MOG-specific chimeric antigen receptors and methods of use

Abstract

The present disclosure relates to chimeric antigen receptors reactive with myelin oligodendrocyte glycoprotein (MOG) and cells expressing the receptors. In particular, the present disclosure relates to regulatory T-cells expressing MOG-reactive chimeric antigen receptors and use thereof for treating neuroinflammation.

Description

MOG-SPECIFIC CHIMERIC ANTIGEN RECEPTORSAND METHODS OF USECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 709,918, filed October 21, 2024, which is incorporated herein by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (237752001840SEQLIST.xml; Size: 73,941 bytes; and Date of Creation: October 20, 2025) is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0003] The present disclosure relates to chimeric antigen receptors reactive with myelin oligodendrocyte glycoprotein (MOG) and cells, such as T-cells, expressing the receptors. In particular, the present disclosure relates to regulatory T-cells expressing MOG-reactive chimeric antigen receptors and use thereof for treating neuroinflammation.BACKGROUND OF THE INVENTION

[0004] Inflammation of the nervous system (neuroinflammation) may be initiated by a variety of conditions, such as infection, injury, toxicity, or autoimmunity. Although the central nervous system is protected in healthy individuals by the blood-brain barrier, it becomes more permeable when compromised. If left unchecked, neuroinflammation can result in the development of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS).

[0005] Demyelinating diseases are characterized by damage to the myelin sheath, which surrounds neurons. Damage to the myelin sheath results in impairment of transmission of electrical impulses along axons and can lead to diminished sensation, movement, and cognition. At this time, cures for demyelinating diseases have not been identified. Instead, treatments are typically focused on the management of symptoms and / or slowing the rate of demyelination.For instance, symptomatic attacks of multiple sclerosis are typically managed by administration of high doses of corticosteroids. While patients may experience short term relief from symptoms, corticosteroids are not beneficial in the long term and are associated with adverse side-effects, including an increase in vulnerability to infections.

[0006] Thus, what is needed in the art are specific therapies for neuroinflammation. In particular, therapies that are more effective and / or have fewer adverse side-effects are desirable. SUMMARY OF THE INVENTION

[0007] The present disclosure relates to chimeric antigen receptors reactive with myelin oligodendrocyte glycoprotein (MOG) and cells, such as T-cells, expressing the receptors. In particular, the present disclosure relates to regulatory T-cells expressing MOG-reactive chimeric antigen receptors and use thereof for treating neuroinflammation.BRIEF DESCRIPTION OF DRAWINGS

[0008] FIG. 1A-1B provide results from Jurkat NFAT firefly luciferase (ffLuc) reporter cell assays used to assess MOG-specific activation of Jurkat T-cells transduced with expression constructs encoding various MOG-reactive CARs and cultured with the indicated dilutions of MOG+ K562 / MOG- K562 target cells. The parental CAR comprising the anti-MOG375 antigenbinding domain (ABD) is referred to as “1413” and the CARS engineered from the parental anti-MOG375 ABD include: 1390, 1391, 1392, 1393 and 1399. FIG. 1A shows activation of anti-MOG CAR expressing T-cells by target cells expressing high levels of MOG. FIG. IB shows activation of anti-MOG CAR expressing T-cells by target cells expressing low amounts of MOG. UTD = untransduced.

[0009] FIG. 2A-2B provide results from Jurkat NFAT firefly luciferase (ffLuc) reporter cell assays used to assess MOG-specific activation of Jurkat T-cells transduced with expression constructs encoding various MOG-reactive CARs and cultured with the indicated dilutions of MOG+ K562 / MOG- K562 target cells. The parental CAR comprising the anti-MOG381 ABD is referred to herein as “1414” and the CARS engineered from the parental anti-MOG381 ABD include: 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409 and 1410. FIG.2A shows activation of anti-MOG CAR expressing T-cells by target cells expressing high levels of MOG. FIG.2B shows activation of anti-MOG CAR expressing T-cells by target cells expressing low amounts of MOG. UTD = untransduced.

[0010] FIG. 3A-3B show the percentages of primary Tregs positive for expression of a protein tag on day 14 after transduction with expression constructs encoding various MOG-reactive CARs upstream of a ribosome skipping peptide and the coding region of the protein tag. By virtue of this expression construct configuration, the percentage of primary Tregs expressing the tag is a measure of efficacy of anti-MOG CAR expression by the transduced cells. FIG.3A shows percentages of transduced Tregs that express anti-MOG CARs derived from the parental anti-MOG 375 ABD sequence. FIG.3B shows percentages of transduced Tregs that express anti-MOG CARs derived from the parental anti-MOG 381 ABD sequence.

[0011] FIG.4A-4D show the level of anti-MOG CAR-Treg activation in response to K562 target cells expressing low levels of MOG. Activation was measured as the percentage of cells (y-axis) that were CD71 -positive (CD71+) and CAR tag positive (Rat Tag+) across a series of target- to-effector cell ratios (x-axis). FIG. 4A and FIG. 4C show activation of anti-MOG Tregs derived from cells of human Donor 1, while FIG.4B and FIG.4D show activation of anti-MOG Tregs derived from cells of human Donor 2.DETAILED DESCRIPTION OF THE INVENTION

[0012] The present disclosure relates to chimeric antigen receptors reactive with myelin oligodendrocyte glycoprotein (MOG) and T-cells expressing the receptors. In particular, the present disclosure relates to regulatory T-cells expressing MOG-reactive chimeric antigen receptors and use thereof for treating neuroinflammation.Definitions

[0013] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0014] As used herein, the terms “antigen,” “immunogen,” and “antibody target,” refer to a molecule, compound, or complex that is recognized by an antibody, i.e., can be bound by the antibody. The term can refer to any molecule that can be recognized by an antibody, e.g., a polypeptide, polynucleotide, carbohydrate, lipid, chemical moiety, or combinations thereof (e.g., phosphorylated or glycosylated polypeptides, etc.). One of skill will understand that the term does not indicate that the molecule is immunogenic in every context, but simply indicates that it can be targeted by an antibody, or an antigen-binding domain derived therefrom.

[0015] As used herein, the term “epitope” refers to the localized site on an antigen that is recognized and bound by an antigen-binding domain of an antibody or fragment derived therefrom. Epitopes can include a few amino acids or portions of a few amino acids, e.g., 5 or 6, or more, e.g., 20 or more amino acids, or portions of those amino acids. In some cases, the epitope includes non-protein components, e.g., from a carbohydrate, nucleic acid, or lipid. In some cases, the epitope is a three-dimensional moiety. Thus, for example, where the target is a protein, the epitope can be comprised of consecutive amino acids, or amino acids from different parts of the protein that are brought into proximity by protein folding (e.g., a discontinuous epitope).

[0016] As used herein, the term “antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene, that specifically bind and recognize an antigen. Typically, the “variable region” contains the antigen-binding region of the antibody (or its functional equivalent) and is most critical in specificity and affinity of binding. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD).

[0017] Antibodies can be of (i) any of the five major classes of immunoglobulins, based on the identity of their heavy-chain constant domains - alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG) and mu (IgM), or (ii) subclasses (isotypes) thereof (E.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2). The light chains can be either lambda or kappa.

[0018] The following are a non-exhaustive list of different antibody forms, all retaining antigen binding activity:(1) whole immunoglobulins (also referred to as “intact” antibodies) (two light chains and two heavy chains, e.g., a tetramer);(2) an immunoglobulin polypeptide (a light chain or a heavy chain);(3) an antibody fragment, such as Fv (a monovalent or bi-valent variable region fragment, and can encompass only the variable regions (e.g., VL and / or VH), Fab (VLCL VHCH), F(ab’)2, Fv (VLVH), SCFV (single chain Fv) (a polypeptide comprising a VL and VH joined by a linker, e.g., a peptide linker), (scFv)2, sc(Fv)2, bispecific sc(Fv)2, bispecific (scFv)2, minibody (sc(FV)2 fused to CH3 domain), diabody (noncovalent dimer of single-chain Fv (scFv) fragment that consists of the heavy chain variable (VH) and light chain variable (VE) regions connected by a smallpeptide linker), triabody is trivalent sc(Fv)3 or trispecific sc(Fv)3;(4) a multivalent antibody (an antibody comprising binding regions that bind two different epitopes or proteins, e.g., “scorpion” antibody;(5) a fusion protein comprising a binding portion of an immunoglobulin fused to another amino acid sequence (such as a fluorescent protein); and(6) heavy chain only antibody or antibody fragment having only two heavy chains and lacking the two light chains usually found in antibodies.

[0019] The phrase “CDR sequence set” as used herein refers to the 3 heavy chain and / or 3 light chain CDRs of a particular antibody or antigen-binding domain. A “light chain” CDR sequence set refers to the light chain CDR sequences. A “heavy chain” CDR sequence set refers to the heavy chain CDR sequences. A “full” CDR sequence set refers to both heavy chain and light chain CDR sequences. CDRs are predicted based on IMGT sequence alignment.

[0020] As used herein, the term “humanized antigen-binding domain” refers to a chimeric antigen-binding domain in which the CDRs, obtained from the VH and VL regions of a non-human antibody having the desired specificity, affinity and capability are grafted to human framework regions. In one embodiment, the framework residues of the humanized antigenbinding domain are modified to refine and optimize the specificity, affinity and capability of the antigen-binding domain.

[0021] As used herein, the term “human antigen-binding domain” refers to an antigenbinding domain of an antibody produced by a human or an antibody having an amino acid sequence corresponding thereto.

[0022] As used herein, an antigen-binding domain “preferentially binds” binds a first antigen relative to a second antigen if it binds the first antigen with greater affinity than it does the second antigen. Preferential binding can be at least any of 2-fold, 5-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold or 1000-fold greater affinity.

[0023] As used herein, an antigen-binding domain “specifically binds” or is “specific for” a target antigen or target group of antigens if it binds the target antigen or each member of the target group of antigens with an affinity of at least any of IxlO-6M, IxlO-7M, IxlO-8M, IxlO-9M, IxlO-10M, IxlO-11M, IxlO-12M, and, for example, binds to the target antigen or each member of the target group of antigens with an affinity that is at least two-fold greater thanits affinity for non-target antigens to which it is being compared. Typically, specific binding is characterized by binding the antigen with sufficient affinity that the antigen-binding domain is useful as a diagnostic to detect the antigen or epitope and / or as a therapeutic agent in targeting the antigen or epitope.

[0024] As used herein, the term “polypeptide” refers to a molecule having a sequence of natural and / or unnatural amino acids connected through peptide bonds. The term “peptide” refers to a short polypeptide, typically no more than 30 amino acids long. The amino acid sequence of a polypeptide is referred to as its “primary structure.” The term “protein” refers to a polypeptide having a secondary, tertiary and / or quaternary structure, e.g., structures stabilized by hydrogen bonds, relationships between secondary structures and structures formed of more than one protein. Proteins can be further modified by other attached moieties such as carbohydrate (glycoproteins), lipids (lipoproteins) phosphate groups (phosphoproteins) and the like.

[0025] As used herein, an amino acid sequence “consists of’ only the amino acids in that sequence.

[0026] As used herein, a first amino acid sequence “consists essentially of’ a second amino acid sequence if the first amino acid sequence (1) comprises the second amino sequence and (2) is no more than 1 , no more than 2 or no more than 3 amino acids longer than the second amino acid sequence.

[0027] As used herein, a first amino acid sequence is a “fragment” of a second amino acid sequence if the second amino acid sequence comprises the first amino acid sequence. In certain embodiments, a first amino acid sequence that is a fragment of a second amino acid sequence may have no more than any of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 fewer amino acids than the second amino acid sequence.

[0028] As used herein, a “functional equivalent” of a reference amino acid sequence is a sequence that is not identical to the reference sequence, but that contains minor alterations such as, for example, insertion, deletion or substitution of one or a few amino acids. A functionally equivalent sequence retains the function (e.g., immunogenicity) of the reference sequence to which it is equivalent. If a functionally equivalent amino acid sequence contains substitution of one or more amino acids with respect to the reference sequence, these will generally be conservative amino acid substitutions.

[0029] As used herein, a “conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue without abolishing the protein’s desired properties. Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R-chain length for one another. See, e.g., Watson, et al., “Molecular Biology of the Gene,” 4thEdition, 1987, The Benjamin / Cummings Pub. Co., Menlo Park, CA, p. 224. Examples of conservative amino acid substitution include the following (Note, some categories are not mutually exclusive):Conservative SubstitutionsType of Amino Acid Substitutable Amino AcidsHydrophilic Ala, Pro, Gly, Glu, Asp, Gin, Asn, Ser, Thr Sulphydryl CysAliphatic (non-polar, hydrophobic) Ala, Vai, He, Leu, Met, Gly, ProBasic Lys, Arg, HisAromatic Phe, Tyr, Trp

[0030] As used herein, the term “substantially identical” refers to identity between a first amino acid sequence that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and / or common functional activity and / or common immunogenicity. For example, amino acid sequences that contain a common structural or antigenic domain having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are termed sufficiently or substantially identical. In the context of nucleotide sequence, the term “substantially identical” is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity, or encode polypeptides having the same immunogenic properties.

[0031] As used herein, a chemical entity, such as a polypeptide, is “substantially pure” or “isolated” if it is the predominant chemical entity of its kind (e.g., of polypeptides) in a composition. This includes the chemical entity representing more than 50%, more than 80%,more than 90%, more than 95%, more than 98%, more than 99%, more than 99.5%, more than 99.9%, or more than 99.99% of the chemical entities of its kind in the composition. A substantially purified fraction is a composition wherein the object species comprises at least about 50% (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition means that about 80% to 90% or more of the macromolecular species present in the composition is the purified species of interest. The object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) if the composition consists essentially of a single macromolecular species. Solvent species, small molecules, stabilizers (e.g., BSA), and elemental ion species are not considered macromolecular species for purposes of this definition.

[0032] The term “sequence identity” as used herein refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions / total number of positions.times.100%). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, 1990, modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215:403, 1990. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score-50,wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. Alternatively, PSLBLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSLBlast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CAB IOS 4:11-17, 1988. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0033] For antibodies and antigen-binding domains, percentage sequence identities can be determined when their sequences are maximally aligned by IMGT. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, multiplied by 100 to convert to percentage.

[0034] Percent amino acid sequence identity may also be determined using the sequence comparison program NCBLBLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997). The NCBLBLAST2 sequence comparison program may be obtained from the National Institute of Health, Bethesda, Md. NCBLBLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask=yes, strand=all, expected occurrences=10, minimum low complexity length= 15 / 5, multi-pass e-value=0.01, constant for multi-pass=25, dropoff for final gapped alignment=25 and scoring matrix=BLOSUM62.

[0035] In situations where NCBLBLAST2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, oragainst a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. The term “nucleic acid sequence” as used herein refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages and includes cDNA. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. It is understood that polynucleotides comprising non-transcribable nucleotide bases may be useful as probes in, for example, hybridization assays. The nucleic acid can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term "nucleic acid" includes the complementary nucleic acid sequences as well as codon optimized or synonymous codon equivalents.

[0036] The term "isolated nucleic acid" as used herein refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized. An isolated nucleic acid is also substantially free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) from which the nucleic acid is derived.

[0037] Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g., 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ionconcentration and temperature (Tm = 81.5°C - 16.6 (LoglO [Na+]) + 0.41(%(G+C) - 600 / 1), or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule a 1% mismatch may be assumed to result in about a 1°C decrease in Tm, for example, if nucleic acid molecules are sought that have a >95% identity, the final wash temperature will be reduced by about 5°C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions. In preferred embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5x sodium chloride / sodium citrate (SSC) / 5x Denhardt’s solution / 1.0% SDS at Tm - 5°C based on the above equation, followed by a wash of 0.2x SSC / 0.1% SDS at 60°C. Moderately stringent hybridization conditions include a washing step in 3x SSC at 42°C. It is understood, however, that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. Additional guidance regarding hybridization conditions may be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in: Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001.

[0038] As used herein, the term “expression construct” refers to a polynucleotide comprising an expression control sequence operatively linked with a heterologous nucleotide sequence (i.e., a sequence to which the expression control sequence is not normally connected to in nature) that is to be the subject of expression. As used herein, the term “expression vector” refers to a polynucleotide comprising an expression construct and sequences sufficient for replication in a host cell or insertion into a host chromosome. Plasmids and viruses are examples of expression vectors. As used herein, the term “expression control sequence” refers to a nucleotide sequence that regulates transcription and / or translation of a nucleotide sequence operatively linked thereto. Expression control sequences include promoters, enhancers, repressors (transcription regulatory sequences) and ribosome binding sites (translation regulatory sequences).

[0039] As used herein, a nucleotide sequence is “operatively linked” with an expression control sequence when the expression control sequence functions in a cell to regulate transcription of the nucleotide sequence. This includes promoting transcription of the nucleotide sequence through an interaction between a polymerase and a promoter.

[0040] The term "vector" as used herein comprises any intermediary vehicle for a nucleic acid molecule which enables said nucleic acid molecule, for example, to be introduced into prokaryotic and / or eukaryotic cells and / or integrated into a genome, and include plasmids, phagemids, bacteriophages or viral vectors such as retroviral based vectors, lentiviral vectors, Adeno Associated viral vectors and the like. The term "plasmid" as used herein generally refers to a construct of extrachromosomal genetic material, usually a circular DNA duplex, which can replicate independently of chromosomal DNA.

[0041] “Transfection” refers to the introduction of new genetic material into a cell. It includes transformation (the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane), transduction (the introduction of foreign DNA by a bacteriophage virus into a host cell) and conjugation.

[0042] As used herein, a “host cell” refers to a recombinant cell comprising an expression construct.

[0043] As used herein, the term “biological sample” refers to a sample containing cells (e.g., cells) or biological molecules derived from cells.

[0044] As used herein, the term terms “therapy,” “treatment,” “therapeutic intervention” and “amelioration” refer to any activity resulting in a reduction in the severity of symptoms. The terms “treat” and “prevent” are not intended to be absolute terms. Treatment and prevention can refer to any delay in onset, amelioration of symptoms, improvement in patient survival, increase in survival time or rate, etc. Treatment and prevention can be complete or partial. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment. In some aspects, the severity of disease is reduced by at least 10%, as compared, e.g., to the individual before administration or to a control individual not undergoing treatment. In some aspects, the severity of disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, no longer detectable using standard diagnostic techniques. "Treating" and "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. "Treating" and "treatment" as used herein also include prophylactic treatment.

[0045] Compositions or methods "comprising" or "including" one or more recited elements may include other elements not specifically recited (e.g., open-ended terms meaningincluding but not limited to). For example, a composition that "comprises" or "includes" an antibody may contain the antibody alone or in combination with other ingredients. In contrast, the phrase “consisting of’ is closed, indicating that such embodiments do not include additional elements. The term "consisting essentially of" refers to the inclusion of recited elements and other elements that do not materially affect the basic and novel characteristics of a claimed combination (e.g., partially closed term). It is understood that aspects and embodiments described herein as “comprising” include “consisting of’ and “consisting essentially of’ embodiments.

[0046] As used herein, the following meanings apply unless otherwise specified. The word “may” is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). The singular forms “a,” “an,” and “the” include plural referents. Thus, for example, reference to “an element” includes a combination of two or more elements, notwithstanding use of other terms and phrases for one or more elements, such as “one or more.”

[0047] The phrase “at least one” includes “one,” “one or more,” “one or a plurality” and “a plurality”. The term “or” is, unless indicated otherwise, non-exclusive, i.e., encompassing both “and” and “or.” The term “any of’ between a modifier and a sequence means that the modifier modifies each member of the sequence. So, for example, the phrase “at least any of 1, 2 or 3” means “at least 1, at least 2 or at least 3”. The term “plurality” in connection with a referent (e.g., object such as a T cell) refers to 3 or more, preferably 10 or more, preferably 100 or more, preferably 1,000 or more, preferably 10,000 or more, preferably 100,000 or more, preferably, 106or more, preferably 107or more, preferably 108or more, preferably 109or more, but not an infinite number of the referent (e.g., preferably less than 1012, preferably less than 1011, and preferably less than 1010).

[0048] The term “about” as used herein in reference to a value, encompasses from 90% to 110% of that value. For instance, the phrase about 20 residues (amino acids) in length to describe a polypeptide linker refers to a polypeptide linker of from 18 to 22 amino acids in length and includes a polypeptide linker of 20 amino acids in length.I. Chimeric Antigen Receptors (CARs)

[0049] “Chimeric antigen receptors” or “CARs” are engineered molecules comprising an optional signal peptide, a target antigen-binding domain, an optional hinge region, a transmembrane domain, an intracellular signaling domain and an optional co-stimulatory domain. CARs are based on the structure of T-cell receptors, which are expressed on T-cells, and are involved in the cell-mediated immune responses. The “target-binding domain” is also referred to herein as an “antigen-binding domain” or “antigen-recognition domain,” and as such the term “target” encompasses an “antigen.”

[0050] So-called “first-generation” CARs had a targeting domain and a CD32, signal transduction domain. So-called “second generation” CARs further included a co-stimulatory domain, such as a CD28 or a 4-1BB domain. So-called “third generation” CARs comprise multiple co-stimulatory domains. So-called “fourth generation” CARs, also referred to as “TRUCKS” are engineered to release a transgenic cytokine upon CAR signaling.

[0051] Chimeric antigen receptors (“CARs”) include the following elements: (1) an optional signal peptide, (2) a target antigen-binding domain, (3) a linker; (4) a transmembrane region; (5) an intracellular domain comprising a signal transduction domain. Optionally, the CAR can include a co-stimulatory (signal transduction) domain. That is, these optional elements can be included in addition to required elements. The target antigen-binding domain is heterologous to at least one of the other domains. That is, the antigen-binding domain does not naturally occur on a T-cell receptor or is not in the same protein as at least one of the other domains of the CAR.

[0052] The “signal peptide” guides the CAR polypeptide through the cell membrane. The “antigen-binding domain” provides binding specificity to the CAR. The antigen-binding domain can bind to a domain of an antibody that binds to the target antigen for a so-called “Universal CAR.” The linker, which may act as a “hinge region” is a flexible connector region, e.g., a natural or synthetic polypeptide, or any other type of molecule, providing structural flexibility and spacing to flanking polypeptide regions. The “transmembrane domain” is a membranespanning protein domain. The “intracellular signaling domain” transmits a signal through a signal transduction domain into the cell upon binding of an antigen by the antigen-binding domain. Such signaling activates an activity of the cell. “Co-stimulatory domains” are accessory signaling domains that further transmit signals.A. Signal Peptide

[0053] A signal peptide that may be present at the N-terminus of a nascent CAR of the present disclosure during expression in a cell can be a signal peptide of any human Type I transmembrane protein or any other signal peptide suitable for translocating the nascent CAR to the surface of a human cell. In some embodiments, the signal peptide is derived from a protein of the human immunoglobulin superfamily. In some embodiments, the signal peptide is derived from a CD4, CD8, CD19, CD28, TCR or immunoglobulin chain.

[0054] An exemplary signal peptide is the GMCSF signal peptide:MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO:1). Another exemplary signal peptide is the CD8alpha signal peptide: MALPVTALLLPLALLLHAARP (SEQ ID NO:2). In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO:1, or SEQ ID NO:2.B. Antigen-Binding Domains

[0055] The antigen-binding domain (ABD) of the CARs of the present disclosure binds to the extracellular domain of myelin oligodendrocyte glycoprotein (MOG). The MOG antigen is found on external lamellae of myelin sheaths in the central nervous system and on the surface of myelinating oligodendrocytes (Pham-Dinh et al., Proc Nat Acad Sci USA, 90:7990-7994, 1993).

[0056] The amino acid sequence of the human MOG isoform alphal precursor is set forth as NCBI Reference Sequence NP_996532, with the extracellular domain spanning residues 30-154, and the immunoglobulin (Ig)-like domain spanning residues 32-144. The amino acid sequence of human MOG is also set forth in UniProt No. Q16653 and the amino acid sequence of murine MOG is set forth as UniProt No. Q61885. In some embodiments, the ABD binds to the Ig-like domain of a human MOG isoform. In some embodiments, the ABD binds to the Ig-like domains of both human MOG and murine MOG.

[0057] In some embodiments, the antigen-binding domain (ABD) comprises a single chain variable fragment (scFV). In some embodiments, such as when the ABD is a scFV, the antigen-binding domain comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain of an antibody, wherein the VH domain comprises complementarityregion (CDR)-Hl, CDR-H2, and CDR-H3 and the VL domain comprises CDR-L1, CDR-L2, and CDR-L3. In some embodiments, the CDRs are as defined using Chothia nomenclature, which can easily be determined for a given sequence using online tools such as the abYsis annotate tool.

[0058] The CDRs and framework regions (FRs) of the heavy chain and light chain variable domains of the anti-MOG381 ABD are set forth in Table I and Table II below.Table I. anti-MOG381 HC SequenceRegion SEQ ID SequenceHFR1 NO:25 QVQLQESGGGLAKPGGSLRLTCAASCDR-H1 NO:26 GFTFSDYHFR2 NO:27 WMSWVRQAPGKGLEWVGEICDR-H2 NO:28 NPDGGRHFR3 NO:29 TNYKDFVKGRFTISRDNAKNTLYLQLNSLKTEDTAIYYCTGCDR-H3 NO:30 AGPTYYFDYHFR4 NO:31 WGQGTLVTVSSASTKNPDVFPLTTable II. anti-MOG381 LC SequenceRegion SEQ ID SequenceLFR1 NO:32 ELVMTQSPSSLFASIGDRVTITCCDR-L1 NO:33 RASQNIRSNLALFR2 NO:34 WYQQKPGKTPRLLIYCDR-L2 NO:35 DASSLX1X2 wherein XI is Q or E, and X2 is P, S or absentLFR3 NO:36 GIPSRFSGSGSGTYYTLTISSLQSDDLATYYCCDR-L3 NO:37 QQGYTTPVTLFR4 NO:38 FGQGTKLEIKRAVAAPSVFIFPPSEDQVKSGTA

[0059] The CDRs and framework regions (FRs) of the heavy chain and light chain variable domains of the anti-MOG375 ABD set forth in Table III and Table IV below.Table III. anti-MOG375 HC SequenceRegion SEQ ID SequenceHFR1 NO:39 QVQLVQSGAEVKKPGASVKVSCKASCDR-H1 NO:40 GYTFTSYHFR2 NO:41 GMQWVRQAPEQGLEWMGWICDR-H2 NO:42 NTNTGGHFR3 NO:43 TSYAQKFQGRVTMTRDASTSTAYMELSSLRPEDTAVYYCARCDR-H3 NO:44 DATRILADVLDYHFR4 NO:45 WGQGTLVTVSSASTKN PDVFTable IV. anti-MOG375 LC SequenceRegion SEQ ID SequenceLFR1 NO:46 E LTLTQSPVTLSLS P KETATLSCCDR-L1 NO:47 RASQSVRSYLALFR2 NO:48 WYQQKPGQAPRLLIYCDR-L2 NO:49 GASTRATLFR3 NQ:50 GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCCDR-L3 NO:51 QQYSSWYTLFR4 NO:52 FGQGTKLEIKRAVAAPSVFIFPPSEDQVKSGTAT

[0060] In some embodiments, the antigen-binding domain is a MOG-binding domain (anti-MOG domain) that comprises: (i) a heavy chain variable (VH) domain comprising the heavy chain CDRs of SEQ ID NO:4 and a light chain variable (VL) domain comprising the light chain CDRs of SEQ ID NO:5; or (ii) a heavy chain variable (VH) domain comprising the heavy chain CDRs of SEQ ID NO:7 and a light chain variable (VL) domain comprising the light chain CDRs of SEQ ID NO: 8. In exemplary embodiments, the antigen-binding domain is a MOG-binding domain that comprises a heavy chain variable (VH) domain comprising the heavy chain CDRs of SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:30, and a light chain variable (VL) domain comprising the light chain CDRs of SEQ ID NO:33, SEQ ID NO:35, and SEQ ID NO:37. In embodiments, the antigen-binding domain is a MOG-binding domain that comprises a heavy chain variable (VH) domain comprising the heavy chain CDRs of SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:30, and a light chain variable (VL) domain comprising the light chain CDRs of SEQ ID NO:33, SEQ ID NO:53 (DASSLQX3, wherein X3 is P or absent), and SEQ ID NO:37. In other exemplary embodiments, the antigen-binding domain is a MOG-binding domain that comprises a heavy chain variable (VH) domain comprising the heavy chain CDRs of SEQ ID NO:40, SEQ ID NO:42, and SEQ ID NO:44, and a light chain variable (VL) domain comprising the light chain CDRs of SEQ ID NO:47, SEQ ID NO:49, and SEQ ID NO:51.

[0061] In some embodiments, the MOG-binding domain comprises a single chain variable fragment (scFv), wherein:> (a) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:64 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:65; or> (b) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:66 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:67; or> (c) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:68 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:69; or> (d) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:70 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:71; or> (e) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:72 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:73; or> (f) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:74 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:75.In some embodiments, the MOG-binding domain comprises a single chain variable fragment (scFv), wherein the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:4 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:5. In some embodiments, the MOG-binding domain comprises a single chain variable fragment (scFv), wherein:> (a) the VH domain comprises the amino acid sequence of SEQ ID NO: 64 and the VL domain comprises the amino acid sequence of SEQ ID NO: 65; or> (b) the VH domain comprises the amino acid sequence of SEQ ID NO:66 and the VL domain comprises the amino acid sequence of SEQ ID NO: 67; or> (c) the VH domain comprises the amino acid sequence of SEQ ID NO: 68 and the VL domain comprises the amino acid sequence of SEQ ID NO: 69; or> (d) the VH domain comprises the amino acid sequence of SEQ ID NO:70 and the VL domain comprises the amino acid sequence of SEQ ID NO:71; or> (e) the VH domain comprises the amino acid sequence of SEQ ID NO:72 and the VL domain comprises the amino acid sequence of SEQ ID NO:73; or> (f) the VH domain comprises the amino acid sequence of SEQ ID NO:74 and the VL domain comprises the amino acid sequence of SEQ ID NO:75.In some embodiments, the MOG-binding domain comprises a single chain variable fragment (scFv), wherein the VH domain comprises the amino acid sequence of SEQ ID NO:4 and the VL domain comprises the amino acid sequence of SEQ ID NO:5.

[0062] In some embodiments, the MOG-binding domain comprises a single chain variable fragment (scFv), wherein the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 62 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:63, or wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 62 and the VL domain comprises the amino acid sequence of SEQ ID NO:63. In some embodiments, the MOG-binding domain comprises a single chain variable fragment (scFv), wherein the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:7 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 8, or wherein the VH domain comprises the amino acid sequence of SEQ ID NO:7 and the VL domain comprises the amino acid sequence of SEQ ID NO: 8.

[0063] The antigen-binding domains of the CARs of the present disclosure comprising a VH and a VL region, preferably are separated by a polypeptide linker. In some embodiments, the antigen-binding domain is arranged as VH-linker-VL. In other embodiments, the antigen-binding domain is arranged as VL-linker-VH. In some embodiments, the polypeptide linker is from about 4 to about 24 amino acids in length. In exemplary embodiments, the linker is a glycine linker comprising the amino acid sequence of: XaXbGGGGSGGGGSGGGGSXc (SEQ ID NO: 9), wherein Xa, Xb and Xc are independently selected from S and absent, for example:GGGGSGGGGSGGGGS (SEQ ID NO:54). In other exemplary embodiments, the linker is aWhitlow linker comprising the amino acid sequence of: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 10). In further exemplary embodiments, the linker is a ABpur linker comprising the amino acid sequence of: ASSGGSTSGSGKPGSGEGSSGSAR (SEQ ID NO: 11).

[0064] In some embodiments, the MOG-binding domain is a single chain variable fragment (scFv) comprising the amino acid sequence of one of the group consisting of SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61, or an amino acid sequence having at least 95% identity to one of the group consisting of SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61. In exemplary embodiments, the MOG-binding domain is a scFv comprising the amino acid sequence of SEQ ID NO:56, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:56. In other exemplary embodiments, the MOG-binding domain is a scFv comprising the amino acid sequence of SEQ ID NO:57, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO:57. In further exemplary embodiments, the MOG-binding domain is a scFv comprising the amino acid sequence of SEQ ID NO:58, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO:58. In additional exemplary embodiments, the MOG-binding domain is a scFv comprising the amino acid sequence of SEQ ID NO:59, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO:59. In some exemplary embodiments, the MOG-binding domain is a scFv comprising the amino acid sequence of SEQ ID NO:60, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO:60. In other exemplary embodiments, the MOG-binding domain is a scFv comprising the amino acid sequence of SEQ ID NO:61, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO:61. In still further exemplary embodiments, the MOG-binding domain is a scFv comprising the amino acid sequence of SEQ ID NO:61, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO:61. In other exemplary embodiments, the MOG-binding domain is a scFv comprising the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO:3.

[0065] In some embodiments, the MOG-binding domain is a single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO:55, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:55. In other exemplary embodiments, the MOG-binding domain is a scFv comprising the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity of SEQ ID NO:6.C. Hinge Region

[0066] In exemplary embodiments, the antigen-binding domain and the transmembrane domain of the CARs of the present disclosure are separated by a hinge region. The “hinge region” is from about 10 to about 20, 30, 40, or 50 amino acids in length. In some embodiments, the hinge region is a CD8a hinge region, a CD28 hinge region or an IgG4 hinge region. In some embodiments, the hinge region is a CD28 hinge region.

[0067] In some embodiments, the hinge region is a CD8a hinge region. In exemplary embodiments, the CD8a hinge region comprises the amino acid sequence of:TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 13), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO: 13.

[0068] In some embodiments, the hinge region is a CD28 hinge region. In exemplary embodiments, the CD28 hinge region comprises the amino acid sequence of: ffiVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 14), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO: 14.

[0069] In some embodiments, the hinge region is an IgG4 hinge region. In exemplary embodiments, the IgG4 hinge region comprises the amino acid sequence of: ESKYGPPCPPCP (SEQ ID NO: 15), or an amino acid sequence differing by one or two amino acids from SEQ ID NO:15.D. Transmembrane Domain

[0070] The hinge region of the CARs of the present disclosure is separated from the intracellular signaling domain by a transmembrane domain of from about 20 to about 30 amino acids in length.

[0071] In some embodiments, the transmembrane domain is a CD8a transmembrane domain. In exemplary embodiments, the CD8a domain comprises the amino acid sequence of: IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 16), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO: 16.

[0072] In some embodiments, the transmembrane domain is a CD28 transmembrane domain. In exemplary embodiments, theCD28 transmembrane domain is a wild type CD28 transmembrane domain, which comprises the amino acid sequence of:FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 17). In some embodiments the CD28 transmembrane domain comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO: 17.

[0073] In some embodiments, the transmembrane domain is a modified CD28 transmembrane domain. In exemplary embodiments, the amino acid sequence of the modified CD28 transmembrane domain comprises an insertion, substitution, and / or deletion relative to SEQ ID NO: 17. In some embodiments, the amino acid sequence of the CD28 transmembrane domain comprises at least one substitution selected from the group consisting of: C165L, Y166L, S167L, T171L (which correspond to C13L, Y14L, S15L, and T19L substitutions of the wildtype CD28 TM sequence of SEQ ID NO: 17). In some embodiments, the modified CD28 transmembrane domain comprises the amino acid sequence of:FWVLVVVGGVLALLLLLVLVAFIIFWV (SEQ ID NO: 18). In some embodiments, the CD28 TM domain comprises the consensus amino acid sequence of:FWVLVVVGGVLAX1X2X3LLVX4VAFIIFWV (SEQ ID NO: 19), wherein Xi is C or L, X2is Y or L, X3 is S or L, and X4 is T or L, and / or wherein at least one of Xi, X2, X3, and X4 is L.E. Intracellular Signaling Domain

[0074] The intracellular signaling domain of the CARs of the present disclosure comprises a CD3zeta signal transduction domain. In some preferred embodiments, the intracellular signaling domain further comprises a co-stimulatory domain.

[0075] In some embodiments, the co-stimulatory domain can be derived from, for example, CD28, 4-1BB, CD2, CD27, CD30, 0X40, CD40, PD-1, PD-L1, PD-L2, ICOS, LFA-1, CD7, LIGHT, NKG2C, B7-H3, CD83L, B7-1 (CD80), B7-2 (CD86), B7-H3, B7-H4 and others. In some embodiments, the CARs of the present disclosure comprise two or more co-stimulatory signaling domains (e.g., CD28 and 4-1BB).

[0076] In some embodiments, the co-stimulatory domain is a CD28 co-stimulatory domain. In exemplary embodiments, the CD28 co-stimulatory domain comprises the amino acid sequence of: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:20), or an amino acid having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:20.

[0077] In some embodiments, the co-stimulatory domain is a 4-1BB co-stimulatory domain. In exemplary embodiments, the 4- IBB co-stimulatory domain comprises the amino acid sequence of: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:21), or an amino acid having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:21.

[0078] In exemplary embodiments, the CD3^ signal transduction domain comprises the amino acid sequence of:RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:22), or an amino acid having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:22.

[0079] In some embodiments, the intracellular signaling domain comprises a CD28 co-stimulatory domain and a CD3^ signal transduction domain. In exemplary embodiments, the intracellular signaling domain comprise the amino acid sequence of SEQ ID NO:23 or an amino acid having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID

[0080] In some embodiments, the intracellular signaling domain comprises a 4- IBB costimulatory domain and a CD3^ signal transduction domain. In exemplary embodiments, the intracellular signaling domain comprise the amino acid sequence of SEQ ID NO:24 or an amino acid having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID NO:24.II. Nucleic Acids Encoding CARs

[0081] Nucleic acid molecules (polynucleotides) encoding CARs of the present disclosure can be isolated molecules or can be included within a cassette or a vector.A. Nucleic Acids

[0082] The present disclosure provides nucleic acid molecules (polynucleotides) comprising a nucleotide sequence that encodes (e.g., comprises the coding region of) a CAR as described herein. The nucleic acid can be in the form of DNA or in the form of RNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or singlestranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand. In some embodiments, the polynucleotides as described herein are isolated.

[0083] For example, nucleic acids can comprise nucleotide sequences that encode the CAR polypeptides described herein or a portion thereof. In some embodiments, the nucleotide sequences encode a polypeptide comprising the amino acid sequence of a specific SEQ ID NO, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity with a specific SEQ ID NO.

[0084] In some embodiments, the polynucleotides are variants that comprise alterations in the coding regions, non-coding regions, or both. In some embodiments, the polynucleotide variants contain alterations that are silent. In other embodiments, the polynucleotide variants comprise substitutions, additions, or deletions, but do not alter the binding properties of the antigen-binding domain of the encoded CAR polypeptide. In some embodiments, the polynucleotide variants contain one or more alterations that do not produce any changes in the amino acid sequence of the CAR. In some embodiments, polynucleotide variants contain “silent” substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host cell.B. Expression Cassettes and Vectors

[0085] The present disclosure further provides expression cassettes and vectors comprising a nucleic acid molecule encoding a CAR described herein. In some embodiments, the nucleic acid molecule can further comprise an expression control sequence operatively linked with the nucleotide sequence encoding the CAR. In some embodiments, the nucleic acid molecule encoding the disclosed CAR can be inserted into an expression vector in operable combination with an expression control sequence appropriate for expression of the disclosed CAR in a desired host cell. Correct assembly can be confirmed by nucleotide sequencing, restriction mapping, and / or expression of the CAR polypeptide in a suitable host cell.

[0086] For example, a polynucleotide can include one or more transcription regulatory elements, such as promoters or enhancers, which, when the polynucleotide is present in a cell, cause the sequence encoding the CAR to be expressed within the cell.

[0087] Nucleic acids disclosed herein can be incorporated into vectors that can be introduced into a host cell. Such vectors include, without limitation, viral vectors and plasmids. Exemplary viral vectors include but are not limited to retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses. Lentiviruses are particularly desirable as they are able to deliver a large amount of genetic material into the genome of a host cell and because they are able to infect non-dividing cells.III. Host Cells

[0088] The present disclosure also provides host cells (e.g., recombinant cells) comprising a nucleic acid molecule, an expression cassette, or an expression vector encoding a CAR described herein. In some embodiments, the host cell is a eukaryotic cell, such as a mammalian cell. In other embodiments, the host cell is a prokaryotic cell, such as a bacterial cell.

[0089] The nucleic acid molecule encoding the disclosed CAR can be delivered to a host cell by transformation, transfection or transduction as is known to one of skill in the art. The resulting recombinant (host) cell can be an immune cell, including but not limited to a T-cell, such as a CD4+ T-cell, CD8alpha+ T-cell, or CD8beta+ T cell. In some embodiments, the T-cell is a Treg cell. In some embodiments, the T-cell is a T helper (Th) cell. In some embodiments, the T-cell is a cytolytic T lymphocyte (CTL). In some embodiments, the recombinant (host) cellcomprising the nucleic acid molecule encoding the disclosed CAR is a lymphocyte (e.g., T-cell, B-cell or NK-cell).

[0090] In some embodiments, the cells expressing the CARs of this disclosure are Treg cells. “Regulatory T cells,” or “Treg cells,” are cells belonging to a specialized subpopulation of T cells that act to suppress immune response, thereby maintaining homeostasis and selftolerance. Tregs are able to inhibit T cell proliferation and cytokine production and play a critical role in preventing autoimmunity. Tregs are characterized by expression of FoxP3.Surface markers for Tregs include CD4, CD25high (high molecular density) and CD1271ow (low molecular density). Mouse and human Tregs express GITR / ATTR, and CTLA-4. Human CD4+FoxP3+ Treg cells can be divided into three sub-populations:(1) CD45RA+CD25+FoxP31ow resting Treg cells,(2) CD45RO+CD25highFoxP3high activated Treg cells, and(3) proinflammatory cytokine-producing CD45RO+CD25+FoxP31ow non-suppressive effector T cells (Teffs).

[0091] In some embodiments, the host cell is an autologous cell (e.g., recombinant progeny of a cell obtained from the intended recipient). That is, the cells to be transformed with the nucleic acids disclosed herein can be cells taken from a subject into whom the recombinant cells are to be administered. In this way, issues of an allogeneic immune response can be mitigated. Even so, in other embodiments, the host cell is an allogeneic cell (e.g., recombinant progeny of a cell obtained from an immunologically distinct individual than the intended recipient). Regardless of their origin, host cells can be expanded ex vivo before administration to a subject.

[0092] Also, the proteins produced by a transformed / recombinant host can be purified according to any suitable method. Such methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags such as hexa-histidine, maltose binding domain, influenza coat sequence and glutathione- S -transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column. In some embodiments, proteins can also be physically characterized using such techniques as proteolysis, high performance liquid chromatography, nuclear magnetic resonance and x-ray crystallography.IV. Compositions

[0093] Also disclosed are pharmaceutical compositions comprising a host cell (e.g., recombinant cell) comprising a nucleic acid molecule encoding and expressing the disclosed CAR polypeptide, and a pharmaceutically acceptable excipient, as well as methods of use in the treatment of a disease or disorder.

[0094] As used herein, the term “pharmaceutical composition” refers to a composition comprising a pharmaceutical agent (e.g., a drug or a recombinant Treg cell as described herein) and a pharmaceutically acceptable excipient.

[0095] As used herein, the term “pharmaceutically acceptable” refers to a compound that is compatible with the other ingredients of a pharmaceutical composition and can be safely administered to a subject. The term is used synonymously with “physiologically acceptable” and “pharmacologically acceptable.” Pharmaceutical compositions and techniques for their preparation and use are known to those of skill in the art in light of the present disclosure. For a detailed listing of suitable pharmacological compositions and techniques for their administration one may refer to texts such as Remington's Pharmaceutical Sciences, 17th ed. 1985; Brunton et al., “Goodman and Gilman’s The Pharmacological Basis of Therapeutics,” McGraw-Hill, 2005; University of the Sciences in Philadelphia (eds.), “Remington: The Science and Practice of Pharmacy,” Lippincott Williams & Wilkins, 2005; and University of the Sciences in Philadelphia (eds.), “Remington: The Principles of Pharmacy Practice,” Lippincott Williams & Wilkins, 2008.

[0096] Pharmaceutical compositions will generally be sterile, at least for human use. A pharmaceutical composition will generally comprise pharmaceutically acceptable excipients for buffering and preservation in storage, and can include buffers for appropriate delivery, depending on the route of administration. Examples of pharmaceutically acceptable excipients include, without limitation, normal (0.9%) saline, phosphate-buffered saline (PBS) Hank’s balanced salt solution (HBSS) and multiple electrolyte solutions.

[0097] Pharmaceutical compositions can be formulated for any route of administration. However, in most embodiments of the present disclosure, the pharmaceutical compositions are formulated for parenteral (e.g., subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, or intracerebroventricular injection, either bolus or infusion) administration.

[0098] Injectable pharmaceutical compositions can comprise a solution of the pharmaceutical agent suspended in a pharmaceutically acceptable excipient, such as an aqueous excipient. Any of a variety of aqueous excipients can be used, e.g., water, buffered water, 0.4% saline, 0.9% isotonic saline, 0.3% glycine, 5% dextrose, and the like, and may include glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. Often, normal buffered saline (135-150 rnM NaCl) will be used. The pharmaceutically acceptable excipients can comprise auxiliary substances to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. In some embodiments, the composition can be formulated for intravenous administration. In some embodiments, the composition can be formulated for intrathecal or intracerebroventricular administration.

[0099] Pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the compositions isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Injection solutions and suspensions can also be prepared from sterile powders, granules, and tablets. In the practice of the present invention, pharmaceutical compositions can be administered, for example, by intravenous, intrathecal or intracerebroventricular infusion. The pharmaceutical compositions can be presented in unit-dose or multi-dose sealed containers.

[0100] Host cells can be cryopreserved. Cryopreservation can include formulating host cells with a cryopreservation agent, such as DMSO. Commercially available media include, for example, CryoStor® and pZerve®, available from Millipore Sigma.

[0101] The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of the pharmaceutical agent given to an individual at each administration. The dose will vary depending on a number of factors, including frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; the route of administration; and the imaging modality of the detectable label (if present). One of skill in the art will recognize that the dose can be modified depending on the above factors or based on therapeutic progress.

[0102] The pharmaceutical composition can be packaged or prepared in unit dosage form. In such form, the pharmaceutical composition is subdivided into unit doses containing appropriate quantities of the pharmaceutical agent, e.g., according to the dose of the pharmaceutical agent or the concentration of the pharmaceutical agent in the pharmaceutical composition. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the pharmaceutical composition. The pharmaceutical composition can, if desired, also contain other compatible therapeutic agents.V. Methods of Use

[0103] Host cells (e.g., recombinant T cells) that express the anti-MOG CARs disclosed herein are useful in the treatment of neuroinflammation. Methods of use comprise administering an effective amount of a pharmaceutical composition of this disclosure comprising anti-MOG CAR-expressing cells, such as CAR-T cells, to a subject in need thereof (e.g., an individual suffering from neuroinflammation). In some methods of the present disclosure, the neuroinflammation is an inflammatory disorder or an autoimmune disease. In some methods of the present disclosure in which the subject has an autoimmune disease or inflammatory disorder, the host cells comprise Treg cells expressing a CAR that binds an antigen associated with the autoimmune disorder or the inflammatory disorder. For instance, in some embodiments, the neuroinflammation is a MOG-associated disease or disorder. In some embodiments, the neuroinflammation is demyelinating disease. In some embodiments, the demyelinating disease is amyotrophic lateral sclerosis (ALS). In other embodiments, the demyelinating disease is multiple sclerosis (MS). In some embodiments, the neuroinflammation comprises neurodegeneration. In some embodiments, the host cells are autologous to the subject.

[0104] As used herein, the term “subject” refers to an individual animal. The term “patient” as used herein refers to a subject under the care or supervision of a health care provider such as a doctor or nurse. Subjects include mammals, such as humans and non-human primates, such as monkeys, as well as dogs, cats, horses, bovines, rabbits, rats, mice, goats, pigs, and other mammalian species. Subjects can also include avians. In some embodiments, the subject is a human patient. A patient can be an individual that is seeking treatment, monitoring, adjustment or modification of an existing therapeutic regimen, etc. Subjects with a disease or disorder can include individuals that have not received treatment, are currently receiving treatment, have had treatment, and those that have discontinued treatment.

[0105] As used herein, the terms “effective amount,” “effective dose,” and “therapeutically effective amount,” refer to an amount of a pharmaceutical agent that is sufficient to generate a desired response, such as reduce or eliminate a sign or symptom of a condition or ameliorate a disorder. In some examples, an “effective amount” is one that treats (including prophylaxis) one or more symptoms and / or underlying causes of any of a disorder or disease and / or prevents progression of a disease. For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of therapeutic effect at least any of 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least any of a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.

[0106] The pharmaceutical composition can be administered by any suitable route, including but not limited to intravenous, intracranially, intrathecally, intracerebroventricularly, subcutaneous, intramuscular or intraperitoneal routes. An example of administration of a pharmaceutical composition includes storing the composition at 10 mg / ml in sterile isotonic aqueous saline solution for injection at 4°C, and diluting it in either 100 ml or 200 ml 0.9% sodium chloride for injection prior to administration to the patient. The pharmaceutical composition is administered by intravenous infusion over the course of 1 hour at a dose of between 0.2 and 10 mg / kg. In other embodiments, the pharmaceutical composition is administered by intravenous infusion over a period of between 15 minutes and 2 hours. In still other embodiments, the administration procedure is via sub-cutaneous bolus injection.

[0107] The dose of the pharmaceutical composition is chosen in order to provide effective therapy for the patient and is in the range of less than 0.1 mg / kg body weight to about 25 mg / kg body weight or in the range 1 mg- 2 g per patient. In some cases, the dose is in the range 1- 100 mg / kg, or approximately 50 mg- 8000 mg / patient. The dose may be repeated at an appropriate frequency which may be in the range once per day to once every three months, depending on the pharmacokinetics of the composition (e.g., half-life of the composition in the circulation) and the pharmacodynamic response (e.g., the duration of the therapeutic effect of the composition). In some embodiments, the in vivo half-life of between about 7 and about 25 days and composition dosing is repeated between once per week and once every 3 months.

[0108] Administration can be periodic. Depending on the route of administration, the dose can be administered, e.g., once every 1, 3, 5, 7, 10, 14, 21, or 28 days or longer (e.g., onceevery 2, 3, 4, or 6 months). In some cases, administration is more frequent, e.g., 2 or 3 times per day. The patient can be monitored to adjust the dosage and frequency of administration depending on therapeutic progress and any adverse side effects, as will be recognized by one of skill in the art.

[0109] Thus, in some embodiments, additional administration is dependent on patient progress, e.g., the patient is monitored between administrations. For example, after the first administration or round of administrations, the patient can be monitored for rate of symptom relief.VI. Kits

[0110] As used herein, the term “kit” refers to a collection of items intended for use together. In some embodiments, the kit comprises an agent and instructions for use thereof. In some embodiments, the kit further comprises a container, such as a vial that contains a composition as disclosed herein. For instance, a kit can include a container, such as a bag or bottle for intravenous, intrathecal, or intracerebroventricular administration, comprising a pharmaceutical composition comprising a plurality of recombinant cells that express a CAR of the present disclosure. In some embodiments, the kit can further comprise a fluidic conduit, such as a plastic tube, with a drip chamber. The drip chamber can communicate through a fluidic conduit with an intravenous needle. The fluidic conduit also can comprise one or more Y-sites and a roller clamp.VII. Enumerated Embodiments1. A chimeric antigen receptor (CAR) comprising an antigen-binding domain, a hinge domain, a transmembrane domain, one or more co-stimulatory domains, and an intracellular signaling domain, wherein the antigen-binding domain specifically binds to extracellular domain of myelin oligodendrocyte glycoprotein (MOG) and comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain and the VL domain comprise complementarity-determining regions (CDRs) from:(a) the VH domain of SEQ ID NO:4 and the VL domain of SEQ ID NO:5; or(b) the VH domain of SEQ ID NO:7 and the VL domain of SEQ ID NO: 8.2. The CAR of embodiment 1, wherein the antigen-binding domain binds to the extracellular domain of human MOG and the extracellular domain of mouse MOG.3. The CAR of embodiment 1 or embodiment 2, wherein:(i) the VH domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:26, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:28, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 30, and(ii) the VL domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:33, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:35, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:37.4. The CAR of embodiment 3, wherein the antigen-binding domain comprises a single chain variable fragment (scFv), and wherein:> (a) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:64 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:65; or> (b) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:66 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:67; or> (c) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:68 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:69; or> (d) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:70 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:71; or> (e) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:72 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:73; or> (f) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:74 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:75.5. The CAR of embodiment 3, wherein the antigen-binding domain comprises a single chain variable fragment (scFv), and wherein:> (a) the VH domain comprises the amino acid sequence of SEQ ID NO: 64 and the VL domain comprises the amino acid sequence of SEQ ID NO: 65; or> (b) the VH domain comprises the amino acid sequence of SEQ ID NO:66 and the VL domain comprises the amino acid sequence of SEQ ID NO: 67; or> (c) the VH domain comprises the amino acid sequence of SEQ ID NO: 68 and the VL domain comprises the amino acid sequence of SEQ ID NO: 69; or> (d) the VH domain comprises the amino acid sequence of SEQ ID NO:70 and the VL domain comprises the amino acid sequence of SEQ ID NO:71; or> (e) the VH domain comprises the amino acid sequence of SEQ ID NO:72 and the VL domain comprises the amino acid sequence of SEQ ID NO:73; or> (f) the VH domain comprises the amino acid sequence of SEQ ID NO:74 and the VL domain comprises the amino acid sequence of SEQ ID NO:75.6. The CAR of embodiment 1 or embodiment 2, wherein:(i) the VH domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:40, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:42, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:44, and(ii) the VL domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:47, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:49, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:51.7. The CAR of embodiment 6, wherein the antigen-binding domain comprises a single chain variable fragment (scFv), and wherein the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 62 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 63.8. The CAR of embodiment 6, wherein the antigen-binding domain comprises a single chain variable fragment (scFv), and wherein the VH domain comprises the amino acid sequence of SEQ ID NO:62 and the VL domain comprises the amino acid sequence of SEQ ID NO:63.9. The CAR of any one of embodiments 3-8, wherein the VH domain and the VL domain are joined by a linker comprising the amino acid sequence of SEQ ID NOV, SEQ ID NOTO, or SEQ ID NO:11.10. The CAR of embodiment 4, wherein the scFv comprises the amino acid sequence of one of the group consisting of SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ IDNO:59, SEQ ID NO:60 and SEQ ID NO:61, or an amino acid sequence having at least 95% identity to one of the group consisting of SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61.11. The CAR of embodiment 7, wherein the scFv comprises the amino acid sequence of SEQ ID NO:55 or an amino acid sequence having at least 95% identity to SEQ ID NO:55.12. The CAR of any one of embodiments 1-11, wherein the intracellular signaling domain comprises a CD3-zeta signal transduction domain.13. The CAR of embodiment 12, wherein the CD3-zeta signal transduction domain comprises the amino acid sequence of SEQ ID NO:22.14. The CAR of embodiment 12 or embodiment 13, wherein the one or more costimulatory domains comprise a CD28 co-stimulatory domain or a 4- IBB co-stimulatory domain.15. The CAR of embodiment 14, wherein the co-stimulatory domain comprises a CD28 co-stimulatory domain.16. The CAR of embodiment 15, wherein the CD28 co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:20.17. The CAR of embodiment 16, wherein an intracellular domain comprising the one or more co-stimulatory domains and the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:23.18. The CAR of any one of embodiments 1-17, wherein the transmembrane domain is a CD8 transmembrane domain or a CD28 transmembrane domain.19. The CAR of embodiment 18, wherein the transmembrane domain is a CD 8 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19.20. The CAR of any one of embodiments 1-19, wherein the hinge domain is a CD8 hinge domain or a CD28 hinge domain.21. The CAR of embodiment 20, wherein the hinge domain is a CD28 hinge domain comprising the amino acid sequence of SEQ ID NO: 14.22. The CAR of any one of embodiments 1-21, further comprising a signal peptide.23. The CAR of embodiment 22, wherein the signal peptide is a granulocytemacrophage colony-stimulating factor receptor alpha (GMCSFRa) signal peptide or a CD8-alpha (CD8a) signal peptide.24. The CAR of embodiment 23, wherein the signal peptide is a GMCSFRa signal peptide comprising the amino acid sequence if SEQ ID NO:1.25. A nucleic acid encoding the CAR of any one of embodiments 1-24.26. An expression vector comprising the nucleic acid of embodiment 25 in operable combination with an expression control sequence.27. The expression vector of embodiment 26, wherein the vector is a viral vector, optionally wherein the viral vector is a lentiviral vector a retroviral vector, an adenoviral vector, or an adeno-associated viral vector.28. The expression vector of embodiment 27, wherein the viral vector is a lentiviral vector.29. The expression vector of embodiment 26, wherein the vector is a plasmid.30. A host cell comprising the expression vector of any one of embodiments 26-29.31. A modified immune cell that has been engineered to express the CAR of any one of embodiments 1-24, wherein the modified immune cell is a modified mammalian T-cell, NK-cell or NKT-cell, optionally wherein the modified immune cell expresses a constitutively- active IL-2 receptor.32. The modified immune cell of embodiment 31 , wherein the modified immune cell is a human T-cell.33. The modified immune cell of embodiment 32, wherein the human T-cell is a regulatory T-(Treg) cell.34. The modified immune cell of embodiment 33, wherein the human Treg cell is CD4+, CD25+ and CD1271O.35. The modified immune cell of embodiment 34, wherein the human Treg cell is FOXP3+ and HELIOS+.36. A pharmaceutical composition comprising a plurality of the modified immune cells of any one of embodiments 31-35, and a pharmaceutically acceptable excipient.37. A method of treating a human subject suffering from neuroinflammation, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 36.38. The method of embodiment 37, wherein the neuroinflammation is an inflammatory disorder or an autoimmune disease.39. The method of embodiment 37, wherein the neuroinflammation is a MOG-associated disease or disorder.40. The method of embodiment 37, wherein the neuroinflammation is a demyelinating disease.41. The method of embodiment 40, wherein the demyelinating disease is amyotrophic lateral sclerosis (ALS) or multiple sclerosis (MS).42. The method of any one of embodiments 37-41, wherein the pharmaceutical composition is administered intravenously.43. A method of treating a human subject suffering from neuroinflammation, the method comprising:(a) isolating T-cells from a biological sample obtained from the subject;(b) enriching the T-cells for T regulatory (Treg) cells;(c) transfecting the enriched Treg cells with an expression vector encoding the CAR of any one of embodiments 1-24 to produce CAR-Treg cells;(d) expanding the CAR-Treg cells to produce a plurality of CAR-Treg cells; and(e) administering the plurality of CAR-Treg cells to the subject.44. The method of embodiment 43, wherein the expansion comprises using anti- CD3 / CD28 coated beads.45. The method of embodiment 43, wherein the expansion does not comprise using anti-CD3 / CD28 coated beads.46. A kit comprising a container containing the pharmaceutical composition of embodiment 36, communicating through a fluidic conduit with a drip chamber, wherein the drip chamber communicates through a fluidic conduit with an intravenous needle.47. The kit of embodiment 46, wherein the container comprises a bag.48. The kit of embodiment 46 or embodiment 47, wherein the fluidic conduit between the container and the needle comprises one or more Y-sites and a roller clamp.49. A method of preparing T-regulatory (Treg) cells expressing a chimeric antigen receptor (CAR), the method comprising:(a) isolating T-cells from a biological sample obtained from a human subject;(b) enriching the T-cells for T regulatory (Treg) cells;(c) transfecting the enriched Treg cells with an expression vector encoding the CAR of any one of embodiments 1-24 to produce CAR-Treg cells; and(d) expanding the CAR-Treg cells to produce a plurality of CAR-Treg cells.50. The method of embodiment 49, wherein the expansion comprises using anti-CD3 / CD28 coated beads.51. The method of embodiment 49, wherein the expansion does not comprise using anti-CD3 / CD28 coated beads.52. The method of any one of embodiment 49-51, wherein the transfection occurs by use of a viral vector, electroporation, heat shock, bacteriophage, sonication, or calcium phosphate.53. The method of any one of embodiments 49-51, wherein the transfection occurs by use of a viral vector.54. A pharmaceutical composition comprising the plurality of the CAR-Treg cells produced by the method of any one of embodiments 49-53, and a pharmaceutically acceptable excipient.55. A method of treating neuroinflammation, the method comprising: administering an effective amount of the pharmaceutical composition of embodiment 54 to a human subject suffering from the neuroinflammation.56. The pharmaceutical composition of embodiment 36 or embodiment 54 for use as a medicament.57. The pharmaceutical composition of embodiment 36 or embodiment 54 for use in a method of treating a neuroinflammation, comprising administering an effective amount of the composition to an individual in need thereof to treat the neuroinflammation, optionally wherein the individual is a human subject.58. Use of a plurality of modified regulatory T (Treg) cells in the manufacture of a medicament for treating a subject suffering from neuroinflammation, wherein the modified Treg cells express the CAR of any one of embodiments 1-24, and wherein the modified Treg cells are CD4+, CD25+ and CD1271O, optionally wherein the neuroinflammation is a demyelinating disease, optionally wherein the demyelinating disease is amyotrophic lateral sclerosis (ALS) or multiple sclerosis (MS).EXAMPLES

[0111] Abbreviations: Ab (antibody); ABD (antigen-binding domain); CAR (chimeric antigen receptor); EGFR (epidermal growth factor receptor); EGFRt (EGFR tag); extracellular domain (EC or ECD); FACS (fluorescence activated cell sorting); ffLuc (firefly luciferase); IL-2 (interleukin 2); MOG (myelin oligodendrocyte glycoprotein); MOI (multiplicity of infection); NFAT (nuclear factor of activated T-cells); Prom, (promoter); scFv (single-chain variable fragment); SR (serum replacement); TM (transmembrane domain); Treg (regulatory T-cell); and WT (wild-type).

[0112] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention, which is defined by the claims.Example 1: Expression of Anti-MOG CARs in Jurkat T-Cells

[0113] In order to provide a source of antigen to test activation of T-cells via anti-MOG CARs, several expression constructs were made and transfected into K562 cells (human myelogenous leukemia cell line). In brief, the expression constructs comprised a coding region of human MOG or murine MOG in operably combination with one of three different promoters: MND (strong promoter), EFla (medium promoter), or UBC (weak promoter). Levels of expression of MOG on transfected K562 cells was assessed by flow cytometry with a fluorochrome-labelled anti-MOG antibody. As expected, levels of MOG expression were greatest when driven by the MND promoter, and least when driven by the UBC promoter, with intermediate levels observed when driven by the EFla promoter.

[0114] Anti-MOG chimeric antigen receptors (CARs) were generated and introduced into a plasmid backbone. Lentivirus for CAR plasmids was then made and titered. A plurality of different anti-MOG CARs were engineered. A first series of CARs was engineered from a parent anti-MOG375 antigen binding-domain comprising the amino acid sequence of SEQ ID NO:6. The parental CAR comprising the anti-MOG375 ABD is referred to herein as “1413”. The CARS engineered from the parental anti-MOG375 ABD include: 1390, 1391, 1392, 1393 and 1399. The amino acid sequence of single chain variable fragment (scFv) comprising the ABD of the 1399 CAR is set forth as SEQ ID NO:55. A second series of CARs was engineered from a parent anti-MOG381 antigen binding-domain comprising the amino acid sequence of SEQ ID NO:3. The parental CAR comprising the anti-MOG381 ABD is referred to herein as “1414”.The CARS engineered from the parental anti-MOG381 ABD include: 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409 and 1410. The amino acid sequence of single chain variable fragment (scFv) comprising the ABD of the 1403, 1404, 1405, 1406, 1408 and 1409 CAR are set forth as SEQ ID NO:56-61, respectively.

[0115] The anti-MOG CAR lentiviral constructs were transduced into a human Jurkat T-cell line that was genetically engineered as an NFAT-firefly luciferase reporter cell line. Thus, upon activation of the reporter cell line by binding of the anti-MOG CAR to the MOG antigen, the nuclear factor of activated T-cells (NF AT) is activated resulting in expression of luciferase. The cells were allowed to recover for 48 hrs before being co-cultured with K562 cells transduced to express high (MND) or low (UBS) levels of MOG. Activation of Jurkat T-cells expressing the anti-MOG CARS by K562 target cells expressing human MOG is shown in FIG. 1A-B for anti-MOG CARS engineered from the anti-MOG375 ABD of the 1413-parental CAR, and shown in FIG.2A-2B for anti-MOG CARS engineered from the anti-MOG381 ABD of the 1414-parental CAR. The 915-medium anti-MOG CAR is a control CAR with a distinct ABD from the anti-MOG375 and anti-MOG381 ABDs, and their derivatives.Example 2: Expression of Anti-MOG CARs in Primary Human Regulatory T-cells

[0116] In Vitro Assays. Regulatory T (Treg) cells were purified from PBMCs. CD25+ cells were enriched by positive selection from PBMCs by magnetic cell sorting (Miltenyi Biotec). CD25+ T-cells were then stained with fluorochrome-labelled mAbs specific for CD4, CD25 and CD127 and sorted by flow cytometry into a population of CD4+ CD25high CD1271ow cells. Purified primary Tregs were expanded via anti-CD3 and anti-CD28 coated Dynabeads at a ratio 1:1 in the presence of IE-2 in T-cell media, Optimizer with 0.5% Serum Replacement. Cells were expanded with fresh IE-2 every 2-3 days. On day 3 of the expansion, Treg cells were transduced with an anti-MOG CAR lenti virus. Cells were restimulated with anti-CD3 and anti-CD28 coated beads on day 9 at a ratio of 1:1. On day 14 of expansion, the cells were debeaded to allow for rest from anti-CD3 and anti-CD28 coated Dynabead-induced activation in RPMI with 10% FBS and IL-2.

[0117] On Day 17, the transduced-primary Tregs were labeled with CFSE and then cocultured in RPMI with 10%FBS and IL-2 in 96 well plates with K562 cells that were engineered to express high levels of human MOG, low levels of human MOG, or control K562 cells that did not express any MOG antigen. After 3 days of co-culture, the cells were then harvested andstained with fluorochrome-labelled mAbs specific for CD4 to gate Tregs independent of K562 cells, for Rat Tag to identify the tag indicative of successful transduction, and for the CD71 activation marker to determine the percentage of anti-MOG CAR-expressing Tregs (i.e., Rat Tag-i- cells) that were activated (i.e., CD71+) by MOG expression on K562 target cells.

[0118] Results from in vitro analysis of primary human Treg cells expressing an anti-MOG CAR are shown in FIG. 3A-3B, and FIG.4A-4D. Levels of expression of the tag and hence the associated anti-MOG CAR in primary Treg cells are shown in FIG.3A-3B. The medium and low anti-MOG CARs are control CARs with distinct ABDs from the anti-MOG375 and anti-MOG381 ABDs, and their derivatives. Levels of activation of primary Treg cells expressing anti-MOG CARS engineered from the anti-MOG375 ABD of the 1413-parental CAR are shown in FIG. 4C-4D. Levels of activation of primary Treg cells expressing anti-MOG CARS engineered from the anti-MOG381 ABD of the 1414-parental CAR are shown in FIG.4A-4B. The 914, 915 and 916 anti-MOG CARs are control CARs with distinct ABDs from the anti-MOG375 and anti-MOG381 ABDs, and their derivatives. These results demonstrate that the anti-MOG CAR expressing human T-cells generated using the methods described in the examples are able to bind to human MOG antigen and effect T cell activation. Interestingly, primary Treg cells expressing some anti-MOG CARs (e.g., 1410 and 1392) were subject to antigen-independent activation (“tonic-signaling”) by parental MOG-negative, K562 cells. In terms of low tonic signaling and MOG-specific activation, anti-MOG CARs 1405, 1406, 1408, 1409 and 1399 were identified as leads for further development.

[0119] In Vivo Assays. Primary Tregs are prepared and transduced with an anti-CAR lentivirus and are then infused via intracerebroventricular injection into immunocompromised NSG mice. Each mouse is to receive IxlO6cells diluted in 5 L of PBS, with 2.5 pL to be infused bilaterally into each ventricle. On days 7, 14, and 28 after infusion, mice are euthanized, perfused, and blood and brain (i.e., central nervous system “CNS”) samples harvested and dissociated into single-cell suspensions using standard protocols. Single-cell suspensions are stained for human CD3, human CD45, and Rat Tag expression and analyzed by flow cytometry. Primary human Treg cells expressing an anti-MOG CAR are expected to persist in the central nervous system of recipient mice.

[0120] It should be understood that the description and the drawings are not intended to limit the invention to the particular form disclosed, but to the contrary, the intention is to cover all modifications, equivalents, and embodiments falling within the spirit and scope of the present invention as defined by the appended claims. Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description and the drawings are to be construed as illustrative only and are for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed or omitted, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims. Headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description.SEQUENCES>SEQ ID NO:1 (GMCSFRa signal peptide) Homo sapiensMLLLVTSLLLCELPHPAFLLIP>SEQ ID NO:2 (CD8a signal peptide) Homo sapiensMALPVTALLLPLALLLHAARP>SEQ ID NO:3 (antiMOG381 scFV VH-VL) artificial QVQLQESGGGLAKPGGSLRLTCAASGFTFSDYWMSWVRQAPGKGLEWVGEINPDGGRTNYKDFVKGRFTI SRDNAKNTLYLQLNSLKTEDTAIYYCTGAGPTYYFDYWGQGTLVTVSSASTKNPDVFPLTGGGGSGGGGSG GGGSELVMTQSPSSLFASIGDRVTITCRASQNIRSNLAWYQQKPGKTPRLLIYDASSLQPGIPSRFSGSGSGTY YTLTISSLQSDDLATYYCQQGYTTPVTFGQGTKLEIKRAVAAPSVFIFPPSEDQVKSGTA>SEQ ID NO:4 (antiMOG381 VH) marmoset QVQLQESGGGLAKPGGSLRLTCAASGFTFSDYWMSWVRQAPGKGLEWVGEINPDGGRTNYKDFVKGRFTI SRDNAKNTLYLQLNSLKTEDTAIYYCTGAGPTYYFDYWGQGTLVTVSSASTKNPDVFPLT>SEQ ID NO:5 (antiMOG381 VL) marmoset ELVMTQSPSSLFASIGDRVTITCRASQNIRSNLAWYQQKPGKTPRLLIYDASSLQPGIPSRFSGSGSGTYYTLTIS SLQSDDLATYYCQQGYTTPVTFGQGTKLEIKRAVAAPSVFIFPPSEDQVKSGTA>SEQ ID NO:6 (antiMOG375 scFV VH-VL) artificial QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGMQWVRQAPEQGLEWMGWINTNTGGTSYAQKFQGRV TMTRDASTSTAYMELSSLRPEDTAVYYCARDATRILADVLDYWGQGTLVTVSSASTKNPDVFGGGGSGGGG SGGGGSELTLTQSPVTLSLSPKETATLSCRASQSVRSYLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSG TDFTLTISSLEPEDFAVYYCQQYSSWYTFGQGTKLEIKRAVAAPSVFIFPPSEDQVKSGTAT>SEQ ID N0:7 (antiMOG375 VH) marmoset QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGMQWVRQAPEQGLEWMGWINTNTGGTSYAQKFQGRV TMTRDASTSTAYMELSSLRPEDTAVYYCARDATRILADVLDYWGQGTLVTVSSASTKNPDVF>SEQ ID N0:8 (antiMOG375 VL) marmoset ELTLTQSPVTLSLSPKETATLSCRASQSVRSYLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTDFTLTIS SLEPEDFAVYYCQQYSSWYTFGQGTKLEIKRAVAAPSVFIFPPSEDQVKSGTAT>SEQ ID N0:9 synthetic glycine linkerXaXbGGGGSGGGGSGGGGSXcXa, Xb and Xc are independently selected from S and absent>SEQ ID NO:10 synthetic Whitlow linkerGSTSGSGKPGSGEGSTKG>SEQ ID NO:11 synthetic ABpur linkerASSGGSTSGSGKPGSGEGSSGSAR> (flexible polypeptide linker motif) synthetic[XaXbXcXdXeXf]n, whereinXa, Xb, Xc, and Xd are independently selected from G and S,Xe and Xf are independently selected from G, S, and absent, andn is 1, 2 or 3.>SEQ ID N0:13 (CD8a Hinge) Homo sapiens TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD>SEQ ID N0:14 (CD28 Hinge) Homo sapiens IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP>SEQ ID N0:15 (lgG4 Hinge "P") Homo sapiensESKYGPPCPPCP>SEQ ID N0:16 (CD8a transmembrane) Homo sapiensIYIWAPLAGTCGVLLLSLVITLYC>SEQ ID NO:17 (CD28 transmembrane WT) Homo sapiens FWVLVVVGGVLACYSLLVTVAFIIFWV>SEQ ID N0:18 (CD28 modified transmembrane domain aa sequence) synthetic FWVLVVVGGVLALLLLLVLVAFIIFWV(C13L, Y14L, S15L and T19L)>SEQ ID N0:19 (generic CD28 transmembrane domain) synthetic FWVLVVVGGVLAX1X2X3LLVX4VAFIIFWVwherein XI is C or L, X2 is Y or L, X3 is S or L, and X4 is T or L>SEQ ID NO:20 (CD28 costimulatory domain) Homo sapiens RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS>SEQ ID NO:21 (4-1BB costimulatory domain) Homo sapiens KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL>SEQ ID NO:22 (CD3zeta intracellular signal transduction domain) Homo sapiens RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR>SEQ ID NO:23 (CD28+CD3zeta) synthetic RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR>SEQ ID NO:24 (4-lBB+CD3zeta) synthetic KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR>SEQ ID NOS:25-52, SEE TABLES l-IV>SEQ ID NO:53 alternate CDR-L2 of 381 LCDASSLQX3 (SEQ ID NO:53), wherein X3 is P or absent>SEQ ID NO:54 (glycine linker) syntheticGGGGSGGGGSGGGGS>SEQ ID NO:55 (antiMOG1399 VH-VL) synthetic QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGMQWVRQAPGQGLEWMGIINTNTGGTSYAQKFQGRVT MTRDTSTSTVYMELSSLRSEDTAVYYCARDATRILADVLDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVL TQSPGTLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLIYGASTRATGIPDRFSGSGSGTDFTLTISRLE PED F A VYYCQQYSS WYTFG QGTK L E I K>SEQ ID NO:56 (antiMOG1403 VH-VL) synthetic EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYWMSWVRQAPGKGLEWVGEINPDGGRTNYAAPVKGRFTI SRDDSKNTLYLQMNSLKTEDTAVYYCTGAGPTYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQ SPSSLSASVGDRVTITCRASQNIRSNLAWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQGYTTPVTFGQGTKLEIK>SEQ ID NO:57 (antiMOG1404 VH-VL) synthetic EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYWMSWVRQAPGKGLEWVGEINPDGGRTNYAAPVKGRFTI SRDDSKNTLYLQMNSLKTEDTAVYYCTGAGPTYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSAIVLTQS PSSLSASVGDRVTITCRASQNIRSNLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTYFTLTISSLQPED FATYYCQQGYTTPVTFGQGTKLEI K>SEQ ID NO:58 (antiMOG1405 VH-VL) synthetic EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYWMSWVRQAPGKGLEWVSEINPDGGRTNYKDFVKGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCTGAGPTYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQS PSSVSASVGDRVTITCRASQNIRSNLAWYQQKPGKAPKLLIYDASSLQPGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQGYTTPVTFGQGTKLEIK>SEQ ID NO:59 (antiMQG1406 VH-VL) synthetic EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYWMSWVRQAPGKGLEWVSEINPDGGRTNYKDFVKGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCTGAGPTYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQS PSSLSASVGDRVTITCRASQNIRSNLAWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQGYTTPVTFGQGTKLEI K>SEQ ID NQ:60 (antiMQG1408 VH-VL) syntheticEVQLVESGGG LVQPGGSLRLSCAASG FTFSDYWMSWVRQAPG KG LVWVSE I N PDGG RTNYADSVKG RFTI SRDNAKNTLYLQMNSLRAEDTAVYYCTGAGPTYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQ SPSSVSASVGDRVTITCRASQNIRSNLAWYQQKPGKAPKLLIYDASSLQPGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQGYTTPVTFGQGTKLEIK>SEQ ID N0:61 (antiMQG1409 VH-VL) syntheticEVQLVESGGG LVQPGGSLRLSCAASG FTFSDYWMSWVRQAPG KG LVWVSE I N PDGG RTNYADSVKG RFTI SRDNAKNTLYLQMNSLRAEDTAVYYCTGAGPTYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQ SPSSLSASVGDRVTITCRASQNIRSNLAWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQGYTTPVTFGQGTKLEIK>SEQ ID NO:62 (antiMOG1399 VH) synthetic QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGMQWVRQAPGQGLEWMGIINTNTGGTSYAQKFQGRVT MTRDTSTSTVYMELSSLRSEDTAVYYCARDATRILADVLDYWGQGTLVTVSS>SEQ ID NO:63 (antiMOG1399 VL) synthetic EIVLTQSPGTLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLIYGASTRATGIPDRFSGSGSGTDFTLTI SRLEPEDFAVYYCQQYSSWYTFGQGTKLEIK>SEQ ID NO:64 (antiMOG1403 VH) synthetic EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYWMSWVRQAPGKGLEWVGEINPDGGRTNYAAPVKGRFTI SRDDSKNTLYLQMNSLKTEDTAVYYCTGAGPTYYFDYWGQGTLVTVSS>SEQ ID NO:65 (antiMOG1403 VL) synthetic DIQMTQSPSSLSASVGDRVTITCRASQNIRSNLAWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQGYTTPVTFGQGTKLEIK>SEQ ID NO:66 (antiMOG1404 VH) synthetic EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYWMSWVRQAPGKGLEWVGEINPDGGRTNYAAPVKGRFTI SRDDSKNTLYLQMNSLKTEDTAVYYCTGAGPTYYFDYWGQGTLVTVSS>SEQ ID NO:67 (antiMOG1404 VL) synthetic AIVLTQSPSSLSASVGDRVTITCRASQNIRSNLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTYFTLTIS SLQPEDFATYYCQQGYTTPVTFGQGTKLEIK>SEQ ID NO:68 (antiMOG1405 VH) synthetic EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYWMSWVRQAPGKGLEWVSEINPDGGRTNYKDFVKGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCTGAGPTYYFDYWGQGTLVTVSS>SEQ ID NO:69 (antiMOG1405 VL) synthetic DIVMTQSPSSVSASVGDRVTITCRASQNIRSNLAWYQQKPGKAPKLLIYDASSLQPGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQGYTTPVTFGQGTKLEIK>SEQ ID NO:70 (antiMOG1406 VH) synthetic EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYWMSWVRQAPGKGLEWVSEINPDGGRTNYKDFVKGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCTGAGPTYYFDYWGQGTLVTVSS>SEQ ID N0:71 (antiMOG1406 VL) synthetic DIQMTQSPSSLSASVGDRVTITCRASQNIRSNLAWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQGYTTPVTFGQGTKLEIK>SEQ ID NO:72 (antiMOG1408 VH) syntheticEVQLVESGGG LVQPGGSLRLSCAASG FTFSDYWMSWVRQAPG KG LVWVSE I N PDGG RTNYADSVKG RFTI SRDNAKNTLYLQMNSLRAEDTAVYYCTGAGPTYYFDYWGQGTLVTVSS>SEQ ID NO:73 (antiMOG1408 VL) synthetic DIVMTQSPSSVSASVGDRVTITCRASQNIRSNLAWYQQKPGKAPKLLIYDASSLQPGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQGYTTPVTFGQGTKLEIK>SEQ ID NO:74 (antiMOG1409 VH) syntheticEVQLVESGGG LVQPGGSLRLSCAASG FTFSDYWMSWVRQAPG KG LVWVSE I N PDGG RTNYADSVKG RFTI SRDNAKNTLYLQMNSLRAEDTAVYYCTGAGPTYYFDYWGQGTLVTVSS>SEQ ID NO:75 (antiMOG1409 VL) synthetic DIQMTQSPSSLSASVGDRVTITCRASQNIRSNLAWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQGYTTPVTFGQGTKLEIK

Claims

CLAIMSWe claim:

1. A chimeric antigen receptor (CAR) comprising an antigen-binding domain, a hinge domain, a transmembrane domain, one or more co-stimulatory domains, and an intracellular signaling domain, wherein the antigen-binding domain specifically binds to extracellular domain of myelin oligodendrocyte glycoprotein (MOG) and comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain and the VL domain comprise complementarity-determining regions (CDRs) from:(a) the VH domain of SEQ ID NO:4 and the VL domain of SEQ ID NO:5; or(b) the VH domain of SEQ ID NO:7 and the VL domain of SEQ ID NO: 8.

2. The CAR of claim 1, wherein the antigen-binding domain binds to the extracellular domain of human MOG and the extracellular domain of mouse MOG.

3. The CAR of claim 1, wherein:(i) the VH domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:26, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:28, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 30, and(ii) the VL domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:33, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:35, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:37.

4. The CAR of claim 3, wherein the antigen-binding domain comprises a single chain variable fragment (scFv), and wherein:> (a) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:64 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:65; or> (b) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:66 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:67; or> (c) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ IDNO:68 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:69; or> (d) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:70 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:71; or> (e) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:72 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:73; or> (f) the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:74 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:75.

5. The CAR of claim 3, wherein the antigen-binding domain comprises a single chain variable fragment (scFv), and wherein:> (a) the VH domain comprises the amino acid sequence of SEQ ID NO: 64 and the VL domain comprises the amino acid sequence of SEQ ID NO: 65; or> (b) the VH domain comprises the amino acid sequence of SEQ ID NO:66 and the VL domain comprises the amino acid sequence of SEQ ID NO: 67; or> (c) the VH domain comprises the amino acid sequence of SEQ ID NO: 68 and the VL domain comprises the amino acid sequence of SEQ ID NO: 69; or> (d) the VH domain comprises the amino acid sequence of SEQ ID NO:70 and the VL domain comprises the amino acid sequence of SEQ ID NO:71; or> (e) the VH domain comprises the amino acid sequence of SEQ ID NO:72 and the VL domain comprises the amino acid sequence of SEQ ID NO:73; or> (f) the VH domain comprises the amino acid sequence of SEQ ID NO:74 and the VL domain comprises the amino acid sequence of SEQ ID NO:75.

6. The CAR of claim 1, wherein:(i) the VH domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:40, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:42, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:44, and(ii) the VL domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ IDNO:47, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:49, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:51.

7. The CAR of claim 6, wherein the antigen-binding domain comprises a single chain variable fragment (scFv), and wherein the VH domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO:62 and the VL domain comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 63.

8. The CAR of claim 6, wherein the antigen-binding domain comprises a single chain variable fragment (scFv), and wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 62 and the VL domain comprises the amino acid sequence of SEQ ID NO: 63.

9. The CAR of claim 3, wherein the VH domain and the VL domain are joined by a linker comprising the amino acid sequence of SEQ ID NOV, SEQ ID NO: 10, or SEQ ID NO: 11.

10. The CAR of claim 4, wherein the scFv comprises the amino acid sequence of one of the group consisting of SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61, or an amino acid sequence having at least 95% identity to one of the group consisting of SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61.

11. The CAR of claim 7, wherein the scFv comprises the amino acid sequence of SEQ ID NO:55 or an amino acid sequence having at least 95% identity to SEQ ID NO:55.

12. The CAR of claim 1, wherein the intracellular signaling domain comprises a CD3-zeta signal transduction domain.

13. The CAR of claim 12, wherein the CD3-zeta signal transduction domain comprises the amino acid sequence of SEQ ID NO:22.

14. The CAR of claim 13, wherein the one or more co-stimulatory domains comprise a CD28 co-stimulatory domain or a 4- IBB co-stimulatory domain.

15. The CAR of claim 14, wherein the one or more co-stimulatory domains comprise a CD28 co-stimulatory domain.

16. The CAR of claim 15, wherein the CD28 co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:20.

17. The CAR of claim 16, wherein an intracellular domain comprising the one or more co-stimulatory domains and the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:23.

18. The CAR of claim 17, wherein the transmembrane domain is a CD 8 transmembrane domain or a CD28 transmembrane domain.

19. The CAR of claim 18, wherein the transmembrane domain is a CD 8 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19.

20. The CAR of claim 19, wherein the hinge domain is a CD8 hinge domain or a CD28 hinge domain.

21. The CAR of claim 20, wherein the hinge domain is a CD28 hinge domain comprising the amino acid sequence of SEQ ID NO: 14.

22. The CAR of claim 1, further comprising a signal peptide.

23. The CAR of claim 22, wherein the signal peptide is a granulocyte-macrophage colony-stimulating factor receptor alpha (GMCSFRa) signal peptide or a CD8-alpha (CD8a) signal peptide.

24. The CAR of claim 23, wherein the signal peptide is a GMCSFRa signal peptide comprising the amino acid sequence if SEQ ID NO:1.

25. A nucleic acid encoding the CAR of claim 1.

26. An expression vector comprising the nucleic acid of claim 25 in operable combination with an expression control sequence.

27. The expression vector of claim 26, wherein the vector is a viral vector, optionally wherein the viral vector is a lentiviral vector a retroviral vector, an adenoviral vector, or an adeno-associated viral vector.

28. The expression vector of claim 27, wherein the viral vector is a lentiviral vector.

29. The expression vector of claim 26, wherein the vector is a plasmid.

30. A host cell comprising the expression vector of claim 26.

31. A modified immune cell that has been engineered to express the CAR of claim 1 , wherein the modified immune cell is a modified mammalian T-cell, NK-cell or NKT-cell, optionally wherein the modified immune cell expresses a constitutively-active IL-2 receptor.

32. The modified immune cell of claim 31, wherein the modified immune cell is a human T-cell.

33. The modified immune cell of claim 32, wherein the human T-cell is a regulatory T-(Treg) cell.

34. The modified immune cell of claim 33, wherein the human Treg cell is CD4+, CD25+ and CD1271O.

35. The modified immune cell of claim 34, wherein the human Treg cell is FOXP3+ and HELIOS+.

36. A pharmaceutical composition comprising a plurality of the modified immune cells of claim 31, and a pharmaceutically acceptable excipient.

37. A method of treating a human subject suffering from neuroinflammation, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 36.

38. The method of claim 37, wherein the neuroinflammation is an inflammatory disorder or an autoimmune disease.

39. The method of claim 37, wherein the neuroinflammation is a MOG-associated disease or disorder.

40. The method of claim 37, wherein the neuroinflammation is a demyelinating disease.

41. The method of claim 40, wherein the demyelinating disease is amyotrophic lateral sclerosis (ALS) or multiple sclerosis (MS).

42. The method of claim 37, wherein the pharmaceutical composition is administered intravenously.

43. A method of treating a human subject suffering from neuroinflammation, the method comprising:(a) isolating T-cells from a biological sample obtained from the subject;(b) enriching the T-cells for T regulatory (Treg) cells;(c) transfecting the enriched Treg cells with an expression vector encoding the CAR of claim 1 to produce CAR-Treg cells;(d) expanding the CAR-Treg cells to produce a plurality of CAR-Treg cells; and(e) administering the plurality of CAR-Treg cells to the subject.

44. The method of claim 43, wherein the expansion comprises using anti-CD3 / CD28 coated beads.

45. The method of claim 43, wherein the expansion does not comprise using anti-CD3 / CD28 coated beads.

46. A kit comprising a container containing the pharmaceutical composition of claim 36, communicating through a fluidic conduit with a drip chamber, wherein the drip chamber communicates through a fluidic conduit with an intravenous needle.

47. The kit of claim 46, wherein the container comprises a bag.

48. The kit of claim 47, wherein the fluidic conduit between the container and the needle comprises one or more Y-sites and a roller clamp.

49. A method of preparing T-regulatory (Treg) cells expressing a chimeric antigen receptor (CAR), the method comprising:(a) isolating T-cells from a biological sample obtained from a human subject;(b) enriching the T-cells for T regulatory (Treg) cells;(c) transfecting the enriched Treg cells with an expression vector encoding the CAR of claim 1 to produce CAR-Treg cells; and(d) expanding the CAR-Treg cells to produce a plurality of CAR-Treg cells.

50. The method of claim 49, wherein the expansion comprises using anti-CD3 / CD28 coated beads.

51. The method of claim 49, wherein the expansion does not comprise using anti-CD3 / CD28 coated beads.

52. The method of claim 49, wherein the transfection occurs by use of a viral vector, electroporation, heat shock, bacteriophage, sonication, or calcium phosphate.

53. The method of claim 49, wherein the transfection occurs by use of a viral vector.

54. A pharmaceutical composition comprising the plurality of the CAR-Treg cells produced by the method of claim 49-53, and a pharmaceutically acceptable excipient.

55. A method of treating neuroinflammation, the method comprising: administering an effective amount of the pharmaceutical composition of claim 54 to a human subject suffering from the neuroinflammation.

56. The pharmaceutical composition of claim 36 or claim 54 for use as a medicament.

57. The pharmaceutical composition of claim 36 or claim 54 for use in a method of treating a neuroinflammation, comprising administering an effective amount of the composition to an individual in need thereof to treat the neuroinflammation, optionally wherein the individual is a human subject.

58. Use of a plurality of modified regulatory T (Treg) cells in the manufacture of a medicament for treating a subject suffering from neuroinflammation, wherein the modified Tregcells express the CAR of any one of claims 1-24, and wherein the modified Treg cells are CD4+, CD25+ and CD1271O, optionally wherein the neuroinflammation is a demyelinating disease, optionally wherein the demyelinating disease is amyotrophic lateral sclerosis (ALS) or multiple sclerosis (MS).

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