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

Abstract

The present invention provides methods and materials for treating mammals (e.g., humans) that have or are at risk of developing a disease or condition characterized by tissue inflammation and / or degeneration. [Solution] For example, mesenchymal stem cells (MSCs) that express tissue-targeting antigen receptors (e.g., chimeric antigen receptors) capable of exerting immunosuppressive effects in targeted tissues are provided, as well as a method using such MSCs.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the benefit of U.S. Provisional Application No. 62 / 932,610, filed on 8 November 2019. The disclosures of the prior application are deemed to be part of the disclosures of this application (and are incorporated into this application by reference).

[0002] background 1. Technical Fields This document relates to methods and materials for using engineered mesenchymal stem cells (MSCs) to treat mammals (e.g., humans) that have or are at risk of developing inflammatory diseases or conditions, and / or have or are at risk of developing degenerative diseases. For example, this document provides engineered MSCs designed to express antigen receptors (e.g., chimeric antigen receptors (CARs)) that have the ability to bind to (e.g., specifically bind to) antigens (e.g., tissue-specific antigens). Such engineered MSCs can exert immunosuppressive effects (e.g., reduce or eliminate immune responses) in targeted tissues and / or regenerate tissue-specific cells. In some cases, MSCs engineered to express antigen receptors that have the ability to bind to antigens (e.g., tissue-specific cells expressing antigens targeted by the MSCs) can also be engineered to express polypeptides that can promote differentiation into tissue-specific cells. For example, MSCs can be engineered to contain nucleic acids encoding polypeptides that can promote tissue-specific cell differentiation and can differentiate into tissue-specific cells (e.g., cardiac cells or neurons). Furthermore, this document also provides a method for treating inflammatory diseases or conditions in mammals (e.g., humans) that have or are at risk of developing such diseases or conditions, by administering one or more MSCs expressing antigen receptors that have the ability to bind to tissue-specific antigens (e.g., specifically). [Background technology]

[0003] 2. Background information Inflammation is a common factor in many diseases. In 2015, an estimated 1.3% (approximately 3 million) of adults in the United States were diagnosed with IBD (either Crohn's disease or ulcerative colitis; Dahlhamer et al., MMWR Morb Mortal Wkly Rep. 2016, Vol. 65 (No. 42): pp. 1166-1169 (2015)). [Overview of the project]

[0004] This document provides methods and materials for treating mammals (e.g., humans) that have or are at risk of developing inflammatory diseases or conditions, and / or have or are at risk of developing degenerative diseases. For example, one or more MSCs designed to express antigen receptors (e.g., CARs) that can bind to tissue-specific antigens (e.g., specifically) can be administered to a mammal to induce an immunosuppressive response in the mammal (e.g., reduce or eliminate an inflammatory immune response). In some cases, a CAR that targets an epithelial-specific antigen (e.g., epithelial cadherin (ECAD, also known as CDH1)) can be expressed by an MSC (e.g., MSC-CAR), allowing the MSC to target epithelial tissue. In other cases, a CAR that targets a nerve-specific antigen (e.g., myelin oligodendrocyte glycoprotein (MOG)) can be expressed by an MSC (e.g., MSC-CAR), allowing the MSC to target nerve tissue. In some cases, CARs that target cardiac-specific antigens (e.g., human epidermal growth factor receptor 2 (HER2)) can be expressed by MSCs, allowing the MSCs to target cardiac tissue. For example, one or more MSCs designed to express CARs that can bind (e.g., specifically bind) to tissue-specific antigens (e.g., epithelial-specific antigens, nerve-specific antigens, or cardiac-specific antigens) can be administered to mammals (e.g., humans) that have (or are at risk of developing) an inflammatory disease or condition (e.g., by adoptive transfer) to treat that inflammatory disease or condition in the mammal. In some cases, one or more MSCs expressing CARs that can bind (e.g., specifically bind) to epithelial-specific antigens (e.g., ECAD) can be administered to mammals (e.g., humans) that have (e.g., are at risk of developing) inflammatory bowel disease (IBD; e.g., colitis) (e.g., by adoptive transfer) to treat that inflammatory bowel disease in the mammal.In some cases, multiple sclerosis can be treated in mammals (e.g., humans) that have or are at risk of developing multiple sclerosis by administering one or more MSCs expressing CARs that can specifically bind to nerve-specific antigens (e.g., MOG) (e.g., by adoptive transfer). In some cases, immune-mediated encephalomyelitis can be treated in mammals (e.g., humans) that have or are at risk of developing immune-mediated encephalomyelitis by administering one or more MSCs expressing CARs that can specifically bind to nerve-specific antigens (e.g., MOG) (e.g., by adoptive transfer). In some cases, myocarditis can be treated in mammals (e.g., humans) that have or are at risk of developing myocarditis by administering one or more MSCs expressing CARs that can specifically bind to cardiac-specific antigens (e.g., HER2) (e.g., by adoptive transfer). For example, MSCs designed to express an antigen receptor (e.g., CAR) capable of binding to a tissue-specific antigen can also be engineered to express a polypeptide that can promote differentiation into tissue-specific cells (e.g., this may include nucleic acids encoding a polypeptide that can promote differentiation into tissue-specific cells). Such engineered MSCs can express polypeptides 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) within the mammal. In some cases, one or more MSCs designed to express an antigen receptor (e.g., CAR) capable of binding to a tissue-specific antigen, and also designed to differentiate into tissue-specific cells, can be administered (e.g., by adoptive transfer) to a mammal (e.g., human) that has or is at risk of developing a degenerative disease to treat that degenerative disease within the mammal.

[0005] As described herein, MSCs (e.g., adipose-derived MSCs) can be engineered to express CARs that target tissue-specific antigens (e.g., antigens expressed on target tissue). For example, MSCs can be engineered to express CARs that target (e.g., bind to) ECADs (e.g., CAR-ECADs) in epithelial tissue within mammals and can be used to treat diseases or disorders characterized by inflammation of epithelial tissue (e.g., colitis). In another example, MSCs can be engineered to express CARs that target (e.g., bind to) MOGs (e.g., CAR-MOGs) in nervous tissue within mammals and can be used to treat diseases or disorders characterized by inflammation of nervous tissue (e.g., multiple sclerosis and immune-mediated encephalomyelitis). In some cases, MSCs can be engineered to express CARs that target (e.g., bind to) antigens expressed on target tissue in order to exert an immunosuppressive effect within the target tissue (e.g., to reduce or eliminate inflammatory immune responses). For example, MSCs expressing the CARs described herein can be used to reduce or eliminate the proliferation of stimulated T cells in targeted tissue (compared to, for example, levels present before administration of such MSCs).

[0006] By having the ability to treat the inflammatory diseases and conditions described herein, clinicians and patients can reduce inflammation within the patient in an effective and efficient manner.

[0007] Generally, one aspect of this document features a method for treating a mammal having colitis. This method may involve, or essentially consist of, administering to the mammal a composition comprising MSCs containing an exogenous nucleic acid encoding a CAR that targets an epithelial-specific antigen, the MSCs expressing the CAR. The mammal may be human. The MSCs may be adipose-derived MSCs. The epithelial-specific antigen may be ECAD. The CAR may contain a single-stranded variable fragment (scFv). The scFv may contain light and heavy chains derived from an anti-CDH1 antibody. The anti-CDH1 antibody may be hSC10.17. The MSCs may be manipulated to express the CAR ex vivo before administration. Symptoms of colitis can be reduced by at least 10 percent. The CAR may contain a CD28 or TLR4 signaling domain.

[0008] In another aspect, one aspect of this document features a method for treating mammals at risk of developing colitis. The method comprises, or may essentially consist of, administering to a mammal a composition comprising MSCs containing an exogenous nucleic acid encoding a CAR that targets an epithelial-specific antigen, the MSCs expressing the CAR. The mammal may be human. The MSCs may be adipose-derived MSCs. The epithelial-specific antigen may be ECAD. The CAR may contain scFv. The scFv may contain light and heavy chains derived from an anti-CDH1 antibody. The anti-CDH1 antibody may be hSC10.17. The MSCs may be manipulated to express the CAR ex vivo before administration. The CAR may contain a CD28 or TLR4 signaling domain.

[0009] In another aspect, one aspect of this document features a method for treating a mammal having multiple sclerosis. The method may include, or may essentially consist of, administering to a mammal a composition comprising MSCs containing an exogenous nucleic acid encoding a CAR that targets a nerve-specific antigen, the MSCs expressing the CAR. The mammal may be human. The MSCs may be adipose-derived MSCs. The nerve-specific antigen may be MOG. The CAR may contain scFv. The scFv may contain light and heavy chains derived from an anti-MOG antibody. The anti-MOG antibody may be 8-18C5. The MSCs may be manipulated to express the CAR ex vivo before administration. The symptoms of multiple sclerosis can be reduced by at least 10 percent. The MSCs may also contain an exogenous nucleic acid encoding a polypeptide that can promote neuronal differentiation, the MSCs expressing the polypeptide. Polypeptides that can promote neuronal differentiation may include 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, or any combination thereof. CAR may contain a CD28 or TLR4 signaling domain.

[0010] In another aspect, one aspect of this document features a method for treating mammals at risk of developing multiple sclerosis. The method comprises, or may essentially consist of, administering to a mammal a composition comprising MSCs containing an exogenous nucleic acid encoding a CAR that targets a nerve-specific antigen, the MSCs expressing the CAR. The mammal may be human. The MSCs may be adipose-derived MSCs. The nerve-specific antigen may be MOG. The CAR may contain scFv. The scFv may contain light and heavy chains derived from an anti-MOG antibody. The anti-MOG antibody may be 8-18C5. The MSCs may be engineered to express the CAR ex vivo before administration. The CAR may contain a CD28 or TLR4 signaling domain.

[0011] In another embodiment, one aspect of this document features a method for treating a mammal having immune-mediated encephalomyelitis. The method may include, or may essentially consist of, administering to a mammal a composition comprising MSCs containing an exogenous nucleic acid encoding a CAR that targets a neuronal specific antigen, the MSCs expressing the CAR. The mammal may be human. The MSCs may be adipose-derived MSCs. The neuronal specific antigen may be MOG. The CAR may contain scFv. The scFv may contain light and heavy chains derived from an anti-MOG antibody. The anti-MOG antibody may be 8-18C5. The MSCs may be manipulated to express the CAR ex vivo before administration. The symptoms of immune-mediated encephalomyelitis can be reduced by at least 10 percent. The MSCs may also contain an exogenous nucleic acid encoding a polypeptide that can promote neuronal differentiation, the MSCs expressing the polypeptide. Polypeptides that can promote neuronal differentiation may include 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, or any combination thereof. CARs may contain CD28 or TLR4 signaling domains.

[0012] In another aspect, one aspect of this document features a method for treating mammals at risk of developing immune-mediated encephalomyelitis. The method comprises, or may essentially consist of, administering to a mammal a composition comprising MSCs containing an exogenous nucleic acid encoding a CAR that targets a neuron-specific antigen, the MSCs expressing the CAR. The mammal may be human. The MSCs may be adipose-derived MSCs. The neuron-specific antigen may be MOG. The CAR may contain scFv. The scFv may contain light and heavy chains derived from an anti-MOG antibody. The anti-MOG antibody may be 8-18C5. The MSCs may be engineered to express the CAR ex vivo before administration. The CAR may contain a CD28 or TLR4 signaling domain.

[0013] In another embodiment, one aspect of this document features a nucleic acid construct encoding a CAR that targets a nerve-specific antigen. The nerve-specific antigen may be MOG. The CAR may contain scFv. The CAR that targets the nerve-specific antigen may be encoded by the nucleic acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7. The nucleic acid construct may also encode a polypeptide that can promote neuronal differentiation. Polypeptides that can promote neuronal differentiation may be 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, or any combination thereof. The CAR may contain a CD28 or TLR4 signaling domain.

[0014] In another aspect, one aspect of this written description features a method of treating a mammal having 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 heart-specific antigen, wherein the MSCs express the CAR. The mammal can be a human. The MSCs can be adipose-derived MSCs. The heart-specific antigen can be HER2. The CAR can include 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 that promotes cardiac cell differentiation, and the MSCs express the polypeptide. The polypeptide that can promote neural differentiation can be a GATA4 polypeptide, a MEF2C polypeptide, a TBX5 polypeptide, an ERRG polypeptide, a MESP1 polypeptide, and any combination thereof. The CAR can include a CD28 or TLR4 signaling domain.

[0015] In another aspect, one aspect of this written description 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 heart-specific antigen, wherein the MSCs express the CAR. The mammal can be a human. The MSCs can be adipose-derived MSCs. The heart-specific antigen can be HER2. The CAR can include an scFv. The MSCs can be engineered to express the CAR ex vivo prior to administration. The CAR can include a CD28 or TLR4 signaling domain.

[0016] In another aspect, one aspect of this written document features a nucleic acid construct encoding a CAR that targets a heart-specific antigen. The heart-specific antigen can be HER2. The CAR can include a scFv. The CAR targeting the heart-specific antigen can be encoded by the nucleic acid sequence shown in SEQ ID NO: 9. The nucleic acid construct can also encode a polypeptide capable of promoting heart cell differentiation. The polypeptide capable of promoting nerve 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 include a CD28 or TLR4 signaling domain.

[0017] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In order to implement the present invention, methods and materials similar or equivalent to those described herein can be used, but appropriate methods and materials are described below. All publications, patent applications, patents, and other references described herein are incorporated herein by reference in their entirety. In case of conflict, the present specification including the definitions will govern. In addition, the materials, methods, and examples are merely illustrative and not intended to be limiting.

[0018] Details of one or more embodiments of the present invention are shown in the accompanying drawings and the following description. Other features, objectives, and advantages of the present invention are apparent from the specification, drawings, and claims.

Brief Description of the Drawings

[0019] [Figure 1] It is a diagram showing enhancement of lentiviral transduction of MSC. [Figure 2] It is a diagram showing MSC expression of CAR19 (MSC19). [Figure 3] It is a diagram showing the stability of MSC-CAR19 expression after passage 10+. [Figure 4]A. Suppression of MSCs during antigen-specific CAR-T cell proliferation. B. Suppression of MSCs during antigen-specific CAR-T cell proliferation. [Figure 5] This is a schematic diagram of an exemplary nucleic acid construct encoding CAR19. [Figure 6] This figure shows the expression of MSCs in CAR-ECAD. [Figure 7] MSCs suppress T cell proliferation, but do not suppress CAR2-T cell proliferation. [Figure 8] MSC-CAR mediated the suppression of T cell proliferation. [Figure 9A-9B] A. MSC-CAR mediated the suppression of activated T cells and CART cells. B. CART19+ NALM6 suppressed antigen-specific CAR-T cell proliferation. [Figure 10A] This figure shows the retention of stem cell properties in MSCs. A. Flow cytometry analysis of CD90. [Figure 10B] This figure shows the retention of stem cell properties in MSCs. Flow cytometry analysis of B.CD105. [Figure 10C] This figure shows the retention of stem cell properties in MSCs. Flow cytometry analysis of C.CD73. [Figure 10D] This figure shows the retention of stem cell properties in MSCs. Flow cytometry analysis of D.CD34. [Figure 10E] This figure shows the retention of stem cell properties in MSCs. Flow cytometry analysis of E.CD45. [Figure 10F] This figure shows the retention of stem cell properties in MSCs. Flow cytometry analysis of F.HLA-DR. [Figure 10G] This figure shows the retention of stem cell properties in MSCs. Flow cytometry analysis of G.CD14. [Figure 11] This figure shows the MSC expression of CAR-MOG. [Figure 12A] A. Nucleic acid sequence encoding an exemplary CAR-ECAD (SEQ ID NO: 1). [Figure 12B] B. An example amino acid sequence of CAR-ECAD (SEQ ID NO: 2). [Figure 13A]A. Nucleic acid sequence encoding an example CAR-MOG (SEQ ID NO: 3). [Figure 13B] B. Amino acid sequence of an example CAR-MOG (SEQ ID NO: 4). [Figure 13C] C. Nucleic acid sequence encoding an example CAR-MOG (SEQ ID NO: 5). [Figure 13D] D. Amino acid sequence of an example CAR-MOG (SEQ ID NO: 6). [Figure 13E] E. Nucleic acid sequence encoding an exemplary CAR-MOG (SEQ ID NO: 7). [Figure 13F] F. Amino acid sequence of an exemplary CAR-MOG (SEQ ID NO: 8). [Figure 14A] A. Nucleic acid sequence encoding an exemplary CAR-HER2 (SEQ ID NO: 9). [Figure 14B] B. An example amino acid sequence of CAR-HER2 (SEQ ID NO: 10). [Figure 15] Figure 15A is a graph plotting the percentage of CAR-positive MSCs after lentiviral transduction with K002 (CD19-targeted CAR) compared with UTD (untransduced MSCs). Figure 15B is a graph plotting the percentage of MSCs expressing markers shown two days after lentiviral transduction with CAR19 compared with untransduced MSCs (UTD). MSCs retain stem cell-specific markers. [Figure 16]This graph plots the absolute number of surviving CD3 cells 5 days after stimulation with CD3 / CD28 beads, after co-culturing with or without MSCs, either with or with MSC-CAR19 (CD28 containing CAR19, K122), or MSC-CAR19 (CD137 containing CAR19, K002). T cells are activated with CD3 / CD28 beads and then co-culturing with different MSC conditions, either in the 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 MSC-CAR19 (containing the CD137 signaling domain) or non-transduced MSCs. T cells, MSCs, and NALM6 cells were cultured in a ratio of 1:0.1:1 E(T cells (effector):MSCs (suppressor):T (tumor)). This demonstrates that if MSC-CAR cells contain a CD28 signaling domain, and if MSCs are activated via CAR upon antigen-specific stimulation, MSC-CAR cells can also suppress T cell proliferation. * indicates a p-value ≤ 0.05. [Figure 17] This graph plots the absolute number of viable MSC cells after 5 days of co-culture of untransduced MSCs (MSC-UTD), MSC-CAR19 (TLR4 containing CAR19, K142), MSC-CAR19 (CD137 containing CAR19, K002), or MSC-CAR19 (CD28 containing CAR19, K122), regardless of the presence or absence of irradiated CD19+ NALM6 cells. The proliferation of CD28-containing MSC-CAR19 is enhanced in the presence of irradiated CD19+ cells but not in their absence, suggesting antigen-specific stimulation of MSC-CAR19 and signal transduction via CD28. Similarly, the proliferation of TLR4-containing MSC-CAR19 is reduced in the presence of irradiated CD19+ cells but not in their absence, suggesting antigen-specific stimulation of MSC-CAR19 and signal transduction via CD28. * represents P ≤ 0.05, and ** represents 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-culturing with or without untransduced MSCs (MSC-UTDs), or with MSC-CAR19 (TLR4 containing CAR19, K142), or without MSCs. T cells are activated with CD3 / CD28 beads and then co-culturing 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 TLR4 signaling domain, K142), but not in the presence of untransduced MSCs. T cells, MSCs, and NALM6 cells were cultured in a ratio of 1:0.1:1 E (T cells (effector):MSC (suppressor):T (tumor)). This demonstrates that MSC-CAR cells can suppress T cell proliferation if they contain a TLR4 signaling domain and if the MSCs are activated via CAR in response to antigen-specific stimulation. ** represents P ≤ 0.01. [Figure 19] Figure 19A is a graph plotting the absolute number of MSCs after 5 days of co-culture with irradiated CD19+ NALM6 cells in a 1:1 ratio of MSC:NALM6. NALM6 cells were included in this culture to stimulate MSC-CAR19 via CAR. Co-culture of CD19+ cells with MSC-CAR19-CD28 resulted in increased proliferation compared to untransduced MSCs (MSC-UTD), or MSC-K002, or MSC-K122, or MSC-K142. This indicates that antigen-specific stimulation of MSC-CAR19 via CAR leads to changes in MSC signaling and proliferation. Figure 19B shows the number of MSCs on days 3 and 5 of the co-culture. [Figure 20] This graph plots the absolute number of MSCs in cultures after transduction using CAR-MSC-E-cadherin or control GFP (MSC-ZSG). Transduction using CAR-E-cadherin-CD28 resulted in reduced MSC growth compared to untransduced MSCs (MSC-UTD) or MSCs transduced with GFP (MSC-ZSG). [Figure 21] This bar graph plots the number of T cells / μL after stimulation with CD3 / CD28 beads and CD19+ cells (to stimulate MSC-CAR) in the presence of MSCs, either by direct contact or in a Transwell experiment. Co-culture of activated T cells and CD19+ cells with MSC-CAR19 (containing the CD28 stimulating domain) resulted in inhibition of T cell proliferation, whether by direct cell contact or in a non-direct contact in a Transwell experiment. This indicates that MSC-CAR exerts its inhibitory function through direct contact, cell-to-cell contact, and the secretion of soluble inhibitors / cytokines. [Figure 22] This graph plots the number of T cells / μL 5 days after stimulation with CD3 / CD28 beads, following co-culture with untransduced MSCs or MSC-CAR19 (CD28 containing CAR19, K122) at a higher E:S:T ratio. T cells are activated with CD3 / CD28 beads and then co-cultured with different MSC conditions, either in the 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 in a 1:1:1 E (T cells (effector):MSC (suppressor):T (tumor)) ratio. This demonstrates that when MSCs are activated via CAR upon antigen-specific stimulation, MSC-CAR cells can suppress T cell proliferation if they contain the CD28 signaling domain at different suppressor-to-T cell ratios. ** represents P ≤ 0.01, and *** represents P ≤ 0.001. [Figure 23]This graph plots the absolute number of surviving CD3 cells 5 days after stimulation with CD3 / CD28 beads, after co-culturing 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 or without E-cadherin + cell line MCF-7. Co-culturing MSC-CAR-E-cadherin with T cells, in the presence of E-cadherin + cell line MCF-7, results in suppression of their antigen-specific proliferation at low effector:suppressor ratios. [Figure 24] Figure 24A is a graph plotting bioluminescence from luciferase+ / E-cadherin+ MCF-7 cells (1 × 10⁶) injected into NSG mice, and then treated with CAR-ECAD T cells in combination with either MSCs, non-transduced MSCs (MSC-UTDs), or MSC-ECAD-CARs possessing a CD28 signaling domain. Mice treated with MSC-ECAD showed greater 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 CAR-ECAD T cells and CD3+ T cells in NSG mice treated with no MSCs, non-transduced MSCs (MSC-UTDs), 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 representative mice. [Modes for carrying out the invention]

[0020] This document provides methods and materials for treating mammals (e.g., humans) that have or are at risk of developing inflammatory diseases or conditions, and / or have or are at risk of developing degenerative diseases. For example, one or more MSCs designed to express antigen receptors (e.g., CARs) that can bind to tissue-specific antigens (e.g., specifically) can be administered to a mammal to induce an immunosuppressive response in the mammal (e.g., reduce or eliminate an inflammatory immune response). In some cases, CARs that target epithelial-specific antigens (e.g., ECADs) can be expressed by MSCs to target epithelial tissue in an effective manner for treating diseases or disorders characterized by inflammation of epithelial tissue (e.g., colitis). For example, MSCs engineered to express CARs that can target (e.g., target and bind to) antigens expressed by epithelial cells (e.g., cell surface antigens) (e.g., epithelial-specific antigens or epithelial antigens) can be administered to mammals (e.g., humans) to treat or delay the progression of diseases or disorders characterized by inflammation and / or degeneration of epithelial tissue, such as colitis, hepatitis, pneumonia, asthma, pancreatitis, pulmonary fibrosis, colonic stenosis, colostomy, glomerulonephritis, renal infarction, or hepatic infarction.

[0021] In some cases, CARs that target nerve-specific antigens (e.g., MOG) can be expressed by MSCs and target MSCs to nerve tissue in an effective manner for treating diseases or disorders characterized by inflammation of nerve tissue (e.g., multiple sclerosis and immune-mediated encephalomyelitis). For example, MSCs engineered to express CARs that can target (e.g., target and bind to) antigens expressed by nerve cells (e.g., cell surface antigens) (e.g., nerve-specific antigens or nerve antigens) can be administered to mammals (e.g., humans) to treat or slow the progression of diseases or disorders characterized by inflammation and / or degeneration of nerve tissue, such as multiple sclerosis, immune-mediated encephalomyelitis, or amyotrophic lateral sclerosis.

[0022] In some cases, CARs that target cardiac-specific antigens (e.g., HER2) can be expressed by MSCs to target MSCs to cardiac tissue in an effective manner for treating diseases or disorders characterized by inflammation of cardiac tissue (e.g., myocarditis). For example, MSCs engineered to express CARs that can target (e.g., target and bind) antigens expressed by cardiac cells (e.g., cell surface antigens) (e.g., cardiac-specific antigens or cardiac antigens) can be administered to mammals (e.g., humans) to treat or slow the progression of diseases or disorders characterized by inflammation and / or degeneration of cardiac tissue, such as myocarditis, myocardial infarction, fever failure, or endocarditis.

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

[0024] In some cases, cartilage antigens (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 osteocyte antigens (e.g., E11 or gp38)) can be administered to mammals (e.g., humans) (e.g., by adoptive transfer) to treat or slow the progression of diseases or disorders characterized by inflammation and / or degeneration of the joints, 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., CAR) that can bind to a tissue-specific antigen (e.g., specifically), can also be engineered to express a polypeptide that can promote differentiation into tissue-specific cells (e.g., cardiac cells or neurons) within a mammal. In some cases, MSCs engineered to express a polypeptide that can promote 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 that can promote 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 that can promote differentiation into any suitable tissue-specific cells (e.g., cardiac cells or neurons) within a mammal. In some cases, MSCs containing nucleic acids encoding a polypeptide that can promote cardiac cell differentiation can differentiate MSCs into cardiac cells. In some cases, MSCs containing nucleic acids encoding a polypeptide that can promote neuronal differentiation can differentiate MSCs into neurons. For example, one or more MSCs designed to express an antigen receptor (e.g., CAR) that can bind to a tissue-specific antigen (e.g., specifically), and also designed to differentiate into tissue-specific cells, can be administered (e.g., by adoptive transfer) to a mammal (e.g., human) that has or is at risk of developing a degenerative disease, to treat that degenerative disease within the mammal. In some cases, one or more MSCs designed to differentiate into cardiac cells (e.g., one or more MSCs containing nucleic acids that can encode polypeptides that can promote cardiac cell differentiation) can be administered (e.g., by adoptive transfer) to a mammal (e.g., human) that has or is at risk of developing a degenerative heart disease (e.g., congestive heart failure (CHF)), to treat that degenerative heart disease within the mammal (e.g., by regenerating cardiac cells within the mammal).In some cases, one or more MSCs designed to differentiate into nerve cells (e.g., one or more MSCs containing nucleic acids capable of encoding polypeptides that can promote neuronal differentiation) can be administered to mammals (e.g., humans) that have or are at risk of developing neurodegenerative diseases (e.g., Parkinson's disease and Alzheimer's disease) to treat those neurodegenerative diseases within the mammal (e.g., by regenerating neurons within the mammal).

[0026] In some cases, MSCs (e.g., MSCs engineered to express CARs as 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, MSCs can be administered to mammals (e.g., humans) to treat or slow the progression of diseases or disorders characterized by cardiac degeneration, such as myocardial infarction or heart failure.

[0027] In some cases, MSCs (e.g., MSCs designed to express CARs as described herein) can be designed to express one or more transcription factors that promote their differentiation into osteocytes (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 (e.g., BMP-2, BMP-3, BMP-5, BMP-7, N-cadherin, NCAN-1, and / or perlecan). In such cases, MSCs can be administered to mammals (e.g., humans) to treat or slow the progression of diseases or disorders characterized by bone degeneration, such as degenerative arthritis or osteogenesis imperfecta.

[0028] MSCs described herein (for example, MSCs that express CARs that target tissue-specific antigens, e.g., epithelial-specific antigens, neuronal-specific antigens, or cardiac-specific antigens, and optionally express polypeptides 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) may be any suitable MSCs. Examples of MSCs that can be used as described herein include, but are not limited to, adipose-derived MSCs, osteoblasts (osteocytes), 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 CARs and optionally express polypeptides that can promote the differentiation of MSCs into tissue-specific cells that target tissue-specific antigens may be adipose-derived MSCs.

[0029] A CAR may include an antigen-binding domain and a signaling domain. The antigen-binding domain may be any suitable antigen-binding domain. In some cases, the antigen-binding domain may include an antibody or fragment thereof that targets an antigen (e.g., a tissue-specific antigen, e.g., an epithelial-specific antigen, a nerve-specific antigen, or a cardiac-specific antigen). Examples of antigen-binding domains, but not limited to, include antigen-binding fragments (Fab), variable regions (VH) of antibody heavy chains, variable regions (VL) of light chains, single-chain variable fragments (scFv), polypeptides, ligands, and cytokines. In some cases, the antigen-binding domain may target (e.g., be able to target and bind) a tissue-specific antigen (e.g., an epithelial-specific antigen, a nerve-specific antigen, or a cardiac-specific antigen). For example, the MSCs described herein may express (e.g., be manipulated 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, MSCs can be engineered to express CARs that can target (e.g., can target and bind) antigens (e.g., cell surface antigens) (e.g., epithelial-specific antigens or epithelial antigens) expressed by epithelial cells in mammals (e.g., mammals that have or are at risk of developing diseases or disorders 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, surfactants, trans tyretin, VAT-1, and vimentin. For example, an MSC-CAR engineered to target epithelial tissue can bind to ECAD. In some cases, an MSC-CAR engineered to express CAR-ECAD can be engineered to target ECAD expressed by epithelial cells in mammals that have or are at risk of developing IBD (e.g., colitis).

[0031] In some cases, MSCs can be engineered to express CARs that can target (e.g., can target and bind) antigens (e.g., cell surface antigens) (e.g., neuron-specific antigens or neuronal antigens) expressed by nerve cells in mammals (e.g., mammals that have or are at risk of developing diseases or disorders characterized by inflammation and / or degeneration of nerve tissue, e.g., multiple sclerosis and immune-mediated encephalomyelitis). If the antigen is a neuron-specific antigen, the neuron-specific antigen can be any suitable neuron-specific antigen. The neuron-specific antigen can be expressed on any suitable type of nerve cell (e.g., sensory neurons, motor neurons, interneurons, glial cells, and oligodendrocytes). The neuron-specific antigen can be expressed on nerve cells in the central nervous system (CNS) and / or peripheral nervous system (PNS). In some cases, the neuron-specific antigen can be a transmembrane protein. Examples of nerve-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 nerve tissue can bind to MOG. In some cases, an MSC-CAR can be engineered to express a CAR-MOG that targets MOG expressed by nerve cells in mammals with or at risk of developing multiple sclerosis. In other cases, an MSC-CAR can be engineered to express a CAR-MOG that targets MOG expressed by nerve cells in mammals with 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., can target and bind to) antigens (e.g., cell surface antigens) (e.g., cardiac-specific antigens or cardiac antigens) expressed by cardiac cells in mammals (e.g., mammals that have or are at risk of developing a disease or disorder characterized by inflammation and / or degeneration of cardiac tissue, e.g., myocarditis). If 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 a CAR-HER2 that targets HER2 expressed by cardiac cells in mammals that have or are at risk of developing myocarditis.

[0033] The signaling domain can be any suitable signaling domain. In some cases, the signaling domain of the CAR used in the MSCs described herein may be an intracellular signaling domain commonly found in T cells or NK cells. Examples of signaling domains that can be used as described herein include, but are not limited to, CD3 zeta signaling domains, CD28 signaling domains, Toll-like receptors (TLRs) (e.g., TLR3 and TLR4), 4-1BB, OX40, ICOS, CD2, and promoters (e.g., specific promoters, e.g., cardiac-specific promoter, neuronal-specific promoter, T-cell-specific promoter, GI, lung-specific promoter, joint-specific promoter, and cartilage-specific promoter). In some cases, the signaling domain of the CAR expressed by the MSCs described herein can induce the MSC to produce one or more cytokines (e.g., anti-inflammatory cytokines) upon CAR activation. Examples of cytokines that can be induced in MSCs by the manipulated CARs of MSCs described herein 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 the CARs described herein (e.g., CARs targeting tissue-specific antigens, e.g., epithelial-specific antigens, nerve-specific antigens, or cardiac-specific antigens) on the surface of the MSCs described herein. For example, nucleic acids encoding the CAR can be introduced into the MSCs. In some cases, nucleic acids encoding the CAR can be introduced into the MSCs by transduction (e.g., viral transduction) or transfection. In some cases, nucleic acids encoding the CAR described herein can be introduced ex vivo into one or more MSCs. For example, ex vivo manipulation of MSCs to express the CAR described herein may include transducing isolated MSCs using a lentiviral vector encoding the CAR. When MSCs are manipulated ex vivo to express the CAR, the MSCs can be obtained from any suitable source (e.g., mammals, e.g., the mammal being treated, or a donor mammal, or a cell line). In some cases, the MSC-CAR can be prepared as described herein (see, for example, Figure 2 and Example 1). For example, by introducing one or more constructs containing nucleic acids encoding CAR (e.g., CAR-targeted ECAD) into MSCs, CAR-ECAD can be expressed in the MSCs, thereby directing the MSCs to epithelial tissue. For example, by introducing one or more constructs containing nucleic acids encoding CAR (e.g., CAR-targeted MOG) into MSCs, CAR-MOG can be expressed in the MSCs, thereby directing the MSCs to nerve tissue. In some cases, MSC-CAR can be prepared as described elsewhere (see, for example, Blat et al., Mol. Ther., vol. 22(no. 5): pp. 1018-28 (2014); MacDonald et al., J. Clin. Invest., vol. 126(no. 4): pp. 1413-24 (2016); and Yoon et al., Blood, vol. 129(no. 2): pp. 238-245 (2017)).

[0035] Furthermore, this specification provides CARs and constructs (e.g., nucleic acid constructs) encoding CARs (e.g., CARs that target tissue-specific antigens, e.g., epithelial-specific antigens, nerve-specific antigens, or cardiac-specific antigens) as described herein. For example, a construct encoding a CAR-targeted ECAD (e.g., CAR-ECAD) may include nucleic acid sequences encoding one or more molecules that bind to ECAD as described herein. In some cases, CAR-ECAD may include 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) may include nucleic acid sequences encoding one or more molecules that bind to MOG as described herein. In some cases, CAR-MOG may include 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) may include nucleic acid sequences encoding one or more molecules that bind to HER2 as described herein. In some cases, CAR-HER2 may 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 may be included in a construct described herein and that may encode one or more molecules that bind to a tissue-specific antigen as described herein include, but are not limited to, those encoding 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 a bold / underlined linker, followed by a heavy chain) TIFF2026086575000002.tif25150 Anti-ECAD scFv clone 6 (SEQ ID NO: 56; light chain, followed by bold / underlined linker, followed by heavy chain) TIFF2026086575000003.tif25150 Anti-ECAD scFv clone 7 (SEQ ID NO: 57; heavy chain, followed by bold / underlined linker, followed by light chain) TIFF2026086575000004.tif25150 Anti-ECAD scFv clone 14 (SEQ ID NO: 58; light chain, followed by bold / underlined linker, followed by heavy chain) TIFF2026086575000005.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 that can bind to a tissue-specific antigen described herein and can be encoded by a nucleic acid sequence that may be included in a construct described herein may have a sequence that deviates from the polypeptide sequence (e.g., a light-chain polypeptide sequence or a heavy-chain polypeptide sequence) shown in any one of SEQ ID NOs. 11-17 (sometimes called a mutant sequence). For example, an amino acid segment that can bind to a tissue-specific antigen described herein and can be encoded by a nucleic acid sequence that may be included in a construct described herein may have at least 80% sequence identity with any one of SEQ ID NOs. 11-17. In some embodiments, an amino acid segment that can bind to a tissue-specific antigen described herein and can be encoded by a nucleic acid sequence that may be included in a construct described herein may have at least 85%, 90%, 95%, or at least 99% sequence identity with any one of SEQ ID NOs. 11-17. The sequence identity percentage is calculated by determining the number of matching positions in the aligned polypeptide sequence, dividing the number of matching positions by the total number of aligned amino acids, and multiplying by 100. Matched positions refer to the positions in the aligned sequences where the same amino acid exists at the same position. The total number of aligned amino acids refers to the minimum number of amino acids in an amino acid segment that can bind to the tissue-specific antigen described herein, are necessary for aligning a second sequence, and do not involve alignment with other sequences (e.g., forced alignment), and can be encoded by a nucleic acid sequence that can be included in the construct described herein. For example, if the amino acid segment that can bind to the 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 chains.The total number of aligned amino acids may correspond to an entire amino acid segment that can bind to a tissue-specific antigen described herein and may be encoded by a nucleic acid sequence that may be included in a construct described herein, or that can bind to a tissue-specific antigen described herein and may be encoded by a nucleic acid sequence that may be included in a construct described herein. Sequences can be aligned using the algorithm described by Altschul et al. (Nucleic Acids Res., Vol. 25: pp. 3389-3402 (1997)), which is incorporated into the BLAST (Basic Local Alignment Search Tool) program, available on the World Wide Web at ncbi.nlm.nih.gov. Sequence identity percentages can be determined by performing a BLAST search or alignment using Altschul et al.'s algorithm. BLASTN is a program used to align and compare the identity of nucleic acid sequences, and BLASTP is a program used to align and compare the identity of amino acid sequences. When using the BLAST program to calculate the percentage of identity between nucleic acid sequences and amino acid segments that can be encoded by other sequences and that may be included in the constructs described herein, the default parameters of each program are used.

[0038] The following are exemplary nucleic acid sequences that may be included in the constructs described herein and that may encode one or more molecules that bind to the tissue-specific antigens described herein:

[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 a bold / underlined linker, followed by a heavy chain) TIFF2026086575000006.tif30150TIFF2026086575000007.tif44150 Anti-ECAD scFv clone 6 (SEQ ID NO: 60; light chain, followed by bold / underlined linker, followed by heavy chain) TIFF2026086575000008.tif71150 Anti-ECAD scFv clone 7 (SEQ ID NO: 61; heavy chain, followed by bold / underlined linker, followed by light chain) TIFF2026086575000009.tif70150 Anti-ECAD scFv clone 14 (SEQ ID NO: 62; light chain, followed by bold / underlined linker, followed by heavy chain) TIFF2026086575000010.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 GAGGTTCAGCTGGGGAGTCTGGCGGTGGCCTGGTGCAGCCCGGGGGCTCTCTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATTAAAGACACCTATATCCACTGGGTGCGTCAGGCTCCGGGTAAGGGCCTGGAGTGGGTTGCAAGGATTTATCCTACGAATGGTTATACTCGTTATGCCGATAGCGTCAAGGGCGTTCACTATAAGCGCAGACACTTCGAAAAAACACAGCCTACCTCCAGAATGAACAGC CTGCGTGCTGAGGACACTGCCGTCTATTATTGTAGCAGATGGGGTGGGGACGGCTTCTATGCTATGGACTACTGGGGTCAAGGTACACTAGTCACCGTCAGCAGCGCTAGCACCAAGGGCACCACGACGCCAGCGCCGCGACCACAACACCGGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT Anti-HER2 antibody GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGGGCGGATAGGGTCACTATCACCTGCCGTGCCAGTCAGGATGTGAATACTGCTGTAGCCTGGTATCAACAGAAACCCGGAAAGGCCCGAAACTGCTGATTTACTCGGCATCCTTCCCTACTCTG GAGTCCCTTCTCGCTTCTCTGGTTCCCGCTCTGGGACGGATTTCACTCTGACCATCAGCTCCCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAACACTATACTACTCTCCGACGTTCGGACAGGGTACCAAGGTGGAGATCAAACGTACCGTGGCGGCCCCA

[0040] The CARs described herein (e.g., CAR-ECAD, CAR-MOG, or CAR-HER2) may also contain one or more additional components. Examples of additional components that may be included in a CAR include, but are not limited to, leader sequences (e.g., CD8 leader sequences), hinges (e.g., CD8 hinges and CD28 hinges), transmembrane domains (e.g., CD8 transmembrane domains and CD28 transmembrane domains), co-stimulatory signaling domains (e.g., those that may increase immunosuppression, e.g., TLR3 signaling domain and / or TLR4 signaling domain), Toll / interleukin-1 receptor / resistance (TIR) ​​interaction sequences (e.g., TLR3 TIR interaction domain and / or TLR4 TIR interaction domain), intracellular TLR domains (e.g., TLR intracellular linkers), and transmembrane domains (e.g., TLR3 transmembrane domain and / or TLR4 transmembrane domains). In some cases, nucleic acids encoding components of a CAR-encoding construct can be separated from nucleic acids encoding other components using one or more linkers. The nucleic acids in a CAR-encoding construct may be present in any suitable order. In some cases, a CAR can be designed to include an scFv that is light-chain to heavy-chain oriented or heavy-chain to light-chain oriented, using any suitable interchain linker. For example, a construct encoding CHD1-CAR can be generated with an scFv in a light-chain to heavy-chain orientation, or with an scFv in a heavy-chain to light-chain orientation. In some cases, a CAR can be designed to include an scFv that is light-chain to heavy-chain oriented or heavy-chain to light-chain oriented, without an interchain linker. Exemplary nucleic acid sequences that may encode one or more additional components that may be contained in a CAR that may be contained in a construct described herein include, but are not limited to, the following amino acid sequences:

[0041] CD8 reader array (sequence number 26) MALPVTALLLPLALLLHAARP Linker (Sequence ID 27) GGGGSGGGGSGGGGS CD8 hinge (Sequence No. 28) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD CD28 hinge (sequence number 29) LEPKSCDKTHTCPPCPDPK TLR long chain (long-chain peptide derived from Toll-like receptors; SEQ ID NO: 46) NDFACTCEHQSFLQWIKDQRQLLVEVERMECATPSDKQGMPVLSLNITCQMNKTI TLR short chain (short-chain peptide derived from Toll-like receptors; 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 incorporated into a construct described herein may have sequences that deviate from the polypeptide sequences shown in any one of SEQ ID NOs. 26-36 and 46-49, and are sometimes referred to as mutant sequences. For example, an additional component that can be encoded by a nucleic acid sequence that can be included in a CAR and incorporated into a construct described herein may have at least 80% sequence identity with any one of SEQ ID NOs. 26-36 and 46-49. In some embodiments, an additional component that can be encoded by a nucleic acid sequence that can be included in a CAR and incorporated into a construct described herein may have at least 85%, 90%, 95%, or at least 99% sequence identity with any one of SEQ ID NOs. 26-36 and 46-49. The sequence identity percentage is calculated by determining the number of matching positions in the aligned polypeptide sequence, dividing the number of matching positions by the total number of aligned amino acids, and multiplying by 100. Matching positions refer to positions in the aligned sequence where identical amino acids exist at the same position. 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 construct described herein, which can be included in the CAR, is necessary for aligning a second sequence, and does not involve alignment with other sequences (e.g., forced alignment). For example, if the additional component that can be included in a CAR and can be included in a construct described herein, which can be encoded by a nucleic acid sequence, is a signaling domain, the total number of aligned amino acids can exclude any transmembrane domain. The total number of aligned amino acids may correspond to an entire amino acid segment of the additional component that can be included in a CAR and can be included in a construct described herein, or to a fragment of an amino acid segment of the additional component that can be included in a CAR and can be included in a construct described herein.Sequences can be aligned using the algorithm described by Altschul et al. (Nucleic Acids Res., Vol. 25: pp. 3389-3402 (1997)), which is incorporated into the BLAST (Basic Local Alignment Search Tool) program, available on the World Wide Web at ncbi.nlm.nih.gov. Using Altschul et al.'s algorithm, a BLAST search or alignment can be performed to determine the sequence identity percentage. BLASTN is a program used for aligning and comparing the identity of nucleic acid sequences, and BLASTP is a program used for aligning and comparing the identity of amino acid sequences. When using the BLAST program to calculate the identity percentage between nucleic acid sequences that can bind to the tissue-specific antigens described herein and that may be included in the constructs described herein, and between amino acid segments that may be encoded by other sequences, the default parameters of each program are used.

[0043] The following are some example nucleic acid sequences of some additional components that may be included in CAR and may be included in the constructs described herein:

[0044] CD8 reader array (sequence number 37) ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG Linker (Sequence ID 38) GGTGGAGGTGGTTCGGGAGGTGGAGGTAGCGGAGGTGGTGGATCT Linker (Sequence ID 50) GGTGGAGGTGGTTCGGGAGGTGGAGGTAGCGGAGGTGGTGGAAGC CD8 hinge (Sequence No. 39) ACCACTACCCCTGCACCGCGACCACCAACACCGGCGCCCACCATTGCGTCGCAGCCTCTGTCCCTGCGCCCAGAAGCATGCCGTCCAGCAGCAGGTGGTGCAGTTCATACTCGTGGTCTGGATTTCGCCTGTGAT CD28 hinge (Sequence number 40) CTCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCGGATCCCAAA TLR length (Sequence ID 51) AACGACTTCGCCTGCACCTGCGAGCACCAGAGCTTCCTGCAGTGGATCAAGGACCAGAGGCAGCTGCTGGTGGAGGTGGAGAGGATGGAGTGCGCCACCCCCAGCGACAAGCAGGGCATGCCCGTGCTGAGCCTGAACATCACCTGCCAGATGAACAAGACCATC TLR short (sequence number 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, the nucleic acid constructs encoding CAR-ECAD described herein can be designed to encode a CD8 reader sequence, an anti-ECAD antibody (e.g., hSC10.17 antibody) heavy chain, a linker, an anti-ECAD antibody (e.g., 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 CAR-ECAD can be designed to encode a CD8 reader containing the amino acid sequence shown in SEQ ID NO: 26, an hSC10.17 heavy chain containing the amino acid sequence shown in SEQ ID NO: 11, a linker containing the amino acid sequence shown in SEQ ID NO: 27, an hSC10.17 light chain containing the amino acid sequence shown in SEQ ID NO: 12, a CD8 hinge containing the amino acid sequence shown in SEQ ID NO: 28, a CD8 transmembrane domain containing the amino acid sequence shown in SEQ ID NO: 30, and a CD3 zeta signaling domain containing the amino acid sequence shown in SEQ ID NO: 33.

[0046] In some cases, the nucleic acid constructs encoding CAR-MOG as described herein can be designed to encode a CD8 reader sequence, 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, a CD8 hinge, a CD28 transmembrane domain, and a CD3 zeta signaling domain. For example, a nucleic acid construct encoding CAR-MOG can be designed to encode a CD8 reader containing the amino acid sequence shown in SEQ ID NO: 26, an 8-18C5 heavy chain containing the amino acid sequence shown in SEQ ID NO: 14, a linker containing the amino acid sequence shown in SEQ ID NO: 27, an 8-18C5 light chain containing the amino acid sequence shown in SEQ ID NO: 15, a CD8 hinge containing the amino acid sequence shown in SEQ ID NO: 28, a CD8 transmembrane domain containing the amino acid sequence shown in SEQ ID NO: 30, and a CD3 zeta signaling domain containing the amino acid sequence shown in SEQ ID NO: 33.

[0047] In some cases, the nucleic acid constructs encoding CAR-HER2 described herein may be designed to encode a CD8 reader 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 CAR-HER2 may be designed to encode a CD8 reader containing the amino acid sequence shown in SEQ ID NO: 26, a 4D5 heavy chain containing the amino acid sequence shown in SEQ ID NO: 16, a linker containing the amino acid sequence shown in SEQ ID NO: 27, a 4D5 light chain containing the amino acid sequence shown in SEQ ID NO: 17, a CD28 hinge containing the amino acid sequence shown in SEQ ID NO: 29, a CD8 transmembrane domain containing the amino acid sequence shown in SEQ ID NO: 30, a 4-1BB signaling domain containing the amino acid sequence shown in SEQ ID NO: 34, and a CD3 zeta signaling domain containing the amino acid sequence shown in SEQ ID NO: 33.

[0048] In some cases, nucleic acid constructs encoding CAR-ECAD, CAR-MOG, or CAR-HER2 as described herein may be designed to encode (a1) an anti-ECAD antibody (e.g., hSC10.17 antibody) heavy chain, a linker, an anti-ECAD antibody (e.g., hSC10.17 antibody) light chain, (a2) an anti-MOG antibody (e.g., 8-18C5 antibody) heavy chain, a linker, an anti-MOG antibody (e.g., 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 an intracellular TLR4 domain.

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

[0050] In some cases, nucleic acid constructs encoding CAR-ECAD, CAR-MOG, or CAR-HER2 as described herein can be designed to encode (a1) an anti-ECAD antibody (e.g., hSC10.17 antibody) heavy chain, a linker, an anti-ECAD antibody (e.g., hSC10.17 antibody) light chain, (a2) an anti-MOG antibody (e.g., 8-18C5 antibody) heavy chain, a linker, an anti-MOG antibody (e.g., 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., the linker encoded by Sequence ID No. 50, (c) a TLR4 transmembrane domain, and (d) a TLR4 signaling domain or an intracellular TLR4 domain.

[0051] In some cases, nucleic acid constructs encoding CAR-ECAD, CAR-MOG, or CAR-HER2 as described herein may be designed to encode (a1) an anti-ECAD antibody (e.g., hSC10.17 antibody) heavy chain, linker, and anti-ECAD antibody (e.g., hSC10.17 antibody) light chain, (a2) an anti-MOG antibody (e.g., 8-18C5 antibody) heavy chain, linker, and anti-MOG antibody (e.g., 8-18C5 antibody) light chain, or (a3) ​​an anti-HER2 antibody (e.g., 4D5) heavy chain, linker, and 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 an intracellular TLR4 domain.

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

[0053] In some cases, nucleic acid constructs encoding CAR-ECAD, CAR-MOG, or CAR-HER2 as described herein can be designed to encode (a1) an anti-ECAD antibody (e.g., hSC10.17 antibody) heavy chain, a linker, an anti-ECAD antibody (e.g., hSC10.17 antibody) light chain, (a2) an anti-MOG antibody (e.g., 8-18C5 antibody) heavy chain, a linker, an anti-MOG antibody (e.g., 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, nucleic acid constructs encoding CAR-ECAD, CAR-MOG, or CAR-HER2 as described herein can be designed to encode (a1) an anti-ECAD antibody (e.g., hSC10.17 antibody) heavy chain, a linker, an anti-ECAD antibody (e.g., hSC10.17 antibody) light chain, (a2) an anti-MOG antibody (e.g., 8-18C5 antibody) heavy chain, a linker, an anti-MOG antibody (e.g., 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, if an MSC designed to express an antigen receptor (e.g., CAR) capable of binding to a tissue-specific antigen (e.g., specifically binding) is also designed to express a polypeptide capable of promoting differentiation into tissue-specific cells, the MSC may contain 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 may encode any polypeptide that can promote the differentiation of the MSC into any type of tissue-specific cell. For example, an MSC may be designed to contain one or more nucleic acids encoding a polypeptide capable of promoting cardiac cell differentiation. A polypeptide capable of promoting cardiac differentiation can promote differentiation into any suitable type of cardiac cell (e.g., cardiomyocyte). Examples of polypeptides capable of promoting cardiac cell differentiation that 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, an MSC may be designed to contain one or more nucleic acids encoding a polypeptide capable of promoting neuronal differentiation. Polypeptides that can promote 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 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 mammals (e.g., humans) that have or are at risk of developing diseases or disorders characterized by tissue inflammation and / or degeneration. 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 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) can be administered (e.g., by adoptive transfer) to mammals that have or are at risk of developing diseases or disorders characterized by tissue inflammation and / or degeneration in order to reduce the severity of inflammation of targeted tissues within the mammal. Mammals can be identified as having or being at risk of developing inflammatory diseases or conditions, and / or having or being at risk of developing degenerative diseases, using any appropriate method. For example, imaging techniques (e.g., ultrasound, computed tomography (CT) scanning) and 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 being at risk of developing inflammatory diseases or conditions, and / or having or being at risk of developing degenerative diseases.When identified as having (or at risk of developing) an inflammatory disease or condition and / or a degenerative disease, one or more MSCs expressing a CAR that targets a tissue-specific antigen and optionally a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells may be administered as described herein to mammals (e.g., humans) (e.g., humans who have or are at risk of developing a disease or disorder characterized by inflammation and / or degeneration of tissues expressing a tissue-specific antigen) that need it, for example, to reduce tissue inflammation by 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent or more. For example, one or more MSCs expressing a polypeptide that can promote the differentiation of MSCs into any type of tissue-specific cell (e.g., cardiac cells or neurons) may be administered (e.g., by adoptive transfer) to mammals (e.g., humans) who have (or are at risk of developing) a degenerative heart disease to treat that degenerative heart disease within the mammal. Mammals can be identified as having or being at risk of developing a degenerative disease by any appropriate method. 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 mammals as having or being at risk of developing a degenerative disease. Once identified as having (or being at risk of developing) a degenerative disease, one or more MSCs expressing polypeptides that can promote differentiation of MSCs into any type of tissue-specific cells can be administered to mammals in need (e.g., humans) (e.g., humans with 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 may 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, cattle, 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 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) can be administered to humans having or at risk of developing a disease or disorder characterized by tissue inflammation and / or degeneration (e.g., by adoptive transfer) for treatment of humans.

[0058] In some cases, mammals (e.g., humans) that have or are at risk of developing diseases or disorders characterized by inflammation and / or degeneration of epithelial tissues can be treated using materials and methods for treating mammals (e.g., humans) that have or are at risk of developing diseases or disorders characterized by inflammation and / or degeneration of epithelial tissues. Examples of diseases and disorders characterized by inflammation and / or degeneration of epithelial tissues 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 mucosa lining the gastrointestinal tract), colofistria, graft-versus-host disease, and inflammatory pneumonia (e.g., characterized by inflammation of lung cells). When treating mammals with colitis (or at risk of developing it), the colitis may be any type of colitis (e.g., ulcerative colitis, clonal colitis, circumventing colitis, ischemic colitis, infectious colitis, fulminant colitis, collagen colitis, chemical colitis, microscopic colitis, lymphocytic colitis, and atypical colitis).

[0059] In some cases, mammals can be identified as having or being at risk of developing a disease or disorder characterized by inflammation and / or degeneration of epithelial tissue (e.g., IBD such as colitis). Mammals can be identified as having or being at risk of developing a disease or disorder characterized by inflammation and / or degeneration of epithelial tissue using any appropriate method. For example, mammals can be identified as having or being at risk of developing a disease or disorder characterized by inflammation and / or degeneration of epithelial tissue using blood tests (e.g., for signs of anemia or infection), laboratory tests (e.g., for leukocytes in mammalian feces), imaging techniques (e.g., colonoscopy, flexible sigmoidoscopy, X-ray, CT scan, CT colonoscopy, and magnetic resonance (MR) colonoscopy), biopsies, skin biopsies, and / or liver function tests. When identified as having (or at risk of developing) a disease or disorder characterized by inflammation and / or degeneration of epithelial tissue, the mammal may be treated by administering (e.g., by adoptive transfer) or by being directed to self-administer one or more MSCs described herein (e.g., MSCs expressing CARs that target epithelial-specific antigens and optionally polypeptides that can promote the differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells) (e.g., by adoptive transfer) to reduce or eliminate inflammation of one or more epithelial tissues within the mammal.

[0060] As described herein, when treating mammals 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) (for example, by administering one or more MSCs expressing a CAR that targets epithelial-specific antigens and optionally a polypeptide that can promote differentiation into tissue-specific cells), one or more MSCs expressing a CAR that targets epithelial-specific antigens and optionally a polypeptide that can promote differentiation of the MSC and / or one or more resident progenitor cells into tissue-specific cells may be effective in reducing the severity of diseases or disorders characterized by inflammation and / or degeneration of epithelial tissue in mammals. When colitis is a disease or disorder characterized by inflammation and / or degeneration of epithelial tissue, reducing the severity of colitis in mammals may include reducing or eliminating one or more symptoms of colitis (e.g., diarrhea, abdominal pain and cramps, rectal pain, rectal bleeding, urgency of defecation, inability to defecate despite urgency, weight loss, fatigue, fever, jaundice, liver failure, abnormal liver test results, dyspnea, skin erythema, and / or peeling). For example, one or more MSCs expressing CARs that target epithelial-specific antigens and optionally polypeptides that can promote the differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells may be administered to mammals (e.g., humans) in need, as described herein, to reduce the severity of one or more symptoms of colitis by, for example, 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 a polypeptide that can promote differentiation into tissue-specific cells (for example, a composition containing one or more MSCs expressing a CAR that targets an epithelial-specific antigen and optionally a polypeptide that can promote differentiation of the MSC and / or one or more resident progenitor cells into tissue-specific cells) may be the sole active ingredient for treating mammals 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) (for example, by administering one or more MSCs expressing a CAR that targets an epithelial-specific antigen and optionally a polypeptide that can promote differentiation of the MSC and / or one or more resident progenitor cells into tissue-specific cells), as described herein.

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

[0063] Materials and methods for treating mammals (e.g., humans) that have or are at risk of developing diseases or disorders characterized by inflammation and / or degeneration of tissues may be used to treat mammals (e.g., humans) that have or are at risk of developing diseases or disorders characterized by inflammation and / or degeneration of nerve tissues. Examples of diseases and disorders characterized by inflammation and / or degeneration of nerve tissues 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 mammals that have or are at risk of developing immune-mediated encephalomyelitis, immune-mediated encephalomyelitis may be any type of immune-mediated encephalomyelitis (e.g., acute disseminated encephalomyelitis (ADEM) and paraneoplastic encephalitis).

[0064] In some cases, mammals may be identified as having or being at risk of developing a disease or disorder characterized by inflammation and / or degeneration of nerve tissue (e.g., multiple sclerosis or immune-mediated encephalomyelitis). Mammals having (or being at risk of developing) a disease or disorder characterized by inflammation and / or degeneration of nerve tissue can be identified using any appropriate method. For example, clinical tests, e.g., blood tests and lumbar punctures (e.g., to examine biomarkers associated with specific diseases or disorders characterized by inflammation and / or degeneration of nerve tissue, e.g., multiple sclerosis, and to examine the presence of antibodies associated with specific diseases or disorders characterized by inflammation and / or degeneration of nerve tissue, e.g., anti-MOG autoantibodies), imaging techniques (e.g., magnetic resonance imaging (MRI); to examine the presence of lesions on the brain and / or spinal cord), cerebrospinal fluid analysis for protein levels, and / or cell counts for specific proteins can be used to identify mammals having or being at risk of developing a disease or disorder characterized by inflammation and / or degeneration of nerve tissue, e.g., multiple sclerosis or immune-mediated encephalomyelitis. When identified as having (or at risk of developing) a disease or disorder characterized by inflammation and / or degeneration of nerve tissue, the mammal may be treated by administering (e.g., by adoptive transfer) or by being directed to self-administer one or more MSCs described herein (e.g., one or more MSCs expressing CARs that target nerve-specific antigens and optionally polypeptides that can promote the differentiation of MSCs and / or one or more resident progenitor cells into neurons) (e.g., by adoptive transfer) (e.g., to reduce or eliminate inflammation in one or more nerve tissues within the mammal and / or to regenerate nerve tissue within the mammal).

[0065] As described herein, when mammals having (or at risk of developing) a disease or disorder characterized by inflammation and / or degeneration of nerve tissue (e.g., multiple sclerosis or immune-mediated encephalomyelitis) are treated (for example, by administering one or more MSCs expressing a CAR that targets nerve-specific antigens and optionally a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into neurons), one or more MSCs expressing a CAR that targets nerve-specific antigens and optionally a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into neurons may be effective in reducing the severity of diseases or disorders characterized by inflammation and / or degeneration of nerve tissue in mammals. If multiple sclerosis is a disease or disorder characterized by inflammation and / or degeneration of nerve tissue, reducing the severity of multiple sclerosis in mammals may 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., occurring with certain neck movements, especially bending the neck forward), tremors, lack of coordination, unsteady gait, partial or complete vision loss, long-term double vision, blurred vision, slurred speech, fatigue, dizziness, tingling or pain in a part of the body, sexual dysfunction, bowel dysfunction, and / or bladder dysfunction) and / or one or more complications associated with multiple sclerosis (e.g., muscle stiffness, muscle spasms, paralysis, memory loss, mood swings, depression, epilepsy, focal weakness, and / or visual impairment).For example, one or more MSCs expressing a CAR that targets a neuron-specific antigen and optionally a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into neurons may be administered as described herein to mammals (e.g., humans) (e.g., humans who have or are at risk of developing a disease or disorder characterized by inflammation and / or degeneration of nerve tissue) that need it to reduce the severity of one or more symptoms of a disease or disorder characterized by inflammation and / or degeneration of nerve tissue, and / or the severity of one or more complications associated with the disease or disorder characterized by inflammation and / or degeneration of nerve tissue, for example by 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 nerve-specific antigen and optionally a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into neurons (e.g., a composition containing one or more MSCs expressing one or more nerve-specific antigens and optionally a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into neurons) may be the sole active ingredient for treating mammals having (or at risk of developing) a disease or disorder characterized by inflammation and / or degeneration of nerve tissue (e.g., multiple sclerosis or immune-mediated encephalomyelitis) (e.g., by administering one or more MSCs expressing a CAR that targets a nerve-specific antigen and optionally a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into neurons), as described herein.

[0067] In some cases, one or more MSCs expressing a CAR that targets a nerve-specific antigen and optionally a polypeptide that can promote 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 that targets a nerve-specific antigen and optionally a polypeptide that can promote 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., therapeutic agents that can be used to treat mammals that have or are at risk of developing a disease or disorder characterized by inflammation and / or degeneration of nerve tissue, and therapeutic agents that can be used to treat inflammation of nerve tissue in mammals). For example, mammals that have (or are at risk of developing) a disease or disorder characterized by inflammation and / or degeneration of nerve 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 CARs that target neuronal specific antigens and optionally polypeptides 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 CARs that target neuronal specific antigens and optionally polypeptides that can promote the differentiation of MSCs and / or one or more resident progenitor cells into neurons can be administered substantially concurrently with one or more additional therapeutic agents that can be used to treat inflammation of nerve tissue in mammals.For example, a composition comprising one or more MSCs expressing a CAR that targets a nerve-specific antigen and optionally a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into neurons may include one or more additional therapeutic agents that can be used to treat inflammation of nerve tissue in mammals. In some cases, one or more MSCs expressing a CAR that targets a nerve-specific antigen and optionally a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into neurons may be administered first, and one or more additional therapeutic agents may be administered second, or vice versa.

[0068] In some cases, mammals (e.g., humans) that have or are at risk of developing diseases or disorders characterized by inflammation and / or degeneration of cardiac tissue can be treated using materials and methods for treating mammals (e.g., humans) that have or are at risk of developing diseases or disorders 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 cardiomyocytes), endocarditis (e.g., characterized by inflammation of endocardinal cells), and heart failure.

[0069] In some cases, mammals can be identified as having or being at risk of developing a disease or disorder characterized by inflammation and / or degeneration of cardiac tissue (e.g., myocarditis). Mammals can be identified as having or being at risk of developing a disease or disorder characterized by inflammation and / or degeneration of cardiac tissue using any appropriate method. For example, electrocardiograms (ECGs; e.g., to detect abnormal rhythms), chest X-rays, MRI (e.g., cardiac MRI), echocardiograms, and clinical tests (e.g., white blood cell and red blood cell counts, and / or blood tests to measure levels of specific enzymes indicating damage to the myocardium and to detect antibodies) can be used to identify mammals having or being at risk of developing a disease or disorder characterized by inflammation and / or degeneration of cardiac tissue, e.g., myocarditis. When identified as having (or at risk of developing) a disease or disorder characterized by inflammation and / or degeneration of cardiac tissue, the mammal may be directed to administer (e.g., by adoptive transfer) or self-administer one or more MSCs described herein (e.g., MSCs expressing CARs that target cardiac-specific antigens and optionally polypeptides that can promote the differentiation of the MSC 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 mammals having (or at risk of developing) a disease or disorder characterized by inflammation and / or degeneration of cardiac tissue as described herein (e.g., by administering one or more MSCs expressing a CAR that targets cardiac-specific antigens and optionally a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into cardiac cells), one or more MSCs expressing a CAR that targets cardiac-specific antigens and optionally a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into cardiac cells may be effective in reducing the severity of diseases or disorders characterized by inflammation and / or degeneration of cardiac tissue in mammals. If myocarditis is a disease or disorder characterized by inflammation and / or degeneration of epithelial tissue, then reducing the severity of myocarditis in mammals may include reducing or eliminating one or more symptoms of myocarditis (e.g., chest pain, arrhythmia, dyspnea, fluid retention (e.g., swelling of the legs, ankles, and feet), fatigue, headache, body aches, joint pain, fever, sore throat, diarrhea, syncope, and / or rapid breathing). For example, one or more MSCs expressing a CAR that targets a cardiac-specific antigen and optionally a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into cardiac cells may be administered to mammals (e.g., humans) (e.g., humans who have or are 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 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) may be the sole active ingredient for treating mammals 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 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 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) may be administered in combination with one or more additional therapeutic agents (e.g., therapeutic agents that can be used to treat mammals that have or are at risk of developing a disease or disorder characterized by inflammation and / or degeneration of cardiac tissue in mammals, and therapeutic agents that can be used to treat inflammation of cardiac tissue in mammals). For example, mammals that have (or are at risk of developing) a disease or disorder characterized by inflammation and / or degeneration of cardiac tissue (e.g., myocarditis) may also be treated with one or more additional therapeutic agents. In some cases, the therapeutic agent may be an anti-inflammatory agent. In some cases, the therapeutic agent may be an immunosuppressant. In some cases, the therapeutic agent may be an angiotensin-converting enzyme (ACE) inhibitor. In some cases, the therapeutic agent may be an angiotensin II receptor blocker (ARB). 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 a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into cardiac cells, as described herein, 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 a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into cardiac cells can be administered substantially concurrently with one or more additional therapeutic agents that can be used to treat inflammation of cardiac tissue in mammals.For example, a composition comprising one or more MSCs expressing a CAR that targets a cardiac-specific antigen and optionally a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into cardiac cells may also include one or more additional therapeutic agents that can be used to treat inflammation of cardiac tissue in mammals. In some cases, one or more MSCs expressing a CAR that targets a cardiac-specific antigen and optionally a polypeptide that can promote the differentiation of MSCs and / or one or more resident progenitor cells into cardiac cells may be administered first, and one or more additional therapeutic agents may be administered second, or vice versa.

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

[0074] This document also provides kits containing one or more materials described herein. In some cases, the kit may include one or more MSCs that express CARs that target tissue-specific antigens and optionally express polypeptides that can promote the differentiation of MSCs and / or one or more resident progenitor cells into tissue-specific cells (for example, a composition containing one or more MSCs that express CARs that target tissue-specific antigens, e.g., epithelial-specific antigens, nerve-specific antigens or cardiac-specific antigens and optionally express polypeptides 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). 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 expressing a CAR that targets a tissue-specific antigen, e.g., epithelial-specific antigen, neuronal-specific antigen, or cardiac-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., 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 that can bind to a tissue-specific antigen, e.g., epithelial-specific antigen, neuronal-specific antigen, or cardiac-specific antigen) (e.g., a nucleic acid construct) can be combined with packaging materials to form a kit. Packaging materials included in such a kit typically contain instructions or labels describing how the composition may be used in adoptive transplantation to treat mammals that have or are at risk of developing a disease or disorder characterized by, for example, inflammation and / or degeneration of the tissues described herein. In some cases, the materials provided in the kits described herein may be used to treat mammals (e.g., humans) that have or are at risk of developing a disease or disorder characterized by inflammation and / or degeneration of the tissues described herein.In some cases, the packaging material included in such a kit may contain instructions and / or labels describing how the compositions described herein may be used. For example, the kit may contain instructions and / or labels describing how the compositions described herein may 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 a kit may contain instructions and / or labels describing how the manipulated MSCs described herein may be used. For example, the packaging material included in such a kit may contain instructions and / or labels describing how the manipulated MSCs described herein may be used in adoption transfer to treat mammals that have or are at risk of developing a disease or disorder characterized by inflammation and / or degeneration of the tissues described herein. In some cases, the kit (e.g., a kit containing instructions and / or labels describing how the manipulated MSCs described herein may be used in adoption transfer) may include materials for use in an adoption transfer procedure.

[0075] The present invention will be further described in the following embodiments, but this will not limit the scope of the invention as described in the claims.

[0076] [Examples] [Example 1] Engineered CAR-MSCs for immunomodulation To determine whether CAR-expressing MSCs can reduce inflammation in target cell populations, we engineered MSCs expressing CD19-targeting CARs (MSC-CAR19).

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

[0078] MSC-CAR19 was created by transduction of MSC with CD19-CAR lentivirus (VSV-G pseudotype).

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

[0080] Adipose-derived mesenchymal stem cells (100-Biotr-0024) were isolated from cultures after 10+ consecutive passages, and CAR19 surface expression was probed. As shown above, 48.5% of the CAR19 transduced cultures retained CAR19 expression, and there was a 20% loss after transduction (Figure 3).

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

[0082] Figure 5 shows the designer's constructs for enhancing MSC trafficking, persistence, and immunomodulation effectiveness.

[0083] [Example 2] Engineered CAR-MSCs for immunomodulation of epithelial tissue We designed MSCs capable of targeting epithelial tissue by manipulating them to express a CAR (MSC-CAR-ECAD) that targets ECAD.

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

[0085] MSCs were transduced with an E-cadherin CAR lentivirus (VSV-G pseudotype) to create MSC-CAR-ECAD. A second-generation CAR construct containing a TLR3 signaling domain and / or a TLR4 signaling domain was used.

[0086] Adipose-derived mesenchymal stem cells (100-Biotr-0024) were passaged into four wells of a six-well plate (100k each). One group was retained as a non-transduction (UTD) negative control. The second well was transduced with GMP-grade pan-VSV, ECAD-recognizing chimeric antigen receptor (CAR-ECAD) lentivirus (approximately 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 the third group, but with 100 μg / ml of enhancer PLUS. Transduction efficiency increased in a concentration-dependent manner with enhancer PLUS (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 antigens. CART19 was activated via CAR (CD19 + When activated (via co-culture with the NALM6 cell line), 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 do not suppress CART cell proliferation.

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

[0089] The MSC phenotype was confirmed using flow cytometry (Figures 10A, 10B, 10C, and 10D). MSCs that retain stem cell characteristics were identified as CD90. + CD105 + CD73 + CD34 - CD45 - CD19 - CD14 -, HLA-DR - should be.

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

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

[0092] Exemplary nucleic acid sequences encoding CAR-MOG (SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7) are shown in Figures 13A, 13C, and 13E. Exemplary amino acid sequences of CAR-MOG (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 immunomodulation of heart tissue MSCs capable of targeting heart tissue were designed by engineering MSCs to express a CAR that targets HER2 (MSC-CAR-HER2).

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

[0095] [Example 5] Transduction efficiency of MSC-CAR19 Lentiviral transduction of MSCs using the CAR19 vector yielded a 60% transduction efficiency compared to non-transduction MSCs (Figure 15A).

[0096] [Example 6] MSCs retain their stem cell properties even after lentiviral transduction using CAR vectors. Flow cytometry analysis of MSC-CAR19 two days after transduction with CAR lentivirus demonstrated their stem cell characteristics and were equivalent to non-transduced MSCs (MSC-UTD). MSC-CAR19 continued to express CD105, CD90, and CD73, while lacking expression of CD34, CD45, HLA-DR, and CD14 (Figure 15B).

[0097] [Example 7] MSC-CAR19 is CD19 + It inhibits T cell proliferation in the presence of the target and exhibits antigen-specific stimulation of MSC-CAR19. T cells were stimulated with CD3 / CD28 beads and, after 24 hours, co-cultured with or without MSCs, either with untransduced MSCs (MSC-UTDs), MSC-CAR19 (CD28 containing CAR19 and K122), or MSC-CAR19 (CD137 containing CAR19 and K002). The CD19-targeting K002 CAR was designed to have a single-chain antibody targeting CD19 derived from FMC63, followed by a CD8 hinge, followed by a CD8 transmembrane domain, and 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 CD19-targeting K122 CAR was designed to have a single-chain antibody targeting CD19 derived 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 (Sequence ID 64).

[0098] As a strategy to stimulate MSC-CAR19 via CAR, T cells and MSCs are treated with culture medium alone or irradiated with CD19. + Co-culture was performed in either the presence or absence of cells. CD3 proliferation 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, and irradiated CD19 + Inhibited in the presence of cells, CD19 + Inhibition was not observed in the absence of cells (Figure 16). T cells, MSCs, and NALM6 cells were cultured in a ratio of 1:0.1:1 E (T cells (effector):MSCs (suppressor):T (tumor)). These results indicate that when CARs contain a CD28 signaling domain, MSC-CARs can suppress T cell proliferation in response to antigen-specific stimulation.

[0099] [Example 8] CD28 signaling uptake enhances MSC-CAR19 proliferation. MSC-CAR19 cells with different stimulation domains react to irradiated CD19 + MSCs were co-cultured with and without the NALM6 cell line, and MSC proliferation was monitored. Incorporation of the CD28 signaling domain resulted in enhanced proliferation of MSC-CAR19 compared to MSC-UTD or MSC-CAR19 incorporating the CD137 stimulating domain. Conversely, proliferation of MSC-CAR19 cells incorporating TLR4 signaling was suppressed. These results indicate that CD28 and TLR4 signaling are involved in the proliferation of MSC-CARs upon antigen-specific stimulation. The CD19-targeting K142 CAR was designed to have a single-chain antibody targeting CD19 derived from FMC63, followed by a CD8 hinge, a TLR4 transmembrane domain, and a TLR4 signaling domain. The amino acid sequence of K142 was as follows: MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT GGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGT SVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIGVSVLSVLVVSVVAVLVYKFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI (Sequence ID 65).

[0100] As a strategy to stimulate MSC-CAR19 via CAR, T cells and MSCs are treated with culture medium alone or irradiated with CD19. + Co-culture was performed in the presence of cells. CD3 proliferation 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. + Inhibited in the presence of cells, CD19 + Inhibition was not observed in the absence of cells (Figure 17). T cells, MSCs, and NALM6 cells were cultured in a ratio of 1:0.1:1 E (T cells (effector):MSC (suppressor):T (tumor)). These results indicate that when the CAR contains a TLR4 stimulating molecule, the MSC-CAR was able to suppress T cell proliferation upon antigen-specific stimulation.

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

[0102] [Example 10] Antigen-specific stimulation of MSC-CARs containing CD28 signaling molecules enhances their proliferation. Untransduced MSCs (MSC-UTD), MSC-CAR19 (CD28 containing CAR19, K122), MSC-CAR19 (CD137 containing CAR19, K002), or MSC-CAR19 (TLR4 containing CAR19, K142) are irradiated with CD19 + MSCs were co-cultured with the NALM6 cell line in a 1:1 ratio. The absolute number of MSCs was counted by flow cytometry using the absolute numbers on days 3 and 5. Antigen-specific stimulation with 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 using CAR results in reduced proliferation. MSCs were transduced with CAR lentivirus or GFP lentivirus on day 1 and then cultured for 15 days. Transduction with CAR-E cadherin resulted in reduced growth compared to untransduced MSCs or MSCs transduced with GFP lentivirus (Figure 20).

[0104] [Example 12] MSC-CAR exerts its inhibitory ability through intercellular contact-mediated and soluble factor-mediated mechanisms. First, T cells were stimulated with CD3 / CD28 beads in a 1:3 ratio (T cells to beads). After 24 hours, they were tested for MSCs and CD19 by either direct contact or Transwell experiment. + Cells (to stimulate MSC-CAR) were cultured together. MSC-CAR19 (containing the CD28 stimulating domain) and activated T cells, and CD19 +Co-culture with cells resulted in inhibition of T cell proliferation in Transwell experiments, whether the two cell groups were in direct or non-direct contact (Figure 21). These results indicate that MSC-CARs exert their inhibitory functions through both direct contact, intercellular contact, and secretion of soluble inhibitors / cytokines.

[0105] [Example 13] MSC-CAR suppresses T cell proliferation upon antigen-specific stimulation. First, T cells were stimulated with CD3 / CD28 beads in a 1:3 ratio. After 24 hours, T cells were cultured with NALM6 at a higher E:S:T ratio with untransduced MSCs or with MSC-CAR19 (CD28 containing CAR19 and K122), or T cells were cultured with NALM6 alone as a control for allogeneic effects. Irradiated CD19 + 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 MSCs (Figure 22). T cells, MSCs, and NALM6 cells were cultured in a 1:1:1 E ratio (T cells (effector):MSC (suppressor):T (tumor)).

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

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

[0108] The K128-CAR, which targets human E-CAD, was designed to have a single-chain antibody derived from clone 14, followed by a CD28 hinge, a CD28 transmembrane domain, and a CD28 signaling domain. The amino acid sequence of K128-CAR was as follows: MALPVTALLLPLALLLHAARPEVQLVQSGGGLVKPGGS-LRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV YYCARAQRQWGAFDYWGQGTLVTVSSEGKSSGSGSESKASSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITG AQAEDEADYYCNSRDSSGNPVFGGGTKLTVLGLEPKSCDKTHTCPPCPDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 66). K129-CAR, which targets human E-CAD, was designed to have a single-chain antibody derived 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, which targets human E-CAD, was designed to have 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 (Sequence ID 70).

[0109] T cells transduced in all three constructs are luciferase + Human E-cadherin + It showed potent killing of MCF-7 cells. Transduced T cells with K129-CAR also showed luciferase + Mouse E-cadherin + ID8 cells and luciferase + / Canine E-Cadherin + These results demonstrate potent killing of MCDK cells. These results indicate that the E-cadherin-targeting CAR is functional and can be used as described herein to produce MSCs containing the E-cadherin-targeting CAR.

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

[0111] [Example 17] Treatment of people with colitis Humans identified as having colitis are administered MSC-CAR-E-cadherin cells intravenously or intra-arterial at a dose of approximately 1 to 2 million cells per kg of body weight. Dose escalation is included, with single doses given every 10 days. Patients are clinically monitored for improvement in colitis symptoms.

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

[0113] [Example 19] Treatment of people with multiple sclerosis Humans identified as having multiple sclerosis are administered MSC-CAR-MOG at a dose of approximately 2 to 10 million cells per kg of body weight. The cells are administered either intravenously or intraventricularly. Multiple doses, including dose escalations, are administered, for example, every 10 days. Patients are clinically monitored for improvement in the symptoms of multiple sclerosis.

[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 confirmed, mice will be treated with either MSC-CAR-MOG or MSC-UTD. The neurological status of the mice will be monitored on a daily basis, and mouse survival will be tracked.

[0115] [Example 21] Treatment of individuals with immune-mediated encephalomyelitis Humans identified as having immune-mediated encephalomyelitis are administered MSC-CAR-MOG at a dose of approximately 2 to 10 million cells per kg of body weight. The cells are administered intravenously or intraventricularly. Multiple doses, including dose escalations, are administered, for example, every 10 days. Patients are clinically monitored for improvement in the symptoms of immune-mediated encephalomyelitis.

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

[0117] [Example 23] Treatment of people with myocarditis Humans identified as having severe or life-threatening myocarditis are administered MSC-CAR-HER2 at a dose of approximately 2 to 10 million cells / kg of body weight. The cells are administered either intravenously or via an intracardiac route. Multiple doses, including dose escalations, are administered, for example, every 10 days. Patients are clinically monitored for improvement in the symptoms of myocarditis.

[0118] [Example 24] MSC-CAR-E-cadherin inhibits the antitumor activity and proliferation of T cells in vivo. Luciferase in NSG mice + / E-cadherin + MCF-7 cell line (1 x 10 6 Cells were transplanted (intravenously). One week later, bioluminescence imaging was performed to confirm engraftment. All mice were treated with E-cadherin CAR T cells (2 × 10⁶). 6 Mice were treated with MSC-CAR-E-cadherin (1 × 10) cells and mice were given MSC-CAR-E-cadherin (1 × 10) 6 ), non-transduced MSCs (MSC-UTD) (1 × 10 6 Mice were randomly assigned to receive treatment with either MSC-CAR-ECAD or a control without MSCs. Disease burden was measured by sequential bioluminescence imaging of the mice. Treatment with MSC-CAR-ECAD resulted in a reduction in the antitumor activity of E-cadherin-targeting 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 tended to reduce T cell proliferation in vivo (Figure 24B). These results indicate that MSC-CAR-ECAD can suppress T cell effector function in this xenograft model.

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

[0120] Other embodiments Although the present invention has been described in detail, it should be understood that the foregoing description is intended to illustrate, and not limit, 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 includes the following embodiments: [Embodiment 1] A method for 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 according to claim 1, wherein the mammal is a human. [Embodiment 3] The method according to any one of claims 1 to 2, wherein the MSC is adipose-derived MSC. [Embodiment 4] The method according to any one of claims 1 to 3, wherein the epithelial-specific antigen is E-cadherin (ECAD). [Embodiment 5] The method according to any one of claims 1 to 4, wherein the CAR includes a single-stranded variable fragment (scFv). [Embodiment 6] The method according to claim 5, wherein the scFv comprises a light chain and a heavy chain derived from an anti-CDH1 antibody. [Embodiment 7] The method according to claim 6, wherein the anti-CDH1 antibody is hSC10.17. [Embodiment 8] The method according to any one of claims 1 to 7, wherein the MSC is operated to express the CAR ex vivo prior to the administration. [Embodiment 9] The method according to any one of claims 1 to 8, wherein the symptoms of colitis are reduced by at least 10 percent. [Embodiment 10] A method for 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 according to claim 10, wherein the mammal is a human. [Embodiment 12] The method according to any one of claims 10 to 11, wherein the MSC is adipose-derived MSC. [Embodiment 13] The method according to any one of claims 10 to 12, wherein the epithelial-specific antigen is E-cadherin (ECAD). [Embodiment 14] The method according to any one of claims 10 to 13, wherein the CAR includes a single-stranded variable fragment (scFv). [Embodiment 15] The method according to claim 14, wherein the scFv comprises a light chain and a heavy chain derived from an anti-CDH1 antibody. [Embodiment 16] The method according to claim 15, wherein the anti-CDH1 antibody is hSC10.17. [Embodiment 17] The method according to any one of claims 10 to 16, wherein the MSC is operated to express the CAR ex vivo prior to the administration. [Embodiment 18] A method for 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 nerve-specific antigen, wherein the MSCs express the CAR. [Embodiment 19] The method according to claim 18, wherein the mammal is a human. [Embodiment 20] The method according to any one of claims 18 to 19, wherein the MSC is adipose-derived MSC. [Embodiment 21] The method according to any one of claims 18 to 20, wherein the nerve-specific antigen is myelin oligodendrocyte glycoprotein (MOG). [Embodiment 22] The method according to claim 21, wherein the CAR includes a single-stranded variable fragment (scFv). [Embodiment 23] The method according to claim 22, wherein the scFv comprises a light chain and a heavy chain derived from an anti-MOG antibody. [Embodiment 24] The method according to claim 23, wherein the anti-MOG antibody is 8-18C5. [Embodiment 25] The method according to any one of claims 18 to 24, wherein the MSC is operated to express the CAR ex vivo prior to the administration. [Embodiment 26] The method according to any one of claims 18 to 25, wherein the symptoms of the multiple sclerosis are reduced by at least 10 percent. [Embodiment 27] The method according to any one of claims 18 to 26, wherein the MSC further comprises an exogenous nucleic acid encoding a polypeptide that can promote neuronal differentiation, and the MSC expresses the polypeptide. [Embodiment 28] The method according to 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 for 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 nerve-specific antigen, wherein the MSCs express the CAR. [Embodiment 30] The method according to claim 29, wherein the mammal is a human. [Embodiment 31] The method according to any one of claims 29 to 30, wherein the MSC is adipose-derived MSC. [Embodiment 32] The method according to any one of claims 29 to 31, wherein the nerve-specific antigen is myelin oligodendrocyte glycoprotein (MOG). [Embodiment 33] The method according to claim 32, wherein the CAR includes a single-stranded variable fragment (scFv). [Embodiment 34] The method according to claim 33, wherein the scFv comprises a light chain and a heavy chain derived from an anti-MOG antibody. [Embodiment 35] The method according to claim 34, wherein the anti-MOG antibody is 8-18C5. [Embodiment 36] The method according to any one of claims 29 to 35, wherein the MSC is operated to express the CAR ex vivo prior to the administration. [Embodiment 37] A method for 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 neuronal specific antigen, wherein the MSCs express the CAR. [Embodiment 38] The method according to claim 37, wherein the mammal is a human. [Embodiment 39] The method according to any one of claims 37 to 38, wherein the MSC is adipose-derived MSC. [Embodiment 40] The method according to any one of claims 37 to 39, wherein the nerve-specific antigen is myelin oligodendrocyte glycoprotein (MOG). [Embodiment 41] The method according to claim 40, wherein the CAR includes a single-stranded variable fragment (scFv). [Embodiment 42] The method according to claim 41, wherein the scFv comprises a light chain and a heavy chain derived from an anti-MOG antibody. [Embodiment 43] The method according to claim 42, wherein the anti-MOG antibody is 8-18C5. [Embodiment 44] The method according to any one of claims 37 to 43, wherein the MSC is operated to express the CAR ex vivo prior to the administration. [Embodiment 45] The method according to 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 according to any one of claims 37 to 45, wherein the MSC further comprises an exogenous nucleic acid encoding a polypeptide that can promote neuronal differentiation, and the MSC expresses the polypeptide. [Embodiment 47] The method according to claim 46, 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 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 neuronal specific antigen, wherein the MSCs express the CAR. [Embodiment 49] The method according to claim 48, wherein the mammal is a human. [Embodiment 50] The method according to any one of claims 48 to 49, wherein the MSC is adipose-derived MSC. [Embodiment 51] The method according to any one of claims 48 to 50, wherein the nerve-specific antigen is myelin oligodendrocyte glycoprotein (MOG). [Embodiment 52] The method according to claim 51, wherein the CAR includes a single-stranded variable fragment (scFv). [Embodiment 53] The method according to claim 52, wherein the scFv comprises a light chain and a heavy chain derived from an anti-MOG antibody. [Embodiment 54] The method according to claim 53, wherein the anti-MOG antibody is 8-18C5. [Embodiment 55] The method according to any one of claims 48 to 54, wherein the MSC is operated to express the CAR ex vivo prior to the administration. [Embodiment 56] A nucleic acid construct encoding a chimeric antigen receptor (CAR) that targets a nerve-specific antigen. [Embodiment 57] The nucleic acid construct according to claim 56, wherein the nerve-specific antigen is myelin oligodendrocyte glycoprotein (MOG). [Embodiment 58] The nucleic acid construct according to any one of claims 56 to 57, wherein the CAR comprises a single-stranded variable fragment (scFv). [Embodiment 59] The nucleic acid construct according to any one of claims 56 to 58, wherein the CAR that targets the nerve-specific 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] The nucleic acid construct according to any one of claims 56 to 59, wherein the nucleic acid construct can also encode a polypeptide that can promote neuronal differentiation. [Embodiment 61] The nucleic acid construct according to claim 60, 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 62] A method for 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 heart-specific antigen, wherein the MSCs express the CAR. [Embodiment 63] The method according to claim 62, wherein the mammal is a human. [Embodiment 64] The method according to any one of claims 62 to 63, wherein the MSC is fat-derived MSC. [Embodiment 65] The method according to any one of claims 62 to 64, wherein the cardiac-specific antigen is HER2. [Embodiment 66] The method according to claim 65, wherein the CAR includes a single-stranded variable fragment (scFv). [Embodiment 67] The method according to any one of claims 62 to 66, wherein the MSC is operated to express the CAR ex vivo prior to the administration. [Embodiment 68] The method according to any one of claims 62 to 67, wherein the symptoms of myocarditis are reduced by at least 10 percent. [Embodiment 69] The method according to any one of claims 62 to 68, wherein the MSC further comprises an exogenous nucleic acid encoding a polypeptide that can promote cardiac cell differentiation, and the MSC expresses the polypeptide. [Embodiment 70] The method according to claim 69, wherein the polypeptide capable of promoting neural differentiation is selected from the group consisting of GATA4 polypeptide, MEF2C polypeptide, TBX5 polypeptide, ERRG polypeptide, MESP1 polypeptide, and any combination thereof. [Embodiment 71] A method for 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 heart-specific antigen, wherein the MSCs express the CAR. [Embodiment 72] The method according to claim 71, wherein the mammal is a human. [Embodiment 73] The method according to any one of claims 71 to 72, wherein the MSC is fat-derived MSC. [Embodiment 74] The method according to any one of claims 71 to 73, wherein the cardiac-specific antigen is HER2. [Embodiment 75] The method according to claim 74, wherein the CAR includes a single-stranded variable fragment (scFv). [Embodiment 76] The method according to any one of claims 71 to 75, wherein the MSC is operated to express the CAR ex vivo prior to the administration. [Embodiment 77] A nucleic acid construct encoding a chimeric antigen receptor (CAR) that targets a heart-specific antigen. [Embodiment 78] The nucleic acid construct according to claim 77, wherein the cardiac-specific antigen is HER2. [Embodiment 79] The nucleic acid construct according to any one of claims 77 to 78, wherein the CAR comprises a single-stranded variable fragment (scFv). [Embodiment 80] The nucleic acid construct according to any one of claims 77 to 79, wherein the CAR that targets the heart-specific antigen is encoded by the nucleic acid sequence shown in Sequence ID No. 9. [Embodiment 81] The nucleic acid construct according to any one of claims 77 to 80, wherein the nucleic acid construct can also encode a polypeptide that can promote cardiac cell differentiation. [Embodiment 82] The nucleic acid construct according to claim 81, wherein the polypeptide capable of promoting neuronal differentiation is selected from the group consisting of GATA4 polypeptide, MEF2C polypeptide, TBX5 polypeptide, ERRG polypeptide, 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 the binding of the CAR to the antigen in the mammal results in the suppression of the immune response in the mammal. [Embodiment 84] The method according to claim 83, wherein the mammal is a human. [Embodiment 85] The method according to any one of claims 83 to 84, wherein the MSC is adipose-derived MSC. [Embodiment 86] The method according to any one of claims 83 to 85, wherein the antigen is E-cadherin (ECAD). [Embodiment 87] The method according to any one of claims 83 to 86, wherein the CAR comprises a single-stranded variable fragment (scFv). [Embodiment 88] The method according to any one of claims 83 to 87, wherein the MSC is operated to express the CAR ex vivo prior to the administration. [Embodiment 89] The method according to 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 according to claim 89, wherein the CAR comprises a CD28 signaling domain. [Embodiment 91] The method according to claim 89, wherein the CAR comprises a TLR4 signaling domain. [Embodiment 92] The nucleic acid according to any one of claims 56 to 61 and 77 to 82, wherein the CAR comprises a CD28 or TLR4 signaling domain. [Embodiment 93] The nucleic acid according to claim 92, wherein the CAR comprises a CD28 signaling domain. [Embodiment 94] The nucleic acid according to claim 92, wherein the CAR comprises a TLR4 signaling domain.

[0121] [Sequence List] SEQUENCE LISTING <110> Mayo Foundation for Medical Education and Research <120> METHODS AND MATERIALS FOR USING ENGINEERED MESENCHYMAL STEM CELLS TO TREAT INFLAMMATORY CONDITIONS AND DEGENERATIVE DISEASES <130> PA26-049 <150> US 62 / 932,610 <151> 2019-11-08 <160> 45 <170> PatentIn version 3.5 <210> 1 <211> 1395 <212> DNA <213> Artificial <220> <223> nucleic acid sequence encoding of CAR-ECAD <400> 1 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccgcagatcc tgttggtgca gtctggacct gagctgaaga agcctggaga gacagtcaag 120 atctcctgca aggcttctaa ttataccttc acagactatg gaatgcactg ggtgaagcag 180 gctccaggaa agggtttaaa gtggatgggc tggataaacc ccaagactgg tgtggcatca 240 tatgcagatg acttcaaggg aagatttgcc ttctctttgg aaacctctgc cagcactgcc 300 tatttgcaga tcaacaacct cgaaaatgag gacacgtcta tatattcttg tgctagattt 360 tttgactact ggggccaagg caccactctc acagtctcct caggaggtgg cggatcaggc 420 ggaggaggca gcggcggagg tggatcagga ggcggagggt cagatatttgt gctgacacag 480 tctccactct ccctgcttgt cagtcttgga gatcaagcct ccatctcttg cagatctagt 540 cagagccttg tacacagtaa tggaaacacc tatttacatt ggtatctgca gaagccaggc 600 cagtctccaa acctcctgat cttcaaagtt tccaaccgat tttctggggt cccagacagg 660 ttcagtggca gtggatcagg gacagatttc acactcagga tcagcagagt ggaggctgag 720 gatctgggag tttatttctg ctctcaaact acacatgtgt ggacgttcgg tggaggcacc 780 aagctggaaa tcaaactcga gcccaaatct tgtgacaaaa ctcacacatg cccaccgtgc 840 ccggatccca aattttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg 900 ctagtaacag tggcctttat tattttctgg gtgaggagta agaggagcag gctcctgcac 960 agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 1020 tatgccccac cacgcgactt cgcagcctat cgctccagag tgaagttcag caggagcgca 1080 gacgcccccg cgtacaagca gggccagaac cagctctata acgagctcaa tctaggacga 1140 agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaaag 1200 ccgagaagga agaaccctca ggaaggcctg tacaatgaac tgcagaaaga taagatggcg 1260 gaggcctaca gtgagattgg gatgaaaggc gagcgccgga ggggcaaggg gcacgatggc 1320 cttaccagg gtctcagtac agccaccaag gacacctacg acgcccttca catgcaggcc 1380 ctgccccctc gctaa 1395 <210> 2 <211> 464 <212> PRT <213> Artificial <220> <223> amino acid sequence of a CAR-ECAD <400> 2 Put Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Wing Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Ile Leu Leu Val Gln Ser Gly Pro Glu Leu 20 25 30 Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Asn Tyr 35 40 45 Thr Phe Thr Asp Tyr Gly Met His Trp Val Lys Gln Ala Pro Gly Lys 50 55 60 Gly Leu Lys Trp Met Gly Trp Ile Asn Pro Lys Thr Gly Val Ala Ser 65 70 75 80 Tyr Ala Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser 85 90 95 Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Glu Asn Glu Asp Thr 100 105 110 Ser Ile Tyr Phe Cys Ala Arg Phe Phe Asp Tyr Trp Gly Gln Gly Thr 115 120 125 Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln 145 150 155 160 Ser Pro Leu Ser Leu Leu Val Ser Leu Gly Asp Gln Ala Ser Ile Ser 165 170 175 Cys Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu 180 185 190 His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Asn Leu Leu Ile Phe 195 200 205 Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 210 215 220 Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu 225 230 235 240 Asp Leu Gly Val Tyr Phe Cys Ser Gln Thr Thr His Val Trp Thr Phe 245 250 255 Gly Gly Gly Thr Lys Leu Glu Ile Lys Leu Glu Pro Lys Ser Cys Asp 260 265 270 Lys Thr His Thr Cys Pro Pro Cys Pro Asp Pro Lys Phe Trp Val Leu 275 280 285 Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val 290 295 300 Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His 305 310 315 320 Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys 325 330 335 His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 340 345 350 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly 355 360 365 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 370 375 380 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 385 390 395 400 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 405 410 415 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 420 425 430 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 435 440 445 Thr Lys Asp Thr Tyr Asp With Leu His Met Gln Ala Leu Pro Pro Arg 450 455 460 <210> 3 <211> 1401 <212> DNA <213> Artificial <220> <223> nucleic acid sequence encoding and CAR-MOG <400> 3 atggcattac cagttaccgc attack cccgctggcat tgctgttaca tgcagcacgt 60 ccggatattg aactgaccca gagtcccagt agcctggccg tgagtgccgg cgagaaagtg 120 accatgagct gcaaagcag ccagagcctg ctgaacagcg gcaaccagaa aaactacctg 180 gcctgtacc agcagaaacc cggcctgcct cctaagctgc tgattacgg cgccagcacc 240 around gcgtgcccga tagattcacc ggctctggct ctggcaccga cttcaccctg 300 accatcagca gcgtgcaggc cgaagacctg gccgtgtact actgccagaa cgaccacagc 360 taccccctga ccttcggcgc cggcaccaag ctggagatca agggtggagg tggttcggga 420 ggtggaggta gcgggaggtgg tggaagcgag gtgaagctgc acgagagcgg cgcaggtctg 480 gtgaagcccg gcgccagcgt ggagatcagc tgcaaggcca ccggctacac cttcagcagc 540 ttctggatcg agtgggtgaa gcagagaccc ggccacggcc tggagtggat cggcgagatc 600 ctgcccggca gaggcagaac caactacaac gagaagttca agggcaaggc caccttcacc 660 gccgagacca gcagcaacac cgcctacatg cagctgagca gcctgaccag cgaggacagc 720 gccgtgtact actgcgccac cggcaacacc atggtgaaca tgccctactg gggccagggc 780 accaccgtga ccgtgagcag cctcgagccc aaatcttgtg acaaaactca cacatgccca 840 ccgtgcccgg atcccaaatt ttgggtgctg gtggtggttg gtggagtcct ggcttgctat 900 agcttgctag taacagtggc ctttattatt ttctgggtga ggagtaagag gagcaggctc 960 ctgcacagtg actacatgaa catgactccc cgccgccccg gtcccaccg caagcattac 1020 cagccctatg ccccaccacg cgacttcgca gcctatcgct ccagagtgaa gttcagcagg 1080 agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1140 ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatggga 1200 ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1260 atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1320 gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1380 caggccctgc cccctcgcta a 1401 <210> 4 <211> 466 <212> PRT <213> Artificial <220> <223> amino acid sequence of a CAR-MOG <400> 4 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Leu 20 25 30 Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln 35 40 45 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln 50 55 60 Gln Lys Pro Gly Leu Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr 65 70 75 80 Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 85 90 95 Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val 100 105 110 Tyr Tyr Cys Gln Asn Asp His Ser Tyr Pro Leu Thr Phe Gly Ala Gly 115 120 125 Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Glu Val Lys Leu His Glu Ser Gly Ala Gly Leu 145 150 155 160 Val Lys Pro Gly Ala Ser Val Glu Ile Ser Cys Lys Ala Thr Gly Tyr 165 170 175 Thr Phe Ser Ser Phe Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His 180 185 190 Gly Leu Glu Trp Ile Gly Glu Ile Leu Pro Gly Arg Gly Arg Thr Asn 195 200 205 Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Phe Thr Ala Glu Thr Ser 210 215 220 Ser Asn Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser 225 230 235 240 Ala Val Tyr Tyr Cys Ala Thr Gly Asn Thr Met Val Asn Met Pro Tyr 245 250 255 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Leu Glu Pro Lys Ser 260 265 270 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Asp Pro Lys Phe Trp 275 280 285 Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val 290 295 300 Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu 305 310 315 320 Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr 325 330 335 Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr 340 345 350 Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys 355 360 365 Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 370 375 380 Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 385 390 395 400 Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu 405 410 415 Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly 420 425 430 Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser 435 440 445 Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro 450 455 460 Pro Arg 465 <210> 5 <211> 1515 <212> DNA <213> Artificial <220> <223> nucleic acid sequence encoding a CAR-MOG <400> 5 atgctcaggc tgctcttggc tctcaactta ttcccttcaa ttcaagtaac aggagaattt ctgatgaccc agaccccgct gagcctgagc gtgagcgcgg gcgaaaaagt gaccatgagc tgcaaaagca gccagagcct gctgaacagc ggcaaccaga aaaactatct ggcgtggtat 240. cagcagaaac cggggcctgcc gccgaaactg ctgatttatg gcgcgagcac ccgcgaaagc ggcgtgccgg atcgctttac cggcagcggc agcggcaccg attttaccct gaccattagc 300 agcgtgcagg cggaagatct ggcggtgtat tattgccaga acgatcatag ctatccgctg acctttggcg cgggcaccaa actggaactg aaacgcgcgg atgcggcgcc gaccggtgga ggtggttcgg gaggtggagg tagcggaggt ggtggaagcc aggtgaaact gcagcagagc 480 ggcgcggac tgatgaacc gggcgcgagc gtggaatta gctgcaaagc gaccggctat 540 acctttagca gcttttggat tgaatgggtg aaacagcgcc cgggccatgg cctggaatgg 600 attggcgaaa ttctgccggg ccgcggccgc accaactata acgaaaaatt taaaggcaaa 660 gcgaccttta ccgcggaaac cagcagcaac accgcgtata tgcagctgag cagcctgacc 720 agcgaagata gcgcggtgta ttattgcgcg accggcaaca ccatggtgaa catgccgtat 780 tggggtcagg gtacaacatt aacagtttct tctgcacgtc agacaacacc gccgagcgtg 840 tatggcgcgg tggaagcggt gccgcgcgat tgcggctgca aaccgtgcat ttgcaccccg 900 gaagtgagca gcggcctcga gcccaaatct tgtgacaaaa ctcacacatg cccaccgtgc 960 ccggatccca aattttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg 1020 ctagtaacag tggcctttat tattttctgg gtgaggagta agaggagcag gctcctgcac 1080 agtgactaca tgaacatgac tcctagacgc cctggtccca cccgcaagca ttaccagccc 1140 tatgccccac cacgcgactt cgcagcctat cgctccagag tgaagttcag caggagcgca 1200 gacgccccg cgtacaagca gggccagaac cagctctata acgagctcaa tctaggacga 1260 agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaaag 1320 ccgagaagga agaaccctca ggaaggcctg tacaatgaac tgcagaaaga taagatggcg 1380 gaggcctaca gtgagattgg gatgaaaggc gagcgccgga ggggcaaggg gcacgatggc 1440 cttaccagg gtctcagtac agccaccaag gacacctacg acgcccttca catgcaggcc 1500 ctgccccctc gctaa 1515 <210> 6 <211> 504 <212> PRT <213> Artificial <220> <223> amino acid sequence of a CAR-MOG <400> 6 Put Leu Arg Leu Leu Leu Ala Leu Donkey Leu Phe Pro Ser Ile Gln Val 1 5 10 15 Thr Gly Glu Phe Leu Met Thr Gln Thr Pro Leu Ser Leu Ser Val Ser 20 25 30 Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu 35 40 45 Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Leu Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys 100 105 110 Gln Asn Asp His Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu 115 120 125 Glu Leu Lys Arg Ala Asp Ala Ala Pro Thr Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Lys Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Met Lys Pro Gly Ala Ser Val Glu Ile Ser Cys Lys 165 170 175 Ala Thr Gly Tyr Thr Phe Ser Ser Phe Trp Ile Glu Trp Val Lys Gln 180 185 190 Arg Pro Gly His Gly Leu Glu Trp Ile Gly Glu Ile Leu Pro Gly Arg 195 200 205 Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Phe Thr 210 215 220 Ala Glu Thr Ser Ser Asn Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr 225 230 235 240 Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Thr Gly Asn Thr Met Val 245 250 255 Asn Met Pro Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala 260 265 270 Arg Gln Thr Thr Pro Pro Ser Val Tyr Gly Ala Val Glu Ala Val Pro 275 280 285 Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Pro Glu Val Ser Ser 290 295 300 Gly Leu Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 305 310 315 320 Pro Asp Pro Lys Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 325 330 335 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg 340 345 350 Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 355 360 365 Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 370 375 380 Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala 385 390 395 400 Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 405 410 415 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 420 425 430 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 435 440 445 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 450 455 460 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 465 470 475 480 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 485 490 495 His Met Gln Ala Leu Pro Pro Arg 500 <210> 7 <211> 1401 <212> DNA <213> Artificial <220> <223> nucleic acid sequence encoding a CAR-MOG <400> 7 atggcattac cagttaccgc attattactg ccgctggcat tgctgttaca tgcagcacgt 60 ccggaggtga agctgcacga gagcggcgca ggtctggtga agcccggcgc cagcgtggag 120 atcagctgca aggccaccgg ctacaccttc agcagcttct ggatcgagtg ggtgaagcag 180 agacccggcc acggcctgga gtggatcggc gagatcctgc ccggcagagg cagaaccaac 240 tacaacgaga agttcaaggg caaggccacc ttcaccgccg agaccagcag caacaccgcc 300 tacatgcagc tgagcagcct gaccagcgag gacagcgccg tgtactactg cgccaccggc 360 aacaccatgg tgaacatgcc ctactggggc cagggcacca ccgtgaccgt gagcagcggt 420 ggaggtggtt cgggaggtgg aggtagcgga ggtggtggaa gcgatattga actgacccag 480 agtcccagta gcctggccgt gagtgccggc gagaaagtga ccatgagctg caaaagcagc 540 cagagcctgc tgaacagcgg caaccagaaa aactacctgg cctggtacca gcagaaaccc 600 ggcctgcctc ctaagctgct gatctacggc gccagcacca gagaaagcgg cgtgcccgat 660 agattcaccg gctctggctc tggcaccgac ttcaccctga ccatcagcag cgtgcaggcc 720 gaagacctgg ctgtctacta ctgccagaac gaccacagct accccctgac cttcggcgcc 780 ggcaccaagc tggagatcaa gctcgagccc aaatcttgtg acaaaactca cacatgccca 840 ccgtgcccgg atcccaaatt ttgggtgctg gtggtggttg gtggagtcct ggcttgctat 900 agcttgctag taacagtggc ctttattatt ttctgggtga ggagtaagag gagcaggctc 960 ctgcacagtg actacatgaa catgactccc cgccgccccg gtcccacccg caagcattac 1020 cagccctatg ccccaccacg cgacttcgca gcctatcgct ccagagtgaa gttcagcagg 1080 agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1140 ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatggga 1200 ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1260 atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1320 gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1380 caggccctgc cccctcgcta a 1401 <210> 8 <211> 466 <212> PRT <213> Artificial <220> <223> amino acid sequence of a CAR-MOG <400> 8 Put Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Wing Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Lys Leu His Glu Ser Gly Ala Gly Leu 20 25 30 Val Lys Pro Gly Ala Ser Val Glu Ile Ser Cys Lys Ala Thr Gly Tyr 35 40 45 Thr Phe Ser Ser Phe Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His 50 55 60 Gly Leu Glu Trp Ile Gly Glu Ile Leu Pro Gly Arg Gly Arg Thr Asn 65 70 75 80 Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Phe Thr Ala Glu Thr Ser 85 90 95 Ser Asn Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser 100 105 110 Ala Val Tyr Tyr Cys Ala Thr Gly Asn Thr Met Val Asn Met Pro Tyr 115 120 125 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln 145 150 155 160 Ser Pro Ser Ser Leu Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser 165 170 175 Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr 180 185 190 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Leu Pro Pro Lys Leu Leu Ile 195 200 205 Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly 210 215 220 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala 225 230 235 240 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp His Ser Tyr Pro Leu 245 250 255 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Leu Glu Pro Lys Ser 260 265 270 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Asp Pro Lys Phe Trp 275 280 285 Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val 290 295 300 Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu 305 310 315 320 Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr 325 330 335 Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr 340 345 350 Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys 355 360 365 Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 370 375 380 Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 385 390 395 400 Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu 405 410 415 Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly 420 425 430 Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser 435 440 445 Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro 450 455 460 Pro Arg 465 <210> 9 <211> 1518 <212> DNA <213> Artificial <220> <223> nucleic acid sequence encoding a CAR-HER2 <400> 9 tctagaatgg ccttaccagt gaccgccttg ctcctgccgc tggccttgct gctccacgcc 60 gccaggccgg atatccagat gacccagtcc ccgagctccc tgtccgcctc tgtgggcgat 120 agggtcacta tcacctgccg tgccagtcag gatgtgaata ctgctgtagc ctggtatcaa 180 cagaaacccg gaaaggcccc gaaactgctg atttactcgg catccttcct ctactctgga 240 gtcccttctc gcttctctgg ttcccgctct gggacggatt tcactctgac catcagctcc 300 ctgcagccgg aagacttcgc aacttattac tgtcagcaac actatactac tcctccgacg 360 ttcggacagg gtaccaaggt ggagatcaaa cgtaccgtgg cggcgccagg aggtggcgga 420 tcaggcggag gaggcagcgg cggaggtgga tcaggaggcg gagggtcaga ggttcagctg 480 gtggagtctg gcggtggcct ggtgcagccc gggggctctc tccgtttgtc ctgtgcagct 540 tctggcttca acattaaaga cacctatatc cactgggtgc gtcaggctcc gggtaagggc 600 ctggagtggg ttgcaaggat ttatcctacg aatggttata ctcgttatgc cgatagcgtc 660 aagggccgtt tcactataag cgcagacact tcgaaaaaca cagcctacct ccagatgaac 720 agcctgcgtg ctgaggacac tgccgtctat tattgtagca gatggggtgg ggacggcttc 780 tatgctatgg actactgggg tcaggtaca ctagtcaccg tcagcgc tagcaccag 840 ggcaccacga cgccagcgcc gcgaccacca acaccggcgc ccaccacacgc gtcgcagcccc 900 ctgtccctgc gcccagaggc gtgccggcca gcggcggggg gcgcagtgca cacgagggggg 960 ctggacttcg cctgtgatat ctacatctgg gcgcccttgg ccggacttg tggggtcctt 1020 ctcctgtcac tggttatcac cctttactgc aaacggggca gaaagaact cctgtatatata 1080 ttcaacaac catttatgag accagtacaa actactcaag aggagatgg ctgtagctgc 1140 cgatttccag agagaga aggattgt gaacgagag tgagttcag caggagcgca 1200 gacgcccccg cgtacaagca gggccagaac cagctctata acgagctca tctaggacga 1260 agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat gggggaaag 1320 ccgagaagga agaccctca gggaggcctg tacaatgaac tgcagaaga agaatggcg 1380 gaggcctaca gtgagattgg gatgaaaggc gagcgccgga ggggcaaggg gcacgatggc 1440 ctttaccagg gtctcagtac agccaccaag gacacctacg acgcccttca catgcaggcc 1500 ctgccccctc gcgtcgac 1518 <210> 10 <211> 506 <212> PRT <213> Artificial <220> <223> amino acid sequence of a CAR-HER2 <400> 10 Ser Arg Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu 1 5 10 15 Leu Leu His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser 20 25 30 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala 35 40 45 Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly 50 55 60 Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly 65 70 75 80 Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu 85 90 95 Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln 100 105 110 Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu 115 120 125 Ile Lys Arg Thr Val Ala Ala Pro Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu 145 150 155 160 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 165 170 175 Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp 180 185 190 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr 195 200 205 Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe 210 215 220 Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn 225 230 235 240 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly 245 250 255 Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 260 265 270 Thr Val Ser Ser Ala Ser Thr Lys Gly Thr Thr Thr Pro Ala Pro Arg 275 280 285 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 290 295 300 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 305 310 315 320 Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 325 330 335 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg 340 345 350 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 355 360 365 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 370 375 380 Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 385 390 395 400 Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 405 410 415 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 420 425 430 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 435 440 445 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 450 455 460 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 465 470 475 480 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 485 490 495 His Met Gln Ala Leu Pro Pro Arg Val Asp 500 505 <210> 11 <211> 113 <212> PRT <213> Artificial <220> <223> amino acid sequence of an anti-ECAD antibody heavy chain <400> 11 Gln Ile Leu Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Asn Tyr Thr Phe Thr Asp Tyr 20 25 30 Gly Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Pro Lys Thr Gly Val Ala Ser Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Glu Asn Glu Asp Thr Ser Ile Tyr Phe Cys 85 90 95 Ala Arg Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser 100 105 110 Ser <210> 12 <211> 112 <212> PRT <213> Artificial <220> <223> amino acid sequence of an anti-ECAD antibody light chain <400> 12 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Ala Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 13 <211> 111 <212> PRT <213> Artificial <220> <223> amino acid sequence of an anti-ECAD antibody light chain <400> 13 Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Leu Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Asn Leu Leu Ile Phe Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Thr 85 90 95 Thr His Val Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 14 <211> 118 <212> PRT <213> Artificial <220> <223> amino acid sequence of an anti-MOG antibody heavy chain <400> 14 Glu Val Lys Leu His Glu Ser Gly Ala Gly Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Glu Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Phe 20 25 30 Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro Gly Arg Gly Arg Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Phe Thr Ala Glu Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Asn Thr Met Val Asn Met Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 15 <211> 113 <212> PRT <213> Artificial <220> <223> amino acid sequence of an anti-MOG antibody light chain <400> 15 Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Leu 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp His Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 16 <211> 170 <212> PRT <213> Artificial <220> <223> amino acid sequence of an anti-HER2 antibody heavy chain <400> 16 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Thr Thr Thr 115 120 125 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 130 135 140 Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 145 150 155 160 His Thr Arg Gly Leu Asp Phe Ala Cys Asp 165 170 <210> 17 <211> 113 <212> PRT <213> Artificial <220> <223> amino acid sequence of an anti-HER2 antibody light chain <400> 17 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro <210> 18 <211> 361 <212> DNA <213> Artificial <220> <223> nucleic acid encoding an anti-ECAD antibody heavy chain <400> 18 gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120 ccagggaagg ggctggagtg ggttgcatac attactacta gaagtagtac catatactac 180 gcagactctg tgaagggccg attcaccatc tccagagaca atgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtac tagagaaccc 300 ctaactggat actatgctat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 360 g 361 <210> 19 <211> 339 <212> DNA <213> Artificial <220> <223> nucleic acid encoding an anti-ECAD antibody heavy chain <400> 19 cagatcctgt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60 tcctgcaagg cttctaatta taccttcaca gactatggaa tgcactgggt gaagcaggct 120 ccaggaaagg gtttaaagtg gatgggctgg ataaacccca agactggtgt ggcatcatat 180 gcagatgact tcaagggaag atttgccttc tctttggaaa cctctgccag cactgcctat 240 ttgcagatca acaacctcga aaatgaggac acgtctatat atttctgtgc tagatttttt 300 gactactggg gccaaggcac cactctcaca gtctcctca 339 <210> 20 <211> 336 <212> DNA <213> Artificial <220> <223> nucleic acid encoding an anti-ECAD antibody light chain <400> 20 gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccttggaca gccggcctcc 60 atctcctgca ggtctagtca aagcatcgta cacagtgatg gaaacaccta cttggaatgg 120 tatcagcaga ggccaggcca atctccaagg cgcctaattt ataaggtttc taaccggttc 180 tctggggtcc cagacagatt cagcggcagt gggtcaggca ctgatttcac actgaaaatc 240 agcagggtgg aggctgagga tgttggggtt attactgctt tcaaggttca catgctccgt 300 ggacgttcgg tggaggcacc aaggtggaaa tcaaac 336 <210> 21 <211> 333 <212> DNA <213> Artificial <220> <223> nucleic acid encoding an anti-ECAD antibody light chain <400> 21 gatattgtgc tgacacagtc tccactctcc ctgcttgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120 tatctgcaga agccaggcca gtctccaaac ctcctgatct tcaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaggatc 240 agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaactac acatgtgtgg 300 acgttcggtg gaggcaccaa gctggaaatc aaa 333 <210> 22 <211> 354 <212> DNA <213> Artificial <220> <223> nucleic acid encoding an anti-MOG antibody heavy chain <400> 22 gaggtgaagc tgcacgagag cggcgcaggt ctggtgaagc ccggcgccag cgtggagatc 60 agctgcaagg ccaccggcta caccttcagc agcttctgga tcgagtgggt gaagcagaga 120 cccggccacg gcctggagtg gatcggcgag atcctgcccg gcagaggcag aaccaactac 180 aacgagaagt tcaagggcaa ggccaccttc accgccgaga ccagcagcaa caccgcctac 240 atgcagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc caccggcaac 300 accatggtga acatgcccta ctggggccag ggcaccaccg tgaccgtgag cagc 354 <210> 23 <211> 339 <212> DNA <213> Artificial <220> <223> nucleic acid encoding an anti-MOG antibody light chain <400> 23 gatattgaac tgacccagag tcccagtagc ctggccgtga gtgccggcga gaaagtgacc 60 atgagctgca aaagcagcca gagcctgctg aacagcggca accagaaaaa ctacctggcc 120 tggtaccagc agaaacccgg cctgcctcct aagctgctga tctacggcgc cagcaccaga 180 gaaagcggcg tgcccgatag attcaccggc tctggctctg gcaccgactt caccctgacc 240 atcagcagcg tgcaggccga agacctggct gtctactact gccagaacga ccacagctac 300 cccctgacct tcggcgccgg caccaagctg gagatcaag 339 <210> 24 <211> 510 <212> DNA <213> Artificial <220> <223> nucleic acid encoding an anti-HER2 antibody heavy chain <400> 24 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc ccgggggctc tctccgtttg 60 tcctgtgcag cttctggctt caacattaaa gacacctata tccactgggt gcgtcaggct 120 ccgggtaagg gcctggagtg ggttgcaagg atttatccta cgaatggtta tactcgttat 180 gccgatagcg tcaagggccg tttcactata agcgcagaca cttcgaaaaa cacagcctac 240 ctccagatga acagcctgcg tgctgaggac actgccgtct attattgtag cagatggggt 300 ggggacggct tctatgctat ggactactgg ggtcaaggta cactagtcac cgtcagcagc 360 gctagcacca agggcaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 420 gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg 480 cacacgaggg ggctggactt cgcctgtgat 510 <210> 25 <211> 339 <212> DNA <213> Artificial <220> <223> nucleic acid encoding an anti-HER2 antibody light chain <400> 25 gatatccaga tgacccagtc cccgagctcc ctgtccgcct ctgtgggcga tagggtcact 60 atcacctgcc gtgccagtca ggatgtgaat actgctgtag cctggtatca acagaaaccc 120 ggaaaggccc cgaaactgct gatttactcg gcatccttcc tctactctgg agtcccttct 180 cgcttctctg gttcccgctc tgggacggat ttcactctga ccatcagctc cctgcagccg 240 gaagacttcg caacttatta ctgtcagcaa cactatacta ctcctccgac gttcggacag 300 ggtaccaagg tggagatcaa acgtaccgtg gcggcgcca 339 <210> 26 <211> 21 <212> PRT <213> Artificial <220> <223> CD8 leader sequence <400> 26 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 27 <211> 15 <212> PRT <213> Artificial <220> <223> linker sequence <400> 27 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 28 <211> 45 <212> PRT <213> Artificial <220> <223> CD8 hinge sequence <400> 28 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 29 <211> 19 <212> PRT <213> Artificial <220> <223> CD28 hinge sequence <400> 29 Leu Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 Asp Pro Lys <210> 30 <211> 24 <212> PRT <213> Artificial <220> <223> CD8 transmembrane domain sequence <400> 30 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> 31 <211> 27 <212> PRT <213> Artificial <220> <223> CD28 transmembrane domain sequence <400> 31 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 32 <211> 41 <212> PRT <213> Artificial <220> <223> CD28 signaling domain sequence <400> 32 Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 33 <211> 112 <212> PRT <213> Artificial <220> <223> CD3zeta signaling domain sequence <400> 33 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 34 <211> 42 <212> PRT <213> Artificial <220> <223> 41BB signaling domain sequence <400> 34 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 35 <211> 151 <212> PRT <213> Artificial <220> <223> TLR3 signaling domain sequence <400> 35 Phe Glu Tyr Ala Tyr Ile Ile His Ala Tyr Lys Asp Lys Asp Trp 1 5 10 15 Val Trp Glu His Phe Ser Ser Met Glu Lys Glu Asp Gln Ser Leu Lys 20 25 30 Phe Cys Leu Glu Glu Arg Asp Phe Glu Ala Gly Val Phe Glu Leu Glu 35 40 45 Ala Ile Val Asn Ser Ile Lys Arg Ser Arg Lys Ile Ile Phe Val Ile 50 55 60 Thr His Leu Leu Lys Asp Pro Leu Cys Lys Arg Phe Lys Val His 65 70 75 80 His Ala Val Gln Gln Ala Ile Glu Gln Asn Leu Asp Ser Ile Ile Leu 85 90 95 Val Phe Leu Glu Glu Ile Pro Asp Tyr Lys Leu Asn His Ala Leu Cys 100 105 110 Leu Arg Arg Gly Met Phe Lys Ser His Cys Ile Leu Asn Trp Pro Val 115 120 125 Gln Lys Glu Arg Ile Gly Ala Phe Arg His Lys Leu Gln Val Ala Leu 130 135 140 Gly Ser Lys Asn Ser Val His 145 150 <210> 36 <211> 168 <212> PRT <213> Artificial <220> <223> TLR4 signaling domain sequence <400> 36 Asn Ile Tyr Asp Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp 1 5 10 15 Val Arg Asn Glu Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe 20 25 30 Gln Leu Cys Leu His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala 35 40 45 Ala Asn Ile Ile His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val 50 55 60 Val Val Ser Gln His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr 65 70 75 80 Glu Ile Ala Gln Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile 85 90 95 Phe Ile Val Leu Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val 100 105 110 Glu Leu Tyr Arg Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp 115 120 125 Ser Val Leu Gly Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu 130 135 140 Leu Asp Gly Lys Ser Trp Asn Pro Glu Gly Thr Val Gly Thr Gly Cys 145 150 155 160 Asn Trp Gln Glu Ala Thr Ser Ile 165 <210> 37 <211> 63 <212> DNA <213> Artificial <220> <223> nucleic acid sequence encoding a CD8 leader sequence <400> 37 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccg 63 <210> 38 <211> 45 <212> DNA <213> Artificial <220> <223> nucleic acid sequence encoding a linker <400> 38 ggtggaggtg gttcgggagg tggaggtagc ggaggtggtg gatct 45 <210> 39 <211> 135 <212> DNA <213> Artificial <220> <223> nucleic acid sequence encoding a CD8 hinge <400> 39 accactaccc ctgcaccgcg accaccaaca ccggcgccca ccattgcgtc gcagcctctg 60 tccctgcgcc cagaagcatg ccgtccagca gcaggtggtg cagttcatac tcgtggtctg 120 gatttcgcct gtgat 135 <210> 40 <211> 57 <212> DNA <213> Artificial <220> <223> nucleic acid sequence encoding a CD28 hinge <400> 40 ctcgagccca aatcttgtga caaaactcac acatgcccac cgtgcccgga tcccaaa 57 <210> 41 <211> 72 <212> DNA <213> Artificial <220> <223> nucleic acid sequence encoding a CD8 transmembrane domain <400> 41 atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60 accctttact gc 72 <210> 42 <211> 81 <212> DNA <213> Artificial <220> <223> nucleic acid sequence encoding a CD28 transmembrane domain <400> 42 ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60 gcctttatta ttttctgggt g 81 <210> 43 <211> 123 <212> DNA <213> Artificial <220> <223> nucleic acid sequence encoding a CD28 signaling domain <400> 43 aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60 gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120 tcc 123 <210> 44 <211> 336 <212> DNA <213> Artificial <220> <223> nucleic acid sequence encoding a CD3zeta signaling domain <400> 44 agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagatagat ggcggaggcc tacagtgaga ttgggatga aggcgagcgc 240 cggaggggca aggggcacga tggctttac cagggtctca gtacagccac caggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgc 336 <210> 45 <211> 126 <212> DNA <213> Artificial <220> <223> nucleic acid sequence encoding and 41BB signaling domain <400> 45 aaacggggca gaagaaact cctgtatata ttcaacaac catttatgag accagtacaa 60 actactcaag aggagatgg ctgtagctgc cgatttccag agagaga aggagatgt 120 failure 126

Claims

1. A method for 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.

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

3. The method according to any one of claims 1 to 2, wherein the MSC is adipose-derived MSC.

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

5. The method according to any one of claims 1 to 4, wherein the CAR includes a single-stranded variable fragment (scFv).

6. The method according to claim 5, wherein the scFv comprises a light chain and a heavy chain derived from an anti-CDH1 antibody.

7. The method according to claim 6, wherein the anti-CDH1 antibody is hSC10.

17.

8. The method according to any one of claims 1 to 7, wherein the MSC is operated to express the CAR ex vivo prior to the administration.

9. The method according to any one of claims 1 to 8, wherein the symptoms of colitis are reduced by at least 10 percent.

10. A method for treating a mammal at risk of developing colitis, the method comprising administering to the mammal a composition comprising mesenchymal stem cells (MSCs) containing an exogenous nucleic acid encoding a chimeric antigen receptor (CAR) that targets an epithelial-specific antigen, wherein the MSCs express the CAR.

11. The method according to claim 10, wherein the mammal is a human.

12. The method according to any one of claims 10 to 11, wherein the MSC is adipose-derived MSC.

13. The method according to any one of claims 10 to 12, wherein the epithelial-specific antigen is E-cadherin (ECAD).

14. The method according to any one of claims 10 to 13, wherein the CAR comprises a single-stranded variable fragment (scFv).

15. The method according to claim 14, wherein the scFv comprises a light chain and a heavy chain derived from an anti-CDH1 antibody.

16. The method according to claim 15, wherein the anti-CDH1 antibody is hSC10.

17.

17. The method according to any one of claims 10 to 16, wherein the MSC is operated to express the CAR ex vivo prior to the administration.

18. A method for 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 nerve-specific antigen, wherein the MSCs express the CAR.

19. The method according to claim 18, wherein the mammal is a human.

20. The method according to any one of claims 18 to 19, wherein the MSC is adipose-derived MSC.

21. The method according to any one of claims 18 to 20, wherein the nerve-specific antigen is myelin oligodendrocyte glycoprotein (MOG).

22. The method according to claim 21, wherein the CAR includes a single-stranded variable fragment (scFv).

23. The method according to claim 22, wherein the scFv comprises a light chain and a heavy chain derived from an anti-MOG antibody.

24. The method according to claim 23, wherein the anti-MOG antibody is 8-18C5.

25. The method according to any one of claims 18 to 24, wherein the MSC is operated to express the CAR ex vivo prior to the administration.

26. The method according to any one of claims 18 to 25, wherein the symptoms of the multiple sclerosis are reduced by at least 10 percent.

27. The method according to any one of claims 18 to 26, wherein the MSC further comprises an exogenous nucleic acid encoding a polypeptide capable of promoting neuronal differentiation, and the MSC expresses the polypeptide.

28. The method according to 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.

29. A method for 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 nerve-specific antigen, wherein the MSCs express the CAR.

30. The method according to claim 29, wherein the mammal is a human.

31. The method according to any one of claims 29 to 30, wherein the MSCs are fat-derived MSCs.

32. The method according to any one of claims 29 to 31, wherein the nerve-specific antigen is myelin oligodendrocyte glycoprotein (MOG).

33. The method according to claim 32, wherein the CAR includes a single-stranded variable fragment (scFv).

34. The method according to claim 33, wherein the scFv comprises a light chain and a heavy chain derived from an anti-MOG antibody.

35. The method according to claim 34, wherein the anti-MOG antibody is 8-18C5.

36. The method according to any one of claims 29 to 35, wherein the MSC is operated to express the CAR ex vivo prior to the administration.

37. A method for 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 nerve-specific antigen, wherein the MSCs express the CAR.

38. The method according to claim 37, wherein the mammal is a human.

39. The method according to any one of claims 37 to 38, wherein the MSC is adipose-derived MSC.

40. The method according to any one of claims 37 to 39, wherein the nerve-specific antigen is myelin oligodendrocyte glycoprotein (MOG).

41. The method according to claim 40, wherein the CAR includes a single-stranded variable fragment (scFv).

42. The method according to claim 41, wherein the scFv comprises a light chain and a heavy chain derived from an anti-MOG antibody.

43. The method according to claim 42, wherein the anti-MOG antibody is 8-18C5.

44. The method according to any one of claims 37 to 43, wherein the MSC is operated to express the CAR ex vivo prior to the administration.

45. The method according to any one of claims 37 to 44, wherein the symptoms of the immune-mediated encephalomyelitis are reduced by at least 10 percent.

46. The method according to any one of claims 37 to 45, wherein the MSC further comprises an exogenous nucleic acid encoding a polypeptide capable of promoting neuronal differentiation, and the MSC expresses the polypeptide.

47. The method according to claim 46, 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.

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) containing an exogenous nucleic acid encoding a chimeric antigen receptor (CAR) that targets a neuronal specific antigen, wherein the MSCs express the CAR.

49. The method according to claim 48, wherein the mammal is a human.

50. The method according to any one of claims 48 to 49, wherein the MSC is adipose-derived MSC.

51. The method according to any one of claims 48 to 50, wherein the nerve-specific antigen is myelin oligodendrocyte glycoprotein (MOG).

52. The method according to claim 51, wherein the CAR includes a single-stranded variable fragment (scFv).

53. The method according to claim 52, wherein the scFv comprises a light chain and a heavy chain derived from an anti-MOG antibody.

54. The method according to claim 53, wherein the anti-MOG antibody is 8-18C5.

55. The method according to any one of claims 48 to 54, wherein the MSC is operated to express the CAR ex vivo prior to the administration.

56. Nucleic acid constructs that encode chimeric antigen receptors (CARs) that target nerve-specific antigens.

57. The nucleic acid construct according to claim 56, wherein the nerve-specific antigen is myelin oligodendrocyte glycoprotein (MOG).

58. The nucleic acid construct according to any one of claims 56 to 57, wherein the CAR comprises a single-stranded variable fragment (scFv).

59. The nucleic acid construct according to any one of claims 56 to 58, wherein the CAR that targets the nerve-specific antigen is encoded by the nucleic acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO:

7.

60. The nucleic acid construct according to any one of claims 56 to 59, wherein the nucleic acid construct can also encode a polypeptide that can promote neuronal differentiation.

61. The nucleic acid construct according to claim 60, 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.

62. A method for 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 heart-specific antigen, wherein the MSCs express the CAR.

63. The method according to claim 62, wherein the mammal is a human.

64. The method according to any one of claims 62 to 63, wherein the MSCs are fat-derived MSCs.

65. The method according to any one of claims 62 to 64, wherein the cardiac-specific antigen is HER2.

66. The method according to claim 65, wherein the CAR includes a single-stranded variable fragment (scFv).

67. The method according to any one of claims 62 to 66, wherein the MSC is operated to express the CAR ex vivo prior to the administration.

68. The method according to any one of claims 62 to 67, wherein the symptoms of myocarditis are reduced by at least 10 percent.

69. The method according to any one of claims 62 to 68, wherein the MSC further comprises an exogenous nucleic acid encoding a polypeptide that can promote cardiac cell differentiation, and the MSC expresses the polypeptide.

70. The method according to claim 69, wherein the polypeptide capable of promoting neural differentiation is selected from the group consisting of GATA4 polypeptide, MEF2C polypeptide, TBX5 polypeptide, ERRG polypeptide, MESP1 polypeptide, and any combination thereof.

71. A method for 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 heart-specific antigen, wherein the MSCs express the CAR.

72. The method according to claim 71, wherein the mammal is a human.

73. The method according to any one of claims 71 to 72, wherein the MSC is adipose-derived MSC.

74. The method according to any one of claims 71 to 73, wherein the cardiac-specific antigen is HER2.

75. The method according to claim 74, wherein the CAR includes a single-stranded variable fragment (scFv).

76. The method according to any one of claims 71 to 75, wherein the MSC is operated to express the CAR ex vivo prior to the administration.

77. A nucleic acid construct that encodes a chimeric antigen receptor (CAR) that targets a cardiac-specific antigen.

78. The nucleic acid construct according to claim 77, wherein the cardiac-specific antigen is HER2.

79. The nucleic acid construct according to any one of claims 77 to 78, wherein the CAR comprises a single-stranded variable fragment (scFv).

80. The nucleic acid construct according to any one of claims 77 to 79, wherein the CAR that targets the heart-specific antigen is encoded by the nucleic acid sequence shown in Sequence ID No.

9.

81. The nucleic acid construct according to any one of claims 77 to 80, wherein the nucleic acid construct can also encode a polypeptide that can promote cardiac cell differentiation.

82. The nucleic acid construct according to claim 81, wherein the polypeptide capable of promoting neuronal differentiation is selected from the group consisting of GATA4 polypeptide, MEF2C polypeptide, TBX5 polypeptide, ERRG polypeptide, MESP1 polypeptide, and any combination thereof.

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 the binding of the CAR to the antigen in the mammal results in the suppression of the immune response in the mammal.

84. The method according to claim 83, wherein the mammal is a human.

85. The method according to any one of claims 83 to 84, wherein the MSC is adipose-derived MSC.

86. The method according to any one of claims 83 to 85, wherein the antigen is E-cadherin (ECAD).

87. The method according to any one of claims 83 to 86, wherein the CAR comprises a single-stranded variable fragment (scFv).

88. The method according to any one of claims 83 to 87, wherein the MSC is operated to express the CAR ex vivo prior to the administration.

89. The method according to any one of claims 1 to 55, 62 to 76, and 83 to 88, wherein the CAR comprises a CD28 or TLR4 signaling domain.

90. The method according to claim 89, wherein the CAR comprises a CD28 signaling domain.

91. The method according to claim 89, wherein the CAR comprises a TLR4 signaling domain.

92. The nucleic acid according to any one of claims 56 to 61 and 77 to 82, wherein the CAR comprises a CD28 or TLR4 signaling domain.

93. The nucleic acid according to claim 92, wherein the CAR comprises a CD28 signaling domain.

94. The nucleic acid according to claim 92, wherein the CAR comprises a TLR4 signaling domain.

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