Genetically engineered placental mucosa related constant T (MAIT) cells and uses thereof

Abstract

One major aspect of the invention relates to a cell composition comprising a population of engineered mucosa-related constant T (MAIT) cells derived from placental tissue expressing an exogenous chimeric antigen receptor (CAR). The invention further discloses a unique placental MAIT cell population, cell compositions comprising the MAIT cell population, and methods of use.

Description

Technical Field

[0001] The present disclosure generally relates to the field of genetic engineering of immune cells. More specifically, the present disclosure provides genetically engineered mucosal-associated invariant T (MAIT) cells from placenta with chimeric antigen receptors (CARs) and uses thereof. Background Art

[0002] Mucosal-associated invariant T (MAIT) cells were first identified in humans, mice, and cattle as a population of αβ T cells enriched in double negative (CD4 - CD8 - ) subgroup, expressing constant Vα7.2-Jα33 T cell receptor (TCR) in humans. The term MAIT is established due to the relative enrichment of these T cells in mucosal tissues. The limiting factor of MAIT cells is the MHC-1b molecule MR1 (MHC-associated protein 1), which presents intermediates of the vitamin B (riboflavin (vitamin B2) and folic acid (vitamin B9)) synthesis pathway to MAIT cells. The potent stimulatory ligands in the riboflavin synthesis pathway include 5-(2-oxopropylamino)-6-D-ribosylaminouracil (5-OP-RU) and 5-(2-oxoethylamino)-6-D-ribosylaminouracil (5-OERU) produced by a variety of bacteria, mycobacteria and yeast during riboflavin (vitamin B2) synthesis. This pathway does not exist in mammals; therefore, its immunodetection allows effective host-pathogen discrimination.

[0003] In more detail, T cells expressing αβ and γδ T cell receptors (TCR) develop from common T progenitor cells in the thymus, and can mature into one of several functionally distinct T cell lineages. Among these, the best studied are conventional T cells, which express αβTCRs that recognize peptide antigens presented by MHC molecules. However, there are several 'unconventional' T cell lineages that also develop in the thymus. Unconventional T cells include CD1-restricted T cells, MHC-associated protein 1 (MR1) restricted T cells and γδT cells, which recognize non-peptide antigens such as lipids, vitamin B metabolites and phosphorus-antigens, respectively. These lineages usually express TCRs with limited diversity compared to the TCRs of conventional T cells, and they are often described as cells that bridge the gap between innate immunity and adaptive immunity. Most unconventional T cells co-express surface antigens associated with natural killer (NK) cells and produce (mount) powerful cytokine responses very quickly after activation. Their distinctive phenotype is established during their development, and they represent a critically important subset of peripheral T cells with unique effector and regulatory roles (Godfrey, D., Uldrich, A., McCluskey, J. et al. The burgeoning family of unconventional T cells. Nat Immunol 16, 1114–1123 (2015). https: / / doi.org / 10.1038 / ni.3298 ).

[0004] Mucosal-associated invariant T (MAIT) cells are innate-like T cells defined by their semi-constant αβ T cell receptor (TCR), which recognizes a small biosynthetic derivative of riboflavin synthesis presented on the restricted molecule major histocompatibility complex (MHC)-associated protein-1 (MR1). As mentioned above, the MAIT cell TCR contains a semi-constant TCR-α chain—usually Vα7.2–Jα33 / 12 / 20 in humans—which is mainly related to the β chain Vβ2 / Vβ13 in humans.

[0005] MAIT cells contrast with conventional T cells, which have highly variable TCRs that are able to target a large number of peptide epitopes produced by viruses, bacteria, and malignant cells. Conventional T cells therefore have precise specificity for a single peptide, and individual clones can undergo large-scale expansion to provide T cell memory. However, upon first encountering a pathogen, the frequency of any single peptide-specific T cell will be very low. In contrast, MAIT cell TCRs provide an innate ability to respond to a specific set of ligands without the need for expansion. The key discovery was the identification of these ligands presented on MR1, which include the potent MAIT cell ligands 5-(2-oxopropylideneamino)-6-D-ribosylaminouracil (5-OP-RU) and 5-(2-oxoethylideneamino)-6-D-ribosylaminouracil (5-OE-RU), which are produced by a wide variety of bacteria, mycobacteria, and yeast during riboflavin (vitamin B2) synthesis (mini review article, Front. Immunol., May 27, 2020, Sec. Mucosal Immunity, Vol. 11–2020( https: / / doi.org / 10.3389 / fimmu.2020.01014 ).

[0006] MAIT cells have been found to recognize bacterially infected cells and produce IFN-γ in response. MAIT cells have been shown to protect mice from bacterial infection. Early phenotypic work established that human MAIT cells are CD8 + or double negative, with predominant CCR7 - Effector memory phenotype, and expressed high levels of CD161. Detailed phenotyping of MAIT cells also demonstrated that they share several features with invariant natural killer T (iNKT) cells, including expression of promyelocytic leukemia zinc finger (PLZF), a transcription factor that controls the innate-like functionality of iNKT cells. Expression of PLZF likewise confers innate-like functionality to MAIT cells, as demonstrated by the ability of the cytokine to induce interferon-γ (IFN-γ) production in the absence of TCR stimulation.

[0007] Subsequent studies have found that human MAIT cells do not express a single constant TCR, but express restricted TCRs containing Vα7.2-Jα33, Vα7.2-Jα12, or Vα7.2-Jα20 that are primarily associated with human β chains Vβ2 / Vβ13. Thus, current understanding considers MAIT cells to be T cells that (a) express a semi-constant Vα7.2-Jα33 / 12 / 20 TCR, (b) are activated by microbial vitamin B antigens presented by MR1 to perform both type 1 and type 17 effector functions, and (c) exhibit innate-like characteristics, controlled by the expression of PLZF, including the ability to be activated by cytokines that are independent of their TCR.

[0008] Several properties of MAIT cells suggest that they play a fundamental role in mammalian immunity. First, MAIT cells have an intrinsic effector-memory phenotype and are typically CD45RA - CD45RO + CD95 Hi CD62L Lo CD44 Hi , with the ability to rapidly secrete several pro-inflammatory cytokines. Secondly, MAIT cells are very abundant in human tissues, usually comprising 1-4% of all T cells in peripheral blood, up to 10% of airway T cells, and 20-40% of liver T cells. In addition, since each TCR recognizes the same ligand, MAIT cells will significantly exceed the number of conventional antigen-specific T cells responding to the cognate antigen in the early stages of the immune response.

[0009] The conservation and abundance of MAIT cells may be explained by their broad functionality, which is attributed to different activation modes, each triggering a distinct transcriptome program. Due to their capacity for diverse functional responses in diverse immunological contexts, these intriguing cells now appear to be multifunctional effectors, at the core of the interface between innate and adaptive immunity.

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

[0011] Chimeric antigen receptors (CARs), also called chimeric immunoreceptors, chimeric T cell receptors, or artificial T cell receptors, are receptor proteins that have been engineered to give T cells a new ability to target specific antigens. Receptors are chimeric in that they combine both antigen binding and T cell activation functions into a single receptor.

[0012] CAR T cell therapy is well known in the art and uses T cells engineered with CAR to treat cancer. The T cells are modified to recognize cancer cells and destroy them. The standard approach is to harvest T cells from the patient, genetically modify them, and then infuse the resulting CAR T cells into the patient to attack their tumor (Fox M (July 12, 2017. "NewGene Therapy for Cancer Offers Hope to ThoseWithNoOptions Left" .NBC News.( https: / / www.nbcnews.com / health / health-news / new-gene-therapy-cancer-offers- hope-those-no-options-left-n741326 ).

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

[0014] There are substantial differences between CAR-T cell therapy and TCR-T cell therapy, as described in the following “ CAR T-Cell Therapy vs.TCR-T Therapy: Differences and Applications-Atlantis Bioscience Pte Ltd .” https: / / atlantis2.e;sbiz.website / the-evolution-of-car-t- cells / (opens%20in%20a%20new%20tab .

[0015] Structurally, CAR T cells are different from TCR-engineered T cells. CAR-T cells are composed of engineered intracellular domains and extracellular domains. The extracellular domain is composed of a single-chain variable fragment (composed of the antigen-binding domain of an antibody) to recognize specific antigens on the surface of the cell to trigger intracellular signaling that causes T cell activation. The maintenance of T cell activation and the survival of CAR T cells are further enhanced by adding a co-stimulatory domain to the intracellular part of the CAR T cell.

[0016] The use of CAR T cells has been previously described in the art. For example, U.S. Application No. 17 / 309,451 discloses a population of T cells expressing a chimeric antigen receptor (CAR), wherein the T cells are placental T cells derived from umbilical cord blood, placental perfusate T cells, or a mixture of the two. The application further discloses a method for treating cancer or symptoms in a patient in need by administering to the patient a certain amount of the above-mentioned T cell population.

[0017] In addition, attempts have been made in the art to use MAIT cells for allogeneic adoptive immunotherapy. U.S. Patent Application No. 17 / 414,689 discloses mucosal-associated constant T (MATT) cells expressing chimeric antigen receptors (CARs) for treating cancer, immune diseases, or infectious diseases in a subject, wherein the MAIT cells are allogeneic to the subject. In this disclosure, MAIT cells are obtained from blood samples (including peripheral blood and umbilical cord blood).

[0018] The present invention takes advantage of the use of CARs and the use of MAIT cells and takes it a step further by providing unique placental CAR-MAIT cells that have improved effector activity and higher survival in the host compared to peripheral blood and cord blood MAIT cells and compared to conventional T cells disclosed in the prior art. Engineering these placental MAIT cells to express CARs allows for a variety of therapeutic uses. Summary of the invention

[0019] In one main aspect, the present invention relates to a cell composition comprising a population of engineered mucosal-associated constant T (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).

[0020] According to an embodiment of the present invention, the cell composition provided herein is suitable for cell therapy. Preferably, but not necessarily, at least 90% of the placental MAIT cells are characterized by expressing TCRVα7.2+ and high levels of CD161 (CD161 高 In some embodiments, the cell composition of the present invention comprises 10 of the engineered placental CAR-MAIT cell population. 9 Up to 10 11 living cells.

[0021] According to the present invention, the exogenous CAR expressed by placental MAIT cells of the cell composition recognizes tumor antigens. In one embodiment, the tumor antigen is mesothelin (MSLN). However, in other optional embodiments, 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, mesothelin, tight junction protein 3, tight junction protein 4, tight junction protein 6, tight junction protein 18.2, ROR1, ROR3, Muc1 or Muc16. In some optional embodiments, the exogenous CAR expressed by placental MAIT cells of the cell composition provided herein recognizes microbial antigens and / or viral antigens. However, in some other optional embodiments, the exogenous CAR expressed by placental MAIT cells of the cell composition provided herein recognizes target molecules expressed on regulatory T cells. In a specific non-limiting embodiment, the target molecule is CCR8. However, in a further embodiment of the present invention, the exogenous CAR expressed by placental MAIT cells of the cell composition specifically recognizes the target molecule expressed on alloreactive lymphocytes. In a specific non-limiting embodiment, the target molecule is CD70. In a further selection, the exogenous CAR expressed by placental MAIT cells of the cell composition described herein recognizes the target molecule expressed on senescent cells. In a specific non-limiting example, the target molecule is urokinase-type plasminogen activator (uPAR). In a further optional embodiment of the present invention, the exogenous CAR expressed by placental MAIT cells of the cell composition recognizes the target molecule expressed on cells associated with fibrotic diseases or conditions. A specific non-limiting example of such a target molecule is fibroblast activation protein (FAP). Optionally, the exogenous CAR expressed by placental MAIT cells of the cell composition recognizes the target molecule expressed on cells associated with autoimmune diseases or disorders. In a specific non-limiting example, the target molecule is expressed on B cells, such as but not limited to CD19, CD20, CD21.In some other optional embodiments of the present invention, the target molecule comprises a pathogenic MHC class II:peptide complex associated with an autoimmune disease, an autoantibody, a B cell antibody targeted by a B cell antibody receptor (BAR), carboxypeptidase H, chromogranin A, glutamate decarboxylase, imogen-38, insulin, insulinoma antigen-2 and 2β, islet-specific glucose-6-phosphatase catalytic subunit-associated protein (IGRP), proinsulin, citrullinated protein, collagen II, heat shock protein, human cartilage glycoprotein, double-stranded DNA, La antigen, nucleosomal histones and ribonucleoproteins (snRNPs), phospholipid-β-2 glycoprotein I complex poly (AD P-ribose) polymerase, Sm antigen of U-1 small nuclear ribonucleoprotein complex, alpha enolase, aquaporin-4, beta-arrestin, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein S100-beta, R1-type reticulin, gastric H+ / K+-ATPase, 21-hydroxylase, 17-hydroxylase, cytochrome P450 side chain cleavage enzyme, CD19, and the engineered MAIT cells bind to the pathogenic MHC associated with the autoimmune disease Class II: peptide complexes, autoantibodies, B cell antibodies targeted by the B cell antibody receptor (BAR), carboxypeptidase H, chromogranin A, glutamate decarboxylase, imogen-38, insulin, insulinoma antigen-2,2β, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proinsulin, citrullinated proteins, collagen II, heat shock proteins, human cartilage glycoprotein, double-stranded DNA, La antigen, nucleosomal histones, nuclear glyconucleoprotein (snRNP), phospholipid-β-2 glycoprotein I complex poly (ADP-ribose) polymerase, Sm antigen of U-1 small nuclear glyconucleoprotein complex, alpha enolase, aquaporin-4, β-arrestin, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein S100-β, R1-type reticulin, gastric H+ / K+-ATPase, 21-hydroxylase, 17-hydroxylase, cytochrome P450 side chain cleavage enzyme, or CD 19. However, in some optional embodiments of the present invention, the autoantibody used in the cell composition is DSG3.

[0022] In some optional embodiments of the present invention, the exogenous chimeric antigen receptor (CAR) expressed by placental MAIT cells of the cell composition provided herein includes: an antigen binding domain configured to specifically recognize tumor antigens; a transmembrane domain anchoring CAR in the cell membrane; at least one co-stimulatory domain that enhances cell signaling and T cell activation; and an intracellular signaling domain responsible for initiating T cell activation in the case of antigen recognition. In a further embodiment, the antigen binding domain of the chimeric antigen receptor (CAR) may include any of the following: a single-chain antibody, a single-chain variable fragment (scFv), a VHH fragment, each of which is configured to recognize a specified tumor antigen. However, in some optional embodiments, the antigen binding domain of the CAR of the cell composition of the present invention can 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, mesothelin, tight junction protein 3, tight junction protein 4, tight junction protein 6, tight junction protein 18.2, ROR1, ROR3, Muc1, or Muc16. In some additional optional embodiments, the costimulatory domain of the CAR of the cell composition of the present invention comprises one or more functional signaling domains derived from OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) or 4-1BB (CD137). In some additional optional embodiments, the intracellular signaling domain of the CAR comprises an intracellular signaling domain of CD3 zeta, FcRgamma, or a functional fragment thereof, each configured to initiate or maintain T cell activation upon antigen recognition.

[0023] However, in some further embodiments of the present invention, the CAR expressed by placental MAIT cells of the cell composition provided herein is encoded by a genetic construct selected from the group consisting of: a monocistronic construct, a bicistronic construct or a tricistronic construct; wherein at least one additional element in the bicistronic or tricistronic construct is selected from the group consisting of: a cytokine (e.g., IL15, IL18), a constitutively active tyrosine kinase (e.g., c-kit or other genetic elements that enhance the survival, proliferation, or effector function of MAIT cells), wherein the cytokine is unmodified or modified, and / or secreted, conditionally active, constitutively active, or expressed in a membrane-bound form.

[0024] In some embodiments, the pre-expanded population of placental MAIT cells provided herein shows a higher percentage of cells expressing low levels of CD45RA and low levels of CD62L compared to the pre-expanded population of MAIT cells derived from peripheral blood, indicating the effector memory phenotype of placental MAIT cells. In some additional embodiments, the pre-expanded population of placental MAIT cells of the cell composition shows a higher percentage of cells expressing CCR5 and / or CCR6 chemokine receptors and a lower percentage of cells expressing CXCR4 compared to the pre-expanded population of MAIT cells derived from peripheral blood. In a further embodiment, the population of placental MAIT cells of the cell composition of the present invention shows a higher percentage of cells expressing granzyme B compared to the population of MAIT cells derived from peripheral blood, indicating the enhanced effect and lytic function of placental MAIT cells. However, in additional embodiments, the pre-expanded population of the placental MAIT cells of the cell composition of the present invention shows a higher percentage of cells expressing perforin compared to the pre-expanded population of MAIT cells derived from peripheral blood, indicating the enhanced effect and lytic function of placental MAIT cells. In additional embodiments, the pre-expansion population of placental MAIT cells of the cell composition exhibits a higher percentage of cells expressing perforin and / or granzyme B compared to the pre-expansion population of conventional T cells derived from peripheral blood, indicating enhanced effector and lytic functions of placental MAIT cells. However, in further embodiments, the pre-expansion population of placental MAIT cells of the disclosed cell composition exhibits a higher percentage of cells expressing perforin and / or granzyme B compared to the pre-expansion population of conventional T cells derived from umbilical cord blood, indicating enhanced effector and lytic functions of placental MAIT cells. In additional embodiments, the pre-expansion population of placental MAIT cells of the cell composition exhibits a higher percentage of cells expressing TNFα compared to the pre-expansion population of conventional T cells derived from umbilical cord blood, indicating enhanced effector functions of placental MAIT cells. In additional embodiments, the pre-expansion population of placental MAIT cells of the cell composition exhibits a higher percentage of cells expressing TNFα compared to the pre-expansion population of conventional T cells derived from peripheral blood conventional T cells, indicating enhanced effector functions of placental MAIT cells. However, in additional embodiments, the population of placental MAIT cells of the cell composition exhibits a higher percentage of cells expressing one or more of the following: perforin, granzyme B, granzyme B and perforin, TNFα, and TNFα and INFγ, compared to a population of placental T cells, indicating enhanced effector and lytic function.According to a further embodiment of the present invention, the pre-expansion population of placental MAIT cells of the cell composition exhibits a higher percentage of cells expressing CD8αα+ compared to the pre-expansion population of cord blood MAIT cells, indicating a mature phenotype of placental MAIT cells.

[0025] In some aspects of the invention, the cell compositions disclosed herein are used for therapies. In some optional embodiments, the cell compositions can be used to treat subjects with tumors or malignancies. Exemplary uses of the cell compositions of the invention are for subjects with tumors such as, but not limited to, blood 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 cancer, skin cancer, and sarcoma.

[0026] In some aspects of the invention, a method of treating a subject having a tumor or malignancy is provided, wherein the method comprises administering to the subject a cell composition according to any optional embodiment as defined above. The tumor treated may be, for example, a blood 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 cancer, skin cancer, and sarcoma. Preferably, but not necessarily, the cell composition used to treat the subject is suitable for cell therapy, and the population comprises at least 10 9 viable cells, of which at least 90% were TCRVα7.2 + CD161 高 .

[0027] In some embodiments, the engineered placental MAIT cells used to treat a subject are allogeneic to the subject. In other options, the engineered placental MAIT cells are partially histocompatible with the subject, or the engineered placental MAIT cells are not histocompatible with the subject.

[0028] In some aspects of the invention, a method of treating a subject infected with a pathogen is provided. The pathogen may be any of a bacterial pathogen, a viral pathogen, or a fungal pathogen, and the method comprises administering to the subject a cell composition according to any suitable optional embodiment as defined above.

[0029] In some additional aspects of the invention, methods of treating a subject suffering from a fibrotic disease or condition, methods of treating a subject suffering from a disease or condition associated with aging, methods of treating a subject with an allogeneic transplant, methods of treating a subject suffering from an autoimmune disease or condition are provided, wherein all of the above methods comprise administering to a subject in need thereof a cell composition according to any suitable optional embodiment as defined above.

[0030] However, according to an additional aspect of the present invention, a method for treating a subject infected with a pathogen is provided, the method comprising administering to the subject a composition comprising a colony of MAIT cells derived from the placenta. The colony of MAIT cells according to this embodiment is not an engineered MAIT cell, and the cell does not express exogenous CAR. The pathogen may be, for example, a bacterial pathogen, a viral pathogen, or a fungal pathogen. In some specific examples, the bacterial pathogen may include Mycobacterium tuberculosis bacteria. In some embodiments, the bacterial pathogen includes antibiotic-resistant bacteria or bacterial treatment-resistant bacteria. In some other specific examples, the fungal pathogen may include invasive Aspergillus. In some further examples, the viral pathogen may include cytomegalovirus, hepatitis B virus, or hepatitis C, and optionally, the infection is the result of postoperative infection. Preferably, but not necessarily, the MAIT cells for treating a subject according to this aspect of the present invention are allogeneic with the subject.

[0031] However, according to an additional aspect of the present invention, there is provided a population of placental mucosa-associated invariant T (MAIT) cells, wherein the placental MAIT cells are at least 90% TCRVα7.2 + CD161 + , and most of the placental MAIT cells are characterized by lack of expression of CD45RA and CCR7 (CD45RA - CCR7 - ), indicating an effector memory phenotype of the cells. Optionally, the population of MAIT cells is engineered placental MAIT cells expressing a chimeric antigen receptor (CAR).

[0032] In some embodiments, the population of placental MAIT cells is further characterized by high or low expression of CD62L. In some additional embodiments, the population of placental MAIT cells is further characterized by high expression of CD69. Optionally, the population of placental MAIT cells can be obtained from intervillous blood (IVB). In addition, the population of placental MAIT cells according to any of the optional embodiments above can be used to prepare a cell composition to be used for cell therapy.

[0033] In some embodiments, the present disclosure provides a population of engineered mucosa-associated invariant T (MAIT) cells comprising an exogenous chimeric antigen receptor (CAR), wherein the MAIT cells are derived from placental intervillous blood (IVB).

[0034] In some embodiments, CAR recognizes tumor antigens. In some embodiments, CAR recognizes target molecules expressed on regulatory T cells. In some embodiments, CAR recognizes target molecules expressed on alloreactive lymphocytes. In some embodiments, CAR recognizes target molecules expressed on autoimmune cells. In some embodiments, CAR recognizes target molecules expressed on senescent cells.

[0035] In some embodiments, the CAR of the engineered IVB MAIT cells comprises an antigen binding domain, a transmembrane domain, and a co-stimulatory domain. The antigen binding domain can be a single-chain antibody or a single-chain antibody fragment such as, but not limited to, a single-chain variable fragment (scFv). In some embodiments, the target of the antigen binding domain includes but is 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, mesothelin, Claudin 3, Claudin 4, Claudin 6, Claudin 18.2, ROR1, ROR2, ROR3, or Muc1, Muc16, Muc-CD.

[0036] In some embodiments, the costimulatory domain of CAR comprises a functional signaling domain derived from OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) or 4-1BB (CD137). In some embodiments, the intracellular signaling domain of CAR comprises an intracellular signaling domain of CD3 zeta, or FcR gamma, or a functional fragment thereof.

[0037] In some embodiments, a pharmaceutical composition is provided, comprising a pharmaceutically acceptable carrier and a population of engineered IVB CAR-MAIT cells disclosed herein.

[0038] In some embodiments, a method of treating a subject having a tumor or malignancy is provided, the method comprising administering to the subject a composition comprising a population of engineered IVB CAR-MAIT cells disclosed herein. In some embodiments, the engineered IVB CAR-MAIT cells are allogeneic to the subject.

[0039] In some other embodiments, the engineered IVB CAR-MAIT cells bind mesothelin (MSLN), and the engineered IVB CAR-MAIT cells bind MSLN.

[0040] In some embodiments, a method for treating a subject suffering from a fibrotic disease or condition is provided, the method comprising administering to the subject a composition comprising a colony of engineered IVB CAR-MAIT cells disclosed herein. In some embodiments, the fibrotic disease or condition comprises cardiac fibrosis. In some embodiments, when the subject suffers from cardiac fibrosis, the engineered IVB CAR-MAIT cells bind to fibroblast activation protein (FAP). In some embodiments, the engineered IVB CAR-MAIT cells are allogeneic to the subject.

[0041] In another embodiment, a method of treating a subject with an allogeneic transplant is provided, the method comprising administering to the subject a composition comprising a population of engineered IVB CAR-MAIT cells disclosed herein, wherein the IVB CAR-MAIT cells bind to a target molecule expressed on an alloreactive lymphocyte. Examples of target molecules expressed on alloreactive lymphocytes include, but are not limited to, CD70, CD19, CD20.

[0042] In some embodiments, a method for treating a subject suffering from a disease or condition associated with aging is provided, the method comprising administering to the subject a composition comprising a population of engineered IVB CAR-MAIT cells disclosed herein. In certain embodiments, the engineered MAIT cells are allogeneic to the subject. In some embodiments, the disease or condition associated with aging comprises chronic inflammation, fibrotic liver disease, atherosclerosis, or diabetes, or a combination thereof. In some embodiments, the IVB CAR-MAIT cells express an exogenous antigen receptor that binds to a target molecule. In some embodiments, the engineered IVB CAR-MAIT cells bind to a urokinase-type plasminogen activator receptor (uPAR), and the engineered IVB CAR-MAIT cells bind to uPAR.

[0043] In some embodiments, a method for treating a subject suffering from an autoimmune disease or condition is provided, the method comprising administering to the subject a composition comprising a population of engineered IVB CAR-MAIT cells disclosed herein. In some embodiments, the engineered IVB CAR-MAIT cells are allogeneic to the subject. In some embodiments, the autoimmune disease or condition comprises systemic lupus erythematosus (SLE), refractory SLE, pemphigus vulgaris, multiple sclerosis, type I diabetes, rheumatoid arthritis, celiac disease, pernicious anemia, inflammatory myopathy, myasthenia gravis, or adrenalitis, or a combination thereof. In some embodiments, the engineered IVB CAR-MAIT cells recognize and bind to a target antigen comprising a pathogenic MHC associated with the autoimmune disease. Class II: peptide complexes, autoantibodies, B cell antibodies targeted by the B cell antibody receptor (BAR), 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 proteins, collagen II, heat shock proteins, human cartilage glycoprotein, double-stranded DNA, La antigen, nucleosomal histones, nuclear In some embodiments, the autoantibody is DSG3.

[0044] In other embodiments, a method of treating a subject infected with a pathogen is provided, the method comprising administering to the subject a composition comprising a population of placental IVB CAR-MAIT cells, wherein the IVB MAIT cells are allogeneic to the subject.

[0045] In certain embodiments, these IVB MAIT cells do not comprise exogenous antigen receptors. In some embodiments, the pathogen is a bacterial pathogen, a viral pathogen, or a fungal pathogen. In certain embodiments, the bacterial pathogen comprises an antibiotic-resistant bacteria or a bacterial treatment-resistant bacteria. In some embodiments, the bacterial pathogen comprises Mycobacterium tuberculosis bacteria. In some embodiments, the fungal pathogen comprises an invasive Aspergillus genus. In some embodiments, the viral pathogen comprises cytomegalovirus, hepatitis B virus, or hepatitis C virus. In some embodiments, the infectious conditions treated by the methods disclosed herein are limited to site-specific infections, for example, wherein the infection is limited to a specific site or organ. In some embodiments, the infection treated by the methods disclosed herein comprises a postoperative infection.

[0046] These and other aspects of engineered placental CAR-MAIT cells and their uses will be understood from the following description of the drawings and detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Some embodiments of engineered IVB MAIT cells and / or IVB CAR-MAIT cells and methods of use thereof are described herein by way of example only with reference to the accompanying drawings. With specific reference now to the drawings in detail, it should be emphasized that the details shown are by way of example and for the purpose of illustrative discussion of embodiments of engineered IVB CAR-MAIT cells and IVB MAIT cells lacking exogenous antigen receptors and therapeutic uses thereof. In this regard, the description using the accompanying drawings makes it apparent to those skilled in the art how embodiments of engineered IVB CAR-MAIT cells, and IVB MAIT cells lacking exogenous antigen receptors, and therapeutic uses thereof may be implemented.

[0048] Figures 1A-1D is a schematic diagram showing the collection of maternal blood from the placenta, wherein Figure 1A Both the fetal and maternal sides of the placenta are shown; Figure 1B The maternal side is shown after removal of the membrane; Figure 1C showing the incision made in the maternal tissue; and Figure 1D Operations performed on a fetal part according to an embodiment of the present invention are shown.

[0049] Placenta 100 comprises a fetal side 101 of the placenta comprising an umbilical cord 102 which is clamped with a clamp 103 to prevent mixing of fetal blood with maternal (IVB) blood, and a maternal side 104 of the placenta ( Figure 1A IVB collection begins with the removal of the surrounding amniotic membrane on the maternal side, exposing the chorionic villi 106 ( Figure 1B Using scissors or any other suitable cutting tool, make a superficial cut in the villi leaf ( Figure 1C). The placenta is then flipped so that the fetal side is facing up. Phosphate buffered saline (PBS) is injected into the decidua basalis, avoiding fetal blood vessels. All maternal blood flowing out of the placenta is collected. Mononuclear cells are then isolated using Lymphoprep (density gradient tubes), and intermediate cell stocks are subsequently frozen.

[0050] Figure 2A Flow cytometric graphs showing activation and proliferation of MAIT cells in a packed bed bioreactor for ten (10) days are presented in accordance with embodiments of the present invention.

[0051] Mononuclear cells from intervillous blood (IVB) were seeded onto ECM-coated The MAIT cells were grown in a packed bed bioreactor containing carriers that simulated the natural environment and promoted the adhesion of APCs. MAIT cell activation was induced by 5-OP-RU and IL-15. The activation and proliferation of MAIT cells were evaluated at the following time points: day zero (0), five (5), seven (7), and ten (10). The MAIT cell population was detected by the expression of CD3, Vα7.2, and CD161 markers. The results showed an increase in the proportion of MAIT cells, starting with CD3 on day zero (0). + 22.63% of the population, and up to 96.26% on the tenth (10) day. In addition, the expression of activation markers CD69 and CD25 increased from day zero (0) to day seven (7), and slightly decreased on day ten (10).

[0052] Figure 2B Presented are flow cytometry graphs of MAIT cell growth after switching cells from a first bioreactor to a second bioreactor on the tenth (10th) day and growing for an additional seven (7) days in the second bioreactor in accordance with an embodiment of the present invention. Cells that reached maximum growth capacity on the tenth day in the first bioreactor were harvested and approximately 30% of these cells were then inoculated into the second bioreactor, which was similar in design to the first bioreactor. The cells were grown for an additional seven (7) days. Flow cytometry results demonstrated that the MAIT percentages were similar during most of the time, but presented a decrease from >90% on day 14 to 82% on day 17. CD69 marker expression was upregulated from 30% to 87% on day 17, indicating that MAIT cells maintained their activation signals, while CD25 gradually decreased as expected, indicating that MAIT cells were activated a short time ago, but had not reached their full proliferation and functional state, suggesting their ability to be reactivated and function in the event of a future target encounter.

[0053] Figure 3 Results of placental IVB MAIT cell activation and expansion according to some optional embodiments of the present invention are shown. Mononuclear cells of IVB were seeded in tissue culture plates, and MAIT cell activation was induced by 5-OP-RU and IL-15. On the third (3) and fifth (5) days after activation, cells were counted and analyzed for MAIT percentage by flow cytometry, and fresh culture medium and IL-15 were added. On the seventh (7) day, cells were separated with magnetic beads and anti-TCR Vα7.2 antibodies to further enrich the MAIT population; cells were cultured for an additional three (3) days in the presence of IL-15. Results show an increase in MAIT percentage and MAIT fold expansion (increase in MAIT absolute numbers relative to the starting number) throughout the culture period.

[0054] Figure 4A-4B Shown on CD3 + The percentage of MAIT cells in placental intervillous blood (IVB) mononuclear cells and peripheral blood mononuclear cells (PBMC) were gated. Figure 4A Display from CD3 + Representative sample of placental IVB MAIT cells gated for population. Figure 4B Shown are CD3 from thirteen (13) IVB donors and eleven (11) PB donors analyzed. + The proportion of placental (IVB) and peripheral blood (PB) MAIT cells. p = 0.0159.

[0055] Figure 5 Shown are immunophenotypic analyses of placental IVB MAIT cells and PB MAIT cells collected from each of six (6) different donors and analyzed on day zero (0).

[0056] Cells were stained according to a standard cell surface marker staining protocol and analyzed by flow cytometry. Cell surface markers of CD45RA and CD62L were used to characterize the cells. As shown, placental IVB MAIT cells had statistically significantly more "effect memory" cells and fewer "terminal differentiation" cells compared to PB MAIT cells. The gating strategy for phenotyping is listed on the right side of the figure. Statistical analysis was performed using GP Prism, t-test comparison. *p<0.05, **p<0.01, ***p<0.001.

[0057] Figure 6A-6BChemokine receptor analysis of placental IVB MAIT cells and PB MAIT cells according to an embodiment of the present invention is shown. To evaluate the chemokine receptor profile of the MAIT population, IVB MAIT cells and PB MAIT cells were collected from six (6) different donors and analyzed for chemokine receptor expression on day zero (0). Cell surface markers of CCR6, CCR5, CXCR4 and CXCR6 were used to characterize the cells. Cells were stained according to standard cell surface marker staining protocols and analyzed by flow cytometry. Statistical analysis was performed using GP Prism, t-test comparisons. *p<0.05, **p<0.01, ***p<0.001. Fig. 6A It was shown that IVB MAIT cells had higher expression of CCR5 and CCR6 chemokine receptors compared to PB MAIT cells. Figure 6B It was shown that IVB MAIT cells had statistically significantly lower levels of CXCR4 and comparable levels of CXCR6 compared to PB MAIT cells. The results further illustrate that the two populations of MAIT cells, IVB-derived and PB-derived, are distinct.

[0058] Figure 7A-7B Shown are the expressions of granzyme B and perforin in placental IVB MAIT cells and in peripheral blood MAIT cells. Fig. 7A Representative samples of both cell types are shown. Figure 7B MAIT cells from placenta IVB and from peripheral blood are shown.

[0059] Cells from each of six (6) donors were stimulated with PMA / ionomycin for four (4) hours and analyzed for intracellular expression of granzyme B and perforin. As shown, placental IVB MAIT cells had statistically significantly higher levels of granzyme B compared to peripheral blood MAIT cells, indicating increased efficacy and lytic capacity of placental IVB MAIT. Cells were gated on MAIT cells. p=0.0001.

[0060] Fig. 8A -8B presents flow cytometry plots of the percentage of MAIT cells in placental intervillous blood (IVB) and matched (same donor) umbilical cord blood (CB) ( Fig. 8A ); and a table summarizing the results obtained in the flow cytometry plots ( FIG. 8B ).

[0061] Cells from three (3) different donors were used as previously described. IVB cells and CB cells were activated similarly. As shown, CB had very few MAIT cells at day zero (0) compared to IVB, and their percentage decreased over time (day 6 / 7). In more detail, IVB MAIT and matched CB MAIT cells were collected according to the protocol described in the following materials and methods. Cells were stained for CD161 and TCR Vα7.2 at day 0 and at day 6 / 7, which represents the MAIT-positive gating strategy. The square gate represents the percentage of MAIT cells. The table describes the actual cell counts. As observed, intervillous blood (IVB) has more MAIT cells than cord blood (CB).

[0062] Fig. 9 Flow cytometry graphs showing the expression of CD8αα+ by placental IVB MAIT cells and CB MAIT cells according to an example of the present invention.

[0063] As shown in the flow cytometry graphs, placental IVB MAIT cells expressed a high frequency of CD8αα+ at a three (3)-fold higher frequency (41.2%) compared to CB MAIT cells (14.2%), indicating a more "antigen experienced" phenotype compared to the naive state present in CB MAIT cells.

[0064] CD8α is a cell surface glycoprotein that can be expressed as a disulfide-linked heterodimer with CD8β or as a homodimer. In contrast to CD8αβ, CD8αα is never expressed in naive cells. CD8αα is expressed on T cells, but is easily induced on strongly activated T cells. In addition, CD8αα can act as other "tuning" molecules to help survival in a similar way to the expression of KIR or other NK cell-related receptors on T cells. MAIT cells from IVB and CB were stained for two CD8 subunits (CD8α and CD8β), and the frequency was measured by flow cytometry. The results showed that IVB MAIT cells had a higher frequency of CD8αα, indicating a more mature and "antigen-experienced" phenotype compared to CB MAIT cells.

[0065] Fig.10 Flow cytometry plots showing the effector phenotype of placental IVB MAIT cells and CBMAIT cells using CD45RO, CD27, CD45RA, CCR7 and CD62L.

[0066] IVB MAITs and matched CB MAITs were collected according to the methods described in the following Materials and Methods. Cell surface markers of CD45RA, CD27, CCR7, CD62L, and CD45RO were used to determine the cell immune phenotype, with CD45RA - / CCR7 - 、CD45RA - / CD62L - 、CD45RA - / CD45RO + and CD27 - / CD45RO + They represent effector memory cells, effector memory cells, activated memory cells, and effector memory phenotypes, respectively. As clearly shown in the flow cytometry graph, IVB MAIT cells lag behind CB MAIT cells because IVB MAIT cells possess an effector memory phenotype while CB MAIT cells present a naive phenotype.

[0067] Fig.11 is the transcriptome analysis of IVB MAIT cells and PB MAIT cells, showing a set of top 50 differentially expressed genes.

[0068] In order to understand the transcriptome differences between MAIT cells derived from different tissues (i.e., PBMC and IVB), cells were extracted and amplified as described in the method, and RNAseq analysis was performed. IVB and PB MAIT cells were collected from three (3) different donors, and RNAseq transcriptome analysis was analyzed on the seventh (7) day. Cells were harvested as described in the method. Heat map of significantly (|log2FC|>1) and BH padj<0.01) differentially expressed protein-coding genes between IVB and PB cell types. Pearson correlation calculations were applied to the normalized values ​​of the count data for clustering. The color scheme represents z scores. As shown, compared with peripheral blood MAIT cells, MAIT originating from IVB has a unique and special transcriptome landscape, meaning that the two MAIT populations are highly different from each other. The names of the top 50 genes are listed in the figure.

[0069] Fig.12Comparison between IVB MAIT cells and PB MAIT cells for Granzyme B secretion is shown. Placental IVB MAIT cells and PB MAIT cells were collected from each of four (4) donors and expanded using standard protocols as described in the Methods section. On day eight (8), cells were activated with Phorbol Myristate Acetate (PMA) plus ionomycin and analyzed for Granzyme B and Perforin secretion using standard intracellular staining protocols. Granzyme B is a serine protease found in the granules of lysed cells. It is secreted together with the pore-forming protein perforin to mediate apoptosis of target cells.

[0070] To evaluate the basal potential of MAIT cells derived from IVB or PB, the collected cells were stimulated with PMA and ionomycin and stained for intracellular granzyme B secretion. As shown, stimulated IVB MAIT cells produced significantly higher levels of granzyme B compared to PB MAIT cells, indicating increased efficacy and lytic capacity of cells derived from IVB. Statistical analysis was performed using GP Prism, t-test comparison. *p<0.05, **p<0.01, ***p<0.001.

[0071] Fig.13 is a graphic illustration of the functional killing characteristics of placental IVB CAR-MAIT cells according to an embodiment of the present invention.

[0072] CAR-MAIT cells were generated according to the standard protocol described in the methods and co-cultured with NSCLC targets overexpressing MSLN and luciferase. Killing was measured by luminescence readout. Different effectors had different levels of CAR + Fraction. Co-cultured with target cells in the presence of CAR + All different culture conditions of CAR-MAIT cells resulted in similar efficacy against target cells, indicating the high and dose-dependent efficacy of CAR-MAIT cells.

[0073] Figures 14A-14B Comparisons between IVB MAIT cells, peripheral blood (PB) maternal T cells, and cord blood (CB) T cells from the same donor (matched blood) are shown. In order to evaluate the differences between MAIT cells and T cells and eliminate donor-to-donor variability, the inventors evaluated the functional differences of different cells originating from the same donor. IVB, PBMCs, and CB were collected as described in the methods and activated with T cell activators on day zero (0). Following the intracellular staining protocol, cells were analyzed using flow cytometry for perforin, granzyme B, IFN-γ, and TNF-α. Figure 14 describes the gating strategy for the analysis. Fig.14A is a flow cytometry graph depicting raw data for perforin and granzyme B; Fig. 14B are flow cytometry graphs depicting raw data for IFN-γ and TNF-α.

[0074] As shown, MAIT cells at day 0 possessed completely different effector molecules compared to conventional T cells. T cells originating from cord blood and peripheral blood had similar characteristics.

[0075] Figures 15A-15B Shown according to Figures 14A-14B Comparison of the gating strategy shown in Figure 1 with the positive MAIT population derived from IVB and the conventional T cell population derived from peripheral maternal blood (MB) and CB cultured using the standard activation protocol for T cells. The examples are examples of two donors, from each of which IVB, peripheral maternal blood (MB) and umbilical cord blood (CB) were obtained.

[0076] To evaluate the differences between MAIT and T cells and eliminate donor variability, on day zero (0), IVB, PBMC and CB were collected as described in the methods and activated and cultured with T cell activators according to standard protocols. Briefly, T cell expansion was performed using TCR activation with CD28 and CD3 antibodies or beads with IL2 cytokine (TransAct). On day ten (10), cells were stained for CD161 and TCR VA7.2 to evaluate the fraction of MAIT positive populations. As observed, the final population was highly purified, up to CD3 only when the specific MAIT expansion protocol was followed. + By applying the standard T cell expansion protocol, the initially detected (before expansion, day 0) MAIT cells did not expand, and the MAIT population detected on day 10 was significantly diluted to as low as CD3 + <1.2% of cells.

[0077] Figures 16A-16B is a graphical illustration depicting a comparison of IVB-MAIT and IVB-T effector functions according to an embodiment of the present invention. Fig.16A is a graphic illustration depicting a comparison of IVB-MAIT and IVB-T effector functions. IVB-MAIT was cultured as described in the methods. From the same starting material (IVB cell harvest), the inventors also cultured T cells using a standard conventional T cell protocol with TransAct and IL2. On the tenth (10th) day, IVB-MAIT cells and IVB-T cells were stimulated with PMA+ionomycin or TransAct, stained for effector molecules such as IFN-γ, TNF-α, perforin and granzyme B and analyzed by flow cytometry. Fig. 16BMAIT cells were shown to have higher levels of perforin and TNF-α compared to T cells derived from the same source.

[0078] Figures 17A-17B Flow cytometry images of placental MAIT cells and placental CAR-MAIT cells, respectively, for comparison of the immunophenotype of cell populations. Cells were expanded for 10 days, stained according to standard cell surface marker protocols, and analyzed by flow cytometry. Compared with "central memory (CM)" cells, the gating strategy of CD45RA and CCR7 showed a statistically significant increase in "effect memory (EM)". Naive and terminally differentiated (TD) cells were almost absent.

[0079] Fig. 17C is a graphic illustration of the results representing the average of 3 different donors analyzed at day 10. The results show that more than 80% of both MAIT and CAR-MAIT cells are effector memory, while the remaining cells are central memory. Naive and terminally differentiated cells are less than 1%. DETAILED DESCRIPTION

[0080] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of engineered mucosal-associated constant T (MAIT) cells and methods of use thereof. Although methods and materials similar or equivalent to those described herein can be used to implement or test embodiments of engineered mucosal-associated constant T (MAIT) cells and methods of use thereof, exemplary methods and / or materials are described below. In the event of a conflict, the patent specification including the definition shall prevail. In addition, materials, methods and examples are illustrative only and are not intended to be necessarily restrictive. Each literature reference or other citation mentioned herein is incorporated herein by reference in its entirety.

[0081] The terms "comprise", "comprises", "comprising", "includes", "including", "having" and their synonyms mean "including but not limited to".

[0082] As used herein, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an enzyme" or "at least one enzyme" may include a plurality of enzymes, including mixtures thereof.

[0083] Unless otherwise indicated, the term "population" when used in conjunction with a particular cell attribute or multiple cell attributes can encompass a collection of cells, at least 70% of which exhibit one or more of the attributes mentioned. In other embodiments, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells in the collection exhibit one or more of the attributes mentioned. In some embodiments, the population is separated from a cell type different from a particular attribute. In some embodiments, the population of placental engineered MAIT cells described herein comprises exogenous attributes.

[0084] With respect to placental-derived cells, unless otherwise indicated, "placenta," "placental," and the like encompass intervillous blood (IVB). Placental-derived MAIT cells are obtained from a maternal source of the placenta. In some embodiments, MAIT cells are obtained from intervillous blood of the placenta. In some embodiments, MAIT cells are obtained from the decidua parietalis of the placenta. In some embodiments, MAIT cells are obtained from the decidua basalis of the placenta. In some embodiments, MAIT cells are not obtained from umbilical cord blood.

[0085] As used herein, the terms "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 a different donor. In some other embodiments, the peripheral blood is matched to intervillous blood and / or matched to umbilical cord blood and received from the mother. These terms can be used interchangeably and have all the same qualities and meanings.

[0086] As used herein, the terms "intervillous blood (IVB)-derived MAIT cells" or "IVB MAIT cells," "placental intervillous blood MAIT," "placental IVB MAIT," and "placental MAIT" as used herein are all synonymous and refer to MAIT cells isolated from the intervillous blood of the placenta, and / or blood obtained from the maternal portion of the placenta.

[0087] As used herein, "MAIT cells" shall broadly encompass MAIT cells from any source including, but not limited to, peripheral blood, umbilical cord blood, and intervillous blood.

[0088] In some embodiments of the population of engineered MAIT cells, the MAIT cells are derived from a maternal blood source of the placenta. In some embodiments of the population of engineered MAIT cells, the MAIT cells are derived from a maternal blood source of the placenta. In some embodiments of the population of engineered MAIT cells, the MAIT cells are obtained from intervillous blood of the placenta. In some embodiments of the population of engineered MAIT cells, the MAIT cells are not obtained from umbilical cord blood.

[0089] In various embodiments, placental cells can be obtained from a full-term placenta. A convenient source of placental tissue is a postpartum placenta (e.g., less than 48 hours after birth); however, various sources of placental tissue or cells may be considered by a technician. In other embodiments, the placenta is used within 24 hours of birth (in some embodiments, while being stored in a physiological buffer), 18 hours, 14 hours, 10 hours, 8 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour. In certain embodiments, the placenta is kept frozen before the harvesting of the cells. In other embodiments, prenatal placental tissue is used. In some embodiments, the donor is 40 years old or younger, in other embodiments 35 years old or younger, yet in other embodiments, the donor can be any woman of childbearing age.

[0090] Methods for isolating MAIT cells from placental intervillous blood (IVB) are well known in the art. An exemplary non-limiting protocol utilizes blood dripping from a placenta lifted with the umbilical cord clamped face down. Such methods have been shown to have very low cross-contamination rates between IVB and cord blood. Those skilled in the art are familiar with methods for checking the purity of cell populations and, if necessary, improving purity using cell sorting, etc.

[0091] Mucosal-associated invariant T (MAIT) cells

[0092] As used herein, mucosal-associated constant T (MAIT) cells include unconventional T cells expressing semi-constant TCRs such as Vα7.2-Jα33 in humans; in some embodiments, they are associated with β chains Vβ2 / Vβ13. In other embodiments, the aforementioned α chains are associated with β chains from the TRBV6 or TRBV20 gene family. In some embodiments, MAIT cells recognize antigens that are limited to non-peptide molecules presented in the context of (non-polymorphic) major histocompatibility complex (MHC) class I-like protein MR1. In some embodiments, the engineered MAIT cells disclosed herein are detectable by staining with MR1-Ag tetramers, which are loaded with, for example, 5-(2-oxopropyleneamino)-6-D-ribosylaminouracil (5-OP-RU); 5-(2-oxoethylideneamino)-6-D-ribosylaminouracil (5-OE-RU); RL-6,7-diMe (PubChem CID168989); RL-6-Me-7-OH (PubChem CID 440869), or diclofenac (PubChem CID 3033). In other embodiments, one of the following compounds may also be used: 6-(1H-indol-3-yl)-7-hydroxy-8-ribitoldioxeteridine or photolumazine III (PLIII); 6-(2-carboxyethyl)-7-hydroxy-8-ribitoldioxeteridine or photolumazine I; 5-hydroxydiclofenac (PubChem CID 3052566); 4-hydroxydiclofenac (PubChem CID 116545); benzbromarone (PubChem CID 2333); cloxacin (PubChem CID 2722); floxuridine (PubChem CID 2723); 5790); galangin (4H-1-benzopyran-4-one, 3,5,7-trihydroxy-2-phenyl or 3,5,7-trihydroxyflavone); or mercaptopurine (PubChem CID 667490) (see, e.g., Corbett et al., Antigen Recognition by MR1-Reactive TCells; MAIT Cells, Metabolites, and Remaining Mysteries. Front Immunol. 11: 1961 (2020), and references cited therein).

[0093] In certain embodiments, 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 population of engineered MAIT cells as described herein.

[0094] Methods for isolating and characterizing placental IVB MAIT cells and other leukocyte subpopulations are known in the art. For illustration only, leukocyte subpopulations can be isolated and / or analyzed using gating strategies as described herein.

[0095] There are quantitative and qualitative differences between MAIT cells from umbilical cord blood and adult blood (see, e.g., Youssef et al., Ontogeny of Human Mucosal-Associated Invariant T Cells and Related T Cell Subsets. JEM 215:459-479 (2018)). Vα7.2 and CD161 staining allow identification of circulating stage 3 MAIT cells at birth. However, Vα7.2+CD161 in umbilical cord blood 高 The fractions may also encompass other T cells that may share a common developmental pathway. In cord blood, MAIT cells express a naive phenotype (CD45RA + / RO - ) and express CD8αβ heterodimers, whereas most of them display a memory phenotype and express CD8αα homodimers in adults. + CD161 高 The very low expansion of T cells could be related to cell-intrinsic characteristics or to the limited availability of MR1-ligands of microbial origin. + CD161 高 T cells expressed significantly lower levels of PLZF than adult MAIT cells, suggesting that cord blood Vα7.2 + CD161 高 The final maturation of T cells requires early activation signals after birth. Similar to conventional CD8 T cells, umbilical cord blood Vα7.2+CD161 高 T cells proliferate strongly after stimulation with PHA, while adult Vα7.2+CD161 高 Compared with mature MAIT cells in adult blood, cord blood Vα7.2 + CD161 高 T cells are unable to display immediate effector functions to bacterially infected cells. This data suggests that despite their inherent ability to proliferate, cord blood Vα7.2 + CD161 高 T cells require functional maturation and / or expansion after birth to acquire detectable effector activity following recognition of microbial-derived antigens.

[0096] Due to umbilical cord blood Vα7.2 + CD161高 Cells show a naive phenotype and intermediate PLZF levels, and compared with mature adult MAIT cells, they cannot quickly produce cytokines or cytotoxic molecules in response to bacterial ligands. Umbilical cord blood MAIT cells are also not responsive to stimulation by exogenous IL-12 and IL-18, despite the high expression of receptors for these cytokines. As Chen et al., Circulating Mucosal-Associated Invariant T Cells in a Large Cohortof Healthy Chinese Individuals From Newborn to Elderly.Front.Immunol., vol.10, article 260 (2019) further show that umbilical cord blood MAIT cells have a naive phenotype and do not secrete IFN-γ, IL17A and TNF-α after in vitro stimulation with PMA / ionomycin. Overall, these data indicate that cord blood MAIT cells are phenotypically and functionally different from MAIT cells derived from adult subjects.

[0097] In some embodiments, the MAIT cells disclosed herein comprise CD161 + Vα7.2 + 、CD4 - CD3 + lymphocytes, which in further embodiments also bind to the MR1-Ag tetramer. In various embodiments, at least 50% of the cells in the population are CD161 + Vα7.2 + 、CD4 - CD3 + In some embodiments, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the cells in the population are CD161 + Vα7.2 + 、CD4 - CD3 + .

[0098] In yet other embodiments, MAIT subsets are utilized, including but not limited to CD8 + cells (a more specific embodiment of which is CD8 + CD4 - cells), CD8 - CD4 - cells, or CD4 + In various embodiments, at least 50% of the cells in the population are CD8 +In other embodiments, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the cells in the population are CD8 + In various embodiments, at least 50% of the cells in the population are CD8 + CD4 - In other embodiments, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the cells in the population are CD8 + CD4 - In various embodiments, at least 50% of the cells in the population are CD8 - CD4 - In other embodiments, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the cells in the population are CD8 - CD4 - .

[0099] In certain embodiments, the MAIT cells disclosed herein are CD45RA - CCR7 - , in some embodiments, also reflects an effector memory phenotype. In other embodiments, CD45RO + is an additional characteristic of effector memory cells. In various embodiments, at least 50% of the cells in the population are CD45RA - CCR7 - In other embodiments, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the cells in the population are CD45RA - CCR7 - In various embodiments, at least 50% of the cells in the population are CD45RO + In other embodiments, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the cells in the population are CD45RO + .

[0100] In certain embodiments, the MAIT cells disclosed herein are CD45RA - CCR7 - CD62L + In various embodiments, at least 50% of the cells in the population are CD45RA - CCR7- CD62L + ; In other embodiments, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%.

[0101] In certain embodiments, the MAIT cells disclosed herein are CD45RA - CCR7 - CD62L - In various embodiments, at least 50% of the cells in the population are CD45RA - CCR7 - CD62L - ; In other embodiments, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%.

[0102] In certain embodiments, the MAIT cells disclosed herein are CD45RO + CCR7 - CD62L + In various embodiments, at least 50% of the cells in the population are CD45RO + CCR7 - CD62L + ; In other embodiments, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%.

[0103] In certain embodiments, the MAIT cells disclosed herein are CD45RO + CCR7 - CD62L - In various embodiments, at least 50% of the cells in the population are CD45RO + CCR7 - CD62L - ; In other embodiments, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%.

[0104] 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. In some embodiments, MAIT cells express interleukin PD-1. In some embodiments, MAIT cells express interleukin (IL) -18Rα, CD127, α4β7 and PD-1. In some embodiments, the cells also express transcription factors promyelocytic leukemia zinc finger (PLZF), RORγt, Helios, detached mesodermin (Eomes) and / or T-box transcription factor (T-bet). In some embodiments, MAIT cells express transcription factors promyelocytic leukemia zinc finger (PLZF), RORγt, Helios, detached mesodermin. In some embodiments, MAIT cells express T-box transcription factors. In some embodiments, MAIT cells express transcription factors promyelocytic leukemia zinc finger (PLZF), RORγt, Helios, eomesodermin, and T-box transcription factors. Alternatively, or in addition, cells express surface markers CD26, CD44, CD69, or CD25; or receptors interleukin 7 receptor (IL-7R), IL-12R, IL-15R, or IL-18R. In some embodiments, cells express inducible T cell co-stimulator (ICOS). Each of the above proteins and each combination thereof represents a separate embodiment.

[0105] In some embodiments, the MAIT cells disclosed herein express genes related to 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 alpha) when stimulated with 5-OP-RU.

[0106] In certain embodiments, placental MAIT cells recognize riboflavin precursor derivatives of microbial origin. In some embodiments, engineered placental CAR-MAIT cells secrete inflammatory cytokines (e.g., interferon-gamma [IFN-g or IFN-γ], tumor necrosis factor alpha [TNF-a or TNF-α], interleukin 17, or colony stimulating factor 2 [CSF2 / GM-CSF]), for example, by recognition of MR-1 ligands or in other embodiments in an MR1-independent manner. In other embodiments, IL-17A, TNF-a, CSF2 or MIP-1 are all secretory. In other embodiments, IL-26, oncostatin M (OSM), or heparin-binding early growth factor (HBEGF) are upregulated when stimulated with IL-12, IL-18, IL-15, or tumor necrosis factor-like protein 1A (TL1A). Alternatively, or in addition, the engineered placental CAR-MAIT cells disclosed herein perform granzyme B-dependent cytotoxicity of target cells when activated. In other embodiments, IFN-γ, perforin, or granzymes are all upregulated.

[0107] In certain embodiments, the methods described herein include in vitro and / or ex vivo expansion and / or enrichment of engineered MAIT cells prior to administration to a subject. In various embodiments, expansion or enrichment is performed before or after the MAIT cells are engineered to express exogenous antigen receptors. In other embodiments, activation or stimulation assays are performed to characterize or determine the quality of engineered MAIT cells. Typically, however, activation requires stimulation of antigen receptors and co-receptors, and combined cytokine treatment. Once the cells are activated, in some embodiments, they can be further expanded using cytokines alone (without returning to a naive state).

[0108] In some embodiments, MAIT cells are expanded in vitro and / or ex vivo for at least about 5 days, in other embodiments 5-10 days; in other embodiments at least 10 days; in other embodiments 10-15 days; in other embodiments at least 15 days; in other embodiments 15-20 days; and in some embodiments, at least 20 days.

[0109] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description of the range format is merely for convenience and brevity, and should not be interpreted as an inflexible limitation on the scope of engineered MAIT cells and their uses. Therefore, the description of the range should be considered to specifically disclose all possible sub-ranges and single numerical values ​​within the range. For example, a description such as a range from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0110] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integer) within the indicated range. The phrases "a range between" a first indicated number and a second indicated number / a range between" a first indicated number and a second indicated number and "a range from" a first indicated number to" a second indicated number / a range from" a first indicated number to" a second indicated number" are used interchangeably herein and are meant to include the first indicated number and the second indicated number and all fractions and integers therebetween.

[0111] Methods for stimulating MAIT cells are known in the art. In one non-limiting embodiment, Hela cells overexpressing human MR1 protein (Hela-hMR1) are washed and incubated with E. coli Dh5α ATCC strain (usually at a multiplicity of infection of 10-100 bacteria per HeLa cell) in antibiotic-free DMEM at 37°C for 30 minutes, washed, and then incubated at 37°C for 2 hours in complete medium with 100 μg / mL gentamicin and 10 μg / mL chloramphenicol. MAIT cells are added for overnight co-culture, and then the cells are harvested and stained for FACS analysis.

[0112] Additional methods for amplifying and / or enriching MAIT cells are known in the art. In a non-limiting embodiment, MAIT cells are incubated with 5ug / mL CpG, 300nM 5-(2-oxopropylideneamino)-6-D-ribosylaminouracil (5-OP-RU), and 50ng / mL human IL-15. In certain embodiments, IL-15 is included to preferentially enhance the expansion of memory T cells.

[0113] In some embodiments, sorted or purified or enriched MAIT cells are stimulated by CD3 / CD28 (e.g., using functionalized beads, marketed under the trademark TransActTM In other embodiments, CD3 / CD28 beads (available under the trademark ClinExVivo TM Non-limiting exemplary protocols include X Vivo-15 TM ( Walkersville, MD), 100 units / mL of IL-2, and 10 ng / mL of IL-7 were mixed with ClinExVivo TM Incubate for 10-14 days. Once activated, MAIT cells can be further expanded in the presence of cytokines in various embodiments with or without the above ligands.

[0114] In other embodiments, MAIT cells are activated in vitro and / or ex vivo in the presence of MAIT cell activating ligands such as 5-OP-RU, 5-amino-4-D-ribosylaminouracil dihydrochloride (5-ARU), 5-(2-oxoethylideneamino)-6-D-ribosylaminouracil (5-OE-RU), 5-amino-6-ribitolamino-2,4-(1H,3H)-pyrimidinedione (5-A-RU), or other riboflavin (vitamin B2)-derivatives. In certain embodiments, the ligand is provided in combination with a cytokine, such as a cytokine mentioned herein. As a non-limiting example, MAIT cells can be expanded in 100 nM 5-OP-RU and 100 IU / mL IL-2 for, for example, 6-17 days. In other embodiments, MAIT cells are expanded and / or activated in vitro and / or ex vivo in the presence of MAIT cell activating drug metabolites (e.g., diclofenac metabolites). Once activated, MAIT cells can be further expanded in the presence of cytokines in various embodiments with or without the above-mentioned ligands.

[0115] In some embodiments, MAIT cells are expanded and / or activated in vitro in the presence of IL-12, IL-15, IL-18, and 5-OP-RU in the range of 10-1000 nM as described in the art, or IL-12+IL-18 or IL-15+IL-18.

[0116] In some embodiments, the engineered MAIT cells disclosed herein produce IL-17 and upregulate the Th17-associated transcription factor RORC (RORγt) upon PMA and ionomycin stimulation, but not upon CD3+CD28 stimulation.

[0117] In some embodiments, the engineered MAIT cells disclosed herein produce IFN-γ and upregulate T-bet upon PMA / ionomycin stimulation. In more specific embodiments, these cells are CD8 + .

[0118] In various embodiments, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70% of a population of engineered MAIT cells disclosed herein express CD69.

[0119] In other embodiments, less than 30%, less than 20%, or less than 10% of the population of MAIT cells disclosed herein express PD-1. In other embodiments, more than 10%, more than 15%, or more than 20% of the population of engineered MAIT cells express CD25. In other embodiments, any combination of two of the above markers are expressed, the percentages of which can be freely combined with each other. In some embodiments, more than 50%, more than 60%, more than 70%, or more than 80% of the MAIT cells are PD-1. - / LAG-3 - , which indicates that the cells do not show T cell exhaustion. In other embodiments, CTLA-4, TIGIT, 2B4, BTLA, CD57, TIM-3, or KLRG-1 are used to detect T cells that show exhaustion.

[0120] In some embodiments, the MAIT cells disclosed herein express CCR2, CCR5, CCR6, CCR9, CXCR4, CXCR3, VLA-4, or CXCR6, or a combination of 2, 3, 4, 5, 6, 7, or all 8 of these receptors. Alternatively, or in addition, MAIT cells express activating receptors, for example, NKG2D, NKp30, NKp44, or NKG2D and NKp30 and NKp44. In some embodiments, MAIT cells express high levels of CXCR4 and moderate levels of CCR9, but express low levels of CXCR2 or do not express CXCR2. Alternatively, or in addition, MAIT cells also express CXCR3. In some embodiments, MAIT cells disclosed herein express more than one cytokine receptor such as IL-7R, IL-12R, IL-15R, IL-18R, and IL-21R, or any combination thereof.

[0121] Exogenous Antigen Receptor

[0122] As used herein, the term "exogenous antigen receptor" includes antigen receptors that are not naturally present on MAIT cells. An example of such a receptor is a chimeric antigen receptor (CAR). The MAIT cells disclosed herein are engineered to express exogenous CAR.

[0123] In certain embodiments, the exogenous antigen receptor is permanently integrated into the engineered IVB-derived MAIT cells. In other embodiments, the IVB-derived MAIT cells are engineered to transiently express the exogenous antigen receptor gene. The term "permanent" is used herein to refer to the insertion of exogenous DNA into the genome of the target cell (which can utilize various viral and non-viral techniques generally known in the art). The term "transient" is used to refer to the engineering of cells to temporarily express the exogenous antigen receptor gene, in some embodiments via mRNA insertion into the cell.

[0124] It will be understood by those skilled in the art that the exogenous antigen receptors disclosed herein comprise, in certain embodiments, an antigen binding domain, which causes the MAIT cells comprising the exogenous antigen receptor to bind to a target molecule, i.e., an antigen of interest. As used herein, the terms "antigen" and "target molecule" are used interchangeably and have all the same qualities and meanings.

[0125] Chimeric Antigen Receptor (CAR)

[0126] As is generally known in the art, chimeric antigen receptor (CAR) is a genetically engineered receptor comprising at least an extracellular antigen binding domain, a hinge domain, a transmembrane domain, a co-stimulatory domain, and a cytoplasmic signaling domain. The engineered receptor can be easily inserted into immune cells such as MAIT cells derived from placenta or IVB and expressed by them according to techniques known in the art. Using CAR, a single receptor can be programmed to both recognize specific antigens and activate immune cells to attack and destroy cells carrying the antigen when bound to the antigen. When these antigens are present on tumor cells, immune cells expressing CAR can target and kill tumor cells.

[0127] In some embodiments, in the engineered placental CAR-MAIT cells described herein, CAR comprises an engineered immune receptor containing (a) an extracellular antigen binding domain optionally connected to a hinge region; (b) a transmembrane region; and (c) an intracellular signaling domain (e.g., CD3zeta or CD3ζ). In some embodiments, the extracellular antigen binding domain comprises a single-chain variable 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.

[0128] In some embodiments, the transmembrane domain can be derived from any of the CD4, CD8, or CD3zeta domains, or from a domain of a co-stimulatory molecule such as CD28.

[0129] In some embodiments, the extracellular antigen binding domain comprises an antigen binding fragment of an antibody. In certain embodiments, a costimulatory domain (non-limiting examples of which are 4-1BB / CD137, OX40 / CD134, and CD28) is also present and in some embodiments inserted into the CD3zeta domain. In some embodiments, CD28 and another costimulatory domain such as 4-1BB or OX40 are inserted.

[0130] In some embodiments, the costimulatory domain of the CAR is designed to provide costimulatory signaling to the activation domain, which then activates at least one of the normal effector functions of the immune cell. The effector function of the T cell may include, but is not limited to, cytolytic activity or auxiliary activity including the secretion of cytokines.

[0131] In certain embodiments, suitable costimulatory domains include, but are not limited to, costimulatory domains of 4-1BB / CD137, CD2, DAP12, ICOS, GITR, FcRγ, CD27, CD28, LFA-1, OX-40, or a combination thereof.

[0132] In some embodiments, intracellular signaling or activation domains can also be incorporated into CAR. For example, CD3 is an element of the T cell receptor on naive conventional T cells and has been shown to be an important intracellular activation element in CAR. In some embodiments, CD3 is CD3-zeta or CD3-epsilon.

[0133] Target Antigen

[0134] In certain embodiments, an exogenous antigen receptor, such as a CAR described herein, directs the engineered MAIT cells to recognize tumor antigens, microbial antigens, pathogens, viral antigens, fungal antigens, bacterial antigens, antigens expressed on Treg cells, antigens expressed on alloreactive 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 conditions.

[0135] In some embodiments, the CAR included as part of the engineered IVB MAIT cell guides the engineered MAIT cell to recognize, activate, proliferate, and lyse target cells in response to CAR-driven antigen recognition, such as, but not limited to, tumor-associated antigens (TAA) or viral antigens. In some embodiments, the CAR included as part of the engineered placenta or IVB MAIT cell guides the engineered MAIT cell to recognize, activate, proliferate, and lyse target cells in response to scFv-driven recognition antigens, such as, but not limited to, tumor-associated antigens (TAA) or viral antigens. As used herein, the terms "CAR-MAIT" and "CAR-MAIT cells" cover IVB-derived MAIT cells that have been engineered to express exogenous CARs, and can be used interchangeably, with all the same qualities and meanings.

[0136] As used herein, "viral antigens" include antigens expressed by viral proteins, including situations where the antigen is currently expressed by a virus or cancer cell (e.g., in the case of an oncogenic protein). Thus, in some embodiments, engineered MAIT cells that recognize viral antigens are used to treat viral infections; or in other embodiments, for the treatment of malignant tumors that express viral antigens. It will be understood by those skilled in the art that recognition of such antigens is typically MHC-independent in the case of CARs.

[0137] Non-limiting examples of viral antigens include the following: hexon or penton, e.g., for treatment of adenovirus; HPV E6; HPV E7; immediate early-1 (IE-1) of 65 kilodaltons (pp65) or tegument phosphoprotein; phosphoprotein), for example, for the treatment of cytomegalovirus (CMV); EBV nuclear antigen 1 (EBNA1), BZLF1, or the product of any of the EBV latent genes LMP1, LMP2, EBNA1, EBNA2, EBNA3A, EBNA3B, or EBNA3C, for example, for the treatment of Epstein-Barr virus (EBV) or lymphoma; VP1 or large T, for example, for the treatment of BK virus (BKV); U11, U14, or U90, for example, for the treatment of human herpes virus 6 (HHV-6); herpes simplex virus-1 (HSV-1) thymidine kinase (HSV-TK), for example, for the treatment of HSV-1.

[0138] In certain embodiments, the CAR target includes a tumor-associated antigen (TAA) or a cancer antigen. As used herein, the term "tumor-associated antigen" or "cancer antigen" includes tumor-specific antigens, whether originally derived from a virus or a normal cell genome. It will be understood by those skilled in the art that the recognition of such antigens is generally MHC-independent in the case of CAR.

[0139] Non-limiting examples of suitable cancer antigens include the following: alpha-fetoprotein; desmosome junction protein / AHNAK S2580F ; BCMA (e.g., for relapsed or refractory multiple myeloma); CD7 (e.g., for treatment of T-cell leukemia or lymphoma); CD19 / 20 / 22 (e.g., for treatment of non-Hodgkin's lymphoma, acute lymphoblastic leukemia, B-cell leukemia, or B-cell lymphoma); CD30 (e.g., for treatment of lymphomas such as Hodgkin's lymphoma); CD33; CD38; CD70 (e.g., for treatment of leukemia, lymphoma, or renal cell carcinoma); CD73; CD123 and CD133 (e.g., for treatment of AML); CS1 (e.g., for treatment of multiple myeloma); c-MET; DR5; Diasialoganglioside GD2 or IL13ra2 (e.g., for treatment of neuroblastoma); EGFR VIII; Epstein-Barr virus; (e.g., for treatment of B-cell malignancies); ERBB2 H473Y and ERBB2IP E805G ; HBV surface antigen; HER2 (e.g., for the treatment of glioblastoma); mesothelin (e.g., for the treatment of solid tumors expressing mesothelin); minor H antigen (HA-1); NKG2DL (e.g., for the treatment of solid tumors or colorectal cancer); prostate-specific membrane antigen (PSMA); TPBG (trophoblast glycoprotein) or 5T4 (e.g., for the treatment of solid tumors such as colorectal cancer, ovarian cancer and gastric cancer, and childhood 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.

[0140] In certain embodiments, or additionally, the engineered MAIT cells disclosed herein are used to target tumor cells expressing MR-1.

[0141] 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 certain embodiments, the engineered MAIT cells disclosed herein are used to treat diseases or infections caused by microbes expressing antigens.

[0142] It will be appreciated by those skilled in the art that microorganisms include bacteria, viruses, fungi, and parasites, wherein disease-causing microorganisms are referred to as pathogens. In some embodiments, as used herein, the term "microorganism antigen" may encompass pathogenic targets, i.e., pathogenic antigens.

[0143] In some embodiments, the exogenous CAR targets a bacterial antigen, which in some embodiments is used to treat a bacterial infection. In certain embodiments, the targeted bacteria can be selected from the group consisting of Nitrospira spp., Nitrosospira spp., Nitrobacter spp., Nitrosomonas spp., Clostridium spp., Bacillus spp., methanogenic archaea, coliforms, Salmonella spp., Bacteroides spp., Staphylococcus spp., Streptococcus spp., Neisseria spp., Haemophilus spp., Bordetella spp., Listeria spp.), Mycobacterium spp., Shigella spp., Pseudomonas spp., Brucella spp., Treponema spp., Mycoplasma spp., Yersinia spp., Vibrionaceae spp., Chlamydia spp., Legionella spp., Escherichia spp., Acinetobacter spp., Burkholderia spp., Thiobacillus spp., Rickettsia spp., Sphingomonas spp. spp.), Francisella spp., Campylobacter spp., and Helicobacter spp.

[0144] In some embodiments, the exogenous CAR targets a fungal antigen, which in some embodiments is used to treat a fungal infection. In one embodiment, the fungal infection is a yeast infection.

[0145] In some embodiments, exogenous CAR targets molecules expressed on regulatory T cells (Treg), and such targeting can lead to the exhaustion of Treg. Treg-related molecules are generally known in the art, such as CCR8.

[0146] In some embodiments, exogenous CAR targets molecules expressed on alloreactive T cells. Such targeting will result in selective elimination of alloreactive T cells without exhausting other non-alloreactive T cells. Exhaustion of alloreactive T cells should improve the durability of response after patient treatment by allogeneic products. Alloreactive T cell targets include, but are not limited to, CD70.

[0147] In some embodiments, the exogenous CAR targets a molecule expressed in a subject suffering from a fibrotic disease such as, but not limited to, cardiac fibrosis, wherein the target antigen may encompass fibroblast activation protein (FAB).

[0148] In some embodiments, exogenous CAR targets molecules expressed in subjects with aging-related diseases or conditions. Aging is a chronic "alarm state" in tissues. The range of such diseases (ailment) is huge, and includes debilitating conditions such as chronic inflammation, fibrotic liver disease, atherosclerosis, and diabetes. Examples of molecules expressed by senescent cells include, but are not limited to, urokinase-type plasminogen activator receptors (uPAR), which can be targeted by uPAR-specific CARs. Targeted senescent CAR T cells are referred to as "senolytic CAR T". Disclosed herein is a "senolytic CAR MAIT" comprising an exogenous antigen receptor for molecules expressed on the cell surface targeting senescent cells. In one embodiment, the target of the engineered MAIT cells disclosed herein is uPAR.

[0149] In some embodiments, the exogenous CAR target molecule disclosed herein expressed in a subject undergoing an allogeneic transplant targets an antigen molecule expressed on alloreactive lymphocytes, such as, but not limited to, CD70.

[0150] In some embodiments, the exogenous CAR target molecules disclosed herein expressed in subjects suffering from autoimmune diseases or disorders target antigen molecules related to those diseases or disorders. Examples of autoimmune diseases include, but are not limited to, systemic lupus erythematosus (SLE), pemphigus vulgaris, multiple sclerosis, type I diabetes, rheumatoid arthritis, celiac disease, pernicious anemia, inflammatory myopathy, myasthenia gravis, or adrenalitis, or a combination thereof, including refractory SLE. In some embodiments, the exogenous antigen receptor associated with an autoimmune disease or disorder includes a pathogenic MHC II class related to an autoimmune disorder: a peptide complex as a target peptide-MHCII chimeric antigen receptor (pMHCII-CAR), a chimeric autoantibody receptor (CAAR) such as DSG3 specific CAAR, CD19- and / or CD20-specific CAR, a B cell antibody receptor (BAR). In other embodiments, the exogenous antigen receptor associated with autoimmune diseases and disorders comprises carboxypeptidase H, chromogranin A, glutamate decarboxylase, Imogen-38, insulin, insulinoma antigen-2 and 2β, islet-specific glucose-6-phosphatase catalytic subunit-associated protein (IGRP), proinsulin, citrullinated proteins, collagen II, heat shock proteins, human cartilage glycoprotein, double-stranded DNA, La antigen, nucleosomal histones, ribonucleoproteins (snRNPs), phospholipid-β-2 glycoprotein I complex poly (ADP-ribose) polymerase, Sm antigen of U-1 small nuclear ribonucleoprotein complex, alpha enolase, aquaporin-4, β-arrestin, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein S100-β; R1-type reticulin, gastric H+ / K+-ATPase, 21-hydroxylase, 17-hydroxylase, and cytochrome P450 side-chain cleavage enzyme, or CD19.

[0151] How to use

[0152] Described herein are methods of using the MAIT and engineered MAIT cells described throughout. In some embodiments, the engineered MAIT cells used in the methods disclosed herein comprise engineered placental MAIT cells. In some embodiments, the engineered MAIT cells used in the methods disclosed herein comprise engineered IVB MAIT cells. In some embodiments, the source of the engineered placenta-derived MAIT cells used in the methods disclosed herein is maternal. In some embodiments, the source of the engineered placenta-derived MAIT cells used in the methods disclosed herein is placental blood from the basal decidua region. In some embodiments, the source of the engineered placenta-derived MAIT cells used in the methods disclosed herein is placental blood from the parietal decidua region. In some embodiments, the source of the engineered placenta-derived MAIT cells used in the methods disclosed herein is placental interchorionic blood. In some embodiments, the source of placental MAIT comprises a combination of placental sources described herein. In some embodiments, the source of placental MAIT used in the methods disclosed herein is not umbilical cord blood.

[0153] In some embodiments, the MAIT cells used in the methods disclosed herein comprise placental MAIT cells. In some embodiments, the MAIT cells used in the methods described herein comprise IVB MAIT cells. In some embodiments, the source of the placental-derived MAIT cells used in the methods disclosed herein is maternal. In some embodiments, the source of the placental-derived MAIT cells used in the methods disclosed herein is placental blood from the decidua basalis region. In some embodiments, the source of the placental-derived MAIT cells used in the methods disclosed herein is placental blood from the decidua parietal region. In some embodiments, the source of the placental-derived MAIT cells used in the methods disclosed herein is placental interchorionic blood. In some embodiments, the source of placental MAIT comprises a combination of placental sources described herein. In some embodiments, the source of placental MAIT used in the methods disclosed herein is not umbilical cord blood.

[0154] In some embodiments, the MAIT cells used in the methods disclosed herein are derived from a maternal blood source, a placental source, or an IVB source, or a combination thereof, wherein the MAIT cells may be engineered to express an exogenous antigen receptor or may not be engineered to express an exogenous antigen receptor. In some embodiments, the MAIT cells are derived from a maternal blood source, a placental source, or an IVB source, or a combination thereof; and are not derived from umbilical cord blood, wherein the MAIT cells may be engineered to express an exogenous antigen receptor or may not be engineered to express an exogenous antigen receptor.

[0155] In some embodiments of the methods of use described herein, the engineered MAIT cells comprise CAR-MAIT cells, wherein the MAIT cells are derived from IVB. In some embodiments of the methods of treatment described herein, the methods comprise administering a population of CAR-MAIT cells to a subject in need thereof.

[0156] In some embodiments, a method of treating a subject having a tumor or malignancy is provided, comprising the step of administering to the subject a population of engineered MAIT cells described herein.

[0157] In other embodiments, a composition or pharmaceutical composition comprising a colony of engineered MAIT cells described herein for treating a subject with a tumor or malignancy is provided. It will be understood by those skilled in the art in view of the present disclosure that tumors, malignancies, and hyperproliferative disorders can be treated with a colony of engineered MAIT cells disclosed herein, particularly in the case where the cells of the tumor, malignancy, or hyperproliferative disorder contain (e.g., express, or contain in other embodiments) recognized antigens. In some embodiments of the method for treating a subject with a tumor or malignancy, the engineered MAIT cells are derived from placental intervillous blood. In some embodiments, when treating a subject with a tumor or malignancy, the engineered MAIT cells are allogeneic to the subject. In some embodiments, when treating a subject with a tumor or malignancy, the engineered MAIT cells comprise engineered CARs for cancer antigens or tumor-associated antigens, such as, but not limited to, those disclosed herein.

[0158] In other embodiments, a method for eliminating alloreactive T cells in a subject in need is provided, comprising administering to the subject a population of engineered MAIT cells described herein. In some embodiments, the subject in which alloreactive T cells need to be eliminated includes a subject with an allogeneic transplant. In some embodiments, engineered MAIT cells are targeted to alloreactive T cells due to the expression of an exogenous CAR targeting a molecule expressed on alloreactive T cells. In some embodiments, the subject may have a condition associated with a transplant such as a bone marrow transplant, a solid organ transplant, or any other allogeneic transplant. In some embodiments of the method for eliminating alloreactive T cells in a subject, engineered MAIT cells are derived from placental interchorionic blood. In some embodiments, engineered MAIT cells are allogeneic with the subject. In some embodiments, the target expressed by engineered MAIT cells on alloreactive lymphocytes. In some embodiments, the target expressed on alloreactive lymphocytes is CD70.

[0159] In other embodiments, a method of eliminating regulatory T cells (T regs) is provided as a method of treating cancer in a subject or improving anti-cancer therapy, comprising administering to the subject a population of engineered MAIT cells described herein. In some embodiments, engineered MAIT cells are targeted to Tregs due to the expression of an exogenous CAR targeting a molecule expressed on Tregs. In some embodiments, the targeting molecule expressed on Tregs is CCR8.

[0160] In some embodiments, a method for treating a subject infected with a pathogen is provided, comprising the step of administering to the subject a population of engineered MAIT cells described herein. In other embodiments, a composition or pharmaceutical composition comprising a population of engineered MAIT cells described herein for treating a subject infected with a pathogen is provided. The pathogen may be a bacterial pathogen, a viral pathogen, or a fungal pathogen generally known in the art. It will be understood by those skilled in the art in view of the present disclosure that various infections can be treated with the engineered MAIT cells disclosed herein, particularly in cases where the pathogenic cells contain (e.g., express, or contain in other embodiments) recognized antigens. In some embodiments of the method for treating a subject infected with a pathogen, the engineered MAIT cells are derived from placental intervillous blood. In some embodiments, when a subject infected with a pathogen is treated with the engineered MAIT cells described herein, the engineered MAIT cells are allogeneic to the subject.

[0161] In some embodiments, a method for treating a subject suffering from a fibrotic disease is provided, comprising the step of administering to the subject a population of engineered MAIT cells described herein. In other embodiments, a composition or pharmaceutical composition comprising a population of engineered MAIT cells described herein for treating a subject suffering from a fibrotic disease is provided. The fibrotic disease may be any fibrotic disease generally known in the art, such as, but not limited to, cardiac fibrosis. In some embodiments of the method for treating a fibrotic disease, the engineered MAIT cells target expressed antigens associated with the fibrotic disease. In some embodiments of the method for treating a fibrotic disease, the engineered MAIT cells target fibroblast activation protein (FAP). In some embodiments of the method for treating a subject suffering from a fibrotic disease, the engineered MAIT cells are derived from placental intervillous blood. In some embodiments, when a subject suffering from a fibrotic disease is treated with the engineered MAIT cells described herein, the engineered MAIT cells are allogeneic to the subject.

[0162] In some embodiments, a method for treating a subject suffering from a disease or condition associated with aging is provided, comprising the step of administering to the subject a population of engineered MAIT cells described herein. In other embodiments, a composition or pharmaceutical composition comprising a population of engineered MAIT cells described herein for treating a subject suffering from a disease or condition associated with aging is provided. A disease or condition associated with aging may be any disease or condition associated with aging generally known in the art, such as, but not limited to, chronic inflammation, fibrotic liver disease, atherosclerosis, or diabetes, or a combination thereof. In some embodiments of the method for treating a disease or condition associated with aging, the engineered MAIT cells target an expressed antigen associated with a disease or condition associated with aging. In some embodiments of the method for treating a disease or condition associated with aging, the engineered MAIT cells target urokinase-type plasminogen activator receptor (uPAR). In some embodiments of the method for treating a subject suffering from a disease or condition associated with aging, the engineered MAIT cells are derived from placental intervillous blood. In some embodiments, when a subject suffering from a disease or condition associated with aging is treated with the engineered MAIT cells described herein, the engineered MAIT cells are allogeneic to the subject.

[0163] In some embodiments, a method for treating a subject suffering from an autoimmune disease or condition is provided, comprising administering to the subject a colony of engineered MAIT cells described herein. In other embodiments, a composition or pharmaceutical composition comprising a colony of engineered MAIT cells described herein for treating a subject suffering from an autoimmune disease or condition is provided. The autoimmune disease or condition may be any autoimmune disease or condition commonly known in the art, such as, but not limited to, systemic lupus erythematosus (SLE), refractory SLE, pemphigus vulgaris, multiple sclerosis, type I diabetes, rheumatoid arthritis, celiac disease, pernicious anemia, inflammatory myopathy, myasthenia gravis, or adrenalitis, or a combination thereof. In some embodiments of the method for treating an autoimmune disease or condition, the engineered MAIT cells target an expressed antigen associated with the autoimmune disease or condition.In some embodiments of the method of treating an autoimmune disease or disorder, the engineered MAIT cells target pathogenic MHC class II:peptide complexes associated with the autoimmune disease, autoantibodies, B cell antibodies targeted by the B cell antibody receptor (BAR), carboxypeptidase H, chromogranin A, glutamate decarboxylase, imogen-38, insulin, insulinoma antigen-2 and 2β, islet-specific glucose-6-phosphatase catalytic subunit-associated protein (IGRP), proinsulin, citrullinated proteins, collagen II, heat shock proteins, human cartilage glycoprotein, double-stranded DNA, La antigen, nucleosomal histones and ribonucleoproteins (snRNPs), phospholipid-β- 2 glycoprotein I complex poly (ADP-ribose) polymerase, Sm antigen of U-1 small nuclear glycoprotein complex, alpha enolase, aquaporin-4, beta-arrestin, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein S100-beta, R1-type reticulin, gastric H+ / K+-ATPase, 21-hydroxylase, 17-hydroxylase, cytochrome P450 side chain cleavage enzyme, CD19, wherein the engineered MAIT cells bind to the pathogenic MHC associated with the autoimmune disease Class II: peptide complexes, autoantibodies, B cell antibodies targeted by B cell antibody receptor (BAR), carboxypeptidase H, chromogranin A, glutamate decarboxylase, imogen-38, insulin, insulinoma antigen-2, 2β, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proinsulin, citrullinated proteins, collagen II, heat shock proteins, human cartilage glycoprotein, double-stranded DNA, La antigen, nucleosomal histones, nuclear glyconucleoprotein (snRNP), phospholipid-β-2 glycoprotein I complex poly (ADP-ribose) polymerase, Sm antigen of U-1 small nuclear glyconucleoprotein complex, alpha enolase, aquaporin-4, β-arrestin, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein S100-β, R1-type reticulin, gastric H+ / K+-ATPase, 21-hydroxylase, 17-hydroxylase, cytochrome P450 side chain cleavage enzyme, or CD19. In some embodiments of the method for treating a subject suffering from an autoimmune disease or condition, the engineered MAIT cells are derived from placental intervillous blood. In some embodiments, when a subject suffering from an autoimmune disease or condition is treated with the engineered MAIT cells described herein, the engineered MAIT cells are allogeneic to the subject.

[0164] In some embodiments, methods of treating a subject in need thereof comprise using any of the engineered MAIT cells described herein, including treating a subject having a tumor or malignancy, a subject infected with a pathogen, a subject suffering from a fibrotic disease or condition, a subject suffering from a disease or disorder associated with aging, a subject having an allogeneic transplant, or a subject suffering from an autoimmune disease or disorder.

[0165] In certain embodiments, methods for treating subjects in need include using placental MAIT cells that have not been engineered to express exogenous antigen receptors. In some embodiments, methods for treating subjects infected with pathogens are provided, comprising administering to the subject a composition comprising a population of MAIT cells derived from the placenta, wherein the MAIT cells do not comprise engineered MAIT cells expressing exogenous antigen receptors. In other embodiments, compositions or pharmaceutical compositions comprising a population of MAIT cells described herein for treating subjects infected with pathogens are provided. In certain embodiments, methods for treating subjects infected with pathogens include treating infectious conditions limited to specific sites or organs. In some embodiments, infections limited to specific sites and or organs include postoperative infections. In some embodiments of the method for treating subjects infected with pathogens, the MAIT cells are allogeneic to the subject.

[0166] In some embodiments, the pathogen is a bacterial pathogen, a viral pathogen, or a fungal pathogen. In some embodiments, the bacterial pathogen comprises an antibiotic-resistant bacteria or a bacterial treatment-resistant bacteria. In some embodiments, the bacterial pathogen comprises Mycobacterium tuberculosis bacteria. In some embodiments, the fungal pathogen comprises an invasive Aspergillus genus. In some embodiments, the viral pathogen comprises cytomegalovirus, hepatitis B virus, or hepatitis C virus. In some embodiments of the method for treating a subject infected with a pathogen, the MAIT cells are derived from placental interchorionic blood. In some embodiments, when a subject infected with a pathogen is treated with the MAIT cells described herein, the MAIT cells are allogeneic with the subject.

[0167] Pharmaceutical composition

[0168] In some embodiments, the composition comprises engineered MAIT cells that express an exogenous antigen receptor. In some embodiments, the composition comprises MAIT cells that do not express an exogenous antigen receptor. As used herein, the terms "composition", "cell composition" and "pharmaceutical composition" are used interchangeably in some embodiments and have the same properties and meanings. In some embodiments, disclosed herein are pharmaceutical compositions for treating conditions or diseases as described herein.

[0169] In some embodiments, disclosed herein is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a population of engineered MAIT cells as described herein. In some embodiments, disclosed herein is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a population of MAIT cells as described herein, wherein the population of MAIT cells has not been engineered to express an exogenous antigen receptor.

[0170] The skilled artisan will appreciate that a "pharmaceutical composition" may encompass a preparation of more than one active ingredient described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0171] The skilled artisan will understand that the phrases "physiologically acceptable carrier", "pharmaceutically acceptable carrier", "physiologically acceptable excipient" and "pharmaceutically acceptable excipient" are used interchangeably and can encompass carriers, excipients or diluents that do not cause significant irritation to an organism and do not abrogate the biological activity and properties of the administered active ingredient.

[0172] Techniques for formulating and administering drugs or pharmaceutical compositions are found in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, most recent edition, which is incorporated herein by reference.

[0173] In some embodiments, the compositions or pharmaceutical compositions disclosed herein are injectable compositions, which are manufactured by adding one or more excipients (e.g., stabilizers and aqueous buffers) to a population of engineered MAIT cells described herein. In some embodiments, the compositions or pharmaceutical compositions disclosed herein are injectable compositions, which are manufactured by adding one or more excipients (e.g., stabilizers and aqueous buffers) to a population of MAIT cells that have not been engineered to express an exogenous antigen receptor.

[0174] Alternatively or additionally, prior to administration to a patient, the engineered MAIT cells described herein have been expanded at least 100-fold, in other embodiments at least 200-fold, in other embodiments at least 400-fold, in other embodiments at least 600-fold, in other embodiments at least 1000-fold, in other embodiments at least 1500-fold, in other embodiments at least 2000-fold, in other embodiments at least 3000-fold, and in still other embodiments at least 5000-fold compared to day 0 of expansion.

[0175] In some embodiments, methods of preparing a pharmaceutical composition comprising the engineered MAIT cells described herein include a step of freezing (eg, cryopreserving) the cells prior to or after isolation, incubation, and / or engineering. Examples of suitable cryogens are generally known in the art.

[0176] In other embodiments, for injection, the engineered MAIT cells disclosed herein can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer, optionally in combination with a medium containing a cryopreservative.

[0177] In other embodiments, a pharmaceutical composition is provided, comprising the engineered MAIT cells disclosed herein, wherein the composition is indicated for treating or ameliorating any of the diseases, disorders and complications mentioned herein, each of which represents a separate embodiment.

[0178] In various embodiments, one can administer the pharmaceutical compositions disclosed herein in a systemic manner as is known in the art. Alternatively, one can administer the pharmaceutical composition locally, for example, via direct injection of the pharmaceutical composition into a tissue region of the patient, such as atrophied muscle in a non-limiting embodiment. In other embodiments, the engineered MAIT cells are administered intramuscularly, intravenously, subcutaneously, or intraperitoneally, each of which is considered to be a separate embodiment. In this regard, "intramuscular" administration includes administration into the muscle tissue of the subject; "subcutaneous" refers to administration just below the skin; and "intravenous" includes administration into the vein of the subject. In some embodiments, the pharmaceutical composition is administered intralymphatically as previously described in the art.

[0179] The effective dosage and schedule for administration of the composition can be determined empirically, and making such determinations is within the skill of the art. If any contraindications occur, the dosage can be adjusted by the individual physician.

[0180] Depending on the severity and responsiveness of the condition to be treated, dosing may be a single administration, or, in other embodiments, multiple administrations, until a relief of the disease state is achieved.

[0181] In certain embodiments, the treatment methods described herein further include lymphodepleting the recipient prior to treatment, or immunosuppression in other embodiments. In other embodiments, reversible lymphodepleting or immunosuppression prolongs the biological half-life of the transplanted cells. Methods for lymphodepleting and immunosuppression of patients are known in the art. In some embodiments, lymphodepleting or immunosuppression is not necessary for the administration of the engineered MAIT cells disclosed herein, at least in part because of their low immunogenicity.

[0182] In various embodiments, the engineered MAIT cells do not need to be implanted into a host for the cells to exert the therapeutic effect, each of which is considered a separate embodiment. In other embodiments, implantation of the engineered MAIT cells is required to exert the effect.

[0183] In certain 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.

[0184] Also disclosed herein are kits and articles of manufacture that relate to reagents useful for practicing the methods disclosed herein. Kits and articles of manufacture may include any reagent or combination of reagents discussed herein or that will be understood to be necessary or beneficial in practicing the disclosed methods, including lymphocytes. In another aspect, kits and articles of manufacture comprise labels, instructions, and packaging materials, for example, for treating the disorders or therapeutic indications mentioned herein.

[0185] It will be appreciated that certain features of the engineered MAIT cells and methods of use thereof described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the engineered MAIT cells and methods of use thereof described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination, or as appropriate in any other described embodiment of the engineered MAIT cells and methods of use thereof.

[0186] Additional Materials and Methods

[0187] IVB Collection

[0188] Placenta donors were recruited after cesarean section at various medical centers in Israel, and written informed consent was obtained from the maternal patients. The umbilical cord was clamped with two clamps to prevent mixing of fetal and maternal blood. The placenta was placed on a large flat plate with the fetal side facing down, and the amnion was removed to expose the chorionic lobes. A small X-shaped incision was made only on the upper part of the chorionic lobes with surgical scissors. The placenta was then turned over, with the fetal side facing up, and PBS was injected into the basal decidua at different points avoiding fetal blood vessels. All maternal venous blood (IVB) flowing out of the placenta was collected. Mononuclear cells were then separated using Lymphoprep (density gradient column), and the intermediate cell stock was subsequently frozen.

[0189] RNAseq and Legend Screening

[0190] Blood and intervillous blood (IVB) donors: PBMCs were obtained from healthy donors recruited from the transfusion laboratory of Sheba Medical Center (Sheba Medical Center, Tel-Hashomer, Israel) in Ramat Gan, Tel Aviv, Israel. IVB samples were harvested from human term placentas collected from healthy individuals (from Bnai Zion Medical Center, Haifa, Israel) who were delivered by planned elective cesarean section after healthy pregnancies. Written informed consent was obtained from the patients.

[0191] MAIT cell and PBMC treatment

[0192] PBMCs and IVB human mononuclear cells (MNCs) were isolated by Lymphoprep density gradient centrifugation. Cells were frozen in 90% FBS + 10% DMSO.

[0193] MAIT cell processing for RNAseq

[0194] Cryopreserved PBMC / IVB MNC were thawed and cultured at 2×10 in 24-well plates using complete Optimizer CTS medium (Gibco) supplemented with Optimizer CTS T cell expansion supplement (Gibco), 2.5% CTS immune cell SR (Gibco), 2 mM Glutamax 100x (Gibco), 100 U / mL Pen / Strep 100x (Gibco), IL-15 (50 ng / mL, Peprotech), and 250 nM 5-OP-RU [prepared by mixing 5-A-RU (Cayman chemical) and methylglyoxal (Merck)]. 6 Every 2-3 days, the cells were cultured with fresh medium containing 50 ng / ml IL-15 at a concentration of 0.5-1 x 10 6 The expanded cells were re-cultured at a concentration of 100 μg / ml to replenish the culture medium. On day 7, the expanded cells were MACS-sorted to enrich for MAIT cells via biotin-conjugated anti-TCR Vα7.2 REafinity antibody (Miltenyi biotech) and anti-biotin microbeads (Miltenyi biotech) labeling.

[0195] PBMC treatment

[0196] PBMCs were obtained from healthy donors recruited at the Transfusion Laboratory of Sheba Medical Center, Ramat Gan, Tel Aviv, Israel. Written informed consent was obtained from the patients. PBMCs were isolated by Lymphoprep density gradient centrifugation, cryopreserved in CryoStor CS10 (BioLife Solutions) and stored in liquid nitrogen vapor phase.

[0197] CAR-T preparation

[0198] Thawed PBMCs were cultured at 1×10 in Optimizer CTS medium (Gibco) supplemented with Optimizer CTS T cell expansion supplement (Gibco), 2 mM Glutamax 100x (Gibco), 100 U / mL Pen / Strep 100x (Gibco), IL-2IS premium grade (Miltenyi Biotech) and supplemented with TransAct anti-CD3 / anti-CD28 polymer nanomatrix (Miltenyi Biotech). 6 / ml density overnight culture. The next day (day 1), LV MSLN-Flag CAR vector (VectorBuilder) was used for transduction at an MOI of 1:10 (for transduced cultures), or no vector was used for transduction as a non-transduced control (NTD control). Transduction enhancers (such as Retronectin or Polybrene) were not used. On day 4, cells were transferred to 24 or 6-well G-Rex cultures (Wilson Wolf, MN, USA) containing 1% HI human AB serum (IMBH), and IL-2IS premium products were added to the cultures every other day. The cultures were harvested on days 12-13. Samples were pulled on days 8 and 12-13 for cell fold expansion calculation and for MSLN-CAR expression testing by CytoFLEX V5-B5-R3 flow cytometer (Beckman) using Whitlow / 218 linker (E3U7Q) rabbit mAb (Cellsignaling technology).

[0199] CAR-MAIT preparation

[0200] Frozen leukocytes from IVB sources were thawed and cultured overnight in OpTmizer CTS medium (Gibco) complete medium containing fetal bovine serum (FBS) and IL-15. The next day, cells were activated for 48 hours using 5-OP-RU (a known activator of MAIT cells). On the 3rd day, cells were counted and OpTmizer CTS medium was updated. On the 5th day, cells were collected and separated using LS columns with antibodies against Vα7.2 and magnetic microbeads (Miltenyi Biotech). The pure MAIT cell fraction (after separation) was incubated for 3 hours and then transduced with LV MSLN-Flag CAR vector (VectorBuilder) at an MOI of 1:10. Transduction was performed on a plate covered with Retronectin (Takara), which was inoculated with virus rotation for 2 hours and incubated overnight with pure MAIT cells. Virus removal was performed by 4 washes, followed by pure MAIT cell counting and culture until days 10-12. Complete OpTmizer medium enriched with IL-15 was added to the cultures every other day.

[0201] Cell surface staining – flow cytometry

[0202] Cells were plated in 96-well microplates and washed with staining buffer containing FBS and EDTA. Cells were then stained with a cocktail of fluorescently labeled antibodies against CD3, TCR Va7.2, and CD161 for detection of MAIT cells and additional antibodies as indicated. Cells were incubated at 4°C for 20 minutes, washed, and resuspended in staining buffer containing 7-AAD viability dye. Stained cells were analyzed on a CytoFlex flow cytometer; results were then analyzed on FlowJo or Kaluza software.

[0203] Intracellular staining – flow cytometry

[0204] Cells were placed in T cell culture medium in 96-well microplates, supplemented with phorbol 12-myristate 13-acetate (PMA) and ionomycin to induce T cell activation, and supplemented with brefeldin A and monensin (BD) to inhibit protein secretion. Cells were incubated at 37°C for 4 hours, then washed with staining buffer and stained with a fixable viability dye, followed by staining with cell surface antibodies as described above. Cells were then fixed and permeabilized with Cytofix / Cytoperm solution (BD) at room temperature for 30 minutes, washed with Perm / Wash buffer (BD), and stained for 30 minutes at 4°C in Perm / Wash buffer containing fluorescently labeled antibodies to intracellular proteins (IFNγ, TNFα, perforin, and granzyme B). Cells were washed and analyzed as described above.

[0205] Luciferase killing assay

[0206] Luc-labeled target cells were inoculated in 96-well plates (1.0E4 per well) and CAR-T or CAR-MAIT cells were added 4 hours later in duplicate. After 18 hours of incubation, luciferase activity was determined using the Bio-Glo luciferase assay system (Promega). Viability was normalized to the maximum RLU signal of a given effector. EC50 was estimated using GraphPad Prism (Sigmoidal (4PL)).

[0207] Example 1

[0208] Isolation, enrichment and expansion of MAIT cells from intervillous blood (IVB)

[0209] The placenta (100) is an organ that includes a fetal side (101) and a maternal side (104) covered with an amnion (105), wherein the fetal side (101) includes the umbilical cord (102) connecting the fetus to the mother. To isolate MAIT cells from IVB, the umbilical cord is first clamped, for example by using a clamp (103) to prevent mixing of fetal blood with maternal (IVB) blood. IVB collection begins with the removal of the amnion surrounding the maternal side, exposing the chorionic villus (106). A superficial cut is made in the chorionic villus using scissors or any other suitable cutting tool. The placenta is then turned over so that the fetal side is facing up, and phosphate-buffered saline (PBS) is injected into the decidua basalis, avoiding fetal blood vessels. All maternal blood that flows out of the placenta is collected. Mononuclear cells are then isolated from the collected maternal blood using Lymphoprep (density gradient column), followed by freezing of the intermediate cell stock ( Figures 1A-1D ).

[0210] Figure 2AFigure 1 presents flow cytometry graphs of activation and proliferation of MAIT cells in a packed bed bioreactor over 10 days according to an embodiment of the present invention. Mononuclear cells from intervillous blood (IVB) were seeded into ECM-coated The MAIT cells were cultured in a packed bed bioreactor containing carriers, which simulates the natural environment and promotes the adhesion of antigen presenting cells (APCs). MAIT cell activation was induced by 5-OP-RU (250 ng / ml) and IL-15 (50 ng / ml). The activation and proliferation of MAIT cells were evaluated at the following time points: days 0, 5, 7 and 0. The MAIT cell population was detected by the expression of CD3, Vα7.2 and CD161 markers. The results showed an increase in the proportion of MAIT cells, starting at 22.63% on day 0 and reaching 96.26% on day 10. In addition, the expression of activation markers CD69 and CD25 increased from day 0 to day 7 and decreased slightly on day 10.

[0211] Figure 2B Flow cytometry graphs of MAIT cell growth after switching cells from a first bioreactor to a second bioreactor on day 10 and growing for an additional 7 days in the second bioreactor are presented in accordance with an embodiment of the present invention. Cells that reached maximum growth capacity on day 10 in the first bioreactor were harvested, and approximately 30% of these cells were then inoculated into a second bioreactor that was similar in design to the first bioreactor. The cells were grown for an additional 7 days. Flow cytometry results demonstrated that the MAIT percentages were similar during most of the time, but presented a decrease from >90% on day 14 to 82% on day 17. CD69 marker expression was upregulated from 30% to 87% on day 17, indicating that MAIT cells maintained their activation signals, while CD25 gradually decreased as expected, indicating that MAIT cells were activated a short time ago, but did not reach their full proliferation and functional state, suggesting their ability to be reactivated and function in the event of a future target encounter.

[0212] From the results, it can be seen that MAIT cells are generally more abundant in placental intervillous blood (IVB) mononuclear cells than in peripheral blood mononuclear cells (PBMCs).

[0213] Figure 3The results of placental IVB MAIT cell activation and expansion according to some optional embodiments of the present invention are shown. In order to obtain a population rich in MAIT cells, IVB-derived mononuclear cells were seeded in tissue culture plates, and MAIT cell activation was induced by 5-OP-RU (250ng / ml) and in the presence of IL-15 (50ng / ml). On days 3 and 5 after activation, the cells were counted and the MAIT percentage was analyzed by flow cytometry, and fresh culture medium and IL-15 were added. After 7 days of culture, MAIT cells accounted for between 78% and 93% of the cells in the CD3+ population, depending on the donor. On day 7, the cells were separated with magnetic beads and anti-TCR Va7.2 antibodies to further enrich the MAIT population. The cells were cultured for another 3 days in the presence of IL-15.

[0214] The results show the percentage increase of MAIT and the fold expansion of MAIT (the increase in the absolute number of MAIT relative to the starting number) during the entire culture period.

[0215] Figure 4A-4B CD3 + Comparison of the percentage of MAIT cells in gated placental intervillous blood (IVB) mononuclear cells and peripheral blood mononuclear cells (PBMCs) was performed. p=0.0159.

[0216] As shown, although the MAIT% varied significantly between donors in both peripheral blood and placenta, on average there were significantly more MAIT cells among the CD3+ cells in IVB compared to peripheral blood.

[0217] Example 2

[0218] Comparison of immunophenotypes between placental IVB MAIT cells and peripheral blood MAIT cells

[0219] The phenotype of MAIT cells from placental IVB and from peripheral blood was analyzed using CD45RA and CD62L markers. Figure 5 Shown is a comparison of immunophenotyping of placental IVB MAIT cells collected from 6 different donors and analyzed on day 0 with PB MAIT cells.

[0220] Cells were stained according to a standard cell surface marker staining protocol and analyzed by flow cytometry. Cell surface markers of CD45RA and CD62L were used to characterize the cells. As shown. Placental IVB MAIT cells have statistically significantly more "effect memory" cells and fewer "terminal differentiation" cells compared to PB-MAIT cells. The gating strategy for phenotyping is listed in the figure on the right. Statistical analysis was performed using GP Prism, t-test comparison. *p<0.05, **p<0.01, ***p<0.001.

[0221] Notably, IVB-derived MAIT cells have a significantly higher proportion of effector memory cells than peripheral blood-derived MAIT cells. Thus, they have fewer terminally differentiated effector cells than peripheral blood-derived MAIT cells. Effector memory cells may be a preferred phenotype for engineered MAIT cells because they live longer than terminal effectors and have better effector function and the ability to migrate to peripheral tissues compared to naive and central memory cells.

[0222] Example 3

[0223] Comparative analysis of chemokine receptor expression in placental IVB MAIT and peripheral blood MAIT cells

[0224] Chemokine receptors mediate the migration of engineered MAIT cells to peripheral tissues and to secondary lymphoid organs; they are also important for the infiltration of engineered MAIT cells into tumors.The expression of chemokine receptors on MAIT cells from IVB relative to MAIT from peripheral blood was analyzed.

[0225] Figure 6A-6B Chemokine receptor analysis of placental IVB MAIT cells and PB MAIT cells according to an embodiment of the present invention is shown. To evaluate the chemokine receptor profile of the MAIT population, IVB MAIT cells and PB MAIT cells were collected from six (6) different donors and analyzed for chemokine receptor expression on day zero (0). Cell surface markers of CCR6, CCR5, CXCR4 and CXCR6 were used to characterize the cells. Cells were stained according to standard cell surface marker staining protocols and analyzed by flow cytometry. Statistical analysis was performed using GP Prism, t-test comparisons. *p<0.05, **p<0.01, ***p<0.001.

[0226] like Fig. 6A As observed in , IVB MAIT cells have higher expression of CCR5 and CCR6 chemokine receptors compared with PB MAIT cells. Figure 6BIt was shown that IVB MAIT cells had statistically significantly lower levels of CXCR4 and comparable levels of CXCR6 compared to PB MAIT cells.

[0227] The results further illustrate that the two populations of MAIT cells, IVB-derived and PB-derived, are distinct and that IVB MAIT cells contain more cells of the "effector memory" phenotype and fewer "terminally differentiated effector" cells compared to PB MAIT cells.

[0228] Example 4

[0229] Comparison of Granzyme B and Perforin Expression in Placental IVB MAIT and Peripheral Blood MAIT in Thawed Conditions

[0230] Perforin and granzyme B are molecules essential for the cytotoxic function of effector lymphocytes such as MAIT cells. Perforin creates pores in target cells, while granzyme B enters target cells through these pores and induces apoptosis. In comparative analysis, it was found that IVB MAIT cells expressed significantly higher levels of both molecules compared to peripheral blood MAIT cells under stimulation with PMA / ionomycin. Fig. 7A Representative samples of both cell types are shown, while Figure 7B MAIT cells from placenta IVB and from peripheral blood are shown.

[0231] Cells from each of the six donors were stimulated with PMA / ionomycin for 4 hours and analyzed for intracellular expression of granzyme B and perforin. As shown, placental IVB MAIT cells had statistically significantly higher levels of granzyme B compared to peripheral blood MAIT cells, indicating increased efficacy and lytic capacity of placental IVB MAIT. Cells were gated on MAIT cells. p=0.0001.

[0232] Example 5

[0233] Comparison of intervillous blood (IVB) MAIT and cord blood (CB) MAIT

[0234] MAIT cells were collected from intervillous blood (IVB) and umbilical cord blood (CB) for comparison of frequency and immunophenotype between the two samples by flow cytometry.

[0235] Fig. 8A -8B presents flow cytometry graphs of the percentage of MAIT cells in placental intervillous blood (IVB) and matched (same donor) CB, and a table summarizing the results obtained in the flow cytometry graphs.

[0236] Cells from 3 different donors were used as previously described. IVB cells and CB cells were activated similarly. As shown, CB had very few MAIT cells at day 0 compared to IVB, and their percentage decreased over time at day 6 / 7. In more detail, IVB MAIT and matched CB MAIT cells were collected according to the scheme described in the following materials and methods. Cells were stained for CD161 and TCR Va7.2 at day 0 and at day 6 / 7, which represents the MAIT positive gating strategy. The square gate represents the percentage of MAIT cells. The table describes the actual cell count. As observed, intervillous blood (IVB) has more MAIT cells than cord blood (CB).

[0237] Fig. 9 Flow cytometry graphs showing the expression of CD8αα+ by placental IVB MAIT cells and CB MAIT cells according to an embodiment of the present invention. Our results show that placental IVB MAIT cells express a high frequency of CD8αα+ at a 3-fold higher frequency (41.2%) compared to CB MAIT cells (14.2%), indicating a more "antigen experienced" phenotype compared to the immature / naive state present in CB MAIT cells.

[0238] CD8α is a cell surface glycoprotein that can be expressed as a disulfide-linked heterodimer or as a homodimer with CD8β. In contrast to CD8αβ, CD8αα is never expressed on naive T cells, but is easily induced on strongly activated T cell receptors (TCR). In addition, in a manner similar to the expression of KIR or other NK cell-related receptors on T, CD8αα behaves like other "tuning" molecules to help survive. MAIT cells from IVB and CB were stained with two CD8 subunits (CD8α and CD8β), and the frequency was measured by flow cytometry. The results clearly show that IVB MAIT cells have a higher frequency of CD8αα, indicating a more "antigen-experienced" phenotype compared to the naive state present in CB MAIT cells.

[0239] Fig.10 Flow cytometry plots showing the effector memory phenotype of placental IVB MAIT cells and CBMAIT cells using CD45RO, CD27, CD45RA, CCR7 and CD62L.

[0240] IVB MAIT and matched CB MAIT were collected according to the methods described in Materials and Methods below. Cell surface markers of CD45RA, CD27, CCR7, CD62L, and CD45RO were used to determine cell immunophenotyping, with CD45RA - / CCR7 -、CD45RA - / CD62L - 、CD45RA - / CD45RO + and CD27 - / CD45RO - Represent effector memory cells, activated effector memory cells and effector memory phenotype, respectively. As clearly shown in the flow cytometry graph, IVB MAIT cells lag behind CB MAIT cells, as IVB MAIT cells have an effector memory phenotype while CB MAIT cells present a naive phenotype.

[0241] In summary, comparison of the percentage of MAIT cells in CB or IVB showed that the frequency of MAIT cells in CB was significantly lower than that in IVB. In addition, the lower frequency of MAIT cells expressing CD8αα in CB suggests that CB MAIT cells are in an immature state relative to MAIT cells in IVB.

[0242] Example 6

[0243] Transcriptome analysis of IVB MAIT cells and PB MAIT cells

[0244] Transcriptome analysis of IVB MAIT cells and PB MAIT cells was performed, which showed a set of top 50 significantly expressed genes ( Fig.11 , the names of genes are listed in the figure). To understand the transcriptome differences between MAIT cells derived from different tissues (i.e., PBMC and IVB), cells were extracted and amplified as previously described, and RNAseq analysis was performed. IVB and PB MAIT cells were collected from three (3) different donors and analyzed on the seventh (7) day for RNAseq transcriptome analysis. Cells were harvested as described in the methods. Heat map of protein coding genes that were significantly (|log2FC|>1) and BH padj<0.01) differentially expressed between IVB and PB cell types. Pearson correlation calculations were applied to the normalized values ​​of the count data for clustering. The color scheme represents z scores.

[0245] As shown in the results, MAITs originating from IVB possess a unique and specific transcriptome landscape compared with peripheral blood MAIT cells, implying that the two MAIT populations are highly distinct from each other.

[0246] Example 7

[0247] Comparison of granzyme B and perforin expression in placental IVB MAIT cells and peripheral blood MAIT cells after expansion

[0248] Granzyme B is a serine protease found in granules of lytic cells. It is secreted together with the pore-forming protein perforin to mediate apoptosis of target cells. A comparison of granzyme B secretion between IVB MAIT cells and PB MAIT cells is shown in Fig.12 .

[0249] Placental IVB MAIT cells and PBMAIT cells were collected from each of the four donors and expanded using standard protocols as previously described in Methods. On day 8, cells were activated with Phorbol Myristate Acetate (PMA) plus ionomycin and analyzed for granzyme B and perforin secretion using standard intracellular staining protocols.

[0250] To evaluate the basal potential of MAIT cells originating from IVB or PB, harvested cells were stimulated with PMA and ionomycin and stained for intracellular granzyme B secretion.

[0251] As shown, stimulated IVB MAIT cells produced significantly higher levels of granzyme B compared to PB MAIT cells, indicating increased efficacy and lytic capacity of cells derived from IVB. Statistical analysis was performed using GP Prism, t-test comparisons. *p<0.05, **p<0.01, ***p<0.001.

[0252] Example 8

[0253] Efficacy of placenta-derived CAR-MAIT cells

[0254] The functional killing characteristics of placental IVB CAR-MAIT cells were analyzed and the results are shown in Fig.13 For this purpose, CAR-MAIT cells were generated according to the standard protocol described in Methods and co-cultured with NSCLC targets overexpressing MSLN and luciferase. Killing was measured by luminescence readout.

[0255] As shown in the figure, different effectors have different levels of CAR + Fraction. Co-cultured with target cells in the presence of CAR + All different culture conditions of CAR-MAIT cells resulted in robust efficacy against target cells, indicating a high and dose-dependent efficacy of placental CAR-MAIT cells.

[0256] Example 9

[0257] Intervillous blood (IVB) MAIT cells, peripheral blood (PB) T cells, and umbilical cord blood from matched donors

[0258] Comparison between (CB)T cells

[0259] Comparisons between IVB MAIT cells, peripheral blood (PB) maternal T cells, and cord blood (CB) T cells from the same donor (matched blood) were performed to evaluate differences between MAIT cells and T cells and to eliminate donor-to-donor variability. The inventors evaluated functional differences in different cells originating from the same donor. IVB, PBMC, and CB were collected as described in the methods and activated with T cell activators on day 0. Following an intracellular staining protocol, cells were analyzed for perforin, granzyme B, IFN-γ, and TNF-α using flow cytometry. Fig.14A is a flow cytometry plot depicting raw data for perforin and granzyme B, and Fig. 14B are flow cytometry graphs depicting raw data for IFN-γ and TNF-α.

[0260] As shown in the results, IVB MAIT cells at day 0 have completely different expression levels of effector molecules compared to conventional T cells, while T cells originating from umbilical cord blood and peripheral blood have similar expression levels of these effector molecules. A graphical summary of the results is shown in Figure 15. In this figure, the percentage of cells from the MAIT population of IVB, from peripheral maternal blood (MB) and the conventional T cell population of CB (cultured using the T cell standard activation protocol) is shown. Figures 14A-14B The gating strategy shown was used for analysis. The examples are for two donors, from each of which IVB, peripheral maternal blood (MB) and umbilical cord blood (CB) were obtained.

[0261] As shown in the results from two independent donors, IVB MAIT cells at day 0 possessed an increased percentage of perforin compared to conventional T cells. + Granzyme B + , perforin + + Granzyme B + (double positive), IFNg + TNFa + IFNg + +TNFa + (Double positive).

[0262] Example 10

[0263] Analysis of placental IVB MAIT and placental IVB T effector function

[0264] On day 0, matched IVB, PB and CB mononuclear cells from the same donor were thawed, activated with T cell activators and cultured according to standard protocols. Briefly, T cell expansion was used by activating TCR with CD28 and CD3 antibodies, or beads (TransAct) and IL2 cytokine.

[0265] On day 10, cells were stained for CD161 and TCR Vα7.2 to evaluate the fraction of the MAIT-positive population. As observed, the specific MAIT expansion protocol resulted in highly enriched >95% MAIT cells, whereas the standard T cell expansion protocol resulted in a negligible MAIT population <1.2%.

[0266] Figures 16A-16B is a graphic illustration depicting a comparison of IVB-MAIT and IVB-T effector functions. IVB-MAIT was cultured as described in the methods. From the same starting material (IVB cell harvest), the inventors also cultured T cells using a standard conventional T cell protocol with TransAct and IL2. On day 10, IVB-MAIT cells and IVB-T cells were stimulated with PMA+ionomycin or TransAct, stained for effector molecules such as IFN-γ, TNF-α, perforin and granzyme B and analyzed by flow cytometry ( Fig.16A ). A graphical summary of the results is shown in Fig. 16B In the present study, we showed that MAIT cells had higher levels of perforin and TNF-α compared with T cells originating from the same source (i.e., placental IVB), indicating that placental IVB MAIT cells have higher effector function compared with placental IVB T cells.

[0267] Embodiment 11

[0268] Placental MAIT and placental CAR-MAIT immunophenotypes

[0269] On day 10, the immunophenotype of placenta-derived MAIT cells and CAR-MAIT cells was evaluated by flow cytometry using a standard cell surface marker protocol, staining with CD45RA and CCR7. Figures 17A-17B As shown in . Leukocytes were isolated from venous blood and decidua basalis of the placenta, activated with 5-OP-RU and expanded until day 5. After isolation of MAIT cells, they were transduced with lentivirus and expanded until day 10.

[0270] Both MAIT cells and engineered CAR-MAIT cells were expanded for 10 days, stained and analyzed via flow cytometry. The gating strategy of CD45RA relative to CCR7 showed a statistically significant increase in "effect memory (EM)" compared to "central memory (CM)" cells. There were almost no naive and terminally differentiated (TD) cells. The results representing the average of 3 different donors analyzed on day 10 ( Fig. 17C ). The results showed that more than 80% of both MAIT and CAR-MAIT cells were effector memory phenotypes, while the remaining cells were central memory phenotypes. Naive and terminally differentiated cells were less than 1%. CD62L staining showed high or low expression. CD69 staining showed continued increased expression after MAIT activation and throughout the expansion period.

[0271] Example 12

[0272] Antibacterial activity of placental MAIT cells – Inhibition of bacterial growth by placental MAIT secretions

[0273] IVB-derived MAIT 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 culture medium containing the MAIT secretome was collected and mixed with live antibiotic-resistant or antibiotic-sensitive bacteria (Gram-negative, such as carbapenem-resistant Escherichia coli or KPC-producing Klebsiella pneumoniae, or Gram-positive, such as methicillin-resistant Staphylococcus aureus, beta-lactam-resistant Streptococcus pneumoniae, or multi-drug-resistant Mycobacterium tuberculosis). After incubation of the bacteria with the collected culture medium for different time points (from a few minutes to 24 hours) in the presence or absence of antibiotics, the bacteria were diluted and plated on agar plates. After 24 hours of plating on agar plates, bacterial colony forming units (CFU) were counted. The number of colonies was compared to the number of colonies grown after incubation of the cells with fresh antibiotic-free medium for 24 h in the presence or absence of antibiotics.

[0274] Example 13

[0275] Antibacterial activity of placental MAIT – killing of cells infected with antibiotic-resistant bacteria

[0276] Infect MR-1 expressing cells (monocytes, macrophages, B cells, HeLa cells, etc.) with live bacteria (Gram-negative, such as carbapenem-resistant E. coli or KPC-producing Klebsiella pneumoniae, or Gram-positive, such as methicillin-resistant Staphylococcus aureus, β-lactam-resistant Streptococcus pneumoniae, or multidrug-resistant Mycobacterium tuberculosis) in antibiotic-free medium for 3 hours. Wash the infected cells and incubate with antibiotics for 1 hour to kill extracellular bacteria. IVB-derived MAIT cells were obtained from placenta and stained with fluorescent dyes (cell proliferation dye eFluor TM 450, etc.) and added to bacterially infected cells at different effector to target ratios for 3 hours and Caspase-3 / 7 detection reagent (CellEvent TM Caspase-3 / 7 detection reagent, etc.). After incubation, the target cells were collected and analyzed by flow cytometry for activated caspase-3 / 7 and compared with the percentage of activated caspase-3 / 7 in target cells infected with bacteria alone. To evaluate the killing of intracellular bacteria, cell lysates of target cells infected with bacteria after co-culture with IVB-derived MAIT were diluted and plated on agar plates. Bacterial CFU were counted 24 hours after plating on agar plates. The number of colonies was compared with the number of colonies grown after incubating cell lysates of target cells infected with bacteria alone for 24 hours.

Claims

1. A cell composition comprising a population of engineered mucosal-associated invariant T (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.

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

4. The cell composition according to any one of the preceding claims, wherein at least 90% of the placental MAIT cells are characterized by the expression of TCRVα7.2+ and high levels of CD161 (CD161 高 ).

5. The cell composition according to any one of the preceding claims, comprising 10% of the engineered placental CAR-MAIT cell population. 9 Up to 10 11 living cells.

6. A cell composition according to any of the preceding claims, wherein the exogenous CAR expressed by placental MAIT cells recognizes a tumor antigen.

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

8. The cell composition of 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, mesothelin, Claudin 3, Claudin 4, Claudin 6, Claudin 18.2, ROR1, ROR3, Muc1 or Muc16.

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

10. A cell composition according to any of the preceding claims, wherein the exogenous CAR expressed by placental MAIT cells recognizes a target molecule expressed on regulatory T cells. The cell composition according to claim 10 , wherein the target molecule is CCR8.

12. A cell composition according to any of the preceding claims, wherein the exogenous CAR expressed by placental MAIT cells specifically recognizes a target molecule expressed on alloreactive lymphocytes. The cell composition according to claim 12 , wherein the target molecule is CD70.

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

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

16. A cell composition according to any of the preceding claims, wherein the exogenous CAR expressed by placental MAIT cells recognizes a target molecule expressed on cells associated with a fibrotic disease or condition. The cell composition according to claim 16 , wherein the target molecule is fibroblast activation protein (FAP).

18. A cellular composition according to any of the preceding claims, wherein the exogenous CAR expressed by placental MAIT cells recognizes a target molecule expressed on cells associated with an autoimmune disease or disorder.

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

20. A cellular composition according to any of the preceding claims, wherein the exogenous chimeric antigen receptor (CAR) expressed by placental MAIT cells comprises: an antigen binding domain configured to specifically recognize a tumor antigen; a transmembrane domain that anchors the CAR in the cell membrane; at least one co-stimulatory domain that enhances cell signaling and T cell activation; and an intracellular signaling domain responsible for initiating T cell activation in the presence of antigen recognition.

21. The cell composition of claim 20, wherein the antigen binding domain of the chimeric antigen receptor (CAR) may comprise any one of the following: a single-chain antibody, a single-chain variable fragment (scFv), a VHH fragment, each of which is configured to recognize a specified tumor antigen.

22. The cell composition of 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, mesothelin, Claudin 3, Claudin 4, Claudin 6, Claudin 18.2, ROR1, ROR3, Muc1, or Muc16.

23. The cell composition of 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) or 4-1BB (CD137).

24. The cell composition of claim 20, wherein the intracellular signaling domain of the CAR comprises an intracellular signaling domain of CD3 zeta, FcR gamma, or a functional fragment thereof, each configured to initiate or maintain T cell activation in the context of antigen recognition.

25. A cellular composition according to any of the preceding claims, wherein 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 or a tricistronic construct; wherein at least one additional element in the bicistronic or tricistronic construct is selected from the group consisting of: a cytokine, a constitutively active tyrosine kinase, wherein the cytokine is unmodified or modified, and / or secreted, conditionally active, constitutively active, or expressed in a membrane-bound form.

26. A cellular composition according to any of the preceding claims, wherein the pre-expansion population of placental MAIT cells exhibits a higher percentage of cells expressing low levels of CD45RA and low levels of CD62L compared to the pre-expansion population of MAIT cells derived from peripheral blood, indicating an effector memory phenotype of placental MAIT cells.

27. A cellular composition according to any of the preceding claims, wherein the pre-expansion population of placental MAIT cells exhibits a higher percentage of cells expressing CCR5 and / or CCR6 chemokine receptors and a lower percentage of cells expressing CXCR4 compared to a pre-expansion population of MAIT cells derived from peripheral blood.

28. A cellular composition according to any of the preceding claims, wherein the population of placental MAIT cells exhibits a higher percentage of cells expressing granzyme B compared to a population of MAIT cells derived from peripheral blood, indicating enhanced effector and lytic function of placental MAIT cells.

29. A cellular composition according to any of the preceding claims, wherein the pre-expansion population of placental MAIT cells exhibits a higher percentage of cells expressing perforin compared to the pre-expansion population of MAIT cells derived from peripheral blood, indicating enhanced effector and lytic function of placental MAIT cells.

30. A cellular composition according to any of the preceding claims, wherein the pre-expansion population of placental MAIT cells exhibits a higher percentage of cells expressing perforin and / or granzyme B compared to the pre-expansion population of conventional T cells derived from peripheral blood, indicating enhanced effector and lytic functions of placental MAIT cells.

31. A cellular composition according to any of the preceding claims, wherein the pre-expansion population of placental MAIT cells exhibits a higher percentage of cells expressing perforin and / or granzyme B compared to the pre-expansion population of conventional T cells derived from umbilical cord blood, indicating enhanced effector and lytic functions of placental MAIT cells.

32. A cellular composition according to any of the preceding claims, wherein the pre-expansion population of placental MAIT cells exhibits a higher percentage of cells expressing TNFα compared to the pre-expansion population of conventional T cells derived from umbilical cord blood, indicating enhanced effector function of placental MAIT cells.

33. A cell composition according to any of the preceding claims, wherein the pre-expansion population of placental MAIT cells exhibits a higher percentage of cells expressing TNFα compared to the pre-expansion population of conventional T cells derived from peripheral blood conventional T cells, indicating enhanced effector function of placental MAIT cells.

34. A cellular composition according to any of the preceding claims, wherein the population of placental MAIT cells exhibits a higher percentage of cells expressing one or more of the following: perforin, granzyme B, granzyme B and perforin, TNFα, and TNFα and INFγ, compared to a population of placental T cells, indicating enhanced effector and lytic function.

35. A cell composition according to any of the preceding claims, wherein the pre-expansion population of placental MAIT cells exhibits a higher percentage of cells expressing CD8αα+ compared to the pre-expansion population of umbilical cord blood MAIT cells, indicating a mature phenotype of placental MAIT cells.

36. A cellular composition according to any one of the preceding claims for use in therapy.

37. A cellular composition for use according to claim 36, wherein the use is for treating a subject having a tumor or malignancy.

38. The cell composition for use according to claim 37, wherein the subject suffers from a tumor selected from the group consisting of blood cancer, melanoma, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, uterine cancer, cervical cancer, bladder cancer, stomach cancer, head and neck cancer, brain cancer, skin cancer and sarcoma.

39. A method of treating a subject having a tumor or malignancy, the method comprising administering to the subject a cellular composition as defined in any one of claims 1 to 35.

40. The method of claim 39, wherein the tumor is selected from the group consisting of blood cancer, melanoma, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, uterine cancer, cervical cancer, bladder cancer, stomach cancer, head and neck cancer, brain cancer, skin cancer, and sarcoma.

41. The method of claim 39 or 40, wherein the cell composition is suitable for cell therapy, and the population comprises at least 10 9 viable cells, of which at least 90% were TCRVα7.2 + CD161 高 .

42. The method of any one of claims 39-41, wherein the engineered placental MAIT cells are allogeneic to the subject.

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

44. A method of treating a subject infected with a pathogen, wherein the pathogen is any of a bacterial pathogen, a viral pathogen, or a fungal pathogen, the method comprising administering to the subject a cellular composition as defined in any one of claims 1-35.

45. A method of treating a subject suffering from a fibrotic disease or condition, the method comprising administering to the subject a cellular composition as defined in any one of claims 1-35.

46. ​​A method of treating a subject suffering from a disease or condition associated with aging, the method comprising administering to the subject a cellular composition as defined in any one of claims 1-35.

47. A method of treating a subject having an allogeneic transplant, the method comprising administering to the subject a cell composition as defined in any one of claims 1-35.

48. A method of treating a subject suffering from an autoimmune disease or disorder, the method comprising administering to the subject a cellular composition as defined in any one of claims 1-35.

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

50. The method of claim 49, wherein the MAIT cells are allogeneic to the subject.

51. The method of claim 49, wherein the pathogen is a bacterial pathogen, and wherein the bacterial pathogen comprises an antibiotic resistant bacteria or a bacterial treatment resistant bacteria.

52. A population of placental mucosa-associated invariant T (MAIT) cells, wherein the placental MAIT cells are at least 90% TCRVα7.2 + CD161 + , and most of the placental MAIT cells are characterized by lack of expression of CD45RA and CCR7 (CD45RA - CCR7 - ), indicating the effector memory phenotype of the cells.

53. A population according to claim 52, wherein the MAIT cells are engineered placental MAIT cells expressing a chimeric antigen receptor (CAR).

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

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

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

57. A cell composition comprising a population of placental MAIT according to any one of claims 52-56.

58. The cell composition of claim 57 for use in therapy.

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