Engineered mesenchymal stromal cells

Abstract

This document relates to methods and materials for using engineered mesenchymal stromal cells (MSCs) to treat mammals (e.g., humans) in need of immunosuppression (e.g., humans at risk of having or having one or more autoimmune diseases (e.g., graft versus host disease (GVHD))). For example, provided are CAR-MSCs (e.g., MSCs expressing chimeric antigen receptors (CARs) having (e.g., engineered to have) elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more transport polypeptides) (e.g., MSCs expressing chimeric antigen receptors (CARs) having the ability to bind to tissue-specific antigens). Also provided are methods of administering one or more CAR-MSCs provided herein to a mammal (e.g., a human) in need of immunosuppression (e.g., a human at risk of having or having one or more autoimmune diseases such as GVHD) to treat the mammal.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. patent application serial number 63 / 421,832, filed on November 2, 2022. The disclosure of the prior application is considered part of the disclosure of the present application and is incorporated by reference thereto.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically as an XML file named "07039-2165WO1.xml." This XML file was created on October 31, 2023, and is 160,000 bytes in size. The material in the XML file is incorporated herein by reference in its entirety. Technical Field

[0005] This article relates to methods and materials for treating mammals (e.g., humans) (e.g., humans with or at risk of one or more autoimmune diseases (e.g., graft-versus-host disease (GVHD))) using engineered mesenchymal stromal cells (MSCs) for the treatment of mammals (e.g., humans) in need of immunosuppression. For example, provided herein are CAR-MSCs (e.g., MSCs expressing chimeric antigen receptors (CARs) with the ability to bind to tissue-specific antigens) having (e.g., engineered to have) elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more transport polypeptides. Such CAR-MSCs can exert an immunosuppressive effect in target tissues (e.g., an immune response (such as an overactive immune response) can be reduced or eliminated). Also provided herein are methods for treating mammals (e.g., humans) in need of immunosuppression (e.g., humans with or at risk of one or more autoimmune diseases (such as GVHD)) by administering elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more transport polypeptides. Background Art

[0006] MSCs are attractive cell therapy candidates because they can home to sites of inflammation (Shi et al., Nat. Rev. Nephrol., 14: 493-507 (2018)) and have immunomodulatory functions that support immune homeostasis (Uccelli et al., Nat. Rev. Immunol., 8: 726-736 (2008)). However, reliable therapeutic effects have not yet been demonstrated in human clinical trials, making MSCs the most studied experimental cell therapy platform in the world, but there is no US FDA-approved therapy (Murata et al., Bone Marrow Transplant., 56: 2355-2366 (2021)).

[0007] The therapeutic effect of MSCs in humans is limited by multiple factors, including 1) poor immunosuppression and 2) inability to adequately home to the target site after administration. People have tried to address these deficiencies separately through strategies such as cytokine regulation (Guess et al. Stem Cells Transl. Med., 6: 1868-1879 (2017)), manipulation of MSC extracellular vesicles (Harrell et al. Cells, 8 (2019)), and genetic modification (Sarkar et al. Blood 118: e184-191 (2011)), but have not yet been able to simultaneously enhance immunosuppression and homing ability. Summary of the Invention

[0008] Provided herein are methods and materials for treating a mammal (e.g., a human) (e.g., a human with or at risk of one or more autoimmune diseases (such as GVHD)) requiring immunosuppression. For example, one or more CAR-MSCs can be administered to a mammal, the CAR-MSC having (e.g., engineered to have) elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more transport polypeptides to induce an immunosuppressive response (e.g., to reduce or eliminate an inflammatory immune response) in a target tissue within the mammal. In some cases, one or more CAR-MSCs can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) having (or at risk of) one or more autoimmune diseases (e.g., GVHD), the CAR-MSC having (e.g., engineered to have) elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more transport polypeptides to treat the mammal. As demonstrated herein, CAR-MSCs with elevated levels of nuclear factor kappa B subunit 1 (NFκB1) polypeptide, Jun proto-oncogene (JUN) polypeptide, transcription factor RelB (RELB) polypeptide, interferon regulatory factor 1 (IRF1) polypeptide, tumor necrosis factor (TNF) α polypeptide, interleukin (IL) -10 polypeptide, fibroblast growth factor (FGF) -2 polypeptide, granulocyte colony-stimulating factor (G-CSF) polypeptide, granulocyte macrophage colony-stimulating factor (GM-CSF) polypeptide, eotaxin polypeptide, galectin 9 (Gal-9) polypeptide, programmed cell death protein 1 (PD-1) polypeptide, T cell immunoglobulin mucin-3 (TIM-3) polypeptide, CXC chemokine receptor (CXCR) 3 polypeptide and / or CXCR4 polypeptide exhibit enhanced immunosuppression and enhanced homing ability to sites of inflammation. It is also demonstrated herein that CAR-MSCs with elevated levels of cytotoxic T lymphocyte-associated protein 4 (CTLA4) polypeptide, Toll-like receptor (TLR) 3 polypeptide, TLR4 polypeptide, TLR9 polypeptide, and / or TNF receptor 2 (TNFR2) polypeptide exhibit enhanced immunosuppression and enhanced homing ability to sites of inflammation.

[0009] In general, the present invention provides, on the one hand, MSCs comprising (1) an exogenous nucleic acid encoding a CAR targeting an epithelial-specific antigen, wherein the MSC expresses the CAR; and (2) an elevated level of a polypeptide selected from the group consisting of an NFκB1 polypeptide, a JUN polypeptide, a RELB polypeptide, an IRF1 polypeptide, a TNFα polypeptide, an IL-10 polypeptide, an FGF-2 polypeptide, a G-CSF polypeptide, a GM-CSF polypeptide, an eotaxin polypeptide, a Gal-9 polypeptide, a PD-1 polypeptide, a TIM-3 polypeptide, a CXCR3 polypeptide, a CXCR4 polypeptide, a CTLA4 polypeptide, a TLR3 polypeptide, a TLR4 polypeptide, a TLR9 polypeptide, and a TNFR2 polypeptide. The MSCs may be human MSCs. The MSCs may be adipose-derived MSCs. The epithelial-specific antigen may be E-cadherin (Ecad). The polypeptide may be an NFκB1 polypeptide, a JUN polypeptide, a RELB polypeptide, or an IRF1 polypeptide. The MSCs may comprise an exogenous nucleic acid encoding an NFκB1 polypeptide, wherein the MSC expresses the NFκB1 polypeptide. The MSC may contain an exogenous nucleic acid encoding a JUN polypeptide, wherein the MSC expresses the JUN polypeptide. The MSC may contain an exogenous nucleic acid encoding a RELB polypeptide, wherein the MSC expresses the RELB polypeptide. The MSC may contain an exogenous nucleic acid encoding an IRF1 polypeptide, wherein the MSC expresses the IRF1 polypeptide. The polypeptide may be a CXCR3 polypeptide or a CXCR4 polypeptide. The MSC may contain an exogenous nucleic acid encoding a CXCR3 polypeptide, wherein the MSC expresses the CXCR3 polypeptide. The MSC may contain an exogenous nucleic acid encoding a CXCR4 polypeptide, wherein the MSC expresses the CXCR4 polypeptide. The polypeptide may be a PD-1 polypeptide, a Gal-9 polypeptide, or a TIM-3 polypeptide. The MSC may contain an exogenous nucleic acid encoding a PD-1 polypeptide, wherein the MSC expresses the PD-1 polypeptide. The MSC may contain an exogenous nucleic acid encoding a Gal-9 polypeptide, wherein the MSC expresses the Gal-9 polypeptide. The MSC may contain an exogenous nucleic acid encoding a TIM-3 polypeptide, wherein the MSC expresses the TIM-3 polypeptide. The polypeptide may be a TNFα polypeptide, an IL-10 polypeptide, or an FGF-2 polypeptide. The MSC may comprise an exogenous nucleic acid encoding a TNFα polypeptide, wherein the MSC expresses the TNFα polypeptide. The MSC may comprise an exogenous nucleic acid encoding an IL-10 polypeptide, wherein the MSC expresses the IL-10 polypeptide. The MSC may comprise an exogenous nucleic acid encoding an FGF-2 polypeptide, wherein the MSC expresses the FGF-2 polypeptide. The CAR may comprise a heavy chain and a light chain, wherein the heavy chain comprises the CDRs set forth in SEQ ID NO:1 and the light chain comprises the CDRs set forth in SEQ ID NO:2. The heavy chain may comprise the amino acid sequence set forth in SEQ ID NO:1, and wherein the light chain may comprise the amino acid sequence set forth in SEQ ID NO:2.The CAR may comprise a heavy chain and a light chain, wherein the heavy chain comprises the CDRs shown in SEQ ID NO: 3, and the light chain comprises the CDRs shown in SEQ ID NO: 4. The heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 3, and wherein the light chain may comprise the amino acid sequence shown in SEQ ID NO: 4.

[0010] In another aspect, described herein are compositions comprising MSCs comprising (1) an exogenous nucleic acid encoding a CAR that targets an epithelial-specific antigen, wherein the MSCs express the CAR; and (2) elevated levels of a polypeptide selected from the group consisting of an NFκB1 polypeptide, a JUN polypeptide, a RELB polypeptide, an IRF1 polypeptide, a TNFα polypeptide, an IL-10 polypeptide, an FGF-2 polypeptide, a G-CSF polypeptide, a GM-CSF polypeptide, an eotaxin polypeptide, a Gal-9 polypeptide, a PD-1 polypeptide, a TIM-3 polypeptide, a CXCR3 polypeptide, a CXCR4 polypeptide, a CTLA4 polypeptide, a TLR3 polypeptide, a TLR4 polypeptide, a TLR9 polypeptide, and a TNFR2 polypeptide.

[0011] In another aspect, a method for treating a mammal suffering from GVHD is described herein. The method may include or consist essentially of the following steps: administering MSCs to a mammal suffering from GVHD, the MSCs comprising (1) an exogenous nucleic acid encoding a CAR targeting an epithelial-specific antigen, wherein the MSCs express the CAR; and (2) an elevated level of a polypeptide selected from the following group: NFκB1 polypeptide, JUN polypeptide, RELB polypeptide, IRF1 polypeptide, TNFα polypeptide, IL-10 polypeptide, FGF-2 polypeptide, G-CSF polypeptide, GM-CSF polypeptide, eotaxin polypeptide, Gal-9 polypeptide, PD-1 polypeptide, TIM-3 polypeptide, CXCR3 polypeptide, CXCR4 polypeptide, CTLA4 polypeptide, TLR3 polypeptide, TLR4 polypeptide, TLR9 polypeptide, and TNFR2 polypeptide. The mammal may be human. The symptoms of GVHD may be reduced by at least 10%. The number of Tregs in the mammal may be increased by at least 10%.

[0012] In another aspect, described herein is a method for suppressing an immune response in a mammal. The method may include or consist essentially of the following steps: administering MSC to a mammal, the MSC comprising (1) an exogenous nucleic acid encoding a CAR targeting an epithelial-specific antigen, wherein the MSC expresses the CAR; and (2) an elevated level of a polypeptide selected from the following groups: NFκB1 polypeptide, JUN polypeptide, RELB polypeptide, IRF1 polypeptide, TNFα polypeptide, IL-10 polypeptide, FGF-2 polypeptide, G-CSF polypeptide, GM-CSF polypeptide, eotaxin polypeptide, Gal-9 polypeptide, PD-1 polypeptide, TIM-3 polypeptide, CXCR3 polypeptide, CXCR4 polypeptide, CTLA4 polypeptide, TLR3 polypeptide, TLR4 polypeptide, TLR9 polypeptide, and TNFR2 polypeptide. The mammal may be human. The number of activated T cells in the mammal may be reduced by at least 10%. The number of Tregs in the mammal may be increased by at least 10%.

[0013] In another aspect, described herein is a method for reducing the number of activated T cells in a mammal. The method may include or consist essentially of the following steps: administering MSC to a mammal, the MSC comprising (1) an exogenous nucleic acid encoding a CAR targeting an epithelial-specific antigen, wherein the MSC expresses the CAR; and (2) an elevated level of a polypeptide selected from the following groups: NFκB1 polypeptide, JUN polypeptide, RELB polypeptide, IRF1 polypeptide, TNFα polypeptide, IL-10 polypeptide, FGF-2 polypeptide, G-CSF polypeptide, GM-CSF polypeptide, eotaxin polypeptide, Gal-9 polypeptide, PD-1 polypeptide, TIM-3 polypeptide, CXCR3 polypeptide, CXCR4 polypeptide, CTLA4 polypeptide, TLR3 polypeptide, TLR4 polypeptide, TLR9 polypeptide, and TNFR2 polypeptide. The mammal may be human. The number of activated T cells in a mammal may be reduced by at least 10%. The number of Tregs in a mammal may be increased by at least 10%.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention relates. Although methods and materials similar or equivalent to those described herein can be used to implement the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. If there is any conflict, this specification (including definitions) shall prevail. In addition, materials, methods and embodiments are only illustrative and are not intended to be limiting.

[0015] The accompanying drawings and the following description further illustrate one or more embodiments of the present invention in detail. Other features, objectives and advantages of the present invention will be apparent from the description, drawings and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figures 1A-1J It was shown that MSCs had been stably transduced to express CAR, and EcCAR-MSCs exhibited superior T cell suppressive effects compared to untransduced (UTD) MSCs. Figure 1A Representative flow cytometry graphs depict CAR19 expression detected by goat anti-mouse IgG (y-axis) after transduction of MSCs with VSV-γ lentiviral particles (with increasing concentrations of protamine sulfate enhancer, 50 μg / mL and 100 μg / mL). Data represent the results of more than three independent experiments using MSC donors from five different organisms. Figure 1B The histogram shows CAR expression of anti-Ecad CAR-MSC (EcCAR-MSC), anti-CD103 T cell integrin CAR-MSC (CD103CAR-MSC) and anti-CD19 CAR MSC (CD19CAR-MSC) positive controls detected by flow cytometry using goat anti-mouse IgG antibody. The data shown represent the mean ± standard deviation of more than 6 independent experiments performed using 5 MSC donors. Figure 1C The graph shows the CAR expression of EcCAR-MSC vs. UTD-MSC 2 days and 11 days after transduction with multiple cell passages by flow cytometry. The data shown represent the mean ± standard deviation (SD) of 3 independent experiments using 3 MSC donors. Statistical analysis was performed by two-way ANOVA (****p≤0.0001) (n=3 per group). Figure 1D The bar graph depicts >90% expression of Ecad-CAR (EcCAR) in MSCs from three different primary biological donors. Data represent the results of >10 independent experiments using MSCs from five different biological donors. The x-axis represents the level of CAR detection using a goat anti-mouse antibody in the APC channel of a flow cytometer. Figure 1E The graph shows the absolute CD3 + Cell counts were performed for quantification. Data shown are mean ± SD of 3 MSC donors. Statistical analysis was performed by two-way ANOVA (*p ≤ 0.05, ****p < 0.0001) (n = 3 per group). Figure 1FThe graph shows the absolute CD3 expression of EcCAR-MSCs, UTD-MSCs, or no MSCs after co-culture with activated T cells in the presence of medium alone or 250 ng / mL soluble Ecad based on flow cytometry. + Cell counts were performed for quantification. Data shown are mean ± SD of a representative experiment using 5 MSC donors. Statistical analysis was performed by two-way ANOVA (no significant difference (ns) = p ≥ 0.05, **p ≤ 0.01) (n = 2 per group). Figure 1G The graph shows the absolute CD3 expression of EcCAR-MSCs, UTD-MSCs, or no MSCs after co-culture with activated T cells in the presence of irradiated Ecad-positive cell line MCF-7. + Cell counts were quantified, and the results showed that antigen-specific activation enhanced T cell suppression. The data shown are the mean ± standard deviation of a representative experiment using 3 different biological MSC donors. Statistical analysis was performed by two-way ANOVA (ns=p≥0.05, *p≤0.05) (n=2 per group). Figure 1H Figure 2 is a graph showing the stemness immunophenotype of UTD-MSC and EcCAR-MSC measured by flow cytometry after staining MSC for CD105, CD90, CD73, CD34, CD45, and CD14. The data shown are the mean ± standard deviation of 4 independent experiments using 4 biological MSC donors. Statistical analysis was performed by two-way ANOVA (ns=p≥0.05) (n=3 per group). Figures 1I-1J The graph shows the up-regulated genes in bulk RNAseq analysis of EcCAR-MSCs compared to UTD-MSCs obtained by CellMarker enhanced gene set enrichment analysis ( Figure 1I ) and downgrade ( Figure 1J ) pathway. Significant genes were selected by differential expression analysis of three biological replicates in each group. The selection criteria were log fold change > 1 or < -1 and adjusted p value < 0.05. The calculated -log 10 The p-values ​​are shown on the x-axis to indicate statistically significant enrichment of all pathways (-log 10 (p<0.05). Data represent the analysis results of MSCs from three different biological donors for each differential expression analysis group.

[0017] Figures 2A-2H EcCAR-MSCs were shown to suppress T cell activity in tumor and GVHD models. Figure 2A Schematic diagram of an exemplary method for evaluating the effect of EcCAR-MSCs on the Nalm6 xenograft tumor model. - / - (NSG) mice were implanted with luciferase+ CD19 + Nalm6 cells (intravenous (iv), 1 x 10 6 Bioluminescence imaging was performed 5 days later to confirm engraftment. All mice subsequently received CD19-targeted CAR T (CART19) cells (1 x 10 6 cells, intravenously injected) and irradiated Ecad + In addition, mice were randomly assigned to receive UTD-MSC or EcCAR-MSC (1x 10 6 The mice were then followed up every two weeks by bioluminescence imaging and their survival was monitored. Figure 2B Figure 2 is a graph of tumor flux after CART19 infusion in the Nalm6 model, comparing EcCAR-MSC, UTD-MSC, and no treatment. Data are shown as mean ± standard error of the mean (SEM) and statistically analyzed by two-way ANOVA (**p≤0.01, ***p≤0.001, ns=p≥0.05) (n=3-4 mice per group). Figure 2C Figure 2 shows the survival results of EcCAR-MSCs compared with UTD-MSCs and controls without MSCs. Statistical analysis was performed by Kaplan-Meier simple survival analysis (*p≤0.05, ns=p≥0.05) (n=3-4 mice per group). Figure 2D Schematic diagram of an exemplary method for evaluating the effect of EcCAR-MSC on GVHD xenograft mouse model. 6 cells, injected intravenously) and EcCAR-MSCs or UTD-MSCs (1 x 10 cells, injected intraperitoneally on days 0, 14, and 28) 6 The mice were then monitored for weight loss, progression of clinical symptoms of GVHD (scored based on weight loss, diarrhea, posture, activity, hair texture, and skin integrity), and survival. Figure 2E Figure 2 shows the percentage weight change of GVHD xenografts relative to baseline after treatment with UTD-MSCs, EcCAR-MSCs, or no treatment. Data are mean ± SD, and statistical analysis was performed by two-way ANOVA (****p≤0.0001, ns=p≥0.05) (n=5 mice per group). Figure 2FRepresentative mouse images (right) and graphs (left) containing GVHD clinical score results after treatment with EcCAR-MSCs, UTD-MSCs, or no MSCs starting from day 50. Data shown are mean ± SD, statistically analyzed by two-way ANOVA (**p ≤ 0.01, ns = p ≥ 0.05) (n = 5 mice per group). Figure 2G Figure 2 is an evaluation of mouse peripheral blood comparing human CD3 between EcCAR-MSC and UTD-MSC treated groups 2 weeks after the first MSC injection by flow cytometry. + The absolute number of T cells. The data shown are mean ± SD, and statistical analysis was performed by unpaired t-test (**p ≤ 0.01) (n = 5 mice per group). Figure 2H The survival results of EcCAR-MSC compared with UTD-MSC and non-MSC control were shown in the figure. Statistical analysis was performed by Kaplan-Meier simple survival analysis (**p≤0.01, ns=p≥0.05) (n≥5 mice per group). Tumor images ( Figures 2A-2C ) represents two independent experiments performed on two different tumor models and two different biological MSC donors. GVHD images ( Figures 2D-2H ) are representative of three independent experiments performed on male and female NSG mice using three different biological PBMC donors and three different biological MSC donors.

[0018] Figures 3A-3E RNAseq analysis revealed antigen-specific activation of EcCAR-MSCs, leading to enrichment of immunosuppressive pathways. Figure 3A The heat map depicts the different gene expression profiles of EcCAR-MSC+Ecad samples, highlighting the functional impact of CAR antigen-specific stimulation by hierarchical clustering of unstimulated EcCAR-MSC, stimulated UTD-MSC+Ecad, and unstimulated UTD-MSC samples. Gene fold-overs are normalized (padj≤0.05) for all samples (n=3 MSC donors per condition). Figure 3B Principal component analysis (PCA) depicting RNAseq transcriptional profiles of six samples from three biological MSC replicates showed clustering by donor only in both unstimulated and stimulated UTD-MSCs. Figure 3C is the PCA of unstimulated and stimulated EcCAR-MSCs, which revealed unique clustering by stimulation group, suggesting CAR-based functional activation. Figure 3DIngenuity Pathway Analysis (IPA) was used to reveal canonical pathways upregulated in stimulated and unstimulated EcCAR-MSCs. Dashed lines mark significantly enriched pathways (p≤0.05) (n=3 donors per group). Figure 3E Comparison chart of standardized gene counts of unstimulated UTD-MSC, stimulated UTD-MSC, unstimulated EcCAR-MSC and stimulated EcCAR-MSC, showing the activation of transcription factor genes NFκB1, JUN, RELB and IRF1 related to CAR (CD28) in stimulated EcCAR-MSC. Data show the mean ± standard deviation of standardized gene counts in each group. Statistical analysis was performed by one-way analysis of variance (**p≤0.01, ***p≤0.001, ****p≤0.0001) (n=3 donors per group).

[0019] Figures 4A-4I EcCAR-MSCs were shown to support enhanced immunosuppression by increasing cytokine secretion, T cell mediation, and upregulating inhibitory surface markers. Figure 4A The graph shows serum cytokine levels (in pg / mL) of IL-10 polypeptide, TNFα polypeptide, G-CSF polypeptide, eotaxin polypeptide, and FGF-2 polypeptide in EcCAR-MSC-treated mice compared to UTD-MSC and untreated xenografts 17 days after administration by multiple assays. The data shown are mean ± SEM and statistically analyzed by conventional one-way ANOVA (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001) (n=4-6 samples per group). Figure 4B Graph showing human T cell subsets in GVHD xenograft mice treated with EcCAR-MSC versus UTD-MSC. Absolute cell counts in peripheral blood of mice were quantified by flow cytometry 2 weeks after the first MSC injection to assess human CD4 + and CD8 + T cell suppression. Data are shown as mean ± SD of the absolute number of cell subsets. Statistical analysis was performed by two-way ANOVA (*p≤0.05, ***p≤0.001) (n=5 per group). Figure 4C Graph showing flow cytometry analysis of human CD4 T cells in mice treated with EcCAR-MSCs versus UTD-MSCs in a GVHD xenograft model. + and CD8 + T cell ratio. Data are shown as mean ± SD of cell subset percentages. Statistical analysis was performed by two-way ANOVA (**p ≤ 0.01) (n = 5 per group). Figure 4DGraph showing the percentage of weight change in GVHD mice from baseline day 0 to day 31, showing that only EcCAR-MSC-treated mice lost weight and GVHD prevention was significantly improved. Data show mean ± SEM of percentage change in body weight. Statistical analysis was performed by two-way ANOVA (***p≤0.001) (n=5 per group). Figure 4E The graph shows that human CD4 + 、CD25 + and CD127 - Treg-like cell subsets. Flow cytometric analysis showed that Treg% in mice treated with EcCAR-MSCs was significantly increased compared to UTD-MSCs. Data shown are mean ± standard deviation, and statistical analysis was performed by unpaired t-test (**p ≤ 0.001) (n = 4 per group). Figure 4F Figure 2 is a T-distributed stochastic neighbor embedding (tSNE) plot of surface marker expression of EcCAR-MSCs and UTD-MSCs stimulated with or without Ecad by time-of-flight flow cytometry (CyTOF). + MCF7 cell line was irradiated to induce Ecad stimulation for 24 hours. tSNE plots of the surface marker profiles in the samples (using unsupervised clustering) showed the presence of four major cell populations: Ecad + Cell lines, UTD-MSCs, EcCAR-MSCs, and Ecad-stimulated EcCAR-MSCs. Figure 4G Figure 3 is a heat map showing cluster surface features, where CXCR3 and PD1 markers uniquely characterize the stimulated EcCAR-MSC population clusters. Figure 4H The graph shows the percentage expression of inhibitory receptor surface markers PD-1 and Gal-9 in UTD-MSCs and EcCAR-MSCs subjected to surface marker evaluation by flow cytometry analysis after stimulation with soluble Ecad and co-culture with PBMCs for 5 days. Figure 4I The graph shows the percentage expression of migratory chemokine surface markers CXCR3 and CXCR4 in UTD-MSCs and EcCAR-MSCs subjected to surface marker assessment by flow cytometry analysis after stimulation with soluble Ecad and co-culture with PBMCs for 5 days. Figures 4H-4I The data shown in are the mean ± SD surface expression % of 3 MSC donors. Statistical analysis was performed by two-way ANOVA (**p≤0.01, ****p≤0.0001) (n=3 per group).

[0020] Figures 5A-5G The homing and safety profiles of EcCAR-MSCs in canine and murine in vivo models are shown. Figure 5ASchematic diagram showing an exemplary method for CAR-MSC manufacturing, safety, and homing analysis in a healthy canine in vivo model. Figure 5B Figure 3 shows an exemplary immunohistochemistry (IHC) analysis of transverse colon tissue stained for Ecad 3 days after EcCAR-MSC administration, demonstrating that human EcCAR-MSCs home to canine Ecad-rich colon. + At different magnifications, IHC analysis showed co-localization of human CD105-positive antibodies (upper panel) and canine Ecad-positive antibodies (lower panel) in canine colon tissue. Figure 5C The graph shows the complete blood cell count level after intraperitoneal injection of EcCAR-MSC. From left to right, including white blood cells (WBC), monocytes, lymphocytes, neutrophils and platelets, short-term (3 days) and long-term (28 days) blood level monitoring was performed after in vivo injection of EcCAR-MSC, compared with baseline. The data shown are the mean ± standard deviation of blood components, and statistical analysis was performed by two-way analysis of variance (ns=p≥0.05 between groups) (n=3 for each experimental group). Figure 5D The graphs from left to right are the levels of total protein, BUN, creatinine, albumin, and alkaline phosphatase monitored short-term (3 days) and long-term (28 days) after in vivo injection of EcCAR-MSCs. The data shown are the mean ± standard deviation of blood components, and statistical analysis was performed by two-way analysis of variance (ns=p≥0.05). Figure 5E Figure 1 is a schematic diagram of an exemplary method for determining the in vivo expansion kinetics and persistence of CAR-MSCs using a luciferase-positive MSC xenograft model. Luciferase-expressing UTD-MSCs or CAR-MSCs were injected into NSG mice pretreated with human PBMCs, with or without irradiated Ecad + MCF7 cells were used as CAR stimulation modality, and mice were tracked daily by continuous bioluminescence imaging. Figure 5F The graph shows bioluminescence imaging flux as a measure of MSC expansion and clearance kinetics after administration of EcCAR-MSCs or UTD-MSCs (with or without Ecad stimulation). Figure 5G The exemplary images show the expansion and clearance of luciferase-positive MSCs in mice from 3 to 24 days after administration. The data shown are mean ± standard deviation (SD), and statistical analysis was performed by two-way ANOVA (ns = p ≥ 0.05) (n = 5 per group).

[0021] Figure 6 Luciferase-tagged canine Ecad + Canine / human cross-reactivity of MCKD cell lines co-cultured with human vs. mouse vs. canine directed EcadCAR T cells. Bioluminescence imaging was performed 24 hours after co-culture. UTD = untransduced.

[0022] Figure 7 A schematic diagram of an exemplary EcCAR-MSC construct is shown, where hmcECAD.6 represents the human, mouse, and canine cross-reactive Ecad antigen binding domain.

[0023] Figure 8A Schematic diagram of an exemplary method for evaluating the effect of EcCAR-MSC on JeKo1 xenograft tumor model. JeKo1 cells (1 x 10 6 cells, injected intravenously) and EcCar-MSCs or UTD-MSCs (1×10 6 The mice were then monitored for weight loss, progression of clinical symptoms of GVHD (scored based on weight loss, diarrhea, posture, activity, hair texture, and skin integrity), and survival. Figure 8B Figure 2 is a graph of tumor flux (photons / second) in NSG mice injected with JeKo1 cells.

[0024] Figure 9A Figure 2 is a graph of differentially expressed genes in unstimulated EcCAR-MSC versus UTD-MSC, Ecad-stimulated versus unstimulated EcCAR-MSC, and Ecad-stimulated versus unstimulated UTD-MSC. Comparison of adjusted p-values ​​<0.01 and ±1-log fold changes, unregulated and downregulated gene counts. Compared with EcCAR-MSC alone, Ecad-stimulated CAR-MSC-induced transcriptional changes included 2362 significant genes (compared to EcCAR-MSC alone) and 3032 significant genes (compared to Ecad-stimulated UTD-MSC). CAR transduction-induced transcriptional changes included 606 significant genes. Ecad stimulation-induced transcriptional changes included 206 significant genes. Figure 9B Figure 3. Ingenuity pathway analysis (IPA) reveals canonical pathways that are upregulated in unstimulated EcCAR-MSCs versus UTD-MSCs. Dashed lines mark enriched pathways (p≤0.05) (n=3 MSC donors per group).

[0025] Figure 10 The graphs from left to right are comparisons of MDC polypeptide, GRO polypeptide, GM-CSF polypeptide, MCP-3 polypeptide, and Flt-3 polypeptide in EcCAR-MSC-treated mice 17 days after administration, compared with UTD-MSC and untreated xenograft tumors by multiple assays. The data shown are mean ± SEM, and statistical analysis was performed by conventional one-way ANOVA (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001) (n=4-6 samples per group).

[0026] Figure 11 for Figure 4F Heat map of surface marker expression clusters in the cell populations constituting the cell clusters identified by CyTOF.

[0027] Figure 12A Figure 2 shows weight changes in healthy dogs after administration of EcCAR-MSCs compared to a control group. No significant differences in weight change were observed between the two groups. Data are presented as mean ± standard deviation of percent weight change relative to baseline, and statistical analysis was performed using standard one-way ANOVA (ns = p ≥ 0.05). Figure 12B Data show H&E-stained cross-sectional tissue sections of canine colon (20x and 40x magnification) after treatment with human EcCAR-MSCs (left) or control (right) 28 days after administration.

[0028] Figure 13 A bar chart showing the use of CAR + FMC63 antibody was used to detect CAR expression in MSCs containing different intracellular signaling domains, including 4-1BB, CD28, TLR, and IFNγ signaling domains, by flow cytometry. The data shown represent the mean ± standard deviation of more than 6 independent experiments performed using 5 MSC donors.

[0029] Figure 14 Schematic diagram containing exemplary mechanisms for enhancing CAR-MSC immunosuppression and migration.

[0030] Figures 15A-15F We show that MSCs are stably transduced to express CAR and maintain stem cell-like characteristics after transduction and stimulation. Figure 15A Representative flow cytometry plots show CAR19 expression in lentiviral transduced MSCs in the presence of increasing concentrations of protamine sulfate enhancer (50 μg / mL and 100 μg / mL, as indicated) compared to untransduced (UTD) MSCs. Data are representative of 6 different biological MSC donors. Figure 15B The histogram shows CAR expression of anti-EcadCAR and anti-CD19 CAR positive controls detected by flow cytometry. The data shown are the mean ± standard deviation (SD) of MSC donors from 6 different organisms. Figure 15C The graph shows CAR expression in EcCAR-MSCs compared to UTD-MSCs, as assessed by flow cytometry 2 and 11 days after transduction and ex vivo expansion. Data are mean ± SD of three independent experiments performed using three MSC donors. Statistical analysis was performed by two-way ANOVA (****p ≤ 0.0001). Figure 15DThe histogram shows EcCAR expression on MSCs from three different primary biological MSC donors. The data represent the results of more than 10 independent experiments using more than 6 different MSC donors. Figure 15E The graph shows the stem cell phenotype of EcCAR-MSCs and UTD-MSCs measured by flow cytometry after staining for CD105, CD90, CD73, CD34, CD45, and CD14 surface markers. The data shown are the mean ± standard deviation of three independent experiments using four biological MSC donors. Statistical analysis was performed using two-way ANOVA (ns = p ≥ 0.05). Figure 15F The two graphs depict bulk RNAseq analysis of upregulated (left) and downregulated (right) phenotypes enriched in EcCAR-MSCs compared to UTD-MSCs using CellMarker enhanced gene set enrichment analysis. Significant genes were selected by differential expression analysis of three independent MSC donors using a ≥1 or ≤-1 log-fold change and an adjusted p-value (padj) of ≤0.05. The dotted line across the x-axis indicates statistically significant enrichment of -log (p ≤ .05) across all pathways.

[0031] Figures 16A-16G It was shown that EcCAR-MSC exhibited excellent primary T cell antigen-specific inhibitory effect in vitro. Figure 16A Is a histogram that shows Ecad + The establishment of NALM6 with a matching Ecad-NALM6 makes Ecad-based cell stimulation suitable for in vitro inhibition assays and in vivo tumor models. Figure 16B The graph shows the CD3 + Absolute number of T cells, wherein soluble Ecad concentration increases (0ng / mL, 250ng / mL and 1000ng / mL). Data shown are the mean ± standard deviation of three independent experiments using three independent MSC donors. Statistical analysis was performed using two-way ANOVA (***p≤0.001). Figure 16C The graph shows the stem cell phenotype of EcCAR-MSCs after stimulation with different concentrations of soluble Ecad (0, 250, or 1000 ng / mL) after staining MSCs for CD73, CD90, CD105, CD14, CD34, and CD45, as determined by flow cytometry. The data shown are mean ± standard deviation using four different MSC donors. Statistical analysis was performed using two-way ANOVA (ns = p ≥ 0.05). Figure 16D 、 16E The graphs of 16F and 16F show that MSCs from three different donors were treated with or without Ecad-based + or Ecad- CD3 T cells were derived from EcCAR-MSC or UTD-MSC after co-culture + Absolute number of T cells. Data shown are mean ± SD. Statistics were performed using two-way ANOVA (n = 2-3 replicates per donor) (*p ≤ 0.05, **p ≤ 0.01). Figure 16G The graph shows the effect of EcCAR-MSC or UTD-MSC in co-culture alone or with Ecad-based + or Ecad - After stimulation and co-culture of cells, the stem cell phenotype was measured by staining MSCs for CD73, CD90, CD105, CD14, CD34, and CD45 and using flow cytometry. The data shown are the mean ± standard deviation from n = 3 MSC donors. Statistical analysis was performed using two-way ANOVA (ns = p ≥ 0.05).

[0032] Figures 17A-17G EcCAR-MSCs were shown to induce excellent immunosuppressive effects in tumor and GvHD xenograft models. Figure 17A To evaluate Ecad + Schematic diagram of an exemplary method for the effect in the Nalm6 xenograft tumor model. + Ecad + or luc + Ecad-NALM6 cells (1x 10 6 cells, intravenously injected) into immunocompromised NOD-SCID-γ - / - (NSG) mice were treated with CAR-T targeting CD19 (CART19) (1x 10 6 Mice were then randomly assigned to receive either untransduced MSCs (UTD-MSCs) or EcCAR-MSCs (1 x 10 6 cells, injected intraperitoneally), and tumor burden was monitored by bioluminescence imaging (BLI), and the survival and health of the mice were monitored. Figure 17B The graph shows Ecad after CART19 infusion in the Nalm6 model + NALM6 tumor flux, comparing UTD-MSCs and EcCAR-MSCs. Data are median ± standard error of the mean (SEM), and statistical analysis was performed using two-way ANOVA (***p ≤ 0.001; n = 4-5 mice per group, 2 independent experiments). Figure 17C The graph shows Ecad after CART19 infusion in the Nalm6 model -NALM6 tumor flux, comparing UTD-MSCs and EcCAR-MSCs. Data shown are median ± SEM, and statistical analysis was performed using two-way ANOVA (ns = p ≥ 0.05; n = 4-5 mice per group, 2 independent experiments). Figure 17D Schematic diagram showing an exemplary method for evaluating the effect of EcCAR-MSC in a human PBMC-induced GvHD xenograft model. Human peripheral blood mononuclear cells (PBMC) (25-30 x 10 6 cells, intravenously) were injected into the rats that had been randomly assigned to receive EcCAR-MSC or UTD-MSC treatment (1 x 10 6 The mice were monitored for weight loss, progression of clinical symptoms of GVHD (scored based on weight loss, diarrhea, posture, activity, hair texture, and skin integrity), and survival. Figure 17E The graph shows the percent weight change of GVHD xenografts relative to baseline after treatment with UTD-MSCs, EcCAR-MSCs, or no treatment. Data are shown as mean ± SEM, and statistical analysis was performed using two-way ANOVA (ns = p ≥ 0.05, *p ≤ 0.05, ****p ≤ 0.0001; n = 5 mice per group, 3 independent experiments). Figure 17F The graph shows the clinical severity scores of GvHD after EcCAR-MSC, UTD-MSC, or no MSC treatment (left), accompanied by representative mouse images (right). Data shown are mean ± SEM, and statistical analysis was performed using two-way ANOVA. (ns = p ≥ 0.05, *p ≤ 0.05; n = 5 mice per group, 3 independent experiments) Figure 17G The graph shows the survival results after EcCAR-MSC treatment versus UTD-MSC and no MSC control. Statistical analysis was performed using Kaplan-Meier survival analysis (ns=p≥0.05, *p≤0.05, **p≤0.01; n=5 mice per group).

[0033] Figures 18A-18G EcCAR-MSCs were shown to exhibit antigen-specific activation and transduction of Ecad in an acute GvHD xenograft model. + Colon target tissue migration. Figure 18A The schematic diagram shows the evaluation of Ecad + Exemplary methods for use in an acute GvHD xenograft model. Immunocompromised NOD-SCID-γ - / - (NSG) mice were first irradiated with 250 cGy to further induce an inflammatory environment. Human PBMC (10-15x10 6 cells, intravenously injected) and luc+ GFP + Ecad-CAR-MSC or luc + GFP + CD19-CAR-MSC (1x10 6 The researchers injected 100 cells (100 cells, intraperitoneally) into mice. The mice were monitored for long-term weight loss, progression of clinical GvHD, and survival. One week after MSC injection, satellite mice were isolated, and MSC localization in the colonic target organ was assessed using bioluminescence imaging and immunofluorescence staining. Figure 18B The graph shows the percentage change in weight relative to baseline of acute GvHD xenografts after treatment with Ecad-CAR-MSCs, CD19-CAR-MSCs, or no treatment. Data are shown as mean ± SEM, and statistical analysis was performed using two-way ANOVA (**p ≤ 0.01, ***p ≤ 0.001; n = 4-5 mice per group, 3 independent experiments). Figure 18C Figure 2 shows the survival results of mice treated with Ecad-CAR-MSCs, CD19-CAR-MSCs, and a control group without MSCs. Statistical analysis was performed using Kaplan-Meier survival analysis (*p≤0.05; n=4-5 mice per group). Figure 18D The graph shows the comparison of human CD3 in peripheral blood of Ecad-CAR-MSC and CD19-CAR-MSC treated mice by flow cytometry 2 weeks after MSC injection. + The absolute number of T cells. The data shown are mean ± standard deviation, and statistical analysis was performed using conventional one-way ANOVA (*p ≤ 0.05; n = 4-5 mice per group, 2 independent experiments). Figure 18E Shows luc + Representative bioluminescence imaging of CD19-CAR-MSC and Ecad-CAR-MSC treated mice (left), and the percentage of MSC flux in the colon relative to the total flux detected in all organs (right). The data shown are mean ± standard deviation, and statistical analysis was performed using an unpaired t-test (*p ≤ 0.05; n = 4-5 mice per group, 2 independent experiments). Figure 18F The graph based on immunofluorescence quantification shows that EcCAR-MSCs and CD19-CAR-MSCs are more sensitive to Ecad + Localization in colon tissue. Data are obtained by MSCs in each field of view image. + The data are shown as mean ± SD, and statistical analysis was performed using conventional one-way ANOVA (**p ≤ 0.01; n = 3 mice per group). Figure 18G GFP +A series of representative immunofluorescence images of mouse colon tissue isolated from an acute GvHD xenograft model 7 days after MSC administration. The colocalization of CD19-CAR-MSC and Ecad-CAR-MSC (row 2) with E-cadherin+ (row 3) colon regions was compared. The nuclei were stained with DAPI and each color channel was displayed to generate a merged image. Images were collected at 40x magnification, capturing 1x cropped areas.

[0034] Figures 19A-19F Showing the activation of antigen-specific immunosuppressive signaling pathways identified in EcCAR-MSCs. Figure 19A The heat map depicts the different gene expression profiles of stimulated EcCAR-MSC+Ecad samples, and the functional impact of CAR antigen-specific stimulation is highlighted by hierarchical clustering of unstimulated EcCAR-MSC, stimulated UTD-MSC+Ecad, and unstimulated UTD-MSC samples. The data shown are gene fold counts normalized for all samples (padj≤0.05) (n=3 MSC donors per condition). Figure 19B Graph showing principal component analysis (PCA) of gene expression profiles of 6 samples in stimulated and unstimulated UTD-MSCs clustered by MSC donor origin. Figure 19C Graph shows principal component analysis (PCA) of gene expression profiles of stimulated EcCAR-MSCs versus unstimulated EcCAR-MSCs and reveals unique clusters by stimulation group. Figure 19D The schematic diagram shows the analysis performed using the Ingenuity Pathway Analysis (IPA) machine learning algorithm, which illustrates the factors activated by stimulated EcCAR-MSCs compared to unstimulated EcCAR-MSCs. Factors such as upstream regulators, canonical pathways, and biological functions are combined to predict meaningful functional effects. The analysis revealed that leukocyte apoptosis (middle) was the most significantly enriched (padj ≤ 0.0001) functional pathway directly associated with all 17 activation molecules predicted in the dataset, including CD28 CAR signaling molecules. Figure 19E yes Figure 16D Figure 2. Graphical representation of IPA revealing canonical pathways upregulated in stimulated versus unstimulated EcCAR-MSCs. The dashed line across the x-axis represents the statistically significant enrichment threshold -log (p≤.05) for all pathways (n=3 MSC donors per group). Figure 19FFigure 2 is a series of graphs comparing normalized gene counts in unstimulated UTD-MSCs, stimulated UTD-MSCs, unstimulated EcCAR-MSCs, and stimulated EcCAR-MSCs. CD28-related transcription factor genes (NFκB1, JUN, RELB, and IRF1) and their downstream effector genes (TRAF1, TLR3, and FYN) were only present in the stimulated EcCAR-MSC group. Data shown are mean ± standard deviation. Gene counts were normalized between groups. Statistical analysis was performed using one-way ANOVA (ns = p ≥ 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

[0035] Figures 20A-20H We showed that stimulation of EcCAR-MSCs resulted in increased cytokine secretion, increased expression of surface markers, and consequently enhanced T cell regulation. Figure 20A Heat map of multiple cytokine assays showing that compared with Ecad - Cell line stimulation, Ecad + Upregulation of cytokines by cell line stimulation of T cells alone, UTD-MSCs, and EcCAR-MSCs. Data shown are fold changes in cytokines measured by multiplex analysis and normalized to the minimum (0) and maximum (100) values ​​for each cytokine (n=2 technical replicates per assay). Figure 20B A series of graphs show that compared to Ecad - Cell line stimulation, UTD-MSC and EcCAR-MSC in Ecad + Expression of inhibitory surface markers in cell lines after stimulation. Data are shown as mean ± SD, and statistical analysis was performed using two-way ANOVA (***p ≤ 0.001, ****p ≤ 0.0001; n = 3 replicates per donor). Figure 20C A series of graphs show the enrichment of serum cytokines (IL-10, TNFα, G-CSF, eotaxin and FGF-2, in pg / mL) in mice with tumor xenografts treated with EcCAR-MSCs at 2 weeks. The data shown are mean ± SEM and statistically analyzed by conventional one-way ANOVA (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001; n = 4-6 mice per group). Figure 20D The graph shows the percentage change in weight compared to baseline, which shows that a single dose of EcCAR-MSC treatment can prevent GvHD-induced weight loss compared to UTD-MSC treatment. The mean ± SEM of the percentage weight change is shown. Statistical analysis was performed using two-way ANOVA (***p ≤ 0.001; n = 5 mice per group). Figure 20E The graph shows the human CD3 in peripheral blood of EcCAR-MSC and UTD-MSC treated mice measured by flow cytometry 2 weeks after the first MSC injection. + The absolute number of T cells. The data shown are mean ± SD, and statistical analysis was performed by unpaired t-test (**p ≤ 0.01; n = 5 mice per group). Figure 20F The graph shows that 2 weeks after EcCAR-MSC treatment, human CD4 + and CD8 + T cell quantification. Data shown are mean ± SD of cell counts. Statistical analysis was performed using two-way ANOVA (*p ≤ 0.05, ***p ≤ 0.001; n = 5 mice per group). Figure 20G The graph shows the human CD4 + T cells and CD8 + Changes in T cell proportions. Data shown are mean ± SD percentages of cell subsets. Statistical analysis was performed using two-way ANOVA (**p ≤ 0.01; n = 5 mice per group). Figure 20H Human CD4 T cells were expressed in GvHD xenograft mice after 4 weeks of MSC treatment. + CD25 + CD127 - Figure 3. Graph of Treg cell subsets. Data shown are mean ± SD of cell percentages. Statistical analysis was performed using an unpaired t-test (***p ≤ 0.001; n = 4 mice per group).

[0036] Figures 21A-21G The CD28 signaling domain in EcCAR-MSCs was shown to enhance immunosuppressive efficacy. Figure 21A Schematic diagram showing an exemplary method for designing EcCAR-MSC constructs for studying the mechanism of action of CAR-MSCs. The constructs contain complete CD28ζ, only CD28, only CD3ζ, and empty (no signaling domain) EcCAR-MSCs. Figure 21B The histogram of CAR expression on MSCs by flow cytometry for CD28ζ, CD28, CD3ζ, and empty EcCAR-MSC constructs is shown. CAR expression percentages are representative of ≥5 independent experiments performed on 4 independent MSC donors. Figure 21C A series of graphs are shown in Ecad + After cell line stimulation, each EcCAR-MSC signaling domain subtype or T cells alone and Ecad + and matching Ecad - When cell lines are co-cultured, CD3 +The degree of T cell suppression. Data shown are the mean ± SD of four primary T cell donors. Statistical analysis was performed using one-way ANOVA (ns = p ≥ 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; n = 3 technical replicates per donor). Figure 21D Schematic diagram showing a representative method for testing the effects of various CAR-MSC signaling domains in an acute GvHD xenograft mouse model. NSG mice were irradiated with 250 cGy to induce an inflammatory environment. Human PBMCs (10-15 x 10 6 cells, intravenously) and one of the following MSC groups were injected into mice: CD28ζ, CD28, CD3ζ or empty EcCAR-MSC, UTD-MSC (1x 10 6 Mice were monitored for long-term weight loss, progression of clinical GvHD symptoms, and survival. Figure 21E The graph shows the percentage weight change relative to baseline in the acute GvHD xenograft model after treatment with each MSC group or without treatment. Data are shown as mean ± SEM and statistically analyzed using two-way ANOVA (ns = p ≥ 0.05, *p ≤ 0.05, **p ≤ 0.01; n = 5-6 mice per group, 2 independent experiments). Figure 21F The graph shows the clinical severity score of GvHD symptom progression after treatment with alternative MSC signaling domains. Data shown are mean ± SEM, and statistical analysis was performed using two-way ANOVA (*p ≤ 0.05; n = 5-6 mice per group, 2 independent experiments). Figure 21G The graph shows the survival outcomes of mice in an acute GvHD xenograft model treated with various EcCAR-MSC subtypes. These data show a trend toward improved survival, but were not statistically significant and are accompanied by expected p-values. Statistical analysis was performed using Kaplan-Meier survival analysis (ns = p ≥ 0.05, *p ≤ 0.05, **p ≤ 0.01; n = 5-6 mice per group).

[0037] Figures 22A-22I The safety and clearance profiles of EcCAR-MSCs across tissues are shown. Figure 22A Schematic diagram showing a representative method to determine the role of EcCAR-MSC in the MSC persistence model. + GFP + UTD-MSC or luc + GFP + CAR-MSCs were injected into NSG mice pretreated with human PBMCs and co-administered with irradiated Ecad +Cells were treated with or without CAR stimulation, and mice were followed by serial bioluminescence imaging (BLI) every two weeks. Figure 22B Luciferase in mice 3 to 24 days after administration + Representative images of MSC expansion and clearing. Figure 22C Graph showing the kinetics of MSC expansion and clearance after administration of EcCAR-MSCs or UTD-MSCs with or without additional Ecad stimulation, as measured by BLI flux. Data shown are mean BLI, and statistical analysis was performed using two-way ANOVA (ns=p≥0.05; n=4-5 mice per group). Figure 22D The graph shows the absolute number of keratinocytes after 24 hours of co-culture with UTD-MSCs or EcCAR-MSCs. The data shown are mean ± SD, and statistical analysis was performed by paired t-test (ns = p ≥ 0.05; n = 3 replicates per donor). Figure 22E The graph shows the expression of Ecad identified on keratinocytes after 24 hours of co-culture with UTD-MSCs or EcCAR-MSCs. The data shown are mean % ± standard deviation, and statistical analysis was performed by paired t-test (ns=p≥0.05; n=3 replicates per donor). Figure 22F Heatmaps showing upregulated cytokines in UTD-MSCs, EcCAR-MSCs, and keratinocytes under baseline and 24-hour co-culture conditions. Data shown are cytokine levels measured by multiplex analysis and normalized to the minimum (0) and maximum (100) values ​​for each cytokine (n = 2 technical replicates per assay). Figure 22G Figure 2 is a graph showing the absolute number of bronchial cells after 24 hours of co-culture with UTD-MSCs or EcCAR-MSCs at different ratios. Data are mean ± standard deviation, and statistical analysis was performed by paired t-test (ns = p ≥ 0.05; n = 3 technical replicates). Figure 22H Graph based on immunofluorescence quantification showing EcCAR-MSC localization versus CD19-CAR-MSC localization in Ecad + Positioning in lung tissue. Data are obtained by MSC of each focal image. + The data are shown as mean ± SD, and statistical analysis was performed using conventional one-way ANOVA (ns = p ≥ 0.05; n = 3 mice per group). Figure 22I Representative images of immunofluorescence imaging show GFP + Mouse bronchial (lung) tissue isolated from an acute GvHD xenograft model 7 days after MSC administration. Ecad expression in CD19-CAR-MSC and Ecad-CAR-MSC (last column) treated mice was compared. +(Second column) Colocalization with bronchial regions. Cell nuclei were stained with DAPI (third column), and each color channel was displayed to generate a merged image. Images were acquired at 40x magnification, and 1x cropped regions were captured. Figures 23A-23D The design and fabrication of the EcCAR construct are shown. Figure 23A This is a representative sequence of an optimized anti-Ecad scFv clone sequence (hmcECAD.6) identified by phage display-generated Ecad screening technology. hmcECAD with cross-reactive affinity for human, mouse, and canine was selected and cloned into the EcCAR construct to generate EcCAR-MSCs. Figure 23B Figure 2 is a representative schematic of the complete EcCAR-MSC construct, which contains the hmcCAD heavy and light chains for binding to Ecad, the CD28 hinge and transmembrane domains for spanning the MSC cell membrane, and the CD28 intracellular signaling domain for immunosuppressive activation, with CD3ζ costimulation typically used to maintain CAR construct stability. Figure 23C The graph shows the results of phage ELISA, which was used to detect the relative affinity of hmcECAD.6 scFv clones to mouse Ecad compared to the FLAG tag positive control. The data shown are all technical replicates (n=3), and statistical analysis was performed using two-way ANOVA (ns=p≥0.05). Figure 23D The diagram shows the expression of human and mouse Ecad in CART cells containing the hmcECAD.6scFv construct. + Dose-dependent killing of tumors (MCF7 and 4T1, respectively). Data shown are all technical replicates (n=3), and statistical analysis was performed using two-way ANOVA (**p≤0.01, ****p≤0.0001).

[0038] Figures 24A-24D An exemplary method for CAR-MSC generation and confirmation of their stemness validation is shown. Figure 24A The schematic diagram shows an exemplary method for generating EcCAR-MSCs in culture after thawing from a cryogenic storage system. Briefly, after in vitro growth in T175 culture flasks, MSCs were digested and quantified for transduction using an optimized lentiviral vector delivery system containing a CAR plasmid and a protamine sulfate enhancer. CAR percentage was confirmed on day 5 by flow cytometric analysis using a goat anti-mouse antibody. Figure 24B The graph shows the expression of EcCAR-MSCs in the presence and absence of Ecad as measured by flow cytometry after surface marker staining. +Figure 3. Stemness phenotype of cell lines stimulated by stimulation, with naive T cells used as a positive control for comparison. Data shown are mean ± SD from three independent MSC donors. Statistical analysis was performed by two-way ANOVA (****p ≤ 0.0001). Figure 24C Representative images comparing baseline UTD-MSC and CAR-MSC morphology with addition of matched Ecad - and Ecad + Comparative results of cell lines (as CAR antigen-specific stimulation). Figure 24D A series of histograms show that the MSC stemness phenotype is characterized by positive CD105, CD90 and CD73 and negative CD34, CD45 and CD14 surface markers. Positive gate selection was established by fluorescence minus (FMO) based on the negative control (top) for quantification of UTD-MSC (middle) and EcCAR-MSC (bottom) positive samples.

[0039] Figures 25A-25G The effect of EcCAR-MSCs on CART effector function in an in vivo tumor model was shown. Figure 25A Ecad in control mice without CART19 cell infusion - NALM6 and Ecad + Comparison of NALM6 tumor flux measurements. Data shown are mean ± SEM, and statistical analysis was performed using two-way ANOVA (ns = p ≥ 0.05; n = 4-5 mice per group). Figure 25B and 25C luc measured by luminescence after 24 h in vitro co-culture with UTD-MSCs or EcCAR-MSCs at different MSC to NALM6 ratios. + Ecad-NALM6( Figure 25B ) and luc + Ecad + NALM6( Figure 25C The data are mean ± SD, and statistical analysis was performed using two-way ANOVA (ns = p ≥ 0.05; n = 2 replicates per group). Figure 25D Schematic diagram showing representative methods for evaluating CART19 in NALM6 and JeKo-1 tumor models. + CD19 + Nalm6 or JeKo-1 cells (1 x 10 6 , intravenous injection), and CART19 (1x 10 6 cells, intravenously injected) and irradiated Ecad +Cell line treatment. Mice were then randomly assigned to receive either UTD-MSCs or EcCAR-MSCs (1 x 10 6 cells, injected intraperitoneally), and monitored by bioluminescence imaging and survival every two weeks. Figure 25E Figure 2 shows the tumor flux after CART19 infusion in the Jeko-1 tumor model, comparing EcCAR-MSC, UTD-MSC, and no MSC treatment. Data are shown as mean ± SEM, and statistical analysis was performed using two-way ANOVA (**p ≤ 0.01; n = 3-4 mice per group). Figure 25F The graph shows the tumor flux measurement results between MSC administration groups after CART19 infusion in the NALM6 model. The data shown are mean ± SEM, and statistical analysis was performed using two-way ANOVA (ns = p ≥ 0.05, ***p ≤ 0.001; n = 3-4 mice per group, 2 independent experiments). Figure 25G The graph shows the survival results of mice treated with EcCAR-MSCs, UTD-MSCs, and a control group without MSCs. Statistical analysis was performed using Kaplan-Meier survival analysis (ns=p≥0.05, *p≤0.05; n=3-4 mice per group, 2 independent experiments).

[0040] Figures 26A-26B An exemplary flow cytometry gating strategy is shown. Figure 26A Figure 2 is an exemplary gating strategy for identifying positive MSC surface markers, validated by FMO and internal controls. MSC populations were identified by size, single cells were selected by diagonal gating, live MSCs were stained with an aqueous fluorophore that can fix live / dead cells to exclude dead cells, and positive cell markers (shown as CAR) were identified based on positive gating of cells of interest staining (validated by internal and FMO controls). + %). Figure 26B For identification of luciferase+ and CAR + Example gating strategy for MSCs to be evaluated in an in vivo clearance experiment. MSCs were transduced with a luciferase-expressing transgene and a CAR construct and then expressed in the presence of luciferase. + The results showed that the transduction of luciferase and CAR was successful, and the double positive (luciferase + CAR + ) transduction rate>50%.

[0041] Figures 27A-27E Bulk RNA sequencing (RNAseq) pathway analysis and cytokine secretion are demonstrated. Figure 27AFigure 24 shows a summary of differentially expressed genes of unstimulated EcCAR-MSC vs. UTD-MSC, Ecad-stimulated EcCAR-MSC vs. unstimulated UTD-MSC, Ecad-stimulated EcCAR-MSC vs. unstimulated UTD-MSC, and Ecad-stimulated EcCAR-MSC vs. UTD-MSC. The data shown are gene counts (adjusted p-value < 0.01, and ± 1-log multiple changes) of up-regulated and down-regulated genes with significant significance in the comparison. Compared with EcCAR-MSC alone, Ecad stimulates the transcriptional changes induced by CAR-MSC to include 2362 significant genes (compared to EcCAR-MSC alone) and 3032 significant genes (compared to Ecad-stimulated UTD-MSC). CAR transduction-induced transcriptional changes include 606 significant genes. Ecad stimulation-induced transcriptional changes include 206 significant genes. Figure 27B The graph shows Ingenuity Pathway Analysis (IPA), revealing canonical pathways upregulated in unstimulated EcCAR-MSCs compared to UTD-MSCs. The dashed line across the x-axis indicates statistically significant enrichment of -log for all pathways (p≤.05) (n=3 MSC donors per group). Figure 27C and 27D for Figure 27B Figure 2. Graphical summary of IPA showing the significant entities activated in unstimulated EcCAR-MSCs versus UTD-MSCs ( Figure 27C ) and the significant entities activated in stimulated EcCAR-MSCs vs. UTD-MSCs ( Figure 27D ). Canonical pathways and activated molecules are used to predict meaningful functional impacts between datasets. Figure 27E The graph shows elevated serum cytokines in the peripheral blood of mice bearing tumor xenografts treated with EcCAR-MSCs compared to UTD-MSCs and controls. Cytokines included macrophage-derived chemokine (MDC), growth-related alpha protein (GRO), granulocyte macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein 3 (MCP-3), and FMS-related tyrosine kinase 3 ligand (Flt-3L) (pg / mL). Data are presented as mean ± standard deviation, and statistical analysis was performed using a multiple t-test (*p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001; n = 4-6 mice per group).

[0042] Figures 28A-28E Shown is the safety profile of EcCAR-MSCs determined using an in vivo canine model. Figure 28ASchematic diagram of an exemplary method for CAR-MSC production and safety analysis in a healthy canine model. EcCAR, which is cross-reactive with human, mouse, and canine Ecad, was transduced into human MSCs via lentivirus and expanded in vitro before intraperitoneal injection into healthy canine subjects. Each subgroup was monitored for hematological and organ toxicity for 28 days. Figure 28B It is a series of complete blood cell count level charts used to evaluate the post-hematopoietic safety of EcCAR-MSC administration. This includes white blood cells, monocytes, lymphocytes, neutrophils and platelets, and short-term (3 days) and long-term (28 days) blood level monitoring after in vivo injection of EcCAR-MSC compared to baseline. The mean ± standard deviation of blood components is shown, and statistical analysis was performed using one-way analysis of variance (ns=p≥0.05; n=3 subjects per experimental group). Figure 28C A series of graphs showing total protein, BUN, creatinine, albumin, and alkaline phosphatase levels for safety confirmation by short-term (3 days) and long-term (28 days) monitoring after in vivo EcCAR-MSC injection. Data shown are mean ± standard deviation, and statistical analysis was performed using one-way ANOVA (ns=p≥0.05; n=3 subjects per experimental group). Figure 28D The graph shows weight changes in healthy dogs after administration of EcCAR-MSCs compared to a control group. No significant differences in weight change were observed between the two groups. Data presented are mean ± SD percent weight change from baseline, statistically analyzed by conventional one-way ANOVA (ns = p ≥ 0.05; n = 3 subjects per experimental group). Figure 28E Representative images of transverse tissue sections of canine colon treated with human EcCAR-MSCs (left) or control (right) at 28 days post-administration and taken at 20x and 40x magnifications by H&E staining.

[0043] Figure 29 This graph shows the EcCAR positivity rate after transduction of mouse adipose-derived MSCs by flow cytometry. Data shown are the mean ± standard deviation of three technical replicates of each mouse cell line. DETAILED DESCRIPTION

[0044] Provided herein are methods and materials for treating a mammal (e.g., a human) (e.g., a human with or with a risk of one or more autoimmune diseases (such as GVHD)) requiring immunosuppression. In some cases, one or more CAR-MSCs can be given to a mammal, wherein the one or more CAR-MSCs have (e.g., engineered to have) elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more transport polypeptides to induce an immunosuppressive response (e.g., to reduce or eliminate an inflammatory immune response) in the target tissue of the mammal. For example, CAR-MSCs can express CARs capable of targeting epithelial-specific antigens (e.g., Ecad) (also referred to as CDH1) to target MSCs to epithelial tissue, and can also have (e.g., engineered to have) elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more transport polypeptides to enhance immunosuppression within the target epithelial tissue. In some cases, CAR-MSC can express a CAR that can target a neural-specific antigen (e.g., myelin oligodendrocyte glycoprotein (MOG)) to target MSC to neural tissue, and can also have (e.g., can be engineered to have) elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more transport polypeptides to enhance immunosuppression within the target neural tissue. For example, a mammal (e.g., a human) having (or at risk of developing) an inflammatory disease or disorder can be given (e.g., by adoptive transfer) one or more CAR-MSCs having (e.g., engineered to have) elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more transport polypeptides to treat one or more autoimmune diseases (e.g., GVHD) in a target tissue (e.g., inflamed tissue) in a mammal.

[0045] With respect to the level of a polypeptide (e.g., an immunosuppressive polypeptide or a trafficking polypeptide), the term "elevated level" as used herein refers to any level above a reference level of the polypeptide. With respect to a polypeptide (e.g., an immunosuppressive polypeptide or a trafficking polypeptide), the term "reference level" as used herein refers to the level of the polypeptide typically observed in MSCs (e.g., CAR-MSCs) that have not been engineered to have elevated levels of the polypeptide described herein.

[0046] MSC as described herein (for example, CAR-MSC with one or more immunosuppressive polypeptides, one or more regeneration polypeptides and / or one or more transport polypeptides of elevated levels) can be any suitable MSC. The example of MSC that can be used as described herein includes but is not limited to MSC derived from fat, MSC derived from bone marrow, MSC derived from placental tissue, MSC derived from pulp tissue, MSC derived from umbilical cord, MSC derived from cord blood, MSC derived from Wharton's jelly, MSC derived from dermis, MSC derived from olfactory mucosa, MSC derived from peripheral blood and MSC derived from amniotic membrane. For example, CAR-MSC with (for example, engineered to have) elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regeneration polypeptides and / or elevated levels of one or more transport polypeptides can be adipose-derived MSC.

[0047] CAR-MSCs provided herein (e.g., CAR-MSCs with elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more transport polypeptides) can express any suitable CAR. CAR can comprise (a) an antigen binding domain, (b) a transmembrane domain, and (c) one or more signal transduction domains.

[0048] CAR-MSC provided herein (for example, with one or more immunosuppressive polypeptides of elevated levels, one or more regeneration polypeptides of elevated levels and / or one or more transport polypeptides of elevated levels) expressed CAR The antigen binding domains can be any suitable antigen binding domains. In some cases, antigen binding domains can include antibodies or fragments thereof that can target antigens (for example, tissue-specific antigens, such as epithelial-specific antigens or nerve-specific antigens). The example of antigen binding domains includes but is not limited to antigen binding fragment (Fab), variable region of antibody heavy chain (VH), variable region of light chain (VL) and single-chain variable fragment (scFv). In some cases, antigen binding domains can target (for example, can be targeted and bound to) tissue-specific antigens (for example, epithelial-specific antigens or nerve-specific antigens). For example, CAR-MSC as described herein can express (for example, can be engineered to express) can be bound to tissue-specific antigens (for example, present on cells within tissues, with minimal expression or no expression of antigens on other cell types) CAR.

[0049] In some cases, provided herein are CAR-MSCs (for example, CAR-MSCs with elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides and / or elevated levels of one or more transport polypeptides) that can be engineered to express CAR, which can target (for example, can target and bind to) an antigen (for example, cell surface antigen) (for example, an epithelial-specific antigen or an epithelial antigen) expressed by epithelial cells in the epithelial tissue of a mammal (for example, a human with or with a risk of one or more autoimmune diseases (such as GVHD)) that needs immunosuppression. The epithelial-specific antigen can be any suitable epithelial-specific antigen. The epithelial-specific antigen can be expressed on any appropriate type of epithelial cell (for example, gastrointestinal cells, such as colon cells and rectal cells, skin cells, lung cells, liver cells, reproductive tract cells, and urinary tract cells). In some cases, the epithelial-specific antigen can be a cell adhesion molecule (CAM). In some cases, the epithelial-specific antigen can be an integrin (for example, intestinal integrin). Examples of epithelial-specific antigens include, but are not limited to, Ecad, CD103, αEβ7, and α4β7. For example, the CAR-MSCs engineered to target epithelial tissue can bind to Ecad. In some cases, CAR-MSCs can be engineered to express CAR-Ecad to target Ecad expressed by mammalian epithelial cells. In some cases, the CARs expressed by the CAR-MSCs provided herein can include an Ecad binding domain. For example, the CARs expressed by the CAR-MSCs provided herein can include an anti-Ecad scFv, the anti-Ecad scFv including a heavy chain and a light chain, the heavy chain containing a CDR as shown in SEQ ID NO: 1, and the light chain containing a CDR as shown in SEQ ID NO: 2. For example, the CARs expressed by the CAR-MSCs provided herein can have an anti-Ecad scFv, the anti-Ecad scFv including a heavy chain containing an amino acid sequence as shown in SEQ ID NO: 1, consisting essentially of an amino acid sequence as shown in SEQ ID NO: 1, or consisting of a heavy chain as shown in SEQ ID NO: 1, and containing an amino acid sequence as shown in SEQ ID NO: 2, consisting essentially of an amino acid sequence as shown in SEQ ID NO: 2, or consisting of a light chain as shown in SEQ ID NO: 2 (see, e.g., Example 2). In some cases, the CAR expressed by the CAR-MSC provided herein can include a CD103 binding domain. For example, the CAR expressed by the CAR-MSC provided herein can include an anti-CD103 scFv, wherein the anti-CD103 scFv includes a heavy chain and a light chain, wherein the heavy chain contains CDRs as shown in SEQ ID NO: 3, and the light chain contains CDRs as shown in SEQ ID NO: 4.For example, the CAR-MSC-expressed CAR provided herein can have an anti-CD103 scFv comprising a heavy chain comprising, consisting essentially of, or consisting of the amino acid sequence as shown in SEQ ID NO: 3, and a light chain comprising, consisting essentially of, or consisting of the amino acid sequence as shown in SEQ ID NO: 4 (see, e.g., Example 2).

[0050] In some cases, the CAR-MSCs provided herein (e.g., CAR-MSCs with elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more transport polypeptides) can be engineered to express CAR that can target (e.g., can target and bind to) antigens (e.g., cell surface antigens) (e.g., neural-specific antigens or neural antigens) expressed by neural cells in the nervous tissue of a mammal (e.g., a human at risk of suffering from or having one or more autoimmune diseases (such as multiple sclerosis)) that requires immunosuppression. When the antigen is a neural-specific antigen, the neural-specific antigen can be any suitable neural-specific antigen. The neural-specific antigen can be expressed on any appropriate type of neural cell (e.g., sensory neurons, motor neurons, interneurons, glial cells, and nerve sheath cells). The neural-specific antigen can be expressed on neural cells of the central nervous system (CNS) and / or the peripheral nervous system (PNS). In some cases, the neural-specific antigen can be a transmembrane protein. In some cases, the neural-specific antigen can target myelin. Examples of neural-specific antigens include, but are not limited to, MOG and AMPA (e.g., antigens on one or more AMPA receptor polypeptides). For example, a CAR-MSC engineered to target neural tissue can bind to MOG. In some cases, a CAR-MSC can be engineered to express CAR-MOG to target MOG expressed by neural cells in mammals suffering from or at risk of multiple sclerosis. In some cases, the CAR expressed by the CAR-MSC provided herein may comprise a MOG antigen binding domain. For example, the CAR expressed by the CAR-MSC provided herein may comprise an anti-MOG scFv comprising a heavy chain and a light chain, the heavy chain comprising the CDRs shown in SEQ ID NO: 5, and the light chain comprising the CDRs shown in SEQ ID NO: 6.

[0051] For example, the CAR-MSCs expressed herein may have an anti-MOG scFv comprising a heavy chain comprising, consisting essentially of, or consisting of an amino acid sequence as shown in SEQ ID NO: 5, and a light chain comprising, consisting essentially of, or consisting of an amino acid sequence as shown in SEQ ID NO: 6 (see, e.g., Example 2). In some cases, the CARs expressed by the CAR-MSCs provided herein may comprise an AMPA antigen binding domain. For example, the CARs expressed by the CAR-MSCs provided herein may comprise an anti-AMPA scFv comprising a heavy chain and a light chain, the heavy chain comprising a CDR as shown in SEQ ID NO: 7, and the light chain comprising a CDR as shown in SEQ ID NO: 8. For example, the CAR-MSC expressed by the CAR provided herein can have an anti-AMPA scFv comprising a heavy chain comprising, consisting essentially of, or consisting of the amino acid sequence as shown in SEQ ID NO: 7, and a light chain comprising, consisting essentially of, or consisting of the amino acid sequence as shown in SEQ ID NO: 8 (see, e.g., Example 2).

[0052] CAR-MSC provided herein (for example, CAR-MSC with one or more immunosuppressive polypeptides of elevated levels, one or more regeneration polypeptides of elevated levels and / or one or more transport polypeptides of elevated levels) expressed) The membrane spaning domain of the CAR can be any suitable membrane spaning domain. The example of the membrane spaning domain that can be used as described herein includes but is not limited to NKG2D membrane spaning domain, CD8 α membrane spaning domain, CD28 membrane spaning domain, IgG4 membrane spaning domain, TNFR membrane spaning domain and TLR membrane spaning domain. In some cases, the membrane spaning domain included in the CAR expressed by CAR-MSC provided herein can be connected (for example, covalently linked) to the adapter polypeptide. In some cases, the CAR expressed by CAR-MSC provided herein can include CD28 membrane spaning domain. For example, the CAR expressed by CAR-MSC provided herein can include CD28 membrane spaning domain, the CD28 membrane spaning domain including as shown in SEQ ID NO:9 amino acid sequence, substantially by as shown in SEQ ID NO:9 amino acid sequence composition or by as shown in SEQ ID NO:9 amino acid sequence composition (see, e.g., Example 2).

[0053] The signal transduction domain of the CAR expressed by CAR-MSC provided herein (for example, one or more immunosuppressive polypeptides with elevated levels, one or more regeneration polypeptides with elevated levels and / or one or more transport polypeptides with elevated levels) can include any suitable one or more signal transduction domains.For example, the CAR expressed by CAR-MSC provided herein can be designed to include one, two, three or four signal transduction domains.When the CAR expressed by CAR-MSC provided herein includes more than one (for example, two, three or four) signal transduction domains, the CAR can include a combination of any suitable signal transduction domains.In some cases, the CAR expressed by CAR-MSC provided herein can be designed to include one or more signal transduction domains typically present in immune cells (for example, lymphocytes, such as TIL, T cells or NK cells).In some cases, the CAR expressed by CAR-MSC provided herein can be designed to include one or more signal transduction domains typically present in MSC (for example, MSC with an immunosuppressive phenotype).In some cases, the CAR expressed by CAR-MSC provided herein can be designed to include one or more regeneration signal transduction domains (for example, to stimulate tissue regeneration). The example of the signal transduction domain that can be used as described herein includes but is not limited to CD3 ζ signal transduction domain, CD28 signal transduction domain, 4-1BB signal transduction domain, OX40 signal transduction domain, TLR3 signal transduction domain, TLR4 signal transduction domain, TIR3 signal transduction domain, TIR4 signal transduction domain, IFNγ signal transduction domain, brain-derived neurotrophic factor (BDNF) signal transduction domain, vascular endothelial-derived growth factor receptor (VEGFR2) signal transduction domain, TLR3 / TIR3 signal transduction domain, TLR4 / TIR4 signal transduction domain, TLR9 / TIR9 signal transduction domain, IFNGR1 signal transduction domain, IL10RA signal transduction domain and TNFR2 signal transduction domain.In some cases, the CAR for CAR-MSC expression provided herein can be designed as the first generation CAR with CD3 ζ signal transduction domain. In some cases, the CAR for CAR-MSC expression provided herein can be designed to have CD28 signal transduction domain and subsequent CD3 ζ signal transduction domain second generation CAR.In some cases, the CAR for CAR-MSC expression provided herein can be designed to have (a) CD28 signal transduction domain and subsequently (b) CD27 signal transduction domain, OX40 signal transduction domain or 4-1BB signal transduction domain and subsequently (c) CD3 ζ signal transduction domain third generation CAR.In some cases, the CAR for CAR-MSC expression provided herein can include CD28 signal transduction domain.For example, the CAR-MSC expressed by the CAR provided herein can include a CD28 signaling domain comprising, consisting essentially of, or consisting of the amino acid sequence shown in SEQ ID NO: 10 (see, e.g., Example 2) and a CD3 zeta signaling domain comprising, consisting essentially of, or consisting of the amino acids shown in SEQ ID NO: 11 (see, e.g., Example 2).

[0054] In some cases, the CAR-MSC provided herein (for example, CAR-MSC with one or more immunosuppressive polypeptides, one or more regeneration polypeptides and / or one or more transport polypeptides of increased levels) expressed may include one or more additional components. The example of additional components that may be included in the CAR expressed by the CAR-MSC provided herein includes but is not limited to a joint, a hinge domain and a detectable marker. When the antigen receptor (for example, CAR) provided herein includes a detectable marker, the detectable marker may be any suitable detectable marker. The example of detectable markers that may be included in the CAR expressed by the CAR-MSC provided herein includes but is not limited to bioluminescent polypeptides (for example, luciferase polypeptides), fluorescent polypeptides (for example, green fluorescent polypeptide (GFP)), sodium-iodine symporter (NIS), SSTR2 polypeptide, PSMA polypeptide, hdCK polypeptide and eDHFR polypeptide.

[0055] In some cases, the CAR expressed by the CAR-MSC provided herein (e.g., CAR-MSC having elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more trafficking polypeptides) can comprise an anti-Ecad scFv, a CD28 transmembrane domain, a CD28 signaling domain, and a CD3ζ signaling domain. For example, the CAR-MSC-expressed CAR provided herein can comprise an anti-Ecad scFv having a heavy chain and a light chain, the heavy chain comprising the CDRs set forth in SEQ ID NO: 1, and the light chain comprising the CDRs set forth in SEQ ID NO: 2 (see, e.g., Example 2), a CD28 transmembrane domain comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 9 (see, e.g., Example 2), a CD28 signaling domain comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 10 (see, e.g., Example 2), and a CD3 zeta signaling domain comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 11 (see, e.g., Example 2).

[0056] Any appropriate method can be used to express CAR described herein (e.g., CAR-MSC with elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more transport polypeptides) on the surface of CAR-MSCs provided herein (e.g., CAR targeting tissue-specific antigens (such as epithelial-specific antigens or neural-specific antigens)). For example, a nucleic acid encoding CAR can be introduced into MSC. In some cases, the nucleic acid encoding CAR can be introduced into MSC by transduction (e.g., viral transduction) or transfection. In some cases, the nucleic acid encoding CAR described herein can be introduced into one or more MSCs in vitro. For example, ex vivo engineering of MSC to express CAR described herein can include transducing isolated MSCs with a vector encoding CAR (e.g., a viral vector, such as a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector). If MSC is engineered in vitro to express CAR, the MSC can be obtained from any suitable source (e.g., a mammal (such as a mammal to be treated or a donor mammal) or a cell line). In some cases, CAR-MSC can be prepared as described herein (see, e.g., Figure 7 and Example 1). For example, by introducing one or more constructs containing nucleic acids encoding CAR (e.g., CAR targeting Ecad) into MSCs, CAR-Ecad can be expressed on MSCs to direct MSCs to epithelial tissue. For example, by introducing one or more constructs containing nucleic acids encoding CAR (e.g., CAR targeting MOG) into MSCs, CAR-MOG can be expressed on MSCs to direct MSCs to neural tissue.

[0057] In some cases, the CAR-MSCs provided herein (e.g., CAR-MSCs with elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more transport polypeptides) expressed CAR can be a CAR described elsewhere. See, e.g., International Patent Application Publication No. WO 2021 / 092577, e.g., page 18, line 16 to page 20, line 25, and page 21, line 29 to page 25, line 11.

[0058] CAR-MSC provided herein (for example, CAR-MSC with one or more immunosuppressive polypeptides, one or more regeneration polypeptides and / or one or more transport polypeptides of elevated levels) can express any appropriate one or more immunosuppressive polypeptides, one or more regeneration polypeptides and / or elevated levels of one or more transport polypeptides. In some cases, the polypeptide (for example, immunosuppressive polypeptide or transport polypeptide) expressed by CAR-MSC provided herein can be an exogenous polypeptide. In some cases, the polypeptide (for example, immunosuppressive polypeptide or transport polypeptide) expressed by CAR-MSC provided herein can be a part of a signal transduction pathway (for example, TNFα signal transduction pathway, TLR signal transduction pathway and IL-10 signal transduction pathway). In some cases, the polypeptide (for example, immunosuppressive polypeptide or transport polypeptide) expressed by CAR-MSC provided herein can be associated with T cell suppression. In some cases, the polypeptide (for example, immunosuppressive polypeptide or transport polypeptide) expressed by CAR-MSC provided herein can be a kinase (for example, tyrosine kinase). In some cases, the polypeptide (for example, immunosuppressive polypeptide or transport polypeptide) expressed by CAR-MSC provided herein can be a transcription factor. In some cases, the polypeptide (for example, immunosuppressive polypeptide or transport polypeptide) expressed by CAR-MSC provided herein can be a chemokine (for example, a homing chemokine). In some cases, the polypeptide (for example, immunosuppressive polypeptide or transport polypeptide) expressed by CAR-MSC provided herein can be a cytokine (for example, an anti-inflammatory cytokine). In some cases, the polypeptide (for example, immunosuppressive polypeptide or transport polypeptide) expressed by CAR-MSC provided herein can be an immunomodulatory cell surface polypeptide. In some cases, the polypeptide (for example, immunosuppressive polypeptide or transport polypeptide) expressed by CAR-MSC provided herein can be related to cell migration (for example, transendothelial migration). Examples of immunosuppressive polypeptides and trafficking polypeptides that the CAR-MSCs provided herein can express (e.g., can be designed to express) include, but are not limited to, NFκB1 polypeptides, JUN polypeptides, RELB polypeptides, IRF1 polypeptides, TNFα polypeptides, IL-10 polypeptides, FGF-2 polypeptides, PD-1 polypeptides, G-CSF polypeptides, GM-CSF polypeptides, eotaxin polypeptides, Gal-9 polypeptides, PD-1 polypeptides, TIM-3 polypeptides, CXCR3 polypeptides, CXCR4 polypeptides, CTLA4 polypeptides, TLR3 polypeptides, TLR4 polypeptides, TLR9 polypeptides, and TNFR2 polypeptides.

[0059] Any appropriate method can be used to increase the level of one or more immunosuppressive polypeptides in CAR-MSC provided herein, increase the level of one or more regeneration polypeptides and / or increase the level of one or more transport polypeptides.In some cases, the nucleic acid (for example, exogenous nucleic acid) of coded polypeptide (for example, immunosuppressive polypeptide or transport polypeptide) can be introduced into CAR-MSC.For example, the exogenous nucleic acid encoding one or more immunosuppressive polypeptides, one or more regeneration polypeptides and / or one or more transport polypeptides can be introduced into MSC by transduction (for example, viral transduction) or transfection.In some cases, the nucleic acid (for example, endogenous nucleic acid) encoding one or more immunosuppressive polypeptides, one or more regeneration polypeptides and / or one or more transport polypeptides can be modified in CAR-MSC to increase the expression level of one or more immunosuppressive polypeptides, one or more regeneration polypeptides and / or one or more transport polypeptides in CAR-MSC. For example, endogenous nucleic acids encoding immunosuppressive polypeptides, regenerative polypeptides, or transport polypeptides within MSCs can be modified by gene editing techniques (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease (Cas) systems, transcription activator-like effector nuclease (TALEN) systems, zinc finger nuclease (ZFN) systems, and base pair editing) to increase the expression level of one or more immunosuppressive polypeptides in CAR-MSCs, increase the expression level of one or more regenerative polypeptides, and / or increase the level of one or more transport polypeptides in CAR-MSCs.

[0060] In some cases, provided herein are CAR-MSCs (for example, CAR-MSCs with elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides and / or elevated levels of one or more transport polypeptides) that can be engineered to have elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides and / or elevated levels of one or more transport polypeptides in vitro. For example, CAR-MSCs are engineered in vitro to have elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides and / or elevated levels of one or more transport polypeptides, including CAR-MSCs transduced with a vector encoding one or more polypeptides (for example, a viral vector, such as a lentiviral vector, a retroviral vector, an adenoviral vector, or an AAV vector). For example, CAR-MSCs are engineered in vitro to have elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides and / or elevated levels of one or more transport polypeptides, including CAR-MSCs transduced with one or more lentiviral vectors encoding gene editing components.

[0061] Examples of NFκB1 polypeptides that can be increased in CAR-MSCs provided herein (e.g., CAR-MSCs having increased levels of one or more immunosuppressive polypeptides, increased levels of one or more regenerative polypeptides, and / or increased levels of one or more transport polypeptides) include those shown in the National Center for Biotechnology Information (NCBI) database, e.g., Accession No. NM_001165412 (version NM_001165412.1), Accession No. NM_001319226 (version NM_001319226.2), and Accession No. NM_001382625 (version NM_001382625.1). In some cases, the NFκB1 polypeptide that can be increased in CAR-MSCs provided herein can have the amino acid sequence shown in SEQ ID NO: 12 (see, e.g., Example 2).

[0062] Examples of JUN polypeptides that can be increased in CAR-MSCs provided herein (e.g., CAR-MSCs having increased levels of one or more immunosuppressive polypeptides, increased levels of one or more regenerative polypeptides, and / or increased levels of one or more transport polypeptides) include those shown in the NCBI database, e.g., Accession No. NM_002228 (version NM_002228.4), Accession No. AL136985 (version AL136985.11), and Accession No. CH471059 (version CH471059.2). In some cases, the JUN polypeptide that can be increased in CAR-MSCs provided herein can have the amino acid sequence shown in SEQ ID NO: 13 (see, e.g., Example 2).

[0063] Examples of RELB polypeptides that can be increased in CAR-MSCs provided herein (e.g., CAR-MSCs having increased levels of one or more immunosuppressive polypeptides, increased levels of one or more regenerative polypeptides, and / or increased levels of one or more transport polypeptides) include those shown in the NCBI database, such as NM_001411087 (version NM_001411087.1) and accession number NM_006509 (version NM_006509.4). In some cases, the RELB polypeptide that can be increased in CAR-MSCs provided herein can have the amino acid sequence shown in SEQ ID NO: 14 (see, e.g., Example 2).

[0064] The example of the IRF1 polypeptide that can be increased in CAR-MSC provided herein (for example, CAR-MSC with increased one or more immunosuppressive polypeptide levels, increased one or more regenerative polypeptide levels and / or increased one or more transport polypeptide levels) includes those shown in the NCBI database, for example, Accession No. NM_001354924 (version NM_001354924.1), Accession No. NM_001354925 (version NM_001354925.1) and Accession No. NM_002198 (version NM_002198.3). In some cases, the IRF1 polypeptide that can be increased in CAR-MSC provided herein can have SEQ ID NO: The amino acid sequence shown in 15 (see, e.g., Example 2).

[0065] Examples of TNFα polypeptides that can be increased in CAR-MSCs provided herein (e.g., CAR-MSCs having increased levels of one or more immunosuppressive polypeptides, increased levels of one or more regenerative polypeptides, and / or increased levels of one or more transport polypeptides) include those shown in the NCBI database, e.g., accession number NM_000594 (version NM_000594.4). In some cases, the TNFα polypeptides that can be increased in CAR-MSCs provided herein can have the amino acid sequence shown in SEQ ID NO: 16 (see, e.g., Example 2).

[0066] Examples of IL-10 polypeptides that can be elevated in CAR-MSCs provided herein (e.g., CAR-MSCs having elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more transport polypeptides) include those shown in the NCBI database, e.g., Accession No. NM_000572 (Version NM_000572.3) and Accession No. NM_001382624 (Version NM_001382624.1). In some cases, the IL-10 polypeptide that can be elevated in CAR-MSCs provided herein can have the amino acid sequence shown in SEQ ID NO: 17 (see, e.g., Example 2).

[0067] Examples of FGF-2 polypeptides that can be increased in CAR-MSCs provided herein (e.g., CAR-MSCs having increased levels of one or more immunosuppressive polypeptides, increased levels of one or more regenerative polypeptides, and / or increased levels of one or more transport polypeptides) include those shown in the NCBI database, e.g., Accession No. NM_001361665 (Version NM_001361665.2) and Accession No. NM_002006 (Version NM_002006.6). In some cases, the FGF-2 polypeptide that can be increased in CAR-MSCs provided herein can have the amino acid sequence shown in SEQ ID NO: 18 (see, e.g., Example 2).

[0068] Examples of G-CSF polypeptides that can be elevated in CAR-MSCs provided herein (e.g., CAR-MSCs having elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more trafficking polypeptides) include those shown in the NCBI database, e.g., Accession No. NM_000759 (Version NM_000759.4), Accession No. NM_001178147 (Version NM_001178147.2), and Accession No. NM_172219 (Version NM_172219.3). In some cases, the G-CSF polypeptide that can be elevated in CAR-MSCs provided herein can have the amino acid sequence shown in SEQ ID NO: 19 (see, e.g., Example 2).

[0069] Examples of GM-CSF polypeptides that can be elevated in CAR-MSCs provided herein (e.g., CAR-MSCs having elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more trafficking polypeptides) include those shown in the NCBI database, e.g., Accession No. NM_000758 (version NM_000758.4), Accession No. AC003959 (version AC003959.1), and Accession No. DQ789232 (version DQ789232.1). In some cases, the GM-CSF polypeptide that can be elevated in CAR-MSCs provided herein can have the amino acid sequence shown in SEQ ID NO: 20 (see, e.g., Example 2).

[0070] The example of the eotaxin polypeptide that can be increased in CAR-MSC provided herein (for example, CAR-MSC with increased levels of one or more immunosuppressive polypeptides, increased levels of one or more regenerative polypeptides and / or increased levels of one or more transport polypeptides) includes those shown in the NCBI database, for example, Accession No. NM_002986 (version NM_002986.3), Accession No. AC003959 (version AC003959.1) and Accession No. CH471062 (version CH471062.2). In some cases, the eotaxin polypeptide that can be increased in CAR-MSC provided herein can have SEQ ID NO: The amino acid sequence shown in 21 (see, e.g., Example 2).

[0071] The examples of Gal-9 polypeptides that can be increased in CAR-MSCs provided herein (e.g., CAR-MSCs with increased levels of one or more immunosuppressive polypeptides, increased levels of one or more regenerative polypeptides, and / or increased levels of one or more transport polypeptides) include those shown in the NCBI database, e.g., Accession No. NM_001330163 (version NM_001330163.2), Accession No. NM_002308 (version NM_002308.4), and Accession No. NM_009587 (version NM_009587.3). In some cases, the Gal-9 polypeptides that can be increased in CAR-MSCs provided herein can have an amino acid sequence shown in SEQ ID NO: 22 (see, e.g., Example 2).

[0072] Examples of PD-1 polypeptides that can be increased in CAR-MSCs provided herein (e.g., CAR-MSCs having increased levels of one or more immunosuppressive polypeptides, increased levels of one or more regenerative polypeptides, and / or increased levels of one or more transport polypeptides) include those shown in the NCBI database, e.g., Accession No. NM_005018 (version NM_005018.3), Accession No. AF363458 (version AF363458.1), and Accession No. EF064716 (version EF064716.1). In some cases, the PD-1 polypeptide that can be increased in CAR-MSCs provided herein can have the amino acid sequence shown in SEQ ID NO: 23 (see, e.g., Example 2).

[0073] Examples of TIM-3 polypeptides that can be increased in CAR-MSCs provided herein (e.g., CAR-MSCs having increased levels of one or more immunosuppressive polypeptides, increased levels of one or more regenerative polypeptides, and / or increased levels of one or more transport polypeptides) include those shown in the NCBI database, e.g., Accession No. NM_032782 (version NM_032782.5), Accession No. CQ834184 (version CQ834184.1), and Accession No. AC011377 (version AC011377.6). In some cases, the TIM-3 polypeptide that can be increased in CAR-MSCs provided herein can have the amino acid sequence shown in SEQ ID NO: 24 (see, e.g., Example 2).

[0074] Examples of CXCR3 polypeptides that can be increased in CAR-MSCs provided herein (e.g., CAR-MSCs having increased levels of one or more immunosuppressive polypeptides, increased levels of one or more regenerative polypeptides, and / or increased levels of one or more transport polypeptides) include those shown in the NCBI database, e.g., Accession No. NM_001142797 (version NM_001142797.2), Accession No. NM_001504 (version NM_001504.2), and Accession No. AB032738 (version AB032738.1). In some cases, the CXCR3 polypeptide that can be increased in CAR-MSCs provided herein can have the amino acid sequence shown in SEQ ID NO: 25 (see, e.g., Example 2).

[0075] Examples of CXCR4 polypeptides that can be increased in CAR-MSCs provided herein (e.g., CAR-MSCs having increased levels of one or more immunosuppressive polypeptides, increased levels of one or more regenerative polypeptides, and / or increased levels of one or more transport polypeptides) include those shown in the NCBI database, e.g., Accession No. NM_001008540 (version NM_001008540.2), Accession No. NM_001348056 (version NM_001348056.2), and Accession No. NM_001348059 (version NM_001348059.2). In some cases, the CXCR4 polypeptide that can be increased in CAR-MSCs provided herein can have the amino acid sequence shown in SEQ ID NO: 26 (see, e.g., Example 2).

[0076] Examples of CTLA4 polypeptides that can be elevated in CAR-MSCs provided herein (e.g., CAR-MSCs with elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more transport polypeptides) include those listed in the NCBI database, e.g., accession numbers NM_001037631 (version NM_001037631.2), NM_001037631 (version NM_001037631.3), and NM_005214 (versions NM_005214.4 and NM_005214.5). In some cases, the CTLA4 polypeptides that can be elevated in CAR-MSCs provided herein can have an amino acid sequence as shown in SEQ ID NO:91 (see, e.g., Example 2).

[0077] Examples of TLR3 polypeptides that can be increased in CAR-MSCs provided herein (e.g., CAR-MSCs with increased levels of one or more immunosuppressive polypeptides, increased levels of one or more regenerative polypeptides, and / or increased levels of one or more transport polypeptides) include those shown in the NCBI database, e.g., accession numbers NM_003265 (versions NM_003265.2 and NM_003265.3), NM_133484 (NM_133484.2), and NM_126166 (version NM_126166.4). In some cases, the TLR3 polypeptides that can be increased in CAR-MSCs provided herein can have the amino acid sequence shown in SEQ ID NO: 92 (see, e.g., Example 2).

[0078] Examples of TLR4 polypeptides that can be elevated in CAR-MSCs provided herein (e.g., CAR-MSCs having elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more transport polypeptides) include those listed in the NCBI database, e.g., accession numbers NM_003266 (version NM_003266.4), NM_138557 (version NM_138557.3), and NM_138554 (version NM_138554.4). In some cases, the TLR4 polypeptides that can be elevated in CAR-MSCs provided herein can have the amino acid sequence shown in SEQ ID NO: 93 (see, e.g., Example 2).

[0079] Examples of TLR9 polypeptides that can be increased in CAR-MSCs provided herein (e.g., CAR-MSCs having increased levels of one or more immunosuppressive polypeptides, increased levels of one or more regenerative polypeptides, and / or increased levels of one or more transport polypeptides) include those shown in the NCBI database, e.g., accession numbers NM_017442 (versions NM_017442.3 and NM_017442.4) and NM_031178 (version NM_031178.2). In some cases, the TLR9 polypeptides that can be increased in CAR-MSCs provided herein can have an amino acid sequence shown in SEQ ID NO: 94 (see, e.g., Example 2).

[0080] Examples of TNFR2 polypeptides that can be elevated in CAR-MSCs provided herein (e.g., CAR-MSCs having elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more trafficking polypeptides) include those shown in the NCBI database, e.g., accession numbers NM_001066 (version NM_001066.2) and NM_011610 (version NM_011610.3). In some cases, the TNFR2 polypeptide that can be elevated in CAR-MSCs provided herein can have the amino acid sequence shown in SEQ ID NO: 95 (see, e.g., Example 2).

[0081] Also provided herein are materials and methods for treating a mammal (e.g., a human being with or with a risk of one or more autoimmune diseases (such as GVHD)) requiring immunosuppression. In some cases, a mammal (e.g., a human being) requiring immunosuppression may suffer from GVHD (or have a risk of suffering from it). For example, one or more CAR-MSCs provided herein (e.g., one or more immunosuppressive polypeptides with elevated levels, one or more regenerative polypeptides with elevated levels, and / or one or more transport polypeptides with elevated levels) can be administered (e.g., by adoptive transfer to) a mammal with one or more autoimmune diseases to reduce the severity of the immune response in the mammal. Any appropriate method can be used to identify a mammal requiring immunosuppression. Once identified as requiring immunosuppression, one or more CAR-MSCs provided herein can be administered to a mammal (e.g., a human being), as described herein, to reduce the immune response in the mammal by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more.

[0082] In some cases, when treating a mammal (e.g., a human) in need of immunosuppression (e.g., a human suffering from or at risk for one or more autoimmune diseases such as GVHD), the treatment can be effective to reduce inflammation in the mammal (e.g., a target tissue in the mammal), as described herein. For example, the methods and materials described herein can be used to reduce inflammation in a mammal suffering from one or more autoimmune diseases (e.g., GVHD) by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

[0083] In some cases, when treating a mammal (e.g., a human) in need of immunosuppression (e.g., a human suffering from or at risk of developing one or more autoimmune diseases (such as GVHD)), as described herein, the treatment can be effective to increase the number of regulatory T cells (Tregs) in the mammal (e.g., a target tissue in the mammal). For example, the methods and materials described herein can be used to increase the number of Tregs in a mammal in need of immunosuppression (e.g., a human suffering from or at risk of developing one or more autoimmune diseases (such as GVHD)), for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more). In some cases, the number of Tregs in the mammal does not decrease.

[0084] In some cases, when treating a mammal in need of immunosuppression (e.g., a human suffering from or at risk for one or more autoimmune diseases such as GVHD), the treatment can be effective to reduce the number of activated T cells (e.g., CD4 T cells) in the mammal (e.g., in a target tissue in the mammal), as described herein. + T cells and / or CD8 + For example, the methods and materials described herein can be used to convert activated T cells (e.g., CD4 T cells) into a population of activated T cells (e.g., CD4 T cells) in a mammal in need of immunosuppression (e.g., a human suffering from or at risk for one or more autoimmune diseases such as GVHD) + T cells and / or CD8 + T cells) is reduced, for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some cases, the number of activated T cells (e.g., CD4 + T cells and / or CD8 + T cells) will not increase in number.

[0085] In some cases, when treating a mammal (e.g., a human) in need of immunosuppression (e.g., a human suffering from or at risk of one or more autoimmune diseases (such as GVHD)), as described herein, the treatment can be effective to reduce the rate of T cell proliferation (e.g., activated T cell proliferation) in the mammal (e.g., in a target tissue in the mammal). For example, the methods and materials described herein can be used to reduce the rate of T cell proliferation in a mammal (e.g., a human suffering from or at risk of one or more autoimmune diseases (such as GVHD)) in need of immunosuppression by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some cases, the rate of T cell proliferation in the mammal does not increase.

[0086] Any suitable mammal requiring immunosuppression (e.g., humans with or at risk of one or more autoimmune diseases (such as GVHD)) can be treated as described herein. Examples of mammals that may require immunosuppression and can be treated as described herein include, but are not limited to, humans, non-human primates (e.g., monkeys), dogs, cats, horses, cattle, pigs, sheep, mice, and rats. For example, one or more CAR-MSCs provided herein (e.g., CAR-MSCs with elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more transport polypeptides) can be administered (e.g., by adoptive transfer) to humans with one or more autoimmune diseases to treat the humans.

[0087] Any appropriate one or more autoimmune diseases (e.g., GVHD) can be treated as described herein. Examples of autoimmune diseases include, but are not limited to, GVHD, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), hepatitis, bronchiolitis obliterans, pneumonia, encephalitis, multiple sclerosis, rheumatoid arthritis, lupus and psoriasis. In some cases, autoimmune disease can be GVHD (e.g., acute GVHD or chronic GVHD). GVHD can be allogeneic GVHD (allo-GVHD) or autologous GVHD (auto-GVHD). GVHD can be any grade of GVHD. In some cases, GVHD can be associated with transplantation (e.g., after transplantation). Transplantation can be allogeneic transplantation or autologous transplantation. When treating a mammal suffering from GVHD (or having a risk of suffering from it), the mammal can receive any type of transplantation (e.g., allogeneic transplantation, such as bone marrow transplantation, stem cell transplantation, and organ transplantation, such as kidney transplantation and liver transplantation).

[0088] In some cases, mammals can be identified as having or having the risk of suffering from one or more autoimmune diseases (e.g., GVHD).Any appropriate method can be used to identify that mammals have or have the risk of suffering from one or more autoimmune diseases (e.g., GVHD).In some cases, mammals can be identified as having GVHD (or having its risk).Any appropriate method for identifying mammals as having GVHD (or having its risk) can be used.Once identified as having GVHD (or having its risk), mammals can be given (e.g., by adoptive transfer) or instructed to self-administer one or more CAR-MSCs provided herein (e.g., one or more immunosuppressive polypeptides with elevated levels, one or more regenerative polypeptides with elevated levels, and / or one or more transport polypeptides with elevated levels) to treat GVHD in mammals.

[0089] In some cases, one or more CAR-MSCs provided herein (e.g., one or more immunosuppressive polypeptides, one or more regenerative polypeptides and / or one or more transport polypeptides with elevated levels) can be administered (e.g., by adoptive transfer) to a mammal with or at risk of GVHD to reduce the severity of GVHD in the mammal. In some cases, reducing the severity of GVHD in a mammal can include alleviating or eliminating one or more symptoms of GVHD (e.g., rash, immune-mediated pneumonia, intestinal inflammation, intestinal mucosal sloughing, severe diarrhea, abdominal pain, nausea, vomiting, and / or elevated bilirubin levels). In some cases, reducing the severity of GVHD in a mammal can include reducing the grade (stage) of GVHD. The grade of GVHD can be assessed as described elsewhere (see, e.g., Jacobsohn et al., Orphanet. J. Rare Dis., 2: 35 (2007)).

[0090] Any appropriate method can be used to administer one or more CAR-MSCs provided herein (e.g., one or more CAR-MSCs with elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more transport polypeptides) to a mammal (e.g., a human) in need of immunosuppression (e.g., a person suffering from or at risk of developing one or more autoimmune diseases such as GVHD). Examples of methods for administering MSCs provided herein to a mammal may include, but are not limited to, injection (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or local injection into an area of ​​inflammation). For example, a composition comprising one or more CAR-MSCs provided herein can be administered to a human by intravenous injection.

[0091] In some cases, one or more CAR-MSCs provided herein (e.g., one or more immunosuppressive polypeptides, one or more regenerative polypeptides and / or one or more transport polypeptides with elevated levels) can be used to treat a mammal (e.g., a human) (e.g., a human with or at risk of one or more autoimmune diseases (such as GVHD)) that needs immunosuppression. For example, a composition comprising one or more CAR-MSCs provided herein (e.g., one or more immunosuppressive polypeptides, one or more regenerative polypeptides and / or one or more transport polypeptides with elevated levels) can include one or more CAR-MSCs provided herein as the sole active agent to suppress an immune response in a mammal (e.g., a human) (e.g., a human with or at risk of one or more autoimmune diseases (such as GVHD)) that needs immunosuppression.

[0092] In some cases, provided herein is one or more CAR-MSC (for example, one or more immunosuppressive polypeptides with elevated levels, one or more regenerative polypeptides and / or one or more transport polypeptides with elevated levels) can be administered in combination with one or more additional agents to treat a mammal (for example, a human) that needs immunosuppression (for example, a human with or with the risk of suffering from one or more autoimmune diseases (such as GVHD)). In some cases, the medicament that can be used to suppress the immune response can be an anti-inflammatory agent. In some cases, the medicament that can be used to suppress the immune response can be an immunosuppressant. The example of the medicament that can be used in combination with one or more CAR-MSC provided herein includes but is not limited to corticosteroids (for example, prednisone and methylprednisolone), azathioprine, mercaptopurine, cyclosporin, infliximab, adalimumab, golimumab and vedolizumab. In some cases, provided herein is one or more CAR-MSC that can be administered substantially simultaneously with one or more additional agents. For example, a composition comprising one or more CAR-MSCs provided herein (e.g., one or more CAR-MSCs with elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more transport polypeptides) may further comprise one or more additional agents that can be used to suppress an immune response in a mammal. In some cases, one or more CAR-MSCs provided herein may be administered first, followed by administration of one or more additional agents, or vice versa.

[0093] In some cases, provided herein are one or more CAR-MSCs (e.g., one or more immunosuppressive polypeptides, one or more regeneration polypeptides and / or one or more transport polypeptides with elevated levels) can be used to treat a mammal (e.g., a human) having a disease or condition not characterized by inflammation. For example, provided herein are CAR-MSCs that can be used to target diseased tissue in a mammal (e.g., a human without immunosuppression) to treat the diseased tissue. In some cases, provided herein are CAR-MSCs that can be used to target degenerative tissue in a mammal (e.g., a human without immunosuppression) to treat tissue degeneration. For example, CAR-MSCs can express CARs that can target neural-specific antigens (e.g., AMPA) to target MSCs to neural tissue in a mammal (e.g., a mammal with a neurodegenerative disease such as amyotrophic lateral sclerosis (ALS)), and include a regeneration signal transduction domain (e.g., BDNF signaling domain), and can also have (e.g., can be engineered to have) elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regeneration polypeptides and / or elevated levels of one or more transport polypeptides to treat the mammal. In some cases, provided herein are CAR-MSCs that can be used for targeting mammals (e.g., humans that do not need immunosuppression) to treat the fibrotic tissue in vivo. For example, CAR-MSCs can express CARs that can target cartilage-specific antigens (e.g., fibroblast activation protein (FAP) or signal transduction lymphocyte activation molecule-7 (SLAMF7, also referred to as CS-1)), to target MSCs to mammals (e.g., mammals with one or more fibrotic diseases (e.g., cardiac fibrosis, liver fibrosis, pulmonary fibrosis, genital fibrosis or skin fibrosis)), and can also have (e.g., can be engineered to have) elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides and / or elevated levels of one or more transport polypeptides to treat the mammal. In some cases, provided herein are CAR-MSCs that can be used for targeting mammals (e.g., humans that do not need immunosuppression) to treat heart disease. For example, CAR-MSCs can express a targetable cardiac-specific antigen (e.g., HER2) to target the MSC to cardiac tissue in a mammal (e.g., a mammal with cardiac inflammation and / or cardiac degeneration), and include a regenerative signaling domain (e.g., a VEGFR2 signaling domain), and can also have (e.g., be engineered to have) elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides, and / or elevated levels of one or more trafficking polypeptides to treat the mammal.

[0094] Also provided herein is a kit comprising one or more materials described herein. In some cases, the kit may include one or more CAR-MSCs provided herein (for example, one or more immunosuppressive polypeptides with elevated levels, one or more regeneration polypeptides and / or one or more transport polypeptides with elevated levels). For example, provided herein is one or more CAR-MSCs that can be combined with packaging materials to form a kit. For example, one or more constructs described herein (for example, nucleic acid constructs) (for example, encoding a construct for a CAR that can bind to a tissue-specific antigen (such as an epithelial-specific antigen), and encoding one or more immunosuppressive polypeptides, one or more regeneration polypeptides and / or one or more transport polypeptides) can be combined with packaging materials to form a kit. The packaging material included in such a kit typically contains instructions or labels describing how to use the composition (for example, for adoptive transplantation) to treat a mammal (for example, suffering from or having one or more autoimmune diseases (such as GVHD)) that needs immunosuppression, as described herein. In some cases, the material provided in the kit described herein can be used to treat a mammal (for example, a human) suffering from GVHD (or having a risk of suffering from it). In some cases, the packaging material included in such a kit can contain instructions and / or labels describing how to use a composition as described herein. For example, the kit may contain instructions and / or labels describing how to use the compositions described herein to express one or more CARs and how to express one or more immunosuppressive polypeptides, one or more regenerative polypeptides and / or one or more transport polypeptides in MSC to engineer the CAR-MSCs provided herein (e.g., CAR-MSCs with elevated levels of one or more immunosuppressive polypeptides, elevated levels of one or more regenerative polypeptides and / or elevated levels of one or more transport polypeptides). In some cases, the packaging material included in such kits may contain instructions and / or labels describing how to use the CAR-MSCs provided herein. For example, the packaging material included in such kits may contain instructions and / or labels describing how to use the CAR-MSCs provided herein in adoptive transfer to treat mammals requiring immunosuppression (e.g., having or having a risk of suffering from one or more autoimmune diseases (such as GVHD)), as described herein. In some cases, a kit (e.g., containing instructions and / or labels describing how the CAR-MSCs provided herein can be used for adoptive transfer) may include materials for adoptive transfer procedures.

[0095] The present invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0096] Example

[0097] Example 1: Chimeric Antigen Receptor Engineering of Adipose-Derived Mesenchymal Stromal Cells (CAR-MSCs) for Enhanced Immunosuppression

[0098] Existing MSC therapies can be ineffective due to poor homing ability and poor immunosuppression after treatment. Lentiviral transduction of MSCs to stably express a CAR construct generates CAR-MSCs. This example describes the design of CAR-MSCs expressing an Ecad-specific scFv to generate anti-Ecad CAR-MSCs (EcCAR-MSCs). Incorporation of a CD28 signaling domain into EcCAR-MSCs enhances T cell suppression in tumor and GVHD models.

[0099] Materials and methods

[0100] Cell lines, primary PBMC / T cells, and primary MSCs

[0101] Primary human adipose-derived (Ad)-MSCs were obtained and cultured using StemXVivo Mesenchymal Stromal Cell Expansion Medium (R&D Systems, Minneapolis, MN). The human epithelial breast cancer cell line MCF7 was purchased from ATCC (CRL-3006, Manassas, VA, USA) and cultured in D10 (DMEM Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS, Millipore Sigma, Ontario, Canada) and 1% penicillin-streptomycin-glutamine (Gibco, Gaithersburg, MD, USA). MCF7 cells were irradiated and confirmed to be Ecad-positive by flow cytometry.

[0102] Mantle cell lymphoma cell line JeKo-1 and acute lymphoblastic leukemia cell line Nalm6 were purchased from ATCC. JeKo-1 and Nalm6 cells were cultured in R10 (RPMI 1640, Gibco, Gaithersburg, Maryland, USA) containing 10% or 20% fetal bovine serum (Millipore Sigma, Ontario, Canada) and 1% penicillin-streptomycin-glutamine (Gibco, Gaithersburg, Maryland, USA). In order to perform in vivo experiments, the cell lines were transduced into luciferase-ZsGreen lentivirus (Addgene, Cambridge, Massachusetts, USA). The cell lines were cultured to 10 generations and fresh cells were thawed every 7-8 weeks. The cell lines were identified by the manufacturer and phenotypes were regularly checked by flow cytometry. Mycoplasma detection was performed on the cell lines every month. Peripheral blood mononuclear cells (PBMCs) were isolated from de-identified normal donor blood apheresis tubes using SepMate tubes (STEMCELL Technologies, Vancouver, Canada). TM Human T cell 80 isolation kit (Stem Cell Technologies) was used to separate T cells by negative selection of magnetic beads.

[0103] Primary T cells and PBMCs were cultured in T cell culture medium containing X-VIVO 15 (Lonza, Walkersville, MD, USA), 10% human serum albumin (Innovative Research, Novi, MI, USA), and 1% penicillin-streptomycin-glutamine (Gibco, Gaithersburg, MD, USA) before in vitro co-culture screening. Freshly isolated human PBMCs were administered intravenously for in vivo experiments.

[0104] CAR design and virus production

[0105] All MSCs were transduced with a lentiviral vector that encodes a specially designed CAR construct under the EF1α promoter. The CART19 construct was used for transduction optimization. The CAR19 used contains a CD19-directed scFv derived from clone FMC-63 and fused with 41BB and CD3ζ signaling domains (FMC63-41BB-ζ). The CAR19 transgene was loaded into the lentivirus by lipofectamine transfection and transfected into HEK-293T cells, followed by standard procedures for lentivirus collection, concentration, and functional titration to generate a VSV-g pseudotyped second-generation lentivirus. Subsequently, the CAR construct was designed to contain a scFv against human / canine Ecad ( Figure 6 ) and carries the CD28 intracellular signaling domain to induce immunomodulatory activation of MSCs ( Figure 7CAR plasmids were generated and sequence-verified by Advgene (Watertown, MA, USA). Pre-seeded Ad-MSCs (250,000 cells / well) were transduced with lentiviral particles at a multiplicity of infection (MOI) of 3 in 6-well plates, and different concentrations of protamine sulfate (25 μg / μL, 50 μg / μL, or 100 μg / μL) were added to optimize CAR expression and MSC proliferation.

[0106] T cell suppression assay

[0107] The ability of CAR-MSC to inhibit activated T cells was tested using T cell suppression assay. Untransduced (UTD)-MSC or EcCAR-MSC (inhibitor) were co-cultured with activated T cells (effectors) and provided with antigen-specific stimulation to CAR-MSC in the presence or absence of soluble Ecad (stimulator). T cells were isolated from PBMC of normal donors using negative selection magnetic beads. In T cell culture medium containing X-VIVO 15 (Lonza, Walkersville, Maryland, USA), 10% human serum albumin (Innovative Research, Novi, Michigan, USA) and 1% penicillin-streptomycin-glutamine (Gibco, Gaithersburg, Maryland, USA), with a 3: 1 ratio of magnetic beads to T cells, CD3 stimulation magnetic beads were used to nonspecifically activate the isolated T cells. After 24 hours, the stimulated T cells (effectors) were co-cultured with UTD-MSC or EcCAR-MSC (inhibitor) alone or with soluble Ecad protein (stimulator) to activate EcCAR-MSC specifically in the CAR-free mesenchymal stromal cell expansion medium (R&D Systems, Minneapolis, Minnesota, USA). As shown, cells were co-cultured with a 1:10 MSC to T cell ratio, with 100,000 T cells per 96-well plate. Then, according to the co-culture time shown in each experiment, cells were collected and analyzed by flow cytometry.

[0108] Multichannel flow cytometry

[0109] Flow cytometry staining was performed. Briefly, MSCs were isolated and cultured, then transduced with CAR to generate CAR-MSCs, which were co-cultured with T cells / PBMC immune cells in 96-well plates for 24 hours and in 6-well plates for long-term culture. After the desired co-culture time, 10 mL / 75 cm 2Surface area of ​​Accutase-treated adherent MSC cells were detached by incubation at 37°C for 10-15 minutes. After detachment, all well contents were centrifuged, washed in flow buffer (PBS, 2% FBS v / v and 1% sodium azide v / v), and stained with the desired antibody cocktail for 15 minutes in the dark. After the final wash step, the cells were analyzed for the desired surface markers and positives were determined by negative gating of fluorescence subtraction (FMO) control wells. Absolute cell counts were obtained using volume measurement. The cells were gated as follows: SSC versus FSC plots for separation of cells based on size and complexity, FSC-H versus FSC-A plots for exclusion of doublets, SSC versus V500 channel LIVE / DEAD aqua fixable dyes for exclusion of dead cells, followed by cell subpopulation characterization based on a pre-designed antibody panel that was optimized and used to stain the samples.

[0110] Anti-human antibodies were purchased from Biolegend, eBioscience, or BD Biosciences (San Diego, California, USA). The preparation of samples for flow cytometry was as described elsewhere (see, e.g., Teshima et al., Biol Biol Blood Marrow Transplant 22: 11-16 (2016)). All antibodies used for staining samples are listed in Table 1. Flow cytometry was performed using a three-laser CytoFLEX (Beckman Coulter, Chaska, Minnesota, USA), which gated cells by singlet recognition and determined live cells by Live / DeadAqua staining (L34966, Thermo Fisher Scientific, Waltham, Massachusetts, USA). All gate selection analyses were performed using FlowJo X10.0.7r2 software (Ashland, Oregon, USA) and Kaluza Analysis software (Indianapolis, Indiana, USA).

[0111] Table 1. Antibodies.

[0112]

[0113]

[0114] In vivo studies in mice

[0115] 6-8 week old female and male immunocompromised NOD-SCID-γ - / - (NSG) mice were purchased from Jackson Laboratories. All cells were injected into the tail vein or intraperitoneal cavity via syringe in 100 μL-200 μL PBS. Mice were imaged using a bioluminescent imager using the Lumina S5 Imaging System (PerkinElmer, Hopkinton, MA, USA) to confirm the presence of luciferase+ tumors in CD19 tumor xenograft models. + Engraftment of Nalm6 / JeKo1 cells or luciferase in MSC persistence xenograft models + Implantation of CAR-MSCs. Imaging was performed 10 minutes after intraperitoneal injection of 10 μL / g D-luciferin (15 mg / mL, Gold Biotechnology, St. Louis, MO, USA). In the tumor mouse model, NSG mice were implanted with CD19 + Luciferase + JeKo-1 or Nalm6 (1x10 6 cells). Engraftment was confirmed by bioluminescence imaging 1-2 weeks after injection. All mice were then treated with irradiated Ecad + MCF-7 cells (5x10 6 cells to stimulate EcCAR-MSCs) and CART19 cells (1x10 6 CD19 cells as therapeutic + Strategy for tumors). Mice were then randomly assigned to receive UTD-MSCs, EcCAR-MSCs, or no treatment based on bioluminescence imaging as a measure of tumor burden (Figure 15). Serial bioluminescence imaging was then performed to assess residual disease and determine the anti-tumor activity of CART19 cells.

[0116] In the GVHD mouse model, allogeneic human PBMC (20-30 x 10 6 GVHD was induced in NSG mice (1×10 cells, intravenously injected) and grouped with the following treatments: 1) UTD-MSCs or 2) CAR-MSCs (1×10 cells, intraperitoneally injected on days 10 and 20). 6cells), and 3) MSC-free control mice (Figure 16). In all experiments, body weight and clinical GVHD scores (including body weight, posture, diarrhea, activity, hair condition and skin integrity) were monitored to understand the progression of GVHD in each experimental group (n=5). Mouse tail vein blood (approximately 100 μL) was collected, of which 70 μL was used for flow cytometry analysis. RBC lysis was performed using 1:10BD FACS lysate (BD Biosciences, San Jose, California, USA). Subsequently, the cells were washed with flow buffer and incubated with a specific antibody mixture in the dark at room temperature, and then flow analyzed using CytoFLEX (Beckman Coulter, Chaska, Minnesota, USA). The remaining blood was centrifuged at 13,000 rpm for 10 minutes to separate serum for cytokine analysis.

[0117] Canine research

[0118] The Ecad-targeting CAR is based on a human / canine cross-reactive Ecad-directed scFv. This scFv was generated using phage display technology and has been shown to cross-react with both canine and human Ecad. Figure 7 EcCAR-MSCs were tested in healthy beagle dogs. The subjects were injected intraperitoneally with EcCAR-MSCs (1 x 10 6 The subjects were followed up daily to monitor body weight, and colon biopsy samples were collected serially on day 3 for detection of human CD105 by immunohistochemistry (IHC). + cells to assess MSC homing to canine Ecad + The condition of cells.

[0119] RNA isolation, sequencing, and analysis

[0120] EcCAR-MSC or UTD-MSC cells were transduced in the presence or absence of 250 ng / mL recombinant human Ecad Fc chimera (Bio-Zinc, San Diego, CA, USA) as a CAR-specific stimulus for 24 hours. After in vitro culture, MSCs were isolated using the QIAGEN miRNeasy micro kit (Cat. No. 217084, QIAGEN, Germantown, MD, USA) for RNA extraction. To ensure the rigor of the results, batch RNA sequencing was performed on MSCs derived from three different biological donors in the UTD-MSC and EcCAR-MSC groups.

[0121] Total RNA was prepared using the SMARTer Stranded Total RNA-seq Kit v2 (Pico-grade Input, Mammalian) (Takara, Mountain View, CA, USA). Total RNA (three samples / lane) was sequenced using an Illumina HiSeq 4000 (Illumina, San Diego, CA, USA). Library preparation and sequencing were performed. The generated FastQC files for the samples were quality checked using FastQC v0.11.8. Adapter sequences were trimmed and removed using Cutadapt v1.18. The adapter removal and quality of the output files were confirmed using FastQC v0.11.8.

[0122] Paired-end reads from the trimmed FastQ files were aligned to the latest human reference genome (GRCh38) downloaded from the NCBI database. Genome index files were constructed and aligned using STAR v2.5.4b. Universal expression counts for each gene were generated using HTSeq (Python 3.6.5). Gene counts were normalized (geometric mean) and differential expression analysis was calculated using DESeq2 (R v3.6.1, R-project.org / ), using an adjusted p-value < 0.05 as the statistical cutoff. Heatmaps were created using pheatmap (cran.r-project.org / web / packages / pheatmap / index.html), and principal component analysis (PCA) was generated using the ggplot2 tool (cran.r-project.org / web / packages / ggplot2 / index.html).

[0123] Gene set enrichment analysis was performed using Enrichr, and protein-protein interaction networks were generated using QIAGEN Ingenuity pathway analysis.

[0124] Cytokine assay

[0125] Cytokine assays were performed on serum samples collected from mice 15 days after MSC treatment or control treatment. Serum was centrifuged at 10,000 x g for 5 minutes to remove debris. Before inoculation, serum was diluted with serum matrix at a ratio of 1:2 and operated according to the manufacturer's protocol of the Milliplex Human Cytokine / Chemokine Magnetic Bead Premix 38Plex Kit (HCYTMAG-60K-PX38, Millipore Sigma, Ontario, Canada). Data were collected using Luminex (Millipore Sigma, Ontario, Canada) and analyzed using Belysa Immunoassay Curve Fitting Software (Millipore Sigma, Ontario, Canada) and Microsoft Excel (Microsoft, Redmond, Washington, USA). Significant results were determined and reported using Prism GraphPad (La Jolla, California, USA).

[0126] Statistical analysis

[0127] In vitro and in vivo experiments were performed using both technical and biological repetitions for appropriate statistical analysis. Two-factor or one-way analysis of variance was used to assess the difference between EcCAR-MSC and UTD-MSC surface marker expression (by expression percentage) and T cell suppression ability (assessed by absolute T cell count) in vitro. ANOVA was also used to determine the significant difference between EcCAR-MSC-treated mice and UTD-MSC-treated mice by IVIS imaging, weight comparison, flow detection of luciferase-positive tumors and luciferase-positive MSCs, and GVHD clinical score comparison. KaplanMeier survival analysis was used to determine the significance of survival outcome differences in tumor and GVHD xenograft models, and Cox regression analysis was used to adjust confounding factors (such as sex). In order to perform multiple comparisons in each analysis (above) described previously, Tukey's multiple comparison test was used as a supplement based on analysis of variance. In order to compare between the two groups, two-tailed unpaired Sigmund's t-test was used instead of analysis of variance. RNA-seq data were processed using the DeSeq2 program, where the raw counts of the samples were normalized (geometric mean) and multiple hypothesis correction was performed using the Benjamini-Hochberg procedure. All relevant statistically significant comparisons are marked with asterisks, corresponding to a significance level below p < 0.05, and 95% confidence intervals are indicated (where *p < 0.05, **p < 0.005, ***p < 0.0005, and ****p < 0.0001). End data points without asterisks indicate no significant difference between groups (ns). Relevant data represent the mean of all data points, and error bars are determined by standard deviation (sd) or standard error of the mean (SEM). Experimental data were analyzed using Prism Graph Pad (La Jolla, CA, USA) and Microsoft Excel (Microsoft, Redmond, WA, USA).

[0128] result

[0129] MSCs were successfully transduced to stably express CAR.

[0130] Using a polycationic enhancer-enhanced lentiviral vector, efficient transduction and stable expression of CAR in MSCs were successfully achieved. To verify the concept of CAR-MSC construction, the transduction of the CAR19 lentiviral vector was optimized. Using protamine sulfate enhancer, the delivery efficiency and cell survival rate of CAR-containing lentiviral particles were optimized. Using protamine sulfate enhancer, a CAR transduction efficiency of >70% was achieved with an MOI of 3 lentivirus ( Figure 1A). Following transduction optimization using CAR19, alternative CAR designs for CAR-MSC autoimmune disease therapy were developed. The primary CAR-MSC construct was directed to Ecad, a ligand expressed on inflammatory intestinal epithelial cells involved in GVHD. Since T cell migration is mediated by the interaction of CD103 (αE integrin) on the surface of T cells with Ecad, which is ubiquitously expressed on all epithelial cells, it was necessary to determine whether redirecting MSCs to Ecad via CAR could protect host epithelial tissue by enhancing the specificity and activation of MSCs at sites of inflammation in GVHD and other intestinal autoimmune diseases. Using phage display, several Ecad-directed scFvs were generated, and one cross-reactive scFv against humans and dogs was identified for testing in a canine model ( Figure 6 Using this clone, an Ecad-specific CAR construct was generated ( Figure 7 ) and transduced into MSCs to generate anti-Ecad CAR-MSCs (EcCAR-MSCs), with a transduction efficiency >90% ( Figure 1B MSCs were derived from 5 different biological MSC donors (Table 2).

[0131] Table 2. MSC donor profile.

[0132] MSC donor gender age Race sample D1 male 48 White people normal D2 female 26 White people Chest cavity concavity D3 male 26 White people Chest cavity concavity D4 male 25 White people normal D5 male 28 White people normal

[0133] To determine whether the immunosuppressive effect of CAR-MSCs could be enhanced by incorporating specific intracellular signaling domains into the CAR, we incorporated CD28 signaling into the CAR-MSC construct design and used a CD28 costimulatory CAR backbone to generate a CAR targeting Ecad using a CD28ζCAR backbone (EcCAR-MSC) ( Figure 7 ).

[0134] The study also demonstrated that EcCAR-MSCs could stably express CAR after multiple passages (>3) in vitro, and achieved a transduction efficiency of >90% in multiple MSC cell donors ( Figures 1C-1D , see Table 3 for a description of the GVHD clinical scoring system). To test the ability of EcCAR-MSC to inhibit activated T cells, a T cell suppression assay was performed. UTD-MSC or EcCAR-MSC (suppressor) were co-cultured with activated T cells (effector). Compared with UTD-MSC, EcCAR-MSC induced superior T cell suppression in a dose-dependent manner ( Figure 1EAntigen-specific activation of EcCAR-MSCs was tested by measuring the inhibition of T cells by EcCAR-MSCs in the presence or absence of Ecad protein to stimulate the anti-Ecad scFv on the CAR. To ensure reproducibility, we used soluble Ecad and Ecad + MCF7 cell line was used as the source of Ecad antigen. Both soluble and cell-specific antigen stimulation of EcCAR-MSCs could enhance the inhibitory effect of T cells ( Figure 1G-1G ).

[0135] Table 3. GVHD clinical scoring system.

[0136]

[0137]

[0138] MSC engineering does not induce differentiation, while CAR-MSCs retain stemness.

[0139] The MSC stem cell phenotype and pathway enrichment were studied to identify whether CAR-MSC engineering induced unnecessary differentiation. According to the minimum standards for MSC stem cell maintenance established by the International Society for Cellular Therapy, flow cytometry was used to study the EcCAR-MSC stem cell phenotype (CD105 + 、CD73 + 、CD90 + 、CD34 - 、CD14 - and CD45 - ) (See, for example, Dominici et al., Cytotherapy 8, 315-317 (2006)). No significant differences were found between CAR-MSCs and UTD-MSCs ( Figure 1H ).

[0140] Differential gene expression analysis of EcCAR-MSCs and UTD-MSCs was performed by bulk RNA sequencing (RNAseq). Gene set enrichment analysis (GSEA) using a gene set amplified by CellMarker showed that adipose-derived stem cells and non-restricted adult stem cell subsets were enriched in EcCAR-MSCs ( Figure 1I In contrast, GSEA analysis showed that differentiated cell lineages (such as smooth muscle, neural or vascular differentiation pathways) were downregulated ( Figure 1J These results suggest that the stem cell phenotype is maintained after CAR-MSC modification, while differentiation induced by transduction or CAR expression is limited.

[0141] EcCAR-MSCs exhibited excellent immunosuppressive effects in an in vivo tumor GVHD model.

[0142] To study the immunosuppressive function of CAR-MSCs in vivo, we used a variety of tumor and GVHD models to ensure the reproducibility and rigor of the results. + Mantle cell lymphoma JeKo-1 and CD19 + Acute lymphoblastic leukemia Nalm6 was used to test whether CAR-MSCs inhibited the potent anti-tumor activity of CART19 cells ( Figure 2A luciferase was injected intravenously into immunocompromised NSG mice. + JeKo-1 or luciferase + A xenograft model was established with Nalm6 cells. Bioluminescence imaging was performed one week later to confirm the success of the transplantation, and the mice were randomly assigned to the CART19 alone treatment group, the CART19 combined with UTD-MSC treatment group, or the CART19 combined with EcCAR-MSC treatment group. Tumor burden was assessed by bioluminescence imaging every two weeks. Mice that received Nalm6 transplantation and CART19 combined with EcCAR-MSC treatment had a higher tumor burden than mice treated with CART19 combined with UTD-MSC or CART19 alone ( Figure 2B ), and the overall survival rate is low ( Figure 2C Similar results were also shown in the Jeko-1 xenograft model using CART19 combined with EcCAR-MSC or UTD-MSC treatment ( Figures 8A-8B Together, these data suggest that EcCAR-MSCs achieve superior effector T cell suppression by increasing tumor burden and decreasing survival.

[0143] Subsequently, a xenograft GVHD model was established by intravenously injecting human PBMCs into NSG mice. In addition to PBMCs, mice were also injected intraperitoneally (ip) with PBS control, UTD-MSCs, or EcCAR-MSCs as GVHD treatment. Additional doses of MSCs or control treatment ( Figure 2D Mice were monitored for the development of clinical GVHD symptoms (e.g., diarrhea, motor function, posture, hair integrity and skin integrity, body weight changes, and overall survival (see Table 3 for a description of the GVHD clinical scoring system). Peripheral blood was collected 14 days after the first MSC treatment and analyzed for human T cell proliferation. EcCAR-MSC treatment prevented weight loss compared to UTD-MSC treatment ( Figure 2E ), improve clinical GVHD ( Figure 2F ), inhibit T cell proliferation ( Figure 2G ) and improve overall survival ( Figure 2HTogether, these results suggest that EcCAR-MSCs enhance T cell suppression, thereby preventing GVHD.

[0144] EcCAR-MSCs showed increased expression of key transcription factors, inhibitory cytokines, and inhibitory surface markers, thereby inducing an immunosuppressive environment.

[0145] To elucidate the mechanisms underlying enhanced immunosuppression by EcCAR-MSCs, the unique transcriptional activation and cytokine secretion profiles of CAR-MSCs and the surface markers characterizing their phenotype were investigated.

[0146] To determine the effects of antigen-specific CAR-MSC stimulation on the MSC transcriptome, RNAseq was performed under the following conditions: UTD-MSC (unstimulated), UTD-MSC (stimulated with soluble Ecad), EcCAR-MSC (unstimulated), and EcCAR-MSC (stimulated with soluble Ecad, CAR-MSC stimulation by CAR).

[0147] To elucidate the intrinsic pathway activation of EcCAR-MSCs and stimulated EcCAR-MSCs, a comprehensive differential gene expression analysis was performed comparing the following: 1) EcCAR-MSCs (unstimulated) versus UTD-MSCs (unstimulated) to reflect intrinsic differences caused by EcCAR transduction and insertion, 2) EcCAR-MSCs (stimulated with soluble Ecad) versus EcCAR-MSCs (unstimulated) to reflect differences caused by antigen-specific stimulation of EcCAR-MSCs by CAR scFv, 3) UTD-MSCs (stimulated with soluble Ecad) versus UTD-MSCs (unstimulated) as a control to evaluate potential Ecad-based stimulation in the absence of Ecad-specific CAR scFv. Unstimulated EcCAR-MSCs and UTD-MSCs (Comparison 1) showed different pathway activation, with 355 genes significantly upregulated (at least 1.5-fold) in EcCAR-MSCs ( Figure 3A and Figure 9).

[0148] Differential gene expression analysis between Ecad-stimulated and unstimulated EcCAR-MSCs revealed more robust transcriptional activation, with 1996 genes upregulated at least 1.5-fold ( Figure 3A The number of upregulated genes was nearly 6-fold higher than that in control group 1, highlighting the functional capacity of CAR-based stimulation in MSCs (Figure 9). PCA of expression profiles between Ecad-stimulated and unstimulated CAR-MSCs revealed clustering based on stimulation status ( Figure 3B ), whereas Ecad-stimulated and unstimulated UTD-MSCs (Comparison 3) were clustered only by MSC biodonors ( Figure 3C GSEA of upregulated pathways characterized by CAR stimulation was strongly correlated with the NFκB-mediated TNFα signaling pathway and the IL-10 anti-inflammatory signaling pathway ( Figure 3D ).

[0149] These pathways are associated with canonical CD28 signaling, inducing nuclear activation of downstream functional cascades through intracellular protein binding to the CD28 src-like domain. Comparison between unstimulated EcCAR-MSCs and UTD-MSCs showed that the changes in signaling pathways were inherent to the introduction of the CAR signaling domain and CAR transduction, rather than to CAR stimulation through its scFv.

[0150] Transcriptomic analysis also showed that the transcription factors NFκB1, JUN, RELB, and IRF1 were increased in EcCAR-MSCs stimulated by Ecad ( Figure 3E ).

[0151] To validate the transcriptomic results, we measured human cytokine levels in the serum of UTD-MSC- and EcCAR-MSC-treated mice. Analysis of serum cytokine composition 17 days after MSC administration supported the RNAseq data, showing that EcCAR-MSC-treated mice had significantly higher levels of TNFα and IL-10 cytokines than UTD-MSC-treated mice. Fibroblast growth factor 2 (FGF-2) levels were also elevated in the serum of EcCAR-MSC-treated mice ( Figure 4A and Figure 10 ).

[0152] To characterize the effects of MSCs on the composition of human T cell subsets in a mouse model, peripheral blood was collected on days 14 and 31 after treatment with UTD-MSCs or EcCAR-MSCs, respectively, in a GVHD xenograft model, where EcCAR-MSCs treatment ameliorated body weight loss (see ). Figure 2D ). Flow cytometry analysis showed that the EcCAR-MSC-treated group had significant human CD4 + and CD8 + T cell suppression, associated with weight maintenance ( Figures 4B-4C ). CD4 + With CD8 + Ratio analysis showed that EcCAR-MSC-treated mice had a significantly higher CD4 + T cell subsets tend to increase and / or CD8 + Cytotoxic T cell subsets tend to decrease ( Figure 4D Long-term blood analysis on day 31 after MSC injection showed that Treg (human CD4+ 、CD25 + 、CD127 - ) cell percentage increased significantly ( Figure 4E ).

[0153] The expression of EcCAR-MSC surface markers was detected using CyTOF, and the results supported the retention of stem cell phenotype and revealed changes in immunosuppressive surface markers and chemokines. Unlike UTD-MSC, resting and Ecad-stimulated EcCAR-MSCs clustered into different groups based on t-distributed random neighbor embedding statistical analysis of surface markers, indicating the presence of functional CAR antigen-specific stimulation ( Figures 4F-4G and 11). To verify whether EcCAR-MSCs retained these unique immunosuppressive surface marker characteristics after co-culture with effector cells, we performed additional surface marker characterization of MSCs co-cultured with PBMCs by flow cytometry. Both EcCAR-MSCs and UTD-MSCs were stimulated with soluble Ecad and co-cultured with PBMCs for 5 days before surface marker assessment. These results showed that surface markers for the inhibitory receptor PD-1 and galectin 9 (Gal-9) were significantly upregulated in EcCAR-MSCs ( Figure 4H ). In stimulated EcCAR-MSCs, CXCR3 and CXCR4 homing chemokines were also significantly increased ( Figure 4I RNA sequencing analysis showed enhanced enrichment of CXCR chemokine receptor binding and leukocyte chemotaxis pathway regulation in EcCAR-MSCs, supporting the enhancement of this chemokine ( Figure 12 ).

[0154] Collectively, these mechanistic analyses suggest that EcCAR-MSCs generate enhanced immunosuppressive functions, especially upon CAR stimulation.

[0155] In a canine model, EcCAR-MSCs homed to the target site and were safe.

[0156] To ensure the homing ability and safety of EcCAR-MSCs, we used a canine model and used canine EcCAR-MSCs with cross-reactivity ( Figure 7 CAR-MSCs were designed using human and canine cross-reactive anti-Ecad scFv antibodies to construct EcCAR-MSCs. EcCAR-MSCs were generated by lentiviral transduction to generate human human MSCs, expanded in vitro, and finally injected intraperitoneally into healthy canine subjects. Immunohistochemical analysis was used to evaluate the expression of canine Ecad mediated by EcCAR-MSC scFv. +Cell homing ability. Hematological and organ toxicity were monitored in subgroups throughout the study by blood assessment. EcCAR-MSCs were administered to healthy beagle dogs via intraperitoneal injection. Colon biopsies were collected 3 and 28 days after EcCAR-MSC administration and canine Ecad was assessed by immunohistochemistry. + Ligand and human CD105 + MSC cells ( Figure 5A ). Human CD105 + MSCs are present in colon tissue, which overlaps with the tissue area where canine Ecad is highly expressed, indicating their target homing ( Figure 5B and 13 ). Then, the toxicity of EcCAR-MSC was evaluated by serial measurement of complete blood count, liver function, renal function and body weight. EcCAR-MSC administration did not cause weight loss, hematopoietic toxicity ( Figure 5D ) or organ toxicity ( Figure 5E ), indicating its strong security profile.

[0157] To study the expansion kinetics and persistence and to further evaluate the safety of CAR-MSCs in vivo, we constructed a luciferase + CAR-MSCs and monitored the expansion, persistence, and clearance of EcCAR-MSCs by serial bioluminescence imaging in the presence and absence of antigen-specific stimulation by irradiating Ecad + MCF-7 cells were administered in combination with Figure 14 ). Human PBMCs were injected into NSG mice, similar to the GVHD xenograft model. Subsequently, the mice received the following treatments: 1) luciferase alone + UTD-MSC; 2) Luciferase + UTD-MSC combined with irradiated Ecad + MCF-7 cells; 3) Luciferase alone + EcCAR-MSC; or 4) luciferase + EcCAR-MSC combined with irradiated Ecad + MCF-7 cells ( Figure 5G ). Subsequently, mice were monitored by serial bioluminescence imaging every 2-3 days to detect MSC expansion and persistence. These studies showed no difference in the clearance time (approximately 25 days) between UTD-MSCs and EcCAR-MSCs, further supporting the safety profile of EcCAR-MSCs (Figures 5H-5I).

[0158] Example 2: Exemplary amino acid sequences

[0159] Exemplary scFv sequences

[0160] Bold and underlined fonts Indicates CDR sequence

[0161] Exemplary anti-Ecad scFv (also known as anti-hmcECAD.6)

[0162] heavy chain

[0163]

[0164] light chain

[0165]

[0166]

[0167] Exemplary anti-CD103 scFv

[0168] heavy chain

[0169]

[0170] light chain

[0171]

[0172] Exemplary anti-MOG scFv

[0173] Heavy chain

[0174]

[0175] light chain

[0176]

[0177] Exemplary anti-AMPA scFv

[0178] Heavy chain

[0179]

[0180]

[0181] light chain

[0182]

[0183] Exemplary anti-HER2 scFv

[0184]

[0185] Exemplary anti-FAP scFv

[0186] Heavy chain

[0187]

[0188] light chain

[0189]

[0190]

[0191] Exemplary anti-CS1 scFv

[0192] Heavy chain

[0193] MGWSIILFLVATATGVHSQVQLQQPGAELVRPGASVKLSCKASGYSFTTYWMNWVKQRPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTA

[0194] (SEQ ID NO:30)

[0195] light chain

[0196]

[0197] Exemplary transmembrane domain sequences

[0198] Exemplary CD28 transmembrane domains

[0199] FWVLVVVGGVLACYSLLVTVAFIIFWV

[0200] (SEQ ID NO:9)

[0201] Exemplary CD8 transmembrane domains

[0202] IYIWAPLAGTCGVLLLSLVITLYC

[0203] (SEQ ID NO:32)

[0204] Exemplary TLR3 transmembrane domains

[0205] FFMINTSILLIFIFIVLLIHF

[0206] (SEQ ID NO:72)

[0207] Exemplary TLR4 transmembrane domains

[0208] IGVSVLSVLVVSVVAVLVY

[0209] (SEQ ID NO:73)

[0210] Exemplary IFNGR1 transmembrane domains

[0211] SLWIPVVAALLLFLVLSLVFI

[0212] (SEQ ID NO:96)

[0213] Exemplary IL10RA transmembrane domains

[0214] VIIFFAFVLLLSGALAYCLAL

[0215] (SEQ ID NO:97)

[0216] Exemplary TLR9 transmembrane domains

[0217] FALSLLAVALGLGVPMLHHLC

[0218] (SEQ ID NO:98)

[0219] Exemplary TNFR2 transmembrane domains

[0220] FALPVGLIVGVTALGLLIIGVVNCVIMTQV

[0221] (SEQ ID NO:99)

[0222] Exemplary signal transduction domain sequences

[0223] Exemplary 4-1BB signaling domains

[0224] (SEQ ID NO:89)

[0225] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0226] Exemplary TLR3 signaling domains

[0227] EGWRISFYWNVSVHRVLGFKEIDRQTEQFEYAAYIIHAYKDKDWVWEHFSSMEKEDQSLKFCLEERDFEAAGVFELEAIVNSIKRSRKIIFVITHHLLKDPLCKRFKVHHAVQQAIEQNLDSIILVFLEEIPDYKLNHALCLRRRGMFKSHCILNWPVQKERIGAFRHKLQVALGSKNSVH

[0228] (SEQ ID NO:74)

[0229] Exemplary TLR3 signaling domains

[0230] WRISFYWNVSVHRVLGFKEIDRQTEQFEYAAYIIHAYKDKDWVWEHFSSMEKEDQSLKFCLEERDFEAAGVFELEAIVNSIKRSRKIIFVITHHLLKDPLCKRFKVHHAVQQAIEQNLDSIILVFLEEIPDYKLNHALCLRRRGMF KSHCILNWPVQKERIGAFRHKLQVALGSKNSVHRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0231] (SEQ ID NO:75)

[0232] Exemplary TLR4 signaling domains

[0233] KFYFHLMLLAGCIKYGRGE

[0234] (SEQ ID NO:76)

[0235] Exemplary TIR4 signaling domains

[0236] NIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKSWNPEGT VGTGCNWQEATSI

[0237] (SEQ ID NO:77)

[0238] Exemplary TLR4 / TIR4 signaling domains

[0239] KFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI

[0240] (SEQ ID NO: 100)

[0241] Exemplary IFNGR1 signaling domains

[0242] CFYIKKINPLKEKSIILPKSLISVVRSATLETKPESKYVSLITSYQPFSLEKEVVCEEPLSPATVPGMHTEDNPGKVEHTEELSSITEVVTTEENIPDVVPGSHLTPIERE SSSPLSSNQSEPGSIALNSYHSRNCSESDHSRNGFDTDSSCLESHSSLSDSEFPPNNKGEIKTEGQELITVIKAPTSFGYDKPHVLVDLLVDDSGKESLIGYRPTEDSKEFS

[0243] (SEQ ID NO: 101)

[0244] Exemplary IFNγ signaling domains

[0245] MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRG

[0246] (SEQ ID NO:78)

[0247] Exemplary IL10RA signaling domains

[0248] QLYVRRRKKLPSVLLFKKPSPFIFISQRPSPETQDTIHPLDEEAFLKVSPELKNLDLHGSTDSGFGSTKPSLQTEEPQFLLPDPHPQADRTLGNREPPVLGDSCSSGSSNSTDSGICLQEPSLSPSTGPTWEQQVGSNSRGQDDSGIDLVQNSEGRAGDTQGGSALGHHSPPEPEVPGEEDPAAVAFQGYLRQTRCAEEKATKTGCLEEESPLTDGLGPKFGRCLVDEAGLHPPALAKGYLKQDPLEMTLASSGAPTGQWNQPTEEWSLLALSSCSDLGISDWSFAHDLAPLGCVAAPGGLLGSFNSDLVTLPLISSLQSSE

[0249] (SEQ ID NO:102)

[0250] Exemplary TLR9 / TIR9 signal transduction domain

[0251] GWDLWYCFHLCLAWLPWRGRQSGRDEDALPYDAFVVFDKTQSAVADWVYNELRGQLEECRGRWALRLCLEERDWLPGKTLFENLWASVYGSRKTLFVLAHTDRVSGLLRASFLLAQQRLLEDRKDVVVLVILSPDGRRSRYVRLRQRLCRQSVLLWPHQPSGQRSFWAQLGMALTRDNHHFYNRNFCQGPTAE

[0252] (SEQ ID NO:103)

[0253] Exemplary TNFR2 signal transduction domain

[0254] KKKPLCLQREAKVPHLPADKARGTQGPEQQHLLITAPSSSSSSLESSASALDRRAPTRNQPQAPGVEASGAGEARASTGSSDSSPGGHGTQVNVTCIVNVCSSSDHSSQCSSQASSTMGDTDSSPSESPKDEQVPFSKEECAFRSQLETPETLLGSTEEKPLPLGVPDAGMKPS

[0255] (SEQ ID NO:104)

[0256] Exemplary CD28 signal transduction domain

[0257] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0258] (SEQ ID NO: 10)

[0259] Exemplary CD3ζ Signaling Domains

[0260] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0261] (SEQ ID NO: 11)

[0262] Exemplary BDNF signaling domains

[0263] KLARHSKFGMKGPASVISNDDDSASPLHHISNGSNTPSSSEGGPDAVIIGMTKIPVIENPQYFGITNSQLKPDTFVQHIKRHNIVLKRELGEGAFGKVFLAECYNLCPEQDKILVAVKTLKDASDNARKDFHREAELLTNLQHEHIVKFYGVCVEGDPLIMVFEYMKHGDLNKFLRAHGPDAVL MAEGNPPTELTQSQMLHIAQQIAAGMVYLASQHFVHRDLATRNCLVGENLLVKIGDFGMSRDVYSTDYYRVGGHTMLPIRWMPPESIMYRKF TTESDVWSLGVVLWEIFTYGKQPWYQLSNNEVIECITQGRVLQRRPRTCPQEVYELMLGCWQREPHMRKNIKGIHTLLQNLAKASPVYLDILG

[0264] (SEQ ID NO:33)

[0265] Exemplary VEGFR2 signaling domains

[0266] LKLGKPLGRGAFGQVIEADAFGIDKTATCRTVAVKMLKEGATHSEHRALMSELKILIHIGHHLNVVNLLGACTKPGGPLMVIVEFCKFGNLSTYLRSKRNEFVPYKTKGARFRQGKDYVGAIPVDLKRRLDSITSSQSSASSGFVEEKSLSDVEEEEAPEDLYK DFLTLEHLICYSFQVAKGMEFLASRKCIHRDLAARNILLSEKNVVKICDFGLARDIYKDPDYVRKGDARLPLKWMAPETIFDRVYTIQSDVWSFGVLLWEIFSLGASPYPGVKIDEEFCRRLKEGTRMRAPDYTTPEMYQTMLDCWHGEPSQRPTFSELVEHLGN

[0267] (SEQ ID NO:79)

[0268] Exemplary hinge domain sequences

[0269] Exemplary CD8 hinge domain

[0270] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0271] (SEQ ID NO:34)

[0272] Exemplary CD28 hinge domain

[0273] LEPKSCDKTHTCPPCPDPK

[0274] (SEQ ID NO:35)

[0275] Exemplary leader sequences

[0276] Exemplary leader polypeptides

[0277] MALPVTALLLPLALLLHAARP

[0278] (SEQ ID NO:36)

[0279] Exemplary linker sequences

[0280] Exemplary linker polypeptides

[0281] EGKSSGSGSESKAS

[0282] (SEQ ID NO:37)

[0283] Exemplary polypeptide sequences

[0284] Exemplary NFκB1 polypeptides

[0285] MAEDDPYLGRPEQMFHLDPSLTHTIFNPEVFQPQMALPTADGPYLQILEQPKQRGFRFRYVCEGPSHGGLPGASSEKNKKSYPQVKICNYVGPAKVIVQLVTNGKNIHLHAHSLVGKHCEDGICTVTAGPKDMVVGFANLGILHVTKKKVFETLEARMTEACIRGYNPGLLVHPDLAYLQAEGGGDRQLGDREKELIRQAALQQTKEMDLSVVRLMFTAFLPDSTGSFTRRLEPVVSDAIYDSKAPNASNLKIVRMDRTAGCVTGGEEIYLLCDKVQKDDIQIRFYEEEENGGVWEGFGDFSPTDVHRQFAIVFKTPKYKDINITKPASVFVQLRRKSDLETSEPKPFLYYPEIKDKEEVQRKRQKLMPNFSDFGGSGAGAGGGGMFGSGGGGTGSTGPGYSFPHYGFPTYGGITFHPGTTKSNAGMKHGTMDTESKKDPEGCDKSDDKNTVNLFGKVIETTEQDQEPSEATVGNGEVTL TYATGTKEESAGVQDNLFLEKAMQLAKRHANALFDYAVTGDVKMLLAVQRHLTAVQDENGDSVLHLAIIHLHSQLVRDLLEVTSGLISDDIINMRNDLYQTPLHLAVITKQEDVVEDLLRAGADLSLLDRLGNSVLHLAAKEGHDKVLSILLKHKKAALLLDHPNGDGLNAIHLAMMSNSLPCLLLLVAAGADVNAQEQKSGRTALHLAVEHDNISLAGCLLLEGDAHVDSTTYDGTTPLHIAAGRGSTRLAALLKAAGADPLVENFEPLYDLDDSWENAGEDEGVVPGTTPLDMATSWQVFDILNGKPYEPEFTSDDLAQGDMKQLAEDVKLQLYKLLEIPDPDKNWATLAQKLGLGILNNAFRLSPAPSKTLMDNYEVSGGTVRELVEALRQMGYTEAIEVIQAASSPVKTTSQAHSLPLSPASTRQQIDELRDSDSVCDSGVETSFRKLSFTESLTSGASLLTLNKMPHDYGQEGPLEGKI

[0286] (SEQ ID NO: 12)

[0287] Exemplary JUN polypeptides

[0288] MAEDDPYLGRPEQMFHLDPSLTHTIFNPEVFQPQMALPTADGPYLQILEQPKQRGFRFRYVCEGPSHGGLPGASSEKNKKSYPQVKICNYVGPAKVIVQLVTNGKNIHLHAHSLVGKHCEDGICTVTAGPKDMVVGFANLGILHVTKKKVFETLEARMTEACIRGYNPGLLVHPDLAYLQAEGGGDRQLGDR EKELRQAALQQTKEMDLSVVRLMFTAFLPDSTGSFTRRLEPVVSDAIYDSKAPNASNLKIVRMDRTAGCVTGGEEEIYLLCDKVQKDDIQIRFYEEEENGGVWEGFGDFSPTDVHRQFAIVFKTPKYKDINITKPASVFVQLRRKSDLETSEPKPFLYYPEIKDKEEVQRKRQKLMPNFSDSFGGGSGAGAG GGGMFGSGGGGGGTGSTGPGYSFPHYGFPTYGGITFHPGTTKSNAGMKHGTMDTESKKDPEGCDKSDDKNTVNLFGKVIETTEQDQEPSEATVGNGEVTLTYATGTKEESAGVQDNLFLEKAMQLAKRHANALFDYAVTGDVKMLLAVQRHLTAVQDENGDSVLHLAIIHLHSQLVRDLLEVTSGLISDDIINMRNDLYQTPLHLAVITKQEDVVEDLLRAGADLSLLDRLGNSVLHLAAKEGHDKVLSILLKHKKAALLLDHPNGDGLNAIHLAMMSNSLPCLLLLVAAGADVNAQEQKSGRTALHLAVEHDNISLAGCLLLEGDAHVDSTTYDGTTPLHIAAGRGSTRLAALLKAAGADPLVENFEPLYDLDDSWENAGEDEG VVPGTTPLDMATSWQVFDILNGKPYEPEFTSDDLLAQGDMKQLAEDVKLQLYKLLEIPDPDKNWATLAQKLGLGILNNAFRLSPAPSKTLMDNYEVSGGTVRELVEALRQMGYTEAIEVIQAASSPVKTTSQAHSLPLSPASTRQQIDELRDSDSVCDSGVETSFRKLSFTESLTSGASLLTLNKMPHDYGQEGPLEGKI

[0289] (SEQ ID NO:13)

[0290] Exemplary RELB polypeptide

[0291] MLRSGPASGPSVPTGRAMPSRRVARPPAAPELGALGSPDLSSLSLAVSRSTDELEIIDEYIKENGFGLDGGQPGPGEGLPRLVSRGAASLSTVTLGPVAPPATPPPWGCPLGRLVSPAPGPGPQPHLVITEQPKQRGMRFRYECEGRSAGSILGESSTEASKTLPAIELRDCGGLREVEVTACLVWKDWPHRVHPHSLVGKDCTDGICRVRLRPHVSPRHSFNNLGIQCVRKKEIEAAIERKIQLGIDPYNAGSLKNHQEVDMNVVRICFQASYRDQQGQMRRMDPVLSEPVYDKKSTNTSELRICRINKESGPCTGGEELYLLCDKVQKEDISVVFSRASWEGRADFSQADVHRQIAIVFKTPPYEDLEIVEPVTVNVFLQRLTDGVCSEPLPFTYLPRDHDSYGVDKKRKRGMPDVLGELNSSDPHGIESKRRKKKPAILDHFLPNHGSGPFLPPSALLPDPDFFSGTVSLPGLEPPGGPDLLDDGFAYDPTAPTLFTMLDLLPPAPPHASAVVCSGGAGAVVGETPGPEPLTLDSYQAPGPGDGGTASLVGSNMFPNHYREAAFGGGLLSPGPEAT

[0292] (SEQ ID NO:14)

[0293] Exemplary IRF1 polypeptide

[0294] YPYDVPDYAGTELGSTMASWSHPQFEKGGGSGGGSGGGSWSHPQFEKAADITSLYKKAGSTMPITRMRMRPWLEMQINSNQIPGLIWINKEEMIFQIPWKHAAKHGWDINKDACLFRSWAIHTGRYKAGEKEPDPKTWKANFRCAMNSLPDIEEVKDQSRNKGSSAVRVYRMLPPLTKNQRKERKSKSSRDAKSKAKRKSCGDSSPDTFSDGLSSSTLPDDHSSYTVPGYMQDLEVEQALTPALSPCAVSSTLPDWHIPVEVVPDSTSDLYNFQVSPMPSTSEATTDEDEEGKLPEDIMKLLEQSEWQPTNVDGKGYLLNEPGVQPTSVYGDFSCKEEPEIDSPGGDIGLSLQRVFTDLKNMDATWLDSLLTPVRLPSIQAIPCAPLDPAFLYKVVT

[0295] (SEQ ID NO:15)

[0296] Exemplary TNFα polypeptide

[0297] MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCL LHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRL

[0298] (SEQ ID NO:16)

[0299] Exemplary IL-10 polypeptide

[0300] MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0301] (SEQ ID NO: 17)

[0302] Exemplary FGF-2 polypeptides

[0303] GGWLYHHSAGPAGRWRQRRVPAGPF*GSEAPVLQKRRLFPAHSPGWPCGRCA*EKRPAHQTATASGGTWCSEHQGCMR*PLSGDERRRPPAGEQMRHRRMFLLRTPGVQQLQHVSLPQIHLLVRGSETHRPVQTGLQNRPWPESDPVPADVREIL

[0304] (SEQ ID NO: 18)

[0305] Exemplary G-CSF polypeptides

[0306] MTPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLCATYKLCHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP

[0307] (SEQ ID NO: 19)

[0308] Exemplary GM-CSF polypeptides

[0309] MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQELPTFLYKVVGSTSGSGKPGSGEGSTKG

[0310] (SEQ ID NO: 20)

[0311] Exemplary eotaxin polypeptides

[0312] MKVSAALLWLLLIAAAFSPQGLAGPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKDICADPKKKWVQDSMKYLDQKSPTPKP

[0313] (SEQ ID NO:21)

[0314] Exemplary Gal-9 polypeptide

[0315] AFSGSQAPYLSPAVPFSGTIQGGLQDGLQITVNGTVLSSSGTRFAVNFQTGFSGNDIAFHFNPRFEDGGYVVCNTRQNGSWGPEERKTHMPFQKGMPFDLCFLVQSSDFKVMVNGILFVQYFHRVPFHRVDTISVNGSVQLSYISFQPPGVWPANPAPITQTVIHTVQSAPGQMFSTPAIPPMMYPHPAYPMPFITTILGGLYPSKSILLSGTVLPSAQRFHINLCSGNHIAFHLNPRFDENAVVRNTQIDNSWGSEERSLPRKMPFVRGQSFSVWILCEAHCLKVAVDGQHLFEYYHRLRNLPTINRLEVGGDIQLTHVQT

[0316] (SEQ ID NO:22)

[0317] Exemplary PD1 polypeptide

[0318] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSIVYASLDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPLTRLQDIKLAVPRARDPPVAT

[0319] (SEQ ID NO:23)

[0320] Exemplary TIM-3 polypeptide

[0321] MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTLQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIGIYIGAGICAGLALALIFGALIFKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQPSQPLGCRFAMP

[0322] (SEQ ID NO:24)

[0323] Exemplary CXCR3 polypeptide

[0324] MVLEVSDHQVLNDAEVAALLENFSSSYDYGENESDSCCTSPPCPQDFSLNFDRAFLPALYSLLFLLGLLGNGAVAAVLLSRRTALSSTDTFLLHLAVADTLLVLTLPLWAVDAAVQWVFGSGLCKVAGALFNINFYAGALLLACISFDRYLNI VHATQLYRRGPPARVTLTCLAVWGLCLLFALPDFIFLSAHHDERLNATHCQYNFPQVGRTALRVLQLVAGFLLPLLVMAYCYAHILAVLLVSRGQRRLRAMRLVVVVVVAFALCWTPYHLVVLVDILMDLGALARNCGRESRVDVAKSVTSGLGYMHCCLNPLLYAFVGVKFRERMWMLLLRLGCPNQRGLQRQPSSSRRDSSWSETSEASYSGL

[0325] (SEQ ID NO:25)

[0326] Exemplary CXCR4 polypeptide

[0327] MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS

[0328] (SEQ ID NO:26)

[0329] Exemplary CTLA4 polypeptide

[0330] MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN

[0331] (SEQ ID NO:91)

[0332] Exemplary TLR3 polypeptide

[0333] MRQTLPCIYFWGGLLPFGMLCASSTTKCTVSHEVADCSHLKLTQVPDDLPTNITVLNLTHNQLRRLPAANFTRYSQLTSLDVGFNTISKLEPELCQKLPMLKVLNLQHNELSQLSDKTFAFCTNLTELHLMSNSIQKIKNNPFVKQKNLITLDLSHNGLSSTKLGTQVQLENLQELLLSNNKIQALKSEELDIFANSSLKKLELSSNQIKEFSPGCFHAIGRLFGLFLNNVQLGPSLTEKLCLELANTSIRNLSLSNSQLSTTSNTTFLGLKWTNLTMLDLSYNNLNVVGNDSFAWLPQLEYFFLEYNNIQHLFSHSLHGLFNVRYLNLKRSFTKQSISLASLPKIDDFSFQWLKCLEHLNMEDNDIPGIKSNMFTGLINLKYLSLSNSFTSLRTLTNETFVSLAHSPLHILNLTKNKIS KIESDAFSWLGHLEVLDLGLNEIGQELTGQEWRGLENIFEIYLSYNKYLQLTRNSFALVPSLQRLMLRRVALKNVDSSPSPFQPLRNLTILDLSNNNIANINDDMLEGLEKLEILDLQHNNLARLWKHANPGGPIYFLKGLSHLHILNLESNGFDEIPVEVFKDLFELKIIDLGLNNLNTLPASVFNNQVSLKSLNLQKNLITSVEKKVFGPAFRNLTELDMRFNPFDCTCESIAWFVNWINETHTNIPELSSHYLCNTPPHYHGFPVRLFDTSSCKDSAPFELFFMINTSILLIFIFIVLLIHFEGWRISFYWNVSVHRVLGFKEIDRQTEQFEYAAYIIHAYKDKDWVWEHFSSMEKEDQSLKFCLEERDFEAGVFELEAIVNSIKRSRKIIFVITHHLLKDPLCKRFKVHHAVQQAIEQNLDSIILVFLEEIPDYKLNHALCLRRGMFKSHCILNWPVQKERIGAFRHKLQVALGSKNSVH

[0334] (SEQ ID NO:92)

[0335] Exemplary TLR4 polypeptide

[0336] MMSASRLAGTLIPAMAFLSCVRPESWEPCVEVVPNITYQCMELNFYKIPDNLPFSTKNLDLSFNPLRHLGSYSFFSFPELQVLDLSRCEIQTIEDGAYQSLSHLSTLILTGNPIQSLALGAFSGLSSLQKLVAVETNLASLENFPIGHLKTLKELNVAHNLIQSFKLPEYFSNLTNLEHLDLSSNKIQSIYCTDLRVLHQMPLLNLSLDLSLNPMNFIQPGAFKEIRLHKLTLRNNFDSLNVMKTCIQGLAGLEVHRLVLGEFRNEGNLEKFDKSALEGLCNLTIEEFRLAYLDYYLDDIIDLFNCLTNVSSFSLVSVTIERVKDFSYNFGWQHLELVNCKFGQFPTLKLKSLKRLTFTSNKGGNAFSEVDLPSLEFLDLSRNGLSFKGCCSQSDFGTTSLKYLDLSFNGVITMSSNFLGLEQLEHLDFQHSNLKQMSEFSVFLSLRNLIYLDISHTHTRVAFNGIFNGLSSLEVLKMAGNSFQENFLPDIFTELRNLTFLDLSQCQLEQLSPTAFNSLSSLQVLNMSHNNFFSLDTFPYKCLNSLQVLDYSLNHIMTSKKQELQHFPSSLAFLNLTQNDFACTCEHQSFLQWIKDQRQLLVEVERMECATPSDKQGMPVLSLNITCQMNKTIIGVSVLSVLVVSVVAVLVYKFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI

[0337] (SEQ ID NO:93)

[0338] Exemplary TLR9 polypeptide

[0339] MGFCRSALHPLSLLVQAIMLAMTLALGTLPAFLPCELQPHGLVNCNWLFLKSVPHFSMAAPRGNVTSLSLSSNRIHHLHDSDFAHLPSLRHLNLKWNCPPVGLSPMHFPCHMTIEPSTFLAVPTLEELNLSYNNIMTVPALPKSLISLSLSHTNILMLDSASLAGLHALRFLFMDGNCYYKNPCRQALEVAPGALLGLGNLTHLSLKYNNLTVVPRNLPSSLEYLLLSYNRIVKLAPEDLANLTALRVLDVGGNCRRCDHAPNPCMECPRHFPQLHPDTFSHLSRLEGLVLKDSSLSWLNASWFRGLGNLRVLDLSENFLYKCITKTKAFQGLTQLRKLNLSFNYQKRVSFAHLSLAPSFGSLVALKELDMHGIFFRSLDETTLRPLARLPMLQTLRLQMNFINQAQLGIFRAFPGLRYVDLSDNRISGASELTATMGEADGGEKVWLQPGDLAPAPVDTPSSEDFRPNCSTLNFTLDLSRNNLVTVQPEMFAQLSHLQCLRLSHNCISQAVNGSQFLPLTGLQVLDLSHNKLDLYHEHSFTELPRLEALDLSYNSQPFGMQGVGHNFSFVAHLRTLRHLSLAHNNIHSQVSQQLCSTSLRALDFSGNALGHMWAEGDLYLHFFQGLSGLIWLDLSQNRLHTLLPQTLRNLPKSLQVLRLRDNYLAFFKWWSLHFLPKLEVLDLAGNQLKALTNGSLPAGTRLRRLDVSCNSISFVAPGFFSKAKELRELNLSANALKTVDHSWFGPLASALQILDVSANPLHCACGAAFMDFLLEVQAAVPGLPSRVKCGSPGQLQGLSIFAQDLRLCLDEALSWDCFALSLLAVALGLGVPMLHHLCGWDLWYCFHLCLAWLPWRGRQSGRDEDALPYDAFVVFDKTQSAVADWVYNELRGQLEECRGRWALRLCLEERDWLPGKTLFENLWASVYGSRKTLFVLAHTDRVSGLLRASFLLAQQRLLEDRKDVVVLVILSPDGRRSRYVRLRQRLCRQSVLLWPHQPSGQRSFWAQLGMALTRDNHHFYNRNFCQGPTAE

[0340] (SEQ ID NO:94)

[0341] Exemplary TNFR2 polypeptides

[0342] MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQN RICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPT PEPSTAPSTSFLLPMGPSPPAEGSTGDFALPVGLIVGVTALGLLIIGVVNCVIMTQVKKKPLCLQREAKVPHLPADKARGTQGPEQQHLLITAPSSSSSSLESSASALDRRAPTR NQPQAPGVEASGAGEARASTGSSDSSPGGHGTQVNVTCIVNVCSSSDHSSQCSSQASSTMGDTDSSPSESPKDEQVPFSKEECAFRSQLETPETLLGSTEEKPLPLGVPDAGMKPS

[0343] (SEQ ID NO:95)

[0344] Example 3: Exemplary Nucleotide Sequences

[0345] Nucleic acids encoding exemplary scFvs

[0346] Nucleic acids encoding exemplary anti-Ecad scFv (also known as anti-hmcECAD.6)

[0347] Heavy chain

[0348] GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTAGTAGTACCATATACTACGTAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGGACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGAGGTCGGGTGTTAGTGGGAGCTCTATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0349] (SEQ ID NO:38)

[0350] Light chain

[0351] CTGCCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATTATGTCTACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTATTGTGCATCATGGGATACCAGCCTGCGTGCCTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT

[0352] (SEQ ID NO:39)

[0353] Nucleic acid encoding an exemplary anti-CD103 scFv

[0354] Heavy chain

[0355] CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCGTCAGTAGTTACTATTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGCCATATCTATTACAGTGGGAATACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAATCAGTTCTCCCTGAAACTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTTTTGTGCGAGAGATAGATGGAATTATTATGATAGTAGTCCCGGCTATTATTATTACTACGGTATGG ACGTCTGGGGCCAAGGGACCACGGTCACCGTCAGCTCA

[0356] (SEQ ID NO:40)

[0357] Light chain

[0358] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCGCCTAATCTTTGCTGCATCCCATTTGCAAAGTGGAGTCCCTTCAAGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGCATAATAGTTCCCCATTCACTTTCGGCCCTGGGACCAGAGTGGATATCAAA

[0359] (SEQ ID NO:41)

[0360] Nucleic acid encoding an exemplary anti-MOG scFv

[0361] Heavy chain

[0362] GAGGTGAAGCTGCACGAGAGCGGCGCAGGTCTGGTGAAGCCCGGCGCCAGCGTGGAGATCAGCTGCAAGGCCACCGGCTACACCTTCAGCAGCTTCTGGATCGAGTGGGTGAAGCAGAGACCCGGCCACGGCCTGGAGTGGATCGGCGAGATCCTGCCCGGCAGAGGCAGAACCAACTACAACGAGAAGTTCAAGGGCAAGGCCACCTTCACCGCCGAGACCAGCAGCAACACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCACCGGCAACACCATGGTGAACATGCCCTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGAGGTGGTTCGGGAGGTGGAGGTAGCGGAGGTGGTGGAAGC

[0363] (SEQ ID NO:42)

[0364] Light chain

[0365] GATATTGAACTGACCCAGAGTCCCAGTAGCCTGGCCGTGAGTGCCGGCGAGAAAGTGACCATGAGCTGCAAAAGCAGCCAGAGCCTGCTGAACAGCGGCAACCAGAAAAACTACCTGGCCTGGTACCAGCAGAAACCCGGCCTGCCTCCTAAGCTGCTGATCTACGGCGCCAGCACCAGAGAAAGCGGCGTGCCCGATAGATTCACCGGCTCTGGCTCTGGCACCGACTTCACCCTGACCATCAGCAGCGTGCAGGCCGAAGACCTGGCTGTCTACTACTGCCAGAACGACCACAGCTACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGATCAAG

[0366] (SEQ ID NO:43)

[0367] Nucleic acid encoding an exemplary anti-HER2 scFv

[0368] GAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAAGATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAAAACACCGCGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCAGCCGCTGGGGCGGCGATGGCTTTTATGCGATGGATTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCGAGCACCAAAGGCCCGAGCGTGTTTCCGCTGGCGCCGAGCAGCAAAAGCACCAGCGGCGGCACCGCGGCGCTGGGCTGCCTGGTGAAAGATTATTTTCCGGAACCGGTGACCGTGAGCTGGAACAGCGGCGCGCTGACCAGCGGCGTGCATACCTTTCCGGCGGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTGGTGACCGTGCCGAGCAGCAGCCTGGGCACCCAGACCTATATTTGCAACGTGAACCATAAACCGAGCAACACCAAAGTGGATAAAAAAGTGGAACCG

[0369] (SEQ ID NO:44)

[0370] Nucleic acid encoding an exemplary anti-FAP scFv

[0371] Heavy chain

[0372] CAGGTGCAGCTCCAGCAGAGTGGCGCAGAGCTCGCTCGCCCAGGCGCTTCTGTGAATCTGAGTTGTAAGGCCTCCGGATATACTTTTACGAACAACGGCATCAACTGGCTGAAGCAGCGGACCGGCCAGGGCCTGGAGTGGATCGGCGAAATATACCCCCGGTCCACAAACACTCTCTATAACGAGAAGTTTAAGGGCAAAGCAACTCTGACCGCGGACAGGTCCTCTAACACAGCCTATATGGAGCTGAGAAGCTTGACGAGTGAGGACTCCGCTGTCTATTTTTGCGCCCGAACTCTGACCGCTCCTTTTGCTTTTTGGGGCCAGGGCACGCTCGTGACCGTAAGTGCG

[0373] (SEQ ID NO:45)

[0374] Light chain

[0375] CAGATCGTCCTGACGCAGTCTCCAGCCATCATGAGCGCCTCACCCGGCGAAAAGGTGACCATGACCTGCTCAGCCTCTTCTGGTGTGAATTTCATGCACTGGTACCAGCAAAAAAGTGGGACCTCCCCTAAAAGGTGGATCTTCGATACCAGCAAACTGGCTTCTGGCGTTCCCGCAAGGTTTAGCGGCTCTGGTTCCGGCACATCATACAGCCTGACGATCAGCAGCATGGAGGCAGAAGACGCAGCTACCTATTACTGCCAGCAATGGAGCTTTAACCCACCTACTTTCGGAGGAGGAACAAAGCTGGAAATAAAAAGA

[0376] (SEQ ID NO:46)

[0377] Nucleic acid encoding an exemplary anti - CS1 scFv

[0378] Heavy chain

[0379] ATGGGATGGAGCTCTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCCAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCGGGGTACTCCTTCACCACCTACTGGATGAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGCATGATTCATCCTTCCGATAGTGAAACTAGGTTAAATCAGAAGTTCAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCC

[0380] (SEQ ID NO:47)

[0381] Light chain

[0382] GACATTGTGATGACCCAGTCTCAGAAATCCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTTATTACTGGTGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAATTACTGATTTACTCGGCATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAATGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCTCTCACTTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0383] (SEQ ID NO:48)

[0384] Nucleic acid encoding an exemplary anti-AMPA scFv

[0385] Heavy chain

[0386]

[0387] (SEQ ID NO:49)

[0388] Light chain

[0389] ATGACCAGCACCCTGCCGTTTAGCCCGCAGGTGAGCACCCCGCGCAGCAAATTTGCGACCATGGAATTTCAGACCCAGGTGCTGATGAGCCTGCTGCTGTGCATGAGCGGCGCGGCGGCGGATGTGGTGATGACCCAGACCCCGCTGACCCTGAGCGTGACCATTGGCCAGCCGGCGAGCATTAGCTGCAAAAGCAGCCAGAGCCTGCTGGATAGCGATGGCAAAACCTATCTGAACTGGCTGCT GCAGCGCCCGGGCCAGAGCCCGAAACGCCTGATTTATCTGGTGAGCAAACTGGATAGCGGCGTGCCGGATCGCTTTACCGGCAGCGGCAGCGGCACCGATTTTACCCTGAAAATTAGCCGCGTGGAAGCGGAAGATCTGGGCGTGTATTATTGCTGGCAGGGCACCCATTTTCCGCAGACCTTTGGCGGCGGCACCAAACTGGAAATTAAACGCGCGCGCGCGGATGCGGCGCCGACCGTGAGCATTTTTCCGCCGAGCAGCGAACAGCTGACCAGCGGCGGCGCGAGCGTGGTGTGCTTTCTGAACAACTTTTATCCGAAAGATATTAACGTGAAATGGAAAATTGATGGCAGCGAACGCCAGAACGGCGTGCTGAACAGCTGGACCGATCAGGATAGCAAAGATAGCACCTATAGCATGAGCAGCACCCTGACCCTGACCAAAGATGAATATGAACGCCATAACAGCTATACCTGCGAAGCGACCCATAAAACCAGCACCAGCCCGATTGTGAAAAGCTTTAACCGCAACGAATGC

[0390] (SEQ ID NO:50)

[0391] Nucleic acid encoding an exemplary transmembrane domain

[0392] Nucleic acid encoding an exemplary CD28 transmembrane domain

[0393] TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTG CTAGTAACAGTGGCCTTTATTATTTTCTGGGTG

[0394] (SEQ ID NO:51)

[0395] Nucleic acids encoding exemplary CD8 transmembrane domains

[0396] ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC

[0397] (SEQ ID NO:52)

[0398] Nucleic acids encoding exemplary TLR3 transmembrane domains

[0399] TTTTTTATGATCAATACGTCTATTCTTCTCATATTTATTTTCATCGTTC TTCTGATTCACTTT

[0400] (SEQ ID NO:80)

[0401] Nucleic acids encoding exemplary TLR4 transmembrane domains

[0402] ATTGGGGGTCTGTCCTAAGCGTGCTGGTTGTTTCCGTGGTTGCCGTT CTGGTATAT

[0403] (SEQ ID NO:81)

[0404] Nucleic acids encoding exemplary IFNGR1 transmembrane domains

[0405] TCTCTTTGGATTCCAGTTGTTGCTGCTTTACTACTCTTTCTAGTGCTTA GCCTGGTATTCATC

[0406] (SEQ ID NO: 105)

[0407] Nucleic acids encoding exemplary IL10RA transmembrane domains

[0408] GTCATCATATTCTTTGCCTTTGTTTTGCTGCTCTCCGGAGCGCTGGCT TACTGCCTCGCGCTC

[0409] (SEQ ID NO: 106)

[0410] Nucleic acids encoding exemplary TLR9 transmembrane domains

[0411] TTTGCCCTCAGTCTTCTGGCTGTAGCTCTCGGGCTTGGCGTCCCTATG CTTCACCACCTGTGT

[0412] (SEQ ID NO: 107)

[0413] Nucleic Acids Encoding Exemplary TNFR2 Transmembrane Domains

[0414] TTCGCTCTTCCAGTTGGACTGATTGTGGGGTTGACAGCCTTGGGTCTA CTAATAATAGGAGTGGTGAACTGTGTCATCATGACCCAGGTG

[0415] (SEQ ID NO: 108)

[0416] Nucleic acids encoding exemplary signal transduction domains

[0417] Nucleic acids encoding exemplary CD28 signaling domains

[0418] AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC

[0419] (SEQ ID NO:53)

[0420] Nucleic Acids Encoding Exemplary CD3ζ Signaling Domains

[0421] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

[0422] (SEQ ID NO:54)

[0423] Nucleic acids encoding exemplary 4-1BB signaling domains

[0424] AAACGGGGCAGAAAGAAACTCCGTATATATTCAAACAACCATTTAT GAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

[0425] (SEQ ID NO:82)

[0426] Nucleic acids encoding exemplary TLR3 signaling domains

[0427] GAGGGGTGGAGGATTTCCTTTTACTGGAATGTGTCTGTGCATAGGGTTTTGGGTTTTAAGGAGATAGATCGCCAAACAGAGCAGTTCGAGTATGCAGCTTATATTATACACGCGTACAAAGATAAGGACTGGGTATGGGAGCATTTCAGTTCCATGGAAAAAGAAGACCAGTCCTTGAAGTTCTGCCTTGAAGAAAGGGACTTCGAGGCCGGGGTTTTTGAGCTGGAAGCGATCGTTAATAGTATTAAGCGGTCACGCAAGATCATCTTCGTAATAACACATCACCTCCTCAAGGATCCACTTTGCAAACGCTTCAAAGTTCACCATGCGGTGCAGCAAGCGATCGAACAGAACCTTGACTCCATAATCCTTGTCTTTCTGGAAGAAATACCTGATTACAAGTTGAATCATGCTCTGTGTCTGCGGCGGGGAATGTTTAAGTCTCATTGTATCCTTAACTGGCCTGTGCAGAAAGAGCGCATTGGGGCGTTTAGGCATAAACTCCAGGTTGCGCTTGGAAGCAAAAATTCCGTTCAT

[0428] (SEQ ID NO:83)

[0429] Nucleic acid encoding an exemplary TIR3 signaling domain

[0430] TGGCGGATCTCTTTCTACTGGAACGTGTCAGTGCATAGAGTCCTCGGGTTCAAAGAGATTGACAGGCAGACGGAACAATTTGAGTATGCAGCGTACATTATCCATGCCTACAAAGATAAAGACTGGGTTTGGGAACATTTCTCCTCCATGGAAAAGGAGGATCAGTCTTTGAAGTTTTGTCTGGAGGAGCGGGACTTTGAGGCGGGAGTGTTCGAGCTTGAGGCCATTGTAAACTCGATCAAGCGGTCCCGTAAAATCATCTTTGTGATAACACACCACCTGTTGAAAGACCCGCTCTGCAAGCGCTTTAAGGTGCATCATGCTGTCCAGCAGGCCATCGAGCAGAATCTGGATTCAATTATTCTGGTGTTTTTAGAAGAGATCCCAGACTATAAGCTGAATCACGCTCTATGTTTGCGTCGAGGCATGTTCAAATCTCATTGCATCTTGAATTGGCCGGTGCAAAAGGAAAGGATTGGTGCTTTTCGGCACAAATTGCAGGTCGCCCTGGGCTCCAAAAACAGTGTTCACAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTC AGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAAG

[0431] (SEQ ID NO:84)

[0432] Nucleic acid encoding an exemplary TLR3 / TIR3 signaling domain

[0433] GAAGGATGGCGAATCTCTTTCTACTGGAACGTGTCTGTTCATAGAGTTTTGGGATTCAAGGAAATAGATCGCCAAACCGAGCAATTCGAGTATGCCGCATATATCATACATGCATATAAGGACAAGGACTGGGTATGGGAACACTTCTCCTCTATGGAGAAAGAAGATCAGTCACTCAAATTCTGCTTGGAGGAACGAGATTTCGAAGCCGGTGTATTTGAGCTTGAAGCAATAGTAAACTCTATAAAAAGAAGTCGAAAAATTATTTTCGTCATCACACATCATCTGTTGAAGGACCCGCTTTGTAAGAGGTTTAAAGTGCATCACGCTGTGCAGCAAGCCATTGAACAAAATCTGGATAGCATCATTCTCGTTTTTCTCGAAGAAATACCCGACTATAAGCTCAATCACGCTTTGTGCTTGCGACGGGGAATGTTCAAGTCCCACTGCATTCTTAACTGGCCAGTTCAAAAGGAACGAATCGGGGCCTTCAGACATAAATTGCAAGTTGCCCTGGGCTCAAAGAACTCAGTGCAC

[0434] (SEQ ID NO:109)

[0435] Nucleic acid encoding an exemplary TLR4 signaling domain

[0436] AAGTTCTATTTCCATCTGATGCTTCTCGCTGGCTGCATAAAGTACGGG AGGGGGGAG

[0437] (SEQ ID NO:85)

[0438] Nucleic acid encoding an exemplary TIR4 signaling domain

[0439] AATATATATGACGCTTTCGTGATCTACTCGAGCCAGGATGAGGACTGGGTTCGCAACGAGCTAGTCAAGAATCTTGAAGAGGGCGTGCCTCCTTTCCAGCTCTGTCTGCATTACCGCGATTTTATTCCTGGGGTGGCCATCGCGGCCAACATCATCCACGAGGGCTTCCATAAATCCAGAAAAGTGATTGTCGTTGTGAGCCAGCATTTCATCCAGTCCAGGTGGTGCATTTTCGAATATGAGATAGCCCAGACCTGGCAGTTTCTTAGCAGTCGGGCTGGGATTATTTTTATCGTGCTGCAGAAGGTTGAAAAGACCCTTTTGCGGCAACAGGTGGAACTGTACCGATTATTATCCCGTAACACTTACTTGGAATGGGAAGACTCAGTTCTCGGACG CCACATTTTCTGGCGCCGGCTCAGGAAGGCCCTGCTGGATGGTAAATCCTGGAACCCCGAGGGGACAGTGGGGACCGGATGTAACTGGCAAGAGGCAACAAGTATA

[0440] (SEQ ID NO:86)

[0441] Nucleic acid encoding an exemplary TLR4 / TIR4 signaling domain

[0442] AAGTTCTATTTCCATCTGATGCTTCTCGCTGGCTGCATAAAGTACGGGAGGGGGGAGAATATATATGACGCTTTCGTGATCTACTCGAGCCAGGATGAGGACTGGGTTCGCAACGAGCTAGTCAAGAATCTTGAAGAGGGCGTGCCTCCTTTCCAGCTCTGTCTGCATTACCGCGATTTTATTCCTGGGGTGGCCATCGCGGCCAACATCATCCACGAGGGCTTCCATAAATCCAGAAAAGTGATTGTCGTTGTGAGCCAGCATTTCATCCAGTCCAGGTGGTGCATTTTCGAATATGAGATAGCCCAGACCTGGCAGTTTCTTAGCAGTCGGGCTGGGATTATTTTTATCGTGCTGCAGAAGGTTGAAAAGACCCTTTTGCGGCAACAGGTGGAACTGTACCGATTATTATCCCGTAACACTTACTTGGAATGGGAAGACTCAGTTCTCGGACGCCACATTTTCTGGCGCCGGCTCAGGAAGGCCCTGCTGGATGGTAAATCCTGGAACCCCGAGGGGACAGTGGGGACCGGATGTAACTGGCAAGAGGCAACAAGTATA

[0443] (SEQ ID NO:110)

[0444] Nucleic acid encoding an exemplary IFNγ signal transduction domain

[0445] ATGAAGTACACTTCTTACATACTCGCCTTCCAGCTTTGTATAGTGTTGGGCAGTCTGGGTTGCTATTGCCAAGATCCTTATGTGAAGGAAGCAGAAAATTTGAAGAAATACTTTAACGCGGGTCATTCTGACGTTGCAGATAATGGAACCTTGTTTTTGGGAATACTTAAAAATTGGAAGGAGGAAAGCGACCGCAAGATCATGCAGTCTCAAATCGTTTCCTTTTATTTTAAACTTTTCAAGAATTTTAAGGACGATCAGTCCATACAGAAATCTGTAGAGACTATCAAAGAGGATATGAACGTAAAGTTTTTTAACAGCAACAAAAAGAAAAGAGATGACTTTGAGAAACTTACGAATTATAGTGTCACCGATCTGAACGTCCAACGCAAAGCAATCCACGAGTTGATTCAAGTTATGGCAGAGCTTTCCCCAGCGGCGAAAACTGGAAAGCGCAAACGATCTCAAATGCTCTTCCGGGGT

[0446] (SEQ ID NO:87)

[0447] Nucleic acid encoding an exemplary IFNGR1 signal transduction domain

[0448]

[0449] (SEQ ID NO: 111)

[0450] Nucleic acids encoding exemplary IFNγ signaling domains

[0451] ATGAAGTACACTTCTTACATACTCGCCTTCCAGCTTTGTATAGTGTTGGGCAGTCTGGGTTGCTATTGCCAAGATCCTTATGTGAAGGAAGCAGAAAATTTGAAGAAATACTTTAACGCGGGTCATTCTGACGTTGCAGATAATGGAA CCTTGTTTTTGGGAATACTTAAAAATTGGAAGGAGGAAAGCGACCGCAAGATCATGCAGTCTCAAATCGTTTCCTTTTATTTTAAACTTTTCAAGAATTTTAAGGACGATCAGTCCATACAGAAATCTGTAGAGACTATCAAAGAGGATATGAACGTAAAGTTTTTT AACAGCAACAAAAAGAAAAGAGATGACTTTGAGAAACTTACGAATTATAGTGTCACCGATCTGAACGTCCAACGCAAAGCAATCCACGAGTTGATTCAAGTTATGGCAGAGCTTTCCCCAGCGGCGAAAACTGGAAAGCGCAAACGATCTCAAATGCTCTTCCGGGGT

[0452] (SEQ ID NO: 112)

[0453] Nucleic Acids Encoding Exemplary IL10RA Signaling Domains

[0454] CAGCTCTATGTCAGAAGGAGAAAAAAGCTCCCGTCCGTGCTTCTTTTTAAGAAGCCTTCCCCGTTCATCTTCATTAGTCAGCGACCGAGCCCTGAGACCCAGGACACGATCCATCCGCTTGATGAAGAGGCGTTTCTTAAGGTAAGCCCTGAACTGAAGAATCTCGATCTGCACGGCAGCACCGACAGTGGATTCGGGTCCACGAAACCCTCACTCCAGACAGAAGAGCCTCAGTTTTTGTTGCCTGATCCTCACCCCCAGGCTGATAGAACGTTGGGTAATCGCGAGCCGCCGGTACTTGGAGACAGCTGCTCATCTGGGTCAAGCAACTCAACGGATTCTGGGATATGTTTGCAGGAACCAAGCCTCTCACCTAGCACTGGGCCAACCTGGGAGCAGCAAGTGGGAAGCAATTCACGGGGGCAGGACGACTCTGGTATAGACCTGGTACAAAACTCTGAGGGACGGGCGGGAGATACTCAAGGTGGTAGCGCCCTGGGACACCACAGCCCACCGGAACCAGAGGTCCCGGGCGAGGAGGACCCTGCAGCGGTGGCGTTTCAGGGATATTTGCGCCAAACCCGCTGTGCTGAGGAGAAGGCTACTAAAACGGGCTGCTTGGAAGAAGAATCTCCGCTCACAGACGGGCTCGGCCCGAAGTTCGGCCGATGTTTGGTGGACGAAGCGGGGCTTCATCCGCCGGCGCTTGCTAAAGGGTACCTGAAGCAAGACCCCTTGGAGATGACCCTGGCCTCCTCCGGGGCGCCTACTGGTCAGTGGAACCAGCCGACCGAGGAATGGTCTCTCCTCGCCCTTTCCAGTTGCTCCGACCTCGGTATATCAGACTGGAGTTTCGCACACGACCTTGCACCCCTCGGCTGTGTAGCCGCCCCAGGAGGCCTTCTTGGCTCATTTAACAGTGACCTCGTTACGCTCCCCCTCATTTCATCTCTGCAATCCTCTGAA

[0455] (SEQ ID NO:113)

[0456] Nucleic acid encoding an exemplary TLR9 / TIR9 signaling domain

[0457] GGCTGGGACCTCTGGTACTGCTTCCACCTGTGCCTGGCCTGGCTTCCCTGGCGGGGGCGGCAAAGTGGGCGAGATGAGGATGCCCTGCCCTACGATGCCTTCGTGGTCTTCGACAAAACGCAGAGCGCAGTGGCAGACTGGGTGTACAACGAGCTTCGGGGGCAGCTGGAGGAGTGCCGTGGGCGCTGGGCACTCCGCCTGTGCCTGGAGGAACGCGACTGGCTGCCTGGCAAAACCCTCTTTGAGAACCTGTGGGCCTCGGTCTATGGCAGCCGCAAGACGCTGTTTGTGCTGGCCCACACGGACCGGGTCAGTGGTCTCTTGCGCGCCAGCTTCCTGCTGGCCCAGCAGCGCCTGCTGGAGGACCGCAAGGACGTCGTGGTGCTGGTGATCCTGAGCCCTGACGGCCGCCGCTCCCGCTACGTGCGGCTGCGCCAGCGCCTCTGCCGCCAGAGTGTCCTCCTCTGGCCCCACCAGCCCAGTGGTCAGCGCAGCTTCTGGGCCCAGCTGGGCATGGCCCTGACCAGGGACAACCACCACTTCTATAACCGGAACTTCTGCCAGGGACCCACGGCCGAATAG

[0458] (SEQ ID NO:114)

[0459] Nucleic acid encoding an exemplary TNFR2 signaling domain

[0460] AAAAAGAAGCCCTTGTGCCTGCAGAGAGAAGCCAAGGTGCCTCACTTGCCTGCCGATAAGGCCCGGGGTACACAGGGCCCCGAGCAGCAGCACCTGCTGATCACAGCGCCGAGCTCCAGCAGCAGCTCCCTGGAGAGCTCGGCCAGTGCGTTGGACAGAAGGGCGCCCACTCGGAACCAGCCACAGGCACCAGGCGTGGAGGCCAGTGGGGCCGGGGAGGCCCGGGCCAGCACCGGGAGCTCAGATTCTTCCCCTGGTGGCCATGGGACCCAGGTCAATGTCACCTGCATCGTGAACGTCTGTAGCAGCTCTGACCACAGCTCACAGTGCTCCTCCCAAGCCAGCTCCACAATGGGAGACACAGATTCCAGCCCCTCGGAGTCCCCGAAGGACGAGCAGGTCCCCTTCTCCAAGGAGGAATGTGCCTTTCGGTCACAGCTGGAGACGCCAGAGACCCTGCTGGGGAGCACCGAAGAGAAGCCCCTGCCCCTTGGAGTGCCTGATGCTGGGATGAAGCCCAGTTAACCAGGCCGGTGTGGGCTGTGTCGTAGCCAAGGTGGGCTGAGCCCTGGCAGGATGACCCTGCGAAG

[0461]

[0462] (SEQ ID NO: 115)

[0463] Nucleic acids encoding exemplary BDNF signaling domains

[0464]

[0465] (SEQ ID NO:55)

[0466] Nucleic acids encoding exemplary VEGFR2 signaling domains

[0467] CTGAAGCTGGGCAAGCCCCTGGGCAGGGGCGCCTTCGGCCAGGTGATCGAGGCCGACGCCTTCGGCATCGACAAGACCGCCACCTGCAGGACCGTGGCCGTGAAGATGCTGAAGGAGGGCGCCACCCACAGCGAGCACAGGGCCCTGATGAGCGAGCTGAAGATCCTGATCCACATCGGCCACCACCTGAACGTGGTGAACCTGCTGGGCGCCTGCACCAAGCCCGGCGGCCCCCTGATGGTGATCGTGGAGTTCTGCAAGTTCGGCAACCTGAGCACCTACCTGAGGAGCAAGAGGAACGAGTTCGTGCCCTACAAGACCAAGGGCGCCAGGTTCAGGCAGGGCAAGGACTACGTGGGCGCCATCCCCGTGGACCTGAAGAGGAGGCTGGACAGCATCACCAGCAGCCAGAGCAGCGCCAGCAGCGGCTTCGTGGAGGAGAAGAGCCTGAGCGACGTGGAGGAGGAGGAGGCCCCCGAGGACCTGTACAAGGACTTCCTGACCCTGGAGCACCTGATCTGCTACAGCTTCCAGGTGGCCAAGGGCATGGAGTTCCTGGCCAGCAGGAAGTGCATCCACAGGGACCTGGCCGCCAGGAACATCCTGCTGAGCGAGAAGAACGTGGTGAAGATCTGCGACTTCGGCCTGGCCAGGGACATCTACAAGGACCCCGACTACGTGAGGAAGGGCGACGCCAGGCTGCCCCTGAAGTGGATGGCCCCCGAGACCATCTTCGACAGGGTGTACACCATCCAGAGCGACGTGTGGAGCTTCGGCGTGCTGCTGTGGGAGATCTTCAGCCTGGGCGCCAGCCCCTACCCCGGCGTGAAGATCGACGAGGAGTTCTGCAGGAGGCTGAAGGAGGGCACCAGGATGAGGGCCCCCGACTACACCACCCCCGAGATGTACCAGACCATGCTGGACTGCTGGCACGGCGAGCCCAGCCAGAGGCCCACCTTCAGCGAGCTGGTGGAGCACCTGGGCAAC

[0468] (SEQ ID NO:88)

[0469] Nucleic acids encoding exemplary hinge domains

[0470] Nucleic acids encoding exemplary CD28 hinge transduction domains

[0471] CTCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCGGATCCCAAA

[0472] (SEQ ID NO:56)

[0473] Nucleic acids encoding exemplary CD8 hinge transduction domains

[0474] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT

[0475] (SEQ ID NO:57)

[0476] Nucleic acids encoding exemplary polypeptides

[0477] Nucleic acids encoding exemplary NFκB1 polypeptides

[0478]

[0479] (SEQ ID NO:58)

[0480] Nucleic acids encoding exemplary JUN polypeptides

[0481]

[0482] (SEQ ID NO:59)

[0483] Nucleic acids encoding exemplary RELB polypeptides

[0484]

[0485] (SEQ ID NO:60)

[0486] Nucleic acids encoding exemplary IRF1 polypeptides

[0487]

[0488] (SEQ ID NO:61)

[0489] Nucleic acids encoding exemplary TNFα polypeptides

[0490] ATGTCTACCGAGTCTATGATTAGGGACGTGGAACTGGCTGAGGAGGCACTGCCCAAAAAAACCGGCGGACCACAGGGCTCTAGGAGATGTCTGTTTCTGTCTCTGTTCTCTTTTCTCATCGTGGCTGGCGCTACAACACTCTTCTGTCTGCTCCATTTCGGCGTGATTGGACCACAGCGAGAGGAATTTCCCCGGGATCTGTCACTCATTTCACCACTGGCACAGGCTGTCCGATCTTCATCTCGGACTCCATCCGACAAACCTGTCGCCCATGTCGTCGCCAACCCACAGGCCGAGGGCCAGCTCCAGTGGCTCAATAGGAGGGCAAACGCTCTGCTCGCCAATGGCGTGGAACTCCGGGATAACCAGCTCGTCGTGCCTAGTGAGGGACTGTACCTCATCTACTCCCAGGTGCTGTTTAAGGGCCAGGGATGTCCTTCTACACATGTGCTGCTCACACACACAATTTCACGGATCGCCGTGTCTTACCAGACTAAAGTCAATCTGCTCTCTGCCATCAAATCCCCATGTCAGCGGGAAACACCTGAGGGCGCTGAGGCTAAACCTTGGTACGAACCCATCTACCTCGGAGGCGTGTTCCAGCTGGAGAAGGGCGATAGACTGA

[0491] (SEQ ID NO:62)

[0492] Nucleic acids encoding exemplary IL-10 polypeptides

[0493] ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTTCTGACAGGCGTAAGGGCGTCACCTGGCCAAGGAACACAGTCAGAGAACAGCTGTACACATTTCCCCGGCAACTTGCCCAATATGCTTAGGGATCTTCGCGATGCCTTCTCACGAGTGAAGACATTTTTTCAGATGAAGGATCAGTTGGATAACCTTTTGCTGAAGGAAAGCCTGCTCGAGGACTTCAAGGGATACCTCGGATGCCAGGCACTGAGCGAAATGATACAGTTCTACCTGGAAGAGGTAATGCCTCAGGCAGAAAATCAGGACCCCGATATTAAAGCTCATGTGAACTCTCTGGGTGAGAACCTGAAAACTCTGAGGCTGAGGCTGCGGAGGTGTCACAGATTCCTGCCATGCGAGAACAAATCAAAAGCCGTCGAACAGGTGAAGAACGCCTTTAACAAACTGCAGGAGAAAGGCATCTATAAAGCGATGAGCGAGTTCGATATTTTCATCAACTACATTGAGGCATACATGACGATGAAAATCCGAAATTAG

[0494] (SEQ ID NO:63)

[0495] Nucleic acid encoding an exemplary FGF-2 polypeptide

[0496] GGCGGCTGGCTCTATCACCACTCTGCCGGCCCTGCCGGAAGATGGCGGCAGCGGCGCGTTCCCGCCGGGCCATTTTAAGGATCCGAAGCGCCTGTAC TGCAAAAACGGCGGCTTTTTCCTGCGCATTCACCCGGATGGCCGTGTGGACGGTGTGCGTGAGAAAAGCGACCCGCACATCAAACTGCAACTGCAAGCGGAGGAACGTGGTGTAGTGAGCATCAAGGGTGTATGCGCTAACCGTTATCTGGCGATGAAAGAAGACGGCCGCCTGCTGGCGAGCAAATGCGTCACCGACGAATGTTTCTTCTTCGAACGCCTGGAGTCCAACAACTACAACACGTATCGCTCCCGCAAATACACCTCTTGGTACGTGGCTCTGAAACGCACCGGCCAGTACAAACTGGGCTCCAAAACCGGCCCTGGCCAGAAAGCGATCCTGTTCCTGCCGATGTCCGCGAAATCCTAA

[0497] (SEQ ID NO:64)

[0498] Nucleic acid encoding an exemplary G-CSF polypeptide

[0499] ATGACTCCTTTGGGTCCAGCTTCTTCCTTGCCTCAATCCTTCTTGTTGAAGTGTTTGGAGCAGGTTAGAAAGATCCAGGGTGATGGTGCTGCTTTGCAAGAGAAGTTGTGTGCTACTTACAAGTTGTGTCACCCAGAAGAGTTGGTTTTGTTGGGTCACTCCTTGGGTATTCCTTGGGCTCCATTGTCCTCTTGTCCATCCCAAGCTTTGCAATTGGCTGGTTGTTTGTCCCAATTGCACTCCGGTTTGTTCTTGTACCAGGGTTTGTTGCAAGCTTTGGAGGGTATTTCTCCAGAGTTGGGTCCAACTTTGGACACATTGCAGTTGGACGTTGCTGACTTCGCTACTACTATCTGGCAACAGATGGAAGAATTGGGTATGGCTCCAGCTTTGCAGCCAACTCAAGGTGCTATGCCAGCTTTTGCTTCTGCTTTCCAGAGAAGAGCTGGTGGTGTTTTGGTTGCTTCTCACTTGCAGTCTTTCTTGGAGGTTTCCTACAGAGTTTTGAGACACTTGGCTCAACCATAA

[0500] (SEQ ID NO:65)

[0501] Nucleic acid encoding an exemplary GM-CSF polypeptide

[0502] ATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTGCACCCGCCCGCTCGCCCAGCCCCAGCACGCAGCCCTGGGAGCATGTGAATGCCATCCAGGAGGCCCGGCGTCTCCTGAACCTGAGTAGAGACACTGCTGCTGAGATGAATGAAACAGTAGAAGTCATCTCAGAAATGTTTGACCTCCAGGAGCCGACCTGCCTACAGACCCGCCTGGAGCTGTACAAGCAGGGCCTGCGGGGCAGCCTCACCAAGCTCAAGGGCCCCTTGACCATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACCCCGGAAACTTCCTGTGCAACCCAGATTATCACCTTTGAAAGTTTCAAAGAGAACCTGAAGGACTTTCTGCTT GTCATCCCCTTTGACTGCTGGGAGCCAGTCCAGGAGTTGCCAACTTTCTTGTACAAAGTGGTTGGCTCCACCAGCGGCAGCGGCAAGCCAGGCTCCGGCGAAGGCAGCACCAAAGGC

[0503] (SEQ ID NO:66)

[0504] Nucleic acid encoding an exemplary Gal-9 polypeptide

[0505] GCCTTCAGCGGTTCCCAGGCTCCCTACCTGAGTCCAGCTGTCCCCTTTTCTGGGACTATTCAAGGAGGTCTCCAGGACGGACTTCAGATCACTGTCAATGGGACCGTTCTCAGCTCCAGTGGAACCAGGTTTGCTGTGAACTTTCAGACTGGCTTCAGTGGAAATGACATTGCCTTCCACTTCAACCCTCGGTTTGAAGATGGAGGGTACGTGGTGTGCAACACGAGGCAGAACGGAAGCTGGGGGCCCGAGGAGAGGAAGACACACATGCCTTTCCAGAAGGGGATGCCCTTTGACCTCTGCTTCCTGGTGCAGAGCTCAGATTTCAAGGTGATGGTGAACGGGATCCTCTTCGTGCAGTACTTCCACCGCGTGCCCTTCCACCGTGTGGACACCATCTCCGTCAATGGCTCTGTGCAGCTGTCCTACATCAGCTTCCAGCCTCCCGGCGTGTGGCCTGCCAACCCGGCTCCCATTACCCAGACAGTCATCCACACAGTGCAGAGCGCCCCTGGACAGATGTTCTCTACTCCCGCCATCCCACCTATGATGTACCCCCACCCCGCCTATCCGATGCCTTTCATCACCACCATTCTGGGAGGGCTGTACCCATCCAAGTCCATCCTCCTGTCAGGCACTGTCCTGCCCAGTGCTCAGAGGTTCCACATCAACCTGTGCTCTGGGAACCACATCGCCTTCCACCTGAACCCCCGTTTTGATGAGAATGCTGTGGTCCGCAACACCCAGATCGACAACTCCTGGGGGTCTGAGGAGCGAAGTCTGCCCCGAAAAATGCCCTTCGTCCGTGGCCAGAGCTTCTCAGTGTGGATCTTGTGTGAAGCTCACTGCCTCAAGGTGGCCGTGGATGGTCAGCACCTGTTTGAATACTACCATCGCCTGAGGAACCTGCCCACCATCAACAGACTGGAAGTGGGGGGCGACATCCAGCTGACCCATGTGCAGACATAG

[0506] (SEQ ID NO:67)

[0507] Nucleic acids encoding exemplary PD1 polypeptides

[0508] ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAA CTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCTTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCTGCTCCCGGGCCGCACGAGGGACAATAGGAGCCAGGCGCACCGGCCAGCCCCTGAAGGAGGACCCCTCAGCCGTGCCTGTGTTCTCTATTGTTTATGCTTCCCTGGATTTCCAGTGGCGAGAGAAGACCCCGGAGCCCCCCGTGCCCTGTGTCCCTGAGCAGACGGAGTATGCCACCATTGTCTTTCCTAGCGGAATGGGCACCTCATCCCCCGCCCGCAGGGGCTCAGCCGACGGCCCTCGGAGTGCCCAGCCACTGAGGCCTGAGGATGGACACTGCTCTTGGCCCCTGACGCGTCTGCAGGATATCAAGCTTGCGGTACCGCGGGCCCGGGATCCACCGGTCGCCACC

[0509] (SEQ ID NO:68)

[0510] Nucleic acids encoding exemplary TIM-3 polypeptides-

[0511] ATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTACTACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAATGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTGCCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACGTGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATACTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAGAATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGGATCCAAATCCCAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACCAGCCAAGGTCACCCCTGCACCGACTCTGCAGAGAGACTTCACTGCAGCCTTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGACACTGGGGAGCCTCCCTGATATAAATCTAACACAAATATCCACATTGGCCAATGAGTTACGGGACTCTAGATTGGCCAATGACTTACGGGACTCTGGAGCAACCATCAGAATAGGCATCTACATCGGAGCAGGGATCTGTGCTGGGCTGGCTCTGGCTCTTATCTTCGGCGCTTTAATTTTCAAATGGTATTCTCATAGCAAAGAGA AGATACAGAATTTAAGCCTCATCTCTTTGGCCAACCTCCCTCCCTCAGGATTGGCAAATGCAGTAGCAGAGGGAATTCGCTCAGAAGAAAACATCTATACCATTGAAGAGAACGTATATGAAGTGGAGGAGCCCAATGAGTATTATTGCTATGTCAGCAGCAGGCAGCAACCCTCACAACCTTTGGGTTGTCGCTTTGCAATGCCATAG

[0512] (SEQ ID NO:69)

[0513] Nucleic acids encoding exemplary CXCR3 polypeptides

[0514]

[0515] (SEQ ID NO:70)

[0516] Nucleic acids encoding exemplary CXCR4 polypeptides

[0517]

[0518] (SEQ ID NO:71)

[0519] Nucleic acids encoding exemplary CTLA4 polypeptides

[0520] ATGGCTTGCCTTGGATTTCAGCGGCACAAGGCTCAGCTGAACCTGGCTACCAGGACCTGGCCCTGCACTCTCCTGTTTTTTCTTCTCTTCATCCCTGTCTTCTGCAAAGCAATGCACGTGGCCCAGCCTGCTGTGGTACTGGCCAGCAGCCGAGGCATCGCCAGCTTTGTGTGTGAGTATGCATCTCCAGGCAAAGCCACTGAGGTCCGGGTGACAGTGCTTCGGCAGGCTGACAGCCAGGTGACTGAAGTCTGTGCGGCAACCTACATGATGGGGAATGAGTTGACCTTCCTAGATGATTCCATCTGCACGGGCACCTCCAGTGGAAATCAAGTGAACCTCACTATCCAAGGACTGAGGGCCATGGACACGGGACTCTACATCTGCAAGGTGGAGCTCATGTACCCACCGCCATACTACCTGGGCATAGGCAACGGAACCCAGATTTATGTAATTGATCCAGAACCGTGCCCAGATTCTGACTTCCTCC

[0521] TCTGGATCCTTGCAGCAGTTAGTTCGGGGTTGTTTTTTTATAGCTTTCTCCTCACAGCTGTTTCTTTGAGCAAAATGCTAAAGAAAAGAAGCCCTCTTACAACAGGGGTCTATGTGAAAATGCCCCCAACAGAGCCAGAATGTGAAAAGCAATTTCAGCCTTATTTTATTCCCATCAATTGA

[0522] (SEQ ID NO:116)

[0523] Nucleic acids encoding exemplary TLR3 polypeptides

[0524]

[0525] (SEQ ID NO: 117)

[0526] Nucleic acids encoding exemplary TLR4 polypeptides

[0527]

[0528] (SEQ ID NO: 118)

[0529] Nucleic acids encoding exemplary TLR9 polypeptides

[0530]

[0531] (SEQ ID NO: 119)

[0532] Nucleic Acids Encoding Exemplary TNFR2 Polypeptides

[0533]

[0534] (SEQ ID NO: 120)

[0535] Example 4: Bioengineering Mesenchymal Stromal Cells with Chimeric Antigen Receptors to Enhance Immunosuppressive Therapy

[0536] This example describes the design and engineering of MSCs to express one or more chimeric antigen receptors (CAR-MSCs) and describes how such CAR-MSCs enhance immunosuppression at antigen-specific inflammatory target sites.

[0537] The results in this example replicate and extend at least some of the results provided in the other examples.

[0538] method

[0539] Cell lines, primary peripheral blood mononuclear cells (PBMCs), primary T cells, and primary mesenchymal stromal cells (MSCs)

[0540] Primary human adipose-derived MSCs were obtained and cultured with StemXVivo Mesenchymal Stromal Cell Expansion Medium (R&D Systems, Minneapolis, MN). Human embryonic kidney 293 (HEK-293T) and human epithelial breast cancer cell line (MCF7) were obtained from ATCC (HTB-22, Manassas, VA, USA) and cultured in D10 (DMEM Gibco, Gaithersburg, MD, USA) medium containing 10% (v / v) fetal bovine serum (FBS, Millipore Sigma, Ontario, Canada) and 1% (v / v) penicillin-streptomycin-glutamine (PSG, Gibco, Gaithersburg, MD, USA). HEK-293T was used for lentiviral production. MCF7 was irradiated and confirmed to be E-cadherin (Ecad) positive by flow cytometry to be used as a form of CAR-based single-chain variable fragment (scFv) stimulation in the in vivo tumor model.

[0541] Normal human primary epidermal keratinocytes were obtained from the epidermis of adolescent or adult foreskin from single or mixed donors and cultured in Keratinocyte Growth Medium (PromoCell, Heidelberg, Germany). Normal human primary bronchial epithelial cells were isolated from the airway epithelium above the lung bifurcation (Lonsard, Cohasset, MN). These cells were then cultured in Airway Epithelial Cell Basal Expansion Medium (PromoCell, Heidelberg, Germany).

[0542] The acute lymphoblastic leukemia cell line NALM6 and the mantle cell lymphoma cell line JeKo-1 were also purchased from ATCC (CRL-3273 and CRL-3006, Manassas, VA, USA, respectively). These cell lines were cultured in R20 and R10 (RPMI 1640, Gibco, Gaithersburg, MD, USA) containing 20% ​​(v / v) or 10% (v / v) fetal bovine serum (Millipore Sigma, Ontario, Canada) and 1% (v / v) PSG (Gibco, Gaithersburg, MD, USA), respectively. For in vivo experiments, the cell lines were transduced with luciferase-ZsGreen lentivirus (Adgene, Cambridge, MA, USA). In addition, NALM6 and / or luciferase + The NALM6 gene is transduced into a lentivirus encoding human Ecad (GeneCopoeia, Rockville, Maryland) as a cell-based Ecad stimulation form. The cell line is cultured to 20 generations, and fresh cells are thawed every 7-8 weeks. The cell line is identified by the manufacturer and the phenotype is regularly checked by flow cytometry. Mycoplasma detection is performed on the cell line every month. PBMCs are separated from normal donor blood apheresis cones using SepMate tubes (Stem Cell Technologies, Vancouver, Canada). T cells are separated by negative selection magnetic beads using EasySep™ human T cell 80 separation kit (Stem Cell Technologies, Vancouver, Canada).

[0543] Primary T cells and PBMCs were cultured in T cell culture medium containing X-VIVO 15 (Lonza, Walkersville, MD, USA), 10% (v / v) human serum albumin (Innovative Research, Novi, MI, USA), and 1% (v / v) PSG (Gibco, Gaithersburg, MD, USA) before in vitro co-culture screening. Freshly isolated human PBMCs were administered intravenously for in vivo experiments.

[0544] scFv phage display library

[0545] The hmcECAD.6scFv sequence was generated and optimized by phage display library screening to enable it to bind to Ecad protein. 9A diverse natural human scFv library was used to screen for recombinant Ecad proteins (Biomolecular Discovery, Rochester, NY). After two rounds of screening against mouse Ecad and one round of screening against a human Ecad Fc chimeric protein (both from Biolgen, San Diego, CA), the hmcECAD.6 scFv clone was identified. Phage ELISA assays showed that this clone reacted with mouse Ecad. The relative affinity of the hmcECAD.6 clone was estimated to be approximately 5 nM compared to the relative affinity of the FLAG antibody. The final hmcECAD.6 sequence (SEQ ID NO: 90) was codon-optimized for cloning its heavy and light chains into the EcCAR plasmid.

[0546] Chimeric Antigen Receptor (CAR) Design and Virus Production

[0547] MSCs are transduced by a lentiviral vector that encodes a specially designed CAR construct under the EF-1α promoter. Transduction optimization uses a CAR (CAR19) construct targeting CD19. See, for example, June et al. (359) Science 359, 1361-1365 (2018). CAR19 comprises a CD19-directed scFv derived from clone FMC-63 and fused with 4-1BB and CD3ζ signal transduction domains (FMC63-41BB-ζ). HEK-293T cells are transfected by lipofectamine to generate VSV-g-pseudotyped second-generation lentivirus carrying a CAR transgene, and then lentivirus collection, concentration, and functional titration are performed according to standard procedures. For example, see Sterner et al., Blood 133, 697-709 (2019) and Sakemura et al., Blood 139 (26): 3708-3721 (2022). Subsequently, the CAR construct was designed to contain a scFv against human / canine Ecad ( Figure 23A ) and carries the CD28ζ intracellular signaling domain to induce immunomodulatory activation of MSCs ( Figure 23B CAR plasmids were generated and sequence-verified. Pre-seeded MSCs (250,000 cells / well) were transduced with lentiviral particles at a multiplicity of infection (MOI) of 3 in 6-well plates supplemented with different concentrations of protamine sulfate (25, 50, and 100 μg / μL) to optimize CAR expression and MSC proliferation.

[0548] T cell suppression assay

[0549] To test the ability of CAR-MSCs to inhibit activated T cells, a T cell inhibition assay was performed. Briefly, untransduced (UTD)-MSCs or EcCAR-MSCs (suppressors) were co-cultured with activated T cells (effectors) in the presence or absence of the following stimuli: 1) soluble Ecad (stimulator) or 2) matched Ecad + and Ecad - Co-cultured in the presence of the NALM6 cell line to provide soluble and cell-based antigen-specific stimulation to the CAR-MSCs. T cells were isolated from PBMCs of normal donors using negative selection magnetic beads. See, for example, Sterner et al. Blood 133, 697-709 (2019). The isolated T cells were non-specifically activated using CD3 / CD28 stimulating magnetic beads (Dynabeads, Invitrogen, Waltham, Massachusetts, USA) at a 1:1 ratio of magnetic beads to T cells in T cell culture medium containing X-VIVO 15 (Lonza, Walkersville, Maryland, USA), 10% (v / v) human serum albumin (Innovative Research, Novi, Michigan, USA) and 1% (v / v) PSG (Gibco, Gaithersburg, Maryland, USA). MSCs were incubated with or without soluble Ecad protein (250ng-1000ng / mL) or matched Ecad + and Ecad - Cell lines (MSC and Ecad + The cells were co-cultured with UTD-MSC or EcCAR-MSC (inhibitor) at a 1: 5 MSC to T cell ratio, with 250,000 T cells per 6-well plate. The cells were then collected and analyzed by flow cytometry according to the co-culture time points shown in each experiment.

[0550] Multiparameter flow cytometry

[0551] Flow cytometry staining was performed. Briefly, MSCs were isolated and cultured, then transduced with CAR to generate CAR-MSCs, which were co-cultured with T cells / PBMCs in 96-well plates for 24-hour assays and in 6-well plates for long-term culture. After the desired co-culture time, 10 mL / 75 cm 2Adherent MSCs were treated with Accutase (STEMCELL Technologies, Vancouver, Canada) for 10-15 minutes at 37°C to allow detachment. After detachment, all well contents were centrifuged and washed with running buffer (phosphate-buffered saline (PBS), 2% (v / v) FBS, and 1% (v / v) sodium azide), then stained with the desired antibodies for 15 minutes at room temperature in the dark. After the final wash step, cells were analyzed for the desired surface markers, and positive results were determined by negative gating on fluorescence minus expression (FMO) control wells. Absolute cell counts were obtained using volumetric measurements. Cells were gated on SSC versus FSC plots to separate cells based on size and complexity; gated on FSC-H versus FSC-A plots to exclude doublets; dead cells were excluded using LIVE / DEAD Aqua stain (Cat. No. L34966, Thermo Fisher Scientific, Waltham, MA, USA); and cell subset characterization was then performed based on a pre-designed antibody panel that was optimized and used to stain the samples.

[0552] Anti-human antibodies were purchased from Bailejin, Electronic Biosciences, or BD Biosciences (San Diego, California, USA). Samples were prepared for flow cytometry analysis. See, for example, Sakemura et al. Blood 139(26):3708-3721(2022). All antibodies used for staining are listed in (Table 4). Flow cytometry was performed using a three-laser CytoFLEX (Beckman Coulter, Chaska, Minnesota, USA), which gated cells by singlet recognition and determined live cells by Live / Dead Aqua dye (Cat. No. L34966, Thermo Fisher Scientific, Waltham, Massachusetts, USA). All gate selection analyses were performed using FlowJo X10.0.7r2 software (BD (Becton Dickenson), Ashland, Oregon, USA) and Kaluza Analysis software (BD, Indianapolis, Indiana, USA).

[0553] Table 4. List of antibodies used for flow cytometry, clone, supplier, and catalog number.

[0554]

[0555]

[0556] In vivo studies in mice

[0557] 6-8 week old female and male immunocompromised NOD-SCID-γ - / - (NSG) mice were purchased from Jackson Laboratory and housed in BSL2 +All cells were injected into the tail vein or intraperitoneal cavity via syringe in 100 μL-200 μL PBS. Mice were imaged using a bioluminescent imager using the Lumina S5 Imaging System (PerkinElmer, Hopkinton, MA, USA) to confirm luciferase expression in cancer xenograft models. + CD19 + Engraftment of Nalm6 / JeKo1 cells or luciferase in MSC persistence xenograft models + Implantation of CAR-MSCs. Imaging was performed 10 minutes after intraperitoneal injection of 10 μL / g D-luciferin (15 mg / mL, Gold Biotechnology, St. Louis, MO, USA). In the Ecad tumor xenograft model, NSG mice were implanted with Ecad + or Ecad - Luciferase + CD19 + NALM6 (1 x 10 intravenous injection) 6 Five days after injection, engraftment was confirmed by BLI and mice were randomly assigned to receive UTD-MSCs or EcCAR-MSCs (1 x 10 6 cells) and CD19-targeted CART cells (CART19) (1 x 10 6 CD19 cells as therapeutics + Tumor Strategy) Figure 17A After CART19 effectively killed the NALM6 tumor burden, mice were re-challenged to receive Ecad + or Ecad - Luciferase + CD19 + NALM6 (1 x 10 intravenous injection) 6 cells) and UTD-MSCs or EcCAR-MSCs (1 x 10 6 Serial BLI was then performed to assess residual disease and determine the Ecad + NALM6 and Ecad - Antitumor activity on NALM6.

[0558] In a complementary tumor xenograft model, NSG mice were implanted with luciferase + CD19 + JeKo-1 or NALM6 (1 x 10 6cells). Depending on the tumor subtype, engraftment was confirmed by BLI 1-2 weeks or 5 days after injection. All mice subsequently received irradiated Ecad + MCF7 cells (5x10 6 cells to stimulate EcCAR-MSCs) and CART19 cells (1x10 6 CD19 cells as therapeutics + Based on BLI as a measure of tumor burden, mice were then randomly assigned to receive UTD-MSCs, EcCAR-MSCs, or no additional treatment ( Figure 25B ). Serial BLI was then performed to assess residual disease and determine the anti-tumor activity of CART19 cells.

[0559] In the graft-versus-host disease (GVHD) model, NSG mice were treated with allogeneic human peripheral blood mononuclear cells (PBMCs) (20-30 x 10 6 cells) to induce GVHD and were treated with UTD-MSCs (1×10 6 cells) or EcCAR-MSCs (1x10 cells intraperitoneally every 3 weeks) 6 cells) treated with Figures 17D-17G In all experiments, body weight and clinical GVHD scores (based on body weight, posture, diarrhea, activity, coat condition, and skin integrity, Table 5) were monitored to understand the progression of GVHD in each experimental group.

[0560] Table 5. GVHD clinical scoring system.

[0561]

[0562] In the acute GVHD model, GVHD can be induced more rapidly by first irradiating NSG mice with a dose of 250 cGy. Allogeneic human PBMCs (10-15 x 10 6 cells, intravenously) were administered to irradiated mice and randomly assigned according to body weight to receive UTD- or CAR-MSC ( Figure 18A luciferase + GFP + CD19-CAR-MSC as luciferase + GFP + EcCAR-MSC-treated controls. MSC localization in organs was detected by BLI and immunofluorescence staining. Allogeneic PBMCs were depleted of B cells using CD19 Pan BCellDynabeads (Invitrogen, Waltham, MA, USA) to ensure the absence of human CD19 in mice. +In all experiments, body weight and clinical GVHD scores were monitored to assess the progression of GVHD in each experimental group, using the same criteria as listed above.

[0563] To evaluate the trafficking and localization of luciferase + anti-Ecad CAR-MSCs and luciferase + anti-CD19 CAR-MSCs, Lumina S5 imaging system (PerkinElmer, Hopkinton, Massachusetts, USA) performed bioluminescence imaging (BLI) imaging on mice. In an acute GVHD xenograft model, one week after injection of MSC, satellite mice were randomly selected for euthanasia and subsequently evaluated for organ flux. Here, organ imaging was performed 10-20 minutes after intraperitoneal injection of 250uL 50mg / mL D-luciferin (Gold Biotech, St. Louis, MO, USA) to detect luciferase in the colon, kidney, liver, lung and spleen relative to whole-body BLI detection. + MSC.

[0564] For all in vivo experiments, mouse tail vein blood (about 100 μL) was collected, of which 70 μL were used for flow cytometry analysis. Red blood cells (RBC) were lysed using 1:10 BD FACS lysate (BD Biosciences, San Jose, California, USA). Subsequently, cells were washed with flow buffer and incubated with a specific antibody mixture at room temperature in the dark, and then flow analyzed using CytoFLEX (Beckman Coulter, Chaska, Minnesota, USA). The remaining blood was centrifuged at 4 ° C for 10 minutes at 13,000 rpm to separate serum for cytokine analysis.

[0565] Immunofluorescence staining

[0566] In an acute GVHD mouse xenograft model, satellite mice were sacrificed 7 days after MSC injection, and organs, including the colon, liver, lung, and heart, were sampled and preserved. Organs were fixed with 4% paraformaldehyde for 48 hours and immediately frozen in Tissue-Tek OCT compound (Sakura, Hayward, CA) for cryosectioning and slide preparation. Tissue slides were then thawed from −80°C storage, washed with PBS, and then incubated with 0.1% Sudan Black (Sigma-Aldrich, Burlington, MA) solution and dissolved in blocking buffer containing 0.3% Triton-X-100 (Thermo Fisher Scientific, Waltham, MA) and 5% bovine serum albumin (Sigma-Aldrich, Burlington, MA). Antigen retrieval was performed by boiling in unmasking solution for 15 minutes. Tissues were incubated with primary antibodies against Ecad (Cat. 144725, Cell Signaling Technology, Danvers, MA) and GFP for 1 hour at room temperature. + CAR-MSCs (Cat. No. Ab183734, Abcam, Boston, MA) were stained. Next, secondary antibodies (Cat. No. A11001, Cat. No. A11012, Thermo Fisher Scientific, Waltham, MA) were incubated for 1 hour at room temperature. Slides were imaged using a Zeiss LSM 980 confocal microscope (Zeiss Group, Oberkochen, Germany) and analyzed using ZEN 2.3 microscopy software (Zeiss Group, Oberkochen, Germany). EcCAR-MSCs, CD19CAR-MSCs, and MSC-free (negative control) groups were compared in terms of Ecad + The localization of GFP in the colonic crypts was determined by averaging five 40x magnification microscopic fields under each condition. + MSC colonic crypts were quantified to calculate localization ( Figures 18F-18G and Figure 21D and 21F ).

[0567] Canine research

[0568] In all experiments, the Ecad-targeting CAR expressed on human MSCs was based on a human / murine / canine cross-reactive scFv. This scFv was generated using phage display technology and has been shown to cross-react with both canine and human Ecad ( Figures 23A-23D ). EcCAR-MSCs in healthy beagle dogs. Subjects received intraperitoneal injection of EcCAR-MSCs (2 x 10 6The subjects were followed up daily, their body weights were monitored, and blood samples were collected serially. Colon biopsies were performed on day 3, and tissue integrity was assessed using hematoxylin-eosin (H&E) staining.

[0569] RNA isolation, sequencing, and analysis

[0570] EcCAR-MSCs or UTD-MSCs were stimulated in culture for 24 hours with or without 250 ng / mL recombinant human E-cadherin Fc chimera (Bio-Zine, San Diego, CA, USA) as a CAR-specific stimulus. After in vitro culture, MSCs were isolated, and RNA was isolated using the QIAGEN RNeasy Plus Mini Kit (Cat. No. 74134, Qiagen, Germantown, MD, USA). To ensure the rigor of the results, bulk RNA sequencing was performed on MSCs derived from three different biological donors in the UTD-MSC and EcCAR-MSC groups.

[0571] Total RNA was prepared using the SMARTer Stranded Total RNA-seq Kit v2 (Pico-grade Input, Mammalian) (Takara, Mountain View, CA, USA). Total RNA (three samples / lane) was sequenced using an Illumina HiSeq 4000 (Illumina, San Diego, CA, USA). Library preparation and sequencing were performed by the Genome Analysis Center, Institute for Medical Genomics (Mayo Clinic, Rochester, MN, USA). The generated FastQC files were quality-checked using FastQC v0.11.8. Adapter sequences were trimmed and removed using Cutadapt v1.18. The output files were verified for adapter removal and quality using FastQC v0.11.8.

[0572] Paired-end reads from trimmed FastQ files were aligned to the latest human reference genome (GRCh38) downloaded from NCBI (ncbi.nlm.nih.gov / ). A genome index file was constructed and aligned using STAR v2.5.4b. Universal expression counts for each gene were generated using HTSeq (Python 3.6.5). Gene counts were normalized (geometric mean), and differential expression analysis was calculated using DESeq2 (R v3.6.1), using an adjusted p-value ≤ 0.05 as a statistical cutoff. Heatmaps were created using pheatmap, and principal component analysis (PCA) was generated using ggplot2 and Prism Graph Pad (La Jolla, CA). Gene set enrichment analysis of cellular phenotypes was performed using the Enrichr Cell Enhanced Gene Set. Canonical pathways, molecules, and protein-protein interaction networks for activation and repression were generated using Ingenuity Pathway Analysis (Qiagen, Redwood City, CA, USA), with stringent p-value and fold-change cutoffs (p ≤ 0.01, ±1 fold change) for differential gene expression comparisons.

[0573] Multiplex cytokine assay

[0574] Cytokine assays were performed on serum samples collected from mice 2 weeks after MSC or control treatment. In vitro cytokine assays were performed on supernatants collected after 24 hours of co-culture with MSCs and stimulators. Debris was removed from the serum or supernatant by centrifugation at 10,000 x g for 5 minutes. Prior to inoculation, serum and / or supernatant were diluted with serum matrix at a ratio of 1:2 and operated according to the manufacturer's protocol of the Milliplex Human Cytokine / Chemokine Magnetic Bead Premix 38Plex Kit (HCYTMAG-60K-PX38, Millipore Sigma, Ontario, Canada). Data were collected using Luminex (Millipore Sigma, Ontario, Canada) and analyzed using Belysa Immunoassay Curve Fitting Software (Millipore Sigma, Ontario, Canada) and Microsoft Excel (Microsoft, Redmond, Washington, USA). Significant results were determined and reported using Prism GraphPad (La Jolla, California, USA).

[0575] Statistical analysis and graphs

[0576] Both in vitro and in vivo experiments were performed with technical and biological replicates to allow for appropriate statistical analysis. Two-way or one-way ANOVA was used to assess differences in surface marker expression (by percentage expression) and T cell suppressive capacity (assessed by absolute T cell counts) between EcCAR-MSCs and UTD-MSCs in vitro. ANOVA was also used to compare the expression of luciferase+tumor and luciferase + IVIS imaging, weight comparison, flow analysis of blood components and GVHD clinical score comparison of MSC were used to determine the significant differences between EcCAR-MSC treated mice and UTD-MSC treated mice in vivo. The Kaplan Meier survival analysis using Cox regression was used to determine the significance of the differences and to adjust the confounding factors (e.g., sex) of the survival results in tumor and GVHD xenograft models. In order to make multiple comparisons between the groups in the analysis described previously, the analysis of variance was supplemented with Tukey's multiple comparison test. In order to compare between the two groups, a two-tailed unpaired Sigmund's t-test was used instead of an analysis of variance.

[0577] RNA-seq data were processed using the DeSeq2 program, where the raw counts of the samples were normalized (geometric mean) and multiple hypothesis correction was performed using the Benjamini-Hochberg procedure. All relevant statistically significant comparisons are marked with asterisks, corresponding to a significance level below p ≤ 0.05, and 95% confidence intervals are indicated (where ns = p ≥ 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001). End data points without asterisks indicate no significant difference between groups (ns). Relevant data represent the mean of all data points, and error bars are determined by standard deviation (SD) or standard error of the mean (SEM). Experimental data were analyzed and created using Prism Graph Pad (La Jolla, CA, USA) and Microsoft Excel (Microsoft, Redmond, WA, USA). Finally, canonical pathway enrichment and molecular networks were generated, and significant findings (*padj≤0.05) were reported by Ingenuity Pathway Analysis (Qiagen, Redwood City, CA, USA).

[0578] result

[0579] MSCs were successfully transduced and stably expressed CAR.

[0580] Using a lentiviral vector with a polycationic enhancer, we achieved efficient transduction of CAR in MSCs. We first optimized the delivery of CAR lentivirus with protamine sulfate, achieving a transduction efficiency of ≥70% in healthy donor adipose-derived MSCs ( Figure 15A). After optimization using standardized CAR vectors, alternative CAR constructs for CAR-MSC immune-mediated disease therapy were developed. The primary CAR-MSC constructs were directed to Ecad, a ligand expressed on inflammatory intestinal epithelial cells involved in xenogeneic graft-versus-host disease (GVHD). GVHD is caused by donor T cells attacking host epithelial tissue, in part due to the interaction of T cell integrins with E-cadherin (Ecad) expressed on the gastrointestinal tract. In order to redirect MSCs to Ecad, which is vulnerable to immune attack, + We constructed multiple anti-Ecad scFv clones by phage display for CAR incorporation. We selected the optimized scFv sequence (hmcECAD.6 (SEQ ID NO: 90), Figures 23A-23B ), which has human, mouse ( Figures 23C-23D ) and canine cross-reactivity, allowing for preclinical testing in animal models.

[0581] To determine whether incorporation of a CAR signaling domain could enhance the immunosuppressive function of MSCs without increasing the risk of cytotoxic T cell conversion, we tested CAR constructs containing the CD28ζ signaling domain. hmcECAD.6 was incorporated into the CAR CD28ζ backbone ( Figure 23B ) to generate EcCAR-MSCs ( Figure 15B EcCAR can maintain stable expression after multiple passages in vitro, and the transduction efficiency in different MSC donors is ≥90% ( Figures 15C-15D and Table 5 ).

[0582] EcCAR-MSCs retain their stemness

[0583] To exclude any unnecessary differentiation induced by CAR-MSC engineering, we determined the phenotype, morphology, and gene expression of EcCAR-MSC stem cells. Based on the MSC stem cell maintenance standards established by the International Society for Cell & Gene Therapy, EcCAR-MSC maintained their stem cell phenotype and morphology, similar to untransduced (UTD)-MSCs ( Figure 15E and Figures 24A-24C We also used bulk RNA sequencing (RNAseq) between EcCAR-MSCs and UTD-MSCs to identify enrichment of phenotypic gene sets. The adipose-derived stem cell phenotype was enriched for upregulated EcCAR-MSC genes, while the differentiated cell lineages were enriched for downregulated genes ( Figure 15F These results support the maintenance of stemness after MSC engineering, while CAR transduction-induced differentiation is limited.

[0584] EcCAR-MSCs exhibit excellent antigen-specific immunosuppressive ability while maintaining stem cell characteristics

[0585] The ability of CAR-MSCs to inhibit T cells was tested in vitro. Briefly, EcCAR-MSCs or UTD-MSCs (inhibitors) were co-cultured with activated T cells (effectors) for 24 hours in the presence or absence of Ecad protein as a strategy to stimulate CAR (stimulator). Soluble Ecad protein and a NALM6 cell line engineered to stably overexpress Ecad ( Figure 16A ) were used as CAR stimulation sources. Compared with UTD-MSC control, soluble Ecad stimulation of EcCAR-MSC resulted in a dose-dependent and superior T cell inhibition ( Figure 16B After stimulation with soluble Ecad, the MSC stem cell phenotype was significantly maintained ( Figure 16C ). In addition, influenced by Ecad + EcCAR-MSCs stimulated by cell lines also exhibited antigen-specific immunosuppression. + cells (not Ecad - After co-culture with WT cells, excellent T cell suppression ( Figures 16D-16F ). Ecad + or Ecad - Stimulation of cell lines also did not affect the stemness or morphology of EcCAR-MSCs ( Figure 16G and Figures 24B-24C EcCAR-MSCs inhibit the anti-tumor activity of CART19 in tumor xenograft models

[0586] Based on these in vitro findings, to determine whether CAR-MSCs inhibit CART19 and enhance tumor growth in an antigen-specific setting, the immunosuppressive effects of CAR-MSCs were evaluated in vivo using tumor and GVHD xenograft models. In the tumor model, the ability of MSCs to inhibit the potent antitumor activity of CD19-targeted T cells (CART19) was measured. Figure 17A In order to provide antigen-specific stimulation to EcCAR-MSC, Ecad + NALM6 cells and Ecad - NALM6 cells were used as control ( Figure 16A ). By delivering - / - (NSG) mice were injected intravenously (iv) with luciferase + Ecad + NALM6 or luciferase + Ecad - NALM6 tumor cell-derived tumor model ( Figure 17A ). It was determined that in immunocompromised mice without additional treatment, Ecad + NALM6 and Ecad - NALM6 growth is similar ( Figure 25A ).

[0587] After implantation, mice were randomly assigned to receive CART19 and UTD-MSC treatment or CART19 and EcCAR-MSC treatment based on tumor burden determined by bioluminescence imaging (BLI), and tumor burden was assessed by BLI every two weeks. The results showed that EcCAR-MSC activated in an Ecad-dependent manner in vivo. Compared with UTD-MSC treatment, EcCAR-MSC treatment of Ecad + NALM6 xenograft mice ( Figure 17B ) would reduce its anti-tumor activity, while in Ecad-NALM6 mice ( Figure 17C ) had no such effect. In NALM6 co-culture, EcCAR-MSC alone did not directly promote leukemia growth ( Figures 25B-25C ).

[0588] The study also found that when irradiated Ecad + When MCF7 co-injection was used as an alternative strategy to stimulate EcCAR-MSCs, EcCAR-MSC treatment significantly impaired their antitumor activity and decreased survival in tumor xenograft models ( Figures 25D-25G These results highlight the potent immunosuppressive potential of EcCAR-MSCs in the presence of Ecad antigen in vivo.

[0589] In a GVHD xenograft model, EcCAR-MSCs improved therapeutic efficacy and induced Ecad + Targeted antigen-specific transport

[0590] The therapeutic effect of EcCAR-MSC was tested in a GVHD xenograft model. A GVHD xenograft model was generated by intravenously injecting human peripheral blood mononuclear cells (PBMC) into NSG mice. See, for example, Ehx et al. Front Immunol 9, 1943 (2018). Mice received intraperitoneal (ip) administration of PBS control, UTD-MSC, or EcCAR-MSC as a form of GVHD treatment ( Figure 17D GVHD progression was recorded by body weight measurement, clinical GVHD symptom scores (Table 4), and survival outcomes. EcCAR-MSC treatment prevented weight loss compared with mice that received UTD-MSC or no MSC treatment ( Figure 17E ), improve clinical GVHD symptoms (including hair, skin integrity, posture and activity) ( Figure 17F ), and improve overall survival ( Figure 17G ).

[0591] To evaluate the effect of anti-Ecad scFv contained in EcCAR-MSC, we compared the efficacy of EcCAR-MSC with that of CAR-MSC containing scFv that is not related to mouse tissue specificity (anti-CD19 CAR-MSC). + Antigen-specific stimulation and trafficking of epithelial cells. + Luciferase + EcCAR-MSCs and GFP + Luciferase + control anti-CD19 CAR-MSC ( Figures 26A-26B ). GFP + Luciferase + EcCAR-MSCs and GFP + Trafficking and efficacy of luciferase + control anti-CD19 CAR-MSCs in a GVHD xenograft model ( Figure 18A ). In this model, mice are irradiated before injection of PBMC and MSC to induce a more severe GVHD phenotype (hereinafter referred to as the "acute GVHD model"), in which mice develop acute GVHD within a week after injection of PBMC. See, for example, Schroeder and DiPersio. Dis. Model Mech. 4, 318-333 (2011). In this acute GVHD model, EcCAR-MSC treatment again significantly reduced the weight loss of mice compared to the control group CD19-CAR-MSC or no MSC ( Figure 18B ) and prolonged the survival rate of mice ( Figure 18C To elucidate the potential mechanism of enhanced efficacy of EcCAR-MSC in this model, peripheral blood was collected from mice two weeks after MSC injection and human T cells were analyzed. Compared with CD19-CAR-MSC and control mice without MSC treatment, EcCAR-MSC administration resulted in enhanced suppression of human T cells in peripheral blood ( Figure 18D One week after CAR-MSC injection, the satellite mice in these experiments were euthanized, their organs were removed, and the presence of CAR-MSCs was analyzed by BLI and immunofluorescence. After MSC treatment, BLI and immunofluorescence evaluation of the colon tissue of mice showed that EcCAR-MSCs were significantly higher in Ecad compared to CD19-CAR-MSCs. + Infiltration was more pronounced in colonic tissue ( Figures 18E-18G Together, these results demonstrate that EcCAR-MSCs possess enhanced immunosuppressive efficacy and target tissue trafficking capacity in a GVHD model.

[0592] Activation of signal transduction pathways, inhibitory cytokines, and surface receptors induces an immunosuppressive phenotype

[0593] To evaluate the mechanism of EcCAR-MSC immunosuppression enhancement, the gene expression, cytokine secretion, and surface marker profiles of stimulated EcCAR-MSCs were investigated. Gene expression profiles were first analyzed by bulk RNA sequencing under the following conditions: UTD-MSCs (unstimulated), UTD-MSCs (stimulated with soluble Ecad as a control), EcCAR-MSCs (unstimulated), and EcCAR-MSCs (stimulated with soluble Ecad to detect antigen-specific CAR stimulation). Pathway enrichment under different conditions was identified using a predictive network generated by Ingenuity Pathway Analysis (IPA) machine learning software.

[0594] To characterize the effects of CAR transduction on MSCs, we compared non-stimulated EcCAR-MSC and UTD-MSC samples. Although 331 genes were significantly upregulated between the two groups, the gene enrichment was mainly attributed to cellular stress response pathways commonly induced by lentiviral transduction ( Figures 27A-27C Notably, these enrichments became less significant in stimulated EcCAR-MSCs compared to UTD-MSCs, which instead showed enrichment of major homeostatic pathways ( Figure 27D Unbiased hierarchical gene clustering analysis across all conditions revealed that the most pronounced gene expression changes were driven by antigen-specific stimulation of EcCAR-MSCs (+Ecad), forming clusters distinct from all other conditions ( Figure 19A ).

[0595] To characterize the effects of CAR antigen-specific CD28ζ stimulation, Ecad-stimulated EcCAR-MSC samples were compared with non-stimulated EcCAR-MSC samples. 1662 upregulated genes were identified to be activated ( Figure 27A ), which supports the idea that CAR transduction alters MSC gene expression, whereas CAR stimulation leads to more pronounced changes in gene activation ( Figure 19A Principal component analysis of gene expression under Ecad stimulation and non-stimulation conditions showed that UTD-MSC samples tended to cluster according to the MSC biodonor from which they were derived ( Figure 19B ), while EcCAR-MSC samples tended to cluster according to Ecad stimulation status ( Figure 19C The classical pathways significantly upregulated by CAR stimulation were strongly associated with MSC anti-inflammatory interleukin (IL-6 and IL-10), NFκB, tumor necrosis factor receptor 2 (TNFR2), and Toll-like receptor (TLR) signaling pathways ( Figures 19D-19EThese pathways each promote the immunosuppressive function of MSCs through cytokine-mediated T cell inhibition, Treg induction, or direct receptor interactions.

[0596] In Ecad-stimulated EcCAR-MSC samples, the gene counts of transcription factors (NFκB1, JUN, RELB, IRF1) and their associated regulatory factors (TRAF1, TLR3 and FYN) were strongly increased ( Figure 19F In summary, these transcription factors, as well as others found after CAR stimulation, have also been identified as direct downstream targets of CD28 stimulatory molecules ( Figure 19D ) and is a regulator of MSC immunosuppressive function. Leukocyte apoptosis was identified as a significant functional enrichment among all signaling molecules contained in the EcCAR-MSC stimulation prediction network ( Figure 19D ).

[0597] To understand how gene activation in EcCAR-MSCs translates into functional immunosuppression, we measured the expression of inhibitory receptors and production of human cytokines following antigen-specific stimulation of EcCAR-MSCs. - EcCAR-MSCs were co-cultured with activated T cells in the presence of cell lines. Antigen-specific stimulation of EcCAR-MSCs (using Ecad + cell lines) resulted in increased secretion of inhibitory cytokines in vitro, such as interleukin 10 (IL-10), interleukin 4 (IL-4), granulocyte colony-stimulating factor (G-CSF), and vascular endothelial growth factor (VEGF) ( Figure 20A Furthermore, the key inhibitory surface markers galectin-9 (Gal-9) and cytotoxic T lymphocyte-associated protein 4 (CTLA4) increased in an antigen-dependent manner in stimulated EcCAR-MSCs ( Figure 20B In the tumor model, human cytokine production was measured in mouse serum after 2 weeks of EcCAR-MSC or UTD-MSC treatment ( Figure 17A In EcCAR-MSC-treated mice, there were elevated levels of cytokines such as IL-10, G-CSF, and eotaxin ( Figure 20C and Figure 27E ).

[0598] Compared with the UTD-MSC-treated group, the GVHD xenograft mice treated with EcCAR-MSC also showed a significant decrease in human CD3 + T cell subtypes were evaluated. Figures 20D-20H ) related to the human CD3 circulating in the peripheral blood of mice treated with EcCAR-MSCs. +The absolute number of T cells was significantly suppressed ( Figure 20E ). Although human CD4 + and CD8 + T cells were suppressed ( Figure 20F ), but in mice treated with EcCAR-MSCs, human CD4 + The proportion of T cells increased ( Figure 20G Peripheral blood analysis 4 weeks after treatment also showed that Treg (human CD4 + 、CD25 + 、CD127 - ) is enriched ( Figure 20H ).

[0599] Together, these findings demonstrate that T-cell suppression is enhanced after antigen-specific stimulation of EcCAR-MSCs compared with unstimulated EcCAR-MSCs and demonstrate that this enhancement is associated with activation of immunosuppressive pathways, increased production of inhibitory cytokines, and upregulation of inhibitory receptors.

[0600] Incorporation of the CD28ζ signaling domain into CAR-MSCs is essential for enhancing immunosuppressive activity

[0601] To determine which components of the CD28ζ signaling domain are necessary for EcCAR-MSC-mediated enhanced immunosuppression after their antigen-specific stimulation, we generated CD28-stimulated EcCAR-MSC (CD28 EcCAR-MSC), CD3ζ-stimulated EcCAR-MSC (CD3ζEcCAR-MSC), and EcCAR-MSC lacking the intracellular domain (empty EcCAR-MSC) and compared them with CD28-CD3ζ-stimulated EcCAR-MSC (CD28ζEcCAR-MSC) ( Figures 21A-21B CD28ζEcCAR-MSC and CD28 EcCAR-MSC showed antigen-specific inhibition of activated T cells, while CD3ζEcCAR-MSC, empty EcCAR-MSC and UTD-MSC did not show this effect ( Figure 21C ), suggesting that CD28 signaling plays a role in the immunosuppressive properties of CAR-MSCs. The effect of CAR signaling domain incorporation was also tested in an acute GVHD xenograft model ( Figure 21D In this model, mice treated with CD28ζ EcCAR-MSCs were protected from weight loss compared to mice treated with CD28, CD3ζ, or an empty signaling domain EcCAR-MSCs ( Figure 21E), and achieved the lowest clinical GVHD symptom severity score ( Figure 17F ) and the longest overall survival ( Figure 17G Treatment with CD28 EcCAR-MSCs improved weight loss and overall survival, which were numerically slightly lower than those with CD28ζ EcCAR-MSCs, but the differences were not statistically significant. These data suggest that although CD28 alone can confer immunosuppressive capacity on EcCAR-MSCs, incorporating CD28ζ into EcCAR-MSCs can enhance the immunosuppressive therapeutic effect.

[0602] EcCAR-MSCs are safe in animal models

[0603] After functional evaluation, studies were performed to ensure the safety of allogeneic EcCAR-MSCs. First, luciferase was used in NSG mice to + EcCAR-MSC and UTD-MSC determine the expansion and clearance of EcCAR-MSC ( Figure 22A and Figure 26A ). In irradiated or non-irradiated human Ecad + The persistence of MSCs was monitored in mice with MCF7 cells to understand the effect of antigen-specific stimulation on MSC clearance. Mice were treated with the following: 1) luciferase alone + UTD-MSC; 2) Luciferase + UTD-MSC Joint Ecad + cells; 3) using luciferase alone + EcCAR-MSC; or 4) luciferase + EcCAR-MSC combined with human Ecad + cell( Figure 22A ). Serial BLI was performed every 2-3 days to quantify MSCs. These studies showed that there was no significant difference in the clearance time (approximately 30 days) between UTD-MSCs and EcCAR-MSCs, regardless of the presence or absence of additional target antigen stimulation, further supporting the safety of EcCAR-MSCs ( Figures 22B-22C ).

[0604] Then, the toxicity of EcCAR-MSCs to normal epithelial tissue expressing Ecad was determined. + Effect of keratinocytes. Compared with UTD-MSC co-culture, there was no significant difference in the survival rate of keratinocytes co-cultured with EcCAR-MSC ( Figure 22D These co-cultures also did not appear to significantly affect Ecad expression on keratinocyte target cells ( Figure 22EEvaluation of co-culture supernatants demonstrated that EcCAR-MSCs were antigen-specifically stimulated, as evidenced by upregulation of cytokines including IL-10, IL-4, and eotaxin ( Figure 22F ).

[0605] Secondly, the effect of EcCAR-MSC on Ecad + Compared with UTD-MSC co-culture, Ecad + Co-culture of bronchial cells with EcCAR-MSCs had no significant effect on the survival rate of bronchial cells ( Figure 22G Furthermore, in a GVHD xenograft model, immunofluorescence evaluation of lungs harvested 1 week after EcCAR-MSC treatment did not reveal trafficking of EcCAR-MSCs to the lungs ( Figures 22H-22I ).

[0606] A canine model was used to evaluate whether administration of human EcCAR-MSCs containing canine cross-reactive scFvs would cause any toxicity ( Figure 23A Healthy dogs received EcCAR-MSCs via intraperitoneal injection and were monitored for safety outcomes and tissue integrity for 28 days ( Figure 28A Toxicity was assessed by serial measurement of complete blood count, liver and kidney function. EcCAR-MSCs did not cause hematopoietic toxicity ( Figure 28B ), organ toxicity ( Figure 28C ), weight loss ( Figure 28D ) or tissue damage ( Figure 28E ), indicating that EcCAR-MSC administration has a good safety profile and is non-toxic.

[0607] Together, these results demonstrate that CAR-MSC generation provides antigen-specific immunosuppressive activation designed to safely enhance MSC therapy.

[0608] Example 5: Treatment of GVHD

[0609] Ecad-CAR-MSCs engineered to express one or more polypeptides selected from the group consisting of NFκB1 polypeptide, JUN polypeptide, RELB polypeptide, IRF1 polypeptide, TNFα polypeptide, IL-10 polypeptide, FGF-2 polypeptide, PD-1 polypeptide, G-CSF polypeptide, GM-CSF polypeptide, eotaxin polypeptide, Gal-9 polypeptide, PD-1 polypeptide, TIM-3 polypeptide, CXCR3 polypeptide, and CXCR4 polypeptide are administered to a human identified as having or at risk of having GVHD. CAR-MSCs engineered to express elevated levels of one or more polypeptides selected from the group consisting of NFκB1 polypeptide, JUN polypeptide, RELB polypeptide, IRF1 polypeptide, TNFα polypeptide, IL-10 polypeptide, FGF-2 polypeptide, PD-1 polypeptide, G-CSF polypeptide, GM-CSF polypeptide, eotaxin polypeptide, Gal-9 polypeptide, PD-1 polypeptide, TIM-3 polypeptide, CXCR3 polypeptide, and CXCR4 polypeptide are administered using intravenous injection. After administration of one or more CAR-MSCs engineered to express elevated levels of one or more polypeptides, the number of activated T cells in the human body is reduced, wherein the one or more polypeptides are selected from the group consisting of NFκB1 polypeptide, JUN polypeptide, RELB polypeptide, IRF1 polypeptide, TNFα polypeptide, IL-10 polypeptide, FGF-2 polypeptide, PD-1 polypeptide, G-CSF polypeptide, GM-CSF polypeptide, eotaxin polypeptide, Gal-9 polypeptide, PD-1 polypeptide, TIM-3 polypeptide, CXCR3 polypeptide, and CXCR4 polypeptide. After administration of one or more CAR-MSCs engineered to express elevated levels of one or more polypeptides, one or more symptoms of GVHD in the human body are alleviated, wherein the one or more polypeptides are selected from the group consisting of NFκB1 polypeptide, JUN polypeptide, RELB polypeptide, IRF1 polypeptide, TNFα polypeptide, IL-10 polypeptide, FGF-2 polypeptide, PD-1 polypeptide, G-CSF polypeptide, GM-CSF polypeptide, eotaxin polypeptide, Gal-9 polypeptide, PD-1 polypeptide, TIM-3 polypeptide, CXCR3 polypeptide, and CXCR4 polypeptide.

[0610] Example 6: Treatment of GVHD

[0611] MSCs engineered to express a CAR targeting an epithelial-specific antigen and engineered to express elevated levels of one or more polypeptides selected from the group consisting of NFκB1 polypeptide, JUN polypeptide, RELB polypeptide, IRF1 polypeptide, TNFα polypeptide, IL-10 polypeptide, FGF-2 polypeptide, PD-1 polypeptide, G-CSF polypeptide, GM-CSF polypeptide, eotaxin polypeptide, Gal-9 polypeptide, PD-1 polypeptide, TIM-3 polypeptide, CXCR3 polypeptide, and CXCR4 polypeptide are administered to a human identified as having or at risk of having GVHD. The MSCs are engineered to express a CAR targeting an epithelial-specific antigen and are engineered to express elevated levels of one or more polypeptides selected from the group consisting of NFκB1 polypeptides, JUN polypeptides, RELB polypeptides, IRF1 polypeptides, TNFα polypeptides, IL-10 polypeptides, FGF-2 polypeptides, PD-1 polypeptides, G-CSF polypeptides, GM-CSF polypeptides, eotaxin polypeptides, Gal-9 polypeptides, PD-1 polypeptides, TIM-3 polypeptides, CXCR3 polypeptides, and CXCR4 polypeptides. Following administration of one or more CARs engineered to express an epithelial-specific antigen and engineered to express elevated levels of one or more polypeptides, the number of activated T cells in the human body is reduced. One or more symptoms of GVHD are alleviated in a human following administration of one or more CARs engineered to express a target epithelial-specific antigen and engineered to express elevated levels of one or more polypeptides selected from the group consisting of an NFκB1 polypeptide, a JUN polypeptide, a RELB polypeptide, an IRF1 polypeptide, a TNFα polypeptide, an IL-10 polypeptide, an FGF-2 polypeptide, a PD-1 polypeptide, a G-CSF polypeptide, a GM-CSF polypeptide, an eotaxin polypeptide, a Gal-9 polypeptide, a PD-1 polypeptide, a TIM-3 polypeptide, a CXCR3 polypeptide, and a CXCR4 polypeptide.

[0612] Other Implementations

[0613] It should be understood that although the invention has been described in conjunction with specific embodiments, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and improvements are within the scope of the claims.

Claims

1. A mesenchymal stromal cell (MSC) comprising (1) an exogenous nucleic acid encoding a chimeric antigen receptor (CAR) targeting an epithelial-specific antigen, wherein the MSC expresses the CAR, and (2) elevated levels of a polypeptide selected from the group consisting of a nuclear factor kappa B subunit 1 (NFκB1) polypeptide, a Jun proto-oncogene (JUN) polypeptide, a transcription factor RelB (RELB) polypeptide, an interferon regulatory factor 1 (IRF1) polypeptide, a tumor necrosis factor (TNF) α polypeptide, an interleukin (IL)-10 polypeptide, a fibroblast growth factor (FGF)-2 polypeptide, a granulocyte colony-stimulating factor (G-CSF) polypeptide, a granulocyte-macrophage colony-stimulating factor (GM-CSF) polypeptide, an eosinophil chemoattractant polypeptide, a galectin 9 (Gal-9) polypeptide, a programmed cell death protein 1 (PD-1) polypeptide, a T cell immunoglobulin mucin-3 (TIM-3) polypeptide, a CXC chemokine receptor (CXCR) 3 polypeptide, and a CXCR4 polypeptide. The MSC according to claim 1 , wherein the MSC is a human MSC. The MSC according to any one of claims 1 to 2, wherein the MSC is an adipose-derived MSC. The MSC according to claim 1 , wherein the epithelial-specific antigen is E-cadherin (Ecad). 5 . The MSC according to claim 1 , wherein the polypeptide is selected from the group consisting of the NFκB1 polypeptide, the JUN polypeptide, the RELB polypeptide, and the IRF1 polypeptide. The MSC according to claim 5 , wherein the MSC comprises an exogenous nucleic acid encoding the NFκB1 polypeptide, wherein the MSC expresses the NFκB1 polypeptide. The MSC according to claim 5 , wherein the MSC comprises an exogenous nucleic acid encoding the JUN polypeptide, wherein the MSC expresses the JUN polypeptide.

8. The MSC according to claim 5, wherein the MSC comprises an exogenous nucleic acid encoding the RELB polypeptide, wherein the MSC expresses the RELB polypeptide. 9 . The MSC according to claim 5 , wherein the MSC comprises an exogenous nucleic acid encoding the IRF1 polypeptide, wherein the MSC expresses the IRF1 polypeptide.

10. The MSC according to any one of claims 1 to 4, wherein the polypeptide is selected from the group consisting of the CXCR3 polypeptide and the CXCR4 polypeptide. The MSC according to claim 10 , wherein the MSC comprises an exogenous nucleic acid encoding the CXCR3 polypeptide, wherein the MSC expresses the CXCR3 polypeptide. 12 . The MSC according to claim 10 , wherein the MSC comprises an exogenous nucleic acid encoding the CXCR4 polypeptide, wherein the MSC expresses the CXCR4 polypeptide.

13. The MSC according to any one of claims 1 to 4, wherein the polypeptide is selected from the group consisting of the PD-1 polypeptide, the Gal-9 polypeptide, and the TIM-3 polypeptide. The MSC according to claim 13 , wherein the MSC comprises an exogenous nucleic acid encoding the PD-1 polypeptide, wherein the MSC expresses the PD-1 polypeptide. 15 . The MSC according to claim 13 , wherein the MSC comprises an exogenous nucleic acid encoding the Gal-9 polypeptide, wherein the MSC expresses the Gal-9 polypeptide.

16. The MSC according to claim 13, wherein the MSC comprises an exogenous nucleic acid encoding the TIM-3 polypeptide, wherein the MSC expresses the TIM-3 polypeptide.

17. The MSC according to any one of claims 1 to 4, wherein the polypeptide is selected from the group consisting of the TNFα polypeptide, the IL-10 polypeptide, and the FGF-2 polypeptide.

18. The MSC of claim 17, wherein the MSC comprises an exogenous nucleic acid encoding the TNFα polypeptide, wherein the MSC expresses the TNFα polypeptide.

19. The MSC of claim 17, wherein the MSC comprises an exogenous nucleic acid encoding the IL-10 polypeptide, wherein the MSC expresses the IL-10 polypeptide.

20. The MSC of claim 17, wherein the MSC comprises an exogenous nucleic acid encoding the FGF-2 polypeptide, wherein the MSC expresses the FGF-2 polypeptide.

21. The MSC according to any one of claims 1-20, wherein the CAR comprises a heavy chain and a light chain, the heavy chain comprising the CDRs shown in SEQ ID NO: 1, and the light chain comprising the CDRs shown in SEQ ID NO:

2.

22. The MSC according to claim 21, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 1, and wherein the light chain comprises the amino acid sequence shown in SEQ ID NO:

2.

23. The MSC according to any one of claims 1-20, wherein the CAR comprises a heavy chain and a light chain, the heavy chain comprising the CDRs shown in SEQ ID NO: 3, and the light chain comprising the CDRs shown in SEQ ID NO:

4.

24. The MSC according to claim 23, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 3, and wherein the light chain comprises the amino acid sequence shown in SEQ ID NO:

4.

25. A composition comprising the MSCs according to any one of claims 1 to 24.

26. A method of treating a mammal suffering from graft-versus-host disease (GVHD), wherein the method comprises administering to the mammal the MSC of any one of claims 1-24 or the composition of claim 25.

27. The method of claim 26, wherein the mammal is a human.

28. The method of any one of claims 26-27, wherein the symptoms of GVHD are reduced by at least 10%.

29. The method of any one of claims 26-27, wherein the number of regulatory T cells (Tregs) in the mammal is increased by at least 10%.

30. A method of suppressing an immune response in a mammal, wherein the method comprises administering to the mammal the MSC according to any one of claims 1 to 24 or the composition according to claim 25.

31. The method of claim 30, wherein the mammal is a human.

32. The method of any one of claims 30-31, wherein the number of activated T cells in the mammal is reduced by at least 10%.

33. The method of any one of claims 30-31, wherein the number of Tregs in the mammal is increased by at least 10%.

34. A method for reducing the number of activated T cells in a mammal, wherein the method comprises administering to the mammal the MSC according to any one of claims 1 to 24 or the composition according to claim 25.

35. The method of claim 34, wherein the mammal is a human.

36. The method of any one of claims 34-35, wherein the number of activated T cells in the mammal is reduced by at least 10%.

37. The method of any one of claims 34-35, wherein the number of Tregs in the mammal is increased by at least 10%.

38. Use of the MSC according to any one of claims 1 to 24 or the composition according to claim 25 for suppressing an immune response in a mammal.

39. Use of the MSC according to any one of claims 1 to 24 or the composition according to claim 25 in the preparation of a medicament for suppressing an immune response in a mammal.

40. Use of the MSC according to any one of claims 1 to 24 or the composition according to claim 25 for suppressing an immune response in a mammal.

41. A mesenchymal stromal cell (MSC) comprising (1) an exogenous nucleic acid encoding a chimeric antigen receptor (CAR) targeting an epithelial-specific antigen, wherein the MSC expresses the CAR, and (2) an elevated level of a polypeptide selected from the group consisting of a nuclear factor kappa B subunit 1 (NFκB1) polypeptide, a Jun proto-oncogene (JUN) polypeptide, a transcription factor RelB (RELB) polypeptide, an interferon regulatory factor 1 (IRF1) polypeptide, a tumor necrosis factor (TNF) α polypeptide, a cytotoxic T lymphocyte-associated protein 4 (CTLA4), a toll-like receptor 3 (TLR3), and a toll-like receptor 4 (TLR4). (TLR4), toll-like receptor 9 (TLR9), tumor necrosis factor receptor 2 (TNFR2) polypeptide, interleukin (IL)-10 polypeptide, fibroblast growth factor (FGF)-2 polypeptide, granulocyte colony-stimulating factor (G-CSF) polypeptide, granulocyte macrophage colony-stimulating factor (GM-CSF) polypeptide, eotaxin polypeptide, galectin 9 (Gal-9) polypeptide, programmed cell death protein 1 (PD-1) polypeptide, T cell immunoglobulin mucin-3 (TIM-3) polypeptide, CXC chemokine receptor (CXCR) 3 polypeptide and CXCR4 polypeptide.

42. The MSC according to claim 41, wherein the MSC is a human MSC.

43. The MSC according to any one of claims 41-42, wherein the MSC is an adipose-derived MSC.

44. The MSC according to any one of claims 41-43, wherein the epithelial-specific antigen is E-cadherin (Ecad).

45. The MSC according to any one of claims 41-44, wherein the polypeptide is selected from the group consisting of the CTLA4 polypeptide, the TLR3 polypeptide, the TLR4 polypeptide, the TLR9 polypeptide, and the TNFR2 polypeptide.

46. The MSC of claim 45, wherein the MSC comprises an exogenous nucleic acid encoding the CTLA4 polypeptide, wherein the MSC expresses the CTLA4 polypeptide.

47. The MSC of claim 45, wherein the MSC comprises an exogenous nucleic acid encoding the TLR3 polypeptide, wherein the MSC expresses the TLR3 polypeptide.

48. The MSC of claim 45, wherein the MSC comprises an exogenous nucleic acid encoding the TLR4 polypeptide, wherein the MSC expresses the TLR4 polypeptide.

49. The MSC of claim 45, wherein the MSC comprises an exogenous nucleic acid encoding the TLR9 polypeptide, wherein the MSC expresses the TLR9 polypeptide.

50. The MSC of claim 45, wherein the polypeptide is selected from the group consisting of: the TNFR2 polypeptide and the TNFR2 polypeptide.

51. The MSC according to any one of claims 41-50, wherein the CAR comprises a heavy chain and a light chain, the heavy chain comprising the CDRs shown in SEQ ID NO: 1, and the light chain comprising the CDRs shown in SEQ ID NO:

2.

52. The MSC according to claim 51, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 1, and wherein the light chain comprises the amino acid sequence shown in SEQ ID NO:

2.

53. The MSC according to any one of claims 41-50, wherein the CAR comprises a heavy chain and a light chain, the heavy chain comprising the CDRs shown in SEQ ID NO: 3, and the light chain comprising the CDRs shown in SEQ ID NO:

4.

54. The MSC according to claim 53, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 3, and wherein the light chain comprises the amino acid sequence shown in SEQ ID NO:

4.

55. A composition comprising the MSCs of any one of claims 41-54.

56. A method of treating a mammal suffering from graft-versus-host disease (GVHD), wherein the method comprises administering to the mammal the MSCs of any one of claims 41-54 or the composition of claim 55.

57. The method of claim 56, wherein the mammal is a human.

58. The method of any one of claims 56-57, wherein the symptoms of GVHD are reduced by at least 10%.

59. The method of any one of claims 56-57, wherein the number of regulatory T cells (Tregs) in the mammal is increased by at least 10%.

60. A method of suppressing an immune response in a mammal, wherein the method comprises administering to the mammal the MSC of any one of claims 41-54 or the composition of claim 55.

61. The method of claim 60, wherein the mammal is a human.

62. The method of any one of claims 60-61, wherein the number of activated T cells in the mammal is reduced by at least 10%.

63. The method of any one of claims 60-61, wherein the number of Tregs in the mammal is increased by at least 10%.

64. A method for reducing the number of activated T cells in a mammal, wherein the method comprises administering to the mammal the MSC of any one of claims 41-54 or the composition of claim 55.

65. The method of claim 64, wherein the mammal is a human.

66. The method of any one of claims 64-65, wherein the number of activated T cells in the mammal is reduced by at least 10%.

67. The method of any one of claims 64-65, wherein the number of Tregs in the mammal is increased by at least 10%.

68. Use of the MSC according to any one of claims 41 to 54 or the composition according to claim 55 for suppressing an immune response in a mammal.

69. Use of the MSC according to any one of claims 41 to 54 or the composition according to claim 55 in the preparation of a medicament for suppressing an immune response in a mammal.

70. Use of the MSC according to any one of claims 41 to 54 or the composition according to claim 55 for suppressing an immune response in a mammal.

Images