NZ788450A - Methods of treating autoimmune disease using allogeneic t cells - Google Patents

Abstract

Provided herein are compositions comprising allogeneic cytotoxic T cells expressing a T cell receptor that specifically binds to an Epstein-Barr virus (EBV) peptide presented on a class I MHC and methods of treating autoimmune diseases with this composition.

Description

MEMODS 0F IREArmGAUTQIMMtL/vn Disease L/SnveALL0GENEYC 1'‘ CELLS RELATED APPLICATEONS This application claims the benefit of priority to US. Provisional hatent ation U} serial number 62/34l,360 tiled May 25, 2(ll6, US Provisional Patent Application serial numher ,326, filed on luly 7, Ztllo, and US. Provisional Patent Application serial numheréSZ/487,8l1l-, filed on April 20, 2M7, each of which is incorporated. by reference in its entirety.

BACKGROUND li’) Autoimmune diseases, such as multiple sclerosis (MS) and systemic autoimmune disease (SAD), and inflammatory bowel disease (lBD) are pathologies arising from abnormal immune response against the body’s own tissue. MS is characterized by the degradation ofthe myelin, a protective lipid shell surrounding nerve fibers, by the body’s own immune cells. SADs are a group of connective tissue diseases with diverse ms that include rheumatoid arthritis (RA), systemic lupus erydhematosus (SLE) and Sjogren’s syndrome (SS). lBDs are a group ofinllamniatory conditions ofthe colon and small intestine that include Crohn’s e, celiac disease, and ulcerative colitis.

Epstein Barr Virus (EBV), also known as human herpesvirus 4, is a ubiquitous herpes virus. Recently, it has heen shown that exposure to EBV can predispose or otherwise play a role in the pathogenesis of autoimmune diseases, including MS, SAD and. lBD. For example, recent studies have shown that individuals diagnosed with MS show higher levels of EBV related proteins in B cells ated in nerve tissue than healthy duals. lt is hypothesized that an increase ofEBV—intected B cells and/or defective elimination of such cells may predispose individuals to such autoimmune es. h.) (I: SUMMARY Provided herein are methods for treating autoimmune diseases (eg, MS, SAD and/or lBD), comprising administering to a subject allogeneic cytotoxic T cells ss) sing a T cell receptor that specifically binds to an EBV peptide presented on a class I Ml-lC. in some embodiments, the class l MllC to which the TCR is restricted is encoded by an HLA allele that is present in the subject. in some embodiments, the method. comprises ing the allogeneic CTLs from a cell bank. in some ments, the EBV e comprises a LMPl peptide or a fragment thereof, a LMPZA peptide or fragment thereof, and/or an EBNAl e or fragment thereof in some embodiments, the EBV peptide comprises a sequence listed in Table l. in certain s, ed herein are methods of treating an autoimmune disease (eg, MS, SAD r lBD), comprising generating allogeneic (Ills that express a T cell UT! receptor that specifically binds to an EBV peptide presented on a class l MllC and then administering the allogeneic C’l‘los to a subject. ln some embodiments, the allogeneic C’l‘los are stored in a cell bank prior to administration to the subject. in some embodiments, the class l Ml-lC to which the TCR is restricted is encoded by an HLA allele that is present in the subject. in some ments, the allogeneic CTLs are generated by incubating a sample comprising neic CTLs (eg. , a FBMC sample) with antigen presenting cells (APCS) presenting an FBV peptide on a class l MHC (e.g a class l MHC d by an HLA allele that is t in the subject), thereby inducing proliferation peptide—specific (fills in the sample. in some embodiments, the APCS are made to present the EBV peptide by incubating them with a nucleic acid. construct (cg. AdELLMFpoly) encoding for the ELEV peptide, thereby inducing the AFCs to present the EBV e. ln some embodiments, the APCs may be B cells, antigen—presenting T cells, dendritic cells, or artificial antigenupresenting cells (6.g a cell line sing C980, (TD83, 4lBBnL and/or CD86, such as al4562 cells). ln some embodiments, the EBV peptide comprises a. LMF l peptide or a fragment thereof, a LMFZEA peptide or fragment thereof, and/or an EBNAl peptide or fragment thereof, in some embodiments, the EBV peptide comprises a ce listed in Table l. in some embodiments, C'l‘lss are ed, (cg ed from a cell bank) for compatibility with the subject prior to administration to the subj ect. in some embodiments, the CTLs are selected if they are restricted through an l-lLA allele shared with the subject (tie, the TCR of the CL'l‘s are restricted to an MHC class 1 protein encoded by a HLA allele that is present in the subject). in some embodiments, the C’l‘lss are selected. itthe Oils and subject share at least 2 (tag at least 3, at least 4, at least 5, at least 6) l-lLA alleles and the CTLs are restricted through a shared l-lLA allele. in some embodiments, the (ills administered to the subject are selected from a cell bank (eg, a CTL bank).

BRIEF DESCRIPNON OF THE DRAWINGS Figure 1 shows improved effector thnction in C’l'L product obtained from healthy (Nb/HE? donors compared, to C'l'll product obtained. from MS patients as measured by fraction of viable lymphocytes that are Cl)? and lFNg'l' ing stimulation (Mann Whitney p value 0.0002).

ED DESCREPTIQN general Provided herein are methods of treating autoimmune disorders (eg, MS, SAD and/or Hill) in a subject using allogeneic CTLs that recognize one or more ofthe EBV epitopes U} described herein, for example. in some embodiments, the method r comprises selecting the allogeneic CTLS from a cell banlt. in some embodiments, the method r comprises making the allogeneic s.

Definitions For convenience, certain terms employed in the cation, examples, and l 0 appended claims are collected here.

The articles “a” and “on” are used herein to refer to one or to more than one (216., to at least one) of the grammatical obj ect of the article, By way of example, “an element” means one t or more than one element.

As used herein, the term “administering“ nieans providing a pharmaceutical agent or LI! composition to a subject, and includes, hut is not limited to, administering by a medical professional and selllalmiriistering. Eluch an agent can contain, for e, peptide described herein, an n presenting cell provided herein and/or a C’l‘L provided herein.

The term “amino acid” is intended to emhraee all molecules, Whether natural or synthetic, which include both an amino timctionality and an acid fiinctionality and capable of being included in a polymer of naturally"occurring amino acids. Exemplary amino acids include naturally~oecurring amino acids; analogs, derivatives and coiigeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing. 'lhe term “binding” or “Detained/rig” refers to an association, which may be a stable association, between two molecules, 6. g. , between a TCR and a peptide/MHC, due to, for example, electrostatic, hydrophobic, ionic and/or en ~hond interactions under physiological ions.

The term “biological ample,” “tissue sample,” or simply “ ample” each refers to a collection of cells obtained from a tissue ofa subject. The source of the tissue sample may be solid tissue, as from a fresh, frozen and/or preserved organ, tissue sample, , or aspirate; blood or any blood constituents, serum, blood; hodily lluids such as al spinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid, urine, saliva, stool, tears, or cells from anytime in gestation or development of the suhject.

As used herein, the term “cyz‘okme” refers to any secreted polypeptide that affects the functions of cells and is a molecule which modulates interactions n cells in the immune, atory or hematopoietic response. A cytolrine includes, but is not d to, monolrines and lymphokines, regardless of which cells produce them, For instance, a ‘4’! monolrine is generally referred to as being produced and secreted by a niorionuclear cell, such as a hage and/or nionocyte. Many other cells however also produce monolrines, such as natural killer cells, fibroblasts, hasophils, phils, endothelial cells, brain ytes, bone marrow strornal cells, epidermal lceratinocytes and B—lyinphocytes. okines are generally referred to as being produced by lymphocjyde cells. Examples of l0 illltES include, but are not limited to, lnterleulrin—l (llcml), lnterleulrin—Z (lL—Z), lnterleulrin-o (Ibo), lnterleulrin—S (Hang), 'l‘umor Necrosis Factor—alpha ('l‘NFa), and Tumor Necrosis Factor beta (TNFB), The term “cpz'tope” means a protein determinant capable of specific binding to an antibody or TCR, Epitopes y consist of chemically active surface groupings of molecules such as amino acids or sugar side chains. n epitopes can be defined by a particular sequence of amino acids to which an antibody is e of binding.

As used herein, the phrase “pharmaceutici'yli acceptable” refers to those agents, conipounds, materials, compositions, and/or dosage forms which are, within the scope of sound niedical 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 able benefit/ii sh ratio, As used herein, the phrase "pharmacecritically—acceptable carrier” means a pliarrnaceutically~acceptahle material, composition or vehicle, such as a liquid or solid tiller, diluent, excipient, or solvent ulating material, involved in carrying or transporting an E‘J U} agent from one organ, or portion of the body, to r organ, or portion ofthe body. Each carrier must be "acceptable” in the sense of being compatible with the other ingredients of the tormulation and not injurious to the patient. Some es of materials which can serve as pharinaceutically—acceptahle carriers include: (l) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch, (3) cellulose, and its tives, such as sodium carhoxyniethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) tale; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil, (l 0) glycols, such as propylene glycol, (l l) polyols, such as glycerin, sorhitol, mannitol and polyethylene ; (l 2) esters, such as ethyl oleate and ethyl laurate; (l3) agar; (l4) buffering agents, such as magnesium ide and um hydroxide, (l5) alginic acid; (l6) n-free water; ( l7) isotonic saline; (l8) Ringer's solution; (l9) ethyl l; (20) pH buffered solutions; (Zl) polyesters, polycarbonates and/or (I: polyanhydrides; and (22) other non~toxic compatible substances employed in pharmaceutical formulations.

The terms ‘jeolynudeofidel’, and “tandem acz'cf’ are used interchangeably 'l‘hey refer to a. polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three—dimensional ll) structure, and may perform any function. The following are non—limiting examples of polynueleotides: coding or non~eoding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyines, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, ed RNA of any sequence, nucleic acid probes, and s, A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. lfpresent, modifications to the nucleotide structure may he imparted before or alter assembly of the polymer. A polynucleotide may he {hither modified, such as by conjugation with a ng component, in all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.

As used herein, a therapeutic that "pre vents" a condition refers to a compound that, when administered to a statistical sample prior to the onset of the disorder or condition, reduces the ence of the disorder or ion in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more ms of the disorder or condition relative to the untreated control sample.

As used herein, fir framing” refers to the ability of a TCR, to bind to a peptide presented on an Ml—lC (a g, class l hill-ll: or class ll lvll-lC). Typically, a TCR ically binds to its peptide/Milli? with an affinity of at least a Kn of about l0"4 M or less, and binds to the predetermined antigen/binding partner with an affinity (as expressed by Kc) that is at least 10 fold less, at least lilt‘i fold less or at least 10th fold less than its affinity for binding to a non—specific and unrelated peptide/"l‘vll-lC complex (eg, one comprising a BSA peptide or a casein peptide).

As used herein, the term “subject” means a human or non-human animal selected for ent or therapy.

WO 03368 e phrases “titertipeufibrillyefiecflw amount" and “ejfi’ctive amount” as used herein means the amount ot‘an agent which is eftective for producing the d therapeutic effect in at least a pulation of cells in a subject at a reasonable /risk ratio applicable to any medical treatment. ‘4’! As used herein, the term “treating” a disease in a subject or “treating” a subject having or suspected of having a disease refers to ting the subject to a pharmaceutical treatment, eg the administration a C'l‘l... described herein, such that at least one symptom of the disease is decreased or prevented from worsening.

The term “ "actor” refers to the means by which a nucleic acid can be propagated l0 and/or transferred between organisms, cells, or ar components. Vectors include plasmids, viruses, iophage, pro—viruses, phagemids, transposons, and artificial somes, and the like, that may or may not be able to replicate mously or integrate into a chromosome ot‘a host cell. hepatitis in certain aspects, provided herein are methods of treating autoimmune disorders (eg, MS, SAD and/or EBB) using allogeneic CTLs sing TCRs that specifically bind to peptides comprising EBV epitopes presented on class l MHC. in some embodiments, provided, herein are methods generating such allogeneic C'l‘Ls, for example, by incubating a sample comprising CTLs (re, a PBMC sample) with antigen-presenting cells (APCs) that present one or more of the EBV epitopes described herein (cg. APCs that present a peptide described herein sing a EBV epitope on a class i MHC complex) in some embodiments, the peptides provided herein comprise a sequence of any EBV viral protein (eg a sequence ofat least 5, 6, 7, 8, 9, lb, ll, l2, l3, l4, l5, l6, l7, l8, l9 or contiguous amino acids of any EBV protein). in some embodiments, the peptides provided, herein comprise no more than 25, 20, l9, l8, l7, l6, l5, l4, l3, l2, ll or 10 contiguous amino acids of the EBV viral protein. in some embodiments, the peptides provided herein comprise a sequence ot‘LMPl (cg). asequence ofat least 5, e, 7, 8., 9, lt‘r, ll, l2, 13, 14, l5, 16, l7, l8, l9 or 20 contiguous amino acids of LMW). in some embodiments, the peptides provided herein se no more than 25, 20, l9, l8, l7, l6, l5, l4, l3, l2, ll or it) contiguous amino acids ot‘Lh/ll’l. An exemplary LMPl amino acid sequence is provided below (SEQ ll) NO: mdldlergpp '7}:": [-1“pp r1 .4 a)0 issyia all llllallfwl yiimsnwugj allvlyafal 0‘, 1—3 mlviiiliif i’H H Hd1 H Cpl galcll lmi Clllialwnl hgqalylgiv lfifgcllvl ,__i 1K} H giwvyfleil wrlgatiwa la i flatfld illliialyl qqnwwtllvd llwlllflai 181 iiwmyyfioor hsdehhhdds 1phiqqatdd ssnhsdsnsn lvsg aoiipplcsfi T41 nigaifodpd nqpqdpdntd dngiqdpdnt ddngphdplp qdpdntddng pqdpdntddn 301 jphdfi“ pnlteevenk ggdrgppsmt dggggdphlp tlllgtsgsg 361 gdddd,ngpv olsyyd in some embodiments, the es provided herein comprise a. sequence 2A (erg, asequence ofat1east 5, 6, 7', 8, 9,10,11,12,13,14,15,16,17,13,19 or 20 contiguous amino acids of LMPZA). in some embodiments, the es provided herein comprise no more than 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 eontiguous amino acids of LMPZA, An exempiai‘y LMFZA amino acid sequence is. provided below (SEQ 11) NO: 2): 1 mgs1emvpmg agpispoodo oqdoggnnsq vonasosdgn tptppnoeer esneeppppy 61 edldwgngdr hsdyqplgnq ops1v1qlcp dgnoq1ppoo ssqn iyeeagrgsm 12 H npvclpviva pylfwlaaia ascftasvst vvtatglals llllaavxss veaaqullt ,__i 0'.) H pvtvltavvt ffaicltwri edpyfnsllf oglq giyvlvmlvl lilayrrrwr u :1; H rltvcggimf lacvlvlivd rvlqlspllo avtvvsmtll llafvlwlss pgglgtlgaa 'u.) CD H wtlvvllics scsscpitki llarlflyal {\J' CD L») O\ ,_a nlfcmllliv agi1filail tewgsgnrty .5. f\) H ;& 03. F1 in some embodiments, the es provided herein comprise a. sequence of FBNA1 (erg, asequence ofat1east 5, 6, 7', 8, 9,10,11,12,13,14,15,16,17,13,19 or 20 h.) (I: contiguous amino acids of EBNAi). in some embodiments, the peptides provided herein se no more than 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 eontiguous amino acids of FBNA1 An, exemp1aiy EBNA], amino acid sequence is provided heiow (SEQ 11) N0: 3): 1 iffhpvgead eggp ppga iqupildpg egpstgprgq gdggrrkkgg 3U 61 wfgkhrgqgg snpkfeniae glrvllarsh vettteegtw vagvayggs ktslynlrtg 121 taiaipqcrl pfem apgpgpquf leesivcyfm vflqthifae Vlhdaikdlv 181 mthpaptcni fddg vdlypwfp m vegaaaegdd gddgdeggdg degeeo‘e in some embodiments, the peptide comprises the sequence of an epitope listed in 'i‘abie 1.

U) 1J1 Table i: Exemplary EBV Virai protein epitnpes CLGGLLTMV FLYALALLL YLLEMLWRL ALWLYsrA LLSAWM‘A L't‘AGFLirL setssEfiisiEi''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''''' FYLFWLAA TYGPVFMCL RRREVRRLRI’ IEDP?FNSF_J IALYLQQ‘MN i 8 MSN'I‘LLSAW 19 VLKDAIKDL 20 RPQKRPSU "3i""""""""""""""""""""""""""""""""""""""" IPQCRLTPL 22 YNLRRGTAL 23 FVYGGSKTSL in some embodiments, the peptides provided herein comprise two or more ofthe EBV epitopes. In some embodiments, the peptides ed herein se at ieast 2, 3 4, U3 5, 6, 7, 3, 9, 10, H, 12, E3, 14, 35, 16, 17, 18, 19 or 20 EBV epitopes. For example, in some embodiments, the peptide ed herein comprises two or more ofthe EBV epitopes connected by linkers (eg. polypeptide linkers). in some embodiments, the ce of the peptides comprises an EBV viral protein sequence except for l or more (cg l, 2, 3, 4, 5, 6, 7, 8, 9, ll) or more) conservative sequence modifications. As used herein, the term rvative sequence modifications” is intended to refer to amino acid modifications that do not significantly af‘ect or alter the U} interaction between a TCR and a peptide ning the amino acid ce presented on an MHC. Such conservative modifications include amino acid substitutions, additions (c g. additions of amino acids to the N or C terminus of the peptide) and deletions (cg, deletions of amino acids from the N or C terminus of the e). Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue l 0 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 (cg lysine, arginine, histidine), acidic side chains (cg, aspartic acid, glutamic acid), uncharged polar side chains (e.g, glycine, asparagine, glutaniine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (cg alanine, valine, leucine, isoleucine, proline, LI! phenylalanine, methionine), beta—branched side chains (c g, threonine, valine, isoleucine) and aromatic side chains (cg, tyrosine, phenylalanine, tryptophan, histidine). lhus, one or more amino acid residues ofthe peptides described herein can be replaced with other amino acid residues from the same side chain family and the altered e can he tested for retention of TCR hinding using methods known in the art. Modifications can he introduced into an antibody by standard techniques known in the art, such as siteudirected mutagenesis and ediated mutagenesis, in some embodiments, the peptides provide herein comprise a ce that is at least 89%, 85%, 90%, 95% or l0(l% identical to an EBV viral protein ce (cg. the sequence ot‘a fragment of an EBV viral protein). To determine the percent identity oftwo amino acid sequences, the sequences are aligned for optimal comparison purposes (cg, gaps can he uced in one or both of a first and a second amino acid sequence for l alignment and nonuidentical sequences can be disregarded for comparison purposes). The amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then the molecules are cal at that position. The percent ty between the two sequences is a on of the number of identical positions shared liy the sequences, taking into account the number of gaps, and the length of each gap, which need to he introduced for optimal alignment of the two sequences. in some embodiments, the peptide is chimeric or fusion e, As used herein, a “chimeric peptide” or “liision peptide” comprises a peptide having a sequence provided herein linlred to a distinct peptide having ce to which it is not linked in nature. For example, the distinct peptide can he fused to the N—terminus or C~terminus of the peptide provided herein either directly, through a peptide bond, or indirectly through a chemical linker. in some embodiments, the peptide of the provided herein is linked to another peptide comprising a distinct EBV epitopes. ln some ments, the peptide provided herein is linked to peptides comprising epitopes from other viral and/or infectious diseases.

A chimeric or hision peptide provided herein can he ed by standard l0 recombinant DNA ques. For example, DNA tragments coding for the different peptide sequences are d together inmframe in ance with conventional techniques, for example by employing blunt—ended or stagger—ended termini for on, restriction enzyme digestion to provide for appropriate termini, fillingnin of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation, in another embodiment, the hision gene can be synthesized by conventional techniques including automated DNA sizers. Alternatively, PCR ication of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two utive gene fragments which can subsequently be annealed and re— amplified to generate a chimeric gene sequence (see, for example, t Protocols in Molecular Biology, Ausuhel et al, eds, John Wiley 63: dons: l992). Moreover, many expression vectors are commercially available that already encode a fusion moiety. e es provided herein can be isolated from cells or tissue s by an appropriate purification scheme using standard protein purification techniques, and can he produced by recombinant DNA techniques, and/or can be chemically synthesized using standard. e synthesis techniques, The peptides described herein can he ed in yotic or eukaryotic host cells by expression of nucleotides encoding a peptidets) of the present invention. Alternatively, such peptides can be synthesized by chemical methods.

Methods for sion of heterologous peptides in recombinant hosts, chemical synthesis of peptides, and in wire translation are well known in the art and are described further in Maniatis et al., Molecular Cloning: A Laboratory Manual @989), 2nd Ed, Cold Spring Harbor, N. ‘17., Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning "l‘echniques (_ l’987), Academic Press, Inc, San Diego, Calif; Merritield, .l, 6.969) I, Am. Chem, Soc. 9l:50l; Chaiken l. M. (l98l) CRC Crit. Rev. m, ll:255; Kaiser et al. (”39) Science 243187; ield, B. 0986) Science 232342, Kent, S, B. ll. 0988) Annu. Rev. Biochem. 57:957; and , R. E. 0980) Sernisynthetic Proteins, Wiley hing, which are incorporated herein by reference. in certain aspects, provided herein are nucleic acid molecules encoding the peptides (I: bed herein. in some embodiments, the nucleic acid molecule is a vector. ln some embodiments, the nucleic acid molecule is a viral vector, such as an adenovinis based sion vector, that comprises the nucleic acid, molecules described herein. In some embodiments, the vector provided herein encodes a plurality of es provided herein (6. g, as a polyepitope). in some embodiments, the vector provided herein encodes at least 2, ll) 3;, 4, 5, 6, 7, 8, 9, l0, ll, l2, l3, l4, l5, l6, l7, l8, l9 or 20 epitopes provided herein (cg. epitopes provided in Table l). ln some embodiments, the vector is AdEl—lelllpoly. The AdEl ~LMPpoly vector encodes a polyepitope of defined C’l‘lc epitopes from LMPl and LMPZ fused to a Gly—Ala —depleted EBNA l sequence. The AdEl —Ll\v/ll)poly vector is described, for example, in Smith at al, Cancer ch 72: l l l6 (ZOl 2); Duraiswaniy er al, Cancer Research 6414839 (2004), and Smith civil, J. Immune] ll7:4897—9(l6, each of which is hereby incorporated by reference.

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 doubleustranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a. viral vector, wherein additional DNA segments may be ligated into the viral . Certain vectors are capable of autonomous replication in a host cell into which they are introduced (cg, bacterial vectors having a bacterial origin of replication, episornal mammalian vectors). thier vectors (cg, flOl‘i— al mammalian vectors) can be integrated into the genome of a. host cell upon introduction into the host cell, and thereby be replicated along with the host genome.

Moreover, certain vectors are e of directing the expression of genes. Eluch s are referred to herein as “recombinant expression vectors” (or simply, “expression s”), In some embodiments, provided herein are nucleic acids operably linked to one or more regulatory ces (cg, a promoter) in an expression vector. in some embodiments, the cell transeribes the nucleic acid provided herein and thereby ses a peptide described herein. The nucleic acid molecule can be integrated into the genome of the cell or it can be extrachremasoinal. in some embodiments, provided herein are cells that contain a nucleic acid described herein tag. a nucleic acid encoding a peptide bed herein). The cell can be, for example, proltaryotic, eultaryotic, mammalian, avian, murine and/or human. in some embodiments, the cell is a mammalian cell. in some embodiments the cell is an APC (eg. an ‘4’! antigenupresenting T cell, a dendritic cell, a B cell, or an aKSbZ cell). In the present methods, a nucleic acid described herein can be administered to the cell, for e, as nucleic acid without delivery vehicle, in combination, with a delivery reagent. 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, cg, the Mirus Transit TKO l0 lipophilic reagent; lipolectin; lipolectamine, celltectin; polycations (e.g, polylysine), atelocollagen, nanoplexes and liposonies. ln some embodiments ofthe methods described. , liposomes are used to deliver a nucleic acid to a cell or subject, Liposomes suitable for use in the methods described herein can he formed from standard vesicle—forming lipids, which generally include neutral or negatively d phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of s such as the desired liposonie size and halfulife of the liposomes in the blood stream. A variety of methods are known for ing liposomes, for example, as described in Szolra et all 0980), Ann Rev ,Biqnizys. Bioeng. 9:467; and US. Pat. Nos. 4,235,871, 4,50l,728, 4,337,028, and 5,0l9,369, the entire disclosures of which are herein incorporated by reference.

AlloechicCils ed herein are methods of treating autoimmune diseases (eg, MS, SAD, Hill) by administering to the subject allogeneic CTLs expressing a T cell or that specifically binds to an EBV peptide presented on a class i MHC. in some ments, the C'l’Ls are from a. cell bank. in some embodiments, the MHC is a class 1 MHC. in some embodiment, the class ll h/ll-lC has an a chain polypeptide that is DMA, l-lLA-DGA, l-ll.;A~DPA, l-lLAuDQA or l-lLAuDRA. in some embodiments, the class ll MHC has a 3 chain polypeptide that is l-lLAnDMB, llLA-DOB, B, B or HLA—DRB. in some embodiments, the s are stored. in a cell library or bank, before they are stered to the subject. in some embodiments, provided herein are APCs that present a peptide described herein (cg a peptide comprising al.1i‘vllll, LMFZA, or EBNAl e sequence). in some embodiments the Ath are B cells, antigen presenting s, dendritic cells, or artificial antigen—presenting cells (6g, aK562 cells).

Dendritic cells for use in the process may be prepared by taking PBMCs from a patient sample and adhering them to c. Generally, the nionocyte population sticks and (I: all other cells can be washed off. The adherent population is then differentiated with lLuel and thCSF to produce monocyte derived dendritic cells. These cells may be matured by the addition of , lie—6, l’GEd and 'lNF—a (which upregulates the important co— stimulatory molecules on the surface of the dendritic cell) and are then transduced with one or more of the peptides provided herein. ll) in some embodiments, the AFC is an artificial antigenmpresenting cell, such as an aKSdZ cell. In some embodiments, the ial nprescnting cells are engineered to express C1380, CD33, “BB—L, and/or CD86. Exemplary artificial antigenpresenting> cells, including aKSoZ cells, are described US. Pat. Pub. No. Zl)03/’Ol47869, which is hereby incorporated by reference. in certain aspects, provided herein are s of generating APCS that present the one or more of the EBV epitopes described herein comprising contacting an APC with a peptide comprising a EBV epitopc and/or with a nucleic acid encoding a EBV e. in some embodiments, the AFCs are irradiated. in some embodiments, the Alle that present a peptide described herein (eg, a, peptide comprising a LlVlPl, LMPZA, or EBNAl epitope sequence). A cell presenting a peptide described herein can be produced by standard techniques lrnown in the art. For e, a cell may be pulsed to encourage peptide uptake. ln some ments, the cells are transfected with a c acid encoding a peptide provided herein. Provided herein are methods o‘l’prodncing antigenupresenting cells (APCs), sing pulsing a cell with the peptides described herein. Exemplary examples of producing antigen presenting cells can be found in WGZOlSGllSl lél, hereby incorporated in its entirety. in some embodiments, provided herein are T cells (cg, CD4 T cells and/or (IDS T cells) that express a TCR (eg, an all TCR or a yd TCR} that recognizes a peptide described herein presented on a lVll-lC in some embodiments, the T cell is a, CD3 T cell (a CTL) that expresses a TCR that recognizes a peptide described herein presented on a class l Ml-EC in some embodiments, the T cell is a C114 T cell (a helper T cell) that recognizes a peptide described herein presented on a class ll MHC.

WO 03368 in some embodiments, provided herein are methods of ting, activating and/or inducing proliferation of T cells (cg CTLs) that recognize one or more of the EBV epitopes described herein. in some embodiments, a sample comprising C'l’Ls (tie, a PEMC sample) is incubated in culture with an APC provided herein (e. g. an APC that presents a peptide UT! comprising a EBV e on a class I MHC complex), in some ments, the APCs are autologous to the subiect from whom the T cells were obtained. in some embodiments, the Alle are not autologous to the subject from whom the T cells were obtained. in some embodiments, the sample containing T cells are incubated Z or more times with APCs provided . in some ments, the T cells are incubated with the APCs in the presence of at least one ne. in some embodiments, the cytolrine is llfil, an7 and/or le l5. Exemplary methods for inducing proliferation of T cells using AFCs are provided, for example, in US. Pat, Pub. No. 20l5,’(”llll7723, which is hereby incorporated by reference. in some embodiments, provided herein are compositions (eig. therapeutic itions) comprising T cells and/or AFCs ed. herein used to treat and/or prevent an autoimmune disease in a subi ect by administering to the t an effective amount of the composition. in some aspects, provided herein are methods oftreating autoimmune disorders using a composition (6.g a pharmaceutical composition, such compositions comprising allogeneie C’l‘lrs). ln some embodiments, the composition includes a ation of le tag, two or more) CTlss provided herein. eutic Methods in, some embodiments, the provided he rein are methods oftreating an autoimmune disorder in a subject by administering to the subject allogeneic CTlss provided herein. ln some embodiments, the allogenic CTLs are selected from a cell bank (cg, a tire—generated third party donor derived bank of epitope~specific CTLs).

E‘J U} In some embodiments, the methods provided herein can he used to treat any autoimmune disease. Examples of autoimmune diseases include, for example. glomerular nephritis, arthritis, dilated cardiomyopathy—like e, ulceous colitis, Sjogren me, Crohn disease, systemic erythematodes, chronic rheumatoid arthritis, juvenile rheumatoid arthritis, Still’s diease, multiple sclerosis, psoriasis, allergic contact dermatitis, polymyositis, pachyderma, periarteritis nodosa, rheumatic fever, vitiligo vulgaris, Behcet disease, Hashimoto disease, Addison disease, dermatomyositis, myasthenia gravis, Reiter syndrome, Graves' disease, anaemia. perniciosa, ity disease, pempliigus, autoimmune thrombopenic purpura, autoimmune hemolytic anemia, active chronic hepatitis, Addison’s disease, anti- olipid syndrome, atopic allergy, autoimmune atrophic gastritis, achlorliydra autoimmune, celiac disease, Cushing’s syndrome, dermatomyositis, discoid lupus erythernatosus, Goodpasture‘s syndrome, oto’s iditis, idiopathic adrenal atrophy, idiopathic thromhocytopenia, insulinwdependent diabetes, Lambert—Eaton syndrome, lupoid U} tis, lymphopenia, mixed connective tissue disease, peniphigoid, pemphigus vulgaris, pernicious anemia, phacogenic uveitis, polyarteritis hodosa, polyglandular autosyndrornes, primary biliary cirrhosis, primary sclerosing cholangitis, Raynaud’s me, relapsing polychondritis, Schmidt‘s syndrome, limited scleroderma (or crest syndrome, sympathetic ophthalmia, systemic lupus erydhematosis, Taltayasu's arteritis, temporal arteritis, l 0 thyr‘otoxicosis, type b insulin resistance, type l diabetes, ulcerative colitis and ‘v ”egeher‘s granulomatosis. in some embodiments, the methods provided herein are used to treat MS. ln some embodiments, the MS is relapsihgnremitting MS, secondary progressive MS, primary progressive MS or progressively ing MS, LI! in some embodiments, the methods provided herein are used to treat a SAD, For e, in certain embodiments, the methods ed herein are used to treat rheumatoid arthritis, systemic lupus erythematosus and/or SiogrenE syndrome. in some embodiments, the methods provided herein are used to treat EBB, For example, in certain embodiments the methods ed herein are used to treat Crohn's e (regional bot 'el disease, eg, inactive and active terms), celiae e leg. inactive or active forms) and/or ulcerative colitis leg, inactive and active forms). in some embodiments, the methods provided herein are used to treat irritable bowel syndrome, copic colitis, lymphocytic—plasmocytic enteritis, coeliac disease, enous colitis, lymphocytic colitis, eosinophilic enterocolitis, indeterminate colitis, infectious colitis ( viral, bacterial or protozoan, cg. amoebic colitis) (e, g., elostridium dilicile colitis), pseudomembranous colitis (necrotizing colitis), iscliemic inflammatory bowel disease, Behcet’s e, sarcoidosis, scleroderma, lBD~associated dysplasia, dysplasia associated masses or lesions, and/or primary selerosing gitis.

Actual dosage levels of the active ingredients in the pharmaceutical itions provided herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, Without being toxic to the patient.

The selected dosage leve will depend upon a variety of factors including the activity ot‘the particular agent employed, the route nistration, the time of administration, the rate ofexcretion or metabolism of the particular nd being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, l health and prior medical history of the patient being treated, and like factors well known in the medical arts. in some embodiments, the method includes selecting allogeneic C'l‘Ls front a cell hanl< (eg. a pie—generated third party donor derived bank of epitope c ). ln some embodiments, the CTLs are selected because they express a TCR cted to a class l Milt: l0 that is encoded by an HLA allele that is present in the subiect. in some ments, the C'l‘lss are selected ifthe C'l‘lss and subject share at least 2. (erg at least 3 at least 4-, at least , at least 6) l-lliA alleles and the CTLs are restricted through a shared HLA . in some embodiments, the method comprises testing the TCR repertoire of the prengenerated third~ party~donormderiyed epitope—specitic T cells (216. allogeneic T cells) with flow cytometry. in some embodiments epitope~specitic T cells are detected using a tetramer assay, an ELISA assay, a western blot assay, a fluorescent microscopy assay, an Edman ation assay and/or a mass spectrometry assay (cg protein sequencing). in some embodiments, the TCR repertoire is analyzed, using a nucleic acid prohe, a nucleic acid amplification assay and/or a sequencing assay. 2% EXAMPLES Examine 1: Generatin, tarry donor derived bank 0 wife re s recific CTLS.

A third party donor derived hanlt ot‘epitope specific CTLS is generated through the targeted identification of donor lymphocyte rnaterial in order to te C’l‘L populations with sufficient scale, breadth of patient lfilLA matching capability, and, target—restricted activity identification of donor material is facilitated by any combination of donor/material genetic annotation or resultant product y characteristics yielded from the ing materials: (a) Donor l-lLA alleles — specific l-llsA alleles may he prioritize and specifically gathered as input al for CTL generation based on y to cover most broadly the targeted patient population and/or the cognate epitopes contained in the stimulating peptide sequences. (b) Capacity for a test aliquot of donor material to expand and/or produce eftectiye cytotoxic capacity following the CTL stimulation protocol.

WO 03368 (c) Epitope/l-llA restriction of stimulated donor material as indicated by cytotoxicity s, or through functional characterization of response as ted by degranulation, cytolrine release, ing assays, or other markers of apoptosis in target cells and/or epitope-specilic stimulation ofthe CTL compartin ent.

(I: (d) Resultant phenotypic profile of {ITL product in a test aliquot as related to sion ofco—stirnulatory molecules, exhaustion markers, differentiation markers, and/or other resultant product characteristics.

Following the parallel or sequential stimulation and generation of CTL comprising ts, each CTL batch or lot is characterized and ted for l-llsA restriction ll) specificity and potency. Characterized lots are then viably re served to allow for reanimation at a. later date. The cumulative cryo—preseiyation of multiple lots generated from distinct donor material with distinct l-lLA allele expression results in a breadth of diversity in HLA restricted activity across the cryompreseived “banlr.” The content of the bank is then ready to be selected and matched to patient characteristics at a future date, such that specific lots can be retrieved and reanimated for the purposes of providing a readily~available therapy with characteristics tailored to that of each patient. specific CTLS.

Patientuspecilic requisitioning ot‘banked products can be accomplished through the ordered and prioritized integration of material characteristics with the patients genetic or disease background. Such a sequence of hierarchical erations may be accomplished through use of an algorithm designed to integrate these inputs and output a matching lot.

This thm can be based on HLA ction, or, when multiple lots are available, matching by HlLA restriction in combination with a series of additional inputs, each appropriately ed and including additional lot and/or patient—specific characteristics and annotations to select the most eftbctive patient~specific lot, or one that most mitigates potential for adverse events. Below is provided an exemplary format for such a requisitioning algorithm.

Allogeneic third party Ls are selected for the subject from the library of available EBVnC’l‘L cell lines. The following steps outline the process to identify the cell lihets) to he used for a subject: l) ln order to match a cell line with a patient, a cell line and patient must share 22 l-lLA loci at high resolution, with at least l HLA locus of the sub} ect or preferentially the subject’s EB V-i- B—cell compartment, ifl<nown, matched to the given C'l‘L cell lines HLA restriction 2) ln order to ain the presence of an adequate cell dose, the presence of sufficient cells from the selected cell line to ster at minimum X cycles at Y CTL/kg actual body weight per dose (n doses per eyele(X)l X cycles = nX doses total, therefrom“, the minimum dose available must be at least nXY X l06 CTL/lrg actual body weight). The m dose may change depending on patient or disease characteristics. 3) if only one cell line is identified according to the previously discussed standards, then that cell line should be used and no firrther selection criteria. are imposed, However, in some cases, there may exist more than one cell line in the CTL library meeting l-lLA matching (l) and minimum dose requirements (2) for a given subject. Among those CTL cell lines, some may have additional HLA allele characteristics, either restricting or defined. in the genotype of the material donor, that may he associated, either ally or through indirect levels of evidence, with decreased clinical mance, as defined by decreased y or increased association with adverse events. lf this is the case, cell lines would be selected. for the lack of this additional HLA allele Additionally, cell lines that have been previously administered to patients and the resulting responses recorded. lhese cell line response data is used to select among CTL cell line s g the requirements of t l) and (2) as follows: 3a) Among cell lines where the previous response rates have been r than a specified cut—off among at least 4 patients d, then the largest esi sting cell dose available in the library is selected. Ifthe donor starting material was used for a subsequent hatch and the same HLA restriction was obtained. as for the first batch, then the response rates for the subsequent hatches sharing the same l-lLA restriction can he assumed to be similarly effective. 3b) if there are no cell lines meeting the criteria in (3a), then among the cell lines meeting the requirements of ( l) and (2), the line with the highest response rate and at least one previous response is selected. 3c) lfthere are no cell lines with a previous response rate, select among cell lines whose lllA ction has been shown previously to elicit responses, prioritizing which cell line by the l-lliA restriction shared with the subject (or subject’s e) with the t previous response. 3d) Finally, if previous requirements cannot be met, the line avoiding l—lliA restrictions with known inadequate response or with sed prevalence or ial (I: ation with decreased clinical performance is selected.

Any patient diagnosed with primary progressive MS (PPMS), secondary progressive MS (Sl’MS), or relapsing remitting MS (RRMS) patients may he treated with EBV {Ellis as long, as there is an available cell line with an l-lLA restriction that matches an l-lliA allele on the patient.

Eramjie .5 Treatment i mm third arrv donor derived CTZ’ZS Patients with relapsing remitting, primary progressive, and secondary progressive MS are treated with third party allogeneic targeted Ls that exhibit cytotoxicity against Bmcells and plasma cells presenting EBNAl, l...Ml’l and l.;Ml’2 antigens, F’atieiits receive four administrations of targeted EBV~CTLs at a dose of 2 x ltd/“7 cells/m2, administered intra I’enously at QZ week intervals (lie. on Days, l, 15, 29, and 43). Patients are assessed for relapse events, serial Gadolinium enhanced brain MRl, and serial lumbar puncture to measure cerehrospinal fluid lgG levels and incidence of oligoelonal hands. The Expanded lity Status Scale (EDSS) is administered to characterize the progression of lity.

Concomitant medications and adverse events are ted to characterize the safety profile oftreatment. The following are indications of efficacy oftreatinent: l) Significant decreases in new nium enhancing lesions, as observed on Mill imaging at monthly visits in RRMS patients when compared to historical controls in a similar patient population. 2) Significant decreases in annualized clinical relapse rates at monthly visits when compared to historical controls in a r patient population. 3) Significant reduction in CSF lgG levels when compared to baseline in primary progressive MS (PPMS), secondary progressive MS (SPMS) and relapsing remitting MS (RRMS) patients. 4) Thirty percent of primary ssive, secondary progressive and relapsing remitting MS patients e lonal bands which had been present at baseline.

) Mild to moderate ement in EDSS scores at 6 and l2 months in primary progressive MS (PPMS), secondary progressive MS (SPMS) and relapsing remitting MS (RELMS) patients. 6) Significant improvement occurring in motor strength in 50% of TERMS, 30% of UT! PPMS, and 259/5 of SPMS patients. 7) 80% ofRRlvlS patients showing no evidence of disease activity at l year compared to historical controls which showed 65%.

Example 4': Treatment 0 ill/IS mitt} third tartv donor dermal?! (7715 34324 1' 88" Patients with relapsing remitting, primary progressive, and secondary progressive MS are treated with adoptive transfer ofthird party donor derived C'l‘ls. Allogeneic latency—2 rgeted cytotoxic T lymphocytes (allogeneic [.2 EBV CTLs), or ATAltllla are l-TLA- matched, m vitromexpandedi antigennspecitic T cells specific for EBV protein ns including latent membrane protein l (lil‘v’llll), Lily/ll); and EBNAL ATAlSS is produced from the peripheral blood niononnclear cells (PBMCs) of healthy EBV seropositive donors A portion of these donor cells become the T cells for imminiotherapy and a portion are the antigen presenting cells (AFC) which are used to stimulate the T cells. The Al’Cs are transduced With a novels recombinant, replication ent adenovirns encoding a tran sgene that expresses a polypeptide protein and ted EBNAl protein loh/leoly) The polyepitope protein includes multiple T-lLA class l—restricted CD8+ T cell epitopes from LMPl and LMPZ as a g of heads? The truncated EBNAT protein excludes the glycine— alanine repeat sequence which interferes with translation and endogenous processing of this protein and maintains the CD'S-+- and CD4+ T cell epi‘topes. Preclinical and al studies have shown that these LMP and EBNAl expressing APCs are highly effective at inducing the rapid expansion of antigen ~specilic T cells from human donors in the presence of eukin~2 . The resulting cell product, ATA l88a is cryopreserved and verified to he T-lLAurestricted with cytotoxic potential and to be without irns infectivity.

Protocol and Dosing Elatients receive 2 cycles of treatment with each cycle ting of a lS—day treatment period (with 3 infiisions, each given approximately '7 days apart, on Days l, 8 lit-2 days}, and l5 {:2 . Alter the third infusion of Cycle l, subjects enter a Zilnday ohse rvation period with approximately weekly , and, alter the third infusion of Cycle .2 subjects enter a follow—up period with l l monthly (every 23 i5 days) visits. er, subjects are observed for at least l year after the first dose of ATAlSS.

The first cohort is treated at a dose ot‘5 X l06 cells, followed by doses of l >< ltll, 2le HF, and 4.0x MW, (in Cohorts 2, 3 and 4, tively). Within Cohorts l to 4, treatment (I: is staggered for the subjects, with an 8~day pause n treatment ofthe first and second subjects and the second and third subjects (eg treatment for the second subject may begin the day after the first subject receives their Day 3 on, if no dose limiting toxicities are observed. Dose limiting toxicities, or Ills/Ts, is a toxicity considered at least possibly related to the administration of ATAlXS. Once the third subject is ed, the remainder ot‘the ll) cohort is ed. Dose escalation from one cohort to the next will occur if no DL’l‘s occur during the first 35 days after the first dose of Cycle l Day l (is, 35rday BL? assessment window) for all 6 subjects in the cohort. lf one subject among the six experiences a BLT within the 35nday assessment window, an additional 3 subjects will he enrolled into that close cohort. If no lel‘s are observed. (within the 35—day assessment ) among the additional 3 subjects, dose tion to the next dose cohort will proceed. if 2 or more of the 9 subjects within a cohort experience DLTs within the 35—day assessment , that dose level will be considered the maximum tolerated dose (MID). The bill) is highest dose studied at which < l in 6 subjects have lel‘. lf all doses have < l in 6, then, the hill) is the highest dose studied. ln on, the previous dose level will be considered the RPZD. RPQB is the ATAl 38 dose selected for phase 2 based on evaluation of all safety, efficacy, and hiomarlrer data collected during dose escalation (216,, Cohorts Ml) with a subject nce of DLTs of < 16.6% during the first 35 days of dosing by the enrolling investigators and sponsor’s medical monitor. it 2 or more ofthe 9 subjects within the lowest dose cohort (Cohort l) experience DL’l‘s within the 35nday assessment window, a lower dose/schedule may be explored in consultation with the sponsor’s medical monitor and the enrolling investigators.

Dose tion will be based on satety assessments, including treatnientuemergent adverse events (llrk‘tl‘sl clinical laboratory data, physical examination, findings, including vital signs, and electrocardiograms (ECG s) after all subjects within a cohort have completed the 35~day BLT assessment window.

The dose expansion (:16. Cohort 5) will he performed at the Rig/El) with no staggering/pausing of treatment between subj ects.

Patients are assessed for relapse events and change from ne in the number of gadolinium (Gd)_enliancing and new or enlarging T2 lesions on brain magnetic resonance imaging (Mill) scans. A'l‘AlStl is selected for each subject based on matching at least 2 human leukocyte antigen (HLA) alleles with at least l Elicia-restricting allele shared n UT! ATAl 83 and the subject. lhe ed Disability Status Scale (EDSS) is administered to characterize the progression of the disease and of disability. Concomitant medications and adverse events are collected. to characterize the safety e of treatment Outcome .Measwes/Smdy ments.“ The following are tions icacy oftreatnient: l0 l.) The change from baseline in the number of hancing and, new or enlarging T2 lesions on brain Mill scans. 2) Decreases in ized clinical relapse rates. 3) Mild to moderate improvement in EDSS scores in primary progressive MS , secondary progressive MS {Sl’h’lS} and ing ing MS (RRMS) patients l3 Patients are evaluated for the frequency; persistence, and expansion of circulating EBV~specitic T cells, and to correlate cellular kinetics with efficacy and safety endpoints.

Additionally, any number of endpoints may be evaluated in the study participants. For example, the change in EBV—deoxyrihonucleic acid (DNA), the change in vitamin US, the change in nuerofilamintsj the change in thl magnetic field transfer ratio (Mm), the change in clinical outcome assessments (cg Multiple Sclerosis impact Scale 29 (MSlS) score, Fatigue Severity Scale (FSS) scorer, Visual Acuity (VA)? and le Sclerosis Functional Composite (MSFC) score»? and the change in iirmiunoglobulin Ci (lgG) index (including quantification of lgG and oligoclonal band (0(33} analysis in serum and cerebral spinal tluid ((78.3)) may be measured by taking a first measurement prior to the administration of T cells 2.5 and, taking additional measurements during or after the study.

Study Population: Up to 42 subjects with RRMS and 6 subjects SPMS with recent disease activity will he enrolled at 6 to l 0 study sites. If no Dljl" occurs in the study; a total of 36 sub} eets will he enrolled (30 with RRMS and 6 with SPMS).

The following are inclusion/exclusion criteria for patients involved in the study. A subject will be considered eligible to participate in this study if all ofthe following are satisfied: l. History of MS? meeting one of the following criteria: RRMS, as defined by the 20m Revised McDonald criteria for the diagnosis of SPMS diagnosed at least 1 year prior to enrollment: with no history of relapses within the year before ing informed consent 2 Positive EBV serology 3. Availability of appropriate partial -inatched and restricted ATAl 83 4. Males and females 18 to 45 years of age . EDSS score ) to 6.5 it) (i. Willing> and able to provide written informed consent A suhj ect will not be eligible to participate in the study if any of the following criteria are met: l. Concurrent serious uncontrolled or unresolved l condition, such as infection, limiting protocol compliance or exposing the sub} ect to unacceptable risl< l5 2. Positive serology and/or nucleic acid g (NAT) for human immunodeficiency virus (HIV) 3. Serology and/or NAT indicating active hepatitis B Virus (HBV) infection or carrier status for HBV (Note: A positive serology tor l-lBV indicating a previous but cleared infection with HBV is not an exclusion ion) 4. Serology and/or NAT ting active hepatitis C Virus (HCV) infection . Positive serology for syphilis or human T cell lymphotropliic Virus l/ll (llTLV) 6. Significant non—malignant disease (eg, severe cardiac or respiratory dysfunction) 7. Uncontrolled psychosis, uncontrolled depression or suicide rislr, substance dependence, or any other psychiatric condition that may compromise the ability to participate in this trial 8. Clinically significant abnormalities of full blood count, renal on, or c function: a. Elevated liver function tests including> total hiliruhin (TRIM) > l5 X the upper limit of normal (ULN; unless t has documented Gilbert’s disease), aspartate 3t) aniinotranstcrase (AST) or alanine arninotransferase (ALT) > 3.0 >11 ULN. ‘0, Sub} ects with both a creatinine > 1.5 / ULN and an estimated creatinine clearance of < 60. mL/min (using the (using the Cockcroft—Gault equation) c, l-lemoglohin < l0 g/dlo; platelet < lOflx ; ahsolute neutrophil count < l.5% ltlg/L 9. Any contraindication to Mill and/or Gd, such as allergy or any object that is reactive to strong static magnetic, pulsed—gradient fields including any metallic fragments or foreign body (eg, sm clip(s), pacemakers, electronic implants, shunts) l0. Prior cancers, except successfully treated nonninelanoma skin cancer or carcinoma in situ, of the cervix, with a 2: 5% chance of recurrence within l2. months l l. lnnnunoinodulatory therapy (apart from short courses of corticosteroids) as follows: a. Any previous ent with a B—cell depleting agent l0 b. Any previous treatment with alerntuzuinah e. Treatment with amer acetate or lFNfi within 4 weeks of providing informed d. Treatment with dinietliyl te within 4 weelrs ofproviding informed consent e. Treatment with fingolirnod within 2 months of providing informed consent f. ent with natalizumah, niethotrexate, azathioprine, or cyclosporine within 6 months of providing informed consent g. Treatment with teriilunoniirle within l2 months of providing informed consent unless patient has completed an accelerated clearance with tyramine h, Treatment with niitoxantrone, cyelophosphamide, cl adrihine, rituxiniah or any other imniunosuppressant or cytotoxic therapy (other than steroids) within l2 months of providing informed. consent, or determined by the investigator to have residual immune suppression from these treatments l2. Antithymocyte globulin or similar antimT cell antibody therapy 5 4 weeks before providing informed t.

E‘J U} 13 Female of childbearing ial unwilling to use a highly effective method of contraception (i. e. , one that results in ncy less than l% per year when used consistently and correctly ), 6g. , implants, iriiectahles, combined oral contraceptives, some terine contraceptive devices, sexual abstinence, or a vasectomized partner while undergoing treatment with ATAl 88 and for 3 months after the last dose.

OR Men with a female partner ofchildhearing potential unwilling to use a highly effective contraceptive measure and/or unwilling to refrain from donating sperm while undergoing treatment with ATAl 83 and for 3 months after the last dose l4. Women who are breastfeeding. l5. Pregnancy. l6. inability to comply with study procedures. l 7'. Previous treatment with EBV T~cell therapy.

Statistical ill/{clitoris ed in the Study Analysis Population All suhi ects enrolled in the study and who e any study product will be included in the efficacy and safety populations. The efii cacy population will be for the primary efficacy analyses, and all analyses of disposition, demographic and baseline disease ll) characteristics. ln order for a subject to he considered evaluable for the analysis of a BET, the subject should have either had a BLT during the 35~day DLT ment window or had completed the 35—day DLT assessment. ttticacasiiahsc» The descriptive statistics will he provided for the efficacy endpoints, and in addition the continuous efficacy endpoints will he analyzed using regression methods.

Safety Analyses Safety assessments will, include all related and unrelated AEs All AEs will be mapped using the Medical Dictionary for Regulatory Activities and graded according to the CTCAE version 4.03. AEs will be ized by the number and percentage of subjects for whom AEs were reported serious versus non~serious, and investigator—reported relationship (unrelated? possibly d, related). Descriptive statistics will he used to ize AE types and frequencies.

Peripheral blood mononuclear cells (PBMCS) were obtained from of healthy EBV seropositive donors (NMDP Donors) or MS patients. A portion of each of these donor cell samples were used for as the source of expanded CTLS and a portion were used as a source the antigen presenting cells (AECs) which used to ate the C'l‘lss. The AFCS were uced with a recombinant, replicationudeticient adenovirus encoding a transgene that expresses a polypeptide protein and truncated EBNAl protein (AdElnLMPpoly). The polyepitope protein ed multiple lfilliA class l~restricted CBS+ T cell es from lsMPl and LMPZB as a c"string of . The truncated EBNAl protein excluded the glycine— alanine repeat sequence which interferes with translation and endogenous processing of this protein and maintains the {IDS-t and CD4+ T cell epi‘topes. The CTL portion ofthe donor cell sample was concultured with the prepared Al’Cs to expand and stimulate CTLs in the sample specific for the EBV es. Following stimulation and generation of CTL ‘4’! comprising products, the CTL hatches were tested for effector function by FACs. As seen in Figure l, the CTL ts generated from healthy donors had a significantly higher percentage of vi ahle lymphocytes that are interferon y (ll‘Ng) expressing and CBS+ compared to CTL products generated from MS patients (Mann Whitney p value of 0.0.002).

These data show that a more robust CTL product with a higher fraction of or CD8 T l0 cells and onal lFNg"‘ C'l‘Ls is ted when healthy donors are used as the source of C'l‘lss for allogenic transfer compared, to when MS patients are used as the source of C'l‘ls for autologous transfer All publications s. patent, applications and sequence accession numbers mentioned herein are hereby incorporated by reference in their entirety as if each individual publication; patent or patent application was specifically and individually indicated to be incorporated by reterence. in case ot‘conl‘lict, the present application, including any definitions herein“, will control.

Those d in the art will recognize, or he able to ascertain using no more than routine experimentation, rnany equivalents to the specific embodiments of the invention described. herein. Such, equivalents are intended. to he encompassed by the ing claims.

Claims (1)

1. What is claimed is: l. A method of treating or preventing an autoimmune disease in a subject, comprising administering to the subject allogeneic cytotoxic T cells (C’l‘Ls) expressing a T cell receptor that cally binds to an EBV peptide presented on a class l h/ll-lC 5 2. The method, of claim l, wherein the class 1 MHC is encoded by an lllA allele that is present in the subject. 3 The method of claim 1 or 2, wherein the autoimmune disease is multiple sclerosis (MS). 4. The method, of claim l, or 2 wherein the autoimmune disease is rheumatoid arthritis l0 (RA). 5. The method of any one of claims l to ll? wherein the neic 0?le are obtained from a cell bank. 6. A method of treating or preventing; an autoimmune e in a subject sing: a) selecting from a cell hank allogeneic xic T cells (Cllas) expressing a l5 T cell receptor that specifically binds to an EBV peptide presented on a class l Ml-lC; b) administering the allogeneic C'l‘Ls to the t. 7. The method ofclaim 6, wherein the class I Ml—lC is d by an HLA allele that is present in the subject. 8. The method of claim 6 or 7, wherein the autoimmune disease is multiple sclerosis 20 (MS) or rheumatoid arthritis (RA). 9. A method of treating or preventing an autoimmune disease in a subject, sing: a) incubating a. sample comprising allogeneie cytotoxic T cells (C'l'lis) with antigen—presenting cells (APCs) presenting an EBV peptide? thereby inducing proliferation of peptide~specific T cells in the sample; 25 b) administering the peptidenspecil‘lc allogeneic CTLs to the subject. l0. The method ofclaim 9, wherein the class I Ml—lC is d by an HLA allele that is t in the subject. ll. A method of treating or preventing an autoimmune disease in a subject, comprising: a) ting antigen-presenting cells (APCS) with a c acid construct encoding for an EBV peptide, thereby inducing the APCs to present an EBV peptide; h) inducing peptidenspecil‘ic CTL proliferation by incubating a sample comprising allogeneie CTlis with the antigen—presenting cells (Ath), y ‘4’! inducing the CTLs to proliferate; and c) administering the peptidenspecil‘ic allogeneic CTLs to the subject. l2. The method of claim l 1, wherein the class i MHC is encoded by an HLA allele that is present in the sub} ect. l3. The method of claim ll or 12, wherein the nucleic acid construct is a viral vector. ii) l4” The method of claim l3, wherein the Viral vector is AdlEl poly. la. The method of any one of claims 9 to l4, wherein the allogeneic C'Tlss are stored in a cell hank before being> administered to the subject. lot The method of any one of claims .9 to l5, wherein the autoimmune disease is multiple sclerosis (MS). 15 l7. The method of any one of claims 9 to l5, wherein the autoimmune disease is rheumatoid arthritis (RA). lit The method of any one of claims .9 to l7, wherein the sample is incubated with one or more cytohines in step (a). l9. The method of any one of claims 9 to l8, wherein the APCs comprise B cells. 29 2.0. The method of any one of claims 9 to l9, wherein the Al’Cs comprise n— presenting T-cells. 2l. The method of any one of claims 9 to 20, wherein the APCs comprise (leiidritic cells. 22. The method of any one of claims 9 to 213 wherein the APCs comprise artificial antigen~presenting cells. 23. The method of claim 22, wherein the artificial antigennpresenting cells are aléi 62 cells. 24. The method of any one of claims 9 to 23, wherein the sample comprises. peripherai hiood mononuclear cells (PBMCS). 25: The method of any one of claims 1 to 24, wherein the EBV peptide ses a LMPl peptide or fragment thereof. U1 26. "the method of any one ofeiaims i to 245 n the EBV peptide comprises a LMPZA peptide or fragment thereof. 27. The method ofany one of claims 1 to 24, wherein the EBV peptide comprises an EBNAE peptide or fragment thereof.