Genetically engineered mucous membrane related constant T (MAIT) cells for adoptive transfer cell therapy

Abstract

The present disclosure relates generally to the field of genetic engineering of immune cells, in particular to mucosa-related constant T (MAIT) cells genetically engineered to express an exogenous T cell receptor (TCR) and uses thereof. More specifically, the present invention relates, in its embodiments, to cell compositions adapted for adoptive transfer cell therapy (ACT), providing improved therapeutic modalities.

Description

Technical Field

[0001] This disclosure generally relates to the field of genetic engineering of immune cells. More specifically, this disclosure provides genetically engineered mucosa-associated inert T (MAIT) cells to express exogenous T cell receptors (TCRs) and their uses. Background Technology

[0002] Mucosa-associated inertial T (MAIT) cells were first identified in humans, mice, and cattle as a population of αβ T cells rich in double-negative (CD4+) T cells. - CD8 - The subgroup of T cells, expressing the constant Vα7.2-Jα33 T cell receptor (TCR) in humans, is known as MAIT. The term MAIT arose from the relative enrichment of these T cells within mucosal tissues. A limiting factor for MAIT cells is the MHC-1b molecule MR1 (MHC-associated protein 1), which presents intermediates of the vitamin B1 (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-OE-RU), produced during riboflavin synthesis by a wide variety of bacteria, mycobacteria, and yeasts. This pathway is absent in mammals; therefore, its immunoassay allows for effective host-pathogen differentiation.

[0003] It has been found that MAIT cells can recognize bacterial-infected cells and produce IFN-γ in response. MAIT cells have been shown to protect mice from bacterial infection. Early phenotypic work has established that human MAIT cells are CD8+ cells. + Or double negative, with major CCR7 - MAIT cells exhibit an effector memory phenotype and express high levels of CD161. Detailed phenotypes of MAIT cells also demonstrate that they share several characteristics with invariant natural killer T (iNKT) cells, including the expression of PLZF (a transcription factor that controls the innate-like function of iNKT cells). PLZF expression similarly confers innate-like function on MAIT cells, as evidenced by the ability of cytokines to induce IFN-γ production in the absence of TCR stimulation.

[0004] Subsequent studies have found that human MAIT cells do not express a single constant TCR, but rather express restricted TCRs, including Vα7.2-Jα33, Vα7.2-Jα12, or Vα7.2-Jα20, primarily associated with a limited repertoire of human β chains such as Vβ2 / Vβ13. Therefore, the current understanding considers MAIT cells as T cells that (a) express a semi-constant Vα7.2-Jα33 / 12 / 20 TCR, (b) are activated by microbial vitamin B metabolite antigens presented by MR1 to perform both type 1 and type 17 effector functions, and (c) exhibit innate-like characteristics controlled by PLZF expression, including the ability to be activated by cytokines independent of their TCRs.

[0005] In contrast to conventional T cells, which possess a highly variable TCR capable of targeting a large number of peptide epitopes produced by viruses, bacteria, and malignant cells, MAIT cells exhibit precise specificity for individual peptides, and single clones can undergo large-scale expansion to provide T cell memory. However, the frequency of any single peptide-specific T cell will be very low upon first encounter with a pathogen. Conversely, the MAIT cell TCR provides an innate-like ability to respond to a specific set of ligands without requiring expansion.

[0006] Several properties of MAIT cells suggest a fundamental role in mammalian immunity. First, MAIT cells possess an intrinsic effector-memory phenotype, typically CD45RA. - CD45RO + CD95 Hi CD62L Lo CD44 Hi MAIT cells have the ability to rapidly secrete several pro-inflammatory cytokines. Secondly, MAIT cells are very abundant in human tissues, typically comprising 1-4% of all T cells in peripheral blood, up to 10% of airway T cells, and 20-40% of hepatic T cells. Furthermore, because each TCR recognizes the same ligand, in the early stages of the immune response, MAIT cells will significantly outnumber conventional antigen-specific T cells responding to homologous antigens.

[0007] Recent studies using single-cell RNA sequencing and immunological techniques have revealed significant heterogeneity in MAIT cells, which encapsulates conventional T cell biology. This significant heterogeneity has been shown to include different CD4 groups. + and CD8 + Lineage, and “killer,” “helper,” and “regulatory” cell phenotypes – indicators of the complex functions of MAIT cells.

[0008] The conservation and abundance of MAIT cells may be explained by their broad range of functions, attributed to different activation modes, each triggering a different transcriptomic program. Due to their ability to respond in diverse functional contexts across a diverse immunological background, these intriguing cells now appear to be multifunctional effectors, occupying a central position at the intersection of innate and adaptive immunity. Three main functions have been described for these cells – antibacterial host defense, antiviral host defense, tissue repair, and homeostasis – but there are likely many other functions yet to be discovered.

[0009] WO 2021 / 113759 discloses a population of T cells expressing chimeric antigen receptors (CARs), wherein the T cells are placental T cells derived from umbilical cord blood, placental perfusion fluid, or a mixture of both. The use of MAIT cells is not mentioned, nor is the isolation of a specific population derived from intervillous blood from the placenta.

[0010] Parrot et al. (JCI Insight. 2021; 6(5):e140074) and Healy et al. (JHEP Reports 2021; vol. 3) reported experiments using human MAIT cells optionally engineered to recognize viral antigens. Specifically, the MAIT cells were obtained from peripheral blood, expanded in culture, and engineered to express hepatitis B virus (HBV)-specific TCRs. These publications disclosed that the ex vivo expanded MAIT cells were shown to be low in CD161 after expansion and to exhibit downregulation of CD161 in response to antigen stimulation. The authors further reported the development of a progressively more differentiated phenotype over time during all of their tested expansion protocols, which could potentially impair the function and survival of adoptively transferred MAIT cells in vivo.

[0011] Some of the inventors and collaborators disclosed in WO 2024 / 023826 an engineered TCR targeting the cancer testis antigen New York esophageal squamous cell carcinoma-1 (NY-ESO-1), which could be used to treat cancer.

[0012] US 11,939,562 discloses a three-dimensional (3D) bioreactor and its method of use for large-scale expansion of immune cells.

[0013] Therefore, it is of interest to further study and develop MAIT cell compositions for various therapeutic applications. Summary of the Invention

[0014] This disclosure generally relates to the field of genetic engineering of immune cells, and more specifically to mucosa-associated inert T (MAIT) cells genetically engineered to express exogenous T cell receptors (TCRs) and their uses. More specifically, the invention relates in its embodiments to cell compositions adapted for adoptive transfer cell therapy (ACT), providing an improved mode of treatment.

[0015] In various embodiments, the compositions and methods according to the invention can be used to treat cancer and other conditions associated with the expression of HLA class I restrictive antigens. As further disclosed herein, the compositions and methods according to the invention are advantageously suitable for use even with respect to patient populations otherwise not considered suitable for treatment with immunotherapy-based methods (e.g., due to primary or acquired resistance).

[0016] In one aspect, a cell composition is provided comprising a population of engineered MAIT cells expressing exogenous TCRs, wherein the MAIT cells are derived from the placenta, and the cell composition optionally further comprises a pharmaceutically acceptable carrier.

[0017] In one embodiment, the MAIT cells are advantageously derived from placental intervillous blood (IVB). In another embodiment, the composition is adapted for adoptive transfer cell therapy (ACT). In yet another embodiment, the composition comprises 10 9 -10 11 The composition comprises live cells from the engineered MAIT cell population. In another embodiment, the composition contains at least 90% TCRVα7.2. + CD161 高 Cells. In another embodiment, the composition is as disclosed and further characterized herein.

[0018] In another embodiment, the TCR recognizes a tumor antigen. In some embodiments, the tumor antigen may be selected from the group consisting of: NY-ESO-1, KRAS, p53, PIK3CA, PTEN, ERBB2 (HER2), AFP, KK-LC-1, RAC1-P29S, LAGE-1A, COL6A3, HA-2, HERV-E, BRAF, gp100, alpha-fetoprotein, desmosome connective protein / AHNAKS2580F, cancer / testis antigen 1, ERBB2H473Y, ERBB2IPE805G, minor H antigen (HA-1), PRAME, TPBG, 5T4, MAGEA1, MAGE-A3 / A6, MAGEA4 / 8, Melan-A / MART-1, NRAS, and Wilms tumor 1 (WT-1). Each possibility represents a separate embodiment of the invention. According to an exemplary embodiment, the tumor antigen is selected from the group consisting of NY-ESO-1 and LAGE-1A. In a specific embodiment, the tumor antigen is NY-ESO-1. In another embodiment, the TCR is capable of specifically binding to an epitope presented by HLA-A2. According to an exemplary embodiment, the TCR comprises a TCRα chain and a TCRβ chain, the TCRα chain comprising CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 2, and CDR3 having the amino acid sequence of SEQ ID NO: 3, and the TCRβ chain comprising CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 6, as shown in Table 1 below. In another specific embodiment, the TCR comprises a TCR α chain and a TCR β chain, the TCR α chain having the amino acid sequence shown in SEQ ID NO: 7, optionally excluding the signal peptide located at positions 1-20 of SEQ ID NO: 7, and the TCR β chain having the amino acid sequence shown in SEQ ID NO: 8, optionally excluding the signal peptide located at positions 1-21 of SEQ ID NO: 8. In another embodiment, the antigen or TCR is as disclosed and further characterized herein.

[0019] In another embodiment, the cellular composition of the present invention is used for therapeutic purposes. In another embodiment, the use is to treat a subject with a tumor or malignant tumor. In another embodiment, the subject has a tumor selected from the group consisting of: 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. Each possibility represents a separate embodiment of the invention. In another embodiment, the subject has a treatment-resistant tumor or is otherwise unsuitable for treatment using immunotherapy comprising chimeric antigen receptor (CAR) T cells and / or therapeutic antibodies. In another embodiment, the use is to treat a subject with a condition associated with the expression of an HLA class I restricted antigen. In another embodiment, the antigen is a low-density antigen characterized by surface expression of fewer than 50 antigen molecules per cell (e.g., epitopes as disclosed herein presented in the context of MHC molecules). In another embodiment, the use is as disclosed and further characterized herein. In another aspect, a cell composition adapted for ACT is provided, the composition comprising a substantially purified population of MAIT cells engineered to express a foreign TCR, wherein the TCR recognizes a cancer testis antigen. In another aspect, a cell composition adapted for ACT is provided, the composition comprising a substantially purified population of MAIT cells engineered to express a foreign TCR, wherein the TCR recognizes a tumor antigen selected from the group consisting of NY-ESO-1 and LAGE-1A. In another embodiment, the TCR comprises a TCR α chain and a TCR β chain, the TCR α chain comprising CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 2, and CDR3 having the amino acid sequence of SEQ ID NO: 3, and the TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 6. In another embodiment, the TCR comprises a TCR α chain and a TCR β chain, the TCR α chain having the amino acid sequence shown in SEQ ID NO: 7, optionally excluding the signal peptide located at positions 1-20 of SEQ ID NO: 7, and the TCR β chain having the amino acid sequence shown in SEQ ID NO: 8, optionally excluding the signal peptide located at positions 1-21 of SEQ ID NO: 8. In another embodiment, the cellular composition is as disclosed and further characterized herein.

[0020] In another aspect, a method is provided for treating a subject with a condition associated with the expression of an HLA class I restricted antigen, the method comprising administering to the subject a cell composition as defined herein. In one embodiment, the cell composition comprises a population of engineered MAIT cells expressing exogenous TCRs, wherein the MAIT cells are derived from the placenta, and the cell composition optionally further comprises a pharmaceutically acceptable carrier. In another embodiment, the cell composition is adapted for ACT and comprises a substantially purified population of MAIT cells engineered to express exogenous TCRs, wherein the TCRs recognize tumor antigens selected from the group consisting of NY-ESO-1 and LAGE-1A.

[0021] In another aspect, the present invention provides a method for treating a subject with a tumor or malignant tumor, the method comprising administering to the subject a cell composition as defined herein. In another aspect, the present invention provides a method for treating a subject with a tumor or malignant tumor, the method comprising administering to the subject a cell composition comprising a population of engineered MAIT cells expressing exogenous TCRs, wherein the MAIT cells are derived from the placenta, and the cell composition optionally further comprises a pharmaceutically acceptable carrier. In another aspect, the present invention provides a method for treating a subject with a tumor or malignant tumor, the method comprising administering to the subject a cell composition adapted for ACT, the composition comprising a substantially purified population of MAIT cells engineered to express exogenous TCRs, wherein the TCRs recognize tumor antigens selected from the group consisting of NY-ESO-1 and LAGE-1A.

[0022] In another aspect, a method is provided for treating a subject with a condition associated with the expression of an HLA class I restriction antigen, the method comprising administering to the subject a cell composition comprising a population of engineered MAIT cells expressing exogenous TCRs and a pharmaceutically acceptable carrier.

[0023] In one embodiment of the method of the present invention, the MAIT cells are derived from the placenta. In another embodiment of the method of the present invention, the MAIT cells are obtained from placental IVB. In another embodiment, the subject has a tumor or malignancy, and the TCR recognizes a tumor antigen expressed by cells of the tumor or malignancy. In another embodiment, the antigen is a cancer testis antigen. In another embodiment, the antigen is selected from the group consisting of NY-ESO-1 and LAGE-1A. In another embodiment, the antigen is NY-ESO-1. In another embodiment, the subject is HLA-A2 positive and has a tumor or malignancy expressing NY-ESO-1 and / or LAGE-1A. In another embodiment, the subject is HLA-A2 positive and has a tumor or malignancy expressing NY-ESO-1 and / or LAGE-1A, and the TCR comprises a TCR α chain and a TCR β chain, the TCR α chain comprising CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 2, and CDR3 having the amino acid sequence of SEQ ID NO: 3, and the TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 6. In another embodiment, the TCR comprises a TCR α chain and a TCR β chain, the TCR α chain having the amino acid sequence as shown in SEQ ID NO: 7, optionally excluding the signal peptide located at positions 1-20 of SEQ ID NO: 7, and the TCR β chain having the amino acid sequence as shown in SEQ ID NO: 8, optionally excluding the signal peptide located at positions 1-21 of SEQ ID NO: 8. In another embodiment, the tumor is selected from the group consisting of: melanoma, myeloma, sarcoma, and bladder tumors, brain tumors, ovarian tumors, lung tumors, breast tumors, synovial tumors, and prostate tumors. Each possibility represents a separate embodiment of the invention. In a particular embodiment, the tumor is melanoma.

[0024] In another embodiment of the method of the invention, the antigen is a low-density antigen characterized by surface expression of fewer than 50 antigen molecules per cell. In another embodiment, the cell composition is adapted to ACT, and the population contains at least 10 9 7.2 live cells, of which at least 90% are TCR-Vα 7.2 + CD161 高In another embodiment, the engineered MAIT cells are allogeneic to the subject. In another embodiment, the engineered MAIT cells are partially tissue-compatible with the subject. In another embodiment, the engineered MAIT cells are not tissue-compatible with the subject. In another embodiment, the subject has a treatment-resistant tumor or is otherwise unsuitable for treatment using immunotherapy comprising CAR T cells and / or therapeutic antibodies. Each possibility represents a separate embodiment of the invention. In another embodiment, the method is as disclosed and further characterized herein.

[0025] In another aspect, the present invention provides a process for producing a cell composition adapted to ACT, the process comprising the following steps:

[0026] a. Obtain a cell population containing MAIT cells.

[0027] b. The cells were incubated in the presence of MAIT cell antigen and IL-15 to generate a population of activated MAIT cells.

[0028] c. The resulting activated MAIT cells were engineered to express exogenous TCR (eTCR).

[0029] d. Expand the resulting engineered MAIT cells to obtain a therapeutically effective amount of the cells, and

[0030] e. Engineered MAIT cells obtained from isolation.

[0031] In another embodiment, the cell population containing MAIT cells is obtained from the placenta. In another embodiment, the cell population containing MAIT cells is obtained from IVB. In another embodiment, step d. (amplification step) is performed to obtain an expansion factor of at least 100-fold. In another embodiment, step e. (isolation step) includes subjecting the resulting cells to positive selection of cells expressing TCRVα7.2 to obtain a substantially purified population of engineered MAIT cells. In another embodiment, the process is as disclosed and further characterized herein.

[0032] In another embodiment, the process provides a cell composition adapted for ACT, the cell composition comprising at least 10 9 The engineered MAIT cells comprised a substantially purified population of live cells, of which at least 90% were TCR-Vα 7.2. + CD161 高 And at least 70% are eTCR +In another embodiment, the process provides a cell composition as disclosed and further characterized herein. In another embodiment, a cell composition produced by said process is provided.

[0033] Other objects, features, and advantages of the present invention will become apparent from the following description and accompanying drawings. Attached Figure Description

[0034] This document describes, by way of example only, some embodiments of engineered mucosa-associated inert T (MAIT) cells and their use with reference to the accompanying drawings. Referring now to the drawings in detail, it should be emphasized that the details shown are for the purpose of illustrating engineered MAIT cells and MAIT cells lacking exogenous antigen receptors, and embodiments for their therapeutic uses. In this regard, the description using the drawings makes it apparent to those skilled in the art how engineered MAIT cells, MAIT cells lacking exogenous antigen receptors, and embodiments for their therapeutic uses can be practiced.

[0035] Figure 1A-1B The results showed that CD3 was detected by flow cytometry. + MAIT was performed on phylogenetic cells in placental intervillous blood (IVB) mononuclear cells and peripheral blood mononuclear cells (PBMCs). Figure 1A Displayed on CD3 + Representative samples of MAIT cells (“MAIT”) from placental IVB were identified by phylogenetic analysis and staining with CD161 and TCR Vα7.2. Figure 1B The CD3 values ​​analyzed from 13 IVB donors and 11 PB donors are shown. + The ratio of MAIT cells in the placenta (IVB) and peripheral blood (PB). p = 0.0159.

[0036] Figure 2A -C shows the phenotype of MAIT cells based on CD62L and CD45RA expression. Figure 2A Representative samples of total T cells from peripheral blood (left), peripheral MAIT cells (middle), and placental (IVB)-derived MAIT cells (right) are shown. Effector memory cells (EM) are highlighted. CM stands for central memory. TemRA stands for terminally differentiated effector cells re-expressing CD45RA. Figure 2B The results show the results of IVB-derived MAIT cells and peripheral blood-derived MAIT cells analyzed from each of the six donors. The percentage of the indicated population in the evaluated MAIT population is shown (EM – left, TemRA – right). Figure 2CSimilarly, the results for IVB-derived MAIT cells (IVB MAIT) and peripheral blood-derived T cells (PB T) (%) are shown.

[0037] Figures 3A-3B This demonstrates the expression of chemokine receptors on peripheral blood MAIT cells or IVB MAIT cells obtained by flow cytometry. Figure 3A MAIT cells from placental IVB and peripheral blood (PB) of six donors are displayed, and the expression of CCR5 (top left), CCR6 (top right), CXCR4 (bottom left), and CXCR6 (bottom right) is analyzed. MAIT cells were gated, and the median fluorescence intensity (MFI) of each marker was analyzed. Figure 3B Presenting the tested MAIT cell population Figure 3A The percentage of each receptor evaluated (in MAIT).

[0038] Figures 4A-4B – Showing granzyme B and perforin in MAIT cells derived from placental IVB and peripheral blood, as well as in total peripheral blood CD3. + Expression in cells. Figure 4A Three cell types are shown (placental IVB – left, PB MAIT – middle, PBCD3). + – A representative sample (right). Figure 5B MAIT cells and total peripheral blood CD3 from placental IVB (IVB MAIT) and peripheral blood (PBMAIT) of each of the six donors are displayed. + Cells (PB T) were stimulated with PMA / ionomycin for 4 hours, and the intracellular expression of granzyme B and perforin was analyzed. This was performed on MAIT cells or CD3 cells. + The cells were gating. p = 0.0001.

[0039] Figures 5A-5B – Shows cytokine production in placental IVB MAIT cells and PB T cells (PB T). Figure 5A This shows representative samples of placental IVB cells (gated in the MAIT population) stained for IFNγ and TNFα, and peripheral blood T cells. Figure 5B This shows a quantitative analysis of cytokine production in placental MAIT cells relative to peripheral T cells (6 donors each, showing the % expression of the tested cytokines in the analyzed cells).

[0040] Figures 6A-6B The expression of MDR1 in IVB MAIT cells and peripheral blood T cells was shown. Figure 6AThe image shows placental IVB cells (gated in the MAIT population, dashed lines) and peripheral blood T cells (PB CD3). + Representative samples (with solid lines). Figure 6B Quantitative analysis of MDR1 protein expression in placental MAIT cells (IVB MAIT) and peripheral T cells (PB T, 6 donors each) was presented, such as... Figure 6A As detailed in the text.

[0041] Figures 7A-7C The MAIT cells showed activation and expansion. Figure 7A This section provides an overview of the expansion process, in which IVB mononuclear cells were seeded in tissue culture plates, and MAIT cell activation was induced by 5-OP-RU (250 ng / ml) and IL-15 (50 ng / ml). On days 3 and 5 post-activation, cells were counted and MAIT% analyzed by flow cytometry, with fresh culture medium and IL-15 added. On day 7, cells were separated using magnetic beads and anti-TCR Vα7.2 antibody to further enrich the MAIT population; cells were then cultured for another 3 days in the presence of IL-15. Results showed an increase in MAIT% throughout the culture period (…). Figure 7B ) and MAIT multiple amplification (MAIT is the increase in absolute number relative to the initial number, Figure 7C ).

[0042] Figures 8A-8B RNAseq RNA expression analysis of amplified placental MAIT cells relative to peripheral T cells. Figure 8A – MAIT and CD8 + A heatmap of genes with significant differential expression (top) among T cells. The three columns on the left represent CD8 from three different donors. + T cells; the three columns on the right represent MAIT cells from three different placentas. The upper section shows the top 23 genes that are expressed in higher values ​​in peripheral T cells than in MAIT cells, and the lower section shows the top 17 genes that are expressed in higher values ​​in MAIT cells than in peripheral T cells. Figure 8B – Shown in MAIT cells relative to normal CD8 + A volcano diagram of genes that are significantly upregulated (right) or downregulated (left) in T cells.

[0043] Figures 9A-9B LegendScreen shows expanded placental MAIT cells and peripheral T cells. TM Protein expression analysis. Figure 9AMedian fluorescence intensity (MFI) of biomarkers was plotted for the two cell populations; for each population, two donors were analyzed, and the mean MFI was plotted. Biomarkers with consistent differential expression were highlighted by black circles or black diamonds (for chemokine receptors). Figure 9B An exemplary flow cytometry histogram showing the selected differentially expressed biomarkers, where IVB MAIT cells are marked by dashed lines, and PB CD8... + T cells are marked with solid lines. Figure 10 The results showed that chemokine receptor expression was higher on IVB MAIT cells (MAIT) compared to conventional T cells (T cells).

[0044] Figure 11 This indicates that MAIT cells have a lower allogeneic profile compared to peripheral blood T cells. Left panel – CD8 in the presence of inactivated MAIT cells (MAIT) or T cells (T). + T-cell alloactivation (measured as CD25) + %) of cells. Unstimulated CD8 + T cells (“unstimulated”) and CD8 cells stimulated by anti-CD3 / CD28 TransAct reagent + T cells (“stimulation”) and CD8 cells incubated with lymphocytes of the same origin + T cells (“self”) were used as a control. Right image – CD8 cells from the same experiment. + T cell proliferation is defined as cells that have undergone more than three divisions.

[0045] Figure 12A-12D – A phenotypic flow cytometry analysis comparing cord blood (CB) MAIT cells and IVB-MAIT cells (IVB). Figure 12A The image shows the expression of TCR Vα7.2 and CD161 in T cells of CB (right panel) and IVB (left panel). The indicated percentage (0.15% vs. 4.75%) is from MAIT cells (derived from CD3). + The percentage of phylum cells. Figure 12B This shows the expression of CD8α and CD8β in MAIT cells in CB (right) and IVB (left). The numbers indicated are the percentage of MAIT cells in IVB or CB. Figure 12C The image shows the expression of CD45RA and CCR7 (top image) and CD45RA and CD62L (bottom image) in MAIT cells of CB (right) and IVB (left). CD45RA + CCR7+ The cells are naïve cells; CD45RA - CCR7 - The cell is an effector memory cell; CD45RA + CD62L + The cell is an immature cell; CD45RA - CD62L - The cells are effector memory cells. The numbers indicated are the percentage of MAIT cells in IVB or CB. Figure 12D The image shows the expression of CD45RA and CD45RO (top image) and CD27 and CD45RO (bottom image) in MAIT cells from CB (right) and IVB (left). CD45RA + CD45RO - The cell is an immature cell; CD45RA - CD45RO + The cell is an activating cell or a memory cell; CD27 + CD45RO + The cell is a central memory cell; CD27 - CD45RO + The cells are effector memory cells. The numbers indicated are the percentage of MAIT cells in IVB or CB.

[0046] Figures 13A-13B Flow cytometry showing engineered MAIT cells. Figure 13A This illustrates the gating strategy for flow cytometry, from single-cell (top) to live cells (middle left) to MAIT cells (made by CD161). + TCRα7.2 + Definition: (right middle) to MAIT NYESO1-TCR (Vβ13.1+) cells (bottom). Figure 13B Flow cytometry of representative placental-derived donors' MAIT cells isolated using TCRα7.2 and transduced with NY-ESO-1 TCRs. Populations exist before and after separation of positive beads.

[0047] Figure 14 The image shows MAIT cells (CD161) from two separate placentas at days 0, 4, 7, and 10 post-activation. + TCRα7.2 + The number of cells.

[0048] Figures 15A-15EThis indicates the levels of the cytokine IFNγ (as measured by intracellular staining and flow cytometry) after co-culturing MAIT NYESO1 TCR cells or untransduced controls with NYESO-1-expressing target cells at a 1:1 effector-to-target ratio for 6 hours. Figure 15A ), IL2 ( Figure 15B ), TNFα ( Figure 15C The expression of ) was observed. The results represent two different placental-derived MAIT cells. Figure 15D Representative images showing MAIT NY-ESO-1 TCR cells co-cultured with T2-ESO or T2-HIV target cells and stained with antibodies against IFNγ, IL2, and TNFα, or untransduced controls. Figure 15E The classification of MAIT NYESO-1 TCR cells based on the expression of 1, 2, or 3 cytokines (IFNγ, IL2, and TNFα) is shown as an indication of their pluripotent status. MAIT 1 and MAIT 2 represent MAIT populations from two different donors.

[0049] Figure 16 The results of an ELISA assay depicting IFNγ secretion after 24 hours of co-culture between MAIT-NYESO1-TCR cells and target cells at a 1:1 ratio are shown. MAIT 1 and MAIT 2 represent MAIT populations from two different donors.

[0050] Figure 17A-17F The activation marker CD137 (as measured by flow cytometry) was observed after co-culturing MAIT NYESO1 TCR cells or untransduced controls with NYESO-1-expressing target cells at a 1:1 effector-to-target ratio for 24 hours. Figures 17A-17B CD69 Figure 17C-17D CD25 Figure 17E-17F The expression of ) is shown in the figure. The figure represents two different MAIT cells (named MAIT 1 and MAIT 2) co-cultured with A375, M624, 526, T2-ESO, or T2-HIV target cells. Flow cytometry plots depict representative MAIT cells co-cultured with T2 target cells. Figure 17A – CD137 expression (%TCR) + CD137 + cell). Figure 17B – Flow cytometry plot of CD137 expression. Figure 17C – CD69 expression (%TCR) + CD69 + cell). Figure 17D – Flow cytometry image of CD69 expression. Figure 17E – CD25 expression (%TCR)+ CD25 + cell). Figure 17F – Flow cytometry image of CD25 expression. Figures 18A-18B Describe the cytotoxic activity of engineered MAIT cells. Figure 18A – Killing assay measured by flow cytometry using the expression of active caspase-3 after co-culturing MAIT cells expressing NY-ESO-1TCR or untransduced (NT) controls with target cells at a 1:1 ratio for 1.5 hours. Figure 18B – A representative flow cytometry image with caspase-3 staining depicts the targeted killing of T2-ESO cells by MAIT 1 NYESO1-TCR cells.

[0051] Figures 19A-19B The expression of CD107, as measured by antibody staining and flow cytometry, is shown after co-culturing MAIT NYESO1 TCR cells or untransduced controls with target cells expressing NYESO-1 at a 1:1 effector-to-target ratio for 6 hours. Figure 19A It is TCR + CD107 + Quantification of cell percentiles. Figure 19B Provide representative images of MAIT NY-ESO-1 TCR cells co-cultured with T2-ESO or T2-HIV target cells and stained with an antibody against CD107, or untransduced controls.

[0052] Figure 20 Results of an ELISA assay quantifying granzyme B (GZMB) secretion after 24 hours of co-culturing MAIT-NYESO1-TCR cells and target cells at a 1:1 ratio. Detailed Implementation

[0053] This disclosure generally relates to the field of genetic engineering of immune cells, and more specifically to mucosa-associated inert T (MAIT) cells genetically engineered to express exogenous T cell receptors (TCRs) and their uses. More specifically, the invention relates in its embodiments to cell compositions adapted for adoptive transfer cell therapy (ACT), providing an improved mode of treatment. In various embodiments, the compositions and methods according to the invention can be used to treat cancer and other conditions associated with the expression of HLA class I restrictive antigens.

[0054] In some embodiments, the present invention relates to compositions and methods utilizing placental MAIT cells, particularly MAIT cells derived from placental intervillous blood (IVB), said MAIT cells being engineered to express exogenous TCRs. In some embodiments, the exogenous TCRs are specific for tumor antigens such as cancer testicular antigens such as NY-ESO-1 and / or LAGE-1A.

[0055] In one aspect, a cell composition is provided comprising a population of engineered MAIT cells expressing exogenous TCRs, wherein the MAIT cells are derived from the placenta, and optionally the cell composition further comprises a pharmaceutically acceptable carrier.

[0056] On the other hand, a cell composition adapted for ACT is provided, the composition comprising a substantially purified population of MAIT cells engineered to express exogenous TCRs, wherein the TCRs recognize tumor antigens selected from the group consisting of NY-ESO-1 and LAGE-1A.

[0057] In another aspect, a method is provided for treating a subject with a condition associated with the expression of HLA class I restrictive antigens, the method comprising administering to the subject a cellular composition as defined herein.

[0058] In another aspect, the present invention provides a method for treating a subject with a tumor or malignant tumor, the method comprising administering to the subject a cell composition as defined herein.

[0059] In another aspect, a method is provided for treating a subject with a condition associated with the expression of an HLA class I restriction antigen, the method comprising administering to the subject a cell composition comprising a population of engineered MAIT cells expressing exogenous TCRs and a pharmaceutically acceptable carrier.

[0060] In another aspect, the present invention provides a process for producing a cell composition adapted to ACT, the process comprising the steps detailed herein.

[0061] These and other aspects are described in more detail below.

[0062] definition

[0063] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which engineered mucosa-associated constant T (MAIT) cells and methods of their use pertain. While similar or equivalent methods and materials may be used to practice or test embodiments of engineered mucosa-associated constant T (MAIT) cells and methods of their use, exemplary methods and / or materials are described below. In case of conflict, the patent specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. Every reference or other citation cited herein is incorporated herein by reference in its entirety.

[0064] The terms “comprise”, “includes”, “have”, and their related terms mean “including but not limited to”.

[0065] As used herein, the singular forms “a / an” and “the” include plural references unless the context clearly specifies otherwise. For example, the terms “a / an enzyme” or “at least one / an enzyme” can include multiple enzymes, including mixtures thereof.

[0066] Unless otherwise stated, the term "collection" when used in conjunction with one or more cell attributes can cover a set of cells in which at least 70% of the cells exhibit one or more of the mentioned attributes. 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 set exhibit one or more of the mentioned attributes. In some embodiments, the population is separated from cell types different from the specific attribute. In some embodiments, the population of engineered MAIT cells described herein includes both endogenous and exogenous attributes. In some embodiments, the population of unengineered MAIT cells expressing exogenous antigen receptors described herein includes both endogenous and exogenous attributes.

[0067] Regarding placental-derived cells, unless otherwise stated, the terms "placenta" and "placental" can encompass various solid or liquid portions of the placenta, such as the decidua parietalis, intervillous blood (IVB), and decidua basalis. Placental-derived MAIT cells can be obtained from the fetus in various embodiments, or from the maternal region of the placenta in other embodiments, or from both regions in other embodiments. In some embodiments, MAIT cells are obtained from placental blood. In some embodiments, MAIT cells are obtained from fetal or maternal blood sources from the placenta. In some embodiments, MAIT cells are obtained from both fetal and maternal blood sources from the placenta. In some embodiments, MAIT cells are obtained from fetal blood sources from the placenta. In some embodiments, MAIT cells are obtained from maternal blood sources from the placenta. In some embodiments, MAIT cells are obtained from intervillous blood from the placenta. In some embodiments, MAIT cells are obtained from the decidua parietalis of the placenta. In some implementations, MAIT cells are obtained from the decidua basalis of the placenta. In some implementations, MAIT cells are not obtained from umbilical cord blood.

[0068] 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). These terms are used interchangeably and have all the same properties and meanings.

[0069] As used herein, the terms “intervillous blood (IVB) derived MAIT cells” or “IVB MAIT cells” include MAIT cells isolated from intervillous blood from the placenta. These terms are used interchangeably and have all the same properties and meanings.

[0070] As used herein, “MAIT cells” will broadly encompass MAIT cells from any source, including but not limited to peripheral blood or intervillous blood.

[0071] In some embodiments of the engineered MAIT cell population, the MAIT cells are derived from a maternal source of the placenta (e.g., tissue and / or blood). In some embodiments of the engineered MAIT cell population, the MAIT cells are derived from maternal blood from the placenta. In some embodiments of the engineered MAIT cell population, the MAIT cells are obtained from intervillous blood from the placenta. In some embodiments of the engineered MAIT cell population, the MAIT cells are not obtained from umbilical cord blood. In some embodiments, the MAIT cells are derived from a maternal source, a placental source, or an IVB source, or a combination thereof. In some embodiments, the MAIT cells are derived from a fetal source, a maternal source, a fetal and maternal source, a placental source, or an IVB source, or a combination thereof; and are not derived from umbilical cord blood. In a particular embodiment, the MAIT cells are derived from IVB.

[0072] In various embodiments, placental cells can be obtained from a full-term placenta or a preterm placenta. In a particular embodiment, placental cells are obtained from a full-term placenta, for example, a placenta from 38-39 weeks of gestation can be conveniently used. A convenient source of placental tissue is the postpartum placenta (e.g., less than 48 hours after birth); however, various sources of placental tissue or cells may be considered by a person skilled in the art. In other embodiments, the placenta is used within 24 hours of birth (in some embodiments, while simultaneously preserved in physiological buffer), 18 hours, 14 hours, 10 hours, 8 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour. In some embodiments, the placenta is kept frozen prior to cell harvesting. In other embodiments, prenatal placental tissue is used. In some embodiments, the donor is 40 years of age or younger, in other embodiments 35 years of age or younger, however, in other embodiments the donor can be any woman of reproductive age.

[0073] Methods for isolating lymphoid cells or MAIT cells from placental intervillous blood (IVB) are well known in the art. Exemplary, non-limiting methods utilize blood dripped from a lifted placenta with the umbilical cord clamped 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, where necessary, using techniques such as cell sorting to enhance purity.

[0074] As used herein, exogenous antigen receptors include antigen receptors that are not naturally present on MAIT cells. An example of such receptors is an exogenous TCR. Typically, the MAIT cells disclosed herein are engineered to express exogenous antigen receptors. In some embodiments, engineered MAIT cells comprise TCR-MAIT cells expressing exogenous TCRs.

[0075] In some embodiments, the exogenous antigen receptor is permanently incorporated into engineered IVB-derived MAIT cells. In other embodiments, IVB-derived MAIT cells are engineered to transiently express the exogenous antigen receptor. "Permanently" herein is used to mean the insertion of exogenous DNA into the genome of a target cell (this can utilize a variety of viral and nonviral techniques generally known in the art). "Transient" is used to mean the engineering of cells to temporarily express a exogenous antigen receptor gene, which, in some embodiments, is inserted into the cell via mRNA.

[0076] Those skilled in the art will understand that the exogenous antigen receptors disclosed herein, in some embodiments, include an antigen-binding domain, which causes MAIT cells containing these exogenous antigen receptors to bind to target molecules (i.e., antigens of interest). As used herein, the terms “antigen” and “target molecule” are used interchangeably and have all the same properties and meanings.

[0077] For any cell surface marker described herein, the terms “positive” or “high,” “dim” or “low,” “or negative,” and all such names are recognized terms used in the practice of the assays and methods described herein. A cell is considered “positive” or “high” for a cell surface marker if it expresses a marker on its cell surface in an amount detectable using methods known to those skilled in the art (such as contacting the cell with an antibody that specifically binds to the marker, followed by flow cytometry analysis of such contacted cells to determine whether the antibody has bound the cell). It should be understood that while cells can express messenger RNA for cell surface markers, for a cell to be considered positive for the assays and methods described herein, it must express the cell surface marker of interest on its surface. If a cell expresses a marker on its cell surface in an amount detectable using methods known to those skilled in the art, it is considered “positive” or “high” for the cell surface marker. If a marker is detected at a level that is not readily apparent (e.g., by contacting cells with an antibody that specifically binds to the marker, followed by flow cytometry analysis of such contacted cells to determine whether the antibody binds to the cells), but another distinct population of cells expresses the marker at a higher level, and at least two distinct populations are generated when flow cytometry analysis is used (e.g., designating “high” or “bright” cells to represent the highest expression level of the marker in said population), then the cell is considered “weak” or “low” for the cell surface marker. Similarly, if a cell does not express a marker at a level sufficient to be detected using methods known to those skilled in the art (e.g., by contacting cells with an antibody that specifically binds to the marker, followed by flow cytometry analysis of such contacted cells to determine whether the antibody binds to the cells), then the cell is considered “negative” for the cell surface marker.

[0078] As used herein, “cell sorting” generally encompasses immunologically based positive and negative selection methods that result in the physical separation of cell types having specific cell surface markers or combinations of markers using antibodies or antibody fragments, or combinations of antibodies or antibody fragments, that specifically recognize the markers. Examples include, but are not limited to, cell sorting performed via fluorescence-activated cell sorting (FACS), magnetic beads, magnetically activated cell sorting (MACS), column-based cell sorting, and immunopanning.

[0079] In some embodiments, this disclosure provides a population of engineered mucosa-associated invariant T (MAIT) cells comprising exogenous antigen receptors, wherein the MAIT cells are derived from the placenta. In some embodiments, the engineered MAIT cells are derived from intervillous blood (IVB) of the placenta. The exogenous antigen receptors of these engineered MAIT cells may be T cell receptors (TCRs). In some embodiments, the TCRs recognize tumor antigens.

[0080] Mucosa-associated constant T (MAIT) cells

[0081] As used herein, mucosa-associated constant T cells (MAIT) include T cells expressing semi-constant TCRs such as Vα7.2-Jα33 in humans; in some embodiments, they are associated with the β chain Vβ2 / Vβ13. In other embodiments, the aforementioned alpha chain is associated with the beta chain from the TRBV6 or TRBV20 gene family. In some embodiments, MAIT cell recognition is limited to antigens of 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, such as those loaded with 5-(2-oxopropylamino)-6-D-ribosylaminouracil (5-OP-RU); 5-(2-oxoethylamino)-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-ribitol dioxane or photolumazine III (PLIII); 6-(2-carboxyethyl)-7-hydroxy-8-ribitol dioxane or photolumazine I; 5-hydroxydiclofenac (PubChem CID 3052566); 4-hydroxydiclofenac (PubChem CID 116545); benzbromarone (PubChem CID 2333); chloroxam (PubChem CID 2722); fluorouracil (PubChem CID 2333); fluorouracil (PubChem CID 2722); fluorouracil (PubChem CID 2333). 5790); galangin (4H-1-benzopyran-4-one, 3,5,7-trihydroxy-2-phenyl or 3,5,7-trihydroxyflavone); or mercaptopurine (PubChem CID 667490) (see, for example, Corbett et al., Antigen Recognition by MR1-Reactive T Cells; MAIT Cells, Metabolites, and Remaining Mysteries. Front Immunol. 11:1961 (2020), and references cited therein).

[0082] Conveniently, in some implementations, MAIT cells may be referred to as TCRVα7.2. + CD161 + Or more commonly TCRVα7.2 +CD161 高 .

[0083] In some embodiments, the MAIT cells disclosed herein are human MAIT cells. In some embodiments, the recipient of the MAIT cells disclosed herein is an allogeneic entity relative to the population of engineered MAIT cells as described herein.

[0084] Methods for isolating and characterizing MAIT cells and other leukocyte subsets are known in the art. For illustrative purposes only, leukocyte subsets may be isolated and / or analyzed using gating strategies as described herein.

[0085] Quantitative and qualitative differences exist between MAIT cells derived from 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 allows for the identification of MAIT cells circulating in phase 3 at birth. However, Vα7.2 in cord blood... + CD161 高 The classification can also encompass other T cells that may share common developmental pathways. In cord blood, MAIT cells exhibit a preterm state and possess an immature phenotype (CD45RA). + / RO - Furthermore, they express CD8αβ heterodimers, while adult MAIT cells are mature, mostly exhibiting a memory phenotype and expressing CD8αα homodimers. Vα 7.2 after birth. + CD161 高 The very low expansion of T cells may be related to intrinsic cellular characteristics or the limited availability of MR1-ligands from microbial sources. (Immature cord blood Vα7.2) + 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 Vα 7.2 + CD161 高 T cells proliferate strongly after stimulation with phytohemagglutinin (PHA), while adult Vα is 7.2. + CD161 高 T cell proliferation efficiency is much lower. Compared to mature MAIT cells in adult blood, umbilical cord blood Vα 7.2+ CD161 高 T cells cannot exhibit immediate effector function towards bacterial-infected cells. These data suggest that, despite their inherent proliferative capacity, umbilical cord blood Vα7.2 + CD161 高 T cells require functional maturation and / or expansion after birth to acquire detectable effector activity after recognizing antigens of microbial origin.

[0086] Due to umbilical cord blood Vα 7.2 + CD161 高 The cells exhibited an immature phenotype and moderate PLZF levels, and compared to mature adult MAIT cells, they did not respond to bacterial ligands by rapidly producing cytokines or cytotoxic molecules. Cord blood MAIT cells also did not respond to stimulation with exogenous IL-12 and IL-18, despite high expression of receptors for these cytokines. Further evidence from Chen et al., Circulating Mucosal-Associated Invariant T Cells in a Large Cohort of Healthy Chinese Individuals From Newborn to Elderly. Front. Immunol., vol.10, article 260 (2019), showed that cord blood MAIT cells possessed an immature phenotype and did not secrete IFN-γ, IL17A, and TNF-α after in vitro stimulation with PMA / ionomycin. Overall, these data suggest that cord blood premature MAIT cells are phenotypically and functionally distinct from mature MAIT cells derived from adult subjects.

[0087] In some embodiments, the MAIT cells disclosed herein contain CD161 + Vα7.2 + CD4 - CD3 + Lymphocytes, in a further embodiment, 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 implementations, 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 + .

[0088] In some implementations, MAIT subgroups are used, including but not limited to CD8. + Cells (with a more specific implementation scheme being CD8) + CD4 - Cells), CD8 - CD4 - Cells, or CD4 + Cells. In various implementations, 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 implementation schemes, 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 implementation schemes, 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 other implementation schemes, no more than 50% of the cells in the population are CD8. - CD4 - In various other implementations, no more than 40%, 30%, 20%, 10%, 5%, 3%, 2%, or 1% of the cells in the population are CD8. - CD4 - In various other implementation schemes, no more than 50% of the cells in the population are CD4. + In various other implementations, no more than 40%, 30%, 20%, 10%, 5%, 3%, 2%, or 1% of the cells in the population are CD4+. + .

[0089] In some implementations, the MAIT cells disclosed herein are additionally CD62L. lo CD122 int CD127 hi CD95 hiIn some embodiments, it indicates the effector memory phenotype. In other embodiments, the MAIT cells disclosed herein are CD45RA. - CCR7 - In some implementations, it also reflects the effect memory phenotype. In other implementations, CD45RO + This is an additional characteristic of effector memory cells. In various implementation schemes, at least 50% of the cells in the population are CD62L. lo CD122 int CD127 hi CD95 hi 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 CD62L. lo CD122 int CD127 hi CD95 hi In other embodiments, at least 5%, 10%, 20%, 30%, or 40% of the cells in the population are CD62L. lo CD122 int CD127 hi CD95 hi In various implementation schemes, 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 other embodiments, at least 5%, 10%, 20%, 30%, or 40% of the cells in the population are CD45RA. - CCR7 - In other embodiments, no more than 40%, 30%, 20%, 10%, or 5% of the cells in the population are CD45RA. - CCR7 - In various implementation schemes, 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. + In other embodiments, at least 5%, 10%, 20%, 30%, or 40% of the cells in the population are CD45RO. + .

[0090] In other embodiments, the MAIT cells disclosed herein are CD45RA. - CCR7 + In some implementations, it reflects the central memory phenotype. In other implementations, CD45RO + This is an additional characteristic of central memory cells. Unless otherwise indicated, "central memory" T cells encompass transitional memory cells (CCR7). - CD45RO + CD28 + CD95 + In various implementation schemes, 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 implementation schemes, 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. + In various implementation schemes, at least 50% of the cells in the population are CCR7. - CD45RO + CD28 + CD95 + 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 CCR7. - CD45RO + CD28 + CD95 + In various other implementations, no more than 50% of the cells in the population are CD45RA. - CCR7 + .

[0091] In other embodiments, the MAIT cells disclosed herein are CD45RA. + CCR7 - In some implementations, it reflects the terminal differentiation (terminal effector) phenotype. In various implementations, 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 other implementations, no more than 50% of the cells in the population are CD45RA. + CCR7 - In various other implementations, no more than 40%, 30%, 20%, 10%, 5%, 3%, 2%, 1%, 0.5%, or 0.2% of the cells in the population are CD45RA. + CCR7 - .

[0092] In other embodiments, the MAIT cells disclosed herein express interleukin (IL)-18Rα, CD127, α4β7, and / or PD-1. In some embodiments, MAIT cells express IL-18Rα, CD127, and α4β7. In some embodiments, MAIT cells express PD-1. In some embodiments, MAIT cells express IL-18Rα, CD127, α4β7, and PD-1. In some embodiments, the cells also express transcription factors promyelocytic leukemia zinc finger (PLZF), RORγt, Helios, amesoleucine (Eomes), and / or T-box transcription factor (T-bet). In some embodiments, MAIT cells express transcription factors PLZF, RORγt, Helios, and amesoleucine. In some embodiments, MAIT cells express T-box transcription factor. In some embodiments, MAIT cells express transcription factors PLZF, RORγt, Helios, amesoleucine, and T-box transcription factor. Optionally or additionally, the cells express the surface markers CD26, CD44, CD69, or CD25; or the receptors interleukin-7 receptor (IL-7R, also known as CD127), IL-12R, IL-15R, or IL-18R. In some embodiments, the cells express inducible T cell co-stimulatory factors (ICOS). Each of the above proteins and each combination thereof represents a separate embodiment. In some embodiments, the MAIT cells used according to the invention are TCRva7.1. + CD26 + .

[0093] In some embodiments, the MAIT cells disclosed herein express genes associated with tissue repair (e.g., transforming growth factor Beta-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.

[0094] In some embodiments, the engineered MAIT cells disclosed herein are capable of reacting with antigens restricted by the MHC class I-associated protein MR1 (i.e., recognizing and reacting with cells presenting MAIT cell antigens in the context of MR1). In other embodiments, the engineered MAIT cells recognize microbial-derived riboflavin precursor derivatives. In some embodiments, the engineered MAIT cells secrete inflammatory cytokines (e.g., interferon-gamma [IFN-g or IFN-γ], tumor necrosis factor α, interleukin-17, or colony-stimulating factor 2 [CSF2 / GM-CSF]) in an MR1-independent manner, for example, through recognition of MR-1 ligands or in other embodiments upon activation. In other embodiments, IL-17A, TNF-α, CSF2, or MIP-1 are secreted forms. In other embodiments, IL-26, OSM (oncostatin M), or heparin-bound early growth factor (HBEGF) is upregulated upon stimulation with IL-12, IL-18, IL-15, or tumor necrosis factor-like protein 1A (TL1A). Optionally or additionally, the engineered MAIT cells disclosed herein exhibit perforin / granzyme and / or granzyme-dependent cytotoxicity of target cells upon activation. In other embodiments, IFN-γ, perforin, granzyme, or granzyme are all upregulated. In another embodiment, IFN-γ, perforin, and granzyme B are upregulated.

[0095] In some implementations, compared to peripheral blood (PB) derived MAIT cells, or in another implementation compared to liver-derived MAIT cells, the specific subtypes mentioned herein (including, but not limited to, CD8) + The levels of these cells were enriched at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, or at least 10-fold in the population of engineered MAIT cells disclosed herein. In other embodiments, compared to peripheral blood (PB) derived MAIT cells, or in another embodiment compared to liver-derived MAIT cells, the specific subtypes mentioned herein (including, but not limited to, CD8+) were enriched. - CD4 -The levels of these cells were enriched at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, or at least 10-fold in the population of engineered MAIT cells disclosed herein. In other embodiments, compared to peripheral blood (PB) derived MAIT cells, or in another embodiment compared to liver-derived MAIT cells, the specific subtypes mentioned herein (including, but not limited to, CD4+) were enriched. + The levels of the cells were enriched by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, or at least 10-fold in the population of engineered MAIT cells disclosed herein.

[0096] In some embodiments, the methods described herein include in vitro expansion and / or enrichment of engineered MAIT cells prior to administration to a subject. In various embodiments, expansion or enrichment is performed before and / or after the MAIT cells are engineered to express a foreign antigen receptor. According to an advantageous embodiment, the process of the present invention includes an enrichment step performed prior to engineering and an expansion step performed after engineering. In other embodiments, activation or stimulation assays are performed to characterize or determine the quality of the engineered MAIT cells. It will be understood that protocols for expanding and activating T cells in vitro may often overlap. However, typically, activation requires stimulation of both the antigen receptor and co-receptor, in combination with cytokine treatment. Once the cells are activated, in some embodiments, they may be further expanded using cytokines alone (without reverting to an naïve state).

[0097] In some embodiments, MAIT cell expansion in vitro takes 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. For example, MAIT cell expansion in vitro takes 7 to 12 days, such as 10 days.

[0098] Throughout this application, various embodiments may be presented in a range format. It should be understood that the range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of engineered MAIT cells and their uses. Therefore, a range description should be considered to specifically disclose all possible subranges and individual numerical values ​​within that range. For example, a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within that range such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0099] Whenever a range of numbers is indicated herein, it means any referenced number (fraction or integer) included within the indicated range. The phrases “range between the first indicated number and the second indicated number” / “range between the first indicated number and the second indicated number” and “range from the first indicated number to the second indicated number” / “range from the first indicated number to the second indicated number” are used interchangeably herein and mean including the first indicated number and the second indicated number, as well as all fractions and integers in between.

[0100] 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 *Escherichia coli* Dh5αATCC strain (typically 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 for 2 hours at 37°C in complete medium containing 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.

[0101] Additional methods for expanding and / or enriching MAIT cells are known in the art. In a non-limiting embodiment, MAIT cells are incubated with 5 μg / mL CpG, 300 nM 5-(2-oxopropylamino)-6-D-ribosylaminouracil (5OP-RU), and 50 ng / mL human IL-15. In some embodiments, IL-15 is included to preferentially enhance the expansion of memory T cells. In some embodiments, IL-15 (e.g., 10-200 ng / ml) and 5-OP-RU (e.g., 100-1000 nM) are conveniently used.

[0102] In some embodiments, MAIT cells are stimulated by CD3 / CD28 in the presence of autologous or allogeneic irradiated PBMCs and IL-2, IL-7, IL-12, IL-18, IL-21, IL-15, or analogs thereof, or combinations thereof (e.g., using functionalized beads or polymers, under the trademark TransAct). TM Commercially available (in vitro activation). In other embodiments, CD3 / CD28 beads (branded ClinExVivo) are used. TM Dynabeads® (clinically available grade) are used with IL-7 and IL-2. Non-limiting exemplary regimens include X Vivo-15. TM(BioWhittaker®, Walkersville, MD), 100 units / mL IL-2 and 10 ng / mL IL-7 in a 3:1 cell:pixel ratio with ClinExVivo TM Dynabeads® are incubated for 10–14 days. In various embodiments with or without the aforementioned ligands, MAIT cells can be further expanded in the presence of cytokines once activated. According to exemplary embodiments, this expansion method can be employed after cells have been engineered to express exogenous TCRs.

[0103] In other embodiments, MAIT cells are activated in vitro in the presence of MAIT cell activation ligands such as 5OP-RU, 5-amino-4-D-ribosylaminouracil dihydrochloride (5-ARU), 5-(2-oxoethyleneamino)-6-D-ribosylaminouracil (5-OE-RU), 5-amino-6-ribitolamino-2,4-(1H,3H)-pyrimidinidone (5-A-RU), or other riboflavin (vitamin B2) derivatives. In some embodiments, the ligands are provided in combination with cytokines such as those mentioned herein. As a non-limiting example, MAIT cells can be expanded, for example, for 6-17 days in 100 nM 5-OP-RU and 100 IU / mL IL-2. In other embodiments, MAIT cells are expanded and / or activated in vitro in the presence of MAIT cell activation drug metabolites (e.g., diclofenac metabolites). In various embodiments with or without the aforementioned ligands, once activated, MAIT cells can be further expanded in the presence of cytokines.

[0104] In some embodiments, MAIT cells are expanded and / or activated in vitro in the presence of IL-12 (2 ng / mL), IL-18 (50 ng / mL) and 10 mM 5-OP-RU, or IL-12 + IL-18 or IL-15 + IL-18, as described in the art.

[0105] In some implementations, the MAIT cells to be engineered produce IL-17 and upregulate the Th17-associated transcription factor RORC (RORγt) upon stimulation with PMA and iomycin, but do not produce IL-17 and do not upregulate the Th17-associated transcription factor RORC (RORγt) upon stimulation with CD3+CD28.

[0106] In some embodiments, the engineered MAIT cells disclosed herein produce IFN-γ and upregulate T-bet upon PMA / iomycin stimulation. In a more specific embodiment, these cells are CD8... + .

[0107] In various embodiments, more than 50%, more than 60%, or more than 70% of the engineered MAIT cell population disclosed herein express CD69. Optionally or additionally, less than 30%, less than 25%, or less than 20% of the engineered MAIT cell population express Ki67.

[0108] In other embodiments, more than 50%, more than 60%, or more than 70% of the engineered MAIT cell population disclosed herein express PD-1. In other embodiments, more than 40%, more than 50%, or more than 60% of the engineered MAIT cell population express CD38. In other embodiments, more than 10%, more than 15%, or more than 20% of the engineered MAIT cell population express CD25. In other embodiments, any combination of two or all three of the above markers is expressed, and the percentages 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 engineered MAIT cells are PD-1. - / LAG-3 - This indicates that the cells did not exhibit T cell exhaustion. In other embodiments, CTLA-4, TIGIT, 2B4, BTLA, CD57, TIM-3, or KLRG-1 are used to detect T cells exhibiting exhaustion. In some embodiments, such cells are removed from or exhausted from the T cell population.

[0109] In some embodiments, MAIT cells do not proliferate significantly in a mixed lymphocyte response (MLR) when incubated with allogeneic cells. Methods for measuring the proliferation of lymphoid cells in response to allogeneic antigen stimulation are known in the art.

[0110] In some embodiments, the MAIT cells disclosed herein (e.g., MAIT cells used to produce cell compositions according to the invention) express CCR2, CCR5, CCR6, CCR9, CXCR4, CXCR3, VLA-4, or CXCR6, or combinations of 2, 3, 4, 5, 6, 7, or all eight of these receptors. Optionally or additionally, MAIT cells express activating receptors, such as NKG2D, NKp30, NKp44, or NKG2D and NKp30 and NKp44. In some embodiments, MAIT cells express high levels of CXCR4 and intermediate levels of CCR9, but low levels of CXCR2 or no CXCR2. Optionally or additionally, MAIT cells also express CXCR3. In some embodiments, the MAIT cells disclosed herein express one or more cytokine receptors, such as IL-7R, IL-12R, IL-15R, IL-18R, and IL-21R, or any combination thereof.

[0111] In some embodiments, when exposed to cells expressing the target antigen, such as in response to riboflavin-producing *E. coli*, the engineered MAIT cells disclosed herein secrete IFN-γ at levels at least 1.2, 1.5, 1.8, 2, 2.5, or 3 times higher than an equivalent number of peripheral blood (PB)-derived MAIT cells. In some embodiments, liver-derived MAIT cells are used instead of PB-derived MAIT cells as the basis for comparison.

[0112] In some embodiments, when exposed to cells expressing the target antigen, such as in response to riboflavin-producing *E. coli*, the engineered MAIT cells disclosed herein release granzymes at levels at least 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 2.5-fold, or 3-fold higher than an equivalent number of PB-derived MAIT cells. In other embodiments, liver-derived MAIT cells are used instead of PB-derived MAIT cells as the basis for comparison.

[0113] In some embodiments, when exposed to cells expressing the target antigen, such as in response to riboflavin-producing *E. coli*, the engineered MAIT cells disclosed herein mediate perforin / granzyme-dependent cytotoxicity of target cells at levels at least 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 2.5-fold, or 3-fold higher than PB-derived MAIT cells (e.g., as measured by an ELISPOT assay of granzyme B). In other embodiments, liver-derived MAIT cells are used instead of PB-derived MAIT cells as a basis for comparison. In some embodiments, cytotoxicity is measured by measuring the number of target cells killed when incubated with a limited dilution of MAIT cells.

[0114] In some embodiments, the engineered MAIT cells disclosed herein secrete XCL1, CCL3, CCL4, and CXCL16. In some embodiments, the engineered MAIT cells disclosed herein secrete at least one of XCL1, CCL3, CCL4, and CXCL16. In some embodiments, the engineered MAIT cells disclosed herein secrete at least two of XCL1, CCL3, CCL4, and CXCL16, or in other embodiments, secrete at least three of them.

[0115] In some embodiments, the engineered MAIT cells disclosed herein secrete at least one of XCL1, CCL3, CCL4, and CXCL16 (each representing a separate embodiment) at levels at least 1.2-fold, at least 1.5-fold, at least 1.8-fold, at least 2-fold, at least 2.5-fold, or at least 3-fold higher than PB-derived MAIT cells; in other embodiments, at least two; in other embodiments, at least three; or in other embodiments, all four. In some embodiments, the level of one of these cytokines, in other embodiments two, in other embodiments three, or in other embodiments all four of XCL1, CCL3, CCL4, and CXCL16 is at least 1.2-fold, at least 1.5-fold, at least 1.8-fold, at least 2-fold, at least 2.5-fold, or at least 3-fold higher than PB-derived MAIT cells, while the levels of the other listed cytokines are not significantly lower than those of PB-derived MAIT cells. In other embodiments, liver-derived MAIT cells are used instead of PB-derived MAIT cells as the basis for comparison.

[0116] In some embodiments, when exposed to cells expressing the target antigen, such as co-incubating with THP-1 cells expressing MR1 infected with Pseudomonas aeruginosa, the engineered MAIT cells disclosed herein secrete / release IL-17 at levels at least 1.2, 1.5, 1.8, 2, 2.5, or 3 times higher than PB-derived MAIT cells. In other embodiments, when exposed to cells expressing the target antigen, the engineered MAIT cells disclosed herein secrete / release IL-22 at levels at least 1.2, 1.5, 1.8, 2, 2.5, or 3 times higher than PB-derived MAIT cells. In other embodiments, when exposed to cells expressing the target antigen, the engineered MAIT cells disclosed herein secrete / release IL-17 and IL-22 at levels at least 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 2.5-fold, or 3-fold higher than PB-derived MAIT cells. In other embodiments, liver-derived MAIT cells are used instead of PB-derived MAIT cells as a basis for comparison.

[0117] In some embodiments, the engineered MAIT cells disclosed herein exhibit antigen-independent activation in response to IL-18, particularly in combination with IL-12, IL-15, and / or type I interferons (e.g., interferon-α / β). In some embodiments, activation is manifested, in particular, through the secretion and upregulated expression of IFN-γ and granzyme B, respectively. In other embodiments, activation is manifested through the upregulated expression of the IL-18 receptor (IL-18R).

[0118] In some embodiments, the engineered MAIT cells disclosed herein exhibit antigen-independent activation in response to a combination of IFN-α / β and IL-12 and IL-18. In some embodiments, activation is manifested, in particular, through the secretion and upregulated expression of IFN-γ and granzyme B, respectively.

[0119] Exogenous T-cell receptor (eTCR)

[0120] In some embodiments, IVB-derived MAIT cells are engineered to express a foreign antigen receptor, which is a foreign, unmodified or modified T-cell receptor (TCR). Such cells include “TCR-engineered” MAIT cells. The production of TCR-engineered MAIT cells expressing a foreign TCR can be achieved by a variety of methods generally known in the art. Some methods are described below for illustrative purposes. As used herein, the terms “TCR-MAIT” and “TCR-MAIT cells” encompass MAIT cells that have been engineered to express a foreign TCR (e.g., IVB-derived MAIT cells) and are used interchangeably, having all the same properties and meanings.

[0121] Non-limiting examples of TCR-engineered cells are described in Debets et al., TCR-engineered T cells to treat tumors: Seeing but not touching? Semin Immunol. 28:10-21 (2016); Ping et al., T-cell receptor-engineered T cells for cancer treatment: current status and future directions. Protein Cell. 9:254-266 (2018), and the references cited therein; as well as U.S. Patent Application Publication Nos. 2020 / 0237820 and 2017 / 0224733, which are incorporated herein by reference.

[0122] As used herein, unmodified TCRs include TCRs isolated from non-MAIT T cells and introduced into MAIT cells, such as those derived from the placenta or IVB, without any modifications for improving expression levels, binding affinity, or other characteristics. Modified TCRs, as used herein, include TCRs isolated from non-MAIT T cells, modified as described herein, and introduced into MAIT cells, such as those derived from the placenta or IVB. Non-limiting examples of methods for modifying TCRs include, but are not limited to, improving TCR affinity, as described in Ohta et al., Improving TCR affinity on 293T cells. J Immunol Methods 466:1–8 (2019). In some embodiments, the TCR has a K+ of greater than 2.5 nanomolar. D The target affinity (reflected by a number less than 2.5 nM). In other embodiments, the TCR has an affinity for the antigen-binding MHC of 10... -4 -10 -6 Between M.

[0123] In some other embodiments, the two TCR protein chains are stabilized by introducing disulfide bonds between the two constant domains. In a further embodiment, the modified TCR has an affinity of 10 for antigen-binding MHC. -10 -10 -12 Between M.

[0124] The TCR is a disulfide-linked, membrane-bound heterodimeric protein expressed on the surface of T cells and is a member of the immunoglobulin superfamily. The TCR binds to MHC / HLA complexed antigenic peptides via its variable region to induce downstream T cell signaling (referred to herein as TCR-mediated signaling). TCRs typically contain highly variable alpha and beta chains complexed with a constant CD3 chain molecule; a minority of TCRs contain variable gamma and delta chains. Both the alpha (α) and beta (β) chains contain two extracellular domains: a variable region (VR) and a constant region (CR). Each variable region (e.g., in the α and β chains) contains three hypervariable regions, also known as “complementarity-determining regions” (CDRs) separated from the framework region (FR). CDR3 is the major CDR responsible for antigen binding. The α and β chains also contain a junctional (J) region. The β chain typically also contains a diversification (D) region between the V and J regions; however, this D region can be considered part of the J region.

[0125] target antigen

[0126] In some implementations, exogenous antigen receptors, such as TCRs as described herein, guide engineered MAIT cells to recognize tumor antigens.

[0127] In some embodiments, TCR targets include tumor-associated antigens (TAAs) or cancer antigens. As used herein, the terms “tumor-associated antigen,” “tumor antigen,” or “cancer antigen” refer to any protein, peptide, or antigen associated with a tumor or tumor cell (carried by, expressed by, produced by, secreted by, etc., by a tumor or tumor cell). Tumor-associated antigens may be associated (almost) only with tumors or tumor cells and not with healthy normal cells, or may be overexpressed (e.g., 50-fold, 100-fold, 1000-fold, or more) in tumor tissues or tumor cells compared to healthy normal tissues or cells. Those skilled in the art will understand that, in the case of exogenous TCRs, recognition of such antigens is typically MHC-dependent. More specifically, the TAA to be targeted by the TCR according to the invention is an antigen capable of being presented (in processed form) by the MHC determinants of tumor cells.

[0128] Non-limiting examples of suitable cancer antigens include the following: NY-ESO-1; alpha-fetoprotein; desmosome connective protein / AHNAK. S2580F (e.g., for relapsed or refractory multiple myeloma); Cancer / Testis antigen 1; CD7 (e.g., for treating T-cell leukemia or lymphoma); c-MET; DR5 (e.g., for treating neuroblastoma); Epstein-Barr virus (e.g., for treating B-cell malignancies); ERBB2 H473Y and ERBB2IP E805G gp100; histone H3 trimethylation; MAGEA1; MAGE-A3 / A6; MAGEA4 / 8; Melan-A / MART-1; minor H antigen (HA-1); (e.g., for the treatment of solid tumors or colorectal cancer); PRAME; TPBG (trophoblast glycoprotein) or 5T4 (e.g., for the treatment of solid tumors such as colorectal cancer, ovarian cancer, and gastric cancer, as well as childhood acute lymphoblastic leukemia (ALL)); TGFβRII frameshift antigen; VEGFR-2; Wilms tumor 1 (WT-1); KRAS-G12V; KRAS-G12D; TP53R175H; AFP; KK-LC-1; RAC1-P29S; PIK3CA; LAGE-1A; P53; COL6A3; HA-2; HERV-E; and BRAF.

[0129] In some embodiments, the TCR is capable of specifically binding to epitopes presented by HLA class I. In some embodiments, the TCR is capable of specifically binding to epitopes presented by HLA-A. In a particular embodiment, the TCR is capable of specifically binding to epitopes presented by HLA-A2. In another embodiment, the TCR recognizes (specifically binds to) a tumor antigen (also referred to herein as a tumor-associated antigen or TAA). In various embodiments, the tumor antigen is selected from the group consisting of: NY-ESO-1, KRAS (e.g., mutant G12C, G12D, and G12V), p53 (e.g., mutant residues R175, G245, R248, R249, R273, and R282), PIK3CA, PTEN, and ERBB2. (HER2), AFP, KK-LC-1, RAC1-P29S, LAGE-1A, COL6A3, HA-2, HERV-E, BRAF, gp100, alpha-fetoprotein, desmosome-associated protein / AHNAKS2580F, cancer / testis antigen 1, ERBB2H473Y, ERBB2IPE805G, minor H antigen (HA-1), PRAME, TPBG, 5T4, MAGEA1, MAGE-A3 / A6, MAGEA4 / 8, Melan-A / MART-1, NRAS, and Wilms tumor 1 (WT-1). In some implementations, the tumor antigen is selected from the group consisting of: NY-ESO-1, KRAS (e.g., mutant G12C, G12D, and G12V), p53 (e.g., mutant residues R175, G245, R248, R249, R273, and R282), PIK3CA, PTEN, and ERBB2. (HER2), AFP, KK-LC-1, RAC1-P29S, LAGE-1A, COL6A3, HA-2, HERV-E, BRAF, gp100, alpha-fetoprotein, desmosome-connecting protein / AHNAKS2580F, cancer / testis antigen 1, ERBB2H473Y, ERBB2IPE805G, minor H antigen (HA-1), NKG2DL, PRAME, TPBG, 5T4, MAGEA1, MAGE-A3 / A6, MAGEA4 / 8, Melan-A / MART-1, NRAS, and Wilms tumor 1 (WT-1).In some embodiments, the tumor antigen is a cancer testis antigen, including but not limited to NY-ESO-1 (CTAG1B), the MAGE family (e.g., MAGEA1, MAGE-A3 / A6, MAGEA4 / 8, MAGEC1, MAGEC3), the BAGE family (e.g., BAGE1, BAGE2), the GAGE ​​family (e.g., GAGE1, GAGE2, GAGE3), the SSX family (e.g., SSX2, SSX4), the STAGE family (e.g., CTAGE-1, CTAGE-5), the PAGE family (e.g., PAGE1, PAGE2, PAGE3), PRAME, cancer / testis antigen 1, and LAGE-1A. In another specific embodiment, the tumor antigen is a mutated KRAS antigen, including but not limited to G12C, G12D, and / or G12V mutations. In yet another specific embodiment, the tumor antigen is a mutated NRAS, including but not limited to codon 12, 13, and 61 mutations. In another specific embodiment, the tumor antigen is a mutated p53 antigen, including but not limited to R175, G245, R248, R249, R273, and / or R282 mutations. In a specific embodiment, the epitope differs from a viral epitope.

[0130] In another embodiment, the tumor antigen is selected from the group consisting of NY-ESO-1 and LAGE-1A. In a particular embodiment, the antigen is NY-ESO-1. Exemplary and advantageous TCRs against human NY-ESO-1 are disclosed and illustrated below.

[0131] In some embodiments, the exogenous antigen receptor targets patient-specific and / or shared tumor neoantigens. As used herein, the term "neoantigen" refers to a peptide not present in normal human tissues and expressed on a tumor of interest. Tumor neoantigens are generally known to those skilled in the art. Examples of shared neoantigens include, but are not limited to, KRAS (e.g., mutant G12C, G12D, and G12V), p53 (e.g., mutant residues R175, G245, R248, R249, R273, and R282), PIK3CA, PTEN, and ERBB2. In another embodiment, the neoantigen is selected from the group consisting of KRAS (e.g., mutant G12C, G12D, and G12V), p53 (e.g., mutant residues R175, G245, R248, R249, R273, and R282), PIK3CA, and PTEN. Optionally or additionally, the engineered MAIT cells disclosed herein are used to target tumor cells expressing MR-1. However, in other embodiments, the cell composition of the present invention is not used to treat tumors expressing MR-1.

[0132] According to other implementation schemes, it is anticipated that a TCR targeting additional antigens will be used. Those skilled in the art will understand that the recognition of such antigens is typically MHC-dependent, and more often HLA class I-dependent.

[0133] In some other embodiments, the TCR may target microbial antigens, pathogens, viral antigens, fungal antigens, or bacterial antigens, each of which represents a separate embodiment of the invention. In other embodiments, the target of the exogenous TCR is a microbial antigen. Examples of microbial antigens include, but are not limited to, viral antigens, bacterial antigens, and fungal antigens. In some embodiments, the engineered MAIT cells disclosed herein are used to treat diseases or infections caused by microbes expressing antigens. Those skilled in the art will understand that microbes include bacteria, viruses, fungi, and parasites, wherein the microorganism that causes disease is called a pathogen. In some embodiments, as used herein, the term "microbial antigen" may encompass a pathogenic target, i.e., a pathogenic antigen.

[0134] In some implementations, exogenous TCRs target bacterial antigens, which are used in some implementations to treat bacterial infections. In some implementations, the targeted bacteria may be selected from the genera *Nitrospira*, *Nitrosospira*, *Nitrobacter*, *Nitrosomonas*, *Clostridium*, *Bacillus*, methanogenic archaea, *coliforms*, *Salmonella*, *Bacteroides*, *Staphylococcus*, *Streptococcus*, *Neisseria*, *Haemophilus*, *Bordetella*, and *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 The genera *Francisella*, *Campylobacter*, and *Helicobacter* are listed as spp.

[0135] In some embodiments, the exogenous TCR targets fungal antigens, which are used in some embodiments to treat fungal infections. In one embodiment, the fungal infection is a yeast infection.

[0136] In another embodiment (e.g., when a placental-derived MAIT such as IVB MAIT is intended to be used), the antigen is a viral antigen. As used herein, "viral antigen" includes antigens expressed by viral proteins, including cases where the antigen is currently expressed by a virus or cancer cells (e.g., in the case of oncogenic proteins). Thus, in some embodiments, engineered MAIT cells that recognize viral antigens are used to treat viral infections; or, in other embodiments, for treating malignancies expressing viral antigens. In other embodiments, the antigen is different from a viral antigen.

[0137] Non-limiting examples of viral antigens include the following: hexon or penton, for example, for the treatment of adenovirus; HPV E6; HPV E7; Immediate Early-1 (IE-1) or a 65 kDalton envelope phosphoprotein (pp65), for example, for the treatment of cytomegalovirus (CMV); EBV nuclear antigen 1 (EBNA1), BZLF1, or a 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 herpesvirus 6 (HHV-6); herpes simplex virus-1 (HSV-1) thymidine kinase (HSV-TK), for example, for the treatment of HSV-1.

[0138] In various embodiments, the antigen is characterized by surface expression of fewer than 200, 100, 80, 70, 50, 40, 30, 20, or 15 molecules per cell. Each possibility represents a separate embodiment of the invention.

[0139] Cancer testicular antigen

[0140] According to some embodiments, the present invention provides compositions and methods comprising a population of engineered MAIT cells expressing exogenous TCRs targeting cancer testis antigens. Cancer testis antigens are proteins that are normally expressed only in human testicular germ cells but are aberrantly expressed in various types of cancer cells. They are characterized by restricted expression in normal adult tissues (primarily in testicular germ cells), while expression in other cells and tissues is generally absent in healthy adults. These antigens can be expressed in a wide variety of tumor types and can be used to induce tumor-specific immunogenicity in cancer patients.

[0141] For example, but not limited to, TAAs including HLA class I restricted epitopes can be derived from cancer testis antigens including: NY-ESO-1 (CTAG1B), MAGE family (e.g., MAGEA1, MAGE-A3 / A6, MAGEA4 / 8, MAGEC1, MAGEC3), BAGE family (e.g., BAGE1, BAGE2), GAGE ​​family (e.g., GAGE1, GAGE2, GAGE3), SSX family (e.g., SSX2, SSX4), STAGE family (e.g., CTAGE-1, CTAGE-5), PAGE family (e.g., PAGE1, PAGE2, PAGE3), PRAME, cancer / testis antigen 1, and LAGE-1A.

[0142] According to a specific embodiment, the cancer testis antigen is selected from the group consisting of NY-ESO-1 and LAGE-1A. In some embodiments, the cellular composition of the present invention comprises a population of engineered MAIT cells expressing an exogenous TCR targeting NY-ESO-1.

[0143] The term “NY-ESO-1” or “New York esophageal squamous cell carcinoma 1” refers to the well-known cancer-testis antigen (CTA), also known as cancer / testis antigen 1B (CTAG1B). The human CTAG1B gene maps to the Xq28 region of the X chromosome and is silenced in normal somatic cells except for the male testes. However, as a result of epigenetic events involving tightly controlled recruitment and sequential interactions of histone deacetylases, histone methyltransferases, DNA methyltransferases, and transcription factors, NY-ESO-1 is aberrantly expressed in many types of cancer cells. The human NY-ESO-1 sequence is shown in accession number NP_001318.1.

[0144] NY-ESO-1 expression has been reported in a wide range of tumor types, including neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, and breast cancer.

[0145] Identifying the various naturally occurring or engineered TCRs of NY-ESO-1 is known in the art, such as those described in Thomas et al. (Front Immunol. 2018, 9:947) and US10201597, WO2020188348, US 20200040358 and US8088379, which are incorporated herein by reference.

[0146] In some implementations, the TCR recognizes epitopes containing amino acids 157-167 of human NY-ESO-1. For example, Robbins et al. (J. Immunol. 2008, 180: 6116–6131) disclosed a variant of the 1G4 TCR, which recognizes HLA-A... 02. Identification of a peptide corresponding to amino acid residues 157-165 (SEQ ID NO: 9) of NY-ESO-1 in the context of class I alleles. Some inventors and collaborators also disclose in WO 2024 / 023826 the recognition of peptides corresponding to amino acid residues 157-165 (SEQ ID NO: 9) of NY-ESO-1 in the context of HLA-A. In the context of 02, engineered TCRs targeting this epitope were developed. The contents of these publications are incorporated herein by reference. In some embodiments, the TCR according to the invention specifically binds to an epitope presented by HLA-A2, said epitope comprising the amino acid sequence of SEQ ID NO: 9 corresponding to residues 157 to 165 of human NY-ESO-1.

[0147] NY-ESO-1 shares sequence homology with its paralog CTAG2 (NY-ESO-2 or LAGE-A1), therefore TCRs can be designed to target both tumor antigens. For example, the peptide epitope in SEQ ID NO: 9 is characteristic of both tumors expressing NY-ESO-1 and tumors expressing LAGE-A1.

[0148] According to a specific embodiment, the TCR of the present invention in HLA-A 0201 and / or HLA-A In the context of 0206, this refers to the peptide epitope of SEQ ID NO: 9. As used herein, the term "HLA-presenting" peptide, epitope, or antigen refers to a peptide capable of specifically binding to an antigen-binding groove of an MHC or a specific allele thereof (e.g., an HLA-A2-presenting epitope is specific to the HLA-A2 allele). Such antigens are commonly referred to in the art as being "restricted" by such MHC. Antigens typically have characteristic sizes and / or chemical compositions – for example, in the case of peptide antigens, characteristic amino acid lengths and anchored residue sets, respectively – enabling them to specifically bind to the antigen-binding groove of a specific MHC haplotype in order to form an MHC / antigen complex with it having an antigen-presenting portion capable of specifically binding to a homologous TCR. For example, for HLA-A2, the anchoring sites are P2 and P9.

[0149] According to an exemplary embodiment, the TCR according to the present invention comprises the complementarity determination region (CDR) sequence shown in Table 1 below, wherein, respectively, CDR1α, CDR2α and CDR3α correspond to the CDR1, CDR2 and CDR3 sequences of the alpha chain, and CDR1β, CDR2β and CDR3β correspond to the CDR1, CDR2 and CDR3 sequences of the beta chain.

[0150] Table 1 – Exemplary CDR sequences against NY-ESO-1 TCR

[0151]

[0152] In a particular embodiment, the tumor antigen is NY-ESO-1. According to an exemplary embodiment, the TCR comprises a TCR α chain and a TCR β chain, the TCR α chain comprising CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 2, and CDR3 having the amino acid sequence of SEQ ID NO: 3, and the TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 6. In a particular embodiment, the TCR comprises a TCR α chain and a TCR β chain, the TCR α chain having the amino acid sequence shown in SEQ ID NO: 7, optionally excluding the signal peptide at positions 1-20 of SEQ ID NO: 7, and the TCR β chain having the amino acid sequence shown in SEQ ID NO: 8, optionally excluding the signal peptide at positions 1-21 of SEQ ID NO: 8. In another embodiment, the TCR comprises a CDR sequence with certain modifications (typically conserved substitutions) compared to the CDR sequence described herein, thereby preserving antigen specificity. In some embodiments, the analogs or derivatives described herein comprise no more than three, no more than two, or no more than one amino acid substitution.

[0153] According to the preferred implementation scheme, the TCR for NY-ESO-1 includes:

[0154] (a) A TCR α chain comprising: i. VR, comprising CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 2 and CDR3 having the amino acid sequence of SEQ ID NO: 3; ii. CR, comprising a cysteine ​​residue at position 47 and an amino acid sequence as shown in SEQ ID NO: 10 at positions 250-254;

[0155] (b) A TCR β chain comprising: i. VR, comprising CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 6; and ii. CR, comprising a cysteine ​​residue at position 57; and

[0156] (c) Multiple interchain disulfide bonds between the α chain and the β chain.

[0157] For example, the amino acid sequences (including the signal peptide, which is underlined) of the full-length α and β chain precursors according to a particularly advantageous embodiment of the invention are shown in SEQ ID NO: 7-8, respectively, as follows:

[0158] MFETLLGLLILWLQLQWVSS KQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLYGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLR DSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (alpha chain precursor, SEQ ID NO: 7).

[0159] MSIGLLCCAALSLLWAGPVNA GVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLA TGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (Beta chain precursor, SEQ ID NO: 8).

[0160] In other embodiments, the TCR may include certain modifications (e.g., substitutions) to the amino acid sequences shown in SEQ ID NO: 7 and 8 that retain a high degree of homology (e.g., greater than 95%, 96%, 97%, 98%, or 99%), as long as the structural elements shown in items (a) to (c) above are maintained. In certain embodiments, the substitutions are conservative substitutions.

[0161] As used herein, the terms “homology” and “sequence identity” refer to the degree of association between two or more amino acid sequences, or two or more nucleic acid sequences, as determined by sequence comparison. Sequence comparison and determination of sequence identity or homology can be accomplished using mathematical algorithms; those skilled in the art will recognize computer programs that can be used to align two sequences and determine the percentage of identity between them. The term “homology” specifically refers to the percentage of amino acid residues or nucleotides in a sequence that are identical to residues in a reference polypeptide or polynucleotide being compared with it, after sequence alignment, and in some embodiments after introducing vacancies as needed to achieve maximum percentage homology, without regard to any conserved substitutions. As used herein, in the context of two or more nucleic acid or polypeptide sequences, the term “% identity,” which can be used interchangeably with “sequence identity,” refers to two or more sequences having a specified percentage of identical nucleotide or amino acid residues, as measured by visual inspection, when comparing and aligning for maximum correspondence, using a suitable sequence comparison algorithm (e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art). Furthermore, regarding sequence identity as used in this paper, the total length of the molecules being compared is also taken into account, so that the degree of identity is calculated over the entire length of the sequence rather than in a localized manner.

[0162] Genetic engineering of MAIT cell populations

[0163] As used herein in conjunction with the terminology of MAIT cell populations, the terms "engineered" and "genetically engineered" are used interchangeably and refer to genetic modifications of the population, whether transient or permanent. Engineered MAIT cells preferably express heterologous molecules, such as the TCR transgenes disclosed herein. Therefore, engineered MAIT cells are distinctly different from naturally occurring cell populations such as primary MAIT cells and from unmodified expanded MAIT cells. Such modifications can be conveniently performed using available recombination methods, which can be further employed with additional techniques such as viral and nonviral transduction, mRNA electroporation, LNPs, transposons, gene editing (e.g., using the CRISPR-Cas9 system).

[0164] Recombinant methods for designing, expressing, and purifying proteins, peptides, and nucleic acid molecules are known in the art (see, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold SpringsHarbor Laboratory, New York). Nucleic acid molecules may include DNA, RNA, or derivatives of DNA or RNA. Isolated nucleic acid sequences encoding polypeptides or peptides may be obtained from their natural sources as complete (i.e., whole) genes or parts thereof. Nucleic acid molecules may also be produced using recombinant DNA techniques (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Nucleic acid sequences include natural nucleic acid sequences and their homologues, including but not limited to modified nucleic acid sequences (where nucleotides have been inserted, deleted, substituted, and / or reversed in a manner that substantially does not interfere with the ability of the nucleic acid molecule to encode a functional product). Polynucleotide or oligonucleotide sequences may be derived from the genetic code of a protein; however, the degeneracy of the code and the exceptions to classical base pairing allowed at the third position of a codon, as given by the so-called "wobble rule," must be considered. Polynucleotides, which contain more or fewer nucleotides, can produce the same or equivalent proteins. Using recombinant production methods, selected host cells (e.g., host cells of microorganisms such as E. coli or yeast) are transformed with a hybrid virus or plasmid DNA vector containing a specific DNA sequence encoding a polypeptide, and the polypeptide is synthesized in the host through transcription and translation of the DNA sequence.

[0165] Such recombination methods can also be used to prepare nucleic acid constructs, particularly expression constructs or vectors for the delivery and expression of exogenous TCRs in suitable host cells (such as in MAIT cell populations), as detailed herein. As is known in the art, constructs comprise the nucleic acid molecules of the present invention and may also include regulatory sequences or selectivity markers. Nucleic acid constructs (also referred to as vectors in some embodiments) may include additional sequences that adapt the vector for replication and integration in prokaryotes, eukaryotes, or optionally both (e.g., shuttle vectors). Furthermore, typical cloning vectors may also contain transcription and translation initiation sequences, transcription and translation terminators, and polyadenylation signals.

[0166] In another embodiment, the nucleic acid construct comprises at least one nucleic acid molecule encoding TCR α and β chains as disclosed herein. In another embodiment, at least one nucleic acid molecule is operatively linked to one or more transcriptional control elements.

[0167] The phrase "operably linked" refers to a nucleic acid sequence linked to a transcriptional control sequence in a manner that enables the molecule to be expressed when transfected (i.e., transformed, transduced, infected, or transfected) into a host cell. Transcriptional control sequences are those that control the initiation, elongation, and termination of transcription. Particularly important transcriptional control sequences are those that control transcription initiation, such as promoters, enhancers, operons, and repressor sequences.

[0168] For example, in expression vectors capable of expressing nucleic acid molecules in human MAIT cells, the expression control sequence operatively linked to the gene product to be expressed includes a promoter and other elements active in human MAIT cells. The promoter may be genomically derived or synthetically produced. A variety of promoters for lymphocytes, including MAIT cells, are well known in the art. For example, the promoter may be constitutive or inducible, wherein induction is associated with activation or maturation at a specific cell type or level. According to an exemplary embodiment, the promoter is the human eukaryotic translation elongation factor 1 α1 promoter, EF1A. Alternatively, many well-known viral promoters are also suitable. Promoters of interest include, but are not limited to, the β-actin promoter, SV40 early and late promoters, immunoglobulin promoters, human cytomegalovirus promoters, retroviral promoters, and the Friend spleen focus-forming virus promoter. The promoter may or may not be associated with an enhancer, wherein the enhancer may be naturally associated with a specific promoter or with different promoters.

[0169] In another embodiment, the invention utilizes an expression vector comprising at least one nucleic acid construct as disclosed herein. As used herein, the term "expression vector" refers to a vector comprising a recombinant polynucleotide containing an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by a host cell or in an in vitro expression system. Expression vectors include suitable vectors known in the art, such as viscera, plasmids (e.g., naked or contained in liposomes) incorporating recombinant polynucleotides, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses). In another embodiment, the vector is a viral vector. In a particular embodiment, the vector is a retroviral vector.

[0170] Suitable viral vectors are known in the art and are commercially available. Exemplary retroviral vectors include, but are not limited to, pMSGV1, pMSCV, pBMN, pQXIX, pBullet, pBabe, pMSGV, pRETRO, pMIGR, pMX, and pRET. Exemplary lentiviral vectors include, but are not limited to, pRRL, pCLX, pLenti, pHR, pLVX, and pCAG. Additional exemplary vectors include, but are not limited to, pBullet, pLSC, adenovirus vectors (including their single and multiple replication-deficient forms) (including, but not limited to, adenovirus type 5 (Ad5) and adenovirus type 35 (Ad35)), and adeno-associated virus (AAV) vectors (including, but not limited to, pAAV and pX601). In another embodiment, the vector is a retroviral vector containing an LTR (MSCV LTR) derived from a mouse stem cell virus, such as a vector derived from pMSGV1 or pMSCV.

[0171] Pharmaceutical Composition

[0172] In some embodiments, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and a population of engineered MAIT cells disclosed herein.

[0173] In some embodiments, the composition comprises engineered MAIT cells expressing a foreign antigen receptor. As used herein, in some embodiments, the terms "composition" and "pharmaceutical composition" are used interchangeably and have all the same properties and meanings. In some embodiments, pharmaceutical compositions for treating conditions or diseases as described herein are disclosed.

[0174] In some embodiments, this document discloses a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a population of engineered MAIT cells as described herein.

[0175] Those skilled in the art will understand that a “pharmaceutical composition” may encompass a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the compound to a living organism.

[0176] Those skilled in the art will understand that the phrases “physiologically acceptable carrier,” “pharmaceutically acceptable carrier,” “physiologically acceptable excipient,” and “pharmaceutically acceptable excipient” are used interchangeably to cover carriers, excipients, or diluents that do not cause significant irritation to organisms and do not eliminate the biological activity and properties of the applied active ingredient.

[0177] Techniques for the formulation and administration of drugs or drug compositions are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

[0178] In some embodiments, the compositions or pharmaceutical compositions disclosed herein are injectable compositions manufactured by adding one or more excipients (e.g., stabilizers and aqueous buffers) to a population of engineered MAIT cells described herein.

[0179] Optionally or additionally, prior to administration to a patient, the engineered MAIT cells described herein have been expanded by 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 other embodiments at least 5000-fold.

[0180] In some embodiments, a method for preparing a pharmaceutical composition comprising the engineered MAIT cells described herein includes the step of freezing (e.g., cryopreservation) the cells before or after isolation, incubation, and / or engineering. Examples of suitable cryogens are generally known in the art.

[0181] 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.

[0182] In other embodiments, a pharmaceutical composition is provided comprising the engineered MAIT cells disclosed herein, wherein the composition is indicated for the treatment or improvement of any of the diseases, conditions and complications mentioned herein, each representing a separate embodiment.

[0183] In various embodiments, the pharmaceutical compositions disclosed herein can be administered in a systemic manner known in the art. Optionally, the pharmaceutical compositions can be administered locally, for example, by direct injection into a tissue region of a patient, such as intratumoral or intralesional administration as in the non-limiting embodiments. In other embodiments, engineered MAIT cells are administered intramuscularly, intravenously, subcutaneously, or intraperitoneally, each of which is considered a separate embodiment. In this regard, "intravenous" includes administration into a vein of the subject. In some embodiments, the pharmaceutical compositions are administered intralymphatically as previously described in the art. For example, routes of administration particularly suitable for adoptive transfer include systemic (e.g., intravenous or intraperitoneal) or local injection (e.g., intratumoral or intrathecal, such as injection into the cerebrospinal fluid of a brain tumor).

[0184] The effective dosage and schedule for administering the composition can be determined empirically, and making such a determination is within the scope of the art. If any contraindications occur, the dosage can be adjusted by an individual physician. Exemplary effective amounts of engineered MAIT cells for use in ACT compositions are provided below.

[0185] Depending on the severity and responsiveness of the condition to be treated, administration may be a single dose, or in other implementation schemes, multiple doses until remission of the disease state is achieved.

[0186] In some embodiments, the treatment methods described herein further include lymphodepleting the recipient prior to treatment, or immunosuppression in other embodiments. In other embodiments, reversible lymphodepletion or immunosuppression prolongs the biological half-life of the transplanted cells. Methods for lymphodepletion and immunosuppression of patients are known in the art. In some embodiments, lymphodepletion or immunosuppression is not necessary for the administration of the engineered MAIT cells disclosed herein, at least in part because of their low immunogenicity.

[0187] In various embodiments, implantation of engineered MAIT cells into the host is not essential for the cells to exert the therapeutic effect, and each is considered a separate implementation. In other embodiments, implantation is essential for the engineered MAIT cells to be effective.

[0188] In some embodiments, the subject treated with 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. In yet another embodiment, the subject does not have an infection (e.g., a viral infection).

[0189] This document also discloses reagent kits and articles of manufacture relating to reagents that can be used to carry out the methods disclosed herein. Reagent kits and articles of manufacture may include any reagents or combinations of reagents discussed herein or which will be understood to be necessary or beneficial in carrying out the disclosed methods, including lymphoid cells. In another aspect, reagent kits and articles of manufacture include labels, instructions for use, and packaging materials, for example, for treating the conditions or indications mentioned herein.

[0190] Adoptive cell transfer therapy (ACT)

[0191] As used herein, unless otherwise stated, the term “adoptive transfer” refers to a form of passive immunotherapy in which a previously sensitized immunotherapy agent (e.g., cells or serum) is transferred to a recipient. The phrases “adoptive cell therapy,” “adoptive cell transfer,” “adoptive immunotherapy,” “adoptive transfer therapy,” “adoptive cell therapy,” and “adoptive cell immunotherapy” are used interchangeably herein to denote a therapeutic or prophylactic regimen or manner in which effector immunocompetent cells are administered (adoptively transferred) to a subject in need to alleviate or improve the development or symptoms of cancer or infectious diseases.

[0192] Adopted cells are typically targeted at tumors or tumor-associated antigens, such as NY-ESO-1. Specifically, the ACT compositions of the present invention comprise an effective amount of MAIT cells engineered to express TCRs as disclosed herein. Therefore, the ACT compositions according to the present invention (also referred to herein as "compositions adapted for ACT") comprise an effective amount of live, engineered cells as disclosed herein, produced under sterile and suitable (e.g., cGMP grade) conditions, said composition to be administered to human subjects as described herein as part of their treatment (e.g., antitumor) regimen. Furthermore, the cells administered in the ACT compositions are typically non-attenuated and capable of proliferating in the recipient subject in the presence of the relevant antigen (e.g., tumor antigen).

[0193] As used herein in conjunction with cell compositions, an effective amount (also referred to herein as a therapeutically effective amount) is an amount sufficient, when administered to a subject, to induce or enhance a beneficial immune response, such as an antitumor response, for example, 10 7 Up to 10 12 One cell or 10 8 Up to 10 12 Cells. Specifically, regarding the ACT compositions of the present invention, the effective amount is sufficient to induce or enhance the immune response in the event of adoptive transfer to a recipient subject, and typically contains at least 10 9 More typically at least 5 × 109 Individual cells, and at most approximately 10 × 10 10 One or more cells, for example 5 × 10⁶ cells provided by a preparation process as disclosed herein. 9 -10×10 9 One living cell.

[0194] In one aspect, a cell composition is provided comprising a population of engineered mucosa-associated inert T (MAIT) cells expressing a extrinsic T cell receptor (TCR), wherein the MAIT cells are derived from the placenta, and the cell composition optionally further comprises a pharmaceutically acceptable carrier.

[0195] In one embodiment, the MAIT cells are derived from placental intervillous blood (IVB). In another embodiment, the composition is adapted for adoptive transfer cell therapy (ACT). In some embodiments, the composition contains at least about 10 7 Or 10 8 The engineered MAIT cell population consists of live cells, typically containing 10 9 -10 11 5×10 9 -10 11 Or 10 10 -10 11 The cells of the aforementioned population.

[0196] According to this disclosure, the cell compositions of the present invention are characterized by unique structural features that provide advantageous functional properties, as will be further described and illustrated below.

[0197] As disclosed herein, the cell compositions according to the invention comprise a population of substantially purified engineered MAIT cells, such that the composition contains less than 10% and typically less than 8%, 7%, 5%, 3%, 2%, 1%, or 0.5% of other cell types. Typically, the cell composition comprises a population of ex vivo expanded MAIT cells engineered to express exogenous TCRs as disclosed herein. In some embodiments, the cell compositions of the invention comprise at least 90% TCRVα7.2. + CD161 + Cells, for example, at least 93%, 95%, 98%, 99% or more. Typically and advantageously, the cell compositions of the present invention contain at least 90% TCRVα7.2. + CD161 高Cells, for example, at least 93%, 95%, 98%, 99% or more. According to a specific embodiment, the surface expression level of CD161 is characterized by a reduction of less than 10%, 7%, 5%, 3%, 2% or 1% (conveniently measured as mean fluorescence intensity by flow cytometry) compared to primary MAIT cells isolated from placental IVB.

[0198] The cell compositions of the present invention are characterized by the expression of exogenous TCRs (eTCRs, which are typically α / β TCRs) on the surface of at least 20% of the cells and typically at least 30%, 40%, or 50% of the cells. According to additional advantageous embodiments demonstrated herein, the inventive preparation process for producing the cell compositions of the present invention is characterized by high surface expression levels of eTCRs. For example, more than 50% and typically at least 55%, 60%, 65%, 70%, 75%, or 80% of the cells may be characterized by surface expression of the eTCRs, for example, 70-80%, 75-85%, 70-90%, or 80%-95% eTCRs. + Cells. In another embodiment, at least 75% and more typically at least 80% or 85% of said cells are CD8 cells. + Cells (e.g., 85-95%, 80-98%, or 90-100%).

[0199] In another embodiment, the cell composition of the present invention is characterized by a significantly enhanced level of one or more markers compared to a control cell composition (such as a corresponding ACT composition of expanded peripheral blood T cells). In some embodiments, at least one marker (characterized by a significantly enhanced level in the composition of the present invention) is selected from the group consisting of CD69, MDR1, siglec-7, and KLRG1, wherein each possibility represents a separate embodiment of the present invention. In some embodiments, at least 5% and typically at least 7%, 10%, 15%, 20%, 30%, 40%, 45%, or 50% of the cells are MDR1. + (e.g., 5-50%, 5-20%, 6-25%, 7-40%, or 10-60%, or in other embodiments, 20-50% or more). Therefore, this is in contrast to expressing very low levels of MDR1. + (usually in CD8) + In contrast to conventional blood-derived T cells (approximately 1%), the cell composition of the present invention is characterized by MDR1. + The cells undergo significant enhancement.

[0200] In another embodiment, the cell composition of the present invention is characterized by a significantly reduced level of one or more markers compared to a control cell composition. In some embodiments, at least one marker (characterized by a significantly reduced level in the composition of the present invention) is selected from the group consisting of CD73, CD45RA, CCR7, and CXCR4, wherein each possibility represents a separate embodiment of the present invention.

[0201] In another embodiment, the cell composition of the present invention is characterized by a significantly enhanced level of one or more cytokines (e.g., IFNγ, TNFα, and / or IL-2) compared to a control cell composition. In another embodiment, the cell composition of the present invention is characterized by a significantly enhanced level of multiple cytokines (e.g., two or three) compared to a control cell composition. In another embodiment, the cell composition of the present invention is characterized by a significantly enhanced level of one or more cytotoxic T cell effector molecules (e.g., perforin and granzyme B) compared to a control cell composition. In another embodiment, the cell composition of the present invention is characterized by a significantly enhanced level of perforin or granzyme B compared to a control cell composition.

[0202] In another embodiment, the cell composition of the present invention is characterized by significantly reduced allo-reactivity compared to the control cell composition. Therefore, without being bound by a specific theory or mechanism of action, the cell composition can be used to induce antigen-specific cytotoxicity of target cells in vivo, while minimizing or delaying the rejection of the adopted MAIT cells by the recipient's immune cells, thereby improving therapeutic efficacy and / or safety.

[0203] In another embodiment, the cell composition of the present invention is further characterized by structural and functional properties (such as surface expression of additional markers) as disclosed herein and illustrated in the examples below. Each possibility represents a separate embodiment of the invention.

[0204] While the MAIT cells of the present invention can be engineered to express additional molecules or elements such as cytokines, chemokines, or their receptors in addition to exogenous TCRs, co-expression of other antigen-specific receptors such as chimeric antigen receptors (CARs) is explicitly excluded. Typically, the cell compositions of the present invention do not substantially contain cells engineered to express CARs.

[0205] On the other hand, a composition adapted for ACT is provided, the composition comprising a substantially purified population of ex vivo expanded MAIT cells engineered to express exogenous TCR (eTCR), the population comprising at least 10 97.2 live cells, of which at least 90% are TCR-Vα 7.2 + CD161 高 .

[0206] In some embodiments, the cell compositions of the present invention are characterized by a significant enhancement in the occurrence of MAIT cells exhibiting a memory phenotype (e.g., effector memory MAIT cells) and / or MAIT cells expressing CD8αα homodimers compared to control cell compositions. In various embodiments, the control cell compositions are prepared using conventional peripheral blood T cells. In another embodiment, the control cell compositions are prepared using umbilical cord blood leukocytes. In yet another embodiment, the control cell compositions are prepared using control cell populations as disclosed herein. Each possibility represents a separate embodiment of the invention.

[0207] In another embodiment, the composition may further comprise additional active ingredients, such as anticancer drugs or cytokines. In a particular embodiment, the anticancer drug is a cancer immunotherapy, such as an immune checkpoint inhibitor.

[0208] In some embodiments, a cell composition adapted for ACT is provided, comprising a therapeutically effective amount of MAIT cells engineered to express a TCR targeting NY-ESO-1, said TCR comprising:

[0209] (a) A TCR α chain comprising: i. VR, comprising CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 2 and CDR3 having the amino acid sequence of SEQ ID NO: 3; ii. CR, comprising a cysteine ​​residue at position 47 and an amino acid sequence as shown in SEQ ID NO: 10 at positions 250-254;

[0210] (b) A TCR β chain comprising: i. VR, comprising CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 6; and ii. CR, comprising a cysteine ​​residue at position 57; and

[0211] (c) Multiple interchain disulfide bonds between the α chain and the β chain.

[0212] In another embodiment, the cell composition is prepared using a process as disclosed herein. In yet another embodiment, a process for preparing the cell composition as disclosed herein is provided.

[0213] The ACT compositions of the present invention can be administered to subjects in need via, for example, intravenous, intraperitoneal, intratumoral, and / or intrathecal routes. In various embodiments, single-dose or multiple-dose administration regimens can be employed. For example, a single-dose administration route includes administration of a therapeutically effective amount of engineered MAIT cells (e.g., 10) disclosed herein. 9 -10 11 An ACT composition comprising approximately 10 cells. A single-dose administration route may include administering the ACT composition once every 3, 4, 5, 6 months or longer (e.g., until remission or another treatment endpoint is achieved). In another embodiment, a single-dose administration route may include a single administration of the ACT composition. In other instances, a multiple-dose administration route may include more frequent administration of the ACT composition containing a lower amount of engineered MAIT cells to achieve therapeutic outcomes. For example, approximately 10 7 -10 8 The dose of an engineered MAIT cell can be administered weekly or every 2, 3, 4, 5, 6, 7 or 8 weeks.

[0214] In another embodiment, the cell composition of the present invention is prepared by a process comprising the following steps:

[0215] a. Obtain a cell population containing MAIT cells.

[0216] b. The cells were incubated in the presence of MAIT cell antigen and IL-15 to generate a population of activated MAIT cells.

[0217] c. The resulting activated MAIT cells were engineered to express exogenous TCR.

[0218] d. Expand the resulting engineered MAIT cells to obtain a therapeutically effective amount of the cells, and

[0219] e. Engineered MAIT cells obtained from isolation.

[0220] In one embodiment, the cell population containing MAIT cells is obtained from the placenta. In another embodiment, the cell population containing MAIT cells is advantageously obtained from IVB. In yet another embodiment, the method may further include subjecting the cell population obtained in step a. to cryopreservation, which may be thawed prior to the incubation step (step b.).

[0221] In another embodiment, the MAIT cell antigen used in step b. is a riboflavin metabolite. In another embodiment, the MAIT cell antigen comprises 5-OP-RU. For example, but not limited to, 10-200 ng / ml IL-15 and 100-1000 nM 5-OP-RU can be used. Typically, step b. is performed in the absence of additional antigens or activators targeting (or specific to) non-MAIT TCRs, such as α / β TCR antigens. In another embodiment, step b. is performed in the absence of anti-CD3 and / or anti-CD28 antibodies. In another embodiment, step b. is performed in the absence of additional antibodies. In another embodiment, step b. is performed in the absence of additional cytokines (such as IL-2). In another embodiment, step b. is performed in the absence of additional T cell activators (such as cytokines and antibodies). In another embodiment, step b. is performed in the absence of feeder cells. In another embodiment, step b. is performed in the presence of only MAIT cell antigens (e.g., 5-OP-RU) and IL-15. According to an exemplary implementation, step b. lasts for 1-5 days or 2-4 days (e.g., 2, 3 or 4 days).

[0222] In various embodiments, the engineering of activated MAIT cells to express exogenous TCRs can be performed by suitable methods, including but not limited to viral and nonviral transduction, transfection, mRNA electroporation, LNP, transposons, gene editing (e.g., using a CRISPR-Cas9 system). In another embodiment, the engineering of activated MAIT cells to express exogenous TCRs is performed by transduction (e.g., by incubating the cells with a viral vector containing a nucleic acid construct encoding an eTCR as disclosed herein). In some embodiments, fibronectin fragments (e.g., retroviruses) or other agents that promote the co-localization of the viral vector and the intended target cells can be used to improve transduction efficiency. By way of non-limiting examples, viral vectors (e.g., retroviral vectors as disclosed herein) can be applied to a surface (e.g., a plate or well) coated with fibronectin or another suitable substance (e.g., using centrifugation) to provide a vector-coated surface, and said coated surface (e.g., using centrifugation) is incubated with MAIT cells generated as disclosed herein.

[0223] In another embodiment, step d. is performed to obtain an amplification factor of at least 100-fold (compared to the number of MAIT cells obtained in step a.). In another embodiment, the amplification factor is at least 120, 150, 200, 300, 350, 400, 500, 600, 700, 800, 900, 1,000, or more. In various embodiments, step d. is performed to obtain 10 9 -10 11 5×10 9 -10 11 Or 10 10 -10 11 The engineered MAIT cells are described. In another embodiment, step d. is performed in the presence of IL-15. In another embodiment, step d. is performed in the absence of additional antibodies. In another embodiment, step d. is performed in the absence of additional cytokines (such as IL-2). In another embodiment, step d. is performed in the absence of additional T cell activators (such as cytokines and antibodies). In another embodiment, step d. is performed in the absence of feeder cells. Typically and advantageously, step d. is performed in the absence of MAIT cell antigens. In another embodiment, step d. is performed in the presence of IL-15 as the only cytokine supporting cell proliferation. In another embodiment, step d. is performed in the presence of IL-15 as the only T cell activator. In other embodiments, a second amplification step may be added after IL-15-mediated amplification (e.g., after 7-10 days) in the presence of additional antigens or activators against α / β TCR antigens (e.g., CD3 and / or CD28 specific antibodies).

[0224] Typically, the process of the present invention further includes positive selection of cells expressing TCRVα7.2 to obtain a population of substantially purified engineered MAIT cells. According to an advantageous embodiment, step e. includes subjecting the resulting cells to positive selection of cells expressing TCRVα7.2 to obtain a population of substantially purified engineered MAIT cells. Optionally, positive selection of cells expressing TCRVα7.2 is performed after step b. and before step c. In another embodiment, step e. is performed within 7-14 days of starting step b., more preferably within 8-12 or 9-11 days, for example within 10 days of starting step b. In some embodiments, positive selection of cells expressing TCRVα7.2 can be performed by cell sorting. In various embodiments, positive selection of cells expressing TCRVα7.2 can be performed using a binding agent (e.g., an antibody) specific to the TCRVα7.2 chain and employing suitable methodologies (such as magnetic bead separation and flow cytometry). For example, step e. can be performed by flow cytometry-based sorting (e.g., by staining cells with an antibody specific to TCRVα7.2 and subjecting the cells to fluorescence-activated cell sorting (FACS)). In another embodiment, magnetic bead separation is performed (e.g., using reagents and kits commercially available, for example, from Miltenyi Biotech). According to non-limiting examples, biotinylated antibodies (such as biotinylated anti-TCR Vα7.2 antibody, TCR Va7.2 anti-human antibody, Biotin REAfinity) can be used in combination with suitable avidin-containing magnetic beads.

[0225] In another embodiment, the process includes large-scale amplification using a bioreactor suitable for large-scale clinical-grade processes. According to a non-limiting example, such as the three-dimensional (3D) bioreactor disclosed in US 11,939,562, which is incorporated herein by reference, such bioreactors can be used, for example, during the amplification step. In some embodiments, amplification is performed using a packed-bed reactor. For example, but not limited to, a reactor containing Fibra-Cel can be readily used. ® A disc-filled bed MiniBio reactor. Advantageously, the disc may be coated with FBS or other suitable coatings to create an ECM-like environment that promotes the attachment and growth of antigen-presenting cells.

[0226] Immunotherapy and special patient groups

[0227] In some embodiments, the cell compositions of the present invention are used for therapeutic purposes. In another embodiment, the cell compositions of the present invention are used for immunotherapy. In yet another embodiment, the cell compositions of the present invention are used to treat a subject with a tumor or malignancy. In some embodiments, the cell compositions of the present invention are used to treat a subject suffering from a condition associated with the expression of HLA class I restrictive antigens.

[0228] As used in this article, a disease or condition characterized by the expression of HLA class I restricted antigens is characterized by the presence of target cells expressing the antigens in the context of HLA class I molecules. Typically, the presence of such target cells contributes to the etiology and / or pathology of the disease or condition.

[0229] In another aspect, a method for treating a subject with a tumor or malignant tumor is provided, the method comprising administering to the subject a cellular composition as defined herein.

[0230] In another embodiment, the tumor or malignant tumor is characterized by the expression of HLA class I restricted epitopes of the tumor antigen. In various embodiments, the tumor antigen is selected from the group consisting of: NY-ESO-1, KRAS, p53, PIK3CA, PTEN, ERBB2 (HER2), AFP, KK-LC-1, RAC1-P29S, LAGE-1A, COL6A3, HA-2, HERV-E, BRAF, gp100, alpha-fetoprotein, desmosome connective protein / AHNAKS2580F, cancer / testis antigen 1, ERBB2H473Y, ERBB2IPE805G, minor H antigen (HA-1), PRAME, TPBG, 5T4, MAGEA1, MAGE-A3 / A6, MAGEA4 / 8, Melan-A / MART-1, NRAS, and Wilms tumor 1 (WT-1). In another embodiment, the tumor is a solid tumor. In another embodiment, the tumor is selected from the group consisting of: 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. In another embodiment (e.g., when the tumor antigen is NY-ESO-1), the tumor may be selected from the group consisting of: melanoma, myeloma, sarcoma, and bladder tumors, brain tumors (e.g., glioblastoma), ovarian tumors, lung tumors, breast tumors, synovial tumors, and prostate tumors. In other embodiments, treatment is intended for hematopoietic tumors such as acute myeloid leukemia (AML). In a particular embodiment, the tumor is melanoma. In another embodiment, the subject has a tumor or malignancy expressing NY-ESO-1. In another embodiment, the subject is HLA-A2 positive. In another embodiment, the subject has a tumor or malignancy expressing NY-ESO-1 and is HLA-A2 positive. In another specific embodiment, the subject does not have hepatoma or hepatocellular carcinoma. In another specific embodiment, the subject does not have liver cancer.

[0231] As disclosed herein, the cell compositions according to the invention are particularly effective even in the treatment of tumors and malignant tumors characterized by low levels of tumor antigen expression.

[0232] As used herein, the term "antigen density" refers to the level of expression of the antigen on the surface of target cells. When mentioned in the context of a cell population, the term indicates the average surface expression of the antigen in the population. The density or amount of a target antigen can be measured by various methods, such as semi-quantitative FACS, immunohistochemistry, or other immunoassays using labeled antibodies against the antigen.

[0233] For example, a high antigen density (e.g., 1,000 antigen molecules or more per cell) of a target antigen (e.g., a tumor antigen expressed by tumor cells) is generally considered sufficient to activate (by binding) a sufficient number of immune cells (including TCR- and CAR-based therapies) expressing receptors specific to said antigen in order to induce a clinically significant immune response. Such an immune response diminishes with decreasing antigen density (e.g., less than 200, 150, 100, 80, 60, 50 antigen molecules or less per cell) and may lead to impaired treatment response or even resistance to treatment.

[0234] Unless otherwise specified in this disclosure, the term "low antigen density" generally refers to the surface expression of fewer than 50 antigen molecules per cell. It should be understood that antigens do not need to be expressed at low density throughout the entire population of target cells to impair a durable therapeutic response. For example, the presence of a population of tumor cells characterized by low tumor antigen density can evade the immune response and ultimately lead to the development of treatment-resistant tumors, even after the initial therapeutic response. For example, low-density expression of the HLA-A-restricted antigen according to the invention comprises the surface expression of fewer than 50 complexes of antigen-derived peptide epitopes presented in the context of HLA-A.

[0235] Therefore, the term "low-density antigen" and related embodiments such as conditions associated with the expression of low-density antigens (e.g., tumors) refer to a situation in which a target cell population is present in a subject to be treated, characterized by the expression of fewer than 50 antigen molecules on the surface of each cell. Examples of low-density antigens to be targeted, in conjunction with embodiments of the invention, include, for example, NY-ESO-1 and other cancer testicular antigens disclosed herein.

[0236] As disclosed herein, the cell compositions according to the invention are particularly effective even in the treatment of tumors and malignancies characterized by low levels of tumor antigen expression. As illustrated herein, the cell compositions of the invention exhibit high cytotoxicity against target cells characterized by low antigen density (e.g., 10-50 antigen molecules per cell) compared to target cells characterized by antigen density higher than 100 antigen molecules per cell. Therefore, the cell compositions of the invention can be advantageously and effectively used even in the treatment of subjects who are additionally unsuitable for treatment with immunotherapies such as CAR T cells and antibody-based therapies (e.g., antibody-drug conjugates, radionuclide therapy) due to insufficient surface expression of disease-associated antigens (e.g., tumor antigens). In some embodiments, the subject has a treatment-resistant tumor in which one or more previous treatment processes (such as immunotherapy as described above) have failed. In another embodiment, the tumor is MDR1 positive.

[0237] For example, this document demonstrates that the cell compositions of the present invention exhibit antigen-specific cell lysis of target cells (e.g., evident through lysed caspase 3), said target cells characterized by an average antigen density of 10-50 antigen molecules per cell. Furthermore, this document surprisingly demonstrates that the compositions of the present invention are particularly effective against tumor cells with a very low antigen density of about 10-30 antigen molecules per cell, compared to tumor cells characterized by an antigen density of about 30-50 antigen molecules per cell.

[0238] In some embodiments, the engineered MAIT cells and the subject are autologous (obtained from the subject to be treated, autologous cell composition). In other embodiments, the engineered MAIT cells and the subject are allogeneic (obtained from the donor, allogeneic cell composition). In some embodiments, the allogeneic cell composition used in the method of the present invention can be administered to the subject in a modified treatment regimen, wherein the subject is provided with multiple administrations of a cell composition (prepared from the same or different donors) that may contain a subtherapeutic amount of engineered cells, so as to co-administer a therapeutic amount of the cells (e.g., a total of 10). 9 -10 11 (individual cells). In some embodiments, the cells have been obtained from a single donor (e.g., from a healthy IVB donor). In other embodiments, the cells have been obtained from multiple donors.

[0239] It should be understood that as long as the exogenous TCR is HLA-matched with the subject to be treated, the allogeneic donor cells do not need to be tissue-compatible with the subject. As disclosed herein, MAIT cells exhibit a significantly reduced likelihood of allogeneic rejection compared to conventional peripheral blood T cells expressing endogenous α / β TCRs. Therefore, donors with partial or complete HLA mismatches can be conveniently used, thereby improving the availability of treatment for various patient populations. In another embodiment, the donor of the engineered MAIT cells of the allogeneic composition is tissue-compatible with the subject. In another embodiment, the donor of the engineered MAIT cells of the allogeneic composition is partially tissue-compatible with the subject. In another embodiment, the donor of the engineered MAIT cells of the allogeneic composition is not tissue-compatible with the subject.

[0240] In another embodiment, the cell composition of the present invention is used to induce antigen-specific cytotoxicity of target cells, wherein an exogenous TCR recognizes a low-density antigen on the cells. In another embodiment, the cell composition of the present invention is used to treat a subject suffering from a condition associated with a low-density antigen, wherein the TCR recognizes the low-density antigen. According to a particular embodiment, the low-density antigen is a tumor antigen as disclosed herein. In a particular embodiment, the low-density antigen is characterized by surface expression of fewer than 50, 40, 30, 20, or 15 molecules per cell. In some other particular embodiments, the low-density antigen is a tumor antigen characterized by surface expression levels as disclosed herein. In another embodiment, the cell composition of the present invention is used for immunotherapy. In another embodiment, the cell composition of the present invention is used for minimizing or delaying host anti-graft response while inducing antigen-specific cytotoxicity of target cells. In other embodiments, the cell compositions and methods of the present invention provide enhanced persistence of the administered cells in a receiving subject compared to a control cell composition (e.g., prepared from peripheral blood T cells). In other embodiments, the cell compositions and methods of the present invention provide prolonged therapeutic activity compared to a control cell composition.

[0241] Additional usage methods

[0242] This document describes the methods of using MAIT and engineered MAIT cells as described throughout. In some embodiments, the engineered MAIT cells used in the methods disclosed herein include engineered placental MAIT cells. In some embodiments, the engineered MAIT cells used in the methods described herein include engineered IVB MAIT cells. In some embodiments, the engineered placental-derived MAIT cells used in the methods disclosed herein are derived from the mother. In some embodiments, the engineered placental-derived MAIT cells used in the methods disclosed herein are derived from the fetus. In some embodiments, the engineered placental-derived MAIT cells used in the methods disclosed herein are derived from both the mother and the fetus. In some embodiments, the engineered placental-derived MAIT cells used in the methods disclosed herein are derived from the placenta from the decidua basalis region. In some embodiments, the engineered placental-derived MAIT cells used in the methods disclosed herein are derived from the placenta from the decidua parietal region. In some embodiments, the engineered placental-derived MAIT cells used in the methods disclosed herein are derived from intervillous blood of the placenta. In some embodiments, the source of placental MAIT includes a combination of placental sources described herein. In some implementations, the placental MAIT used in the methods disclosed herein is not derived from umbilical cord blood.

[0243] In some embodiments, the MAIT cells used in the methods disclosed herein include placental MAIT cells. In some embodiments, the MAIT cells used in the methods described herein include IVB MAIT cells. In some embodiments, the placental-derived MAIT cells used in the methods disclosed herein are derived from the mother. In some embodiments, the placental-derived MAIT cells used in the methods disclosed herein are derived from the fetus. In some embodiments, the placental-derived MAIT cells used in the methods disclosed herein are derived from both the mother and the fetus. In some embodiments, the placental-derived MAIT cells used in the methods disclosed herein are derived from the placenta in the decidua basalis region. In some embodiments, the placental-derived MAIT cells used in the methods disclosed herein are derived from the placenta in the decidua parietal region. In some embodiments, the placental-derived MAIT cells used in the methods disclosed herein are derived from intervillous blood. In some embodiments, the source of placental MAIT includes 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.

[0244] In some embodiments, the MAIT cells used in the methods disclosed herein are derived from fetal blood, maternal blood, fetal and maternal blood, placental blood, or IVB blood, or combinations thereof, wherein the MAIT cells may be engineered to express exogenous antigen receptors or may not be engineered to express exogenous antigen receptors. In some embodiments, the MAIT cells are derived from fetal blood, maternal blood, fetal and maternal blood, placental blood, or IVB blood, or combinations thereof; and are not derived from umbilical cord blood, wherein the MAIT cells may be engineered to express exogenous antigen receptors or may not be engineered to express exogenous antigen receptors.

[0245] In some embodiments of the methods of use described herein, engineered MAIT cells include TCR-MAIT cells, wherein the MAIT cells are derived from IVB. In some embodiments of the treatment methods described herein, the method includes administering a population of TCR-MAIT cells to a subject in need.

[0246] In some embodiments, a method of treating a subject with a tumor or malignancy is provided, the method comprising administering to the subject a population of engineered MAIT cells as described herein. In some embodiments, a method of treating a subject with a tumor or malignancy is provided, the method comprising administering to the subject a composition comprising a population of engineered MAIT cells disclosed herein. In some embodiments, the engineered MAIT cells are allogeneic to the subject.

[0247] In other embodiments, a composition or pharmaceutical composition comprising a population of engineered MAIT cells described herein is provided for treating a subject with a tumor or malignancy. Based on this disclosure, those skilled in the art will understand that the population of engineered MAIT cells disclosed herein can be used to treat tumors, malignancies, and hyperproliferative disorders, particularly where the cells of the tumor, malignancy, or hyperproliferative disorder include (e.g., express, or contain in other embodiments) a recognized antigen. In some embodiments of the method of 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 TCRs targeting cancer antigens or tumor-associated antigens, such as, but not limited to, those disclosed herein.

[0248] In some embodiments, a method of treating a subject infected with a pathogen is provided, the method comprising administering to the subject a population of engineered MAIT cells as described herein. In other embodiments, a composition or pharmaceutical composition comprising a population of engineered MAIT cells as described herein is provided for treating a subject infected with a pathogen. The pathogen may be a bacterial pathogen, a viral pathogen, or a fungal pathogen commonly known in the art. Based on this disclosure, those skilled in the art will understand that various infections can be treated with the engineered MAIT cells disclosed herein, particularly where the pathogenic cells include (e.g., express, or contain) a recognized antigen. In some embodiments of the method of 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 as described herein, the engineered MAIT cells are allogeneic to the subject.

[0249] In some implementations, methods of treating subjects in need include using any of the engineered MAIT cells described herein, including treating subjects with tumors or malignancies, or subjects infected with pathogens.

[0250] In some implementations, the exogenous TCR of engineered MAIT cells includes an antigen-binding domain, wherein the antigens include NY-ESO-1, KRAS (mutant G12C, G12D, and G12V), P53 (residues R175, G245, R248, R249, R273, and R282), PIK3CA, PTEN, ERBB2, KRAS-G12V, KRAS-G12D, and TP53. R175H, AFP, KK-LC-1, RAC1-P29S, PIK3CA, LAGE-1A, P53, COL6A3, HA-2, HERV-E, BRAF, alpha-fetoprotein; desmosome connective protein / AHNAKS2580F; cancer / testis antigen 1; Epstein-Barr virus, ERBB2H473Y and ERBB2IPE805G; HBV surface antigen; minor H antigen (HA-1); PRAME, TPBG, 5T4, Wilms tumor 1 (WT-1), gp100, MAGEA1; MAGE-A3 / A6; MAGEA4 / 8; or Melan-A / MART-1.

[0251] The following examples are presented to illustrate some embodiments of the invention more fully. However, they should not in any way be construed as limiting the broad scope of the invention.

[0252] Example

[0253] Materials and methods

[0254] Blood mononuclear cells were isolated from human placental intervillous blood (IVB) and filtered using the density gradient medium Lymphoprep. TM (Ficoll) was used for separation. Red blood cells were depleted using RBC X1 lysis buffer. The separated population was cryopreserved in HI-FBS and dimethyl sulfoxide (DMSO) cryoprotectant. To analyze MAIT percentage and phenotype, cells were thawed, stained for MAIT markers and phenotypic markers (CD62L, CD45RA), and analyzed by flow cytometry. In some samples, MAIT% was analyzed before and after cryopreservation (no difference was observed). To compare MAIT% in IVB peripheral blood mononuclear cells (PBMCs), PBMCs were isolated from peripheral blood of unrelated healthy donors and similar analyses were performed.

[0255] To generate MAIT cells, IVB mononuclear cells were thawed and MAIT cell activation was induced by 5-OP-RU (250 ng / ml) and IL-15 (50 ng / ml). On days 3 and 5 post-activation, cells were counted and the MAIT% was analyzed by flow cytometry, with fresh culture medium and IL-15 added. On day 7, cells were separated using magnetic beads and anti-TCR Vα7.2 antibody to further enrich the MAIT population; and cells were cultured for another 3 days in the presence of IL-15. In some embodiments, the culture medium and conditions included supplementation with 2.5% CTS. TM Immune cell serum substitute (Gibco) TM CTS TM OpTmizer TM T cell expansion in serum-free medium at 37°C and 5% CO2.

[0256] For cell surface staining by flow cytometry, cells were placed 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 detecting MAIT cells and a specified additional antibody. Cells were incubated at 4°C for 20 min, washed, and resuspended in staining buffer containing 7-AAD active dye. Stained cells were analyzed on a CytoFlex flow cytometer; results were then analyzed on FlowJo or Kaluza software.

[0257] For intracellular staining by flow cytometry, cells were placed in T-cell culture medium in 96-well microplates supplemented with phorbol 12-tetradecanoate 13-acetate (PMA) and iomycin 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 viable staining agent, 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 at 4°C for 30 minutes in Perm / Wash buffer containing fluorescently labeled antibodies against intracellular proteins (IFNγ, TNFα, perforin, and granzyme B). Cells were washed and analyzed as described above.

[0258] Example 1. MAIT cells are generally more abundant in placental IVB mononuclear cells than in peripheral blood mononuclear cells (PBMCs).

[0259] IVB and blood samples were obtained from healthy donors and separated using a Ficoll gradient to isolate mononuclear cells as described above. The resulting cells were stained with antibodies as described above and analyzed by flow cytometry. IVB and PBMC CD3 were obtained from placental IVB and PBMCs from 13 and 11 (respectively) unrelated donors. + In cells, compare the percentage of MAIT cells as defined by the expression of TCRva7.2 and CD161. Figure 1A-1B As shown, although the proportion of MAIT cells (CD3+) is high in both peripheral blood and placenta, + The percentage of CD3 in IVB varies between donors, but on average, it is higher than that in peripheral blood. + A significantly higher number of MAIT cells were present in the cells.

[0260] Example 2. Phenotypic differences among placental MAIT cells, peripheral MAIT cells, and peripheral T cells.

[0261] Phenotypic analysis of T cells and MAIT cells from placental IVB and peripheral blood (PB) was performed using CD62L-specific and CD45RA-specific antibodies. Figure 2A-2CAs can be seen, unlike conventional T cells, which still contain a large number of naive and central memory cells, MAIT cells express low levels of CD62L and are therefore primarily effector memory cells or terminally differentiated effector (TemRA) cells. Notably, IVB-derived MAIT cells have a significantly higher proportion of effector memory cells than peripheral blood-derived MAIT cells. Therefore, they have fewer terminally differentiated effector cells compared to peripheral blood-derived MAIT cells. Figure 2A-2B The results also showed that IVB MAIT cells had lower CD45RA levels compared to PB MAIT cells (p = 0.035), therefore the IVB MAIT cell phenotype is characterized by less TemRA and more effector memory. Effector memory cells may be a preferred phenotype for engineered T cells, including MAIT cells, because they have a longer lifespan than terminal effectors, and at the same time, they exhibit better effector function and the ability to migrate to peripheral tissues compared to naive and central memory cells. Furthermore, as in... Figure 2C As can be seen, compared with conventional peripheral blood T cells (PB T), placental (IVB) MAIT cells contain a significantly larger population of effector memory cells, while the mean difference in the abundance of terminal effectors did not reach statistical significance.

[0262] Example 3. Compared with MAIT cells derived from peripheral blood, the expression of chemokine receptors was higher in MAIT cells derived from IVB.

[0263] Chemokine receptors mediate the migration of engineered T cells to peripheral tissues and secondary lymphoid organs; they are also important for engineered T cell infiltration into tumors. The expression of chemokine receptors was analyzed on MAIT cells derived from IVB and MAIT cells derived from peripheral blood (primary MAIT cells as described in Examples 1-2). Placental MAIT cells showed significantly higher levels of CCR5 and lower levels of CXCR4, while CCR6 was also higher in IVB MAIT cells, although the difference did not reach statistical significance. CXCR6 expression was similar in MAIT cells from both sources. Figures 3A-3B Increased CCR5 on IVB MAIT cells is expected to improve their transport to solid tumors. Reduced CXCR4 may have the same effect, as CXCR4 directs T cells to the bone marrow. In some solid tumors, the CXCR4 ligand SDF1 is highly expressed in the matrix, isolating T cells and preventing them from infiltrating the tumor core. Therefore, pharmacological inhibition of CXCR4 has been shown to improve the infiltration of cytotoxic T cells into tumors and to synergize with PD1 blockade in animal and in vitro models of solid tumors.

[0264] Example 4. Functional comparison among MAIT cells from IVB, peripheral blood MAIT cells, and peripheral blood T cells.

[0265] Perforin and granzyme B are essential molecules for the cytotoxic function of effector lymphocytes such as T cells (including MAIT cells). Perforin creates pores in target cells, while granzyme B enters the target cells through these pores and induces apoptosis.

[0266] To evaluate perforin and granzyme B expression in different cell populations, cells isolated as described in Example 1 above were stimulated with PMA and iomycin for 4 hours in the presence of a protein secretion inhibitor. Cells were then permeabilized, stained for MAIT markers or CD3 and test molecules, and analyzed by flow cytometry. Results are presented in… Figures 4A-4B middle.

[0267] In comparative analysis, it was found that, under stimulation with PMA and iomycin, IVB MAIT cells expressed significantly higher levels of both effector molecules compared to peripheral blood MAIT cells. Figures 4A-4B Furthermore, the results showed that compared to conventional T cells (PB CD3), + In contrast, MAIT cells contain significantly more cells that produce perforin, granzyme B, or both.

[0268] Similarly, the expression of cytokines (IFNγ, TNFα, IL2) in cells was analyzed, and the results were presented in... Figures 5A-5B As can be seen, the MAIT population exhibits significantly higher IFNγ production compared to the conventional T cell population. + TNFα (TNFα) + ) or both (IFNγ) + / TNFα + Furthermore, MAIT cells from two sources (IVB and blood) were found to produce cytokines at similar levels, suggesting that MAIT cells have a high cytotoxic potential in patients treated with engineered MAIT cells without triggering / inducing cytokine release syndrome (CRS).

[0269] Therefore, the results indicate that MAIT cells are more cytotoxic and act as a stronger effector compared to conventional peripheral blood T cells.

[0270] Finally, the expression of multidrug resistance 1 (MDR1) was similarly examined by flow cytometry. The results are presented in… Figures 6A-6B Among them Figure 6A This displays representative samples of placental IVB cells (gated in the MAIT population) and peripheral blood T cells, and Figure 6B This is a quantitative phenotypic analysis of MDR1 protein expression on placental MAIT cells and peripheral T cells (each from 6 donors).

[0271] As can be seen, placental MAIT cells express significantly higher levels of MDR1. Therefore, MAIT cells may be more resistant to chemotherapy and other drugs used in the treatment of patients requiring adoptive transfer therapy.

[0272] Example 5. Phenotypic characterization of expanded MAIT cells.

[0273] MAIT cells were enriched by activating placental IVB cells with 5-OP-RU.

[0274] To obtain a population rich in MAIT cells, IVB-derived mononuclear cells were activated with the MAIT homolog 5-OP-RU and cultured in the presence of IL-15. After 7 days of culture, depending on the donor, MAIT cells represented CD3+. + The number of cells in the population was between 78% and 93%, and the total fold increase of MAIT cells was 46-112 times. Figure 7A When the MAIT% was below 90%, cells were further enriched using anti-TCR Va7.2 antibody and magnetic beads. Cells were cultured until day 10. Results are presented in... Figures 7B-7C middle.

[0275] Since the production of the T-cell composition ultimately requires the expansion of activated T cells, expanded placental MAIT cells were compared with expanded conventional T cells. For this purpose, placental IVB mononuclear cells were activated with 5-OP-RU and cultured in the presence of IL15 to enrich the MAIT population. On day 7 post-activation, MAIT cells were isolated using anti-TCR Va7.2 antibody and magnetic beads. Simultaneously, PBMCs were immobilized with anti-CD3 and anti-CD28 antibodies (TransAct). TM Activate and culture with IL-2; on day 7 after activation, isolate CD8 using anti-CD8 antibody and magnetic beads. + T cells. MAIT cells were inoculated with CD8. + T cells were compared to exclude CD4. + With CD8 + The difference between T cells is because MAIT is mainly CD8 + And it has a very low CD4 + A population was established. Three donors were used for each cell type. RNA was isolated from each sample and sequenced (RNA-seq). The results are presented in... Figures 8A-8B middle.

[0276] As can be seen from the results, multiple genes in MAIT cells differ from those in conventional CD8. + Peripheral blood T cells show persistent differences, with some being higher in MAIT and others higher in regular T cells.

[0277] To analyze gene expression at the protein level, expanded MAIT cells and conventional T cells were tested for expression in LegendScreen. TM Expression of ~360 proteins on the BioLegend group. Cells were activated and expanded for 10 days, then MAIT and CD8 were separated using magnetic beads. + T cells. Purified cells of each cell type were stained with anti-CD45 antibodies with different fluorophores, washed, and combined, then used with PE-conjugated antibody groups (LegendScreen) according to the manufacturer's instructions. TM The analysis was performed. The experiment was repeated with another pair of donors. Gating of MAIT and T cells was performed based on the CD45 fluorophore. The mean median fluorescence intensity (MFI) of each marker was calculated between the two donors; each marker was plotted with its mean MFI in MAIT cells (X-axis) and mean MFI in peripheral CD8+ T cells (Y-axis).

[0278] The results are presented in Figures 9A-9B In the middle. As can be seen, although most markers did not differ between the two cell types in terms of intensity or percentage of positive cells, but with CD8. + Compared to T cells, some markers were higher or lower in MAIT cells (shown in black). Notably, many chemokine receptors were differentially expressed on expanded MAIT cells (shown in black diamonds), with CCR2, CCR5, CCR6, and CXCR6 being higher in MAIT cells, while CCR7 and CXCR4 were higher in conventional CD8+ cells. + The higher level of MAIT cells compared to T cells suggests that MAIT cells have a better potential to transport to tissues (including solid tumors), while conventional T cells are better able to transport to lymph nodes and bone marrow.

[0279] As in Figure 9B As can be seen, compared with conventional T cells, the expression of the following exemplary markers was significantly higher in MAIT cells: CD69, MDR1, siglec-7, and KLRG1. Furthermore, compared with conventional T cells, the expression of the following exemplary markers was significantly lower in MAIT cells: CD73, CD45RA, CCR7, and CXCR4. Therefore, MAIT cells maintain significantly differential expression of various markers even after in vitro expansion.

[0280] To evaluate the additional donors in LegendScreenTM The results were analyzed by staining expanded MAIT and T cells against chemokine receptors and performing flow cytometry. In this experiment, placental IVB cells were activated on day 0 as described above; on day 5, cells were isolated with anti-TCR Va7.2 antibody to enrich the MAIT population, and then the purified MAIT cells were cultured with IL-15 until day 12. Simultaneously, PBMCs were analyzed via TransAct. TM Cells were activated and cultured with IL-2 until day 12. Three donors were used for each cell type. Cells were then stained against the cytokine receptor group and analyzed by flow cytometry.

[0281] Figure 10 The results presented showed that, compared with expanded conventional T cells, the expression of chemokine receptors, including CCR2, CCR5, CCR6 and CXCR6, was significantly higher in expanded MAIT cells, while the expression of CXCR4 was comparable.

[0282] Example 6. MAIT cells have lower allogeneic characteristics compared to T cells.

[0283] A major complexity in using allogeneic cell compositions is that they can be rejected by the recipient host's immune system, particularly by peripheral blood T cells. Therefore, MAIT cells and conventional T cells are compared in terms of their ability to induce allo-rejection from T cells derived from unrelated donors.

[0284] For this purpose, expanded MAIT cells and T cells (as described in Example 5 above) were pretreated with mitomycin C to induce proliferation arrest and co-cultured with T cells from unrelated donors (“responder cells”) in a one-way mixed lymphocyte reaction (MLR). Potential allogeneic rejection was evaluated by the expression of the activation marker CD25 on the responder cells or by their proliferation rate.

[0285] The results are presented in Figure 11 As can be seen, although responding cells respond to MAIT cells, the response is significantly weaker than that to conventional T cells, indicating that MAIT cells have lower allogeneic rejection sensitivity.

[0286] It is worth noting that, such as Figure 9B As shown, compared with the amplified conventional CD8 +Compared to T cells, MDR1 remains significantly upregulated in expanded MAIT cells. Therefore, the use of MAIT cell compositions containing in vitro expanded MAIT cells can provide enhanced retention and / or prolonged therapeutic activity, even when administered to patients undergoing treatment regimens involving chemotherapy or other cancer therapies.

[0287] Example 7. Comparison of IVB-derived MAIT cells and MAIT cells derived from umbilical cord blood.

[0288] MAIT cells were collected from intervillous blood (IVB) and umbilical cord blood (CB) for frequency and immunophenotypic comparison between the two samples by flow cytometry. Figure 12A As shown, very few MAIT cells were present in CB compared to those in IVB (0.15% vs. 4.75% CD3). + (Assuming phylogenetic cells). The data presented in this paper show a high frequency of CD8αβ (immature) cell subsets in CB MAIT cells, accompanied by a relatively low percentage of CD8αα (mature) cells, indicating the immature / juvenile state of CB MAIT cells compared to IVB-MAIT cells. Figure 12B Therefore, compared to MAIT cells in IVB, which primarily exhibit an effector memory phenotype, CB MAIT cells display an immature phenotype (CD45RA). + CCR7 + CD45RA + CD62L + CD45RA + CD45RO - ()( Figure 12C-12D ).

[0289] In summary, a 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. The lower frequency of CD8αα-expressing MAIT cells in CB indicates that CB MAIT cells are in an immature state relative to the mature MAIT cells in IVB. The increased frequency of effector memory MAIT cells observed in IVB, as reflected by the high percentage of cells expressing the markers CD45RA, CCR7, CD62L, and CD27 in CB MAIT cells, compared to the immature state of effector memory MAIT cells in CB, further suggests that the isolated IVB MAIT cell population described herein does not exhibit significant contamination (if any) of MAIT cells from CB.

[0290] Example 8. Generation of MAIT cells expressing exogenous TCR (eTCR).

[0291] To generate MAIT cells expressing exogenous TCR, a gene encoding HLA-A was used. A nucleic acid construct for an engineered TCR that recognizes NY-ESO-1 in a Class I background is described. The TCR construct encodes human α and β chains having amino acid sequences as shown in SEQ ID NO: 7 and 8, respectively. The construct comprises nucleic acid sequences encoding the α and β chains as shown in SEQ ID NO: 11 and 12, respectively, linked via a P2A linker containing a 5' furin recognition site. The encoded TCR is named “NY-ESO-1 TCR” or “eTCR” herein. The resulting TCR was HLA-A 0201 and / or HLA-A Identify the peptide epitope of SEQ ID NO: 9 in the context of 0206.

[0292] The DNA sequence encoding the eTCR chain was cloned into the MSGV-1 retroviral expression vector, which is a derivative of the mouse stem cell virus (MSCV) splice-gag vector (using pMSGV with long terminal repeats (LTRs) of MSCV).

[0293] In order to produce NY-ESO-1-TCR containing placental origin + The composition of a population of MAIT cells is subjected to the following steps:

[0294] 1. Isolate lymphocytes from the IVB of the human placenta as described above and cryopreserve them for further use (isolation step).

[0295] 2. Thaw the cells and culture them for at least 3 days in lymphocyte culture medium supplemented with IL-15 (50 ng / ml) and 5-OP-RU (250 ng / ml) (MAIT enrichment and activation steps).

[0296] 3. The activated cells are then transduced using a retroviral vector expressing eTCR or by sham transduction (NT, incubating cells under the same conditions in the absence of a viral vector) (transduction steps).

[0297] 4. Grow the cells in culture for a total of 10 days in the presence of IL-15 (expansion step).

[0298] 5. On day 10, MAIT cells were isolated by positive selection of cells expressing TCRVα7.2 (purification step).

[0299] At the end of the amplification step (step 4), the amplification rate and stability of the vector expression were evaluated. After isolation (step 5), various experiments were performed to evaluate the functional properties of the resulting composition.

[0300] MAIT cells were isolated as described above and stained against the markers CD161 and TCRVα7.2 to identify the percentage of cells exhibiting the MAIT phenotype. Figure 13A This paper describes the flow cytometry gating strategy used to evaluate MAIT cell phenotypes and eTCR expression, such as as measured by the Vβ13.1 antibody. Representative results are presented in [the table / document / etc.]. Figure 13B middle.

[0301] The results showed that, following the above process, the three batches tested for MAIT cell isolation exhibited CD161. + TCRα7.2 + Expression increased from 25%, 5%, and 27% to 90%, 83%, and 94%, respectively. These purified MAIT cells expressed high levels of exogenous NY-ESO-1 TCR (73%, 76%, and 81%, respectively). Figure 13B These results demonstrate that the process can produce products containing eTCR with high efficiency and specificity. + Composition of a population of MAIT cells.

[0302] In addition, such as in Figure 14 As can be seen, in two separate batches, such as MAIT cells counted on days 0, 4, 7, and 10, the cell counts showed a 275-fold and 102-fold increase, respectively, indicating high amplification and transduction capacity suitable for clinical use.

[0303] In contrast, alternative production protocols in which the transduction step precedes the MAIT enrichment and activation steps result in lower cell survival and overall expansion rates.

[0304] Example 9. eTCR + HLA-I-restricted cytokine response in MAIT cells.

[0305] To functionally test the resulting cell population, the composition produced as described above was evaluated as follows. After isolation on day 10, cytokine production and secretion (Example 9), as well as their activation and cytotoxicity levels (Example 10), were assessed.

[0306] To test cytokine production and secretion, cells generated as described above were co-cultured with various target cells expressing different levels of NY-ESO-1 (A375, M624, and T2 cells loaded with 1 µg / ml of NY-ESO-1 peptide) or various target cells negative for NY-ESO-1 (M526 and T2 cells loaded with HIV peptide). For cytokine production capacity, the intracellular expression of IFNγ, IL-2, and TNFα in cells co-cultured with target cells for 6 hours was analyzed. This was performed using eBioscience. TM Intracellular fixation and permeabilization buffer set and corresponding anti-cytokine antibodies from Biolegend were used in experiments, and intracellular staining was employed. Results are depicted in [the following figures / images]. Figure 15A , 15B And in 15C.

[0307] As in Figures 15A-15C As can be seen, in two different MAIT batches, cells expressing eTCR, rather than the untransduced (NT) control, showed cytokine expression upon recognizing their target antigen NY-ESO-1. As can be further seen, cytokine expression increased in a dose-dependent manner relative to antigen expression on the target, as evident from the differential responses to cell types expressing different levels of antigen. In co-culture with T2 cells loaded with the NY-ESO-1 peptide (T2-ESO), the cytokine levels measured in eTCR-expressing MAIT cells increased by up to 88% relative to IFNγ. Figure 15A ), for IL-2 increased to a maximum of 19% ( Figure 15B ), and for TNFα increased to a maximum of 65% ( Figure 15C ).

[0308] Depicting eTCRs stained simultaneously with IL-2 and IFNγ or TNFα + A representative image of MAIT cells is presented in Figure 15D In this study, it can be observed that, upon recognizing the target antigen NY-ESO-1, a population of cells co-expressing multiple cytokines can be identified. The eTCR model demonstrates the multifunctional state of these cells. + The simultaneous expression of multiple cytokines in MAIT cells further demonstrates... Figure 15E In the study, the results showed that over 50% of the cells expressed at least two of these cytokines, and at least 7% of them expressed all three of the tested cytokines.

[0309] To further quantify IFNγ secretion from MAIT NY-ESO-1 TCR cells, an ELISA was performed on the supernatant of MAIT cells co-cultured with target cells for 24 hours, according to the manufacturer's instructions (Human IFN-gamma DuoSet ELISA, R&D, DY285B-05). Results are presented as follows: Figure 16 In the middle. As can be seen, in two separate batches, eTCR + MAIT cells secreted high levels of IFNγ when co-cultured with A375, M624, and T2 cells loaded with NY-ESO-1. When co-cultured with T2-ESO, the secretion levels of the two MAIT batches reached significant levels of 25,200 pg / ml and 14,900 pg / ml, respectively. Figure 16 ).

[0310] In summary, these results demonstrate that MAIT cells transduced to express NY-ESO-1-specific TCRs are capable of antigen-induced cytokine expression and secretion. Furthermore, the observation of a multifunctional state suggests that these cells possess high functional potential.

[0311] Example 10. Redirecting Antigen Response – Activation Markers and Cytotoxicity

[0312] To test the activation of the MAIT NY-ESO-1 TCR, cells were co-cultured with various target cells expressing NY-ESO-1 (A375, M624, and T2 cells loaded with NY-ESO-1 peptide) or various target cells negative for NY-ESO-1 (M526 and T2 cells loaded with HIV peptide). After 24 hours of incubation, the surface expression of the markers CD137, CD69, and CD25 was measured by flow cytometry. The results are shown in... Figure 17A-17F middle.

[0313] As can be seen, only when eTCR + When MAIT cells were co-cultured with target cells expressing NY-ESO-1, the activation markers CD137 and CD25 showed a specific increase. The highest levels of these markers were obtained when co-cultured with T2-ESO cells, reaching 73% for CD137 and 79% for CD25. Figures 17A-17B (17E-17F). When co-cultured with T2-ESO cells, the marker CD69 in eTCR under target recognition + The percentage of MAIT cells also increased from 35% at baseline to 91%. Figure 17C-17D These results indicate that the NY-ESO-1 specific TCR introduced into MAIT cells can functionally activate these cells and induce them to express corresponding T cell activation markers.

[0314] Next, measure eTCR + MAIT cells demonstrate cytotoxic potential against various cell lines. This was achieved through interaction with eTCR. + Alternatively, the expression of active cysteine-3 on target cells after co-culturing with MAIT cells can be used to evaluate cytotoxicity. Two batches of MAIT cells were tested, and the results are presented in [Table data would be inserted here]. Figures 18A-18B As can be seen, in both batches, MAIT cells expressing NY-ESO-1TCR exhibited up to 38% caspase-3 expression in NY-ESO-1 positive target cells, while the highest killing effect was observed in T2-ESO and A375 cells. Interestingly, M624 cells expressing higher levels of NY-ESO-1 also showed significant killing effect, but to a lesser extent than A375 cells expressing lower levels of the antigen. Figures 18A-18B ).

[0315] In addition to direct killing, CD107 surface expression (as an indicator of degranulation) and granzyme B secretion (GZMB) were used to evaluate cytotoxicity. Results are presented in... Figures 19A-19B (CD107) and Figure 20 (Granzyme B)

[0316] As can be seen, in identifying NY-ESO-1 + In the case of cells, in eTCR + Increased CD107 was detected in MAIT cells. After co-culturing with T2-ESO cells, an average of 43% eTCR was achieved. + The MAIT group was positive for CD107. Figures 19A-19B In addition, two batches of eTCRs were co-cultured with T2-ESO. + In MAIT cells, granzyme B is secreted in large quantities, reaching a maximum level of 25,000 pg / ml. Figure 20 ).

[0317] In summary, these results demonstrate that MAIT cells expressing NY-ESO-1 TCR have acquired the ability to target and directly and specifically lyse cells expressing the HLA class I restriction antigen (i.e., NY-ESO-1). This document discloses a particularly efficient process for producing improved cell compositions for ACT, characterized by unique and advantageous properties.

[0318] It is noteworthy that the cell composition of the present invention does not require high levels of antigen presentation to exert effective antigen-induced cytotoxicity. For example, M624 cells are known to present moderately low levels of the NY-ESO-1 epitope (approximately 30-50 complexes per cell) in the presence of HLA-A2. In contrast, A375 cells are known to present lower levels of HLA-A2-presented NY-ESO-1 epitopes (approximately 10-30 complexes per cell), while 526 cells are characterized by levels of HLA-A2-presented NY-ESO-1 epitopes below the detectability threshold (and insufficient for effective TCR-mediated activation). In the experiments described herein, MAIT cells transduced by NY-ESO-1-specific TCRs were specifically activated by A375 and M624 cells expressing low levels of antigen (~10-50 antigen molecules per cell) and were able to effectively kill these cells (as is evident, as through the expression of cleaved caspase 3 in the target and indirectly through the expression of activation markers on TCR-transduced MAIT cells).

[0319] It is worth noting that, although eTCR + Cell activation was enhanced in the presence of increased antigen levels (as evident from activation markers and cytokine secretion in the presence of M624 cells compared to A375 cells), but specific cleavage was unexpectedly enhanced in A375 cells compared to M624 cells. In other words, the discovery of eTCR was surprising. + The direct cytotoxicity of MAIT cells is particularly effective against tumor cells expressing (physiologically significant) low levels of NY-ESO-1 than against tumor cells expressing high levels of NY-ESO-1.

[0320] Example 11. Isolation and expansion of MAIT cells from intervillous blood (IVB) in a three-dimensional (3D) system.

[0321] To generate MAIT cells in a 3D system, a 0.5L packed-bed MiniBio reactor was assembled, containing 2.5 grams of Fibra-Cel. ® The tray was then sterilized in an autoclave for 30 minutes by steam sterilization at 122.5°C and 1 bar above atmospheric pressure. The MiniBio reactor was then connected to the Applikon MiniBio control station. The Fibra-Cel... ® The plate was incubated with RPMI-1640 supplemented with 10% HI-FBS at 37°C for approximately 24 hours. During incubation, serum proteins and Fibra-Cel... ® The hydrophilic end groups on the disc interact electrostatically and produce an ECM coating on Fibra-Cel® that mimics the natural cellular environment.

[0322] 300 ± 20×10 6 4 cells were thawed to a solution supplemented with 1% L-glutamine 200mM and 0.1% gentamicin 50mg / ml. ® In Nutri-T GMP medium, thawed cells were diluted to 1×10⁻⁶. 6 The target concentration was [number of cells / ml]. The prepared cell suspension was seeded into a bioreactor system set to the following conditions: 37°C, 80% DO, pH 7.4, and stirring at 100 rpm, to a final volume of 300 ml. With the cells seeded, they were distributed within the bioreactor between the packed bed and the “external” environment, and a decrease in cell concentration was observed 3 hours after seeding.

[0323] Three hours after inoculation, MAIT cells were activated via their T-cell receptors (TCRs). Activation via the TCR pathway requires co-stimulatory signals, including recognition of microbial-derived riboflavin metabolites presented on the MHC class I-like molecule MR1, and co-stimulation via CD28, TLR agonists, bacterial products, or cytokines. The initial cell population isolated from human placental IVB included various antigen-presenting cells (APCs), such as dendritic cells, monocytes, and B cells, which can activate MAIT cells via MR1. 5-OP-RU, a microbial-derived riboflavin intermediate, was added to the growth medium at a concentration of 250 nM to present TCRs to MAIT cells via APCs on MR1.

[0324] During the growth phase, the growth medium and cell suspension were sampled daily to measure pH, cell concentration (via Vi-Cell), and cellular metabolic activity based on nutrient consumption (via Cedex). TM (Bioanalyzer), and via flow cytometry (CytoFLEX) TM Cell population distribution. Cells were grown in a packed-bed bioreactor for 10 days, with the culture medium being replenished at 5%, 5.2%, and 100% on days 3, 5, and 7, respectively. Cells were harvested from the packed-bed bioreactor on day 10, as maximum growth capacity had been achieved. The total cell count reached 1089 × 10⁶ cells / day. 6 94% of MAIT cells (Vα7.2 positive, high CD161) indicated a fold increase of 43.5%. The proportions of different cell populations in the culture and their changes over time are shown in Table 3.

[0325] After harvesting cells from the bioreactor on day 10 of culture, further cell expansion was examined. An additional 0.5L packed-bed MiniBio reactor was assembled, and a sterile system containing 2.5g Fibra-Cel® discs was prepared as before, pre-incubated at 37°C for approximately 24 hours with RPMI-1640 supplemented with 10% HI-FBS to provide ECM coating on the Fibra-Cel® discs and create a natural environment for reseeded cells. After ECM coating, the packed-bed bioreactor was pre-equilibrated for culture with growth medium consisting of 4Cell® Nutri-T GMP supplemented with 1% L-glutamine 200mM and 0.1% 50mg / ml gentamicin. 295 × 10⁶ cells were cultured. 6 The harvested cells were inoculated into growth medium and diluted to ~1×10⁻⁶. 6 The target concentration was [number of cells / ml]. The prepared cell suspension was seeded into a bioreactor system set to the following conditions: 37°C, 80% DO, pH 7.4, and stirring at 100 rpm, to a final volume of 300 ml. IL-15 at a concentration of 50 ng / ml was added to the growth medium to induce MAIT cell expansion.

[0326] During the growth phase, growth medium and cell suspension were sampled daily to measure pH, cell concentration (via Vi-Cell), cellular metabolic activity based on nutrient consumption (via Cedex Bioanalyzer), and flow cytometry (CytoFLEX). TM Cell population distribution was determined. Cells were grown in a packed-bed bioreactor for an additional 7 days, with the culture medium being refreshed at 26.5% and 100% on days 12 and 14, respectively. Cells were harvested from the packed-bed bioreactor on day 17. The total cell count reached 490 × 10⁻⁶. 6 88% of MAIT cells (Vα7.2 positive, high CD161) indicated a fold increase of 1.5. The relative percentage of MAIT cells in the culture and its change over time are shown in Table 4.

[0327] Table 2 below summarizes the initial and final total number of viable cells during the growth period, their relative population share, and fold expansion.

[0328] Table 2 – Cell characteristics during the growth phase

[0329]

[0330] As shown in the table above, all fold expansion results were greater than 1, indicating cell expansion. Furthermore, the table shows the shift in cell population balance, which reached over 94% of the target cells (MAIT cells after 10 days of growth) at the end of the examined growth phase. The second growth phase of MAIT showed a slight decrease in the percentage of MAIT cells (from 94% to 86%).

[0331] Table 3 – Activation and proliferation of MAIT cells over 10 days in a packed-bed bioreactor

[0332]

[0333] The results presented in Table 3 show an increase in the proportion of MAIT cells, starting at 22.63% on day 0 and reaching as high as 96.26% on day 10. Furthermore, the expression of activation markers CD69 and CD25 increased from day 0 to day 7 and decreased on day 10. "Cells in a single cell" represents a population of cells that were phylogenized for further analysis.

[0334] Table 4 – MAIT cell activation and proliferation after transition from bioreactor 1 to bioreactor 2

[0335]

[0336] The results presented in Table 4 indicate that the percentage of MAIT remained similar for most of the time period, but decreased from >90% on day 14 to 82% on day 17. CD69 marker expression was upregulated from 30% to 87% on day 17, suggesting that MAIT cells maintained their activation signals, while CD25 gradually decreased.

[0337] Furthermore, the expression of chemokine receptors was examined during the activation and expansion of MAIT cells as described in Example 5, and in MAIT cells grown in the 3D bioreactor system as described above. It was found that MAIT cells maintained high expression of tissue homing chemokine receptors, including CCR5, CCR6, and CXCR6, on days 10 and 17 post-activation, and maintained the same levels in MAIT cells cultured in the 3D system.

[0338] Example 12 – Comparative example of engineered T cells – IVB MAIT and PBMC-derived.

[0339] IBV-derived MAIT cells and donor-matched PBMC-derived control T cells (collected from the same donor) were obtained from placenta and blood of three healthy female donors. As described above, cells from each sample were transduced using an NY-ESO-1 specific eTCR construct and expanded for 10 days. The structural and functional parameters of the transduced cells were evaluated and compared at different time points, as detailed below.

[0340] On day 0 (before transduction and expansion), cells were incubated with antibodies labeled against various differentiation markers CD45RA, CCR7, CD62L (differentiation group) and exhaustion markers PD1, TIM3, LAG3, CTLA4 (exhaustion group), and evaluated by flow cytometry as described above. Activation and cytotoxicity markers 41BB, CD25, CD69, CD107, and GZMB were also evaluated by flow cytometry (activation and cytotoxicity FACS group). Furthermore, transcription factors RORγt, t-bet, and EOMES were evaluated by nuclear staining with antibodies and flow cytometry (transcriptomics). Transduction efficiency and purity were also evaluated by flow cytometry after transduction and isolation using eTCR, MAIT, and T cell-specific antibodies.

[0341] On day 10 (post-amplification), the resulting cell compositions were evaluated again using differentiation, exhaustion, activation and cytotoxicity FACS, and transcriptomics.

[0342] Furthermore, essentially as described above, functional assays were performed by co-culturing the resulting cells with target cells (T2, A375, 526, and M624 cells loaded with NY-ESO-1 peptide). The following functional assays were performed:

[0343] Intracellular staining of cytokines IL-2, IFNγ, and TNFα.

[0344] Specific killing – staining of caspase 3 in target cells.

[0345] Activation and cytotoxicity of FACS group – 41BB, CD25, CD69, CD107 and GZMB.

[0346] ELISA for cytokine secretion – IFNγ, GZMB.

[0347] The foregoing description of the specific embodiments so fully reveals the general nature of the invention that others can readily modify and / or adapt them to various applications of such particular embodiments by applying present knowledge, without excessive experimentation and without departing from the general concept. Therefore, such adaptations and modifications should and are intended to be understood as being within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the wording or terminology used herein is for descriptive purposes and not for limitation. Various alternative forms may be taken for the means, materials, and steps used to perform the various disclosed functions without departing from the invention.

Claims

1. A cell composition comprising a population of engineered mucosa-associated inert T (MAIT) cells expressing a extrinsic T cell receptor (TCR), wherein the MAIT cells are derived from the placenta, and the cell composition optionally further comprises a pharmaceutically acceptable carrier.

2. The cell composition of claim 1, wherein the MAIT cells are derived from intervillous blood (IVB) of the placenta.

3. The cell composition according to claim 1 or 2, which is adapted for adoptive transfer cell therapy (ACT).

4. The cell composition according to any one of the preceding claims, comprising 10 9 -10 11 Live cells of the engineered MAIT cell population.

5. The cell composition according to any one of the preceding claims, comprising at least 90% TCRVα7.2 + CD161 高 cell.

6. The cell composition according to any one of the preceding claims, wherein the TCR recognizes a tumor antigen.

7. The cell composition according to claim 6, wherein the tumor antigen is selected from the group consisting of: NY-ESO-1, KRAS, p53, PIK3CA, PTEN, ERBB2 (HER2), AFP, KK-LC-1, RAC1-P29S, LAGE-1A, COL6A3, HA-2, HERV-E, BRAF, gp100, alpha-fetoprotein, desmosome connective protein / AHNAKS2580F, cancer / testis antigen 1, ERBB2H473Y, ERBB2IPE805G, minor H antigen (HA-1), PRAME, PSMA, TPBG, 5T4, MAGEA1, MAGE-A3 / A6, MAGEA4 / 8, Melan-A / MART-1, NRAS, and Wilms tumor 1 (WT-1).

8. The cell composition according to claim 7, wherein the tumor antigen is selected from the group consisting of NY-ESO-1 and LAGE-1A.

9. The cell composition according to any one of the preceding claims, wherein the TCR is capable of specifically binding to an epitope presented by HLA-A2.

10. The cell composition of claim 9, wherein the TCR comprises a TCR α chain and a TCR β chain, the TCR α chain comprising CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 2, and CDR3 having the amino acid sequence of SEQ ID NO: 3, and the TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO:

6.

11. The cell composition of claim 11, wherein the TCR comprises a TCR α chain and a TCR β chain, the TCR α chain having an amino acid sequence as shown in SEQ ID NO: 7, optionally excluding the signal peptide located at positions 1-20 of SEQ ID NO: 7, and the TCR β chain having an amino acid sequence as shown in SEQ ID NO: 8, optionally excluding the signal peptide located at positions 1-21 of SEQ ID NO:

8.

12. The cell composition according to any one of the preceding claims, for use in a therapeutic manner.

13. The cell composition for use according to claim 12, wherein the use is for treating a subject with a tumor or malignant tumor.

14. The cell composition for use according to claim 13, wherein the subject suffers from a tumor selected from the group consisting of: 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.

15. The cell composition for use according to claim 13 or 14, wherein the subject suffers from a therapeutically resistant tumor or is otherwise unsuitable for treatment with immunotherapy comprising chimeric antigen receptor (CAR) T cells and / or therapeutic antibodies.

16. The cell composition of claim 15 for use, wherein the use is for treating a subject suffering from a condition associated with the expression of an HLA class I restrictive antigen.

17. The cell composition for use according to any one of claims 12 to 16, wherein the antigen is a low-density antigen characterized by surface expression of less than 50 antigen molecules per cell.

18. A cell composition adapted for adoptive transfer cell therapy (ACT), the composition comprising a substantially purified population of mucosa-associated inert T (MAIT) cells engineered to express a extrinsic T cell receptor (TCR), wherein the TCR recognizes a tumor antigen selected from the group consisting of NY-ESO-1 and LAGE-1A.

19. The cell composition of claim 18, wherein the TCR comprises a TCR α chain and a TCR β chain, the TCR α chain comprising CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 2, and CDR3 having the amino acid sequence of SEQ ID NO: 3, and the TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO:

6.

20. The cell composition of claim 19, wherein the TCR comprises a TCR α chain and a TCR β chain, the TCR α chain having an amino acid sequence as shown in SEQ ID NO: 7, optionally excluding the signal peptide located at positions 1-20 of SEQ ID NO: 7, and the TCR β chain having an amino acid sequence as shown in SEQ ID NO: 8, optionally excluding the signal peptide located at positions 1-21 of SEQ ID NO:

8.

21. A method of treating a subject with a condition associated with the expression of an HLA class I restrictive antigen, the method comprising administering to the subject a cellular composition as defined in any one of claims 1-11.

22. A method of treating a subject with a tumor or malignant tumor, the method comprising administering to the subject a cellular composition as defined in any one of claims 6-11 and 18-20.

23. A method of treating a subject with a condition associated with the expression of an HLA class I restrictive antigen, the method comprising administering to the subject a cell composition comprising a population of engineered mucosa-associated inert T (MAIT) cells and a pharmaceutically acceptable carrier, the engineered mucosa-associated inert T (MAIT) cells expressing a extrinsic T cell receptor (TCR).

24. The method of claim 23, wherein the MAIT cells are obtained from intervillous blood (IVB) of the placenta.

25. The method of claim 23 or 24, wherein the subject has a tumor or malignancy, and wherein the TCR recognizes a tumor antigen expressed by cells of the tumor or malignancy.

26. The method of claim 25, wherein the antigen is selected from the group consisting of NY-ESO-1 and LAGE-1A.

27. The method of claim 26, wherein the antigen is NY-ESO-1.

28. The method of claim 27, wherein the subject is HLA-A2 positive and has a tumor or malignancy expressing NY-ESO-1 and / or LAGE-1A, and the TCR comprises a TCR α chain and a TCR β chain, the TCR α chain comprising CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 2, and CDR3 having the amino acid sequence of SEQ ID NO: 3, and the TCR β chain comprising CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO:

6.

29. The method of claim 28, wherein the TCR comprises a TCR α chain and a TCR β chain, the TCR α chain having an amino acid sequence as shown in SEQ ID NO: 7, optionally excluding the signal peptide located at positions 1-20 of SEQ ID NO: 7, and the TCR β chain having an amino acid sequence as shown in SEQ ID NO: 8, optionally excluding the signal peptide located at positions 1-21 of SEQ ID NO:

8.

30. The method of claim 28 or 29, wherein the tumor is selected from the group consisting of: melanoma, myeloma, sarcoma, and bladder tumor, brain tumor, ovarian tumor, lung tumor, breast tumor, synovial tumor and prostate tumor.

31. The method of claim 30, wherein the tumor is a melanoma.

32. The method according to any one of claims 23-31, wherein the antigen is a low-density antigen characterized by surface expression of less than 50 antigen molecules per cell.

33. The method according to any one of claims 23-32, wherein the cell composition is adapted for adoptive transfer cell therapy (ACT), and the population comprises at least 10 9 7.2 live cells, of which at least 90% are TCR-Vα 7.2 + CD161 高 .

34. The method according to any one of claims 23-33, wherein the engineered MAIT cells are allogeneic to the subject.

35. The method of claim 34, wherein the engineered MAIT cells are partially tissue-compatible with the subject, or wherein the engineered MAIT cells are not tissue-compatible with the subject.

36. The method according to any one of claims 23-35, wherein the subject suffers from a treatment-resistant tumor or is otherwise unsuitable for treatment with immunotherapy comprising chimeric antigen receptor (CAR) T cells and / or therapeutic antibodies.

37. A process for producing a cell composition adapted for adoptive transfer cell therapy (ACT), the process comprising the following steps: a. Obtain a cell population containing MAIT cells. b. The cells were incubated in the presence of MAIT cell antigen and IL-15 to generate a population of activated MAIT cells. c. The resulting activated MAIT cells were engineered to express exogenous TCR (eTCR). d. Expand the resulting engineered MAIT cells to obtain a therapeutically effective amount of the cells, and e. Engineered MAIT cells obtained from isolation.

38. The process of claim 37, wherein the cell population comprising MAIT cells is obtained from placental intervillous blood (IVB).

39. The process according to claim 37 or 38, wherein step d is performed to obtain an amplification factor of at least 100 times.

40. The process according to any one of claims 37-39, wherein step e. comprises subjecting the resulting cells to positive selection of cells expressing TCRVα7.2 in order to obtain a population of substantially purified engineered MAIT cells.

41. The process according to any one of claims 37-40, which provides a cell composition adapted for ACT, said cell composition comprising at least 10 9 The engineered MAIT cells comprised a substantially purified population of live cells, of which at least 90% were TCR-Vα 7.

2. + CD161 高 And at least 70% are eTCR + .

42. A cell composition produced by the process according to any one of claims 37-41.

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