Genetically modified placenta-derived mucosal-associated invariant T cells (MAIT cells) and their applications

Abstract

In one of its main embodiments, the present invention relates to a cell composition comprising a population of modified mucosa-associated invariant T cells (MAIT cells) derived from placental tissue that express exogenous chimeric antigen receptors (CARs). The present invention further discloses a unique placental MAIT cell population, a cell composition comprising the MAIT cell population, and methods of using the same.

Description

[Technical Field]

[0001] This disclosure relates to the field of genetic modification technologies for immune cells in general. Specifically, this disclosure provides placental-derived genetically modified mucosal-associated invariant T cells (MAIT cells) possessing chimeric antigen receptors (CARs) and their applications. [Background technology]

[0002] Mucosa-associated invariant T cells (MAIT cells) are double-negative (CD4) T cells that express the invariant Vα7.2-Jα33 T cell receptor (TCR) in humans, mice, and cattle. - CD8 - They were initially identified as a population of αβT cells rich in a subset. The term MAIT was defined by the relatively abundant presence of these T cells in mucosal tissues. The limiting factor for MAIT cells is the MHC-lb molecule MR1 (MHC-associated molecule 1), which presents intermediates of the vitamin B (both riboflavin (vitamin B2) and folic acid (vitamin B9)) synthesis pathway to MAIT cells. Potent stimulant ligands for the riboflavin synthesis pathway include 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) and 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OERU), which are produced by various bacteria, mycobacteria, and yeasts during the process of riboflavin (vitamin B2) synthesis. This pathway is not present in mammals, and its immunodetection allows for effective host-pathogen differentiation.

[0003] More specifically, T cells expressing αβ and γδ T cell receptors (TCRs) differentiate from common T cell progenitor cells in the thymus and can mature into one of several functionally distinct T cell lineages. The most well-studied of these is the conventional T cell, which expresses the αβ TCR, recognizing peptide antigens presented by MHC molecules. On the other hand, several other "atypical" T cell lineages develop in the thymus. These atypical T cells include CDl-restricted T cells, MHC-related molecule 1 (MR1)-restricted T cells, and γδ T cells, which recognize non-peptide antigens such as lipids, vitamin B metabolites, and phosphate antigens, respectively. These lineages typically express TCRs with less diversity compared to conventional T cells and are well known as cells that bridge innate and adaptive immunity. Many atypical T cells co-express surface antigens associated with natural killer (NK) cells and exhibit a very rapid and potent cytokine response after activation. Their characteristic phenotypes are established during development and constitute a crucial subset of peripheral T cells with unique effector functions and regulatory roles (Non-Patent Literature 1).

[0004] Mucosa-associated invariant T cells (MAIT cells) are innate immune-like T cells defined by a semi-variant αβ T cell receptor (TCR) that recognizes small biosynthetic derivatives of riboflavin synthesis presented on the restriction molecule major histocompatibility complex (MHC)-associated protein-1 (MR1). As mentioned above, the MAIT cell TCR contains a semi-variant TCR-α chain, typically Vα7.2-Jα33 / 12 / 20 in humans, and is primarily associated with the β chain Vβ2 / Vβ13 in humans.

[0005] MAIT cells are in contrast to conventional T cells, which have a highly variable TCR and can target a vast number of peptide epitopes produced by viruses, bacteria, and malignant cells. Therefore, conventional T cells have extremely high specificity for individual peptides, and individual clones proliferate in large numbers, leading to T cell memory. On the other hand, the frequency of appearance of individual peptide-specific T cells is very low during the first encounter with a pathogen. In contrast, MAIT cell TCRs possess an innate ability to respond to specific ligands without the need for proliferation. One important discovery was the identification of these ligands presented by MR1, including the potent MAIT cell ligands 5-(2-oxopropyrideneamino)-6-D-ribitylaminouracil (5-OP-RU) and 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU). These are produced by a wide variety of bacteria, mycobacteria, and yeasts during riboflavin (vitamin B2) synthesis (Non-Patent Literature 2).

[0006] MAIT cells have been shown to recognize bacteria-infected cells and respond by producing IFN-γ. MAIT cells have been demonstrated to protect mice from bacterial infection. Early phenotypic studies have shown that human MAIT cells are CD8 + Alternatively, it may be double negative, mainly CCR7 - It was established that MAIT cells possess an effector memory phenotype and express CD161 at high levels. Furthermore, detailed phenotypic analysis of MAIT cells revealed that they share several characteristics with invariant natural killer T (iNKT) cells, including the expression of promyelocytic leukemia Zn finger (PLZF), a transcription factor that regulates the innate immune-like function of iNKT cells. PLZF expression similarly confers innate immunity-like function to MAIT cells, as evidenced by the cytokine's ability to induce interferon-γ (IFN-γ) production even without TCR stimulation.

[0007] Subsequent studies have shown that human MAIT cells do not express a single invariant TCR, but rather express a restricted TCR that includes Vα7.2-Jα33, Vα7.2-Jα12, or Vα7.2-Jα20, which are mainly associated with human β-chain Vβ2 / Vβ13. Thus, current understanding is that MAIT cells are T cells with innate characteristics that (a) express a semi-invariant Vα7.2-Jα33 / 12 / 20 TCR, (b) are activated by microbial vitamin B antigens presented by MR1, execute both type 1 and type 17 effector functions, and (c) are regulated by the expression of PLZF, which includes the ability to be activated by cytokines independent of the TCR.

[0008] Some properties of MAIT cells suggest a fundamental role in mammalian immunity. First, MAIT cells have an intrinsic effector memory phenotype, usually CD45RA - CD45RO + CD95 Hi CD62L Lo CD44 Hi and have the ability to rapidly secrete several inflammatory cytokines. Second, MAIT cells are very abundant in human tissues and usually account for 1 - 4% of total T cells in peripheral blood, 10% of airway T cells, and 20 - 40% of liver T cells. Furthermore, because each TCR recognizes the same ligand, MAIT cells significantly outnumber conventional antigen-specific T cells that respond to cognate antigens early in the immune response.

[0009] The conserved and abundant presence of MAIT cells can be attributed to their ability to perform diverse functions through different activation modes that each induce a distinct transcriptome program. Due to their ability to show diverse functional responses in diverse immunological situations, this interesting cell is currently considered a central multifunctional effector at the interface of innate and adaptive immunity.

[0010] Chimeric antigen receptors (CARs) are a type of genetically modified receptor that is attached to immune cells, typically T cells, in cancer treatment. These receptors allow T cells to recognize and target specific antigens on cancer cells. CAR-T cell therapy has shown promising results in the treatment of certain types of cancer.

[0011] Chimeric antigen receptors (CARs), also known as chimeric immune receptors, chimeric T cell receptors, or modified T cell receptors, are receptor proteins that have been modified to confer a new ability to target specific antigens to T cells. A receptor is considered chimeric in that it integrates antigen-binding and T cell activation functions into a single receptor.

[0012] CAR-T cell therapy is a well-known technique in this field, using CAR-modified T cells to treat cancer. The T cells are modified to recognize and destroy cancer cells. The standard procedure involves collecting T cells from a patient, genetically modifying them, and then injecting the resulting CAR-T cells into the patient to attack the tumor (Non-Patent Literature 3).

[0013] CAR-T cells can be generated autologously from T cells in the patient's own blood, or allogeneically from T cells of a donor. Once isolated, the T cells are genetically modified to express specific CARs that program the T cells to target antigens present on the surface of tumor cells. The CAR helps the T cells recognize specific cancer cell antigens and activate them to kill cancer cells.

[0014] There are significant differences between CAR-T cell therapy and TCR-T cell therapy, which are described in the following document (Non-Patent Literature 4).

[0015] Structurally, CAR-T cells and TCR-modified T cells are different. CAR-T cells consist of modified intracellular and extracellular domains. The extracellular domain is composed of single-chain variable region fragments (including antibody antigen-binding domains) that recognize specific antigens on the cell surface and trigger intracellular signaling leading to T cell activation. Maintaining T cell activation and the survival of CAR-T cells are further enhanced by adding a costimulatory domain to the intracellular portion of the CAR-T cell.

[0016] The use of CAR-T cells has been previously described in the art. For example, Patent Document 1 discloses a population of T cells expressing a chimeric antigen receptor (CAR), in which the T cells are placental T cells derived from umbilical cord blood, placental irrigation fluid T cells, or a mixture of both. This application further discloses a method for treating cancer or symptoms in patients requiring treatment by administering a certain amount of the above-mentioned population of T cells to the patient.

[0017] Furthermore, attempts to use MAIT cells in allogeneic adoptive immunotherapy have been made in the art. Patent Document 2 discloses mucosa-associated invariant T cells (MAIT cells) expressing chimeric antigen receptors (CARs) for the treatment of cancer, immune diseases, or infections in subjects, wherein the MAIT cells are allogeneic with respect to the subject. In this disclosure, MAIT cells are collected from blood samples (including peripheral blood and umbilical cord blood). [Prior art documents] [Patent Documents]

[0018] [Patent Document 1] U.S. Patent Application No. 17 / 309,451 [Patent Document 2] U.S. Patent Application No. 17 / 414,689 [Non-patent literature]

[0019] [Non-Patent Document 1] Godfrey, D., Uldrich, A., McCluskey, J. et a1. The burgeoning family of unconventional T cells. Nat Immunol 16, 1114-1123 (2015). https: / / doi.org / 10.1038 / ni. 3298 [Non-Patent Document 2] MINI REVIEW article, Front. Immunol., 27 May 2020, Sec. Mucosal Immunity, Volume 11 - 2020 (https: / / doi.org / 10.3389 / fimmu.2020.01014) [Non-Patent Document 3] Fox M(July 12, 2017,"New Gene Therapy for Cancer Offers Hope to Those With No Options Left".NBC News.(https: / / www.nbcnews.com / hea1th / hea1th-news / new-gene-therapy-cancer-offers-hope-those-no-options-1eft-n741326)) [Non-Patent Document 4] “CAR T-Cell Therapy vs. TCR-T Therapy: The Differences and Applications - Atlantis Bioscience Pte Ltd.” https: / / atlantis2.e;sbiz.website / the-evolution-of-car-t-cells / (opens%20in%20a%20new%20tab) [Overview of the project]

[0020] This invention advances the use of CARs and MAIT cells by providing unique placental CAR-MAIT cells that exhibit improved effector activity and high viability in the host compared to MAIT cells from peripheral blood or umbilical cord blood, and compared to conventional T cells as disclosed in the prior art. By engineering placental MAIT cells to express CARs, various therapeutic applications become possible.

[0021] In one primary embodiment, the present invention is directed to a cell composition comprising a population of modified mucosa-associated invariant T cells (MAIT cells) expressing an exogenous chimeric antigen receptor (CAR), wherein the MAIT cells are derived from placental tissue, and optionally, the composition further comprises a pharmaceutically acceptable carrier. In some optional embodiments, the population of placental MAIT cells is obtained from intervillous blood (IVB).

[0022] According to embodiments of the present invention, the cell compositions described herein are suitable for cell therapy. More than 90% of placental MAIT cells are TCRVα7.2 + and high level CD161 (CD161 high Characterized by the expression of ) is not essential but preferred. In some embodiments, the cell composition of the present invention comprises a population of modified placental CAR-MAIT cells. 9 from 10 11 Contains individual living cells.

[0023] According to the present invention, exogenous CARs expressed by placental MAIT cells of the cell composition recognize tumor antigens. In one embodiment, this tumor antigen is mesoserin (MSLN). In any other embodiment, the tumor antigen is selected from the group consisting of CD19, B7-H6, CD20, CD22, CD33, CD38, CD70, CD123, BCMA, CLL1, CD7, CS1, CEA, AFP, PSMA, GPC3, GD2, EGFRVIII, CXCR5, NKG2D, HER2, mesoserin, claudin 3, claudin 4, claudin 6, claudin 18.2, ROR1, ROR3, Muc1, and Mucl6. In some optional embodiments, exogenous CARs expressed by placental MAIT cells of the cell composition described herein recognize microbial antigens and / or viral antigens. Furthermore, in several other optional embodiments, the exogenous CAR expressed by placental MAIT cells of the cell composition described herein recognizes a target molecule expressed on regulatory T cells. In a specific non-limiting embodiment, the target molecule is CCR8. Moreover, in a further embodiment of the present invention, the exogenous CAR expressed by placental MAIT cells of the cell composition specifically recognizes a target molecule expressed on alloreactive lymphocytes. In a specific non-limiting embodiment, the target molecule is CD70. In a further option, the exogenous CAR expressed by placental MAIT cells of the cell composition described herein recognizes a target molecule expressed on senescent cells. In a specific non-limiting example, the target molecule is urokinase plasminogen activator (uPAR). In a further optional embodiment of the present invention, exogenous CARs expressed by placental MAIT cells of the cell composition recognize target molecules expressed on cells associated with fibrotic diseases or pathological conditions. A specific, non-limiting example of such target molecules is fibroblast-activating protein (FAP). Optionally, exogenous CARs expressed by placental MAIT cells of the cell composition recognize target molecules expressed on cells associated with autoimmune diseases or disorders. In certain non-limiting examples, target molecules are expressed on B cells, such as CD19, CD20, and CD21, but are not limited to these. In some other optional embodiments of the present invention, the target molecule is a pathogenic MHC class II:peptide complex associated with autoimmune diseases, autoantibodies, B cell antibodies targeted by B cell antibody receptors (BARs), carboxypeptidase H, chromogranin A, glutamate decarboxylase, imogen-38, insulin, insulinoma antigens-2 and 2β, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proinsulin, citrullinated proteins, collagen N II, heat shock protein, human cartilage glycoprotein, double-stranded DNA, La antigen, nucleosome histone and ribonucleoprotein (snRNP), phosphatidyl lipid-β-2 glycoprotein I complex, poly(ADP-ribose) polymerase, Sm antigen of U1 small ribonucleoprotein complex, α-enolase, aquaporin 4, β-arrestin, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein S100-β, R1 type reticulin, gastric H + / K + -The modified MAIT cells contain ATPase, 21-hydroxylase, 17-hydroxylase, cytochrome P450 side-chain cleavage enzyme and CD19, and the modified MAIT cells contain pathogenic MHC class II:peptide complexes associated with the autoimmune disease, autoantibodies, B cell antibodies targeted by B cell antibody receptors (BARs), carboxypeptidase H, chromogranin A, glutamate decarboxylase, imogen-38, insulin, insulinoma antigen-2, 2β, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proi Nsrin, citrullinated protein, collagen II, heat shock protein, human cartilage glycoprotein, double-stranded DNA, La antigen, nucleosome histone, ribonucleoprotein (snRNP), phosphatidyllipid-β-2 glycoprotein I complex, poly(ADP-ribose) polymerase, Sm antigen of U1 small ribonucleoprotein complex, α-enolase, aquaporin 4, β-arrestin, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein S100-β, R1 type reticulin, gastric H + / K + -Contains ATPase, 21-hydroxylase, 17-hydroxylase, cytochrome P450 side-chain cleavage enzyme, or CD19. In some of the embodiments of the present invention, the autoantibody used in the cell composition is DSG3.

[0024] In some optional embodiments of the present invention, the exogenous chimeric antigen receptor (CAR) expressed by placental MAIT cells of the cell composition described herein comprises an antigen-binding domain configured to specifically recognize a tumor antigen, a transmembrane domain for immobilizing the CAR to the cell membrane, one or more costimulatory domains for enhancing cell signaling and T cell activation, and an intracellular signaling domain that plays a role in initiating T cell activation upon antigen recognition. In further embodiments, the antigen-binding domain of the chimeric antigen receptor (CAR) may comprise any of a single-chain antibody, a single-chain variable region fragment (scFv), and a VHH fragment, each configured to recognize a designated tumor antigen. Furthermore, in some optional embodiments, the antigen-binding domain of the CAR of the cell composition of the present invention may specifically bind to one or more antigens selected from the group consisting of CD19, B7-H6, CD20, CD22, CD33, CD38, CD70, CD123, BCMA, CLL1, CD7, CS1, CEA, AFP, PSMA, GPC3, GD2, EGFRVIII, CXCR5, NKG2D, HER2, mesoserine, claudin 3, claudin 4, claudin 6, claudin 18.2, ROR1, ROR3, Muc1, and Muc16. In any other embodiment, the co-stimulatory domain of the CAR in the cell composition of the present invention may include one or more functional signaling domains derived from OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). In any other embodiment, the intracellular signaling domain of CAR may include any of the intracellular signaling domains of CD3ζ, FcRγ, or their functional fragments, each configured to initiate or maintain T cell activation upon antigen recognition.

[0025] Furthermore, in some further embodiments of the present invention, the CAR expressed by placental MAIT cells of the cell composition shown herein may be encoded by a gene construct selected from the group consisting of monocistronic constructs, bicistronic constructs, and tricistronic constructs, one or more additional elements included in the bicistronic construct or tricistronic construct may be selected from the group consisting of cytokines (e.g., IL-15, IL-18), constitutively active tyrosine kinases (e.g., c-kit or other gene elements that promote the survival, proliferation, or effector function of MAIT cells), and the cytokine may be unmodified or modified, and may be expressed in secreted, conditionally active, constitutively active, or membrane-bound form.

[0026] In some embodiments, pre-grown populations of placental MAIT cells from the cell compositions described herein exhibit a higher proportion of cells expressing low levels of CD45RA and CD62L, and show the effector memory phenotype of placental MAIT cells, compared to pre-grown populations of peripheral blood-derived MAIT cells.

[0027] In some additional embodiments, a pre-grown population of placental MAIT cells from the cell composition shows a higher proportion of cells expressing CCR5 and / or CCR6 chemokine receptors and a lower proportion of cells expressing CXCR4 compared to a pre-grown population of peripheral blood-derived MAIT cells. In further embodiments, a population of placental MAIT cells from the cell composition of the present invention shows a higher proportion of cells expressing granzyme B compared to a population of peripheral blood-derived MAIT cells, indicating enhanced effector and lysis functions of placental MAIT cells. In additional embodiments, a pre-grown population of placental MAIT cells from the cell composition of the present invention shows a higher proportion of cells expressing perforin compared to a pre-grown population of peripheral blood-derived MAIT cells, indicating enhanced effector and lysis functions of placental MAIT cells. In additional embodiments, a pre-grown population of placental MAIT cells from the cell composition shows a higher proportion of cells expressing perforin and / or granzyme B compared to a pre-grown population of conventional T cells derived from peripheral blood, indicating enhanced effector and lysis functions of placental MAIT cells. Furthermore, in a further embodiment, a pre-grown population of placental MAIT cells from the disclosed cell composition showed a higher proportion of cells expressing perforin and / or granzyme B compared to a pre-grown conventional T cell population derived from umbilical cord blood, indicating that the effector function and lysis function of placental MAIT cells are enhanced. In an additional embodiment, a pre-grown population of placental MAIT cells from the cell composition showed a higher proportion of cells expressing TNFα compared to a pre-grown population of conventional T cells derived from umbilical cord blood, indicating that the effector function of placental MAIT cells is enhanced. In an additional embodiment, a pre-grown population of placental MAIT cells from the cell composition showed a higher proportion of cells expressing TNFα compared to a pre-grown population of conventional T cells derived from peripheral blood, indicating that the effector function of placental MAIT cells is enhanced. In an additional embodiment, the population of placental MAIT cells in the cell composition has a higher proportion of cells expressing one or more of perforin, granzyme B, granzyme B, perforin, TNFα, TNFα, and INFγ compared to the population of placental T cells, and exhibits enhanced effector function and lysis function. According to a further embodiment of the present invention, a pre-proliferated population of placental MAIT cells of the cell composition is compared to a pre-proliferated population of umbilical cord blood MAIT cells with respect to CD8αα + The proportion of cells expressing this gene is high, and they exhibit the mature phenotype of placental MAIT cells.

[0028] In some aspects of the present invention, the cell compositions disclosed herein are for therapeutic use. In some arbitrary embodiments, the cell compositions may be used to treat subjects having tumors or malignant tumors. Examples of the use of the cell compositions of the present invention include, but are not limited to, use in subjects suffering from tumors such as hematological cancers, melanomas, breast cancers, colon cancers, kidney cancers, liver cancers, lung cancers, ovarian cancers, pancreatic cancers, prostate cancers, uterine cancers, cervical cancers, bladder cancers, gastric cancers, head and neck cancers, brain tumors, skin cancers, and sarcomas.

[0029] In some embodiments of the present invention, a method is provided for treating a subject having a tumor or malignant tumor, the method comprising administering a cell composition according to any of the embodiments described above to the subject. The tumors to be treated may be, for example, hematological cancers, melanomas, breast cancers, colon cancers, kidney cancers, liver cancers, lung cancers, ovarian cancers, pancreatic cancers, prostate cancers, uterine cancers, cervical cancers, bladder cancers, gastric cancers, head and neck cancers, brain tumors, skin cancers, and sarcomas. Although not essential, the cell composition for treating the subject is adapted for cell therapy, and the population thereof is 10 9 It consists of more than 1 living cell, of which more than 90% is TCRVα7.2 + CD161 high That is the case.

[0030] In some embodiments, the modified placental MAIT cells used to treat a subject are allogeneic to the subject. In other options, the modified placental MAIT cells are partially histocompatible with the subject or are not histocompatible with the subject.

[0031] In some embodiments of the present invention, a method is provided for treating a subject infected with a pathogen. For example, the pathogen may be a bacterial pathogen, a viral pathogen, or a fungal pathogen, and the method comprises administering a cell composition according to any preferred embodiment described above to the subject.

[0032] In some additional aspects of the present invention, methods are provided for treating subjects suffering from fibrotic diseases or conditions, for treating subjects suffering from age-related diseases or conditions, for treating subjects who have undergone allografting, and for treating subjects suffering from autoimmune diseases or disorders, all of which involve administering a cell composition according to any preferred embodiment as defined above to the subject in need.

[0033] Furthermore, according to an additional aspect of the present invention, a method is provided for treating a subject infected with a pathogen, the method comprising administering to the subject a composition comprising a population of placental-derived MAIT cells. The population of MAIT cells according to this embodiment is not modified MAIT cells, and the cells do not express exogenous CARs. The pathogen is, for example, a bacterial pathogen, a viral pathogen, or a fungal pathogen. In some specific examples, the bacterial pathogen may include Mycobacterium tuberculosis. In some embodiments, the bacterial pathogen includes antibiotic-resistant bacteria or bacteria-resistant bacteria. In some other specific examples, the fungal pathogen may consist of invasive Aspergillus. In some further examples, the viral pathogen includes cytomegalovirus, hepatitis B virus, or hepatitis C virus, and optionally the infection is post-surgical infection. Although not essential, it is preferable that the MAIT cells used to treat a subject according to this aspect of the present invention are allogeneic to the subject.

[0034] Furthermore, an additional aspect of the present invention provides a population of placental mucosa-associated invariant T cells (MAIT cells), wherein more than 90% of these placental MAIT cells are TCRVα7.2 + CD161 + Furthermore, the majority of the placental MAIT cells are CD45RA and CCR7(CD45RA - CCR7 - Characterized by the absence of expression of ), the MAIT cells exhibit an effector memory phenotype. Optionally, this population of MAIT cells may be modified placental MAIT cells expressing a chimeric antigen receptor (CAR).

[0035] In some embodiments, this placental MAIT cell population is further characterized by high or low expression of CD62L. In some additional embodiments, this placental MAIT cell population is further characterized by high expression of CD69. Optionally, this placental MAIT cell population may be obtained from intervillous blood (IVB). Furthermore, this placental MAIT cell population according to any of the above embodiments may be used to prepare cell compositions for use in cell therapy.

[0036] In some embodiments, the disclosure provides a population of modified mucosa-associated invariant T cells (MAIT cells) containing exogenous chimeric antigen receptors (CARs), wherein the MAIT cells are derived from placental intervillous blood (IVB).

[0037] In some embodiments, CARs recognize tumor antigens. CARs recognize target molecules expressed on regulatory T cells. CARs recognize target molecules expressed on alloreactive lymphocytes. CARs recognize target molecules expressed on autoimmune cells. CARs recognize target molecules expressed on senescent cells.

[0038] In some embodiments, the CAR of modified IVBMAIT cells includes an antigen-binding domain, a transmembrane domain, and a costimulatory domain. The antigen-binding domain may be, but is not limited to, a single-chain antibody or a single-chain antibody fragment, for example, a single-chain variable region fragment (scFv). Targets of the antigen-binding domain include, but are not limited to, 5T4, ALK, AXL, B7-H6, CD5, CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD80, CD89, CEA, cMET, CLEC14a, CD123, CD133, CDH17, BCMA, CLL1, CXCR5, CS1, CEA, AFP, PSMA, PSCA, FAP, FHSR, GPC3, GD2, EGFR, EGFRVIII, EpCAM, NKG2D, HER2, IL13Rα2, TEM8, TGF, mesoserine, claudin 3, claudin 4, claudin 6, claudin 18.2, ROR1, ROR2, ROR3, or Muc1, Muc16, Muc-CD.

[0039] In some embodiments, the co-stimulatory domain of CAR includes a functional signaling domain derived from OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), or 4-1BB (CD137). The intracellular signaling domain of CAR includes an intracellular signaling domain of CD3ζ or FcRγ, or functional fragments thereof.

[0040] In some embodiments, pharmaceutical compositions are provided that comprise a pharmaceutically acceptable carrier and a population of modified IVB-CAR-MAIT cells as disclosed herein.

[0041] In some embodiments, a method is provided for treating a subject having a tumor or malignant tumor, comprising administering to the subject a composition comprising a population of modified IVB-CAR-MAIT cells as disclosed herein. The modified IVB-CAR-MAIT cells are allogeneic to the subject.

[0042] In some other embodiments, modified IVB-CAR-MAIT cells bind to mesoserine (MSLN).

[0043] In some embodiments, a method is provided for treating a subject suffering from a disease or condition, comprising administering to the subject a composition comprising a population of modified IVBCAR-MAIT cells as disclosed herein. The fibrotic disease or condition includes cardiac fibrosis. If the subject suffers from cardiac fibrosis, the modified IVBCAR-MAIT cells bind to fibroblast-activating protein (FAP). The modified IVBCAR-MAIT cells are allogeneic to the subject.

[0044] In another embodiment, a method is provided for treating an allogeneically transplanted subject, comprising administering to the subject a composition comprising a population of modified IVBCAR-MAIT cells as disclosed herein, wherein the IVBCAR-MAIT cells bind to target molecules expressed on allogeneically reactive lymphocytes. Examples of target molecules expressed on allogeneically reactive lymphocytes include, but are not limited to, CD70, CD19, and CD20.

[0045] In some embodiments, methods are provided for treating subjects suffering from age-related diseases or conditions, comprising administering a composition comprising a population of modified IVBCAR-MAIT cells as disclosed herein to the subject. In some embodiments, the modified MAIT cells are allogeneic to the subject. Age-related diseases or conditions include chronic inflammation, fibrous liver disease, atherosclerosis, diabetes mellitus, or a combination thereof. IVBCAR-MAIT cells express exogenous antigen receptors that bind to target molecules. Modified IVBCAR-MAIT cells bind to urokinase plasminogen activator receptor (uPAR).

[0046] In some embodiments, methods are provided for treating subjects suffering from autoimmune diseases or disorders, comprising administering to the subjects a composition comprising a population of modified IVBCAR-MAIT cells as disclosed herein. Modified IVBCAR-MAIT cells are allogeneic to the aforementioned subjects. Autoimmune diseases or disorders include systemic lupus erythematosus (SLE), refractory SLE, pemphigus vulgaris, multiple sclerosis, type 1 diabetes mellitus, rheumatoid arthritis, celiac disease, pernicious anemia, inflammatory myopathy, myasthenia gravis, adrenalitis, or combinations thereof. Modified IVBCAR-MAIT cells are allogeneic to the aforementioned autoimmune diseases, including pathogenic MHC class II:peptide complexes, autoantibodies, B-cell antibodies targeted by B-cell antibody receptors (BARs), carboxypeptidase H, chromogranin A, glutamate decarboxylase, imogen-38, insulin, insulinoma antigens-2 and 2β, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proinsulin, citrullinated proteins, and collagen. Gen II, heat shock protein, human cartilage glycoprotein, double-stranded DNA, La antigen, nucleosome histone, ribonucleoprotein (snRNP), phospholipid-β-2 glycoprotein I complex, poly(ADP-ribose) polymerase, Sm antigen of U-1 small ribonucleoprotein complex, α-enolase, aquaporin 4, β-arrestin, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein, S100-β, Rl-type reticulin, gastric H + / K + - It recognizes and binds to a target antigen including ATPase, 21-hydroxylase, 17-hydroxylase, cytochrome P450 side-chain cleavage enzyme, or CD19. In one embodiment, the autoantibody is DSG3.

[0047] In another embodiment, a method is provided for treating a subject infected with a pathogen, comprising administering to the subject a composition comprising a population of placental IVBCAR-MAIT cells, wherein the IVBMAIT cells are allogeneic to the subject.

[0048] In some embodiments, these IVBMAIT cells do not contain exogenous antigen receptors. In some embodiments, the pathogen is a bacterial pathogen, a viral pathogen, or a fungal pathogen. Bacterial pathogens include antibiotic-resistant or bacterial treatment-resistant bacteria. Bacterial pathogens include Mycobacterium tuberculosis. Fungal pathogens include invasive Aspergillus. Viral pathogens include cytomegalovirus, hepatitis B virus, or hepatitis C virus. The infectious conditions treated by the methods disclosed herein are limited to site-specific infections, such as those in which the infection is confined to a particular site or organ. Infections treated by the methods disclosed herein include postoperative infections.

[0049] The above and other aspects of the modified placental CAR-MAIT cells and their uses will be understood from the drawings and detailed description. [Brief explanation of the drawing]

[0050] Some embodiments of modified IVBMAIT cells and / or IVBCAR-MAIT cells and methods of using them are described herein only as examples, with reference to the accompanying drawings. In describing the details with reference to the drawings, it should be emphasized that each item shown is intended to illustrate the modified IVBCAR-MAIT cells, IVBMAIT cells lacking exogenous antigen receptors, and embodiments of their therapeutic applications. In this regard, by reading this description together with the drawings, those skilled in the art will be able to see how to carry out the modified IVBCAR-MAIT cells, IVBMAIT cells lacking exogenous antigen receptors, and embodiments of their therapeutic applications.

[0051] Figures 1A to 1D are schematic diagrams illustrating the collection of maternal blood from the placenta. [Figure 1A] The diagram illustrates both the fetal and maternal sides of the placenta. [Figure 1B] The diagram shows the parent body after the membrane has been removed. [Figure 1C]The incision made in the maternal tissue is illustrated. [Figure 1D] Modifications made to the fetal portion according to embodiments of the present invention are illustrated.

[0052] The placenta 100 includes the fetal side 101 of the placenta, which contains the umbilical cord 102 clamped with a clip 103 to prevent mixing of fetal blood and maternal (IVB) blood, and the maternal side 104 of the placenta, which is covered with the amniotic membrane 105 (Figure 1A). IVB collection begins by removing the amniotic membrane surrounding the maternal side to expose the cotyledon 106 (Figure 1B). A shallow incision is made in the cotyledon using scissors or other appropriate cutting instrument (Figure 1C). The placenta is then inverted so that the fetal side faces upward. Phosphate-buffered saline (PBS) is injected into the basal decidua, away from the fetal blood vessels. All maternal blood flowing out of the placenta is collected. Mononuclear cells are then separated using Lymphoprep (density gradient column), and the intermediate cell stock is subsequently cryopreserved.

[0053] Figure 2A shows a flow cytometry plot of MAIT cell activation and proliferation over 10 days in a packed-bed bioreactor according to an embodiment of the present invention. [Figure 2A(1)] The flow cytometry plot for day 0 is shown. [Figure 2A(2)] The flow cytometry plot for day 5 is shown. [Figure 2A(3)] The flow cytometry plot for day 7 is shown. [Figure 2A(4)] The flow cytometry plot for day 10 is shown.

[0054] Mononuclear cells derived from intervillous blood (IVB) were inoculated into a filled-bed bioreactor containing an ECM-coated Fibra-CelR carrier, which mimics the natural environment and promotes antigen-presenting cell (APC) adhesion. MAIT cell activation was induced by 5-OP-RU and IL-15. MAIT cell activation and proliferation were assessed at several time points: day 0 (0), day 5 (5), day 7 (7), and day 10 (10). MAIT cell populations were detected by the expression of CD3, Vα7.2, and CD161 markers. The results showed an increase in the proportion of MAIT cells, rising from 22.63% of the CD3+ population on day 0 to 96.26% on day 10 (10). Furthermore, the expression of activation markers CD69 and CD25 increased from day 0 to day 7 and then slightly decreased on day 10.

[0055] Figure 2B shows a flow cytometry plot of MAIT cells that were transferred from the first bioreactor to the second bioreactor on day 10, according to an embodiment of the present invention, and then grown in the second bioreactor for a further 7 days. [Figure 2B(1)] The flow cytometry plot is shown 10 days after sampling. [Figure 2B(2)] The flow cytometry plot is shown 12 days after sampling. [Figure 2B(3)] The flow cytometry plot is shown 14 days after sampling. [Figure 2B(4)] The flow cytometry plot is shown 17 days after sampling.

[0056] Cells that reached maximum growth capacity by day 10 were collected from the first bioreactor, and approximately 30% of these cells were seeded into a second bioreactor with a similar design to the first bioreactor. These cells were cultured for a further 7 days. Flow cytometry results showed that the proportion of MAIT cells remained relatively constant for most of the period, but decreased from over 90% on day 14 to 82% on day 17. CD69 marker expression increased from 30% to 87% on day 17, indicating that MAIT cells maintained their activation signal. On the other hand, CD25 gradually decreased as expected, indicating that MAIT cells were recently activated but had not yet reached full proliferation and functional state, suggesting their ability to reactivate and function upon encountering future targets.

[0057] Figure 3 shows the results of activation and proliferation of placental IVBMAIT cells according to several optional embodiments of the present invention. [Figure 3(1)] The results from day 0 to day 7 are shown. [Figure 3(2)] The results from day 7 to day 10 are shown below.

[0058] IVB mononuclear cells were seeded into tissue culture plates, and MAIT cell activation was induced with 5-OP-RU and IL-15. On days 3 (3) and 5 (5) after activation, cells were counted, the proportion of MAIT cells was analyzed by flow cytometry, and fresh medium and IL-15 were added. On day 7, cells were separated using magnetic beads and anti-TCRVα7.2 antibody to further enrich the MAIT population. The cells were then cultured for a further 3 days (3) in the presence of IL-15. The results show an increase in the proportion of MAIT cells and the proliferation rate of MAIT cells (increase in the absolute number of MAIT cells relative to the initial number) during the culture period.

[0059] Figures 4A and 4B show the proportion of MAIT cells in placental intervillous blood (IVB)-derived mononuclear cells and peripheral blood mononuclear cells (PBMCs) that were gated for CD3+ cells. [Figure 4A] This is a representative sample of MAIT cells collected from placental IVB, with the CD3+ population being the target group for gated analysis. [Figure 4B] This shows the percentages of CD3+ placental (IVB) and peripheral blood (PB) MAIT cells analyzed from 13 IVB donors (13) and 11 PB donors (11). p=0.0159.

[0060] Figure 5 shows the immunophenotypic analysis of placental IVBMAIT cells and PBMAIT cells collected from six different donors (6) and analyzed on day 0. [Figure 5(1)] This is the result of the analysis of placental IVBMAIT cells. [Figure 5(2)] This is the result of the analysis of PBMAIT cells. [Figure 5(3)]This is the result of the analysis of placental IVBMAIT cells and PBMAIT cells.

[0061] Cells were stained according to a standard cell surface marker staining protocol and analyzed by flow cytometry. Cell characterization was performed using the cell surface markers CD45RA and CD62L. As shown in the figure, placental IVBMAIT cells had a statistically significantly higher proportion of "effector memory cells" and fewer "terminally differentiated" cells compared to PBMAIT cells. The gating strategy for phenotypic analysis is shown in the right panel. Statistical analysis was performed using GP Prism, and comparisons were made using t-tests. *p<0.05, **p<0.01, ***p<0.001.

[0062] Figures 6A and 6B show chemokine receptor analysis of placental IVBMAIT cells and PBMAIT cells according to embodiments of the present invention. [Figure 6A(1)] This is the result of the analysis of placental IVBMAIT cells. [Figure 6A(2)] This is the result of the analysis of PBMAIT cells. [Figure 6A(3)] This is the result of the analysis of placental IVBMAIT cells and PBMAIT cells. [Figure 6B(1)] This is the result of the analysis of placental IVBMAIT cells. [Figure 6B(2)] This is the result of the analysis of PBMAIT cells. [Figure 6B(3)] This is the result of the analysis of placental IVBMAIT cells and PBMAIT cells.

[0063] To evaluate the chemokine receptor profile of the MAIT population, IVBMAIT and PBMAIT cells were collected from six different donors on day 0, and chemokine receptor expression was analyzed. Cell surface markers CCR6, CCR5, CXCR4, and CXCR6 were used to characterize the cells. Cells were stained according to a standard cell surface marker staining protocol and analyzed by flow cytometry. Statistical analysis was performed using GP Prism, and comparisons were made by t-test (*p<0.05, **p<0.01, ***p<0.001). Figure 6A shows that IVB-MAIT cells express higher levels of CCR5 and CCR6 chemokine receptors compared to PB-MAIT cells. Figure 6B shows that IVB-MAIT cells have statistically significantly lower levels of CXCR4 and similar levels of CXCR6 compared to PB-MAIT cells. These results further illustrate the differences between the two MAIT cell populations, IVB-derived and PB-derived.

[0064] Figures 7A and 7B show the expression of granzyme B and perforin in placental IVBMAIT cells and peripheral blood MAIT cells. [Figure 7A] Representative samples of both cell types are shown. [Figure 7B] This shows MAIT cells derived from placental IVB and peripheral blood.

[0065] Cells from six donors (6) were stimulated with PMA / ionomycin for 4 hours (4), and intracellular expression of granzyme B and perforin was analyzed. As shown in the figure, placental IVBMAIT cells had statistically significantly higher levels of granzyme B compared to peripheral blood MAIT cells, indicating increased effector and lytic capacity of placental IVBMAIT cells. Cells were gated by MAIT cells. p=0.0001.

[0066] Figures 8A and 8B are flow cytometry plots (Figure 8A) and a table (Figure 8B) summarizing the results obtained from the flow cytometry plots of MAIT cells in placental intervillous blood (IVB) and umbilical cord blood (CB) from the same donor. [Figure 8A(1)] This is a flow cytometry plot of the proportion of MAIT cells. [Figure 8A(2)] Flow cytometry plot of the proportion of MAIT cells. [Figure 8B] This table summarizes the results obtained from the flow cytometry plot.

[0067] Cells were collected from three different donors (3) in the same manner as described above. IVB cells and CB cells were activated similarly. As shown in the figure, on day 0, CB cells had significantly fewer MAIT cells compared to IVB cells, and this proportion decreased over time (days 6 / 7). More specifically, MAIT cells from IVB and corresponding MAIT cells from CB were collected according to the protocol described in Materials and Methods below. Cells were stained for CD161 and TCRVα7.2 on day 0 and day 6 / 7, which indicates MAIT + This shows the gating strategy. The gates enclosed in squares represent the proportion of MAIT cells. The table shows the actual cell counts. Observations show that intervillous blood (IVB) has a significantly higher concentration of MAIT cells compared to umbilical cord blood (CB).

[0068] [Figure 9] This is a flow cytometry plot of CD8αα+ expression in placental IVBMAIT cells and CBMAIT cells according to an embodiment of the present invention.

[0069] As shown in the flow cytometry plot, placental IVBMAIT cells were approximately three times (41.2%) more likely to contain CD8αα compared to CBMAIT cells (14.2%). + The high frequency of expression indicates a more "antigen-experienced" phenotype compared to the naive state present in CBMAIT cells.

[0070] CD8α is a cell surface glycoprotein that can be expressed as a heterodimer disulfide-bonded to CD8β or as a homodimer. In contrast to CD8αβ, CD8αα is not expressed on naive T cells but is readily induced on strongly activated T cells. Furthermore, like other "regulatory" molecules, CD8αα may function to aid cell survival in a manner similar to how KIR and other NK cell-associated receptors are expressed on T cells. MAIT cells derived from IVB and CB cells were stained for the two subunits of CD8, CD8α and CD8β, and their frequencies were measured by flow cytometry. The results showed that IVBMAIT cells had a higher frequency of CD8αα and exhibited a more mature "antigen-experienced" phenotype compared to CBMAIT cells.

[0071] Figure 10 shows flow cytometry plots of placental IVBMAIT cells and CBMAIT cells for effector phenotypes using CD45RO, CD27, CD45RA, CCR7, and CD62L. [Figure 10(1)] The above flow cytometry plot is shown. [Figure 10(2)] The above flow cytometry plot is shown.

[0072] IVBMAIT and the corresponding CBMAIT were collected according to the methods described in the Materials and Methods section below. The cell surface markers CD45RA, CD27, CCR7, CD62L, and CD45RO were used to determine the immunophenotype of the cells. Here, CD45RA - / CCR7 - CD45RA - / CD62L - CD45RA - / CD45RO + , and CD27 - / CD45RO + These represent effector memory cells, activated memory cells, and the effector memory phenotype, respectively. As clearly shown in the flow cytometry plot, IVBMAIT cells differ from CBMAIT cells in that IVBMAIT cells exhibit the effector memory phenotype, while CBMAIT cells exhibit the naive phenotype.

[0073] [Figure 11] Transcriptome analysis of IVBMAIT cells and PBMAIT cells reveals the top 50 gene sets with differing expression levels.

[0074] To understand the transcriptome differences between MAIT cells derived from different tissues, namely PBMCs and IVBs, cells were extracted and grown, and RNA sequencing analysis was performed as described above. MAIT cells derived from IVBs and PBs were collected from three different donors (3), and RNA sequencing analysis was performed on day 7 (7). Cells were collected as described in the "Methods" section. Heatmaps were created for protein-coding genes that showed significant expression differences (|log2FC|>1 and BH-adjusted p-value <0.01) between the IVB and PB cell types. Clustering was performed by applying Pearson correlation to the normalized values ​​of the count data. The color scheme represents the z-score. As shown in the diagram, IVB-derived MAIT cells have a unique and distinctive transcriptome compared to peripheral blood MAIT cells, meaning that both are extremely unique MAIT populations. The names of the top 50 genes are listed in the diagram.

[0075] Figure 12 shows a comparison of IVBMAIT cells and PBMAIT cells regarding granzyme B secretion. [Figure 12(1)] Comparison result 1. [Figure 12(2)] Comparison result 2. [Figure 12(3)] Comparison result 3. [Figure 12(4)] Comparison result 4. [Figure 12(5)] This is a summary of the comparison results.

[0076] Placental IVBMAIT cells and PBMAIT cells were collected from four donors (4) and grown using a standard protocol as described in the "Methods" section. On day 8 (8), the cells were activated with phorbol myristate acetate (PMA) and ionomycin, and the secretion of granzyme B and perforin was analyzed using a standard intracellular staining protocol. Granzyme B is a serine protease present in the granules of lysed cells. It is secreted together with perforin, a protein that forms pores in the membrane, to induce apoptosis (cell death) in target cells.

[0077] To evaluate the basal activity of IVB or PB-derived MAIT cells, collected cells were stimulated with PMA and ionomycin, and intracellular granzyme B secretion was confirmed by staining. As shown in the figure, stimulated IVB-derived MAIT cells secreted significantly higher levels of granzyme B compared to PB-derived MAIT cells, indicating increased effector and lytic activity of IVB-derived cells. Statistical analysis was performed using GP Prism, and comparisons were conducted using t-tests. * p<0.05, ** p<0.01, *** p<0.001).

[0078] [Figure 13] This graph shows the functional killing properties of placental IVBCAR-MAIT cells according to an embodiment of the present invention.

[0079] CAR-MAIT cells were prepared according to the standard protocol described in the "Methods" section and co-cultured with NSCLC targets overexpressing MSLN and luciferase. The killing function was measured by luminescence readout. Each effect pedal is CAR + The fraction levels are different. Co-culture with target cells is CAR +The tests were performed within the effector:target (E:T) ratio range determined by the fraction. Similar efficacy against target cells was obtained under all CAR-MAIT cell culture conditions, demonstrating the high and dose-dependent efficacy of CAR-MAIT cells.

[0080] Figures 14A and 14B show a comparison of IVBMAIT cells, peripheral blood (PB) mother T cells, and umbilical cord blood (CB) T cells from the same donor (matched blood). To evaluate the differences between MAIT cells and T cells and to eliminate variability between donors, the inventors evaluated the functional differences between different cells derived from the same donor. IVB, PBMCs, and CB cells were collected as described in the "Methods" section and activated with T cell activators on day 0 (0). Cells were analyzed for perforin, granzyme B, IFN-γ, and TNF-α using flow cytometry according to an intracellular staining protocol. Figure 14 shows the gating strategy for the analysis. [Figure 14A(1)] This is a plot of raw data for perforin and granzyme B obtained by flow cytometry. [Figure 14A(2)] Similarly, this is a plot obtained using flow cytometry. [Figure 14B(1)] This is a plot of raw IFN-γ and TNF-α data obtained using flow cytometry. [Figure 14B(2)] Similarly, this is a plot obtained using flow cytometry.

[0081] As shown in the diagram, MAIT cells on day 0 have effector molecules that are completely different from conventional T cells. T cells derived from umbilical cord blood and peripheral blood have similar characteristics.

[0082] Figures 15A and 15B show a comparison between the IVB-derived positive MAIT population and conventional T cell populations derived from peripheral breast blood (MB) and CB, cultured using the T cell standard activation protocol according to the gating strategy shown in Figures 14A and 14B. [Figure 15A(1)] The above comparison result is 1. [Figure 15A(2)] The above comparison result is 2. [Figure 15A(3)] The above comparison result is 3. [Figure 15B(1)] The above comparison result is 3. [Figure 15B(2)] The above comparison result is 4. [Figure 15B(3)] The result of the above comparison is 5.

[0083] In this example, IVB, peripheral maternal blood (MB), and umbilical cord blood (CB) were obtained from two donors, respectively.

[0084] To evaluate the differences between MAIT and T cells and to eliminate donor variability, IVB, PBMC, and CB cells were collected on day 0 (0) as described in the "Methods" section, activated with a T cell activator according to a standard protocol, and cultured. Briefly, T cell proliferation is achieved by activating the TCR using CD28 and CD3 antibodies, or TransAct beads supplemented with IL-2 cytokine. On day 10 (10), the cells were stained with CD161 and TCRVα7.2, and the proportion of the MAIT-positive population was evaluated. CD3 + It was found that MAIT cells were obtained from the cells with a high purity of over 95%. On the other hand, when a standard T cell proliferation protocol was applied, the MAIT cells initially detected (pre-proliferation, day 0) did not proliferate, and the MAIT population detected on day 10 was CD3 + It is greatly diluted to less than 1.2% of the cells.

[0085] Figures 16A and 16B are graphs illustrating a comparison of the effector functions of IVB-MAIT and IVB-T according to embodiments of the present invention. Figure 16A is a graph illustrating a comparison of the effector functions of the IVB-MAIT and IVB-T. [Figure 16A(1)] The above comparison result is 1. [Figure 16A(2)] The above comparison result is 2. [Figure 16A(3)] The above comparison result is 3. [Figure 16A(4)] The above comparison result is 4. [Figure 16A(5)] The result of the above comparison is 5.

[0086] IVB-MAIT cells were cultured as described in the "Methods" section. From the same raw materials (IVB cells), the inventors also cultured T cells using a conventional standard T cell protocol with TransAct and IL-2. On day 10 (10), IVB-MAIT cells and IVB-T cells were stimulated with PMA + ionomycin or TransAct, and then stained for effector molecules such as IFN-γ, TNF-α, perforin, and granzyme B, and analyzed by flow cytometry. [Figure 16B] This study demonstrates that MAIT cells possess higher levels of perforin and TNF-α compared to T cells of the same origin.

[0087] [Figure 17A] This is a flow cytometry plot of placental MAIT cells. [Figure 17B] This is a flow cytometry plot of placental CAR-MAIT cells.

[0088] The immunophenotypes of each cell population were compared. Cells were cultured for 10 days, stained according to a standard cell surface marker protocol, and analyzed by flow cytometry. The CD45RA vs. CCR7 gating strategy showed a statistically significant increase in "effector memory (EM)" cells compared to "central memory (CM)" cells. Naive and terminally differentiated (TD) cells were rarely observed.

[0089] [Figure 17C] This graph shows the average results of three different donors analyzed on day 10.

[0090] In both MAIT and CAR-MAIT cells, over 80% of cells exhibited effector memory, while the remaining cells exhibited central memory. Naive cells and terminally differentiated cells exhibited less than 1% effector memory. [Modes for carrying out the invention]

[0091] Unless otherwise defined, all technical and / or scientific terms used herein are synonymous with those generally understood by those skilled in the art concerning modified mucosa-associated invariant T (MAIT) cells and their uses. Similar or equivalent methods and materials may be used in the implementation or testing of embodiments of modified mucosa-associated invariant T (MAIT) cells and their uses, but exemplary methods and / or materials are described below. In case of any conflict, the patent specification, including definitions, shall prevail. Furthermore, the materials, methods, and examples are illustrative and not necessarily intended to be limiting. Each reference or other citation cited herein is incorporated herein by reference in its entirety.

[0092] The words "comprise," "comprises," "comprising," "includes," and "having," and their conjugations, all mean "to include, but not limited to."

[0093] In this specification, singular expressions such as "a, an" and "the" may refer to plural unless the context clearly indicates otherwise. For example, the expression "one enzyme" or "at least one enzyme" may mean multiple enzymes or a mixture thereof.

[0094] Unless otherwise indicated, the term “population,” when used in conjunction with a specific cell attribute(s), encompasses a collection of cells where 70% or more exhibit that attribute(s). In other embodiments, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% or more of the cells in the collection exhibit that attribute(s). In some embodiments, the population is isolated from cell types that do not possess the specific attribute. In some embodiments, the population of placental modified MAIT cells described herein includes exogenous attributes.

[0095] Unless otherwise specified, "placenta," "of the placenta," etc., in reference to placental-derived cells include intervillous blood (IVB). Placental-derived MAIT cells are obtained from the maternal source of the placenta. In some embodiments, MAIT cells are obtained from the intervillous blood of the placenta. In some embodiments, MAIT cells are obtained from the parietal decidua of the placenta. In some embodiments, MAIT cells are obtained from the basal decidua of the placenta. In some embodiments, MAIT cells are not obtained from umbilical cord blood.

[0096] As used herein, “peripheral blood-derived MAIT cells,” “peripheral blood MAIT cells,” or “peripheral MAIT cells” include MAIT cells isolated from peripheral blood (PB). In some embodiments, the peripheral blood is obtained from different donors. In some other embodiments, the peripheral blood is equivalent to intervillous blood and / or umbilical cord blood and is collected from the mother. All these terms have the same nature and meaning and can be used interchangeably.

[0097] As used herein, the terms “intervillous blood (IVB) derived MAIT cells” or “IVBMAIT cells,” “placental intervillous blood MAIT,” “placental IVBMAIT,” and “placental MAIT” all have the same meaning and refer to MAIT cells isolated from intervillous blood of the placenta and / or blood obtained from the maternal portion of the placenta.

[0098] As used herein, "MAIT cells" broadly encompass MAIT cells from any source, including peripheral blood, umbilical cord blood, and intervillous blood, but are not limited to these.

[0099] In some embodiments of the modified MAIT cell population, the MAIT cells are derived from maternal blood from the placenta. In some embodiments of the modified MAIT cell population, the MAIT cells are obtained from intervillous blood from the placenta. In some embodiments of the modified MAIT cell population, the MAIT cells are not obtained from umbilical cord blood.

[0100] Placental cells can be obtained from a full-term placenta in various embodiments. A convenient source of placental tissue is the postpartum placenta (e.g., less than 48 hours after birth), but various sources of placental tissue or cells will be conceivable to those skilled in the art. In other embodiments, the placenta is used within 24 hours (in some embodiments, while still stored in physiological buffer), 18 hours, 14 hours, 10 hours, 8 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour after birth. In some embodiments, the placenta is refrigerated before cell collection. In other embodiments, prepartum placental tissue is used. In some embodiments, the donor is under 40 years of age, in other embodiments under 35 years of age, while in other embodiments, the donor may be any woman of childbearing age.

[0101] Methods for isolating MAIT cells from placental intervillous blood (IVB) are generally known in the art. Exemplary and non-limiting protocols utilize blood dripping from a placenta held upright with a clamped umbilical cord. Such methods involve lifting the placenta with the umbilical cord facing downwards and allowing blood to drip from it. These methods have been shown to exhibit very little cross-contamination between IVB and umbilical cord blood. Those skilled in the art are familiar with methods for verifying and, if necessary, improving the purity of the cell population using cell sorting.

[0102] <Mucosal-associated invariant T (MAIT) cells> The mucosa-associated invariant T (MAIT) cells used herein include non-conventional T cells expressing semi-invariant T cell receptors (TCRs) such as Vα7.2-Jα33 (in humans). In some embodiments, these are associated with β-chains, Vβ2 / Vβ13. In other embodiments, the aforementioned α-chains are associated with β-chains derived from the TRBV6 or TRBV20 gene family. MAIT cells recognize antigens limited to non-peptide molecules presented in the context of the (non-polymorphic) major histocompatibility complex (MHC) class I-like protein MR1. The modified MAIT cells disclosed herein can be detected, for example, by staining with MR1-Ag tetramers loaded with 5-(2-oxopropyrideneamino)-6-D-ribitylaminouracil (5-OP-RU), 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU), RL-6,7-diMe (PubChemCID168989), RL-6-Me-7-OH (PubChemCID440869), or diclofenac (PubChemCID3033). In other embodiments, any of the following compounds may also be used: namely, 6-(1H-indole-3-yl)-7-hydroxy-8-ribitylrumazine or photolumazine III (PLIII), 6-(2-carboxyethyl)-7-hydroxy-8-ribitylrumazine or photolumazine I, 5-hydroxydiclofenac (PubChemCID3052566), 4-hydroxydiclofenac (PubChemCID116545), benzbromarone (PubChemCID2333), chloroxin (PubChemCID2722), phloxyuridine (PubChemCID5790), galangin (4H-1-benzopyran-4-one, 3,5,7-trihydroxy-2-phenyl, or 3,5,7-trihydroxyflavone), or mercaptopurine (PubChemCID667490). (For example, see Corbett et al., Antigen Recognition by MR1-Reactive T Cells; MAIT Cells, Metabolites, and Remaining Mysteries, Front Immunol. 11:1961 (2020) and the literature cited therein).

[0103] In one embodiment, the placental MAIT cells disclosed herein are human MAIT cells. In some embodiments, the MAIT cells disclosed herein are allogeneic with respect to the recipient of the modified MAIT cell population, as described herein.

[0104] Methods for isolating and characterizing placental IVBMAIT cells and other leukocyte subpopulations are known in the art. Leukocyte subpopulations can be isolated and / or analyzed using gating strategies such as those described herein, solely for illustrative purposes.

[0105] There are quantitative and qualitative differences between MAIT cells derived from umbilical cord blood and adult blood (see, for example, Youssef et a1., Ontogeny of Human Mucosal-Associated Invariant T Cells and Related T Cell Subset. JEM 215:459-479 (2018)). Staining for Vα7.2 and CD161 can identify stage 3 MAIT cells circulating at birth. However, Vα7.2 in umbilical cord blood... + CD161 high The fraction may also include other T cells that are thought to share a common developmental pathway. In umbilical cord blood, MAIT cells exhibit a naive phenotype (CD45RA). + / RO - They express ) and CD8αβ heterodimer, but most adults exhibit a memory phenotype and express CD8αα homodimer. Postnatal Vα7.2 + CD161 high The extremely low proliferation of T cells may be related to cell-specific characteristics or to the limited availability of microbial-derived MR1 ligands. (Naive umbilical cord blood Vα7.2) + CD161 high T cells expressed significantly lower levels of PLZF than adult MAIT cells, suggesting that umbilical cord blood Vα7.2 + CD161 high The study suggested that early postnatal activation signals are necessary for the final maturation of T cells. (Umbilical cord blood Vα7.2) + CD161 high T cells, like conventional CD8 T cells, proliferate strongly after stimulation with PHA, but adult Vα7.2 + CD161 high T cell proliferation efficiency is much lower. In contrast to mature MAIT cells in adult blood, umbilical cord blood Vα7.2 + CD161 high T cells cannot exhibit immediate effector function against bacterial-infected cells. From this data, umbilical cord blood Vα7.2 + CD161 highAlthough T cells inherently possess proliferative capacity, it has been shown that they cannot acquire detectable effector activity after recognizing microbial antigens unless they undergo functional maturation and / or proliferation after birth.

[0106] Umbilical cord blood Vα7.2 + CD161 high Because the cells exhibit a naive phenotype and intermediate PLZF levels, they cannot rapidly produce cytokines or cytotoxic molecules in response to bacterial ligands, in contrast to mature adult MAIT cells. Furthermore, despite high expression of receptors for exogenous IL-12 and IL-18, umbilical cord blood MAIT cells do not respond to stimulation by these cytokines. In addition, as shown in Chen et al., Circulating Mucosal-Associated Invariant T Cells in a Large Cohort of Healthy Chinese Individuals From Newborn to Elderly. Front.Immunol., vol.10, article 260 (2019), umbilical cord blood MAIT cells have a naive phenotype and do not secrete IFN-γ, IL17A, or TNF-α after in vitro stimulation with PMA / ionomycin. In summary, these data indicate that umbilical cord blood MAIT cells differ phenotypically and functionally from MAIT cells derived from adult subjects.

[0107] In some embodiments, the MAIT cells disclosed herein are CD161 + Vα7.2 + CD4 - CD3 + They consist of lymphocytes, which also bind to the MRL-Ag tetramer. In various embodiments, more than 50% of the cells in a population are CD161 + Vα7.2 + CD4 - CD3 +is. In some embodiments, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more of the cells in the population are CD161 + Vα7.2 + , CD4 - CD3 + .

[0108] In some further embodiments, but not limited thereto, CD8 + cells (more specific embodiments are CD8 + CD4 - cells), CD8 - CD4 - cells, or CD4 + cells are included, and a MAIT subset is utilized. In various embodiments, 50% or more of the cells in the population are CD8 + . 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more of the cells in the population are CD8 + . 50% or more of the cells in the population are CD8 + CD4 - . 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more of the cells in the population are CD8 + CD4 - . 50% or more of the cells in the population are CD8 - CD4 - . 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more of the cells in the population are CD8 - CD4 ― .

[0109] In one embodiment, the MAIT cells disclosed herein are CD45RA - CCR7 - , which also, in some embodiments, reflects an effector memory phenotype. In other embodiments, CD45RO +is an additional feature of effector memory cells. More than 50% of the cells in the population are CD45RA - CCR7 - and more than 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the cells in the population are CD45RA - CCR7 - In various embodiments, more than 50% of the cells in the population are CD45RO + and more than 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the cells in the population are CD45RO + In certain embodiments, the MAIT cells disclosed herein are CD45RA

[0110] CCR7 - CCR7 - CD62L + In various embodiments, more than 50% of the cells in the population are CD45RA - CCR7 - CD62L + and in other embodiments, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more.

[0111] In certain embodiments, the MAIT cells disclosed herein are CD45RA - CCR7 - CD62L - In various embodiments, more than 50% of the cells in the population are CD45RA - CCR7 - CD62L - and in other embodiments, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more.

[0112] In certain embodiments, the MAIT cells disclosed herein are CD45RO + CCR7 - CD62L +In various embodiments, more than 50% of the cells in the population are CD45RO. + CCR7 - CD62L + In other embodiments, the percentages are 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more.

[0113] In one embodiment, the MAIT cells disclosed herein are CD45RO + CCR7 - CD62L - In various embodiments, more than 50% of the cells in the population are CD45RO. + CCR7 - CD62L - In other embodiments, the percentages are 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more.

[0114] In other embodiments, the MAIT cells disclosed herein express interleukin (IL)-18Rα, CD127, α4β7, and / or PD-1. In some embodiments, MAIT cells express interleukin (IL)-18Rα, CD127, and α4β7. MAIT cells express interleukin PD-1. MAIT cells express interleukin (IL)-18Rα, CD127, α4β7, and PD-1. In some embodiments, the cells also express the transcription factors promyelocytic leukemia Zn finger (PLZF), RORγt, Helios, Eomesodermin (Eomes), and / or T-box transcription factor (T-bet). In some embodiments, MAIT cells express promyelocytic leukemia Zn finger (PLZF), RORγt, Helios, and Eomesodermin. MAIT cells express T-box transcription factor. MAIT cells express the transcription factors promyelocytic leukemia zinc finger (PLZF), RORγt, Helios, Eomesodermin, and the T-box transcription factor. In other embodiments, or additionally, cells express the surface markers CD26, CD44, CD69, or CD25, or the receptors interleukin-7 receptor (IL-7R), IL-12R, IL-15R, or IL-18R. Cells express the inducible T cell costimulatory molecule (ICOS). Each of the above proteins, and each combination thereof, represents a distinct embodiment.

[0115] In some embodiments, MAIT cells disclosed herein express genes associated with tissue repair (e.g., transforming growth factor β-1, platelet-derived growth factor subunit B, or matrix metallopeptidase) or angiogenesis (e.g., granulocyte-macrophage colony-stimulating factor, vascular endothelial growth factor, or hypoxia-inducible factor 1 subunit α) upon stimulation with 5-OP-RU.

[0116] In one embodiment, placental MAIT cells recognize riboflavin precursor derivatives of microbial origin. In some embodiments, modified placental CAR-MAIT cells, upon activation, secrete inflammatory cytokines (e.g., interferon-γ [IFN-γ], tumor necrosis factor α [TNF-α], interleukin-17, or colony-stimulating factor 2 [CSF2 / GM-CSF]) for example, by recognition of an MR-1 ligand, or in other embodiments, independently of MR1. IL-17A, TNF-α, CSF2, or MIP-1 are all secreted. IL-26, oncostatin M (OSM), or heparin-binding early growth factor (HBEGF) are increased upon stimulation with IL-12, IL-18, IL-15, or tumor necrosis factor-like protein 1A (TL1A). Alternatively, or additionally, the modified placental CAR-MAIT cells disclosed herein exhibit granzyme B-dependent cytotoxicity to target cells upon activation. In other embodiments, IFN-γ, perforin, or granzyme B are all increased.

[0117] In some embodiments, the methods described herein include growing and / or enriching modified MAIT cells in vitro and / or ex vivo before administration to a subject. In various embodiments, the growth or enrichment is performed either before or after modifying the MAIT cells to express exogenous antigen receptors. Activation assays or stimulation assays are performed to characterize or determine the quality of the modified MAIT cells. However, activation usually requires stimulation of antigen receptors or co-receptors in combination with cytokine treatment. Once the cells are activated, in some embodiments, the cells can be further grown by cytokine alone (without returning them to a naive state).

[0118] In some embodiments, MAIT cells are grown in vitro and / or ex vivo for about 5 days or more, in other embodiments for 5 to 10 days, in yet another embodiment for 10 days or more, in yet another embodiment for 10 to 15 days, in yet another embodiment for 15 days or more, in yet another embodiment for 15 to 20 days, and in some embodiments for 20 days or more.

[0119] Throughout this application, various embodiments may be presented in range form. It should be understood that range form descriptions are merely for convenience and conciseness, and should not be interpreted as inflexible limitations on the scope of modified MAIT cells and their uses. Therefore, a range description should be considered to specifically disclose all possible sub-ranges, as well as the individual numbers within that range. For example, a range description such as 1 to 6 should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, and 3 to 6, along with the individual numbers within that range, e.g., 1, 2, 3, 4, 5, 6. This applies regardless of the breadth of the range.

[0120] Where a numerical range is indicated herein, it means that all numerical values ​​(whether fractions or integers) within the indicated range are included. In this specification, the expressions "range between a first numerical value and a second numerical value" and "range from a first numerical value to a second numerical value" are used interchangeably and mean that the first numerical value and the second numerical value, as well as all fractions and integers between them, are included.

[0121] Methods for stimulating MAIT cells are known in the art. In one non-limiting embodiment, HeLa cells overexpressing human MR1 protein (Hela-hMRl) are washed and incubated with Escherichia coli Dh5αATCC strain (typically with a bacterial infection multiplicity of 10 to 100 cells per HeLa cell) in antibiotic-free DMEM at 37°C for 30 minutes. After washing, the cells are incubated at 37°C for 2 hours in complete medium supplemented with 100 μg / mL gentamicin and 10 μg / mL chloramphenicol. MAIT cells are added and co-cultured overnight. The cells are then harvested, stained, and subjected to FACS analysis.

[0122] Other methods for growing and / or enriching MAIT cells are also known in the art. In one non-limiting embodiment, MAIT cells are incubated with 5 μg / mL CpG, 300 nM 5-(2-oxopropyrideneamino)-6-D-ribitylaminouracil (5-OP-RU), and 50 ng / mL human IL-15. In one embodiment, IL-15 is included to preferentially promote the proliferation of memory T cells.

[0123] In some embodiments, selected, purified, or concentrated MAIT cells are stimulated in vitro and / or ex vivo in the presence of autologous or allogeneically irradiated PBMCs and IL-2, IL-7, IL-12, IL-18, IL-21, IL-15 or analogs thereof, or combinations thereof, with CD3 / CD28 stimulation (e.g., TransAct). TM Activated by functionalized beads (commercially available under the trademark of [company name]). In other embodiments, CD3 / CD28 beads (ClinExVivo TM Use XVivo-15 (marketed as a clinical-grade product under the Dynabeads trademark) together with IL-7 and IL-2. Non-exclusive exemplary protocols include XVivo-15. TM (BioWhittaker, Walkersville, MD), using 100 units / mL of IL-2 and 10 ng / mL of IL-7, ClinExVivo TMThis involves incubating Dynabeads in a cell:bead ratio of 3:1 for 10–14 days. In various embodiments, once activated, MAIT cells can be further proliferated in the presence of cytokines, with or without the aforementioned ligands.

[0124] In other embodiments, MAIT cells are activated in vitro and / or ex vivo in the presence of MAIT cell activation ligands such as 5-OP-RU, 5-amino-4-D-ribitylaminouracil dihydrochloride (5-ARU), 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU), and 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione (5-A-RU), or other riboflavin (vitamin B2) derivatives. In some embodiments, the ligand(s) are provided in combination with cytokines, e.g., cytokines mentioned herein. As a non-limiting example, MAIT cells can be grown for, for example, 6 to 17 days using 100 nM 5-OP-RU and 100 IU / mL IL-2. In other embodiments, MAIT cells are proliferated and / or activated in vitro and / or ex vivo in the presence of MAIT cell-activating drug metabolites, such as diclofenac metabolites. In various embodiments, once activated, MAIT cells can be further proliferated in the presence of cytokines, with or without the aforementioned ligands.

[0125] In some embodiments, MAIT cells are grown and / or activated in vitro in the presence of IL-12, IL-15, IL-18 and 5-OP-RUs ranging from 10 to 1000 nM, or IL-12 + IL-18, or IL-15 + IL-18, as described in the literature of the art.

[0126] In some embodiments, the modified MAIT cells disclosed herein produce IL-17 upon PMA and ionomycin stimulation and increase the expression level of the Thl7-related transcription factor RORC(RORγt), but do not increase it after CD3+CD28 stimulation.

[0127] In some embodiments, the modified MAIT cells disclosed herein produce IFN-γ and increase T-bet expression upon PMA / ionomycin stimulation. In more specific embodiments, these cells are CD8 + That is the case.

[0128] In various embodiments, more than 30%, more than 40%, more than 50%, more than 60%, or more than 70% of the population of modified MAIT cells disclosed herein express CD69.

[0129] In other embodiments, less than 30%, less than 20%, or less than 10% of the MAIT cell population disclosed herein express PD-1. In other embodiments, more than 10%, more than 15%, or more than 20% of the modified MAIT cell population express CD25. In other embodiments, any combination of two of the above markers is expressed, and their proportions can be freely combined. In some embodiments, more than 50%, more than 60%, more than 70%, or more than 80% of the MAIT cells express PD-1. - / LAG-3 - The expression of indicates that the cell does not show T cell exhaustion. In other embodiments, CTLA-4, TIGIT, 2B4, BTLA, CD57, TIM-3, or KLRG-1 are used to detect exhausted T cells.

[0130] In some embodiments, the MAIT cells disclosed herein express CCR2, CCR5, CCR6, CCR9, CXCR4, CXCR3, VLA-4, or CXCR6, or any combination of two, three, four, five, six, seven, or eight of these receptors.

[0131] Alternatively or additionally, MAIT cells express activating receptors such as NKG2D, NKp30, NKp44, or NKG2D, NKp30, and NKp44. In some embodiments, MAIT cells express high levels of CXCR4 and moderate levels of CCR9, but low or no expression of CXCR2. Alternatively or additionally, MAIT cells also express CXCR3. In some embodiments, the MAIT cells disclosed herein express one or more cytokine receptors such as IL-7R, IL-12R, IL-15R, IL-18R, and IL-21R, or any combination thereof.

[0132] <Exogenous antigen receptors> As used herein, the term “exogenous antigen receptor” includes antigen receptors that do not naturally occur on MAIT cells. Examples of such receptors include chimeric antigen receptors (CARs). The MAIT cells disclosed herein are modified to express exogenous CARs.

[0133] In one embodiment, the exogenous antigen receptor is permanently incorporated into the modified IVB-derived MAIT cells. In another embodiment, the IVB-derived MAIT cells are modified to transiently express the exogenous antigen receptor gene. The term “permanent” is used herein to indicate the insertion of exogenous DNA into the genome of a target cell (various viral and nonviral techniques commonly known in the art may be utilized). The term “transient” is used in some embodiments to indicate that cells are modified to transiently express the exogenous antigen receptor gene by inserting mRNA into them.

[0134] Those skilled in the art will understand that, in some embodiments, the exogenous antigen receptors disclosed herein include an antigen-binding domain that binds to a MAIT cell containing the exogenous antigen receptor in order to bind to a target molecule, i.e., an antigen of interest. As used herein, the terms “antigen” and “target molecule” may be used interchangeably as they have the same nature and meaning.

[0135] <Chimera antigen receptor (CAR)> As is commonly known in the art, chimeric antigen receptors (CARs) are genetically modified receptors that include at least an extracellular antigen-binding domain, a hinge domain, a transmembrane domain, a costimulatory domain, and a cytoplasmic signaling domain. These modified receptors can be readily introduced and expressed in immune cells such as placental cells and IVB-derived MAIT cells according to techniques known in the art. Using CARs, a single receptor can be programmed to recognize a specific antigen and, upon binding to that antigen, activate immune cells to attack and destroy cells possessing that antigen. When these antigens are present on tumor cells, immune cells expressing CARs can target and kill the tumor cells.

[0136] In some embodiments, in the modified placental CAR-MAIT cells described herein, the CAR comprises a modified immune receptor comprising (a) an extracellular antigen-binding domain optionally linked to a hinge region, (b) a transmembrane region, and (c) an intracellular signaling domain (e.g., CD3 zeta or CD3ζ). In some embodiments, the extracellular antigen-binding domain comprises a single-chain variable region fragment (scFv). In some embodiments, the extracellular antigen-binding domain comprises an antigen-binding antibody fragment. In some embodiments, the extracellular antigen-binding domain comprises a single-chain antibody.

[0137] In some embodiments, the transmembrane domain is derived from either the CD4, CD8, or CD3ζ domain, or from a co-stimulatory molecule such as CD28.

[0138] In some embodiments, the extracellular antigen-binding domain contains an antigen-binding fragment of the antibody. In some embodiments, a co-stimulatory domain (non-limiting examples being 4-1BB / CD137, OX40 / CD134, and CD28) is also present and, in some embodiments, is inserted into the CD3 zeta domain. In some embodiments, both CD28 and another co-stimulatory domain, e.g., 4-1BB or OX40, are inserted.

[0139] In some embodiments, the co-stimulatory domain of a CAR is designed to provide a co-stimulatory signal to an activating domain, which activates one or more of the normal effector functions of immune cells. Effector functions of T cells include, but are not limited to, helper activities such as cytolytic activity and cytokine secretion.

[0140] In one embodiment, suitable co-stimulatory domains include, but are not limited to, 4-1BB / CD137, CD2, DAP12, ICOS, GITR, FcRγ, CD27, CD28, LFA-1, OX-40, or combinations thereof.

[0141] Intracellular signaling domains or activation domains can also be incorporated into CARs in some embodiments. For example, CD3 is a component of the native conventional T cell receptor on T cells and has been shown to be an important intracellular activating element in CARs. In some embodiments, CD3 is CD3-zeta or CD3-epsilon.

[0142] <Target antigen> In one embodiment, for example, an exogenous antigen receptor such as a CAR described herein is used to guide modified MAIT cells to recognize tumor antigens, microbial antigens, pathogens, viral antigens, fungal antigens, bacterial antigens, antigens expressed on Treg cells, antigens expressed on heteroreactive lymphocytes, antigens expressed on senescent cells, antigens expressed on cells associated with fibrotic diseases or conditions, or antigens expressed on cells associated with autoimmune diseases or disorders.

[0143] In some embodiments, CARs included as part of modified IVB-MAIT cells guide the modified MAIT cells to recognize, activate, proliferate, and lyse target cells in response to CAR-driven recognition of antigens such as tumor-associated antigens (TAAs) or viral antigens, for example, but not limited to these. In some embodiments, CARs included as part of modified placental MAIT cells or IVB-MAIT cells guide the modified MAIT cells to recognize, activate, proliferate, and lyse target cells in response to scFv-driven recognition of antigens such as tumor-associated antigens (TAAs) or viral antigens, for example, but not limited to these. As used herein, the terms "CAR-MAIT" and "CAR-MAIT cells" encompass IVB-derived MAIT cells modified to express exogenous CARs, and all have the same properties and meanings and can be used interchangeably.

[0144] As used herein, “viral antigen” includes antigens expressed by viral proteins, and includes scenarios in which the antigen is currently expressed by either viral cells or cancer cells (e.g., in the case of oncogenic proteins). Thus, in some embodiments, modified MAIT cells that recognize viral antigens are used to treat viral infections, or in other embodiments, they are used to treat malignancies that express viral antigens. In the case of CARs, it will be understood by those skilled in the art that the recognition of such antigens is typically MHC-independent.

[0145] Non-limiting examples of viral antigens include, for example, hexon or penton, HPVE6, HPVE7 for the treatment of adenovirus; for example, immediate early 1 (IE-1) or 65 kilodalton tegument phosphorylated protein (pp65) for the treatment of cytomegalovirus (CMV); for example, EBV nuclear antigen 1 (EBNA1), BZLF1, or any of the EBV latent genes LMP1, LMP2, EBNA1, EBNA2, EBNA3A, EBNA3B, or EBNA3C for the treatment of Epstein-Barr virus (EBV) or lymphoma; for example, VP1 or large T for the treatment of BK virus (BKV); for example, U11, U14, or U90 for the treatment of human herpesvirus 6 (HHV-6); and for example, HSV-1 thymidine kinase (HSV-TK) for the treatment of herpes simplex virus type 1 (HSV-1).

[0146] In some embodiments, the CAR target includes a tumor-associated antigen (TAA) or cancer-associated antigen. As used herein, the terms “tumor-associated antigen” or “cancer antigen” include tumor-specific antigens, whether originally derived from a virus or from a normal cell genome. In the case of CARs, it will be understood by those skilled in the art that the recognition of such antigens is typically MHC-independent.

[0147] Non-limiting examples of appropriate cancer antigens include: alpha-fetoprotein, desmoyoquine / AHNAK S2580FBCMA (e.g., for the treatment of relapsed or refractory multiple myeloma), CD7 (e.g., for the treatment of T-cell leukemia or lymphoma), CD19 / 20 / 22 (e.g., for the treatment of non-Hodgkin lymphoma, acute lymphoblastic leukemia, B-cell leukemia or B-cell lymphoma), CD30 (e.g., for the treatment of lymphomas such as Hodgkin lymphoma), CD33, CD38, CD70 (e.g., for the treatment of leukemia, lymphoma or renal cell carcinoma), CD73, CD123 and CD133 (e.g., for the treatment of AML), CS1 (e.g., for the treatment of multiple myeloma), c-MET, DR5, diasialoganglioside GD2 or IL13ra2 (e.g., for the treatment of neuroblastoma), EGFRVIII, Epstein-Barr virus (e.g., for the treatment of B-cell malignancies), ERBB2 H473Y and ERBB2IP E805G HBV surface antigen, HER2 (e.g., for the treatment of glioblastoma), mesoserine (e.g., for the treatment of solid tumors expressing mesoserine), minor H antigen (HA-1), NKG2DL (e.g., for the treatment of solid tumors or colorectal cancer), prostate-specific membrane antigen (PSMA), TPBG (human chorionic glycoprotein) or 5T4 (e.g., for the treatment of solid tumors such as colorectal cancer, ovarian cancer and gastric cancer, and pediatric acute lymphoblastic leukemia (ALL)), TGFβRII frameshift antigen, VEGFR-2, Wilms tumor 1 (WT-1), GPC3, ROR1, RAC1-P29S, COL6A3, HA-2, claudin 18.2, MUC16, GPRC5D, HERV-E, Muc1, claudin 3, claudin 4 and claudin 6.

[0148] In one embodiment, or additionally, the modified MAIT cells disclosed herein are used to target tumor cells expressing MR-1.

[0149] In other embodiments, the CAR target is a microbial antigen. Examples of microbial antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, and protozoan antigens. In one embodiment, the modified MAIT cells disclosed herein are used to treat a disease or infection caused by a microorganism expressing the antigen.

[0150] Those skilled in the art will understand that microorganisms include bacteria, viruses, fungi, and parasites, and that microorganisms that cause disease are called pathogens. In some embodiments, the term “microbial antigen” as used herein may encompass pathogenic targets, i.e., pathogenic antigens.

[0151] In some embodiments, exogenous CARs target bacterial antigens, which in some embodiments are used to treat bacterial infections. In some embodiments, the targeted bacteria include the genera Nitrospira, Nitrosospira, Nitrobacter, Nitrosomonas, Clostridium, Bacillus, methanogenic archaea, coliforms, Salmonella, Bacteroides, Staphylococcus, Streptococcus, Neisseria, Haemophilus, Bordetella, Listeria, Mycobacterium, and Shigella. The genera may be selected from: Ra, Pseudomonas, Brucella, Treponema, Mycoplasma, Yersinia, Vibrionaceae, Chlamydia, Legionella, Escherichia, Acinetobacter, Burkholderia, Thiobacillus, Rickettsia, Sphinomonas, Francisella, Campylobacter, and Helicobacter.

[0152] In some embodiments, exogenous CARs target fungal antigens, which are used in some embodiments to treat fungal infections. In one embodiment, the fungal infection is a yeast infection.

[0153] In some embodiments, exogenous CARs target molecules expressed on regulatory T cells (Tregs), and such targeting can lead to Treg depletion. Treg-related molecules are commonly known in the art, such as CCR8.

[0154] In some embodiments, exogenous CARs target molecules expressed on allogeneic T cells. Such targeting selectively eliminates allogeneic T cells without depleting other non-allogeneic T cells. Depletion of allogeneic T cells is thought to improve the persistence of the response after treatment with allogeneic drugs. CD70 is one example of an allogeneic T cell target, though not an exhaustive one.

[0155] In some embodiments, the exogenous CAR targets molecules expressed in subjects suffering from fibrotic diseases such as cardiac fibrosis, for example, but not limited to these, and the target antigen may include fibroblast-activating proteins (FAPs).

[0156] In some embodiments, exogenous CARs target molecules expressed in subjects suffering from age-related diseases or conditions. Aging is a chronic “alarm state” in tissues. The range of such diseases is broad and includes debilitating conditions such as chronic inflammation, fibrotic liver disease, atherosclerosis, and diabetes. Examples of molecules expressed by senescent cells, but not limited to, include urokinase plasminogen activator receptor (uPAR), which can be targeted by uPAR-specific CARs. CAR-T cells that target aging are called “senescent cell-clearing CAR-Ts.” Disclosed herein are “senescent cell-clearing CAR-MAIT” cells that include exogenous antigen receptors that target molecules expressed on the cell surface of senescent cells. In one embodiment, the target of the modified MAIT cells disclosed herein is uPAR.

[0157] In some embodiments, the exogenous CAR targets a molecule disclosed herein that is expressed in an allografted subject, which is an antigen molecule expressed on alloreactive lymphocytes, such as CD70, but is not limited thereto.

[0158] In some embodiments, the exogenous CAR targets a molecule disclosed herein that is expressed in a subject suffering from an autoimmune disease or disorder, the molecule being an antigen molecule associated with the disease or disorder. Examples of autoimmune diseases, but not limited to, include systemic lupus erythematosus (SLE), including refractory SLE, pemphigus vulgaris, multiple sclerosis, type 1 diabetes mellitus, rheumatoid arthritis, celiac disease, pernicious anemia, inflammatory myopathy, myasthenia gravis, or adrenal inflammatory disease, or a combination thereof. In some embodiments, the exogenous antigen receptor associated with an autoimmune disease or disorder includes peptide-MHCII chimeric antigen receptors (pMHCII-CARs) that target pathogenic MHC class II:peptide complexes associated with autoimmune diseases, chimeric autoantibody receptors such as DSG3-specific CAARs, CD19 and / or CD20-specific CARs, and B-cell antibody receptors (BARs). In other embodiments, exogenous antigen receptors associated with autoimmune diseases and disorders include carboxypeptidase H, chromogranin A, glutamate decarboxylase, Imogen-38, insulin, insulinoma antigen-2 and 2β, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proinsulin, citrullinated protein, collagen II, heat shock protein, human cartilage glycoprotein, double-stranded DNA, La antigen, nucleosome histone, ribonucleotide protein (snRNP), phospholipid-β-2 glycoprotein I complex, poly(ADP-ribose) polymerase, Sm antigen of U1 small ribonucleoprotein complex, α-enolase, aquaporin 4, β-arrestin, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein, S100-β, RI-type reticulin, and gastric H + / K + - Containing ATPase, 21-hydroxylase, 17-hydroxylase, and cytochrome P450 side-chain cleavage enzyme, or CD19.

[0159] <How to use> This specification discloses methods of using MAIT cells and modified MAIT cells as described throughout. In some embodiments, the modified MAIT cells used in the methods disclosed herein include modified placental MAIT cells. In some embodiments, the modified MAIT cells used in the methods disclosed herein include IVBMAIT cells. In some embodiments, the modified placental-derived MAIT cells used in the methods disclosed herein are maternal-derived. In some embodiments, the modified placental-derived MAIT cells used in the methods disclosed herein are derived from placental blood in the basal decidual region. In some embodiments, the modified placental-derived MAIT cells used in the methods disclosed herein are derived from placental blood in the parietal decidual region. In some embodiments, the modified placental-derived MAIT cells used in the methods disclosed herein are derived from intervilli blood. In some embodiments, placental MAIT cells include a combination of the multiple placental-derived sources described herein. In some embodiments, placental MAIT cells used in the methods disclosed herein are not derived from umbilical cord blood.

[0160] In some embodiments, the MAIT cells used in the methods disclosed herein include placental MAIT cells. In some embodiments, the MAIT cells used in the methods disclosed herein include IVBMAIT cells. In some embodiments, the placental-derived MAIT cells used in the methods disclosed herein are maternal-derived. In some embodiments, the placental-derived MAIT cells used in the methods disclosed herein are derived from placental blood from the basal decidual region. In some embodiments, the placental-derived MAIT cells used in the methods disclosed herein are derived from placental blood from the parietal decidual region. In some embodiments, the placental-derived MAIT cells used in the methods disclosed herein are derived from intervilli blood. In some embodiments, the placental MAIT cells include the combination of placental sources described herein. In some embodiments, the placental MAIT cells used in the methods disclosed herein are not derived from umbilical cord blood.

[0161] In some embodiments, the MAIT cells used in the methods disclosed herein are derived from maternal blood, placenta, or IVB, or a combination thereof, and may or may not be modified to express exogenous antigen receptors.

[0162] In some embodiments of the methods of use described herein, the modified MAIT cells include CAR-MAIT cells, and the MAIT cells are derived from IVB. In some embodiments of the therapeutic methods described herein, the method includes administering a population of CAR-MAIT cells to a subject requiring treatment.

[0163] In some embodiments, a method is provided for treating a subject having a tumor or malignant tumor, the method comprising the step of administering a population of modified MAIT cells described herein to the subject.

[0164] In other embodiments, compositions or pharmaceutical compositions comprising a population of modified MAIT cells described herein for treating subjects having tumors or malignancies are provided. Those skilled in the art will understand, in light of this disclosure, that tumors, malignancies, and hyperproliferative disorders may be treated with the population of modified MAIT cells disclosed herein, in particular, when the cells of the tumor, malignancy, or hyperproliferative disorder contain an antigen that is recognized (e.g., expressing the antigen, or, in other embodiments, containing the antigen). In some embodiments of methods for treating subjects having tumors or malignancies, the modified MAIT cells are derived from intervillous blood of the placenta. In some embodiments, when treating subjects having tumors or malignancies, the modified MAIT cells are allogeneic to the subject. In some embodiments, when treating subjects having tumors or malignancies, the modified MAIT cells include modified CARs directed to cancer antigens or tumor-associated antigens, for example, but not limited to those disclosed herein.

[0165] In other embodiments, a method is provided for eliminating allogeneic-reactive T cells in a subject requiring such elimination, comprising the step of administering a population of modified MAIT cells described herein to the subject. In some embodiments, subjects requiring elimination of allogeneic-reactive T cells include subjects who have undergone allogeneic transplantation. In some embodiments, the modified MAIT cells target allogeneic-reactive T cells by expressing exogenous CARs that target molecules expressed on allogeneic-reactive T cells. In some embodiments, the subject may have a transplant-related condition such as bone marrow transplantation, organ transplantation, or any other allogeneic transplantation. In some embodiments of the method for eliminating allogeneic-reactive T cells in a subject, the modified MAIT cells are derived from placental intervillous blood. In some embodiments, the modified MAIT cells are allogeneic to the subject. In some embodiments, the modified MAIT cells have a target expressed on allogeneic-reactive lymphocytes. In some embodiments, the target expressed on allogeneic-reactive lymphocytes is CD70.

[0166] In other embodiments, a method is provided for eliminating regulatory T cells (Tregs) as a method for treating cancer or improving an anti-cancer treatment (one or more) in a subject, the method comprising the step of administering a population of modified MAIT cells described herein to the subject. In some embodiments, the modified MAIT cells target Tregs by expressing exogenous CARs that target molecules expressed on Tregs. In some embodiments, the target molecule expressed on Tregs is CCR8.

[0167] In some embodiments, a method is provided for treating a subject infected with a pathogen, comprising the step of administering a population of modified MAIT cells described herein to the subject. In other embodiments, a composition or pharmaceutical composition comprising a population of modified MAIT cells described herein is provided for treating a subject infected with a pathogen. The pathogen may be a bacterial pathogen, a viral pathogen, or a fungal pathogen commonly known in the art. Those skilled in the art will understand in light of this disclosure that a variety of infectious diseases can be treated with the modified MAIT cells disclosed herein, in particular when the pathogenic cells have an antigen to be recognized (for example, if they express an antigen, or in other embodiments if they contain an antigen). In some embodiments of a method for treating a subject infected with a pathogen, the modified MAIT cells are derived from intervillous blood of the placenta. In some embodiments, when treating a subject infected with a pathogen with the modified MAIT cells described herein, the modified MAIT cells are allogeneic to the subject.

[0168] In some embodiments, a method is provided for treating a subject suffering from a fibrotic disease, comprising the step of administering a population of modified MAIT cells described herein to the subject. In other embodiments, a composition or pharmaceutical composition comprising a population of modified MAIT cells described herein for treating a subject suffering from a fibrotic disease is provided. The fibrotic disease may be any fibrotic disease commonly known in the art, including, but not limited to, myocardial fibrosis. In some embodiments of the method for treating a fibrotic disease, the modified MAIT cells target expressed antigens associated with the fibrotic disease. In some embodiments of the method for treating a fibrotic disease, the modified MAIT cells target fibroblast-activating protein (FAP). In some embodiments of the method for treating a subject suffering from a fibrotic disease, the modified MAIT cells are derived from intervillous blood of the placenta. In some embodiments, when treating a subject suffering from a fibrotic disease with the modified MAIT cells described herein, the modified MAIT cells are allogeneic to the subject.

[0169] In some embodiments, a method is provided for treating a subject suffering from an age-related disease or condition, comprising the step of administering a population of modified MAIT cells described herein to the subject. In other embodiments, a composition or pharmaceutical composition comprising a population of modified MAIT cells described herein is provided for treating a subject suffering from an age-related disease or condition. The age-related disease or condition may be any age-related disease or condition commonly known in the art, such as, but not limited to, chronic inflammation, hepatic fibrosis, atherosclerosis, diabetes mellitus, or a combination thereof. In some embodiments of the method for treating an age-related disease or condition, the modified MAIT cells target expressed antigens associated with the age-related disease or condition. In some embodiments of the method for treating an age-related disease or condition, the modified MAIT cells target urokinase plasminogen activator receptor (uPAR). In some embodiments of the method for treating a subject suffering from an age-related disease or condition, the modified MAIT cells are derived from intervillous blood of the placenta. In some embodiments, when a subject suffering from an age-related disease or condition is treated with the modified MAIT cells described herein, the modified MAIT cells are allogeneic to the subject.

[0170] In some embodiments, a method is provided for treating a subject suffering from an autoimmune disease or disorder, comprising the step of administering a population of modified MAIT cells described herein to the subject. In other embodiments, a composition or pharmaceutical composition comprising a population of modified MAIT cells described herein is provided for treating a subject suffering from an autoimmune disease or disorder. The autoimmune disease or disorder may be any autoimmune disease or disorder commonly known in the art, and may include, for example, but are not limited to, systemic lupus erythematosus (SLE), refractory SLE, pemphigus vulgaris, multiple sclerosis, type 1 diabetes mellitus, rheumatoid arthritis, celiac disease, pernicious anemia, inflammatory myopathy, myasthenia gravis, or adrenal inflammatory disease, or a combination thereof. In some embodiments of the method for treating an autoimmune disease or disorder, the modified MAIT cells target expressed antigens associated with the autoimmune disease or disorder. In some embodiments of methods for treating autoimmune diseases or disorders, modified MAIT cells are targeted by pathogenic MHC class II:peptide complexes associated with autoimmune diseases, autoantibodies, B cell antibodies targeted by B cell antibody receptors (BARs), carboxypeptidase H, chromogranin A, glutamate decarboxylase, imogen-38, insulin, insulinoma antigens-2 and 2β, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proinsulin, and citrullinated proteins. Citrate, collagen II, heat shock protein, human cartilage glycoprotein, double-stranded DNA, La antigen, nucleosome histone and ribonucleoprotein (snRNP), phospholipid-β-2 glycoprotein I complex, poly(ADP-ribose) polymerase, Sm antigen of U1 small ribonucleoprotein complex, α-enolase, aquaporin 4, β-arrestin, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein S100-β, R1 type reticulin, gastric H + / K +-Targets ATPase, 21-hydroxylase, 17-hydroxylase, cytochrome P450 side-chain cleavage enzyme and CD19. The modified MAIT cells target pathogenic MHC class II:peptide complexes associated with the autoimmune disease, autoantibodies, B cell antibodies targeted by B cell antibody receptors (BARs), carboxypeptidase H, chromogranin A, glutamate decarboxylase, imogen-38, insulin, insulinoma antigens-2 and 2β, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proinsulin, citrullinated protein, collagen II, and he Shock protein, human cartilage glycoprotein, double-stranded DNA, La antigen, nucleosome histone, ribonucleoprotein (snRNP), phospholipid-β-2 glycoprotein I complex, poly(ADP-ribose) polymerase, Sm antigen of U1 small ribonucleoprotein complex, α-enolase, aquaporin 4, β-arrestin, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein S100-β, R1 type reticulin, gastric H + / K + - It binds to ATPase, 21-hydroxylase, 17-hydroxylase, cytochrome P450 side-chain cleavage enzyme, or CD19. In some embodiments of methods for treating subjects suffering from autoimmune diseases or disorders, the modified MAIT cells are derived from intervillous blood of the placenta. In some embodiments, when a subject suffering from an autoimmune disease or disorder is treated with the modified MAIT cells described herein, the modified MAIT cells are allogeneic to the subject.

[0171] In some embodiments, a method for treating a subject in need of treatment, comprising the use of any of the modified MAIT cells described herein, the method comprising treating a subject having a tumor or malignant tumor, a subject infected with a pathogen, a subject suffering from a fibrotic disease or condition, a subject suffering from an age-related disease or condition, a subject who has undergone an allograft, or a subject suffering from an autoimmune disease or disorder.

[0172] In some embodiments, a method for treating a subject in need of treatment includes using placental MAIT cells that have not been modified to express exogenous antigen receptors. In some embodiments, a method is provided for treating a subject infected with a pathogen, comprising administering to the subject a composition comprising a population of placental-derived MAIT cells, wherein the MAIT cells do not include modified MAIT cells that express exogenous antigen receptors. In other embodiments, a composition or pharmaceutical composition comprising the population of MAIT cells described herein for treating a subject infected with a pathogen is provided. In some embodiments, a method of use for treating a subject infected with a pathogen includes treating an infectious condition limited to a specific site or organ. In some embodiments, an infection limited to a specific site and / or organ includes a postoperative infection. In some embodiments of a method for treating a subject infected with a pathogen, the MAIT cells are allogeneic to the subject.

[0173] In some embodiments, the pathogen is a bacterial pathogen, a viral pathogen, or a fungal pathogen. In some embodiments, the bacterial pathogen includes antibiotic-resistant bacteria or bacteria resistant to treatment. In some embodiments, the bacterial pathogen includes Mycobacterium tuberculosis. In some embodiments, the fungal pathogen includes invasive Aspergillus. In some embodiments, the viral pathogen includes cytomegalovirus, hepatitis B virus, or hepatitis C virus. In some embodiments of a method for treating a subject infected with a pathogen, the MAIT cells are derived from intervilli blood of the placenta. In some embodiments, when a subject infected with a pathogen is treated with the MAIT cells described herein, the MAIT cells are allogeneic to the subject.

[0174] <Pharmaceutical composition> In some embodiments, the composition comprises modified MAIT cells that express a foreign antigen receptor. In some embodiments, the composition comprises MAIT cells that do not express a foreign antigen receptor. As used herein, the terms "composition," "cell composition," and "pharmaceutical composition" have, in some embodiments, the same nature and meaning and are used interchangeably with each other. In some embodiments, pharmaceutical compositions for the treatment of the conditions or diseases described herein are disclosed herein.

[0175] In some embodiments, pharmaceutical compositions are disclosed herein that comprise a pharmaceutically acceptable carrier and a population of the modified MAIT cells described herein. In some embodiments, pharmaceutical compositions are disclosed herein that comprise a pharmaceutically acceptable carrier and a population of the MAIT cells described herein that have not been modified to express a foreign antigen receptor.

[0176] One of ordinary skill in the art will understand that a "pharmaceutical composition" can include preparations of one or more active ingredients described herein and other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate the administration of a compound to a living organism.

[0177] One of ordinary skill in the art will understand that the terms "physiologically acceptable carrier," "pharmaceutically acceptable carrier," "physiologically acceptable excipient," and "pharmaceutically acceptable excipient" include carriers, excipients, or diluents that do not give a significant stimulus to an organism and do not impair the biological activity and properties of the active ingredient being administered, and can be used interchangeably with each other.

[0178] Techniques for the formulation and administration of drugs or pharmaceutical compositions are described in the latest edition of Remington’s Pharmaceutical Sciences, published by Mack Publishing Co. (Easton, Pennsylvania), which is incorporated herein by reference.

[0179] In some embodiments, the compositions or pharmaceutical compositions disclosed herein are injectable compositions prepared by adding one or more excipients, such as stabilizers and aqueous buffers, to a population of modified MAIT cells as described herein. In some embodiments, the compositions or pharmaceutical compositions disclosed herein are injectable compositions prepared by adding one or more excipients, such as stabilizers and aqueous buffers, to a population of MAIT cells that have not been modified to express exogenous antigen receptors.

[0180] Alternatively or additionally, the modified MAIT cells disclosed herein are grown to at least 100 times, at least 200 times in other embodiments, at least 400 times in other embodiments, at least 600 times in other embodiments, at least 1000 times in other embodiments, at least 1500 times in other embodiments, at least 2000 times in other embodiments, at least 3000 times in other embodiments, and at least 5000 times in yet other embodiments, compared to the time of growth initiation (day 0), before administration to a patient.

[0181] In some embodiments, a method for preparing a pharmaceutical composition comprising the modified MAIT cells disclosed herein includes a step of freezing the cells, e.g., cryopreservation, which may be performed before or after separation, incubation, and / or modification. Examples of suitable cryotherapy agents are generally known in the art.

[0182] In other embodiments, for injection, the modified MAIT cells disclosed herein may be formulated in aqueous solution, preferably in a physiologically suitable buffer such as Hank's solution, Ringer's solution, or physiological salt buffer, or optionally in combination with a culture medium containing a cryopreservative.

[0183] In other embodiments, pharmaceutical compositions comprising modified MAIT cells as disclosed herein are provided, which are necessary for treating or improving any of the diseases, disorders, or complications described herein, each disease, disorder, or complication constituting a separate embodiment.

[0184] In various embodiments, the pharmaceutical compositions disclosed herein can be administered systemically in manner commonly known in the art. Alternatively, for example, the pharmaceutical compositions may be administered topically, for example, by direct injection into a patient's tissue area such as atrophied muscle, in a non-limiting embodiment. In other embodiments, the modified MAIT cells are administered intramuscularly, intravenously, subcutaneously, or intraperitoneally, each of which is considered a separate embodiment. In this regard, “intramuscular” administration includes administration into the muscle tissue of the subject, “subcutaneous” administration includes administration just below the skin, and “intravenous” includes administration into the vein of the subject. In some embodiments, the pharmaceutical compositions are administered intralymphatically, as shown in the prior art.

[0185] The effective dosage and schedule for administering the composition may be determined empirically, and such determination is within the scope of the art of those skilled in the art. The dosage may be adjusted by individual physicians if there are any contraindications.

[0186] Depending on the severity and responsiveness of the condition being treated, administration may be a single dose or, in other embodiments, multiple doses, until the disease state is alleviated.

[0187] In some embodiments, the therapeutic methods described herein include lymphocyte depletion of the recipient, or in other embodiments, immunosuppression, prior to treatment. In other embodiments, reversible lymphocyte depletion or immunosuppression extends the biological half-life of the transplanted cells. Methods of lymphocyte depletion and immunosuppression in patients are known in the art. In some embodiments, lymphocyte depletion or immunosuppression is not required when administering the modified MAIT cells disclosed herein, at least in part due to their low immunogenicity.

[0188] In various embodiments, engraftment of modified MAIT cells in the host is not essential for the cells to exert the described therapeutic effects, and each effect is considered a separate embodiment. In other embodiments, engraftment is required for the modified MAIT cells to exert their effects.

[0189] In some embodiments, the subject treated by the methods and compositions described herein has a tumor. In other embodiments, the subject has a bacterial infection. In some embodiments, the subject has a viral infection. In some embodiments, the subject has a fungal infection. In some embodiments, the subject is human.

[0190] Furthermore, kits and products relating to reagents usable for carrying out the methods disclosed herein are also disclosed. The kits and products may include reagents or combinations of reagents containing any lymphoid cells discussed herein or understood by those skilled in the art to be necessary or useful for carrying out the methods disclosed herein. In other words, the kits and products include labels, instructions for use, and packaging materials for use in the treatment of diseases or therapeutic targets described herein, for example.

[0191] Certain features of modified MAIT cells and their uses are described as separate embodiments for clarity, but it should be understood that they may also be provided in combination in a single embodiment. Conversely, various features of modified MAIT cells and their uses described as a single embodiment for simplification may be provided separately, or as any suitable subcombination, or in a suitable form in any other described embodiment of modified MAIT cells and their uses.

[0192] <Additional materials and methods> IVB collection Placental donors were recruited after cesarean sections at various medical centers in Israel, and written informed consent was obtained from the patients. The umbilical cord was secured with two clips to prevent mixing of fetal and maternal blood. The placenta was placed on a large plate with the fetal side down, and the amnion was removed to expose the cotyledons. A small X-shaped incision was made with surgical scissors only at the top of the cotyledons. The placenta was then inverted with the fetal side up, and PBS was injected into the basal decidua at different sites away from the fetal blood vessels. All maternal intervillous blood (IVB) that flowed out of the placenta was collected, and mononuclear cells were isolated using Lymphoprep (density gradient column). The intermediate cell stock was then cryopreserved.

[0193] RNA sequencing and Legend screen Blood and intervillous blood (IVB) donors: PBMCs were collected from healthy donors recruited at the transfusion laboratory of Sheba Medical Center in Tel Ha Shomer, Israel. IVB samples were collected from full-term human placentas from healthy women who had a normal pregnancy and delivered by planned elective cesarean section (Bnei Zion Medical Center, Haifa, Israel). Written informed consent was obtained from the patients.

[0194] Treatment of MAIT cells and PBMCs Human mononuclear cells (MNCs) derived from PBMCs and IVBs were isolated by Lymphoprep density gradient centrifugation. The cells were frozen in 90% FBS + 10% DMSO.

[0195] RNA sequencing of MAIT cells Thaw the frozen PBMC / IVB-derived MNCs and prepare 2 × 10⁻⁶ 6They were cultured in a 24-well plate at a concentration of cells / mL in complete OpTmizer CTS medium (Gibco). The medium was supplemented with Optmizer CTS T cell growth supplement (Gibco), 2.5% CTS Immune cell SR (Gibco), 2 mM Glutamax 100× (Gibco), 100 U / mL Pen / Strep 100× (Gibco), IL-15 (50 ng / mL, Peprotech), and 250 nM 5-OP-RU (prepared by mixing 5-A-RU (Cayman Chemical) and methylglyoxal (Merck)). Every 2 - 3 days, the proliferating cells were reseeded at a concentration of 0.5 - 1×10 6 cells / mL into fresh medium containing 50 ng / mL of IL-15 to change the medium. On day 7, the proliferated cells were labeled with a biotinylated anti-TCRVα7.2 REAfinity antibody (Miltenyi Biotech) and anti-biotin microbeads (Miltenyi Biotech) by the MACS method to enrich MAIT cells.

[0196] Treatment of PBMC PBMCs were obtained from healthy donors recruited at the transfusion laboratory of the Sheba Medical Center in Tel Hashomer, Israel, with written informed consent from the patients. PBMCs were isolated by Lymphoprep density gradient centrifugation, cryopreserved in CryoStor CS10 (BioLife Solutions), and stored in the liquid nitrogen vapor phase.

[0197] Preparation of CAR-T The thawed PBMCs were cultured in OpTmizer CTS medium (Gibco) supplemented with OpTmizer CTS T cell growth supplement (Gibco), 2 mM Glutamax 100× (Gibco), 100 U / mL Pen / Strep 100× (Gibco), IL-2 IS premium grade (Miltenyi Biotech), and TransAct anti-CD3 / anti-CD28 polymeric nanomatrix (Miltenyi Biotech) at a concentration of 1×10 6Cells were cultured overnight at a density of / mL. Transduction was performed the following day (day 1) by introducing the LV MSLN-Flag CAR vector (VectorBuilder) at an MOI of 1:10 (transduction culture), or as a non-transduction control (NTD Ctrl) without vector addition. Transduction enhancers (such as Retronectin or Polybrene) were not used. On day 4, cells were transferred to 24-well or 6-well G-Rex cultures (Wilson Wolf, MN, USA) containing 1% HI human AB serum (IMBH), and IL-2 IS premium grade was added to the cultures every other day. The cultures were harvested on days 12-13. Samples were taken on days 8 and 12-13, and the cell proliferation rate was calculated and MSLN-CAR expression was detected. MSLN-CAR expression was evaluated using Whitlow / 218 linker (E3U7Q) Rabbit mAb (rabbit monoclonal antibody) (Cell Signaling Technology) by CytoFLEX V5-B5-R3 flow cytometry (Beckman).

[0198] Preparation of CAR-MAIT IVB-derived frozen leukocytes were thawed and cultured overnight in complete OpTmizer CTS medium (Gibco) containing fetal bovine serum (FBS) and IL-15. The following day, the cells were activated for 48 hours using 5-OP-RU (known as an activator for MAIT cells). On day 3, the cells were counted and the OpTmizer CTS medium was replaced with fresh cells. On day 5, the cells were harvested and separated using an LS column containing antibody against Vα7.2 and an LS column containing magnetic microbeads (Miltenyi Biotech). The pure MAIT cell fraction (after separation) was incubated for 3 hours, after which the LV MSLN-Flag CAR vector (VectorBuilder) was introduced at an MOI of 1:10. Transduction was performed on a plate coated with retronectin (Takara), the virus was spinocured for 2 hours, and then incubated overnight with the pure MAIT cells. Virus removal was performed by four washes, after which pure MAIT cells were counted and cultured for 10–12 days. Complete OpTmizer medium containing IL-15 was added to the culture every other day.

[0199] Cell surface staining - flow cytometry Cells were placed in a 96-well microplate and washed with a staining buffer containing FBS and EDTA. The cells were then stained with a cocktail of fluorescently labeled antibodies against CD3, TCRVα7.2, and CD161 for detection of MAIT cells, along with additional antibodies as indicated. The cells were incubated at 4°C for 20 minutes, washed, and resuspended in a staining buffer containing 7-AAD viability dye. Stained cells were analyzed by CytoFlex flow cytometry, and the results were analyzed using FlowJo or Kaluza software.

[0200] Intracellular staining - flow cytometry Cells were placed in a T cell medium supplemented with Phorbol12-myristate13-acetate (PMA) and ionomycin to induce T cell activation, and brefelzin A and monensin (BD) to suppress protein secretion, and then placed in a 96-well microplate. After incubation at 37°C for 4 hours, the cells were washed with staining buffer, then stained with a fixable viability dye, and subsequently stained with cell surface antibodies as described above. The cells were then fixed and permeabilized with Cytofix / Cytoperm solution (BD) at room temperature for 30 minutes and stained in Perm / Wash buffer containing fluorescently labeled antibodies for intracellular proteins (IFNγ, TNFα, perforin, granzyme B) at 4°C for 30 minutes. The cells were washed and analyzed as described above.

[0201] Luciferase Killing Assay Luc-labeled target cells were seeded in 96-well plates (1.0E4 cells per well), and CAR-T cells or CAR-MAIT cells were added to two wells after 4 hours. After 18 hours of incubation, luciferase activity was measured using the Bio-Glo Luciferase Assay system (Promega). Viability was normalized by the maximum RLU signal of a given effector. EC50 was estimated using GraphPad Prism (Sigmoidal (4PL)).

[0202] <Example 1> Isolation, concentration, and proliferation of MAIT cells from intervillous blood (IVB). The placenta (100) is an organ that includes a fetal side (101) containing the umbilical cord (102) connecting the fetus and the mother, and a maternal side (104) covered by the amniotic membrane (105). To isolate MAIT cells from the IVB, the umbilical cord is first clamped using, for example, a clip (103) to prevent the fetal blood and maternal (IVB) blood from mixing. IVB collection begins by removing the amniotic membrane covering the maternal side to expose the cotyledons (106). A shallow incision is made in the cotyledons using scissors or other appropriate cutting tools. The placenta is then inverted so that the fetal side is facing upward, and phosphate-buffered saline (PBS) is injected into the basal decidua away from the fetal blood vessels. All maternal blood that flows out of the placenta is collected. Subsequently, mononuclear cells are isolated from the collected maternal blood using Lymphoprep (density gradient column), and an intermediate cell stock is frozen (Figures 1A-1D).

[0203] Figure 2A shows a flow cytometry plot of MAIT cell activation and proliferation over 10 days in a packed-bed bioreactor according to an embodiment of the present invention. Mononuclear cells from intervillous blood (IVB) were seeded in a packed-bed bioreactor containing an ECM-coated Fibra-CelR carrier that mimics the natural environment and promotes antigen-presenting cell (APC) adhesion. MAIT cell activation was induced by 5-OP-RU (250 ng / mL) and IL-15 (50 ng / mL). MAIT cell activation and proliferation were evaluated at several time points on days 0, 5, 7, and 10. The MAIT cell population was detected by the expression of the CD3, Vα7.2, and CD161 markers. As a result, the percentage of MAIT cells increased from 22.63% on day 0 to 96.26% on day 10. Furthermore, the expression of the activation markers CD69 and CD25 increased from day 0 to day 7 and slightly decreased on day 10.

[0204] Figure 2B shows a flow cytometry plot of MAIT cell proliferation after translocation of MAIT cells from a first bioreactor to a second bioreactor on day 10, and subsequent proliferation in the second bioreactor for a further 7 days, according to an embodiment of the present invention. Cells that reached maximum proliferation capacity by day 10 in the first bioreactor were harvested, and approximately 30% of these cells were seeded in a second bioreactor similarly designed to the first bioreactor. These cells were cultured for a further 7 days. Flow cytometry results showed that the percentage of MAIT cells remained relatively constant for most of the period, but decreased from over 90% on day 14 to 82% on day 17. CD69 marker expression increased from 30% to 87% on day 17, indicating that MAIT cells maintained their activation signal, while CD25 gradually decreased as predicted, indicating that MAIT cells were recently activated but had not yet reached their full proliferation and functional state, suggesting their ability to reactivate and function upon future encounters with targets.

[0205] The results show that MAIT cells are generally more abundant in mononuclear cells derived from placental intervillous blood (IVB) than in peripheral blood mononuclear cells (PBMCs).

[0206] Figure 3 shows the results of activation and proliferation of placental IVBMAIT cells in several optional embodiments of the present invention. To obtain a population rich in MAIT cells, mononuclear cells derived from IVB were seeded on tissue culture plates, and activation of MAIT cells was induced in the presence of 5-OP-RU (250 ng / mL) and IL-15 (50 ng / mL). Cells were counted on days 3 and 5 after activation, the proportion of MAIT cells was analyzed by flow cytometry, and fresh medium and IL-15 were added. By day 7 of culture, MAIT cells accounted for 78% to 93% of the cells in the CD3+ population, depending on the donor. On day 7, cells were separated using magnetic beads and anti-TCRVα7.2 antibody to further enrich the MAIT population. Cells were cultured for a further 3 days (up to day 10) in the presence of IL-15.

[0207] The results show an increase in the proportion of MAIT cells, the proliferation rate of MAIT cells, and an increase in the absolute number of MAIT cells relative to the initial number throughout the culture period.

[0208] Figures 4A and 4B show the results of comparing the proportion of MAIT cells in placental intervillous blood (IVB) mononuclear cells gated on CD3+ cells from 10 and 11 unrelated donors, respectively, with the proportion in peripheral blood mononuclear cells (PBMCs). p=0.0159.

[0209] As shown in the figure, the proportion of MAIT cells varies considerably among donors in both peripheral blood and placenta, but on average, the proportion of MAIT cells among CD3+ cells in IVB is significantly higher than in peripheral blood.

[0210] <Example 2> Immunophenotype of placental IVBMAIT cells compared to peripheral blood MAIT cells The phenotypes of placental IVB MAIT cells and peripheral blood MAIT cells were analyzed using CD45RA and CD62L markers. Figure 5 shows the immunophenotypic analysis of placental IVB MAIT cells and PB MAIT cells collected from six different donors and analyzed on day 0.

[0211] Cells were stained according to a standard cell surface marker staining protocol and analyzed by flow cytometry. The cell surface markers CD45RA and CD62L were used to characterize the cells. As shown in the figure, placental IVBMAIT cells had a statistically significantly higher proportion of "effector" memory cells and fewer "terminally differentiated" cells compared to PBMAIT cells. The gating strategy for phenotypic analysis is shown in the right panel. Statistical analysis was performed using GP Prism, and comparisons were made using t-tests. *p<0.05, **p<0.01, ***p<0.001.

[0212] Notably, IVB-derived MAIT cells had a significantly higher proportion of effector memory cells than peripheral blood-derived MAIT cells. Consequently, IVB-derived MAIT cells had a lower proportion of terminally differentiated effector cells than peripheral blood-derived MAIT cells. Effector memory cells may be a desirable phenotype for modified MAIT cells because they have a longer lifespan than terminal effector cells and possess superior effector function and migration ability to peripheral tissues compared to naive cells and central memory cells.

[0213] <Example 3> Chemokine receptor expression analysis in placental IVBMAIT cells compared to peripheral blood MAIT cells. Chemokine receptors may mediate the migration of modified MAIT cells to peripheral tissues and secondary lymphoid organs, and may also be important for modified MAIT cells to invade tumors. We analyzed the expression of chemokine receptors in IVB MAIT cells and MAIT cells from peripheral blood.

[0214] Figures 6A and 6B show chemokine receptor analysis of placental IVBMAIT cells and PBMAIT cells according to an embodiment of the present invention. To evaluate the chemokine receptor profile of the MAIT population, IVBMAIT cells and PBMAIT cells were collected from six different donors (6), and chemokine receptor expression was analyzed on day 0. Cell surface markers CCR6, CCR5, CXCR4, and CXCR6 were used to characterize the cells. Cells were stained according to a standard cell surface marker staining protocol and analyzed by flow cytometry. Statistical analysis was performed using GP Prism, and comparisons were made by t-test. *p<0.05, **p<0.01, ***p<0.001.

[0215] As observed in Figure 6A, IVBMAIT cells show higher expression of CCR5 and CCR6 chemokine receptors compared to PBMAIT cells. Furthermore, Figure 6B shows that IVBMAIT cells have statistically significantly lower levels of CXCR4 and similar levels of CXCR6 compared to PBMAIT cells.

[0216] The results further indicate that the two MAIT cell populations, IVB-derived and PB-derived, are distinct, with IVB-MAIT cells containing more cells exhibiting the "effector memory" phenotype and fewer "terminally differentiated effector" cells compared to PB-MAIT cells.

[0217] <Example 4> Granzyme B and perforin expression in placental IVBMAIT cells compared to thawed peripheral blood MAIT cells Perforin and granzyme B are essential molecules for the cytotoxic function of effector lymphocytes. Perforin creates pores in target cells, and granzyme B enters these pores to induce apoptosis. Comparative analysis showed that IVB-MAIT cells expressed both molecules at significantly higher levels when stimulated with PMA / ionomycin compared to peripheral blood MAIT cells. Figure 7A shows representative samples of both cell types, and Figure 7B shows placental IVB and peripheral blood MAIT cells.

[0218] Cells from six donors were stimulated with PMA / ionomycin for 4 hours, and intracellular expression of granzyme B and perforin was analyzed. As shown in the figure, placental IVBMAIT cells had statistically significantly higher levels of granzyme B compared to peripheral blood MAIT cells, indicating increased effector and lytic capacity of placental IVBMAIT. The cells were gated to MAIT cells. p=0.0001.

[0219] <Example 5> Intervillous blood (IVB) MAIT compared to umbilical cord blood (CB) MAIT MAIT cells were collected from intervillous blood (IVB) and umbilical cord blood (CB), and flow cytometry was used to compare the frequency and immunophenotype of the two samples.

[0220] Figures 8A and 8B show flow cytometry plots of the percentage of MAIT cells in placental IVB and matching (same donor) CB cells, and a table summarizing the results obtained from the flow cytometry plots.

[0221] As previously mentioned, cells were collected from three different donors. IVB and CB cells were similarly activated. As shown in the figure, CB had significantly fewer MAIT cells compared to IVB on day 0, and this proportion decreased further by day 6 / 7. More specifically, IVB MAIT cells and matching CB MAIT cells were collected according to the protocol described in the Materials and Methods section above. Cells were stained with CD161 and TCRVα7.2 on day 0 and day 6 / 7, indicating a gating strategy for MAIT cells. The square gates indicate the proportion of MAIT cells. The table lists the actual cell counts. As observed, intervillous blood (IVB) contains far more MAIT cells than umbilical cord blood (CB).

[0222] Figure 9 shows CD8αα in placental IVBMAIT cells and CBMAIT cells according to an example of the present invention. + The flow cytometry plot shows the expression of CD8αα in placental IVBMAIT cells. Our results indicate that placental IVBMAIT cells express CD8αα + The cells express this gene at a high frequency, with a frequency (41.2%) being approximately three times higher than that of CBMAIT cells (14.2%), indicating a more "antigen-experienced" phenotype compared to the immature / naive state observed in CBMAIT cells.

[0223] CD8α is a cell surface glycoprotein that can be expressed as a disulfide-bonded heterodimer or homodimer with CD8β. In contrast to CD8αβ, CD8αα is not expressed in naive T cells and is readily induced at strongly activated T cell receptors (TCRs). Furthermore, like other "regulatory" molecules, CD8αα may function to aid survival in a similar manner to how KIR and other NK cell-associated receptors are expressed on T cells. IVB and CB-derived MAIT cells were stained with the two subunits of CD8, namely CD8α and CD8β, and their frequencies were measured by flow cytometry. The results showed high frequency expression of CD8αα in IVB-MAIT cells, indicating a more "antigen-experienced" phenotype compared to the naive state observed in CB-MAIT cells.

[0224] Figure 10 shows flow cytometry plots illustrating the effector memory phenotypes of placental IVBMAIT cells and CBMAIT cells using CD45RO, CD27, CD45RA, CCR7, and CD62L.

[0225] IVBMAIT cells and matching CBMAIT cells were collected according to the methods described in the "Materials and Methods" section below. The immunophenotype of the cells was determined using cell surface markers for CD45RA, CD27, CCR7, CD62L, and CD45RO. - / CCR7 - CD45RA - / CD62L - CD45RA - / CD45RO + and CD27 - / CD45RO - Each phenotype represents an effector memory cell, an activated effector memory cell, and an effector memory phenotype, respectively. As is clear from the flow cytometry plot, IVBMAIT cells differ from CBMAIT cells in that IVBMAIT cells have an effector memory phenotype, while CBMAIT cells exhibit a naive phenotype.

[0226] In summary, a comparison of the proportion of MAIT cells in CB and IVB revealed that the frequency of MAIT cells in CB was significantly lower than in IVB. Furthermore, the lower frequency of CD8αα-expressing MAIT cells in CB suggests that CBMAIT cells are in a more immature state compared to IVBMAIT cells.

[0227] <Example 6> Transcriptome analysis of IVBMAIT cells and PBMAIT cells Transcriptome analysis was performed on IVB-MAIT cells and PB-MAIT cells, and the top 50 gene sets that were significantly expressed are shown (Figure 11, gene names are indicated in the figure). To understand the transcriptome differences between MAIT cells from different tissues, namely PBMCs and IVB-derived cells, cells were extracted and grown as described above, and RNA sequencing analysis was performed. MAIT cells from IVB and PB were collected from three donors (3) each, and RNA sequencing analysis was performed on day 7 (7). Cells were collected as described in the "Methods" section. Heatmaps were created for protein-coding genes that showed significant expression differences (|log2FC|>1 and BH-adjusted p-value <0.01) between IVB and PB cell types. Clustering was performed by applying Pearson correlation to the normalized values ​​of the count data. The color scheme represents the z-score.

[0228] As these results show, IVB-derived MAIT cells have a unique and distinctive transcriptome compared to peripheral blood MAIT cells, meaning that the two are extremely distinct MAIT populations.

[0229] <Example 7> Expression of granzyme B and perforin in placental IVBMAIT cells compared to post-proliferation peripheral blood MAIT cells Granzyme B is a serine protease present in the granules of lysed cells. It is secreted along with perforin, a pore-forming protein, to mediate apoptosis in target cells. Figure 12 shows a comparison of granzyme B secretion levels between IVBMAIT cells and PBMAIT cells.

[0230] Placental IVBMAIT cells and PBMAIT cells were collected from four donors, and proliferated using the standard protocol described in the "Methods" section. On day 8, the cells were activated with Phorbol Myristate Acetate (PMA) and ionomycin, and the secretion of granzyme B and perforin was analyzed using a standard intracellular staining protocol.

[0231] To evaluate the fundamental potential of IVB or PB-derived MAIT cells, collected cells were stimulated with PMA and ionomycin, and staining was performed to detect intracellular granzyme B secretion.

[0232] As shown in the figure, stimulated IVBMAIT cells produced significantly higher levels of granzyme B compared to PBMAIT cells, indicating higher effector and lytic activity of IVB-derived cells. Statistical analysis was performed using t-tests with GP Prism. *p<0.05, **p<0.01, ***p<0.001.

[0233] <Example 8> Effectiveness of placenta-derived CAR-MAIT cells The functional killing properties of placental IVBCAR-MAIT cells were analyzed, and the results are shown in Figure 13. For this purpose, CAR-MAIT cells were prepared according to the standard protocol described in the "Methods" section and co-cultured with NSCLC targets overexpressing MSLN and luciferase. Killing function was measured by luminescence readout.

[0234] As shown in the graph, each effect pedal is CAR + The fraction levels are different. Co-culture with target cells is CAR +The tests were performed within the effector:target (E:T) ratio range determined by the fraction. Robust efficacy against target cells was achieved under all CAR-MAIT cell culture conditions, demonstrating the high and dose-dependent efficacy of placental CAR-MAIT cells.

[0235] <Example 9> Comparison of intervillous blood (IVB) MAIT cells, peripheral blood (PB) T cells, and umbilical cord blood (CB) T cells derived from matching donors. To evaluate the differences between MAIT cells and T cells and to eliminate donor variability, IVBMAIT cells, peripheral blood (PB) maternal T cells, and umbilical cord blood (CB) T cells derived from the same donor (matched donor) were compared. The inventors evaluated the functional differences between the various cell types derived from the same donor. IVB, PBMC, and CB cells were collected as described in the "Methods" section and activated with a T cell activator on day 0. After staining according to the intracellular staining protocol, perforin, granzyme B, IFN-γ, and TNF-α were analyzed by flow cytometry. Figure 14A is a flow cytometry plot showing the raw data for perforin and granzyme B, and Figure 14B is a flow cytometry plot showing the raw data for IFN-γ and TNF-α.

[0236] As these results show, IVB-MAIT cells on day 0 exhibit completely different expression levels of effector molecules compared to conventional T cells, while T cells derived from umbilical cord blood and peripheral blood show similar levels of expression of these effector molecules. Figures 15A and 15B graph the results for two donors. These figures show the proportion of cells from the MAIT population from IVB, the conventional T cell population from peripheral maternal blood (MB), and the conventional T cell population from CB (cultured using the T cell standard activation protocol). The analysis was performed according to the gating strategy shown in Figures 14A and 14B. This example is from two donors, from whom IVB, peripheral maternal blood (MB), and umbilical cord blood (CB) were obtained.

[0237] As shown in the results from two independent donors, IVBMAIT cells on day 0 showed superior perforin levels compared to conventional T cells. + Granzyme B + perforin + +Granzyme B + (double positive), IFNγ + , TNFα + , and IFNγ + +TNFα + The rate of (double positive) cases was high.

[0238] <Example 10> Placental IVBMAIT and Placental IVBT Effector Functional Analysis Matching IVB, PB, and CB mononuclear cells obtained from the same donor were thawed on day 0 and activated and cultured according to a standard protocol using T cell activators. Simply put, T cell proliferation is achieved by activating the TCR using CD28 and CD3 antibodies, or TransAct beads supplemented with IL-2 cytokines.

[0239] On day 10, cells were stained with CD161 and TCRVα7.2, and the MAIT-positive population fraction was evaluated. As observed, the specific MAIT cell proliferation protocol yielded a highly enriched MAIT cell population of over 95%, while the standard T cell proliferation protocol yielded only a very small MAIT cell population of less than 1.2%.

[0240] Figures 16A and 16B show a comparison of the effector functions of IVBMAIT and IVBT cells. IVBMAIT cells were cultured as described in the "Methods" section. From the same source (IVB cells), the inventors also cultured T cells using a standard conventional T cell protocol with TransAct and IL-2. On day 10, IVBMAIT and IVBT cells were stimulated with PMA + ionomycin or TransAct, stained for effector molecules such as IFN-γ, TNF-α, perforin, and granzyme B, and then analyzed by flow cytometry (Figure 16A). A graphical summary of the results is shown in Figure 16B, showing that MAIT cells have higher levels of perforin and TNF-α compared to T cells from the same source, i.e., placental IVB-derived cells. This indicates that placental IVBMAIT cells have higher effector function compared to placental IVBT cells.

[0241] <Example 11> Immunophenotypes of placental MAIT and placental CAR-MAIT The immunophenotypes of placental-derived MAIT cells and placental-derived CAR-MAIT cells were evaluated on day 10 using flow cytometry staining with CD45RA and CCR7 according to a standard cell surface marker protocol. As shown in Figures 17A and 17B, leukocytes were isolated from placental intervillous blood and basal decidua, activated with 5-OP-RU, and grown until day 5. After isolating MAIT cells, they were introduced using lentivirus and grown until day 10.

[0242] Both MAIT cells and modified CAR-MAIT cells were grown for 10 days, then stained and analyzed by flow cytometry. The CD45RA vs. CCR7 gating strategy showed a statistically significant increase in "effector memory (EM)" cells compared to "central memory (CM)" cells. Naive and terminally differentiated (TD) cells were rarely observed. These results are the mean values ​​from three different donors analyzed on day 10 (Figure 17C). The results showed that over 80% of MAIT and CAR-MAIT cells exhibited the effector memory phenotype, while the remaining cells exhibited the central memory phenotype. Naive and terminally differentiated cells accounted for less than 1%. CD62L staining showed high or low expression. CD69 staining consistently showed increased expression throughout the growth period after MAIT activation.

[0243] <Example 12> Antibacterial activity of placental MAIT cells - Inhibition of bacterial growth by placental MAIT secretions IVBMAIT cells are obtained from the placenta and co-cultured with MR-1 expressing cells (monocytes, macrophages, B cells, cell lines, etc.) loaded with MR-1 ligands (5-OP-RU, 5-OE-RU, etc.) or immobilized bacteria in antibiotic-free medium. After 24-72 hours, the medium containing the MAIT secretome is collected and mixed with antibiotic-resistant or antibiotic-sensitive live bacteria (e.g., Gram-negative bacteria such as carbapenem-resistant Escherichia coli or KPC-producing Klebsiella pneumoniae, or Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus, β-lactam-resistant Streptococcus pneumoniae, or multidrug-resistant Mycobacterium tuberculosis). The collected medium and bacteria are incubated for different periods, from a few minutes to 24 hours, in or without antibiotics, after which the bacteria are diluted and seeded onto agar plates. After seeding the bacteria onto the agar plates, bacterial colony-forming units (CFUs) are counted 24 hours later. The number of colonies is compared to the number of colonies that formed after 24 hours when bacteria were incubated in a fresh medium without antibiotics, with or without antibiotics. <Example 13> Antibacterial activity of placenta-derived MAIT cells - killing cells infected with antibiotic-resistant bacteria Cells expressing MR-1 (monocytes, macrophages, B cells, HeLa cells, etc.) are infected with live bacteria (Gram-negative bacteria such as carbapenem-resistant Escherichia coli or KPC-producing Klebsiella pneumoniae, and Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus, β-lactam-resistant Streptococcus pneumoniae, or multidrug-resistant Mycobacterium tuberculosis) and cultured in antibiotic-free medium for 3 hours. The infected cells are washed and incubated with antibiotics for 1 hour to kill extracellular bacteria. IVB-derived MAIT cells obtained from the placenta are then treated with a fluorescent dye (Cell Proliferation Dye eFluor). TM Label with 450 (etc.), vary the ratio of effector to target, and add to bacterial-infected cells for 3 hours. During this time, add Caspase-3 / 7 detection reagent (CellEvent) during the last 30 minutes. TM Add a Caspase-3 / 7 Detection Reagent (e.g.). After incubation, harvest the target cells and analyze the activated caspase-3 / 7 by flow cytometry, comparing the percentage with that of bacterial-infected target cells alone. To evaluate intracellular bacterial killing, dilute the cell lysate of bacterial-infected target cells after co-culture with IVB-derived MAIT cells and seed it on an agar plate. Bacterial colony-forming units (CFUs) are counted 24 hours after seeding on the agar plate. Compare the number of colonies with that of bacterial-infected target cells alone after incubation.

Claims

1. A cell composition comprising a population of modified mucosal-associated invariant T cells (MAIT cells) expressing exogenous chimeric antigen receptors (CARs), The MAIT cells are placental MAIT cells derived from placental tissue, A cell composition comprising a pharmaceutically acceptable carrier as deemed appropriate.

2. The cell composition according to claim 1, wherein the population of placental MAIT cells is obtained from intervillous blood (IVB).

3. A cell composition according to claim 1 or 2, adapted for cell therapy.

4. More than 90% of the placental MAIT cells were TCRVα7.2 ＋ and high level CD161 (CD161 high A cell composition according to any one of claims 1 to 3, which expresses ).

5. A population of modified placental CAR-MAIT cells ９ ~10 １１ A cell composition according to any one of claims 1 to 4, comprising a viable cell.

6. The cell composition according to any one of claims 1 to 5, wherein the exogenous CAR expressed by the placental MAIT cells recognizes a tumor antigen.

7. The cell composition according to claim 6, wherein the tumor antigen is mesoserine (MSLN).

8. The cell composition according to claim 6, wherein the tumor antigen is selected from the group consisting of CD19, B7-H6, CD20, CD22, CD33, CD38, CD70, CD123, BCMA, CLL1, CD7, CS1, CEA, AFP, PSMA, GPC3, GD2, EGFRVIII, CXCR5, NKG2D, HER2, mesoserine, claudin 3, claudin 4, claudin 6, claudin 18.2, ROR1, ROR3, Mucl, and Mucl6.

9. The cell composition according to any one of claims 1 to 8, wherein the exogenous CAR expressed by the placental MAIT cells recognizes microbial antigens and / or viral antigens.

10. The cell composition according to any one of claims 1 to 9, wherein the exogenous CAR expressed by the placental MAIT cells recognizes a target molecule expressed on regulatory T cells.

11. The cell composition according to claim 10, wherein the target molecule is CCR8.

12. The cell composition according to any one of claims 1 to 11, wherein the exogenous CAR expressed by the placental MAIT cells specifically recognizes target molecules expressed on alloreactive lymphocytes.

13. The cell composition according to claim 12, wherein the target molecule is CD70.

14. The cell composition according to any one of claims 1 to 13, wherein the exogenous CAR expressed by the placental MAIT cells recognizes a target molecule expressed on senescent cells.

15. The cell composition according to claim 14, wherein the target molecule is a urokinase-type plasminogen activator (uPAR).

16. The cell composition according to any one of claims 1 to 15, wherein the exogenous CAR expressed by the placental MAIT cells recognizes a target molecule expressed on cells associated with fibrotic disease or pathological condition.

17. The cell composition according to claim 16, wherein the target molecule is fibroblast-activating protein (FAP).

18. The cell composition according to any one of claims 1 to 17, wherein the exogenous CAR expressed by the placental MAIT cells recognizes a target molecule expressed on cells associated with autoimmune diseases or disorders.

19. The cell composition according to claim 18, wherein the target molecule is expressed on B cells.

20. The cell composition according to any one of claims 1 to 19, wherein the exogenous chimeric antigen receptor (CAR) expressed by the placental MAIT cells comprises an antigen-binding domain configured to specifically recognize a tumor antigen, a transmembrane domain for immobilizing the CAR to the cell membrane, one or more costimulatory domains for enhancing cell signaling and T cell activation, and an intracellular signaling domain that plays a role in initiating T cell activation upon antigen recognition.

21. The cell composition according to claim 20, wherein the antigen-binding domain of the chimeric antigen receptor (CAR) comprises one of a single-chain antibody, a single-chain variable region fragment (scFv), or a VHH fragment, each configured to recognize a designated tumor antigen.

22. The cell composition according to claim 20, wherein the antigen-binding domain of the CAR specifically binds to one or more antigens selected from the group consisting of CD19, B7-H6, CD20, CD22, CD33, CD38, CD70, CD123, BCMA, CLL1, CD7, CS1, CEA, AFP, PSMA, GPC3, GD2, EGFRVIII, CXCR5, NKG2D, HER2, mesoserine, claudin 3, claudin 4, claudin 6, claudin 18.2, ROR1, ROR3, Mucl, and Mucl6.

23. The cell composition according to claim 20, wherein the co-stimulatory domain of the CAR comprises one or more functional signaling domains derived from OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).

24. The cell composition according to claim 20, wherein the intracellular signaling domain of CAR comprises any of the intracellular signaling domains of CD3ζ, FcRγ, or their functional fragments, each configured to initiate or maintain T cell activation upon antigen recognition.

25. The CAR expressed by the placental MAIT cells is encoded by a gene construct selected from the group consisting of a monocistronic construct, a bicistronic construct, and a tricistronic construct. One or more additional elements in the bicistronic construct or the tricistronic construct are selected from the group consisting of cytokines and constitutively active tyrosine kinases. The cell composition according to any one of claims 1 to 24, wherein the cytokine is either unmodified or modified, and / or expressed in a secreted, conditionally activated, constitutively activated, or membrane-bound form.

26. The cell composition according to any one of claims 1 to 25, wherein the population of placental MAIT cells that has been proliferated in advance has a higher proportion of cells expressing low levels of CD45RA and CD62L compared to the population of peripheral blood-derived MAIT cells that has been proliferated in advance, and exhibits the effector memory phenotype of the placental MAIT cells.

27. The cell composition according to any one of claims 1 to 26, wherein the population of placental MAIT cells that has been proliferated in advance has a higher proportion of cells expressing CCR5 and / or CCR6 chemokine receptors and a lower proportion of cells expressing CXCR4 compared to the population of peripheral blood-derived MAIT cells that has been proliferated in advance.

28. The cell composition according to any one of claims 1 to 27, wherein the population of placental MAIT cells has a higher proportion of cells expressing granzyme B compared to the population of MAIT cells derived from peripheral blood, and exhibits enhanced effector function and lysis function of the placental MAIT cells.

29. The cell composition according to any one of claims 1 to 28, wherein the population of placental MAIT cells that has been proliferated in advance has a higher proportion of cells expressing perforin compared with the population of peripheral blood-derived MAIT cells that has been proliferated in advance, and exhibits enhanced effector function and lysis function of the placental MAIT cells.

30. The cell composition according to any one of claims 1 to 29, wherein the population of placental MAIT cells that has been proliferated in advance has a higher proportion of cells expressing perforin and / or granzyme B compared to the population of conventional T cells derived from peripheral blood that has been proliferated in advance, and exhibits enhanced effector function and lysis function of the placental MAIT cells.

31. The cell composition according to any one of claims 1 to 30, wherein the population of placental MAIT cells that has been proliferated in advance has a higher proportion of cells expressing perforin and / or granzyme B compared to the population of conventional T cells derived from umbilical cord blood that has been proliferated in advance, and exhibits enhanced effector function and lysis function of the placental MAIT cells.

32. The cell composition according to any one of claims 1 to 31, wherein the population of placental MAIT cells that has been proliferated in advance has a higher proportion of cells expressing TNFα compared to the population of conventional T cells derived from umbilical cord blood that has been proliferated in advance, indicating an enhancement of the effector function of the placental MAIT cells.

33. The cell composition according to any one of claims 1 to 32, wherein the population of placental MAIT cells that has been proliferated in advance has a higher proportion of cells expressing TNFα compared to the population of conventional T cells derived from peripheral blood that has been proliferated in advance, indicating an enhancement of the effector function of the placental MAIT cells.

34. The cell composition according to any one of claims 1 to 33, wherein the population of placental MAIT cells has a higher proportion of cells expressing one or more of perforin, granzyme B, granzyme B, perforin, TNFα, TNFα, and IFNγ compared to the population of placental T cells, and exhibits enhanced effector function and lysis function.

35. The pre-propagated population of placental MAIT cells showed a higher CD8αα compared to the pre-propagated population of umbilical cord blood MAIT cells. ＋ The cell composition according to any one of claims 1 to 34, wherein the proportion of cells expressing is high and exhibits the mature phenotype of the placental MAIT cells.

36. A cell composition according to any one of claims 1 to 35, for use in therapeutic purposes.

37. The cell composition used according to claim 36, wherein the use is to treat a subject having a tumor or malignant tumor.

38. The cell composition used according to claim 37, wherein the subject is a subject suffering from a tumor selected from the group consisting of hematological cancer, melanoma, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, uterine cancer, cervical cancer, bladder cancer, gastric cancer, head and neck cancer, brain tumor, skin cancer, and sarcoma.

39. A method for treating a subject having a tumor or malignant tumor, comprising the step of administering to the subject a cell composition according to any one of claims 1 to 35.

40. The method according to claim 39, wherein the tumor is selected from the group consisting of hematological cancers, melanomas, breast cancers, colon cancers, kidney cancers, liver cancers, lung cancers, ovarian cancers, pancreatic cancers, prostate cancers, uterine cancers, cervical cancers, bladder cancers, gastric cancers, head and neck cancers, brain tumors, skin cancers, and sarcomas.

41. The cell composition is suitable for cell therapy. The aforementioned group of MAIT cells is 10 ９ It contains more than 1 living cell, of which more than 90% are TCRVα7.2 ＋ CD161 high The method according to claim 39 or 40.

42. The method according to any one of claims 39 to 41, wherein the modified placental MAIT cells are allogeneic with respect to the subject.

43. The method according to any one of claims 39 to 41, wherein the modified placental MAIT cells are partially histocompatible with the subject or are not histocompatible with the subject.

44. A method for treating a subject infected with a pathogen, wherein the pathogen is a bacterial pathogen, a viral pathogen, or a fungal pathogen. A method comprising the step of administering the cell composition according to any one of claims 1 to 35 to the subject.

45. A method for treating a subject suffering from a fibrotic disease or condition, comprising the step of administering to the subject a cell composition according to any one of claims 1 to 35.

46. A method for treating a subject suffering from an aging-related disease or condition, comprising the step of administering to the subject a cell composition according to any one of claims 1 to 35.

47. A method for treating a subject that has undergone allogeneic transplantation, comprising the step of administering to the subject the cell composition described in any one of claims 1 to 35.

48. A method for treating a subject suffering from an autoimmune disease or disorder, comprising the step of administering to the subject a cell composition according to any one of claims 1 to 35.

49. A method for treating a subject infected with a pathogen, comprising the step of administering to the subject a composition containing a population of placenta-derived MAIT cells.

50. The method according to claim 49, wherein the MAIT cells are allogeneic with respect to the subject.

51. The aforementioned pathogen is a bacterial pathogen, The method according to claim 49, wherein the bacterial pathogen includes antibiotic-resistant bacteria or bacteria-resistant bacteria.

52. A population of placental mucosa-associated invariant T cells (MAIT cells), More than 90% of placental MAIT cells are TCRVα7.2 ＋ CD161 ＋ and The majority of the placental MAIT cells are CD45RA and CCR7 (CD45RA ― CCR7 ― A cell population characterized by the absence of expression of ), exhibiting the effector memory phenotype of the placental MAIT cells.

53. The cell population according to claim 52, wherein the MAIT cells are modified placental MAIT cells that express a chimeric antigen receptor (CAR).

54. The cell population according to claim 52 or 53, wherein the placental MAIT cells are further characterized by high or low expression of CD62L.

55. The cell population according to claim 52 or 53, wherein the placental MAIT cells are further characterized by high expression of CD69.

56. The cell population according to any one of claims 52 to 55, wherein the placental MAIT cells are obtained from intervillous blood (IVB).

57. A cell composition comprising the cell population described in any one of claims 52 to 56.

58. A cell composition according to claim 57, for use in therapeutic purposes.

Images