system, endocrine system, as well as the skin and other connective tissues, eyes, blood and It can affect blood vessels. Examples of autoimmune diseases include, but are not limited to, pemphigus vulgaris, Hashimoto's disease, Systemic lupus erythematosus, Sjögren's syndrome, Graves' disease, scleroderma, rheumatoid arthritis, This includes multiple sclerosis, myasthenia gravis, and diabetes mellitus.

[0098] In certain embodiments, CAR-MAIT cells and / or pharmaceutical compositions are used for the treatment of cancer. It is intended for use in the following contexts. Therefore, the present invention also applies to subjects where this is necessary. A method to inhibit the growth of cancer and / or in patients in need of such inhibition of cancer formation and / or This concerns methods for preventing metastasis.

[0099] In certain embodiments, CAR-MAIT cells are used to treat cancer recurrence. It is.

[0100] In particular, cancer can be a solid tumor or a hematopoietic carcinoma.

[0101] In particular, cancer is a solid tumor such as sarcoma and cell tumor, which includes fibrosarcoma, myxosarcoma, Liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synoviomas, mesotheliomas, Ewing's sarcomas, smooth muscle Sarcoma, rhabdomyosarcoma, colon cancer, lymphoid tumors, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, liver cancer Cystic carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, brown cell carcinoma Sebaceous gland tumors, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic lung cancer, renal cell carcinoma, hepatocellular carcinoma, bile ductus Tubal carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular cancer, seminoma, bladder cancer, melanoma , and central nervous system tumors (gliomas, etc. (brainstem gliomas and mixed gliomas, etc.)), glioblastomas (pleomorphic Also known as glioblastoma, astrocytoma, central nervous system lymphoma, germ cell tumor, medulloblastoma, Schwann cell tumor, craniopharyngioma, ependymoma, pineal gland tumor, hemangioblastoma, acoustic neuroma, oligodendritis This includes tumors, meningiomas, neuroblastomas, retinoblastomas, and brain metastases.

[0102] Hematopoietic carcinoma is cancer of the blood or bone marrow. An example of blood (i.e., hematogenous) carcinoma is acute white blood cancer. Blood diseases (acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic leukemia, pre- Myeloid, myelomonocytic, monocytic and erythroleukemia, etc.), chronic leukemia (chronic myeloid (granulocytic) (Leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia, etc.), polycythemia vera, lymphoma, Dikin's disease, non-Hodgkin lymphoma (painless and high-grade forms), multiple myeloma, Walden Ström's hypergammaglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and This includes leukemia, including spinal cord dysplasia.

[0103] In certain embodiments, the disease is a hematological malignancy, more preferably leukemia, lymphoma or It is myeloma.

[0104] In certain embodiments, MAIT cells are administered to immunocompromised subjects. In a host, allogeneic cells may be rejected by the host's immune system. Therefore, To prevent interspecies rejection of CAR MAIT cells, the host immune system is preferably... , to effectively suppress or impair.

[0105] In particular, CAR-MAIT cells are administered after pretreatment to induce immunodeficiency in the target patient.

[0106] In particular, pretreatment may involve myeloablative or non-myeloablative treatment, or lymphocyte apheresis chemotherapy. That is the case.

[0107] In particular, pretreatment involves chemotherapy and / or radiotherapy and / or lymphocyte depletion antibodies.

[0108] Immunosuppressants may be used during pretreatment, such as azathioprine and methotrexate. Xate, 5-fluorouracil, cyclophosphamide, fludarabine, pentostatin Romidepsin (FR901228), anti-CD52 (CAMPATH, alemtuzumab) ), anti-CD3, anti-CD20 (rituximab) antibody or other antibody therapy, cyclosporine These include FK506, rapamycin, mycophenolic acid, and steroids.

[0109] In a particular embodiment, the disease is a hematological malignancy, and MAIT cells are hematopoietic stem cells ( It is administered before hematopoietic stem cell (HSC) transplantation or immediately after hematopoietic stem cell (HSC) transplantation.

[0110] In particular, CAR-MAIT cells are used in hematological malignancies at the minimal residual disease (MRD) stage. It is for therapeutic purposes.

[0111] Minimal residual disease (MRD) refers to cancer cells that remain in the body after treatment and are a major cause of recurrence. This refers to leukemia cancer cells. In particular, the term minimal residual disease (MRD) refers to conventional cells It is used to describe low-level diseases that cannot be detected by morphology (e.g., Brue ggemann and Kotrova; Blood Advances 2017 (See 1:2456-2466).

[0112] In particular, CAR-MAIT cells are effective in treating residual leukemic or disseminated tumors within the therapeutic concentration range. It is used to remove cells. CAR-MAIT cells are used in patients with hematological malignancies. As a bridge to transplantation, or as a treatment for minimal residual disease (MRD) or relapse after hematopoietic stem cell transplantation. It can be used as a treatment (or as an adjunct therapy for high-risk patients).

[0113] CAR-MAIT cell administration is any conventional method, including injection, infusion, intracellular transplantation, or transplantation. The method may be used. The compositions described herein may be administered to the patient by intravenous injection or by tumor It may be administered intraulsively, intranodulate, or intraperitoneally. In one embodiment, the cell composition is preferably It is administered by intravenous infusion. [Examples]

[0114] [Materials and Methods] (Flow cytometry) Peripheral blood mononuclear cells from healthy donors are collected using the Ficol density gradient centrifugation method (Eurobio). ) Isolate and use immediately or freeze. Multi-parameter 10 color flow filter Thread metric analysis was performed for 15 minutes at 4°C using the following antibody combination: anti-CD4 5. Krome Orange, anti-CD3 ECD, anti-CD8β PE or ECD, anti-T CR Vα24 PE Cy7 (all from Beckman Coulter); Anti-T CR Vα7.2 FITC or APC (clone 3C10), anti-CD161 Pe rCP Cy5.5 or BV421, anti-CD4 AF700 (Biolegend); Anti-CD161 APC, anti-CD3 APC Vio770 (Miltenyi Biot ec); Anti-CD4 APC AF750 (Invitrogen); Anti-CD3 Pe rCP (BD Biosciences); Zombie aqua live / de ad (BioLegend Inc.). Data totaling at least 100,000 events. Navios flow cytometer (Beckman Co.) used to collect data at Ivegate Acquired by Ulter Inc. The gate is isotype and fluorescence minus one (FM). O) Confirmed by staining. The gating method was CD45 paired side scattering, and MAIT cells. CD3 + CD4 - CD161 high Vα7.2 + It was confirmed to be a cell. The absolute number, in the same sample, CountBright Absolute Co The calculations were performed using nting Beads (Invitrogen). The analysis was performed using FlowJo software.

[0115] (Mixed lymphocyte reaction) Responder PBMCs are carboxyfluorescein succinimidyl ester (CF Labeled with SE) 1 μM, 2 × 10 5 Responder cells labeled with CFSE were divided into 2×1 0 5 With or in conjunction with irradiated allogeneic or autologous (negative control) PBMCs Was cultured for 6 days in a flat-bottom 96-well plate. Responding T cells were identified by gating on live CD3 cells at the end of the culture + period. Proliferation of normal T (Tconv) and MAIT cells was quantified by CFSE dilution (%CF SE cells). low

[0116] (Adoptive transfer of xenogeneic cells) NSG mice (Jackson laboratory, Bar Harbor, MI ) were housed in the pathogen-free animal facility of the Institut de Recherche Saint-Louis (Par is). Irradiation (1. 3 Gy) of 8- to 10-week-old female NSG was performed 24 hours before injecting 3 × 10 6 human PBMCs into the caudal vein. Mice were evaluated for survival daily and weighed weekly. The development of acute GVHD was monitored based on weight loss, posture (hunchback) and decreased activity. Mice were euthanized when weight loss fell below 15% (mean ± 45 days). Peripheral blood, spleen, bone marrow, liver, lung and intestine were collected and tissues were mechanically dissociated and the single cell suspension was filtered through a cell strainer (70 μ m, BD Biosciences). The proportion of CD3 T cells and MAIT cells in human (CD45 + ) leukocytes was determined by flow cytometry at 45 days post-transplantation in the early inoculation.

[0117] (In vitro increase of MAIT cells) PBMCs (10 6 / ml) from healthy volunteers were inoculated into round-bottom 96 wells and in R PMI-10% human serum with 300 nM synthetic 5-OP-RU (Institut C​​ In urie, by chemical synthesis of 5-A-RU (Department of Chemistry, F. Schmidt), methylgly oxal (manufactured by mixing at the same molar concentration as methyglyoxal) and cultured in the presence of 100 IU / mL of IL-2. After 6 days, the cells were counted, and as described above the viability and the number of MAIT cells were evaluated by flow cytometry. The cells were grown with 100 nM of 5-OP-RU and 100 IU / mL of IL-2 every 3-4 days until day 1 7.

[0118] (Transduction of MAIT cells) CD3 T cells were immunomagnetically purified from PBMCs (human CD3 + T C ell Isolation Kit, Miltenyi Biotec) and activated with anti-CD3 / CD28 beads for 24 hours (1:1 ratio, Dynabeads Human T-Activator CD3 / CD28, ThermoFisher Scient ific). They were transduced with a lentiviral vector encoding a tumor antigen-specific CAR and a fluorescent reporter protein (blue fluorescent protein, BFP) to enable monitoring of CAR+ T cells. The cells were resuspended in fresh X-VIVO 15 medium (Lonza) supplemented with 5% human serum and 100 IU / mL IL- 2. The transduction efficiency was determined 6 days later by flow cytometry as the percentage of BFP

[0119] cells among normal CD + 3 T cells and MAIT cells.

[0120] (In vitro efficacy of CAR-MAIT cells) 5 × 10 expressing an antigen targeted by CAR​4 Luciferase-expressing tumor cells, Fluorescence-activated cell sorting of CAR-T cells, CAR-MAIT cells, or pseudo-phenotypes Along with the introduced MAIT cells, various E / T ratios were used in 96-well microplates. The cells were incubated for 4 hours. Target cell viability was quantified by measuring luminescence intensity.

[0121] [result] 1. MAIT cells do not proliferate in vitro in mixed lymphocyte reaction (MLR). MAIT cells recognize allogeneic cells and contribute to allogeneic immune responses: in vitro possibility To examine the sex, flow cytometry based on the MLR method was used to examine the sex of a third party of the same species. The proliferation of MAIT cells was quantified in the presence of heterogeneous cells. As shown in Figure 1 (A and B), M Unlike normal CD3 T cells, AIT cells do not proliferate in response to allogeneic cells or It demonstrated minimal growth.

[0122] 2. MAIT cells are not involved in GVHD induction in humanized mouse models. To investigate the in vivo potential of MAIT cells as a contributing factor to acute GVHD, PBMC transplantation in irradiated immunodeficient NOD-Scid-IL-2Rγnull(NS) G) Used in mice (Figure 2). In this model, xenophobic and lymphopenic environments were used. The engraftment, increase, and activation of human T lymphocytes can lead to symptoms of acute xenograft-versus-host disease (GVHD). (±45 days after transplantation) Infiltration of human cells into various tissues, ultimately leading to death.

[0123] 3.10 6 Irradiated (1,3 Gy) NSG mice injected with human PBMCs were subjected to GVH. Monitor for the progression of D (weight loss, hunched posture, wrinkled fur, decreased activity). When the weight loss was less than 15% (average 45 days after transplantation), the mice were sacrificed. Human CD45 in the peripheral blood, spleen, bone marrow, liver, colon and lung of the diseased mice + white blood cell flow cytometry analysis could not detect the presence of MAITs. On the contrary, a large accumulation of normal CD3 T cells was accompanied (Figure 2).

[0124] In summary, such results indicate that MAITs are less likely to cause an alloreaction, opening the way to use MA IT cells as a lymphocyte source for universal allogeneic T cell expression.

[0125] 3. Effective MAIT cell expansion and lentiviral CAR transduction A prerequisite for using MAIT cells as a new CAR-T cell source is that MAIT cells can expand to a sufficient amount in vitro and can be efficiently transduced by a lentiviral vector. Selectively, MAIT cells were cultured in vitro under experimental conditions for increasing them from healthy donor PB MCs. Repeated stimulation with 5-OP-RU and IL-2 for 21 days selectively enriched MAIT cells (MAIT cells among CD3 T cells exceeded 80% in 21 days), and the absolute number of MAIT cells increased 2400-fold over the culture period (Figure 3A and Figure 3B).

[0126] Apart from the MAIT cell expansion treatment, it was determined that lentiviral CAR transduction of MAIT cells is as effective as that of normal CD3 T cells. As shown in Figure 4, anti-CD3 / CD28-stimulated CD3 T cells transduced MAIT cells with CAR-BFP lentivirus containing GFP at the same ratio as that of CD3 T cells. The + M AIT cells.

[0127] 4. CAR-MAIT cells effectively kill related target cells, similar to CAR-T cells. Let To demonstrate the in vitro efficacy of CAR-MAIT cells, relevant CAR targets were developed. We conducted cell death experiments on the tumor cells present. 5×10 4 Luciferase-expressing target cells , CAR-MAIT, CAR-T or non-transduced MAIT(NT) effector cells and Each cell was incubated for 24 hours at various effector / target (E / T) ratios. The survival rate of CAR-MAIT cells was quantified by measuring the fluorescence intensity of the surviving target cells. However, non-transduced MAIT cells (NTs) target tumors in the same way as normal CAR-T cells. It effectively killed them with a low E:T ratio.

Claims

1. Mucosa-associated invariant T (MAIT) cells that express chimeric antigen receptors (CARs).

2. The mucosa-associated invariant T (MAIT) cells according to claim 1, which are allogeneic.

3. The aforementioned chimeric antigen receptor (CAR) is an antigen that specifically binds to or recognizes a target antigen. Mucosa-associated invariant T (MAIT) cells according to claim 1, comprising a binding domain.

4. The aforementioned antigen-binding domain is an antibody, according to claim 3, mucosal-associated invariant T(M AIT) cells.

5. The mucosa-associated invariant T according to claim 3, wherein the antigen-binding domain is scFv. (MAIT) cells.

6. The CAR binds to tumor-associated antigen (TAA), as described in claim 3. Riant T (MAIT) cells.

7. The CAR is any TAA expressed on the surface of tumor cells, preferably CD19, GD 2, EGFR, CD20, CD22, CD33, CD138, CD52, CD30, RO R1, HER2, EpCAM, MUC-1, MUC5AC, BCMA, CD38, SLA MF7 / CS1, CD123, IL-13Ra2, HER2, LeY, MUC16 or P The mucosa-related invariant T (MAIT) particle according to claim 6, which specifically binds to SMA. Cell.

8. The aforementioned CAR is an intracellular oncolytic protein or intracellular tumor-associated antigen, particularly WT-1, N Y-ESO-1, MAGE, PRAME, RAS, mesothelin, c-Met, CEA, C Claim 6, which specifically targets SPG-4, EBNA3C, CA-125, or GPA7. Mucosa-associated invariant T (MAIT) cells as described above.

9. The CAR is between the antigen-binding domain and the transmembrane domain and / or the transmembrane domain The mucosal-associated injection according to claim 1, comprising a hinge sequence between the main and cytoplasmic domains. Variant T (MAIT) cells.

10. The aforementioned hinge has an IgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region, and an Ig A small number of G4 Fc regions, IgE Fc regions, IgM Fc regions, or IgA Fc regions Mucosal-related invariants according to claim 9, which are derived from or include both. T (MAIT) cells.

11. The aforementioned hinge contains IgG1, IgG2, IgG3 within the CH2 and CH3 domains. Including at least a portion of the IgG4, IgE, IgM, or IgA immunoglobulin Fc region, Mucosa-associated invariant T (MAIT) cells according to claim 9.

12. The CAR is a first-generation CAR, according to any one of claims 1 to 11. Associated invariant T (MAIT) cells.

13. The CAR is a second-generation CAR, according to any one of claims 1 to 11. Associated invariant T (MAIT) cells.

14. The CAR is a third-generation CAR, according to any one of claims 1 to 11. Associated invariant T (MAIT) cells.

15. The CAR is a fourth-generation CAR, according to any one of claims 1 to 11. Associated invariant T (MAIT) cells.

16. A method for producing CAR-MATT cells, MAIT cells are obtained from cell cultures or from blood samples from individual subjects or blood banks. Steps to prepare, The steps include isolating the MAIT cells, The steps of activating and increasing the MAIT cells, CAR-MAT, which includes the steps of transduction and selection of the MAIT cells. A method for producing T cells.

17. The isolation is performed by affinity or immunoaffinity-based isolation, as described in claim 16. A method for producing CAR-MAIT cells.

18. The MAIT cells are treated with anti-Vα7.2 antibody, anti-IL18Rα, anti-CD161, or anti- The CAR-MAIT product according to claim 16 is positively isolated using a CD26 antibody. A method for producing cells.

19. The aforementioned MAIT cells are irradiated PBMCs from the autologous or allogeneic lineage, as well as IL-2 and IL-2. CD3 in the presence of IL-7, IL-12, IL-18 and / or IL-15 cytokines / CD28 stimulation increases in vitro, or 5-OP-RU and / Alternatively, it can be increased in vitro in the presence of MAIT cell-activating ligands such as 5-OE-RU. A method for producing CAR-MAIT cells according to claim 16, wherein the cells are either activated or otherwise activated.

20. The C according to claim 16, wherein a nucleic acid construct encoding CAR is introduced into the MAIT cells. A method for producing AR-MAIT cells.

21. A pharmaceutical or veterinary combination comprising CAR-MAIT according to any one of claims 1 to 15. A finished product.

22. The pharmaceutical or veterinary composition according to claim 21 for parenteral or intravenous administration.

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