Engineered mesenchymal stromal cells

Abstract

This document relates to methods and materials for using engineered mesenchymal stromal cells (MSCs) to treat 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 (e.g., graft versus host disease (GVHD))). For example, provided are CAR-MSCs (e.g., MSCs expressing a chimeric antigen receptor (CAR) capable of binding to a tissue-specific antigen), which have (e.g., have been engineered to have) high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides. Similarly, provided are methods of administering one or more CAR-MSCs described herein to 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 (e.g., GVHD)) to treat the mammal.

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 421,832, filed November 2, 2022. The disclosure of the prior application is considered part of the disclosure of this application and is incorporated by reference into the disclosure of this application.

[0002] Sequence Listing This application contains a Sequence Listing, which has been submitted electronically as an XML file entitled "07039-2165WO1.xml". The XML file is 160,000 bytes in size, created on October 31, 2023. The contents of the XML file are incorporated herein by reference in their entirety.

[0003] Technical Field This document relates to methods and materials for using engineered mesenchymal stromal cells (MSCs) in mammals (e.g., humans) in need of immunosuppression (e.g., humans suffering from or at risk of developing one or more autoimmune diseases, e.g., graft-versus-host disease (GVHD)). For example, this document provides CAR-MSCs (e.g., MSCs expressing a chimeric antigen receptor (CAR) capable of binding to a tissue-specific antigen) that have (e.g., have been engineered to have) high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or one or more distribution polypeptides. Such CAR-MSCs can exert an immunosuppressive effect (e.g., reduce or eliminate an immune response (e.g., hyperactivation of an immune response)) in a target tissue. This document also provides methods of administering one or more CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides to 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 (e.g., GVHD)) to treat the mammal. [Background technology]

[0004] Background information MSCs are attractive cell therapy candidates due to their trophic ability, which allows them to home to sites of inflammation (Shi et al., Nat. Rev. Nephrol., 14:493-507 (2018)) and to perform immunomodulatory functions supporting immune homeostasis (Uccelli et al., Nat. Rev. Immunol., 8:726-736 (2008)). However, reliable therapeutic efficacy has not been demonstrated in human clinical trials, and although MSCs have become the most studied form of experimental cell therapy worldwide, they have yet to yield a US FDA-approved treatment (Murata et al., Bone Marrow Transplant., 56:2355-2366 (2021)).

[0005] The therapeutic efficacy of MSCs in humans is limited by several factors, including suboptimal immunosuppression and poor homing to target sites immediately after administration. Strategies to address these shortcomings individually through cytokine modulation (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)) have been attempted, but have not yet succeeded in simultaneously enhancing both immunosuppression and homing capabilities. Summary of the Invention [Problem to be solved by the invention]

[0006] This document provides methods and materials for treating mammals (e.g., humans) in need of immunosuppression, e.g., humans suffering from or at risk of developing one or more autoimmune diseases (e.g., GVHD). [Means for solving the problem]

[0007] For example, one or more CAR-MSCs with (e.g., engineered to have) high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides can be administered to a mammal to induce an immunosuppressive response (e.g., reducing or eliminating an inflammatory immune response) in a target tissue in the mammal. In some aspects, one or more CAR-MSCs with (e.g., engineered to have) high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) suffering from (or at risk of developing) one or more autoimmune diseases (e.g., GVHD) to treat the mammal. As described herein, CAR-MSCs with high 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) alpha 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 exhibited both enhanced immunosuppression and enhanced homing capability at sites of inflammation. Also as described herein, CAR-MSCs with high 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 exhibited both enhanced immunosuppression and enhanced homing ability at sites of inflammation.

[0008] In general, one embodiment of this document features MSCs comprising: (1) an exogenous nucleic acid encoding a CAR targeting 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. 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 MSC may comprise an exogenous nucleic acid encoding an NFκB1 polypeptide, wherein the MSC expresses the NFκB1 polypeptide. The MSC may comprise an exogenous nucleic acid encoding a JUN polypeptide, wherein the MSC expresses the JUN polypeptide. The MSC may comprise an exogenous nucleic acid encoding a RELB polypeptide, wherein the MSC expresses the RELB polypeptide. The MSC may comprise 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 comprise an exogenous nucleic acid encoding a CXCR3 polypeptide, wherein the MSC expresses the CRCR3 polypeptide. The MSC may comprise an exogenous nucleic acid encoding a CXCR4 polypeptide, wherein the MSC expresses the CRCR4 polypeptide. The polypeptide may be a PD-1 polypeptide, a Gal-9 polypeptide, or a TIM-3 polypeptide. The MSC may comprise an exogenous nucleic acid encoding a PD-1 polypeptide, wherein the MSC expresses the PD-1 polypeptide. The MSCs may comprise an exogenous nucleic acid encoding a Gal-9 polypeptide, wherein the MSCs express the Gal-9 polypeptide.The MSC may comprise 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 comprising a CDR set forth in SEQ ID NO: 1 and a light chain comprising a CDR set forth in SEQ ID NO: 2. The heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 1, and the light chain may comprise the amino acid sequence set forth in SEQ ID NO: 2. The CAR may comprise a heavy chain comprising a CDR set forth in SEQ ID NO: 3 and a light chain comprising a CDR set forth in SEQ ID NO: 4. The heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 3, and the light chain may comprise the amino acid sequence set forth in SEQ ID NO: 4.

[0009] In another aspect, this document features a composition that includes MSCs, the MSCs comprising: (1) 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.

[0010] In another aspect, this document features a method of treating a mammal suffering from GVHD. The method may include, or may consist essentially of, administering to a mammal suffering from GVHD MSCs comprising: (1) an exogenous nucleic acid encoding a CAR targeting 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. The mammal may be a human. Symptoms of GVHD may be reduced by at least 10 percent. The number of Tregs in the mammal may be increased by at least 10 percent.

[0011] In another aspect, this document features a method of suppressing an immune response in a mammal. The method may include, or may consist essentially of, administering to the mammal MSCs comprising: (1) an exogenous nucleic acid encoding a CAR targeting 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. The mammal may be a human. The number of activated T cells in the mammal may be reduced by at least 10 percent. The number of Tregs in the mammal may be increased by at least 10 percent.

[0012] In another aspect, this document features a method for reducing the number of activated T cells in a mammal. The method may include, or may consist essentially of, administering to the mammal MSCs, the MSCs comprising: (1) an exogenous nucleic acid encoding a CAR targeting 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. The mammal may be a human. The number of activated T cells in the mammal may be reduced by at least 10 percent. The number of Tregs in the mammal may be increased by at least 10 percent.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein may be used to practice the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are merely illustrative and not intended to be limiting.

[0014] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0015] [Figure 1A]Figures 1A-1J show that MSCs were stably transduced to express CAR, and EcCAR-MSCs exhibited superior T cell suppression compared to untransduced (UTD) MSCs. Figure 1A shows a representative flow cytometry plot depicting CAR19 expression detected with goat anti-mouse IgG (y-axis) after transduction of MSCs with VSV-γ lentiviral particles in combination with increasing concentrations of protamine sulfate enhancer (50 μg / mL and 100 μg / mL). Data are representative of >3 independent experiments using 5 different biological MSC donors. [Figure 1B] Figures 1A-1J show that MSCs were stably transduced to express CARs, and EcCAR-MSCs exhibited superior T cell suppression compared to untransduced (UTD) MSCs. Figure 1B shows histograms of CAR expression in anti-Ecad CAR-MSCs (EcCAR-MSCs), anti-CD103 T cell integrin CAR-MSCs (CD103CAR-MSCs), and anti-CD19 CAR-MSCs (CD19CAR-MSCs) positive controls, as detected by flow cytometry using a goat anti-mouse IgG antibody. Data shown are mean values ​​± SD, representative of >6 independent experiments using five MSC donors. [Figure 1C] Figures 1A-1J show that MSCs were stably transduced to express CAR, and EcCAR-MSCs exhibited superior T cell suppression compared to untransduced (UTD) MSCs. Figure 1C shows a graph depicting CAR expression in EcCAR-MSCs compared to UTD-MSCs by flow cytometry 2 and 11 days after multiple cell passages following transduction. Data presented are mean ± standard deviation (SD) values ​​representative of three independent experiments using three MSC donors. Statistical analysis was performed by two-way analysis of variance (ANOVA) (****p ≤ 0.0001) (n = 3 per group). [Figure 1D]Figures 1A-1J show that MSCs were stably transduced to express CAR, and EcCAR-MSCs exhibited superior T cell suppression compared to untransduced (UTD) MSCs. Figure 1D is a histogram depicting >90% expression of Ecad-CAR (EcCAR) on MSCs across three different primary biological donors. Data are representative of >10 independent experiments using five different MSC donors. The x-axis represents the level of CAR detection with goat anti-mouse in the APC channel of the flow cytometry instrument. [Figure 1E] Figures 1A-1J show that MSCs were stably transduced to express CAR, and EcCAR-MSCs exhibited superior T cell suppression compared with untransduced (UTD) MSCs. Figure 1E shows a graph of flow cytometry-based quantification of the absolute number of CD3+ cells after coculture of activated T cells with EcCAR-MSCs, UTD-MSCs, or no MSCs at MSC:T cell ratios of 1:5 and 1:10. Data presented are mean ± SD from three MSC donors. Statistical analysis was performed by two-way ANOVA (*p ≤ 0.05, ****p < 0.0001) (n = 3 per group). [Figure 1F] Figures 1A-1J show that MSCs were stably transduced to express CAR, and EcCAR-MSCs exhibited superior T cell suppression compared with untransduced (UTD) MSCs. Figure 1F shows a graph of flow cytometry-based quantification of the absolute number of CD3-positive cells after co-culture of activated T cells with EcCAR-MSCs, UTD-MSCs, or no MSCs in the presence of medium alone or 250 ng / mL soluble Ecad. Data shown are the mean ± SD of a representative experiment using five MSC donors. Statistical analysis was performed by two-way ANOVA. (Not significant (ns) p ≥ 0.05, **p ≤ 0.01) (n = 2 per group). [Figure 1G]Figures 1A-1J show that MSCs were stably transduced to express CAR, and EcCAR-MSCs exhibited superior T cell suppression compared with untransduced (UTD) MSCs. Figure 1G shows a graph quantified by flow cytometry of the absolute number of CD3+ cells after coculture of activated T cells with EcCAR-MSCs, UTD-MSCs, or no MSCs in the presence of the irradiated Ecad-positive cell line MCF-7. Antigen-specific activation enhances T cell suppression. Data shown are the mean ± SD of a representative experiment using three different biological MSC donors. Statistical analysis was performed by two-way ANOVA (n = 2 per group). (ns = p ≥ 0.05, *p ≤ 0.05). [Figure 1H] Figures 1A-1J show that MSCs were stably transduced to express CAR, and EcCAR-MSCs exhibited superior T cell suppression compared to untransduced (UTD) MSCs. Figure 1H shows a graph depicting the stem cell immunological phenotype of UTD-MSCs and EcCAR-MSCs, measured by flow cytometry after staining MSCs with CD105, CD90, CD73, CD34, CD45, and CD14. Data presented are the mean ± SD of four independent experiments from four biological MSC donors. Statistical analysis was performed by two-way ANOVA (ns = p ≥ 0.05) (n = 3 per group). [Figure 1I]Figures 1A-1J show that MSCs were stably transduced to express CAR, and EcCAR-MSCs exhibited superior T cell suppression compared with untransduced (UTD) MSCs. Figures 1I-1J contain graphs of elevated (Figure 1I) and decreased (Figure 1J) pathways in bulk RNA-seq analysis of EcCAR-MSCs compared with UTD-MSCs by CellMarker Augmented gene set enrichment analysis. Significant genes were selected from genes exhibiting a log fold change of >1 or <-1 and an adjusted p-value of <0.05 in expression change analysis of three biological replicates per group. The calculated -log10 p-values ​​are shown on the x-axis, indicating that all pathways were statistically significantly enriched (-log10 (p < 0.05)). Data represent analyses of three different biological MSC donors per expression change analysis group. [Figure 1J] Figures 1A-1J show that MSCs were stably transduced to express CAR, and EcCAR-MSCs exhibited superior T cell suppression compared with untransduced (UTD) MSCs. Figures 1I-1J contain graphs of pathways elevated (Figure 1I) and decreased (Figure 1J) in bulk RNA-seq analysis of EcCAR-MSCs compared with UTD-MSCs in CellMarker Augmented gene set enrichment analysis. Significant genes were selected from genes exhibiting a log fold change of >1 or <-1 and an adjusted p-value of <0.05 in expression change analysis of three biological replicates per group. The calculated -log10 p-values ​​are shown on the x-axis, indicating that all pathways were statistically significantly enriched (-log10 (p < 0.05)). Data represent analyses of three different biological MSC donors per expression change analysis group. [Figure 2A]Figures 2A-2H show that EcCAR-MSCs suppress T cell activity in tumor and GVHD models. Figure 2A is a schematic diagram of an example method for evaluating the effect of EcCAR-MSCs on a Nalm6 xenograft tumor model. Luciferase-positive, CD19-positive Nalm6 cells (1 × 10 cells intravenously (iv)) were implanted into immunodeficient NOD-SCID-γ- / - (NSG) mice. Bioluminescence imaging was performed 5 days later to confirm implantation. All mice were then treated with CD19-targeted CAR T (CART19) cells (1 × 10 cells intravenously) and irradiated Ecad-positive cell line MCF-7. Additionally, mice were randomized and treated with UTD-MSCs or EcCAR-MSCs (1 × 10 cells intraperitoneally (ip)). Mice were then examined for bioluminescence imaging and survival every two weeks. Tumor figures (Figures 2A-2C) are representative of two independent experiments with two different tumor models and two different biological MSC donors. GVHD figures (Figures 2D-2H) are representative of three independent experiments with three different biological PBMC donors and three different biological MSC donors, performed in both male and female NSG mice. [Figure 2B] Figures 2A-2H show that EcCAR-MSCs suppress T cell activity in tumor and GVHD models. Figure 2B shows graphs of tumor flux after CART19 injection in the Nalm6 model, comparing EcCAR-MSCs, UTD-MSCs, and no treatment. Data are shown as mean ± standard error of mean (SEM) values ​​(n = 3-4 mice per group) with statistical analysis by two-way ANOVA (**p ≤ 0.01, ***p ≤ 0.001, ns = p ≥ 0.05). Tumor figures (Figures 2A-2C) are representative of two independent experiments with two different tumor models and two different biological MSC donors. GVHD figures (Figures 2D-2H) are representative of three independent experiments with three different biological PBMC donors and three different biological MSC donors, performed in both male and female NSG mice. [Figure 2C]Figures 2A-2H show that EcCAR-MSCs suppress T cell activity in tumor and GVHD models. Figure 2C shows the survival results of EcCAR-MSCs compared with UTD-MSCs and MSC-free controls. Statistical analysis was performed by Kaplan-Meier simple survival analysis. (*p ≤ 0.05, ns = p ≥ 0.05) (n = 3-4 mice per group). The tumor figures (Figures 2A-2C) are representative of two independent experiments with two different tumor models and two different biological MSC donors. The GVHD figures (Figures 2D-2H) are representative of three independent experiments with three different biological PBMC donors and three different biological MSC donors, performed on both male and female NSG mice. [Figure 2D] Figures 2A-2H show that EcCAR-MSCs suppress T cell activity in tumor and GVHD models. Figure 2D is a schematic diagram of an example method for evaluating the effect of EcCAR-MSCs in a mouse xenograft GVHD model. NSG mice were injected with human PBMCs (25-30 × 10 cells intravenously) along with either EcCAR-MSCs or UTD-MSCs (1 × 10 cells intraperitoneally on days 0, 14, and 28). Mice were then examined for weight loss, the development of clinical signs of GVHD (weight loss, diarrhea, posture, activity, coat, and skin integrity scores), and survival. Tumor figures (Figures 2A-2C) are representative of two independent experiments using two different tumor models and two different biological MSC donors. The GVHD figures (Figures 2D-2H) are representative of three independent experiments with three different biological PBMC donors and three different biological MSC donors, performed in both male and female NSG mice. [Figure 2E]Figures 2A-2H show that EcCAR-MSCs suppress T cell activity in tumor and GVHD models. Figure 2E shows a graph of the percent body weight change from baseline in GVHD xenografts after treatment with UTD-MSCs, EcCAR-MSCs, or no treatment. Data are shown as mean ± SD (n = 5 mice per group) with statistical analysis performed by two-way ANOVA (****p ≤ 0.0001, ns = p ≥ 0.05). The tumor figures (Figures 2A-2C) are representative of two independent experiments with two different tumor models and two different biological MSC donors. The GVHD figures (Figures 2D-2H) are representative of three independent experiments with three different biological PBMC donors and three different biological MSC donors, performed in both male and female NSG mice. [Figure 2F] Figures 2A-2H show that EcCAR-MSCs suppress T cell activity in tumor and GVHD models. Figure 2F includes representative mouse photographs (right) and graphs of GVHD clinical score calculations (left) 50 days after treatment with EcCAR-MSCs, UTD-MSCs, or no MSCs. Data are shown as mean ± SD with statistical analysis performed by two-way ANOVA (**p ≤ 0.01, ns = p ≥ 0.05) (n = 5 mice per group). Tumor figures (Figures 2A-2C) are representative of two independent experiments with two different tumor models and two different biological MSC donors. GVHD figures (Figures 2D-2H) are representative of three independent experiments with three different biological PBMC donors and three different biological MSC donors, performed on both male and female NSG mice. [Figure 2G]Figures 2A-2H show that EcCAR-MSCs suppress T cell activity in tumor and GVHD models. Figure 2G is a graph of mouse peripheral blood evaluation comparing the absolute number of human CD3-positive T cells between EcCAR-MSC and UTD-MSC treatment groups by flow cytometry 2 weeks after the first injection of MSCs. Data presented are mean ± SD with statistical analysis performed by unpaired t-test (**p≦0.01) (n = 5 mice per group). Figures for tumors (Figures 2A-2C) are representative of two independent experiments with two different tumor models and two different biological MSC donors. Figures for GVHD (Figures 2D-2H) are representative of three independent experiments with three different biological PBMC donors and three different biological MSC donors, performed in both male and female NSG mice. [Figure 2H] Figures 2A-2H show that EcCAR-MSCs suppress T cell activity in tumor and GVHD models. Figure 2H is a graph of survival results after treatment with EcCAR-MSCs compared with UTD-MSCs and MSC-free controls. Statistical analysis was performed by Kaplan-Meier simple survival analysis (**p ≦ 0.01, ns = p ≧ 0.05) (n ≧ 5 mice per group). Tumor figures (Figures 2A-2C) are representative of two independent experiments with two different tumor models and two different biological MSC donors. GVHD figures (Figures 2D-2H) are representative of three independent experiments with three different biological PBMC donors and three different biological MSC donors, performed on both male and female NSG mice. [Figure 3A]Figures 3A-3E show RNA-seq analysis revealing that antigen-specific activation of EcCAR-MSCs leads to enrichment of immunosuppressive pathways. Figure 3A is a heatmap depicting the unique gene expression profile of EcCAR-MSC + Ecad samples, highlighting the functional impact of CAR antigen-specific stimulation through hierarchical clustering of unstimulated EcCAR-MSC, stimulated UTD-MSC + Ecad, and unstimulated UTD-MSC samples. Normalized gene fold counts are shown for all samples (padj ≤ 0.05) (n = 3 MSC donors per condition). [Figure 3B] Figures 3A-3E show RNA-seq analysis revealing that antigen-specific activation of EcCAR-MSCs leads to enrichment of immunosuppressive pathways. Figure 3B shows a PCA depicting the RNA-seq transcript profiles of six samples from three biological MSC replicates, demonstrating that unstimulated and stimulated UTD-MSCs form clusters only by donor. [Figure 3C] Figures 3A-3E show RNA-seq analysis revealing that antigen-specific activation of EcCAR-MSCs leads to enrichment of immunosuppressive pathways. Figure 3C shows PCA of unstimulated and stimulated EcCAR-MSCs, revealing that the stimulated group forms a unique cluster, indicating CAR-based functional activation. [Figure 3D] Figures 3A-3E show RNA-seq analysis revealing that antigen-specific activation of EcCAR-MSCs leads to enrichment of immunosuppressive pathways. Figure 3D uses Ingenuity Pathway Analysis (IPA) to reveal elevated canonical pathways in stimulated vs. unstimulated EcCAR-MSCs. Dotted lines indicate significantly enriched pathways (p ≤ 0.05) (n = 3 donors per group). [Figure 3E]Figures 3A-3E show RNA-seq analysis revealing that antigen-specific activation of EcCAR-MSCs leads to enrichment of immunosuppressive pathways. Figure 3E includes graphs comparing normalized gene counts between unstimulated UTD-MSCs, stimulated UTD-MSCs, unstimulated EcCAR-MSCs, and stimulated EcCAR-MSCs, showing activation of CAR (CD28)-associated transcription factor genes NFκB1, JUN, RELB, and IRF1 in stimulated EcCAR-MSCs. Data presented are mean ± SD for normalized gene counts between groups. Statistical analysis was performed by one-way ANOVA (**p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001) (n = 3 donors per group). [Figure 4A] Figures 4A-4I show that EcCAR-MSCs support enhanced immunosuppression through increased cytokine secretion, T cell mediation, and elevated inhibitory surface markers. Figure 4A includes graphs comparing serum cytokine levels (pg / ml) of IL-10 polypeptide, TNFα polypeptide, G-CSF polypeptide, eotaxin polypeptide, and FGF-2 polypeptide in EcCAR-MSC-treated mice with UTD-MSC and no treatment xenografts by combined analysis 17 days after administration. Data presented are mean ± SEM (n = 4-6 samples per group) with statistical analysis performed by standard one-way ANOVA (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). [Figure 4B]Figures 4A-4I show that EcCAR-MSCs support enhanced immunosuppression through increased cytokine secretion, T cell-T cell interactions, and elevated inhibitory surface markers. Figure 4B shows graphs of human T cell subsets in EcCAR-MSC-treated mice vs. UTD-MSC-treated mice from GVHD xenografts. Quantitative results of human CD4+ and CD8+ T cell suppression are depicted by absolute cell counts in mouse peripheral blood 2 weeks after the first MSC infusion by flow cytometry. Data presented are mean ± SD for absolute numbers of cell subsets. Statistical analysis was performed by two-way ANOVA (*p ≤ 0.05, ***p ≤ 0.001) (n = 5 per group). [Figure 4C] Figures 4A-4I show that EcCAR-MSCs support enhanced immunosuppression through increased cytokine secretion, T cell-T cell interactions, and elevated inhibitory surface markers. Figure 4C is a graph comparing the ratio of CD4-positive cells to human CD8-positive T cells in mice treated with EcCAR-MSCs with UTD-MSCs in GVHD xenografts by flow cytometry. Data presented are mean ± SD for percentages of cell subsets. Statistical analysis was performed by two-way ANOVA (**p ≦ 0.01) (n = 5 per group). [Figure 4D] Figures 4A-4I show that EcCAR-MSCs support enhanced immunosuppression through increased cytokine secretion, T cell-cell interactions, and elevated inhibitory surface markers. Figure 4D is a graph showing the percent weight change from baseline 0 to day 31 in GVHD mice, demonstrating that weight loss was significantly attenuated and GVHD was prevented only in EcCAR-MSC-treated mice. Data shown are mean ± SEM for percent weight change. Statistical analysis was performed by two-way ANOVA (***p ≦ 0.001) (n = 5 per group). [Figure 4E]Figures 4A-4I show that EcCAR-MSCs support enhanced immunosuppression through increased cytokine secretion, T cell-cell interactions, and elevated inhibitory surface markers. Figure 4E shows graphs of human CD4+, CD25+, and CD127-negative Treg-like cell subsets 4 weeks after PBMC administration and MSC treatment. Flow cytometry analysis revealed a significant increase in the percentage of Tregs in EcCAR-MSC-treated mice compared with UTD-MSCs. Data presented are mean ± SD for percentages of cell subsets. Statistical analysis was performed by unpaired t-test (***p ≤ 0.001) (n = 4 per group). [Figure 4F] Figures 4A-4I show that EcCAR-MSCs support enhanced immunosuppression through increased cytokine secretion, T cell-cell interactions, and elevated inhibitory surface markers. Figure 4F shows T-distribution stochastic neighbor embedding (tSNE) plots of surface marker expression by flow cytometry time-of-flight (CyTOF) for Ecad-stimulated and unstimulated EcCAR-MSCs and UTD-MSCs. Ecad stimulation was induced by irradiating the Ecad-positive cell line MCF-7 for 24 hours prior to CyTOF analysis. tSNE plots of surface marker profiles across samples by unsupervised clustering revealed the presence of four major cell populations: Ecad-positive cell lines, UTD-MSCs, EcCAR-MSCs, and Ecad-stimulated EcCAR-MSCs. [Figure 4G] Figures 4A-4I show that EcCAR-MSCs support enhanced immunosuppression through increased cytokine secretion, T cell-cell interactions, and elevated inhibitory surface markers. Figure 4G shows a heat map depicting cluster surface characteristics by CXCR3 and PD1 markers, characterizing stimulated EcCAR-MSC population clusters as unique. [Figure 4H]Figures 4A-4I show that EcCAR-MSCs support enhanced immunosuppression through increased cytokine secretion, T cell-T cell interactions, and elevated inhibitory surface markers. Figure 4H shows graphs analyzing the percent expression of inhibitory receptor surface markers PD-1 and Gal-9 by flow cytometry for UTD-MSCs and EcCAR-MSCs stimulated with soluble Ecad and cocultured with PBMCs for 5 days before surface marker assessment. Data presented in Figures 4H-4I are mean ± SD for percent surface expression representative of three MSC donors. Statistical analysis was performed by two-way ANOVA (**p ≤ 0.01, ****p ≤ 0.0001) (n = 3 per group). [Figure 4I] Figures 4A-4I show that EcCAR-MSCs support enhanced immunosuppression through increased cytokine secretion, T cell-cell interactions, and elevated inhibitory surface markers. Figure 4I shows graphs analyzing the percent expression of the infiltrating chemokine surface markers CXCR3 and CXCR4 by flow cytometry for UTD-MSCs and EcCAR-MSCs stimulated with soluble Ecad and cocultured with PBMCs for 5 days before surface marker assessment. Data presented in Figures 4H-4I are mean ± SD for percent surface expression representative of three MSC donors. Statistical analysis was performed by two-way ANOVA (**p ≤ 0.01, ****p ≤ 0.0001) (n = 3 per group). [Figure 5A] Figures 5A-5G show the homing and safety characteristics of EcCAR-MSCs in in vivo dog and mouse models. Figure 5A is a schematic diagram of an example method for analyzing the production, safety, and homing of CAR-MSCs in an in vivo healthy dog ​​model. [Figure 5B]Figures 5A-5G show the homing and safety characteristics of EcCAR-MSCs in in vivo dog and mouse models. Figure 5B shows examples of immunohistochemical (IHC) analysis of transverse colon tissue stained with Ecad 3 days after EcCAR-MSC administration, demonstrating that human EcCAR-MSCs home to canine Ecad-positive tissues. Human CD105-positive antibodies (top) and canine Ecad-positive antibodies (bottom) show colocalization in IHC analysis of canine colon tissue under various magnifications. [Figure 5C] Figures 5A-5G show the homing and safety characteristics of EcCAR-MSCs in in vivo dog and mouse models. Figure 5C includes graphs of whole blood count levels after intraperitoneal administration of EcCAR-MSCs. From left to right, the graphs include blood level tests of white blood cells (WBCs), monocytes, lymphocytes, neutrophils, and platelets compared to baseline values ​​for short-term (3 days) and long-term (28 days) in vivo EcCAR-MSC injections. Data presented are mean ± SD for blood compositions, with statistical analysis performed by two-way ANOVA (ns = p ≧ 0.05 between groups) (n = 3 per experimental group). [Figure 5D] Figures 5A-5G show the homing and safety characteristics of EcCAR-MSCs in in vivo dog and mouse models. Figure 5D, from left to right, includes graphs of total protein, BUN, creatinine, albumin, and alkaline phosphatase levels tested short-term (3 days) and long-term (28 days) after in vivo EcCAR-MSC infusion. Data presented are mean ± SD for blood composition, with statistical analysis performed by two-way ANOVA (ns = p ≧ 0.05). [Figure 5E]Figures 5A-5G show the homing and safety characteristics of EcCAR-MSCs in in vivo dog and mouse models. Figure 5E is a schematic diagram of an example method for determining the growth kinetics and persistence of CAR-MSCs in vivo using a luciferase-positive MSC xenograft model. NSG mice previously injected with human PBMCs were injected with luciferase-expressing UTD-MSCs or CAR-MSCs in the presence or absence of irradiated Ecad-positive MCF7 cells as a CAR-stimulating agent. Mice were then photographed daily for bioluminescence imaging. [Figure 5F] Figures 5A-5G show the homing and safety characteristics of EcCAR-MSCs in in vivo and mouse models. Figure 5F is a graph of bioluminescence intensity as an index of MSC proliferation and elimination kinetics after administration of EcCAR-MSCs or UTD-MSCs in the presence or absence of Ecad stimulation. [Figure 5G] Figures 5A-5G show the homing and safety characteristics of EcCAR-MSCs in in vivo and mouse models. Figure 5G shows exemplary images showing the proliferation and disappearance of luciferase-positive MSCs in mice 3 to 24 days after administration. Data presented are mean ± standard deviation (sd) with statistical analysis performed by two-way ANOVA (ns = p ≧ 0.05) (n = 5 per group). [Figure 6] Figure 6 shows canine / human cross-reactivity of luciferase canine Ecad-positive MCKD cell lines co-cultured with human vs. mouse vs. canine-tropic Ecad CAR T cells. Bioluminescence imaging was performed 24 hours after co-culture. UTD = untransduced. [Figure 7] FIG. 7 depicts a schematic diagram of an example EcCAR-MSC construct, where hmcECAD.6 represents the human, mouse, and canine cross-reactive Ecad antigen-binding domain. [Figure 8A]Figure 8A is a schematic diagram of an example method for evaluating the effect of EcCAR-MSCs in a JeKo1 cell xenograft tumor model. JeKo1 cells (1 × 10 cells intravenously) were co-injected into NSG mice with either EcCar-MSCs or UTD-MSCs (1 × 10 cells intraperitoneally on days 0, 14, and 28). Mice were then examined for weight loss, the development of clinical signs of GVHD (weight loss, diarrhea, posture, activity, coat, and skin integrity scores), and survival. [Figure 8B] FIG. 8B is a graph of tumor luminescence intensity (photons / second) in NSG mice injected with JeKo1 cells. [Figure 9A] Figure 9A is a graph of gene expression changes in unstimulated EcCAR-MSCs vs. UTD-MSCs, Ecad-stimulated vs. unstimulated EcCAR-MSCs, and Ecad-stimulated vs. unstimulated UTD-MSCs. Data represent the counts of elevated and decreased genes in the comparison, with an adjusted p-value < 0.01 and ± 1-log fold change. Transcriptional changes induced by Ecad stimulation of CAR-MSCs included 2,362 significant genes compared to EcCAR-MSCs alone and 3,032 significant genes compared to UTD-MSCs stimulated with Ecad. Transcriptional changes induced by CAR transduction included 606 significant genes. Transcriptional changes induced by Ecad stimulation included 206 significant genes. [Figure 9B] Figure 9B shows that Ingenuity Pathway Analysis (IPA) revealed elevated canonical pathway activity in unstimulated EcCAR-MSCs vs. UTD-MSCs. Dotted lines represent enriched pathways (p ≦ 0.05) (n = 3 MSC donors per group). [Figure 10]Figure 10 includes graphs showing, from left to right, MDC polypeptide, GRO polypeptide, GM-CSF polypeptide, MCP-3 polypeptide, and Flt-3 polypeptide in EcCAR-MSC-treated mice compared with xenografts of UTD-MSC and no treatment in a combined analysis 17 days after administration. Data presented are mean ± SEM with statistical analysis performed by standard one-way ANOVA (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001) (n = 4-6 samples per group). [Figure 11] FIG. 11 is a heat map of the clustering of surface marker expression in the cell populations that make up the cell clusters identified from CyTOF in FIG. 4F. [Figure 12A] Figure 12A shows the weight change in healthy dogs after EcCAR-MSC administration compared to controls. No significant difference in weight change was found between groups. Data are presented as mean ± SD for % weight change from baseline, with statistical analysis performed by standard one-way ANOVA (ns = p ≧ 0.05). [Figure 12B] FIG. 12B contains data representing H&E stained canine transverse colon tissue sections at 20× and 40× magnification from treatment with human EcCAR-MSCs (left) or control (right) after 28 days of administration. [Figure 13] Figure 13 shows histograms depicting CAR expression on MSCs, including histograms of various intracellular signaling domains (including 4-1BB, CD28, TLR, and IFNγ signaling domains) detected by flow cytometry using the CAR+ FMC63 antibody. Data presented are mean values ​​± SD for a representative set of >6 independent experiments using 5 MSC donors. [Figure 14] Figure 14 includes a schematic diagram of an example mechanism for enhancing immunosuppression and distribution of CAR-MSCs. [Figure 15A]Figures 15A-15F show that MSCs are stably transduced to express CAR and maintain stem cell-like properties after transduction and stimulation. Figure 15A is a representative flow cytometry plot depicting CAR19 expression after lentiviral transduction of MSCs with increasing concentrations of protamine sulfate enhancer (50 μg / mL and 100 μg / mL as shown) compared to untransduced (UTD) MSCs. Data are representative using six different biological MSC donors. [Figure 15B] Figures 15A-15F show that MSCs were stably transduced to express CARs and maintained stem cell-like properties after transduction and stimulation. Figure 15B shows a histogram depicting CAR expression of the anti-Ecad CAR and anti-CD19 CAR positive control as detected by flow cytometry. Data presented are the mean ± standard deviation (sd) of six different biological MSC donors. [Figure 15C] Figures 15A-15F show that MSCs are stably transduced to express CAR and maintain stem cell-like properties after transduction and stimulation. Figure 15C is a graph showing CAR expression in EcCAR-MSCs compared to UTD-MSCs by flow cytometry 2 and 11 days after transduction and ex vivo expansion. Data shown are mean ± SD representative values ​​from three independent experiments using three MSC donors. Statistical analysis was performed by two-way ANOVA (****p≦0.0001). [Figure 15D] Figures 15A-15F show that MSCs are stably transduced to express CAR and maintain stem cell-like properties after transduction and stimulation. Figure 15D is a histogram showing EcCAR expression on MSCs across three different primary biological MSC donors. Data are representative of >10 independent experiments using six different MSC donors. [Figure 15E]Figures 15A-15F show that MSCs were stably transduced to express CAR and maintained stem cell-like properties after transduction and stimulation. Figure 15E is a graph depicting the stem phenotype of EcCAR-MSCs and UTD-MSCs, as measured by flow cytometry after staining MSCs with CD105, CD90, CD73, CD34, CD45, and CD14 surface markers. Data presented are the mean ± SD of three independent experiments from four biological MSC donors. Statistical analysis was performed by two-way ANOVA (ns = p≧0.05). [Figure 15F] Figures 15A-15F show that MSCs were stably transduced to express CAR and maintained stem cell-like properties after transduction and stimulation. Figure 15F depicts two graphs of bulk RNA-seq analysis showing increased (left panel) and decreased (right panel) phenotypic enrichment in EcCAR-MSCs compared to UTD-MSCs by CellMarker Augmented gene set enrichment analysis. Significant genes were selected from expression change analysis of three individual MSC donors with a log fold change of ≥ 1 or ≤ -1 and an adjusted p-value (padj) of ≤ 0.05. The dotted line on the x-axis indicates that all pathways were statistically significantly enriched (-log(p ≤ 0.05)). [Figure 16A] Figures 16A-16G show that EcCAR-MSCs demonstrate superior antigen-specific suppression of primary T cells in vitro. Figure 16A is a histogram showing the establishment of Ecad-positive NALM6 and its counterpart, Ecad-negative NALM6, making cell-based Ecad stimulation suitable for use in in vitro suppression analysis and in in vivo tumor models. [Figure 16B]Figures 16A-16G show that EcCAR-MSCs demonstrate superior antigen-specific suppression of primary T cells in vitro. Figure 16B is a graph of the absolute number of CD3-positive T cells after co-culture with EcCAR-MSCs or UTD-MSCs in the presence of increasing concentrations of soluble Ecad (0 ng / mL, 250 ng / mL, and 1000 ng / mL). Data presented are the mean ± SD for three independent experiments using three individual MSC donors. Statistical analysis was performed by two-way ANOVA (***p≦0.001). [Figure 16C] Figures 16A-16G show that EcCAR-MSCs demonstrate superior antigen-specific suppression of primary T cells in vitro. Figure 16C shows a graph of the stemness phenotype of EcCAR-MSCs measured by flow cytometry after staining MSCs with CD73, CD90, CD105, CD14, CD34, and CD45 under increasing soluble Ecad stimulation (0, 250, or 1000 ng / mL). Data presented are mean values ​​± SD from four different MSC donors. Statistical analysis was performed using two-way ANOVA (ns = p≧0.05). [Figure 16D] Figures 16A-16G show that EcCAR-MSCs demonstrate superior antigen-specific suppression of primary T cells in vitro. Figures 16D, 16E, and 16F are graphs of the absolute number of CD3-positive T cells after co-culture of MSCs from three different donors as EcCAR-MSCs or UTD-MSCs derived from Ecad-positive or Ecad-negative cells with or without cell-based stimulation. Data presented are mean ± SD. Statistical analysis was performed using two-way ANOVA (n = 2-3 replicates per donor) (*p ≤ 0.05, **p ≤ 0.01). [Figure 16E]Figures 16A-16G show that EcCAR-MSCs demonstrate superior antigen-specific suppression of primary T cells in vitro. Figures 16D, 16E, and 16F are graphs of the absolute number of CD3-positive T cells after co-culture of MSCs from three different donors as EcCAR-MSCs or UTD-MSCs derived from Ecad-positive or Ecad-negative cells with or without cell-based stimulation. Data presented are mean ± SD. Statistical analysis was performed using two-way ANOVA (n = 2-3 replicates per donor) (*p ≤ 0.05, **p ≤ 0.01). [Figure 16F] Figures 16A-16G show that EcCAR-MSCs demonstrate superior antigen-specific suppression of primary T cells in vitro. Figures 16D, 16E, and 16F are graphs of the absolute number of CD3-positive T cells after co-culture of MSCs from three different donors as EcCAR-MSCs or UTD-MSCs derived from Ecad-positive or Ecad-negative cells with or without cell-based stimulation. Data presented are mean ± SD. Statistical analysis was performed using two-way ANOVA (n = 2-3 replicates per donor) (*p ≤ 0.05, **p ≤ 0.01). [Figure 16G] Figures 16A-16G show that EcCAR-MSCs demonstrate superior antigen-specific suppression of primary T cells in vitro. Figure 16G shows a graph of the stemness phenotype of EcCAR-MSCs or UTD-MSCs, measured by flow cytometry after staining MSCs with CD73, CD90, CD105, CD14, CD34, and CD45 following coculture alone or with Ecad-positive or Ecad-negative cell-based stimuli. Data presented are the mean ± SD for results from three (n = 3) MSC donors. Statistical analysis was performed using two-way ANOVA (ns = p > 0.05). [Figure 17A]Figures 17A-17G show that EcCAR-MSCs induce potent immunosuppression in tumor and GvHD xenograft models. Figure 17A is a schematic diagram of an example method for assessing the effect on an Ecad-positive Nalm6 xenograft tumor model. Immunodeficient NOD-SCID-γ- / - (NSG) mice were implanted with luciferase (luc)-positive Ecad-positive or luc-positive Ecad-negative NALM6 cells (1 × 10 cells intravenously) and treated with CD19-targeting CAR T (CART19) (1 × 10 cells intravenously). Mice were then randomized and received untransduced MSCs (UTD-MSCs) or EcCAR-MSCs (1 × 10 cells intraperitoneally). Tumor burden was assessed by bioluminescence imaging (BLI), and survival and health were assessed. [Figure 17B] Figures 17A-17G show that EcCAR-MSCs induce superior immunosuppression in tumor and GvHD xenograft models. Figure 17B is a graph of the luminescence intensity of Ecad-positive NALM6 tumors after CART19 injection in a Nalm6 model, comparing UTD-MSCs and EcCAR-MSCs. Data presented are median ± standard error of the mean (SEM) with statistical analysis using two-way ANOVA (***p ≦ 0.001; n = 4-5 mice per group, two independent experiments). [Figure 17C] Figures 17A-17G show that EcCAR-MSCs induce superior immunosuppression in tumor and GvHD xenograft models. Figure 17C is a graph of the luminescence intensity of Ecad-negative NALM6 tumors after CART19 injection in a Nalm6 model, comparing UTD-MSCs and EcCAR-MSCs. Data presented are median ± SEM with statistical analysis using two-way ANOVA (ns = p≧0.05; n = 4-5 mice per group, two independent experiments). [Figure 17D]Figures 17A-17G show that EcCAR-MSCs induce potent immunosuppression in tumor and GvHD xenograft models. Figure 17D is a schematic diagram of an example method for evaluating the effect of EcCAR-MSCs in a human PBMC-induced GvHD xenograft model. NSG mice were randomized and injected with human peripheral blood mononuclear cells (PBMCs) (25-30 × 10 cells intravenously) and treated with EcCAR-MSCs or UTD-MSCs (1 × 10 cells intraperitoneally). Mice were examined for weight loss, the development of clinical GvHD symptoms (scores based on weight loss, diarrhea, posture, activity, coat, and skin integrity), and survival. [Figure 17E] Figures 17A-17G show that EcCAR-MSCs induce excellent immunosuppression in tumor and GvHD xenograft models. Figure 17E is a graph of the percent body weight change from baseline in GvHD xenografts after treatment with UTD-MSCs, EcCAR-MSCs, or no treatment. Data presented are mean ± SEM with statistical analysis using two-way ANOVA (ns = p≧0.05, *p≦0.05, ****p≦0.0001; n = 5 mice per group, 3 independent experiments). [Figure 17F] Figures 17A-17G show that EcCAR-MSCs induce excellent immunosuppression in tumor and GvHD xenograft models. Figure 17F shows a graph of GvHD clinical severity scores (left) and a representative mouse photograph (right) after treatment with EcCAR-MSCs, UTD-MSCs, or no MSCs. Data presented are mean ± SEM with statistical analysis using two-way ANOVA (ns = p≧0.05, *p≦0.05; n = 5 mice per group, 3 independent experiments). [Figure 17G] Figures 17A to 17G show that EcCAR-MSCs induce excellent immunosuppression in tumor and GvHD xenograft models. Figure 17G shows a graph of survival results after treatment with EcCAR-MSCs compared to UTD-MSCs and MSC-free controls. Statistical analysis was performed using Kaplan-Meier survival analysis (ns = p≧0.05, *p≦0.05, **p≦0.01; n=5 mice per group). [Figure 18A]Figures 18A-18G show that EcCAR-MSCs exhibit antigen-specific activation and distribution to Ecad-positive colonic target tissues in an acute GvHD xenograft model. Figure 18A is a schematic diagram of an example method for assessing the effects of Ecad-positive acute GvHD xenograft models. First, immunodeficient NOD-SCID-γ- / - (NSG) mice were irradiated with 250 cGy to further induce an inflammatory environment. Human PBMCs (10-15 × 10 cells intravenously) were injected into the mice along with either luciferase-positive GFP-negative Ecad-CAR-MSCs or luciferase-positive GFP-positive CD19-CAR-MSCs (1 × 10 cells intraperitoneally). Mice were examined for long-term weight loss, clinical GvHD progression, and survival. One week after MSC injection, satellite mice were isolated, and MSC localization to the colonic target organ was assessed by bioluminescence imaging and immunofluorescence staining. [Figure 18B] Figures 18A-18G show that EcCAR-MSCs exhibit antigen-specific activation and distribution to Ecad-positive colon target tissues in an acute GvHD xenograft model. Figure 18B is a graph of percent body weight change from baseline in acute GvHD xenografts after treatment with Ecad-CAR-MSCs, CD19-CAR-MSCs, or no treatment. Data presented are mean ± SEM with statistical analysis using two-way ANOVA (**p≦0.01, ***p≦0.001; n=4-5 mice per group, 3 independent experiments). [Figure 18C] Figures 18A-18G show that EcCAR-MSCs exhibit antigen-specific activation and distribution to Ecad-positive colonic target tissues in an acute GvHD xenograft model. Figure 18C shows a graph of survival results after treatment with Ecad-CAR-MSCs compared to CD19-CAR-MSCs and MSC-free controls. Statistical analysis was performed using Kaplan-Meier survival analysis (*p≦0.05; n=4-5 mice per group). [Figure 18D]Figures 18A-18G show that EcCAR-MSCs demonstrate antigen-specific activation and distribution to Ecad-positive colon target tissues in an acute GvHD xenograft model. Figure 18D is a graph of the absolute number of human CD3-positive T cells in peripheral blood, comparing Ecad-CAR-MSC and CD19-CAR-MSC treated mice by flow cytometry 2 weeks after MSC infusion. Data presented are mean ± SD, with statistical analysis performed using a standard one-way ANOVA (*p≦0.05; n=4-5 mice per group, two independent experiments). [Figure 18E] Figures 18A-18G show that EcCAR-MSCs exhibit antigen-specific activation and distribution to Ecad-positive colonic target tissues in an acute GvHD xenograft model. Figure 18E shows representative bioluminescence images (left panel) of mice treated with luciferase-positive CD19-CAR-MSCs and Ecad-CAR-MSCs, and the percentage of colonic MSC luminescence intensity relative to the total luminescence intensity detected throughout all organs (right panel). Data presented are mean ± SD, with statistical analysis performed using an unpaired t-test (*p≦0.05; n=4-5 mice per group, two independent experiments). [Figure 18F] Figures 18A-18G show that EcCAR-MSCs exhibit antigen-specific activation and distribution to Ecad-positive colonic target tissues in an acute GvHD xenograft model. Figure 18F is a graph of immunofluorescence-based quantification of EcCAR-MSCs compared to CD19-CAR-MSC localization to Ecad-positive colonic tissues. Data were determined by the percentage of MSC-positive colonic crypts per focal image. Data presented are mean ± SD, with statistical analysis using standard one-way ANOVA (**p≦0.01; n=3 mice per group). [Figure 18G]Figures 18A-18G show that EcCAR-MSCs exhibit antigen-specific activation and distribution to Ecad-positive colonic target tissues in an acute GvHD xenograft model. Figure 18G is a series of representative immunofluorescence images of mouse colon tissue isolated from an acute GvHD xenograft model 7 days after GFP-positive MSC administration. Colocalization (row 2) with E-cadherin-positive colonic regions (row 3) is compared between CD19-CAR-MSCs and Ecad-CAR-MSCs. Cell nuclei were stained with DAPI, and each color channel is shown to create a merged image. Images were acquired by photographing 1× cropped areas at 40× magnification. [Figure 19A] Figures 19A-19F show that activation of antigen-specific immunosuppressive signaling pathways was identified in EcCAR-MSCs. Figure 19A is a heat map depicting the unique gene expression profile of stimulated EcCAR-MSC + Ecad samples, highlighting the functional impact of CAR antigen-specific stimulation through hierarchical clustering of unstimulated EcCAR-MSC, stimulated UTD-MSC + Ecad, and unstimulated UTD-MSC samples. Data presented are normalized gene fold counts for all samples (padj ≦ 0.05) (n = 3 MSC donors per condition). [Figure 19B] Figures 19A-19F show that activation of antigen-specific immunosuppressive signaling pathways was identified in EcCAR-MSCs. Figure 19B shows a graph of principal component analysis (PCA) of gene expression profiles among six sample clusters by MSC donor derived from stimulation compared to unstimulated UTD-MSCs. [Figure 19C] Figures 19A to 19F show that activation of antigen-specific immunosuppressive signaling pathways was identified in EcCAR-MSCs. Figure 19C is a graph of PCA of gene expression profiles of stimulated compared to unstimulated EcCAR-MSCs, revealing that each stimulation group formed a unique cluster. [Figure 19D]Figures 19A-19F show that activation of antigen-specific immunosuppressive signaling pathways was identified in EcCAR-MSCs. Figure 19D is a schematic summary of the analysis using the Ingenuity Pathway Analysis (IPA) machine learning algorithm, illustrating factors activated upon stimulation of EcCAR-MSCs compared to unstimulated EcCAR-MSCs. Factors including upstream regulators, canonical pathways, and biological functions were combined to predict significant functional impacts. The analysis revealed that leukocyte apoptosis (center) was the most significantly enriched (padj ≤ 0.0001) functional pathway, and this pathway was directly linked to all 17 predicted activating molecules in the dataset, including the CD28 CAR signaling molecule. [Figure 19E] Figures 19A-19F show that activation of antigen-specific immunosuppressive signaling pathways was identified in EcCAR-MSCs. Figure 19E is a graphical representation of the IPA in Figure 16D, revealing that the canonical pathway is elevated in stimulated EcCAR-MSCs compared to unstimulated EcCAR-MSCs. The dotted line on the x-axis represents statistically significant enrichment for all pathways (-log(p≦0.05)) (n=3 MSC donors per group). [Figure 19F] Figures 19A-19F show that activation of antigen-specific immunosuppressive signaling pathways was identified in EcCAR-MSCs. Figure 19F is a series of graphs comparing normalized gene counts between unstimulated UTD-MSCs, stimulated UTD-MSCs, unstimulated EcCAR-MSCs, and stimulated EcCAR-MSCs. CD28-related transcription factor genes (NFκB1, JUN, RELB, and IRF1) and downstream effector genes (TRAF1, TLR3, and FYN) were elevated only in the stimulated EcCAR-MSC group. Data are shown as mean ± SD. 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). [Figure 20A]Figures 20A-20H show that stimulation with EcCAR-MSCs resulted in increased cytokine secretion and surface marker expression, resulting in T cell regulation. Figure 20A is a heat map showing the cytokines elevated when T cells alone, UTD-MSCs, and EcCAR-MSCs were stimulated with an Ecad-positive cell line compared to when stimulated with an Ecad-negative cell line, as determined by multiple cytokine analysis. Data presented are normalized to the minimum (0) and maximum (100) values ​​for each cytokine for the degree of cytokine fold change measured by multiple cytokine analysis (n=2 technical replicates per analysis). [Figure 20B] Figures 20A-20H show that stimulation with EcCAR-MSCs resulted in increased cytokine secretion, surface marker expression, and consequent T cell regulation. Figure 20B is a series of graphs showing inhibitory surface marker expression between UTD-MSCs and EcCAR-MSCs stimulated with an Ecad-positive cell line compared to stimulation with an Ecad-negative cell line. Data presented are mean ± SD, with statistical analysis performed using two-way ANOVA (***p≦0.001, ****p≦0.0001; n=3 replicates per donor). [Figure 20C] Figures 20A-20H show that stimulation with EcCAR-MSCs resulted in increased cytokine secretion and surface marker expression, resulting in T cell regulation. Figure 20C is a series of graphs of concentrated serum cytokines (IL-10, TNFα, G-CSF, eotaxin, and FGF-2 (pg / mL)) at week 2 in EcCAR-MSC-treated mice from tumor xenografts. Data presented are mean ± SEM with statistical analysis using standard one-way ANOVA (*p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001; n=4-6 mice per group). [Figure 20D]Figures 20A-20H show that stimulation with EcCAR-MSCs resulted in increased cytokine secretion and surface marker expression, resulting in T cell regulation. Figure 20D is a graph of the percent weight change compared to baseline, demonstrating that GvHD-induced weight loss was prevented after a single treatment with EcCAR-MSCs compared to UTD-MSCs. Percent weight change values ​​are shown as mean ± SEM. Statistical analysis was performed using two-way ANOVA (***p≦0.001; n=5 mice per group). [Figure 20E] Figures 20A-20H show that stimulation with EcCAR-MSCs resulted in increased cytokine secretion and surface marker expression, resulting in T cell regulation. Figure 20E is a graph of the absolute number of human CD3-positive T cells in the peripheral blood of mice treated with EcCAR-MSCs and UTD-MSCs, measured by flow cytometry 2 weeks after the first MSC injection. Data presented are mean ± SD, with statistical analysis performed using an unpaired t-test (**p≦0.01; n=5 mice per group). [Figure 20F] Figures 20A-20H show that stimulation with EcCAR-MSCs resulted in increased cytokine secretion and surface marker expression, resulting in T cell regulation. Figure 20F shows a graph of human CD4+ and CD8+ T cells quantified in mouse peripheral blood 2 weeks after EcCAR-MSC treatment. Data presented are mean ± SD for cell counts. Statistical analysis was performed using two-way ANOVA (*p≦0.05, ***p≦0.001; n=5 mice per group). [Figure 20G] Figures 20A-20H show that stimulation with EcCAR-MSCs resulted in increased cytokine secretion and surface marker expression, resulting in T cell regulation. Figure 20G is a graph showing the change in the proportion of human CD4+ and CD8+ T cells in EcCAR-MSC-treated mice compared to UTD-MSC-treated mice. Data presented are mean ± SD for percentages of cell subsets. Statistical analysis was performed using two-way ANOVA (**p≦0.01; n=5 mice per group). [Figure 20H] Figures 20A-20H show that stimulation with EcCAR-MSCs resulted in increased cytokine secretion and surface marker expression, resulting in T cell regulation. Figure 20H shows a graph of human CD4+CD25+CD127-Treg cell subsets in GvHD xenografted mice 4 weeks after MSC treatment. Data presented are mean ± SD for % of cells. Statistical analysis was performed using an unpaired t-test (***p≦0.001; n=4 mice per group). [Figure 21A] Figures 21A-21G show that the CD28 signaling domain in EcCAR-MSCs increases the immunosuppressive effect. Figure 21A is a schematic diagram of an example method for designing EcCAR-MSC constructs used to investigate the mechanism of action of CAR-MSCs. Constructs included full-length CD28ζ, CD28-only, CD3ζ-only, and null (signaling domain-lacking) EcCAR-MSCs. [Figure 21B] Figures 21A-21G show that the CD28 signaling domain in EcCAR-MSCs increases the immunosuppressive effect. Figure 21B shows histograms of CAR expression on MSCs detected by flow cytometry for CD28ζ, CD28, CD3ζ, and null EcCAR-MSC constructs. % CAR expression is shown, representing ≥5 independent experiments for four independent MSC donors. [Figure 21C] Figures 21A-21G show that the CD28 signaling domain in EcCAR-MSCs increases the immunosuppressive effect. Figure 21C is a series of graphs showing the degree of CD3+ T cell suppression after culturing each EcCAR-MSC signaling domain subtype or T cells alone with Ecad-positive and paired Ecad-negative cell lines and stimulating with the Ecad-positive cell line. Data shown are means ± SD, representative 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]Figures 21A-21G show that the CD28 signaling domain in EcCAR-MSCs enhances immunosuppressive effects. Figure 21D is a schematic diagram of 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. Mice were injected with human PBMCs (10-15 × 10 cells intravenously) along with one of the following MSC groups: CD28ζ, CD28, CD3ζ, or null EcCAR-MSCs; UTD-MSCs (1 × 10 cells intraperitoneally); or "no MSC" treatment. Mice were examined for long-term weight loss, development of clinical GvHD symptoms, and survival. [Figure 21E] Figures 21A-21G show that the CD28 signaling domain in EcCAR-MSCs increases the immunosuppressive effect. Figure 21E shows a graph of the percent body weight change from baseline in acute GvHD xenografts after treatment with or without any of the MSC groups. Data presented are mean ± SEM with statistical analysis using two-way ANOVA (ns = p≧0.05, *p≦0.05, **p≦0.01; n = 5-6 mice per group, two independent experiments). [Figure 21F] Figures 21A-21G show that the CD28 signaling domain in EcCAR-MSCs increases the immunosuppressive effect. Figure 21F shows a graph of GvHD clinical severity score calculations for symptom progression after treatment with alternative MSC signaling domains. Data presented are mean ± SEM with statistical analysis using two-way ANOVA (*p≦0.05; n=5-6 mice per group, two independent experiments). [Figure 21G]Figures 21A-21G show that the CD28 signaling domain in EcCAR-MSCs enhances the immunosuppressive effect. Figure 21G shows a graph of survival results in an acute GvHD xenograft mouse model after treatment with either EcCAR-MSC subtype. Data depicting a trend toward prolonged survival were included in the estimated p-value measurement, even though they were not statistically significant. 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). [Figure 22A] Figures 22A-22I show the safety and elimination profiles of EcCAR-MSCs across tissues. Figure 22A is a schematic diagram of a representative method for determining the efficacy of EcCAR-MSCs in an MSC persistence model. NSG mice pre-treated with human PBMCs were injected with either luciferase-positive, GFP-positive UTD-MSCs or luciferase-positive, GFP-positive CAR-MSCs, with or without CAR stimulation via co-administration of irradiated Ecad-positive cells. Mice were then photographed biweekly by bioluminescence imaging (BLI). [Figure 22B] Figures 22A-22I show the safety and elimination profile of EcCAR-MSCs across tissues. Figure 22B shows representative images of the proliferation and elimination of luciferase-positive MSCs in mice 3 to 24 days after administration. [Figure 22C] Figures 22A-22I show the safety and elimination profile of EcCAR-MSCs across tissues. Figure 22C shows graphs of MSC proliferation and elimination kinetics measured by BLI luminescence intensity after administration of EcCAR-MSCs or UTD-MSCs in the presence or absence of additional Ecad stimulation. Data presented are the mean BLI values ​​followed by statistical analysis using two-way ANOVA (ns = p≧0.05; n=4-5 mice per group). [Figure 22D]Figures 22A-22I show the safety and clearance profile of EcCAR-MSC across tissues. Figure 22D shows a graph of the absolute number of keratinocytes after 24 hours of co-culture with UTD-MSC or EcCAR-MSC. Data presented are mean ± SD with statistical analysis using a paired t-test (ns = p≧0.05; n = 3 replicates per donor). [Figure 22E] Figures 22A-22I show the safety and clearance profile of EcCAR-MSCs across tissues. Figure 22E shows a graph of Ecad expression identified in keratinocytes after 24 hours of co-culture with UTD-MSCs or EcCAR-MSCs. Data presented are % mean ± SD with statistical analysis using a paired t-test (ns = p≧0.05; n = 3 replicates per donor). [Figure 22F] Figures 22A-22I show the safety and clearance profile of EcCAR-MSCs across tissues. Figure 22F is a heat map showing cytokines elevated from baseline in UTD-MSCs, EcCAR-MSCs, and keratinocytes after 24 hours of co-culture. Data shown are normalized to the minimum (0) and maximum (100) values ​​per cytokine for cytokine levels measured by composite analysis (n=2 technical replicates per analysis). [Figure 22G] Figures 22A-22I show the safety and clearance profile of EcCAR-MSCs across tissues. Figure 22G shows a graph of the absolute number of bronchial cells after 24 hours of co-culture with UTD-MSCs or EcCAR-MSCs at various ratios. Data presented are mean ± SD with statistical analysis using a paired t-test (ns = p≧0.05; n = 3 technical repeats). [Figure 22H]Figures 22A-22I depict the safety and clearance profile of EcCAR-MSCs across tissues. Figure 22H is a graph of immunofluorescence-based quantification of EcCAR-MSC localization compared to CD19-CAR-MSC localization to Ecad-positive lung tissue. Data were determined by % MSC-positive area per focal image. Data presented are mean ± SD with statistical analysis using standard one-way ANOVA (ns = p≧0.05; n=3 mice per group). [Figure 22I] Figures 22A-22I show the safety and clearance profile of EcCAR-MSCs across tissues. Figure 22I shows representative immunofluorescence images of mouse bronchial (lung) tissue isolated from an acute GvHD xenograft model 7 days after administration of GFP-positive MSCs. Colocalization of Ecad-positive (second row) bronchial regions is compared between CD19-CAR-MSC and Ecad-CAR-MSC (bottom row) treated mice. Cell nuclei were stained with DAPI (third row), and each color channel is shown to create a merged image. Images were acquired by photographing 1× cropped areas at 40× magnification. [Figure 23A] Figures 23A-23D show the design and production of EcCAR constructs. Figure 23A shows a representative sequence of an optimized anti-Ecad scFv clone sequence (hmcECAD.6) identified through phage display generation for Ecad selection. hmcECAD was selected for human, mouse, and canine cross-reactivity affinity and cloned into the EcCAR construct to generate EcCAR-MSCs. [Figure 23B] Figures 23A-23D show the design and production of EcCAR constructs. Figure 23B is a representative schematic diagram of a full-length EcCAR-MSC construct, which contains the hmcCAD heavy and light chains that bind to Ecad, the CD28 hinge and transmembrane domains that penetrate the MSC cell membrane, and the CD28 intracellular signaling domain that activates immunosuppression together with CD3ζ costimulation, which are commonly utilized for CAR construct stabilization. [Figure 23C]Figures 23A-23D show the design and production of EcCAR constructs. Figure 23C shows a graph of a phage ELISA used to verify the relative affinity of the hmcECAD.6 scFv clone for mouse Ecad, compared to a FLAG-tagged positive control. Data shown are all technical replicates (n=3) with statistical analysis using two-way ANOVA (ns=p≧0.05). [Figure 23D] Figures 23A-23D show the design and production of EcCAR constructs. Figure 23D is a graph showing the dose-dependent killing effect of CART cells containing the hmcECAD.6 scFv construct on human and mouse Ecad-positive tumors (MCF7 and 4T1, respectively). Data shown are all technical replicates (n=3) with statistical analysis using two-way ANOVA (**p≦0.01, ****p≦0.0001). [Figure 24A] Figures 24A-24D depict an example method for generating and validating CAR-MSCs, along with confirmation of stemness. Figure 24A is a schematic diagram of an example method for generating EcCAR-MSCs in culture after thawing from a cryopreservation system. Briefly, after in vitro expansion in T175 flasks, MSCs are enzymatically digested and quantified for transduction with an optimized CAR plasmid containing a lentiviral vector delivery system and a protamine sulfate enhancer. Validation of CAR% is completed by day 5 through flow cytometry analysis with a goat anti-mouse antibody. [Figure 24B] Figures 24A-24D show an example of the method and validation of CAR-MSC generation, along with confirmation of stemness. Figure 24B is a graph of the stemness phenotype of EcCAR-MSCs measured by flow cytometry after staining for surface markers, in the presence or absence of Ecad-positive cell line stimulation, and an additional positive control measurement using primary naive T cells. Data presented are mean values ​​± SD from three individual MSC donors. Statistical analysis was performed using two-way ANOVA (****p≦0.0001). [Figure 24C]Figures 24A-24D show examples of the method and validation of CAR-MSC generation, along with confirmation of stemness. Figure 24C shows representative images of morphological comparisons of UTD-MSC and CAR-MSC from baseline after the addition of paired Ecad-negative and Ecad-positive cell lines as a means of CAR antigen-specific stimulation. [Figure 24D] Figures 24A-24D show an example of the method and validation of CAR-MSC generation, along with confirmation of stemness. Figure 24D is a series of histograms characterizing the stem phenotype of MSCs by positive CD105, CD90, and CD73 surface markers and negative CD34, CD45, and CD14 surface markers. Positive gating was established by the Fluorescence Minus One (FMO) method for the negative control (top row) for positive sample quantification of UTD-MSCs (center row) and EcCAR-MSCs (bottom row). [Figure 25A] Figures 25A-25G show the effect of EcCAR-MSCs on CART effector function in an in vivo tumor model. Figure 25A is a graph comparing tumor luminescence intensity measurements of Ecad-negative NALM6 and Ecad-positive NALM6 between control mice without CART19 cell injection. Data presented are mean ± SEM with statistical analysis using two-way ANOVA (ns = p≧0.05; n = 4-5 mice per group). [Figure 25B] Figures 25A-25G show the effect of EcCAR-MSCs on CART effector function in an in vivo tumor model. Figures 25B and 25C are graphs of the relative levels of luciferase-positive, Ecad-negative NALM6 (Figure 25B) and luciferase-positive, Ecad-positive NALM6 (Figure 25C) measured by luminescence intensity 24 hours after in vitro co-culture of UTD-MSCs or EcCAR-MSCs at various MSC:NALM6 ratios. Data presented are mean ± SD, with statistical analysis performed using two-way ANOVA (ns = p≧0.05; n = 2 replicates per group). [Figure 25C]Figures 25A-25G show the effect of EcCAR-MSCs on CART effector function in an in vivo tumor model. Figures 25B and 25C are graphs of the relative levels of luciferase-positive, Ecad-negative NALM6 (Figure 25B) and luciferase-positive, Ecad-positive NALM6 (Figure 25C) measured by luminescence intensity 24 hours after in vitro co-culture of UTD-MSCs or EcCAR-MSCs at various MSC:NALM6 ratios. Data presented are mean ± SD, with statistical analysis performed using two-way ANOVA (ns = p≧0.05; n = 2 replicates per group). [Figure 25D] Figures 25A-25G show the effect of EcCAR-MSCs on CART effector function in an in vivo tumor model. Figure 25D shows a representative schematic diagram of the method for evaluating CART19 in NALM6 and JeKo-1 tumor models. NSG mice were implanted with luciferase-positive, CD19-positive NALM6 or JeKo-1 cells (1 × 10 cells intravenously) and treated with CART19 (1 × 10 cells intravenously) and an irradiated Ecad-positive cell line. Mice were then randomized and received UTD-MSCs or EcCAR-MSCs (1 × 10 cells intraperitoneally), and bioluminescence imaging and survival were monitored every two weeks. [Figure 25E] Figures 25A-25G show the effect of EcCAR-MSCs on CART effector function in an in vivo tumor model. Figure 25E is a graph of tumor luminescence intensity after CART19 injection in the Jeko-1 tumor model, comparing EcCAR-MSCs, UTD-MSCs, or no MSC treatment. Data presented are mean ± SEM with statistical analysis by two-way ANOVA (**p≦0.01; n=3-4 mice per group). [Figure 25F]Figures 25A-25G show the effect of EcCAR-MSCs on CART effector function in an in vivo tumor model. Figure 25F shows a graph of tumor luminescence intensity measurements between MSC-treated groups after CART19 injection in the NALM6 model. Data presented are mean ± SEM with statistical analysis using two-way ANOVA (ns = p≧0.05, ***p≦0.001; n = 3-4 mice per group, two independent experiments). [Figure 25G] Figures 25A to 25G show the effect of EcCAR-MSCs on CART effector function in an in vivo tumor model. Figure 25G shows a graph of the survival results of mice treated with EcCAR-MSCs compared to UTD-MSCs and MSC-free control groups. Statistical analysis was performed using Kaplan-Meier survival analysis (ns = p≧0.05, *p≦0.05; n = 3 to 4 mice per group, two independent experiments). [Figure 26A] Figures 26A-26B show an example of a flow cytometry gating strategy. Figure 26A shows an example of the gating strategy used to identify positive MSC surface markers confirmed by FMO controls and internal standards. MSC populations are identified by size, single-cell diagonal gating is used to select, viable MSCs are stained with a live / dead fixable aqua fluorophore to remove dead cells, and positive cell markers (represented as CAR+%) are identified based on positive gating of cells of interest (confirmed by internal standards and FMO controls). [Figure 26B] Figures 26A-26B show an example of a flow cytometry gating method. Figure 26B shows an example of the gating method used to identify luciferase-positive and CAR-positive MSCs in an in vivo depletion experiment. MSCs were similarly transduced with a luciferase-expressing transgene and a CAR construct and compared with luciferase-positive MSCs and UTD-MSC controls. The results show that luciferase and CAR transduction were successful, with both luciferase and CAR positivity rates >50% after in vivo intraperitoneal injection. [Figure 27A]Figures 27A-27E show bulk RNA-seq pathway analysis and cytokine secretion. Figure 27A is a graph showing a summary of gene expression changes, comparing unstimulated EcCAR-MSCs with UTD-MSCs, Ecad stimulation with unstimulated EcCAR-MSCs, Ecad stimulation with unstimulated UTD-MSCs, and Ecad-stimulated EcCAR-MSCs with UTD-MSCs. Data presented are gene counts of significantly elevated and decreased genes within comparisons, with adjusted p-values ​​of <0.01 and ±1-log fold changes. Transcriptional changes induced by Ecad stimulation of CAR-MSCs included 2,362 significant genes compared to EcCAR-MSCs alone and 3,032 significant genes compared to Ecad-stimulated UTD-MSCs. Transcriptional changes induced by CAR transduction included 606 significant genes. Transcriptional changes induced by Ecad stimulation included 206 significant genes. [Figure 27B] Figures 27A-27E show bulk RNA-seq pathway analysis and cytokine secretion. Figure 27B shows an Ingenuity Pathway Analysis (IPA) graph demonstrating elevated canonical pathway activity in unstimulated EcCAR-MSCs compared to UTD-MSCs. The dotted line on the x-axis represents statistically significant enrichment for all pathways (-log(p≦0.05)) (n=3 MSC donors per group). [Figure 27C] Figures 27A-27E show bulk RNA-seq pathway analysis and cytokine secretion. Figures 27C and 27D are graphical summaries of the IPA in Figure 27B, illustrating significantly activated EcCAR-MSCs vs. UTD-MSCs (Figure 27C) and stimulated EcCAR-MSCs compared to UTD-MSCs (Figure 27D). Canonical pathways and activated molecules were used to predict significant functional impact across datasets. [Figure 27D]Figures 27A-27E show bulk RNA-seq pathway analysis and cytokine secretion. Figures 27C and 27D are graphical summaries of the IPA in Figure 27B, illustrating significantly activated EcCAR-MSCs vs. UTD-MSCs (Figure 27C) and stimulated EcCAR-MSCs compared to UTD-MSCs (Figure 27D). Canonical pathways and activated molecules were used to predict significant functional impact across datasets. [Figure 27E] Figures 27A-27E show bulk RNA-seq pathway analysis and cytokine secretion. Figure 27E is a graph showing elevated serum cytokines found in peripheral blood from tumor xenograft mice treated with EcCAR-MSCs compared to UTD-MSCs and controls. Cytokines include macrophage-derived chemokine (MDC), growth-associated alpha protein (GRO), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein 3 (MCP-3), and FLT-3L (Follicle-Related Tyrosine Phosphate 3 Ligand) (pg / mL). Data are presented as mean ± SD, with statistical analysis determined by multiple t-test (*p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001; n = 4-6 mice per group). [Figure 28A] Figures 28A-28E show the safety profile of EcCAR-MSCs determined using an in vivo canine model. Figure 28A is a schematic diagram of an example 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 lentivirally transduced into human MSCs and subsequently expanded in vitro for intraperitoneal injection into healthy canine subjects. Subgroups were tested for hematologic and organ toxicity over 28 days. [Figure 28B]Figures 28A-28E show the safety profile of EcCAR-MSCs determined using an in vivo model. Figure 28B shows a series of graphs of whole blood count levels to determine hematopoietic safety after EcCAR-MSC administration. The figures include blood level tests of leukocytes, monocytes, lymphocytes, neutrophils, and platelets compared to baseline values ​​short-term (3 days) and long-term (28 days) after in vivo EcCAR-MSC infusion. Mean values ​​± SD for blood composition are shown, along with statistical analysis by one-way ANOVA (ns = p≧0.05; n = 3 subjects per experimental group). [Figure 28C] Figures 28A-28E show the safety profile of EcCAR-MSCs determined using an in vivo model. Figure 28C shows a series of graphs of total protein, BUN, creatinine, albumin, and alkaline phosphatase levels at short-term (3 days) and long-term (28 days) examinations after in vivo EcCAR-MSC infusion, plotted to verify safety. Data presented are mean ± SD, with statistical analysis performed using one-way ANOVA (ns = p≧0.05; n = 3 subjects per experimental group). [Figure 28D] Figures 28A-28E show the safety profile of EcCAR-MSCs determined using an in vivo model. Figure 28D is a graph of body weight change in healthy dogs after EcCAR-MSC administration compared to controls. No significant differences in body weight change were found between groups. Data presented are mean % body weight change from baseline ± SD, with statistical analysis performed by standard one-way ANOVA (ns = p > 0.05), (ns = p > 0.05; n = 3 subjects per experimental group). [Figure 28E] Figures 28A-28E show the safety profile of EcCAR-MSCs determined using an in vivo model. Figure 28E shows representative images (20x and 40x magnification) of H&E-stained canine transverse colon tissue sections from treatment with human EcCAR-MSCs (left) or control (right) after 28 days of administration. [Figure 29]Figure 29 is a graph depicting the percent EcCAR positivity after transduction of mouse adipose-derived MSCs as determined by flow cytometry. Data presented are the mean ± SD for a representative replicate of three technical mouse cell lines. DETAILED DESCRIPTION OF THE INVENTION

[0016] Description of the Invention This document provides methods and materials for the treatment of 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 (e.g., GVHD)). In some embodiments, one or more CAR-MSCs having (e.g., engineered to have) high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides can be administered to a mammal to induce an immunosuppressive response (e.g., reduce or eliminate an inflammatory immune response) in a target tissue in the mammal. For example, the CAR-MSCs can express a CAR that can target an epithelial-specific antigen (e.g., Ecad (also called CDH1)) to target the MSCs to epithelial tissue, and can further have (e.g., engineered to have) high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides to enhance immunosuppression in the target epithelial tissue. In some embodiments, the CAR-MSCs may express a CAR capable of targeting a neural-specific antigen (e.g., myelin oligodendrocyte glycoprotein (MOG)) to target the MSCs to neural tissue, and may further have (e.g., be engineered to have) high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides to enhance immunosuppression in the target neural tissue. For example, one or more CAR-MSCs having (e.g., engineered to have) high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides can be administered to a mammal (e.g., a human) suffering from (or at risk of developing) an inflammatory disease or condition to treat one or more autoimmune diseases (e.g., GVHD) in a target tissue (e.g., an inflamed tissue) in the mammal.

[0017] The term "high level," when used herein with reference to the level of a polypeptide (e.g., an immunosuppressive polypeptide or a distribution polypeptide), refers to any level that is higher than a reference level of that polypeptide. The term "reference level," when used herein with reference to a polypeptide (e.g., an immunosuppressive polypeptide or a distribution polypeptide), refers to the level of that polypeptide that is typically observed in MSCs (e.g., CAR-MSCs) that have not been engineered to have high levels of that polypeptide as described herein.

[0018] The MSCs described herein (e.g., CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) can be any suitable MSCs. Examples of MSCs that may be used as described herein include, but are not limited to, adipose-derived MSCs, bone marrow-derived MSCs, placental tissue-derived MSCs, dental pulp tissue-derived MSCs, umbilical cord-derived MSCs, umbilical cord blood-derived MSCs, Wharton's jelly-derived MSCs, dermis-derived MSCs, olfactory mucosa-derived MSCs, peripheral blood-derived MSCs, and amniotic membrane-derived MSCs. For example, CAR-MSCs having (e.g., engineered to have) high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides can be adipose-derived MSCs.

[0019] The CAR-MSCs described herein (e.g., CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) may express any suitable CAR. The CAR may include (a) an antigen-binding domain, (b) a transmembrane domain, and (c) one or more signaling domains.

[0020] The antigen-binding domain of a CAR expressed by a CAR-MSC described herein (e.g., a CAR-MSC having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) can be any suitable antigen-binding domain. In some aspects, the antigen-binding domain can comprise an antibody or fragment thereof that targets an antigen (e.g., a tissue-specific antigen (e.g., an epithelial-specific antigen or a neural-specific antigen)). Examples of antigen-binding domains include, but are not limited to, an antigen-binding fragment (Fab), a variable region of an antibody heavy chain (VH), a variable region of a light chain (VL), and a single-chain variable fragment (scFv). In some aspects, the antigen-binding domain can target (e.g., target and bind to) a tissue-specific antigen (e.g., an epithelial-specific antigen or a neural-specific antigen). For example, a CAR-MSC described herein can express (e.g., be engineered to express) a CAR that can bind to a tissue-specific antigen (e.g., an antigen present on cells in a tissue with minimal or no expression on other cell types).

[0021] In some aspects, a CAR-MSC described herein (e.g., a CAR-MSC having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) can be engineered to express a CAR that can target (e.g., can target and bind to) an antigen (e.g., a cell surface antigen) (e.g., an epithelial-specific antigen or epithelial antigen) expressed by epithelial cells of a mammal in need of immunosuppression in epithelial tissue (e.g., a human suffering from or at risk of developing one or more autoimmune diseases (e.g., GVHD)). The epithelial-specific antigen can be any suitable epithelial-specific antigen. The epithelial-specific antigen can be expressed on any suitable type of epithelial cell (e.g., gastrointestinal cells (e.g., colon and rectal cells), skin cells, lung cells, liver cells, reproductive tract cells, and urinary tract cells). In some aspects, the epithelial-specific antigen can be a cell adhesion molecule (CAM). In some aspects, the epithelial-specific antigen can be an integrin (e.g., an intestinal integrin). Examples of epithelial-specific antigens include, but are not limited to, Ecad, CD103, αEβ7, and α4β7. For example, CAR-MSCs engineered to target epithelial tissues may bind to Ecad. In some embodiments, CAR-MSCs may be engineered to express CAR-Ecad, which targets Ecad expressed by mammalian epithelial cells. In some embodiments, CARs expressed by CAR-MSCs described herein may comprise an Ecad-binding domain. For example, CARs expressed by CAR-MSCs described herein may comprise an anti-Ecad scFv comprising a heavy chain comprising a CDR set forth in SEQ ID NO: 1 and a light chain comprising a CDR set forth in SEQ ID NO: 2. For example, CARs expressed by CAR-MSCs described herein may have an anti-Ecad scFv comprising a heavy chain comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 1, and a light chain comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 2 (see, e.g., Example 2). In some embodiments, CARs expressed by CAR-MSCs described herein may comprise a CD103-binding domain.For example, a CAR expressed by a CAR-MSC described herein may comprise an anti-CD103 scFv comprising a heavy chain comprising the CDRs set forth in SEQ ID NO: 3 and a light chain comprising the CDRs set forth in SEQ ID NO: 4. For example, a CAR expressed by a CAR-MSC described herein may have an anti-CD103 scFv comprising a heavy chain comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a light chain comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 4 (see, e.g., Example 2).

[0022] In some aspects, the CAR-MSCs described herein (e.g., CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) can be engineered to express a CAR that can target (e.g., target and bind to) an antigen (e.g., a cell surface antigen) (e.g., a neuro-specific antigen or neuro-antigen) expressed by neural cells of a mammal in need of immunosuppression in neural tissue (e.g., a human suffering from or at risk of developing one or more autoimmune diseases (e.g., multiple sclerosis)). When the antigen is a neuro-specific antigen, the neuro-specific antigen can be any suitable neuro-specific antigen. The neuro-specific antigen can be expressed on any suitable type of neural cell (e.g., sensory neurons, motor neurons, interneurons, glial cells, and nerve sheaths). The neuro-specific antigen can be expressed on neural cells of the central nervous system (CNS) and / or peripheral nervous system (PNS). In some aspects, the neuro-specific antigen can be a transmembrane protein. In some embodiments, the neuro-specific antigen may target myelin. Examples of neuro-specific antigens include, but are not limited to, MOG and AMPA (e.g., antigens of one or more AMPA receptor polypeptides). For example, a CAR-MSC engineered to target neural tissue may bind to MOG. In some embodiments, a CAR-MSC may be engineered to express CAR-MOG to target MOG expressed by neural cells in a mammal suffering from or at risk of developing multiple sclerosis. In some embodiments, a CAR expressed by a CAR-MSC described herein may comprise a MOG antigen-binding domain. For example, a CAR expressed by a CAR-MSC described herein may comprise an anti-MOG scFv comprising a heavy chain comprising the CDRs set forth in SEQ ID NO:5 and a light chain comprising the CDRs set forth in SEQ ID NO:6. For example, a CAR expressed by a CAR-MSC described herein may have an anti-MOG scFv comprising a heavy chain comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a light chain comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 6 (see, e.g., Example 2).In some aspects, a CAR expressed by a CAR-MSC described herein may comprise an AMPA antigen-binding domain. For example, a CAR expressed by a CAR-MSC described herein may comprise an anti-AMPA scFv comprising a heavy chain comprising the CDR set forth in SEQ ID NO: 7 and a light chain comprising the CDR set forth in SEQ ID NO: 8. For example, a CAR expressed by a CAR-MSC described herein may have an anti-AMPA scFv comprising a heavy chain comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 7, and a light chain comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 8 (see, e.g., Example 2).

[0023] The transmembrane domain of a CAR expressed by a CAR-MSC described herein (e.g., a CAR-MSC having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) can be any suitable transmembrane domain. Examples of transmembrane domains that may be used as described herein include, but are not limited to, an NKG2D transmembrane domain, a CD8α transmembrane domain, a CD28 transmembrane domain, an IgG4 transmembrane domain, a TNFR transmembrane domain, and a TLR transmembrane domain. In some embodiments, a transmembrane domain included in a CAR expressed by a CAR-MSC described herein can be linked (e.g., covalently linked) to an adaptor polypeptide. In some embodiments, a CAR expressed by a CAR-MSC described herein can comprise a CD28 transmembrane domain. For example, a CAR expressed by a CAR-MSC described herein can comprise 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).

[0024] The signaling domain of a CAR expressed by a CAR-MSC described herein (e.g., a CAR-MSC with high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) may comprise any suitable signaling domain or domains. For example, a CAR expressed by a CAR-MSC described herein may be designed to comprise one, two, three, or four signaling domains. When a CAR expressed by a CAR-MSC described herein comprises more than one (e.g., two, three, or four) signaling domains, the CAR may comprise any suitable combination of signaling domains. In some aspects, a CAR expressed by a CAR-MSC described herein may be designed to comprise one or more signaling domains normally found in immune cells (e.g., lymphocytes (e.g., TILs, T cells, or NK cells)). In some aspects, a CAR expressed by a CAR-MSC described herein may be designed to comprise one or more signaling domains normally found in MSCs (e.g., MSCs with an immunosuppressive phenotype). In some aspects, the CARs expressed by the CAR-MSCs described herein may be designed to include one or more regenerative signaling domains (e.g., to stimulate tissue regeneration). Examples of signaling domains that may be used as described herein include, but are not limited to, a CD3zeta signaling domain, a CD28 signaling domain, a 4-1BB signaling domain, an OX40 signaling domain, a TLR3 signaling domain, a TLR4 signaling domain, a TIR3 signaling domain, a TIR4 signaling domain, an IFNγ signaling domain, a brain-derived neurotrophic factor (BDNF) signaling domain, a vascular endothelial-derived growth factor receptor (VEGFR2) signaling domain, a TLR3 / TIR3 signaling domain, a TLR4 / TIR4 signaling domain, a TLR9 / TIR9 signaling domain, an IFNGR1 signaling domain, an IL10RA signaling domain, and a TNFR2 signaling domain.In some embodiments, the CAR expressed by the CAR-MSCs described herein may be designed to be a first-generation CAR with a CD3ζ signaling domain. In some embodiments, the CAR expressed by the CAR-MSCs described herein may be designed to be a second-generation CAR with a CD28 signaling domain followed by a CD3ζ signaling domain. In some embodiments, the CAR expressed by the CAR-MSCs described herein may be designed to be a third-generation CAR with (a) a CD28 signaling domain followed by (b) a CD27 signaling domain, an OX40 signaling domain, or a 4-1BB signaling domain followed by (c) a CD3ζ signaling domain. In some embodiments, the CAR expressed by the CAR-MSCs described herein may comprise a CD28 signaling domain. For example, the CAR expressed by the CAR-MSCs described herein may comprise 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 CD3ζ 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).

[0025] In some aspects, a CAR expressed by a CAR-MSC described herein (e.g., a CAR-MSC having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) may comprise one or more additional elements. Examples of additional elements that a CAR expressed by a CAR-MSC described herein may comprise include, but are not limited to, a linker, a hinge domain, and a detectable marker. When an antigen receptor (e.g., a CAR) described herein comprises a detectable marker, the detectable marker may be any suitable detectable marker. Examples of detectable markers that a CAR expressed by a CAR-MSC described herein may comprise include, but are not limited to, a bioluminescent polypeptide (e.g., a luciferase polypeptide), a fluorescent polypeptide (e.g., a green fluorescent polypeptide (GFP)), a sodium-iodide symporter (NIS), an SSTR2 polypeptide, a PSMA polypeptide, a hdCK polypeptide, and an eDHFR polypeptide.

[0026] In some aspects, a CAR expressed by a CAR-MSC described herein (e.g., a CAR-MSC having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) may comprise an anti-Ecad scFv, a CD28 transmembrane domain, a CD28 signaling domain, and a CD3ζ signaling domain. For example, a CAR expressed by a CAR-MSC described herein may comprise an anti-Ecad scFv having a heavy chain comprising the CDR set forth in SEQ ID NO: 1 and a light chain comprising the CDR 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 CD3ζ 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).

[0027] Any suitable method may be used to express a CAR described herein (e.g., a CAR that targets a tissue-specific antigen (e.g., an epithelial-specific antigen or a neural-specific antigen)) on the surface of a CAR-MSC described herein (e.g., a CAR-MSC having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides). For example, a nucleic acid encoding a CAR may be introduced into the MSC. In some embodiments, a nucleic acid encoding a CAR may be introduced into the MSC by transduction (e.g., viral transduction) or transfection. In some embodiments, a nucleic acid encoding a CAR described herein may be introduced into one or more MSCs ex vivo. For example, ex vivo manipulation of MSCs to express a CAR described herein may include transducing isolated MSCs with a vector encoding a CAR (e.g., a viral vector (e.g., a lentiviral vector, retroviral vector, adenoviral vector, or adeno-associated viral (AAV) vector)). When MSCs are engineered ex vivo to express a CAR, the MSCs may be obtained from any suitable source (e.g., a mammal (e.g., a mammal receiving treatment or a donor mammal) or a cell line). In some embodiments, CAR-MSCs may be prepared as described herein (see, e.g., Figure 7 and Example 1). For example, by introducing into the MSCs one or more constructs comprising a nucleic acid encoding a CAR (e.g., an Ecad-targeting CAR), CAR-Ecad can be expressed on the MSCs to direct the MSCs to epithelial tissue. For example, by introducing into the MSCs one or more constructs comprising a nucleic acid encoding a CAR (e.g., an MOG-targeting CAR), CAR-MOG can be expressed on the MSCs to direct the MSCs to neural tissue.

[0028] In some aspects, the CAR expressed by the CAR-MSCs described herein (e.g., CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) 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.

[0029] CAR-MSCs described herein (e.g., CAR-MSCs with high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) may express any suitable one or more immunosuppressive polypeptides, one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides. In some aspects, a polypeptide (e.g., an immunosuppressive polypeptide or a distribution polypeptide) expressed by a CAR-MSC described herein may be an exogenous polypeptide. In some aspects, a polypeptide (e.g., an immunosuppressive polypeptide or a distribution polypeptide) expressed by a CAR-MSC described herein may be part of a signaling pathway (e.g., the TNFα signaling pathway, the TLR signaling pathway, and the IL-10 signaling pathway). In some aspects, a polypeptide (e.g., an immunosuppressive polypeptide or a distribution polypeptide) expressed by a CAR-MSC described herein may be associated with T-cell suppression. In some aspects, a polypeptide (e.g., an immunosuppressive polypeptide or a distribution polypeptide) expressed by a CAR-MSC described herein may be a kinase (e.g., a tyrosine kinase). In some aspects, a polypeptide (e.g., an immunosuppressive polypeptide or a distribution polypeptide) expressed by a CAR-MSC described herein can be a transcription factor. In some aspects, a polypeptide (e.g., an immunosuppressive polypeptide or a distribution polypeptide) expressed by a CAR-MSC described herein can be a chemokine (e.g., a homing chemokine). In some aspects, a polypeptide (e.g., an immunosuppressive polypeptide or a distribution polypeptide) expressed by a CAR-MSC described herein can be a cytokine (e.g., an anti-inflammatory cytokine). In some aspects, a polypeptide (e.g., an immunosuppressive polypeptide or a distribution polypeptide) expressed by a CAR-MSC described herein can be an immunomodulatory cell surface polypeptide. In some aspects, a polypeptide (e.g., an immunosuppressive polypeptide or a distribution polypeptide) expressed by a CAR-MSC described herein can be associated with cell invasion (e.g., transendothelial invasion).Examples of immunosuppressive or distribution polypeptides that the CAR-MSCs described herein may express (e.g., may be designed to express) include, but are not limited to, 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, a CXCR4 polypeptide, a CTLA4 polypeptide, a TLR3 polypeptide, a TLR4 polypeptide, a TLR9 polypeptide, and a TNFR2 polypeptide.

[0030] Any suitable method may be used to increase the level of one or more immunosuppressive polypeptides, to increase the level of one or more regenerative polypeptides, and / or to increase the level of one or more distribution polypeptides in the CAR-MSCs described herein. In some embodiments, a nucleic acid (e.g., an exogenous nucleic acid) encoding a polypeptide (e.g., an immunosuppressive polypeptide or a distribution polypeptide) may be introduced into the CAR-MSC. For example, an exogenous nucleic acid encoding one or more immunosuppressive polypeptides, one or more regenerative polypeptides, and / or one or more distribution polypeptides may be introduced into the MSC by transduction (e.g., viral transduction) or transfection. In some embodiments, a nucleic acid (e.g., an exogenous nucleic acid) encoding one or more immunosuppressive polypeptides, one or more regenerative polypeptides, and / or one or more distribution polypeptides may be modified in the CAR-MSC to increase the expression level of one or more immunosuppressive polypeptides, one or more regenerative polypeptides, and / or one or more distribution polypeptides in the CAR-MSC. For example, in MSCs, exogenous nucleic acids encoding immunosuppressive polypeptides, regenerative polypeptides, or distribution polypeptides may be modified by gene editing techniques (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease (Cas) systems, transcription activator-like effector nucleases (TALEN) systems, zinc finger nuclease (ZFN) systems, and base pair editing) to increase the expression level of one or more immunosuppressive polypeptides, increase the expression level of one or more regenerative polypeptides, and / or increase the level of one or more distribution polypeptides in CAR-MSCs.

[0031] In some aspects, CAR-MSCs described herein (e.g., CAR-MSCs with high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) may be engineered ex vivo to have high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides. For example, ex vivo engineering of CAR-MSCs to have high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides may include transducing isolated CAR-MSCs with a vector (e.g., a viral vector (e.g., a lentiviral vector, retroviral vector, adenoviral vector, or AAV vector)) encoding the polypeptide(s). For example, ex vivo manipulation of CAR-MSCs to have high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides may include transducing isolated CAR-MSCs with one or more lentiviral vectors encoding gene editing elements.

[0032] Examples of NFκB1 polypeptides that may be elevated in the CAR-MSCs described herein (e.g., CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) include polypeptides set forth in the National Center for Biotechnology Information (NCBI) database, for example, under 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 aspects, an NFκB1 polypeptide that may be elevated in the CAR-MSCs described herein may have the amino acid sequence set forth in SEQ ID NO: 12 (see, e.g., Example 2).

[0033] Examples of JUN polypeptides that may be elevated in the CAR-MSCs described herein (e.g., CAR-MSCs with high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) include polypeptides set forth in the NCBI database, for example, in Accession No. NM_002228 (Version NM_002228.4), Accession No. AL136985 (Version AL136985.11), and Accession No. CH471059 (Version CH471059.2). In some aspects, a JUN polypeptide that may be elevated in the CAR-MSCs described herein may have the amino acid sequence set forth in SEQ ID NO: 13 (see, e.g., Example 2).

[0034] Examples of RELB polypeptides that may be elevated in the CAR-MSCs described herein (e.g., CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) include polypeptides set forth in the NCBI database, for example, under accession numbers NM_001411087 (version NM_001411087.1) and NM_006509 (version NM_006509.4). In some aspects, a RELB polypeptide that may be elevated in the CAR-MSCs described herein may have the amino acid sequence set forth in SEQ ID NO: 14 (see, e.g., Example 2).

[0035] Examples of IRF1 polypeptides that may be elevated in the CAR-MSCs described herein (e.g., CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) include polypeptides set forth in the NCBI database, for example, in 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 aspects, an IRF1 polypeptide that may be elevated in the CAR-MSCs described herein may have the amino acid sequence set forth in SEQ ID NO: 15 (see, e.g., Example 2).

[0036] Examples of TNFα polypeptides that may be elevated in the CAR-MSCs described 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 distribution polypeptides) include polypeptides set forth in the NCBI database, e.g., under accession number NM_000594 (version NM_000594.4). In some aspects, a TNFα polypeptide that may be elevated in the CAR-MSCs described herein may have the amino acid sequence set forth in SEQ ID NO: 16 (see, e.g., Example 2).

[0037] Examples of IL-10 polypeptides that can be elevated in the CAR-MSCs described herein (e.g., CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) include polypeptides set forth in the NCBI database, for example, in Accession No. NM_000572 (Version NM_000572.3) and Accession No. NM_001382624 (Version NM_001382624.1). In some aspects, an IL-10 polypeptide that can be elevated in the CAR-MSCs described herein may have the amino acid sequence set forth in SEQ ID NO: 17 (see, e.g., Example 2).

[0038] Examples of FGF-2 polypeptides that may be elevated in the CAR-MSCs described 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 distribution polypeptides) include polypeptides set forth in the NCBI database, for example, in Accession No. NM_001361665 (Version NM_001361665.2) and Accession No. NM_002006 (Version NM_002006.6). In some aspects, an FGF-2 polypeptide that may be elevated in the CAR-MSCs described herein may have the amino acid sequence set forth in SEQ ID NO: 18 (see, e.g., Example 2).

[0039] Examples of G-CSF polypeptides that can be elevated in the CAR-MSCs described herein (e.g., CAR-MSCs with high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) include polypeptides set forth in the NCBI database, for example, at accession number NM_000759 (version NM_000759.4), accession number NM_001178147 (version NM_001178147.2), and accession number NM_172219 (version NM_172219.3). In some aspects, a G-CSF polypeptide that can be elevated in the CAR-MSCs described herein may have the amino acid sequence set forth in SEQ ID NO: 19 (see, e.g., Example 2).

[0040] Examples of GM-CSF polypeptides that may be elevated in the CAR-MSCs described 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 distribution polypeptides) include polypeptides set forth in the NCBI database, for example, in Accession No. NM_000758 (Version NM_000758.4), Accession No. AC003959 (Version AC003959.1), and Accession No. DQ789232 (Version DQ789232.1). In some aspects, a GM-CSF polypeptide that may be elevated in the CAR-MSCs described herein may have the amino acid sequence set forth in SEQ ID NO: 20 (see, e.g., Example 2).

[0041] Examples of eotaxin polypeptides that may be elevated in the CAR-MSCs described 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 distribution polypeptides) include polypeptides set forth in the NCBI database, for example, in Accession No. NM_002986 (Version NM_002986.3), Accession No. AC003959 (Version AC003959.1), and Accession No. CH471062 (Version CH471062.2). In some aspects, an eotaxin polypeptide that may be elevated in the CAR-MSCs described herein may have the amino acid sequence set forth in SEQ ID NO: 21 (see, e.g., Example 2).

[0042] Examples of Gal-9 polypeptides that may be elevated in the CAR-MSCs described 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 distribution polypeptides) include polypeptides set forth in the NCBI database, for example, at 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 aspects, a Gal-9 polypeptide that may be elevated in the CAR-MSCs described herein may have the amino acid sequence set forth in SEQ ID NO: 22 (see, e.g., Example 2).

[0043] Examples of PD-1 polypeptides that may be elevated in the CAR-MSCs described 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 distribution polypeptides) include polypeptides set forth in the NCBI database, for example, in Accession No. NM_005018 (Version NM_005018.3), Accession No. AF363458 (Version AF363458.1), and Accession No. EF064716 (Version EF064716.1). In some aspects, a PD-1 polypeptide that may be elevated in the CAR-MSCs described herein may have the amino acid sequence set forth in SEQ ID NO: 23 (see, e.g., Example 2).

[0044] Examples of TIM-3 polypeptides that may be elevated in the CAR-MSCs described 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 distribution polypeptides) include polypeptides set forth in the NCBI database, for example, in Accession No. NM_032782 (Version NM_032782.5), Accession No. CQ834184 (Version CQ834184.1), and Accession No. AC011377 (Version AC011377.6). In some aspects, a TIM-3 polypeptide that may be elevated in the CAR-MSCs described herein may have the amino acid sequence set forth in SEQ ID NO: 24 (see, e.g., Example 2).

[0045] Examples of CXCR3 polypeptides that may be elevated in the CAR-MSCs described herein (e.g., CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) include polypeptides set forth in the NCBI database, for example, in 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 aspects, a CXCR3 polypeptide that may be elevated in the CAR-MSCs described herein may have the amino acid sequence set forth in SEQ ID NO: 25 (see, e.g., Example 2).

[0046] Examples of CXCR4 polypeptides that may be elevated in the CAR-MSCs described herein (e.g., CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) include polypeptides set forth in the NCBI database, for example, in 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 aspects, a CXCR4 polypeptide that may be elevated in the CAR-MSCs described herein may have the amino acid sequence set forth in SEQ ID NO: 26 (see, e.g., Example 2).

[0047] Examples of CTLA4 polypeptides that may be elevated in the CAR-MSCs described 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 distribution polypeptides) include polypeptides set forth in the NCBI database, for example, under 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 aspects, a CTLA4 polypeptide that may be elevated in the CAR-MSCs described herein may have the amino acid sequence set forth in SEQ ID NO: 91 (see, e.g., Example 2).

[0048] Examples of TLR3 polypeptides that may be elevated in the CAR-MSCs described herein (e.g., CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) include polypeptides set forth in the NCBI database, e.g., under 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 aspects, a TLR3 polypeptide that may be elevated in the CAR-MSCs described herein may have the amino acid sequence set forth in SEQ ID NO: 92 (see, e.g., Example 2).

[0049] Examples of TLR4 polypeptides that may be elevated in the CAR-MSCs described 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 distribution polypeptides) include polypeptides set forth in the NCBI database, e.g., under accession numbers NM_003266 (version NM_003266.4), NM_138557 (version NM_138557.3), and NM_138554 (version NM_138554.4). In some aspects, a TLR4 polypeptide that may be elevated in the CAR-MSCs described herein may have the amino acid sequence set forth in SEQ ID NO: 93 (see, e.g., Example 2).

[0050] Examples of TLR9 polypeptides that may be elevated in the CAR-MSCs described 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 distribution polypeptides) include polypeptides set forth in the NCBI database, for example, under accession numbers NM_017442 (versions NM_017442.3 and NM_017442.4) and NM_031178 (version NM_031178.2). In some aspects, a TLR9 polypeptide that may be elevated in the CAR-MSCs described herein may have the amino acid sequence set forth in SEQ ID NO: 94 (see, e.g., Example 2).

[0051] Examples of TNFR2 polypeptides that may be elevated in the CAR-MSCs described herein (e.g., CAR-MSCs with high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) include polypeptides set forth in the NCBI database, for example, under accession numbers NM_001066 (version NM_001066.2) and NM_011610 (version NM_011610.3). In some aspects, a TNFR2 polypeptide that may be elevated in the CAR-MSCs described herein may have the amino acid sequence set forth in SEQ ID NO: 95 (see, e.g., Example 2).

[0052] This document also provides materials and methods for treating a mammal in need of immunosuppression (e.g., a human suffering from or at risk of developing one or more autoimmune diseases (e.g., GVHD)). In some aspects, a mammal (e.g., a human) in need of immunosuppression can suffer from (or be at risk of developing) GVHD. For example, one or more CAR-MSCs described herein (e.g., one or more CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) can be administered (e.g., by adoptive transfer) to a mammal having one or more autoimmune diseases to reduce the severity of the immune response in the mammal. Any suitable method may be used to identify a mammal as a subject in need of immunosuppression. Once identified as a subject in need of immunosuppression, one or more CAR-MSCs described herein can be administered to a mammal (e.g., a human) 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 percent.

[0053] In some aspects, when treating a mammal (e.g., a human) in need of immunosuppression as described herein (e.g., a human suffering from or at risk of developing one or more autoimmune diseases (e.g., GVHD)), the treatment can be effective in reducing inflammation 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 inflammation in a mammal suffering from one or more autoimmune disorders (e.g., GVHD) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.

[0054] In some aspects, 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 (e.g., GVHD)) as described herein, the treatment can be effective to increase the number of regulatory T cells (Tregs) in the mammal (e.g., in a target tissue in the mammal). For example, the methods and materials described herein can be used to increase the number of Tregs present in a mammal in need of immunosuppression (e.g., a human suffering from or at risk of developing one or more autoimmune diseases (e.g., GVHD)) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some aspects, the number of Tregs present in the mammal is not reduced.

[0055] In some aspects, 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 (e.g., GVHD)) as described herein, the treatment can be effective in reducing the number of activated T cells (e.g., CD4+ T cells and / or CD8+ T cells) 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 number of activated T cells (e.g., CD4+ T cells and / or CD8+ T cells) present in a mammal in need of immunosuppression (e.g., a human suffering from or at risk of developing one or more autoimmune diseases (e.g., GVHD)) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some aspects, the number of activated T cells (e.g., CD4+ T cells and / or CD8+ T cells) present in the mammal is not increased.

[0056] In some aspects, 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 (e.g., GVHD)) as described herein, the treatment can be effective in decreasing the rate of T cell proliferation (e.g., proliferation of activated T cells) in the mammal (e.g., in a target tissue in the mammal). For example, the methods and materials described herein can be used to decrease the rate of T cell proliferation in a mammal in need of immunosuppression (e.g., a human suffering from or at risk of developing one or more autoimmune diseases (e.g., GVHD)) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some aspects, the rate of T cell proliferation in the mammal is not increased.

[0057] Any suitable mammal in need of immunosuppression (e.g., a human suffering from or at risk of developing one or more autoimmune diseases (e.g., GVHD)) can be treated as described herein. Examples of mammals that may require immunosuppression and that can be treated as described herein include, but are not limited to, humans, non-human primates (e.g., monkeys), dogs, cats, horses, cows, pigs, sheep, mice, and rats. For example, one or more CAR-MSCs described herein (e.g., one or more CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) can be administered (e.g., by adoptive transfer) to a human suffering from one or more autoimmune diseases to treat the human.

[0058] Any suitable 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 aspects, the autoimmune disease can be GVHD (e.g., acute GVHD or chronic GVHD). The GVHD can be allogeneic GVHD (alloGVHD) or autologous GVHD (autoGVHD). The GVHD can be any stage of GVHD. In some aspects, the GVHD can be associated with a transplant (e.g., post-transplant). The transplant can be an allogeneic transplant or an autologous transplant. When treating a mammal suffering from (or at risk of developing) GVHD, the mammal may undergo any type of transplant, including allogeneic transplants, such as bone marrow transplants, stem cell transplants, and organ transplants, such as kidney and liver transplants.

[0059] In some aspects, a mammal can be identified as suffering from or at risk of developing one or more autoimmune diseases (e.g., GVHD). Any suitable method may be used to identify a mammal suffering from or at risk of developing one or more autoimmune diseases (e.g., GVHD). In some aspects, a mammal can be identified as suffering from (or a subject at risk of developing) GVHD. Any suitable method may be used to identify a mammal suffering from (or a subject at risk of developing) GVHD. After being identified as suffering from (or a subject at risk of developing) GVHD, the mammal can be administered (e.g., by adoptive transfer) or instructed for self-administration with one or more CAR-MSCs described herein (e.g., one or more CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) to treat GVHD in the mammal.

[0060] In some aspects, one or more CAR-MSCs described herein (e.g., one or more CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) can be administered (e.g., by adoptive transfer) to a mammal suffering from or at risk of developing GVHD to reduce the severity of GVHD in the mammal. In some aspects, reducing the severity of GVHD in a mammal can include reducing or eliminating one or more symptoms of GVHD (e.g., skin rash, immune-mediated pneumonia, intestinal inflammation, desquamation of the intestinal mucosa, severe diarrhea, abdominal pain, nausea, vomiting, and / or elevated bilirubin levels). In some aspects, reducing the severity of GVHD in a mammal can include reducing the stage of GVHD. The stage of GVHD may be assessed as described elsewhere (see, e.g., Jacobsohn et al., Orphanet. J. Rare Dis., 2:35 (2007)).

[0061] Any suitable method may be used to administer one or more CAR-MSCs described herein (e.g., one or more CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) to 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 (e.g., GVHD)). Exemplary methods of administering MSCs described herein to a mammal may include, but are not limited to, injection (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous injection, or local injection at a site of inflammation). For example, a composition comprising one or more CAR-MSCs described herein may be administered to a human by intravenous injection.

[0062] In some aspects, one or more CAR-MSCs described herein (e.g., one or more CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) may be the sole active ingredient for 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 (e.g., GVHD)). For example, a composition comprising one or more CAR-MSCs described herein (e.g., one or more CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) may comprise one or more CAR-MSCs described herein as the sole active ingredient for suppressing an immune response in 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 (e.g., GVHD)).

[0063] In some aspects, one or more CAR-MSCs described herein (e.g., one or more CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) may be administered in combination with one or more additional agents that can be used to treat 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 (e.g., GVHD)). In some aspects, an agent that can be used to suppress an immune response can be anti-inflammatory. In some aspects, an agent that can be used to suppress an immune response can be an immunosuppressant. Examples of agents that can be used in combination with one or more CAR-MSCs described herein include, but are not limited to, corticosteroids (e.g., prednisone and methylprednisolone), azathioprine, mercaptopurine, cyclosporine, infliximab, adalimumab, golimumab, and vedolizumab. In some embodiments, one or more CAR-MSCs described herein may be administered substantially simultaneously with one or more additional agents. For example, a composition comprising one or more CAR-MSCs described herein (e.g., one or more CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) may include one or more additional agents that can be used to suppress an immune response in a mammal. In some embodiments, one or more CAR-MSCs described herein may be administered a first time and one or more additional agents may be administered a second time, or vice versa.

[0064] In some aspects, one or more CAR-MSCs described herein (e.g., one or more CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides) can be used to treat a mammal (e.g., a human) suffering from a disease or disorder not characterized by inflammation. For example, a CAR-MSC described herein can be used to target diseased tissue in a mammal (e.g., a human not requiring immunosuppression) to treat the diseased tissue. In some aspects, a CAR-MSC described herein can be used to target degenerated tissue in a mammal (e.g., a human not requiring immunosuppression) to treat the degenerated tissue. For example, the CAR-MSCs may express a CAR capable of targeting a neural-specific antigen (e.g., AMPA) to target the MSCs to neural tissue in a mammal (e.g., a mammal suffering from a neurodegenerative disease (e.g., amyotrophic lateral sclerosis (ALS))), and may include a regenerative signaling domain (e.g., a BDNF signaling domain), and may further have (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 distribution polypeptides, thereby treating the mammal. In some aspects, the CAR-MSCs described herein may be used to target fibrotic tissue in a mammal (e.g., a human not requiring immunosuppression) to treat fibrotic tissue. For example, CAR-MSCs may express a CAR capable of targeting a cartilage-specific antigen (e.g., fibroblast activation protein (FAP) or signaling lymphocyte activation molecule 7 (SLAMF7, also called CS-1)) to target MSCs to fibrotic tissue in a mammal (e.g., a mammal suffering from one or more fibrotic diseases (e.g., cardiac fibrosis, liver fibrosis, pulmonary fibrosis, genital fibrosis, or skin fibrosis)), and may further have (or be engineered to have) high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides, thereby treating the mammal.In some aspects, the CAR-MSCs described herein may be used to target cardiac tissue in a mammal (e.g., a human not requiring immunosuppression) to treat cardiac disease. For example, the CAR-MSCs may express a CAR capable of targeting a cardiac-specific antigen (e.g., HER2) and include a regenerative signaling domain (e.g., a VEGFR2 signaling domain) to target MSCs to cardiac tissue in a mammal (e.g., a mammal suffering from cardiac inflammation and / or cardiac degeneration), and may further have (or be engineered to have) high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides, thereby treating the mammal.

[0065] This document also provides kits that include one or more materials described herein. In some aspects, the kits can include one or more CAR-MSCs described herein (e.g., one or more CAR-MSCs having high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides). For example, one or more CAR-MSCs described herein can be combined with packaging materials to form a kit. For example, one or more constructs (e.g., nucleic acid constructs) described herein (e.g., a construct encoding a CAR capable of binding to a tissue-specific antigen (e.g., an epithelial-specific antigen) and a construct encoding one or more immunosuppressive polypeptides, one or more regenerative polypeptides, and / or one or more distribution polypeptides) can be combined with packaging materials to form a kit. The packaging materials included in such kits typically include instructions or a label describing how the compositions can be used, for example, in adoptive transfer to treat a mammal in need of immunosuppression (e.g., a human suffering from or at risk of developing one or more autoimmune diseases (e.g., GVHD)). In some aspects, the materials provided in the kits described herein can be used to treat a mammal (e.g., a human) suffering from (or at risk of developing) GVHD as described herein. In some aspects, the packaging materials included in such kits may include instructions and / or a label describing how the compositions described herein can be used.For example, a kit may include instructions and / or a label describing how a composition described herein can be used to express one or more CARs, as well as methods for expressing one or more immunosuppressive polypeptides, one or more regenerative polypeptides, and / or one or more distribution polypeptides in MSCs to engineer CAR-MSCs described herein (e.g., CAR-MSCs with high levels of one or more immunosuppressive polypeptides, high levels of one or more regenerative polypeptides, and / or high levels of one or more distribution polypeptides). In some aspects, the packaging material included in such a kit may include instructions and / or a label describing how a CAR-MSC described herein can be used. For example, the packaging material included in such a kit may include instructions and / or a label describing how a CAR-MSC described herein can be used in adoptive transfer to treat a mammal in need of immunosuppression as described herein (e.g., suffering from or at risk of developing one or more autoimmune diseases (e.g., GVHD)). In some aspects, kits (e.g., kits that include instructions and / or labels describing how the CAR-MSCs described herein can be used for adoptive transfer) may include materials for use in the adoptive transfer procedure.

[0066] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0067] (Example) Example 1: Chimeric Antigen Receptor Engineering of Adipose-Derived Mesenchymal Stromal Cells (CAR-MSC) for Enhanced Immunosuppression Current MSC therapies can be ineffective due to suboptimal homing and immunosuppressive capacity after therapeutic administration. MSCs were transduced with lentivirus to stably express a CAR construct to generate 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 the CD28 signaling domain into EcCAR-MSCs resulted in improved T cell suppression in both tumor and GVHD models.

[0068] Materials and Methods Cell lines, primary PBMC / T cells and primary MSCs Primary human adipose-derived (Ad) MSCs were obtained and cultured in StemXVivo Mesenchymal Stromal Cell Growth Medium (R&D Systems, Minneapolis, MN). Human epithelial breast cancer cell line MCF7 was obtained from ATCC (CRL-3006, Manassas, VA, USA) and cultured in D10 (DMEM Gibco, Gaithersburg, MD, US) with 10% fetal bovine serum (FBS, MilliporeSigma, Ontario, Canada) and 1% penicillin-streptomycin-glutamine (Gibco, Gaithersburg, MD, US). MCF7 cells were irradiated and confirmed to be Ecad positive by flow cytometry.

[0069] The mantle cell lymphoma cell line JeKo-1 and the acute lymphoblastic leukemia cell line Nalm6 were purchased from ATCC. JeKo-1 and Nalm6 cells were cultured in R10 (RPMI 1640, Gibco, Gaithersburg, MD, USA) with 10% or 20% FBS (Millipore Sigma, Ontario, Canada), respectively, and 1% penicillin-streptomycin-glutamine (Gibco, Gaithersburg, MD, USA). For use in in vivo experiments, the cell lines were transduced with luciferase-ZsGreen lentivirus (Addgene, Cambridge, MA, USA). Cell lines were cultured for up to 10 passages, with fresh aliquots thawed every 7–8 weeks. Cell lines were authenticated by the manufacturer and regularly phenotyped by flow cytometry. Cell lines were tested monthly for mycoplasma infection. Peripheral blood mononuclear cells (PBMCs) were isolated from anonymized healthy donor blood apheresis cones using SepMate tubes (STEMCELL Technologies, Vancouver, Canada). T cells were isolated by negative magnetic bead selection using the EasySep™ Human T Cell 80 Isolation Kit (STEMCELL Technologies).

[0070] Primary T cells and PBMCs were cultured in T cell 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 selection for in vitro coculture. Freshly isolated human PBMCs were infused intravenously for in vivo experiments.

[0071] CAR design and virus production All MSCs were transduced with lentiviral vectors encoding specifically designed CAR constructs downstream of the EF1α promoter. Transduction optimization was performed using the CART19 construct. The CAR19 used contained a CD19-directed scFv derived from the FMC-63 clone fused to the 41BB and CD3ζ signaling domains (FMC63-41BB-ζ). VSV-g-pseudotyped second-generation lentivirus was generated by transfecting HEK-293T cells with the CAR19 transgene via Lipofectamine transfection, followed by routine lentiviral recovery, concentration, and functional titer assay. To induce MSC immunomodulatory activation, the CAR construct was designed to contain a human / canine Ecad scFv (Figure 6) and the CD28 intracellular signaling domain (Figure 7). CAR plasmids were generated and sequence verified by Addgene (Watertown, MA, USA). To optimize CAR expression and MSC proliferation, pre-seeded Ad-MSCs (at a concentration of 250,000 cells / well) were transduced in 6-well plates with lentiviral particles at a multiplicity of infection (MOI) of 3 and various concentrations of protamine sulfate (25 μg / μL, 50 μg / μL, or 100 μg / μL).

[0072] T cell suppression assay The ability of CAR-MSCs to inhibit activated T cells was examined using a T cell suppression assay. To provide antigen-specific stimulation to CAR-MSCs, untransduced (UTD)-MSCs or EcCAR-MSCs (suppressors) were co-cultured with activated T cells (effectors) in the presence or absence of soluble Ecad (stimulator). T cells were isolated from PBMCs of healthy donors using negative selection magnetic beads. Isolated T cells were nonspecifically activated using CD3 stimulator beads at a 3:1 ratio of beads to T cells in T cell 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). After 24 hours, stimulated T cells (effectors) were co-cultured with UTD-MSCs or EcCAR-MSCs (suppressors) alone or with soluble Ecad protein (stimulator) to specifically activate EcCAR-MSCs via CAR in StemXVivo serum-free mesenchymal stromal cell growth medium (R&D Systems, Minneapolis, MN, USA). Cells were co-cultured as described above with 100,000 T cells per 96-well plate at a 1:10 MSC:T cell ratio. After different co-culture times as indicated in individual experiments, cells were harvested and analyzed by flow cytometry.

[0073] Multichannel Flow Cytometry Staining was performed for flow cytometry. Briefly, MSCs were isolated and cultured, then transduced with CAR to generate CAR-MSCs and co-cultured with T cells / PBMC immune cells in 96-well plates for 24 hours or in 6-well plates for longer-term culture. After the desired co-culture period, adherent MSC cells were cultured in 75cm plates. 2Cells were detached with 10 mL of Accutase per surface area and incubated at 37°C for 10–15 minutes. After detachment, all well contents were centrifuged, washed with flow buffer (PBS, 2% FBS v / v, and 1% sodium azide v / v), and stained with the desired antibody mixture for 15 minutes in the dark. After the final wash step, cells were analyzed for the desired surface markers, and positivity was determined through negative gating with Fluorescence Minus One (FMO) control wells. Absolute cell counts were obtained using volumetric measurements. Cells were gated using the following: SSC vs. FSC plots for cell separation by size and complexity, FSC-H vs. FSC-A plots to remove doublets, and SSC vs. V500 channel live / dead fixable aqua stain to remove dead cells. Cell subset characterization was performed based on a predesigned antibody panel optimized for the stained samples and used.

[0074] Anti-human antibodies were purchased from Biolegend, eBioscience, or BD Biosciences (San Diego, CA, USA). Samples were prepared for flow cytometry as described elsewhere (see, e.g., Teshima et al., Biol Blood Marrow Transplant 22:11-16 (2016)). All antibodies used to stain samples are listed in Table 1. Flow cytometry was performed on a three-laser CytoFLEX (Beckman Coulter, Chaska, MN, USA) using singlet gating, and live cells were determined using Live / Dead Aqua staining (L34966, Thermo Fisher Scientific, Waltham, MA, USA). All gating analyses were performed using FlowJo X10.0.7r2 software (Ashland, OR, USA) and Kaluza Analysis software (Indianapolis, Indiana, USA).

[0075] Table 1. Antibodies [Table 1]

[0076] In vivo mouse studies 6-8 week old female and male immunodeficient NOD-SCID-γ mice - / - NSG mice were obtained from Jackson Laboratories. All cells were injected via tail vein injection or intraperitoneal injection in 100-200 μL of PBS via a syringe. Images of the mice were taken with a bioluminescence imager using an IVIS® Lumina S5 Imaging System (PerkinElmer, Hopkinton, MA, USA) to verify the engraftment of luciferase-positive tumor models (CD19-positive Nalm6 / JeKo1 cells) in cancer xenograft models, or luciferase-positive CAR-MSC cells in MSC-persistence xenograft models. Images were taken 10 minutes after intraperitoneal injection of 10 μL / g of D-luciferin (15 mg / mL, Gold Biotechnology, St. Louis, MO, USA). For the mouse tumor models, NSG mice were engrafted with CD19-positive luciferase-positive JeKo-1 cells or Nalm6 cells (1 × 10 6 (5 × 10 cells were administered intravenously). Engraftment was confirmed by bioluminescence imaging 1–2 weeks after injection. All mice were then injected with irradiated Ecad-positive MCF-7 cells (5 × 10 cells to stimulate EcCAR-MSCs). 6 cells) and CART19 cells (1 x 10 cells administered intraperitoneally) as a strategy to treat CD19-positive tumors. 6 Mice were then randomized based on bioluminescence imaging, an indicator of tumor burden, to receive UTD-MSCs, EcCAR-MSCs, or no additional treatment (Figure 15). Serial bioluminescence imaging was performed sequentially to assess residual disease and determine the antitumor activity of CART19 cells.

[0077] In a GVHD mouse model, allogeneic human PBMCs (20–30 × 10 6GVHD was induced in NSG mice by intravenous injection of 1) UTD-MSCs or 2) CAR-MSCs (1 × 10 cells on days 10 and 20). 6 Mice were treated with either MSCs or MSCs intraperitoneally (intraperitoneally), and 3) control mice were also treated without MSCs (Figure 16). Body weight and clinical GVHD scoring (considering body weight, posture, diarrhea, activity level, coat condition, and skin integrity) were examined for GVHD progression in each experimental group (n = 5). Blood was collected from the mice via tail vein bleeding (approximately 100 μL), and 70 μL of blood was used for flow cytometry analysis. RBC lysis was performed using 1:10 BD FACS Lyse buffer (BD Biosciences, San Jose, CA, USA). Cells were then washed with flow buffer and incubated with specific antibody mixtures at room temperature in the dark before flow analysis using a CytoFLEX (Beckman Coulter, Chaska, MN, USA). The remaining blood was centrifuged at 13,000 rpm for 10 minutes, and serum was separated for cytokine analysis.

[0078] Canine studies The Ecad-targeting CAR was based on a human / canine cross-reactive Ecad-directed scFv. This scFv was generated using a phage display method, and cross-reactivity with both canine and human Ecad was verified (Figure 7). EcCAR-MSCs were tested in healthy beagle dogs. Subjects received EcCAR-MSCs (1 × 10 6 Cells / kg) were injected intraperitoneally. Subjects were observed daily, body weight was checked, and on day 3, colon biopsies were performed and serial blood samples were drawn to examine human CD105-positive cells by IHC and to assess MSC homing to canine Ecad-positive cells.

[0079] RNA isolation, sequencing and analysis EcCAR-MSC or UTD-MSC cells were transduced with or without 250 ng / mL of recombinant human Ecad Fc chimera (BioLegend, San Diego, CA, USA) as a CAR-specific stimulus for 24 hours. After in vitro culture, MSCs were detached for RNA isolation using a QIAGEN miRNeasy micro kit (product number #217084, QIAGEN, Germantown, MD, USA). To ensure the rigor of the results, bulk RNA sequencing was performed on MSCs from three different biological donor replicates in both the UTD-MSC and EcCAR-MSC groups.

[0080] Total RNA was prepared using the SMARTer stranded total RNA-seq kit v2, Pico input mammalian (Takara, Mountain View, CA, USA). Total RNA (three samples per lane) was sequenced on an Illumina HiSeq 4000 (Illumina, San Diego, CA, USA). Library preparation and sequencing were performed. The FastQC files generated for each sample were quality checked using FastQC v0.11.8. Cutadapt v1.18 was used to trim and remove adapter sequences. The generated files were verified for adapter removal and quality using FastQC v0.11.8.

[0081] Paired fragment reads from the trimmed FastQ files were mapped to the latest human reference genome (GRCh38) downloaded from the NCBI database. A genome index file was constructed and assigned using STAR v2.5.4b. Gene expression counts for each gene were generated using HTSeq (Python 3.6.5). Gene counts were normalized (geometric mean), and expression change analysis was calculated using DESeq2 (R v3.6.1, R-project.org / ) using an adjusted p-value of <0.05 as the statistical cutoff. Heatmaps were generated using pheatmap (cran.r-project.org / web / packages / pheatmap / index.html) with PCA generated using the ggplot2 tools (cran.r-project.org / web / packages / ggplot2 / index.html).

[0082] Gene set enrichment analysis was performed using Enrichr. Protein-protein interaction networks were generated using QIAGEN Ingenuity Pathway Analysis.

[0083] Cytokine analysis Cytokine analysis was performed on serum samples collected from mice 15 days after MSC or control treatment. Serum was centrifuged at 10,000 × g for 5 minutes to remove precipitate. Serum was diluted 1:2 with serum matrix before plating and according to the manufacturer's instructions for the Milliplex Human Cytokine / Chemokine Magnetic Bead Premixed 38 Plex 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, WA, USA). Significant differences were determined and reported using Prism Graph Pad (La Jolla, CA, USA).

[0084] statistical analysis To perform appropriate statistical analysis, in vitro and in vivo experiments were performed with technical and biological replicates. Two-way or one-way ANOVA was used to assess differences in surface marker expression between in vitro EcCAR-MSCs and UTD-MSCs by percent expression and T cell suppressive capacity by assessing absolute T cell counts. ANOVA was also used to determine significant differences between in vivo EcCAR-MSC-treated and UTD-MSC-treated mice through comparison of luciferase-positive tumors and luciferase-positive MSCs, IVIS imaging, body weight comparisons, blood composition by flow cytometry, and GVHD clinical scores. Kaplan-Meier survival analysis was used to determine significant differences in survival outcomes in both tumor and GVHD xenograft models, and Cox regression analysis was used to adjust for confounding factors (e.g., gender). To allow for multiple comparisons between each individual group in each of the aforementioned analyses (above), Tukey's multiple comparison test was used as a supplement to the ANOVA-based analysis. For comparisons between two groups, a two-tailed unpaired Student's t-test was used instead of wayside analysis of variance. RNA-seq data were processed using the DeSeq2 program, where raw counts were normalized (geometric mean) between samples, and the Benjamini-Hochberg procedure was used to correct for multiple hypothesis testing. All relevant statistically significant comparisons are indicated with an asterisk, corresponding to a significance level below p<0.05 (*p<0.05, **p<0.005, ***p<0.0005, and ****p<0.0001) with a 95% confidence interval. The final data point without an asterisk indicates no significant difference (ns) between groups. Relevant data are plotted as the mean of all data points, with either the standard deviation (sd) or standard error of the mean (SEM) used to determine error bars. Experimental data were analyzed using Prism Graph Pad (La Jolla, CA, USA) and Microsoft Excel (Microsoft, Redmond, WA, USA).

[0085] result MSCs are successfully transduced and stably express the CAR. Using a lentiviral vector enhanced with a polyvalent cation enhancer, we established efficient transduction of MSCs to stably express a CAR. Transduction using the CAR19 lentiviral vector was optimized as a proof-of-concept for generating CAR-MSCs. Delivery of CAR-containing lentiviral particles was optimized with a protamine sulfate enhancer for efficiency and cell survival. Using a protamine sulfate enhancer and a lentivirus with an MOI of 3, we consistently achieved CAR transduction efficiencies >70% (Figure 1A). Following transduction optimization using CAR19, we developed an alternative CAR design for CAR-MSC-based autoimmune disease treatment. The primary CAR-MSC construct was directed against Ecad, a ligand expressed on inflammatory intestinal epithelial cells associated with GVHD. Because T cell migration is mediated by the interaction of Ecad, which is ubiquitously expressed on all epithelial cells, with CD103 (αE integrin) on the T cell surface, we determined whether redirecting MSCs to Ecad via CARs would protect host epithelial tissues by enhancing MSC specificity and activation at inflammatory sites of GVHD and other intestinal autoimmune diseases. Using phage display, we generated several Ecad-directed scFvs and identified a human- and canine-cross-reactive scFv to enable validation in a canine model (Figure 6). This clone was used to generate an Ecad-specific CAR construct (Figure 7) and transduced into MSCs to generate anti-Ecad CAR-MSCs (EcCAR-MSCs) with a transduction efficiency of >90% (Figure 1B). MSCs were derived from five different biological MSC donors (Table 2).

[0086] Table 2. MSC donor profile [Table 2]

[0087] To determine whether the immunosuppressive effect of CAR-MSCs could be enhanced through the inclusion of specific intracellular signaling domains within the CAR, a CD28 costimulatory CAR scaffold was used to incorporate the CD28 signaling domain into the CAR-MSC construct design, resulting in the creation of an Ecad-targeted CAR (EcCAR-MSC) using the CD28ζ CAR scaffold (Figure 7).

[0088] We demonstrated that stable CAR expression in EcCAR-MSCs was possible over multiple ex vivo passages (>3), and that transduction efficiencies of >90% were achieved across multiple MSC donors (Figure 1C-D; see Table 3 for a description of the GVHD clinical scoring system). To verify the suppressive ability of EcCAR-MSCs to activated T cells, we performed a T cell suppression assay. UTD-MSCs or EcCAR-MSCs (suppressors) were cocultured with activated T cells (effectors). EcCAR-MSCs induced superior T cell suppression in a dose-dependent manner compared with UTD-MSCs (Figure 1E). We verified the antigen-specific activation of EcCAR-MSCs by measuring T cell suppression in the presence or absence of Ecad protein, which stimulates the anti-Ecad scFv on the CAR. To ensure reproducibility, both soluble Ecad and an Ecad-positive MCF7 cell line were used as sources of Ecad antigen. Stimulation of both soluble and cell-derived antigen-specific EcCAR-MSCs resulted in enhanced T cell suppression ( Figures 1G-1G ).

[0089] Table 3. GVHD Clinical Scoring System [Table 3]

[0090] MSC manipulation does not induce differentiation, and CAR-MSCs retain stemness. To identify unwanted differentiation induced by CAR-MSC manipulation, we verified the stem phenotype and pathway enrichment analysis of EcCAR-MSCs. Using flow cytometry, the stem phenotype of EcCAR-MSCs (CD105+, CD73+, CD90+, CD34-, CD14-, and CD45-) was verified based on the minimal criteria for maintaining MSC stemness set by the International Society for Cell Therapy (see, e.g., Dominici, et al., Cytotherapy 8, 315-317 (2006)). No significant differences were observed between CAR-MSCs and UTD-MSCs (Figure 1H).

[0091] Gene expression change analysis was performed comparing EcCAR-MSCs and UTD-MSCs by bulk RNA sequencing (RNAseq). Gene set enrichment analysis (GSEA) using the CellMarker Augmented gene set revealed enrichment of adipose-derived stem cells and non-committed somatic stem cell subsets in EcCAR-MSCs (Figure 1I). Conversely, differentiation pathways for differentiated cell lineages, such as smooth muscle, neural, or vascular, were downregulated by GSEA (Figure 1J). These results indicate that differentiation induced by transduction or CAR expression is limited and that the stem phenotype is maintained after CAR-MSC manipulation.

[0092] EcCAR-MSCs exhibit excellent immunosuppression in in vivo tumor and GVHD models. To verify the immunosuppressive potential of CAR-MSCs in vivo, we used multiple tumor and GVHD models to ensure reproducibility and rigor of our results. We used the CD19-positive mantle cell lymphoma JeKo-1 and CD19-positive acute lymphoblastic leukemia Nalm6 tumor xenograft models to test whether CAR-MSCs suppressed the potential antitumor activity of CART19 cells (Figure 2A). Either luciferase-positive JeKo-1 or luciferase-positive Nalm6 cells were intravenously administered into immunodeficient NSG mice to establish xenograft models. Bioluminescence imaging was performed one week later to confirm engraftment, and mice were randomized and treated with CART19 alone, CART19 in combination with UTD-MSCs, or CART19 in combination with EcCAR-MSCs. Tumor burden was assessed via biweekly bioluminescence imaging. Mice engrafted with Nalm6 and treated with CART19 and EcCAR-MSCs showed higher tumor burden (Figure 2B) and decreased overall survival compared to mice treated with CART19 and UTD-MSCs or CART19 alone (Figure 2C). Similar results were demonstrated when Jeko-1 xenografts were used and treated with CART19 and EcCAR-MSCs or UTD-MSCs (Figures 8A-8B). Taken together, these data demonstrate superior suppression of effector T cells by EcCAR-MSCs through increased tumor burden and decreased survival outcomes.

[0093] Next, a xenograft GVHD model was established by intravenously administering human PBMCs to NSG mice. In addition to PBMCs, mice received PBS control, UTD-MSCs, or EcCAR-MSCs via intraperitoneal (ip) injection as GVHD treatment. Additional MSC or control treatments were administered intraperitoneally every two weeks (Figure 2D). Mice were examined for the development of clinical GVHD symptoms, such as diarrhea, motor function, posture, coat and skin integrity, weight change, 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 human T cell proliferation was analyzed. Compared to UTD-MSC treatment, EcCAR-MSC treatment prevented weight loss (Figure 2E), alleviated clinical GVHD (Figure 2F), suppressed T cell proliferation (Figure 2G), and improved overall survival (Figure 2H). Taken together, these results indicate that EcCAR-MSCs enhance T cell suppression, leading to the prevention of GVHD.

[0094] EcCAR-MSCs exhibit increased expression of critical transcription factors, suppressive cytokines, and inhibitory surface markers that induce an immunosuppressive environment. To elucidate the mechanism of enhanced EcCAR-MSC immunosuppression, CAR-MSCs were examined for their unique transcriptional activation, cytokine secretion profile, and surface markers that characterize their phenotype.

[0095] To determine the effect of antigen-specific CAR-MSC stimulation on the MSC transcriptome, RNAseq was performed on the following conditions: UTD-MSC (unstimulated), UTD-MSC (stimulated with soluble Ecad), EcCAR-MSC (unstimulated), and EcCAR-MSC (stimulated with soluble Ecad to stimulate CAR-MSC via CAR).

[0096] To elucidate the activation of intrinsic pathways in EcCAR-MSCs and stimulated EcCAR-MSCs, comprehensive gene expression change analysis was performed comparing: 1) EcCAR-MSCs (unstimulated) vs. UTD-MSCs (unstimulated) (to account for intrinsic differences induced by EcCAR transduction and insertion), 2) EcCAR-MSCs (stimulated with soluble Ecad) vs. EcCAR-MSCs (unstimulated) (to account for differences induced by antigen-specific stimulation of EcCAR-MSCs via CAR scFv), and 3) UTD-MSCs (stimulated with soluble Ecad) vs. UTD-MSCs (unstimulated) (to provide a control for potential Ecad-derived stimulation in the absence of Ecad-specific CAR scFv). Unstimulated EcCAR-MSCs and UTD-MSCs (Comparison 1) revealed activation of unique pathways in EcCAR-MSCs, with significant increases (at least 1.5-fold) in 355 genes (Figure 3A and Figure 9).

[0097] Gene expression change analysis between Ecad-stimulated and unstimulated EcCAR-MSCs revealed greater transcriptional activation, with at least a 1.5-fold increase in 1,996 genes (Figure 3A). This increase of nearly 6-fold more genes compared to Comparison 1 highlights the functional potential of CAR-derived stimulation in MSCs (Figure 9). PCA of expression profiles between Ecad-stimulated and unstimulated CAR-MSCs demonstrated clustering based on stimulation status (Figure 3B), whereas Ecad-stimulated and unstimulated UTD-MSCs (Comparison 3) clustered solely by MSC biological donor (Figure 3C). GSEA of activated pathways characterized by CAR stimulation was more strongly associated with the NFκB-mediated TNFα signaling pathway and the IL-10 anti-inflammatory signaling pathway (Figure 3D).

[0098] These pathways are related to classical CD28 signaling, which induces activation of downstream functional cascades in the nucleus through binding of intracellular proteins to the CD28 src-like domain. Comparison between unstimulated EcCAR-MSCs and UTD-MSCs suggested alterations in signaling pathways that are specific to the incorporation of the CAR signaling domain and CAR transduction, rather than CAR stimulation via its scFv.

[0099] Transcriptome analysis similarly demonstrated elevated NFκB1, JUN, RELB, and IRF1 transcription factors in Ecad-stimulated EcCAR-MSCs ( Fig. 3E ).

[0100] To validate our transcriptional findings, we measured human cytokines in the serum of UTD-MSC- and EcCAR-MSC-treated mice. Analysis of serum cytokine composition 17 days after MSC administration confirmed the RNA-seq data, showing a significant increase in TNFα and IL-10 cytokines in EcCAR-MSC-treated mice compared with UTD-MSC-treated mice. Fibroblast growth factor 2 (FGF-2) was also elevated in the serum of EcCAR-MSC-treated mice (Figures 4A and 10).

[0101] To characterize the effect of MSCs on human T cell subset composition in mouse studies, peripheral blood samples were collected on days 14 and 31 after treatment with UTD-MSCs or EcCAR-MSCs in a GVHD xenograft model (body weight loss was attenuated after treatment with EcCAR-MSCs) (see Figure 2D). Flow cytometry analysis revealed significant suppression of CD4+ and CD8+ human T cells in the EcCAR-MSC-treated group, correlating with the maintenance of body weight measurements (Figures 4B-4C). Analysis of the CD4+ to CD8+ ratio revealed a bias toward an increase in the CD4+ T cell subset and / or a decrease in the CD8+ cytotoxic T cell subset in EcCAR-MSC-treated mice compared with UTD-MSC-treated mice (Figure 4D). Long-term analysis of blood on day 31 after MSC administration revealed a significant increase in the percentage of Treg (human CD4+, CD25+, CD127-negative) cells in the EcCAR-MSC-treated group (Figure 4E).

[0102] EcCAR-MSC surface marker expression was examined by CyTOF, confirming the preservation of the stem phenotype and revealing changes in immunosuppressive surface markers and chemokines. Unlike UTD-MSCs, resting and Ecad-stimulated EcCAR-MSCs were classified into distinct populations by surface marker-based t-distribution stochastic neighborhood embedding statistical analysis, indicating functional CAR antigen-specific stimulation (Figures 4F–4G and 11). To verify whether this unique immunosuppressive surface marker profile was maintained in EcCAR-MSCs after coculture with effector cells, additional surface marker characterization via flow cytometry was performed on MSCs cocultured with PBMCs. Prior to surface marker evaluation, EcCAR-MSCs and UTD-MSCs were stimulated with soluble Ecad and cocultured with PBMCs for 5 days. These results demonstrated an increase in the unique EcCAR-MSC surface markers of the inhibitory receptors PD-1 and galectin-9 (Gal-9) (Figure 4H). CXCR3 and CXCR4 homing chemokines were also elevated in stimulated EcCAR-MSCs (Figure 4I). This chemokine elevation was supported by RNA-seq analysis, which showed enrichment of regulatory pathways for CXCR chemokine receptor binding and leukocyte chemotaxis pathways in EcCAR-MSCs (Figure S12).

[0103] Taken together, these mechanistic assays demonstrated enhanced immunosuppressive functions inherent to EcCAR-MSC generation, particularly upon CAR stimulation.

[0104] EcCAR-MSCs home to target sites and are safe in a canine model. A canine model was used using canine cross-reactive EcCAR-MSCs to ensure both the homing ability and safety of EcCAR-MSC administration (Figure 7). CAR-MSCs were engineered with human and canine cross-reactive anti-Ecad scFvs to generate EcCAR-MSCs. Human MSCs were transduced with lentivirus to produce EcCAR-MSCs, which were then expanded in vitro until intraperitoneal injection into healthy canine subjects. EcCAR-MSC scFv-mediated homing to canine Ecad-positive cells was evaluated through immunohistochemical staining analysis. Blood and organ toxicity was examined in subgroups throughout the study through hematological evaluation. Healthy beagle dogs were administered EcCAR-MSCs via intraperitoneal injection. Colon biopsies were collected 3 and 28 days after EcCAR-MSC administration, and canine Ecad-positive ligand and human CD105-positive MSCs were evaluated by immunohistochemical staining (Figure 5A). Human CD105-positive MSCs were present in colon tissue, overlapping with tissue areas with high canine Ecad expression, indicating homing to the target site (Figures 5B and 13). Next, EcCAR-MSC toxicity was assessed through serial measurements of whole blood counts, liver function, renal function, and body weight. EcCAR-MSC administration was not associated with weight loss, hematopoietic toxicity (Figure 5D), or organ toxicity (Figure 5E), demonstrating a high safety profile.

[0105] To further characterize the safety of CAR-MSCs in vivo and verify their proliferation kinetics and persistence, we generated luciferase-positive CAR-MSCs and administered them in combination with irradiated Ecad-positive MCF-7 cells. The proliferation, persistence, and elimination of EcCAR-MSCs were examined by sequential bioluminescence imaging in the presence and absence of antigen-specific stimulation (Figure 14). Similar to the GVHD xenograft model, human PBMCs were administered to NSG mice. Mice were then treated with 1) luciferase-positive UTD-MSCs alone, 2) luciferase-positive UTD-MSCs in combination with irradiated Ecad-positive MCF-7 cells, 3) luciferase-positive EcCAR-MSCs alone, or 4) luciferase-positive EcCAR-MSCs in combination with irradiated Ecad-positive MCF-7 cells (Figure 5G). Mice were then examined by sequential bioluminescence imaging every 2–3 days to detect MSC proliferation and persistence. These studies revealed no difference between the clearance time of UTD-MSCs and EcCAR-MSCs (~25 days), further supporting the safety profile of EcCAR-MSCs (Figures 5H-5I).

[0106] Example 2: Examples of amino acid sequences

[0107] Example of scFv sequence The CDR sequences are shown in bold and underlined type.

[0108] Example of anti-Ecad scFv (also called anti-hmcECAD.6) heavy chain TIFF2025538131000004.tif22158 (SEQ ID NO: 1) Light chain TIFF2025538131000005.tif15158 (SEQ ID NO: 2)

[0109] Example of anti-CD103 scFv heavy chain TIFF2025538131000006.tif22158 (SEQ ID NO: 3) Light chain TIFF2025538131000007.tif16156 (SEQ ID NO: 4)

[0110] Examples of anti-MOG scFv heavy chain TIFF2025538131000008.tif21159 (SEQ ID NO: 5) Light chain TIFF2025538131000009.tif16157 (SEQ ID NO: 6)

[0111] Examples of anti-AMPA scFv heavy chain TIFF2025538131000010.tif65159 (SEQ ID NO: 7) Light chain TIFF2025538131000011.tif23157TIFF2025538131000012.tif15158 (SEQ ID NO: 8)

[0112] Examples of anti-HER2 scFv TIFF2025538131000013.tif29159 (SEQ ID NO: 27)

[0113] Examples of anti-FAP scFv heavy chain TIFF2025538131000014.tif21158 (SEQ ID NO: 28) Light chain TIFF2025538131000015.tif15157 (SEQ ID NO: 29)

[0114] Examples of anti-CS1 scFv heavy chain MGWSSIILFLVATATGVHSQVQLQQPGAELVRPGASVKLSCKASGYSFTTYWMNWVKQRPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTA (SEQ ID NO: 30) Light chain TIFF2025538131000016.tif16157 (SEQ ID NO: 31)

[0115] Examples of transmembrane domain sequences

[0116] Example of CD28 transmembrane domain FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 9)

[0117] Example of a CD8 transmembrane domain IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 32)

[0118] Example of a TLR3 transmembrane domain FFMINTSILLIFIFIVLLIHF (SEQ ID NO: 72)

[0119] Examples of TLR4 transmembrane domains IGVSVLSVLVVSVVAVLVY (SEQ ID NO: 73)

[0120] Example of IFNGR1 transmembrane domain SLWIPVVAALLLFLVLSLVFI (SEQ ID NO: 96)

[0121] Example of IL10RA transmembrane domain VIIFFAFVLLLSGALAYCLAL (SEQ ID NO: 97)

[0122] Examples of TLR9 transmembrane domains FALSLLAVALGLGVPMLHHLC (SEQ ID NO: 98)

[0123] Examples of TNFR2 transmembrane domains FALPVGLIVGVTALGLLIIGVVNCVIMTQV (SEQ ID NO: 99)

[0124] Example signaling domain sequences

[0125] Examples of 4-1BB signaling domains (SEQ ID NO: 89) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0126] Examples of TLR3 signaling domains EGWRISFYWNVSVHRVLGFKEIDRQTEQFEYAAYIIHAYKDKDWVWEHFSSMEKEDQSLKFCLEERDFEAGVFELEAIVNSIKRSRKIIFVITHHLLKDPLCKRFKVHHAVQQAIEQNLDSIILVFLEEIPDYKLNHALCLRRGMFKSHCILNWPVQKERIGAFRHKLQVALGSKNSVH (SEQ ID NO: 74)

[0127] Examples of TIR3 signaling domains WRISFYWNVSVHRVLGFKEIDRQTEQFEYAAYIIHAYKDKDWVWEHFSSMEKEDQSLKFCLEERDFEAGVFELEAIVNSIKRSRKIIFVITHHLLKDPLCKRFKVHHAVQQAIEQNLDSIILVFLEEIPDYKLNHALCLRRGMF KSHCILNWPVQKERIGAFRHKLQVALGSKNSVHRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 75)

[0128] Examples of TLR4 signaling domains KFYFHLMLLAGCIKYGRGE (SEQ ID NO: 76)

[0129] Examples of TIR4 signaling domains NIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI (SEQ ID NO: 77)

[0130] Examples of TLR4 / TIR4 signaling domains KFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRIFFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI (SEQ ID NO: 100)

[0131] Examples of IFNGR1 signaling domains CFYIKKINPLKEKSIILPKSLISVVRSATLETKPESKYVSLITSYQPFSLEKEVVCEEPLSPATVPGMHTEDNPGKVEHTEELSSITEVVTTEENIPDVVPGSHLTPIERE SSSPLSSNQSEPGSIALNSYHSRNCSESDHSRNGFDTDSSCLESHSSLSDSEFPPNNKGEIKTEGQELITVIKAPTSFGYDKPHVLVDLLVDDSGKESLIGYRPTEDSKEFS (SEQ ID NO: 101)

[0132] Examples of IFNγ signaling domains MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRG (SEQ ID NO: 78)

[0133] Examples of IL10RA signaling domains QLYVRRRKKLPSVLLFKKPSPFIFISQRPSPETQDTIHPLDEEAFLKVSPELKNLDLHGSTDSGFGSTKPSLQTEEPQFLLPDPHPQADRTLGNREPPVLGDSCSGSSNSTDSGICLQEPSLSPSTGPTWEQQVGSNSRGQDDSGIDLVQNSEGRAGDTQ GGSALGHHSPPEPEVPGEEDPAAVAFQGYLRQTRCAEEKATKTGCLEEESPLTDGLGPKFGRCLVDEAGLHPPALAKGYLKQDPLEMTLASSGAPTGQWNQPTEEWSLLALSSCSDLGISDWSFAHDLAPLGCVAAPGGLLGSFNSDLVTLPLISSLQSSE (SEQ ID NO: 102)

[0134] Examples of TLR9 / TIR9 signaling domains GWDLWYCFHLCLAWLPWRGRQSGRDEDALPYDAFVVFDKTQSAVADWVYNELRGQLEECRGRWALRLCLEERDWLPGKTLFENLWASVYGSRKTLFVLAHTDRVSGLLRASFLLAQQRLLEDRKDVVVLVILSPDGRRSRYVRLRQRLCRQSVLLWPHQPSGQRSFWAQLGMALTRDNHHFYNRNFCQGPTAE (SEQ ID NO: 103)

[0135] Examples of TNFR2 signaling domains KKKPLCLQREAKVPHLPADKARGTQGPEQQHLLITAPSSSSSLESSASALDRRAPTRNQPQAPGVEASGAGEARASTGSSDSSPGGHGTQVNVTCIVNVCSSSDHSSQCSSQASSTMGDTDSSPSESPKDEQVPFSKEECAFRSQLETPETLLGSTEEKPLPLGVPDAGMKPS (SEQ ID NO: 104)

[0136] Examples of CD28 signaling domains RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 10)

[0137] Examples of CD3ζ signaling domains RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 11)

[0138] Examples of BDNF signaling domains KLARHSKFGMKGPASVISNDDDSASPLHHISNGSNTPSSSEGGPDAVIIGMTKIPVIENPQYFGITNSQLKPDTFVQHIKRHNIVLKRELGEGAFGKVFLAECYNLCPEQDKILVAVKTLKDASDNARKDFHREAELLTNLQHEHIVKFYGVCVEGDPLIMVFEYMKHGDLNKFLRAHGPDAVL MAEGNPPTELTQSQMLHIAQQIAAGMVYLASQHFVHRDLATRNCLVGENLLVKIGDFGMSRDVYSTDYYRVGGHTMLPIRWMPPESIMYRKFTTESDVWSLGVVLWEIFTYGKQPWYQLSNNEVIECITQGRVLQRPRTCPQEVYELMLGCWQREPHMRKNIKGIHTLLQNLAKASPVYLDILG (SEQ ID NO: 33)

[0139] Examples of VEGFR2 signaling domains LKLGKPLGRGAFGQVIEADAFGIDKTATCRTVAVKMLKEGATHSEHRALMSELKILIHIGHHLNVVNLLGACTKPGGPLMVIVEFCKFGNLSTYLRSKRNEFVPYKTKGARFRQGKDYVGAIPVDLKRRLDSITSSQSSASSGFVEEKSLSDVEEEEAPEDLYK DFLTLEHLICYSFQVAKGMEFLASRKCIHRDLAARNILLSEKNVVKICDFGLARDIYKDPDYVRKGDARLPLKWMAPETIFDRVYTIQSDVWSFGVLLWEIFSLGASPYPGVKIDEEFCRRLKEGTRMRAPDYTTPEMYQTMLDCWHGEPSQRPTFSELVEHLGN (SEQ ID NO: 79)

[0140] Example of a hinge domain sequence

[0141] Example of a CD8 hinge domain TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 34)

[0142] CD28 hinge domain example LEPKSCDKTHTCPPCPDPK (SEQ ID NO: 35)

[0143] Example of a leader sequence

[0144] Examples of leader polypeptides MALPVTALLLPLALLLHAARP (SEQ ID NO: 36)

[0145] Linker sequence examples

[0146] Examples of linker polypeptides EGKSSGSGSESKAS (SEQ ID NO: 37)

[0147] Example Polypeptide Sequences

[0148] Examples of NFκB1 Polypeptides MAEDDPYLGRPEQMFHLDPSLTHTIFNPEVFQPQMALPTADGPYLQILEQPKQRGFRFRYVCEGPSHGGLPGASSEKNKKSYPQVKICNYVGPAKVIVQLVTNGKNIHLHAHSLVGKHCEDGICTVTAGPKDMVVGFANLGILHVTKKKVFETLEARMTEACIRGYNPGLLVHPDLAYLQAEGGGDRQLGDREKELIRQAALQQTKEMDLSVVRLMFTAFLPDSTGSFTRRLEPVVSDAIYD SKAPNASNLKIVRMDRTAGCVTGGEEIYLLCDKVQKDDIQIRFYEEEENGGVWEGFGDFSPDTVHRQFAIVFKTPKYKDINITKPASVFVQLRRKSDLETSEPKPFLYYPEIKDKEEVQRKRQKLMPNFSDSFGGGSGAGAGGGGMFGSGGGGGGTGSTGPGYSFPHYGFPTYGGITFHPGTTKSNAGMCHGTMDTESKKDPEGCDKSDDKNTVNLFGKVIETTEQDQEPSEATVGNGEVTL TYATGTKEESAGVQDNLFLEKAMQLAKRHANALFDYAVTGDVKMLLAVQRHLTAVQDENGDSVLHLAIIHLHSQLVRDLLEVTSGLISDDIINMRNDLYQTPLHLAVITKQEDVVEDLLRAGADLSLLDRLGNSVLHLAAKEGHDKVLSILLKHKKAALLLDHPNGDGLNAIHLAMMSNSLPCLLLVAAGADVNAQEQKSGRTALHLAVEHDNISLAGCLLLEGDAHVDSTTYDGTTPLHI AAGRGSTRLAALLKAAGADPLVENFEPLYDLDDSWENAGEDEGVVPGTTPLDMATSWQVFDILNGKPYEPEFTSDDLLAQGDMKQLAEDVKLQLYKLLEIPDPDKNWATLAQKLGLGILNNAFRLSPAPSKTLMDNYEVSGGTVRELVEALRQMGYTEAIEVIQAASSPVKTTSQAHSLPLSPASTRQQIDELRDSDSVCDSGVETSFRKLSFTESLTSGASLLTLNKMPHDYGQEGPLEGKI (query number 12)

[0149] Examples of JUN Polypeptides MAEDDPYLGRPEQMFHLDPSLTHTIFNPEVFQPQMALPTADGPYLQILEQPKQRGFRFRYVCEGPSHGGLPGASSEKNKKSYPQVKICNYVGPAKVIVQLVTNGKNIHLHAHSLVGKHCEDGICTVTAGPKDMVVGFANLGILHVTKKKVFETLEARMTEACIRGYNPGLLVHPDLAYLQAEGGGDRQLGDREKELIRQAALQQTKEMDLSVVRLMFTAFLPDSTGSFTRRLEPVVSDAIYD SKAPNASNLKIVRMDRTAGCVTGGEEIYLLCDKVQKDDIQIRFYEEEENGGVWEGFGDFSPDTVHRQFAIVFKTPKYKDINITKPASVFVQLRRKSDLETSEPKPFLYYPEIKDKEEVQRKRQKLMPNFSDSFGGGSGAGAGGGGMFGSGGGGGGTGSTGPGYSFPHYGFPTYGGITFHPGTTKSNAGMCHGTMDTESKKDPEGCDKSDDKNTVNLFGKVIETTEQDQEPSEATVGNGEVTL TYATGTKEESAGVQDNLFLEKAMQLAKRHANALFDYAVTGDVKMLLAVQRHLTAVQDENGDSVLHLAIIHLHSQLVRDLLEVTSGLISDDIINMRNDLYQTPLHLAVITKQEDVVEDLLRAGADLSLLDRLGNSVLHLAAKEGHDKVLSILLKHKKAALLLDHPNGDGLNAIHLAMMSNSLPCLLLVAAGADVNAQEQKSGRTALHLAVEHDNISLAGCLLLEGDAHVDSTTYDGTTPLHI AAGRGSTRLAALLKAAGADPLVENFEPLYDLDDSWENAGEDEGVVPGTTPLDMATSWQVFDILNGKPYEPEFTSDDLLAQGDMKQLAEDVKLQLYKLLEIPDPDKNWATLAQKLGLGILNNAFRLSPAPSKTLMDNYEVSGGTVRELVEALRQMGYTEAIEVIQAASSPVKTTSQAHSLPLSPASTRQQIDELRDSDSVCDSGVETSFRKLSFTESLTSGASLLTLNKMPHDYGQEGPLEGKI (query number 13)

[0150] Examples of RELB Polypeptides MLRSGPASGPSVPTGRAMPSRRVARPPAAPELGALGSPDLSSLSLAVSRSTDELEIIDEYIKENGFGLDGGQPGPGEGLPRLVSRGAASLSTVTLGPVAPPATPPPWGCPLGRLVSPAPGPGPQPHLVITEQPKQRGMRFRYEC EGRSAGSILGESSTEASKTLPAIELRDCGGLREVEVTACLVWKDWPHRVHPHSLVGKDCTDGICRVRLRPHVSPRHSFNNLGIQCVRKKEIEAAIERKIQLGIDPYNAGSLKNHQEVDMNVVRICFQASYRDQQGQMRRMDPVLS EPVYDKKSTNTSELRICRINKESGPCTGGEELYLLCDKVQKEDISVVFSRASWEGRADFSQADVHRQIAIVFKTPPYEDLEIVEPVTVNVFLQRLTDGVCSEPLPFTYLPRDHDSYGVDKKRKRGMPDVLGELNSSDPHGIESKR RKKKPAILDHFLPNHGSGPFLPPSALLPDPDFFSGTVSLPGLEPPGGPDLLDDGFAYDPTAPTLFTMLDLLPAPPHASAVVCSGGAGAVVGETPGPEPLTLDSYQAPGPGDGGTASLVGSNMFPNHYREAAFGGGLLSPGPEAT (SEQ ID NO: 14)

[0151] Examples of IRF1 Polypeptides YPYDVPDYAGTELGSTMASWSHPQFEKGGGSGGGSGGGSWSHPQFEKAADITSLYKKAGSTMPITRMRMRPWLEMQINSNQIPGLIWINKEEMIFQIPWKHAAKHGWDINKDACLFRSWAIHTGRYKAGEKEPDPKTWKANFRCAMNSLPDIEEVKDQSRNKGSSAVRVYRMLPPLTKNQRKERKSKSSRDAKSKAKR KSCGDSSPDTFSDGLSSSTLPDDHSSYTVPGYMQDLEVEQALTPALSPCAVSSTLPDWHIPEVVVPDSTSDLYNFQVSPMPSTSEATTDEDEEGKLPEDIMKLLEQSEWQPTNVDGKGYLLNEPGVQPTSVYGDFSCKEEPEIDSPGGDIGLSLQRVFTDLKNMDATWLDSLLTPVRLPSIQAIPCAPLDPAFLYKVVT (SEQ ID NO: 15)

[0152] Examples of TNFα Polypeptides MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRL (SEQ ID NO: 16)

[0153] Examples of IL-10 Polypeptides MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 17)

[0154] Examples of FGF-2 Polypeptides GGWLYHHSAGPAGRWRQRRVPAGPF*GSEAPVLQKRRLFPAHSPGWPCGRCA*EKRPAHQTATASGGTWCSEHQGCMR*PLSGDERRRPPAGEQMRHRRMFLLRTPGVQQLQHVSLPQIHLLVRGSETHRPVQTGLQNRPWPESDPVPADVREIL (SEQ ID NO: 18)

[0155] Examples of G-CSF Polypeptides MTPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLCATYKLCHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP (SEQ ID NO: 19)

[0156] Examples of GM-CSF Polypeptides MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQELPTFLYKVVGSTSGSGKPGSGEGSTKG (SEQ ID NO: 20)

[0157] Examples of Eotaxin Polypeptides MKVSAALLWLLLIAAAFSPQGLAGPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKDICADPKKKWVQDSMKYLDQKSPTPKP (SEQ ID NO: 21)

[0158] Examples of Gal-9 Polypeptides AFSGSQAPYLSPAVPFSGTIQGGLQDGLQITVNGTVLSSSGTRFAVNFQTGFSGNDIAFHFNPRFEDGGYVVCNTRQNGSWGPEERKTHMPFQKGMPFDLCFLVQSSDFKVMVNGILFVQYFHRVPFHRVDTISVNGSVQLSYISFQPPGVWPANPAPITQ TVIHTVQSAPGQMFSTPAIPPMMYPHPAYPMPFITTILGGLYPSKSILLSGTVLPSAQRFHINLCSGNHIAFHLNPRFDENAVVRNTQIDNSWGSEERSLPRKMPFVRGQSFSVWILCEAHCLKVAVDGQHLFEYYHRLRNLPTINRLEVGGDIQLTHVQT (SEQ ID NO: 22)

[0159] Examples of PD1 polypeptides MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDHHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTA HPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSIVYASLDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPLTRLQDIKLAVPRARDPPVAT (SEQ ID NO: 23)

[0160] Examples of TIM-3 Polypeptides MFSHLPFDCVLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTLQRDFTAAFPR MLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIGIYIGAGICAGLALALIFGALIFKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQPSQPLGCRFAMP (SEQ ID NO: 24)

[0161] Examples of CXCR3 Polypeptides MVLEVSDHQVLNDAEVAALLENFSSSYDYGENESDSCCTSPPCPQDFSLNFDRAFLPALYSLLFLLGLLGNGAVAAVLLSRRTALSSTDTFLLHLAVADTLLVLTLPLWAVDAAVQWVFGSGLCKVAGALFNINFYAGALLLACISFDRYLNIVHATQLYRRGPPARVTLTCLAVWGLCLLFAL PDFIFLSAHHDERLNATHCQYNFPQVGRTALRVLQLVAGFLLPLLVMAYCYAHILAVLLVSRGQRRLRAMRLVVVVVVAFALCWTPYHLVVLVDILMDLGALARNCGRESRVDVAKSVTSGLGYMHCCLNPLLYAFVGVKFRERMWMLLLRLGCPNQRGLQRQPSSSRRDSSWSETSEASYSGL (SEQ ID NO: 25)

[0162] Examples of CXCR4 Polypeptides MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFAN VSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS (SEQ ID NO: 26)

[0163] Examples of CTLA4 Polypeptides MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQ VNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 91)

[0164] Examples of TLR3 Polypeptides MRQTLPCIYFWGGLLPFGMLCASSTTKCTVSHEVADCSHLKLTQVPDDLPTNITVLNLTHNQLRRLPAANFTRYSQLTSLDVGFNTISKLEPELCQKLPMLKVLNLQHNELSQ LSDKTFAFCTNLTELHLMSNSIQKIKNNPFVKQKNLITLDLSHNGLSSTKLGTQVQLENLQELLLSNNKIQALKSEELDIFANSSLKKLELSSNQIKEFSPGCFHAIGRLFGL FLNNVQLGPSLTEKLCLELANTSIRNLSLSNSQLSTTSNTTFLGLKWTNLTMLDLSYNNLNVVGNDSFAWLPQLEYFFLEYNNIQHLFSHSLHGLFNVRYLNLKRSFTKQSIS LASLPKIDDFSFQWLKCLEHLNMEDNDIPGIKSNMFTGLINLKYLSLSNSFTSLRTLTNETFVSLAHSPLHILNLTKNKISKIESDAFSWLGHLEVLDLGLNEIGQELTGQEW RGLENIFEIYLSYNKYLQLTRNSFALVPSLQRLMLRRVALKNVDSSPSPFQPLRNLTILDLSNNNIANINDDMLEGLEKLEILDLQHNNLARLWKHANPGGPIYFLKGLSHLH ILNLESNGFDEIPVEVFKDLFELKIIDLGLNNLNTLPASVFNNQVSLKSLNLQKNLITSVEKKVFGPAFRNLTELDMRFNPFDCTCESIAWFVNWINETHTNIPELSSHYLCN TPPHYHGFPVRLFDTSSCKDSAPFELFFMINTSILLIFIFIVLLIHFEGWRISFYWNVSVHRVLGFKEIDRQTEQFEYAAYIIHAYKDKDWVWEHFSSMEKEDQSLKFCLEER DFEAGVFELEAIVNSIKRSRKIIFVITHHLLKDPLCKRFKVHHAVQQAIEQNLDSIILVFLEEIPDYKLNHALCLRRGMFKSHCILNWPVQKERIGAFRHKLQVALGSKNSVH (SEQ ID NO: 92)

[0165] Examples of TLR4 Polypeptides MMSASRLAGTLIPAMAFLSCVRPESWEPCVEVVPNITYQCMELNFYKIPDNLPFSTKNLDLSFNPLRHLGSYSFFSFPELQVLDLSRCEIQTIEDGAYQSLSHLSTLILTGNPIQSLALGAFSGLSSLQKLVAVETNLASLENFPIGHLKTLKELNVAHNLIQSFKLPEYFSNLTNLEHLDLSSNKIQSIYCTDLRVLHQMPLLNLSLD LSLNPMNFIQPGAFKEIRLHKLTLRNNFDSLNVMKTCIQGLAGLEVHRLVLGEFRNEGNLEKFDKSALEGLCNLTIEEFRLAYLDYYLDDIIDLFNCLTNVSSFSLVSVTIERVKDFSYNFGWQHLELVNCKFGQFPTLKLKSLKRLTFTSNKGGNAFSEVDLPSLEFLDLSRNGLSFKGCCSQSDFGTTSLKYLDLSFNGVITMSSNFL GLEQLEHLDFQHSNLKQMSEFSVFLSLRNLIYLDISHTHTRVAFNGIFNGLSSLEVLKMAGNSFQENFLPDIFTELRNLTFLDLSQCQLEQLSPTAFNSLSSLQV LNMSHNNFFSLDTFPYKCLNSLQVLDYSLNHIMTSKKQELQHFPSSLAFLNLTQNDFACTCEHQSFLQWIKDQRQLLVEVERMECATPSDKQGMPVLSLNITCQM NKTIIGVSVLSVLVVSVVAVLVYKFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVI VVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI (SEQ ID NO: 93)

[0166] Examples of TLR9 Polypeptides (SEQ ID NO: 94)

[0167] Examples of TNFR2 Polypeptides MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQN RICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPT PEPSTAPSTSFLLPMGPSPPAEGSTGDFALPVGLIVGVTALGLLIIGVVNCVIMTQVKKKPLCLQREAKVPHLPADKARGTQGPEQQHLLITAPSSSSSSLESSASALDRRAPTR NQPQAPGVEASGAGEARASTGSSDSSPGGHGTQVNVTCIVNVCSSSDHSSQCSSQASSTMGDTDSSPSESPKDEQVPFSKEECAFRSQLETPETLLGSTEEKPLPLGVPDAGMKPS (SEQ ID NO: 95)

[0168] Example 3: Example nucleotide sequences

[0169] Nucleic acids encoding examples of scFvs Nucleic acids encoding exemplary anti-Ecad scFv (also called anti-hmcECAD.6) heavy chain GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTAGTAGTACCATATACTACGTAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGGACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGAGGTCGGGTGTTAGTGGGAGCTCTATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 38) Light chain CTGCCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATTATGTCTACTGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTATTGTGCATCATGGGATACCAGCCTGCGTGCCTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT (SEQ ID NO: 39)

[0170] Nucleic acid encoding an example of anti-CD103 scFv Heavy chain CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCGTCAGTAGTTACTATTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGCCATATCTATTACAGTGGGAATACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAATCAGTTCTCCCTGAAACTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTTTTGTGCGAGAGATAGATGGAATTATTATGATAGTAGTCCCGGCTATTATTATTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCAGCTCA (SEQ ID NO: 40) Light chain GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCGCCTAATCTTTGCTGCATCCCATTTGCAAAGTGGAGTCCCTTCAAGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGCATAATAGTTCCCCATTCACTTTCGGCCCTGGGACCAGAGTGGATATCAAA (SEQ ID NO: 41)

[0171] Nucleic acid encoding an example of anti-MOG scFv Heavy chain GAGGTGAAGCTGCACGAGAGCGGCGCAGGTCTGGTGAAGCCCGGCGCCAGCGTGGAGATCAGCTGCAAGGCCACCGGCTACACCTTCAGCAGCTTCTGGATCGAGTGGGTGAAGCAGAGACCCGGCCACGGCCTGGAGTGGATCGGCGAGATCCTGCCCGGCAGAGGCAGAACCAACTACAACGAGAAGTTCAAGGGCAAGGCCACCTTCACCGCCGAGACCAGCAGCAACACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCACCGGCAACACCATGGTGAACATGCCCTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGTGGAGGTGGTTCGGGAGGTGGAGGTAGCGGAGGTGGTGGAAGC (SEQ ID NO: 42) Light chain GATATTGAACTGACCCAGAGTCCCAGTAGCCTGGCCGTGAGTGCCGGCGAGAAAGTGACCATGAGCTGCAAAAGCAGCCAGAGCCTGCTGAACAGCGGCAACCAGAAAAACTACCTGGCCTGGTACCAGCAGAAACCCGGCCTGCCTCCTAAGCTGCTGATCTACGGCGCCAGCACCAGAGAAAGCGGCGTGCCCGATAGATTCACCGGCTCTGGCTCTGGCACCGACTTCACCCTGACCATCAGCAGCGTGCAGGCCGAAGACCTGGCTGTCTACTACTGCCAGAACGACCACAGCTACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGATCAAG (SEQ ID NO: 43)

[0172] Nucleic acid encoding an example of anti-HER2 scFv GAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAAGATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAAAACACCGCGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCAGCCGCTGGGGCGGCGATGGCTTTTATGCGATGGATTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCGAGCACCAAAGGCCCGAGCGTGTTTCCGCTGGCGCCGAGCAGCAAAAGCACCAGCGGCGGCACCGCGGCGCTGGGCTGCCTGGTGAAAGATTATTTTCCGGAACCGGTGACCGTGAGCTGGAACAGCGGCGCGCTGACCAGCGGCGTGCATACCTTTCCGGCGGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTGGTGACCGTGCCGAGCAGCAGCCTGGGCACCCAGACCTATATTTGCAACGTGAACCATAAACCGAGCAACACCAAAGTGGATAAAAAAGTGGAACCG (SEQ ID NO: ; 44)

[0173] Nucleic acid encoding an example of an anti - FAP scFv Heavy chain CAGGTGCAGCTCCAGCAGAGTGGCGCAGAGCTCGCTCGCCCAGGCGCTTCTGTGAATCTGAGTTGTAAGGCCTCCGGATATACTTTTACGAACAACGGCATCAACTGGCTGAAGCAGCGGACCGGCCAGGGCCTGGAGTGGATCGGCGAAATATACCCCCGGTCCACAAACACTCTCTATAACGAGAAGTTTAAGGGCAAAGCAACTCTGACCGCGGACAGGTCCTCTAACACAGCCTATATGGAGCTGAGAAGCTTGACGAGTGAGGACTCCGCTGTCTATTTTTGCGCCCGAACTCTGACCGCTCCTTTTGCTTTTTGGGGCCAGGGCACGCTCGTGACCGTAAGTGCG (SEQ ID NO: 45) Light chain CAGATCGTCCTGACGCAGTCTCCAGCCATCATGAGCGCCTCACCCGGCGAAAAGGTGACCATGACCTGCTCAGCCTCTTCTGGTGTGAATTTCATGCACTGGTACCAGCAAAAAAGTGGGACCTCCCCTAAAAGGTGGATCTTCGATACCAGCAAACTGGCTTCTGGCGTTCCCGCAAGGTTTAGCGGCTCTGGTTCCGGCACATCATACAGCCTGACGATCAGCAGCATGGAGGCAGAAGACGCAGCTACCTATTACTGCCAGCAATGGAGCTTTAACCCACCTACTTTCGGAGGAGGAACAAAGCTGGAAATAAAAAGA (SEQ ID NO: 46)

[0174] Nucleic acid encoding an example of anti-CS1 scFv Heavy chain ATGGGATGGAGCTCTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCCAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCGGGGTACTCCTTCACCACCTACTGGATGAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGCATGATTCATCCTTCCGATAGTGAAACTAGGTTAAATCAGAAGTTCAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCC (SEQ ID NO: 47) Light chain GACATTGTGATGACCCAGTCTCAGAAATCCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTTATTACTGGTGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAATTACTGATTTACTCGGCATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAATGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCTCTCACTTTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO: 48)

[0175] Nucleic acid encoding an example of anti-AMPA scFv Heavy chain (SEQ ID NO: 49) Light chain ATGACCAGCACCCTGCCGTTTAGCCCGCAGGTGAGCACCCCGCGCAGCAAATTTGCGACCATGGAATTTCAGACCCAGGTGCTGATGAGCCTGCTGCTGTGCATGAGCGGCGCGGCGGCGGATGTGGTGATGACCCAGACCCCGCTGACCCTGAGCGTGACCATTGGCCAGCCGGCGAGCATTAGCTGCAAAAGCAGCCAGAGCCTGCTGGATAGCGATGGCAAAACCTATCTGAACTGGCTGCTGCAGCGCCCGGGCCAGAGCCCGAAACGCCTGATTTATCTGGTGAGCAAACTGGATAGCGGCGTGCCGGATCGCTTTACCGGCAGCGGCAGCGGCACCGATTTTACCCTGAAAATTAGCCGCGTGGAAGCGGAAGATCTGGGCGTGTATTATTGCTGGCAGGGCACCCATTTTCCGCAGACCTTTGGCGGCGGCACCAAACTGGAAATTAAACGCGCGCGCGCGGATGCGGCGCCGACCGTGAGCATTTTTCCGCCGAGCAGCGAACAGCTGACCAGCGGCGGCGCGAGCGTGGTGTGCTTTCTGAACAACTTTTATCCGAAAGATATTAACGTGAAATGGAAAATTGATGGCAGCGAACGCCAGAACGGCGTGCTGAACAGCTGGACCGATCAGGATAGCAAAGATAGCACCTATAGCATGAGCAGCACCCTGACCCTGACCAAAGATGAATATGAACGCCATAACAGCTATACCTGCGAAGCGACCCATAAAACCAGCACCAGCCCGATTGTGAAAAGCTTTAACCGCAACGAATGC (SEQ ID NO: 50)<​​​​​​​​TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (SEQ ID NO: 51)

[0178] Nucleic acids encoding examples of CD8 transmembrane domains ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC (SEQ ID NO: 52)

[0179] Nucleic acids encoding examples of TLR3 transmembrane domains TTTTTTATGATCAAATACGTCTATTCTTCTCATATTTATTTTCATCGTTCTTCTGATTCACTTT (SEQ ID NO: 80)

[0180] Nucleic acids encoding examples of TLR4 transmembrane domains ATTGGGGTGTCTGTCCTAAGCGTGCTGGTTGTTTCCGTGGTTGCCGTTCTGGTATAT (SEQ ID NO: 81)

[0181] Nucleic acids encoding examples of IFNGR1 transmembrane domains TCTCTTTGGATTCCAGTTGTTGCTGCTTTACTACTCTTTCTAGTGCTTAGCCTGGTATTCATC (SEQ ID NO: 105)

[0182] Nucleic acids encoding exemplary IL10RA transmembrane domains GTCATCATATTCTTTGCCTTTGTTTTGCTGCTCTCCGGAGCGCTGGCTTACTGCCTCGCGCTC (SEQ ID NO: 106)

[0183] Nucleic acids encoding examples of TLR9 transmembrane domains TTTGCCCTCAGTCTTCTGGCTGTAGCTCTCGGGCTTGGCGTCCCTATGCTTCACCACCTGTGT (SEQ ID NO: 107)

[0184] Nucleic acids encoding examples of TNFR2 transmembrane domains TTCGCTCTTCCAGTTGGACTGATTGTGGGTGTGACAGCCTTGGGTCTACTAATAATAGGAGTGGTGAACTGTGTCATCATGACCCAGGTG (SEQ ID NO: 108)

[0185] Nucleic acids encoding exemplary signaling domains

[0186] Nucleic Acids Encoding Exemplary CD28 Signaling Domains AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC (SEQ ID NO: 53)

[0187] Nucleic Acids Encoding Examples of CD3ζ Signaling Domains AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 54)

[0188] Nucleic acids encoding exemplary 4-1BB signaling domains AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 82)

[0189] Nucleic Acids Encoding Exemplary TLR3 Signaling Domains GAGGGGTGGAGGATTTCCTTTTACTGGAATGTGTCTGTGCATAGGGTTTTGGGTTTTAAGGAGATAGATCGCCAAACAGAGCAGTTCGAGTATGCAGCTTATATTATACACGCGTACAAAGATAAGGACTGGGT ATGGGAGCATTTCAGTTCCATGAAAAAAGAAGACCAGTCCTTGAAGTTCTGCCTTGAAGAAAGGGACTTCGAGGCCGGGGTTTTTGAGCTGGAAGCGATCGTTAATAGTATTAAGCGGTCACGCAAGATCATCT TCGTAATAACACATCACCTCCTCAAGGATCCACTTTGCAAACGCTTCAAAGTTCACCATGCGGTGCAGCAAGCGATCGAACAGAACCTTGACTCCATAATCCTTGTCTTTCTGGAAGAAATACCTGATTACAAG TTGAATCATGCTCTGTGTCTGCGGCGGGGAATGTTTAAGTCTCATTGTATCCTTAACTGGCCTGTGCAGAAAGAGCGCATTGGGGCGTTTAGGCATAAACTCCAGGTTGCGCTTGGAAGCAAAAATTCCGTTCAT (SEQ ID NO: 83)

[0190] Nucleic Acids Encoding Exemplary TIR3 Signaling Domains TGGCGGATCTCTTTCTACTGGAACGTGTCAGTGCATAGAGTCCTCGGGTTCAAAGAGATTGACAGGCAGACGGAACAATTTGAGTATGCAGCGTACATTATCCATGCCTACAAAGATAAAGACTGGGTTTGGGAACATTTCTCCTCCATGGAAAAGGAGGATCAGTCTTTGAAGTTTTGTCTGGAGGAGCGGGACTTTGAGGCGGGAGTGTTCGAGCTTGAGGCCATTGTAAACTCGATCAAGCGGTCCCGTAAAATCATCTTTGTGATAACACACCACCTGTTGAAAGACCCGCTCTGCAAGCGCTTTAAGGTGCATCATGCTGTCCAGCAGGCCATCGAGCAGAATCTGGATTCAATTATTCTGGTGTTTTTAGAAGAGATCCCAGACTATAAGCTGAATCACGCTCTATGTTTGCGTCGAGGCATGTTCAAATCTCATTGCATCTTGAATTGGCCGGTGCAAAAGGAAAGGATTGGTGCTTTTCGGCACAAATTGCAGGTCGCCCTGGGCTCCAAAAACAGTGTTCACAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAAG (SEQ ID NO: 84)

[0191] Nucleic acid encoding an example of the TLR3 / TIR3 signaling domain GAAGGATGGCGAATCTCTTTCTACTGGAACGTGTCTGTTCATAGAGTTTTGGGATTCAAGGAAATAGATCGCCAAACCGAGCAATTCGAGTATGCCGCATATATCATACATGCATATAAGGACAAGGACTGGGT ATGGGAACACTTCTCCTCTATGGAGAAAGAAGATCAGTCACTCAAATTCTGCTTGGAGGAACGAGATTTCGAAGCCGGTGTATTTGAGCTTGAAGCAATAGTAAACTCTATAAAAAGAAGTCGAAAAATTATTT TCGTCATCACACATCATCTGTTGAAGGACCCGCTTTGTAAGAGGTTTAAAGTGCATCACGCTGTGCAGCAAGCCATTGAACAAAATCTGGATAGCATCATTCTCGTTTTTCTCGAAGAAATACCCGACTATAAG CTCAATCACGCTTTGTGCTTGCGACGGGGAATGTTCAAGTCCCACTGCATTCTTAACTGGCCAGTTCAAAAGGAACGAATCGGGGCCTTCAGACATAAATTGCAAGTTGCCCTGGGCTCAAAGAACTCAGTGCAC (SEQ ID NO: 109)

[0192] Nucleic Acids Encoding Exemplary TLR4 Signaling Domains AAGTTCTATTTCCATCTGATGCTTCTCGCTGGCTGCATAAAGTACGGGAGGGGGGAG (SEQ ID NO: 85)

[0193] Nucleic Acids Encoding Exemplary TIR4 Signaling Domains AATATATATGACGCTTTCGTGATCTACTCGAGCCAGGATGAGGACTGGGTTCGCAACGAGCTAGTCAAGAATCTTGAAGAGGGCGTGCCTCCTTTCCAGCTCTGTCTGCATTACCGCGATTTTATTCCTGGGGTGGCCATCGCGGCCAACATCATCCACGAGGGCTTCCATAAATCCAGAAAAGTGATTGTCGTTGTGAGCCAGCATTTCATCCAGTCCAGGTGGTGCATTTTCGAATATGAGATAGCCCAGACCTGGCAGTTTCTTAGCAGTCGGGCTGGGATTATTTTTATCGTGCTGCAGAAGGTTGAAAAGACCCTTTTGCGGCAACAGGTGGAACTGTACCGATTATTATCCCGTAACACTTACTTGGAATGGGAAGACTCAGTTCTCGGACGCCACATTTTCTGGCGCCGGCTCAGGAAGGCCCTGCTGGATGGTAAATCCTGGAACCCCGAGGGGACAGTGGGGACCGGATGTAACTGGCAAGAGGCAACAAGTATA (SEQ ID NO: 86)

[0194] Nucleic acid encoding an example of the TLR4 / TIR4 signaling domain AAGTTCTATTTCCATCTGATGCTTCTCGCTGGCTGCATAAAGTACGGGAGGGGGGAGAATATATATGACGCTTTCGTGATCTACTCGAGCCAGGATGAGGACTGGGTTCGCAACGAGCTAGTCAAGAATCTTGAAGAGGG CGTGCCTCCTTTCCAGCTCTGTCTGCATTACCGCGATTTTATTCCTGGGGTGGCCATCGCGGCCAACATCATCCACGAGGGCTTCCATAAATCCAGAAAAGTGATTGTCGTTGTGAGCCAGCATTTCATCCAGTCCAGGT GGTGCATTTTCGAATATGAGATAGCCCAGACCTGGCAGTTTCTTAGCAGTCGGGCTGGGATTATTTTTATCGTGCTGCAGAAGGTTGAAAAGACCCTTTTGCGGCAACAGGTGGAACTGTACCGATTATTATCCCGTAAC ACTTACTTGGAATGGGAAGACTCAGTTCTCGGACGCCACATTTTCTGGCGCCGGCTCAGGAAGGCCCTGCTGGATGGTAAATCCTGGAACCCCGAGGGGACAGTGGGGACCGGATGTAACTGGCAAGAGGCAACAAGTATA (SEQ ID NO: 110)

[0195] Nucleic acids encoding exemplary IFNγ signaling domains ATGAAGTACACTTCTTACATACTCGCCTTCCAGCTTTGTATAGTGTTGGGCAGTCTGGGTTGCTATTGCCAAGATCCTTATGTGAAGGAAGCAGAAAATTTGAAGAAATACTTTAACGCGGGTCATTCTGACGTTGCAGATAATGGAACCTTGTTTTTGGGAATACTTAAAAATTGGAAGGAGGAAAGCGACCGCAAGATCATGCAGTCTCAAATCGTTTCCTTTTATTTTAAACTTTTCAAGAATTTTAAGGACGATCAGTCCATACAGAAATCTGTAGAGACTATCAAAGAGGATATGAACGTAAAGTTTTTTAACAGCAACAAAAAGAAAAGAGATGACTTTGAGAAACTTACGAATTATAGTGTCACCGATCTGAACGTCCAACGCAAAGCAATCCACGAGTTGATTCAAGTTATGGCAGAGCTTTCCCCAGCGGCGAAAACTGGAAAGCGCAAACGATCTCAAATGCTCTTCCGGGGT (SEQ ID NO: 87)

[0196] Nucleic acid encoding an example of the IFNGR1 signaling domain (SEQ ID NO: 111)

[0197] Nucleic acids encoding exemplary IFNγ signaling domains ATGAAGTACACTTCTTACATACTCGCCTTCCAGCTTTGTATAGTGTTGGGCAGTCTGGGTTGCTATTGCCAAGATCCTTATGTGAAGGAAGCAGAAAATTTGAAGAAATACTTTAACGCGGGTCATTCTGACGTTGCAGATAATGGAACCTTGTTTTTGGGAATACTTAAAAATTGGAAGGAGGAAAGCGACCGCAAGATCATGCAGTCTCAAATCGTTTCCTTTTATTTTAAACTTTTCA AGAATTTTAAGGACGATCAGTCCATACAGAAATCTGTAGAGACTATCAAAGAGGATATGAACGTAAAGTTTTTTAACAGCAACAAAAAGAAAAGAGATGACTTTGAGAAACTTACGAATTATAGTGTCACCGATCTGAACGTCCAACGCAAAGCAATCCACGAGTTGATTCAAGTTATGGCAGAGCTTTCCCCAGCGGCGAAAACTGGAAAGCGCAAACGATCTCAAATGCTCTTCCGGGGT (SEQ ID NO: 112)

[0198] Nucleic acids encoding exemplary IL10RA signaling domains CAGCTCTATGTCAGAAGGAGAAAAAAGCTCCCGTCCGTGCTTCTTTTTAAGAAGCCTTCCCCGTTCATCTTCATTAGTCAGCGACCGAGCCCTGAGACCCAGGACACGATCCATCCGCTTGATGAAGAGGCGTTTCTTAAGGTAAGCCCTGAACTGAAGAATCTCGATCTGCACGGCAGCACCGACAGTGGATTCGGGTCCACGAAACCCTCACTCCAGACAGAAGAGCCTCAGTTTTTGTTGCCTGATCCTCACCCCCAGGCTGATAGAACGTTGGGTAATCGCGAGCCGCCGGTACTTGGAGACAGCTGCTCATCTGGGTCAAGCAACTCAACGGATTCTGGGATATGTTTGCAGGAACCAAGCCTCTCACCTAGCACTGGGCCAACCTGGGAGCAGCAAGTGGGAAGCAATTCACGGGGGCAGGACGACTCTGGTATAGACCTGGTACAAAACTCTGAGGGACGGGCGGGAGATACTCAAGGTGGTAGCGCCCTGGGACACCACAGCCCACCGGAACCAGAGGTCCCGGGCGAGGAGGACCCTGCAGCGGTGGCGTTTCAGGGATATTTGCGCCAAACCCGCTGTGCTGAGGAGAAGGCTACTAAAACGGGCTGCTTGGAAGAAGAATCTCCGCTCACAGACGGGCTCGGCCCGAAGTTCGGCCGATGTTTGGTGGACGAAGCGGGGCTTCATCCGCCGGCGCTTGCTAAAGGGTACCTGAAGCAAGACCCCTTGGAGATGACCCTGGCCTCCTCCGGGGCGCCTACTGGTCAGTGGAACCAGCCGACCGAGGAATGGTCTCTCCTCGCCCTTTCCAGTTGCTCCGACCTCGGTATATCAGACTGGAGTTTCGCACACGACCTTGCACCCCTCGGCTGTGTAGCCGCCCCAGGAGGCCTTCTTGGCTCATTTAACAGTGACCTCGTTACGCTCCCCCTCATTTCATCTCTGCAATCCTCTGAA (SEQ ID NO: 113)

[0199] Nucleic acid encoding an example of a TLR9 / TIR9 signaling domain GGCTGGGACCTCTGGTACTGCTTCCACCTGTGCCTGGCCTGGCTTCCCTGGCGGGGGCGGCAAAGTGGGCGAGATGAGGATGCCCTGCCCTACGATGCCTTCGTGGTCTTCGACAAAACGCAGAGCGCAGTGGCAGACTGGGTGTACAACGAGCTTCGGGGGCAGCTGGAGGAGTGCCGTGGGCGCTGGGCACTCCGCCTGTGCCTGGAGGAACGCGACTGGCTGCCTGGCAAAACCCTCTTTGAGAACCTGTGGGCCTCGGTCTATGGCAGCCGCAAGACGCTGTTTGTGCTGGCCCACACGGACCGGGTCAGTGGTCTCTTGCGCGCCAGCTTCCTGCTGGCCCAGCAGCGCCTGCTGGAGGACCGCAAGGACGTCGTGGTGCTGGTGATCCTGAGCCCTGACGGCCGCCGCTCCCGCTACGTGCGGCTGCGCCAGCGCCTCTGCCGCCAGAGTGTCCTCCTCTGGCCCCACCAGCCCAGTGGTCAGCGCAGCTTCTGGGCCCAGCTGGGCATGGCCCTGACCAGGGACAACCACCACTTCTATAACCGGAACTTCTGCCAGGGACCCACGGCCGAATAG (SEQ ID NO: 114)

[0200] Nucleic acid encoding an example of a TNFR2 signaling domain AAAAAGAAGCCCTTGTGCCTGCAGAGAGAAGCCAAGGTGCCTCACTTGCCTGCCGATAAGGCCCGGGGTACACAGGGCCCCGAGCAGCAGCACCTGCTGATCACAGCGCCGAGCTCCAGCAGCAGCTCCCTGGAGAGCTCGGCCAGTGCGTTGGACAGAAGGGCGCCCACTCGGAACCAGCCACAGGCACCAGGCGTGGAGGCCAGTGGGGCCGGGGAGGCCCGGGCCAGCACCGGGAGCTCAGATTCTTCCCCTGGTGGCCATGGGACCCAGGTCAATGTCACCTGCATCGTGAACGTCTGTAGCAGCTCTGACCACAGCTCACAGTGCTCCTCCCAAGCCAGCTCCACAATGGGAGACACAGATTCCAGCCCCTCGGAGTCCCCGAAGGACGAGCAGGTCCCCTTCTCCAAGGAGGAATGTGCCTTTCGGTCACAGCTGGAGACGCCAGAGACCCTGCTGGGGAGCACCGAAGAGAAGCCCCTGCCCCTTGGAGTGCCTGATGCTGGGATGAAGCCCAGTTAACCAGGCCGGTGTGGGCTGTGTCGTAGCCAAGGTGGGCTGAGCCCTGGCAGGATGACCCTGCGAAG (SEQ ID NO: 115)

[0201] Nucleic acids encoding examples of BDNF signaling domains (SEQ ID NO: 55)

[0202] Nucleic Acids Encoding Exemplary VEGFR2 Signaling Domains CTGAAGCTGGGCAAGCCCCTGGGCAGGGGCGCCTTCGGCCAGGTGATCGAGGCCGACGCCTTCGGCATCGACAAGACCGCCACCTGCAGGACCGTGGCCGTGAAGATGCTGAAGGAGGGCGCCACCCACAGCGAGCACAGGGCCCTGATGAGCGAGCTGAAGATCCTGATCCACATCGGCCACCACCTGAACGTGGTGAACCTGCTGGGCGCCTGCACCAAGCCCGGCGGCCCCCTGATGGTGATCGTGGAGTTCTGCAAGTTCGGCAACCTGAGCACCTACCTGAGGAGCAAGAGGAACGAGTTCGTGCCCTACAAGACCAAGGGCGCCAGGTTCAGGCAGGGCAAGGACTACGTGGGCGCCATCCCCGTGGACCTGAAGAGGAGGCTGGACAGCATCACCAGCAGCCAGAGCAGCGCCAGCAGCGGCTTCGTGGAGGAGAAGAGCCTGAGCGACGTGGAGGAGGAGGAGGCCCCCGAGGACCTGTACAAGGACTTCCTGACCCTGGAGCACCTGATCTGCTACAGCTTCCAGGTGGCCAAGGGCATGGAGTTCCTGGCCAGCAGGAAGTGCATCCACAGGGACCTGGCCGCCAGGAACATCCTGCTGAGCGAGAAGAACGTGGTGAAGATCTGCGACTTCGGCCTGGCCAGGGACATCTACAAGGACCCCGACTACGTGAGGAAGGGCGACGCCAGGCTGCCCCTGAAGTGGATGGCCCCCGAGACCATCTTCGACAGGGTGTACACCATCCAGAGCGACGTGTGGAGCTTCGGCGTGCTGCTGTGGGAGATCTTCAGCCTGGGCGCCAGCCCCTACCCCGGCGTGAAGATCGACGAGGAGTTCTGCAGGAGGCTGAAGGAGGGCACCAGGATGAGGGCCCCCGACTACACCACCCCCGAGATGTACCAGACCATGCTGGACTGCTGGCACGGCGAGCCCAGCCAGAGGCCCACCTTCAGCGAGCTGGTGGAGCACCTGGGCAAC (SEQ ID NO: 88)

[0203] Nucleic acids encoding examples of hinge domains

[0204] Nucleic acids encoding examples of CD28 hinge domains CTCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCGGATCCCAAA (SEQ ID NO: 56)

[0205] Nucleic acids encoding examples of CD8 hinge domains ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 57)

[0206] Nucleic acids encoding exemplary polypeptides

[0207] Nucleic acids encoding exemplary NFκB1 polypeptides (SEQ ID NO: 58)

[0208] Nucleic Acids Encoding Exemplary JUN Polypeptides (SEQ ID NO: 59)

[0209] Nucleic Acids Encoding Examples of RELB Polypeptides (SEQ ID NO: 60)

[0210] Nucleic Acids Encoding Examples of IRF1 Polypeptides (SEQ ID NO: 61)

[0211] Nucleic Acids Encoding Exemplary TNFα Polypeptides ATGTCTACCGAGTCTATGATTAGGGACGTGGAACTGGCTGAGGAGGCACTGCCCAAAAAAACCGGCGGACCACAGGGCTCTAGGAGATGTCTGTTTCTGTCTCTGTTCTCTTTTCTCATCGTGGCTGGCGCTACAACACTCTTCTGTCTGCTCCAT TTCGGCGTGATTGGACCACAGCGAGAGGAATTTCCCCGGATCTGTCACTCATTTCACCACTGGCACAGGCTGTCCGATCTTCATCTCGGACTCCATCCGACAAACCTGTCGCCCATGTCGTCGCCAACCCACAGGCCGAGGGCCAGCTCCAGTGG CTCAATAGGAGGGCAAACGCTCTGCTCGCCAATGGCGTGGAACTCCGGGATAACCAGCTCGTCGTGCCTAGTGAGGGACTGTACCTCATCTACTCCCAGGTGCTGTTTAAGGGCCAGGGATGTCCTTCTACACATGTGCTGCTCACACACAATT TCACGGATCGCCGTGTCTTACCAGACTAAAGTCAATCTGCTCTCTGCCATCAAATCCCCATGTCACGGGAAACACCTGAGGGCGCTGAGGCTAAACCTTGGTACGAACCCATCTACCTCGGAGGCGTGTTCCAGCTGGAGAAGGGCGATAGACTGA (SEQ ID NO: 62)

[0212] Nucleic acids encoding exemplary IL-10 polypeptides ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTTCTGACAGGCGTAAGGGCGTCACCTGGCCAAGGAACACAGTCAGAGAACAGCTGTACACATTTCCCCGGCAACTTGCCCAATATGCTTAGGGATCTTCG CGATGCCTTCTCACGAGTGAAGACATTTTTTCAGATGAAGGATCAGTTGGATAACCTTTTGCTGAAGGAAAGCCTGCTCGAGGACTTCAAGGGATACCTCGGATGCCAGGCACTGAGCGAAATGATACAGTTCT ACCTGGAAGAGGTAATGCCTCAGGCAGAAAATCAGGACCCCGATATTAAAGCTCATGTGAACTCTCTGGGTGAGAACCTGAAAACTCTGAGGCTGAGGCTGCGGAGGTGTCACAGATTCCTGCCATGCGAGAAC AAATCAAAAGCCGTCGAACAGGTGAAGAACGCCTTTAACAAACTGCAGGAGAAAGGCATCTATAAAGCGATGAGCGAGTTCGATATTTTCATCAACTACATTGAGGCATACATGACGATGAAAATCCGAAATTAG (SEQ ID NO: 63)

[0213] Nucleic Acids Encoding Exemplary FGF-2 Polypeptides GGCGGCTGGCTCTATCACCACTCTGCCGGCCCTGCCGGAAGATGGCGGCAGCGGCGCGTTCCCGCCGGGCCATTTTAAGGATCCGAAGCGCCTGTACTGCAAAAACGGCGGCTTTTTCCTGCGCATTCACCCGGATGGCCGTGTGGACGGTGTGCGTGAGAAAAGCGACCCGCACATCAAACTGCAACTGCAAGCGGAGGAACGTGGTGTAGTGAGCATCAAGGGTGTATGCGCTAACCGTTATCTGGCGATGAAAGAAGACGGCCGCCTGCTGGCGAGCAAATGCGTCACCGACGAATGTTTCTTCTTCGAACGCCTGGAGTCCAACAACTACAACACGTATCGCTCCCGCAAATACACCTCTTGGTACGTGGCTCTGAAACGCACCGGCCAGTACAAACTGGGCTCCAAAACCGGCCCTGGCCAGAAAGCGATCCTGTTCCTGCCGATGTCCGCGAAATCCTAA (SEQ ID NO: 64)

[0214] Nucleic acid encoding an example of a G-CSF polypeptide ATGACTCCTTTGGGTCCAGCTTCTTCCTTGCCTCAATCCTTCTTGTTGAAGTGTTTGGAGCAGGTTAGAAAGATCCAGGGTGATGGTGCTGCTTTGCAAGAGAAGTTGTGTGCTACTTACAAGTTGTGTCAC CCAGAAGAGTTGGTTTTGTTGGGTCACTCCTTGGGTATTCCTTGGGCTCCATTGTCCTCTTGTCCATCCCAAGCTTTGCAATTGGCTGGTTGTTTGTCCCAATTGCACTCCGGTTTGTTCTTGTACCAGGGT TTGTTGCAAGCTTTGGAGGGTATTTCTCCAGAGTTGGGTCCAACTTTGGACACATTGCAGTTGGACGTTGCTGACTTCGCTACTACTATCTGGCAACAGATGGAAGAATTGGGTATGGCTCCAGCTTTGCAG CCAACTCAAGGTGCTATGCCAGCTTTTGCTTCTGCTTTCCAGAGAAGAGCTGGTGGTGTTTTGGTTGCTTCTCACTTGCAGTCTTTCTTGGAGGTTTCCTACAGAGTTTTGAGACACTTGGCTCAACCATAA (SEQ ID NO: 65)

[0215] Nucleic acids encoding exemplary GM-CSF polypeptides ATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTGCACCCGCCCGCTCGCCCAGCCCCAGCACGCAGCCCTGGGAGCATGTGAATGCCATCCAGGAGGCCCGGCGTCTCCTGAACCTGAGTAGAGACACTGCTGCTGAGATGAATGAAACAGTAGAAGTCATCTCAGAAATGTTTGACCTCCAGGAGCCGACCTGCCTACAGACCCGCCTGGAGCTGTACAAGCAGGGCCTGCGGGGCAGCCTCACCAAGCTCAAGGGCCCCTTGACCATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACCCCGGAAACTTCCTGTGCAACCCAGATTATCACCTTTGAAAGTTTCAAAGAGAACCTGAAGGACTTTCTGCTTGTCATCCCCTTTGACTGCTGGGAGCCAGTCCAGGAGTTGCCAACTTTCTTGTACAAAGTGGTTGGCTCCACCAGCGGCAGCGGCAAGCCAGGCTCCGGCGAAGGCAGCACCAAAGGC (SEQ ID NO: 66)

[0216] Nucleic acid encoding an example of a Gal-9 polypeptide GCCTTCAGCGGTTCCCAGGCTCCCTACCTGAGTCCAGCTGTCCCCTTTTCTGGGACTATTCAAGGAGGTCTCCAGGACGGACTTCAGATCACTGTCAATGGGACCGTTCTCAGCTCCAGTGGAACCAGGTTTGCTGTGAACTTTCAGACTGGCTTCAGTGGAAATGACATTGCCTTCCACTTCAACCCTCGGTTTGAAGATGGAGGGTACGTGGTGTGCAACACGAGGCAGAACGGAAGCTGGGGGCCCGAGGAGAGGAAGACACACATGCCTTTCCAGAAGGGGATGCCCTTTGACCTCTGCTTCCTGGTGCAGAGCTCAGATTTCAAGGTGATGGTGAACGGGATCCTCTTCGTGCAGTACTTCCACCGCGTGCCCTTCCACCGTGTGGACACCATCTCCGTCAATGGCTCTGTGCAGCTGTCCTACATCAGCTTCCAGCCTCCCGGCGTGTGGCCTGCCAACCCGGCTCCCATTACCCAGACAGTCATCCACACAGTGCAGAGCGCCCCTGGACAGATGTTCTCTACTCCCGCCATCCCACCTATGATGTACCCCCACCCCGCCTATCCGATGCCTTTCATCACCACCATTCTGGGAGGGCTGTACCCATCCAAGTCCATCCTCCTGTCAGGCACTGTCCTGCCCAGTGCTCAGAGGTTCCACATCAACCTGTGCTCTGGGAACCACATCGCCTTCCACCTGAACCCCCGTTTTGATGAGAATGCTGTGGTCCGCAACACCCAGATCGACAACTCCTGGGGGTCTGAGGAGCGAAGTCTGCCCCGAAAAATGCCCTTCGTCCGTGGCCAGAGCTTCTCAGTGTGGATCTTGTGTGAAGCTCACTGCCTCAAGGTGGCCGTGGATGGTCAGCACCTGTTTGAATACTACCATCGCCTGAGGAACCTGCCCACCATCAACAGACTGGAAGTGGGGGGCGACATCCAGCTGACCCATGTGCAGACATAG (SEQ ID NO: 67)

[0217] Nucleic acid encoding an example of a PD1 polypeptide ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCTTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCTGCTCCCGGGCCGCACGAGGGACAATAGGAGCCAGGCGCACCGGCCAGCCCCTGAAGGAGGACCCCTCAGCCGTGCCTGTGTTCTCTATTGTTTATGCTTCCCTGGATTTCCAGTGGCGAGAGAAGACCCCGGAGCCCCCCGTGCCCTGTGTCCCTGAGCAGACGGAGTATGCCACCATTGTCTTTCCTAGCGGAATGGGCACCTCATCCCCCGCCCGCAGGGGCTCAGCCGACGGCCCTCGGAGTGCCCAGCCACTGAGGCCTGAGGATGGACACTGCTCTTGGCCCCTGACGCGTCTGCAGGATATCAAGCTTGCGGTACCGCGGGCCCGGGATCCACCGGTCGCCACC (SEQ ID NO: 68)

[0218] Nucleic acid encoding an example of TIM-3 polypeptide ATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTACTACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAATGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTGCCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACGTGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATACTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAGAATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGGATCCAAATCCCAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACCAGCCAAGGTCACCCCTGCACCGACTCTGCAGAGAGACTTCACTGCAGCCTTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGACACTGGGGAGCCTCCCTGATATAAATCTAACACAAATATCCACATTGGCCAATGAGTTACGGGACTCTAGATTGGCCAATGACTTACGGGACTCTGGAGCAACCATCAGAATAGGCATCTACATCGGAGCAGGGATCTGTGCTGGGCTGGCTCTGGCTCTTATCTTCGGCGCTTTAATTTTCAAATGGTATTCTCATAGCAAAGAGAAGATACAGAATTTAAGCCTCATCTCTTTGGCCAACCTCCCTCCCTCAGGATTGGCAAATGCAGTAGCAGAGGGAATTCGCTCAGAAGAAAACATCTATACCATTGAAGAGAACGTATATGAAGTGGAGGAGCCCAATGAGTATTATTGCTATGTCAGCAGCAGGCAGCAACCCTCACAACCTTTGGGTTGTCGCTTTGCAATGCCATAG (SEQ ID NO: 69)

[0219] Nucleic acids encoding exemplary CXCR3 polypeptides (SEQ ID NO: 70)

[0220] Nucleic acids encoding exemplary CXCR4 polypeptides (SEQ ID NO: 71)

[0221] Nucleic acid encoding an example of a CTLA4 polypeptide ATGGCTTGCCTTGGATTTCAGCGGCACAAGGCTCAGCTGAACCTGGCTACCAGGACCTGGCCCTGCACTCTCCTGTTTTTTCTTCTCTTCATCCCTGTCTTCTGCAAAGCAATGCACGTGGCCCAGCCTGCTGTGGTACTGGCCAGCAGCCGAGGCATCGCCAGCTTTGTGTGTGAGTATGCATCTCCAGGCAAAGCCACTGAGGTCCGGGTGACAGTGCTTCGGCAGGCTGACAGCCAGGTGACTGAAGTCTGTGCGGCAACCTACATGATGGGGAATGAGTTGACCTTCCTAGATGATTCCATCTGCACGGGCACCTCCAGTGGAAATCAAGTGAACCTCACTATCCAAGGACTGAGGGCCATGGACACGGGACTCTACATCTGCAAGGTGGAGCTCATGTACCCACCGCCATACTACCTGGGCATAGGCAACGGAACCCAGATTTATGTAATTGATCCAGAACCGTGCCCAGATTCTGACTTCCTCC TCTGGATCCTTGCAGCAGTTAGTTCGGGGTTGTTTTTTTATAGCTTTCTCCTCACAGCTGTTTCTTTGAGCAAAATGCTAAAGAAAAGAAGCCCTCTTACAACAGGGGTCTATGTGAAAATGCCCCCAACAGAGCCAGAATGTGAAAAGCAATTTCAGCCTTATTTTATTCCCATCAATTGA (SEQ ID NO: 116)

[0222] Nucleic acid encoding an example of a TLR3 polypeptide (SEQ ID NO: 117)

[0223] Nucleic Acids Encoding Exemplary TLR4 Polypeptides (SEQ ID NO: 118)

[0224] Nucleic Acids Encoding Exemplary TLR9 Polypeptides (SEQ ID NO: 119)

[0225] Nucleic Acids Encoding Exemplary TNFR2 Polypeptides (SEQ ID NO: 120)

[0226] Example 4: Biological engineering of mesenchymal stromal cells bearing chimeric antigen receptors to enhance the efficacy of immunosuppressive treatments This example describes the design and engineering of MSCs expressing one or more chimeric antigen receptors (CAR-MSCs) and how such CAR-MSCs can enhance immunosuppression at targeted sites of antigen-specific inflammation.

[0227] The results of this example restate and extend at least some of the results provided in other examples.

[0228] method Cell lines, primary peripheral blood mononuclear cells (PBMC), primary T cells and primary mesenchymal stromal cells (MSC) Primary human adipose-derived MSCs were obtained and cultured in StemXVivo Mesenchymal Stem Cell Growth 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, US) containing 10% (v / v) fetal bovine serum (FBS, MilliporeSigma, Ontario, Canada) and 1% (v / v) penicillin streptomycin-glutamine (PSG, Gibco, Gaithersburg, MD, US). HEK-293T cells were used for lentivirus production. MCF7 were irradiated and confirmed to be E-cadherin (Ecad) positive by flow cytometry for use as a stimulatory format for CAR-based single-chain variable fragment (scFv) in the in vivo tumor model described above.

[0229] Primary normal human keratinocytes were derived from the epidermis of young or adult donors from single or pooled donors and cultured in Keratinocyte Growth Medium (PromoCell, Heidelberg, Germany). Primary normal human bronchial epithelial cells were isolated from the supraclavicular airway epithelial layer of the lung (Lonza, Cohasset, MN). These cells were then cultured in Airway Epithelial Cell Basal Growth Medium (PromoCell, Heidelberg, Germany).

[0230] The acute lymphoblastic leukemia cell line NALM6 and mantle cell lymphoma cell line JeKo-1 were also purchased from ATCC (CRL-3273 and CRL-3006, respectively, Manassas, VA, USA). These cell lines were cultured in R20 and R10 (RPMI 1640, Gibco, Gaithersburg, MD, USA) containing 20% ​​(v / v) or 10% (v / v) FBS (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 (Addgene, Cambridge, MA, USA). Additionally, NALM6 and / or luciferase-positive NALM6 were transduced with lentivirus encoding human Ecad as cell-derived Ecad-stimulating forms (GeneCopoeia, Rockville, MD). Cell lines were cultured for up to 20 passages, with fresh aliquots thawed every 7–8 weeks. Cell lines were authenticated by the manufacturer and routinely phenotyped by flow cytometry. Cell lines were tested monthly for mycoplasma infection. PBMCs were isolated from anonymized healthy donor blood apheresis cones using SepMate tubes (STEMCELL Technologies, Vancouver, Canada). T cells were isolated by negative selection magnetic beads using the EasySep™ Human T Cell 80 Isolation Kit (STEMCELL Technologies, Vancouver, Canada).

[0231] Primary T cells and PBMCs were cultured in T cell 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 selection for in vitro coculture. Freshly isolated PBMCs were infused intravenously for in vivo experiments.

[0232] scFv phage display library Through phage display library selection, the hmcECAD.6 scFv sequence was generated and optimized for binding to the Ecad protein. Approximately 2 x 10 9 A naive human scFv library with a diversity of 1000 was used for selection against recombinant Ecad protein (Biomolecular Discovery, Rochester, NY). After two rounds of selection against mouse Ecad and another round of panning against human Ecad Fc chimeric protein (both from BioLegend, San Diego, CA), the hmcECAD.6 scFv clone was identified. Phage ELISA showed that this clone reacted with mouse Ecad. The relative affinity of the hmcECAD.6 clone to the FLAG antibody was estimated to be approximately 5 nM. The final hmcECAD.6 sequence (SEQ ID NO: 90) was codon-optimized for cloning of the heavy and light chains into the EcCAR plasmid.

[0233] Chimeric antigen receptor (CAR) design and virus production MSCs were transduced with a lentiviral vector encoding a specifically designed CAR construct downstream of the EF-1α promoter. Transduction optimization was performed using a CD19-targeted CAR (CAR19) construct. See, for example, June et al., Science 359, 1361-1365 (2018). CAR19 is composed of a CD19-directed scFv derived from the FMC-63 clone fused to the 41BB and CD3ζ signaling domains (FMC63-41BB-ζ). VSV-g-pseudotyped second-generation lentivirus carrying the CAR transgene was generated via Lipofectamine transfection into HEK-293T cells, followed by standard procedures for lentiviral recovery, concentration, and functional titer determination. See, for example, Sterner et al. Blood 133, 697-709 (2019) and Sakemura et al. Blood 139(26):3708-3721 (2022). To induce MSC immunomodulatory activation (Figure 23B), a CAR construct was designed containing an scFv against human / canine Ecad (Figure 23A) and the CD28ζ intracellular signaling domain. The CAR plasmid was generated and sequence verified. To optimize CAR expression and MSC proliferation, pre-seeded MSCs (at a concentration of 250,000 cells / well) were transduced with lentiviral particles at a multiplicity of infection (MOI) of 3 in 6-well plates with various concentrations of protamine sulfate (25, 50, or 100 μg / μL).

[0234] T cell suppression assay To verify the ability of CAR-MSCs to inhibit activated T cells, a T cell suppression assay was used. Briefly, to provide both soluble and cell-derived antigen-specific stimulation to CAR-MSCs, untransduced (UTD) MSCs or EcCAR-MSCs (suppressors) were co-cultured with activated T cells (effectors) in the presence or absence of 1) soluble Ecad (stimulator) or 2) paired Ecad-positive and Ecad-negative NALM6 cell lines. T cells were isolated from PBMCs of healthy donors using negative selection magnetic beads. See, for example, Sterner et al. Blood 133, 697-709 (2019). Isolated T cells were nonspecifically activated using CD3 / CD28 stimulatory beads (Dynabeads, Invitrogen, Waltham, MA, USA) at a 1:1 bead-to-T cell ratio in T cell 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). MSCs were preincubated in the presence or absence of soluble Ecad protein (250–1000 ng / mL) or paired Ecad-positive and Ecad-negative cell lines (MSC to Ecad-positive cell ratio: 1:1) (stimulators), and EcCAR-MSCs were specifically activated through the CAR in StemXVivo serum-free mesenchymal stem cell growth medium (R&D Systems, Minneapolis, MN, USA). After 24 h, stimulated T cells (effectors) were cocultured with UTD-MSCs or EcCAR-MSCs (suppressors). Cells were cocultured at 250,000 T cells per 6-well plate at a 1:5 MSC to T cell ratio. After different coculture time points, as indicated in individual experiments, cells were harvested and analyzed by flow cytometry.

[0235] Multiparameter Flow Cytometry Staining was performed for flow cytometry. Briefly, MSCs were isolated and cultured, then transduced with CAR to generate CAR-MSCs and co-cultured with T cells / PBMCs in 96-well plates for 24-hour analysis or in 6-well plates for longer-term culture. After the desired co-culture period, adherent MSCs were cultured in 75cm plates. 2 Cells were detached with 10 mL of Accutase (STEMCELL Technologies, Vancouver, Canada) per surface area and incubated at 37°C for 10–15 minutes. After detachment, all well contents were centrifuged, washed with flow buffer (phosphate-buffered saline (PBS), 2% (v / v) FBS, and 1% (v / v) sodium azide), and 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 positivity was determined through negative gating with Fluorescence Minus One (FMO) control wells. Absolute cell counts were obtained using volumetric measurements. Cells were gated on an SSC vs. FSC plot for cell separation by size and complexity, an FSC-H vs. FSC-A plot to remove doublets, and live / dead Aqua staining (product number L34966, Thermo Fisher Scientific, Waltham, MA, USA) to remove dead cells. Cell subset characterization was then performed based on a predesigned antibody panel optimized for the stained samples.

[0236] Anti-human antibodies were purchased from Biolegend, eBioscience, or BD Biosciences (San Diego, CA, USA). Samples were prepared for flow cytometry. 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 on a 3-laser CytoFLEX (Beckman Coulter, Chaska, MN, USA) using singlet gating, and live cells were determined using Live / Dead Aqua staining (product number L34966, Thermo Fisher Scientific, Waltham, MA, USA). All gating analyses were performed using FlowJo X10.0.7r2 software (Becton Dickenson, Ashland, OR, USA) or Kaluza Analysis software (Beckman Coulter, Indianapolis, Indiana, USA).

[0237] Table 4. List of antibodies used for flow cytometry, including clones, vendors, and part numbers. [Table 4]

[0238] In vivo mouse studies Six- to eight-week-old female and male immunodeficient NOD-SCID-γ mice housed in a BSL2+ animal facility - / -NSG mice were obtained from Jackson Laboratories. All cells were injected via tail vein, intravenously, or intraperitoneally in 100-200 μL of PBS via a syringe. Mice were imaged by bioluminescence imaging (BLI) using an IVIS® Lumina S5 imaging system (PerkinElmer, Hopkinton, MA, USA) to confirm the engraftment of luciferase-positive CD19-positive Nalm6 / JeKo-1 cells in the cancer xenograft model or luciferase-positive CAR-MSCs in the MSC-persistence xenograft model. Images were taken 10 minutes after intraperitoneal injection of 10 μL / g of D-luciferin (15 mg / mL, Gold Biotechnology, St. Louis, MO, USA). For the Ecad tumor xenograft model, NSG mice were injected with Ecad-positive or Ecad-negative luciferase-positive CD19-positive NALM6 (1 × 10 6 Mice were transplanted with 1 × 10 cells (intravenously administered). Engraftment was confirmed by BLI 5 days after injection, and mice were randomized to receive CD19-targeted CART cells (CART19) (1 × 10 cells administered intravenously as a strategy to treat CD19-positive tumors). 6 cells) and UTD-MSCs or EcCAR-MSCs (1 × 10 6 Immediately after the NALM6 tumor burden was effectively killed by CART19, mice were rechallenged with either Ecad-positive or Ecad-negative luciferase-positive CD19-positive NALM6 (1 × 10 cells administered intraperitoneally) (Figure 17A). 6 cells) and UTD-MSCs or EcCAR-MSCs (1 × 10 6 NALM6 mice were then given CART19 cells intraperitoneally (IP). Serial BLI was performed to assess residual disease and determine the antitumor activity of CART19 cells on Ecad-positive and Ecad-negative NALM6 mice.

[0239] In a complementary tumor model, NSG mice were inoculated with luciferase-positive, CD19-positive JeKo-1 or NALM6 (1 × 10 6Mice were then transplanted with irradiated Ecad-positive MCF7 cells (5 × 10 cells administered intravenously) into the tumor. Depending on the tumor subtype, transplantation was confirmed by BLI 1–2 weeks or 5 days after injection. All mice were then injected with irradiated Ecad-positive MCF7 cells (5 × 10 cells administered intravenously to stimulate EcCAR-MSCs). 6 cells) and CART19 (1 × 10 cells administered intraperitoneally as a strategy to treat CD19-negative tumors. 6 Mice were then randomized based on BLI as an indicator of tumor burden and received UTD-MSCs, EcCAR-MSCs, or no additional treatment (Figure 25B). Serial BLI was performed to assess residual disease and determine the antitumor activity of CART19 cells.

[0240] In a graft-versus-host disease (GVHD) model, allogeneic human PBMCs (20–30 × 10 6 GVHD was induced in NSG mice by intravenous injection of UTD-MSCs (1 × 10 6 cells intraperitoneally every 3 weeks) or EcCAR-MSCs (1 × 10 6 Mice were treated with 1000 cells intraperitoneally every 3 weeks (Figures 17D-17G). Body weight and clinical GVHD scoring (based on body weight, posture, diarrhea, activity, coat condition, and skin integrity, Table 5) were monitored for GVHD progression in each experimental group in all experiments.

[0241] Table 5. GVHD Clinical Scoring System [Table 5]

[0242] In the acute GVHD model, NSG mice were first irradiated with a dose of 250 cGy to induce GVHD more rapidly. Then, allogeneic human PBMCs (10–15 × 10 6Cells were administered intravenously to irradiated mice, after which the groups were randomized by body weight to receive either UTD-MSCs or CAR-MSCs (Figure 18A). Luciferase-positive GFP-positive CD19-CAR-MSCs served as a control for luciferase-positive GFP-positive EcCAR-MSC treatment. Localization of MSCs in organs was detected by BLI and immunofluorescence staining. To ensure the absence of human CD19-positive cells stimulating CD19CAR-MSCs in mice, B cells were removed from allogeneic PBMCs using CD19 Pan B Cell Dynabeads (Invitrogen, Waltham, MA, USA). Body weight and clinical GVHD scoring were examined for each experimental group for GVHD progression in all experiments using the same criteria listed above.

[0243] To assess the distribution and localization of luciferase-positive anti-Ecad CAR-MSCs and luciferase-positive anti-CD19 CAR-MSCs, mice were imaged by bioluminescence imaging (BLI) using an IVIS® Lumina S5 imaging system (PerkinElmer, Hopkinton, MA, USA). In the acute GVHD xenograft model, one week after MSC injection, preliminary mice were randomly selected and euthanized, and organ bioluminescence intensity assessment was performed. Here, organ imaging was performed 10–20 minutes after intraperitoneal injection of 250 μL of 50 mg / mL D-luciferin (Gold Biotechnology, St. Louis, MO, USA). Luciferase-positive MSCs were detected in the colon, kidney, liver, lung, and spleen for systemic BLI detection.

[0244] For all in vivo experiments, mouse blood was collected by tail vein bleeding (approximately 100 μL), and 70 μL of blood was used for flow cytometry analysis. Red blood cell (RBC) lysis was performed using 1:10 BD FACS Lyse buffer (BD Biosciences, San Jose, CA, USA). Cells were then washed with flow buffer and incubated with their specific antibody mixtures in the dark at room temperature before flow analysis using a CytoFLEX (Beckman Coulter, Chaska, MN, USA). The remaining blood was centrifuged at 13,000 rpm at 4°C for 10 minutes, and serum was separated for chemokine analysis.

[0245] Immunofluorescence staining In an acute GVHD mouse xenograft model, preliminary mice were sacrificed 7 days after MSC injection, and organs, including the colon, liver, lungs, and heart, were collected and preserved. Organs were fixed in 4% paraformaldehyde for 48 hours and immediately frozen in Tissue-Tek OCT Compound (Sakura, Hayward, CA) for cryosectioning and slide preparation. Next, tissue slides were thawed from -80°C storage, washed with PBS, and incubated in 0.1% Sudan Black (Sigma-Aldrich, Burlington, MA) solution. The slides were then solubilized in blocking buffer containing 0.3% Triton-X-100 (Thermo Fisher Scientific, Waltham, MA, USA) and 5% bovine serum albumin (Sigma-Aldrich, Burlington, MA). Antigen retrieval was performed by boiling in retrieval solution for 15 minutes. Tissues were incubated with primary antibodies (Cat. No. 144725, Cell Signaling Technology, Danvers, MA) for 1 hour at room temperature to stain for Ecad (Cat. No. 144725) and GFP-positive CAR-MSCs (Cat. No. Ab183734, Abcam, Boston, MA). Secondary antibodies (Cat. No. A11001, Cat. No. A11012, Thermo Fisher Scientific, Waltham, MA, USA) were then incubated across the slides for 1 hour at room temperature. Slides were photographed with a Zeiss LSM 980 Confocal Microscope (Zeiss Group, Oberkochen, Germany) and analyzed using ZEN 2.3 Microscopy Software (Zeiss Group, Oberkochen, Germany). MSC localization to Ecad-positive colonic crypts was compared between EcCAR-MSCs, CD19CAR-MSCs, and a negative control group without MSCs. Across each condition, colonic crypts containing GFP-positive MSCs were quantified from an average of more than five 40x magnification microscopic fields, and localization was calculated (Figures 18F-18G, Figures 21D and 21F).

[0246] Canine studies In all experiments, the Ecad-targeting CAR expressed on human MSCs was based on a human / mouse / canine cross-reactive scFv. This scFv was generated using phage display, and cross-reactivity to both canine and human Ecad was confirmed (Figures 23A-23D). EcCAR-MSCs in healthy beagle dogs. Subjects received EcCAR-MSCs (2 × 10 6 Cells / kg) were injected intraperitoneally. Subjects were observed daily, body weights were checked, and serial blood collections were performed. Colon biopsies were performed on day 3 and tissue integrity was assessed by hematoxylin and eosin (H&E).

[0247] RNA isolation, sequencing and analysis EcCAR-MSCs or UTD-MSCs were stimulated with or without 250 ng / mL recombinant human E-cadherin Fc chimera (BioLegend, San Diego, CA, USA) as a CAR-specific stimulation for 24 hours. After in vitro culture, MSCs were detached and RNA was isolated using a QIAGEN RNeasy Plus Mini Kit (product number 74134, QIAGEN, Germantown, MD, USA). To ensure the rigor of the results, bulk RNA sequencing was performed on MSCs from three different biological donor replicates in both the UTD-MSC and EcCAR-MSC groups.

[0248] Total RNA was prepared using the SMARTer stranded total RNA-seq kit v2, Pico input mammalian (Takara, Mountain View, CA, USA). Total RNA (three samples per lane) was sequenced on an Illumina HiSeq 4000 (Illumina, San Diego, CA, USA). Library preparation and sequencing were performed by the Medical Genome Facility Genome Analysis Core (Mayo Clinic, Rochester, MN, USA). Quality checks of the fastqc files generated for each sample were performed using FastQC v0.11.8. Cutadapt v1.18 was used to trim and remove adapter sequences. The generated files were verified for adapter removal and quality using FastQC v0.11.8.

[0249] Paired fragment reads from the trimmed fastq files were mapped to the latest human reference genome (GRCh38) downloaded from NCBI (ncbi.nlm.nih.gov / ). A genome index file was constructed and assigned using STAR v2.5.4b. Gene expression counts for each gene were generated using HTSeq (Python 3.6.5). Gene counts were normalized (geometric mean), and expression change analysis was calculated using DESeq2 (R v3.6.1) using an adjusted p-value of ≤0.05 as the statistical cutoff. Heatmaps were generated using pheatmap, with PCA generated using ggplot2 and Prism Graph Pad (La Jolla, CA). Gene set enrichment analysis for cellular phenotypes was performed using the Enrichr Cell Augmented Gene set. Activated and inhibited canonical pathways, molecules, and protein-protein interaction networks 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 comparison of gene expression changes.

[0250] Comprehensive cytokine analysis Cytokine analysis was performed on mouse serum samples collected 2 weeks after MSC or control treatment. In vitro cytokine analysis was performed on supernatants collected 24 hours after co-culture with MSCs and stimulators. Serum or supernatants were centrifuged at 10,000 × g for 5 minutes to remove precipitates. Serum and / or supernatants were diluted 1:2 with serum matrix before plating and according to the manufacturer's instructions for the Milliplex Human Cytokine / Chemokine Magnetic Bead Premixed 38 Plex 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, WA, USA). Significant differences were determined and reported using Prism Graph Pad (La Jolla, CA, USA).

[0251] Statistical analysis and figures To perform appropriate statistical analysis, in vitro and in vivo experiments were performed using technical and biological replicates. Two-way or one-way ANOVA was used to assess differences in surface marker expression and T cell suppressive capacity between in vitro EcCAR-MSCs and UTD-MSCs by evaluating percent expression (%) and absolute T cell counts. Similarly, ANOVA was used to determine significant differences between in vivo EcCAR-MSC-treated and UTD-MSC-treated mice through IVIS imaging of luciferase-positive tumors and luciferase-positive MSCs, weight comparisons, blood flow analysis, and GVHD clinical score comparisons. Kaplan-Meier survival analysis was used with Cox regression analysis to determine significant differences in survival outcomes in tumor and GVHD xenograft models, adjusting for confounding factors (e.g., gender). To allow for multiple comparisons between each individual group in the aforementioned analyses, ANOVA was supplemented with Tukey's multiple comparison test. For comparison between two groups, a two-tailed unpaired Student's t-test was used instead of a way analysis of variance.

[0252] RNA-seq data were processed using the DeSeq2 program, where raw counts were normalized (geometric mean) between samples, and the Benjamini-Hochberg method was used for multiple hypothesis testing correction. All relevant statistically significant comparisons are indicated by asterisks, corresponding to significance levels below p ≤ 0.05 (ns = p ≥ 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001), indicating a 95% confidence interval. The final data point without an asterisk indicates no significant difference (ns) between groups. Relevant data are plotted as the mean of all data points, with either the standard deviation (SD) or standard error of the mean (SEM) used to determine error bars. Experimental data figures were analyzed and generated using Prism Graph Pad (La Jolla, CA, USA) and Microsoft Excel (Microsoft, Redmond, WA, USA). Finally, canonical pathway enrichment and molecular networks were generated with significant differences (*padj ≤ 0.05) reported by Ingenuity Pathway Analysis (QIAGEN, Redwood City, CA, USA).

[0253] result MSCs are successfully transduced and stably express the CAR. We established efficient transduction of CARs into MSCs using lentiviral vectors enhanced with polyvalent cation enhancers. Initially, we optimized delivery of CAR-loaded lentiviruses using protamine sulfate, achieving transduction efficiencies of ≥70% in adipose-derived MSCs from healthy donors (Figure 15A). Following optimization using conventional CAR vectors, we developed alternative CAR constructs for disease treatment via CAR-MSC immunity. The primary CAR-MSC constructs were directed against Ecad, a ligand expressed on inflammatory intestinal epithelial cells associated with graft-versus-host disease (GVHD). GVHD occurs when donor T cells attack host epithelial tissues, in part through interaction between T cell integrins and E-cadherin (Ecad), which is expressed on the gastrointestinal tract. To redirect MSCs to Ecad-positive tissues susceptible to immune attack, we generated several anti-Ecad scFv clones for CAR delivery by phage display. An optimized scFv sequence (hmcECAD.6 (SEQ ID NO: 90), Figures 23A-23B) was selected with human, mouse (Figures 23C-23D) and dog cross-reactivity to enable preclinical studies in animal models.

[0254] To determine whether the inclusion of a CAR signaling domain enhances the immunosuppressive function of MSCs without risking their conversion into cytotoxic T cells, we tested a CAR construct containing the CD28ζ signaling domain. hmcECAD.6 was incorporated into the CAR CD28ζ scaffold (Figure 23B) to generate EcCAR-MSCs (Figure 15B). Stable EcCAR expression was maintained over multiple ex vivo passages across various MSC donors, with transduction efficiencies of ≥90% (Figures 15C-15D and Table 5).

[0255] EcCAR-MSCs retain their stemness. To eliminate any unwanted differentiation induced by CAR-MSC manipulation, the stem phenotype, morphology, and gene expression of EcCAR-MSCs were determined. Based on the International Society for Cell and Gene Therapy's MSC stemness maintenance criteria set, EcCAR-MSCs maintained their stem phenotype and morphology similar to untransduced (UTD)-MSCs (Figure 15E and Figures 24A-24C). Bulk RNA sequencing (RNAseq) was also used to identify phenotypic gene set enrichment between EcCAR-MSCs and UTD-MSCs. The adipose-derived stem cell phenotype was enriched in the expanded gene group in EcCAR-MSCs, while differentiated cell lineages were enriched in the decreased gene group (Figure 15F). These results support the limited differentiation induced by CAR transduction and the maintenance of stemness after MSC manipulation.

[0256] EcCAR-MSCs maintain stemness while exhibiting excellent antigen-specific immunosuppression. Next, the T cell suppressive capacity of CAR-MSCs was examined in vitro. Briefly, EcCAR-MSCs or UTD-MSCs (suppressors) were cocultured with activated T cells (effectors) for 24 hours in the presence or absence of Ecad protein as a CAR stimulation (stimulator) strategy. Soluble Ecad protein and the NALM6 cell line engineered to stably overexpress Ecad (Figure 16A) were both used as CAR stimulators. Stimulation of EcCAR-MSCs with soluble Ecad resulted in superior T cell suppression in a dose-dependent manner compared with UTD-MSC controls (Figure 16B). The MSC stem phenotype was significantly maintained after stimulation with soluble Ecad (Figure 16C). Additionally, EcCAR-MSCs stimulated with an Ecad-positive cell line also exhibited antigen-specific immunosuppression. Across multiple MSC donors, superior T cell suppression was achieved after coculture of EcCAR-MSCs with Ecad-positive, but not Ecad-negative, cells (Figures 16D-16F). Stimulation with Ecad-positive or Ecad-negative cell lines did not affect the stemness properties or morphology of EcCAR-MSCs (Figures 16G and 24B-24C).

[0257] EcCAR-MSCs suppress the antitumor activity of CART19 in tumor xenograft models. Based on these in vitro findings, we evaluated the immunosuppression of CAR-MSCs in vivo through tumor and GVHD xenograft models to determine whether CAR-MSCs suppress CART19 and enhance tumor growth under antigen-specific conditions. In tumor models, we measured the ability of MSCs to suppress the potential antitumor activity of CD19-targeted T cells (CART19) (Figure 17A). Ecad-positive NALM6 cells were used to provide antigen-specific stimulation to EcCAR-MSCs, and Ecad-negative NALM6 cells served as a control (Figure 16A). Immunodeficient NOD-SCID-γ - / - Tumor models were established by intravenous (iv) administration of luciferase-positive, Ecad-positive NALM6 or luciferase-positive, Ecad-negative NALM6 tumor cells into (NSG) mice (Figure 17A). Without additional treatment, Ecad-positive and Ecad-negative NALM6 tumors grew similarly in immunodeficient mice (Figure 25A).

[0258] After transplantation, mice were randomized based on tumor burden, as determined by bioluminescence imaging (BLI), and treated with CART19 and UTD-MSCs or CART19 and EcCAR-MSCs. Tumor burden was assessed biweekly via BLI. Results demonstrated Ecad-dependent activation of EcCAR-MSCs in vivo. Treatment of Ecad-positive NALM6 xenografted mice (Figure 17B), but not Ecad-negative NALM6 xenografted mice (Figure 17C), with EcCAR-MSCs resulted in diminished antitumor activity compared with UTD-MSC treatment. EcCAR-MSCs alone in coculture with NALM6 did not directly promote leukemia cell proliferation (Figures 25B-25C).

[0259] As an alternative strategy to stimulate EcCAR-MSCs, we found that co-injection of irradiated Ecad-positive MCF7 cells with EcCAR-MSCs significantly reduced their antitumor activity and survival rate in tumor xenograft models (Figures 25D-25G). These results highlight the immunosuppressive potential of EcCAR-MSCs in the presence of Ecad antigen in vivo.

[0260] In a GVHD xenograft model, EcCAR-MSCs improve therapeutic outcomes and exhibit antigen-specific distribution to Ecad-positive target sites. The therapeutic effect of EcCAR-MSCs was examined in a GVHD xenograft model. A GVHD xenograft model was established by intravenous administration of human peripheral blood mononuclear cells (PBMCs) into NSG mice. See, for example, Ehx et al. Front Immunol 9, 1943 (2018). Mice were treated with PBS control, UTD-MSCs, or EcCAR-MSCs intraperitoneally (ip) (Figure 17D). The progression of GVHD was recorded through weight measurement, clinical GVHD symptom scoring (Table 4), and survival outcomes. EcCAR-MSC treatment prevented weight loss (Figure 17E), alleviated clinical GVHD symptoms (including fur, skin integrity, posture, and activity) (Figure 17F), and improved overall survival (Figure 17G) compared with mice treated with UTD-MSCs or no MSCs.

[0261] To evaluate the influence of the anti-Ecad scFv contained in EcCAR-MSCs, we compared the effects of EcCAR-MSCs with CAR-MSCs containing an scFv that is not specific for mouse tissue (anti-CD19-CAR-MSCs). Specifically, we evaluated the antigen-specific stimulation and distribution of EcCAR-MSCs to Ecad-positive epithelial tissue in the colon of GVHD xenografted mice. GFP-positive, luciferase-positive EcCAR-MSCs and a GFP-positive, luciferase-positive anti-CD19 CAR-MSC control were generated (Figures 26A-26B). The distribution and effects of GFP-positive, luciferase-positive EcCAR-MSCs and a GFP-positive, luciferase-positive anti-CD19-CAR-MSC control in a GVHD xenograft model are shown in Figure 18A. In this model, mice were irradiated prior to PBMC and MSC infusion to induce a more severe GVHD phenotype (referred to as the "acute GVHD model"), and within one week of PBMC infusion, the mice developed acute GVHD. See, e.g., Schroeder and DiPersio. Dis. Model Mech. 4, 318-333 (2011). In this acute GVHD model, EcCAR-MSC treatment again significantly reduced weight loss (Figure 18B) and prolonged survival (Figure 18C) in mice compared with the CD19-CAR-MSC control or no MSCs. To elucidate the mechanism underlying the enhanced efficacy of EcCAR-MSC in this model, peripheral blood from mice was collected 2 weeks after MSC infusion, and human T cells were analyzed. Administration of EcCAR-MSCs resulted in increased human T cell suppression in the peripheral blood compared to CD19-CAR-MSCs and control mice without MSC treatment (Figure 18D). One week after CAR-MSC infusion, preliminary mice from these experiments were euthanized, organs were harvested, and the presence of CAR-MSCs was analyzed by both BLI and immunofluorescence. Both BLI and immunofluorescence assessment of mouse colon tissue after MSC treatment demonstrated superior infiltration of EcCAR-MSCs into Ecad-positive colon tissue compared to CD19-CAR-MSCs (Figures 18E-18G). Together, these results demonstrate the enhanced immunosuppressive effect and target tissue distribution of EcCAR-MSCs in a GVHD model.

[0262] EcCAR-MSC stimulation induces an immunosuppressive phenotype through activation of signaling pathways, inhibitory cytokines, and surface receptors. To elucidate the mechanism of enhanced immunosuppression in EcCAR-MSCs, we investigated the gene expression, cytokine secretion, and surface marker profiles of stimulated EcCAR-MSCs. First, gene expression profiles were analyzed through bulk RNA sequencing across 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 verify antigen-specific CAR stimulation). Pathway enrichment between conditions was identified through predictive networks generated in Ingenuity Pathway Analysis (IPA) machine learning software.

[0263] To characterize the effects of CAR transduction on MSCs, we compared unstimulated EcCAR-MSC vs. UTD-MSC samples. While 331 genes were significantly elevated between the groups, gene enrichment was largely attributable to cellular stress response pathways, typically induced by lentiviral transduction (Figures 27A-27C). Notably, these enrichments were no longer significant between stimulated EcCAR-MSC vs. UTD-MSC, revealing instead a higher concentration of homeostatic pathway enrichment (Figure 27D). Unbiased hierarchical gene clustering across all conditions indicated that the most significant changes in gene expression were caused by antigen-specific EcCAR-MSC (+Ecad) stimulation, forming a distinct cluster from all other conditions (Figure 19A).

[0264] To characterize the effects of CAR antigen-specific CD28ζ stimulation, Ecad-stimulated EcCAR-MSC samples were compared with unstimulated EcCAR-MSC samples. We identified elevated gene activation for 1,662 genes (Figure 27A), confirming that while CAR transduction alters MSC gene expression, CAR stimulation results in greater changes to gene activation (Figure 19A). Analysis of the principal components of gene expression across Ecad-stimulated and unstimulated conditions revealed that UTD-MSC samples tended to cluster by their MSC biological donor origin (Figure 19B), whereas EcCAR-MSC samples tended to cluster instead by Ecad stimulation status (Figure 19C). Canonical pathways significantly elevated by CAR stimulation were strongly associated with anti-inflammatory interleukin (IL-6 and IL-10), NFκB, tumor necrosis factor receptor 2 (TNFR2), and toll-like receptor (TLR) signaling pathways in MSCs (Figures 19D-19E). Each of these pathways contributes to the immunosuppressive function of MSCs, either through cytokine-mediated T cell suppression, induction of Tregs, or direct receptor interaction.

[0265] Gene counts of transcription factors (NFκB1, JUN, RELB, IRF1) and associated regulators (TRAF1, TLR3, and FYN) were strongly elevated in Ecad-stimulated EcCAR-MSC samples (Figure 19F). At the same time, these transcription factors, especially those identified after CAR stimulation, were also identified as direct downstream targets of CD28 stimulatory molecules (Figure 19D) and as regulators of the immunosuppressive function of MSCs. Among all signaling molecules included in the predictive network of EcCAR-MSC stimulation, leukocyte apoptosis was identified as a significant functional enrichment (Figure 19D).

[0266] To understand how gene activation in EcCAR-MSCs can lead to functional immunosuppression, we measured the expression of inhibitory receptors and human cytokine production after antigen-specific stimulation of EcCAR-MSCs. Antigen-specific stimulation of EcCAR-MSCs (with an Ecad-positive cell line) resulted in increased secretion of inhibitory cytokines, such as interleukin-10 (IL-10), interleukin-4 (IL-4), granulocyte-colony-stimulating factor (G-CSF), and vascular endothelial growth factor (VEGF), in vitro compared with coculture of EcCAR-MSCs with activated T cells in the presence of an Ecad-negative cell line (Figure 20A). Additionally, key inhibitory surface markers, galectin-9 (Gal-9) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4), were increased in stimulated EcCAR-MSCs in an antigen-dependent manner (Figure 20B). In the tumor model, human cytokine production was measured in mouse serum 2 weeks after treatment with EcCAR-MSC or UTD-MSC (Figure 17A). Increases in IL-10, G-CSF, and eotaxin, among other cytokines, were found in EcCAR-MSC-treated mice (Figures 20C and 27E).

[0267] Human CD3+ T cell subtypes were also assessed in the peripheral blood of GVHD xenograft mice treated with EcCAR-MSCs compared with UTD-MSCs. Correlated with significant prevention of weight loss (Figures 20D–20H), EcCAR-MSC-treated mice demonstrated a significant suppression of the absolute number of human CD3+ T cells circulating in the peripheral blood (Figure 20E). While both human CD4+ and CD8+ T cells were suppressed in EcCAR-MSC-treated mice (Figure 20F), we identified an increased proportion of human CD4+ T cells in EcCAR-MSC-treated mice after 2 weeks of treatment (Figure 20G). Peripheral blood analysis after 4 weeks of treatment similarly demonstrated enrichment of Tregs (human CD4+, CD25+, CD127-) in EcCAR-MSC-treated mice compared with UTD-MSC-treated mice (Figure 20H).

[0268] Taken together, these results demonstrated enhanced T cell suppression after antigen-specific stimulation of EcCAR-MSCs compared with unstimulated EcCAR-MSCs and demonstrated an association with activation of immunosuppressive pathways, increased inhibitory cytokine production, and upregulation of inhibitory receptors.

[0269] Incorporation of the CD28ζ signaling domain into CAR-MSCs is required for enhanced immunosuppressive activity. To determine which elements of the CD28ζ signaling domain are required to drive enhanced immunosuppression via EcCAR-MSCs after antigen-specific stimulation, we generated CD28-stimulated EcCAR-MSCs (CD28 EcCAR-MSCs), CD3ζ-stimulated EcCAR-MSCs (CD3ζ EcCAR-MSCs), and EcCAR-MSCs lacking the intracellular domain (null EcCAR-MSCs) and compared them with CD28-CD3ζ-stimulated EcCAR-MSCs (CD28ζ EcCAR-MSCs) (Figures 21A-21B). CD28ζ EcCAR-MSCs and CD28 EcCAR-MSCs exhibited antigen-specific suppression of activated T cells, whereas CD3ζ EcCAR-MSCs, null EcCAR-MSCs, and UTD-MSCs did not (Figure 21C), demonstrating the role of CD28 signaling in the immunosuppressive properties of CAR-MSCs. The effect of incorporating the CAR signaling domain was also examined in an acute GVHD xenograft model (Figure 21D). In this model, when compared with mice treated with EcCAR-MSCs lacking CD28, CD3ζ, or the signaling domain, treatment with CD28ζ EcCAR-MSCs prevented weight loss (Figure 21E), resulted in the lowest clinical GVHD severity score (Figure 17F), and the longest overall survival (Figure 17G). CD28 EcCAR-MSC treatment also resulted in attenuation of weight loss and overall survival, which was numerically inferior to CD28ζ EcCAR-MSCs, but not statistically different. These data suggest that while the CD28 domain alone is responsible for the immunosuppressive capacity of EcCAR-MSCs, incorporating CD28ζ into EcCAR-MSCs enhances the outcome of immunosuppressive treatment.

[0270] EcCAR-MSCs are safe in animal models. After functional verification, studies were conducted to ensure the safety of allogeneic EcCAR-MSCs. First, the proliferation and loss of EcCAR-MSCs in NSG mice were defined using luciferase-positive EcCAR-MSCs and UTD-MSCs (Figures 22A and 26A). To understand the effect of antigen-specific stimulation on MSC loss, MSC persistence was examined in mice in the presence or absence of irradiated human Ecad-positive MCF7 cells. Mice were treated with 1) luciferase-positive UTD-MSCs alone, 2) luciferase-positive UTD-MSCs in combination with human Ecad-positive cells, 3) luciferase-positive EcCAR-MSCs alone, or 4) luciferase-positive EcCAR-MSCs in combination with human Ecad-positive cells (Figure 22A). Sequential BLI was performed every 2–3 days to quantify MSCs. These studies revealed no significant difference between the clearance time (~30 days) of UTD-MSCs and EcCAR-MSCs in the presence or absence of additional target antigen stimulation, further supporting the safety profile of EcCAR-MSCs (Figures 22B-22C).

[0271] Next, we determined the toxicity of EcCAR-MSCs to normal epithelial tissues expressing Ecad. First, we determined the effect of EcCAR-MSCs on Ecad-positive keratinocytes from healthy donors. Coculture of EcCAR-MSCs with keratinocytes did not result in a significant difference in keratinocyte survival compared to coculture with UTD-MSCs (Figure 22D). These cocultures also did not significantly affect Ecad expression on keratinocyte target cells (Figure 22E). Evaluation of coculture supernatants demonstrated antigen-specific stimulation by EcCAR-MSCs, as evidenced by elevated cytokines including IL-10, IL-4, and eotaxin (Figure 22F).

[0272] Second, we determined the toxicity of EcCAR-MSCs to Ecad-positive bronchial cells. Coculture of Ecad-positive bronchial cells with EcCAR-MSCs did not significantly affect bronchial cell survival compared with coculture with UTD-MSCs (Figure 22G). Furthermore, in a GVHD xenograft model, immunofluorescence evaluation of lungs harvested 1 week after EcCAR-MSC treatment revealed no distribution of EcCAR-MSCs to the lungs (Figures 22H-22I).

[0273] A canine model was used to evaluate whether administration of human EcCAR-MSCs containing canine cross-reactive scFvs was associated with any toxicity (Figure 23A). Healthy dogs were given EcCAR-MSCs via intraperitoneal injection, and safety outcomes and tissue integrity were examined over a 28-day period (Figure 28A). Toxicity was assessed by serial measurements of whole blood counts, liver, and kidney function. EcCAR-MSCs were not associated with hematopoietic toxicity (Figure 28B), organ toxicity (Figure 28C), weight loss (Figure 28D), or tissue damage (Figure 28E), demonstrating a high safety profile and the absence of toxicity induced by EcCAR-MSC administration.

[0274] Taken together, these results demonstrate that generation of CAR-MSCs provides antigen-specific activated immunosuppression designed to safely enhance MSC therapy.

[0275] Example 5: GVHD treatment Ecad-CAR-MSCs engineered to express one or more polypeptides selected from 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 suffering from or at risk of developing GVHD. CAR-MSCs engineered to express high levels of one or more polypeptides selected from 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. Following administration of one or more CAR-MSCs engineered to express high levels of one or more polypeptides selected from 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, the number of activated T cells in a human is reduced.Following administration of one or more CAR-MSCs engineered to express high levels of one or more polypeptides selected from 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, one or more symptoms of GVHD are reduced in a human.

[0276] Example 6: GVHD treatment MSCs engineered to express a CAR that targets an epithelial-specific antigen and engineered to express high levels of one or more polypeptides selected from 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 are administered to a human identified as suffering from or at risk of developing GVHD. MSCs engineered to express a CAR that targets an epithelial-specific antigen and engineered to express high levels of one or more polypeptides selected from 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 are administered using intravenous injection. The number of activated T cells in a human is reduced following administration of one or more MSCs engineered to express a CAR that targets an epithelial-specific antigen and engineered to express high levels of one or more polypeptides selected from 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.Following administration of one or more MSCs engineered to express a CAR that targets an epithelial-specific antigen and engineered to express high levels of one or more polypeptides selected from 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, one or more symptoms of GVHD are reduced in a human.

[0277] Other embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the above description is illustrative of, but not limiting, the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. Mesenchymal stromal cells (MSCs), including: (1) an exogenous nucleic acid encoding a chimeric antigen receptor (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 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) alpha 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.

2. The MSC according to claim 1, wherein the MSC is a human MSC.

3. The MSCs according to any one of claims 1 to 2, wherein the MSCs are adipose-derived MSCs.

4. The MSC according to any one of claims 1 to 3, wherein the epithelial-specific antigen is E-cadherin (Ecad).

5. The MSC according to any one of claims 1 to 4, wherein the polypeptide is selected from the group consisting of the NFκB1 polypeptide, the JUN polypeptide, the RELB polypeptide and the IRF1 polypeptide.

6. The MSC according to claim 5 , wherein the MSC comprises an exogenous nucleic acid encoding the NFκB1 polypeptide, and the MSC expresses the NFκB1 polypeptide.

7. The MSC of claim 5 , wherein the MSC comprises an exogenous nucleic acid encoding the JUN polypeptide, and 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, and the MSC expresses the RELB polypeptide.

9. The MSC of claim 5, wherein the MSC comprises an exogenous nucleic acid encoding the IRF1 polypeptide and 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.

11. The MSC according to claim 10, wherein the MSC comprises an exogenous nucleic acid encoding the CXCR3 polypeptide, and 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 and 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.

14. The MSC of claim 13 , wherein the MSC comprises an exogenous nucleic acid encoding the PD-1 polypeptide, and the MSC expresses the PD-1 polypeptide.

15. The MSC of claim 13, wherein the MSC comprises an exogenous nucleic acid encoding the Gal-9 polypeptide, and the MSC expresses the Gal-9 polypeptide.

16. The MSC of claim 13, wherein the MSC comprises an exogenous nucleic acid encoding the TIM-3 polypeptide and 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, and 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, and 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, and the MSC expresses the FGF-2 polypeptide.

21. The MSC of any one of claims 1 to 20, wherein the CAR comprises a heavy chain comprising the CDR set forth in SEQ ID NO: 1 and a light chain comprising the CDR set forth in SEQ ID NO:

2.

22. The MSCs of claim 21 , wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 1 and the light chain comprises the amino acid sequence set forth in SEQ ID NO:

2.

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

4.

24. The MSCs of claim 23, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 3 and the light chain comprises the amino acid sequence set forth in SEQ ID NO:

4.

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

26. 26. A method for treating a mammal with graft-versus-host disease (GVHD), said method comprising administering to said mammal MSCs according to any one of claims 1 to 24 or the composition according to claim 25.

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

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

29. 28. The method of any one of claims 26 to 27, wherein the number of regulatory T cells (Treg) in the mammal is increased by at least 10 percent.

30. 26. A method for suppressing an immune response in a mammal, said method comprising the step of administering to said mammal MSCs described in any one of claims 1 to 24 or the composition described in claim 25.

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

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

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

34. 26. A method for reducing the number of activated T cells in a mammal, said method comprising the step of administering to said mammal MSCs according to any one of claims 1 to 24 or the composition according to claim 25.

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

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

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

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

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

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

41. Mesenchymal stromal cells (MSCs), including: (1) an exogenous nucleic acid encoding a chimeric antigen receptor (CAR) that targets an epithelial-specific antigen, wherein the MSCs express the CAR; and (2) 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) alpha polypeptide, cytotoxic T lymphocyte-associated protein 4 (CTLA4), toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), toll-like receptor 9 (TLR9), tumor necrosis factor receptor 2 (TNFR2) polypeptide, interleukin (IL)-10 polypeptide and high levels of a polypeptide selected from the group consisting of: 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 eotaxin 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 C-X-C chemokine receptor (CXCR) 3 polypeptide, and a CXCR4 polypeptide.

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

43. The MSCs according to any one of claims 41 to 42, wherein the MSCs are adipose-derived MSCs.

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

45. The MSC of any one of claims 41 to 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, and the MSC expresses the CTLA4 polypeptide.

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

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

49. The MSC of claim 45, wherein the MSC comprises an exogenous nucleic acid encoding the TLR9 polypeptide, and 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 of any one of claims 41 to 50, wherein the CAR comprises a heavy chain comprising the CDR set forth in SEQ ID NO: 1 and a light chain comprising the CDR set forth in SEQ ID NO:

2.

52. The MSCs of claim 51, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 1 and the light chain comprises the amino acid sequence set forth in SEQ ID NO:

2.

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

4.

54. The MSCs of claim 53, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 3 and the light chain comprises the amino acid sequence set forth in SEQ ID NO:

4.

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

56. 56. A method for treating a mammal with graft-versus-host disease (GVHD), said method comprising administering to said mammal MSCs of any one of claims 41 to 54 or a composition of claim 55.

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

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

59. 58. The method of any one of claims 56 to 57, wherein the number of regulatory T cells (Treg) in the mammal is increased by at least 10 percent.

60. 56. A method for suppressing an immune response in a mammal, said method comprising the step of administering to said mammal MSCs according to any one of claims 41 to 54 or the composition according to claim 55.

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

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

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

64. 56. A method for reducing the number of activated T cells in a mammal, said method comprising the step of administering to said mammal MSCs according to any one of claims 41 to 54 or the composition according to claim 55.

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

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

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

68. Use of the MSCs 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. 56. The MSCs of any one of claims 41 to 54 or the composition of claim 55 for use in the preparation of a medicament for suppressing an immune response in a mammal.

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

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