METHODS AND MATERIALS FOR USING ENGINEERED MESENCHYMAL STEM CELLS TO TREAT INFLAMMATORY CONDITIONS AND DEGENERATIVE DISEASES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No. 62/932,610, filed November 8, 2019. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.

BACKGROUND

1. Technical Field

This document relates to methods and materials for using engineered mesenchymal stem cells (MSCs) to treat a mammal ( e.g . , a human) having, or at risk of developing, an inflammatory disease or condition and/or having, or at risk of developing, a degenerative disease. For example, this document provides engineered MSCs designed to express an antigen receptor (e.g. , a chimeric antigen receptor (CAR)) having the ability to bind (e.g., bind specifically) to an antigen (e.g., a tissue-specific antigen). Such engineered MSCs can exert an immunosuppressive effect (e.g., can reduce or eliminate an immune response) in a targeted tissue and/or can regenerate tissue specific cells. In some cases, an MSC engineered to express an antigen receptor having the ability to bind to an antigen (e.g., a tissue specific cell that expresses the antigen targeted by the MSC) also can be engineered to express a polypeptide that can promote differentiation into a tissue specific cell. For example, a MSC can be designed to include nucleic acid encoding a polypeptide that can promote tissue specific cell differentiation and can differentiate into a tissue specific cell (e.g. , a cardiac cell or a neuron). This document also provides methods for administering one or more MSCs expressing an antigen receptor having the ability to bind (e.g., specifically bind) a tissue-specific antigen to a mammal (e.g., a human) having, or at risk of developing, an inflammatory disease or condition to treat that inflammatory disease or condition within the mammal. 2. Background Information

Inflammation is a common factor in many diseases. In 2015, an estimated 1.3% of U.S. adults (about 3 million people) reported being diagnosed with IBD (either Crohn’s disease or ulcerative colitis; Dahlhamer et al., MMWRMorb Mortal Wkly Rep. 2016, 65(42): 1166-1169 (2015)).

SUMMARY

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

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

Having the ability to treat inflammatory diseases and conditions as described herein can allow clinicians and patients to reduce inflammation within patients in an effective and efficient manner.

In general, one aspect of this document features methods for treating a mammal having colitis. The methods can include, or consist essentially of, administering to said mammal a composition including MSCs containing exogenous nucleic acid encoding a CAR targeting an epithelial-specific antigen, where the MSCs express the CAR. The mammal can be a human. The MSCs can be adipose derived-MSCs. The epithelial- specific antigen can be ECAD. The CAR can include a single chain variable fragment (scFv). The scFv can include a light chain and a heavy chain from an anti-CDHl antibody. The anti-CDHl antibody can be hSC10.17. The MSCs, prior to the administration, can be engineered to express the CAR ex vivo. A symptom of the colitis can be reduced at least 10 percent. The CAR can include a CD28 or TLR4 signaling domain.

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

In another aspect, one aspect of this document features methods for treating a mammal having multiple sclerosis. The methods can include, or consist essentially of, administering to the mammal a composition including MSCs containing exogenous nucleic acid encoding a CAR targeting a neural-specific antigen, where the MSCs express the CAR. The mammal can be a human. The MSCs can be adipose derived-MSCs. The neural-specific antigen can be MOG The CAR can include a scFv. The scFv can include a light chain and a heavy chain from an anti-MOG antibody. The anti-MOG antibody can be 8-18C5. The MSCs, prior to the administration, can be engineered to express the CAR ex vivo. A symptom of the multiple sclerosis can be reduced at least 10 percent. The MSCs also can contain exogenous nucleic acid encoding a polypeptide that can promote neural differentiation, where the MSCs express the polypeptide. The polypeptide that can promote neural cell differentiation can be a Oct3/4 polypeptide, a Klf4 polypeptide, a Sox2 polypeptide, a Glisl polypeptide, a cDMyc polypeptide, a BMP4 polypeptide, a WNT polypeptide, a FGF2 polypeptide, a SHH polypeptide, a Soxll polypeptide, a Sox2 polypeptide, a Sox3 polypeptide, aZicl polypeptide, aZic2 polypeptide, a lrxl polypeptide, a lrx2 polypeptide, alrx3 polypeptide, aFoxD4 polypeptide, aMKx2.5 polypeptide, a cTnT polypeptide, or any combinations thereof. The CAR can include a CD28 or TLR4 signaling domain.

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

In another aspect, one aspect of this document features methods for treating a mammal having immune mediated encephalomyelitis. The methods can include, or consist essentially of, administering to the mammal a composition including MSCs containing exogenous nucleic acid encoding a CAR targeting a neural-specific antigen, where the MSCs express the CAR. The mammal can be a human. The MSCs can be adipose derived-MSCs. The neural-specific antigen can be MOG The CAR can include a scFv. The scFv can include a light chain and a heavy chain from an anti-MOG antibody. The anti-MOG antibody can be 8-18C5. The MSCs, prior to the administration, can be engineered to express the CAR ex vivo. A symptom of the immune mediated encephalomyelitis can be reduced at least 10 percent. The MSCs also can contain exogenous nucleic acid encoding a polypeptide that can promote neural differentiation, where the MSCs express the polypeptide. The polypeptide that can promote neural differentiation can be a Oct3/4 polypeptide, a Klf4 polypeptide, a Sox2 polypeptide, a Glisl polypeptide, a cDMyc polypeptide, a BMP4 polypeptide, a WNT polypeptide, a FGF2 polypeptide, a SHH polypeptide, a Soxll polypeptide, a Sox2 polypeptide, a Sox3 polypeptide, aZicl polypeptide, aZic2 polypeptide, a lrxl polypeptide, a lrx2 polypeptide, alrx3 polypeptide, aFoxD4 polypeptide, aMKx2.5 polypeptide, a cTnT polypeptide, or any combinations thereof. The CAR can include a CD28 or TLR4 signaling domain.

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

In another aspect, one aspect of this document features nucleic acid constructs encoding a CAR targeting a neural-specific antigen. The neural-specific antigen can be MOG The CAR can include a scFv. The CAR targeting the neural-specific antigen can be encoded by a nucleic acid sequence set forth in SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7. The nucleic acid construct also can encode a polypeptide that can promote neural differentiation. The polypeptide that can promote neural differentiation can be a Oct3/4 polypeptide, aKlf4 polypeptide, a Sox2 polypeptide, a Glisl polypeptide, a cDMyc polypeptide, a BMP4 polypeptide, a WNT polypeptide, a FGF2 polypeptide, a SHH polypeptide, a Soxll polypeptide, a Sox2 polypeptide, a Sox3 polypeptide, aZicl polypeptide, a Zic2 polypeptide, a Irxl polypeptide, a Irx2 polypeptide, a Irx3 polypeptide, a FoxD4 polypeptide, aMKx2.5 polypeptide, a cTnT polypeptide, or any combinations thereof. The CAR can include a CD28 or TLR4 signaling domain.

In another aspect, one aspect of this document features methods for treating a mammal having myocarditis. The methods can include, or consist essentially of, administering to the mammal a composition including MSCs containing exogenous nucleic acid encoding a CAR targeting a cardiac-specific antigen, where the MSCs express the CAR. The mammal can be a human. The MSCs can be adipose derived- MSCs. The cardiac-specific antigen can be HER2. The CAR can include a scFv. The MSCs, prior to the administration, can be engineered to express the CAR ex vivo. A symptom of the myocarditis can be reduced at least 10 percent. The MSCs also can contain exogenous nucleic acid encoding a polypeptide that can promote cardiac cell differentiation, where the MSCs express the polypeptide. The polypeptide that can promote neural differentiation can be a GATA4 polypeptide, a MEF2C polypeptide, a TBX5 polypeptide, a ERRG polypeptide, aMESPl polypeptide, and any combinations thereof. The CAR can include a CD28 or TLR4 signaling domain.

In another aspect, one aspect of this document features methods for treating a mammal at risk of developing myocarditis. The methods can include, or consist essentially of, administering to the mammal a composition including MSCs containing exogenous nucleic acid encoding a CAR targeting a cardiac-specific antigen, where the MSCs express the CAR. The mammal can be a human. The MSCs can be adipose derived-MSCs. The cardiac-specific antigen can be HER2. The CAR can include a scFv. The MSCs, prior to the administration, can be engineered to express the CAR ex vivo.

The CAR can include a CD28 or TLR4 signaling domain.

In another aspect, one aspect of this document features nucleic acid constructs encoding a CAR targeting a cardiac-specific antigen. The cardiac-specific antigen can be HER2. The CAR can include a scFv. The CAR targeting said cardiac-specific antigen can be encoded by a nucleic acid sequence set forth in SEQ ID NO:9. The nucleic acid construct also can encode a polypeptide that can promote cardiac cell differentiation. The polypeptide that can promote neural differentiation can be a GATA4 polypeptide, a MEF2C polypeptide, aTBX5 polypeptide, a ERRG polypeptide, aMESPl polypeptide, or any combinations thereof. The CAR can include a CD28 or TLR4 signaling domain.

Unless otherwise defined, 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. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. DESCRIPTION OF THE DRAWINGS

Figure 1. Enhanced Lentiviral Transduction of MSCs.

Figure 2. MSC expression of CAR19 (MSC19).

Figure 3. Stability of MSC-CAR19 expression after passage 10+.

Figures 4A-4B. A. MSC suppression of antigen specific CAR-T cell proliferation. B. MSC suppression of antigen specific CAR-T cell proliferation.

Figure 5. Schematic representations of nucleic acid constructs encoding exemplary CAR19s.

Figure 6. MSC expression of CAR- EC AD.

Figure 7. MSCs suppress T cell proliferation but not CAR-T cell proliferation. Figure 8. MSC-CAR mediated suppression of T-cell proliferation.

Figures 9A-9B. A. MSC-CAR mediated suppression of activated T-cell and CART-cells. B. Suppression of antigen-specific CAR-T proliferation by CART19 + NALM6.

Figures 10A-10G. Sternness retention of MSCs. A. Flow cytometry analysis of CD90. B. Flow cytometry analysis of CD105. C. Flow cytometry analysis of CD73. D.

Flow cytometry analysis of CD34. E. Flow cytometry analysis of CD45. F. Flow cytometry analysis of HLA-DR. G. Flow cytometry analysis of CD 14.

Figure 11. MSC expression of CAR-MOG.

Figures 12A-12B. A. Nucleic acid sequence (SEQ ID NO:l) encoding an exemplary CAR-ECAD. B. Amino acid sequence (SEQ ID NO:2) of an exemplary CAR- ECAD.

Figures 13A-13F. A. Nucleic acid sequence (SEQ ID NO:3) encoding an exemplary CAR-MOG. B. Amino acid sequence (SEQ ID NO:4) of an exemplary CAR- MOG. C. Nucleic acid sequence (SEQ ID NO:5) encoding an exemplary CAR-MOG. D. Amino acid sequence (SEQ ID NO:6) of an exemplary CAR-MOG. E. Nucleic acid sequence (SEQ ID NO:7) encoding an exemplary CAR-MOG. F. Amino acid sequence (SEQ ID NO:8) of an exemplary CAR-MOG. Figures 14A-14B. A. Nucleic acid sequence (SEQ ID NO:9) encoding an exemplary CAR-HER2. B. Amino acid sequence (SEQ ID NO: 10) of an exemplary CAR-HER2.

Figure 15A is a graph plotting the percent of MSCs that are CAR positive following lentivirus transduction of MSC with K002 (CD19 directed CAR) compared to UTD (untransduced MSC). Figure 15B is a graph plotting the percent of MSCs expressing the indicated marker two days after lentivirus transduction with CAR19 compared to untransduced MSC (UTD). The MSCs retain markers of sternness.

Figure 16 is a graph plotting the absolute number of live CD3 cells after 5 days of stimulation with CD3/CD28 beads, following co-culture with untransduced MSC (MSC- UTD), or MSC-CAR19 (CD28 containing CAR19, K122), or MSC-CAR19 (CD137 containing CAR19, K002), or with no MSCs. T cells are activated with CD3/CD28 beads and then co-cultured with different MSC conditions either in medium alone, or in the presence of irradiated CD19 ⁺ cells as a strategy to stimulate MSC-CAR19 through the CAR. Proliferation of CD3 is inhibited in the presence of MSC-CAR19 (containing CD28 signaling domain), but not in the presence of MSC-CAR19 (containing CD137 signaling domain), or un-transduced MSCs. T cells, MSCs, and NALM6 cells were cultured at ratio of 1:0.1 :1 E (T cells (effectors):MSCs (suppressors):T (tumor). This demonstrates that MSC-CAR cells are able to suppress T cell proliferation when MSC-CAR cells contain a CD28 signaling domain and when the MSCs are activated through the CAR upon antigen specific stimulation. * represents p value < 0.05.

Figure 17 is a graph plotting the absolute number of live MSC cells after 5 days of co-culture of untransduced MSC (MSC-UTD), or MSC-CAR19 (TLR4 containing CAR19, K142), or MSC-CAR19 (CD137 containing CAR19, K002), or MSC-CAR19 (CD28 containing CAR19, K122), with or without the irradiated CD19 ⁺ NALM6 cells. Proliferation of MSC-CAR19 containing CD28 is enhanced in the presence but not in the absence of irradiated CD19 ⁺ cells, suggesting antigen specific stimulation of MSC- CAR^ and signaling through CD28. Similarly, proliferation of MSC-CAR19 containing TLR4 is decreased in the presence but not in the absence of irradiated CD19 ⁺ cells, suggesting antigen specific stimulation of MSC-CAR19 and signaling through CD28. * represents P < 0.05; ** represents P < 0.01.

Figure 18 is a graph plotting the absolute number of live CD3 cells after 5 days of stimulation with CD3/CD28 beads, following co-culture with untransduced MSC (MSC- UTD), or MSC-CAR19 (TLR4 containing CAR19, K142), or with no MSCs. T cells are activated with CD3/CD28 beads and then co-cultured with different MSC conditions either in medium alone, or in the presence of irradiated CD19 ⁺ cells as a strategy to stimulate MSC-CAR19 through the CAR. Proliferation of CD3 is inhibited in the presence of MSC-CAR19 (containing TLR4 signaling domain, K142), but not in the presence of un-transduced MSCs. T cells, MSCs, and NALM6 cells were cultured at ratio of 1:0.1 :1 E (T cells (effectors):MSCs (suppressors):T (tumor). This demonstrates that MSC-CAR cells are able to suppress T cell proliferation when MSC-CAR cells contain a TLR4 signaling domain and when the MSCs are activated through the CAR upon antigen specific stimulation. ** represents P < 0.01.

Figure 19A is a graph plotting the absolute number of MSCs after 5 days of co culture with irradiated CD19 ⁺ NALM6 cells at 1:1 ratio of MSC:NALM6. NALM6 cells are included in this culture in order to stimulate MSC-CAR19 through the CAR. A co culture of CD19 ⁺ cells with MSC-CAR19-CD28 led to enhanced proliferation, compared to untransduced MSC (MSC-UTD), or MSC-K002, or MSC-K122, or MSC-K142. This indicates that antigen specific stimulation of MSC-CAR19 through the CAR results in signaling and altered proliferation of MSCs. Figure 19B represents the MSC count on days 3 and 5 of the co-culture.

Figure 20 is a graph plotting the absolute number of MSCs in cultures following their transduction with CAR-MSC-E-cadherin or with control GFP (MSC-ZSG). Transduction with CAR-E-cadherin-CD28 led to reduced MSC proliferation, compared to untransduced MSC (MSC-UTD), or MSC-transduced with GFP (MSC-ZSG).

Figure 21 is a bar graph plotting the number of T cells per pL, following their stimulation with CD3/CD28 beads in the presence of MSCs, and CD19 ⁺ cells (to stimulate MSC-CARs), either in direct contact or in transwell experiments. A co-culture with MSC-CAR19 (containing CD28 stimulatory domain) with activated T cells, and CD19 ⁺ cells led to inhibition of T cell proliferation both when cells were in direct contact or not in direct contact in a trans-well experiment. This indicates that MSC-CAR exert their suppressive functions both through direct, cell to cell contact and through secretion of soluble inhibitory factor s/cytokines.

Figure 22 is a graph plotting the number of T cells per pL after 5 days of 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 medium alone, or in the presence of irradiated CD19 ⁺ cells as a strategy to stimulate MSC-CAR19 through the CAR. Proliferation of CD3 is inhibited in the presence of MSC-CAR19 (containing CD28 signaling domain), but not in the presence of un-transduced MSCs. T cells, MSCs, and NALM6 cells were cultured at ratio of 1 : 1 : 1 E (T cells (effectors):MSCs (suppressors):T (tumor). This demonstrates that MSC-CAR cells are able to suppress T cell proliferation when MSC-CAR cells contain a CD28 signaling domain, at different suppressor to T cell ratios, when the MSCs are activated through the CAR upon antigen specific stimulation. ** represents P < 0.01; *** represents P < 0.001.

Figures 23A-23B are graphs plotting the absolute number of live CD3 cells after 5 days of stimulation with CD3/CD28 beads, following co-culture with untransduced MSC (MSC-UTD, Figure 24A), or MSC-CAR- E-cadherin (CD28 containing CAR-E-cadherin, Figure 24B), in the presence or absence of the E-cadherin ⁺ cell line MCF-7, at different effector: suppressor (E:S) ratios. The co-culture of MSC-CAR-E-cadherin with T cells results in suppression of their antigen specific proliferation in the presence of the E- cadhertiC cell line MCF-7, at a low effector: suppressor ratio.

Figure 24A is a graph plotting bioluminescence from luciferase ⁺ /E-cadherin ⁺ MCF-7 cells (lxlO ⁶ ) that were injected into NSG mice that were then treated with CAR- ECAD T cells in combination with either no MSCs, untransduced MSCs (MSC-UTD), or MSC-ECAD-CAR with CD28 signaling domain. Mice treated with MSC-ECAD exhibited more bioluminescence demonstrating that the MSC-ECAD inhibited the antitumor effects of the CAR-ECAD T cells. Figure 24B is a bar graph plotting the absolute number of CD3 ⁺ T cells in the NSG mice treated with CAR-ECAD T cells and either no MSCs, untransduced MSCs (MSC-UTD), or MSC-ECAD at day 3.

Figure 25 A is a graph plotting bioluminescence from luciferase ⁺ MSC-CAR-E- cadherin cells that were administered into immunocompromised NSG mice. Figure 25B contains representative images of the bioluminescence from representative mice.

DETAILED DESCRIPTION

This document provides methods and materials involved in treating a mammal ( e.g ., a human) having, or at risk of developing, an inflammatory disease or condition and/or having, or at risk of developing, a degenerative disease. For example, one or more MSCs designed to express an antigen receptor (e.g., a CAR) capable of binding (e.g., specifically binding) to a tissue-specific antigen can be administered to a mammal to induce an immunosuppressive response (e.g., to reduce or eliminate an inflammatory immune response) within the mammal. In some cases, a CAR targeting an epithelial- specific antigen (e.g. , EC AD) can be expressed by a MSC to target the MSC to epithelial tissues in a manner effective to treat a disease or disorder characterized by inflammation of an epithelial tissue (e.g., colitis). For example, MSCs engineered to express a CAR that can target (e.g., can target and bind to) an antigen (e.g., a cell surface antigen) expressed by epithelial cells (e.g., an epithelial-specific antigen or an epithelial antigen) can be administered to a mammal (e.g., a human) to treat or slow the progression of a disease or disorder characterized by inflammation and/or degeneration of epithelial tissue such as colitis, hepatitis, pneumonitis, asthma, pancreatitis, pulmonary fibrosis, colon strictures, colon fistulas, glomerulonephritis, renal infarction, or liver infarction.

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

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

In some cases, one or more MSCs designed to express a CAR capable of binding (e.g., specifically binding) to a tissue-specific antigen (e.g., an epithelial-specific antigen) can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) having (or at risk of developing) an inflammatory disease or condition to treat that inflammatory disease or condition within the mammal. In some cases, one or more MSCs expressing a CAR capable of binding (e.g., specifically binding) to an epithelial-specific antigen (e.g., ECAD) can be administered (e.g. , by adoptive transfer) to a mammal (e.g. , a human) having, or at risk of developing, an 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 capable of binding (e.g., specifically binding) to a neural- specific antigen (e.g., MOG) can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) having, or at risk of developing, multiple sclerosis to treat that multiple sclerosis within the mammal. In some cases, one or more MSCs expressing a CAR capable of binding (e.g., specifically binding) to a neural-specific antigen (e.g., MOG) can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) having, or at risk of developing, immune mediated encephalomyelitis to treat that immune mediated encephalomyelitis within the mammal. In some cases, one or more MSCs expressing a CAR capable of binding (e.g., specifically binding) to a cardiac-specific antigen (e.g., HER2) can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) having, or at risk of developing, myocarditis to treat that myocarditis within the mammal.

In some cases, one or more MSCs expressing a CAR capable of binding (e.g., specifically binding) to a cartilage antigen (e.g., CH65, human cartilage glycoprotein-39 (HC gp-39), CD44, thymocyte anti gen- 1 (Thy-1), CD90, CD24, lymphocyte function- associated antigen-3 (LFA-3) or to an osteocyte antigen (e.g., Ell or gp38) can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) to treat or slow the progression of a disease or disorder characterized by inflammation and/or degeneration of joints such as inflammatory arthritis, degenerative arthritis, autoimmune spondyloarthritis, psoriatic arthritis, osteogenesis imperfecta, or metachromatic leukodystrophy.

In some cases, MSCs designed to express an antigen receptor (e.g., a CAR) capable of binding (e.g., specifically binding) to a tissue-specific antigen, also can be engineered to express a polypeptide that can promote differentiation into a tissue specific cell (e.g., a cardiac cell or a neuron) within the mammal. In some cases, an MSC designed to express a polypeptide that can promote differentiation can promote differentiation of a cell (e.g., the MSC and/or one or more resident progenitor cells) into a tissue specific cell (e.g., a cardiac cell or a neuron) within the mammal. In some cases, an MSC designed to express a polypeptide that can promote differentiation can promote differentiation of a resident cell (e.g., a resident progenitor cell) into a tissue specific cell (e.g., a cardiac cell or a neuron) within the mammal. Such engineered MSCs that can express a polypeptide that can express a polypeptide that can promote differentiation into any appropriate tissue specific cell (e.g., a cardiac cell or a neuron) within the mammal.

In some cases, a MSC including nucleic acid encoding a polypeptide that can promote cardiac cell differentiation can differentiate the MSC into a cardiac cell. In some cases, a MSC including nucleic acid encoding a polypeptide that can promote neural differentiation can differentiate the MSC into a neuron. For example, one or more MSCs designed to express an antigen receptor (e.g., a CAR) capable of binding (e.g., specifically binding) to a tissue-specific antigen, and also designed to differentiate into a tissue specific cell can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) having, or at risk of developing, a degenerative disease to treat that degenerative disease within the mammal. In some cases, one or more MSCs designed to differentiate into a cardiac cell (e.g., one or more MSCs including nucleic acid that can encode a polypeptide that can promote cardiac cell differentiation can be administered (e.g., by adoptive transfer) to a mammal (e.g. , a human) having, or at risk of developing, a degenerative heart disease (e.g., congestive heart failure (CHF)) to treat 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 a neuron (e.g., one or more MSCs including nucleic acid that can encode a polypeptide that can promote neural differentiation can be administered (e.g., by adoptive transfer) to a mammal (e.g., a human) having, or at risk of developing, a neurodegenerative disease (e.g., Parkinson’s disease and Alzheimer’s disease) to treat that neurodegenerative disease within the mammal (e.g., by regenerating neurons within the mammal).

In some cases, MSCs (e.g., MSCs engineered to express a CAR as described herein) can be designed to express one or more transcription factors to force their differentiation into a cardiomyocyte (e.g., BMP -4, FGF-4, FGF-basic, and/or TGF -beta). In such cases, the MSCs can be administered to a mammal (e.g., a human) to treat or slow the progression of a disease or disorder characterized by degeneration of the heart such as myocardial infarction or heart failure. In some cases, MSCs (e.g., MSCs engineered to express a CAR as described herein) can be designed to express one or more transcription factors to force their differentiation into an osteocyte (e.g., BMP -2, BMP -4, BMP-6, FGF-basic, TGF-beta, PTH, and/or WntlOb) or one or more transcription factors to force their differentiation into chrondrocytes (e.g., BMP -2, BMP-3, BMP-5, BMP-7, N-Cadherin, NCAN-1, and/or Perlecan). In such cases, the MSCs can be administered to a mammal (e.g., a human) to treat or slow the progression of a disease or disorder characterized by degeneration of the bones such as degenerative arthritis or osteogenesis imperfect.

A MSC described herein (e.g. , a MSC expressing a CAR targeting a tissue- specific antigen such as an epithelial-specific antigen, a neural-specific antigen, or a cardiac-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a tissue specific cell such as a cardiac cell or a neuron) can be any appropriate MSC. Examples of MSCs that can be used as described herein include, without limitation, adipose derived MSCs, osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells), adipocytes (fat cells), neuronal precursor stem cells (neurons), dental pulp derived MSCs, cord blood derived MSCs, and umbilical cord derived MSCs. For example, a MSC expressing a CAR and, optionally, expressing a polypeptide that can promote differentiation into a tissue specific cell targeting a tissue-specific antigen can be an adipose derived MSC.

A CAR can include an antigen-binding domain and a signaling domain. An antigen-binding domain can be any appropriate antigen-binding domain. In some cases, an antigen-binding domain can include an antibody or a fragment thereof that targets an antigen (e.g., a tissue-specific antigen such as an epithelial-specific antigen, a neural- specific antigen, or a cardiac-specific antigen). Examples of antigen-binding domains include, without limitation, an antigen-binding fragment (Fab), a variable region of an antibody heavy (VH) chain, a variable region of a light (VL) chain, a single chain variable fragment (scFv), a polypeptide, a ligand, and a cytokine. In some cases, an antigen-binding domain can target (e.g., can target and bind to) a tissue-specific antigen (e.g., an epithelial-specific antigen, a neural-specific antigen, or a cardiac-specific antigen). For example, a MSC described herein can express (e.g., can be engineered to express) a CAR that can bind to a tissue- specific antigen (e.g. , an antigen present on cells within a tissue with minimal, or no, expression on other cell types). In some cases, a MSC can be engineered to express a CAR that can target (e.g., can target and bind to) an antigen (e.g., a cell surface antigen) expressed by epithelial cells (e.g., an epithelial-specific antigen or an epithelial antigen) in a mammal (e.g., a mammal having, or at risk of developing, a disease or disorder characterized by inflammation and/or degeneration of an epithelial tissue such as colitis). An epithelial- specific antigen can be any appropriate epithelial-specific antigen. An epithelial-specific antigen can be expressed on any appropriate type of epithelial cell (e.g., gastrointestinal tract cells such as colon cells and rectal cells, skin cells, lung cells, and liver cells). In some cases, an epithelial-specific antigen can be a cell adhesion molecule (CAM). Examples of epithelial-specific antigens include, without limitation, EC AD, CD103, hSC10.17, hSClO.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, pl6, p63, p-Cadherin, PSA, surfactant, Transthyretin, VAT-1, and Vimentin. For example, a MSC-CAR engineered to target epithelial tissues can bind to ECAD. In some cases, a MSC-CAR can be engineered to express a CAR- ECAD to target ECAD expressed by epithelial cells in a mammal having, or at risk of developing, an IBD (e.g., colitis).

In some cases, a MSC can be engineered to express a CAR that can target (e.g., can target and bind to) an antigen (e.g., a cell surface antigen) expressed by neural cells (e.g., a neural-specific antigen or a neural antigen) in a mammal (e.g. , a mammal having, or at risk of developing, a disease or disorder characterized by inflammation and/or degeneration of a neural tissue such as multiple sclerosis and immune mediated encephalomyelitis). When an antigen is a neural-specific antigen, the neural-specific antigen can be any appropriate neural-specific antigen. A neural-specific antigen can be expressed on any appropriate type of neural cell (e.g., sensory neurons, motor neurons, interneurons, glial cells, and oligodendrocytes). A neural-specific antigen can be expressed on a neural cell in the central nervous system (CNS) and/or in the peripheral nervous system (PNS). In some cases, a neural-specific antigen can be a transmembrane protein. Examples of neural-specific antigens include, without limitation, MOG, MBP, PDGF receptor alpha, OSP, SOX10, Olig 1, Olig 2, Olig 3, and NG2. For example, a MSC-CAR engineered to target neural tissues can bind to MOG In some cases, a MSC- CAR can be engineered to express a CAR-MOG to target MOG expressed by neural cells in a mammal having, or at risk of developing, multiple sclerosis. In some cases, a MSC- CAR can be engineered to express a CAR-MOG to target MOG expressed by neural cells in a mammal having, or at risk of developing, immune mediated encephalomyelitis.

In some cases, a MSC can be engineered to express a CAR that can target (e.g., can target and bind to) an antigen (e.g., a cell surface antigen) expressed by cardiac cells (e.g., a cardiac-specific antigen or a cardiac antigen) in a mammal (e.g., a mammal having, or at risk of developing, a disease or disorder characterized by inflammation and/or degeneration of a cardiac tissue such as myocarditis). When an antigen is a cardiac-specific antigen, the cardiac-specific antigen can be any appropriate cardiac- specific antigen. A cardiac-specific antigen can be expressed on any appropriate type of cardiac cell (e.g, cardiomyocytes and endocardial cells). An example of a cardiac- specific antigen includes, without limitation, HER2. For example, a MSC-CAR engineered to target cardiac cells can bind to HER2. In some cases, a MSC-CAR can be engineered to express a CAR-HER2 to target HER2 expressed by cardiac cells in a mammal having, or at risk of developing, myocarditis.

A signaling domain can be any appropriate signaling domain. In some cases, a signaling domain of a CAR to be used within a MSC as described herein can be an intracellular signaling domain normally found within T cells or NK cells. Examples of signaling domains that can be used as described herein include, without limitation, CD3zeta signaling domains, CD28 signaling domains, Toll-like receptors (TLRs) (e.g., TLR3 and TLR4), 4-1 BB, 0X40, ICOS, CD2, and promotors (e.g, specific promotors such as cardiac specific promotors, neural specific promotors, T cell specific promotors, GI, pulmonary specific promotors, joint specific promotors, and cartilage specific promotors). In some cases, a signaling domain of a CAR expressed by a MSC as described herein can, upon activation of the CAR, induce the MSC to produce one or more cytokines (e.g., anti-inflammatory cytokines). Examples of cytokines that an engineered CAR of a MSC described herein can induce the MSC to produce include, without limitation, IFN-B, IL-10, IL-4, IL-2, MIP1B, IFNg, MIPla, GM-CSF, IL-6, TNFa, and TNFb.

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

Also provided herein are CARs and constructs (e.g. , nucleic acid constructs) encoding CARs described herein (e.g., CARs targeting a tissue-specific antigen such as an epithelial-specific antigen, a neural-specific antigen, or a cardiac-specific antigen).

For example, a construct encoding a CAR targeting ECAD (e.g., a CAR-ECAD) can include a nucleic acid sequence encoding one or more molecules that bind ECAD described herein. In some cases, a CAR-ECAD can include an anti-EC AD antibody (e.g., hSC10.17 and hSClO.178) heavy chain and an anti-ECAD antibody (e.g., hSC10.17 and hSClO.178) light chain. For example, a construct encoding a CAR targeting MOG (e.g., a CAR-MOG) can include a nucleic acid sequence encoding one or more molecules that bind MOG described herein. In some cases, a CAR-MOG can 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 targeting HER2 (e.g., a CAR-HER2) can include a nucleic acid sequence encoding one or more molecules that bind HER2 described herein. In some cases, a CAR-HER2 can include an anti-HER2 antibody (e.g, 4D5) heavy chain and an anti-ECAD antibody (e.g., 4D5) light chain. Exemplary nucleic acid sequences that can encode one or more molecules that bind a tissue-specific antigen described herein that can be included in a construct described herein include, without limitation, those that encode the following amino acid sequences: anti-ECAD antibody heavy chain (SEQ ID NO:ll)

QILLV Q S GPELKKP GET VKIS CKASNYTFTD Y GMHWVKQ AP GKGLKWMGWINP KTGVASYADDFKGRFAFSLETSASTAYLQINNLENEDTSIYFCARFFDYWGQGTTL TVSS anti-ECAD antibody light chain (SEQ ID NO: 12)

DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSDGNTYLEWYQQRPGQSPRRLIYKV

SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHAPWTFGGGTKVEI anti-ECAD antibody light chain (SEQ ID NO: 13)

DIVLTQSPLSLLVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPNLLIFKVS NRF S GVPDRF S GS GS GTDFTLRIS RYE AEDLGVYF C S QTTH V WTF GGGTKLEIK anti-ECAD scFv clone 5 (SEQ ID NO:55; light chain followed by linker in bold/underlined followed by heavy chain)

QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGHYPHWFQQRPGQAPKSLIYDIRS KY S STP ARE S GSLLGGKAALT V S GV QPEDEAE YY CLLYY GGAQ VF GGGTKLTVL GEGKSSGSGSESKASEVOLVESGAEVKKPGASVKVSCKASGYMFTGYFLHWVR QAPGQGLEWMGWINPNSGDTNYPQKFQGRVTMTRDTSITTAYMELSRLTSNDT AMYYCARRAISLVRGIISNQFDPWGQGTLVTVSS anti-ECAD scFv clone 6 (SEQ ID NO:56; light chain followed by linker in bold/underlined followed by heavy chain)

LPVLTQPP S ASGTPGQRVTISC SGS S SNIGSNYVYWY QQLPGTAPKLLIYRNNQRP SGVPDRFSGSKSGTSASLAISGLQSEDEADYYCASWDTSLRAWVF GGGTKLTVL GEGKSSGSGSESKASEVOLVOSGGGLVOPGGSLRLSCAASGFTFSSYSMNWVRO APGKGLEWVS YIS S S S STIYYVDS VKGRFTIS RDNAKNSLYLQMDS LRAEDTAVY Y C ARGGRVL V GALFD YW GQGTL VT V S S anti-ECAD scFv clone 7 (SEQ ID NO:57; heavy chain followed by linker in bold/underlined followed by light chain)

EV QL VE S GTD VKKP GAS VT V S CKAS GYTFT AYYIHW VRQ APGQGLEWMGWINP YSGASNYAQKFLGRVTMTRDTSTNTVYMQLSSLRASDTAMYYCAKAACGSNG CYMREFDYWGOGTLVTVSSSEGKSSGSGSESKASDIVMTOSPLSLPVTLGOPASI SCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDF TLKISRVEAEDV GIYY CMQGTYWPGAF GQGTKVDIK anti-ECAD scFv clone 14 (SEQ ID NO:58; light chain followed by linker in bold/underlined followed by heavy chain) SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPS GIPDRF S GS S S GNTASLTIT GAQ AEDE AD YY CNSRD S S GNP VF GGGTKLTVLGEG KSSGSGSESKASEV QLVQ S GGGLVKPGGSLRL S C AAS GFTF SD YYM S WIRQ APG KGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCA RAQRQ W GAFD Y W GQGTLVT V S S anti-MOG antibody heavy chain (SEQ ID NO: 14)

EVKLHESGAGLVKPGASVEISCKATGYTFSSFWIEWVKQRPGHGLEWIGEILPGR GRTNYNEKFKGKATFTAETS SNTAYMQLS SLTSEDSAVYY CATGNTMVNMPYWG QGTTVTVSS anti-MOG antibody light chain (SEQ ID NO:15)

DIELTQSPSSLAVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGLPPKLLIY

GASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSYPLTFGAGTKLE

IK anti-HER2 antibody heavy chain (SEQ ID NO: 16)

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTN

GYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMD

YWGQGTLVTVSSASTKGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG

LDFACD anti-HER2 antibody light chain (SEQ ID NO: 17)

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYS

GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAP

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

Exemplary nucleic acid sequences that can encode one or more molecules that bind a tissue-specific antigen described herein that can be included in a construct described herein are as follows. anti-ECAD antibody heavy chain (SEQ ID NO:18)

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCC TGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACT GGGTCC GCC AGGCTCC AGGGAAGGGGCT GGAGT GGGTT GC ATAC ATTACTACT AGAAGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCC AGAGACAATGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCG AGGACACGGCTGTGTATTACTGTACTAGAGAACCCCTAACTGGATACTATGCTA TGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAG anti-ECAD antibody heavy chain (SEQ ID NO:19)

CAGATCCTGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAG TCAAGATCTCCTGCAAGGCTTCTAATTATACCTTCACAGACTATGGAATGCACT GGGT GA AGC AGGCT C C AGGA A AGGGTTTA A AGT GGAT GGGCT GGATA A AC C C C AAGACT GGT GT GGC AT C ATAT GC AGAT GACTT C AAGGGAAGATTT GC CTTCTC TTTGGAAACCTCTGCCAGCACTGCCTATTTGC AGATCAACAACCTCGAAAATG AGGAC AC GTCTATATATTT CT GT GCTAGATTTTTT GACTACT GGGGCC AAGGC A CCACTCTCACAGTCT CCTCA anti-ECAD antibody light chain (SEQ ID NO:20) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCC GGCCTCCATCTCCTGCAGGTCTAGTCAAAGCATCGTACACAGTGATGGAAACA CCTACTTGGAATGGTATCAGCAGAGGCCAGGCCAATCTCCAAGGCGCCTAATTT ATAAGGTTTCTAACCGGTTCTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGG T C AGGC ACT GATTT C AC ACT GAAAAT C AGC AGGGT GGAGGCT GAGGAT GTT G GGGTTATTACTGCTTT C AAGGTTC AC AT GCTCCGT GGACGTTCGGTGGAGGC A CC AAGGT GGAAAT C AAAC anti-ECAD antibody light chain (SEQ ID NO:21)

GATATTGTGCTGACACAGTCTCCACTCTCCCTGCTTGTCAGTCTTGGAGATCAA GCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACC TATTTACATTGGTATCTGCAGAAGCCAGGCCAGTCTCCAAACCTCCTGATCTTC AAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATC AGGGACAGATTTCACACTCAGGATCAGCAGAGTGGAGGCTGAGGATCTGGGA GTTTATTTCTGCTCTCAAACTACACATGTGTGGACGTTCGGTGGAGGCACCAA GCT GGAA AT C AAA anti-ECAD scFv clone 5 (SEQ ID NO:59; light chain followed by linker in bold/underlined followed by heavy chain)

CAGGCTGTGGTGACCCAGGAGCCCTCATTGACTGTGTCCCCAGGAGGGACAG TCACTCTCACCTGTGGCTCCAGCACTGGAGCTGTCACCAGTGGTCATTATCCC CACTGGTTCCAGCAGAGGCCTGGCCAGGCCCCCAAGTCACTGATTTATGATAT AAGGAGCAAATACTCCTCAACACCTGCCCGGTTCTCAGGCTCCCTCCTTGGGG GCAAAGCTGCCCTGACAGTGTCAGGTGTGCAGCCTGAGGACGAGGCTGAATA TT ACT GCCT GCT CT ACT AT GGT GGT GCT C AGGT GTTCGGCGGGGGGACC AAGC TGACCGTCCTAGGTGAGGGTAAATCTTCCGGATCTGGTTCCGAATCCAAA GCTAGCGAGGT GC AGCT GGT GGAGT CT GGGGCT GAGGT GAAGAAGCCTGGG GCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACATGTTCACCGGCTACTT TCTGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGG ATCAACCCTAACAGTGGTGACACAAACTATCCACAGAAATTTCAGGGCAGGG TCACCATGACCAGGGACACGTCCATCACCACAGCCTACATGGAACTGAGCAG GCTGACTTCCAACGACACGGCCATGTACTATTGTGCGAGGAGGGCTATTTCTC TGGTTCGGGGAATTATTAGCAACCAGTTCGACCCCTGGGGCCAGGGAACCCT GGTCACCGTCTCCTCA anti-ECAD scFv clone 6 (SEQ ID NO:60; light chain followed by linker in bold/underlined followed by heavy chain)

CTGCCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG TCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATTATGTCTAC TGGTACCAGCAACTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATA ATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACC TCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTA TTGTGCATCATGGGATACCAGCCTGCGTGCCTGGGTGTTCGGCGGAGGGACC AAGCTGACCGTCCTAGGTGAGGGTAAATCTTCCGGATCTGGTTCCGAATC CAAAGCTAGCGAGGT GC AGCT GGT GC AGT CT GGGGGAGGCTT GGT AC AGCC TGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCT ATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTC ATACATTAGTAGTAGTAGTAGTACCATATACTACGTAGACTCTGTGAAGGGC CGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGG ACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGAGGTCG GGTGTTAGTGGGAGCTCTATTTGACTACTGGGGCCAGGGAACCCTGGTCACC GTCTCCTCA anti-ECAD scFv clone 7 (SEQ ID NO:61; heavy chain followed by linker in bold/underlined followed by light chain)

GAGGTGCAGCTGGTGGAGTCTGGGACTGATGTGAAGAAGCCTGGGGCCTCAG TGACGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGCCTACTATATCCAC TGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACC CTTACAGTGGTGCCTCAAACTATGCACAGAAGTTTCTGGGCCGGGTCACCATG ACCAGGGACACGTCCACCAACACAGTCTACATGCAGTTGAGCAGTCTGAGGG CCTCGGACACCGCCATGTATTACTGTGCGAAAGCGGCTTGTGGTAGTAACGG CTGCTATATGAGGGAGTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCT CCTCAAGCGAGGGTAAATCTTCCGGATCTGGTTCCGAATCCAAAGCTAGC GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCC GGCCTCCATCTCCTGCAGGTCTAGTCAAAGTCTCGTATACAGTGATGGAAACA CCTACTTGAATTGGTTTCAGCAGAGGCCAGGCCAATCTCCAAGGCGCCTAATT TATAAGGTTTCTAACCGGGACTCTGGGGTCCCAGACAGATTCAGCGGCAGTG GGT C AGGC ACT GATTT C AC ACT GAAAAT C AGC AGGGT GGAGGCT GAGGAT GT TGGGATTTATTACTGCATGCAAGGTACATACTGGCCGGGCGCTTTTGGCCAGG GGACC AAGGT GGAC AT C AAA anti-ECAD scFv clone 14 (SEQ ID NO:62; light chain followed by linker in bold/underlined followed by heavy chain)

TCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGT C AGGAT C AC ATGC C AAGGAGAC AGCCT C AGAAGCT ATT AT GC GAGCT GGT AC CAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACC GGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGC TTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTA ACTCCCGGGACAGCAGTGGTAACCCGGTATTCGGCGGAGGGACCAAGCTCAC CGTCCTAGGTGAGGGTAAATCTTCCGGATCTGGTTCCGAATCCAAAGCTA GCGAGGT GC AGCT GGT GC AGT CT GGGGGAGGCTT GGT C AAGCCTGGAGGGT C CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGA GCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAG TAGTAGTGGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACC ATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGA GAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGCCC AACGGCAGTGGGG GGCCTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA anti-MOG antibody heavy chain (SEQ ID NO:22)

GAGGTGAAGCTGCACGAGAGCGGCGCAGGTCTGGTGAAGCCCGGCGCCAGC GTGGAGATCAGCTGCAAGGCCACCGGCTACACCTTCAGCAGCTTCTGGATCGA GTGGGTGAAGCAGAGACCCGGCCACGGCCTGGAGTGGATCGGCGAGATCCTG CCCGGC AGAGGC AGAAC C AACTAC AACGAGAAGTT C AAGGGC AAGGCC AC C TTCACCGCCGAGACCAGCAGCAACACCGCCTACATGCAGCTGAGCAGCCTGA CCAGCGAGGACAGCGCCGTGTACTACTGCGCCACCGGCAACACCATGGTGAA CATGCCCTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC anti-MOG antibody light chain (SEQ ID NO:23)

GATATTGAACTGACCCAGAGTCCCAGTAGCCTGGCCGTGAGTGCCGGCGAGA AAGTGACCATGAGCTGCAAAAGCAGCCAGAGCCTGCTGAACAGCGGCAACCA GAAAAACTACCTGGCCTGGTACCAGCAGAAACCCGGCCTGCCTCCTAAGCTGC TGATCTACGGCGCCAGCACCAGAGAAAGCGGCGTGCCCGATAGATTCACCGGC TCTGGCTCTGGCACCGACTTCACCCTGACCATCAGCAGCGTGCAGGCCGAAG ACCTGGCtGTcTACTACTGCCAGAACGACCACAGCTACCCCCTGACCTTCGGCG CCGGC AC C AAGCT GGAGAT C AAG anti-HER2 antibody heavy chain (SEQ ID NO:24)

GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCCGGGGGCTCTC TCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATTAAAGACACCTATATCCACT GGGTGCGTCAGGCTCCGGGTAAGGGCCTGGAGTGGGTTGCAAGGATTTATCCT ACGAATGGTTATACTCGTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGC GCAGACACTTCGAAAAACACAGCCTACCTCCAGATGAACAGCCTGCGTGCTG AGGAC ACT GC CGT CTATTATT GTAGC AGAT GGGGT GGGGACGGCTT CTAT GCTA TGGACTACTGGGGTCAAGGTACACTAGTCACCGTCAGCAGCGCTAGCACCAA GGGCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCG TCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCG

CAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT anti-HER2 antibody light chain (SEQ ID NO:25)

GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAG

GGTCACTATCACCTGCCGTGCCAGTCAGGATGTGAATACTGCTGTAGCCTGGTA

TCAACAGAAACCCGGAAAGGCCCCGAAACTGCTGATTTACTCGGCATCCTTCC

TCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTTCCCGCTCTGGGACGGATTTCA

CTCTGACCATCAGCTCCCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAG

C AAC ACTATACTACT C CTCC GACGTTC GGAC AGGGTACC AAGGT GGAGAT C AA

ACGTACCGTGGCGGCGCCA

A CAR described herein (e.g., a CAR-ECAD, a CAR-MOG, or a CAR-HER2) also can include one or more additional components. Examples of additional components that can be included in a CAR include, without limitation, a leader sequence (e.g., a CD8 leader sequence), a hinge (e.g., a CD8 hinge and a CD28 hinge), a transmembrane domain (e.g., a CD8 transmembrane domain and a CD28 transmembrane domain), a co stimulatory signaling domain (e.g., that may increase immunosuppression such as a TLR3 signaling domain and/or a TLR4 signaling domain), a Toll/interleukin- 1 receptor/resistance (TIR) interaction sequence (e.g., a TLR3 TIR-interaction domain and/or a TLR4 TIR-interaction domain), a TLR intracellular domain (e.g., a TLR intracellular linker), and a TLR transmembrane domain (e.g., a TLR 3 transmembrane domain and/or a TLR4 transmembrane domain). In some cases, nucleic acid encoding a component of a construct encoding a CAR can be separated from nucleic acid encoding another component using one or more linkers. Nucleic acids in a construct encoding a CAR can be present in any appropriate order. In some cases, a CAR can be designed to include a scFv that is in a light chain to heavy chain orientation or in a heavy chain to light chain orientation using any appropriate linker between the chains. For example, constructs encoding a CHD1-CAR can be generated in a light to heavy chain orientation of the scFv or in a heavy to light chain orientation of the scFv. In some cases, a CAR can be designed to include a scFv that is in a light chain to heavy chain orientation or in a heavy chain to light chain orientation without a linker between the chains. Exemplary nucleic acid sequences that can encode one or more additional components that can be included in a CAR that can be included in a construct described herein include, without limitation, those that encode the following amino acid sequences: CD8 leader sequence (SEQ ID NO:26)

MALPVTALLLPLALLLHAARP

Linker (SEQ ID NO:27) GGGGS GGGGS GGGGS

CD8 hinge (SEQ ID NO:28)

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD CD28 hinge (SEQ ID NO:29)

LEPKSCDKTHTCPPCPDPK

TLR long (long peptide derived from toll like receptor; SEQ ID NO:46)

NDFACTCEHQSFLQWIKDQRQLLVEVERMECATPSDKQGMPVLSLNITCQMNKTI

TLR short (short peptide derived from toll like receptor; SEQ ID NO:47)

MNKTI

CD8 transmembrane domain (SEQ ID NO:30) IYIWAPL AGTCGVLLLS LVITLY C

CD28 transmembrane domain (SEQ ID NO:31)

FWVLVVV GGVL ACY SLLVTVAFIIFWV TLR4 transmembrane domain (SEQ ID NO:48)

IGVSVLSVLVVSVVAVLVY

CD28 signaling domain (SEQ ID NO:32)

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD3zeta signaling domain (SEQ ID NO:33)

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN

PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM

QALPPR

4-1BB (CD137) signaling domain (SEQ ID NO:34)

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

TLR3 signaling domain (SEQ ID NO:35) FEYAAYUHAYKDKDWVWEHFSSMEKEDQSLKFCLEERDFEAGVFELEAIVNSIK RSRKIIFVITHHLLKDPLCKRFKVHHAVQQAIEQNLDSIILVFLEEIPDYKLNHALCL RRGMFKSHCILNWPVQKERIGAFRHKLQVALGSKNSVH

TLR4 signaling domain (SEQ ID NO:36) NIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGF HKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVE LYRLLS RNTYLEWED S VLGRHIF WRRLRKALLDGKSWNPEGTV GT GCNW QE AT SI TLR4 intracellular domain (SEQ ID NO:49)

KFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCL HYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRA GIIF I VLQKVEKTLLRQ Q VEL YRLL SRNTYLEWED S VLGRHIF WRRLRKALLD GK SWNPEGTVGTGCNWQEATSI

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

Exemplary nucleic acid sequences for some additional components that can be included in a CAR and can be included in a construct described herein are as follows. CD8 leader sequence (SEQ ID NO:37)

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCC

GCCAGGCCG

Linker (SEQ ID NO:38)

GGT GGAGGT GGTT C GGGAGGT GGAGGTAGC GGAGGT GGT GGAT CT

Linker (SEQ ID NO:50)

GGT GGAGGT GGTT C GGGAGGT GGAGGT AGC GGAGGT GGT GGA AGC

CD8 hinge (SEQ ID NO:39)

ACCACTACCCCTGCACCGCGACCACCAACACCGGCGCCCACCATTGCGTCGCA

GCCTCTGTCCCTGCGCCCAGAAGCATGCCGTCCAGCAGCAGGTGGTGCAGTTC

ATACTCGTGGTCTGGATTTCGCCTGTGAT

CD28 hinge (SEQ ID NO:40)

CTCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCGGATCC

CAAA

TLR long (SEQ ID NO:51)

AACGACTTCGCCTGCACCTGCGAGCACCAGAGCTTCCTGCAGTGGATCAAGG

ACCAGAGGCAGCTGCTGGTGGAGGTGGAGAGGATGGAGTGCGCCACCCCCA

GCGACAAGCAGGGCATGCCCGTGCTGAGCCTGAACATCACCTGCCAGATGAA

CAAGACCATC

TLR short (SEQ ID NO:52)

ATGAACAAGACCATC

CD8 transmembrane domain (SEQ ID NO:41)

ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTG

GTTATCACCCTTTACTGC CD28 transmembrane domain (SEQ ID NO:42)

TTTTGGGT GCT GGT GGT GGTT GGT GGAGTCCT GGCTT GCTATAGCTT GCTAGTA AC AGT GGC CTTTATTATTTT CT GGGT G TLR4 transmembrane domain (SEQ ID NO:53)

ATTGGGGTGTCTGTCCTAAGCGTGCTGGTTGTTTCCGTGGTTGCCGTTCTGGTA

TAT

CD28 signaling domain (SEQ ID NO:43) AGGAGTAAGAGGAGCAGGCTCCTGC ACAGTGACTACATGAAC ATGACTCCCC GCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGAC TTCGCAGCCTATCGCTCC

CD3zeta signaling domain (SEQ ID NO:44) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAG AACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTT GGAC AAGAGACGT GGC CGGGACC CT GAGAT GGGGGGAAAGCCGAGAAGGAA GAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAG GCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCAC GATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCT TCACATGCAGGCCCTGCCCCCTCGC

4-1BB (CD137) signaling domain (SEQ ID NO:45)

AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGAC CAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGA AGAAGAAGGAGGAT GT GAACT G

TLR4 intracellular domain (SEQ ID NO:54)

AAGTT CT ATTTCC AT CT GAT GCTT CTCGCT GGCT GC AT AAAGT ACGGGAGGGG GGAGAATATATATGACGCTTTCGTGATCTACTCGAGCCAGGATGAGGACTGG GTTCGCAACGAGCTAGTCAAGAATCTTGAAGAGGGCGTGCCTCCTTTCCAGCT CTGTCTGCATTACCGCGATTTTATTCCTGGGGTGGCCATCGCGGCCAACATCA TCCACGAGGGCTTCCATAAATCCAGAAAAGTGATTGTCGTTGTGAGCCAGCA TTTCATCCAGTCCAGGTGGTGCATTTTCGAATATGAGATAGCCCAGACCTGGC AGTTT CTT AGC AGTCGGGCT GGGATT ATTTTT ATC GT GCTGC AGAAGGTT GAA AAGACCCTTTTGCGGCAACAGGTGGAACTGTACCGATTATTATCCCGTAACAC TTACTTGGAATGGGAAGACTCAGTTCTCGGACGCCACATTTTCTGGCGCCGGC TCAGGAAGGCCCTGCTGGATGGTAAATCCTGGAACCCCGAGGGGACAGTGGG GACCGGAT GT AACT GGC AAGAGGC AAC AAGT AT A

In some cases, a nucleic acid construct encoding a CAR-ECAD described herein can be designed to encode a CD8 leader sequence, an anti-EC AD antibody (e.g., a hSC10.17 antibody) heavy chain, a linker, an anti-ECAD antibody (e.g., a hSC10.17 antibody) light chain, a CD8 hinge, a CD8 transmembrane domain, and a CD3zeta signaling domain. For example, a nucleic acid construct encoding a CAR-ECAD can be designed to encode a CD8 leader including the amino acid sequence set forth in SEQ ID NO:26, a hSC10.17 heavy chain including the amino acid sequence set forth in SEQ ID NO: 11, a linker including the amino acid sequence set forth in SEQ ID NO:27, a hSC10.17 light chain including the amino acid sequence set forth in SEQ ID NO: 12, a CD8 hinge including the amino acid sequence set forth in SEQ ID NO:28, a CD8 transmembrane domain including the amino acid sequence set forth in SEQ ID NO:30, and a CD3zeta signaling domain including the amino acid sequence set forth in SEQ ID NO:33.

In some cases, a nucleic acid construct encoding a CAR-MOG described herein can be designed to encode a CD8 leader sequence, an anti-MOG antibody (e.g., a 8-18C5 antibody) heavy chain, a linker, an anti-MOG antibody (e.g., a 8-18C5 antibody) light chain, a CD8 hinge, a CD28 transmembrane domain, and a CD3zeta signaling domain.

For example, a nucleic acid construct encoding a CAR-MOG can be designed to encode a CD8 leader including the amino acid sequence set forth in SEQ ID NO: 26, a 8-18C5 heavy chain including an amino acid sequence set forth SEQ ID NO:14, a linker including the amino acid sequence set forth in SEQ ID NO:27, a 8-18C5 light chain including the amino acid sequence set forth in SEQ ID NO: 15, a CD8 hinge including the amino acid sequence set forth in SEQ ID NO:28, a CD8 transmembrane domain including the amino acid sequence set forth in SEQ ID NO:30, and a CD3zeta signaling domain including the amino acid sequence set forth in SEQ ID NO:33. In some cases, a nucleic acid construct encoding a CAR-HER2 described herein can be designed to encode a CD8 leader sequence, an anti-HER2 antibody (e.g., 4D5) heavy chain, a linker, an anti-HER2 antibody (e.g., 4D5) light chain, a CD8 hinge, a CD28 transmembrane domain, a 4-1 BB signaling domain, and a CD3zeta signaling domain. For example, a nucleic acid construct encoding a CAR-HER2 can be designed to encode a CD8 leader including the amino acid sequence set forth in SEQ ID NO:26, a 4D5 heavy chain including the amino acid sequence set forth in SEQ ID NO: 16, a linker including the amino acid sequence set forth in SEQ ID NO:27, a 4D5 light chain including the amino acid sequence set forth in SEQ ID NO: 17, a CD28 hinge including the amino acid sequence set forth in SEQ ID NO:29, a CD8 transmembrane domain including the amino acid sequence set forth in SEQ ID NO:30, a4-lBB signaling domain including the amino acid sequence set forth in SEQ ID NO:34, and a CD3zeta signaling domain including the amino acid sequence set forth in SEQ ID NO:33.

In some cases, a nucleic acid construct encoding a CAR-ECAD described herein, a CAR-MOG described herein, or a CAR-HER2 described herein can be designed to encode (al) an anti-ECAD antibody (e.g., ahSC10.17 antibody) heavy chain, a linker, an anti-ECAD antibody (e.g., ahSC10.17 antibody) light chain, (a2) an anti-MOG antibody (e.g., a 8-18C5 antibody) heavy chain, a linker, an anti-MOG antibody (e.g., a 8-18C5 antibody) light chain, or (a3) an anti-HER2 antibody (e.g., 4D5) heavy chain, a linker, an anti-HER2 antibody (e.g., 4D5) light chain followed by (b) a CD8 hinge, (c) a TLR4 transmembrane domain, and (d) a TLR4 signaling domain or a TLR4 intracellular domain.

In some cases, a nucleic acid construct encoding a CAR-ECAD described herein, a CAR-MOG described herein, or a CAR-HER2 described herein can be designed to encode (al) an anti-ECAD antibody (e.g., ahSC10.17 antibody) heavy chain, a linker, an anti-ECAD antibody (e.g., ahSC10.17 antibody) light chain, (a2) an anti-MOG antibody (e.g., a 8-18C5 antibody) heavy chain, a linker, an anti-MOG antibody (e.g., a 8-18C5 antibody) light chain, or (a3) an anti-HER2 antibody (e.g., 4D5) heavy chain, a linker, an anti-HER2 antibody (e.g., 4D5) light chain followed by (b) a CD8 hinge, (c) a TLR4 transmembrane domain, (d) a TLR4 signaling domain or a TLR4 intracellular domain, and (e) a CD3zeta signaling domain.

In some cases, a nucleic acid construct encoding a CAR-ECAD described herein, a CAR-MOG described herein, or a CAR-HER2 described herein can be designed to encode (al) an anti-ECAD antibody (e.g., ahSC10.17 antibody) heavy chain, a linker, an anti-ECAD antibody (e.g., ahSC10.17 antibody) light chain, (a2) an anti-MOG antibody (e.g., a 8-18C5 antibody) heavy chain, a linker, an anti-MOG antibody (e.g., a 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 such as a linker encoded by SEQ ID NO:50, (c) a TLR4 transmembrane domain, and (d) a TLR4 signaling domain or a TLR4 intracellular domain.

In some cases, a nucleic acid construct encoding a CAR-ECAD described herein, a CAR-MOG described herein, or a CAR-HER2 described herein can be designed to encode (al) an anti-ECAD antibody (e.g., ahSC10.17 antibody) heavy chain, a linker, an anti-ECAD antibody (e.g., ahSC10.17 antibody) light chain, (a2) an anti-MOG antibody (e.g., a 8-18C5 antibody) heavy chain, a linker, an anti-MOG antibody (e.g., a 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) an TLR long, (c) a TLR4 transmembrane domain, and (d) a TLR4 signaling domain or a TLR4 intracellular domain.

In some cases, a nucleic acid construct encoding a CAR-ECAD described herein, a CAR-MOG described herein, or a CAR-HER2 described herein can be designed to encode (al) an anti-ECAD antibody (e.g., ahSC10.17 antibody) heavy chain, a linker, an anti-ECAD antibody (e.g., ahSC10.17 antibody) light chain, (a2) an anti-MOG antibody (e.g., a 8-18C5 antibody) heavy chain, a linker, an anti-MOG antibody (e.g., a 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) an TLR short, (c) a TLR4 transmembrane domain, (d) a TLR4 signaling domain or a TLR4 intracellular domain, and (e) a CD3zeta signaling domain.

In some cases, a nucleic acid construct encoding a CAR-ECAD described herein, a CAR-MOG described herein, or a CAR-HER2 described herein can be designed to encode (al) an anti-ECAD antibody (e.g., ahSC10.17 antibody) heavy chain, a linker, an anti-ECAD antibody (e.g., ahSC10.17 antibody) light chain, (a2) an anti-MOG antibody (e.g., a 8-18C5 antibody) heavy chain, a linker, an anti-MOG antibody (e.g., a 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. In some cases, a nucleic acid construct encoding a CAR-ECAD described herein, a CAR-MOG described herein, or a CAR-HER2 described herein can be designed to encode (al) an anti-ECAD antibody (e.g., ahSC10.17 antibody) heavy chain, a linker, an anti-ECAD antibody (e.g., ahSC10.17 antibody) light chain, (a2) an anti-MOG antibody (e.g., a 8-18C5 antibody) heavy chain, a linker, an anti-MOG antibody (e.g., a 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 CD3zeta signaling domain.

In cases where a MSC designed to express an antigen receptor (e.g., a CAR) capable of binding (e.g., specifically binding) to a tissue-specific antigen is also designed to express a polypeptide that can promote differentiation into a tissue specific cell, the MSC can include one or more nucleic acids encoding a polypeptide that can promote differentiation into a tissue specific cell. Nucleic acid that can encode a polypeptide that can promote tissue specific cell differentiation can encode any polypeptide that can promote a MSC to differentiate into any type of tissue specific cell. For example, a MSC can be designed to include one or more nucleic acids encoding a polypeptide that can promote cardiac cell differentiation. A polypeptide that can promote cardiac differentiation can promote differentiation into any appropriate type of cardiac cell (e.g., cardiomyocytes). Examples of polypeptides that can promote cardiac cell differentiation and can be included in a MSC as described herein include, without limitation, a GATA4 polypeptide, a MEF2C polypeptide, a TBX5 polypeptide, a ERRG polypeptide, and a MESP1 polypeptide. For example, a MSC can be designed to include one or more nucleic acids encoding a polypeptide that can promote neural differentiation. A polypeptide that can promote neural differentiation can promote differentiation into any appropriate type of neuron (e.g., sensory neurons, motor neurons, intemeurons, oligodendrocytes, astrocytes, and glial cells). Examples of polypeptides that can promote neural differentiation and can be included in a MSC as described herein include, without limitation, a Oct3/4 polypeptide, a Klf4 polypeptide, a Sox2 polypeptide, a Glisl polypeptide, a cDMyc polypeptide, a BMP4 polypeptide, a WNT polypeptide, a FGF2 polypeptide, a SHH polypeptide, a Soxll polypeptide, a Sox2 polypeptide, a Sox3 polypeptide, aZicl polypeptide, aZic2 polypeptide, a lrxl polypeptide, alrx2 polypeptide, a Irx3 polypeptide, aFoxD4 polypeptide, aMKx2.5 polypeptide, and a cTnT polypeptide.

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

Any appropriate mammal having, or at risk of developing, a disease or disorder characterized by inflammation and/or degeneration of a tissue can be treated as described herein. Examples of mammals that can have a disease or disorder characterized by inflammation and/or degeneration of a tissue and can be treated as described herein include, without limitation, humans, non-human primates such as monkeys, dogs, cats, horses, cows, pigs, sheep, mice, and rats. For example, one or more MSCs expressing a CAR targeting a tissue-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a tissue specific cell (e.g., a composition containing one or more MSCs expressing a CAR targeting a tissue-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a tissue specific cell) can be administered (e.g., by adoptive transfer) to humans having, or at risk of developing, a disease or disorder characterized by inflammation and/or degeneration of a tissue to treat the human.

In some cases, the materials and methods for treating mammals (e.g., humans) having, or at risk of developing, a disease or disorder characterized by inflammation and/or degeneration of a tissue can be used to treat a mammal (e.g., a human) having, or at risk of developing, a disease or disorder characterized by inflammation and/or degeneration of an epithelial tissue. Examples of diseases and disorders characterized by inflammation and/or degeneration of an epithelial tissue include, without limitation, an IBD such as colitis (e.g, characterized by inflammation of colon cells 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 mucous membranes lining the digestive tract), colonic fistula, graft versus host disease, and inflammatory pneumonitis (e.g., characterized by inflammation of lung cells). When treating a mammal having (or at risk of developing) colitis, the colitis can be any type of colitis (e.g., ulcerative colitis, Crohn’s colitis, diversion colitis, ischemic colitis, infectious colitis, fulminant colitis, collagenous colitis, chemical colitis, microscopic colitis, lymphocytic colitis, and atypical colitis).

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

When treating a mammal having (or at risk of developing) a disease or disorder characterized by inflammation and/or degeneration of an epithelial tissue (e.g., an IBD such as colitis) as described herein (e.g., by administering one or more MSCs expressing a CAR targeting an epithelial-specific antigen and, optionally, expressing a polypeptide that can promote differentiation into a tissue specific cell), the one or more MSCs expressing a CAR targeting an epithelial-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a tissue specific cell can be effective to reduce the severity of the disease or disorder characterized by inflammation and/or degeneration of an epithelial tissue in the mammal. In cases where the disease or disorder characterized by inflammation and/or degeneration of an epithelial tissue is colitis, reducing the severity of colitis in a mammal can include reducing or eliminating one or more symptoms of colitis (e.g., diarrhea, abdominal pain and cramping, rectal pain, rectal bleeding, urgency to defecate, inability to defecate despite urgency, weight loss, fatigue, fever, jaundice, liver failure, abnormal liver tests, respiratory distress, skin erythema, and/or desquamation).

For example, one or more MSCs expressing a CAR targeting an epithelial-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a tissue specific cell can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having, or at risk of developing, colitis) 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, or more percent.

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

In some cases, one or more MSCs expressing a CAR targeting an epithelial- specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a tissue specific cell (e.g., a composition containing one or more MSCs expressing a CAR targeting an epithelial- specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a tissue specific cell) can be administered in combination with one or more additional therapeutic agents (e.g., therapeutic agents that can be used to treat a mammal having or at risk of developing a disease or disorder characterized by inflammation and/or degeneration of an epithelial tissue and therapeutic agents that can be used for treating inflammation of an epithelial tissue within a mammal). For example, a mammal having (or at risk of developing) a disease or disorder characterized by inflammation and/or degeneration of an epithelial tissue (e.g. , an IBD such as colitis) also can be treated with one or more additional therapeutic agents. In some cases, a therapeutic agent can be an anti-inflammatory. In some cases, a therapeutic agent can be an immunosuppressant. In some cases, a therapeutic agent can be a T cell such as a T cell expressing a CAR (a CAR-T cell). Examples of therapeutic agents that can be used in combination with one or more MSCs expressing a CAR targeting an epithelial-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a tissue specific cell described herein include, without limitation, a CAR-T cell (e.g., a CART19 cell) 5 -aminosalicylates (e.g., sulfasalazine, mesalamine, balsalazide, and olsalazine), corticosteroids (e.g., prednisone and methylprednisolone), azathioprine, mercaptopurine, cyclosporine, infliximab, adalimumab, golimumab, vedolizumab, antibiotics, anti-diarrheal medications (e.g., loperamide), pain relievers (e.g., acetaminophen), and iron supplements. In some cases, one or more MSCs expressing a CAR targeting an epithelial-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a tissue specific cell can be administered at substantially the same time as one or more additional therapeutic agents that can be used for treating inflammation of an epithelial tissue within a mammal. For example, a composition including one or more MSCs expressing a CAR targeting an epithelial-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a tissue specific cell also can include one or more additional therapeutic agents that can be used for treating inflammation of an epithelial tissue within a mammal. In some cases, one or more MSCs expressing a CAR targeting an epithelial-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a tissue specific cell can be administered first, and the one or more additional therapeutic agents administered second, or vice versa. In some cases, the materials and methods for treating mammals (e.g., humans) having, or at risk of developing, a disease or disorder characterized by inflammation and/or degeneration of a tissue can be used to treat a mammal (e.g., a human) having, or at risk of developing, a disease or disorder characterized by inflammation and/or degeneration of a neural tissue. Examples of diseases and disorders characterized by inflammation and/or degeneration of a neural tissue include, without limitation, multiple sclerosis (e.g., characterized by inflammation of neurons in the brain and/or spinal cord), encephalomyelitis such as immune mediated encephalomyelitis (e.g., characterized by inflammation of the CNS), and paraneoplastic encephalitis (e.g., characterized by multifocal inflammation of the CNS). When treating a mammal having, or at risk of developing, immune mediated encephalomyelitis, the immune mediated encephalomyelitis can be any type of immune mediated encephalomyelitis (e.g., acute disseminated encephalomyelitis (ADEM), and paraneoplastic encephalitis).

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

When treating a mammal having (or at risk of developing) a disease or disorder characterized by inflammation and/or degeneration of a neural tissue (e.g., multiple sclerosis or immune mediated encephalomyelitis) as described herein (e.g., by administering one or more MSCs expressing a CAR targeting a neural-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a neuron), the one or more MSCs expressing a CAR targeting a neural-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a neuron can be effective to reduce the severity of the disease or disorder characterized by inflammation and/or degeneration of a neural tissue in the mammal. In cases where the disease or disorder characterized by inflammation and/or degeneration of a neural tissue is multiple sclerosis, reducing the severity of multiple sclerosis in a mammal can include reducing or eliminating one or more symptoms of multiple sclerosis (e.g., numbness or weakness in one or more limbs, electric-shock sensations (e.g., that occur with certain neck movements, especially bending the neck forward), tremor, lack of coordination, unsteady gait, partial or complete loss of vision, prolonged double vision, blurry vision, slurred speech, fatigue, dizziness, tingling or pain in parts of the body, problems with sexual function, problems with bowel function, and/or problems with bladder function) and/or one or more complications associate with multiple sclerosis (e.g., muscle stiffness, muscle spasms, paralysis, forgetfulness, mood swings, depression, epilepsy, focal weakness, and/or vision defects). For example, one or more MSCs expressing a CAR targeting a neural-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a neuron can be administered to a mammal (e.g. , a human) in need thereof (e.g., a human having, or at risk of developing, a disease or disorder characterized by inflammation and/or degeneration of a neural tissue) as described herein to reduce the severity of one or more symptoms of a disease or disorder characterized by inflammation and/or degeneration of a neural tissue and/or one or more complications associated with a disease or disorder characterized by inflammation and/or degeneration of a neural tissue by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, one or more MSCs expressing a CAR targeting a neural-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a neuron (e.g., a composition containing one or more MSCs expressing one or more neural-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a neuron) can be the sole active ingredient for treating a mammal having (or at risk of developing) a disease or disorder characterized by inflammation and/or degeneration of a neural tissue (e.g., multiple sclerosis or immune mediated encephalomyelitis) as described herein (e.g., by administering one or more MSCs expressing a CAR targeting a neural-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a neuron).

In some cases, one or more MSCs expressing a CAR targeting a neural-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a neuron (e.g., a composition containing one or more MSCs expressing a CAR targeting a neural-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a neuron) can be administered in combination with one or more additional therapeutic agents (e.g. , therapeutic agents that can be used to treat a mammal having or at risk of developing a disease or disorder characterized by inflammation and/or degeneration of a neural tissue and therapeutic agents that can be used for treating inflammation of a neural tissue within a mammal).. For example, a mammal having (or at risk of developing) a disease or disorder characterized by inflammation and/or degeneration of a neural tissue (e.g., multiple sclerosis or immune mediated encephalomyelitis) also can be treated with one or more additional therapeutic agents. Examples of therapeutic agents that can be used in combination with one or more MSCs expressing a CAR targeting a neural-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a neuron described herein include, without limitation, corticosteroids (e.g., prednisone and methylprednisolone), ocrelizumab, beta interferons, glatiramer acetate, fingolimod, dimethyl fumarate, teriflunomide, siponimod, natalizumab, alemtuzumab, mitoxantrone, baclofen, tizanidine, amantadine, modafmil, methylphenidate, dalfampridine, and immunoglobulins. In some cases, one or more MSCs expressing a CAR targeting a neural-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a neuron can be administered at substantially the same time as one or more additional therapeutic agents that can be used for treating inflammation of a neural tissue within a mammal. For example, a composition including one or more MSCs expressing a CAR targeting a neural-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a neuron also can include one or more additional therapeutic agents that can be used for treating inflammation of a neural tissue within a mammal. In some cases, one or more MSCs expressing a CAR targeting a neural-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a neuron can be administered first, and the one or more additional therapeutic agents administered second, or vice versa.

In some cases, the materials and methods for treating mammals (e.g., humans) having, or at risk of developing, a disease or disorder characterized by inflammation and/or degeneration of a tissue can be used to treat a mammal (e.g., a human) having, or at risk of developing, a disease or disorder characterized by inflammation and/or degeneration of a cardiac tissue. Examples of diseases and disorders characterized by inflammation and/or degeneration of a cardiac tissue include, without limitation, myocarditis (e.g., characterized by inflammation of cardiomyocytes), endocarditis (e.g., characterized by inflammation of endocardium cells), and cardiac failure.

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

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

In some cases, one or more MSCs expressing a CAR targeting a cardiac-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a cardiac cell (e.g., a composition containing one or more MSCs expressing a CAR targeting a cardiac-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a cardiac cell) can be the sole active ingredient for treating a mammal having (or at risk of developing) a disease or disorder characterized by inflammation and/or degeneration of a cardiac tissue (e.g., myocarditis) as described herein (e.g., by administering one or more MSCs expressing a CAR targeting a cardiac-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a cardiac cell).

In some cases, one or more MSCs expressing a CAR targeting a cardiac-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a cardiac cell (e.g., a composition containing one or more MSCs expressing a CAR targeting a cardiac-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a cardiac cell) can be administered in combination with one or more additional therapeutic agents (e.g., therapeutic agents that can be used to treat a mammal having or at risk of developing a disease or disorder characterized by inflammation and/or degeneration of a cardiac tissue and therapeutic agents that can be used for treating inflammation of a cardiac tissue within a mammal). For example, a mammal having (or at risk of developing) a disease or disorder characterized by inflammation and/or degeneration of a cardiac tissue (e.g., myocarditis) also can be treated with one or more additional therapeutic agents. In some cases, a therapeutic agent can be an anti-inflammatory. In some cases, a therapeutic agent can be an immunosuppressant. In some cases, a therapeutic agent can be an angiotensin converting enzyme (ACE) inhibitor. In some cases, a therapeutic agent can 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 targeting a cardiac-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a cardiac cell described herein include, without limitation, enalapril, captopril, lisinopril, ramipril, losartan, valsartan, metoprolol, bisoprolol, and carvedilol. In some cases, one or more MSCs expressing a CAR targeting a cardiac-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a cardiac cell can be administered at substantially the same time as one or more additional therapeutic agents that can be used for treating inflammation of a cardiac tissue within a mammal. For example, a composition including one or more MSCs expressing a CAR targeting a cardiac-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a cardiac cell also can include one or more additional therapeutic agents that can be used for treating inflammation of a cardiac tissue within a mammal. In some cases, one or more MSCs expressing a CAR targeting a cardiac-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a cardiac cell can be administered first, and the one or more additional therapeutic agents administered second, or vice versa.

Any appropriate method can be used to administer one or more MSCs expressing a CAR targeting a tissue-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a tissue specific cell (e.g., a composition containing one or more MSCs expressing a CAR targeting a tissue-specific antigen such as an epithelial-specific antigen, a neural-specific antigen, or a cardiac-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a tissue specific cell such as a cardiac cell or a neuron) to a mammal (e.g. , a human) in need thereof (e.g., a human having, or at risk of developing, a disease or disorder characterized by inflammation and/or degeneration of a tissue expressing a tissue-specific antigen). Examples of methods of administering MSCs described herein to a mammal can include, without limitation, injection (e.g., intravenous, intradermal, intramuscular, or subcutaneous injection). For example, a composition including one or more MSCs expressing a CAR targeting a tissue-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a tissue specific cell can be administered to a human by intravenous injection.

This document also provides kits containing one or more materials described herein. In some cases, a kit can include one or more MSCs expressing a CAR targeting a tissue-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a tissue specific cell (e.g., a composition containing one or more MSCs expressing a CAR targeting a tissue-specific antigen such as an epithelial-specific antigen, a neural-specific antigen, or a cardiac-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a tissue specific cell such as a cardiac cell or a neuron). For example, one or more MSCs expressing a CAR targeting a tissue-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a tissue specific cell (e.g., a composition containing one or more MSCs expressing a CAR targeting a tissue-specific antigen such as an epithelial-specific antigen, a neural-specific antigen, or a cardiac-specific antigen and, optionally, expressing a polypeptide that can promote differentiation of the MSC and/or one or more resident progenitor cells into a tissue specific cell such as a cardiac cell or a neuron) can be combined with packaging material to form a kit. For example, one or more constructs (e.g., nucleic acid constructs) described herein (e.g., encoding a CAR that can bind a tissue-specific antigen such as an epithelial-specific antigen, a neural-specific antigen, or a cardiac-specific antigen) can be combined with packaging material to form a kit. The packaging material included in such a kit typically contains instructions or a label describing how the composition can be used, for example, in an adoptive transfer to treat a mammal having, or at risk of developing, a disease or disorder characterized by inflammation and/or degeneration of a tissue as described herein. In some cases, materials provided in kits described herein can be used for treating mammals (e.g., humans) having, or at risk of developing, a disease or disorder characterized by inflammation and/or degeneration of a tissue as described herein. In some cases, the packaging material included in such a kit can contain instructions and/or a label describing how a composition described herein can be used. For example, a kit can contain instructions and/or a label describing how a composition described herein can be used to express one or more CARs in MSCs to engineer the MSCs to express CARs that bind 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 can contain instructions and/or a label describing how the engineered MSCs described herein can be used. For example, the packaging material included in such a kit can contain instructions and/or a label describing how the engineered MSCs described herein can be used in adoptive transfer to treat a mammal having, or at risk of developing, a disease or disorder characterized by inflammation and/or degeneration of a tissue as described herein. In some cases, a kit (e.g., a kit containing instructions and/or a label describing how the engineered MSCs described herein can be used in adoptive transfer) can include materials for use in an adoptive transfer procedure. The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1: Engineered CAR-MSCs for Immunomodulation

To determine if MSCs expressing CARs could reduce inflammation in a target cell population, MSCs expressing CARs targeting CD19 (MSC-CAR19s) were engineered.

Adipose derived-mesenchymal stem cells (100-Biotr-0024) were passaged into 3 wells of a 6 well plate (100k each). One group was left as an untransduced (UTD) negative control. The second well was transduced with Luciferase-ZsGreen lentivirus (~MOI 3). The third group was transduced with the same MOI and lentivirus but with 100 pg/ml of a protamine sulfate solution transduction “enhancer PLUS” system. The efficiency of transduction increased >20% upon use of our “enhancer PLUS” (Figure 1).

MSCs were transduced with CD19-CAR lentivirus (VSV-G pseudo-typed) to generate MSC-CARl 9.

Adipose derived-mesenchymal stem cells (100-Biotr-0024) were passaged into 4 wells of a 6 well plate (100k each). One group was left as an untransduced (UTD) negative control. The second well was transduced with our GMP-grade, pan-VSV, CD 19 recognizing-Chimeric Antigen Receptor (CAR19) lentivirus (~MOI 3). The third group was transduced with the same MOI and lentivirus in addition to 50 pg/ml Enhancer PLUS. The fourth group is identical to the third but 100 pg/ml of enhancer PLUS was used. The efficiency of transduction increased upon use of enhancer PLUS (Figure 2).

Adipose derived-mesenchymal stem cells (100-Biotr-0024) were taken from culture after consecutive 10+ passages and probed for surface expression of CAR19. As displayed above, 48.5% of the CAR19 transduced culture retained CAR19 expression, a loss of 20% since transduction (Figure 3).

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

Designer constructs for the enhancement of MSC trafficking, persistence, and efficacy for immunomodulation are shown in Figure 5. Example 2: Engineered CAR-MSCs for Immunomodulation of Epithelial Tissues

MSCs that can target epithelial tissues were designed by engineering MSCs to express CARs targeting ECAD (MSC-CAR-ECADs).

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

MSCs were transduced with E-cadherin CAR lentivirus (VSV-G pseudo-typed) to generate MSC-CAR-ECAD. A second generation CAR construct including a TLR3 signaling domain and/or a TLR4 signaling domain was used.

Adipose derived-mesenchymal stem cells (100-Biotr-0024) were passaged into 4 wells of a 6 well plate (100k each). One group was left as a, untransduced (UTD) negative control. The second well was transduced with our GMP-grade, pan-VSV, ECAD recognizing-Chimeric Antigen Receptor (CAR-ECAD) lentivirus (~MOI 3). The third group was transduced with the same MOI and lentivirus in addition to 50 pg/ml Enhancer PLUS. The fourth group is identical to the third but 100 pg/ml of enhancer PLUS was used. The efficiency of transduction increased in an enhancer PLUS concentration dependent manner (Figure 6).

WT-MSCs significantly reduced the proliferative capacity of stimulated T-cells but not stimulated CART cells. When T cells were stimulated with nonspecific stimulus (PMA/IONO) and cultures in the presence of MSCs, proliferation was significantly inhibited. However, WT-MSCs were ineffective in suppressing CAR specific T-cell proliferation when activated through their antigen. When CART 19 are activated through the CAR (through a co-culture with CD19+ cell line NALM6), in the presence of MSCs, antigen specific proliferation was not inhibited (Figure 5 and Figure 6). Thus, adipose derived-mesenchymal stem cells suppress naive and CD3XCD28 T-cell proliferation but do not suppress CART-cell proliferation.

MSC-CAR cells exhibit more profound immunosuppressive effect when stimulated through the CAR, compared to wild type MSC or unstimulated MSCs. CD3+ T cells, stimulated CD3+ cells, or stimulated CART19 (stimulated through the CAR) were cultured in media alone, with wild type MSC, with MSCCAR19, with MSC-CAR- ECAD, or with MSC-CAR-ECAD with MCF-7 (ECAD expressing cell line). All conditions with MSC inhibited T cell proliferation. Stimulated MSC-CAR inhibited

CAR-T cell proliferation more profoundly in the presence of their target antigens. MSC- CAR-ECAD inhibition of CART 19 proliferation was more profound in the presence of MFC-7 cell line, compared to MSC-CAR-ECAD inhibition of CART19 proliferation in the absence of MCF-7 cell line (Figures 7A and 7B). Thus, adipose derived- mesenchymal stem cells, when engineered to express CAR-ECAD, suppress CART-cell proliferation in an antigen dependent manner.

Flow cytometry was used to confirm the MSC phenotype (Figures 10A, 10B, IOC, and 10D). MSCs that maintain sternness should be CD90 ⁺ , CD105 ⁺ , CD73 ⁺ , CD34 . CD45 , CD19 , CD 14-, HLA-DR .

Example 3: Engineered CAR-MSCs for Immunomodulation of the Central Nervous System

MSCs that can target neural tissues were designed by engineering MSCs to express CARs targeting MOG (MSC-CAR-MOGs).

Lentiviruses encoding CAR-MOGs were transduced into MSCs. Expression of CAR-MOGs in the MSCs was determined by comparison with an untransduced (UTD) MSC population. Transduction was performed with or without protamine sulfate solution (~70 pg/ml) for enhancement of transduction efficiency. CAR-MOGs were expressed on the surface of -78% mesenchymal stem cells with specificity towards MOG (Figure 11).

Nucleic acid sequences (SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7) encoding an exemplary CAR-MOG are shown in Figures 13A, 13C, and 13E. Amino acid sequences (SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8) of exemplary CAR- MOGs are shown in Figures 13B, 13D, and 13F.

Example 4: Engineered CAR-MSCs for Immunomodulation of Cardiac Tissue

MSCs that can target cardiac tissues were designed by engineering MSCs to express CARs targeting HER2 (MSC-CAR-HER2s).

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

Example 5: Transduction efficiency ofMSC-CAR19

Lentivirus transduction of MSCs with CAR19 vectors result in a transduction efficiency of 60% compared to untransduced MSCs (Figure 15 A). Example 6: MSCs retain their sternness after lentiviral transduction with a CAR vector Flow cytometric analysis of MSC-CAR19, two days after their transduction with the CAR lentivirus, indicate their sternness, which was comparable to untransduced MSCs (MSC-UTD). MSC-CAR19 continued to express CD 105, CD90, CD73, and lack expression of CD34, CD45, HLA-DR, and CD 14 (Figure 15B).

Example 7: MSC-CAR19 inhibits T cell proliferation in the presence of CD19 ⁺ targets, indicating antigen specific stimulation ofMSC-CAR19

T cells were stimulated with CD3/CD28 beads, and 24 hours later were co cultured with untransduced MSC (MSC-UTD), or MSC-CAR19 (CD28 containing CAR19, K122), or MSC-CAR19 (CD137 containing CAR19, K002), or with no MSCs. The K002 CAR, which targets CD 19, was designed to have single chain antibody targeting CD19, derived from FMC63, followed by a CD8 hinge, followed by a CD8 transmembrane domain, followed by a 4-1BB (CD137) signaling domain. The amino acid sequence of K002 was as follows:

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLN

WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQ

GNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCT

V S GV S LPD Y GV S WIRQPPRKGLE WLGVIW GSETTYYN S ALKS RLTIIKDN S KS Q V FLKMNSLQTDDTAIYY C AKHYYY GGS Y AMDYWGQGTS VTV S STTTP APRPPTP APTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL YCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP AYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAY SEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR (SEQ ID NO:63). The K122 CAR, which targets CD19, was designed to have single chain antibody targeting CD 19, 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:

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLN

WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQ

GNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCT

V S GV S LPD Y GV S WIRQPPRKGLE WLGVIW GSETTYYN S ALKS RLTIIKDN S KS Q V FLKMNSLQTDDT AIYY C AKHY YY GGS Y AMD YW GQGTS VT V S SLEPKS CDKTHT CPPCPDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR PGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLY QGLSTATKDTYDALHMQALPPR (SEQ ID NO:64).

T cells and MSCs were co-cultured either in medium alone, or in the presence of irradiated CD19 ⁺ cells as a strategy to stimulate MSC-CAR19 through the CAR. Proliferation of CD3 was inhibited in the presence of MSC-CAR19 (containing CD28 signaling domain), but not in the presence of MSC-CAR19 (containing CD137 signaling domain), or un-transduced MSCs, and in the presence of irradiated CD19 ⁺ cells, but not in the absence of CD19 ⁺ cells (Figure 16). T cells, MSCs, and NALM6 cells were cultured at ratio of 1:0.1 :1 E (T cells (effectors):MSCs (suppressors):T (tumor). These results indicate that MSC-CARs were able to suppress T cell proliferation upon antigen specific stimulation, when the CAR contained a CD28 signaling domain

Example 8: Incorporation of CD28 signaling enhances the proliferation ofMSC-CAR19

MSC-CAR19 cells with different stimulatory domains were co-cultured with or without the irradiated CD19 ⁺ cell line NALM6 and MSC growth was monitored. The incorporation of CD28 signaling domain resulted in enhanced proliferation of MSC- CAR19 compared to SC-UTD or MSC-CAR19 that incorporated CD137 stimulatory domain. On the other hand, the proliferation of MSC-CAR19 cells that incorporate TLR4 signaling was suppressed. These results demonstrate that CD28 and TLR4 signaling are involved in proliferation of MSC-CAR upon antigen specific stimulation. The K142 CAR, which targets CD 19, was designed to have single chain antibody targeting CD 19, derived from FMC63, followed by a CD8 hinge, followed by a TLR4 transmembrane domain, followed by a TLR4 signaling domain. The amino acid sequence of K142 was as follows:

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLN WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQ GNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCT V S GV S LPD Y GV S WIRQPPRKGLE WLGVIW GSETTYYN S ALKS RLTIIKDN S KS Q V FLKMNSLQTDDT AIYY C AKHYYY GGS Y AMDYWGQGTS VTV S STTTP APRPPTP APTI AS QPLS LRPE ACRP AAGGA VHTRGLDF ACDIGV S VLS VL V V S V V A VL VYKF YFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLH YRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAG IIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKS WNPEGTVGTGCNWQEATSI (SEQ ID NO:65).

T cells and MSCs were co-cultured either in medium alone, or in the presence of irradiated CD19 ⁺ cells as a strategy to stimulate MSC-CAR19 through the CAR. Proliferation of CD3 was inhibited in the presence of MSC-CAR19 (containing TLR4 signaling domain), but not in the presence of MSC-CAR19 (containing CD137 signaling domain), or un-transduced MSCs, and in the presence of irradiated CD19 ⁺ cells, but not in the absence of CD19 ⁺ cells (Figure 17). T cells, MSCs, andNALM6 cells were cultured at ratio of 1:0.1 :1 E (T cells (effectors):MSCs (suppressors):T (tumor). These results indicate that MSC-CARs were able to suppress T cell proliferation upon antigen specific stimulation, when the CAR contained TLR4 stimulatory molecule.

Example 9: MSC-CAR19 inhibits T cell proliferation in the presence of CD19 ⁺ targets, indicating antigen specific stimulation ofMSC-CAR19

T cells were stimulated with CD3/CD28 beads, and 24 hours later were co cultured with untransduced MSC (MSC-UTD), or MSC-CAR19 (TLR4 containing CAR19, K142), or with no MSCs, and in the presence of irradiated CD19 ⁺ cells as a strategy to stimulate MSC-CAR19 through the CAR. Proliferation of CD3 was inhibited in the presence of MSC-CAR19 (containing TLR4 signaling domain), but not in the presence of un-transduced MSCs (Figure 18). T cells, MSCs, and NALM6 cells were cultured at ratio of 1:0.1 :1 E (T cells (effectors):MSCs (suppressors):T (tumor). These results indicate that MSC-CARs were able to suppress T cell proliferation upon antigen specific stimulation, when the CAR contained TLR4 stimulatory molecule.

Example 10: Antigen specific stimulation of MSC-CARs containing CD28 signaling molecule enhances their proliferation

Un-transduced MSC (MSC-UTD), MSC-CAR19 (CD28 containing CAR19, K122), MSC-CAR19 (CD137 containing CAR19, K002), or MSC-CAR19 (TLR4 containing CAR19, K142) were co-cultured with the irradiated CD19 ⁺ cell line NALM6 at 1 :1 ratio. The absolute number of MSCs was counted by flow cytometry using absolute counts on days 3 and 5. Antigen specific stimulation of MSC-CAR19 (containing CD28) led to increased MSC proliferation (Figures 19A and 19B). These results demonstrate that CD28 signaling via a CAR contributes to MSC-CAR growth.

Example 11: Lentiviral transduction ofMSCs with CAR results in reduced proliferation MSCs were transduced with CAR lentiviruses or GFP lentiviruses on day 1 and followed in culture for 15 days. Transduction with CAR-Ecadherin resulted in reduced expansion compared to untransduced MSCs or MSCs transduced with GFP lentivirus (Figure 20).

Example 12: MSC-CAR exert their suppressive abilities through cell to cell contact mediated and soluble factor mediated mechanisms

T cells were first stimulated with CD3/CD28 beads at 1:3 (T cell to bead) ratio.

24 hours later, they were cultured with MSCs and CD19 ⁺ cells (to stimulate MSC-CARs), either in direct contact or in transwell experiments. A co-culture with MSC-CAR19 (containing CD28 stimulatory domain) with activated T cells, and CD19 ⁺ cells led to inhibition of T cell proliferation both when cells were in direct contact or not in direct contact in a trans-well experiment (Figure 21). These results demonstrate that MSC-CAR exert their suppressive functions through both direct, cell to cell contact and through secretion of soluble inhibitory factors/cytokines.

Example 13: MSC-CAR suppress T cell proliferation upon antigen specific stimulation T cells were first stimulated with CD3/CD28 beads at a 1 :3 ratio. 24 hours later,

T cells were cultured with untransduced MSCs, or MSC-CAR19 (CD28 containing CAR19, K122) at higher E:S:T ratio with NALM6, or T cells were cultured with NALM6 alone as a control for allogeneic effect. The addition of irradiated CD19 ⁺ cells was to stimulate MSC-CAR19 through the CAR. Proliferation of CD3 was inhibited in the presence of MSC-CAR19 (containing CD28 signaling domain), but not in the presence of un-transduced MSCs (Figure 22). T cells, MSCs, and NALM6 cells were cultured at ratio of 1:1:1 E (T cells (effectors):MSCs (suppressors):T (tumor).

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

Example 15: Designing CAR constructs targeting E-cadherin using scFvs

Four scFvs, clones 5, 6, 7, and 14, were identified as having binding affinity for human E-CAD. Three of these (clones 6, 7, and 14) were used to design CARs targeting E-CAD.

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, followed by a CD28 transmembrane domain, followed by a CD28 signaling domain. The amino acid sequence of K128 -CAR was as follows:

M ALP VT ALLLPL ALLLH AARPEV QL VQ S GGGL VKPGGS -

LRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRD NAKNSLYLQMN S LRAEDT AVYY C ARAQRQ W GAFD YW GQGTL VT V S S EGKS S G SGSESKASSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVI Y GKNNRP SGIPDRFSGSSS GNT AS LTIT GAQ AEDE AD Y Y CN S RD S S GNP VF GGGT KLTVLGLEPKSCDKTHTCPPCPDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSK RSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:66). The 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-

ALLLPLALLLHAARPEVQLVQS GGGLV QPGGSLRLSCAASGFTFS SY SMNWVRQ APGKGLEWVS YIS S S S STIYYVDS VKGRFTIS RDNAKNSLYLQMDS LRAEDTAVY Y C ARGGRVLV GALFD YW GQGTL VT V S SEGKS S GS GS ESKASLP VLTQPP S AS GTP

GQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGT

SASLAISGLQSEDEADYYCASWDTSLRAWVFGGGTKLTVLGLEPKSCDKTHTCP

PCPDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPG

PTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYD

VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG

HDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:67). The 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-

VQLVESGTDVKKPGASVTVSCKASGYTFTAYYIHWVRQAPGQGLEWMGWINPY SGASNYAQKFLGRVTMTRDTSTNTVYMQLSSLRASDTAMYYCAKAACGSNGC YMREFDYWGQGTLVTVSSSEGKSSGSGSESKASDIVMTQSPLSLPVTLGQPASISC RS S QS LVY S DGNTYLNWFQQRP GQ S PRRLI YKV SNRD S GVPDRF S GS GS GTDFTL KISRVEAEDVGIYYCMQGTYWPGAFGQGTKVDIKLEPKSCDKTHTCPPCPDPKF WVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHY QPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKM AEAY SEIGMKGERRRGKGHDGLY Q GLSTATKDTYDALHMQALPPR (SEQ ID NO:70).

T cells transduced with all three constructs exhibited potent killing against the luciferase ⁺ /human E-cadherin ⁺ MCF-7 cells. T cells transduced with K129-CAR also exhibited potent killing against the luciferase ⁺ /mouse E-cadherin ⁺ ID8 cells and luciferase ⁺ /canine E-cadherin ⁺ MCDK cells. These results demonstrate that CARs targeting E-cadherin are functional and can be used as described herein to create MSCs having CARs targeting E-cadherin.

Example 16: Treating or preventing colitis

To create a mouse model for xenograft colitis, NSG mice are treated with PBMCs, and their weight is monitored over time. Mice develop colitis within about 30-40 days, associated with weight loss. At this time point, satellite mice are euthanized, and colon tissue is harvested and examined for the infiltration of lymphocytes. Mice are then treated with MSC-UTD, or different MSC-CAR-E-cadherin cells (derived from various scFv clones) and daily weight is monitored. Thirty days later, mice are euthanized, and T cell infiltration into the colon is measured by flow cytometry and compared between mice treated with MSC-UTD and MSC-CAR-E-cadherin to confirm the treatment of colitis.

Example 17: Treating a human having colitis

A human is identified as having colitis and is administered MSC-CAR-E-cadherin cells at a dose of about 1 to about 2 million cells/kg of body weight intravenously or intra-arterially. A dose escalation is included with one dose given every 10 days.

Patients are monitored clinically for improvement of symptoms of colitis.

Example 18: Treating multiple sclerosis

Using the experimental autoimmune encephalomyelitis (EAE) mouse model, the efficacy of MSC-CAR-MOG is confirmed. After neuro-encephalitis is established, mice are treated with MSC-CAR-MOG or MSC-UTD. Neurological status of the mice is monitored on daily basis, and mice are followed for survival.

Example 19: Treating a human having multiple sclerosis

A human is identified as having multiple sclerosis and is administered MSC- CAR-MOG at a dose of about 2 to about 10 million cells/kg of body weight. The cells are administered either intravenously or intraventricularly. A dose escalation is included, and multiple doses are administered, for example, every 10 days. Patients are monitored clinically for improvement of symptoms of multiple sclerosis.

Example 20: Treating immune mediated encephalomyelitis

Using the experimental autoimmune encephalomyelitis (EAE) mouse model, the efficacy of MSC-CAR-MOG is confirmed. After neuro-encephalitis is established, mice are treated with MSC-CAR-MOG or MSC-UTD. Neurological status of the mice is monitored on daily basis, and mice are followed for survival.

Example 21: Treating a human having immune mediated encephalomyelitis

A human is identified as having immune mediated encephalomyelitis and is administered MSC-CAR-MOG at a dose of about 2 to about 10 million cells/kg of body weight. Cells are administered either intravenously or intraventricularly. A dose escalation is included, and multiple doses are administered, for example, every 10 days. Patients are monitored clinically for improvement of symptoms of immune mediated encephalomyelitis.

Example 22: Treating myocarditis

Using an experimental mouse myocarditis model, induced by infection with coxsackievirus B3, mice are treated with MSC-UTD or MSC-CAR-HER2. Mice are followed clinically and for survival. At the completion of the experiment, mice are euthanized, and infiltration of T cells into the heart is determined by flow cytometry.

Example 23: Treating a human having myocarditis

A human is identified as having severe or life threatening myocarditis and is administered MSC-CAR-HER2 at a dose of about 2 to about 10 million cells/kg of body weight. Cells are administered either intravenously or via intra-cardiac route. A dose escalation is included, and multiple doses are administered, for example, every 10 days. Patients are monitored clinically for improvement of symptoms of myocarditis.

Example 24: MSC-CAR-E-cadherin suppresses T cell antitumor activity and their proliferation in vivo

NSG mice were engrafted with the luciferase ⁺ /E-cadherin ⁺ MCF-7 cell line (lxl 0 ⁶ cells intravenously). One week later, bioluminescent imaging was performed to confirm engraftment. All mice were treated with E-cadherin CAR T cells (2 x 10 ⁶ cell), and mice were randomized to treatment with MSC-CAR-E-cadherin (1 xlO ⁶ ), untransduced MSC (MSC-UTD) (1 xlO ⁶ ), or no MSC control. Mice were followed with serial bioluminescent imaging to measure disease burden. Treatment with MSC-CAR-ECAD resulted in reduced anti-tumor activity of E-cadherin directed CAR T cells (Figure 24 A). Peripheral blood flow cytometry on day 7 post treatment with CAR T cells and CAR- MSC cells was performed. Treatment with MSC-CAR-E-Cadherin led to a trend to reduced T cell proliferation in vivo (Figure 24B). These results demonstrate that MSC- CAR-E-CAD are able to suppress T cell effector functions in this xenograft model.

In another experiment, luciferase ⁺ MSC-CAR-E-cadherin cells were generated and injected intraperitoneally into immunocompromised NSG mice. Serial bioluminescent imaging was performed to determine the persistence of MSC-CAR-E- cadherin. MSC-CAR-E-cadherin cells were found to persist in vivo for over 10 days (Figures 25A and 25B). These results demonstrate that MSC-CAR cells are able to persist in vivo, for example, within xenograft models.

OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.