Major histocompatibility complex-based chimeric receptors and uses thereof for treating autoimmune diseases

Abstract

To provide major histocompatibility complex-based chimeric receptor (MHC-CAR) for use in targeting autoreactive immune cells.SOLUTION: Disclosed is a major histocompatibility complex (MHC)-based chimeric receptor (CAR) comprising: (i) an extracellular domain of a MHC molecule conjugated to an antigen peptide from an antigen involved in an autoimmune disease; and (ii) a cytoplasmic signaling domain, at least one co-stimulating domain or a combination thereof.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Related Applications This application claims the benefit of the filing date of U.S. Provisional Application No. 62 / 584,449, filed November 10, 2017, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Autoimmune diseases are characterized by abnormal immune responses against self-antigens, leading to tissue damage or destruction. Multiple sclerosis (MS) is an autoimmune disease of the central nervous system in which activated autoreactive T cells invade the blood-brain barrier and initiate an inflammatory response that leads to myelin destruction and axonal loss. Although the etiology of MS, the mechanisms associated with its onset and progression, and the determination of its outcome remain to be elucidated, all available evidence suggests that therapies that specifically target pathological immune cells involved in MS would have improved therapeutic outcomes over available therapies. Reinhard et al., Proceedings of the National Academy of Sciences, 101(suppl 2):14599-14606; 2004. This strategy can be extended to other immune disorders with similar mechanisms, including rheumatoid arthritis. Carol et al., Nature Reviews Immunology, 2(2):85-95, 2002.

[0003] The major histocompatibility complex (MHC), known in humans as human leukocyte antigen (HLA), is a set of cell surface proteins essential for the immune system to recognize foreign agents. The MHC complex binds to antigens derived from pathogens and presents them to T cells, which then become activated, leading to the elimination of cells presenting the foreign antigens. The MHC complex also presents intact, and in some cases misfolded, host-derived proteins to B cells, which can induce the autoantibody response characteristic of autoimmune disorders. Jiang et al., International immunology, 25(4):235-246 (2013), and Busch et al., The EMBO journal, 15(2):418, (1996). Summary of the Invention

[0004] In an aspect, the present disclosure features a major histocompatibility complex (MHC)-based chimeric receptor (CAR), comprising (i) an extracellular domain of an MHC molecule conjugated to an antigenic peptide from an antigen involved in an autoimmune disease, and (ii) a cytoplasmic signaling domain, at least one costimulatory domain, or a combination thereof. The MHC-based CAR may further comprise a hinge domain located between (i) and (ii). The antigenic peptide depends on the autoimmune disorder and may be from myelin basic protein (MBP), proteolipid protein (PLP), insulin, glutamic acid decarboxylase, or additional exemplary autoantigens as described in Table 1.

[0005] In some examples, the MHC-based chimeric receptor comprises at least one costimulatory domain, which may be a costimulatory domain from 4-1BB (CD137), a costimulatory domain from CD28, or a combination thereof. In other examples, the MHC-based chimeric receptor as described herein may not comprise a cytoplasmic signaling domain. Alternatively or additionally, the MHC-CAR comprises a cytoplasmic signaling domain of CD3ζ.

[0006] In some embodiments, the MHC molecule in the MHC-CAR is a class I MHC, e.g., a human class I MHC. In some cases, the extracellular domain of the chimeric receptor comprises the extracellular domain of the alpha chain of class I MHC fused to an antigenic peptide. For example, the chimeric receptor may be a fusion polypeptide comprising (i) the extracellular domain of a class I MHC molecule, and (ii) a cytoplasmic domain, at least one costimulatory domain, or a combination thereof. In one example, the chimeric receptor is a fusion polypeptide, and the fusion polypeptide comprises, from N-terminus to C-terminus, a signal peptide, a first peptide linker, an antigenic peptide, a second peptide linker, an extracellular domain of a macroglobulin, a third peptide linker, a class I MHC molecule, a transmembrane domain, at least one costimulatory domain, and CD3zeta.

[0007] In other embodiments, the MHC-based chimeric receptor as described herein comprises a class II MHC (e.g., human MHC II) or a portion thereof. Such a chimeric receptor may comprise a first polypeptide comprising an extracellular domain of a first MHC class II, and a second polypeptide comprising an extracellular domain of a beta chain of a second MHC class II, and an antigenic peptide is fused to either the first or second polypeptide, and either the first or second polypeptide further comprises a cytoplasmic signaling domain, at least one costimulatory domain, or a combination thereof. In some examples, the chimeric receptor may be a fusion polypeptide comprising (i) an extracellular domain of an alpha chain of a first MHC class II molecule, (ii) an extracellular domain of a beta chain of a second MHC class II molecule, (iii) an antigenic peptide, and (iv) a cytoplasmic signaling domain, at least one costimulatory domain, or a combination thereof. In some examples, the antigen protein may not be linked to MHC class II and may instead be expressed as a separate fusion polypeptide with an alternative signal peptide (e.g., from CD150, i.e., MDPKGLLSLTFVLFLSLAFG (SEQ ID NO: 388)). In some examples, the first MHC class II is HLA-DRA*1010. Alternatively or additionally, the second MHC class II is HLA-DRB1*1501.

[0008] In another aspect, the disclosure features a nucleic acid or set of nucleic acids that generally encodes any of the MHC-based chimeric receptors described herein. In some cases, the nucleic acid or set of nucleic acids may be located in one or more vectors, e.g., viral vectors.

[0009] Furthermore, the present disclosure provides a genetically modified immune cell (e.g., T cell), expressing any of the MHC-based chimeric receptors described herein. In some cases, the activity of endogenous T cell receptor (TCR) can be suppressed, which may be achieved by mutating or deleting the alpha chain of endogenous TCR, the beta chain of endogenous TCR, or both to inhibit the surface expression of endogenous TCR. Alternatively or additionally, the expression of endogenous CD52 can be inhibited.

[0010] In some embodiments, the genetically modified immune cells as described herein may further express a suicide gene (e.g., RQR8), a marker gene (e.g., GFP), or both. If necessary, the immune cells may be further modified for delivery and retention to lymph nodes or tertiary lymphoid organs. For example, the immune cells may be further engineered to overexpress VAP-1, L-selectin, CCR7, CXCR5, or a combination thereof. In some cases, the expression of endogenous sphingosine-1-phosphate receptor 1 may be inhibited in the genetically modified immune cells.

[0011] In some embodiments, immune cells can be engineered to migrate to sites of inflammation using, for example, chemokine receptors such as CCR6 (e.g., to the site of Th17 cells), CXCR3 or CXCR4 (e.g., to the site of plasma cells), or through membrane-bound antigen-targeting antibodies. Alternatively or additionally, genetically modified immune cells may further comprise genetic modifications that result in blocking PD-1 signaling. If necessary and the disorder is particularly severe, MHC-CAR cells can also be designed to remove or inactivate bystander B cells (using CD19 or CD20-CARs) or plasma cells (using CS1-CARs and / or CS1 knockouts).

[0012] In some embodiments, the genetically modified immune cells as described herein may be regulatory T cells, and the regulatory T cells may be CD25+ and optionally CD4+. In some cases, the regulatory T cells may be derived from CD25++CD45R+ T cells isolated from peripheral blood mononuclear cells or umbilical cord blood. In other cases, the regulatory T cells may include a transgene encoding CD25. Any Treg cell disclosed herein may further express a chimeric receptor specific for CD19, a chimeric receptor specific for CS-1, or both. Alternatively or additionally, the regulatory T cells may further express CCR6, CXCR5, PD-1, or a combination thereof. In some cases, the regulatory T cells may present an antibody specific for MOG.

[0013] In yet another aspect, the present disclosure provides a method of suppressing autoreactive immune cells in a subject having an autoimmune disease (e.g., multiple sclerosis). The method may include administering to the subject an effective amount of a genetically modified immune cell as described herein, which may be a T cell.

[0014] In some embodiments, the genetically modified immune cells are autologous cells. In other embodiments, the genetically modified immune cells are allogeneic cells. Any genetically modified immune cells may be administered to the lymph nodes of the subject. In some cases, the subject is receiving a therapy that includes an antibody specific for CD52.

[0015] In some embodiments, the subject is a human patient having or at risk for multiple sclerosis, and the genetically modified T cells are Treg cells or cytotoxic lymphocytes (CTLs) as described herein.

[0016] In some examples, the human patient is an early stage MS patient, and the Treg cells express MHC-CAR and have one or more of the following genetic modifications: (i) PD-L1 and / or PD-1 knockout, (ii) surface expression of CCR6 and / or CXCR5, (iii) surface presentation of an antibody or antigen-binding fragment thereof specific for MOG, and (iv) surface expression of a chimeric receptor targeting CD19. In some examples, the patient may be initially, simultaneously, or alternately treated with cytotoxic CTLs having the same types of modifications.

[0017] In some examples, the human patient has relapsing-remitting MS or early stage progressive MS, and the Treg cells express MHC-CAR and have one or more of the following modifications: (i) surface display of an antibody or antigen-binding fragment specific for MOG, and (ii) surface expression of CCR6. In some examples, the patient may be initially, simultaneously, or alternately treated with cytotoxic CTLs having the same type of modification.

[0018] In some examples, the human patient has relapsing-remitting MS or early stage progressive MS, and the Treg cells express MHC-CAR and have one or more of the following modifications: (i) surface expression of a chimeric receptor targeting CD19, and (ii) surface expression of CXCR5. In some examples, the patient may be initially, simultaneously, or alternately treated with CTLs having the same modifications.

[0019] In some cases, the human patient has a chronic progressive form of MS and the Treg cells express MHC-CAR and have one or more of the following modifications: (i) surface expression of a chimeric receptor targeting CS-1, and (ii) surface expression of the agents CXCR4, CCR6, and / or CXCR5. In some cases, the patient may be treated initially, simultaneously, or alternating with CTLs having the same modifications.

[0020] In some embodiments, the subject is a human patient having or at risk for systemic lupus erythematosus, rheumatoid arthritis, juvenile idiopathic arthritis (also known as juvenile idiopathic arthritis), Sjogren's syndrome, systemic sclerosis, ankylosing spondylitis, type 1 diabetes, autoimmune thyroid disease (Graves' disease and Hashimoto's disease), multiple sclerosis myasthenia gravis, inflammatory bowel disease (Crohn's disease or ulcerative colitis), psoriasis, or a disease described in Table 1, and the genetically modified T cells are Treg cells and / or CTLs as described herein.

[0021] In some examples, the human patient is an early stage patient with any of the autoimmune disorders described herein (e.g., those listed in Table 1), and the Treg cells express MHC-CAR and have one or more of the following genetic modifications: (i) PD-L1 and / or PD-1 knockout, (ii) surface expression of CCR6 and / or CXCR5, (iii) surface presentation of an antibody or antigen-binding fragment thereof specific for a relevant protein listed as an autoantigen in Table 1 for that autoimmune disorder, and (iv) surface expression of a chimeric receptor targeting CD19. In some examples, the patient may be initially, simultaneously, or alternatingly treated with CTLs having the same modifications.

[0022] In some examples, the human patient has a moderately severe pathology of any autoimmune disorder as described herein (e.g., those listed in Table 1), and the Treg cells express MHC-CAR and have one or more of the following modifications: (i) surface display of an antibody or antigen-binding fragment specific for the relevant protein listed as an autoantigen in Table 1 for that autoimmune disorder, and (ii) surface expression of CCR6. In some examples, the patient may be treated initially, simultaneously, or alternating with CTLs having the same modifications.

[0023] In some examples, the human patient has a moderately severe pathology of any autoimmune disorder as described herein (e.g., those listed in Table 1), and the Treg cells express MHC-CAR and have one or more of the following modifications: (i) surface expression of a chimeric receptor targeting CD19, and (ii) surface expression of CXCR5. In some examples, the patient may be treated initially, simultaneously, or alternately with CTLs having the same modifications.

[0024] In some examples, the human patient has a severe pathology of any of the autoimmune disorders described herein (e.g., those listed in Table 1), and the Treg cells express MHC-CAR and have one or more of the following modifications: (i) surface expression of a chimeric receptor targeting CS-1, and (ii) surface expression of an agent targeting CXCR4, CCR6, and / or CXCR5. In some examples, the patient may be treated initially, simultaneously, or alternately with CTLs having the same modifications.

[0025] Also within the scope of the present disclosure are pharmaceutical compositions for use in the treatment of autoimmune diseases, compositions comprising genetically modified immune cells expressing MHC-CAR as described herein, such as Treg cells, and a pharma- ceutically acceptable carrier, as well as the use of such genetically modified immune cells for the manufacture of a medicament for use in the treatment of a targeted autoimmune disease.

[0026] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the invention will be apparent from the following drawings and detailed description of some embodiments, as well as from the appended claims. [Brief description of the drawings]

[0027] [Figure 1] 1 is a schematic diagram of an exemplary design of a lentiviral expression vector for expression of an antigen-specific T cell receptor (TCR). The designs of mRNA and multicistronic mRNA are similar. [Diagram 2]2 is a schematic diagram of MHC-CAR T cells expressing various designs of MHC-CAR as shown in conjugate with myelin basic protein (MBP) peptide to target pathological T cells involved in multiple sclerosis (MS). Optionally, the MHC-CAR T cells may have endogenous TCR and / or CD52 knocked out. The MHC-CAR T cells may further express RQR8 on the cell surface. [Diagram 3] Figure 3 is a schematic diagram of an exemplary design for various MHC-CAR constructs. An exemplary MHC-CAR construct may have two subunits: an alpha chain containing a leader sequence, a DRA*1010 domain, and a cytoplasmic domain, and a beta chain containing a leader sequence from HLA-DRB1*1501, a peptide from MBP, and a domain from HLA-DRB1*1501. The DNA used to generate the mRNA contains a single strand or is multicistronic and separated by an orthogonal 2A sequence. RQR8 and eBFP (or GFP) are used for both cell control and labeling. [Figure 4] FIG. 4 is a schematic diagram of an exemplary design of expression cassettes for various MHC-CAR constructs containing MBP peptides, which may further include eBFP (or GFP) or RQR8. [Diagram 5] 5 is a schematic diagram of an exemplary design of a lentiviral expression construct for MHC-CAR and optionally a label protein such as eBFP and / or RQR8. Such expression cassettes are small enough to be included in one lentiviral package. [Figure 6] Figure 6 is a schematic diagram of an exemplary design of an MHC-CAR, which may contain multiple sites for tagging. Site 1 is an HLA-DR antibody binding site for cases where native HLA-DR is not expressed or is due to CIITA editing. Sites 2 and 3 are potential insertion sites for polyhistidine tag motifs. Sites 4 and 5 represent RQR and RQR8, respectively. [Figure 7] 7 is a schematic diagram of an exemplary design of an MHC class II moiety linked to an antigenic peptide. "N" refers to the N-terminus of the polypeptide. The dot in the filled circle refers to the antigenic peptide. [Figure 8A] Figures 8A and 8B are schematic diagrams of exemplary constructs of MHC class I. Figure 8A shows an exemplary design of an MHC class I moiety linked to an antigenic peptide. "N" refers to the N-terminus of the polypeptide. The dot in the filled circle refers to the antigenic peptide. [Figure 8B] FIG. 8B shows an exemplary expression cassette for an MHC class I CAR construct. [Figure 9] FIG. 9 shows exemplary costimulatory domains and combinations thereof for constructing MHC-CARs and for consideration of cytokine co-expression. [Figure 10-1] FIG. 10 shows exemplary single-chain and multi-chain MHC class I and class II MHC-CAR constructs, including multi-chain MHC-CAR constructs containing both MHC class I and class II components. [Figure 10-2] Same as above. [Figure 11] FIG. 11 is a plot showing CD3 expression on primary human stimulated CD3+ T cells (TCELL-0028) transfected with mRNA encoding Cas9 ("Cas9 alone") or mRNA encoding Cas9 ("Cas9 alone") and an sgRNA targeting the gene encoding the T cell receptor alpha domain (TRAC) ("Cas9+TRAC gRNA"). [Figure 12] 12 is a plot showing relative cell survival in a killing assay. HEK-293 cells transfected with Construct 1, Construct 2, or both Constructs 1 and 2 were incubated with medium alone, Rituximab, complement, or both Rituximab and complement. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Autoreactive T cells (e.g., for myelin components involved in multiple sclerosis) are present in normal individuals. A large part of the determinant of disease induction lies in the class of immune response that occurs when these autoreactive T cells are triggered in autoimmune patients (e.g., MS patients). The generation of pathological autoreactive T cells favors both specific major histocompatibility complex (MHC) and non-MHC genes, which determine the protein sequences and class of immune response to which the individual responds.

[0029] Once an immune attack is launched against an initiating autoantigen (e.g., myelin antigen in MS), spreading of reactivity to other autoantigens occurs; i.e., when T cells attack one autoantigen (e.g., brain proteins in MS), other structures are damaged and they can sensitize additional T cells to attack other targets, in a process called "epitope spreading," which is a process shared by all autoimmune disorders and common to disease responses in general.

[0030] B cells are a normal component of the immune response in early disease lesions, e.g., active MS lesions, triggered by early autoreactive attacks. Accumulation of B cells occurs as packed aggregates or ectopic B cell follicles. Serafini et al., Brain Pathol. 14: 164-144 (2004);Wekerle, Autoimmunity, 50:1, 57-60 (2017);and Probstel, et al., International journal of molecular sciences, 16(7), pp.16576-16592 (2015). In MS, B cells were reported to be found in the brain and spinal cord of RR, SP, and P stage MS patients. Therapeutic treatments that target B cells either directly or indirectly have proven beneficial in the treatment of autoimmune diseases such as MS. Wekerle, 2017.

[0031] Both CD4+ and CD8+ T cells are present in MS lesions and are believed to play a central role in disease development. Increased frequencies of myelin-reactive (MBP, PLP, and MOG) CD4 and CD8 cells are found in MS patients compared to healthy controls. Cao, et al., Sci. Transl. Med. 7 (287), 287ra74 (2014);Martin, et al., J. Exp. Med. 173 (Jan 1, 1991);Ota, et al., Nature 346, 183 (Jul 12, 1990);Pette, et al., Neurology 40, 1770 (1990);and Raddassi, et al., J. Immunol. 187, 1039 (2011).

[0032] IFN-gamma producing Th1 and Th17 cells are uniquely pathogenic. Factors that favor the development of Th1 cells are elevated in MS patients and are also induced by viral infection: gamma interferon; IL-12. Nearly all treatments that affect the immune system and help MS decrease Th1 responses and increase Th2 and TH3 responses. Th17 cells are present at sites of tissue inflammation and are involved in autoimmune / chronic inflammatory conditions. Th17 producing CD4 and CD8 cells are increased in lesions, blood, and CSF of patients. [Tzartos 2008; Matusevicius 1999; Bruchlacher-Waldert 2009]. CCR6 and CD161 on Th17 cells are presumed to be homing molecules to inflamed tissues [Cosmi, 2008].

[0033] Th17 cells are also involved in a number of other autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, juvenile idiopathic arthritis (also known as juvenile idiopathic arthritis), Sjogren's syndrome, systemic sclerosis, ankylosing spondylitis, type 1 diabetes, autoimmune thyroid disease (Graves' disease and Hashimoto's disease), myasthenia gravis, inflammatory bowel disease (Crohn's disease or ulcerative colitis), and psoriasis. Tabarkiewicz et al., Archivum immunologiae et therapiae experimentalis, 63(6):435-449 (2015).

[0034] The ultimate goal of any treatment for autoimmune disease is complete suppression of pathology. In the case of multiple sclerosis and other autoimmune disorders, pathological lymphocytes (both B and T cells, and, if necessary, plasma cells for very severe cases) are expected to be eliminated or controlled to stop the disease process, and interventions at different stages of disease progression require different cellular targets, and therefore therapeutic cells.

[0035] Disclosed herein is a major histocompatibility complex (MHC)-based chimeric receptor (MHC-CAR) for targeting autoreactive immune cells, such as autoreactive T cells. The MHC-CAR as described herein comprises one or more MHC polypeptides or their extracellular domains and one or more cell signaling domains, such as a cytoplasmic signaling domain (e.g., from CD3ζ), at least one costimulatory domain (e.g., from 4-1BB or CD28), or both. The MHC-CAR may further comprise an antigen peptide from an autoantigen or a foreign antigen that mimics an autoantigen in eliciting an autoimmune response. Also herein are nucleic acids encoding MHC-CARs, vectors carrying same, and engineered immune cells, such as T cells and natural killer (NK) cells, that express MHC-CARs. Such engineered immune cells can be used to target autoreactive immune cells, thereby obtaining benefits in the treatment of autoimmune diseases involving autoreactive immune cells.

[0036] Also disclosed herein are genetically modified regulatory T (Treg) cells expressing MHC-based chimeric receptors as disclosed herein. Such Treg cells may be further modified with additional genetic engineering, in addition to the chimeric receptor(s) targeting T cell and / or B cell surface markers, for example, to target specific tissue sites (e.g., lymph nodes or sites of inflammation) or to modulate immune responses (e.g., checkpoint modulation). Genetically modified Treg cells may be used to inhibit pathology in early stages of the target disease, to control disease progression in mid-stages of the disease (e.g., relapsing or remitting MS), or to suppress pathology in later stages of the disease (e.g., chronic progressive MS), for example, via induction of cytotoxicity of pathological CD8+ T cells.

[0037] I. Major Histocompatibility Complex (MHC)-based Chimeric Receptors (MHC-CARs) The MHC-based chimeric receptor (MHC-CAR) described herein comprises an MHC moiety conjugated to an antigenic peptide (e.g., misfolded) and at least one cell signaling moiety, which may be a cytoplasmic signaling domain (e.g., that of CD3ζ), one or more costimulatory domains (e.g., that of 4-1BB or CD28), or a combination thereof. In some cases, the antigenic peptide may be part of a fusion polypeptide of the MHC-CAR. In other cases, the antigenic peptide forms a complex with the MHC-CAR without forming a fusion polypeptide with the MHC-CAR. As used herein, the term "conjugated" means that at least two components are physically associated via a covalent bond or via a non-covalent interaction.

[0038] In some examples, the MHC-CAR may be a single fusion polypeptide containing an MHC portion, an antigenic peptide, and at least one cell signaling portion. Such a single fusion polypeptide may form a complex with an endogenous cell membrane protein (e.g., β-microglobulin) when expressed in a suitable immune cell.

[0039] In other examples, the MHC-CAR described herein may be a multi-chain protein complex, e.g., a heterodimer, comprising one polypeptide comprising an antigenic peptide. In some cases, the antigenic peptide or polypeptide may be expressed as separate polypeptides, which may form a complex (e.g., a trimer) with an MHC component. The antigenic polypeptide may be a misfolded antigenic protein that binds to MHC. Optionally, the MHC-CAR may further comprise a hinge domain, which may be adjacent to the antigenic peptide and / or MHC moiety, an N-terminal signal peptide, and / or one or more tagging sites, e.g., a histidine protein tag and / or an RQR domain that additionally acts as a kill switch site.

[0040] (i) Components of MHC-CAR (a) MHC part The MHC-CAR constructs disclosed herein include an MHC portion, which may include one or more MHC polypeptides or extracellular domains thereof. The MHC portion may be derived from a suitable source, such as a human or non-human mammal (e.g., monkey, mouse, rat, rabbit, pig, etc.). In some cases, the MHC portion is from a human MHC molecule (also known as HLA). In some cases, the domain that interacts with molecules from other cells (TCR or BCR) is from a human MHC molecule. There are mainly two classes of MHC molecules, MHC class I molecules and MHC class II molecules, both of which can be used to construct the MHC-CARs described herein. Sequences of MHC class I and class II molecules from various species (e.g., humans, non-human primates, canines, fish, ovids, bovines, equines, hogs, murines, and galli) are available from public genetic datasets, such as the IPD-MHC database and the IMGT / HLA database provided by EMBL-EBI and the dbMHC database provided by the National Center for Biotechnology Information (NCBI).

[0041] MHC class I molecules are heterodimers containing an alpha chain and β-microglobulin. The extracellular domain of the alpha chain includes three subdomains, α1, α2, and α3. In some embodiments, the MHC portion may include the alpha chain of the MHC class I molecule, or its extracellular domain, such as the α1 domain, the α2 domain, the α3 domain, or a combination thereof. The MHC class I molecule may be a human HLA-A molecule, a human HLA-B molecule, or a human HLA-C molecule. In some cases, the alpha chain of the MHC class I molecule may be fused with β-microglobulin to produce a single-chain fusion protein. In some examples, the MHC class I portion is from HLA A3, which may be used in conjunction with a PLP peptide. Honma et al., J. Neuroimmunol. 73:7-14 (1997). In other examples, the MHC class I is from HLA A2, which may be used in conjunction with the same PLP peptide and the presentation of a viral peptide such as TAX. TAX is a molecular mimic of the protein tax or p40 (Genbank accession number BAB20130.1) of a human neuronal protein involved in diseases such as rheumatoid arthritis, system lupus erythematosus, and Sjogren's syndrome, and from the HTLV-1 virus. Garboczi, et al. The Journal of Immunology, 157(12):5403-5410, 1996. Quaresma, et al., 2015. Viruses, 8(1):5 2015. Class I proteins and peptides may additionally contain modifications to allow for more robust peptide loading, such as the substitution of the invariant tyrosine at position 84 of the heavy chain with an alanine, or alternatively, the tyrosine at position 84 may be replaced by a cysteine, such that the second position of the peptide-β2m linker may be substituted to create a disulfide trap. Hansen et al. Trends in immunology, 31(10):363 (2010).

[0042] Similar to MHC class I molecules, MHC class II molecules are also heterodimers consisting of two homologous peptides, α chain and β chain. The extracellular domain of each of the α chain and β chain contains two subdomains, α1 / α2, and β1 / β2. When MHC class II molecules are used to construct MHC-CARs, the MHC portion may include two subunits, one containing an α chain or a portion thereof, e.g., its extracellular domain (e.g., α1, α2, or both), and the other containing a b chain or a portion thereof, e.g., its extracellular domain (e.g., β1, β2, or both). When only the regions that interact with other cell types are used (i.e., α1 and β1), specific amino acid modifications may be required to enhance folding of the mini-MHC; see mini-sequences with shaded regions and Birnbaum et al. The MHC class II molecule may be a human HLA DP molecule, a human HLA DM molecule, a human HLA DOA molecule, a human HLA DOB molecule, a human HLA DQ molecule, or a human HLA DR molecule. In some examples, the MHC class II molecule is a human HLA DR molecule, for example, HLA DR*1501.

[0043] (b) Antigenic peptides The antigenic peptides of the MHC-CARs described herein are antigenic peptides that can be recognized by pathogenic immune cells (e.g., autoreactive T cells or B cells) involved in autoimmune diseases. When presented by a suitable MHC molecule, such antigenic peptides interact with the antigen-specific T cell receptors of pathogenic T cells, leading to downstream immune responses.

[0044] In some cases, specific antigen peptides can be designed for a specific autoimmune disease patient, such as an MS patient, using methods known in the art. Programs such as NetMHC allow for the personalized design of antigen peptides specific to patient MHC and have been used to develop personalized cancer vaccines. Hacohen et al., Cancer immunology research, 1(1):11-15 (2013). Personalized CAR T and Treg therapy for autoimmune disorders is also within the scope of this disclosure. For disorders with very strong MHC associations (e.g., MS), personalized therapy can be utilized to treat large classes of patients at different stages of the disease. Recent studies have also demonstrated that class II MHC and especially HLA, involved in autoimmune disorders, can present whole antigen proteins, not just processed peptides. Jiang et al., International immunology, 25(4):235-246, (2013). These MHC-protein complexes appear to induce autoantibody production in autoimmune disorders, including autoantibodies specific to those with a particular autoimmune disorder, in addition to antibodies that do not bind to properly folded proteins. The present inventors believe that the presentation of antigenic proteins in MHC-CARs may provide a specific route to eliminate or inactivate autoimmune-specific B cells, such as those in MS, which give rise to oligoclonal bands whose specificity to proteins has not been elucidated despite many intense attempts.Owens et al., Annals of neurology, 65(60):639-649, 2009;Chastre et al., New England Journal of Medicine, 374(15):1495-1496, 2016;Housley et al., Clinical Immunology, 161(1):51-58, 2015;Larman et al., 2013. Journal of autoimmunity, 43:1-9, 2013.If the antigen protein does not bind to MHC, that particular MHC-CAR will not be expressed, but as a Treg or CTL it can still play a bystander role in regulating the immune response as part of a patient-specific population of MHC-CAR T and Treg cells with different specificities, depending on other characteristics.

[0045] The antigenic peptides used in the present invention may be fragments of autoantigens involved in autoimmune diseases, such as myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and proteolipid protein (PLP) involved in multiple sclerosis, insulin and glutamic acid decarboxylase (GAD) involved in type I diabetes, tryptase involved in rheumatoid arthritis (RA), and proteins included in Table 1 below. Alternatively, the antigenic peptides may be fragments of pathogenic proteins, such as viral or bacterial proteins, that are highly homologous to autoantigens involved in autoimmune diseases. Such antigenic peptides may also target pathogenic T cells. If necessary, the antigenic peptides may be antigenic proteins or protein fragments (typically misfolded) that can be expressed separately and directly bound to the MHC portion of the MHC-CAR described herein. In the natural state (bound to MHC, but not to the MHC portion of the MHC-CAR), such antigenic protein / MHC complexes stimulate pathogenic B cells to produce autoantibodies.IgGH or rheumatoid factor in rheumatoid arthritis (Jin et al., Proceedings of the National Academy of Sciences, 111(10):3787-3792, 2014), β2-glycoprotein I in antiphospholipid syndrome (Tanimura et al., Blood, 125(18):2835-2844, 2015) and recurrent miscarriage (Tanimura et al., Placenta, 46:108, 2016), GM-CSF in autoimmune pulmonary alveolar proteinosis (Hamano et al., ALVEOLAR MACROPHAGE BIOLOGY B32:A3147-A3147, 2016), tyrosinase in vitiligo (Arase et al. Journal of Dermatological Science, 84(1):e87, For proteins such as erythrocyte segregation (Ischemia, 2016), and myeloperoxidase in microscopic polyangiitis (Hiwa et al., Arthritis & Rheumatology, 69(10):2069-2080, 2017), HLA-mediated surface presentation of misfolded variant / HLA complexes and, in some cases, autoantibody binding, may occur.

[0046] Antigen peptides for use in the MHC-CARs described herein may contain up to 20 amino acid residues, the extracellular domain of an antigen protein, or the full-length antigen protein. When used in conjunction with an MHC class I portion, the antigen peptide may be 8-10 amino acids in length. Such antigen peptides fit well into the peptide-binding site of an MHC class I molecule. Antigen peptides used in conjunction with an MHC class II portion may be longer, for example, containing 15-24 amino acid residues or up to the full-length of an antigen protein, because the antigen-binding groove of an MHC class II molecule is open at both ends, whereas the corresponding antigen-binding groove on a class I molecule is usually closed at each end. The open antigen-binding groove of MHC class II molecules involved in autoimmune disorders may also frequently present intact (but e.g., misfolded) antigen proteins or splice variants. Jiang et al., International immunology, 25(4):235-246, 2013.

[0047] In some examples, a fragment of human MBP is used to construct the MHC-CAR described herein. An exemplary amino acid sequence of human MBP is provided below: MASQKRPSQRHGSKYLATASTMDHAARHGFLPRHRDTGILDSIGRFFGGDRGAPKRGSGKVPWLKPGRSPLPSHARSQPGLCNMYKDSHHPARTAHYGSLPQKSHGRTQDENPVVHFFKNIVTPRTPPPSQGKGRGLSLSRFSWGAEGQRPGFGYGGRASDYKSAHKGFKGVDAQGTLSKIFKLGGRDSRSGSPMARRHHHHHH (SEQ ID NO: 1)

[0048] Exemplary MBP antigenic peptides include, but are not limited to, the following: GSKYLATASTMDHARHGFLPRHRDTGILDSIGRFFGGDRG (SEQ ID NO: 2), KYLATASTMDHARHGFLPRH (SEQ ID NO: 3), ATASTMDHARHGFLPRHRDTGIL (SEQ ID NO: 4), RDTGILDSIGRFFGGDRGAP (SEQ ID NO: 5), IGRFFGGDRGAPKRGSGKDSHHPARTAHY (SEQ ID NO: 6), APKRGSGKDSHHAARTAHY (SEQ ID NO: 7), GSGKDSHHPAR TAHYGSLPQ (SEQ ID NO: 8), HHPARTAHYGSLPQKSHGR (SEQ ID NO: 9), HAARTAHYGSLPQKSQGHR (SEQ ID NO: 10), SLPQKSHGRTQDENPVVHF (SEQ ID NO: 11), PQDENPVVHFFKNIVTPRTP (SEQ ID NO: 12), TQDENPVVHFFKNIVTPRTP (SEQ ID NO: 13), QDENPVVHFFKNIVTPRTP (SEQ ID NO: 14), DENPVVHFFKNIVTPRTPP (SEQ ID NO: 15), SEQ ID NO:15), ENPVVHFFKNIVTPR (SEQ ID NO:16), ENPVVHFFKNIVTPRTP (SEQ ID NO:17), ENPVVHFFKNIVTP (SEQ ID NO:18), NPVVHFFKNIVTPRTPPPSQ (SEQ ID NO:19), VVHFFKNIVTPRT (SEQ ID NO:20), VVHFFKNIVTPRTPPPSQGK (SEQ ID NO:21), KNIVTPRTPPPSQGKGRGL (SEQ ID NO:22), PSQGKGRGLSLSRFSWGAE (SEQ ID NO:23), No.:23), GKGRGLSLSRFSWGAEGQRP (SEQ ID NO:24), LSRFSWGAEGQRPGFGYGG (SEQ ID NO:25), QRPGFGYGGRASDYKSAHK (SEQ ID NO:26), ASDYKSAHKGFKGVDAQGT (SEQ ID NO:27), FKGVDAQGTLSKIFKLGGR (SEQ ID NO:28), VDAQGTLSKIFKLGGRDSRS (SEQ ID NO:29), and SKIFKLGGRDSRSGSPMARR (SEQ ID NO:30).

[0049] An exemplary nucleic acid sequence encoding the MBP antigenic peptide of SEQ ID NO:15 is provided below: GATGAGAATCCCGTGGTTCATTTTTTTAAGAACATCGTCACACCGCGCACCCCACCTG (SEQ ID NO: 411)

[0050] Particular examples include MBP13-32, MBP89-101, MBP83-99, MBP111-129, or MBP146-170.

[0051] Exemplary amino acid sequences for human myelin oligodendrocyte glycoprotein, proteolipid protein, and myelin associated glycoprotein are provided below: >CAA52617.1 Myelin oligodendrocyte glycoprotein [Homo sapiens] MASLSRPSLPSCLCSFLLLLLLQVSSSYAGQFRVIGPRHPIRALVGDEVELPCISPGKNATGMEVGWYRPPFSRVVHLYRNGKDQDGDQAPEYRGRTELLKDAIGEGKVTLRIRNVRFSDEGGFTCFFRDHSYQEEAAMELKVEDPFYWVSPGVLVLLAVLPVLLLQITVGLVFLCLQYRLRGKLRAEIENLHRTFDPHFLRVPCWKITLFVIVPVLGPLVALIICYNWLHRRLAGQFLEELRNPF (SEQ ID NO: 31)

[0052] Exemplary MOG antigenic peptides include MOG1-20 or MOG35-55. >AAA60117.1 Proteolipid Protein [Homo sapiens] [ka]

[0053] Exemplary antigenic fragments of PLP are underlined and bolded. Other examples include PLP139-151(4) or PLP178-191. >AAH93045.1 Myelin-associated glycoprotein [Homo sapiens] (SEQ ID NO:33)

[0054] Table 1 below provides additional exemplary autoantigens associated with other autoimmune diseases. Table 1. Autoantigens in various autoimmune disorders [Table 1] TIFF2025019209000004.tif200157TIFF2025019209000005.tif196157TIFF2025019209000006.tif194157TIFF20250192090 00007.tif183157TIFF2025019209000008.tif197157TIFF2025019209000009.tif204157TIFF2025019209000010.tif194157

[0055] Table 2 below provides HLAs and classes commonly associated with autoimmune disorders, however, in exemplary cases, the HLA or portions of the HLA are patient-specific and are derived from high resolution sequences or serological equivalents of patients suffering from the disorder.

[0056] Table 2. HLA types and classes commonly associated with autoimmune diseases [Table 2] TIFF2025019209000012.tif199157TIFF2025019209000013.tif196157TIFF2025019209000014.tif197157TIFF2025019209000015.tif164157

[0057] In some embodiments, the antigenic peptides used in the present invention are HLA-DR*1501, e.g., the GAD peptide TYEIAPVFVLLFYVTLKKMR (SEQ ID NO: 34) (involved in type I diabetes), the MBP peptides listed above, the following MPP peptides (involved in MS) LLECCARCLVGAPFASLVATGLCFFGVALFC (SEQ ID NO: 35), LVGAPFASLVATGLCFFGVA (SEQ ID NO: 36), FGVALFCGCEVEALTGTEKLIETYFSKNYQD (SEQ ID NO: 37), LFCGCG HEALTGTEKLIETY (SEQ ID NO: 38), TGTEKLIETYFSKNYQDYEY (SEQ ID NO: 39), TGTEKLIETYFSKNYQDYEYL (SEQ ID NO: 40), YFSKNYQDYEYLINVIHAFQYVIYGTASFFFL (SEQ ID NO: 41), GTASFFFLYGALLLAYGFYTTGAVRQIFGDYK (SEQ ID NO: 42), LYGALLLAEGFYTTGAVRQI (SEQ ID NO: 43), FYYTTGAVRQIFGDYKTTICG (SEQ ID NO: 44), AVRQIFGDYKTTICGKGL SATV (SEQ ID NO: 45), RQIFGDYKTTCGKGLSATVTGGQKGRGSRGQ (SEQ ID NO: 46), KGLSATVTGGQKGRGYRGQH (SEQ ID NO: 47), QKGRGSRGQHQAHSLERVCH (SEQ ID NO: 48), KGRGSRGQHQAHSLERVCHCLGCWLGHPDKFV (SEQ ID NO: 49), LGHPDKFVGITYALTVVWLLVFACSAVPVYIY (SEQ ID NO: 50), SAVPVYIYFNTWTTCQSIAAPCKTSASIGTLC (SEQ ID NO: 51), AVPVYIY It is associated with FNTWTTCQSIAFP (SEQ ID NO:52), WTTCQSIAFPSKTSASIGSL (SEQ ID NO:53), SASIGTLCADARMYGVLPWNAFFGKVCGSNLL (SEQ ID NO:54), KVCGSNLLSICKTAEFQMTFHLFIAAFVGAAA (SEQ ID NO:55), AAFVGAAATLVSLLTFMIAATYNFAVLKLMGR (SEQ ID NO:56), MIAATYNFAVLKLMGRGTKF (SEQ ID NO:57), and MAATYNFAVLKLMGRFTKF (SEQ ID NO:58).

[0058] In some embodiments, the antigen peptide or polypeptide is patient-specific and designed for the patient's MHC. For example, a doctor can diagnose a patient with an autoimmune disorder and determine the severity of the disease. The patient's class I (HLA-A, B, and C) and II (HLA-DR, DQ, DP) regions can be typed, which can now be done at high resolution using DNA sequencing and comparison with a reference database (https: / / www.ebi.ac.uk / ipd / imgt / hla / ). The class I and II MHC of patients with the strongest evidence of autoimmune involvement can be identified for the disorder. Those known to be associated with a particular autoimmune disorder can be used as a reference. See, for example, Table 1 and Table 2. The strongest evidence-based antigens are identified for the disorder (iedb.org / ) and Table 1. A set of personalized peptides (cbs.dtu.dk / services / NetMHC / or cbs.dtu.dk / services / NetMHCII / ) and protein targets (for class II) expected to bind to the patient's autoimmune-associated MHC can be identified.

[0059] To allow targeting of pathogenic immune cells, individualized MHC-CAR lentivirus or mRNA can be prepared for the patient. The individualized lentivirus is used to prepare autologous or allogeneic T cells (CTL and / or Treg) that can be combined with receptor or cell modification to allow combination therapy with additional therapy, desired interaction with pathogenic cells, route control to desired location (to interact with pathogenic inflammatory or inflammation-producing cells), or secretion of cytokines (to reduce inflammation).

[0060] (c) Costimulatory Signaling Domains Many immune cells require costimulation in addition to stimulating antigen-specific signals to activate cell effector functions, in addition to promoting cell proliferation, differentiation and survival. The MHC-CARs described herein may include one or more costimulatory signaling domains. The term "costimulatory signaling domain" as used herein refers to at least a portion of a protein that mediates intracellular signaling to induce an immune response, such as an effector function. The costimulatory signaling domain of the MHC-CARs described herein may be a cytoplasmic signaling domain from a costimulatory protein that transmits a signal and modulates a response mediated by an immune cell, such as a T cell, a NK cell, a macrophage, a neutrophil, or an eosinophil.

[0061] Activation of a costimulatory signaling domain in a host cell (e.g., an immune cell) may induce the cell to increase or decrease cytokine production and secretion, phagocytic properties, proliferation, differentiation, survival, and / or cytotoxicity. The costimulatory signaling domain of any costimulatory molecule may be suitable for use in the MHC-CARs described herein. Examples of costimulatory signaling domains for use in chimeric receptors can be the cytoplasmic signaling domains of costimulatory proteins, including, but not limited to, members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, CD28, CTLA-4, ICOS / CD278, or PD-1); members of the TNF superfamily (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, CD40 / TNFRSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, OX40 / TNFRSF4, OX40 ligand / TNFSF4, or TNF-alpha); and other molecules such as FRB, which allow costimulation to be induced only in the presence of a particular drug molecule, and FKBF (but now in the context of a unique heterodimeric MHC-CAR). Wu et al., Science, 350(6258):aab4077, 2015. In some embodiments, any cytoplasmic signaling domain of a costimulatory protein may be used in receptors that target inactive bystander B cells (e.g., with CD19 or CD20-CARs) or plasma cells (e.g., with CS1-CARs and / or CS1 knockouts).

[0062] In some cases, MHC-CARs may include combinations (e.g., two or three) of costimulatory domains, which may be from the same or different costimulatory receptors. Examples include CD28+4-1BB, CD28+FRB, CD28+FKBF, or 4-1BB+FRB. See also FIG. 9. In some examples, MHC-CARs include a costimulatory domain from CD28, a costimulatory domain from 4-1BB, or both. In some embodiments, the costimulatory domain is preceded by a short linker. For example, for class II MHC-CARs, the short linker may be TS (i.e., MHC internal linker), and for class I MHC-CARs, the short linker may be PG.

[0063] In some cases, the MHC-CAR construct described herein may not include a costimulatory domain. Alternatively, it may contain non-traditional elements, such as TALEN nucleases, activators, or repressors, which may now be implemented in clinically applicable lentiviral formats using recoded or non-repeat-containing TAL domains, linked to the single-chain MHC-CAR through a membrane domain derived from Notch.

[0064] Exemplary costimulatory domains for use in the MHC-CARs described herein include, but are not limited to, the following: 41BB intracellular domain: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 59) 41BBe intracellular domain: pg KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELaha (SEQ ID NO: 60) CD28 intracellular domain: RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 61) CD28e intracellular domain: pg RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSaha (SEQ ID NO: 62) FRB: EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRI (SEQ ID NO: 63) FBP with linker (GSSS)4-EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRI-(GSSS)3 (SEQ ID NO: 64) FKRB GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 65) FKBP with linker (GSSS)4-GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE-(GSSS)3 (SEQ ID NO: 66)

[0065] (d) Cytoplasmic signaling domains Any cytoplasmic signaling domain containing an immunoreceptor tyrosine-based activation motif (ITAM) can be used to construct the chimeric receptors described herein. An "ITAM" as used herein is a conserved protein motif that is commonly present in the tail portion of signaling molecules expressed in many immune cells. The motif may include two repeats of the amino acid sequence YxxL / I separated by 6-8 amino acids, where each x is independently any amino acid that creates the conserved motif YxxL / Ix(6-8)YxxL / I. In some examples, the cytoplasmic signaling domain containing an ITAM is that of CD3ζ. In some examples, the MHC-CAR does not include a costimulatory domain and the cytoplasmic signaling domain is preceded by a short linker. For example, for class II MHC-CARs, the short linker may be TS (i.e., MHC internal linker). For example, for class I MHC-CARs, the short linker may be PG. In some cases, the linker may be AHA or absent, such as in certain cases where a costimulatory domain precedes the signaling domain.

[0066] In some embodiments, the MHC-CAR may not contain a cytoplasmic signaling domain, such as that of CD3ζ. Such CD3ζ-free MHC-CARs have an inhibitory effect on target cells or induce the death of target cells. Moisini, et al., The Journal of Immunology, 180(5), pp.3601-3611.

[0067] An exemplary cytoplasmic signaling domain from CD3ζ is provided below: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 67)

[0068] Exemplary nucleic acid sequences encoding the cytoplasmic signaling domain from CD3ζ are provided below: AGAGTAAAGTTTTCCCGAAGTGCGGACGCTCCCGCGTATCAGCAAGGTCAAAACCAGCTTTACAACGAACTGAACTTGGGACGACGCGAAGAGTACGATGTTCTTGATAAGCGGAGAGGGCGATCCCGAAATGGGGGAAAGCCTCGGAGGAAGAACCCACAAGAAGGCCT TTATAATGAACTGCAGAAGGACAAGATGGCGGAGGCGTATTCCGAAATAGGCATGAAGGGTGAACGGAGGAGAGGAAAGGGACATGACGGACTTTATCAAGGATTGTCTACCGCAACTAAAGACACCTATGACGCGTTGCACATGCAGGCTCTCCCTCCGAGA (SEQ ID NO: 410) CGGGTCAAATTTAGCAGATCCGCTGACGCACCGGCCTACCAGCAGGGCCAGAACCAACTCTACAACGAGCTGAATCTCGGCCGACGGGAAGAGTATGACGTACTCGACAAGCGGAGAGGTCGAGACCCTGAGATGGGCGGTAACCGAGACGGAAAAATCCCCAAGAGGGTCT TTATAATGAACTCCAGAAGGATAAGATGGCTGAAGCCTATTCTGAGATAGGGATGAAAGGCGAGCGGCGGAGGGGTAAGGGCCATGATGGCCTTTACCAGGGACTCTCCACGGCAACCAAAGATACTTACGACGCCCTTCACATGCAAGCCCTCCCGCCACGC (SEQ ID NO: 422)

[0069] (e) Additional Ingredients The MHC-CARs described herein may optionally further comprise one or more of the following components: a hinge domain, a transmembrane domain, a signal (leader) peptide, and a peptide linker.

[0070] In some cases, an antigenic peptide may be linked to the hinge peptide to enhance the immune targeting activity of the resulting MHC-CAR and / or to reduce the antibody response by the target cell to the MHC-TCR complex. In some cases, the MHC-CAR containing the hinge peptide may not include a cytoplasmic domain (e.g., does not include a CD3ζ domain). The MHC-CAR construct containing the hinge peptide may also include an MHC class I portion. The hinge domain may contain about 10 to 100 amino acids, e.g., 15 to 75 amino acids, 20 to 50 amino acids, or 30 to 60 amino acids. In some embodiments, the hinge domain may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids in length.

[0071] In some examples, the following peptide linkers may be used in class I MHC-CARs: MHCI linker 1: GGGSGGGGSGGGGS (SEQ ID NO: 68) MHCI linker 2: GGGGGGSGGSGGSGG (SEQ ID NO: 69) MHCI linker 3: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 70) MHCI linker 4: GGGSGGGGSGGGGS (SEQ ID NO: 68)

[0072] Exemplary peptide linkers for class II MHC-CARs can be GSGSGSGS (MHCII linker 1; SEQ ID NO:72), GGGSGGGGSGGGGS (MHC II linker II; SEQ ID NO:68), GGGSGGGGSGGGS (SEQ ID NO:400), or those described herein as MHCI linkers (i.e., MHCI linkers 1-4). Exemplary pre-peptide linkers for class II MHC-CARs can be AS or GS or 1 or 2 copies of either AS or GS.

[0073] An exemplary nucleic acid sequence encoding the peptide linker provided by SEQ ID NO:400 is provided below. GGGGGAGGCGGATCTGGCGGAGGCGGGAGTGGAGGCTCA (SEQ ID NO: 401)

[0074] The hinge peptide for use in the MHC-CAR described herein may be derived from a naturally occurring receptor. Any protein hinge domain known in the art to contain a hinge domain is suitable for use in the chimeric receptor described herein. In some embodiments, the hinge domain is a portion of the hinge domain of CD8α, for example, a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8α. Alternatively, it may be a synthetic peptide.

[0075] Exemplary hinge domains include IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO:73), and IWAPLAGICVALLLSLIITLI (SEQ ID NO:74). Additional examples are provided below: FKBP / FRB-CD8 Hinge: [ka] GS short hinge: GGGGSGGGGSGGGGS (SEQ ID NO: 68) GS long hinge: GGGGSGGGGSGGGGSGGGGGSGGGGGSGGGGGSGGGGGGSGGGGGS (SEQ ID NO: 76) H2-Kb hinge: LRWEPPSTVSNM (SEQ ID NO: 77) HLA-A2 hinge: LRWEPSSQPTIPI (SEQ ID NO: 78) HLA-A3 hinge: LRWELSSQPTIPI (SEQ ID NO: 79) DAP10 hinge: QTTPGERSSLPAFYPGTSGSCSGCGSLSLP (SEQ ID NO: 80) DAP10 hinge with linker: (GSSS)4QTTPGERSSLPAFYPGTSGSCSGCGSLSLP (SEQ ID NO:81) DAP12 hinge: LRPVQAQAQSDCSCSTVSP (SEQ ID NO: 82) DAP12 hinge with linker: (GSSS)4LRPVQAQAQSDCSCSTVSP (SEQ ID NO:83) FcIgGIIIa hinge: GLAVSTISSFFPPGYQ ​​(SEQ ID NO: 84) CD8α hinge: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 85) IgGl Hinge: EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 86) DRA*0101 hinge: EFDAPSPLPETTE (SEQ ID NO: 87) DRB1*1501 Hinge: VEWRARSESAQSK (SEQ ID NO: 88)

[0076] An exemplary nucleic acid sequence encoding the DRA*0101 hinge is provided below. GAGTTCGACGCCCCATCACCGCTTCCAGAAACGACTGAA (SEQ ID NO: 417)

[0077] An exemplary nucleic acid sequence encoding the DRB1*1501 hinge is provided below. GTTGAGTGGAGGGCGCGGTCAGAGAGCGCACAATCTAAA (SEQ ID NO: 404)

[0078] In some embodiments, the MHC-CAR construct described herein further comprises a transmembrane domain. Any transmembrane domain for use in MHC-CAR can be in any form known in the art. As used herein, "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. The transmembrane domain suitable for use in the chimeric receptor used in the present invention may be obtained from a naturally occurring protein. Alternatively, it may be a synthetic, non-naturally occurring protein segment, such as a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

[0079] Transmembrane domains are classified based on the three-dimensional structure of the transmembrane domain. For example, the transmembrane domain may form an alpha helix, a complex of more than one alpha helix, a beta barrel, or any other stable structure that can penetrate the phospholipid bilayer of a cell. Furthermore, the transmembrane domain may additionally or alternatively be classified based on the topology of the transmembrane domain, including the number of times the transmembrane domain penetrates the membrane and the orientation of the protein. For example, a single-pass transmembrane protein penetrates the cell membrane once, and a multiple-pass transmembrane protein penetrates the cell membrane at least twice (e.g., 2, 3, 4, 5, 6, 7 or more times).

[0080] Membrane proteins can be defined as type I, type II, or type III depending on their termini and the topology of the transmembrane segment(s) relative to the inside and outside of the cell. Type I membrane proteins have a single transmembrane region and are oriented such that the N-terminus of the protein is on the extracellular side of the lipid bilayer of the cell and the C-terminus of the protein is on the cytoplasmic side. Type II membrane proteins also have a single transmembrane region, but are oriented such that the C-terminus of the protein is on the extracellular side of the lipid bilayer of the cell and the N-terminus of the protein is on the cytoplasmic side. Type III membrane proteins have multiple transmembrane segments and may be further subclassified based on the number of transmembrane segments and the location of the N-terminus and C-terminus.

[0081] In some embodiments, the transmembrane domain of the MHC-CAR described herein is derived from a type I single-pass membrane protein, such as CD8α, CD8β, 4-1BB / CD137, or CD28. Transmembrane domains from multiple-pass membrane proteins may also be suitable for use in the chimeric receptors described herein. The multiple-pass membrane protein may comprise a complex (at least 2, 3, 4, 5, 6, 7 or more) alpha-helix or beta-sheet structure. Preferably, the N-terminus and C-terminus of the multiple-pass membrane protein are on opposite sides of the lipid bilayer, e.g., the N-terminus of the protein is on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is on the extracellular side. One or more helical passages from the multiple-pass membrane protein can be used to construct the chimeric receptor variants described herein.

[0082] Exemplary transmembrane domains for use in the construction of the MHC-CAR constructs described herein are provided below: CD8a transmembrane domain: IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:89) HLA-A2 transmembrane domain: VGIIAGLVLFGAVITGAVVAAVMW (SEQ ID NO: 90) HLA-A3 transmembrane domain: VGIIAGLVLLGAVITGAVVAAVMW (SEQ ID NO: 91) CD3 zeta transmembrane domain: LCYLLDGILFIYGVILTALFL (SEQ ID NO: 92) DR*1501 transmembrane domain: MLSGVGGFVLGLLFLGAGLFI (SEQ ID NO:93) DR*1501e transmembrane domain: MLSGVGGFVLGLLFLGAGLFIYFRNQ (SEQ ID NO: 94) DRA*0101 transmembrane domain: NVVCALGLTVGLVGIIIGTIFII (SEQ ID NO: 416) DRA*0101e transmembrane domain: NVVCALGLTVGLVGIIIGTIFIIKGL (SEQ ID NO: 418)

[0083] Exemplary nucleic acid sequences encoding the DR*1501e transmembrane domain are provided below: ATGCTGTCAGGAGTAGGCGGATTTGTACTCGGACTCCTCTTTTGGCGCTGGGTTGTTTATCTACTTTAGAAACCAA (SEQ ID NO: 406)

[0084] An exemplary nucleic acid sequence encoding the DRA*0101e transmembrane domain is provided below: AACGTTGTCTGCGCTCTTGGCCTGACAGTGGGCCTGGTAGGCATTATTATCGGGACCATCTTTATCATCAAAGGTTTG (SEQ ID NO: 419) Notch transmembrane domain: ILDYSFTGGAGRDIPPPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRR (SEQ ID NO: 95) Notch 2 transmembrane domain: PCVGSNPCYNQGTCEPTSENPFYRCLCPAKFNGLLCHILDYSFTGGAGRDIPPPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRR (SEQ ID NO: 96)

[0085] In some embodiments, the MHC-CAR may also include a signal peptide (also known as a signal sequence or leader peptide) at the N-terminus of the polypeptide. In general, a signal sequence is a peptide sequence that targets a polypeptide to a desired site in a cell. In some embodiments, the signal sequence targets the MHC-CAR to the secretory pathway of a cell, allowing the incorporation and anchoring of the MHC-CAR into the lipid bilayer. Signal sequences, including naturally occurring protein signal sequences or synthetic non-naturally occurring signal sequences, compatible for use in the chimeric receptors described herein will be apparent to those skilled in the art. In some embodiments, the signal sequence is from CD8α. In some embodiments, the signal sequence is from CD28 (e.g., MLRLLLALNLFPSIQVTG (SEQ ID NO:97)).

[0086] Exemplary signal peptides include, but are not limited to, beta-2-microglobulin signal peptide (e.g., MSRSVALAVLALLSLSGLEA (SEQ ID NO: 98)), HLA A3 signal peptide (e.g., MAVMAPRTLLLLLSGALALTQTWA (SEQ ID NO: 99), or), DRA*0101 signal peptide (e.g., MAISGVPVLGFFIIAVLMSAQESWA (SEQ ID NO: 100)), DRB1*1501 signal peptide (e.g., MVCLKLPGGSCMTALTVTLMVLSSPLAL (SEQ ID NO: 101)), and DRB5 signal peptide (e.g., MVCLKLPGGSYMAKLTVTLMVLSSPLALA (SEQ ID NO: 102)). Exemplary signal peptides may be followed by a flexible pre-peptide linker, such as AS, GS, ASAS, GSGS. In some embodiments, a flexible pre-peptide linker is used when the signal peptide is class II and is followed by the peptide to be introduced. Any construct encoding an MHC-CAR described herein may comprise a nucleic acid sequence encoding any of the pre-peptide linkers described above, for example, the AS may be encoded by the nucleic acid sequence GCATCT and the TS may be encoded by the nucleic acid sequence ACAAGT.

[0087] An exemplary nucleic acid sequence encoding the beta-2-microglobulin signal peptide is provided below: ATGGTATGCTTGAAGCTCCCGGGCGGGTCCTGCATGACCGCTCTCACTGTTACTCTTATGGTCCTTAGTTCACCGCTTGCCCTG (SEQ ID NO: 397) ATGGCAATATCTGGTGTTCCTGTCCTCGGGTTTTTTATCATAGCCGTACTGATGTCAGCACAGGAATCATGGGCG (SEQ ID NO: 414)

[0088] In some embodiments, the MHC-CARs described herein may include one or more peptide linkers between other components as described herein. Examples include (Gly x Ser) n linkers (where x and n can independently be integers between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or greater). In some examples, the peptide linker is (Gly4Ser) n (SEQ ID NO: 103) (n can be an integer from 3 to 20). Specific examples include (Gly4Ser)3 (SEQ ID NO: 68), (Gly4Ser)6 (SEQ ID NO: 69), (Gly4Ser)9 (SEQ ID NO: 76), (Gly4Ser) 12 (SEQ ID NO: 105), and (Gly4Ser) 15 (SEQ ID NO: 106).

[0089] (ii) MHC-CAR configuration The MHC-CAR constructs disclosed herein, comprising one or more components described herein, may be configured in any suitable format. Exemplary MHC class I and MHC class II constructs are provided in Figures 7 and 8.

[0090] An MHC-CAR construct containing an MHC class I moiety as described herein may be a single fusion polypeptide that includes an MHC class I moiety, an antigenic peptide, and a signaling domain (e.g., a costimulatory domain, a cytoplasmic signaling domain, or a combination thereof), and optionally one or more of the additional components described herein. See, for example, FIG. 8. In some examples, the MHC class I CAR construct contains a hinge domain adjacent to the antigenic peptide. The MHC class I CAR may not contain β2-microglobulin (b2m). When expressed on the cell surface, such an MHC-CAR may form a heterodimer with endogenous b2m. Alternatively, the MHC class I CAR may also include b2m, which may be fused to an alpha chain to produce a single polypeptide. In some cases, the MHC class I CAR may contain two subunits, one of which may include an alpha chain or a portion thereof (e.g., an extracellular domain), and the other of which may include a b2m or a portion thereof (e.g., an extracellular domain). In some examples, the antigenic peptide may be fused to the alpha chain. In other examples, the antigenic peptide may be fused to b2m. Optionally, the MHC class I CAR may contain a peptide linker between the two components. An example is provided in Figure 8B.

[0091] In some examples, the MHC-CAR comprises a class I molecule or a portion thereof, such as HLA A3 or HLA A2, and an antigenic peptide suitable for presentation by the class I molecule (e.g., the PLP fragment KLIETYFSK (SEQ ID NO: 107) or the TAX fragment LLFGYPVYV (SEQ ID NO: 108)). Optionally, the MHC-CAR may further comprise b2m. Alternatively, the b2m molecule may be expressed separately from the class I MHC-CAR. Examples of class I molecules and b2m sequences are provided below: HLA A2: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLT (SEQ ID NO: 109) HLA A3: GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGYYNQSEAGSHTIQIMYGCDVGSDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHEAEQLRAYLDGTCVEWLRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLT (SEQ ID NO: 110) Micro-extension of the above: LRWE (SEQ ID NO: 111) H-2K b HLA A2 with alpha 3 domain (underlined / italicized) [ka] H-2K b HLA A3 with alpha 3 domain (underlined / italicized) [ka] Micro-extension of the above: LRWE (SEQ ID NO: 111) Beta-2-microglobulin (human): IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 114) Beta-2-microglobulin (mouse): IQKTPQIQVYSRHPPENGKPNILNCYVTQFHPPHIEIQMLKNGKKIPKVEMSDMSFSKDWSFYILAHTEFTPTETDTYACRVKHASMAEPKTVYWDRDM (SEQ ID NO: 115)

[0092] Figure 7 provides a number of exemplary designs of MHC class II CAR constructs. Typically, MHC class II CAR constructs contain two subunits, one containing an alpha chain or a portion thereof (e.g., extracellular domain) and the other containing a beta chain or a portion thereof (e.g., extracellular domain). The antigenic peptide can be fused to either the alpha chain or the beta chain. In some cases, MHC class II CARs can also be in the format of a single fusion polypeptide, in which the alpha chain and the beta chain are fused to form a single polypeptide. The alpha chain and the beta chain of the MHC class II CAR can be derived from the same MHC class II molecule. Alternatively, they can be from different MHC class II molecules. For example, an MHC class II CAR can contain an alpha chain from HLA DRA*1010 and a beta chain from HLA DRB1*1501, which can be fused to an antigenic peptide such as MBP peptide.

[0093] In some examples, the MHC-CAR comprises a class II molecule or a portion thereof, such as DRB1*1501 or DRA*0101, and an antigenic peptide suitable for presentation by the class II molecule (e.g., the MBP fragment DENPVVHFFKNIVTPRTPP (SEQ ID NO: 15)). Examples of class II molecule sequences are provided below; DRB1*1501: GDTRPRFLWQPKRECHFFNGTERVRFLDRYFYNQEESVRFDSDVGEFRAVTELGRPDAEYWNSQKDILEQARAAVDTYCRHNYGVVESFTVQRRVQPKVTVYPSKTQPLQHHNLLVCSVSGFYPGSIEVRWFLNGQEEKAGMVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSVTSPLT (SEQ ID NO: 117) DRA*0101 IKEEHVIIQAEFYLNPDQSGEFMFDFDGDEIFHVDMAKKETVWRLEEFGRFASFEAQGALANIAVDKANLEIMTKRSNYTPITNVPPEVTVLTNSPVELREPNVLICFIDKFTPPVVNVTWLRNGKPVTTGVSETVFLPREDHLFRKFHYLPFLPSTEDVYDCRVEHWGLDEPLLKHW (SEQ ID NO: 118) DRB1*1501 human / IA-Dbeta mouse (mutated residues are bolded and underlined) [ka] DRA*0101 human / IA-D alpha mouse (mutated residues are bolded and underlined) [ka] DR-2 ベータ Mini (mutated residues are bolded and underlined) [ka] DR-2 アルファ mini [ka] An exemplary nucleic acid sequence encoding DRB1*1501 is provided below: (SEQ ID NO:402) An exemplary nucleic acid sequence encoding DRA*0101 is provided below: (SEQ ID NO:415)

[0094] Any of the MHC class I and MHC class II constructs described herein can be further fused to one or more signaling domains and, optionally, one or more additional components. In some cases, the MHC-CAR constructs described herein do not include a signaling domain.

[0095] Preferably, the MHC-CAR as described herein contains a matched MHC portion and an antigen peptide, such as an MHC molecule that presents an antigen peptide or a homologous analog in a natural state.In some cases, the MHC-CAR as described herein may contain an alpha or beta chain from HLA DRB1*1501 and an antigen peptide associated with this HLA allele, such as the MBP peptide and others described herein.The association between antigen peptides involved in autoimmune disease and specific HLA alleles is well known in the art or can be identified through routine practice, such as library screening.

[0096] One exemplary MHC-CAR may have the following formula (+ / - means that the particular component is optional):

[0097] Single chain (MHC class I or II + peptide) (+ / - hinge) + single chain CD28 / 4-1BB (+ / - dileucine motif) (+ / - CD3 zeta)). (Additional short peptide linkers can be added between the components as previously described.)

[0098] Other exemplary MHC-CAR designs (single and multi-chain) are shown in Figure 10. In the case of multi-chain constructs, one or more short hinges may be used to enhance successful expression of the MHC-CAR. In addition, it may be desirable to replace parts of the structure with conserved domains from mouse domains to prevent cross-reactivity. Note that in some cases, the internal domain may only be attached to one of the chains.

[0099] The amino acid sequences of the MHC-CAR binding (MBP presenting) TCRs are provided below: TCR Alpha MBP: METLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATAAVGGFKTIFGAGTRLFVKANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 123) TCR Beta MBP MLLLLLLLGLAGSGLGAVVSQHPSWVISKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSARDLTSGANNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYSLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 124) TCR Alpha Class I mamllgasvl ilwlqpdwvn sqqkndd QQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNADGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAAMEGAQKLVFGQGTRLTINPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS Cdvklve ksfetdtnln fqnlsvigfr illlkvagfn llmtlrlwss (SEQ ID NO: 125) TCR Beta Class I: msigllccaa lsllwagpv NAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYPGGGFYEQYFGPGTRLTVTEDLKNVFP PEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD cgftse syqqgvlsat ilyeillgka tlyavlvsal vlmamvkrkd srg (SEQ ID NO: 126)

[0100] The amino acid sequences of exemplary CD19-targeting CAR constructs are provided below (note that these designs contain a 4-1BB domain that may be replaced by a cd28 domain): 4G7-CAR version 1: MALPVTALLLPLALLLHAARPEVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYC ARGTYYYGSRVFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTAGTKLELKR SDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 127) 4G7-CAR version 2: METDTLLLWV LLLWVPGSTG EVQLQQSGPE LIKPGASVKM SCKASGYTFT SYVMHWVKQK PGQGLEWIGY INPYNDGTKY NEKFKGKATL TSDKSSSTAY MELSSLTSED SAVYYCARGT YYYGSRVFDY WGQGTTLTVS SGGGGSGGGG SGGGGSDIVM TQAAPSIPVT PGESVSISCR SSKSLLNSNG NTYLYWFLQR PGQSPQLLIY RMSNLASGVP DRFSGSGSGT AFTLRISRVE AEDVGVYYCM QHLEYPFTFG AGTKLELKRS DPTTTPAPRP PTPAPTIASQ PLSLRPEACR PAAGGAVHTR GLDFACDIY I WAPLAGTCGV LLLSLVIT LY CKRGRKKLLY IFKQPFMRPV QTTQEEDGCS CRFPEEEEGG CE LRVKFSRS ADAPAYQQGQ NQLYNELNLG RREEYDVLDK RRGRDPEMGG KPRRKNPQEG LYNELQKDKM AEAYSEIGMK GERRRGKGHD GLYQGLSTAT KDTYDALHMQ ALPPR (SEQ ID NO: 128)

[0101] The nucleic acid sequences of exemplary CD19-targeting CAR constructs are provided below. 4G7-CAR version 2

[0102] MHC-CAR1 Part B (MHC-DRB CAR) - MHC-CAR1 containing HLA DRB1*1501 (signal peptide), MBP peptide, HLA DRB1*1501 (outer, hinge, transmembrane) CD3ζ (cytoplasmic signaling domain) is provided below: MVCLKLPGGSCMTALTVTLMVLSSPLALASDENPVVHFFKNIVTPRTPPGGGGSGGGGSGGSGDTRPRFLWQPKRECHFFNGTERVRFLDRYFYNQEESVRFDSDVGEFRAVTELGRPDAEYWNSQKDILEQARAAVDTYCRHNYGVVESFTVQRRVQPKVTVYPSKTQPLQHHNLLVCSVSGFYPGSIEVRWFLNGQEEKAGMVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSVTSPLTVEWRARSESAQSKMLSGVGGFVLGLLFLGAGLFIYFRNQTSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (Sequence Number: 412)

[0103] An exemplary nucleic acid sequence encoding MHC_CAR1 containing MHC-CAR1 part B (MHC-DRB CAR)-HLA DRB1*1501 (signal peptide), MBP peptide, HLA DRB1*1501 (outer, hinge, transmembrane) CD3ζ (cytoplasmic signaling domain) is provided below:

[0104] MHC-CAR1 Part A (MHC-DRA CAR) MHC-CAR containing HLA-DRA*1010 (signal peptide, outer, hinge, transmembrane), CD3ζ (cytoplasmic signaling domain) is provided below: MAISGVPVLGFFIIAVLMSAQESWAIKEEHVIIQAEFYLNPDQSGEFMFDFDGDEIFHVDMAKKETVWRLEEFGRFASFEAQGALANIAVDKANLEIMTKRSNYTPITNVPPEVTVLTNSPVELREPNVLICFIDKFTPPVVNVTWLRNGKPVTTGVSETVFLPREDHLFRKFHYLPFLPSTEDVYDCRVEHWGLDEPLLKHWEFDAPSPLPETTENVVCALGLTVGLVGIIIGTIFIIKGLTSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 423)

[0105] Exemplary nucleic acid sequences encoding MHC-CAR1 Part A (MHC-DRA CAR) HLA-DRA*1010 (signal peptide, outer, hinge, transmembrane), CD3ζ (cytoplasmic signaling domain) are provided below:

[0106] Construct 1 (CD19 CAR and CCR6 region) is provided below: (SEQ ID NO:425)

[0107] Construct 1 (CD19 CAR, CCR6, GFP region) is provided to:

[0108] An exemplary nucleic acid sequence encoding Construct 1 (CD19 CAR, CCR6, GFP region) is provided below:

[0109] Construct 2 MHC CAR regions (MHC-CAR1 partial B, MHC-CAR1 partial A regions) are provided below: (SEQ ID NO:431)

[0110] Exemplary nucleic acid sequences encoding Construct 2 MHC CAR regions (MHC-CAR1 part B, MHC-CAR1 part A) are provided below:

[0111] Construct 2 kill switch and MHC CAR regions (RQR8, MHC-CAR1 partial B, MHC-CAR1 partial A regions) are provided below: (SEQ ID NO:211)

[0112] Exemplary nucleic acid sequences encoding Construct 2 kill switch and MHC CAR regions (RQR8, MHC-CAR1 partial B, MHC-CAR1 partial A regions) are provided below:

[0113] Construct 2 (RQR8, MHC-CAR1 part B, MHC-CAR1 part A, GFP region) is provided below.

[0114] An exemplary nucleic acid sequence encoding Construct 2 (RQR8, MHC-CAR1 part B, MHC-CAR1 part A, GFP region) is provided below:

[0115] The amino acid sequences of exemplary CS-1-targeting CAR constructs are provided below (note that these designs contain 4-1BB, which may be replaced by a CD28 domain): Anti-CS1-CAR-v1 (Luc63-V1 CAR): MALPVTALLLPLALLLHAARPEVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYTPSLKDKFIISRDNAKNTLYLQMSKVRSE DTALYYCARPDGNYWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSITCKASQDVGIAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSG SGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTKLEIKGLAVSTISSFFPPGYQKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 129) Anti-CS1-CAR-v2 (Luc63-V2 CAR): MALPVTALLLPLALLLHAARPEVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCARPDGNYWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSITCKASQDVGIAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 130) Anti-CS1-CAR-v3 (Luc63-V3 CAR): MALPVTALLLPLALLLHAARPEVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCARPDGNYWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSITCKASQDVGIAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTKLEIKEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 131) Anti-CS1-CAR-v4 (Luc90-V1 CAR): MALPVTALLLPLALLLHAARPQVQLQQPGAELVRPGASVKLSCKASGYSFTTYWMNWVKQRPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCARSTMIATRAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSQKSMSTSVGDRVSITCKASQDVITGVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISNVQAEDLAVYYCQQHYSTPLTFGAGTKLELKGLAVSTISSFFPPGYQKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 132) Anti-CS1-CAR-v5 (Luc90-V2 CAR): MALPVTALLLPLALLLHAARPQVQLQQPGAELVRPGASVKLSCKASGYSFTTYWMNWVKQRPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCARSTMIATRAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSQKSMSTSVGDRVSITCKASQDVITGVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISNVQAEDLAVYYCQQHYSTPLTFGAGTKLELKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 133) Anti-CS1-CAR-v6 (Luc90-V3 CAR): MALPVTALLLPLALLLHAARPQVQLQQPGAELVRPGASVKLSCKASGYSFTTYWMNWVKQRPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCARSTMIATRAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSQKSMSTSVGDRVSITCKASQDVITGVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISNVQAEDLAVYYCQQHYSTPLTFGAGTKLELKEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 134) Anti-CA1-CAR-v7 (Luc34-V1 CAR): MALPVTALLLPLALLLHAARPQVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYCARGKVYYGSNPFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGS GSGKDYTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLEIKGLAVSTISSFFPPGYQKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(sequence number: 135) Anti-CS1-CAR-v8(Luc34-V2 CAR): MALPVTALLLPLALLLHAARPQVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCARGKVYYGSNPFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 136) Anti-CS1-CAR-v9 (Luc34-V3 CAR): MALPVTALLLPLALLLHAARPQVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCARGKVYYGSNPFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLEIKEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 137) Anti-CS1-CAR-v10 (LucX1-V1 CAR) MALPVTALLLPLALLLHAARPQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGQGLEWIGRIYPGDGDTKYNGKFKGKATLTADKSSSTAYMQLSSLTSVDSAVYFCARSTMIATGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSETTVTQSPASLSMAIGEKVTIRCITSTDIDDDMNWYQQKPGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNLPLTFGGGTKLEIKGLAVSTISSFFPPGYQKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 138) Anti-CS1-CAR-v11 (LucX1-V2 CAR) MALPVTALLLPLALLLHAARPQVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCARGKVYYGSNPFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 139) Anti-CS1-CAR-v12 (LucX1-V3 CAR): MALPVTALLLPLALLLHAARPQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGQGLEWIGRIYPGDGDTKYNGKFKGKATLTADKSSSTAYMQLSSLTSVDSAVYFCARSTMIATGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSETTVTQSPASLSMAIGEKVTIRCITSTDIDDDMNWYQQKPGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNLPLTFGGGTKLEIKEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 140) Anti-CS1-CAR-v13 (LucX2-V1 CAR): MALPVTALLLPLALLLHAARPQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGQGLEWIGRIYPGDGDTKYNGKFKGKATLTADKSSSTAYMQLSSLTSVDSAVYFCARSTMIATGAMDYWGQGTSVTVSSGGGGSGGGGSGGGSDIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSG SGTDFTFTISSVQAEDLAVYYCQQHYSTPPYTFGGGTKLEIKGLAVSTISSFFPPGYQKRGRKKLLYIFKQPFMRPVQTTQEEDGCCRFPEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(sequence number: 141) Anti-CS1-CAR-v14(LucX2-V2 CAR): MALPVTALLLPLALLLHAARPQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGQGLEWIGRIYPGDGDTKYNGKFKGKATLTADKSSSTAYMQLSSLTSVDSAVYFCARSTMIATGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPPYTFGGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFADIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 142) Anti-CS1-CAR-v15 (LucX2-V3 CAR): MALPVTALLLPLALLLHAARPQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGQGLEWIGRIYPGDGDTKYNGKFKGKATLTADKSSSTAYMQLSSLTSVDSAVYFCARSTMIATGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPPYTFGGGTKLEIKEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 143)

[0116] (iii) Preparation of MHC-CAR Any MHC-CAR construct described herein can be prepared by conventional methods, such as recombinant techniques. The method of preparing a chimeric receptor in the present invention involves the generation of a nucleic acid or set of nucleic acids that encodes or collectively encodes an MHC-CAR construct (comprising a single polypeptide or two subunits). In some embodiments, the nucleic acid also encodes a self-cleaving peptide (e.g., P2A, T2A, or E2A peptide) between the coding sequences for the two subunits of the MHC-CAR, or between the coding sequence for the MHC-CAR and the coding sequence for another gene that is co-expressed with the MHC-CAR in the host cell (see discussion below).

[0117] The sequence of each component of the MHC-CAR may be obtained via routine techniques, for example, PCR amplification from any one of a variety of sources known in the art. In some embodiments, the sequence of one or more components of the MHC-CAR is obtained from a human cell. Alternatively, the sequence of one or more components of the MHC-CAR may be synthesized. The sequence of each component (e.g., domain) may be linked directly or indirectly (e.g., using a nucleic acid sequence encoding a peptide linker) using methods such as PCR amplification or ligation to form a nucleic acid sequence encoding the MHC-CAR. Alternatively, the nucleic acid encoding the MHC-CAR may be synthesized. In some embodiments, the nucleic acid is DNA. In other embodiments, the nucleic acid is RNA.

[0118] Any MHC-CAR protein, its encoding nucleic acid, and expression vector carrying the nucleic acid are all within the scope of this disclosure and can be mixed with a pharma- ceutically acceptable carrier to form a pharmaceutical composition, which is also within the scope of this disclosure. By "acceptable" is meant that the carrier is compatible with the active components of the composition (e.g., nucleic acid, vector, cell, or therapeutic antibody) and does not negatively affect the subject to which the composition(s) are administered. Any pharmaceutical composition used in the methods of the present invention may contain a pharma- ceutically acceptable carrier, excipient, or stabilizer in lyophilized or aqueous form.

[0119] Pharmaceutically acceptable carriers, including buffers, are well known in the art and may include phosphate, citric acid, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and / or non-ionic surfactants. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover.

[0120] II. Engineered immune cells expressing MHC-CAR (i) MHC-CAR expressing immune cells Immune cells expressing MHC-CARs described herein provide a specific population of cells that can recognize pathogenic cells (e.g., autoreactive T cells) involved in autoimmune disease via MHC / peptide-TCR engagement. Interaction between the MHC-peptide portion of the MHC-CAR and the cognate TCR on the pathogenic cell activates the MHC-CAR-expressing immune cell via the signaling domain(s) of the MHC-CAR (optionally by localizing cell membrane signaling molecules of the immune cell), leading to proliferation and / or effector function of the MHC-CAR-expressing immune cell, which then eliminates the pathogenic cell. The immune cell can be a T cell, a NK cell, a macrophage, a neutrophil, an eosinophil, or any combination thereof. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a NK cell. Specific examples are provided in the Examples below.

[0121] The population of immune cells can be obtained from any source, such as peripheral blood mononuclear cells (PBMC), bone marrow, tissue, such as spleen, lymph nodes, thymus, tumor tissue, or established cell lines. A suitable source for obtaining the desired immune cell type is clear to the skilled artisan. In some embodiments, the population of immune cells is derived from PBMC. The desired immune cell type (e.g., T cells, NK cells, macrophages, neutrophils, eosinophils, or any combination thereof) can be expanded in the population of cells obtained by incubating the cells with stimulatory molecules, for example, anti-CD3 and anti-CD28 antibodies can be used for T cell expansion.

[0122] To construct immune cells expressing any of the MHC-CAR constructs described herein, expression vectors for stable or transient expression of the chimeric receptor constructs may be constructed via conventional methods as described herein and introduced into immune host cells. For example, the nucleic acid encoding the MHC-CAR may be cloned into a suitable expression vector, such as a viral vector (e.g., a lentiviral vector) operably linked to a suitable promoter. The nucleic acid and the vector may be contacted with a restriction enzyme under suitable conditions to create complementary ends on each molecule that can pair with each other and can be linked using a ligase. Alternatively, a synthetic nucleic acid linker may be ligated to the end of the nucleic acid encoding the chimeric receptor. The synthetic linker may contain a nucleic acid sequence that corresponds to a specific restriction site in the vector. The choice of expression vector / plasmid / viral vector depends on the type of host cell for expression of the chimeric receptor, but should be suitable for integration and replication in eukaryotic cells.

[0123] Various promoters can be used for the expression of the MHC-CAR constructs described herein, including but not limited to cytomegalovirus (CMV) intermediate early promoter, viral LTR, such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Simian Virus 40 (SV40) early promoter, herpes simplex tk virus promoter. Additional promoters for the expression of chimeric receptors include any promoter that is constitutively active in immune cells. Alternatively, any regulatable promoter may be used so that expression can be modulated in immune cells.

[0124] Additionally, the vector may contain, for example, some or all of the following: a selectable marker gene, e.g., a neomycin gene for selection of stable or transient transfectants in a host cell; an enhancer / promoter sequence from the immediate early gene of human CMV for high levels of transcription; a transcription termination and RNA processing signal from SV40 for mRNA stability; an SV40 polyoma origin of replication and ColE1 for proper episomal replication; an internal ribosome binding site (IRES), a universal multiple cloning site; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; a "suicide switch" or "suicide gene" that, when induced, causes death of cells harboring the vector (e.g., an inducible caspase such as HSV thymidine kinase, iCasp9), as well as a reporter gene for assessing expression of MHC-CAR.

[0125] In some embodiments, the marker / selection / suicide molecule for use in the present disclosure may be used for killing with rituximab and / or for selection with QBEND. Philip et al., Blood 124(8):1277-87; 2014). One example is RQR8, which contains the rituximab mimotope and the QBEND-10 epitope. Exemplary sequences are provided below: MGTSLLCWMALCLLGADHADACPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSPAPPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV (SEQ ID NO: 144) MGTSLLCWMALCLLGADHADACPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV (SEQ ID NO: 394)

[0126] An exemplary nucleic acid sequence encoding RQR8 is provided below. ATGGGTACTTCACTGTTGTGCTGGATGGCACTTTGTCTTTTGGGTGCCGATCATGCTGATGCATGTCCGTACTCCAATCCTAGCCTGTGCTCCGGGGGGGGAGGGAGTGAACTCCCTACACAGGGAACCTTCTCTAATGTCTCCACCAACGTCTCCCCTGCAAAACCGACCACAACAGCTTGCCCCTATAGTAACCCTTCCCTCTGTAGTGGAGGGGGGGGTTCACCTGCTCCACGCCCT CCTACCCCCGCGCCAACGATCGCGTCACAACCGCTCAGTCTTAGGCCGGAAGCCTGTAGGCCAGCGCTGGCGGTGCGGTTCATACGCGGGGATTGGATTTTGCCTGCGACATTTACATT TGGGCTCCGCTGGCCGGTACTTGTGGGGTATTGCTGTTGTCTCTTGTTATTACGCTTTATTGCAATCACAGGAACAGGCGACGAGTATGCAAATGCCCGCGGCCCGTCGTG (SEQ ID NO: 395)

[0127] In another example, the following exemplary RQR sequence tag can be attached to an MHC-CAR construct as disclosed herein: [ka] The bold fragment is the rituximab minotope and the underlined / italicized fragment is the QBEND-10 epitope.

[0128] Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.Any vector containing the nucleic acid sequence encoding the MHC-CAR construct described herein is also within the scope of the present disclosure.Such vectors may be delivered to host immune cells by suitable methods.Methods for delivering vectors to immune cells are well known in the art and may include DNA electroporation, RNA electroporation, transfection reagents such as liposomes, or viral transduction.In some embodiments, the vector for expression of MHC-CAR is delivered to host cells by viral transduction. Exemplary viral methods for delivery include recombinant retroviruses (see, e.g., PCT International Publication Nos. WO 90 / 07936, WO 94 / 03622, WO 93 / 25698, WO 93 / 25234, WO 93 / 11230, WO 93 / 10218, WO 91 / 02805, U.S. Patent Nos. 5,219,740 and 4,777,127, GB Patent Nos. 5,219,740 and 5,219,740, and ... 00,651 and EP Patent No. 0345242), alphavirus-based vectors, and adeno-associated virus (AAV) vectors (see, for example, PCT International Publication Nos. WO94 / 12649, WO93 / 03769, WO93 / 19191, WO94 / 28938, WO95 / 11984 and WO95 / 00655). In some embodiments, the vector for expression of the chimeric receptor is a retrovirus. In some embodiments, the vector for expression of the chimeric receptor is a lentivirus.

[0129] In instances where the vector encoding the chimeric receptor is introduced into the host cell using a viral vector, viral particles capable of infecting immune cells and carrying the vector may be produced by any method known in the art, for example, as can be found in PCT Application Nos. WO1991 / 002805A2, WO1998 / 009271A1, and U.S. Patent No. 6,194,191. Viral particles may be harvested from cell culture supernatants and isolated and / or purified prior to contacting the viral particles with immune cells.

[0130] After introduction of any MHC-CAR-encoding vector provided herein into the host cell, the cell is cultured under conditions that allow expression of the chimeric receptor. In examples where the nucleic acid encoding the MHC-CAR is regulated by a regulatable promoter, the host cell is cultured under conditions where the regulatable promoter is activated. In some embodiments, the promoter is an inducible promoter, and the immune cells are cultured in the presence of an inducer molecule or under conditions where the inducer molecule is produced. The determination of whether the MHC-CAR is expressed is clear to the skilled artisan and may be assessed by any known method, for example, detection of chimeric receptor-encoding mRNA by quantitative reverse transcriptase PCR (qRT-PCR) or detection of chimeric receptor protein by methods including Western blotting, fluorescence microscopy, and flow cytometry. See also the examples below. Alternatively, expression of the MHC-CAR may occur in vivo after the immune cells are administered to a subject.

[0131] Alternatively, expression of the MHC-CAR construct in any immune cell disclosed herein can be achieved by introducing an RNA molecule encoding the MHC-CAR construct. Such RNA molecules can be prepared by in vitro transcription or chemical synthesis. The RNA molecules can then be introduced into suitable host cells, such as immune cells (e.g., T cells, NK cells, macrophages, neutrophils, eosinophils, or any combination thereof), for example, by electroporation. For example, RNA molecules can be synthesized and introduced into host immune cells according to the methods described in Rabinovich et al., Human Gene Therapy, 17:1027-1035 and WO WO2013 / 040557.

[0132] Methods of preparing host immune cells expressing any of the MHC-CARs described herein may include expanding the host immune cells ex vivo. Expanding the host immune cells may involve any method that results in an increase in the number of cells expressing the MHC-CAR, such as allowing the host cells to expand or stimulating the host cells to expand. Methods of stimulating the expansion of host cells depend on the type of host cells used for expression of the chimeric receptor and will be apparent to one of skill in the art. In some embodiments, the host immune cells expressing any of the MHC-CARs described herein may be expanded ex vivo prior to administration to a subject.

[0133] (ii) Additional genetic modifications One or more additional genetic modifications may be introduced into the host immune cells before, simultaneously with, or after transfection of the MHC-CAR construction. For example, one or more markers and / or suicide genes may be introduced into the host immune cells. Examples include green fluorescent protein (GFP), enhanced blue fluorescent protein (eBFP), and RQR genes such as RQR8 (a small marker / suicide gene for T cells that combines target epitopes from CD34 and CD20. Philip et al., Blood 124(8):1277-87; 2014). Such markers / suicide genes may be constructed in one expression cassette with the MHC-CAR components.

[0134] An example of the amino acid sequence of GFP is provided below: VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO: 427)

[0135] An example of a nucleic acid sequence encoding GFP is provided below: (query number:428)

[0136] In some cases, endogenous TCR (alpha chain, beta chain, or both) can be destroyed so that host immune cells do not express endogenous TCR. Deficiency of endogenous TCR can avoid undesired T cell activation. Alternatively or additionally, certain cell surface receptors can be knocked out. Such surface receptors can be target receptors for disease treatment, for example, CD52, which is a target for MS treatment. Knocking out such target receptors from MHC-CAR immune cells allows the combination of MHC-CAR immune cells with therapeutic agents specific for the target receptor (e.g., anti-CD52 antibodies such as alemtuzumab).

[0137] In some embodiments, host immune cells may be modified with synthetic surface proteins to enhance retention in specific organs or tissues, such as lymph nodes, tertiary lymphoid organs, or sites of inflammation. Doing so allows the modified immune cells to access target pathogenic cells while minimizing lethal off-target effects resulting from the passage of the blood-brain barrier or the free migration of immune cells through peripheral blood. Cells early in the T cell differentiation pathway (e.g., naive, stem cell memory, and central memory T cells) freely migrate to lymph nodes. As differentiation progresses, most effector T cells leave the lymph nodes. Pathological immune cells may also migrate and accumulate at sites of inflammation. Treatment with activated CAR-T cells has numerous undesirable effects when they react with undesirable targets. Interaction with cardiac tissue can be lethal to cardiac proteins, and passage through the brain can lead to lethal cerebral edema. Recently, progress has been made in treating the brain cancer glioblastoma using lower doses of CAR T therapy than in systemic treatment for cancer using CD19 CAR T therapy. Brown et al., New England Journal of Medicine, 375(26):2561-2569, 2016. Resolution of inflammation has the potential to transform a pathological environment into a protective one. Gagliani et al., Nature, 523(7559):221-225, 2015.

[0138] Introduction of one or more lymph node retention proteins into immune cells can enhance the retention of immune cells in lymph nodes, and while immune cells still have access to target pathogenic cells, the above-mentioned undesirable effects can be significantly reduced. Naive lymphocytes enter lymph nodes via high endothelial venules (HEVs). Thus, expression or overexpression of proteins involved in HEV anchoring and / or entry can promote immune cells to enter lymph nodes. Exemplary lymph node retention proteins include, but are not limited to, CCR7 (chemokine receptor), MECA79 (peripheral lymph node addressin), vascular adhesion protein-1 (VAP-1) and CD62 (selectin, a family of cell adhesion molecules). Azzi et al., Blood 124(4):476-477, 2016;Streeter et al., J. Cell. Biol. 107:1853-186; 1988;Michie et al., Amer, J. Path. 143:1688-1698; 1993;Berg et al., J. Cell. Biol. 114:343-349; 1991;Berg et al., Nature 366:695-698; 1993;and Hemmerich et al., J. Exp. Med. 180:2219-2226; 1994. Alternatively, genes encoding proteins involved in lymphocyte egress from the thymus and lymphoid organs (e.g., sphingosine-1-phosphate receptor-1 or S1P) can be knocked out of immune cells.

[0139] Chemokine and adhesion receptors that facilitate trafficking to sites of inflammation may also bring MHC-CAR immune cells into contact with pathogenic cells that propagate immune disease. [Barreiro et al., Cardiovascular research, 86(2):174-182, 2010] See Tables 3 and 4. Receptors involved in the localization of immune cells that propagate inflammation include receptors (i.e., CXCR5, CCR7, CCR6) that are localized in tertiary lymphoid organs (where CXCL13, CCL19, CCL20, CCL21 are expressed).

[0140] One or more genes encoding proteins involved in targeting of other organs / tissues, e.g., the brain / CNS, bone marrow, pancreas, intestine, liver, lung, spleen, and / or thymus, may be introduced into or knocked out of the immune cell.

[0141] By virus-induced or transient RNA-mediated expression in therapeutic cells (optionally combined with knockout of endogenous genes), the genes (genes in Tables 3 and 4) may be used to route either Treg or CTL cells to the desired location or to treat / eliminate the desired cells. Barreiro, et al. Cardiovascular research, 86(2):174-182, 2010. If mRNA transfection is utilized, it may allow expression of chemokines or adhesion receptors for one week. Wang and Riviere. Molecular Therapy-Oncolytics, 3:16015 2016.

[0142] Table 3. Chemokine receptors and their natural contexts [Table 3] TIFF2025019209000025.tif171157

[0143] The amino acid sequence of CCR6 provided by AAC51124.1 is shown below: MSGESMNFSDVFDSSEDYFVSVNTSYYSVDSEMLLCSLQEVRQFSRLFVPIAYSLICVFGLLGNILVVITFAFYKKARSMTDVYLLNMAIADILFVLTLPFWAVSHATGAWVFSNATCKLLKGIYAINFNCGMLLLTCISMDRRYIAIVQATKSFRLRSRTLPRSKIICLVVWGLSVIISSSTFVFNQKYNTQGSDVCEPKYQTVSEPIRWKLLMLGLELLFGFFIPLMFMIFCYTFIVKTLVQAQNSKRHKAIRVIIAVVLVFLACQIPHNMVLLVTAANLGKMNRSCQSEKLIGYTKTVTEVLAFLHCCLNPVLYAFIGQKFRNYFLKILKDLWCVRRKYKSSGFSCAGRYSENISRQTSETADNDNASSFTM (SEQ ID NO: 391)

[0144] An exemplary nucleic acid sequence for CCR6 is shown below.

[0145] Table 4. Adhesion receptors and their natural contexts [Table 4] TIFF2025019209000027.tif150157

[0146] Alternatively or additionally, genes that can enhance immune cell function, e.g., proliferation, cytotoxicity, etc., can also be introduced into or knocked out of the immune cells. Examples include TNF / TNFR2 overexpression (due to short-lived but more effective CD8 T cells), gld (FasL mutant; due to lymphoproliferation; CTLs do not kill via the Fas-FasL pathway); lpr (Fas mutant; due to upregulation of FasL - target cells resistant to FasL-mediated apoptosis); deficiency of granzyme B* (delayed nuclear apoptotic changes in target cells); deficiency of granzymes A and B* (delayed nuclear apoptotic changes in target cells); deficiency of perforin (complete absence of granule-mediated apoptosis); deficiency of perforin and FasL (defective granule-mediated and Fas-mediated apoptosis); deficiency of cathepsin C (dipeptidyl-peptidase I) (does not produce active granzymes and some hematopoietic serine proteases); underexpression of FAS (CD95); and / or overexpression of FASL.

[0147] Table 5 below lists the additional genetic modifications of MHC-CAR T cells or combination therapies described herein and the associated benefits resulting therefrom.

[0148] Table 5. Genetic modification or combination treatments and their benefits [Table 5] TIFF2025019209000029.tif102157

[0149] In some embodiments, genetic modifications leading to PD blockade may be introduced into immune cells expressing MHC-CAR as described herein. Such modifications include one or more of PD-1 knockout, PD-L1 or L2 overexpression, or PD-L1 knockout. PD blockade may be combined with immune inhibitors (e.g., knockout of CTLA-4, TIM-3, LAG3, TIGIT, IDO, or arginase, or CTLA-4Ig secretion), immune stimulants (e.g., anti-OX40, anti-CD137, IL-2, TLR ligands, or STING), and / or kinase inhibitors (e.g., Braf inhibitors or MEK inhibitors).

[0150] Table 6 below provides exemplary genetic modifications for PD blockade, immune inhibitors, cell death receptors, immune stimulants, toll-like receptors, kinase inhibition, master regulators, cytokine signaling, reducing cell interaction, and drug interaction-related editing. The table also provides GenBank accession numbers of sequences that can be used for expression / overexpression, as well as target sequences for guide RNA using Cas9 in T cells. Genome editing using gRNA is performed through transduction of lentivirus (lentiCRISPRv2) containing the desired gRNA and Streptococcus pyogenes Cas9 nuclease. This can be performed as an alternative to the delivery of TALEN RNA in the protocol. Sanjana, et al. Nature methods, 11(8):783-784, 2014.

[0151] Table 6. Exemplary genes for genetic modification and corresponding Cas9-mediated editing [Table 6] TIFF2025019209000031.tif197157TIFF2025019209000032.tif197157TIFF2025019209000033.t if194157TIFF2025019209000034.tif202157TIFF2025019209000035.tif198157TIFF20250192090 00036.tif198157TIFF2025019209000037.tif197157TIFF2025019209000038.tif204157TIFF202 5019209000039.tif196157TIFF2025019209000040.tif195157TIFF2025019209000041.tif145157

[0152] The following table provides protein sequences as well as target sequences for TALEN editing. Table 7. Primarily TALEN-mediated editing [Table 7] TIFF2025019209000043.tif190157TIFF2025019209000044.tif194157TIFF2025019209000045.tif188157TIFF2025019209000046.tif19415 7TIFF2025019209000047.tif196157TIFF2025019209000048.tif198157TIFF2025019209000049.tif203157TIFF2025019209000050.tif48157

[0153] (iii) Exemplary Genetic Modification Approaches Any conventional genetic modification approach can be used to genetically modify immune cells in the manner described herein. In some embodiments, genetic modification is performed using genome editing. "Genome editing" refers to a method of modifying the genome, including any protein-coding or non-coding nucleotide sequence of an organism, to knock out the expression of a target gene. In general, genome editing methods involve the use of endonucleases that can cut the nucleic acid of the genome, for example, at the nucleotide sequence that is targeted. Repair of double-strand breaks in the genome can be repaired to introduce mutations and / or exogenous nucleic acid can be inserted into the site that is targeted.

[0154] Genome editing methods are generally classified based on the type of endonuclease involved in generating double-stranded breaks in the target nucleic acid. These methods include the use of zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), meganucleases, and CRISPR / Cas systems.

[0155] In some cases, genetic modification of immune cells as described herein is performed using TALEN technology known in the art. TALENs are engineered restriction enzymes that can specifically bind and cleave a desired target DNA molecule. TALENs typically contain a transcription activator-like effector (TALE) DNA binding domain fused to a DNA cleavage domain. The DNA binding domain may contain a highly conserved 33-34 amino acid sequence with a 2 amino acid RVD (repeat variable dipeptide motif) that differs at positions 12 and 13. The RVD motif determines the binding specificity to a nucleic acid sequence and can be engineered according to methods known to those skilled in the art to specifically bind to a desired DNA sequence (e.g., Juillerat, et al. (January 2015). Scientific reports, 5; Miller et.al. (February 2011). Nature Biotechnology 29 (2): 143-8; Zhang et.al. (February 2011). Nature Biotechnology 29 (2): 149-53; Geiβler, et al., Boch, (2011), PLoS ONE 6 (5): e19509; Boch (February 2011). Nature Biotechnology 29 (2): 135-6; Boch, et.al. (December 2009). Science 326 (5959): 1509-12; and Moscou et al, (December 2009). Science 326 (5959): 1501. The DNA cleavage domain may be derived from FokI endonuclease, which is active in many different cell types. The FokI domain functions as a dimer and requires two constructs with unique DNA binding domains for sites in the target genome in the appropriate orientation and spacing. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between two individual TALEN binding sites appear to be important parameters for achieving high levels of activity.Miller et al. (2011) Nature Biotech. 29: 143-8.

[0156] TALENs specific to sequences in target genes of interest (e.g., TCR, CD52, MHC, and others described herein) can be constructed using any method known in the art, including a variety of schemes using modular components. Zhang et al. (2011) Nature Biotech. 29: 149-53; Geibler et al. (2011) PLoS ONE 6: e19509.

[0157] TALENs specific to a target gene of interest can be used in cells to generate double-strand breaks (DSBs). If the repair mechanism improperly repairs the break via non-homologous end joining, a mutation can be introduced at the break site. For example, improper repair can introduce a frameshift mutation. Alternatively, a foreign DNA molecule with a desired sequence can be introduced into a cell together with TALENs. Depending on the sequence of the foreign DNA and the chromosomal sequence, this process can be used to correct defects or introduce DNA fragments into a target gene of interest, or to introduce such defects into endogenous genes, thus reducing the expression of the target gene.

[0158] In some cases, genetic modification of immune cells as described herein is performed using CRISPR technology (CRISPR / Cas system) as known in the art. Such modifications may include deletions or mutations of sequences in a target gene of interest can be constructed using the CRISPR-Cas system, where the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system is an engineered, non-naturally occurring CRISPR-Cas system. The present disclosure utilizes a CRISPR / Cas system that hybridizes to a target sequence in a target gene of interest, where the CRISPR Cas system comprises a Cas endonuclease and an engineered crRNA / tracrRNA (or single guide RNA ("sgRNA"). In some embodiments, the CRISPR / Cas system comprises a crRNA and does not comprise a tracrRNA sequence. The CRISPR / Cas complex can bind to a lineage-specific protein polynucleotide and allow cleavage of the protein polynucleotide, thereby modifying the polynucleotide.

[0159] The CRISPR Cas system of the present disclosure may bind to and / or cleave a region of interest within a target gene of interest, within or adjacent to the gene, such as a leader sequence, trailer sequence, or intron, or within a non-transcribed region either upstream or downstream of the coding region. The guide RNA (gRNA) used in the present disclosure may be designed such that the gRNA directs the binding of the Cas enzyme-gRNA complex to a predetermined cleavage site (target site) in the genome. The cleavage site may be selected to release a fragment containing a region of unknown sequence, or a region containing SNPs, nucleotide insertions, nucleotide deletions, recombinations, and the like. Cleavage of a gene region may include cleaving one or both strands at the location of the target sequence by the Cas enzyme. In one embodiment, such cleavage may result in a decrease in transcription of the target gene. In another embodiment, cleavage may further include repairing the cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, where repair results in an insertion, deletion, or replacement of one or more nucleotides of the target polynucleotide.

[0160] The terms "gRNA", "guide RNA" and "CRISPR guide sequence" may be used interchangeably throughout and refer to a nucleic acid that comprises a sequence that determines the specificity of the Cas DNA-binding protein of a CRISPR / Cas system. The gRNA hybridizes (is partially or fully complementary to) a target nucleic acid sequence in the genome of a host cell. The gRNA or the portion thereof that hybridizes to the target nucleic acid may be 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length. In some embodiments, the gRNA sequence that hybridizes to the target nucleic acid is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the gRNA sequence that hybridizes to the target nucleic acid is 10-30, or 15-25 nucleotides in length.

[0161] In addition to the sequence that binds to the target nucleic acid, in some embodiments, gRNA also comprises a scaffold sequence. The expression of gRNA that codes for both the sequence that is complementary to the target nucleic acid and the scaffold sequence can have the dual function of both binding (hybridizing) to the target nucleic acid and localizing endonuclease to the target nucleic acid, resulting in site-specific CRISPR activity. In some embodiments, such chimeric gRNA can be referred to as single guide RNA (sgRNA).

[0162] As used herein, "scaffold sequence", also referred to as tracrRNA, refers to a nucleic acid sequence that localizes Cas endonuclease to a target nucleic acid that is bound (hybridized) to a complementary gRNA sequence. Any scaffold sequence that includes at least one stem-loop structure and localizes endonuclease may be used in the genetic elements and vectors described herein. Exemplary scaffold sequences are clear to those skilled in the art and can be found, for example, in Jinek, et al. Science (2012) 337(6096):816-821, Ran, et al. Nature Protocols (2013) 8:2281-2308, PCT Application No. WO2014 / 093694, and PCT Application No. WO2013 / 176772. In some embodiments, the CRISPR-Cas system does not include a tracrRNA sequence.

[0163] In some embodiments, the gRNA sequence does not include the scaffold sequence, and the scaffold sequence is expressed as a separate transcript. In such embodiments, the gRNA sequence further includes an additional sequence that is complementary to a portion of the scaffold sequence and that binds (hybridizes) to the scaffold sequence to localize the endonuclease to the target nucleic acid.

[0164] In some embodiments, the gRNA sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary to the target nucleic acid (see also U.S. Pat. No. 8,697,359, which is incorporated by reference for its teaching of gRNA sequence complementarity with target polynucleotide sequences). It has been demonstrated that mismatches between CRISPR guide sequences and target nucleic acids near the 3' end of the target nucleic acid can abolish nuclease cleavage activity (Upadhyay, et al. Genes Genome Genetics (2013) 3(12):2233-2238). In some embodiments, the gRNA sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary to the 3' end of the target nucleic acid (e.g., the last 5, 6, 7, 8, 9, or 10 nucleotides of the 3' end of the target nucleic acid).

[0165] Exemplary sgRNA sequences are provided herein, including both modified and unmodified sgRNAs, that target T cell receptor alpha constant (TRAC) gene.As will be apparent to those skilled in the art, the selection of sgRNA sequence may depend on factors such as the number of predicted on-target and / or off-target binding sites.In some embodiments, sgRNA sequence is selected to maximize potential on-target sites and minimize potential off-target sites.

[0166] In some embodiments, the Cas endonuclease is a Cas9 nuclease (or a variant thereof) or a Cpfl nuclease (or a variant thereof). The Cas9 endonuclease cleaves the double-stranded DNA of the target nucleic acid resulting in blunt ends, while cleavage with a Cpfl nuclease results in sticky ends of the nucleic acid.

[0167] Generally, the target nucleic acid is flanked by a protospacer adjacent motif (PAM) at the 3' or 5' side, which may interact with the endonuclease and further participate in targeting the endonuclease activity to the target nucleic acid. It is generally believed that the PAM sequence flanking the target nucleic acid depends on the endonuclease and the source from which the endonuclease is derived. For example, for the Cas9 endonuclease from Streptococcus pyogenes, the PAM sequence is NGG, but the PAM sequences NAG and NGA may be recognized with lower efficiency. For the Cas9 endonuclease from Staphylococcus aureus, the PAM sequence is NNGRRT. For the Cas9 endonuclease from Neisseria meningitidis, the PAM sequence is NNNNGATT. For the Cas9 endonuclease from Streptococcus thermophilus, StlCas9 dSt3Cas9, the PAM sequence is NNAGAAW and NGGNG, respectively. For the Cas9 endonuclease from Treponema denticola, the PAM sequence is NAAAAC. In some embodiments, the Cas endonuclease is a Cpfl nuclease. In contrast to the Cas9 endonuclease, the Cpfl endonuclease generally does not require a tracrRNA sequence and recognizes the PAM sequence located at the 5' end of the target nucleic acid. For the Cpfl nuclease, the PAM sequence is TTTN. In some embodiments, the Cas endonuclease is MAD7 (also referred to as Cpfl nuclease from Eubacterium rectale) and the PAM sequence is YTTTN.

[0168] In some embodiments, genetically engineering the cell also includes introducing a Cas endonuclease, or a nucleic acid sequence encoding it (e.g., an mRNA encoding a Cas endonuclease), into the cell. In some embodiments, the nucleic acid encoding the Cas endonuclease and the gRNA are provided on the same nucleic acid (e.g., a vector). In some embodiments, the nucleic acid encoding the Cas endonuclease and the gRNA are provided on different nucleic acids (e.g., different vectors). In some embodiments, the Cas endonuclease is provided as an mRNA encoding the Cas endonuclease, and the gRNA is provided as a modified gRNA molecule. Alternatively or additionally, the Cas endonuclease may be provided or introduced into the cell in the form of a protein.

[0169] In some embodiments, the Cas endonuclease is a Cas9 enzyme or a variant thereof. In some embodiments, the Cas9 endonuclease is derived from Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitidis, Streptococcus thermophilus, or Treponema denticola. In some embodiments, the nucleotide sequence encoding the Cas endonuclease may be codon optimized for expression in a host cell. In some embodiments, the endonuclease is a Cas9 homolog or ortholog.

[0170] In some embodiments, the nucleotide sequence encoding the Cas9 endonuclease is further modified to change the activity of the protein. In some embodiments, the Cas9 endonuclease is modified to inactivate one or more catalytic residues of the endonuclease. In some embodiments, the Cas9 endonuclease is modified to inactivate one of the catalytic residues of the endonuclease and is referred to as "nickases" or "Cas9n". The Cas9 nickase endonuclease cleaves one DNA strand of the target nucleic acid. In some embodiments, the methods described herein involve two separate cleavage reactions, one Cas9 nickase directed to cleave one DNA strand of the target nucleic acid, and one Cas9 nickase directed to cleave a second DNA strand of the target nucleic acid.

[0171] (iv) MHC-CAR regulatory T cells (Treg) Any MHC-CAR expressing T cell disclosed herein may be a regulatory T cell (Treg), which may mimic the immune modulating activity of follicular regulatory cells. As used herein, regulatory T cells or Treg cells, also known as suppressor T cells, refer to a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and / or prevent autoimmune disease. Treg cells function as immune suppressors to suppress or downregulate the induction and / or proliferation of effector T cells, such as pathological CD4+ and / or CD8+ cells, involved in autoimmune disease.

[0172] The genetically modified Treg cells disclosed herein express one or more of the biomarkers naturally associated with Treg cells, such as CD4, FOXP3, CD25, CD45R (e.g., CD45RA or CD45RO), or a combination thereof. Treg cells may be prepared using (or derived from) peripheral blood mononuclear cells (PBMCs) isolated from a suitable donor (e.g., a human patient to be treated). Methods for isolating subpopulations of Treg cells from PBMCs are well known in the art, such as cell sorting. In addition to suitable MHC-CAR constructs, expression vectors for other genetic modifications (e.g., those described herein) may be introduced into Treg subpopulations via methods as described herein or other methods known in the art. Alternatively, genetically modified Treg cells may be prepared by introducing a transgene encoding CD25 and / or other Treg cell markers into suitable T cells, which may be further modified to introduce an expression cassette for an MHC-CAR and optionally other genetic modifications as described herein.

[0173] In some embodiments, the genetically modified Treg cells may be further modified to present (e.g., surface expressed or surface bound) molecules that target specific types of pathological cells (e.g., CD4+ cells or CD8+ cells) and / or to present molecules that target specific tissue sites (e.g., lymph nodes or sites of inflammation).

[0174] In some examples, the genetically modified Treg cells further express a chimeric receptor (CAR) that includes an extracellular domain, such as a single chain antibody (scFv) specific for a B cell surface marker, e.g., CD19. Alternatively or additionally, the Treg cells may further express a chimeric receptor that includes an extracellular domain (e.g., scFv) specific for a T cell surface marker, e.g., CS-1. Such a chimeric receptor may be a cell surface receptor that includes an extracellular domain, a transmembrane domain, and a cytoplasmic domain (e.g., a costimulatory domain, a cytoplasmic signaling domain, e.g., CD3ζ, or a combination thereof) in a combination that is not naturally found together on a single protein.

[0175] Treg cells may further present molecules that target lymph nodes and / or germinal centers, such as CXCR5, and / or molecules that target inflammatory sites, such as CCR6. Targeting germinal center B cells (GC B cells) may be mediated, at least in part, by a specialized helper T cell subset, CXCR5highPD-1high T follicular helper (TFH) cells. Foxp3+ Tregs can be converted to be TFH repressors through expression of Bcl6 and SAP-mediated interaction with B cells. The resulting follicular regulatory T cells (TFR) are expected to share characteristics of both TFH and Treg cells, localize to germinal centers, and regulate the size of the TFH cell population and germinal centers in vivo.

[0176] Additionally, the Treg cells disclosed herein may comprise one or more of the additional genetic modifications as described herein, eg, a checkpoint molecule knockout.

[0177] Treg cells expressing B cell or T cell specific CARs may target pathological B cells and / or T cells involved in autoimmune diseases. For example, genetically modified Treg cells as described herein are expected to exhibit functions similar to follicular regulatory cells, for example, targeting B cells, T cells, and / or dendritic cells, thereby, for example, secreting inhibitory cytokines that can downregulate B cell stimulation, inhibit germinal center activation, induce cytolysis of (GC B cells (e.g., Il-10 and TGF-beta), Tfh (through MHC CAR) and GC) B (e.g., through CD19 CAR), and / or mechanically disrupt signaling to GC B cells or T follicular helper (Tfh) cells (e.g., through binding to GC B and MHC peptide Tfh). Alternatively or additionally, Treg cells may be capable of engaging, or in some cases physically blocking, both helper T cells, B cells, and / or antigen presenting cells.

[0178] III. Application of MHC-CAR-expressing immune cells in immunotherapy Host immune cells expressing the MHC-CARs (encoding nucleic acids or vectors comprising same) described herein are useful for targeting and elimination of pathogenic cells involved in autoimmune diseases such as MS, type I diabetes, lupus, rheumatoid arthritis, etc. In some embodiments, the subject is a mammal, e.g., a human, monkey, mouse, rabbit, or domesticated mammal. In some embodiments, the subject is a human, e.g., a human patient having, suspected of having, or at risk of having an autoimmune disease (e.g., MS).

[0179] The MHC-CAR expressing immune cells can be mixed with a pharma- ceutically acceptable carrier to form a pharmaceutical composition, which is also within the scope of the present disclosure. To carry out the methods described herein, an effective amount of immune cells expressing any of the MHC-CAR constructs described herein can be administered to a subject in need of treatment. The immune cells can be autologous to the subject, i.e., the immune cells are obtained from a subject in need of treatment, genetically engineered for expression of an MHC-CAR construct and optionally contain one or more additional genetic modifications as described herein, and then administered to the same subject. Administration of autologous cells to a subject can result in reduced rejection of the immune cells compared to administration of non-autologous cells. Alternatively, the immune cells are allogeneic, i.e., the cells are obtained from a first subject, genetically engineered for expression of an MHC-CAR construct, and administered to a second subject of the same species but different from the first subject. For example, allogeneic immune cells can be derived from a human donor and administered to a human recipient different from the donor.

[0180] In some embodiments, immune cells are combined with therapeutic agents for target immune diseases, such as alemtuzumab for treating MS. Such immunotherapy is used to treat, alleviate or reduce the symptoms of the target immune disease for which immunotherapy is considered to be useful in the subject.

[0181] The effectiveness of MHC-CAR immunotherapy may be evaluated by any method known in the art and apparent to a skilled medical professional. For example, the effectiveness of immunotherapy may be evaluated by subject survival and / or reduction of disease symptoms in a subject.

[0182] In some embodiments, immune cells expressing any of the MHC-CARs disclosed herein are administered to a subject who has been or is being treated with a therapeutic agent for an autoimmune disease. Immune cells expressing any one of the MHC-CARs disclosed herein may be administered in combination with a therapeutic agent. For example, the immune cells may be administered to a human subject simultaneously with the therapeutic agent. Alternatively, the immune cells may be administered to a human subject during the course of treatment with the therapeutic agent. In some examples, the immune cells and the therapeutic agent may be administered to a human subject at least 4 hours apart, for example, at least 12 hours apart, at least 1 day apart, at least 3 days apart, at least 1 week apart, at least 2 weeks apart, or at least 1 month apart.

[0183] To carry out the methods disclosed herein, an effective amount of immune cells expressing MHC-CAR or compositions thereof may be administered to a subject (e.g., a human MS patient) in need of treatment via a suitable route, such as intravenous administration. Any immune cells expressing MHC-CAR or compositions thereof may be administered to a subject in an effective amount. As used herein, an effective amount refers to the amount of each agent (e.g., immune cells expressing MHC-CAR or compositions thereof) that confers a therapeutic effect on the subject upon administration. Determining whether the amount of cells or compositions described herein achieves a therapeutic effect is apparent to one of skill in the art. As will be recognized by one of skill in the art, an effective amount will vary depending on the particular condition being treated, the severity of the condition, individual patient parameters including age, physical condition, height, sex, and weight, duration of treatment, the nature of concurrent therapy (if any), the particular route of administration, and similar factors within the knowledge and expertise of the medical practitioner. In some embodiments, an effective amount relieves, alleviates, ameliorates, improves, reduces, or delays the progression of any disease or disorder symptoms in the subject. In some embodiments, the subject is a human. In some embodiments, the subject is a human cancer patient.

[0184] In some embodiments, the subject is a human patient suffering from an autoimmune disease, which is characterized by an abnormal immune response that attacks normal body parts. Examples of autoimmune diseases include multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, juvenile idiopathic arthritis (also known as juvenile idiopathic arthritis), Sjogren's syndrome, systemic sclerosis, ankylosing spondylitis, type 1 diabetes, autoimmune thyroid disease (Graves' disease and Hashimoto's disease), multiple sclerosis myasthenia gravis, inflammatory bowel disease (Crohn's disease or ulcerative colitis), psoriasis, or diseases listed in Table 1.

[0185] Generally, there are multiple stages in the immune cascade that autoimmune disease can be affected.There is a continuum of interventions that can be made possible by the editing combinations that the methods disclosed herein include.For example, in addition to MHC-CAR, Treg cells that present a distinct set of surface molecules can be used to treat autoimmune disease at different stages.

[0186] In the early stages of many immune disorders, including MS, there is an unexplained defect in the regulatory mechanisms and / or tolerance induction in MS, and repeated attacks on the nervous system by T cells begin. Treg cells expressing suitable MHC-CARs and anti-CD19 CARs (optionally with other genetic modifications as described herein) may be used for intervention.

[0187] The advantage of autoreactive Treg cells is their ability to act as "bystander" suppressors and attenuate inflammation in a site-specific manner in response to cognate antigens expressed locally by diseased tissues. Induction of regulatory T cells (by autoantigens) can suppress disease progression even in the presence of various autoantigens (or even when the initiating / primary) autoantigen is unknown. Tregs can migrate relatively freely to inhibit T cells and B cells and prevent them from returning to an inflammatory environment. These autoreactive Tregs are advantageous in their ability to act as bystander suppressors and attenuate inflammation in a site-specific manner in response to cognate antigens expressed locally by diseased tissues.

[0188] Thus, genetically modified Treg cells may be engineered to mimic the suppressive function of autoreactive Treg cells, e.g., knocking out PD-L1 / PD-1, presenting scFvs targeting CCR6 and / or MOG to route to sites of inflammation, and expressing suitable MHC-CARs and / or anti-CD19-CARs. Alternatively, Treg cells may be engineered to, for example, knock out PD-L1 / PD-1, present CXCR5 to route to germinal centers and / or ectopic lymph nodes, and express a suitable MHC-CAR and / or anti-CD19-CAR. These types of Treg cells interact with pathogenic cells at the site of inflammation. These therapeutic agents may block pathogenic interactions and / or alleviate the inflammatory environment. They may be used in early disease stages (to inhibit pathogenicity) or after cytotoxic therapy (to prevent a return to the inflammatory environment).

[0189] Relapsing-remitting MS (mid-stage) is naturally self-regulated, and treatments that increase these natural regulatory mechanisms help control the disease process. In successful disease treatment, there is a shift from Th1 cells to Th2 and Th3 cells, as well as the emergence of other regulatory cells. At this stage, therapeutic targets include both pathogenic B cells and pathogenic T cells. Treg cells for treating such mid-stage disease may express a suitable MHC-CAR as described herein, and an additional CAR that targets B cells (e.g., anti-CD19 CAR) or T cells, such as CD8+ cells. Treg cells may further present CXCR5 or may not include CXCR5 targeting. Treg cells expressing anti-CD19 CAR may be used to eliminate B cells in germinal centers.

[0190] When MS changes from relapsing-remitting to chronic progressive (late stage), T cells enter a state of chronic activation and degenerative processes occur. Aggressive treatment with cytotoxic CD8+ T cells requires CAR augmentation that is sufficiently cytotoxic. At this point, treatment may shift from being driven primarily by Treg cells to being driven by MHC-CAR CD8+ T cells and even MHC-CAR CS-1 cells. The ultimate goal remains the same, to suppress pathology through enhanced cytotoxicity by bystander effects. Engineered T cells for use in this disease stage may express a suitable MHC-CAR and an additional CAR that targets pathological T cells involved in the later stages of the disease, such as CD8+ T cells. In some cases, the additional CAR may target CS-1 (also known as SLAMF7), which is a glycoprotein expressed on CD8+ T cells. CS1 is a promising antigen that can be used to target and kill CD8+ T cells and plasma cells.CS1-CAR T cells secrete more IFN-gamma as well as IL-2, express higher levels of the activation marker CD69, have a higher capacity for degranulation, and show enhanced cytotoxicity.Anti-CS1 CAR targets CD8+ T cells.Genetically modified T cells may further present molecules for bone marrow targeting of plasma cells, such as CXCR4, and for targeting to inflamed tissues using CXCR3.

[0191] Hiepe et al., Nature Reviews Rheumatology, 7(3):170-178, 2011. Examples include targeting plasma cells in lupus.

[0192] IV. Kits for therapeutic use The present disclosure also provides kits for the use of MHC-CAR-expressing immune cells for use in suppressing pathogenic immune cells, such as autoreactive T cells in autoimmunity. Such kits may include one or more containers containing a composition comprising immune cells expressing a MAR-CAR (such as those described herein) and a pharma- ceutically acceptable carrier.

[0193] In some embodiments, the kit may include instructions for use in any of the methods described herein. The included instructions may include instructions for administering MHC-CAR-expressing immune cells to a subject in need of treatment, such as an MS patient. The kit may further include instructions for selecting a suitable subject for treatment based on identifying whether the subject is in need of treatment. In some embodiments, the instructions include instructions for administering immune cells to a subject in need of treatment.

[0194] The instructions for use of the immune cells expressing MHC-CAR described herein generally include information regarding dosage, administration schedule, and administration route for the intended treatment. The container may be a unit dose, bulk package (e.g., a multi-dose package) or less than a unit dose. The instructions provided in the kit of the present disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical composition is used to treat, delay the onset, and / or alleviate a disease or disorder in a subject.

[0195] The kit provided herein is in suitable packaging.Suitable packaging includes, but is not limited to, vials, bottles, jars, and flexible packaging.Packaging for use in combination with a specific device is also envisioned.The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle).The container may also have a sterile access port.At least one active agent in the pharmaceutical composition is an immune cell that expresses MHC-CAR as described herein.

[0196] The kit may optionally provide additional components such as buffers and instructional information. Typically, the kit includes a container and a label or package insert(s) on or associated with the container. In some embodiments, the disclosure provides an article of manufacture that includes the contents of the kit described above.

[0197] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are therefore to be construed as merely illustrative, and not limiting in any way to the remainder of the disclosure. All publications referenced herein are incorporated by reference for the purpose or subject matter referenced herein. EXAMPLES

[0198] The following example focuses on the development of a cellular immunotherapy that utilizes chimeric receptors to selectively redirect therapeutic T cells against myelin basic protein (MBP)-specific T lymphocytes involved in MS.

[16] The results of this program can support the further development of redirected therapeutic T cells capable of opposing pathological self-specific T lymphocytes, and further validate the humanized MBP-DR2 chimeric receptor as a therapeutic target in MS.

[29]

[0199] Example 1: Construction of modified T cells specific for myelin basic protein Constructs for antigen-specific T cell receptor (TCR) targeting MBP-loaded major histocompatibility complex-chimeric antigen receptor (MHC-CAR) are designed for reintroduction into cells with TCR knockout for assay. The design of the TCR construct is based on antigen-specific TCRs with published structures and TCR expression constructs that have been validated in human cell lines [17, 46, 51, 52]. Green fluorescent protein (eGFP) or luciferase are genetically encoded for labeling

[28] . An exemplary lentiviral expression vector containing the TCR construct and reporting gene is provided in Figure 1.

[0200] Several cell lines were selected to test TCR expression and activity, including Jurkat E6-1 (a control line expressing the TCR), Jurkat J.RT3-T3.5 (a line lacking TCRb), SupT1 (a line with an impaired TCRa), and primary human T cells (containing a diverse population of TCR clonotypes).

[0201] Jurkat E6-1 cells are an established human T lymphocyte cell line from peripheral blood. It is used as a control cell line expressing TCR [15, 18].

[0202] Jurkat RT3-T3.5 lacks TCRβ due to a mutation that precludes expression of the TCR β chain. It also does not express surface CD3 and does not produce T cell receptor α,β heterodimers. It is therefore used to validate T cell receptor gene transfer [1, 6, 21, 49].

[0203] SupT1 is a human lymphoblastoid line that expresses multiple T lineage markers, encodes a non-functional receptor, and is used because it does not express TCRα

[49] .

[0204] PMBC-derived primary human T cells contain a diverse repertoire of TCR clonotypes.

[0205] Lentiviral vectors containing antigen-specific TCRs as shown in Figure 1 are used to transduce cancer cell lines lacking at least one TCR chain and then assessed for expression using fluorescence activated cell sorting (FACs). In cancer cell lines containing antigen-specific TCRs, luciferase is added to the cell line following successful stable expression to enable its use in mouse studies.

[0206] mRNA, multicistronic mRNA, and TCR constructs for lentiviral transduction proceed straightforwardly via labeling-based screening with genetically encoded eGFP and anti-TCR or anti-CD3 antibodies [3, 19].

[0207] Example 2: Construction of MHC-based chimeric receptors (MHC-CARs) and T cells expressing them (i) Design of MHC-CAR construct We have generated receptors for adoptive cell therapy that genetically link the MBP 84-102 epitope to the human leukocyte antigen HLADR2, which either incorporates or lacks a chimeric intracellular signaling domain

[29] . The antigen-major histocompatibility complex (Ag-MHC) domain serves as a receptor and binds to the TCR of MBP-specific target cells. Ag-MHC-CARs have been validated in preclinical mouse models with CD3-ζ (i.e., first generation signaling domains) and may be optionally combined with additional costimulatory signaling domains (i.e., second or third generation signaling domains) for efficacy in humans, following the methodology provided in [9, 25]. A schematic diagram of the various designs of MHC-CARs is provided in Figure 2.

[0208] MHC-CAR is designed based on the structure of HLA-DR and combined with various internal cytoplasmic costimulatory domains. MHC-CAR has two subunits: (i) an α chain containing a leader sequence, a DRA*1010 domain, and a cytoplasmic domain, and (ii) a β chain containing a leader sequence (from HLA-DRB1*1501), a peptide (DENPVVHFFKNIVTPRTPP (SEQ ID NO: 15) from myelin basic protein), a domain (from HLA-DRB1*1501), and a cytoplasmic signal domain, e.g., CD3z

[29] . Figure 3. The DNA used to generate the mRNA either contains a single strand (shown as an embodiment in Figure 4) or is multicistronic and separated by an orthogonal 2A sequence as shown in Figure 5. Genetically encoded eBFP or RQR8 is introduced into cells for cell labeling or to provide a mechanism for depletion [4, 5, 37, 40]. Figures 3 and 5.

[0209] As shown in Figure 6, the designed MHC-CAR has multiple sites for tagging. Site 1 is an HLA-DR antibody binding site for cases where native HLA-DR is not expressed due to CIITA editing [26, 38]. Sites 2 and 3 are potential insertion sites for polyhistidine tag motifs

[24] . Sites 4 and 5 represent RQR and RQR8, respectively

[37] . To promote the safety of therapeutic MHC-CARs, the genetically encoded RQR allows for rapid depletion of MHC-CAR T cells by administration of rituximab, a chimeric monoclonal antibody therapy used off-label in the treatment of severe MS, as multiple sclerosis has been shown to affect blood-brain barrier permeability

[42] .

[0210] (ii) Construction of T cell lines expressing MHC-CAR A number of cell lines, discussed below, will be selected to test the MHC-CAR constructs. Assays will then be continued in human T cells to establish a clinically translatable protocol.

[0211] K56 cells lack class I and class II MHC, allowing tag-free validation of HLA-DR expression using antibodies (HLA-DR is a component of MHC-CAR)

[45] . The same lentiviral constructs are then used in PMBC-derived T cells, allowing evaluation of MHC-CAR expression relative to RQR8 expression using flow cytometry

[23] .

[0212] KM-H2 is a human Hodgkin's lymphoma line that can be used as an HLA-DR positive control line [34, 44]. As mentioned above, Jurkat E6-1 conditionally expresses HLA-DR upon delivery of CIITA and can be used to evaluate CIITA TALENs when a CIITA knockout is used

[33] .

[0213] In addition, primary human T cells can be used. PMBC-derived primary human T cells are purified and enriched from whole blood, then activated. Transduction follows enrichment. The cells are incubated with recombinant human interleukin-2 (and / or IL-7 and / or IL-15) [7, 12, 43]. Based on preclinical studies and the anticipated therapeutic course, the desired cell type is CD8+ T cells with molecular and functional characteristics of stem cell memory TSCM, central memory TCM, and naive cell TN [11, 22, 32, 35]. Antibody staining allows immunotyping of the cells

[27] .

[0214] Initial constructs containing permutations of signaling domains (CD3z, 41BB, CD28) are expressed in K562 cells using mRNA, multicistronic mRNA, and lentiviral strategies. The lack of MHC class I and II in the cell lines allows for tag-free validation of MHC-CAR expression using HLA-DR antibodies, following a strategy that has been successfully used

[29] . Quantification of expression efficiency in more clinically relevant cell lines, including therapeutic T cells, depends on the construct and is measured using a genetically encoded fluorescent reporter (BFP) or antibody staining of the RQR site, polyhistidine-(HIS)-tag, or MHCCAR (using HLA-DR antibodies if CIITA is inactivated using TALENs). Labeling sites are indicated in Figure 6. To test MHC-CAR delivery rates as potential therapeutic vectors, the BFP is removed from the construct, retaining the RQR to provide a depletion control. Expression rates of clinically relevant constructs are measured using antibody staining after editing.

[0215] Transcription activator-like effector technology (TALEN) can also be used to prepare T cells expressing MHC-CAR. Human T cell lines are activated, transfected with TALEN, and transfected or transduced with MHC-CAR. They are then stained and analyzed by flow cytometry to evaluate TALEN gene inactivation and MHC-CAR expression.

[0216] Once expression of the construct in the desired cell line is verified, TALEN transduction into human T cell lines (TCRa and CD52 or CIITA as described above) is performed. After the TALEN is introduced into the activated human T cell line, the MHC-CAR is introduced into the same T cell line for evaluation. This order of human T cell modification prevents accidental sibling killing of MHC-modified cells due to the natural TCR

[10] . The activity of the TALEN-edited MHC-CAR T cells is confirmed.

[0217] (iii) Transcription activator-like effector (TALEN)-mediated modification of MHC-CAR-expressing T cells Human T cells were selected to confirm the inactivation of TCRa, and CD52 or CIITA genes by TALENs, and then TALENs were used to evaluate in combination with MHC-CAR. Transcription activator-like effectors (TALENs) bind to DNA in a sequence-specific manner. The DNA-binding domain contains a highly conserved 33-34 amino acid sequence with a distinct two-amino acid RVD (repeat variable dipeptide motif) that confers specific nucleotide recognition [23, 31].

[0218] Mutations in either the α or β chain of the TCR are sufficient to inhibit surface TCR expression [15, 36]. TALENs are used to inhibit expression of two genes in therapeutic cells without introducing a growth advantage through undesired translocations

[39]

[47] . To prevent graft-versus-host disease (GvHD) and enable the generation of allogeneic therapy, TCR expression is inhibited through TRAC-targeted TALENs [15, 39, 47].

[0219] Deletion of CD52 can be performed for alemtuzumab compatibility. Alemtuzumab is a humanized anti-CD52 IgG1 monoclonal antibody that targets and depletes circulating T and B lymphocytes

[41] . Alemtuzumab can be used as rescue therapy or first-line drug in severe onset MS

[50] and is co-administered in human patient populations. TALEN knockout makes MHC-CAR compatible with concurrent treatment in patients [14, 39]. In addition, deletion of CIITA can be performed for characterization of HLA-DR in MHC-CAR. CIITA is a protein-coding gene essential for transcriptional activity of the HLA class II promoter

[26] . The knockout allows direct measurement of HLA-DR using antibodies for characterization of MHC-CAR expression [38, 53]. Inhibition of CD52 expression allows for co-treatment with Lemtrada (alemtuzumab), an FDA-approved treatment for multiple sclerosis

[42] . Alemtuzumab is also used as a lymphodepleting / lymphoid suppressant agent to aid in the engraftment of CAR T therapy

[39] . CAR T cell therapy engineered with TRAC and CD52-targeting TALENs is currently being tested in clinical trials [38, 40, 43].

[0220] TALENs that inactivate MHC class II transactivator of T cell proliferation (CIITA) instead of CD52 are expected to inhibit HLA-DR expression as inactivation of CIITA

[26]

[38] , thus allowing direct identification of MHC-CAR-containing cells by HLA-DR antibody staining.

[0221] Validated TALENs (TRAC: TTGTCCCACAGATATCCagaaccctgaccctgCCGTGTACCAGCTGAGA (SEQ ID NO: 376); CD52: TTCCTCCTACTCACCATcagcctcctggttatGGTACAGGTAAGAGCAA (SEQ ID NO: 371); CIITA: TTCCCTCCCAGGCAGCTCacagtgtgccaccaTGGAGTTGGGGCCCCTA (SEQ ID NO: 366)) were obtained from Cellectis or designed to target previously validated sites [38, 43].

[0222] Human T cells are activated and electroporated with mRNA encoding three different TALEN variants: TCR-alpha constant chain, CD52, and CIITA. Cells are surface stained with anti-CD3 or anti-TCR (TCR-alpha constant chain), anti-CD52 (CD52), or anti-HLA-DR (CIITA), and then analyzed by flow cytometry.

[0223] Revalidate the ability of previously validated TALENs against inactive target genes in TALEN expression assays

[39] . Re-perform translocation and off-target studies and confirm the results with several whole genome sequences. GUIDE-seq can be used as an alternative to whole genome sequencing to confirm on- and off-target editing [8].

[0224] Edit one or more of the following endogenous genes to reduce interaction with other cells: TCR (through the TCR alpha or beta chain; to reduce targeting to unwanted cells), CIITA (modulates expression of MHC class II genes; target cells to taster the deletion), B7-1 (CD80) and / or B7-2 (CD86) knockout, and b2m (modulates expression of MHC class I genes) along with NKG2D ligand or UL18.

[0225] Additionally, one or more of the following genes can be edited to modify the function of interacting cells: PD-L1+ / -CTLA4-Ig overexpression; PD-L1 / 2 overexpression+ / -PD-1 knockout; FasL overexpression+ / -Fas knockout; Galectin 9 overexpression+ / -TIM3 knockout; and / or PVT / CD155 overexpression+ / -TIGIT knockout. Defects in PD-1, Fas, TIm3, TIGIT predispose patients to autoimmunity. Some drugs may restore function (e.g., Tim3, glatiramer acetate, and IFN-beta). If autologous cells from patients with autoimmune disease are utilized, they may require patient-specific correction of defective genes that affect CTL and Treg function. Their personal mutation sets may also determine whether CTL or Treg cells are most therapeutically relevant and whether any cell modifications will be effective if either autologous or allogeneic cells are used.

[0226] Additionally, the following edits may alter cell location and / or function (e.g., to make them more follicular regulatory cell-like): - MHC-CAR-(FOX3P)-(CS1 or CD19 CAR) / (CS-1 knockout required for CS-1 CAR) - MHC-CAR-(FOX3P)-(CCR7 or CXCR5)

[0227] Additionally, the following edits may enable combination therapies for autoimmune diseases (e.g., MS-specific therapies). - RQR tag: engineered T cells can be deleted using Rituximab (kill switch). A tag for the newly approved anti-CD20 antibody Ocrevus can be generated. - Rapamycin switch: inactive / active CAR only when patient is treated with rapamycin (tacrolimus) - CD52 knockout: Allows pre-treatment with Lemtrada (alemtuzumab) to reduce the number of immune cells present - VLA-4 knockout: Can be treated with Tysabri to migrate pathogenic immune cells to the periphery, but the engineered cells are forced to stay there (may not be ideal for patients with established MS as cells stuck in the brain / spinal cord, but simultaneous mRNA expression of VLA-4 may provide temporary access to those locations) - Secretion of IL-6 antibody (Toclizumab) from engineered T cells: This drug is useful when engineered T cells need access to the brain and spinal cord, but cannot access these locations due to the BBB.

[0228] Example 3: Investigation of MHC-CAR activity (i) Preparation of primary T cells expressing MHC-CAR Primary T cells can be prepared as follows: T cells are isolated from peripheral blood mononuclear cells (EasySep Human T Cell Enrichment Kit, Stemcell Technologies) and activated with (X-VIVO 15 medium, Lonza; 20mg / ml Il-2, Miltenyi; 5% human AB serum, Seralab) (Dynabeads Human T-Activator CD3 / CD28, Life Technologies). A suitable MHC-CAR construct containing MBP antigen peptide is introduced into primary T cells using conventional methods. Surface expression of the MHC-CAR construct is verified by FACS and antibody staining.

[0229] (ii) In vitro MHC-CAR activity test Once the trafficking and expression of the constructs (using mRNA, multicistronic mRNA, lentivirus) have been verified, activity studies are performed in vitro. All studies are performed at different effector:target (E:F) cell concentrations. In vitro studies provide an initial assessment of the MHC-CAR signaling domains and T cell subsets.

[0230] (iii) Evaluation of signal transduction domains by IL-2 production 24 hours after electroporation, human T cells transiently engineered with MHC-CAR are stimulated with plate-bound HLA-DR antibodies to determine whether the MHC-CAR (containing various signaling domains) is functional. IL-2 production is measured 24 hours later using the StemCell IL-2 ELISA kit. This test provides a rapid assay as to whether the variant should be re-engineered or abandoned

[29] .

[0231] (iv) Interaction of MHC-CAR cells and pathogenic TCR cells through proliferation assays Target cell lines transiently expressing the TCR are magnetically sorted for TCR expression 24 h after electroporation and irradiated to test whether engagement of the MHC-CAR with the antigen-specific TCR stimulates proliferation of MHC-CAR containing T cells. Alternatively, target cell lines stably expressing the TCR are irradiated. Irradiated cells presenting (+ / - antigen-specific) TCR are incubated with CFSE-labeled MHCCAR cells and proliferation is measured after culture at different T:E ratios

[30] .

[0232] (v) Degranulation assay CAR T cells are labeled with an epitope on RQR (not expressed on the target cells used) or through eBFP instead of a T cell marker. Assays are performed on cell lines with transient or stable expression (an example of transient expression is described). 24 hours after electroporation, MHC-CAR human T cells with either RQR8 or BFP are co-cultured with target (antigen-specific TCR SupT1 or Jurkat) or control (+ / -TCR Jurkat or TCR- SupT1) cells for 6 hours. Transiently expressing (and later stably expressing) target cells are electroporated with antigen-specific TCR and sorted using CD3 magnetic beads after electroporation. RQR+ or BFP+ MHC-CAR T cells (identified using anti-rituximab, QBEnd10 antibodies) are analyzed by flow cytometry to detect expression of the degranulation marker CD107a on their surface [2].

[0233] (vi) Cytokine secretion assay Assays are performed on cell lines with transient or stable expression (describe transient expression). Human T cells transiently expressing MHC-CAR are assessed for cytokine secretion after co-culture with target cells 24 hours after electroporation. Human T cells transiently expressing MHC-CAR are co-cultured with target (antigen-specific TCR-containing Jurkat or SupT1 cells) or control (+ / - non-antigen-specific Jurkat or SupT1) cells for 24 hours. Antigen-specific TCR are then killed by irradiation prior to assay. Supernatants are collected and analyzed using a TH1 / TH2 cytokine cytometric bead array kit to quantify cytokines produced by the T cells

[13] . MHC-CAR T cells produce IFN and other cytokines in co-culture with antigen-specific TCR-expressing target cells but not in co-culture with control cells.

[0234] (vii) IFNγ release assay Cells expressing various levels of MHC-CAR are incubated with irradiated TCR T cells 24 hours after transfection. The co-cultures are maintained for 24 hours. After incubation and centrifugation, the supernatants are tested using IFNγ detection by ELISA.

[0235] (viii) Cytotoxicity assay TCR T cells are incubated with the therapeutic MHC-CAR as well as control cells. Target and control cells are labeled with a fluorescent intracellular dye (CFSE or Cell Trace Violet) prior to co-culture with MHC-CAR T cells. Co-cultures are incubated for 4 hours. After this incubation period, cells are labeled with a fixable viability dye and analyzed by flow cytometry. The survival of each cell population (target or negative control) is determined and the percentage of specific cell lysis is calculated. Cytotoxicity assays are performed 48 hours after transduction.

[0236] (ix) Inhibition assay PBMCs are co-cultured with irradiated or mitomycin-treated engineered T cells expressing the MHC-CAR construct. As a control, PBMCs are co-cultured with irradiated or mitomycin-treated engineered T cells not expressing the MHC-CAR construct. After 7 days, cell proliferation from human patient donor A is measured by XTT colorimetric assay or CFSE dilution (FACS analysis). Cell proliferation is observed in the control, but no or limited cell proliferation is expected when engineered T cells express secreted FP. The results from this experiment aim to show that proliferation of alloreactive T cells is inhibited when MHC-CAR expressing T cells express FP.

[0237] (x) Multiplication To test whether engagement of MHC-CAR with antigen-specific TCR stimulates proliferation of MHC-CAR-containing T cells, target cell lines transiently expressing TCR are magnetically sorted for TCR expression 24 hours after electroporation and irradiated. Alternatively, target cell lines stably expressing TCR are irradiated. Irradiated cells presenting (+ / - antigen-specific) TCR are incubated with CFSE-labeled MHC-CAR cells and proliferation is measured after culture at different T:E ratios.

[0238] (xi) In vivo testing MHC-CARs (with mouse and human MHC) in mouse T cells have previously shown therapeutic efficacy in experimental allergic encephalomyelitis, a mouse model for multiple sclerosis [25, 29]. Here we test whether MHC-CARs in human cells can target human TCRs in human T cell lines using an in vivo mouse model similar to that in previous CAR T preclinical studies

[39] . In vivo testing will allow further evaluation of MHC-CAR signaling domains and T cell subsets.

[0239] As shown in Figure 7, the in vivo activity of MHC-CAR T cells can be validated in a mouse xenograft model.

[0240] Immunodefficient NOG mice are intravenously injected with antigen-specific TCR-luciferase expressing T cells as a MS xenograft mouse model. The mice are then given intravenous doses of MHC-CAR T cells tested at different doses 2 or 7 days after injection with the tumor cell line. Intravenous injection with T cells not transduced with the CAR lentiviral vector serves as a control. To follow the expansion growth of TCR-luciferase expressing cells in different animals, the bioluminescence signal is determined on the day of T cell injection (D0), D7, 14, 21, 28 and 40 after T cell injection

[39] .

[0241] CAR T cells with similar background modifications to those presented here (TALEN to inactivate the native TCR and minimize graft-versus-host, TALEN to inactivate CD52 and allow for co-treatment with alemtuzumab, and RQR8 to allow for depletion) have been previously validated in mouse models and will now be used in the UCART19 clinical trial

[39] .

[0242] (xii) Verification of the kill switch Complement-dependent cytotoxicity (CDC)-mediated susceptibility is examined by exposing transduced T cells to 25% baby rabbit complement (AbD Serotec) for 4 hours with or without pharmaceutical complement (rituximab, Tysabri, or alemtuzumab). Miltenyi CD34 magnetic bead-selected transduced RQR8 T cells are compared against a population of similarly treated Q8-transduced T cells to demonstrate the specificity of CDC-mediated deletion. Further investigation of CDC assay parameters was achieved through a time / dose titration assay using RQR8-transduced T cells incubated with 12.5, 25, 50, and 100 mg / mL pharmaceutical complement and time point evaluation ranging from 1 to 120 min.

[0243] Example 4: Regulatory T cells (Treg) expressing MHC-CAR Therapies that antigen-specifically target pathological T lymphocytes involved in multiple sclerosis and other autoimmune diseases are expected to have an improved therapeutic index compared to antigen-nonspecific therapies. This example provides an exemplary cellular immunotherapy that uses a chimeric antigen receptor to selectively redirect therapeutic T cells to myelin basic protein-specific T lymphocytes involved in MS.

[0244] Selection, transduction, and expansion of Treg cells CD4+ T cells are isolated from PMBCs via RosetteSep (STEMCELL Technologies) and enriched for CD25+ cells (Miltenyi Biotec), then sorted by FACS into live CD4+CD45RO-CD45RA+CD25+ Treg and CD4+CD45RO-CD45RA+CD25- control T cells. Sorted T cells are stimulated with artificial APCs (aAPCs) loaded with αCD3 mAb in 1,000 U / ml or 100 U / mL IL-2 for Treg or non-reg control T, respectively. One day later, cells are transduced with lentivirus. On day 7, ΔNGFR+ cells are purified using magnetic selection (Miltenyi Biotec), then restimulated with aAPCs as above and expanded for 6-7 days.

[0245] Flow cytometry: For phenotypic analysis, cells were stained with fixable viability dyes (FVD) (65-0865-14 and 65-0866-14, eBioscience) and stained with FOXP3 / Transcription Factor Staining Buffer Set (eBioscience) before fixation / permeabilization for surface markers, followed by staining for intracellular proteins. For analysis of cytokine production, cells were stimulated with 10 ng / ml PMA and 500 ng / ml ionomycin (all Sigma-Aldrich) in the presence of Brefeldin A (10 μg / ml) for 4 h. Samples were analyzed by flow cytometry.

[0246] Microscopy: PBMCs are labeled with PKH26 or PKH67 (Sigma-Aldrich, PKH26GL-1KT and PKH67GL-1KT) and Tregs with cell proliferation dye (CPD) eFluor450 (eBiosciences, 65-0842-85) and then suspended in 1.5% rat tail collagen type I 3D gel (Ibidi) composed of 1× DMEM and 10% FCS according to the manufacturer's general 3D gel protocol. The cell suspension is pipetted into a μ-Slide Chemotaxis3D and polymerized for 30 min in a humidified incubator (Tokai Hit) at 35° C. and 5% CO2 on a Leica TCS SP8 confocal microscope. The outer chamber is filled with 1× DMEM and images are recorded every 2 min for 3 h using a ×10 / 0.30 objective. eFluor450, PKH67, and PKH26 are excited at 405 nm, 488 nm, and 561 nm, and fluorescence emission is collected at 415-470 nm, 495-525 nm, and 570-650 nm, respectively. The number of interactions between CAR-Tregs and either target or control cells is quantified every 20 min. Immobile cells were excluded from the analysis. The total number of each labeled cell type per field of view can be counted using the analyze particles function in ImageJ (imagej.nih.gov / ij / ).

[0247] Analysis of Treg-specific hypomethylated regions (TSDRs) Stable Treg expression of Foxp3 is associated with selective demethylation of the TSDR within the Foxp3 locus. To test for stable expression, DNA from frozen T cell pellets was isolated using the DNeasy Blood and Tissue Kit (QIAGEN) and bisulfite converted using the EZ Direct Kit (Zymo Research). PCR of BisDNA was performed using the Human FOXP3 Kit (Epigen DX) and prepared for pyrosequencing using PyroMark buffer (QIAGEN) and then assayed on a Biotage PyroMark Q96 MD pyrosequencer (QIAGEN). Results were calculated using Pyro Q-CpG software (Biotage).

[0248] Cytokine production To measure cytokine production, T cell lines were stimulated with K562 cells (1 K562:2 T cells) for 48 h. Supernatants were collected and cytokine concentrations were determined and analyzed using the Human Th1 / Th2 / Th17 Cytokine Kit (BD Biosciences).

[0249] Inhibition of MHC CAR-specific proliferation To test whether target-specific Tregs were also able to suppress CD4+ T cell proliferation, MHC CAR-specific CD4+ T clones are isolated. Epstein-Barr virus-transformed B lymphablastoid cell lines were transduced with MHC-CAR using lentivirus. EBV cell lines were grown overnight, irradiated with 150 Gy, and co-cultured with CPD-labeled MHC CAR-specific CD4+ T clones in the absence or presence of CAR-expressing Tregs or conventional T cells. Proliferation was determined after 4 days, and the percentage of suppression of MHC CAR-specific clones was calculated using the percentage of proliferation as follows: (100-[(% of proliferated MHC CAR+ test) / (% of proliferated MHC CAR alone)]×100).

[0250] Once the trafficking and expression of the constructs (using mRNA, multicistronic mRNA, and / or lentivirus) has been verified, activity studies will begin in vitro. All studies will be performed at different effector:target (E:F) cell concentrations. In vitro studies are expected to provide an initial assessment of the MHC-CAR signaling domains and T cell subsets.

[0251] Transient or lentiviral expression of chemokine and adhesion receptors in T cells Receptors are expressed in human T cells after electroporation of mono / polycistronic mRNA or lentiviral transduction. Receptor expression is analyzed using flow cytometry. In summary, 5 × 10 T cells preactivated for several days (3–5) with anti-CD3 / CD28 coated beads and IL2 were cultured in 100% HO cells. 6 T cells were resuspended in cytoporation buffer T and electroporated with 45 μg of mRNA. 24 hours after electroporation, human T cells engineered with polycistronic mRNA encoding a multi-chain CAR were labeled with the fixable viability dye eFluor-780 and PE-conjugated goat anti-mouse IgG F(ab')2 fragment specific and analyzed by flow cytometry. Alternatively, receptors were vectorized and expressed in lentivirus and analyzed similarly.

[0252] In vitro chemotaxis assay Transduced T cells were used in chemotaxis assays as previously described [Burkle et al., Blood, 110(9):3316-3325, 2007; Wu and Hwang, Journal of immunology, 168(10):5096, 2002.; Singh et al., Journal of immunology, 180(1):214-221, 2008.; Ryu et al., Molecules and Cells, 39.12:898-908, 2016.]. Cells (approximately 20,000 cells in medium, 1 million cells, 5 × 10 6100 µL / mL) was placed in duplicate on top of a 5 µm pore size filter, while media with and without chemokine was placed in the lower chamber. After 30 min, 1 h, 3 h, 5 hr, and 24 hr at 37°C, the migrated cells that had fallen to the bottom of the plate were treated as follows:

[0253] A. Images were taken using a 4x objective. Three random fields from each of two wells were counted using Image Pro Plus (Media Cybernetics, Silver Spring, MD). Three independent experiments were performed with similar results.

[0254] B. 400 μL of the cell suspension was added to 100 μL of a solution containing 4×10−7 M FITC-labeled phalloidin, 0.5 mg / mL 1-alpha-lysophosphatidylcholine (both from Sigma, St Louis, MO), and 7% formaldehyde in phosphate-buffered saline (PBS). Fixed cells were analyzed by flow cytometry on a FACSCalibur, and all time points are plotted against the average relative trend of samples before addition of chemokine.

[0255] C. Cells in the lower chamber were counted using a Countess II FL (Thermo Fisher Scientific, USA) or the OD value at 450 nm was measured using a Versamax microplate reader (Molecular Devices).

[0256] Example 5: Expression of MHC-based chimeric receptors (MHC-CAR) in HEK293 cells Constructs encoding MHC-CARs were constructed as discussed in Example 2 and evaluated for expression in HEK293 cells. Briefly, construct 1 comprises an EF1 alpha short promoter, CD19 CAR (4G7-CAR), CCR6, and GFP (provided by SEQ ID NO:426); construct 2 comprises an EF1 alpha short promoter, RQR8, MHC-CAR1 part B MHC-CAR1 part A, and GFP (provided by SEQ ID NO:409).

[0257] Construct 1, 2, or media control (non-transfected) were transfected into HEK293 cells and cultured. Cells were assessed for expression by microscopy based on GFP expression. A population of GF positive cells was observed in the groups transfected with Construct 1 or Construct 2. Cells were also assessed for expression of the components encoded by the constructs by flow cytometry. Tables 8 and 9.

[0258] Expression of construct 1 For detection of CCR6, cells were incubated with anti-CCR6 monoclonal antibody conjugated to APC (17-1969-42, eBioscience), and for detection of CD19, cells were incubated with biotinylated CD19 (Acro CD9-H8259, Acro Biosystems) followed by streptavidin-PE (405203, BioLegend®).

[0259] Table 8: Expression of Construct 1 [Table 8]

[0260] Expression of construct 2 For detection of CD34 expression from epitopes contained in RQR8, cells were incubated with anti-CD34 APC-conjugated monoclonal antibody, and for detection of MHC-CAR expression, cells were incubated with anti-HLA-DR antibody.

[0261] Table 9: Expression of Construct 2 [Table 9]

[0262] Nucleic acid sequence of construct 1 (SEQ ID NO:426) Nucleic acid sequence of construct 2 (SEQ ID NO:409) [ka]

[0263] Example 6: Expression of CD3 Expression of CD3 was assessed to determine the efficacy of the CRISPR method targeting the T cell receptor alpha constant (TRAC) gene. Briefly, sgRNA targeting the TRAC gene was generated by amplifying the target site using a forward primer (5'-AGCGCTCTCGTACAGAGTTGG-3' (SEQ ID NO: 385)) and a reverse primer (5'-AAAAAAAGCACCGACTCGGTGCC-3' (SEQ ID NO: 386).

[0264] The unmodified sgRNA has the nucleic acid sequence: 5'- GAG AAU CAA AAU CGG UGA AUG UUU UAG AGC UAG AAA UAG CAA GUU AAA AUA AGG CUA GUC CGU UAU CAA CUU GAA AAA GUG GCA CCG AGU CGG UGC UUU U -3 (SEQ ID NO: 384) Provided by.

[0265] The modified sgRNA has the nucleic acid sequence: 5'- 2'OMe(G(ps)A(ps)G(ps))AAU CAA AAU CGG UGA AUG UUU UAG AGC UAG AAA UAG CAA GUU AAA AUA AGG CUA GUC CGU UAU CAA CUU GAA AAA GUG GCA CCG AGU CGG UGC 2'OMe(U(ps)U(ps)U(ps)U -3'(SEQ ID NO:387) Provided by: 2'OMe = 2'O)-methyl RNA and ps = phosphorothioate.

[0266] Primary human stimulated CD3+ T cells were transfected with mRNA encoding Cas9 (Cas9 alone) or both mRNA encoding Cas9 and sgRNA targeting the TRAC gene. Seven days after transfection, CD3 expression was assessed by flow cytometry. Cells were incubated with anti-CD3-APC antibody (clone OKT3; BioLegend® product number 317318) at a dilution of 1:100. As shown in Figure 11, transfection of mRNA encoding Cas9 and sgRNA targeting the TRAC gene resulted in a significant reduction in CD3 expression.

[0267] Example 7: Validation of the kill switch The efficacy of the RQR8 killing switch encoded in exemplary construct 2 was evaluated using a cell viability assay. Briefly, HEK cells were transfected with medium alone, construct 1 (SEQ ID NO:X), construct 2 (encoding a rituximab-mediated RQR8 killing switch; SEQ ID NO:X), or both construct 1 and construct 2. Transfected HEK293 cells were harvested, counted, and resuspended at 1×10^6 cells / mL. 300uL of cell suspension was transferred to each of four wells of a 48-well tissue culture plate. 100uL of complete medium and 4uL of rituximab were added to the second well, and 100uL of freshly prepared baby rabbit complement and 4uL of rituximab were added to the fourth well. Plates were incubated for 2, 4, or 24 hours. The assay was terminated by adding 1 uL of chilled Annexin V buffer (150 mM NaCl, 10 mM HEPES, 10 mM CaCl) and then samples were transferred to pre-prepared flow cytometry tubes containing 3 mL of Annexin V buffer.

[0268] Samples were collected by centrifugation and any residual buffer was blotted off with paper towels. Samples were then stained with 1 uL of Annexin V APC, vortexed and placed under soft light for 15 minutes. Samples were then washed with Annexin V buffer and 5 uL of propidium iodide was added per mL of buffer and placed on ice immediately after final suspension until flow cytometry was performed.

[0269] The percentage of GFP-positive cells was quantified to quantify the killing induced by the RQR8 killing switch. As shown in Figure 12, incubation with complement alone resulted in some cell death, but this reduction in survival was observed in all cell groups. Cell death was observed in cells expressing construct 2 or both constructs 1 and 2 in the presence of a combination of rituximab and complement, indicating specific RQR8-mediated cell death.

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[0271] Other embodiments All of the features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0272] From the above description, those skilled in the art can easily grasp the essential features of the present invention, and can make various modifications and improvements to the present invention to adapt it to various usages and conditions without departing from the spirit and scope thereof. Therefore, other embodiments are also within the scope of the claims.

Claims

1. 1. A major histocompatibility complex (MHC)-based chimeric receptor, (i) a first polypeptide comprising an extracellular domain of an alpha chain of a first MHC class II; and (ii) a second polypeptide comprising an extracellular domain of a beta chain of a second MHC class II. Including, An antigenic peptide is fused to either the first polypeptide or the second polypeptide, Either the first polypeptide or the second polypeptide, but not both, further comprises a cytoplasmic signaling domain. MHC-based chimeric receptors.

2. The MHC-based chimeric receptor of claim 1 , wherein the first polypeptide and / or the second polypeptide further comprises at least one costimulatory domain.

3. The MHC-based chimeric receptor of claim 2, wherein the at least one costimulatory domain is a costimulatory domain from 4-1BB, a costimulatory domain from CD28, or a combination thereof.

4. The MHC-based chimeric receptor of any one of claims 1 to 3, further comprising a hinge domain located between the antigenic peptide and the extracellular domain of MHC class II fused to the antigenic peptide.

5. The MHC-based chimeric receptor of any one of claims 1 to 4, wherein the cytoplasmic signaling domain is the cytoplasmic signaling domain of CD3ζ.

6. 6. The MHC-based chimeric receptor of any one of claims 1 to 5, wherein the antigenic peptide is an antigenic peptide from myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), insulin, glutamic acid decarboxylase, or those listed in Table 1.

7. said first MHC class II is HLA-DRA*1010; and / or The second MHC class II is HLA-DRB1*1501; The MHC-based chimeric receptor according to any one of claims 1 to 6.

8. A nucleic acid or set of nucleic acids encoding the MHC-based chimeric receptor according to any one of claims 1 to 7.

9. The nucleic acid or set of nucleic acids according to claim 8, wherein the nucleic acid or set of nucleic acids is located in a viral vector.

10. A genetically modified immune cell expressing the MHC-based chimeric receptor of any one of claims 1 to 7.

11. The genetically modified immune cell of claim 10, which is a T cell.

12. Features: (a) endogenous T cell receptor (TCR) activity is suppressed; (b) the alpha chain of the endogenous TCR, the beta chain of the endogenous TCR, or both, are mutated or deleted so as to inhibit surface expression of the endogenous TCR; (c) expression of endogenous CD52 is inhibited; (d) further expressing a suicide gene, a marker gene; or (e) has been further modified or engineered to exhibit one or more of the following characteristics: (I) delivery to and retention in lymph nodes; (II) overexpressing VAP-1, L-selectin, CCR7, or a combination thereof; (III) express one or more surface molecules for delivery to and retention in tertiary or ectopic lymph nodes; (IV) expression of endogenous sphingosine-1-phosphate receptor 1 is inhibited; (V) having an IL6ST knockout, an IL6R knockout, or both; (VI) expressing or overexpressing a chemokine receptor; (VII) expressing or overexpressing an adhesion receptor, and (VIII) showing blockade of PD-1 signaling; The genetically modified immune cell of claim 11, comprising one or more of the following:

13. the suicide gene is RQR8, and / or The marker gene is a fluorescent protein gene. The genetically modified immune cell of claim 12.

14. The chemokine receptor is involved in the migration of pathological cells into inflamed tissues. said adhesion receptor is involved in the trafficking of pathological immune cells to areas of inflammation; and / or The blockade of PD-1 signaling is by PD-1 knockout, PD-L1 knockout, or a combination thereof; A genetically modified immune cell according to claim 12 or 13.

15. the chemokine receptor comprises CCR5, CXCR3, CCR4, CCR3, CCR6, CXCR3, CXCR4, CXCR5, or a combination thereof; or The adhesion receptor is VLA-4, α ４ β １ , α ４ β ７、 α Ｌ β ２ or combinations thereof, The genetically modified immune cell of claim 14.

16. the immune cells are regulatory T cells that are CD25+; or The immune cells are cytotoxic lymphocytes that are cytotoxic T cells. The genetically modified immune cell according to any one of claims 10 to 15.

17. The regulatory T cells are derived from CD25++CD45R+ T cells isolated from peripheral blood mononuclear cells; The cytotoxic T cells are derived from CD8+ T cells isolated from peripheral blood mononuclear cells; The regulatory T cells comprise a transgene encoding CD25; and / or the regulatory T cells comprise a transgene encoding FoxP3; The genetically modified immune cell of claim 16.

18. the regulatory T cells or the cytotoxic T cells further express a chimeric receptor specific for CD19, a chimeric receptor specific for CS-1, or both; the regulatory T cells or the cytotoxic T cells further express CCR6, CXCR5, PD-1, or a combination thereof; or the regulatory T cells or the cytotoxic T cells present antibodies specific to self-antigens and / or secrete anti-inflammatory cytokines; The genetically modified immune cell of claim 17.

19. 19. The genetically modified immune cell of claim 18, wherein the regulatory T cells or the cytotoxic T cells present an autoantigen that is an autoantigen listed in Table 1.

20. The genetically modified immune cell of claim 19, wherein the autoantigen is MOG.

21. 19. The genetically modified immune cell of claim 18, wherein the regulatory T cells or the cytotoxic T cells secrete anti-inflammatory cytokines comprising IL-35, IL-10, and / or TGF-beta.

22. The genetically modified immune cell of any one of claims 10 to 21 for use in treating an autoimmune disease in a subject.

23. 23. The genetically modified immune cell of claim 22, wherein the autoimmune disease is multiple sclerosis.

24. the subject is receiving a therapy comprising an antibody specific for CD52; or The subject is a human patient having or at risk of having multiple sclerosis, and the genetically modified immune cells are regulatory T cells or cytotoxic T cells according to any one of claims 16 to 21. The genetically modified immune cell of claim 23.

25. A genetically modified immune cell according to any one of claims 16 to 21 for use in treating an autoimmune disease in a subject, comprising: (A) the subject is a human patient with early stage MS, and the regulatory T cells or cytotoxic T cells express the MHC-based chimeric receptor and have the following genetic modifications: (i) PD-L1 and / or PD-1 knockout, (ii) surface expression of CCR6 and / or CXCR5; (iii) surface display of an antibody or antigen-binding fragment thereof specific for MOG; and (iv) Surface expression of a chimeric receptor targeting CD19 having one or more of: (B) the subject is a human patient with relapsing-remitting MS or early stage progressive, and the regulatory T cells or cytotoxic T cells express the MHC-based chimeric receptor and have the following modifications: (i) surface expression of a chimeric receptor that targets CD19, and (ii) Surface expression of CXCR5 having one or more of: (C) the subject is a human patient with relapsing-remitting MS or early stage progressive, and the regulatory T cells or cytotoxic T cells express the MHC-based chimeric receptor and have the following modifications: (i) surface display of an antibody or antigen-binding fragment thereof specific for MOG; and (ii) Surface expression of CCR6 or (D) the subject is a human patient with a chronic progressive form of MS, and the regulatory T cells or cytotoxic T cells express the MHC-based chimeric receptor and have the following modifications: (i) surface expression of a chimeric receptor that targets CS-1; and (ii) Surface expression of agents targeting CXCR4 and / or CXCR3 having one or more of: Genetically modified immune cells.

26. 22. The genetically modified immune cell of any one of claims 16 to 21 for use in treating an autoimmune disease in a subject, the genetically modified immune cell comprising both said regulatory T cells and said cytotoxic T cells.

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