Methods and materials using engineered mesenchymal stem cells to treat inflammatory conditions and degenerative diseases

Abstract

This document relates to methods and materials involved in treating mammals (e.g., humans) having or at risk of developing a disease or condition characterized by inflammation and / or degeneration of tissue. For example, provided herein are mesenchymal stem cells (MSCs) that express tissue-targeting antigen receptors (e.g., chimeric antigen receptors) that can exert immunosuppressive effects in targeted tissues, and methods of using such MSCs.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 932,610, filed November 8, 2019. The disclosure of the prior application is considered part of (and is incorporated by reference into) the disclosure of this application.

[0002] background 1. Technical Field This document relates to methods and materials for using engineered mesenchymal stem cells (MSCs) to treat mammals (e.g., humans) having or at risk of developing an inflammatory disease or condition and / or having or at risk of developing a degenerative disease. For example, this document provides engineered MSCs designed to express an antigen receptor (e.g., a chimeric antigen receptor (CAR)) capable of binding (e.g., specifically binding) to an antigen (e.g., a tissue-specific antigen). Such engineered MSCs can exert an immunosuppressive effect (e.g., reduce or eliminate an immune response) in a targeted tissue and / or regenerate tissue-specific cells. In some cases, MSCs engineered to express an antigen receptor capable of binding to an antigen (e.g., tissue-specific cells expressing an antigen targeted by the MSC) can also be engineered to express a polypeptide capable of promoting differentiation into tissue-specific cells. For example, MSCs can be engineered to contain a nucleic acid encoding a polypeptide capable of promoting tissue-specific cell differentiation and capable of differentiating into tissue-specific cells (e.g., cardiac cells or neurons). This document also provides methods of treating an inflammatory disease or condition in a mammal (e.g., a human) having or at risk of developing an inflammatory disease or condition by administering to the mammal one or more MSCs that express an antigen receptor capable of binding (e.g., specifically binding) to a tissue-specific antigen. [Background technology]

[0003] 2. Background information Inflammation is a common factor in many diseases. In 2015, an estimated 1.3% of U.S. adults (approximately 3 million people) were reported to have been diagnosed with IBD (either Crohn's disease or ulcerative colitis; Dahlhamer et al., MMWR Morb Mortal Wkly Rep. 2016, 65(42):1166-1169 (2015)). Summary of the Invention

[0004] This document provides methods and materials involved in treating a mammal (e.g., a human) having or at risk of developing an inflammatory disease or condition and / or having or at risk of developing a degenerative disease. For example, one or more MSCs engineered to express an antigen receptor (e.g., a CAR) capable of binding (e.g., specifically binding) to a tissue-specific antigen can be administered to the mammal to induce an immunosuppressive response (e.g., reduce or eliminate an inflammatory immune response) in the mammal. In some cases, a CAR targeting an epithelial-specific antigen (e.g., epithelial cadherin (ECAD, also known as CDH1)) can be expressed by MSCs (e.g., MSC-CARs) to target the MSCs to epithelial tissues. In some cases, a CAR targeting a neural-specific antigen (e.g., myelin oligodendrocyte glycoprotein (MOG)) can be expressed by MSCs (e.g., MSC-CARs) to target the MSCs to neural tissues. In some cases, a CAR targeting a cardiac-specific antigen (e.g., human epidermal growth factor receptor 2 (HER2)) can be expressed by MSCs to target the MSCs to cardiac tissue. For example, one or more MSCs engineered to express a CAR capable of binding to (e.g., specifically binding to) a tissue-specific antigen (e.g., an epithelial-specific antigen, a neural-specific antigen, or a cardiac-specific antigen) can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) having (or at risk of developing) an inflammatory disease or condition to treat the inflammatory disease or condition in the mammal. In some cases, one or more MSCs expressing a CAR capable of binding to (e.g., specifically binding to) an epithelial-specific antigen (e.g., ECAD) can be administered to a mammal (e.g., a human) having or at risk of developing inflammatory bowel disease (IBD; e.g., colitis) to treat the inflammatory bowel disease in the mammal (e.g., a human) (e.g., by adoptive transfer).In some cases, one or more MSCs expressing a CAR capable of binding (e.g., specifically binding) to a neuro-specific antigen (e.g., MOG) can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) having or at risk of developing multiple sclerosis to treat the multiple sclerosis in the mammal. In some cases, one or more MSCs expressing a CAR capable of binding (e.g., specifically binding) to a neuro-specific antigen (e.g., MOG) can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) having or at risk of developing immune-mediated encephalomyelitis to treat the immune-mediated encephalomyelitis in the mammal. In some cases, one or more MSCs expressing a CAR capable of binding (e.g., specifically binding) to a cardiac-specific antigen (e.g., HER2) can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) having or at risk of developing myocarditis to treat the myocarditis in the mammal. For example, MSCs engineered to express an antigen receptor (e.g., a CAR) capable of binding (e.g., specifically binding) to a tissue-specific antigen can also be engineered to express a polypeptide capable of promoting differentiation into a tissue-specific cell (e.g., can comprise a nucleic acid encoding a polypeptide capable of promoting differentiation into a tissue-specific cell). Such engineered MSCs can express a polypeptide capable of promoting MSC differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells (e.g., cardiac cells or neurons) within a mammal. In some cases, one or more MSCs engineered to express an antigen receptor (e.g., a CAR) capable of binding (e.g., specifically binding) to a tissue-specific antigen and engineered to differentiate into tissue-specific cells can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) having or at risk of developing a degenerative disease to treat the degenerative disease in the mammal.

[0005] As demonstrated herein, MSCs (e.g., adipose-derived MSCs) can be engineered to express a CAR that targets a tissue-specific antigen (e.g., an antigen expressed on a target tissue). For example, MSCs can be engineered to express a CAR that targets (e.g., binds to) ECAD in epithelial tissues (e.g., CAR-ECAD) in a mammal and can be used to treat diseases or disorders characterized by inflammation of epithelial tissues (e.g., colitis). In another example, MSCs can be engineered to express a CAR that targets (e.g., binds to) MOG in neural tissues (e.g., CAR-MOG) in a mammal and can be used to treat diseases or disorders characterized by inflammation of neural tissues (e.g., multiple sclerosis and immune-mediated encephalomyelitis). In some cases, MSCs can be engineered to express a CAR that targets (e.g., binds to) an antigen expressed on a target tissue to exert an immunosuppressive effect in the target tissue (e.g., to reduce or eliminate an inflammatory immune response). For example, MSCs expressing a CAR described herein can be used to reduce or eliminate the proliferation of stimulated T cells in a targeted tissue (e.g., compared to levels present prior to administration of such MSCs).

[0006] Having the ability to treat inflammatory diseases and conditions as described herein allows clinicians and patients to reduce inflammation in patients in an effective and efficient manner.

[0007] In general, one aspect of this document features a method of treating a mammal with colitis. The method can include or consist essentially of administering to the mammal a composition comprising MSCs containing exogenous nucleic acid encoding a CAR targeting an epithelial-specific antigen, wherein the MSCs express the CAR. The mammal can be human. The MSCs can be adipose-derived MSCs. The epithelial-specific antigen can be ECAD. The CAR can comprise a single-chain variable fragment (scFv). The scFv can comprise a light chain and a heavy chain from an anti-CDH1 antibody. The anti-CDH1 antibody can be hSC10.17. The MSCs can be engineered to express the CAR ex vivo prior to administration. Symptoms of colitis can be reduced by at least 10 percent. The CAR can comprise a CD28 or TLR4 signaling domain.

[0008] In another aspect, one aspect of this document features a method of treating a mammal at risk of developing colitis. The method can include or consist essentially of administering to the mammal a composition comprising MSCs containing an exogenous nucleic acid encoding a CAR targeting an epithelial-specific antigen, wherein the MSCs express the CAR. The mammal can be a human. The MSCs can be adipose-derived MSCs. The epithelial-specific antigen can be ECAD. The CAR can comprise an scFv. The scFv can comprise a light chain and a heavy chain from an anti-CDH1 antibody. The anti-CDH1 antibody can be hSC10.17. The MSCs can be engineered to express a CAR ex vivo prior to administration. The CAR can comprise a CD28 or TLR4 signaling domain.

[0009] In another aspect, one aspect of this document features a method for treating a mammal with multiple sclerosis. The method can include or consist essentially of administering to the mammal a composition comprising MSCs containing an exogenous nucleic acid encoding a CAR targeting a neurospecific antigen, wherein the MSCs express the CAR. The mammal can be human. The MSCs can be adipose-derived MSCs. The neurospecific antigen can be MOG. The CAR can include an scFv. The scFv can include a light chain and a heavy chain from an anti-MOG antibody. The anti-MOG antibody can be 8-18C5. The MSCs can be engineered to express the CAR ex vivo prior to administration. Symptoms of multiple sclerosis can be reduced by at least 10 percent. The MSCs can also contain an exogenous nucleic acid encoding a polypeptide capable of promoting neural differentiation, wherein the MSCs express the polypeptide. The polypeptide capable of promoting neural cell differentiation can be an Oct3 / 4 polypeptide, a Klf4 polypeptide, a Sox2 polypeptide, a Glis1 polypeptide, a c-Myc polypeptide, a BMP4 polypeptide, a WNT polypeptide, an FGF2 polypeptide, an SHH polypeptide, a Sox11 polypeptide, a Sox2 polypeptide, a Sox3 polypeptide, a Zic1 polypeptide, a Zic2 polypeptide, an Irx1 polypeptide, an Irx2 polypeptide, an Irx3 polypeptide, a FoxD4 polypeptide, an MKx2.5 polypeptide, a cTnT polypeptide, or any combination thereof. The CAR can comprise a CD28 or TLR4 signaling domain.

[0010] In another aspect, one aspect of this document features a method of treating a mammal at risk of developing multiple sclerosis. The method can include or consist essentially of administering to the mammal a composition comprising MSCs containing an exogenous nucleic acid encoding a CAR targeting a neuro-specific antigen, wherein the MSCs express the CAR. The mammal can be a human. The MSCs can be adipose-derived MSCs. The neuro-specific antigen can be MOG. The CAR can comprise an scFv. The scFv can comprise a light chain and a heavy chain from an anti-MOG antibody. The anti-MOG antibody can be 8-18C5. The MSCs can be engineered to express a CAR ex vivo prior to administration. The CAR can comprise a CD28 or TLR4 signaling domain.

[0011] In another aspect, one aspect of this document features a method for treating a mammal with immune-mediated encephalomyelitis. The method can include or consist essentially of administering to the mammal a composition comprising MSCs containing an exogenous nucleic acid encoding a CAR targeting a neurospecific antigen, wherein the MSCs express the CAR. The mammal can be human. The MSCs can be adipose-derived MSCs. The neurospecific antigen can be MOG. The CAR can include an scFv. The scFv can include a light chain and a heavy chain from an anti-MOG antibody. The anti-MOG antibody can be 8-18C5. The MSCs can be engineered to express the CAR ex vivo prior to administration. Symptoms of immune-mediated encephalomyelitis can be reduced by at least 10 percent. The MSCs can also contain an exogenous nucleic acid encoding a polypeptide capable of promoting neural differentiation, wherein the MSCs express the polypeptide. The polypeptide capable of promoting neural differentiation can be an Oct3 / 4 polypeptide, a Klf4 polypeptide, a Sox2 polypeptide, a Glis1 polypeptide, a c-Myc polypeptide, a BMP4 polypeptide, a WNT polypeptide, an FGF2 polypeptide, an SHH polypeptide, a Sox11 polypeptide, a Sox2 polypeptide, a Sox3 polypeptide, a Zic1 polypeptide, a Zic2 polypeptide, an Irx1 polypeptide, an Irx2 polypeptide, an Irx3 polypeptide, a FoxD4 polypeptide, an MKx2.5 polypeptide, a cTnT polypeptide, or any combination thereof. The CAR can comprise a CD28 or TLR4 signaling domain.

[0012] In another aspect, one aspect of this document features a method of treating a mammal at risk of developing immune-mediated encephalomyelitis. The method can include or consist essentially of administering to the mammal a composition comprising MSCs containing an exogenous nucleic acid encoding a CAR targeting a neurospecific antigen, wherein the MSCs express the CAR. The mammal can be a human. The MSCs can be adipose-derived MSCs. The neurospecific antigen can be MOG. The CAR can comprise an scFv. The scFv can comprise a light chain and a heavy chain from an anti-MOG antibody. The anti-MOG antibody can be 8-18C5. The MSCs can be engineered to express a CAR ex vivo prior to administration. The CAR can comprise a CD28 or TLR4 signaling domain.

[0013] In another aspect, one embodiment of the present document features a nucleic acid construct encoding a CAR targeting a neurospecific antigen. The neurospecific antigen can be MOG. The CAR can comprise an scFv. The CAR targeting a neurospecific antigen can be encoded by the nucleic acid sequence set forth in SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7. The nucleic acid construct can also encode a polypeptide capable of promoting neural differentiation. The polypeptide capable of promoting neural differentiation can be an Oct3 / 4 polypeptide, a Klf4 polypeptide, a Sox2 polypeptide, a Glis1 polypeptide, a c-Myc polypeptide, a BMP4 polypeptide, a WNT polypeptide, an FGF2 polypeptide, an SHH polypeptide, a Sox11 polypeptide, a Sox2 polypeptide, a Sox3 polypeptide, a Zic1 polypeptide, a Zic2 polypeptide, an Irx1 polypeptide, an Irx2 polypeptide, an Irx3 polypeptide, a FoxD4 polypeptide, an MKx2.5 polypeptide, a cTnT polypeptide, or any combination thereof. The CAR can comprise a CD28 or TLR4 signaling domain.

[0014] In another aspect, one aspect of this document features a method for treating a mammal with myocarditis. The method can include or consist essentially of administering to the mammal a composition comprising MSCs containing an exogenous nucleic acid encoding a CAR targeting a cardiac-specific antigen, wherein the MSCs express the CAR. The mammal can be human. The MSCs can be adipose-derived MSCs. The cardiac-specific antigen can be HER2. The CAR can comprise an scFv. The MSCs can be engineered to express the CAR ex vivo prior to administration. The symptoms of myocarditis can be reduced by at least 10 percent. The MSCs can also contain an exogenous nucleic acid encoding a polypeptide capable of promoting cardiac cell differentiation, wherein the MSCs express the polypeptide. The polypeptide capable of promoting neural differentiation can be a GATA4 polypeptide, a MEF2C polypeptide, a TBX5 polypeptide, an ERRG polypeptide, a MESP1 polypeptide, or any combination thereof. The CAR can comprise a CD28 or TLR4 signaling domain.

[0015] In another aspect, one aspect of this document features a method of treating a mammal at risk of developing myocarditis. The method can include or consist essentially of administering to the mammal a composition comprising MSCs containing an exogenous nucleic acid encoding a CAR that targets a cardiac-specific antigen, wherein the MSCs express the CAR. The mammal can be a human. The MSCs can be adipose-derived MSCs. The cardiac-specific antigen can be HER2. The CAR can comprise an scFv. The MSCs can be engineered to express the CAR ex vivo prior to administration. The CAR can comprise a CD28 or TLR4 signaling domain.

[0016] In another aspect, one embodiment of the present document features a nucleic acid construct encoding a CAR targeting a cardiac-specific antigen. The cardiac-specific antigen can be HER2. The CAR can comprise an scFv. The CAR targeting the cardiac-specific antigen can be encoded by the nucleic acid sequence set forth in SEQ ID NO: 9. The nucleic acid construct can also encode a polypeptide capable of promoting cardiac cell differentiation. The polypeptide capable of promoting neural differentiation can be a GATA4 polypeptide, a MEF2C polypeptide, a TBX5 polypeptide, an ERRG polypeptide, a MESP1 polypeptide, or any combination thereof. The CAR can comprise a CD28 or TLR4 signaling domain.

[0017] 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 can 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.

[0018] 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]

[0019] [Figure 1] FIG. 1 shows enhanced lentiviral transduction of MSCs. [Figure 2] FIG. 1 shows MSC expression of CAR19 (MSC19). [Figure 3] FIG. 1 shows the stability of MSC-CAR19 expression after passage 10+. [Figure 4]A. MSC suppression of antigen-specific CAR-T cell proliferation. B. MSC suppression of antigen-specific CAR-T cell proliferation. [Figure 5] FIG. 1 is a schematic diagram of an exemplary CAR19-encoding nucleic acid construct. [Figure 6] FIG. 1 shows MSC expression of CAR-ECAD. [Figure 7] MSCs suppress T cell proliferation but not CAR2 T cell proliferation. [Figure 8] MSC-CAR mediated suppression of T cell proliferation. [Figure 9A-9B] A. MSC-CAR mediated suppression of activated T cells and CART cells. B. Suppression of antigen-specific CAR-T proliferation by CART19+ NALM6. [Figure 10A] Figure 1 shows stemness maintenance of MSCs A. Flow cytometry analysis of CD90. [Figure 10B] B. Flow cytometry analysis of CD105. [Figure 10C] C. Flow cytometry analysis of CD73. [Figure 10D] Figure 1 shows the stemness maintenance of MSCs D. Flow cytometry analysis of CD34. [Figure 10E] Figure 1 shows stemness maintenance of MSCs E. Flow cytometry analysis of CD45. [Figure 10F] Figure 1 shows the maintenance of stemness of MSCs F. Flow cytometry analysis of HLA-DR. [Figure 10G] Figure 1 shows stemness maintenance of MSCs. G. Flow cytometry analysis of CD14. [Figure 11] FIG. 1 shows MSC expression of CAR-MOG. [Figure 12A] A. An exemplary CAR-ECAD-encoding nucleic acid sequence (SEQ ID NO: 1). [Figure 12B] B. Amino acid sequence of an exemplary CAR-ECAD (SEQ ID NO:2). [Figure 13A]A. An exemplary CAR-MOG-encoding nucleic acid sequence (SEQ ID NO: 3). [Figure 13B] B. Amino acid sequence of an exemplary CAR-MOG (SEQ ID NO: 4). [Figure 13C] C. An exemplary nucleic acid sequence encoding CAR-MOG (SEQ ID NO: 5). [Figure 13D] D. Amino acid sequence of an exemplary CAR-MOG (SEQ ID NO: 6). [Figure 13E] E. An exemplary nucleic acid sequence encoding CAR-MOG (SEQ ID NO: 7). [Figure 13F] F. Amino acid sequence of an exemplary CAR-MOG (SEQ ID NO: 8). [Figure 14A] A. An exemplary nucleic acid sequence encoding CAR-HER2 (SEQ ID NO: 9). [Figure 14B] B. Amino acid sequence of an exemplary CAR-HER2 (SEQ ID NO: 10). [Figure 15] Figure 15A is a graph plotting the percentage of MSCs that are CAR-positive after lentiviral transduction of MSCs with K002 (CD19-directed CAR) compared to UTD (untransduced MSCs). Figure 15B is a graph plotting the percentage of MSCs expressing the indicated markers two days after lentiviral transduction with CAR19 compared to untransduced MSCs (UTD). MSCs retain markers of stemness. [Figure 16]Figure 1 shows a graph plotting the absolute number of viable CD3 T cells 5 days after stimulation with CD3 / CD28 beads after co-culture with untransduced MSCs (MSC-UTD), or MSC-CAR19 (CAR19, CD28 containing K122), or MSC-CAR19 (CAR19, CD137 containing K002), or without MSCs. T cells are activated with CD3 / CD28 beads and then co-cultured with different MSC conditions, either with medium alone or in the presence of irradiated CD19+ cells, as a strategy to stimulate MSC-CAR19 via the CAR. CD3 proliferation is inhibited in the presence of MSC-CAR19 (containing the CD28 signaling domain), but not in the presence of MSC-CAR19 (containing the CD137 signaling domain) or untransduced MSCs. T cells, MSCs, and NALM6 cells were cultured at a ratio of 1:0.1:1 (E (T cells (effector): MSCs (suppressor): T (tumor)). This demonstrates that when MSC-CAR cells contain the CD28 signaling domain and when MSCs are activated via the CAR upon antigen-specific stimulation, MSC-CAR cells can also suppress T cell proliferation. * denotes a p-value ≦0.05. [Figure 17] Figure 1 shows the absolute number of viable MSC cells after 5 days of coculture of untransduced MSCs (MSC-UTD), or MSC-CAR19 (CAR19, TLR4 containing K142), or MSC-CAR19 (CAR19, CD137 containing K002), or MSC-CAR19 (CAR19, CD28 containing K122) with or without irradiated CD19+ NALM6 cells. The proliferation of CD28-containing MSC-CAR19 expanded in the presence of irradiated CD19+ cells but not in their absence, suggesting antigen-specific stimulation of MSC-CAR19 and signaling via CD28. Similarly, the proliferation of TLR4-containing MSC-CAR19 decreased in the presence of irradiated CD19+ cells but not in their absence, suggesting antigen-specific stimulation of MSC-CAR19 and signaling via CD28. * denotes P ≤ 0.05, ** denotes P ≤ 0.01. [Figure 18] This graph plots the absolute number of viable CD3+ cells 5 days after stimulation with CD3 / CD28 beads after co-culture with untransduced MSCs (MSC-UTD) or MSC-CAR19 (CAR19, TLR4 containing K142) or without MSCs. T cells were activated with CD3 / CD28 beads and then co-cultured with different MSC conditions in medium alone or in the presence of irradiated CD19+ cells as a strategy to stimulate MSC-CAR19 via CAR. CD3+ proliferation was inhibited in the presence of MSC-CAR19 (containing the TLR4 signaling domain, K142), but not in the presence of untransduced MSCs. T cells, MSCs, and NALM6 cells were cultured at a ratio of 1:0.1:1 (E:T cells (effector):MSCs (suppressor):T (tumor)). This demonstrates that MSC-CAR cells can suppress T cell proliferation when they contain the TLR4 signaling domain and when MSCs are activated via the CAR upon antigen-specific stimulation. ** indicates P≦0.01. [Figure 19] Figure 19A is a graph plotting the absolute number of MSCs after 5 days of coculture with irradiated CD19+ NALM6 cells at a 1:1 ratio of MSC:NALM6. NALM6 cells are included in this culture to stimulate MSC-CAR19 via CAR. Coculture of CD19+ cells with MSC-CAR19-CD28 resulted in enhanced proliferation compared to untransduced MSCs (MSC-UTD), MSC-K002, MSC-K122, or MSC-K142. This indicates that antigen-specific stimulation of MSC-CAR19 via CAR results in altered MSC signaling and proliferation. Figure 19B shows the number of MSCs on days 3 and 5 of coculture. [Figure 20] 1 is a graph plotting the absolute number of MSCs in culture after transduction with CAR-MSC-E-cadherin or control GFP (MSC-ZSG). Transduction with CAR-E-cadherin-CD28 resulted in reduced MSC proliferation compared to untransduced MSCs (MSC-UTD) or MSCs transduced with GFP (MSC-ZSG). [Figure 21]

[0033] Figure 1 is a bar graph plotting the number of T cells / μL after stimulation with CD3 / CD28 beads and CD19+ cells (to stimulate MSC-CARs) in the presence of MSCs in either direct contact or transwell experiments. Co-culture of activated T cells with MSC-CAR19 (containing the CD28 stimulatory domain) and CD19+ cells resulted in inhibition of T cell proliferation, both when the cells were in direct contact or without direct contact in transwell experiments. This indicates that MSC-CARs exert their suppressive function through direct cell-to-cell contact and through the secretion of soluble inhibitory factors / cytokines. [Figure 22] This graph plots the number of T cells / μL 5 days after stimulation with CD3 / CD28 beads after co-culture with untransduced MSCs or MSC-CAR19 (CAR19, CD28 containing K122) at higher E:S:T ratios. T cells were activated with CD3 / CD28 beads and then co-cultured with different MSC conditions, either in medium alone or in the presence of irradiated CD19+ cells, as a strategy to stimulate MSC-CAR19 via CAR. CD3 proliferation is inhibited in the presence of MSC-CAR19 (containing the CD28 signaling domain), but not in the presence of untransduced MSCs. T cells, MSCs, and NALM6 cells were cultured at a 1:1:1 E (T cells (effector): MSCs (suppressor): T (tumor)) ratio. This demonstrates that when MSCs are activated via CAR upon antigen-specific stimulation, MSC-CAR cells containing the CD28 signaling domain can suppress T cell proliferation at different suppressor-to-T cell ratios. ** represents P<0.01, and *** represents P<0.001. [Figure 23]Figure 24A is a graph plotting the absolute number of viable CD3+ cells 5 days after stimulation with CD3 / CD28 beads after coculture with untransduced MSCs (MSC-UTD, Figure 24A) or MSC-CARE-cadherin (CD28 containing CAR-E-cadherin, Figure 24B) at different effector:suppressor (E:S) ratios in the presence or absence of the E-cadherin+ cell line MCF-7. Coculture of MSC-CAR-E-cadherin with T cells results in the suppression of their antigen-specific proliferation in the presence of the E-cadherin+ cell line MCF-7 at low effector:suppressor ratios. [Figure 24] Figure 24A is a graph plotting bioluminescence from luciferase+ / E-cadherin+ MCF-7 cells (1 x 106) injected into NSG mice and then treated with CAR-ECAD T cells in combination with either MSCs, untransduced MSCs (MSC-UTD), or MSC-ECAD-CAR with a CD28 signaling domain. Mice treated with MSC-ECAD exhibited more bioluminescence, demonstrating that MSC-ECAD inhibits the antitumor effect of CAR-ECAD T cells. Figure 24B is a bar graph plotting the absolute number of CD3+ T cells in NSG mice treated with CAR-ECAD T cells and either no MSCs, untransduced MSCs (MSC-UTD), or MSC-ECAD on day 3. [Figure 25] Figure 25A is a graph plotting bioluminescence from luciferase+ MSC-CAR-E-cadherin cells administered to immunodeficient NSG mice. Figure 25B contains representative images of bioluminescence from a representative mouse. DETAILED DESCRIPTION OF THE INVENTION

[0020] This document provides methods and materials involved in treating a mammal (e.g., a human) having or at risk of developing an inflammatory disease or condition and / or having or at risk of developing a degenerative disease. For example, one or more MSCs engineered to express an antigen receptor (e.g., a CAR) capable of binding (e.g., specifically binding) to a tissue-specific antigen can be administered to the mammal to induce an immunosuppressive response (e.g., reduce or eliminate an inflammatory immune response) in the mammal. In some cases, a CAR targeting an epithelial-specific antigen (e.g., ECAD) can be expressed by the MSCs to target the MSCs to epithelial tissue in a manner effective to treat a disease or disorder characterized by inflammation of the epithelial tissue (e.g., colitis). For example, MSCs engineered to express a CAR capable of targeting (e.g., capable of targeting and binding to) an antigen (e.g., a cell surface antigen) expressed by an epithelial cell (e.g., an epithelial-specific antigen or an epithelial antigen) can be administered to a mammal (e.g., a human) to treat or delay the progression of a disease or disorder characterized by inflammation and / or degeneration of epithelial tissue, such as colitis, hepatitis, pneumonia, asthma, pancreatitis, pulmonary fibrosis, colonic stricture, colonic fistula, glomerulonephritis, renal infarction, or hepatic infarction.

[0021] In some cases, a CAR that targets a neural-specific antigen (e.g., MOG) can be expressed by MSCs to target the MSCs to neural tissue in a manner effective to treat diseases or disorders characterized by inflammation of neural tissue (e.g., multiple sclerosis and immune-mediated encephalomyelitis). For example, MSCs engineered to express a CAR that can target (e.g., target and bind to) an antigen (e.g., a cell surface antigen) expressed by neural cells (e.g., a neural-specific antigen or neural antigen) can be administered to a mammal (e.g., a human) to treat or slow the progression of a disease or disorder characterized by inflammation and / or degeneration of neural tissue, such as multiple sclerosis, immune-mediated encephalomyelitis, or amyotrophic lateral sclerosis.

[0022] In some cases, a CAR that targets a cardiac-specific antigen (e.g., HER2) can be expressed by MSCs to target the MSCs to cardiac tissue in a manner effective to treat a disease or disorder characterized by inflammation of cardiac tissue (e.g., myocarditis). For example, MSCs engineered to express a CAR that can target (e.g., target and bind to) an antigen (e.g., a cell surface antigen) expressed by cardiac cells (e.g., a cardiac-specific antigen or cardiac antigen) can be administered to a mammal (e.g., a human) to treat or slow the progression of a disease or disorder characterized by inflammation and / or degeneration of cardiac tissue, such as myocarditis, myocardial infarction, heat failure, or endocarditis.

[0023] In some cases, one or more MSCs engineered to express a CAR capable of binding (e.g., specifically binding) to a tissue-specific antigen (e.g., an epithelial-specific antigen) can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) having (or at risk of developing) an inflammatory disease or condition to treat the inflammatory disease or condition in the mammal. In some cases, one or more MSCs expressing a CAR capable of binding (e.g., specifically binding) to an epithelial-specific antigen (e.g., ECAD) can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) having or at risk of developing inflammatory bowel disease (IBD; e.g., colitis) to treat the inflammatory bowel disease in the mammal. In some cases, one or more MSCs expressing a CAR capable of binding (e.g., specifically binding) to a neuro-specific antigen (e.g., MOG) can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) having or at risk of developing multiple sclerosis to treat the multiple sclerosis in the mammal. In some cases, one or more MSCs expressing a CAR capable of binding (e.g., specifically binding) to a neuro-specific antigen (e.g., MOG) can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) having or at risk of developing immune-mediated encephalomyelitis to treat the immune-mediated encephalomyelitis in the mammal. In some cases, one or more MSCs expressing a CAR capable of binding (e.g., specifically binding) to a cardiac-specific antigen (e.g., HER2) can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) having or at risk of developing myocarditis to treat the myocarditis in the mammal.

[0024] In some cases, a cartilage antigen (e.g., CH65, human cartilage glycoprotein-39 (HC gp-39), CD44, thymocyte antigen-1 (Thy-1), CD90, CD24, lymphocyte function-associated antigen-3 (LFA-3), or a bone cell antigen (e.g., E11 or gp38)) can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) to treat or slow the progression of a disease or disorder characterized by joint inflammation and / or degeneration, such as inflammatory arthritis, degenerative arthritis, autoimmune spondyloarthritis, psoriatic arthritis, osteogenesis imperfecta, or metachromatic leukodystrophy.

[0025] In some cases, MSCs engineered to express an antigen receptor (e.g., a CAR) capable of binding (e.g., specifically binding) to a tissue-specific antigen can also be engineered to express a polypeptide capable of promoting differentiation into tissue-specific cells (e.g., cardiac cells or neurons) within a mammal. In some cases, MSCs engineered to express a polypeptide capable of promoting differentiation can promote the differentiation of cells (e.g., MSCs and / or one or more resident progenitor cells) into tissue-specific cells (e.g., cardiac cells or neurons) within a mammal. In some cases, MSCs engineered to express a polypeptide capable of promoting differentiation can promote the differentiation of resident cells (e.g., resident progenitor cells) into tissue-specific cells (e.g., cardiac cells or neurons) within a mammal. Such engineered MSCs can express a polypeptide capable of promoting differentiation into any suitable tissue-specific cell (e.g., cardiac cells or neurons) within a mammal. In some cases, MSCs comprising a nucleic acid encoding a polypeptide capable of promoting cardiac cell differentiation can differentiate the MSCs into cardiac cells. In some cases, MSCs comprising a nucleic acid encoding a polypeptide capable of promoting neural differentiation can differentiate the MSCs into neurons. For example, one or more MSCs engineered to express an antigen receptor (e.g., a CAR) capable of binding (e.g., capable of specifically binding) a tissue-specific antigen and also engineered to differentiate into tissue-specific cells can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) having or at risk of developing a degenerative disease to treat the degenerative disease in the mammal. In some cases, one or more MSCs engineered to differentiate into cardiac cells (e.g., one or more MSCs comprising a nucleic acid capable of encoding a polypeptide capable of promoting cardiac cell differentiation) can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) having or at risk of developing a degenerative heart disease (e.g., congestive heart failure (CHF)) to treat the degenerative heart disease in the mammal (e.g., by regenerating cardiac cells in the mammal).In some cases, one or more MSCs engineered to differentiate into neural cells (e.g., one or more MSCs comprising a nucleic acid capable of encoding a polypeptide capable of promoting neural differentiation) can be administered to a mammal (e.g., a human) having or at risk of developing a neurodegenerative disease (e.g., Parkinson's disease and Alzheimer's disease) to treat the neurodegenerative disease in the mammal (e.g., by regenerating neurons in the mammal).

[0026] In some cases, MSCs (e.g., MSCs engineered to express a CAR described herein) can be designed to express one or more transcription factors to promote differentiation into cardiomyocytes (e.g., BMP-4, FGF-4, FGF-basic, and / or TGF-β). In such cases, the MSCs can be administered to a mammal (e.g., a human) to treat or slow the progression of a disease or disorder characterized by cardiac degeneration, such as myocardial infarction or heart failure.

[0027] In some cases, MSCs (e.g., MSCs engineered to express a CAR described herein) can be engineered to express one or more transcription factors that promote their differentiation into bone cells (e.g., BMP-2, BMP-4, BMP-6, FGF-basic, TGF-beta, PTH, and / or Wnt10b), or one or more transcription factors that promote their differentiation into chondrocytes (BMP-2, BMP-3, BMP-5, BMP-7, N-cadherin, NCAN-1, and / or perlecan). In such cases, the MSCs can be administered to a mammal (e.g., a human) to treat or delay the progression of a disease or disorder characterized by bone degeneration, e.g., degenerative arthritis or osteogenesis imperfecta.

[0028] The MSCs described herein (e.g., MSCs that express a CAR that targets a tissue-specific antigen, e.g., an epithelial-specific antigen, a neural-specific antigen, or a cardiac-specific antigen, and optionally express a polypeptide capable of promoting differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells, e.g., cardiac cells or neurons) can be any suitable MSCs. Examples of MSCs that can be used as described herein include, but are not limited to, adipose-derived MSCs, osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells), adipocytes (fat cells), neural progenitor stem cells (neurons), dental pulp-derived MSCs, umbilical cord blood-derived MSCs, and umbilical cord-derived MSCs. For example, MSCs that express a CAR and optionally express a polypeptide capable of promoting differentiation into tissue-specific cells that target a tissue-specific antigen can be adipose-derived MSCs.

[0029] The CAR can comprise an antigen-binding domain and a signaling domain. The antigen-binding domain can be any suitable antigen-binding domain. In some cases, the antigen-binding domain can comprise an antibody or fragment thereof that targets an antigen (e.g., a tissue-specific antigen, such as an epithelial-specific antigen, a neural-specific antigen, or a cardiac-specific antigen). Examples of antigen-binding domains include, but are not limited to, antigen-binding fragments (Fab), variable regions of antibody heavy chains (VH), variable regions of light chains (VL), single-chain variable fragments (scFv), polypeptides, ligands, and cytokines. In some cases, the antigen-binding domain can target (e.g., target and bind to) a tissue-specific antigen (e.g., an epithelial-specific antigen, a neural-specific antigen, or a cardiac-specific antigen). For example, the MSCs described herein can express (e.g., can be engineered to express) a CAR that can bind to a tissue-specific antigen (e.g., an antigen present on cells in a tissue that is minimally expressed or not expressed on other cell types).

[0030] In some cases, the MSCs 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 in a mammal (e.g., a mammal having or at risk of developing a disease or disorder characterized by inflammation and / or degeneration of epithelial tissue, e.g., colitis). 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, and liver cells). In some cases, the epithelial-specific antigen can be a cell adhesion molecule (CAM). Examples of epithelial-specific antigens include, but are not limited to, ECAD, CD103, hSC10.17, hSC10.178, CD234, EPCAM, EMA, MUC1, cytokeratin, CA125, ALCAM, HLA, desmin, Eputheliam antigen antibody, CD227, ESA, galactin 3, GGT, HLA-DR, lectin, LAMP-1, MMR, MOC-31, p16, p63, p-cadherin, PSA, surfactant, transthyretin, VAT-1, and vimentin. For example, an MSC-CAR engineered to target epithelial tissue can bind to ECAD. In some cases, an MSC-CAR can be engineered to express CAR-ECAD to target ECAD expressed by epithelial cells in a mammal having or at risk of developing IBD (e.g., colitis).

[0031] In some cases, the MSCs can be engineered to express a CAR that can target (e.g., target and bind to) an antigen (e.g., a cell surface antigen) expressed by a neural cell (e.g., a neural-specific antigen or neural antigen) in a mammal (e.g., a mammal having or at risk of developing a disease or disorder characterized by inflammation and / or degeneration of neural tissue, e.g., multiple sclerosis and immune-mediated encephalomyelitis). When the antigen is a neural-specific antigen, the neural-specific antigen can be any suitable neural-specific antigen. The neural-specific antigen can be expressed on any suitable type of neural cell (e.g., sensory neurons, motor neurons, interneurons, glial cells, and oligodendrocytes). The neural-specific antigen can be expressed on neural cells of the central nervous system (CNS) and / or peripheral nervous system (PNS). In some cases, the neural-specific antigen can be a transmembrane protein. Examples of neural-specific antigens include, but are not limited to, MOG, MBP, PDGF receptor alpha, OSP, SOX10, Olig 1, Olig 2, Olig 3, and NG2. For example, an MSC-CAR engineered to target neural tissue can bind to MOG. In some cases, an MSC-CAR can be engineered to express CAR-MOG, which targets MOG expressed by neural cells in a mammal having or at risk of developing multiple sclerosis. In some cases, an MSC-CAR can be engineered to express CAR-MOG, which targets MOG expressed by neural cells in a mammal having or at risk of developing immune-mediated encephalomyelitis.

[0032] In some cases, MSCs can be engineered to express a CAR that can target (e.g., target and bind to) an antigen (e.g., a cell surface antigen) expressed by cardiac cells (e.g., a cardiac-specific antigen or cardiac antigen) in a mammal (e.g., a mammal having or at risk of developing a disease or disorder characterized by inflammation and / or degeneration of cardiac tissue, e.g., myocarditis). When the antigen is a cardiac-specific antigen, the cardiac-specific antigen can be any suitable cardiac-specific antigen. The cardiac-specific antigen can be expressed on any suitable type of cardiac cell (e.g., cardiomyocytes and endocardial cells). Examples of cardiac-specific antigens include, but are not limited to, HER2. For example, an MSC-CAR engineered to target cardiac cells can bind to HER2. In some cases, an MSC-CAR can be engineered to express CAR-HER2, which targets HER2 expressed by cardiac cells in a mammal having or at risk of developing myocarditis.

[0033] The signaling domain can be any suitable signaling domain. In some cases, the signaling domain of a CAR used in the MSCs described herein can be an intracellular signaling domain normally found in T cells or NK cells. Examples of signaling domains that can be used as described herein include, but are not limited to, a CD3 zeta signaling domain, a CD28 signaling domain, a Toll-like receptor (TLR) (e.g., TLR3 and TLR4), 4-1BB, OX40, ICOS, CD2, and a promoter (e.g., a specific promoter, such as a cardiac-specific promoter, a neural-specific promoter, a T cell-specific promoter, a GI, a lung-specific promoter, a joint-specific promoter, and a cartilage-specific promoter). In some cases, the signaling domain of a CAR expressed by an MSC described herein can induce the MSC to produce one or more cytokines (e.g., an anti-inflammatory cytokine) upon activation of the CAR. Examples of cytokines that the engineered CARs of MSCs described herein can induce MSCs to produce include, but are not limited to, IFN-B, IL-10, IL-4, IL-2, MIP1B, IFNg, MIP1a, GM-CSF, IL-6, TNFa, and TNFb.

[0034] Any suitable method can be used to express a CAR described herein (e.g., a CAR that targets a tissue-specific antigen, e.g., an epithelial-specific antigen, a neural-specific antigen, or a cardiac-specific antigen) on the surface of an MSC described herein. For example, a nucleic acid encoding a CAR can be introduced into an MSC. In some cases, a nucleic acid encoding a CAR can be introduced into an MSC by transduction (e.g., viral transduction) or transfection. In some cases, a nucleic acid encoding a CAR described herein can be introduced into one or more MSCs ex vivo. For example, ex vivo manipulation of MSCs to express a CAR described herein can include transducing isolated MSCs with a lentiviral vector encoding a CAR. When MSCs are manipulated ex vivo to express a CAR, the MSCs can be obtained from any suitable source (e.g., a mammal, e.g., the mammal to be treated, or a donor mammal, or a cell line). In some cases, an MSC-CAR can be prepared as described herein (see, e.g., Figure 2 and Example 1). For example, by introducing one or more constructs containing a nucleic acid encoding a CAR (e.g., CAR-targeted ECAD) into MSCs, CAR-ECAD can be expressed in MSCs to direct MSCs to epithelial tissue. For example, by introducing one or more constructs containing a nucleic acid encoding a CAR (e.g., CAR-targeted MOG) into MSCs, CAR-MOG can be expressed in MSCs to direct MSCs to neural tissue. In some cases, MSC-CARs can be prepared as described elsewhere (see, for example, Blat et al., Mol. Ther., 22(5):1018-28 (2014); MacDonald et al., J. Clin. Invest., 126(4):1413-24 (2016); and Yoon et al., Blood, 129(2):238-245 (2017)).

[0035] Also provided herein are CARs and constructs (e.g., nucleic acid constructs) encoding the CARs described herein (e.g., CARs that target tissue-specific antigens, e.g., epithelial-specific antigens, neural-specific antigens, or cardiac-specific antigens). For example, a construct encoding a CAR-targeted ECAD (e.g., CAR-ECAD) can comprise a nucleic acid sequence encoding one or more molecules that bind to ECAD described herein. In some cases, a CAR-ECAD can comprise an anti-ECAD antibody (e.g., hSC10.17 and hSC10.178) heavy chain and an anti-ECAD antibody (e.g., hSC10.17 and hSC10.178) light chain. For example, a construct encoding a CAR-targeted MOG (e.g., CAR-MOG) can comprise a nucleic acid sequence encoding one or more molecules that bind to MOG described herein. In some cases, a CAR-MOG can comprise an anti-MOG antibody (e.g., 8-18C5) heavy chain and an anti-MOG antibody (e.g., 8-18C5) light chain. For example, a construct encoding a CAR that targets HER2 (e.g., CAR-HER2) can include a nucleic acid sequence encoding one or more molecules that bind to HER2 as described herein. In some cases, CAR-HER2 can include an anti-HER2 antibody (e.g., 4D5) heavy chain and an anti-ECAD antibody (e.g., 4D5) light chain. Exemplary nucleic acid sequences that can encode one or more molecules that bind to tissue-specific antigens as described herein and that can be included in the constructs described herein include, but are not limited to, those that encode the following amino acid sequences:

[0036] Anti-ECAD antibody heavy chain (SEQ ID NO: 11) QILLVQSGPELKKPGETVKISCKASNYTFTDYGMHWVKQAPGKGLKWMGWINPKTGVASYADDFKGRFAFSLETSASTAYLQINNLENEDTSIYFCARFFDYWGQGTTLTVSS Anti-ECAD antibody light chain (SEQ ID NO: 12) DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSDGNTYLEWYQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHAPWTFGGGTKVEIK Anti-ECAD antibody light chain (SEQ ID NO: 13) DIVLTQSPLSLLVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPNLLIFKVSNRFSGVPDRFSGSGSGTDFTLRISRVEAEDLGVYFCSQTTHVWTFGGGTKLEIK Anti-ECAD scFv clone 5 (SEQ ID NO: 55; light chain followed by bold / underlined linker followed by heavy chain) TIFF0007817159000001.tif25150 Anti-ECAD scFv clone 6 (SEQ ID NO: 56; light chain followed by bold / underlined linker followed by heavy chain) TIFF0007817159000002.tif25150 Anti-ECAD scFv clone 7 (SEQ ID NO: 57; heavy chain followed by bold / underlined linker followed by light chain) TIFF0007817159000003.tif25150 Anti-ECAD scFv clone 14 (SEQ ID NO: 58; light chain followed by bold / underlined linker followed by heavy chain) TIFF0007817159000004.tif25150 Anti-MOG antibody heavy chain (SEQ ID NO: 14) EVKLHESGAGLVKPGASVEISCKATGYTFSSFWIEWVKQRPGHGLEWIGEILPGRGRTNYNEKFKGKATFTAETSSNTAYMQLSSLTSEDSAVYYCATGNTMVNMPYWGQGTTVTVSS Anti-MOG antibody light chain (SEQ ID NO: 15) DIELTQSPSSLAVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGLPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSYPLTFGAGTKLEIK Anti-HER2 antibody heavy chain (SEQ ID NO: 16) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD Anti-HER2 antibody light chain (SEQ ID NO: 17) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAP

[0037] In some cases, an amino acid segment capable of binding to a tissue-specific antigen described herein and encoded by a nucleic acid sequence that can be included in a construct described herein can have a sequence (sometimes referred to as a variant sequence) that deviates from the polypeptide sequence (e.g., the light chain polypeptide sequence or the heavy chain polypeptide sequence) set forth in any one of SEQ ID NOS: 11-17. For example, an amino acid segment capable of binding to a tissue-specific antigen described herein and encoded by a nucleic acid sequence that can be included in a construct described herein can have at least 80% sequence identity to any one of SEQ ID NOS: 11-17. In some embodiments, an amino acid segment capable of binding to a tissue-specific antigen described herein and encoded by a nucleic acid sequence that can be included in a construct described herein can have at least 85% sequence identity, 90% sequence identity, 95% sequence identity, or at least 99% sequence identity to any one of SEQ ID NOS: 11-17. The percent sequence identity is calculated by determining the number of matching positions in the aligned polypeptide sequences, dividing each of the number of matching positions by the total number of aligned amino acids, and multiplying by 100. A matched position refers to a position where the same amino acid exists at the same position in the aligned sequences. The total number of aligned amino acids refers to the minimum number of amino acids in an amino acid segment that can bind to a tissue-specific antigen described herein and can be encoded by a nucleic acid sequence that can be included in the construct described herein, which is necessary to align a second sequence and does not include alignment with other sequences (e.g., forced alignment). For example, if an amino acid segment that can bind to a tissue-specific antigen described herein and can be encoded by a nucleic acid sequence that can be included in the construct described herein is a heavy chain, the total number of aligned amino acids can exclude any light chain.The total number of aligned amino acids may correspond to the entire amino acid segment that can bind to the tissue-specific antigens described herein and that can be included in the constructs described herein, or that can be encoded by a nucleic acid sequence that can bind to the tissue-specific antigens described herein and that can be included in the constructs described herein. Sequences can be aligned using the algorithm described by Altschul et al. (Nucleic Acids Res., 25:3389-3402 (1997)), which has been incorporated into the BLAST (Basic Local Alignment Search Tool) program, available on the World Wide Web at ncbi.nlm.nih.gov. BLAST searches or alignments can be performed using the Altschul et al. algorithm to determine percent sequence identity. BLASTN is a program used to align and compare identity between nucleic acid sequences, and BLASTP is a program used to align and compare identity between amino acid sequences. When using BLAST programs to calculate percent identity between an amino acid segment that can bind to a tissue-specific antigen described herein and that can be encoded by a nucleic acid sequence and another sequence that can be included in a construct described herein, the default parameters of the respective programs are used.

[0038] Exemplary nucleic acid sequences that can encode one or more molecules that bind to the tissue-specific antigens described herein that can be included in the constructs described herein are as follows:

[0039] Anti-ECAD antibody heavy chain (SEQ ID NO: 18) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGCATACATTACTACTAGAAGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTACTAGAGAACCCCTAACTGGATACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAG Anti-ECAD antibody heavy chain (SEQ ID NO: 19) CAGATCCTGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTAATTATACCTTCACAGACTATGGAATGCACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACCCCAAGACTGGTGTGGCATCATATGCAGATGACTTCAAGGGAAGATTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCGAAAATGAGGACACGTCTATATATTTCTGTGCTAGATTTTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA Anti-ECAD antibody light chain (SEQ ID NO: 20) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCATCGTACACAGTGATGGAAACACCTACTTGGAATGGTATCAGCAGAGGCCAGGCCAATCTCCAAGGCGCCTAATTTATAAGGTT TCTAACCGGTTCTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGGGTTATTACTGCTTTCAAGGTTCACATGCTCCGTGGACGTTCGGTGGAGGCACCAAGGTGGAAATCAAAC Anti-ECAD antibody light chain (SEQ ID NO: 21) GATATTGTGCTGACACAGTCTCCACTCTCCCTGCTTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTATCTGCAGAAGCCAGGCCAGTCTCCAAACCTCCTGATCTTCAAAG TTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAGGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAACTACACATGTGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA Anti-ECAD scFv clone 5 (SEQ ID NO: 59; light chain followed by bold / underlined linker followed by heavy chain) TIFF0007817159000005.tif30150TIFF0007817159000006.tif44150Anti-ECAD scFv clone 6 (SEQ ID NO: 60; light chain followed by bold / underlined linker followed by heavy chain) TIFF0007817159000007.tif71150 Anti-ECAD scFv clone 7 (SEQ ID NO: 61; heavy chain followed by bold / underlined linker followed by light chain) TIFF0007817159000008.tif70150 Anti-ECAD scFv clone 14 (SEQ ID NO: 62; light chain followed by bold / underlined linker followed by heavy chain) TIFF0007817159000009.tif66150 Anti-MOG antibody heavy chain (SEQ ID NO: 22) GAGGTGAAGCTGCACGAGAGCGGCGCAGGTCTGGTGAAGCCCGGCGCCAGCGTGGAGATCAGCTGCAAGGCCACCGGCTACACCTTCAGCAGCTTCTGGATCGAGTGGGTGAAGCAGAGACCCGGCCACGGCCTGGAGTGGATCGGCGAGATCCTGCCCGGCAGAGGCAGAACCAAC TACAACGAGAAGTTCAAGGGCAAGGCCACCTTCACCGCCGAGACCAGCAGCAACACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCACCGGCAACACCATGGTGAACATGCCCTACTGGGCCAGGGCACCACCGTGACCGTGAGCAGC Anti-MOG antibody light chain (SEQ ID NO: 23) GATATTGAACTGACCCAGAGTCCCAGTAGCCTGGCCGTGAGTGCCGGCGAGAAAGTGACCATGAGCTGCAAAAGCAGCCAGAGCCTGCTGAACAGCGGCAACCAGAAAAACTACCTGGCCTGGTACCAGCAGAAACCCGGCCTGCCTCCTAAGCTGCTGATCTACGGCG CCAGCACCAGAGAAAGCGGCGTGCCCGATAGATTCACCGGCTCTGGCTCTGGCACCGACTTCACCCTGACCATCAGCAGCGTGCAGGCCGAAGACCTGGCtGTcTACTACTGCCAGAACGACCACAGCTACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGATCAAG Anti-HER2 antibody GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCCGGGGGCTCTCCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATTAAAGACACCTATATCCACTGGGTGCGTCAGGCTCCGGGTAAGGGCCTGGAGTGGGTTGCAAGGATTTATCCTACGAATGGTTATACTCGTTATGCCGATAGCGTCAAGGGCGTTCACTATAAGCGCAGACACTTCGAAAAAACACAGCCTACCTCCAGAATGAACAGC CTGCGTGCTGAGGACACTGCCGTCTATTATTGTAGCAGATGGGGTGGGGACGGCTTCTATGCTATGGACTACTGGGGTCAAGGTACACTAGTCACCGTCAGCAGCGCTAGCACCAAGGGCACCACGACGCCAGCGCCGCGACCACAACACCGGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT Anti-HER2 antibody GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGGGCGGATAGGGTCACTATCACCTGCCGTGCCAGTCAGGATGTGAATACTGCTGTAGCCTGGTATCAACAGAAACCCGGAAAGGCCCGAAACTGCTGATTTACTCGGCATCCTTCCCTACTCTG GAGTCCCTTCTCGCTTCTCTGGTTCCCGCTCTGGGACGGATTTCACTCTGACCATCAGCTCCCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACACTATACTACTCTCCGACGTTCGGACAGGGTACCAAGGTGGAGATCAAACGTACCGTGGCGGCCCCA

[0040] The CARs described herein (e.g., CAR-ECAD, CAR-MOG, or CAR-HER2) can also comprise one or more additional components. Examples of additional components that can be included in a CAR include, but are not limited to, a leader sequence (e.g., a CD8 leader sequence), a hinge (e.g., a CD8 hinge and a CD28 hinge), a transmembrane domain (e.g., a CD8 transmembrane domain and a CD28 transmembrane domain), a costimulatory signaling domain (e.g., one that may increase immunosuppression, such as a TLR3 signaling domain and / or a TLR4 signaling domain), a Toll / interleukin-1 receptor / resistance (TIR) ​​interacting sequence (e.g., a TLR3 TIR interacting domain and / or a TLR4 TIR interacting domain), a TLR intracellular domain (e.g., a TLR intracellular linker), and a TLR transmembrane domain (e.g., a TLR3 transmembrane domain and / or a TLR4 transmembrane domain). In some cases, nucleic acids encoding a component of a CAR-encoding construct can be separated from nucleic acids encoding another component using one or more linkers. The nucleic acids in a CAR-encoding construct can be present in any suitable order. In some cases, a CAR can be designed to include scFvs in a light to heavy chain orientation or a heavy to light chain orientation, using any suitable linker between the chains. For example, a construct encoding a CHD1-CAR can be generated in a light to heavy chain orientation of the scFv or a heavy to light chain orientation of the scFv. In some cases, a CAR can be designed to include scFvs in a light to heavy chain orientation or a heavy to light chain orientation without a linker between the chains. Exemplary nucleic acid sequences that can encode one or more additional components that can be included in a CAR that can be included in the constructs described herein include, but are not limited to, those that encode the following amino acid sequences:

[0041] CD8 leader sequence (SEQ ID NO: 26) MALPVTALLLPLALLLHAARP Linker (SEQ ID NO: 27) GGGGSGGGGSGGGGS CD8 hinge (SEQ ID NO: 28) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD CD28 hinge (SEQ ID NO: 29) LEPKSCDKTHTCPPCPDPK TLR long chain (long peptide derived from Toll-like receptor; SEQ ID NO: 46) NDFACTCEHQSFLQWIKDQRQLLVEVERMECATPSDKQGMPVLSLNITCQMNKTI TLR short chain (short peptide derived from Toll-like receptor; SEQ ID NO: 47) MNKTI CD8 transmembrane domain (SEQ ID NO: 30) IYIWAPLAGTCGVLLLSLVITLYC CD28 transmembrane domain (SEQ ID NO: 31) FWVLVVVGGVLACYSLLVTVAFIIFWV TLR4 transmembrane domain (SEQ ID NO: 48) IGVSVLSVLVVSVVAVLVY CD28 signaling domain (SEQ ID NO: 32) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD3 zeta signaling domain (SEQ ID NO: 33) RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 4-1BB (CD137) signaling domain (SEQ ID NO: 34) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL TLR3 signaling domain (SEQ ID NO: 35) FEYAAYIIHAYKDKDWVWEHFSSMEKEDQSLKFCLEERDFEAAGVFELEAIVNSIKRSRKIIFVITHHLLKDPLCKRFKVHHAVQQAIEQNLDSIILVFLEEIPDYKLNHALCLRRGMFKSHCILNWPVQKERIGAFRHKLQVALGSKNSVH TLR4 signaling domain (SEQ ID NO: 36) NIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI TLR4 intracellular domain (SEQ ID NO: 49) KFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRIFFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI

[0042] In some cases, additional components that can be included in a CAR and that can be included in the constructs described herein can have sequences that deviate from the polypeptide sequence set forth in any one of SEQ ID NOS: 26-36 and 46-49, and are sometimes referred to as variant sequences. For example, additional components that can be included in a CAR and that can be encoded by a nucleic acid sequence that can be included in the constructs described herein can have at least 80% sequence identity to any one of SEQ ID NOS: 26-36 and 46-49. In some embodiments, additional components that can be included in a CAR and that can be encoded by a nucleic acid sequence that can be included in the constructs described herein can have at least 85%, 90%, 95%, or at least 99% sequence identity to any one of SEQ ID NOS: 26-36 and 46-49. The percent sequence identity is calculated by determining the number of matched positions in the aligned polypeptide sequences, dividing the number of matched positions by the total number of aligned amino acids, and multiplying by 100. A matched position refers to a position where an identical amino acid is present at the same position in the aligned sequences. The total number of aligned amino acids refers to the minimum number of amino acids in an additional component that can be included in a CAR and encoded by a nucleic acid sequence that can be included in a construct described herein that is necessary to align a second sequence and does not include alignments (e.g., forced alignments) with other sequences. For example, if the additional component that can be included in a CAR and encoded by a nucleic acid sequence that can be included in a construct described herein is a signaling domain, the total number of aligned amino acids can exclude any transmembrane domain. The total number of aligned amino acids can correspond to the entire amino acid segment of the additional component that can be included in a CAR and encoded by a nucleic acid sequence that can be included in a construct described herein, or can correspond to a fragment of the amino acid segment of the additional component that can be included in a CAR and encoded by a nucleic acid sequence that can be included in a construct described herein.Sequences can be aligned using the algorithm described by Altschul et al. (Nucleic Acids Res., 25:3389-3402 (1997)), which has been incorporated into the BLAST (Basic Local Alignment Search Tool) program, available on the World Wide Web at ncbi.nlm.nih.gov. BLAST searches or alignments can be performed using the Altschul et al. algorithm to determine percent sequence identity. BLASTN is a program used to align and compare identity between nucleic acid sequences, and BLASTP is a program used to align and compare identity between amino acid sequences. When using BLAST programs to calculate percent identity between an amino acid segment that can bind to a tissue-specific antigen described herein and that can be encoded by another sequence and that can be included in the constructs described herein, the default parameters of the respective programs are used.

[0043] Exemplary nucleic acid sequences of some additional components that can be included in a CAR and that can be included in the constructs described herein are as follows:

[0044] CD8 leader sequence (SEQ ID NO: 37) ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG Linker (SEQ ID NO: 38) GGTGGAGGTGGTTCGGGAGGTGGAGGTAGCGGAGGTGGTGGATCT Linker (SEQ ID NO: 50) GGTGGAGGTGGTTCGGGAGGTGGAGGTAGCGGAGGTGGTGGAAGC CD8 hinge (SEQ ID NO: 39) ACCACTACCCCTGCACCGCGACCACCAACACCGGCGCCCACCATTGCGTCGCAGCCTCTGTCCCTGCGCCCAGAAGCATGCCGTCCAGCAGCAGGTGGTGCAGTTCATACTCGTGGTCTGGATTTCGCCTGTGAT CD28 hinge (SEQ ID NO: 40) CTCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCGGATCCCAAA TLR length (SEQ ID NO: 51) AACGACTTCGCCTGCACCTGCGAGCACCAGAGCTTCCTGCAGTGGATCAAGGACCAGAGGCAGCTGCTGGTGGAGGTGGAGAGGATGGAGTGCGCCACCCCCAGCGACAAGCAGGGCATGCCCGTGCTGAGCCTGAACATCACCTGCCAGATGAACAAGACCATC TLR short (SEQ ID NO: 52) ATGAACAAGACCATC CD8 transmembrane domain (SEQ ID NO: 41) ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC CD28 transmembrane domain (SEQ ID NO: 42) TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG TLR4 transmembrane domain (SEQ ID NO: 53) ATTGGGGTGTCTGTCCTAAGCGTGCTGGTTGTTTCCGTGGTTGCCGTTCTGGTATAT CD28 signaling domain (SEQ ID NO: 43) AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC CD3 zeta signaling domain (SEQ ID NO: 44) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC 4-1BB (CD137) signaling domain (SEQ ID NO: 45) AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG TLR4 intracellular domain (SEQ ID NO: 54) AAGTTCTATTTCCATCTGATGCTTCTCGCTGGCTGCATAAAGTACGGGAGGGGGGAGAATATATATGACGCTTTCGTGATCTACTCGAGCCAGGATGAGGACTGGGTTCGCAACGAGCTAGTCAAGAATCTTGAAGAGGGCGTGCCTCCTTTCCAGCTCTGTCTGCATTACCGCGATTTTATTCCTGGGGTGGCCATCGCGGCCAACATCATCCACGAGGGCTTCCATAAATCCAGAAAAGTGATTGTCGTTGTGAGCCAGCATTTCATCCAGTCCAGGTGGTGCATTTTCGAATATGAGATAGCCCAGACCTGGCAGTTTCTTAGCAGTCGGGCTGGGATTATTTTTATCGTGCTGCAGAAGGTTGAAAAGACCCTTTTGCGGCAACAGGTGGAACTGTACCGATTATTATCCCGTAACACTTACTTGGAATGGGAAGACTCAGTTCTCGGACGCCACATTTTCTGGCGCCGGCTCAGGAAGGCCCTGCTGGATGGTAAATCCTGGAACCCCGAGGGGACAGTGGGGACCGGATGTAACTGGCAAGAGGCAACAAGTATA

[0045] In some cases, a nucleic acid construct encoding a CAR-ECAD described herein can be designed to encode a CD8 leader sequence, an anti-ECAD antibody (e.g., an hSC10.17 antibody) heavy chain, a linker, an anti-ECAD antibody (e.g., an hSC10.17 antibody) light chain, a CD8 hinge, a CD8 transmembrane domain, and a CD3 zeta signaling domain. For example, a nucleic acid construct encoding a CAR-ECAD can be designed to encode a CD8 leader comprising the amino acid sequence set forth in SEQ ID NO:26, an hSC10.17 heavy chain comprising the amino acid sequence set forth in SEQ ID NO:11, a linker comprising the amino acid sequence set forth in SEQ ID NO:27, an hSC10.17 light chain comprising the amino acid sequence set forth in SEQ ID NO:12, a CD8 hinge comprising the amino acid sequence set forth in SEQ ID NO:28, a CD8 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:30, and a CD3 zeta signaling domain comprising the amino acid sequence set forth in SEQ ID NO:33.

[0046] In some cases, a nucleic acid construct encoding a CAR-MOG described herein can be designed to encode a CD8 leader sequence, an anti-MOG antibody (e.g., 8-18C5 antibody) heavy chain, a linker, an anti-MOG antibody (e.g., 8-18C5 antibody) light chain, a CD8 hinge, a CD28 transmembrane domain, and a CD3 zeta signaling domain. For example, a nucleic acid construct encoding a CAR-MOG can be designed to encode a CD8 leader comprising the amino acid sequence set forth in SEQ ID NO:26, an 8-18C5 heavy chain comprising the amino acid sequence set forth in SEQ ID NO:14, a linker comprising the amino acid sequence set forth in SEQ ID NO:27, an 8-18C5 light chain comprising the amino acid sequence set forth in SEQ ID NO:15, a CD8 hinge comprising the amino acid sequence set forth in SEQ ID NO:28, a CD8 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:30, and a CD3 zeta signaling domain comprising the amino acid sequence set forth in SEQ ID NO:33.

[0047] In some cases, a nucleic acid construct encoding a CAR-HER2 described herein can be designed to encode a CD8 leader sequence, an anti-HER2 antibody (e.g., 4D5) heavy chain, a linker, an anti-HER2 antibody (e.g., 4D5) light chain, a CD8 hinge, a CD28 transmembrane domain, a 4-1BB signaling domain, and a CD3 zeta signaling domain. For example, a nucleic acid construct encoding a CAR-HER2 can be designed to encode a CD8 leader comprising the amino acid sequence set forth in SEQ ID NO:26, a 4D5 heavy chain comprising the amino acid sequence set forth in SEQ ID NO:16, a linker comprising the amino acid sequence set forth in SEQ ID NO:27, a 4D5 light chain comprising the amino acid sequence set forth in SEQ ID NO:17, a CD28 hinge comprising the amino acid sequence set forth in SEQ ID NO:29, a CD8 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:30, a 4-1BB signaling domain comprising the amino acid sequence set forth in SEQ ID NO:34, and a CD3 zeta signaling domain comprising the amino acid sequence set forth in SEQ ID NO:33.

[0048] In some cases, a nucleic acid construct encoding a CAR-ECAD described herein, a CAR-MOG described herein, or a CAR-HER2 described herein can be designed to encode (a1) an anti-ECAD antibody (e.g., an hSC10.17 antibody) heavy chain, a linker, an anti-ECAD antibody (e.g., an hSC10.17 antibody) light chain, (a2) an anti-MOG antibody (e.g., an 8-18C5 antibody) heavy chain, a linker, an anti-MOG antibody (e.g., an 8-18C5 antibody) light chain, or (a3) ​​an anti-HER2 antibody (e.g., 4D5) heavy chain, a linker, an anti-HER2 antibody (e.g., 4D5) light chain, followed by (b) a CD8 hinge, (c) a TLR4 transmembrane domain, and (d) a TLR4 signaling domain or a TLR4 intracellular domain.

[0049] In some cases, a nucleic acid construct encoding a CAR-ECAD described herein, a CAR-MOG described herein, or a CAR-HER2 described herein can be designed to encode (a1) an anti-ECAD antibody (e.g., an hSC10.17 antibody) heavy chain, a linker, an anti-ECAD antibody (e.g., an hSC10.17 antibody) light chain, (a2) an anti-MOG antibody (e.g., an 8-18C5 antibody) heavy chain, a linker, an anti-MOG antibody (e.g., an 8-18C5 antibody) light chain, or (a3) ​​an anti-HER2 antibody (e.g., 4D5) heavy chain, a linker, an anti-HER2 antibody (e.g., 4D5) light chain, followed by (b) a CD8 hinge, (c) a TLR4 transmembrane domain, (d) a TLR4 signaling domain or a TLR4 intracellular domain, and (e) a CD3 zeta signaling domain.

[0050] In some cases, a nucleic acid construct encoding a CAR-ECAD described herein, a CAR-MOG described herein, or a CAR-HER2 described herein can be designed to encode (a1) an anti-ECAD antibody (e.g., an hSC10.17 antibody) heavy chain, a linker, an anti-ECAD antibody (e.g., an hSC10.17 antibody) light chain, (a2) an anti-MOG antibody (e.g., an 8-18C5 antibody) heavy chain, a linker, an anti-MOG antibody (e.g., an 8-18C5 antibody) light chain, or (a3) ​​an anti-HER2 antibody (e.g., 4D5) heavy chain, a linker, an anti-HER2 antibody (e.g., 4D5) light chain, followed by (b) a linker, e.g., a linker encoded by SEQ ID NO: 50, (c) a TLR4 transmembrane domain, and (d) a TLR4 signaling domain or a TLR4 intracellular domain.

[0051] In some cases, a nucleic acid construct encoding a CAR-ECAD described herein, a CAR-MOG described herein, or a CAR-HER2 described herein can be designed to encode (a1) an anti-ECAD antibody (e.g., an hSC10.17 antibody) heavy chain, a linker, an anti-ECAD antibody (e.g., an hSC10.17 antibody) light chain, (a2) an anti-MOG antibody (e.g., an 8-18C5 antibody) heavy chain, a linker, an anti-MOG antibody (e.g., an 8-18C5 antibody) light chain, or (a3) ​​an anti-HER2 antibody (e.g., 4D5) heavy chain, a linker, an anti-HER2 antibody (e.g., 4D5) light chain, followed by (b) a TLR long chain, (c) a TLR4 transmembrane domain, and (d) a TLR4 signaling domain or a TLR4 intracellular domain.

[0052] In some cases, a nucleic acid construct encoding a CAR-ECAD described herein, a CAR-MOG described herein, or a CAR-HER2 described herein can be designed to encode (a1) an anti-ECAD antibody (e.g., an hSC10.17 antibody) heavy chain, a linker, an anti-ECAD antibody (e.g., an hSC10.17 antibody) light chain, (a2) an anti-MOG antibody (e.g., an 8-18C5 antibody) heavy chain, a linker, an anti-MOG antibody (e.g., an 8-18C5 antibody) light chain, or (a3) ​​an anti-HER2 antibody (e.g., 4D5) heavy chain, a linker, an anti-HER2 antibody (e.g., 4D5) light chain, followed by (b) a TLR short chain, (c) a TLR4 transmembrane domain, (d) a TLR4 signaling domain or a TLR4 intracellular domain, and (e) a CD3 zeta signaling domain.

[0053] In some cases, a nucleic acid construct encoding a CAR-ECAD described herein, a CAR-MOG described herein, or a CAR-HER2 described herein can be designed to encode (a1) an anti-ECAD antibody (e.g., an hSC10.17 antibody) heavy chain, a linker, an anti-ECAD antibody (e.g., an hSC10.17 antibody) light chain, (a2) an anti-MOG antibody (e.g., an 8-18C5 antibody) heavy chain, a linker, an anti-MOG antibody (e.g., an 8-18C5 antibody) light chain, or (a3) ​​an anti-HER2 antibody (e.g., 4D5) heavy chain, a linker, an anti-HER2 antibody (e.g., 4D5) light chain, followed by (b) a CD28 hinge, (c) a CD28 transmembrane domain, and (d) a CD28 signaling domain.

[0054] In some cases, a nucleic acid construct encoding a CAR-ECAD described herein, a CAR-MOG described herein, or a CAR-HER2 described herein can be designed to encode (a1) an anti-ECAD antibody (e.g., an hSC10.17 antibody) heavy chain, a linker, an anti-ECAD antibody (e.g., an hSC10.17 antibody) light chain, (a2) an anti-MOG antibody (e.g., an 8-18C5 antibody) heavy chain, a linker, an anti-MOG antibody (e.g., an 8-18C5 antibody) light chain, or (a3) ​​an anti-HER2 antibody (e.g., 4D5) heavy chain, a linker, an anti-HER2 antibody (e.g., 4D5) light chain, followed by (b) a CD28 hinge, (c) a CD28 transmembrane domain, (d) a CD28 signaling domain, and (e) a CD3 zeta signaling domain.

[0055] Furthermore, when MSCs designed to express an antigen receptor (e.g., a CAR) capable of binding (e.g., specifically binding) to a tissue-specific antigen are also designed to express a polypeptide capable of promoting differentiation into tissue-specific cells, the MSCs can comprise one or more nucleic acids encoding a polypeptide capable of promoting differentiation into tissue-specific cells. The nucleic acid capable of encoding a polypeptide capable of promoting tissue-specific cell differentiation can encode any polypeptide capable of promoting the differentiation of MSCs into any type of tissue-specific cell. For example, MSCs can be designed to comprise one or more nucleic acids encoding a polypeptide capable of promoting cardiac cell differentiation. The polypeptide capable of promoting cardiac differentiation can promote differentiation into any suitable type of cardiac cell (e.g., cardiomyocyte). Examples of polypeptides that can promote cardiac cell differentiation and can be included in the MSCs described herein include, but are not limited to, GATA4 polypeptide, MEF2C polypeptide, TBX5 polypeptide, ERRG polypeptide, and MESP1 polypeptide. For example, MSCs can be designed to comprise one or more nucleic acids encoding a polypeptide capable of promoting neural differentiation. Polypeptides capable of promoting neural differentiation can promote differentiation into any suitable type of neuron (e.g., sensory neurons, motor neurons, interneurons, oligodendrocytes, astrocytes, and glial cells). Examples of polypeptides that can promote neural differentiation and can be included in the MSCs described herein include, but are not limited to, Oct3 / 4 polypeptide, Klf4 polypeptide, Sox2 polypeptide, Glis1 polypeptide, c-Myc polypeptide, BMP4 polypeptide, WNT polypeptide, FGF2 polypeptide, SHH polypeptide, Sox11 polypeptide, Sox2 polypeptide, Sox3 polypeptide, Zic1 polypeptide, Zic2 polypeptide, Irx1 polypeptide, Irx2 polypeptide, Irx3 polypeptide, FoxD4 polypeptide, MKx2.5 polypeptide, and cTnT polypeptide.

[0056] This document also provides materials and methods for treating a mammal (e.g., a human) having or at risk of developing a disease or disorder characterized by tissue inflammation and / or degeneration. For example, one or more MSCs expressing a CAR targeting a tissue-specific antigen and optionally expressing a polypeptide capable of promoting the differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells (e.g., a composition containing one or more MSCs expressing a CAR targeting a tissue-specific antigen and optionally expressing a polypeptide capable of promoting the differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells) can be administered (e.g., by adoptive transfer) to a mammal having or at risk of developing a disease or disorder characterized by tissue inflammation and / or degeneration to reduce the severity of inflammation in the targeted tissue within the mammal. Any suitable method can be used to identify a mammal as having or at risk of developing an inflammatory disease or condition and / or as having or at risk of developing a degenerative disease. For example, imaging techniques (e.g., ultrasound, computed tomography (CT) scanning), clinical tests (e.g., blood tests for inflammatory markers, e.g., erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and / or plasma viscosity (PV)) can be used to identify mammals as having or at risk of developing an inflammatory disease or condition and / or having or at risk of developing a degenerative disease.Upon identification as having (or at risk of developing) an inflammatory disease or condition and / or having (or at risk of developing) a degenerative disease, one or more MSCs expressing a CAR that targets a tissue-specific antigen, and optionally expressing a polypeptide capable of promoting differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells, can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having or at risk of developing a disease or disorder characterized by inflammation and / or degeneration of a tissue that expresses the tissue-specific antigen) as described herein to reduce inflammation in the tissue by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent or more. For example, one or more MSCs expressing a polypeptide capable of promoting differentiation of MSCs into any type of tissue-specific cell (e.g., cardiac cells or neurons) can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) having (or at risk of developing) a degenerative heart disease to treat the degenerative heart disease in the mammal. Any suitable method can be used to identify a mammal as having or at risk of developing a degenerative disease. For example, imaging techniques (e.g., ultrasound, computed tomography (CT) scanning) and / or clinical tests (e.g., blood tests for genetic markers) can be used to identify a mammal as having or at risk of developing a degenerative disease. Once identified as having (or at risk of developing) a degenerative disease, one or more MSCs expressing a polypeptide that can promote the differentiation of MSCs into any type of tissue-specific cell can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having or at risk of developing a degenerative disease) as described herein to increase the number of tissue-specific cells in the tissue by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent or more.

[0057] Any suitable mammal having or at risk of developing a disease or disorder characterized by tissue inflammation and / or degeneration can be treated as described herein. Examples of mammals that can have a disease or disorder characterized by tissue inflammation and / or degeneration and can be treated as described herein include, but are not limited to, humans, non-human primates, such as monkeys, dogs, cats, horses, cows, pigs, sheep, mice, and rats. For example, one or more MSCs expressing a CAR that targets a tissue-specific antigen and optionally expressing a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells (e.g., a composition containing one or more MSCs that express a CAR that targets a tissue-specific antigen and optionally expressing a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells) can be administered (e.g., by adoptive transfer) to a human having or at risk of developing a disease or disorder characterized by tissue inflammation and / or degeneration to treat the human.

[0058] In some cases, materials and methods for treating a mammal (e.g., a human) having or at risk of developing a disease or disorder characterized by inflammation and / or degeneration of tissue can be used to treat a mammal (e.g., a human) having or at risk of developing a disease or disorder characterized by inflammation and / or degeneration of epithelial tissue. Examples of diseases and disorders characterized by inflammation and / or degeneration of epithelial tissue include, but are not limited to, IBD, e.g., colitis (e.g., characterized by inflammation of colon and / or rectal cells), hepatitis (e.g., characterized by inflammation of liver cells), cholangitis (e.g., characterized by inflammation of bile duct cells), dermatitis (e.g., severe dermatitis characterized by inflammation of skin cells), mucositis (e.g., severe mucositis characterized by inflammation of the mucous membrane lining the digestive tract), colonic fistula, graft-versus-host disease, and inflammatory pneumonia (e.g., characterized by inflammation of lung cells). When treating a mammal having (or at risk of developing) colitis, the colitis can be any type of colitis (e.g., ulcerative colitis, Crohn's colitis, diversional colitis, ischemic colitis, infectious colitis, fulminant colitis, collagenous colitis, chemical colitis, microscopic colitis, lymphocytic colitis, and atypical colitis).

[0059] In some cases, a mammal can be identified as having or at risk of developing a disease or disorder characterized by inflammation and / or degeneration of epithelial tissue (e.g., an IBD such as colitis). Any suitable method can be used to identify a mammal as having or at risk of developing a disease or disorder characterized by inflammation and / or degeneration of epithelial tissue. For example, blood tests (e.g., for signs of anemia or infection), laboratory tests (e.g., for white blood cells in the mammal's stool), imaging techniques (e.g., colonoscopy, flexible sigmoidoscopy, X-ray, CT scan, CT enteroscopy, and magnetic resonance (MR) enteroscopy), biopsy, skin biopsy, and / or liver function tests can be used to identify a mammal as having or at risk of developing a disease or disorder characterized by inflammation of epithelial tissue, e.g., colitis. Once identified as having (or at risk of developing) a disease or disorder characterized by inflammation and / or degeneration of epithelial tissue, a mammal can be administered (e.g., by adoptive transfer) or instructed to self-administer one or more MSCs described herein (e.g., MSCs that express a CAR that targets an epithelial-specific antigen and, optionally, express a polypeptide that can promote differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells) to treat the mammal (e.g., to reduce or eliminate inflammation of one or more epithelial tissues within the mammal).

[0060] As described herein, when treating a mammal having (or at risk of developing) a disease or disorder characterized by inflammation and / or degeneration of epithelial tissue (e.g., IBD, e.g., colitis) (e.g., by administering one or more MSCs that express a CAR that targets an epithelial-specific antigen and optionally express a polypeptide that can promote differentiation into tissue-specific cells), the one or more MSCs that express a CAR that targets an epithelial-specific antigen and optionally express a polypeptide that can promote differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells can be effective in reducing the severity of the disease or disorder characterized by inflammation and / or degeneration of epithelial tissue in the mammal. Where the disease or disorder characterized by inflammation and / or degeneration of epithelial tissue is colitis, reducing the severity of colitis in a mammal can include reducing or eliminating one or more symptoms of colitis (e.g., diarrhea, abdominal pain and cramps, rectal pain, rectal bleeding, urgency to defecate, inability to defecate despite urgency, weight loss, fatigue, fever, jaundice, liver failure, abnormal liver test results, difficulty breathing, skin erythema, and / or peeling). For example, one or more MSCs expressing a CAR that targets an epithelial-specific antigen, and optionally expressing a polypeptide capable of promoting the differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells, can be administered to a mammal (e.g., a human) in need thereof, as described herein, to reduce the severity of one or more symptoms of colitis, for example, by 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent or more.

[0061] In some cases, one or more MSCs expressing a CAR that targets an epithelial-specific antigen and optionally expressing a polypeptide that can promote differentiation into tissue-specific cells (e.g., a composition containing one or more MSCs expressing a CAR that targets an epithelial-specific antigen and optionally expressing a polypeptide that can promote differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells) can be the only active ingredient for treating a mammal having (or at risk of developing) a disease or disorder characterized by inflammation and / or degeneration of epithelial tissue (e.g., IBD, e.g., colitis) as described herein (e.g., by administering one or more MSCs expressing a CAR that targets an epithelial-specific antigen and optionally expressing a polypeptide that can promote differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells).

[0062] In some cases, one or more MSCs expressing a CAR targeting an epithelial-specific antigen and optionally expressing a polypeptide capable of promoting the differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells (e.g., a composition containing one or more MSCs expressing a CAR targeting an epithelial-specific antigen and optionally expressing a polypeptide capable of promoting the differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells) can be administered in combination with one or more additional therapeutic agents (e.g., a therapeutic agent that can be used to treat a mammal having or at risk of developing a disease or disorder characterized by inflammation and / or degeneration of epithelial tissue within the mammal, and a therapeutic agent that can be used to treat inflammation of epithelial tissue within the mammal). For example, a mammal having (or at risk of developing) a disease or disorder characterized by inflammation and / or degeneration of epithelial tissue (e.g., IBD, e.g., colitis) can also be treated with one or more additional therapeutic agents. In some cases, the therapeutic agent can be an anti-inflammatory agent. In some cases, the therapeutic agent can be an immunosuppressant. In some cases, the therapeutic agent can be a T cell, e.g., a CAR-expressing T cell (CAR-T cell). Examples of therapeutic agents that can be used in combination with one or more MSCs expressing a CAR that targets an epithelial-specific antigen, and optionally expressing a polypeptide that can promote differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells described herein, include, but are not limited to, CAR-T cells (e.g., CART19 cells), 5-aminosalicylates (e.g., sulfasalazine, mesalamine, balsalazide, and olsalazine), corticosteroids (e.g., prednisone and methylprednisolone), azathioprine, mercaptopurine, cyclosporine, infliximab, adalimumab, golimumab, vedolizumab, antibiotics, antidiarrheals (e.g., loperamide), painkillers (e.g., acetaminophen), and iron supplements.In some cases, one or more MSCs expressing a CAR targeting an epithelial-specific antigen and optionally expressing a polypeptide capable of promoting the differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells can be administered substantially simultaneously with one or more additional therapeutic agents that can be used to treat inflammation of epithelial tissue in a mammal. For example, a composition comprising one or more MSCs expressing a CAR targeting an epithelial-specific antigen and optionally expressing a polypeptide capable of promoting the differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells can also include one or more additional therapeutic agents that can be used to treat inflammation of epithelial tissue in a mammal. In some cases, one or more MSCs expressing a CAR targeting an epithelial-specific antigen and optionally expressing a polypeptide capable of promoting the differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells can be administered first, and one or more additional therapeutic agents can be administered second, or vice versa.

[0063] In some cases, materials and methods for treating a mammal (e.g., a human) having or at risk of developing a disease or disorder characterized by inflammation and / or degeneration of tissue can be used to treat a mammal (e.g., a human) having or at risk of developing a disease or disorder characterized by inflammation and / or degeneration of nervous tissue. Examples of diseases and disorders characterized by inflammation and / or degeneration of nervous tissue include, but are not limited to, multiple sclerosis (e.g., characterized by inflammation of neurons in the brain and / or spinal cord), encephalomyelitis, e.g., immune-mediated encephalomyelitis (e.g., characterized by inflammation of the CNS), and paraneoplastic encephalitis (e.g., characterized by multifocal inflammation of the CNS). When treating a mammal having or at risk of developing immune-mediated encephalomyelitis, the immune-mediated encephalomyelitis can be any type of immune-mediated encephalomyelitis (e.g., acute disseminated encephalomyelitis (ADEM) and paraneoplastic encephalitis).

[0064] In some cases, a mammal can be identified as having or at risk for developing a disease or disorder characterized by inflammation and / or degeneration of nervous tissue (e.g., multiple sclerosis or immune-mediated encephalomyelitis). Any suitable method can be used to identify a mammal having (or at risk for developing) a disease or disorder characterized by inflammation and / or degeneration of nervous tissue. For example, clinical tests, such as blood tests and lumbar punctures (e.g., to check for biomarkers associated with a particular disease or disorder characterized by inflammation and / or degeneration of nervous tissue, e.g., multiple sclerosis, and to check for the presence of antibodies associated with a particular disease or disorder characterized by inflammation and / or degeneration of nervous tissue, e.g., anti-MOG autoantibodies), imaging techniques (e.g., magnetic resonance imaging (MRI); to check for the presence of lesions on the brain and / or spinal cord), spinal fluid analysis for protein levels, and / or cell counts for specific proteins, can be used to identify a mammal as having or at risk for developing a disease or disorder characterized by inflammation and / or degeneration of nervous tissue, e.g., multiple sclerosis or immune-mediated encephalomyelitis. Upon identification as having (or at risk of developing) a disease or disorder characterized by inflammation and / or degeneration of neural tissue, a mammal can be administered (e.g., by adoptive transfer) or instructed to self-administer one or more MSCs described herein (e.g., one or more MSCs expressing a CAR that targets a neural-specific antigen, and optionally expressing a polypeptide that can promote differentiation of MSCs and / or one or more resident progenitor cells into neurons) to treat the mammal (e.g., to reduce or eliminate inflammation of one or more neural tissues within the mammal and / or to regenerate neural tissue within the mammal).

[0065] As described herein, when treating a mammal having (or at risk of developing) a disease or disorder characterized by inflammation and / or degeneration of nervous tissue (e.g., multiple sclerosis or immune-mediated encephalomyelitis) (e.g., by administering one or more MSCs that express a CAR that targets a neural-specific antigen and optionally expressing a polypeptide that can promote differentiation of MSCs and / or one or more resident progenitor cells into neurons), the one or more MSCs that express a CAR that targets a neural-specific antigen and optionally expressing a polypeptide that can promote differentiation of MSCs and / or one or more resident progenitor cells into neurons can be effective in reducing the severity of the disease or disorder characterized by inflammation and / or degeneration of nervous tissue in the mammal. Where the disease or disorder characterized by inflammation and / or degeneration of nervous tissue is multiple sclerosis, reducing the severity of multiple sclerosis in a mammal can include reducing or eliminating one or more symptoms of multiple sclerosis (e.g., numbness or weakness in one or more limbs, electric shock sensations (e.g., caused by certain neck movements, particularly bending the neck forward), tremors, lack of coordination, unsteady gait, partial or complete vision loss, prolonged double vision, blurred vision, slurred speech, fatigue, dizziness, tingling or pain in parts of the body, sexual function problems, bowel function problems, and / or bladder function problems) and / or one or more complications associated with multiple sclerosis (e.g., muscle stiffness, muscle spasms, paralysis, forgetfulness, mood swings, depression, epilepsy, focal weakness, and / or vision impairment).For example, one or more MSCs expressing a CAR that targets a neural-specific antigen, and optionally expressing a polypeptide that can promote differentiation of MSCs and / or one or more resident progenitor cells into neurons, can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having or at risk of developing a disease or disorder characterized by inflammation and / or degeneration of nervous tissue) as described herein to reduce the severity of one or more symptoms of a disease or disorder characterized by inflammation and / or degeneration of nervous tissue and / or the severity of one or more complications associated with a disease or disorder characterized by inflammation and / or degeneration of nervous tissue by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent or more.

[0066] In some cases, one or more MSCs expressing a CAR that targets a neural-specific antigen, and optionally expressing a polypeptide that can promote differentiation of MSCs and / or one or more resident progenitor cells into neurons (e.g., a composition containing one or more MSCs that express one or more neural-specific antigens, and optionally expressing a polypeptide that can promote differentiation of MSCs and / or one or more resident progenitor cells into neurons), as described herein, can be the only active ingredient for treating a mammal having (or at risk of developing) a disease or disorder characterized by inflammation and / or degeneration of nervous tissue (e.g., multiple sclerosis or immune-mediated encephalomyelitis) (e.g., by administering one or more MSCs expressing a CAR that targets a neural-specific antigen, and optionally expressing a polypeptide that can promote differentiation of MSCs and / or one or more resident progenitor cells into neurons).

[0067] In some cases, one or more MSCs expressing a CAR targeting a neural-specific antigen and optionally expressing a polypeptide capable of promoting the differentiation of MSCs and / or one or more resident progenitor cells into neurons (e.g., a composition containing one or more MSCs expressing a CAR targeting a neural-specific antigen and optionally expressing a polypeptide capable of promoting the differentiation of MSCs and / or one or more resident progenitor cells into neurons) can be administered in combination with one or more additional therapeutic agents (e.g., a therapeutic agent that can be used to treat a mammal having or at risk of developing a disease or disorder characterized by inflammation and / or degeneration of neural tissue, and a therapeutic agent that can be used to treat inflammation of neural tissue in a mammal). For example, a mammal having (or at risk of developing) a disease or disorder characterized by inflammation and / or degeneration of neural tissue (e.g., multiple sclerosis or immune-mediated encephalomyelitis) can also be treated with one or more additional therapeutic agents. Examples of therapeutic agents that can be used in combination with one or more MSCs expressing a CAR that targets a neural-specific antigen, and optionally expressing a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into neurons as described herein, include, but are not limited to, corticosteroids (e.g., prednisone and methylprednisolone), ocrelizumab, beta interferon, glatiramer acetate, fingolimod, dimethyl fumarate, teriflunomide, siponimod, natalizumab, alemtuzumab, mitoxantrone, baclofen, tizanidine, amantadine, modafinil, methylphenidate, dalfampridine, and immunoglobulins. In some cases, one or more MSCs expressing a CAR that targets a neural-specific antigen, and optionally expressing a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into neurons, can be administered substantially simultaneously with one or more additional therapeutic agents that can be used to treat inflammation of nervous tissue in a mammal.For example, a composition comprising one or more MSCs expressing a CAR that targets a neural-specific antigen and optionally expressing a polypeptide that can promote differentiation of MSCs and / or one or more resident progenitor cells into neurons can include one or more additional therapeutic agents that can be used to treat inflammation of neural tissue in a mammal. In some cases, the one or more MSCs expressing a CAR that targets a neural-specific antigen and optionally expressing a polypeptide that can promote differentiation of MSCs and / or one or more resident progenitor cells into neurons can be administered first, and the one or more additional therapeutic agents can be administered second, or vice versa.

[0068] In some cases, materials and methods for treating a mammal (e.g., a human) having or at risk of developing a disease or disorder characterized by inflammation and / or degeneration of tissue can be used to treat a mammal (e.g., a human) having or at risk of developing a disease or disorder characterized by inflammation and / or degeneration of cardiac tissue. Examples of diseases and disorders characterized by inflammation and / or degeneration of cardiac tissue include, but are not limited to, myocarditis (e.g., characterized by inflammation of cardiac muscle cells), endocarditis (e.g., characterized by inflammation of endocardial cells), and heart failure.

[0069] In some cases, a mammal can be identified as having or at risk of developing a disease or disorder characterized by inflammation and / or degeneration of cardiac tissue (e.g., myocarditis). Any suitable method can be used to identify a mammal as having or at risk of developing a disease or disorder characterized by inflammation and / or degeneration of cardiac tissue. For example, an electrocardiogram (ECG; e.g., to detect abnormal rhythms), a chest X-ray, an MRI (e.g., cardiac MRI), an echocardiogram, and laboratory tests (e.g., blood tests to measure white and red blood cell counts and / or levels of certain enzymes indicative of damage to the heart muscle and to detect antibodies) can be used to identify a mammal as having or at risk of developing a disease or disorder characterized by inflammation and / or degeneration of cardiac tissue, e.g., myocarditis. Upon identification as having (or at risk of developing) a disease or disorder characterized by inflammation and / or degeneration of cardiac tissue, a mammal can be administered (e.g., by adoptive transfer) or instructed to self-administer one or more MSCs described herein (e.g., MSCs that express a CAR that targets a cardiac-specific antigen and, optionally, express a polypeptide that can promote differentiation of MSCs and / or one or more resident progenitor cells into cardiac cells) to treat the mammal (e.g., to reduce or eliminate inflammation of one or more cardiac tissues within the mammal).

[0070] When treating a mammal having (or at risk of developing) a disease or disorder characterized by inflammation and / or degeneration of cardiac tissue (e.g., myocarditis) described herein (e.g., by administering one or more MSCs that express a CAR that targets a cardiac-specific antigen and optionally express a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into cardiac cells), the one or more MSCs that express a CAR that targets a cardiac-specific antigen and optionally express a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into cardiac cells can be effective in reducing the severity of the disease or disorder characterized by inflammation and / or degeneration of cardiac tissue in the mammal. Where the disease or disorder characterized by inflammation and / or degeneration of epithelial tissue is myocarditis, reducing the severity of the myocarditis in a mammal can include reducing or eliminating one or more symptoms of myocarditis (e.g., chest pain, arrhythmia, shortness of breath, fluid retention (e.g., accompanied by swelling of the legs, ankles, or feet), fatigue, headache, body aches, joint pain, fever, sore throat, diarrhea, fainting, and / or rapid breathing). For example, one or more MSCs expressing a CAR that targets a cardiac-specific antigen, and optionally expressing a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into cardiac cells, can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having or at risk of developing myocarditis) as described herein to reduce the severity of one or more symptoms of myocarditis by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent or more.

[0071] In some cases, one or more MSCs expressing a CAR that targets a cardiac-specific antigen, and optionally expressing a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into cardiac cells (e.g., a composition comprising one or more MSCs that express a CAR that targets a cardiac-specific antigen, and optionally expressing a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into cardiac cells) can be the only active ingredient for treating a mammal having (or at risk of developing) a disease or disorder characterized by inflammation and / or degeneration of cardiac tissue (e.g., myocarditis).

[0072] In some cases, one or more MSCs expressing a CAR targeting a cardiac-specific antigen and optionally expressing a polypeptide capable of promoting the differentiation of MSCs and / or one or more resident progenitor cells into cardiac cells (e.g., a composition comprising one or more MSCs expressing a CAR targeting a cardiac-specific antigen and optionally expressing a polypeptide capable of promoting the differentiation of MSCs and / or one or more resident progenitor cells into cardiac cells) can be administered in combination with one or more additional therapeutic agents (e.g., a therapeutic agent that can be used to treat a mammal that has or is at risk of developing a disease or disorder characterized by inflammation and / or degeneration of cardiac tissue in the mammal, and a therapeutic agent that can be used to treat inflammation of cardiac tissue in the mammal). For example, a mammal that has (or is at risk of developing) a disease or disorder characterized by inflammation and / or degeneration of cardiac tissue (e.g., myocarditis) can also be treated with one or more additional therapeutic agents. In some cases, the therapeutic agent can be an anti-inflammatory agent. In some cases, the therapeutic agent can be an immunosuppressant. In some cases, the therapeutic agent can be an angiotensin-converting enzyme (ACE) inhibitor. In some cases, the therapeutic agent can be an angiotensin II receptor blocker (ARB). As described herein, examples of therapeutic agents that can be used in combination with one or more MSCs expressing a CAR that targets a cardiac-specific antigen and, optionally, expressing a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into cardiac cells include, but are not limited to, enalapril, captopril, lisinopril, ramipril, losartan, valsartan, metoprolol, bisoprolol, and carvedilol. In some cases, one or more MSCs expressing a CAR that targets a cardiac-specific antigen and, optionally, expressing a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into cardiac cells can be administered substantially simultaneously with one or more additional therapeutic agents that can be used to treat inflammation of cardiac tissue in a mammal.For example, a composition comprising one or more MSCs expressing a CAR that targets a cardiac-specific antigen and optionally expressing a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into cardiac cells can also include one or more additional therapeutic agents that can be used to treat inflammation of cardiac tissue in a mammal. In some cases, the one or more MSCs expressing a CAR that targets a cardiac-specific antigen and optionally expressing a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into cardiac cells can be administered first, and the one or more additional therapeutic agents can be administered second, or vice versa.

[0073] Any suitable method can be used to administer one or more MSCs that express a CAR that targets a tissue-specific antigen, and optionally express a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells (e.g., a composition comprising one or more MSCs that express a CAR that targets a tissue-specific antigen, e.g., an epithelial-specific antigen, a neural-specific antigen, or a cardiac-specific antigen, and optionally express a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells, e.g., cardiac cells or neurons) to a mammal (e.g., a human) in need thereof (e.g., a human having or at risk of developing a disease or disorder characterized by inflammation and / or degeneration of a tissue that expresses the tissue-specific antigen). Examples of methods of administering MSCs to a mammal described herein include, but are not limited to, injection (e.g., intravenous, intradermal, intramuscular, or subcutaneous injection). For example, a composition comprising one or more MSCs expressing a CAR that targets a tissue-specific antigen, and optionally expressing a polypeptide that can promote differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells, can be administered to a human by intravenous injection.

[0074] This document also provides kits containing one or more of the materials described herein. In some cases, the kits can include one or more MSCs that express a CAR that targets a tissue-specific antigen, and optionally express a polypeptide that can promote differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells (e.g., a composition containing one or more MSCs that express a CAR that targets a tissue-specific antigen, e.g., an epithelial-specific antigen, a neural-specific antigen, or a cardiac-specific antigen, and optionally express a polypeptide that can promote differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells, e.g., cardiac cells or neurons). For example, one or more MSCs expressing a CAR targeting a tissue-specific antigen and optionally expressing a polypeptide capable of promoting differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells (e.g., a composition containing one or more MSCs expressing a CAR targeting a tissue-specific antigen, e.g., an epithelial-specific antigen, a neural-specific antigen, or a cardiac-specific antigen, and optionally expressing a polypeptide capable of promoting differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells, e.g., cardiac cells or neurons) can be combined with packaging materials to form a kit. For example, one or more constructs described herein (e.g., encoding a CAR capable of binding to a tissue-specific antigen, e.g., an epithelial-specific antigen, a neural-specific antigen, or a cardiac-specific antigen) (e.g., a nucleic acid construct) can be combined with packaging materials to form a kit. The packaging materials included in such kits typically contain instructions or a label describing how the composition can be used in adoptive transfer, for example, to treat a mammal having or at risk of developing a disease or disorder characterized by inflammation and / or degeneration of a tissue as described herein. In some cases, the materials provided in the kits described herein can be used to treat a mammal (e.g., a human) having or at risk of developing a disease or disorder characterized by inflammation and / or degeneration of tissue as described herein.In some cases, the packaging material included in such kits can contain instructions and / or labels describing how the compositions described herein can be used. For example, the kits can contain instructions and / or labels describing how the compositions described herein can be used to express one or more CARs in MSCs to express CARs that bind to one or more epithelial-specific antigens (e.g., CAR-ECAD, CAR-MOG, or CAR-HER2). In some cases, the packaging material included in such kits can contain instructions and / or labels describing how the engineered MSCs described herein can be used. For example, the packaging material included in such kits can contain instructions and / or labels describing how the engineered MSCs described herein can be used in adoptive transfer to treat a mammal having or at risk of developing a disease or disorder characterized by tissue inflammation and / or degeneration as described herein. In some cases, the kit (e.g., a kit containing instructions and / or labels describing how the engineered MSCs described herein can be used in adoptive transfer) can include materials for use in adoptive transfer procedures.

[0075] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0076] [Example] [Example 1] Engineered CAR-MSCs for immune modification To determine whether CAR-expressing MSCs can reduce inflammation in target cell populations, MSCs expressing a CAR that targets CD19 (MSC-CAR19) were engineered.

[0077] Adipose-derived mesenchymal stem cells (100-Biotr-0024) were passaged into three wells of a 6-well plate (100k each). One group was left as an untransduced (UTD) negative control. The second well was transduced with luciferase-ZsGreen lentivirus (approximate MOI 3). The third group was transduced with the same MOI and lentivirus, but with 100 μg / ml protamine sulfate solution transduction "Enhancer PLUS" system. Transduction efficiency increased by >20% using our "Enhancer PLUS" system (Figure 1).

[0078] MSCs were transduced with CD19-CAR lentivirus (VSV-G pseudotyped) to generate MSC-CAR19.

[0079] Adipose-derived mesenchymal stem cells (100-Biotr-0024) were passaged into four wells of a six-well plate (100k each). Group 1 served as an untransduced (UTD) negative control. The second well was transduced with GMP-grade pan-VSV, CD19-recognizing chimeric antigen receptor (CAR19) lentivirus (approximate MOI 3). The third group was transduced with the same MOI and lentivirus, in addition to 50 μg / ml of Enhancer PLUS. The fourth group was identical to Group 3, but with 100 μg / ml of Enhancer PLUS. The transduction efficiency increased with the use of Enhancer PLUS (Figure 2).

[0080] Adipose-derived mesenchymal stem cells (100-Biotr-0024) were harvested from culture after 10+ serial passages and probed for surface expression of CAR19. As presented above, 48.5% of CAR19-transduced cultures retained CAR19 expression, with a 20% loss from transduction (Figure 3).

[0081] Adipose-derived mesenchymal stem cells (100-Biotr-0024), when engineered to express CAR19, suppress CAR T cell proliferation. T cell proliferation can be further suppressed by increasing the MSC:T cell ratio (Figures 4A and 4B).

[0082] Designer constructs for enhancing MSC trafficking, persistence, and efficacy of immunomodulation are shown in Figure 5 .

[0083] [Example 2] Engineered CAR-MSCs for immune modification of epithelial tissues MSCs capable of targeting epithelial tissues were engineered by engineering MSCs to express a CAR that targets ECAD (MSC-CAR-ECAD).

[0084] An exemplary nucleic acid sequence encoding CAR-ECAD (SEQ ID NO: 1) is shown in Figure 12A. An exemplary amino acid sequence of CAR-ECAD (SEQ ID NO: 2) is shown in Figure 12B.

[0085] MSCs were transduced with E-cadherin CAR lentivirus (VSV-G pseudotyped) to generate MSC-CAR-ECAD. Second-generation CAR constructs containing TLR3 and / or TLR4 signaling domains were used.

[0086] Adipose-derived mesenchymal stem cells (100-Biotr-0024) were passaged into four wells of a six-well plate (100k each). Group 1 served as an untransduced (UTD) negative control. The second well was transduced with GMP-grade pan-VSV, ECAD-recognizing chimeric antigen receptor (CAR-ECAD) lentivirus (approximate MOI 3). The third group was transduced with the same MOI and lentivirus, but with 50 μg / ml Enhancer PLUS. The fourth group was identical to Group 3, but with 100 μg / ml Enhancer PLUS. The transduction efficiency increased in an Enhancer PLUS concentration-dependent manner (Figure 6).

[0087] WT-MSCs significantly reduced the proliferative capacity of stimulated T cells, but not stimulated CART cells. When T cells were stimulated with nonspecific stimuli (PMA / IONO) and cultured in the presence of MSCs, proliferation was significantly inhibited. However, WT-MSCs were ineffective in suppressing CAR-specific T cell proliferation when activated via their antigen. CART19 was expressed via CAR (CD19 + When activated (via coculture with the cell line NALM6), antigen-specific proliferation was not inhibited in the presence of MSCs (Figures 5 and 6). Thus, adipose-derived mesenchymal stem cells suppress naive CD3xCD28 T cell proliferation, but not CART cell proliferation.

[0088] MSC-CAR cells exhibited stronger immunosuppressive effects when stimulated via CAR compared with wild-type MSCs or unstimulated MSCs. CD3+ T cells, stimulated CD3+ cells, or stimulated CART19 (stimulated via CAR) were cultured with medium alone, wild-type MSCs, MSCCAR19, MSC-CAR-ECAD, or MSC-CAR-ECAD with MCF-7 (an ECAD-expressing cell line). All conditions with MSCs inhibited T cell proliferation. Stimulated MSC-CARs more strongly inhibited CAR-T cell proliferation in the presence of their target antigen. MSC-CAR-ECAD inhibition of CART19 proliferation was stronger in the presence of MCF-7 cell line compared with MSC-CAR-ECAD inhibition of CART19 proliferation in the absence of MCF-7 cell line (Figures 7A and 7B). Thus, adipose-derived mesenchymal stem cells, when engineered to express CAR-ECAD, suppress CART cell proliferation in an antigen-dependent manner.

[0089] Flow cytometry was used to confirm the MSC phenotype (Figures 10A, 10B, 10C, and 10D). MSCs that retained stemness expressed CD90 + , CD105 + , CD73 + , CD34 - , CD45 - , CD19 - , CD14 -, HLA-DR - It should be.

[0090] [Example 3] Engineered CAR-MSCs for immune modulation of the central nervous system MSCs capable of targeting neural tissue were engineered by engineering MSCs to express a CAR that targets MOG (MSC-CAR-MOG).

[0091] MSCs were transduced with a lentivirus encoding CAR-MOG. CAR-MOG expression in MSCs was determined by comparison with a non-transduced (UTD) MSC population. Transduction was performed with or without protamine sulfate solution (approximately 70 μg / ml) to enhance transduction efficiency. CAR-MOG was expressed on the surface of approximately 78% of mesenchymal stem cells, with specificity for MOG (Figure 11).

[0092] Exemplary CAR-MOG-encoding nucleic acid sequences (SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7) are shown in Figures 13A, 13C, and 13E. Exemplary CAR-MOG amino acid sequences (SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8) are shown in Figures 13B, 13D, and 13F.

[0093] [Example 4] Engineered CAR-MSCs for immune modification of cardiac tissue MSCs capable of targeting cardiac tissue were engineered by engineering MSCs to express a CAR that targets HER2 (MSC-CAR-HER2).

[0094] The nucleic acid sequence encoding an exemplary CAR-HER2 (SEQ ID NO: 9) is shown in Figure 14A. The amino acid sequence of an exemplary CAR-HER2 (SEQ ID NO: 10) is shown in Figure 14B.

[0095] [Example 5] Transduction efficiency of MSC-CAR19 Lentiviral transduction of MSCs with CAR19 vectors results in a transduction efficiency of 60% compared to non-transduced MSCs (Figure 15A).

[0096] [Example 6] MSCs retain stemness after lentiviral transduction with CAR vectors Flow cytometry analysis of MSC-CAR19 2 days after transduction with CAR lentivirus demonstrated their stemness, comparable to that of untransduced MSCs (MSC-UTD). MSC-CAR19 continued to express CD105, CD90, and CD73, but lacked expression of CD34, CD45, HLA-DR, and CD14 (Figure 15B).

[0097] [Example 7] MSC-CAR19 is a CD19 + Inhibition of T cell proliferation in the presence of target, demonstrating antigen-specific stimulation of MSC-CAR19 T cells were stimulated with CD3 / CD28 beads and, 24 hours later, co-cultured with untransduced MSCs (MSC-UTD), MSC-CAR19 (CAR19, CD28 containing K122), or MSC-CAR19 (CAR19, CD137 containing K002), or without MSCs. The CD19-targeting K002 CAR was designed with a CD19-targeting single-chain antibody derived from FMC63, followed by a CD8 hinge, followed by a CD8 transmembrane domain, followed by a 4-1BB (CD137) signaling domain. The amino acid sequence of K002 was as follows: MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGG GTKLEITGGGGSGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYY YGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 63). The K122 CAR targeting CD19 was designed with a single chain antibody targeting CD19 from FMC63, followed by a CD28 hinge, followed by a CD28 transmembrane domain, followed by a CD28 signaling domain. The amino acid sequence of K122 was as follows: MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSLEPKSCDKTHTCPPCPDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 64).

[0098] As a strategy to stimulate MSC-CAR19 via CAR, T cells and MSCs were incubated with medium alone or with irradiated CD19 + The proliferation of CD3 was inhibited in the presence of MSC-CAR19 (containing the CD28 signaling domain), but not in the presence of MSC-CAR19 (containing the CD137 signaling domain) or non-transduced MSCs, whereas the proliferation of irradiated CD19 + was inhibited in the presence of CD19 cells + There was no inhibition in the absence of cells (Figure 16). T cells, MSCs, and NALM6 cells were cultured at a ratio of 1:0.1:1 E (T cells (effector): MSCs (suppressor): T (tumor)). These results indicate that when the CAR contains the CD28 signaling domain, the MSC-CAR was able to suppress T cell proliferation upon antigen-specific stimulation.

[0099] [Example 8] Incorporation of CD28 signaling enhances MSC-CAR19 proliferation MSC-CAR19 cells with different stimulatory domains were able to express irradiated CD19 + MSCs were co-cultured with or without the cell line NALM6, and proliferation was monitored. Incorporation of the CD28 signaling domain resulted in enhanced proliferation of MSC-CAR19 compared with MSC-UTD or CD137 stimulatory domain-incorporated MSC-CAR19 cells. Meanwhile, proliferation of MSC-CAR19 cells incorporating the TLR4 signaling domain was suppressed. These results indicate that CD28 and TLR4 signaling are involved in MSC-CAR proliferation upon antigen-specific stimulation. The CD19-targeting K142 CAR was designed with a CD19-targeting single-chain antibody derived from FMC63, followed by a CD8 hinge, followed by a TLR4 transmembrane domain, followed by a TLR4 signaling domain. The amino acid sequence of K142 was as follows: MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT GGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGT SVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIGVSVLSVLVVSVVAVLVYKFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI (SEQ ID NO: 65).

[0100] As a strategy to stimulate MSC-CAR19 via CAR, T cells and MSCs were incubated with medium alone or with irradiated CD19 + The proliferation of CD3 was inhibited in the presence of MSC-CAR19 (containing the TLR4 signaling domain), but not in the presence of MSC-CAR19 (containing the CD137 signaling domain) or non-transduced MSCs, whereas the proliferation of irradiated CD19 + was inhibited in the presence of CD19 cells + There was no inhibition in the absence of T cells (Figure 17). T cells, MSCs, and NALM6 cells were cultured at a ratio of 1:0.1:1 E (T cells (effector): MSCs (suppressor): T (tumor)). These results indicate that when the CAR contains a TLR4 stimulatory molecule, the MSC-CAR was able to suppress T cell proliferation upon antigen-specific stimulation.

[0101] [Example 9] MSC-CAR19 is a CD19 + Inhibition of T cell proliferation in the presence of target, demonstrating antigen-specific stimulation of MSC-CAR19 T cells were stimulated with CD3 / CD28 beads and 24 hours later, either with non-transduced MSCs (MSC-UTD), or MSC-CAR19 (CAR19, TLR4 containing K142), or without MSCs, and irradiated CD19 as a strategy to stimulate MSC-CAR19 via CAR. + The cells were co-cultured in the presence of MSC-CAR19 (containing the TLR4 signaling domain) but not in the presence of non-transduced MSCs. CD3 proliferation was inhibited in the presence of MSC-CAR19 (containing the TLR4 signaling domain), but not in the presence of non-transduced MSCs (Figure 18). T cells, MSCs, and NALM6 cells were cultured at a ratio of 1:0.1:1 E (T cells (effector): MSCs (suppressor): T (tumor)). These results indicate that when the CAR contains a TLR4 stimulatory molecule, the MSC-CAR was able to suppress T cell proliferation upon antigen-specific stimulation.

[0102] [Example 10] Antigen-specific stimulation of MSC-CARs containing CD28 signaling molecules enhances their proliferation Non-transduced MSCs (MSC-UTD), MSC-CAR19 (CAR19, CD28 containing K122), MSC-CAR19 (CAR19, CD137 containing K002), or MSC-CAR19 (CAR19, TLR4 containing K142) were transduced with irradiated CD19 + MSCs were co-cultured with the cell line NALM6 at a 1:1 ratio. The absolute number of MSCs was counted by flow cytometry using absolute counts on days 3 and 5. Antigen-specific stimulation of MSC-CAR19 (containing CD28) resulted in increased MSC proliferation (Figures 19A and 19B). These results indicate that CD28 signaling via CAR contributes to MSC-CAR proliferation.

[0103] [Example 11] Lentiviral transduction of MSCs with CAR results in reduced proliferation MSCs were transduced with CAR or GFP lentivirus on day 1 and then cultured for 15 days. Transduction with CAR-E cadherin resulted in reduced proliferation compared to non-transduced MSCs or MSCs transduced with GFP lentivirus (Figure 20).

[0104] [Example 12] MSC-CARs exert their suppressive capacity through cell-cell contact- and soluble factor-mediated mechanisms First, T cells were stimulated with CD3 / CD28 beads at a ratio of 1:3 (T cells to beads). After 24 hours, they were then stimulated with MSCs and CD19 beads either in direct contact or transwell experiments. + MSC-CAR19 (containing the CD28 stimulatory domain) was cultured with activated T cells and CD19 cells (to stimulate MSC-CAR). +Co-culture with MSC-CARs resulted in inhibition of T cell proliferation in transwell experiments, whether or not both cells were in direct contact (Figure 21). These results indicate that MSC-CARs exert their suppressive function through both direct cell-cell contact and secretion of soluble inhibitory factors / cytokines.

[0105] [Example 13] MSC-CAR suppresses T cell proliferation upon antigen-specific stimulation First, T cells were stimulated with CD3 / CD28 beads at a ratio of 1:3. After 24 hours, T cells were cultured with non-transduced MSCs or MSC-CAR19 (CAR19, CD28 containing K122) at a higher E:S:T ratio with NALM6, or T cells were cultured with NALM6 alone as a control for allogeneic effects. + The addition of cells stimulated MSC-CAR19 via CAR. CD3 proliferation was inhibited in the presence of MSC-CAR19 (containing the CD28 signaling domain), but not in the presence of non-transduced MSC (Figure 22). T cells, MSCs, and NALM6 cells were cultured at a ratio of 1:1:1 E (T cells (effector): MSCs (suppressor): T (tumor)).

[0106] [Example 14] MSC-CAR-E-cadherin cells suppress T cell proliferation upon antigen-specific stimulation at low E:T ratios First, T cells were stimulated with CD3 / CD28 beads at a 1:3 ratio. After 24 hours, activated T cells were cultured with untransduced MSCs (MSC-UTD, Figure 23A) or MSC-CAR-E-cadherin (CAR-E-cadherin containing CD28, Figure 23B) at different effector:suppressor (E:S) ratios in the presence or absence of the E-cadherin+ cell line MCF-7. Coculture of T cells with MSC-CAR-E-cadherin resulted in the suppression of their antigen-specific proliferation at a low effector:suppressor ratio in the presence of the E-cadherin+ cell line MCF-7. These results indicate that antigen-specific stimulation of MSC-CAR-E-cadherin containing the CD28 signaling domain results in enhanced suppressive capacity of T cells.

[0107] [Example 15] Design of CAR constructs targeting E-cadherin using scFvs Four scFvs, clones 5, 6, 7, and 14, were identified as having binding affinity for human E-CAD, and three of these (clones 6, 7, and 14) were used to design CARs targeting E-CAD.

[0108] K128-CAR, which targets human E-CAD, was designed with a single-chain antibody derived from clone 14, followed by a CD28 hinge, followed by a CD28 transmembrane domain, followed by a CD28 signaling domain. The amino acid sequence of K128-CAR was as follows: MALPVTALLLPLALLLHAARPEVQLVQSGGGLVKPGGS-LRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV YYCARAQRQWGAFDYWGQGTLVTVSSEGKSSGSGSESKASSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITG AQAEDEADYYCNSRDSSGNPVFGGGTKLTVLGLEPKSCDKTHTCPPCPDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 66). K129-CAR targeting human E-CAD was designed with a single chain antibody from clone 6, followed by a CD28 hinge, followed by a CD28 transmembrane domain, followed by a CD28 signaling domain. The amino acid sequence of K129-CAR was as follows: MALPVT-ALLLPLALLLHAARPEVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYVDSVKGRFTISRDNAKNSLYLQMDSLRAEDTAVY YCARGGRVLVGALFDYWGQGTLVTVSSEGKSSGSGSESKASLPVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAI SGLQSEDEADYYCASWDTSLRAWVFGGGTKLTVLGLEPKSCDKTHTCPPCPDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 67). K130-CAR targeting human E-CAD was designed with a single-chain antibody derived from clone 7, followed by a CD28 hinge, followed by a CD28 transmembrane domain, followed by a CD28 signaling domain. The amino acid sequence of K130-CAR was as follows: MALPVTALLLPLALLLHAARPE-VQLVESGTDVKKPGASVTVSCKASGYTFTAYYIHWVRQAPGQGLEWMGWINPYSGASNYAQKFLGRVTMTRDTSTNTVYMQLSSLRASDTAMYYC AKAACGSNGCYMREFDYWGQGTLVTVSSSEGKSSGSGSESKASDIVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTD FTLKISRVEAEDVGIYYCMQGTYWPGAFGQGTKVDIKLEPKSCDKTHTCPPCPDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 70).

[0109] T cells transduced with all three constructs expressed luciferase + / human E-cadherin + K129-CAR-transduced T cells also showed potent killing of MCF-7 cells. + / Mouse E-cadherin + ID8 cells and luciferase + / Canine E-cadherin + These results demonstrate that E-cadherin-targeting CARs are functional and can be used as described herein to generate MSCs bearing E-cadherin-targeting CARs.

[0110] [Example 16] Treatment or prevention of colitis To generate a mouse model of xenograft colitis, NSG mice were treated with PBMCs and their body weight was monitored over time. Mice developed colitis within approximately 30–40 days, accompanied by weight loss. At this time, satellite mice were euthanized, colon tissue was collected, and lymphocyte infiltration was examined. Next, mice were treated with MSC-UTD or different MSC-CAR-E-cadherin cells (derived from various scFv clones), and their body weight was monitored daily. After 30 days, mice were euthanized, and T cell infiltration into the colon was measured by flow cytometry and compared between mice treated with MSC-UTD and MSC-CAR-E-cadherin to confirm the treatment of colitis.

[0111] [Example 17] Treatment of humans with colitis A human is identified as having colitis and is administered MSC-CAR-E-cadherin cells intravenously or intra-arterially at a dose of approximately 1 to 2 million cells per kg of body weight. Dose escalation is included, with one dose given every 10 days. The patient is clinically monitored for improvement of colitis symptoms.

[0112] [Example 18] Treatment of Multiple Sclerosis The efficacy of MSC-CAR-MOG will be confirmed using an experimental autoimmune encephalomyelitis (EAE) mouse model. After neuroencephalitis is established, mice will be treated with MSC-CAR-MOG or MSC-UTD. The neurological status of the mice will be monitored daily, and their survival will be tracked.

[0113] [Example 19] Treatment of People with Multiple Sclerosis A human is identified as having multiple sclerosis and is administered MSC-CAR-MOG at a dose of about 2 to about 10 million cells per kg of body weight. The cells are administered either intravenously or intracerebroventricularly. Multiple doses, including dose escalation, are administered, for example, every 10 days. The patient is clinically monitored for improvement of multiple sclerosis symptoms.

[0114] [Example 20] Treatment of immune-mediated encephalomyelitis The efficacy of MSC-CAR-MOG will be confirmed using an experimental autoimmune encephalomyelitis (EAE) mouse model. After neuroencephalitis is established, mice will be treated with MSC-CAR-MOG or MSC-UTD. The neurological status of the mice will be monitored daily, and their survival will be tracked.

[0115] [Example 21] Treatment of humans with immune-mediated encephalomyelitis A human is identified as having immune-mediated encephalomyelitis and is administered MSC-CAR-MOG at a dose of about 2 to about 10 million cells per kg of body weight. The cells are administered intravenously or intracerebroventricularly. Multiple doses, including dose escalation, are administered, for example, every 10 days. The patient is clinically monitored for improvement of immune-mediated encephalomyelitis symptoms.

[0116] [Example 22] Treatment of myocarditis Using an experimental murine myocarditis model induced by infection with Coxsackievirus B3, mice are treated with MSC-UTD or MSC-CAR-HER2. Mice are followed clinically and for survival. At the end of the experiment, mice are euthanized, and T cell infiltration into the heart is measured by flow cytometry.

[0117] [Example 23] Treatment of humans with myocarditis A human is identified as having severe or life-threatening myocarditis and is administered MSC-CAR-HER2 at a dose of about 2 to about 10 million cells / kg of body weight. The cells are administered via either intravenous or intracardiac routes. Multiple doses, including dose escalation, are administered, for example, every 10 days. The patient is clinically monitored for improvement of the symptoms of myocarditis.

[0118] [Example 24] MSC-CAR-E-cadherin suppresses the antitumor activity of T cells and their proliferation in vivo Luciferase in NSG mice + / E-cadherin + MCF-7 cell line (1 x 10 6 All mice were transplanted with E-cadherin CAR T cells (2 × 10 cells, intravenous). One week later, bioluminescence imaging was performed to confirm engraftment. 6 cells), and mice were treated with MSC-CAR-E-cadherin (1 × 10 6 ), non-transduced MSCs (MSC-UTD) (1 × 10 6 MSC-CAR-ECAD treatment resulted in a reduction in the antitumor activity of E-cadherin-directed CAR T cells (Figure 24A). Peripheral blood flow cytometry was performed 7 days after treatment with CAR T cells and CAR-MSC cells. Treatment with MSC-CAR-E-cadherin resulted in a trend toward reduced T cell proliferation in vivo (Figure 24B). These results indicate that MSC-CAR-E-CAD can suppress T cell effector function in this xenograft model.

[0119] In another experiment, luciferase + MSC-CAR-E-cadherin cells were generated and injected intraperitoneally into immunodeficient NSG mice. To measure the persistence of MSC-CAR-E-cadherin, continuous bioluminescence imaging was performed. MSC-CAR-E-cadherin cells were found to persist in vivo for more than 10 days (Figures 25A and 25B). These results indicate that MSC-CAR cells can persist in vivo, for example, in a xenograft model.

[0120] Other embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to be illustrative, but not limiting, of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. For example, the present invention encompasses the following embodiments: [Embodiment 1] A method of treating a mammal having colitis, the method comprising administering to the mammal a composition comprising mesenchymal stem cells (MSCs) comprising an exogenous nucleic acid encoding a chimeric antigen receptor (CAR) that targets an epithelial-specific antigen, wherein the MSCs express the CAR. [Embodiment 2] The method of claim 1, wherein the mammal is a human. [Embodiment 3] The method of any one of claims 1 to 2, wherein the MSCs are adipose-derived MSCs. [Embodiment 4] The method of any one of claims 1 to 3, wherein the epithelial-specific antigen is E-cadherin (ECAD). [Embodiment 5] The method of any one of claims 1 to 4, wherein the CAR comprises a single-chain variable fragment (scFv). [Embodiment 6] The method of claim 5, wherein the scFv comprises a light chain and a heavy chain derived from an anti-CDH1 antibody. [Embodiment 7] The method described in claim 6, wherein the anti-CDH1 antibody is hSC10.17. [Embodiment 8] The method of any one of claims 1 to 7, wherein the MSCs are engineered to express the CAR ex vivo prior to the administration. [Embodiment 9] The method of any one of claims 1 to 8, wherein the colitis symptoms are reduced by at least 10 percent. [Embodiment 10] A method of treating a mammal at risk of developing colitis, the method comprising administering to the mammal a composition comprising mesenchymal stem cells (MSCs) comprising an exogenous nucleic acid encoding a chimeric antigen receptor (CAR) that targets an epithelial-specific antigen, wherein the MSCs express the CAR. [Embodiment 11] The method of claim 10, wherein the mammal is a human. [Embodiment 12] The method of any one of claims 10 to 11, wherein the MSCs are adipose-derived MSCs. [Embodiment 13] The method of any one of claims 10 to 12, wherein the epithelial-specific antigen is E-cadherin (ECAD). [Embodiment 14] The method of any one of claims 10 to 13, wherein the CAR comprises a single-chain variable fragment (scFv). [Embodiment 15] The method described in claim 14, wherein the scFv comprises a light chain and a heavy chain derived from an anti-CDH1 antibody. [Embodiment 16] The method described in claim 15, wherein the anti-CDH1 antibody is hSC10.17. [Embodiment 17] The method of any one of claims 10 to 16, wherein the MSCs are engineered to express the CAR ex vivo prior to the administration. [Embodiment 18] A method of treating a mammal having multiple sclerosis, the method comprising administering to the mammal a composition comprising mesenchymal stem cells (MSCs) comprising an exogenous nucleic acid encoding a chimeric antigen receptor (CAR) that targets a neurospecific antigen, wherein the MSCs express the CAR. [Embodiment 19] The method of claim 18, wherein the mammal is a human. [Embodiment 20] The method described in any one of claims 18 to 19, wherein the MSCs are adipose-derived MSCs. [Embodiment 21] The method of any of claims 18 to 20, wherein the neuro-specific antigen is myelin oligodendrocyte glycoprotein (MOG). [Embodiment 22] The method of claim 21, wherein the CAR comprises a single-chain variable fragment (scFv). [Embodiment 23] The method of claim 22, wherein the scFv comprises a light chain and a heavy chain derived from an anti-MOG antibody. [Embodiment 24] The method of claim 23, wherein the anti-MOG antibody is 8-18C5. [Embodiment 25] The method of any one of claims 18 to 24, wherein the MSCs are engineered to express the CAR ex vivo prior to the administration. [Embodiment 26] The method of any one of claims 18 to 25, wherein the symptoms of multiple sclerosis are reduced by at least 10 percent. [Embodiment 27] The method described in any one of claims 18 to 26, wherein the MSCs further comprise an exogenous nucleic acid encoding a polypeptide capable of promoting neural differentiation, and the MSCs express the polypeptide. [Embodiment 28] The method of claim 27, wherein the polypeptide capable of promoting neuronal differentiation is selected from the group consisting of Oct3 / 4 polypeptide, Klf4 polypeptide, Sox2 polypeptide, Glis1 polypeptide, c-Myc polypeptide, BMP4 polypeptide, WNT polypeptide, FGF2 polypeptide, SHH polypeptide, Sox11 polypeptide, Sox2 polypeptide, Sox3 polypeptide, Zic1 polypeptide, Zic2 polypeptide, Irx1 polypeptide, Irx2 polypeptide, Irx3 polypeptide, FoxD4 polypeptide, MKx2.5 polypeptide, cTnT polypeptide, and any combination thereof. [Embodiment 29] A method of treating a mammal at risk of developing multiple sclerosis, the method comprising administering to the mammal a composition comprising mesenchymal stem cells (MSCs) comprising an exogenous nucleic acid encoding a chimeric antigen receptor (CAR) that targets a neurospecific antigen, wherein the MSCs express the CAR. [Embodiment 30] The method of claim 29, wherein the mammal is a human. [Embodiment 31] The method described in any one of claims 29 to 30, wherein the MSCs are adipose-derived MSCs. [Embodiment 32] The method of any one of claims 29 to 31, wherein the neuro-specific antigen is myelin oligodendrocyte glycoprotein (MOG). [Embodiment 33] The method of claim 32, wherein the CAR comprises a single-chain variable fragment (scFv). [Embodiment 34] The method of claim 33, wherein the scFv comprises a light chain and a heavy chain derived from an anti-MOG antibody. [Embodiment 35] The method of claim 34, wherein the anti-MOG antibody is 8-18C5. [Embodiment 36] The method of any one of claims 29 to 35, wherein the MSCs are engineered to express the CAR ex vivo prior to the administration. [Embodiment 37] A method of treating a mammal having immune-mediated encephalomyelitis, the method comprising administering to the mammal a composition comprising mesenchymal stem cells (MSCs) comprising an exogenous nucleic acid encoding a chimeric antigen receptor (CAR) that targets a neurospecific antigen, wherein the MSCs express the CAR. [Embodiment 38] The method of claim 37, wherein the mammal is a human. [Embodiment 39] A method according to any one of claims 37 to 38, wherein the MSCs are adipose-derived MSCs. [Embodiment 40] The method of any of claims 37 to 39, wherein the neurospecific antigen is myelin oligodendrocyte glycoprotein (MOG). [Embodiment 41] The method of claim 40, wherein the CAR comprises a single-chain variable fragment (scFv). [Embodiment 42] The method of claim 41, wherein the scFv comprises a light chain and a heavy chain derived from an anti-MOG antibody. [Embodiment 43] The method of claim 42, wherein the anti-MOG antibody is 8-18C5. [Embodiment 44] The method of any of claims 37 to 43, wherein the MSCs are engineered to express the CAR ex vivo prior to the administration. [Embodiment 45] The method of any one of claims 37 to 44, wherein the symptoms of immune-mediated encephalomyelitis are reduced by at least 10 percent. [Embodiment 46] The method described in any one of claims 37 to 45, wherein the MSCs further comprise an exogenous nucleic acid encoding a polypeptide capable of promoting neural differentiation, and the MSCs express the polypeptide. [Embodiment 47] The method of claim 46, wherein the polypeptide capable of promoting neural differentiation is selected from the group consisting of Oct3 / 4 polypeptide, Klf4 polypeptide, Sox2 polypeptide, Glis1 polypeptide, c-Myc polypeptide, BMP4 polypeptide, WNT polypeptide, FGF2 polypeptide, SHH polypeptide, Sox11 polypeptide, Sox2 polypeptide, Sox3 polypeptide, Zic1 polypeptide, Zic2 polypeptide, Irx1 polypeptide, Irx2 polypeptide, Irx3 polypeptide, FoxD4 polypeptide, MKx2.5 polypeptide, cTnT polypeptide, and any combination thereof. [Embodiment 48] A method for treating a mammal at risk of developing immune-mediated encephalomyelitis, the method comprising administering to the mammal a composition comprising mesenchymal stem cells (MSCs) comprising an exogenous nucleic acid encoding a chimeric antigen receptor (CAR) that targets a neurospecific antigen, wherein the MSCs express the CAR. [Embodiment 49] The method of claim 48, wherein the mammal is a human. [Embodiment 50] The method described in any one of claims 48 to 49, wherein the MSCs are adipose-derived MSCs. [Embodiment 51] The method of any of claims 48 to 50, wherein the neuro-specific antigen is myelin oligodendrocyte glycoprotein (MOG). [Embodiment 52] The method of claim 51, wherein the CAR comprises a single-chain variable fragment (scFv). [Embodiment 53] The method of claim 52, wherein the scFv comprises a light chain and a heavy chain derived from an anti-MOG antibody. [Embodiment 54] The method of claim 53, wherein the anti-MOG antibody is 8-18C5. [Embodiment 55] The method of any one of claims 48 to 54, wherein the MSCs are engineered to express the CAR ex vivo prior to said administration. [Embodiment 56] A nucleic acid construct encoding a chimeric antigen receptor (CAR) that targets a neurospecific antigen. [Embodiment 57] The nucleic acid construct of claim 56, wherein the neuro-specific antigen is myelin oligodendrocyte glycoprotein (MOG). [Embodiment 58] A nucleic acid construct described in any one of claims 56 to 57, wherein the CAR comprises a single-chain variable fragment (scFv). [Embodiment 59] A nucleic acid construct described in any of claims 56 to 58, wherein the CAR targeting the neurospecific antigen is encoded by the nucleic acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7. [Embodiment 60] A nucleic acid construct described in any of claims 56 to 59, wherein the nucleic acid construct is also capable of encoding a polypeptide capable of promoting neural differentiation. [Embodiment 61] The nucleic acid construct of claim 60, wherein the polypeptide capable of promoting neural differentiation is selected from the group consisting of Oct3 / 4 polypeptide, Klf4 polypeptide, Sox2 polypeptide, Glis1 polypeptide, c-Myc polypeptide, BMP4 polypeptide, WNT polypeptide, FGF2 polypeptide, SHH polypeptide, Sox11 polypeptide, Sox2 polypeptide, Sox3 polypeptide, Zic1 polypeptide, Zic2 polypeptide, Irx1 polypeptide, Irx2 polypeptide, Irx3 polypeptide, FoxD4 polypeptide, MKx2.5 polypeptide, cTnT polypeptide, and any combination thereof. [Embodiment 62] A method of treating a mammal having myocarditis, the method comprising administering to the mammal a composition comprising mesenchymal stem cells (MSCs) comprising an exogenous nucleic acid encoding a chimeric antigen receptor (CAR) that targets a cardiac-specific antigen, wherein the MSCs express the CAR. [Embodiment 63] The method of claim 62, wherein the mammal is a human. [Embodiment 64] The method described in any one of claims 62 to 63, wherein the MSCs are adipose-derived MSCs. [Embodiment 65] The method described in any one of claims 62 to 64, wherein the cardiac-specific antigen is HER2. [Embodiment 66] The method of claim 65, wherein the CAR comprises a single-chain variable fragment (scFv). [Embodiment 67] The method of any of claims 62 to 66, wherein the MSCs are engineered to express the CAR ex vivo prior to said administration. [Embodiment 68] The method of any of claims 62 to 67, wherein the symptoms of myocarditis are reduced by at least 10 percent. [Embodiment 69] The method described in any one of claims 62 to 68, wherein the MSCs further comprise an exogenous nucleic acid encoding a polypeptide capable of promoting cardiac cell differentiation, and the MSCs express the polypeptide. [Embodiment 70] The method described in claim 69, wherein the polypeptide capable of promoting neural differentiation is selected from the group consisting of a GATA4 polypeptide, a MEF2C polypeptide, a TBX5 polypeptide, an ERRG polypeptide, a MESP1 polypeptide, and any combination thereof. [Embodiment 71] A method of treating a mammal at risk of developing myocarditis, the method comprising administering to the mammal a composition comprising mesenchymal stem cells (MSCs) comprising an exogenous nucleic acid encoding a chimeric antigen receptor (CAR) that targets a cardiac-specific antigen, wherein the MSCs express the CAR. [Embodiment 72] The method of claim 71, wherein the mammal is a human. [Embodiment 73] The method described in any one of claims 71 to 72, wherein the MSCs are adipose-derived MSCs. [Embodiment 74] The method of any one of claims 71 to 73, wherein the cardiac-specific antigen is HER2. [Embodiment 75] The method of claim 74, wherein the CAR comprises a single-chain variable fragment (scFv). [Embodiment 76] The method of any one of claims 71 to 75, wherein the MSCs are engineered to express the CAR ex vivo prior to said administration. [Embodiment 77] A nucleic acid construct encoding a chimeric antigen receptor (CAR) that targets a cardiac-specific antigen. [Embodiment 78] The nucleic acid construct of claim 77, wherein the cardiac-specific antigen is HER2. [Embodiment 79] A nucleic acid construct described in any one of claims 77 to 78, wherein the CAR comprises a single-chain variable fragment (scFv). [Embodiment 80] A nucleic acid construct described in any of claims 77 to 79, wherein the CAR targeting the cardiac-specific antigen is encoded by the nucleic acid sequence shown in SEQ ID NO: 9. [Embodiment 81] A nucleic acid construct described in any of claims 77 to 80, wherein the nucleic acid construct is also capable of encoding a polypeptide capable of promoting cardiac cell differentiation. [Embodiment 82] The nucleic acid construct of claim 81, wherein the polypeptide capable of promoting neural differentiation is selected from the group consisting of a GATA4 polypeptide, a MEF2C polypeptide, a TBX5 polypeptide, an ERRG polypeptide, a MESP1 polypeptide, and any combination thereof. [Embodiment 83] A method for treating an inflammatory disease or condition or a degenerative disease or condition in a mammal, the method comprising administering to the mammal a composition comprising mesenchymal stem cells (MSCs) comprising an exogenous nucleic acid encoding a chimeric antigen receptor (CAR) that targets an antigen expressed in the mammal, wherein the MSCs express the CAR, and binding of the CAR to the antigen in the mammal results in suppression of an immune response in the mammal. [Embodiment 84] The method of claim 83, wherein the mammal is a human. [Embodiment 85] The method of any one of claims 83 to 84, wherein the MSCs are adipose-derived MSCs. [Embodiment 86] The method described in any one of claims 83 to 85, wherein the antigen is E-cadherin (ECAD). [Embodiment 87] The method of any one of claims 83 to 86, wherein the CAR comprises a single-chain variable fragment (scFv). [Embodiment 88] The method of any of claims 83 to 87, wherein the MSCs are engineered to express the CAR ex vivo prior to said administration. [Embodiment 89] The method of any one of claims 1 to 55, 62 to 76, and 83 to 88, wherein the CAR comprises a CD28 or TLR4 signaling domain. [Embodiment 90] The method of claim 89, wherein the CAR comprises a CD28 signaling domain. [Embodiment 91] The method of claim 89, wherein the CAR comprises a TLR4 signaling domain. [Embodiment 92] The nucleic acid of any of claims 56 to 61 and 77 to 82, wherein the CAR comprises a CD28 or TLR4 signaling domain. [Embodiment 93] The nucleic acid of claim 92, wherein the CAR comprises a CD28 signaling domain. [Embodiment 94] The nucleic acid of claim 92, wherein the CAR comprises a TLR4 signaling domain.

Claims

1. 1. A composition for treating a mammal having or at risk of developing colitis, the composition comprising mesenchymal stem cells (MSCs) comprising an exogenous nucleic acid encoding a chimeric antigen receptor (CAR) that targets an epithelial-specific antigen, wherein the MSCs express the CAR, the epithelial-specific antigen is E-cadherin (ECAD), and the CAR comprises a single-chain variable fragment (scFv).

2. The composition of claim 1, wherein the mammal is a human.

3. 3. The composition of claim 1, wherein the MSCs are adipose-derived MSCs.

4. The composition of any one of claims 1 to 3, wherein the scFv comprises a light chain and a heavy chain derived from an anti-CDH1 antibody.

5. The composition of claim 4, wherein the anti-CDH1 antibody is clone 6.

6. The composition of claim 5, wherein the CAR comprises the amino acid sequence set forth in SEQ ID NO:

67.

7. The composition of claim 4, wherein the anti-CDH1 antibody is hSC10.

17.

8. 8. The composition of any one of claims 1 to 7, wherein the MSCs are engineered to express the CAR ex vivo prior to administration of the composition.

9. 9. The composition of any one of claims 1 to 8, wherein the mammal has colitis and the symptoms of the colitis are reduced by at least 10 percent.

10. 10. The composition of any one of claims 1 to 9, wherein the CAR comprises a CD28 or TLR4 signaling domain.

11. The composition of claim 10, wherein the CAR comprises a CD28 signaling domain.

12. 11. The composition of claim 10, wherein the CAR comprises a TLR4 signaling domain.

13. A composition described in any one of claims 1 to 12 for inhibiting T cell proliferation, thereby treating a mammal having colitis or at risk of developing colitis.

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