NZ751506B2 - Immunotherapy for polyomaviruses - Google Patents

Abstract

Provided herein are methods and compositions related to polyomavirus epitopes useful in the treatment of cancer or a polyomavirus infection.

Description

L74 APYFGRPOLY051A VIELI'SES RELATED APPLICATIONS This application claims the bene?t of priority to U .S, Provisional Patent Application serial number 62/3 85456, ?led September 9, 20l6, which is herein incorporated by reference UT in its entirety.

BACKGROUND l’olyornavirnses are nbiq nitous viruses that infect a wide range of mammalian species. Currently, more than l2 distinct human polyomavirns species have been ?ed, including BK polyomavirtis (BK‘V), John Cunningham polyoinavirus (ECV), and Mer‘kel cell avirns (MCV).

Most human polyoinaviruses diseases are acquired in childhood, though clinically apparent diseases in irnniunocompetent hosts are generally rare. BKV and JCV viruses typically remain latent possibly in the lymphoid organs, neuronal tissue, and kidney.

However, under the circumstances of inimunosnppression, both JCV and BliV reactivate and may progress to signi?cant organ disease. For example, BK‘V is tirotheliotorpic and reactivation of BKV causes a fonn of interstitial nephritis, known as BK polyornaviruses associated nephropathy, which is typically associated with higli graft loss when not ized early. ropic lC virus may enter the "brain and cause progressive innltifocal leukoencephalopathy, a demyelinating disease of the central nervous system with a high ity rate. Various polyornaviruses have also been associated with different forms of cancer. For example, MCV has been associated with Merltel cell oma, a rare but aggressive form of skin cancer. "there are no known effective antiviral agents for treatment of polyoniavirusesi Thus, new therapies are needed to treat and prevent polyoniavirus infections and/or naviIns—associated cancer.

Provided herein are compositions and methods related to polyoinavirus epitopes (eg epitopes listed in Tables 1, 2, 3, 4 and/or 5) that are recognized by T lymphocytes (6g, cytotoxic T lymphocytes (C’l‘Ls) andfor helper T lymphocytes) and that are useful in the prevention and/or treatment of a avirus infection (eg. a BKV, JCV, or MCV virns infection), and/or cancer {8. g. a polyomavims associated , such as a BKV, JCV, or MCV associated cancer). In some ments, the compositions and methods relate to Bli‘v’ epitopes (eg, the epitopes listed in Table l). ln some embodiments, the compositions and, methods provided herein relate to JCV epitopes (eg the epitopes listed. in 'l‘able 2). ln some embodiments, the compositions and methods relate to hybrid es that incomorate sequence variations found within a viral strain and/or across d viral strains tag, the epitopes listed in Table 3). in certain aspects, provided herein is a protein (eg, an isolated n) sing one or more epitopes from one or more BKV antigens (9g, epitopes from LTA, STA or VPl viral antigens, such as the epitopes listed in Table l), one or more ECV antigens ((eg. epitopes from LTAQ, S'I'A or Vl’l viral antigens, such as the epitopes listed in Table 2) and/or one or more hybrid epitopes (eg, the epitopes listed in Table 3). In some embodiments, the polypeptide comprises a plurality of such epitopes. in some ments, the polypeptide further ses an intervening amino acid sequence between at least two of the plurality of es. in some embodiments, the protein is capable of eliciting an immune response upon administration to a subject (6.53., a mammalian subject, such as a human subject}. in some embodiments, the epitopes are selected to provide broad coverage ofthe human population. in some embodiments, the es have HlA class l restrictions to HLA~AL —A2, ~1A3, ~Al l, —A23, ~A24, ~A26, —A29, ~ A30, ~87, —B8, 5827, - B35, 4338, - B40, 4341, —B44, —B5 l, 4356, 4357 or 358. in some embodiments, the epitopes have l-lLA class ll ctions to HLiAnDl’, —DM, "DOA, "DOB, "DQ, or —DR. in some embodiments, the epitopes have HLA class ll restrictions to HIA~DRB or -l)QB. In some embodiments, the protein ses, consists essentially of or consists ofepitope amino acid sequences set forth in SEQ lD NOS: 5, e, 36, 41 and 42. in some embodiments, provided herein is a pharmaceutical composition comprising a protein provided herein. in certain aspects, provided herein is a nucleic acid (eg. an isolated nucleic acid) encoding a protein disclosed herein. in some embodiments, ed herein is an expression construct comprising such a nucleic acid. in some embodiments, provided herein is a host cell comprising such an expression con struct. ln certain aspects provided herein is a method 3-3 of producing> an isolated protein comprising expressing the isolated protein in the host cell of provided herein and at least partly purilying the isolated protein. ln some embodiments, provided herein is a pharmaceutical composition comprising a c acid provided herein.

In certain aspects, provided herein is a T lymphocyte (eg, a. an isolated T lymphocyte, a CD4+- T cyte, a (338+ T lymphocyte) comprising a T cell receptor (TCR‘) that speci?cally binds to an epitope described herein presented on an HLA (cg, a class l HLA, a class ll l-lLA). in certain embodiments, provided herein is a method of ing BK virus-specific T lymphocytes for adoptive immunotherapy, including: (i) contacting one or more cells isolated from a subject, wherein the one or more cells comprise T lymphocytes, with an antigen presenting cell presenting an epitope provided herein; and (ii) culturing the one or more cells under conditions such that BK Virus—speci?c T— lymphocytes are expanded from said one or more cells. In speci?c embodiments, culturing the one or more cells is performed in the presence oflL—Zl, in some embodiments, the cells are cultured. in the presence ofat least l, 2, 3, Al, 5, 6, 7, 8, 9, 10, ll, l2, l3, id, 15, l6, l7, l8, l9, 20, 2l 29 or 30 rig/ml lL—Zl ln some embodiments, the , 22, 23, 24, 25, 26, 27, 28, , cells are cultured in no more than 30, 35, 4t), 45, 5t}, 60, 7t), 80, 90 or 100 ng/ml lL—Zl. In some embodiments, the cells are cultured in lOUin, 2040, 25—35 or about 3% rig/ml lL—Zl. in some embodiments, the cells are cultured in 30 l lL-Zl. ln certain embodiments, compared to expansion in the absence of lL—Zl, expansion in the presence oflL—Zl results in an increase in the ratio of absolute number ofpolyomavirus—speci?c CD8 T cells to the te number of polyomavirus~specific CD/l T cells in the ed population of T lymphocytes. in certain embodiments, provided herein is a method oftreating or preventing a polyomavirus infection (eg, a BKV, ECV or MCV infection) and/or treating a polyomaviriis~associated cancer (tag, a REV-associated, l’CV—assooiated or hcl'CV—associated cancer} and/or inducing a T—lymphocyte immune se in a t comprising administering to the subject a protein, nucleic acid, T cell or pharmaceutical composition provided herein. ln some embodiments, the sub} ect is a mammal in some ments, the subject is a human. In some embodiments, the subject is immunocompromised, In certain aspects, provided herein is a method of detecting a BK virus infection in a subject, the method comprising detecting the presence ofBKVmspeci?c T lymphocytes by ting T cytes ed. from the subject with the isolated n provided herein. ln some embodiments, the method further comprising treating the BK virus infection in the subject according to a method described herein. in some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is immunocompromise . in certain aspects, provided herein are methods of ng a cancer in a subject (tag. a polyomaviius~associated cancer, such as a , 1CV—, or sociated cancer). in some embodiments, the method comprises administering to the subject a pharmaceutical composition comprising cytotoxic T cells {CTLS) comprising T cell receptors (TCRs) that recognize one or more (tag, 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, 31) or more) ofthe epitopes listed in Table 1, Table 2 and/or e 3. in some embodiments, the subject expresses a human. leukocyte antigen (1-1LA) to which the one or more es is restricted. in some embodiments, the CTLs are autologous to the subject. 1n some embodiments, the CTLS are not autologous to the subject. in some ments, the CTLs are obtained from a CTL library or bank, In some embodiments, the method. ses administering to the subject a vaccine composition comprising one ormore (tag, at least l, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) ofthe epitopes listed in Table 1, Table 2 and/or 'l'ab1e 3. in some embodiments, the method comprises administering to the .15 subject a pharmaceutical composition antigen presenting ce11s (APCs) presenting one or more (cg, at least l, 2, 3, 4, 5, 6, 7, 8, 9,111,11,12,13,14,15,16,17,18.19, 21), 21, 22, 23, 24, 25, 26, 2‘7, 28, 29, 3C1 or more) of the epitopes listed in Table 1, Table 2 and/or Table 3. 1n some embodiments, the subject expresses a. human leukocyte antigen (HLA) to which the one or more es is restricted. in certain aspects, provided herein are methods of treating a polyomaviius infection (eg. a BKV, MCV, or JCV infection) in a subject. In some embodiments the subject is immunocompromised. in some embodiments, the method comprises administering to the subject a pharmaceutical composition comprising CTLs comprising TCRs that recognize one or more (cg, at least 1, 2, 3, 4, 5, 6, ’7, 8, 9,10,11,12,13,14,15,16,1’7,18. 19, 2G, 21, 22, 23, 2.4-, 25, 26, ‘27, 28, 29, 30 or more) of the epitopes listed in Table 1, Table 2 and/or Table 3. in some embodiments, the subject expresses a l-lLA to which the one or more epitopes is restricted. in some embodiments, the CTLs are autologous to the subject. In some embodiments, the CTLS are not antelogous to the subject. in some embodiments, the CTLS are obtained from a C11, library or bank in some embodiments, the method comprises 3-3 steiing to the t a vaccine composition comprising one or more (tag, 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, 211, 29, 30 or more) ofthe epitopes listed in Table 1, Table 2 and/or Table 3. ln some embodiments, the method comprises administering to the subject a pharmaceutical composition n presenting cells (APCS) presenting one or more (6.5;. at least 1, 2, 3, 4, , 6, 7, 8, 9,it),ll,12,l3,14,15,16,l7,18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) of the es listed in Table 1, Table 2 and/or Table 3. ln some embodiments, the subject expresses human leukocyte antigens (111.121) to which the one or more epitopes is restricted. in some aspects, provided herein is a population of C'l‘los sing '1' cell receptors (TCRS) that recognize one or more (eg. at least 1, 2 3, 4, 5, 6, 7, 8, ’9, 10, 11, 12, l3, 14, 15, 16, 17, 18. 19, 20, 21, 22, 2'3, 24, 25, 26, 27, 28, 29, 30 or more) ofthe epitopes listed in Table 1, Table 2 and/or Table 3.

In some aspects, provided herein is a population ot‘APCs presenting one or more (eg, at least 1, 2, 3, 4, 5, 6, 7, 8,9,10, 11,12, 13, 1/41, 15, 16, 17,18. 19, 20, 21, 22, 23, 24, , 26, 2",", 28, 29, 30 or more) oftlie es listed in Table 1, Table 2 and/or Table 3. ln some embodiments, the APCs comprise B cells, antigen—presenting T cells, dendritic cells and/or artificial antigenmpresenting cells, such as aK562 cells. in some aspects, the antigen" presenting cells (cg Z cells) express C1381), C1383, 41138—1, and/or (11386. 1n some embodiments, provided herein are methods of treating or preventing cancer (eg, a polyomavirns associated , such as a BKV, JCV, or MCV associated cancer) and/or a polyoniaviins (cg, BKV, JVK, or MCV) infection in a. subject comprising administering the APCs described herein to a t. in some aspects, provided herein is a polypeptide comprising one or more (eg, at least l, ‘2, 3, 51-, 5, (3, 7, 8, 9,10,11,12,13,14,15,16,17,18.19, 21), 2.1, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) of the epitopes listed, in. Table 1., Table 2. and/or "Table 3. in certain aspects, provided herein is a nucleic acid molecule tag. a DNA molecule or an RNA le) encoding a polypeptide comprising one or more (8.3:. 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, 2-4, 25, 26, 27, 28, 29, 3O ormore) of the epitopes listed in Table 1, Table 2 and/or Table 3, In some embodiments, the nucleic acid molecule is a vector (eg, an adenoviral vector). In some embodiments, provided herein are vaccine compositions comprising a polypeptide and/or a nucleic acid molecule described herein.

In some embodiments, ed herein are methods of generating, activating and/or inducing proliferation of polyomavirnsnpeci?c CTLs (cg, BKV speci?c or JCV c C'l‘liskomprising contacting CTLs with Al’Cs that present one or more (cg, at least 1, 2, 3, 4, :3, 6, 7, 8, 9,1(l,ll,12,l3,14,15,16,17,18.19, 20, 21, 22, 23, 241, 2:3, 26, 27, 28, 29, 3O or more) ofthe epitopes listed. in Table 1, Table 2 and/or Table 3. in some embodiments, the CTLs are ted with APCs in vitro. In some embodiments, the APCs comprise 13 cells, antigen—presenting '1‘ cells, dendritic cells and/or artificial antigen-presenting cells, such as aKSéSZ cells. in some s, the n~presenting cells (eg, 211662 cells) express CDSO, CD83, 411384;, and/or CD86. in some embodiments, the CTLs are contacted to the APCs in the presence of one or more cytokines. in some ments, provided, herein are methods of generating Al’Cs that present epitopes ed herein comprising contacting APCs with a polypeptide comprising one or more (an, at least 1, 2, 3, 4, 5, 6, 7, 8, 9,1(1),11,12,13,14,15,16,17,18.l9, 2t), 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) ofthe epitopes listed in Table 1, Table 2 and/or Table 3 and/or a nucleic acid encoding a polypeptide comprising one or more (eg at least 1, 2 3, 4, 5, 6, 7, 8, 9,10,11,12,l3,14,15,l(§,17,18.19, 20, 21, 22, 2'3, 24, 25, 26, 27, 28, 29, 30 or more} of the epitopes listed in Table 1, Table 2 and/or Table 3 . ln some embodiments, the APCs express HLA to which the one or more epitopes is restricted. .15 in some embodiments, the one or more epitopes comprise an epitope shared by two or more polyornaviruses. in some embodiments, the shared epitope comprises a region of sequence gy between the at least two polyomaviruses, and the region of ce homology is at least 3 4, 5, 6 or 7 amino acids across the full length ofthe e sequence. 1n some embodiments, the two polyomaviruses are BKV and .lCV. in some embodiments, the at least three amino acids are LLL.

In other aspects, provided herein is a method of identi?/ing a subject suitable fora method of treatment provided herein (e.g administration of C'l‘lls, Al’Cs, or e compositions provided herein) comprising isolating a sample from the subject (e. g, a blood or tumor sample) and detecting the presence of an e provided herein, or a nucleic acid encoding an epitope provided herein. lri certain embodiment, the subject is ?ed as suitable for a method of treatment provided herein if the subject expresses an 1114A to which one or more of the epitopes described herein are restricted. In some embodiments, the subject identi?ed as being suitable for a method of treatment provided herein is treated using the method of ent. 3-3 BRIEF DESCRI?TION OF THE DRAWINGS Figure 1 shows the in vitro expansion of BliV speci?c T cells. The dot blots show the detectable expression of 1FN «y by BKV specific T cells after growing the PBMCS with BKV antigens and CMV is shown as a positive control.

Figure 2 shows the T cell response to BKV antigens. The graphs show the overall T cell response to BKV antigens in healthy individuals.

Figure 3 shows peptide matrix for large T antigen (LTA), as well as the composition of the peptide pools following the matrix format.

Figure 4 is a flow chart showing the s of epitope mapping bed herein.

Figure 5 has live panels and shows epitope mapping ofHLA 8* 39 epitope. Panel A shows FACTS blot for the {138* T cell response to S'l'A OPP. Panel B shows S'l‘A pep pools 4 and l0 responded when overlayed on the matrix showed STAZZ peptide to be the common peptide among the pools. lCS assay with ST22 stimulation showed a response which is shown in the FACS hlot next to the matrix. Panel C shows fine e mapping by trimming the amino acids from either side of the S'l‘IZLZ peptide. Panel l.) shows the responding peptides from trimming process are titrated to see the most immunogenic section of the peptide which showed Vl—lCPCMLCQL to be the epitope sequence. Panel B shows antigen presentation assay using the peptide loaded HLA restricted LCLS showing the epitope to be HLA 8*39 restricted Figure 6 shows transcriptional regulators in BKV and CMV speci?c T cells. The histogram shows the comparison of CMV and BKV ic T cells for the expression oi"? bet, Eomes, Granzyme l3 and perforin Histogram lines shows the expression in CMV speci?c T cells and BKV speci?c T cells as indicated.

Figure 7 shows in Vitro expansion of BKV-speci?c T cells following stimulation with pooled BKV epitopes (see Table 1). PBMC from y volunteers were stimulated with synthetic BKV es for l h and then cultured for l2~l4 days in the presence different cytokine combinations. These included an2 (Ming/ml), lL—Zl (Bilng/ml), ll_.’7 ( lOng/ml), lLlZ { lOng/ml) and/or lLlS ( l). Blé‘v’ speci?city oftliese "l" cells was assessed using rd. intracellular cytolrine assays.

Figure 8 shows consensus sequence alignments between BKV and JCV LTA, STA and VPl amino acid sequences.

Figure 9 shows sus sequence alignments between BKV and MCV LTA, STA and Vl’l amino acid sequences.

Figure 10 shows the transcriptional factor and or le profile of BKV speci?c T cells grown in the presence of lL2 or 1L2 and lLZl. The frequencies of granzynie high and "1" bet high cells were higher in cells grown in the presence of lL—Z and lL~2l Figure 11 shows the lFN~y expression of CD4 and CD3 T cells grown in the presence of lL—Z or lLZ and lL—Bl and analysed for the specificity using BKV es.

Figure 12 shows the number of CD4 and CD8 cells after e in the presence of lle or ill—2 and llel, "lhe total number ofBKV speci?c CD4+ T cells was reduced, in the cultures grown in the presence of lL~2 and lL-2l compared to cultures grown in 112 alone.

Figure 13 shows that the percentage of C925+ cells in both Cllllt’t+ and CD4+ T cell populations was higher in the T cells grown in the presence of lid-2 alone compared to cells grown in presence of lL—Z and lL—Z 1.

Figure 14 shows neuropilinl expression on {TIM-13325hi CDlZC’mw cells (Treg cells), Figure 15 shows representative lFNm'y sion data from exemplary epitopes that Show BKV/JCV cross—reactivity.

Figure in shows representative lFN—y expression data from cells expanded using a .lCV epitope and recalled using various concentrations of either the .lCV epitope or the corresponding BKV epitope.

DETAILED PTION General l’royided herein are compositions and methods related. to polyomavitus epitopes (a: s, epitopes listed in Tables l, 2, 3, 4 and/or 5) that are recognized by T lymphocytes tag, cytotoxic T lymphocytes (CTLs) and/or helper T lymphocytes) and that are useful in the tion and/or treatment of a polyomavirtts infection (eg. a BKV, lCV, or MCV S infection), and/or cancer (e. g., a polyoniayims associated. cancer, such as a BKV, JCV , or MCV ated cancer). In some embodiments, the compositions and methods relate to BKV epitopes (cg, the epitopes listed in Table l). in some embodiments, the compositions and, methods provided herein relate to lCV epitopes (eg the epitopes . in Table 2). in some embodiments, the compositions and methods relate to hybrids epitopes that ass variations found within or across BKV and lCV epitopes (e. g, the epitopes listed in Table 3-3 Definitions For convenience, certain terms employed in the speci?cation, examples, and appended claims are collected here.

The attic es "a" and "cm" are used herein to refer to one or to more than one tie. to at least one) of the grammatical object of the article. Byway of e, "an element" means one element or more than one element.

As used herein, the term istering" means providing a. pharmaceutical agent or composition to a subject, and includes, but is not limited to, stering by a medical professional and self—administering. Such an agent can contain, for example, peptide described herein, an antigen presenting cell provided herein. and/or a CTL provided, herein.

The term "amino acid" is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of in; O being included in a polymer ofnaturallymoccurring amino acids. Exemplary amino acids include naturally—occurring amino acids; s, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing.

The term ng" or "interacting" refers to an association, which may be a stable association, between two molecules, cg. , between a TCR and a peptide/llLA, due to, for ;._.\ L." example, electrostatic, hydrophobic, ionic and/or hydrogen—bond interactions under physiological conditions. A TCR "recognizes" a T cell epitope that it is capable ofbindmg to when the e is presented on an appropriate HLA.

The term "biological sample," "tissue sample," or simply "sample" catch refers to a collection of cells ed from a tissue of a sub} ect. The source of the tissue sample in ay e solid tissue, as from afresh, frozen and/or preserved organ, tissue sample, biopsy, or aspirate; blood or any blood constituents, serum, blood, bodily ?uids such as cerebral spinal fluid, ic ?uid, peritoneal. ?uid or interstitial fluid, urine, saliva, stool, tears; or cells from any time in gestation or pment of the subject.

As used herein, the term "cancer" includes, but is not d to, solid tumors and blood, borne tumors The term cancer es diseases of the skin, tissues, organs, hone, cartilage, blood and vessels. The term "cancer" ?irther encompasses primary and metastatic cancers.

The term "homologous" as used herein, refers to sequence similarity (eg a nucleic acid, or amino acid. sequence) between two regions of the same sequence strand or between regions of two different ce strands, The term "homologous" may also be used to refer to sequence similarity n two regions of the same sequence strand or between regions regions is occupied by the same amino acid residue, then the regions are homologous at that position. A ?rst region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two s is expre ssed in terms of the proportion of nucleotide or amino g cid residue positions of the two s that are occupied. by the same nucleotide or amino acid e. By way of example, a region having the nucleotide sequence SKATTGCOL-l' and a. region having the nucleotide sequence 5*5l‘i/3i'l‘iC‘iGC—3E share 50% homology. ably, the first region comprises a ?rst portion and the second region comprises a second portion, whereby, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 959/5 of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions ofeach ofthe portions are occupied by the same nucleotide residue.

The term ed " refers to material that has been removed from its natural state or otherwise been subjected to human manipulation, isolated material may be substantially or essentially free from components that ly any it in its natural state, or may be manipulated so as to be in an ial state together with components that normally accompany it in its natural state.

The term ‘jveprz'a’el’ refers to a e or polypeptide, in certain embodiments prepared from recombinant DNA or RNA, or of synthetic origin, or some combination f, which (i) is not associated with proteins that it is normally found with in nature, (2) is isolated from the cell in which it normally occurs, (3) is isolated free of other proteins from the same cellular source, (4) is expressed by a cell from a ditl‘erent species, or (5) does not occur in nature.

The term "epitape" means a protein determinant capable ot‘specitic binding to an antibody or TCR. Epitopes usually consist of chemically active surface groupings of les 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 capable of binding.

As used herein, the phrase ‘tharmaceulicaily acceptable" refers to those agents, compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and 3-3 s Without excessive toxicity, irritation, ic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein, the phrase "pharmaceuocular-acceptable carrier" means a ceutically—acceptable material, composition or vehicle, such as a liquid or solid tiller, diluent, excipient, or t encapsulating material, involved in carrying or transporting an agent from one organ, or portion ofthe body, to another organ, or n of the body. 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 es of materials which can serve as pharmaceutically-acceptahle 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 carboxymethyl ose, ethyl ose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) tale; (8) excipients, such as cocoa butter and suppository axes; (9) oils, such as peanut oil, cottonseed oil, saf?oi 'er oil, sesame oil, olive oil, corn oil i-= O and soybean oil; (10) glycols, such as ene glycol; (l l) polyols, such as glycerin, sor‘oitol, mannitol and polyethylene glycol; (_ l2) estersr such as ethyl oleate and ethyl laurate; ( l3) agar; ( l 4) buffering agents such as magnesium hydroxide and aluminum hydroxide; (l5) alginic acid; (l6) pyrogen—free water; (17) isotonic saline; l: l8) Ringer‘s solution; (l 9) ethyl alcohol; (20) pH huilered solutions; (El) polyesters, polycar‘honates and/or ;._.\ L." polyanhydrides; and. (2.2) other non —toxic compatible substances employed in pharmaceutical formulations.

The terms ‘polynucleonde"; and "nucleic (.1 car" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyrihonucleotides or rihonucleotides, or analogs thereof. Polynucleotides may ha. e any dimen sional structure, and may perform any function. The following are non—limiting examples of polynucleotides: coding or nonncoding regions ot‘a gene or gene fragment, loci (locus) de?ned from linkage analysis; exons, lntrons; messenger RNA (mRN A), transfer RNA; ribosomal RNA, ribozymes, cDNA; recombinant poly/nucleotides; branched poly/nucleotides, plasmids, s, isolated DNA of any sequence, isolated RNA of any sequence; nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as ated nuc eotides and nucleotide analogs. If present, cations to the nucleotide ure may he imparted before or after assembly ot‘the polymer. A polynucleotide may he further modified, such as by conjugation with a labeling component. In all nucleic acid sequences provided ; U nucleotides are interchangeable with ’1‘ nucleotides.

As used herein, a therapeutic that "prevents" a ion refers to a compound that, when administered to a statistical sample prior to the onset of the disorder or condition, reduces the occurrence ot‘the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or s the severity of one or more symptoms of the disorder or condition relative to the ted control sample.

As used herein, "specz?c g" refers to the ability of an antibody to bind to a predetermined antigen or the ability of a peptide to bind to its predetermined binding partner.

Typically, an antibody or peptide speci?cally binds to its ermined antigen or binding partner with an af?nity corresponding to a lie ot‘ahout l0'7 M or less, and binds to the predetermined antigen/binding partner with an affinity (as expressed by Kn) that is at least ll") fold less, at least lOO fold less or at least ltlllO fold less than its affinity for binding to a non—speci?c and unrelated antigen/binding partner (e.g., BSA, casein).

As used herein, the term "subject" means a human or nonnhuman animal selected for ent or y.

The phrases "therapezt?nally—effective amount" and ive amount" as used herein means the amount of an agent which is effective for producing the desired therapeutic effect in at least a suhnpopulation of cells in a subject at a reasonable bene?t/risk ratio applicable to any medical treatment.

"Treating" a disease in a subject or "treating" a subject having a disease refers to subjecting the suhj ect to a pharmaceutical treatment, eg , the administration ofa drug, such that at least one symptom ofthe e is decreased, or prevented. from worsening.

The term "vector" refers to the means by which a nucleic acid can be propagated and/or transferred n organisms, cells, or cellular components. Vectors include plasmids, viruses, bacteriophage, pi‘omviruses, phagemids, transposons, and arti?cial somes, and, the like, that may or may not be able to replicate autonomously or integrate into a some of a host cell. ?nances in certain embodiments provided herein are methods and compositions related BK‘V epitopes, .lCV epitopes, MCV epitopes and/or es that comprise sequences homologous between BKV, .lCV and/or MCV es that are recognized CTLs when presented on an limit, In certain embodiments, the epitopes described herein are useful in the prevention and/or treatment of a polyornavirus infection (e. g. a BKV, lCV, or MCV viral infections) and/or cancer tag, a polyontavims associated cancer expressing an epitope provided ) and/or for the generation of pharmaceutical agents (6'. g. CTLs and/or APCS) that are useful in the prevention and/or treatment of a polyomavirus infection (cg, a BKV, JCV, or MCV viral infections) and/or cancer (62g. 3 polyomavirus associated. cancer expressing an epitope provided herein). in certain embodiments, the epitope is a BKV epitope listed in Table 1, and/or a 36V epitope listed in Table 2. In some embodiments, the e is a hybrid epitope comprising amino acids from, both 21. BEN epitope and. a homologous lCV epitope and/or amino acid variants found within different BKV or .lCV [insert appropriate noun here}.

Exemplary hybrid epitopes are lismcl in Table 3. In some embodiments, the compositions and s provided herein r comprise an l‘vflCV epitope (eg a MCV epitope homologous to an epilope listed in Tables L3). ln some embodiments, the compositions and methods described herein further relate to epitopes from addition viruses, such as EBV, CMV, or ADV. In some embodiments, the epitopes are HLA class lurestrictecl T cell es. in other embodiments, the es are HLA class ll—resiricted T cell epitopes.

Table l: Exemplary EKV HLA class I and class il—restrieted T cell epitopes Epitooe Antigen l-iLA Restriction SEQ ED N().: _______________________________________________________________________________________________________________________________________________________________________________________________________ 'l'l’PK LTA A * l l, AVDTVLAKK A* l l C‘Y’CIDCF'I‘Q A*24 AYL LTA/STA 3* 0'7/B*08 .......................................................................................................................................................................................................

FPLCPUI‘LY S'I‘A. B*35 TLYCKEWPI hll(\()V&(k/l)l>€¥ VHCPCMLCQL srA \Riissimmi"Zilliiiifi"""""""""""""""""""""""""""""""""""""""""""""""""" MELMDLLGL FFAVGGDPLEM ‘1’ClDCFTlQ/EWV TPl-lRl-lRVSA LLLGMYLEF l?GITSML VP 1 3*40 i 7 ARIPM’NI..- B* 2. 7 i 8 VKNPYPISFLL CW* ()7 l 9 QAVIDTVLAKK A* 1 i 20 MLT(E/D)RFN}-HL A* 02 21 ELERVFEPV A*02:0f. 22 AIT(E/'Q)VECFL A* ()2 : 01 23 (R/K)I..DSE1SMY' A* O i SVKVNLEKi-i (NELMVVEAVTL A" ()2 LPGDPDMIRYIDRQG AM/AZWBWBE’L) LEVKTG-VDAITEVEC A24/A29/B7/B39 "£3EESE{EEii?'?éiéiéé??é?é"""""""""""""""""""""""XZil?liiéié?éé"""""""""""""""""""""""""" VRVFDGTEQ A24/AZQ/B7/B39 G'I‘QQXVRGLARYFKER DRE 1 * 1. 1/8 FKIRLRKRS DRE 1 * 1 1 REAYLRKCKEFHPDK ,f,‘ DRE 1 * i3 WDEDLFCE-EEDMFASD DQBS * ()1 CFTQVVFGLDLTEETL ‘ DRE 1 * 03/04 (3GBEDKMKRMNTLYK LTA/‘STA DRE 1 * 13 KMKRYVFNTLYKKMEQD ETA/STA DRBE * 13 FNVi’Ki?KYW’LFKGPI DRE 1 * 15 RRY‘NLFKGPIDSGKT DRE 1 * 'i S VGPLCKADSLYVSAA ND* AYLDKNNAYPVEC‘WI DMIRYIDRQGQLQTK SQHSWK m 121 LLIKGGVEV *ND: Net defineii Table 2: Exempiary epitape sequences from JCV hamulngaus t0 BKV epitepe sequences Epitope" Antigen PILA Restriction SEQ ED NO; EPKMRKAYL LTA/STA B*()7,I’B*08 FPPNSDT‘E.,Y STA 3*35 MELMDLLGL FFSVGGEALEI; YCEDCFRQW " ‘ . .

R‘VSA; LLMGMYLQF VFLLMGMYLDF 3:E(E/G)SIQGGL LPGDPDMMRYXDEQLG LEVKEGVDSITEVEC DXCGMFENQSG?gQW DAQVEEVRVFEG’IEF HLA. AMIAZ9/8 7/1339 66 --------------------------------------- AAKQ A* 1 1 67 MMV/M?E/Q/GRFNQL J A*02 68 LLLIWFEQ’V A*02:01 67 A* 24 RGL§RYFKmLRKR? RKAYLEKCKELHPDK DRBms 77 WDEDLFCHEQMFASD ‘A DQBSWE 78 CFQQWFGQDLTQEAL LTA STA mama/04 79 GGDEDKMKRMTNELYK DRE 3 * 13 80 KMKRMNELYKKMEQQ DRE 1* 13 8 i LngggKRXWLFKGPIDSGKT P 1 DRBNS $2 NI)M 84 NEW 85 ND* * 86 "QEEEQEEEEEE"""""""""""""""""""""""""""""""""""""""""LFEE"""""""""""""""""""""""""m"""""""""""""""" FPMTLYC 3*35 125 I..-LIK_GGVEV ND"? ND* 12.6 *Amino acid residues which are variant fmm the BKV epitapss are bowed and underlined.

"Na defined Tahie 3: Exempiary e sequenws fmm JCV/BKV hybrid epitspe sequences ....................................................................................................................................................................................................................

HLA SEQ ID Epitope* Restrictian Na: gDiK}S(Q/K)HSTPP ' ' AVD’I'VMAKM CYCLIEQDCFWQ MPMMRKAYL FWL/l’ P/N 3/3 DTLY mLYCKEu/Pgm mm;WWI/VWK/Vm/GWWCY VHCPC M'I’ UM C YCMDCFGJ’RKQ/E?7 LLMQNWLMF VQMLLMGMYLMF MEWSIWGGL TEngiM/L} ja/VHgS/Lywiguwg LPGDPDMgI/Mgk?FWD(WK)gQ/Y)G- VPI A24/A29/B7/B3 i 9 Dg’l/V} ZGgL/MEQQN(SiR?G-{T/SZQQW VPI A24/A29/B7/B3 : c; QAVDTVMAKM A*1i c. "gag?Ragggg'agyg'"7g;Hga?g"g7;;;'-;;""""" : gAI/SIiSV‘WQgQX/ECFI, ??ismy sxl’mxmLEng-g AYLMKCKEM : : GHQQWRGLMRYFKgiivggwg; VP} ‘ :a. 1/8 RGLMRYFKg‘I/VggR/‘QELRKRM LTA DRE! 1 1/3 aa/DEDLFCI-iEmMFASD LTA DQBS*01 CFMQWFGMDLTMEML EMA/STA EDRBm/m .

GGDEDKMKRMNMIXK LTA DRE}, 1 3 ,.

Kuxmmimmmumwmmmu Egg Vii PKMRWJLFKGPIDSGKT VP} DREW 15 g R/K I..FKGPIDSGKT * *Not defined in some embodiments, provided herein are peptides comprising one or more of the epitopes from Table 1, Table 2 and/or Table 3. in some embodiments, the peptides disclosed herein are full length viral proteins (eg. full length BKV, JCV and/or MCV proteins). In U"! some embodiments, the peptide is not a titlhlengtli viral protein (e. 3;, not a full length BliV, JCV and/or MCV n), In. some embodiments, the peptides disclosed herein comprise BKV and lCV epitopes with sequence homology (rag. epitopes listed in Tables l-3). In some embodiments, the peptides disclosed herein comprise less than 130, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15 or l0 contiguous amino acids of a Viral protein. In some embodiments, the peptides sed herein comprise two or more ofthe epitopes listed in Table l, "fable 2 and/or Table 3. For example, in some embodiments, the peptide disclosed herein ses two or more of the epitopes listed in Table 1, Table 2 and/or Table 3 connected by polypeptide linkers. in some embodiments, the peptide provided herein comprises at least l, 2, 3, 4, 5, 6, 7, 8, 9, l0, ll, l2, l3, 14, 15, l6, l7, l8, l9, 20, El, 22, 2'3, 24, 25, 26, or 27 epitopes tag at least i, 2, 3, 4, 5, 6, ’7, 3,9, 10, ll,l2, l3,l4, lilo, 17,18, l9, 2t}, Zl, 22, 23, 24, 25, 26, or 2.7 of the epitopes listed in Table l, Table 2 and/or Table 3). ln certain aspects, provided herein is a. polypeptide and/or protein (eg, an isolated polypeptide or protein) comprising a plurality of epitopes from one or more BKV or JCV ns (6g, epitopes from LTA, STA or VPl viral antigens, such as the es listed in 2'3 Tables I, 2 or 3}. in some embodiments, the polypeptide or protein further comprises an intervening; amino acid sequence n at least two of the plurality of epitopes. lri some embodiments, the intervening amino acids or amino acid sequences are some liberation amino acids or amino acid ces. Non—limiting examples of proteasonie liberation amino acids or amino acid sequences are or comprise AD, K or R. In some embodiments, the intervening amino acids or amino acid sequence are TAP recognition motifs. Typically, TAP recognition motifs may conform to the tbllowing formula: {R/N?/QIW'IYln where n is any integer >_: l. Nornlimiting examples of TAP recognition motifs include Rl‘W, RQW, NTW and NQ‘Y} ln some embodiments, the es provided, herein are linked or joined by the proteasome liberation amino acid sequence and, optionally, the TAP recognition motif at the carboxyl terminus of each epitope.

In some embodiments, the polypeptides provided herein further comprise epitopes from and at least one additional virus (a g, Epstein Barr virus (EBV), cytomegalovirus (CMV), and/or adenovirus (ADVD. in some embodiments the peptides comprise epitopes two or more viruses. in some embodiments the peptides comprise epitopes three or more viruses, in some embodiments the peptides se epitopes four or more viruses, in some embodiments the peptides comprise es five or more viruses. For example, in some embodiments the peptides comprise sequences from at least two, three, four or ?ve of JCV, in; O BK‘V, MCV, EBV, CMV and/or ADV. in some embodiments, provided herein is a. polyepitope protein (119., a single chain of amino acid residues comprising multiple T cell es not linked in nature) comprising two or more of the epitopes described herein. in some embodiments, the T cell epitopes in the polyepitope pro ein are connected via an amino acid linker, ln some embodiments, the '1‘ cell ;._.\ L." epitopes in the polyepitope protein are directly linked. Without. intervening amino acids. es of polyepitope proteins, methods of ting polyepitope proteins, and vectors encoding polyepitope proteins can be found in Dasari errr[.,1ti’oleczilar lhempy — Methods 42 Clinic .17 Development (2.0%) 3 £60.58, which is hereby incorporated by reference in its entirety. in some embodiments, the compositions and methods ed herein comprise or relate to naturally occurring variants of the es listed in Tables 1 and/or 2. For example, in some embodiments, provided herein is a polyepitope protein that comprises two or more (cg, at least 3, 4, 5, 6, 7, 8, 9 or 10) lly occurring variants of an epitope listed in Table l and/or 'l‘able 2. in some embodiments, the sequence of the epitopes ed herein have a sequence disclosed herein except for l or more (tag. l 2, 3, 4, 5, 6, 7, 8, 9, id or more) conservative sequence modi?cations. As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not signi?cantly affect or alter the interaction between a TCR and a peptide containing the amino acid sequence presented on an HLA. Such conservative modifications include amino acid substitutions, ons (cg, 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). vative amino acid substitutions are ones in which the 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 de?ned 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 (a g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (£3.31, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta—branched side chains (cg, threonine, valine, isoleucine) and ic side chains (cg, tyrosine, phenylalanine, tryptophan, histidine) Thus, one or more amino acid residues of the peptides described herein can be ed with other amino acid residues from the same side chain family and the altered peptide can he tested for retention ofTCR binding using methods known in the art. Modifications can be introduced into an antibody by rd techniques known in the art, such as site—directed mutagenesis and ?CR—mediated inutagenesis. in some aspects, provided herein are cells that present one or more peptide described herein (9, g. a peptide comprising an epitope listed in Table l, 'l‘able 2 and/or Table 3). In some embodiments, the cell is a mammalian cell. in some embodiments the cell is an antigen—presenting cell (AFC) (eg, an antigenupresenting T-cell, a dendritic cell, a B cell, a macrophage or am cial antigen—presenting cell, such as aKS 62 cell). A cell presenting a peptide described herein can be produced by standard techniques known in the art. For example, a cell may e pulsed to encourage peptide uptake. in some ments, the cells are transfected with a nucleic acid encoding a peptide provided herein. In some aspects, provided herein are methods of producing antige presenting cells (Al’Cs), sing pulsing a cell with the peptides described herein. Exeniplaiy examples of producing antigen— presenting cells can he found in WOZOBngl l4, hereby incorporated in its entirety.

The peptides provided herein can be ed from cells or tissue sources by an appropriate purification scheme using standard. protein puri?cation ques, can be produced by recombinant DNA ques, and/or can be chemically synthesized using rd peptide synthesis ques. The peptides described herein can be produced in prokaryotic or eukaryotic host cells by expression of nucleotides encoding a e(s) of the present invention. Alternatively, such peptides can be sized by chemical methods, 3-3 Methods for expression of heterologous peptides in recombinant hosts, chemical synthesis of peptides, and m vim; translation are well known in the art and are described r in Maniatis et al, Molecular Cloning: A Laboratory Manual (1989), 2nd Ed, Cold Spring Harbor, N Y, Berger and Kimniel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques ( l987), Academic Press, Inc, San Diego, Calif.; Menitield, . 0969) J. Am. Chem. Soc. 9i :SOl; Chaiken l M. (l98l) CRC Crit. Rev. Biochem. ; Kaiser et al. (1989) e 243:187; Merri?eld, B. (1986) e 2321342; Kent, S. B. H. @988) Annu. Rev. Biochern. ; and. Offordl R. E 0980) Semisynthetic llroteinsl Wiley Publishing, which are incorporated herein by reference.

Provided herein are c acid molecules that encode the es and peptides described herein. The nucleic acids may be present, for example, in whole cells: in a cell in; O lysate, or in a partially purified or substantially pure form. A nucleic acid molecule described herein can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, oligonucleotides corresponding to the nucleotide sequence of one or more of the epitopes listed in Tables 1, 2, or 3 can be prepared by standard synthetic techniques, ice, using an automated DNA synthesizer. ;._.\ L." in some embodimentsl provided herein are vectors (erg a viral vector", such as an adenovirus based expression vector) that contain the nucleic acid molecules described herein. A viral vector may contain additional DNA segments may be d into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (cg, bacterial vectors having a bacterial origin of ation, episomal mammalian vectors). Other vectors (cg, non-episornal mammalian s) can be integrated into the genome of a host cell upon introduction into the host cell, and y be replicated along with the host genome. er, certain vectors are capable of directing the expression of genes. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). in some embodiments, provided herein are nucleic acids operable linked to one or more regulatory sequences (eg, a promoter) in an expression vector in some embodiments the cell transcribes the nucleic acid provided herein and thereby ses an dy/9 antigen binding fragment thereof or e described herein.

The nucleic acid molecule can be integrated into the genome ol‘the cell or it can be extrachromosoinal.

In some embodiments, the nucleic acid vectors or recombinant adenovinises provided herein encode one or more epitopes listed in Tables 1, 2, and/or 3. For example, the nucleic acid vectors or inant adenoviruses may consist of one or mo ’e epitopes from the same table (6.0 one or more epitopes from Table (7’9 l, one or more epitopes from Table 2, or one or more epitopes from Table 3). Or, the nucleic acid vectors or recombinant iruses may consist of one or more epitopes from the same table (e. 53., Table l), and one or more epitopes from a different table (cg Table 2). In some embodiments, the nucleic acid s or recombinant adenoviruses provided herein encode for no more than 20, 19, l8, l7, l6, l5, 14, 13, 12, l I, ll), 9, 3, 7, 6, 5, 4, 3, 2 or 1 amino acids in addition to the epitopes listed in es l, 2, or 3. in some embodiments, the nucleic acid vectors comprise nucleic acid sequences that have undergone codon optimization. ln such embodiments, a coding sequence is constructed by varying the codons in each nucleic acid used to assemble the coding sequence. In general, ll) a method to identity a nucleotide ce that optimizes codon usages for production of a peptide comprises at least the following steps (a) through (c). in step (a), oligoniers are ed encoding portions of the polypeptide containing degenerate forms of the codon for an tunino acid encoded in the ns, with the oligomers extended to provide ?anking coding sequences with overlapping sequences. in step (b), the oligoniers are treated to effect assembly ofthe coding sequence for the peptide. The reassembled peptide is included in an expression system that is operably linked to control sequences to effect its expression. in step (c), the sion system is transfected into a culture of compatible host cells. in smp (d), the colonies obtained from the transformed host cells are tested for levels of tion of the polypeptide. in step (e), at least one colony with the highest or a. satisfactory production of the ptide is obtained from the expression system. The sequence ofthe portion of the expression system that encodes the protein is determined. Further description of codon optimization is provided in. US. Patent Publication nuniher US20l0/O35 768, which is incorporated by reference in its entirety.

AntiaenPIescntmaMls in some aspects, provided herein are APCS that present (tag. on HLA) one or more T cell epitopes provided herein (ergo one or more T cell epitopes listed in Table l, Table 2 and/or ’I’able 3). in some embodiments, the HLA is a class l HLA. in some ments, the HLA is a class ll HLA. in some nicnts, the class l HLA has an or chain polypeptide 3-3 that is HLA-A, l-lLA-B, l-lLA-C, l-lLA~E, l-lLA—F, l-lLAsg, l-lLA~K or l-lLAJo. in some embodiment, the class ll l-lLA has an or chain ptide that is l-lLA~Dl‘le, l-lLA—DOA, HLA—Dl’A, A or l—lLA-DRA. in some embodiments, the class II MHLA has a [5 chain polypeptide that is l-lLA—DMB, l-lLA~DOB, l-lLA—DPB, Plinth-DEBS or l-lLA—DRB ln some embodiments, APCS present at least l, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, l3, ML, 15, 1.6, l7, l8,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 3l, 32, 33, 34, 35, 36, 37 or 38 T cell epitopes (eg. at least i, 2, 3, 4, 5, 6, 7, 8, 9,lO,ll,l2,13,14,15,16,l7,l8,l9, 20, 2l, 22, 23, 24, 25, 26, 27', 28, 29, 30, 3l, 32, 33, 34, 35, 36, 37 or 38 39 T cell epitopes Table l, Table 2 and/or Table 3), in some embodiments, the Al’Cs are B cells, n presenting ’ll—cells, dendritic cells, or arti?cial antige ., presenting; cells (t2. ;., aKSéZ cells). Dendritic cells for use in the process maybe prepared by taking PBMCS from a patient sample and adhering them to plastic. Generally the inonocyte population sticks and all other cells can be washed off. The adherent population is then differentiated with lL—4 and GlVl—CSF to produce incnocyte derived. dendritic cells. These cells may be matured by the on of Ill—ll}, Ill—6, PGE—l and TNF—d (which npregnlates the important coustimnlatory molecules on the surface of the dendritic cell) and are then contacted with a recombinant iins described herein. in some embodiments, the Al’C is an arti?cial antigen—presenting cell, such as an alifl 62 cell. In some embodiments, the arti?cial antigen—presenting cells are engineered to express CD80, CD83, L, and/or CD86. Exemplary arti?cial antigen—presenting cells, including al§562 cells, are described US. Pat. Pub. No. 200319147869, which is hereby incorporated by reference. ln certain aspects, provided herein are methods of generating APCs that present the two or more of the T cell epitopes described herein comprising contacting an APC with a nucleic acid vector and/or recombinant adenovirnses ng T cell epitopes described herein and/or with a polyepitope produced by the c acid, vectors or recombinant adenovinises described herein. in some ments, the APCs are irradiated. ln certain aspects, provided herein are T cells and tions of T cells (cg CD4 T cells and/or CD8 T cells) that express a TCR (e. 53., an all TCR or a yd TCR) that recognize a peptide described herein (eg, an epitope listed in Table l, Table 2 and/or Table 3;) presented on HLA. In some embodiments, the T cell is a CBS T cell (a C'l'li) that expresses a TCR that recognizes a peptide described herein presented on a class l l-lLA, In some ments, the T cell is a CDLl T cell (a helper T cell) that izes a peptide described herein presented on a class ll HLA. in some aspects, provided herein are methods of generating, activating and/or inducing proliferation of T cells e g, CTLs) that recognize one or more of the epitopes described herein. in some ments, a sample sing ?lls (in, a PBMC ) is incubated in culture with an AFC provided herein (eg, an APC that presents a peptide comprising a BKV and/or lCV epitope described herein on a class ll-lLA complex). ln some embodiments, the sample containing T cells are ted 2 or more times with Al’Cs provided herein. ln some embodiments, the T cells are incubated with. the APCs in. the presence of at least one cytolzine. in some embodiments, the cytolrine is ill—4, ill—7 and/or lL- l5. Exemplary methods for inducing proliferation of T cells using APCs are provided, for example, in US. Pat. Pub. No. 2015/00l7723, which is hereby orated by reterence. in some aspects, provided herein is a population of CTLS collectively comprising T cell receptors that recognize one or more T cell epitopes (e. g, one or more of the T cell epitopes listed in Table l, Table 2 and/or Table 3). In some embodiments, the CTLs ize two or more T cell epitopes from Table 1, Table 2 and/or Table 3. In some embodiments, the population of C'l'lis collectively comprise T cell receptors that recognize T cell epitopes from any combination of HIV, BKV, MCV, EBV, CMV, ADV and/or from other viruses. in some embodiments, the population of CTLs collectively comprise T cell receptors that recognize at least 1, 2, 3, 4, 5, o, 7, 8, 9, ll), ll, l2, l3, l4, l5, l6, l7, 13, l9, , 21, 22, 2'3, 24, 25, 26, 27, 28, 29, 30, Si, 32, '33, 34, 35 36, 37 or 38 T cell es (eg, at least 1, 2, 3, 4, 5, 6., or 7 T cell es from Table l and/or at least l, 2, 3, 4, 5, 6, 7, 8, 9, ll), ll, l2, l3, l4, ii 16, 17, 18, 19, 20, 21, 2.2, 23, 24, 25, 26, 27, 28, 2.9, or 30 ofthe epitopes . in Table l, Table 2 and/or Table 3). in some aspects, provided herein are methods of presenting or treating a polyomavirus infection (eg, a BKV, lCV, or MCV infection) or cancer (23., a polyomavirus associated. cancer, such as a BKV , lVC, or hllC'V associated cancer) in a subject comprising administering, to a subject, compositions (cg. therapeutic itions) comprising the nucleic acid vector described herein, peptides produced by the nucleic acid vector described herein, CTLS and/or APCS provided herein (agx, comprising the c acid vector described herein) and a pharmaceutically acceptable carrier. in some embodiments, the CTLs and/or APCs are not antologous to the subject. In some embodiments, the T cells and/or APCs are autologous to the subject. ln some embodiments, the T cells and/or Al’Cs are stored in a cell bank before they are administered to the subject.

Pharmaceutical Compositions In some s, provided herein is a composition (eg a ceutical composition, such as a vaccine composition), containing a peptide (cg, comprising an e from Table l), nucleic acid, nucleic acid vector, recombinant adenovirus, antibody, CTL, or an APC describe herein formulated together with a phannaceutically acceptable carrier, as well as methods oftreating cancer (eg, a avirus associated , such as a BKV, lVC, or h/lC'V ated cancer) or a polyomavirus infection (eg, a BKV, 343V, MCV, CMV, EBV, or ADV ion) using such pharmaceutical compositions in some embodiments, the composition includes a combination of le (9g, two or more) agents in; O provided herein. in some embodiments, the pharmaceutical composition ?irtlier comprises an adjuvant. As used herein, the term "atelier/ant" broadly refers to an agent that affects an logical or physiological response in a patient or t. For example, an adjuvant might increase the presence of an antigen over time or to an area of interest like a tumor, ;._.\ L." help absorb an antigen—presenting cell antigen, te macrophages and lymphocytes and support the production of cytol permit a smaller dose of an immune interacting agent to increase the effectiveness or safety of a. particular dose ofthe immune interacting agent. For example, an adjuvant might prevent T cell exhaustion and thus increase the effectiveness or safety of a particular immune interacting agent. es ofadjuvants include, but are not limited to, an immune modulatory protein, Adjuvant 65, o—GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, p~Glucan Peptide, CpG DNA, Gill—(3100, lipid A, lipopolysaccliatide, Lipovant, Montanide, N—acetylurnurzmiyl—L—alanyl—D—isoglutamine, Parn3CSK4, quil A and trehalose dimycolate.

Methods of preparing these formulations or compositions include bringing into association an agent bed herein with the carrier and, optionally, one or more accessory ingredients. in general, the formulations are ed by uniformly and intimately bringing into association an agent described herein with liquid carriers, or ?nely divided solid carriers, or both, and, then, if necessary, shaping the product.

Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more agents described herein in combination with one or more pharmaceutically—acceptable sterile isotonic aqueous or eous solutions, dispersions, suspensions or emulsions, or sterile powders which may he reconstituted into sterile injectahle solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, bullets, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or ning agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as ol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and inj ectable c esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of tants.

Regardless of the route of administration selected, the agents ofthe present invention, which may be used in a suitable hydrated form, and/or the ceutical itions of the present invention, are formulated into pharmaceuticallyuaccentable dosage forms by conventional methods known to those of skill in the art.

Therargeutic Methods in certain s, provided herein are methods of treating and/or ting cancer (eg a polyomayirusvassociated cancer, such as a BKV~, lCV—, or Mill/associated cancer) or a polyomavims infection (tag, a BKV, JCV, or MCV infection). in some embodiments, the method comprises administering to the subject pharmaceutical composition comprising a GIL, AFC, polypeptide and/or nucleic acid molecule bed herein. in some embodiments, tl e subject treated is compromised. For example, in some embodiments, the subject has a T cell de?ciency. in some embodiments, the subject has leukemia, lymphoma or multiple myeloma. In some embodiments, the subject is infected with HlV and/or has AIDS, in some embodiments, the subject has undergone a tissue, organ and/or bone marrow transplant. ln some embodiments, the sub} ect is being administered immunosuppressive drugs. in some embodiments, the t has undergone and/or is undergoing chemotherapy. In some embodiments, the subject has undergone and/or is undergoing radiation therapy. in some embodiments, the subject has cancer. in some embodiments, the methods described herein may be used to treat any cancerous or tire—cancerous tumor. ln some ments, the cancer expresses one or more ofthe BKV, MCV or JCV epitopes provided herein (eg. the BKV or .lCV epitopes listed in Tables l, 2, or 3). In some embodiments, the cancer is Merkel cell carcinoma. In some embodiments, the cancer includes a solid tumor.

Cancers that may be treated by methods and compositions provided herein include, but are not limited to, cancer cells from the bladder, blood? bone, bone marrow; brain, breast, colon; esophagus", gastrointestine, gum; head, kidney; liver, lung, nasopnarynx, neck, ovary, prostate, skin, stomach, testis, tongue, or nteins. In on, the cancer may speci?cally lie oftlic ing histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell. carcinoma; small cell carcinoma; papillary carcinoma; us cell carcinoma; oepitlielial carcinoma; basal cell carcinoma; atrix carcinoma; transitional cell oma; papillary transitional in; O cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcmoma; liepatocellnlar carcinoma; combined hepatocellular carcinoma and. cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoicl tumor, malignant; hi'ancliiolo—alyeolar adenocarcinoma; papillary adenocarcinoma; chromopliobe carcinoma; ;._.\ L." acidopliil carcinoma; oxypliilic adenocarcinoina; 'basoplnl carcinoma; clear cell arcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine arcinoma; ous adenocarcinoma; ceinminous adenocarcinoma; pidernioid carcinoma; cy stadenocarcinoma; papillary cyst‘adenocarcinonia; papillary serous cystadenocarcinoma; mncmous cystarlenocarcinonia; mocinons iadenoearcmoma; Signet ring cell carcinoma; in?ltrating duct oma; medullary carcnoma; lobnlar carcinoma; inflammatory carcinoma; mammary paget's disease; acinar cell carcinoma; quamous carcinoma; adenocarcinoma w/squamous asia; malignant tliymoma; malignant ovarian stromal tumor; malignant tliecoma; malignant granulosa cell tumor; and, ant roblastoma; sertoli cell carcinoma; malignant leydig cell tumor; malignant lipid cell tumor; ant paraganglioma; ant extra~mammary paraganglioma; pheochromocytoma; gloniangiosarcoma; ant melanoma; amelanotic melanoma; super?cial spreading melanoma; malignant ma in giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant ?brous liistiocytoma; myxosarcoma; liposarcoma; leiornyosarcoma; rhabdoniyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarccma; stromal sarcoma; malignant mixed tumor; mnllerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarconia; malignant liymoma; malignant hrenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant rnesothelioma; (1y sgerminoma; embryonal carcinoma; malignant teratorna; malignant stnima ; choriocarcinoma; malignant mesonephroma; hemangiosarcoma; malignant hemangioen(lothelionia; kaposi's sarcoma; malignant hemangioperi cytonia; lympliangiosarcoma; osteosarcoma; juxtacortical osteosareoma; chondrosarcoma; malignant chonclrohlastoma; mesenchymal cliondrosarcoma; giant cell tumor e; e ving’s sarcoma; malignant odontogenic tumor; ameloblastic odontosareoma; malignant blastoma; arneloblastic fibrosarconia; malignant pinealoma; choralorna; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; ?brillary astrocytoma; astrohlastoma; gliohlastoma; oligodendroglioma; oligodenrlrohlastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneurohlastoma; neurohiastoma; i‘etinoblastoma; olfactory neurogenic tumor; malignant meningioma; neuro?brosareoma; malignant neurilemmoma; malignant granular cell tumor; ant lymphoma; Hodgkin’s disease; l-lodgkin's lymphoma; paragranuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; ular malignant lymphoma; mycosis fungoides; other speci?ed non—l—lodgkin's mas; malignant histiocj?osis; multiple ma; mast cell sarcoma; immunoprolilerative small intestinal disease; leukemia; lymphoid ia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; niyeloirl leukemia; hasophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryohlastic leukemia; myeloid sarcoma; and hairy cell leukemia. in some embodiments; the subject is also administered an anti-Viral drug that inhibits BKV or JCV ation. For example; in some embodiments; the subject is administered ganciclovir; valganciclovir; foscamet; ciclofovir; vir, formivirsen; marihavir; BAY 38» 4766 or GWZ?5l75X. in some embodiments; the t. is also administered an immune checkpoint inhibitor. immune Checkpoint inhibition broadly refers to inhibiting the checkpoints that cancer cells can produce to prevent or clownregulate an immune response. Examples of immune checkpoint proteins include, but are not d to, C'l'LALl; PD—l; ; l’D—LZ; AZAR; B7-llf3; 3%ch BTLA; KlR, LAGS; 'l‘llVl—3 or A; immune checkpoint 3-3 inhibitors can be antibodies or antigen binding fragments thereof that bind to and t an immune checkpoint protein. Examples of immune checkpoint inhibitors include; but are not limited to, mab; peinhrolizumab; pidilizumab; AMP-224, AMP~5 14; STE—Al 1 it); WO 49165 2017/050686 KER-042, EKG—7446, BMS~936559, MEDl-4736, MSBJlOZO'Il 3C, AUR—Ol 2 and STl— Al Oltl.

In some embodiments, a composition provided herein is stered prophylactically to prevent cancer and/or a BKV , MCV or JCV infection. in some embodiments the composition may be administered prior to or after the detection of cancer cells or BIO/n, MCVn or JC‘Vminfccted cells in a subject. in some embodiments, after administration of a composition comprising peptides nucleic acids, C'I'Ls, and/or Al’Cs described herein, a prriintlaniinatory' response is induced. The proin?anim atory immune response comprises production of proint‘lammatory cytokines and/or chemokines, for example, interferon gamma (iFN «7) and/or interleukin 2 (lL—Z).

Conjunctive therapy es sequential, simultaneous and separate, and/or co— stration ofthe active compounds in such a way that the therapeutic: effects of the first agent administered have not entirely disappeared when the subsequent treatment is administered. in some ments, the second agent maybe contomiulated with the first agent or be formulated in a separate pharmaceutical composition In some aspects, provided herein is a method ofidentitying a t suitable for a therapy provided herein (cg. s oftreating a BKV, JCV, or MCV infection and/or cancer in a subj ect comprising administe ring to the subject a pharmaceutical composition provided herein). in some embodiments, the method comprises isolating a sample from the subject (cg a blood sample, a tissue , a tumor sample) and detecting the presence of an epitope listed in Tables l or 2 in the sample. in some embodiments the e is detected using an ELISA assay a n blot assay, a FACS assay: a ?uorescent microscopy assay: an Edman degradation assay and/or a mass spectrometry assay (cg, protein sequencing). in some embodiments, the presence of the BKV or JCV epitope is detected by detecting a nucleic acid encoding the BKV. MCV or lCV epitope. in some embodiments, the nucleic acid encoding the BKV, MCV or JCV epitope is detected using a nucleic acid probe, a nucleic acid cation assay and/or a sequencing assay in some embodiments, the method comprises HLA typing of the t. in some embodiments, the suhject is fied as suitable for treatment With a method. provided herein if the subject expresses an l-iLA to which an epitope provided herein is restricted. in some embodiments, the methods provided herein further comprise treating the fied subject using a therapeutic method provided herein toga, by administering to the subject a pharmaceutical composition provided herein). in some embodiments the subject is administered a composition comprising CTLs described herein, wherein the CTLs comprise TCRs that recognize an epitope provided herein that is HLA restricted to an HLA expressed by the subject. In some embodiments the subject is administered a composition comprising a polypeptide comprising an e provided herein that is HLA restricted to an Hl_,A expressed by the subject. in some embodiments the subject is administered a composition comprising an APC presenting a polypeptide comprising an epitope provided herein that is HLA restricted to an HLA expressed by the subj ect. in some embodiments the subject is administered a composition comprising> an c acid encoding a ptide comprising an e provided herein that is l-lLA restricted to an HLA expressed by the subject.

EXAMPLES Exam 19 f: CD8+ T6612 resim‘zses are directed towards [114 and SE4 while CD4+ Tcelz’ res cases are directed towards LTA VP] and 5714 PBMCS from healthy volunteers were incubated with BK'V Ol’l’s and cultured these cells for 14 days in the presence of lL~2 and T cell growth factor (TCGF), On day 14, these T cell es were assessed for SKY—speci?city using 105% assay. Figure 1 shows that m Wire culture of T cells with BKV peptides for l4 days resulted. in ion of virus—speci?c T cells. in some cases? these expansions were comparable to CMV—speei?c T cells. A detailed y of the T cell assays based on in vitro expanded T cells is presented in Figure 2. These initial analyses y showed that CBS+ T cell responses were predominantly directed towards LTA and STA, while CD4" T cell responses were directed towards LTA, VPl and STA. To validate these ations, T cell assays were repeated in 50 volunteers (including many volunteers from the first set of assays) and a summary of this analysis are presented in Figure 2. Consistent with the data presented in Figure 2., dominant (IDS+ and CD4+ T cell responses were detected. towards ETA, STA and VP} antigens Exam {6 2: further Clzaitacz'erz'zi-izion of 1’ ’ cell responses in order to terize the T cell ses directed. towards these ns and precisely map the HLA class i and class ll-restricted T cell responses, individual overlapping peptides (l 5 aa long pping by ll) an) were sourced for LTA, STA and VPl proteins for T cell epitope mapping. A two—dimensional peptide matrix was used to distribute all individual peptides into small overlapping peptide pools. The matrix is set up in the way that eaeh peptide occurs once on the ordinate (Figure 3). These peptide pools were used. in TCS assays. After the K33 analysis, T cell response to the e pools was compared with the matrix to fy dual peptides. These individual peptides were r assessed for T cell expansion and lCS analysis to identify ial BK. Once the l5nier peptide was identi?ed, further mimimalization of the epitope sequence was carried out to identify the optimal T cell epitope sequence. The l5 mer peptide sequences were trimmed from both N— and C~terminus to a minimal. of 9 as long peptides. Once the minimal peptide sequence was identified, further confirmation was carried out using ng dose titration lCS assay After mapping minimal epitope sequence, the l-lLA restriction of the epitope was identified by stimulating T cells using peptide loaded HLAnmatched and mismatched LCLs. The complete process of e mapping is shown in the flowchart provided. in Figure 4.

Representative data from one of the BKV epitope mapping s is shown in Figure 5. Data presented in Figure 5, Panel A shows that BKV—speciiic T cells from healthy volunteer H26 recognized STA 0P9. in order to map the T cell epitope thither analysis was carried out using sub pools of STA peptides (ll pools) designed based. on the two dimensional matrix shown in Figure 3. intracellular cytoltine analysis based on STA peptides showed that pools 4 and 10 were ef?ciently recognized by Cl) 8+ T cells, which when overlayed on to the matrix layout showed S'TAZZ peptide as the common peptide sequence among the responding pools (Figure 5, Panel B). The peptide trimming> process showed PCMLCQL to he the T cell epitope (Figure 5, Panel C and D). The l-lLA restriction analysis using the HLA matched LCLs showed Vl—lCPCML-CQL to he an l-lLA B*39— restricted epitope {Figure 5, Panel. E) Similar epitope mapping process was carried out for other CD4" and CBS+ T cell epitopes. The list of CBS" and €134" BKV epitopes mapped during this study is listed in Table 4 and 5, respectively.

Table 4: CD8+ epitopes CD8 es e HLA Restriction SEQ ID NO; LPLMRKAYL L'l‘A/S'I'A 3* 07/8 * 08 mpm gm 5 WWW 6 Ema-Day gm 7 VHCPCMLCQL 3* 39 8 NREESMELMDL LTA/‘STA 8*49 9 I.;GL LITA/STA B* 4-0 I 0 FFAVGGDPLEM STA 3*40 1 1 fcu>c?ré§>¥ 37m 3*57 1* TPHRHRVSA E§mmmmmmm" '\/'15'I_{LI_..GMYLEF IEESEQGGL "EEKEQEEEME:_________________________________________________________________________________________________________________________________________________________________ ARIPLPNL VKNP‘H'PESFLL QAVDTVLAKK "MMEEEEE_______________________________________________________________________________________________________________________________________________________________ LLLEWFRPV .f?IEYEQEmemmmmmmmmmm- .mmmmu?igggammmmmmm:mmmmmmmmmm SMY A* ()1 §m§@g%§mmmmmmm?;4mmmm AYLRKCKEF AMI/AZWBE’B" 33 LPGDPDMIRYIDRQG ....................................................................................................................................................................................................< AM'IAzg/BWB" LEV’K'E‘G‘V'DAITEVEC [A24/iA29/IB7/839 3: 1 ESQV’EEV’RV’FDGTEQ ‘VPI Table 52 (1134+ es .........................................................................................................................................................................................................

CD4 es Enitope Antigen HLA Restriction SEQ ED NO; .......................................................................................................................................................................................................

GTQQWRGLARYFKZR VPl DRE 1* 1 1/8 RGLARYFKIRLRKRS DEB 1* l 1 RKAYLRKCKEFE—iPDK DRE 1 * l3 CHEDMFASD DQBS * Oi CFTQW’FGLDLTEETL one} res/o4 GGDEDKMKRMNTLYK L’l‘AfS'I'A KMKRNTNTLYKKMEQD LTA/STA DRE 1 * l3 FNVPKEQQYWLFKGPI L'I'A DRE 1 * l5 RRYVVLFKGPIDSGKT DRE} * l 5 AYIDKRNAY?VECVVI ND’i‘ nMiRYinaoGoLoTK VP} Exam [6 3: Pro 'iiz'mi wzciz'onai and ahenot' ic characteristics (27"BKV sneci?c T cells in health ‘ individuals and trams [am recz' rents in recent years the T—box transcription factors {T—bet) and Eomesoderniin ) have been shown to play important roles in determining the fate of CD8+ T cells during infection. High levels ot‘T—bet are ated with the cytotoxic T ceil differentiation and unregulation of m and Granzynie B in antigen speci?c cells. A high level ot‘Eoines is associated with. the long term. memory fonnation. It has been seen in various snidies that their cooperative expression is critical for infection control. in mouse studies it has also been shown that the deletions of either ofthe transcription factors have resulted in failure to control infection. Hence it is critical to study the sion oi‘tliese transcription factors which could help in the understanding the phenotypie characterization. of the T cells and T cell entiation during both acute and chronic viral infections. The expression patterns of T—bet and Eonies in BK‘V speci?c 1" cells is not yet been understood and the analysis ofthe transcription factors on these T cells may enable a deeper understanding on the differentiation ofBKV specific T cells A detailed study on the functional characteristics of T cells could also lead to development of effective immunotherapy for BKV associated diseases. An initial set of ments have started to study the transcriptional s on the ' ‘ cells which regulate the differentiation ofthe T cells. The expression of "f—‘oeh Eomes, perform and granzyme B were assayed on the BKV specific T cells and CMV specific T cells using ES The initial analysis showed a medium to low level of T bet expression in BKV specific '1" cells while high. levels of"l‘~bet was seen with CMV ic T cells (Figure 6). Also, very low expression of Homes was found with BKV speci?c T cells in comparison to the CMV speci?c T cells. Low levels ‘forin and gi'anzyrne B was also seen with BKV speci?c T cells. This preliniinaiy data suggests that BKV specific T cells could be functionally low in effector function, Hence driving the effector function V speci?c CTLs will he the focus ofmy study which could help in developing an effective adoptive T cell inim unotherapy.

Exam 7142 4: BKV-S eel it: 1" cell expansion.

BKV—speeitic T cells were expanded in Wire following ation with pooled BKV epitopes. ically, PBMC from healthy volunteers were stimulated with synthetic BKV peptides (Table l) for l hour and then cultured for lZ~l4 days in the presence ent cytokine combinations, including lL—Z (lllng/nil), lL—‘Zl (Sting/nil), lit/7 ( l Orig/ml), lLlZ (l Orig/ml) and/or lLl 5 (l Orig/ml). The BKV specificity of the extianded T cells was assessed using standard intracellular cytokine assays e 7).

Examvle 5: Generation 0 "Consensus Alinnmems To identify JC virus epitopes homologous to BKV epitopes described herein, the NCBl Blastp ce alignment program was used to align the amino acid sequences of the BKV and JCV Large T Antigen (ETA) n, Small T Antigen protein (STA), and VPl protein, respectively: and homologous sequences were identified (Figure 8, epitopes highlighted). The NCBI Blastp sequence ent program was also used to align the amino acid sequences ofthe BKV and thV VPl protein to identify homologous sequences 3-3 (Figure 9) F‘ ' "e (3st ansion ofCTLs in the oresence M1202] BK‘V specific T cells were generated using the PBMCs from healthy donors. PBMCS were stimulated in VHFO with respective BKV peptide pools at a concentration of l ugi’nil and incubated at 37°C, 65% C02 for an hour. The cells were then washed and split into two to he cultured in two conditions. A part of the cells was grown in the Rift medium (RPMI + % FCS) ning 30 ngfnil oflLZl (Milteyni Biotech Ltd) in 24 well plates incubated at 37°C, 6.5% (‘02. Another part of the cells was ted with RlO medium without ill—2; l.

The cultures grown in both conditions were mented with Rift medium containing recombinant interleukin—2 (Charles River Laboratory, hill-l, l} SA) at 20 lU/ml on day 2 and then supplemented with media containing an2 every 3 days thereafter until day 29.

T cells in the cultures were counted and. required amount of cells were used for an lF‘N—y intracellular cytokine (lCS) assay while the remaining cells were cryopreserved in liquid nitrogen. Approximately 2le5 of CTLs were added to a 96 well V—bottom plate. Cells were stimulated with respective peptides at a concentration of 1 gig i’inl in thl medium containing Golgiplug Brefeldin A (El) l’hamiingen, San Diego, CA) and incubated, at 37°C, 6.5% CO2 for four hours. BKV speci?c T cells were recalled with both BKV peptide and its respective JCV variant and vice versa for JCV speci?c T cells. After incubation, the cells were washed with PBS containing 2% hBS (wash ) and. the pellet was rcsuspended in 50 all of wash buffer containing "TC-conjugated D4 and PerCP—CyiS conjugated anti-CD8 antibodies and incubated at 40C for 30 minutes. Cells were then washed twice with PBS, fixed and pernieabilized with Cytolix/Cytoperin solution (Bl) l’liarniingen) for 20 mins. Cells were then. washed and incubated, with PE— arrti—lFN—y antibody diluted in Perniwasli buffer at 4°C for 30 s. Stained cells were washed twice with Peirnwash butler, resuspencled in PBS containing l% paratoimaldeliytle and acquired using using a El) LSR Fortessa, Post—acquisition analysis was conducted using Flowlo re ('l'reeStar). lFN—y sion of the cell populations is provided in Figure l l, while the numbers (£1394 and CD8 cells in the expanded cultures is provided in Figure l2.

The effect of the presence oflL~21 in the culture on transcription factor and effector molecule sion was . Approximately leOS of C’l‘lis grown in both ions were added to a 96 well V~hottom plate. The cells were washed. with PBS containing 2% PBS (wash buffer) and the pellet was re suspended in it) uL ofPBS containing l iii of respective APC conjugated BKV speci?c dextramer and incubated ark/10C for 20 minutes.

Cells were then added with PE—Cy’] CD4 and V3300 CD8 antibodies and incubated at 4°C for minutes, Alter incubation, cells were washed twice with PBS, ?xed and penneahilized with transcription factor x/CYlOpBHH solution (BB Pharrningen) for l hour. Cells were then washed and incubated with eflour7l0— anti—homes antibody, AFloll—conjugated—anti— GranzyineB, Zl ated antivl’erforin and Pl?) conjugated antim'lhet dies diluted in Permwash buffer at 49C for 30 minutes. Stained cells were washed twice with Perinwash buffer, resuspended in PBS containing l% paraihrinaldehyde and acquired using using a BI) LSR sa. cquisition analysis was conducted using Flowlo so?ware ('l‘reeStar).

Transcription factor and effector molecule expression in the cell populations shown in Figure The effect oi‘tlie presence oi‘anZl on expansion of regulatory T cells was also tested. Approximately 2x ll)5 of C’l‘Ls grown in both conditions were added to a 96 well V— bottom plate. The cells were washed with hBS containing 2% PBS (wash buffer) and the pellet was resuspended in 50 uL of PBS containing FlTC conjugated anti—CD39 Paci?c blue conjugated anti—CD4, PEcy’?‘ conjugated antinCDZS, Plinconjugated antinneuropilinl. and BV786 conjugated anti—Cl) 127 antibodies and incubated at 40C for 30 minutes. Cells were then mixed with PEuCy7 Clint and V590 CD8 dies and incubated at 4°C for 30 s. After tion, Cells were washed twice with PBS, ?xed and pei‘meahilized with Foxl’S Cyto?x/Cytopcrm solution (chioscienees Ltd) for l hour. Cells were then washed and incubated with APC conjugated anti-FoxP3 antibody diluted in Perniwash buffer at 4°C for 30 minutes. Stained cells were washed twice with Pennwash butler, resuspended in PBS containing l% parathrinalclehyde and acquired using a El) LSR Fortessa. l’ostnacquisition analysis was conducted using Flowlo software ('l‘reeStar). The presence of regulatoiy T cells in the cell populations is shown in Figures 13 and l4‘ .lCV variants for mapped BKV epitopes were synthesized. BKV and JCV specific T cells were generated using the PBMCs from healthy donors. PBMCS were washed and resuspended in RlU (Rl’Ml +10% FCS). The cells were then stimulated in vim; with tive BKV and 36V peptide separately at a tration of l gig/ml and incubated at 3-3 37°C, 6.5% (X): for an hour. The cells were then washed and grown for l4 days in 24 well plates incubated at 37°C, 6.5% (202. The cultures were mented with Rlll medium containing recombinant interleukin-2 (Charles River Laboratory, Nil-l, USA) at 20 lU/rnl on day 2 and then supplemented with Rl fl medium containing ilk/’3 every three days thereafter until day l4. On day 14, T cells in the cultures were counted using the Trypan Blue exclusion method and required amount of cells were used for an lFN—y intracellular cytol (lCS) assay while the ing cells were cryopreserved in liquid en.

T cell cross reactivity was ined, by measuring 1?wa expression following '1‘ cell nulation with BKV or JCV epitopesi Approximately 2x10S of CTlis were added to a 96 well V-liottoni plate. Cells were stimulated with respective peptides at a concentration of l gig /nil in Bill medium containing Golgiplug Breteldin A (Bl) l’liamingen San Diego, CA) and incubated at 37°C, 65% C02 for four liours. BKV ic T cells were recalled with both BKV peptide and its respective JCV variant and vice versa for JCV specific T cells. After incubation, the cells were washed with PBS containing 2% PBS (wasli butter) and the pellet was resuspended in 5‘) pl; ofwash buffer containing MIC-conjugated, anti— CD4 and PerCP~Cy5 .5 conjugated anti-CD8 antibodies and incubated at 40C for 30 minutes.

Cells were then washed twice with PBS ?xed and pernieabilized with Cyto?X/Cytopenn solution (ED l’narrningen) for 20 niins. Cells were then washed and incubated with PE— anti— ll‘N-y antibody d in Perniwash buffer at 4°C for 30 minutes. Stained cells were washed twice with sh butler, ended in PBS containing l"?/£i parafornialdenyde and acquired using using a Bl) LSR Fortessa. Pest—acquisition analysis was conducted using Flowlo software eStar), Representative lFN—y expression data is shown in Figure l5.

The peptides that responded both in BKV and .lCV specific T cells were further analysed for avidity using limiting dose titration assay. The peptides were titrated l0 fold starting from l pg frnl upto a concentration of lil'5 pg ,I’rnl. These titrated peptides were then used, to recall the BKV and JCV speci?c C'l‘Ls in standard lFNmy intracellular cytokine assay. Representative titration assay data is shown in Figure 16.

Epitope crossmreactivity is provided in Table 6 (for {708 epitopes) and Table 7 (for CD4 epitopes).

Table 6: BK‘WJCV Cross—reactivity (if exemplary CD'S epitopes.

C98 eito es tnnv and JCV BKV se. uence JCV so. uenee -reaetiviti NREESMELMDL Witness/inm/int 'es malarialitter i‘v‘lEl’sl‘i/llllil?li es SQHSTPPKK lsonsrernn lYes ETN-ERHRVSA E-ERVSA es EAVIm/TLAKK AVDTVAAEQ 0 rmpmzwrpmmm gum 2%may S'E’L‘v w .zucy ?mcmcgm CYCFDCFRQ Eye; DCFTi w ELLIKGGVEV EAETEVECFL WJLMWEAVTV ELLLGMYLEF aRKML BKV and JCV Cross~ reactivitv (’TQQV‘ MIME" FKIR GSOOWRGLSRYFKVO RGLARYFKIRLRKRS {GLSRYFKVOLRKRR CHEDMFASD WEED!19KYBiif?i-{EliSD REQAYLRKCKEFE-{f-‘DK RK4YEKKCKELHPDK GGDEBKR/EKRIXIENTLYK GC§£>EiDIGMKfM£NPZYK KMKRNWTIIYKKMEQD IQMKRIS-QTFEYIQGMCEQG 2E1?.3}?RRQEL.E§S§EE........ ‘ RRYWLP .G'i’EfBSGK'i" CF11 WFGLDLTEETL {ZEROWE'(3CELL"75.41:, AYLDKNNAYPVEC‘WI : EIDKNKAYPVECWV VGPLCKADSLYVSAA VGPLCKGDAY,YLEMV

Claims (11)

Claims

1. A method of expanding BK virus-specific T cells for adoptive immunotherapy, the method comprising: (i) contacting a population of T cytes obtained from a subject with a peptide pool comprising at least five peptides that have the amino acid sequences of SEQ ID NO: 87-120; and (ii) culturing the population of T cells under conditions such that BK virus-specific T- cells are expanded from the population of T cells.

2. The method of claim 1 wherein the peptide pool further comprises peptides that have the amino acid sequences of SEQ ID NO: 2, 4, 5, 6, 15, 22, 23, 34, 36, 39, 40 and 41.

3. Use of the expanded BK virus-specific T lymphocytes of claim 1 or claim 2 in the manufacture of a medicament for treating or preventing a polyomavirus infection in a human t.

4. The use of claim 3, n the polyomavirus infection is a BKV infection.

5. Use of the expanded BK virus-specific T lymphocytes of claim 1 or claim 2 in the manufacture of a medicament for treating or preventing a polyomavirus-associated cancer in a human subject.

6. The use of claim 5, wherein the avirus-associated cancer is a BKV associated cancer.

7. The use or the method any one of claims 3 to 6, wherein the subject is immunocompromised.

8. Use of a pharmaceutical ition comprising xic T cells (CTLs) comprising T cell receptors (TCRs) that recognize five or more epitopes, with the epitopes having amino acid sequences according to SEQ ID NO: 87-120in the manufacture of a medicament for treating or preventing a polyomavirus-associated cancer in a human subject.

9. The use of claim 8, wherein the one or more epitopes further comprises the epitopes having amino acid sequences ed from the group consisting of the amino acid sequences of SEQ ID NO: 2, 4, 5, 6, 15, 22, 23, 34, 36, 39, 40 and 41.

10. The use of claim 8 or claim 9, wherein the polyomavirus is a BK virus (BKV).

11. Use of cytotoxic T cells (CTLs) comprising T cell receptors (TCRs) that recognize five or more epitopes, with the epitopes having amino acid sequences according to SEQ ID NO: 87-