Methods for treating multiple sclerosis using autologous T cells

Abstract

Provided herein are methods for treating multiple sclerosis in a subject or for selecting a subject for adoptive immunotherapy, comprising autologous cytotoxic T cells that express a T cell receptor that specifically binds to Epstein-Barr virus (EBV) or that express CD107a, TNF, IFN-γ, or IL-2. [Selection diagram] None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 448,707, filed January 20, 2017, and U.S. Provisional Patent Application No. 62 / 576,349, filed October 24, 2017, each of which is incorporated herein by reference in its entirety. [Background technology]

[0002] Autoimmune diseases, such as multiple sclerosis (MS), are conditions that result from an abnormal immune response against the body's own tissues. MS is characterized by the breakdown of myelin, the protective lipid shell that surrounds nerve fibers, by the body's own immune cells.

[0003] Epstein-Barr virus (EBV), also known as human herpesvirus 4, is a ubiquitous herpesvirus. It has recently been shown that exposure to EBV may predispose to or otherwise play a role in the pathogenesis of autoimmune diseases, including MS. For example, recent studies have shown that individuals diagnosed with MS exhibit higher levels of EBV-associated proteins in aggregated B cells in neural tissue than healthy individuals. It has been hypothesized that an increase in EBV-infected B cells and / or incomplete elimination of such cells may predispose individuals to multiple sclerosis. Summary of the Invention

[0004] Provided herein are methods of treating MS (e.g., relapsing-remitting MS, secondary progressive MS, primary progressive MS, or progressive relapsing MS) comprising administering to the subject autologous T cells (e.g., cytotoxic T cells, or CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on MHC (e.g., class I MHC). In some embodiments, the MS is primary progressive MS. In some embodiments, the method comprises administering to the subject autologous T cells (e.g., CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC, thereby ameliorating or stabilizing MS symptoms in the subject. Also provided herein are methods of reducing anti-EBV IgG levels in the CSF of a subject with MS by administering to the subject autologous T cells (e.g., CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC molecules.

[0005] In some embodiments, at least 1%, at least 5%, at least 10%, at least 15%, or at least 20% of the CTLs express CD107a, IFNγ, TNF, or IL-2. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the CTLs express CD107a, IFNγ, TNF, and IL-2. At least 5%, at least 7%, at least 8%, at least 10%, at least 15%, at least 20%, or at least 30% of the CTLs are EBV-reactive.

[0006] In some embodiments, the EBV peptide comprises an LMP1 peptide or a fragment thereof, an LMP2A peptide or a fragment thereof, and / or an EBNA1 peptide or a fragment thereof. In some embodiments, the EBV peptide comprises a sequence listed in Table 1. In certain aspects, provided herein are methods of treating multiple sclerosis (e.g., relapsing-remitting MS, secondary progressive MS, primary progressive MS, or progressive relapsing MS) comprising isolating a sample containing T cells (e.g., CTLs) from a subject, generating T cells that express a T cell receptor that specifically binds to an EBV peptide presented on MHC (e.g., class I MHC), and then administering the T cells to the subject. In some embodiments, the T cells are generated by incubating a sample containing autologous T cells (e.g., a PBMC sample) with antigen-presenting cells (APCs) that present an EBV peptide on MHC (e.g., class I MHC), thereby inducing proliferation of peptide-specific T cells (e.g., peptide-specific autologous CTLs) in the sample. In some embodiments, APCs can be made to present EBV peptides by incubating them with a nucleic acid construct encoding the EBV peptide (e.g., AdE1-LMPpoly), thereby inducing the APCs to present the EBV peptide. In some embodiments, the APCs can be B cells, antigen-presenting T cells, dendritic cells, or artificial antigen-presenting cells (e.g., a cell line expressing CD80, CD83, 41BB-L, and / or CD86, such as aK562 cells). In some embodiments, the method further includes analyzing the expression of CD107a, IFNγ, TNF, or IL-2 by the expanded peptide-specific autologous CTLs, and administering the peptide-specific autologous CTLs to the subject if at least 1%, at least 5%, at least 10%, at least 15%, or at least 20% of the expanded peptide-specific autologous CTLs express CD107a, IFNγ, TNF, or IL-2. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the peptide-specific autologous CTLs express CD107a, IFNγ, TNF, and IL-2.At least 5%, at least 7%, at least 8%, at least 10%, at least 15%, at least 20%, or at least 30% of the peptide-specific autologous CTLs may have EBV reactivity.

[0007] In some embodiments, the EBV peptide comprises an LMP1 peptide or a fragment thereof, an LMP2A peptide or a fragment thereof, and / or an EBNA1 peptide or a fragment thereof. In some embodiments, the EBV peptide comprises a sequence listed in Table 1. In some embodiments, the MS is primary progressive MS.

[0008] In some embodiments, 5×10 6 , 1×10 7 , 1.5×10 7 or 2 x 10 7 In some embodiments, an initial dose of T cells (e.g., autologous CTLs) is administered, followed by one or more additional doses of T cells (e.g., autologous CTLs), e.g., at gradually increasing doses over the course of treatment. In some embodiments, two or more, three or more, four or more, or five or more doses are administered. The amount of T cells (e.g., autologous CTLs) may vary from the initial dose to the additional doses. For example, a lower dose may be administered first, followed by a higher dose. In some embodiments, at least one, at least two, at least three, at least four, or at least five doses are administered to the subject. The doses may be administered weekly or biweekly. In some embodiments, the subject does not suffer any adverse effects as a result of the administration of T cells (e.g., autologous CTLs). In some embodiments, the method includes administering four doses of successively greater numbers of CTLs. In some embodiments, the method includes administering 5×10 of the first dose. 6 CTLs, second dose 1 x 10 7 CTLs, third dose 1.5 x 10 7 CTLs, and the fourth dose 2 x 10 7 The method includes administering CTLs.

[0009] In some embodiments, the method further comprises obtaining a first sample of cerebrospinal fluid (CSF) from the subject, analyzing the amount of anti-EBV IgG in the CSF in the first sample (preferably before CTL administration), obtaining a second sample of CSF from the subject after a period of time (preferably after CTL administration), analyzing the relative amount of anti-EBV IgG in the CSF in the second sample, and evaluating the efficacy of adoptive immunotherapy in a subject with multiple sclerosis by indicating that the amount of anti-EBV IgG in the second sample is lower than that in the first sample, indicating stable and / or non-progressing disease. The period of time can be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 3 months, 6 months, or 1 year.

[0010] In some embodiments, the subject undergoes a diagnostic test, such as an EDSS test. In some embodiments, the subject undergoes an EDSS score before and after administration of the T cells. After administration of the T cells, the EDSS score may remain the same, or the EDSS score may be reduced (e.g., to at least 0.5 or at least 1.0).

[0011] In some embodiments, provided herein are methods of selecting a subject for adoptive immunotherapy by obtaining a sample comprising T cells (e.g., CTLs) from a subject, isolating autologous T cells, determining the EBV reactivity of the autologous T cells in the sample, and selecting the subject for adoptive immunotherapy if at least a threshold percentage (e.g., at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80%) of the autologous T cells are EBV reactive.

[0012] In some embodiments, provided herein are methods of selecting a subject for adoptive immunotherapy by obtaining a sample comprising T cells (e.g., CTLs) from a subject, isolating autologous T cells, determining CD107a, IFNγ, TNF, and / or IL-2 expression on the autologous T cells, and selecting the subject for adoptive immunotherapy if at least a threshold percentage (e.g., at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80%) of the autologous T cells express CD107a, IFNγ, TNF, and / or IL-2. In some embodiments, the subject has MS (eg, relapsing-remitting MS, secondary progressive MS, primary progressive MS, or progressive relapsing MS). [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1 contains two panels (A-B) showing CSF samples from the same patient before and after autologous EBV-specific T-cell therapy after the first course of treatment 4 years ago and after retreatment this year at the current time point. (A) CSF IgG index (with a horizontal dotted line indicating the upper limit of the normal range). (B) Intrathecal IgG production (IgG(loc)) was calculated using the formula from Reiber and Felgenhauer (16): IgG(loc) (mg / L) = {(CSF IgG ÷ serum IgG) - [0.8 × (√((CSF albumin ÷ serum albumin)² + 15))] + 1.8} × serum IgG. Vertical lines indicate successive T-cell infusions of 5 × 10⁶, 1 × 10⁶, 1.5 × 10⁶, and 2 × 10⁶ cells. [Figure 2A] Figure 2 has two parts (A-B) showing the correlation between EBV-specific CD8+ T cell reactivity with T cell products and clinical response to T cell therapy. [Figure 2B]Figure 2 has two parts (A-B) showing the correlation between EBV-specific CD8+ T cell reactivity of T cell products and clinical response to T cell therapy. [Figure 3] Figure 3 shows disease activity on brain MRI. DETAILED DESCRIPTION OF THE INVENTION

[0014] General Provided herein are methods of treating multiple sclerosis (e.g., relapsing-remitting MS, secondary progressive MS, primary progressive MS, or progressive relapsing MS) in a subject using autologous T cells (e.g., CTLs) that recognize one or more EBV epitopes (e.g., EBV epitopes disclosed herein). In some embodiments, the method further comprises isolating a sample containing T cells from the subject, incubating the T cells with APCs that present an EBV peptide (e.g., an EBV peptide disclosed herein), and generating T cells that recognize the EBV peptide presented on MHC. Also provided herein are methods of assessing the efficacy of adoptive immunotherapy in a subject with multiple sclerosis by obtaining samples of cerebrospinal fluid (CSF) from the subject both before and after administration of the T cells and analyzing the relative amount of anti-EBV IgG in the CSF.

[0015] definition For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0016] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0017] As used herein, the term "administer" means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administration by a healthcare professional and self-administration. Such agents may include, for example, a peptide described herein, an antigen-presenting cell provided herein, and / or a T cell provided herein.

[0018] The term "amino acid" is intended to encompass all molecules, natural or synthetic, that contain both amino and acid functional groups and that can be included in a polymer of natural amino acids. Exemplary amino acids include natural amino acids, their analogs, derivatives and congeners, amino acid analogs with variant side chains, and all stereoisomers of any of the above.

[0019] The terms "bind" or "interact" refer to an association, which can be a stable association, between two molecules, e.g., a TCR and a peptide / MHC, e.g., by electrostatic, hydrophobic, ionic and / or hydrogen bonding interactions under physiological conditions.

[0020] The terms "biological sample," "tissue sample," or simply "sample" each refer to a collection of cells obtained from a subject's tissue. The source of a tissue sample can be solid tissue such as from a fresh, frozen, and / or preserved organ, tissue sample, biopsy, or aspirate, blood or any blood component, serum, blood, bodily fluids such as spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid, urine, saliva, feces, tears, or cells from any point in a subject's pregnancy or development.

[0021] As used herein, the term "cytokine" refers to any secreted polypeptide that affects cellular function and regulates cell-cell interactions in immune, inflammatory, or hematopoietic responses. Cytokines include, but are not limited to, monokines and lymphokines, regardless of which cells produce them. For example, monokines are generally said to be produced and secreted by mononuclear cells, such as macrophages and / or monocytes. However, many other cells, such as natural killer cells, fibroblasts, basophils, neutrophils, endothelial cells, brain astrocytes, bone marrow stromal cells, epidermal keratinocytes, and B lymphocytes, also produce monokines. Lymphokines are generally said to be produced by lymphocytes. Examples of cytokines include, but are not limited to, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor alpha (TNFα), and tumor necrosis factor beta (TNFβ).

[0022] The term "epitope" refers to a protein determinant capable of specific binding to an antibody or TCR. Epitopes usually consist of chemically active surface groups of molecules, such as amino acids or sugar side chains. A particular epitope can be defined by a particular sequence of amino acids to which an antibody can bind.

[0023] As used herein, the phrase "pharmaceutically acceptable" refers to agents, compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0024] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or material encapsulating a solvent, that is involved in carrying or transporting from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) starches, such as corn starch and potato starch; (11) starches, such as corn starch and potato starch; (12) starches, such as corn starch and potato starch; (13) starches, such as corn starch and potato starch; (14) starches, such as corn starch and potato starch; (15) starches, such as corn starch and potato starch; (16) starches, such as corn starch and potato starch; (17) starches, such as corn starch and potato starch; (18) starches, such as corn starch and potato starch; (19) starches, such as corn starch and potato starch; (20) starches, such as corn starch and potato starch; (21) starches, such as corn starch and potato starch; (22) starches, such as corn starch and potato starch; (23) starches, such as corn starch and potato starch; (24) starches, such as corn starch and potato starch; (25) starches, such as corn starch and potato starch; (26) starches, such as corn starch and potato starch; (27) starches, such as corn starch and potato starch; (28) starches, such as corn starch and potato (10) glycols, such as propylene glycol, (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) esters, such as ethyl oleate and ethyl laurate, (13) agar, (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffered solutions, (21) polyesters, polycarbonates, and / or polyanhydrides, and (22) other non-toxic compatible materials used in pharmaceutical formulations.

[0025] The terms "polynucleotide" and "nucleic acid" are used interchangeably. They refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides, ribonucleotides, or their analogs. Polynucleotides may have any three-dimensional structure and may perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci determined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure, if present, may be imparted before or after assembly of the polymer. Polynucleotides may be further modified, such as by conjugation with a labeling component. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.

[0026] As used herein, a therapeutic agent that "prevents" a condition refers to a compound that, when administered to a statistical sample prior to the onset of the disorder or condition, reduces the occurrence of the disorder or condition in the treated sample compared to an untreated control sample, or delays the onset of or reduces the severity of one or more symptoms of the disorder or condition compared to an untreated control sample.

[0027] As used herein, "specific binding" refers to the ability of a TCR to bind to a peptide presented on an MHC (e.g., class I MHC or class II MHC). Typically, a TCR binds to a peptide of at least about 10 -4 K below M Dand with an affinity (K) that is at least 10-fold less, at least 100-fold less, or at least 1000-fold less than the affinity for binding to a nonspecific and unrelated peptide / MHC complex (e.g., one containing a BSA peptide or a casein peptide). D The target antigen / binding partner binds to the target antigen / binding partner via the ATP-binding domain (e.g., as represented by

[0028] As used herein, the term "subject" means a human or non-human animal selected for treatment or therapy.

[0029] As used herein, the phrases "therapeutically effective amount" and "effective amount" mean an amount of an agent effective to produce a desired therapeutic effect in at least a subpopulation of cells in a subject, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0030] As used herein, the term "treating" a disease in a subject or a subject having or suspected of having a disease refers to administering a pharmaceutical treatment to a subject, such as administering a CTL as described herein, to reduce or prevent at least one symptom of the disease from worsening.

[0031] The term "vector" refers to a means by which nucleic acids can be propagated and / or transferred between organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophages, proviruses, phagemids, transposons, and artificial chromosomes, which may or may not replicate autonomously, or which may integrate into a host cell chromosome.

[0032] peptide In some embodiments, provided herein are methods of treating multiple sclerosis (e.g., relapsing-remitting MS, secondary progressive MS, primary progressive MS, or progressive relapsing MS) using autologous T cells (e.g., CTLs) that express a TCR that specifically binds to a peptide comprising an EBV epitope presented on MHC (e.g., class I MHC). In some embodiments, provided herein are methods of generating such autologous T cells, e.g., by incubating a sample comprising T cells (i.e., autologous T cells) with antigen-presenting cells (APCs) that present one or more of the EBV epitopes described herein (e.g., APCs that present a peptide described herein that comprises an EBV epitope on a class I MHC complex).

[0033] In some embodiments, the peptides provided herein comprise a sequence of any EBV viral protein (e.g., a sequence of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids of any EBV protein). In some embodiments, the peptides provided herein comprise no more than 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 consecutive amino acids of an EBV viral protein.

[0034] In some embodiments, the peptides provided herein include a sequence of LMP1 (e.g., a sequence of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids of LMP1). In some embodiments, the peptides provided herein include no more than 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 consecutive amino acids of LMP1. An exemplary LMP1 amino acid sequence is provided below (SEQ ID NO: 1): 1 mdldlergpp gprrpprgpp lssyialall llllallfwl yiimsnwtgg allvlyafal 61 mlviilliif ifrrdllcpl galcllllmi tlllialwnl hgqalylgiv lfifgcllvl 121 giwvyfleil wrlgatiwql lafflaffld illliialyl qqnwwtllvd llwlllflai 181 liwmyyhgqr hsdehhhdds lphpqqatdd ssnhsdsnsn egrhhllvsg agdapplcsq 241 nlgapgggpd ngpqdpdntd dngpqdpdnt ddngphdplp qdpdntddng pqdpdntddn 301 gphdplphnp sdsagndggp pnlteevenk ggdrgppsmt dggggdphhlp tlllgtsgsg 361 gddddphgpv qlsyyd

[0035] In some embodiments, the peptides provided herein include a sequence of LMP2A (e.g., a sequence of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids of LMP2A). In some embodiments, the peptides provided herein include no more than 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 consecutive amino acids of LMP2A. An exemplary LMP2A amino acid sequence is provided below (SEQ ID NO: 2): 1 mgslemvpmg agppspggdp dgddggnnsq ypsasgsdgn tptppndeer esneeppppy 61 edldwgngdr hsdyqplgnq dpslylglqh dgndglpppp ysprddssqh iyeeagrgsm 121 npvclpviva pylfwlaaia ascftasvst vvtatglals llllaavass yaaaqrkllt 181 pvtvltavvt ffaicltwri edppfnsllf allaaagglq giyvlvmlvl lilayrrrwr 241 rltvcggimf lacvlvlivd avlqlspllg avtvvsmtll llafvlwlss pgglgtlgaa 301 lltlaaalal laslilgtln lttmfllmll wtlvvllics scsscpltki llarlflyal 361 allllasali aggsilqtnf kslsstefip nlfcmllliv agilfilail tewgsgnrty 421 gpvfmclggl ltmvagavwl tvmtntllsa wiltagflif ligfalfgvi rccryccyyc 481 ltleseerpp tpyrntv

[0036] In some embodiments, the peptides provided herein comprise a sequence of EBNA1 (e.g., a sequence of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids of EBNA1). In some embodiments, the peptides provided herein comprise no more than 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 consecutive amino acids of EBNA1. An exemplary EBNA1 amino acid sequence is provided below (SEQ ID NO: 3): 1 pffhpvgead yfeylqeggp dgepdvppga ieqgpaddpg egpstgprgq gdggrrkkgg 61 wfgkhrgqgg snpkfeniae glrvllarsh vertteegtw vagvfvyggs ktslynlrrg 121 talaipqcrl tplsrlpfgm apgpgpqpgp lresivcyfm vflqthfae vlkdaikdlv 181 mtkpaptcni kvtvcsfddg vdlppwfppm vegaaaegdd gddgdeggdg degeegqe

[0037] In some embodiments, the peptide comprises the sequence of an epitope listed in Table 1.

[0038] [Table 1]

[0039] In some embodiments, the peptides provided herein comprise two or more EBV epitopes. In some embodiments, the peptides provided herein comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 EBV epitopes. For example, in some embodiments, the peptides provided herein comprise two or more EBV epitopes linked by a linker (e.g., a polypeptide linker).

[0040] In some embodiments, the sequence of the peptide comprises an EBV viral protein sequence except for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) conservative sequence modifications. As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the interaction between a TCR and a peptide containing the amino acid sequence presented on an MHC. Such conservative modifications include amino acid substitutions, additions (e.g., addition of an amino acid to the N- or C-terminus of the peptide), and deletions (e.g., deletion of an amino acid from the N- or C-terminus of the peptide). A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the peptides described herein can be substituted with other amino acid residues from the same side chain family, and the altered peptides can be tested for retention of TCR binding using methods known in the art. Modifications can be introduced into antibodies by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0041] In some embodiments, the peptides provided herein include sequences that are at least 80%, 85%, 90%, 95%, or 100% identical to an EBV viral protein sequence (e.g., the sequence of a fragment of an EBV viral protein). To determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). The amino acid residues at corresponding amino acid positions are then compared. If a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.

[0042] In some embodiments, the peptide is a chimeric peptide or a fusion peptide. As used herein, a "chimeric peptide" or "fusion peptide" includes a peptide having a sequence provided herein linked to a separate peptide having an essentially unlinked sequence. For example, the separate peptide can be fused to the N-terminus or C-terminus of a peptide provided herein directly via a peptide bond or indirectly via a chemical linker. In some embodiments, a peptide provided herein is linked to another peptide comprising a separate EBV epitope. In some embodiments, a peptide provided herein is linked to a peptide comprising an epitope from another viral disease and / or infectious disease.

[0043] The chimeric or fusion peptides provided herein can be produced by standard recombinant DNA techniques. For example, DNA fragments encoding different peptide sequences can be ligated in-frame according to conventional techniques, e.g., by using blunt or staggered ends for ligation, restriction enzyme digestion to provide appropriate ends, filling in sticky ends as needed, alkaline phosphatase treatment to avoid undesired ligations, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques, including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that generate complementary overhangs between two consecutive gene fragments, followed by annealing and reamplified to generate a chimeric gene sequence (see, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons: 1992). Furthermore, numerous expression vectors already encoding fusion moieties are commercially available.

[0044] The peptides provided herein can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques, can be produced by recombinant DNA technology, and / or can be chemically synthesized using standard peptide synthesis techniques. The peptides described herein can be produced in prokaryotic or eukaryotic host cells by expression of nucleotides encoding the peptide(s) of the invention. Alternatively, such peptides can be synthesized by chemical methods. Methods for expression of heterologous peptides in recombinant hosts, chemical synthesis of peptides, and in vitro translation are well known in the art and can be found in further publications such as Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd ed., Cold Spring Harbor, NY; Berger and Kimmel, Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif.; Merrifield, J. (1969) J. Am. Chem. Soc. 91:501; Chaiken IM (1981) CRC Crit. Rev. Biochem. 11:255; Kaiser et al. (1989) Science 243:187; Merrifield, B. (1986) Science 232:342; Kent, SBH (1988) Annu. Rev. Biochem. 57:957, Offord, RE (1980) Semisynthetic Proteins, Wiley Publishing.

[0045] In certain aspects, provided herein are nucleic acid molecules encoding the peptides described herein. In some embodiments, the nucleic acid molecule is a vector. In some embodiments, the nucleic acid molecule is a viral vector, such as an adenovirus-based expression vector, comprising the nucleic acid molecule described herein. In some embodiments, the vector provided herein encodes multiple epitopes provided herein (e.g., as a polyepitope). In some embodiments, the vector provided herein encodes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 epitopes provided herein (e.g., epitopes provided in Table 1).

[0046] In some embodiments, the vector is AdE1-LMPpoly, which encodes a polyepitope of defined CTL epitopes from LMP1 and LMP2 fused to a Gly-Ala repeat-deficient EBNA1 sequence. The AdE1-LMPpoly vector is described, for example, in Smith et al., Cancer Research 72:1116 (2012); Duraiswamy et al., Cancer Research 64:1483-9 (2004); and Smith et al., J. Immunol 117:4897-906, each of which is incorporated herein by reference.

[0047] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors can replicate autonomously in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication, episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genome. Furthermore, certain vectors can direct the expression of genes. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In some embodiments, provided herein is a nucleic acid operably linked to one or more regulatory sequences (e.g., promoters) in an expression vector. In some embodiments, the cell transcribes the nucleic acid provided herein, thereby expressing the peptides described herein. The nucleic acid molecule can be integrated into the genome of the cell, or it can be extrachromosomal.

[0048] In some embodiments, provided herein are cells containing a nucleic acid described herein (e.g., a nucleic acid encoding a peptide described herein). The cell can be, for example, prokaryotic, eukaryotic, mammalian, avian, murine, and / or human. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an APC (e.g., an antigen-presenting T cell, a dendritic cell, a B cell, or an aK562 cell). In the methods, the nucleic acid described herein can be administered to the cell in combination with a delivery reagent, e.g., as a nucleic acid without a delivery vehicle. In some embodiments, any nucleic acid delivery method known in the art can be used in the methods described herein. Suitable delivery reagents include, but are not limited to, Mirus Transit TKO lipophilic reagent, lipofectin, lipofectamine, cellfectin, polycations (e.g., polylysine), atelocollagen, nanoplexes, and liposomes. In some embodiments of the methods described herein, liposomes are used to deliver the nucleic acid to a cell or a subject. Liposomes suitable for use in the methods described herein can be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and sterols, such as cholesterol.The selection of lipids is generally guided by factors such as the desired liposome size and the half-life of the liposomes in the bloodstream.Various methods for preparing liposomes are known, and are described, for example, in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng. 9:467, and U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028 and 5,019,369, the disclosures of which are incorporated herein by reference in their entirety.

[0049] autologous T cells Provided herein are methods of treating multiple sclerosis (e.g., relapsing-remitting MS, secondary progressive MS, primary progressive MS, or progressive relapsing MS) by administering to a subject autologous T cells (e.g., CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on MHC. In some embodiments, the MHC is class I MHC. In some embodiments, the MHC is class II MHC.

[0050] In some embodiments, provided herein are APCs that present a peptide described herein (e.g., a peptide comprising an LMP1, LMP2A, or EBNA1 epitope sequence). In some embodiments, the APC is a B cell, an antigen-presenting T cell, a dendritic cell, or an artificial antigen-presenting cell (e.g., aK562 cell).

[0051] Dendritic cells for use in this process can be prepared by collecting PBMCs from a patient sample and allowing them to adhere to plastic. Generally, the monocyte population remains, allowing all other cells to be washed away. The adherent cell population is then differentiated with IL-4 and GM-CSF to produce monocyte-derived dendritic cells. These cells can be matured by the addition of IL-1β, IL-6, PGE-1, and TNF-α (which upregulates important costimulatory molecules on the surface of dendritic cells), and then transduced with one or more of the peptides provided herein.

[0052] In some embodiments, the APCs are artificial antigen-presenting cells, such as aK562 cells. In some embodiments, the artificial antigen-presenting cells are engineered to express CD80, CD83, 41BB-L, and / or CD86. Examples of artificial antigen-presenting cells, such as aK562 cells, are described in U.S. Patent Application Publication No. 2003 / 0147869, which is incorporated herein by reference.

[0053] In certain aspects, provided herein are methods for generating APCs that present one or more EBV epitopes, comprising contacting the APCs with peptides comprising an EBV epitope described herein and / or nucleic acids encoding the EBV epitopes. In some embodiments, the APCs are irradiated. In some embodiments, the APCs present a peptide described herein (e.g., a peptide comprising an LMP1, LMP2A, or EBNA1 epitope sequence). Cells that present the peptides described herein can be produced by standard techniques known in the art. For example, cells can be pulsed to promote peptide uptake. In some embodiments, cells are transfected with nucleic acids encoding the peptides provided herein. Provided herein are methods for producing antigen-presenting cells (APCs), comprising pulsing cells with the peptides described herein. Illustrative examples of producing antigen-presenting cells can be found in WO2013088114, incorporated herein in its entirety.

[0054] In some embodiments, provided herein are T cells (e.g., CD4 T cells and / or CD8 T cells) that express a TCR (e.g., an αβ TCR or a γδ TCR) that recognizes a peptide described herein presented on MHC. In some embodiments, the T cells are CD8 T cells (e.g., CTLs) that express a TCR that recognizes a peptide described herein presented on class I MHC. In some embodiments, the T cells are CD4 T cells (e.g., helper T cells) that recognize a peptide described herein presented on class II MHC.

[0055] In some embodiments, provided herein are methods for generating, activating, and / or inducing proliferation of T cells (e.g., autologous CTLs) that recognize one or more of the EBV epitopes described herein. In some embodiments, a sample containing autologous T cells (i.e., a PBMC sample) is incubated in culture with an APC provided herein (e.g., an APC that presents a peptide comprising an EBV epitope on a class I MHC complex). In some embodiments, the APC is autologous to the subject from which the T cells were obtained. In some embodiments, the APC is not autologous to the subject from which the T cells were obtained. In some embodiments, a sample containing T cells is incubated with an APC provided herein more than once. In some embodiments, the T cells are incubated with the APC in the presence of at least one cytokine. In some embodiments, the cytokine is IL-4, IL-7, and / or IL-15. Exemplary methods for inducing proliferation of T cells using APCs are provided, for example, in U.S. Patent Application Publication No. 2015 / 0017723, which is incorporated herein by reference.

[0056] In some embodiments, provided herein are compositions (e.g., therapeutic compositions) comprising the T cells and / or APCs provided herein, used to treat and / or prevent multiple sclerosis in a subject by administering an effective amount of the composition to the subject. In some aspects, provided herein are methods of treating multiple sclerosis using a composition (e.g., a pharmaceutical composition, such as a composition comprising autologous CTLs). In some embodiments, the composition comprises a combination of multiple (e.g., two or more) CTLs provided herein.

[0057] Treatment method In some embodiments, provided herein are methods for treating MS in a subject (e.g., primary progressive MS) by administering to the subject autologous T cells (e.g., autologous CTLs) provided herein. In some embodiments, the MS is relapsing-remitting MS, secondary progressive MS, primary progressive MS, or progressive relapsing MS. In some embodiments, autologous T cells are isolated from a peripheral blood mononuclear cell sample. Biomarker expression by autologous T cells can be assessed by any suitable method, for example, flow cytometry. In some embodiments, autologous T cells are stimulated with a vector comprising an EBV viral peptide (e.g., AdE1-LMPpoly). In some embodiments, autologous T cells are stimulated with a viral vector and sorted by flow cytometry. For example, autologous T cells can be surface stained according to the protocol exemplified in Example 2. In some embodiments, autologous T cells are incubated with one or more antibodies specific for CD107A, followed by sorting by flow cytometry. In some embodiments, autologous T cells are incubated with one or more antibodies that bind to intracellular cytokines, such as antibodies specific for IFNγ, IL-2, and / or TNF. In some embodiments, autologous T cells are incubated with antibodies against intracellular cytokines and then sorted by flow cytometry.

[0058] In some embodiments, provided herein are methods of selecting a subject for adoptive immunotherapy by obtaining a PMBC sample from a subject, isolating autologous T cells, determining the EBV reactivity of the autologous T cells, and selecting the subject for adoptive immunotherapy if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the autologous T cells are EBV reactive.

[0059] In some embodiments, provided herein are methods of selecting a subject for adoptive immunotherapy by obtaining a sample comprising T cells (e.g., CTLs) from a subject, isolating autologous T cells, determining CD107A expression on the autologous T cells, and selecting the subject for adoptive immunotherapy if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the autologous T cells express CD107A.

[0060] In some embodiments, provided herein are methods of selecting a subject for adoptive immunotherapy by obtaining a sample comprising T cells (e.g., CTLs) from a subject, isolating autologous T cells, determining IFNγ expression of the autologous T cells, and selecting the subject for adoptive immunotherapy if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the autologous T cells express IFNγ.

[0061] In some embodiments, provided herein are methods of selecting a subject for adoptive immunotherapy by obtaining a sample comprising T cells (e.g., CTLs) from a subject, isolating autologous T cells, determining TNF expression in the autologous T cells, and selecting the subject for adoptive immunotherapy if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the autologous T cells express TNF.

[0062] In some embodiments, provided herein are methods of selecting a subject for adoptive immunotherapy by obtaining a sample comprising T cells (e.g., CTLs) from a subject, isolating autologous T cells, determining IL-2 expression of the autologous T cells, and selecting the subject for adoptive immunotherapy if at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the autologous T cells express IL-2.

[0063] In some embodiments, the method further includes obtaining a sample comprising T cells from the subject (e.g., obtaining a PBMC sample from the subject). In some embodiments, autologous T cells (e.g., CD4+ T cells or CD8+ T cells) are isolated from the sample. In some embodiments, the sample is composed mostly or entirely of autologous T cells.

[0064] Provided herein are methods for treating or preventing multiple sclerosis (MS) in a subject, comprising administering to the subject autologous cytotoxic T cells (CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the T cells (e.g., CTLs) in the sample express CD107A. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the T cells (e.g., CTLs) in the sample express IFNγ. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the T cells (e.g., CTLs) in the sample express TNF. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, or 80% of the T cells (e.g., CTLs) in the sample express IL-2.

[0065] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 12 , 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107A and IFNγ.

[0066] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 12 , 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107A and TNF.

[0067] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 12 , 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107A and IL-2.

[0068] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 12 %, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express IFNγ and TNF.

[0069] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 12 %, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express IFNγ and IL-2.

[0070] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 12 %, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express TNF and IL-2.

[0071] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 12 6%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express IFNγ, TNF, and IL-2.

[0072] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 12 %, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107A, TNF, and IL-2.

[0073] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 12 %, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107A, IFNγ, and IL-2.

[0074] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 12 %, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107A, IFNγ, and TNF.

[0075] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 12 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107A, IFNγ, TNF, and IL-2.

[0076] In some embodiments, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, , 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% are EBV reactive.

[0077] T cell biomarker expression and / or EBV reactivity may be measured and / or analyzed either before or after T cell expansion (proliferation) using the nucleic acid constructs disclosed herein, the polypeptides disclosed herein or the APCs.

[0078] In some aspects, provided herein are methods of treating or preventing MS in a subject by incubating antigen-presenting cells (APCs) with a nucleic acid construct encoding an EBV peptide, thereby inducing the APCs to present the EBV peptide and inducing proliferation of peptide-specific T cells (e.g., CTLs), or by incubating a sample containing autologous T cells (e.g., CTLs) with antigen-presenting cells (APCs), thereby inducing proliferation of the autologous T cells (e.g., CTLs), and administering the peptide-specific autologous T cells (e.g., CTLs) to the subject. In some embodiments, EBV reactivity and biomarker expression are quantified before stimulating autologous T cells with a viral vector (e.g., a viral vector disclosed herein) and / or APCs (e.g., APCs disclosed herein). Alternatively or additionally, EBV reactivity and biomarker expression may be quantified after stimulating autologous T cells with a viral vector (e.g., a viral vector disclosed herein) and / or APCs (e.g., APCs transfected with a viral vector disclosed herein). In some embodiments, EBV reactivity is measured by quantifying the percentage of T cells in a sample that express CD107A. In some embodiments, EBV reactivity is measured by quantifying the percentage of T cells in a sample that express IFNγ. In some embodiments, EBV reactivity is measured by quantifying the percentage of T cells in a sample that express TNF. In some embodiments, EBV reactivity is measured by quantifying the percentage of T cells in a sample that express IL-2. In some embodiments, EBV reactivity is measured as the percentage of T cells that express multiple biomarkers (e.g., two or more, preferably all four, of CD107A, IFNγ, TNF, and IL-2). In some embodiments, EBV reactivity is calculated by quantifying the percentage of autologous T cells in a sample that express CD107A, IFNγ, TNF, and IL-2. T cells may be isolated from a sample (e.g., a PBMC sample or a sample containing T cells) either before or after determining the percentage of EBV reactivity. Thus, in some embodiments, EBV reactivity is the percentage of T cells with the desired characteristics in a sample containing mostly T cells.

[0079] In some embodiments, EBV reactivity is measured by quantifying the percentage of CD8+ lymphocytes in a sample that express CD107A. In some embodiments, EBV reactivity is measured by quantifying the percentage of CD8+ lymphocytes in a sample that express IFNγ. In some embodiments, EBV reactivity is measured by quantifying the percentage of CD8+ lymphocytes in a sample that express TNF. In some embodiments, EBV reactivity is measured by quantifying the percentage of CD8+ lymphocytes in a sample that express IL-2. In some embodiments, EBV reactivity is measured as the percentage of CD8+ lymphocytes that express multiple biomarkers (e.g., two or more, preferably all four, of CD107A, IFNγ, TNF, and IL-2). CD8+ lymphocytes can be isolated from a sample (e.g., a PBMC sample or a CD8+ lymphocyte sample) either before or after quantifying the percentage of EBV reactivity. Thus, in some embodiments, EBV reactivity is the percentage of CD8+ lymphocytes with the desired characteristics in a sample containing mostly or entirely CD8+ lymphocytes.

[0080] In some embodiments, EBV reactivity is measured by quantifying the percentage of CD3+ lymphocytes in a sample that express CD107A. In some embodiments, EBV reactivity is measured by quantifying the percentage of CD3+ lymphocytes in a sample that express IFNγ. In some embodiments, EBV reactivity is measured by quantifying the percentage of CD3+ lymphocytes in a sample that express TNF. In some embodiments, EBV reactivity is measured by quantifying the percentage of CD3+ lymphocytes in a sample that express IL-2. In some embodiments, EBV reactivity is measured as the percentage of CD3+ lymphocytes that express multiple biomarkers (e.g., two or more, preferably all four, of CD107A, IFNγ, TNF, and IL-2). CD3+ lymphocytes can be isolated from a sample (e.g., a PBMC sample or a CD3+ lymphocyte sample) either before or after quantifying the percentage of EBV reactivity. Thus, in some embodiments, EBV reactivity is the percentage of CD3+ lymphocytes with the desired characteristics in a sample containing mostly CD3+ lymphocytes.

[0081] In some embodiments, the method comprises analyzing the expression of CD107a, IFNγ, TNF or IL-2 by expanded peptide-specific autologous T cells (e.g., CTLs), and determining whether at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, If 2%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107a, IFNγ, TNF, or IL-2, the method further comprises administering peptide-specific autologous T cells (e.g., CTLs) to the subject.

[0082] In some embodiments, the method includes analyzing expression of CD107a and TNF by expanded peptide-specific autologous T cells (e.g., CTLs), and determining whether at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, If 2%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107a and TNF, the method further comprises administering peptide-specific autologous T cells (e.g., CTLs) to the subject.

[0083] In some embodiments, the method comprises analyzing expression of CD107a and IFNγ by expanded peptide-specific autologous T cells (e.g., CTLs), and determining whether at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115 If 2%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107a and IFNγ, further comprising administering peptide-specific autologous T cells (e.g., CTLs) to the subject.

[0084] In some embodiments, the method includes analyzing expression of CD107a and IL-2 by expanded peptide-specific autologous T cells (e.g., CTLs), and determining whether at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115 If 2%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107a and IL-2, the method further comprises administering peptide-specific autologous T cells (e.g., CTLs) to the subject.

[0085] In some embodiments, the method includes analyzing the expression of TNF and IL-2 by expanded peptide-specific autologous T cells (e.g., CTLs), and determining whether at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, If 2%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express TNF and IL-2, further comprising administering peptide-specific autologous T cells (e.g., CTLs) to the subject.

[0086] In some embodiments, the method comprises analyzing expression of IFNγ and IL-2 by expanded peptide-specific autologous T cells (e.g., CTLs), and determining whether at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, If 2%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express IFNγ and IL-2, further comprising administering peptide-specific autologous T cells (e.g., CTLs) to the subject.

[0087] In some embodiments, the method comprises analyzing expression of IFNγ and TNF by expanded peptide-specific autologous T cells (e.g., CTLs), and determining whether at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 1 If 2%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express IFNγ and TNF, further comprising administering peptide-specific autologous T cells (e.g., CTLs) to the subject.

[0088] In some embodiments, the method includes analyzing expression of CD107a, IFNγ, and TNF by expanded peptide-specific autologous T cells (e.g., CTLs), and determining whether at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, If 2%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107a, IFNγ, and TNF, the method further comprises administering peptide-specific autologous T cells (e.g., CTLs) to the subject.

[0089] In some embodiments, the method includes analyzing expression of CD107a, IFNγ, and IL-2 by expanded peptide-specific autologous T cells (e.g., CTLs), and determining whether at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 11 If 2%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107a, IFNγ, and IL-2, the method further comprises administering peptide-specific autologous T cells (e.g., CTLs) to the subject.

[0090] In some embodiments, the method includes analyzing expression of CD107a, IL-2, and TNF by expanded peptide-specific autologous T cells (e.g., CTLs), and determining whether at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, If 2%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107a, IL-2, and TNF, the method further comprises administering peptide-specific autologous T cells (e.g., CTLs) to the subject.

[0091] In some embodiments, the method includes analyzing expression of IFNγ, IL-2, and TNF by expanded peptide-specific autologous T cells (e.g., CTLs), and determining whether at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, If 2%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express IFNγ, IL-2, and TNF, further comprising administering peptide-specific autologous T cells (e.g., CTLs) to the subject.

[0092] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 12 If 9%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107a, IFNγ, TNF, and IL-2, autologous T cells (e.g., CTLs) are administered to the subject.

[0093] Peptide-specific autologous T cells (e.g., CTLs) account for at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, , 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% may be EBV reactive.

[0094] In some embodiments, about 1 x 10 per dose of T cells 5 ~Approx. 1×10 8 In some embodiments, about 1 x 10 T cells are administered to the subject per dose of T cells. 6 ~Approx. 1×10 7 In some embodiments, 5 x 10 T cells are administered to the subject. 6 , 1×10 7 , 1.5×10 7 , or 2 × 10 7T cells (e.g., CTLs) are administered to the subject. Multiple doses may be administered to the subject. In some embodiments, an initial dose of T cells (e.g., autologous CTLs) is administered, followed by one or more additional doses of T cells (e.g., autologous CTLs), e.g., at escalating doses along the course of treatment. In some embodiments, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more doses are administered. The subject may also receive additional doses that are the same as or different from the initial dose. For example, a low dose may be administered, followed by a higher dose. Doses may be administered daily, twice weekly, weekly, every other week, monthly, every two months, every three months, or every six months. In some embodiments, the subject does not experience any adverse effects as a result of the administration of the T cells (e.g., autologous CTLs).

[0095] In some embodiments, the method further comprises obtaining a first sample of cerebrospinal fluid (CSF) from the subject, analyzing the amount of anti-EBV IgG in the CSF in the first sample (preferably before CTL administration), obtaining a second sample of CSF from the subject after a period of time (preferably after CTL administration), analyzing the amount of anti-EBV IgG in the CSF in the second sample, and evaluating the efficacy of adoptive immunotherapy in a subject with multiple sclerosis if the amount of anti-EBV IgG in the second sample is lower than in the first sample, indicating stable disease and / or no progression. Additional samples of CSF may be obtained and compared with the previous sample. Also provided herein are methods for reducing anti-EBV IgG levels in CSF in a subject with MS by administering to the subject autologous T cells (e.g., CTLs) expressing a T cell receptor that specifically binds to an EBV peptide presented on class I MHC. The reduction in anti-EBV IgG levels in CSF may be measured by the CSF IgG index. In some embodiments, CSF IgG levels may be calculated by the Reiber and Felgenhauer formula (i.e., see Figure 1). Anti-EBV IgG levels may be reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% following administration of the T cells.

[0096] In some embodiments, the methods include improving or stabilizing symptoms of MS in a subject (e.g., vision loss, visual acuity loss, loss or reduced manual dexterity, increased fatigue, and / or urinary urgency) by administering to the subject autologous T cells (e.g., CTLs, such as peptide-specific autologous CTLs described herein) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC. Also provided herein are methods of improving vision, improving color vision, or stabilizing visual decline in a subject with MS, comprising administering to the subject autologous T cells (e.g., CTLs, such as peptide-specific autologous CTLs described herein). In some embodiments, provided herein are methods of improving motor skills, balance, or manual dexterity in a subject with MS, comprising administering to the subject autologous T cells described herein. Also provided herein are methods of improving sleep in a subject, comprising administering to the subject autologous T cells (e.g., CTLs, such as peptide-specific autologous CTLs described herein).

[0097] In some embodiments, the subject undergoes a diagnostic test, such as the EDSS. In some embodiments, the subject undergoes an EDSS test and receives an EDSS score before and / or after administration of the T cells. After administration of the T cells, the EDSS score may remain the same, or the EDSS score may be reduced (e.g., by at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0).

[0098] The various methods disclosed herein can be methods of improving gait, vision, balance, cognition, or other symptoms in a subject (e.g., a subject with multiple sclerosis), and / or methods of improving the Multiple Sclerosis Functional Composite (MSFC), EDSS, or MSSS score in a subject (e.g., a subject with multiple sclerosis). Accordingly, in certain embodiments, treatment methods disclosed herein include methods of stabilizing or improving a disability or symptom (e.g., motor skills / balance / manual dexterity, sleep, visual or color vision, fatigue, urinary urgency) in a patient, such that the patient's disability score (as measured by these tests or another suitable test) after 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 3 months, 6 months, 1 year, or 2 years of treatment is at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 25%, at least about 40%, at least about 50%, or even at least about 60% higher than the patient's EDSS score before T cell therapy.

[0099] For example, a subject's EDSS score may be tested, e.g., by assessing the subject's performance on the test at various time points (e.g., 0 months (baseline), 1 month, 2 months, 3 months, 6 months, 1 year, and 2 years). In certain embodiments, if there is a recorded decrease in the subject's score after administration of the T cells, then the MS is considered stable and / or not progressing. In other embodiments, if there is no increase or decrease in the subject's score after administration of the T cells, then the MS is considered stable and / or not progressing. EDSS testing may be repeated at any time from the start of CTL treatment (e.g., at 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months after the start of CTL treatment) to assess whether the treatment has slowed or stopped further deterioration of motor skills / balance / manual dexterity, improved sleep, improved visual or color vision, fatigue, and urinary urgency.

[0100] In some embodiments, the progression of gait impairment can be assessed using a gait test, for example, by assessing the subject's ability to walk a 25-foot walk at different time points. In certain embodiments, if there is no recorded deterioration in gait, the subject is then considered to have no progressive deterioration in gait. For patients who have not already received T cell therapy, subjects who exhibit progressive gait impairment are initiated into treatment with T cells (e.g., CTLs). The gait test can be repeated (e.g., at 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, and 3 months after the start of treatment) to assess whether the treatment has slowed or stopped further deterioration of walking ability (e.g., as measured by the gait test).

[0101] Whether there has been an improvement in cognitive outcome associated with MS treatment, i.e., slowing of cognitive decline, stabilization of cognitive decline, or improvement in cognitive function, can be assessed using the PASAT (e.g., PASAT 2 or PASAT 3) or SDMT tests, or using the MS-COG test (see Erlanger et al., J Neuro Sci 340: 123-129 (2014)).

[0102] Actual dosage levels of the active ingredients in the pharmaceutical compositions provided herein may be varied to achieve an amount, composition, and method of administration of the active ingredients that is not toxic to the patient and is effective to achieve the desired therapeutic response for a particular patient.

[0103] The selected dosage level will depend upon a variety of factors, such as the activity of the particular agent used, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular compound used, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical arts. The present invention encompasses, for example, the following embodiments: [Embodiment 1] A method for treating or preventing multiple sclerosis (MS) in a subject, comprising administering to the subject autologous cytotoxic T cells (CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC. [Embodiment 2] The method of embodiment 1, wherein at least 5% of the CTLs express CD107a. [Embodiment 3] The method of embodiment 1, wherein at least 10% of the CTLs express CD107a. [Embodiment 4] The method of embodiment 1, wherein at least 15% of the CTLs express CD107a. [Embodiment 5] The method of embodiment 1, wherein at least 20% of the CTLs express CD107a. [Embodiment 6] The method of any one of embodiments 1 to 5, wherein at least 5% of the CTLs express IFNγ. [Embodiment 7] The method of embodiment 6, wherein at least 10% of the CTLs express IFNγ. [Embodiment 8] The method of embodiment 7, wherein at least 15% of the CTLs express IFNγ. [Embodiment 9] The method of embodiment 8, wherein at least 20% of the CTLs express IFNγ. [Embodiment 10] The method of any one of embodiments 1 to 9, wherein at least 5% of the CTLs express TNF. [Embodiment 11] The method of embodiment 10, wherein at least 10% of the CTLs express TNF. [Embodiment 12] The method of embodiment 11, wherein at least 15% of the CTLs express TNF. [Embodiment 13] The method of embodiment 12, wherein at least 20% of the CTLs express TNF. [Embodiment 14] The method of any one of embodiments 1 to 13, wherein at least 1% of the CTLs express IL-2. [Embodiment 15] The method of embodiment 14, wherein at least 5% of the CTLs express IL-2. [Embodiment 16] The method of embodiment 15, wherein at least 10% of the CTLs express IL-2. [Embodiment 17] The method of embodiment 16, wherein at least 15% of the CTLs express IL-2. [Embodiment 18] The method of embodiment 1, wherein at least 30% of the CTLs express CD107a, IFNγ, TNFα, and IL-2. [Embodiment 19] The method of embodiment 1, wherein at least 40% of the CTLs express CD107a, IFNγ, TNFα, and IL-2. [Embodiment 20] The method of embodiment 1, wherein at least 50% of the CTLs express CD107a, IFNγ, TNFα, and IL-2. [Embodiment 21] The method of embodiment 1, wherein at least 70% of the CTLs express CD107a, IFNγ, TNFα, and IL-2. [Embodiment 22] The method of any one of embodiments 1 to 21, wherein the CTLs have at least 5% EBV reactivity. [Embodiment 23] The method of embodiment 22, wherein the CTLs have an EBV reactivity of at least 7%. [Embodiment 24] The method of embodiment 23, wherein the CTLs have an EBV reactivity of at least 10%. [Embodiment 25] The method of embodiment 24, wherein the CTLs have an EBV reactivity of at least 15%. [Embodiment 26] The method of embodiment 25, wherein the CTLs have an EBV reactivity of at least 20%. [Embodiment 27] The method of embodiment 26, wherein the CTLs have an EBV reactivity of at least 30%. [Embodiment 28] A method of treating or preventing multiple sclerosis (MS) in a subject, comprising: (a) isolating from a subject a sample containing cytotoxic T cells (CTLs) that express T cell receptors that specifically bind to EBV peptides presented on class I MHC; (b) administering CTLs to a subject; A method comprising: [Embodiment 29] A method of treating or preventing MS in a subject, comprising: (a) incubating a sample containing autologous cytotoxic T cells (CTLs) with antigen-presenting cells (APCs) that present EBV peptides, thereby inducing proliferation of peptide-specific T cells in the sample; (b) administering peptide-specific autologous CTLs to the subject; A method comprising: [Embodiment 30] A method of treating or preventing MS in a subject, comprising: (a) incubating antigen-presenting cells (APCs) with a nucleic acid construct encoding an EBV peptide, thereby inducing the APCs to present the EBV peptide; (b) inducing peptide-specific CTL proliferation by incubating the sample containing autologous CTLs with antigen-presenting cells (APCs), thereby inducing the autologous CTLs to proliferate; and (c) administering peptide-specific autologous CTLs to the subject; A method comprising: [Embodiment 31] The method described in embodiment 30, wherein the nucleic acid construct is a viral vector. [Embodiment 32] The method described in embodiment 31, wherein the viral vector is AdE1-LMPpoly. [Embodiment 33] The method described in any one of embodiments 29 to 32, further comprising analyzing the expression of CD107a by the expanded peptide-specific autologous CTLs, and administering the peptide-specific autologous CTLs to a subject if at least 5% of the expanded peptide-specific autologous CTLs express CD107a. [Embodiment 34] The method of embodiment 33, wherein if at least 10% of the expanded peptide-specific autologous CTLs in the sample express CD107a, the CTLs are administered. [Embodiment 35] The method of embodiment 33 or 34, wherein if at least 15% of the expanded peptide-specific autologous CTLs in the sample express CD107a, the CTLs are administered. [Embodiment 36] The method of any one of embodiments 33 to 35, wherein if at least 20% of the expanded peptide-specific autologous CTLs in the sample express CD107A, the CTLs are administered. [Embodiment 37] The method described in any one of embodiments 29 to 36, further comprising analyzing the expression of IFNγ by the expanded peptide-specific autologous CTLs, and administering the peptide-specific autologous CTLs to a subject if at least 5% of the expanded peptide-specific autologous CTLs express IFNγg. [Embodiment 38] The method of embodiment 37, wherein if at least 10% of the expanded peptide-specific autologous CTLs in the sample express IFNγ, the CTLs are administered. [Embodiment 39] The method of embodiment 37 or 38, wherein if at least 15% of the expanded peptide-specific autologous CTLs in the sample express IFNγ, the CTLs are administered. [Embodiment 40] The method of any one of embodiments 37 to 39, wherein if at least 20% of the expanded peptide-specific autologous CTLs in the sample express IFNγ, the CTLs are administered. [Embodiment 41] The method described in any one of embodiments 29 to 40, further comprising analyzing the expression of TNF by the expanded peptide-specific autologous CTLs, and administering the peptide-specific autologous CTLs to a subject if at least 5% of the expanded peptide-specific autologous CTLs express TNF. [Embodiment 42] The method of embodiment 41, wherein if at least 10% of the expanded peptide-specific autologous CTLs in the sample express TNF, the CTLs are administered. [Embodiment 43] The method of embodiment 41 or 42, wherein if at least 15% of the expanded peptide-specific autologous CTLs in the sample express TNF, the CTLs are administered. [Embodiment 44] The method of any one of embodiments 41 to 43, wherein if at least 20% of the expanded peptide-specific autologous CTLs in the sample express TNF, the CTLs are administered. [Embodiment 45] The method described in any one of embodiments 29 to 44, further comprising analyzing the expression of IL-2 by the expanded peptide-specific autologous CTLs, and administering the peptide-specific autologous CTLs to a subject if at least 1% of the expanded peptide-specific autologous CTLs express IL-2. [Embodiment 46] The method of embodiment 45, wherein if at least 5% of the peptide-specific autologous CTLs in the sample express IL-2, the CTLs are administered. [Embodiment 47] The method of embodiment 45 or 46, wherein if at least 10% of the peptide-specific autologous CTLs in the sample express IL-2, the CTLs are administered. [Embodiment 48] The method of any one of embodiments 45 to 47, wherein if at least 15% of the expanded peptide-specific autologous CTLs in the sample express IL-2, the CTLs are administered. [Embodiment 49] The method described in any one of embodiments 29 to 32, further comprising analyzing the expression of CD107a, TNF, IFNγ, and IL-2 by the expanded peptide-specific autologous CTLs, and administering the peptide-specific autologous CTLs to the subject if at least 20% of the expanded peptide-specific autologous CTLs express CD107a, TNF, IFNγ, and IL-2. [Embodiment 50] The method of embodiment 49, wherein the CTLs are administered if at least 30% of the expanded peptide-specific autologous CTLs in the sample express CD107a, TNF, IFNγ, and IL-2. [Embodiment 51] The method of embodiment 49 or 50, wherein the CTLs are administered if at least 40% of the expanded peptide-specific autologous CTLs in the sample express CD107a, TNF, IFNγ, and IL-2. [Embodiment 52] The method of any one of embodiments 49 to 51, wherein the CTLs are administered if at least 50% of the expanded peptide-specific autologous CTLs in the sample express CD107a, TNF, IFNγ, and IL-2. [Embodiment 53] The method described in any one of embodiments 29 to 52, further comprising analyzing the EBV reactivity of the expanded peptide-specific autologous CTLs, and administering the peptide-specific autologous CTLs to the subject if the reactivity exceeds a predetermined threshold. [Embodiment 54] The method described in embodiment 53, wherein the threshold is 5%. [Embodiment 55] The method described in embodiment 53, wherein the threshold is 7%. [Embodiment 56] The method described in embodiment 53, wherein the threshold is 10%. [Embodiment 57] The method described in embodiment 53, wherein the threshold is 20%. [Embodiment 58] The method described in embodiment 53, wherein the threshold is 30%. [Embodiment 59] The method described in embodiment 53, wherein the threshold is 50%. [Embodiment 60] The method of any one of embodiments 29 to 59, wherein the sample is incubated with one or more cytokines in step (a). [Embodiment 61] The method of any one of embodiments 29 to 60, wherein the APC comprises a B cell. [Embodiment 62] The method described in any one of embodiments 29 to 61, wherein the APC comprises an antigen-presenting T cell. [Embodiment 63] The method described in any one of embodiments 29 to 62, wherein the APC comprises a dendritic cell. [Embodiment 64] The method described in any one of embodiments 29 to 63, wherein the APC comprises an artificial antigen-presenting cell. [Embodiment 65] The method described in embodiment 64, wherein the artificial antigen-presenting cells are aK562 cells. [Embodiment 66] The method described in any one of embodiments 28 to 65, wherein the sample comprises peripheral blood mononuclear cells (PBMCs). [Embodiment 67] The method of any one of embodiments 1 to 66, further comprising obtaining a sample from a subject. [Embodiment 68] The method of any one of embodiments 1 to 67, wherein the EBV peptide comprises an amino acid sequence listed in Table 1. [Embodiment 69] A method according to any one of embodiments 1 to 67, wherein the EBV peptide comprises an LMP1 peptide or a fragment thereof. [Embodiment 70] A method according to any one of embodiments 1 to 67, wherein the EBV peptide comprises an LMP2A peptide or a fragment thereof. [Embodiment 71] A method according to any one of embodiments 1 to 67, wherein the EBV peptide comprises an EBNA1 peptide or a fragment thereof. [Embodiment 72] One dose of about 5 x 10 6 72. The method of any preceding embodiment, comprising administering to the subject the CTLs. [Embodiment 73] One dose of about 1 x 10 7 72. The method of any preceding embodiment, comprising administering to the subject the CTLs. [Embodiment 74] One dose of about 1.5 x 10 7 72. The method of any preceding embodiment, comprising administering to the subject the CTLs. [Embodiment 75] One dose of about 2 x 10 7 72. The method of any preceding embodiment, comprising administering to the subject the CTLs. [Embodiment 76] The method of any one of embodiments 1 to 75, wherein multiple doses of CTLs are administered to the subject, for example, in escalating doses. [Embodiment 77] The method of embodiment 76, wherein the dose is administered weekly. [Embodiment 78] The method of embodiment 76, wherein the dose is administered every other week. [Embodiment 79] The method of embodiment 78, comprising administering four doses of successively greater numbers of CTLs. [Embodiment 80] 5 x 10 6 First dose of CTLs: 1 x 10 7 Second dose of CTLs, 1.5 x 10 7 a third dose of CTLs, and 2 x 10 7 80. The method of embodiment 79, comprising administering a fourth dose of the CTLs. [Embodiment 81] The method of any one of embodiments 1 to 80, wherein the subject does not experience significant adverse effects as a result of CTL administration.

[0082]

[0083] [Embodiment 82] The method further comprises assessing the efficacy of adoptive immunotherapy in a subject with multiple sclerosis, (a) obtaining a first sample of cerebrospinal fluid (CSF) from a subject; (b) determining the amount of anti-EBV IgG in the CSF in the first sample before CTL administration; (c) after a period of time, obtaining a second sample of CSF from the subject after CTL administration; (d) determining the amount of anti-EBV IgG in the CSF in the second sample; Including, The method wherein the disease is stable and / or not progressing if the amount of anti-EBV IgG in the second sample is lower than in the first sample. [Embodiment 83] The method of any one of embodiments 1 to 82, wherein the MS is relapsing-remitting MS, secondary progressive MS, primary progressive MS, or progressive relapsing MS. [Embodiment 84] The method of embodiment 83, wherein the MS is primary progressive MS. [Embodiment 85] The method of any one of embodiments 1 to 84, wherein the subject's motor skills / balance / manual dexterity, sleep, eyesight or color vision, fatigue, and / or urinary urgency are improved after CTL administration. [Embodiment 86] The method of any one of embodiments 1 to 85, wherein the subject's EDSS score remains the same after CTL administration. [Embodiment 87] The method of any one of embodiments 1 to 85, wherein the subject's EDSS score is reduced by at least 0.5 after CTL administration. [Embodiment 88] The method of any one of embodiments 1 to 85, wherein the subject's EDSS score is reduced by at least 1.0 after CTL administration. [Embodiment 89] A method for reducing anti-EBV IgG levels in the CSF in a subject with MS, comprising administering to the subject autologous cytotoxic T cells (CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC. [Embodiment 90] A method for improving vision, improving color vision, or stabilizing vision loss in a subject with MS, comprising administering to the subject autologous cytotoxic T cells (CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC. [Embodiment 91] A method for improving motor skills, balance, or manual dexterity in a subject with MS, comprising administering to the subject autologous cytotoxic T cells (CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC. [Embodiment 92] A method for improving sleep in a subject with MS, comprising administering to the subject autologous cytotoxic T cells (CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC. [Embodiment 93] A method for treating or preventing fatigue in a subject with MS, comprising administering to the subject autologous cytotoxic T cells (CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC. [Embodiment 94] A method for treating or preventing urinary urgency in a subject with MS, comprising administering to the subject autologous cytotoxic T cells (CTLs) that express a T cell receptor that specifically binds to an EBV peptide presented on class I MHC. [Embodiment 95] The method of any one of embodiments 1 to 94, wherein the MS is relapsing-remitting MS. [Embodiment 96] The method of any one of embodiments 1 to 94, wherein the MS is secondary progressive MS. [Embodiment 97] The method of any one of embodiments 1 to 94, wherein the MS is primary progressive MS. [Embodiment 98] The method of any one of embodiments 1 to 94, wherein the MS is primary progressive progressive relapsing MS.

[0099] A method for selecting a subject for adoptive immunotherapy, comprising: (a) obtaining a sample comprising T cells from a subject; (b) isolating autologous T cells in the sample; (c) determining the EBV reactivity of autologous T cells in the sample and selecting the subject for adoptive immunotherapy if at least a threshold percentage (%) of the autologous T cells are EBV reactive; A method comprising: [Embodiment 100] The method described in embodiment 99, wherein the threshold is 1%. [Embodiment 101] The method described in embodiment 99, wherein the threshold is 2%. [Embodiment 102] The method described in embodiment 99, wherein the threshold is 3%. [Embodiment 103] The method described in embodiment 99, wherein the threshold is 5%. [Embodiment 104] The method described in embodiment 99, wherein the threshold is 10%. [Embodiment 105] The method described in embodiment 99, wherein the threshold is 20%.

[00197] [Embodiment 106] A method for selecting a subject for adoptive immunotherapy, comprising: (a) obtaining a sample comprising T cells from a subject; (b) isolating autologous T cells in the sample; (c) determining the percentage (%) of autologous T cells in the sample that express CD107A, and selecting the subject for adoptive immunotherapy if at least a certain percentage (%) of the autologous T cells express CD107A. [Embodiment 107] The method described in embodiment 106, wherein the subject is selected for adoptive immunotherapy if at least 1% of autologous T cells express CD107A. [Embodiment 108] The method of embodiment 106, wherein the subject is selected for adoptive immunotherapy if at least 3% of autologous T cells express CD107A. [Embodiment 109] The method described in embodiment 106, wherein the subject is selected for adoptive immunotherapy if at least 5% of autologous T cells express CD107A. [Embodiment 110] The method described in embodiment 106, wherein the subject is selected for adoptive immunotherapy if at least 10% of autologous T cells express CD107A.

[0092] [Embodiment 111] A method for selecting a subject for adoptive immunotherapy, comprising: (a) obtaining a sample comprising T cells from a subject; (b) isolating autologous T cells in the sample; (c) determining the percentage of autologous T cells in the sample that express TNF, and selecting the subject for adoptive immunotherapy if at least a certain percentage of the autologous T cells express TNF. A method comprising: [Embodiment 112] The method described in embodiment 111, wherein the subject is selected for adoptive immunotherapy if at least 1% of autologous T cells express TNF. [Embodiment 113] The method described in embodiment 111, wherein the subject is selected for adoptive immunotherapy if at least 3% of autologous T cells express TNF. [Embodiment 114] The method described in embodiment 111, wherein the subject is selected for adoptive immunotherapy if at least 5% of autologous T cells express TNF. [Embodiment 115] The method described in embodiment 111, wherein the subject is selected for adoptive immunotherapy if at least 10% of autologous T cells express TNF.

[00199] [Embodiment 116] A method for selecting a subject for adoptive immunotherapy, comprising: (a) obtaining a sample comprising T cells from a subject; (b) isolating autologous T cells in the sample; (c) determining the percentage of autologous T cells in the sample that express IFNγ, and selecting the subject for adoptive immunotherapy if at least a certain percentage of the autologous T cells express IFNγ. A method comprising: [Embodiment 117] The method described in embodiment 116, wherein the subject is selected for adoptive immunotherapy if at least 1% of autologous T cells express IFNγ. [Embodiment 118] The method described in embodiment 116, wherein the subject is selected for adoptive immunotherapy if at least 3% of autologous T cells express IFNγ. [Embodiment 119] The method described in embodiment 116, wherein the subject is selected for adoptive immunotherapy if at least 5% of autologous T cells express IFNγ. [Embodiment 120] The method described in embodiment 116, wherein the subject is selected for adoptive immunotherapy if at least 10% of autologous T cells express IFNγ.

[00199] [Embodiment 121] A method for selecting a subject for adoptive immunotherapy, comprising: (a) obtaining a sample comprising T cells from a subject; (b) isolating autologous T cells in the sample; (c) determining the percentage of autologous T cells in the sample that express IL-2, and selecting the subject for adoptive immunotherapy if at least a certain percentage of the autologous T cells express IL-2. A method comprising: [Embodiment 122] The method described in embodiment 116, wherein the subject is selected for adoptive immunotherapy if at least 1% of autologous T cells express IL-2. [Embodiment 123] The method described in embodiment 116, wherein the subject is selected for adoptive immunotherapy if at least 3% of autologous T cells express IL-2. [Embodiment 124] The method described in embodiment 116, wherein the subject is selected for adoptive immunotherapy if at least 5% of autologous T cells express IL-2. [Embodiment 125] The method described in embodiment 116, wherein the subject is selected for adoptive immunotherapy if at least 10% of autologous T cells express IL-2.

[00199] [Embodiment 126] A method for selecting a subject for adoptive immunotherapy, comprising: (a) obtaining a PMBC sample from a subject; (b) isolating autologous T cells in the sample; (c) determining the percentage of autologous T cells in the sample that express CD107a, IFNγ, TNFα, and IL-2, and selecting the subject for adoptive immunotherapy if at least a certain percentage of the autologous T cells express CD107a, IFNγ, TNFα, and IL-2. A method comprising: [Embodiment 127] The method described in embodiment 126, wherein the subject is selected for adoptive immunotherapy if at least 1% of autologous T cells express CD107a, IFNγ, TNFα, and IL-2. [Embodiment 128] The method described in embodiment 126, wherein the subject is selected for adoptive immunotherapy if at least 3% of autologous T cells express CD107a, IFNγ, TNFα, and IL-2. [Embodiment 129] The method described in embodiment 126, wherein the subject is selected for adoptive immunotherapy if at least 5% of autologous T cells express CD107a, IFNγ, TNFα, and IL-2. [Embodiment 130] The method described in embodiment 126, wherein the subject is selected for adoptive immunotherapy if at least 10% of the autologous T cells express CD107a, IFNγ, TNFα, and IL-2.

[0104] [Example 1] A study of multiple treatments with EBV-specific T cell therapy for MS patients and MS Participants provided a 200-400 mL blood sample. Peripheral blood mononuclear cells from this sample were used for laboratory generation of autologous latent membrane protein (LMP1&2) / Epstein-Barr virus nuclear antigen 1 (EBNA1)-specific T cells suspended in clinical-grade saline. The investigational drug was produced by stimulation with gamma-irradiated autologous peripheral blood mononuclear cells infected with the recombinant adenoviral vector AdE1-LMPpoly, an adenoviral vector encoding multiple CD8+ T cell epitopes from EBV nuclear antigen-1 (EBNA1), latent membrane protein 1 (LMP1), and LMP2A. T cell cultures were then evaluated for cell yield, viability, and T cell frequency. It typically takes approximately 5 weeks from collection of the 200-400 mL blood sample to the first cell administration.

[0105] Each patient received their own T cells stimulated ex vivo to enhance reactivity against EBNA1, LMP1, and LMP2A and were followed for 26 weeks. Patients received T cell therapy intravenously every 2 weeks. Each dose was given once, with the first dose consisting of 5 x 10 6 T cells, followed by 1 × 10 7 pieces, 1.5×10 7 pieces, 2×10 7 The dose was 100 cells. A total of 4 doses were given over 8 weeks. Each dose of cells was administered via intravenous line infusion, allowing for a slow administration of T cells into the blood rather than a bolus of cells.

[0106] Thirteen patients were enrolled. Three patients discontinued before receiving cell therapy: one due to an unrelated diagnosis of malignancy and two due to an inability to generate EBV-specific T cells. The remaining 10 patients each received four T cell infusions per protocol (Table 1 below).

[0107] Autologous EBV-CTLs were well tolerated, and no serious adverse events (AEs) were observed. Only one patient experienced a related or possibly related AE, a transient grade 1 "altered taste" attributed to the DMSO vehicle. No grade 4 or 5 AEs were reported.

[0108] Six patients experienced symptomatic and objective clinical improvement, which began 2 to 14 weeks after the first infusion (Table 1). Decreased fatigue was a prominent feature in patients with clinical improvement. A correlation between EBV reactivity and clinical response was observed. Patients receiving T cells with higher EBV reactivity experienced greater clinical responses. Six patients in this study received T cells with EBV reactivity of 7% or greater. Five of these patients experienced clinical improvement, and three patients experienced improvement in EDSS scores. Four patients received T cells with EBV reactivity of 3% or less. Of these, only one showed clinical improvement, one showed worsening of EDSS, and two reported no change. Clinical improvement or lack thereof over the 6-month study period also correlated with biomarkers of EBV-specific T cell function of the administered T cells (Figure 2). Compared with patients who did not experience benefit, patients who experienced clinical benefit received treatment with significantly enriched EBV-specific CD8+ T cells expressing CD107a, IFNγ, and TNFα. Furthermore, clinical benefit correlated with the polyfunctionality of administered T cells (expression of CD107a, IFNγ, TNFα, and IL-2).

[0109] Autologous T-cell therapy for MS patients in this study was safe and well tolerated, with no serious AEs and the only treatment-related AE being dysgeusia, likely related to the DMSO in the formulation rather than the T cells.

[0110] Clinical improvements in SPMS and PPMS patients were from a relatively fixed baseline (up to 5 years) and did not represent resolution of acute MS inflammation. Of six patients who received T cells with EBV reactivity ≥7%, five showed clinical improvement, and three patients improved their EDSS scores.

[0111] Consistent with the hypothesized mechanism of action of EBV-specific T cells, there appears to be a dose-response relationship between EBV reactivity of T cell products and clinical improvement.

[0112] Decreased fatigue was a consistent and prominent feature of responders. Fatigue is the most disabling symptom of MS and can often precede the onset of clinical MS by years.

[0113] Our data add to the growing body of evidence for a pathogenic role of EBV infection in MS. Because T cells have access to all CNS compartments, T cell therapy targeting exclusively EBV-infected B cells represents a novel therapeutic approach that may offer favorable safety and durable efficacy.

[0114] [Example 2] Exemplary methods for multiparameter intracellular cytokine staining and degranulation analysis of LMP- and EBNA1-specific T cells 1. Dilute the LMP / EBNA1 CD8 pepmix (100 μg / ml stock), EBNA1 pepmix (100 μg / ml stock), and any HLA-matched peptide epitopes (200 μg / ml stock) to be tested to 2 μg / ml in RPMI 1640-10% FCS (for a final assay concentration of 1 μg / ml). 2. Dilute the cell stimulation cocktail 1:50 in RPMI 1640-10% FCS (this will result in a final dilution of 1:100 in the assay). NOTE: eBioscience cell stimulation cocktail is diluted 1 in 5 with RPMI to a stock concentration of 100x and stored at -20 °C. 3. Add 100 μL of the appropriate pepmix, peptide epitope, or cell stimulation cocktail, or 100 μL of RPMI1640-10% FCS (no peptide control) to the appropriate wells of a 96-well V-bottom plate. 4. PBMCs or T cells were cultured at 5 x 10 in RPMI1640-10% FCS. 6 Dilute to cells / mL (this is 5 x 10 per assay) 5 cells are obtained). 5. Add GolgiPlug (Brefeldin A) to the cells to a final ratio of 2 μL / mL (resulting in a final concentration of GolgiPlug in the assay of 1 μL / mL). 6. Add GolgiStop (Monensin) to the cells to a final ratio of 1.4 μL / mL of cells (resulting in a final concentration of GolgiStop in the assay of 0.7 μL / mL). 7. Add FITC-conjugated anti-CD107a to the cells to a final ratio of 50 μL / mL (final volume of anti-CD107a is 5 μL / test). 8. Add 100 μL of cell suspension per well to the required wells of a 96-well V-bottom plate. 9. Incubate at 37°C / 6.5% CO2 for 4 hours. 10. Centrifuge the plate at 2300 rpm for 2 minutes. 11. Discard the supernatant. Wash the cells by adding 200 μL of PBS-2% FCS per well and centrifuge the plate for 2 minutes at 2300 rpm. Repeat this step. 12. Wash the cells twice with 200 μL of PBS-2% FCS per well and centrifuge the tubes at 1000 g (2300 rpm) for 2 minutes.

[0115] Surface cell antigen staining 13. Resuspend cells in 50 μL / well of PBS-2% FCS containing 0.125 μL perCP-Cy5.5-conjugated anti-CD8, 0.25 μL Pacific Blue-conjugated anti-CD4, and 0.2 μL Live / Dead Near IR. Incubate at 4° C. for 30 minutes. 14. Centrifuge the plate at 2300 rpm for 2 minutes. Discard the supernatant. Add 200 μL of PBS-2% FCS per well to wash the cells. Centrifuge the plate at 2300 rpm for 2 minutes. Repeat this step. 15. Resuspend cells in 100 μL / well of BD cytofix / cytoperm solution and incubate at 4°C for 20 minutes. 16. Centrifuge the plate at 2300 rpm for 2 minutes. Discard the supernatant. Wash the cells by adding 200 μL per well of BD Perm / Wash. Centrifuge the plate at 2300 rpm for 2 minutes. Repeat this step.

[0116] Intracellular cytokine staining 17. Resuspend fixed / permeabilized cells in 50 μL / well of BD Perm / Wash solution containing 1 μL PE-conjugated anti-IL-2, 1 μL AF700-conjugated anti-IFNγ, and 0.25 μL APC-conjugated anti-TNF. Incubate at 4°C for 30 minutes. 18. Centrifuge the plate at 2300 rpm for 2 minutes. Discard the supernatant. Add 200 μL of Perm / Wash per well to wash the cells. Centrifuge the plate at 2300 rpm for 2 minutes. Repeat this step. 19. Resuspend cells in 200 μL of PBS-2% paraformaldehyde and store at 4°C. 20. Acquire cells using a BD Fortessa. Analyze cytokine production / degranulation using Flow Jo software.

[0117] [Table 2] JPEG0007726625000003.jpg207121JPEG0007726625000004.jpg21861

Claims

1. A composition comprising autologous cytotoxic T cells (CTLs) expressing a T cell receptor that specifically binds to an EBV peptide presented on class I MHC, for use in treating or preventing multiple sclerosis (MS) in a subject, wherein at least 42% of the CTLs express each of IFNγ, CD107a, IL-2, and TNF.

2. The composition of claim 1, wherein at least 50% or at least 70% of the CTLs express IFNγ, CD107a, IL-2, and TNF.

3. The composition of claim 1, wherein at least 50% of the CTLs express each of IFNγ, CD107a, IL-2, and TNF.

4. The composition of claim 1, wherein at least 60% of the CTLs express each of IFNγ, CD107a, IL-2, and TNF.

5. A composition described in any one of claims 1 to 4, wherein, before use, the CTL is incubated with antigen-presenting cells (APCs) that present EBV peptides, thereby inducing the proliferation of autologous EBV peptide-specific CTLs.

6. The composition of any one of claims 1 to 5, wherein the EBV peptide comprises any of the amino acid sequences set forth in SEQ ID NOs: 4 to 26.

7. Approximately 5 × 10 per dose 6 Approximately 1 x 10 CTLs 7 CTLs, approximately 1.5 x 10 7 CTLs, or approximately 2 x 10 7 The composition according to any one of claims 1 to 6, which is intended to be administered to a subject.

8. The composition of any one of claims 1 to 7, wherein treating or preventing MS in a subject comprises reducing anti-EBV IgG levels in the CSF in a subject with MS.

9. 9. The composition of any one of claims 1 to 8, wherein the MS is relapsing-remitting MS, secondary progressive MS, primary progressive MS, or progressive relapsing MS.

10. Use of autologous cytotoxic T cells (CTLs) expressing a T cell receptor that specifically binds to an EBV peptide presented on class I MHC, in the manufacture of a medicament for the treatment or prevention of multiple sclerosis (MS) in a subject, wherein at least 42% of the CTLs express each of IFNγ, CD107a, IL-2, and TNF.

11. The use according to claim 10, wherein at least 50% or at least 70% of the CTLs express IFNγ, CD107a, IL-2, and TNF.

12. The use according to claim 10, wherein at least 50% of the CTLs express each of IFNγ, CD107a, IL-2, and TNF.

13. The use according to claim 10, wherein at least 60% of the CTLs express each of IFNγ, CD107a, IL-2, and TNF.

14. 1. Use of autologous cytotoxic T cells (CTLs) in the manufacture of a medicament for use in treating or preventing MS in a subject, the CTLs comprising: Incubating a sample containing autologous cytotoxic T cells (CTLs) with antigen-presenting cells (APCs) that present EBV peptides, thereby inducing proliferation of peptide-specific T cells in the sample. and wherein the peptide-specific autologous CTLs are prepared by a method comprising the steps of:

15. The use described in claim 14, wherein the peptide-specific autologous CTLs are for administration to the subject when at least 50% of the expanded peptide-specific autologous CTLs express each of IFNγ, CD107a, IL-2, and TNF.

16. The use described in claim 14, wherein the peptide-specific autologous CTLs are for administration to the subject when at least 60% of the expanded peptide-specific autologous CTLs express each of IFNγ, CD107a, IL-2, and TNF.

17. 17. The use according to any one of claims 14 to 16, wherein the APC comprises a B cell, an antigen-presenting T cell, a dendritic cell, or an artificial antigen-presenting cell.

18. 18. The use according to claim 17, wherein the artificial antigen-presenting cells are aK562 cells.

19. The use according to any one of claims 10 to 18, wherein the EBV peptide comprises any of the amino acid sequences shown in SEQ ID NOs: 4 to 26.

20. Approximately 5 × 10 per dose 6 Approximately 1 x 10 CTLs 7 CTLs, approximately 1.5 x 10 7 CTLs, or approximately 2 x 10 7 The use according to any one of claims 10 to 19, wherein the CTLs are to be administered to a subject.

21. 21. The use according to any one of claims 10 to 20, wherein treating or preventing MS in a subject comprises reducing anti-EBV IgG levels in the CSF of a subject with MS.

22. 22. The use according to any one of claims 10 to 21, wherein the MS is relapsing-remitting MS, secondary progressive MS, primary progressive MS, or progressive relapsing MS.

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